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<strong>Environmental</strong> <strong>and</strong><br />
<strong>Molecular</strong><br />
<strong>Mutagenesis</strong><br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
An international journal speciplipng jp :~<br />
• <strong>Molecular</strong> mechanisms of mutaganesis<br />
• DNA dama e <strong>and</strong> repair '?<br />
• Genetic an d cytogenetic methodology<br />
• Screening for mutagens <strong>and</strong> antimutagens<br />
• Mutagenic hazards for humans ;<br />
• Genetic toxicology <strong>and</strong>'risk assessment<br />
ISSN 0893-6692
<strong>Environmental</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Mutagenesis</strong><br />
JOURNAL OF THE ENVIRONMENTAL MUTAGEN SOCIETY<br />
OFFICERS, ENVIRONMENTAL MUTAGEN SOCIETY<br />
President President-Elect<br />
S.M. Galloway R .J . Preston<br />
COUNCILORS<br />
J.W. Allen A.V. Carrano<br />
D. Anderson D .A . Casciano<br />
M. Bloom D.M . DeMarini<br />
B .E. Butterworth J .M . Gentile<br />
EDITOR-IN-CHIEF<br />
Richard J. Albertini<br />
Vermont Regional Cancer Center<br />
Burlington, Vermont<br />
REVIEWS EDITORS<br />
Rosalie K . Elespuru<br />
NCI-Frederick Cancer Research Facility<br />
Frederick, Maryl<strong>and</strong><br />
Barry W . Glickman<br />
York University, Toronto, Ontario, Canada<br />
Michael D . Shelby<br />
National Institute of <strong>Environmental</strong> Health Sciences,<br />
Research Triangle Pork, North Carolina<br />
Raymond R . Tice<br />
Brookhaven National Laboratory, Upton, New York<br />
EDITORIAL BOARD<br />
Secretary<br />
R .K . Elespuru<br />
P.C. Hanowolt<br />
D . Jacobson-Kram<br />
B .H . Margolin<br />
B .C . Myhr<br />
Treasurer<br />
J .T. MacGregor<br />
P. Ostrosky<br />
M . Plewo<br />
C .A. Schreiner<br />
J .S . Wasson<br />
ASSOCIATE EDITOR<br />
J . Patrick O'Neill<br />
Vermont Regional Cancer Center<br />
Burlington, Vermont<br />
STATISTICAL EDITOR<br />
Barry H . Margolin<br />
University of North Carolina, Chapel Hill, North Carolina<br />
BOOK REVIEW EDITOR<br />
James M . Gentile<br />
Hope College, Holl<strong>and</strong>, Michigan<br />
REFERENCE EDITORS<br />
Elizabeth S . Von Halle<br />
Oak Ridge National Laboratory, Oak Ridge, Tennessee<br />
Kathleen Mavournin<br />
Oak Ridge Notional Laboratory, Oak Ridge, Tennessee<br />
Seymour Abrahamson Sheila M . Galloway James T. MacGregor Herbert S . Rosenkranz<br />
University of Wisconsin Merck Institute for Therapeutic U .S . Department of Agriculture Case Western Reserve University<br />
Madison, Wisconsin Research Berkeley, California Clevel<strong>and</strong>, Ohio<br />
John Ashby Imperial<br />
Chemical Industries PLC<br />
Alderley Pork, Cheshire,<br />
United Kingdom<br />
Herman E . Brackman<br />
Illinois State University<br />
Normal, Illinois<br />
Anthony Carrano<br />
Lawrence Livermore Laboratory<br />
Livermore, California<br />
June H . Carver<br />
West Point, Pennsylvania<br />
Mary Esther Goulden<br />
University of Texas<br />
Dallas, Texas<br />
Philip E . Hartman<br />
The Johns Hopkins University<br />
Baltimore , Maryl<strong>and</strong><br />
George R . Hoffmann<br />
Centre Nationole de la Recherche<br />
Scientifique<br />
Strasbourg, France<br />
Donald G. MacPhee<br />
La Trobe University<br />
Bundooro, Australia<br />
Toijiro Matsushima<br />
University of Tokyo<br />
Tokyo,Japon<br />
J . Justin McCormick<br />
Michigan State University East Lansing, Michigan<br />
Robin W. Morgan<br />
Gary A . Sega<br />
Oak Ridge Notional Laboratory<br />
Oak Ridge, Tennessee<br />
Richard B . Setlow<br />
Brookhaven Notionol Laborotory<br />
Upton, New York<br />
Thomas R. Skopek<br />
Chemical Industry Institute of<br />
Toxicology<br />
Research Triangle Park,<br />
North Carolina<br />
Chevron <strong>Environmental</strong> Health Center<br />
University of Delaware<br />
Richmond, California<br />
Vicki L. Dellorco<br />
<strong>Environmental</strong> Protection Agency<br />
Washington, DC<br />
David M . DeMorini<br />
<strong>Environmental</strong> Protection Agency<br />
Research Triangle Park,<br />
North Carolina<br />
Henry E . Holden<br />
Pfizer Central Research<br />
Groton, Connecticut<br />
Joseph R . L<strong>and</strong>olph<br />
University of Southern California<br />
School of Medicine<br />
Los Angeles, California<br />
Newark, Delaware<br />
Kristien E . Mortelmons<br />
SRI International<br />
Menlo Park, California<br />
Earle R . Nestmonn<br />
CanTox Inc ., Oakville<br />
Ontario,<br />
Canada<br />
Sheldon Wolff<br />
University of California<br />
Son Francisco, California<br />
Friedrich E . Wurgler<br />
University<br />
of Zurich<br />
Schwerzenboch, Switzerlond<br />
Errol Zeiger<br />
Notionol Institute of <strong>Environmental</strong><br />
Eric Eisenstadt L . Gayle Littlefield Miahael J . Prival Health Sciences<br />
Harvard School of Public Health Oak Ridge Associated Universities Food <strong>and</strong> Drug Administration Research Triangle Pork,<br />
Boston, Massachusetts Oak Ridge, Tennessee Washington, DC North Carolina<br />
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<strong>Environmental</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Mutagenesis</strong> (ISSN 0893-6692 ; Coden EMMUEG) is published monthly except February, June, October, December by Alan R . Liss, Inc . . 41 East 11th<br />
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Subscription Price: Volumes 13 <strong>and</strong> 14, 1989, 8 issues, $180 in US . $212 outside US. All subscriptions outside North America will be sent by air. Payments must be made in US dollars<br />
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Contents/ Life Sciences ISI (BIOMED) • Index Medicus • BIOSIS-Data base • Exceipta Medica • Cambridge Scientific Abstracts . ISI-Soviet Union • Chcmicat Abstracts .<br />
The palrf on which rhrs,journas is printed adheres to the requirements for library/archival stability . Printed in the United States of America . CCopyright 1989 Alan R . Liss_ Inc_<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
<strong>Environmental</strong> <strong>and</strong> <strong>Molecular</strong> <strong>Mutagenesis</strong> Volume 14, Supplement 15 : 1-252 (1989)<br />
Abstracts of the<br />
Fifth International Conference<br />
on<br />
<strong>Environmental</strong> Mutagens<br />
Case Western Reserve University<br />
Clevel<strong>and</strong>, Ohio<br />
J u ly 10-15, 1989<br />
National Organizing <strong>and</strong> Program Committee Members<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Frederick J . deSerres, Chairman<br />
Richard J. Albertini John Mirsalis<br />
David Brusick R . Julian Preston<br />
Eugene L. Elmore Sheila Galloway<br />
Mortimer L. Mendelsohn Herbert Rosenkranz
ABSTRACTS<br />
I THE KINETICS OF SODIUM FLUORIDE-INDUCED ENDOREDUPLICATION IN CHINESE NAMSTER OVARY<br />
CELLS . M .J . Aardema,'D .P . Gibson, R .A . LeBoeuf . The Procter & Gamble Company,<br />
Cincinnati, OH 45239 (USA) .<br />
We previously reported endoreduplication in Chinese hamster ovary (CHO) cells<br />
exposed to high doses of NaF (Mut . Rea ., 1989) . To inveatigate further the induction<br />
<strong>and</strong> the mechanism of NaF-induced endoreduplication, we conducted kinetic studies after<br />
NaF exposure . With a 4 h exposure to 1 .276 mg/ml NaF, the highest percentage of<br />
endoreduplicated cells in the culture was observed 20 h after exposure . When cells<br />
were exposed to 20 yM BrdU for 1 h or 2 h before NaF treatment, the endoreduplicated<br />
cells harvested 20 h after exposure had 2-6 or 2-7 differentially stained chromosomes,<br />
respectively, or had no differential staining . The differential staining pattern corresponded<br />
to late replicating chromosomes . This indicates that cells which were sensitive<br />
to the induction of endoreduplication were in late S or G2 of the cell cycle .<br />
To examine the timing of the DNA synthesis phase leading to <strong>and</strong>oreduplication, cells<br />
were pulse labeled with tritiated thymidine for 15 min at 2 h intervals following NaF<br />
exposure <strong>and</strong> harvested 20 h after NaF treatment . The results indicated that DNA<br />
synthesis leading ~o endoreduplication lasts at least 18 h <strong>and</strong> is followed by a normal<br />
2 h G2 phase . The delayed appearance of endoreduplicated cells, the sensitivity of<br />
S/G2 cells, <strong>and</strong> the long endoreduplication DNA synthesis phase observed in our studies<br />
are very similar to that reported for other agents (e .g . cytosine arabinoside) which<br />
are proposed to induce endoreduplication by inhibition of DNA synthesis . These results<br />
are consistent with a mechanism of NaF-induced endoreduplication by an inhibition of<br />
DNA synthesis as we previously suggested for NaF-induced chromosome aberrations .<br />
L<br />
COMPARATIVE MUTAGENICITY TESTING OF A DRUG CANDIDATE : BROPIRIMINE, A<br />
POTENTIALLY POWERFUL ANTIVIRAL COMPOUND . C.S . Aaron, A . Thilagar, V . Kumaroo,<br />
T. Petry, J . Mayo, D . Branstetter, J . Mazurek, The Upjohn company, Kalamazoo, MI ; Sitek<br />
Research Laboratories, Rockville, MD .<br />
Comprehensive genetic risk assessment of potential drugs involves more detailed analysis than the<br />
assignment of positive or nagative values to the outcome of a battery of tests . Bropirimina was subjected<br />
to a variety of qenotoxicity ssssYs <strong>and</strong> presents a complex risk assessment problam. Bropirimine was<br />
negative in the Ames sssay,<br />
. UDS, rst micronudeus assay <strong>and</strong> the LS178Y TKt mouse<br />
lymphoma assa Ho, wever slka induced the line elution a compound stronyIy non-Iinear incraase In chromosome<br />
aberrations (CA)y(as high as 42 % of the cells had aberrations at S00 pg/ml) in CHO ceils . The CA were only<br />
observed in the presence of inetabolic activation (S 9) . Extensive experiments were carried out in order to<br />
evaluate this observation . For exampie, chromosome aberration induction in LS17gY cells was<br />
demonstrated, despite the absence of mutagenesis in this assay . Furthermore, gene mutations (HPRT)<br />
were demonstrated in CHO cells over the same dose range which yielded CA . Several studies were carried<br />
out to characterize the influence of metabolism . Heat inactivation of the S-9 failed to eliminate<br />
chromosome aberration induction . Furthermore, no significant quantities of metabolites were formed !n<br />
vitro by S-9 acting on bropirimine. No binding to DNA or protein could be detected following metabolism<br />
of bropirimine by S•9 in vitro . These latter experiments suggest that the positive findings In in vitro<br />
systems may be due to enhanced transport (caused by interadion with protein) coupled to deleterious<br />
effects on chromatin structure caused by the unchanged drug . This drug did not cause rat bons marrow<br />
CA or micronucleus formation, but did induce significant frequencies of mouse micronucleus formation . In<br />
addition, bropirimine induced transofrmation of Balb 3T3 cells . This array of genetic toxicology findings is<br />
similar to an alternative therapy acylrovir . The risk assessment process for drugs such as bropirimine must<br />
incorporate a balancing of relative risks .<br />
3<br />
COMPARATIVE MUTAGENICITY TESTING OF A DRUG CANDIDATE : U-68,553B .<br />
EVALUATION OF U-68,553B REVEALS AN UNUSUAL ABILITY TO BREAK SPECIFIC CHO<br />
CHROMOSOMES AND LACK OF IN VIVO GENOTOXICITY . C .S . Aaron, A. Thilapar, V .<br />
Kumaroo, J . Mazurek, J . Mirsalis, K. Steinmetz, L . Stankowski, R . Sorp The Upjohn<br />
Company, Kalamazoo, MI ; SITEK Research Laboratories, Rockviile, MD ; SRI, Menlo Park,<br />
CA; Pharmakon Research International, Inc . Waverly, PA .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 3<br />
Notes
4 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes U•68,5538 is a novel drug under development by The Upjohn Company . This compound was marginally<br />
positive in the Ames assay In the presence of S•9 <strong>and</strong> in In vitro cytopenetics in CHO cells In the absence of S-<br />
9 . The result of testing in the In vitro UDS assay In rat hepatocytes was a clear positive . The compound was<br />
not mutagenic in the mouse micronucleus test <strong>and</strong> was negative in both the CHO/HPRT <strong>and</strong> ASS2/xvRT<br />
mammalian cell mutation assays . A variety of follow-up studies has revealed that the In vitro findings<br />
probably do not reflect a 9enotoxic hazard . For exampie, no evidence of Induction of chromosome<br />
aberrations in vivo was observed In rat bone marrow . Furthermore, the kinds of aberrations Induced In vitro<br />
were primarily chromosome breaks <strong>and</strong> appeared to occur selectively in a few chromosomes . We followed<br />
up this observation by G-bsndinq the chromosomes <strong>and</strong> determining the distribution of breaks in the<br />
9enome . In fact, approximately 90% [207/234) of all the chromosomal aberrations were breaks in the three<br />
longest chromosomes in the complement, with a larye majority in Chromosome 3. These breaks appear to<br />
occur at discrete locations In the chromosomes that are broken . Finally, the In vitro UDS findings were<br />
pursued by treatment of rats In vivo (the so-called "in vivolin vitro UDS") <strong>and</strong> there was no UDS observed In<br />
vivo . Thus, the genetic toxicology profile of U-68,S63t yields several positive In vitro findings which are not<br />
relevant to In vivo hazard . The assessment of risk due to this compound is clearly an example of the need for<br />
rational risk assessment when in vitro tests give positive results . The full data <strong>and</strong> evaluation of the results<br />
will be presented in the context of the risk assessment .<br />
IN VIVO GENOTOXICITY OF CHEMICALS WHEN ADMINISTERED IN DIPPERENT SOLVENT/CARRIERS .<br />
N .G . Abbott <strong>and</strong> A .F tlcFee, Oak Ridge Associated Oniversities, Oak Ridge, TN (USA)<br />
Soae ehemieal coapounds to be tested for mutagen/eareinogen potency are insoluble in<br />
both the aqueous <strong>and</strong> oil solvents eomaonly used for in vivo adainistration . Diaethyl<br />
sulfoxide (DMSO) has often been used for injecting such compounds, but its in vivo<br />
behavior <strong>and</strong> possible interaetion with the solute have besn questioned . We quantified<br />
sister ehroaatid exchanges (SCE) in marrow leukoeytes <strong>and</strong> aieronuelei (MN) in<br />
polychromatic erythrocytes (pCE) of mice after injections of compounds having various<br />
degrees of solubility in phosphate-buffered saline (PBS), corn oil (00), <strong>and</strong> DHSO .<br />
Equal quantities of chemicals were adainistered as solutions or mechanical suspensions<br />
in each of the carriers, <strong>and</strong> aice vsre killed 24 hr later . SCEs were scored in 25<br />
metaphases from each of 4 mice psr treatment, <strong>and</strong> fluorescence microscopy was used to<br />
enuoerate MN among 2,000 FCE in each of 5 aice per treatment . SCEs induced by 10mg/kg<br />
of 7,12-diaethylbensanthracene (DqBA) were significantly higher when administration was<br />
in CO solution than in PBS suspension, <strong>and</strong> even higher when in DNSO solution . MN<br />
induction was significantly higher following administration of 50 ag/kg of DMBA in OD<br />
or DMSO solutions than with PBS suspension . The number of SCE induced by<br />
diglycidylresorcinol ether showed a relationship to ita solubility in the various<br />
carriers, whereas MN induetion was increased most sharply when injeetions were in DMSO .<br />
Doses of 1,2-dibroao-3-chloropropane in DNS0 solution repeatedly produced much higher<br />
nuabers of SCE than the same amounts given in CO solution or PBS suspension . The data<br />
identify some compounds which express greater genotoxicity when administered in DpSO<br />
solution <strong>and</strong> some with a possible chemical interaction with DdSO . Supported by NIEBS<br />
Interagency Agreement Y01-ES-20100 <strong>and</strong> DOE/0RA0 Contract DE-ACOS-760R00033 .<br />
5<br />
METABOLIC ACTIVATION OF Me1Q TO A MUTAGEN BY HEPATIC PREPARATIONS FROM DIFFERENT RAT<br />
STRAINS . Amal Abu-Shakra, Cellular <strong>and</strong> Genetic Toxicology Branch, National Institute<br />
of <strong>Environmental</strong> Health Sciences, Research Triangle Park, MC 27709 .<br />
MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]Quinoline) was activated to a mutagen in<br />
the Ames test by hepatic microsomal preparations from Aroclor- pretreated Mistar .<br />
Sprague-Dawley <strong>and</strong> Fischer rats . NeIQ-Rwtapenicity was catalysed by cytochromes P448,<br />
which are induced by Aroclor . Microsomes from the uninduced rats were deficient In<br />
the cytochromes P448 <strong>and</strong> could not activate MeIQ . Purified microsomes from induced<br />
rats, regardless of the strain, activated MeIQ to give 860-920 rev/ng . This strong<br />
mutagenic response was enhanced when the microsomes were supplemented with their<br />
respective cytosolic fractions, to generate the •resuspended• $9 . Strain differences<br />
became apparent with this procedure . Resuspension caused Fischer-S9 to be noticeably<br />
weaker than Wistar-S9 <strong>and</strong> Sprague-Dawley-S9 . Because the strains were similar in their<br />
microsome-mediated responses, it seemed that differences in S9-activation could be<br />
attributable to the cytosol . However, in a cross-over study, where the cytosol of one<br />
strain was mixed with the microsomes of the other, Fischer-cytosol mixed with Wistaror<br />
Sprague-Dawley-microsomes provided stronger MeIQ-activation than did Fischercytosol<br />
with Fischer-microsomes . Also, Wistar-<strong>and</strong> Sprague-Dawley-cytosol, which were<br />
excellent enhancers of their respective mlcrosane-mediated responses, failed to<br />
enhance that of the Fischer-microsomes . Therefore, strain differences are not only due<br />
to different levels of cytosolic activity, but are affected by tnteractions between<br />
the cytosol <strong>and</strong> the microsome-generated metabolites .<br />
4
6 1989 EMS Abstracts 5<br />
COhiPA:jArIVi; N:LTAGT13S13 O? VITAVAC . N . A3hikari an3 LS, 3rover, Notes<br />
school of Life Sciences, Guru Nanak Dev University, Amritsar, In31a .<br />
Vitavax (carboxin), an important systomic fungicide, has an annual<br />
consumption in In3ia of about 80 M .T, Reports on its differential<br />
mutaienic response in in vitrq, (both with animal an3 plant microEomal<br />
activation) an3 Ip, vivo bioassays are scarce . Iience a comparison of<br />
its genotoxicity in Jn y~~ (Ames test) an3 in_ vivo chromosomal<br />
aberrations an3 MN in3uction in bone marrow cells of rats was done .<br />
Neqative control (DMSO) an3 appropriate positive controls (ao3itm azi3e-<br />
TA100r NPD-TA97al FMS-rats) were usej concurrently. In Ames test, the<br />
pestici3e was teste3 both in the presence an3 absence of S9 mix an3 two<br />
S14s (see3ling homogenaW of Zea mavs an3 Btasg1a over a range of 7<br />
log concentrations . However, it faile3 to elicit any positive<br />
response + S3/S14s. C ytogene tic 3amage in rats was screene3 for thre e<br />
9oses ( 1/40 LDS , 1/20 LD an3 1/10 LD ) with the abarrant cells<br />
ranging from 4,90 to 16 .90Oper cent . Fa"q the MN test, three 3ose<br />
levels, 1/4 MTD (maximum tolerate3 9ose) , 1/2 MTD an3 bITO were testa3 .<br />
Micronucleate3 pCEs range3 from 3. 60-11 .60/1000 PCSS . Significant<br />
cytog=netic 3amage in both the assays was observe3 at the two highest<br />
3oses teste3. Though vitavax 3i3 not elicit any significant response<br />
in Anas assay, yet its ability to in3uce an appreciable clastogenic<br />
3ama :3e warrants further stu3ies .<br />
7<br />
SURVEILLANCE OF TOBACCO-ARECA NUT CHEWERS WITH CYTOGENETIC MARKERS .<br />
Adhvaryu S .G ., Dave B .J ., Trivedi A .H ., Cell Biology Div ., Gujarat Cancer Research<br />
Institute, Ahmedabad, 380016, INDIA .<br />
Role of tobacco-areca nut chewing in causation of oral cancer <strong>and</strong> oral submucous<br />
fibrosis (SMF) is well documented, however, thorough human studies are rare . In<br />
the present study; (i) controls not consuming tobacco or areca nut in any form, (ii)<br />
chewers with no appreciable change in buccal mucosa (nor .chw .), (iii) chewers with<br />
oral SMF <strong>and</strong> (iv) chewers with oral cancer, have been studied for frequencies of<br />
sister chromatid exchanges (SCE) & chromosome aberrations (CA) in lymphocytes <strong>and</strong><br />
micronuclei (MN) in exfoliated buccal mucosa cells . The study design was expected to<br />
provide information about genotoxic effects of the habit on target as well as nontarget<br />
tissue . The observations were :<br />
Frequency Of :- SCE/cell C .A ./cell % MN cells .<br />
Controls 6 .185 0 .050 0 .193<br />
Nor .Chw . 7 .219 0 .097 0 .740<br />
Or . SMF Pts . 7 .617 0 .127 0 .753<br />
Oral Ca . Pts . 8 .500 0 .144 -<br />
Values obtained for<br />
all three groups of<br />
chewers were significantly<br />
higher with p
6 1989 EMS Abstracts<br />
Notes GENETIC TOXICOLOGY OF FOOD PRODUCTS<br />
H .U . Aeschbacher, Nestld Research Centre, Nestec Ltd .<br />
Lausanne 26 (Switzerl<strong>and</strong>) .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
9<br />
Vers-chez-les-Blanc CH-1000<br />
There is evidence from epidemiological studies that food is involved in cancer<br />
etiology . Such a relationship has not generally been established for non<br />
carcinogenic .genotoxic food components . This might be due in part to the difficulty to<br />
provide evidence for genetic defects in man but might also be due to the fact that a<br />
variety of food components which are positive In vitro are not harmful to man .<br />
For instance, a number of compounds such as some vitamins, plant phenolics, etc are<br />
mutagenic in vitro due to their antioxidant activity but might as a matter of fact<br />
protect man from cancer . Food components also affect other mechanisms e .g . adsorption<br />
to fiber, alteration of gut flora, etc, which can hardly be taken into account by In<br />
vitro test systems .<br />
It is therefore essential to permit for a correct risk assessment to thoroughly<br />
investigate food-borne compounds with possible mutagenic or carcinogenic potential .<br />
SYNEYISTIC GENOTOXF EFFECT OF.3 SMOKING AtiD LEAD. YR Ahulal, Talitha T<br />
RaJah , B DineshKumar , NV Prasad , K Kashyap , K Krishnaswamy . 1)Dept . of<br />
Genetics, Osmania University,2)National Institute of Nutrition 3)Govt Printing Press<br />
4)Bureau of Polic . R6D, Hyderabad, INDIA .<br />
Both smoking (S) <strong>and</strong> lead (Pb) have been shown to be genotoxic when tested<br />
alone . Since the results of the studies on the Interacting effects between S <strong>and</strong><br />
Pb exposure are controversial, the present stud y was undertaken . Eighteen adult<br />
male workers (8 of whom were smokers) exposed to Pb In a printing press <strong>and</strong><br />
25 controls were included in our study . Blood Pb levels were determined in 15<br />
of the exposV subjects . Atmospheric Pb levels were also determined . Atmospheric<br />
Pb (3 .1qjig/m ) was significantly higher as compared to the unpolluted environment<br />
(0 .5ug/m ) . Blood Pb level was also significantly elevated (41 .55i5 .91,ug/dl) as<br />
compared to controls (23 .6:3 .29,ug/dl) . Chromosome aberrations (CA) (structural+<br />
numerical) were increased significantly in those who were exposed to Pb (15 .78%)<br />
as compared to controls (6 .7%) . Amongst the Pb exposed workers, there was no<br />
difference in the frequency of CA between smokers <strong>and</strong> nonsmokers (14 .16 vs<br />
14 .70%)i however smokers had a significantly higher frequency of micronuclei<br />
than nonsmokers (0 .85 vs 0 .44%) suggesting that 8 In combination with Pb is<br />
clastogenic leading to cell death . The mitotic Index was also depressed in Pb<br />
exposed smokers indicating that Pb In combination with smoking is cytotoxic .<br />
Our results suggest that smoking interacts synergistically with Pb in causing genetic<br />
damage .<br />
Financial Assistance from ICMR is acknowledged .<br />
NUCLEOID SEDIMENTATION ANALYSIS OF DNA DAMAGE PRODUCED BY NUTAGENS IN RAT LYMPHOCYTES .<br />
Anane Aidoo, Ning Gao <strong>and</strong> Robert H . Heflich, National Center for Toxicological Research,<br />
Jefferson, AR (USA), <strong>and</strong> Drug Inspection Institute of Inner Mongolia, Huhhot (PRC)<br />
Assays based on detecting DNA damage in lymphocytes are used to monitor exposure to<br />
potential genotoxic chemicals in vivo . In this study, we have examined the ability of<br />
the model genotoxic agents, 2-acetylaminofluorene (2-AAF), benzo(a)pyrene (BP) <strong>and</strong><br />
several of their metabolites to produce DNA damage in rat lymphocytes . Lymphocytea,<br />
isolated from the spleens of male Fischer 344 rats, were exposed to the agents for<br />
one hour In serum-free medium . DNA damage was determined by the sedimentation rate of<br />
nucleoids from the treated cells as compared with the appropriate controls . The<br />
relative efficiency of producing DNA damage in rat lymphocytes by BP <strong>and</strong> its metabolites<br />
was : BP anti-7,8-diol-9,10-epoxide > BP 4,5-oxide > BP trans-7,8-dihydrodiol a<br />
BP, with the latter two compounds eliciting essentially no damage . For 2-AAF <strong>and</strong> its<br />
derivatives, the order of potency was : N-acetoxy-2-AAF > N-hydroxy-2-aminofluorene<br />
(N-hydroxy-2AF) a N-hydroxy-2-AAF > fluorene 3- 2-AF a 2-MF . The latter three compounds<br />
produced very little damage . Paraoxon eliminated the DNA damage produced by Nacetoxy-2-AAF,<br />
but both paraoxon <strong>and</strong> salicylamide had no affect on N-hydroxy-2-AAFinduced<br />
damage . Rat lymphocytes also mediated a mutagenic response with N-hydroxy-<br />
2-AAF in Salmonella ~t himuriu~m TA98 . These results indicate that rat lymphocyte<br />
nucleoid seation detecS DNA damage produced by reactive metabolites of 2-AAF<br />
<strong>and</strong> BP . While rat lymphocytes were capable of metabolizing N-acetoxy <strong>and</strong> N-hydroxy-<br />
2-AAF to DNA-damaging species, they displayed little ability for activating the parent<br />
compounds or BP trans-7,8-dihydrodiol .<br />
10<br />
11
12 1989 EMS Abstracts 7<br />
THEORETICAL BASIS OF THE TRANSPLACENTAL CARCINOGENICITY AND TERATOGENICITY OF DIMETHY-<br />
LNITROSAMINE AND N-ETHYL-N-NITROSOUREA . Dunstan A .A . Akintonwa, Department of Biochemistry,<br />
College of Medical Sciences, University of Calabar, Calabar - Nigeria .<br />
Dimethylnitrosamine <strong>and</strong> N-ethyl-N-nitrosourea are mutagens in the Ames test <strong>and</strong> they<br />
are carcinogens whilst thalidomide is a teratogen <strong>and</strong> neither a mutagen nor a carcinogen<br />
. The evidence that nitrosamines are not active in transplacental carcinogenesie<br />
except when administered late in pregnancy has been challenged . Mechanism of transplacental<br />
carcinogenesis of dimethylnitrosamine (DMNA) <strong>and</strong> N-ethyl-N-nitrosourea (ENU)<br />
has been presented based on the presence of monooxygenases (EC1 .14 .14 <strong>and</strong> EC1 .14 :15)<br />
<strong>and</strong> smooth endoplasmic reticulum <strong>and</strong> rough endoplasmic reticulum in 13 week <strong>and</strong> 16<br />
week old human foetal livers . Plausible mechanism for the teratogenicity of ENU based<br />
on chelating propensity of its metabolites has been presented . Probable mechanism for<br />
the basis of the2#eratogenicity 21 a metabolite of thalidomide based on chelation<br />
with calcium (Ca ) <strong>and</strong> zinc (Zn ) comparable to similare chelation of ethylene<br />
diaminetetraacetate (EDTA) has also been presented . From the molecular basis of carcinogenesis<br />
<strong>and</strong> teratogenesis DMNA was found to be negative (-) as a theoretical (T)<br />
<strong>and</strong> experimental (E) traneplacental carcinogen in rodent <strong>and</strong> (-) <strong>and</strong> positive (+) for<br />
T if the maternal metabolite or the intact DMNA passes through the placenta respectively<br />
<strong>and</strong> not known (?) for (E) in human . For ENU, (+) for (T) <strong>and</strong> (E) in rodent<br />
<strong>and</strong> (+) for (T) <strong>and</strong> (?) for (E) for human . In terstogenesis for DMNA, (-) for (T) <strong>and</strong><br />
(E) in rodent <strong>and</strong> (-) for (T) <strong>and</strong> (?) for (E) in human were obtained . Por ENU, (+)<br />
for (T) <strong>and</strong> (E) in rodent <strong>and</strong> (+) <strong>and</strong> (?) for (T) <strong>and</strong> (E) respectively were obtained .<br />
13<br />
COMPARISON OF CTTOGBNETIC, SPRT uD GLLCOP80RIN A 8?UDIL9 IN A-B01D $URYIYORS<br />
Mitoshi Akiyama, M .D .,?, Seishi Hyoisumi, Ph .~ .,1, Yuko Hirai, Ph .D .?,<br />
Masayuki Hakoda, M .D .,1, Nori Nak~aura, Ph .D . <strong>and</strong> Akio A . Awa, Ph .D .<br />
Department of Radiobiology <strong>and</strong> Department of Genetics,<br />
Radiation Effects Research Foundation, Hiroshima 732, Japan<br />
It is well known that somatic mutation are induced by various environmental<br />
genotoxic substances including ionizing radiation . At present, only a few methods<br />
are available for measuring the frequency of in vivo mutants in human somatic<br />
cells . One such method is to detect the mutant lymphocytes lacking HPRT, <strong>and</strong><br />
second method is to detect the variant erythrocytes lacking surface protein,<br />
glycoprotein A (GPA), third is the HLA somatic mutation method recently developed<br />
by Morley et al . The frequencies of somatic mutation were measured by using the<br />
former two methods in atomic bomb survivors in Hiroshima, <strong>and</strong> were found to be<br />
significantly correlated with the DS 86 estimated dose <strong>and</strong>, as well, to the<br />
chromosome aberration frequency of lymphocytes, respectively . However, the<br />
variant frequency from HPRT <strong>and</strong> GPA study are not correlated significantly with<br />
each other . One possible reason for this lack of correlation may be in the fact<br />
that the lymphocytes bearing sex-linked HPRT mutations, presumably large<br />
deletions (based on other studies) ∎ay have been at greater selective<br />
disadvantage than erythrocytes containing glycophorin mutations .<br />
14<br />
hurt MUTATIONS IN VIVO IN HUMAN T-LYMP110CYTES : FREQUBNCIES, SPECTRA AND CLONALITY, R .J .<br />
Albertini, J .P . O'Neill, J .A . Nicklas, M .J . McGinniss, L . Recio, <strong>and</strong> T .R . gkopek, VRCC<br />
Genetics Lab, Univ . of Vermont . Burlington, VT <strong>and</strong> CIIT, Research Triangle Park, NC .<br />
hort mutations in vivo in human T-lymphocytes reveal (i) mutant frequency values, (!i)<br />
v v mutational spectra, (iii) pra- versus post-thymic origin of the !n vivo mutations<br />
<strong>and</strong> (iv) clonality of )= mutants . Mean mutant frequency values differ by ten-fold<br />
between newborns <strong>and</strong> young adults, being 0 .64 t 0 .41 x 10-6 for 45 placental blood samples<br />
<strong>and</strong> 6 .5 ± 4 .8 x 10-6 for a large cohort of adults . h= mutational spectra also differ<br />
between newborns <strong>and</strong> adults ; more than 85% of the newborn mutations show deletions of h=<br />
exons 2 <strong>and</strong> 3 while only 151 of 326 b= mutants from normal adults show b= structural<br />
alterations detectable by Southern blot analyses . In adults, no type of alteration<br />
predominates . Deletions are common <strong>and</strong> deletion breakpoints are distributed r<strong>and</strong>omly<br />
along the length of the gene . Direct sequencing studies of adult b= mutants are<br />
determining the actual mutations tn mutants with normal Southern blots . bQ=j, mutations<br />
in newborns <strong>and</strong> adults differ also in their cells of origin with those recovered from<br />
newborns tending to arise in pre-thymie cells while those recovered from adults tending<br />
to arise in post-thymic T-cells . Iterative analyses of 1)= alterations <strong>and</strong> TCR gene<br />
rearrangement patterns in wild type <strong>and</strong> b= mutant isolates from adults reveal clonality<br />
among the mutant but not the wild type isolates . •Doublets•, 'triplets' <strong>and</strong> higher orders<br />
of clonality are routinely observed among an individual's recovered mutants . This has<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes
8 1989 EMS Abstracts<br />
Notes led to the hypothesis that proliferating rather then "resting" T-lyaphocytes are<br />
preferentially susceptible to gsne mutations in vivo . This may have significant<br />
implications for interpreting human in vivo mutagenicity studies . Research supported by<br />
NCI CA30688 <strong>and</strong> DOE FG0287ER60502 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
INFLUENCE OF DIFFERENT FACTORS ON THE INDUCTION or KALSEGREGATION IN<br />
SACCHAROMYCES CEREVISIAE D61 .M BY BAVISTAN .<br />
Silvio Albertini, Pharmaceutical Research, Department of Toxicology,<br />
F .Hoffmann-La Roche o Co ., Ltd, CH-4002 Basel, Switzerl<strong>and</strong> .<br />
Bavistan is known as a potent inducer of malsegregation in Sacharom ces<br />
cerevisiae . with both st<strong>and</strong>ard protocols used with S . cerev s ae D .M<br />
overnight incubation <strong>and</strong> cold treatment protocol, respectively)<br />
bavistan induced malsegregants in the same range . The frequencies obtained<br />
were not influenced by the plating volume used on selective medium<br />
. The higher the YEP content the more distinct the toxicity <strong>and</strong> the<br />
induction of malsegregants was . The physiological temperature for<br />
yeasts of 28°C led to a more pronounced induction than 33°C <strong>and</strong> 37°C . A<br />
clear observable induction by bavistan was detectable after 8 hours<br />
incubation (overnight protocol) <strong>and</strong> 1 .5 hours of the second incubation<br />
period (cold treatment protocol), respectively .<br />
Comparison of the growth of mixed cultures of red, cycloheximide<br />
sensitive cells <strong>and</strong> white, cycloheximide resistant, leucine auxotrophic<br />
cells prepared at different ratios revealed, that there is a strong<br />
selection towards red cells <strong>and</strong> against the malsegregants .<br />
PLASKID COPY NUMBER AND MUTANT FREQUENCIES IN S .TYPHIMURIUM TA102<br />
Silvio Albertini <strong>and</strong> Elmar Gocke, Pharmaceutical Research, Department<br />
o Tox co ogy, c .Hoffmann-La Roche i Co . Ltd, CH-4002 Basel,Switserl<strong>and</strong> .<br />
Tetracycline <strong>and</strong> chloramphenicol increase the number of mutant colonies<br />
of strain TA102, which carries the reverting gene on the plasmid pAQl .<br />
Determination of the plasmid content by agarose gel analysis shows that<br />
the increase of the mutant colony number is paralleled closely by an<br />
increase of the number of pAQl plasmids per cell, indicating that the<br />
two compounds do not increase the frequency of mutants "per gene", but<br />
only enhance the number of genes at which mutations can occur . Thus,<br />
not considering the molecular processes could result in mistakenly<br />
attributing the increase in the number of mutants per plate (respective<br />
to the number of mutants per cell) to a mutagenic activity of the antibiotics<br />
. An other antibiotic, kanamycin, acts by reducing the translation<br />
fidelity which leads to read through of the ochre stop codon <strong>and</strong><br />
thereby to an accumulation of enough functional enzyme so that enhanced<br />
cellular growth is possible .<br />
These different mechanisms all mimic mutagenic responses with some<br />
analogies to the effect of growth enhancing compounds (e .g . complex<br />
mixtures containing histidine) . It is discussed whether such false<br />
positive results are likely to occur in routine testing with TA102 <strong>and</strong><br />
how to avoid possible misinterpretation .<br />
17<br />
INDIRECT EFFECT OF SOME DNA DAMAGING AGENTS ON LAMBDA RECOMBINATION . D . Alcintara-Dfaz<br />
<strong>and</strong> N . Brefia-Valle, Instituto Nacional de lnvsstigaciones Nucleares, Kixico, D .F . (NE-<br />
IQCO) .<br />
Recombinant progeny of undamaged lambda phage, increases up to four times when infecting<br />
E . coli hosts previously irradiated with UV light. A similar effect occurs in<br />
the mutant recA-441 (tif-1) SOS induced by hest, but so far none could be detected in<br />
lexA (ind-) bacteria (Alcfintara <strong>and</strong> BreBa, data submitted for publicatioa) . All these<br />
results suggeat an SOS-dependent stimulation of undamaged lambda recombination . The<br />
present work shows more data supporting the former view . Phage crosses were carried<br />
out according to st<strong>and</strong>ard procedures in hosts with distinct repair capabilities treated<br />
with either UV or three other genotoxicants . An increase of 7 times the normal recombinant<br />
progeny was found in umuC cells exposed to 50 J/m2 of W light, a fact which<br />
to our view proves that these ara true recombinants <strong>and</strong> not mutants due to the well<br />
known W effect . On the other h<strong>and</strong> the experiments using FRlC, l04S <strong>and</strong> MJNG as recombina<br />
tion stimulants show that whereas no affect could be found in treated E . coli lexA<br />
(ind-) hosts, a significant increase in phage recombinaits occurred whan Tnfecting<br />
15<br />
16
1989 EMS Abstracts<br />
wild type cells exposed to either chemical . We conclude that this is due to some recom Notes<br />
binational event(s) associated in some way to SOS induction . Nevertheless, in the case<br />
of the chemicals we cannot exclude the possibility that at least part of the effect<br />
could be adscribed to phage DNA damage by the agent itself . For these reason further<br />
experiments on the subject are being carried on presentely .<br />
Acknow)edgements : We wish to thank Mrs . Rufina Gbmez <strong>and</strong> Mrs . Guadalupe Martinez<br />
for technical assistance .<br />
18<br />
METABOLISM OF HETEROCYCLIC AMINES IN COOKED FOOD .<br />
J . Alex<strong>and</strong>er, H . Wallin, G . Becher, J .A . Holme <strong>and</strong> A . Mikalsen .<br />
Dept . of Toxicology, Natl . Inst . Publ . Health, Geitmyrsvn .75, N-0462 Oslo 4, NORWAY<br />
Metabolic fate of ingested heterocyclic amines involves both metabolic activation to<br />
prnximate or ultimate mutagens/carcinogens <strong>and</strong> detoxication reactions . Metabolic pathways<br />
of aminoamidazoazaarenes (AIA) were studied in isolated rat hepatocytes as well<br />
as microsomes <strong>and</strong> a reconstituted P450 system . Isolated hepatocytes contain both oxidative-<br />
<strong>and</strong> conjugation enzymes for xenobiotics <strong>and</strong> modulation of inetatalism can easily<br />
be done with enzyme inducers <strong>and</strong> inhibitors . Radiolabelled parent ea :pouuxi <strong>and</strong> cofactor<br />
in conjugation reaction are useful .Metabolites are excreted into the incubation buffer<br />
which is far less carplex than bile or urine, making metabolite isolation for chemical<br />
characterization (e .g . MS <strong>and</strong> NhIIt) less cotnplicated . Formation of maonarplecular adduct<br />
(i .e . with DNA or proteins) <strong>and</strong> genotoxic events in the hepatocyte(e .g . UDB, DNA-str<strong>and</strong><br />
breaks) or in coincubated bacteria or cells are good indicators ofinetabolic activation .<br />
P450 dependent activation with N-hydroxylation of the exocyclic amino group is moet<br />
inportant <strong>and</strong> further activation can also take place to more reactive products (e .g .<br />
O-actetyl or O-prolyl esters) . But estrification to semistabile products (e .g . 0-glucuronides)<br />
may also occur . Formation of glutathione conjugates have also been noted .<br />
Important detoxication pathways for the I¢coffpounds are, provided low P450 activity,<br />
direct conjugation of the amino group to sulphate or glucuronic acid, while N-acetylation<br />
seems much less important . With high P450 activity ring hydmxylations followed<br />
by conjugation to glucuronic acid <strong>and</strong> sulphate are major detoxication pathways both<br />
for the IQ-campounds <strong>and</strong> PhIP . P45OBNF performs both activation <strong>and</strong> detoxication reactions,<br />
while P450b is inactive .<br />
19<br />
REPAIR OF O6-METHYT.GUANINE AND MNNG INDUCED MUTAGENESIS IN LIVER<br />
EPITHELIAL CELLS . N .K . Alvi, P .G . Foiles <strong>and</strong> G .M . Williams . American<br />
Health Foundation, Valhalla, NY 10595 USA .<br />
Methylation of guanine at the 06 position is considered to be a promutagenic<br />
lesion in mammalian DNA . An assay measuring mutations at the<br />
hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in adult<br />
rat liver epithelial cells (ART~,) was employed to study the mutagenic<br />
effects of O-methylguanine (O MeG) resulting from N-methyl-N'-nitro-Nnitrosoguanidine<br />
(MNNG) egposure . ARL cells were pretreated witg a non<br />
toxic dose of exogenous O MeG (0 .6 mM for 4 hr .) to deplete of Omethylguanine<br />
methyltransferase (MT) . Subsequently, cells were exposed<br />
to the various doses of MNNG . A dose dependent induction of 6-thioguanine<br />
resistnt (TGr) mutations was evident at lower levels of MNNG<br />
treatment in o6MeG pretreated <strong>and</strong> MeG pretreated cultures . ARL cells<br />
partially depleted of MT activity still repaired about 60t of 06MeG<br />
present in DNA as compared to untreated c~ltures . An immunoslot study<br />
employing a mouse antibody specific for O feG confirmed that the ARL<br />
cells were hi hly proficient in removing O MeG from their DNA . The<br />
kinetics of OgMeG elimination indicated that the half life of O6MeG<br />
repair in ARL cells was 1 hour <strong>and</strong> 4 hours respectively at two different<br />
doses of MNNG treatment .<br />
20<br />
A PROSPECTIVE STUDY COMPARING 6-TNIOGUANINE-RESISTANT VARIANT FREQUENCIES WITH CBROMO-<br />
SOME ABERRATION FREQUENCIES IN LYMPHOCYTES FROM RADIOTHERAPY AND CBEMOTf1ERAPY PATIENTS .<br />
M .M . Ammenheuserl, J .B . Ward, Jr .l, W .W . Aul, <strong>and</strong> J .A . Bel1i2, 1Departeent of Preventive<br />
Medicine <strong>and</strong> Community Health . 2Department of Radiation Therapy, The University of<br />
Texas Medical Branch, Galveston, TX 77550 (USA) .<br />
We used the autoradiographic 6-thioguanine-resistant (TGr) somatic cell sLtation<br />
assay to determine the frequency (Vf) <strong>and</strong> timing of appearance of variant T-lymphocytes<br />
from 6 patients receiving pelvic x-irradiation for uterine or bladder cancer . The<br />
patients received 180cGy/day, 5 days/week for 5 weeks . Blood samples were obtained<br />
before treatment <strong>and</strong> at weekly intervals during treatment . An aliquot of each sample<br />
was also analyzed for chromosome aberrations . The mean TGr Vf increased from 3 .47 x<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
9
10 1989 EMS Abstracts<br />
Notes 10-6 pre-treatment to 9 .43 x 10-6 2 weeks after6beginning radiotherapy, b~ was significantly<br />
higher (p
0 .40, <strong>and</strong> 0 .60% EMS in phosphate buffer to maintain pH 8 for 4 hr at 30'C . The treated<br />
teeds were washed with running water for 4 hr, sown on blotter 's<strong>and</strong>wiches', <strong>and</strong> placed<br />
n a growth chamber with a temperature of 30'C . Eight days after the treatment, the<br />
seedling height was measured, seedlings pere planted individually in soil contained in<br />
polyethylene bags, transplanted to the field after 6 weeks, <strong>and</strong> grown to maturity .<br />
Seeds from self-pollinated M plants were planted for the M2 progeny . The dose of 0 .30,<br />
0 .45, <strong>and</strong> 0 .60% reduced the lercentage of germination to 95 .6, 92 .2, <strong>and</strong> 66 .7X respectively<br />
. Seedling height of those treated with 0 .15% was 83 .2% while those treated with<br />
0 .30, 0 .45, <strong>and</strong> 0 .60% were 76 .2 . 58 .4, <strong>and</strong> 49 .2% of control respectively . The survival<br />
percentage of 6-week-old M plants treated with 0 .15, 0 .30, 0 .45, <strong>and</strong> 0 .60% were 98 .7,<br />
89 .1, 88 .3, <strong>and</strong> 57 .1% of cAntrol respectively . Nine M tricotyledonous seedlings were<br />
observed, two of which were grown successfully to maturity . Although the effects of<br />
the treatment on the percentage of germination, seedling height, <strong>and</strong> survival of M1<br />
plants were significant, except for the tricotyledonous plants with short stature <strong>and</strong><br />
small leaves, genetic effects of the treatment which could have been manifested in the<br />
form of chlorophyll mutations <strong>and</strong> other types of mutations in the M2 plants, were not<br />
observed . Ricinus communis L . which is possibly a polyploid, has probably a certain<br />
mechanism of buffering some genetic changes .<br />
24<br />
4nt tFFECTS OF GnM4n KAD1AT10N AND >:THYL Mk:'1'NANESULFONATk UN NUSTO( : LINI:KIA<br />
A .'1' . Aranez, lnstitute of Biology, University of the 1'hilippines, Uiliman tYhilippinss)<br />
Nostoc is a blue-green alga which is more closely related to bacteria than other<br />
algal groups . The objectives ot this study were to determine the etfects of gamma<br />
radiation <strong>and</strong> ethyl metnaneeulfonate (l+MS) on general morphology of calls, the ttequency<br />
of heterocysts, <strong>and</strong> the growth rate . Finely divided suspension of colonies of<br />
Nostoc suspended in distilled water were treatad with 75, 150, 300, <strong>and</strong> 45U kr gamma<br />
radiation . Anotner set was treated with U .6, 1 .2, 1 .8, <strong>and</strong> 2 .41 e:MS in phosphate<br />
butter at pN 8 for 4 hr at 3U-l: . Morphological changes produced by both treatments<br />
were enlarged <strong>and</strong> abnormally shaped vegetative cells, enlarged heterocysts, <strong>and</strong> chalns<br />
ot heterocysts . tlranched filaments were observed in Noatoc treated with gamma radiation<br />
. An increase in the number of beterocytts was observed in both treatments . Seven<br />
weeks atter the treatments, except for the presence of some elongated cslls <strong>and</strong> cells<br />
that were not in perfect alignment in Nostoc subjected to high doses ot gamma radiation<br />
<strong>and</strong> tP1S, treated Noatoc appeared as normal . keduction in growth rates were observed<br />
in the first week after treatment for algae treated with gamma radiation <strong>and</strong> the first<br />
two weeks, for algae treated with EMS . Afterwards, growth rates of treated Nostoc<br />
ihcreased . Seven weeks after the treatment, the population densities or the treated<br />
<strong>and</strong> untreated Nostoc were more or less similar . Organisms treated with U .b <strong>and</strong> 1 .Yx<br />
r:MS solution lost the capacity to form large aggregates for 7 days <strong>and</strong> those subjected<br />
to 1 .8 <strong>and</strong> 2 .4s formed small aggregates 10 days attsr the treatment . Nostoe was<br />
observed as not very sensitive to gamma radiation <strong>and</strong> CMS . (This study was funded by<br />
the Natural Science Nesearch institute, University of the Yhitippines)<br />
25<br />
TRANSMISSIBLE GENETIC EFFECTS IN MICE FOLLOWING POST-MEIOTIC EXPOSURE<br />
TO METHYL METHANESULFONATE<br />
M. Aravindakshan <strong>and</strong> P.S. Chauhan, <strong>Molecular</strong> Biology <strong>and</strong> Agriculture Division, Bhabha<br />
Atonic Research Centre, Banbey 400085,India .<br />
Majority of chenical mutagens unlike ionizing radiation induce genetic danage specifically<br />
in post-meiotic phase of epehnatogenesis . It is therefore inportant to investigate heritable<br />
effects arising from mutagenic exposure of post-meiotic germ cells . In the present study<br />
trananissible genetic effects followirnl post{neiotic exposure of mice to methyl methanesulfonate<br />
(MMS) have been investigated. Adult Swiss male mice, injected Lp. with 50 mg/kg<br />
of MMS or the solvent were individually paired with virgin fenales fran 7-13 days posttreaen<br />
ent, the m ost sensitive period of MMS m utagenesis in m ice . The fern ales, r<strong>and</strong>om ised<br />
into two groups were either evaluated for the induction of dominant lethals at r~ ~d tertn<br />
pregnancy or allowed to deliver to obtain subsequent generation . Based on two tndepenGent<br />
experinants, the incidence of daninant lethal mutations was 51 .9% in MMS treated -nice<br />
against 5.0% in controls. A higher incidence of post-implantation lethal mutations (PLM)<br />
in the test group (23.3%) as compared with controls (7 .5%) was observed in the Flgeneration .<br />
The mutation frequency was 15.5% <strong>and</strong> 5.9% respectively in test <strong>and</strong> control groups In<br />
the F2generation. However, in the F3g eneration, the incidence of PLM was canp arable<br />
in the control (7 .1%) <strong>and</strong> test group (7 .2%). Daninant lethal mutations are self I'mitirg<br />
as these are el'minated fran the progeny . However, these observations clearly demonstrate<br />
that post{n eiotic exposure of m ale m ice to MMS induce heritable genetic alterations which<br />
express as lethals in the early ernbryonic development in the subsequent generations <strong>and</strong><br />
that these effects are gradually elininated<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 11<br />
Notes
12 1989 EMS Abstracts 26<br />
Notes Temporal control of AP endonuclease !n human flbroblasts <strong>and</strong> Blooms syndrome cells . P .<br />
Arenaz, S . Trevizo, P . Anaya, V . Pelaez <strong>and</strong> L . Winkfield, Dept . Biology, University of<br />
Texas El Paso, El Paso, TX (USA)<br />
Previous reports have indicated that DNA repair <strong>and</strong> replication are coordinately<br />
regulated within the defined context of the cell cycle <strong>and</strong> that this temporal<br />
regulation acts as a screen to ensure replicative fidelity . Certain putative DNA<br />
repair deficient syndromes exhibit an aberrant pattern of gene expression of several<br />
DNA repair enzymes . We have investigated the temporal regulation of AP endonuclease<br />
in human fibroblasts <strong>and</strong> in Bloom's syndrome cells . Celie were synchronized by either<br />
serum starvation or hydroxyurea <strong>and</strong> cells collected at various time points after<br />
release from the block . DNA repair <strong>and</strong> replicative parameters were assayed using cell<br />
extracts . AP endonuclease activity, measured in serum synchronized cells was maximal<br />
at 18 h, 3 h prior to DNA synthesis <strong>and</strong> one hour prior to the fnduction of uracil DNA<br />
g)ycosylase . This same pattern was observed for AP endonuclease activity after<br />
hydroxyurea synchronization . In Bloom's syndrome cells, AP endonuclease was again<br />
Induced prior to DNA synthesis . However, as reported before uracil DNA glycosylase<br />
was induced concomitant with DNA synthesis . These data suggest that AP endonuclease<br />
ia Induced within the defined context of the cell cycle in both normal human<br />
fibroblasts <strong>and</strong> Bloom's cells . Furthermore, 1t appears that AP endonucleaoe <strong>and</strong><br />
uracil DNA glycosylase are not concomitantly controlled <strong>and</strong> suggests that there may be<br />
different control signals involved in the induction of these enzymes .<br />
Supported in part by NIH grant RR08012 <strong>and</strong> a grant from the University of Texas at E1<br />
Paso .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
CHARACTERIZATION OF A DIRECT-ACTING MUTAGEN FORMED FROM N-NITROSOPIPERIDINE AND<br />
PHOSPHATE WITH NEAR-ULTRAVIOLET LIGHT IRRADIATION<br />
Sakae Arimoto, Hiromi Shimada, Satoko Ukawal, Masataka Mochizukil <strong>and</strong> Hikoya Hayatsu<br />
Faculty of Pharmaceutical Sciences, Okayama University . Tsushima, Okayama 700, <strong>and</strong><br />
1Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo 105, Japan<br />
Previously we found that direct-acting autagens can be formed from N-nitrosodialkylamines<br />
on exposure to UVA . We have now isolated the product formed from N-nitrosopiperidine<br />
(NPIP) <strong>and</strong> investigated its structure . NPIP (40 mM) in 20 mM sodium phosphate<br />
at pH 7 .4 was irradiated with UVA (313-400 nm, 10 yN/mm2) for 3 hr . Direct-acting<br />
mutagenicity of the solution on S . typhimurium TA 1535 was 44000 His+ revertants for<br />
1 mmole equivalent of NPIP . The reaction mixture was freeze-dried <strong>and</strong> the residue was<br />
extracted with methanol . The methanol extract was evaporated to dryness under reduced<br />
pressure, <strong>and</strong> the residue was fractionated by HPLC on an ODS column . Direct-acting<br />
mutagenicity was observed only in one UV-absorbing peak . The compound in this peak<br />
fraction was identical to an authentic sample of a-phosphonooxy-NPIP in terms of the<br />
retention time in HPLC, UV spectrum, sensitivity to phosphatase <strong>and</strong> the mutagenic<br />
potency as determined on the basis of A231 units . Moreover, the 1H-NMR spectrum of the<br />
isolated compound was identical to that of the authentic specimen . Thus, it waa<br />
established that N-nitrosodialkylamines can be transformed into their 0-hydroxy phosphate<br />
esters by the action of near UV light in the presence of inorganic phosphate .<br />
This reaction represents a new, non-enzymatic activation of promutagenic N-nitrosodialkylamines<br />
.<br />
28<br />
ANALYSIS OF MUTATIONS INDUCED BY NEAR-UV RADIATION . J .D . Armstrong, M . Glattke, L .<br />
Kohalmi <strong>and</strong> B .A . Kunz, Microbiology Department, The University of Manitoba, Winnipeg,<br />
Manitoba, Canada R3T 2N2<br />
Ultraviolet (UV) wavelengths found in solar radiation are autagenic <strong>and</strong> carcinogenic<br />
. Attenuation by atmospheric ozone limits the total amount of solar UV in our<br />
environment <strong>and</strong> restricts incident solar UV wavelengths to the naar-W (NUV : 300-400<br />
nm) region so that virtually no UVC (200-280 nm) wavelengths are present . To begin<br />
probing the mechanism(s) responsible for solar UV mutagenesis, we have used DNA sequencing<br />
to characterize 120 autetlons induced in the SUP4-o gene of yeast by NUV<br />
<strong>and</strong> have compared the resulting spectrum to that for 185 UVC-induced mutations . In<br />
both cases, single base-pair substitutions accounted for approx . 90% of the induced<br />
mutations but the fraction of double-pair changes was 3-fold greater for NW <strong>and</strong> all<br />
double mutations were t<strong>and</strong>em events compared to only 330 for UVC . For each agent,<br />
approx . 90% of the substitutions were transitions but NUV induced substantially more<br />
G•C -> A•T events than UVC (88t vs . 681, respectively) . Of the substitutions that<br />
could be assigned to the 5' or 3' base of particular dipyrimidines, 800 of those induced<br />
by NUV occurred at the 3' base of S'-TC-3' or 5'-CC-3' sites compared to 60%<br />
for UVC . In addition, S'-TT-3' sites that were hotapots for UVC were not targets for<br />
NUV mutagenesis although hotspots at 5'-TC-3' sites coincided . Wavelengths involved<br />
in photoreactivation of cyclobutane dimers or photolysis of [6-41 photoproducts, are<br />
present in NUV radiation . On this basis, our data suggest that either (6-41 photophotoproducts,<br />
or photoproducts resulting from photolysis of these lesions, are the<br />
major premutational NW lesions . (Supported by NSERC Canada)<br />
27
29 1989 EMS Abstracts<br />
TRANSFECTION WITH HUMAN STOMACH DNA AND BiCNU RESISTANCE OF CHO CELLS Notes<br />
I .I . Arzimanoglou, C . Troungos, <strong>and</strong> S . Kyrtopoulos, N .H .R .F ., Athens 11635, Greece<br />
The prem,tagenic, precarcinogenic alkylation lesion 06-alkylguanine is repaired<br />
in repair-proficient cells by the enzyme O6-alkylguanine-DNA-alkyltranaferase (06-AGT),<br />
an enzyme which in E .coli can be induced under conditions known as "adaptation" . The<br />
phenomenon of adaptation has not been clearly demonstrated in eucaryotic cells . We<br />
have exposed CHO cells (with very low 06-AGT activity) to low concentrations of ?RiNG<br />
<strong>and</strong> then tested them for 06-AGT induction . No such induction was achieved . However,<br />
following such pretreatment, a reduction in SCEs induction by a challenge dose of NNU<br />
was observed, suggesting the induction of protective mechanism other than 06-AGT . In<br />
an attempt to examine the expression of an eucaryotic ACT gene, we co-transfected<br />
CHO cells with human DNA, <strong>and</strong> the bacterial neomycin gene, which is enclosed in<br />
Cosmid Homer-6 . Control cells were transfected only with the latter .G418-rasistant<br />
colonies were continuously cultured for a number of cycles of further selection with<br />
the cross-linking, cytostatic, alkylating agent BiCNU (Carmustine) . 06-AGT has been<br />
demonstrated to confer resistance, against the toxic effects of the cytostatic drug<br />
BiCNU . The main results of the study are the following : 1) Following selection with<br />
BiCNU, colonies with increased amounts of AGT were obtained from both transfected<br />
<strong>and</strong> control cells, 2) While the average increase in 06-AGT levels was roughly the same<br />
in colonies derived from transfected <strong>and</strong> control cells, larger number of BiCNU -<br />
resistant colonies were obtained from transfected cells, 3) Cells containing increased<br />
06-ACT, showed increased resistance against BiCNU <strong>and</strong> decreased mutability (to thioguanine-resistance)<br />
by HNNG .<br />
30<br />
FUTURE DEVELOPI[ENY OF SHORT TERM TESTS<br />
J . Ashby . ICI Central Toxicology Laboratory, Alderley Park, Cheshire .<br />
Two models of chemically-induced rodent carcinogenicity are currently competing for<br />
attention . The first requires that all carcinogens induce cancer by virtue of their<br />
assumed ability to modify directly DNA structure or functlon, a property that<br />
presumably can be determ3ned in vitro given appropriate assays . The second model<br />
recognizes the potential impoTr7Rf~'Zff biologlcal disturbances lnduced in rodents by<br />
the administration of chemicals . These disturbances may be due to the chemical's<br />
overt toxicity or to its ability to induce subtle changes in tissue hoaeostasis <strong>and</strong><br />
gene expression . If the chemical administration is chronic the associated biological<br />
changes may assume pseudo- permanence <strong>and</strong> lead to modification of tumor lncidences in<br />
the treated animal at death, in particular to 'carcinogenic' increases . If the<br />
chemical is also genotoxic, then truly irreversible changes in gane expression .ay<br />
accompany the temporal changes, <strong>and</strong> this could lead to more overt carcinogenic<br />
consequences . This second model fits the available facts of rodent carcinogenicity<br />
better than does the first model, however, the first model is sufficlent to account<br />
for the large majority of trans-species <strong>and</strong> multiple site carcinogens, ie, those<br />
carcinogens generally considered most likely to pose a hazard to humans are genotoxie<br />
in vitro . Progress in this field will depend upon which of these two models is<br />
accep e for development . If the goal is to detect DNA modifying carcinogens, such as<br />
benzpyrene, then the only need !s to organize <strong>and</strong> refine current techniques <strong>and</strong><br />
testinh strategies . In contrast, if the requirement is to predict all future rodent<br />
carcinogens, then other disciplines must be invoked to underst<strong>and</strong> the nature <strong>and</strong><br />
significance of the many non-specific toxicities elicited by chemicals in rodents .<br />
From such studies may emerge useful predictive assays for non-genotoxic rodent<br />
carcinokens such as saccharin <strong>and</strong> limonene .<br />
31<br />
THE SPONTANEOUS MUTATION SPECTRUM IN H13mp2 PHAGE<br />
Gen-ichi Atsumll, Keiko Matsumotol, Tadayoshi Besshol, Kazuo Negishi2<br />
<strong>and</strong> Hikoya Hayatsul<br />
1 Faculty of Pharmaceutical Sciences . Okayama University, Tsushima, Okayama 700<br />
2 Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan<br />
Naturally occurring mutations include almost all conceivable changes in DNA<br />
sequences . These r<strong>and</strong>om genetic changes are likely to be harmful to organisms ; genetic<br />
defects <strong>and</strong> cancer are believed to be caused by mutations . We have analyzed spontaneous<br />
mutations in the phage M13mp2 lac promoter-lac Za region ; the forward mutations in the<br />
lac promoter-lac Za region that is necessary for a-complementation to restore the Bgalactosidase<br />
activity in E. coli NR9099 were analyzed . Mutant a-complementationdeficient<br />
phages were detected as colorless or pale blue plaques among the wild blue<br />
plaques in the plates containing X-gal <strong>and</strong> IPTG . Viral single str<strong>and</strong>ed DNA was prepared<br />
from the isolated mutant plaques <strong>and</strong> sequenced by chain termination procedure of Sanger<br />
et al . using a sequencing kit with a-32P-dCTP . The frequency of spontaneous mutation<br />
was 4 .7 x 10-5 (55/1170000) . Of the 55 DNA samples, sequence changes were detected in<br />
30 samples . The spectrum of these changes comprised transitions, transverslons,<br />
multiple base-deletions, -additions, single base-deletions, <strong>and</strong> -additions . About a<br />
half of these mutations were single base additions <strong>and</strong> deletions in several hot spots .<br />
Analysis of the local DNA sequences suggests that the intensity of these hot spots<br />
depends on structural features of the DNA, i .e ., runs of more than four identical bases .<br />
The cause of these spontaneous mutations is, therefore, suggested to be slippage errors<br />
by DNA polymerase of the host bacterium E . coli .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
13<br />
0<br />
0
14 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
IN UTERO AND MALE MEDIATED-EFFECTS OF 1,2-DIBROlIO-3-CHLOROPROPANE IN RATS . W.W. Au,<br />
D . Walker <strong>and</strong> M .S . Legator . Department of Preventive Medicine <strong>and</strong> Community Health,<br />
University of Texas Medical Branch, Galveston, Texas 77550, USA .<br />
In utero <strong>and</strong> male-mediated effects after oral exposure to Sprague Dawley rate to 1,<br />
2-dibromo-3-chloropropane (DBCP) were investigated . DBCP was administered to timedpregnant<br />
females on days 11 <strong>and</strong> 15 of gestation at single doses of 1, 10, 50 <strong>and</strong> 150<br />
mg/kg . Calls were harvested g hours after treatment . Chromatid breaks <strong>and</strong> exchange<br />
figures were significantly elevated in pooled whole-embryo cell suspensions <strong>and</strong> in<br />
pooled fetal liver cells from females treated on day 15 . A doae related response was<br />
observed, even at the lowest concentration . In the dominant lethal test, DBCP was<br />
administered in doses of 1, 10 <strong>and</strong> 50 mg/kg for 5 consecutive days, with 5 males in<br />
each group . The males were each mated to 2 untreated females 4 <strong>and</strong> 5 weeks later . In<br />
the combined 4 <strong>and</strong> S week results, a dose-responsive effect was seen in the dominant<br />
lethal index (number of live fetuses in treated compared to nontreated females) . In<br />
addition to the dominant lethal effect using the same protocol but allowing the pups<br />
to go to term, a post natal effect was found . A significant number of new born rats<br />
died within 24 hours after birth . This increase in post natal death was dose dependent,<br />
with a effect found at the lowest concentration used, 1 mg/kg/day for 5 days .<br />
AFLATOXIN ADDUCTS AS A MEASURE OF EXPOSURE .<br />
Autrup, H ., Fibiger Institute, Danish Cancer Society, DK-2100 Copenhagen<br />
Denmark .<br />
Aflatoxin 81, a potent human liver carcinogen, is metabolized by cytochrome<br />
P-450 oxidases to several metabolites, including the electrophilic<br />
8,9-epoxide . This metabolite will react with in nucleic acids <strong>and</strong><br />
proteins . The major DNA adduct is formed in the reaction between the<br />
N-7 position of guanine <strong>and</strong> the epoxide . This adduct is unstable, <strong>and</strong><br />
would either ringopen to the more stable AFB-FAPY adduct or depurinate .<br />
The latter product is excreted in urine . Human exposure to aflatoxin<br />
has been determined by measuring the amount of AFS-FAPY in human liver<br />
samples from China, <strong>and</strong> by detecting the depurinated product in urine<br />
collected in Kenya . The sensitivity of both assays is 5 adducts per<br />
1oE7 nucleotides . Aflatoxin 8,9-epoxide also reacts with serum albumin,<br />
<strong>and</strong> the major adduct is formed with lysine, This adduct is detected in<br />
blood collected in China, <strong>and</strong> a positive association between exposure<br />
level <strong>and</strong> binding to serum albumin has been established . Three different<br />
methological approached with equal sensitivity <strong>and</strong> specificity have<br />
been developed to assess the biological active dose of aflatoxin Bl in<br />
humans .<br />
jLl SITU DETECTION OF DNA DAMAGE IN SINGLE CELLS OR TISSUE SECTIONS BY QUANTITATIVE<br />
IMMUNOFLUORESCENCE MICROSCOPY<br />
RA Baan, L Roza, GP van der Schans, MWM van Loon <strong>and</strong> CJM van der Wulp<br />
TNO Medical Biological Laboratory, P0 Box 45, 2280 M, Rijswijk, The Netherl<strong>and</strong>s<br />
The interaction of reactive chemical species with DNA - leading to the formation of DNA<br />
adducts - is considered to be an important step in mutation <strong>and</strong> cancer initiation . To<br />
study the correlation between the presence of DNA adducts <strong>and</strong> mutation induction, methods<br />
are required to detect <strong>and</strong> quantify DNA damage . Correlations between DNA lesions <strong>and</strong><br />
biological effects may be used to develop rlsk-asses,ment procedures . <strong>Molecular</strong> dosimetry<br />
of DNA damage can be used to estimate the extent of genotoxic exposure . .<br />
The present work is aimed at the application of issiunoohemical methods to assess gsnotoxic<br />
damage in a limited number of cells or at the single-cell level . This approach involves<br />
the development of antibodies specific for a particular DNA lesion, <strong>and</strong> the use of such<br />
antibodies for detection of DNA damage in fixed calls or tissue sections by laser-scan<br />
immunofluorescence microscopy (IFM) . The fluorescence is quantified by analog-digital<br />
conversion <strong>and</strong> data-processing in a Microdutch 100 computer with the software package<br />
TCL-image . The IFN technique has been or will be used for various purposes :<br />
the study of induction <strong>and</strong> removal of thymine dimers, with dimsr-specific antibodies,<br />
in UV-irradiated cultured human fibroblasts or skin ∎ections .<br />
analysis of benzo(a)pyrene(BP)-DNA adducts in human white blood cells with antibodies<br />
specific for the BP-daoxyguanosine adduct, to monitor exposure to polycyclic aromatics .<br />
- detection of DNA damage induced in germ cells by ionising radiation, with antibodies<br />
specific for single-str<strong>and</strong>ed regions in DNA, to study repair processes in such cells .<br />
50869 3526<br />
32<br />
33<br />
34
35 1989 EMS Abstracts 1 5<br />
A CYTOGBf7BTIC STUDYlIN CARBON PLACK SXPOSED JNDIVIDUALS OF TYRB INDUSTRY Notes<br />
P .P.Babu, V .S.Prasad , K .Ram Rao <strong>and</strong> Y .R .Ahuja . (1) Dept. of Gestetica .Osmania<br />
University . Hyderabad-500 007 .India. (2) Deputy Civil Surgeon . ESI Disptsttsary.<br />
Moulali, Secunderabad, India .<br />
Carbon black (CB) is of substantial industrial importance <strong>and</strong> is used in the<br />
manufacture of rubber <strong>and</strong> leather . Several studies have been done to investigate<br />
the mutagenic effect of CB. However, the evidence for the mutagenicity of CB is<br />
still considered to be controversial . A cytogenetic study was undertaken in the<br />
workers occuptaionally exposed to CB, over a period of 2 to 8 years. In the tyre<br />
Industry . For comparison age <strong>and</strong> sex matched controls were taken from the<br />
unexposed population . Peripheral blood samples were collected from 28 CB exposed<br />
workers <strong>and</strong> 15 controls, <strong>and</strong> cultured in RPMI 1640 medium for 48 hours . Chromosomal<br />
aberrations were scored in 100 metaphases from each subject . There was<br />
a significant increase in the frequency of chromosome aberrations in the workers<br />
exposed to industrial CB (5 .07%) as compared to the unexposed controls (2 .27%) .<br />
The present study indicates that CB is a genotoxic agent .<br />
36<br />
Financial Assistance from ICMR is acknowledged .<br />
STAGE-SPECIFIC SYNAPTONEMAL CQMPLEX AND CHROMQSOZtE DAMAGE INDUCED BY X-RAYS IN MALE<br />
MOUSE GERM CELLS . L .C . Backeri <strong>and</strong> J .W . AllenL . IEHRT, P .O. Box 12199, RTP, NC, 27709 ;<br />
2U . S . EPA, RTP, NC 27711 . (th1 . .b .DS.et do .s neo n .e .sssly e fl et U .S . stA oolsoy . )<br />
The synaptonemal complex (SC) comprises the proteituceous axes of paired homologous<br />
chromosomes at meiotic prophase <strong>and</strong> is involved in pairing, crossing over, <strong>and</strong><br />
segregation. SC analysis allows the observation of damage induced prior to <strong>and</strong> during<br />
meiosis. In the present study, male C57BL/6J nice were exposed to 0, 2, <strong>and</strong> 4 Gy of X<br />
rays. Harvest times were chosen to assess specific damage induced in different germ<br />
cell stages. 4 animals/dose were harvested at each time point <strong>and</strong> the results were<br />
based on 100 cells/animal . Within 2-4 hours of radiation exposure, there was a 4-fold<br />
increase (compared to controls) in SC breakage ; damage was observed in all prophase<br />
stages but was most prevalent in mid-pachytene cells . 4 days following exposure (at<br />
DNA synthesis [Sj), there were significant increases in SC breaks (3 .5-fold) <strong>and</strong><br />
rearrangements (16/animal compared to 0 for controls) . Spermatocytes (Hls) showed<br />
significant increases in both chromosome <strong>and</strong> chromatid damage on days 9 <strong>and</strong> 11<br />
following exposure . Overall damage was most extensive on day 9(zygotene exposure)<br />
with >60 chromosome rearrangements (CRs) <strong>and</strong> 42 chromosome breaks (CBrs)/nouse<br />
compared to 0 .5 CR <strong>and</strong> 1 .0 CBr/mouse in controls . On day 11 (premeiotic S exposure),<br />
there were 9 CRs <strong>and</strong> 49 CBrs/mouse . Consistent with the literature, zygotene was more<br />
sensitive to the induction of damage that results in CRs than S phase was . on the<br />
basis of the above data <strong>and</strong> the trends observed in preliminary analyses of day 1 postexposure<br />
SCs, SC analysis is a sensitive method with which to detect damage that may<br />
be expressed as chromosome aberrations at metaphase . The types of damage induced by<br />
radiation, including rearrangements qualitatively different from those we have<br />
observed with chemical treatments, should prove useful for further studies of the<br />
relationships between specific types of SC damage <strong>and</strong> chromosome aberrations .<br />
37<br />
ANALYSIS OF DIESEL PARTICULATE MATTER AND VAPOR PHASE SAMPLES USING THE<br />
MICROSUSPENSION ASSAY. S. L. Stoltz <strong>and</strong> $. Z. Bag)liy, Michigan Technological University,<br />
Houghton, MI (USA)<br />
Organic extracts of diesel particulate matter <strong>and</strong> vapor phase (from XAD-2 resin) samples were assayed<br />
using the Salmonella microsuspension (MS) assay (Kado et at ., Mutat . Res . 121, 1983, 25) to determine if<br />
increased sensitivity could be obtained compared to the st<strong>and</strong>ard plate incorporation (STD) Ames assay,<br />
particularly with the vapor phase extracts which typically show little muts~enicity in the STD assay . The<br />
MS assay uses a 90 minute preincubation with 10-fold more cells (1X10 cells) <strong>and</strong> 20-fold leu sample<br />
volume (5 µl) than the STD assay; after the preincubation, the MS assay proceeds as for the STD assay .<br />
In this study, 100 pl <strong>and</strong> S pl aliquots of the same dilutions were used in the STD <strong>and</strong> MS assays,<br />
respectively . The MS assay with TA98 <strong>and</strong> the particulate <strong>and</strong> vapor phase extracts was 10 <strong>and</strong> 30-times,<br />
respectively, more sensitive than the STD assay. This increase In sensitivity was related to more<br />
proximate interaction bewteen cells <strong>and</strong> sample <strong>and</strong> not, for example, to sample interactions with Os<br />
during the preincubation period as MS assays +/- Os had no difference in responses . With the MS assay,<br />
tester strains TA98NR <strong>and</strong> TA98 l,8-DNPs had similar increases in sensitivity with the particulate<br />
extracts <strong>and</strong> mutagenicity due to nitroarenes was detected for the first time in the vapor phase extracts .<br />
Based on repeat analysis of the same diesel samples, the MS assay was as repeatable as the STD may . I.e.,<br />
C.V.'s S 15% . The MS assay was also modified for use with smaller (60XI5-mm) petri dishes to save on<br />
assay costs, with no loss in assay sensitivity or repeatability; the small dish MS assay uses 10 mL bottom<br />
agar <strong>and</strong> 0 .7 mL top agar, containing 90 nmoles histidine <strong>and</strong> biotin. The increased sensitivity of the MS<br />
assay not only allows for detection of mutagenicity with use of 20-fold less diesel extract mass, but<br />
mutagenicity, including that due to nitroarenes, can also be detected in vapor phase samples having little<br />
activity in the STD assay. (Supported in part by contract No . 87-6 from the Health Effects Institute .)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
16 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
ELECTROPHILICITY'OF NONGENOTOXIC CARCINOGENS AND GENOTOXIC NONCARCINOGENS AS MEASURED<br />
BY THE ke TEST. G . Bakale <strong>and</strong> R .D . McCreary, Case Western Reserve University,<br />
Clevel<strong>and</strong>, OH (USA)<br />
The results of applying a physico-chemical short-term carcinogen-screeaing test,<br />
the ke test, to probe the elctrophilic properties of nonganotoxic carcinogens <strong>and</strong><br />
genotoxic noncarcinogens will be presented . The electrophilicity of a test chemical<br />
as measured by the ke test is based upon the reaction rate constant, ke, at which<br />
excess electrons attach to the chemical in a nonpolar liquid (e .g., cyclohexane) ; a<br />
diffusion-controlled ke is regarded as a positive indication that the test chemical<br />
is an electrophile, whereas the ke of a chemical that is less than diffusioncontrolled<br />
indicates an activation barrier to attachment <strong>and</strong> a non-electrophilic test<br />
chemical . The pulse-conductivity syste∎ used to measure ke's in the nanosecond time<br />
regime will be described as well as the results of screening with the ke test those<br />
chemicals that yield conflicting rodent carcinogeniclty <strong>and</strong> bacterial mutagenicity as<br />
determined, respectively, by National Toxicology Program (NTP) long-term animal<br />
studies <strong>and</strong> by the Ames Salmonella test . Of 47 chemicals that are NTP rodent<br />
carcinogens but which yield negative Ames test reponses, 26 are k-test<br />
electrophiles. Of 23 chemicals that are noncarcinogenic in the NTP animal teits but<br />
are mutagenic to the Ames Salmonella strains, 17 also yield positive electrophilicity<br />
responses in the ke test . The implications of the ke-electrophilicity/bacterial<br />
mutagenicity/rodent carcinogenicity rel}tionship to short-term screening of<br />
carcinogens will be discussed as well as the rationale for the k test yielding<br />
positive electrophilicity responses to procareinogens that 4have not been<br />
metabolically activated .<br />
PAN OBTAIIPRD BY HPLC FRACTIOd1ATION OF DIRSEL-SQGINg-EXU1UST-PARTICLE ERTRACTS ARE NOT<br />
ACTIVATED BY 59 TISSUE HOMOGHiATE . James C . Ball <strong>and</strong> Irving Salmeen, Research Staff,<br />
Ford Motor Company, Dearborn, MI 41821-2053 .<br />
Diesel-sngine-exhaust-particle extracts are active in the Ames assay without the<br />
addition of S9 . However, the interpretation of the indirect-acting mutagenicity (i .e .<br />
mutagenicity in the presence of S9 tissue hosogenate) of these samples is difficult<br />
because of the unknown effect of the S9 enzymes on the direct-acting mutagens . lie<br />
carried out Ames assays on an HPLC-fractionated methylene chloride extract of a<br />
diessl-engine-exhaust-particle aample using both TA98 <strong>and</strong> TA100 with <strong>and</strong> without the<br />
addition of exogenous tissue homogenate . These resulta (shown below) suggest that the<br />
"classic" PAN fraction (e .g . pyrene, chrysene, <strong>and</strong> benzo(a)pyrene) is not mutagenic<br />
even with the addition of exogenous metabolizing enzymes <strong>and</strong> cofactors . These results<br />
have implications for the interpretation of Ames assays of diesel-engine-exhaustparticle<br />
extracts .<br />
Bacterial Unfract . Fraction Number ; Rev/ug<br />
Strain Extract 1 2 3 4 5 6 7 8<br />
TA98 ; -S9 13 nm Q .8 5 180 66 20 39 6<br />
+S9 8 nm znl 27 120 56 15 60 4<br />
TA100 ;-59 1S mm Tm 7 130 70 21 38 8<br />
_ +S9 11 r~m nl SO 130 42 22 31 6<br />
1nm-not mutagenic ; nl~non-linear <strong>and</strong> less than 2x spont . rev .<br />
F.FFECT OF PROTEIN A ON DRUG META90LISIKG ENZYMES<br />
M .R .Bansal <strong>and</strong> Deepika Khanna<br />
Department of Biophysics, Panjab University, Ch<strong>and</strong>igarh 160 014, India<br />
The phase I <strong>and</strong> phase II enzyme systems are responsible for conversion of the<br />
carcinogen into a reactive metabolite <strong>and</strong> for its detoxification . Protein A is<br />
known to regenerate cytochrome P-450 activity . To study the effect of protein A<br />
on drug metabolising enzymes, female Swiss Porten rata were fed Ja'3A (24 mg in<br />
olive oil) which caused 50% tumor incidence after five months . The palpable<br />
tumor-bearing rats <strong>and</strong> the DMBA-fed rata without any morphological sign of tumor<br />
were treated with 12 ug protein A in normal saline s .c . twice a week for 6 weeks .<br />
Cytochrame P-450 levels increased significantly after protein A treatment whereas<br />
there was no significant change in cytochrome b5 activity . DM3A-fed rate revealed<br />
increased glutathione <strong>and</strong> glutathione-S-transferase activities . Protein A<br />
edministration to non-tumor bearing rate showed that glutathione levels decreaseo<br />
<strong>and</strong> glutathione-S-transferase activity increased . However, no significant change<br />
in phase II system was observed in tumor-bearing rats treated with protein A .<br />
It is concluded that cytochrome P-450 activities are regenerated by protein A<br />
<strong>and</strong> hence metabolism of the carcinogen .<br />
38<br />
39<br />
40
41 1989 EMS Abstracts 17<br />
THE MUTAGENIC IMPACT OF X-RAY TREATMENT ON ELECTROPHORETICALLY EXPRESSED<br />
LOCI IN THE MOUSE<br />
Lois B . Barnettt, Raymond A . Poppz . <strong>and</strong> Susan E . Lewist<br />
tResearch Triangle Institute . P .O . Box 12194 . Research Triangle Park, NC 27709 USA<br />
z0ak Ridge National Laboratory . P .O . Box Y . Oak Ridge, TN 37830 USA<br />
The induction of mutations by various doses of x-rays has been studied in the<br />
electrophoretic specific locus test in the mouse . Both female <strong>and</strong> male germ cells<br />
were studied . In order to study effects on specific germ cell stages, matings to<br />
produce (C57BL/6J x DBA/2J)F1 hybrid mice for electrophoretic analysis were made<br />
at appropriate times following x-ray treatment . When C576L/6J females are the<br />
treated parent . two visible loci (brown <strong>and</strong> dilute) can be studied in addition to<br />
the electrophoretic loci . The comparison of visible <strong>and</strong> electrophoretic loci in<br />
C57BL/6J females treated with 300R shows that the visible loci appear to be more<br />
mutable at this dose than are the electrophoretic loci . Hhile spermatogonia<br />
treated with a single dose of 300R gave higher frequencies of mutations on a per<br />
locus basis than did the 600R dose, the effects on electrophoretically expressed<br />
loci in all studies were less than in the visible specific locus test (supported<br />
in part by Contract No . Nol-ES-2-5012 . NIEHS) .<br />
42 THE SOS RESPONSE AND INDUCED MUTAGENESIS . ) .R . Battista, C.E . Donnelly, T. Ohta, V.<br />
lgras, <strong>and</strong> Graham C. Walker . Massachusetts Institute of Technology, Cambridge, MA 02139<br />
The products of the umuD <strong>and</strong> umuC genes are required for most UV <strong>and</strong> chemical mutagenesis in<br />
Escherichia colr. The genes are organized in an operon that is repressed by LexA <strong>and</strong> regulated as part of<br />
the SOS response. We have noted that UmuD shares amino acid similarities with the LexA protein of E .<br />
colr <strong>and</strong> also with gp45 of bacteriophage T4 <strong>and</strong> that UmuC shares amino acid similarities with gp44 <strong>and</strong><br />
gp62 of T4 . We have carried out a series of genetic experiments that indicate that RecA-mediated<br />
cleavage of UmuD at its Cyss4-Glyss bond activates UmuD for its role in mutagenesis <strong>and</strong> that the<br />
COOH-terminal fragment of UmuD is both necessary <strong>and</strong> sufficient for that role . Other genetic<br />
experiments indicate that the primary role of SereO in UmuD function is to act as a nucleophile in the<br />
RecA-mediated cleavage reaction <strong>and</strong> that RecA has a third role in mutagenesis besides mediating the<br />
cleavage of LexA <strong>and</strong> UmuD. Their similarities to the T4 DNA polymerase accessory proteins, gp4S,<br />
gp44, <strong>and</strong> gp62, suggest possible roles for UmuD <strong>and</strong> UmuC in mutagenesis that are supported by<br />
preliminary evidence . Our observation that groEL <strong>and</strong> groES mutations suppress the cold-sensitivity of<br />
DNA replication caused by umuDC overexpression led to the discovery that groEL <strong>and</strong> groES mutants<br />
are largely nonmutable with UV. The fact that this nonmutability of groEL <strong>and</strong> groES mutants can be<br />
suppressed by overexpression of the umuDC operon suggests that these 'chaperone' proteins may affect<br />
the ability of UmuD <strong>and</strong> UmuC to exert their functions in mutagenesis .<br />
43<br />
ROLE OF RECOMBINANT CYTOCHROME P4501A2 IN THE METABOLISM AND GENOTOXICITY OF FOOD<br />
DERIVED MUTAGENS . N . Battula, H .A .J . Schut', E .G . Snyderwine <strong>and</strong> S .S . Thorgeirsson,<br />
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD<br />
20892 ' Department of Pathology, Medical College of Ohio, Toledo, OH 43699<br />
To circumvent the inherent difficulties of purification <strong>and</strong> specificity in<br />
reconstituted cytochrome P450 systems, we have constructed several types of recombinant<br />
viruses containing the coding DNA sequences for individual P450s to efficiently<br />
transfer the P450s into mammalian cells including human cells . Analysis of cell lines<br />
containing the introduced sequences for cytochrome P4501A2 indicate that the enzyme<br />
is constitutively expressed in its natural microsomal environment . These cell lines<br />
have permitted us to carry out measurements of the biotransformation of substrates j„0<br />
jJyt under physiological conditions . To determine the role of P4501A2 in the<br />
metabolism of carcinogenic heterocyclic ar~lamines, the DNA of the exposed cells was<br />
analyzed for DNA-carcinogen adducts by P-postlabeling . DNA of IQ (2-amino-3methylimidazo[4,5-flquinoline)<br />
exposed cells showed five specific DNA-IQ adducts . The<br />
finger prints of the adducts formed in cells were chromatographically indistinguishable<br />
from those formed in rat <strong>and</strong> mouse liver after in vivo administration of IQ . Similar<br />
analysis with AF <strong>and</strong> AAF indicate specific adducts with the cell DNA but showed minor<br />
differences with the in vivo adducts . This novel approach of stably expressing<br />
specific P450s in situ <strong>and</strong> their catalytic measurements provide an excellent method<br />
to study the biotransformation of substrates <strong>and</strong> their role in toxicity, mutagenesis,<br />
<strong>and</strong> carcinogenesis .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes
18 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
DNA ADDUCT FORMATION IN RELATION TO TUMORIOENESIS IN MICE<br />
CONTINUOUSLY ADMINISTERED 2-ACETYLAMINOFLUORENE OR 4-AMINO-<br />
RIPHENYL Frederick A . Bel<strong>and</strong>, Nancy F . Fuperton, M . Matilde Marques. William B .<br />
Melchior, Jr., Beverly A . Smith <strong>and</strong> eMiriam C . Poirier National Center for<br />
Toxicological Research, Jefferson, Arkansas 72079 <strong>and</strong> •National Cancer Institute,<br />
Bethesda, Maryl<strong>and</strong> 20892<br />
Continuous administration of 2-acetylaminofluorene 12-AAFI or 4-aminobiphenyl (4•<br />
ABP/ to female BALB/c mice results in the formation of liver <strong>and</strong> bladder tumors . We<br />
have compared the relationship between the concentration of DNA adducts in these target<br />
tissues following one month of treatment <strong>and</strong> the reported tumor incidences after two<br />
years of dosing . One adduct, N-ldeoxyguanosin-8 y1F2•aminofluorene (dO-C8-AF1, was detected<br />
after 2-AAF feeding, <strong>and</strong> at each of the seven doses . the adduct concentration in<br />
the bladder was two- to four-fold higher than in the liver . Two major adducts, one of which<br />
was N-ldeoxyguanoain-8 y11-4-aminobiphenyl ldO-CB-ABPI, were found in mice treated with<br />
4-ABP, <strong>and</strong> In contrast to 2-AAF, the adduct levels were five- to eight-fold higher In the<br />
liver than in the bladder. At doses producing equivalent liver tumor incidences, hepatic<br />
4-ABP adduct levels were higher than those induced by 2-MF, which suggests that<br />
2-AAF adducts are more efficient at inducing tumors than 4-ABP adducts . In order to<br />
investigate differential processing of these adduct. . pBR322 DNA, modified with dO-C8-<br />
A13P <strong>and</strong> d0-C8-AF . was transformed Into 8OS-repair induced F. coH. Although both<br />
adducts induced a similar pattern of transversion <strong>and</strong> transition mutations, d0-C8-AF also<br />
caused deletions . Thus, thee turoorigenic <strong>and</strong> mutagenic potential of aromatic amine adducts<br />
appear to be compound specific .<br />
USE OF THE POLYMERASE CHAIN REACTION TO AMPLIFY A SEGMENT OF THE ~$ GENE OF<br />
Salmonella FOR DNA SEQUENCE ANALYSIS . Douglas A . gelll, Jaaes E . Lae2, tvnhi <strong>and</strong> David M<br />
. DeMarini . 1National Research Council, U .S . EPA, RTP, NC 27711 ; 2Duke<br />
University Medical Center, Durham, NC 27710 ; <strong>and</strong> 3U .S . EPA, RTP, NC 27711 (U .S .A .) .<br />
Previous DNA sequence analyses of revertants of the hisD303l gene of $ . t,yphimurium<br />
TA98 or TA1538 have employed either a colony hybridisation technique (Cebula et al . .<br />
Environ . Mol . Mutagen . 11, Suppl . 11 :21 ; 1988) or traditional cloning procedures<br />
(Fuscoe et al ., Mutat . Res . 201 :241 ; 1988) . pa have explored the polymerase chain<br />
reaction (PCR) to see if it might permit more rapid processing of revertants for DNA<br />
sequence analysis than do the other methods . Briefly, a revertant is streaked onto<br />
minimal medium supplemented with biotin . After grovth, a colony is boiled for 5 min<br />
in 200 yl of TE buffer <strong>and</strong> then centrifuged for 5 min . The supernatant contains the<br />
Salmonella genomic DNA used for the PCR . The two amplification primers (AP) flank a<br />
327-bp segment that contains the hi,gD3Q52 mutation approximately in the center . The<br />
sequence of AP1 is : 5' GTC TGA ACT ACT GGT CAT CG 3' ; AP2 is : CCC TOG TM TCG CAT<br />
CCA CC . The reaction is performed essentially as described for Taq polymerase in the<br />
Perkin Elmer Cetua GeneAmp KitTM using 10 µl of Salmonella genomic DNA/200-pl reaction<br />
volume . Amplification of ds-DNA is obtained using a 1 :1 ratio of the primers (50<br />
pmoles of each) <strong>and</strong> 30 cycles of an automatic thermocycler ; amplification of ss-DNA is<br />
obtained using a 1 :100 (0 .5 :50 pmoles) ratio of primers <strong>and</strong> 36 cycles . The amplified<br />
DNA is purified by ultrafiltration in dH20 in an Amicon CentriconTM 30 microconcentrator,<br />
followed by resn• .p,na3on in 9 pl of sequencing buffer . The feasibility<br />
of employing nested primers for sequencing the amplified DNA is being evaluated .<br />
(Abstract of proposed presentation that does not necessaril'y reflect U .S . EPA policy,)<br />
VIDEO MICROSCOPY AND DIGITAL IMAGE PROCESSING OF PRENATAL BRAIN CELLS EZPOSED<br />
TO LEAD NITRATE . Maxwell A . Bampong, Biology Department, Norfolk, VA 23504 .<br />
The effect of glutamic acid on lead-induced morphological changes io cultured<br />
brain cells derived from prenatal mouse, was examined . Fluorescence microscopy,<br />
video-enhanced/intensified microscopy <strong>and</strong> digital image processing were used to<br />
monitor dose-dependent, qualitative <strong>and</strong> quantitative changes in cells exposed to<br />
lead nitrate <strong>and</strong> incubated in the presence or absence of 102 glutamic acid . Leadtreated<br />
cells without concurrent glutamic acid exposure were characterized by<br />
cell rounding, spreading <strong>and</strong> extensive cytoplasmic vacuolation . Thirty ∎inute<br />
exposure to S ug/ml of rhodamine 123, resulted in fluoreicently stained cytoplasm<br />
as well as nuclei . Video-intensified microscopy confirmed lead-induced<br />
degenerated nuclear membrane . Other morphological changes observed to be associated<br />
with lead exposure included dendrite retraction, globular shaped cells<br />
with the attending increased shape factor (a fraction for estimating the amouni<br />
by which a cell varies from a circle), <strong>and</strong> increased total cell area . Theai<br />
stereological changes in lead-treated calls, were dose-dependent . Cells incubated<br />
concurrently in lead <strong>and</strong> glutamic acid medium, regardless of lead concentration,<br />
were mostly spindle in shape, bi- or multipolar, with very low shape factor<br />
values . These data from fluorescence <strong>and</strong> video-intensified microscopy suggest<br />
that intracellular membranes were the primary targets of lead action . Intracellular<br />
digestion leading to extensive vacuolation was effected by lead impact on<br />
lyaosome, surface membrane disruption was responsible for changes in cell shape<br />
<strong>and</strong> the appearance of rhodamine 123 in nuclear region resulted from lead induced<br />
nuclear membrane degeneration .<br />
44<br />
45<br />
46
47<br />
TUMORIGENESIS PROFILES IN THE NCI/NTP DATA BASE<br />
R . Benigni<br />
Istituto Superiore di Sanita'-Lab . Tossicologia Comparata ed Ecotossicologia<br />
Rome - Italy<br />
The tumor profiles of 139 chemicals,resulted to induce cancer in rodents in the NCI/NTP<br />
experimentation, were analysed by multivariate statistical methods . The study pointed<br />
to four main aggregations of the chemicals . Benzene had a profile non classifiable in<br />
any of the four classes . From the point of view of risk assessment, two classes of car-<br />
cinogens ( composed by only 7 <strong>and</strong> 9 chemicals with peculiar profiles ) were associated<br />
with Ames test mutagenicity . No apparent association with Ames test result was observed<br />
for the two other classes, that included the large majority of chemicals . Thus, the cl-<br />
assification of carcinogens based on the whole spectrum of hystopathological evidences<br />
did not parallel the repartition between Ames positive <strong>and</strong> Ames negative chemicals .<br />
This suggests that the case for "primary" carcinogens ( genotoxic, <strong>and</strong> assumed to pose<br />
a greater risk ), <strong>and</strong> "secondary" carcinogens ( presumably producing the carcinogenic<br />
effects via non genotoxic mechanisms ) is rather speculative, <strong>and</strong> at present cannot be<br />
taken as a basis for risk management .<br />
48<br />
MUTAGENICITY STUDIES ON SIX PESTICIDES IN lIICE .<br />
D .K .Benova, I . Roupova, A . Yagova, A . Vuglenov, M . Bineva' . Institute of Nuclear Medicine,<br />
Radiobiology <strong>and</strong> Radiation Hygiene, Sofia 1756, Bulgaria . *Cell . Biol . Lab ., Gen . Biol .<br />
Dept ., University of Plovdiv, Plovdiv, Bulgaria .<br />
Six pesticides widely used in agriculture were studied for their Sp vivo mutagenic<br />
activities . They were : (a) insecticides - Vaztak (Pyrethroid), Dursban (Organophosphate) ;<br />
(b) fungicide - Rubigan (Pyrimidine) ; <strong>and</strong> (c) herbicides - Cliphozat (N-Phosopho-methylglycine),<br />
Stomp (Nitroaniline) <strong>and</strong> Diquat (Dipyridilium) . The production of polychromatic<br />
erythrocytes with micronuclei at different times after oral administration of an 1/2 LD50<br />
dose was examined . All pesticides except Gliphozat are mutagens with Rubigan the weakest .<br />
Doses of 1/4 <strong>and</strong> 1/8 of the LDsa ware found to be ineffective .<br />
The frequency of chromosome aberrations in mouse bone marrow cells after administration<br />
of Stomp or Diquat in doses of 1/2 the LDyo were also examined . A tendency of increasing<br />
numbers of hyperdiploid calls <strong>and</strong> acentric fragments was observed only with Stomp in the<br />
late sampling hours (96 <strong>and</strong> 120h) .<br />
The mutagenic effect of selected pestides is discussed . The data suggest that these<br />
chemicals are (at least Stomp <strong>and</strong> Diquat) more aneugens than clastogens .<br />
49<br />
UNSCHEDULED DNA SYNTHESIS (UDS) AND DNA ADDUCTS IN HEPATOCYTES AND GERM CELLS OF<br />
2-ACETYLAMINOFLUORENE (2AAF)-EXPOSED F-344 RATS . K .S . Bentley, G .T . Arce,<br />
K . R<strong>and</strong>erath, P .K . Working, D .A . Agostinelli, T . Smith-Oliver, <strong>and</strong> B .E . Butterworth,<br />
Haskell Laboratory, E .I . du Pont de Nemours 3 Co ., Newark, DE (USA), Baylor College of<br />
Medicine, Houston . TX (USA), <strong>and</strong> CIIT, Research Triangle Park, NC (USA) .<br />
In vivo/in vitro hepatocyte <strong>and</strong> spermatocyte UDS assays are used to quantitate<br />
chemicalTyinduced DNA repair in liver <strong>and</strong> germ cells . The ability of these assays to<br />
detect DNA damage was compared to quantities of DNA adducts . Male F-344 rats were<br />
exposed by gavage to 0, 0 .5, 5, 50, or 250 mg/kg 2AAF . Twelve hours later, primary<br />
hepatocyte <strong>and</strong> speSmatocyte cultures were prepared <strong>and</strong> the cells were incubated in<br />
medium containing H-thymidine . UDS was measured by autoradiography as net nuclear<br />
grains (NG) . DNA was isolat~ from the remaining cell suspensions <strong>and</strong> 2AAF-DNA<br />
adducts were evaluated by the P-postlabeling method . A dose-related increase in NG<br />
was observed in hepatocytes ; but a positive UDS response was not detected until a dose<br />
of 5 mg/kg was reached . Both N-(deoxyguanosin-B yl)-2-aminofluorene (dG-C8-AF) <strong>and</strong><br />
N-(deoxyguan%sin-8-yl)~AAF (dG-C8-AAF) were detected at doses as low as 5 mg/kg<br />
(approx . 10 <strong>and</strong> 10' adducts/nucl4otide, respectively) . Only dG-C8-AF could be<br />
quantitated at 0 .5 mg/kg (approx . 10- adducts/nucleotide) . A correlation between DNA<br />
adducts <strong>and</strong> the UDS response was observed . In spermatocytes, UDS was detected only at<br />
250 mg/kg . Unlike the liver, only dG-C8-AF was detected in the germ cells . It was<br />
present at doses as low as 5 mg/kg (
20 1989 EMS Abstracts<br />
Notes URINARY BIOMARKERS 0F ENDOGENOUS DNA DAMAC+E<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
D .S . Bergtold <strong>and</strong> M .G . Simic<br />
National Institute of St<strong>and</strong>ards <strong>and</strong> Te chnoloNy, Gaithersburg, MD 20899<br />
The biological consequences of damage to DNA by exogenous agents have been focal points<br />
of enormous numbers of studies. Damage to DNA by <strong>and</strong>oRenous processes, in contrast,<br />
has been investigated to a much smaller extent due to intrinsic difficulties, e .q .,<br />
lack of controls, low levels of damage, lack of suitable biomarkera, etc. Using gas<br />
chromatography/mass spectroscopy, we have developed absolute measurements of oxidative<br />
urinary biomarkers based on the original work of Ames <strong>and</strong> coworkers. The urinary<br />
levels of products (thymine glycol, 8-hydroxyguanine, their nucleoside moieties, <strong>and</strong><br />
others) have been correlated in different species to their metabolic rates, longevity,<br />
<strong>and</strong> size . Since many of the observed urinary biomarkers are identical to •OH radical<br />
reaction products with DNA bases, gamma irradiation has been used to elucidate the<br />
underlying mechanisms by which metabolic biomsrkers eriae .<br />
L .F .Bernini, A .T .Nstarajan . A .H .M .Schrauder-Rotteveel, P .C .Giordano, J .S .Ploem<br />
<strong>and</strong> A .Tates .<br />
Depart . of Human Genatics . Dept . of Radiation Genetics <strong>and</strong> Chemical <strong>Mutagenesis</strong>,<br />
Dept . of Histochemistry <strong>and</strong> Cytochemistry, University of Leiden, Sylvius<br />
Laboratoria, Wassenaarsavag 72, Leiden, The Netherl<strong>and</strong>s .<br />
ASSAY FOR SOMATIC MUTATIONS OF HUMAN HB}IOGLOBINS .<br />
With the purpose of monitoring by cytoimmunocheaical methods somatic mutations<br />
of globin genes, we have raised in rabbits monospecific polyclonal antisera<br />
against a number of abnormal hemoglobins . Rare mutant calls are identified on<br />
peripheral blood smears by indirect issmmofluorescence <strong>and</strong> counted by an<br />
automatic scanning microscope especially constructed for the examination of Hb<br />
abeorbance <strong>and</strong> FITC fluorescence . With the aid of a suitable adapter, glass<br />
slides of 8x8 cm . containing about fifty million red cell acan be screened in<br />
three hours . All fluorescent objects found on the smear undergo a computer<br />
directed pattern recognition analysis . Only those objects having the size <strong>and</strong> the<br />
shape of a red cell <strong>and</strong> absorbing at 413 nm are eventually classified as mutants<br />
<strong>and</strong> checked through direct examination by an expariented observer . On the<br />
average, five Hb S cells/10 • erythrocytes have been found in the peripheral<br />
blood of healthy adult individuals . When, in the sam. person, three different<br />
mutations are monitored simultaneously with a mixture of the relative antisera, a<br />
correspondent increase of the frequency of mutant cells is observed . We report<br />
<strong>and</strong> comment the results obtained with this assay in people exposed to genotoxic<br />
agents or carriers of inherited abnormalities of DNA repair .<br />
READTHROUGH OF CHEMICALLY INDUCED DAMAGES IN DNA DURING KLENOW FRAGMENT-MEDIATED<br />
DNA SYNTHESIS .<br />
Tadayoshi Beeshol, Kazuo Negishi2 <strong>and</strong> Hikoya Hayatsul<br />
1Faculty of Pharmaceutical Sciences, 2Gene Research Center, Okayama University,<br />
Tsushima, Okayama 700, Japan<br />
In E . coli, DNA damages inducible by UV, X-ray irradiation <strong>and</strong> many chemical<br />
carcinogens block DNA replication <strong>and</strong> exert "SOS response" . A characteristic event<br />
accompanying this phenomenon is an enhanced mutation . A possible way to elevate the<br />
mutation rate is "translesion" DNA synthesis ; but its mechanism remains unclear . We<br />
have shown the direct role of RecA protein in such a translesion DNA synthesis, with<br />
N4-amino-5,6-dihydrocytosine-6-sulfonate residues as the DNA base damage . This<br />
modification can be formed on single str<strong>and</strong>ed M13mp2 DNA by treatment with a bisulfitehydrazine<br />
mixture . From the analysis of in vitro DNA chain elongation products using<br />
sequencing gels, we were able to show that this damage can block DNA chain elongation<br />
at one nucleotide before the damage . In the presanse of RecA protein, Rlenow fragment<br />
was able to readthrough this damage ; but in the presense of dGMP, an inhibitor for<br />
exonuclease activity, the DNA chain elongation remained to be blocked . A high rate of<br />
dCMP/dCTP turnover during the translesion DNA synthesis was observed, as detected by<br />
chromatographic analysis of the nucleotides in the reaction mixture . Therefore,<br />
enhanced exonuclease activity <strong>and</strong> the binding of RecA protein to damaged DNA may affect<br />
the processivity of the polymerase <strong>and</strong> this effect may be important in the translesion<br />
DNA synthesis .<br />
50<br />
51<br />
52<br />
tn<br />
m<br />
CO<br />
Oh<br />
t0<br />
W<br />
tn W<br />
N
53 1989 EMS Abstracts<br />
ESTIM4TION OF CYTOGENETIC Di4M4GE DUE TO ENVIRONMENTAL MUTAGENS BY Notes<br />
STUDYING CHROMOSOMAL ABERRATIONS AND MICRONUCLEI<br />
Bharati Bhatt <strong>and</strong> Sreedevi Balakrishnan, <strong>Molecular</strong> Biology <strong>and</strong> Agriculture Division,<br />
Bhabha Atonic Research Centre, Banbay 400085, India .<br />
Using lymphocyte culture method, da~e response curve was established in vitro for<br />
dicentrics by expoaing whole blood to Co gamma ray doses ranging fro .m 0L_'f-Gy to<br />
2.00 Gy, in our laboratory . It has enabled us to estinate equivalent whole body exposure<br />
dose in a number of cases of suspected over-exposure to radiation during the last 13 years,<br />
most of which were either trivial or negative . Recently sinilar dose response curve Is<br />
also established for m icronuclei in bi-nucleated cells, obtained by blocking cytokinesis<br />
u in cytochalasin B. This is a quick m ethod <strong>and</strong> can be of great asset in case of a m ishap<br />
w~e~ large nunber of cases have to be studied. Besides being quick <strong>and</strong> cheap It does<br />
not require an expert scorer to score m icronuclei, which are easy to identify . These two<br />
methods are evaluated in in vivo studies of exposures to other envirormental mutagens .<br />
54<br />
MODULATION OF MUTAGENICITY BY PLANT FLAVONOIDS<br />
R .R . Bhattacharya, Biochemistry Division, Bhabha Atomic Research Centre, Bombay<br />
400 085, INDIA<br />
The flavonoids are interesting natural compounds which are regularly consumed<br />
by humans through diet containing fruits <strong>and</strong> vegetables, <strong>and</strong> several of them<br />
possess anticarcinogenic property . Using structurally different flavonoids it<br />
was observed that polyhydroxylated flavonols were highly efficient in modulating<br />
mutagenicity of carcinogens, expressed in Salmonella tester strains . These<br />
compounds were, more effective towards aflatoxin B1~ (AFB ), a carcinogen requiring<br />
metabolic activation for its action, than towaYAs N-1ethyl-N'-nitro-N-nitrosoguanidine<br />
(MNNG), a direct acting carcinogen . Methylation of hydroxyl groups<br />
reduced the activity, although glycosylation did not . Isoflavone group was also<br />
effective for both carcinogens . Saturation introduced into the ry-pyrone ring of<br />
flavone nucleus (flavanone) rendered the flavonoid inactive for modulating AFB1<br />
mutagenicity, but increased the antimutagenic potency for MNNG . Dose-response<br />
data, calculated on molar basis, revealed exceptional activity for kaempferol <strong>and</strong><br />
rutin towards AFBt , <strong>and</strong> for naringin towards MNNG . These flavonoids acted either<br />
by interfering with the enzymatic activation leading to DNA adduct formation, as<br />
in the case of AFBl, or by preventing passage of the carcinogen into bacterial<br />
cell <strong>and</strong>/or by altering some cellular process, as in the case of MNNG .<br />
55<br />
CARCINOGEN-INDUCED HOMOLOGOUS RECOMBINATION BETWEEN DUPLICATED GENES STABLY<br />
INTEGRATED WITHIN THE GENOME OF MAFPVILIAN CELLS, INCLUDING NORMALLY-REPAIRING AND<br />
REPAIR-DEFICIENT HUMAN CELLS . N .P . Bhattacharyya, T . Tsujimura, Y . Wang, J .J .<br />
McCormick <strong>and</strong> V .M . Maher, Carcinogenesis Laboratory, Michigan State UniversTfy,<br />
asnsing, MI 48824 (USA)<br />
We have been studying carcinogen-induced homologous recombination in a tk- mouse<br />
L cell line which contains a single integrated plasmid which duplicat-3 copies<br />
of the Herpes tk gene, each containing an 8bp Xhol site inserted in a different<br />
place <strong>and</strong> with tTie neo gene located in the interven~ng sequence . Only by undergoing<br />
a productive recomFFnational event between the two non-functional tk genes can<br />
a functional gene product be made <strong>and</strong> the recombinant be selected witT-HAT medium .<br />
With this system, we determined that the frequency of recombination induced by<br />
UV radiation or a series of mutagens that form multi-ringed DNA adducts is linearly<br />
related to the number of DNA adducts or photoproducts . The majority of the events<br />
are consistent with non-reciprocal transfer of genetic material, i .e ., gene<br />
conversion . To examine the mechanisms involved <strong>and</strong> the role of DNA repair, we<br />
have transfected human cells which have normal rates of 06-alkylguanine DNA repair<br />
<strong>and</strong> nucleotide excision repair or which are deficient in one or both processes<br />
with the plasmid carrying the Htk genes or a related plasmid carrying duplicated<br />
copies of the gene for hygromycin resistance . Using these 2 systems, we have found<br />
that there is no difference in the rate of spontaneous recombination among the<br />
human cell lines, but nucleotide repair=proficient cells have a lower frequency<br />
of recombination induced by UV <strong>and</strong> adducts than repair-deficient cells . (Supported<br />
by Grants from the NCI <strong>and</strong> by Contract 87-2 from the HEI .)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
21
22 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
56<br />
STUDIES ON MUTAGENIC AND ANTIMUTAGENIC EFFECTS OF COBALT (1l) CHLORIDE<br />
IN SWISS MICE<br />
H .N . Bhilwade, R.C. Chaubey <strong>and</strong> P.S. Chauhan, <strong>Molecular</strong> Biology <strong>and</strong> Agriculture Division,<br />
Bhabha Atonic Research Centre, Bo-nbay 400085 .<br />
Cobalt (1l) chloride has been reported to inhibit the m utagenic effects of ionizing radiation<br />
aaj chan ical m utagens in prokeryotic systen s . Lack of a s'm ilar study in m amm als, which<br />
are more relevant for investigations on genotoxic prevention in man, prvnpted us to examine<br />
the ability of cobalt (11) chloride to modify the clastogenicityof a chemical mutagen methyl<br />
m ethanesulfonate (MMS) <strong>and</strong> ionizing radiation (gamm a rays) using bone m arrow m icronucleated<br />
cells in mice. Cobalt (Im) chloride at a single dose of 15.0 mg/kg provoked a mild increase<br />
in the frequency of micronucleated polychro~natic erythrocytes (MN-PCEs ` over controls<br />
which increased further at 30 <strong>and</strong> 60 mg/kg dose (p< 0.05). There was, however, $ lack<br />
of dose-response relationship . Pretreahn ent of m ice with a single dose of 45 m g/kg of<br />
cobalt (ll) chloride reduced the frequency of MMS induced MN-PCts, significantly . Exposure<br />
of mice to 15.0, 30.0, 45.0 <strong>and</strong> 60.0 mg/kg of cobalt (lI) chloride showed a dose dependent<br />
decrease in the levels of MN-PCEs in cvnparison with MMS alone. A moderate but significant<br />
(p e 0 .05) reduction in the frequency of MN-PCEs in gamm a Irradiated (1 .0 Gy) m ice was<br />
also observed at 30 .0 mg/kg of cobalt (1I) chloride. The multiple dosage reginen of cobalt<br />
(il) chloride were not of much consequence .<br />
NUTAGENICITY OF ORGANIDINo AND ITS MAJOR COMPONENT, 3-IODO-1,2-PROPANEDIOL<br />
J .B .Bisho ,K.L .Mitt,E .Zeiyer,J .Mason,N .D .Shelby, <strong>and</strong> J .E .French, NIEHS,RTP,N .C .<br />
ryan inm [5634-39-9], is an expectorant found in OTC cough preparations . In<br />
NTP 2-year rodent studies, it induced leukemias <strong>and</strong> thyroid tumors in male rats,<br />
<strong>and</strong> pituitary <strong>and</strong> harderian gl<strong>and</strong> tumors in female mice . Organidin is mutagenic in<br />
bacterial <strong>and</strong> mamnalian cells . In contrast to the patented formulation, Jameson, et<br />
al .(1988) reported that Organidin consists of 33% 3-iodo-1,2-propanediol (IPO), 17%<br />
glycerol, 40% polymers of glycerol, iodo-plycerol <strong>and</strong> numerous<br />
other components, of which -10% were isomeric p-dioxanes . Jones (1975) purported<br />
metabolism of IPD to the mutagenic epoxide, glycidol ; therefore, we have examined<br />
the in vitro <strong>and</strong> in vivo genetic toxicity of Organidin, IPD <strong>and</strong> glycidol to better<br />
underst<strong>and</strong> the bases of Organidin's mutagenic <strong>and</strong> carcinogenic activity . None of<br />
the 3 chemicals required exogenous metabolic activation in the bacterial strains or<br />
mammalian cells in which they were active . Organidin was muta9enic in base substitution<br />
strains of Salmonella (TA100 <strong>and</strong> TA1535) but not in frame shift strains<br />
(TA97 <strong>and</strong> TA98) ; glycidol was mutagenic in both base pair <strong>and</strong> frame shift strains<br />
(TA100, TA1535, TA1537, TA97 <strong>and</strong> TA98) (Cantor et al .1986) ; IPD, like Organidin,<br />
was mutagenic in TA100 but not TA98 . All 3 chemicals induced SLRL mutations In<br />
Drosophila (Glycidol>IPD>Orqanidin) . Glycidol <strong>and</strong> Organidin both induced ABS <strong>and</strong><br />
SCE in CHO cells (Glyctdol>Or9anidin) . IPD is on test In CHO . Glycidol was weakly<br />
positive in a bone marrow micronucleus test using B6C3F1 mice, but IPD <strong>and</strong><br />
Organidin were negative . The relevance of these responses to underst<strong>and</strong>ing the<br />
mutagenic <strong>and</strong> carcinogenic effects of Organidin will be discussed .<br />
NONACTIVATED MVfAGENICITY OF CHEMICALLY NITRATED OILS CORRELATES VITH S-9-DEfENDEPr['<br />
ACTIVITY OF THE UNMODIFIED OIL IN THE AMES ASSAY . G .R . Blackburn, R .A . Deitch , S .E .<br />
IRVIN , C .A . Schreiner, <strong>and</strong> C .R . Mackerer , Mobil <strong>Environmental</strong> & Health Science<br />
Laboratory, P .O . Box 1029, Princeton, NJ 08540 .<br />
Previous studies In this laboratory have shown that modifications to the sampledelivery<br />
<strong>and</strong> activation procedures in the Ames Assay lead to a substantial increase<br />
in the correlation between mutagenic <strong>and</strong> dermal carcinogenic potency of petroleumderived<br />
materials . An adjunct method, providing equal predictability, but requiring<br />
no S-9 preparation, <strong>and</strong> hence no sacrifice of animals, relies upon preliminary<br />
chemical nitration of oil samples, followed by assay in atrain TA98 . One hundred ul<br />
aliquots of oil are nitrated in 70 X(v/v) nitric acid at 80°C, for 90 ∎in . The<br />
nitrated derivatives are extracted into dichloromethane, concentrated, <strong>and</strong> resolubilized<br />
In DMSO for testing . Doses equivalent to 2, 1, 0 .5, 0 .1, <strong>and</strong> 0 .05 ug<br />
oil/plate generate dose-response curves with slopes ranging from 0 to 500 revertants/ug<br />
. The correlation between slopes from the nitration method <strong>and</strong> the S-9<br />
dependent assay for 30 oils is 0 .85 (r), <strong>and</strong> between the former <strong>and</strong> the relative<br />
carcinogenic potency (reciprocal of the mean latent period to tumor formation) is<br />
0 .92 (27 oils) . Neither method provides reliable predictability for oils boiling<br />
under 500°F, apparently because many of these materials are not genotoxici the nitration<br />
method is further limited at the high end of the distillation range for<br />
petroleum, i .e . fractions boiling above about 900•F . The simplicity of the method<br />
makes possible the evaluation of as many as 20 oils in one assay, at very low rnet<br />
per sample .<br />
57<br />
58
59<br />
MODIFICATION OF 7,8-BENZOFLAVONE METABOLISM IN HAMSTER HEPATIC BUT NOT RENAL MICROSOMES<br />
BY LIVER TUMOR INDUCING AGENTS . G . Blaich, A .M . Tritscher, <strong>and</strong> M . Metsler . Institute of<br />
Toxicology, University of Wurzburg, Versbacher Str . 9, D-8700 Wurzburg, West Germany .<br />
Male Syrian golden hamsters chronically exposed to certain synthetic estrogens, e .g .<br />
diethylstilbestrol (DES) or 17u-ethinylestradiol (EE2), <strong>and</strong> fed a diet containing 7,8benzoflavone<br />
(BF) develop a high incidence (80-100%) of tumors in the liver but not in<br />
the kidney after 6-8 months . No hepatic tumors have been observed in animals treated<br />
with estrogen or BF alone . In order to clarify the role of BF metabolism in the mechanism<br />
of this hepatotumorigenesis, the effects of pretreatment of male Syrian golden hamsters<br />
with BF, with DES, with EE2, with BF plus DES, <strong>and</strong> with SF plus EE2 on the metabolism<br />
of 14C-BF were studied in hepatic <strong>and</strong> renal microsomes in vitro . Whereas hepatic<br />
microsomes from animals treated with DES or EE2 produced the same pattern of BF metabolites<br />
as control microsomes, a marked quantitative <strong>and</strong> qualitative alteration was observed<br />
after pretreatment with BF, with BF plus DES, <strong>and</strong> with BF plus EE2 : the metabolic<br />
rate was increased <strong>and</strong> several new metabolites were formed which were not observed with<br />
control hepatic microsomes . These metabolites, which were tentatively identified as BF<br />
dihydrodiol <strong>and</strong> di- <strong>and</strong> tri-hydroxy-BFa, were not formed in control or pretreated renal<br />
microsomes . Non-extractable binding of radioactivity to hepatic microsomal protein was<br />
observed but did not exhibit as pronounced a dependence on pretreatment as did the pattern<br />
of BF metabolites . It is concluded that BF induces its own oxidative metabolism to<br />
reactive intermediates in the hamster liver which may play an important role in hepatic<br />
tumor formation following prolonged treatment with BF plus DES or EE2 . It is proposed<br />
that BF acts as initiator <strong>and</strong> the estrogen as promotor in this animal tumor model .<br />
Support of this study by the Deutsche Forschungsgemeinschaft is acknowledged .<br />
60<br />
SALMONELLA MUTAGENICITY AND RODENT CARCINOGENICITY : QSAR<br />
B .W . Blake, K . Enslein, V .K . Combar, H .H . Borgstedt<br />
Health Designs, Inc ., 183 East Main Street, Rochester, NY 14604<br />
Discriminant analysis (DA) equations have been developed to achieve (1) a division of<br />
the carcinogens into genotoxic (Ames positive) <strong>and</strong> non-genotoxir groups, <strong>and</strong> (2) a<br />
division of the non-genotoxic compounds into carcinogenic <strong>and</strong> non-carcinogenic ones,<br />
solely on the basis of quantitative structure-activity relationships (QSAR) . An<br />
equation comprising eight sigma charge descriptors, two molecular connectivity indices,<br />
two kappa shape descriptors, <strong>and</strong> one substructure descriptor achieved discrimination<br />
between 56 genotoxic <strong>and</strong> 43 non-genotoxic carcinogens with an accuracy of 96 .7% .<br />
Another equation comprising eight sigma charge descriptors, three MCI's, one kappa<br />
shape desciptor, <strong>and</strong> twelve substructural descriptors achieved discrimination between<br />
39 non-genotoxic carcinogens <strong>and</strong> 48 non-genotoxic non-carcinogens with an accuracy of<br />
96 .4% . The QSAR models are suitable for the classification of genotoxic <strong>and</strong> nongenotoxic<br />
carcinogens in the absence of experimental data .<br />
61<br />
OSAR PREDICTION Of SALMONELLA MUTAGENICITY ASSAY RESULTS<br />
B .W . Blake, K . Enslein, V .K . Combar, H .H . Borgstedt<br />
Health Designs, Inc ., 183 East Main Street., Rochester, NY 14604<br />
An equation which discriminates Salmonella mutagens from non-mutagens based on<br />
quantitative structure-activity relationships (QSAR), including sigma charges, threedimensional<br />
shape descriptors, kappa shape indices, molecular connectivity indices, <strong>and</strong><br />
substructural fragments was developed from :<br />
1 . The results of 795 Salmonella mutagenicity assays performed up to 1979, previously<br />
modeled with an accuracy of 94 .7% .<br />
2 . The results of 383 Salmonella/microsome mutagenicity assays recently reviewed by a<br />
Gene-Tox panel .<br />
3 . The results of Salmonella mutagenicity assays listed by Ashby <strong>and</strong> Tennant (1988),<br />
Mutation Research 204, 17-115 .<br />
The resulting equation permits predicition of Salmonella mutagenicity on unassayed<br />
compounds based on structure .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 23<br />
Notes
24 1989 EMS Abstracts 6 2<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes THE AUGER ELEGTRON DOSIMETRY OF INDIUM IN THE NUCLEUS OF V79 CELLS . D.H .<br />
Blakey', J.R. McLean', G .R. Douglas', D . Wilkinson', <strong>and</strong> J.M . Bayley', '<strong>Mutagenesis</strong> Section, Bureau of<br />
Chemical Hazards, <strong>and</strong> 'RadioPharmaceuticals Section, Bureau of Medical Devices, Department of National<br />
Health <strong>and</strong> Welfare, Ottawa, Canada, K1A 0I2.<br />
Many radiopharmaceuticals or their metabolites distribute to intracellular sites where the release of Auger<br />
electrons can damage radiosensitive structures such as DNA . The effect of intracellular "'udium eadne on<br />
chromosomal aberrations <strong>and</strong> cell killing was defined in terms of equivalent "Cobalt doses . About 30% of<br />
cell-associated "'In oxine was found in the nucleus <strong>and</strong> 7 .5% in the DNA. Based on the assumption that<br />
these effects result only from radioactivity in the cell nucleus, the radiation dose to a cell was estimated by<br />
chromosomal aberration analysis to be 9 .6 x 10' Gy/decay <strong>and</strong>, by the colony assay for cell survival to be<br />
10.1 x 10' Gy/decay. The dose based on chromosomal aberrations is probably underestimated, since data<br />
from cells with pulverized chromosomes were excluded from the calculations . Furthermore, the cellular<br />
radiation dose could be as high as 13 .0 x 10' Gy/decay if only the DNA-associated radioactivity was presumed<br />
to contribute to the biological endpoint . Using the method deaeribedby NCRP (Public . 63), the average<br />
radiation dose to the cell nucleus from introcxllular "'In was estimated to be about 3.3 x 10' Gy/decay.<br />
Accordingly, based on the biological dose estimates in this study, the relative biological effectiveness (RBE)<br />
for intracellular "'In was estimated to be as high as 3.1 (i.e. 10.1/3.3). Moreover, if the DNA-associated<br />
radioactivity is assumed to be the sole contributor to cellular damage, the RBE could be even higher .<br />
Virtually all of the energy absorbed in the nucleus would be from the nuclear-associated Auger electron, since<br />
thepe netrating emissions (x- <strong>and</strong> gamma rays, <strong>and</strong> internal conversion electrons) ~ntn'bute only 6 .1 x 1001<br />
Gy/decay. It would, therefore, appear that the conventional method of organ dosimetry, which presumes a<br />
uniform distribution of energy throughout the target volume, is inappropriate when applied to intracellular<br />
Auger electron emitting compounds such as "'In .<br />
NUTAGENICITY AND ANTIMDTACENICITY TESTING OF SIX CHEMICALS ASSOCIATED WITH<br />
THE PUNGENT PROPERTIES OF SPECIFIC SPICES. E. D. Elevins <strong>and</strong> Asliyati Asian,<br />
School of Public 6 Allied Health, Dept. of Health Sciencea, East Tennessee State<br />
Univeraity, Johnson City, Tli 37614.<br />
Six compounds, capsaiein, thymol, borneol . <strong>and</strong> allyl were screened for<br />
mutagenic activity using Salmonella t~hiauriom strains TA97, TA98 <strong>and</strong> TA100,<br />
with <strong>and</strong> without 89 metabolic activation. A11 six oompounds are associated with<br />
the pungent properties of some specific spices . It was observed that capsaicin<br />
was mutagenic using strain TA100 in the presence of 89. Capaaiein, found in the<br />
spice Cansicum annum, was detected <strong>and</strong> quantified using thin layer <strong>and</strong> gas<br />
chrosutographic techniques . The presence of an antimutagenic factor(s) in C.<br />
annum that could suppress the nutagenicity of capsaicin was detected. When the<br />
mutagens capsaicin <strong>and</strong> 2-aminoanthracene were assayed in the presence of C .<br />
annum acetone extract, using strain TA100 with 89 metabolic activation, the<br />
nutagenic response of both the mutagens were reduced by approxisutely 50%.<br />
Assaying capsaicin <strong>and</strong> 2-aminoanthracene in the presence of chlorophyll, the<br />
mutagenic response of the two smtagene were reduced by less than 401 . From this<br />
observation it was inferred that chloropbyll can successfully suppress the<br />
mutagenicity activities of capsaicin <strong>and</strong> 2-astinoanthracene, together with other<br />
antimutagenic factors that were present in the acetone extract of C. annum.<br />
THE DETECTION OF 1WTAGENECITY OF CKMfICAL FACTCRS IN INDUSTRY<br />
N. P.Boabko v<br />
Institute of Medical (ienetios ASS USSR, Mosco w<br />
The paper generalizes the results of the control of mutagenio effect<br />
of chemioals in real industry conditions (some plants) in the USSR . To<br />
detect possible mutageneoity danger of industry chemicals for a man oytogenetio<br />
analysis was made regarding the oooupational group of workers<br />
being in contact with chloroprene lead,dioetbyl phtalat dimethyl aoetamide,benao(a)<br />
pyrene,trichlorfon,d~methyl sulplute formaidelyde,ethylene<br />
ozide,vinyl chloride <strong>and</strong> also rubber industry wor>
65 1989 EMS Abstracts 25<br />
investigation of Styrene Oxide-DNA Adducts <strong>and</strong> Their Detection in Notes<br />
Workers Exposed to Styrene . W . J . Bodell, K . Pongracz, S . Kaur, A . L .<br />
Burlingame, S . F . Liu <strong>and</strong> S . M. Rappaport Brain Tumor Research Center<br />
<strong>and</strong> Mass Spectrometry Facility, Univ . of California, San Francisco <strong>and</strong><br />
School of Public Health, Univ . of California, Berkeley<br />
Styrene Oxide (SO), a metabolite of the industrial chemical styrsna<br />
is mutagenic <strong>and</strong> carcinogenic in test systems . The identification of<br />
DNA adducts formed by SO may lead to an underst<strong>and</strong>ing of the genotoxic<br />
mechanisms of SO <strong>and</strong> quantitation of the SO-DNA add~~ts may provide a<br />
molecular dosimeter for human exposure to styrene . P-postlabeling of<br />
DNA reacted with SO resulted in the detection of six adducts .<br />
Chromatography st<strong>and</strong>ards were synthesized to allow assignment of the<br />
structures . Adducts ; <strong>and</strong> Z, are diaddu5ts resulting from the reaction<br />
of two molecules of SO with guanne (N ,C-8) . Adducts g <strong>and</strong> A are<br />
isomers of SO reacted with2the 04 position of guanine . Adduct I re~}lts<br />
from aralkylation at the N-site of guanine . We have extended the Ppostlabeling<br />
measurements to mononuclear cells isolated from the blood<br />
of workers exposed to styrene . Styrene exposure varied from less then 1<br />
ppm (low) to 20 ppm (high) . Several SO-DNA adducts were detected in the<br />
high exposure group . These preliminary results suggest that there may<br />
be a correlation between styrene exposure <strong>and</strong> levels of SO-DNA adducts<br />
in human mononuclear cells . (Calif . Toxic Substances FUND, RR01614 <strong>and</strong><br />
P42-ES04705<br />
66<br />
ANEUPLOIDY INDUCTION BY ALKYLATED BASES AND NUCLEOSIDES IN MAMMALIAN CELLS .<br />
S . Bonatti, G . Cercignanni, M . De Ferrari, P .L . Ipata, M . Rocco, M .G . Tozzi, S . Viaggi,<br />
<strong>and</strong> A . Abbond<strong>and</strong>olo, National Institute for Research on Cancer, Genova (Italy), University<br />
of Genova (Italy), LMD of CNR, Pisa (Italy), <strong>and</strong> University of Pisa, (Italy)<br />
The mechanism by which alkylating agents induce aneuploidy is not know . We discovered<br />
(Bonatti et al ., 1986) that 01-ethylguanine (O6etG) is a powerful inducer of aneuploidy<br />
<strong>and</strong> polyploidy when given to mammalian cells as a free base . To ascertain whether the in_<br />
duction of aneuploidy is a peculiarity of 06etG or is shared by other alkylated bates,<br />
the effect of O'meG, 7meG, 7atG, 3meA, 3meC <strong>and</strong> of unmodified bases was tested in human<br />
lymphocytes . It resulted that : (i) hypodiploidy was induced at similar frequencies by a .il<br />
tested compounds ; (ii) alkylated bases were more active than unmodified bases in inducing<br />
hyperdiploidy ; (iii) a high polyploidy induction was observed with 06etG . Since knwledge<br />
of the metabolic fate of the alkylated bases into the mammalian cell seemed essential, a<br />
number of enzymatic reactions were studied . We found that O4meG (i) is not degraded by<br />
guanase, that readily deaminates guanine to xanthine ; (ii) is not demethylated by adenosine<br />
deaminase that demethylates 04 methylguano$ine (O6meGuo) ; (iii)_is not converted to<br />
O6MeGuo by purine nuclaoside phosphorylase ; (iv) is not converted to O6meGMP by HPRT . On<br />
the other h<strong>and</strong>, O'meG was efficiently converted to O'meGuo by bacterial adenosine phosphorylase<br />
. In conclusion, the induction of aneuploidy was a general feature of alkylatad<br />
bases <strong>and</strong>, to some extent, of normal bases . The mechanism of induction, whether by spaci_<br />
fic alkylated products, by nucleotide pool imbalance, or other, remains to be clarified .<br />
67<br />
INHIBITION OF RADIOGENIC TRANSFORMATION BY a-LAPACHONE . David A . Boothman <strong>and</strong> Arthur<br />
B . Pardee, Division of Cell Growth & Regulation (D-810A), DAna-Farber Cancer<br />
Institute, 44 Binney Street, Boston, MA 02115 .<br />
p-lapachone is a potent inhibitor of DNA repair in mammalian cells . It activates<br />
topoisomerase I . We show that p-lapachone can prevent the radiogenic transformation<br />
of CHEF/18A cells . Potentially lethal DNA damage repair (PLDR) occurs while cells<br />
are held in medium containing low serum prior to replating . PLDR processes permitted<br />
survival recovery, but also drastically increased the number of foci/plate (i .e .,<br />
transformation) of CHEF/18A cells . By blocking PLDR with P-lapachone both survival<br />
recovery <strong>and</strong> enhanced transformation were prevented . At equivalent survival levels,<br />
exposure of X-irradiated cells to Q-lapachone resulted in an 8-fold decrease in the<br />
number of foci/dish as compared to the number of transformants produced in<br />
X-irradiated cells following PLDR .<br />
Early PLDR-derived increases in transformation may be the result of error-prone<br />
genetic rearrangements dependent upon topoisomerase I, which are thereby prevented<br />
by p-lapachone . a-Lapachone decreased the rejoining of DNA str<strong>and</strong> breaks <strong>and</strong> also<br />
appeared to produce additional double str<strong>and</strong> breaks in X-irradlated calls during<br />
PLDR . We hypothesize that the activation of topoisomerasa I by (i-lapachone may<br />
convert repairable single str<strong>and</strong> DNA breaks into the more repair-resistant double<br />
str<strong>and</strong> breaks, thereby preventing PLDR <strong>and</strong> radiogenic transformation . These results<br />
suggest a novel direction for the development of new anticarcinogenic agents .<br />
Supported by grant CA 22427 to Arthur B . Pardee from the National Cancer Inst .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
26 1989 EMS Abstracts 68<br />
Notes MITOD8PRL88IVE El7ECT8 OF PJ1R11QUAT Ill 71LLIU![ CEPA .<br />
R . A . Boroffice . Department of Biological <strong>and</strong> Chemical Sciences, Lagos<br />
State University, Ojo .<br />
Cells of Allium ceoa root meristems were exposed to different<br />
concentrations of paraquat (5, 10, 25, <strong>and</strong> 50mg/i) . There was a doserelated<br />
reduction in mitotic index which was directly associated with<br />
length of exposure . There was a relative reduction in mitotic index in<br />
treatments allowed recovery periods .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
SCREFNING FOR BASE MUPATICNS IN THE HPRT AND PAH LflCUS USING THE POLYMFRASE CHAIN<br />
REACfION (PCR) IN COMIDINATICN WITH DENATURING GRADIENT GEL ELDCIROPHORESIS (DGGE) .<br />
A .-L . BOrresen,l E . Hbvig,l B . S . Smrensen,l H . Vrieling2 <strong>and</strong> A . Brogger .l<br />
Inst, Cancer Research, The Norwegian Radium Hospital, Oslo, Norway<br />
2) Dept . of Rai . Gen . <strong>and</strong> Chen . <strong>Mutagenesis</strong>, Univ . of Leiden, The Netherl<strong>and</strong>s<br />
We have u9ej the polymerase chain reaction (pCR) <strong>and</strong> denaturing gradient gel<br />
electrophoresis (DGGE) to screen for base mutations in the PAH (phenylalamin<br />
hydroxylase) locus <strong>and</strong> the HPRT (Hypottanthine guanine phospho-ribosyl transferase)<br />
locus . For the PAH locus a 245 base pair fragnent containing exon 12 with flanking<br />
intronic sequences was amplifie8 . In this DNA sequence a single base substitution has<br />
beert found in 30% of the Norwegian PKU patients . This mutation gives a different<br />
melting profile of the 245 bp fragments resulting in a different migration on DGGE .<br />
The hu3e amplificaton of the genamic DNA fragments by PCR allows detection by direct<br />
EtBr staining of the gel making a rapid <strong>and</strong> easy method to screen for this PKU<br />
mutation in newborns .<br />
For the HPRT locus exon 3 was amplified fran a collection of 13 W <strong>and</strong> ENU induced<br />
mouse <strong>and</strong> hamster mutants, all shown to contain a mutation in the 3rd exon after<br />
sequencing . 8 of these mutants could be detected on DGGE after PCR . A collection of<br />
12 unknown HPRT mutants induced by porphyrins plus W were screened for mutations in<br />
exon 3 using these methods . No deletions of exon 3 were fourd, <strong>and</strong> no base mutations<br />
in the lower melting domain of exon 3 have been found so far . The use of PCR in<br />
combination with DG3E is a rapid <strong>and</strong> applicable method for screening both known <strong>and</strong><br />
unknown mutations, inherited or induced in the lower melting domain of a given<br />
DNA-sequence .<br />
MEJ-41 AND OTHER REPAIR GENES OF DROSOPHILA . James B . Boyd <strong>and</strong> Satnam S . Bangs. Department of<br />
Genetics, University of California . Davis CA 95616<br />
Genetic studies of repair deficient mutants in Drosophila have revealed the existence of extensive overlap in the<br />
functions that participate in DNA repair, recombination <strong>and</strong> synthesis . Mutants in several genes are known, for<br />
example, to be deficient in both DNA repair <strong>and</strong> meiotk recombinadon . A forward genetie approach is currently<br />
being employed in this laboratory to clone the repeir related genea nui-9 <strong>and</strong> ntef-I1 . Mutants at these loci are<br />
highly pleiotropic in that they influettce both meiotic reeombbttdon <strong>and</strong> a broad tpectrttm of DNA repair responses .<br />
Yamamoto <strong>and</strong> Mason have succeeded in mutageniztng both ioci In crosaes that mobilize transposable P elements .<br />
A combination of in siru hybridization <strong>and</strong> tevusiott atulysis has established that the recovered mutants were indeed<br />
induced by transposon insertions. Chromosome walking has been employed to recover all of the etei-41 gene .<br />
Transcriptionally active sequences adjacent to the P insertioa sites have been used to isolate a 2 .2 kb embryonic<br />
cDNA clone whose homology extends over 14 kb of the ohrumosomal walk . That cDNA represents a mei-01<br />
transcript because It exhibits homology to genomio sequences on both sides of the P insertion sites . Norihern blots<br />
prepared with poly(A)+ RNA from both embryos <strong>and</strong> adult females have Identified a single 2.2 kb transcript with<br />
homology to the cDNA clone .<br />
EVALUATION OF GENOTOXICFIY OF N-NfTROSODIBENZYlA1vIINE IN CHINESE<br />
HAMSTER V79 CELIS AND IN SALMONEIdA . B.G. Boyes. C.O . Rogers, N .P. Sen<br />
<strong>and</strong> T.I . Matula, Toxicology Research <strong>and</strong> Food Research Divisions . Food Directorate<br />
<strong>and</strong><br />
. Health <strong>and</strong> Welfare Canada .<br />
Ottawa~OntariolCANADAI• sion, Drugs Directorate<br />
Health concerns have arisen due to the formation of N-nitrasodibenzylamine<br />
(NDBzA) in pork processed in a new type of rubber netting . While NDBzA was<br />
reported to be non-carcinogenic in an early in vivo study (Druckrey ct 2 .<br />
Krebsforsch .69:103, 1967), the potent carclnogenicity of related nitrasamines eg . Nnitroso-n-dlbutylamine<br />
<strong>and</strong> N-nitrosodiethylamtne) prompted evahxation this<br />
compound for genotoxicity in y= in both Chinese hamster V79 cells <strong>and</strong> In<br />
Salmonella . Concentrations up to 25 Ng/ml were tested in V79 cells with <strong>and</strong><br />
without activation by rat or hamster hepatocytes . Under any of these conditions<br />
there was no significant elevation of sister-chromatid exchange levels . Mutation to<br />
6-thioguanine resistance was also negative, except for a weakpoaitive in only one of<br />
three replicate experiments, at 25 pg/ml, <strong>and</strong> only In the absence of hepatocytes<br />
which was considered not biologically siArti9cant . At concentrations up to 1000<br />
69<br />
70<br />
71
N ml in the Salmonella assay using pre-incubation protocol . NDBzA was negative<br />
A 98 . <strong>and</strong> in TA 100 with rat S9 . but was positive at the highest dose in TA 100<br />
with hamster S9 . <strong>and</strong> more strongly with Aroclor 1254-induced hamster S9 . When<br />
activated bY rat or hamster hepatocytes . as opposed to S9. NDBzA was negative with<br />
all tester strains. Thus . NDBzA appeared to be weakly mutagenic to Salm Qp!~8 but<br />
was apparently non-genotoxic to V79 cells with or without activation by rat or<br />
hamster hepatocytes.<br />
72<br />
HUMAN SPERM CHROMOSOMES : ANALYSIS OF STRUCTURAL ABERRATIONS AND DNA<br />
REPLICATION . B . F. Br<strong>and</strong>riff, L . A . Gordon <strong>and</strong> A . V. Carrano . Lawrence Livermore<br />
National Laboratory, University of Callfornia, Livermore, CA .<br />
Structural aberrations similar to those Identified In human sperm chromosomes by<br />
cytogenetic analysis following fusion of sperm with hamster eggs may contribute to some<br />
rare human heritable genetic diseases, as well as to unexplained Infertility <strong>and</strong> early<br />
embryonic losses . We analyzed structural aberrations observed in 6000 sperm<br />
chromosomal complements from 24 men . The most frequentiy observed aberrations<br />
were chromosome breaks <strong>and</strong> fragments . In lonpltudinal studies, frequencies were<br />
stable over many months or even years for Individual men . Egg culture conditions,<br />
numbers of chromosome complements processed per egg, <strong>and</strong> different sperm<br />
pretreatments had no effect on frequencies <strong>and</strong> types of aberrations . We examined DNA<br />
replication following sperm-egg fusion by detectinp Incorporated bromodeoxyuridine<br />
with Immunocytochemical methods . Human sperm chromatin was available as template<br />
soon after entry Into eggs, before full pronuclear development was achieved . This early<br />
availability may be an expression of a more labile packaging In the human sperm head<br />
compared to rodent sperm chromatin, <strong>and</strong> may suggest one mechanism leadinp to the<br />
higher frequencies of structural aberrations In human compared to rodent sperm<br />
chromosomes . Work performed by the Lawrence Livermore National Laboratory under<br />
the auspices of the US Department of Energy under contract 6W-7405-ENG-48 .<br />
73<br />
BASIC MECHANISMS OF ENVIRONMENTAL MUTAGENESIS ~<br />
Bryn A . Bridges, MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR,<br />
Gt . Britain .<br />
Most types of DNA damage do not inevitably result,in the induction of mutations .<br />
The latter usually arise during the operation of cellular processes . These cellular<br />
processes are most easily studied in microorganisms <strong>and</strong> have led to the formulation of<br />
mechanistic models for error-prone DNA repair pathways, as well as for error-free DNA<br />
repair <strong>and</strong> damage tolerance pathways, <strong>and</strong> for fidelity mechanisms that eliminate polymerization<br />
errors even after they have occurred . Many of these pathways are induced<br />
by DNA damage <strong>and</strong> some function poorly or not at all in the absence of a significant<br />
level of mutagen . Current studies are seeking to discover the extent to which mechanisms<br />
established in bacteria <strong>and</strong> other microorganisms also operate in human <strong>and</strong><br />
other mammalian cells . Sometimes mammalian processes show a similarity which reflects<br />
a mechanistic similarityp sometimes they mask differences that are far from trivial .<br />
In bacteria many if not most induced mutations arise by a process that requires<br />
proteins specified by the umuD <strong>and</strong> C genes . Partially homologous genes have been<br />
reported in bacteriophage T4, where they code for processivity proteins interacting<br />
with DNA polymerase, <strong>and</strong> in the eukaryote Schizosaccharomyces pomb . It is possible<br />
that such proteins operate throughout the evolutionary scale, giving further justification<br />
for mechanistic work with microorganisms .<br />
74<br />
THE CENTROMERE AND ANEUPLOIDYi DRUG-INDUCED FRAGMENTATION AND DETACHMENT OF KINETOCHORES<br />
OF MAMMALIAN CHROMOSOMES . B .R . Brinkley, R .P . Ziakowski, S .L. McCune <strong>and</strong> R .D .<br />
Balczon . Department of Cell Biology <strong>and</strong> Anatomy . University of Alabama at Birmingham,<br />
UAB Station, Birmingham, AL 35294 .<br />
The centromere, a specialized region of inetaphase chromosomes required for normal<br />
partitioning of the eukaryotic genome, has been implicated in aberrant chromosome<br />
distribution leading to aneuploidy . We have identified a unique type of drug-induced<br />
chromosome damage involving the kinetochore, a component of the centromere required<br />
for the attachment of spindle microtubules to chromosomes . Chinese hamster ovary<br />
(CHO) cells synchronized at the G1/S phase of the cell cycle with 2 mM bydroxyures<br />
for 20 h <strong>and</strong> subsequently treated with 5 mN caffeine for 2-6 h entered mitosis without<br />
completing DNA synthesis (Schlegel <strong>and</strong> Pardee, Science 232i1264, 1986) . A similar<br />
effect was induced when either Mitomycin C (1 .0 pg/ml) or Bleomycin (5 yg/ml) was<br />
used instead of caffeine in the above protocol . We termed these mitotic cells with<br />
unreplicated genomes (MUGs) <strong>and</strong> found that pre-S phase entry into ∎itosis was<br />
accompanied by the detachment of kinetochores from prematurely condensed, highly<br />
fragmented chromosomes . Using video microscopy, fluorescent image analysis, <strong>and</strong><br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts<br />
Notes<br />
27
28 1989 EMS Abstracts<br />
Notes electron microscopy, we determined that the detached elements were actually<br />
aub-fragments of the unreplicated kinetochore . These fragments are short repeated<br />
egments of 30 nm chromatin fibers which bind MTs <strong>and</strong> undergo the complete repertoire<br />
of mitotic movements . Based upon these studies, a model for kinetochore structure<br />
will be presented which explains new aspects of centromere structure, evolution <strong>and</strong><br />
involvement in aneuploidy .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
MOLECULAR ANALYSIS OF IN VIVO, SOMATIC MUTATIONS OF THE HPRT<br />
GENE IN MOUSE AND MAN, Eleanor C . Brinson, Karolyn Burkhart-<br />
Schultz, I M• l "Io, <strong>and</strong> Cheryl L . Strout, Biomedical Sciences<br />
Division, Lawrence'.ivermore National Laboratory, Livermore, CA 94550 (USA)<br />
To analyze factors that affect the spectrum of mutations in mammalian cells in<br />
vivo, we are studying mutations of the hypoxanthine phosphoribosyltransferase<br />
gene (hprt) in lymphocytes of both mouse <strong>and</strong> man . Gbmparison of mutations<br />
detected in these systems with mutations detected in other in vivo <strong>and</strong> in vitro<br />
systems will lead to better underst<strong>and</strong>ing of the effect that species, tissue, locus,<br />
selection method, <strong>and</strong> sequence have on the spectra of both spontaneous <strong>and</strong><br />
mutagen induced mutations . Particularly in the mouse we are able to manipulate<br />
factors that may affect the mutations recovered . in the mouse model, using<br />
restriction fragment length analyses, we have detected a high proportion of<br />
deletions among spontaneous mutations of the hprt gene . In nondeleted mutants<br />
we have detected base substitution <strong>and</strong> frameshift mutations of the coding regions<br />
by sequencing exon DNA, after amplification of genomic DNA using ahe<br />
polymerase chain reaction (PCR). Studies of mutants isolated from irradiated mice<br />
are in progress . Interspecies comparisons of spontaneous mutations are being<br />
initiated with human lymphocytes by sequencing PCR amplified hprt cDNA. This<br />
work performed under the auspicies of the U.S. Department of Energy by<br />
Lawrence Livermore National Laboratory under contract number W-7405-ENG~48,<br />
in part supported by an Interagenc A reement (Y01-ES-80171) between the<br />
National Institute of <strong>Environmental</strong> Healt~ Sciences <strong>and</strong> DOE .<br />
QICTOX .`C rlM BIOCMIICAL ACPIVITIES OF SCt'lE C3BACIMED ElHANES .<br />
Brorizetti G ., I•Iorichetti E ., Del Carratore R., Rosellini D ., Paolini M., Cantelli Forti<br />
G., Grilli S . <strong>and</strong> Vellosi R .<br />
Istituto de A9utagenesi e Differenziamento, CtJR, Pisa<br />
Istituto de Farmaoologia, Istituto di Canoerologia, Universita di Bologna, ITALY<br />
1,1,1,2,-4+etrachloroethane (TIM), Pentachlaroethane (PCE) <strong>and</strong> Hexmchloroethane (F1CE)<br />
have a wide range of applications in industrial processes as solvents, degreasers ard<br />
aooeLerators in rubber vulcanization . 7he increasirg production of these caipoucrls in<br />
the past few years led to major attention in un8erst<strong>and</strong>ing their effects on huren health .<br />
In this work, TiCT:, PCE <strong>and</strong> HCE were tested in D7 strain of yeast-Sacctwr -Cerevisiae<br />
in suspension test with <strong>and</strong> withcut a mentnalian activation system S . TDCE, P(E~r Hf~<br />
gave positive results on cells harvested fraa logarithmic
1989 EMS Abstracts<br />
aberrations/cell, respectively . Alpha-particles <strong>and</strong> X-rays given alone resulted in a Notes<br />
linear increase in micronuclei/binucleated cell with respective slopes of 0 .77 1 0 .08<br />
<strong>and</strong> 0 .20 t 0 .05 micronuclei/binucleated cell/Gy . When 1 .0 Gy of a-dose was given<br />
simultaneously with 0 .0, 0 .75, 1 .5, or 3 .0 Gy of X-rays, the slope of the combined<br />
exposure dose-response curve for the X-ray induced nicronuclei had a slope similar to<br />
that observed after exposure to a-particles alone (0 .74 2 0 .05 micronuclei/binucleated<br />
cell/Gy) . These data sets both suggest a synergistic interaction between a- <strong>and</strong> Xray-induced<br />
damage . Regardless of exposure sequence, when alpha <strong>and</strong> X-ray exposures<br />
were separated in time by 1/2, 2, or 6 hrs, synergistic interactions between the<br />
damage induced by both exposures was no longer evident . These data demonstrate that<br />
X-rays <strong>and</strong> a-particles can interact <strong>and</strong> that repair of the chromosome damage involved<br />
in the interaction is very rapid . (Research sponsored by the U .S . Department of<br />
Energy's Office of Health <strong>and</strong> <strong>Environmental</strong> Research under Contract DE-AC04-76EV01013 .)<br />
78<br />
ORGAN-SPECIFIC GENOTOXIC EFFECTS OF CHEMICALS : THE USE OF ALKALINE ELUTION TO<br />
DETECT DNA DAMAGE IN VARIOUS ORGANS OF IN VIVO-EXPOSED ANIMALS<br />
G . Brunborg, ] .A . Holme, EJ Sederlund <strong>and</strong> E . Dybing, Department of Toxicology, National Institute of<br />
Public Health, Oslo, Norway<br />
The existence of genotoxic chemicals with marked organ specificity indicates that chemicals should be<br />
tested in more than one organ or cell type . We have developed techniques for detecting aad comparing<br />
DNA damage in different organs of experimental animals after in vivo administration of chemicals .<br />
Exposed anim als are anesthetized, organs are re moved, cooled rapidly in cold buffer, <strong>and</strong> processed as<br />
follows: The liver, kidney, testis, lung or spleen is minced with scissors aad forced through a stainless<br />
steel screen + one layer of cotton gauze . The stomach, small or large intestines or the urine bladder is<br />
rinsed, opened, the epithelial cells scraped off with a glass slide, <strong>and</strong> homogenized in a glass homogenizer .<br />
The brain is homogenized directly . The femur is opened, <strong>and</strong> bone marrow cells are rinsed out <strong>and</strong><br />
homogenized . All samples are then centrifuged <strong>and</strong> counted . The procedure takes about 1 hour for 16<br />
samples. Two million cells or nuclei are loaded on an automated alkaline elution system (Brunborg et al .,<br />
Anal . Biochem . 1Z, 522-536, 1988) .<br />
Experiments with 1,2-dibromo-3-chloropropane (DBCP) (10 mg/kg i .p ., 1 br) demonstrate highly<br />
variable effects in the various organs : Maximum DNA damage was observed in the liver, kidney <strong>and</strong><br />
duodenum, with intermediate damage in the lung, spleen, brain, testis <strong>and</strong> stomach . Higher doses (40<br />
mg/kg) were required to produce significant damage in bone marrow <strong>and</strong> colon . DBCP has been shown<br />
to induce tumors in rat liver, kidney <strong>and</strong> stomach .<br />
T he data indicate that an evaluation of the genotoxic properties of a chemical by means of short-term<br />
tests should involve several organs . We are currently utilizing the methods described to study the<br />
genotoxicity of various dietary mutagens .<br />
79<br />
THE EFFECTS OP MBTBAPYRILBNB oN IN VIVO SCE AND IN VITRO CBROMOSOME ABERRATION<br />
INDUCTION. J . Brunny, D . Kindig, an-d f.Garriott, LThy tesaarch Laboratories, 8li<br />
Lilly <strong>and</strong> Company, Greenfield, IN 46140<br />
The antihistamine methapyrilene hydrochloride (MP) has been shovn to be a rat<br />
liver carcinogen ; however, ∎ixed positive <strong>and</strong> negative results exist in short term<br />
genetic toxicology assays . To date, the only in vivo data available on MP are<br />
negative results from two unscheduled DNA synthe®Ts studies using rat (Mirsalis et<br />
al ., Environ . Mutagen . 7, Suppl . 3 :73, 1985 ; Steinmetz et al ., Carcinogenesis 9t95§,<br />
1988) <strong>and</strong> souse (Steinmetz et al ., op . cit .) hepatocytes <strong>and</strong> from an SCE assay in<br />
Fischer 344 rats (Iype et al ., Cancer Res . 42t4614, 1982) . Results are presented<br />
here from an in vivo SCS assay in male CD-1 Uce . Intravenous doses of 2 .5, 5, 10,<br />
<strong>and</strong> 20 mg/kg oT IffP vere administered <strong>and</strong> bone marrow harvested 21 hr later . The<br />
incidence of SCE was recorded <strong>and</strong> ranged from 3 .2 to 4 .3 SCE/metaphase, which was not<br />
significantly different from solvent controls . Because of positive findings in the<br />
L5178Y mouse lymphoma assay in which an increase in small colony mutants was observed<br />
<strong>and</strong> chromosome damage confirmed (Blazak et al ., Environ . Mutagen .• Bt229, 1986), MP<br />
vas also tested for its ability to induce cTiromosome aberrations in v1tro in cultured<br />
Chinese hamster ovary cells . Doses of 375, 450, <strong>and</strong> 550 yg/al of MP were tested both<br />
vith <strong>and</strong> without an S-9 activation system . The percent aberrations ranged from 0 to<br />
2 percent in the activated assay <strong>and</strong> from 0 to 3 percent in the nonactivated assay .<br />
These results were not significantly different from solvent controls . The negative<br />
results from these two assays lend further support to the theory that NP is carcinogenic<br />
through a nongenotoxic mechanism .<br />
80<br />
FffiFRE HAVE WE BFEN? 74fE IAST 20 YFJtRS OF FNVtRMlOWPAL 1'1t1DWMMIS<br />
~<br />
Dnvid Brvsiak. Hazleton Laboratories, U .S .A .<br />
N<br />
It is wr#Miile to assess the peaks an! valleys of the road travelled by<br />
tp<br />
the discipline of envirormental autagenesis daa'ing the past 20 years . /'<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
tr<br />
m<br />
00<br />
Q1<br />
29
30 1989 EMS Abstracts<br />
Notes WitTbut question, this discipline has ocntributed significanGly to the<br />
protection of the genetic integrity of fuRazti+e ganesaticre of humans . In<br />
additicn, basic researth infcrmatim devuloped by members of this field<br />
during the past two decedes has lead to a clearer mechanistic<br />
tadezstarxiirg of the prrooesees of cancer, cellular diffezantiation <strong>and</strong><br />
aging . mnowledge bases within the applied scienoes of inedicine <strong>and</strong><br />
taxicology would be poorer today if it w+ .re not for research into mutation,<br />
aa repair, ssrleic acid biodwemistsy <strong>and</strong> da+omosmis funct.ions carried azt<br />
under the domain of genetic toocicity .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
qhile valleys have been enoaa*ared along the road travelled since 1969,<br />
their existence has generally resulted in renewed snthusiasm <strong>and</strong> scientific<br />
vigor . Any scientific discipline which tails to subject itself to critical<br />
assesseent for fear that it will reveal possible flaws or lead to &Ange,<br />
is docmed to be replaced. Ths field of erwiratnental mltagenesis is<br />
forWnate to hn+e had a lartre manber of creative <strong>and</strong> dedicated visionaries<br />
leading it Liuu4i i .i .ia uailwylny aua7 ew.iuig jwciery .<br />
A COMPUTER-ASSISTED PROCEDURE FOR THE ASSEMBLY AND ANALYSIS OF SHORT-TERM<br />
GENOTOXICITY TEST DATA<br />
D .J . Brusick, P . Lohman, M . Mendelsohn, M . Waters, S . Nesnow, D . Moore, F . de Serres,<br />
J . Ashby, B . Matte- r . T: Matsushima, ICPENC, Subcommittee 1 .<br />
Determining the genetic hazard of a chemical is generally approached by using an<br />
assortment of tests for measuring the DNA reactivity of a chemical or its resultant<br />
genotoxicity . Over 100 short-term tests employing a wide diversity of species <strong>and</strong><br />
genetic mechanisms have been used to measure genetic hazard . To date, attempts to<br />
achieve a st<strong>and</strong>ard test battery for defining genetic hazard have not been successful .<br />
Consequently, testing for genetic hazard involves the use of test batteries with<br />
variable types <strong>and</strong> numbers of assays . This increases the difficulties of interpreting<br />
data sets since the data sets are often filled with inconsistent responses from<br />
diverse types of assays .<br />
Several years ago, the International Commission for Protection Against <strong>Environmental</strong><br />
Mutagens <strong>and</strong> Carcinogens (ICPEMC) established a Committee to establish a method to<br />
compile <strong>and</strong> interpret diverse short-term test data . The Committee has produced a<br />
quantitative weight-of-evidence approach that combines test data using certain<br />
parameters such as dose, replication <strong>and</strong> metabolic capacity into a series of scores<br />
for test type, test class test family <strong>and</strong> an overall score that defines the total<br />
weight-of-evidence regard ;ng the genetic hazard of the agent . •<br />
SISTER CHROMATID EXCHANGE AND MICRONUCLEUS ANALYSIS IN RAT PERIPHERAL<br />
BLOOD LYMPHOCYTES AFTIR IN VIVO EIPOSURE TO BENZO(A)gYREVE . M .F .<br />
Bryanta, G .L. Eregson , P . Kwanyuen , <strong>and</strong> A .D. Kligerman , EHRT,Inc,<br />
RTP, NC 27709, <strong>and</strong> U .S . EPA, RTP, NC 27711 (USA) .<br />
In an effort to determine the persistence of sister chromatid<br />
exchange (SCE) <strong>and</strong> micronucleus (MN) induction following exposure to a<br />
polycyclic aromatic hydrocarbon known to exist at hazardous waste<br />
cleanup sites, experiments were conducted in peripheral blood<br />
lymphocytes (PBLs) of male Sprague-Dawley rats injected ip . with<br />
0, 100, or 250 mg benzo(a)pyrene/kg . Peripheral blood was removed by<br />
cardiac puncture from each rat at 1, 2, 5, 7, 14, <strong>and</strong> 21 days after<br />
injection. Isolated PBLs from three animals/dose/harvest time were<br />
cultured, according to previously published methods, for analysis of<br />
SCEs in second-division cells <strong>and</strong> MN in cytochalasin B-blocked<br />
binucleated lymphocytes. Both the SCE <strong>and</strong> MN frequencies remained<br />
elevated for 21 days post-injection . These results suggest that rat<br />
PBLs containing SCE <strong>and</strong> MN inducing lesions remain viable for at least<br />
three weeks . Due to variable SCE frequencies in isolated PBLs among<br />
replicate animals, a 56 day study was undertaken in which whole blood<br />
was used for SCE analysis . Results will be presented comparing SCE<br />
responses in whole blood <strong>and</strong> isolated PBLs .<br />
(This abstract does not necessarily reflect US EPA policy .)<br />
50869 3542<br />
81<br />
82
83<br />
FREQUENCIES OF CHROMOSSOMAL ABERRATIONS AND MICRONUCLEUS IN RODENTS COLECTED IN COAL-<br />
FIELD AND TOBACCO CULTURE REGION - CRICIOMA - SC - BRAZIL .Angela M . S . Bueno, Jeanete<br />
M . S . Agostini, Karina Gaidzinsk, Riroko Nitta, Josane Moreira, Ivan Brognoli -<br />
Departamento de Biologia - UFSC - Florianopolis - SC - Brazil - 88049 .<br />
Criciuma - SC - is a town situated in the coal-field of South Brazil . The present work<br />
has been done in the Basin of Sangao River, one of the local rivers that receiv the<br />
rejectes of the coal washing, like mercury, cadmium, lead, etc . in wich bank exist many<br />
proprieties of tobacco cultures where various organophosphorate <strong>and</strong> carbamate agrotoxics<br />
are used . Our purpose in this area is to try to detect <strong>and</strong> estimate the possible action<br />
of the environmental pollution in the organisms directly exposed to it - in this case<br />
rodents collected in the local - through the study of the frequency of chromossomal<br />
aberrations <strong>and</strong> micronucleus in bone marrow . Parallely the same methodology has been<br />
used for rodents collected in regions free from these polluter factors to set up a<br />
control group . With this finality we delimited two collect points in the Basin of Sangao<br />
River (A <strong>and</strong> B), one point out of this area (C) <strong>and</strong> some points in Florian6polis 040Km<br />
from Criciuma) free from the action of the above mentionate polluters . The points C <strong>and</strong><br />
D are considered control points . Our parcial results have showed the following<br />
percentage for chromossomal aberration : A-3,62x, .B-1,30x, C-none, <strong>and</strong> D-1,46x, <strong>and</strong> the<br />
following results for micronucleus analisis : 20% . for Criciuma's points <strong>and</strong> 10x .for the<br />
control group .For the meantime we can say that these results suggest that this<br />
methodology can be applied to detect the action of the environmental pollution in the<br />
organisms . (FINEP) .<br />
84<br />
STUDIES ON A GENOTO%ICITY TEST USING P . PHOSPHOREUM<br />
Anthony A . Bulich, Mary Grace Schreibner, Ser-Eon , Charles C . Walbourn<br />
Microbics Corporation, 2232 Rutherford Roa , ars a, CA 92008<br />
Chemicals are essential for modern society, <strong>and</strong> proper application of them has<br />
greatly improved general living conditions . However, the chemical wastes, <strong>and</strong> the<br />
by-products of these wastes, are a major cause of environmental pollution . In order<br />
to monitor <strong>and</strong> control these pollutants, simple <strong>and</strong> fast tests are needed . Following<br />
the introduction of the Microtox (R) Acute Toxicity Test, we now propose a test for<br />
genotoxic compounds (Mutatox (TM)) . The test uses a dark mutant of luminous bacteria<br />
<strong>and</strong> determines the ability of the tested agent or sample to restore the luminescent<br />
state . Genotoxic agents which are mutagens, DNA damaging agents, DNA synthesis<br />
inhibitors, <strong>and</strong> DNA intercalating agents are detected with this test . This one-step<br />
test can be performed as an overnight assay, under non-sterile conditions . Fifty<br />
pure chemicals have been tested with the proposed method, <strong>and</strong> the results correlate<br />
vell with published data obtained with the Ames test . The predictive value of this<br />
method for carcinogenicity is also comparable to the Salmonella assay . Validation of<br />
the test with an additional 50 extensively studied chemicals from the National<br />
Toxicology Program is underway . The results from this study will be presented .<br />
85<br />
MOUSE MODEL FOR SOlATIC MUTATION AT TH$ HPRT GENE : MOLECULAR AND CELLULAR<br />
ANALYSES, Ic . Burkhart-Schults, C .L . Strout, <strong>and</strong> I_ H . Jenaa, Lawrence<br />
Livermore National Laboratory, Livesmore, CA 94550 (USA)<br />
We are using the mouse to study factors that affect the frequency <strong>and</strong><br />
molecular nature of somatic mutations that occur in vivo . We have<br />
detected differential induction of thioquanine resistant mutants in thymus<br />
<strong>and</strong> spleen T lymphocytes ; tissue, mutaqen <strong>and</strong> age dependent patterns of<br />
mutant frequency with time ; <strong>and</strong> persistence of up to one year for both<br />
ethylnitrosourea (sNU) <strong>and</strong> radiation induced mutants . Whereas the mutant<br />
frequency increased linearly up to 72mg ZNO/kQ, <strong>and</strong> was not reduced by<br />
fractionation of ENt1 dose, it increased curvilinearly to 400 cOy 1,7Ce <strong>and</strong><br />
was reduced by radiation dose fractionation . The molecular nature of<br />
spontaneous <strong>and</strong> induced mutations is being studied by Southern analysis<br />
<strong>and</strong> by polym.rase chain reaction based methods . Soon after acute exposure<br />
of young adult, male mice to radiation the mutation spectrum is enriched<br />
two-fold for total deletions of the hypoxanthine phosphoribosyltransferase<br />
(hprt) locus, whereas after ZNO no deletions were detected . The recovery<br />
of many deletions, of frameshifts, <strong>and</strong> of 3 different base substitutions<br />
affecting the protein synthesis initiation codon, indicate that selection<br />
of mutants with 2 .5 mq/ml thioguanine is highly stringent <strong>and</strong> may limit<br />
the spectrum of point mutations recovered . This work performed under the<br />
auspicies of the U .S . Department of inerqy by Lawrence Livermore National<br />
Laboratory under contract number N-7405-iNG-48 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 31<br />
Notes
32 1989 EMS Abstracts<br />
Notes IN VIVO DETECTION OF UDS IN THE RAT GASTRIC NUCOSA 86<br />
B . Burlinson, D .G . .Gatehouse <strong>and</strong> D .J . Tweats, Dept . Genetic <strong>and</strong> Reproductive Tox .,<br />
Glaxo Group Res . Ltd ., Ware, Herts, Engl<strong>and</strong> .<br />
An assay is needed to detect genotoxicity in vivo in tissues other than the liver<br />
or bone marrow . For the pharmaceutical industry the most obvious tissue to<br />
investigate is the stomach <strong>and</strong> although there are UDS assays already described for<br />
this organ they are relatively insensitive or require the use of hydroxyurea . An<br />
assay has been developed in which account is taken of the morphology of the gastric<br />
mucosa to enable the isolation of non-S-phase cells <strong>and</strong> the measurement of UDS by<br />
scintillation counting without the need of hydroxyurea . The assay is sensitive <strong>and</strong><br />
has a stable control background, (209 * 83 dpa/ vg DNA (11 .34)) . The gastric<br />
carcinogen ICPNG was detectable at doses down to 12 .6mg/kg (316 t 87 dpm/ vg DNA<br />
(N :5)) . Indomethacin a non-genotoxic gastric lrritant, was inactive at doses known<br />
to induce moderate cellular damage indicating that false positive responses due to<br />
gastric irritancy should not occur . The value of this assay is clearly demonstrated<br />
by the results obtained with epichlorhydrin . This compound is a direct acting,<br />
forestomach carcinogen which is Ames positive but which is not detectable in the bone<br />
marrow micronucleus test or the in vivo liver UDS assay . It was however, easily<br />
detected in the stomach UDS assay at 50 mg/kg (LD50 .2S0 mg/kg) . From the data<br />
obtained so far this assay appears to be a rapid <strong>and</strong> reliable method of measuring UDS<br />
in gastric mucosa . It can be used in cases where, becacse of the pharmacokinetics or<br />
lability of the compound, negative results obtained using the marrow or liver as the<br />
indicator tissue offer little confidence . It is also of use for investigating drug<br />
nitrosation in vivo, this aspect of a test is currently being investigated .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
COHPLFMENTATION GBOUP ASSIGl0KM1TS FOR W-SENSITIVB CH0 CELIS . D .B . BUSCH, L.H. -"<br />
THCMON, AND G . ADAIR, Armed Forces Inatitute of Pathology, Washington, D .C. (USA),<br />
Lawrence Livermore National laboratory, Livermore, CA (USA), <strong>and</strong> University of Texas<br />
System Cancer Center, Smithville, TZ (USA)<br />
These studies were performed in order to determine the number of oomplementation<br />
groups of W-sensitive CH0 cells, <strong>and</strong> to investigate the effeot of mutagen treatment<br />
of the parental lines on the distribution of CH0 W mutant groups . Parental cells<br />
included E-rey sensitive (EM9), mitoaqoin-C (MM) sensitive (M05), <strong>and</strong> wild type<br />
(AA8) CH0 cells . Using a rapid screening procedure, 166 of approximatel,y 284 W<br />
mutant CH0 cells were assigned to a total of six groups . Over 90% of the assigned<br />
mutants were in the first two groups, with group 1 predominating after frameshift<br />
mutagen ICR-170 treatment, <strong>and</strong> class 2 predominating after use of miesense mutagena .<br />
The relative scarcity of group 2 mutants following ICR-170 is consistent with the<br />
hypothesis that the gene damaged in group 2 mutants, VX,2-, is an essential gene . The<br />
effect of mutagen treatment on the yield of mutant groups demonstrates the potential<br />
value of varying the mutagen treatment during mutant hunts . Our isolation of W<br />
mutants of X-ray sensitive, MAiC sensitive, <strong>and</strong> (in one case) W-sensitive CH0 oells<br />
shows that mammalian cells can yield viable double mutants in mutagen sensitivity .<br />
Work supported by the National Institutes of Health (grant numbers GM22021 <strong>and</strong><br />
R1O0961) <strong>and</strong> performed under the auspices of the U.S . Department of Energy by<br />
Iewrence Berkeley laboratory grant number 7134700 <strong>and</strong> by the Lawrence Livermore<br />
National Isboratory under contract W-7405-11110-48 .<br />
RIiR 71gsEgsMENT OF s00o 1WTJ10sNgt<br />
by Lait Dusk<br />
Toxicology Laboratory, National tood Administration, box 622, 8-75126 Uppsala, Sweden .<br />
There is little doubt that pyrido-iaidasoles/indolss <strong>and</strong> imidasoasaarenes ferasd during<br />
normal cooking are eapable of producing cancer in man, sinoe they induoa malignant<br />
tumours at aultiple sites in both sexes of rats <strong>and</strong> mios . ginoa very few food autagens<br />
have been tested in anwlti-dose experiments, the possibility to perform hi9h-to-lov doss<br />
extrapolations is Ii .ited . Usinq the simieluantitativ. TDSO approach, sinql"ose<br />
exparisents can be used to provide rough estisutes of the potency in animals . Such<br />
calculations 4ive TDSO values in the 1-100 mg/kg dose range, indioatino a moderate carelnoqenio<br />
potency in high-dose animal experiments . It is not known whether this is true<br />
for low doses or if man <strong>and</strong> animals are equally sensitive . At present, the axposura of<br />
humans to food autagens cannot be adequately detar .ined, since Quantitativs data are<br />
∎canty . A very rough estimate from the available data suq0ests that humans might be<br />
exposed to 1 Y9 of oooked food autaq .ns/kq bw . Combining thes* very rough exposure data<br />
with the eQually rough cancer potency estiaates <strong>and</strong> assuming that aan <strong>and</strong> rodents are<br />
equally sensitive, that the dose-rasponse relationship is linear, it saams unlikely<br />
that the cooked food autaqens tssted so far represent a major threat to human health .<br />
However, the carcinogenicity of the muta9ens present at the highest levels in food have<br />
not yet been detersin.d <strong>and</strong> virtually nothing is known about aodifyiaq dietary factors<br />
<strong>and</strong> differences in sensitivity between animals <strong>and</strong> man . The need for furthar resaaroh<br />
in order to provide a data base for a swre meaningful human risk assessment will be<br />
disoussad .<br />
50869 3544<br />
88
89<br />
HUMAN LIVER CYTOCHROME P-450P IS PRIMARILY RESPONSIBLE FOR CAFFEINE 3-DEMETHYLATION<br />
AND CARCINOGENIC ARYLAMINE N-eXIDATION . Mary Ann Butler, Masahiko Iwasaki*, F . Peter<br />
Guengerich*, <strong>and</strong> Fred F . Kaglubar . National Center for Toxicological Research,<br />
Jefferson, AR 72079 aerbilt University School of Medicine, Nashville, TN 37232<br />
Aromatic amines are well known as occupational carcinogens <strong>and</strong> are also found in<br />
cooked foods, tobacco smoke, synthetic fuels, <strong>and</strong> agrochemicals . The metabolic<br />
N-oxidation of several primary arylamines, which is generally regarded as an initial<br />
activation step leading to carcinogenesis, recently has been shown to be catalyzed<br />
primarily by rat cytochrome P-450I F-G <strong>and</strong> by its human ortholog, cytochrome P-450<br />
We now report that human liver aic~osomal caffeine (CF) 3-demethylation, the initift<br />
major step in CF biotransformation in humans, is selectively catalyzed by P-450PA . CF<br />
3-demethylation was highly correlated with 4-aminobiphenyl (ABP) N-oxidation (r - 0 .99,<br />
p
34 1989 EMS Abstracts<br />
Notes exposed to fractionated doses of 6000 1=raya (1 .7 Gy weekly <strong>and</strong> an accumulated<br />
dose of 6 .8 (}y) . Mice were sacrificed at various time during 1-<br />
195 days after irradiation . Some changes in histopathology, cytogenetics<br />
ultrastructure <strong>and</strong> trace element were observed . The results indicated<br />
that radiation could induce thymoma in treated mice (66 .3%), none of it<br />
occurred in control . By histopathological <strong>and</strong> ultrastructural studies,<br />
4 steps appeared clearly after irradiations thymic cortex atrophy, hyperplasia,<br />
precancerous <strong>and</strong> cancerous phenomena . Some round viruslike<br />
particles could be seen in lymphoma cells of exposed mice . The most frequent<br />
type of chromosomal aberration was reciprocal translocation <strong>and</strong><br />
the abnormal clones appeared in some cells of thymus, spleen <strong>and</strong> tone<br />
marrow in exposed mice . But there was no significant difference on the<br />
frequency of SCE between two groups . Both the contents of Zn <strong>and</strong> Cu<br />
have risen after irradiation . The results suggest radiation itself is a<br />
potential mutagen, radiation induced mutation in the target cells may<br />
be responsible for the development of tumor <strong>and</strong> leukemia in an irradiation<br />
subject .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
METABOLIC ACTIVATION OF 6-NITROCBRYSENE IN ISOLATED RAT HEPATOCYTES DERIVED FROM<br />
UNINDUCED AND INDUCED SPRAGUE-DAWLEY RAT LIVERS . D .A . Casciano, K .B . Delclos,<br />
R.P . Walker, <strong>and</strong> J .G . Shaddock . National Center for Toxicological Research,<br />
Jefferson, AR 72079 .<br />
It has previously been shown that 6-nitrochrysene (6-NC) can be activated to<br />
electrophilic species capable of reacting with DNA through metabolic pathways that<br />
form N-hydroxy-6-aminochrysene (N-hydroxy-AC) or trans-1,2-hydroxy-1,2-dihydroxy-6aminochrysene<br />
(AC-1,2-dihydrodiol) . We have alsoemonstrated that when N-hydroxy-AC<br />
is reacted with DNA in cell-free systems, three major adducts are formed, N-(deoxyinosin-8-yl)-6-aminochrysene<br />
(I) . 5-(deoxyguanosin-N2-yl)-6-aminochrysene (II), <strong>and</strong> N-<br />
(deoxyguanosin-8-yl)-6-aminochrysene (III) ; while an as yet unidentified adduct (IV)<br />
is formed from the in vitro reaction of a microsomal metabolite of AC-1,2-dihydrodiol .<br />
In order to test the influence of microsomal enzyme induction on the activation pathway<br />
in an intact cell system, 6-NC was incubated with freshly isolated hepatocytes<br />
from rats that were either untreated or pretreated with phenobarbital (PB), 3-methylcholanthrene<br />
(3-MC) or Aroclor 1254 (ARO) . Hepatocytea isolated from untreated or PBtreated<br />
rats formed adducts derived from N-hydroxy-AC (predominately I . II, <strong>and</strong> III),<br />
whereas hepatocytes isolated from rats pretreated with 3-MC or ARO formed adducts<br />
derived from AC-1,2-dihydrodiol (predominately adduct IV) . Our results provide the<br />
first clear demonstration that exposure to inducers of drug metabolizing enzymes can<br />
profoundly alter the qualitative pattern of adducts formed in the DNA of cells treated<br />
with a nitro PAH .<br />
USE OF POLYMERASE CHAIN REACTION (PCR), DIRECT SEQUENCING, AND DNA<br />
COLONY HYBRIDIZATION IN THE CHARACTERIZATION OF SdIJKONBI .L4<br />
?YPHIMURIUM REVERTANTS. T.A. Cebula <strong>and</strong> W.H. Koch. Food <strong>and</strong> Drug Administration,<br />
200 C Street, S.W., Washington, D.C. 20204 .<br />
The S. typbLnuriuas reverse mutation assay Is widely used to assess the mutagenicity <strong>and</strong><br />
potential carcinogenicity of a large variety of chemical compounds . Elucidation of the molecular<br />
events <strong>and</strong>erlying the reversion of the kLsG46, 6ttD30S2, <strong>and</strong> bisC3076 mutations by well<br />
characterized carcinogenic <strong>and</strong> mutagenic agents should provide valuable insights Into the<br />
mechanistic relationship between bacterial mutagenesls <strong>and</strong> oncogenic potential in higher<br />
organisms . Thus, we have developed a battery of synthetic DNA probes, discriminating single<br />
base-pair mismatches, for use in DNA colony hybrldiatbn to study spontaneous <strong>and</strong> mutageninduced<br />
revertants of S. &phimu.Lrsi strains harboring each of the aforementioned mutations .<br />
Using PCR, we have selectively amplified a single 400 bp fragment encompassing the bLsC3076<br />
locus from total genomic DNA of strain TA2637 . Direct sequencing of this fragment showed the<br />
mutation to be a GG Insertion in a run of four C residues ; DNA sequence near the hisC3076<br />
locus Is S'- ...TTAAAAGTGATCGCCCC=ATCCGCTIT...4'. Southern analysis, using synthetic<br />
probes (18 men) designed to discriminate between rrild-type <strong>and</strong> hisC3076 DNA, confirmed the<br />
sequence tindings . We are currently using PCR <strong>and</strong> direct sequencing to characterize mutant<br />
sequences of hisD3052 <strong>and</strong> hisC3076 revertants unidentitled by the probing analysis . Thus far,<br />
approximately 1000 A1s* revertants have been characterized using these combined methods .<br />
50869 3546<br />
93<br />
94
95 1989 EMS Abstracts<br />
COMPARISON OF AMBIENT AIR MUTACENICITY DETECTED WITH TRADESCANTIA STAMEN Notes<br />
HAIRS AFTER CHERNOBYL ACCIDENT AND ONE YEAR LATER<br />
Antonina Cebulska-Vasilevska Radiobiology Department, Institute of Nuclear<br />
Physics, 152 Radzikovskiego, 31-342 Cracov, POLAND<br />
This paper presents the results of the research on mutagenic effect of ambient<br />
air in the Cracow area . Reported studies were condueted first in May 1986,<br />
after the Chernobyl accident . For comparison, second studies were performed in<br />
various sites within the city in the Spring of 1987 . Counts were made of<br />
stunted hairs <strong>and</strong> pink cells in the stamen hairs of Tradescantia clone 4430 .<br />
Mutation scored since llth day after the beginning of exposure vera used as a<br />
measure of mutation effect caused by the exposure . The mean mutation<br />
frequencies measured for investigated periods were 0 .41 <strong>and</strong> 0 .17 nut/100<br />
hairs respectively . Mutation frequency levels observed after the Chernobyl<br />
accident show a correlation with iodine-131 <strong>and</strong> cesium-137 activity measured<br />
in the air at that time . Although, results obtained in 1987 show on the<br />
average the significant decrease of the ambient air mutagenieity, but the<br />
variation betveen levels of mutations caused by chemical pollution <strong>and</strong><br />
observed in the Spring 1987 in different sites of the Cracov area is rather<br />
high (0 .09 to 0 .31 mut/ 100 hairs) . It also indicates some correlation with<br />
S02 contents in the air . Comparison between biological effects observed in<br />
those two periods demonstrates the importance of chemical pollutants<br />
mutagenicity .<br />
96<br />
INFLUENCE OF SOME ENVIRONMENTAL FACTORS ON DOSE RESPONSE CURVE FOR SOMATIC<br />
MUTATIONS IN TRADESCANTIA .<br />
Antonina Cebulska-Vasilevska+, Robert Rorzeniovski***Radiobiology Department,<br />
Institute of Nuclear Physics,152 Radzikowskiego, 31-342 Cracov,<br />
**Civil Engineering Department, Technical University of Cracow, L-1<br />
24 Varszavska Street, 31-155 Cracov, POLAND<br />
Somatic mutations <strong>and</strong> cell lethality observed in the stamen hairs of the<br />
heterosygous for the flower color clones of Tradescantia have often been used<br />
as a biological plant test system . Due to its high sensitivity to radiation<br />
<strong>and</strong> chemicals the system is particularly suitable for environmental studies .<br />
The radiation dose response curve for the somatie sAtations in Tradescantia<br />
is vall described by linear quadratic model . Presented paper shows hov an<br />
alteration of the biophysical processes caused by some environmental agents<br />
may influence the mutation dose-effect relationship . There are presented<br />
different dose-response curves obtained by modification of the various<br />
parameters describing DNA repair process efficiency . Results obtained both, in<br />
previous studies with sodium fluoride as a possible repair processes<br />
inhibitor, <strong>and</strong> also in recent studies with magnesium contents changed through<br />
the use of dolomite sdded to the soil, are discussed <strong>and</strong> explained by<br />
presented dose response curves analysis .<br />
97<br />
COMPARATIVE STUDIES ON THE MICRONUCLEUS ASSAY IN CYTOKINESIS-BLOCKED BINUCLEATED AND<br />
CONVENTIONAL MONONUCLEATED METHODS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES .<br />
Channarayappa, J . Nath <strong>and</strong> T . Ong, West Virginia University <strong>and</strong> Division of<br />
Respiratory Disease Studies, National Institute for Occupational Safety <strong>and</strong> Health,<br />
Morgantown, WV 26505 .<br />
Bone marrow as well as peripheral blood lymphocytes <strong>and</strong> cultured manmalisn cells<br />
have been used in the micronucleus assay . However, if the measurement of micronuclei<br />
is to be used as a reliable indicator of damage due to chromosomal break or spindle<br />
dysfunction, scoring of micronuclei must be restricted to cells which have undergone<br />
division . Several attempts have been made to discriminate divided from nondivided<br />
cells . Among them, the cytokinesis-blocked binucleated cell (CB) method of Fenech<br />
<strong>and</strong> Morley (Mutation Res . 147 :29, 1985) is being used by several investigators . In<br />
this study, we determined the optimum concentration of cytochalasin B (CYB) for the<br />
induction of a maximum number of binucleated cells in human peripheral blood<br />
lymphocytes (PBLS) <strong>and</strong> compared the efficacy of CD method with the conventional<br />
mononucleus (CM) method for scoring micronuclei after human PBLS were treated with<br />
mitomycin C <strong>and</strong> cyclophosphamide . The results showed that 3 pg CYB/ml was an<br />
optimum concentration for the induction of binucleated cells without any significant<br />
toxic effect <strong>and</strong> that the frequency of micronuclei in the CB method was higher than<br />
that in the CM method for both chemicals . Thus, it appears that scoring of<br />
micronuclei in binucleated cells is a usefyl method for measuring chrosasomal damage<br />
in human PBLS .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
35
36 1989 EMS Abstracts 98<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes MUTANTS DEFECTIVE IN POLY(ADP-RIBOSE) NETABOLISN EXHIBIT DIVERGENT PATTERNS IN<br />
SUSCEPTIBILITY TOWARDS DIFFERENT DNA DAMAGING AGENTS . S . Chatterjee, N .F . Cheng,<br />
S .J . Berger <strong>and</strong> N .A . Berger, Irel<strong>and</strong> Cancer Center, Case Western Reserve University,<br />
Clevel<strong>and</strong>, Ohio 44106 .<br />
We have developed two groups of mutant cell lines from V79 Chinese hamster cells<br />
capable of proliferating with impaired poly(ADP-rtbose) metabolism . One group<br />
consists of ADPRT 54 <strong>and</strong> ADPRT 351 which are defective in poly(ADP-ribose)<br />
polymerase activity while the other consists of N2, N3 <strong>and</strong> N4 which can grow stably<br />
in the absence of free nicotinamide in the mediun with total NAD levels of 1 .5 -<br />
3 .0% of that found in parental V79 cells grown in camplete medium . Thus, in the<br />
latter group of cells, poly(ADP- N bose) synthesis is restricted due to limited<br />
availability of the substrate MAD . Since poly(ADP-ribose) polymerase has been<br />
implicated in DNA repair we examined the susceptibility of these mutants towards<br />
various kinds of DNA damaging agents <strong>and</strong> topoisomerase II inhibitors . Our studies<br />
show that these mutants are resistant to topoisomerase II targeted drugs such as<br />
VP-16 <strong>and</strong> m-ANSA. In contrast, these mutants showed increased sensitivity to NNNG,<br />
bleomycin <strong>and</strong> X-rays . Preliminary studies show that ADPRT 351 cells repair X-ray<br />
induced DNA str<strong>and</strong> breaks more slowly than do V79 cells . This phenomenon could<br />
account for their increased sensitivity to X-rays . A detailed account of the<br />
susceptibility of the mutants <strong>and</strong> Y79 ce11s towards a variety of DNA damaging agents<br />
<strong>and</strong> possible role of poly(ADP-ribose) polymerase in DNA repair will be presented .<br />
MJhDCZ7iAR ANALYSIS OF HPRT MUTATICNS 3N Y79 CRINESE HAMSTFR CEL1S<br />
M. A . Chaudhry <strong>and</strong> Margaret Fbx, Paterson Institute for Cancer Research,<br />
Nanchester, M20 .9BX .<br />
To underst<strong>and</strong> basic mectusnisms of mutation at the HPRT locus in Chinese<br />
hamster cells, spontaneous, Xray, MM14 <strong>and</strong> (F74S) nutants have been independently<br />
isolated . 9 Spontaneous, 36 MMS, 20 Xray, <strong>and</strong> 20 IIdS mutants were analysed by<br />
Southern blotting <strong>and</strong> more limited number Northern analysis . None of the<br />
spontaneous mutants showed changes detectable by Southern analysis . 14/36 M<br />
induced, 9/20 Xray induced, <strong>and</strong> 0/20 FMS induced mutants showed oomplete deletion<br />
of HPRT DNm sequences . The remainder showed no detectable changes . Northern<br />
analysis of the 9 spontaneous mutants indicated 6/9 produced no detectable HPT .T<br />
mRNA,, also in 3/7 MMS induced mutants no mFM was detectable . In a further two MNS<br />
induced mutants the level of niessage was much reduced oaipared with wild-type<br />
levels as indicated by similar levels of APRT message in all cell lines . sJmA frae<br />
further 14 induced mutants was analysed by dot blotting <strong>and</strong> was much reduced in one<br />
Xray <strong>and</strong> absent in one Eti4 induced mutant . HPRT mRNn frae 14 mutants has been<br />
amplified using the polymarase chain reaction . In three mutants in which sTM was<br />
undetectable on Northern analysis low levels were detectable after in vitro<br />
amplification . To date the site of the nutatien has been identified by direct UNA<br />
sequencing in four of these mutants . The sutations ware oonsistent with the<br />
lmowm mechanism of action of the autagens . The high proportion of deletions<br />
produced by Xrays <strong>and</strong> MPiS probably explains their inefficiency as mutagens .<br />
100<br />
LONG-DISTANCE TRANSPORTATION AND RAPID PROPAGATION TECHNIQUE FOR PLANTS<br />
OF TRADESCANTIA PALUDOSA<br />
Kui-2hang Chen <strong>and</strong> Gui-Ying Peng . Guangxi Institute of Botany,<br />
Yanshan, Guilin, Guangxij PEOPLE'S REPUBLIC OF CHINA<br />
Tradescantia Micznnucleus (Trad-DLS1) Assay, established by Dr . Te-Hsiu Ma, is a<br />
well lnown, simple <strong>and</strong> effective technique for monitoring mutagens in the envirocment .<br />
Sinoe it was introduoed into China in 1980, it has been used for in situ monitoring<br />
of many sites . In 1986, it was registered in the regulation of Erniir+amental Matiitoring<br />
Technology by the State Environrental Protection Bureau of China . Because<br />
Tradesoantia clone 03 can be repraiuoed only with vegetative propagation, the longdistance<br />
transportation of plants would be a barrier to increasing the appli,ed range .<br />
To overoome these difficulties <strong>and</strong> keep plants living during lang-distanoe transportation<br />
<strong>and</strong> to obtain a large number of plants with the same original micronucleus<br />
rate in the pollen mother cells in short time assay, a series of experimsnts have<br />
been doee . These show that selecting a plant whose pollen mother cells oontain low<br />
original micronucleus rate <strong>and</strong> adopt3ng rapid propagation methods of the test tube<br />
plants , the white buds are suitable for long-distance transportation<br />
99<br />
101<br />
A PRACTICAL TECHNIQUE FOR SHIPPING AND RAPID PROPAGATION OF TRADESCANTIA PLANTS UNDER<br />
POLLUTION-FREE CONDITION, Rulshaaa C_I1gG* <strong>and</strong> Guiying Peng*, Guangxi Institute of<br />
botany, Yanshan, Guilin, PRO (Introd . by T . H . Na) Departsent of Biological Sciences,<br />
Western Illinois University, Haoosb, IL (USA)<br />
50869 3548
Tradescantia-Micronucleus (Trad-MCN) bioassay is an efficient test for genotoxioity<br />
of environmental pollutants . It is currently under the validation prooess for the<br />
International Program on Chemical Safety, WHO, UN for possible world-wide adoption for<br />
chemical testing . Large population of Tradescantia clone #4430 has been vegetatively<br />
propagated in the field <strong>and</strong> greenhouse in Guangxi Institute of Botany where the<br />
climate <strong>and</strong> day length are suitable for the natural growth of this plant all year<br />
round . Our Institute could serve as the supply house of this plant material for all<br />
corners of the world . In order to maintain the genetic purity of this plant during<br />
long distance shipping <strong>and</strong> rapid propagation of a large quantity, we developed a<br />
practical technique to meet thie dem<strong>and</strong> . First, young plant buds (usually whitish in<br />
color <strong>and</strong> about 0 .5 cm long) which are beneath the soil surface are selected from the<br />
low background stocks . The selected buds are then transplanted in a sterile<br />
pollutant-free medium in a container <strong>and</strong> kept in the dark for shipping . These plant<br />
buds can be kept alive in the shipping package for as long as a month duration . The<br />
plant buds will turn into normal green plantlets after exposure to the light <strong>and</strong> ready<br />
to be transplanted into field or greenhouse . The new plants will reach maturity Sn<br />
about a month . The plants shipped in this fashion could minimize the possible genetic<br />
change during shipping <strong>and</strong> gain easy passage of quarantine stations at the<br />
international boundaries .<br />
102<br />
A NEW TW0-DINENTIONAL ANALYSIS OF DNA FRAGMENTS : TEMPERATURE GRADIENT GEL<br />
ELECTROPHORESIS . Y .Chen, J .Fu, G .FAN, <strong>and</strong> B .Liu . Vest China University of<br />
Medical Sciences . Chengdu, Sichuan (China)<br />
A new technique, nased teeperature gradient gel electrophoresis (TGGE) .<br />
used for sequence dependent separation of DNA fragments has been developed .<br />
If double helical DNA is partially ∎elting in polyacrylamide gels, its<br />
electrophoretic ∎obility undergoes a sharp transition, resulting in a<br />
large reduction in ∎obility . In the present experiment, the transition that<br />
was effected at a uniform concentration of denaturant, formamide . in a<br />
temperature gradient formed during elect.rophoresis . Each restrictive<br />
fragment of pBR322-DNA <strong>and</strong> bacteriaphage lambda-DNA exhibited the ∎obility<br />
transition at a particular temperature . The sudden retardation of fragments<br />
moving forward in the gel depended upon nucteotide composition <strong>and</strong> sequence .<br />
rather than the length . When combined with length dependent electrophoresis<br />
in the perpendicular direction . TGGE provided a two dlsentional separation<br />
of DNA restrictive fragments . The resolving power of the new system was<br />
demonstrated by the clear resolution of over 80 fragments of the restrictive<br />
entyme Hinfl digest of lambda-DNA . This technique would find its appiic:ation<br />
in ∎any fields .<br />
103<br />
RADICAL-MEDIATED SITE-SPECIFIC CROSSLINKING OF NUCLEAR MATRIX PROTEIN AND DNA . S .M .<br />
Chiu, L .Y . Xue, L .R. Friedman, <strong>and</strong> N .L . ole nick . Departments of Radiology <strong>and</strong><br />
<strong>Environmental</strong> Health Sciences, Case Western Reserve University, Clevel<strong>and</strong>, ON (USA) .<br />
Ionizing radiation-induced DNA-protein crosslinks (DPC), as assayed by<br />
nitrocellulose filter-binding, are formed preferentially between domains of mammalian<br />
chromosomal DNA enriched in active sequences <strong>and</strong> normal DNA-binding proteins of the<br />
nuclear matrix (Chiu et al ., Radiat . Res . 107, 24 . 1986). In an effort to explore the<br />
mechanism for the microheterogeneity, we have fractionated V79 cells <strong>and</strong> nuclei both<br />
before <strong>and</strong> after irradiation. When intact cells were irradiated, the same radiation<br />
dose response was obtained if the assay of DPC was subsequently conducted on cells,<br />
nuclei, or nuclear matrix, suggesting that all of the components of DPC are retained<br />
in the nuclear matrix. The same result was obtained for irradiation of nuclei <strong>and</strong><br />
assay of nuclei or nuclear matrix . However, the formation of DPC reached an early<br />
maximum when nuclear matrix wae irradiated . Nuclear matrix was isolated by extraction<br />
with lithium diiodosalicylate (LIS) . A. analyzed by SDS-PAGE, a similar set of<br />
proteins was found in matrices prepared by LIS or by extraction in 2 N NaCl. In the<br />
case of LIS extraction, nuclei were first stabilised by incubation in 0 .5 ∎M Cu804 .<br />
The radiation dose-response for formation of DPC in these nuclei was 4-6 timea greater<br />
than in untreated nuclei . Tests for the presence of Cu++ or other trace metals <strong>and</strong><br />
for the participation of Fenton-type teactions suggest a poseible explanation for the<br />
selective formation of DPC at nuclear matrix sites, i .e ., the generation of hydroxyl<br />
radicals or other oxidative radicals in the vicinity of preferential sites of binding<br />
of metal ions . (Supported by NIH Grants RO1CA15378 <strong>and</strong> P30CA43703 .)<br />
104<br />
ISOLATION AND PARTIAL CHARACTERIZATION OF RAD4 GENE OF Saccharomyces cerevlsiae THAT<br />
CAN BE PROPAGATED IN S .coli WITHOUT INACTIVATION . I .S .Choi, J .B .Kim, <strong>and</strong> S .D .Park,<br />
Department of Zoology, College of Natural Sciences . Seoul National University, Seoul<br />
151-742, South Korea<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts<br />
Notes<br />
37
38 1989 EMS Abstracts<br />
Notes The RAD4 gene requiied for incision of UV-induced excision repair in Saccharomyces<br />
cerevtsiae has been isolated by phenotypic complementation of a UV-sensitive rad4-4<br />
mutant with yeast genomic library amplified In E .coli (Yoon et al .,1985) . The yeast<br />
insert in recombinant plasmid designated as pPCl was isolated in the 2 .6Kb BglII-gamHI<br />
fragment in the aubcloned pPC100 <strong>and</strong> contained a functional RAD4, but not a suppressor<br />
tRNA gene as determined by restriction mapping <strong>and</strong> DNA-tRNA hybridization . In addition,<br />
the observation that all different plasmids harboring yeast insert conferred UV<br />
resistance to rad4-4 cells to a similar level regardless of their copy number, strongly<br />
suggests that our cloned gene does not contain a suppressor . Northern blot analysis<br />
with pPC100 as the probe indicates that the insert DNA containing the entire RAD4 gene<br />
can hybridize with transcripts of 1 .2Kb <strong>and</strong> 2 .3Kb in length . Stationary-phase cultures<br />
of E .co1i BL21(DE3) strain transformed with plasmids harboring the cloned RAD4 gene<br />
showed a considerable delay in the resumption of exponential growth <strong>and</strong> a dramatic<br />
reduction in the production of host proteins . Moreover, the overexpressed Rad4 protein<br />
estimated as 89Kd by 2-D gel analysis revealed a toxic effect In Z .coli, suggesting<br />
that the Rad4 protein interferes with normal growth control in 6 .co1t cells .<br />
105<br />
USE OF THE BALB/c-3T3 ACAR SUSPENSION ASSAY TO DETECT CHEMICAL-INDUCED TRANSFORMATION .<br />
M . A . Cifone, L . Custer <strong>and</strong> E .J . Matthews, Hazleton Laboratories America, Inc .,<br />
Kensington, Maryl<strong>and</strong> .<br />
An alternate endpoint to morphological transformation In the BALB/c-3T3 transformation<br />
assay has been described . In this assay, the formation of colonies in agar was<br />
measured after chemical treatment . Approximately 50,000 cells were seeded on day 0 <strong>and</strong><br />
treated on day 1 with a chemical for 24 hours . The cells were then kept in logarithmic<br />
phase for 11 to 13 days <strong>and</strong> plated in 0 .4% Noble agar . Four weeks later the colonies<br />
were fixed with 2 .5% glutaraldehyde <strong>and</strong> colonies with diameters greater than 0 .1 mm were<br />
automatically counted using an Olympus Q2 1mage analyzer . Fifteen chemicals were<br />
investigated In this system <strong>and</strong> activities were compared to the presence or absence of<br />
structural alerts, <strong>and</strong> published results obtained in the st<strong>and</strong>ard BALB/c-3T3<br />
transformation assay, several in vitro genotoxicity assays, <strong>and</strong> rodent bioassay . The<br />
chemicals studied included four genotoxic carcinogens, six genotoxic nonearcinogens <strong>and</strong><br />
five nongenotoxic carcinogens . The results with the agar suspension assay closely<br />
correlated with the results obtained with the st<strong>and</strong>ard BALBe/-3T3 transformation assay .<br />
However, in some cases the agar suspension assay appeared to be les∎ sensitive . Some<br />
chemicals gave an equivocal or negative response In the agar suspension assay <strong>and</strong> ware<br />
weakly active in the st<strong>and</strong>ard transformation assay . While the BALB/c-3T3 agar<br />
suspension assay is labor intensive <strong>and</strong> not appropriate for routine screening, it<br />
measures an endpoint that is crucial to the transformation process <strong>and</strong> can be used to<br />
answer mechanistic questions .<br />
(supported by NIEHS Contract No . N01-ES-65150) .<br />
PARALLEL DETECTION OF SCE, DNA ADDUCTS AND PROTEIN ADDUCTS IN MICE DOSED WITH THE<br />
CARCINOGEN 2-ACETYLAMINOFLUORENE .<br />
106<br />
G .Citro, R .Zito, A .Verdina, R .Galati, R .Benigni, P .Leopardi, A .Zijno, <strong>and</strong> R .Crebelli,<br />
Istituto Superiore di SanitB <strong>and</strong> Istituto Regina Elena, Rome (Italy) .<br />
In order to explore the interrelationships among DNA adducts, protein adducts <strong>and</strong><br />
SCE simultaneously induced in the complexity of in vivo situation, an investigation<br />
was carried out in Swiss mice treated with the carcinogen 2-acetylaminofluorene (2AAF) .<br />
Groups of 4 male mice were treated either by gastric intubation (at 25,50 <strong>and</strong> 100 mg/kg<br />
on 4 consecutive days) or by chronic dietary exposure (2 months feeding with a satura-<br />
ted 2AAF water solution) . For each animal the frequency of SCE in bone marrow cells,<br />
as well as the levels of binding of 2AAF to liver <strong>and</strong> spleen DNA <strong>and</strong> blood proteins we-<br />
re determined by means of cytogenetic techniques <strong>and</strong> competitive immunoassays, respect .<br />
The results obtained highlight a dose-related trend in SCE rates after acute exposure,<br />
with wide interindividual variance wich was significantly related to individual levels<br />
of liver DNA adducta . Conversely, the amount of covalent binding to spleen DNA proved<br />
to be related only to individual levels of binding to blood proteins . Such picture was<br />
modulated by the schedule of treatment : chronic 2AAF exposure in fact produced relati-<br />
vely higher levels of binding to spleen than to liver DNA (about 1,000 <strong>and</strong> 500 adducts/<br />
million nucleotides, respect .) <strong>and</strong> did not increase significantly SCE rates in bone<br />
marrow .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
50869 3550
STRUCTURE ACTIVITY ANALYSIS OF AZO DYES AND RELATED CONPOUNDS . L .D . Claxtonl, D .B .<br />
Walsh2, J . Esancy3, <strong>and</strong> H . Freeman3, l<strong>Environmental</strong> Protection Agency, Research<br />
Triangle Park, NC (USA), 2<strong>Environmental</strong> Health Research <strong>and</strong> Testing, Inc ., Research<br />
Triangle Park, NC (USA), <strong>and</strong> 3North Carolina State University, Raleigh, NC (USA) .<br />
Azo dyes represent a significant group of industrial dyes . The carcinogenicity of<br />
some azo dyes have been known for decades . However, debates persist as to the role of<br />
metabolism <strong>and</strong> various substructural units in the genotoxicity of these azo dyes . For<br />
example, although one of the most prevalent metabolic pathways implicated in the<br />
activity of azo dyes is azo reduction, not all reductive cleavage products are<br />
mutagenic . A computerized SAR system, ADAPT, was used to analyze two separate data<br />
bases : a group of azo dyes <strong>and</strong> a group of reductive cleavage products . The data used<br />
for both data bases was the response when tested in the Salmonella reversion bioassay .<br />
A compound was considered mutagenic if there was a positive response in any of the<br />
st<strong>and</strong>ard five tester strains either with or without metabolic activation . However, a<br />
compound was considered nonmutagenic only if there was no response in any of the<br />
strains with or without metabolic activation . The azo dye data base consists of 25<br />
mutagens <strong>and</strong> 13 nonmutagens . A set of molecular descriptors, including substructural<br />
descriptors, was selected that successfully classified the mutagens from the<br />
nonmutagens . These molecular descriptors were used to predict the mutagenicity of a<br />
group of azo dyes not included in the data base . The reductive cleavage data base<br />
consists of 48 mutagens <strong>and</strong> 13 nonmutagens . Structure activity relationships of both<br />
data bases, as well as their predictive capability, will be discussed . This is an<br />
abstract of a proposed presentation <strong>and</strong> does not necessarily reflect EPA policy .<br />
108<br />
IN VIVO MICRONUCLEUS TESTS IN MOUSE . COPPARISON OF TFE SENSITIVITY OF THREE TARGET ORGANS<br />
(BONE MARROW, LIVER AND TESTIS) TO SIX CARCINOGENS .<br />
I .Cliet, E .Fournier, C .Melcion <strong>and</strong> A .Cordler . Dbpartement de Toxicologie, Centre de<br />
Recherches de Vitry, RhBne-Poulenc SantB, 13 Qual Jules Guesde, 94400 Vitry sur Seine,<br />
France .<br />
In vivo somatic chromosome mutation is usually carried out using the bone marrow<br />
micronucleus (BMM) test In mouse, which is considered of predictive value In the study of<br />
clastogenicity In the germ cell line . However, it has been reported that the sensitivity<br />
of the BMM test !s insufficient to detect unstable compounds or short-lived metabolites<br />
<strong>and</strong> the use of target cells with metabolic activity (hepatocytes <strong>and</strong> spermatids) has been<br />
questioned . In order to analyze In vivo micronucleus lnduction, particularly In celis with<br />
metabolic enzyme activity, we compared the sensitivity of somatic <strong>and</strong> germ cell lines<br />
towards six carcinogens In the bone marrow, liver <strong>and</strong> .spermatld micronucleus test In<br />
mouse . Five procarcinogens, with a complex metabolic pattern : dlmethylnitrosamine (DMN),<br />
diethyinitrosamine (DEN), 1-1-dimethyihydrazine (1-1-DMW), 4-aminophenol (4-APOL) <strong>and</strong><br />
4-aminobiphenyl (4-ABPYL) <strong>and</strong> one direct unstable mutagen, a-proplolactone (BPL) were<br />
tested . DMN, DEN, 1-1DMH, BPL were not detected by the BMM test due to their lnstability,<br />
whereas 4-APOL <strong>and</strong> 4-ABPYL were clearly positive . All six carcinogens were detected In the<br />
mouse liver <strong>and</strong> spermatid micronucleus test <strong>and</strong> induced clear clastogenic effects . In<br />
conclusion this study demonstrates the importance of organ-specificity studies . Moreover,<br />
the results of the liver <strong>and</strong> spermatid micronucleus tests show that the BMM test cannot<br />
replace clastogenicity evaluation in the germ cell line .<br />
109<br />
MAMMALIAN CELL MUTAGENESIS: A MAJOR ROLE FOR NON-DNA TARGETS . D . Clive,<br />
Wellcome Research Laboratories, Research Triangle Park, NC 27709 USA<br />
Evidence is accumulating for major non-DNA targets for specific locus mutageniclty In mammalian<br />
cells involving heritable DNA alterations . Such a possibility (a) Is consistent with radial-loop models<br />
of chromosome structure In mammalian cells (Marsden <strong>and</strong> Laemmll, Cell17 : 849, 1979) ; (b) is<br />
based upon non-DNA structural components of the eukaryotic chromosome (e .g., topolsomerase is ; TP)<br />
(Gaulden, <strong>Mutagenesis</strong> 2(1987) 357 ; Evans et al., Mutation Res. 217 : 53, 1989) ; (c) invokes<br />
genetic alterations quantitatively larger than, <strong>and</strong> mechanistically different from, classical point<br />
mutations as a major class of gene mutations (Y<strong>and</strong>ell et al ., Som. Cell Mol. Gen . 12 : 255, 1987;<br />
Applegate <strong>and</strong> Hozier, Banbury Report #28 : 213, 1987) which (d) are of the types seen In rodent <strong>and</strong><br />
human tumors (All et al ., Science 236 : 933, 1987) ; (e) attributes the relative Insensitivity of some<br />
genotoxicity tests to the wrong chromosomal architecture (prokaryotes, including the Ames assay), the<br />
wrong chemistry (DNA repair synthesis incapable of detecting TP redimerization) or the wrong<br />
endpoint (dominant or X-linked loci fail to detect large scale recombination events) ; (f) explains the<br />
higher sensitivities of other assays (SCE assays, chromosome aberration tests, L517gY/fk +/- mouse<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
40 1989 EMS Abstracts<br />
Notes lymphoma assay [MtA)) ; <strong>and</strong> (g) Implies the existence of penotoxicJcarcinopenic chemical structures<br />
different from those Implicated in DNA reactivity . The cytopenet)c <strong>and</strong> molecular mutational spectra of a<br />
number of chemically diverse mutagens In the MLA Indicates that only a few mutagens (e .y., EMS,<br />
2-amino-6N-hyd(oxyadenine) Induce a significant proportion of subgenic DNA alterations at the<br />
heterozypous tk locus, whereas most Induce predominantly DNA alterations which are at least many<br />
kilobases (<strong>and</strong> probably multigenic) in extent (Glover et al ., these abstracts) . These results will be<br />
discussed In terms of penotoxicity models <strong>and</strong> mechanisms which Indude non-DNA targets .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
110<br />
MUTAGENIC ACTIVITY OF EXTRACTS FROM ROLLING MINERAL OILS AND LEATHER SAMPLES .<br />
E . Cionferot, P . Venier=, M . iordanz <strong>and</strong> A .G . Levis= . t Inst . Occup . Health<br />
University of Padova (ITALY) 2 Dept. of Biology, University of Padova (ITALY) .<br />
Unused crank case oils are norsally negative in the Salmonella/sicrosose assay<br />
but become strongly sutagenic after use due to the enrichment of their PAH content<br />
resulting from engine cosbustion processes . On the other h<strong>and</strong> refined rolling oils<br />
lack sutagenic activity even after prolonged use because of the low temperature<br />
working conditions . Finished leather probably contains naturally occurring tannins,<br />
but other chemical compounds used in the tanning <strong>and</strong> refining processes may also<br />
play a part in determining the carcinogenicity of soae leather dusts . We evaluated<br />
the sutagenicity (in the Saleonella/ .icroso.e assay) of extracts fro . a aineral oil<br />
esulsion used in a rolling ∎ill <strong>and</strong> from finished leather . ' The oil emulsion was<br />
suspended in DMSO <strong>and</strong> extracted with CHzCl= . The extracts fros the unused oil were<br />
negative, while after prolonged use sutagens directly active on Salmonella strains<br />
TA98 <strong>and</strong> TAIOO were found (8-10 ind . rev ./ .g eq . of extract) . Relatively low<br />
concentrations of oil (10 ∎g/plate) were found to inhibit totally the .utagenicity<br />
of 5 pg of benzo(a)pyrene assayed in the presence of S9 . " After treatment in a<br />
Soxhiet apparatus with toluene or ethanol the extracts from a leather widely used in<br />
the furniture <strong>and</strong> dresssaking Industries were mutagenic on strain TA98 of 3,<br />
j,yphisurius in the absence of S9 ∎ix . The analysis of the extracts from leather at<br />
various intersediate stages of processing showed that the mutagenic activity<br />
appeared after the coloration process . The responsible compound was identified to be<br />
an azodye (Color Index : Acid Brown 83, .utagenic potency about 4<br />
revertants/sicrogras) . SUPPORTED BY C .N .R . p .f . "ONCOLOGIA" .<br />
111<br />
THE MUTAGENICITY OF 2-AMINO-N6-HYDROxYADENINE (ASA) TO L517BY TK+/- CELLS MEASURED<br />
BY THE INDUCTION OF TRIFLUOROTHYMIDINE (TPT), 6-TBIOGUANINE (6TG) AND OUABAIN (OUA)<br />
RESISTANCE AND THE INDUCTION OF MICRONUCLEI .<br />
Jane Cole, Frances Richmond <strong>and</strong> Bryn Bridaes, MRC Cell Mutation Unit, University of<br />
Sussex, Brighton BN1 9RR, Gt . Britain .<br />
L5178Y TK+/- (3 .7 .2c) cells were treated with AHA at concentrations ranging from<br />
0 .005 to 2 .Oµq ml-1 (100 - 15% survival) . Samples were plated on day 0 after treatment<br />
to determine the toxic effect, on days 2 <strong>and</strong> 3 to determine TFT <strong>and</strong> OUA mutant<br />
frequency <strong>and</strong> on day 7 to determine 6TG mutant frequency . All platinqs were done by<br />
the microtiter method of Cole at al . (1986, Mutaqenesis 1, 157-167) . Slides were<br />
prepared 24h <strong>and</strong> 48h after treatment to determine the induction of micronuclai (MN) .<br />
Positive controls (ethyl or methylmethanesulphonate) were included in every experiment<br />
. Over the non-toxic range (0 .01 - 0 .04µq/ml, 80 - 100% survival) there was<br />
approximately linear induction of TFT, OUA <strong>and</strong> 6TG resistance . At toxic concentrations,<br />
TFTR continued to increase with dose, 6TG resistance plateaued at around 0 .5µq<br />
/ml, <strong>and</strong> a peak of OUAR mutants was observed at 0 .25µq/ml, at higher doses there was<br />
a marked decrease in OUA resistance . Only small increases in the percentage of MN<br />
were observed .<br />
112<br />
A NEW METHOD FOR PREPARATION OF METAPHASES FROM THE G .I . TRACT OF RODENTS . Barbara<br />
Coles, Leslie McSparrin, Judith Horvath, <strong>and</strong> William S . Barnes, Department of Biology,<br />
Clarion University of PA, Clarion, PA .<br />
Research has frequently been h<strong>and</strong>icapped by the lack of a good experimental<br />
system to study the events of colon carcinogenesis . Carcinogenic activity can<br />
be identified by induction of SCE, but it remains difficult to obtain good yields<br />
of mitotic cells from the G .I . tract . In our protocol, 35 g . male C57BL/6 mice<br />
were implanted with a 25 mg agar-coated BrdUrd tablet . Twenty-three hours later,<br />
they were injected i .p . with 10 mg/kg coichicine . Twenty-five hours after BrdUrd<br />
adminsitration, animals were anesthetized <strong>and</strong> a loop of small intestine was exposed<br />
from the body cavity . The loop was cut from both ends, being careful not to cut<br />
any blood vessels, <strong>and</strong> fecal material was flushed out . The segment was then treated<br />
sequentially with two incubations of 0 .5mM EDTA, followed by two incubations of<br />
collagenase in 10mM HEPES (750 units/ml .) . The segment was removed from the animal .<br />
50869 3552<br />
,
everted, <strong>and</strong> vortexed vigorously in 3% glacial acetic acid to remove mucosal cells .<br />
Preparation of metaphase spreads <strong>and</strong> sister chromatid differentiation were done<br />
according to st<strong>and</strong>ard techniques . This technique yields many M~ metaphases with a median<br />
of approximately 30 chromosomes/metaphase . Various refine6Tenta are being investigated<br />
. We are also adapting this method to the large bowel of P344 rats . Supported<br />
by the Clarion University Foundation <strong>and</strong> a SSHE Professional Development Award .<br />
113<br />
EVALUATION OF THE GENOTOXICITY OF CARBOFURAN IN HUMAN PERIPHERAL BLOOD<br />
LYMPHOCYTES . Ilce Mara de S . Colus, Catarina S . Takahashi <strong>and</strong> Elza T . Sa<br />
kamoto-Hojo (FUEL-PR, FFCLRP-USP, IBILCE-SJRP-UNESP) . Brasil<br />
The pesticide Carbofuran is a carbamate specifically used for the<br />
treatment of seeds from grains such as wheat, corn <strong>and</strong> rice, among others .<br />
The genotoxicity of the compound was tested in vitro in terms of induction<br />
of chromosome aberrations <strong>and</strong> SCE in hutfan petip :eia~ blood lymphocytes<br />
submitted to concentrations of 4 .13 x 10 <strong>and</strong> 4 .13 x 10 jug/ml . The drug was<br />
added when the culture was initiated <strong>and</strong> allowed to act until fixation<br />
time, which was 48 hours later for chromosome aberration test <strong>and</strong> 72 hours<br />
for SCE test . The percentage of cells with chromosome aberrttions did not<br />
differ among groups : 3,62 4 or the control <strong>and</strong> the 4 .13 x 10 concentration<br />
<strong>and</strong> 5 .8% for the 4 .13 x 10 concentration of the product . Similarly, the<br />
mean number of SCE/cell did not diffber among the three groups : 10,65 for<br />
the control,-211 .20 for the 4 .13 x 10 pg/ml eoncentration <strong>and</strong> 11 .07 for<br />
the 4 .13 x 10 pg/ml concentration . The results agree with those reported<br />
in the literature, which indicate that Carbofuran is not mutagenic in microorganisms,<br />
in the SLRL test using Drosophila, or in human fibroblasts . Turba<br />
genic effects of Carbofuran have been reported in Ilordeum vuZgare <strong>and</strong> in<br />
AZZium cepa . Since the insecticide Carbofuran proved to be ineffective in inducing<br />
a genotoxic effect on human lymphocytes, <strong>and</strong> considering that the cultures<br />
usually exhibit a low metabolizing ability, we may conclude that the pa=<br />
rental compound did not show clastogenic activity on the test system used .<br />
114<br />
MOLtCOLAR MLCalUlIBM OF OZNLTIC NLCON7INatION : .<br />
rORMATION aND RISOLOTION Or IOLLIDIIY JOMCTIONS IN VISRO .<br />
Edward C . Conley, Berndt Milller <strong>and</strong> Stephen C . West<br />
Imperial Cancer Research flund, Clare Hall Laboratories, South Misms, Retts EN6 IID, D .R .<br />
A central internediate in the process of qenetic reconbination is a Holliday junction in<br />
which two DNA helices are int.rlinked by a reciproul str<strong>and</strong> crossover . Holliday junctions ray<br />
be formed in vitro by the enzymatic action of the F. coli RecA protein . Rach gains access to<br />
duplex DNA by nucleation from a short sinql.-strended gap, to form a spiral nucleoprotein<br />
fil>ment which is capable of interaction with homoloqous duplex DNII. Any part of the filamant,<br />
whether it contains single- or double-str<strong>and</strong>ad DNA, is capable of pairing with the homoloyous<br />
duplex . Homaloqous contacts between reqions of duplex DNA lead to an increase in the initial<br />
rate <strong>and</strong> final extent of joint molecule formation . !]tparimants indicate that pairing is<br />
facilitated by the formation of nascent synaptic internrdiates between duplex DtA seQuences .<br />
Duplex-duplex pairing reactions involve underwindinq of the double helix relative to normal B-<br />
form DNA .In an ATP-dependsnt reaction, RecA protein drives the reciprocal exchanqe of D1A str<strong>and</strong>s<br />
:n polar fashion from a gapped region into <strong>and</strong> along four str<strong>and</strong>s of DNA,so forming a Holliday<br />
;unction . When T4 endonuclease VII i. added to the reaction mixture, the Holliday junctions<br />
formsd by RecA protein are resolved to form heteroduplex DNh . Cleavage occurred by cutting in<br />
either of the two possible orientations . Thus, recombinant DNA mol .cules may be formed in an in<br />
vitro system by the combined action of a recombinase <strong>and</strong> a r .solvase protein .<br />
115<br />
THE E .E .C . GENOMIC MUTATION PROGRAM : RESULTS WITH ASPERGILLUS NIDULANS<br />
R .Crebelli, G .Conti, L .Conti, <strong>and</strong> A .Carere, Istituto Superiore di Sanitli,Rome(Italy)<br />
In the framework of the coordinated E .E .C . Genomic Mutation Program for the evalua-<br />
tion of current methodologies for the detection of aneuploidy, a set of nine reference<br />
compounds (colchicine, econazole, chloral hydrate, hydroquinone, diazepam, thiabenda-<br />
zole, cadmium chloride, thimerosal <strong>and</strong> pyrimetamine) were assayed in Aspergillus nidulans<br />
diploid strain P1 . The following chemicals proved to increase significantly the<br />
frequency o,f:~hole chromosome segregants : econazole (active dose range : 0 .1-0 .2 /lg/ml),<br />
chloral hydrate (6-10 mM), hydroquinone (1-3 mM), thiabendazole (12-40 mM), thimerosal<br />
(0 .035-0 .40 Ng/ml) . In the case of chloral hydrate, thiabendazole end thimerosal no<br />
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4<br />
1989 EMS Abstracts 41<br />
Notes
42 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes concurrent increase in mitotic cross-overs was observed, suggesting the disturbance<br />
of chromosome segregation as primary genetic event . Conversely, in the case of hydroquinone<br />
the secondary origin of chromosome malsegregants was demonstrated, whereas<br />
additional assays are required to disclose the nature of mitotic segregants produced<br />
by econazole treatments . Finally, colchicine, diazepam, cadmium chloride <strong>and</strong> pyrimeta-<br />
mine were assayed in a wide range of doses with completely negative results . (Work<br />
partially granted by the E .E .C . contract n . EV4C-0044-I(a) ) .<br />
116<br />
THE DEVELOPMENT OF A PANEL OF HUMAN CELL LINES EXPRESSING SPECIFIC HUMAN<br />
CYTOCHROME P450 cDNAS<br />
C . L . Crespi <strong>and</strong> B . W . Penman, Gentest Corporation, Woburn, MA (USA)<br />
We have previously reported a development of human lymphoblastold cell lines which express<br />
tranfected human cytochrome P450IIA3 <strong>and</strong> microsomal epoxide hydrolase cDNA's . We have<br />
now isolated additional cDNA's encoding human cytochrome P4501IE1, P450IIC9 <strong>and</strong> P450IIIA4<br />
using oligonucleotide probes . The cDNA's were sized <strong>and</strong> the DNA sequence of the 5' end of<br />
the isolate was determined. The cDNA's had the following properties :<br />
P450 Form cDNA Size DNA Sequence HomologV Coding Sequence<br />
fIE1 1 .7 kb 97% 11 bp short<br />
11C9 1 .9 kb 100% 32 bp short<br />
IIIA4 2 .1 kb 99% Complete<br />
The IIE1 <strong>and</strong> IIC9 Isolates which did not contain complete coding sequences were completed<br />
by the addition of synthetic oligonucleotides. The complete cDNA's are being introduced into<br />
a pHEBo based expression vector system under the control of the herpes simplex virus thymidine<br />
kinase gene promoter <strong>and</strong> polyadenylation signal . Recombinant cell lines expressing these<br />
cytochrome P450 cDNA's are being constructed . These cell lines, in addition to cell lines<br />
expressing human cytochrome P450IIA3 <strong>and</strong> P4S01A1, should provide a useful panel of cell lines<br />
evaluating the ability of specific human cytochrome P450 forms to activate chemical mutagens .<br />
117<br />
ASSESSMENT OF THE MUTAGENIC EFFECT OF MATERNAL FACTORS ON HUMAN CHORICNIC VILLI BY<br />
MICRONUCLEUS TEST<br />
Y .Q . Cui, Z .W . Dong, S .B . Liu, S .C . Chang, Y . Wang, X .Y . Ji, National Research<br />
Institute for Family Planning, Beijing, People's Republic of China .<br />
Rapid determination of DNA damage by micronucleus test is well accepted . Animal bone<br />
marrow cells or human peripheral lymphocytes used in most studies could not directly<br />
reflect the influence of the mutagenic effect on the offsprings by environmental<br />
factors . Although the cytogenetic effect of human chorionic villi chromaomes are used<br />
in prenatal diagnosis, human chorionic villi sdcronucleus test to detect directly the<br />
mutagenic effect of environmental factors has not been reported in the literature .<br />
Direct determination of human chorionic villi sdcronuclei (CVlIIi) was established by<br />
us, to study the mutagenic effect of mother's age, gravidity, abortion history, contraception,<br />
smoking <strong>and</strong> drinking on the offsprings . Cross investigation <strong>and</strong> micronucleus<br />
test were used in 507 couples undergoing artificial abortion . Micronuclei were scored<br />
according to Countryman's st<strong>and</strong>ard . 2, 000 interphases were observed in each subject<br />
for CVMN frequency . Aresine transformation {aresine (Sqr(P)1) was used in transforming<br />
CVMN frequency :<strong>and</strong> the analysis of variance were used for statistics . No correlation<br />
between CVM4 frequency <strong>and</strong> mother's age, gravidity, gestation age, abortion history,<br />
<strong>and</strong> contraception was found . Neither smoking nor drinking habit was found among the<br />
women of this study . The CVlA1 frequency of husb<strong>and</strong> smoking was 0 .76+0 .06%., of husb<strong>and</strong><br />
non-smoking-drinking was 0 .55t0 .06X., of husb<strong>and</strong> drinking was 0 .57t0 .20X ., of husb<strong>and</strong><br />
smoking <strong>and</strong> drinking was 0 .79* 0 .08X . . There was a statistical difference in CVMN<br />
frequency between husb<strong>and</strong> smoking <strong>and</strong> non-saaking . No significant difference was<br />
found between husb<strong>and</strong> drinking <strong>and</strong> non-drinking .<br />
118<br />
METHYLAZOMETHANOL ACETATE (MAMoAC), A STABLE FORH OF THE ULTIMATE MUTAGEN FROM<br />
CYCASIN IS ACTIVATED BY PORCINE LIVER ESTERASE . Michael L . Cunningham <strong>and</strong> Timothy F .<br />
McMahon, Systemic Toxicology Branch, National Institutes of Health, NIEHS,<br />
Research Triangle Park, North Carolina, 27709<br />
Nethylazomethanol (HAH) is the short-lived toxic <strong>and</strong> carcinogenic aglycone of cycasin,<br />
a natural component of the cycad plant . These plants may be consumed by people in the<br />
South Pacific isl<strong>and</strong>s either as foodstuff or in medicinal preparations . Thg<br />
spontaneous decomposition of MAN to reactive methyl carbonium ions above 0 C makes<br />
the study of its mutagenic effects difficult . Hovever, we have used the stable ethyl<br />
50869 3554
1989 EMS Abstracts<br />
ester of I41M, 1L1M acetate (MAMoAC) in combination with porcine liver esteraee Notes<br />
(Carboxylic-ester hydrolase ; EC 3 .1 .1 .1) to study the mutagenic activity of NAM .<br />
MAMoAC produces a dose-dependant increase in the mutation rate in Salmonella<br />
typhimurium His G46 at concentrations of 2 .5-30 micromoles/plate following<br />
preincubation at 30oC for 90 minutes . Addition of porcine liver esterase increased<br />
the mutation rate from 14 to 2200 <strong>and</strong> from 375 to 5800 revertants per plate at 2 .5<br />
<strong>and</strong> 5 .0 micromoles/plate, respectively . This increased mutagenicity was dependant on<br />
esterase concentration (optimum 63 micrograms protein/ml) . The preincubation time<br />
was also critical for expression of the mutagenicity of the MAMoAC/esterasa reaction .<br />
The dose-response curve was shifted to steeper slopes by increasing the preincubation<br />
time up to 90 minutes . Our results indicate that the MAMoAC/esterase system is a<br />
convenient model with which to study the mutagenicity of 14AM .<br />
119<br />
ANALYSIS OF MULTIPLE CONGENITAL ANOMALIES /ICPEMC/<br />
Andrew Czeizel<br />
par men o uman Genetics <strong>and</strong> Teratology, National Institute of<br />
Hygiene, Budapest, Hungary<br />
Three indicator conditions of offspring : sentinel anomalies, Down<br />
syndrome <strong>and</strong> unidentified multiple oongenital anomalies /UIICAs/ are<br />
being evaluated within the Hungarian population-based surveillance<br />
of germinal mutations . Ul[CAs are possible indioators of germinal<br />
dominant gene <strong>and</strong> ohromosomal mutations . The component oongenital<br />
abnormalities of UMCAs were classified into 45 groups . These<br />
component congenital abnormalities were reduced to pairs A pair is<br />
a set of two independent component congenital abnormalities in index<br />
patients with two or more congenital abnormalities . Baseline figures<br />
of all component congenital abnormality pairs in 3,722 UMCAs were<br />
determined in the study period 1973-1982 . The observed annual data<br />
after 1982 were compared with expected occurrences based on baseline<br />
figures . This pair-wise evaluation of component congenital<br />
abnormalities within U11[CAs seems to be an adequate surveillance<br />
method to detect any time cluster of congenital abnormalit' pairs<br />
due to environmental factors including germinal mutagens . The<br />
Hungarian data will be presented concerning tpe Chernobyl nuclear<br />
power accident .<br />
120<br />
SJMAN MEIUPIC CHRObDSCMAL IIANW(£ IN ItffERCIIB MhIIES AE To ImCPIQI .<br />
T .V . Darmdaran an9 K .M . Marinuthu, Department of Genetics, P .(; .Institute of<br />
Basic Me icaT-Sciences, University of Madras,Taramani, Medras 600 113,MIA .<br />
lwenty one infertile males were studied using mitotic, meiotic <strong>and</strong><br />
histological techniques . Syphilis (2), lynphogranulana venerum (2) <strong>and</strong><br />
nonspecific chronic epididymo orchitis (17) were the various infections<br />
observed . Meiotic studies fran normal healthy men (11) formed the control<br />
data . Changes in the meiotic percentage cell dietribution pattern (MPM) t .es<br />
correlated with meiotic chromeanal anamolies . There was no change in WD<br />
in 2 patients, wtro had shawefl micronuclei of the spermatids <strong>and</strong> increased<br />
frequency of mitotic chrormeane aberrations . Altered MI (msiotic met.aphase<br />
I) <strong>and</strong> M II (meiotic metaphase II) values were noted in 2 patients who have<br />
sho.ed separation difficulties of MI <strong>and</strong> M II . Slight increase in SPM<br />
(Sbermatogonial metaphase) values were noted in two patients . Highly<br />
increased SPM value vss noticed with loss of architecture of bivalents <strong>and</strong><br />
M II elements were saen in a patient . Thus, the results abtained so far<br />
irdicate that though the actual mrJde of action could not be fully undertood<br />
the fact that the meiotic studies fro :n normal healthy control mmles did not<br />
shar any of these ananaliess oonfizm the possible clastogenic <strong>and</strong> autagenic<br />
action of these microbes on meiotic system .<br />
121<br />
MATERNAL UPTAKE AND TRANSFER TO EGGS OF A PROMUTAGEN<br />
(CYCLOPHOSPHAMIDE) BY AN ESTUARINE FISH, CYPRINODON VAR/EQATUS. C.B .<br />
Daniels, D . McMillin <strong>and</strong> J .C . Means . Institute for <strong>Environmental</strong> Studies, Louisiana State<br />
University, Baton Rouge, LA 70803 .<br />
The cytogenetic <strong>and</strong> mutagenic properties of cyclophosphamide (CP) have been shown to<br />
be a function of its metabolism to 4-hydroxycyclophosphamide . We have Initiated research to<br />
determine the availability of cyclophosphamide to developing eggs <strong>and</strong> to characterize the<br />
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43
44 1989 EMS Abstracts<br />
Notes major DNA adducts formed In an estuarine fish, C. varlgQAtus. as the result of exposure to<br />
radioiabeled i14CJ cyclophosphamide . Mature females were allowed to swim In tx10-3M CP<br />
for 5d then placed In tanks of clean water along with a mature male . The female was allowed<br />
to depurate for a total of 5d then sacrificed <strong>and</strong> analyzed for radioactivity(body w/o ovaries<br />
<strong>and</strong> ovarian tissue) by counting on a Packard TRI-CARB 1500 iiquid scintillation counter . All<br />
eggs spawned during this period were collected <strong>and</strong> assayed for radioactivity . A subsample<br />
of the eggs collected were subjected to cetyltrimethylammonlum bromide for the Isolation of<br />
DNA (Murry <strong>and</strong> Thompson, 1980) <strong>and</strong> the major adducts formed characterized by high<br />
performance liquid chromatography (Benson <strong>and</strong> Garner, 1987) . Kinetic data for the uptake<br />
into adults <strong>and</strong> transfer of cyclophosphamide to eggs will be presented <strong>and</strong> contrasted to the<br />
kinetics of uptake of CP in eggs directly from aqueous media . Data on the molecular binding<br />
of cyclophosphamide to cellular DNA <strong>and</strong> chromosome aberration frequencies will also be<br />
presented .<br />
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122<br />
COMBINED jjj VIVO DNA BINDING AND TUMOR DOSE-RESPONSE STUDIES TO INVESTIGATE THE<br />
MOLECULAR DOSIMETRY CONCEPT . R . Dashwood, P . Lovel<strong>and</strong> . A . Fong, J . Hendricks <strong>and</strong><br />
G . Bailey, Oregon State University, Corvallis, OR 97331 . (USA) .<br />
Swenberg et al . (Prog .exp .Tumor Res .11 :42,1987) have discussed the possibility of<br />
using " . the concentration of DNA adducts . . as a better exposure index for quantitative<br />
risk assessment than . .the chemical's exposure concentration" . To evaluate this<br />
molecular dosimetry concept, we have undertaken concomitant jD vivo DNA binding <strong>and</strong><br />
tumor dose response (d-r) studies in rainbow trout . In one approach, -10 000 animals<br />
were exposed both to the carcinogen aflatoxin B1 (AFB1) in the dose-range 10-320 ppb<br />
in diet, <strong>and</strong> to 5 doae levels of a natural anti-carcinogen known to reduce AFB1-DNA<br />
binding in trout (indole-3-carbinol, I3C) . When each data-point from the 12 mo . tumor<br />
d-r analyses was plotted with its corresponding AFB1-DNA binding data-point (logit %<br />
tumor response vs (log) AFB1-DNA binding), a linear relationship was observed at each<br />
13C dose-level . At I3C doses
1989 EMS Abstracts<br />
high incidence of tumors in sun-exposed skin. The defect In early steps of excision repair of XP cells Notes<br />
leads to hypermutability towards UV-mimicking agents . DNA from 8 XP tumors were screened for<br />
activated transforming genes using 3T3 transfection . In 2 skin tumors Isolated from a XP child, an<br />
activated N-M oncogene was detected . Synthetic oligonucieotide probes were used to characterize the<br />
mutation in the La8 gene. Both tumors were found to be mutated In the 61st N-Lal codon from gin to his .<br />
The mutation was accompanied by an Increase in the level of N-tg6, specific mRNA <strong>and</strong> in one<br />
transformant, by the alteration of the p21 protein . In the same tumors, c-mTv .c amplification <strong>and</strong> over<br />
transcription, <strong>and</strong> Ha-LSS gene rearrangement <strong>and</strong> amplification were also detected . Amplified <strong>and</strong>/or<br />
rearranged Ha-LaS gene sequences were detected in 10 other XP tumors . The normal skin fibroblasts<br />
from XP patients show normal pattern levels of N-L&I, c-1,Dy0 <strong>and</strong> Ha-1aS sequences . It Is proposed that<br />
the presence of several oncogene alterations In the same tumor could be due to the high amount of UV<br />
-induced DNA lesions found in the exposed skin cells, In the absence of efficient repair . An activated KI-<br />
L,U was detected in a new XP tumor which contains a mutation In the 12 th codon, probably due to<br />
replication errors at a thymine dimer resulting in a substitution of glycine by valine . We are at present<br />
analysing several XP tumors for ras p21 expression which together with screening by PCR<br />
amplification should help further identify the activated oncogenes in XP tumors .<br />
MECHANISMS OF INHIBITORS OF GENOTOXICITY . RELEVANCE IN PREVENTIVE MEDICINE . Silvio De Flora,<br />
institute of Hygiene <strong>and</strong> Preventive Medicine, University of Genoa, via Pastore 1 . 1-16132 Genoa (Italy)<br />
Inhibition of genotoxic effects in germ <strong>and</strong> somatic cells provides a fundamental strategy, complementary<br />
to the control of environmental <strong>and</strong> lifestyle risk factors, aiming at extending life expectancy <strong>and</strong> at<br />
preventing mutation-related pathologic conditions, first of all cancer . Moreover, some measures against<br />
mutagens <strong>and</strong> carcinogens counteract certain agents, like those causing oxidative damage to the cell, which<br />
are also involved in the pathogenesis of other chronic-degenerative diseases . Unfortunately, due to the<br />
double-edged nature of many physiologic protective factors <strong>and</strong> to the multiple biologic properties shared<br />
by several inhibitors, it is very difficult to stimulate the host defense machinery without inducing adverse<br />
effects . This renders m<strong>and</strong>atory not only to assess the efficacy <strong>and</strong> safety of putative inhibitors In various<br />
experimental systems, but even more to underst<strong>and</strong> <strong>and</strong> elucidate their mechanisms . Genotoxicity can be<br />
inhibited in extracellular environments, either by hampering the uptake of mutagens, or by preventing<br />
their endogenous formation, or by deactivating them before penetration into metabolizing or target cells.<br />
Thereafter, genotoxicity can be inhibited by blocking reactive chemical species <strong>and</strong> by modulating<br />
intracellular mechanisms, such as cell replication, metabolic activation- <strong>and</strong> detoxification pathways, DNA<br />
functions <strong>and</strong> repair processes . At later stages, inhibitors can -act within initiated celis by preventing<br />
tumor promotion <strong>and</strong>/or progression by a variety of mechanisms . Certain inhibitors work through multiple<br />
mechanisms, which warrants a larger spectrum of efficacy . Moreover, inhibitors displaying complementary<br />
mechanisms or acting in different cell compartments can be conveniently associated for a combined<br />
chemoprevention. In any case, the exploitment of protective dietary factors <strong>and</strong>/or pharmacologic agents in<br />
humans is advisable only on the basis of careful risk-benefit analyses .<br />
De Flora, S . (Ed .), Role <strong>and</strong> Mechanisms of Inhibitors in Prevention of Mutation <strong>and</strong> Cancer, Mutation Re,t .<br />
202 : 277-446 (1988) .<br />
126<br />
STIIOY OF PROCEDURES FOR THE DESTRUCTION OF FOUR ALRYLATING AGENTS .<br />
p, Moo . M .P .t, M . Castagnero2 . M . Lagett <strong>and</strong> G . Dumknilt<br />
I l,boratoire de Microbiologie, FacultE de Pharmacie . 27 ilct Jean Moulin, 13385<br />
1Ir,rp~eille Cedex 5, France .<br />
^_ tnil' 1es Canc.tro¢Anea do 1'Environnement et Facteurs d'Hote, IARC, 150 fburs<br />
Aihr•rf-Thomas, 69372 Lyon Cedex 8, France .<br />
nestrnction procedures of dimeth,vl sulfate (DMS), diethyl sulfate (DES), methvl<br />
w1hsnFealfonate (!P]S) <strong>and</strong> ethyl methanesulfonate (EMS) were studied . Destructinn<br />
.nr; t-rformed by adding the alkylating agents at once to a flask containing 1N<br />
N,nH, IN NHaOH, IM Na2C03 <strong>and</strong> IN NazSOa . Determination of the alkylating ngeuts:<br />
•Inrinc the destruction process was performed by the derivation of p-nitrophenoxide<br />
<strong>and</strong> the resulting p-nitroanisole <strong>and</strong> p-nitrophenetole were separatetl hy high<br />
pprfnrm-mce liryuid chromatography . Mutagenic activity of the destruction prodacts<br />
,.•,e eva].uated by the Ames test using Salmonella tester strains TA 97, TA 98 . TA<br />
100 nn .l TA 102 . The kinetics of destruction followed an time-deirrrolvnt<br />
F-:prnenlinl relationship with all the solutions . Solutions of 1M Na280 . shnwvd eh•<br />
Iiiph-st ^nt,RCity of destruction of the four alkyknting agents . Half-time lit•es ~if<br />
nHS . neS, !PdS <strong>and</strong> EMS were 0 .12 ∎in, 1 .2F ∎in, 0 .60 ∎iu <strong>and</strong> 5 .26 ∎in respert i••-1•<br />
in IM NnzSn3 . No mutnqenic aotivity eould be detected following t .he .v .ml•l+•re<br />
d-stru••lion of the qlk,-lstiog n :;ents in 1M Na2S03 . Destrttction of lhese nikYlstinn<br />
agents in the workplace is discussed .<br />
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45
46 1989 EMS Abstracts 127<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes HETEROZYGOUS EFFECTS OF X-RAY-INDUCEO SPECIFIC LOCUS MUTATIONS RESULTING FROM<br />
GENE/POINT MUTATION AND MULTILOCUS DELETION IN THE dQ,-3 REGION OF ye(irgspQEicEass_ .<br />
F .J . de Serresa• L .K . Overtona <strong>and</strong> B .M . Sadlerb aCenter for Life Sciences <strong>and</strong><br />
Toxicology• bCenter for Bioorganic Chemistry . Chemistry <strong>and</strong> Life Sciences . Research<br />
Triangle Institute . Research Triangle Park . NC 27709 (USA)<br />
Previous studies (de Serres, Mutation Res . Z)Q . 281 . 1989) on X-ray-induced<br />
p_d9D_ine-3 (gQm3) mutants induced in a two-component heterokaryon (H-12) of y . cr_ .a_ssa<br />
showed that they were not recessive <strong>and</strong> had heterozygous effects in terms of markedly<br />
reduced linear growth rates . H-12 is heterozygous for mutants at 3 closely linked<br />
loci, gQ-3A• ad-JQ <strong>and</strong> .n_ig-3 (nicotinamide-requiring) ; thus, X-ray-induced mutants in<br />
this region can be of 5 different genotypes : Sg-IA . i1o.-3@ . .4.Q~A AQ $@• 9~~94.Q 3_@<br />
n_ic-Z, <strong>and</strong> g .d_-36 pic-2 . All 5 classes can result from multilocus deletion (g_d,=$+R)o<br />
gene/point mutations (_#d_3R) are usually restricted to the first 2 classes . Present<br />
studiQs were pQrformed to determine whether single-locus mutations of either type<br />
(ad-3R or ad-3`R) sho~v heterozygous effecIA . For example . qi(-~A.IR mutants were<br />
combined with an ad_3a mutant or an pd- @ mutant, <strong>and</strong> the lin ar growth r tes of<br />
the resulting forced dikaryons (aq-jA7~ + q~ .jBR, or aQ;~I$ + ~) were<br />
determined . Ad-3AIR utants showed more pConounced heterozygous effects in combination<br />
with an ad-381~ mutant thin an ad_-9oR mutant ; similar results were found with<br />
_Q-381R mutants . Only 1/30 aQ_3AR showed a heterozygous effect• in contrast to larger<br />
numbers of __d_-_~B R_~BR mutants . Mutants at the AA-3B locus show allelic complementation,<br />
<strong>and</strong> the level of heterozygous effect is higher among noncomplementing mutants than<br />
among complementing mutants with either nonpolarized or polarized patterns .<br />
EVALUATION OF THE EFFECTS 07 CHERNOBYL IN WESTERN EUROPE<br />
by p . ~E WALS, H . DOLE, F . BERTRAND, M .F . LECNAT<br />
EUROCAT Csntral Registry <strong>and</strong> Department of Epidemiology,<br />
Catholic University of Louvain, Brussels, Belgium .<br />
128<br />
Following the Chernobyl accident there was widespread public concern over the possible<br />
mutagenic <strong>and</strong> teratogenic effects of the radiological contamination in the<br />
countries of Western Europe . Assesament of the radiological exposure of the population<br />
in the countries of the EEC .as made by the V .II . National Radiological<br />
Protection Board indicating excess average effective doses to adult individuals in<br />
the first year ranging between 0 .2 pSv in Portugal <strong>and</strong> 130-300 Y8v in Nest-•Gesmany .<br />
Italy <strong>and</strong> Greece . Data from the EIIROCAT sussillance system covering 19 re=ions in<br />
12 countries were used to compare tha frequency of chromosomal syndromds <strong>and</strong> central<br />
nervous system malformations in exposed <strong>and</strong> non-eYposed fetusas . The prelisinary<br />
results do not indicate any increase but a small cluster 'of apina bifida in Odenae<br />
(Denmark) . Other studies were carried out in Finl<strong>and</strong>, Sweden <strong>and</strong> Hungary with negative<br />
results concerning indicators of teratogenic or mutagenie effects . A cluster of<br />
Down syndrome cases in West-Berlin <strong>and</strong> of neural tube defects in Bursa, Turkey have<br />
been reported but relationship with the contamination is not established . Indueed<br />
abortions <strong>and</strong> postponement of conceptions vere two measutable psychological conasquences<br />
of the accident .<br />
EVALUATION OF THE EFFECTS OF CHERNOBYL IN WESTERN EUROPE<br />
by P . DE WALS, H . DOLE, F . BERTRAND, N .P. LECNAT<br />
EUROCAT Central Registry <strong>and</strong> Department of Epideaiology,<br />
Catholic University of Louvain, Brussels, Belgium .<br />
129<br />
Following the Chernobyl accident there was ridespread public concern over the possible<br />
autagenic <strong>and</strong> terstogenic effects of the radiological contamination in the<br />
countries of Western Europe . Assessment of the radiological exposure of the population<br />
in the countries of the EEC was made by the U .K . National Radiological<br />
Protection Board indicating excess average effective doses to adult individuals in<br />
the first year ranging between 0 .2 pSv in Portugal <strong>and</strong> 150-300 vSv in West-Germany .<br />
Italy <strong>and</strong> Greece . Data from the EUROCAT surveillance system covering 19 regions in<br />
12 countries were used to compare the frequency of chromosomal syndromes <strong>and</strong> central<br />
nervous system malformations in exposed <strong>and</strong> non-exposed fetuses . The prelimioary<br />
results do not indicate any increase but a small cluster of spina bifida in Odense<br />
(Denmark) . Other studies were carried out in Finl<strong>and</strong>, Sweden <strong>and</strong> Hungary with negative<br />
results concerning indicatora of teratogenic or mutagenic effects . A cluster of<br />
Down syndrome cases in West-Berlin <strong>and</strong> of neural tube defects in Bursa, Turkey have<br />
been reported but relationship with the contamination is not established . Induced<br />
abortions <strong>and</strong> postponement of conceptions were two measurable psychological consequenees<br />
of the accident .<br />
50869 3558
130<br />
ANALYSIS OF MICHAEL2ADDITION-TYPJ COMPOUNDS Ij MQUSE LYMPHOMA CELLS . K .L . Dearfieldl,<br />
K . Harrington-Brock , C2L . Doerr , M .M . Moore . U .S . <strong>Environmental</strong> Protection Agency,<br />
Washington, DC 20460 ; E5vironmental Health Research <strong>and</strong> Testing, Inc ., Research<br />
Triangle Park, NC 27709 ; U .S . <strong>Environmental</strong> Protection Agency, Research Triangle Park,<br />
NC 27711 .<br />
The mutagenicity <strong>and</strong> clastogenicity of some Michael addition-type compounds including<br />
acrylamide, several acrylates <strong>and</strong> vinyl sulfones have been evaluated . These compounds<br />
have terminal double bonds that suggest they may be substrates for epoxide formation ;<br />
however, unlike epoxides, they are apparently inactive in the Salmonella assay :'In mammalian<br />
cells, they appear to act via a direct acting clastogenic mechanism . Our work<br />
with L5178Y mouse lymphoma cells demonstrates that acrylamide, several acrylates <strong>and</strong><br />
methacrylates <strong>and</strong> vinyl sulfones (vinyl sulfone <strong>and</strong> methyl vinyl sulfone) without exogenous<br />
activation induce increases in the number of small colony tk-deficient mutants .<br />
This suggests a clastogenic mechanism which was confirmed by demonstrating increases in<br />
aberration <strong>and</strong> micronucleus frequencies . With exogenous metabolic activation systems<br />
(S9), the activity of these compounds may be altered, e .g . activity increased . in<br />
methacrylates (probably due to recruitment of epoxide formation) <strong>and</strong> decreased in<br />
acrylates (alternate detoxification pathways via S9 may decrease the potent response) .<br />
Since these compounds are known to deplete glutathione, phorone, a model glutathione<br />
depleter, will be examined . The results suggest that the direct-acting Michael addition<br />
mechanism may have activity relevant to genetic toxicity . Since acrylamide is a potent<br />
germ cell mutagen, this has importance in terms of heritable risk . (This is an abstract<br />
of a proposed presentation <strong>and</strong> does not necessarily reflect U .S . EPA policy .)<br />
131<br />
POTEtr"I'IAL EPA/OPP uUfAGENICITY TESTING REQUIREMENTS-GUIDELINES REVISION . K .L . Dearfield<br />
U .S . <strong>Environmental</strong> Protection Agency, Office of Pesticide Programs, Washington DC 20460<br />
The USEPA's Office of Pesticide Programs (OPP) is currently revising its Pesticide<br />
Assessment Guidelines - Subdivision F dealing with the requirements for mutagenicity<br />
testing . Currently, to register a pesticide with the OPP, mutagenicity testing must be<br />
performed in a minimum of three tests, one each in the categories for gene mutation,<br />
structural chromosomal aberrations <strong>and</strong> other genotoxic effects . The OPP's purposes for<br />
mutagenicity testing are to detect, with appropriate assay methods, the capacity of a<br />
chemical to alter genetic material in cells <strong>and</strong> to incorporate these findings in i) the<br />
assessment of heritable genetic alterations of concern to humans, ii) the<br />
weight-of-evidence approach for an oncogenicity classification of a chemical when<br />
bioassay results are available, end iii) the decision to trigger an oncogenicity study<br />
if such a study is conditionally required . The auggestAd revised mutagenicity testing<br />
begins with an initial battery : Salmonella, mouse lymphoma (tk locus, small <strong>and</strong> large<br />
colony counts), <strong>and</strong> in vivo cytogenetic assays . Other tests appropriate to the test<br />
chemical may be substituted upon discussion with the OPP . Testing to confirm results<br />
from the initial battery, such as with another in vivo organ or tissue, may be<br />
necessary . A weight-of-evidence approach will be used to reach a decision for the need<br />
for further testing . For a heritable concern, tests such as dominant lethal,<br />
cytogenetics in germ cells, specific locus <strong>and</strong>/or heritable translocation assays may be<br />
required . It should be recognized that these are potential revisions <strong>and</strong> have not<br />
undergone complete Agency <strong>and</strong> policy review . As such, they are not to be considered<br />
current EPA/OPP policy .<br />
132 OXIDATIVE DNA DAMAGE : REPAIR AND INDUCIBLE CELLULAR RESPONSES<br />
g . Dgmplg, D.S . Chen, J.T . Greenberg, J .D . Levin, P. Monach <strong>and</strong> S .C . Popoff, Department of<br />
Biochemistry <strong>and</strong> <strong>Molecular</strong> Biology, Harvard University, Cambridge, Mass, 02138 .<br />
Escherichia coil can respond to different kinds of oxidative stress by activating multicomponent<br />
systems that can prevent damages, regulate cell division, repair DNA lesions, <strong>and</strong> Increase NADPH<br />
production, as well as a large number of proteins of unknown function . These irxluctions are triggered<br />
by nontoxic levels of certain agents (e .g ., H202 or superoxide-generating agents) <strong>and</strong> confer elevated<br />
cellular resistance to higher levels of the same agents, <strong>and</strong> are therefore termed 'adaptive responses"<br />
to reflect their possible roles in evolution . We have been studying the complex response of E. coli to<br />
low levels of oxidative damage . Redox-cycling agents such as the quinone menadbne oonvey single<br />
electrons from NADPH or NADH to molecular oxygen, generating intracellular superoxide radical 021,<br />
which can then be converted to H202 by superoxide dismulase . Redox-cycling triggers the elevated<br />
synthesis of >70 polypeptides, of which about half are inducible by H202 directly . Consequently, E .<br />
coli possesses at least two distinct reponses to oxidative stress, one triggered by peroxide <strong>and</strong> at least<br />
part of the rest activated by superoxide . We have isolated menadione-resistant bacterial mutants that<br />
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1989 EMS Abstracts 47<br />
Notes
48 1989 EMS Abstracts<br />
Notes express constitutively 10 proteins that are usually indudbie by Or . These proteins Include three<br />
known enzymes : Mn-containing superoxide dismutase (to destroy Or), giucose-B-phosphate<br />
dehydrogenase (to generate NADPH), <strong>and</strong> DNA endonuclease IV, which acts on a variety of oxidative<br />
damages In DNA . These soxR mutants express Increased resistance to a variety of oxidative agents, <strong>and</strong><br />
these elevated resistances are only partly dependent on the sodA-encoded superoxide dismutase <strong>and</strong> the<br />
n/o-enooded endonuclease IV . Consequently, the functions of the soxR regulon contribute In multiple<br />
ways to cellular resistance, reflecting the complex modes of ceil damage caused by free radicals .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
133<br />
HAVE THE COCA LEAVES AND BASIC COCAINE CONSUMERS A HIGH FREQUENCY OF CHROMO<br />
SOME MUTATIONS . J . Descallleaux, M .L . Guwaro, L .A . Rodrrguez,M . Paredes, <strong>and</strong> M . Abarca.<br />
Lab . Genet . Hum ., Fae . Ctenclas Biol6gioas, UNMSM, P .O . Box 14-0010, L(ma-PerG .<br />
The number of coca leaves (CL) <strong>and</strong> basic cocaine (BC) oonsumers in the peruvian <strong>and</strong>eon region (CL)<br />
<strong>and</strong> the capital ctty intiabitants (BC) (s In constant raise, (CEDRO,1987), the wnrks reported In relation<br />
with the pharmacology effects of these hobitudes are oonhoverstals In the CL case, but all of<br />
them hove appointed the BC as a very toxlc drug to the human health . We have selected two samples<br />
of routinely consumers of CL <strong>and</strong> BC with their respective controls, In all of them, we have obtalned<br />
4-6 ml of periptieral b lood to m ade a lymphory te culture <strong>and</strong> measure the chromosome aberro -<br />
tions (CA) In first-dlvision metaphase oalls, <strong>and</strong> 5CE In seoond-diviston metaphase cells ; <strong>and</strong> exfolia<br />
te cells from the oral m uoosa In order to estimate the frequency of mteronuclsi .<br />
The results obtained In CL consumers showed a minor frequency of CA <strong>and</strong> SCE than the control -<br />
group suggesting that the CL hove on enti-mutagenle property that may be a consequence of t he<br />
high level of vitamine A(Pio6n-Rebtegu1,1976) knowing that this vitamine has on anti-mutogenle pro<br />
perty (Grubbs et a1 .,1977) . But the mieronuelel frequency was higher In CL consumers, In this relation<br />
it'is important to consider tha• the "ehoecheo" habitude (CL chswing) enolose the use of the<br />
cal (CoO) alcaline <strong>and</strong> caustic substance, that would be the responstble of the aberront nuelel . In<br />
the BC consumers, although our first results m ust be eonsidered as prei(minary, mainly because of the<br />
s mall number of patients e xamined, they are showing an Inerese frequency of CA <strong>and</strong> SCE In the con<br />
sumers than In the control group .<br />
MUTAGENICITY OF TWO GIUCOCORTIC0I0St DEXAYiETFWSONE AND PREDNISOLONE<br />
H .S . Dhillon <strong>and</strong> J .R . Singh, School of Life Sciences, Human Genetics Laboratory,<br />
Guru Nanak Dev University, Amritsar (India)<br />
Glucocorticoids are therapeutically very important as they possess strong<br />
antiallergic <strong>and</strong> immunusuppressive properties . Keeping in view the mechanism of<br />
action of these medicines which involves the interection of the drug with DNA<br />
via specific receptor proteins, the mutagenic propertisa of two glucocorticoids -<br />
Dexamethasone <strong>and</strong> Prednisolone - were investigated using Ames Salmonelly/S-9 test<br />
<strong>and</strong> host mediated assay with mice <strong>and</strong> Salmonella tester strains (TA97e, TA98, TA<br />
100 <strong>and</strong> TA1535) There was no significant increass or decre4se in the nueber of<br />
His+ revertests/plate either with or without S-9 mix at the doses rangIng from<br />
1 ug4to 1x10 ug/plate in the Ames test . Ho"r at higher doses (Sx10 ug <strong>and</strong><br />
1x10 ug/plete) the growth <strong>and</strong> number of His revertants/plate were inhibited<br />
significantly . In the host mediated assay doses of the drugs tested were 1, 10<br />
<strong>and</strong> 100 mg/kg bw . No significant positive results were obtained with either of<br />
the drug, indicating that observed non-mutagenicity in the Salmonella straina is<br />
not because of the insufficient metabolic degradation of the glucocorticoids in<br />
the in vitro system but due to their inability to interact with the prokeryotic<br />
DNA .<br />
COMPARATIVE EFFICACY OF ASCORBIC ACID AND EXTRACT FROM<br />
PHYLLANTHUS Mii BLICA FRUIT IN MODIFYING METAL CLASTOGENICITY<br />
H .Dhir, S .Ghosh, Sharmila Ghosh, A .ohosh, A .K . Ghosh, S . Palit .<br />
G . Talukder, A . Sharma, Human Genetics Unit, Centre for Advanced<br />
Study Cell rx Chromosome Researeh, Department Botany, University<br />
of Calcutta, Calcutta 700019, India .<br />
134<br />
135<br />
Fruits of Ph 1 an hus emblica L . contain Vitamin C <strong>and</strong> are Used<br />
extensively n many me c na preparations of Ayurvedic <strong>and</strong> Unani<br />
systems of medicine . Aqueous extravts of the fruit were fed to<br />
laboratory mice for different periods to observe if the clasto- 'P<br />
50869 3560
genicity of well known cytotoxic metals like lead, cadmium, tin,<br />
cobalt, nickel, aluminum, barium <strong>and</strong> cesium could be modified by<br />
the extract . Oral gavage of the mice with the extract for 7 days<br />
prior to intraperitoneal injection of the metal salts, significantly<br />
reduced the frequencies of chromosomal aberrations, gaps <strong>and</strong><br />
rearrangements induced by the metals when compared with control<br />
mice given the metal alone <strong>and</strong> not previously fed the extract .<br />
substitution of the fruit extract with an equivalent amount of<br />
ascorbic acid as that presebt in the extract, also reduced the<br />
frequency of chromosomal aberrations induced by the metals, but<br />
to a lesser extent than the fruit extract . On the other h<strong>and</strong>,<br />
following treatment with some of the higher doses of the metals<br />
used, ascorbic acid increased the clastogenic'effects . The greater<br />
efficacy of the •fr1dt, .Axtrdct can .be ii:LL'.lbuted to_ .the combined action .<br />
136<br />
REVISED GUIDELINES OF UK COMMITTEE ON MUTAGENICITY (COM) 1989<br />
DR DIGGLE AND DR FIELDER (DEPT HEALTH LONDON)<br />
The COM is an expert advisory Committee, chaired by Professor B Bridges, set up<br />
to advise UK Government Departments on the mutagenic risk of substances . Their first<br />
guidelines on testing (1982) have now been updated, a revised strategy being<br />
recommended . Initial studies (Stage I) consist of in vitro screening designed to<br />
ensure a high probability of detecting mutagenic poten-fia-T .7-7ests should be done to<br />
the best available protocols, <strong>and</strong> results confirmed 1n an lndependent experiment . Two<br />
tests are routinely required, a bacterial assay for gene mutation <strong>and</strong> a test for<br />
clastogenicity tn mammalian cells, except where human exposure is expected to be<br />
extensive or sustained <strong>and</strong> difficult to avoid, when a test for gene mutation in<br />
mammalian cells is also necessary . This may be followed by 1n vivo studies (Stage<br />
II) . It is not felt justifiable to use in vivo studies for gene-screening <strong>and</strong> 1f<br />
the in vitro tests are negative no further lesting would normally be required . The<br />
excep-tTon--Ts substances where relatively high exposure, or moderate but prolonged<br />
exposure, is anticipated . An assay for chromosome damage in bone marrow would then be<br />
recommended . This may also be required for substances mutagenic tn vitro, to see<br />
whether the activity may be expressed In vivo . If negative one or more further<br />
in vivo assays tn a different tissue Teg Ztver, gut) may give the necessary<br />
reassurance (in the light of other toxicological data)•that the substance Is unlikely<br />
to be genotoxic to man . The most appropriate test(s) need to be determined on a<br />
case-by-case basis . Stage III consists of in vivo tests for germ cell effect, <strong>and</strong> is<br />
only necessary when a risk assessment of herTl :abTe effects ts justified .<br />
137<br />
CHEMISTRY OF DNA ALKYLATION AND ARAIXYIATION . Anthony Dipple, BRI-Basic Research<br />
Program, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 .<br />
Since the mutagenic properties of many carcinogens depend upon their metabolic<br />
conversion to chemically reactive metabolites, chemical reactivity can be considered<br />
to be the 'essence' of mutagenic/carcinogenic activity for these compounds . Agents<br />
with different chemical reactivities modify different sites on DNA bases . For<br />
example, simple alkylating agents preferentially modify the ring nitrogen at the 7position<br />
of guanine residues whereas alkylnitrosoureas modify both the 0s- <strong>and</strong> the 7position,<br />
<strong>and</strong> polycyclic aralkylating agents modify the exocyclic N=-site on guanine<br />
residues almost exclusively . In order to underst<strong>and</strong> the basis for these changes in<br />
site selectivity with changes in reactivity of the agent, extensive studies of site<br />
specificity in guanosine modification by benzylating agents, which attack the 7-, 0'<strong>and</strong><br />
N2-positions of guanosine, have been undertaken . More recently, an optically<br />
active benzylating agent, styrene oxide, has been examined so that the<br />
stereochemistry of various guanosine alkylation products gives insight into the<br />
differences in mechanism through which alkylation at different sites occurs . These<br />
investigations indicate that both the degree of ionic character in the reagent (i .e .<br />
the Ssl or Ss2 character] <strong>and</strong> the nature of the ionic intermediate (i .e . whether the<br />
charge on the reaction center is localized (hard) or dalocalized (soft)) determine<br />
the sites of alkylation on guanine residues . Research sponsored by the NCI, DHHS,<br />
under contract No . NO1-CO-74101 with Bionetics Research, Inc .<br />
138<br />
ANALYSIS OF MUTATION INDUCTION IN VIVO AND IN VITRO WITH AN SV40-BASED SHUTTLE<br />
VECTOR. K . Dixon, J . Hauser, M . Carty, N . Zernik, E . Roilides, J . Carr, <strong>and</strong> A . S .<br />
Levine, Section on Viruses <strong>and</strong> Cellular Biology, NICHD, NIH, Bethesda MD 20892<br />
We have used the SV40-basad shuttle vector, p2189, to study mechanisms of<br />
mutagenesis in mammalian cells . When the vector DNA is treated with either UV<br />
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1989 EMS Abstracts 49<br />
Notes
50 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes radiation or benzo[a]pyrene diolepoxide <strong>and</strong> then allowed to replicate in CV-1 monkey<br />
cells, mutations are induced in the mutagenesis target gene (the bacterial auoF gene)<br />
of the vector . DNA sequence analysis of these mutations reveals that the two<br />
mutagens induce very different spectra of mutations . By comparing the mutational<br />
spectra with the spectra of damage induced by the two agents, we have been able to<br />
deduce cellular mutational mechanisms . Recently, we constructed a series of<br />
polyomavirus-based vectors that replicate in rodent cells to facilitate the analysis<br />
of the role that cellular DNA repair processes play in mutational specificity . With<br />
these vectors we have begun to analyse the specificity of UV mutagenesis in repairdeficient<br />
mouse cells . We have also studied mutagenesis in an in vitro DNA<br />
replication system . The p2189 vector can be completely replicated in this cell-free<br />
system in the presence of SV40 T-antigen . Undamaged vector DNA is replicated with<br />
high fidelity in this system . UV-damaged vector DNA is also replicated, but less<br />
efficiently . It appears that mutagenesis occurs as a consequence of replication of<br />
the UV-damaged templates in vitro . Preliminary results suggest that the spectra of<br />
mutations induced by UV in vitro resemble those induced in vivo . Thus, the in vitro<br />
system may prove to be particularly useful in the molecular analysis of mutation<br />
induction in mammalian cells .<br />
139<br />
STABLE EXPRESSION OF P450IIB1 AND P450IA1 CDNA IN V79 CHINESE HAMSTER<br />
CELLS APPLIED TO MUTAGENICITY TESTING<br />
J . Doehmer, S . Satish, H .R .Glatt, <strong>and</strong> F . Oesch<br />
Institut fUr Toxikologie, Johannes Gutenberg-Universit8t,<br />
D-6500 Mainz, FRG<br />
cDNA encoding P450IA1 <strong>and</strong> P450II81 were recombined with the SV 40<br />
eukaryotic vector . The recombinant plasmids were transferred into V79<br />
Chinese hamster cells by the calcium/phosphate co-precipitation<br />
procedure using the neomycin phosphotransferase gene as selective<br />
marker . Several cell clones were obtained . Cell clones were<br />
characterized by Southern-, Northern-, <strong>and</strong> Western-blotting . The<br />
enzymatic acttivity was determined using P450 specific substrates like<br />
7-pentoxyresorufin in the case of P450IIB1 <strong>and</strong> 7-ethoxycoumarin in the<br />
case of P4501A1 . Specific activities were found to be in the same range<br />
typical for uninduced liver . Cytotoxicity <strong>and</strong> mutagenicity studies<br />
revealed that P450 producing cells respond to the exposure of<br />
cyclophosphamide, benzo(a]pyrene, <strong>and</strong> trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene<br />
in a dose dependent manner . The mutation rate<br />
also correlated with the different levels of activity contained in the<br />
various cell lines . So far, the enzymatic activity remained stable in<br />
these cell lines for more than one year .<br />
The project is supported by the Deutsche Forschungsgemeinschaft (SFB<br />
302 "Kontrollfaktoren der Tumorentstehung") .<br />
140<br />
EFFECT OF SIMULATED ACTIVATED SLUDGE TREATMENT USING BENCH-SCALE BIOREACTORS ON THE<br />
MUTAGENICITY OF MUNICIPAL WASTEWATER . J . U . Doerger, J . R . Meier, R . A . Dobbs <strong>and</strong> G .<br />
N . Stelma, U . S . <strong>Environmental</strong> Protection Agency, Cincinnati, Ohio, 45268 .<br />
Previous studies of mutagens in municipal wastewater have shown that following<br />
activated sludge treatment the level of extractable organic matter had decreased .<br />
However, the level of mutagenic activity had not decreased substantially with treatment .<br />
Two possible explanations for this finding were postulated : the bioreaction process<br />
increased the number or potency of the mutagens, or the process removed non-mutagenic<br />
compounds more efficiently than it removed autagens . In order to have a controlled<br />
system for comparing pre- <strong>and</strong> post-treatment samples, bench-scale bioreaction studies<br />
were performed . Primary effluent was inoculated with aerobic organisms from the aeration<br />
basin <strong>and</strong> incubated at 23°C with aeration for 24 hrs . The extracts were evaporated<br />
<strong>and</strong> redissolved in DMSO for subsequent autagenicity testing using the Ames Salmonella<br />
assay with tester strain TA98 with <strong>and</strong> without S9 activation . The results of the<br />
bench-scale bioreactor were consistent with data derived from treatment plant studies .<br />
Total mutagenic activity was not significantly changed with incubation ; however, the<br />
specific mutagenic activity (his+ revertants/mg) of post-incubation extract did increase<br />
approximately 50% . The weight of the extracts decreased 35% . Viable cell counts of<br />
the inoculated wastewater indicated 40-fold increase after the incubation . These data<br />
suggest that the compounds responsible for mutagenicity in a municipal wastewater are<br />
less biodegradable by activated sludge treatment than the bulk of the extracted compounds<br />
. Consequently, improved biological <strong>and</strong>/or chemical-physical processes may be<br />
required to remove the mutagens . (This abstract does not necessarily reflect EPA<br />
policy) .<br />
50869 3562
141<br />
CORRELATION BETWEEN CHROMOSOME ABERRATION FREQUENCY AND SMALL-COLONY TK-DEFICIENT<br />
MUTANT FREQUENCY IN L5178Y/TK+/- -3 .7 .2C CELLS. C .L . Doerrl <strong>and</strong> M .M . Moore2 .<br />
l<strong>Environmental</strong> Health Research <strong>and</strong> Testing, Inc ., Research Triangle Park, NC 27709<br />
USA ; 2U .S . <strong>Environmental</strong> Potection Agency, Research Triangle Park, NC 27711 USA .<br />
The L5178Y mouse lymphoma assay quantitates gene mutation at ths thymidine kinase<br />
locus . Karyotypic <strong>and</strong> molecular analyses of mutants have shown an association<br />
between small-colony phenotype <strong>and</strong> chromosomal events . We have bean evaluating the<br />
association of small-colony induction with gross aberration analysis to determine if<br />
the mouse lymphoma assay can be used directly to determine potential clastogenicity<br />
of test compounds . Based on data from 36 compounds, we find a clear association<br />
between clastogenicity <strong>and</strong> small-colony induction . There is, as expected, no simple<br />
mathematical correlation between the two endpoints . Many gross aberrations are<br />
incompatible with cell survival <strong>and</strong> colony formation, <strong>and</strong> some small-colony TK<br />
mutants are products of events which cannot be scored visually as chromosome<br />
aberrations when small changes in some essential DNA sequence(s) results in slow<br />
growth . Based on these analyses <strong>and</strong> previous studies, we fsel that the small-colony<br />
TK mutant frequency is the most useful measure of the clastogenicity of test<br />
compounds . It reflects cytogenetic events compatible with long-term cell viability,<br />
<strong>and</strong> these are the types of events important in human disease . (This is an abstract<br />
of a proposed presentation <strong>and</strong> does not necessarily reflect U .S . EPA policy .)<br />
142<br />
SULFOTRANSFERASE AND SULFATE LEVELS VARY THE ACTIVATION OF2-P.MIW-3-METHYLIMIDA7A(4,5-<br />
f)QUINOLINE (IQ)<br />
P . Dolara, M . Lodovici, P . Grassi<br />
Department of Pharmacology, University of Florence, Viale Morgagni, 65, Florence,Italy<br />
The in vitro binding of 3H-IQ to herring sperm DNA was measured in the presence of<br />
S9 <strong>and</strong> microsomes from Wistar rat livers . Both S9 <strong>and</strong> microsomes from control rats<br />
catalyzed DNA binding of IQ, <strong>and</strong> a considerable increase in binding was observed using<br />
livers from PCB treated animals . DNA binding was totally blocked by pentachlorophenol<br />
<strong>and</strong> 2,6-dichloronitrophenol, a specific inhibitor of sulfotransferaae . A mod-<br />
est increase in DNA binding was observed with 3'-phosphoadenosine-5'-phosphosulfate .<br />
Incubation of IQ in media with high sulfate concentrations increased DNA binding in<br />
vitro . An increase of IQ binding was also observed in vivo by varying sulfate pools<br />
in rats with the administration of sulfite in drlnking'water . The data demonstrate a<br />
role for sulfotransferase in the activation of IQ, <strong>and</strong> suggest a possible role of<br />
dietary sulfite in modulating the activation process of IQ .<br />
143<br />
DETERMINATION OF 4-AMINOBIPHENYL HEMOGLOBIN ADDUCTS AS A MEASURE OF ACTIVATION OF<br />
AROMATIC AMINES IN HUMANS<br />
P . Dolara, G . Moneti, M . Salvadori .<br />
Department of Pharmacology, University of Florence, V .le Morgagni, 65, Florence, Italy<br />
The levels of hemoglobin (Rb) adducts of 4-aminobiphenyl (4-ABP) in smokers are a<br />
function of the exposure to cigarette smoke <strong>and</strong> of the activation of aromatic amines<br />
to reactive metabolites . We measured the Hb adduct of 4-ABP in humans with massspectrometry,<br />
after hydrolysis of Rb <strong>and</strong> extraction with methylene chloride . Methy-<br />
lene chloride is preferable to hexane, used in published methods, because of better<br />
recovery of 4-ABP . The exposure to cigarette smoke was quantified by measuring the<br />
24 h excretion of a long-lived metabolite of nicotine (cotinine) . Cotinine levels are<br />
poorly correlated with self reported smoking habits . A good correlation was observed<br />
between 24 h cotinine urinary excretion <strong>and</strong> the levels of 4-ABP adducts to Hb . The<br />
levels of 4-ABP in non-smokers were never inferior to 50 pg/ g Hb<br />
. The correlation of<br />
cotinine urinary levels <strong>and</strong> 4-ABP adducts to Hb is defined by a line, the slope of<br />
which is a function of metabolic activation of aromatic amines<br />
. The method was applied<br />
to smoking controls <strong>and</strong> urinary cancer patients <strong>and</strong> gave a good estimate of the<br />
capability of different subjects to activate aromatic amines .<br />
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1989 EMS Abstracts 51<br />
Notes
52 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
144<br />
COMPARATIVE STUDIES* ON THE GENOTORIC POTENTIAL OF SIDESTREAN SMOKE FROM CIGARETTES<br />
WHICH BURN OR ONLY HEAT TOBACCO . D . J . Doolittle, C . K . Lee, G . T . Burger <strong>and</strong> A . V .<br />
Hayes, R . J . Reynolds <strong>Tobacco</strong> Company, Bowman Gray Technical Center, Winston-Salem,<br />
NC 27102<br />
The in vitro genotoxic activity of sidestream cigarette smoke (SSCS) from cigarettes<br />
which heat but do not burn tobacco was compared to that of SSCS from cigarettes<br />
which burn tobacco . SSCS from five cigarettes were compared . Three of the<br />
cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available<br />
ultra-low tar br<strong>and</strong> (ULT) <strong>and</strong> a commercially available ultra-low tar menthol<br />
br<strong>and</strong> (ULT-menthol)) burn tobacco while two of the cigarettes [a regular (TEST) <strong>and</strong> a<br />
menthol (TEST-menthol)] heat tobacco . SSCS from all cigarettes was collected by<br />
identical techniques, which involve collecting particulate matter on Cambridge filter<br />
pads <strong>and</strong> combining with the vapor-phase materials collected by bubbling the smoke<br />
through DHSO . All samples were simultaneously evaluated at identical concentrations<br />
in the test battery . SSCSs from 1R4F . ULT <strong>and</strong> ULT-menthol cigarettes were positive<br />
in the Ames bacterial mutation assay while TEST <strong>and</strong> TEST-menthol SSCS was negative .<br />
SSCS from 1R4F, ULT <strong>and</strong> ULT-menthol cigarettes was positive in the CHO-chromosome<br />
aberration assay <strong>and</strong> in the CHO-sister chromatid exchange assay while TEST <strong>and</strong><br />
TEST-menthol SSCSa were negative in both assays . 1R4F, ULT <strong>and</strong> ULT-menthol SSCSs<br />
were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST<br />
<strong>and</strong> TEST-menthol SSCSs were devoid of activity in this assay . These results demonstrate<br />
that sidestream smoke from the TEST <strong>and</strong> TEST-menthol cigarettes was not<br />
genotoxic under conditions in which sidestream smoke from 1R4F, ULT <strong>and</strong> ULT-menthol<br />
cigarettes were genotoxic in a concentration-dependent manner .<br />
145<br />
THE EVOLUTION OF MUTATION RATES : PROSPECTS FOR ANTIMUTAGENESIS .<br />
John W. Drake, Laboratory of <strong>Molecular</strong> Genetics, National Institute of <strong>Environmental</strong><br />
Health Sciences, Research Triangle Park, NC (USA)<br />
Arguments can be adduced for the operation of forces that would either increase or<br />
decrease mutation rates in the course of evolution . In microbial systems, an equilibrium<br />
might be established reflecting the advantages of higher mutation rates that generate<br />
adaptive mutations <strong>and</strong> the disadvantages of lower mutation rates that generate deleterious<br />
mutations . In more intensively sexual systems, the balance might be between the costs in<br />
time, materials <strong>and</strong> energy between reducing mutation rates <strong>and</strong> the costs of bearing the<br />
load of deleterious mutations . Classical experiments bear on both of these possibilities . It<br />
is also likely that antimutagenesis by either genetic modification or pharmaceutical intervention<br />
is a tenuous goal. This notion is superficially contradicted by diverse reports of<br />
antimutator mutations <strong>and</strong> antimutagenic chemicals, most of which are helpful in underst<strong>and</strong>ing<br />
mutational processes but are illusory in promising ways to lower mutation rates in<br />
genetically optimized animal <strong>and</strong> plant germlines or in higher primates .<br />
146<br />
Comparison of the clastogenic action of mutagens in the presence <strong>and</strong><br />
absence of bromodeoxyuridine .<br />
Joachim H . Dresp<br />
Pharmaceutical Research, Department of Toxicology<br />
F . Hoffmann-La Roche i Co . Ltd, CH-4002 Basel, Switzerl<strong>and</strong><br />
The incorporation of the base analogon bromodeoxyuridine (BrdUrd) into<br />
replicating DNA allows a distinction between cells which are in their<br />
first or second division after initiation of the cell culture .<br />
Up to now the question whether or not the presence of BrdUrd influences<br />
the experimentally induced rate of chromosomal aberrations in human<br />
peripheral lymphocytes cannot be answered unequivocally .<br />
Experiments with different xenobiotics were carried out . The results<br />
illustrate the benefits <strong>and</strong> the disadvantages of the Brdurd-labelling<br />
technipue .<br />
50869 3564
147<br />
SEMPERVIRINE : A NON-MUTAGENIC ANTI-TUMOR ALKALOID FROM GELSEMIUM<br />
ELEGANS<br />
Du,. .Y .-X .' Wu, Z .-L .', Liang, W .-Z .', Chen, H .-H .•, Liang, X .-Re•<br />
Departments of Hygienee <strong>and</strong> Microblologye, Guangzhou Medical College,<br />
195 Dongfeng Rd ., Guangzhou 510182, The People's Republic of China .<br />
Gelsemium Elegans Benth, a medicinal herb grown in South China <strong>and</strong><br />
used locally as a roborans for pigs <strong>and</strong> sheeps, is seveNly toxic to<br />
humans . Recently however, Sempervirine, an alkaloid extracted from<br />
Gelsemiun elegans has shown promise as an anti- tumor agent in cancers<br />
of the liver, esophagus <strong>and</strong> stomach . In this study we have examined<br />
the in vitro effects of sempervirlne on tumor cell lines A-549,<br />
PLC/PRF/2 <strong>and</strong> CNE-2 from lung, liver <strong>and</strong> nasopharynx respectively . At<br />
drug concentrations between 100 to 400 yg/ml a significant decrease in<br />
cell proliferation was observed for all three cell lines examined .<br />
Whereas no effect was observed for in vitro mutagenicity assays using<br />
bacterial strains TAs7, TAss, TAioo <strong>and</strong> TAio : at drug concentrations<br />
between 2 to 500 Ng/ml in the presence or absence of a S-9 fraction .<br />
Chromosome abberation assays with CHL cells exposed for 24 or 48 hours<br />
at five drug concentrations between 5 to 100 pg/ml showed no difference<br />
as compared to controls. The micronucleus test with NIH mice at<br />
dosages of 1/2LDso, 1/5LDso <strong>and</strong> 1/10LDso showed no statistical<br />
difference (P>0 .05) in the ratio of polychromatic erythrocytes to<br />
normocytic erythrocytes as compared to controls .<br />
148<br />
TRAHSGENIC ANIMALS EXPRESSING THE BACTERIAL 06ALKYLGAUNINE-DNA ALKYLTRANSlERASE<br />
GENE : A MODEL TO STUDY THE ROLE OF DNA REPAIR IN THE CARCINOGENBSIS OF N-NITROSO<br />
COMPOUNDS. L .L . Dumenco, D .W . Clapp, I .K . Lim, S . Kesssn, C . Donovan, 3 . Warman,<br />
N . Gorodetzkaya, J . Yun, T . Wagner, R .W . Hanson, S .L . Gerson . Irel<strong>and</strong> Cancer<br />
Center, Dept of Biochem <strong>and</strong> Med, Clevel<strong>and</strong>, OH, 44106, <strong>and</strong> Edison Biotech Center<br />
Ohio Univ Athens, OH, 457016<br />
The DNA repair protein 0 alkylgaunine-DNA alkyltransferase is a critical<br />
factor controlling tissue specific tumor induction following nitrosamine <strong>and</strong><br />
nitrosourea exposure in animals . We designed a chimerio gene consisting of 340 bp<br />
of the promotor-regulatory region of the rat phosphoenolpyruvate carboxy kinass<br />
(GTP) (PEPCK) gene linked to the ads gane coding for the ~~} alkyltransferase .<br />
The PEPCK promotor is highly inducible in transganic animals by a high protein diet<br />
or serotonin treatment <strong>and</strong> is primarily expressed in kldney <strong>and</strong> liver . Using this<br />
chimeric gene, two haterozygous transgenic founder animals were obtained .<br />
Offspring had two-fold increased alkyltransferasa activity in liver <strong>and</strong> kidney <strong>and</strong><br />
ada gene expression as confirmed by Northern analysis . The ~ gene was inducible<br />
in the liver <strong>and</strong> kidney by a high protein diet <strong>and</strong>/or serotonin . ?olloving<br />
induction, total alkyltransf.rase activity was increased five-fold above background<br />
in liver <strong>and</strong> at least two-fold in kidne~r . These transgenic mice will allow us to<br />
determine whether efficient repair of 0 alkylguanine-DNA adducts by high levels<br />
of alkyltransferase activity can decrease the tissue specific carcinogenicity of<br />
N-nitroso compounds .<br />
149<br />
EXPRESSION OF XRCC1 IN HUMAN TUMOR CELL LINES .<br />
E . Dunphy, R .R . Weichselbaum, L .H . Thompson <strong>and</strong> J .L . Schwartz,<br />
University of Chicago, Chicago, IL <strong>and</strong> Lawrence Livermore National<br />
Laboratory, Livermore, CA (USA) .<br />
XRCC1 is a gene whose expression complements the defect in the<br />
mutagen-sensitive Chinese hamster cell line EM9 . The XRCC1 gene product<br />
is believed to be involved in DNA single-str<strong>and</strong> break rejoining <strong>and</strong> SCE<br />
induction . Absence of this gene product confers sensitivity to<br />
ionizing radiation, monofunctional alkylating agents <strong>and</strong> halogenated<br />
pyrimidines . In this study, the expression of XRCC1 was examined in a<br />
variety of human tumor cell lines . These cell lines have widely<br />
different radiosensitivities which might, in part, be a function of<br />
xRCC1 expression . Twenty-five human squamous cell carcinomas <strong>and</strong><br />
sarcomas were examined . The radiosensitivities (DO) ranged from about<br />
1 .0 Gy to 2 .7 Gy . The expression of XRCC1 was variable, being either<br />
normal (compared to nontransformed fibroblasts) or somewhat elevated .<br />
One cell line had a much reduced level of expression . There was no<br />
relation between expression of XRCC1 <strong>and</strong> radiosensitivity nor-between<br />
XRCC1 expression <strong>and</strong> the repair of DNA single-str<strong>and</strong> breaks (as<br />
measured by DNA alkaline elution in a subset of the 25 tumor cell<br />
lines) . Therefore, variations in the expression of XRCCl do not<br />
underlie differences in human tumor radiosensitivity or the repair of<br />
radiation-induced DNA damage . We are presently analyzing SCE frequency<br />
<strong>and</strong> monofunctional alkylating agent sensitivity in these lines .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 53<br />
Notes
54 1989 EMS Abstracts 150<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes ANEUPLOIDY DETECTION BY ANALYSIS OF INTERPHASE NUCLEI USING CHROMOSOMESPECIFIC DNA<br />
PROBES . D .A. Eastmond <strong>and</strong> D. Pinkel, Lawrence Livermore National Laboratory . LNermore, CA (USA)<br />
Fluorescence In situ hybridization with probes for chromosome-specifio DNA sequences (usually<br />
centromeric) results In brightly fluorescent spots In Interphase nuclei at the position of the target DNA<br />
sequences . The number of spots In a nucleus therefore corresponds to the number of copies of the<br />
chromosome that are present . Eight thous<strong>and</strong> Interphase nuclei from untreated 72 hr lymphooyte<br />
cultures were examined following hybridization with probes for chromosomes 1, 7 . 9 or 17 . The<br />
frequencies of nuclei containing 0, 1, 2, 3 <strong>and</strong> 4 hybridlzation spots were 0 .0024, 0.079, 0.915,<br />
0 .0024 <strong>and</strong> 0 .001, respectively . Based on these frequencies, scoring 1000-2000 cells should allow<br />
detection of aneuplold cells with a 0 .013 frequency of hyperdiploldy or a 0 .11 frequency of hypodiploidy<br />
for a specific chromosome of interest (a - 0.05, S- 0.80) . This difference In test sensitivity Is related<br />
to the higher frequency of cells with one apparent spot . A comparison of the ratio of spot to nuclear area<br />
in the two dimensional Images used for this analysis Indicates that an overlap of the two spots probably<br />
accounts for the high frequency of apparent monosomy observed in normal cells . Using a chromosome 9<br />
probe, colchicine (0 .1 µM), vincristine (0.06 µM) <strong>and</strong> DES (30 µM) treatments were shown to Induce<br />
hyperdipiokiy for this chromosome at frequencies of 0 .05, 0.02 <strong>and</strong> 0 .36, respectively. The use of this<br />
technique should facilitate a more rapid Identification of aneupioidy-Inducing environmental agents .<br />
Work performed under the auspices of the U .S . Department of Energy by the Lawrence Livermore<br />
National Laboratory under contract number W-7405-ENCi-48 with additional support from the<br />
Alex<strong>and</strong>er Hollaender Postdoctoral Fellowship (D .A.E.) .<br />
ANTIMUTAGENIC ACTIVITY OF VEGETABLES HARVESTED DURING DIFFERENT CONDITIONS .<br />
J . Ebata, <strong>and</strong> T . Inoue, Osaka City University, Osaka (JAPAN)<br />
151<br />
By employing streptomycin-dependent strain SD100 of Salmonella typhtmurtum (T .Kada<br />
K . Aoki, <strong>and</strong> T . Sugimura,Envtronmental <strong>Mutagenesis</strong> 5,pp .9-15(1983)),the antimutagenicity<br />
of homogenates of fresh vegetables has been examined for AF2 . In this paper,<br />
the influences of the growing conditions on the antimutagenicity of 5 kinds of<br />
vegetables cultured in open fields of Agricultural Experiment Station on Nara Prefecture<br />
were studied . The antimutagenic activities were compared with vegetables<br />
having suitable maturity <strong>and</strong> nearly of the same size . The antimutagenicity of<br />
spinach, 8ptnacia oleracea L .cv .'Orion' increased summer< autumn< winter . The activity<br />
of cabbage, Brassica oleracea L . var . capltata L . cv .'Shutoku' was more potent<br />
during the autumn harvest than in summer <strong>and</strong> more in the outer layer than the inner<br />
for both harvests . In the case of eggplants, Solanun aelnyena L . cv .'Senryou', the<br />
maximum activity was found in September during harvest time (July - October) . The<br />
activity of cucumbers, Cucumis sativus L . cv .'Ryokusui' <strong>and</strong> 'Sachikaze' increased in<br />
the order of low
153<br />
Clastogenic effects of ethyl-diamminedichbroplatinum in mice .<br />
1 .Induction of ehromosomal aberration in Bone marrow cells .<br />
A .E1-Tarras , A,N,Sharaf <strong>and</strong> N,A .Abdalla,<br />
Dept . of Genetics, Fac . of Agric ., Cairo University, Cairo, Egypt .<br />
The in vivo clastogenic effect of the anticancer drug ethyldiamminedich)<br />
.roplatinum was investigated as chromosomal abersations<br />
(CA) in the bone marrow cells of mice . Studies was carried out on F1<br />
mice of the cross C3HX101 aged 12-14 weeks <strong>and</strong> weighted 25-30 gm .<br />
The drug concentrations were 1,2 .5, 5 .0 .10,15 <strong>and</strong> 20 mg/kg at<br />
duration time of 6,12 <strong>and</strong> 18 hours <strong>and</strong> the number of cells tested per<br />
group is 500 cells . Results showed that 10 mg/kg dose was lethal<br />
between 3 <strong>and</strong> 6 days while 15 <strong>and</strong> 20 mg/kg doses lethality oooured<br />
within 24 hours . The percentage of cells containing CA without gap<br />
for the doses 1,2,5 <strong>and</strong> 5 mg for 12 hours were 2,25%, 5 .25% <strong>and</strong> 8 .8%<br />
respectively. However at 5 mg dose the CA persentage was 4 .2 at 6<br />
hour interval while it was 2 .2, at 18 hours . These results indicate<br />
that the clastogenicity of ethyl-platinum depends upon the stage of<br />
cell cycle (S .p?iase) <strong>and</strong> it had a dose-dependant . Calculting the<br />
doubling dose (DD) for clostogenic effects of ethyl-platinum in BM<br />
was 0 .5 mg/kg . (Y a 0 .8 + 1 .6 D) .<br />
154<br />
POTENTIATING EFFECTS OF CAFFEINE ON MUTAGENICITY AND TERATOGENICITY<br />
OF ALKYLATING AGENTS . M .M . E1-Zawahri <strong>and</strong> L .K . A1-Ghaith, Department of<br />
Zoology, Faculty of Science, Kuwait University (Kuwait) .<br />
A set of experiments was carried out to study the effect of caffeine<br />
on the mutagenic <strong>and</strong> carcinogenic activities of EMS <strong>and</strong> MMS . Males<br />
of D. melanogaster were fed or injected with 0 .5% caPfeine in sa11ne~~<br />
before or after their treatment with a solution of 0 .1 mM EMS or M`15<br />
in 10% sucrose for a period of 48h . The effects studied were dominant<br />
lethals <strong>and</strong> sex-linked recessive lethals . Pre-treatment with caffeine<br />
injection revealed a significant increase in the frequency of dominant<br />
lethals but not of sex-linked recessive lethals induced by EMS or MMS .<br />
Post-treatment of males with caffeine injection did not alter the frequency<br />
of either of the two types of lethal mutations induced by MMS<br />
or EMS . uhen 0-10h old males emerged from lardae reared on agar containing<br />
0 .5% caffeine were treated with a solution of 0 .1 mM EMS or MMS in<br />
10% sucrose, the frequencies of both dominant- lethals <strong>and</strong> sex-linked<br />
recessive lethals significantly increased than of those untreated with<br />
caffeine . These results indicate that caffeine may inhibit the repair<br />
mechanism(s) of genetic damage induced by alkylating mutagens <strong>and</strong> this<br />
may increase the mutagenic <strong>and</strong> teratogenic potentialities of such chemical<br />
agents .<br />
155<br />
AUTOMATION OF SCREENING ASSAYS FOR DNA DAMAGING AGENTS WHICH INDUCE THE SOS<br />
RESPONSE . R .K . Elespuru . Basic Research Program, NCI-Frederick Cancer<br />
Research Facility, Frederick, MD 21701 .<br />
The induction of the SOS response in E . coli is a physiological response to<br />
DNA damage in which 20 or more genes are turned on . Unlike single gene<br />
mutation, SOS induction is not a rare event <strong>and</strong> may be sean to occur in the<br />
majority of an induced population . SOS induction may be monitored biochemically,<br />
therefore, in a matter of hours after the event . Monitoring of<br />
SOS-inducible genes has been facilitated by the fusion of these genes to jME,<br />
the product of which, B-galactosidase, is expressed upon induction <strong>and</strong> is<br />
easily monitored . Among SOS-inducible genes which have been fused to JM7,<br />
<strong>and</strong> used for screening assays are lambda phage, &U <strong>and</strong> yQy . Two types of<br />
colorimetric substrate-cleavage assays for B-galactosidase have been developed<br />
which may be automated for screening purposes . One uses a soluble substrate ;<br />
a miniaturied version of this quantitative assay may be performed in a∎ingle<br />
microtiter well <strong>and</strong> may be automated using dispensers <strong>and</strong> plate readers . The<br />
other, a spot test assay, uses a substrate generating an insoluble reaction<br />
product ; this assay may be run like a semi-quantitative •dot blot• on petri<br />
dishes to a dilution endpoint . For screening, several hundred samples may be<br />
processed simultaneously on 243mm bioassay plates containing lawns of bacteria<br />
<strong>and</strong> activating enzymes . Research sponsored by the National Cancer Institute .<br />
DHHS, under contract No . NO1-CO-74101 with Bionetics Research, Inc .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 55<br />
Notes
56 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes ALTERATIONS IN l41NG-INDUCED MUTATIONS AT SPECIFIC LOCI IN E• COLI i1U3610 UNDER<br />
CONDITIONS OF THE "ADAPTIVE RESPONSE" . R .K . Elespuru <strong>and</strong> L .L . Stupar . Basic<br />
Research Program, NCI-Frederick Cancer Research Facillty, Frederick, MD 21701 .<br />
156<br />
Treatment of EU coli with low concentrations of alkylating agents results in the<br />
induction of the "adaptive response", an alkylation damage-specific DNA repair<br />
response . Increased levels of two enzyses, an 0s-alkylguanine alkyltransferase, <strong>and</strong><br />
a 3-aethyladenlne glycosylase, are associated with this response . Among guanine<br />
lesions, the transferase is specific for, <strong>and</strong> removes stoichiometrically, alkyl<br />
groups at the 0s position . We have exmined the spectrum of mutations lnduced by<br />
N-methyl-N'-nitro-N-nitrosoguanidine (NNNG) at 8 monitorable sites in E . coli IJU3610<br />
under normal <strong>and</strong> adaptive conditions . The level of mutations at three monitorabie<br />
GC-AT transition sites was reduced by 90-95% under adaptive conditions, implicating<br />
the 0s-methylguanlne lesion as the causative agent in the generation of these<br />
mutations . A substantial loss of mutants was also observed at two putative AT•GC<br />
transition sites, consistent with the existence of 04-aethylthyaine as a premutatlonal<br />
lesion at these sites (0'-aethylthyaine has been reported to be a<br />
substrate of the transferase) . Little or no change was observed in mutation<br />
frequency at two TA+AT transversion sites . Increases at these sites might have been<br />
expected via glycosylase removal of 3-aethyladenine lesions <strong>and</strong> generation of<br />
apurinic sites . However, unlike the transferasa, th.re is a high constitutive level<br />
of 3-MA glycosylase in normal E . coli . Action of the induced enzyme could have been<br />
masked by the constitutive activity . Research sponsored by National Cancer<br />
Institute, DHHS, under contract No . NO1-CO-74101 with Bionetics Research, Inc .<br />
157<br />
SOLl181LQATION OF PARTICULATE CHROMIUM COMPOIlnIDS AND tTS RELEVANCE TO CYTOTOXICITY<br />
AND MORPHOLOGICAL TRANSFORMATION OF SYRIAN FNMSTER EMBRYO (SHE) CELLS.<br />
Z . Elias, O . Poirot, M .C . DaniBre, F. Terzetp, O . Schneider <strong>and</strong> F . Baruthio, I .N .R .S .,<br />
54501 V<strong>and</strong>oeuvre (France)<br />
From previous experiments concerning water-Insoluble or poorly soluble Cr(VI) compounds,<br />
we have found differences In cytotoxiclty <strong>and</strong> transforming potency among some of them . In the<br />
present study we examine the possible relevance of the extracellular solubilization <strong>and</strong><br />
intracellular level of Cr accumulation to cytotoxicity end morphological transformation of SHE<br />
cells Induced by particulate Cr compounds . Ca, Sr, Zn <strong>and</strong> Pb chromatea, of inedium, slight <strong>and</strong><br />
scarcely water solubilities, were tested . In two parallel experiments, the cells were treated with<br />
supernatants or with suspensions of the Cr compounds . The cloning efficiency <strong>and</strong> the<br />
transformation frequency for each compound were determined after 7 days of exposure .<br />
Measurements of Cr In complete medium alone . In cell culture conditions <strong>and</strong> In counted cells were<br />
made by electrothermal atomic absorption spectrometry . The results showed that : (1) Incubation<br />
In cell culture conditions significantly increased the solubilization process ; (2) Intracellular Cr<br />
concentration was directly related to Cr treatment concentration ; (3) oytotoxidty was dependent<br />
on the extracellular solubillzed Cr ; (4) transformation frequency induced by Ca, Sr <strong>and</strong> Zn<br />
chromates correlated to the intracetlular soluble Cr concentration ; (5) Pb=• lons could play a<br />
role In transforming activity of Pb chromate . The results suggest that the oytotoxicity <strong>and</strong><br />
transformation are distinct processes <strong>and</strong> depend, among other factors, on the stte <strong>and</strong> the kinetics<br />
of particle dissolution .<br />
158<br />
EVALUATION OF THE BIOLUMINESCENCE ASSAYS AS SCREENS FOR GENOTOXIC CHEMICALS .<br />
Eugene Elmore, NSI Technology Services Corporation, P .O . Box 12313, Research Triangle<br />
Park, North Carolina 27709<br />
The need for rapid <strong>and</strong> cost efficient screens for muteyens <strong>and</strong> other toxicants has<br />
increased dramatically over the past few years . This increase is due In part chemical<br />
manufacturing <strong>and</strong> the need for monitoring of effluents <strong>and</strong> clean up activities at hazardous<br />
waste sites . The bioluminescence test was first proposed for screening<br />
genotoxic agents by Ulitzur et al . (Mutat . Res . 74, 113-124, 1980) . Bioluminsscence<br />
tests measure the ability of the test chemicals to restore luminescence to dark<br />
mutants of various Photobacterium species, probably by derepression of the luminescence<br />
operon . A variety of chemicals with known mutagenic or carcinogenic activity<br />
have been evaluated <strong>and</strong> the bioluminescence assay has been shown to be responsive to<br />
direct mutagens lncluding point <strong>and</strong> frameshift mutayens, DNA-damsplny agents, DNAintercalating<br />
agents, <strong>and</strong> DNA synthesis Inhibitors . The results correlate very well<br />
with the published data obtained with the Ames assay . The results of a coded validation<br />
study using chemicals provided by the National Toxicology Program In the<br />
Microbics Mutatox11 Assay, which uses dark mutants of the luminous bacteria, P . phosphoraeum,<br />
will be reviewed .
159 1989 EMS Abstracts<br />
Notes<br />
VALUE-OF-INFORMATION ANALYSIS OF TESTING STRATEGIES . F .K . Ennever, H .S . Rosenkranz,<br />
L .B . Lave, <strong>and</strong> G .S . Omenn, Case Western Reserve University . Clevel<strong>and</strong> . OH (USA),<br />
Carnegie-Mellon University . Pittsburgh . PA (USA), <strong>and</strong> University of Washington .<br />
Seattle . WA (USA) .<br />
The goal of reducing the incidence of cancers caused by environmental exposures<br />
requires strategies for identifying hazards among >50.000 chemicals in commerce <strong>and</strong><br />
industry . Our value-of-information framework analyzes strategies for classifying<br />
chemicals as human carcinogens or non-carcinogens . Any classification will have false<br />
negatives (FN) : falsely exonerating a human carcinogen ; false positives (FP) ; falsely<br />
indicting a human non-carcinogen ; true negatives ; <strong>and</strong> true positives . One extreme<br />
strategy treats all chemicals as non-carcinogena, as existing chemicals generally are<br />
treated ; FP = 0 <strong>and</strong> FN = c, where c is the proportion of carcinogens among those<br />
chemicals . A second extreme strategy treats all chemicals as carcinogens ; PN - 0 <strong>and</strong><br />
FP = 1 - c . A third strategy classifies chemicals on the basis of results of<br />
structure-activity analyses, short-term tests, <strong>and</strong>/or rodent cancer bioassays ; PN -<br />
(1 - p)c, where p is the weighted sensitivity of the tests, <strong>and</strong> PP =(1 - q)(l - c),<br />
where q is the weighted specificity of the tests . Using reasonable estimates of c, p,<br />
q, the cost of testing, <strong>and</strong> the societal costs of PN (needless cancers) <strong>and</strong> FP (loss<br />
of the use of a chemical), our model has shown that in many cases the lifetime rodent<br />
bioassay is not the most cost-effective way to classify chemicals as human carcinogens<br />
or non-carcinogens . Our most recent work has focused on the influence of uncertainty<br />
in parameter estimation <strong>and</strong> on deriving a sequential testing strategy with decision<br />
rules for when to classify a chemical without further testing .<br />
160<br />
COVALENT BINDING TO MICROTUBULAR PROTEINS AS A POSSIBLE CAUSE OF THE ANEUPLOIDY AND<br />
MICRONUCLEUS FORMATION INDUCED BY CARCINOGENIC ESTROGENS AND OTHER CARCINOGENS .<br />
B . Epe, U .H . Harttig, D . Schiffmann, <strong>and</strong> M. Metzler, Institute of Toxicology, University<br />
of Wiirzburg, Versbacher Strasse 9, D-8700 Wurzburg, Fad . Rep . Germany .<br />
Certain estrogens, e .g . diethylstilbestrol (DES), transform Syrian hamster embryo<br />
fibroblasts neoplastically in vitro without producing detectable DNA damage or structural<br />
chromosomal abnormalities . Instead, induction of aneuploidy <strong>and</strong> micronucleus formation<br />
was observed <strong>and</strong> was associated with cell transformation . In order to elucidate<br />
the biochemical mechanisms of aneuploidy induction, we have studied the interaction of<br />
several radioactively labeled estrogens <strong>and</strong> their metabolites with tubulin in a cellfree<br />
system . We observed highly specific covalent bindiijg to the carboxy terminal<br />
domain of /1-tubulin of those estrogens which undergo peroxidase-mediated quinone formation<br />
(DES, indenestrol A <strong>and</strong> the catechol estrogens 2-hydroxyestradiol <strong>and</strong> 2-hydroxy-<br />
17a-ethinylestradiol) . Estrogens which do not form peroxidative quinone metabolites<br />
(hexestrol, estradiol, 17K-ethinylestradiol) did not bind covalently . Covalent binding<br />
was not inhibited by sulfhydryl reagents nor by colchicine, podophyllotoxin, taxol or<br />
vinblastine . Specific covalent binding to tubulin was also obtained with hydroquinone,<br />
a major metabolite of the non-mutagenic carcinogen benzene, upon peroxidase-mediated<br />
oxidation . Electron microscopy of the microtubulee formed from tubulin coupled to DES<br />
or hydroquinone shoved clear abnormalities indicating that covalent tubulin binding<br />
has functional consequences . Therefore, we propose covalent binding to tubulin as an<br />
early biochemical lesion in the mechanism of aneuploidy induction <strong>and</strong> neoplastlc cell<br />
transformation by non-DNA-damaging carcinogens .<br />
Supported by Deutsche Forschungsgemeinschaft <strong>and</strong> BMJFFG .<br />
161<br />
SELECTION OF AN EAPERIMENTAI. PROTOCOL FOR SCREENING TEST AGENTS IN THE MOUSE BONE<br />
MARROW MICRONUCLEUS TEST . G .L . Erexsonl, J .L . Hustonl*, R .M . Boehml*, D . Gulati2,<br />
<strong>and</strong> M .D . Shelby3 . l<strong>Environmental</strong> Health Rea . 6 Testing, Inc ., P .O . Box 12199, RTP, NC<br />
27709 <strong>and</strong> 22514 Regency Road, Lexington, KY 40503 ; 3NIERS . NTP, Box 12874, RTP, NC .<br />
Due to methodological variations in the mouse bone marrow micronucleus (!D1) test ;<br />
one universal treatment protocol for in vivo chemical exposure is desirable . A common<br />
protocol among laboratories would simplify comparison of MN data . Mitomycin C(14/C)<br />
<strong>and</strong> 7 .12-dimethylbenzanthracena (DMBA) were tested i .p . at doses of 0, 0 .5 . 1, <strong>and</strong> 1 .3<br />
mg MMC/kg <strong>and</strong> 0, 25 . 50, <strong>and</strong> 100 mg DMBA/kg using three different protocols . Male<br />
B6C3F1 mice (9 to 14 weeks of age, 5 mice/dose) were treated with either (1) one<br />
exposure with harvests for bone marrow polychromatic erythrocytes (PCEs) at 24, 48, or<br />
72 h post-treatment ; (2) two exposures separated by 24 h with harvests at 24 or 48 h<br />
after the second treatment ; or (3) three exposures separated by 24 h with one harvest<br />
time at 24 h after the third treatment . For comparison, DMBA was administered also by<br />
gavage but only for the three-treatment protocol at the same doses used for the 1 .p .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
57
58 1989 EMS Abstracts<br />
Notes injections (0, 25, 50, <strong>and</strong> 100 mg/kg) . Bone marrow smears were stained in acridine<br />
orange (pH - 7 .4) <strong>and</strong> 2000 PCEs/souss were scored for NN-containing PCEs . The percent<br />
PCEs was determined by counting 200 consecutive PCEs <strong>and</strong> normochromatics . Statistical<br />
analyeas revealed that the three-treatment protocol vas superior . Therefore, this<br />
protocol was selected by NIP for use in in vivo rodent bone marrow MN testing .<br />
Additional experiments were done using this protocol to select a positive control dose<br />
for both MMC <strong>and</strong> DNBA to be used in testing coded compounds . The positive control<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
doses chosen are 0 .2 mg l4lC/kg <strong>and</strong> 12 .5 mg DNSA/kg which lnduce about 8!Q4-PCEs/3000<br />
PCEs . .[This research was supported by a contract fro's NTP, /NOI-ES-85208) .<br />
162<br />
DEVELOPMENTAL AND GENETIC TOXICITY OF SHORT-CH4IN CARBOXYLIC ACIDS .<br />
A . Esposito, G . Corsale, G .G . Giordano, <strong>and</strong> 0 . Pagano<br />
Istituto Nazionale Tumori, 1-80131 Naples, Italy<br />
The genetic <strong>and</strong> developmental toxicity of acidic pollutants was reported previously<br />
(Pagano at al ., 1985a,b) ; a weak acid-induced teratogenic action was reported by Nau &<br />
Scott (1986) on mammalian embryos, whereas the amidic analogues tested were ineffec-<br />
tive on embryogenesis . A series of short-chain carboxylic acids (as sodium propionate,<br />
malonate, <strong>and</strong> valproate), <strong>and</strong> their amidic analogues have been investigated for their<br />
action on sea urchin (P .lividus) early development <strong>and</strong> fertilizatlon . Embryos were exposed<br />
to acids (or amides) at levels ranging from 10-'M to 10-'M, without any detect-<br />
able shift of medium pH ; the endpoint consisted of changes in the frequencies of :<br />
a) developmental defects, <strong>and</strong> b) cytogenetic abnormalities . Sperm pretreatment experi-<br />
ments led to the following outcomes : 1 . changes in fertilizstion success ; ii . alte :a-<br />
tions of offspring quality, including mortality, developmental defects, <strong>and</strong> cytogenet-<br />
ic abnormalities . The results showed that the acids exerted developmental toxicity at<br />
levels ranging from 10-'to l0-°M, whereas their amidic analogues were ineffective up<br />
to 10-= M . Cytogenetic analysis <strong>and</strong> offspring quality confirmed acid-induced genetic<br />
damage, as reported previously . The data provide further evidence for the role of<br />
acidic pollutants <strong>and</strong> drugs in affecting cell division <strong>and</strong> differentiation . (Supported<br />
by the Italian Ministry of Health) .<br />
(;ESiETIC REST FOR 7[PM P%WI'E17S ATID COCAACIIIO(04.S<br />
Rudolf Fahrig, Department of Genetics, Frauntafer-Institut f(ir Tbotikologie ia ;d<br />
Aerosolforschung, Ha:ne%rer 61, F .R. Gezmany<br />
163<br />
Escpariments with yeast <strong>and</strong> with mioe show that tumor prcmoters <strong>and</strong> cocarcinogens are<br />
genetically active when given in eombinatien with a nutagen . The activity is indeperr<br />
dent of the nutagen/carcinogen used but specific for a given eocarcinogen or tumor<br />
praroter . 4fiere are striking similarities between the eacurrenoe of specific genetic<br />
effects <strong>and</strong> specific effects in carcinogenicity tests :<br />
1 . Cocarcinogens were ornutagenic .<br />
2 . 2lanor promoters which are anticarcinogenic if given simultaneously with the carcinogen<br />
were oorecaabinogenic <strong>and</strong> antinutagenio .<br />
3 . Rtiamr promoters which can bA transforns+d into eocur.irwgcets reverted their genetic<br />
effects fsrom corecambinogenicity to oortutagenicity upon metabolic activatien .<br />
4 . Substances which are t:mor promoters as well as cocarcinogens were also comecaebinogens<br />
<strong>and</strong> cenutagens .<br />
The hypothesis presented offers plausible explanatiens for the meny divergent effects<br />
of these substances in carcinogenicity tests . It therefare seems desirable to establish<br />
the test procedure deacribed as stazt-tean tests for turor promoters <strong>and</strong> eocancisrogens<br />
.<br />
MULTIPLE ENOPOINT MJTATIONAL ANALYSIS IN TFE MOUSE<br />
Jack Favor, GSF-Institut filr Slugetiergenetik, D-8042 Neuherberg, Germany, F .R .<br />
164<br />
Two main classes of mutational event from the wildtype allele are possible, loss<br />
or gain type mutations . The type of mutational event is important in determining the<br />
mutation rate observed <strong>and</strong> the effects of such mutant alleles when they occur in a<br />
diploid, r<strong>and</strong>omly mating population. A loss event represents the loss of functional<br />
gene product . Such a mutation may result from a wide spectrum of DNA alterations<br />
ranging from deletion to point mutation, <strong>and</strong> should have no phenotypic effect when<br />
occurring as a heterozygote . A gain type mutational event represents an abnormally<br />
functioning gene product, is likely due to a defective gene product or a misregulated<br />
50869 3570<br />
t
1989 EMS Abstracts<br />
normal gene product, <strong>and</strong> would result in an altered phenotype when occurrirg as a Notes<br />
heterozygote . Comparative mutagenicity data in the mouse are available for four<br />
genetic endpoints in which recovered mutations are genetically confirmed : recessive<br />
mutations at seven specific loci, dominant cataract alleles, enzyme electrophoretic<br />
variants <strong>and</strong> enzyme activity alleles . Results indicate the mutation rate to be an<br />
order of magnitude higher for those genetic endpoints which screen for loss events<br />
than for those genetic endpoints which screen for altered gene products . Further, the<br />
ratio gain/loss mutational yield is increased for ENU mutagenic treatment as compared<br />
to radiation, which is due to a shift in the spectrum of DNA alterations to point<br />
mutations as compared to mainly deletions by radiation . Thus, the observed<br />
sensitivity to mutation induction is dependent upon the type of mutational event<br />
screened as well as the type of mutagenic treatment employed .<br />
165<br />
THE ROLE OF PHOTOSYNTHESIS IN PROMUTAGEN ACTIVATION BY PLANT SYSTEMS . G . Fedorvicz,<br />
J . Day, G . J . Gentile <strong>and</strong> J . M . Gentile . Hope College, Holl<strong>and</strong>, MI ., 49423 (USA) .<br />
We investigated the ability of photosynthetic <strong>and</strong> non-photosynthetic cotton<br />
suspension cell cultures for the ability to activate 2-aminofluorine (2AF) <strong>and</strong> a<br />
contaminant of 4-nitro-o-phenylenediamine (NOPX) into forms mutagenic to Salmonella<br />
tyhpimurium using the plant cell/microbe coincubation assay . The activation potential<br />
of each cell line was compared under varying light conditions both in the presence <strong>and</strong><br />
absence of a potent inhibitor of photosynthesis (3-(3,4-dichlorophenyl)-1,1-demethylurea<br />
(DCMU) <strong>and</strong> as a function of the plant-cell growth cycle . NOPX was activated<br />
to the same degree by both cell lines under both light <strong>and</strong> dark regimes, <strong>and</strong> late-log<br />
phase cultures proved most effective for activation . DCMU, which is not mutagenic to<br />
Salmonella, had no effect on NOPX activation by either cell line under any test<br />
conditions . Experiments with 2AF indicated that this compound was preferentially<br />
activated by non-photosynthetic cells which were harvested from early to mid-logphase<br />
of growth, independent of DCMU treatment . In general, photosynthetic cells<br />
proved non-responsive under all treatment conditions . We are continuing to intestigate<br />
the activation potential of our photosynthetic cell line under more stringent<br />
photosynthetic-inhibitory conditions . If is feasible that calls which are actively<br />
photosynthesizing, or cells that maintain an intact photosynthetic apparatus, may<br />
not rely on certain enzymes complexes for general metabolic functions (e .g .,P450related<br />
enzymes) . Therefore, substrates which require such enzyme complexes for<br />
activation (2AF) would not be metabolized while substrate+e which require other enzyme<br />
complexes (NOPX) would be activated by these cells . Support from NSF Grt .BB5-8712566 .<br />
166<br />
MUTAGENICITY OF BURNT GUN PROPELLANTS. J.S . Felton, P. Lewis, M .G . Knize,<br />
<strong>and</strong> G . Millerl, Biomedical Sciences Division <strong>and</strong> lHazards Control Dept ., Lawrence Livermore<br />
National Laboratory, P .O . Box 5507, Livermore, CA 94550 .<br />
The use of the Ames/Salmonella assay as a workplace monitoring method is a long-st<strong>and</strong>ingpractxx at<br />
LLNL. This practice has led to the discovery of very mutagenic soot in <strong>and</strong> around a 4 inch test gu<br />
Examination of a rag used to clean the barrel of the gun revealed 87,000 TA98 (+S9) revertants/30 cm~<br />
of the cloth. Analysis of the residue in the gun breech after fuing 2140 g HPC95 <strong>and</strong> 34 g H870<br />
propellants showed 24 x 106 rev/g residue . Open-air burning of HPC95, H870, M-1, <strong>and</strong> M-6<br />
propellants (all of which are not mutagenic before burning) gave 3800, 3500,16,600, <strong>and</strong> 5700 revhng<br />
(-S9) of residue . The presence of S9 reduced the response by -50% . Samples from cloth2ttsed to clean a<br />
22 caliber pistol <strong>and</strong> rifle <strong>and</strong> a 12 gauge shotgun showed 289, 1494, <strong>and</strong> 2375 rev/6 cm (TA98, +S9),<br />
respectively . Two S9 requiring <strong>and</strong> 1 direct acting mutagenic components have been separated by<br />
HPLC . Subsequent mass spectral anal sis <strong>and</strong> NMR analysis have not given additional structural<br />
information due to small amounts of purified material available after the final clean-up . Estimated specific<br />
mutagenic activity is >100,000 rev/µg . Chromatographic behavior <strong>and</strong> specific Ames/Salmoneila<br />
response in TA98 suggest aromatic amutes are responsible for the mutagenic activity . It appears that the<br />
propellant components, nitrocellulose, nitroglycerine, diphenylamine, potassium nitrate, phthalates, <strong>and</strong><br />
graphite (present in various concentrations), when heated may produce nitroarornatics in the open-air<br />
burning <strong>and</strong> aromatic amines in the reduced atmosphere of the guns ; not unlike production of potent<br />
mutagenic aromatics from diesel exhaust <strong>and</strong> cooked meat . (Work done under auspices of the U .S.DOE<br />
by LLNL under contract No . W-7405-ENG-48) .<br />
167<br />
THE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY : BIOLOGICAL DOSIMETRY IN CANCER PATIENTS<br />
FOLLOWING IN VIVO FRACTIONATED EXPOSURE TO IONISING RADIATION . Fenech M .*, Denham J .**<br />
Francis W .** <strong>and</strong> Morley A .A .*** . *Bianedicine <strong>and</strong> Health Prog ., Australian Nuclear<br />
Science <strong>and</strong> Technology Organisation, New Illawara Rd, Lucas Heights, NSW, Australia ;<br />
**Dept . of Radiotherapy, Royal Adelaide Hospital, Nth . Terrace, Adelaide, SA,<br />
Australia ; *** Dept . of Haematology, Flinders Med . Centre, Bedford Park, SA, Australia .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
59
60 1989 EMS Abstracts<br />
Notes A prospective study was campleted on micronucleus (FP() induction in cytokinesisblocked<br />
(CB) lymphocytes in eleven cancer patients undergoing radiotherapy . This<br />
study was performed to evaluate the CB micronucleus assay as an in vivo dosimeter .<br />
Measurements before .during <strong>and</strong> at the end of therapy showed that-TFere was a clear<br />
dose-related response in MM induction in all the patients <strong>and</strong> that the extent of<br />
induction (between 59 .0 <strong>and</strong> 578 .0 MN/1000 CB cells) was directly proportional to the<br />
estimated equivalent whole body dose . Measurements were also performed after completion<br />
of therapy to estimate the rate of decline in MH frequency . These values were<br />
expressed as a percentage of the values at the end of therapy with the results<br />
showing that MN frquencies (mean + 1 s .e .) dropped to 91% (+ 11) after 3 months, to<br />
72% (+ 13) after 6 months, to 57~(+ 10) after 12 months . Measurements made 24<br />
months post-treatment showed that MR frequencies only returned to base-line levels<br />
in two of the four patients studied . The other two patients retained very high<br />
MN frequencies (212 .9 <strong>and</strong> 223 .3 MN/1000 CB gells) . These reults suggest that a state<br />
of chromosome instability (loss or breakage) may have been induced In the surviving<br />
lymphocytes of the latter patients .<br />
168<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
ANTIb1LfA0ENIC eCTIVITISS OF CHLOHOPHYLLIh<br />
Pko-ohaen 'r`en,YLn-Jiob Ch<strong>and</strong>,Ji ..g-A1 ..6 Chu,Xiao-yinS Cha_~, <strong>and</strong> Luaaa<br />
Fac~,Shar.g~i ..i lusL :Lui:e or Oecupa, .oual health in Chem .oal Indusi,ry<br />
Shsnghai(PK Chi.na)<br />
6hlorophyllin,the sodium <strong>and</strong> copper salt of ohlorophyll,was tested<br />
for• its ability to Ii,hibit the mutagenic activitv of soc•s chericsl<br />
pr•oducts(2-1 :oroartobenzimidezol, 0-Nitrouniline, 0-Phsnvlenedismine),<br />
extrations of fr :ed beef <strong>and</strong> <strong>and</strong> red wine ooneentratfons of tap<br />
wai~r #:nd knov.n mutagens(Duunomyoin, 2-AFj in TA98 of SaiTonslla<br />
typhimurium . Complete inhibition of these were obtainel with l .cSm_¢<br />
cf chlorophyllin per plate . The antimutaRenlc activit,y of chlorcphyllin<br />
was heat-stuble . The results indicate that cn3orsphyllin is<br />
potentially useful as sin ant .'mutagenic agent .<br />
169<br />
NEW Mf7D(1LAIVRS OF MMOXICITY IN YFAST CIIJ .S<br />
L.R . Fbrguson <strong>and</strong> B .C . Baguley, Cancer Jbsearch Laboratory. University of Auckl<strong>and</strong><br />
Medical Sohool, Private Bag, New Zeal<strong>and</strong> .<br />
We havee previously shown that verapsmil, a calciun antagonist which is )nown to<br />
reverse multidzug resistance in memnalian oe11s, reduoes the ability of a nnrber<br />
of DNA intercalating agents to induoe mitochondrial "petite" nutations in yeast<br />
eells . We have developed an assay system enploying Sacchn:nrtyces oeaevisiae D6 <strong>and</strong><br />
a strongly basic analogue of the antileukemia agent amsacrine to search for other<br />
conpounds which reduce mitochondrial nutagenesis . 'Petite' induction by the amsacrine<br />
analogus can be reduced fram 80% to less than 104 by the oo-addition of appropriate<br />
concentrations of some cartpounds . ZMieen 80, chloroquine <strong>and</strong> cyclosporin A<br />
were found to be highly effective . The most likely axplanation for the <strong>and</strong>ulation<br />
of mutagenic activity is through the inhibiticn of m,itocriondrial nptake of the DNA<br />
intercalating mutagen . This principle could be of inpox'tanoe in liroitiag mitochondrial<br />
mutagenesis in rtemmalian cells .<br />
170<br />
DETECTION OF GENE MUTATIONS IN MOUSE SPERM WITH POLYMERASE CHAIN<br />
REACTION (PCR) . G . Ficsor, L . C . Ginsberg J . F . Klepetka <strong>and</strong> T . P .<br />
McManus . Western Michigan University, Kalamazoo, MI (USA)<br />
To detect base-pair substitutions <strong>and</strong> small deletions in sperm, PCR<br />
was used to amplify a 228 base-pair segment of the PGf:2 gene of mice .<br />
The amplified DNA ran as a single baud on a 1 .4% agarose gel<br />
corresponding to its expected size . Dot blot hybridization demonstrated<br />
that we could detect a single base pair difference between the PGK-2a<br />
<strong>and</strong> PGK-2b alleles by binding of the appropriate 21 mer probe under<br />
stringent conditions . To detect a rare event such as a new gene mutation<br />
in a single sperm amongst thous<strong>and</strong>s <strong>and</strong> even millions of non-mutant<br />
sperm the PCR alone is of limited help since it amplifies both the<br />
mutant <strong>and</strong> non-mutant DNA resulting in more DNA, with the mutant<br />
sequence still a minor component . We attempted to solve this problem<br />
by restriction digesting sperm DNA before amplification with Hinc II<br />
which cuts the normal DNA sequence to be amplified in two . If a basepair<br />
substitution mutation or small deletion is present in the }(incll<br />
sequence, that target DNA will not be cut by HinclI <strong>and</strong> will be<br />
available for amplification by PCR . As a consequence the amplified DNA<br />
will be enriched for mutant DNA sequences . The amplified DNA then can<br />
be analyzed for the presence <strong>and</strong> amount of mutant DNA sequences .<br />
Supported by NIH grant 1 R15 HD21171-O1A1 <strong>and</strong> by a Western Michigan<br />
University Faculty Research Fellowship <strong>and</strong> Grant .<br />
50869 3572
171<br />
CYTOLOGICALEFFECTS OF ALUMINIUM IN PLANT ROOTS<br />
G . Fiskesjt9, Institute of Genetics, University of Ltatd, Sweden<br />
Structures of a new type ("A1-stnictures") have been discovered in the oyEoplasta of<br />
root cells of certain plants treated with altzainitmt ions (A13+) . Material leaches out<br />
frotn the nuclei particularly of root cap cells, forming oblong structures one in each<br />
cell, eventually dividing into two equal-sized structures, one at each end of the cell<br />
with the nucleus in between . Feulgen/Light Green staining of the Al-structures indicates<br />
that they possibly are connected with IaiA <strong>and</strong> rwcleoli(Fiskesjt5 1983) . Light<strong>and</strong><br />
transtnission electron microscopy of sectioned material of Al-treated cells<br />
yielded new aspects on the structure of cytoplasm <strong>and</strong> cell arambrane(Fiakesj8 et al .<br />
1989, in press) . The current acidification of forest soils induces increase of Al in<br />
solution in the soil . A13+ ions in concentrations around 20 mg/L cause severe damage<br />
to Allitnn roots, <strong>and</strong> concentrations of this strength have actually been fotatd in forest<br />
soil solutions when pH decreases towards 4 . The specific Al-structures which<br />
were first found in Alliua roots after A1-treatments, have later been found also when<br />
Alliun roots were grown in Al-rich forest soil solutiens(Berggren & Fiskesj8 1987) .<br />
Al-structures have been found in two Allium species(oepa <strong>and</strong> schoemphrasum) <strong>and</strong> in<br />
growth-restricted forest roots after A1-treatments(e .g . Picea abies, Fagus silvatica)<br />
(Fiskesj8, in preparation) . Thus, A13+ ions undoubtedly ootttribute to forest damage<br />
by interfering with root cell na:tabolism . The Al-structures may be a general response<br />
of plants to Al . kbether the Al-structures actually oontain Al, <strong>and</strong> whether there is<br />
a oonnection between the formation of the structures <strong>and</strong> the function of the nucleoli<br />
are questions which remain to be answered .<br />
172<br />
DNA-DAMAGE INDUCIBLE GENES IN MAMMALIAN CELLS, Albert J . Fornace Jr., N .C .I .,<br />
N .I .H ., Bethesda, MD 20892 .<br />
Based on the results of others in bacteria <strong>and</strong> yeast, many genes would be expected to be DNA-damage<br />
inducible (DDI) in mammalian cells ; some may represent specific responses to DNA damage, while others<br />
may be general stress responses to cell injury . A variety of mammalian genes, such as metallothionein,<br />
collagenase, c jos, ubiquitin, <strong>and</strong> B-polymerasel, have been found to be DDI by our group <strong>and</strong>/or other<br />
investigators . Most of these examples probably represent general stress responses since they were induced<br />
by unrelated agents such as heat shock <strong>and</strong>/or activators of protdin kinase C. However, B•polymerase<br />
mRNA was found to be specifically induced only by alkylating agents <strong>and</strong> similar agents that produce DNA<br />
damage repaired by a mechanism involving B-polym erasel . The 8-polymerase gene had several properties<br />
in common with bacterial genes that are specifically DDI : low abundance, rapid induction of 2-10 fold, <strong>and</strong><br />
induction specific for DNA damage . An approach to isolate cDNA clones of other such DDI genes was<br />
developed using hybridization subtraction at low ratios of RNA :cDNA2. 49 different cDNA clones were<br />
isolated that coded for transcripts rapidly induced 2-28 fold by UV radiation in Chinese hamster cells2 .<br />
Many of these transcripts were induced only by DNA-damaging agents ; these DDI cDNA clones were<br />
divided into 2 classes. In Class I, only UV radiation <strong>and</strong> other UV-mimetic agents were effective inducing<br />
agents, while in Class II other base damaging agents such as alkylating agents were also inducing agents .<br />
Characterization of individual DDI cDNA clones will be presented including evidence that a Class I<br />
member (DDlA18) encodes a nucleic acid single str<strong>and</strong> binding protein, <strong>and</strong> that several Class II genes are<br />
coordinately regulated <strong>and</strong> may represent members of the same rqulon . These results support the<br />
conclusion that multiple transcripts in mammalian cells are specifically induced by DNA damaging agents,<br />
Ind that their protein products may be involved in the cellular response to such damage .<br />
Fornace AJ. Jr ., Zmudka, B .Z., Holl<strong>and</strong>er, M .C., <strong>and</strong> Wilson, S .H .: Molec. Cell . Biol . 9: 851-853,1989 .<br />
2 Fornace AJ. Jr ., Schakh, H., <strong>and</strong> Alamo, I. Jr.: Proc . NaO . Acad . Sci . USA 85 : 8800-8804,1988.<br />
173<br />
ACCUMULATION OF DNA SINGLE-STRAND BREARS AND POSSIBLY ARTIPACfUAL IIDS RESPONSE IN RAT<br />
HEPATOCYTES BY DIFLOXACIN . F . L . Fort, X . A . Cifone, <strong>and</strong> R . 0 . Curren, Abbott Lahe,<br />
Abbott Park, IL, Razleton Labs, Rensington, MD <strong>and</strong> Microbiological Associates,<br />
Rockville, MI<br />
Difloxacin is a quinolone antibacterial which has been found positive for<br />
unscheduled DNA synthesis (UDS) activity in rat hepatocyte cultures . Skare, et al .<br />
(Mutat . Res . 172 : 77-87, 1986) reported an artifactual UDS response with sodium<br />
fluoride presumably due to precipitahle complex formation involving fluorine ion <strong>and</strong><br />
[3N]thymidine triphosphate . Since difloxacin has low aqueous solubility <strong>and</strong> is difluorinated,<br />
it is possible that the positive UDS response might be artifactual by a<br />
similar mechanism . As a preliminary test of this hypothesis, difloxacin was tested in<br />
a modified UDS protocol in which the culture medium containing drug was filtered prior<br />
to treatment of hepatocyte cultures . Under these conditions difloxacin did not induce<br />
a UDS response even though the drug concentration after filtration was equal or higher<br />
than that when a UDS response was obtained without filtration . Therefore, the UDS<br />
response may be artifactual ; further studies are necessary to prove this . To further<br />
test the mechanism for the UDS response, hepatocyte cultures treated with difloxacin<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 61<br />
Notes
62 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes were analyzed for DNA single-str<strong>and</strong> breakage by alkaline elution . A dose-related<br />
positive response for DNA single-str<strong>and</strong> breaks was obtained . These results indicate<br />
that difloxacin causes an accumulation of single-str<strong>and</strong> breaks in hepatocyte DNA . It<br />
is possible that accumulation of DNA single-str<strong>and</strong> breaks may result from the action<br />
of this drug as a topoisomerase inhibitor, but this accumulation may be unrelated to<br />
the apparently artifactual DDS response .<br />
ATRAZINE AND THE GENOTOXICITY OF ITS METABOLITES<br />
Franekic . J . . G . Hulina . J . Kniewald <strong>and</strong> M . Alabevic<br />
Faculty of Food Technology <strong>and</strong> Biotechnology . Zagreb . Yugoslavia<br />
174<br />
Tne aim of our study was to examine genotoxicity of atrazine <strong>and</strong> its metabolites<br />
deethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine) <strong>and</strong> deisopropylatrazine<br />
(2-chloro-4-ethylamino-6-amino-s-triazine) detected by t .1 .c . <strong>and</strong> gas chromatography,<br />
<strong>and</strong> structurally identified by mass speetrometry in the kidney . brain <strong>and</strong><br />
liver of male rats treated with atrazine .<br />
Experiments were performed with microbial test-systems with Salmonella t himurium<br />
strains TA100 <strong>and</strong> TA98 (plate-incorporation assay <strong>and</strong> preineu a ion met an<br />
with Saccharomyces cerevisiae D7 .<br />
In the plate incorporation assay atrazine was negative in both strains (TA100 <strong>and</strong><br />
TA98) : deethylatrazine was positive in strain TA100 <strong>and</strong> deisopropylatrazine was positive<br />
in strain TA98 . Results of preincubation method indicated that all examined<br />
substances are genotoxic <strong>and</strong> toxic . In S . cerevisiae D7 only deethylatrazine showed<br />
recombinogenic effect. -<br />
175<br />
FIVE COMPOUNDS WITH ANTIVIRAL ACTIVITY TESTED FOR THEIR RESPECTIVE<br />
GENOTOXIC POTENCIES IN THE DROSOPHILA SOMATIC MUTATION AND<br />
RECOMBINATION TEST (SMART) . H . Frei <strong>and</strong> F .E. Wiirgler, Institute of<br />
Toxicology, ETH <strong>and</strong> University of Zurich, Schwerzenbach, Switzerl<strong>and</strong> .<br />
The two potential anti-AIDS drugs azidodeo:ythymidine (AZT) <strong>and</strong><br />
dideoxycytidine (DDC) were tested for their respective genotoxicity<br />
in the Somatic Mutation And Recombination Test (SMART) of Drosophila .<br />
Both nucleoside analogs apparently interfere with DNA synthesis since<br />
mutant twin spots (IDtdh <strong>and</strong> f1ta) as well as single spots (mFih or<br />
fjZ2) were induced in the wing disc cells of animals which were<br />
trans-heterozygous for the two recessive markers . ThA compounds were<br />
fed for 48h to 3rd-instar larvae . Sibs from the same cultures which<br />
were heterozygous for the marker QKh <strong>and</strong> the recombination-suppressing<br />
balancer-chromosome TM3 allowed to evaluate mutagenicity separately<br />
in the absence of recombination . Overall, AZT was 30-50x less<br />
genotoxic than DDC . With DDC, recombination clearly predominated,<br />
whereas with AZT, recombination was only moderately more frequent<br />
than mutation . Three other antiviral agents, i .e . ribavirin, phosphonoformic<br />
acid, <strong>and</strong> particularly acyclovir also had stronger genotoxicity<br />
than AZT . Thus, SMART is not only useful for rapid screening,<br />
but also allows to assess genotoxicity quantitatively <strong>and</strong> to take<br />
into account basically different endpoints .<br />
176<br />
CHARACTERIZATION AND EXPRESSION OF EURARYOTIC GENES REQUIRED FOR NUCLEOTIDE EXCISION<br />
REPAIR :YEAST AS A MDDEL SYSTEM . Errol C . Friedberg, Lee Bardwell, A . Jane Cooper,<br />
Itzik Harosh, Wolfram Siede <strong>and</strong> Jae-Mahn Song, Department of Pathology, Stanford<br />
University School of Medicine, Stanford, CA 94305 .<br />
In the yeast Saccharomyces cerevisias at least 10 genes are involved in the<br />
removal of bulky base adducts . Five of these (RAD1, RAD2, RAD3, RAD4 <strong>and</strong> RADIO)<br />
are absolutely required for damage-specific recognition <strong>and</strong> incision of DNA . These 5<br />
genes have been cloned by phenotypic complementation . Their nucleotide sequences<br />
predict expression of proteins with calculated molecular weights of 126 .2 kDa<br />
(Radl), 117 .7 kDa (Rad2), 89 .7 kDa (Rad3), 87 .1 kDa (Rad4) <strong>and</strong> 24 .3 kDa (Rad10) . The<br />
cloned genes have been tailo4ed into vectors for overexpression in yeast <strong>and</strong> E .<br />
coli . The RAD3 gene is multifunctional . In addition to its role in nucleotide<br />
excision repair RAD3 is an essential gene . Furthermore, certain rad3 mutant<br />
alleles confer a phenotype of increased spontaneous mutation <strong>and</strong> increased mitotic<br />
recombination . Rad3 protein has been purified to apparent homogeneity . The purified<br />
50869 3574
protein is a DNA-dependent ATPase with DNA helicase activity . The role of this<br />
ATPase/helicase in the various RAD3 functions identified remains to be established .<br />
The RAD2 gene is DNA damage-inducible . Following exposure to a variety of DNAdamaging<br />
agents steady-state levels of RAD2 mRNA increase -3-6 fold . Induction is<br />
positively regulated . Cis-acting sequences required for induction have been<br />
identified by deletion mapping <strong>and</strong> mutants have been isolated that are defective in<br />
induction of RAD2 . The amino acid sequences of the RAD10 <strong>and</strong> RAD3 genes share<br />
homology with the human excision repair genes ERCC1 <strong>and</strong> ERCC2 respectively .<br />
Additionally, the RAD10 gene partially camplements the phenotype of mammalian cells<br />
defective in the ERCCI gene . Thus, genes for NER are apparently conserved in<br />
eukaryotes .<br />
177<br />
IN VIVO EVALUATION OF CYCLOACTIVE AND CLASTOGENIC EFFECTS OF BEET ROOT COLORS . N .C . Froes <strong>and</strong> N . V. Garcia,<br />
TEILC-'UNESP, Seo Jose do Rio Preto, S ;o Paulo, Brazil .<br />
The recent development of food industry has made food additives a relevant cause of human cancer,<br />
particularlydue to the autagenic activity of several synthetic food colors . The knowledge of mutagenic<br />
effect of natural food colors gives scientific support for the replacement of synthetic by natural ones .<br />
Two different forms of beet root colors were tested, the form I without nitrate residues <strong>and</strong> the form II1<br />
with nitrate residue . The coloring active principle of both extracts is the betanin pigment which gives the<br />
characteristic beet root purple color . llatagenic effects were tested in vivo . The experiments were carried<br />
out with bone marrow cells of males <strong>and</strong> females Rattus ~norvergi~cus var . star six-seven weeks old, 90-100g<br />
of weight, orally treated with both forms for one wee , w-3tTi- 0 .02 mg <strong>and</strong> 5 .0 mg of betanin per 100 g of body<br />
weight per day ; the bone marrow material was collected at the 8th day . For the bone marrow cell experiments<br />
two different control groups were established : a negative group of untreated animals <strong>and</strong> a positive group<br />
of animals treated with 5 .0 mg of cyclophosphamide per 100 g of body weight 24 hours before sacrifice .<br />
Mitotic index (MI), chromosome anomalies (CA) <strong>and</strong> micronuclei frequencies were recorded . Slides of bone<br />
marrow were analysed in blind tests . There were no differences in MI, micronuclei, <strong>and</strong> CA frequencies, when<br />
the treated samples were compared with the negative control . The positive control presented a gross MI<br />
reduction <strong>and</strong> an increased micronucleus <strong>and</strong> CA frequency. These results suggest that aiie both forms of the<br />
compounds were metabolized <strong>and</strong> inactivated in non-mutagenic derivates, justifying the absence of S vivo<br />
cycloactive <strong>and</strong> clastogenic effects . The present results seem to support that beet root color is a sa er<br />
alternative food additive . Economic <strong>and</strong> technical restraints were not considered .<br />
178<br />
PREDICTION OF POSSIBLE CARCINOGENS, TUMOR-PROMOTERS AND ANTI-TUMOR<br />
PROMOTERS IN THE GLANDULAR STOMACH . C . Furihata <strong>and</strong> T . Matsushima,<br />
Institute of Medical Science, University of Tok~o, Tokyo (Japan)<br />
An in vivo short-term assay method was developed for prediction of<br />
carcinogens, tumor-promoters <strong>and</strong> anti-tumor promoters in the gl<strong>and</strong>ular<br />
stomach . In this method, test chemicals are administered to male F344<br />
rats . Then tumor-initiating activity is assayed by measuring inductions<br />
of unscheduled DNA synthesis (UDS) <strong>and</strong> DNA str<strong>and</strong> scission (by alkaline<br />
elution method), <strong>and</strong> tumor-promoting activity is determined by measuring<br />
induction of ornithine decarboxylase (ODC) activity <strong>and</strong> stimulation of<br />
replicative DNA synthesis (RDS) in the gl<strong>and</strong>ular stomach mucosa. By<br />
this method five possible gl<strong>and</strong>ular stomach carcinogens, glyoxal,<br />
methylglyoxal, diacetyl, 3-diazo-N-nitrosobamethan <strong>and</strong> 1-nitrosoindole-<br />
3-acetonitrile were identified . Hickory smoke condensate, with or<br />
without treatment with nitrite, was found to be a possible gl<strong>and</strong>ular<br />
stomach carcinogen . In addition, 20 possible gl<strong>and</strong>ular stomach tumor<br />
promoters were identified, including various sodium salts, potassium<br />
salts, <strong>and</strong> an ammonium salt of food additives <strong>and</strong> bile acids . CaC12 was<br />
found to be a possible anti-tumor promoter in the gl<strong>and</strong>ular stomach : its<br />
administration inhibited stimulation of replicative DNA synthesis<br />
induced by subsequent administration of NaCl, a tumor promoter in the<br />
gl<strong>and</strong>ular stomach .<br />
179<br />
IN VIVO MUTAGENICITY TESTS ON POLYPLOID INDUCERS<br />
Furukawa,A ., Ohuchida,A . <strong>and</strong> Wierzba,K .*<br />
Drug Safety Research Lab . <strong>and</strong> * Biological Research Lab ., Taiho Pharmaceutical<br />
Co .,LTD . . Kawauchi,Tokushima 771-01 Japan .<br />
The micronucleus (MN) <strong>and</strong> a chromosomal aberration (CA) tests were used to<br />
study in vivo the mutagenicity of polyploid inducers in mouse bone marrow cells .<br />
Five chemicals, e .g . ethyl vanillin, narcotine, p-nitrotoluene,<br />
diethylstilbestrol, thiabendazole, were used as specific in vitro polyploid<br />
inducers (1) . These chemicals were suspended in olive oil <strong>and</strong> were injected<br />
intraperitoneally to BDF1 male mice (8 veek-old, 23 .2-28 .5g) . In pilot PQN-test,<br />
frequencies of micronucleated polychromatic erythrocyte (MNPCEs) varied from 0<br />
to 0 .4% depending on the dose <strong>and</strong> sampling time . The main test was carried out<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 63<br />
Notes
64 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Note s 24 hour after treatment using 5 animals per group . The test compounds did not<br />
increase the frequencies of MNFCEs . The preliminary in vivo CA-tests were<br />
carried out at 6, 24, 48hours after administration . The frequencies of polyploid<br />
were 0-3 .5% <strong>and</strong> were not different from control . Furthermore, the effect of<br />
thes chemicals on polymerization of tubulin was examined . These compounds did<br />
not inhibit specifically tubulin polymerization to microtubules . Therefore, in<br />
vitro induced polyploid can not be mediated through the microtubular system .<br />
(1)Ishidate,M . et a1(1988) Mutation Rea . 195 151-213 .<br />
180<br />
MUTAGENIC ACTIVITY, DNA BREAKAGE AND SOMATIC MUTATION OF ARENEQUINONES .<br />
H . Furukawa, K . Kavai, T . Miyazawa <strong>and</strong> T . Sato, Meijo University, Nagoya(Japan), Tohoku<br />
University, Sendai(Japan), <strong>and</strong> Inaba Biophoton Project, Research Development<br />
Corporation of Japan, Sendai(Japan) .<br />
it was reported that arenequinone was synthesized photochemically by sunlight<br />
irradiation from corresponding arenas . The mutagenic activity of 3,6-banzola)pyrenequlnone,<br />
1,6-benzota]pyrenequinone, 1,6-pyranequinone <strong>and</strong> 1,8-pyrenequinone are 44, 77,<br />
91 <strong>and</strong> 298 revertants on Salmonella typhimwrdun TA100 without S-9mix . . It was<br />
considered there must be the other mechanism with the exception of epoxide formation .<br />
Futhermore breakage of DNA such as pBR322 or A phage DNA by 1,6-pyrenequinone or 1,8pyrenequinone<br />
were suppressed by active oxygen scavenger such as buthylhydroxytoluene,<br />
glutathione, a-tocopherol, sorbitol <strong>and</strong> L-aacorbic acid in vitro . Therefore we<br />
considered that both mutagenic activity <strong>and</strong> DNA breakage activity were caused by active<br />
oxygen molecules <strong>and</strong> the like . Small single spot frequency in Drosophila wing spot test<br />
by 1,6-pyrenequinone, 1,8-pyrenequinone, 1,6-bento(a]pyrenequinone <strong>and</strong> 3,6-benzo(aJpyrenequinone<br />
were risen about two fold of control's frequency . Then it was considered<br />
active oxygen molecules caused the somatic mutation of Droeoph{la melanOgaster . For the<br />
reason above mentioned phenomena, we considered that the planar ring surface of arenequinone<br />
molecules are x-electron deficient, <strong>and</strong> toxic arenequinones contain<br />
considerable radical structures in the resonance formulae . And we would like to<br />
emphasize the urbane airborn particulate containes a few arenequinones <strong>and</strong> toxic<br />
arenequinones should be formed by sunlight irradiation from corresponding arenes .<br />
TRANSPOSITION : EFFRCTg AND MECtlAMISMS<br />
David J . Galas <strong>Molecular</strong> Biology, University of Southern California, Los Angeles, CA<br />
90089<br />
Transposition of mobile genetic elements is apparently a universal phenomenon<br />
occurring in the genomes of all organisms . Certainly in all the experimental organisms<br />
common to the genetics laboratory, they have been extensively characterized . The list<br />
begins with maize, of course (McClintock, 1956), <strong>and</strong> includes Drosophila, 8aecharomyces<br />
Caenorhabditis <strong>and</strong> several different types of bacteria . The genetic effects of mobile<br />
elements are diverse <strong>and</strong> include the induction of insertion mutations, deletions,<br />
inversions <strong>and</strong> other rearrangements, <strong>and</strong> the turning off <strong>and</strong> on of gene expression . In<br />
this talk, I will discuss several examples of the ways in which the mobile elements can<br />
cause these effects . I will draw examples from observations <strong>and</strong> experiments in several<br />
systems, but will focus on bacterial systems in discussing specific mechanisms . The<br />
bacterial insertion sequence, IS1, has been studied to determine the molecular<br />
mechanisms of the transposition process, the component parts of the molecular<br />
apparatus, <strong>and</strong> to dissect the specificity determinants of the protein <strong>and</strong> DNA<br />
components .<br />
GFW=CITY OF VANADIUM CCt1QC[[RNID6<br />
A . GaLLI, L . GIHCKDR, R . DEL CARFA2WE, C . DELIA CPDCE <strong>and</strong> G . BI+DNZETTI<br />
Instituto di Mutagenesi e DifferenziamenYa CIIIR PISA ITALY<br />
181<br />
182<br />
Acmnnium Metavanadate <strong>and</strong> Vanadyl Sulfate were tested for their ability to induce mitotic<br />
gene oonversion <strong>and</strong> point reverse uutatian in the D7 strain of S . oerevisiae . Metavanadate<br />
increased the convertant <strong>and</strong> revertant frequencies <strong>and</strong> the highest ac v ty was observed<br />
without metabolic activation . This indioates that S9 hepatic fraction <strong>and</strong> cells fron logphase<br />
oantainirg a high level of cytochreme P-450 biotransform vanadate probably reducing<br />
it to vanadyl . Vanadyl did not induce any genetic effects in the same experimental conditions<br />
An increase of gene conversion <strong>and</strong> point mitation was induced by vanadyl in cells<br />
fraa log-phase, suggesting that these cells are able to axidate vanadyl to vanadate . To<br />
explain our results, we hypothized that matavanadate is the genetoxically active form <strong>and</strong><br />
the monooxygenase system is involved in biotransfotmntion of varadiun, particularly reduoing<br />
vanadate or oxidatiry vanadyl . To confirm this hypothesis, years cells harvested fram<br />
lod-phase with high level of cytochrane P-450 were treated with metavanadate <strong>and</strong> vanadyl<br />
50869 3576
in the presenoe of specific inhibitors of cytochrome P-450 . In these eorditions, vanadyl<br />
genotwcicity renaiz>aa unaffected, while the presenoe of inhibitors determined an increase<br />
in mitotic yene conversion an3 point reverse mutation irr3uaed by vanadate . Therefore,<br />
tcrmooxygenase system cytochrome P-450 depertflent is probably involved in the vanaditm tretabolism<br />
reducirg vanadate, but not oxidating vanadyl ..<br />
183<br />
DIFFEREhTIAL MUTAGENIC RESPOOiSE CF A SYtVCi-ic .~l'IC pYR::THRCID, DELTAMETHR4tv,<br />
IN SUB-MAN:MALIAN AND MAMNALIAN TEST SYSTfVIS . G . G<strong>and</strong>hi, J .b . Chowt9'tury,<br />
P .K .Sareen <strong>and</strong> I .S .Grover, Scliool of Life Scit•aces,GNDU,Amritsar,India .<br />
Deltamethrin is one of the most commonly used pesticides in North<br />
India . Mence,•-tts genoto•ricity was studied for germ-cell mutagenesis in<br />
Dromvhil~ <strong>and</strong> for clastogenicity in the mouse bone-marrow !• :0 test . LM<br />
(solvent) exhibited mutation rates comparable to the norn :al control v luas<br />
while EMS elicited a positive response in all the test assays . il.iA<br />
were exposed to media containing varying concentrations of the insecticide<br />
(0 .2, 0 .4, 0 .6, 0 .8 <strong>and</strong> 1 .0 ppn) for the induction of dominant<br />
lethals . A steady but non-significant increase in lethality fr(m 32 .58:4<br />
at 0 .20 ppm to 5O .05X at 1 .00 ppm was ob served . Also none of the concentrations<br />
i nduced significa nt SLRLs (2 .45%) even at the highest eoncent ration<br />
tested (0 .80 pgo) . Cytogenetic damage in mice was screened for three<br />
ip administered doses (32 .50, 162 .50 <strong>and</strong> 300 .00 tng/kg b .w), selected from<br />
estimated LD50 (325 mg/),-.g b .w) . significant genetic damage (1 .26 <strong>and</strong><br />
'1 .35% micronucleated PCES) was observed at the two higher doses . The PCE/<br />
tCZ<br />
ratio demonstrated a significant ir.crease in the percentage of pCES,<br />
at lower doses sig nifying a stimulatcry eff ect of deltamethrin . Though,<br />
deltameth rin showed no germ-cell mutagenesis in ro• :nnhd .11, yet it acted<br />
as a strong clastogerl/sPindle ir4fibitor in the mouse . Its differential<br />
response has rather made it desirable to investigate its ; genotoxicity in<br />
oth er systems too .<br />
184<br />
RFGULA'TION OF NUM,AN DNA GLYCOSYLASFS 'rdAT INT'fTA'TF gAgfy FXCISION RFPAIR OF OXii)ATTVE<br />
PYHf,lIjL2F MODIFICA'TTONS . Tapan Cang-il y <strong>and</strong> aahum J . Duker, Temple University<br />
School of Nndicine, Philadelphia, PA (OSA)<br />
DNA oxid,rrive damap,ea are among the most frequPOt~ tyoes encountered in the<br />
lifetime of a cell . TheaPe can result from ionizing or gamma radiation <strong>and</strong> froaa<br />
activated oxygen species Renerated from metabolic procesees . Fxciaion of oxidized<br />
bases from DNA of hurnan cella ia initiated by DNA qlycosylaees . The oxidized<br />
t7yminrc moiety 5-hydroxyrethyluracil is removed frorn DNA by 5-hydroxyerethyluracil-<br />
DNA e,lycosylase . A redoxyendonuclease, active against a wide variety of substrates,<br />
has ;lycosylic activity aF,ai .ist many modified DNA pyrimidiiea . We studied<br />
re.gul .ation of these enzymes in proliferating human cella . Both glycosylases were<br />
assayed by measurement of direct telease of modified free bases from their DNA<br />
substrates . Serum-atimulated JI-38 Sunan cells were the sources of enzyme<br />
a .tiviries assayed in crude extracts as a function of cell division . 5hydroxyaret.hyluracil-DNA<br />
qlycosylase activity did not vary significantly during the<br />
cell cycle . ny contrast, the glycosylic activity of the redoxyendonucleaee<br />
increased four-fold as a function of cell growth . Maximum stimulation was obtained<br />
during peak DNA synthesis . Tnis enzyme activity increased once again aa thn cella<br />
entered a second growth cycle . This ia similar to the stimulation observed for<br />
uracil-UNA glycosylase in a aynchronous cell population . Theae results indicate<br />
that the glycosylasea that initiate base excision repair of oxidised DNA are not<br />
coor3inately induced during the cell cycle .<br />
185<br />
LATE EFFECTS OF FEMALE SEX HORMONES<br />
han Fengeing, )laog Hsnyfug, <strong>and</strong> Yu Yannao, Dept . of Blological Effect of<br />
Radislion, Laboratory of tnduslrial Nygiens, Ministry of Public Health . I<br />
Xinkang Street, Desheng.enr,l, Beijing, (China)<br />
Inj . Hydroxyprugesterone Co . (containing hydroxyprogesterons capruste (P)<br />
250 •g <strong>and</strong> estradiol valerate (E) S sfgi .l of vehit•ls, .EP) was Isjecled<br />
intrseuscularly into fe .ale Vister rats <strong>and</strong> different strains of rtee of both<br />
sexes with doses of 5 to 100 ti .es that used Is huans ones or twice a month for<br />
10 to 24 ti .es . In so .e experl .ents EP was co .bined with •hole-body 3 Gy ga. .a<br />
radiation once or twice . The purpose of this work was to detsr .ine whether F•P<br />
would possess carcleogenleity or not <strong>and</strong> whether synergistic cercinogeeicily<br />
would exist when EP had been ad .lnistered In coebination of ga . .a radletion .<br />
Results show that EP has ubvious carcloogenicity/tu .our lncidence was siegnifi-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 65<br />
Notes
66 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes cenlly increased . HI'• also has the sffect of laducing relrugrade infeclion or<br />
urugenital sysles . F.Y enhanced the gassa ray carcisogenieily, thereby<br />
increasing the tumour incidence or the ratio of sa1)gnant to benign tuaosrs .<br />
Mhen added into the sedium, 1iY,fi or P aloae •as able to directty lnduce<br />
matignant transformation of souse esbtyo cetls in vitro . it caa partly explain<br />
the reasan •hy the aajur lyps of tusors ladueed by 8Y in various atralae of<br />
sire ua : qaitr difforeat as a result of observing ihe distribution of<br />
trltialed estradlol receptor cosplexes in various t)ssues <strong>and</strong> organs by<br />
nutoradlography .<br />
186<br />
HUMAN GENOTOXICITY IN PHOSPHINE-EXPOSED APPLICATORS . V . Garry, T . Danzl, J . Griffith,<br />
R . Nelson, E . Whorton, University of Minnesota, Minneapolis, MN (USA), <strong>and</strong> U .S . EPA,<br />
Research Triangle Park, NC (USA) .<br />
Fumigation of grain is a world-wide agricultural practice . In this effort, we<br />
examined the health histories of more than 400 persons who may have come in contact<br />
with fumigants <strong>and</strong> pesticides . We identified a small group of workers who perform<br />
fumigant application . We then undertook an integrated human study of fumigant<br />
applicators exposed to phosphine, one of the most common fumigants . Evidence for<br />
qenotoxicity was expressed in terms of sister chromatid exchanges <strong>and</strong> chromosome<br />
aberrations in b<strong>and</strong>ed <strong>and</strong> non-b<strong>and</strong>ed preparations . These data were coupled with<br />
personal breathing zone sampling for phosphine . As a group, applicators (n-24) show<br />
significantly increased chromosome aberrations compared to control subjects (n-24) .<br />
Workers exposed to phosphine alone (n-9) have increased chromatid gaps/deletions<br />
compared to all other groups . Workers earlier exposed to phosphine or to phosphine<br />
<strong>and</strong> other pesticides have significantly increased chromosome rearrangements,<br />
including chromosome translocations (11/12) compared to controls (2/10) . Chromosome<br />
breakpoints identified in the exposed workers seem to cluster in certain oncogene<br />
regions of specific chromosomes . In vitro studies of phosphine suggest that<br />
phosphine or a phosphine-generated product(s) crosslinks DNA as studied by alkaline<br />
elution procedures . Further work indicates that the mechanism of qenotoxicity may<br />
be related to peroxidase inhibition .<br />
187<br />
A IL TRDUSTRIAL AND UK PERSPECTIVE ON SHORT TER_M TEQTING .<br />
DG Gatehouse, Genetic <strong>and</strong> Reproductive Toxicology Department, Glaxo Group Research<br />
Lta ., Ware, Herts .<br />
The appropriate use of short-term tests for the screening of carcinogenic agents<br />
is currently under review . In the UK, the DHSS Committee on Mutagenicity (COM) has<br />
been revising its guidelines with a view to publication later this year . To<br />
compliment this the UKEMS are revising their recommendations for the conduct of the<br />
main categories of short-term tests . In these revisions it is recognised that a<br />
limited number of in vitro tests carried out exhaustively but with a degree of<br />
flexibility is the most effective strategy for priaary screening . Mammalian gene<br />
mutation assays have been retained in the revised DiiSS CON Guidelines, aa some<br />
"unique" mammalian cell mutagens have been identified by the recent R!P study .<br />
However, the credibility of these data is being contested <strong>and</strong> further studies are<br />
required to resolve this . There is still considerable debate on the role of<br />
short-term in vivo tests ie . confirmatory or screeningt . Their use as screens may<br />
still be essential when complex metabolic activation processes are required . If used<br />
in a confirmatory role, there is accumulating evidence that organ-site specificity<br />
requires that more than one tissue should be examined to eliminate false negative<br />
results . It is possible that species-specificity might also be an important<br />
consideration when designing experimental protocols . Finally the need for an<br />
additional test(s) to detect "aneugenic" agents has still to be decided . Some<br />
potential aneugens may be detectable using the existing assays (eg micronucleus teat<br />
<strong>and</strong> in vitro cytogenetic assays) . Further validation data are required before any<br />
firm decisions can be made .<br />
188<br />
DATA AND RATIONALE FOR A MODEL THAT EXPLAINS THE VARYING FREQUENCY OF ANEUFLOID CHILDREN<br />
WITH MATERKAL AGE (THE J-SHAPED CURVE) . M .E . Caulden, Radiology Department, University<br />
of Texas Southwestern Medical Center, Dallas, TX 75235<br />
The majority of aneuploid children are born to older women <strong>and</strong> result from nondisjunction<br />
at first meiotio division in the ovary . What ovarian condition promotes<br />
aneuploidy induction? I propose that it As decreased miorooiroulation around follicles,<br />
leading to deficient 0p supply <strong>and</strong> a concomitant inorease in C02 <strong>and</strong> anaerobic produots<br />
such as lactic acid, whioh lower pH . We have found that exposure of somatic cells in<br />
vitro for 1 h, 38°C to C02 or acid medium (pH
1989 EMS Abstracts<br />
causes dissociation of 1-2 chromosomes, comparable to that caused by 0 .025 ug/ml Coloe- Notes<br />
mid, producing aneuploid daughter eelle . The follicle ie avascular ; the oooyte in an<br />
immature follicle is probably hypoxic because the capillaries in the surrounding theca<br />
are few . Development of capillaries around a maturing follicle is determined by sex<br />
hormones <strong>and</strong> angiogenlo factors, whose levels may be lower in the very young <strong>and</strong> the<br />
older woman, <strong>and</strong> also occasionally in one of intermediate age . Exchange of gases<br />
<strong>and</strong> other substances must take place through granulosa oelle <strong>and</strong> follioular fluid, so<br />
the oocyte 02-C02 balance is dependent on an ample blood supply . Thus, a compromised<br />
microciroulation could account for aneuploidy incidence in women of any reproductive<br />
age, the frequency varying with the probability of events leading to reduced development<br />
<strong>and</strong>/or funotion of the critical perifollioular capillary bed . The testioular tubule is<br />
also avascular, so small localized regions of reduced circulation could result in•<br />
aneuploidy . Lagging angiogenesis has been shown to cause bypoxic regions in tumors, so<br />
reduced pH could be responsible for some of the aneuploidy seen in practically all<br />
advanced tumors . Experiments in progress with mouae oooytes will be reported .<br />
189<br />
SYMPOSIUM : CEOtOMOSOME ABERRATIONS :IMECHANISMS<br />
MICRODOSIMETRY, LET AND CkQt0t4DSOMAL ABERRATICNS .<br />
Charles R . Geard, Radiological Research Laboratory, College of Physicians <strong>and</strong><br />
Surgeons o Co umbia university, New York, N .Y . U .S .A .<br />
Microdosimetry deals with the statistical fluctuations of energy deposition in<br />
small volumes of irradiated matter . When applied to the cell nucleus or parts<br />
thereof, the frequencies <strong>and</strong> intensities of different ionizing radiations can<br />
be related to the induction of individual chromosomal changes . That is, a<br />
relationship can be established between track based energy deposits <strong>and</strong> the<br />
probability of lesion induction <strong>and</strong> interaction . It has been long established<br />
that as linear energy transfer (LET) increases so does the likelihood of<br />
biological effect . Currently this is particularly pertinent for the alpha<br />
particle cellular traversals from radon daughters . At environmental levels of<br />
radon, individual cells are very rarely likely to encountermmore than one<br />
alpha particle . Therefore it is necessary to evaluate thromosomal changes in<br />
individual cells on a per particle basis . To attain this end microdosimetric<br />
evaluations of track events <strong>and</strong> of energy transfers in sub-nuclear volumes are<br />
necessary in conjunction with morphometric assessments of cellular targets .<br />
Over the LET range consistent with radon daughter alpha particles there is a<br />
non constant probability of aberration induction both in terms of frequencies<br />
<strong>and</strong> types of aberations . Hence assuming an equivalent status for these alpha<br />
particles which are of profound societal concern is inappropriate .<br />
190<br />
USE OF CYTOGENETIC STUDIES IN ASSESSING THE INVOLVEMENT OF MUTAGENIC AGENTS IN<br />
PRELEUKAEMIC SYNDROMES AND ACUTE LEUKAEMIA . A .D .Geddes <strong>and</strong> A .Jacobs . Department of<br />
Haematology, University of Wales College of Medicine, Cardiff, U .K .<br />
The involvement of mutagenic/carcinogenic agents in the induction of preleukaemia<br />
<strong>and</strong> acute leukaemia has been known for some time . Exposure may occur as a result of<br />
chemo/radiotherapy or from occupational or environmental sources . Secondary leukaemias<br />
are generally rapidly progressive with poor response to normal therapeutic regimes <strong>and</strong><br />
short survival . Cytogenetic characteristics include a high incidence of clonal<br />
karyotypic abnormalities in the bone marrow (>85a) ; a high level of aneuploidy,<br />
particularly chromosome loss ; high incidence of complex rearrangements including<br />
unstable configurations such as rings, dicentrics, double minutes, etc <strong>and</strong> specific<br />
involvement of certain chromosomes particularly 5 <strong>and</strong> 7 (66-90% of cases) . Cytogenetic<br />
studies are therfore useful in the identification of mutagen induced disease <strong>and</strong> have<br />
been used in Cardiff over the last 3 years to assess the potential involvement of<br />
mutagenic agents in new cases of preleukaemia <strong>and</strong> leukaemia with no apparent history<br />
of therapeutic or occupational exposure as well as monitoring those patients with a<br />
history of prior therapy for other disorders or of occupational exposure to potentially<br />
mutagenic agents . Studies included investigation of clonal karyotypic abnormalities<br />
<strong>and</strong> aneuploidy in bone marrow <strong>and</strong> assessment of chromosome aberration levels in both<br />
bone marrow <strong>and</strong> peripheral blood . Although such studies cannot prove a direct causative<br />
role for mutagens in the development of preleukaemia/leukaemia they can provide<br />
indications of mutagenic involvement <strong>and</strong> they may also assist in defining groups <strong>and</strong><br />
individuals at risk .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
67
;<br />
68<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts<br />
Notes MOUSE MODELS FOR UNDERSTARDING HUMAN DEVELOPNffirfAL ANOMALIES<br />
Valderico N . Generoso<br />
Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077 (USA)<br />
191<br />
Mutaganesis research in mice has a long tradition of addressing the problems of<br />
human genetic risk <strong>and</strong> of enriching our knowledge of basic mammalian biology . In line<br />
with this tradition, we are using mouse models in order to underst<strong>and</strong> the origin <strong>and</strong><br />
pathogenesis of certain classes of human developmental anomalies . One such model<br />
involves the study of the developmental defects that are caused by chemically induced<br />
chromosomal rearrangements <strong>and</strong> imbalances . Of interest are the specific chromosomes<br />
involved, the nature of breakpoints, the teiotic segregation that produces unbalanced<br />
segregants that survive to late gestation, <strong>and</strong> the sequence of changes that are<br />
observed in the pathogenesis of the defects . Another model involves the study of<br />
developmental anomalies that are produced subsequent to exposure of zygotes to certain<br />
mucagens . The fetal malformations produced in these studies are generally similar to<br />
the majority of human malformations, for which the etiology is largely unknown . The<br />
evidence suggests a genetic basis for the mouse fetal anomalies, but of a type that is<br />
different from conventional gene mutations <strong>and</strong> ehromosome aberrations .<br />
Research sponsored jointly by the National Toxicology Program under NIEHS Interagency<br />
Agreement Y01-ES-20085 <strong>and</strong> the OHER, U .S . DOE under contract DE-AC05-840R21400 with the<br />
Martin Marietta Energy Systems, Inc .<br />
192<br />
COMPARISON OF THE GENOTOXIC EFFECTS OF 1- AND 2- NITROPROPANE IN THE RAT<br />
E . George, B . Burlinson <strong>and</strong> D .G . Gatehouse, Dept . Genetic <strong>and</strong> Reproductive Tox .,<br />
Glaxo Group Res . Ltd ., Ware, Herts, Engl<strong>and</strong> .<br />
2-nitropropane is a potent rat liver carcinogen, whilst the 1-isomer is<br />
non-carcinogenic in rodents . Although the 2-isomer induces UDS in the rat liver,<br />
uniformly negative results have been obtained in the mouse micronucleus test . The<br />
inability of the latter to discriminate between the carcinogenic <strong>and</strong> non-carcinogenic<br />
isomers may reflect either species-specific genotoxicity in vivo ; an organospecific<br />
effect of the 2-isomer or an end-point specific effect .<br />
To determine which of these factors precluded detection of 2-nitropropane in the<br />
mouse micronucleus test, studies were carried out in the rat in which micronucleus<br />
induction (bone marrov <strong>and</strong> liver) <strong>and</strong> UDS induction (liver) were measured after oral<br />
treatment with each compound . 2-nitropropane was found to induce UDS in rat liver,<br />
whilat the 1-isomer was negative, thus confirming earlier studies using the<br />
intraperitoneal route . In the bone-marrov micronucleus test, small increases were<br />
obtained with individual animals, however group means fell within the historical<br />
control range <strong>and</strong> the results were considered negative . In the liver micronucleus<br />
test, 2-nitropropane induced a highly significant response . Therefore the negative<br />
mouse micronucleus test results reported for 2-nitropropane were not due to species<br />
specificity, or end-point specificity ; but instead were a reflection of the<br />
organospecific genotoxicity of this compound in vivo . These data provide further<br />
evidence that bone marrov assays are insufficient for the detection of all genotoxic<br />
carcinogens in vivo, indicating the need for a second tissue, although the choice of<br />
end-point may be of less critical importanae .<br />
193<br />
A CHROMOSOME STUDY OF 387 REFERRED CASES HITH VARII3D GENETIC DISORDERS .<br />
M .A.Ghalib, G .S.Issac, A .Jyothy <strong>and</strong> O .S .Reddy, Institute of Genetics <strong>and</strong> Hospital<br />
for Genetic Diseases, Osmania University, Begumpet, Hyderabad . A .P . (INDIA) .<br />
This paper describes the result of chromosome study carried out on 387 cases<br />
suspected of chromosomal abnormalities . They were referred during the period<br />
from January 1987 to July 1988 to the Cytogenetics Division of the Institute of<br />
Genetics <strong>and</strong> Hospital for Genetic Diseases from the various districts of the State<br />
of Andhra Pradesh, India . They Include congenital anomalies (134), am biguous<br />
external genitalia (29) . Down's syndrome (101) . Klinefelter's phenotype (17) .<br />
Turner's phenotype (12), <strong>and</strong> primary <strong>and</strong> secondary amenorrhea (94) cases .<br />
Out of these 387 cases . 119 were found to have chromosomal abnormalities, a<br />
frequency of 30 .75% of abnormal karyotypes, leaving 268 with normal keryotypes .<br />
Autosomal aberrations were detected in 99 cases <strong>and</strong> the remaining 19 cases had<br />
sex-chromosome abnormalities . The frequency of 30 .75% in a referred population<br />
for chromosomal aberrations is considered high when compared to similar reports<br />
from other countries . Factors related to the parents that might have a predisposition<br />
in the aetiology of the aneuploidies will be discussed . Acknowledgements .<br />
The first author would like to thank the Rector . University of Aden, Government<br />
of P .D .R . of Yemen <strong>and</strong> the Education Officer . Ministry of Human Resources Development<br />
. Govt . of India fo~ financial support . The authors acknowledge the Director<br />
of the Institute for providing facilities .<br />
50869 3580
194 1989 EMS Abstracts 69<br />
Notes<br />
II+cLUEhCE OF iiALIDI%IC ACID ON THE KILLING AND h1GTATION IiJDliCED BY UV<br />
LIGiIT AP+D M~tiNG IN DENSITY INHIBITED V79 CELLb<br />
Rita Gaosh(Datta), S. at a ^ <strong>and</strong> G . Bnaumik, 8aha Institute<br />
of Nuclear ?nyeics, I ~, alt ake, Calcutta-700 064 .<br />
Density inhibited plateau phase V79 cells after treatment with UV<br />
light or N-metnyl-N'-nitro-N-nitrosoguanidine(13NNG) exhibited improvement<br />
in survival accompanied by lowering of mutant yield (resistance<br />
to 6-tnioguanine) on delay in trypsinization (20h) . If nalidixic acid,<br />
an inhibitor of topoisomerase activity was present during tae period of<br />
delay, survival levels were similar to tnose obtained on immediate<br />
trypainization For mutational analysis, UV fluence was va 1ed from<br />
4J/m` to 20J/m! ; corresponding mutant frequencies (per 10~ viable<br />
cells) were 3 .0+0 .5 <strong>and</strong> 16 .9+0 .8 at these two fluences on immediate<br />
trypsinization . On delayed trypsinization, tnesp values decreased to<br />
1 .8+0 .6 <strong>and</strong> 13 .0+0 .5 respectively <strong>and</strong> again increased to 5 .5+0 .8 <strong>and</strong><br />
21 .a+0 .8 when nalidixic acid was present . In case of JSYtteG, tpe doses<br />
varied from 0 .5µg/ml to 2 .Oµg/ml f3r lh treatment time ; the ccrresoonding<br />
mutant frequencies (per 10 viable cells) were 7 .5+0 .5 <strong>and</strong><br />
32 .5t2•5 at these doses on immediate trypsinization . If tnB trypsinization<br />
was delayed, the corresponding values were 4 .5±1 .0 <strong>and</strong> 23 .3+2 .2<br />
respectively . However, wnen nalidixic acid was present during thle<br />
delay period, tue values were 8 .5+1 .2 <strong>and</strong> 38 .8+2 .0 . The results indicate<br />
the involvement of topoieomerase in repair of potentially lethal<br />
damage .<br />
195<br />
IN SITU EVALUATION OF POTENTIAL GENETIC HAZARDS FROM CHEMICAL WASTE SITES .<br />
B .S . Gill, J . Rice <strong>and</strong> S .S . S<strong>and</strong>hu . EHRT, Research Triangle Park, NC 27709 <strong>and</strong> EPA,<br />
Research Triangle Park, NC 27709 .<br />
In situ monitoring of biological effects from chemical waste sites provides hazard<br />
assessment under the complexities of natural environmental conditions . For such studies,<br />
plant assays are cost effective <strong>and</strong> are ideal for preliminary investigations .<br />
The studies reported here were initiated at The Fairway Six pesticide site in Aberdeen,<br />
North Carolina <strong>and</strong> Palmetto Wood Preserving site in Dixiana . South Carolina using Tradescantia<br />
micronucleus <strong>and</strong> maize vaxy locus assays . The chemical analyses of soil<br />
samples from these sites indicate concentration of lindane (17385 ug/kg), beta BHC<br />
(12645 ug/kg), <strong>and</strong> heptachlor (378 ug/kg) at four feet dbpths at the Aberdeen site <strong>and</strong><br />
arsenic (1292 mg/kg), chromium (1444 mg/kg), <strong>and</strong> copper (924 mg/kg) on the surface at<br />
the Dixiana site . Results of Tradescantia micronucleus assays shoved significantly<br />
higher frequencies of micronuclei from the contaminated plots as compared to the control<br />
plots . Toxic effects for maize growth were observed on contaminated plots at<br />
Dixiana site . The soil samples collected from these sites were analyzed in the laboratory<br />
for their biological effects using Vicia root tip, wheat aneuploidy, <strong>and</strong><br />
Tradescatia micronucleus assays . Preliminary evidence confirms the in situ findings .<br />
The sites have now been cleaned . The in situ <strong>and</strong> laboratory experiments vill be<br />
repeated to evaluate the efficacy of the remedial operations . This is an abstract of<br />
a proposed presentation <strong>and</strong> does not necessarily reflect EPA policy .<br />
196<br />
OXIDATIVE STRESS-INDUCED GENETIC INSTABILITY IN CULTURED CHINESE HAMSTER CELLS<br />
J .J .P . Gille, C .G .M . van Berkel, F .A .J .M van de Klundert, <strong>and</strong> H . JoenSe . Institute of<br />
Human Genetics, Free University, P .O . Box 7161, 1007 MC Amsterdam, The Netherl<strong>and</strong>s .<br />
Two different inducera of oxidative stress, i .e ., normobaric hyperoxia <strong>and</strong> H202<br />
were compared with respect to their ability to induce str<strong>and</strong> breaks <strong>and</strong> gene muta-<br />
tions .<br />
Normobaric hyperoxia (1 atm ., 984 02) was found to be a very weak inducer of DNA<br />
single-str<strong>and</strong> breaks <strong>and</strong> alkali-labile lesions, whereas H202 was found to induce large<br />
amounts of str<strong>and</strong> breaks . Iron-chelators were unable to afford protection against the<br />
clastogenic <strong>and</strong> SCE-inducing effects of hyperoxia, whereas it is known from the<br />
literature that they do protect Chinese hamster cells against the induction of DNA<br />
str<strong>and</strong> breaks <strong>and</strong> SCEs by H202 . From these results, together with the knowledge that<br />
most OH radical-induced DNA lesions are detectable by alkaline elution, it is suggested<br />
that the induction of genetic damage by hyperoxia is, in contrast to H202, probably<br />
not OH radical mediated .<br />
Data on the mutagenicity of hyperoxia <strong>and</strong> H202 on three loci (aprt, hprt, <strong>and</strong><br />
xprt) will be presented . Conclusions with respect to the type of mutation (base<br />
substitutions <strong>and</strong> small deletions v .e . large deletions) induced by the different<br />
sources of oxidative stress will be discussed .<br />
Supported by grant 87-10 from the Netherl<strong>and</strong>s Cancer Foundation .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
70 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
197<br />
LIVER PROTEIN EXPRESSION IN FETAL MICE HOMOZYGOUS OR RETEROZYGOUS FOR CHROMOSOMAL<br />
DELETIONS AROUND THE ALBINO LOCUS . C .B .Giosetti, M .A .Geazsell, S .L .Tollaksen, <strong>and</strong><br />
S .Gluecksohn-Waelsch, Argonne National Laboratory, Argonne, IL (USA) <strong>and</strong> Albert<br />
Einstein College of Medicine, Bronx, NY (USA)<br />
Liver protein expression was examined in fetal mice homozygous or heterozygoua<br />
for the c3N or c14CoS deletions in the albino locus region of chromosome 7 to identify<br />
apecific gene products that correlate with the dedeted ene s,~ ences . Liver<br />
proteins from vildtype (ceh/cch)~ heterosygous (co /c3 or cc14 ~), <strong>and</strong> homozygous<br />
(c3H/c3H or c1~'CoS/c14C0S) fetal mice (18/19 days gestation) were separated by<br />
two-dimensional electrophoresis (2DE) . The Coomassie Blue-stained protein patterns<br />
were screened for both qualitative <strong>and</strong> quantitative protein differences by using the<br />
Tycho analysis system . In the 2DE patterns from c3H homozygotes, 20 proteins were<br />
missing that were present in equal abundance in patterns of c3H heterosygous <strong>and</strong><br />
h4~o~gous wildtype littermates . However, the same 20 proteins were present in the<br />
C1 homozygotes . In addition to the 20 qualitative protein differences, five<br />
proteins were effected quantitatively in the c3H homozygotes (four decreased <strong>and</strong> one<br />
increased in expression) relative to both heterosygous <strong>and</strong> homozygous vitdtype<br />
littermates . The identity of quantitative expression in deletion heterozygotes <strong>and</strong><br />
wildtype homozygotes supports previous interpretations of experimental findings<br />
suggesting that the albino deletions involve loss of one or more regulatory genes .<br />
This work was supported by the U .S .DOE, ONER, contract W-31-109-ENG-38, NIH grant<br />
GM27250, American Cancer Society grant CD38 .<br />
COMPARATIVE MUTAGENICITY AND GENOTOXICITY OF THE ANTIPARASITIC DRUGS .<br />
MEBENDAZOLE . FLUBENDAZOLE AND AN OXIME DERIVATIVE OF FLUBENDAZOLE<br />
A .K . Girl, S . Osorio, J .E . Sinsheimer, D .S . Mise <strong>and</strong> L .B . Townsend .<br />
College of Pharmacy . University of Michigan . Ann Arbor . MI 48109 . USA .<br />
As part of a program to develop compounds with macrofilaricidal activity . we<br />
have tested the ∎utagenicity in Salmonella, <strong>and</strong> the In vivo genotoxicity to<br />
bone- marrow cells in ∎ice of the commercially available anthelaintic drugs<br />
Mebendazole <strong>and</strong> Flubendazole as well as an oxiae prodrug of Flubendazole . This<br />
information was sought to develop a base line for the further preparation of<br />
∎ore efficient .acrofilaricidal compounds without eenotoxiclty . Mutagenicity<br />
was established only for TA 98 with the presence of $9 at high doses for<br />
Mebendazole <strong>and</strong> at lower levels for the oxime . Mutagenicity was not observed<br />
for Flubendazole under the same conditions . A dose related increase in sister<br />
chroaatid exchanges (SCE) was found In vivo for Mebendazole <strong>and</strong> Flubendazole .<br />
There were comparable significant (pNPO>NPGEtTCPO> control . When the genotoxic<br />
effects of two of their metabolic precursors, 1-allylnaphthalene (AN) <strong>and</strong><br />
trichloropropylene (TCP) were also carried out In vivo . AN was genotoxic in bone<br />
marrow but not TCP . The ∎uch shorter half life of TCPO is a factor in<br />
explaining the lower SCE <strong>and</strong> CA results than would be predicted by chemical<br />
alkylation rates or by the preincubation version of the Ames test . DNA str<strong>and</strong><br />
breaks in liver were used as a test requiring less In vivo exposure ti .e for a<br />
genotoxicity comparison of the four epoxides <strong>and</strong> the two precursors . A<br />
significant increase in x of unwound DNA was observed for all the compounds<br />
except AN at high doses . TCPO, the least gsnotoxic in bone marrow . Is indicated<br />
to have the greatest liver toxicity after 4 b exposure while NOE shows the most<br />
toxicity after 6 h . Supported by grant RO1 ES0334S . NIENB .<br />
50869 3582<br />
198<br />
199
200 1989 EMS Abstracts 71<br />
Glickman, B .W., MUTATIONAL SPECIFICITY AS A WINDOW ON THE MECHANISMS Notes<br />
OF MUTATION, Biology Department, York University,4700 Keele Street,<br />
Toronto, Canada M3J 1P3<br />
The development of cloning <strong>and</strong> sequencing technology has permitted the examination<br />
of the nature of mutation at the molecular level . These studies have in many cases<br />
confirmed the targeted nature of mutation <strong>and</strong> hence, provided insights into the lesions<br />
responsible. Mutational spectra obtained following treatment with a variety of agents<br />
also confirm that mutation is non-r<strong>and</strong>om . In some cases this appears to be related to<br />
the initial deposition of damage, in others, it likely reflects differences in the efficiency<br />
of repair at different sites . Spectra may also reveal a component of mutation that<br />
reflects the influence of the local DNA sequence on the accuracy of repair or replication<br />
past a DNA lesion . Studies with a diverse series of agents over a broad ran*e of doses<br />
in several repair deficient backgrounds have contributed to our current view of the<br />
mechanisms of mutation. This lecture will concentrate on the kinds of lessons that<br />
might be )earned from the study of mutational spectra . In particular, we will examine the<br />
mutational specificity of a series of alkylating agents <strong>and</strong> discuss the basis of their<br />
mutational specificity in light of both the expected lesions <strong>and</strong> the role for DNA repair<br />
in the avoidance <strong>and</strong>/or fixation of mutation .<br />
201<br />
MOLECULAR SPECTRA OF L5178Y/tk'/' MUTANTS INDUCED BY DIVERSE MUTAGENS . P .<br />
Gbver, R . Krehl <strong>and</strong> D. Clive, Wellcome Research Laboratories, Research Triangle Park, NC 27709<br />
U5A<br />
Southern blot analyses were performed on DNA from at least 10 large <strong>and</strong> 10 small colony tk-Imutants<br />
induced by each of 10 mutagens [2-amino-N6-hydroxyadenlne (AHA), EMS, MMS, 2-AAF,<br />
methotrexate (Mtx), caffeine, methapyrilene (MP), m-AMSA, hycanthone methanesulfonate,<br />
procarbazine (Proc)) . Two molecular mutant genotypes were recognized upon digestion with Nco-1 <strong>and</strong><br />
subsequent probing with 1 .1 kb cDNA Insert from plasmid pMtk 4 (ATCC N37556) : (1) no detectable<br />
alteration <strong>and</strong> (2) absence of the newly mutated tk allele as Indicated by the absence of the 6 .2 kb<br />
fragment (Applegate <strong>and</strong> Hozier, Banbury #28 : page 213, 1987) . In combination with the previously<br />
established chromosomal nature of most small colony tk -I' mutants (Moore et al ., 1985), this<br />
pemritted the classification of these 10 mutagens according to the relative proportions of each of 4<br />
classes of genetic damage they Induced . AHA <strong>and</strong> EMS gave mutational spectra consistent with their point<br />
mutational effects In other systems . The other 8 mutagens Induced mostly small colony mutants, most of<br />
which had lost the entire tk allele . Mtx Induced high frequenciei of large colony mutants at the rk<br />
locus, mostly lacking the tk allele, <strong>and</strong> was weakly or nonmutagenic at the hemizygous hprt locus . Four<br />
mutagens--Mtx, caffeine, MP <strong>and</strong> Proc--lack structural alerts for DNA reactivity (Ashby <strong>and</strong> Tennant,<br />
Mutation Res ., 204 : 17-115, 1988) Implying a major class of non-DNA targets for mutagenicity in<br />
mammalian cells (Clive, these abstracts) . The mutagenicity spectra for 2-AAF . Hyc <strong>and</strong> caffeine are<br />
quite similar, raising the possibility that the DNA adducts of 2-AAF <strong>and</strong> the Intercalating activity of Hyc<br />
may not be their principle mechanisms of mutagenicity In mammalian cells .<br />
202<br />
EVALUATION OF THE CLASTOGENIC AND MUTAGENIC POTENTIAL OF DIETHYLENETRIAMINE (DETA)<br />
B . Bhaskar Gollapudi, V . Ann Linscombe, <strong>and</strong> Anil K . Sinha, The Dow Chemical Company,<br />
Health <strong>and</strong> <strong>Environmental</strong> Sciences, Freeport, TX 77541<br />
DETA (C .A .S . N111-40-0), a colorless/yellowish hygroscopic liquid, is widely used<br />
in the production of paper wet-strength resins, epoxy-curing agents, chelating<br />
agents, lubricating oil <strong>and</strong> fuel additives, surfactants, <strong>and</strong> corrosion inhibitors .<br />
The clastogenic potential of DETA was evaluated in vitro by treating Chinese hamster<br />
ovary cells in culture for 4 h with 250, 833, <strong>and</strong> 2500 pg/ml DETA in the presence<br />
<strong>and</strong> absence of a metabolic activation system (Aroclor 1254-induced rat liver S-9) .<br />
The treatments did not induce significant increases in the chromosomal aberration<br />
frequency in cells harvested 18 h after termination of the treatment<br />
. DETA, when<br />
ad .inistered by oral gavage to CD-1 mice at dose levels of 85, 283, <strong>and</strong> 850 mg/kg<br />
body weight, did not significantly increase the frequency of micronucleated bone<br />
marrow polychromatic erythrocytes at any of the three sampling times (24, 48, or 72<br />
h) . The mutagenic potential of DETA was evaluated in the male germ cells of<br />
Drosophila melanogaster by the sex-linked recessive lethal (SLRL) assay . Adult<br />
Canton-S males were allowed to feed on treatment solutions containing 60 mM DETA for<br />
22-24 h . The treated post-meiotic male germ cells were sampled in three broods of<br />
3, 2, <strong>and</strong> 2 days each . The treatment did not significantly increase the frequency<br />
of SLRL mutations . Thus, DETA failed to induce a genotoxic response in any of the<br />
test systems employed .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
72 1989 EMS Abstracts 203<br />
Notes GENETIC TOXICITY TESTING OF CHLORPYRIFOS AND ITS MAJOR METABOLITE<br />
B . B . Gollapudi, J . A . Zempel, A . L . Mendrala, V . A . Linscombe, M . L . McClintock,<br />
<strong>and</strong> A . K . Sinha, The Dow Chemical Company, Health <strong>and</strong> <strong>Environmental</strong> Sciences,<br />
Freeport, TX 77541, <strong>and</strong> Mammalian <strong>and</strong> <strong>Environmental</strong> Toxicology Research Laboratory,<br />
Midl<strong>and</strong>, MI 48674 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
The genotoxic potential of the organophosphate insecticide chlorpyrifos [Q,Qdiethyl-Q-(3,5,6-trichloro-2-<br />
pyridyl)phosphorothioate, C .A .S . ! 2921-88-2j <strong>and</strong> its<br />
major metabolite, 3,5,6-trichloro-2-pyridinol (C .A .S . I 6515-38-4) were evaluated in<br />
a battery of short-term assays consistiny of Ames, UDS, CHO/HGPRT, <strong>and</strong> the mouse<br />
bone marrow micronucleus tests . In the Anes test, the chemicals did not elicit a<br />
positive response in the Salmonella typhimurium tester strains TA98, TA100, TA1535,<br />
TA1537, <strong>and</strong> TA1538 either in the presence or absence of an externally supplied<br />
metabolic activation system (Aroclor 1254-induced rat liver S9) . Hepatocytes<br />
collected from male Fischer 344 rats (11-15 weeks old) were treated in vitro with<br />
the test chemicals in the presence of H-thymidine <strong>and</strong> the extent of UDS was<br />
quantitated by autoradiography . Neither chemical induced a positive UDS response .<br />
No significant increases in the frequency of TGr colonies were observed in the<br />
CHO/HGPRT assay after treatment of CHO-K' -8H4 cells with the test chemicals either<br />
in the presence or absence of S9 . Administration of the test chemicals by oral<br />
gavage to CD-I mice did not increase the frequency of micronucleated bone marrow<br />
polychromatic erythrocytes . These results suggest lack of genotoxic potential for<br />
the test chemicals .<br />
204<br />
MOLECULAR ORBITAL AND GEOMETRICAL STRUCTURE DESCRIPTORS IN GSAR STUDIES : MUTAGENICITY<br />
OF SOME AMINO- AND NITRO-BENZENES<br />
V .K . Gombar, K . Enalein<br />
Health Designs, Inc ., 183 East. Main Street, Rochester, NY 14604<br />
Topology-based structure quantifiers are commonly employed In QSAR studies for the<br />
ease <strong>and</strong> speed of their computation . They, however, by definition, do not encode<br />
structural information pertaining to the spatial arrangement of atoms . A study has been<br />
conducted to investigate the importance of descriptors derived from three-dimensional<br />
atomic coordinates in QSAR models . A set of 43 amino- <strong>and</strong> nitro-benzenes (X- 0-NYZ)<br />
tested for mutagenic response of Salmonella tester strain TA100 (rat liver S-9, Aroclor<br />
1254 induced) has been selected for the study . Twenty-aix of these were labeled +ve <strong>and</strong><br />
17 -vP . The geometry-based parameters investigated Include shape parameters viz .<br />
molecular surface area <strong>and</strong> volume, principal moments of inertia, etc . <strong>and</strong> electronic<br />
parameters such as Atomic charges, dipole moment, orbital energies, etc . Topological<br />
shape descriptors K (kappa) give a poor correlation (n = 40 ; m- 5 ; r z 0 .486) . A<br />
marginal improvement is observed with group surface area <strong>and</strong> volume (n : 40 ; m c 5 ;<br />
r = 0 .583) . However, the two types combined yield a significant improvement (n a 40 ;<br />
m = 8 ; r = 0 .738) . Similar comparison of QSAR equations obtained using other electronic<br />
<strong>and</strong> geometrical descriptors will also be presented .<br />
205<br />
DEVE)APMRNT OF' AN IMMUNOASSAY TO MONITOR EXPOSURK TO REN7.O(n)PYRENH BY MHASUREMF.NT OF<br />
URINARY METABOLITES M . Gomes, T . Vo--Dinh <strong>and</strong> R . M . Santclla, Oak Ridge National<br />
Laborat.ories, Oak Ridge, TN (USA) <strong>and</strong> Columbia University, New York, NY (USA)<br />
The aim oi' this study was to develop an i.munoassay to monitor human exposure to<br />
benzo(a)pyrene (Bt') by meaaure.ment of BP <strong>and</strong> its metabolites in urine . A monoclonal<br />
antibody was produced from the spleen cells of Balb/c mice immunized with 6-amino-BP<br />
covalently coupled to bovine serum albumin . The most sensitive antibody, 4U5, was<br />
characterized in terms of sensitivity <strong>and</strong> specificity by competitive enzyme-linked<br />
immunosorbentt assay (ELISA) . The antibody recognizes BP (50% inhibition at 4 pmolo) as<br />
well as a number of BP metabolites . With BP phenols including 1-OH, 3-OH, 4-OH <strong>and</strong> 5-<br />
OH, 50% inhib .ition occurs at. 20 . 90, 60, <strong>and</strong> Hpmol, respectively . With BP-1,8--diol<br />
<strong>and</strong> 9,10-diol <strong>and</strong> 7,6,9,10-tetraol, 50% Lnhibition is at 1 .4, 4 .5 <strong>and</strong> 1 .0 paolo,<br />
respectively . Crossreactivity was also seen with several other polycyclic aromatic<br />
hydrocarbons (PAHs) including pyrene (60% inhibition at 1 .6pmol), 1-aminopyrene (60%<br />
inhibition at 0 .49pmo1) <strong>and</strong> 7,12-dimethylbenz(n)anthrecene (50% inhibition at 67pmol) .<br />
These results indicate that this assay will detect multiple PAH metabolites .<br />
Validation of the assay was carried outt on ∎ice treated with [3H) RP . Urine was<br />
treated with beta--glucuronidase <strong>and</strong> arylsulfatase <strong>and</strong> BP <strong>and</strong> its metabolites isolated<br />
by affinity chromatography on ScpPek cartridges . Samples were counted <strong>and</strong> analyzed by<br />
competitive ELISA using BP in the st<strong>and</strong>ard curve . These studies indicated that the<br />
imaunoassay detected about 54% of the adducte measured by radioactivity . This assay<br />
will be used to analyze urines from populations with high exposure to PAHs including<br />
BP . Because of the broad specificity of the antibody, absolute quantitation of PAH<br />
metabolites may not be possible . This work was supported by a grant from NIOSH-02622 .<br />
50869 3584
206<br />
METABOLISM AND ACTIVATION OF FOOD MUTAGENS<br />
N .J . Gooderham, B.P. Murray, S. Murray, A .R . Boobis <strong>and</strong> D.S. Davies. Department of Clinical Pharmacology,<br />
Royal Postgraduate Medical School, London, W12 ONN, UK .<br />
There is considerable evidence that diet plays a major role in the aetiology of cancer in man . In addition<br />
to naturally occurring carcinogens present in food, a number of heteroaromatic amine carcinogens are known<br />
to be formed during the cooking process . These include imidazoquinoline/ lmidazoquinoxaline (IQ<br />
compounds) which are of particular interest due to their high mutagenic potency . Consumption of cooked be te<br />
containing the IQ compound 2-amino-3,8-dimethytitnidazo[4,5-fiquinoxaline (MeIQx) at levels of 0 .8 -<br />
2 .sng/g beef, resulted in systemic exposure, evidenced by urinary excretion of 1 .3 - 4 .9% of the dose as<br />
unchanged amine . Studies in animals have shown that MelQx is almost completely absorbed but then undergoes<br />
extensive metaboli m, with only small amounts of unchanged amine excreted in urine . After administration<br />
(oral, ip or iv) of [i4C)Me1Qx (2 - 60mg/kg) to mice, 25 - 35% of radioactivity was excreted In urine <strong>and</strong> a<br />
further 35-45% was eliminated in faeces within 24 h . Urinary metabolites of MelQx included N-acetylsted,<br />
N-sulphated, N-glucuronidated, ring N2-demethylated, C8-hydroxylated <strong>and</strong> ring CS-hydroxylated<br />
derivatives . IQ compounds are readily activated by liver microsomal fractions to derivatives which are<br />
genotoxic in the Ames Salmonella typhimurium test . Human liver microsomes are particularly efficient at<br />
activating these amines. The primary activated product of microsomal oxidation is thought to be the exocyclic<br />
amino N-hydroxy derivative . Studies of hepatic microsomal activation of IQ compounds, employing<br />
monoclonal antibodies <strong>and</strong> specific chemical inhibitors, have shown that they are converted to mutagenic<br />
derivatives by a monooxygenase system predominantly involving the cytochrome P-450 isozyme orthologous<br />
to rat P-450d .<br />
207<br />
MUfAL2I1S IN 1QNYAN '1RADITIQiAI. MICIId: PtEC3IIATiQS .<br />
H .N.B . Copalan <strong>and</strong> A.N . WairimA, University of Nairobi, Department of Botany, P .O. Haet 30197, Nairobi<br />
(Kenya)<br />
Several plant praducts are used in Kenyan traditianal medicine which is popular both in rural <strong>and</strong><br />
urban centres . 7his form of inedicine is gaining raamenum due to goverrnrntal support <strong>and</strong> active<br />
interest in it from both physicians acd pharmacologists . Several of the plant producta are in camen<br />
usage . Houever, their effects have not been vigorausly tested . In the current study, the fresh sap fram<br />
Aloe graminicola, ard the sethanol extrazts of Amona aenegelensia, Centella asiatica, Msess lanceolat :a,<br />
sine africana ~d I rica salisifolia Which are all extensively used in Benyat tr~aditional medicine,<br />
were tested for their garotoxicity using the Nmes test <strong>and</strong> the Vicia faba root meristem assay . TA 104<br />
yielded the highest nuaaer of revertants vith soet extracts . TA 102 detected aame but not as<br />
effectively as TA 104 . A reduction in nuober of revertants at higher does uas noticed . Apart from<br />
M. lanceolata, all other plant extracts induced abrotael metaphaaes in V . faba root taeristem cells,<br />
after 2 hrs of treatrrent . After recovery, only the extracta from C.aas'iatTc-a-, M . africata <strong>and</strong><br />
M . salisifolia caused statistically significant increase in chromosoae aberrations . Fcesh extract of<br />
A . graminicola induced micracuclei formation after recovery . 'ilau several plants used in itenyan<br />
craditional medicine appear to erfiibit m¢agatic potential . Further atudies using pirified etaaacts<br />
are needed before recanendations regarding eotuinued use can be made•<br />
* lbrk foras part of the requirarents for the degree of Master of Science of the University of<br />
Nairobi .<br />
208<br />
A FORWARD Ml1TATIONAL SYSTEM TO DF.TECT FRAMESHIFTS WIT1i1N A IRO BASB-PA1R TARdI:T<br />
OF F.schcrichia cnlr. Alasdair J.F C)ordon, Jennifer A. Halliday, Michael J . Horsfall <strong>and</strong> Barry W .<br />
Olickman . Dept. of Biology, York University Toronto Canada M3J 1P3 .<br />
A forward mutational system is described which is based on the acquisition of lacl negative<br />
dominant properties as the resolt of secondary frameshift mutation (of either sign) within that region of<br />
the lacl gene that encodes the DNA-binding domain of the lac repressor . Strains containing both I'd<br />
(dominant-negative) <strong>and</strong> wild-type alleles of the lacl gene exhibit constitutive synthesis of Qgalactosidase<br />
<strong>and</strong> thereby grow on agar plates in which the non-Inducing sugar phenyl-i3•D-galaetoaide is<br />
the sole carbon source . A consequence of -1 frameshifts in the initial 180 base-pain of lacl is the inframe<br />
creation of translation termination signals. Translation reinitiation at codons Va123, Met42 or<br />
Leu62, results in repressor fragments that confer the 1'd phenotype since the ability to aggregate fa<br />
function of the core domain) is not impaired . However, the first in-frame stop codon which results from<br />
a+ I frameshift is at buse-pair 277 t1legl/Lyag4Y <strong>and</strong> therefore translational reinitiation will not produce<br />
the intact core domain required for an 1'd phenotype . Utilising an F7acl factor with a +A frameshift at<br />
position 46 (recessive negative) we have devised a test which specifically selects irattteahifts as events<br />
that yield the I-d phenotype. Within the 181) base-pair target frameahifts of rither sign 1+ I or -1) cnn be<br />
detected ; -1 events restore the reading frame of the core domain ; +1 events in conjunction with +A46<br />
exhinit the con.equences of a-1 frameshift. In either aae, some portion of the DNA-hinding domain will<br />
be non-functional <strong>and</strong> hence the repressor will be 1-d . This selection system In combination with routine<br />
cloning <strong>and</strong> sequencing technologies will facilitate the rapid colliection <strong>and</strong> characterisation of the large<br />
numbers of mutations required for studies of mutational specificity .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts<br />
Notes<br />
73
74 1989 EMS Abstracts 209<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Note s METHYL ISOCYANATE'EXPOSURE IN BHOPAL HAS DAMAGED PLANT AND HUMAN CELLS<br />
H .K .GOSWANI, PUNEET, RAJEEV GOSWAMI, NANOJ CHANDORKAR AND L .K .SENGUPTA,<br />
DEPARTMENT OF GENETICS, BHOPAL UNIVERSITY, BHOPAL 462 026, INDIA .<br />
A muttidLsciplinaay etudy conducted 6ince the ga4eou4 expoeuae on the<br />
2nd Dec .1984, haa pnoved beyond doubt that methyl ieocyanate (I/IC) hae b4ought about<br />
biotogical damagee . The aeeed.ament o6 the potential damage6 by SCE 6aequencEee <strong>and</strong><br />
chaomoeomal abe4aatione in human lymphocyte cultuaea have been eetimated a6tea etudying<br />
200 peneona by Giemoa b<strong>and</strong>ing . In oadeR to eompute4ibe the data on epeeisic metaphade<br />
chROmoAome4, 100 micaophotogaaphe, each baom ga4 expoeed <strong>and</strong> otheRwiee no4eal donoita<br />
weae uaed . Thie gave a highly etatietiealty aigni6ieant aatio that chaomoeome 9,<br />
11 <strong>and</strong> 16 aae mo4t paedominantly ab6eeted .<br />
The MIC expoauae have baought about ala4ming changea in haemogtobin .<br />
Exten,6ive .6tudiee on moae than 1,400 peaeon4 by cellogel electaophoaetic eepenation<br />
have aevealed the 11ae o6 Foetat haemoglobin (HbF) in adult aamplea .<br />
Tideue cultuae etudie4 os Datbea ia aieeoo, have ehown dib6eaential gaowth<br />
in MIC expoeed explant when compaaed to eon ao undentical eultuae condEtion3 .<br />
One o6 the modt impoatant in6eaeneee, theae6oae, appeaad to be that Nethyl<br />
idocyanate might be mutagenic undea ceatain conditione .<br />
210<br />
MUTATION ASSAY FOR PERSONAL AIRBORNE PARTICULATE SAMPLES BY A HIGHLY SENSITIVE ULTRA-<br />
MICRO FORWARD-MUTATION METHOD. S . Goto ; Y . Takagi ; H . Murata ; J . Levtas ; <strong>and</strong><br />
H . Matsushita ; 1National Institute of Public Health, Minato-ku, Tokyo (Japan),<br />
zSchool of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa (Japan), <strong>and</strong><br />
3 Health Effects Research Laboratory, Research Triangle Park, NC (U .S .A .) .<br />
Actual sensitivity of forward-mutation method using SaUnonsZEa typh*merium TM677<br />
was improved remarkably by the modification of preincubation step, where 10 yil of the<br />
solution containing tester strain <strong>and</strong> a test chemical was incubated at 37'C for 2 hr .<br />
Actual sensitivity of this ultramicro forward-mutation method was about 10 times higher<br />
than the micro forward-mutation method (volume of the mixed solution in the preincubation<br />
step : 100 p1) <strong>and</strong> about 100 times higher than the original method (1000 ) :1) . This<br />
method can measure mutagenicity of extracts from airborne particulates obtained by only<br />
3 cubic meter air sampling . This method was applied to the survey of mutagenicity of<br />
airborne particulate samples collected by personal air sampler at 1 .5 - 2 1/min for 24<br />
hr . Mutagenic activity of these personal air samples was in the following order ; smoker<br />
(10 cig/sampling), smoker(6 cig .), passive smoker(5 cig .) <strong>and</strong> non-smoker/non-passive<br />
smoker during sampling, irrespective of presence <strong>and</strong> absence of S9 mix . It was also<br />
found that mutagenic activity of airborne particulates collected in Washington D .C .,<br />
U .S .A . was higher in the test condition with S9 mix than without S9 mix . On the other<br />
h<strong>and</strong>, mutagenic activity of airborne particulates collected in Tokyo, Japan was nearly<br />
same or higher in the test condition without S9 mix than with S9 mix, suggesting<br />
difference in species of mutagens in both the airborne samples .<br />
211<br />
EFFECT OF METHYL ISOCYANATE ON PROLIFERATICN AND DI6'FEREIJfIATICN OF MOUSE MUSCLE<br />
CELLS IN VITRO<br />
Shobha GoTyFe-, -School of Life Sciences, Jawaharlal Nehru University, New Mehrauli<br />
road, New Delhi-110067, India .<br />
One of the striking features of the Bhopal disaster in which several thous<strong>and</strong><br />
people were killed or injured was the lack of toxicological information on the<br />
causal agent methyl isocyanate (MIC) . This incident has raised new concern<br />
over MIC as a toxicant . To ascertain the morphological <strong>and</strong> functional sequalae<br />
of a single MIC exposure to the mamealian skeletal muscle, the neonatal rat<br />
muscle in vitro was exposed to different concentrations of MIC ranging from<br />
0 .025 -6.5~~ ml medium . The drug was suspended in the growth medium . It<br />
was added to the cultures at the end of days 1 to 6 . The effects of MIC were<br />
dose dependent . Fusion of myoblasta was inhibited at 0 .125 <strong>and</strong> 0 .25 yl, <strong>and</strong><br />
at 0 .5 u1 cells were completely destroyed . Lower concentrations of MIC allowed<br />
cell fusion to occur, but the total cell count was significantly low . This<br />
would suggest either an effect on muscle differentiation or a selective toxicity<br />
towards myoblasta .<br />
212<br />
ANALYSIS OF STRUCTURALLY RELATED COMPOUNDS IN THE DROSOPHILA WING<br />
SOMATIC MUTATION AND RECOMBINATION TEST (SMART) . U . Graf, A . Alonso<br />
Moraga <strong>and</strong> N . van Schaik, Institute of Toxicology, ETH <strong>and</strong> University<br />
of Zurich, Schwerzenbach, Switzerl<strong>and</strong>, Department of Genetics,<br />
Faculty of Sciences, University of Cordoba, Spain, <strong>and</strong> Department of<br />
Genetics, University of the Witwatersr<strong>and</strong>, Johannesburg, South Africa .<br />
50869 3586
1989 EMS Abstracts<br />
The test for somatic mutation <strong>and</strong> recombination in cells of the Notes<br />
wing imaginal disc of Drosophila melanoyaster makes use of the two<br />
recessive markers Dltitl (multiple wing hairs) <strong>and</strong> = (flare) on the<br />
left arm of chromosome 3 . In cells trans-heterotygous for the two<br />
markers, mutant clones can be induced which differentiate into somatic<br />
spots on the wings of adult flies . Twin spots, consisting of an MWh<br />
<strong>and</strong> aLIX clone, result from mitotic recombination ; mwh (<strong>and</strong> f-U)<br />
single spots are due to point mutation, deletion, or mitotic recombination<br />
. Compounds are tested by acute (2 to 6 h) or chronic (48 h)<br />
feeding of 3-day-old larvae . Three different groups of structurally<br />
related compounds have been tested : Alkylating agents (nitrogen mustard<br />
<strong>and</strong> half mustard, three N-nitroso-guanidines), 5-nitrofurans<br />
(nifurtimox <strong>and</strong> eight related compounds), <strong>and</strong> tricyclic antidepressants<br />
(amitriptyline, desipramine, imipramine, nortriptyline, protriptyline)<br />
. The results obtained demonstrate that the Drosophila wing<br />
spot test is ideally suited for this type of study due to its fast <strong>and</strong><br />
easy performance .<br />
213<br />
T:fE USE OF TRADESCANTIA AND VICIA FASA BIOASSAYS FOR THE IN p7 DETECTIO1 OF<br />
MUTAGEdS IN AN AQUATIC ENVIRONMEiVT . Ff . F . Grantl, H . G . Lee , D .M . Logan <strong>and</strong> M .F .<br />
Salamone3, IMcgill University, Montreal (Canada), 2York University, North York<br />
(Canada), <strong>and</strong> Ministry of the Environment, Toronto (Canada)<br />
Most studies with plant bioassays have been carried out in the laboratory, or<br />
under atmospheric in situ conditions . The primary purpose of this study was to gain<br />
some experience with plant mutagenicity bioaasays under in situ aquatic conditions .<br />
The assay systems teated were the Tradescantia stamen hair <strong>and</strong> micronucleus assays<br />
for the detection of gene mutations <strong>and</strong> chromosomal aberrations, respectively, <strong>and</strong><br />
the Vicia faba bioassay system which detects chromosomal aberrations in root tips .<br />
The assays were used to test the effluent from a pulp <strong>and</strong> paper mill locatqd on the<br />
north shore of Lake Superior . Assays were performed in a creek containing raw<br />
effluent <strong>and</strong> in a bay of Lake Superior into which the creek emptied . All in situ<br />
treatipents were carried out for 24 hours . On the creek, 11 .5 km from the source, the<br />
effluent was toxic to the Vicia faba roots as expresse# by a reduction in the mitotic<br />
index . The effluent in the creek induced a significant increase in the number of<br />
stamen hair mutants, micronuclei, <strong>and</strong> chromosome aberrations . Likewise in the bay,<br />
witn the exception of the two most distant sites, the creek effluent also induced<br />
increases in stamen hair mutants, micronuclei, <strong>and</strong> chromosome aberrations .<br />
Tradescantia tested in the laboratory several days later with water from the test<br />
sites, demonstrated decreased mutagenicity compared to the in situ tests . This<br />
suggests that at least part of the mutagenic fraction is unstable <strong>and</strong> indicates the<br />
need for aquatic testing either in situ or as soon after sampling as possible .<br />
214<br />
THE CARCINOGENICITY INFORMATION DATABASE OF ENVIRONMENTAL SUBSTANCES (CIDES) AND THE<br />
ASSESSMENT OF POSSIBLE HUMAN RISK . Mildred R . Green, Technical Database Services,<br />
Inc ., 10 Columbus Circle, New York, NY 10019<br />
The Carcinogenicity Information Database of <strong>Environmental</strong> Substances (CIDES) contains<br />
numeric data on the effects of approximately 1000 known or suspected environmental<br />
carcinogens : cancer-relevant data covering a wide variety of long- <strong>and</strong> short-term<br />
tests <strong>and</strong> physical/environmental property data . The carcinogenicity data are organized<br />
into six categories - whole animal carcinogenesis, mammalian call lines, mammalian<br />
tissues or cells, bacterial tests, all other assays <strong>and</strong> human epidemiological<br />
studies . Searches can be directed to specific areas of interest, e .g ., species, route<br />
of entry or assay type . A unique feature in CIDES is that the collected studies are<br />
analyzed to provide a rating, the Carcinogenicity Ratio(CR), summarising the likelihood<br />
<strong>and</strong> the weight of the evidence(w) that a substance is a carcinogen . The CR is the<br />
means of the percent positive studies weighted by a factor that varies with teat category<br />
; the w is based upon the extent of the testing . When thie approach was applied<br />
to known human carcinogens, the CR was consistently above 50 . For example, for bensidine<br />
<strong>and</strong> 2-naphthylamine the calculated CR/v ratios summarizing all the evidence included<br />
in CIDES were 84 .9/94 .0 <strong>and</strong> 65 .6/83 .0 respectively . However, CR/v values may<br />
vary as new studies are added to the database . This analysis can be used to identify<br />
compounds for which data on human exposure are not available but whose test results<br />
indicate that they are likely to be carcinogenic, <strong>and</strong> also, compounds which should be<br />
tested furthtfi to improve the weight of the evidence . CIDES will be available through<br />
the Numerica online service of Technical Database Services in the summer of 1989 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
75
76 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
215<br />
THE CARCINOGENICITY PREDICTOR : AN ONLINE ANALYSIS BASED UPON THE RESULTS OF SHORT-TERM<br />
GENOTOXICITY TESTS . Mildred R . Green, Technical Database Services Inc ., 10 Colusobus<br />
Circle, New York, NY 10019<br />
The Carcinogenicity Predictor is a computerized package based upon the CPBSTM research<br />
model developed at Case Western Reserve University(CWRU) by H . S . Rosenkranz <strong>and</strong><br />
others . The program calculates the probability that a chemical is carcinogenic from<br />
results of short-term genotoxic assays . The user identifies one or more tests in a<br />
battery, indicates the results (positive or negative) for each one, <strong>and</strong> specifies the<br />
prior probability of carcinogenicity for the chemical in question . The prediction of<br />
probability results from a Bayeslan analysis of the information input <strong>and</strong> of data quantifying<br />
the sensitivity <strong>and</strong> specificity of each assay which is stored in the package .<br />
The sensitivity <strong>and</strong> specificity values reflect the performance of each genotoxic test<br />
in distinguishing between known carcinogens <strong>and</strong> noncarcinogens . Currently, the Carcinogenicity<br />
Predictor contains a database of sensitivities <strong>and</strong> specificities for 32 different<br />
assays ; updated values will be added as a result of further research at CSiRU .<br />
However, users have the opportunity to create their own databases if they want to use<br />
other values for a particular test . Calculations may be obtained at a single value of<br />
prior probability (the default of 0 .5 or some other value chosen by the user) or as a<br />
graph <strong>and</strong> a table of predicted probabilities of carcinogenicity at 11 values of the<br />
prior probability from 0 .0 to 1 .0 . Recent enhancements to the package include the development<br />
of a user interface which permits the evaluation of genetic toxicity via remote<br />
accew The new version of the Carcinogenicity Predictor is available on the<br />
Numerica online service of Technical Database Services .<br />
216<br />
COMPLEMENTATION ANALYSIS OF X RAY SENSITIVElARA-C RESISTANT HAMSTER CELL<br />
(AraC 213) - HUMAN ATAXIA TELANGIECTASIA HYBRIDS . Gloria Greer <strong>and</strong> R. Julian Preston,<br />
Univ. of Tennessee Biomed. Grad. Sch . <strong>and</strong> Biology Division, ORNL, Oak Ridge, TN 37831 .<br />
We have recently isolated an ara-C resistant X ray sensitive cell line, AraCa 213. Initial studies had been<br />
performed with the cell line Ara 2 .1 (derived from a CHO-KI cell line) which was screened for X ray<br />
sensitivity after selection for ara-C resistance at a concentration of S x 10' M . This mutant cell line was<br />
mutagenized a second time with EMS <strong>and</strong> selected at a concentration of 10° M ara-C in an effort to isolate<br />
a mutant which would exhibit an increase in X ray sensitivity . Thiss was found to be the case . This hamster<br />
mutant cell line (AraC 213) shows an increased sensitivity to X ray-induced chromatid-type aberrations for<br />
G, exposures, as is also the case with ata :aa telangiectasia (AT) cells. Other characteristics of AraC" 213<br />
are similar also to those of AT. Therefore, cell fusion etperiments were performed using K1-CHO hgprt'<br />
or AraC` 213 with a wild type human lymphoblastoid cell line (GM606), or an AT lymphoblastoid cell line<br />
(GM717). Complementation analysis of the hamster-human hybrids was assessed by colony forming ability<br />
after exposure to X rays. Preliminary experiments show that the CHO-AT hybrid has the same sensitivity<br />
as the normal hamster cells, while the AraC` 213-AT hybrid showed no complementation ; it was still X ray<br />
sensitive . These results suggest that there might be a common repair deficiency in the ara-C resistant mutant<br />
<strong>and</strong> the AT cells. Additional experiments will further characterize the defects such that AraCR 213 can be<br />
used to isolate the human repair gene that reverts the ara-C resistance phenotype <strong>and</strong> also the AT X ray<br />
sensitivity . (Operated by Martin Marietta Energy Systems, Inc . under contract DE-ACOS-840R21400 with the<br />
U. S. Dept. of Energy. GG is supported by NIH-CA08104-13) .<br />
SITE-SPECIFIC MUTAGENESIS . Arthur P. Grollman, SUNY at Stony Brook . Stony Brook. NY<br />
11794.<br />
Chemical methods have been developed by which nucleoside adducts <strong>and</strong> related lesions<br />
may be incorporated at defined positions in DNA. Biological systems have been devised which<br />
allow mutagenic spectra to be determined, site-specifically, in bacteria <strong>and</strong> mammalian cells as<br />
follows : A duplex oligodeoxynucleotide containing an adduct or other lesion is ligated into an<br />
SV40-based shuttle plasmid vector, then used to transform bacteria or transfect simian kidney<br />
(COS) cells. Mutations are fixed during plasmid replication . Progeny DNA. recovered from a<br />
COS cell lysate, is amplified in F. coG, then digested by a restriction enzyme known to cleave within<br />
a unique nucleotide sequence that includes the site of adduct modification. Circular DNA, obtained<br />
from mutant plasmids, is amplified preferentially by transforming competent strains of E coJl .<br />
Specific mutations are identified by oligonucleotide hybridization techniques. The advantages of<br />
this experimental system include the homogeneity of input DNA, lack of bias in the procedure used<br />
for detection of mutations <strong>and</strong> the potential for systematically altering base sequence in the vicinity<br />
of the adduct . This approach has been used to establish the mutagenic specificity of abasic sites,<br />
selected bulky <strong>and</strong> exocyclic DNA adducts <strong>and</strong> to explore molecular mechanisms Involved in<br />
chemical mutagenesis .<br />
217
218<br />
CYTOGENETIC ACTION OF METHYLMERCURY CHLORIDE IN HUMAN LYMPHO-<br />
CYTES,<br />
Helena Groot de Restrepo <strong>and</strong> Luz A . Carvajal de Gil . Laboratorio de Genbtica<br />
Hurnana, Universidad de los Andes, A .A . 4976, Bogot3, Colombia .<br />
An in vitro study was carried out to analyse the effects of inethylmercury chloride<br />
(MM) in hurnan lymphocyte txaltures . Analyses of cell cycle kinetics, chromosome<br />
aberrations <strong>and</strong> sister chromatid exchange (SCE) were performed . The results<br />
show a dose effect relationship in tlie cell proliferative processes . No<br />
statistically difference was observed for the frequency of SCE in the cultures exposed<br />
to different concentrations of MM (dose-response curve) non in the exposed<br />
<strong>and</strong> control cultures (blood from 10 donors) . However the dose-response curve<br />
at one of the concentrations of MM used (1 .26 ug/ml) show a significant increase<br />
of SCE <strong>and</strong> a higher replication index (RI) . A significant increase in the frequency<br />
of chromatid type aberrations was found in the MM exposed group .<br />
219<br />
INSERTIONAL MUTAGENESIS OF HUMAN CELLS USING RETROVIRUS SHUTTLE VECTORS<br />
Andrew J . Grosovskyt, Linda S . Rosst, Barry W. Glickmanz <strong>and</strong> Adonis Sk<strong>and</strong>alis2, tUniversity of<br />
California, Riverside, California 92521 <strong>and</strong> 2York University, Toronto, Ontario M3J 1P3<br />
Insertional mutagenesis is a powerful approach for identifying novel genes with an identifiable phenotype<br />
because genes of interest may be simultaneously inactivated <strong>and</strong> tagged for cloning as a consequence of the<br />
insertion . Although this strategy has been widely used in prokaryotes <strong>and</strong> lower eukaryotes, its applicability<br />
in mammalian systems has been limited by the lack of suitable insertion elements . Retrovirus based shuttle<br />
vectors are, however, well suited for this role since they infect a broad range of host cells at high efficiency,<br />
integrate into the host genome at low copy number <strong>and</strong> at r<strong>and</strong>om, <strong>and</strong> encode selectable markers within the<br />
vector genome.<br />
We present here a characterization of insertional mutagenesis of the human B lymphoblastoid cell line<br />
TK6 following infection with the retrovirus shuttle vecto pZipNeo . TK6 cultures were exposed to pZipNeo<br />
infection for periods ranging from 12 to 72 hours . A population which contained stably integrated provirus<br />
was selected by exposing the infected population to G418 ; G418 resistance is encoded by the neo glne carried<br />
in the vector genome . The infection efficiency in the exposed population, as estimated by determining the<br />
frequency of G418 resistance in a clonal assay, ranged up to 3% in cells exposed for 72 hours. Insertion<br />
mutation frequency was monitored using several endogenous selectable markers available in TK6 cells (aprt,<br />
hpri, tk, Na'/K- ATPase) . An exposure time dependant induction vyas seen for the first three markers .<br />
Induction ranged from 6 to 30 fold following a 72 hour infection period . A small induction was also<br />
observed at the Na'/K" ATPase following 24 hours of infection ; this may be attributable to effects on gene<br />
expression or gene amplification associated with retroviral integration . '<br />
Experiments are now being conducted to determine the percentage of mutants recovered following<br />
retrovirus exposure which are directly attributable to disruption of the coding sequence<br />
by a provirus, <strong>and</strong> to determine the provirus copy number after various infection periods .<br />
220<br />
G---7CTOXIC :T'_ Or FESTICL0:S N•TD P :J :a'T_^ SY31'»2ii<br />
: . :;, Gt~)v:: .2, School of Life Sciences, 3uru Nanak -'ev University,<br />
q,-,rit ;ar-143005, In3ia .<br />
Several short term assays encompassing all the maior tax>nomic groups<br />
hav' been rsaommen3e3. Although plant 3ystems are being use3<br />
successfull_v by a ntmtber of workers, yet the scepticism about it<br />
Persists . A comparative account of the cp_notnxicity of 20 pestici3es<br />
workz3 out by cytogenetic assays in root meristems <strong>and</strong> pollen mother<br />
cells in &li_~rn ceDa/Hor3_e_tlm W19 ra , Ln<br />
chromosomal aberration<br />
<strong>and</strong> microzuclel assays in bone marrow calls in rats an .9 histidine<br />
r=version assa :• in j~1lmonel_l:a_ tyaUmu~j
78 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
221<br />
SEL3CPIVE ANTIMUTAUENIC ACTZVITY GF TERKNALIA 1C~E9J~L! IN SALhiONEL14<br />
TyyUlhUk2= . I .S .GRLVER <strong>and</strong> Saroj bAl,p . School of Lite Sciences, Guru<br />
Nanak Lev University, Amritsar-143005, India .<br />
Tecmiralin chebula is a eommon medicinal plant whose fruit is used in<br />
"Ayurvedic" <strong>and</strong> "Unani' system of medicine as caaninative, exFectorant<br />
<strong>and</strong> gastric disorders . Antimutagenicity of its water <strong>and</strong> chloroform<br />
extracts of dried f wits was determined against two direct acting<br />
mutagens, sodium azide <strong>and</strong> 4-nitro-o-phecylenediamine (NPD) in strains<br />
TA1o0 <strong>and</strong> TA1535 <strong>and</strong> TA97a <strong>and</strong> TA98 respectively <strong>and</strong> S9 dependent<br />
mutagen 2-aminofluorene (2-AF) in TA97a <strong>and</strong> TA9S of ~ .S,ynhimurium . It was'<br />
obse rved that water ext ract s reduced NrL i nduced hi a revertant s i n both<br />
TA97a <strong>and</strong> TA98 significantly but did not have any perceptible effect<br />
against sodium az ;de induced his+ revertant in TA100 <strong>and</strong> TA1535 of<br />
.Z,tvl)hlm yrium, The pre-incubation studies where the extract was incubated<br />
at 370C for 30 minutes with the said mutegen prior to plating enhanced<br />
the inhibitory effect . Autoclaving the water extract reduced its effect<br />
but the reduction was not significant . The water extracts inhibited signi .<br />
ficantly 2-AF reduced his+ revertants too in all the strains tested but<br />
its effect inTA98 was striking as it reduced the revertants to spontaneous<br />
level . No inhibitory effect was observed in any of the strains <strong>and</strong> against<br />
all the test mutagens with chloroform extract . It appears that inhibitory<br />
factor (antimutagen) is water soluble, heat stable <strong>and</strong> desnutagen .<br />
COMYARISON OF TF1E ANTIMITTAUENIC EFFECT ( .F WACER Ey.TRAGTS OF TWU<br />
DIFFEFtEhT VARIETIES OF INLIAN CGUSEbF :NIQf . I .S .GRGVER 6, AjpLMD liaur,<br />
School of Life Sciences,Guru Nanak l :ev University, Amrit :.;ar, 1NDIA .<br />
Indian gooseberry (Llnbiica Officinalis) is a very comnon medicinal<br />
plant <strong>and</strong> its fruit is thv richest source of Vitamin C . Nntimutagenicity<br />
ot the wate r extracts ot its two difterent varletiess was<br />
tested against two direct acting.mutagens, sodium azido <strong>and</strong> 4-nltrcL<br />
o-phenylenediaminr in TA1535 <strong>and</strong> 'l'A98 respectively using thn Ames<br />
test . water extract of var .-I reduced the his+ revertants in TA98<br />
upto 5r/. but its effect was not significant in TA1535 . Pre-incubation<br />
of the extract with the mutagen at 370C for 30 min did~not have a<br />
significant eff--ct in TA98 but reduction'of his+ revertants in TA1535<br />
was . enhanced by pre-incubating the extract with sodium azide . Auto-oclaving<br />
of ext ract reduced its inhibitory effect in TA98 but promoted<br />
its effect in TA1535 . Water ext ract of var .-II showed a maximum of<br />
52% inhibition in the sodium ezide inLluced frequency of his+<br />
revertants in TA1535 but its effect was corvparatively less in TA98 .<br />
P re-incubation of the extracts with the mutagen increased the<br />
inhibitory effect in both the strains . 1Heating of the extracts did<br />
rnt have -a significant effect on its inhibitory activity . Thus it<br />
may be concluded from the results that both th ese varieties may have<br />
different nutagenic facto rs .<br />
222<br />
A GENETIC ASSAY FOR TNE DETECTION OF INEUPLOIDY AN? CLASTOGENICITY USING A<br />
NONOCBRONOSONAL HYBRID CELL LINE . R . 6ud1 , R .S . Athwal . <strong>and</strong> S .S . S<strong>and</strong>hu2 . 1Nsw<br />
Jersey Medical School, Newark, NJ 07103 USA ; 2U .S . <strong>Environmental</strong> Protection Agency,<br />
RTP, NC 27711 USA .<br />
We have developed an assay for induced chromosomal anomalies based on the<br />
quantitation of loss of a chromosome or chromosomal segment by growth of cells in<br />
selection media . For this purpose, a mouse/human hybrid cell line containing human<br />
chromosome 5 as the only human component has been produced . This chromosome S<br />
carries two dominant selectable markers, Ecogpt, <strong>and</strong> a gene for sensitivity to<br />
diptheria toxin (DTs) . Cultivation of these cells in DNEN containing mycophenolic<br />
acid <strong>and</strong> xanthine (NX medium) selects for Ecogpt <strong>and</strong> thus for the retention of<br />
chromosome 5 . The growth of these cells in the medium containing 6•thioguanine <strong>and</strong><br />
diptheria toxin (DT) gives the frequency of chromosome loss . Similarly, growth of<br />
hybrid cells in MX medium containing DT (10'10l1) selects for the cells that have lost<br />
the segment of chromosome 5 carrying DTs gene while retaining Ecogpt . This provides<br />
a method to quantitate induced chromosome breaks . Preliminary results using model<br />
compounds show a dose-related response for induced aneuploidy <strong>and</strong> chromosome breaks .<br />
The same cell line can also be used to quantitate induced point mutations by growth<br />
in the medium containing ouabain (10-3M) . A unified genetic assay for multiple<br />
endpoints will enable the evaluation of test chemicals at doses relevant to human<br />
exposure . (This is an abstract of a proposed presentation <strong>and</strong> does not necessarily<br />
reflect U .S . EPA policy .)<br />
50869 3590<br />
223
224 1989 EMS Abstracts 79<br />
ACTIVATION OF MUTAGENS BY HUMAN CYTOCHROME P-450 ENZYMES . F .P . Guengerich, T. Notes<br />
Shimada, M . Iwasakl. <strong>and</strong> M .V. Martin. Dept. of Biochemistry <strong>and</strong> Center in <strong>Molecular</strong> Toxicology .<br />
V<strong>and</strong>erbilt University, Nashville . Tennessee 37232<br />
Cytochrome P-450 (P-450) enzymes have been implicated in the In otbro bloactivation of mutagens<br />
<strong>and</strong> carcinogens (Cancer Res . 48. 2946, 1988) . The specificity of a series of the major human liver P-450<br />
enzymes towards a series of pro-mutagens was examined tn vitro using the approaches of enzyme<br />
reconstitution, correlation with marker activities in liver samples . selecUve inhibition <strong>and</strong><br />
sttmulation, <strong>and</strong> Immuno-inhibition . The endpoint used was activation of a chimeric umuC'lacZcontaining<br />
plasmid (pSK1002) in Salmonella typhtmurtum TA1535, which is Indicative of DNA<br />
alkylation. P-45OpA, the low Km phenacetin O-deethylase . is the major enzyme involved in the<br />
activation of 2-aminoanthracene . 2-acetylaminofluorene . 4-amtnobiphenyl. 2-amlrtoAuorene, <strong>and</strong> the<br />
food pyrrolysates Glu-P-1, Glu-P-2, IQ, Me1Q, MEIQx, <strong>and</strong> Trp-P-2 . P-45oNF . the nifedipine oxidase, is<br />
the major enzyme involved in the activation of 6-aminochrysene, trls(2,3-dibrompropyl)phosphate,<br />
aflatoxins B1 <strong>and</strong> G1 . stertgmatocystin, benzo(a)pyrene-7,8-diol . benzo(b)tluoranthene-9 .10-diol, <strong>and</strong><br />
7,12-dlmethyl-benz(a)anthracene-3 .4-diol. The umu system is not responsive to N-nitroaamines but<br />
other studies (Cancer Res. 88, 1499 . 1988) suggest that P-450J is involved in the activation of<br />
N.N-dimethyl, N-benzyl-N-methyl-, N-butyl-N-methyl- . <strong>and</strong> N,N-diethylnltrosamine. Our studies<br />
suggest that human P-45ODB (debrisoquine 4-hydroxylase) . P-450Mp (mephenytoin 4'-hydroxylase, <strong>and</strong><br />
P-45OTB (tolbutamide hydroxylase) do not contribute to the activation of the compounds studied . Rat<br />
P-450 enzymes are generally not unreasonable models for human orthologs, although a number of<br />
results suggest cauuon in making interspectes comparisons among P-450 gene products with regard to<br />
catalytic specificity. (Supported in part by USPHS grants CA44353 <strong>and</strong> ES00267)<br />
225<br />
MUTAGENICITY OF NITROSCANATE AND ITS PUTATIVE METABOLITES IN<br />
SAIA4JNELLA MJfATION ASSAY<br />
_R .L . Gupta, I .P. Kaur <strong>and</strong> T .R . Juneja, Department of Pharmaceutical<br />
Sciences, Panjab University, Ch<strong>and</strong>igarh-160014, INDIA .<br />
Nitroscanate, 4-Nitrodiphenyl ether, an anthelmintic drug, belongs<br />
to nitroarenes which have been reported to possess mutagenlcity<br />
<strong>and</strong> carcinogenicity <strong>and</strong> their toxicity is considered to be mediated<br />
through metabolic reduction of the nitro group . We have investigated<br />
nitroscanate (1) <strong>and</strong> its hydrolytic product amino (2) <strong>and</strong> the corresponding<br />
acetamido (3) including its reduction products formohydroxamic<br />
acid (4), nitroso (5), hydroxylamine (6) <strong>and</strong> its N-acetyl<br />
(7) <strong>and</strong> N,O-diacetyl (8) for their mutagenicity in TA98,TA98NR<br />
(nitroreductase deficient) <strong>and</strong> TA98/1,8DNP 6 (arylhydroxylamine<br />
esterifying deficient) . Nitroscanate was direct acting mutagen to<br />
only TA98 suggesting that bacterial nitroreductases <strong>and</strong> esterifyinenzymes are necessary for its activation in vitro<br />
. However, 4,9<br />
<strong>and</strong> 6 showed direct mutagenicity to TA98 <strong>and</strong> . TA98NR <strong>and</strong> the activity<br />
increased after mammalian S9 activation . In contrast, strain TA98/1,-<br />
8DNP6 was markedly resistant to these compounds . It would, therefore,<br />
appear that arylhydroxylamine esterifying bacterial enzyme Is<br />
necessary for their activation . To conclude that nitroscanate <strong>and</strong><br />
its hydrolytic product are mutagenic only after reduction <strong>and</strong> subsequent<br />
esterification .<br />
226<br />
Mtl1'ATIONAL SPECIFICITIES OF N-NITRC50 COMPOUBIDS• J .B. t;uttenplan ., Dept . of<br />
Biochemistry ., N .Y . University Dental School,, <strong>and</strong> Dept . of <strong>Environmental</strong> Medicine,,<br />
N.Y . University Medical School,~ New York,, NY (U .S .A .) .<br />
The mutational specificities of a group of N-nitroso compounds were examined in<br />
Salmonella using the system of Levin <strong>and</strong> Ames (Environ . Mutag .#, 8., 9-28 (1986) which<br />
assays the six different point mutations . All strains were uvrB <strong>and</strong> harbored pia1101 .<br />
Direct acting N-nitroso-N-alkyl derivatives of ureas anitroguanidines .. with<br />
increasing chain length ., were compared . Additionally, N-nitcosodimethylamine (NDMA)<br />
<strong>and</strong> nitrosomethylphenylamine [nitrosanethylaniline (NMA)j were studied . Consistent<br />
with previous reports, methylnitrosourea (IRiU) induced predominantly GC--AT<br />
transitions . Ethylnitrosourea induced mainly three base changes, . GC-AT,, AT--GC <strong>and</strong><br />
AT-GC with only the latter two detected at low doses . Both the propyl <strong>and</strong> butyl<br />
con4ounds also induced these three mutations,, but the fraction resulting from AT--CG<br />
transversions was somewhat reduced . The fraction of GC-CG tcansversions ., although<br />
small, also increased with chain length . The fractions of GC-TA, <strong>and</strong> AT-TA<br />
transversions increased with chain length <strong>and</strong> were quite significant for the butyl<br />
compound . NMA induced exclusively AT--CG transversions . NDMA surprisingly exhibited<br />
a much different profile than MNU at the doses examined . It induced both AT-TA <strong>and</strong><br />
GC-AT base changes when metabolized by rat or hamster S-9 fraction . Bowever ., when<br />
metabolized by microsomes, it exhibited the same specificity as lRNU . This study<br />
demonstrates (1) that even relatively similar compounds have their own unique<br />
mutational profiles <strong>and</strong> (2) that the comparison of mutational profiles can be<br />
valuable to a tool in determining whether similar compounds induce mutations by<br />
coamon or different pathways . Supported by Grant #ES03332 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
80 1989 EMS Abstracts 227<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes AN OPTIMIZED PROCEDURE FOR CULTURING RAT LYMPHOCYTES FOR IN VITRO CYTOGENETIC<br />
SCREENING . P.J. G azie <strong>and</strong> Y. OaWro, O.D. SearM & Co .. Skokle, IL .<br />
Treatment prooedures <strong>and</strong> culture conditbns were optimized for the tn vitro treatment of peripheral blood<br />
lymphocytes (PBLs) of rats for oyto0enstb evaluation . Approxktttlaly 12-16m1 of blood was aolMded by cardisc<br />
puncture Into heparinl2ed VACUTAOJERTN tubes . Whole blood cultures <strong>and</strong> FboR4epaated ytnphooyla wan<br />
cuUured at 37'C for 48 h In RPMI-1824 medium supplemented with 20%fetal bovine sennn, L-pkAamMn (2 mM) .<br />
peniciuiNstreptomydn <strong>and</strong> the mitogen, ConcanavaNrt A(Con A : 6.7 Npht* . Various a>aure aondtbns were<br />
tested Including the addition of agents whidt have been reported to Increase blaslopenaN of ymphooytes .<br />
The addition of UCI (0.3 mM) to cultine medium aotltatNtq Con A did not slpNfioarMy Yanaa the ntlbfb Index<br />
relative to the use of Con A alone . However, the addition of 2-mathoxyelharal (2-ME ; 0 .9 µM) to medium<br />
contalninp Con A produced an 8-told karease In the mitotio Index compared to that observed with Con A alotw .<br />
M approxhnate 6-fold krorase tn numbent of mltotio oaNS was observed with FbDDN-teparaled cultures compared<br />
to whole blood cultures oodaininp both 2•ME <strong>and</strong> Con A . The effect of c.u density on tM mltadc kdex was<br />
studied by culturing Flooll-separated PBL's at densltNs of I X 108, 1 .6 X 106, <strong>and</strong> 2 X 106 oa6s per culture .<br />
Maximum mitotic Indices (6-7%) were achieved when 1 .6 X 108 PBLS were cultured In RPMI medium<br />
supplemented with Con A, <strong>and</strong> 2-ME for 48 h . In addition, cell cycle kinatkn were defined using<br />
5-bromodeoxyuridine In the culture medium to dineretttlale tha propottbtr of cells that had undarpona 1, 2 or 3<br />
cen .divisions over a defined period of 6me . TheN sabNs Indicated an avlrape oeM division tlme of approxknatey<br />
14-16 h after an InRial delay of 24-28 h . TrlelhylensrnslatNne (TEM ; 0.06 mphnq, a dUect-actUq daslopen, was<br />
added to the culture medium for 4 h after a 24 h cuMura period . Lymphocytes were harvested after a total of 48 h<br />
(final 3 h with coloemid) In culture <strong>and</strong> evaluated for chromoaome damage . Approximately 37% of the<br />
TEM-treated cells were aberrant compared to oottirol levels of 3-4% . Additional known dastopans <strong>and</strong><br />
non-clastogens are being tested to evaluate thk test aystem for roupne aonankp of potential dastooens .<br />
POINT ttUTATIOMS IM THE K-RAS ONCOGRa,tt IY DMA-SAHPLSS FROIt LUNG CANCER PATIENTS<br />
228<br />
Hackman, P ., Husaafvel-Pursiainen, K ., Mttila, S ., KalliomYki, P .-L ., HeikkilY, L .,<br />
Hattila, S . & Vainio, H ., Institute of Occupational Health, Topeliuksenkatu 41 aA,<br />
00250 Helsinki, Finl<strong>and</strong><br />
Activation of the cellular onco6enes in the rn-Sene family has been implicated<br />
in many types of human piali6nancies . In this work we are studyina the mutational<br />
activation of K_ras-onco6enes in lunS cancer patients . Peroperational lung tissues<br />
as well as blood samples from 21 lunb cancer patients, were obtained from the<br />
Helsinki Universlty Central Hospital . DnA was isolated from tumour tissue as well as<br />
from normal peripheral lung tissue, <strong>and</strong> from wfiite blood cells <strong>and</strong> stored freeze<br />
dri•ed . All of these patients were smokers or ex smokers . Of the 21 lung cancers<br />
twelve were squamous cell carcinomas, six were adenocarcinomas <strong>and</strong> three small cell<br />
carcinomas . Fourteen of these patients had a history of occupational exposure to<br />
asbestos . DNA-sequences from samples of human DNA were amplified using the<br />
PCR-technique (Polymerase Chain Reaction) . The amplimers used in this reaction were<br />
identical with sequences situated on both sides of the human K-ras-oncoSene exon 1 .<br />
Codons 12 <strong>and</strong> 13 which are located in this exon have been reported to be amon6 the<br />
critical points for the mutational activation of the K_ras-onco6ene . Sxperiments are<br />
in proaress to analyse the amplified DHA for specific point mutations by<br />
hybridization using specific 20 bp oli`onucleotldes . Wild-type oli6onucleotides as<br />
well as at least 6 "mutated" oli6onucleotides differinS in one base pair/codon are<br />
used in these experiments .<br />
229<br />
THE ORIGIN OF MIlTA[JT3 . Bany 0 . Hall . Dept. of Biology, University of Rochester, Rochester, NY .<br />
Spontaneous mutations are assumed to occur r<strong>and</strong>omly, condntxxtsly, <strong>and</strong> without regard to their utility .<br />
Two studies from my labaatory suggest that some mutations occur more often when they are advantageous<br />
than they do when they are irrelevant to the well being of the cell . In both cases these spontaneous ad hoc<br />
mutations occur in aged, nutritionally depleted, colonies of BschertchJa coll . In the first study excision of a<br />
mobile DNA element, IS103, from within the bglF gene is required for expression of that gene which then<br />
permits utilization of the 8-glucoside sugar salicin . The excision event is undetectable (Q x 10-12 per cell<br />
division) in cultures of growing cells, but it occurs at frequencies as high as 10'1 in aged colonies on<br />
MacConkey plates if, <strong>and</strong> only if, salicin is present in the plate . In the second study reversion of point<br />
mutations within the trp operon of E . coli occur 15 to 25 fold morefrequently In aged colonies on<br />
tryptophan depleted medium than they do in growing cells . A variety of trivial explanations for these<br />
observations are ruled out, <strong>and</strong> it is concluded that the vast bulk of the mutants recovered in these<br />
experiments are the result of mutations that would not have occurred under non-selective conditions by<br />
chance alone. It now seems likely that spontaneous mutation rates are not inherent properties of organisau,<br />
but instead that they are subject to modulation by normally encountered envtronmental conditions .<br />
Furthermore, the probability of a particular mutation arising may depend strongly upon the selective<br />
advantage conferred by the mutation.<br />
50869 3592
230 1989 EMS Abstracts<br />
TFIE EFFDLT OF DIETAFB! B1diSSIMS SPAWiS CN 7~ GENOTWQCTIY OF AFLi~T03mi, Notes<br />
D AND 2-ACLZYLAIIDFiDl7RFIJE . C .M . Hatniltcnt, R .M . Iblmest . ~ P .<br />
Bakke~, K .E . Garinf , K.L. Steirmetz', G .S . Steaws<strong>and</strong>2, <strong>and</strong> J .C . llirsalist . SRI<br />
International, Menlo Park, CA, <strong>and</strong> 2Corne11 University, Geneva, NY .<br />
Dietary oortstmgtion of civcifervus vegetables, especially lmmsels sprouts, have<br />
been shown to decrease hepatocarcinogerticity of aflatAxin 13~ (AFBi) in rats, but the<br />
medhanisn of this effect is tatlvfam . We e~mined the ef ect of di bzussels<br />
sprouts (~ Q, L . ) on tha gertotwdci of senrezal krtv.m carcirogens in<br />
male Fisdaer-344 zats . Rats wer+a fed a basal diet~80) or a brussels sprouts amerded<br />
diet (SSAD ; 20% freeze-dried btussels ) for apQrwtimately 3 weeks prior to<br />
testinq . Ur~3iedu .led ON71 is was aieesured in ilepatocytes expesed to<br />
AFSi, dimethylnitrosamine ~j~ ~-acetylaminofhsoresfe (2-AA ) . BD rat<br />
exposed to 0 .0005, 0 .001, <strong>and</strong> 0 .005 µq/ml yielded -0.9, 6 .2, <strong>and</strong> 22 .0<br />
~g~rau~is/twcleus (NG) . respectively, with 24, 54, <strong>and</strong> 92% of eells in repair (tIIt,<br />
t
82 1989 EMS Abstracts<br />
Notes Research supported by National Institute of General Medical Sciences (GM 09901) <strong>and</strong> by an<br />
Outst<strong>and</strong>ing Investigator Award from the National Cancer Institute (CA 44349) . References: Bohr VA,<br />
Phillips DH, Hanawalt PC, Cancer Res 47 :6426 . 1987 ; Hanawalt PC, Environ Health Pers 76 :9, 1987;<br />
Mellon I, Spivak G, Hanawalt PC, Cell 31 :241, 1987 ; Vos J-M, Hanawalt PC, Cell 30 :1789, 1987;<br />
Scicchitano DS, Hanawalt PC, Proc Nail Acad Se! USA : in press; Ho L, Bohr VA, Hanawalt PC, Mo!<br />
Cellu 8iol: in press.<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
233<br />
ANTAGONISTIC EFFECT OF ASPARAGIIS ON DPIIi-INDUCED NICRONUCLEI AND APOPTOSIS IN THE COLON<br />
CRYPT CELLS OF NICE . S . Hanxiao, Z . Qingfan, A . Yuhui, F . Shuli, D . Guirong, Y . Bin,<br />
<strong>and</strong> W . Yongjun . Dept . of Pathology, Henan Medical Univ ., Zhengzhou, Henan, P .R . China .<br />
The effect of asparagus on micronuclei <strong>and</strong> apoptosis in the colon crypt epithelial<br />
cells of mouse induced by Dimethylhydrazina (Dt41) was studied . Four groups (5 mice/group)<br />
were intraperitoneally injected with D141 solution, among which three groups were treated<br />
with oral liquid asparagus in the volume of 0 .1, 0 .5 or 1 ml per mouse, respectively, by<br />
stomach intubation one hour prior to injection . The fifth group was injected with 1mM<br />
EDTA intraperitoneally as the control . Twenty four hours after injection, all animals<br />
were sacrificed . The maximum number of micronuclei <strong>and</strong> apoptosis were observed in the<br />
DMH alone group . All three groups of oral liquid asparagus mice had lower incidence of<br />
micronuclei <strong>and</strong> apoptosis than Dt4t alone treated animals, <strong>and</strong> showed a positive doseeffect<br />
relationship . The results indicated that asparagus has antagonistic effect on the<br />
mutagenicity of colonic carcinogens .<br />
TABLE . Number of Micronuclei <strong>and</strong> Apoptosis in Colon Crypt Cells (p < 0 .01)<br />
Mean M of lt .A_/ Mean N of lK .A ./<br />
Cx=t of Each Nouse Crypt of Each Grouo<br />
Crouv 1 2 3 4 5<br />
DMH 4 .05 3 .80 3 .50 4 .15 4 .45 3 .99<br />
1 ml 0 .80 0 .70 0 .70 1 .00 0 .60 0 .76<br />
0 .5 ml 1 .90 1 .85 1 .15 1 .35 1 .50 1 .55<br />
0 .1 ml 2 .85 2 .30 3 .20 2 .55 2 .65 2 .71<br />
EDTA 0 .45 0 .30 0 .65 0 .35 0 .45 0 .42<br />
234<br />
MUTAGENICITY STUDIES ON FENITROTHION IN BACTERIA AND MAMMALIAN CELLS .<br />
M . Hara, S . Kogiso, F . Yamada, M . Kawamoto, A . Yoshitake <strong>and</strong> J .<br />
Miyamoto, Biochemistry <strong>and</strong> Toxicology Laboratory, Sumitomo Chemical<br />
Co ., Ltd ., Osaka, Japan .<br />
The mutagenicity of fenitrothion was determined in strains of<br />
Salmonella ~t h~imurium <strong>and</strong> Escherichia coli . Fenitrothion was found to<br />
be non-mutagenic in Salmone la t mur~um strains of TA98, TA1535 <strong>and</strong><br />
TA1537 <strong>and</strong> in Escher c WP uvrA both with <strong>and</strong> without S9 mix,<br />
while weak mutage t~y was observ-eT-only in TA100 strain <strong>and</strong> enhanced<br />
by the addition of S9 mix . The mutagenicity observed in the TA100<br />
strain was not expressed in a nitroreductase-deficient strain, TA100<br />
NR, <strong>and</strong> decreased in a transacetylase-deficient~strain TA100 1,8-DNPG<br />
The mutagenicity of fenitrothion was also examined by a gene mutation<br />
assay using the gene for hypoxanthine-guanine phosphoribosyltrangferase<br />
(hgprt) in V79 Chinese hamster lung cells . Fenitrothion<br />
did not induce any increment of 6-thioguanine-resistant mutant cells<br />
at doses ranging from 0 .01 to 0 .3 mM regardless of the presence or<br />
absence of S9 mix . The results suggest that reduction of fenitrothion<br />
by a bacterial nitroreductase of TA100 to an active form is essential<br />
for the expression of the mutagenicity of fenitrothion in TA100 <strong>and</strong><br />
that a bacterial transacetylase of TA100 also has an important role in<br />
the process of mutagenic activation .<br />
235<br />
EVALUATION OF SEVERAL ANTINEOPLASTIC DRUGS IN THE IN VITRO UNSCHEDULED DNA<br />
SYNTHESIS ASSAY. P.R. Harbach, S .K . Wiser. A .L. Smith <strong>and</strong> C .S. Aaron . The Upjohn Co .,<br />
Kalamazoo, MI 49007<br />
The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes is an<br />
essential part of the genetic toxicity at The Upjohn Company (TUC) . This assay detects the<br />
repair of damage to rat hepatocyteDNA caused by a wide variety of mutagens <strong>and</strong><br />
carcinogens. Primary hepatocytes were dissociated from the liver <strong>and</strong> placed into<br />
monolayer culture which was incubated with the test <strong>and</strong> control compounds <strong>and</strong><br />
labelled thymidine for 16-20 hours . The cultures were fixed, <strong>and</strong> the incorporation of<br />
thymidine was measured by autoradiogrsphy . The results were expressed as net<br />
grains/nucleus (NG) . We have tested several antineoplastic dru s in the UDS assay :<br />
tetraplatin (U-77,233) cis-platinum, m-AMSA, CC- 1065, U-73,97~<strong>and</strong> menogaril . The<br />
latter three compounds were developed by TUC . U-73,975 is an analog of CC-106S which<br />
50869 3594
has been reported as strongly $enotoxic in other systems . Both compounds were<br />
extremely potent DNA-damaging agents, inducin8 > +29 NG at 30 <strong>and</strong> 10<br />
nanograms/mI, respectively . Tetraplatin was strongly positive : + 6.47 to + 58 .26 NG at 3-<br />
30 ug/ml . Cisplatin also induced UDS with similar cytotoxicity to tetraplatin . Menogaril<br />
<strong>and</strong> m-AMSA were tested up to toxic or near toxic doses <strong>and</strong> were both negative . Based<br />
on these observations, anticancer drugs differ dramatically in their ability to induce<br />
unscheduled DNA synthesis .<br />
236<br />
CORRELATION OF RESULTS FROM THREE GENETIC TOXICOLOGY TESTS WITH RESULTS<br />
FROM ONCOGENICITY ASSAYS . M . C . Harnois*, T . Sofuni, <strong>and</strong> M . Ishidate,<br />
Jr ., National Institute of Hygienic Sciences, Setagaya-ku, Tokyo<br />
(Japan), *Fellow, Japan Foundation for Promotion of Cancer Research .<br />
Literature data (1965-85) from the in vitro clastogenicity (IVC)<br />
test (951 chemicals) were compared with databases on the S~ .~tSr.p~himurium<br />
point mutation (STPM) test (316 chemicals),the bone marrow micronu~eus<br />
(BMM) test (105 chemicals) <strong>and</strong> oncogenicity (0) tests (177 chemicals) .<br />
Of the IVC+, 41% were STPM- <strong>and</strong> 55% were BMM- . Of the few IVCsubstances,<br />
5/18 were STPM+ <strong>and</strong> 2/10 BMM+ . There were no 0- chemicals<br />
when all three mutagenicity tests were + . Chemicals negative in all<br />
three tests were either 0- (pyrene, methionine) or had an unresolved<br />
oncogenic status by the criteria used (diazepam, phenanthrene) .<br />
Chemicals IVC+ <strong>and</strong> 0+ but STPM- in our data were benzene,<br />
hexamethylphosphoramide, mitomycin C, urethane (BMM+) <strong>and</strong> actinomycin<br />
D, diethylstilboestrol, griseofulvin, lead acetate (BMM) .<br />
Dibutylnitrosamine <strong>and</strong> ethylene thiourea were BMM- <strong>and</strong> IVC- but STPM+<br />
<strong>and</strong> 0+ . The data indicate that no one test was adequate to demonstrate<br />
genotoxic effect or predict oncogenic effectl the chromosome assays<br />
<strong>and</strong> STPM test were complementary in both instances . (Ref . M . Ishidate,<br />
Jr . et al . : Mutation Res ., 195, 151-213, 1988)<br />
237<br />
GENOTORICITY OF 2-AMINO-N6-NYDROEYADENINE (AMA) T6 LS178Y/TK+/- -3 .7 .2C OUSE<br />
LYtlPHOMA CELLS . K . Narrington-Brockl P . Glover2 P . Poorman-Allen2 C . Doerr~, R .<br />
Krehl2, D . Clive2 J . Nozier3 <strong>and</strong> M .N . Moore4 . lEnvironmontal Health Research <strong>and</strong><br />
Testing, Inc ., RTP, NC 27709 USA ; 2Wellcome Research Laboratories RTP, NC 27709 USA ;<br />
3Florida Institute of Technology, Melbourne, FL 32901 USA ; 4U .S . <strong>Environmental</strong><br />
Protection Agency, RTP, NC 27711 USA .<br />
Studies in a two-component heterokaryon of Neurofpora Srassa have shown AMA to be a<br />
potent inducer of adenine-3 point mutations ; none of the induced mutants were found<br />
to be multilocus deletions (Overton et al ., Environ . Mutagen ., 5•501-502, 1983) . An<br />
international collaborative study has been initiated by do Serres to underst<strong>and</strong> the<br />
types of mutations induced by AHA in higher eukaryotic organisms . Using L5178Y/TK+/-<br />
-3 .7 .2C mouse lymphoma cells we have found AAA to be a potent inducer of TK-/-<br />
mutants (70 ng/ml induced approximately 1600 mutants/106 survivors ; survival - 19X) .<br />
The majority of the colonies were large colonies ; however, a significant number of<br />
small colonies was also induced . AMA induced a moderate (as compared to ethyl<br />
methanesulfonate) response at the heort locus (40 ng/ml induced approximately 200<br />
mutants/106 survivors) . The induced mutant frequency at the ouabain-resistance<br />
marker was slightly lower (70 ng/ml induced approximately 135 mutants/106 survivors) .<br />
Consistent with the induction of small-colony TK mutants, AAA was found to be<br />
clastogenic . From these studies it appears that in addition to point mutations, AMA<br />
may be capable of inducing chromosomal events in mammalian cells that are not<br />
possible in the two component heterokaryon of Neurospora . B<strong>and</strong>ed karyotype <strong>and</strong><br />
molecular analysis will be used to evaluate the type of genetic events induced by<br />
AHA . (This abstract does not necessarily reflect U .S . EPA policy .)<br />
238<br />
ROLE OF ONCOGENES AND TUMOR SUPPRESSOR GENES IN HUMAN LUNG CARCINOGENESIS . Curtis C .<br />
Harris, Laboratory of Hunan Carcinogenesis, National Cancer Institute, Bethesda, MD<br />
20892 .<br />
Five families of proto-oncogenes, ras, raf, fur, un <strong>and</strong> c have so far been<br />
associated with human lung cancer . Human ~nch~ al ~thelicells in vitro are<br />
being used to investigate the functional role of these specific oncogenes growth<br />
regulatory genes in carcinogenesis <strong>and</strong> tumor progression . Using the protoplast<br />
fusion method for high frequency gene transfection, the v-Ha-ras oncogene initiates a<br />
cascade of events in the normal human bronchial cells leading'To their apparent<br />
immortality, decreased responsiveness to inducers of squamous differentiation,<br />
aneuploidy, <strong>and</strong> tumorigenicity with metastasis in athymic nude mice . Transfection of<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 83<br />
Notes
84 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes the SV-40 T antigen gene leads to nontumorigenic cell lines that have a nearly normal<br />
pathway of terminal squamous differentiation <strong>and</strong> can be readily transformed to<br />
malignant cells by transfected Ha-ras . N-ras or K1-ras . The combination of<br />
transfected c-myc c <strong>and</strong> c-raf-1 will aTso cause neoplastic transformation of human<br />
bronchial epitheTial cetls that exhibit some phenotypic traits found in small cell<br />
carcinomas . These <strong>and</strong> other results indicate that proto-oncogenes dysregulate<br />
pathways of growth <strong>and</strong> differentiation of human bronchial epithelial cells <strong>and</strong> play<br />
an important role in human lung carcinogenesis . Allelic deletion <strong>and</strong> somatic cell<br />
genetic analyses suggest that tumor suppressor genes may be present on chromosomes<br />
3p, llp, 13q <strong>and</strong> 17p . These studies also indicate that tumor suppressor genes may<br />
play a dominant role in lung carcinogenesis <strong>and</strong> provide in vitro model systems for<br />
isolating these genes by subtraction library <strong>and</strong> insertlonaT-mu-Tagenesis approaches .<br />
INVESTIGATION OF SPONTANEOUS TRANSFORMATION OF BALB/c-3T3 CELLS . S .B . Harris <strong>and</strong><br />
E .J . Matthews, Hazleton Laboratories America, Inc ., Kensington, Maryl<strong>and</strong> .<br />
239<br />
Spontaneous transformation (ST) was investigated using the A31, I-13 clone of chemical<br />
transformable BALB/c-3T9 cells . All experiments were performed with one lot of<br />
fetal bovine serum, <strong>and</strong> one cryopreserved pool of cells . ST was shown to include a<br />
continuum of Type I, 11, <strong>and</strong> III foci of diffarent sizes <strong>and</strong> morphologies, <strong>and</strong> the<br />
appearance of foci in individual dishes in an experiment was not normally distributed<br />
. A log, mathematical transformation of the ST data was shown to convert it to<br />
a normal distr1bution . Detection of ST was determined to be influeneed by several<br />
different experimental parameters . First, ST was expressed in proportion to the<br />
tog „ of the surface area of the culture vessels over the range of 36 to 160 mm in<br />
diameter . Second, ST was significantly different for cells derived from different<br />
ampules of cells . Third, pre-existing spontaneous transformants were occasionally<br />
observed in experiments, <strong>and</strong> these foci were detected in cultures seeded at cell densities<br />
> 3 .2 x 10• ce11s/60 mm dish . In contrast, two experimental parameters did<br />
not effect ST . First, the level of ST was shown to be expressed independently of the<br />
initial seeding density of the cell cultures . Seeond, ST was relatively constant for<br />
10 passages <strong>and</strong> up to 60 population doublings . Taken together, these data permitted<br />
an estimation of the frequency of spontaneous transformants of 0 .64 x 10-6 for a<br />
contact-inhibited monolayer of cells in a 60 mm dish . Investigations were supported<br />
by NIEHS Contract NO1-ES-66160 .<br />
240<br />
NATURALLY OCCURRING . IMIDAZOLE-CONTAINING ANTIMUTAGENS : ERGOTHIONEINE AND CARNOSINE .<br />
Philip E . Hartman, Department of Biology, The Johns Hopkins University, Baltimore,<br />
MD .<br />
Ergothioneine (2-thiol-L-histidine betaine) has properties both of a thlol <strong>and</strong><br />
of a thione ; it is present in millimolar ooncentrations in fungi synthesizing it <strong>and</strong><br />
in particular tissues of animals ingesting it . Ergothioneine !s oxidized <strong>and</strong><br />
catalyzes the oxidation of reduoed glutathione to the disulfide in the presence of<br />
H=0, (reviewed in Hartman PE 1989 Meth Enzymols in press) . Ergothioneine protects<br />
baeteriophages from Y-irradiation (Hartman et al 1988 Radiat Rea 114 :319) <strong>and</strong><br />
decreases mutagenicity of t-butylhydroperoxide <strong>and</strong> products of nltrosated spermidine<br />
for Salmonella (Hartman Z <strong>and</strong> Hartman PE 1987 Environ Moleo Mutagen 10 :3) .<br />
Ergo one ne lessens lipid peroxidation in animal systems (Kawano at al 1983 Chem<br />
Pharm Bull 31 :1676, 1682) -- Carnosine (6-alanyl-L-histidine) is present in<br />
millimolar amounts in striated musolea of humans <strong>and</strong> other animals (Crush 1970 Comp<br />
Biochem Physiol 34 :3 ; Parkhouse at ai 1985 J Appl Physiol 58s14) . Carnoaine quenches<br />
singlet oxygen <strong>and</strong> traps lipid peroxyl radicals more effeotively than does free<br />
L-histidine (Dahl at al 1988 Photochem Photobiol 47 :357 ; Kohen at al 1988 Proo Natl<br />
Aead Sei USA 85 :3175 ; Hartman Z at al these prooeedings), proteots baoteriophages<br />
from Y-irradiation (Hartman at al 198~ op oit), <strong>and</strong> prevents induction of lipid<br />
peroxidation in muscle sarcoplasmio retioultso (Dupin at al 1987 Biochemistry<br />
52 :672) . Honocarnosine (Y-aminobutyryl-L-hiatldine) exhibite similar prQteotive<br />
actiona (Kricheskaya et al 1985 Vopr Med Khim 31(4) :75 : Kohen at al 1985 0 oit) .<br />
--These prevalent <strong>and</strong> naturally occurring imidazole compounds warrant furtRer<br />
investigation as to their possible protective actions against a range of chemioal<br />
mutagens .<br />
241<br />
C-TERMINAL HISTIDINE DIPEPTIDES AS EFFECTIVE SCAVENGERS OF SINGLET MOLECULAR OXYGEN .<br />
Zlata Hartman, Philip E . Hartman <strong>and</strong> Katherine T . Ault, Department of Biology, The<br />
Johns Hopkins University, Baltimore, MD 21218 USA .<br />
Exogenous singlet oxygen ('0,) ia highly oytotoxic for Salmonella (Dahl at al 1988<br />
Photochem Photobiol 47 :357) but non-mutagenio (Dahl at al-T986'Mdt Res 201 :127) .<br />
Free deoxyguanosine is degraded by ainglet oxygen but ia about 40-fold less sensitive<br />
when in single-str<strong>and</strong>ed DNA <strong>and</strong> 1000-fold less sensitive when in duplex DNA (E<br />
Hildebr<strong>and</strong> 1989 PhD Thesis, Johns Hopkins University) . The imidazole, L-histidine, a<br />
50869 3596
scavenger of singlet oxygen (Bellus 1979 Adv Photoohem 11 :105), reacts with singlet<br />
oxygen about 8-fold more rapidly than does free deoxyguanosine (Hildebr<strong>and</strong>, op cit) .<br />
Comparative studies were carried out on over 30 imidazole-containing compounds using a<br />
modification of the RNO bleaching assay for ainglet oxygen of Krali6 (of Verlhac et al<br />
1984 Nouv J Chim 8 :401) . L-Carnosine (B-alanyl-L-hiatidine), the imidazole compound<br />
prevalent in human striated muscle (Parkhouse et al 1985 J Appl Physiol 58 :14),<br />
scavenges singlet oxygen 2- to 3-fold faster than does L-histidine (also see Dahl et<br />
al, op cit) . We find similar high efficiencies for other dipeptidea with oarboxyterminal<br />
histidyls . These dipeptides are roughly twice as effective as analogous<br />
dipeptides with histidine at the amino terminus . A number of other imidazole<br />
containing compounds either have low solubility, do not exhibit increasing efficiencies<br />
with increasing concentrations over the range tested (1 .5 - 15 mM), or lose their<br />
measured capacity for quenching of singlet oxyaen uDon Grolonged ex ofure to oxidants .<br />
L-Carnosine may be a near-optimal molecule that doubly serves as a gu fer <strong>and</strong> as a<br />
defense against oxidative damage .<br />
242<br />
ENHANCEMENT OF GENOTOXICITY BY LEAD .<br />
A . Hartwig, R . Schlepegrell, <strong>and</strong> D . Beyersmann, University of Bremen, Bremen (F .R .G .)<br />
Inorganic lead compounds are considered as suspected carcinogens ; however, the mode<br />
of action is not well understood . Part of the contradictory results in short-term<br />
assays might be due to differences in bioavailability, since we found a'marked dependency<br />
of lead cytotoxicity <strong>and</strong> uptake on cell type <strong>and</strong> medium composition . Because<br />
mutagenicity <strong>and</strong> transforming ability are not accompanied by direct DNA damage (1), we<br />
investigated whether the genotoxic action of lead is due to rather indirect effects by<br />
interfering with genetic control <strong>and</strong> repair mechanisms, using UV as a st<strong>and</strong>ard mutagen .<br />
Pb(II) is comutagenic with UV in the V79 HGPRT-assay . Even though there is only a weak<br />
induction of SCE's in the same cell line by lead alone, we find a pronounced enhancement<br />
of UV-induced SCE's . Pb(II) did not produce DNA str<strong>and</strong> breaks itself but increased<br />
the number of breaks generated during repair of UV damage, indicating an inhibition of<br />
the polymerisation or the ligation step of excision repair . We conclude that the genotoxicity<br />
of lead compounds is in part due to interference with repair processes .<br />
Reference : .<br />
1 . Zelikoff, J .T ., Li, J .H ., Hartwig, A . Wang, X .W ., Costa, M . <strong>and</strong> Rosaman, T .G . (1988)<br />
Carcinogenesis 9, 1727 - 1732 .<br />
243<br />
FLOW CYTOMETRIC MICRONUCLEUS TEST WITH MOUSE BONE MARROW AND PERIPHERAL<br />
BLOOD ERYTHROCYTFS<br />
Makoto Hayashil .2, Hannu Norppa2, Toshio Sofunil <strong>and</strong> Motoi Ishidate. Irl<br />
1Divisioo of <strong>Mutagenesis</strong>, Biological Safety Research Center, National Institute of Hygienic Sciences,<br />
1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158 Japan<br />
2Mutagen Laboratory, Department of Industry Hygiene <strong>and</strong> Toxicology, Institute of Occupational Healtb,<br />
Topeliuksenkatu 41 aA, SF-00250 Helsinki, Finl<strong>and</strong><br />
Flow cytometry was applied to the micronucleus test with mouse bone marrow (BM) <strong>and</strong> peripheral<br />
blood (PB) erythrocytes. BM cells were fixed with 1% glutaraldehyde in 1/20 M phosphate buffer at pH<br />
6 .8 <strong>and</strong> stored in 70% ethanol . PB erythrocytes were sphered <strong>and</strong> fixed with 1% glutaraldehyde containing<br />
0 .03 mg/ml of sodium dodecyl sulfate in 1/20 M phosphate buffer at pH 6 .8. PB cells were also treated<br />
with RNase to reduce noise from RNA-eoataining erythrocytes . The cells were stained with DAPI, <strong>and</strong><br />
50000 erythrocytes were analyzed in an EPICS V flow cytometer using a UV laser operating at 150-200<br />
mW. Data from the flow cytometer were further analyzed by a computer program Including model fitting<br />
to estimate the frequency of micronueleated erythrocytea . For each sample 1000-2000 srythrocytea were<br />
also analyzed from Giemsa-stained smears microscopically . There was a good correlation between the<br />
flow cytometric <strong>and</strong> microscopical measurements after treatment with mitomycia C (BM,PB), benzene<br />
(BM,PB), 6-mercaptopurine (PB), benzo[aJpyrene (PB), N-ethyl-N-nitrosourea (PB), bromodichloromethane<br />
(PB), <strong>and</strong> potassium chromate (PB) . A repeated experiment with mitomycin C also showed good<br />
reproducibility for the flow cytometric measumeat of mieronuelai In BM . Generally, st<strong>and</strong>ard deviations<br />
were smaller by the flow cytometric method than by the manual method probably because of the different<br />
sample sizes (50000 erythrocytes for flow cytometry <strong>and</strong> 1000-2000 erythrocytes for manual analysis) .<br />
244<br />
MONITORING OF FOOD MUTAGENS<br />
Hikoya Hayatsu<br />
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan<br />
Monitoring of food for mutagenicity requires a simple, practical way of preparing<br />
testable samples . We have been using the blue cotton method for this purpose . Blue<br />
cotton is absorbent cotton bearing covalently linked copper phthalocyaaine trisulfonate .<br />
Since this lig<strong>and</strong> has a selective affinity to polycyclic compounds, adsorption of mutagens<br />
having polycyclic structures takes place when food extracts in aqueous media are<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
4<br />
1989 EMS Abstracts 85<br />
Notes<br />
/
86 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes treated with blue cotton . Elution of the cotton with organic solvents, most efficiently<br />
with ammoniacal methanol, results in recovery of the adsorbed polycyclics . With this<br />
method, a high concentration extent is generally obtainable, <strong>and</strong> the entire work needs<br />
only short periods of time <strong>and</strong> little labor . Since mutagenic heterocyclic amines <strong>and</strong><br />
polycyclic aromatic hydrocarbons are efficiently adsorbable to blue cotton, the use of<br />
this method allows monitoring of these classes of mutagens in food . It has been shown<br />
that many proteinaceous foods that had undergone heating processes contain mutagens . in<br />
most cases the heterocyclic aminee ; e .g ., katsuobushi, a heat-dried bonito-fish meat,<br />
which is one of the everyday food items in Japan, has been shown to contain MeIQx . We<br />
have also observed that some shell fish in their raw state contain mutagens . We have<br />
recently improved the method by introducing blue rayon, an adsorbent more powerful than<br />
blue cotton . The blue-cotton <strong>and</strong> -rayon techniques can also be used for isolating foodmutagen<br />
metabolites in excretions <strong>and</strong> body fluids, <strong>and</strong> for monitoring mutagens in<br />
environmental waters <strong>and</strong> airs . (Supported by grants from the Ministry of Health <strong>and</strong><br />
Welfare, Japan)<br />
245<br />
GENOTOXICITY OF INITIATING CARCINOGENS IN THE SKIN .<br />
S . He <strong>and</strong> R .S .U . Baker, National Institute of Occupational Health & Safety, Building<br />
A27, University of Sydney . NSW 2006 (Australia) .<br />
Nicronucleus(MN) induction can be evaluated in keratlnocvtes followinR application<br />
of chemicals onto the dorsal skin surface of hairless HRA/Skh mice . Previous studies<br />
have indicated that animals of this strain are sensitive to chemical induction of<br />
akin cancer, <strong>and</strong> also that the active alkylating carcinogen, triethylenemelamine, can<br />
be readily detected as a genotoxicant in keratinocytes using this procedure . For<br />
chemicals requiring in vivo metabolism, it was first necessary to establish an<br />
appropriate sampling time between chemical treatment <strong>and</strong> removal of skin for in vitro<br />
studies . With 7,12-dimethylbenz[a]anthracene (DNBA), MN induction reached maximal<br />
values at 12, 24 <strong>and</strong> 48h post-treatment . A sampling time of 24h was therefore<br />
selected for all chemicals . Dose-related increases in HN were detected with the<br />
initiating carcinogens DMBA, benzo[a]pyrane (BP), chrysene <strong>and</strong> urethane while pyrene<br />
failed to induce MN at subtoxic doses up to 2 .5mg/mouse . Significant MN induction<br />
(p
247<br />
GESVMXICITY OF MRGANIC MEFiLURH<br />
HEIMI, S . ; EIrSFEFII, M. ; EL-ZYAT H . & M76TAFA M.H . Institute of Graduate Studies<br />
<strong>and</strong> FEsearch, University of Alex<strong>and</strong>ria, A exa a, Egypt .<br />
Genotoxic effects of HgC1 were tested using a battery of tests . In Vicia faba<br />
the predaminant cytogenetic iffects in msiosis ware the formntion of ~~i,<br />
structural chranoscme aberrations, <strong>and</strong> abnormal ctuamsc :ne distribution . In<br />
Saccharanyces oerevisiae, strains were different in their sensitivity to HgC1 , the<br />
nor JD strain was 4 <strong>and</strong> 1 .4 ppn when ex.oosure was for 3 <strong>and</strong> 5 hours res~ectively,<br />
Qe the ID5p values for strain D7 were 1 .4 a,rri 0 .6 pZ m. Log-phase cells were more<br />
sensitive th3n those of stationary phase . Mitotic gene conversion at HIS4 locus in<br />
strain JD1 <strong>and</strong> TRP5 locus in strain D7 was suppressed by HgC1 . In AsMElus imnersus<br />
(Pasadena strains the older the strain the more seensitive R was to HgCr'-CUFUrrg<br />
in distilled water, oomplete liquid , axrl cortQlete solid media supplementW with HgC12<br />
gave different results . Meiotic gene eonversion frequency in + x m crosses was not<br />
affected, only direction of eonversias showed significant change in the favour of<br />
wildtype allele . It was proposed that HgCl influenced mis-match repair mechanism<br />
rather than hybrid DNA formation freduenc .y? However, HgCl increased crossing-over<br />
frequency - except at 1 pfm - significantly. ZWo hypothe2s were proposed to explain<br />
such effect :one is based on the fate of half-chiasmata <strong>and</strong> their resolution, the<br />
other eonsiders the physical arrangement of chranatids . With respect to forward<br />
mutation frequency, HgC1 significantly lowered the spontaneous frequency of asoospore<br />
colour mutations . Probab~y, the inhibition of enzymes involved in the repair of<br />
premutational lesions by FigC12 caused such reduction .<br />
248<br />
EFFECT OF PHENOLIC ANTIOXIDANTS ON BENZO(A)PYRENE METABOLISM, GENOTOXICI-<br />
TY AND ITS METABOLITES BINDING TO BACTERIAL DNA IN SOS CHROMOTEST .<br />
E .E . Hennig, K .K . Demkowicz-DobrzaAski, <strong>and</strong> L . Dock, Medical Academy,<br />
02-097 Warsaw (Pol<strong>and</strong>), <strong>and</strong> The National Institute of <strong>Environmental</strong> Medicine,<br />
S-104 01 Stockholm (Sweden)<br />
The effect of butylated hydroxyanisole (BHA) <strong>and</strong> butylated hydroxytoluene<br />
(BHT) has been studied on benzo(a)pyrene (BP) metabolism, .genotoxicity<br />
<strong>and</strong> BP metabolites binding to bacterial DNA . BP activation has been<br />
performed using S9 fractions from the liver of mice fed a diet containing<br />
BHA or BHT . Both BHA <strong>and</strong> BHT treatment slightly enhanced total BP metabolism<br />
<strong>and</strong> markedly increased water-soluble BP nyetabolites formation as was<br />
indicated by the estimation of the distribution of organic extractable<br />
<strong>and</strong> water-soluble metabolites formed in the presence of S9 fractions . The<br />
HPLC analysis of BP metabolic profile, in the presence of antioxidantsmodified<br />
fractions, shows significant decrease of 9-hydroxyBP formation .<br />
The bacterial test SOS Chromotest was used to study the genotoxicity of<br />
BP . Formation of BP metabolite adducts in Escherichia coli DNA was analysed<br />
by HPLC procedure . There was indicated a strong inhibition of BP genotoxicity<br />
when the S9 fraction from BHT-fed mice was used for BP activation<br />
. BHA had only a moderate, not significant, inhibitory effect . Our<br />
results indicate that there existed a clear correlation between antioxidants<br />
effect on BP genotoxicity <strong>and</strong> total BP metabolites binding to bacterial<br />
DNA .<br />
249<br />
STRUCTURE-ACTIVITY RELATIONSHIPS INVOLVED IN THE GENOTOXICITY OF ANALOGS OF PYRVINIUM<br />
IN YEAST AND BACTERIA . U .G .G . Hennig <strong>and</strong> R .C . von Borstel, Department of Genetics .<br />
University of Alberta, Edmonton, Alberta (Canada) T6G 2E9<br />
The structural requirements for the mutagenic action of pyrvlniue were investigated<br />
with structural analogs . The genotoxicity of these analogs was studied in diploid (D5<br />
<strong>and</strong> 07) <strong>and</strong> haploil (XV1B5-14C, XY718-1A, <strong>and</strong> •1854-1A) strains of Saooharomyoee<br />
cereviaiae . Substitution with a methyl group at the 6-position of pyrviniuln did not<br />
affect the mutagenicity ; the 6-methyl-analog induced frameshift <strong>and</strong> base-substitution<br />
mutations just as readily . However, the 6-methyl-analog induced both transitions <strong>and</strong><br />
transversions, whereas pyrvinium induced only transitions . With the 6-chloro-analog,<br />
the toxicity <strong>and</strong> mutagenicity were diminished but detectable levels of frameshifts <strong>and</strong><br />
transitions were observed . Pyrvinium <strong>and</strong> the 6-chloro-analog induced frameshifts <strong>and</strong><br />
transitions, whereas the 6-methyl-analog induced frameshifts, transitions, <strong>and</strong><br />
transversions . The hydroxylation of the methyl-substituent of the 6-methyl-analog<br />
probably results in a mutagen that is as active as pyrvlniun itself . A second DNA<br />
binding site is the cationic site at the methylated ring nitrogen of pyrvinium . This<br />
is evident fram the diminished, yet not abolished, genotoxicity of the chloro-analog .<br />
A methyl- or dimethylamino-substituent at the 6-position <strong>and</strong> the cationic site at the<br />
methylated ring nitrogen are required for mutagenic activity . The reversion spectra<br />
for SaZmoneLLa typh{muri.um (strains TA97, 98, 100, <strong>and</strong> 102) indicate that the chloro<strong>and</strong><br />
methyl-analogs are mutagenic in all strains (-S9 <strong>and</strong> +S9) . Pyrvinium pamoate <strong>and</strong><br />
pyrviniun iodide are frameshift mutagens (-S9 <strong>and</strong> +S9) but activation is required for<br />
them to induce base-substitution mutations . (Research supported by NHRDP <strong>and</strong> AHFMR) .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 87<br />
Notes
88 1989 EMS Abstracts 250<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes EFFECTS OF PROGFSTERONE AND ESTRADIOL ON PROLIFFRATION OF HUMAN L1'MPFDCYTFS IN VITRO .<br />
L .A . Herrera M ., R . Montero M ., <strong>and</strong> P . Ostrosky-Wegman . Instituto de InvestTgacones<br />
Biom6dicas, UNAM, Apdo . Postal 70228, 14sxico 04510 D .F . Mexico .<br />
During a study of lymphocyte cell-cycle kinetics we found variability between individuals<br />
<strong>and</strong> between samples of the same individual taken at different times . Trying to<br />
underst<strong>and</strong> some of the factors that could be involved in those variations, we thought<br />
over the relationship between menstrual cycle <strong>and</strong> cell vroliferation . In vivo studies<br />
on blood from 6 women, sampled every week during 3 months, didn't show any cTar effect<br />
that could be directly correlated with their menstrual cycles . Since many other factors<br />
could be interfering in our studies, we evaluated in vitro effects of estradiol<br />
<strong>and</strong> progesterone over cell proliferation kinetics (CPK) ard'Ft'totic Index (MI) on<br />
human lymphocytes both from men <strong>and</strong> from women on day 1 of menstrual cycle (lowest<br />
concentrations of respective hormones) . The two hormones were added to the cultures<br />
individually or simultaneously, 2 h after stimulation with PHA . Lowest doses of each<br />
hormone showed an inhibitory effect on CPK <strong>and</strong> MI . When the hormones were added<br />
simultaneously, the t of cells in 3th or more divisions decreased at doses similar to<br />
those found in menstrual <strong>and</strong> lutheal phases, whereas it increased at doses similar to<br />
the ovulatory phase . The MI of lymphocytes from women donors decreased at all doses<br />
of the hormones, whereas those of men donors decreased only at doses similar to menstrual<br />
phase . The effects of hormones on lymphocyte cell cycle should be further<br />
studied mainly in relation to the recently described increased sensitivity to genotoxic<br />
dama?e on lymphocytes from both pregnant women <strong>and</strong> women taking oral hormonal<br />
contraceptives, <strong>and</strong> to the well known increased sensitivity to infections of pregnant<br />
women .<br />
PROTOCOL EVALUATION FOR THE MICRONUCLEUS TEST . C . Hilliard, R . Tice, <strong>and</strong> M.D .<br />
Shelby*, Integrated Laboratory Systems, P0 Box 13501, Research Triangle Park, NC<br />
27709 <strong>and</strong> *NIEHS . P0 Box 12233, Research Triangle Park, NC 27709 .<br />
251<br />
Using dimethylbenzanthracene (DNBA) <strong>and</strong> benzidine (B2D), the efficacy of three<br />
different in vivo micronuclei (MN) protocols were evaluated, using polychromatic<br />
erythrocytes (PCE) sampled In both bone marrow <strong>and</strong> peripheral blood . B6C3F1 male<br />
mice (9-12 weeks of age ; 5 mice per group) were Injected IP with DMBA (25, 50, 100<br />
mg/kg) or with BZD (50 . 100, 200, 300 mg/kg) on 1, 2, or 3 consecutive days, with<br />
bone marrow <strong>and</strong> peripheral blood smears being made at 24, 48, <strong>and</strong>/or 72 hours after<br />
the first injection . Slides were stained with acridine orange <strong>and</strong> 2000 PCE scored<br />
per tissue per animal for the presence of MN . DMBA induced a significant increase in<br />
MN-PCE at all sample times in both tissues using all three protocols, with the yield<br />
of MN-PCE at 48 <strong>and</strong> 72 hr being independent of treatment protocol <strong>and</strong> tissue . BZD<br />
induced a positive response in bone marrow <strong>and</strong> peripheral blood only when injected 2<br />
or 3 times <strong>and</strong> sampled at 72 hr after the first injection . When administered by<br />
gavage, a significant increase in MN-PCE was induced by BZD in bone marrow 24 hr<br />
after a single treatment, with increased activity being demonstrated after 3 daily<br />
injections . These data indicate that a treatment protocol based on multiple<br />
injections eliminates the need for multiple sampling times, minimizes the number of<br />
animals <strong>and</strong> scoring time, <strong>and</strong> simplifies the statistical analysis . Also, the lack of<br />
a significant difference in MN levels between bone marrow <strong>and</strong> peripheral blood<br />
suggest that either tissue can be used following this protocol . Supported by NTP<br />
contract N01-ES-85209 .<br />
252<br />
RELATIONSHIPS BETWEEN STRUCTURE OF NITRATED ARENES AND THEIR MUTAGENICITY IN SALMONEL-<br />
LA TYPHIMURIUH ; 2- AND 2,7-NITRO SUBSTITUTED FLUORENE, PHENANTHRENE AND PYRENE<br />
T. Nirayama, T . Watanabe, Y . Fujioka, S . Ozasa, <strong>and</strong> S . Pukui, Kyoto Pharmaceutical<br />
University, Kyoto (Japan)<br />
In order to elucidate the mechanisms of autagenic activation of nitroarenes, we<br />
studied the relationships between the autagenic potency <strong>and</strong> chemical structure of 2nitro-<br />
<strong>and</strong> 2,7-dinitro-arenes including nitrated fluorene (F1), dihydrophenanthrene<br />
(DNPh), phenanthrene (Ph), tetrahydropyrene (TBPy), dihydropyrene (DHPy) <strong>and</strong> pyrene<br />
(Py) together with 9-N02-Ph, 1-N02-Py <strong>and</strong> 1,3-diN02-Py . The autagenicity tests were<br />
carried out on Salmonella typhimurium TA98, TA98NR <strong>and</strong> TA98/1,8-DNP6 in the absence of<br />
S9 mix . The order of mutability of mononitro- <strong>and</strong> dinitro-arenes in TA98 is as given<br />
below : 2-NOy-THPy
253 1989 EMS Abstracts<br />
EFFECT OF THE RELATIVE HUMIDITY ON THE FORMATION OF NITROPYRENES BY TME PHOTOCHEMICAL Notes<br />
REACTION OF PYRENE WITH NITROGEN OXIDES<br />
Yoshiharu Hisamatsu, Kazutoshi Sugita, <strong>and</strong> Hidetsuru Matsushita The Institute of<br />
Public Health, 6-1 Shirokanedai 4-chome Minato-ku Tokyo 108, Japan<br />
The 2-nitropyrene, which have not been identified in direct emissions, in collected<br />
ambient particulate organic matter have been idetified <strong>and</strong> quantified . We have investigated<br />
the effect of the relative humidity (r .h .) to study the atmospheric transformation<br />
of pyrene to 2-nitropyrene, which is direct-acting mutagen, by the photochemical<br />
reaction with nitrogen dioxide .<br />
The photochemical reaction products of pyrene with nitrogen dioxide under various<br />
humidity of the air which is used to dilute nitrogen dioxide gas were fractionated to<br />
analyze for mononitro-pyrene by HPLC . The partial fractions corresponding to mononitro<br />
pyrene were analyzed using GC-MS, <strong>and</strong> the molecular ions m/z 247([M]+) together with<br />
the characteristic fragment ions, m/z 217([M-NO]+) <strong>and</strong> 201([M-N02]+), were monitored .<br />
The 2-nitropyrene has been identified in the photochemical reaction products of<br />
pyrene with nitrogen dioxide below 20% r .h . of air, but it has not been identified<br />
in the reaction products of them under no light irradiation . The photochemical<br />
reaction products were the most mutagenic below 20% r .h . of air for Salmonella typhimurium<br />
strain TA98 in the absence of S9 mix , <strong>and</strong> the mutagenic activities of them<br />
decreased with the increase of air .<br />
254<br />
DIRECT MEASUREMENT OF CHROMOSOME REPAIR BY PREMATURE CHROMOSOME CONDENSATION .<br />
Walter N . Hittelman . Department of Medical Oncology . University of Texas M . D .<br />
Anderson Cancer Center, Houston, Texas, USA 77030 .<br />
Chromosome damage in interphase cells is generally visualized when the cell<br />
reaches mitosis <strong>and</strong> the chromosomes condense . However, several events might have<br />
occurred between the time of genetic insult in interphase <strong>and</strong> chromosome<br />
visualization at mitosis, including repair of chromosome damage <strong>and</strong> delay of•damaged<br />
cells from reaching mitosis . In addition, chromosome damage in non-dividing call<br />
populations cannot be visualized by conventional techniques due to the lack of<br />
mitoses . The technique of premature chromosome condensation overcomes these<br />
problems by allowing the direct visualization of interphase chromosomes . Thus<br />
chromosome damage <strong>and</strong> its repair can be directly measured in interphase cells . We<br />
<strong>and</strong> others have used this approach to better underst<strong>and</strong> . .the underlying basis for<br />
chromosome damage <strong>and</strong> its repair as well as to characterize repair capabilities of<br />
various radiation <strong>and</strong> drug-sensitive <strong>and</strong> resistant cells . For example, chromosome<br />
repair in normal fibroblasts <strong>and</strong> lymphocytes exhibit a fast <strong>and</strong> a slow component of<br />
chromosome repair, while mature granulocytes show little chromosome repair . The<br />
fast component can be inhibited by hypertonic salt while the slow component is<br />
inhibited by inhibitors of protein or DNA synthesis . Radiation-sensitive cell lines<br />
such as LS178Y-S <strong>and</strong> Ataxia telangiectasia exhibit partial deficiencies in both the<br />
fast <strong>and</strong> slow components of chromosome repair . The ability to directly measure<br />
chromosome repair by the technique of premature chromosome condensation can now be<br />
applied to better underst<strong>and</strong> the in vivo interaction of environmental mutagens with<br />
tissues within the body .<br />
255<br />
THE EBV-Xy1E SMJ111E : ITS OODE'D2111RI0ii MD MJfATIaiAL SPDCIFICITY IN Hu7AN CELL .<br />
X . K. Hong, X. L . Wang,X .F . Qiu <strong>and</strong> J .L.Hsueh, Institute of Genetics, Fudan University,Sha*si (Qdna)<br />
A n.rber of studies recently have been reported in which shuttle vector plasmids were used to study<br />
mitational specificity in memmalian cells . Shuttle vector systems have becc.re an inportant tool in the study<br />
of nutational mechanisms in higher organisms . We have developed a shuttle vector system for studying mr<br />
tational specificity, as a mutational target, the shuttle vector contains the EB virus origin <strong>and</strong> E . coli<br />
Xy1E gene, which codes for the enzyme Catechol : oxygen 2,3-oxidoreductase . '!he vector which we constructed<br />
nared pFV891, is derived fram plasmid p1CfA2, pTG503, p'iCfiO'+ <strong>and</strong> pACi3 . The bacterial gene XylE, carried on<br />
a shuttle vector, is introduced into human cultured cells by transfection <strong>and</strong> allowed to replicated autonomously<br />
in the cell nucleus . During the replication period of the vector, the cells are exposed to a sut .gan,<br />
an increased in nutation frequency above the spontaneous background is readily obtained . Mitations form in<br />
the shuttle vector DNA in the memrelian rucleus . DNA is extracted <strong>and</strong> introduced into bacteria . Colonies<br />
that express Xy1E becan; yellow within seconds after selection plates are sprayed with Catechol, while cells<br />
were transformed by the shuttle vector in which the XylE gene were mutated didn't change the colour . Therefore,<br />
induced sutants can be rapidly isolated <strong>and</strong> characterized . Tne sensitive colour assay offers an approch<br />
to develop this shuttle vector for a wide variety of host organisms . Use of the EBV-Xy1E shuttle vector<br />
should permit determination of the mrtagenic specificity of a wide range of muagens <strong>and</strong> carcinogens in hr-<br />
rmn cells .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
89
90 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
256<br />
THE EFFECT OF THYMIDINE ANALOGUES ON RESTRICTION ENDONUCt .EASE INDUCED<br />
CHROMOSOME ABERRATIONS. OJ. Hook. R.J . Preston, University of Tennessee Biomedical Graduate Sclwol,<br />
<strong>and</strong> Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 .<br />
The thymidine analogue CIdU has been used to analyze the Importance of reoombination-like repair on the<br />
frequency <strong>and</strong> type of chromosome aberrations (CAs) inductd by X rays . The results of these experiments<br />
demonstrated that the level of CAs induced by X rays eould be enhanced <strong>and</strong> the proportion of exchange type<br />
aberrations Increased when CIdU was present in the template during 03 repair . CIdU Incorporation b a useful<br />
method of analyzing the Importance of excision repair on the formation of CAs Induced in Or 7ype 11 restriction<br />
endonucleases (REa) have Iken shown to induce CAs in mammalian cells in all parts of the cell cycle Including O, .<br />
We propose to test the bypotbeses that repair of RE induee double str<strong>and</strong> cuts involves more than simple ligation<br />
<strong>and</strong> the extent to which induced CAs are the result of lack of repair of the Induced double str<strong>and</strong> cuts as opposed<br />
to misrepair. CHO cells grown in the presence of CIdU were treated with Sau 3A1 at concentrations of 10 <strong>and</strong> 25<br />
units per 100 pl using the 'osmolytic shock' protocol . The cells were grown In the presence of CIdU for 26 hrs<br />
before treatment <strong>and</strong> harvested 4 hrs after treatment . Astoradiography has shown that the majority of cells<br />
harvested at 4 bn post-treatment were In O= or M when treated . The presence of CIdU (concentration 30yM)<br />
resulted In a reduction in the level of deletion type CAs <strong>and</strong> a doubling of the number of exchange type CAs above<br />
that expected from additivity. No Increase over additivity was observed when CHO cells were grown in the presence<br />
of BrdU. In conclusion, RE induced double str<strong>and</strong> cuts are, in part, repaired by a mechanism that Involves a<br />
resynthesis step . By altering the likelihood of misrepalr the damage that would have been converted Into deletion<br />
type aberrations was converted into exchange type aberrationt . (Research sponsored by the Office of Health <strong>and</strong><br />
<strong>Environmental</strong> Reaearch, U. S. Department of Energy under contract DE-ACUS-840R21400 with the Martin Marietta<br />
Energy Systems, Inc .)<br />
257<br />
RESTRICTION ENZYMI
259 1989 EMS Abstracts<br />
THE INDUCTION OF CHROMOSOME ABERRATIONS IN MOUSE BONE MARROW AND CH0 CELLS BY THE DNA Notes<br />
TOPOISOMERASE INHIBITORS CAMPTOTHECIN AND m-AMSA . D .R . Hovardl L .C . Backerl, J .A .<br />
Campbelll, D .M . DeMarini2, J .W . Allen2, 1EHRT, RTP, NC 27709 ; 2U .S . EPA, RTP, NC 27711 .<br />
Topoisomerases are enzymes that control supercoiling, breakage, <strong>and</strong> reunion of DNA<br />
str<strong>and</strong>s . There is evidence to suggest that two antitumor drugs, camptothecin (CAMP)<br />
<strong>and</strong> amsacrine (ID-ANSA), inhibit topoisomerase activity by binding to the DNAtopoisomerase<br />
complex <strong>and</strong> preventing reunion of the broken DNA str<strong>and</strong>s . a-AMSA binds<br />
to topoisomerase II to induce double-str<strong>and</strong> breaks in DNA . CAMP inhibits the activity<br />
of topoisomerase I, inducing single-str<strong>and</strong> DNA breaks . Although y-AMSA induces<br />
chromosome aberrations (CAs), CAMP has not been characterized for this effect . In the<br />
present experiments, CAMP <strong>and</strong> m-AMSA were compared for their capacities to induce<br />
chromosome- <strong>and</strong> chromatid-type aberrations in mouse bone marrow <strong>and</strong> CH0 cells . Male<br />
mice were exposed by i .p . injection to 0, 0 .5, 1 .5, or 3 .0 mg/kg of CAMP or a-AMSA<br />
dissolved in DMSO . Four animals/dose <strong>and</strong> 100 cells/animal were scored for CAs . Both<br />
chemicals induced approximately 50 chromatid-type <strong>and</strong> 10 chromosome-type aberrations<br />
per animal at the highest dose . CHO cells were incubated in medium containing either<br />
CAMP or g-AMSA at 0, 10, 50, or 100 ng/ml in 0 .5% DMSO . Cells were harvested at 16-18<br />
h <strong>and</strong> 100 cells/dose scored for CAs . At 100 ng/ml, a-AMSA induced 0 .56 chromatid-type<br />
<strong>and</strong> 4 .95 chromosome-type aberrations/cell ; CAMP induced 0 .44 chromatid- <strong>and</strong> 1 .33<br />
chromosome-type aberrations/cell . In summary, jD vivo analyses did not reveal<br />
qualitative or quantitative differences in clastogenic activity between a-AMSA <strong>and</strong><br />
CAMP ; both induced predominantly chromatid-type aberrations . In contrast, both<br />
chemicals induced more chromosome-type aberrations jn vitro ; ID-AMSA was more potent<br />
than CAMP in producing this effect . (Seis .bsts .et do.s aet n.e.. . .rily r .fl.ct U .S . eM youey .)<br />
260<br />
METABOLISM AND MUTAGENICITY OF 1-NITROPYRENE, 3-NITROFLUORANTHENE AND<br />
1,8-DINITROPYRENE IN SELECTED STRAINS OF SALMONELLA TYPRIb1URIUM. Paul C .<br />
Howard <strong>and</strong> Elena C . McCoy, Department of <strong>Environmental</strong> Health Sciences, Case<br />
Western Reserve University School of Medicine, Clevel<strong>and</strong>, OH (USA) 44106<br />
The metabolism of three nitrated polycyclic aromatic hydrocarbons (1nitropyrene,<br />
3-nitrofluoranthene, 1,8-dinitropyrene) were determined in selected<br />
derivatives of the Salmonella typhimurium bacteria used in the widely used<br />
reversion assay (TA98, TA98/1,8DNP6, TA100, TA100NR, Tn5-1012), <strong>and</strong> contrasted to<br />
the mutagenicity of the chemicals in these bacteria. As expected, only<br />
nitroreductive metabolism was detected with the three chgmicals . In all cases, the<br />
nitroreductive metabolism of 3-nitrofluoranthene <strong>and</strong> 1,8-dinitropyrene were twice<br />
the rate of the metabolism of 1-nitropyrene. The lack of mutagenicity of several<br />
of the chemicals in some of the bacteria do not correlate with nitroreduction, <strong>and</strong><br />
can be attributed to the loss of the bacterial arylhydroxylamine O-esterificase .<br />
However, in several other cases, the loss of mutagenicity of chemicals is apparently<br />
caused by differing expressions of multiple nitroreductases . Supported in part by<br />
grant ES-03648 from the NIH .<br />
261<br />
IN VITRA ASSAYS OF IN VIW E}PCSURE TO CY=PHOSPFm!-IIDE AND BENZO(a)PYItENE : INDCK,TIGN<br />
OF SISTER-C3ffZ0IWZD E}OQHANGE4 CF HUMAN PERIPIERAL LYMPHOCYRFS BY CHElffCT+L E7lPOBID<br />
NOUSE BIOOD<br />
You-Chiu Hu, M .D. <strong>and</strong> Lia Ping<br />
Cancer Research Lab ., Hunan Medical University, Charr3sha 410078, P .R. China<br />
A method was devised to assay the genotoxic potential of cycLophosphmnide (CY)<br />
<strong>and</strong> benzq7yrene (BP) . Mice were exposed to different doses of CY <strong>and</strong> HP, blood from<br />
the drug-exposed mice was added to human ly::phocyte cultures <strong>and</strong> the sister clu-emstic<br />
exc'ianges (SCE) of 1ymPlxx.ytes were assayed . CY at doses of 0 .2, 0 .4, <strong>and</strong> 0 .8 mM,<br />
BP at doses of 0 .08, 0 .16 <strong>and</strong> 0 .32 mM were ip injected to mice . 20 minutes post ip<br />
injection, 0 .4 ml blood fran each exposed m0use was added to a phyt9henagglutinin<br />
(PHA) stimulated human lymphoc.yte culture <strong>and</strong> the SCE's of human 1ynQhocytss were<br />
assayed . Normal saline or D6ND was ip injected as zero dose . In the control group,<br />
CY <strong>and</strong> BP at different doses were added directly to the PHA-stimulated human lytsphocyte<br />
cultures oontai .cting Brdurd . Exposed mouse blood induoed a dose-dependent SCE<br />
increase in hunan lymphocytes at all tested doses . The CY tested group, the increase<br />
of SCE/cell over base line were : 0 .2mM, 19 .81 ; 0 .4 mN1, 41 .97 ; 0 .8mM, 66 .74 . BP<br />
treated group showed similar results : 0 .8mM, 23 .06 ; 0 .16mN., 34 .30 ; 0 .32 mM, 55 .62,<br />
indicating the presence of direct-acting SCE-in8uciry metabolites of CY <strong>and</strong> BP in<br />
r.ice blood . in both control groups, the increase of SCE were nsgligible . This<br />
In Vi',tb assay-In VIvO exposure technique may be a simpl,e <strong>and</strong> useful system for<br />
assaying the In Vivo genotoxicity of a chetnical .<br />
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92 1989 EMS Abstracts<br />
Notes<br />
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262<br />
SHURT-:r9Ji TES"C FCR FRF:DICTICN OF CARCINUGF24S AND/UR YAU11UT8RS<br />
?(uanq Fianjun, S .M .Chen <strong>and</strong> F .Lin<br />
Nat . S.ar.t . Contr . Phara . & Biol . Prod . lseijing, China<br />
e T.a7sr7 sch9me of mutagrnicity has boen established for investigation of thirty<br />
Eninaa.e herbs <strong>and</strong> pbarmaceutieals . Tnroe amrays applied were within the schemes<br />
c :,.te :tinle gene mutation in Awes tsntt detent:ng ohroa,oso .al aberration in CHL<br />
(Ch :nese hamster fibroblast cell line) teat :n vitro <strong>and</strong> in oiaronuoleus in vivo .<br />
Sy aix nas been applied in vitro . Mutaganioity of unknovn drugs in CBL teat, 37 .5%<br />
druge (9/24) induc-d chromosomal aberrations, 4•17yi drugs (1/24) shown inconcluaive<br />
(sle 58 .3% drugs (14/24) didn't induce chro®osoeal aberrations . In aieronueleus<br />
teatn, V,3% drup,e (6/2 ;) induced mioronuoleuted yolychrooatie erythroeytes (MNYCE)<br />
incrvr :ng, 73 .5% dritn (,17/23) shown Y.N" vas in nosmal . in /wea teats, the ∎utation<br />
nus-~bere of 4 strains varied within nosmal fur 24 drup.e . In wutagenicity study on 30<br />
druf*s, cytogenetic study has ehom that suta&nr, <strong>and</strong>/or caroinogens induoed<br />
chroc!osowal aberrations in mammalian eells in CHL <strong>and</strong> wioronuelsue tests . Howver,<br />
mutap+ns <strong>and</strong>/or carcinogens dldn't induee gene autation in AsNa testa, especially,<br />
horm,ne <strong>and</strong> antinotaboliea druRe . The battery ecneote of mutagenioity, especially,<br />
:wong GNL <strong>and</strong> mloronuoloua test, hae been prov .d being effeotive, sensitive, accurate<br />
<strong>and</strong> rpecific, but, Ames test seem *neffective, unaensitive <strong>and</strong> tnacourste .<br />
263<br />
MV:ACFaS 111 DRUGS<br />
Na.ang Nianjun, J,it .Neng, S .ll,Chen <strong>and</strong> D .q .bi<br />
Nat . Snst, Contr . Thans, & Biol . Prod . ]bijing, China<br />
an our elrtwg+nicity study on thirty drugs, cytogenetio study has shown that<br />
mute,w9ca arxl/or caroinogens induced chroaosomal aberrations in sesuealian oells in<br />
C3IL <strong>and</strong> sicronucleus teste . Nine drugs have been proved to be sutagsns . Among nine<br />
auta."•-is, one indirect mutagens <strong>and</strong> eight direct sutagene <strong>and</strong>/or promoters were<br />
discovered . 8561 (Coumarin from Ruta, graveoleus L), no ohromososal aberrations in<br />
C~:;, in culture when the cells vere directly treated for 24h <strong>and</strong> 48 h . While<br />
chror.ozenal aba*rations wers ∎ore prominently with S9 six <strong>and</strong> showed clearly dosedepenier.t<br />
relation . Tr^ break <strong>and</strong> exchange at .errations reached at 93% <strong>and</strong> 69%<br />
(72 . ;nc,/nl) respectively . Folloving five airt-ot .utsgena induced chromoaomal<br />
s.berritions withcut 69 mix <strong>and</strong> also showed the dose-dependent relation . The rate of<br />
c5roonaosal aberrations <strong>and</strong> nieronueleated Yolychromatio erythrocytes in Canciolovir<br />
vne ?t& (0 .5u ./al) <strong>and</strong> 16.67J4 . (Bi~Ong/kr'), iiarrsngtonine 47% (o .1y5ug/al) <strong>and</strong> 18 .05xe<br />
10 .4j :ng/Ir.g), Boanharringtonine 25% (n .1y5ug/v:i <strong>and</strong> 10 .940 (0 .625mg/kg),Desciclwir<br />
+1% (2 . ;?mt;/m1) <strong>and</strong> 10 .5%e (50W'g/kg), <strong>and</strong> Sulbactum NV)L (4 .59®g/ml) <strong>and</strong> 14 .9% .<br />
(± .04r:kg) . AnQther three drugs induc-d polyploid aberrations in CHL test,The rat .e<br />
of po::yplo.id ar,orrations for three druo reached very high levels Flub<strong>and</strong>osole 95%<br />
ea 0 .?bug./vl, 8stinylestrsdial 8YY. at 12 .5ug/sl, Hezoesterol 40A at 15ug/ml, The<br />
ei£~e :i .e to ind-n po2yploid aberrntiooc wan like dlnthyl,tilbestral .<br />
264<br />
TI$ $BFfiCT OF HR7fACHLOR09@iZ6NE AND DDT ON RfiPlODIQCTIPi OUT00lS :4 OF RURAL M0lRN.<br />
X . HUARG, S . VANG, X . sAN, BNTIRCIIlWlAL 1MAL'!8 I118TI1111Ti, ZtVI4liC lEDICAL DPIYSRSITY<br />
,RANGZHOa, CBINA<br />
It was founded that 11BC <strong>and</strong> DDT levels in paddy field soil,surfaoe water <strong>and</strong> foods<br />
in xizin village dropped rapidly after stopping use of these pesticides at the end of<br />
1982 . The present study was to investigate whether the reproductive outoors of vos»n<br />
in the sera wre related to the use of IdiO <strong>and</strong> DDT . During 1981-1986 a total of 995<br />
pairs of mother <strong>and</strong> child tnre underwent medical surveilanoe . 'me detailed questionnaire<br />
<strong>and</strong> examination mainly concerned rternal reproductive events <strong>and</strong> congenital aalforsrtione<br />
. The paraosters adopted in the investieation weret inoidenoes of spontaneous<br />
abortione,of gestation period ( 37 weeks or > 42 veska,of neonate birth wight < 2500 g,<br />
congenital malfor .ations <strong>and</strong> perinatal deaths. 'me results obtained in 1981 end 1982<br />
while llHC <strong>and</strong> DDT were being used didn't differ statistically from those in 1983-1986<br />
while ZXC <strong>and</strong> DDT had been banned(P> 0 .56) . 4fter l'41C <strong>and</strong> DDT vas banned wC level in<br />
breast milk of the village woman dropped fso∎ 0 .8639 pps W982 to 0 .5671 ppa in 1985<br />
<strong>and</strong> DDT from 0 .4279 ppm in 1982 to 0 .0731 ppn in 1985 Hspeotitnly, Fnrthesmore,regreseion<br />
lines analyses between each inoidence of the abov* reproduotive events in the area<br />
<strong>and</strong> 1qiC <strong>and</strong> DDT levels in breast milk in the same year were conducted respeotively . No<br />
signifioant difference was found . 'lhat is to say,the inoidences of abnorrl reproduotive<br />
outooss in Xi=in village remained the sar level after the reduction of ths<br />
. lkiC <strong>and</strong> conclusion i<strong>and</strong> abnotr~l this<br />
avestigatioxsi is that no association between the~ use of<br />
reproductive outoomss in Lixin village vas observed .
265 1989 EMS Abstracts<br />
MUTAGENICITIES OF THAI FOLKLORE MEDICINES AND THEIR NITROSATION PRODUCTS Notes<br />
C . Ieamworapong, K . Kangsadalumpai <strong>and</strong> W . Rojanapo, National Cancer Institute, Bangkok<br />
<strong>and</strong> Institute of Nutrition, Nakornpathom, Thail<strong>and</strong> .<br />
Crude ethanol extracts from 10 commonly used Thai folklore medicinal plants namely<br />
Cassia alata Linn, Casstia angusti.foiia Vahl, Carthamua tinctoriue Linn, Centeila asiatica<br />
Urban, Andrographis paniculata Nees, Cassia fi.stula Linn, Curcwna domeattica Val,<br />
Curcumazedcartia Rosc, Cyperus rotundus Linn <strong>and</strong> Orosylum indiown Vent were prepared <strong>and</strong><br />
tested for their mutagenic activity using the Salmonella/microsome mutagenicity tast .<br />
The first 4 plant extracts, i .e . C . alata, C . angusttifoltia, C. tinotorius <strong>and</strong> C . asfattiea<br />
exhibited mutagenic activity preferably to strain TA 98 only in the presence of<br />
S-9 mix . However, an extract of C . angustifolia showed highest mutagenicity <strong>and</strong> it was<br />
also mutagenic to strain TA 100 when tested in the presence of S-9 mix . After nitrosation<br />
of the above 10 plant extracts with nitrite under acidic condition, extracts from<br />
A . paniculata, C . rotundus <strong>and</strong> Oro2ylum indioum became mutagenic towards both strain<br />
TA 98 <strong>and</strong> TA 100 either tested in the presence or absence of S-9 mix . In addition,<br />
extracts of C. alata, C . angusttifolia <strong>and</strong> C . ttinctortius in which all of them were found<br />
to be mutagenic were also mutagenic towards both tester strains after nitrosation . The<br />
mutagenicities of these nitrosation products were markedly stronger than those of<br />
extracts before nitrosation . Results in the present study demonstrate that some commonly<br />
used Thai folklore medicinal plants contains mutagen especially C . angusttif0ltia<br />
which is now widely used as laxative drug . More interestingly, these results indicate<br />
that some of these plant extracts especially C . alata <strong>and</strong> 0 . tindiOLm contain chemicaUs)<br />
capable of being nitrosated to become strongly mutagenic compounds .<br />
266<br />
LUMINOL . AN INHIBITOR OF POLY (ADP-R I BOSE) POLYMERASE, IS A STRONG INDUCER OF SISTER<br />
CHROMATID EXCHANGES (SCEs) .<br />
T . Ikushima, Research Reactor Inst .,Kyoto Univ ., Kumatori, Osaka 590-04 (Japan)<br />
SCEs provide one of sensitive short-term tests for screening environmental<br />
mutagens . The disturbance of the programmmed replication timing of replicons in the<br />
processes of DNA replication is thought to be involved in the SCE formation<br />
(Ikushima, T . 1980, Annu . Rep . Res . Reactor Inst ., Kyoto Univ .13, 67) . Its has been<br />
suggested that there is a positive correlation between the inhibition of<br />
poly(ADP-ribose) polymerase <strong>and</strong> SCE induction . The specific loss of the oncogenes<br />
induced by inhibition of the enzyme has been recently shown in the transformants Jq<br />
vitro. Here, SCE inducibility of luminol . (5-atino-2,j3-dihydro-l,4-phtalazinedione :<br />
Sigma), a potent inhibitor of the enzyme, has been tested in cultured Chinese<br />
hamster V79 cells . The actively growing cells were treated with luminol for the<br />
two cell cycles, the last one, or the pre-one cycle at various concentrations during<br />
bromodeoxyuridine-labelling, <strong>and</strong> SCEs were detected by FPG method in<br />
colcemid-arrested metaphase chromosomes . Very high SCE frequencies were obtained<br />
after treatments with low concentrations (e .g . 82 SCEs/cell at 0 .5 mM) . Luminol was<br />
more than four times as effective to induce SCEs as 3-aminobezamide, a NAD site<br />
inhibitor of the enzyme . No enhancement of SCE level was observed after the<br />
pre-cycle treatment, in contrast with other strong SCE-inducers such as cis-platin<br />
or mytomycin C . SCEs might be formed during DNA replication by inhibition of<br />
poly(ADP-ribosyl)ation, accompanying the gene amplification or elimination .<br />
267<br />
SUPPRESSIVE EFFECT OF VANILLIN ON MICRONUCLEI INDUCED BY MITOMYCIN C OR X-RAYS .<br />
T .Inouye, Y .F .Sasakl, H .Imanishi, M .Wetanabe, T .Kato, K .Matsumoto, T .Ohta <strong>and</strong><br />
Y .Shirasu, Institute of <strong>Environmental</strong> Toxicology, Kodaira, Tokyo 187, (Japan)<br />
Vanillin is a component of vanilla essence flavour . We previously reported the<br />
antimutaQenlc effect of vanillin on mutagenesis in bacteria(1) <strong>and</strong> its anticlastogenic<br />
activity in a cytogenetice study ueing cultured cells(2) . In order to<br />
find out in vivo anticlastogenic effect of vanillin, bone marrow micronucleous test<br />
was conducted. BDF1 male mice at 7 weeks old were treated with mitomycin C(MMC) or<br />
ionizing radiation . Thereafter vanillin at 500 mg/kg was given orally . Bone marrow<br />
cells were sampled 24 h after injection of 2 mg/kg MMC . In the experiments the time<br />
interval between MMC injection <strong>and</strong> vanillin treatment was varied, a significant<br />
reduction in the frequencies of micronucleated polychromatic erythrocytes(MN-PCEs) by<br />
38-50/ was observed from 6 to 9 h after YlAC injection . The frequencies of IQ)-PCEe<br />
induced by X-rays (200R, 150kV, 5mA) plus vanillin treatment was less than those of<br />
X-rays only (mean.SD :3 .1a1 .1/ vs 4 .97+0 .95), when bone marrow cells were sampled 24 h<br />
after X-irradfetion . When sampled at 15 h, however, the similar frequencies were<br />
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93
94 1989 EMS Abstracts<br />
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Notes observed (3 .1+1 .75'vs 3 .6+1 .1) . These results indicate that vanillin is also<br />
effective in vivo syeteme <strong>and</strong> that vanillin may act at <strong>and</strong> before the S period of the<br />
cell cycle. 1 Ohta et al ., Food Chem . Toxicol . 24,51(1986) . (2)Sasaki at al .,<br />
Mutation Res ., 191,193(1987) .<br />
268<br />
IN VITRO CLASTOGENICITY OF 951 CHEMICAL SUBSTANCES . M . Ishidate,<br />
Jr ., M . C . Harnois*, <strong>and</strong> T . Sofuni, National Institute of Hygienic<br />
Sciences, Setagaya-ku, Tokyo (Japan), *Fellow, Japan Foundation<br />
for Promotion of Cancer Research .<br />
A literature review of 1964-1985 publications on the in vitro<br />
clastogenicity of chemical substances resulted in data from 240<br />
reports on 951 substances . Of these 951, 447 were consistently<br />
positive either with or without activationj 417 were negative<br />
without activation but not tested with activationi 30 were<br />
consistently negative when tested both waysi <strong>and</strong> 53 gave variable<br />
results . The variability was related to cell type, presence of<br />
activation mechanisms <strong>and</strong> treatment schedule . Addition of an<br />
activation system reduced the variation among different cell typesr<br />
no one cell type appeared to be superior for testing all clastogens .<br />
The conce%tration at which substayes tested positive ranged from<br />
4 .3 x 10 (trenimon) to 6 .9 x 10 mM (acetone) . Generally, there<br />
was an inverse relationship between the concentration required<br />
to induce aberrations <strong>and</strong> the frequency of exchange-type aberrations<br />
produced by a chemical at its maximum effect dose . The relevance<br />
of tests conducted at high concentrations is considered, <strong>and</strong> caution<br />
urged in the interpretation of test results obtained under<br />
physiologically stressful conditions .(Mutation Res .,195, 151, 1988)<br />
MOTAGENICITY AND ANTIHUTAGENICITY IN AIR-BORN PARTICULATES<br />
269<br />
Hiroko Iwado1'2 . Mitsuko Naitol Hikoya Hayatsu2, lOkayama Prefectural Research Center<br />
of <strong>Environmental</strong> <strong>and</strong> Public Health, Uchio, Okayama 701-01, 2Faculty of Pharmaceutical<br />
Sciences, Okayama University, Tsushima, Okayama 700 (Japan)<br />
Air-borne particulates collected by a high-volume air sampler in a suburban area of<br />
Okayama City were extracted with methanol under ultrasonic vibration . The residue<br />
obtained on evaporating the solvent was dissolved in a small amount of dimethylsulfoxide<br />
<strong>and</strong> the solution was divided into two portions . One was submitted as such to<br />
the Ames test for assaying mutagenicity (sample 1) . Another portion was suspended in a<br />
large volume of water <strong>and</strong> treated with blue cotton to adsorb polycyclic compounds to<br />
the cotton . A methanol-ammonia eluate of the blue cotton (sample 2) <strong>and</strong> the aqueous<br />
portion that remained after the blue cotton treatment, after evaporation to dryness<br />
(sample 3), were also examined . 1 shoved mutagenicity towards S . typhimurium TA98 in<br />
the presence of S9-mix in a dose-responding linearity up to 27 m3 air-volume equivalent ;<br />
however, the colony number stayed constant in the dose range between 27-270 >l3 . In<br />
contrast, 2 gave a linear dose-response up to 270 m3 . Although the numbers of<br />
revertant colonies found were about 2-times greater with 1 than vith .2 in the range<br />
lower than 27 m3, the numbers obtained with 2 were much greater than those with 1 in<br />
the higher dose range . These results suggest that antimutagenic factors were present<br />
in 1 . In fact, when 3 was mixed with 2, a strong suppression of the mutagenicity was<br />
observed . The antimu[agenic factors pieaent in 3 were capable of inhibiting the<br />
mutagenicity of benzo(a)pyrene <strong>and</strong> that of 2-nitrofluorene . (Supported by Nippon Life<br />
Insurance Foundation .)<br />
270<br />
INFLUENCE OF UMUC ON EMS-INDUCED YUTAOEBESIS IN E . COLI DEFICIENT IN<br />
MISMATCH REPAIEE- C . Janion, <strong>and</strong> E . Orsesiuk, InetiTaTe- of Biochemistry<br />
<strong>and</strong> Biophysics, Polish Academy of Sciences, Warsaw, Pol<strong>and</strong> .<br />
we have compared mutagenio aetivit <strong>and</strong> speeifieity of EMS (ethyl<br />
methaneaulfonate) in ;gc~ 2497( +~~ ) <strong>and</strong> its derivative<br />
strains : EC2401(mutS+~um v _~ Y1(mu uC') <strong>and</strong> EC2402( ut~S'umuC-) . It<br />
has been found tE~"-t s ite of mu at on (based on ezam3nation of the<br />
phenotypes of Arg+ revertants) <strong>and</strong> the dutagenio specificity (based on<br />
ability of the Arg+ revertants to support growth of aaber <strong>and</strong> ochre T4<br />
bacteriophages) depends on um C, but only in the mutS-- mismatch repair<br />
de icient strain . The maJor+it~'of EIB-induoed Arg'r'~3vertants of AB<br />
24~7, EC2401 <strong>and</strong> EC2402, show the phenotype - Arg+His-Thr ; whereas<br />
the majority of EMS-induced Arg+ rewertants of Y1 show the phenotype<br />
50869 3606
Arg'His*Thr* .Examination of the pattern of amber <strong>and</strong> oohre T4 baoteriophage<br />
suppression indicate that the reversion to Arg+His Thr in AB<br />
2497 may be the result of su B <strong>and</strong> ~eu Eoc suppressor formation (whioh<br />
^an arise by a GC 4 AT tranion)•re~as the reversion to Arg*Hie•<br />
rhr* may be the result of g1tY or fxs-4 suppressor formation (which<br />
can arise by a GC (or AT) -~- TA traneversion) The frequency of El13-induced<br />
Arg* reversion is little dependent either on umuC or mutS .<br />
The mechanism of the different specificity of EUS-in ueed m-0s$ione<br />
will be discussed .<br />
271<br />
MULTIPLE END POINTS FOR SOMATIC MUTATIONS IN HUMANS PROVIDE<br />
COMPLEMENTARY VIEWS FOR BIODOSIMETRY, GENOTOXICITY, AND<br />
HEALTH RISKS<br />
R .H . Jensen, W .L. Bigbee, <strong>and</strong> R.G . Langlois, Biomedical Sciences Division,<br />
Lawrence Livermore National Laboratory, Livermore, CA 94550<br />
Recent technologic developments now allow us to measure genotoxic effects of human<br />
exposure to mutagenic phenomena In four different ways--HPRT mutations in lymphocytes,<br />
HLA mutations on the surface of lymphocytes, hemoglobin variant mutations In<br />
erythrocytes, <strong>and</strong> glyoophorin A mutations on the surface of erythrocytes . Each of these<br />
assay methods shows advantages <strong>and</strong> disadvantages In being used to monitor for<br />
exposure of individuals to mutagenic phenomena such as toxic chemicais or Ionizing<br />
radiation . For example, the techniques used for the lymphocyte-based assays can be<br />
extended to isolate nuclear DNA <strong>and</strong> characterize the mutagenic changes that have<br />
occurred, while erythrocytes contain no DNA for such analyses . On the other h<strong>and</strong>, the<br />
erythrocyte-based assays are probing a tissue in which differentiation Is straight forward<br />
<strong>and</strong> clearly delineated, whereas lymphocytes display complex <strong>and</strong> muRi-organ<br />
developmental pathways . Thus, using combinations of these assays (<strong>and</strong> others as they<br />
develop) for each individual should furnish a set of monitoring data that can be broadly<br />
interpreted to provide biodosimetrks Information <strong>and</strong> potential estimates of cancer risk . The<br />
impact of obtaining such information is high enough to justify the challenge of performing<br />
mufti-end point analyses on populations at high risk for mutagenic exposure .<br />
272<br />
GpNOTOXICI7Y OF 23 C}ICMCAIS TO S GFREVLSIAE<br />
li ang Z„oshu et at<br />
A .-parennt of Biology. Sooond Mitlifary Medical thriwsity, Shanghai, CMna<br />
23 kinds or danicals vNne used to evaluate the edidettcy of S ans4sias as a toot in the assay of genotopns<br />
The growing cells ar S aaevimac D7 wero treatod with thatdcels at 28°C for 6 houtz As te~oAOd eonte known<br />
mutagens such as MNNG, 4-NQO <strong>and</strong> hydroxytmea oould inoease the firqtta>cy of ttp oarvertants _ to<br />
3-10 t6rts, <strong>and</strong> we also found that same pvnulagw such as cyclopl~osp}rvnidc <strong>and</strong>trypon btue could iitacase<br />
the froqua~y to s-10 times without any exogatotn adivation 'iltis itd'~caled that the yeast may<br />
cndogawuclY me~aboli~e prcmutagens into teadlre intanndiatea lt wat sepottod that mme eatdttopens had a<br />
false-ncgaGw iesportst in Amca trst, twl in our experinrnt 3l evas shatm lhat Q 1-Q 3•/% anihne muld lncrease<br />
Ux frcqua~y ot gene oonNersion in1tp Ioats to 1-3 4 tirrrs, 15-30 tng/ml aiodnic anhydride 1 .4-A 1 tanea,<br />
10-80 mM trypan biue 1-4 Gntca, Q 12-Q 4 M thioutt8l S-7 . S timx atd a2 -Q S•/. prhott Iettachlatide<br />
2 . 5-10 tirnes AdditionaRy , the intertupt prooodt~ was tssod to aaoen the indttoaa or ehrornason~e<br />
mahr~a fion The growing odls of S oaetidae D6L M t+ere irtated with diatticah at 28'C for 4 hottn, then<br />
at 0 C for 16 hours <strong>and</strong> again ,at 28°C for 4 twun brloee piatod wilh the tmditun oontaininnY Q S µg/m)<br />
cydoheuamide The fioqtrawy ot the while lettdne-ra1 oolonra tepresetttod Ihat of chrarnosatte loss It<br />
was shown that some chanicais such as methyl nntitae,jlate~oould catne ctunsttasorne loes We aostdudod that<br />
the S annisiae has a unique tac~~ulrcss in the aaay of gs,otapts<br />
273<br />
ANTIMUPAGENICITY OF CHINESE MEDICiNES .<br />
Tiang Zuoshu, WangUngh ua <strong>and</strong> Chen Zhongfu<br />
Dcpartmrnt of Biaiogy. Seoord Military Modiesl Lhtiversity <strong>and</strong> Institute of Gcnctics, Fudan L)niversity, Shan ;ha't<br />
Chma<br />
In order to study the antimutaganaty of Cldnese modidncs, the Inductest was uud to saern the inh'hiton of SO6<br />
re;ponsc homar~ang 601rnds of Clmiete rnodidr~, as it has tzen known that SOS response ptays an important role<br />
m mutagrnese. A IItu paper disc with 10 yt of 0. 1 mg/mt of Mitomydn C (MMC) <strong>and</strong> another one with the estract<br />
of Qiinese momcnie wce put 1 an apart on the surfem of the top agar containing both the Absogc* E[xa6<br />
acII ( GY5027) <strong>and</strong> the udieator E aiticd (GY4015) . Aller the plate was incubated at 3NC for 8 hours, phage<br />
plaques appeared around the MMC diu, <strong>and</strong> a partial afipse of SOS hile'hition ( with roplaque or darcased ntmber<br />
of plaque) would appear bdwern the two disc if the Ci :nae iiiediaie estract in tho disc had an inhibitory edax on<br />
SOS response The results showod that 11 kinds of Chineae nmbdnes had such an eQoct. Sane of than were further<br />
studiod with SOS dvamotest. In this test, the growing od af E ca6 PQ37 was exposed to U K <strong>and</strong> incvbated in LB<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 95<br />
Notes
96 1989 EMS Abstracts<br />
6<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
:d witli the atract cf C]i6rae emdidne at 3TC for 2 boun, thepn ~eh(ye ~activity 11 -Balactoaidau was tottad which<br />
L~daa~ea~ : the S06~set~pon io t~~ Sv : e~t td I0a/ft af tlrc~aonlr~d rapo~ivebr. aythe +~icneo o-<br />
OW 1 (rec~A441) te o~'% SOe~ <strong>and</strong> 60e/. ~ the~aaurob but had not eda(on SOB rctw~a~k gene app osion i~ E6<br />
a.df QW1107 ( laulSl )(arore tlrn 9SK of tbe controi) . So it tu+mtm that thtae l~e nn&ars coetaui the<br />
ialr~iton of RxA proteaoe <strong>and</strong> nay be utod u antinutayr~u <strong>and</strong>/a antxarrirwgau .<br />
274<br />
CNRONOSORE ABERRATIORS IR NUNAR LYNPNOCYTES AS AR IROICATOR OF RADIATIOR DANACE<br />
Jin Cuithen, Institute of Radiation Nedicine, Beijing, China<br />
The analyses of chroaosoae aberrations in huean lymphocytes have shown their<br />
usability for the estioation of individual doses of over-exposed persons in radiation<br />
accidents <strong>and</strong> for the assesseent of their late radiation effects . Our laboratory<br />
has been involved in the cytogenetic studies for radiation accidents in<br />
China . The aain results can be suosarited as follows : 1) Under the circumstances<br />
of single acute exposure, the vast oajority of aberrations observed soon after<br />
the exposure were of unstable types . Among thes, the incidence of dicentries<br />
plus rings could be used to ∎ake reliable dose estisation . The results were eonsistent<br />
with the clinical courses of the vioties . 2) If the blood culture ras<br />
done s .veral eonths after the exposura, the aberration frequencies could not<br />
be used as a biological dosimeter, but it still showed sooe general correlation<br />
vith radiation dose previously received . 3) Follor-up studies on over-exposed<br />
peraons indicated that most of the remaining aberrations were of stable types .<br />
4) The chroeosoae analyses for victims following protracted exposure lasted half<br />
year exhibited evidence that the frequencies of unstable types were coeparable<br />
vith that of stable types . 5) G-b<strong>and</strong>ed chromosome analyses carrying out on 5<br />
victies, vho received accidental over-exposure several years ago, showed a nonr<strong>and</strong>o∎<br />
distribution of break-points . They vere eainly clustered in soae chroeosoees<br />
<strong>and</strong> vere preferentially located in the regions Sq3, 6q2, 9q3, 14q3, etc . Soae<br />
break-points were near or at the map sites of the known oncogenes .<br />
275<br />
TEMPERATURE EFFECTS ON RADIATION-INDUCED CHROHOSOHAL ABERRATIONS IN THE PRESENCE OR<br />
ABSEN*E OF DIMETHYL SULFOXIDE . E .E . Joiner, L . G . Littlefie~d, S .P Colyer* <strong>and</strong> E .L .<br />
Frome 1, Oak Ridge Assoc . Univer ., <strong>and</strong> Oak Ridge Nat 1 . Lab . , Oak Ridge, TN (USA)<br />
Studies of the dose response relationship for dicentrics in human lynphocytes<br />
exposed to X-rays at 37°C or 4°C demonstrated that radiation temperature acts as a<br />
dose-modifying factor by influencing the for .ation of chromatin lesions that lead to<br />
aberrations (Gumrich st al ., Int . J . Rad . Eiol ., 49 :665-672, 1986) . We employed DNSO<br />
as a model OH radical scavenger in experiments to determine to what extent radiation<br />
temperature modifies the proportions of aberrations resulting from DNA damage indueed<br />
by direct ionisations vs OH radical attack . Human whole blood was maintained at<br />
temperatures of 4°C vs 37°C in the presence or absence of 1M DHSO during exposures to<br />
1 .04, 2 .08 <strong>and</strong> 4 .17 Gy X-radiation . Lymphocytes were washed <strong>and</strong> cultured for 48 hr .,<br />
<strong>and</strong> aberrations were scored in lst division metaphases . Dicentric yields in cells<br />
irradiated at 37°C in the presence of Dtt30 shoved reductions of 650 compared to<br />
yields observed in unprotected cells . For lymphocytes irradiated in the absence of<br />
DNSO, dicentric yields were 411 lover following exposures at 4°C than the yields<br />
observed at 37°C . In contrast, when cells protected by Dt4S0 were irradiated,<br />
dicentric yields at 4°C were only 211 lover than those observed at 37°C . These<br />
preliminary results suggest that the effects of cold temperature (4°C) on the yields<br />
of radiation-induced chromosomal exchange aberrations in human whole blood<br />
lymphocytes are primarily due to the inhibltion of the indirect radiochemical actions<br />
of OH radicals on DNA targets . Supported by U .S . DOE/ORAU Contract<br />
DE-AC05-760R00033 .<br />
276<br />
MOLECULAR ANALYSIS OF RADON-INDUCED MUTANTS R .F. Jostesl, R .A . Giesi,<br />
T .L . Morganl, E .N . Fleck:, K .P . Gasperi, <strong>and</strong> F .T .Crossl . lPacific Northwest<br />
Laboratory, Richl<strong>and</strong>, Washington <strong>and</strong> $WhitRian College, Nalla Walla, Washington .<br />
An in vitro system for exposing RutRtiaalian cells to radon gas <strong>and</strong> its daughters has<br />
been developed in our laboratory . Cells are exposed in spinner flasks <strong>and</strong> the doses<br />
reported here are to the cell culture medium . Radon-induced mutations at the HGPRT<br />
locus in Chinese hamster ovary cells show a linear dose response with an induced<br />
frequency of 1 .0 x 10-o mutations per viable cell per c6y . To date we have isolated<br />
35 mutants for molecular analysis . Southern blot analysis of DNA from 21 radoninduced<br />
HGPRT- cell lines indicated that 11 (52k) were caused by a complete deletion<br />
of the gene . Three mutations (14%) showed altered b<strong>and</strong>in~ patterns indicative of<br />
subtotal deletions <strong>and</strong> the remaining 7 mutations (34%) conta ned changes undetectable<br />
by this analysis . (Work supported by the U .S . Department of Energy under Contract<br />
DE-AC06-76RL0 1830) .<br />
50869 3608
277 1989 EMS Abstracts 97<br />
Notes<br />
DEVELOPMENT OF A SENSITIVE HUMAN EPITHELIAL CELL LINE FOR MUTAGEN SCREENING . Y .S .<br />
Jou, C .C . Chang, <strong>and</strong> J .E . Trosko, Department of Pediatrics <strong>and</strong> Human Development,<br />
Michigan State University, East Lansing, MI 48824 .<br />
An ideal assay for human mutagens should be relevant (related to mutations in human<br />
cells), sensitive (able to detect low-dose mutagens) <strong>and</strong> informative (revealing genetic<br />
alterations at the molecular level) . Toward developing a cell line with these attributes,<br />
we have reactivated the hypoxanthine-guanine phosphoribosyl transferase<br />
(HGPRT) gene on an inactive x-chromosome by 5-azacytidine treatment in a 6-TGr human<br />
teratocarcinoma cell line (46 ch . ; xx ; t 15/20) . In contrast to the normal human cell<br />
line, the reactivated HGPRT gene is on a non-essential x-chromosome . Therefore, deletions<br />
or mutations of essential genes on the non-essential chromosome associated with<br />
the HGPRT gene mutation would not affect the survival of a 6-TGr mutant . After exposure<br />
to mutagnes, a higher frequency of induced 6-TGr mutants would be expected . After<br />
treatment with 5-azacytidine, HATr colonies were readily recovered . Most of these<br />
clones are revertible to 6-TGr cells at hi gh frequency . Few stable clones, however,<br />
were also recovered . Our preliminary studies using one of these stable clones indicate<br />
that the cell line had a 50-100 fold increase in x-ray induced 6-TGr mutant compared<br />
to the parental HATr cell line . In conjunction with molecular analysis using<br />
polymerase chain reaction amplification <strong>and</strong> DNA sequencing techniques, the cell line<br />
might become an ideal assay for human mutagens . (Research supported by a NCI grant<br />
CA21104-11) .<br />
278<br />
A CYTOGENcTIC STUDY ON SUBJECTS PRESENTING AIENORRNEA . STERILITY AND RPROOUCTIVE FAILURE .<br />
A . Jvothv, G .S . Isaec, A,Shobhe Rani . C. Kususa Kumeri <strong>and</strong> P.P .Raddv .<br />
Institute of Genetics . Hospital for Genetic Diseases . Owanie Universitv, Beouaoet . Hvderebad-S00 016.<br />
A .P . India. '<br />
A B s T R A C T<br />
Chromosoae analysis plays a vital rola in spaculatinq the etioloqY of cases presanting s .enorrhsa . ster!-<br />
litv <strong>and</strong> reproductive failure . The present study is tharsfore aieedrto lnvestipate the role <strong>and</strong> distribution<br />
of chromosose abnormalities in causing thssa conditions . A total of 1575 twale subjects presenting<br />
enenorrhea . sterility <strong>and</strong> reproductive fsilure usre investigated for .chrososae abnormalities . These<br />
include cases of primary awenorrhea (37S) secondary wenorrhw ( 100), sterility (75) <strong>and</strong> reproductive<br />
failure ( 325) .<br />
The subjects were thoroughly examinsd <strong>and</strong> the history of the patient <strong>and</strong> her family rera recorded . Chroaosae<br />
preparations wre ∎ade according to the ∎odified ∎sthod of Moorhead atal (1960 ) <strong>and</strong> b<strong>and</strong>ed<br />
according to Seabright (1971) . Other staining techniques (CBG), Ag NOR etc .) wre employed wherever<br />
necessary . The typs of abnormalities detected include 45X ; 4SX/46XX ; 45X/46XY; 43X/47X)0(i 4V/46X, frsgi<br />
45X/46XX/46XY ; 46XX/46 XY ; 4SX/46xx/47XXX ; 46XY ( Phenotypic females) ; 47xXX ; 46XX, 13 p <strong>and</strong> 46 XX,<br />
16p* The abnormalities ldentified suggest the need for routine chrososose survey among patients with<br />
the above clinical syapta s . These studies will help !n the accurate diagnosis <strong>and</strong> eanapesent of such<br />
clinical entities .<br />
279<br />
BOTRAN AND BLEOMYCIN INDUCED CROSSING OVER AND ANEUPLOIDY IN ASPERGILLUS NIDULANS<br />
WHICH RESULTS IN DIFFERENT PATTERNS OF MITOTIC SEGRECANTS . E . Kifer, Department of<br />
Biology, McGill University, 1205 Dr . Penfiald Ave ., Montreal, P .Q . Canada H3A 151 .<br />
Botran <strong>and</strong> bleomycin reduce growth <strong>and</strong> increase mitotic segregation of recessive<br />
colour markers in diploid heterozygous tester strains of Aspergillus . In both cases,<br />
segregants are mainly diploid crossovers <strong>and</strong> haploids . The latter were especially<br />
frequent on botran media, <strong>and</strong> at low concentration aneuploids, mainly disomics for<br />
chromosome III, also formed discernible patches of coloured conidia . In addition,<br />
diploid segregants showed high levels of coincidence of crossing over, segregation in<br />
three or even four chromosome arms being not uncommon . On the other h<strong>and</strong>, treatment<br />
of germinating conidia which were plated onto complete medium resulted in few large<br />
crossover sectors but produced abnormal colonies in both cases . With increasing<br />
concentrations of bleomycin, these were especially evident, increasing from 25 to 75%<br />
among survivors . On replating, up to two thirds of them could be identified as<br />
aneuploids, the majority being genuine trisomics . These results suggest that both<br />
botran <strong>and</strong> bleomycin induce crossing over <strong>and</strong> malsegregation . (Supported by<br />
Strategic Grant 0032242 from the Natural Science <strong>and</strong> Engineering Research Council of<br />
Canada) .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
.
98 1989 EMS Abstracts 280<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes MOLECULAR SPECfRUM ANALYSIS OF FRAMESHIFT MUTATIONS IN YEAST D . Kalirawcki,<br />
KM. Mottus, M.J. Plewa, <strong>and</strong> RW. Larimer', Institute for <strong>Environmental</strong> Studiea, University of Illiaois,<br />
Urbana, IL 61801, 'Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 .<br />
The purpose of this study is to recover sequence information at the HIS4A region on chromosome III<br />
from a large number of independently arising, htr• spontaneous <strong>and</strong> 4 nitroquinoline-N-oAde (4NQ0)<br />
induced revertants of Saccharomyces temirk+e . We constructed a shuttle vector, pMP4, that contains a<br />
438 bp deletion encompassing the +G framahift mutation, hls4 :38 . A large number of hit4- .38 spontaneous<br />
revertants were obtained ; a r<strong>and</strong>om sample of 200 spontaneous <strong>and</strong> 4NQO induoed revertants<br />
were screened for suppressor mutations <strong>and</strong> are being analpsed . Each revertant is separately transformed<br />
with linear pMP4 that contains the 438 bp gap <strong>and</strong> the Infocmation from the yeast chromosome is copied<br />
onto the plasmid by a gene conversion process known as double,str<strong>and</strong> gap repair. Since the gap extends<br />
3' beyond the original +1 frameshift mutation, a new 1.3 kb SaII restriction site appears in the repaired<br />
pMP4 . We have recovered <strong>and</strong> sequenced chromosomal information from over 40 spontaneous his4-38<br />
revertants . The 4NQO induced revertants were produced at a concentration of 2.3 µghnl <strong>and</strong> are being<br />
screened using the same procedure . This approach permits the isolation of DNA sequence information<br />
of a mutation from an unmodified eukaryotic chromosome. We will define the distribution of bases<br />
involved in mutation leading to reversion at his4- .Id The use of double str<strong>and</strong> gap repair allows the<br />
analysis of the molecular spectrum of these revertants <strong>and</strong> will provide insight to the mechanisms<br />
underlying spontaneous <strong>and</strong> 4NQO•induced frameshift mutation In eukaryotes . Funded by the<br />
Interdisciplinary Program in <strong>Environmental</strong> 7bbcology .<br />
281<br />
S'PuLY UiV 1'cir . .:c.. .dJiOk OF MUTA(3ENS FROM MJNICIPAL INCINP:RATORS BY MhnfiS<br />
OF AMES ASSAY .<br />
A~.Ka~miya,:~agoya City <strong>Environmental</strong> Pollution Research Institute,Chudochc<br />
nNTiamiku, Nagoya City(Japan), Y .Ose <strong>and</strong> T .Sato,Gifu Pharmaceutical<br />
University,Mitahira-higashi, 3ifu City(Japan)<br />
The mutagenicity <strong>and</strong> mutagens of fly ash,emission gas <strong>and</strong> wastewater<br />
in municipal incinerators have been investigated by Ames assay <strong>and</strong> chem•ical<br />
analysis . A negative relationship was obtained for complete combustion<br />
<strong>and</strong> a positive one for incomplete combustion . About 90% of all<br />
mutagens produced in a incinerator are released into atmosphere as emission<br />
gas,<strong>and</strong> 10% are disposed in the electrostatic precipitator <strong>and</strong><br />
wastewater treatment plants . 1,6-Dinitropyrene(DNP) as direct acting<br />
mutagen was detected in emission gas from incomplete combustion . The gac<br />
phase photochemical reaction of oyrene with (vOs gas was examined in a<br />
quartz vessel with various reaction times <strong>and</strong> temperature . 1-NP was<br />
readily formed from pyrene in the absence of light irradiation <strong>and</strong> low<br />
temperature, but the formation of 1,6-DNP is dependent on light irradiation,<br />
temperature <strong>and</strong> nitric acid . Most of mutagens in wastewater<br />
treatment olants are not decomposed by normal aeration times, but are<br />
removed by adsorption onto suspendid solid . Total revertants per minute<br />
from the emitted gases corresoonded to that from the exhaust of 1700 -<br />
3000 motor vehicles . The mutagens emitted from total municipal incinerators<br />
in Japan 1985 were estimated to be 16 .3ton as benzo(a)pyrene .<br />
282<br />
ON THE VALIDATION OF TME SYSTFM OF ASPER(iILLUS FOR TESTING IINIFAM1FNfAL ANEIIGIIdS<br />
A . Kappas<br />
National Research Center "Aeaacritus", Athens, Greece .<br />
Chemically-induced malsegregation of chranosones is detected in two types of diploid<br />
strains of the ascamycete As r illus nidulans . One detects euploid mitotic<br />
segregants as products of either ma segregat on non-disjunction) or mitotic crossing-<br />
-over . The other detects aneuploids as unstable abnormal segregants <strong>and</strong> can distinguish<br />
between primary aneuploidy of whole chrat,oscmes <strong>and</strong> secondary spontaneous aneuploidy<br />
resulting from primary chrotaosome breakage . We developed a system which<br />
belongs to the first type of strains in which we can also detect chromosome breakage<br />
<strong>and</strong> it is possible to identify whether segregants can be the result of secondary spon<br />
taneous malsegregation of chraaosemes with terminal deletions . In this system we have<br />
been able to use the metabolic activation technique with an S9 mix with microsamal<br />
fraction from rats . In a coordinated progratmne of the EBC countries with the aim of<br />
developing <strong>and</strong> validating proper test system(s) for the regulation of environmental<br />
chemicals which can induce genaaic mutations, a variety of chemicals selected on the<br />
basis of their ability to interact with several cellular targets have been investigated<br />
in A . nidulans <strong>and</strong> many of them, including chloral hydrate, hydroquinone, thiabendazole<br />
grise'o uTvin, benomyl, were detected as aneugens .<br />
Supported by Grant EV4V-003S-GR from the EBC .<br />
50869 3610
283 1989 EMS Abstracts 99<br />
SPECIES COMPARISONS REGARDING COMPARATIVE METABOLISM OF TWO STRUCTURALLY SIMILAR Notes<br />
NITROPHENYLENEDIAMINES (HC BLUE 1 AND HC BLUE 2) . F . Kari, S . Driscoll, C . Parker,<br />
K . Rudo, K . Tomer, <strong>and</strong> R . Langenbach ; National Inst u e of <strong>Environmental</strong> Health<br />
Sciences, Research Triangle Park, NC 27709 . Chronic evaluations reveal that HC Blue<br />
1 causes hepatocellular carcinomas in mice, but a structural analog, HC Blue 2 is not<br />
carcinogenic in mice . Neither of these chemicals are carcinogenic in rats . The<br />
bioassay results of this carcinogen/non-carcinogen pair mirror the species- <strong>and</strong><br />
organ- specificity obtained with 23 congeners which have been evaluated under<br />
similar conditions . Accordingly, comparative metabolism studies were done to elucidate<br />
mechanisms for their discordant carcinogenic potencies <strong>and</strong> species-specificity .<br />
Urinary recovery of radiolabel following oral administration of the parent compounds<br />
was equivalent for both compounds in mice <strong>and</strong> rats . Conversion of parent compounds<br />
to metabolites was quantitatively comparable indicating systemic availability is not<br />
responsible for the discordant potencies . Metabolism of these two compounds (200pM)<br />
by hepatocytes isolated from mice <strong>and</strong> rats yields metabolic profiles similar to their<br />
respective in vivo profiles . HPLC separation shows that in mice HC Blue 1 Is metabolized<br />
to five major metabolites while the non-carcinogen HC Blue 2 yields only one<br />
major metabolite . Thermospray LC/MS analysis of HC Blue 1 metabolites from mice provides<br />
tentative evidence for nitroreduction, demethylation <strong>and</strong> conjugation<br />
(acetylation <strong>and</strong> glucuronidatton) . In the non-susceptible rat, HC Blue 1 produces<br />
three metabolites similar to those found in mice, but two metabolites produced in<br />
mice are notably decreased or absent in rats . Qualitative differences in metabolism<br />
of these compounds may contribute to their different carcinogenic potencies <strong>and</strong><br />
species specificity .<br />
284<br />
INDUCTION OF CHROMOSOME-TYPE ABERRATION AT ZYGOTE FOLLOWING CHLORAMBUCIL ADMINISTRATION<br />
IN MALE MICE, M .Katoh <strong>and</strong> R .P .A .Valdivia, Food <strong>and</strong> Drug Safety Center, Hadano,<br />
Kanagawa (JAPAN), <strong>and</strong> University of Chile, Santiago (CHILE)<br />
Katoh et al . (1986) reported that most alkylating chemicals which are shown dominant<br />
lethals in postmeiotic male germ cells induce chromosome-type aberrations at zygotes<br />
<strong>and</strong> these agents associated with the inductions of heritable tranalocations~in offspring<br />
. Chlorambucll, which is an alkylating agent of the nitrogen mustard type,<br />
induced the high frequencies of dominant lethals in male germ cells except late spermstids<br />
(personal communication by W .M .Generoso) . This dominant lethal's pattern is the<br />
complete opposite of that observed by acrylamide which 7bhoved sperm protamina alkylation<br />
but not of DNA alkylation (Shelby et al .1986, Saga et a1 .1988) . Also, cytogenetic<br />
analyses by acrylamide demonstrated chromosome-type aberration (this result<br />
is presented by R .P .A .Valdivia in FIFTH ICEM 1989) . In the present study, cytogenetic<br />
analyses by chlorambucil were undertaken to clarify the mechanisms for chromosome<br />
aberrations in male germ cells of mice . Male mice were given single intraperitoneal<br />
injections of 25, 12 <strong>and</strong> 6 mg/kg of chlorambucil <strong>and</strong> then cytogenetic analyses at<br />
zygotes were carried out for 5 weeks after injection . The frequency pattern of zygotes<br />
with chromosome aberrations was found to correlate with that of dominant lethal . The<br />
sensitive germ cell stages were demonstrated in late spermatocytas, early .permatids<br />
<strong>and</strong> late spermatozoa . Late spermatid stage was ruled out . In these sensitive stages,<br />
chlorambucil induced chromosome-type aberrations . These evidences show that the induction<br />
mechanisms of chromosome-type aberration by chlorambucil is different from that<br />
of sperm protamine alkylating agents such as acrylamide <strong>and</strong> methyl methanesulfonate .<br />
285<br />
MUTAGENICITY TEST FOR GASEOUS AND VAPOUROUS COMPONENT OF COMBUSTION EXHAUST . A .Kawai',<br />
S .Goto', O .Endo', H .Matsushita=, 'Japan Automobile Research Institute, Tsukuba,<br />
Ibaraki,(Japan), 'National Institute of Public Health, Minato-ku,Tokyo (Japan) .<br />
Many reports have been published for the sutagenicity of particulate component of various<br />
combustion exhausts, but few for the mutagenicity of gas/vapour cosponent of these exhausts .<br />
We devised apparatus for sutagenicity assay of gas/vapour co.pounds <strong>and</strong> obtained a suitable<br />
test condition for this assay . Particle-free test gas was prepared by passing a diluted<br />
combustion exhaust through quartz fibre or similar filter, collected in a Tedra bag (200 l),<br />
<strong>and</strong> diluted with fresh air . The test gas was introduced into a Pyrex glass chasber(3 or 8 L)<br />
in which 10 or 16 test plates having tester strain on the surface were placed inversely .<br />
Following test condition was selected fros various kinds of test results as a suitable one<br />
for autagenicity assay ; 0 .251 /ein for flow rate of test gas, 2-8 hr for exposure time, <strong>and</strong> 37<br />
'C for the temperature of chamber . Mutagenicity test was carried out for the particle-tree<br />
exhaust gas from diesel engine,kerosene heator <strong>and</strong> side-streas of cigarette smoking,using<br />
Salmonella typhisurius TA98 <strong>and</strong> TA100 . E .Coli WP2uvrA/pKM101 was also used for the test of<br />
cigarette smoke . Gas/vapour component of diesel exhaust gave positive sutagenic responses for<br />
TA100 with <strong>and</strong> without S9 ∎ix <strong>and</strong> for TA98 with 39 six . The component of kerosene heater<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
100 1989 EMS Abstracts<br />
Notes exhaust gave positive responses for both the tester strains with <strong>and</strong> without S9 ∎ix . The<br />
component of side-stream saoke of cigarette gave positive responses for TA100 <strong>and</strong><br />
WP2uvrA/pKM101 with <strong>and</strong> without S9 ∎ix, but negative for TA98 . It was also found that<br />
∎utagenic potency of all these gas/vapour co .ponents were higher than those of particulate<br />
components of these combustion exhausts, respectively .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
286<br />
: yylencke3ERD>NIST~ ~f E~ V BTE~e~E ~l~ of<br />
CHRON C ETONAL OF KTNKelseyt~~M . B eN-t. J<br />
Radiobidopy <strong>and</strong> 2Occupationai OSUR Health Program, Harvard School of Public Heafth, Boston, MA; 3Lab. of Radbbioioqy<br />
ind <strong>Environmental</strong> Health <strong>and</strong> Dept of Epidemiology <strong>and</strong> International Heahh, UnN . of CelMornia, San Francisco. CA,<br />
NIOSH, Cincinnati, OH .<br />
Ethylene oxide (ETO) Is a potent, m0r0% ;hcftW DNA alkylating agent that Is commonly encountered in the<br />
workplace . Cytopenetfo studies of an"s <strong>and</strong> human workers exposed by kihelatkxt to ETO have found elevated<br />
levels of cytopenetic damage In their peripheral blood lymphocytes . We have studied a cohort of 38 adult male<br />
monkeys that were exposed to 0, SOpprn <strong>and</strong> 100ppm concentrations of ETO by Infbiation from 1979 to 1981 . The<br />
lymphocytes from these animals had elevated levels of sUter chromatid exchange (SCE) knmediately upon cessation<br />
of exposure which persisted 8-7 years after cessation of exposure . Ths persistence of the SCE was attributable to a<br />
subpopulation of cells whlch were prefererkiatty detected at early t:ytopenstlo harvest times. To determine q the<br />
presence of this subpopuiatkxi of eeUs with elevated SCE was assockted with karyatypio change In stem cells we<br />
have b<strong>and</strong>ed <strong>and</strong> karyotyped 20-25 bone marrow ceAs from 2 aNmab exposed to 100ppm, 2 exposed to 60ppm <strong>and</strong> 4<br />
controls. In each animal, all of the karyotypes were normal 42 X Y, suggesting that there b not an exposure-Induced<br />
common stem ceil karyotypic alteration in the csits from exposed animals .<br />
This work has been supported by CCV902885 from the CDC .<br />
HETEROCYCLIC ANINE MUTAGENS IN ROASTED COFFEE BEANS AND BREWED COFFEE<br />
Kiyomi Kikugawa, Tetsuta Kato <strong>and</strong> Shinya Takahashi<br />
Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, JAPAN<br />
287<br />
Roasted coffee beans (hot air-roasted <strong>and</strong> charcoal-roasted) contained at least six<br />
heterocyclic amine mutagens generated during roasting coffee beans . They were extracted<br />
by methanol/ammonium hydroxide from the beans, <strong>and</strong> purified by partition in<br />
chloroform/hydrochloric acid <strong>and</strong> in chloroform/alkaline water, <strong>and</strong> finally by blue<br />
cotton adsorption . They were separated into six mutagenic fractions by high pressure<br />
liquid chromatography . One of the mutagenic fractions was identified as 2-amino-3,4dimethylimidazo[4,5-f)quinoline<br />
(MeIQ) . The other five mutagenic fractions were<br />
unknown heterocyclic amine-like mutagens . The MeIQ contents in roasted coffee beans<br />
were 0 .16 ng/10 g (hot air-roasted) <strong>and</strong> 0 .32 ng/10 g (charcoal-roasted) . These<br />
heterocyclic amine mutagens were tightly adsorbed to coffee bean fiber containing<br />
hemicellulose, <strong>and</strong> the mutagena could be hardly eluted into brewed coffee by general<br />
percolation with boiling water . Substances that could destroy these mutagens were<br />
found in brewed coffee . The substances were found to be water-soluble high molecular<br />
weight polyphenolics . They could destroy the mutagens in brewed coffee in the<br />
presence of dissolved dioxygen . Thus, even if a small amount of the mutagens were<br />
eluted from roasted beans into brewed coffee, the mutagens could be destroyed by<br />
these intrinsic substances in brewed coffee .<br />
288<br />
MUTAGENIC ACTIVITY OF IiAILLARD REACTION PRODUCTS FROM CARBOHYDBATES AND PROTEINS .<br />
N .Kinae, li.Yamashita, S .Kamiya, <strong>and</strong> S .Esaki. School of Food Nutritional Sciences,<br />
University of Shizuoka, 395 Yada, Shizuoka 422(Japan)<br />
It has been demonstrated that several reaction mixtures of carbohydrates <strong>and</strong> amino<br />
compounds such as ammonia, amines, amino acids, show mutagenic <strong>and</strong>/or antiaatagenic<br />
activity toward certain bacteria . Howver, there is few reports concerning the mutagenicity<br />
of the reaction mixtures of carbohydrates <strong>and</strong> paptides, or proteins .<br />
We tried to determine the mutagenic activity of the reaction products from carbohydrates<br />
<strong>and</strong> proteins . A mixture of 50-fold carbohydrate(D-arabinose, D-ribose, D-xylose,<br />
D-glucose, D-fructose, lactose) <strong>and</strong> protein(bovine serum albumin :BSA, human serum<br />
albumin :HSA) was dissolved in phosphate buffered saline(PBS, pH7 .4) <strong>and</strong> incubated at<br />
37-50'C . After 1-2 months, the browning solution was dialyzed <strong>and</strong> then lyophilized<br />
against distilled water . The residue was dissolved in PBS or dimethylsulfoxide <strong>and</strong><br />
submitted to the Ames test . Hydrolysis of the lyophillsate was carried out in 6N HC1 at<br />
110'C for 12 hrs . The hydrolyzate which has fluorescence was also examined the mutagenic<br />
activity .<br />
Several reaction products(BSA-Glu, HSA-Glu, HSA Ara, HSA-Lac) <strong>and</strong> their hydrolysates<br />
showed weak mutagenic activity(80-1370 revs/10 mg) toward ji .t»himuriun TA100<br />
without metabolic activation . Some of them may be contained in daily foods <strong>and</strong> in our<br />
tissues .<br />
50869 3612
289<br />
CHARACTERISTIC PEROXIDASE-MEDIATED DNA-ADDUCTS OF BENZO(a)PYRENE AND OF DIESEL<br />
EXHAUST PARTICULATE EXTRACT .<br />
R . Kind, D . Wild, D. Henschler, Institute of Toxicology, University of WOrzburg,<br />
Wurzburg, Fed . Rep . of Germany<br />
It is our aim to elucidate the potential of peroxidases for the metabolic<br />
activation of Diesel motor exhaust components . While the monooxygenase (MO)dependent<br />
activation of PAHs <strong>and</strong> Diesel emissions <strong>and</strong> the resulting genotoxic<br />
effects have been investigated frequently, little is known about the peroxidasedependent<br />
activation . We have used benzo(a)pyrene (HaP) <strong>and</strong> Diesel particulate<br />
extract (DPF~)2<strong>and</strong> 2 techniques to assay peroxidase-mediated reactive metabolites<br />
: 1 . P-postlabeling assays for DNA binding metabolites <strong>and</strong> 2 . Salmonella/peroxidase<br />
assays for mutagenic metabolites . Horse radish peroxidase <strong>and</strong><br />
bovine lactoperoxidase were used. For the adduct studies, calf-thymua DNA was<br />
exposed to BaP or DPE, peroxidase <strong>and</strong> H202. The DNA was analyzed for adducts by<br />
postlabeling, 2-dimensional TLC on PEI-cellulose <strong>and</strong> autoradiography . For comparison,<br />
analogous experiments were performed using MOs (PCB-induced rat liver<br />
S9 <strong>and</strong> NADPH) . Peroxidase-dependent BaP adduct spots were clearly demonstrated .<br />
Similarly, DPE was activated by peroxidases <strong>and</strong> produced prominent adducts <strong>and</strong> a<br />
diffuse adduct zone . A different adduct-pattern was seen following activation by<br />
MO . On the other h<strong>and</strong>, peroxidase-mediated mutagenic effects were not found . We<br />
conclude that peroxidases produce characteristic DNA-binding metabolites which<br />
are probably short-lived <strong>and</strong> cannot reach the Salmonella DNA target . These<br />
metabolites may however be relevant for cells with endogenous peroxidase activity.<br />
* Supported by BMFT <strong>and</strong> FAT *<br />
290<br />
DIPSffiNTLBZDANTOIM IS NEGATIVE IN A bATTER2 OF SHORT SBM ClTOGBN6IIC ASSAYS .<br />
D . Kindig, J . Beyers*, J . Erunny, J . Parton, <strong>and</strong> M . Garriott, Lilly Research<br />
Laboratories, Eli Lilly <strong>and</strong> Company, Greenfield, IN 46140<br />
5,5-Diphenylhydantoin (DPH) is an antiepileptic drug associated vith an<br />
increase in malformations in offspring of vomen vho took DPH during pregnancy . When<br />
DPH has been tested in genetic toxicology studies, the•results have been varied .<br />
Positive <strong>and</strong> negative results have been reported for in vivo <strong>and</strong> in vitro chromosome<br />
aberration (CAB) assays, in vivo <strong>and</strong> in vitro sister cTiromatid exc7isngi TSCE)<br />
assays, <strong>and</strong> in vivo micronucieus tests (W. This report presents results obtained<br />
from a battery oF Lests performed in a single cytogenetics laboratory . DPA vas<br />
tested in the in vitro CAB assay using Chinese hamster ovary cells at doses of 200,<br />
250, <strong>and</strong> 300 ug7mwfih <strong>and</strong> vithout an S-9 activation system . The percentage of<br />
aberrations ranged from 2 to 4 <strong>and</strong> from 0 to 1 vith <strong>and</strong> vithout activation,<br />
respectively, <strong>and</strong> there vas no significant increase in aberrant cells vhen compared<br />
vith the solvent control . The SCE assay used intraperitoneal doses of 5, 10, 20,<br />
<strong>and</strong> 40 mg/kg <strong>and</strong> bone marrovi cells from CD-1 female mice vere harvested 21 hours<br />
after dosing . The incidence of SCEs ranged from 3 .7 to 4 .5 SCEs per metaphase .<br />
There vas no increase in the number of SCEs in DPB-treated animals vhen compared to<br />
solvent controls . For evaluation in the MNT, intraperitoneal doses of 10, 20, <strong>and</strong><br />
40 mg/kg vere administered to ule <strong>and</strong> female CD-1 mice . Bone marrov vas extracted<br />
from the femurs 24 hours after dosing . The frequency of micronucleated<br />
polychromatic erythrocytes ranged from 1 .4 to 2 .6 <strong>and</strong> did not reflect an inerease<br />
over the solvent controls . The results from this battery of tests indicate that the<br />
knnvn tPintnqen, DPH, is nonmutagenic .<br />
291<br />
INVESTIGATIONS ON THE EXTENT 0! TESTING REQUIRED TO EXCLUDE NON-CL)1STOGEtiB IN ROUTINE<br />
GHROMOSOHAL ABERRATION TESTS . D .J . Kirkl<strong>and</strong>, M . Ishidate Jr, D . Gatehouae <strong>and</strong><br />
C . Richardson . Microtest Research Limited, York, UK ; National Institute of Hygienic<br />
Sciences, Tokyo, Japan ; Glaxo Group Research, Ware, Herts, UK <strong>and</strong> ICI Central Toxicology<br />
Laboratory, Macclesfield, Cheshire, UK .<br />
A number of clastogens seem to require 48 hr treatments - 8-9 in CFRL cells to produce<br />
a positive response . 4 chemicals from Ishidate's Data Book (Elsevier, 1988) which<br />
were negative at the same doses after 24 hr - 8-9, were retested at the same or higher<br />
doses, but also with 6 hr treatment (- <strong>and</strong> + S-9) followed by 18 hr recovery (6-18),<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 101<br />
Notes
102 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes to assess the importance of toxicity <strong>and</strong> variations to protocol . 3 of the chemicals<br />
were also tested in similar protocol variations (except 3-21 inatead of 6-18) in human<br />
lymphocytes (HL) . The fourth, maleic anhydride, became positive in a 6-18 protocol<br />
+ S-9 . Sodium dehydroacetate became positive at higher dosea in CHL cells after 24 hours<br />
-S-9 . In HL it was only positive after 24 hours -3-9, when doses giving > 50% toxicity<br />
could be found . Cocaine hydrochloride became positive in all 4 phases in CML cells .<br />
In HL it required doses > 10 mM to get similar positive results . 6-mercaptopurine<br />
became positive - <strong>and</strong> . S-9 in 6-18 protocols in CHL cells on a second retest when some<br />
doses giving > 50% toxicity were used . In ML, it was such swre positive over 48 hours<br />
-S-9 than 24 hours but was negative at much higher doses in 3-21 protocols . The results<br />
show that (i) "difficult" clastogens can be detected in HL as well as CHL cells ; (ii)<br />
prolonged treatments are not always necessary if ad .quate toxicity (>50%) is achieved ;<br />
(iii) higher doses are generally needed in pulse treatment/recovery protocols, <strong>and</strong> may<br />
not achieve adequate toxicity even at doses > 10mM .<br />
292<br />
X-RAY MUTAGENESIS OF A GPT TARGET IN CHINESE HAMSTER V79 CELLS YIELDS SMALL COLONY<br />
MUTANTS . C .S . Klein <strong>and</strong> T .G . Rossman (Presented by E .T . Snow) . Institute of Evironmental<br />
Medicine, NYU Medical Center, P .O .Box 817, Tuxedo, NY 10987 (USA) .<br />
The E . coli ct gene encoding xanthine-guanine phosphoribosyl transferase has<br />
been stably transfected into HPRT' Chinese hamster V79 cells . One cell line (g12)<br />
maintains a low spontaneous mutation frequency (2 x 10-5), <strong>and</strong> is useful for comparative<br />
mutagenesis studies (ykt vs . hprt) . Alkylating agents such as N-methyl-N'nitro-N-nitrosoguanidine<br />
(MNNG) <strong>and</strong> P-propiolactone (BPL) are equally mutagenic to<br />
the ypt <strong>and</strong> hprt loci at doses (MNNG, 0-2 )ig/ml ; BPL, 0-2 mM) which yield greater<br />
than 50% survival of the cells . UV (0-8 J/M2) is 3-4 times more mutagenic at 2pt<br />
than hprt in V79 cells at relatively non-toxic doses . The y~t locus of g12 transfectants<br />
also appears to be more (3-4x) sensitive than the endogenous hprt to x-ray<br />
(0-750 rads) induced mutations . This data is in agreement with previously reported<br />
x-ray mutagenesis in gpt+ CHO (Stankowski <strong>and</strong> Hsie, 1986, Radiat . Res . 105 :37) . An<br />
abundance of small qpt mutant colonies were found in g12 cells at high x-ray doses<br />
which are not seen in gpt mutants of CHO . Small colony mutants in mouse lymphoma<br />
cells are believed to arise from cells which have sustained chromosomal <strong>and</strong> deletion<br />
mutations at the tk locus . Deletions, especially multilocus deletions, are difficult<br />
to study in most mammalian assays since they are generally lethal events . The data<br />
suggest that 912 cells may be useful for studying clastogen-induced mutagenesis .<br />
Further studies are underway to define the mechanisms of small colony formation in<br />
these cells .<br />
293<br />
S9UDIES ON '1HE II-ID(1f.TICN OF CY10Qt4ZE P450 LSO@Pl)R['S BY 2-AMIPD-1 (4 .5bJP1QtIDI)4E<br />
(phIp) Alm 2-MQP43 .8-0D6DOQ.IIOAllip(4,5-f)%RNID(ALIM (MeIQc) IN VARIOUS OHfM RY!(<br />
MME RAIS .<br />
M. Kleman, E. (iservik . P. LinderJ:os ard J .-A. Qstafasoo. DepartmaK of Medical Nutriticn, Raxolinmka<br />
Institute, Stockhulm, SYweden .<br />
7he mrtasenic ard caninaserdc hetemcyclic amires fonaed in proteinrich food upan eookin6 are<br />
all deperdent an metabolic activation for the formation of their ultismtely active fonre . It was<br />
shown by previom imestisators, that rat cytoduvne P450 forms C . d ard the canstitutive P450 msle<br />
in vitiv performs this activation. fie aubJect of the present imestibatien 4ms to stt* if the<br />
food proMutasens phlP <strong>and</strong> MeIQc have the ability to irduce cytochrome P450 isoe :aymes <strong>and</strong> thus to<br />
ird:re their an activation . 'Rmee dnses of 50 uR MeIQc or phlP per kb b .w . were siven to sale<br />
Wistar rats on three consecutive days . Betairphtoflawne (BNF) aas given aa a positive eontrol at<br />
40 ms per lg b.w . <strong>and</strong> 0 .9% NaCI as negative control . On the fonrth day all ani :rels were sacrificed<br />
by decapitation ard lurg, forestomach, stomach, smsll intestine, liver ard kidney aere talmt .<br />
MicYosrnrs vrre prepared fiam all orsems ard inmdiately froaen at -70'C . 9000 & sapernatant (S-9)<br />
was also prepared from the livers . 1fie microsants were used In liestern inmrnblots a6aiist antibodies<br />
raised in rabbit to cytochYam P450,,,o srd cytorlaarc i450PR. A positive response uss<br />
obtained with liver microsares frao the MeIt* treated ardxsls a6ainst the anti<br />
Measurn~ents of EUrncy~on :fin4deetlrylase activity cordinped the irductiVe effect o Me•<br />
cytoch- KS%s in liver <strong>and</strong> sh4wed a sigdflcantly increased activity of these cytoc]:roee PW<br />
forms also in the iddneys of the MeIQc irduced rats . 7he liver S-9 aanples were used in hms test<br />
an strain TA98 a6ainst MelW <strong>and</strong> Aflatodn BI . Slaprisirgly. the phlp induced livers ah4wed a<br />
significantly increased capecity to activate MeIQc, ahile the MeIQc liver S 9 :s were fnt different<br />
fiom the controls .<br />
50869 3614
294 1989 EMS Abstracts<br />
THE PERSISTENCE OF DICENTRIC CHROMOSOMES IN MURINE SPLENIC AND PERIPHERAL BLOOD Notes<br />
LYMPHOCYTES (PBLs) FOLLOWING IN VIVO ry-IRRADIATION . A .D . K1lgermanl E .C . Halperin2<br />
G .L . Erexson3, <strong>and</strong> G . Honor42 . lU .S . <strong>Environmental</strong> Protection Agency, Research<br />
Triangle Park, NC ; 2Duke University Medical Center, Durham, NC ; 3<strong>Environmental</strong> Health<br />
Research <strong>and</strong> Testing, Inc ., Research Triangle Park, NC .<br />
In developing extrapolation models for human genetic toxicology studies, it is<br />
important to determine the persistence of the damage in the target cells in both the<br />
human <strong>and</strong> the animal model . The persistence of lesions is primarily determined by<br />
three processes : DNA repair, the rate of cell cycling, <strong>and</strong> cell lifespan . In this<br />
study, we determined the persistence of dicentric chromosomes with paired fragments in<br />
splenic lymphocytes <strong>and</strong> PBLs following y-radiation as a measure of the lifespan of<br />
these target cells . Thirty-six male C57B1/6 mice were whole-body ry-irradiated (WBI)<br />
with 3 Gy using a linear accelerator . Blood <strong>and</strong> spleens were removed from each of 4<br />
mice within 30 min, <strong>and</strong> then on days 1,3,7,14,28,56, <strong>and</strong> 112 after WBI . Both B- <strong>and</strong> Tlymphocytes<br />
from the spleen <strong>and</strong> T-lymphocytes from the peripheral blood were cultured<br />
for one cell division for analysis of dicentric prevalence . Decay curves for Tlymphocytes<br />
from the spleen <strong>and</strong> peripheral blood were similar describing the average<br />
lifespan of a T-cell to be 25 <strong>and</strong> 18 days, respectively . The shape of the B-cell decay<br />
curve was very different from those of the T-cells, showing little change from day 0<br />
through 7(40X of the cells contain at least one dicentric with a fragment) . At day<br />
14, 90% of the cells with dicentrics have disappeared . However, for both B- <strong>and</strong> Tlymphocytes,<br />
there exists a small population (approximately 2%) of long-lived cells 112<br />
days after WBI that contain dicentrics with fragments . [This abstract does not ratl .ot 6vA policyl<br />
295<br />
COMPARING THE STRUCTURAL DETERMINANTS OF MUTAGENICITY IN THE GENE TOX AND NATIONAL<br />
TOXICOLOGY PROGRAM DATA BASES . Gilles Klopman <strong>and</strong> Herbert S . Rosenkranz, Departments<br />
of Chemistry <strong>and</strong> <strong>Environmental</strong> Health Sciences, Case Western Reserve University,<br />
Clevel<strong>and</strong>, Ohio 44106. `<br />
The Gene-Tox (GT) <strong>and</strong> National Toxicology Program (NTP) Salmonella mutagenieity<br />
data bases have been widely analyzed with respect to their performances as predictors<br />
of carcinogenicity . The two data bases differ greatly : in GT, mutagens predominate<br />
(80%) prevalence), <strong>and</strong> 88% of the subgroup also tested for carcinogenicity, was<br />
carcinogenic . In NTP, on the other h<strong>and</strong>, only 54% of the ehemicals are mutagens . The<br />
prevalence of carcinogens is 67% but 40% of them are not mutagenic . The two data sets<br />
were each analyzed by CASE, the Computer Automated Structure Evaluation method . It<br />
was found that there was a highly significant overlap among the structural<br />
determinants responsible for the mutagenicity in each data set, even though there was<br />
insignificant overlap of the chemicals in GT <strong>and</strong> NTP .<br />
These findings indicate that each data set can be used for mechanistic studies<br />
<strong>and</strong> that in future studies they can be merged . The results, using two independently<br />
obtained data bases, prove that there is a unique structural basis for mutagenicity .<br />
296<br />
DE"rERmnoTION OF DAlfAGFNIC EFFFICiS IN LIVER .S, SPIFMr INfESmaI. CZxA'FSRS, BzACD SERA<br />
AIID URIHE OF NIItIDA7AIE TRF7ITID fYQCE WITH E . OOf,I K-12 STRAINS<br />
Siegfried Knascdiller<br />
Institut of Experimental Carx :er Research, University of Innslaruck<br />
Fritz-Pregl-StraBe 3/VII, 6020 Innsbruck, AUSTRIA<br />
Intrasanguirteous Host Mediated Assays were performed to ereasure the induction of valine<br />
<strong>and</strong> nalidixic acid resistant imutants in E .eoli K-12 oslls reeovered fran livers <strong>and</strong><br />
spleens of mice treated orally with the antischistoeanal drug Niridazole (dose range<br />
500 - 125 mg/bo8y weight. Following an exposese tine of 180 minr a substantial dose<br />
dependent mutagenic respalse was fotimd in both or+gans with tYte nitr+oz'eductase pa'oficient<br />
parental strain E .eoli K-12 343/113 whereas with a niridazole nitrodreductase<br />
deficient derivative 343/113 NIR 200 (which resists the toocic action of 200 pg Niridazole<br />
per ml agar median), no c]ear sutagenic effects were detectable under identical<br />
experimental ootditions . In additienr urine samples, extracts of intestinal eontents<br />
<strong>and</strong> blood sera of niridazole treated animals were tested in liquid suspension assays•<br />
with the reductase proficient strain pronounced activities were fatald in all camsr<br />
whereas with the reductase deficient strain, positive results were restricted to experiments<br />
with urine smnples . These latter findings stx3qest that nutaqenio nletabolites<br />
formed by nkTmlalian enzymes <strong>and</strong>/oz nitsnreductiea by intestinal mieroorqattisire at ooncentratiens<br />
wich are to low to enable their detectien under the present in vivo oonditicns<br />
are concentrated by ultrafiltration <strong>and</strong> excreted via the renal system .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
103
104 1989 EMS Abstracts<br />
Notes CARCINOGENESIS OF FOODS . lb Knudsen, DVM, Institute of Toxicology,<br />
National Food Agency, Msrkhmj Bygade 19 . DK 2860 Smborg (Cph .),<br />
Denmark .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Food is a major determinant of human health <strong>and</strong> disease, not only from<br />
a nutritional point of view, but also from a toxicological point of view .<br />
Thus food toxicology is not only a scientific discipline to deal with the<br />
toxicology of food additives but also a ma jor source of knowledge about<br />
impact of major <strong>and</strong> minor food constituents on human health . The<br />
Institute of Toxicology has established a testing programme to deal with<br />
these matters covering natural fibers in the protection against colon<br />
cancer, possible interaction between urethane <strong>and</strong> alcohol in carcinogenesis,<br />
food mutagens as a human health factor . The paper will discuss<br />
our recent progress within these areas of research .<br />
INDUCTION OF CHROMOSOME CHANGES BY METAL COMPOUNDS<br />
IN CULTURED CHO CELLS .<br />
TS. Kochhar, B . Leonard, K . Harris, W . Moody<br />
Kentucky State University, Frankfort, KY 40601<br />
Several metal compounds have been identified as potential carcinogens through<br />
occupational exposure as well as in the laboratory . Since chromosome changes such as<br />
abberations <strong>and</strong> sister-chromatid exchange have been linked to DNA damage, the<br />
influence of certain metal salts on these parameters was assessed in cultured CHO cells .<br />
The cells were treated with CdCI2, Cr0„ HgCI= <strong>and</strong> N1CIz for 24 h . The concentrations<br />
used for aberration assay were 10'7 -10" M <strong>and</strong> for SCE assay it was 10-s-10's M . It<br />
was observed that all compounds enhanced . various types of chromosomal abnormalities<br />
compared to the controls . CdCl2 seemed to have the maximum effect as the cultures<br />
treated with 10-7 <strong>and</strong> 10-s M of this compound revealed a considerable number of<br />
chromosomes with two or more centromeres . Other increase in abnormalities were the<br />
ring chromosomes, chromatid breaks, chromatid exchanges <strong>and</strong> the stickiness of the<br />
chromosomes . CdCI„ Cr03 <strong>and</strong> NIC1 also enhanced SCE frequencies . In higher<br />
concontrations of these compounds (10'~ <strong>and</strong> 10-s M) SCE/cell Incrpsed more than twofold<br />
compared to the BrdU controls . HsCI= was relatively ineffective in raising the SCE<br />
rates . These studies indicate a definite link between the metals <strong>and</strong> the mammalian<br />
chromosomep-which calls for further detailed studiq . (5upported by NIH-MgRS RR<br />
08124 grant .)<br />
297<br />
298<br />
299<br />
DNA SEQUENCE ANALYSIS OF MUTATIONS INDUCED IN YEAST BY EXCESS THYMIDYLATE : EVIDENCE<br />
FOR NEXT•NUCLEOTIDE EFFECTS . S .E . Kohalmi <strong>and</strong> B .A . Kunz, Microbiology Department, The<br />
University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2<br />
In thymidylate (dTNP) auxotrophs of the yeast Saccharomvcas cerevisiaa, intracellular<br />
dTTP levels can be modulated by varying the concentration of dTMP in the growth<br />
medium . Provision of excess dTMP is mutagenic in yeast . Recently, yeast DNA polymerase<br />
III was found to have a 3' -> 5' exonuclease activity capable of proofreading in<br />
vitro . Thus, mutations induced by excess dTMP could be due to misincorporation of the<br />
nucleotide <strong>and</strong> the misincorporation frequency might be enhanced by next-nucleotide<br />
effects reducing the efficacy of the 3' -> 5' proofreading activity . On this basis,<br />
<strong>and</strong> since dTTP is a positive effector of GDP reduction In yeast, excess dTMP would be<br />
expected to cause increases in substitution by T <strong>and</strong> in the proportion of events at<br />
sites flanked by a 3' T or C . To test these possibilities, we are using DNA sequence<br />
analysis of mutations induced by excess dTMP in the SUP4-o gene of yeast . To date .<br />
100 induced mutations have been characterized <strong>and</strong> the spectrum <strong>and</strong> distribution of<br />
changes have been compared to those for 306 spontaneous mutations . We have determined<br />
that : 1 . 86% of the induced substitutions involve replacement by T whereas only 64%<br />
of the spontaneous changes do ; 2 . approximately 90% of the induced mutations occur<br />
at sites with a 3' flanking T or G vs . 73% for the spontaneous mutations . The results<br />
argue that dTMP misincorporation is responsible for the induced mutations <strong>and</strong> suggest<br />
that a proofreading function is part of the yeast DNA replication complex In vivo .<br />
Currently, we are analyzing additional mutants . (Supported by NSERC Canada)<br />
300<br />
THE USE OF LAbIDDA PHAGE SHUTTLE VECTORS IN TRANSGENIC MICE FOR<br />
DEVELOPMENT OF A SHORT TERM MUTAGENICITY ASSAY<br />
Kohler, S .W ., Provost, G .S ., Kretz, P .L ., Sorge, J ., Huse, W .D . <strong>and</strong><br />
Short . J .M . Stratagene, 11099 North Torrey Pines Road, La Jolla, CA<br />
92037 .<br />
A short term mutagenesis assay has been developed which will permit<br />
analysis of suspected genotoxic substances in whole animals .<br />
Transgenic mice containing a stably integrated bacteriophage lambda<br />
shuttle vector can be rescued at high efficiency (>1000 pfu/µg of<br />
genomic DNA/integrated copy) from isolated genomic DNA with In vitro<br />
lambda packaging extracts . This rescue efficiency allows the recovery<br />
of over one million lambda phage genomes per tissue, a level<br />
50869 3616
1989 EMS Abstract s<br />
sufficient for measurement of the spontaneous mutation rates in whole Notes<br />
animals . Mutations are detected by monitoring the activity of a<br />
P galactosidase target gene contained within the lambda genome . The<br />
high rescue efficiency is accomplished through the use of methylated<br />
cytosine restriction deficient (mcr ) lambda packaging extracts <strong>and</strong><br />
plating strains. This mutagenesis assay will permit comparison of<br />
mutation rates between different tissues in whole animals . In<br />
addition, the shuttle vector is being modified to include the in vivo<br />
excision features of the lambda ZAP cloning vector. This will<br />
facilitate the DNA sequencing for identification of the sequence<br />
specificity of genotoxic substances .<br />
30 1<br />
SCHISTOSOMIASIS DRUGS : AN ICPEMC STUDY<br />
P .G .N. Kramers (1), J .M. Gentile (2), B.J .A .M. Cryseels (3), P . Jordan (4), N. Katz<br />
(5), K .E . Mott (6), J .J . Mulvihill (7), J .L. Seed (8), <strong>and</strong> H. Frohberg (9) ; (1)<br />
National Institute of Public Health <strong>and</strong> <strong>Environmental</strong> Protection, P .O. Box 1, 3720 BA<br />
Bilthoven, (2) Holl<strong>and</strong>, MI, (3) Leiden, The Netherl<strong>and</strong>s, (4) Ware, UK, (5) Belo<br />
Horizonte, Brazil, (6) Geneva, Switzerl<strong>and</strong>, (7) Bethesda, Md, (8), Frederick, Md, (9)<br />
Darmstadt, PRG .<br />
One of the interests of ICPEMC is to identify situations in which the possible<br />
induction of inherited defects in man by mutagen exposure could actually be studied .<br />
The large-scale use of mutagenic drugs in field programmes against Schistosomiasis,<br />
mainly during the seventies, was considered a possible case . An ICPEMC task group<br />
approached the problem by (1) updating the genetic toxicology data base for the<br />
various antischistosomal drugs known, <strong>and</strong> (2) searching for possible study areas .<br />
Expertise was combined from genetic toxicology, mutation epidemiology <strong>and</strong> tropical<br />
medicine . It was considered that : (a) hycanthone is the best c<strong>and</strong>idate drug, if any ;<br />
(b) local demography would render the reliable tracking of substantial numbers of<br />
offspring of treated persons an almost impossible task (c) in most endemic countries<br />
proper diagnosis <strong>and</strong> registration of inherited defects is largely lacking ; (d) as<br />
estimated from animal experiments the sample sizes needed to show an effect, if any,<br />
would be very large; <strong>and</strong> (a) since non-mutagenic antischistosomal drugs are now in<br />
use, the problem is of low priority in endemic countries that are usually short in<br />
medical resources . Thus, studying offspring of hycanthone-trsated people to<br />
demonstrate the mutagenic potential of the drug in manais not a viable enterprise .<br />
302<br />
THE ROLE OF PROTEIN KINASE C IN SIGNAL TRANSDUCTION AND CELLULAR TRANSFORMATION . R.S .<br />
Krauss, G .M . Housey . W .W.-L. Rsiao, M .D. Johnson, S .A. Rotenberg <strong>and</strong> I .B. Weinstein .<br />
Comprehensive Cancer Center, Columbia University, New York, N .Y. 10032 .<br />
We have utilized a retroviral expression vector to construct a series of rodent embryo<br />
fibroblast cell lines that stably overexpress a full length cDNA encoding rat protein<br />
kinase Cgj (PKC) . Rat 6(R6) cells that contain 20-53 fold higher PKC enzyme activity<br />
than control cells show several disturbances in growth control, including the<br />
ability to form small colonies in soft agar <strong>and</strong> tumors in nude mice . R6-PKC-3, a line<br />
with a 53-fold increase in PKC activity, in hypersensitive to transformation by transfection<br />
with an activated ras oncogene, as measured by the formation of large colonies<br />
in soft agar . Recently, we have developed lines of C31i/10T1j cells that stably overproduce<br />
PKC . 10Tk-PKC-4, a line with an 11-fold increase in PKC activity relative to control<br />
cells (but with a specific PKC activity similiar to the R6-PKC lines), is morphologically<br />
altered, grows to 4-fold higher saturation density <strong>and</strong> has reduced adhesiveness<br />
. When grown in 2 .5% calf serum <strong>and</strong> 100 ng/ml TPA, these cells form numerous large,<br />
dense foci . However, unlike the R6-PKC cell lines, neither 10TIS-PKC-4 calle nor calls<br />
from the TPA-induced foci are capable of growth in soft agar, <strong>and</strong> they are non-tumorigenic<br />
in nude mice . These R6 <strong>and</strong> 10711 cell lines should be useful for investigating<br />
molecular events in tumor promotion <strong>and</strong> multistage carcinogenesis . We have also utilized<br />
these cell lines as a source for obtaining enzyme preparations that are highly enriched<br />
for PKCS1, thus facilitating biochemical analysis of this PKC isoform . The implications<br />
of these studies with respect to the role of PKC in carcinogenesis will be<br />
discussed . Supported by NIH grants CA02656 <strong>and</strong> CA08346 .<br />
303<br />
REASSESSMENT OF SELECT NTP COMPOUNDS IN THE L5178Y/tk*/- MOUSE LYMPHOMA<br />
ASSAY. R. Krehla <strong>and</strong> D . Clivea, aWellcome Research Laboratories, Research Triangle Park, NC<br />
27709 USA<br />
Strong conclusions have been drawn about the performance of 4 short term tests based on summary<br />
(+, -) evaluations of the NTP-sponsored studies on 73 rodent carcinogens <strong>and</strong> noncarcinogens (Tennant<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
105
106 1989 EMS Abstracts<br />
Notes et a!. , Science, 236 : 933, 1987) . Thus, despite an earlier critique of these studies (Ray et al .,<br />
Environ . Molec . Mutagen., 511 : 85, 1988), Ashby (<strong>Mutagenesis</strong>, 3 : 483, 1988) has suggested the<br />
existence of 19 nonclastogenic mutagens based on these evaluations . To date (February, 1989), the<br />
L5178Y/tk +/- mouse lymphoma assay (MLA) data on most of these 73 compounds have not been<br />
published . For these reasons an effort was made to retest In a single laboratory two subsets of those<br />
chemicals : those which had yielded marginal positive or negative results, <strong>and</strong> those proposed as possible<br />
nonclastogenic mutagens . Six of the former (allyl Isovalerate, allyl Isothiocyanate, benzene, ethyl<br />
acrylate, eugenol <strong>and</strong> geranyl acetate) <strong>and</strong> 7 of the latter (benzoln, benzyl acetate, chlorobenzene,<br />
cinnamyl anthranilate, 1,2-dichlorobenzene, geranyl acetate (also on the marginal response list) <strong>and</strong><br />
sulfisoxazole) have been completed to date . Our results differ In 4 ways from those of the NTP : (1) we<br />
saw little of the Inter- <strong>and</strong> intra-experimental variability seen In the original NTP studies ; (2) our<br />
active dose ranges differed markedly for several compounds from those obtained in one of the contract<br />
labs ; (3) we generally obtained significantly stronger mutagenic responses than did that same contract<br />
lab, consistent with their weak MMS positive control responses : <strong>and</strong> (4) NTP's positive result for one<br />
chemical (benzyl acetate) In the absence of S9 was not seen In these studies . The significance of these<br />
results for test protocols, data evaluation, quality control, <strong>and</strong> assay correlations will be discussed .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
304<br />
KINETICS OF DIMETNYLNITROSAMINE(Dl4d)-INDUCED MICRONUCLEUS (IQ1) FORMATION IN MOUSE BONE<br />
MARROW AND SPLEEN CELLS . G . Krishna, M .L . Kropko, <strong>and</strong> J .C . Theiss, Parke-Davis Pharm .<br />
Res . Div ., Warner-Lambert Co ., Ann Arbor, MI (USA)<br />
The MN aasay has been extensively used in routine mutagen/carcinogen screening<br />
programs to detect agents which cause chromosomal breakage <strong>and</strong> spindle dysfunction .<br />
DMN is a known mammalian carcinogen <strong>and</strong> has been tested in both mouse <strong>and</strong> rat bone<br />
marrow for its ability to induce MN . For D!'Qd, both positive <strong>and</strong> negative MN assay<br />
results have been reported which may be due to differences in bone marrow sampling<br />
times . The present study was disigned to obtain information on the kinetics of MN<br />
formation following DMN treatment In mice . Groups of 3 to 5 CD1 male mice were<br />
injected once i .p . with 50 or 100 mg/kg DMN . Both bone ∎arrow <strong>and</strong> spleen cells were<br />
isolated at various sacrifice time-points (6, 12, 24, 36 . 48, <strong>and</strong> 60 h post-treatment)<br />
<strong>and</strong> processed for MN analysis according to established procedures . Cyclophosphamide<br />
(CP, 40 mg/kg) was used as a positive control <strong>and</strong> saline served as the vehicle<br />
control . In the study, the vehicle control group had 0 .6 <strong>and</strong> 0 .9 !Q1/1000<br />
polychromatic erythrocytes (PCEs) in bone .arrow <strong>and</strong> spleen cells, respectively . DMN<br />
at 50 mg/kg, caused 3 .8, 7 .8, 8 .5 <strong>and</strong> 10 .2 !Qi/1000 PCEs in bone marrow <strong>and</strong> 8 .0, 9 .2,<br />
19 .3 <strong>and</strong> 32 .8 AQJ/1000 PCEs in spleen at 12, 24, 36 <strong>and</strong> 48 h sacrifice times,<br />
respectively . A similar time-related elevation of MN frequency was noted for 100<br />
mg/kg DtQI . At each sacrifice time-point, spleen cells had a higher MN frequency<br />
than bone marrow cells . In general . DMN caused a decrease in the proportion of PCEs<br />
to normochromatic erythrocytes, suggesting toxicity . Thus, this study demonstrates<br />
the clastogenic activity of DMN in both bone marrow <strong>and</strong> spleen cells of mice <strong>and</strong> shows<br />
a time-related pattern in the elevation of DMN-induced MN frequency .<br />
MI1rAGFr18, CARCItOMNS AND ANfI'1SkDUR AMTI5 IN '14tE INDIAN DIET<br />
ARtM KRISf3171K[k1AR <strong>and</strong> V .M .SIVARN+Al62I<br />
(Isotope Division, Cancer Institute,t ryr, Madras 600 020, INDIA)<br />
The role of spices <strong>and</strong> other plant products wide consumed in India either<br />
in the diet or in indigenous medicine, in relation to the alimentary cancers (oral,<br />
gastric <strong>and</strong> oesophagal cancers) prevalent here, has been investigated with laboratory<br />
animals . Most of the spices are found to be very well tolerated, even when<br />
fed in large doses, with little change in physical appearance, growth rate or behaviour<br />
.Cinnamon actually increases the growth rate . Out of the 25 plant products<br />
screened,nine induced the carcinogen-detoxifying enzyme, glutathione-S-transferase,<br />
in the st.omach, liver <strong>and</strong> oesophagus sufficiently high to be considered protective<br />
agents . Ttrey are cunin (cLminum num) <strong>and</strong> poppy (Papaver aonniferrum) seeds ;<br />
drumstick (Moringa oleifera), mana li (§21anun ni rwn) <strong>and</strong> (Ocinum basilicum)<br />
leaves ; neem (Azadirachta indica) flowers ; asafoetida (Ferula asafoetida),<br />
k<strong>and</strong>anthippili (pi lon ) <strong>and</strong> turmeric ((lrrctnna 1a a)9 Eight o t~.e . ,<br />
excepting neem flowers, suppress the geno c e ects of 3,4-benzo(a)pyrene, when<br />
simultaneously fed, cumin <strong>and</strong> poppy seeds being most effective . Feeding emblica<br />
(phyllanthus emblica) decreases glutathione-S-transferase possibly a tumour-promoter<br />
. Ames test reveals fried mustard, per<strong>and</strong>ai (Cissus anr laris) <strong>and</strong> oil<br />
of calamus (Acorus calartus) to be mutagenic ; tamarind (Tamar s indica) non-mutagenic<br />
; while poppy s are antimutagenic .<br />
~<br />
m<br />
OD<br />
01<br />
W<br />
01<br />
F+<br />
00<br />
305
306 1989 EMS Abstracts<br />
TFLE USE OF EGDCTORATE FOR M7NITDRING oCCUPATIQQ L P70?OBURE<br />
Notes<br />
A . Krmkje, Department of Botany, The University of Trondheim, Norway<br />
The aim was to investigate if mutagens are biologically available . The workplace<br />
investigated is the top of the batteries in a coke plant, where relatively high<br />
concentrations of polycyclic arrnwtic hydrocarbons (PAHs) are usually measured in the<br />
air. The study is aimed at group exposure . The 4 groups studied are control nonsmkers,<br />
control smokers, battery naumnkers <strong>and</strong> battery smoke.rs . The workers gave their samples<br />
within half an hour of finishing the workshift <strong>and</strong> after the night's sleep - "morning<br />
expectorate" . The samples from each group were pooled, hydrolysed with alkaline<br />
methanol <strong>and</strong> extracted with cyclohexane . The S . typhitturium strains TA98 <strong>and</strong> TA100<br />
rx:re used in the Salmonella/micro®ane-test . Liver <strong>and</strong> lung hanogenates were used as<br />
metabolic activation systems . The expectorate fran exposed workers - mostly fran<br />
smokers, but also to a certain extent fran nonsmkexs - were mutagenic ; however, the<br />
control samples fran both smokers <strong>and</strong> nonamokers were not . The positive results<br />
produced by expectorate samples from the exposed smokers suggest a synergistic<br />
relationship between exposure to air pollution of the working attrosphere <strong>and</strong> smoking .<br />
The analysis of the "morning expectorate" shnws that inhaled pollution with potential<br />
nrtagens is not effectively removed from the respiratory system during the night . The<br />
salmonella-test used on expectorate samples may be an effective assay in certain types<br />
of industries, viz . where the atmsphere contains partieulates acting as carriers for<br />
mutagens apt to be eluated by the body fluids or cellular membranes .<br />
307<br />
BACTERIAL MUTAGENICITY ASSESSMENT OF STRUCTURALLY-RELATED QUINOLINE<br />
THIAZOLAMINE COMPOUNDS, POTENTIAL ANTIPSYCHOTIC DRUGS . M .L . Kropko, J .C .<br />
Jaen, S .A . Wold, B .W . Caprathe, L.D . Wise, <strong>and</strong> J .C . Theiss, Parke-Davis Pharm .<br />
Res . Div ., Warner-Lambert Co ., Ann Arbor, MI(USA)<br />
Quinoline thiazolamines have potential for antipsychotic activity . In a<br />
routine Ames assay (TA98, TA100, TA1S35, TA1537, TA153B, S9t), PD 123403 was<br />
found to be mutagenic to TA98 (peak response of 14-fold background at 32<br />
ug/plate) <strong>and</strong> TA1538 (peak response of 19-fold background at 100 ug/plate)<br />
in the presence of S9 only . This frameshift mutagen is a quinoline<br />
thiazolamine comprised of fused pyridine, cyclohexane <strong>and</strong> aminothiazole rings .<br />
To provide structure-activity information for development of a non-mutagenic<br />
antipsychotic drug in this chemical class, structural analogs were synthesized<br />
<strong>and</strong> screened for mutagenicity using TA1538 with S9 . Methylation of the<br />
cyclohexane ring gave a weak mutagen (peak response of t .4-fold background at<br />
316 ug/plate) . Mutagenic activity was completely lost (maximum dose tested of<br />
10,000 ug/plate) either upon removal of the amine from the aminothiazole ring,<br />
upon ethyl substitution of the nitrogen in the partially reduced pyridine<br />
ring, upon saturation of the pyridine ring with hydrogen, or upon<br />
removal of the cyclohexane ring . These results suggest that oxidation of<br />
PD123403 by S9 may take place at the cyclohexane ring, producing a mutagenic<br />
fully aromatic amine compound . Thus, modifications to this quinoline<br />
thiazolamine which prevent oxidation to a tricyclic heteroaromatic amine,<br />
while at the same time not interfering with pharmacological activity, should<br />
result in a non-mutagenic antipsychotic .<br />
308<br />
TOXICITY OF ENDRIN ( A CHLORINATED HYDROCARBON ) ON THYROID HORMONE<br />
FORMATION OF A TELEOST . DHYANENDRA KUMAR,Department of Zoology,Jagjiwan<br />
College,Magadh University, ARRAH,Bihar,INDIA .<br />
Hypofunction of thyroid due to environmental pollutants may be mediated<br />
through the inhibition of thyroid peroxidase enzyme as this enzyme from<br />
pronephric (head) kidney <strong>and</strong> pharyngeal sources is readily inactivated<br />
by pesticides . <strong>Environmental</strong> pollutants such as organochlorine <strong>and</strong><br />
carbondisulphide considerably depress thyroid function in fish .<br />
5'lhen Anbas testudineus,Bloch (a teleest) was exposed to 48 hr TLM<br />
concentration (0 .05 ppm ) of endrin, formation of MIT (Monoiodotyrosine)<br />
<strong>and</strong> ,IT (Diiodotyrosine) by head kidney soluble supernatatnt fraction<br />
were markedly depressed . Foemation of T(Thyroxine) was found to be<br />
similarly inhibited due,to endrin pollution . Per cent of inhibition<br />
due to exposure of 48 hr TLM to endrin was found to De 53 .6,56 .06 <strong>and</strong><br />
50 .4'; for the formation of MIT,DIT <strong>and</strong> T4 respectively . Inhitory<br />
effect of endrin could effectively reversed by Cytochrome c .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
107
108 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
THE AMPHIBIAN (BAI4A pjpgNS ) LENS AS A BIOLOGICAL MONITOR OF WATERBORNE<br />
XENOBIOTICS . J.S. Kung, C,$,Geard <strong>and</strong> B.V. Worgul, Eye Radiation <strong>and</strong> <strong>Environmental</strong> Research<br />
Laboratory <strong>and</strong> Radiological Research Laboratory, College of Physicians <strong>and</strong> Surgeons of Columbia<br />
University, New York, NY<br />
309<br />
$gpa p,jpj= shows promise as a biological monitor of environmental mutagens . As vertebrates, they<br />
are able to enaymatically activate promutagens to their genotoxic metabolites . Being aquatic, they inhabit<br />
waters that are polluted by domestic sewage <strong>and</strong> industrial waste containing waterbotne xenobiotics which<br />
maY chronically concentrate in their tissues . Since the products of all kns epithelial divisions are<br />
matntained within the lens for the entire lifespan of the animal, the lens may serve as a useful long-term<br />
indicator of genotoxicity. Frogs were exposed through the ambient environment to Benzo(a)pYrene (BP), a<br />
promutagen, 2 .5, 5.0, 10.0 <strong>and</strong> 20.0 µg/ml, <strong>and</strong> to Ethyl methanesulfonate (EMS), a direct-acting<br />
clastogen, 25, 50,100 <strong>and</strong> 200 µg/ml, by partial immersion for a period of one week . Following sacrifice<br />
of the animals, lens epithelial whole-mounts were prepared. The epithelia were stained by the Feulgen<br />
technique to permit visualization <strong>and</strong> quantitative analysis of nuclear DNA content . An increase in the<br />
premitouc 02 population was found with increasing concentrations of either mutagen . An inverse dose<br />
relationship with mitoses suggests that these mutagens may exert a premitotic block in the cell cycle .<br />
Micronuclei frequency, a measure of genotoxicity, was found to be elevated with both mutagens in a nondose-dependent<br />
fashion. A block in the cell cycle may well account for this since micronuclei production is<br />
a mitosis-dependent event . $= may prove to be valuable in the study of environmental mutagens since<br />
they are easily h<strong>and</strong>led, adaptable to laboratory conditions, ubiquitously distributed <strong>and</strong> amenable to short<br />
term genotoxicity assays .<br />
Supported by Grant EY 02648 from the ?dEI .<br />
310<br />
THE SUp4-o SYSTEM FOR ANALYSIS 0F MUTATIONAL SPECIFICITY IN YEAST . B .A . Kunz, J .D .<br />
Armstrong, M . Clattke, S .E . Kohalmi <strong>and</strong> J .R .A . Mis, Microbiology Department, The<br />
University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2<br />
Knowledge of the DNA sequence alterations produced by mutagens, as well as their<br />
frequency <strong>and</strong> location within a gene, can provide valuable clues about the DNA damage<br />
responsible <strong>and</strong> how it is processed into mutations . To obtain detailed mutational<br />
specificity data, we developed a shuttle vector system wherein mutations in<br />
the tRNA suppressor gene &UP4-o of the yeast Saccharomvcas cereviaiae can be easily<br />
analyzed to determine the DNA sequence alterations involved . In this system, the<br />
SUP4-o gene is carried on a plasmid that mimics the behavior of yeast chromosomes . To<br />
date, more than 2,000 SUp4-o mutants have been characterized . The spontaneous mutation<br />
frequency is typical for chromosomal genes in yeast <strong>and</strong> all 6 possible base-pair<br />
substitutions (single <strong>and</strong> double changes), deletions of various lengths, base-pair<br />
insertions, duplications, transposon insertions <strong>and</strong> more complex events arise spontaneously<br />
. Single base-pair changes have been found at 68 of the 75 exon sites, <strong>and</strong><br />
at 2 of the 14 intron positions, within the gene . The system can be used in the<br />
forward mode to analyze mutational specificity or as a reversion assay to provide<br />
additional information on site-specific mutagenesis . In addition, isogenic error-free<br />
repair deficient derivatives have been constructed to allow assessment of the role of<br />
DNA repair in mutagenesis . The $Up4-o system <strong>and</strong> its use will be described <strong>and</strong><br />
results obtained in SUP4-o will be compared with data available for mammalian shuttle<br />
vector systems . (Supported by NSERC Canada)<br />
311<br />
CHARACTERIZATION OF THE YEAST rad52 MUTATOR EFFECT BY DNA SEQUENCING . B .A . Kunz, S .E .<br />
Kohalmi, J .D . Armstrong, <strong>and</strong> M . Clattke, Microbiology Department, The University of<br />
Manitoba, Winnipeg, Manitoba, Canada R3T 2N2<br />
Defects in the $aD52 gene of the yeast Saccharomvices carevisiae confer a mutator<br />
phenotype . This effect was characterized by sequencing a collection of 238 spontaneous<br />
SUP4-o mutations arising in a gDd52 strain . The resulting mutational spectrum<br />
was compared to that derived for an isogenlc wild-type strain (222 mutations analyzed)<br />
. This comparison revealed that the mutator phenotype was associated with an<br />
increase In the frequency of base-pair substitutions . All possible types of substitution<br />
were detected but there was a decrease in the fraction of A-T -> G•C transitions<br />
<strong>and</strong> an increase In the proportion of G-C -> C-G transversions . These changes<br />
were large enough to cause a two-fold greater preference for substitutions at G•C<br />
sites In the rad52 strain despite a decrease In the fraction of G-C -> T-A transversions<br />
. Base-pair changes occurred at fever sites In the tgd5Z strain but the mutated<br />
sites included several that vere not detected In the $OQ52 background . Only two of<br />
the four sites most freqdently mutated In the tyM strain were also prominent In the<br />
wild-type strain <strong>and</strong> mutation frequencies at almnst all sites common to both strains<br />
were greater for the X{¢n derivative . Although single base-pair deletions occurred<br />
at similar frequencies in the two strains, several classes of mutation that were<br />
recovered in the wild-type background including multiple base-pair deletions, insertions<br />
of the yeast transposable element Ty, <strong>and</strong> more complex changes, were not recovered<br />
in the tgdn strain . Our results suggest that the XAM mutator effect is not<br />
due defects in repair of abasic sites or base mismatches . (Supported by NSERC Canada)<br />
S0869 3620
312<br />
ANTIMUTAGENIC EFFECTS OF GLUTATHIONE-S-TRANSFERASE INDUCTION IN E . COLI .<br />
S . Kuo <strong>and</strong> D .M . Shankel, Department of Microbiology, The University of Kansas,<br />
Lawrence, Kansas 66045 .<br />
The glutathione-S-transferases are dimeric enzymes which mediate the detoxification<br />
of potentially mutagenic electrophiles via conjugation to the tripeptide<br />
y-glutamyl cysteinyl glycine (glutathione) . Glutathione-S-transferases are also<br />
present in a variety of bacteria . Using the E . coli K12ND160 system, it was observed<br />
that pretreatment of E . coli with butylated hydroxy anisole (BHA) results in a<br />
decrease in the frequency of induced revertants caused by ethylmethanesulfonate,<br />
nitrofurazone, quinacrine <strong>and</strong> hydrogen peroxide . This reduction in induced mutants<br />
occurred concomitant with an increase in activity of glutathione-S-transferase in<br />
the organism . Thus this enzyme may play an important role in the protective arsenal<br />
of E . coli against potentially genotoxic agents . The significance of these results<br />
<strong>and</strong> related findings will be discussed as they relate to the mechanism of antimutagenesis<br />
.<br />
313<br />
BIOLOGICAL SIGNIFICANCE OF INDUCED ENDOREDUPLICATION . A . Lafi <strong>and</strong> J . M . Parry, School<br />
of Biological Sciences, University College of Swansea, Singleton Park, Swansea SA2 8PP .<br />
Several authors have demonstrated that exposure of cultured cells to a number of<br />
chemical agents results to an increase in endoreduplication . Here we present<br />
comparative data on the ability of tobacco particulate matter (TPM), derived from<br />
three cigarette types, to induce endoreduplication . A dose related increase was<br />
observed but there were no consistently significant differences related to the tar/<br />
nicotine levels per cigarette used . In cells allowed to recover from TPM treatment<br />
there was an increase in polyploidy <strong>and</strong> a decrease in endoreduplication suggesting<br />
that the increase in polyploidy observed was at least partly due to the recovery of<br />
cells from endoreduplication . Chromosome aberrations were induced in all three types<br />
of cells (diploids, endoreduplicated <strong>and</strong> polyploids) . However, the levels of all<br />
types of aberrations were higher in the endoreduplicating cells when compared to<br />
diploids <strong>and</strong> polyploids . This was a very unexpected resuit since it has previously<br />
been shown that endoreduplicating cells (when compared to diploid <strong>and</strong> ordinary<br />
tetraploid metaphases) are seen to contain a decreased frequency of SCEs (Speit . G .,<br />
Vogel, W . <strong>and</strong> Mehnert, K . 1985, Chromosoma, 89, 79-84) ; The presence of cells with<br />
different ploidy levels may have a significant effect upon the capacity of some<br />
chromosome abnormalities to be transmitted to progeny cultures .<br />
314<br />
IN VTW G[fEPIC =C1TY FFOIUCfl[S FYR TfQ3tAP1 ;iRTC HOOlW PCFMAAITQIS. Robert S . Lake, Safety<br />
Evaluation Center, Schering Corporation, Lafayette, NJ 07848 .<br />
Therapeutic proteins (cytokinas, gtvvth factors <strong>and</strong> imniwnndulatory polypeptides) pose tw special<br />
problem for routine in vitro safety testirg . (1) Mode of action is usuall,y species <strong>and</strong> cell-type<br />
specific such that the protein itself is rot acutely toxic <strong>and</strong> (2) clinically used dng fomailation<br />
excipients can modify test system regpor~ses . Dng substanoe is usuall;y ptrsented for testing as<br />
lyophilized active in for+iulatio ;tis as siaple as saline or as oanplex as mixtures with buffers, bulking<br />
agents, stabilizers, antioxidents, ard wettitg ageits . Ideally, the first ptoblem can be dealt with by<br />
the use of primate tissues or oetl types laaun to retain appropriate receptors . For qualiq+ control<br />
pucposes, sub4 .msn system (such as the Mrs Test) are atill weful for detectirg potential activity of<br />
excipients, contaminants, or biottac.tfonmatien-degtadation products . The seoatd problem neoassitates<br />
exteruive pilot trials to establish tolerated dose wlueas of fotsilatien vithout the active protein<br />
(placebo control solution or control vehicle) <strong>and</strong> subsequently cantrolling for toequal vehicle effects in<br />
all dose ard control groups. This latter approach involves adjusting all treataent carditiom to the<br />
level (v/v) of vehicle control solution used in the high-dose gtroup . If the vehicle is t .duly toxic to<br />
backgroud endpoints or interferes with positive control respas es, thet the foraulated therapeutic<br />
protein canot be tested . In effect, since the active protein is nort-tcodc, the ssscian tolerated dose<br />
level is set or deterndned by the level of vehicle control solution tolerated by negstive aod/or positive<br />
control groups . Failure to <strong>and</strong>ify study desigrts can lesd to false negatives if the formulation<br />
conpone.nts suppress S9 sietabolisn or expcesaion of sutation . False positives could ocas- if spontaneam<br />
(backgr<strong>and</strong>) levels are erimoed or cytotcedcities from other systea emipaknts are eliminated . Such<br />
forau]ation effects have been observed in bacterial wtasgenaais, huaan l,ysptwcyte qtagenetics, CfA-tCPRP<br />
<strong>and</strong> rat hepqtocyte UDS studies .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 109<br />
Notes
110 1989 EMS Abstracts 315<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes MDTATION3 IIJDI= IN = 3WJ GEIiE OF E . ooli BY l-Nr1Sa08O-8-YiI=PYft@lE : T}IE<br />
INF11JF2KE OF PTA4IID 01 AND EXCISICN RtRAIIt CN THE *IPATICNAL SPECIS3BI . I .B .<br />
Inmbartl, A.J.E. Gatd, B.W. Gliclaaatl2, D .R . MoCalla, 11dcMastet' University,<br />
Namilton, Ontario <strong>and</strong> 2York University, Toionto, Ontario (Canada) .<br />
1,8-Dinitropyrene is an envizotmental oonRaminar :t MR :id : is mutageni.c in bacteria<br />
<strong>and</strong> cultured mmuaalian cells, ard aast;inogenic in rodents . This oacpo:atid is<br />
activated in vivo by nitsoraduction to 1-nitxneo-8-nitrvpyrei:e (1,8-NONP), <strong>and</strong> then<br />
to the hydroxylamine vhich, followinq soet.ylaticn, reacts with D2ZA . The major 17la<br />
adduct fozmed is 1-N-(2'- T :A txatwrrexsions oonsistent with preferential<br />
misinoorporatian of adenine during error pmne bypass of a bulky lesion . Deletions<br />
are detected in all strains, but are itduoed above the spontaneous frequency only<br />
in the e UvrB, p1C+D01 baclognound .<br />
316<br />
M'M(AF2~ 1~1TAGQIESIS ~ RtIE l~I ~ QF~',,,gJi . I .B. Imnbertl, T .A .<br />
Chin1, D .W . Bryant , B .W . Gliclcna~, D.R. MoCalla yMddaster University, Hamilton,<br />
Ontario (Canada) <strong>and</strong> 2York University, Toronto, Ontario (Canada) .<br />
AF2 is a 5-nitrofaran derivative vhich is :nrtagenic in baateria followitg<br />
activation by endogenas nitx+areduataoes. Pr+avious studies in our laboratory have<br />
suggested that AF2 mrtagenesis is depeneent on the irduction of SOB repair<br />
ftaretiens . In oider to gain greater in4iqht into the mec3 :aaimn by Vhidi AF2 induces,<br />
mutations, we have examined the nutaticnmi specificity of this oaipo :rd in the 11aI<br />
gene of E,gol . Traatment of a ntNrB, P1CU01 host with 1 UM AF2 yielded a nutatian<br />
frequency 300 X that of tastreated oontrols at 20% survival . Mztatiore whicli<br />
occurred in the first 180 base pairs of the episaml JWI gene were ctiaracterized<br />
by L'ta sequenoe analysis . Of the 144 autants in the oollectian, 125 are base<br />
substituticns with trareversicns a :trunberinq txansitions by about 2 to 1 . Ninetytwo<br />
peroent of the base substitutions oaas at G :C beas pairs (62 G :C -> T :A<br />
traneversions, 43 G :C -> A :T transitions, 10 G :C -C :G transveraions) . Base<br />
substitutions ooatr 3 .1 times more frequently at 5'-Pyrimidine-G than 5'-Parine-G<br />
sites . A mec3anism consistent with these results would be the error psnne<br />
imorporation of nucleoti8es across ftnm an apurinic site with an order of<br />
preference of adenine > thymine > guanine . Of the 11 framec+hift mrtatians recovered<br />
in this study, 10 ooour at one hotspot whicA irNolves the loes of the G fraa the<br />
sequence 5'-TTIGCDO-3'<br />
. A further characteriatio of this collection is of several oanplex mutatians irnrolvirg multiple closely spaced ( pn<br />
apart) mrtationnl events .<br />
317<br />
INVITRO DETOXIFICATION OF MX (3-CHLORO-6-(DICHLOROMETHYL)-5-HYDROXY-2-(5H)-F1IRANONE) .<br />
S . Lampelo, T . Vartiainen <strong>and</strong> S . LSt,jBnen, National Public Health Institute, Dept .<br />
Environm . Hyg . Tox . P .O .B . 95, Kuopio <strong>and</strong> University of Kuopio, Dept . Chem . P .0 .0 .21,<br />
Kuopio, Finl<strong>and</strong><br />
MX is a strong bacterial mutagen found in drinking water . In this study the<br />
changes in the mutagenicity of synthesized MX were investigated after its preincubation<br />
with liver 59 (L-S9), human placental S-9 (P-S9), vitamin-C <strong>and</strong> their combination<br />
utilizing Ames" Salmonella test . The effect of serum albumin on the mutagenicity<br />
of MX was also studied . The etability of MX mutagenicity when diluted in<br />
water <strong>and</strong> buffer with <strong>and</strong> without vitamin C was followed during storage at room<br />
temperature .<br />
MX lost its mutagenicity in the presence of P-S9 or L-S9 . Preincubation with vitamin<br />
C (10 ug) enhanced MX mutagenicity whereas adding of P-S9 or L-59 into this mixture<br />
caused a drastic decrease in the mutagenicity . However, no inactivation of the mutogenicity<br />
occurred when P-S9 was incubated with vitamin C before adding MX into thre<br />
mixture . In all cases L-S9 was a very potent inactivator . Albumin decreased the<br />
mutagenicity of MX when added onto the plate but low concentrations of albumin in<br />
the preincubation mixture caused an increase in MX mutagenicity . MX was very stable<br />
when stored in buffered C-vitamine solution whereas it lost its mutagenicity in<br />
water <strong>and</strong> buffer during two weeks .<br />
These results suggest that inactivation of MX might be due to enzymatic systems but<br />
also to an unspecific binding to proteins . The antioxidents might protect the<br />
reactive genotoxic sites in MX-molecule .<br />
50869 3622
318 1989 EMS Abstracts 111<br />
IMPROVERD METABOLIC CAPABILITY BY DRUG METABOLIZING GENE TRANSFECTION. Notes<br />
R . ~Lan~enbach', C . Crespi', R . Davies', P . Smith', <strong>and</strong> K . Rudo', 'National<br />
Tnstitu~Environment Health Sciences, Research Triangle Park, NC 27709 <strong>and</strong><br />
'Genetest, Woburn, MA, 01801 .<br />
The technique of gene transfection was employed to introduce the cDNA coding<br />
for specific cytochrome P450 enzymes into transformable C3H/10TYe mouse embryo<br />
ce11s <strong>and</strong> mutable human AHH-1 lymphoblastoid cells . The long term objective of<br />
this work is to enhance the metabolic capability of these cells for exploring the<br />
role of cellular metabolism in genotoxic <strong>and</strong> nongenotoxic mechanisms of carcinogenesis<br />
. C3H/10TS4 cells containing transfected rat cytochrome P450IIB1 (P450b)<br />
were more sensitive to the cytotoxic effects of acetylaminofluorene <strong>and</strong> dimethylnitrosamine<br />
than parental cells . Transfection of either human cytochrome P450IA1<br />
(methyl cholanthrene inducible form) or cytochrome P450IIA2 increased the mutagenic<br />
response of AHH-1 cells to aflatoxin B1 <strong>and</strong> dimethyl-/diethylnitrosamine,<br />
respectively . The manipulation of drug metabolizing genes provides an experimental<br />
strategy for delineating initial stages of carcinogenesis <strong>and</strong>/or mutagenesis by<br />
toxic agents in mammalian cells .<br />
319<br />
RECENT DEVELOPMENTS IN THE GLYCOPHORIN A ASSAY FOR SOMATIC CELL<br />
MUTATIONS IN HUMANS . Richard G . Langlois, William L . Bigbee, <strong>and</strong> Ronald H. Jensen,<br />
Biomedical Sciences Div., Lawrence Livermore National Laboratory, Livermore, CA (USA) .<br />
The glycophorin A (GPA) assay utilizes Immunofluorescent labeling <strong>and</strong> flow cytometry to<br />
identify <strong>and</strong> enumerate rare erythrocytes which lack the expression of one allelic form of<br />
GPA presumably due to mutations in erythroid precursor cells . Variant cells with both<br />
hemizygous <strong>and</strong> homozygous phenotypes are observed in normal individuals at a<br />
background frequency of about 10 per million normal cells . Transient Increases in variant<br />
frequency (VF) were observed in cancer patients during chemotherapy, but VFs returned to<br />
near normal values six months after therapy was completed . Significant dose-depehdent<br />
increases in VF were observed in A-bomb survivors suggesting persistent stem cell effects<br />
from radiation damage . Elevated spontaneous VFs have also been observed In individuats<br />
with some cancer-prone syndromes . Hemizygous VFs were elevated about 10-fold in<br />
individuals with ataxia telangiectasia, while approximately 60-fold elevations in both<br />
hemizygous <strong>and</strong> homozygous VFs were observed In Individuals with Bloom's syndrome .<br />
VFs for individuals with xeroderma pigmentosum, In contrast, consistently fell within the<br />
range of normal individuals . A new cell labeling method has been developed to allow the<br />
assay to be performed on a single-beam flow cytometer .Work performed under the auspices<br />
of the U .S . Dept . of Energy by the Lawrence Livermore Nat . Lab. under Contract No. W-<br />
7405-ENG-48 with support from the U .S. E .P .A. Grant R-811819-02-0 <strong>and</strong> the U .S .<br />
N .I .E .H .S . Interaqemcv Agreement NIH-ES•83-14 .<br />
320<br />
ONCOCENE CHANCES IN CHEMICALLY-TRANSFORHED RODENT RESPIRATORY EPITHELIAL CELLS .<br />
J .A . Lasleyl, S .J . Austinl, N .S . Schorschinskyl, D .K . Beeman2, <strong>and</strong> M .J . Mass2,<br />
l<strong>Environmental</strong> Health Research <strong>and</strong> Testing, Inc ., Research Triangle Park, NC 27709<br />
<strong>and</strong> 2Carcinogenesis <strong>and</strong> Metabolism Branch, MD-68, U .S .E .P .A ., Research Triangle<br />
Park, NC 27711<br />
Much evidence has accumulated linking alterations in oncogenes <strong>and</strong> the development<br />
of cancer . However, most of these studies have utilized tumors, which are<br />
late stages in the neoplastic process . We utilized the rat tracheal epithelial<br />
(RTE) cell transformation system to observe the participation of oncogenes in comparatively<br />
early stages of cell transformation in vitro . Eight cell lines <strong>and</strong><br />
normal cells were studied for alterations in K-ras . H-ras, <strong>and</strong> c-mvc . These lines<br />
were transformed in culture with the carcinogens benzo(a)pyrene,<br />
7,12-dimethylbenz(a)anthracene (DMBA), <strong>and</strong>/or the tumor promoter 12-0tetradecanoylphorbol-l3-acetate<br />
. Southern blot hybridizations indicated no<br />
amplification of H-ras, K-ras, or c-wc in the linas studied . We did not find<br />
alterations in H-ras codon 61 that have been found in DMBA-induced tumors in vivo .<br />
H-ras probing of Hpa II digests could distinguish normal from transformed cells .<br />
One cell line had 2 types of o-wc RFLPs when digested with Bam HI or Hind III, <strong>and</strong><br />
also exhibited methylation changes in CCGG sequences compared with normal cells .<br />
Northern analysis showed elevated H-ras <strong>and</strong> c-vic expression in some transformed<br />
cells . We conclude that transformed RTE cells can demonstrate a number of oncogene<br />
alterations, however, the relationship of each to the transformed state requires<br />
further study . This is an abstract of a proposed presentation <strong>and</strong> does not<br />
constitute EPA policy .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A<br />
r
112 1989 EMS Abstracts 321<br />
Notes ENHANCED MORPHOLOGICAL AND MALIGNANT TRANSFORMATION OF SYRIAN HAMSTER EMBRYO CELLS<br />
CULTURED AT pH 6 .70. R .A . LeBoeuf <strong>and</strong> G .A . Kerckaert, The Procter & Gamble Company,<br />
Miami Valley Laboratories, P .O . Box 398707, Cincinnati, OH 45239 .<br />
Studies conducted in our laboratory have focused on the development of a cell<br />
transformation model for investigation of mechanisms of carcinogenesis <strong>and</strong> the assessmnt<br />
of carcinogenic potential of chemicals . Our initial studies indicated that proliferation<br />
of SHE cells is optimal at pR 6 .70 compared to pH 7 .35 which has been used<br />
historically for Syrian hamster embryo (SHE) cell culture . Subsequent inter-laboratory<br />
studies demonstrated that the frequency of morphological transformation (MT)<br />
induced by several carcinogens (both DNA reactive <strong>and</strong> non-DNA reactive) from different<br />
chemical classes is greater at pH 6 .70 compared to pH 7 .35 . All carcinogens tested<br />
have induced a statistically significant increase in MT compared to concurrent pH 6 .70<br />
controls whereas non-carcinogens do not cause a statistically significant increase in<br />
MT frequency . In order to determine the relevance of the MT phenotype, we have characterized<br />
the malignant potential of SHE cells derived from colonies of different<br />
morphological phenotypes transformed at pH 6 .70 . Fifteen to 30% of the MT colonies<br />
induced by the carcinogens 3-methylcholanthrence <strong>and</strong> benzo(a)pyrene give rise to<br />
established cell lines . 85% of these lines progress to the malignant phenotype in an<br />
apparent step-wise manner . In contrast, no morphologically normal colonies from<br />
control cultures <strong>and</strong> less than 3X from carcinogen treated cultures escape senescence<br />
upon cell isolation . These results will be discussed in terms of the use of early<br />
passage SHE cells cultured at pH 6 .70 for both carcinogen screening <strong>and</strong> mechanistic<br />
studies .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
322<br />
COMPARATIVE GENOTOXICITY TESTING OF MAINSTREAM WHOLE SMOKE FROM CIGARETTES WHICH<br />
BURN OR ONLY HEAT TOBACCO . C . K . Lee, D . J . Doolittle, C . T . Burger <strong>and</strong> A . W .<br />
Hayes, R . J . Reynolds <strong>Tobacco</strong> Company, Bowman Gray Technical Center, Winston-Salem,<br />
NC 27102<br />
Mainstream whole smoke (MWS) from test cigarettes which heat but do not burn<br />
tobacco was compared for its genotoxio potential to that from a reference cigarette<br />
which burns tobacco . MWS was collected from lR4F, a Kentucky reference cigarette,<br />
<strong>and</strong> two test cigarettes, one with regular flavor <strong>and</strong> the other with menthol flavor .<br />
The cigarettes were smoked on a smoking machine under st<strong>and</strong>ard conditions <strong>and</strong> the<br />
particulate phase was collected on the Cambridge filter pad <strong>and</strong> the vapor phase<br />
passing through the pad was bubbled into a DMSO trap . The filter pad <strong>and</strong> DMSO in<br />
the trap was combined <strong>and</strong> extracted with additional DMSO to obtain MWS . Identical<br />
concentrations of MWS were tested with all samples to make a direct comparison on<br />
their genotoxic properties . MWS of 1R4F registered positive results in the Ames<br />
assay in the presence of metabolic activation but negative results in the absence of<br />
activation . The frequencies of both sister chromatid exchanges <strong>and</strong> chromosome<br />
aberrations were significantly increased in Chinese hamster ovary cells exposed to<br />
1R4F with <strong>and</strong> without metabolic activation . The MWS from the test cigarettes, with<br />
either regular or menthol flavor, did not induce a positive result in any of these<br />
in vitro assays in the concentration range tested . The MWS from 1R4F was cytotoxic<br />
at higher concentration range, while MWS from test cigarettes was not . In summary,<br />
the MWS from test cigarettes was neither genotoxic nor eytotoxic under conditions<br />
where MWS of 1R4F was genotoxic or cytotoxic in a concentration-dependent manner .<br />
INITIATION OF DNA SYNTHFSIS WITH ALTERED PHOSPHATIDYL INOSITOL METABOLISM<br />
Myung Ae Lee, Hyeong Ok Lee <strong>and</strong> Cheol . Joe<br />
Department of Biology, Korea Institute of Technology<br />
Taejon 302-338, Korea<br />
323<br />
Intracellular inositol lipid hydrolysis involved in the initiation of DNA synthesis<br />
was examined using chromatographic analysis . NIH 3T3 cells metabolically labeled with<br />
3H-inositol were growth-arrested by serum starvation for 14 hours followed by 3mM hydroxyurea<br />
treatment for 6 hours <strong>and</strong> were allowed to initaiate DNA synthesis in the normal<br />
media . The increased breakdown of phosphatidyl inositides into inositol 1,4,5-triphosphate<br />
<strong>and</strong> di~cylglycerol was observed with the maximal DNA synthesis rate which was<br />
measured by the H-thymidine incorporatio~ for 30 minutes during the progression of cell<br />
cycle into S phase . The incorpJoration of H into phosphatidyl inositol-4-phosphate <strong>and</strong><br />
inositol-1,4-bisphosphate in H-myo-inositol labeled cells was markedly increased with<br />
the initiation of DNA synthesis . These data suggest that increased phosphatidyl inositol<br />
turnover is associated with the initiation of DNA synthesis, <strong>and</strong> this process is in<br />
close relationship with the phosphorylation <strong>and</strong> hydrolysis of phosphatidyl inositides .<br />
50869 3624
324<br />
DEVELOPMENT OF A PROCEDURE FOR QUANTITATION OF MUTATIONS AT THE NGPRT LOCUS IN<br />
CHINESE HAMSTER OVARY (CHO) CELLS WITH EXPHESSION AND SELECTION IN SEMISOLID CULTURE<br />
NEDIUM . P .S . Lee, K .Y . Henry, <strong>and</strong> C .J . Rudd, SRI International, Menlo Park, CA (USA) .<br />
We are developing a modified procedure for quantitation of mutations at the HGPRT<br />
locus to reduce the cost <strong>and</strong> potential errors associated with the suhculturing of<br />
cells during the expression period . In established procedures, CH0 cells usually<br />
require subculturing every 2 to 3 days for 7 days prior to treatment with the selective<br />
agent, 6-thioguanine (TG) . Colonies of TG-resistant CH0 cells can be selected<br />
either attached to tissue culture dishes or suspended in semisolid medium . In our<br />
laboratory, the cloning efficiencies of unselected cells <strong>and</strong> the numbers of mutant<br />
colonies were comparable under the two conditions . Using a modified procedure, we<br />
eliminated the need for subculturing during the expression period by growing lower<br />
densities of cells in semisolid medium after the induction of mutations . Mutant<br />
colonies were selected after 4 to 7 days by adding TG (final concentration 30 uM) as<br />
an overlay with additional medium . The colonies were counted after a total of 14 or<br />
more days incubation . The recovery of HGPRT- cells in reoonstruetion experiments was<br />
equivalent in the presence or absence of wild-type (HGPRT+) cells under these conditions<br />
. Small HGPRT- colonies could be distinguished from non-viable microcolonies of<br />
HGPRT• cells by staining with Thiozolyl Blue (MTT, 2 .5 mg/dish) . By growing the<br />
cells in semisolid medium for the duration of the experiment, olonal populations were<br />
maintained . Therefore, with the modified procedure, the number of TG-reslstant colonies<br />
should more closely represent the number of cells with a mutation at the HGPRT<br />
locus at the beginning of the expression period . This inereased accuracy, <strong>and</strong> the<br />
savings in labor <strong>and</strong> materials costs, are clear advantages of the modified protocol .<br />
325 THE ELECTROPHORETIC SPECIFIC LOCUS TEST IN THE MOUSE AND ANIMAL NOOELS<br />
OF HUMAN DISEASE<br />
Susan E . Lewis<br />
Research Triangle Institute . P . 0 . Box 12194, Research Triangle Park . NC 27709<br />
The mutagenicity of x-rays <strong>and</strong> a variety of chemicals have now been studied<br />
in the electrophoretic specific locus system . Comparisons of mutation frequencies<br />
in the treated groups in the electrophoretic specifiE locus tests . with those in<br />
the visible specific locus test . will be made . Electrophoretic screeniny has<br />
identified a number of electrophoretically detectable mutations, both spontaneous<br />
<strong>and</strong> induced by various agents . The underlying molecular basis for, <strong>and</strong> the physiological<br />
impact of selected mutations are discussed . Although animals homoZyflous<br />
for null alleles at many of the loci under study appear to be perfectly<br />
healthy . certain others are either not viable or impaired . Mouse models of human<br />
diseases have been recovered in the course of this <strong>and</strong> other biochemical screening<br />
programs . These are discussed as to their potential utility in experimental therapeutics<br />
<strong>and</strong> the relative susceptibility of the relevant loci to mutagenic change .<br />
(Supported in part by Contract No . N01-ES-2-5012 . NIEHS .)<br />
326<br />
Hypothesis : Modification of Oncogene Expression as a Major<br />
Mechanism of Action of "Nongenotoxic" Carcinogens<br />
A . P . Li, Monsanto Company <strong>Environmental</strong> Health Laboratory, 645<br />
ewstead, St . Louis, MO 63110 .<br />
Recent findings in cancer research have consistently associated<br />
mutations in oncogenes with carcinogenesis, therefore supporting<br />
the relevance of genetic damage in this process . However,<br />
findings in genetic toxicology now point out that a large variety<br />
of carcinogens, especially hepatocarcinogens, do not have<br />
genotoxicity readily measured using conventional assays (e .g .<br />
Ames test) . Examination of the biological effects of these<br />
"nongenotoxic" carcinogens include the following : a . Cell<br />
proliferation, either via a mitogenic effect (e .g . phenobarbital)<br />
or cytotoxicity-induced compensatory cell division (e .g . CC14) ;<br />
b . Enzyme induction, such as P450 (e .g . PCBs) or peroxisome<br />
induction (e .g . clofibrate) ; c . Alterations in DNA methylation<br />
(e .g . choline-deficient diet) . I hypothesize that these events<br />
share one common phenomenon which is key to carcinogenesis :<br />
Induction of oncogene expression . Cell proliferation is known to<br />
lead to sequential expression of several oncogenes which are not<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 113<br />
Notes
114 1989 EMS Abstracts<br />
Notes expzessed in quiescent calls . Enzyme induction may be a<br />
pleiotropic lnduetion effect on gene expraasions of the inducers .<br />
oNA methylation has been linked to alterations of gene<br />
expression . Induction of oncogene expression leads to the<br />
expression of aberrant oncogene products in "preinitiated" cells<br />
which are essential for their ultimate development into tumors .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
327<br />
1+iUTA(iENETIC EFFECT OF 15 C0IPOUNDS IN DROJOPHILA Al1BIIPI+OIDY '1S8TIACt .<br />
Li Hua1y1, Ca1 0on®min, Huang Jintang <strong>and</strong> Nan$ Zinmin, Department of<br />
Tozicoiogy, Second Military Medical Ooilege, 200433 8hanghai, P .R . CHINA<br />
In order to determine the sensitivity of Drosophila aneuploidy detecting<br />
syatem, the females (yw/yw) <strong>and</strong> the malea (yB/Yy) are used in our<br />
tests . The treatment sex male <strong>and</strong>/or lemale . The treatment stage lsrva<br />
<strong>and</strong>/or adult . The treatment route feeding method . The brood schedule of<br />
2-3-3 day broods ia carried out in all teats . Tested chemicals inoludes<br />
(1) Colcemid, (2) Colohioine, (3) Catieine, (4) Aotinomyoin D!( 5)Mett~yl<br />
methaneaullonate, (6) 5-Flurodeoxyuridine, (7) Proflavine, (8)0y0lophosphamide,<br />
(9) Mitomyoin-C, (10) Meprobamate (11) 8orbitor, (12)Urethane,<br />
(13) N-Methyl-N=nitroN-nitroaoguanidine, ~14) Sodium lluoride, (15) 5-<br />
Bromodeox}ruridine . Normal test described by Margolin et al (1983) are<br />
used in examine data for statistical aignifioanoe . The compounds which<br />
aignilicant difference at the 5% level between the treated <strong>and</strong> oontrol<br />
lrequencies of chromosome gain in treated d'inolude (1,2,5,7,9) <strong>and</strong> (3,<br />
4) in treated Q . The compounds which significant difference at the 5%<br />
level of chromosome loss in treated d inolude (4,6,7,8,9,11,12,13,14,15)<br />
<strong>and</strong> (1) in treated Q . U! 15 compounds tested for non-diajtimotion <strong>and</strong><br />
loas, only six (4,5,8,9 .12,13) have definitive oaroinogeneais olaaaif ication<br />
(a11 positive) . F ve (4 8,g,12,13) of these were significant di!lere<br />
oe for loas, three ~4 5 .9) o3 these rrere aignilioant di!lerence !or<br />
non-disjunction . Senaitivi~y ia 835: <strong>and</strong> 50>+, respectively.<br />
328<br />
MECHANISM OF ARSENITE INHIBITION OF REPAIR OF N-METHYL-N-NITROSOUREA-INDUCED DNA DAMAGE<br />
J .-H . Li <strong>and</strong> T.G . Rossman<br />
Institute of <strong>Environmental</strong> Medicine<br />
NYU Medical Center, NY, NY 10016<br />
Although inorganic arsenic compounds are known human carcinogens, they are not<br />
mutagens . The lack of arsenic mutagenicity has led us to study its comutagenicity .<br />
We have found that sodium arsenite at relatively nontoxic concentrations (5 uM for<br />
24 hours or 10 aM for 3 hours) is comutagenic with N-methyl-N-nitrosourea (MNU) at<br />
the hprt locus in Chinese hamster V79 cells . A nick translation assay, which measures<br />
DNA str<strong>and</strong> breaks in permeabilized cells, was utilized to show that str<strong>and</strong> breaks<br />
resulting from MNU or its repair accumulate in the presence of arsenite . MNU-induced<br />
poly (ADP-ribose) synthesis, measured by the incorporation of [3H]-NAD in the permeabilized<br />
cells, was also increased by post-treatment of the cells with araenite . This<br />
supports the hypothesis that arsenite inhibits the completion of DNA repair . The<br />
accumulated str<strong>and</strong> breaks in the presence of arsenite are probably not due to direct<br />
inhibition of DNA polymerase alpha, the presumed repair polymerase of MNU-induced DNA<br />
lesions, since very high concentrations of arsenite (about 7 .5 mM for 50% inhibition)<br />
are needed to inhibit this enzyme . We thus suggest that the repair of MNU-induced DNA<br />
lesions may be inhibited by arsenite by interfering with the ligation step .<br />
Supported by grant CA29258 from National Cancer Institute .<br />
GiNOlOIICMY Of ORCANIC gXIRAClS Q f3ggT foILS IN fICBI DIfZRIClS<br />
IN NgBo NANIN , pNILIppIlt3<br />
. .!s~ liaaoo . V . 1 . Isotiasds aad N . V . gotuysa , Iwtitute of<br />
stsy, o sge of Saiewoe, Uaiwrsity of tka pkilippias ., Dilioaa ,<br />
Quesoa City , pkilippiass<br />
Seventy-five per eeat of air pollutiee !a lMtro Naaila is ooatributed by<br />
emaissieas LrN sre tha 500,000 motor velioles operstiag daily . A awbsr of<br />
th.se vehicles are disssel powered vbiek oaa expose tlr urksa populatiea to<br />
diesel exlrusts wbiek eaatsia polyoyelie aresrtie Wreearkooa which posseas<br />
kvmaa eareimejaale poteatial . Tbase pelyeyelie aremetie Iqdreesr6elM esa<br />
aeeu .,late in street oils . orpaie extracts trem sieved soil Nmples trom<br />
lodustrial, oasrroial sad reaidsatisl areas ia eiakt distriets around Nstro<br />
Maaila - Nakati, Narikias, pasia, !sa Juaa, IMadaluyoaa, Csiata, Wleaswla<br />
<strong>and</strong> parsasque - were studied wlag tlr Rsa assay, bost-msdiated assay aad<br />
50g69 3626<br />
329
1989 EMS Abstracts<br />
.feronucl.us tast. Mo direct DNA darsiai capacity aas observed of tAs Organic Notes<br />
axtracts of strsst soil sa.plss from all eight dist:iets . AowesY . Mutagsoieiq<br />
aftsr rtatiolic acti .atien <strong>and</strong> ckro .osesn brsakia= effects wrs exhibited by<br />
orpnic oztraeta of street soil sasplos fro . industrial <strong>and</strong> oe.mreial areas<br />
from four districts . Q"otoxicity wws correlated with ttr prsssncs of<br />
bsnso(a)pyr .as .<br />
330<br />
ASBES'T06 PSSOCIATID III.IS=NP HESOTF>ELICNGi : GCNSISTLNP CFROMOG0t9iL QiF,NM IN 1S2Ctt<br />
CELL LIAES FROtd SIX PATIENIS .<br />
Lirviairiaa, K .I ., PalirrShlun3, K . Tanmilehto, L ., 1Mattson, K ., Jantunen, K ., Husgafvel-<br />
Pursiainen, K . Insti . of Occupational Health <strong>and</strong> 1 Univ . Hospital, Helsinki, FICIIAND<br />
Exposure to asbestos fibers is lanwri to be associated with malignant mesotheliema in<br />
at least 80% of the tumor cases . Karyotypic studies of m3sothelioma tissues have<br />
revealed a variety of changes involving different duomosares . A consistent kazyotypic<br />
abnormality oa :mon to niesotheliomas has not been reported previously . We have chracterized<br />
cytogenetically seven mesothelio:na cell lines <strong>and</strong> a short-term eulture whic3i have<br />
been established in our laboratory from ttsnor tissue or pleural fluid sanples fram six<br />
patients with pleural malignant mesothelioma . All the cases of our study have a heavy<br />
asbestos exposure history . Complex structural <strong>and</strong> ntmerical abnannalities were observed<br />
in all the cultures studied. Excess chromatid material of the short atm of diranosams 5<br />
was consistently seen in the cells of five patients . In four of the cases, monoea :y of<br />
c:lrcc,nsane 13 was observed . Additionally, double minute chrarosaees or harogeniously<br />
staining regions were frequently noted . The observed specific absormalities eottman to<br />
several patients, e .g . the excess of the p-ana of chrarosans 5 <strong>and</strong> monosony of chratrosare<br />
13 suggest the importanee of these karyotypic changes in the development of malignant<br />
nesotheliana . We have shawm that asbestos <strong>and</strong> glass fibers are able to induce ctu :anosanal<br />
damage in human primary mesothelial cell cultures in vitro . The possible association of<br />
these findings with the kaYyotypic changes in turor cells remains to be solved .<br />
331<br />
A STUDY ON THE WORKERS OF COKING PLANT WITH MICRONUCLEUS (MN) FREQUENCY .<br />
FRAGILE SITES (FRR .) AND SISTER CHROMATID EXCHRNGE (SCE)<br />
Liu Yongchang . Shanxi Cancer InstitutQ,Taiyuan .Shanxi (PRC)<br />
In this study, thrQa experiments had been done in coking workers <strong>and</strong><br />
in normal control .ThQy were divided into 5 groups according to different<br />
testing spots : 1 . Workers of coking workshop ; 2 . Other workars . 3 .Staffs<br />
who were not in the workshop . 4 . The individuals of the steel institute .<br />
S .Normal control :ThQ results were as follow : 1 .Ths :data of MN frQquancw<br />
in sequence were 3 .13. 3 .25, 2 .21 . 2 .10, 0 .44 : 2 . The sequence of<br />
chromosome aberration (CR) were 4 .13, 5 .05, 1 .50 . 2 .?2 . 0 .20 . 3 . The<br />
sequence of SCE were 9 .60. . 8 .58 . 8 .88' . 6 .32 . 6 .40 .ThQra were significant<br />
differences among all these data . The concentration of Bap detected at<br />
this 5 spots list as logarithmic valua* 2 .38, 1 .78 . 1 .00 . 0 .59 . E .41,<br />
respectivnlu . Compared these data with MN . CA <strong>and</strong> SCE by means of<br />
correlation tast- the results showed that all were positive correlations<br />
TharQ were 251 fra. at all . 60% in the chromosome group A <strong>and</strong> 22X in<br />
group B . Of the 113 identified fra . . 34 .5X were inheritable (3p14) <strong>and</strong><br />
47 .78% were corresponding to the nonr<strong>and</strong>om carcinoma fra . .<br />
332<br />
CHROMATIN ALTERATIONS DURING EXCISION REPAIR<br />
M. Ljungman <strong>and</strong> G . Ahnstrbm, Department of Radiobiology, Arrheniua Laboratories for Natural Sdeneea,<br />
University of Stockholm, S-10961 Stockbolm,Sweden .<br />
DNA excision repair in mammalian cells scema to be associated with chromatin modification events . Cells<br />
exposed to ultraviolet light appear to undergo a general type of chromatin modification while methylmethane<br />
sulphonate treatment induce a more localized modification . It has been our Intention to further investigate the<br />
mechanismc of these chromatinmodityingeventsinmammalian ceDa .Inapreviousttudy, kinedaofrepairinduced<br />
DNA str<strong>and</strong> breaks were followed in Y79 hamster cell cultures treated with methylmethana aulphonate (Id1vIS) .<br />
The shapes of the DNA str<strong>and</strong> break curves when adding the drug 3-aminobenramide suggest that poly(ADPriboso)polymerase<br />
was involved in locally modifjdog the chromatin prior to the rejoining step . Thia modification<br />
might be required in order for the ligase to gain access to the repair site. To study ehromatin alterations associated<br />
with nucleotide excision repair in human fibroblasts, we used the DNA-damagiog agents bleomycin, gamma<br />
radiation <strong>and</strong>8-methoxypsoralen .Ithasbeenshownthatthese agents react preferentiallywith DNA in open regions<br />
of the chromatin. Thus, they are suitable for talnng'snapahots' of the chromatin, revealing how open its structure<br />
is at any particular time . Results obtained on cells undergoing repair after UV-'uradution indicate that the<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
115
116 1989 EMS Abstracts<br />
Notes chromatin ia becoming altered by a time-dependent process. After 10-20 min of poet-UV incubation at 37T~ the<br />
relative DNA-damaging effects (measured as DNA str<strong>and</strong> breaks or DNA cross-links) of bleomycin, gamma<br />
radiation <strong>and</strong> 8-methoxypsoralen was increased 50% . However, after 60 mm the eHectswere back to control levels .<br />
These alterations might reflect events preparing the chromatin for the repair of W-induoed DNA damages .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
333<br />
LACK OP INDUCTION OF SCEs IN HUMAN LYMPHOCYTES EXPOSED TO MONOENERGETIC NEUTRONS<br />
L .G . Littlsfield, E .L . Proae*I, E .E. Joiner, apd S .P . Colysrt, Oak Ridge Associated<br />
Universities <strong>and</strong> Oak Ridge National Laboratoryl, Oak Ridge, TN (USA)<br />
Low-LET X- or gamaa rays induce few (if any) DNA lesions in boasn lymphocytes that<br />
are axpressed as SCEs . Recently, however, increased SCEs have bsen observed in<br />
lymphocytes exposed to 42 MsV d-Ee neutrons pro .pting the suggestion that the REE for<br />
SCE induction by higb-LET radiation suy be effectively infinite (J . Savage, M .<br />
Holloway, Sr . J . Radiol . 61 :231, 1988) . We conducted studiss to determine whether<br />
aonoenergetic neutrons induce DNA lesions that lead to SCE formation, <strong>and</strong> if so,<br />
whether such damage is modifiable by radioprotsctive cheaicals that selectively<br />
scavenge OH radicals . Busan lymphocytes suspended in culture sedium with or without<br />
1M DMSO were exposed to 5 doses each of 6 .0 <strong>and</strong> .44 M.V neutrons at RARAP . Chromosome<br />
aberrations <strong>and</strong> SCEs were evaluated in 1at or 2nd division staphases from 48br<br />
cultures . As expected, the two high-LET radiations ware considerably more efficient<br />
in inducing aberrations than X-rays, with maxisum calculated RBEs of 6 <strong>and</strong> 16 for<br />
dieantric induction by the 6 .0 <strong>and</strong> .44 NeV neutrons . Eased on results of curve<br />
fitting data, we estimated that OH radical aisdiated lesions in DNA contribute 402 <strong>and</strong><br />
15% . respectively, of the dose dependency for aberrations resulting from exposures to<br />
the 6 .0 <strong>and</strong> .44 MsV neutrons . In contrast, SCE frequencies showed no correlation with<br />
neutron energy, radiation dose, nor presence or absence of radioprotector during<br />
exposure . We conclude that while high LET radiations are extremely efficient in<br />
inducing DNA lesions that lead to chroaoso .e aberrations, sonoensrgetic 6 .0 or .44 M .V<br />
neutrons do not induce DNA lesions that lead to SCEs . Supported by DOE contract<br />
DOE-AC05-760R00033 .<br />
334<br />
MUTAGP.NICITY SLREENING OF WATER EXTRACT8 PROM 102 XIKIIS OF CRUDE DRUGR IK CHIKF.SE RF.DICINC9<br />
Liu Dexiana , Yin Xuojun<br />
Laboratory of Medical Gonetics of Kestern Region Hospital,Orweehi,Xinjit-ig ,<br />
- People's Republic of China<br />
In an a!tenpt to explore the autagonicity of the Chinese Medicinal herbs,102 crudo drx s :.n nu-.ber,<br />
eeployed comm,~only as traditional Chineso medicines,vore chosen <strong>and</strong> boiled,<strong>and</strong> then uaod tlieir obtained<br />
water extracts as the sample of aasay .Each extract vas assayed by Ames bioaa.ay system with TA 38 <strong>and</strong><br />
TA 100 strains .At aame time,tho intreperitoneal injection in mice vaa aivon with the ditferent doses .<br />
<strong>and</strong> obaerved their ehromoaomal aberration (CA) <strong>and</strong> micronucleir (PC3KF) .Aaes test revealed thut,p<br />
extracts of the crude drugs (for instsnos,Astr .galus Meaberaneous Ege,,3ophora Japonica L.<strong>and</strong> xuceh~+la<br />
Ulnoidea Oliv .) vore poaitivs (3/102 ;2 .97t) .Aatragalua Kenberancous Ege in the presence of E9 mix vith<br />
strain TA 98 could induce tho number of revertant colonies to increase threo tieos (40mr,/platc) enre<br />
than spontaneous revertant colonies ;Sopho{~a Japonica L .in the presence or in tha abscnce ol £9 aix vith<br />
strain TA 98 could induce the number of revertant colonies to increase nine to ton point two tiscc<br />
(40mg/ylste') more than spontaneous ones (in those samples' probably eontainin~ certain frnaer .lift r.utegena)<br />
;Eucommia Ulaoidss Oliv .in the presence or in the absence of 59 aix with strain TA 100 could<br />
induce the number of revertant colonies to increase three to four timee (40ag/plati) morro than sppntaneua<br />
ones (in these samples' probably containing mutagenie factor indueir,g base chantoa in DNA)a ihe<br />
increasement of the number of revertant colonies inducing by these 3 hinds of erude drugs van eort'olated<br />
to incoparation into four dosea<br />
.Yith CA <strong>and</strong> PCEKN asaays in mice,the postive results voro riven to<br />
13 water extracts of the crude drugs (13/102;12 .7¢),suoh as,Astragalus 1(embranaceus BE,Euconnia<br />
tRaoides Oliv.,Sophora Japonica L.,Datura Metal L .,Artemisia Capillaris Thunb.,Rehmennia Olutinosa<br />
(Oaertn .)Liboach,Carthsaue Tinctoriu L .,Forsythia Suspensa (Thunb .) Yahl,Paeonia Suffruticosa Andr .,<br />
Platycodon Or<strong>and</strong>iflorum (Jacq .)A .D.C .,Cinnanomum Cassia Presl,Kotopterygiua Incisum Ting .<strong>and</strong> Sophora<br />
Flavescens Ait .The CA <strong>and</strong> PCEMN induced by these drugs rere positive correlation to injecting medicines<br />
into intraperitoneal cavity in nice .<br />
The results have shown that the Aaes assay <strong>and</strong> the miee test (CA <strong>and</strong> PCE?4f) were positive results<br />
(23%;3/13) .The false negative rate of Ames aasqy was higher than the CA <strong>and</strong> PCEMK if the aberration of<br />
sucaryotio cells was taken .as assay eriteria .<br />
A QUANTITATIVE ASSAY OF DNA POLYMERASE-a IN SITU AT SINGLE CELLS USING FLUORESCENCE<br />
PSEUDO-COLOR IMAGE AND ACAS 470 .<br />
P .K . Liu, B . Goudreau, <strong>and</strong> G .S . Hsu, Case Western Reserve University, Clevel<strong>and</strong>, OH<br />
(USA), <strong>and</strong> Chia-Cheng Chang, Michigan State University, E . Lansing, MI . (USA)<br />
Aphidicolin is a speoifio inhibitor of DNA polymerase-o <strong>and</strong> 8 in eukaryotio<br />
oells . Because of the specificity of this inhibitor, it is potentially a useful<br />
probe for the detailed studies of the function of these polymerases . A DNA<br />
polys,erase-o mutant isolated on the basis of resistance to aphidioolin has been<br />
described (Proo . Natl . Aoad . Soi .,USA 80 :797-801, 1983) . We <strong>and</strong> others have<br />
isolated 4 variants which exhibit hypersensitivities to aphidicolin (Aphho) from<br />
Chinese hamster V79/743X fibroblasts (In : DNA Replication <strong>and</strong> <strong>Mutagenesis</strong>, ASM . Pp .<br />
163-172, 1988) . These variants are designated aphhs-1, aphhs-2, aphhs-3 <strong>and</strong> aphna-<br />
4 . We reported here results of studies involving i®unoohemieal characterization .<br />
The Aphhs phenotype in all mutants was stable for at least 30 days in the absence<br />
50869 3628<br />
335
1989 EMS Abstracts 117<br />
of selection pressure . The dCTP pools in the 743X <strong>and</strong> Aphhs cell lines were not Notes<br />
significantly different . The level of total DNA polymerase activity in crude<br />
extract from aphhs-2 clone was 30% of that observed in the parental clone . We<br />
developed a method to quantitate DNA polymerase-a antigen at single oells in situ<br />
using monoclonal antibody SJK 132-20 <strong>and</strong> fluorescence pseudocolor image . We found<br />
that the antigen of DNA polymerase-a in aphhs-2 was 40 - 50% of that in the<br />
parental 743X cells . The underproduction of the antigen of DNA polymerase-o<br />
provides a basis for the observed Aphhs phenotype . A possible mechanism for the<br />
underproduction of DNA polymerase-a in aphhs-2 clone is presented . (Supported by<br />
grants from NSF : DCB 8600659 <strong>and</strong> from CWRU : RIF-Liu to PKL) .<br />
336<br />
4ICR0NU,C(LEI INDUC!? BY FORMALDEHYI~E IN ERYTHROCYTES OF MICE, F . P . Loaroal, G . G .<br />
Arreola , A . Perez <strong>and</strong> T . H . Ma , Centro de Eatudios Aoalemicoe sobre Contaminacion<br />
Anbiental, Universidad Autonoma de Queretaro, QRO Mexico, Institute for <strong>Environmental</strong><br />
Mamagement <strong>and</strong> Department of Biological Sciences, Western Illinois University, Macomb,<br />
IL 61455 (USA)<br />
Subchronic doses of formaldehyde were tested for the clastogenioity using Mouse-<br />
Peripheral Erythrocyte-Micronucleus bioassay . Young (6 weeks old) female white aioe<br />
(CD-7) were divided into groups of 5, <strong>and</strong> administered biweekly with 5 mg/&g, 10<br />
mg/Kg, <strong>and</strong> 15 mg/Kg of formaldehyde (diluted with saline solution) through intraperitoneal<br />
injection for a period of 3 months . Peripheral erythrocytes were collected<br />
repeatedly from the tail monthly from the beginning of the experiment . Blood smears<br />
were double stained with hematoxylin <strong>and</strong> Giemsa for micronuclei in the peripheral<br />
erythrocytes . Micronuclei frequencies were scored (10,000 cell per slide) from each<br />
of the treated <strong>and</strong> control (saline solution) groups . Significantly higher frequencies<br />
of micronuclei in all the treated groups (around 0 .42) than that in the control<br />
(around 0 .2%) groups were noted in the first month blood samples . The differences of<br />
micronuclei frequencies in the blood samples of the second <strong>and</strong> third months were<br />
reduced to the insignificant levels (around 0 .1% <strong>and</strong> 0 .2%) . Whether this was due to<br />
the aging effect or adaptation to the chronic exposure or sexual specificity requires<br />
further investigation . Results of similar studies conducted earlier in .male mice<br />
showed no decline of MCN frequencies at the end of the third month .<br />
337<br />
The Role of Carcinogen DNA Adduct Structure in the Induation of Nutations .<br />
L .L . Loechler,1 M . Benaautti,2 A .K . Basu,2 C .L . Green,2 <strong>and</strong> J .M . Lssigmann2 ;<br />
1Boston University, Boston, MA 02215 ;<br />
2Massachueetts Institute of Technology, Cambridge, MA 02139 .<br />
Carcinogens induce cancer by reacting with DNA to form DNA adducts, which are<br />
processed by cells to yield mutations ; particular mutations in proto-oncogenes can<br />
lead to their activation to oncogenes . One of the key questions in earoinogenesis<br />
is : what are the mechanism by which carcinogen DNA adducts induce mutations? To<br />
answer this question, the mutations induced by specific DNA adducts in vivo must be<br />
known, <strong>and</strong> rationale for the mutations that each induces must be der vd. Using a<br />
combination of chemical synthetic <strong>and</strong> recombinant DNA techniques, individual DNA<br />
adducts have been built into several vectors in vitro, these vectors placed into<br />
bacterial cells, <strong>and</strong> the mutations induced in v vo n progeny vectors by each<br />
individual adduct determined . Adducts to be considered include 06-Methylguanine<br />
(o6MeGua), which is produced when carcinogenic methylating agents react with DNA, <strong>and</strong><br />
Thymine Glycol (TG), which is produced both by ionizing radiation <strong>and</strong> through<br />
oxidative pathways . O6MeGua induces G to A mutations, <strong>and</strong> TG induces T to C<br />
mutations . Proposed mechanisms of mutagenesie for each adduct will be discussed,<br />
including the use of molecular modeling techniques to interpret the results with TG .<br />
Preliminary data on the mutations induced by the major adduct formed in DNA from<br />
activated benro(a)pyrene (i .e ., BP-N2-Gua) will also be discussed .<br />
338<br />
COMMON AND UNCOMMON INDOOR SOURCES OF MUTAGENIC AEROSOL PARTICULATE MATTER .<br />
G6ran Ldfroth <strong>and</strong> Charlotte Stensman, Nordic School of Public Health,<br />
S-402 42 Gothenburg (Sweden)<br />
A number of pyrolysis precesses, which are performed indoors, have been investigated<br />
with respect to the emission of aerosol particulate matter <strong>and</strong> its mutagenic activity<br />
in the Ames Salmonella assay with the plate incorporation <strong>and</strong> microsuspension<br />
methods . The emission of the gaseous pollutants carbon monoxide, isoprene <strong>and</strong> benzene<br />
was also determined . Processes studied include smoking (sidestream) of tobacco <strong>and</strong><br />
herbal cigarettes, mosquito coil <strong>and</strong> incense burning <strong>and</strong> frying of minced, lean pork .<br />
Expressed in the unit of mg per 9 material, the emission of aerosol particulate matter<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
a
118 1989 EMS Abstracts<br />
Notes was about 0 .06 for frying, 20-30 for smoking <strong>and</strong> 50-60 for mosquito coil <strong>and</strong> incense<br />
burning . The mutagenic activity was highest in the presence of S9 <strong>and</strong> the response,<br />
expressed as TA98 revertants per ug particulate matter, was in the microsuspension<br />
assay 1-2 .5 for mosquito coil <strong>and</strong> incense smoke <strong>and</strong> 6-8 for sidestream cigarette smoke<br />
<strong>and</strong> frying fumes . With the exception of frying, all the other processes, which burn<br />
vegetable materials, emitted the gaseous pollutants . Benzene emission was about the<br />
same, 0 .4-0 .5 mg per g material, for all processes . Isoprene <strong>and</strong> carbon monoxide<br />
emission varied depending on process with tobacco cigarette smoking giving the highest<br />
isoprene emission <strong>and</strong> incense burning the highest carbon monoxide emission . It is<br />
apparent that both common activities, as smoking <strong>and</strong> frying, as well as uncommon<br />
activities, as incense <strong>and</strong> mosquito coil burning, can cause indoor air pollution .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
339<br />
MUTAGENIC EFFECTS OF SODIUM AZIDE ON THREE VARIETIES OF PEPPER (CAPSICUM ANUUM L .<br />
C .S . Longid, Institute of Biology, University of the Philippines, i iman Phil .)<br />
The objectives of this study were to determine the mutagenic effects of sodlum azide<br />
on the M1 generation of three varieties of pepper <strong>and</strong> to study the frequency of chlorophyll<br />
muti .tions in the M2 . Saeds of California wonder (CW), Long slim (L8) <strong>and</strong> Chinese<br />
(C) were soaked in water for two hours befose treatment in 0 .12, 0 .25, 0 .50 <strong>and</strong><br />
0 .75 mM sodium azide at pH 3 for two hours at 30 C . The criteria used to assess the<br />
mutaganic effects of azide in the !1~ included germination percentage, seedling <strong>and</strong> plant<br />
height, saad set, number of leaves ~ar plant <strong>and</strong> presence of chimeras . The frequency<br />
of chlorophyll mutations was determined in the M2 . Sodium asida induced a dose-effact<br />
relationship in the indices used for mutagenicity except in the number of leaves par<br />
plant, which did iiot show any relationship between the number of leaves per plant <strong>and</strong><br />
the dosage of the mutagen . The most frequent chlorophyll deficient mutations were the<br />
viridisr, followed by chlorina, xantha <strong>and</strong> albina . Very few xantha <strong>and</strong> albina were<br />
observed . Sodium azide was observed to be mitagenic in the three varieties of pepper<br />
studied .<br />
340<br />
RESPONSES OF PEPPER VARIETIES AND THEIR FI HYBRIDS TO ETHYL METHANESULFONATE<br />
C .S . Longid <strong>and</strong> J .D . Soriano, Institute of Biology, University of the Philippines,<br />
Diliman (Philippines)<br />
Seeds of California wonder (CW), Long Slim (LS) <strong>and</strong> Chinese (C) varieties of pepper<br />
(~Ca e~icum Anuum L .) were treated with 0 .3% <strong>and</strong> 0 .6% ethyl methanesulfonate (EMS) at pH<br />
9 tol at3U°C . The objectives of this study were to determine the effects of EMS on<br />
three pepper varieties <strong>and</strong> to study the chemo-sansitivity to EMS of the F crosses of<br />
chemo-reeistant <strong>and</strong> the chemo-sensitive varieties . Data obtained in the Mi showed decrease<br />
in germination percentage, shoot length, plant height, seed set <strong>and</strong> survival<br />
percentage . Somatic chimeras were obtained . In the M2 seedlings, chlorophyll-deficiant<br />
mutants induced in decreasing frequency were viridis, xantha <strong>and</strong> albina . The shoot<br />
length response of a species is considered an important measure of its reaction to mutagenic<br />
treatment, since it is based on the degree of biological damage . Hence, in this<br />
study, C whose shoot length at 30 days was reduced by 49 .68% is considered chemo-sensitive<br />
while LS <strong>and</strong> CW which had a 27 .48% <strong>and</strong> 29 .20% reduction in shoot length respectively<br />
are considered chemo-resistant . Crosses were made batween the LS <strong>and</strong> C varieties .<br />
Their F1 hybrids were treated with 0 .6% EMS . Same biological effects as in the .Ml were<br />
obtained in the F2 seedlings . The three varieties of pepper showed varying responsas<br />
to EMS, with only one variety (C) which was very sensitive to its effects . Their F1<br />
hybrids were not very sensitive to EMS . (This study is a portion of the senior author's<br />
dissertation presented for a Ph . D . in Botany, University of the Philippines in Diliman,<br />
Philippines .)<br />
341<br />
•iTUDIES ON TEE EFFECTS OF GAMMA RADIATION ON KALANCHOE PINNATA Ys:hS .<br />
C .S . Longid, Institute of Biology, University of the Philippines, Diliman,(Phil .)<br />
Kalanchoe innata Pers ., Kataka-taka (Pil .), life plant (~g .) is a vegetatively<br />
propagatscf species, which gives rise to plantlets at the leaf wargins . the objectives<br />
of this study were to determine the effects of gamma radiation on the regeneration o :<br />
leaves, plant height, general morphology <strong>and</strong> the frequency of chlorophyll mutation@ ir<br />
the VM2 generation of Kalanchoe . Leaves from plants propagated from a single plant war : .<br />
washed, dabbed dry <strong>and</strong> were subjected to different doses of radiation . One set was<br />
exposed to 1000r per six minutes <strong>and</strong> the rest at doses of 2000r to 6000r per hour .<br />
The biological effects of gamma radiation on this plant were studied using different<br />
criteria such as percentage of reganuration per leaf, plant height, types <strong>and</strong> fraquenc7•<br />
of chlorophyll mutations . Percentage of regeneration was markedly reduced at 2000r .<br />
Plnntlet growth was also decreased . Reduction increased linearly with increasing radiation<br />
dose . Chlorophyll mutant plantlets obtained were dark green (viridis),yallow-green<br />
50869 3630
1989 EMS Abstracts<br />
(chlorina), yellow (xantha), <strong>and</strong> white (albina) . Changes in number of shoots <strong>and</strong> ir Notes<br />
ieaf features such as abnormal serratione <strong>and</strong> cordate apices were seen . Somatic an,<br />
Renetic abnormallties were induced by gamma irradiation of Kalanchoe leaves at doser<br />
of 1000 per six minutes <strong>and</strong> 2000r to 6000r per hour . The greatest frequency of chlo<br />
rophyll mutations was recorded at moderate doses of 3000r <strong>and</strong> 4000r . Kalanchoe is sen<br />
3ltive to radiation <strong>and</strong> it is seen in the reduction of regenaration decreased plan' .<br />
qrowth, presence of morphological abnormalities which all increased linearly with in•<br />
creasing dose <strong>and</strong> the presense of chlorophyll mutati .one .<br />
342<br />
TERATOTOGENIC AND GENOTOXIC EFFECTS OF ETHYLENE GLYCOL (EG)<br />
Yin Longzhan, Liu Zheng, Shi Lihua, Bo Kemin .<br />
Shenyang Res . Institute of Industrial Hygiene <strong>and</strong> Occupational Disease,<br />
Shenyang, P .R CHINA<br />
Ethylene glycol was administered to pregnant Wistar rats during 10 days<br />
from the 6th to 15th day orally by a stomach tube at dose levels of 253,<br />
638, 858, 1073, <strong>and</strong> 1595mg/Kg . The fetuses' mean body weight <strong>and</strong> crownrump<br />
length in the 858-1595mg/Kg groups were significantly less than the<br />
control group (p 0 .01) . 1 .8-43 .6% of fetuses among these groups presented<br />
gastroschisis, exencephalia, meningoencephalocele, harelip <strong>and</strong> rib malformation<br />
. Malformation frequencies (mainly bastroschisis) showed a doseresponse<br />
relationship . 638mg/Kg was non-teratogenetic . 858mg/Kg was<br />
threshold teratogenetic <strong>and</strong> 1073mg/Kg was distinctly teratogenetic .<br />
We employed subcutaneous implantation of Agar-coated BrdU tablets in ICR/<br />
JCL mouse for SCE analysis . The results showed no significant difference<br />
in bone marrow cells SCE between control <strong>and</strong> EG groups treated with<br />
1/50 LD50- 1/5 LD50 of EG (EC LD50m7 .26g/KG . No significant difference<br />
in chromosome aberation in mouse bone-marrow cells was found between<br />
control <strong>and</strong> EG groups, when doses of 253-1595mg/Kg were used . The frequencies<br />
of micronuclei in "Kun-Ming" mouse palychromatic erythrocytes<br />
showed no significant difference between control <strong>and</strong> EG groups, with<br />
253 - 1595mg/Kg doses . At 5-500 ug/plate doses with salmonella typhirium<br />
TA98 <strong>and</strong> TA 100, no significant mutation increase was found .<br />
343<br />
EVIDENCE FOR A PCN-P450 ENZYME IN CHICKENS AND COMPARISON OF ITS DEVELOPMENf TO THAT<br />
OF OTHER PB-INDUCIBLE FORMS . N .A . Lorr, S .E . Bloom, S .S . Park, H .V . Gelboin . H .<br />
Miller, <strong>and</strong> F .K . Friedman . Cornell University . Ithaca, NY (USA) <strong>and</strong> National Cancer<br />
Institute, Bethesda, MD 20205 (USA)<br />
The sensitivity of a developing organism to mutagens is dependent in part on the<br />
state of development of the cytochrome P450 enzyme system . In previous studies, we<br />
have shown that the chicken embryo is capable of activating a wide spectrum of xenobiotics<br />
to DNA damaging metabolites . In order to compare the chicken P450 system to<br />
that of the rat, 4 monoclonal antibodies (MAbs) to purified rat liver P450s, including<br />
those from 3-methylcholanthrene, phenobarbital (PB), ethanol, <strong>and</strong> pregnenolone-<br />
16-a-carbonitrile (PCN) treated rats, were used to lmmunodetect proteins in chicken<br />
liver microsomes after blotting from SDS-PAGE . Only the MAb against PCN-inducible<br />
P450 reacted with chicken liver microsomes . The immunodetected protein was most<br />
predominant at 1 day posthatch (DPH) while such lower levels were observed in the<br />
embryo <strong>and</strong> at 36 DPH . PB <strong>and</strong> dexamethasone were both effective inducers of this<br />
protein . Chicken liver microsomal erythromycin demethylase, a characteristic activity<br />
of rat PCN-inducible P450, had a similar developmental profile <strong>and</strong> inducibility<br />
to that of the immunodetected protein with a high degree of augmentation at 1 DPH<br />
compared to that in the embryo end at 36 DPH while aldrin epoxldase, <strong>and</strong> benzphetamine,<br />
ethylmorphine, <strong>and</strong> eminopyrine demethylase were more similar to each other in<br />
development <strong>and</strong> induction <strong>and</strong> were less well correlated with the immunodetected<br />
protein . This evidence suggests the presence in chicken liver of at least two types<br />
of P450, one a form related to the PCN-inducible P450 family . This result agrees with<br />
sequence information suggesting the early evolution of this form . (NIEHS ES03499)<br />
344<br />
SYNTHESIS OF [2-14C]3-CHLORO-4-(DICHLOROMETHYL)-5-HYDROXY-2(5H)-<br />
FURANONE (MX) .<br />
S . Ltltjtlnen, A . Eakelinen, P. Kauranen, T . Vartiainen , Dept . of<br />
Chemistry, Univ . of Kuopio, P .O .B . 6, SF-70211 Kuopio, Finl<strong>and</strong> <strong>and</strong><br />
*National Public Health Institute, Dept . of Environ. Hygiene <strong>and</strong><br />
Toxicology, P .O .B . 95, SF-70701 Kuopio, Finl<strong>and</strong> .<br />
It is well documented that MX is a significant contributor to the<br />
mutagenity of drinking water . To evaluate the potential health risks<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
,<br />
Ln<br />
m<br />
OD<br />
~<br />
10<br />
W<br />
41<br />
W<br />
Ir<br />
119
120 1989 EMS Abstracts<br />
Notes associated with the drinking water consumption, investigations of<br />
the toxicological properties of MX are necessary . We now report a<br />
convenient method for the preparation of 14C labelled MX needed in<br />
those investigations . Bromo[1-14 C] aoetic acid (Ameraham) was first<br />
converted to its methyl ester by general procedure with diazomethane<br />
. Reaction of methyl bromoacetate with triphenylphosphine <strong>and</strong><br />
treatment of the adduct with base gave 14C labelled carbomethoxymethylenetriphenylphosFhorane<br />
. Starting from this compound <strong>and</strong><br />
tetrachloroacetone [2-1 C] MX was then obtained in five steps using<br />
the method of Padmapriya et al .l . The total yield was about 10 ~ .<br />
The method is also suitable for the synthesis of (2-13C] MX .<br />
REFERENCE : 1 . A .A : Padmapriya, G . Just <strong>and</strong> N .G . Lewis . Can . J . Chem .<br />
63 (1985) 828 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
345<br />
TRADESCANTIA-MICRONUCLEUS TESTS ON CLASTOGENS AND IN SITU MONITORING, Te-Esiu Ma,<br />
Institute for <strong>Environmental</strong> Management <strong>and</strong> Department of Biological Sciences, Western<br />
Illinois University, Macomb, IL 61455 USA<br />
Tradesoantia-Mioronucleus (Trad-MCN) bioassay is a highly sensitive test for<br />
chemical clastogens of gaseous or liquid forms <strong>and</strong> nuclear radiatlon . Its high<br />
sensitivity is attributed to the high degree of synchrony <strong>and</strong> fragility of the meiotio<br />
chromosomes which are the targets of exposure . Quantitative data of chromosome damage<br />
in terms of micronuclei frequency can be obtained from the synohronised tetrads (4cell<br />
stage of the meiotic pollen mother cells) 36-48 hr after the exposure . The<br />
microslides were prepared using the aoeto-carmine squash method . Since the<br />
establishment of this bioassay, about 130 chemicals were tested . These include<br />
carcinogene <strong>and</strong> mutagens, common beverages, common toxic compounds, non-prescription<br />
drugs, house-hold chemicals <strong>and</strong> radioisotopee . Among these agents, 50% of them gave<br />
positive responses at relatively low dosages (uM - mM) . Trad-MCN test is an unique<br />
monitoring bioassay which can detect olastogens in the air on the site of pollution<br />
without using the air condensates, <strong>and</strong> olaetogens in the polluted water directly<br />
without using the sediments or concentrates . More than 30 different air pllution<br />
sites <strong>and</strong> 20 sites of polluted water sources were ∎onitored in the US, Canada, Mexico<br />
<strong>and</strong> People's Republic of China . Both surface water <strong>and</strong> ground water sources yielded<br />
seasonal positive responses when tested through the year, Brackish water <strong>and</strong> diluted<br />
eeawater samples from the polluted sites were all positive as compared with the<br />
oontrol group .<br />
346<br />
PROFICIENCY OF TRE T~tAD-NCN 1MAGE ANI~SSIS BYST~1 FOR SCZORING MCN FfjEQ>~ENCY AND DATA<br />
PROCESSING, T . H . Ma , J . Zu , W . Iia , I . Jong , W . Sun <strong>and</strong> G . Lin-, Institute for<br />
<strong>Environmental</strong> Management <strong>and</strong> Depe~tment of Biological Sciences, Western Illinois<br />
Oniveraitu, Macomb, IL 61455 USA, 'Department of Computer Science, Fudan University,<br />
Shanghai, PRC, <strong>and</strong> 3Institute of Oceanology, Academia Sinioa, Qingdao, PRC .<br />
Tradeecantia-Micronucleus (Trad-MCN) bioassay is an efficient short term test for<br />
genotoxioity of pollutants . In order to increase the efficiency <strong>and</strong> to st<strong>and</strong>ardize the<br />
MCN scoring process, an automated scoring system was developed using the principle of<br />
image analysis in computer science . This assemblage is called the Tradescantia-<br />
Micronucleus Image Analysis (Trad-MCNIA) System ." Results scored by Trad-MCNIA system<br />
was compared with that scored by human observer for its proficiency, i .e . the accuracy<br />
<strong>and</strong> speed . A set of low MCN frequency (lese than 10 MCN/100 tetrads) slides prepared<br />
from EMS treated materials <strong>and</strong> a set of high MCN frequency (more than 50 MCN/100<br />
tetrads) slide prepared from X-ray treated materials were used for this study . In low<br />
frequency slides, The Trad-MCNIA system scored about the same value ae human<br />
observers . In high frequency slides, MON frequencies scored by the System was lower<br />
than that scored by human observers . This desorepanoy was corrected by increasing the<br />
power of the objective of the microscope in the Trad-MCNIA System from 10 X to 20 I .<br />
The MCN frequencies scored by the System was about 90x of that scored by human<br />
observer at the present time . The scoring speed of the System was about 4 times ae<br />
fast as that of the human observer, <strong>and</strong> the data can be processed <strong>and</strong> statistically<br />
analyzed immediately after scores were recorded . Further improvement can be made by<br />
upgrading the video camera <strong>and</strong> the computer speed .
347<br />
CLASTOGENICITY OF HEPTACRLOR ON ERYTHROBLASTS OF THE MOUSE, T. $z l,in, J . J . Temenak,<br />
$ . C . Oh, E . Zhou <strong>and</strong> T . D . Chen, Institute for <strong>Environmental</strong> Management <strong>and</strong><br />
Department of Biological Sciences, Western Illinois IIniveraity, Macomb, IL 61455<br />
(USA)<br />
Neptachlor, an agent commonly found in the uncontrolled industrial waste site, was<br />
tested for its clastogenicity using Mouse Peripheral Erythrooyte-MSoronucleus<br />
bioassay . The chemical was first dissolve in ethanol <strong>and</strong> diluted with tapwater to the<br />
final concentrations of 0 .5 <strong>and</strong> 5 .0 uM to feed the mice . Fifteen Balb white mioe were<br />
divided into 3 groups <strong>and</strong> two groups were fed with the heptachlor solution <strong>and</strong> one<br />
group with tapwater as the control . Animals were treated with these chronic low doses<br />
of heptachlor for 3 weeks, with a weekly change of fresh aolutions . Two mice were<br />
died during the second week in the 0 .5 uM treated group, <strong>and</strong> 1 died during the third<br />
week in the 5 .0 uM treated group . Peripheral blood was collected from their tails 7<br />
days after the end of treatment . Two blood smear slides were made from each the the<br />
mice <strong>and</strong> stained with hematoxylin <strong>and</strong> Giemea to reveal mioronuolei in both<br />
polychromatic <strong>and</strong> normochromatio erythrocytes . Mioronuclei (MCN) frequencies were<br />
scored from 10,000 cells in each elide . Preliminary results show that the means <strong>and</strong><br />
the st<strong>and</strong>ard errors of the control group , 0 .5 aM <strong>and</strong> 5 .0 uld treated groups were around<br />
0 .7 (+0 .3) ; 1 .7 (+0 .4) ; <strong>and</strong> 1 .4 (+0 .1) MCM/1000 cells respectively. This indicates<br />
that heptachlor is toxic <strong>and</strong> also exhibits clastogenicity to the ohromoeomes of<br />
erythroblasts of the mouse .<br />
348<br />
MICRONUCLEATED ERYTHROCYTES AS A MARKER OF CYTOGENETIC DAMAGE IN MAN . J .T . MacGreaor,<br />
Department of Biochemistry, University of California, Berkeley, CA (USA)<br />
Splenectomized individuals constitute a unique population for studies of chromosomal<br />
damage in man . Because micronucleated erythrocytes derived from damaged erythroblasts<br />
remain in peripheral blood with a lifespan approximately equal to that of normal<br />
erythrocytes, the incidence of micronucleated cells in peripheral blood can be used as an<br />
index of chromosomal damage in nucleated precursor cells . The target cell population is<br />
rapidly dividing in vivo, <strong>and</strong> therefore is sensitive to agents <strong>and</strong> conditiohs which act<br />
during cell division . This is a significant advantage for studies of conditions such as<br />
transient nutritional imbalances, to which nondividing cells (e .g .,lymphocytes) are<br />
relatively insensitive . Two distinct age populations of erythrocytes can be monitored : 1)<br />
newly-formed erythrocytes, identified by their RNA-positive staining characteristic, which<br />
reflect damage occurring 4-6 days prior to sampling, <strong>and</strong> 2) RNA-negative erythrocytes,<br />
which reflect the average damage over the 4-month period prior to sampling (corresponding<br />
to the turnover time of the mature erythrocyte pool) . Studies to date have established that<br />
micronucleated cell frequencies increase dramatically following treatment with clastogenic<br />
drugs, <strong>and</strong> that the kinetics of the observed increases in erythrocyte subclasses parallel<br />
the kinetics of erythrocyte formation <strong>and</strong> turnover . Studies of environmental factors<br />
associated with modification of the spontaneous frequency of micronucleated cells have<br />
established significant associations between observed frequencies <strong>and</strong> caffeinated beverage<br />
consumption, folate status, calcium supplement consumption in women, <strong>and</strong> consumption of<br />
antioxidant vitamin supplements . Age was strongly associated with the micronucleated cell<br />
frequency when other factors were not considered, but not when adjusted for the above<br />
factors .<br />
349<br />
AMPLIFICATION OF mRNA OF THE HPRT GENE FROM LYSATES OF MUTANT HUMAN CELLS AND DIRECT<br />
DNA SEQUENCING TO DETERMINE THE SPECTRA OF MUTATIONS INDUCED BY CARCINOGENS . V .M.<br />
Maher, J .-L . Yang, <strong>and</strong> J .J . McCormick, Carcinogenesis Laboratory, Michigan State<br />
University, East Lansing, MI 48824 (USA)<br />
Strong evidence points to mutation induction as one mechanism by which changes<br />
are introduced into normal cells to convert them into cancer cells . To underst<strong>and</strong><br />
the mechanisms by which mutations are induced in human cells by carcinogens, we<br />
are determining the kinds <strong>and</strong> spectra of mutations induced in the coding region<br />
of the hypoxanthine(guanine)phosphoribosyltransferase (HPRT) gene . This region,<br />
composed of 654 bp, represents nine exons from a 44 kbp gene . To be able to analyze<br />
a large number of independent mutants rapidly <strong>and</strong> economically, we have optimized<br />
the conditions for copying mRNA directly from lysates of a small number of cells<br />
(e .g ., 200) from a thioguanine resistant clone using reverse transcriptase <strong>and</strong><br />
oligo(dT)1P_18 primers . Then two 20-mer primers, specific for the cDNA of the<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 121<br />
Notes
122 1989 EMS Abstracts<br />
Notes HPRT gene, are used to amplify the first <strong>and</strong> second str<strong>and</strong> cDNA 5 x 107 fold during<br />
30 cycles of polymerase chain reaction (PCR) . The product (2 to 10 ng) is then<br />
purified by ultrafiltration, diluted 1 :1000, <strong>and</strong> subjected to an additional 30<br />
cycles of PCR, using two 20-mer primers located just interior to the first set .<br />
The product (-10 ug) is then sequenced directly using three end-labeled sequencing<br />
primers <strong>and</strong> Sequenase . With this system, we have sequenced 26 BPDE-induced mutants<br />
<strong>and</strong> found that 24/26 involved base substitutions . 97% of these involved G•C,<br />
predominantly G-C--*T•A, distributed throughout the 9 exons but with many located<br />
in exon 3 . (Supported by NCI Grant CA21253 .)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
I<br />
350<br />
ANEUPLOIDY FREQUENCIES IN MOUSE METAPHASE II OOCYTES AND FIRST-CLEAVAGE ZYGOTES<br />
FOLLOWING ORAL DOSAGES OF COLCHICINE . John B . Mailheal, Zhi Ping Yuanl, <strong>and</strong> M .J .<br />
Aardema2 . lDepartment of Obstetrics <strong>and</strong> Gynecology, L .S .U . Medical Center,<br />
Shreveport, LA 71130 ; 2The Proctor <strong>and</strong> Gamble Company, Cincinnati, OH 45247 .<br />
Certain chemicals can interact with cellular organelles responsible for orderly<br />
chromosome segregation <strong>and</strong> enhance the frequency of aneuploidy . Our objective was to<br />
determine the proportion of colchicine-induced aneuploid metaphase II (MII) oocytes<br />
that were transmitted to first-cleavage (lCl) zygotes . Female, CD-1 mice received 7 .5<br />
I .U . PMS <strong>and</strong> 5 .0 I .U . HCG was given 48 h later . Colchicine (2, 3, or 4 mg/kg) or<br />
sterile distilled water (solvent) was given by oral intubation iamnediately following<br />
HCG . MII oocytes were collected 16 h post HCG, whereas 1C1 zygotes were collected 16<br />
h post mating . The proportions (<strong>and</strong> percentages) of hyperploid MII oocytes were 3/216<br />
(1 .4), 81/512 (15 .8), 71/411 (17 .3), <strong>and</strong> 98/413 (23 .7) for control, 2 .0 mg/kg, 3 .0<br />
mg/kg, <strong>and</strong> 4 .0 mg/kg, respectively . Whereas, the proportions of hyperploid 1C1<br />
zygotes were 2/320 (0 .6), 23/364 (6 .3), 68/372 (18 .3), <strong>and</strong> 98/337 (29 .1) for control,<br />
2 .0 mg/kg, 3 .0 mg/kg, <strong>and</strong> 4 .0 mg/kg, respectively . The proportions of hyperploid<br />
cells differed (P0 .05) between MII oocytes <strong>and</strong> lCl zygotes in controls, <strong>and</strong> the 3 .0 mg/kg <strong>and</strong> 4 .0<br />
mg/kg groups . •the level of hyperploidy was greater (P
1989 EMS Abstracts 123<br />
nucleation, micronucleation, chromosome structure or number . To further define the Notes<br />
action of chrysotile asbestos upon human cells, asbestos effects were examined in<br />
cultures of the human lung tumor cell lines A549, Calu-1, KNS62, <strong>and</strong> A1188 . Asbesto3<br />
induced 50% <strong>and</strong> 100% cytostasis of HBE cells at treatment concentrations of til ug/cm<br />
<strong>and</strong> 4 yg/cm , respectively . In contrast, three to thirty-fold higher concentrations<br />
of asbestos were required to inhibit tumor cell growth . Maximal growth inhibition was<br />
cell line dependent <strong>and</strong> ranged from only 20% to 50% . Asbestos produced only two-fold<br />
elevations of binucleation (spontaneous binucleation rate '% .2X) in HBE, KNS62, <strong>and</strong><br />
A1188 cells . In contrast, dose-dependent elevations of binucleation (up to 15-fold)<br />
were observed in cultures of A549 <strong>and</strong> Calu-1 . These results are consistent with observations<br />
that asbestos amplifies the effects of human lung carcinogens such as<br />
cigarette smoke <strong>and</strong> suggest that this synergy may in part be mediated by 1) asbestosinduced<br />
inhibition of HBE cell growth that permits clonal expansion of pre-existing<br />
carcinogen-induced lesions <strong>and</strong>/or 2) enhanced susceptibility of some of these lesions<br />
to asbestos-induced cellular events that facilitate neoplastic progression .<br />
353<br />
ISSUES IN THE EVALUATION OF SHORT-TERM TEST PERFORMANCE AND TESTING STRATEGIES .<br />
Barry H . Margolin, University of North Carolina at Chapel Hill, Chapel Hill, NC .<br />
The formal definitions of statistical independence <strong>and</strong> dependence are reviewed<br />
within the context of construction of batteries of genetic toxicity assays for<br />
prediction of carcinogenicity . The existing empirical evidence in support of independence<br />
or dependence among assays is examined <strong>and</strong> the impact of statistical dependence<br />
on carcinogenicity prediction systems is explored in depth . Finally, carcinogenicity<br />
screening policies based upon short-term tests are reviewed <strong>and</strong> studied for their<br />
sensitivity to assumptions or inferences regarding statistical independence or dependence<br />
among tests .<br />
354<br />
HUMAN SPERM CHROMOSOME COMPLEMENTS, EFFECTS OF DONOR AGE, FREEZING AND SEGREGATION<br />
IN TRANSLOCATION AND INVERSION CARRIERS . Renee H . Martin, Division of Medical<br />
Genetics, Department of Pediatrics, University of Calgary <strong>and</strong> Medical Genetics<br />
Clinic, Alberta Children's Hospital, Calgary, Alberta, Cpnada T2T 5C7<br />
We have studied the effect of age on the frequency of sperm chromosomal<br />
abnormalities in 30 normal donors, stratified by age into 6 age groups (20-24,<br />
25-29, 30-34, 35-39, 40-44, 45+) . Data from newborns had suggested a possible<br />
increased risk of Down syndrome with paternal age but we found no inereased risk<br />
of disomic sperm with advanced patornal age . In contrast, the frequency of<br />
hyperhaploid (n+l) sperm decreased while the frequency of sperm with structural<br />
chromosomal abnormalities increased with age . To assess the effects of sperm<br />
cryopreservation, ejaculates from 10 normal men were split <strong>and</strong> studied pre- <strong>and</strong><br />
post-freezing . A minimum of 100 sperm karyotypes were studied for each donor .<br />
There was no significant difference in the frequency of numerical chromosomal<br />
abnormalities (using a conservative estimate of aneuploidy, 2 x hyperhaploid sperm)<br />
or structural chromosomal abnormalities before <strong>and</strong> after freezing . There was no<br />
evidence for donor heterogeneity . The sex ratios were also not, affected by<br />
cryopreservation <strong>and</strong> did not differ significantly from 50% . Studies on 23 men<br />
with constitutional chromosomal abnormalities have demonstrated dramatic variations<br />
in the frequency of chromosomally unbalanced sperm from 0% to 77% . This information<br />
is useful in elucidating basic principles of how rearranged chromosomes segregate<br />
during meiosis <strong>and</strong> also in providing more accurate genetic counselling for these<br />
men .<br />
355<br />
MUTAGENICITY OF NITROARENES BY NEW TESTER STRAINS, TA100/PY0216, TA100/PY0219, TA98/PYO<br />
216 AND TA98/PYG219 . H . Matsushita, 0 . Endo, H . Katsushita,Jr ., M . Kochizuki, M .<br />
vatanabe, <strong>and</strong> M . Ishidate, Jr ., National Institute of Public Health, Kinato-ku, Tokyo,<br />
(Japan), Eyoriteu College of Pharmacy, Minato-ku, Tokyo (Japan), <strong>and</strong> National Institute<br />
of Hygienic Sciences, Setagaya-ku, Tokyo (Japan) . Mutagenicity of nitroarenes, potent<br />
environmental mutagene, was tested by new tester strains, Salmonella typhimurium TA<br />
100/PYG216, TA100/PYG219, TA98/PYG216 <strong>and</strong> TA98/PY0219 as well as the parent strains,<br />
where TA100/PYG216 <strong>and</strong> TA98/PYG216 have nitroreductase activity <strong>and</strong> TA100/PY0219 <strong>and</strong> TA<br />
98/PYG219 have acetyltraneferase activity . Mutagenicity test was carried out by the<br />
pre-incubation method in the presence <strong>and</strong> absence of S9 mix . Nitroarenes tested were<br />
21 nitro-derivatives of benzene, biphenyl, naphthalene, anthracene, fluorene <strong>and</strong> pyrene,<br />
in which 8 dinitropyrenes were included . The mutagenic activity was generally higher<br />
in the absence of S9 mix than in the presence of S9 mix for all ∎trains tested . Mutagenic<br />
activity of each nitroarene was generally in the order of TA100/PYG219, TA100/PYG<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
124 1989 EMS Abstracts<br />
Notes 216 <strong>and</strong> TA100 irrespective of presence <strong>and</strong> absence of 89 ∎ix . The activity for TA300/<br />
PY0219 was about 10 to 600 times higher than TA100 in the abeence of S9 mix . In the<br />
case of TA98 strains, the order of mutagenic activity was complicated, but TA98/PYG216<br />
or 219 gave generally higher mutagenic activity than TA98 . For example, ratio of mutagenic<br />
activity for TA98/PYG219 to that for TA98 ranged from 245 to 630 (mean :430) for<br />
3 kinds of mono-nitropyrenes, from 6 to 370 (mean : 78) for 8 dinitropyrenes in the<br />
absence of S9 mix . The ratio between TA98/PY0216 <strong>and</strong> TA98 ranged from 130 to 590 (mean :<br />
350) for 3 nitropyrenes <strong>and</strong> from 0 .1 to 89 (mean : 18) for 8 dinitropyrenes in the absence<br />
of S9 mix . These results demonstrate clearly the usefulness of theme strains for<br />
the detection of nitroarenes in the environment .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
MODIFICATION OP SALMONELLA MUTATION TEST AND ITS APPLICATION TO ALKYL HYDRAZINES<br />
H . Natsushita,Jr ., K . Yamamoto, M . Mochizuki, O .Endo, <strong>and</strong> H . Matsushita, IKyoritsu<br />
College of Pharmacy, Minato-ku, Tokyo (Japan), <strong>and</strong> National Institute of Public<br />
Health, Minato-ku, Tokyo (Japan) .<br />
356<br />
Many environmental hydrazines are carcinogenic . However, information on their mutagenicity<br />
is few <strong>and</strong> the mutagenic activity reported 1s generally very low in contrast<br />
of their carcinogenicity . We modified the mutagenicity test procedure mainly on the<br />
pre-culture conditions using Salmonella typhimurium strains TA100 <strong>and</strong> TA102, <strong>and</strong><br />
applied the modified method to the survey of mutagenicity of 12 alkylhydrazines :<br />
four 1,1-dialkylhydrazinea, four 1,2-dlalkylhydrazines <strong>and</strong> four monoalkylhydrazines<br />
with alkyl group from methyl to butyl . In this method, 10 out of 12 alkylhydrazines<br />
were detected as positive in the strain TA100, <strong>and</strong> all the 12 hydrazines were positive<br />
in the strain TA102 . Mutagenic activity obtained here were generally stronger<br />
than those reported hitherto . The mutagenic activity was stronger in the absence of<br />
metabolic activation, <strong>and</strong> the presence of 59 mix reduced remarkably the mutagenic<br />
activity . The inhibition by S9 mix was proved to be due to the capturing of mutagens<br />
by protein of S9, since bovine serum albumin also inhibited the mutagenicity of alkylhydrazines<br />
tested . The procedure developed here is useful in detection of many kinds<br />
of environmental mutagens, <strong>and</strong> also in elucidating mechamisms of mutagenesis <strong>and</strong><br />
carcinogenesis of alkylhydrazines .<br />
COMPARISON OF TWO DIFFERENT PROTOCOLS FOR DETECTION OF CHEMICAL-INDUCED<br />
TRANSFORMATION OF BALB/c-3T3 CELLS . E .J . Matthews, Hazleton Laboratories America,<br />
Inc ., Kensington, Maryl<strong>and</strong> .<br />
In 1983, the NTP furnished this laboratory 55 coded chemicals for testing in a st<strong>and</strong>ard<br />
transformation protocol using the A-31, 1-13 BALB/c-3T3 cells . This investiyation<br />
revealed that the st<strong>and</strong>ard assay was insensitive : 4 chemicals were active, 6 had<br />
limited evidence of activity, <strong>and</strong> 45 were inactive . Recently 61 of these chemicals<br />
were retested in a modified protocol : 26 chemicals were active, 6 had limited evidence<br />
of activity, <strong>and</strong> 20 were inactive . Neither the st<strong>and</strong>ard nor the modified protocol<br />
used an exogenous activation system . The enhanced sensitivity of the second<br />
protocol was attributed to several procedural changes . First, the initiat seeding<br />
density was increased from j to 3 .2 x 104 celts/60 mm dish . Second, the treatment<br />
time was reduced from U to 48-hours <strong>and</strong> treatments were begun dav-2 versus O,av-1<br />
after seeding . Third, the method of measuring chemical cytotoxicity changed from a<br />
st<strong>and</strong>ard clonal survival assay employing M wild type (wt) cells to a co-culture<br />
assay using 3 .2 x 104 wt <strong>and</strong> 100 ouabain-resistant ce11s . Additional assay improvements<br />
will be discussed, including : changing the positive control [3-methylcholanthrene<br />
to benzo(a)pyrene], <strong>and</strong> an alteration of the method of dosing . Finally, many<br />
NTP chemicals had limited solubility in water . This problem was diminished for many<br />
chemicals by dissolving them in an organic solvent at high concentrations <strong>and</strong><br />
diluting them 100-fold into medium supplemented with a SX concentration of Pluronic<br />
F68 (1 .25k w/v) . Investigations were supported by NIEHS Contract N01-ES-65150 .<br />
TRANSFORMATION WITH BALB/c-3T3 CELLS . fv J . Matthews, Hazleton Laboratories America,<br />
Inc ., 5516 Nicholson Lane, Kensington, Maryl<strong>and</strong> .<br />
Chemical-induced transformation of A-31,I-13 BALB/c-3T3 cells was investipated using<br />
a modified procedure . Chemical-induced cytotoxicity was measured using a clonal survival<br />
assay employing co-cultures of 200 ouabain-resistant <strong>and</strong> 3 .2 x 104 wild type<br />
(wt) cells . The transformation assay used vessels seeded with 3 .2 x 104 wt eells<br />
(DAY 0) <strong>and</strong> 48 hour chemical treatments were started on Dav-2 . Chemicals with solubility<br />
problems in water were dissolved at high concentrations in an organic solvent<br />
<strong>and</strong> diluted 100-fold into culture medium supplemented with Pluronic F68 (1 .25k w/v)<br />
50869 3636<br />
357<br />
358
1989 EMS Abstracts<br />
<strong>and</strong> then 5-fold into culture dishes . Cultures were incubated 28-days <strong>and</strong> evaluated Notes<br />
for the presence of Types I-III foci . All experiments were conducted in the sene<br />
of any exogenous activation system . Seventy carcinogens <strong>and</strong> 58 noncareinoaens were<br />
tested in two or more transformation experiments . Carcinogens included 43 chemicals<br />
that were relatively cytotoxic to the BALB/c-3T3 cells (LD < 2 .0mM), <strong>and</strong> 27 noncvtotoxic<br />
chemicals (LOse > 2 .0mM) . Similarly, noncarcinogens included 27 cytotoxic<br />
chemicals <strong>and</strong> 31 noncytotoxic chemicals . All chemicals were evaluated at cytotoxic<br />
treatment doses . Transforming activities of the 128 chemicals will be compared to<br />
their reported structural alerts <strong>and</strong> flenotoxic activities in four in vi r assays :<br />
including Salmonella, mouse lymphoma (TK+/-), <strong>and</strong> Chinese hamster ovary sister chromatid<br />
exchanges <strong>and</strong> chromosomal aberrations . Chemieal-induced activities detected<br />
in the presence <strong>and</strong> absence of an S9 activation system will be discussed separately .<br />
Experimental investigations were supported by NIEHS Contract N01-ES-65150 .<br />
359<br />
TISSUE SPECIFICITY OF THE MUTAGENICITY OF 1,8-DINITROPYRENE IN A MOUSE-MEDIATED<br />
ASSAY . M .A . McCartney, S . Knasmfiller, E .C . McCoy <strong>and</strong> H .S . Rosenkranz, Department of<br />
<strong>Environmental</strong> Health Sciences, School of Medicine, Case Western Reserve University,<br />
Clevel<strong>and</strong>, Ohio 44106 .<br />
1,8-Dinitropyrene (1,8-DNP) is a highly mutagenio environmental contaminant which<br />
is also carcinogenic to rodents . DNA adduct formation, mutagenioity <strong>and</strong> presumably<br />
carcinogenicity are dependent upon nitroreduction to the corresponding<br />
arylhydroxylamine followed by 0-esterification by a transacetylase . Bacteria<br />
(Salmonella typhimuriurn TA98/1,8-DNPs) deficient in this transacetylase do not<br />
exhibit mutagenicity when exposed to 1,8-DNP . In a mouse-mediated assay (MMA) 1,8-DNP<br />
induced mutations in S . typhimurium TA98 recovered from liver <strong>and</strong> spleen of treated<br />
animals . However, when the MMA was performed using S . typhimuriurn TA98/1,8-DNPa 1,8-<br />
DNP-induced mutants could be recovered only from the spleen but not from the liver .<br />
The relevance of these findings to the tissue-specificity of 1,8-DNP will be<br />
addressed .<br />
360<br />
MALIGNANT TRANSFORMATION OF HUMAN FIBROBLASTS BY• ONCOGENE TRANSFECTION OR<br />
CARCINOGENS. J .J . McCormick <strong>and</strong> V .M . Maher, Carcinogenesis Laboratory, Michigan<br />
State University, East Lansing, MI 48824 (USA)<br />
Data indicate that carcinogen exposure is the cause of most human tumors, but<br />
human cells in culture have not been successfully transformed to malignancy by<br />
exposure to chemical carcinogens or radiation . One possible explanation for this<br />
failure is inability to recognize the phenotypes of carcinogen-treated cells that<br />
have undergone intermediate changes, so they can be exp<strong>and</strong>ed <strong>and</strong> exposed a second<br />
time to cause further changes . To Identify possible intermediates, we transfected<br />
diploid human fibroblasts with oncogenes known to be active in cells derived from<br />
fibrosarcomas <strong>and</strong> determined the phenotypes they produced . H- or N-ras oncogenes<br />
flanked by suitable enhancer <strong>and</strong> promoter sequences caused the celfs to acquire<br />
many characteristics of malignant cells, but not to acquire an infinite life span<br />
or form tumors . When we transfected these ras oncogenes in the same constructions,<br />
or a viral K-ras oncogene, into an infinite -Me span, near-diploid, non-tumori genic<br />
cell strain developed in this laboratory (MSU-l .l cells), distinct foci of<br />
morphologically-altered, anchorage independent, <strong>and</strong> growth factor independent cells<br />
were found which formed progressively-growing, invasive malignant sarcomas in athymic<br />
mice . These cells expressed the p2ls of the transfected ras genes . Transfection<br />
of two other infinite life span human cell lines with the~T-ras oncogene in the<br />
same construction also yielded malignant cells . We are currenETy using carcinogen<br />
treatment to activate cellular proto-oncogenes of the MSU-1 .1 cells <strong>and</strong> have<br />
succeeded in malignantly transforming cells . (Supported by DOE Grant 60524, NCI<br />
Grant CA21289 <strong>and</strong> NIEHS Contract ES65152 .)<br />
361<br />
HEPATIC AND LUNG MICROSOMAL METABOLISM OF ENVIRONMENTAL POLLUTANTS : EFFECTS OF !!1<br />
INDUCER PRETREATMENT ON THE METABOLISM OF 1-NITROPYRENE, 3-NITROFLUORANTENE AND m<br />
NICOTINE . 00<br />
G .D . McCoy , D . R . Koop <strong>and</strong> P . C . Howard, Case Western Reserve University, School of<br />
Medicine, Clevel<strong>and</strong>, OH 44106 tp<br />
The ability of microsomes isolated from adult male <strong>and</strong> female New Zeal<strong>and</strong><br />
rabbits to metabolize 1-nitropyrene (i-NP) , 3-nitrofluranthene (3-NF) <strong>and</strong> nicotine W<br />
(N) has been studied . Hepatic <strong>and</strong> lung microsomes were isolated from animals 01<br />
pretreated with either 1-nitropyrene, P-napthoflavone (J)-nf) or phenobarbital (Pb). W<br />
J<br />
The C-oxidation of 1-NP, 3-NF <strong>and</strong> N were significantly increased only in mlcrosomes<br />
from phenobarbital pretreated animals . In contrast, both Pb <strong>and</strong> 0-nf pretreatment<br />
significantly decreased the rates of nicotine N'-oxidation . Our previous studies<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
125
126 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes have shown that reconstitution experiments utilizing purified rabbit cytochrome P-<br />
450 isozymea that all three xenobiotics are preferentially metabolized by isozyme<br />
3b (IIC3) a constitutive form thus far found only in rabbits . The ability of Pb to<br />
induce the metabolism of 1-NP, 3-NF <strong>and</strong> N was also anticipated since Ssozyme 2(II<br />
81) one of the major forms induoed by phenobarbital also exhibited significant<br />
activity with these compounds in reconstitution studies . Signifioant metabolism of<br />
all three xenobiotios occurred in pulmonary miorosomes . Since environmental<br />
exposure to these three xenoblotlos will most likely be via inhalatlon <strong>and</strong> that<br />
common pathways of C-oxidation are involved in their metabolism, significant<br />
interactions between the three can be expected when present concurrently in complex<br />
mixtures . Supported in part by grants ES 03648 <strong>and</strong> AA 07219 .<br />
362<br />
COMPARISON OF-MUTAGENICITY OF TEN CHEMICAL MUTAGENS WITH THE SALMONELLA<br />
(AMES) ASSAY, UMU TEST, AND SOS CHROMOTEST . Audrey E . McDaniels,<br />
Antolin L . Reyes, <strong>and</strong> Larry J . Wymer, U .S . <strong>Environmental</strong> Protection<br />
Agency, <strong>Environmental</strong> Monitoring Systems Laboratory, Microbiology<br />
Research Division, Cincinnati, Ohio 45268 .<br />
With the greater number of environmental samples examined each year<br />
for mutagens, there is an increased need for methods that are less time<br />
consuming yet do not sacrifice sensitivity or reproducibility . Two alternative<br />
methods to the well established Ames test are the Umu test <strong>and</strong><br />
SOS chromotest . These two colorimetric assays are based on production<br />
of B-galactosidase in response to DNA damage . The three methods were compared<br />
in 5 independent experiments . Dose response curves were obtained<br />
using 10 chemical mutagens representing 6 different classes of mutagens .<br />
Sensitivities, expressed as minimum significant doses (MSD), <strong>and</strong> precisions<br />
were also measured . Ames test MSDa ranged from 0 .002 pg to 2 .65 pg<br />
per plate for Salmonella ~ty himuri~um strains TA100 <strong>and</strong> TA98 . SOS chromotest<br />
MSDs rangedom ~0 2 p~ g to 6 . .9 pg per assay, <strong>and</strong> Umu test MSDa<br />
from 0 .005 pg to 0 .39 pg per assay . The range of precisions among the<br />
assays for all mutagens extended from 0 .07 to 0 .19 . The results of this<br />
study indicated the Umu test was either equivalent to or significantly<br />
better than the Ames test in sensitivity <strong>and</strong> precision for all of the<br />
chemicals examined . The SOS chromotest presented toxicity <strong>and</strong> solubility<br />
problems not observed with the Ames <strong>and</strong> Umu tests .<br />
363<br />
CYTOGENETIC EVALUATION OF THE IN VIVO GENOTOEICITY OF THREE QUINOLINE COMPOUNDS<br />
A .F . McFea <strong>and</strong> K .W . Lowe, Oak Ridge Associated Universities, Oak Ridge, TN (USA)<br />
Quinoline-derived compounds are widely used in medicine <strong>and</strong> industry as antiseptics,<br />
solvents, dyes, <strong>and</strong> components of various chemical processes . In vitro assays of their<br />
genotoxicity have yielded varying indications of potency, <strong>and</strong> few studies of their in<br />
vivo activity have been reported . Ye implanted male 86C3F1 aice with BrdU tablets <strong>and</strong><br />
1 hr later gave injections of quinoline or 8-bydroxyquinoline in corn oil solution, or<br />
4-nitroquinoline-l-oxide (4NQ0)dissolved in DMSO . Each was tested at its maximum<br />
tolerated dose (MTD) <strong>and</strong> at 0 .5 <strong>and</strong> 0 .25 MTD . Chromosome aberrations were scored in 50<br />
first-division metaphases from each of 8 mice per dose level at 17 hr post-treatment,<br />
<strong>and</strong> SCEs in 25 second-division metaphases from 4 mice per level at 23 br . Compounds<br />
showing no effect on an endpoint were further tested for aberrations at 36 hr, <strong>and</strong> for<br />
SCEs at 42 hr . Unusually low control values resulted in a significant p-value for<br />
aberration induction by quinoline at 17 hr ; however, a repeat study showed no effect .<br />
Quinoline also showed no effect on aberration levels at 36 hr, no elevation of SCEs at<br />
either 23 or 42 br, <strong>and</strong> no indication of an effect on the rate of csll proliferation .<br />
8-bydroxyquinoline was also without effect on either aberrations or SCEs at any<br />
post-treatment time, although a modest depression of call proliferation rates occurred<br />
at the higher dose levels . 4NQO was a very potent inducer of both aberrations <strong>and</strong><br />
SCEs, causing a significant increase in aberration rate at 30mg/kg, <strong>and</strong> in SCE at<br />
15mg/kg ; a depression of cell proliferation was also evident . Both quinoline <strong>and</strong><br />
8-hydroxyquinoline seea to be without in vivo genotoxic activity, whereas 4NQO retains<br />
a significant potency under in vivo conditions . Supported by NIEHS Interagency<br />
Agreement Y01-ES-20100 <strong>and</strong> DOE/ORAU Contract DE-ACG5-760R00033 .<br />
IN DITRO MAMMALIAN CELL CENOTOXICITY ASSAYS : THEIR USE AND INTERPRETATION .<br />
Douglas McGregor, Dept . of Toxicology <strong>and</strong> Safety Assessment, Soehringer<br />
Ingelheim Pharmaceuticals, Inc ., Ridgefield, CT 06877, USA .<br />
To address the interest in identifying all aspects of genetic toxicity <strong>and</strong> its<br />
relevance to Man, more than 100 different test methods have been developed .<br />
50869 3638<br />
364
Few are commonly used . Mammalian cell assays hold a special position in this<br />
Irategy because of the hroad spectrum of damage that is theoretically<br />
deteccable with them, but which is not acceasable to prokaryotic assays .<br />
Reasons for this privilege include the higher order structure of mammalian<br />
chromosomes, the different repair mechanisms <strong>and</strong> the different chemical <strong>and</strong><br />
metabolic reactions possible in mammalian as compared with prokaryotic cells .<br />
Fundamental to the acceptability or otherwise of mammalian cell assays is<br />
their success in rodent carcinogen prediction, although they also have value<br />
in biochemistry . Their primary function will be discussed <strong>and</strong> it will be<br />
concluded that certain assays have reasonable sensitivity (eg ., mouse lymphoma<br />
cell tk locus <strong>and</strong> sister-chromatid exchange assays), while others have higher<br />
specificity (eg ., hprt locus <strong>and</strong> primary hepatocyte unscheduled DNA synthesis<br />
assays) . Chromosomal abberition assays show moderate sensitivity <strong>and</strong><br />
specificity . These properties of the assays can lead to proposals that some<br />
of them should be discontinued or that, if retained, their use shoul~ be<br />
-d bv scientific <strong>and</strong> ethical objectives .<br />
365<br />
UV-INDUCED CYTOGENETIC DAMAGE IN WILD-TYPE AND THYMIDINE KINASE DEFICIENT FRIEND MOUSE<br />
ERYTHROLEUKAEMIA CELLS . V .J .McKelvey <strong>and</strong> P .G .McKenna, Biomedical Sciencee Research<br />
Centre, University of Ulater, Coleraine BT52 1SA, N .Irel<strong>and</strong> .<br />
Deficiency of the salvage pathway enzyme thymidine kinase (TK) in Friend mouse<br />
erythroleukaemia cells results in increased sensitivity to cell killing <strong>and</strong> mutagenesis<br />
following UV-irradiation (McKenna,P .G . <strong>and</strong> Hickey .I . (1981) Cell Biol .Int .Reps . 5 :555) .<br />
Work with other malignant cell lines indicates that TK deficiency only confers eZevated<br />
sensitivity in those cell lines which are normally proficient in DNA excision repair as<br />
evidenced by their ability to undergo unscheduled DNA synthesis (UDS) (McKenna,P .G .,<br />
Yasseen,A,A . <strong>and</strong> McKelvey,V .J . (1985) Somat .Cell Mo1 .Genet . 11 :239) . Further work has<br />
shown that TK deficiency in Friend cells does not inhibit UDS dccurring (McKenna,P .G .<br />
<strong>and</strong> McKelvey,V .J . (1986) Somat .Cell Mol .Genet . 12 :325) . In this study vild-type clone<br />
707 Friend cells <strong>and</strong> two TK deficient eubclonea 707BUE <strong>and</strong> 707BUF, having TK activities<br />
of 1 .4% <strong>and</strong> 0 .7% that of wild-type cells respectively, were examined for cytogenetic<br />
aberrations following UV-irradiation . Three doses of UY light were used, namely 2 .4,<br />
4 .8 <strong>and</strong> 7 .2 J/m <strong>and</strong> UV-irradiated cultures were harvested for chromosome spreads at 15<br />
hours following treatment . Fifty r<strong>and</strong>omly selected chromosome spreads per treated <strong>and</strong><br />
control culture were scored for thirteen types of cytogenetic aberrations . The<br />
frequency of very severely damaged (pulverised) cells was observed to be considerably<br />
greater in each of the two TK deficient aubclones,707BUE <strong>and</strong> 7078UF, relative to wildtype<br />
clone 707 cells, for each UV treatment examined . Increased UV-aensitivity in the<br />
TK deficient subclones was also reflected in the total aberration frequencies exhibited<br />
by the 3 cell types . The imp rtance of thymidine kinase for accurate DNA repair of UVinduced<br />
damage ia indicated .(The support of the Ulster Cancer Foundation is acknowledge40<br />
366<br />
METABOLISM OF FOOD MUTAGENS WITH PURIFIED AND cDNAl EXPRESSED CSCfOCMROMES P-450 . M+<br />
McManus, W .M . Byrgess, M .E . Ver3nese, J .S . Felton , M .G . Rnize , E .G . Snyderwine ,<br />
L .C . Quattrochi <strong>and</strong> R .Y . Tukey . Department of Clinical Pharmacology, 2Flinders<br />
University, Australia, Lawre3ce Livermore National Laboratory U .S .A ., National<br />
Institute of Health, U .S .A ., & University of California, San Diego, U .S .A .<br />
We have investigated the specificity of six purified forms of rabbit liver cytochrome<br />
P-450 to activate the food derived haterocyclic amines . IQ <strong>and</strong> PhIP, to mutagens<br />
in the Ames test . The polcyclic hydrocarbon inducible isozymes Forms 4 <strong>and</strong> 6<br />
were efficient activators of both these compounds whereas, Forms 2,3b,3c <strong>and</strong> 5 were<br />
inactive in metabolizing IQ <strong>and</strong> PhIP to mutagens . The number of revertants produced<br />
in the Ames test per 10 pmol of Form 4 with IQ <strong>and</strong> PhIP as substrates were 136, 160<br />
<strong>and</strong> 1,521, respectively . In the presence of 10 pmol of Form 6 <strong>and</strong> IQ as substrate<br />
16,000 revertants were obtained, whereas PhIP gave 4,577 revertants . When MeIQ, MeIQx<br />
<strong>and</strong> DiMeIQx were used as substrates Form 4 was at least 3-fold more efficient than<br />
Form 6 in activating these compounds . The gene of the human equivalent of the rabbit<br />
cytochrome P-450 Form 6(P450IA1) <strong>and</strong> a human cDNA equivalent to Form 4(P450IA2) have<br />
been expressed in Coa-1 cells <strong>and</strong> their capacity to activate the heterocyclic amines<br />
IQ, PhIP,MeIQ,MeIQx <strong>and</strong> DiMeIQx was determined . Both the human P4SOIA1 <strong>and</strong> P450IA2<br />
were capable of activating these amines to mutagens . The order of mutagenicity using<br />
either P450IA1 or P450IA2 expressed cell lysates as the activation source were MeIQx ><br />
IQ > DiMeIQx > MeIQx > PhIP, respectively . The IC50's for a-naphthoflavone inhibition<br />
of the expressed human8P450IA1 <strong>and</strong> P450IA2 activities with IQ as a substrate in the<br />
Ames test were 2 x 10 <strong>and</strong> 2 x 10- N, respectively . These data indicate that the<br />
human P4501A subfamily is functionally similar to its rabbit counterpart .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 127<br />
Notes
128 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
367<br />
DHYDRO7CY-2(5H)~F~gT~Ip ONE (MX ~FJ I~R ~Na ier`, A .ID . CDsAna 1o13-FH B .RDaniel1C K~~Schenck1,<br />
M . F . Ske~ly2 <strong>and</strong> S . L . HuangZ lU .S . <strong>Environmental</strong> Protection Agency, Cincinnati,<br />
OH 45268, <strong>Environmental</strong> Health Research <strong>and</strong> Testing, Cincinnati, OH 45245 NC 27709 .<br />
MX is a potent bacterial mutagen <strong>and</strong> mammalian cell clastogen that forms in drinking<br />
water during water chlorination . Concern over potential health hazards stems from the<br />
finding that !DC is a major contributor to the mutagenic activity of drinking water samples .<br />
The present work was done to obtain preliminary information on the nature of the DNA<br />
damage which accounts for the potent genotoxic activity of MX . DNA adduct formation<br />
was examined in Salmonella tyohimurium TA100 cells, primary rat hepatocytes, nd in a<br />
rat liver embryonic cell line (Clone 9) . DNA adducts were anal~zed by the 3~P-postlabeling<br />
method of R<strong>and</strong>erath <strong>and</strong> Gupta . Mutation frequency (his revertants) was also<br />
determined for the TA100 cells . The Salmonella cells were exposed to !D( concentrations<br />
of 0, 1 <strong>and</strong> 3 pg/ml for 30 min . at 37°C whereas the mammalian cells were exposed for 6<br />
hr to concentrations of 0, 1, 5, 10 <strong>and</strong> 50 pg/ml . Mutation induction was linear over<br />
this dose range in the Salmonelia cells, whereas higher concentrations were toxic . The<br />
mutation frequency was 1 x 10- per pg/al . A dose-dependent increase in DNA adduct<br />
formation was observed for all three cell types . In each case a single major adduct<br />
appeared to be formed . The levels of #dducts at equivalent doses were similar in the<br />
two mammalian cell types (ca . 2 per 10 DNA bases at the 10 pg/al dose) . A comparable<br />
adduct level was observed at 1 yg/ml in the Salmonella calls . Further work to characterize<br />
the DNA adduct formed by !IX is needed to elucidate the role of this lesion in<br />
the genotoxic action of this compound . (This abstract does not necessarily reflect EPA<br />
policy) .<br />
368<br />
MUTAGENS IN CHLORINATED WATER . J .R . Meier, Health Effects Research Laboratory,<br />
U .S . <strong>Environmental</strong> Protection Agency, Cincinnati, OH 45268<br />
Over the past decade, substantial evidence has accumulated to show the widespread<br />
presence of genotoxins in drinking water . The sources of genotoxic contaminants can<br />
be generally classified into three groups ; contaminants of the raw water, chemicals<br />
added or formed during water treatment, <strong>and</strong> chemicals formed or unintentionally added<br />
during distribution . In many cases, the genotoxic activity can be directly attributed<br />
to the chlorination stage of water treatment . The genotoxic activity appears to originate<br />
primarily from reactions of chlorine with humic substances in the source waters .<br />
Cenotoxic activity in drinking water concentrates has been most frequently demonstrated<br />
using bacterial mutagenicity tests but results with mammalian cell assays are generally<br />
consistent with the findings from bacterial assays . There is currently no evidence<br />
for genotoxic damage following in vivo exposure, although little work has been done in<br />
this area . Organic acids appear to account for most of the bacterial mutagenicity <strong>and</strong><br />
recovery of these compounds from water requires a sample acidification step prior to<br />
extraction . Recently, one class of acid cospounds, the chlorinated hydroxyfuranones,<br />
was found to be responsible for a major part of the mutagenic activity . Approaches<br />
for drinking water treatment aimed at reduction of genotoxins in drinking water include<br />
granular activated carbon (GAC) filtration, chemical destruction, <strong>and</strong> the use of alternative<br />
means of disinfection (i .e ., ozone, chlorine dioxide, <strong>and</strong> monochloroamine) .<br />
The question of how best to minimize exposure to genotoxins in drinking water while<br />
maintaining a microbiologically safe water remains to be resolved . (This abstract<br />
does not necessarily reflect EPA policy) .<br />
369<br />
HERITABLE VARIATION IN THE RESPONSE OF A CLINICALLY NORIIAL, AU!tAN POPULATION TO ION-<br />
IZING RADIATION . T . Merz, D .Y . Harrison, L .A . Corey, Medical College of Virginia,<br />
Virginia Commonwealth University, Richmond, VA (USA)<br />
This is a study of the inheritance of variability in the response of clinically<br />
normal individuals to ionizing radiation . The micronucleus assay is used to measure<br />
response <strong>and</strong> since micronuclei frequencies are dependent on cell proliferation, cell<br />
growth kinetics are also considered . Then twin method is used to determine whether<br />
there is a heritable component of variation in the response of cells from clinically<br />
normal individuals . Ten pairs of monozygotic twins were examined for their responses<br />
to radiation . The variation of the response of twins within a pair is compared<br />
to the variation between pairs of twins . An analysis of variance does indlcate<br />
that there is considerable variation in observed micronuclei frequency . Nost<br />
of the variability can be accounted for by the differences between twin pairs . The<br />
large interpair variation compared to the intrapair variation demonstrates that<br />
twin micronuclei production is more alike (correlation of 0 .92) than non-twins . It<br />
is suggestive of a genetic influence on mlcronuclel production .
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370 1989 EMS Abstracts<br />
MOLECULAR PATTERNS OF APRT GENE REARRANGEMENTS . M . MEUTH, G . SARGENT, C. MILES, AND Notes<br />
G . PHEAR, IMPERIAL CANCER RESEARCH FUND, Clare Hall Laboratories, South Mimms, Herts .<br />
EN6 3LD, U .K .<br />
Deletions <strong>and</strong> other gene rearrangements appear to be an important step in the<br />
process of oncogenesis <strong>and</strong> are a result of many forms of DNA damage, but very little<br />
is known of the mechanisms responsible for these mutations . We have been studying<br />
gene rearrangements at the hamster adenine vhosnhoribosvl transferase . (anrt)locus with<br />
the intention of identifying sequence featurea <strong>and</strong> functions involved in such<br />
alterations . A striking feature of deletions at aprt is the directionality of the<br />
mutations . Deletion breakpoints frequently occur within aprt <strong>and</strong> often upstream of<br />
the locus but rarely downstream . This suggests that an essential function or structure<br />
lies downstream from the locus <strong>and</strong> limits the mutations recoverable . On the other h<strong>and</strong><br />
this directionality aids molecular analysis by providing a "tag" for deletion junction<br />
fragments allowing their cloning or recovery by the polymerase chain reaction . Many<br />
types of rearrangements (both small <strong>and</strong> very large deletions as well as several<br />
insertion mutants) have now been characterized at base sequence level . These<br />
alterations have a number of distinctive properties which will be discussed in detail .<br />
371<br />
INSTABILITY OF CHROMOSOMES CONTAINING AMPLIFIED REGIONS IN CHINESE BAMSTER CELLS .<br />
M . Miele, S . Bonatti, G . Fronza, L . Ottaggio, S . Yiaggi,<strong>and</strong> A . Abbond<strong>and</strong>olo, National In<br />
stitute for Research on Cancer, Genova (Italy), University-of Genova (Italy), <strong>and</strong> UO of<br />
CNR, Pisa (Italy)<br />
With the aim to study the effect of gross morphological modifications on chromosome<br />
stability, the behaviour of chromosomes carrying amplified CAD (carbamyl phosphate synthetase-aspartate<br />
transcarbamylase-dihydroorotase) or DH7R (dihydrofolate reductase) genas<br />
was studied in V79 Chinese hamster cell derivatives resistant to PALA (N-phospohacetyl-<br />
-L-aspartate) <strong>and</strong> MTX (methotrexate), respectively . In both metaphase chromosomes <strong>and</strong> in_<br />
terphase nuclei, amplified regions were localized by in s1 u hybridisation . In MTX-resis_<br />
tant cells, the amplification bearing chromosomes was lagging behind at anaphase <strong>and</strong> gaw<br />
rise in interphase nuclei to bud-shaped formations . Apparently, these buds could eventually<br />
separate as micronuclei . In both MTX- <strong>and</strong> PALA- resistant cells, micronuelei in in_<br />
terphase <strong>and</strong> displaced chromosomes in metaphase, both containing amplified DNA, were ob<br />
served . The presence of chromosomes in micronuclei was confirmed by fluorescent staining<br />
with antikinetochore antibodies . Finally, amplification,bearing dicentric chromosomes<br />
were found at high frequencies in both drug-resistant call lines . All together, these ob<br />
servations indicate that the presence of an amplified region makes chromosomes unstabla,<br />
since : (i) they tend to be excluded from cells, <strong>and</strong> (ii) they rearrange sare frequently<br />
than normal chromosomes .<br />
372<br />
SPONTANEOUS AND IN VITRO RADIATION-INDUCED CHROMOSOME ABERRATIONS IN HUMAN SPERMATOZOA :<br />
APPLICATION OF A NEW METHOD<br />
Mikamo, K., Kamiguchi, Y. <strong>and</strong> Tateno, H . : Department of Biological Sciences, Asahikawa<br />
Medical College, Asahikawa 078, JAPAN<br />
Chromosomes of the spermatozoon can be analyzed only after they replicate <strong>and</strong> become<br />
condensed in the ootid as male pronuclear chromosomes . Therefore, difficulty of using<br />
human oocytes had long been a severe limitation for the human sperm chromosome study .<br />
Fortunately, however, development of the interspecific in vitro fertilization system<br />
using zona-free hamster oocytes made It possible to carry out a large scale study of<br />
human sperm chromosomes without relying upon human oocytes . In the present talk, we<br />
describe briefly the procedure of our lmproved method <strong>and</strong> the results thereby obtained<br />
in the in vitro experiments . (1) Spontaneous incidences of human sperm chromosome<br />
aberrations in a total of 9280 spermatozoa from 87 samples of 26 men. Incidences of<br />
aneuploidy <strong>and</strong> structural anomaly were 1 .35 Z(hyperhaploidy, 0.68 x; hypohaploidy,<br />
0.67 x) <strong>and</strong> 13.9 x, respectively. The latter incidence varied considerably among the<br />
donors, ranging from 3.6 x to 21 .5 2. (2) Radiation (X-, y- <strong>and</strong> 8-rays)-induced human<br />
sperm chromosome aberrations in a total of 6974 spermatozoa from 57 samples of 12 men .<br />
Incidences of spermatozoa with structural chromosome aberrations increased linearly<br />
with increase of radiation dosage . The slope of the dose-effect equation was nearly<br />
the same between the three kinds of radiation . The incidence of breakage-type<br />
aberrations was far higher than that of exchange-type aberrations, both of them showing<br />
linear dose-dependent increases .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
129
130 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
373<br />
Notes 'itD; AOIE OF C1.T2, PRALiFFItATZCN IN CfmCAL CARCIIvmw7s . Jan C. Mirsalis . SRT<br />
International, Menlo Park, c1.<br />
Most agents that irxh :oe increases in celi proliferaticn have been st :oan to be<br />
effective tumor praootoss in liver <strong>and</strong> othar tissuasf indeea, the cnrcim9enicity of<br />
~r ~ be entu+rx:ed by partial hepatectony followirg c~ioal exposAme .<br />
grvwirg evidsnoe ~ t oall proliferation alone, in the abmenoe of<br />
e:mger:ous initiation, may also irduoe an incn .vased incidsnoa of liver t:moors in<br />
rodents, Ixutioularly mice . We have investigated the role of chaai~<br />
1 mechanians includirq pxtaocticn of spantnneasly initiated tumors, altered<br />
gena zegulation, la~.ion of RA, ar activatacn of<br />
fhis mec3ianism of h~ycancinogenesis will enable batter estima~ of t~ ris~k .~<br />
374<br />
MUTATIONAL SPECIFICITY OF CIS-PLATIN IN YEAST . J .R .A . His <strong>and</strong> g .A . Kunz, Microbiology<br />
Department, The University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2<br />
The chemotherapeutic agent cisplatin Jois-diamminedichloroplatinum(II)) produces<br />
DNA monoadducts <strong>and</strong> crosslinks <strong>and</strong> is mutagenic but the DNA sequence changes caused<br />
by this agent in eukaryotic cells have not been characterized . We are using DNA<br />
sequence analysis of mutations induced in the yeast suppressor tRNA gene SUP4-o to<br />
assay the mutational specificity of cisplatin . =-o mutants were selected following<br />
cisplatin treatment that reduced survival by 701 <strong>and</strong> increased the mutation frequency<br />
five-fold . 100 independent cisplatin-induced mutants have been characterized to date .<br />
Although the frequency of induction was relatively low, the spectrum of induced mutations<br />
differed from the spontaneous spectrum . Single base-pair substitutions constituted<br />
a smaller fraction (62%) of the total mutations <strong>and</strong> G-C -> A-T, C-C -> C-C <strong>and</strong><br />
C•C -> T•A events each accounted for approximately 301 of the base-pair changes identified<br />
. The substitutions occured predominantly at dipurines <strong>and</strong> where changes can be<br />
assigned to specific dipurines, 90% (36/40) were found at 5'-GG-3' or 5'-CA-3' sites .<br />
The fraction of single base-pair deletions induced by cisplatin was 10-fold greater<br />
(30% of the total mutations) than observed spontaneously <strong>and</strong> the majority (75%) of<br />
these events are found in a run of 5 C-C pairs . In addition, a small fraction (3/100)<br />
of non-t<strong>and</strong>em double events involving base-pair substitutions <strong>and</strong>/or deletions were<br />
recovered . Taken collectively, our results suggest that both monoadducts <strong>and</strong><br />
crosslinks may play roles in determining the mutational specificity of cisplatin .<br />
Currently, we are analyzing additional induced mutants . (Supported by NSERC Canada)<br />
375<br />
THE RELATIVE ROLES OF PHARMACOKINETICS AND ORGAN SPECIFIC ME'fABOLISK IN THE<br />
SELECTIVITY OF CYCLOPHOSPHAMIDE-INDUCED IIQIUNE CELL DAMAGE IN VIVO . R .R . Misra <strong>and</strong><br />
S .E . Bloom, Cornell University, Ithaca, NY (USA)<br />
In avian embryos bursectomy is achieved after subchronic administration of cyclophosphamide<br />
(CP) . However, the mechanism(s) by which organ-directed toxicity is<br />
achieved has not yet been elucidated . To this end, studies of xenobiotic metabolism in<br />
bursal <strong>and</strong> thymic cell fractions were undertaken . Three assays of mixed-function<br />
oxidase activity, as well as an assay of alkylation potential, were employed to detect<br />
differences between the abilities of bursal versus thymic micrososes to activate CP .<br />
Additionally, an aldehyde dehydrogenase (AD) assay was used to monitor differences in<br />
cytosolic detoxification activity . Compared to the liver, constitutive levels of P450<br />
activity were quite low in the bursa <strong>and</strong> thymus, <strong>and</strong> of the two lymphoid organs<br />
tested, the thymus exhibited higher levels of P450 activity . Alkylating activity was<br />
clearly demonstrated in hepatic microsomes, but fell below our limit of detection for<br />
bursal <strong>and</strong> thymic fractions . Similarly, iemune-organ AD levels were approximately<br />
one-tenth as high as those of the liver, <strong>and</strong> between lymphoid tissues, no significant<br />
difference in AD activity was apparent . The toxicokinetics of systemically ad .inistered<br />
CP as well as in vitro binding of the activated compound to lymphoid cells, were<br />
also examined . Results from these latter experiments indicate that in the intact<br />
animal, higher concentrations of CP <strong>and</strong>/or activated metabolite reach the bursa as<br />
compared to the thymus but that in vitro, no significant differences in binding occur .<br />
Our'findings suggest that while drug distribution patterns may be involved, differences<br />
in xenobiotic metabolism are probably not a major determinant in the selectivity<br />
of CP-induced immune-organ damage . (Supported by NIEHS ES03499 .)<br />
50869 3644
376 1989 EMS Abstracts<br />
CLONING OF DNA REPAIR GENES IN HAEMOPHILUS INFLUENZAE RD Notes<br />
R. Mody, V.P. Joshi <strong>and</strong> N.K. Notani, ion e ica 3roua BFi-fiFa AtQn ic Research Centre,<br />
B3nbay 400085, India.<br />
We have reported a recanbinational DNA repair systan which Is more clearly manifest<br />
in strains like Uvr1 in which excision repair is deficient . With another UV-sensitive strain<br />
Mbo2 also, we now observe much more of recQnbinational repair than is noted in a wild<br />
type strain. We have earlier reported cloning of uvrl gene on an 11 .3 kb insert. The<br />
plasrid pKuvrl fully cQnplanents Uvrl strain but not a Uvr2 strain . We now report cloning<br />
of mbo2 gene on a 17 .7 kb insert which fully canplanents the UV-sensitivity of Mbo2<br />
strain but not of Uvrl strain indicating that uvrl <strong>and</strong> mbo2 are not allelic . EcoRl cutting<br />
produces two fragm ents from the 17 .7 kb insert, the Terger one of 13 kb anChe sn aller<br />
one of 4.7 kb . Both these fragn ents have been subctoned 13 kb fragm ent subclone does<br />
not conpianent the UV-sensitiwty of Mbo2 strain but 4 .7 kb subclone gives a partial camplem<br />
entation. It is inferred that atleast san e of the genetic infotm ation for expressing<br />
UV-resistance is carried on 4 .7 kb fragm ent.<br />
377<br />
ETHENO-ADDUCT PRODUCTION BY CARCINOGENIC AND ENDOGENOUS AGENTS<br />
Ruth A . Modzelewski, Mary K . Conner, Noriko Kawatani, Departments of Biostatistics <strong>and</strong><br />
Industrial <strong>Environmental</strong> Health Sciences, Graduate School of Public Health, University<br />
of Pittsburgh, PA ., 15162 (USA)<br />
Ethyl carbamate (EC) is a carcinogen which produces highly fluorescent etheno-adenine<br />
(c-A) adducts in RNA . Its active metabolite is presumed to be an analogue of<br />
chloroacetaldehyde . The abundant adenylate pool is an obvious target for c-A adduct<br />
formation . Several e-A derivatives are potent inducers of SCEs <strong>and</strong> multiple complex<br />
chromosomal aberrations .<br />
Choline is an essential dietary element, cell membrane component, <strong>and</strong> a biochemical<br />
substrate . Arsenocholine (AsCh) is a arsenic analogue of choline found in seafood, including<br />
shrimp, crab, <strong>and</strong> some fish . Simple oxidation of the hydroxyl, moiety of choline<br />
or its arsenic analogue produces an analogue of chloroacetaldehyde .<br />
EC, Choline, <strong>and</strong> AsCh yielded incredibly similar fluorescent chromatogramS in extracts<br />
of murine red blood cells exposed in vivo . HPLC elution times of several fluorescent<br />
peaks correspond to those of st<strong>and</strong>ard c-adenine derivatives . The significance<br />
<strong>and</strong> long term consequences of c-A adduct production by carcinogens <strong>and</strong> endogenous<br />
agents remains to be elucidated . Supported by : BRSG 2 S07 RR05451-27, Biomedical Research<br />
Support Grant Program, NIH, <strong>and</strong> Center for <strong>Environmental</strong> Epidemiology, University<br />
of Pittsburgh, EPA CR 812761 .<br />
378<br />
METHODS FOR SCREENING FOR GERMINAL MUTATIONS: DETECTION OF "NON-POINT"<br />
MUTATIONS USING A MODIFICATION OF THE "RFLP" ANALYSIS STRATEGY . H . W .<br />
Mohrenweiser <strong>and</strong> B . A . Perry, Biomedical Sciences Division, Lawrence Livermore National Laboratory,<br />
Livermore CA 94550<br />
Insertions, deletions <strong>and</strong> rearrangements (I/D/R) of the DNA of the human genome have been identified<br />
during molecular analysis of both de novo germinal mutations <strong>and</strong> inherited variants causing genetic diseases .<br />
This class of molecular alteration in DNA structure is expected to predominate among radiation induced<br />
mutations. A modified restriction enzyme site (RFLP) mapping strategy, using only a single restriction<br />
enzyme digestion <strong>and</strong> then repetitively analyzing each sample with a series of probes for different loci, has<br />
the potential to screen a significant portion of the genome in each prob<strong>and</strong> for heritable, "non-point"<br />
mutations . Results from screening 130 unrelated Caucasian individuals for variation at 40 independent loci,<br />
using this RFLP mapping strategy, indicate the frequency of rare, inherited I/D/R variants is 3 .1 variants/<br />
1000 loci screened. A prototype mutation screening experiment that involves screening DNA from 50<br />
independent, clonally derived human lymphoblastoid cell ltnes, established in the absence of selective criteria<br />
following exposure of cell cultures to a mutagenic agent, has been initiated . Two ap nt mutations have<br />
been identified during the initial scteening of the DNA from these cell lines with sevet~NA probes, one in<br />
the progeny of ENU treated cells <strong>and</strong> one in a cell line established following exposure of the parental cells to<br />
X-ray. Confirmation of these apparent mutations <strong>and</strong> analysis of the molecular alteration in DNA structure is<br />
in progress, as is continued screening of the DNA from these cells with probes for additional loci . It should<br />
be possible with only minor enhancements of this RFLP mapping strategy to generate a significant data base<br />
regarding the frequency of germinal I/D/R mutations in an exposed population . Workperfotmmed under<br />
auspices of the US DOE by the Lawrence Livermore National Laboratory ; contract No.W-7d05-ENO-48<br />
379<br />
METABOLISM AND GENOTOXIC EFFECTS, IN VIVO . OF A MARKER FOR NfIRO-PAH . 2-NlIRO-<br />
FLUORENE . COMMONLY FOUND IN THE ENVIRON1vIENf .<br />
L . MBllert, J . Raftert . S . Tbmqulstt . B . BeIJe9, R ToRgArdt . T. Mldtvedt2 . M . Cortie2 <strong>and</strong> J-A .<br />
Gustafssont, Departments of Medical Nutritlonl <strong>and</strong> Medical Microbial Ecology2. ISarolinaka<br />
Institute <strong>and</strong> Department of Genetic <strong>and</strong> Cellular Toxicology3 . University of Stockholm .<br />
Stockholm. Sweden<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
131
132 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes Durtng incomplete combustion of organic matter there is a formation of po)ycyclic aromatic<br />
hydrocarbons IPAH) which can react with nitrogenoxides, with the formation of nttro-PAH'a<br />
as a result, a reaction which Is catalyzed by a low ptl . 2-Nitrotluorene (`IF), a marker for nftro-<br />
PAH, is in vivo metabolized via two different routes . After inhalation there ts a formation of<br />
potent mutagenic metabolites . OH-NF's, which are distributed In the body. After oral administration.<br />
NF is reduced to the amine, a reaction mediated by the intestinal mtcrollora . <strong>and</strong><br />
further acetylated to 2-acetylaminoAuorene (AAF), a potent carcinogen . Further ringhydroxyladon<br />
of AAF leads to detoxification <strong>and</strong> excretion .<br />
Induction of cytochrome P450 c .d affects the metabolism tn that more OH-NF'a are formed . As<br />
a consequence, more mutagenic metabolites are found In the circulation . The liver excretes OH-<br />
NF's as . In terms of mutagenictty. , totally harmless glucuronide conjugates . When these<br />
conjugates are excreted via the bUe . Intestinal beta-glucuronidase can liberate direct-acting<br />
mutagens tn the intestine . Thus, inhalation of NF can lead to formation of potent mutagens in<br />
the mtesttne.<br />
NF induces DNA-repair. In vivo, <strong>and</strong> is an tnttlator <strong>and</strong> a weak promotor, measured as formation<br />
of preneoplastic lesions in the liver. Risk estlmates, by two different methods . Indicate<br />
that nltro-PAH's, extrapolated from the marker NF. can expose humans to a cancer risk .<br />
380<br />
POINT hlJTATION AND CYTOGENETIC A'MLYSIS OA' L1TiPFDCY7ES FROM NH .RtOCYSTICFtCOTIC PATIFATS<br />
TRFATID WITH PRAZIWANTFL, R . Montero , D . Valencia , F . Moreno *, *f . S<strong>and</strong>oval * <strong>and</strong><br />
P . Ostrosky-Wegman . Instituto de Investivaciones Riomddicas, U .N.A .N ., Hosnital de<br />
Psnecialidades, Centro MBdico la Raza . Ando . Postal 70228, C .P . 04510, Wxico, n .F .<br />
Evaluation of genotoxic effects of praziouantel on neurocysticercotic piv. lyrtmhocytes<br />
showed an increased frequency of poliploids with res±+ect to non-infected animals <strong>and</strong><br />
also a greater suscentibility to clastogenic damaqe . When same study was intented on<br />
humans we met a major problem on choosing adequate controls for neurocysticercotic<br />
patients since they are exnosed to several mutagenic agents before the correct diagnose<br />
is done ; these include com?wtarized axial tanopranhy, anticonvulsants, anesthetics,<br />
antiinflamnatory druqs <strong>and</strong> antibiotics . Our controls, therefore, include non-infected<br />
persons in one h<strong>and</strong> <strong>and</strong> neurocysticercotic natients exoosed to different combinations<br />
of treatments, all of them before they received the nraziauantel treatment, on the<br />
other h<strong>and</strong> . The terminal noints studied were : structural <strong>and</strong> numerical chronosomal<br />
aberrations, sister chromatid exchanges, cell cycle kinetics <strong>and</strong> 6-thiopuanine resistant<br />
lymhocyte frequency . Results show that neurocysticercotic status, which involves<br />
exposure to the agents mentioned, causes retardation on cell cycle kinetics ;<br />
besides, hprt assay results show that in some aatients there is also an increase on<br />
point mutations . After praziquantel treatment it was found that cell cycle kinetics<br />
return to normal values .<br />
THE COMPARISON OF MUTAGEN-INDUCED THYMIDINE KINASE (TK) MUTANT FREQUENCIES IN HUMAN<br />
AND MOUSE LYMPHOMA TESTING CELLS . M .M . Moorel, K . Harrington-Brock2 L . Parker2 .<br />
1U .S . <strong>Environmental</strong> Protection Agency, Research Triangle Park, NC 27711 USA ;<br />
2<strong>Environmental</strong> Health Research <strong>and</strong> Testing, Inc ., Research Triangle Park, NC 27709<br />
USA .<br />
The TK6 line of human lymphoblastoid cells can be used to detect mutants at the<br />
heterozygous thymidine kinas* (1k) locus . Little et al . (1987, Banbury Report 28 :<br />
Mammalian Cell <strong>Mutagenesis</strong>, p . 225) have reported that a class of slow-growing TK<br />
mutants can be recovered <strong>and</strong> that at least some of these mutants may result from<br />
mitotic recombination . These results are similar ta our findings for small-colony<br />
TK-deficient mutants of mouse lymphoma cells . We wished to make quantitative<br />
comparisons between the induced mutant frequency in the human <strong>and</strong> mouse lymphoma<br />
cells . Slow-growing mutants are difficult to recover <strong>and</strong> count using the procedures<br />
st<strong>and</strong>ardly used with the TK6 cell line . We are itnrestigating modifications which<br />
might optimize growth <strong>and</strong> quantitation of slow-6roving mutants . Modifications<br />
include using 24 well plates rather than 96 well plates <strong>and</strong> plating cells at 1x103<br />
to 5x103 rather than 4x104 cells per well . Using these procedures, we are comparing<br />
the ICR-170-, EMS-, <strong>and</strong> MMS-induced TK mutant frequencies in human <strong>and</strong> mouse<br />
lymphoma cells . (This 1s an abstract of a proposed presentation <strong>and</strong> does not<br />
necessarily reflect U .S . EPA policy .)<br />
381<br />
382<br />
IN VIVO REPAIR DURING G~ OF GA!!IA RAYS INDUCED LESIONS ELICITING SISTER CHRQIATID E%<br />
CHANGES (SCEs) IN MURINE SALIVARY GLAND CELLS .<br />
Pedro Morales-Ramfrez, Teresita Vallarino-Kelly <strong>and</strong> Re ina Rodr2 uez-Reyes .<br />
Instituto Nacional de Investigaciones Nucleares, Mgxico, D .F ., I~XICO) .<br />
The in vivo ability of mouse cells to repair gamma radiation induced lesions capable<br />
of eliciting SCEs was examined . The experimental protocol was done in mouse salivary<br />
gl<strong>and</strong> cells induced to parasynchronical division by isoproterenol (1 umole/gm bd<br />
wt) . Two groups of mice were irradiated with 0 .38 Gy in a 6OCo source (Vick Rad) at a<br />
50869 3646
dose rate of 6 .9 Gy/min either at early or late GI, followed of a round of division in<br />
presence of bromodeoxyuridine (BrdU) dose of 0 .5 mg/gm bd wt . There was a significant<br />
decrease in SCE frequency at early Gj with respect to those irradiated at late G1 . The<br />
se data suggest that BrdU substituted cells are able to repair about fifty percent of<br />
the lesions induced by gamma radiation during G1 . Also the adequeate BrdU dose to obtain<br />
a clear differentiation of sister chromatids <strong>and</strong> the capacity of ga- a radiation<br />
to induce SCEs in salivary gl<strong>and</strong> cells were determined . The BrdU dose which permits a<br />
good differentiation of sister chromatids (0 .3 mg/g bd wt) was one third of that required<br />
in bone marrow cells . The gaama radiation induced a significant increase of --<br />
SCEs with a similar efficiency to that obtained in bone marrow .<br />
Acknowledgments : We wish to thank Jorge Mercader N ., Angel Reyes P ., Perfecto Aguilar<br />
V ., Felipe Beltran B . <strong>and</strong> Enrique FernEndez V., for their excellent technical assistance<br />
.<br />
383<br />
RESTRICI7ON ENZYMES AND CYTOGENETIC DAMAGE . W.F. Morgan, H .W. Chung. J .W. Phillips <strong>and</strong> RA<br />
Winegar, Laboratory of Radiobiology <strong>and</strong> <strong>Environmental</strong> Health, University of California, San Fraacisco, CA (USA)<br />
Bacterial restriction endonucleases recognize specific, rather short sequences of DNA as binding sites <strong>and</strong> produce<br />
either blunt-ended or cohesive-ended DNA double-str<strong>and</strong> breaks . Eleetroporatioe Is a rapid <strong>and</strong> extremely efficient<br />
method for pcrmeabilning Chinese hamster ovary (CHO) cells, permitting the introduction of restrictioa enzymes into<br />
exponentially growing cells. We have used restriction enzymes with different recognition sequences <strong>and</strong> different<br />
cutting frequencies to generate double-str<strong>and</strong> breaks in CHO cells <strong>and</strong> have examined the role of these breaks in<br />
cytogenetic damage. Restriction enzymes electroporated into cells readily induced chromosome aberratioos at all<br />
stages of the cell cycle. Enzymes generating blunt-ended DNA double-str<strong>and</strong> breaks induced more chromosome<br />
damage compared with enzymes generating coheaive-ended breaks . When restriction enzymes were eledroporated<br />
into exponentially growing cells during the second replication cycle in bromodeoxyuridise, <strong>and</strong> sister chromatid<br />
exchanges (SCEs) analyzed at the subsequent mitosis, we found no inaease in SCE frequency . Many enzymes<br />
induced aberrant metaphase chromosomes, but SCE frequency was not increased in those cells. These results indicate<br />
that in our h<strong>and</strong>s, restriction enzyme-induced DNA double-str<strong>and</strong> breaks give rise to chromosome abenaNons, but do<br />
not lead to SCE formation . Work supported by the Office of Health <strong>and</strong> <strong>Environmental</strong> Research of the US . Dept . of<br />
Energy under contract DE-AC03-76-SF01012 .<br />
384 .<br />
GENOTOXICITY IN RODENT HEPATOCYTES AND CARCINOGENICITY OF ANTHRAQUINONES .<br />
H . Mori, N . Yoshimi, S . Sugie, H . Iwata, T . Tanaka, <strong>and</strong> K . Kawai, Gifu University School<br />
of Medicine, Gifu (Japan), <strong>and</strong> Chukyo Women's University, Ohbu, Aichi (Japan)<br />
A large number of anthraquinones <strong>and</strong> their derivatives have been isolated from higher<br />
plants <strong>and</strong> fungi, <strong>and</strong> some of them have been widely used as colorants in food, cosmetics,<br />
hair dyes <strong>and</strong> textiles . Luteoskyrin <strong>and</strong> rugulosin isolated from Penicillium isl<strong>and</strong>icum<br />
are known as hepatocarcinogenic anthraquinoids . Emodin from the samerun-gus<br />
has been shown to be mutagenic in bacterial mutagenicity assay . We have demonstrated<br />
genotoxicity of some anthraquinones such as 1,8-dihydroxyanthraquinone (chrysazin)<br />
which has been used as a popular laxative, in the hepatocyte/DNA repair assay . In the<br />
genotoxicity assay, 1,2-, 1,4-, <strong>and</strong> 1,5-dihydroxyanthraquinones were negative, although<br />
1-hydroxyanthraquinone <strong>and</strong> 1,8-dihydroxyanthraquinone generated clearly positive res-<br />
ponse of DNA repair, suggesting a mutual relationship between the genotoxicity <strong>and</strong> structures<br />
of anthraquinones . Carcinogenicity testing of chryeazin was done using rats<br />
<strong>and</strong> mice . In rats, chrysazin was tumorigenic to cecum <strong>and</strong> colon . In mice, the chemical<br />
showed carcinogenic potentials in the large bowel <strong>and</strong> liver . Carcinogenicity of 1-hydroxyanthraquinone<br />
was also examined in rats . Carcinogenicity of this naturally occurring<br />
anthraquinone was manifested in cecum, colon <strong>and</strong> liver . The carcinogenic effect of<br />
1-hydroxyanthraquinone appeared to be stronger than chrysazin . The result was agreement<br />
with that of the genotoxicity assay with hepatocytes . It appears that genotoxicity in<br />
rodent hepatocytes is more consistent with mammalian carcinogenesis than bacterial<br />
mutagenicity for these anthraquinone chemicals .<br />
385<br />
EVALUATION OF CLASTOGENICITY OF NON-PHYSIOLOGICAL PHs IN CULTURED MAMiALIAN CELLS .<br />
MORITA, T ., TAKEDA, K ., <strong>and</strong> OKUMURA, R ., Tokyo Research Laboratories,<br />
NIPPON GLAXO LTD ., Nerima-ku, Tokyo, Japan .<br />
Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried<br />
out of formic acid, acetic acid, lactic acid <strong>and</strong> a mixture of S9 <strong>and</strong> NaOH, <strong>and</strong> the<br />
relationship between pHs of the media <strong>and</strong> their clastogenic activity was examined .<br />
The medium used was Ham"s F12 supplemented with 17 mM NaHC03 <strong>and</strong> lOx FCS . All of<br />
these acids induced chromosomal aberrations at the initial pH of ca . 6 .0 or below<br />
(10-14 mM of each acid) either in the presence or absence of S9 mix . Exposures of<br />
cells to pH 5 .7 or below (12-16 mM of each acid) were found toxic . In the culture<br />
media which were first acidified with each of these acids <strong>and</strong> then neutralized to<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 133<br />
Notes
134 1989 EMS Abstracts<br />
Notes pH 6 .4 or pH 7 .2'with NaOH, no clastogenic activity was observed . Using F12 medium<br />
supplemented with 34 mM NaHC03, or 30 mM HEPES as buffer, we observed metaphases<br />
at doses up to 25 or 30 mM of these acids <strong>and</strong> no clastogenic activity at 20-25 mM<br />
or below . A mixture of S9 (final 10X) <strong>and</strong> NaOH induced chromosomal aberrations at<br />
initial pH of ca . 10 .8 . When the mixture was incubated at 37°C for 1 hr <strong>and</strong> then<br />
neutralized with HC1 to pH 9 .3 or pH 7 .4 . it showed no clastogenic activity . The<br />
above results show that these acids <strong>and</strong> base are non-clastogenic <strong>and</strong> thus the<br />
pseud-positive reactions attributable to non-physiological pH conditions could be<br />
precluded by either neutralization of the treatment medium or enhancement of the<br />
buffering ability .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
386<br />
HUMAN SOMATIC MUTATIONS AT THE AUTOSOMAL HLA-A LOCUS<br />
A .A . Morley <strong>and</strong> D .R . Turner, Haematology Department, Flinders Medical Centre,<br />
Adelaide, Australia .<br />
Human lymphocytes mutated at the HLA-A locus were isolated by immunoselection<br />
using monoclonal anti-HLA-A2 or -A3 antibody <strong>and</strong> complement followed by cloning in<br />
micropslates . Mutant frequency in 21 normal individuals, with mean age 35, was 3 .0<br />
x 10- <strong>and</strong> in 10 elderly individuals with mean age 78 it was significantly higher<br />
at 7 .2 x 10-s . Analysis of the T-lymphocyte receptor gene in 98 mutants showed<br />
that ~
1989 EMS Abstracts<br />
at the later time points . Relative cloning ability was determined by exposing P3 Notes<br />
cells to concentrations of 0 .1 to 1 .0 VM BRdU or CLdU for 7 days . Each toxicant<br />
reduced cloning ability from 100% to less than 1%, although CLdU was more efficient on<br />
a concentration basis than BRdU . Not only were the toxic effects of CLdU greater than<br />
those of BRdU, but also its ability to perturb the cell-cycle .<br />
389<br />
INSECTICIDES AND CAF2CZNOGQI METAHOLIZING ENZYMES<br />
M .H . MU6'i'AFA AND A .H . Etrlt(%aEIDY . Institue of Graduate Studies <strong>and</strong> Research,<br />
Alria University, alex<strong>and</strong>ria, Egypt .<br />
The effect of various insecticides on the activities of sane carcirtgertntt:tabolizing<br />
enzymes <strong>and</strong> the eytocliratr` P-450 of rat liver microsomes were studied . Organoc.hlorine<br />
insecticides, Lindane, DDT <strong>and</strong> Endrin significantly increased the activity<br />
of arylhydrocarbon hydroxylase (AHH) . Pretreatment of rats with the synthetic pyrethroids,<br />
fenvalerate insignificantly increased the enzyme activity . Pretreatment with<br />
carbaryl, a carbamate derivative <strong>and</strong> Ditnethoate, an orgartophosphrotls oatQottrd did not<br />
affect the AHH activity . Our data showed that all the tested insecticides which altered<br />
the AHH activity have similar effect on tht: cy''.,ochrane P-450 oontent . The data revw<br />
ealed the ability of various insecticides to increase the atsotlrtt of cytochrattta P-450<br />
<strong>and</strong> the AHH activity which ttssy increase the nutagenic <strong>and</strong> carcinogenic activity of<br />
benzo-a-pyrene, since such an activity is dependent an the induction of cytocYurome<br />
P-450 dependent AHH . A second enzyme was also investigated, dintethylnitrosamine (DM4)<br />
demethylase I <strong>and</strong> II . Pretreatntutt of rats with Lindane, DDT <strong>and</strong> Endrin decreased the<br />
activity of Dm-demethylase I <strong>and</strong> increased the activity of DMN-danethylase II .<br />
Fenvalerate <strong>and</strong> carbamate pretreatment groduoed insignificant change an both enzymes .<br />
Pretreatment with Dimethoate inhibited the oxidative N-darethylaticn of DMD1 . Since<br />
the metabolism of carcinogens by cytochrrnie P-450 dependent mx0aager>ares often<br />
facilitates their elimination or activiation, alteration of norntal tuetabolicpn t2xaays<br />
of these carcinogens by various insecticides may alter the intensity <strong>and</strong> duratiort or<br />
action of these environmental carcinogens .<br />
390<br />
GE\OTOXIC POTENTIALITIES OF ::-NITRU-METIOXYNAP T8O ('2,1-b) FURAN (R 7000)<br />
IN RELATION TO Ti{E GENERAL PROBLEM .<br />
J . Moutschen, J . Gilot-Delhalle, M . Moutschen-Dahmen, <strong>and</strong> F . London,<br />
University of Liige, Sart-Tilman 1322, D-4000 Liege (BELGIUM)<br />
Mutagenic activity of R'7000 was tested on two strains of Sehizoe? ccharomyces<br />
pombe ade 7 <strong>and</strong> ade 6 (at doses ranging from 0 .0Gto 32 . .0 M) .<br />
It was different in both strains . R 7000 showed clastogeniciSy in Nigella<br />
damascena chromosomes at doses ranging from 0 . 12 to 32 .t0-5M for seeds<br />
<strong>and</strong> 0 . 12 to 2 .10-5M for root tips . Treatments of dry seeds revealed the<br />
sensitivity of GO whereas treatments of presoaked seeds showed the aensivity<br />
of Gl, S <strong>and</strong> G': . These results were confirmed by treatments of<br />
growing root tips with a much higher sensitivity of the later phases of<br />
the mitotic cycle . These treatments yielded a high amount of gaps (G'<strong>and</strong><br />
G") . The results are in agreement with those obtained after treatments<br />
of animal cells . Additionally, some treatments induced strong stathmokinetic<br />
effects analysed in details . The spectrum of induced chromosome<br />
damage shows similarities with the spectrum of lesions induced by cytosine-arabinoside<br />
<strong>and</strong> other related substances, wich suggests some mechanisms<br />
of action . In conclusion, R 7000 belongs to the class of substan-,<br />
ces with multiple genotoxic effects, <strong>and</strong> its position in the important<br />
class of furans has to be revaluated .<br />
391<br />
AN SAR STUDY OF THE MUTAGENICITY OF PAH COMPOUNDS IN SALMONELLA<br />
TYPHIMURIUM . Susan R . Moyer <strong>and</strong> Peter C . Jurs, Chemistry Department, Penn State University,<br />
University Park PA 16802<br />
Many Polycyclic Aromatic Hydrocarbons <strong>and</strong> their derivatives have been shown to be potent<br />
mutagens in Salmonella ryphirnuritun via the Ames test . A group of 110 PAHs (48 active <strong>and</strong> 64<br />
inactive) were obtained from the literature <strong>and</strong> were used to carry out a structure-activity relationship<br />
study. All of the compounds were tested by LaVoie <strong>and</strong> coworkers using S . ryphimtvitun TA100 with<br />
metabolic activation . The molecular structures were entered into the ADAPT software system by<br />
sketching them on a computer terminal . Three-dimensional molecular models were constructed using<br />
molecular mechanics . Then, topological, geometrical, <strong>and</strong> electronic molecular structure descriptors<br />
were calculated for each compound . Specific structural features were encoded by using substructure-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
135
136 1989 EMS Abstracts<br />
Notes based descriptors that focus on details of the molecular structures . The descriptors were analyzed<br />
statistically in order to develop a minimal set to characterize the compounds . Pattern recognition<br />
techniques were used to classify the compounds as active or inactive based on the structural<br />
descriptors. A training set of 80 compounds (40 active <strong>and</strong> 40 inactive) was classified almost perfectly<br />
using 15 descriptors, <strong>and</strong> the remaining compounds formed a prediction set of unknowns . Studies in<br />
progress are aimed at refining our SAR model for PAH mutagenicity .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
CLONING ILLID PARTIAL C11WCP6RIZATION OF RftE H(A7)1N IXCISION RLPAIR GENB ERCC-5 .<br />
J .S . Mudgett, G .F . Strniste, <strong>and</strong> ri .A. MacInnes, Genetics Group, LS-3, M886, Los<br />
A,lamos National Laboratory, Los Alamos, t39 87545 (iJSA) .<br />
The human excision repair gene DtCC-5 was identified <strong>and</strong> isolated by complementation<br />
of the W-sensitive Chiness hamster ovary cell mtutant UV-135 <strong>and</strong><br />
subsequent cosmid library construction <strong>and</strong> analysis . Genomic DtA from repair<br />
proficient human skin fibroblasts was ligated to pSV2gpt <strong>and</strong> transferred into<br />
W-sensitive Ctq cells of complementation group 5(W-135) . DNA from a primary<br />
MAX (aycophenolic acid, adenine, xanthine) resistant, W-resistant cell line was<br />
transferred into W-135 cells to isolate a secondary Mhe, W cell line, from<br />
which tertiary MaX`, i)V' cell lines were isolated by gene transfer . Cotuid<br />
clones which contained inserted human sequences wsre obtained from a 20X genumic<br />
library of the secondary transformant . None of thse individual (101) cosmid<br />
clones isolated <strong>and</strong> tested were able to ca .plement the repair defect, but<br />
specific overlapping pairs of cosmids co-transfected into UV-135 cells yielded<br />
transformnnts at high frequency which exhibited wild-type W resistance, suggestit~g<br />
that the repair gene is as large or greater than the cosmid insert size<br />
(-40 kb) . The overlap between the appropriate cosmid clones, <strong>and</strong> the coaplete<br />
contiguous human genomic insert, will be napped to identify the sise <strong>and</strong> functional<br />
boundaries of the human nNA repair gene . Unique huswt sequence probes<br />
derived from these specific cosmid clones were used to : 1) demonstrate a positive<br />
hybridization correlation with genomic tx4A from independent UV-resistant<br />
transformants <strong>and</strong> human cell hybrids ; 2) to confirm the previous assignment of<br />
ERCC-5 on chromosome 13 ; <strong>and</strong> 3) to screen c0Nuh libraries . (Supported by the<br />
U .S . Department of Snerqy under c^ntract W-7405-lN".-36)<br />
392<br />
393<br />
IN VIVO GENOTOXICITY OF TERTIARY BUTYL HYDROQUINONE IN GERM CELLS OP MALB<br />
NICE . A . Mukher,)ee <strong>and</strong> A . Sharma, Centre for Advanced Study in Cell <strong>and</strong> Chromosome<br />
Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road,<br />
Calcutta 700 019 (INDIA)<br />
Effect of tertiary butyl hydroQuinone (doses of 10, 20 <strong>and</strong> 100 mg/kg/day during<br />
14 days) on germ cells of male mice were investigated . The mode of application was<br />
stomach intubation . The germ cell stages analysed were spermatids (for the heritable<br />
effects) <strong>and</strong> differentiating <strong>and</strong> stem-cell spermatogonia (for direct effects) . A lack<br />
of heritable translocations, sperm abnormalities was demonstrated in F males originating<br />
from treated P males . Significant effects in treated males were found with respect<br />
to . (1) sex-chromosomal univalency in the diakinesis-metaphase I stage after the<br />
treatment of stem spermatogonia, (2) sperm-head abnormalities after treatment of<br />
differentiating spermatogonia <strong>and</strong> (3) fertility after treatment of spermatids .<br />
394<br />
MICRONUCLEI INDUCED BY TERTIARY BUTYL HYDROQUINONE (AN ANTIOXIDANT) IN BONE MARROW<br />
CELLS OF MICE . Anita Mukherjee . Human Genetics Unit, Centre for Advanced Study<br />
in Cell <strong>and</strong> Chromosome Research, Department of Botany, University of Calcutta,<br />
35 Ballygunge Circular Road, Calcutta 700 019 . India .<br />
Tertiary butyl hydroquinone (TBHQ) a phenolic antioxidant was administered to<br />
Swiss albino male mice of 20, 50, 100 <strong>and</strong> 200 mg/kg as a single (i .p .) injection .<br />
Corn oil <strong>and</strong> cyclophosphamide were used as the vehicle (negative) <strong>and</strong> reference<br />
(positive) control respectively . Samples of bone marrow were examined for the<br />
incidence of micronuclei in the poiychromatie erythrocytes at 24 hours after dosing .<br />
A positive dose response effect in the micronuclei freQuency was observed following<br />
the Cochran Armitage Trend Test . 50, 100 <strong>and</strong> 200 mg/kg of TBHQ caused an increase<br />
in the incidence of micronuclei compared to the negative vehicle control . Cycl,ophoephamide<br />
caused a significant increase in the incidence of micronuclei .<br />
50869 3650
395 1989 EMS Abstracts<br />
THE ROLE OF NUCLEAR MATRIX IN REPAIR Notes<br />
L.H .F. Mullenders', J . Venema', A.•van Hoffen', L. Mayne', A .T . Natarajan' <strong>and</strong> AA. van Zeel<strong>and</strong>") Department<br />
of Radiation Genetics <strong>and</strong> Chemical <strong>Mutagenesis</strong>, University of Leiden, The Netherl<strong>and</strong>s ; p) Centre of Medical<br />
Research, University of Sussex, U.K .<br />
The organization of the eukaryotic genome into a series of loops anchored to the nuclear matrix bas been<br />
related to functional compartimentalization of the nucleus in order to facilitate processes such as replication <strong>and</strong><br />
transcription. We have investigated the role of the nuclear matrix in processing UV-induced damage in human<br />
fibroblasts . Pulse-labelling of normal fibroblasta followed by enzymatic digestion of DNA-nuclear matrix complexes<br />
revealed a time <strong>and</strong> dose dependent preferential repair of nuclear matrix associated DNA . Pulse-ehase experiments<br />
showed no evidence for compartimentalization of excision repair at the nuclear matrix Le, lesions do not require prior<br />
attachment in order to be repaired . The results favour a model of preferential repair of DNA sequences permanently<br />
associated to the nuclear matrix . Pronounced differences in distribution pattern of repaired sites in DNA-nuclear<br />
matrix complexes were found among normal <strong>and</strong> UV-sensitive cells exposed to UV-'vradiation . Xeroderma<br />
pigmentosum group C (XP-C) efficiently repaired nuclear matrix associated DNA, but not regions of the genome<br />
further extended into thc DNA loops . In Cockayne's syndrome (CS) cells the reversed situation was found : efficient<br />
repair of loop DNA <strong>and</strong> inefficient repair of nuclear matrix-associated DNA . These differences in distribution oan<br />
be correlated to efficiencies in repair of UV-damage in transcriptionally active genes . Despite the low overall repair<br />
XP-C cells were found to be as proficient in repair of pyrimidine dimera from the ADA <strong>and</strong> DHFR gene as normal<br />
fibroblasts. The efficient removal of pyrimidine dimers from active genes was found to be absent in CS cells . The<br />
results suggest the existence of two independent repair pathways directed towards repair of pyrimidine dimers in<br />
either active or inactive DNA .<br />
396<br />
SENTINEL AND OTHER MUTATIONAL EFFECTS IN OFFSPRING OF CANCER SURVIVORS . John J .<br />
Mulvihill, Clinical Epidemiology Branch, National Cancer Institute, Bethesda, MD, USA<br />
To date, no agent has been documented to cause germ cell mutation in human beings,<br />
with the possible exception of radiation causing abnormal meiotic chromosomes in<br />
testes . For studies in humans, mutation epidemiologists prefer the cohort approach,<br />
start ing with an exposed population <strong>and</strong> looking for mutations that may be expressed<br />
in offspring as variants in health, chromosomes, proteins, or nucleic acids . Currently<br />
patients with cancer are the cohort exposed to the largest doses of potential<br />
mutagens, i .e ., radiotherapy <strong>and</strong> drugs . In 12 large studies with over 825 p,atients<br />
<strong>and</strong> 1573 pregnancies, 46 (4%) of 1240 liveborns had a major birth defect, a'rate comparable<br />
to that in the general population . One of these was a classic sentinel phenotype,<br />
i .e ., a new sporadic case of a dominant mendelian syndrome . In collaboration<br />
with 5 U .S . cancer registries, we interviewed a retrospRctive cohort of 2383 patients<br />
diagnosed with cancer under age 20 years, from 1945 thrbugh 1975 . Records were sought<br />
to verify major genetic disease, defined as a cytogenetic or single gene disorder or<br />
1 of 15 isolated birth defects . In 2308 offspring of survivors, 5 had a chromosomal<br />
syndrome, 11 had a single gene disorder, <strong>and</strong> 62 had at least one major malformation .<br />
Among 4722 offspring of sibling controls, the respective numbers were 7, 12, <strong>and</strong> 127,<br />
nonsignificant differences . 7% of the parents of the offspring with possibly new<br />
mutations received potentially mutagenic therapy, compared with 12% of parents of<br />
normal children . Since pregnancy in or by cancer survivors is still a rare event,<br />
future efforts to document germ cell mutation may be best studied through international<br />
cooperation coupled with diverse laboratory measures of mutation .<br />
397<br />
MMC INDUCED SCE IN DIVIDING AND NONDIVIDING HUMAN PURIFIED LYMPHOCYTES AND<br />
CHINESE HAMSTER OVARY CELLS . H . Murli, Department of <strong>Molecular</strong> <strong>and</strong> Cellular<br />
Services, Hazleton Labs, Kensington, MD .<br />
Repair of MMC induced lesions that result in SCE was studied in purified human<br />
lymphocytes (PHL) <strong>and</strong> in Chinese hamster ovary cells (CHO) . PHL at 0~ <strong>and</strong><br />
confluent cultures of CHO cells were treated with MMC for two hours <strong>and</strong> held<br />
in G, or confluent for 0, 18, 25, or 48 hours . PHL received PHA <strong>and</strong> BrdUrd<br />
after the appropriate interval <strong>and</strong> were recultured for -72 hours . CHO cells<br />
were split after the appropriate interval <strong>and</strong> recultured for -26 hours with<br />
BrdUrd . SCE frequencies were similar for all the cultures with both cell<br />
types . Thus PHL <strong>and</strong> CHO cells were incapable of repair of MMC induced SCE<br />
damage under nonreplicating conditions . CHO cells were cultured in log phase<br />
for -14 hours after MMC exposure before Brdurd was added or confluent cultures<br />
exposed to MMC were split after 0 or 24 hours, cultured for 14 hours before<br />
BrdUrd was added <strong>and</strong> recultured for -25 hours . SCE frequencies were near<br />
control levels for all exposure conditions indicating repair of MMC induced<br />
lesions . PHL were treated with MMC, PHA was added 0 or 24 hours later, <strong>and</strong><br />
BrdUrd was added 48 or 72 hours later, respectively . Again SCE frequencies in<br />
all these cultures were near control levels . MMC induced SCE in PHL were<br />
tested under differing exposure conditions to PHA <strong>and</strong> BrdUrd . These experiments<br />
indicate that MMC induced lesions are repaired only after one round of<br />
DNA replication .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
137<br />
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138 1989 EMS Abstracts<br />
Notes ETHICS AND THE ALLOCATION OF RISK : SCIENCE AND VALUES<br />
Thomas H. Murray, Ph .D .<br />
Center for Biomedical Ethics<br />
School of Nedicine<br />
Case Western Reserve University<br />
Clevel<strong>and</strong> OH 44106<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A large number of ethical questions posed by research on<br />
environmental mutagenesis need to be sorted out. They include<br />
research on human subjects, the responsible use of information,<br />
privacy <strong>and</strong> others . This paper will focus on an ethical question<br />
with considerable social importance : How should scientific findings<br />
<strong>and</strong> moral values be used in making public policy decisions about<br />
the control of mutagenic substances in the environment <strong>and</strong> in the<br />
workplace? The values that guide judgments about the relative<br />
certainty of scientific claims differ from the st<strong>and</strong>ards that are<br />
<strong>and</strong> should be used in making public policies that ought to be<br />
informed by scientific findings. The struggle over regulation of<br />
asbestos will form a case study of the interplay between science<br />
<strong>and</strong> values in the making of public policy on toxic substances .<br />
Such social decisions pose unavoidable questions about the<br />
allocation of risk, which are also ethical questions, specifically<br />
questions about justice .<br />
398<br />
399<br />
APPLICATION OF THE 32P-TECHNIQUL+ TO DETECT DNA-ADDUCTS BY CISPLATI N<br />
R . Mustonen <strong>and</strong> K . Hemminki . Institute of Occupational Health .<br />
Helsinki . Finl<strong>and</strong><br />
The main reaction products of Sjg-diamminedichloroplatinum (II),<br />
cisplatin. with DNA are intrastr<strong>and</strong> cross-links with dGpdG <strong>and</strong> . to a<br />
lesser extent, dApdG . Platinum can be conveniently measured by atomic<br />
absorption spectroscopy but with patient white blood cell DNA this<br />
technique is presently at the limit of its sensitivity . We have<br />
therefore applied the 32P-postlabelling technique to the DNA adducts<br />
of cisplatin . The st<strong>and</strong>ard compound, intramolecular crosslink Pt-dGpdG<br />
is labelled with T4 polynucleotide kinase <strong>and</strong> Y-32P-ATP, <strong>and</strong> the<br />
products are separated by PEI-TLC <strong>and</strong> visualized by autoradiography .<br />
st<strong>and</strong>ards containing a 5'-phosphate group are used as internal<br />
st<strong>and</strong>ards . The phosphorylation of the cisplatin-adducts is a slow<br />
process as compared to that of polycyclic adducts . but the levels of<br />
adducts detected with the st<strong>and</strong>ard compounds are presently belo w<br />
100 fmol . Adducts are also detected in DNA platinated vitro <strong>and</strong><br />
digested enzymatically to nucledtides .<br />
CATECHIN AS AN ANTIMUTAGEN AND ANTICARCINOGEN .<br />
M. Nagabhushan, Northwestern Universit , Department of Pathology, 303<br />
E. Chicago Ave., Chicago, IL 60611, U~A .<br />
Betelquid chewing with or without tobacco is correlated with high incidence of oral<br />
cancer in India. Betelquconsists of betelnut, betelleaf, catechu, slaked lime <strong>and</strong> spices .<br />
Catechu is a nonawtagenic constituent of betelquid contain catechin as the principal<br />
compound. Catechu extract <strong>and</strong> catechin inhibits the muugeniciry of tobacco, masheri, bidi<br />
<strong>and</strong> cigarette smoke condensates, smoked meat charred <strong>and</strong> noncharred extracts, benzo(a)<br />
pyrene (BP) <strong>and</strong> dimethylbenz(a) anthracene (DMBA) in a dose dependent manner in<br />
salmonella/microsome test. Catechin inhibits the activity of Cyt P 450 <strong>and</strong> has no effect<br />
on GST, but increased (1SH content in rat liver. Simultaneous treatment of catechin<br />
prevents the mutagenicity of BP <strong>and</strong> DMBA metabolitis but pre or post-tteatment of<br />
bacteria with catechin has no effect on the mutagenicity. Catechin also inhibited the in vitro<br />
binding of 3HBP metabolitis to calf thymus DNA . Oral administration of catechin inhibits<br />
the incidence of BP-induced forestomach tumors in female swiss mice . This study<br />
indicates that catechu in betelquid may act as an antimutagen <strong>and</strong> anHcarcinogen <strong>and</strong> may<br />
suppress the mutageniclcarcinogenic potential of other betelquid ingnedients .<br />
400
401 1989 EMS Abstracts 139<br />
CARCINOGEN INHIBITION OF HAMSTER BUCCAL POUCH EPITHELIAL CELL Notes<br />
REPLICATION jj`[ VITRO M . Nagabhushan, P. Polverini <strong>and</strong> D . Solt . Northwestern<br />
University Department of Pathology, 303 E . Chicago Ave ., Chicago, Il 60611<br />
Precancerous lesions induced in rodent liver with chemical carcinogens are<br />
resistant to the inhibitory effect which these agents exert on liver cell replication .<br />
This functional property of "resistance to cytotoxicity" has been used to detect<br />
very early precancerous liver cell populations . We wish to apply this same strategy<br />
to detect <strong>and</strong> characterize precancerous epithelial cell populations in hamster<br />
buccal pouch(HBP) -- a tissue which is structurally <strong>and</strong> functionally similar to human<br />
oral mucosa . As a first step toward this goal we have developed an approach<br />
to monitor HBP epithelial replication, <strong>and</strong> its inhibition, upon exposure to carcinogens<br />
L vitro . In a representative experiment, fragments of HBP were incubated<br />
in supplemented media for 24 hrs in the presence <strong>and</strong> absence of 7,12-dimethylbenzanthracene(DMBA)<br />
or methylbenzylnitrosamine(MBN). [3H]Tdr was added to<br />
the media for the final 2 hrs of incubation <strong>and</strong> acid insoluble counts were determined<br />
. At concentrations of 0 .15, 0.30, <strong>and</strong> 0 .60 mM, DMBA <strong>and</strong> MBN inhibited<br />
[3H]Tdr incorporation by 35%, 76%, 87% <strong>and</strong> 91%, 93%, <strong>and</strong> 98% respectively . We<br />
conclude that this approach, combined with autoradiography, may be useful for<br />
in vi detection of precancerous HBP lesions resistant to chemical carcinogens .<br />
402<br />
INHIBITORS OF NITROSATION IN VITRO . M . Nagabhushan,<br />
Northwestern University, Department of Pathology, 303 E . Chicago Ave.,<br />
Chicago, IL 60611 .<br />
Nitroso-compounds are mutagenic/carcinogenic compounds formed by the<br />
interaction (nitrosation) of amine/arnide <strong>and</strong> nitrites . Human stomach maintains acid pH, is<br />
the most probable site of formation of nitroso-compounds . Fish, preserved meat,<br />
vegetables <strong>and</strong> drinking water are rich sources of amines/amides <strong>and</strong> nitrites . Several<br />
inhibitors of nitrosadon are known to be present in human diet . In the present study some<br />
of the inhibitors of nitrosation of tnethylur ea <strong>and</strong> fish extract by sodium nitrite at pH 3 .6<br />
<strong>and</strong> 2.0 are reported. Salmonella strain TAIOO <strong>and</strong> TA1535 are used to monitor the<br />
formation of mutagenic nitroso-compounds . The extracts (principles) of turmeric<br />
(curcumins), betel leaf (eugenol, hydroxychavicol), tea (Tannic acid) <strong>and</strong> catechu (catechin)<br />
exhibit dose dependent inhibition of nitrosation . The simultaneous treatment of inhibitor<br />
with precursors is essential for the inhibition . Pre or post-treatment of inhibitor does not<br />
modify the mutagenicity of nitroso - compounds. In case of phenolics pm - hydroxy<br />
group is essential for nitrosation inhibition . The nitrosation inhibition by phenolics in<br />
through the scavenging of nitrite ions from the media, thus making it non-available for<br />
nitrosation.<br />
403<br />
TRENDS IN ENVIRONtWNTAL CARCINOGENESIS<br />
B. Nagarajan, Cancer Institute, Madras - 600 020 INDIA<br />
rlost cancer is caused or promoted by life style <strong>and</strong> environmental<br />
factors . Carcinogenesis is a multiple process involving initiation, promotion<br />
<strong>and</strong> progression . Identification of several risk factors helps<br />
evolve effective approaches to control <strong>and</strong> contain the disease . In our<br />
laboratory, we carried out in-depth studies on hexachlorocyclohexane<br />
(ttCH), a commonly used pesticide . It is a hepatotoxic compound with<br />
potential tumorigenecity that induced gamma glutamyl transpeptidase<br />
positive foci, mostly in periportal hepatocytes . A complete carcinogen<br />
such as dimethylaminoazobenzene hits selected target cells <strong>and</strong> only that<br />
clone's cells proliferate to malignancy . In HCH treated cells, marker<br />
analysis both for differentiation <strong>and</strong> proliferation established an<br />
epigenetic promoter component .<br />
Clofibrate is an extensively used hypolipidemic drug . Cytogenic<br />
studies on drug-treated rat bone marrow cells <strong>and</strong> human lymphocytes in<br />
vitro showed minimal damage that repaired to normal on withdrawal of the<br />
drug . Biochemical studies also agreed with this observation . However,<br />
if the cells were pre-exposed to an initiator such as diethylnitrosamine<br />
<strong>and</strong> followed with clofibrate, they tend to exhibit tumorigenecity . A<br />
similar hazard risk assessment is warranted in the use of halogenated<br />
hydrocarbons .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
F<br />
140 1989 EMS Abstracts<br />
Notes EFFECT OF GLUTATHIONE ON MITOMYCIN-C IN SWISS ALBINO MICE AND HUMAN LY1pNDCYTES<br />
RITA NARAM . D . GsethanJali <strong>and</strong> P .P. R@My<br />
Institute of Genetics Hospital for Genetic Disaases . owania Universitv . Baauaoet . Hvd.rabad-300 016 .<br />
A.P . India .<br />
AWIMOT<br />
Mito,yc in .C (MMC) is a potent antibiotic with autayenic <strong>and</strong> antitumor activity presusably through<br />
its covalent binding to DNA. MMC was tested for its autapanic potential in the presanca of plutethiona<br />
(GSH)in invivo <strong>and</strong> invitro systas .<br />
404<br />
Swiss albino .ice were fed orally with 2s0/Kp . b .w. M(C <strong>and</strong> 20.40 .80 <strong>and</strong> 160 ap/kR b .w . GSH slmultaneously<br />
to four groups of anLsals . The doses were administered at 0 hrs <strong>and</strong> 24 hrs . 6 hrs after the second<br />
dose the animals wra killed <strong>and</strong> aicronucleus test was dona as described by Schsid (1973) . Ll.phocyte<br />
cultures ware initiated fros healthy donors <strong>and</strong> 0 .2 uylUl MMC <strong>and</strong> 2 .5 . 5 .0 . 10 .0. 10 .0 <strong>and</strong> 20 .0 up/al<br />
GSH was added simultaneously to four se-ts of cultures . 72 hrs after treataant the cultures were tenli-<br />
neted <strong>and</strong> slides ware prepared as described by Moorhead et al ( 1960) . Thare was a significant decreese<br />
in the frequency of aicronuclei !n young erythrocytes <strong>and</strong> chroaosasal ebearrations in lyaphocytes<br />
treated with MMC in casbination with OSH when coapered to MMC alone .<br />
405<br />
EFFECT OF NICOTINAMIDE ON HEPATOTOXICITY ASSOCIATED WITH CARBON TETRACHLORIDE,<br />
BROMOBEN2ENE AND ALLYL ALCOHOL, L .M .Narurkar, J .P .Ramat, S .J .D'Souza, R .Krishnamoorthy<br />
& M .V .Narurkar, Biochemistry Division, Bhabha Atomic Research Centre,<br />
Bombay, India .<br />
Recurrent toxicity with cell necrosis resulting in regenerative stimuli has been<br />
proposed as one of the mechanisms of tumour promotion . Tumour promoters which are<br />
usually hepatotoxic agents may bring about selective inhibition of the cytochrome<br />
P-450 dependent microsomal drug metabolizing enzymes . It was observed that administration<br />
of tumour promoting <strong>and</strong> hepatotoxic agents such as carbon tetrachloride<br />
(0 .2 ml/kg .b .w .), bromobenzene (0 .2 ml/kg . b .w .) <strong>and</strong> allyl alcohol (30 mg/kg . b .w .)to<br />
rats brought about a significant inhibition of hepatic mixed function oxidase<br />
(MFO) system, while the conjugating UDP-glucuronosyl transferase activity was<br />
increased . The serum glutamate-oxaloacetate transaminase <strong>and</strong> serum glutamatepyruvate<br />
transaminase activities were also highly increased followin~ administration<br />
of these toxic agents . Further, when nicotinamide (250 mgs/kg . b .w. , an endobiotic<br />
shown in our laboratory to be an inducer of MFO, was administered simultaneously to<br />
rats along with hepatotoxic agents in independent experiments, the biochemical toxic<br />
manifestations were significantly modified . Besides the MF0 inducing ability,<br />
nicotinamide has been shown to bring about stabilization of polysomes as evidenced<br />
by marked reduction in monomers <strong>and</strong> dimers with simultaneous increase in the heavier<br />
aggregates, increase in total polysomal RNA accomganied by decreased alkaline RNase<br />
activity as well as enhanced incorporation of 1 C-leucine pulse in the ribosomal<br />
aggregates .<br />
406<br />
COMPARISON OF SISTER CHROMATID EXCHANGES IN SPLEEN AND THYMIC LYMPHOCYTES FROM AKR,<br />
B6D2F1 AND CBA MICE FOLLOWING IN VIVO EXPOSURE TO N-NITROSO-N-METHYLUREA . R .E . Neft,<br />
H .M . Schol <strong>and</strong> D .A . Casciano, National Center for Toxicological Research, Jefferson .<br />
AR 72079<br />
In order to evaluate the ability of the in vivo/in vitro murine lymphocyte sisterchromatid<br />
exchange (SCE) assay to predict carcnicity . SCE induction by N-nitro-<br />
N-methylurea (MNU) was studied in spleen <strong>and</strong> thymus lymphocytes from ARR mice which<br />
are highly susceptible to MNU-produced thymomas, CBA mice which are much less sensitive<br />
to induction of thymomas by MNU, <strong>and</strong> B6D2F1 mice . Following a single i .p . injection<br />
of 0 .033, 0 .066, 0 .131 mmol/kg MNU or PBS (vehicle control), clear dose-related<br />
increases in SCE were observed in LPS-stisulated spleen lymphocytes <strong>and</strong> Con A-stimulated<br />
spleen <strong>and</strong> thymus lymphocytes from ARR, CBA <strong>and</strong> B6D2F1 mice at 1 <strong>and</strong> 24 hours<br />
post-exposure . In general, MNU-induced SCE were higher in Con A-stimulated spleen<br />
lymphocytes compared to LPS-stimulated spleen lymphocytes <strong>and</strong> Con A-stimulated thymas<br />
lymphocytes from each mouse strain . On the whole, MNU-produced SCE were lower in<br />
AKR <strong>and</strong> CBA spleens than in B6D2F1 spleens . In addition, for the most part, l4iUinduced<br />
SCE levels in thymus lymphocytes from all three strains of mice were similar .<br />
In the present study, differences in MNU-induced genotoxicity in ARR, CBA <strong>and</strong> B6D2F1<br />
thymus lymphocytes could not be ascertained by use of the in vivo/in vitro SCE assay .<br />
(<br />
r<br />
F 50869 3654<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
407 1989 EMS Abstracts 141<br />
GENOTOXICITIES OF N-NITROSAMINES ON DROSOPHILA AND A SEARCH FOR THE INHIBITORS OF THE Notes<br />
TOXICITIES<br />
Tomoe Negishi, Teruko Shiotani <strong>and</strong> Hikoya Hayatsu<br />
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan<br />
N-Nitrosamines are potent carcinogens for rodents, <strong>and</strong> their possible rr_levance to<br />
human cancer has been intensively studied . In spite of the strong carcinogenic activities,<br />
this class of compounds show only weak mutagenicity in the Ames bacterial tests .<br />
We have now found strong genotoxicities for several N-nitroso compounds using the Drosophila<br />
wing spot test . The test detects the activity of a given compound to cause gene<br />
mutations <strong>and</strong> recombinations in the larval somatic cells . N-Methyl-N-nitrosourea (MNU)<br />
<strong>and</strong> N-nitrosodimethylamine (NDMA) gave strong responses in this system, corresponding<br />
to their potent carcinogenicities . Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)<br />
is a strong mutagen against bacteria, its genotoxicity on Drosophila was moderate . The<br />
order of genotoxicities of these compounds in Drosophila was as follows ; MNU> NDMA 3-Nnitrosomorpholine<br />
> N-nitrosopiperidine Z N-nitrosodiethylamine > MNNG > N-nitrosopyrrolidine<br />
. Non-carcinogenic N-nitrosodiphenylamine <strong>and</strong> N-nitrosoproline were not genotoxic<br />
to Drosophila . Since this Drosophila system seems to be suitable for the detection of<br />
N-nitrosamine-mediated genotoxicities, it may be expected that this system is useful<br />
to search for modulators of the genotoxicity . We examined the inhibitory potential of<br />
several vitamins on the NDMA-mediated genotoxicity in Drosophila . We observed, however,<br />
no suppressive effect in 6-carotene, vitamin B1, vitamin C or vitamin D2 on the genotoxicity<br />
of NDMA .<br />
408<br />
EFFECT OF POLYUNSATURATED FATTY ACID ON DMH INDUCED COLON CANCER .<br />
Nehru, B ., Kaur, R ., <strong>and</strong> Bansal, M .P .<br />
Deptt . of Biophysics, Panjab University, Ch<strong>and</strong>igarh-160014, India .<br />
The :e is a strong positive correlation between the dietary fat consurption <strong>and</strong> the<br />
prevalence of cancer of breast, colon, prostate <strong>and</strong> pancreas . The underlined mechanism<br />
are quite complex, multifactorial <strong>and</strong> different in different types of cancer . The<br />
present study was undertaken to study the role of unsaturated fatty acid in the initiation<br />
of colon cancer by a chemical carcinogen 1,2 Dimethyl-hydrazine administered<br />
in a dose of 20 mg/ g body weight / week subcutaneously . Three levels of dietary fat<br />
manipulation was used 10% fat, 20% fat, <strong>and</strong> 30% of the total calore . Marked alteration<br />
were seen in the drug metabolizing enzyme of colon follgwing a high fat dietary regime .<br />
The observations were similar when DMH was given along with the high fat diet . The<br />
cytochrone P 450 content showed a 4 fold increase in the high fat group . A decrease<br />
in phase II enzyme namely glutathione <strong>and</strong> glutathione-S-transferase was noted .<br />
409<br />
A MULTI-FACTOR RANKING SCHEME FOR COMPARING THE CARCINOGENIC ACTIVITY OF CHEMICALS .<br />
S . Nesnow, Carcinogenesis <strong>and</strong> Metabolism Branch, U .S . <strong>Environmental</strong> Protection Agency,<br />
Research Triangle Park, NC 27711 USA<br />
This activity scheme uses as its base, dose potency measured as TD50 (Gold et al .,<br />
Environ . Health Perspect ., 67, 161-200, 1986) The TD,r0 is converted into an inverse<br />
log scale, a decile scale, <strong>and</strong> then adjusted by weighting factors that describe other<br />
parameters of carcinogenic activity . These factors include positive or negative<br />
weightings for the induction of tumors at tissues or organs associated with high<br />
historical control tumor incidences ; the induction of tumors at multiple sites ; the<br />
induction of tumors in both sexes of the species ; <strong>and</strong> the induction of tumors in more<br />
than one species . In order to construct a measure to express the inactivity of<br />
chemicals as inducers of cancer, a measure analogous to the TD50 has been developed :<br />
the highest average daily dose or HADD . The HADD is the highest average daily dose in<br />
mg chemical/kg body weight administered in a chronic cancer study, <strong>and</strong> that did not<br />
induce a statistically significant increase in tumors . HADD values were converted to<br />
log decile units <strong>and</strong> adjusted by weighting factors relating to inactivity in both<br />
sexes of a species, <strong>and</strong> the inactivity in more than one species . Three activity<br />
ranking schemes were developed using a 142-chemical data set from NTP studies : the<br />
Carcinogen Activity-F344 Rat, an activity scheme based on cancer data obtained with<br />
the F344 rat ; the Carcinogen Activity-B6C3F1 Mouse, an activity scheme based on cancer<br />
data obtained with the B6C3F1 mouse, <strong>and</strong> the Carcinogen Activity-Combined, an activity<br />
scheme based on selecting data from both the F344 rat <strong>and</strong> the B6C3F1 mouse .<br />
This is an abstract of a propos .d pnfantation <strong>and</strong> does not nflact EPA policy .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
142 1989 EMS Abstracts _ 410<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
NOteS- "" y'"RECOMMENDED 1TAMM PROTOCOLS BASED ON A SURVEY OF CURRENT PRACTICES IN GENOTOXICITY<br />
TESTING IABOR{RORIES . E .R . Nestmann(1), S .H .H . Swierenga, R .L . Brillinger(1), <strong>and</strong><br />
J .P .W. Gilman ~.Directorate, Health Protection Branch, Ottawa, <strong>and</strong> (1) CanTox<br />
Inc ., 627 Lyons lane, Suite 200, Oakville, (Canada)<br />
The most commonly used genotoxicity assays for cultured mammalian cells are<br />
mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, <strong>and</strong> cell<br />
transformation . Since their inception, protocols for these assays have been modified<br />
in various laboratories . It has been observed that minor but potentially significant<br />
method modifications frequently remain unpublished (Swierenga et al ., J . Tiss . Cult .<br />
Meth . 8 :7, 1983) but should be considered in the development of st<strong>and</strong>ard protocols .<br />
The present study was undertaken to determine the current "state of the art" for these<br />
tests . Detailed questionnaires on culture conditions <strong>and</strong> testing protocols for both<br />
stock <strong>and</strong> test cell populations were designed with the assistance of an international<br />
advisory committee <strong>and</strong> sent to all research <strong>and</strong> contract laboratories that could be<br />
identified in Canada, USA, <strong>and</strong> Europe . Responses from 425 completed questionnaires<br />
were analyzed to determine the most commonly used approach <strong>and</strong> modifications for each<br />
procedural step . As expected, the results show a large degree of interlaboratory<br />
variation . Detailed protocols for conducting each assay have been prepared <strong>and</strong><br />
include : stepwise instructions, precautionary measures <strong>and</strong> practical solutions to<br />
common problems associated with each assay ; recipes for media <strong>and</strong> solutions ; formulas<br />
for quantifying genotoxic responses ; reference lists of related assays ; guidelines for<br />
interpretation ; <strong>and</strong> discussions of the applications, advantages <strong>and</strong> disadvantages of<br />
each test .<br />
411<br />
CRITERIA FOR ASSESSMENT OF GENOTOXICITY DATA WITH RESPECT TO IN VIVO MUTAGENICITY AND<br />
MECHANISM OF CARCINOGENICITY . E .R . Nestmann <strong>and</strong> L .D . Kier, CanTox Inc ., Oakville<br />
(Canada), <strong>and</strong> Monsanto Co ., St . Louis (U .S .A .)<br />
Evaluation of genotoxicity databases often is complicated by a mixture of positive<br />
<strong>and</strong> negative results . We have identified general criteria that can be used in a<br />
"weight of evidence" assessment . Evidence for Jja y1yQ genotoxicity is strenothened by<br />
any or all of the following : induction of genetically stable effects (i .e ., mutation)<br />
rather than indicator endpoints of DNA damage (e .g ., SCE, UDS) ; activity for multiple<br />
endpoints ; in vivo (rather than only in vitro) effects ; induction by appropriate route<br />
of exposure <strong>and</strong> at not overtly toxic doses ; structure <strong>and</strong> pattern of activity related<br />
to known mammalian mutagens ; strong responses . The case for j,p yjys genotoxicity is<br />
weakened by : negative in vivo results ; positive results only In vitro, especially<br />
activity that is reduced or eliminated in the presence of metabolic fractions ; activity<br />
that is only observed under conditions known to cause artifacts (e .g ., high<br />
cytotoxicity or osmolality, low pH) . Evidence for carcinogenesis through a genotoxic<br />
mechanism is strengthened by : consideration of the above criteria for in vivo<br />
genotoxicity ; correlation of in vivo genotoxicity <strong>and</strong> carcinogenicity findings (e .g,<br />
tissue or species specificity ; route) ; similar pattern of response as chemically<br />
related carcinogens . Evidence for a nongenotoxic mechanism is strengthened by :<br />
activity only in tests with low specificity ; other evidence for nongenotoxic or<br />
promoter mechanism of carcinogenesis . (Supported in part by the AIHC, CMA <strong>and</strong><br />
ILSI/RSI ; contributions of the AIHC Mutagenicity Subcommittee are gratefully<br />
acknowledged .)<br />
412<br />
MOLECULAR ANALYSIS OF Zl= MUTATIONS ARISING jg VIVO IN HUMAN T-LYMPHOCYTES, J .A . Nicklas,<br />
T .C . Hunter, J .P . O'Neill, L . Recio, D . Simpson, T .R . Skopek, <strong>and</strong> R .J . Albertini, VRCC<br />
Genetics Lab, Univ . of Vermont, Burlington, VT <strong>and</strong> CIIT, Research Triangle Park, NC .<br />
hort <strong>and</strong> T-cell receptor (TCR) Southern blot analyses were performed on 96 wild type<br />
<strong>and</strong> 330 somatic h= mutant clones from three normal individuals . h= analysis showed<br />
that 16 .0, 16 .5 <strong>and</strong> 9 .6% of the mutations had visible b= structural alterations in the<br />
3 individuals, respectively . The breakpoints of the deletion mutations were spread across<br />
the gene in proportion to length (r- .94) with 0 .73 breaks/kb, allowing an estimate of the<br />
nearest flanking vital genes of 18kb 5' <strong>and</strong> 26 .5kb 3' . TCR analysis determined that 91,<br />
85 <strong>and</strong> 85% of the mutants were i .ndependent T celi clones (i .e . had different TCR gene<br />
rearrangements with TCRj8 or y gane probes) . Doublets, triplets, quadruplets <strong>and</strong> one<br />
nonamer ∎et of sibling clones were observed . The average persistence of a clone was<br />
2 .8,2 .9 <strong>and</strong> 1 month, respectively although the nonamer siblings persist after 2h years .<br />
We have previously described the extensive proliferation of a T call clone in another<br />
normal individual resulting in an elevated mutant frequency with greater than 90% sibling<br />
clones . One individual had 6 mutant clones with the saue apparent exon 2-3 deletion <strong>and</strong><br />
new size axon 1 fragment ; by TCR analysis 4 are sibling clones while 2 share only TCRB<br />
gene rearrangements . This latter fact suggests an intrathymic mutation or extra-thymic<br />
TCRy gene rearrangement . This same apparent deletion was found in a mutant from another<br />
individual suggesting a"hotspot" for mutation . We found two cases of an apparent TCR<br />
clone (i .e . the same TCR gene rearrangements) containing 2 different b= mutations which<br />
is evidence for sensitivity of a dividing TCR clone to mutation . We are currently<br />
sequencing the mutants which did not show hp= gene alterations to define a spectrum of<br />
spontaneous" somatic mutation in man . Supported by NCI CA30688 .<br />
50869 3656
413 ~ 1989 EMS Abstracts 143<br />
DNA SEQUENCE CHARACTERIZATION OF ALKYLATION-INDUCED YERMILION Y{TPANTg IN DROSOPBILA. Notes<br />
t( .J .11 . Nivard, A .PastinkY<strong>and</strong> E .1f .Voge1, University of Leiden, Leiden (The Netherl<strong>and</strong>s) .<br />
The aim of this study hsi been the characterization of base sequenoe changes induced<br />
by ethylnitrosourea (ENU), ethylmethanesulfonate (Et(8), <strong>and</strong> methylaetbanesulfonate<br />
(MMS) at the vezm{Zion locus of Drosophila, after treatment of postmeiotie male germ<br />
cells . The analysis included consideration of the position of the carcinogen on the<br />
potency scale for carcinogenicity (TU50 in relation to initial alkylation pattern ;<br />
collaboration with Drs .B .Bartsch <strong>and</strong> A .Barbin, IARC, Lyon), <strong>and</strong> alteration of the relative<br />
distribution of primary DNA lesions . Sequence analysis of mutants was performed<br />
using the recombinational screening method by 8eed in combination with dideoxy-sequencing,<br />
<strong>and</strong> the PCR method . All vermtZion mutatlons induced by ENU <strong>and</strong> EtE were due to<br />
base-pair changes, <strong>and</strong> all 22 mutations induced by EIIS represented OC : AT transitions .<br />
With ENU, 29 nucleotide substitutions were found in 26 mutants (3 mutants had 2 basepair<br />
changes) . Of these mutations, 23/29 (79%) were transitions <strong>and</strong> 6/29 (21%) transversions<br />
. In 18/29 (62%) of the mutations tiC : AT transitions were observed . Our results<br />
support the view that 08-ethylguanine is the premutagenic lesion in DrOsophila after<br />
treatment with EYS <strong>and</strong> ENU . In the latter case, other products of O-ethylation (02EtT,<br />
04EtT) seem to be involved . Characterization by DNA sequencing of 40 mutations induced<br />
by MMS revealed a decidedly different spectrum in relation to EMS <strong>and</strong> ENU : 21/40<br />
mutations (53%) were tranversions, 8/40 (20%) deletions <strong>and</strong> 10/40 (25%) transitions .<br />
Misrepair <strong>and</strong> indirect miscoding seem the predominant mechanisms of action of high S<br />
value type carcinogens . It is this type of genotoxin for which strongest hypermutabllity<br />
effects are obtained in an excision repair defective background .<br />
414<br />
NA REPAIR TESTS ON FOOD ADT)ITIVBS<br />
N'ichiko Non :,ka<br />
iaboratory of Food Chemistry, Faculty of Agriculture,<br />
Kyushu University, ?iunzoka, Japan .<br />
The liquid Bacillus subtilis rec-assays were carried out 09 25 food<br />
additives, all of which are eurrently used in Japan . Briefl .y, the overniBht<br />
culture of . subtilis strains, H17 eLnd ;d45, was mixed with test<br />
solutions <strong>and</strong> incubated for 30min at 37'C . After treatment, viable<br />
cells aere co•.i .Vltec2 <strong>and</strong> the ratio of 5M, .• stu'vi,va.l, coace:ltr,itions were<br />
calculated . 18 additives ((acetone, L-asoorbic acid, bettsoio acid, ethyl<br />
acetate, Food Red No .2(tanaranth), Food 3ed No .3(erythrosine), Food Red<br />
No .106(acid red), Food Yello•,v :To .h(tartrazine), glycerin, hexane, pota,ssium<br />
sorbate, propylene rlycol, nropyl gel3e.te, sacchexin sodium,<br />
socitvn benzonte, sodium dehydro3cetate, sodium nitrate, sodium sl-te .rtrste)<br />
ehovaed D :•', damaging potential e.lthough they had been negative in<br />
the ~ .~nes test . Seven additives (erythorbic acid, Food Green No .3(Fast<br />
Green FCP), hydrogen peroxide, potassium bromate, soflium nitrite, cacao<br />
;i&nent, cochineal) showed D'Ta damaging potential, which had been positive<br />
in the Ames test . These s+tr~-eRi ; ''s,+." ~; the ltj,tt' Bs subtilis<br />
rec-=- se : y 1-~ y be more sensitive than the Ames test . -<br />
415<br />
THE MICRONUCLEUS ASSAY IN LYMPHOCYTES.<br />
H . Norppa, M . Hayashi, <strong>and</strong> J . Miki-Paakkanen, Institute of Occupational Health, Topeliuksenkatu 41 aA,<br />
SF-00250 Helsinki, Finl<strong>and</strong> .<br />
Cytogenetic monitoring of human populations exposed to genotoxic agents has almost exclusively<br />
relied on the use of peripheral lymphocytas . The sensitivity of the lymphocyte culture system In detecting<br />
in vivo exposure may not always be as good as desired, but it could be improved by more extensive<br />
analyses, method development <strong>and</strong> automation . In the human lymphocyte micronucleus assay, a major<br />
break-through has been the use of cytochalasin B, introduced by Fenecb <strong>and</strong> Morley, to idenNfy cells<br />
that have divided once in the culture . The recognition of these eells by the presence of two nuclei has<br />
abolished the earlier problems experienced with differences in cell growth <strong>and</strong> haa made the scoring of<br />
micronuclei in lymphocytes more accurate . Our experience on human monitoring studies, however, suggests<br />
that - in order to catch the cells after their first division - cytochalasin B treatment has to begin<br />
considerably earlier than originally suggested . As cytochalasin B appears to have some toxic effects on<br />
the lymphocytes, it would be good to have an alternative way to recognize the relevant eell population .<br />
At our laboratory, we have been developing a technique based on S-bromodeoxyuridine (BrdU)<br />
incorporation combined with a monoclonal antibody against BrdU-DNA, in order to produce a method that<br />
could be used as a basis for automated scoring of micronuclei by image analysis . BrdU can also be used<br />
without the antibody by applying a differential staining technique, but the antibody method is more<br />
specific <strong>and</strong> more suitable for automation, because only labelled nuclei are visible. For both of these<br />
approaches, the timing of BrdU treatment <strong>and</strong> cell harvest are ths critical points . It Is probable that<br />
micronucleus analysis in lymphocytes, with its rapid scoring <strong>and</strong> suitability for automation, will be an<br />
important alternative for the traditional analysis of metaphase chromosome aberrations in monitoring<br />
human exposure to genotoxins as well as in screening of elastogens in vitro .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
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144 1989 EMS Abstracts - • s --<br />
0http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
416<br />
NOteS ^ ENVIRONMEN?AL MaU AGENESIS RESEARCH IN DEVELOPING COUNTRIES<br />
N .K . Notani, Biorcal Group, Bhabha Atanic Research Centre, Borbay 400085, India<br />
Indian reseaTcFfers-~trave wide interests <strong>and</strong> work on problens ranging from DNA repair,<br />
gene cloning, mutagens <strong>and</strong> entinutagens, test-syste•ns to nature of heritable variation in<br />
man, etc. Environ•nental Mutagen Society of India affiliated to IAEMS provides a major<br />
annual forun within the country <strong>and</strong> has also periodically organized courses, both international<br />
<strong>and</strong> national, for teaching of new techniques. Besides, it has cosponsored m eetings in related<br />
areas with other societies. In our Biorrsedical Group which m ay be considered as representative<br />
of research activities in the country has projects on mutagenicity testing, modification of<br />
mutagenicity by antrnutagens, mutation surveillance in control <strong>and</strong> high background radiation<br />
areas, cloning of DNA repair genes, Restriction Fragnent Length Pol)morphisn analysis<br />
<strong>and</strong> ostrn eiotic expogure leading to transn issible genetic dan age . Two of the researches<br />
are ~escribed below : i) DNA repair <strong>and</strong> gene cloning : We have self-cloned two genes, uvrl<br />
<strong>and</strong> mbo2, in Haenophi(us Tnt uenzae, u g e s carried on an 11 .3kb insert. This OIm1<br />
clone fully canple•nents UV-sensitive strain Uvr1- . A directedtnutagenesis system was<br />
developed which then induces mutations only in the 11 .3kb insert (Kanade <strong>and</strong> Notani,1987) .<br />
Another DNA repair gene mbo2 has also been cloned which fully canplenents UV-sensitive<br />
Mbo2- strain but not UvrT~strain . Subcloning of two EcoRl fraqnents yield clones; only<br />
the snaller of which partially canple•nents UV-sensitivity of Mbo2- strain (Mody, Joshi<br />
<strong>and</strong> Notani, 1989). ii) Newborn surve of o ulations residi in control <strong>and</strong> hi h bac round<br />
radiation areas: Georg e et ai have exa~n ne new orns for wn syn ro•ne <strong>and</strong><br />
'R>;L'h-06TSi1S88- date for Incidence related to m aternal age . Although, the data fror high<br />
background radiation areas are linited, these nevertheless see•n to be sinilar in incidence<br />
to the control area .<br />
CROSS ADAPTIVE RESPONSE BETVEEN HYDROGEN PEROXIDE AND ALDEHYDES IN E . croli. .<br />
T . NUNOSHIBA, M . HASHIMOTO, <strong>and</strong> H . NISHIOKA, Biochemistry . Doshisha University, Kyoto(Japan)<br />
417<br />
The phenomenon of the adaptive response that the bacterial cells acquire resistance to killing<br />
effect of a lethal dose of hydrogen peroxide(H202) . when they have been pretreated with<br />
its sublethal dose . has been noticed . Ve report here the existence of the adaptive response<br />
between H202 <strong>and</strong> aldehyde compounds . The phenomenon was na .ed by us as "Cross Adaptive<br />
Response" . The cells of E .2Qjj VP2 pretreated with a sublethal dose (30 or 80µM) of HzOz for<br />
30.in at 3TC acquired remarkable resistance to killing effect of for .aidehyde(FA) challenge<br />
at the concentration of B .M . The resistance acquirement due to pretreatment with a low concentration<br />
of H202 was similarly observed in the cases of challenge against the other aldehyde<br />
compounds such as chloroacetaldehyde . glutaraidehyde, glyoxal <strong>and</strong> methyl glyoxal . To elucidate<br />
the .echanism of the "Cross Adaptive Response", the effect of pretreatment with Hz02 on<br />
FA-sensitivity of ji . i strains with different DNA repair capacities was examined . The acquire.ent<br />
of the resistance was observed in VP7uvrA[V1f.l1] <strong>and</strong> ZA12[ug&] . but not in ZA80<br />
[recA] <strong>and</strong> CM561[IexA] . These results suggest that the "Cross Adaptive Response" Ray be attributed<br />
to induction of SOS genes coding some enzymes which are involved in reco .bination<br />
repair . the other unknown DNA repairs <strong>and</strong> aldehyde decomposition .<br />
418<br />
Cenotoxic effects of creosote oils : a relationship between ssutagenicity <strong>and</strong> the concentrations<br />
of A-S ring polycyclic aromatic hydrocarbons<br />
Yylund, L ., Heikkili, P ., Ji[rventaus, H ., Suhonen, S . . Hesso, A ., Himeill . !( . .<br />
Linnainsiaa, K . <strong>and</strong> Sorsa, tl .<br />
Institute of Occupational Health, Topeliuksenkatu •laA, Sy-00250 Helsinki, rinl<strong>and</strong><br />
Pour creosote oils iaported to Finl<strong>and</strong> from different countries were tested using<br />
the Ames test, the SCS-test with CHg-c .lis <strong>and</strong> an automated version of the SOS chrosotest<br />
. All creosote oils were positive using $ . tvohimurius TA98tS9aix . With strain<br />
TA100aS9aix only two of the creosote oils were positive . The SCS-test showed results<br />
similar to the Ames test results with strain TA98i89aix . The two oils that were positive<br />
both with TA98 <strong>and</strong> TAlOO were also positive in the 808 chromotest .The taped plate<br />
assay for compounds evaporating at 37•C gave negative results for all the four creosote<br />
oils . The creosote oils wre fractionated by distillation <strong>and</strong> the fractions were<br />
tested using the Ames test . Mostly, the wtagenic activities were observed in fractions<br />
with high-boiling compounds . The cheaical compositions of the creosote oils were<br />
determined using OC/!tS-techniques . It was found that the wtagenic activities with<br />
TA98a89mix correlated well (r .0 .96, p
419<br />
,<br />
AN SVALUATION OF TBB C90/BGPRT MUTATION ASSAY INVOLVING SOSPSNSION CULTURES AND SOFT<br />
AGAR CLONING : RESULTS FOR 33 CHBKICALS . T . Oberly, M . Rexroat, s . Devsey, <strong>and</strong> K .<br />
Richardson . Lilly Reseatrh Laboratories, Rli Lilly & Co ., Creenfield, IN 46140 .<br />
The Chinese hamster ovary cell assay (C80) which measures forward mutation at<br />
the BGPRT locus is utilized in several laboratories for the detection of mutagens . A<br />
procedure involving treatment of CHO cells in suspension culture <strong>and</strong> mutant selection<br />
in soft agar cloning has been developed (Oberly et al ., Mut . Res ., 18St99, 1987) . In<br />
order to evaluate the effectiveness of these modHications, ff-chemMals representing<br />
six chemical classes were tested, <strong>and</strong> the results were compared to findings obtained<br />
with the additional assays "ployed in the battery of tests . A positive response was<br />
obtained with 20 chemicals, all of_vhich are recognized sutagens . Of the 14<br />
compounds that produced negative results, 4 were considered to be sutagens <strong>and</strong>/or<br />
carcinogens . For example, the rat hepatocarcinogen methapyrilene was negative in the<br />
CHO assay, <strong>and</strong> this finding agreed with all six test systems in the battery . In<br />
contrast, the suspect carcinogen, 4-nitrobiphenyl, was negative in the C80 assay but<br />
positive in 3 of 5 other test systems . Twelve of the compounds mentioned in this<br />
report have been previously tested in other laboratories, <strong>and</strong> as a comparison, the<br />
results reveal strong agreement between laboratories . These findings confirm that<br />
the use of suspension cultures <strong>and</strong> soft-agar cloning in the CBO assay maintains the<br />
sensitivity of the test in the discrimination of mutagens <strong>and</strong> is a viable alternative<br />
to the traditional monolayer procedure of O'Neill et al . (Hut . Res ., 59 :109, 1977) .<br />
In addition, the modified CHO assay has contributeT-to an overalrTsptovement in the<br />
cost effectiveness for the assay which will be discussed .<br />
420<br />
SISTER-CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS INDUCED BY<br />
CISPLATIN IN CULTURED HUMAN LYMPHOCYTES AND INHIBITORY EFFECTS BY<br />
SODIUM THIOSULFATE .<br />
T .Ohe, S .Tsuda*, Y .Sakata <strong>and</strong> T .Abe, Department of Hygiene <strong>and</strong> *Medicine,<br />
Kyoto Prefectural University of Medicine, Kyoto 602(Japan)<br />
Cisplatin(CDDP) is one of the most commonly used cytotoxic chemotherapeutic<br />
agents, but has serious side-effects including renal <strong>and</strong><br />
haematological toxicities . On the other h<strong>and</strong>, it is known that CDDP<br />
therapy combined with STS brings effective relief from such severe sideeffects<br />
. In the present experiment, we examined the cytotoxicity,<br />
SCEs <strong>and</strong> chromosome aberrations induced by CDDP in cultured human<br />
lymphocytes, <strong>and</strong> in vitro interaction of CDDP4<strong>and</strong> STS on the frequencies<br />
of SCEs <strong>and</strong> chromosome aberrations .<br />
The mitotic index decreased ablyptly at 2 x_610-6 M <strong>and</strong> the yield of<br />
SCES was dose-related between 10- <strong>and</strong> 4 x 10 M . A dose-dependent<br />
increasg in frequency of chromosome aberra~ions w_is also found ug to<br />
4 x 10 M . Simultaneous treatment of 10 - 10 M STS <strong>and</strong> 10 M CDDP<br />
significantly reduced the number of CDDP-induced SCEs . Furthermore,<br />
treatment of cells with STS 3-hr later after CDDP administration<br />
significantly prevented toxicities as revealed by SCEs <strong>and</strong> chromosome<br />
aberrations .<br />
421<br />
RELATION OF LIVER C:.RCItiOt~.A TO ENVIRONME.NTAL FdC':ORS IN A TROPICAL RAIN-BELT CITY .<br />
:,.E . Ohwovoriole <strong>and</strong> G .C.E, Okeke, Dept . of Medicine, College of Medicine, University<br />
of Lagos, LAGOS, ATGERIA .<br />
Carcinoma of the liver is the commonest malignancy in many parts of tropical<br />
Africa . The reasons for its high prevalence in many countries are not certain but<br />
there are pieces of epidemiological evidence from other countries that environmental<br />
factors may play a dominant role in the aetiology of liver cancer in the tropics .<br />
Here we present an analysis of environmental factors in 104 patients with carcinoma<br />
of the liver seen in a large tropical hospital in the rain belt of Nigeria . There<br />
was a male predominance . Over 300 of the patients had historical or biochemical<br />
evidence of serum hepatitis (hepatitis B virus infections) . About 30 to 40 percent<br />
respectively gave a fairly strong history of alcohol <strong>and</strong> tobacco . A large rnanber of<br />
the patients admitted to ingestion of local herbs in the form of concoctions . The<br />
occurrence of ingestions of peanuts was no higher than in hospital controls .<br />
<strong>Environmental</strong> factors appear to contribute significantly to the occurrEnce of<br />
carcinoma in Lagos like in the rest of tropical Africa . However the sex ratio <strong>and</strong><br />
peak age differ somewhat from that reported from the northern part of the country .<br />
The aetiology of liver carcinoma appears multifactorial . There is need to study<br />
further the role of co-carcinogens or other mutagens besides hepatitis B virus in the<br />
causation of liver carcinoma .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 145<br />
Notes
146 1989 EMS Abstracts .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
- r~s -<br />
Notes - NOLECUIJ1It~YSIS Y OF N-HYDROXY-2-ACETYLMINOFUlONF1V~-INUCED MVfATIGNS IN<br />
CHIN~SE CELLS LnCRING DiA ERCISION REPAIR . R .T. Okinaka, S .L . Ansick,<br />
<strong>and</strong> G .F . StrQis~i..Genetics Group, Life Sciences Division, Los Alamos National<br />
Laboratory, LZSs-amos, M 87545 (USA) .<br />
It is generally believed that damage to DNA can be fixed as heritable mutations<br />
that are a conssquence of errors in repair or replication through the<br />
damaged sites . In order to better underst<strong>and</strong> the role of DNA replication in the<br />
fixation of mutation, we have employed in our studies a DNA excision repairdeficient<br />
Chinese hamster cell line (CH0 W-5) . initial experiments were<br />
designed to define the magnitude of possible deletion mutations induced at the<br />
X-linked hypoxanthine-guanosine phosphoribosyl transferese (HPRT) locus<br />
N-hydroxy-2-acetylaminofluorene (N-OH-AlLLr) using restriction enzyme digesti~<br />
Southern blotting <strong>and</strong> probing techniques (with a cloned hamster V79 cell HPRT<br />
cDNiA) . However, our observations indicate that none of the N-OH-AAF-induced<br />
UV-5 mutants analyzed to date contain large deletions or rearrangements at this<br />
genetic locus . In order to define the molecular nature of these mutations, we<br />
have recently employed a modification of the polymerase chain reaction (PCR)<br />
technique in conjunction with direct sequencing strategies . Preliminary results<br />
indicate that some of these mutants contain truncated HPRT s*As, possibly the<br />
consequence of small deletion events or processing errors in introNexon<br />
splicing . (This research is supported by the U .S . Department of Ehergy under<br />
contract W-7405-ENC--36)<br />
422<br />
423<br />
OVEP.VIEW OF IN VIVO MATQIALIAN TESTING SYSTEMS . F . B . Oleson, Bristol-Myers Company,<br />
Syracuse, New Yorc-(USA) .<br />
Both somatic <strong>and</strong> germ celle can be targets for genetic damage . In vivo mammalian<br />
assays for both types of damage will be described . Germ cell assays incfude spermatocyte<br />
cytogenetics or the new biochemical specific locus assay (currently under<br />
validation), specific locus <strong>and</strong> heritable translocation assays, <strong>and</strong> the rodent<br />
dominant lethal assay . Somatic cell assays have historically focused on the bone<br />
marrow, including cytogenetic studies for chromosomal aberration (CA) or, more<br />
recently, the erythrocyte micronucleus test . Other in vivo tissue-specific genotoxicity<br />
assays will briefly discussed, including 1) sistar c4rcomatid exchange (SCE)<br />
in bone marrow, 2) in vivo/in vitro hepatocyte DNA repair, 3) host mediated <strong>and</strong> 4)<br />
rodent lymphocyte CATSCE tests . Promising new test systems will also be presented<br />
such as the use of tranegenic mice for studies of oneogene activation <strong>and</strong> the single<br />
cell gel (SCG) DNA fragment electrophoresis assay . International regulatory<br />
guidelines for drug <strong>and</strong> chemical safety evaluation uniformly recommend one in vivo<br />
study be performed as part of a battery of mutagenicity tests . Bone marrow CK-or<br />
micronucleus assays are the recommended systems . Recent validation studies support<br />
the micronucleus test as the primary in vivo screen . The sensitivity <strong>and</strong> efficiency<br />
of these systems have been improved Sy moaffication of dosing/sampling time design,<br />
use of automated scoring techniques <strong>and</strong> the use of mouse peripheral blood for<br />
micronucleus testing . Finally, the rationale <strong>and</strong> scientific justification for the<br />
integration of multiple genetic toxicology endpoints into st<strong>and</strong>ard subacute (7- or<br />
30-day) rodent toxicology studies will be outlined .<br />
424<br />
AUTOMATION OF MOUSE PERIPHERAL BLOOD MICRONUCLEUS SCORING . F .B . Oleson . S .M . Getman,<br />
H . Mahran <strong>and</strong> G . Jenkinson, Bristol-Myers Company, Syracuse, NY (USA), <strong>and</strong> Cambridge<br />
Instruments, Inc ., Deerfield, IL (USA) <strong>and</strong> Cambridge (UK) .<br />
Procedures for the automated scoring of fluorescent stained mouse peripheral blood<br />
smears have been developed . . Peripheral blood smears stained with acridine orange are<br />
analyzed under high resolution microscopy in combination with a Cambridge Instrumants<br />
Quantimet 520 Image Analyzer . Orange-fluorescing polychromatic erythrocytes (PCEs)<br />
<strong>and</strong> yellow-fluorescing micronuclei are enumerated via the image analyzer . The total<br />
erythrocytes scanned in all fields analyzed is automatically estimated using a<br />
st<strong>and</strong>ard per field cell count . A motorized stage <strong>and</strong> autofocus allow for the rapid .<br />
analysis of individual fields for the total number of PCEs <strong>and</strong> micronucleated PCEs<br />
(MN-PCEs) . For each slide, the percent !Qi-PCEs <strong>and</strong> percent PCEs in total erythrocytes<br />
scanned are calculated <strong>and</strong> automatically tabulated by computer . Validation<br />
data comparing manual <strong>and</strong> automated micronucleus scoring will be presented including<br />
estimates of automated counting errors in the MN-PCE <strong>and</strong> PCE frequencies . Counting<br />
speed, limitations <strong>and</strong> problems with automated procedures will be discussed . Manual<br />
peripheral blood micronuclaus scoring for a st<strong>and</strong>ard study using 30 animals (1000<br />
PCEs/animal) <strong>and</strong> one evaluation time can be reduced from appror.imately 10 days to 2-3<br />
days using these automated procedures with no significant difference in results .<br />
These procedures should provide for improved assay sensitivity <strong>and</strong> reliability since<br />
appreciably more cells can be scored per animal than could be reasonably accomplished<br />
via manual scoring techniques .<br />
50869 3660
425<br />
r<br />
GENOTOXIC AND EPIGENETIC MECHANISMS OF CHEMICAL CARCINOGENESIS s IN VITRO AND IN VIVO<br />
STUDIES . L . Orfila, C . La ~ne, I . Chouroulinkov . Cancer Research Institute, C .N .R .S .,<br />
94802 Villejuif Cedex, France .<br />
Genotoxicity of chemicals ia generally considered as the only mechanism of carcinogenesis<br />
. However, it was found chemicals which are mutagenic <strong>and</strong> carcinogenic, or which<br />
are mutagenic but not carcinogenic, <strong>and</strong> finally which are not mutagenic but carcinogenic,<br />
such as TPA (PMA) . It is obvious that epigenetic mechanisms of carcinogenesis<br />
are clearly involved . The question is to know the'respective importance of each of<br />
those mechanisms in carcinogenesis . To give some answer,we have studied the effects of<br />
benzo(a)pyrene (BaP) 12-0-dimethylbenz(aYaT :thracene (DMBA), TPA (PMA),4-NQO, 3Me-4-NQO,<br />
testosterone <strong>and</strong> trembolone in the Syrian hamster embryo (SHE) cell transformation<br />
system, induction of ODC <strong>and</strong> epidermal hyperplasia as well as evaluation of BaP-DNA<br />
covalent binding in SHE <strong>and</strong> epidermal cells . Moreover, dexamethasone (D)040) was used<br />
as inhibitor of epigenetic effects in the different systems . The results showed that<br />
DXMO inhibits cell transformation,ODC <strong>and</strong> hyperplasia induction without inhibition of<br />
BaP-DNA covalent binding . From these results <strong>and</strong> data from long term skin tests we can<br />
classify the chemical carcinogens in 3 classes : with double activity,genotoxic <strong>and</strong><br />
epigenetic activity (BaP,DMBA,4-NQO), only epigenetic activity (TPA,testosterone,<br />
trembolone) <strong>and</strong> only genotoxic activity (3Me-4-NQO) . The genotoxic effect appears to<br />
play a less important role in carcinogenesis than the epigenetic one .<br />
426<br />
IMMUNOHISTOCHEMICAL LOCALIZATION OF DNA-ADDUCTS FORMED DURING DNA R6PLICATION .<br />
Ofelia A . Olivero <strong>and</strong> Miriam Poirier . Laboratory of Cellular Carcinogenesis <strong>and</strong> Tumor<br />
Promotion, National Cancer Institute . Bethesda, MD 20892 .<br />
It has been previously reported that synchronized CHO cells exposed to H-acetoxyacetylaminoflurene(N-Ac-AAF)show<br />
a consistent pattern of preferential adduct formation<br />
in specific regions of metaphase chromosomes . Preliminary experiments suggested that<br />
chromosomal adduct localization correlated with the phase of the cell cycle in which<br />
the exposure took place . To further investigate this phenomenon, shake synchronized<br />
CHO cells were given simultaneous non toxic doses of 5-bromodeoxyuridine(BraU)<strong>and</strong><br />
N-AC-AAF for 15 <strong>and</strong> 30 min periods respectively at times either early or late during<br />
the 8 hours of S phase . Cells were allowed to complete replication <strong>and</strong> were arrested<br />
in metaphase with colchicine . Metaphase chromosome spreads were processed for immunofluorescence<br />
using anti-BrdU antisera <strong>and</strong> a fluorescein :ltagged second antibody to localize<br />
sites of HrdU incorporation by DNA replicative synthesis . An antiserum specific<br />
for guanosin-(8-yl)-aminofluorene <strong>and</strong> a streptavidin-biotin second antibody system<br />
tagged with Texas red were used to localize DNA-adducts .The immunofluorescence staining<br />
showed evidence of adduct formation throughout the whole of each chromosome, but bright<br />
spots indicated regions of high adduct concentration . The BrdU incorporation sites were<br />
coincident with the areas of high adduct concentration, indicating a correlation of DNA<br />
damage <strong>and</strong> DNA replication . These results suggest that the most concentrated DNA-adduct<br />
formation is a cell cycle dependent phenomenon <strong>and</strong> occurs in those regions of the<br />
chromosome that were replicating during carcinogen exposure .<br />
427<br />
FORMATION OF MUTAGFNIC COMPOUNDS BY INTESTINAL BACTSRIA<br />
T .Osawa, M .Namiki, S .Kawakishi, K .Suzuki <strong>and</strong> T .Mitsuoka, Dept . of Food Science &<br />
Technology, Nagoya University, Chikusa, Nagoya 464-01(Japan), <strong>and</strong> The Institute of<br />
Physical <strong>and</strong> Chemical Research, Wako, Saitama 350-01(Japan) .<br />
In the past several years much attention has been given to the correlation between<br />
intestinal microflora <strong>and</strong> the formation of colonic cancer . One area of specific<br />
interest has been the correlation of microflora to cocarcinogens produced from<br />
dietary components through interference with intestinal bacteria, however, no clear<br />
evidence for this correlation has yet been obtained .<br />
Preliminary experiments showed that high mutagenic activity exists in the cellfree<br />
homogenates prepared from the six strains of intestinal bacteria in the fortyfive<br />
strains . High DNA damaging activity has been observed in the ethyl acetate<br />
extracts obtained from Streptococcus faecium IB 37 <strong>and</strong> Veillonella parvula ATCC 10790<br />
as shown by the differential growth inhibition between the Bacillus subtilis strains<br />
H17 rec+ <strong>and</strong> M45 rec- . After a large scale incubation of the strain S . faecium IB<br />
37, isolation <strong>and</strong> purification of the active principles have been carried out .<br />
Instrumental analyses confirmed the structures of two of DNA-damaging substances as<br />
indole <strong>and</strong> streptindle, respectively . Streptindole is the first genotoxic di-indole<br />
derivative found in the metabolites produced by intestinal bacteria . Streptindole<br />
was found to be negative by bacterial reversion assays with Salmonella typhimurium<br />
strains TA98, TA100 <strong>and</strong> TA104, however, positive in the colonic nuclear aberration<br />
assay using C57BL/6J mouse .<br />
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1989 EMS Abstracts 147<br />
Notes
148 1989 EMS Abstracts<br />
- .<br />
Notes -<br />
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428<br />
dv~ -- _ . _•<br />
.+ CENOTOXIC AC9MT#S06F MUCONDIALDEHYDE . Y . 0ehiro I C .E . Piper,l S .C . Gad,1 E .<br />
Rohrbacher,l 1r-S . Balwierz,l S .G . Soelter,l C . Nitzi <strong>and</strong> B .D . Goldstein .2 G .D . Searle<br />
& Co .,l Skokie, IL jMW77 <strong>and</strong> Dept . of <strong>Environmental</strong> <strong>and</strong> Community Medicine,2<br />
University of-4d nd Dentistry of New Jersey, Piscataway, NJ 08854 .<br />
Trans, trans-mucondialdehyde 1s a benzene metabolite that is speculated to cause<br />
leukemia in humans . We have examined its genotoxic activities using the Ames test, a<br />
CHO/HGPRT/micronucleus combined end-point assay <strong>and</strong> the rat primary hepatocyte UDS<br />
assay . In the first Ames experiment, TA1535, TA100, TA1538, TA98 <strong>and</strong> TA97 strains were<br />
treated with mucondialdehyde at six concentrations ranging from 0 .5 to 100 Ug/plate .<br />
The high dose of 100 yg/plate was toxic to the cells <strong>and</strong> there were indications of<br />
activity with TA100 <strong>and</strong> TA97 at 10 <strong>and</strong> 50 pg/plate . A second experiment using test<br />
concentrations from 5 to 70 Ug/plate showed a dose-related increase with TA100, but<br />
the response did not reach twice the value of the solvent control . However . TA97<br />
showed dose-related mutagenic activity that exceeded two times the solvent control at<br />
25 yg/plate <strong>and</strong> higher without S9, <strong>and</strong> at 60 ug/plate <strong>and</strong> higher with S9 . Mucondialdehyde<br />
was toxic at concentrations above 5 yg/ml in the rat primary hepatocyte UDS<br />
assay <strong>and</strong> there was no evidence of induction of DNA repair at lower test concentrations<br />
. Likewise, mucondialdehyde was toxic (17% survival at 0 .8 yg/ml), but negative<br />
for induction of HGPRT mutations in CHO cells, at concentrations from 0 .1 to<br />
0 .8 ug/ml without S9 . Cells from this experiment examined for micronucleus induction<br />
showed a dose-related effect, with significant clastogenic activity detected at 0 .4,<br />
0 .6 <strong>and</strong> 0 .8 ug/ml . Further studies with mucondialdehyde are ongoing, including in vivo<br />
micronucleus assays . The results to date indicate that mucondialdehyde is toxic,<br />
mutagenic, <strong>and</strong> clastogenic in vitro .<br />
429<br />
MODIFICATION OF AFLATOZIN B, INDUCED LYg00gNESIS IT ANTIBODIES . O .A . Osowole <strong>and</strong> A .0 .<br />
Uwaifo . Biochemistry Department, University of Ibadan, Ibadan, Nigeria .<br />
Lysogeneais, a consequence of induction of the SOS pathway, results from chromosomal<br />
DNA lesions persisting after constitutive repair . Lysogenic induction tests in<br />
prokaryotes are a good measure of the carcinogenicity <strong>and</strong> mutagenicity of a given<br />
carcinogen . The effect of antiserum against aflatoxin B1-bovine serum albumin complex on<br />
aflatoxin B1 induced lysogenesia was studied using Escherichia SAi Ku . Antibody against<br />
aflatoxin Bi-bovina serum albumin complex was obtained after a single intradermal multiple<br />
site injection of water in oil emulsion of the complex (66 .67yg/ml) into rabbits . The<br />
antiserum used was obtained in the seventh week after isseunization . The results obtained<br />
ahowed a marked reduction in the degree of lysogenesis induced by aflatoxin 81 after the<br />
addition of the antiserum to the reaction medium prior to microsomal enzyme activation<br />
of aflatoxin B1 . The result also showed that there was no detectable effects of the<br />
antibody when the antiserum was added after aflatoxin 31 activation . This suggests that<br />
the antibody in the aflatoxin B1-bovine serum albumin antiserum could interact with<br />
aflatoxin B1 prior to its activation . It seems probable therefore, that an immune<br />
protective effect may be exerted if the antibody intervenes before activation . Results<br />
from equilibrium dialysis study of the interactfon of purified immunoglobulin G from the<br />
antisera with aflatoxin 31 showed the average number of binding sites on the antibody<br />
molecule for aflatoxin B1 being 1 .65t0 .27 with a F1' of 5 .40 Kcal/mole while the average<br />
association constant was 1 .81f0 .19x10s 3 . These results indicate that the antibody has<br />
high affinity for aflatoxin B1 . A possible explanation for the reduction in aflatoxin B1<br />
induced lysogenesis is that antibody binding to aflatoxin B1 reduces epoxide formation .<br />
430<br />
X-RAY-INDUCED MULTILOCUS DELETIONS IN THE g4--3. REGION OF A TWO-COMPONENT HETEROKARYON<br />
OF Neurosoora rr Up COVERING THE 31174 AND hj;-l LOCI LACK THE WILD-TYPE hi;_.$ DNA IN<br />
RESTRICTION ENZYME ANALYSES . L .K . Overton . J .S . Dubins . R .R . Cobb <strong>and</strong> ~,Z ~g ~grres .<br />
Center for Life Sciences <strong>and</strong> Toxicology . Chemistry <strong>and</strong> Life Sciences . Research<br />
Triangle Institute, Research Triangle Park, NC 27709 (USA)<br />
4 mutant 1-226-565 results from a 1 .8 kb insertion in the hjgj- locus <strong>and</strong> as a<br />
consequence has an altered b<strong>and</strong>ing pattern upon Ps_% I digestion <strong>and</strong> hybridization to<br />
the hi,U- probe pNH6O (Oubins et al ., Mutations Res ., submitted) . After such<br />
treatment fragments of 1 .4 . 4 .9 . <strong>and</strong> 7 .9 kb were seen . Similar treatment of the DNA<br />
from the wild-type strain 74-0R23-1A showed fragments of 1 .4 . 4 .9 . <strong>and</strong> 5 .9 kb . A<br />
series of 30 X-ray-induced pA,1 mutants (genotype $cj:,3A jg=20) induced in a twocomponent<br />
heterokaryon (N-12) of " . Srassa have also been shown to cover the two<br />
proximal loci . IJ,i_;=1 <strong>and</strong> 1vs-4, (de Serres . Mutation Res . . in press) . These mutants<br />
have been subjected to restriction enzyme analysis to determine whether they resulted<br />
from interstitial deletion . The DNA from forced dikaryons between each of the 30 AQ-<br />
,3A ,gq=J@ mutants <strong>and</strong> mutant 1-226-565 was analyzed for the presence of "wild-type"<br />
DNA that should be missing if these 30 mutants resulted from deletions . The<br />
5 .9 kb fragment was missing in 27/30 forced dikaryons analyzed, indicating that the<br />
"wild-type" his-$ locus was ∎issing in these 27 mutants ; 2/30 mutants showed a partial<br />
50869 3662
;<br />
deletion of his-_3 . In the remaining forced dikaryon, the 5 .9 kb fragment was present<br />
in addition to a 2 .6 kb fpagment . Thus, the latter 3/30 Ad;_39 pdZR mutants probably<br />
resulted from multiple 4obus mutation . Thus, data from these studies demonstrated<br />
that the majority of X-ray-induced mutants classified as presumptive multilocus<br />
deletions by classical genetic tests are actual interstitial deletions .<br />
431<br />
Ai r"'UPIAIDY IN MOUSE 00CYTES AND 0NE-C°L .L ZYGOTf.'S AFfF:R ORAL DOSAGES OF GRIS. .OFULVIN<br />
F .?acchie :rotti,C .Tiveron,F .Marchetti,B .Bassani,Lab .Toxicology,FN'cA Casaccia,Roma,Italy<br />
Griseofulvin (GF) has been characterized as a mitoclastic agent in :namralian cell<br />
cultures probably causing spindle disrL4)tion by inhibition of cmrbination between .nicro<br />
tubules <strong>and</strong> microtubule associated proteins . Doses of 200, 666, 1332 or 2000 mg per kg<br />
of body weight dissolved in olive oil have been administered by gavage to young superovulated<br />
BC3F1 female mice either at the time of hunan chorionic gonadotrophin (ECG)<br />
injection or 2 h later . Metaphases of secondary oocytes or one-cell zygotes have been<br />
prepared 18 or 42 h (overnight cultivation in colchicine added mediun included) after<br />
HCG respectively . Cytogenetic analysis of secondary oocytes showed that when GF was<br />
given at the same time of ICG a dose-dependent induction of meiotic arrest was observed<br />
without any evidence of aneiploidy in the oocytes which had corrpleted the first meiotic<br />
division . When the same dose (2000 mg per kg) was given 2 h later a reduction by a<br />
factor of 3 .6 of the meiotic arrest was observed, while a preliminary analysis showed<br />
that 23 out of 40 (57 .5 %) analyzed metaphases were aneuploid . Observatims of one-cell<br />
zygotes showed that approximately all the oocytes which had been arrested at the first<br />
meiotic metaphase underwent irregular fertilization <strong>and</strong> gave rise to polyploid zygotes<br />
or zygotes with an MI or MII arrested female pronucleus . These data suggest the inportance<br />
of treatment time during meiotic progression as a flmction of the way of chemical<br />
adninistration <strong>and</strong> of the cellular target <strong>and</strong> mechanism .s of aneuploidy irxSuction .<br />
432<br />
PREDICTING CARCINOGENIC POTENCY : THE USE 0f A BATTERY<br />
OF GENOTOXIC AND ACUTE TOXICITY ASSAY RESULTS<br />
S . Richter Pack<br />
C .C . Travis<br />
Oak Ridge National Laboratory<br />
Recent studies focusing on the ability of short-term mutagenicity assays to<br />
predict the potency of positive carcinogens have found equivocal correlations<br />
between mutagenic potency <strong>and</strong> cancer potency . The squivocal correlation may be<br />
due to a failure of the short-term tests to account for the ability of some<br />
compounds to act as cancer promoters . It is difficult to assess the extent that<br />
a measure of promotional ability will increase the predictivity of short-term<br />
test batteries since an adequate short-term test for promotion is not available .<br />
We propose using acute toxicity data (LDSO) as a surrogate measure of promotional<br />
activity . The suggested biological basis for this relationship is that tissue<br />
damage <strong>and</strong> consequent cell proliferation may be a surrogate measure for tumor<br />
promotion . We have found that including an assay for cytotoxicity in a shortterm<br />
test battery results in an increase in cancer potency predictivity to<br />
roughly 0 .84, as opposed to 0 .40 for the Ames test alone .<br />
433<br />
MUTAGENICITY OP THE HUMAN CARCINOGEN TREOSULPBAN IN SALMONELLA . D . A . Pa ano<br />
<strong>and</strong> E . Zeiger, Cellular <strong>and</strong> Genetic Toxicology Branch, National Inst tute o<br />
<strong>Environmental</strong> Health Sciences, Research Triangle Park, NC 27709 (USA)<br />
Treosulphan (TREO) is an alkylating agent that is used for the treatment of<br />
ovarian cancer <strong>and</strong> has been shown to be a human carcinogen . As part of a<br />
study of the mutagenic effects of human carcinogens, TREO <strong>and</strong> its hydrolysis<br />
products, diepoxybutane (DEB) <strong>and</strong> methanesulfonic acid (NSA), vere tested for<br />
mutagenicity in Salmonella . TREO <strong>and</strong> DEB were direct acting mutagens in<br />
strain TA1535 . TREO induced 116 rev/plate at a dose of 1 .87 umols <strong>and</strong> DEB<br />
induced 50 rev/plate at 0 .88 umols . The use of a preincubation protocol or<br />
the addition of rat-liver S9 caused a slight enhancement of TREO mutagenicity,<br />
but a decrease in DEB mutagenicity . NSA was not mutagenic or toxic up to 128<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 149<br />
.<br />
Notes
150 1989 EMS Abstracts - -~~ ._ - . s~<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes umols/plate in tha .presence or absence of S9 . TREO is knovn to hydrolyse to<br />
DES <strong>and</strong> MSA vf tlr~lsaasing pH . TREO mutagenicity vas tested at pH 6 .0, 7 .0,<br />
<strong>and</strong> 8 .0 <strong>and</strong> shovn to be p8-dependent, vith the highest autagenic response <strong>and</strong><br />
the least toxicity seen at pH 6 .0 ; the lovest response <strong>and</strong> the greatest<br />
toxicity vas seen at pH 8 .0 . DEB mutagenicity vas unaffected by pH, <strong>and</strong> its<br />
autagenicity patterns vere similar to TREO at pH 8 .0 . At this pH, the<br />
hydrolysis of TREO to DEB is rapid . It vould be expected that the<br />
mutagenicity of DEB, rapidly produced from TREO (at pH 8 .0), vould be similar<br />
to DEB directly applied . The nonmutagenicity of NSA eliminates it from<br />
consideration as the active hydrolysis product . These results are consistent<br />
vith the conclusion that TREO is mutagenic through its nonenzyaatic hydrolysis<br />
to DEB .<br />
434<br />
BIOLOGICAL AND ANALYTICAL MONITORING OF RIVER WATER POLLUTION IN CAMPANIA, ITALY .<br />
G .Pagano, G .Melluso ; G .Corsale, A .Esposito, M .Ouida: <strong>and</strong> G .G .Giordano, Ystituto<br />
Nazionale Tumori, 1-80131 Naples, <strong>and</strong> * Dipartimento di Fisiologia Generale ed<br />
Ambientale, Facoltg di Scienze . Universitl di Napoli, 1-80134 Naples, Italy<br />
Several emissions of industrial, agricultural, <strong>and</strong> domestic sewage affect the Sarno<br />
river <strong>and</strong> the drainage system of the Volturno river-Regi Lagni in the Campania region,<br />
Italy . The possible consequences on marine biota near the mouths of the two rivers<br />
<strong>and</strong> the Regi Lagni have been investigated by biweekly sampling of river water from ten<br />
sites ; some experiments were performed by daily or circadian sampling from two selec-<br />
ted sitea . Biological monitoring was performed by testing river water samples diluted<br />
in seawater (10- to 10-') on sea urchin embryos <strong>and</strong> sperm, <strong>and</strong> scoring the outcomes<br />
in terms of : a) developmental toxicity ; b) fertilization success ; c) cytogenetic ab-<br />
normalities . Chemical analysis was carried out on water <strong>and</strong> sediment, <strong>and</strong> included<br />
several pollution parameters, as : i . COD <strong>and</strong> BOD, ; ii . NO=, N0„ <strong>and</strong> NH ; ; iii . some<br />
selected inorganice [Cd(II) ; Cr(III) <strong>and</strong> (VI) ; As(III) <strong>and</strong> (V)] ; iv . some selected<br />
organics (PAH's, PCB's, some pesticides) . The results showed : 1) the presence of re-<br />
markable pollution <strong>and</strong> toxicity at the mouths of the Sarno river <strong>and</strong> the Regi Lagni,<br />
whereas the Volturno river resulted to be less affected ; 2) daily <strong>and</strong> circadian<br />
changes of pollutant levels ; 3) a good agreement of biological vs . analytical data .<br />
(Partly supported by WHO, <strong>and</strong> by the Italian Ministry of Health) .<br />
435<br />
FANCONI'S ANEMIA (FA) LYMPHOBLASTS BELONGING TO COMPIEMENTATICN GROUPS<br />
"A" AND "B" ARE HYPOMUTABLE FOLLOWING TREATMENT WITH PHOTOACTIVATED<br />
PSORALENS . D . Pa ad oulo, B . Porfirio <strong>and</strong> E . Moustacchi . Institut Curie,<br />
Section de B o og e, UA 1292 CNRS, 26 rue d'Ulm, 75231 Paris cedex 05,<br />
France .<br />
Fanconi's anemia (FA), an eutoscmal•recessive disease, beldngs to the<br />
group of human genetic disorders cha;acterized by chranosaaal instability<br />
<strong>and</strong> a predisposition to malignancy . At the cellular level, FA cells<br />
are hypersensitive to DNA cross-linking agents, a feature likely to be<br />
related to a defect in the processing of lesions induced by these<br />
agents . Two genetic complementatien groups have been recently described<br />
"A" <strong>and</strong> "B" . We have previously demonstrated that FA group "A" cells<br />
are sensitive to cross-links <strong>and</strong> monoadducts (MA) induced by photoactivated<br />
bifunctional psoralens, whereas FA group "B" cells are eapecially<br />
sensitive to MA induced by these agents . In the present study, we measured<br />
in normal <strong>and</strong> FA lymphoblasts the frequency of 6TGr <strong>and</strong> ouar mutations<br />
induced by 8-methoxypsoralen or trimethyl,4,5',8,psoralen in the<br />
presence of 365 nm radiation . We observed that FA cell lines fraa both<br />
complementation groups are clearly hypaoutable for the two loci considered<br />
. The FA group "A" cells are almost immutable . The molecular analysis<br />
of the spectrum of HGPRT- mutants induced by photoactivated psoralens is<br />
in progress .<br />
436<br />
FISH CELL LINE DEB.IVEJ FROlf THE FIN OF TBE CEN(RAL MUDHINJOM/ (UMBRA L=) : A SUITABLE<br />
MC)DEL FOR CLASTOGENICITY ASSAY . E .-H . Park, J .-S . Lee, A .-K. Yi <strong>and</strong> H . Etch,<br />
Department of Biology, College of Sciences, Hanyang University, Seoul 133-791 (Korea),<br />
<strong>and</strong> Division of Biology, National Institute of Radiological Sciences, Chiba 260 (Japan)<br />
A cell line (ULF-23HU) from the fin of the central madminnow (UMbrA UML) was<br />
characterized <strong>and</strong> tested for its suitability to aseess cytogenetic damages induced by<br />
chemicals in fish . . Cells of this line exhibit a fibroblaat-like appearance <strong>and</strong> grew<br />
50869 3664
: 1989 EMS Abstracts<br />
optimally at 25 'C in ttSg TC-199 medium containing 10% fetal bovine serum, but slower Notes<br />
growth continued down ta 4°C, where they could be stored for prolonged periods . They<br />
had a 32-h cell cycle time laken up by a 20-h S period as determined by the<br />
autoradiographic analysis of the fraction of labeled mitosis . 'Itre cultures showed<br />
relatively high mitotic index (0 .84-2 .35%) during exponential growth phase lasting about<br />
7 d . Karyological analysis of the cells at the different subculture passages revealed<br />
constant chromosome modal number of 23 consisting of metacentric or submetacentric<br />
chromosomes which were primarily similar to those of in vivo cells except one additional<br />
chromosome . The spontaneous-sister chromatid exchange rate was 5 .3 per metaphase . When<br />
ULF-23HU cells were exposed to N-methyl-N'-mitro-N-nitoreoguanindine, mitomycin C,<br />
N-methyl-N-nitrosourea, 4-nitroquinolin-N-oxide <strong>and</strong> cie-platinum (II) diamine dichloride<br />
dose-dependent increases in sister chromatid exchanges were clearly detected .<br />
FLthermore, ULF-23HU cells responded to the indirectly acting mutagens (aflatoxin B1,<br />
cyclophosphamide <strong>and</strong> 4-aminoazobenzene) without adding a drug activation system . These<br />
rrsurts offered the high possibility of the use of this cell line as a suitable in vitro<br />
model for clastogenicity studies in fish .<br />
437<br />
CFUtOMOSOME CHANGES IN CELL TRANSFORMANTS INDUCED BY GENOTOXIC AND NON-GENOTOXIC AGENTS .<br />
E . M . Parry, J;M . Parry, T . Issa, M . Kadhim, R . Porter, D . Devi, School of Biological<br />
Sciences, University College of Swansea, Swansea SA2 8PP .<br />
Syrian hamster dermal (SRD) cultures exposed to carcinogens undergo progressive<br />
changes from primary isolation via the formation of immortal cultures to the production<br />
of transformed tumourigenic clones (Newbold et al 1982) . Thase culture rhanges may be<br />
induced by treatments with agents characterised as acting predominantly by mechanisms<br />
involving DNA interactions (genotoxic) <strong>and</strong> by a variety of non-genotoxins . We have been<br />
investigating the chromosomal (numerical <strong>and</strong> structural) <strong>and</strong> division characteristics<br />
(eg mitotic spindle fidelity) of SHD cultures exposed to both genotoxins <strong>and</strong> nongenotoxins<br />
. Immortal cultures which escape culture senescence (normally occurs after 15<br />
population doublings) generally show the presence of significant numbers of polyploid<br />
cells <strong>and</strong> high levels of spindle fidelity . Treatment of immortal cultures with<br />
carcinogens results in the progressive loss of chromosebes (predominantly from the<br />
polyploid population) resulting in the formation of hyperdiploids <strong>and</strong> the production of<br />
fully transformed colonies . Karyotype analysis of transformed SHD cultures has<br />
demonstrated a variety of aberrations with a transloc,ation of chromosome 11 being the<br />
most common event . This translocation is found in all the cells of clones transformed<br />
with alkylating agents <strong>and</strong> benzo(a)pyrene <strong>and</strong> initially at a low frequency in cultures<br />
transformed with diethylstilboestrol . With further subculturing, individual clones<br />
progress to become homozygous for the translocated chromosome 11 . Our data sugfest that<br />
the SHD system provides a suitable model for the study of the mechanism of action of<br />
both genotoxins <strong>and</strong> non-genotoxins . Newbold, R . F ., Overe11,R . W . <strong>and</strong> Connell, J . R .<br />
(1982), Nature ?Q9, 633-635 .<br />
438<br />
THE DETECTION OF ANEUGENS USING YEASTS AND CULTURED MA64fALIAN CELLS . J . M . Parry, E . M .<br />
Parry, T . Warr, A . Lynch, S . James . Biological Sciences, U .C . Swansea SA2 8PP .<br />
The EEC's aneuploidy project involves a study of a range of assays using a common<br />
series of chemicals . The core chemicals selected for their ranPe of cellular activities<br />
consists of colchicine (COL), econazole (EZ), Chloral hydrate (CR), hydroquinone (HQ),<br />
diazepam (DZ), thiabendazole (TB), cadmium chloride (CD), tbimerosal (TM),<br />
pyrimethamine (PY) <strong>and</strong> vinblastine (VB) . These chemicals are being tested in assays<br />
ranging from in vitro polymerisation of tubulin to the induction of numerical<br />
chromosome changes in the germ cells of intact rodents . We have been studying the 10<br />
chemicals to measure their ability to induce chroswsome loss in yeast D6, cell division<br />
aberrations, chromosome number changes <strong>and</strong> micronuclei formation in primary <strong>and</strong><br />
immortal Chinese hamster cultures using a variety of protocols . In yeast D6, five'of<br />
the chemicals namely EZ, TM, TB, HQ <strong>and</strong> CH produced significant increases in chromosome<br />
loss, CD, PY <strong>and</strong> DZ produced only marginal increases in monosozd <strong>and</strong> COL <strong>and</strong> VB were<br />
inactive . These data suggest that fungi are at best only capable of detecting a<br />
fraction of those chemicals which are capable of inducing aneuploidy in mammalian cells<br />
(Parry <strong>and</strong> Parry (1988) . In our h<strong>and</strong>s the most time <strong>and</strong> cost effective in vitro assay<br />
for detecting potential aneugens was the measurement of aberrations of aell division<br />
using procedures which differentially stain the spindle <strong>and</strong> chromosomes (Parry et al<br />
1985) . This assay was capable of detecting induced division abnormalities after<br />
exposure to all the test chemicals using relatively simple protocols <strong>and</strong> in a variety<br />
of cell types . Parry E . M . et al (1985) Mutation Res . 150, 369-381 . Parry E . M . <strong>and</strong><br />
Parry, J . M . (1988) Aneuploidy, Part B : n uction <strong>and</strong> 3et Svstems, 169-1g8 . Alan R .<br />
Liss Inc . New York .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
151
152 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
439<br />
THE HXPULSIQM OF MICRONUCLAI P~OM POLTCHROMATIC nTTHROClTAS OP MOUSB DONS MAAROp IN<br />
VIVO. J . Parto~ J . Deyers , <strong>and</strong> M . Carriott . Lilly Research Laboratories, sIT<br />
~flly <strong>and</strong> Co .'~y;'LTeenfield, IN 46140 .<br />
The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome<br />
damage produced by test articles in polychromatic erythrocytes (PCTj o3-bone marrov .<br />
Compounds that are clastogens, such as cyclophosphamide (CP), induce ∎icronuclei<br />
(MN) that are smaller than MN induced by compounds that are spindle poisons, such as<br />
demecolcine (DE) (Hogstedt <strong>and</strong> Karlason, Mut . Aes . 1S6t229, 1985/ Tas~.moto <strong>and</strong><br />
Kikuchi, Mut . Res . 71s127, 1980) . In vitro studies con ucted by Nito at al . (Mut .<br />
Res . 207 :5, T88)ihoved that siitomyc n <strong>and</strong> vincristine caused a dose-TepenNint<br />
rnluct3on of MN in mouse cell line L-929 but when the ∎icronucleated cells vere then<br />
treated with cytochalasin B, the rate of ∎icronucleated cells was reduced 31-39X due<br />
to MN extrusion . The eurrent study shows 1) MN are expelled from PCs in vivo <strong>and</strong> 2)<br />
expulsion is dependent upon ∎icronucleus sise . Male <strong>and</strong> female CD-1 ml-e : vere given<br />
0 .5, 1, 5, <strong>and</strong> 10 mg/kg of DE or 100 mg/kg of CP . The bone marrov was harvested 24<br />
<strong>and</strong> 48 hr after dosing <strong>and</strong> evaluated for micronucleated PCE . Expelled MN still<br />
attached to the PCE cell surface vere also counted . The clastogen, CP, produced MN<br />
that ranged in size from 0 .5 to 2 .8 ym' with a mean of 1 .5 vm' <strong>and</strong> no expelled MN<br />
were observed . The spindle poison, Dg, induced ∎icronuclei that ranged in sise from<br />
0 .8 to 7 .9 un' . MN retained within the PCE ranged from 0 .8 to 3 .4 ym' with a mean<br />
of 1 .9 um' while expelled MN ranged from 2 .1 to 7 .9 Ym' with a mean of 4 .3 um' . MN<br />
less than 2 .1 ym' are retained within the PCE <strong>and</strong> MN greater than 3 .4 ym' are<br />
expelled . MN ranging from 2 .1 to 3 .4 Ym' in size may or may not be extruded .<br />
440<br />
THE U .S . ENVIRONMENTAL PROTECTION AGENCY'S INTEGRATED RISK INFORMATION SYSTEM, J . Patterson,<br />
J . Swartout, R . Schoeny, R . Picardi, U . S . <strong>Environmental</strong> Protection Agency,<br />
Office of Health <strong>and</strong> <strong>Environmental</strong> Assessment, Cincinnati, Ohio <strong>and</strong> Washington, D .C .<br />
The Integrated Risk Information System (IRIS) is an electronic information system<br />
developed by the U .S . <strong>Environmental</strong> Protection Agency (EPA) containing data related to<br />
health risk assessment . IRIS is the primary vehicle for communication of chronic<br />
health hazard assessments representing EPA consensua after comprehensive review by<br />
intra-Agency work groups . This work group review includes an examination of the<br />
weight of evidence that an agent is likely to be a human carcinogen as well as a consideration<br />
of the validity of a quantitative risk estimate of carcinogenicity . Quantitative<br />
estimates of potential for noncancer health effects are reviewed by a separate<br />
group . The primary intent of IRIS is to provide guidance to EPA personnel in<br />
making risk management decisions . IRIS contains chemical-specific information in summary<br />
format for over 360 agents . The information is structured to provide a description<br />
of the basis for the hazard assessment <strong>and</strong> a discussion of the uncertainties in<br />
that assessment . An IRIS chemical file is initiated when consensus is reached on an<br />
assessment for carcinogenic or noncarcinogenic endpoints <strong>and</strong> contains the summary for<br />
that aseessment . Other information, such as Drinking Water Health Advisories, regulatory<br />
actions, acute toxicity, <strong>and</strong> physical-chemical properties, is added as available<br />
<strong>and</strong> as resources permit . IRIS is available to the general public by telecommunications<br />
link with a commercial carrier, <strong>and</strong> to Public Health Foundation members through<br />
the Public Health Network . Access through the National Library of Medicine's TOXNET<br />
is planned .<br />
441<br />
ANALYSIS OF ELECTROPHORETICALLY DETECTED MUTATIONS IN THE MOUSE, AND COMPARISON OF THE<br />
ELECTROPHORETIC AND SPECIFIC LOCUS MUTATION RESPONSE .<br />
J . Peters <strong>and</strong> S .T . 8a11, N .R .C . Radiobioloqy Unit, Chilton, Didcot, Oxon OXII 0RD (UK)<br />
Mutations occurring in mouse spermatogonial stem cells after exposure to<br />
ethylnitrosourea (ENU) or X-rays have been scored, both by the visible specific locus<br />
test <strong>and</strong> an electrophoretic test . One aim of the experiment was to compare mutation<br />
rates found by each method, <strong>and</strong> a second aim was to investigate the characteristics of<br />
the induced mutations . Significant differences in mutation rates per locus for<br />
recessive visible <strong>and</strong> electrophoretically detectable markers have not been found yet .<br />
Five independent null mutations of glucose phosphate iso .erase-1 were discovered<br />
among the offspring of mice treated with 250 mq/ky ENU . Heterosyyotes are fully<br />
viable <strong>and</strong> fertile, but homozygotes are not known . One homoayqote dies at about<br />
8 .5 days oost coitum (West et al ., in prep .) . Each mutation results in complete loss<br />
of gene product in all adult tissue~i t¢~ted, as judged by quantitative assay <strong>and</strong><br />
electrophoresis, except one, Goi-lsD-mltl . This determines a polypeptide which can be<br />
found only in adult testis, <strong>and</strong> embryonic stages . Another mutant allele Gui-1sh:au<br />
also codes for a polypeptide which is only seen in embryos . Possibly the mutant<br />
polypeptides are unstable, <strong>and</strong> thus can be detected only in rapidly dividing tissues .<br />
<strong>Molecular</strong> analysis by Southern blotting has failed to show 4ifferences between DNA<br />
from the mutants <strong>and</strong> from the wild types goi-tsa <strong>and</strong> Goi-ts . These findings are in<br />
agreement with the suggestion that ENU induced mutations are mainly intragenic<br />
changes .<br />
50869 3666
442 = 1989 EMS Abstracts 153<br />
Notes<br />
,<br />
HUMAN DNA ADDUCTS DUE TO SMOKING AND OTHER CARCINOGEN EXPOSURES .<br />
D .H . Phillips, Institute oftancer Research, Chester Beatty Labs, London, UK .<br />
Human exposure to complex mixtures of potentially carcinogenic polycyclic<br />
aromatic hydrocarbons (PAH) has been monitored by 32P-postlabelling analysis of<br />
DNA isolated from human tissues . The presence of aromatic adducts in DNA from<br />
peripheral lung has been detected at levels proportional to the numbers of<br />
cigarettes smoked by the individuals . The complexity of the adduct patterns<br />
indicated that adducts were formed by-$ large number of different compounds . Lung<br />
DNA from former smokers of several years' abstinence generally showed a level of<br />
adducts similar to that in DNA from non-smokers . Analysis of bronchial DNA also<br />
revealed evidence of smoking-related adducts . Adduct levels in peripheral blood<br />
lymphocyte DNA did not differentiate between smokers <strong>and</strong> non-smokers .<br />
Occupational exposure to PAH amongst iron foundry <strong>and</strong> coke oven workers was,<br />
however, evident from the elevated levels of PAH-DNA adducts in their lymphocyte<br />
DNA compared to levels in unexposed control subjects . Exposure of human skin to<br />
PAH, monitored in psoriasis patients receiving topical applications of coal-tar <strong>and</strong><br />
juniper tar, leads to the formation of adducts in this tissue . Corroborative studies<br />
on the formation of adducts in experimental animals provide further evidence of<br />
the potential carcinogenic risk to humans of exposure to these PAH mixtures .<br />
443<br />
CHROMOSOME-SPECIFIC PROBES IN CYfOGENETICS . D. Pinkel, J. Gray, R. Segraves, J. Lucas, W-L.<br />
Kuo, B . Trask, L C. Yu, D. Eastmond, M. Poggensee. Lawrence Livermore National Laboratory,<br />
Livermore Ca . 94550 .<br />
Cytogenetic analysis now relies primarily on staining procedures that produce patterns of b<strong>and</strong>s on<br />
metaphase chromosomes. Analysis of these b<strong>and</strong>s by skilled observers permits chromosome identification<br />
<strong>and</strong> recognition of structural <strong>and</strong> numerical aberrations. However the proce4ures are time<br />
consuming <strong>and</strong> limited to mitotic cells . Recent developoments in chromosome stainEng by in situ<br />
hybridization promise to extend cytogenetic analysis to new areas . Since staining is based on DNA<br />
sequence, the staining pattern can be tailored by the choice of probes, <strong>and</strong> information can be obtained<br />
from complex settings such as interphase nuclei . Two classes oj probes have proven useful . The first<br />
are probes for chromosome-specific repetitive sequences . These sequences occur in compact clusters,<br />
frequently near the centromeres . Specific probes for approximately 2/3 of the human chromosomes<br />
are known. Nuclei hybridized with these probes show a compact spot at the location of the target<br />
sequence, permitting identification of aneuplold cells . The second class of probes, composite probes,<br />
consist of collections of probes which produce a desired staining pattern on chromosomes . Composite<br />
probes capable of staining a specific chromosome type over its entire length are now available for all of<br />
the human chromosomes. They permit rapid detection of structural abnormalities such as translocations<br />
in metaphase spreads <strong>and</strong> interphase nuclei. In situ hybridization with probes of both types<br />
has allowed us to detect aneuploidy induced by in vitro chemical exposure, trisomy 21 <strong>and</strong> other<br />
important prenatal numerical abnormalities, <strong>and</strong> translocations induced by in vitro <strong>and</strong> in vivo<br />
radiation exposures . Work performed under the auspices of the USDOE by the LLNL under contract<br />
W-7405 ENG-48 <strong>and</strong> USPHS grants CA45919, GM25076, <strong>and</strong> HD17665 .<br />
444<br />
ENHANCED REACTIVATION AND NUTAGENESIS OF ADE 2 VIRUS IN CARCINOGEN -<br />
PRETREATED BELA CELLS<br />
Stelios N . Piperakis<br />
Institute'of Biology, N .R .C . "Democritos"<br />
Agia Paraskevi, Athens - Greece<br />
Enhanced reactivation of a UV - irradiated mammalian virus by pretreatment<br />
of susceptible host cells with a number of agents has now been<br />
found to exist in different systems . In many studies with mammaliancells<br />
an increased degree of viral mutagenesis has also been found with increasing<br />
damage to the virus,but there is still disagreement as to whether<br />
this mutagenesis is further enhanced by pretreatment of the cells .<br />
In the present study an investigation has been undertaken in the Ade 2-<br />
Hela system . Hela cells were treated with the agents sodium arsenite,<br />
EMS, <strong>and</strong> aflatoxin Bl<strong>and</strong> the enhanced reactivation <strong>and</strong> enhanced mutagenesis<br />
of UV - irradiated Ade 2 was compared to the enhanced reactivation<br />
<strong>and</strong> enhanced mutagenesis obtained by UV, heat <strong>and</strong> MMS .<br />
The results show that the enhanced reactivation of UV-irradiated Ade 2<br />
observed in Hela cells pre-treated with these agents is not due (at<br />
least under the used experimental conditions) to the induction of an<br />
error - prone DNA repair mechanism .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
154 1989 EMS Abstracts 445<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
NOteS._ , .,; . ROLE OB Ai.TW4~D HEPATIC FOCI IN THE STAGES OF CARCINOGENESIS. n P't<br />
~ Yvonne Dt9t~an, Mark Pyron, Chris Laufer, <strong>and</strong> Tahir Rlzvi<br />
.<br />
. McArdle Laboratory Research, UmverWof Wisconsin, Madison, Wisconsin 53706<br />
Hepatoaltrcittogettrsis in the rat can be divided into the stages of initiation, promotion, <strong>and</strong> progression<br />
. Initiadon by a DNA-alkylating chemical results in the appcarance of individual hepatocytes<br />
<strong>and</strong> their progeny exhibiting altered gene expression of specific tnarker(s) . The stage of promotion is<br />
induced by exo~enous <strong>and</strong>/or endogenous chemicals, usually non-DNA-reactive, which stimulate<br />
both the re licatton rate <strong>and</strong> reversibly altered gene expression of clones of initiated cells . However,<br />
different chemical classes of promoting agents induce populations of altered hepatic foci (AHF)<br />
which, in general, exhibit different phenotypic characterls 'ttcs, unique to the class of chemical promoting<br />
agents . Furthetmore, when multiple markers are utilized to score the phenotypes of the<br />
clonally exp<strong>and</strong>ed inidated cell populations, all possible phenotypic combinations within the constraints<br />
of the action of the promoting agent itself are represented in the AHF populadons . The stage<br />
of progression, when not induced simultaneously with~ge of initiation, may be heralded by the<br />
appearance of phenotypic heterogeneity within individual AHF in contrast to the phenotyp ic homogeneity<br />
of most or all AHF during the stage of promotion . It is in the stage of progression that karyotypic<br />
changes, proto-oncogene activation, <strong>and</strong> malignant neoplasia occur either predominantly or<br />
exclusively. Progressor agents capable of inducing this stage of neoplastic development are characterized<br />
ubiquitously by their clastogenic effects . Based on this rtwdel, point mutations <strong>and</strong> clastogenic<br />
effects appear to play critical but different roles at the sta es of initiation <strong>and</strong> progression in the<br />
development of hepatic neoplasia respectively. (Supported ~y grants from the National Cancer<br />
Institute: CA-07175, -22484, -43700 <strong>and</strong> a contract from the National Toxicology Program : ES-3-<br />
5024.)<br />
446<br />
DOUBLE-STRAND GAP PLASMIDS DO NOT ALTER THE RATE OF MUTATION OR GENE<br />
CONVERSION IN Saccharornyres cerrvisiac . MJ . Plewa, S . 7>tylor, A. Kumar <strong>and</strong> R. Carr. Institute for<br />
<strong>Environmental</strong> Studies <strong>and</strong> the Department of Microbiology, University of Illinois, Urbana, 61801 USA .<br />
We are developing a system to locate <strong>and</strong> recover information from specific mutant sites in eukaryotic<br />
chromosomes . We are studying the mutational spectrum of forward mutation at CANl by the doublestr<strong>and</strong><br />
gap repair of a series of yeast shuttle vectors . The advantage of this approach is that the<br />
information copied onto the gapped plasmid occurs after the mutational event on the chromosome . We<br />
constructed a progenitor shuttle vector - pMPS - which contains amp' . E. coli Ori, LEUZ GlNI, <strong>and</strong><br />
the yeast 2µ Ori. Yeast that are resistant to canavanine (canl') become sensitive to canavanine after<br />
transformation with pMPS. However, it is important to determine if linear, double-str<strong>and</strong> gap plasmid can<br />
modify the rate of forward mutation at GlNl on chromosome V or gene conversion within the yeast<br />
genome. Linear pMHIS8 (a vector used in the construction of pMP5) at concentrations of 0, 1, 2 .5. 5<br />
<strong>and</strong> 10 µg were added to competent yeast cells (strain XY729) under transforming conditions . Forward<br />
mutation at CANI <strong>and</strong> cell toxicity were measured . There was no difference in the frequency of canl'<br />
cells among the groups with a mean value (t SE) of 39 t 4 .0 mutants per 3.5 x 10' competent cells<br />
plated . The same concentrations of linear pMH15S were added to competent D7 cells <strong>and</strong> gene conversion<br />
was measured between trpS-12 <strong>and</strong> apS-27 heteroalleles . Again there was no difference in the gene<br />
conversion frequency at dpS with a mean value of 750 s 20 .3 convertants per 3 .4 x 10' competent cells<br />
plated . These controls indicate that the use of double-str<strong>and</strong> gap shuttle vectors does not affect the<br />
processes of mutation or gene conversion in competent cells .<br />
447<br />
THE AC'ITVATION OF PROMUTAGENS BY PLANT CELL SYSTEMS. Michael J. Plewa . Institute<br />
for <strong>Environmental</strong> Studies, University of Illinois, Urbana, IL 61801 USA .<br />
Plant activation is the process by which promutagenic agents are activated into mutagens by plant<br />
systems. Many promutagens are activated by the familiar mammalian microsomal monooxygenase systems<br />
as well as by plants . However, several environmentally important agents are preferentially activated by<br />
plant cells. Plants have become a reservoir for the deposition <strong>and</strong> accumulation of environmental<br />
xenobiotics . With the widespread use of agricultural chemicals on crop plants <strong>and</strong> with the global exposure<br />
of plants to pollutants, the possibility that plant-activated agents may be introduced into the human food<br />
chain is a cause of concern . Due to these <strong>and</strong> other concerns, envitonmentaliy relevant agents should be<br />
evaluated with plant assays. The plant celUmicrobe coincubation assay uses cultured plant cell suspensions<br />
as the activating system <strong>and</strong> bacteria or yeast cells as the genetic indicator organism . After a treatment<br />
time, the microbes are plated on selective medium. In this way the activation system <strong>and</strong> the genetic<br />
system can be independently studied. In addition the viability of the plant cells <strong>and</strong> the microbial cells can<br />
be independently determined so that the toxicity of a test agent can be evaluated . We have employed<br />
cultured tobacco, cotton, carrot, maize <strong>and</strong> 7Yadescanlia cells to study the activation of test agents <strong>and</strong><br />
complex environmental mixtures . In addition to screening, this assay is being used in basic research to<br />
elucidate the biochemical mechanisms of plant activation. Data using model monocyclic <strong>and</strong> polycyclic<br />
aromatic amines will be used to identify developmental <strong>and</strong> biochemical pathways involved in plant<br />
activation. This research supported by an OSWR grant <strong>and</strong> USAF grant N88-AF P-0511 .<br />
50869 3668
448 ' r 1989 EMS Abstracts 155<br />
VITAMLN C INTAKE DECRKAS .ES CHROMOSOME DAMACE IN GENETIC INSTABILITY ASSAY<br />
Pohl, H . <strong>and</strong> Reidv . J .1!<br />
Genetics Branch, Division of Environm,mtal Health Laboratory Sciences, Center for<br />
<strong>Environmental</strong> Health <strong>and</strong> Injury Control, Centers for Disease Control, Atlanta, CA<br />
30333, USA<br />
It has been suggested that some people are predisposed to cancer as a result<br />
of genetic instability <strong>and</strong> this instability can be detected by exposing their cells<br />
to a clastogen in vitro . We know very little, however, about person-specific<br />
factors other than genetic instability sbat might influence this assay . We report<br />
here that the amount of chromosome damage induced in human lymphocytes by an<br />
exposure to bleomycin during the last 5 hours of cell culture significantly<br />
decreased after the blood donors took 1 g of vitamin C per day for 2 weeks .<br />
Similar changes were not seen in lymphocytes from control individuals sampled at<br />
the same time but not taking vitamin C supplements . The initial study involved two<br />
persons who took vitamin C <strong>and</strong> two controls who did not take the vitamin . Results<br />
showed little variation among triplicate flasks <strong>and</strong> little variation over a month<br />
in the controls . Results were confirmed in a second experiment with eight people<br />
(p< 0 .01) . Each person was his own control . In addition, two laboratory controls<br />
(individuals not taking vitamin C) showed no differences during the experiment .<br />
From this limited study, we suggoet that it would be prudent to consider dietary<br />
<strong>and</strong> perhaps other lifestyle factors in the interpretation of results from this <strong>and</strong><br />
related assays for genetic instability .<br />
449<br />
ORGAN SPECIFIC GENOTOXICITY AND CARCINOGENICITY B .L. Pool, P.Schmezer, A.<br />
Tompa`, S.Y. Brendler. Institute for Toxicology <strong>and</strong> Chemotherapy, German Cancer<br />
Research Center, INF 280, 6900 Heidelberg, FRG . 'National Institute of Occupational<br />
Health, Nagyvarad T€r 2, Budapest, Hungary .<br />
The factors governing the unique organ specific carcinogenic effects of Nnitrosamines<br />
are largely unknown . Feasible mechanisms include (a) organ specific<br />
metabolism (b) pharmacokinetics (c) peraistance of DNA damage. The role of each<br />
pathway is being investigated for nitrosamines carcinogenic in hepatic or extrahepatic<br />
tissues . Induced DNA single str<strong>and</strong> breaks were determined in primary rat cells of<br />
liver, lung, kidney, testes, thymus gl<strong>and</strong> <strong>and</strong> blood . (a) Organ specific metabolism was<br />
not apparent in vitro using hepatocytes, since hepatotropic compounds were less<br />
genotoxic than extrahepatic carcinogens . (b) PharmAcokinetics were monitored by<br />
determining toxic <strong>and</strong> genotoxic effects in intact cells isolated from treated animals .<br />
This approach has the advantage that DNA damage arising from toxicity can be<br />
excluded, <strong>and</strong> that it is very sensitive . It was shown that < 1 mg/kg of the liver<br />
carcinogen N-Nitrosodimethylamine is genotoxic in the liver ; > 2 mg/kg are active in<br />
lung <strong>and</strong> kidney . No genotoxicity is detectable in cells of thymus gl<strong>and</strong> or testes . (e)<br />
Persistance of DNA damage was assessed after 1, 4, <strong>and</strong> 16 h exposure, <strong>and</strong> was<br />
found to be greater in liver than in lung . Therefore, this compound's organ specificity<br />
in carcinogenicity <strong>and</strong> in vivo genotoxicity agree well . This sensitive, versatile (multiple<br />
organs), rapid (48 h) <strong>and</strong> efficient (few animals) in vivo method can identify tissues<br />
which may be susceptible to carcinogenesis. The value of this approach to study<br />
toxicokinetics of genotoxic compounds is stressed. Results with additional nitrosamines<br />
<strong>and</strong> new approaches to monitor remote target organs will be presented .<br />
450<br />
GENETIC TOXICOLOGY OF THIAARENES B .L . Pool, P .Klein, P. Schmezer, S .Y. Brendler,<br />
J.R. Schlehofer', <strong>and</strong> G. Grimmer", Institute for Toxicology <strong>and</strong> Chemotherapy,<br />
`Institute for Virus Research, German Cancer Research Center, INF 280, 6900 Heidelberg,<br />
°Institute for Biochemistry of <strong>Environmental</strong> Carcinogens, 2070 Grosshansdorf, FRG .<br />
Three isomeric polycyclic aromatic sulfur heterocyclics (thiaarenes) have been<br />
assessed for genotoxicity. An evaluation of thiaarenes is important, since they are<br />
significant constituents of synthetic fuels . Compounds of this class may be structurally<br />
similar to several carcinogenic polycyclic aromatic hydrocarbons, but they have not<br />
been similarly well investigated for adverse biological effects . Benso(b)naphtho(2,1d)thiophene<br />
is a sulfur-containing analog to chrysene <strong>and</strong> the other two isomers are<br />
different by the position of their sulfur substitution (1,2-d <strong>and</strong> 2,3-d isomers) .<br />
Following results were obtained : (a) In S . t,vphimurium TA98, mutagenic activity was<br />
observed for 2 of the lsomers (2,1-d = 2,3-d; 1,2-d was negative) with st<strong>and</strong>ard<br />
metabolic activation by S9 from Aroclor pretreated rats . Stimulation of the system for<br />
glucuronyl acid conjugation did not reveal enhanced mutagenicity . (b) DNA Single<br />
str<strong>and</strong> breaks were not observed in metabolically competent primary hepatocytes nor in<br />
a Chinese hamster cell line. (c) Using the same cell line, integrated SV40-DNA was<br />
shown to be amplified by two compounds (1,2-d ∎ 2,3-d; 2,1-d was not tested) . Results<br />
of ongoing studies will be presented : (d) In a novel system, the amplification of Adeno-<br />
Associated Virus DNA is being measured in primary Syrian hamster embryo cells (SHE)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes
156 1989 EMS Abstracts<br />
_ ._--_-_ .<br />
NOteS - ~-F•infected -by
454 © 1989 EMS Abstracts<br />
CYTOGENETICS IN ENVIRONMENTAL MUTAGENESIS . R. Julian Preston . Biology Division, Oak<br />
Ridge National Laboratory, Qak Ridge, TN 37831<br />
It is 50 years since Karl Sax demonstrated that x rays could induce chromosome aberrations, <strong>and</strong> that the<br />
rc~pcrose was dose-dependent. In the period since•we have learned that a whole range of chemical agents<br />
c: n also induce similar alterations. It is also apparent that specific chromosome alterations arc as .cociated<br />
with many birth defects <strong>and</strong> with tumor initiation <strong>and</strong>/or progression . Important corollaries of these<br />
observations are can we determine which agents are mutagenic (or carcinogenic) <strong>and</strong> can we estimate the<br />
risk (genetic <strong>and</strong> somatic) from exposure to such agents? The answers are still equivocal . This stems from<br />
the fact that the mechanism of induction of chrotnDrame aberrations has not been determined, <strong>and</strong> the role<br />
of specific aberrations in the induction of birth defects <strong>and</strong> cancer are not generally known . Many new<br />
techniques have become available in the past few years to help in this endeavor . It is possible for example.<br />
to characterize chromosomes by using specific DNA probes, to identify <strong>and</strong> sequence chromosome breakpc<br />
ints, <strong>and</strong> to identify chromosomal regions by sophisticated b<strong>and</strong>ing techniques <strong>and</strong> in situ hybridization .<br />
These methods can be used to determine if agents present in the environment can induce specilic<br />
chromosome aberrations, not just aberrations in general . It is also of importance to determine if there is<br />
a range of sensitivities to aberration induction within the population <strong>and</strong> the bases for this, (e .g ., replication<br />
fidelity, repair competence, <strong>and</strong> chromosome fragility) . The incorporation of molecular techniques into shortterm<br />
assays (in vitro <strong>and</strong> in vivo) <strong>and</strong> population monitoring studies will enhance their utility, <strong>and</strong> hopefully<br />
allow truly predictive information to be obtained . Operated by Martin Marietta Energy Systems, Inc . under<br />
contract DE-ACA5-840R21400 with the U . S. Dept. of Energy .<br />
455<br />
RESTRICTION ENDONUCLEASE INDUCED CHROMOSOME DAMAGE : A MODEL FOR IONIZING<br />
RADIATION AND A PROBE OF INTERCHANGE HOTSPOTS . R.J . Preston, G .J. Hook, <strong>and</strong> G.J. Horesovsky,<br />
University of Tennessee Biomedical Graduate School, <strong>and</strong> Biology Division, Oak Ridge National Laboratory, Oak<br />
Ridge, TN 37831 .<br />
Type II restriction endonucleases (REs) are capable of recognizing specific DNA sequences <strong>and</strong> indbcing blunt<br />
or cohesive ended double str<strong>and</strong> cuts at these sites . Since only one type of DNA damage is induced by REs they<br />
represent a good model system for studying the importance of double str<strong>and</strong> breaks in the formation of CAs (CAs) .<br />
In addition since the cuts are persumabley at a specific location it should be possible to produce very specific types<br />
of CAs. Using cell electroporation we have designed experimental protocols by which the validity of RE induce<br />
damage as a model system, specifically for radiation damage, as well as the feasibility of looking at specific<br />
chromosome interactions can be tested. Cell electroporation has the advantage over other techniques used to<br />
introduce RE into cells in that very low doses of RE can be used, <strong>and</strong> .a greater percentage of the cells exhibit<br />
damage. Experiments have been carried out in CHO cells using the blunt end cutter Rsa I <strong>and</strong> the four base<br />
cohesive end cutter Sau 3A1 . The CHO cells were harvested 6 <strong>and</strong> 22 hrs after treatment so that cells treated in<br />
G= <strong>and</strong> G, would be sampled . The induction of chromosome abemations by concentrations of 1-g0 units for Rsa<br />
I <strong>and</strong> 5-30 units for Sau 3AI have been analyzed . As reported by others, REs induce a much higher percentage of<br />
exchange type aberrations than is found for X irradiation at similar levels of deletions . Both Rsa I <strong>and</strong> Sau 3AI are<br />
capable of inducing CAs In 01 <strong>and</strong> G= at all the concentrations tested. The shape of dose response curves are<br />
different from those reported elsewhere in that they are non-linear . For both Rsa I <strong>and</strong> Sau 3AI the dose response<br />
curves are biphasic ending in a plateau . No straightforward correlation can be made between double str<strong>and</strong> cuts<br />
induced by REs <strong>and</strong> double str<strong>and</strong> breaks induced by X irradiation . (Research sponsored by the Office of Health<br />
<strong>and</strong> <strong>Environmental</strong> Research, U . S. Department of Energy under contract DE-AO0S-840R21400 with the Martin<br />
Marietta Energy Systems, Inc. GJ Horesovsky is supported by NIH-CA 09104-13)<br />
456<br />
COMPARATIVE ANTIMUTAGENICITY OF Cff1 .OROPHYLLIN AND FIVE COMRtON ANTIMUTAGENS AGAINST<br />
FOUR LABORATORY I4UTAGENS IN Salmonella typhimurium STRAINS TA98 AND TA100 .<br />
K . Pupatwibul <strong>and</strong> N . E . Brockman, Illinois State University, Normal, IL (USA)<br />
Chlorophyllin (C1Q.) is a potent inhibitor of the mutagenlcity of 10 complex<br />
mixtures in Salmonella typhimurium strain TA98 (Ong, Whong, Stewart <strong>and</strong> Brockausn,<br />
1986) . Furthermore, CNL is more effective than 4 common antimutagens (B-carotene<br />
<strong>and</strong> vitamins A, C, <strong>and</strong> E) against the mutagenicity of 5 of these complex mixtures<br />
in TA98 (Ong, Whong, Stewart <strong>and</strong> Brockman, 1989) . Based on these results, we have<br />
initiated in S . typhimurium strains TA98 <strong>and</strong> TA100 a comparative study of the<br />
antimutagenic activity of CHL <strong>and</strong> commonly used antimutagens against the<br />
mutagenicity of laboratory mutagens with different mutagenic mechanisms ; we will<br />
extend this comparative study to other short-term tests for genetic toxicity . We<br />
report here our results on the antimutagenic activities of CHL . B-carotene,<br />
retinoic acid, <strong>and</strong> vitamins A, C, <strong>and</strong> E against the mutagenicity of N-methyl-N'nitro-N-nitrosoguanidine<br />
(MOING) <strong>and</strong> 6-N-hydroxylaminopurine in TA100 <strong>and</strong> of<br />
aflatoxin BI <strong>and</strong> the acridine mustard ICR-170 in TA98 . Vitamin A inhibited the<br />
mutagenicity of all 4 mutagens, <strong>and</strong> CHL inhibited the mutagenicity of all of the<br />
mutagens except PINNG : Retinoic acid <strong>and</strong> 8-caroteae inhibited the mutagenicity only<br />
of aflatoxin B . Dose-response curves of antimutagenic activity will be presented .<br />
(H .E .B . acknowledges the support of the U .S . EPA Distinguished Visiting Scientist<br />
Program .)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes<br />
157<br />
I
158 1989 EMS Abstracts 457<br />
Notes ~ -" IN VIVO IN<br />
'I<br />
TI~DS-TEST ON RAT HEPATOCYTFS EXPERIENCES IN TESTING XENOBiO-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
TICS AFTER SINGLE OR REPETITIVE TREATMENT .<br />
E .C . Puri, Th . _krtnowd D . MOlier, Ciba-Geigy Limited ., CH-4002 Basle (Switzerl<strong>and</strong>)<br />
The in vivo-in vitro DNA-repair test gained significance during the last years <strong>and</strong> was also performed<br />
in our laboratories for testing of chemical products as well as of the following reference compounds : 4aminobiphenyl<br />
(ABP), benzo(a)pyrene (BaP), dimethylnitrosamine (DMN), methylmethanesulphonate<br />
(MMS) <strong>and</strong> nafenopin (NAF). To investigate the effects of an enzymatic induetion of the liver a repetitive<br />
treatment with two of these compounds was also carried out . Therefore, compounds were administered<br />
either once (all five compounds), or repetitively (BaP, NAP). The preparation of hepatocytes<br />
(perfusion technique) followed 2 hours after the single application of ABP (200 mg/kg), DMN (15 mg/kg)<br />
or MMS (100 mg/kg) <strong>and</strong> 4 hours after the single application of BaP (100 mg/kg) <strong>and</strong> either 2 or 12<br />
hours after the single application of NAF (200 mg/kg). For the repetitive treatment the animals was given<br />
either BaP at a dose of 20 mg/kg or NAF at a dose of $00 ppm administered in the food on seven consecutive<br />
days, followed at the eighth day by a dose of 100 mg/kg BaP 4 hours before isolation of hepatocytes<br />
or by a dose of 200 mg/kg NAF either 2 or 12 hours before Isolation of hepatocytes . NAF did<br />
not induce DNA-repair synthesis under any of the treatment conditions . Several experiments with ABP .<br />
DMN <strong>and</strong> MMS yielded all clear positive results . BaP yielded a clear positive result after repetitive treatment<br />
. These positive results obtained with BsP after enzymatic liver induction by the test substance itself<br />
are noteworthy, inasmuch as earlier in vivo-in vitro experiments with the same substance on rat<br />
hepatocytes (Mirsalis et al ., 1982, Environm . <strong>Mutagenesis</strong> 4, 553), rat pancreatic cells (Steinmetz <strong>and</strong><br />
Mirsalis, 1984, Environm. <strong>Mutagenesis</strong> 6, 321) <strong>and</strong> rat tracheal epithelial cells (Doolittle <strong>and</strong> Butterworth,<br />
1984, Carcinogenesis S, 773), all performed without prior enzymatic induction, yielded negative results .<br />
458<br />
SOMiATIC NUTATIUN AND rtaCOMtlINA'TI0N TrST 0F FUnArYnINID0Nl ;, A Nr:W ANTIFILAaIAL AGEiVT,<br />
IN Dti0S0YHILA hh:LANOGASTan<br />
nei-Li Qian <strong>and</strong> A . F:iche<br />
Shanghai Institute of Pharmaceutical Industry, 1320 Beijing Xi rtoad, Shanghai, China<br />
Safety Assessment, Astra AS, S-15185, StidertRlje, Sweden<br />
The somatic mutation <strong>and</strong> recombination wing spot test in Drosophila melanogaater<br />
was used to evaluate the genotoxicity of Fluapyrimidone . Third inatar larvae, transhetero2ygous<br />
for recessive wing trichome mutationa were f6II food/test compound mixturea<br />
. A saturated solution or dilutions containing the test compound was added to<br />
Instant medium (cxp 1) <strong>and</strong> powdered Furapyrimidone was added to Yeast-Torula (&xp 2)<br />
at percentages of 0 .0j-S . F'urapyrimidone did not cause a significant change in the<br />
mutant spot frequency for any ty ;,e of spots in l:xp 1 . In Fxp 2, the frequency of large<br />
<strong>and</strong> twin spots was increased in flies treated with 0 .SdB Furapyrimidone . However<br />
in repeated experiment the differences were statistically insignificant . Fwrapyrimidone<br />
was positive on TA 100 <strong>and</strong> TA 98 at 0 .1 <strong>and</strong> 1 ntg/plate in Salmonella%microsome<br />
system for mutagenicity test but teat on Sex-linked recessive lethal test in Drosophila<br />
melanogaster Furapyrimidone showed no evidence of mutagenic potential .<br />
Acknowledgmente : This work was done at Safety Assessment, Astra AB . We wish to thank<br />
K . S<strong>and</strong>elin <strong>and</strong> G . idexell for their technical assistance .<br />
459<br />
1SICROt1UCLEUS TEST IN VICIA PAnA ROOT TIPS : INITAOR1tICITT OP COAL TAR PITCH . Z . Qingfan,<br />
ChangFuju, <strong>and</strong> Z . Qingxia . Dept . of Pathology, Henan Medical University . Zhengzhou,<br />
Henan, P .R . China .<br />
The effect of three different processed (high, moderate, low temperature) coal tar<br />
pitches on the induction of micronuclei in yjglg ZAbg root tips was studied . The results<br />
indicated that CTP had potent mutagenicity on'the root tips of y1gjA I&a, but the<br />
mutagenicity may vary in different kinds of CTP .<br />
TABLE . Frequencies of Micronucleated cells in }(jsL j4g root tips induced by CTP<br />
1oa1 ~ T,<br />
ar Pitch Croilp - maan3SD (a)<br />
Control Grouo<br />
Concentration (mg/ml) j, OZa =s<br />
0 .324 14 .33t0 .88 36 .33±4 .44 16 .33t0 .88<br />
(16 13 14) (38 43 38) (16 18 15)<br />
6 .828 39 .67t1 .76 59 .00±3 .46 26 .33t2 .91 1 .67±0 .67<br />
(39 43 37) (53 65 59) (27 21 31) ( 3 1 1)<br />
10 .0 60 .33±8 .08 82 .00t9 .61 60 .67±6 .49<br />
(49 76 56) (63 94 89) (58 73 51)<br />
These results are consistent with the study on rat lung cancera induced by<br />
intratracheal instillations of CTP, which were made by us previoualy, Through the<br />
experiment, we consider that the micronucleus test in yicig ZAg can be used as a new<br />
alarm system to detect envirorueental pollution such as CTP, <strong>and</strong> that this test has the<br />
advantages of sensitivity, reliability, low cost <strong>and</strong> manageability .<br />
50869 3672
460<br />
_<br />
DETECTION OP MICRONUCLSI IN PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH ESOPHAGEAL<br />
CANCER AND IMPROVEMENT OF THE METHOD . 2 . Qingfan, F . Shuli, <strong>and</strong> Y . Bin . Dept . of<br />
Pathology, Henan Medicaltniversity, 2hengzhou, Henan, P .R . China .<br />
Thirty-four cases of esophageal cancer were chosen for this study . Their age ranges<br />
from 30 to 75 . The diagnosis of the patients were confirmed by cytological examination<br />
in our department . They had not received radiotherapy, chemotherapy or other<br />
immunosuppressive agents recently . Thirty-four healthy persons were taken as the control .<br />
The method consists of taking 2 to 3 drops of blood from the ear lobe of the examined<br />
person, treating the blood sample with Tris-ammonium chloride buffer solution to hemolyze<br />
the red cells <strong>and</strong> then using the simplifled cytocentrifuge developed by the author to<br />
prepare the smears, the white cells can be concentrated <strong>and</strong> spread monolayerly onto a<br />
small area of the microscope slide, <strong>and</strong> the morphological details of the cells can be well<br />
preserved .' The background of the smear is clear, which facilitates scoring the<br />
micronuclei in lymphocytes . The normal range of micronuclei in lymphocytes in our study<br />
was 0-3 0/00 . It shows that the results of our improved method are similar to those with<br />
the ordinary method in which blood was taken by venous puncture . The improved method is<br />
not only fast <strong>and</strong> reliable, but liable to be accepted by the persona examined . The mean<br />
frequency of the micronuclei in the peripheral blood lymphocytes of the patients with<br />
esophageal cancer was 2 .8 0/00 as compared with 0 .832 0/00 in the control . The difference<br />
is statistically significant (p < 0 .001), but there is no marked specificity for the<br />
diagnosis of esophageal cancer, because the frequency in half of the patients with<br />
esophageal cancer is below 2 0/00, which superimposes with the control .<br />
461 The SOS Chromotest : an analysLs from published data on 430 chemio3ls.<br />
P . Quillardet, E. Touati <strong>and</strong> M. Hofnung . Institut Pasteur, UPMTG - CNRS UA271 - INSERM<br />
U 163 - Paris France .<br />
We have made use of an E. coli strain carrying a fusion of gene lacZ to gene 4fu1, one of the<br />
SOS gene, to devise a rapid assay for genotoxins : the SOS Chromotest (Quillardet et al ., Proc. Natl .<br />
Acad. Sci ., 79 : 5971, 1982 ; Mutation Res . 147: 65, 1985). The assay is performed in few hours <strong>and</strong><br />
involves simple enzymatic assays . It allows to classify compounds according to their SOS inducing<br />
potency (SOSIP), defined as their ability to induce the expression af the sfGl : aacZ fusion. To day,<br />
works from a number of laboratories using the SOS Chromotest have been published . We have<br />
reviewed data obtained with the SOS Chromotest on 430 chemicals issued from 40 publications arising<br />
from 20 different laboratories . This led us to evaluate further the potential of the SOS Chromotest to<br />
detect carcinogens <strong>and</strong> to compare its response to that of the Salmonella / microsome assay . The results<br />
confirm that in addition to its remarkable simplicity, the SOS Chromotest is a powerful method to<br />
detect <strong>and</strong> evaluate genotoxic agents<br />
462<br />
COOPERATIVE EFFECTS IN ASSAYS OF CHEMICAL MIXTURES OF ATMOSPHERIC POLLUTANTS .<br />
A .S . Raj <strong>and</strong> D .M . Logan . York University, North York, Ontario, Canada M3J 1P3<br />
Exposure to pollutants in natural atmospheres usually involves complex mixtures<br />
of chemicals rather than a single pollutant . In such cases risk assessment is<br />
difficult if not impossible because the chemicals may interact in several<br />
unpredictable ways . The object of this research is to measure chemical interactions<br />
in certain bioassaya <strong>and</strong> define quantitatively their cooperativity . Nine common<br />
atmospheric pollutants (4 promutagens, 1 direct acting mutagen <strong>and</strong> 4 non mutagens)<br />
have been tested alone <strong>and</strong> in combinations of two <strong>and</strong> three in the Ames <strong>and</strong> micronucleus<br />
assays . For each of the combinations a strictly additive dose response has<br />
been calculated <strong>and</strong> compared with the actual response . In the Ames assay most<br />
mixtures produce fewer revertants than predicted <strong>and</strong> this is not due to S9<br />
limitation . In cases where the predicted response is biphasic ag . BaP <strong>and</strong> DMBA due<br />
to different threshold dosages, the actual response is not biphasic but parallels<br />
that of the lower threshold chemical . In a few mixtures (three or more chemicals)<br />
inclusion of the direct acting mutagen 1-nitropyrene produces a highly synergietic<br />
response ie . more than 5 times the additive response . Weighting factors have been<br />
calculated for each chemical tested which can be used to predict the response of<br />
new mixtures of the chemicals . This approach should prove valuable in predicting<br />
the risk due to environmental pollutants .<br />
This research was supported by a grant from the Ontario Ministry of the Environment .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 159<br />
Notes
160 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
463<br />
NULEES" "' °°''tYNERGISTIC-AL$qNReTOXICIIY OF ALCOH9L AND LB IN PRINTING PRESS 11ORKBRS .<br />
Talitha T Rajgh , NV Prasad , K Kashyap , YR AhuJeAq .1)Dept of Genetics, Osmania<br />
University . 2)C~,o_v~rlnting Press, 3)Bureau of Police . RBD, Hyderebad, INDIA .<br />
Lead (Pb) has been shown to have a mutagenic potential but reports on the<br />
mutagenicity of alcohol (A) are controversial . Since there are no reports available<br />
on the effect of Pb exposure in combination with A it was decided to study the<br />
cytogenetic effects of this combination . Air sampling was done to estimate the content<br />
of Pb In the working atmospherf~ The atmospheric Pb level was found to be elevited<br />
in the printing press (3 .144g/m ) as compared to the background value (0 .5A.g/m ) .<br />
Peripheral blood lymphocytes were cultured for 48 hrs from 20 adult males (10<br />
of whom were A consumers) working in a printing press . These individuals were<br />
exposed to Pb for more than 5 years . Twenty one healthy matched controls were<br />
also included . Chromosome aberrations (CA) were analysed from 100 metaphases<br />
per subject . A consumers did not show a significant increase in the frequency<br />
of CA (structual+numerical) as com pared to the control group (11 .35 vs 7 .54%) .<br />
But subjects exposed to Pb showed a significant increase In genotoxicity as compared<br />
to the controls (17 .60 vs 7 .54%) . When a comparison was made between alcohol<br />
consumers <strong>and</strong> nonconsumers within the Pb exposed group, there was a higher frequency<br />
of CA In the consumers than the nonconsumers (20 .72 vs 17 .60%) . This finding<br />
suggests that Pb <strong>and</strong> A interact In a synergistic manner In inducing genetic damage .<br />
Financial Assistance from ICMR Ss acknowledged .<br />
464<br />
MODULATION OF GENOTOXICITY, Claes Ramel, Department of Genetic <strong>and</strong> Cellular ToxicologK<br />
Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden .<br />
In the absence of any principle breakthrough in cancer therapy, cancer prevention<br />
must rely on hindrance of induction of malignant growth . Chemoprevention of cancer by<br />
means of anticarcinogenic agents is one way towards this goal . This approach is favoured<br />
by the multistage process of carcinogenicity, where each step can be subjected<br />
to modulation by various agents . The critical role of genetic alterations In carcinogenicity,<br />
revealed by activation of oncogenes <strong>and</strong> inactivation of antioncogenes, focuses<br />
the attention on genotoxicity in this context . Modulation of genotoxicity can be<br />
achieved at two levels - by preventing chemical induction of mutations <strong>and</strong> by affecting<br />
the expression <strong>and</strong> regulation of altered genes . It has been shown that chemical<br />
induction of mutations can be modified by a large variety of chemicals acting at<br />
different levels - from the initial exposure to genotoxic chemicals to interactions<br />
with DNA <strong>and</strong> the establishment of mutations . The development of neoplasm comprise<br />
alterations of cellular messengers <strong>and</strong> interaction of gene products . The elucidation<br />
of this cellular signal system opens new prospects for chemoprevention of cancer by<br />
interference with the expression <strong>and</strong> regulation of genes involved In this system .<br />
UV-INDUCED TNYMIDINE INOORPORATION IN A CLINICAL VARIANT OF XERJDER4A PIGMENTOSW<br />
A . Shobha Rani . A . Jyothy, C.Kususe Kuearl, M . SuJetha, P.P .Raddy, <strong>and</strong> O .S.Rsddi.<br />
Institute of Geiwtics . Hoapital for Ganetie Diseasae, Ossenle Universitv . Beuumpet,HVderabad-S00 016 .<br />
A . P. Ind ia .<br />
ABSTRACT<br />
Xerodeima piymentosus Ss a rare autosoaal reaassive disease In which patients develop abnorael pigmentation<br />
<strong>and</strong> salipnancies !n the areas of the skin ezposed to sunlipht . These patients are unable to repair<br />
normally a certain type of UV induced daaape !n their DNA . The present lnvestl9etion is carried out<br />
to sae Sf there is any Japsinssnt in the repair process In this clinical variant with only occul .r senlfastation<br />
without any cutaneous manifestation .<br />
For the scintillation eeasurewent of raoelr . we used leukocvte ooouletions contelnina 1Xio cells . The<br />
cells vsre suspended In 1∎1 culture media with 205 serus . The cells were lrradiated with UV <strong>and</strong> then<br />
HTdR was added <strong>and</strong> reincubated for 24 hrs ., at 37oC. The cells rsre washed In saline followed by Sf <strong>and</strong><br />
10% TCA ( trichloroacatie acid) <strong>and</strong> finally In chilled es thenol . 0 .1 •1 of triton X-100 was added to<br />
dissolve the pellet <strong>and</strong> transferred to scintillation vials after which the activity was measured by<br />
B-counter . UV-induced thyaidlne incroporation rate 1s decreased to 20-37% In the affected individuels<br />
as compered to their unaffected sib (64%) parents ( 100% ) <strong>and</strong> controls (100f) lndicatin0 that ths<br />
rapair mechenis+. !n this clinical variant 1s Upaired .<br />
50869 3674<br />
465
466<br />
I)ISCRE:TE PRODADILITY MODELS IN MAMMALIAN MUTAGENESIS<br />
I~ .Ilamnanth fvra.~ DeparlrqpeFt of Genetics . Osmania University,<br />
Ilyderabad - 500 007 (A .P .) India .<br />
One of the objectives of genetic toxicology is to critically evaluate the<br />
mutagenicity of various industrial chemicals <strong>and</strong> environmental pollutants using<br />
dominant lethal system in mice . T he statistical problem arising out of such<br />
situations is to Identify the probability distribution of the variables underconsideration<br />
to assess the significance of the lethality . Preliminary distributions<br />
like binomial, <strong>and</strong> poissori have beeft' suggested by Haseman <strong>and</strong> Soares (1976)<br />
for the distribution of lethality in plants <strong>and</strong> mice <strong>and</strong> are currently in vogue .<br />
However, these distributions have been found to be inadequate . The complexity<br />
of the biological phenomenon envisages the possible role of the com pound<br />
probability distributions . Based on series of experiments conducted in our<br />
laboratory on dominant lethals in mice a new discrete compound probability model<br />
has been constructed . The study suggests that the model fits well for evaluating<br />
even weaker mutagens thus making it appropriate for wider application in the<br />
field of environmental mutagonesis .<br />
467<br />
PREVENTION OF GENETIC DANAGE INDUCED BY BENZO(a)PYRENE IN NICE BY PROSTAGLANDINS<br />
K .P .Rao, K .Sridevi, <strong>and</strong> K .V .Ch<strong>and</strong>rika, Department of Genetics, Osmania University,<br />
Hyderabad-500007, INDIA .<br />
The present investigation is undertaken to see the possible antimutagenic action of<br />
Prostagl<strong>and</strong>in E1(PGE1) against Benzo(a)pyrene (BP) induced genetic damage in bone marrow<br />
cells of mice by micronucleus test . Recent investigations revealed that certain<br />
mutagens/carcinogens are known to influence the production of PGs <strong>and</strong> thus suggesting that<br />
an altered PG system could be responsible for mutation <strong>and</strong> cancer . Hence, two experiments<br />
using micronucleus test in bone marrow cells of mic$ were ~arried out . In the first<br />
experiment the effect of different doses of P~El (10- to 10- N) on BP induced genetic<br />
damage was studied . The effect of PGE1 (10- M) in relation to cell cycle against BP<br />
induced genetic damage was studied in the second experiment . The results obtained from the<br />
above studies substantiates the possible role of PGa in the pathogenesis of mutagenesis <strong>and</strong><br />
carcinogenesis .<br />
468<br />
M1p1/LATIeN OF RADIATIGN-IMJ11C® GEMETIC DAMIIGE BY 2-0EO7fY-0-GLlIC06E IN TISSIR :S OF TRIGD)ELLA FOE<br />
K.V.S . RAO. G . Jayaraman <strong>and</strong> P .M . Gopinath . Department of Genetics . PGIBMS. llniwrsity of Madras, Tara .ani,<br />
Madras - 600 113 . India . Modulatory effect of 2-deoxy-D-Olucwe (2-OG) on radiation induced genetic<br />
damage has been assessed in the cells-of fenu0reek . Trioonells foenua-oraeoue . 2-DG . a Oluoose antioete-<br />
bolite <strong>and</strong> an inhibitor of glycolysis was reported to exert differential action in irradiated normal <strong>and</strong><br />
neoplastic mammalian cells . Mhile I,t potentiates radiation induced genetic damage in tumor cells . !t<br />
reduces siailer damage in noraal cells . In t1a present study roots of fenu0reek were lrradiatW with<br />
ganee rays <strong>and</strong> post-treated with 2-nG . Cytological preparations obtained from the roots on recovery were<br />
scored to record aitotic indices . instances of ∎icronuclei <strong>and</strong> ∎itotic anomalies . Roots <strong>and</strong> calli were<br />
cultured in aediua containing 2-DG to record data on growth kinetics . DNA extracted from these tissues<br />
was irradiated both In the presence <strong>and</strong> absence of 2-DG <strong>and</strong> subjected to spectrophotoaetric characterization<br />
. For corroborative date . DNA from calf thyaus <strong>and</strong> Salaonella tvohieuriua was employed . 2-DG was<br />
found to reduce the incidence of radiation induced ∎icronuclei snd mitotic anoealies !n seedling snd<br />
cultured roots . Reduction of mitotic activity by 2-DG was significant . 2-DG Inhibited proliferation of<br />
cultured tissues . Spectrophotometric characterization of Irradisted DNA in the presence/absence of 2-DG<br />
reveals no direct interaction with DNA in v tr . DNA of calf thymus <strong>and</strong> ;; . tv6hiaurium also exhibited<br />
similar observations . From the observations It is concluded that 2-DG causes reduction !n radiation-<br />
induced genetic damage also in plant cells . 2-DG is found to be a eitotic inhibitor <strong>and</strong> reduces the growth<br />
of celli <strong>and</strong> cultured excised roots . 2-DG did not show direct Interaction with DNA,lrn vitro .<br />
A<br />
1989 EMS Abstracts 161<br />
Notes<br />
469 hEOSEMOGENIC RI83C PREDICTION OF TFE CYTOBTATIC TREIT!'lEliT<br />
Tibor Raposa ?i .D ., <strong>and</strong> Judith Yirkonyi, n .D . Setttmelweis Univ . Ned .<br />
6chool,Ill . bept .of Internal riedicine, 1121 .~udapest,EBtv6s u. 12 .Eung .<br />
The genotoxicity of wide range of cytostatice, commonly used in the<br />
therapy of lymphonas <strong>and</strong> solid tumors wae measured in the " in vivo -<br />
in vitro " SCE assay . A comparison was them made to analyse the rela- r<br />
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i;<br />
I
162 1989 EMS Abstracts<br />
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NOtES'- "-'"'fionship-bgt_t+e~he genotoxicity <strong>and</strong> carcinogenicity of the same cytotoxic<br />
agents . The model for this purpose has been the thorough literature<br />
surve' oLt herapy induced leukemias during the period from 1930<br />
to 1988 . Over 130_o secondary acute leukemias are included . It is established<br />
that the cytostatic compounds with strong BCE induction formed<br />
the basis off the chemotherapy of more than 9d1i of all the primary malignancies<br />
, which were later followed by an acute leukemia of the induced<br />
type . On the contrary, only a few secondary leukemias were repotked<br />
after the administration of an " 8CE negative " cytostatic treatment<br />
schedule . This observation supports the conclusion t at the BCE assay<br />
is a good indicator of the genotoxicity <strong>and</strong> carcinogenicity of different<br />
cytostatic agents, <strong>and</strong> as such it can reasonably be used in planing<br />
new therapeutical trials with low leukemogenic activity but also<br />
high clinical efficacy, in malignancies which have a high probability<br />
for cure .<br />
470<br />
NfIATIOYAL SPEY:IFIJITY OF 2-(:YANJE'D(YtZNE 07QDE IN HIlMN LV1PfOB[ .ASA)ID CELLS . L . Redo, D. Slspson, J .<br />
Cochrane, VLiber <strong>and</strong> T.R. Sknpak . Chmdcal IMustry Institute of Toxicology . Research Triangle Park, NC<br />
(USA) <strong>and</strong> `liatvatd School of Public Health, Boston, ?U (USA)<br />
The proposed sutagenic metabolite of the rat carcirogee acrylanitrile, 2-cyanethylene oxide (AND), is<br />
nutaganic at both the hunan tk <strong>and</strong> prt loci . To develop a better urdeist<strong>and</strong>ing of its or:clanism of action<br />
in human cella, our grcup has determined the specific It(A sequsnce alterations irdueed by ANJ treataent .<br />
Analysis of several tk-/- <strong>and</strong> lrt- mutants by Southern blot liybridisaticn shawed that sost of the iMuced<br />
mutants had no detectable alterations . A collection of brt- mutaixs were further characterized by dideoery<br />
sequercing of cloned 1rt cINA . Point mutatiare in the lct codin6 region Wore obsetved in 9 of 17 lrtautants<br />
; 5 accua-ced at AT base pairs <strong>and</strong> 4 at OC base pairs . 8/17 ct- mutarxs displayed aberrant splicing<br />
of trt mRNA, resulting in the loss of single <strong>and</strong> maltiple ebxm, as tiiall as alternative splicing at a<br />
cryptic splice site vithin aeon 9 . Southern blot analysis of aitants wdth single axmn losses revealed no<br />
visible alterations . Analysis of one mutaix missing afmas 3-6 in its sRNA did reveal a visible deletion in<br />
its gecnodc 1NA. The intron/exon jurcticns of three aitanta (one sd,th somn 7 loss, one adth exan 8 loss,<br />
<strong>and</strong> one mutant exhibdting cryptic splicing in e:mn 9) tiere PCR aplified fram gemedc INA <strong>and</strong> analyzed by<br />
Southern blot using ema-specific probes . The amos missing ftcm the rt aRNA were present in the $enomdc<br />
rt aequence . The pertinent intton/exoa regions of l,xt genosd .c INA fram a nRant rdth axon 8 loss <strong>and</strong> a<br />
mrtant exhibiting cryptic splicing in a:mn 9 uare cloned into M13op19 <strong>and</strong> sequenced . Point sutations in the<br />
conceneus splice acceptor site of ewon 8(AT+fA) ard cmn 9(ATKiC) were observed . These observations<br />
indicate that AND icduces primarily point .utations in hmmn cells at both AT <strong>and</strong> OC base pairs, <strong>and</strong> that<br />
concensus splice acceptor sites are prone to ®rtageneeis . This wrk also suggests that there are at least<br />
two pramutagentc lesions irduced by AA1) .<br />
471<br />
MOLECULAR ANALYSIS OF HPRT- T-LYMPHOCYTES DERIVED FROM AN IN VIVO CLONAL<br />
AMPLIFNATION . L . Recio, D . Simpson, J . Cochrane, T . Skopek, J .A . Nicklaaa, J .P .<br />
O'Neill , <strong>and</strong> R .J . Albertinia) . Chemical Industry Institute of Toxicology, RTP . NC<br />
(USA) <strong>and</strong> the aUniversity of Vermont, Burlington, VT (USA) .<br />
T-lymphocytes rearrange their T-cell receptor (TCR) genes during differentiation<br />
in the thymus <strong>and</strong> then pass the unique TCR gene patterns to their clonal descendents .<br />
Analysis of TCR gene rearrangements has been used to establish the clonal relationship<br />
of hprt- T-lymphocytes isolated from the peripheral blood of humans (<strong>Mutagenesis</strong><br />
2 :341) . A female subject has been identified with an extremely high <strong>and</strong> increasing<br />
frequency of 6-thioguanine-resistant T-lymphocytes . The majority (>92X) of these<br />
mutants display the same TCR pattern <strong>and</strong> therefore must be sibs (Environ . Mol . Hutat .<br />
12 :271) . To develop a better underst<strong>and</strong>ing of the genesis of these mutants we have<br />
isolated <strong>and</strong> sequenced hprt cDNA from 15 mutant clones displaying the same TCR<br />
pattern . All 15 mutants were missing exon 6 from the hcrt message, suggesting that<br />
this mutation was an early event during the clonal expansion process . One mutant was<br />
also missing axon 8 in addition to exon 6 . These results demonstrate that clonal<br />
expansion of mutants in vivo can seriously affect both the frequency <strong>and</strong> spectrum of<br />
hprt- mutants observed 1n vivo . The preliminary finding of one mutant possessing two<br />
separate alterations also suggests the possibility of extreme genetic instability in<br />
this population of dividing T-lymphocytes .<br />
POSITIVE CORRELATION BETWEEN SISTER CHROHATLD KXCHANCB AND COTININf! IN SMOKERS<br />
Reidy, J .A ., Chen . A .T .L ., Spiorto, F .W ., Waymack, P .P ., Jr ., <strong>and</strong> Smith, S .J .<br />
Division of <strong>Environmental</strong> Health Laboratory Sciences . Center for Rnvironmental<br />
Health <strong>and</strong> Injury Control, Centers for Disease Control, Atlanta, CA 30333, USA<br />
Resulta of numerous studies have shown that smokers have a higher<br />
incidence of sister chromatid exchange (SC6) in their lymphocytes <strong>and</strong> higher<br />
levels of cotinine (a metabolite of nicotine) in their serum than nonsmokers .<br />
50869 3676<br />
472
t<br />
SCe <strong>and</strong> serum cotinine were determined for 17 smokers . SCE for each person<br />
was determined by analyong 100 second-division lymphocytes from whole blood<br />
cultures . SCE per persoiC ranged from 4 .54 to 10 .16 exchanges per coll .<br />
Cotinine values were determined in duplicate for blood drawn at the same<br />
time . These values were between 13 <strong>and</strong> 500 ng/ml . In this study, we found a<br />
positive correlation (R .0 .53, p- 0 .03) between SCE <strong>and</strong> serum cotinine . Larger<br />
studies are needed to confirm this finding .<br />
473<br />
4Hk: lNTEGENICITY STUDY OF 69'NINGOCOCCAI: I'OLYSACCIIARIED-TETANUS TOXOIL CONJUGATE<br />
Ren Neiyue, Zhao Hong, tang Yaozhone, <strong>and</strong> Li genqing, Shanghai Institute of Biological<br />
Products, Shanghai, China<br />
Mer.ingococcal group A'polyaaccharide-tetanus toxoid conjugate was combined with po-<br />
lysaccharide <strong>and</strong> protein so as to increase the immunogenicity of both the polysaccheride<br />
<strong>and</strong> tetanus toxoid in the guinea-pigs <strong>and</strong> mice . It In hopeful that the conjugate<br />
will be used widely in human beings, eapecielly in very young children In Chine . Here<br />
we examined the mutagenicity of the conjugate for Ames assay, micronucleus test <strong>and</strong><br />
chromosome aberration analysis in Chinese Han .ater Lung cells . The result indicated th-<br />
at the conjugate could not increase remarkarly either the revertants of four test strains<br />
( TA97, TA98 . TA100, <strong>and</strong> TA102 ) with of without 89 mix In Ames asaay at the con-<br />
centration range of 0 .2-125 ug/plate, or the rate of micronucleus after treatment for<br />
2t hours in the polyghromatic erthrocytes of tho bone mRrrow cells in mice at the con-<br />
centration range of 48-1200 ug/I(g body weight . The result also indicated that half of<br />
the cells was inhirited In vitro when the roriugete was et the concentration if 0 .92<br />
ug/ml <strong>and</strong> the conjugate could not increase remarkably the rate of chromosome aberrati-<br />
on after treFtmertt for u hours with S9 mix or for 24 or 48 hours without 59 mix at the<br />
concentration range of 0 .25-1 .0iug/ml in chromosorre aberration anelysis . It wss belie-<br />
ved that the conjugate hae no mutegenicity in our leb. f<br />
474 •<br />
THE LACK OF DNA HOMOLOGY IN PAIRS OF DNA DIVERGENT CHROMOSOMES SENSITIZES THEN TO<br />
LOSS BY DNA DAMAGE . M .A . Resnick <strong>and</strong> T .Nilsson-Tillgren . Yeast Genetics Group,<br />
National Inst . Environmen-EaT~eafth Sciences, Research Triangle Park, NC, USA ;<br />
Inst . of Genetics, U . Copenhagen, Denmark<br />
Chromosomal DNA is considered a priori to be a target for production of numerical<br />
(whole chromosome) aneuploidy <strong>and</strong> DNA repair would be expected to play a role .<br />
Using the yeast Saccharom ces cerevisiae, we have addressed the importance of<br />
recombinational repa r requ re or ouble-str<strong>and</strong> break [DSB] repair) in the<br />
maintenance of complete chromosomes . Specifically, aneuploidy induction by Ionizing<br />
radiation has been examined in diploids which had either one chromosome III<br />
or chromosome V replaced by a DNA divergent (homoeologous) chromosome from S .<br />
carlsber ensis . While they are functionally equivalent, the lack of precise DNA<br />
omo oof chromosome III or all of V was expected to prevent recombinational<br />
repair . The absence of recombinational repair (presumably of DSBs) in the<br />
divergent chromosomes results in aneuploidy levels of 5 to 15% at nonlethal doses<br />
<strong>and</strong> a low level of rearrangements . The induction appears to level off suggesting<br />
alternative ways of dealing with double-str<strong>and</strong> breaks . For homologous chromosomes,<br />
the aneuploidy frequency is 20 to 50 X lower . Based on genetic <strong>and</strong> physical analyses,<br />
the aneuploidy is due to chromosome loss, not chromosome deletions nor<br />
malsegregation . Thus, the absence of opportunity for homologous recombinational<br />
repair of double-str<strong>and</strong> damage results in chromosome loss . We suggest that nonhomologous<br />
regions of otherwise homologous chromosomes may be important targets for<br />
the induction of aneuploidy . The relevance of these observations to those in mammalian<br />
cells will be discussed .<br />
475<br />
BIOMOHITORIPG OF INDIVIDUALS OCCUPATIONALLY EIpOSED TO AROMATIC A11IwES .<br />
L .R .Ribeiro,D .M .F .Salvadori,E .M .M .Cerqueira,H .S .Barbosa,M .D .M .Oliveira <strong>and</strong> A .R .Silva .<br />
Universidade Federal da Bahia, Salvador, BA (BRASIL) .<br />
Several reports show high frequency of genetic damage <strong>and</strong> high incidence of urinary<br />
bladder cancer due to the presence of mutagenic <strong>and</strong> carcinogenic products in the urinary<br />
tract,apecially the aromatic amines .The genetic <strong>and</strong> carcinogenic risks of workers occupationally<br />
exposed to aromatic amines <strong>and</strong> the presumed antimutagenic <strong>and</strong> anticarcinogenic<br />
potential of provitamin a-carotene are being studied at the production plant at<br />
the Petrochemical Industrial Complex of CamaSari,BA,Brazi1 .30 male individuals exposed<br />
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1989 EMS Abstracts 163<br />
Notes
164 1989 EMS Abstracts<br />
~ Notes<br />
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~<br />
to aromatic amines <strong>and</strong> 30 controls were exaeined .The clastogenic studies were carried '<br />
out vith micronucleus test on exfoliated cells from the urinary bladder <strong>and</strong> the careinQ<br />
genicity was evaluated through the cytopathological analysis of these cells . Urinary<br />
bladder cells were recovered by centrifuging the urine <strong>and</strong> pipetting the asdiASnted eelis<br />
onto microscopic slides .Air-dried-alides were Giemsa-atained to micronuclei analysis <strong>and</strong><br />
Papanicolau-stained to cytopathological analysis .Tbe possible correlation between preneoplasic<br />
cells <strong>and</strong> the frequency of micronuclei is being evaluated,whereas the possible<br />
effects of age,smoking habits,alcobol <strong>and</strong> coffee drinking are also discussed .The aim of<br />
the study presented here was to verify if a combined application of the micrawleus test<br />
<strong>and</strong> the cytopathological analysis in exfoliated urothelial cells could be used to identify<br />
population groups of high risk for urinary bladder cancer <strong>and</strong> to verify the antiinitagenic<br />
<strong>and</strong> anticarcinogenic 3-carotene supplementation effeet .The potential for this method<br />
to be used for noninvasive monitoring will be discussed .This work was supported by<br />
FINEP, CNPq <strong>and</strong> ROCHE .<br />
476<br />
CHROMOSOME CHANGES DURING NEOPLASTIC PROGRESSION IN RAT TRACHEAL EPITHELIAL (RTE)<br />
CELLS . K . Rithidech, D . G . Thomassen, <strong>and</strong> A . L . Brooks, Lovelace Inhalation<br />
Toxicology Research Institute, P .O . Box 5890, Albuquerque, New Mexico 87185 .<br />
Chromosome abnormalities are usually observed in tumor cells . Since it is not<br />
known if these abnormalities are the cause or consequence of tumor development, it is<br />
important to characterize chromosomal changes at all stages of progression . The<br />
purpose of this study was to analyze chromosomal changes in RTE cells at preneoplastic<br />
<strong>and</strong> neoplastic stages of tumorigenesis . RTE cells were exposed to 6 Gy of X-rays or<br />
N-methyl-N-nitro-N-nitrosoguanidine (MiNG) . Preneoplastic enhanced growth variants<br />
(EGVs), the first recognizable step in tumor progression of RTE cells to neoplasia,<br />
were isolated using a selective medium . Eighteen EGVs were isolated 35 days after<br />
exposure . All EGVa from passage 6 were evaluated for chromosomal changes <strong>and</strong> were<br />
injected into nude mice to test for their tumoriganicity . Five X-ray-induced ECVs<br />
have been analyzed . Four of the five cell lines were hyparploid <strong>and</strong> one was<br />
hypoploid . G-b<strong>and</strong>ing revealed unique chrosasomal alterations in each EGV . The<br />
specific type of changes were deletions (1q), isochromosomes (9q), translocations<br />
(5 . 12), marker chromosome/, <strong>and</strong> double minutes . Nost of the hyperploid EGVs possessed<br />
at least one extra copy of chromosome 1 . These results suggest that specific<br />
chromosomal changes can be detected in early stages of carcinogenesis . Cytogenetic<br />
studies of the remaining EGVs <strong>and</strong> of tumor cells produced in nude mice by these EGVs<br />
are underway . Results from these studies will help determine the role of chromosomal<br />
changet in tumor progression . (Research sponsored by the U .S . Department of Energy's<br />
Office of Health <strong>and</strong> <strong>Environmental</strong> Research under Contract No . DE-AC04-76EV01013 .)<br />
477<br />
CTTOGENETIC EVALUATION 0F THE IN VIVO GENOTOXICITT OF SUPERLBTNAL DOSES OF DIOXIN .<br />
S .D . Robertson <strong>and</strong> A .F . McFee, Oak Ridge Associated Universities, Oak Ridge, TN (USA) .<br />
The halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibanso-p-dioxin has<br />
received considerable attention as a highly toxic environmental pollutant . It !s a<br />
positive carcinogen in some test systems <strong>and</strong> a potent teratogen . Cytogenetic findings<br />
following various sub-lethal doses have ranged from no observable effect on either<br />
chromosome aberration or SCE induetion to modest but significant ineresses in the<br />
ineidence of aberrations . We administered single intraparitoneal lnjeetions of 250,<br />
500 <strong>and</strong> 1,000 ug/kg (lOx the acute lethal dose) to ule B6C3F1 mice <strong>and</strong> scored<br />
chromosome aberrations in marrow cells of 8 aice/treatment 17 hr later, <strong>and</strong> sister<br />
chromatid exchanges in 4 mice at 23 hr post-treataent . One-tail trend test analyses<br />
of the data indicated no significant change in the level of SCEs= the percent of calls<br />
containing aberrations was significantly incraased by the two lower doses but not the<br />
highest level . Sfnce dioxin is slowly excreted froa liver <strong>and</strong> fatty tissue storage<br />
<strong>and</strong> lethality of even very high dosas fs not expressed for about 2 weeks, !t was<br />
meaningful to study longer treatment-to-evaluation times . SCgs among 5 mice per group<br />
showed a significant lncruse (p< .05, t test) at 8 days after 1,000 vg/kg doses, but<br />
not at 2 or 4 days ; however, a repeat trial found no elevation of SCBs at 2, 4, 6, or<br />
8 days . The proportions of polycbro .atic erythrocytes baaring ∎icronucla! in the<br />
marrow of treated ∎ice were not different from controls at 1, 3, or 8 days, but<br />
micronuclei in peripheral blood erythroeytes were elevated at 3 days . We conclude<br />
that significant increases in cytogenetlc lesions are not consistently produced by<br />
superlethal doses of dioxin . Supported by NIRRS Interagency Agreement T01-ES-20100<br />
<strong>and</strong> DOE/ORAU Contract DE-AC05-760R00033 .<br />
~<br />
m<br />
CO<br />
Oh<br />
%O<br />
W MJ<br />
CO
478<br />
_<br />
1989 EMS Abstracts<br />
CHLOROPHYLLIN IS AN ANTIMITAGEN IN DROSOPflIIA ItEIANOGASTRRf Notes<br />
Dra . R . Rodriguez-Arnaiz <strong>and</strong> S . Zimmering . Universidad Nacional Autonoma de Mexico,<br />
Coyoacan 04510, Mexico, D:f. MEXICO .<br />
In Drosoohila Melanogaster chlorophyllin was tested for its ability to inhibit or<br />
reduce SLRL using the $ggg method <strong>and</strong> CS males . A brooding scheme of 0-2 <strong>and</strong> 3-5 days<br />
was employed . Two groups of experiments were run . First, males were fed with DMN (75ppm<br />
for 48 hours) <strong>and</strong> then they were injected with a solution of 0 .5% chlorophyllin sodium<br />
salt in 5% sucrose solution . A second group of males was injected with the 0 .5% solution<br />
of chlorophyllin <strong>and</strong> the fed DMN at 75ppm for 48 hours . The negative control were only<br />
injected with chlorophyllin <strong>and</strong> the positive control only fed DMN . The SLRLT was run as<br />
usual . Results obtained show a decrease in the number <strong>and</strong> proportion of sex-linked<br />
recessive lethals in groups treated with DI4d+chlorophyllin <strong>and</strong> chlorophyllin+DMN .<br />
479<br />
Antomatic Evaluation <strong>and</strong> Comparison of Micronuclel Frequencies in Bone Marrow<br />
<strong>and</strong> in Peripheral Blood of Rats Treated with Various Model Clastogens .<br />
F. Romagna <strong>and</strong> A . Papapetropoulos . Drug Safety Assessment/fosieology, S<strong>and</strong>oz<br />
Ltd., CH-4002 Basle, Switzerl<strong>and</strong>.<br />
A new technology is presented which offers high quality slides suitable for fullyautomated<br />
micronuclei scoring by computerized image analysis <strong>and</strong> In addition, allows<br />
the enrichment of immature peripheral blood erythrocytes by using step•gradient<br />
centrifugation . Repeated dosing of rats for four consecutive days led to a steady state<br />
level of micronuclei Induction In bone marrow as well as in peripheral blood. In the<br />
latter, maximal response was delayed. In contrast, no elevated micronuclei frequencies<br />
could be seen in the mature erythrocyte population Indicating that micronuelelIn this<br />
cell compartiment were mainly removed by spleenic function . Theae results are In<br />
contrast to observations made in several mouse strains, where accumulation of<br />
micronuclei in mature erythrocytea does occur. Our data clearly demonstrated that the<br />
immature erythrocyte population of rat peripheral blood Is a highly sensitive system<br />
for micronucleus testing <strong>and</strong> that a steady state level with maximal drug response can<br />
be achieved by using a multiple dose regimen . It is hoped that this study may encourage<br />
further evaluation of the rat peripheral blood micronucleus test fdr routine purposes<br />
in genetic toxicology .<br />
480<br />
GENOTOXIC AND NON-GENOTOXIC CARCINOGENS CAN BE IDENTIFIED AND PREDICTED BY CASE, AN<br />
ARTIFICIAL INTELLIGENCE SYSTEM . Herbert S . Rosenkranz, Dept . of <strong>Environmental</strong> Health<br />
Sciences, School of Medicine, Case Western Reserve Univ ., Clevel<strong>and</strong>, Ohio 44106<br />
Analysis of short-term test <strong>and</strong> animal carcinogenicity results indicates that<br />
there are genotoxic carcinogens (GC) which are characterized by the ability to induce<br />
cancers in mice <strong>and</strong> rats, at multiple sites <strong>and</strong> in both genders, <strong>and</strong> there are nongenotoxic<br />
carcinogens (NGC) which are species- <strong>and</strong> site-specifio <strong>and</strong> may be<br />
restricted to a single gender . NGCs cannot be differentiated from non-genotoxic noncarcinogens<br />
by short-term teats or "structural alerts" . CASE, the Computer Automated<br />
Structure Evaluation system Was applied to this situation <strong>and</strong> a number of findings<br />
were made :<br />
1 . CASE was successful in h<strong>and</strong>ling non-congeneric SalmoneUamutagenieity data bases .<br />
The structural determinants (biophores) identified could be used to prediet<br />
mutagenicity <strong>and</strong> to study mechanisms of mutagenicity . In spite of many differences<br />
in the composition of the NTP <strong>and</strong> Gene-Tox data bases, the same major biophores were<br />
identified in both .<br />
2 . Analysis of the NTP carcinogenicity data base revealed that CASE was highly<br />
effective in classifying carcinogens <strong>and</strong> non-oaroinogens (sensitivity, 1,00 ;<br />
specificity, >0 .86) . Additionally, CASE allowed the recognition of NGCs based upon<br />
unique structural features . CASE identified three types of oaroirwgen-speoifio<br />
biophores : (a) those' specific for GCs (primarily eleotrophilio or potentially<br />
electrophilic biophores) ; (b) NGC-specifio biophorea <strong>and</strong> (a) non-eleotrophilio<br />
biophores shared by some GCs <strong>and</strong> NGCa . These findings reveal an unexpected structural<br />
commonality among NGCs <strong>and</strong> permits a systematic study of the basis of their action .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
r<br />
165
166 1989 EMS Abstracts _ • r -_ . . _. 481<br />
NOtE S<br />
~J•!<br />
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--=iw-<br />
^ ~ PREDICTINC' lRtTAGENICITY AND MUTAGENIC NECHANISNS USING STRUCTURAL CONCEPTS AND<br />
ARTIFICIAL INTELLI E~NCE . H .S . Rosenkranz <strong>and</strong> G . Klopman, Departments of <strong>Environmental</strong><br />
Health Soienoes,_ad1=hemistry, Case Western Reserve Univ ., Clevel<strong>and</strong>, Ohio 44106 .<br />
Heretofore structure activity relationships (SAR) have been confined to highly<br />
oongenerie data bases (i .e ., specific chemical classes) . Thus the study of individual<br />
molecules or small groups of molecules has not generally been possible due to the<br />
paucity of data . The CASE (Computer Automated Structure Evaluation) method developed<br />
by us is an expert system which is completely automatic <strong>and</strong> self-learning . We have<br />
demonstrated that it is further unique in that it can use non-congeneric data bases<br />
<strong>and</strong> still be highly predictive of mutagenioity . In the present study a subset of the<br />
Gene-Tox Sa/rnonella mutagenicity data base consisting of 808 chemicals was analyzed by<br />
CASE <strong>and</strong> the generated structural determinants were used to study the basis of the<br />
mutagenicity of a series of agents . Thus, it was shown that for phenylazoaniline<br />
dyes retention of the azo moiety is essential for mutagenicity . For 1-amino-2naphthol-derived<br />
azo dyes, on the other h<strong>and</strong>, reductive cleavage of the azo bond is<br />
required for activity . The basis of the inactivation of azo dyes <strong>and</strong> polyeyolie<br />
aromatic hydrocarbons by sulfonation was also elucidated <strong>and</strong> it was demonstrated that<br />
only certain sites are targets for inaotivation by sulfonation .<br />
It will be demonstrated that CASE can be used to design molecules retaining their<br />
beneficial properties but of greatly reduced mutagenioity .<br />
MODULATION OF GENOTOXIC EFFECTS IN HUMANS . M.P. Rosin <strong>and</strong> A .M Gilbert,<br />
Carcinogenesis Unit. School of Kinesiology, Simon Fraser University . Burnaby, B.C. <strong>and</strong> British<br />
Columbia Cancer Research Centre . Vancouver, B.C ., Canada<br />
482<br />
Human populations are constantly exposed to a multiplicity of environmental agents, the<br />
interactions of which can have a profound effect on cancer risk. This paper describes studies<br />
validating the micronucleus test on exfoliated cells (MEC test) as a technique for studying the<br />
interplay of carcinogens, cocarcinogens, genetics <strong>and</strong> dietln a biological response with relevance<br />
to cancer, chromosome breakage in target epithelial sites. The initial studies described in this<br />
paper involved carcinogen-exposed populations in which combinations of poor diet, alcohol, <strong>and</strong><br />
tobacco chewing were shown to interact to increase MEC frequencies in the oral cavity . In these<br />
populations, oral supplementation with beta-carotene <strong>and</strong>/or vitamin A resulted in a reduction in<br />
MEC frequencies even when exposure to tobacco/alcohol remained unchanged . Recent studies<br />
are aimed at incorporating genetic factors (defective DNA repair processes, spontaneous<br />
chromosomal instability) into these models . The population currently being examined is<br />
comprised of patients with ataxia-telangiectasia (A-T), a cancer-predisposing syndrome<br />
characterized by a hypersensitivity of cultured cells to the action of free radical-producing<br />
chemicals <strong>and</strong> spontaneous chromosomal Instability . Our initial observations indicate that MEC<br />
frequencies are elevated in A-T patients <strong>and</strong> in some of the parents of such patients (at risk for<br />
breast cancer) . These studies suggest that the MEC test may be one approach by which the<br />
complex interactions of environmental <strong>and</strong> genetic factors can be delineated .<br />
483<br />
0°-METNYLGUANINE-DNA-HETHYLTRANSFERASE ACTIVITY IN SURGICAL SPECIMENS FROM HIGH GRADE<br />
HUMAN MALIGNANT GLIOMAS .<br />
0 . Rossi, G . Arena, G . Frosina, G . Fronza, A . Sobrero, S .L . Gentile, E . Bruzzone, N . Bal_<br />
dini, C . Silvestro, <strong>and</strong> A . Abbond<strong>and</strong>olo, National Institute for Research on Cancer, Genova<br />
(Italy), University of Genova (Italy), <strong>and</strong> Neurosurgical Clinic, S . Martino Hospital,<br />
Genova (Italy)<br />
Chloroethylnitrosoureas (CENUs) are used, usually in combination with radiotherapy,<br />
in the clinical treatment of brain tumors . As pointed out by R .W . Kohn (1987), "It seeas<br />
likely that only tumors consisting predominantly of transferase-deficient cells would be<br />
potentially responsive to chloroethylnitrosoureas . Tumor tissues could be assayed for<br />
guanine-06-alkyltransferase, <strong>and</strong> treatment with these drugs could be confined to patients<br />
bearing transferase-deficinet tumors" . MT-deficiancy has been found to be relatively fre_<br />
quent among cell lines derived from human brain tumors but has not been demonstrated so<br />
far, to our knowledge, in tumor tissues . We have started a study to measure the MT aetiv_<br />
ity in surgical specimens from high grade human malignant glio®as, with the dual aim to<br />
(i), know whether lack of activity can be demonstrated in these tumors, <strong>and</strong> (ii), relate<br />
the measured levels of MT to the histology of the tumors <strong>and</strong> to the response of patients<br />
to chemotherapy with 1-(2-chloroethyl)-3-cyclohaxyl-l-nitrosourea (CCNU) . To date, 12<br />
gliomas have been assayed . In 11 tumors, MT activities ranging from 30 to 150 fmoles/mg<br />
protein have been measured . The only negative specimen derived from a patient who had re<br />
ceived radiotherapy before surgery . At the present stage of the study&therefore, we<br />
have no unequivocal evidence for the existence of MT-deficient gliomas .<br />
50869 3680
484<br />
_<br />
IMUrID RE.VEFtSION OF A SPONfAt~OUS POINT NATPATICN WITHIN THE CHINESE HAFISTFR HPRT<br />
GENE~ Yrz~ ~~ 1<br />
B J F Rossiter ~DCMuzny , C T Caskey1 <strong>and</strong> r( ~, 2 Inst 1 for <strong>Molecular</strong><br />
Genetics, Baylor College of Medicine, Houston :Texas 77030, USA, jDept of Biochem<br />
Genetics,Paterson Institute for Cancer Research, Manchester .<br />
Spontaneous HPRT mutants can revert spontaneously at different frequencies by<br />
anplification, point autation at the site of the original mutation or at a second<br />
site . HPRT mutants can also revert at widely different frequencies on exosure to<br />
alkane sulptnnates or alkyl nitrosaueas . The lowest frequencies (5 x 10 50pghnl<br />
1rIDtu1 were observed in mutants e .g . TG15_arith normal amounts of nozmal sized HPRT<br />
mRm . Two other mutants with no detectable HPRT s04A revert at a tenfold higher<br />
frequency . A cDNA library was made fzan 4G15 in gtlO <strong>and</strong> a full length clone<br />
isolated after screening with HPRT cDNA. Sequencing revealed an A -> G transition<br />
which results in the substitution of glycine for aspartic acid at position 134 .<br />
Allele specific screening of in vitro amplified DNA frem wild-type cells, TG15 <strong>and</strong><br />
four independent M induced revertant clones showed that all the had regained the<br />
wild-type sequence . Thus the point mutation in the TG15 is responsible for<br />
inactivation of the HPAT enzyme . Amino acid 134 is thought to lie within the<br />
catalytic domain . These results <strong>and</strong> those in which a truncated form of the E coli<br />
ada gene transfected <strong>and</strong> expressed in TG15 (FCa <strong>and</strong> Margison, <strong>Mutagenesis</strong> 3, 409,<br />
1988) Eeduced the induced revertant frequency are consistent with the conclusion<br />
that 0 nmethylguanine is the promutagenic lesion <strong>and</strong> reversion is due to a point<br />
mutation at the site of the original lesion .<br />
485<br />
BETA-CA~~TENE IN AQUEOUS DISPERSION AS A SCAVENGER OF<br />
H~O2/CU /ASCORBATE GENERATED FREE RADICALS :EFFECTS OF PARTIAL<br />
PRESSURES OF OXYGEN . EJ .Rousseaus, AJ . Davison & M.P. Rosin . Bioenergetics Research<br />
Lab . Faculty of Applied Sciences, School of Kinesiology, Simon Fraser University, Burnaby, B.C .<br />
V5A 1S6 <strong>and</strong> BC Cancer Research Centre, 601 W 10th Ave ., Vancouver, B .C. VSZ 1L3<br />
We report on the antioxidant capacities of beta-carotene In aqueous dispersion as a<br />
scavenger of hydroxyl radicals under varying partial ressures of oxygen . Hydroxyl radicals<br />
(generated by hydrogen peroxide In the presence of oopper <strong>and</strong> asoorbate) pxid¢ed betacarotene<br />
resulting in a loss of absorbance at 460nm . A mixture of 0 .1mM Cu +, 0.5mM<br />
ascorbate, <strong>and</strong> 2.5mM H2050 iIn Hepes buffer at pH 7.4 <strong>and</strong> 25~, was used, oxidizing 2 .76ug<br />
beta-carotene/min . Each ofthe active ingredients was essential. If any were omitted, the rate of<br />
bleaching of beta-carotene decreased by more than 20% . This system was studied under high<br />
oxygen(62%), normoxic(21%), <strong>and</strong> low oxygen(5%), each with four concentrations of H 02<br />
(2 .5, 5, 7 .5, <strong>and</strong> 1omM) . Rates of bleaching of beta-carotene under 20% oxygen were a~f<br />
average of 15% <strong>and</strong> 30% higher than 62% <strong>and</strong> 5% pressures of oxygen . Thus beta-carotene is<br />
at its most efficient as an antioxidant at intermediate partial pressures of oxygen, In partial<br />
agreement with other workers who have shown enhanced protection at p02 below ambient<br />
levels. Beta-carotene is capable of scavenging hydroxyl radicals . The ability of beta-carotene to<br />
function as an antioxidant against free radicals could help define a mechanism by which betacarotene<br />
acts a chemopreventative agent .<br />
486<br />
TRADESCANTIA-MICRONUCLEUS BIOASSAY ON CI~STOGENICITY OB WASTEWATER AN{~ I~i SITU AIR<br />
MONITORING, E . F . Ruia1, E . R . Valtierra , S. U . Lecona , <strong>and</strong> T . H . Ma , 'Centro de<br />
Estudios Academ~cos sobre Contaminacion Ambientl, IInivereidad Autonoma de Queretaro,<br />
QRO, Mexico, Institute for <strong>Environmental</strong> Management <strong>and</strong> Department of Biological<br />
Sciences, Western Illinois University, Macomb, IL (USA)<br />
Tradescantia-Micronucleus (Trad-MCN) bioassay is known to be a highly sensitive short<br />
term test for clastogenioity of water directly without concentration <strong>and</strong> on site<br />
monitoring of clastogenicity of air pollutants without collecting air condensates .<br />
Results can be obtained within 36 - 48 hr . Trad-MCN bioassay was applied to<br />
wastewater samples collected from the canal at a point about 8 kilometers down stream<br />
of the industrial zone of Queretaro City , Mexico . Four liters of water samples<br />
were collected monthly for tests in the year of 1987 <strong>and</strong> part of the years of of<br />
1986 <strong>and</strong> 1988, for the clastogenicity of the water through the rainy <strong>and</strong> dry seasons .<br />
On site monitoring of the clastogenicity of the air with Trad-MCN test was done at<br />
three locations in the city . The clastogenicity which was reflected by the<br />
frequencies of micronuclei in the meiotic pollen mother cells was always higher in the<br />
wastewater treated groups than the negative control group group using tapwater . When<br />
the fluctuated MCN frequencies were compared with the monthly rainfall records, the<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 167<br />
Notes
168 1989 EMS Abstracts<br />
Notes peak olaetog+nj&.tty-seems to be related to the runoff of the pollutants from the upper<br />
atream of:ftlte 0smF1: Five on site monitoring tripe were made at each of the three<br />
locatione, -U e . Conalep, Flores Magon (induetrial sone) Belles Aetee (Downtown) in May<br />
<strong>and</strong> June, 1988~Positive resulte were obtained in ∎ore than 60% of the tests<br />
conducted .. -~a--r-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
487<br />
PATTERNS OF MUTATIONAL SENSITIVITY IN POST-STEM-CELL STAGES OF MOUSE<br />
SPERMATOGENESIS AND IN ZYGOTES : 'I'HE GENERATION OF DELETION MUTANTS AND<br />
MOSAICS . Liane B . Russell, Oak Ridge National Laboratory . Oak Ridge, TN. 37831-8077.<br />
Because the majority of conclusive results in the mouse specific-locus test (SLT), are for to#onial<br />
stem cells, we are engaged in enlarging the SLT data base for post-stem-cell stages (from differentiating<br />
spermatogonia to mature spermatozoa), each of which spans a relatively brief time interval . Chemicals differ<br />
markedly in their relative effects on different post-stem-cell stages, both with regard to mutation rate <strong>and</strong> to<br />
productivity (determined by dominant-lethal incidence <strong>and</strong> germ-cell death) . Mutations induced by chemicals<br />
that have their greatest mutagenic effect in premeiodc (but post-stem-cell) stages appear to be smaller genetic<br />
lesions than mutations induced by chemicals that are most effective in postmeiottc stages . The correlation<br />
between patterns of germ-cell-stage sensitivity for specific-locus mutations <strong>and</strong> dominant lethals, which has<br />
been noted for some chemicals, does not hold for others . Of all post-stem-cell mutagens chlorambucil<br />
(CHL) is the mos~ effective : we have shown that exposure of early spermadds to only 10 mg CHIIkg<br />
induces 1 .3 x 10' mutations per locus . Almost all such mutations have proved to be deletions or other<br />
structural changes, which are highly valuable tools for molecular mapping studies . -- The zygote may be<br />
regarded as the final germ-cell stage, since maternal <strong>and</strong> paternal genomes are still separate . We have found<br />
that ethylnitrosourea (ENU) produces a very high frequency of pritttarily small genetic lesions in zygotes,<br />
most resulting mutants being mosaics. Mosaics can provide very useful biological tools for developmental<br />
studies . Thus . CHL administered to early spermatids, <strong>and</strong> ENU to zygotes, may be the mutagenic<br />
treatments of choice for generating maximum frequencies of, respectively, large-lesion whole-body mutants<br />
<strong>and</strong> smaU-lesion mosaic mutants . [Research 'p indy sponsored by the Office of Health <strong>and</strong> <strong>Environmental</strong><br />
Research, U .S . DOE contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc ., <strong>and</strong> by the<br />
National Institute of <strong>Environmental</strong> Health Sciences under IAG No . 222Y01-ES-10067 .)<br />
488<br />
DOSE REPETITION GREATLY INCREASES THE MUTAGENIC EFFECTIVENESS OF ENU IN<br />
MOUSE SPERMATOGONIA : ADDITIONAL DATA . W. L . Russell, P. R. Hunsicker, <strong>and</strong> S . C.<br />
Maddux, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077 .<br />
The maximum practicable single dose of ethylnitrosounea (ENU) that can be~g~ven intrapertioneaUy co<br />
mice in mutagenic studies is appro ximately 250 mg/kg . We reported earlier (Hitotsumachi et al ., 1985,<br />
Proc. Natl. Acad. Sci. USA, 82 : 6619-6621) that the effectiveness of ENU in inducing mutations at<br />
specific loci in stem-cell spermatogonia of mice could be increased above the level obtained with 250<br />
mg/kg by using 4 doses of 100 mg/kg spaced at weekly intervals . The increase obtained was 2.2 times,<br />
<strong>and</strong> was statistically significant (P, one-tailed - 0 .02), even when calculations were adjusted for the<br />
occurrence of clusters . However, since this adjustment is only a rough approxitnadon, <strong>and</strong> since the<br />
protocol of repeated doses is an attractive one for other investigators desiring a high mutation rate, we<br />
decided to increase our sample size by doing a teplicate experiment . A total of 37 mutations was obtained<br />
in 3428 offspring from treated stem-cell spetmatogonia. Clusters were again uent, but there were at<br />
least 20 independent mutations . The mutadon frequency is almost identical wI that obtained earlier .<br />
Furthermore, the significance of the difference of the combined data from the mutation frequency obtained<br />
with a single dose of 250 mg/kg is now more convincing (P, one-tailed - 0 .003), <strong>and</strong> the recommendation<br />
to other investigators of rhe value of the dose-repetition protocol can now be made with more confidence .<br />
As was emphasized in our earlier publication, the mutation frequency obtained is of a magnitude that<br />
seemed out of reach a few years ago, being 36 times the highest reported for pr .carbazine, the most<br />
effective chemical mutagen known for stem-cell spermatogonla before ENU had been tested . [Research<br />
jointly sponsored by the Office of Health <strong>and</strong> <strong>Environmental</strong> Research, U .S . DOE contract DE-AC05-<br />
840R21400 with Martin Marietta Energy Systems, Inc ., <strong>and</strong> by the National Institute of <strong>Environmental</strong><br />
Health Science under IAG No. 222Y01-ES-10067 .]<br />
489<br />
CONCENTRATION OF MUTAGENIC COMPONENTS IN RIVER WATER BY USE OF THE BLUE RAYON METHOD<br />
Hiroshi Sakamotol, Katsuhiko Nakamuroz Yasuyoshi Sayato2 <strong>and</strong> Hikoya Hayatsut<br />
F~ulty of Pharmaceutlcal Sciences, Okayama University, Taushima, Okayama 700<br />
2Faculty of Pharmaceutical Sciences, Seteunan University, Hirakata, Osaka 537-01, Japan<br />
Activated carbon <strong>and</strong> %AD-resin are widely used for concentration of organic mutagens<br />
in river water . In these procedures, a large volume of water has to be taken out of<br />
the river <strong>and</strong> processed . Use of the blue rayon method gives facilty for concentrating<br />
mutagens with polycyclic structures from river waters . In this method, blue rayon is<br />
allowed to st<strong>and</strong> for a day in the river to make contact with the flowing water, <strong>and</strong><br />
compounds adsorbed to the rayon 1s then eluted <strong>and</strong> assayed for mutagenicity . In this<br />
way, we have measured the mutagenicity of the water of River Yodo in Osaka, <strong>and</strong> that of<br />
the tributary rivers Katsura, Kisu <strong>and</strong> Uji in Kyoto . The mutagenicity is assayed with<br />
50869 3682
1989 EMS Abstracts<br />
S . typhimurium TA98 <strong>and</strong> TA100, with <strong>and</strong> without metabolic activation . The samples from Notes<br />
the Katsura showed the highest mutagenic activity (3500 revertants with TA98, . +59 ; 87<br />
revertants with TA9B, -S9~ 141 revertants with TA100, +S9 ; negative with TA100, -S9,<br />
per 0 .1-g equivalent of'bIue rayon) . By further measurement at several different sites<br />
of this river, we found that the effluent from a waste-water treatment plant in Kyoto<br />
City is the possible source of the mutagenic pollution . When the samples collected<br />
from this river in the winter <strong>and</strong> summer of 1988 were fractionated by TLC, the mutagenic<br />
activity in TA98 with metabolic activation was found predominantly in single identical<br />
zones . It is suggested that River Katsura is constantly polluted with certain<br />
frameshift promutagens of polycyclic structures .<br />
490<br />
SALMONELLA MUTAGENICITY TEST ON VARIOUS KIND OF IRRADIATED SUGARS AND AMIlIO ACIDS<br />
K . Sakamoto, K . Kanazashi, S . Iwahara, K .Takatori, <strong>and</strong> R . Aibara, Hatano Research<br />
Institute, Food <strong>and</strong> Drug Safety Center, Hadano, [anagawa (Japan)<br />
Recently, various kind of irradiated foods were studied in mutagenicity tests .<br />
Every food comprises variety of components, but the volume of samples that can be<br />
used by these test systems is limited . Accordingly, even if certain components of<br />
the irradiated food should undergo mutagenic changes, it would be impossible to<br />
detect such changes if the volume of them are smaller than the detectable limit of<br />
the test systems .<br />
For this reason, it is necessary to study mutagenicity on each irradiated food<br />
components . And if the test results indicate the formation of mutagens in component<br />
by irradiation, we should evaluate the genetic toxicity of irradiated foods with the<br />
test results in due consideration of : 1) irradiation dose, 2) ratio of mutagenic<br />
products formed from the irradiated food components, 3) strength of mutagenic<br />
activity, 4) proportion of components in food changeable to mutagens <strong>and</strong> 5) possibility<br />
of elimination of the mutagenic products .<br />
We studied Salmonella/mammalian microsome mutagenicity test on various kinds ofsugars<br />
<strong>and</strong> amino acids irradiated in the dry condition <strong>and</strong> in liquid solution with<br />
gamma ray of maximum dosage upto 10 kGy . As a result, weak mutagenic activity was<br />
detected in the 5% solution of cystein <strong>and</strong> 10% solution of arabinose irradilted at<br />
the maximum dosage of 10 kGy, with metabolic activation system . The results indicate<br />
that the mutagenic products from each compound hardly forms in irradiated foods .<br />
491<br />
CLASTOGENIC EFFECT OF ELLIPTICINE ON DIFFERENT PHASES OF THE CELL CYCLE OF<br />
CULTURED HUMAN LYMPHOCYTES AND ITS SYNERGISTIC ACTION WITH INHIBITORSOF<br />
DNA REPAIR AT G2 . Elza T . Sakamoto-Hojo <strong>and</strong> Catarina S . Takahashi'(IBILCE-<br />
S . Jose Rio PreEo-UNESP <strong>and</strong> FFCL de Ribeir`ao Preto-USP, BRASIL) .<br />
Ellipticine, a pyridocarbazole alkaloid, has shown an antitumor effect<br />
on several types of experimental <strong>and</strong> human tumors, being an intercalating<br />
substance whose mutagenicity has been demonstrated in several systems .<br />
To characterize the mechanism of action of this compound at the cell cycle<br />
level, human lymphocyte cultures from 2 healthy donors were treated with<br />
3 pg/ml ellipticine in 30-minute pulses during different phases of the<br />
cycle <strong>and</strong> analyzed for chromosome aberrations <strong>and</strong> sister chromatid exchanges<br />
. The G2 phase was more sensitive in terms of induction of aberrations,<br />
followed by S <strong>and</strong> G1 . The 24-h treatment (S phase) induced a four<br />
fold increase compared to th8 control . SCE induction was significant only<br />
at G1, when SCE frequency doubled_3The effect of lymphocytg post-treatment<br />
with inhibitors of DNA repair (10 M caffeine <strong>and</strong> 5 x 10 M 1-B-D-arabino<br />
furanosylcytosine) was also tested by adding 3 yg/ml ellipticine at G in<br />
30-minute pulses <strong>and</strong> the inhibitors immediately afterward during the2last<br />
3 h before harvesting the cultures . In the two experiments performed on<br />
blood from the 2 donors there was a moderate effect of chromosome aberration<br />
potentiation (about 2-3 times), i .e ., ellipticine bad a synergistic<br />
effect with both inhibitors in terms of chromosome aberration induction .<br />
492<br />
THE ABSENCE OF PAH HYDROXYLATION ACTIVITY IN TOBACCO CALLUS S9 . M . F . Salamone',<br />
S . Richard', C . J . Gentile', J . M . Gentiles, <strong>and</strong> D . A . Rokosh', 'Ministry of the<br />
Environment, Toronto, Canada <strong>and</strong> *Hope College, Holl<strong>and</strong>, Michigan .<br />
Microsomal enzymes (S9) from four week old photosynthetic <strong>and</strong> nonphotosynthetic<br />
tobacco (Nicotiana tobacum) callus cultures were prepared by two separate laboratories .<br />
The ability of each callus S9 to activate 2-aminofluorene (2AF) <strong>and</strong> benzo(a)pyrene<br />
(BaP) was then tested with the Ames fluctuation <strong>and</strong> plate incorporation assays . With<br />
each assay the callus S9 preparations from both laboratories increased the mutagenic<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
.<br />
169<br />
Y
170 1989 EMS Abstracts<br />
NotQs__ . w_ . _<br />
,.+ .,tct)vity of:~Af, ~not BaP . Each S9 was then tested for 2,5-diphenyloxazole (PPO)<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
" hydroxylationtsictivity in a fluorometric mixed function oxidase (MFO) assay modified<br />
from Niebert <strong>and</strong> G oin . No PPO hydroxylation activity was observed with any of the<br />
callus S9 prep_%ra In parallel experiments, rat liver S9 activated both 2AF <strong>and</strong><br />
BaP, <strong>and</strong> exhibited strong HFO activity . Subsequently, nonphotosynthetic tobacco<br />
callus was grown on Aroclor 1254 <strong>and</strong> 3-methylcholanthrene enriched callus media to<br />
determine whether the hydroxylation enzymes could be induced . S9 preparations from<br />
these callus cultures also were negative for BaP activation <strong>and</strong> for MFO activity . An<br />
electron capture-gas chromatographic analysis of the callus grown on the Aroclor<br />
enriched media, indicated that Aroclor 1254 was being incorporated into the growing<br />
callus . These results suggest that callus from Nicotiana tobacum may not possess<br />
sufficient quantities of the enzymes necessary to ydroxylate PANs (PPO <strong>and</strong> BaP) <strong>and</strong><br />
that these enzymes, if present, are not readily induced .<br />
493<br />
CYTOGENETIC EFFECT 0F PLANT USED IN CATTLE FOOD AND IN POPULAR MEDICINE .<br />
D .M .F .Salvadori*,A .R .Silva*,M .D .M .Oliveira*,A .R .P .L .Bautista**,<strong>and</strong> L .R .Ribeiro* .<br />
*Univeraidade Federal da Bahia, Salvador,BA (BRASIL),**Empresa de Pesquisa Agropecuaria<br />
da Bahia, Salvador, BA (BRASIL) .<br />
Indi¢ofera suffruticosa is one of the plants widely used as animal food,ard its ecanmic<br />
importance is due to the fact that the greater part of the cattle are fed with it .It is<br />
also used as a medicinal plant in Brazil .The main compounds present in this plant are the<br />
pyrolizidine alkaliods, A mutagenic activity of these alkaloids <strong>and</strong> their capacity to<br />
induce liver tumor in man <strong>and</strong> in experimental animals have been observed .In this present<br />
study,teste were made on the ability of aqueous <strong>and</strong> hexanic leaf extract of Indiaofera<br />
suffruticosa to induce chromosome aberrations in mouse bone marrow ce1ls .Groups of male<br />
mice were injected intraperitoneally with 3 different concentrations of aqueous leaf<br />
extract, 0 .312, 0 .625 <strong>and</strong> 1 .250mg/kg body wt . which correspond,respectively to 6 .25,12 .5<br />
<strong>and</strong> 25 .0% of the lethal dose (LD100)<strong>and</strong> with 3 different concentrations of hexanic leaf<br />
extract (0 .625, 1 .250 <strong>and</strong> 2 .500mg/kg body wt .),respectively 12 .5, 25 .0 <strong>and</strong> 50 .0% of the<br />
LD100• 24h after the treatment the animals were killed <strong>and</strong> bone marrow metaphase cells<br />
were prepared .No significant increase in the frequency of cells with chremoaare aberrations<br />
was observed between negative control (4 .7%) <strong>and</strong> the groups treated with aqueous leaf<br />
extract (3 .0, 5 .0 <strong>and</strong> 5 .0% respectively) . For hexanic leaf extract the results obtained<br />
were 5 .7, 3 .3 <strong>and</strong> 4 .0% . respectively . A,light increase (5 .7%) was observed only in the<br />
lower concentration as compared to negative control (4 .8x) .Tbeae results showed that the<br />
response decrease with increasing dose . Further experiments are in progress to examine<br />
the clastogenic effects of the other plants in Brazil . FINEP, CNPq end COMCITEC .<br />
494<br />
MUTAGENICITY ACTIVITY OF AIRBORNE PARTICULATE EXTRACTS FROM SAO PAULO, BRAZIL .<br />
PRELIMINARY RESULTS . P .S . Sanchez, G .U . Valent, M .C .L .S . Coelho, C .A . Coimbrao, C . Alon<br />
so <strong>and</strong> M .I .2 . Sato . Companhia de Tecnologia de Saneamento Ambiental (CETESB) . Sao Paulo,<br />
Brazil, CEP 05489 .<br />
It is well recognized that airborne particulate extractable organic matter may<br />
contain compounds which have both mutagenic <strong>and</strong> carcinogenic activity . The objetive of<br />
this study was to evaluate the mutagenicity of airborne particulate from Sao Paulo, in<br />
urban <strong>and</strong> industrial Segions, where the incidence of respiratory diseases is high .<br />
Air volumes of 2000 m were collected in glass-fiber filters using a high - volume<br />
(Hi-vol) sampler in a 24 h period . Filters were extracted twice by ultrasonication<br />
with 100 mL of 1 :1 mixture of methanol <strong>and</strong> dichloromethane . The extracts were filtered<br />
in AP20 membrane <strong>and</strong> evaporated to dryness under vaccum <strong>and</strong> dry nitrogen . After<br />
evaluation of dry weight, the extracts were dissolved in DMSO <strong>and</strong> assayed for<br />
mutagenicity by Ames Test, using S. typJiimu4tua strains TA98 <strong>and</strong> TAJ00, in the presence<br />
<strong>and</strong> absence of S-9 fraction . Results were expressed in revertents/m <strong>and</strong> revertents/yg<br />
of extractable material . Preliminary results showed the presence of mutagenic compounds,<br />
capable of inducing such frameshift mutation as base pair substitution, in several urban<br />
extracts sampled . The results suggest the necessity of carrying out more detailed<br />
studies about mutagenicity of particulate air pollutants in this area, which will be<br />
useful in evaluating future changes in air quality in Sao Paulo, as well as provide<br />
subsidies in the establishment of goals <strong>and</strong> criteria to an efficient action in air<br />
pollution control .<br />
~<br />
m<br />
Cfl<br />
Ot<br />
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495 - 1989 EMS Abstracts<br />
IONIZING RADIATION AND GENETIC RISKS : CURRENT STATUS NO `eS<br />
K . Sankaranarayanan, Daf'artment of Radiation Genetics <strong>and</strong> Chemical Hutagenesis,<br />
Sylvius Laboratories, State University of Leiden, Leiden, The Netherl<strong>and</strong>s<br />
Estimates of the risk of genetic disease in the progeny of parents or populations<br />
exposed to ionizing radiai>on are made on the basis of mutation data from animal<br />
experiments <strong>and</strong> using a number of assumptions to bridge the gap Tetween induced<br />
mutation <strong>and</strong> genetic disease in man . The natural prevalence of genetic <strong>and</strong> multifactorial<br />
diseases in the population provides not only a framework for risk perception,<br />
but also is used in the act¢ffl calculattde of risks .<br />
Current estimates of genetic risks relate primarily to expected increases in the<br />
frequencies of those diseases with Mendelian patterns of inheritance ; their natural<br />
prevalence is of the order of 1 .25% . However, for multifactorial diseases, whose<br />
natural life-time prevalence is well over 50%, no reliable estimates of risk can be<br />
made in view of the fact that the relationship between mutation <strong>and</strong> disease is not<br />
well-understood . Animal studies using multifactorial traits as indicators of genetic<br />
damage <strong>and</strong> the genetic studies of the Hiroshima <strong>and</strong> Nagasaki populations (in the latter<br />
two of the indicators used are multifactorial) have failed to demonstrate significant<br />
adverse effects as a result of radiation exposures . However, as is well-known,<br />
mutation studies with experimental mammals have provided good evidence for the induction<br />
of mutations . The possible reasons for these discrepancies will be briefly<br />
discussed . Additionally, the relevance of knowledge on the nature of spontaneous <strong>and</strong><br />
radiation-induced mutations in the context of risk estimation will be considered .<br />
496<br />
IMMUNOLOGIC METHONS FOR THE DETECTION OF CARCINOGEN ADDUCTS IN HUMANS<br />
R . M . Santella, Columbia University New York, NY<br />
Monoclonal ant,ibodies have been developed which recognize a number of<br />
carcinogen-DNA <strong>and</strong> protein adducts . These antibodies can be used in highly<br />
sensitive competitive enzyme linked immunosorbent assays (ELISA) to detect femtomole<br />
levels of adducts in human samples . With the most sensitive antibodies, DNK adducts<br />
in the range of 1/l0s nucleotides can be measured . In addition, antibodies to DNA<br />
adducts can be used to investigate localization of adducts in specific cell types .<br />
We have used antibodies recognizing the major edduct of benzo(a)pyrene (BP) to<br />
monitor adducts in lymphocyte DNA of foundry workers <strong>and</strong> smokers end nonsmokers .<br />
Adducts in lung tissue of cancer patients <strong>and</strong> placental tissue of smokers <strong>and</strong><br />
nonsmokers have also be analyzed . Because of antibody crossreactivity with<br />
structurally related adducts of other polycyclic aromatic hydrocarbons, this assay<br />
is not specific for BP adducts . Monoclonal antibodies against 8-methoxypsorelen-•DNA<br />
adducts have been used to monitor adducts in psoriasis patients treated with this<br />
chemotherapeutic agent . Immunofluoreacence staining of skin biopsies from patients<br />
demonstrated adduct localization to epidermal cells . Studies with antibodies to<br />
aflatoxin-Ri-DNA adducts were used to detect elevated levels of adducts in liver<br />
tissue from Taiwanese hopatocellular cancer patients . Adduct detection in humans is<br />
now established as a viable method for determination of exposure to certain chemical<br />
carcinogens . The relationship of adduct. measurements to risk requires further<br />
investigation .<br />
497<br />
CYrOGEMETIC RISK ASSES9Etfr OF OPERATI011 T/EATRE PERSOI~EL<br />
S .T . SANIHIYA . V. Padsrani <strong>and</strong> A. Raassh. Departsant of Genetics . University of Madras, Madras - 600 113<br />
(India) . Occupational health risk to personnel working in operation theatres have been extensively<br />
studied . Although results are controversial . many agree that they constitute a potentially risk group <strong>and</strong><br />
needs to be periodically aonitored . This is particularly relevant, to countries,Yhere safety NasurN<br />
at work places receive less priority than desired . Therefore, the present work is contemplated to assess<br />
cytoyenetic risk on anaesthetists <strong>and</strong> supportive staff aeployed in a major referral hospital of Madras .<br />
The study group consisted of 16 snaasthetists <strong>and</strong> 4 theatre assistants serving for 1-33 years (13 .50 s<br />
7 .6) in several surgical theatres, which do not have any scavenging device . N40, ether <strong>and</strong> halothane are<br />
being used aither singly or in combination . Control group (n - 20) consisted of persons with different<br />
occupational set up . satched for possible confounding variablas . Cytopenetic damage !s assessad in terms<br />
of chroaososal aberrations (CA) <strong>and</strong> sister chromatid exchanges (SCE) observed !n 48 <strong>and</strong> 72h lysphocyte<br />
cultures of peripheral blood . Stp-rise regression analysis was performed taking duration of service (xl),<br />
ege (x )u sex (x3) <strong>and</strong> saokinp status (x4) as indep<strong>and</strong>ent variables <strong>and</strong> risk aeasures (f setaphuas with<br />
<strong>and</strong> vi~hout gaps - CA (G .) <strong>and</strong> CA (G -) <strong>and</strong> SCE/cell) as dependent variables using SPSS . Only xl !s the<br />
significant determinant of the variation in CA . This accounted for 609 upvards of the variation . For<br />
SCE . both x) <strong>and</strong> x3 are significant detersinants. xl alone explained eef of the variation <strong>and</strong> both together<br />
accounted for about 915 of the variation !n SCE . 8ased on these findings, it is concluded that persons<br />
working in theatres devoid of scavenging measures are at potential risk of genetic damage which appears<br />
to increase with duration of service .<br />
analysis .<br />
Thanks to our Prof .P .M.Gopinath for encouragement <strong>and</strong> Dr .M .Laks)ranan . CMC. Vellore for Computer<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
171
172 1989 EMS Abstracts<br />
Notes '" rULTRA-VIOLET-.1NDUCED MUTATION SPECTRA ON SIMIAN VIRUS 40 GENES AFTER DNA<br />
TRANSFECTION OR VIRUS INFECTION .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A. SARASIN,. Az■LtOftENBEROER <strong>and</strong> F. BOURRE.<br />
498<br />
Laboratory of <strong>Molecular</strong> Genetics, Institut de Recherches Scientifiques our Is Cancer, B .P. n° 8, 94802 -<br />
VILLEJUIF (France) .<br />
The analysis of induced mutations In mammalian cells has become a major goal for the underst<strong>and</strong>ing of<br />
the carcinogenesis Initiation . Due to the complexity of the cNlular genome, the use of small <strong>and</strong> easily<br />
manipulatable DNA probes, depending on cellular enzymic machinery, is highly desirable . Two such systems<br />
have been developed : shuttle vectors <strong>and</strong> animal virus mutants . We have used the latter one with either natural<br />
SV40 or modified SV40.<br />
Simian virus 40 (SV40) has been used after being treated In vitro with ultraviolet light (UV) . Our<br />
mutation assay is based upon the reversion of a temperature-sensitive (ts) growth at 411C to a wild-type<br />
growth phenotype. UV Irradiation of is 8V40 DNA leads to the Induction of temperaturalndependent phenotypic<br />
revenants which are due to singie-base substitutions located opposite potential UV-Induced DNA lesions . This<br />
mutagenesis appears therefore to be targeted opposite lesions . Treatment of UV-irradiated DNA with the E . coli<br />
photolyase increased virus survival <strong>and</strong> strongly decreased virus mutagenesis . The mutation spectrum is<br />
different after photoreactivatfon implying that pyrimidine dimers <strong>and</strong> Py(8-4)Py photoproducts are both<br />
premutagenic lesions .<br />
UV-irradiated or unirradiated SV40 virions or SV40 naked DNA have been used with the same<br />
phenotypic reversion assay . It appeared that the transfection step, used with naked DNA, <strong>and</strong> the Infection lead<br />
to different results for mutagenesis . Indeed the UV-tnduced mutation spectra were similar but not Identical<br />
while the spontaneous mutation spectra were different regarding the location of mutation hot spots .<br />
GERMINAL IMPRINTING IN CHROMOSOME MUTATION<br />
M. S. Sasakl, Radiation Biology Center, Kyoto University, Kyoto (Japan )<br />
499<br />
There is now a growing body of evidence indicating that the non-Robertsonlan de vo<br />
chromosome mutations have a strong bias toward paternal origin . Such non-r<strong>and</strong>omness has<br />
been implicated as a reflection of differential susceptibility to errors in meiotic process<br />
between males <strong>and</strong> females. However, we meet some difficulties with this idea . Deletion<br />
or loss-of-function mutation of retinoblastoma susceptibility (RB) gene has a critical role in<br />
the development of retinoblastoma <strong>and</strong> also presumably Dsteosarcoma . Investigation on the<br />
parental origin of de novo chromosome mutations involving- RB gene In retinoblastoma<br />
patients showed that the germinal mutations were predominantly paternal origin, providing<br />
an additional evidence for the paternal melotic errors. In the non-hereditary sporadic<br />
osteosarcoma, the Initial mutation is presumed to be somatic origin . The mutation of<br />
tumor auppressing gene is often expressed by a subsequent loss of a large piece, or all, of<br />
the chromosome which harbors the normal allele. When the parental origin of chromosomes<br />
was identified with polymorphic loci, 12 out of 13 tumors showed loss of maternally<br />
derived chromosome 13 alleles, indicating that the Inltial somatic mutations were also<br />
non-r<strong>and</strong>om <strong>and</strong> occurred predominantly on paternally derived chromosome 13 . The<br />
preferential Involvement of paternal chromosomes both in germinal <strong>and</strong> somatic mutations<br />
rather suggests the involvement of germinal imprinting In the mutational susceptibility .<br />
500<br />
CYMIAGICAL ACfIVTTY OF NATURALLY OOCURRING 1K)GS --- S7UDIFS CN PFRILIA EICIRACPS<br />
Mieko Sasaki, 4bkyo Metr.Res.Iab .P,H.,3-24-1 Hyakunin-cho,Shinjuku-ku,7bkyo 169,Japan<br />
Naturally occurring substances have been widely used as drugs <strong>and</strong> food additives .<br />
Toxicology of these substances, however, is not studied enough because aLnost all of<br />
them are carplex chemicals <strong>and</strong> their activities are difficult to be examined . On the<br />
other h<strong>and</strong>, anti-mutagenicity or anti-wrcinogenicity has been found in some of these<br />
materials. Recently, Chinese medicine (naturally occurring drugs) has been interested<br />
<strong>and</strong> considered again in Japan, as a therapy for canoer or chronic diseases . The<br />
effects of these drugs have not been established enough yet, <strong>and</strong> the qualitative <strong>and</strong><br />
quantitative studies bacome necessary now .<br />
Perillae (leaf of Perilla frutescens var.acuta) is one of popular herbs in Japan,<br />
<strong>and</strong> has been used as a food for expecting a good smell <strong>and</strong> anti-spoiled effect, <strong>and</strong><br />
it is mixed into the Chinese medicine for armron cold concerning In its anti-allergic<br />
effect. The main active earnponent of perillae is known as perillaldehyde, <strong>and</strong><br />
another toxic derivative, perilla ketone, is a potent puLronary edematogenic agent<br />
in farm animals. Biological activity of the ether-, ethanol-, water-extracts from<br />
leaves of perilla <strong>and</strong> purified 1-perillaldehyde was investigated in cultured CAD-K1<br />
cells. Chemical analysis of these samples was practiced by capillary OC or HPLC .<br />
Aberratiens of chratnsone were increased in cells treated with the ether-extract ,<br />
<strong>and</strong> weak mutagenicity was also observed. The correlation of cytological activities<br />
to chemical analysis has been exadlLned, <strong>and</strong> the other biological effect, such as an<br />
effect on inaraua-earpetent cells, has been studied to estimate the Chinese medicine .<br />
50869 3686
501<br />
ANTIMUTAGENIC FACTORS IN VARIOUS AQUATIC PLANTS .<br />
T . Sato, Y . Ose, H . Nagase <strong>and</strong> H . Kito, Gifu Pharmaceutical University,<br />
Gifu City, Gifu 502 (•dlrpan)<br />
Various aquatic plants were collected from Nagara River system <strong>and</strong><br />
the inhibitory effect of water extracts of the plants on mutagen-induced<br />
mutations were investigated by the Ames test (Salmonella tYChimurium<br />
TA100 <strong>and</strong> 98) . Smartweed, curled pondweed, Tuiopean cul- -grass an'a<br />
grass-wrack pondweed showed strong antimutagenic effect on benzo(a]pyrene<br />
(B[a]P), but had not for AF-2 . For 2-nitrofluorene (2-NF),<br />
curled pondweed, Europea.n cut-graju <strong>and</strong> grass-wrack pondweed had the<br />
antimutagenic effect, but smartweed had not . These antimutagenic factors<br />
were heat-resistant . The molecular weight of antimutagenic factor in<br />
curled pondweed <strong>and</strong> grass-wrack pondweed was above 300,000 . Four<br />
extracts did not show bioantimutagenicity . The active factors of curled<br />
pondweed, smartweed <strong>and</strong> grass-wrack pondweed acted as desmutagens .<br />
However, the active factor of European cut-grass did not show activity<br />
by desmutagen test, <strong>and</strong> not inhibit spontaneous mutation <strong>and</strong> S9 mix .<br />
The contribution of chlorophyll on antimutagenesis was little . B[a]P<br />
was treated by various amounts of the extracts <strong>and</strong> extracted by ethyl<br />
acetate . The extracted B[a]P decreased with the amount of plant<br />
extract . Almost all of adsorbed B[a]P was released with twice 20 min<br />
ultrasonication . By these results, it became clear that different<br />
antimutagenic factors exist in various aquatic plants .<br />
502<br />
APPROACHES TO DNA METHODS FOR THE DETECTION OF HERITABLE MUTATIONS IN HUMANS .<br />
C . Satoh, K . Hiyama, N . Takahashi <strong>and</strong> M . Kodaira .<br />
Radiation Effects Research Foundation . Hiroshima 732, Japan<br />
.<br />
We have examined feasibility of ribonuclease A cleavage at mismatches in RNA :DNA<br />
duplexes (RNase A method) <strong>and</strong> denaturing gradient gel electrophoresis (DGGE) of<br />
RNA :DNA duplexes for a study determining ns~leotide mutation rates . Identical<br />
RNA :DNA duplexes made by hybridization of P-labeled RNA probes <strong>and</strong> target DNAs<br />
were used in both techniques . Employing the RNase A method, 10 types of mismatches<br />
were examined in duplexes less than 771 bp made from cloned human $-globin genes <strong>and</strong><br />
8 of them were cleaved . Deletion <strong>and</strong> insertion of 1, 4, 5 <strong>and</strong> 10 bp were detected .<br />
A polymorphic substitution of T to C at position 666 of IVS2 of P-globin gene was<br />
detected in genomic DNAs of 59 Japanese after amplification by polymerase chain<br />
reaction (PCR). Using DGGE, 8 types of mismatches were examined <strong>and</strong> all of them<br />
were detected . The deletions <strong>and</strong> insertions detected by the RNase A cleavage method<br />
were also detected. Three different polymorphic substitutions <strong>and</strong> a variable-length<br />
polymorphism [(ATTTT)n, n - 4 . 5, 6, 7] in the globin genes of 59 Japanese were<br />
detected in the genomic DNAs without amplifica~ion . Any large program screening for<br />
mutations requires examining of the order of 10 person .probe determinations. We<br />
are studying ways to optimize efficiency <strong>and</strong> cost effectiveness. These 139lude the<br />
use of multiple probes on a single DGGE gel <strong>and</strong> eliminating the need for P-labeled<br />
probes by the PCR technique .<br />
503<br />
MECHANISMS OF CHROMOSObIE ABERRATION<br />
John R .K . Savage, HtC Radiobioloqy Unit, Chilton, Didcot, Oxon . OX11 ORD . U .K .<br />
The subject of mechanisms has to be considered at several levels <strong>and</strong> not confined<br />
just to the initial molecular lesions <strong>and</strong> their repair/misrepair . Although there are<br />
many kinds of lesions which can be introduced by a wide variety of agents <strong>and</strong> there<br />
are many proposed pathways by which a cell can deal with them, there is only a<br />
limited number of ways in which "rejoining" can occur to produce structural changes<br />
visible in chromosomes at metaphase . This makes chromosomal aberrations a somewhat<br />
non-specific end point . Irrespective of the lesion types <strong>and</strong> their products, at some<br />
stage in time, some of them must come into "physical contact" if exchange is to<br />
occur . Is such contact predisposed? Are all regions vulnerable? Nhat is the<br />
relationship to <strong>and</strong> influence of chromatin structure <strong>and</strong> intranuclear architecture to<br />
such events? The many orders of magnitude difference between the molecular events<br />
<strong>and</strong> the observed result must not be forgotten, for the complex process of chromosome<br />
condensation <strong>and</strong> packing which intervenes, leads to aberration modification <strong>and</strong><br />
disguise . Some mechanism must also exist to cope with entanglement, for even in the<br />
most complicated of,aberrations, this is a very rare phenomenon . Comments <strong>and</strong><br />
observations will be made on these <strong>and</strong> other topics within the context of<br />
"mechanisms" .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 173<br />
Notes
174 1989 EMS Abstracts<br />
Notes - .+•FNTERLABOR/kTGRyoldMPARISON OF THE 32P-POSTLABELLING ASSAY FOR<br />
AROMATIC DNIFADDUCTS IN WHITE BLOOD CELLS OF IRON FOUNDRY WORKERS .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
K .Savela 1 . Heme~kil D .H .Phillips2 . A .Hewer2 K .L .Putman3 .<br />
K .R<strong>and</strong>erath3-'-f""-Tn titute of Occupational Health . Topeliuksenkatu<br />
41aA . SF-00250 Helsinki Finl<strong>and</strong> . 2 . Chester Beatty Laboratories .<br />
Institute of Cancer Research . Fulham Road . London SW3 6JB . UK . 3 .<br />
Department of Pharmacology . Baylor College of Medicine . Houston . TX<br />
77030 USA<br />
The 32P-postlabelling method was compared independently in three<br />
laboratories in the analysis of human DNA samples, isolated from white<br />
blood cells of iron foundry workers . who were occupationally exposed<br />
to polycyclic aromatic hydrocarbons . The levels of aromatic DNA adducts<br />
from the exposed foundry workers in the three laboratories varied<br />
between 9 .2 +23 <strong>and</strong> 26+43 <strong>and</strong> from the controls between 1 .7t0 .7 <strong>and</strong><br />
3 .1±1 .7 adducts per log nucleotides . No effect of smoking was<br />
observed in the present study . Each laboratory found large interindividual<br />
variations in the level of adducts . Good correlations were<br />
found between the results of the 32P-postlabelling assays carried<br />
out in the three laboratories ; the correlation coefficients between<br />
laboratories 1 <strong>and</strong> 2 . 1 <strong>and</strong> 3 <strong>and</strong> 2 <strong>and</strong> 3 were 0 .61 0 .62 . <strong>and</strong> 0 .45<br />
respectively, all being statistically highly significant .<br />
504<br />
505<br />
THE EFFECT OF CHWRINE ON THE MUTAGENICITY OF 3-CHLORO-4-(DICHLOROMER'HYW-5-HYDROXY-2(SH)-<br />
FURANONE (MX) AND ITS RECOVERY FROM NATER SAMPLES . K. M . Schenck, J . R . Meier, H . P .<br />
Ringh<strong>and</strong> <strong>and</strong> F C . Kopfler, U .S . <strong>Environmental</strong> Protection Agency, Cincinnati, OH 45268 .<br />
MX has been shown to contribute significantly to the mutagenic activity of a number<br />
of chlorinated drinking water samples . Preliminary studies on the recovery of MX by XAD<br />
resin concentration showed substantially lower recoveries fran tap water samples than<br />
from granular activated carbon (GAC)-filtered distilled water . These results prcmpted<br />
us to examine the effects of residual chlorine on the recovery of MK . GACrfiltered<br />
distilled water containing 0 to 3 mg/1 chlorine was spiked with MS at 50 rg/1 . The<br />
samples were concentrated 4000-fold on columns containing XAD-8 over XAD-2, <strong>and</strong> then<br />
tested for mutagenicity in the SaLnonellq/microsame assay using TA100 without S9 activation<br />
. The results showed that the percent of spiked MX recovered, as determined by the<br />
level of mutagenic activity present, decreased as the concentration of chlorine increased<br />
. This effect could be explained by the reaction of chlorine with MX . To examine<br />
this directly, the mutagenicity <strong>and</strong> UV absorbance of M8 in the presence of chlorine<br />
were monitored over time using high reactant concentrations (1-20 mg/1 for MXf 1-120<br />
mg/1 for chlorine) . At 1, 2, or 3 mg/1 chlorine, the decrease in the mutagenicity of<br />
Huc over time was significantly greater than that observed in the absence of chlorine .<br />
The concentration of MX . as determined by its absorbance at 250 nm, also decreased in<br />
the presence of chlorine . This decrease was both time <strong>and</strong> dose dependent . These results<br />
suggest that the level of MX present in tap water depends not only on the amount<br />
of MX produced by the chlorination of humic substances but also on the rate of degradation<br />
with residual chlorine. (Abstract does not necessarily reflect EPA policy) .<br />
506<br />
SYSTEMIC GENOTOXIC AND TOXIC COMBINATION EFFECTS OF N-NITROSODIMETHYLAMINE<br />
AND SO2 IN THE LIVER FOLLOWING INHALATION EXPOSURE . Schmezer,P. Pool,B .L .<br />
Liegibel,U . Zeller,W.J. Klein,R.G . Institute for Toxicology <strong>and</strong> Chemotherapy, German<br />
Cancer Research Center, INF 280, 6900 Heidelberg, FRO .<br />
A series of studies is being performed to determine which effects the environmental<br />
pollutant S02 may have on the rat liver carcinogen NDMA . Here, short-term in vivo data<br />
on systemic combination effects of SOz <strong>and</strong> NDMA are presented . Our previous studies<br />
had shown that pretreatment (inhalation) of Sprague-Dawle rats with 60ppm 802 for<br />
two weeks reduced the amount of DNA-single str<strong>and</strong> breaks ~SSB) observed in explanted<br />
hepatocytes incubated in vitro with NDMA. The str<strong>and</strong> breakage induced by 25-50<br />
pMoles/2x10s hepatocytes was reduced by apr. 20% . Accordingly, we have now<br />
investigated wether this decrease in genotoxicity caused by S0i is also apparent when<br />
both S02 <strong>and</strong> NDMA are administered simultaneously in viva We used a sensitive shortterm<br />
in vivo assay with which DNA-SSB are detected in intact cells isolated from treated<br />
animals . Inhalation exposure with NDMA alone revealed astonishingly low doses to be<br />
genotoxic in the liver. The doses were similar to those found to be effective after<br />
gavage . The inconstant respiratory frequency of the animals, however, made exact<br />
dosing of low NDMA levels difficult . Therefore, short-term oral NDMA application was<br />
preferred as exposure route for the combination experiments . We used lh in vivo<br />
exposure to NDMA at doses of 0 .5-1 mg/kg . This mode of application resulted in<br />
comparable amounts of str<strong>and</strong> breakage as the 25-50 pMoles NDMA induced in 2404<br />
hepatocytes in vltro. So far, the results of the combination treatment indicate that in<br />
contrast to the in vitro situation, the in vivo genotoxicity of NDMA was not affected by<br />
a two week SO2 treatment (10 <strong>and</strong> SOppm) . This observation as well as the possible<br />
impact of the formed sulfite (product of SOt + water) on the intracellular ATP level of<br />
the explanted hepatocytes will be discussed .<br />
50869 3688
507<br />
DIETHYLSTILBESTROL i(DES) INDUCES CHROMOSOME STICKINESS IN SYRIAN<br />
HAMSTER (SHE) FIBROBLASTS .<br />
R . Schnitzler, ' . Sch~ fmann <strong>and</strong> M . Metzler, Institute of Toxicology,<br />
University of Wtlrzburg, Versbacher Str . 9, W . Germany<br />
The genotoxic effects of estrogens are still a matter of debate . Existing<br />
data suggest that numerical chromosome changes rather than gene<br />
mutations are involved in estrogen induced neoplastic transformation . We<br />
have shown previously that DES induces micronuclei (MN) in a nonsynchronized<br />
cell population already after 1-2 hrs, whereas agents causing gene<br />
mutations (i .e . .4-NQO)_iriduce MN formation not before 12-18 hrs . This<br />
supports the hypothesis of a DES in3uced disturbance of the mitotic apparatus<br />
. - We report here that DES induces chromosome stickiness during<br />
early mitosis in SHE cells without measurable alterations of the mitotic<br />
index . After 7 hrs of DES treatment (40 }tM) mitotic cells showed a spheric<br />
aggregation of the chromosomes . The spindle apparatus was found to<br />
be morphologically altered in comparison to control cells . In many cases<br />
we observed single chromosomes separated from the chromosome aggregation<br />
None of them was connected to the mitotic spindle apparatus . In addition<br />
the centrioles were located on top of the aggregate <strong>and</strong> in some cells<br />
only one centriole could be detected . - These findings suggest that DES<br />
interacts with the protein matrix of mitotic chromosomes, presumably<br />
topoisomerase II . This interaction <strong>and</strong> the observed disturbance of . the<br />
spindle formation should result in numerical chromosomal aberrations <strong>and</strong><br />
therefore could be the main reason for the formation of DES induced MN .<br />
508<br />
REPEATABILITY OF SPONTANEOUS AND CHEMICALLY-INDUCED SISTER CHROMATID<br />
EXCHANGE (SCE) ~4ND CHROMO$MAL BREAKAC (CB) IN UNRELATED INDIVI~UALS . S,<br />
Schwartz , L . Lasher , S.S. Wasserman , <strong>and</strong> M.M . Cohen ~ . Divi~on of Human Genetics . Division of<br />
Geographic Medicine, University of Maryl<strong>and</strong> School of Medicine, Medical Biotechnology Center of the<br />
Maryl<strong>and</strong> Biotechnology Institute, Baltimore, Maryl<strong>and</strong> 21201 .<br />
It is well-established that considerable variability in the frequency of sister chromatid exchange (SCE) <strong>and</strong><br />
chromosomal breakage (CB) exists within <strong>and</strong> among populations. Lymphocyte cultures from 27 individuals<br />
.e ere examined, on 2-3 occasions during a 6-month -2 year interval, for spontaneous <strong>and</strong> clast en-induced<br />
SCEs <strong>and</strong> CB following exposure to mitomycin C (MMC), bleomycin (BLM), streptonigrin~SN) <strong>and</strong> 4n<br />
itroquinoline- I -oxide (4NQO) . These experiments were performed to assess the reliability of single SCE <strong>and</strong><br />
C B observations in evaluating an individual's response to environmental exposure . Data analysis utilizing a twoway<br />
nested analysis of covariance for each parameter (SCE or CB) yielded information concerning variability<br />
among the individuals as well as among the different cultures of a givenberson . These data clearly demonstrate<br />
that the interindividual variation of both spontaneous <strong>and</strong> induced SCEs <strong>and</strong> induced CB is significantly greater<br />
than among the cultures of the same person (pc0.01). A similar difference was not found for spontaneous<br />
chromosome breakage . For spontaneous SCEa. SN- <strong>and</strong> BLM-induced SCEa, spontaneous CB <strong>and</strong> 4NQOinduced<br />
CB, variation among sampling dates was not significantly greater than the variation within cultures ;<br />
Khile for MMC-<strong>and</strong> 4NQO-induced SCEs <strong>and</strong> MMC- . BLM-, <strong>and</strong> SN-induced CB, variation among sampling<br />
dates was significantly greater thin within sampling dates (p
176 1989 EMS Abstracts _ 510<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes --~~ gEQUENC'F.-iEtifALeR'M OF MUTATION INDUCED IN THE lac/ GENE OF Escherichla colr<br />
BY 2-AMINOPtJRINE . W .E . SCHY. AND B .W. GLICKMAN, Dept. of Biology, York<br />
University. 471N) Keelt*i, Toronto. Ontario, Canada M3J 1P3<br />
We have founzl`tl ~ r.t vses of mutational spectra to be a useful approach In<br />
elucidating mechanisms of inutation induction . To this end, we have characterized at the<br />
DNA sequence Ievel . '~-o-tminopurine (2-AP) induced mutations that lie within the first 180<br />
bp of the /acl gene (1-~ mutants) of Eschericlria cole. A saturated culture of NR 3835<br />
(wtld-t)•pe) cells was diluted in 25(1 Ed (if LB/2AP (600 q/ml) so that each well of a 96well<br />
mtcrotitrr dish contained less than 1(N) cells. Following overnight growth at 37oC<br />
<strong>and</strong> washing. appropriate dilutions were plated directly onto plates containing LB to<br />
determine survtval . <strong>and</strong> plates containing pbenyI .It .D-galactopyranoside (Pgal) as the sole<br />
carhim source tt) select Iacl' n~utants. One Incl- mutant was seleeted from each well .<br />
Mutants were mapped <strong>and</strong> I' mutants were selected for sequence analysis . Of the 42<br />
mutants at 14 sites that have been seyuenced thus far, all are transition mutations .<br />
Approximately 2-fold ax many mutants were G :C => A:T than A :T => G:C transitions ; .<br />
howevrr. when hotspot sitrs werr excluded from the analysis, transition mutation in<br />
eithcr direction apprars te~ hr ryually likely . With res(+ect to site specificity, we noted a<br />
pruminrnt hotspot at pc~sitiun e3 (G :C ==• A :T). This site Is surrounded by 5 neighboring<br />
G :C hase pairs . which may serve to stabilize a 2•AP :C mismatch . This regional sequence<br />
specificity is consistent with previous observations associating hotspot sites with adjacent<br />
Cr :C' huse pairs .<br />
511<br />
DEOXYNUCLEOTIDE TRIPHOSPHATE POOLS AND THE SPECIFICITY OF MUTATION FOR LACI IN E . COLI .<br />
W .D . Sedwick, M .L . Veigl, S . Schneiter, <strong>and</strong> S . Mollis, Dept . of Medicine,Case Western<br />
Reserve Univ . Clevel<strong>and</strong>, OH 44106<br />
The mutational specificity of trimethoprim (TMP) for the Lacl gene of E . coli was<br />
investigated . Mutants (500 each) were isolated for analysis from cultures which were<br />
treated 4 hours with 5 uM trimethoprim (TMP) or TMP + 100 uM thymidine (TdR), followed<br />
by a 4 hour recovery period which allowed resolution of the filanmentation seen only in<br />
the cultures treated with TMP alone . The frequency of mutation in lcI increased 10<strong>and</strong><br />
2-fold under the two conditions, respectively . All isolated mutants were probed<br />
for occurrence of mutation at two of the most common sites represented in the spontaneous<br />
spectrum, the frameshift "hot spot" where the thrice repeated sequence, CTtk ;, is<br />
frequently duplicated or deleted, <strong>and</strong> the +6 position in the operator, the site of a<br />
recurrent transition of T-A -> C-G . Frame shift "hot spot" mutants represented only 20%<br />
with TMP <strong>and</strong> 43% with TMP-TdR vs >66% in the spontaneous background (Shaaper et al, JMB,<br />
189 :273, 1986) . The high frequency mutation in the operator represented 22% <strong>and</strong> 18% of<br />
the total mutants in the TMP <strong>and</strong> TMP-TdR sets, respectively, vs 3% in the spontaneous<br />
collection . About 60 mutants mapped to the first 200 base pairs of 1aal were carried on<br />
for DNA sequence analysis from both TMP <strong>and</strong> TMP-TdR treated cultures . An overall<br />
increase in single base nonsense mutations at mber_ <strong>and</strong> ochre sites was not observed, but<br />
the predominant isolates from TNP-treated cultures were T-A -> C-G transitions clustered<br />
at several different sites . A high frequency of A-T -> C-G <strong>and</strong> G-C -> C-G transversions<br />
was also observed . Preliminary analysis of the TMP-TdR mutants supports a similar<br />
pattern of base substitution mutations . The results of these experiments will be<br />
presented with correlative DNA triphosphate pool analyses .<br />
512<br />
CONTRASTING RESULTS IN HEPATOCYTE UDS ASSAYS USING AUTORADIOGRAPHY 0R LIQUID<br />
SCINTILLATION COUNTING e b r, C .Meli <strong>and</strong> R .Forster, Dept . Genetic Toxicology,<br />
Life Science Research Roma ox co ogy Centre, 00040 POMEZIA, Rome, Italy<br />
Contrasting findings in rat hepatocyte UDS assays performed using liquid<br />
scintillation counting after nuclear separatlon (LSC) or by autoradiographlc<br />
estimation of radiolabel uptake (ARG) have been observed with several products<br />
received for routine testing . Further experiments were with product X, which gave<br />
reproducible increases in UJS of up to 110% over control values in the LSC method . In<br />
the ARG method, negative results were obtained ; both cytoplasmic <strong>and</strong> nuclear grain<br />
counts remained similar to control values at all treatment-levels . Possible<br />
explanations for these results were examined : (1) The responses obtained with the<br />
positive control (2-acetylaminofluorene) suggested that the ARG method is the more<br />
sensitive method of detection . (ii) The percentage of cells in S-phase was determined<br />
in the autoradiographic preparations . There was no lncrease in S-phase cells which<br />
could explain the results obtained in the LSC method, but calculations show that the<br />
sensitivity of S-phase scoring may be insufficient to detect increases which will<br />
result in positive responses in LSC . (ii1) The experimental methods follow the same<br />
tine-plan <strong>and</strong> kinetic differences cannot explain the contrasting results . (iv)<br />
Parallel LSC assays were perfot7ned with or without hydroxyurea treatment ; product X<br />
produced increases in radiolabel uptake 1n the LSC method both in the absence <strong>and</strong><br />
presence of HU, indicating that the observed UDS did not result from the swtagenic<br />
properties of HU . At present the basis of the differing results between the LSC <strong>and</strong><br />
ARG methods cannot be satisfactorily explained .<br />
50869 3690
513<br />
PROTAMINES IN GERM ZELL MUTAGENESIS, Gary A . Sega, Biology Division, ORNL, Oak<br />
Ridge, TN 37831 .<br />
t<br />
A number of chemicals are'powerful mutagens in late spermatids <strong>and</strong> early spermatozoa stages of the<br />
mouse . Among these chemicals are ethyl methanesulfonate, methyl methanesulfonate, ethylene oxide,<br />
<strong>and</strong> acrylamide . We have found that all four of these chemicals bind at much higher levels in the late<br />
spermatids <strong>and</strong> early spermatozoa stages than in other stages . 11te increased binding in the sensitive<br />
stages has been shown not to be the result of increased alkylation of DNA but rather to alkylation of the<br />
protamine present in these stages . The sulfhydryl (-SH) groups present in the cysteine residues of<br />
immature protamine in the sensitive stages are susceptible to aUcylation by these chemicals . We have<br />
hypothesized that such alkylationVrevents nomsal disulfide bond formation in the protamine of the<br />
developing sperm <strong>and</strong> leads to stresses that break the sperm chromatin, with resulting genetic damage to<br />
the Fl progeny. Clearly, not all mutagenic chemicals act by the above mechanism, <strong>and</strong> other molecular<br />
targets may be important in other germ-cell staps. However, our observations of how some chemicals<br />
bind strongly to sperm protamine in mammals gives a new dimension to our underst<strong>and</strong>ing of mutational<br />
processes in mammalian germ cells . [Research jointly sponsored by the Office of Health <strong>and</strong><br />
<strong>Environmental</strong> Research, U .S . DOE contract DE-ACOS-84OR21400 with Martin Marietta Energy<br />
Systems, Inc ., <strong>and</strong> by the National Institute of <strong>Environmental</strong> Health Science under IAG No . 222Y01-<br />
ES-10067 .]<br />
514<br />
POTENTIAL ROLE OF GENOTOXICITY INDUCED BY GANMA IRRADIATION OF SPODOPTERA LITURA IN<br />
ITS MANAGEMENT . S .S .Sehgal, <strong>and</strong> R .K .Seth, Department of Zoology, n ver y'8T-UEtihi,<br />
Delhi-110007 (INDIA)<br />
In accomplishing the laboratory evaluation of the biological effects of gamma irradiation<br />
of a polyphagous lepidopteran pest, Spodoptern litura, it was observed that<br />
as compared with other insects, Spodopteru required a very high dose (25,000 rad)<br />
of gamma irradiation to produce a complete sterility . However, a gamma dose of 13,000<br />
rad that induced a partial sterility (50%) in irradiated insects produced,progeny which<br />
not only exhibited an increased sterility (ca .80%) but also a higher degree of sexual<br />
competitiveness . The holokinetic nature of chromosomes could be a conceptual cytogenetic<br />
explanation for these results . The fragments of irradiated holokinetic chromosomes<br />
were probably represented in heterozyqous conditiorl in the offsprings, behaved as translocation<br />
heterozygotes during meiosls in F-1 generafion which gave rise to aneuploid<br />
gametes that caused dominant lethality in the next generation . The higher dose of gamma<br />
irradiation caused some physiological effects like female infecundity, inability to<br />
mate, reduced life span, malformations <strong>and</strong> sex-ratio skewed in favor of males . The<br />
excess of males was probably caused by meiotic drive of maleness locus in this moth<br />
<strong>and</strong> this phenomenon appears to have potential for the genetic control method for this<br />
pest . Indeed, it is conceivable to identify <strong>and</strong> 'engineer' the deleterious genetic<br />
mechanisms (or the genntoxicit .y)-induced by gamma irradiatinn, that might be transmitted<br />
by the released moths to regulate the field population of this major pest in India .<br />
515<br />
MECHANISNS OF CAFFEINE INHIBITION OF DNA REPAIR IN E . COLI . Christopher P. Selby <strong>and</strong><br />
Aziz Sancar, Department of Biochemistry, University of North Carolina at Chapel Hill,<br />
Chapel Hill, NC 27599 .<br />
Caffeine inhibits excision repair <strong>and</strong> photoreactivation in E . coli in vivo . We<br />
used purified E . coli enzymes <strong>and</strong> DNasel footprinting to study the mechanism of<br />
inhibition . We do observe inhibition of ABC excinuclease <strong>and</strong> DNA photolyase by<br />
caffeine in vitro . ABC excinuclease catalyses early events of excision repair :<br />
recognition of damaged DNA <strong>and</strong> incision of the phosphodiester backbone on both sides<br />
of the damage . The UvrA subunit is involved in damage recognition . Using an<br />
oligonucleotide with a unique psoralen adduct, UvrA protects 33 base pairs<br />
surrounding the adduct from DNaseI digestion . In the presence of caffeine, the<br />
DNasel footprint of UvrA covers the entire oligonucleotide ; thus, caffeine promotes<br />
the binding of UvrA to undamaged DNA . UvrA subunits "trapped" by caffeine would be<br />
unable to catalyze repair . Photolyase binds to pyrimidine dimers, <strong>and</strong> upon<br />
irradiation of the enzyme-dimer complex reverses the dimer . Using an oligonucleotide<br />
with a unique thymine dimer, we found that caffeine binds specifically to the thymine<br />
dimer <strong>and</strong> interferes with binding of photolyase to its substrate . Thus caffeine<br />
inhibits the two repair systems in E . coli by entirely different mechanisms, by<br />
promoting the nonspecific binding of the nucleotide excision repair enzyme <strong>and</strong><br />
interfering with specific binding of the photoreactivating enzyme . Supported by<br />
grants from the NIH (GN32833) <strong>and</strong> NCI (5 T32 CA09156) .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 177<br />
Notes
178 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
_ 516<br />
Notes tes -- . .-DOMINANT SKBLB/Mtff MUTATION METHODS ARE BEING USED TO DETERMINE WHETHER<br />
DOMINANT MUTATIONS ACCOUNT FOR THE MANY ABNORMAL FETUSES FOUND<br />
FOLLOWING EXPOOF ZYGOTES TO ETHYLNITROSOUREA (ENU) . P. B . Selby, W. M.<br />
Generoso, G. D. Re , W. McKinley, Jr., <strong>and</strong> K. T. Cain, Biology Division, Oak Ridge National<br />
Laboratory. P.O. Box 2009, M.S . 8077, Oak Ridge, TN (USA)<br />
Generoso et al . (Mutat. Res. 176:269-274 <strong>and</strong> 199 :175-181) discovered that there are remarkable increases<br />
in the incidence of developmental abnormalities in fetuses after early rygotic stages are exposed to any one of<br />
several chemicals, including ENU . Russell et al . (PNAS 85 :9167-9170) found that a 50 mg/kg i .p.<br />
injection of ENU induces approximately 8 times more specific-locus mutations when It is administered<br />
2 .5-3 h after mating instead of 5-6 h after mating. We Injected female mice with 40 mi ;/hz; of ENU (i .p .) at<br />
either 2 .5 or 5 h after mating . Offspring are being examined for externally visible altered phenotypes <strong>and</strong> for<br />
skeletal damage. The frequencies of mice wtih significant external findtng;(e.g., a missing eye, shortened<br />
tail, misshapen head), among mice living 3 weeks or longer, were 8/207 (3 .9%) <strong>and</strong> 0/156 in the 2 .5-h <strong>and</strong><br />
5-h groups, respectively . The frequency at 2 .5 h is statistically significantly higher, <strong>and</strong> this difference, in<br />
view of the spectfc-locus results, suggests that dominant gene mutations may be responsible for the<br />
externally visible altered phenotypes. Results of detailed skeletal analyses will be presented, as will<br />
preliminary results from breeding tests . Since dominant skeletal mutations are much more common than<br />
dominant visible mutations, it is expected that a high frequency of dominant skeletal mutations will be found<br />
if induced dominant mutations are responsible for the high frequency of abnormal fetuses . [Research jointly<br />
sponsored by NIEHS under Interagency Agreement Y01-ES-10067 <strong>and</strong> the Office of Health <strong>and</strong><br />
<strong>Environmental</strong> Research, U .S . Deparanent of Energy under contract DE-ACOS-84OR21400 with Martin<br />
Marietta Energy Systems, Inc .]<br />
517<br />
SUGGESTIONS FOR IMPROVING THE DIRECT METHOD OF GENETIC RISK ESTIMATION. P. B .<br />
Selby . Biology Division, Oak Ridge National Laboratory, P .O. Box 2009, M .S. 8077, Oak Ridge, TN<br />
(USA)<br />
One of the methods used by committees for quantitative genetic risk estimation for radiation is based upon<br />
a measure of induced phenotypic damage (usually malformed skeletons or cataracts) in the offspring of<br />
irradiated mice . This method, which has been called the direct method, has several distinct advanta*es over<br />
the doubling-dose method of genetic risk estimation. Much of the emphasis to date has been on induced<br />
mutations that were proved to be transmitted to later penerations . However, If these are the only mutations<br />
that are included in mutation frequencies, as is sometimes recommended, the following important classes of<br />
mutations causing dominant damage are overlooked : (1) mutations causing sterility, (2) mutations causing<br />
death before breeding tests can be completed, <strong>and</strong> (3) mutations with such low penetrance that transmission is<br />
not confirmed in the relatively small numbers of offs'pring that can easily be raised in a breeding test .<br />
Accordingly, it seems that the ideal method for collecting data to be used for genetic risk estimation is to<br />
identify all of the first-generation offspring that exhibit any phenotypes that would be clinically important if<br />
they were to occur in people . The induced mutation frequency could be calculated by subtracting the<br />
frequency of offspring with clinically important phenotypes in the control group from that in the experimental<br />
group . Various other suggestions will also be presented for improving our ability to estimate genetic risk by<br />
the direct method . [Research sponsored by the Office of Health <strong>and</strong> <strong>Environmental</strong> Research, U .S .<br />
Department of Energy under contract DE-ACO5-84OR21400 with Martin Marietta Energy Systems, Inc .]<br />
518<br />
INFLUENCE OF OXIDANT STATE ON UV AND FINNO INDUCED MUTATION IN V79 CELLS-<br />
Sharmila Sengupta <strong>and</strong> 1 u a , Saha Institute of<br />
Nuclear Physics, I/AF, alt ce, a outta- 00 064, India .<br />
Present day idea that oxidant state could be involved in carcinogenesie<br />
makes it relevant to study the influence of sueh condition on the<br />
mutation induction by W light <strong>and</strong> Mt+110 (N-methyl-N'-nitro-N-nitroeoguanidine)<br />
. In our experiments, H20p aae been used to create the oxidant<br />
state in V79 cells . The dose of H2 02 was in the non-toxio region ; for<br />
tnis particular cell-line, it was 0 .9µg/ml . The end-point selected for<br />
our mutation analysis was resistance to the drug 6-thioguanine . Oxidant<br />
state itself did not create any mutation above the background level of<br />
0 .33±0 .08 per 105 viable cells . For UV-light, different fluences ranging<br />
frotn 4J/ms to 20J/m were usp~d to get the mutations wnieh varied from<br />
8 .0+12 to 25 .60+7 .40 per 10 viable cells . For MNNG, the doses used<br />
were in the region 0 .2µg/ml to 0 .6µg/ml <strong>and</strong> the correponding mutation<br />
frequencies varied between 5 .0+1 .0 to 32•5+4 .2 per 101 viable cells . The<br />
creation of oxidant state altered the value ; in case of MNNO, these<br />
raaged between 12 .8+2 .3 to 56 .5+3•6 per M1 viable cells <strong>and</strong> in case of<br />
UV light between 8 .7+1 .4 to 22 .3f8 .2 per 105 viable cells at the dose<br />
ranges mentioned . It-wae seen that such a condition enhanced mutation<br />
induction by NNN6 but had no effect on mutation by UV-light . Clearly,<br />
the oxidant state, if involved in oarcinogeneais does not produce any<br />
extra effect for the 254 nm UV-light but the situation is different in<br />
case of chemical agents like MNO .<br />
50869 3692
519<br />
XRS-5 CELLS ARE NOT 47PERSENSITIVE TO X-RAY-INDUCED MUTATION .<br />
J . D . Shadley, M . Toohill, J . Whitlock, J . Rotmensch <strong>and</strong> J . L .<br />
Schwartz, University-qpf Chicago Medical Center, Chicago, IL (USA)<br />
The Chinese hamster ovary (CHO) cell line xrs-5 has been shown<br />
to be hypersensitive to the cytotoxic effects of X-rays compared<br />
to the parental line K1 . In agreement with previous reports on<br />
this cell line, we find that xrs-5 cells are : 1) 3 fold more<br />
sensitive to the cytotoxic effects of X-rays (D10) than K1) 2)<br />
rejoin only 60% of their gamma-ray-induced double str<strong>and</strong> breaks<br />
compared to over 90% for Kl ; <strong>and</strong> 3) 7 fold more sensitive to Xray-induced<br />
G2 chromatid-aberratiotTS than Kl . Given its<br />
hypersensitivity to the above endpoints, we tested to see if xrs-<br />
5 cells are also hypersensistive to X-ray-induced HGPRT mutation<br />
compared to K1 . Xrs-5 cells gave similar 6-thioguanine resistant<br />
induced mutation frequencies per Gy dose (1 .9 x 10-5 for xrs-5<br />
vs 1 .8 x 10-5 for Kl) . When based on equal survival levels,<br />
either DO or D10, X-rays induce only one-half to one-third<br />
the frequency of 6-TG resistance in xrs-5 compared to K1 . Thus,<br />
xrs-5 cells are as mutable if not less mutable than K1 cells . If<br />
the deficiency in double-str<strong>and</strong> break rejoining is in part<br />
responsible for the hypersensitivity to X-ray-induced cytoxicity<br />
<strong>and</strong> chromosomal aberrations, then it does not appear to affect<br />
mutation induction . Research supported by Department of Energy<br />
DE-FG02-88ER60661 .<br />
520<br />
ExxAMCED ASSAYS DETECT INCREASED CNROMOSOME DAMAGE ARD SIST[R CNR0N0S0ME ExCNANG[S IN<br />
xER01N ADDICIS . D .A .Shaf .r, V .G .tlunbar, A .Falek, R .N .Don .hoe, J .J .Maddan . E~ery Univ .<br />
<strong>and</strong> GeorRis Mental Xeatth Inst ., Atlanta, GA 30306<br />
To refine previous studies of chromosome damage (CD) <strong>and</strong> sister chromatid exchanSes<br />
(SCE) in heroin addicts, we applled eethods re developed to enhance detection of the<br />
cytopenetic effects of lor-l w .l radlation expesure in hospital rerkers . For CD<br />
analysis, our TFC• .nhanced proeedure oonslsted of traatment at setup with 1x10•7N FdU<br />
<strong>and</strong> Lx10-SM dT to Inhibit thymidytate synthetase <strong>and</strong> to satisfy that induced<br />
require .ent, <strong>and</strong> treat .ent in G with oaffein . (2 .2 ∎M) to inhibit repair syfthesis .<br />
For SCE enhaneewent, only caffel ne was used but traat~ant ras ext<strong>and</strong>od traek thru S<br />
phase (19 hrs before harvest) . UsinO s at<strong>and</strong>ard <strong>and</strong> an enhaneed CD <strong>and</strong> fCE eulture per<br />
subject, blood samples rera evaluated from 20 street heroin addicts <strong>and</strong> 22 controls .<br />
St<strong>and</strong>ard 2-day CD <strong>and</strong> 3-day SC[ assays showed insiGnificant sonotoxic Increases in<br />
addicts vhile the enhanced CD <strong>and</strong> SCE assays showed highly significant Increases . Most<br />
CD events were in the for∎ of ehromatid <strong>and</strong> ehromososa iruka . There rere no rings <strong>and</strong><br />
a fev dicentrics Yare only observed in the TiC-enhanead eultures . AltheuGA<br />
quadrlradlals are usually rare, 10 vere found In addict TFC•cultures <strong>and</strong> 3 In control<br />
TFC-cultures . V(th the st<strong>and</strong>ard CD assay, the level of chromosome breaks per 100 e .lls<br />
~as .727 for controls <strong>and</strong> 1 .OS6 for addicts (not siGntf .) . Yith the TFC-enhancad assay,<br />
the same e usure shored t .i63 chrosose .e bruks for eentrols <strong>and</strong> 5 .163 fer .ddlet .<br />
(highly signif . p< .0001) . A hiGhly siGnifiaant dtfferenca ras also observed for<br />
chroeatid dasa9e with the TFC-anhancad assay although such damage ts not typloally<br />
considered indicatlve of J„a vlvo chro .oso .e daea0e (chro .atid breaks per 100 callst<br />
16 .793 for eontrols ; 4 l .191 for addicta) . The SCE data Gave sio ilar si0nif/eant<br />
differences . st<strong>and</strong>ard SCE cultures showed 10 .892 SCE/cell for eontrola <strong>and</strong> 11 .732<br />
SCE/cell for addicts . Vith CAF enhanca .ent thare rara 13 .0! SC[/oell for controls <strong>and</strong><br />
17 .05 SCE/cell for addiets (p< .006) . Tha abeve flndlnGa lnd/eats that tAe deteetlen Of<br />
ICEWICD <strong>and</strong> SCE affeces can be siGnifleantly anAane .d by the use of tAass nar pree .durss .<br />
The findinG of increased chromatid damage also auGGU ts a ner interpretation of CO<br />
effects since chro .atld effects •ust have occurred poat S•phase . Perhaps exposure to<br />
cheoicals, druGS <strong>and</strong> environ .ental agents ∎ay not only leave a residue of DNA <strong>and</strong>/or<br />
chromosome damage but also an indueed sensitivity to further Genotoxic damage .<br />
521<br />
ACTIVITY OF DINITROPYRENES IN THE INTRASANGUINOUS HOST MEDIATED ASSAY .<br />
A .B .Shah, I .R .Rowl<strong>and</strong> <strong>and</strong> R .D .Combes, School of Biological Sciences,<br />
Portsmouth Polytechnic, U .K ., B .I .B .R .A ., U .K ., <strong>and</strong> I .R .I . Ltd ., U .K .<br />
Dinitropyrenes, present in polluted air, are potent direct-acting<br />
mutagens in Salmonella tvnhimurium TA 98 . This mutagenicity is markedly<br />
reduced in the presence of mammalian hepatic S9 or microsomes . Since<br />
most in vitro test systems do not adequately simulate conditions<br />
encountered in the intact animal, we have investigated dinitropyrene<br />
mutagenicity to Salmonella in the host mediated assay . 1,8-<br />
Dinitropyrene (1,8-DNP) given 2.2. to BALB/c mice induced a weak<br />
mutagenic effect in S . typhimurium TA 98 recovered from the liver 1<br />
hour after J,y . administration ; over the entire dose range tested no<br />
toxicity to bacterial cells was detected . Mutation induction in vivo<br />
was dose related with maximum response (71t9 revertants/plate) at lmg<br />
DNP/kg body weight. This optimum dose however, was non-mutagenic to<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 179<br />
Notes<br />
I
180 1989 EMS Abstracts e .<br />
._J . _ _- -`<br />
. . --<br />
.y . woo<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
NOteS strains TA 9'BDNP (arylhydroxylamine esterase deficient) or TA 98NR/DNP<br />
(nitroreductasel,, <strong>and</strong> arylhydroxylamine esterase deficient) . 1,3-<br />
Dinitropyren8---EFRF-1,6-dinitropyrene wer weakly mutagenic to TA 98 at<br />
doses similar to 1,8-DNP . Studies with [14C] 1,8-DNP showed that 1 hour<br />
after oral dosing (lmg/kg), over 100nq of DNP were present in the liver<br />
(w 0 .73% dose) . However, only about 5 .5ng were present in the bacterial<br />
pellet, suggesting that hepatic components, in vivo as in vitro, bind<br />
to DNP, thus preventing its interaction with Salmonella .<br />
522<br />
1,8-DINITROPYRENE : DISTRIBUTION AND EXCRETION IN THE MOUSE .<br />
A .B .Shah, I .R.Rowl<strong>and</strong> <strong>and</strong> R .D .Combes, School of Biological Sciences,<br />
Portsmouth Polytechnic, U .K ., B .I .B .R .A ., U .K ., <strong>and</strong> I .R .I . Ltd ., U .K .<br />
Despite considerable interest in the toxicology of 1,8-dinitropyrene<br />
(DNP), little is known of the toxicokinetics of this mutagen . We have,<br />
therefore, investigated the distribution nd excretion of DNP following<br />
oral administration of a single dose of C-DNP (0 .25mg/kg) to female<br />
BALB/c mice . At known times (2,4,6,24,48,72,96,168, <strong>and</strong> 216 hours)<br />
after dosing, mice were killed, various tissues removed <strong>and</strong> their<br />
radioactivity determined. The results indicate a slow uptake of DNP<br />
into the bloodstream <strong>and</strong> other tissues . Maximum amounts were present in<br />
the bloodstream, liver <strong>and</strong> kidneys after 6 hours, representing 0 .274,<br />
2 .9% <strong>and</strong> 0 .21% of the radioactive dose . The radioactivity in these<br />
tissues decreased after 24 hours to 0 .1%, 1 .6% <strong>and</strong> 0 .12% respectively,<br />
after which there was a gradual decrease amongst these <strong>and</strong> all the<br />
other tissues studied . Most of the excretion was within 48 hours of<br />
dosing. At 72 hours, radioactivity recovered from all the tissues<br />
studied <strong>and</strong> urine <strong>and</strong> faeces amounted to
_<br />
presence of .c_,lyDhimuriu_.n TA 98, TA 98NR <strong>and</strong> TA 98/1,8DNP6 . Cytosol, (0.8 mg/plate) as the sole<br />
exogenous enzyme fraction, enlUnced the mutagenicity of 2- <strong>and</strong> 3-nitrofluoranthene (2-NP <strong>and</strong> 3-NF) <strong>and</strong><br />
1,3 dinitrofluoranthene (1,3-I7NF) in the three strains 2- to 9-fold over that observed in its absence . However,<br />
the number of revertants with 1,2 dinitrofluoranthene (1,2-DNF) was decreased 70% by cytosol. The<br />
inclusion of microsomes alone (0.2 mg/plate) decreased the mutagenic response by 57-98% . The addition of<br />
both cytosol <strong>and</strong> microsomes, to reconstitute S9, resulted in a marked decrease In the mutagenicity of the<br />
compounds . In a related study, the mutagenicity of three dinitropyrenes, 1,3-, 1,6- <strong>and</strong> 1,8-dinitropyrene<br />
(1,3-, 1,6- <strong>and</strong> 1,8-DNP) was also rnarkedly enhanced by cytosol m TA 100. This was especialily noticeable<br />
when 1,3 DNP was the substrate . Amongst other possibilities, these findings suggest that cytosol may contain<br />
enzymes more potent than the constitutive bacterial .naroroductases <strong>and</strong>/or acetylases in the activation of<br />
nitroarenes to mutagens or that the bacterial enzymes are rate limiting components of the system . Microsomes<br />
contain deactivating enzymes <strong>and</strong> when combined with cytosol to reconstitute S9, the deactivation potential of<br />
the microsomes overshadows the activation potential of the cytosol <strong>and</strong> a decreased mutagenic response is<br />
expressed .<br />
525<br />
ALTEltATIONS IN DNA CONTENT AND CHRONOciOHE COMPOSITION AS<br />
RELATED TO AGEING IN MAMMALS<br />
A . Sharma, S,Sen, G . Talukder, Human Genetics Unit, Centre for Advanced $<br />
Study in Cell & Chromosome Kesearch, Department of Botany, University<br />
of Calcutta,' Calcutta 700019, India<br />
In view of the hypothesis that DNA is involved in the process of<br />
ageing, the present investigation was undertaken to find out the<br />
relationship between DNA content <strong>and</strong> progressive ageing in ratss<br />
in vivo <strong>and</strong> human leucocyte cultures in ~itrQ Chromosome studies<br />
from bone marrow cells of Kattus norvea-Lcus of .twelve different age<br />
groups showed a significant increase in the frequency of hypodiploid<br />
cells with ageing . This increase was, however, not seen in the .gonadal<br />
cells . A similar enhancement was recorded in the number of hypodiploid<br />
cells in the cultured lymphocytes from sex-mrtched human subjects of<br />
ten different age groups . DNA estimated in situ in bone marrow nuclei<br />
did not change to a significant extent with ~ncieased age . The observations<br />
made in proliferating human lymphocytes supported those made on rats<br />
in vivo . DNA contents of differentiated tissues, including brain, lung,<br />
lve lr, heart <strong>and</strong> kidney from fetal to adult rats showed an organ-specific<br />
variation(within the limits of statistical significance) . It may be<br />
due to controlled DNA amplification during differentiation <strong>and</strong><br />
maturation of the tissues .<br />
526<br />
GENOTOXIC EFFECTS OF INDU3TKIALISATION IN SELECTED POPULATIONS<br />
A .Sharma, A. Raychoudhury, A . Banerjee, M .De, T .Das, M.Joardar,<br />
K . Agarwal, A .K . itoy, S . Maity, G alukder, Human Genetics Unit .<br />
Centre for Advanced Study in Cel & Chromosome Research, Department<br />
of Botany, University of Calcutta, Calcutta 700019, India .<br />
A comparison was carried out between selected populations from the<br />
Eastern India, who had been exposed, directly <strong>and</strong> indirectly, to<br />
industrialisation for 10 to 40 years <strong>and</strong> non-exposed populations<br />
from totally rural areas . The endpoints observed were blood genetic<br />
markers <strong>and</strong> cytogenetic parameters . In 1648 blood samples from<br />
healthy male age-matched donors, the frequency of abnormal variants<br />
of lipoprotein <strong>and</strong> hemoglobin was significantly higher in the<br />
exposed populations . There was no appreciable difference, however .<br />
in the incidence of markers not related to life style or diet, like<br />
haptoglobin, transferrin, acid phosphatase, lactate dehydrogenase .<br />
ABO system <strong>and</strong> Rh factor . The frequencies of chromosomal aberrations,<br />
micronuclei <strong>and</strong> sister chromatid exchanges were significantly higher<br />
in leucocyte cultures set up from 437 individuals tested from the<br />
exposed group, as compared with corresponding cultures from persons<br />
belonging to the non-exposed group . The difference between persons<br />
who had been exposed directly <strong>and</strong> those who had been exposed<br />
indirectly was not statisticilly significant .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 181<br />
Notes
182 1989 EMS Abstracts 527<br />
NOteS<br />
. .. ...<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
..<br />
IN SITU DETECTION OF GENOTOXICITY - POSSIBILITIES AND MEASUREMENTS C .B .S .R . S harma<br />
Salim Ali School of~cology, Pondicherry University, Pondicherry 605 001, India<br />
±i- -<br />
Three approaches exist to monitor the genotoxicity in the polluted ecosystems - (a)<br />
to screen the plants <strong>and</strong> animals in the habitats for genotoxic symptoms in mitotic <strong>and</strong>/<br />
or meiotic tissues, (b) to monitor impacts on specific endpoints in the st<strong>and</strong>ard test<br />
systems maintained in situ <strong>and</strong> (c) to test the in situ habitat samples like water, soil<br />
<strong>and</strong> sediments in the laboratory for genotoxicity i-n the experimental systems . While<br />
reviewing these possibilities, measurements are presented to suggest that some herbaceous<br />
terrestrial weeds, aquatic plants, ipsect populations <strong>and</strong> crop systems have<br />
potential to reveal the in situ genotoxicity of the habitats - (1) Of the 30 weeds<br />
screened from two habitaTs periodically inundated by polymer factory effluents <strong>and</strong><br />
sewage Croton, Justicia, Cassia, Parthenium, Cleome <strong>and</strong> Solanum have shown significant<br />
meiotic karyotoxicity 0 .~3-9: an po len aterllity 1- ..) . (2) Eichornia,<br />
inhabiting ponds variously polluted by effluents from homesteads, dyestu f <strong>and</strong> polymer<br />
factories <strong>and</strong> washates from transport systems,has shown mitotic karyotoxicity in<br />
significant frequencies (1 .2-12 .6%) (3) _Chroto on~u_s inhabiting the ground cover<br />
around sewage ponds revealed highly signi ica~tic karyotoxicity (3 .49-10 .2%) .<br />
(4) Meristem Assays <strong>and</strong> progeny tests involving Allium <strong>and</strong> Hordeum irrigated by sewage<br />
<strong>and</strong> polluted groundwater or maintained in the sewage slud9e- Fiave all shown genotoxic<br />
symptoms to various degrees . Some of the weeds of terrestrial <strong>and</strong> aquatic ecosystems<br />
<strong>and</strong> local insects can therefore be sensitive indicators of the ubiquitious genotoxins<br />
in our ecosystems .<br />
USE OF NATIVE PLANTS AND INSECTS AS INDICATORS OF in situ CENOTOXICITY . Sharma,<br />
C .B .S .R ., Panneerselvam, N ., Arumikkili K ., Raiendran M ., Meenakahi N .D ., Gowri R .,<br />
Salim Ali School of Ecology, Pondirherry University, Pondicherry 605 001 .<br />
Plants <strong>and</strong> insects inhabiting the polluted areas in the cities of Vishakhapatnam,<br />
Madurai, Coimbatore <strong>and</strong> Mysore of Southern India are assayed for genotoxicity . The<br />
pollution was due to polymer factory effluents in Vishakhapatnam <strong>and</strong> sewage in<br />
others . The results are as follows . In Vishakhapatnam <strong>and</strong> Madurai 30 herbs are<br />
acreened for meiotic aberrations <strong>and</strong> Pollen fertility . Twelve of these have shown<br />
significant effects - The frequencies of abnormal cells ranged between 0 .87 to 23 .9A<br />
<strong>and</strong> sterility between 12% to 95% . Among the more sensitive plants ares Croton<br />
bon l<strong>and</strong>ianum, Juaticia sim lex, Cassia tore, Terosia ur urea, Cleome v scosa,<br />
ar hen um ystero o us a~n ?olanum ind~m . Tn t~Fe Eic orn a ro~aystem, !~<br />
frequency of karyotoxicity atT~iahakhapatnsm ranged between ~3 end 12 .6, at<br />
Coimbatore between 3 .2 <strong>and</strong> 8 .5, at Madurai between 3 .0 <strong>and</strong> 8 .9 <strong>and</strong> at Mysore between<br />
2 .1 <strong>and</strong> 5 .8 . In situ analysis of the insect Chroto nus asusaurei inhabiting two<br />
sewage ponds in Combatore, revealed meiotic ary~ty ranging between 3 .49 <strong>and</strong><br />
10 .20 at one pond <strong>and</strong> between 6 .99 <strong>and</strong> 9 .0 in the other . These studies suggest that<br />
in situ inhabitants can indicate habitat genotoxicity especlally the water hyacinth,<br />
rl-chernia <strong>and</strong> the grasshopper, Chrotogonus .<br />
WATER HYACINTH (Eichornia crasaipes) IS AN EXCELLENT in situ MONITOR OF AQUATIC<br />
GENOTOXINS . C .B .S .R . Sharma, Salim Ali School of Ecology, Pondicherry University,<br />
Pondicherry 605 001, India .<br />
The ubiquitous occurrence of water hyacinth (Eichornia crasaipes) prompted us<br />
to explore the possibility of its use as a monitor of aquatic genotoxins . Herewith<br />
reported are the results from five cities of India where Eichornia's responses are<br />
monitored from aquatic habitats with different histories . The cities are : Vishakhapatnam,<br />
Madurai, Coimbatore, Mysore <strong>and</strong> Pondicherry . The assay is restricted to<br />
root systems . The endpoints scored aree clastogeny (fragments, micronuclei <strong>and</strong><br />
bridges) <strong>and</strong> turbagsay(vagrancy <strong>and</strong> micronuclei) . The highest karyotoxic frequency<br />
was obtained in the populations inhabiting waters contaminated by a polymer factory<br />
effluents at Vishakhapatnam (3 .5*0 .25 to 12 .6*0 .64) followed by automobile washates<br />
plus sewage at Coimbatore (3 .2*0 .16 to 8 .5*0 .78) ; dye stuff effluents plus sewage at<br />
Madurai (3 .0 :0 .22 to 8 .9s0 .56)g untreated sewage at Mysore (2 .1*0 .19 to 5 .8*1 .1)<br />
<strong>and</strong> roadside runoffs at Pondicherry (1 .2*0 .12 to 2 .6*0 .5) . The background frequency<br />
has ranged between 0 .89*0 .09 to 1 .02 :0 .15 . Thus the complex mixtures in the water<br />
bodies causing karyotoxicity have in them effluents from homesteads, dyestuff <strong>and</strong><br />
polymer factories, washates from the traffic-busy roads <strong>and</strong> automobile workshops<br />
suggesting propensity of genotoxic exposure in the concerned human populations .<br />
These preliminary studies, suggest that Eichornis can serve aa an excellent in situ<br />
biomonitor of habitat genotoxins .<br />
50869 3696<br />
528<br />
529
530 ©<br />
GENOTOXICITY EVALUATION OF CHLORTETRACYCLINE<br />
R .K . Sharma, K .A . Traul,-C . Caterson, J . Harbell, A . Thilagar, D . Putnam, American<br />
Cyanamid Co ., Princeton,~--NJ ; Sitek Research Labs ., Rockville, MD ; Microbiological<br />
Associates, Bethesda, MD .<br />
Chlortetracycline-HC1 (CTC-HC1), a broad-spectrum antibiotic, was tested in a<br />
battery of short term j.D vitro (microbial <strong>and</strong> mammalian point mutation, <strong>and</strong><br />
unscheduled DNA synthesis) <strong>and</strong> JjI y.jy4 (rat bone marrow chromosome aberrations)<br />
tests . Microbial <strong>and</strong> mammalian point mutation testing was done with <strong>and</strong> without rat<br />
S9 metabolic activation ; all tests had concurrent positive <strong>and</strong> negative controls . The<br />
microbial mutagenesis assay .jn J . tyohi~yyrium <strong>and</strong> E . Sg]j was conducted up to a dose<br />
of 10 pg/plate, in the presence <strong>and</strong> absence of S9, limited by toxicity . No increase<br />
in revertants per plate was observed . In the CHO/HGPRT mammalian gene mutation assay,<br />
doses (based on solubility <strong>and</strong> cytotoxicity) of 100,80,60,40 <strong>and</strong> 20 pg/ml (+S9) <strong>and</strong><br />
125,100,75,50, <strong>and</strong> 25 pg/ml (-S9) were found to be nonmutagenic . In the rat (Sprague<br />
Dawley) bone marrow cyto enetic test, CTC-HC1 was administered by oral gavage at<br />
5,000, 2,500, <strong>and</strong> 500 mgYkg in males <strong>and</strong> females, based on a range-finding study .<br />
Bone marrow was harvested at 12,24 <strong>and</strong> 36 hours after treatment . CTC-HCl induced no<br />
increases in chromosomal aberrations in either sex at any dose or time point . In the<br />
rat hepatocyte primary culture/DNA repair (UDS) test, doses tested ranged from 25 to<br />
75 pg/ml, limited by cytotoxicity . None of the doses produced a mean grain count of<br />
more than five compared to vehicle control . Positive controls in each test system<br />
produced significant positive responses . It is concluded that Chlortetracycline-HC1<br />
is not mutagenic in these test systems .<br />
531<br />
GENOTOXICITY EVALUATION OF SULFAMETHAZINE<br />
R .K . Sharma, K .A . Traul, C . Caterson, E . Johnson, R .R . Young, J .L . Ivett . American<br />
Cyanamid Co ., Princeton, NJ ; Hazleton Laboratories America, Inc ., Kensington, MD .<br />
Sulfamethazine (SULMET), a bacteriostatic antibiotic, was evaluated for genotoxic<br />
potential in a battery of short term j,n yitro (microbial <strong>and</strong> mammalian point<br />
mutation) <strong>and</strong> jIl yjys (rat bone marrow chromosome aberrations) tests . Microbial <strong>and</strong><br />
mammalian point mutation testing was done in the absence <strong>and</strong> presence of rat S9<br />
metabolic activation ; all tests had concurrent positive <strong>and</strong> negative contro)s .<br />
The microbial mutagenesis assay in a . tvohimurium <strong>and</strong> F . & Q]j was conducted up to a<br />
dose of 10 µg/plate, in the presence <strong>and</strong> absence of S9, limited by toxicity . No<br />
increase in revertants per plate was observed . In the CHO/HGPRT mammalian gene<br />
mutation assay, doses (based on solubility <strong>and</strong> cyto~oxicity) ranging from 0 .5 to<br />
7 .0 mg/ml with <strong>and</strong> without metabolic activation, were found to be nonmutagenic . In<br />
the rat (Sprague Dawley) bone marrow cytogenetic test, SULMET was administered by<br />
oral gavage at 3,000, 1,500, <strong>and</strong> 750 mg/kg in males <strong>and</strong> females, based on a rangefinding<br />
study . Bone marrow was harvested at 6, 18 <strong>and</strong> 30 hours after treatment .<br />
SULMET induced no increases in percentages of chromosomally aberrant cells or in the<br />
frequency of aberrations per cell per animal in either sex at any dose or time point .<br />
Positive controls in each test system produced significant positive responses . It is<br />
concluded that Sulfamethazine is not mutagenic in these test systems .<br />
532<br />
MONITORING THYMIDINE CATABOLISM IN COMPLEX NATURAL SYSTEMS : A SIMPLE, NOVEL METHOD<br />
FOR ECOTOXICITY ASSESSMENT<br />
T . Shaw, D .G . MacPhee 6 R.H . Smillie*, Departments of Microbiology, La Trobe<br />
University <strong>and</strong> Biochemiatry, University of Melbourne* (Australia)<br />
Thymidine is one of the most important molecules in biology . Numerous bioassays<br />
depend on measurement of incorporation of radiolabelled thymidine into acid-insoluble<br />
macromolecules, usually presumed - often without justification - to be DNA . In the<br />
majority of steady-state biological systems, <strong>and</strong> in particular in natural ecosystems<br />
where cell growth <strong>and</strong> turnover are slow, thymidine catabolism <strong>and</strong> incorporation into<br />
non-DNA macromolecules is almost invariably quantitatively more important than its<br />
anabolism <strong>and</strong> incorporation into DNA . Consequently, we investigated the influence<br />
of toxic compounds on thymidine catabolism in various biological systems . Our work<br />
demonstrates that (1) meaeurement of thymidine catabolism is extremely simple even<br />
in complex systems, because at saturating concentrations of exogenous thymidine, its<br />
catabolites diffuse into the extracellular environment at a faster rate than they can<br />
be re-utilised, <strong>and</strong> (2) that in stable aquatic microenvironments, the rate of catabolism<br />
of exogenously supplied thymidine is influenced in a time- <strong>and</strong> dose-dependent<br />
fashion by an enormous variety of compounds including "classical" cytotoxins <strong>and</strong><br />
genotoxins, as well as other compounds not usually considered to be in either of these<br />
categories . We conclude that monitoring thymidine catabolism yields useful information<br />
about the metabolic state of all systems investigated <strong>and</strong> seems to show great<br />
potential as an indicator of toxicity .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 183<br />
Notes
184 1989 EMS Abstracts- 533<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes IWEVALUATION`bE'IIJM RODENT CYTOGENETIC TEST RESULTS COMPARED TO IN VITRO<br />
TESTS AND CARCIAbGENICITY . Michael 0 . Shelby, Cellular <strong>and</strong> Genetic Toxicology<br />
Branch, National I te of <strong>Environmental</strong> Health Sciences, Research Triangle<br />
Park, NC 27709 j~SA<br />
The role of in vivo short-term tests for genetic toxicity in testing schemes<br />
intended to detect potential chemical carcinogens has long been a matter of<br />
discussion . Whether in vivo tests should be part of an initial screening battery<br />
or should only be conducted on chemicals selected by in vitro tests has never been<br />
agreed . Over the past 7 years, a substantial data base of in vivo cytogenetic test<br />
results has been developed through NTP contract studies . Preliminary analyses<br />
have shown that in vivo tests for abearations <strong>and</strong> SCE perform as well or better<br />
than in vitro tests in identifying carcinogens <strong>and</strong> in discriminating between carcinogens<br />
<strong>and</strong> noncarcinogens . Published results <strong>and</strong> data acquired through NTP for<br />
the mouse micronucleus test are particularly encouraging . It appears that the<br />
micronucleus test, using either bone marrow or blood to score micronucleated<br />
erythrocytes, performs as well or better than tests for aberrations or SCE in bone<br />
marrow cells <strong>and</strong> at a considerable saving in resources . Use of the micronucleus<br />
test in place of other in vivo cytogenetic assays in initial testing efforts is<br />
under consideration . The results of chromosomal aberration, SCE <strong>and</strong> micronucleus<br />
tests on a variety of carcinogens <strong>and</strong> noncarcinogens <strong>and</strong> the relative performances<br />
of in vivo <strong>and</strong> in vitro tests will be presented .<br />
534<br />
BACKGROUND FREQUENCY OF HYPERPLOIDY IN BONE MARROW CELLS OF MALE CHINESE HAMSTERS<br />
CW Sheu, JK Lee, CA Arras . RL Jones <strong>and</strong> KS Lavappa, CFSAN/FDA, Washington D .C . (USA)<br />
Chinese hamsters, with 22 distinctive individual chromosomes, are an ideal species<br />
for aneuploid analysis . In a series of studies, the hyperploidy frequency in bone<br />
marrow cells of 154 male hamsters was determined . The animals were given a single ip<br />
injection of solvent or a potential aneuploidy-inducing chemical . Ten animals were<br />
used per dose <strong>and</strong> bone marrow was removed at intervals of 6-96 hr . Slides were coded<br />
<strong>and</strong> 50-100 metaphases were analyzed per animal . A metaphase with more than 22 chromosomes<br />
was classified as a hyperploid cell, <strong>and</strong> the data were evaluated using a onetailed<br />
Fisher's Exact test . In three experiments, the frequencies of hyperploid cells<br />
were 0 .43, 0 .91 <strong>and</strong> 1 .14% for the control groups . The combined controls had 17 hyperploid<br />
cells per 2,656 metaphases prepared from 32 hamsters, giving an average frequency<br />
of 0 .64% . The hyperploidy frequencies of treated groups ranged from 0 .50 to 1 .25%,<br />
<strong>and</strong> there was no significant increase in any treated group over its concurrent control<br />
with the exception of one group treated with vincristine at 0 .75 mg/kg . The observed<br />
increase was not significant when compared to the pooled controls . The treated groups<br />
when combined had 65 hyperploid cells per 7,355 metaphases from 90 hamsters, giving an<br />
average frequency of 0 .88% ; this value was not significantly different from that of<br />
the combined controls . In a fourth experiment, the hyperploidy frequencies based on<br />
800 metaphases from 8 animals per group were 3 .75% for the controls <strong>and</strong> 3 .13-4 .52% for<br />
the treated groups . These values were significantly higher than those given above .<br />
The discrepancy appeared to be related to the batch of animals <strong>and</strong> illustrates the<br />
importance of incorporating concurrent controls in assays for aneuploidy .<br />
535<br />
INDUCTION OF HICR01NCL6I IN RAT BOS6 MARROW BY S6LBCTION CHSHICALS . ! . Shi <strong>and</strong> T . OnB,<br />
Division of Respiratory Disease Studies, National Institute for ocoupational Safety<br />
<strong>and</strong> Health . HorBantown, WV (USA)<br />
A large number of chemicals have been tested for the induction of micronuclei in<br />
mouse bone marrow cells . Many of these studies were designed to determine proper<br />
sampling times <strong>and</strong> peak responses . Such studies <strong>and</strong> infors,ation, however, are rather<br />
limited for the rat bone marrow cells . Bfforts have been made in our laboratory to<br />
determine the dose <strong>and</strong> sampling time responses of micronuclei after Sprague Dawley<br />
rats were treated with triethylenem.lamine, mitomycin C, vincristin, <strong>and</strong> dimathylbensanthracene<br />
by a single intraperitoneal injection . Three concentrations were tested for<br />
each compound . Animals were sacrificed 24, 48 <strong>and</strong> 72 hrs after chemical treatment .<br />
The procedure of slide preparation <strong>and</strong> staining followed that of Schmid (Mutation Res .<br />
31 :9, 1975) . The number of micronucleated polychrom.tic erythrocytes among 2000<br />
polychromatic erythrocytes (PC6s) <strong>and</strong> the ratio of PCB to norsochroastic erythrocytes<br />
were determined for each animal . The results showed that all four compounds caused<br />
micronucleus formations in a dose related manner . The peak response sampling tisw is<br />
dependent on the chemical as well as the concentration of chemical . In all cases .<br />
however, an increase in the micronuclested PCis can be detected 24 hrs after chemical<br />
treatment . These results seem to indicate that two sampling times, 24 <strong>and</strong> 48 hrs,<br />
would be adequate for the micronucleus assay usin8 rat bone marrow cells .<br />
50869 3698
536 r<br />
THE COLLABORATIVE STUDY ON MOUSE SPOT TEST IN JAPAN .<br />
x .TUtikawa, T .Shibuya, S .}litotsumachi, T .Ohshima <strong>and</strong> A . Wakata, The Collaborative Study<br />
Group for the Mouse pot Test, Hadano, Kanagawa 257 (JAPAN)<br />
The collaborative study on mouse spot test (MST) was carried out in 11 different<br />
laboratory in Japan . Mouse strain used in this study were male PW (established by<br />
Tutikawa <strong>and</strong> Harada) <strong>and</strong> six different females ; C57BL/6 (B6), four 810 congenic lines<br />
(B10, B10A, B10BR <strong>and</strong> BlOD2) <strong>and</strong> KYG . The test campound employed was N-ethyl-N-nitrosourea<br />
(ENU) . ENU was dissolved in phosphate buffer (pH 6 .0) <strong>and</strong> intraperitoneally<br />
treated to female mice at dag_10 .5 of pregnancy . The observations on the F1 mice were<br />
done on the following characterst recessive color spots (RS), white midventral spots<br />
(WMVS), misdifferentiation spots (MDS) <strong>and</strong> external malformations at their weaning<br />
period .<br />
The incidence of RS lineally increased as dose of ENU in all matings . These results<br />
were clearly different from that of mouse specific locus test (Russell et al,<br />
1982) . These observations may suggest that repair capacity is deficient in melanoblast<br />
cells in mouse embryos . In the case of KYG, the incidence of RS induced by ENU<br />
was always higher than that in the other matings .<br />
The fertility rate in plug-proved females was extremely low in 86 females that the<br />
other females mated .<br />
From these results, we recommend to use male PW <strong>and</strong> female 810 on MST in Japan .<br />
537<br />
N-ETHYL-N-NITROSOUREA INDUCED RECESSIVE MUTATIONS IN MOUSE PRIMORDIAL GERM CELLS .<br />
T .Shibuya, N .Horiya, H .Matsuda, T .Hara, M .Ratoh <strong>and</strong> T .Murota, Hatano Research Institute<br />
Food <strong>and</strong> Drug Safety Center, Hadano, Kanagawa, <strong>and</strong> Mitsubishi Kasei Co, Yokohama(JAPAN)<br />
Previously we found that N-ethyl-N-nitrosourea (ENU) induced recessive mutation in<br />
somatic cells <strong>and</strong> killed primordial germ cells (PGC) in mouse embryos (Shibuga at al,<br />
1982) . We therefore carried out a specific locus test (SLT) on mouse PGC with ENU .<br />
Male <strong>and</strong> female C3H/He mice were mated <strong>and</strong> pregnant females obtained were treated<br />
with 30 or 50 mg/kg ENU at day 10 .5 of pregnancy . The F1 male mice obtained were mated<br />
with tester female PW mice after they were grown . The obtained mice of the next qeneration<br />
were checked for their coat•color <strong>and</strong> ear shape to evaluate whether or not PGC<br />
had been mutated by ENU . The fertility of male mice was 99% <strong>and</strong> 38% at 30 or 50 mg/kg<br />
ENU, respectively, much higher than the value in female mice .<br />
Three mutants were obtained in the 30 mg/kg group fram 5,823 offspring, <strong>and</strong> the two<br />
of them were recovered fram the same male, showing cluster mutations . Fifteen mutants<br />
were gained from 2,837 offspring in the 50 mg/kg group, <strong>and</strong> most of those had originated<br />
from cluster mutations . All mutants thus obtained are viable under hanozygous<br />
conditions . The high viability of mutant genes under homozygous condition is similar<br />
to that of an examination of the effect of ENU on stem-cell spermatogonia .<br />
SLT in mouse PGC at different developmental stages (8 .5 <strong>and</strong> 13 .5 day of development)<br />
by ENU are now in progress . Until now, mutants are obtained fram both experiments .<br />
It may be concluded that early stage of PGC is a sensitive stage to ENU in mice .<br />
538<br />
GENOTOXICITY OF BENZENE AND ITS METABOLITES<br />
H . Shimada, S . Itoh <strong>and</strong> S . Takayama<br />
Research Institute of Daiichi Seiyaku Co ., Ltd ., Tokyo (JAPAN)<br />
Benzene is known to have clastogenicity <strong>and</strong> myelotoxicity in animals, though which<br />
metabolites cause these damages remain unclear . We investigated the relationships<br />
between DNA single-str<strong>and</strong> breaks (SSBs) <strong>and</strong> ∎icronuclei formation induced benzene <strong>and</strong><br />
its metabolites in vivo <strong>and</strong> in vitro . Male ddY strain ∎ice were singly administered po<br />
with benzene <strong>and</strong> killed at 12 to 72 h later . It was found that the SSBs in the bone<br />
marrow cells was markedly increased at 36 h, <strong>and</strong> the ∎icronuclei frequency was markedly<br />
increased at 42 h after the administration . In the ∎icronucleus test of the bensene<br />
metabolites, phenol <strong>and</strong> hydroquinone induced ∎icronuclei but other metabolites (muconic<br />
acid, hydroxyhydroquinone, 1 .4-benzoquinone, 2 .2'-biphenol . 4 .4'-biphenol <strong>and</strong> catechol)<br />
did not . In vitro studies, hydroquinone, 1 .4-benzoquinone, 2 .2'-biphenol <strong>and</strong> hydroxyhydroquinone<br />
induced SSBs in the bone marrow cells, while hydroquinone <strong>and</strong> 2,2'-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 185<br />
Notes
186 1989 EMS Abstracts. - --<br />
. ~ - .n„:-.<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Note s biphenol failed to induce SSBs in the Chinese hasster cells . The SSBs was detected in<br />
the -Chinesa _44nAfte-cells after the treatment of hydroquinone <strong>and</strong> peroxidase<br />
simultaneously . These results suggest that the Induction of ∎icronuclei by benzene<br />
follows after SSBs, <strong>and</strong> the several metabolites play an important role to Induce<br />
micronuclei <strong>and</strong> myelotoxicity through localized setaboliss within the bone marro . such<br />
as peroxidase-aediated metaboliss .<br />
SISTER CHROMATID EXCHANGES IN LYMPHOCYTES FRON INFANTS WITH DOWNS'SYNDROFg .<br />
E .K .Shubber, H .A .Hamamy, <strong>and</strong> B .M .A . AL-Allak, Dept . of <strong>Molecular</strong> biology, Nuclear<br />
Research Center, P .O .Box 765, Baghdad (IRAQ), <strong>and</strong> AL-Nustansiriya Medical College,<br />
Baghdad, (IRAQ) .<br />
This study investigated the relationship of sister chromatid exchanges (SCE)<br />
539<br />
frequency between normal <strong>and</strong> Downs syndrome infants ^nd their parents .•Blood .lymphocytes<br />
from 12 infants (3 females <strong>and</strong> 9 males) with Down's s'mdrome were cultured in RPHI-1640<br />
medium containing 10 ug/ml of brocodeoxyuridine for 63h . The mean frequency of SCE per<br />
metaphase for the patients (both sexes) was 9 .220 .8 which was significantly higher<br />
(P
earlier separation of the centromeres wes also noticed . The clestogenicity <strong>and</strong><br />
cytotoxicity of the druge"was further substantiated by in vivo essays with mouse<br />
bone marrow cells where it caused dose dependent increase of chromosomal aberrations,<br />
sister chrometid exchanges <strong>and</strong> micronuclei . Hydracortisone, therefore,<br />
appears to be highly potent steroidal drug capable of directly attacking the<br />
genetic material inspite of its negative response in the Ames teat,<br />
542<br />
MUTAGENICITY IN SALMONELLA AND SISTER CHROMATID EXCHANGE IN MICE FOR BAY-REGION<br />
SYAL AND ANTI-DIOL EPOXIDES OF 1,4-DIMETHYLPHENANTHRENE<br />
J .E . Sinsheimerl . A .K . Gir11, E .A . Messerlyl, K-Y . Jung2 <strong>and</strong> M . Koreeda2,<br />
1College of Pharmacy <strong>and</strong> 2Department of Chemistry, University of Michigan . Ann<br />
Arbor, MI 48109, USA .<br />
Dose-response relationships for (±)-7a,8s-dihydroxy-5p .6p-epoxy-1 .4-dimethyl-<br />
5,6,7,8-tetrahydrophenanthrene <strong>and</strong> its Sa,Ba-epoxy diastereomer, the syn- <strong>and</strong><br />
anti-diol epoxides of 1,4-dimethyl-phenanthrene respectively, have been tested<br />
for their mutagenicity In Salmonella strains TA98 <strong>and</strong> TA100 <strong>and</strong> for their In vivo<br />
sister chromatid exchange In the bone-marrow cells of mice . Both isomers were<br />
mutagenic In the nmole per plate range with the syn isomer being In the order of<br />
fifteen times more active with TA98 <strong>and</strong> five times more mutagenic In TA100 than<br />
its anti isomer . The anti lsomer was more genotoxic In the sister chromatid<br />
exchange assay than the syn isomer . Stetistically significant results were<br />
obtained as low as 1 .5 mg/kg body weight for the anti isomer <strong>and</strong> 3 mg/kg for the<br />
syn isomer . The present study supports the inclusion of methyl-substituted bayregion<br />
diol epoxides In the concept that the syn isomer, with quasi-diequatorial<br />
hydroxyl groups, contributes to the genotoxicity of the parent polycyclic<br />
aromatic hydrocarbons of those compounds which form hindered bay-region diol<br />
epoxides . Supported by grants R01ES0334S . NIEHS <strong>and</strong> R01CA25185, NCI .<br />
543<br />
THE ROLE OF PROTEIN ADDUCTS IN THE STUDY OF CHEMICAL CI4RCINOGENESIS . P . L . Skipper<br />
<strong>and</strong> S . R . Tannenbaum, Massachusetts Institute of Technology, Cambridge, MA 02139 .<br />
The reaction of carcinogens with proteins is an incidental but apparently inevitable<br />
outcome of the process of metabolic activation to ultimate carcinogens ; thus far no<br />
etiological role has been ascribed to these adducts . Nevertheless, they provide an<br />
excellent historical record of exposure <strong>and</strong> subsequent metabolic events which may be<br />
exploited to several ends . Firstly, they may be productively used in exposure<br />
assessment . In this category fall such applications as exposure monitoring, definition<br />
of exposed populations, <strong>and</strong> identification of cryptic exposures . Cigarette smoking,<br />
as well as exposure to environmental tobacco smoke, have been monitored by analysis<br />
of hemoglobin-bound 3- <strong>and</strong> 4-aminobiphenyl . These studies also demonstrated that<br />
cigarette smokers are a distinct group defined by five- to ten-fold higher adduct<br />
levels than nonsmokers . Similar evaluation of the contribution of cigarette smoking<br />
to benzo[a7pyrene exposure has been conducted . Secondly, protein adducts are useful<br />
in assessing the role of individual differences in biochemical response within a<br />
population exposed to relatively similar amounts of a carcinogen . Thus, hemoglobin<br />
adducts of 4-aminobiphenyl in smokers have been used to assess the significance of<br />
acetylator phenotype on the activation of this carcinogen through hydroxylamine<br />
formation . Finally, subject to many caveats, protein adducts have the potential for<br />
improving assessment of risk . Factors which affect the feasibility of realizing this<br />
potential will be discussed . This work was supported by the National Institutes of<br />
Health (grant nos . ESO0597, ES02109, ES01640) <strong>and</strong> the American Cancer Society (grant<br />
no . SIG 10-II) .<br />
544<br />
CORRELATION OF DNA ADDUCTS WITH SPECIFIC ALTERATIONS IN DNA SEQUENCEt IMPLICATIONS FOR<br />
CANCER RESEARCH . T .R . Skopek . Chemical Industry Institute of Toxicology, Research<br />
Triangle Park, NC (USA)<br />
A variety of DNA alterations have been observed in the activation of oncogenes <strong>and</strong><br />
the inactivation of tumor suppressor genes . These include point mutations in protein<br />
coding regions, point mutations affecting aRNA splicing, gene rearrangements, gene<br />
amplification, <strong>and</strong> losa of hemisygousity . <strong>Molecular</strong> biology techniques are presently<br />
being used to learn how different chemical mutagens can effect these specific<br />
alterations in DNA . Knowledge of the DNA adducts <strong>and</strong> sequence alterations induced by<br />
chemical mutagens'permits the correlation of specific adducts with specific mutations ;<br />
this in turn can be used to infer a mutagen's ability to effect specific base changes<br />
commonly associated with oncogene/suppressor gene alterations . Once identified,<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 187<br />
Notes<br />
Ir
188 1989 EMS Abstracts<br />
NOteS " : ..<br />
promutagenic adiucts can be studied as biologically relevant endpoints in tumor<br />
induction studies (~t e of adduct formation, persistence, repair), as well as in<br />
epidemiology-stddiei:3Md risk assessment calculations . Examples using model compounds<br />
(alkylating agents, acrylonitrile) will be presented to illustrate the importance of<br />
determining the types of mutations induced by chemicals <strong>and</strong> determining promutagenic<br />
adducts .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
545<br />
MOLECULAR ANALYSES OF A NOVEL LESCH-NYHAN SYNDROME MUTATION (hcstHon r ) 81 USE OP<br />
T-LYHPHOJYTE CULTU~ES . T .R~ Skogek, L . Recig, D . SimpsonD L . ba4f~ire , S .$ .<br />
Nelancon , H . Ogier , J .P . 0 Neill , H .T . Falta , J .A . Nicklas , <strong>and</strong> R .J . A1$ertini .<br />
Chemical Industry Institute of Toticology, Research Triangle Park, NC (USA) ; Hospital<br />
Saint Justine, Hontreal, Canada ; University of Vermont, Burlinton, VT (USA) .<br />
The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative<br />
Lesch-Nyhan individuals <strong>and</strong> their parents was determined by a cell cloning assay to<br />
quantify the frequency of thioguanine resistant mutants . The results confirm the<br />
Lesch-Nyhan diagnosis <strong>and</strong> demonstrate that the mother has an elevated mutant frequency<br />
consistent with being heterozygous for an hprt mutation . Mass cultures of Tlymphocytes<br />
from both the children <strong>and</strong> their mother, as well as cultures of hDrt<br />
mutant clones from the mother, were employed as sources of aRNA for cDNA sequence<br />
analysis . These hprt mutants show a single base substitution (T + C transition) at<br />
position 269 (exon 3) . The predicted amino acid change is the substitution of<br />
threonine for methionineS7 . We term this new Leach-Nyhan mutation hertHontreal'<br />
546<br />
ROLE OF METABOLISM IN BENZENE-INDUCED MYELOTOXICITY AND LEUXEMOGENESIS<br />
Smith, M .T., Yager, J .W ., Robertson, M .L ., <strong>and</strong> Eastmond, D .A .' School of Public Health,<br />
University of California, Berkeley, CA 94720 <strong>and</strong> "Biomedical Sciences Division,<br />
Lawrence Livermore National Laboratory, Livermore, CA 94550 .<br />
The principal metabolitea of benzene include phenol, hydroquinone, catechol <strong>and</strong><br />
trans, trans-muconic acid . The repeated co-administration of two of these metabolites,<br />
phenol <strong>and</strong> hydroquinone, produces bone marrow toxicity in mice similar to that caused<br />
by benzene . In parallel studies, phenol has been shown to stimulate the conversion of<br />
hydroquinone to the highly toxic 1,4-benzoquinone by myeloperoxidase, the major peroxidase<br />
enzyme in the bone marrow . A mechanism of benzene-induced myelotoxicity involving<br />
the accumulation of phenol <strong>and</strong> hydroquinone in the bone marrow <strong>and</strong> subsequent<br />
stimulation of peroxidase-dependent 1,4-bensoquinone formation has therefore been proposed<br />
. The same metabolites may also be responsible for benzene's genotoxic <strong>and</strong> leukemogenic<br />
effects . In order to study this further, we have determined the aneuploidyinducing<br />
<strong>and</strong> clastogenic properties of beniena's phenolic metabolites using a micronucleus<br />
assay in cytokinesis-blocked human lymphocytes . These studies revealed that<br />
hydroquinone was by far the most effective inducer of clastogenicity <strong>and</strong> the only<br />
phenolic metabolite which caused a consistent, dose-related increase in aneuploid<br />
cells . Interestingly, these effects were inhibited by the inclusion of ascorbic acid<br />
in the incubation medium . These results suggest that an oxidation product of hydroquinone<br />
is the metabolite responsible for the aneuploidy <strong>and</strong> clastogenicity observed<br />
following occupational benzene exposure <strong>and</strong> thus potentially benzene-induced leukemia .<br />
Supported by NIH grants P42 ES04705 <strong>and</strong> P30 9801896 . Work performed in part under the<br />
auspices of US DOE by the LLNL under contract no . W-7405-ENG48 .<br />
547<br />
PROTEIN CONTENT OF TOBACCO CELLS IN RELATION TO THE PLANT AC'TIVATION OF<br />
m-PHENYLENEDIAMINE AND 2-AMINOFLUORENE. S .R. Smith, M .M . Verdier, ED . Wagner <strong>and</strong><br />
MJ. Plewa. Institute for <strong>Environmental</strong> Studies, University of Illinois, Urbana, IL 61801 USA.<br />
'Ibbacco cell suspension cultures activate the promutagens m-phenylenediamine <strong>and</strong> 2-aminoQuorene<br />
with his• reversion of Sa/mondla 7jphimurium strain TA98 as the genetic end point . 7b help identify the<br />
biochemical mechanism involved, a growth curve was established for these cells by first Inoculating several<br />
flasks with 3 g each from a 7-day culture <strong>and</strong> then measuring fresh weight at approodmately 12-h Intervals<br />
for 2 weeks. At the same time, protein content was analyzed using the Bio-Rad protein assay which is<br />
based on the absorbance of the dye Coomassic Brilliant Blue 0-250 that shifts to 595 nm upon binding<br />
to protein. Fresh tobacco cells were titered, sonicated, <strong>and</strong> centrifuged, leaving the extracted protein in<br />
the supernatant fluid. Frozen cells were also analyzed but gave iaconsistent results . A st<strong>and</strong>ard curve was<br />
determined using bovine gamma-globulin for each 24-h sample . After a lag phase of 3 to 4 days, the cells<br />
entered log phase ; stationary phase was reached by day 7 . Protein content, however, did not follow the<br />
50869 3702
growth curve, instead peaking just before the cells entered log phase . Data from preUminary activation<br />
studies using tobacco cells at diffeyent growth stages showed that lag-phase cells activated 2-aminofluorene<br />
better than those in log or stationary phase . Although the maximum 2-aminofluorene activation coincided<br />
with peak protein content, late log-phase cells were most competent in m-phenylenediamine activation,<br />
suggesting that tobacco cells activate 2-aminofluorene <strong>and</strong> m-phcnylenediamine through different metabolic<br />
pathways. Research funded in part by a WRC grant <strong>and</strong> USAF grant #88-AF-P-0511 .<br />
548<br />
THE POLY (ADP-RIBOSE) POLYMERASE GENE : DIRECT OR INDIRECT INVOLVSNIIirfP DJ DNA REPAIR AND<br />
MAllONANCYI<br />
Smulson, M .E .. Chemey, B ., Haque, J ., Huppi, C., Kang. V., Marr. K .. Mohrart . Z., Simpson. S ., <strong>and</strong> 8hatla, K .<br />
Dept . of Biochemistry, Georgetown University School of Medicine, Washington, D .C .<br />
Poly (ADP-ribose) polymerase Is a nuclear enzyme which appears to play a key role in modulating the<br />
repair of damaged DNA mammalian calls . The catalytic activity of the purified enzyme is stringently<br />
dependent upon the presence of str<strong>and</strong> breaks in DNA . The polymerase ls rapidly modulated In response to<br />
mutagen treatment, probably representing one of the very early responses to DNA damage . We have evaluated<br />
the role of endogenously <strong>and</strong> exogenously Induced DNA str<strong>and</strong> breaks on the transcriptional control of this<br />
enzyme <strong>and</strong> also studied the expression of this gane during the eeU cycle as well as during differentiation,<br />
both biological processes were endogenous discontinuous regions of DNA, <strong>and</strong> possibly str<strong>and</strong> breaks are<br />
operative. Experiments using the polymerase cDNA In an expression vector indieate an Increased rate of<br />
DNA repair In DNA damaged Cos cells, transiently transfeeted to overexpress the protein .<br />
We have also mapped the human ehromowmtl location(s) of this gane . It has often been suggested that<br />
cancer is associated with abnormal recombination events <strong>and</strong> mutations . We hypothesked that if any member<br />
of the potymerase gene family or polymerase-related genes were necessary for reducing inappropriate<br />
«combinationc, its loss might be associated with at least some human cancers. Tbe existence of a deletion<br />
polymorphism in one of the polymerase genes, located on chromosome 13q32-ter, allowed us to test whether<br />
deletion on both alleles on this gene correlates with inoreased tumor Incidence . There was a marked inereaso<br />
(4x) in the frequency of the deleted allele in tumor DNA ; allelic loss was also noted. These results suggest<br />
that deletions in the polymerase, or a gene linked close to it on chromosome 13 may operate to promote the<br />
development of some types of cancer<br />
. 'r<br />
549 A<br />
CHROMIUM(III) BINDS TO SINGLE-STRANDED DNA, INCREASES DNA POLYMERASE PROCESSIVITY,<br />
AND INDUCES MUTAGENESIS IN E . COLI . E .T . Snow, L .S . Xu, <strong>and</strong> M .D . Cohent Institute of<br />
<strong>Environmental</strong> Medicine, NYU Medical Center, P .O . Box 817, Tuxedo, NY (USA) .<br />
Chromium is a suspected human carcinogen, but the mechanism of chromium-induced<br />
carcinogenesis is unknown . Chromate ions are readily taken up by cells <strong>and</strong> are<br />
reduced intracellularly via unstable Cr(V) <strong>and</strong> Cr(IV) intermediates to stable Cr(II1)<br />
species . During this process DNA damage is produced <strong>and</strong> point mutations are induced<br />
in many target genes . However, it is not known how Cr produces mutagenic damage or<br />
if damage is produced indirectly by oxidative processes . Cr(III), the most stable<br />
form of Cr, has been implicated in chromium toxicityt but, most Cr(III) species do not<br />
cross cell membranes <strong>and</strong> are neither mutagenic nor carcinogenic in vivo . We have<br />
investigated the genotoxicity of Cr(III) in vitro using a DNA replication assay <strong>and</strong><br />
in vivo by transfecting Cr(III)-treated DNA into competent E . coli <strong>and</strong> scoring for<br />
survival <strong>and</strong> mutagenesis . Single-str<strong>and</strong>ed (as) M13 DNA was treated with 1 to 10 yM<br />
CrC13 (0 .5 - 24 hr, 370) <strong>and</strong> excess Cr removed by gel exclusion chromatography . The<br />
DNA was primed with a 5'-32P-sequencing primer <strong>and</strong> primer extension was carried out<br />
with DNA polymerase I(Klenow) or polymerase a-primase . Very low doses of Cr(III)<br />
(1 t0 5 uM) increased the processivity of both polymerases although 10 uM Cr(III) was<br />
inhibitory . When transfected into JM101 E . coli, Cr-treated es M13mp2 showed a dosedependent<br />
increase in mutation frequency at the lac2„ gene . These results suggest<br />
that Cr(III) alters the structure of the DNA template increasing the binding strength<br />
of DNA polymerases <strong>and</strong> decreasing the fidelity of DNA replication . These<br />
interactions may account, in part, for the mutagenicity of chromium ions in vivo .<br />
550<br />
A COMPARISON OF CHROMOSOME ABERRATION INDUCTION IN THE CHO AND CHL CELL<br />
SYSTEMS BY 25 CHEMICALS . T . Sofuni, A . Matsuoka, M . Sawada, M . Ishidate<br />
Jr ., <strong>and</strong> M . D . Shelby, National Institute of Hygienic Sciences,<br />
Setagaya-ku, Tokyo (Japan), <strong>and</strong> National Institute of <strong>Environmental</strong><br />
Health Sciences, Research Triangle Park, NC (USA)<br />
The present study was conducted to compare the results of chromosome<br />
aberration tests using the CHL <strong>and</strong> CHO cell systems on 25 test chemi-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 189
190 1989 EMS Abstracts<br />
_ _ .~-- __<br />
Notes •`!'ca1s . In"~e HL system, cells were exposed to the test chemical for<br />
24h <strong>and</strong> 48h without S9 mix <strong>and</strong> for 6h with S9 mix . In the CHO system,<br />
cells were eo~or 10 .5-20 .5h without S9 mix <strong>and</strong> for 2h with S9 mix .<br />
In the absence of S9 mix, 6(24%) out of 25 test chemicals showed diffarent<br />
results : 4 were positive only in CHL, 2 were positive only in CHO .<br />
In the presence of S9 mix, 11(44%) chemicals showed different resultss<br />
8 were positive only in CHL, 3 were positive only in CHO . According<br />
to the combined results with <strong>and</strong> without S9 mix, 10(40%) showed qualitatively<br />
inconsistent results between two test systems . One of the<br />
reasons for these inconsistent results may be the differences in the<br />
experimental protocol used . However, a possibility that, even by the<br />
same experimental protocol, chromosome aberration induction by some<br />
chemicals miqht be qualitatively <strong>and</strong>/or quantitatively different<br />
between the CHL <strong>and</strong> CHO cell systems, still remains .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
551<br />
FURTHER CHARACTERIZATION OF A METHYL IigTRANESULFONATE-RESISTANT MUTANT FROM A CHINESE<br />
HAMSTER CELL LINE : ANALYSIS OF ITS HYPERSENSITIVITY TO UV<br />
A . Sono <strong>and</strong> K . Sakaguchi<br />
Division of Toxicology, Research Center, TOYO JOZO Co . Ltd .(Japan) <strong>and</strong> Department of<br />
Genetics, University of California, Davis(USA)<br />
A Chinese hamster mutant which was highly resistant to methyl mefhanesulfonate (MfS),<br />
referred to as lQ1Sr-1, was found to be hypersensitive to UV . 141S -1 was twice higher<br />
sensitivity to UV in cell killing than the parental line (Don D-6) . The increase was<br />
S-phase specific in the cell cycle . Ca€feine could enhance the UV-lethality of Don<br />
D-6 . However, the survival curve of NMS -1, irrespective of the presence of caffeine,<br />
was almost the same as that for Don D-6 with caffeine . The mutant might be deficient<br />
in post-replicational repair . It also accompanied with higher induction rate of<br />
sister-chromatid exchange (SCE) <strong>and</strong> chromosomal aberration . The UV-lethality of I41Sr-<br />
1 appeared to relate to the increase rate of lethal chromosomal aberrations, such as<br />
chromatid breaks <strong>and</strong> exchanges . Although inhibition of protein synthesis enhanced the<br />
hypersensitivity to UV <strong>and</strong> reduced the acquired resistancy to HHS, it could antagonize<br />
the induction of UV-induced SCE . Inhibition of DNA synthesis affected synergistically<br />
only on the UV-lethality . At the 502 lethal dose of tN, MMSr-1 was unexpectedly much<br />
less mutable than Don D-6 . This phenomenon makes it very difficult to explain only by<br />
a defect in the post-replicational repair system, but may be explanable by adding<br />
another system defect, so called "error-prone repair" which is known only in E . coli .<br />
From these facts, the possible defective feature for this mutant was discussed .<br />
PRCBIEbS Il1 CYTOGENE'PIC SCIIZVEIISd1PKE OF PECPLE '<br />
Sorsa, M ., institute of Occupatiorlal Health, Helsinki, FINIAND<br />
552<br />
Sanatic chrmrosanle damage has been used for some deosdes already as indicator<br />
of exposure of specific grotlps of humans to clastogenic or genotoxic agents . The<br />
niain rationale in the approach is that dmmsge ebserved in the genetic material of<br />
human cells represents or reflects initial events in a process, which may eventually<br />
lead to ill health manifestations . lhus cytoyenetic surveillance has the potential<br />
to serve as an early indicator, enabling prevention of adverse effects .<br />
Several krowm <strong>and</strong> unoontrollable confoiuti3ers have been reoorded in population<br />
studies ; with proper preplanning most of these can be avoided . Since cytogenetic<br />
biamonitorin3 studies are always tedious <strong>and</strong> difficult, experimental identification<br />
of the ahrvnceane damaginy potential of the exposing agent(s) must be considered a<br />
prereyuisite to their performance .<br />
A11 stu3ies concerning humans directly also involve ethical aspects <strong>and</strong> the<br />
riyht-to-know of the persons giving the saaples . T41e planning stage of the study<br />
should also involve an action design for possible decrease of the exposures of the<br />
yroup, if necessary. Otherwise, the preventive nature of cytiogenetic surveillance<br />
is lost .<br />
553<br />
SUB-LETHAL ACIDIFICATION : ITS ROLE IN CHRONIC HEALTH EFFECTS .<br />
C .L . Soskolne ; 0 . Pagano, <strong>and</strong> 0 .0 . Oiordano, Istituto Nazionale Tumori, 80131 Naples .<br />
Italy, <strong>and</strong> •University of Alberta, Edmonton, Canada T60 203 .<br />
Mineral acids in the workplace, ae well aa in the general environment represent<br />
a recognized health concern as yet mainly focussed on lethal or acute outcomes .<br />
Over the past decade, long-term workplace exposure to high concentrations of sulfuric<br />
<strong>and</strong> other acid miste have been shown to be associated with respiratory tract cancers,<br />
50869 3704
1989 EMS Abstracts<br />
~<br />
especially laryngeal carcinomas (Soskolne et al ., 1984 ; 1989 ; Beaumont et al ., 1987). Notes<br />
Toxicologic evidence supgorts these human data <strong>and</strong> lncludes : a) in vivo <strong>and</strong> in vitro<br />
cytogenetic abnormaliti6s following a sub-lethal decrease of extra-cellular pH ;<br />
b) developmental toxicity as induced by acidic contaminants or drugs ; o) a crucial<br />
role for pH in cell cycle, mitosis, <strong>and</strong> differentiation . The underlying biologic<br />
mechanisms that explain adverse health outcomes include pH modulation of toxicity<br />
for a number of xenobiotics (including carcinogens, genotoxins, <strong>and</strong> teratogens), <strong>and</strong><br />
low pH-induced changes of celle involving, for example, alterations in mitotic<br />
apparatus <strong>and</strong> enzyme regulation . A•multi-disciplinary effort is prompted in order to<br />
investigate the role(s) of environmental acids <strong>and</strong> acidic drugs in genotoxicity<br />
<strong>and</strong> carcinogenesis . (Supported by the Italian Ministry of Health) .<br />
554<br />
MITOMICIN C-INDUCED DOMINANT LETHAL MUTA't1VNS IN SYERMATOCYI'IiS ANS NOT DUE 1'0 CELL-<br />
KILLING . J .P . Soto, F P va ivia, M .M . Orellana, N .M . Lafuente <strong>and</strong> H . Katob .<br />
Facultad Odontologia, U . de Chile . STGO (CHILE) <strong>and</strong> Hatano Research Institute,<br />
F .D .S .C ., Kanagawa (JAPAN) .<br />
Mitosicin C(MC) is well known to induce doainant lethal autations in souse early spersatids <strong>and</strong><br />
speraatocytes but has not effect in late spereatids <strong>and</strong> spersatozoa (Ehling, 1971) <strong>and</strong> Kratochvilova<br />
(1973) reported that MC cause high percent of unfertilized ova when MC-treatea anisals were sated at<br />
intervals corresponding to treated speraatocytes, suggesting a strong cell-killing effect of MC to<br />
speraatocytes . Histological studies <strong>and</strong> sperm head abnorsalities test ssre done to deteraine whether<br />
MC-induced failure of fertilization in speraatocytes is attributable to cell-killing effect . 9 weeks<br />
oid sale C('i aice were intraperitoneally (i .p) injected with 5 sg MC/kg . At 4 days intervals over a<br />
7-week period following treataent, cauaal epididiaal spera <strong>and</strong> testes of 3 aniaals were recovered .<br />
The nuaber of abnoraal spera head was 61 to 80 in the tiee points ranging froa 20 to 32 days post<br />
treataent <strong>and</strong> increased to -200 in the tiae points 36 to 49 days after treatsent . At ail others,earliers,<br />
tise points it was around the control nwber, i .e ., -10 abnorsal speras/1000 spera per anieal . The<br />
abnoreal sperm frequency to tiae after treataent parallel the unfertilized ova frequency curve for<br />
MC . In cross sections of testes tubules of these saae treated sice there was not evidences of cell-killing<br />
in sperwtocytes ; therefore, spersatocytes developed to spersatids but there was faulty diffirentiation<br />
of spersatozoa, rich appears to be a significant cause of failure of fertilization in sperutocytes .<br />
555<br />
ANTIMUTAGENIC EFFECT OF PROSTAGLANDINE IN `HUMAN LYMPHOCYTE CULTURES<br />
Sridevl,K . . K .P .Rao <strong>and</strong> K .V .Ch<strong>and</strong>rika . Depariment of Genetics . Osmania University .<br />
Ilyderabad-S00 007 . INDIA .<br />
Prostagl<strong>and</strong>in E .(PGE) . is a derivative of -linolenic acid, an essential fatty<br />
acid . PGE was tested for its antimutagenic activity !n invitro human lymphocyte<br />
cultures . korbol myristate acetate (PMA) at 200ng/ml was used as the clastogen .<br />
Whole blood cu,;tures were set up with Tc 1919 medium . Both the clastogen (PMA)<br />
<strong>and</strong> PGE1 (10 M) were added at the synthesis phase of the culture (48 hrs) .<br />
Cultures were harvested at 72 hours by hypotonic treatment followed by fixation .<br />
Slides were prepared <strong>and</strong> 100 metaphases were scored for various chromosomal<br />
aberrations like breaks . translocations . dicentrics etc . Three individual samples<br />
were studied to minimize the sample variation . The frequency of chromosomal<br />
aberrations was reduced with PGEI in PMA treated cultures, suggesting the protective<br />
role of PGE3 .<br />
556<br />
DETECTION OF MAMMALIAN CELL MD3AGENESIS IN AS52 CELLS . L .F . Stankowski, Jr ., W .G .<br />
Tuman, M.J . Bieszczad <strong>and</strong> K .F . McLaren, Pharmakon Research International, Inc .,<br />
Waverly, pA 18471<br />
The AS52/XPRT assay is quite similar to the more familiar CHO/HPRT assay with<br />
respect to its low spbntaneous mutant frequency <strong>and</strong> cytotoxic response to a variety of<br />
agents . AS52 cells contain a single, functional, stably-integrated copy of the E . coli<br />
qpt gene in a CHO cell line cont=ining a partial deletion of hprt, <strong>and</strong> have been used<br />
to detect mutation of 5Wt (to TG ) under conditions identical to those for hprt in<br />
CHO-KI-BH4 cells . Based upon the demonstrated or presumed carcinogenicity of<br />
approximately 40 agents compared concurrently in the two assays, the AS52/XPRT <strong>and</strong><br />
CHO/HPRT assays exhibit sensitivities of 100 <strong>and</strong> 80%, <strong>and</strong> specificities of 94 <strong>and</strong> 100%,<br />
respectively . Minor modifications further improve/simplify the AS52/XPRT assay . For<br />
example, recalculation of survival data in terms of relative cell viability [(relative<br />
cell density on Day) (relative cloning efficiency)1 provides a more accurate estimate<br />
of cytotoxicity . Transfer of AS52 cells from MPA to XAT medium for 24 hours prior to<br />
treatment prevents the poor growth occasionally observed after transfer to<br />
unsupplemented Ham's F12 . The 7-day phenotypic expression time can be reduced, due to<br />
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191
192 1989 EMS Abstracts<br />
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0<br />
.~<br />
OteS- 'the F~gradual decii3se~n population doubling time observed over the past few years .<br />
(This decrease is likely responsible for the somewhat higher spontaneous mutant<br />
frequencies recent,}y„~ved .) In addition, the use of semi-attached, or other<br />
subculture conditions without trypsinization, contributes to a further reduction in<br />
phenotypic expression time . These data lend further support to the use of AS52 cells<br />
as a more sensitive alternative to the CHO/HPRT assay .<br />
557<br />
FLUORESCENT LIGHT NUTAGENESIS IN SALMONELIJ~T~ ~RINORIUM AND 8SCldERICNIA COLI AS A MODEL<br />
FOR SUNLIGNT-INDUCF.D GENOTOXICITY . L .F . Sta owsnk ki, Jr ., D .B . Say, N .J . Sieszcsad,<br />
W .G . Tuman, K .F . McLaren <strong>and</strong> D .J . Mecca, Pharmakon Research International, Inc .,<br />
Waverly, PA 18471<br />
During evaluation of a photoactivated therapeutic agent, it was serendipitously<br />
observed that fluorespnt light alone yas quite mutagenio to some tester strains used<br />
in the Salmonella his <strong>and</strong> E . coli tM reversion assays . The strains were exposed (in<br />
a plate incorporation assay, without petri dish lids) to eight unshielded/unfiltered<br />
40-watt cool white fluorescent bulbs in a laminar flow hood . Treatment 4der thes;<br />
conditions for up to three hours produced time-dependent increases in his <strong>and</strong> trp<br />
reversion frequencies in strains TA98 <strong>and</strong> WP2 urA (3- to 6-fold) <strong>and</strong> strains TA100 <strong>and</strong><br />
WP2 uvrA pKM101 (10- to 15-fold), with a plateau generally observed after a 90- to<br />
120-minute exposure . Smaller/variable increases occurred in strains TA1S35 <strong>and</strong> TA1537,<br />
but none were observed in strains TA1538, TA102 or WP2 . Filtering the light through<br />
polystyrene lids did not completely abolish its mutagenicity observed in strains TA100<br />
<strong>and</strong> WP2 uvrA pKtt101 under st<strong>and</strong>ard plate incorporation conditions (without lids) . The<br />
presence of a photoactivated promutagen in the nutrient/top agars or overnight culture<br />
medium seems remote, since treatment in microtiter wells after washing <strong>and</strong> resuspension<br />
in phosphate buffer still elicits a mutagenic response . Thus, these cosmion tester<br />
strains may provide the basis for a simple, sensitive, inexpensive model to study<br />
genetic damage induced by natural sunlight, as indicated by the ability of consumer<br />
sunscreens to reduce the mutational response observed under st<strong>and</strong>ard plate<br />
incorporation conditions .<br />
MUTAGENESIS BY GLUTATHIONE AND OTHER THIOLS : MECHANISM AND RELEVANCE TO<br />
HEPATOCARCINOGENESIS . A .A . Stark, D .A . Pagano, <strong>and</strong> E . Zelger. Department of<br />
Biochemistry, Tel-Aviv n vOsTEy, Ramat-Aviv 69978, Tel-Aviv (Israel) ;<br />
Cellular <strong>and</strong> Genetic Toxicity Branch, N .I .E .H .S ., Research Triangle Park, NC<br />
27709 (USA) .<br />
The mechanism of glutathione (GSH) <strong>and</strong> other thiols' mutagenesis was<br />
Investigated (Stark at al ., Mut . Res . 177 :45-52, 1987 ; Carcinogenesis<br />
9 :771-777, 1988) . The activation of GSH to a bacterial mutagen 1s catalyzed<br />
by a single enzyme, y-glutamyltranspeptidase (GGT) . GGT cleaves the Yqlutamyl<br />
residue from GSH to form the reactive dipeptide cystetny191yctne<br />
(CG), which is mutagenic 1n the absence of GGT . The mutagenicity of GSH, CG,<br />
<strong>and</strong> other thiols depends on the pKa of the -SH group <strong>and</strong> the pH of the<br />
medtum . GSH <strong>and</strong> CG mutagenesis requires molecular oxygen, is enhanced by<br />
1ron, <strong>and</strong> Inhibited by lron chelators, radical scavengers, <strong>and</strong><br />
H2O2-metabollztng enzymes . Autoxidation of the thiolate anion generates the<br />
(pen)ultimate mutagen H202 . GSH can also drive lipid paroxldatlon in vitro<br />
<strong>and</strong> tn cultured cells tn the presence of GGT . Elevated GGT levels are often<br />
found in hepatic preneoplastlc foci . The high probability of their<br />
progression to hepatocarcinomas may thus be due to the formation of oxygen<br />
radicals by the GSH-GGT system tn the proximity of these foci .<br />
558<br />
559<br />
DEVELOPMENT OF MONOCLONAL ANTIBODIES RECOGNIZING 4-AMINOBIPHENYL-(SUANOSINE<br />
M . Stefanidis, D . W . Roberts, F . F . Xadlubar <strong>and</strong> R .M . Santella, National Center for<br />
Toxicological Research, Jefferson, AR (USA) <strong>and</strong> Columbia University, New York, NY (USA)<br />
Several aromal.ic aminee, including 4-aminobiphenyl, (4-ABP), have been found in<br />
cigarette smoke <strong>and</strong> are recognized as potent human carcinogens . Exposure to aromatic<br />
animns is believed to be a contributing factor to the increased incidence of bladder<br />
cancer in cigarette smokers . Previously, a polyclonal antibody was developed against<br />
the major 4-ABP--DNA adduct (Cancer Res . 48 :6336,1988) <strong>and</strong> used to monitor adducts in<br />
lung <strong>and</strong> bladder epithelial DNA of smokers (IARC publications i89,p .166, 1988) . To<br />
develop monoclonal antibodies to this adduct, BALB/c mice were issiunized with 4-ABPguanosine<br />
coupled to keyhole limpet hemocyanin . The two most sensitive clones, 6D4 end<br />
10114, were characterized as to sensitivity <strong>and</strong> specificity by competitive enzyme<br />
linked immunoaorbent assay (ELISA) . For both antibodies, 50% inhibitor concentration<br />
is in the range of 1 pmol of adduct . Neither antibody crossreacts with unmodified<br />
50869 3706
guanosine at the highest lonr.entration tested (1 .50 nmol/well) . Antibody 1oB4<br />
re•rofnizod the 4-Al1}'-guHnosine adducl, with similar sensit .ivity when it is present. in<br />
d~rn atured DNA or as the moaoadduct . A rompetitive ELTSA with fluorescence endpoint<br />
d~_•tecl.ion wus developed lo increase assay svnsitivlty (SOt inhibition at . 400 fmol) .<br />
SPnsitivity can be further increased by enzymatic digestion of DNA <strong>and</strong> isolation of<br />
ndducte by immunoaffinit .y chromcd ottraphy or HPLC before analysis . These antibodies<br />
will be used to monitor adduct levels in biological samples from smokers <strong>and</strong><br />
nonsmokers <strong>and</strong> r•valunted as a marker of evpusru-e• to aromat.ic aminos . This work wns<br />
supported by a grant from NCI-CA21111 .<br />
560<br />
BLEOMYCIN-INDUCED ABASIC SITES WITH CLOSELY OPPOSED STRAND BREAKS : STRUCTURE,<br />
SEQUENCE SPECIFICITY AND ROLE IN MUTAGENESIS R .J . Steighner <strong>and</strong> L .F . Povirk, Department<br />
of Pharmacology <strong>and</strong> Toxicology, Medical College of Virginia, Richmond, VA 23298<br />
Bleomycin-induced mutations in the lambda cI gene occur preferentially at hotspots<br />
which share the sequence C-G-C-C . When an end-labeled restriction fragment of _cI DNA<br />
was treated with bleomycin, <strong>and</strong> the resulting abasic (AP) sites were cleaved with<br />
putrescine, discrete shorter double-str<strong>and</strong>ed fragments were produced, whose<br />
electrophoretic mobilities were consistent with putrescine-dependent double-str<strong>and</strong><br />
cleavage at or near each of the mutational hotspots . The shorter double-str<strong>and</strong>ed<br />
fragments were eluted, denatured <strong>and</strong> run on sequencing gels . Analysis of cleavage<br />
sites <strong>and</strong> termini in each str<strong>and</strong> indicated that bivalent lesions consisting of a<br />
str<strong>and</strong> break at the C residue in the C-G-C-C sequence, plus an AP site at the G<br />
residue directly opposite, were formed by bleomycin at each hotspot . In separate<br />
experiments, bleomycin-induced mutagenesis of repackaged lambda phage was reduced 2to<br />
4-fold by treatment of the DNA with putrescine prior to repackaging . This<br />
reduction in mutation frequency may be attributable to cleavage of AP sites with<br />
directly opposed breaks, since endonuclease IV (which cleaved only "nonopposed" AP<br />
sites) had no effect . Our results strongly suggest that AP sites with directly<br />
opposed breaks are intermediates in bleomycin-induced mutagenesis . The potent<br />
mutagenicity of these lesions is probably attributable to the simultaneous loss of<br />
coding information at the same position in both DNA str<strong>and</strong>s . The sequence<br />
specificity for formation of these lesions was distinctly different from thsyt of<br />
bleomycin-induced single-str<strong>and</strong> breaks .<br />
561<br />
MUTAGENIC EFFECTS OF PHTHALATE ESTERS AND ASSESSMENT OF RISKS POSED<br />
TO THE ENVIRONMENT<br />
K . Strobel <strong>and</strong> T . Grummt, Research Institute for yyg iene <strong>and</strong> Microbiology,<br />
GDR - 9933 Bad Elster, H .-Heine-StraBe 12 (GDR)<br />
with an annual production of about one billion pounds phthalste esters<br />
are in wide use . In some plastic materials they make up to 50 %<br />
of volume . By now they are widely dispersed in the environment . Knowledge<br />
on mutagenic effects of phthalate esters is incomplete <strong>and</strong> partly<br />
controversial . Therefore, we investigated the mutagenic activity of<br />
i8 phthalate esters in the AMES-test <strong>and</strong> in cell cultures (FAF-celle<br />
of Chinese hamsters) . Mutagenic effects were detected in one or both<br />
test systems for a number of these substances . Their toxic, <strong>and</strong> partly<br />
mutagenic, end/or carcinogenic potential as well se the fact, that<br />
concentrations of e .g . DEHP <strong>and</strong> di-(n-butyl)phthelate in water samples<br />
<strong>and</strong> sediment were higher than those of DDT end PCBs, have to be considered<br />
in risk assessment for phthalate esters . The hazards which are<br />
posed by this group of substances to the environment give rise for the<br />
conclusion that guideline values for their presence in drinking water<br />
have to be elaborated, aspecielly in view of the revision of WHO Drinking<br />
water Guidelines which is being performed at present .<br />
562<br />
INHIBITORY EFFECT OF ASPARAGUS ON DMH-INDUCED MICRONUCLEI AND APOPTOSIS IN THE COLON<br />
CRYPT CELLS OF MICE . H . Sun, Q . Zhu, S . Fu, Y . An, G . Dou, B . Yang <strong>and</strong> Y . Wang . Henan<br />
Medical University, Zhengzhou, Henan 450052, People's Republic of China<br />
By assaying of micronuclei <strong>and</strong> apoptosis in the colon crypt cells of C57 mice, we<br />
studied the potential inhibitory effect of asparagus toward the intestinal carcinogen<br />
1,2-dimethylhydrazine (DMH) . The original liquid squeezed from fresh asparagus<br />
was applied as inhibitor in this study . Three groups (5 mice/group) were treated with<br />
asparagud liquid in the dosage of 0 .1 ml, 0 .5 ml <strong>and</strong> 1 .0 ml/ mouse respectively by<br />
stomach intubation, one hour before DMH were injected intraperitoneally . Mice injected<br />
with DMH only were positive control <strong>and</strong> mice injected with EDTA only were negative<br />
control . Twenty-four hours after infection with DMH, all the animals were sacrified .<br />
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1989 EMS Abstracts 193<br />
Notes
194 1989 EMS-Abstracts~ '"-aF`<br />
Notes<br />
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Sections of paraf%n-eiedded colo rolls were routinely made, <strong>and</strong> DNA damages were<br />
revealed by Feulgen"s'faMeg . Micronuclei <strong>and</strong> apoptosis were scored in 20 complete<br />
crypts for each sample with oil immersion microscope <strong>and</strong> the mean number of micronuclei<br />
<strong>and</strong> apoptosis of one crypt of each group was caculated . We found that the frequencies<br />
of micronuclei <strong>and</strong> apoptosis in the groups treated with asparagus liquid<br />
were obviously lower than in the group only in ected DMH . The number of micronuclei<br />
<strong>and</strong> apoptosis was 3 .99/crypt in only DMH treated group but was 2 .71-0 .76/crypt in<br />
groups treated both with DMH <strong>and</strong> asparagus . A clear dose-response relation was<br />
observed with different doses of asparagus liqutd . Our result indicated that asparagus<br />
might reduce the mutagenecity <strong>and</strong> carcinogenecity of DMH to colon crpt cells of<br />
mice .<br />
563<br />
EVALUATION OF GENOTOXIC POTENTIAL OF COMMONLY USED INSECTICIDES USING<br />
BARLEY (HORDEUM VULGARE) TEST SYSTEMS<br />
T .Suryakumari <strong>and</strong> K .Vaidyanath, Andhra University, Waltair (A .P) <strong>and</strong> Osmania<br />
University, Hyderabad-500 007 (A .P .) India .<br />
The modern agricultural practices entail intensive application of pesticides for<br />
plant protection, which are potential genotoxicants . To assess the mutagenic potential<br />
of Ekalux (00-diethyl 0-quinoxalin-2-yl phosphorothioate <strong>and</strong> Rogor (00-dimethyl<br />
S-methyl carbamoyl methyl phosphorodithiote) a study was made on barley using<br />
multiple end points . The cytogenetic end point consisted of screening for cytotoxic,<br />
chromotoxic <strong>and</strong> clastogenic effects on somatic chromosomes . The scoring for forward<br />
mutation at waxy locus (Wx -~ wx), chlorophyll deficient seedings !n M, <strong>and</strong><br />
polygenic variability in M, generations, constituted germinal mutation assay . The<br />
frequency <strong>and</strong> spectrum of cytological abnormalities observed in the study indicated<br />
mitodepressive, clastogenic <strong>and</strong> chromotoxic effects of the insecticides . The high<br />
frequency of sterile pollen <strong>and</strong> chlorophyll mutations elicited suggests mutagenic<br />
potential . However, both the insecticides failed to induce mutations at the specific<br />
locus . The polygenic variability noted in M . generation also suggests that Ekalux<br />
<strong>and</strong> Rogor are capable of inducing mutations for quantitative traits . The positive<br />
correlation observed between effects on somatic <strong>and</strong> germinal mutation suggests that<br />
any one or a com bination of assays can be used profitably for m onitor)ng genotoxic<br />
effect of environmental pollutants .<br />
DNA Repatr Induced by Various MutaQens in Rat Xepatocyte Primary Cultures<br />
Measured tn the Presence of Hydroxyurea, OuonasoJe or Aphidicolin<br />
W . Suter <strong>and</strong> F . Romagna, WSP Toxicology, S<strong>and</strong>oz Ltd ., CH 4002 Basel,<br />
Switzerl<strong>and</strong><br />
The goal of the present study was to find a selective inhibitor of DNA replication In rat<br />
hepatocyte primary cultures to replace hydroxyurea (HUI, which has been found to be<br />
genotoxic. Two agents were chosen for this study, ouanasole IoUI 18-amino-lH-1,2,4triazolel,<br />
which like hydroxyurea . is an inhibitor of ribonucleotide reductase, <strong>and</strong><br />
aphidicolin (API, an Inhibitor of DNA polymerase a. Using the nuclei procedure developed<br />
by Althaua (Cancer Res . 42, 8010-3016, 19821, DNA repair induced by UV irradiation<br />
. MNNO. MNU, H,O,, 6-hydroxydopamine. 4-nitroquinolirro-N-oxide, 2acetylaminofluorene,<br />
benzo/alpyrene, cyclophosphamide <strong>and</strong> aflatoxin B, was<br />
measured in the presence of either 10 mM hydroxyurea, 15 mM tuanazole or 0 .015<br />
mM aphidicolin . The latter was found to inhibit DNA repair in addition to its effect on<br />
DNA replication . In the presence of guanazole <strong>and</strong> hydroxyurea the overall sensitivity<br />
of the detection of DNA repair was similar . Aflatoxin B, <strong>and</strong> MNU were slightly more<br />
active In the presence of hydroxyurea, while MNNO was more eaily detected in the<br />
presence of guanazole.<br />
Thus . guanazole can be used in hepatocyte primary cultures instead of hydroxyurea for<br />
selective suppression of DNA replication .<br />
564<br />
SCRUTINY OFPROTOCOI S r-OR Ti il's MICRONUCLEUS TEST WITH MICE ---ACI IIEVEMENTS<br />
IlY CSGMT/JEMS .MMS, CSGMT, prcccnters : S . Sutou (Itoham Foods Inc ., Tokyo), H .<br />
Shimada (Daiichi Seiyaku Co .. Ltd., Tokyo), A . Wakata (Yamanouchi Pharm . Co ., Ltd .,<br />
Tokyo), T. Awogi (Oisuka Iharm. Co ., Lld ., Tokushima), A . Ohuchida (Taiho Pharm . Co.,<br />
Ltd., Tokushima), M . llayashi <strong>and</strong> 1' . Sofuni (Natl . Ins(. Ilygienic Sci ., Tokyo)<br />
The Mammalian Mutagenicity Study Group of the Environmontal Mulagcn Society of<br />
Japan (JEMS/MMS) has organized a collaborative study group for the micronucleus test<br />
50869 3708<br />
565
f 1989 EMS Abstracts 195<br />
Notes<br />
(CSGMT) to scrutinize protoc,Qts <strong>and</strong> to reach a st<strong>and</strong>ard protocol so that all participants<br />
can obtain data under optimal conditions, <strong>and</strong> thus results can be compared regardless<br />
of time, place, <strong>and</strong> person . Firstly, we examined sex-related difference in the test <strong>and</strong><br />
suggested that the use of male mice is sufficient for general screening (Mutation Res .,<br />
172, 151, 1986) . Secondly, we examined strain-related difference <strong>and</strong> concluded that<br />
any strain could be used as a tester, though MS/Ae tended to show the highest<br />
response, especially at higher doses (op . cit ., 204, 307, 1988) . Thirdly, administration<br />
route (ip <strong>and</strong> po)-related difference was exaaained ; the results will be presented<br />
separately in the present meeting . Fourthly, dosing times <strong>and</strong> sampling timing are now<br />
under study . Some preliminary results are presented <strong>and</strong> discussed .<br />
566<br />
MATERNAL INHERITANCE OF ms GENE, S . Sutou <strong>and</strong> S . Sato<br />
(Central Research Institute, Itoham Foods Inc ., 1-6-21 Mita, Meguro,<br />
Tokyo, 153 Japan)<br />
MS/Ae mice, which are mutagen-sensitive in both the dominant lethal<br />
test <strong>and</strong> micronucleus test, <strong>and</strong> CD-1 mice, which are the parental strain of<br />
MS/Ae, were mated in all four possible combinations . Both male <strong>and</strong><br />
female offspring were subjected to the micronucleus test using mitomycin<br />
C(MMC), colchicine (Col), <strong>and</strong> 6-mercaptopurine (6-MP). Col showed<br />
equivocal results . However, MMC <strong>and</strong> 6-MP clearly showed differential<br />
responses in that both male <strong>and</strong> female offspring from CD-1 dams had<br />
lower incidences of micronucleated polychromatic erythrocytes than those<br />
from MS/Ae dams regardless of sire strains . In addition, body weights of '<br />
offspring from MS/Ae dams were lower than those from CD-1 dams<br />
regardless of sires . These results strongly suggest that 1he ms gene (or<br />
genes), which regulates the characteristics of MS/Ae, is maternally inherited .<br />
567<br />
IN VITRO tdICRONOCLEI IN THE MOUSE MAMMARY EPITSSLIAL C$LIS GROWN UNDER<br />
SERIIM-FREE CULTURE CONDITIONS<br />
Kunio ,Suzuki <strong>and</strong> Tomotari Mitsuoka, Animal <strong>and</strong> Cellular Systems<br />
Laboratory, <strong>and</strong> Frontier Research Programs, The Institute of Physical<br />
<strong>and</strong> Chemical Research (RIKEN), Wako, Saitama 351-01, Japan .<br />
Although epidemiological studies suggest that dietary factors are<br />
associated with breast carcinogenesis, the specific carcinogen involved<br />
in cancer of the breast remains to be identified experimentally . In an<br />
attempt to establish an in vitro short-term test for breast carcinogens,<br />
we studied several known carcinogens tested by micronucleus assay in the<br />
primary cultures of mouse mammary epithelial cells . Mammary epithelial<br />
cells from 2 month old ICR virgin mice were cultured on collagen gel in<br />
serum-free Ham's F-12/Dulbecco's modified Eagle's medium supplemented<br />
with insulin, bovine serum albumin, epidermal growth factor, transferrin<br />
<strong>and</strong> cholera toxin . At day 6 of culture, known chemical carcinogens were<br />
added to the cultures, <strong>and</strong> the number of micronuclei per 1,000 cells was<br />
scored at 24 hr after treatment . Two breast carcinogens, g-methyl-gnitrosourea<br />
(MNU) <strong>and</strong> 7,12-dimethylbenz[a]anthracene (DMBA), increased<br />
the micronuclei incidence in the cultures . The cells grown in this<br />
serum-free medium metabolized DPIBAA to the activated form . These results<br />
suggest that the .quantitation of micronuclei in the mouse mammary<br />
epithelial cells cultured under serum-free conditions might be used as<br />
an in vitro short-term screen for breast carcinogens .<br />
568<br />
CANCER PROMOTERS AND THEIR EFFECT ON THE MUTAGENICITY OF MUTACARCINOGENS<br />
Dalisay Chionglo Sy, U .of Santo Tomas, Fac .of Medicine,Dept of Biochem .<br />
Studies have indicated the cancer-promoting activities of phenobarbital(pheno)<br />
<strong>and</strong> saccharin (sacl . The possibility that their effects occur<br />
thru enhancement of a pre-existing mutagenic condition was tested using<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
196 1989 EMS Abstracts<br />
~ . . .r -- . ..<br />
Notes- -<br />
.$c~ .-{~im id's Micromuel~eu.cs Test . Nine to 10 weeks old Strong A male mice were<br />
separately treated i,p with 2 mutacarcinogens : dimethylnitrosamine(DMN),<br />
10 mg per kg_$_V,_~mitomycin C (mito C),3 mg per kg BW . An hour after<br />
injection of the mutacarcinogens,pheno,90 *g per kg BW <strong>and</strong> sac,2 .S gm per<br />
kg BW were given to the animals treated with mutacarcinogens via the<br />
same route . Pheno was injected to the DMN-treated group <strong>and</strong> sac to the<br />
group receiving mito C . Bone marrow cells from femora of animals were<br />
isolated using fetal calf serum for suspension . Cells were collected by<br />
low speed centrifugation <strong>and</strong> smears prepared . Slides were stained using<br />
May Grunwald-Giemsa stain, Animals that received pheno <strong>and</strong> sac after injection<br />
with mutacarcinogens showed -inereased production of micronuclei .<br />
The mito C-sac combination showed peak micronuclei formation when sac was<br />
given 3h after injection of mito C . Statistical analysis showed significance<br />
at p= .05 . Results agree with earlier studies showing increased germ<br />
cell toxicity of DMN <strong>and</strong> mito C after treatment with pheno <strong>and</strong> sac . Both<br />
findings suggest that pheno <strong>and</strong> sac could exert their cancer-promoting<br />
actions by enhancing the mutagenicity of mutacarcinogens in somatic as<br />
well as germ cells . Whether all cancer promoters act in like manner<br />
remains to be seen,<br />
GENOTOXIC ACTIVITIES OF 3-CHLOROPROPIONIC ACID AND RELATED COMPOUNDS<br />
M . SZEGEDI<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Chemical Works of Gedeon Richter Ltd . Microbiological Research Laboratory<br />
H-1475 Budapest, 10 . P .O .B . 27 . Hungary<br />
The 3-cloropropionic acid (3CPA) has a strong genetoxic activity<br />
in Salmonella typhimurium TA 1535 nd TA 100 strains . We have tested<br />
a group of chemicals - withouth metabolic activation - in the E .coli<br />
SOS chromotest . They were similar to 3CPA different in the lenght of<br />
the carbon chain <strong>and</strong> the subtituens as follows : propionic acid,<br />
propionyl chloride, 2-chloropropionic acid, 3-chloropropionic acid,<br />
2-chloropropionyl chloride, 3-chloropropionyl chloride, chloroacetic<br />
acid, 2-chioropropane, 1-chlorobutane, 4-chloro-l-butanol, 1-chloropropane,<br />
1-chloro-2-propanol, 3-chloro-propionitrile,<br />
3-chloropropionitrile . The experiments were carried out in BIOSCREEN<br />
Analyzing System using BIDSOS program (LABSYSTEMS Ltd .) . Positive<br />
response was given only by 3CPA <strong>and</strong> 3-chlorpropionY1 chloride<br />
suggesting that C1-CH2-CH2C0- is resposible for activity .<br />
569<br />
570<br />
EVALUATION OF THE CLASTOGENIC ACTIVITY OF ROCBAGAN (BENZNIDAZOLE) IN<br />
MAMMALIAN SYSTEMS .C .S .Takahashi,S .C .Souaa <strong>and</strong> S .J .Santos, F .F .C .L .R .P .,<br />
USp <strong>and</strong> F .M .R .P .,USP,Ribeirao Preto,Sao Paulo,Brazil .<br />
Rochagan, a drug whose active compound is benznidazole(N-benzil-2 ni<br />
tro-l-imidazolacetamida), is used for the treatment of Chagas'disease<br />
<strong>and</strong> has been effective as a trypanosomicide . The drug was tested for<br />
clastogenic activity in Wistar rats treated by gavage at doses of 50 to<br />
1000 mg/kg body weight administered three times at 8 h intervals <strong>and</strong> in<br />
human lymphocyte cultures at doses of 250 pg/ml culture medium .Six rats<br />
were used in each treatment <strong>and</strong> sacrificed 6, 12 <strong>and</strong> 18 h after the<br />
last treatment . One hundred bone marrow metaphase cells per animal were<br />
analyzed for chromosome aberrations,showing frequencies similar to control<br />
values for all treatments (0 .30 to 1 .66Z) . For the in vitro test,<br />
blood was obtained from 5 healthy donors <strong>and</strong> benznidazole wasa ded at<br />
the beginning of culture .One hundred metapbases per individual were ana<br />
lyzed for chromosome aberrations <strong>and</strong> 50 metaphases for SCE . The frequen<br />
cies of chromosome aberrations <strong>and</strong> SCE were 3 .2x <strong>and</strong> 8 .01 SCE/cell in<br />
control cultures <strong>and</strong> of 62 <strong>and</strong> 13 .89 SCE/cell in treated cultures .A<br />
slight increase in SCE was observed in the treated group .The lack of<br />
positive results could be explained by the absence of reduction products<br />
. These reactive metabolites were observed under anaerobic conditions<br />
which does not occur in the systems used .<br />
50869 3710
571<br />
: 1989 EMS Abstracts 197<br />
Notes<br />
COMPARATIVE CLASrOGENIC EFFECTS OF ORGANIC AND INOROANIC TIN SALTS<br />
IN VITRO JP:.<br />
Geeta TALUKDER,Ban~ B<strong>and</strong>ana Ohosh,Archana Sharma<br />
Human Genet cs Unit,Centre for Advanced Study in Cell a Chromosome<br />
Research,Department of eotany,University of Calcutta,35 BallY9unj<br />
Circular Road,Calcutta 70001l .India .<br />
The clastogenic response of human lymphocytes ;S was studied<br />
following the administration of stannic chloriae <strong>and</strong> 2oiuq/ml)<strong>and</strong><br />
trimethyl tin(0 .5 <strong>and</strong> 7juy/ml) .Blood was obtained from healthy donors<br />
of both sexes of six age groups .The end points screened after 72 hours<br />
were mitotic index(MI),chromosomal aberrations(CA)both with <strong>and</strong><br />
without gaps <strong>and</strong> micronuclei(MN),Sister chromatid exchanges(SCE) <strong>and</strong><br />
cell cycle kinetics(c:CK)were recorded after both 48 <strong>and</strong> 72 hours in<br />
culture . Both tin salts,analysed statistically,increased the frequency<br />
of CA,MN <strong>and</strong> SCE to a statistically significant level <strong>and</strong> depressed<br />
MI <strong>and</strong> CCK when compared to the untreated control . The effects were<br />
directly proportional to the concentrations used . The percentages of<br />
MN <strong>and</strong> CA were significantly higher in cultures from donors of older<br />
age groups(above 50 years) as compared to the lower ones . There was<br />
no difference between the relative effects of the two salts or<br />
between the cultures from the two sexes . SCE was significantly higher<br />
in teeated cases <strong>and</strong> in blood from persons who smoked .<br />
572<br />
Cytogenetic study of gossypol acetate<br />
Tan Yongbu, Zhang Zhongshu <strong>and</strong> Wang Renli<br />
Shanghai Institute of Planned Parenthood Research,<br />
2200-4 Xietu Road, Shanghai PRC, 200032 ,<br />
r<br />
Gossypol, a yellowish phenolic compound isolated from seeds, stems <strong>and</strong> roots<br />
of cotten plant,has been advocated by the Chinese scientists as a antifertility<br />
agent for males . A<br />
Clinical trials indicated that the antifertility effect of gossypol was<br />
exellent, but occasional cases of hypokalemic paralysis might occur in the<br />
course of its administration .<br />
In the present work the effects of gossypol on the concentration of some of<br />
micronucleus, chromosomal abrration, SCE, gene mutation in somatic/reproductive<br />
cells <strong>and</strong> dominant lethal mutation, dose-response relationships are observed in<br />
rats, mice <strong>and</strong> men .<br />
The results indicate that gossypol of antifertility agent for males at lower<br />
dosages may be safe, but it should not be neglected that higher dosages might<br />
disturb or damage the DNA synthesis .<br />
573<br />
In situ detection of induced mutations with chemicals by Tradescantia<br />
TANO, S . Faculty of Agriculture, The University of Tokyo, Bunkyo-ku,<br />
Tokyo 113, Japan<br />
Tradescantia stamen hair cells are highly sensitive to the induction<br />
of somatic mutations by chemical <strong>and</strong> physical mutagens . Five kinds of<br />
chemical mutagens (EMS, NMU, NEU, NDEA, <strong>and</strong> NDMA) were tested for the induction<br />
of somatic mutations with special regards to the dose-response<br />
relationships <strong>and</strong> minimum effective dose . Three different kinds of doseresponse<br />
relationships were obtained depending on the variety of chemicals<br />
. Linear dose-response was observed with EMS treatment . In the case<br />
of NDEA, the mutation frequency increased sharply at the low dose range,<br />
but it saturated at the high dose range . In the case of NDMA, there was<br />
a slow increase at the low dose range <strong>and</strong> a sharp increase at the high<br />
dose range . Similar differences in dose-response relationships between<br />
ethylating agent (NEU) <strong>and</strong> methylating agent (NMU) were also observed as<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
198 1989 EMS Abstracts<br />
_,_-- - .-<br />
Notes "`i`A"'the case bgtw&WINDEA <strong>and</strong> NDMA . With regard to detectability of minimum<br />
dose foEr a chemical using Tradescantia stamen hair system, this depends<br />
upon the kigl'6of chemical <strong>and</strong> test clones . It may be stated that<br />
an effective-8ase`for inducing mutations is in the order of picograms of<br />
chemicals per flower . Therefore, Tradescantia may be possible to use as<br />
one of the indicators for risk evaluation of chemical mutagens .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
MUTAGENIC EFFECTS OF ENVIRONMENTAL RADON<br />
Tavera L ., BalcBzar M ., de la Rosa M . E ., 2immering S .*<br />
ININ . Sierra MoJada N447 . 2 y 3er . pisos . Col . Lomas Barrilaco C .P .11010 .MEXICO<br />
*Brown University, Providence U .S .A .<br />
574<br />
The internal radon progeny exposure exhibits a distinct maximum dose In the respiratory<br />
tract Inducing lung cancer .<br />
From epidemiological data available only a statistical significance Increase of cancer<br />
frequency with Increasing background radiation can be deduced . Because of these data<br />
are given for different ages <strong>and</strong> radiation environments a Radon Chamber was desloned to<br />
exposure bioassays for searching effects other than cancer induced by radon exposure .<br />
The radon atmosphere in the chamber reach 97% of saturation after 19 days, however it<br />
is possible to determine an equivalent average radon concentration by means of Solid<br />
State Track Detectors . The Radon daughters assessment Is also possible with these detectors<br />
.<br />
Samples of Drosophila exposured in this Radon Chamber have shown inter-<strong>and</strong>-Intraspecie<br />
differences in sensibility when fecundity <strong>and</strong> viability were measured . Additionally a<br />
very typical aberrant phenotype was induced in D . melanogaster wild-type .<br />
575<br />
GENOTOXICITY ASSESSMENT OF QUINAPRIL, A NEW ANTIHYPERTENSIVE DRUG . J .C . Theiss, N .L .<br />
Kropko, G . Krishna, <strong>and</strong> V . Ciaravino, Parke-Davis pharm . Res . Div ., Warner-Lambert Co .,<br />
Ann Arbor, MI(USA)<br />
Quinapril was assessed for genotoxicity in a variety of test systems . This drug was<br />
not mutagenic toward S . typhimurium (10,000 ug/plate highest dose tested) <strong>and</strong> did not<br />
increase the mutant or SCE frequency in V79 cells (1400 ug/ml highest concentration<br />
tested) . Quinapril did not induce micronuclei in mice (1430 mg/kg high dose-80% mouse<br />
LD50) nor did it induce structural chromosomal aberrations (SCAs) in rats (2130 mg/kg<br />
high dose-rat LD10) . Thus, quinapril was not clastogenic in vivo at doses far in excess<br />
of the maximum human dose of 1 mg/kg . There was a slight but statistically significant<br />
dose-related increase in SCAs in V79 cells treated with quinapril for 3 hours which<br />
occurred in the presence of S9 only <strong>and</strong> at 18 hours post-exposure only (12, 18, 24, <strong>and</strong><br />
40 hour time points) . The maximun frequency of cells with SCAs detected (6t) was within<br />
the historical control range for this laboratory (0-6 .31 cells with SCAs, n-31) <strong>and</strong> the<br />
minimum concentration at which an increase in SCAs occurred (1400 ug/ml) was cytotoxic<br />
(
ackground peak respons ) . SCA frequency was increased by four quinolones, PD 117579<br />
(10 ug/ml minimal clasto enic concentration, 4 .5-fold background peak response) . PD<br />
117962 (45 ug/ml minimaclastogenic concentration, 9 .3-fold background peak response),<br />
ciprofloxacin (400 ug/ml minimal clastogenic concentration, 8 .9-fold background peak<br />
response), <strong>and</strong> ofloxacin (1800 ug/ml minimal clastogenic concentration, 6 .3-fold<br />
background peak response) . Norfloxacin <strong>and</strong> nalidixic acid were negative for all three<br />
endpoints . Thus, the SCA test is the moat sensitive of the three endpoints assessed .<br />
However, literature reports indicate that the two quinolones that increased SCA<br />
frequency in vitro only at high concentrations (ciprofloxacin, ofloxacin) failed to<br />
exhibit clastogenic activ-ity-in animals yr man, indicating that these high<br />
concentration in vitro effects may be of limited biological significance . Thus, an in<br />
vitro assessment of the clastogenic activity of quinolones should be coupled with an in<br />
vivo test to assess the biological significance of any in vitro activity .<br />
577<br />
Measurement of Mutational Spectra in Human Tissues<br />
William G . Thilly, Phouthone Keohavong, Neal Cariello, John Hanekamp<br />
Center for <strong>Environmental</strong> Health Sciences<br />
MIT E18-666 Cambridge, MA 02139<br />
Application of mutational spectra to diagnose the causes of genetic change in humans<br />
will be discussed . Recent advances in technology, including PCR improvements,<br />
designed to obtain such spectra from human cells <strong>and</strong> tissues will be reviewed .<br />
Topics to be covered are signal/noise requirements, multi-copy sequences <strong>and</strong> means<br />
to obtain spontaneous spectra from individuals .<br />
Sponsored by U .S . Department of Energy Office of Health <strong>and</strong> <strong>Environmental</strong> Research<br />
<strong>and</strong> the U .S . National Institute of <strong>Environmental</strong> Health Sciences. •<br />
578<br />
LACK OE' CYTOGENETLC EFE'E:CTS IN BONE MARROW OF TWO STRAINS OF MICE CHRONICALLY EXPOSfSD<br />
TO CLGARETTE SMOKE . M .A . Thomas*, D . Gulati**, J .P . Wojciechowski**, P . Sabharwal**,<br />
<strong>and</strong> C .G . Gairola* . *Graduate Center for Toxicology, <strong>and</strong> <strong>Tobacco</strong> <strong>and</strong> Health Research<br />
Institute, University of Kentucky, <strong>and</strong> **Rnvironmental Health Research <strong>and</strong> Testing,<br />
Inc ., Lexington, KY .<br />
This study was conducted to determine if chronic exposure to cigarette smoke induces<br />
cytogenetic damage in the bone marrow of aryl hydrocarbon hydroxylase- (AHH) inducible<br />
(C57BL/6J) <strong>and</strong> non-inducible (DBA) strains of mice . Using a nose only exposure system<br />
groups of female mice were chronically exposed (50 - 70 weeks) twice daily to mainstream<br />
(ttS) or sidestream (SS) cigarette smoke, from high-tar/high-nieotine University<br />
of Kentucky Reference cigarettes (2R1) . Room (RC) <strong>and</strong> sham control (SH) groups were<br />
also examined . Pulmonary AHH activity was increased 2-3 fold in C57BL but not DBA<br />
mice . Blood carboxyhemoglobin (%COHb) levels were increased approximately 11 <strong>and</strong> 22<br />
fold for MS <strong>and</strong> SS groups, respectively, in both strains . Average intake values for<br />
smoke total particulate matter were 17 .1 <strong>and</strong> 6 .9 mg/kg per exposure-session, for MS<strong>and</strong><br />
SS- exposed groups, respectively . These dats confirmed effective inhalation of<br />
smoke by exposed animals . Two cytogenetlc endpoints, sister-chromatid exchange (8Cg)<br />
<strong>and</strong> micronucleus (MN) formation, were examined in isolated bone marrow cells . tlnder<br />
these exposure conditions neither MS nor SS cigarette smoke induced an increase in<br />
frequency of SCE or MN in either strain of mice . (Supported by KTRB 41031) .<br />
579<br />
GENETIC ANALYSIS OF HUMAN DNA REPAIR GENES, L. H . Thompson, C . A . Weber,<br />
K .W. Brookman, N .J . Jones, E .P . Salazar, <strong>and</strong> M .J. Sicilianot, Biomedical Sciences Division, Lawrence<br />
Livermore National Laboratory, P.O. Box 5507, Livermore, CA 94550 <strong>and</strong> tDepartment of <strong>Molecular</strong><br />
Genetics, University of Texas System Cancer Center, Houston TX 77030<br />
Human genes that control DNA repair were identified by complementing repau-deficient hamster mutant<br />
lines with human chromasomes in hybrid cells or by transfecting with human DNA . Nucleodde excision<br />
repair (NER) genes, which control UV radiation sensidviry, are located on human chromasomes 2,13,16,<br />
<strong>and</strong> 19 . A total of eight complementadon groups of NER genes are now identified among rodent mutants .<br />
The human ERCC2 (Excision Repair Crass Complementing) gene, which corrects CHO mutant WS <strong>and</strong><br />
was previously cloned in cosmids, was used to isolate homologous cDNA ciones from Okayama's pcD2<br />
expression library. Nucleotide sequencing of an incomplete cDNA clone <strong>and</strong> ERCC2 S' genomic<br />
sequences identified an open reading frame . The protein coding region of ERCC2 has a atrildng 5296 a a .<br />
identity with the RAD3 gene in yeast This similanty suggests conservadon of function <strong>and</strong> the possibiliry<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A<br />
1989 EMS Abstracts 199<br />
Notes
200 1989 EMS Abstracts<br />
Notes . ,,that.ERCC2 is requiiedAsreeU viability as well as repair, as in the case of RAD3 . The gross underrepresentation<br />
of CHO mutants recovered in the ERCCZ complementation group upon treatment with the<br />
frame shift agent ICR170rther suggests an essential function. Two other human genes, XRCCJ (X-ray<br />
Repair Cross Compl XRCC2, which help repair Ionizing radiation damage, were assigned to<br />
chromosomes 19 <strong>and</strong> 7, respectively . Analysis of cosmid clones of XRCCJ shows that this gene, which is<br />
33 kb in length efficiently corrects CHO mutant EM9 . The phenotype of EM9 is defective rejoinin~ of<br />
str<strong>and</strong> breaks <strong>and</strong> greatly elevated sister chromatid exchange (SCE) . The cDNA clone pXRI-30, obtauted<br />
from the pcD2 library, gave stable but incomplete correction of EM9 . Nucleodde sequencing showed that<br />
this clone lacked 26 nucleotides of protein coding region. The XRCCI protein, which appears to be 633<br />
a.a. in length, remains to be identified in terms of itsbiochemicai function in repair <strong>and</strong> SCE . This work<br />
was done under the auspices of the U.S . Dept. of Energy by 1.LN1, under contract No . W-7405-ENO-48 .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
580<br />
MOLECULAR APPROACHES TO CARCINOGENESIS : ONCOGENE INDUCED TRANSFORMATION AND LINEAGE<br />
SWITCHING OF RAT LIVER EPITHELIAL CELLS . Snorri S . Thorgeirsson, Laboratory of<br />
Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute,<br />
National Institutes of Health, Bethesda, MD 20892<br />
Tumors produced by a chemically transformed rat liver epithelial (RLE) cell line<br />
<strong>and</strong> its single cell-derived clonal subpopulations demonstrate wide-ranging morphological<br />
presentations including carcinomas, sarcomas, "mixed epithelial-mesenchymal"<br />
tumors, <strong>and</strong> undifferentiated tumors (Am . J . Pathol . 127 : 168-181, 1987) . To address<br />
the question of heterogeneity of tumors er ve rom transformed RLE cells, we have<br />
used recombinant retroviruses containing the following transforming oncogenes :<br />
v-raf (3611-MSV), v-raf/v-myc (J2), v- c(J5), <strong>and</strong> v-Ha-ras (pRNR16) . A11 of the<br />
oncogenes, with the exception of v-styc~J5), were efficient transforming agents in<br />
the RLE cells . Tumors derived from the v-raf- <strong>and</strong>, to a lesser extent, those from<br />
v-Ha-ras-transformed RLE cells showed mixed epithelial-mesenchymal morphology,<br />
whereas the combination of v-raf/v-myc (J2) consistently produced differentiated<br />
trabecular carcinomas . These data suggest that the lineage commitment of the RLE<br />
cells can be perturbed by a single transforming oncogene <strong>and</strong> that different tumor<br />
types derived from these cells may reflect the expression of a selective oncogene<br />
or a combination of oncogenes .<br />
581<br />
METABOLISM OF1-NITROPYRENE TO A MUTAGEN IN CAO CELLS BY NITROREDUCTION . Janice R .<br />
Thornton-Manning, Beverly A . Smith, Roberta A . Mittelstaedt, Frederick A . Bel<strong>and</strong> <strong>and</strong><br />
Robert H . Heflich, University of Utah, Salt Lake City, UT (USA), <strong>and</strong> National Center<br />
for Toxicological Research, Jefferson, AR (USA)<br />
Nitroreduction of 1-nitropyrene (1-NP), a tumorigenic environmental contaminant, to<br />
N-hydroxy-l-aminopyrene (N-hydroxy-l-AP) is an important pathway for DNA adduct formation<br />
in vivo . In previous studies, treating Chinese hamster ovary (CHO) cells with<br />
1-NP for up to 5 hr produced low levels of a DNA adduct indicative of nitroraduction,<br />
but no mutants were induced . In this study, CHO-KI-SH4 cells <strong>and</strong> repair-deficient CHO<br />
UV5 cells were incubated with 40 uM 1-NP for up to 24 hr . While none of the CHO-R1<br />
treatments induced mutants, treatment of UV5 cslls for 12 <strong>and</strong> 24 hr induced 5 <strong>and</strong> 12<br />
mutants/106 clonable cells (different from control at p
ultraviolet irradiation o'f DT resistance in cultures of the transformed,<br />
xeroderma pigmentosum complementation group-A cell line XP20S(SV40), <strong>and</strong> of<br />
the Chinese hamster ovary ~CHO) repair-defective mutant 43-3B . In both<br />
cultures, there was a do~e-dependent increase in the frequency of toxinresistant<br />
cells . On an equal-dose basis, higher frequencies were observed in<br />
the repair-deficient cell lines than in their respective repair-proficient<br />
controls, GM637(SV40) <strong>and</strong> CHO-9 . At equal levels of survival, however, the<br />
frequencies of DTs cells were similar in the repair- defi~ient <strong>and</strong> proficient<br />
cells . These result provide further evidence that the DT" cells detected by<br />
the autoradiographic assay are indeed mutants . They also indicate a simple<br />
method of studying various aspects of mutation in cells that, because of e .g .<br />
defective DNA-repair, are hard to clone <strong>and</strong> use in a colony assay . (Work<br />
supported by the Israel Cancer Association .)<br />
583<br />
SOFTWARE DEVELOPMENT FOR MICRONUCLEUS (MN) SCORING . R .R . Tice, J .T . MacGre9or*, C .<br />
Campfield, A . Pellom, L . Williams** <strong>and</strong> C .H . Nauman**, Integrated Laboratory Systems,<br />
PO Box 13501, Res . Tri . Park, NC 27709, *Toxicology Consulting Services . Inc ., 383<br />
Diablo Rd, Suite 100, Danville, CA 94526, <strong>and</strong> **EPA/EMSL, P0 Box 89193, Las Vegas, NV<br />
89193 .<br />
Based on input from a panel of experts in the field, a software program, suitable<br />
for an IBM compatible PC, has been developed for the collection <strong>and</strong> analysis of<br />
micronuclei data obtained from in vtvo test systems . Experimental design information<br />
<strong>and</strong> MN data obtained on a number of cell types are easily entered using a series of<br />
menus, statistically analyzed by a variety of statistical models <strong>and</strong> presented both<br />
in tabular <strong>and</strong> graphic form . In the statistical analysis, the data are evaluated for<br />
scorer, sex, treatment, sample time, <strong>and</strong> animal effects . The use of this software<br />
will help to st<strong>and</strong>ardize in vivo MN data analysis <strong>and</strong> presentation . Although the<br />
research described in this article has been supported by the U .S . EPA through<br />
contract number 68-C8-0068 to Integrated Laboratory Systems, it has not been<br />
subjected to Agency review <strong>and</strong> therefore does not necessarily reflect the views of<br />
the Agency <strong>and</strong> no official endorsement should be inferred .<br />
.<br />
584 •<br />
ANALYSIS OF MOUSE MICRONUCLEI BY HIGH SPEED FLOW CYTOMETRY .<br />
A. M . Tometsko, Litron Laboratories, Rochester, N. Y . (USA)<br />
The mouse micronucleus assay is widely used to evaluate the clastogenic activity of<br />
chemicals . The essay involves scoring the number of cells containing micronuclei (MNS) In<br />
either blood or bone marrow preparations using fluorescent or bright field microscopy . The<br />
conventional assay is labor intensive end tedlus, <strong>and</strong> requires many hours of microscopic<br />
examination to complete an assay. High speed flow cytometry provides the means for<br />
analyzing fluorescent cells as they pass through a laser beam at 2,000 cell per second <strong>and</strong><br />
permits the analysis of 30-50 times more cells then manual screening tn e shorter time. For<br />
FCM analysis, the cells are stained with fluorescent dyes which effectively label cellular<br />
nucleic acids (e .g. MNs) . Experiments will be presented which highlight the distribution of<br />
micronucleated cells in peripheral blood of normal <strong>and</strong> ctasto0en treated mice. The location of<br />
MNs in different blood cell populations will be shown <strong>and</strong> will provide en indicetion of the<br />
feasibility of developing an automated FCM based analysis protocol• The distribution of MNcells<br />
will be presented using histograms <strong>and</strong> bivariate graphs. Correlations will be made<br />
between manual scoring <strong>and</strong> analysis by high speed flow cytometry .<br />
585<br />
COMPARISON OF IN VIVO SOMATIC CELL MUTATION, CHROlie/SOME ABERRATION, SISTER CHROMATID<br />
EXCHANGE, MICRONUCLEI FORMATION AND URINE MUTAGENICITY IN STEEL FOUNDRY WORKERS . D .J .<br />
Tomkins, D .R . McCalla, <strong>and</strong> E .S . Gibson, McMaster University <strong>and</strong> Dofasco Inc .,Hamilton,<br />
Ontario, Canada .<br />
Preliminary results of genetic toxicologic monitoring of 125 steel foundry workers<br />
with a higher rate of lung cancer have been reported (Environ . Mutag . 7(S .3) : 33,1985) .<br />
Chromosome aberration (CA), sister chromatid exchange (SCE), <strong>and</strong> morning <strong>and</strong> afternoon<br />
urine mutagenicity (MUTAM,MUTPM), but not micronuclei (HN), were all significantly<br />
elevated with smoking (p- .001, .004, .008, .005 respectively), but not with occupation .<br />
The somatic cell mutation test (SCMT) has now been completed for 37 workers <strong>and</strong> the<br />
statistical analysis did not show any significant effects of smoking or occupation .<br />
In the case of CA, there was a significant interaction between smoking <strong>and</strong> occupation,<br />
particularly when gaps were not included in the total number of aberrations per cell<br />
(p- .01) . When specific types of abnormalities were analyzed separately, breaks<br />
appeared to be the moat important contributors to the effect of the interaction . Two<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 201<br />
Notes
202 1989 EMS Abstracts<br />
Notes important conf.unieta -were identified : age was positively correlated with CA, !IN <strong>and</strong><br />
. . ° .-aelayed/proloapediEWl'division in 48 hr culturas, <strong>and</strong> drug exposure significantly<br />
affected IiUTPW<strong>and</strong> CA . The different genetic tests were often correlated with each<br />
other . In partic . ,HUTAH was positively correlated with CA (p- .02), MUT'PN with CA,<br />
SCE, <strong>and</strong> SCH1 , .04, .01 respectively) . On the other h<strong>and</strong>, SCE <strong>and</strong> SCMf may<br />
have been negatively correlated (p- .05), <strong>and</strong> there was no correlation between CA, SCE<br />
<strong>and</strong> ALN . This suggests that urine mutagenicity may measure the metabolic products of<br />
a genotoxic exposure, but that the types of genetic damage detected by the lymphocyte<br />
tests may vary from subject to subject .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
MAMMALIAN DNA LIGASE I : THE MOLECULAR DEFECT IN BLOOI,fS SYNDROME<br />
Alan E . Tomidnson, Dena D. Lasko, Ams E . WNia <strong>and</strong> Tarnas Lirqalt<br />
rnperml Caneer Research Fund, Clare Hall Laborabries, South t,6mms, Herts ., EN6 SLD, U.K.<br />
Mammalian cells contain two clistinct DNA i0ases. The major aclivity In proUteratkp oeNS <strong>and</strong> therefore prohabfy the enzyme<br />
involved in DNA replioation Is the NOh mdealar weipAt enzyme. DNA Npue I. A 1dOkd brm of frs enzyme has been purilkd b<br />
~ ope~ ~~ thymus <strong>and</strong> tunan oeos . Theae en:ymes tww been characwised <strong>and</strong> poytbrol antlbod~e ses talaed apakrot<br />
DNA Ipases from the bede:bphapea T4 <strong>and</strong> T7, yeasts.J.wevelas <strong>and</strong><strong>and</strong> vaccM4a vkus tonlain conserved<br />
repions of amrw add sequence . Poydonai anbbodes have a t sidue peptlde representing one athese<br />
conserved raqons <strong>and</strong> these anobot
mutagenesis by DMN increasaaLwith S-9 level . 3-MCA muta9enesis reflected a 2AAF-like kinetic at low<br />
exposure, but gave results reminiscent of DMN at higher exposure levels . Mutation frequencies (X10s)<br />
varied as follows :<br />
~1 u~I/mIS-9 ~uI/m IS•9<br />
DMN (400 ug/ma) - 2i~ iT~<br />
2AAF (40 uy7ml) S07 26S<br />
3MCA (10 ul/ml) 280 358<br />
(0 .625 ut/ml) 144 61<br />
Control (Average) 51 .54<br />
The data presented here support ou r choice of 20-40 ul S-9/ml for a 4 hr . treatment of L5178Vi cells in this<br />
assay. However, the response of 2 AAF <strong>and</strong> the biphasic response of 3•MCA is an indication of the care<br />
needed in choosing the optimum en zyme (S-9) concentration for routine or mechanistic investigations .<br />
589<br />
MUTAGENICITY AND COMUTAGENICITY STUDIES ON THREE NOOTROPII+St<br />
PIRACETAM, ANIRACETAM AND HUPERZIIQ A . Z .H . Tu, Y .Y . Wang <strong>and</strong> W .D .<br />
Tang . Institute of Materia Medica, Academia Sinica,Shanghai, China .<br />
The mutagenicity <strong>and</strong> comutagenicity of 3 nootropils, piracetam,<br />
aniracetam <strong>and</strong> hupersin A, were determined at vrious concentration<br />
using S t himurium as the tester stains . The plate-incorporation<br />
essay wea con uc e both in the absence <strong>and</strong> presence of an aroolorinduced<br />
rat-liver S9 mix . A 30-min preinoubation teat protocol waa<br />
used for experiments with Sq . None of them is mutagenic towara theae<br />
tester stains without or with S9 . All of three drugs did not increased<br />
the revertants incueed by p-nitroquinoline in TA98 <strong>and</strong> by MINS<br />
in TA100. Aniracetam at 2 .5 mg/plate decreased the mutagen-indueed<br />
revertans in TA98, it might be of the toxicity of aniraoetam .<br />
Aniracetam at bigher concentration exhibited an inhibition effect<br />
on S t himurium . The micronuoleus teata of 3 nootropile were<br />
con3uc~3n 3ux mice at the dosage from 1/8 to 1/2 LD50 . All of<br />
them did not effect on the frequency of micronucleated polychromatic<br />
erythrocytes .<br />
590<br />
KINETOCHORES IN MICRONUCLEI: DEVELOPMENT OF A.SIMPLE AND RAPID METHOD TO<br />
IDENTIFY EXPOSURE TO ANEUPLOIDY-INDUCING AGENTS . J .D. Tucker <strong>and</strong> D.A . Eastmond .<br />
Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550 .<br />
The identification of agents causing aneuploidy in mammalian cells is currently limited due to<br />
the labor intensive nature of traditional cytogenetic analyses . We have developed a new <strong>and</strong> simple<br />
method to identify exposure to aneuploidy-inducing agents (aneuploidogens) . The assay involves the<br />
induction of micronuclei in cytokinesis-blocked cells <strong>and</strong> the use of an antibody with a specificity of<br />
> 99% for kinetochore regions . Micronuclei with one or more ceneromeres are indicat)ve of cells with<br />
a high potentia) for aneuploidy. Staining of the kinetochores is achleved with the use of a<br />
fluorescein-conjugated seeond antibody <strong>and</strong> the nuclei are atained with DAPL With the simultaneous<br />
use of phase contrast <strong>and</strong> DAPI excitation, it waa possible to ldentify both the cell membrane <strong>and</strong> the<br />
nuclei while scoring . Cella with mlcronuclei were txamined for the presence of fluorescein label to<br />
determine whether the m)cronuclens contained a kinetochore. Every agent tested produced a doserelated<br />
Increase in the frequency of micronucleattd cells . In human peripheral lymphocytes, the<br />
micronucleated cella indueed by the aneuplo)dogens eolehicine, vlncrlatine sulfate <strong>and</strong><br />
diethylstilbestrol contafned kinetochore-positive m)cronuclef 92%, 87%, <strong>and</strong> 76% of the time,<br />
respectively. In contrast, the micronucleated celis induced by the potent clastogens Ionizing radiation<br />
<strong>and</strong> sodium arsenite contained kinetochore-positive micronucfe) only 3% <strong>and</strong> 19% of thq time,<br />
respectively. In Chinese hamster ovary cells, the micronncleated cells induced by the aneuploidogena<br />
benomyl <strong>and</strong> vinblastine sulfate contained kinetochore-positive miuonuclti 92% <strong>and</strong> 94% o£ the<br />
time, respectively . With methyl methanesulfonate, however, only 11% contained a kinetochore .<br />
These results indicate that this simple <strong>and</strong> ra id procedure can discriminate between aneuploidogens<br />
<strong>and</strong> clastogens, <strong>and</strong> may allow mort rap)d identillcation of exposure to aneuploidogens both in vitro<br />
<strong>and</strong> in vivo. Work performed under the auspices of the U.S . DOE by Lawrence Livermore National<br />
Laboratory under contract No . 7405-ENG-48, with additional support from the Alex<strong>and</strong>er Hollaender<br />
Distinguished Postdoctoral Fellowship (D .A.E .) .<br />
591<br />
SMOKE EXPOSURE, AGE, SEX, RACE, AND POTENTIATION AS VARIABLES AFFECTING SISTER<br />
CHROMATID EXCHANGE INDUCTION IN NUMANS . D .A . Tulis, J .K . Smollinger, <strong>and</strong> W .H .<br />
McKenzie, North Carolina State University, Raleigh, NC (USA) .<br />
In vitro cytogenetic analysis was performed on peripheral lymphocytes of 49<br />
passively smoking children, ages 6 mo to 5 yrs . Mean SCE for non-smokers (7 .60 i 0 .46)<br />
was not significantly different (p > 0 .746) from mean SCE for passive smokers (7 .85 3<br />
0 .39) . However, passively smoking children demonstrated a highly significant<br />
(p < 0 .001) SCE increase to in vitro a-naphthoflavone (ANF) exposure, while the nonsmoking<br />
children showed a much lower but etill significant SCE increase to in vitro<br />
ANF exposure (p < 0 .03) . These results suggest that ANF has the potential to magnify<br />
<strong>and</strong> therefore detect an SCE insult experienced by passively smoking children . Age,<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 203<br />
Notes<br />
r
204 1989 EMS Abstracts<br />
Notes ,<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
_~.~t-- - .-<br />
r sex, <strong>and</strong> race~nf3uences were also investigated for their effects on SCE . A<br />
significant (p < 0 .001) SCE age effect was observed, with SCE frequency increasing<br />
with age .- A a?l,gniftsnt (p < 0 .008) SCE sex effect was also observed, with average<br />
female SCE (8 .76 t 0 .26) higher than the average male SCE (7 .85 t 0 .23) . However, no<br />
significant (p > 0 .94) SCE race effect was detected, although the SCE race * smoking<br />
interaction was significant (p < 0 .01) . Urinary cotinine was determined to be highly<br />
correlated (r - 0 .70) with the number of cigarettes smoked by the children's parents,<br />
<strong>and</strong> was used as an estimator of the actual amount of smoke inhaled by the children .<br />
This continuing study is presently investigating the role of ANF as a potentiator of<br />
SCE induction in non-smoking, passively smoking, <strong>and</strong> actively smoking young adults,<br />
<strong>and</strong> is also using cotinine as an indicator of cumulative smoke exposure for each<br />
subject .<br />
592<br />
ACETYLATION AND ACTIVATION OF 2-AMINO-3,8-DRAUCHYLIIIMAZO[4,5-f]QU1NOXALINE<br />
(MeIQx) TO DNA REACTIVE SPECIES IN SALMONELLA BACTERIA . K . W . Turteltaub, B. E.<br />
Watkins, <strong>and</strong> J. S. Felton, Biomedical Sciences Division, Lawrence Livermore National Laboratory, P .O.<br />
Box 5507, Livermore, CA 94550 .<br />
The aminoimidoazaarenes (AIAs), a group of heterocyclic amines found in cooked meat, require<br />
metabolic activation to express genotoxicity in a number of in vitro assays, including the Arnes/SalmoneUa<br />
test. The AlAs are mutagenic to Salmonella strains TA98 <strong>and</strong> TA98NR (nitroreductase deficient) ; but not<br />
to TA98/1,8DNP6 (acetyltransferase deficient), suggesting a requirement for the O-acetyltransferase .<br />
Asan et al. (Carcino enesis, 1987, 8 :1589) have found DNA adducts In Salmonella strains exposed to<br />
the quinoline AIA, IQ . We examined DNA adducts generated in Salmonella following exposure to 2amino-3,8-dimethylimidazo[4,5-f)quinoxaline<br />
(MeIQx) by 32P-posalabeling to access the role of bacterial<br />
acetylation in the activation of one of the quinoxaline AlAs. DNA was isolated from Salmonella TA98,<br />
TA98NR, <strong>and</strong> TA98/1,8DNP6 foUowing exposure to MeIQx, in the presence of Aroclor-induced mouseliver<br />
microsomes, for 4 hr . Four adducts were found in Salmonella TA98 <strong>and</strong> TA98NR . The adducts<br />
formed in these strains were judged to be idendcal by eoo-chromatography . No significant difference was<br />
found in adduct frequencies between these two strains . The same 4 adducts were also detected in<br />
Salmonella strain TA98/1,gDNP6 but at significantly lower levels . Synthetic azido-MelQx was reacted<br />
with calf thymus DNA <strong>and</strong> produced identical adduct profiles to Salmonella DNA as did mice given<br />
MeIQx . These data support the belief that formation of an N-acetoxy AIA intermediate is required to<br />
generated a DNA-reactive species. These data also support the hypo thesis that a single pathway,<br />
independent of species, is responsible for the generation of an ekctrophilic (DNA-reacdve) AIA<br />
(Work preformed under the auspices of the U .S . DOE by the LLNL under contract No. W-7405-F~8<br />
<strong>and</strong> supported by EAG NIEHS 222Y01-ES-10063).<br />
593<br />
FSTABL•ISHED OR PRIMARY CELL LINES FOR CYTOGEHETIC TESTS?<br />
DJ Tweats <strong>and</strong> DG Gatehouse, Glaxo Group Research Ltd ., Ware, Herts, Engl<strong>and</strong> .<br />
When choosing on a cell line for cytogenetic tssts, investigators take into<br />
account the degree of validation ; chromosome stability (in terms of amoku of<br />
chromosomes) ; low spontaneous aberration frequencyl donor variation for lymphocyta<br />
cultures etc . One parameter that is not often taken into account is that astablishei<br />
cell lines e .g . Chinese hamster V79, CH0 etc contain comprehensively re-arrangul<br />
karyotypes involving deletions, additions, inversions, translocations etc. It<br />
appears that many of these changes occurred at the time these cells became<br />
established, but it is clear that sublinea are progressively changing <strong>and</strong> diverging .<br />
In cells with such rearrangements genes concerned with DNA repair, cell division<br />
regulation, chromosome stability etc may well function abnormally due to 'position<br />
effects' or gene dosage effects, as for the activation of oncogenes . Such changes<br />
may influence the response of a particular subline to clastogens . There is a paucity<br />
of comparative tests on clastogens in different cell lines, or sublines . However<br />
discordant results have been obtained both due to differences in protocol <strong>and</strong> to<br />
inherent differences in the cells themselves . Primary cells e .g . human lymphocytes<br />
do not have the problem of tearranged <strong>and</strong> diverging karyotype . However, these cell<br />
are terminally differentiated <strong>and</strong> have to be induced to divide in culture, also donor<br />
variation can result in at least quantitative variations in response . However, ou<br />
oalance, the case for using primary lines appears to be stronger than the case fo•<br />
established lines . This poster will review the karyotypic changes that have occurred<br />
in the CHO <strong>and</strong> V79 sublines since their isolation <strong>and</strong> the results of testing with the<br />
same chemicals with both types of line .<br />
594<br />
USE Of INTERPHASE ANALYSYS TO DETECT CHEMICALLY INDUCED ANEUPLOIDY IN CULTURED CELLS .<br />
Vagnarelli P . . De Sario A . . Raismndi E.. Scariolo S. <strong>and</strong> De Csrli L .<br />
Dipartimento di Genetica e Microbiologia. Universita di Pavia . via S.Epitaaio 14 . Pavia . Italy.<br />
Ia rilu hybridization with cnromosome-specda DNA probes has Wen exploited for developmq an is ritro assay for<br />
chemically inducea aneuploidy . The following probes have been tested : T*7 which idenGfies a 100 fold repeated fraqment in<br />
50869 3718
'ne oencentromer c region of the Tchromosome : 011123. complementarv to a DNA sequence repeated 50 times in thee<br />
penrentromenc region of chromosome 9 : pNlt}it, honwiogous to the N-Myc oncogene <strong>and</strong> specific for chromosome 1 in the<br />
Neuroblastoma cell line LAN-1, wheWthe oncogene is amplified 50 times . /e situ hybridization experiments have been<br />
cerrred out on human lymphocytes with Y97 <strong>and</strong> Op23 <strong>and</strong> on Neuroblastoma cell hnes with oNb19-21 . Preliminary<br />
exper ments, performed on untreated cultures, to test the efficiency of the probes . showed an exceedingly high proportion of<br />
hvpodiplo,d nuclei due to technical artifacts . Therefore hyperdiploiov has been taken as a reliable index of induced sneuploidv .<br />
In order to check the validity of the assay we analyzed four known aneuploidy inducers, iumely fknomyl (8E), 6rieeofulvln<br />
!nF 1 . Chloral hydrate (CH), Diethvlstiibestroi (DES) <strong>and</strong> a putative carcinogenic agent NitrilTnacebcAcid INTAI . A significant<br />
increase in the percentage of hyperdiploid nuclei has been found with 8E . 6F, CH <strong>and</strong> DES ; a dose-related effect has been<br />
revealed w,ti` CH <strong>and</strong> DES . NTA did not show any effect in the induction of aneuplmdv . To improve the efficiency of the method<br />
we have compared the conventional aRO'f ddiographlc proCeDUre with that based on immunofluorescence . To this purpose<br />
parallel experiments heve been carried out with radioactive <strong>and</strong> biotmvlated probes using DES as a test compound . The nonrIclioaCtlve<br />
technique turned Out to be more sensitive <strong>and</strong> therefore more suitable for screening new compounds . The positive<br />
response obtained with agents that induce chromosome number variation by different mechanisms of action indicates that the<br />
rriethod we have devroloped may be of general application for testing aneuploidy Inducers is riua Moreover, the possibility of<br />
scoring large cell samples makes interphase analysis suitable for tne cytogenetic monitoring of exposed populations .<br />
595<br />
APPRAISAL OF GENOTOXIC EFFECTS OF AGROCHEMICALS IN HIGHER PLANTS USING IN VIVO AND<br />
IN VITRO END POINTS .<br />
K .Vaidyanath <strong>and</strong> T .Suryakumari, Department of Genetics, Osmania University,<br />
Hyderabad - 500 007 (A .P .) India .<br />
The use of different agrochemicals to control diseases, pests <strong>and</strong> weeds is<br />
an essential component of the modern agricultural technology . Though economic advantages<br />
of such practice is appreciated, it is belived that exposure to agrochemicals<br />
has many genetic consequences . To have a realistic appraisal of genotoxicity, three<br />
commonly used Chemicals viz ., Ethylene dibromide, Phenyl mercury acetate <strong>and</strong> Ekalux,<br />
have been studied using multiple end points like, somatic chromosomal aberrations,<br />
heritable germinal mutations at specific <strong>and</strong> non-specific locus, <strong>and</strong> in vitro growth<br />
of callus cultures . Significant frequency of chromosomal aberrations, -c-h1orophyll<br />
deficient seedlings in M. generation, specific locus mutations at waxy locus<br />
(Wx - wx), pollen sterility, polygenic variability in H3 generation <strong>and</strong> iphibition<br />
of callus growth have been observed . The overall results of the study suggests that<br />
any one or combination of genetic end points could be employed for the effective<br />
screening of environmental mutagens .<br />
596<br />
ACRYLAMIDE-1NDUCED CHROMOSONE-T1iPE ABBRRAT'IONS IN SPERMiOGENIC STAGES EVALUATED<br />
IN THE FIRST CLEAVAGE ME'1'AYHASES IN '1'Hh MOUSI : . N .Y . Vald>lvia, N .M . Lafuente <strong>and</strong><br />
M . Katoh, Fac . Odontoloqia, U . de Chile, bTGO (CHILIi) <strong>and</strong> Hatano Research Instztute,<br />
F .D .S .C ., Kanagawa (JAPAN) .<br />
Because of the evidences reported by Seqa, et al . (19`" Annual Meeting BMS) that acr7laaide (AA)<br />
binding to protasine of speraioqenic stages in the souse appears to be correlated with the pattern<br />
of genetic dasage oroduced by this cneaical in late speraatids <strong>and</strong> early speraatoza stages (Shelby,<br />
et a1 ., 1986) cytogenatic evaluation of these sase sensitive stages was perforaed in sarlir cleavage<br />
of souse eabryos . Adult sale M sice were intraperitoneally (i .p.) injected with 150 sq AA/kq <strong>and</strong><br />
aated to untreated fesales at intervals ranging fros 6 to 9 <strong>and</strong> 10 to 13 days after tratsent . The<br />
plugged fesales sere i .p . injected with colchicine <strong>and</strong> the fert:lized ova were collected to the first<br />
cleavage aitosis, at sich tiae the male chroaosou cosplesent su analyzed for strsctaral ohrosososal<br />
da.age . The chroaosose-t)
206 1989 EMS Abstracts . r -- - .-<br />
Notes ,~ ~ ~ g~ .•~ . ..<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
of~-the adducts:Z in target tissues is ecarse . We visualized the DNA adducts Osmethylguanine<br />
<strong>and</strong> 7- ethylguanine in tissue sections of rats <strong>and</strong> hamsters six hours<br />
after treatment-asic3ff= .ag (i .v .) resp . 100 mg (s .c .) NNK/kg . Adducts were observed<br />
in all target tissues for tumor formation except rat liver <strong>and</strong> pancreas . Nuclear<br />
staining was strongest in the nasal cavity, particularly in cells of Bowman gl<strong>and</strong>s .<br />
Staining was weaker in sustentacular <strong>and</strong> basal cells of the olfactory epithelium,<br />
the respiratory epithelium <strong>and</strong> serous gl<strong>and</strong>s . DNA adducts were also present in the<br />
trachea (epithelial <strong>and</strong> gl<strong>and</strong>ular cells) <strong>and</strong> in the bronchiolar lining of the lung<br />
(Clara cells) . Accumulation of adducts was observed after twice weekly applications<br />
of NNK for 8 weeks (rats : 3 .5 mg/kg, i .v . ; hamsters : 10 mg/kg, s .c ., each) in Bowman<br />
gl<strong>and</strong>s, serous gl<strong>and</strong>s <strong>and</strong> respiratary epithelium of the nasal cavity <strong>and</strong> in Clarti"<br />
cells . These adducts disappeared within 4 weeks after the cessation of NNK<br />
treatment . The immunocytochemical approach permits to study the distributiun,<br />
accumulation, persistence <strong>and</strong> repair of DNA adducts at the level of the single cell .<br />
One of the potential applications is the detection of adducts in human tissues, e .g .<br />
in buccal or bronchiolar cells of tobacco users .<br />
METABOLISM OF DIETARY OENOTOXIN9 BY THE RUMAN COLONIC MICROFLORA R .L . Van Tassellt,<br />
D .G .I Kingeton2 <strong>and</strong> T .D . Yilkinst . Departments of Anaerobic Miorobiologyt <strong>and</strong><br />
Chemistry2 . Virginia Polytechnic Institute <strong>and</strong> State University Blacksburg, VA . 24061<br />
598<br />
The microflora of the human colon is a complex ecosystem of anaerobic bacteria<br />
which have the capability of enaymatically transforming a variety of dietary (or<br />
biliary) compounds to genotoxic metabolites . In the past, most investigators studying<br />
the interplay between diet <strong>and</strong> colonic flora <strong>and</strong> its role in the etiology of cancers<br />
focused on the reductive <strong>and</strong> glycosidie potential of the bacterial enzymes - many of<br />
which reverse the oxidative <strong>and</strong> conjugative reactions performed by the liver . Recent<br />
work in our laboratory has focused on the metabolism of two relatively new classes of<br />
genotoxine, the heterocyclic amines (pyrolysis carcinogens) <strong>and</strong> the fecapentaenes .<br />
The heterocyclic amines, which originate from fried or broiled proteinaoeous foods,<br />
normally require activation by the liver before being potent mutagens or carcinogens .<br />
However, the "IQ" subclass (IQ . MeIQ <strong>and</strong> MeIQx) can be activated in the colon by<br />
Eubacterium <strong>and</strong> Clostridium spp . to a 7-hydroxy form which is directly mutagenic <strong>and</strong><br />
thus may react directly with the adjaoent oolonic epithelium . The fecapentaenes<br />
(conjugated ether lipids) are produced in the colon by 8aoteroides epp . from<br />
polyunsaturated ether phospholipids (plasmalogens) whose origin is unknown . The<br />
fecapentaenes are potent direct-aoting genotoxins that are detected in the feces of<br />
over 75% of individuals on normal western diets . Although there is no direct evidence<br />
that the 7-hydroxy "IQ" compounds or the fecapentaenes influence risk for colon<br />
cancer, the potency <strong>and</strong> prevalence of these bacterial metabolites is cause for concern<br />
- they may be carcinogens or somehow affect risk levels for cancers, particularly<br />
colo-rectal cancer, by some other means .<br />
599<br />
INFLUENCE OF DNA EXCISION REPAIR ON UV-INDUCED MUTATION SPECTRA IN CHINESE<br />
HAMSTER CELLS. H . Vricfing, M .L. van Rooijen . J . Venema, M .Z. Zdaenicka, J.W.IM. Simons, , L.H .F.<br />
Mullenders, P .H .M . Lobman <strong>and</strong> AA. van Zeel<strong>and</strong>. Department of Radiation Genetics <strong>and</strong> Chemical <strong>Mutagenesis</strong>,<br />
State University of Leiden, P .O . Box 9503, 2300 RA Lciden, The Netherl<strong>and</strong>s .<br />
The molecular nature of mutations induced by UV light was investigated in Aprt mutants from V79 Chinese<br />
hamster cells isolated after exposure to a high (12 J/ms) or a low (2 J/mY) dose . The nature of point mutations in<br />
hpn exon sequences was determined by sequence analysis of in virro amplified Apx CDNA (PCR method) . Among<br />
the mutants analyzed all possible classes of base pair changes were present, 70% being transversions . Since all<br />
mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UVinduced<br />
photoproducts at these sites . At the low dose g0% of the mutations were caused by photoproducts in nontranscribed<br />
str<strong>and</strong> of the hpit gene . This was 64% at the high dose . We propose that the str<strong>and</strong> bias for mutation<br />
induction towards the non-transcribed str<strong>and</strong> is caused by preferential repair of photoproducts from the transcribed<br />
str<strong>and</strong> of the Apn gene . Removal of pyrimidino dimers from an ig kb EooRI fisgment of the hprt gene was<br />
determined after digestion with T4 endonuclease V <strong>and</strong> analysis on alkaline agarose gels. Pyrimidine dimer removal<br />
in the Aprt gene was 80% in 24 hours whereas this was 18% in the genome overall . UV-induced mutations were<br />
also analyzed in a repair deficient cell line V-Hi, which is a UV sensitive derivative of V79 . UV-induced mutation<br />
induction in V-Hl was 7 times higher per unit dase than in normal V79 cells. All UV-iaduced (2 J/ms) single base<br />
pair changes in V-Hl were GC to AT transitions . Furthermore, 90% of the base pair changes were caused by<br />
photoproducta in the transcribed str<strong>and</strong> of the Irpr gese . Removal of pyrimidine dimers from the hpt gene is<br />
completely deficient in V-HI cells. We propose that most UV-induced mutations in V-Hi are caused by pyria8dine<br />
duners <strong>and</strong> that in normal V79 cells a relatively large part of the mutations are caused by 6-4 photoproducts . The<br />
extreme str<strong>and</strong> specificity of mutation induction in V-Hl might be due to differences in fidelity of DNA replication<br />
of the leading <strong>and</strong> the lagging str<strong>and</strong> .
600<br />
MONOCLONAL ANTIBOSY IMMUNOASSAYS FOR COOKING-INDUCED MEAT<br />
MUTAGENS .<br />
Martin V<strong>and</strong>erlaan, Bruce Watkiss ; Mona Hwang, Mark Knize <strong>and</strong> James Felton. Biomedical<br />
Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550<br />
Fifteen monoclonal antibodies have been produced that recognize five ne (AIA)<br />
mutagens formed in cooked meats (IQ, MeIQ, MeIQx, DiMeIQx, <strong>and</strong> Fhlp). These antibodies are<br />
being used to quatttifiy individual AIAs in cooked meats, to identify inunutachemically crosrteac6ve<br />
new AIAs, <strong>and</strong> to quan c'fy AIAs <strong>and</strong> their metabolites in human urine . Extracts of well-done cooked<br />
beef were separated on HPLC <strong>and</strong> competition enzyme linked ittununoaorbent assays (ELISAs) were<br />
conducted on each HPLC fraction using the panel of-antibodies . The plot of intmunochemical activity<br />
vs . HPLC retention time forms an "immunogram," similar to the mone familiar "mutagnm ' plot of<br />
mutagenic activity vs . retention time. Each antibody shows a unique immunogram . Peaks in<br />
inununochemica) activity can be associated with known AIA tnutagani, <strong>and</strong> withpteviously<br />
unidentified chemicals which immunochemically cross-neact with the antibodies . These findings<br />
suggest that the known AlAs are part of a larger family of immunochemically similar compounds<br />
produced by cooking . Immuno-affinity chromatography is being used to isoLte <strong>and</strong> identify these<br />
new AIA-like compounds . The antibodies provide a structure-based means of identifying new AIAs<br />
that complements the previously used bacterial mutageatc activity-based methods . Analysis of urine<br />
from people on vegetarian <strong>and</strong> well-done beef diets s6ows both mutagenic <strong>and</strong> intmunochemicallypositive<br />
material is associated with the diet high in AIAs .<br />
Research supported by NCIgrants ROl CA40811-03 <strong>and</strong> CA48446-02 <strong>and</strong> performed at LLNL under<br />
contract W-7405-ENG-48 with the Department of Energy .<br />
601<br />
MUTAGENICITY OF DRINKING WATERS IN FINLAND . T . Vartiainen, National<br />
Public Health Institute, Dept . Environm . Hyg . Tox . P .O .Box 95, 70701<br />
Kuopio, Finl<strong>and</strong><br />
Mutagenic activities of drinking <strong>and</strong> raw waters from the major<br />
waterworks, covering about 60% of all tap water distributed in Finl<strong>and</strong>,<br />
were tested with the Ames Salmonella tvphimurium assay using tester<br />
strains TA100, TA98, <strong>and</strong> TA97 . The highest yield of organic mytagenic<br />
impurities from these waters was obtained from acidic water samples by<br />
continuous extraction with diethyl ether or by XAD adsorption . Mutagenic<br />
activity of drinking water was dependent on the amount of organic<br />
matter (<strong>and</strong> ammonia) present in water <strong>and</strong> thrA quality <strong>and</strong> quantity of<br />
disinfectant used ; mutagenic activity could be approximated by an equation<br />
of the type A(1-e-kO), where A <strong>and</strong> k are constants <strong>and</strong> c is a parameter<br />
that depends on total organic carbon, the chlorine dose, <strong>and</strong> the<br />
ammonia concentration . The main mutagens in humus-rich chlorinated<br />
drinking water were acidic polar compounds detectable without metabolic<br />
activation . The highly mutagenic compound 3-chloro-4-(dichloromethyl)-<br />
5-hydroxy-2(5H)-furanone (MX) <strong>and</strong> its geometric isomer E-2-chloro-3-<br />
(dichloromethyl)-4-oxo-butenoic acid (E-MX) were detected in all extracts<br />
that exhibited mutagenicity . The concentrations of MX in the mutagenic<br />
drinking water concentrates ranged from 5 to 67 ng/l <strong>and</strong> this<br />
compound was responsible for 15 - 57% (average 33%) of the observed mutagenicity<br />
. Linear correlations were observed between mutagenic activity<br />
in TA100 <strong>and</strong> concentrations of MX <strong>and</strong> E-MX .<br />
602<br />
ENHANCEMENT OF BLEOMYCIN TOXICITY AND MUTATION AT THE APRT LOCUS IN CHO CELLS<br />
M .L . Veigl, J . Bhatt <strong>and</strong> W .D . Sedwick, Dept . of Medicine, Case Western Reserve<br />
Univ . Clevel<strong>and</strong>, OH 44106<br />
In drug combinations causing greater than 95% toxicity for CHO cells, the<br />
calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide<br />
(w13), is synergistic with bleomycin in clonogenic assays as analyzed by the<br />
method of Chou <strong>and</strong> Talalay (Adv . Enz . Reg ., 22, 27-55, 1984) . At concentrations<br />
causing less toxicity, however, this drug combination interacts in an<br />
antagonistic manner . It has been suggested that calmodulin antagonists may<br />
interfere with DNA repair processes responding to bleomycin-induced DNA damage<br />
. In these experiments, the effect of W13 on mutation caused by bleomycin<br />
was investigated by analyzing the impact of this drug combination on<br />
mutation in the adenine phosphoribosyl transferase gene (aprt) in cells that<br />
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1989 EMS Abstracts 207<br />
Notes<br />
l11<br />
m i=<br />
00<br />
Ya<br />
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J<br />
N<br />
I+<br />
,,<br />
I<br />
i<br />
I
208 1989 EMS Abstracts<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
NOteS- 'were either hemfa`ygoSs or heterozygous at this gene locus . The calmodulin<br />
antagonist, W13, at concentrations between 20 <strong>and</strong> 40 uM increased mutation in<br />
the aprt gene of_tIffig,Epzygous CHO cell line, D423, several fold over that<br />
observed with bleomycin alone . The analysis of this drug interaction <strong>and</strong> preliminary<br />
characterization of the specificity of these mutants will be<br />
presented .<br />
603 -<br />
DNA REPAIR IN HIGHER PLANTS . J .Velemfnskjr, Institute of Experimental Botany,<br />
Czechoslovak Academy of Sciences, Vltavskd 17, 150 00 Praha 5,Czechoslovakia<br />
.<br />
Since 1972 several repair pathways analogous to that known in other<br />
pro- <strong>and</strong> eukaryotic models have been described also in organs <strong>and</strong> cells<br />
of a few mono- <strong>and</strong> dicotyledonous plant species . They include an excision<br />
DNA repair after the action of alkylating agents <strong>and</strong> UV light, repair of<br />
ionizing radiation-induced DNA single str<strong>and</strong> breaks <strong>and</strong> bleomycin-induced<br />
double str<strong>and</strong> breaks as well as recovery of short daughter DNA str<strong>and</strong>s<br />
synthesized on templates damaged by alkylating agents . Several DNA-glycosylases,<br />
UV- <strong>and</strong> AP-endonucleases have been isolated <strong>and</strong> the role of DNApolymerases<br />
<strong>and</strong> DNA-ligases in the repair processes in plants has been<br />
investigated . Special topics in plants rpresent the repair of spontaneous<br />
DNA lesions accumulated in seeds during their ageing, repair capacity of<br />
pollen grains <strong>and</strong> chloroplasts <strong>and</strong> the clastogenic adaptation - an analogy<br />
of adaptive response at the chromosome level . At the absence of repair<br />
deficient mutants in plants, metabolic inhibitors <strong>and</strong> artificial conditions<br />
like storage of mutagen-treated seeds help to elucidate the role<br />
of repair pathways in the mutagenic efficiency . A new approach in determining<br />
the involvement of individual DNA lesions in the mutagenic processes<br />
in plants represents the transfer of an E .coli repair gene ada into<br />
tobacco that enhances resistance of Jobacco oe s against the a-cTion of<br />
alkylating agents interacting with 0 in DNA guanine .<br />
THE ROLE OF TRANSCRIPTION IN UV-INDUCED EXCISION REPAIR<br />
J . Venema, L .H .F . Mullendere, A . van Hoffen <strong>and</strong> A .A . van Zeal<strong>and</strong> . Department of<br />
Radiation Genetics <strong>and</strong> Chemical <strong>Mutagenesis</strong> . State University of Leiden, The<br />
Netherl<strong>and</strong>s .<br />
Preferential repair of active genes following exposure to UV-light (254 nm) is now<br />
a well established feature of mamaulian cells . We have shown that in normal human<br />
fibroblasts the transcriptionally active adenosine d .aminase (ADA) gene is faster <strong>and</strong><br />
more efficiently repaired than the genoma overall . The inactive 754 locus is repaired<br />
to the saao extent as the genome overall .<br />
We have extended these studies by assessing the role of transcription in excision<br />
repair . First, we have analyzed repair in the ADA gene with str<strong>and</strong> specific probes . We<br />
show that in a fragment comprising the 3' end of the gene there is no difference in<br />
repair of both str<strong>and</strong>s . A second approach was made by using a cell strain derived from<br />
a patient with severe combined immunodeficiency (SCID) which is homozygous for a<br />
deletion in the promoter region of the ADA gene . In primary fibroblasts no transcription<br />
of the ADA gene is detectable by Northern blotting' . Our results show that repair in<br />
two adjacent fragments of the ADA gane is similar to that in normal human cells . We are<br />
currently investigating str<strong>and</strong> specificity of repair in this cell strain .<br />
1 . Berkvens, Th .M . et al . (1987), Nucl . Acids Res . 15, 9365-9378 .<br />
OVERVIEW OF BACTERIAL TE8TIN0 FOR MUTA08NICITY<br />
S. VenNt, Institute of Csncsr Research: Royal Marsdsn Hosplul, 8udton, Sumy SM2 IfNO, United Kkqdom<br />
604<br />
605<br />
it Is dlHlcuM, N not Imposslble to say anything about bsdsrld gsnotoxdolty tats which has not bsw<br />
sak( already. The discovery of oncogsns activation by point mutation (a:9., bass-pak substitution) has phrsn<br />
a naw leass of life to bacterial mulagsnetiol.b. Point mutations are paRbulsrly easy to detact in the<br />
S.Imonslla/mkxosoms assay, which Is by far the most widely used mutation assay aver dsvbsd . A search on<br />
ToxNne using -Ames" as the key word resulted In 7,'00 oBatloia In February 1ssY. The Ama test has bssn Ln<br />
subjected to criticism (both Informed <strong>and</strong> uninformsd), natimwl <strong>and</strong> International ragdstlon (blind, or not 0<br />
bl<strong>and</strong>), with easy, difficult or knpossibls chemksis . lt has nsisbd this global bsttsrkp with remarkable polas, Co<br />
<strong>and</strong> Is more or tas unchanged from the form 11 took In the wJd4wvonfts when M Mt emerged . Despite ths O%<br />
appearance of a vaMty of alternative tests (aE ., nvans•nwlatbn In straba of EsohMdda ood WP2; t0<br />
fluctuation tes(s ; forward mutation assays; methods, suoh as ths 808 CfKOmotsst, which employ Induction<br />
of the 808 DNA-prooastrp network as s signal of DNA danrgr dllbrsnllsl taodoKy In npat profbisnt <strong>and</strong> J<br />
N<br />
N
epalr-deflcient bacteria) this assay will continue to form the basis of all screening programmes for potential<br />
mutagenicity <strong>and</strong> carcinogenlcaPt . In addition to the role of the Ames test In screening pure chemicals, It Is<br />
used for InvestlgaUng the mutag7nlclty of complex mixtures, both of chemical <strong>and</strong> biological otipin . Virtually<br />
every kind of bodily secretion <strong>and</strong> excretion has been eub)eoted to scrutiny, the outst<strong>and</strong>ing discovery being<br />
the detection of mutagenic activity In the urlne of smokers. Despite the vast quantities of bHormatlon on how<br />
to conduct the S.Imonella/mIcrosome test, purnab <strong>and</strong> regulatory authorities still ncelw data based on<br />
inadequate tests, or Inadequate data based on adequate tests, or various combinatlons of these defects .<br />
606<br />
STUDY OF TtE ACTION OF AN EXTRACT OF Stryphnodendron obovatim BENTH SEEDS ON DIFFERENT<br />
BIOLOGICAL SYSTEhS .<br />
V .E .P .Vicentini-Dias, C .S .Takahashi, Univ .Est .Maringa-PR, F.F .C .L .R .P .-Univ .SP (Brazil) .<br />
Naturall products of plant origin have been extensively used in human therapeutic .<br />
Stryphnodendron obovatun BENTH, popularly called 'Barbatim'eo" in Brazil, is used in folk<br />
medicine against hemorrhage, leucorrhea, diarrhea, hemia <strong>and</strong> chronic ulcer, but the<br />
lethal effects in cattle that ingest its pods are acccxtQanied by signs of photosensitization<br />
<strong>and</strong> liver damage . In view of these effects, we investigated the action of S .<br />
obovatun BFMH seeds on three test systems . An aqueous seed extraot at concentrations<br />
ranging frorn 5 .3 to 23 .8 mg/ml of water drastically inhibited cell division from 16 to<br />
1% after 24 h of treatment in Alliun cepa root tip cells, with no recovery of division<br />
after a period of 6 <strong>and</strong> 24 h in water. Cell spindle formation was also inhibited, leading<br />
to the appearance of colchicine metaphases (2 .5%) . In bone marrow cells of wistar rats<br />
treated "in vivo" for 24 h, the extract (1 .1 mg/g body weight) did not induce a statistically<br />
significant increase in the frequencies of chromqsome aberrations (2 to 4%) .<br />
In hianan peripheral blood lynphocytes treated in culture (0 .7 µg/ml culture mediun) there<br />
was no increase in the frequencies of chronasome aberrations or in mean nunber of sister<br />
chromatid exchanges (about 7 .5 SCEs/cell) . Even though a clastogenic effect was not observed<br />
in nwnalian cells, treatments with the highest concentrations showed a certain<br />
cytotoxic effect .<br />
r<br />
607<br />
GENOTOXIC ACTIVITY OF 1,3-BUTADIENE, NITROGEN DIOXIDE AND THEIR PHOTOCHEMICAL REACTION<br />
PRODUCTS .<br />
K . Victorina M . Stf,hlberga, L . Buskb H . Cederbergc <strong>and</strong> J . Ma~nussonc, aInstltute of<br />
<strong>Environmental</strong> Medicine, Box 60208, S-104 01 STOCKHOLM . Sweden . Svedlsh Food<br />
Administration, Box 622, S-751 26 UPPSALA, Sweden . cUniverstty of Stockholm,<br />
S-106 91 STOCKHOLM, Sweden .<br />
A small exposure system has been developed for mutagenicity studies of gases . UVirradiated<br />
mixtures of nitrogen dioxide <strong>and</strong> alkenes gave rise to mutagenic effects in<br />
Salmonella, strain TA100, in the order 1,3-butadtene > propene > ethene . A directacting<br />
mutagenic effect was evident after 40 min reaction time <strong>and</strong> 6 h exposure time at<br />
such low reactant concentrations as 0 .25 ppm each of butediene <strong>and</strong> nitrogen dioxide .<br />
Butadiene in itself was not mutagenic at 0 .5 - 20 % . Nitrogen dioxide was slightly<br />
mutagenic but also bacteriotoxic at 10 - 15 ppm . In vivo experiments were performed<br />
with the mouse bone-marrow micronucleus assay <strong>and</strong> the somatic mutation <strong>and</strong> recombination<br />
test in Drosophila (the wing spot test) . Butadiene alone was not mutagenic in<br />
Drosophila at 1 X, but induced micronuclei in mice at 10 ppm during 23 h . Nitrogen<br />
dioxide was not genotoxic tn either test system . The photochemical reaction products<br />
were toxic but not mutagenic at high doses tn Droso hila (1000 ppm butadiene + 50 ppm<br />
NO2 + UV), <strong>and</strong> not genotoxic in mouse bone-marrov highest non-toxic mixture 10 ppm<br />
butadiene + 10 pm N02 + UV) . The in vivo experiments thus did not confirm the strong<br />
direct-acting mutagenic activity of the photochemical products in Salmonella .<br />
608<br />
GENOTOXIC RESPONSE IN GROWING AND STATIONARY PHASE YEAST CELLS .<br />
Vierling, Th ., Boehringer Mannheim GmbH, S<strong>and</strong>hofer StraBe 116,<br />
D-6800 Mannheim 31, Federal Republic of Germany<br />
Yeasts are known to provide suitable systems for the assessment of<br />
genotoxic potentials of chemicals . The diploid Saccharomyces<br />
cerevisiae strain MP1 allows for screening of intergenic <strong>and</strong><br />
interallelic recombination <strong>and</strong> of forward mutation . In order to•<br />
investigate intrinsic differences in sensitivity of the Saccharomyces<br />
cerevisia,e MP1 testing system, the genetic responses which<br />
were obtained after treatment of proliferating or stationary phase<br />
cells with known genotoxic agents were compared . It was found that<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A<br />
1989 EMS Abstracts 209<br />
Notes
210 1989 EMS Abstracts,<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes stationary phi[se cells were more severely affected by triethylene<br />
melamine, cyclophyWhamide <strong>and</strong> acridine orange treatment, whilst<br />
the qenotoxia :%f?8'dCY of sodium nitite <strong>and</strong> ethyl-nitro-nitrosoguanidine<br />
were slightly stronger when exposure took place during cell<br />
proliferation . The genetic activity of aminofluorene was expressed<br />
only in growing cells . Sterigmatocystin induced comparable qenotoxic<br />
responses under both treatment conditions . The obtained data<br />
support the necessity of considering both physiological conditions<br />
for the assessment of substance induced genetic alterations in<br />
yeasts. _<br />
ADAPTIVE RESPONSE OF HUMAN BLOOD LYMPROCYTES TO LOW CONCENTRATIONS OF BLEOMYCIN .<br />
VIJAYALAXMI . & W . BURKART .<br />
Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerl<strong>and</strong> .<br />
609<br />
Human peripheral blood lymphocytes cultured in the presence of low (adaptive)<br />
concentrations of bleomycin (BLM), 0 .01 to 0 .1 vg/ml, for 48 hours <strong>and</strong> then treated<br />
with a high (challenge) concentration of the same agent (1 .5 Ug/ml), became significantly<br />
less sensitive to the induction of chromosomel damage than those which did not<br />
receive the pre-treatment with BLM . They responded with lower frequencies of chromatid<br />
<strong>and</strong> ioschromatid breaks . Lymphocytes pre-treated with low concentrations of BLM also<br />
showed significant reduction in chromatid <strong>and</strong> chromosome breaks when challenged with<br />
1 .5 Gy X-rays, as compared with those which were not adapted to BLM . These results lend<br />
support to the operation of an inducible "adaptive repair" in human blood lymphocytes<br />
which offers resistance <strong>and</strong> cross-resistance to the induction of chromosomal damage by<br />
the same or similar DNA damaging agents . The adaptive repair process was found to be<br />
negated when 3-aminobenzamide, an inhibitor of poly (ADP) polymerase, was added to the<br />
cultures immediately after the challenge treatment with BLM or X-rays . The magnitude<br />
of negation in the adaptive response was greater in the case of lymphocytes challenged<br />
with X-rays as compared with those challenged with BLM .<br />
610<br />
ISOLATION AND STRUCTURE ELUCIDATION OF fAUTAGENS FROfA ROASTED SEEDS OF<br />
MORINGA OLEIFERA<br />
Vi esePfor, I . ., C .Y .L . Sylienco, F . Dayrit, P . Finch ; Institute of<br />
Chem .,Univ . of the Phil . ; Dept . of Chem, Ateneo U . <strong>and</strong> Dept . of Chem .,<br />
R .H .B .N .C . (Univ . of London), U .K .<br />
Several mutaqens have been isolated from the roasted seeds of<br />
tAorince oleifera, commonly known as drumstick or horseradish tree .<br />
The extracts of the roasted seeds were purified by solvent partition<br />
end liquid chromatopraphy . The structures of the mutepens were 13<br />
elucidated by spectral methods, includinp FT-IR, EI-IAS, 1H-NAiR, C-<br />
NmR, <strong>and</strong> 2D-COSY . The Micronucleus Test, an in vivo method, using<br />
albino mice as test system, was used for monitoring the mutapenicity<br />
of the various fractions end for studying the structure-activity<br />
correlations . The structure of an isolated now mutagen has been<br />
elucidated ea 4(oC,-L-rhamnosyloxy)phenylacetonitrile . A number of<br />
biosyntheticelly end chemically related compounds were also isolated .<br />
MUTAGENIC ACTIVITY OF CHILLT-PASTES AND TSEIR INGREDIENTS .<br />
U .Vinitketkumnuen, M .Suttajit, <strong>and</strong> T .Matsushim, Dept .of Biochemistry, Fac .of<br />
Medicine, Chiang Mai University, Chiang Mai 50002, Thail<strong>and</strong> . <strong>and</strong> Dept .of <strong>Molecular</strong><br />
Oncology, Inst .of Medical Science, University of Tokyo, Tokyo 108, Japan .<br />
In Thail<strong>and</strong>, different kinds of chilli-paste are commonly used as an appetizing<br />
seasoning for cooking . lie have surveyed the iutagenic activity in some kinds of<br />
chilli-paste <strong>and</strong> their ingredients such as dry-chilli, shallot, garlic, cori<strong>and</strong>er<br />
seeds, caraway seeds, lemon grass, leach lime <strong>and</strong> greater galangal by Salmonella<br />
mutation assay . It was found that some but not all preparations of commercial<br />
chilli-paste <strong>and</strong> several ingredients vere mutagenic in the Salmonella typhimuriua<br />
strain TA98 <strong>and</strong> TA100 with <strong>and</strong> without metabolic activation . The mutagenicity was<br />
not due to food preservative (sodium benzeate) but to some other ingredients used<br />
in chilli-paste preparation . Shallot, exhibited unequivocal mutagenic activity to<br />
both tester strains with <strong>and</strong> without 89 mix, greater galangal was weakly mutagenic<br />
toward TA100 with 39 miz . Chilli, itself <strong>and</strong> capsaicin, the pungent principle<br />
611
- 612<br />
of chilli, were not mutagenic in Salmonella typhimurium TA98 <strong>and</strong> TA100 with <strong>and</strong><br />
without S9 mix . The mutagenic component(s) in shallot <strong>and</strong> greater galangal will be<br />
further isolated <strong>and</strong> chaxa~cterized .<br />
This study was supported by the China Medical Board Grant, Fac .of Medicine,<br />
Chiang Mai University .<br />
nYVCSncw77omTOwARDS THE Srwra)Aantzw11MorZNB ausaYAtlAxs,TOTTti4i wmr uu+PIDt@rraaC6stnAws<br />
W . VoDcner• . E .W . M3ller• <strong>and</strong> H .G . Miltenburger, Institute for Zoology, Technical University, 6100 Darmstadt, F .R.G .<br />
•Preunt address: Cytotest Cell Research (CCR) GmbH, 6101 RoBdorf, F .R.G.<br />
The mammalian spot test (MST), developed by Russell <strong>and</strong> Major (Generfea I2161-175, 1957), is a procedure for the<br />
detection of chemically induced mutations (especially gene mutations) <strong>and</strong>/or reeombinational processes (Fahrf$<br />
Funkt .Brol.Med5:287-297, 1985) in somatic cells of the mouse. The method uses an exposure of embryonic eella ja utero<br />
via treatment of pregnant females with mutagens . This can lead to an alteration of wild type aUe4 at different coat colour<br />
genes in pigment precursor cells of the beterozygous Fl-embryos . Under the precondition of several subsequent<br />
divisions of the genetically altered precursor cell the recessive phenotype can be expressed, constituting a spot of<br />
changed colour on a black (nonagouti) background of the F~-~mouse fur<br />
The aim of our investigations was to compare the suitability of different . crosaes . We expected that the use of a cross<br />
DBA x NMRI, could minimize the efforts to reach satisfactory Fl-numbers. A possible disadvantage resulting from a<br />
smaller set of genetic markers as compared to the uosses T x liT <strong>and</strong> T x C57BI could not be excluded. We performed<br />
experiments to determine the spontaneous spot rates <strong>and</strong> to quantify potential sensttiviry differences between the crosses<br />
T x C57BU61 <strong>and</strong> DBA x NMRI using the well known point mutagen ethylnitrosourea (ENU) . Neither the spontaneous<br />
nor the ENU-induced spot frequencies did sub.taatially differ between the ctosses . However, a much lower number of<br />
pregnant females was required to reach the recommended sample dzea per experimental group of 200 - 300 Fl-anintala<br />
(Braun, Russel4 Schdteich ; Mutation Research D7:155 161, 1982) in the cross DBA x NMRI . In additional experiments we<br />
exposed embryos carrying only one or two heterozygous markers in various combinations to an ENU-treatment . It was<br />
proven by these tests that the value of detectable genetically relevant spots depended on the marker used . The sum of<br />
spot rates at the loci brown, dilute <strong>and</strong> albino equalled the value from a comparable treatment of the hybrids of the cross<br />
DBA x NMRI in which these markers are involved <strong>and</strong> was in the same range as known from the cross T x C57B1 .<br />
In conclusion the cross DBA x NMR] can be mommended jor the routine <strong>and</strong> st<strong>and</strong>ardtzed pedormance ojthe MST.<br />
613<br />
COMPARISON OF THE PLANT CELL ACTIVATION OF TWU PROMUTAGENS USINC3 ENZYME<br />
INHIBITORS . E .D. Wagner, M .M. Verdier <strong>and</strong> MJ . Plewa . Institute for <strong>Environmental</strong> Studies, University<br />
of Illinois, Urbana, IL 61801 USA .<br />
We are studying the mechanisms involved in the plant activation of 2-aminofluorene (2AF) <strong>and</strong> mphenylenediamine<br />
(m-PDA). Tbbacco cells (TX1) were the activating system <strong>and</strong> reversion in SaGnonelta<br />
ryphimurium (TA98) was the genetic endpoint . Specific inhibitors were introduced into the coincubation<br />
mixture of TX1 cells at mid-log phase, TA98 cells <strong>and</strong> a constant amount of 50 µM 2AF or 500 pM m-<br />
PDA. The endpoints of mutation induction, plant cell viability, <strong>and</strong> bacteria viability were assayed .<br />
Diethyldithiocarbamate, a metal chelator, inhibited the activation of both 2AF <strong>and</strong> m-PDA with no<br />
concomitant decrease in cell viability . Likewise acetaminophen, a peroxddase inhibitor, also reduced the<br />
activation of both 2AF <strong>and</strong> m-PDA . Metyrapone, an inhibitor of cytochrome P-450, had no effect on either<br />
the activation of 2AF or m-PDA, but was toxic . (+)-Catechin enhanced the plant activation of 2AF at low<br />
concentrations <strong>and</strong> inhibited activation at higher concentrations . (+)-Catechin reduced the activation of m-<br />
PDA Low concentrations of 7,8-bcnzoflavone (a cytochrome P-448 inhibitor) reduced the number of plantactivated<br />
2AF-induced TA98 revertants . 7,8-benzoflavone had no effect on m-PDA activation . Methimazole,<br />
a high-affinity flavin-containing monooxygenase substrate did not influence 2AF activation but did inhibit<br />
the activation of m-PDA . We conclude that cytochrome P-450 is not involved in 2AF or m-PDA activation.<br />
A peroxidase pathway plays a role in the activation of both promutagens. In addition, a cytochrome R448type<br />
N-hydroxylase is implicated in 2AF activation, whereas a flavin-containing monooxygenase pathway is<br />
important in m-PDA activation. Research funded in part by USAF grant tM88-AF-P-0511 .<br />
614<br />
IDENTIFICATION OF FOOD MUTAGENS . K. Wakabayashi, National Cancer Center Research<br />
Institute, Tokyo (Japan)<br />
Identification of mutagens in food is the first step in determining their<br />
contribution to human cancer development . By using mutagenicity in Salmonella ss a<br />
monitoring system, we have found <strong>and</strong> identified many mutagens <strong>and</strong> nitrosatable<br />
mutagen precursors in foods .<br />
Various kinds of heterocyclic aminea were identified as mutagens in cooked meat<br />
<strong>and</strong> fish, <strong>and</strong> nine of the compounds were proven to be carcinogenic in rodents . IQ<br />
was shown to be carcinogenic in monkeys . Carcinogenic Trp-P-I <strong>and</strong> Trp-P-2 were found<br />
to be very potent inhibitors of monoamine oxidase . Some heterocyclic amines were<br />
detected in human brain, blood <strong>and</strong> urine . Thus, heterocyclic'amines may be involved<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 211<br />
Notes<br />
r
212 1989 EMS Abstract s<br />
Notes -fn the devel.ofle>srtf6t only of human cancer but also of other disorders, including<br />
neural diseaser<br />
Phenol <strong>and</strong> initle derivatives were identified as nitrosatable mutaRen precursors<br />
in soybean fer16et4rat7e'n products <strong>and</strong> vegetables . Broiled meat <strong>and</strong> fish also contained<br />
nitrosatable precursors . Pher.olic derivatives reacted with nitrite to produce<br />
mutagenic diazo compounds . 3-Diazotyramine formed from tyramine <strong>and</strong> nitrite was<br />
carcinogenic to rate . In the case of indoles . N-1 <strong>and</strong>/or C-3 nitrosated compounds<br />
were formed . 1-Nitrosoindole-3-acetonitrile formed DNA adducts in both the forestomach<br />
<strong>and</strong> gl<strong>and</strong>ular stomach of rats . A carcinogenicity teat of this ritrosoindole is<br />
ongoing .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
61 5<br />
DIFFERENCE BETWEEN INTRAPERITONEAL AND ORAL GAVAGE APPLICATION IN THE MICRONUCLEUS TEST<br />
The Collaborative Study Group for the Micronucleus Test, CSGMT/JEMS-MMS, Organizers : A .<br />
Wakata(Yamanouchi Pharm . Co., Ltd . . Tokyo) N . Hayashi(Natl . Inst . Hygienic Sci . .Tokyo),<br />
S. Sutou(Itoham Foods Inc ., Tokyo), H. Shimada(Daiichi Seiyaku Co ., Ltd., Tokyo) . S.Sato<br />
(Japan <strong>Tobacco</strong> Inc ., Kanagaea), <strong>and</strong> Y .F. Sasaki(lnst . of Env . Toxicol . . Tokyo)<br />
As a collaborative study of CSGMT/JEMS MMS by 34 participating organizations, the ip<br />
<strong>and</strong> po administration routes were compared using 2 mouse strains . MS/Ae <strong>and</strong> CD-1 . <strong>and</strong> 17<br />
chemicals with various modes of action (Ara-C . 6-MP, B[ajP . DMBA . 2AAF . Phenacetin . CYP.<br />
EMS . ENU . MMS . MMC, COL . VINC, KBrO3, KrCrO„ Benzene <strong>and</strong> PCZ). On the basis of the<br />
findings of an acute toxicity <strong>and</strong> a pilot experiment for dose <strong>and</strong> sampling time, a full<br />
scale experiments were performed on each chemical . Almost all chemicals shored a<br />
positive response in ■icronucleus induction by both routes of administration in both<br />
mouse strains . Contradictory outcomes were obtained between the ip <strong>and</strong> po routes on<br />
potassium chromate in both strains (ip :positive, po :negative) . In the CD-1 ■ice, benzene<br />
remarkably induced ■icronuclei when administered po, but gave only a marginal response<br />
when administered ip . Generally the chemicals induced ■icronuclei at lower dose levels<br />
(mg/kg) by the ip route . This tendency, hotever, was decreased or even reversed when<br />
dose was expressed as percentage of the LD$ . . Although the ip route, an artificial<br />
exposure route, is useful to detect the inducibility of ■icronuclei of test chemical per<br />
se at a small dose, the po route seems sensitive <strong>and</strong> valuable enough to evaluate the<br />
test chemicals . When the dose levels of chemicals are adjusted on the basis of the<br />
LD, ., both routes are acceptable as routes of administration in the ■icronucleus test .<br />
CHARACTLRIZATION OF METABOLITES OF<br />
2-AMINO-3,8 DIMETHYL-IMIDAZO(4,S-f)QUINOXALINE .<br />
HlYsa Wallin, lera A. Holme, Oeorg Becher <strong>and</strong> Jan Alex<strong>and</strong>er.<br />
Department of Toxiwlogy, National Institute of Public Health,<br />
N-0462 Oslo Norway.<br />
2-Amino-3,8-dimethylimidaxo(4,5-J)quinoxaline was metabolized by Isolated liver<br />
celle from PCB treated rata to at least 10 metabolites . The five major metabolites<br />
which were also major metabolites excreted from the rat were etructuraily determined .<br />
Three of the metabolites contained sulfate, on evidence of the Incorporation of<br />
radioactive sulphur <strong>and</strong> the requirement of tultotraasferase for their fotmation .<br />
Chemical synthesis, NMR, UV <strong>and</strong> mast tpeciroseopy revealed that the major<br />
metabolite was 4 or S-MeIQx-sulfate (formed from 4 or S-hydroxy-MeIQx) . The<br />
other two were MeIQz=2-sulfamate <strong>and</strong> 8-hydroxymethyl-MdQx-S-sulfate. Two of<br />
the metabolitea displayed the protons from a glucuronie acid group . The were<br />
ideatified 4 or S-O-glucoaiduriayl-MeIQx (formed from 4 or S-hydroxy-MeIQx) <strong>and</strong><br />
N-gWcosidurlnyl-MeIQx. We also obtained evidence for the formation of a glutathione<br />
conjugate .<br />
ELIfA FOR FOOD MUTAGEN S<br />
Hlkaa Willis, Karis:,6rEpltierj, 014 Jerges Roulaad, Otorj Becher <strong>and</strong> Jaa Akxaader .<br />
Depfrtmest of Toxisology, Natioaal Ieetitnte of Public Health ,<br />
N-0462 Oab Norway .<br />
To aeseu the risk !oVamiaqimldazo =,azaareaee (AIA) to human popelatiow it Is aeatuery to<br />
know more about the kveL of t>,eee compounds Is common foods . We'beHeve that immssoawys<br />
♦dll provide both the ipeoificity <strong>and</strong> the sensitivity seeded for this task <strong>and</strong> may be rnn Is<br />
larae seriet with relative little work effort .<br />
Our object was to develop its t?LISA for several different AIA. We therefors prepated astigeas<br />
61 6<br />
61 7
whicb carry the eommoa regioMe kaowa carcinogenic AIA :s. Tbus, trana-2•amiso-S(•2•urboxy)etheayl-1•metbyl•beaz(midazole<br />
was synthesized aad coupled to ovalbumia aad boviae albumia .<br />
Rabbits wert (tqmuaized with tbe bovine albumla•baptea eoajugate, aad aa eompetitivt enayme<br />
immaaoasuay was developed os the reaction of the antibodies witb the ovalbumia-baptem In<br />
microtiter ptat6f .<br />
Fifty percent ishibidoa was demoostrated at 10 pmo11Q, 50 pmol MeIQ, S00 pmo17,8-diMelQx,<br />
<strong>and</strong> 2500 pmol Me1Qx, 4,8-diMeIQz aad PhIP . No iahlbitioa was seea for 1 umol ereatiae ot<br />
ereatiaise .<br />
618 OBSERVATION ON RENAL HYPERPLASIA CAUSED BY UNILATERAL NEPHRF.CIC)MY<br />
IN MICE AND ITS SIGNIFICANCE IN SEARCH FOR ANIMAL TESTS FOR<br />
CHEMICAL CARCINOGENS<br />
Da Wang<br />
(Shanghai Institute of Biochemistry, Academia Sinica)<br />
A series of histological <strong>and</strong> anatomical changes of different stages was compared<br />
after unilateral nephrectomy in mice between the remained kidney <strong>and</strong> the<br />
sham-operated kidney . Results of the experiment revealed that after unilateral<br />
nephrectomy in ICR mice a series of changes occurred in the remained kidney :<br />
there were increase in weight, increase in thicknesses of renal cortex <strong>and</strong><br />
medulla, increases in size of the renal corpuscle, increases in number of cell<br />
in the glomerulus, hyperplasia of epithelial cells in the renal tubule, dilatation<br />
of renal blood vessels <strong>and</strong> stromal hyperplasia . Administration of chemical<br />
carcinogens to the hypertrophic <strong>and</strong> hyperplastic kidney may aggravate<br />
cell hyperplasia <strong>and</strong> degeneration in the remained kidney, <strong>and</strong> promote the<br />
development of tumors . The shortening of induction period of tumors after unilateral<br />
nephrectomy might help to supply a better model for chronic induction<br />
of animal experiment in vivo .<br />
619 •<br />
MUTAGENIC AND ANTIMUTAGENIC EFFECTS OF 9-AMINOACRIDINE IN THE YEAST SACCNAROYNYCSS<br />
CsRevISZns . Q . Wang, U .G .G . Hennig, <strong>and</strong> R .C . von Borstel, Department of Genetics,<br />
University of Alberta, Edmonton, Alberta (Canada) T6GJE9<br />
Certain chemicals exhibit the property of reducing the spontaneous mutation rates<br />
in the yeast SaooharoVoee oerev{eiae . In systems for forward mutations (canavanine<br />
sensitivity to resistance) <strong>and</strong> reversions (histidine auxotrophy to prototrophy),<br />
9-aminoacridine was reported to be antimutagenic during mitosis (Magni et at . 1964,<br />
!autat . Ree . 1 : 227-230 ; Pugllsi 1967, Mutat . Rea . 4 : 289-294) . Also, there appears to<br />
be a base sequence specificity involved in the binding of 9-aminoacridine to ONA<br />
(Young et aZ . 1980, Prod . NatZ . Acad . Soi . (USA) 77 : 6453-6457) . The effects of antimutagenic<br />
agents can be quite different on various loci . We have found that 9-aminoacridine<br />
has antimutagenic effects on the hom3-10 frameshift allele but not on the<br />
hisl-7 <strong>and</strong> arg4-17 base substitution alleles in strain XV185-14C of yeast . Mutation<br />
rate measurements were made in growinaq cells of yeast with the 1000-compartment<br />
fluctuation test (von Borstel 1978, in Methods in Cell Biology", Vol . 20, Academic<br />
Press, pp . 1-24) . For the hom3-10 allele, 9-aminoacridine, at concentrations of 10-20<br />
ug/ml, increased the mutation rate 4-fold (mutagenic) <strong>and</strong> at 50-100 ug/ml it decreased<br />
the mutation rate 2-fold (antimutagenic) . The concentration range of 50-100 ug/ml<br />
causes a delay in cell generation time (from 1 .8 hr to 2 .7 hr), which is a decrease in<br />
the rate of cell division . These results implicate an interaction between DNA repair<br />
<strong>and</strong> the delay in the cell cycle in the mechanisr of antimutagenesis when chemicals are<br />
involved . (This research was supported by a grant from the Natural Sciences <strong>and</strong><br />
Engineering Research Council of Canada) .<br />
620<br />
EFFECTS OF CHEMICAL 1eDTAGENESIS ON TREATINO ZYOOTES OF PIANTS . Wang Qinnan . Institute of<br />
Genetics, Academia Sinioca, Beijing .<br />
According to the theory of mosaicism, some investigators have considered that treatment<br />
of zygotes may afford an opportunity to increase the mutation rate . Therefore we have<br />
designed experiments to study the effect of treating zygotes with a chemical autagen .<br />
We used wheat zygotes <strong>and</strong> dry seeds <strong>and</strong> the mutagen EMS . In addition, the wheat zygotes<br />
were given bleomycin (BLM) post-treatment so as to enhance the induction of mutations .<br />
The results obtained indicated that treatment of zygotes with EMS at any stage is<br />
superior in terms of creating mutations as compared to the control . The treatment of<br />
zygotes inducas much higher mutation ratios in the eS3 generation as compared with treating<br />
dry seeds, <strong>and</strong> brings about a broader mutational spectrum . In accordance with the effects<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 213<br />
Notes<br />
t71 F9a<br />
m F,<br />
00<br />
I:<br />
1'
214 1989 EMS Abstracts<br />
---~- .~-- _-<br />
Notes-<br />
of' mutation in ~e [f, the different mutation induction methods used in the experiments<br />
can be arranged in the following sequence : treating zygotes at late stage > treating<br />
zygotes at early a~a~treating dry seeds . The results indicated also that the zygotes<br />
receiving the BL4 post-treatment seem to be equipped with an imperfect DNA-repair system<br />
of enzymes because their mutation ratio is not obviously higher than that of the variant<br />
zygotes not receiving Bt2t post-treatment .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
- 621--<br />
A cTUDY OF SCE IivDUC'PION BY TRIPTi•,'RYGIUM HYPOGLAUCUM(LEVM . ) HUTCH IN<br />
HUMAN PERIPHERAL BLOOD LYMPHOCYTES<br />
Wang Xu, Zhou Rumin <strong>and</strong> Ceo Neng . Department of Biology, Yunnan<br />
Normal University, Kunming, Yunnan, Peo le's Republic of China .<br />
Tripterygium hypoglaucum (Level .) Hutch (pTHH) is a fChinese tr<strong>and</strong>itional<br />
medicine which has been used for the treatment of various<br />
diseases such as systemic lupus erythematosus, rheumatoid arthritis etc .<br />
The medicine showed antifertility effects both in rat <strong>and</strong> human during<br />
the medicine taken period . No other cytotoxic effects were reported .<br />
Our work aimed at the cytogenetic research of THH . The presented<br />
work emploged the sister chromatid exchanges (SCEs) to assess its<br />
genotoxic effects in human peripheral blood lymphocytes .<br />
Blood for all the experiments were obtained from both healthy donors<br />
<strong>and</strong> patients cured with TH .9 for over 90 days <strong>and</strong> treated by THH with<br />
the doses from 200 to 0 .63mg/ml in vitro . The blood from every donor<br />
were cultured in two ways of presencing 69 mix or not .<br />
The results indicated THH didn't induce SCE siRgnificantly in human<br />
lymphocytes from healthy donors <strong>and</strong> patients cured with THH before<br />
sampling . S9 had no effects on SCE induction by THH in all the cases .<br />
However, THH showed a strong cell cycle delay effects when higher<br />
treating doses from 200 to 10mg/ml were used in vitro .<br />
Our results suggest that THH could be a human anti-baby medicine .<br />
Further research about its other cytogenetic <strong>and</strong> cytotoxic effects are<br />
being done in our lab .<br />
622<br />
STUDIES ON THE NUTAGENTC . CARCINOGFNIC AND TI]tATOGDU C EFFSCTS OF l,2-UD101 AND ETEPA<br />
Wang Zhi-qlao, Li Xiu-yin', tlen Si-zhes, Liao Nia'-yaaq<br />
The studies of mutayeaic effects of 1,E-UD101 <strong>and</strong> setepa were investi'ated by Anes test,the bone<br />
narrow micronucleus test of sice,saalysis of bone marrow chranosome aberration of rats,analysls of<br />
chromosome aberration <strong>and</strong> SCE test In bn .aa peripheral blood lymphocytes, analysis of chromosone<br />
aberration <strong>and</strong> SCE of sice spersatoyenous cetls, the do .inant lethal test of sice . The results<br />
showed 1,=-UDIDf was re0ative <strong>and</strong> metepa was positive In all test systens . Nice I drinkiny water<br />
freely for one year which contained 1,2-I1DIIX at the coaceatratlon of 30-300my/1 highly Induced<br />
beaangiaatous lesions of liver <strong>and</strong> adeaotous changes of Lung . The lumoriyenicity of inetepa did<br />
not be induced at the concentration of 1-10n9i1 during experinrat of one year .The teratoyenic<br />
test of rats showed setepa maialy Induced limb ∎alfornation whea It was i .p . ad.laistered at<br />
the dosage of 30sy/kg.Tle malformation frequency was 9f% .aad 1 .=-UDlDI had so teratoyenic effects .<br />
The further study with rabbits showed metepa had so terato'enic effects . The results of the<br />
study Indicated that 1,2-UDIOi ∎ibd belong to epioenetic carcinogen <strong>and</strong> netepa might belong<br />
to carcinogen with mutaoenesis .<br />
623<br />
INDUCTION OF THIOGUANINE RESISTANT MOUSE LYMPHOCYTE VARIANTS BY SUB-<br />
CHRONIC LoW DOSE INHALATION EXPOSURE TO BENZENE . J . B. Ward Jr ., M . M .<br />
Ammenheuser, D . L . Morris, V . M . S . Ramanujam, <strong>and</strong> M . S . Legator Dept .<br />
of Preventive Medicine <strong>and</strong> Community Health University of Texas Medical<br />
Branch, Galveston, TX 77550, USA .<br />
Spleen lymphocytes of CD-1 male <strong>and</strong> female mice exposed to benzene<br />
vapor were evaluated for the frequency of thioguanine resistant variants<br />
(Vf) . The nice were exposed, by inhalation, 22 hours per day for six<br />
weeks to benzene concentrations of zero (air only), 40, 100, <strong>and</strong> 1,000<br />
parts per billion (ppb) or were housed in st<strong>and</strong>ard cages (cage<br />
controls) . An autoradiographic assay for Vf was used with cryopreserved<br />
cells from three animals of each sex at each exposure . The mean Vts'were<br />
zero, 7 .04 ; 40 ppb, 26 .25 ; 100 ppb, 52 .10 ; 1,000 ppb, 20 .39 <strong>and</strong> cage<br />
controls 13 .25 . Vts in females were consistently higher than in males in<br />
benzene exposed animals but not in controls . Confidence intervals (95%)<br />
50869 3728
i<br />
on means at 0, 40, <strong>and</strong> 100 ppb did not overlap . Because the increased<br />
Vfs could have been due to increased in vivo cell cycling, four<br />
conditions were evaf'aated in which the frequency of cycling cells was<br />
determined without In vitro mitogenic stimulation . Benzene exposure did<br />
not stimulate cell cycling . Because the autoradiographic assay does not<br />
permit the recovery of variant cells for confirmation of mutant genotype<br />
these results must be interpreted cautiously . However, they do suggest<br />
that in vivo benzene exposure at low levels may be mutagenic to motLse<br />
spleen lymphocytes . Supported by the Texas Air Control Board .(•vf x 10 )<br />
624<br />
0-VANILLIN ENHANCES MUTAGENESIS INDUCED BY MNNG IN ESCHERICHIA COLI .<br />
K . Watanabe, T . Ohta, T . Kato, M . Watanabe <strong>and</strong> Y . Shirasu, Institute of <strong>Environmental</strong><br />
Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan)<br />
2-hydroxy-3-methoxybenzaldehyde (o-vanillin), the antimutagenic effect of which has<br />
been reported on mutagenesis induced by 4-nitroquinoline 1-oxide (4NQO) in Escherichia<br />
coli WP2s, enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in<br />
the same strain . In order to investigate the mechanism of the enhancing effect of ovanillin<br />
against MNNG-induced mutagenesis in E . coli, we have carried out experiments .<br />
Among 7 derivatives of o-vanillin, 2-hydroxy-3-ethoxybenzaldehyde, o-hydroxybenzaldehyde<br />
<strong>and</strong> m-methoxybenialdehyde showed an enhancing effect on MNNG-induced<br />
mutagenesis in E . c-o -li WP2s . A remarkable enhancement of mutagenesis provoked by Nmethyl-N-nitrosourea,<br />
a methylating agent, was also observed on semi-enriched minimal<br />
agar plates containing o-vanillin in E . coli WP2s . On the contrary, o-vanillin<br />
suppressed greatly furylfuramide- <strong>and</strong> 4NQ0-induced mutagenesis <strong>and</strong> showed a slight<br />
suppressing effect against mutagenesis induced by methylmethanesulfonate, N-ethyl-N'nitro-N-nitrosoguanidine<br />
<strong>and</strong> N-ethyl-N-nitrosourea . In an investigation employing the<br />
various repair-deficient mutants of E . coli B/r, clear enhancement effects by ovanillin<br />
were observed in wild-type atrain E . coli B/r WP2 <strong>and</strong> its 4 mutants, WP2s<br />
uvrA, ZA60 recA, ZA12 umuC <strong>and</strong> ZA160 polA, whereas a weak enhancement was observed in<br />
E. coli ZA180 ada-5, ZA113 alkA <strong>and</strong> ZA130 alkB, all of which cannot induce the<br />
aZcapEive response o alkyla~fon damage . TFiese results may suggest that o-vanillin<br />
inhibits the inducible adaptive response. •<br />
625<br />
NEW MODIFIED STRAINS OF SALMONELLA TYPHIMURIUM TA98, TA100, VERY SENSITIVE TO<br />
NITROARENES AND AROMATIC AMINES BY CLONING OF ACETYLTRANSFERASE GENE .<br />
M . Watanabe, M . Ishidate, Jr ., <strong>and</strong> T . Nohmi, National Institute of Hygienic<br />
Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)<br />
Acetyl-CoA :N-hydroxyarylamine 0-acetyltransferase ia known ae an enzyme<br />
involved in the intracellular metabolic activation of mutagenic nitroarenes <strong>and</strong><br />
aromatic amines . The acetyltranaferase gene of S . t yphimurium TA1538 was cloned<br />
into pBR322 (Biochem . Biophys . Res . Commun . 147, 974-979 (1987)), <strong>and</strong> the<br />
plasmid harboring the gene (pYG219) was introduced into TA98 <strong>and</strong> TA100 . The<br />
resulting strains, YG1024 (- TA98(pYG219)) <strong>and</strong> YG1029 (= TA100(pYG219)), had a<br />
higher isoniazid- <strong>and</strong> 2-aminofluorene-N-acetyltraneferaee activity 100 timee<br />
more than the original strain, TA1538(pBR322) . They showed an extremely high<br />
mutagenic response to 2-nitrofluorene, 1,8-dinitropyrene, Glu-P-1(+S9) <strong>and</strong> 2aminoanthracene(+S9)<br />
. The YG1024 was more sensitive to these chemicals than<br />
TA1538/1,8-DNP(pYG121 or 122) strains which we previously established, since the<br />
YG1024 has pKM101 <strong>and</strong> more acetyltransferase activity . These results indicate<br />
that the new strains are useful for the detection of mutagenic nitroarenes <strong>and</strong><br />
aromatic amines .<br />
This work was supported by a Grant-in-Aid from Japan Health Sciences Foundation .<br />
626<br />
NITROARENE SENSITIVE SALMONELLA TYPHIMURIUM STRAINS YG1021 AND YG1026 WITH A<br />
HIGH NITROREDUCTASE ACTIVITY, DERIVED FROM TA98 AND TA100, RESPECTIVELY .<br />
M . Watanabe, M . Ishidate, Jr ., <strong>and</strong> T . Nohmi, National Institute of Hygienic<br />
Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)<br />
"Classical nitroreductaae" is known as an enzyme involved in the<br />
intracellular metabolic activation of mutagenic nitroarenes . The nitro :eductase<br />
gene of Salmonella t himurium TA1538 was cloned into pBR322 (Biochem . Biophys .<br />
Res . Commun . 147, 974-979 1987)), <strong>and</strong> the plasmid harboring the gene (pYG216)<br />
was introduced into TA98 <strong>and</strong> TA100 . The resulting strains, YG1021 (-<br />
TA98(pYG216)) <strong>and</strong> YG1026 (- TA100(pYG216)), had about 50 times higher<br />
nitrofurazone-reductase activity than TA1538 containing pBR322, <strong>and</strong> were<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
•<br />
1989 EMS Abstracts 215<br />
Notes
216 1989 EMS Abstracts<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
. . ~<br />
--<br />
_ . .-<br />
Aztiemely aensi~iVe ~the mutagenio action of 2-nltrofluorene, 1-nitropyrene<br />
<strong>and</strong> 2-nitronaphthalene . The YG1021 was more sensitive to these chemicals than<br />
TA1538NR(pYG111) gtrjAli~w_hich we previously established, since the YG1021 has<br />
pKM101 . Theae reanlts indicate that the new etrains permit the efficient<br />
detection of mutagenic nitroarenea in the environment .<br />
This work was supported by a Grant-in-Aid from Japan Health Sciences Foundation .<br />
- 627<br />
A SURVEY OF pCTIVITY PROFILES OF A(~TIMUTAGENS/ANTICARCINOGENS .' Waters, M .1, Brady,<br />
A ., Stack, F .2, <strong>and</strong> Brockman, H .3 ; U .S . Environ . Prot . Agency, Res . Tri . Park, NC ;<br />
2Environ . Hlth . Res . & Testing, RTP, NC ; 3111 . State Univ ., Normal, IL (USA) .<br />
Experimental evidence suggests that chemical mutagenesis <strong>and</strong>•carcinogenesis are<br />
related phenomena such that identification of causative agents <strong>and</strong> protection from<br />
exposure might prevent certain human cancers <strong>and</strong> related diseases . Agents that<br />
inhibit the processes of mutagenesis <strong>and</strong> carcinogenesis, particularly naturally<br />
occurring agents, might be expected to exert a primary protective effect . This paper<br />
will consider the use of short-term bioassays to identify antimutagenic <strong>and</strong><br />
anticarcinogenic substances <strong>and</strong> to classify them [cf . Mutation Res . 168 (1986) 47-65]<br />
according to the locus of their rotective influence, i .e ., intracellular or<br />
extracellular, <strong>and</strong> putative mechanismrs) of action . In the extracellular environment,<br />
inhibition of formation or uptake of mutagens <strong>and</strong> inactivation of promutagenic or<br />
mutagenic species are examples of antimutagenic mechanisms . Intracellularly,<br />
antimutagenic substances have been described as "scavengers" of radicals, "blocking<br />
agents" (involving at least 3 different mechanisms), <strong>and</strong> "suppressing agents" .<br />
Additional intracellular mechanisms include alterations in DNA repair processes <strong>and</strong>/or<br />
modification of the genotoxic response to the mutagen/carcinogen . The presentation<br />
format will be based on the genetic activity profile (GAP) methodology developed by<br />
the authors . The GAPs of known mutagens/carcinogens will be presented together wi ht<br />
newly designed profiles for antimutageni/anticarcinogens according to the<br />
classification scheme outlined above . This is an abstract of a proposed<br />
presentation <strong>and</strong> does not necessarily reflect EPA policy .<br />
628<br />
MOLECULAR CHARACTERIZATION OF ERCC2- A HUMAN NUCLEOTIDE EXCISION REPAIR GENE WITH<br />
HOMOLOGY TO THE YEAST RAD3 REPAIR GENE . C.A . Weber, E .P . Salazar, <strong>and</strong> LH. Thompson, Biomedical<br />
Sciences Division, Lawrence Livermore National Laborabry, Livermore, CA (USA)<br />
The UV-sensltNe Chinese hamster ovary (CHO) cell line mutant UV5 has a defect In the Incision step<br />
of nucleotide excision repair. This same process is defeotive in cells from patients with the cancer-prone<br />
genetic disorder xeroderma pigmentosum (XP) . Genomlc clones of the human ERCC2 (Exdslon gepair<br />
~Qross .QomplemenUng) gene that fully complement the UV5 repair defect in a stable manner have been<br />
isoiated <strong>and</strong> characterized (Weber et al ., 1988 . Mol. Cell. Biol . 8 :1137-1146) . The genomic clones<br />
were used to Isolate nine ERCC2 clones from the pcD2 human cDNA expression library (obtained from H .<br />
Okayama) . One of the cDNA clones, pER2-14 (inserl size . 2.6 kb), was found to confer transient UVresistance<br />
of an intermediate level to UV5 cells . The nucleolide sequence of pER2•14 <strong>and</strong> genomic 5'<strong>and</strong><br />
3'-flanking regions was determined . Consensus regulatory signals were Identified for a GC box, CAAT<br />
box, TATA promoter, <strong>and</strong> polyadenylation sNe . Translation of the open reading frame <strong>and</strong> comparison<br />
with sequenced yeast nucleotide excision repair genes revealed a striking homology between ERCC2 <strong>and</strong><br />
RAD3 (-52% Identical ; 760 aa vs . 778 aa) . This comparison, along with examination of the nucleotide<br />
sequence for splice junctions, also revealed that pER2-14 contains part of Intron 1 <strong>and</strong> produces a<br />
protein that is 24 amino acids short at the amino terminus (738 aa) . This presumably accounts for the<br />
transient nature of the UV-resistance conferred to UV5 alis by pER2-14 . The RAD3 protein has both a<br />
repair <strong>and</strong> an essential function <strong>and</strong> has both a single-str<strong>and</strong>ed DNA-dependent ATPase activity <strong>and</strong> an<br />
ATP-dependent helicase activity . Construction of a cDNA clone with genomic 5'•flankinp sequences that<br />
will produce a full-length protein Is underway . This clone will be used to test for correction of the<br />
repair defect in the CHO UV5 mutant <strong>and</strong> In the XP complsmentatkan groups. Work performed under the<br />
auspices of the U .S . Department of Energy by LLNL under contract NW-7405-ENG-48 .<br />
MECHANISMS AND PREVENTION OF FORMATION, AND METABOLISM OF FOOD<br />
MUTAGENS/CARCINOGEN 2-AMINO-3-METHYLIMIDAZO[4,5-]QUINOLINE (IQ) .<br />
J .H . Weisburger, R .C . Jones, H .J . Luke, T . Vavrek, American Health<br />
Foundation, Valhalla, NY 10595 USA .<br />
We have postulated that IQ <strong>and</strong> related imidasoasaarenes, formed<br />
during frying or broiling of meat <strong>and</strong> fish, may be the genotoxic<br />
carcinogens associated with important types of cancer in humans, like<br />
in the breast, colon or pancreas (Prev . Med . 16 :586,1987) . Their<br />
formation appears to involve Maillard reactions, with creatinine as<br />
critical reactant . In laboratory models involving high temperature<br />
50869 3730<br />
629
1989 EMS Abstracts 217<br />
reflux of glycine, glucose <strong>and</strong> creatinine, or in realistic frying of Notes<br />
meat, competition with creatinine by L-tryptophan, L-proline, or both<br />
has lowered the formation of IQs considerably . This approach provides<br />
a feasible, practical means of inhibiting the production of IQs . The<br />
metabolism of IQ in rats yields evidence of N-acetylation, N-demethylation,<br />
hydroxylation on carbon 5 (excreted as O-glucuronide <strong>and</strong> 0sulfate)<br />
<strong>and</strong> intestinal flora-mediated formation of 7-OH-IQ . Xanthineoxidase<br />
reduction of 2-NO2-IQ yielded intermediates reacting with alfthymus<br />
DNA, <strong>and</strong> giving 5 products, visualized upon hydrolysis by 3~Ppostlabeling<br />
. Microsomal metabolism of IQ yielded the same 5 products,<br />
CAd242i? anathOeAr~21~11fs~rNCampuntg`(supported in part of USPHS grants<br />
630<br />
DICHLOROMETHANE-INDUCED CYTOGENETIC DAMAGE IN HICE . B . Westbrook-Collinsl, J .W .<br />
Allenl, A .D . Kligermanl, J .A . Campbell2, G .L . Erexson2, F . Kari3, <strong>and</strong> E . 2eiger3 .<br />
1U .S . <strong>Environmental</strong> Protection Agency, RTP, NC 27711 USA ; 2<strong>Environmental</strong> Health<br />
Research <strong>and</strong> Testing, Inc ., RTP, NC 27709 USA ; 3National Institute of <strong>Environmental</strong><br />
Health Sciences, RTP, NC 27709 USA .<br />
Dichloromethane (DCM) or methylene chloride is a widely used industrial solvent<br />
which causes lung <strong>and</strong> liver tumors in inhalation-exposed B6C3F1 mice (NTP, 1986) . In<br />
the present studies, chromosome damage was studied in female 86C3F1 mice exposed to<br />
DCN by subcutaneous or inhalation treatments . After a single subcutaneous injection<br />
of either 2,500 or 5,000 mg/kg DCM, no increases in frequencies of sister chromatid<br />
exchanges (SCEs) or chromosome aberrations (CAS) in bone marrow cells were observed .<br />
Inhalation exposure to DCM for 10 days at doses of 4,000 or 8,000 ppm resulted in<br />
significant increases in frequencies of SCEs in lung cells <strong>and</strong> peripheral blood<br />
lymphocytes, CAs in lung <strong>and</strong> bone marrow cells, <strong>and</strong> micronuclei (MN) in peripheral<br />
blood erythrocytes . The highest levels of increased damage ware observed for lung<br />
cell CAs <strong>and</strong> blood erythrocyte MN (approximately two times control frequencies) <strong>and</strong><br />
bone marrow CAs (approximately four times control frequency) after 10 days .<br />
Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but<br />
significant increases in lung cell SCEs <strong>and</strong> peripheral blood erythrocyte MNr . It was<br />
concluded that DCM is clastogenic following in vivo inhalation exposure . This<br />
finding suggests that genotoxicity may play a role in the carcinogenic properties of<br />
DCM in B6C3F1 female mice . (This is an abstract of a proposed presentation <strong>and</strong> does<br />
not necessarily reflect U .S . EPA policy.) A<br />
631<br />
CHARACTERIZATION OF THE IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY USIYG PRIMARY LUNG<br />
CELLS OF THE RAT . W-2 . Whong, J .D . Stewart, <strong>and</strong> T . OnB, Division of Respiratory<br />
Disease Studies, National Institute for Occupational Safety <strong>and</strong> Health, Morgantown,<br />
WV (USA)<br />
Since genotoxic agents can be present in the environment as gases, vapors, <strong>and</strong><br />
airborne particles, inhalation becomes an important exposure route with the lung as a<br />
major target for such genotoxicants . Therefore, primary lung cells, which retain<br />
most of their original metabolic ability, may provide a useful cell system for evaluating<br />
the pulmonary genotoxicity of chemicals with different genetic endpoints .<br />
Previously, we had established the micronucleus <strong>and</strong> sister ehromitid exchange assays<br />
in rat primary lung-cell cultures . In the present study, effort was made to incorporate<br />
the in vitro unscheduled DNA synthesis (UDS) assay in the same cell culture<br />
system . The autoradiograph method employed for detecting UDS was characterized, <strong>and</strong><br />
the assay was tested with a direct <strong>and</strong> an indirect-acting genotoxicants in both<br />
alveolar macrophages <strong>and</strong> primary lung cells from rats . Data of a tisas oourse study<br />
revealed that the optimal radioactive labeling was achieved after a 16-b incubation<br />
with 3H-thymidine . Hydroxyurea at the concentration of 20 mlK effectively inhibited<br />
the regular DNA synthesis . With this protocol, a dose-dependent UDS was induced by<br />
Y-methyl-N'-nitroso-N-guanidine <strong>and</strong> 2-aminoanthracene in both rat alveolar sucrophages<br />
<strong>and</strong> primary lung cells . These results indicate that primary rat lung cells in<br />
culture possess DNA repair ability <strong>and</strong> that the UDS assay may be useful for assessing<br />
the genotoxic effect of chemicals in this cell system .<br />
632<br />
:.h-LCCUS LItdI:E.') 'lI^7';'-M?C_-S IPT IN')UC_"OTT OF SCE 'nY °-1tT?C ;a ;w`MTE IN<br />
f•:CUS : aaE 3^.^ .TE:. IN "IN VIVO" AS CC".?:'AYED TC "IN VI^_RO" T--S'S . S .M,<br />
WLelgosz Pnd A .L.P?vlak, Inatitute of Hrnman GPnetics, ^olirh Ac^de^!y<br />
of Sci,~nces, u_.7_9trzeszynskn 32, 60-479 ?ozn .!)A (Polend)<br />
The e°fect of alleles of Ah lecur on the induction of SC- - as •tudied<br />
in C57316 <strong>and</strong> in DBA2 mice +.•r.eeted t .a cs ' . :ith ben~o(^!pyr^nc (EY,<br />
1C0 m,- -,Pr 1cg, p .o . ) . ='or mea-r.^em^nt^ of ch-n!;eo in fre-uency of SCE<br />
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218 1989 EMS Abstracts . - --<br />
:<br />
Notes ~ t . :o pro':oco?.s '-re u^ed : 1 . for "in v!vo" n-,n-uremcnt In hone m~arrorr<br />
ec' .1c 50 .mg V)iAte of rromo9.coxyuri3inc (BrdU) •.•rre implant^d 2Q4 h beio--'c<br />
'"±'~-ir~ o`"^n3'~^1^ ; 2 . `.or "in vitro" ~+e?-ttrement ^plecn 1ymFhocvtee<br />
t•rere cu].tvre :? ~~Str °r' (6/ .tz ~er ml) . The induction of SCy in<br />
~-tre^.ted ^.^7LJ.C ^nd ^7A^ mice "in vivo" :,^s 1 .21 ^nd ti .07-fold, roo-<br />
~°etively . "In vitro" th^ re~ictive v^?t .ies •-erc 1,^9 <strong>and</strong> 2 .73 .<br />
The ob~-erved ?nter^trpin differences In in3action of SC2, were much<br />
Qre^.t^r 7n hone n .r=ov~ "in v3vo" ^ .- compared to ^rleen 1•m?hoc?rte3<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
ttin vi t'"o" "2re A+rr„r^•tCe T~a•r re .%tlt frOm ~^.~ f^Ct fi .^t At+_ring 'tin<br />
vi tro" teqt cel?.r P_o not e :~o'rcd tc =1, a- c•ell ?^ from hicher fre-<br />
^uencieo of SC2 !n ce].13 culturQd " .'.n v!tro" . "_'!•ir• mey Indicate th2t<br />
z.so in c;m V+.e 'in vivo" tent3 !b :° aasa^ament of 7henotype rt Ah locus<br />
(e . - . antip••r
635 =<br />
a.<br />
1989 EMS Abstracts 219<br />
Notes<br />
EVALUATION OF FURAN-INDUCED GENOTOXICITY AND CELL PROLIFERATION IN RAT AND HOUSE HEP-<br />
ATOCYTES . D .H . Wilson, <strong>and</strong>,. .B .E . Butterworth, CIIT, Research Triangle Park, NC (USA) .<br />
Initial observations from bioassays by the National Toxicology Program indicate<br />
that furan produces hepatocellular carcinomas in male F344 rats <strong>and</strong> in male <strong>and</strong> female<br />
B6C3F1 mice . Although furan is negative in the Ames test, the profile of autations<br />
produced in oncogenes from furan-induced tumors are different from those of spontaneous<br />
tumors (Reynolds et al, Science 237, 1309 (1987)) . To investigate the possible<br />
mechanisms by which furan causes tumors, genotoxicity, as indicated by DNA repair, <strong>and</strong><br />
chemically-induced cell proliferation were examined . Prijary hepatocyte cultures<br />
from male F344 rats were incubated with faran <strong>and</strong> 10 LCi/ml H-thymidine . DNA repair<br />
as unscheduled DNA synthesis (UDS) was quantitated autoradiographically as not grains<br />
per nucleus (NG) . Concentrations from 1 YH to 10 aH furan yielded no UDS in vitro . To<br />
examine UDS in vivo, furan was administered by gavage to male rats<br />
(5, 30 <strong>and</strong> 100 mg/kg) <strong>and</strong> to male B6C3F1 mice (10 . 50, 100 <strong>and</strong> 200 ag/kg) twelve hours<br />
prior to isolation of hepatocytes . No increase in NG was observed in hepatocytea from<br />
furan-treated animals compared to r3ontrols . Cell proliferation in vivo was evaluated<br />
by autoradiographic analysis of H-thymidine incorporation in cultures of primary<br />
hepatocytes isolated 36 hours after a single gavage administration of furan (30 mg/kg)<br />
to male rats . Hepatocytes from vehicle treated rats had a labeling index (LI) of<br />
0 .2+0 .1X while that of furan-treated rats was 17+5% . Taken together, available dats<br />
suggest that the distinct profile of oncogenes from furan-induced tumors may have<br />
resulted from enhanced susceptibility of those genes to mutations related to forced<br />
cell proliferation rather than from direct mutagenesis by furan . Puran-induced cell<br />
proliferation may also have been a driving force in tumor promotion <strong>and</strong> progression .<br />
636<br />
THE CYTOGENETIC EFFECI'S OF INTRODUCING RESTRICTION ENZYMES INTO CHO CELLS<br />
USING ELECTROPORATION. RA Winegar H.W. Chung, J .W. Phillips <strong>and</strong> W.F. Morgan, Laboratory of<br />
Radiobiology <strong>and</strong> <strong>Environmental</strong> Health, University of California, San Francisco, CA (USA)<br />
Studies using restriction enzymes to examine the mechanisms of cytogenetic damage have been hampered<br />
by the inefficiency of available permeabilization techniques . We have used cell electroporation with great<br />
success to permeabilize Chinese hamster ovary (CHO) cells for the introduction of restriction enzyptes . Cells<br />
electroporated in the presence of restriction enzymes show an extremely high frequency (>90%) of aberrant<br />
njetaphases as well as a dramatic decrease in cell survival. Electroporation itself caused no increase in<br />
aberrant chromosomes <strong>and</strong> had only a slight effect on cell survival . The isoschaomer pair Msp I <strong>and</strong> Hpa II<br />
was used to determine whether restriction enzymes act with the same sprcificity within a cell as in vitro . Both<br />
enzymes recognize the DNA sequence CCGG, but whereas Hpa II will not cleave this sequence if the internal<br />
cytosine is methylated, Msp I will cleave regardless of the methylation status of the internal cytosine . As<br />
expected, since this sequence is heavily methylated in mammalian cells, Msp I was greatly more effective than<br />
Hpa II at inducing chromosome aberrations . Although restriction enzymes are very effective at inducing<br />
chromosome aberrations, experiments using a large number of different enzymes <strong>and</strong> several different harvest<br />
times indicated that restriction enzymes do not induce sister chromatid exchanges . This is consistent with<br />
previous reports that agents that produce double-str<strong>and</strong> breaks do not induce sister chromatid exchanges .<br />
Work supported by the Office of Health <strong>and</strong> <strong>Environmental</strong> Research of the U .S. Dept of Energy under<br />
contract DE-AC03-76•SF01012 .<br />
637<br />
ISOLATION OF cONAs ASSOCIATED WITH TUMOR SUPPRESSOR GENE FUNCTION IN SYRIAN HAMSTER<br />
EMBRYO CELLS . R .W . Wiseman, J .C . Montgomery, J . Hosoi, E .W . Hou, J .E . Bisi, <strong>and</strong><br />
J .C . Barrett, Laboratory of <strong>Molecular</strong> Carcinogenesis, National Institute of <strong>Environmental</strong><br />
Health Sciences, Research Triangle Park, NC 27709 .<br />
Loss of a tumor suppressor gene function appears to be a critical step in Syrian<br />
hamster embryo (SHE) cell neoplastic transformation in vitro . In order to underst<strong>and</strong><br />
the function of these genes, we have isolated preneoplastic clonal variants<br />
from 2 imnortal SHE cell lines that have either retained (supB+) or lost (supB-) the<br />
ability to suppress the tumorigenicity of the BP6T tumor line in cell hybrids . cONA<br />
probes prepared from polyA+ RNA of supB+ <strong>and</strong> supB- cells have been used to screen a<br />
supB+ Lambda Zap cDNA-library for clones that are preferentially expressed in supB+<br />
cells . cONAs for at least 4 independent genes have been isolated, <strong>and</strong> DNA sequence<br />
analyses revealed that 2 of these encode proal(II) <strong>and</strong> al(IX) collagens, which are<br />
normally expressed in chondrocytes . Another cDNA was unrelated to any known gene<br />
while the fourth cDNA was homologous to the mouse H19 gene, which is developmentally<br />
regulated in liver <strong>and</strong> muscle cells . In complementary studies for the identification<br />
of human tumor suppressor genes . Syrian hamster tumor cells have been utilized as<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
220 1989 EMS Abstracts, . ~ --<br />
. : "'<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
. . . , .. . j. _ - .,r~ ~<br />
NOtES gene transfer recipients for a normal human fibroblast cDNA library prepared in the<br />
pc02 expression y~ect~ Using a low serum/BUDR/nUV light selection, several Independent<br />
cDNA-induce$BM revertants have been isolated from cultures transfected<br />
with the pcD2 cDNA library that were greater than 1000-fold suppressed for anchorage<br />
independence relative to the parental BHKA tumor cells . Progress in characterization<br />
of these cONAs associated with tumor suppressor gene function will be presented .<br />
- 638<br />
SEIECPION OF VF111CL.E MD THE AOfRE OF M14MSTRATICt!1 IN T1B; MD(JSE BfltE MARRQW<br />
MICRONUCLEUS TEST . J .P . Wojciechowski+, D .K . Gulatil, <strong>and</strong> M D . Shelb jy<br />
l<strong>Environmental</strong> Health Research <strong>and</strong> Testing, Inc ., Lexington, KY ~itre National<br />
Institute of Environnental Health Sciences, Research Triangle Park, NC .<br />
Two critical factors that influenoe the swrimum exposure of the target tissue<br />
with the test material in the bone marrow micranucleus test are the route of<br />
administration <strong>and</strong> the vehicle . The purpose of this study was to determine the<br />
extent to which vehicle <strong>and</strong> route of administration affect level of effect .<br />
Dimethylbenza(a)anthraoene (DNIDR), N,N'-methylenebisacrylamide (MR),<br />
Nynethylolacrylamide (NOA), <strong>and</strong> 4-aminobiphenyl (ABP) were tested using a variety of<br />
vehicles <strong>and</strong> two different routes of ad+ninistration . DlIBA suspended in corn oil <strong>and</strong><br />
administered via lP at 10 ug/kg caused a 2 .9 told increase in the incidence of<br />
micronucleated polychronatic erythrocytes (Md-FCE) over the vehicle control . D)a3A<br />
dissolved in Dr9O (lOmq/kg) via lF resulted in an 11 .3 fold increase in t4}-FCE . l81R<br />
in DMSO via lF had no effect on Mi-FM (p>0 .1) at up to SOsiq/kg . MBA dissolved in<br />
PBS (50mg/kg) caused a 16 fold increase in FQ}-PCE's . 143ii dissolved/suspended in<br />
PBS, corn oil, or D6s90 <strong>and</strong> administered via lP at 112 .5 sq/kg gave a negative<br />
response for M-FCE. MON in PBS via gavage caused a weak but significant increase<br />
(p
transformation . The BALB/c 3T3 clone A31-1-13 cells were exposed to coal dust<br />
extract for 72 hrs . At th:--end of treatment, the coal dust extract was removed, <strong>and</strong><br />
cells were cultivated for 4 weeks with twice weekly medium change . Type III foci<br />
were scored after fixation <strong>and</strong> staining . Results showed that coal dust extracts,<br />
both nitrosated <strong>and</strong> nonnitrosated, induced eell transformation in a dose response<br />
manner . However, the transformation frequency was much higher with the nitrosated<br />
than the nonnitrosated coal dust extracts . All transformed eell lines derived from<br />
coal dust extract-induced foci showed characteristics of malignant transformation .<br />
The transforming activity of coal dust extract decreased when chlorophyllin was<br />
incorporated into the treatment solution .--These results seem to support the coal<br />
mine dust hypothesis for the causation of gastric cancer in coal miners <strong>and</strong> indicate<br />
that chlorophyllin possesses an antitransformation activity .<br />
641<br />
RE6P:ARCiI ON MUTAGENICITY OF POLLUT'r.D WATER TREAT6IENT WITH FIOLOGICAL OXIDATION<br />
:+u Guopine, Hong Huacheng, Lu Ying, <strong>and</strong> Yue Shenling, ShanPhai Municipal Waterworks<br />
Co . Shanfhei, China<br />
In this paper, the efficiency of different water purification processes in<br />
:emovinF organic muteger.s from raw water was introduced . We use initial "0"--<br />
ozonation ; "T"-- trcditional rrocess ( comFiro+ion of coa!-ul .;tion,sedimentetion<br />
c : .d filtration ) ; "C1"--chlorinQtion ; "F"-- bio_*iltratlon <strong>and</strong> "C"--bioactive<br />
cerbon filtration . Thus, the 4 test systems (• combined processes) are 07C1,<br />
fi9`Cl, FTCC1, <strong>and</strong> BTOCC1 .<br />
Reeults showed that OT <strong>and</strong> FT processes were not al~le to chanre the mute'enic<br />
nctivities of w~ter . One phenomenon a•e Pre interested in ie that the nurber<br />
_' t^e revertents of the e:nters treated by OT <strong>and</strong> FT processes is approxime.•el;•<br />
identical . However, after chlorination, the numFer of OTC1 effluent raised so<br />
rapiely that it beceme more than thet of rew w .
222 1989 EMS Abstracts «_- ~,,,E:- - -<br />
Notes gation of the best pez'imental conditions of mouse testes<br />
chro :aosotnal assajr . .~..~the first part of experiment according<br />
to an orthogonal design,three factors including dosa„e<br />
of colchicine,effective time of colchicine .nd time of<br />
sucrifice of mouse(8time point~ during 24 hoars) are stuuied<br />
at c: nixin- level of L 32 (8x4 ) design . t)e criteria<br />
adooted is the number of metaphase I emer . ;,ed under every<br />
1J'!,) fields of hi„h power version . In the sen - .' r :rt :~.' .<br />
experiment,Tl,I mice were treated with :Zitomycit . J <strong>and</strong> then<br />
sacrificed separately after 1,3,j,9,9 t.nd 11 days,the aberration<br />
frequency of 100 metaphase I was an,3ljsed of each<br />
,nouse . The result demu .sir :.tnd that no stu;istical si ;;nificant<br />
effect upon the frequoncy o° ^let-tphase i were found<br />
b,j tlte three factors mentioned sbove <strong>and</strong> -ire-itive result<br />
could be abt-,ined it 5-1} d--i,is n-fter treltmer.t,but the hi ;-hest<br />
r-~te o f chroc,osor :r_il .berr .ition is found at tce 1 1 th dkj .<br />
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THE RESEARCH OF LETHAL Nl!TATION OF<br />
SODII'N FLUORIDE PSI\G SEX-LIM1'KF .D RECESSIVF LETHAL (S1 .RL)<br />
B .Q .Xieo, N .C,2hu <strong>and</strong> Q .X .2hang<br />
Guuugzhou Health <strong>and</strong> Anti-epidemic Station,Guengzhou,GuangDong,China<br />
F :uurirle is an e>sential element for human beings,overta{ .ing <strong>and</strong> undertaking<br />
wilt rarnse some tadr effect .Nany countries have increased fluoride content in<br />
drinking water . The purposr of this research is to determine if fluoride can<br />
imrse leUral mutntion under certain dosage .lhe test use two strains of Drosophiln<br />
melauognster, Oregon K <strong>and</strong> Ba>r .The e .
~<br />
test,in vivo gnotQxicity test, we found th t GMA i eley tt ~{' the<br />
sper. bner itY lreq p¢Y t e Qer . 1~ f∎i ~lasal~ P~3?2<br />
~<strong>and</strong>oa~y ~~~ed ~ithiM an~ transere~ to np1~for ~~n~ orsat on .<br />
e re at we trans qrsat on e f1c'e cy ease0 rev a s<br />
~ resp nse rel t ~sh~D, nt~~o~ T A .p ic[ ~ stent<br />
sTel racy li e nsqiti e)c~<strong>and</strong> Ac~~T~r~v th /n er~ta~iS~ty F)ave n<br />
rhich terd through an~jt otrect g bas~ of ~aaase a~e~&s o lu<strong>and</strong>sinCuce~ieet of terharor-ID4rlonel ~eD~ayr utesen<br />
~ 647<br />
aN ToXICITY AND t41fAMMITY OF CFDtCftII[M RICH BR6TaER's YEAST<br />
Li Xili, Bai Cheng Jiang <strong>and</strong> Shen Juen, Div'n of Toxicology, Tianjin Medical College<br />
Schwarz (1957) has postulated that trivalent chromium is an active eonstituent of<br />
glueose tolerance factor (GTF) . Mertz (1969) denoted that animals with a deficiency<br />
of trivalent chromium could result in damage of glucose tolerance or develop<br />
diabetes, hyperlipemia <strong>and</strong> arteriosclerosis . The supplementation of chromium is<br />
able to invert or prevent the pherxmena above mentioned . It is reported by China<br />
Air Force Hospital that a supplesre .nt of 100mg/day of trivalent chraniunrrich yeast<br />
to patients of diabetes <strong>and</strong> hyperlipemia induced an evident inprovecrent of glucose<br />
tolerance <strong>and</strong> significant decrease of plasma lipids after several months . The<br />
purpose of the present paper is to study the probably toxicity <strong>and</strong> nutagenicity of<br />
of chrenium rich brewer's yeast . The results show that the W50 of chromium yeast<br />
in rats <strong>and</strong> mice are all above 21 .5 g/kg, categorized as non-toxic <strong>and</strong> obtained<br />
negative responses to micronucleus test, muse-sperm tmrptalogy test, Ames test<br />
(TA98, 100, 97, 102), Reo-Assay, inductest <strong>and</strong> chromotest of SOS systan . We consider<br />
it to be acceptable to use brewer's yeast as carrier of trivalent chromium<br />
to be the source of chromium supplesnentation for certain populations deficient in<br />
chromiun .<br />
648 ,<br />
TRANSPLACENTAL GENOTOXICITY OF TRIETHYLENEMELAMINE, BENZENE AND VINBLASTINE IN HICE .<br />
S .C . Xing, X . Shi, Z-L . Wu, J-K . Chen, Z,Ona <strong>and</strong> W-Z . Whong, Division of Respiratory<br />
Disease Studies, National Institute for Occupational Safety <strong>and</strong> Health, Morgantown,<br />
WV (USA) A<br />
Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene <strong>and</strong><br />
vinblastine on maternal mice <strong>and</strong> their fetuses hsve been investigated in our<br />
laboratory . CD1 mice of 12-14 days gestation were exposed to TEM, bensene <strong>and</strong><br />
vinblastine twice by intraperitoneal injection at a 24-h interval <strong>and</strong> sacrificed 40<br />
hours after the first injection . Maternal bone marrow <strong>and</strong> fetal livers (2 to 4) from<br />
each pregnant mouse were obtained for the micronucleus <strong>and</strong> the SCE analyses .<br />
Significant dose-response increases in both micronucleus <strong>and</strong> SCE following the<br />
treatment of TEM were found in maternal bone marrow <strong>and</strong> fetal liver cells . Benzene<br />
at the highest dose (1 .5 ml/kg) also caused a significant increase in micronuclei <strong>and</strong><br />
SCEs in both maternal bone marrow <strong>and</strong> fetal liver cells . The data showed that the<br />
embryonic genotoxic effect of TEM was much higher than that of benzene in both<br />
genetic endpoints <strong>and</strong> that the frequency of micronucleus induced by benzene was<br />
higher in fetal liver than in maternal bone marrow cells . Vinbiastine, a spindle<br />
poisons agent, induced micronucleus formations but not SCEs . Micronucleus induction<br />
by vinblastine was 7 folds greater in maternal bone marrow than in fetal liver<br />
cells . All three chemicals showed cytotoxicity in maternal bone marrow cells, but<br />
not in fetal liver cells except TEM, which showed a weak toxicity in fetal liver<br />
cells in the micronucleus assay . These results indicate that TEM, bensene, <strong>and</strong><br />
vinblastine are transplacental genotoxicants in mice .<br />
649<br />
DELETION SCREENING AT THE CH ~cSE HAMS E hprt LOCUS USING THE POL -<br />
MERASE C~AIN REACTION2 7~ . Xu~I , Y . Yu~~~, A .W . Hsiel, C .T . Caskey , B .<br />
Rossiter , R .A . Gibbs I . Department of Preventive Medicine <strong>and</strong><br />
CommuTity Health, The University of Texas Medical Branch, Galveston2 TX<br />
77550 Institute of <strong>Molecular</strong> Genetics, Baylor College of Medicine ,<br />
Laboratories for Genetic Services, Inc .,3 Houston, TX 77030 .<br />
We have developed a rapid screening method using the polymerase chain<br />
reaction (PCR) for detecting deletions at the hypoxanthine-guanine<br />
phosphoribosyltransferase (hprt) locus in Chinese hamster cells, CHO-K1-<br />
BH4 <strong>and</strong> V79 . DNA was extracted from the HPRT-deficient mutants <strong>and</strong> two<br />
primer sets were used to amplify 274-bp <strong>and</strong> 334-bp fragments containing<br />
the exon 3 <strong>and</strong> exon 9 coding sequence, respectively . The PCR product was<br />
directly analyzed by electrophoresis on agarose gels stained with<br />
ethidium bromide . Using this assay, we analyzed 39 independently derived<br />
HPRT mutants . Four out of ten spontaneous mutants showed deletions at<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 223<br />
Notes
224 1989 EMS Abstracts - f<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
r - _ - . -<br />
F -<br />
Notes exon 9 on1y . UYtraolet-light-induced mutants produced predominantly<br />
wild-type amplific ion patterns (10/14), <strong>and</strong> X-ray induced mostly<br />
deletion patterns .15) in either exon 9 or exon 3 . These observations<br />
are consistent with previous data from Southern analyses . Deletions<br />
occur most frequently at the 3'end of the gene, suggesting the possible<br />
existence of hot spot(s) for deletions in this region . Supported in part<br />
by Laboratories for Genetic Services (LGS) <strong>and</strong> an EPA Distinguished<br />
visiting Scientists Award to AWH .<br />
COMPARATIVE STUDIES ON SEVERAL INDICES OF NUCLEAR DAMAGE IN HUMAN<br />
PERIPHERAL LYMPHOCYTES . K .X . Xue, S . Wang, P . Zhuo, G .J .Ma, Cancer Institute<br />
of Jiangsu Province, Nanjing ( China )<br />
In order to use approperiately NAT <strong>and</strong> explore possibility of use of the test in<br />
human cells, authers used Y-rays as mutagenic <strong>and</strong> carcinogenic irradiated whole blood<br />
in vitro, prepared directly smears of isolated lymphocytes, studied comparatively<br />
dose-response relationship of 4 types of nuclear damage . main results of the study<br />
are given as follows :<br />
1 . In doses from 0 to 3 Grays MNF ( freqency of micronucleus ), INF ( freqency of<br />
Irregular nucleus), KNF (frequency of karyorrhetic nucleus), anC PNF ( frequency of<br />
pyknotic nucleus) increase along with increase of irradiated doses . At 5 grays INF<br />
still increases, but MNF,KNF <strong>and</strong> PNF decrease .<br />
2 . In doses from 0 to 3 grays regression equation of 4 indices of nuclear damage<br />
are as follows ( D means dose,r means correlative coefficient) :<br />
MNF : Y-0 .3433+0 .1052D,r-0 .9128,p 0 .05 ; INF : Y-7 .2178+2 .1817D,r-0 .8846,p 0 .05 ;<br />
KNF : Y-0 .6462+0 .2014D,r-0 .7286,p 0 .05 ; PNF : Y=0 .2774+0 .0728C,r-0 .5484,p 0 .05 .<br />
Finally according to synthetical analysis of correlative coefficient, intercept,<br />
regression coefficient <strong>and</strong> feasibility of indices of nuclear damage,we could<br />
consider it suitable that nuclear anomalies in human peripheral lymphocytes include<br />
I' .'ir,INF <strong>and</strong> KNF .<br />
650<br />
651<br />
SISTER CHROMATID EXCHANGES INDUCED IN PERIPHERAL LYMPHOCYTES OF F10RXERS EXPOSED TO LOW<br />
CONCENTRATIONS OF STYRENE . J .W . Yager, S .M . Rappaport, <strong>and</strong> W .M . Paradisin, University<br />
of California, Berkeley, CA (USA)<br />
Following inhalation, styrene is absorbed <strong>and</strong> metabolized to styrene-7,8-oxide (SO)<br />
which is mutagenic in in vitro test systems ; SO also induces sister chromatid exchanges<br />
(SCEs) <strong>and</strong> chromosomal aberrations in human lymphocytes in vitro . However,<br />
previous occupational studies have not shown increases in lymphocyte SCES of workers<br />
exposed to styrene at 8-hour time weighted average (TWA) concentrations below about<br />
40-50 ppm . In this study, relevant exposure data <strong>and</strong> biological measures were obtained<br />
longitudinally during one year at a boat manufacturing facility that utilizes styrene<br />
as a solvent in preparation of reinforced plastics materials . 8KC Model 530 solid<br />
sorbent diffusion badges were used to measure full-shift breathing zone styrene concentrations<br />
up to 7 times over the year on the same 46 individual workers . Styrene in<br />
exhaled air was also determined up to 3 times per day . Blood was obtained for SCE<br />
analyses ; 80 cells were scored per sample . Health <strong>and</strong> occupational histories were obtained<br />
. Mean TWA exposure was 64 .2 m/N3 t 71 .5 (or 15 .1 ppm * 16 .8) styrene in air .<br />
Overall mean SCE was 6 .41 t 0 .96 per cell (rangee 4 .73 - 9 .47) . Smokers comprised<br />
54% of the population <strong>and</strong> smoked an average of 9 .1 cigarettes per day . Statistical<br />
analysis utilizing a bivariate linear regression model indicates that both smoking <strong>and</strong><br />
exposure to styrene contribute significantly to an increase in SCEs (P < 0 .000011 A~<br />
0 .62) . Most strikingly, it can also be shown that these two factors contribute to the<br />
increase in SCEs relatively free of the influence of one another . Results indicate<br />
that SCEs may be induced at rather lower styrene exposure concentrations than have<br />
previously been reported .<br />
652<br />
THE ROLE OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION DURING MULTISTAGE CARCINOGENESIS<br />
Hiroshi Yamasaki, International Agency for Research on Cancer, 150 cours Albert Thomas,<br />
69372 Lyon cedex 08, France .<br />
An important role of gap junctional intercellular communication is to maintain<br />
homeostasis of cells within a given tissue . Since carcinogenesis involves the breakdown<br />
of homeostasis in the tissue, it is conceivable that altered gap junctional<br />
intercellular communication is involved in the multistage carcinogenesia process . A<br />
possible role of blocked intercellular communication during tumor promotion is strongly<br />
supported by the fact that various tumor promoting agents inhibit gap junctional communication<br />
in cultured cells . We have recently extended this finding to in-vivo ; the<br />
administration of liver-specific tumor promoter phenobarbital reduced theeveT of gap<br />
50869 3738
junction protein mRNA in liver, but not in the kidney or in the stomach . Gap junctional<br />
co®unication also plays W important role in the behavior of cancer calls . These<br />
cells can attain inhibition of intercellular communication with surrounding normal<br />
cells by one of two different vays : 1) Decrease of intrinsic gap junctional intercellular<br />
communication among themselves ; 2) Maintainanca of their intrinsic intercellular<br />
communication capacity, but with selective inhibition of communication with<br />
surrounding normal cells . Taken together, available evidence suggests that altered<br />
gap junctional intercellular communication plays an important role in the process of<br />
carcinogenesis <strong>and</strong> the maintainance of the phenotype of tumor calls . Supported in<br />
part by NCI Grant No . ROL CA40534. ' --<br />
653<br />
COMPARING THE FREQUENCY AND SPECTRA OF MUTATIONS INDUCED WHEN DNA CONTAINING<br />
COVALENTLY-BOUND RESIDUES OF POLYCYCLIC AROMATIC CARCINOGENS REPLICATES IN HUMAN<br />
CELLS . J .-L . Yang, M .C .-M . Mah, V .M . Maher, <strong>and</strong> J .J . McCormick, Carcinogenesis<br />
Laboratory, Michigan State University, ast Lansing, MI (USA)<br />
To gain insight into the mechanisms by which carcinogens induce mutations in<br />
human cells, we are using a shuttle vector, pZ189, carrying a bacterial suppressor<br />
tRNA (juRF) as the target for mutations Induced when the plasmid replicates in<br />
human cells . We have treated the plasmid with 7,8-diol-9,10-epoxide of<br />
benzo[a]pyrene (BPDE), 1-nitrosopyrene (1-NOP), <strong>and</strong> N-acetoxy-2-acetylamnofluorene<br />
(N-AcO-AAF) <strong>and</strong> determined the frequency <strong>and</strong> spectra of mutations induced . BPDE<br />
binds principally to the N2 position of guanine, 1-NOP binds to CS guanine, <strong>and</strong><br />
N-AcO-AAF forms deacetylated AF adducts at C8 guanine . To obtain AF adducts In<br />
vitro, we used the trifluoro derivative . Each agent caused a linear increase in<br />
the frequency of supF mutants as a functi Qn of DNA adducts formed, reachi qg<br />
frequencies as high as 20 x 10'4 to 40 x 10-4, above a background of 1 .4 x 10-4 .<br />
When compared on the basis of adducts formed, BPDE was 4 times more mutagenic than<br />
the other 2 carcinogens . This difference may reflect intrinsic differences in<br />
the nature of the adducts <strong>and</strong>/or their location in the gene, but may also reflect<br />
differences in rate of removal of particular adducts . The majority of mutants<br />
from untreated plasmids involved large deletions or insertions . Almost all of<br />
those from treated plasmids involved base substitutions, mainly ` G-C T•A<br />
transversions, but each agent produced its own spectrum of mutations . The hot<br />
spots for mutations could not be explained merely by hot spots for adduct formation .<br />
(Supported by NIH Grant CA21253 <strong>and</strong> by Contract #87-2 Arom the HEI .)<br />
654<br />
MUTAGENESIS BY DNA CROSSLINK PRODUCED AT MULTI-CLONING SITE OF pUC19<br />
Fumio Yatagai, Sumiyu Hachiya, Kiyomi Nakajima (The Inst . Phys . Chem .<br />
Res . Saitama351-01 JAPAN) <strong>and</strong> Barry W . Glickman (York University .<br />
Toronto M3J 1p3, CANADA)<br />
To elucidate mutation induced by site-specific DNA damage, we selected<br />
muticloning-site of plasmid as the target . This site seems relatively<br />
insensitive for selecting base substitution events, but it is very<br />
useful for the detection of frameshift, deletion, <strong>and</strong> rearrangement<br />
events . Following the treatment of pUC19 with UVA (365nm,<br />
1 .5mw/cm2)plus 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT, 50<br />
ug/ml), the 51 bp of EcoRI-HindlII fragment containing the crosslink<br />
was recovered from a 20% denaturating polyacrylamide gel . This fragment<br />
was ligated into the large EcoRk-HindlII fragment (2635bp) <strong>and</strong><br />
used to transform E,_c411 JM105 . Amp transformants were rtecovered<br />
using pre-UV (254nm, 5J/m ) irradiated E~ DII11 host . TrAnsformation<br />
efficiency was about 13% . Approximately 5 .6% of the Amp transformants<br />
recovered following UVA plus HMT carried i ;gZ mutations . Dideoxy<br />
sequencing analysis of these mutations revealed three classes :<br />
1)minus T frameshift at the EcoRl site, 2) deletion of 302 bp (315-<br />
616), <strong>and</strong> 3) rearrangements at position 186 (involving intact 51bptarget<br />
sequence) . These results indicate the involvement of crosslinks<br />
in the induction of these SOS-dependent mutations .<br />
655<br />
SiUffiES ON THE GENOTOXICI'IY OF DLSIr'FFCI'ANTS WfA-I SOS CMOMOTFS'f<br />
Yin Muquan, Qxn Yao[u, WangAng<br />
Departma:t o[ Toxxx:Iogy, Second IvFititary Medical Ucriwrsty, Shanghai, China<br />
The 906 O:ramotest is a sapd, teqiricing os+ly a singlo stnsin <strong>and</strong> a'mp1e oolotanctric enzyme as~ay,<br />
<strong>and</strong> it doea nottoqwrc survival of tho testa stn~ thus it ooud be wed to d'etact the patooddty af<br />
di9nfoctanl In the peraent peptr we sttxjiod the grnoto)ddty of 14 disinfoctants utdud~ng<br />
fm :mldchyd4 glutaraidehYde, ethylaie oxide, sodium diclilaoisocyanurate, peraoetic add, hydropen<br />
prioxid5 etltianoi, isoprophl aloohol, carbobc add lysd brarno 8asttainum, wdi=<br />
diddon :socyu :urate mucUmq fungieide <strong>and</strong> Giangtistt using the SOS dromotest . RasuNs Bnta sttdy<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 225<br />
Notes<br />
n
226 1989 EMS Abstracts . r --<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes ~' ~~~ts trataa in uris wcpavnent hare no lrigher galactas~e acbviry .<br />
Among thrac 14 oa1y ethy~e oXdde <strong>and</strong> C3iangMu gaw posihee nsults We ddmod th<br />
tcst campo~md to bo ; wkn tk fl-galactaddase activiry was ineeavod 2-toid over ahe beckgro~md<br />
lerd Ths • rado of ethylrne obdo was 2.39 at ooooernradon 800 ppm in PQ35 witlr<br />
out metabolic anivation mix4ae (S9 ma) . Asd Cuaaglisu was 2 83 at oon=tration 19 ;q/ml in<br />
PQ37 witYwut S9 mhc <strong>and</strong> 2 43 at mime oonornuation in PQ3S with S9 mix cf the nr<br />
ahs fmm SOS ctuernotat <strong>and</strong> Ames tat mealod a strrnhg aoncadanoe• S~~vai advan•<br />
tapes of SOS Chromoteat was discuseod<br />
dIii; ASS 3S : :T :T 0" 37 :0 .'^ :;TC": I^.As4 :'lIT'T. C-RO:`.'1S(1a,.^<br />
. . 3 1, 177 :r:~l' 113<br />
}~„ - :iu-12.n, ,ao rin, V;, S•'Ao-lin, '.'!is,, -''t,e<br />
-ie$e^rQl, I,' .~i 1 1ate, "Ile ;'inina 1'v Oo C},oc'<br />
1<br />
._ ^.C' . . , .<br />
Ii? 0?.°^er i:o 4et v - .I 2. n^l4 T94e3R^,en' om 4tn n' n" y p .Pfef`•I S 7f<br />
~ ;oto ctc c'. :-c^ 1- -)., -e?°r, ce71 e, r me-od for r, r• n nn^-.p nc+ PT 9ri^Cp-<br />
`io, of 7o+e^ i.^ .,ou^e . . . '!eqcr-ihe•' '•i^ ci^ocqAt,r.P tG 'fec'ive<br />
`o n ^,s,re a. '-i~'• m•ccesa ra+-e of ~ooA ^1i.ren of c rn~o~o~e,<br />
306 net-'-a.^ic e,!- ^niteb'e forr o'-roTn-o^e proprrr'i•on tn 774 .<br />
one-cetl z7r^;ote^ ar-re o`)t^inei . ^.hro-noso•e3 of 754 cPt_1-s in 3nA<br />
e^Tr ca'1 be •,nnt•r?c4e c' .rn ;o^o~P ^i ;des -,•er-e al-^o * . ~e i.n'o ^nrt ^--ba-+A<br />
for ^n^1 .^, ; .s . 'ex c-•-°o .,oso e C'r,,lt?,-en.' fCr r,^'•tnq in "e<br />
9 :•C(: a? •r . .-e'1•vle•le-1)tc1 ,4-•, .i',,tt,2nlot<br />
J'. ;'S3iA-p i,i-Ct .Y,7„~~_o . • .n, .,_ranuinone A .rP.^.•'.U .f.f ;, C^~ .'ti10n<br />
l. e-r^.7.v.p ' . :el A+t' t'v'ic net'.•o'i ,<br />
n-.-e1i,i•.•^r,r t',e -e .I^oO i .s recor<br />
;~end:ri o. .e of evn1'ie ., :nz mr' :z--en 'o •cerw cells of ma-melian<br />
.<br />
656<br />
THE EXTREMELY POTENT CLASTOGENS, U-73,975 AND U-71,184 IN IN VIVO INDUCTION OF<br />
CHROMOSOMAL ABERRATIONS IN BONE MARROW CELLS OF CD-1 MICE . R . Yu, C . Aaron,<br />
S . Wiser <strong>and</strong> N. Wicnienski . The Upjohn Company, Kalamazoo, Michigan, USA .<br />
U-73,975 is a member of a novel anticancer drug family under development by The<br />
Upjohn Company . These materials are structurally related to CC-1065 <strong>and</strong> have been<br />
found to be excellent bacterial <strong>and</strong> mammalian mutagens <strong>and</strong> exhibit highly selective<br />
binding to AT regions of DNA (Envir . Mol . Mutag . 11, su pl• 11 . 2, 1988) . U-73,975 <strong>and</strong> U-<br />
71,184 were administered in a single I .P . dose at the folPowing doses : U-73,975, 1, S <strong>and</strong><br />
10 ug/kg : U-71,184, 3, 6 <strong>and</strong> 10 ug/kg . Three animals per sex per group were treated with<br />
U-73,975 <strong>and</strong> 5 males per group were treated with U-'1,184 . 24 hours after exposure, the<br />
percent of cells with chromosomal aberrations were as follows :<br />
U-73 975 Male F ~inaie U-71 1 4 Male<br />
u-g`~ ~:6-t 3.5 _5.3 t 3.1 • 3 ylky T3Tt 2 .3***<br />
5ug/kg 16.0t4.0*** 31 .3± 6.4*** 6ug/kg 12 .41 4 .6***<br />
10 ug/kg 34.7t5.0*** 45.3t12.9••* 9ug/ky 19 .2t10 .3**•<br />
The percentages compare with untreated control frequency of about 1 X <strong>and</strong> a mitomycin<br />
C(MMC) induced frequency of 60-70% at 3 .5 mg/kg . Thus, these compounds are several<br />
hundred times more potent than MMC in this model . There were decreases in<br />
chromosomal aberrations observed with Increasing time of exposure . In summary, the<br />
compounds are potent chromosome breaking agents in rodent bone marrow cells, a<br />
property shared by many anticancer agents . This study is part of a broader cytogenetic<br />
evaluation of CC-1065 analogs (* P
prevented by ADPRT inhibitors,3-aminobenzamide or nicotinamide,which were shown to<br />
exert no influence on,the NAD lowering due to NAD biosynthesis blockade . (4) In pnaphthoflavon,a<br />
c tocFrome P450 isozyme inducer,induced or unindnced human amnion<br />
FL cells,AFB1,B(a~P,pyrene,2-AAF,9,10-dimethylanthracene <strong>and</strong> sthylcarbamate could<br />
induce the decrease of cellular NAD mediated by ADPRT aotivation,4-4.LF,anthracene,<br />
isopropyl N-(3-chlorophenyl)-carbamate,(i-propiolactone,Y-butyrolactone,cyclophosphamide<br />
<strong>and</strong> safrol couldnot . This preliminary validation results indicate that this<br />
new assay is specific for detecting DNA damage with a sensitivity <strong>and</strong> specificity<br />
at nearly the same grade as well va idated UDS assay with rat liver S9 system for<br />
metabolic activation of p,romutagens~p_rocarcinogens .<br />
659<br />
INITIATION OF CHEMICAL CARCINOGENESIS A ND HYPOMETHYLATION OF CELLULAR DNA REVISITEDe<br />
Y .N .Yu,Z .Z .Shi,<strong>and</strong> X .R .Chen,Zhejiang Medical University,Hangzhou(China)<br />
•The Project supported by National Natural Science Foundation of China<br />
The extent of enzymatic methylation of newly synthesized DNA labelled with 3H-TdR<br />
prior to harvesting in human amnion FL cell line after carcino en (two nongenotoxic,<br />
5-azacytidine <strong>and</strong> L-ethionine <strong>and</strong> two genotoxic, MNNG <strong>and</strong> AFB1~ exposure wes analyer<br />
ed by the digestibility with restriction endonuclease HpaII . The weight average<br />
lengths (Lw) of HpaII digest of the DNA isolated from the -azacytidin . (2 x 10-6M<br />
1-day exposure, DNA isolated at the 5th day after exposure) <strong>and</strong> L-ethionine (2 x<br />
10-3M 9-day exposure, DNA isolated at the time of the end of exposure) treated cells<br />
were significantly lower than those of the controle (Lws8 .0 • 0 .1 kb va 10 .9 i 1 .Okb<br />
p< 0 .01 <strong>and</strong> 9 .8 . 0 .3 kb vs 10 .6 + 0 .3 kb p< 0 .05 respectively) . While those from<br />
the cells exposed to MNNG (5 x 10-6M 1-day exposure, DNA isolated at the 5th day after<br />
treatment <strong>and</strong> AFB1 (2 .5 x 10-5M 1-day exposure, DNA isolated at the 6th day after<br />
treatment) were not different from those of the controls (Lwt 11 . a 0 .7 kb vs<br />
10 .6 . 0 .8 kb <strong>and</strong> 10 .6 + 0 .5 kb vs 10 .4 + 0 .7 kb p> 0 .05 respectively~ . Our results<br />
suggest thpt the stable <strong>and</strong> heritable alteration of inethylatlon patterns in cellular<br />
DNA may be a specific mechanism of carcinogenic initiation by some, but not all<br />
kinds of carcinogens, <strong>and</strong> the hypomethylation of DNA frequently seen in tumor tissues<br />
may be formed during the process of initiation as well as promotion <strong>and</strong>/ or progression<br />
phase of carcinogenesis. •<br />
660 "<br />
BACILLUS SUBTILIS RED-ASSAY AND MUTAGENICITY DETECTION OF METAL COMPOUNDS<br />
Wang Yuzhi <strong>and</strong> Tang Tianbao ; Shenyang Res . Institute of Industrial<br />
Hygiene <strong>and</strong> Occupational Disease, Shenyang, P .R . CHIA<br />
The bacterial systems have become efficient <strong>and</strong> sensitive methods<br />
for screening mutagenicity <strong>and</strong> carcinogenicity of chemical compounds .<br />
in this study, B . subtilis Red-assay was selected to test metal compounds<br />
with Hiran's spore method . The spores of Rec+ <strong>and</strong> Rec- strains<br />
of B . subtilis were prepared in agar <strong>and</strong> a paper disk, impregnated<br />
with the chemical solution placed on the surface . After 20 hours incubation<br />
at 37 C, diameters of inhibition zones are measured with each<br />
strain . A difference of -3mm is a positive effect . In order to<br />
compare their sensitivity, the spore <strong>and</strong> vegetative cell rec-essays<br />
were performed with chmicals <strong>and</strong> metals with or without S9 metabolic<br />
activation . The results show that use of spores in place of vegetative<br />
cells increased the detective sensitivity 7- to 10-fold . In 24 hours<br />
incubation at 37°C, BeSOq <strong>and</strong> V205are negative, but in incubation at<br />
4°C for 24 hours <strong>and</strong> at 37°C for another 20 hours, positive results<br />
are obtained . The B . subtilis spore rec-assay is one of the systems<br />
which is least expensive, rapid <strong>and</strong> non-laborious .<br />
661<br />
OBSERVATION OF THE EYES IN 53 WORKERS OCCUPATIONALLY EXPOSED TO METHANOL<br />
SuJuan-Zang .<br />
Research Institution of Labour Health Sciences in LiaoNing (China)<br />
The methanol is a solvent of chemical industry . Acute poisoning of<br />
methsnol may injure the nervous systems <strong>and</strong> the eyes . Harm of the eyes<br />
is retrobulbar optic neuritis, optic neuritis <strong>and</strong> intraocular neuritis .<br />
cases of acute poisoning of methanol have been reported, however, obser_<br />
vation of the eyes in workers occupationally exposed to merhanol of lower<br />
concentration <strong>and</strong> long term contact are rather rare . In this paper<br />
contrast of'the eyes check in 53 workers occupationally exported to me -<br />
thanol <strong>and</strong> 49 workers not exposed to it were reported .The lowest concentration<br />
of methanol in air of the manufacture workshops of Dawa Chemistry<br />
Factory in Liaoning China is 1 .2rng/m' <strong>and</strong> the tallest concentration<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 227<br />
Notes
228 1989 EMS Abstracts K~J<br />
Notes is 165 .5/m: Chockjft-ophthalmology include history,. functional examination<br />
of the eyes <strong>and</strong> physical examination of the eyes . The check results<br />
of these two groups are similar(P :Q,05) . Because every worker will<br />
change this work frequently, the contacting time, to methanol is short<br />
for every one, the eyes of workers occupationally exposed to methanol<br />
do not are harmed .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
STRATEGIES FOR THE IDENTIFICATION OF RODENT CARCINOGENS BY IN VITRO<br />
SHORT-TERM TESTS . Errol Zeiger, Cellular <strong>and</strong> Genetic Toxicology Branch,<br />
National Institute of Envlronmental Health Sciences, Research Triangle<br />
Park, NC, 27709 (USA)<br />
The effectiveness of four short-term tests for genetic toxicity<br />
(induction of mutations in Salmonella (SAL) <strong>and</strong> mouse lymphoma L5178Y<br />
(MLA) cells, <strong>and</strong> induction of sister chromattd exchanges (SCE) <strong>and</strong><br />
chromosome aberrations (ABS) In Chinese hamster ovary cells) for<br />
predicting rodent carcinogenicity was examined . A total of 114 chemicals,<br />
including 73 previously evaluated, that had been tested for carcinogenicity<br />
by the National Toxicology Program were evaluated . The results with the<br />
114 chemicals confirm <strong>and</strong> extend the results previously reported with 73<br />
chemicals (Tennant et al ., Science 236 :933-941,1987) . Salmonella had the<br />
lowest sensitivity ( .48) <strong>and</strong> the highest speclficity ( .91), whereas mouse<br />
lymphoma had the highest sensitivity ( .70) <strong>and</strong> lowest speclfictty ( .40) . The<br />
concordances of the tests was .66, .61, .59, <strong>and</strong> .58, for SAL, ABS, SCE, <strong>and</strong><br />
MLA, respectively . Salmonella had the highest positive predictivity ( .89) .<br />
None of the other tests complemented Salmonella, <strong>and</strong> no combination of the<br />
four tests was more effective than Salmonella, alone, for predicting<br />
carcinogenicity .<br />
I9HIBITION Od Tr:6TICUlrin DNA SYNTNiSIS BY CNchIOAL MUThGc:NS<br />
8ui-Juan Zhang, Ghi-iien We1 <strong>and</strong> Yu-Zhen Zhu<br />
Shanghal Institute of Yharmaceutical Industry<br />
1320 Beijing Xi 8oad, Shanghai, China<br />
Friedman <strong>and</strong> staub reported the measurement of the inhibition of testicular DNA<br />
662<br />
663<br />
synthesie by chemical carcinogens <strong>and</strong> mutagens in sale mice <strong>and</strong> proposed this method<br />
as a simple <strong>and</strong> effective in vivo mammalian screening test . The testicular tissue of<br />
the mice is capable of utilizing the salvage pathway efficiently when synthesis of<br />
thymidine is inhibited . It is possible to distinguish between DNA-dasaging agent <strong>and</strong><br />
non-DNA-damaging agent . We now report on the results obtained with eight chemicals .<br />
Thio-Tepa, Cyclophosphamide <strong>and</strong> Adriamycin were DNA damaging agents . Fluorouracil <strong>and</strong><br />
Methotrexate were non-DNA-damaaging agents . Fluapyrimidone, Aurapromide <strong>and</strong> Nonylphe-<br />
noxypolyethoxyethanol showed no evidence of genotoxicity .<br />
664<br />
THE EFFECT OF 5-AZhCYTIDIHE ON REVERSION 0F ISOLATED HPHT MUTANTS IN V79 CHINESE<br />
HAMSTER CELLS . L-H . Zhang <strong>and</strong> D . Jenssen, University of Stockholm, Sweden . The<br />
purpose of the present study was to investigate rare types of spontaneously<br />
occurring mutational events . Reverse mutation analysis using medium Containing<br />
L-asaserine (HhsT) of 60 independently isolated HPET- mutants from four groups,<br />
11 spontaneous mutants, 11 EMS-induced mutants <strong>and</strong>, 38 1010-induced mutants with or<br />
without hydro :yurea pretreatment, showed that over 70% of mutant clones of each<br />
group could be reverted either spontaneously or induced by treatments with Ei1U,<br />
ICS191 or S-asacytidine (SAC), indicated to be caused by point mutations . Two of<br />
the revertible mutant clones of spontaneous origin wer* found to be resistant to<br />
HAT but not HA&T medium . The interestinq result of this study was that these two<br />
eTGrHATr mutants were the only mutants isolated which could be affected by SAC<br />
with a significant increase in the reversion frequency . Chromosome aberration<br />
analysis did not indicate any enhancement in aberration frequency in the<br />
X-chromosome by 5AC treatment . Studies on the mutagenicity at 00k-locus indicated<br />
that the SAC- <strong>and</strong> ENU-induced mutation frequencies in these 2 mutants were<br />
50869 3742
comparable with the effedEa in the parent wild type cell line . Their cellular<br />
incorporation of H3-hypoxanthine was enhanced in the presence of aminopterin,<br />
but decreased with L-asaserine indicating that they were PRPP mutants . According<br />
to these results, we suggest that reversion of these 2 6TGrAATr mutants may<br />
occur by a gene amplification mechanism <strong>and</strong> that this process may be facilitated<br />
by 5AC treatment .<br />
665<br />
MUTAGENICITY AND CARCINOGENICITY OF NITROSATED FISH SAUCE<br />
R .F .Zhang, D .J .Deng, Y .Chen, H .Y .Wu, S .Jin, S .X .Zhu <strong>and</strong> Y .P .Liu<br />
Beijing Institute for Cancer Research, Beijing, China<br />
Fish sauce, a traditional daily use seasoning, was collected from<br />
Changle county in Fujian province, where the mortality of gastric cancer<br />
is the highest in China . To evaluate the risk of taking fish sauce for<br />
the high incidence of this disease in that area, mutagenicity <strong>and</strong> carcinogenicity<br />
of the fish sauce were stidied . By the Ames test(S . typhimurium<br />
TA100) <strong>and</strong> in vitro SCE <strong>and</strong> micronucleus tests in V79 cell 11ne,<br />
direct mutagenicity(-S9) appeared in the ethyl acetate extract after<br />
nitrosation with NaN02 . A dose response relationship was obtained . The<br />
highest response was observed in the extracts of acidic nitrosation(pH<br />
2 .0) <strong>and</strong> the samples from the villa e with the highest mortality of<br />
gastric cancer . After 0,1m1 extract~corresponding to 1 .Oml of nitrosated<br />
fish sauce) was given to each of the studied new born Wistar rats by<br />
gava e for 3 consecutive days, marked dysplasia was observed at the 4th<br />
week~in 5/5 rats) <strong>and</strong> adenocarcinoma developed(in 1/5 rat) at the 16th<br />
week in the gl<strong>and</strong>ular stomach . It indicated that some nitrosable precursors<br />
of direct-acting mutagenic/carcinogenic N-nitroso compounds must be<br />
contained in the fish sauce <strong>and</strong> it may play important role, after nitrosation,<br />
in the causes of gastric cancer <strong>and</strong> its precancerous lesions of<br />
stomach in Changle county residents . Detection of these compounds is now<br />
undertaking. f<br />
666 "<br />
A METHOD TO GET THE BACTERIOPHACE SPOT CLEAR IN INDIRECT SOS TEST<br />
Zhao Zezhen, Wel Lirhent Haans Mlntlt Hebet Cancer Instltutes P .R .China<br />
It has been provod that, among oe many methods te check the mutagens<br />
In environment, Indirect SOS Test, I,e, bacterlephage Inductlon methed,<br />
Is a quick, sensitive <strong>and</strong> reliable ene . The methed is te mix CY6027s a<br />
lysesenic bacterlat with GY4016s a Indlcatin{ bacteria which Is sensitive<br />
to the bacterlophage released by Gy60279 In a sub-solid culture<br />
medium according te a certain preportlon . While meeting mutatens, the<br />
lysogenic bacteria wlll release bacterlephase dissolution indicating<br />
bacteria, then bacterlophate spat will appear on the culture medlum<br />
which Is surrounded by muddy bacterlafur, However, the centrast between<br />
bacterlophage spot <strong>and</strong> bacterlafur Is net cloar enough te observe <strong>and</strong>,<br />
especlally, to take photo or make lantern slide . In light ef the prlnciple<br />
that, in the course of grewthr colour bacillus Gy4016 <strong>and</strong> Gy6027<br />
can break down glucose <strong>and</strong> produce acid which can make the Indlcater<br />
change colour . We make an improvement on the original methed, that los<br />
add 2m1 of 2% disinfected (low pound) glucose selutlen <strong>and</strong> iml of 0,6%<br />
disinfected bromecresel purple solution te 60m1 of ineiting sub-salid<br />
culture medlumq then put the two test bacteria liquid to the mixture<br />
selutlon, which will make the culture medium chan{e to blue coleur .<br />
Other test steps are simillar te the original, Comparing the new method<br />
with the orisinalt the new test results show to Increase the contrast<br />
between bacterlephage spot <strong>and</strong> bacterlafur . And though It, we have succeeded<br />
in taking phste <strong>and</strong> making lantern slide,<br />
667<br />
OBSERVwTION OF THE EYES ON FIFTY WORKERS OCCUPATIONALLY<br />
EXPOSBD TO DIMETHYL SULFATE<br />
rien~iang-Zhao .Electricity centre Hospital in NE China .Shenyang(China)<br />
ime.thyl sulfate is an important raw material of chemical industry .<br />
Its decomposing products methanol <strong>and</strong> sulphuris acid may impair cornea,<br />
conjunctiva etc . Cases of acute poisoning of dimethyl sulfate have<br />
been reported,however,observation of the eyes in workers occupationally<br />
exposed to dimethyl sulfate of lower concentration for a long time<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts<br />
Notes<br />
229<br />
" : .<br />
I<br />
t
230 1989 EMS Abstracts . . .- --<br />
Notes<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
.._~ ,.mercontacting<br />
is rather rare . In this paper,the involving worker include<br />
35 males <strong>and</strong> 15-41 ips,<strong>and</strong> the control group includs 40 workers no<br />
exposed to it .Coricentration of dimethyl sulfate in air of the manufacture<br />
workshop of Petroleum Chemistry Factory in LiaoNing in Ghina is<br />
2 .89-o .07mg/um .Check of ophthalmology include history,functional examination<br />
<strong>and</strong> physical examination of the eyes .In history,many involving<br />
workers have vision blurred,eyepains,lacrimation,photesthesia etc .The<br />
most obvious sign is conjunctival congestion <strong>and</strong> it resed with contacting<br />
time .By statistics,vision blurred <strong>and</strong> conjunctival congestion of<br />
the workers occupationally exposed to . .dimethyl sulfate are higher than<br />
other workerslPc,0,05 <strong>and</strong> P
nutber of surviving fetuses decreased significantly with increases in irradiation<br />
dose, while resorption rsbes increased . Vfien miee were irradiated for 5 consecutive<br />
days during the preiu+plantation period, the number of surviving fetuses was less than<br />
in controls but litter weights increased faster within one month of birth, <strong>and</strong> mice<br />
fran treated groups remained larger than those from control during 1h years observation<br />
. The carcinogenic effect of irradiation to embryos showed an incidence of<br />
leukemia 4 .9 times higher in treated versus control animals . Studies of irradiation<br />
induced malformations <strong>and</strong> carosnogegenesis were also conducted by observing inmediate<br />
<strong>and</strong> late effects of single or fractionated low dose irradiations . The nssnber of<br />
living fetuses was significaritly reduoed'with increasing radiation dose . The frequency<br />
of rnalfornmations in external appearance increased with radiation doses . The imnediate<br />
ef :ects in the single irradiation group were different fran those in the fractionated<br />
irradiation groups . In general, all effects fraa single irrad3ati .ans were more significant<br />
than those in the fractionated irradiation group, except for the frequency<br />
of cranioschisis . Litters of mice in the fractionated irradiation group were observed<br />
for late effects for 2 years . Litter weights were lower than in controls <strong>and</strong> the incidenee<br />
of leukania was 2 .5 times higher in the treated group . Late effects were also<br />
studied after 50R of irradiation given at 13-17 days gestation . Litter weights were<br />
lpwer . ThP incidence of tumr was 8 times higher .<br />
671<br />
L .-T . ., ..F n, T', ;="'T~?TY nF L' CTDI!(- :<br />
Lh^u '-hu-,`Zhan~ J+a, -5{a Shu-Zheng, Lfa~Cu+-e,?nd L+an X+n,-3uan ;<br />
health <strong>and</strong> ;nt+-ez+damjc Center -f Henan =r-v{nce, Zhan3zh-u, P .RC .<br />
Jan-derni ]uc+dun, kjn•.i -f tra~+t+-nat Ch+ne :e med+c+nes, had been<br />
used as a t-n+c f-r th-us<strong>and</strong>s -r" ye,rs . T- evaluate tha Wh-les-mene~!s -f<br />
3d•±
232 1989 EMS Abstracts<br />
. . . :-- .<br />
NOtE'S immunosuppresive agents . Two to three drops of blood were taken from the ear lobes of<br />
examined persons . TM&,blood was then incubated with 1 .0-2 .0 ml Tris-ammonium chloride<br />
buffer solution 'to'renxTlyze red blood cells . Monolayer cell smears were made with<br />
cytocentrifuge <strong>and</strong> Feulgen stain was applied to detect DNA content . The morphological<br />
observation <strong>and</strong> micronuclei counting were done with light microscope . We found that<br />
the frequencies of micronuclei of lymphocytes in healthy persons were 0 .82 per<br />
thous<strong>and</strong> cells but in patients with esophageal carcinoma were 2 .88 per thous<strong>and</strong> . The<br />
difference of frequencies of micronuclei of lymphocytes between healthy persons <strong>and</strong><br />
patients with esophageal carcinoma was statastically significant (PA0 .001) . The<br />
method we used to detect micronuclei of lymphocytes was fast <strong>and</strong> reliable <strong>and</strong> easily<br />
accepted by patients examined .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
674<br />
4IT' STU7Y i :+CCU' ; L :^_ IO° OF 2.L' :1 .. . . : IN Gr'.Rh: CSLLS ON INDUCTIi . :i<br />
OF ~.lDICG-_? ;~TC7.L^.OLCGICAL 3F :^r:CT .<br />
Zhu Bhoup n~, heov Siyinq, -ad lP-n :^- Liuyi, Suzhou Nedical College, Suzhou(China)<br />
T~j purpose of the present study is to ascertain r,omp,?~rative retontion of 147Pn<br />
o„- Cs in qerm cells on induction of chromnsome aberraM ns of sparmatogonium <strong>and</strong><br />
eb :iormall+.i .e in sperm . Results it .dicated that after iv Pm throu,h 50 d observstion<br />
. The rEtr.nt~oPC~~a~s in tastis were well describad by an exponential expression<br />
13~(t)=0 .1872xe- , where the retention ^=1C5 ( : . 'lhlle th3 .fat~~ntion data of<br />
Cs in t•?stiF w•er^ described by e-n expreswlofi : =tlt).O,OC15za , where the<br />
retention T, was on}X75 .2 d, 3xperimsnts indicated that the retention value aQJ4thc<br />
abs.irption ose of Pn i?A°a is ree sirnificantly high in comparism with C s .<br />
Internal conta.nins!ion of ?~ or Ce can be induced chromosome aberrations in<br />
germ cells <strong>and</strong> abnormalltiT17in spaj~b Atnona the type of chromosome aberrations of<br />
apermato-onium induc,~d bv _m or Ce includin- ^a~, chromatid breakage, chromosome<br />
brea ::age, <strong>and</strong> tranalocation, as woll as poly :'-oid spermatogonium . },oreover,<br />
the chromosoma aberrft3on rctt*s <strong>and</strong> pol,T,:loid cells were elevated when the periods<br />
of conta^:ination of Pm :rora prolon,? !d . ,1t th : semt_ t#" ehromoso:ae fragment <strong>and</strong><br />
If~nalocations of primary spermatocyte also induced by Pm . By compaTign with<br />
:m, ho-ver, the induction of cytogenetic effects on germ cells by a wke<br />
quite low.<br />
675<br />
INDUC'TION OF MUTATION IN F-'aihcrichin cofi BY DIMETHYISULFATE IS INFLUENCED BY SOS :<br />
OBSERVA'11ONS OF THE MUTATIONAL SPF.C IFICI'IY OF DMS AND E7HYL ETHANESULFONATE<br />
IN WILD-TYPE AND UmuC STRAINS . Maria Zieknaka, Janet Smylie, Jiso Iian Li <strong>and</strong> Barry W . Olickman .<br />
Dept . of Biology. York llniversity, Toronto Canada M3J 1P3 .<br />
In this study we have undertaken to examine how mutagenesis resulting from EMS <strong>and</strong> DMS is<br />
influenced by the error-prone misrepair pathway of E. rofi . We Investigated the mutational specificity of<br />
these alkylating agents by DNA sequencing <strong>and</strong> oligonucleotide probing methodologies In the first 180<br />
base pairs of the Inrl gene of F. rnfi in wild-type <strong>and</strong> UmuC strains . Consistent with the established<br />
alkylating ability of these agents, the O :C _> A :T transition dominated the resulting spedra . EMS<br />
elicited an identical mutagenic response in the two strairo ; after treatment with 3mM KMS there was a<br />
15-fold increase in mutation frequency at 57% survivaL Mutational specificity of EMS in the l1muC<br />
background was identical to that found in the wild-type strain ; O:C - > A :T transitions accounted for<br />
over 96% of the mutational events in each spectrum <strong>and</strong> their distribution was identical . DMS<br />
mutagenicity decreased in the UmuC background from a 128-fold increase in mutation frequency at 14%<br />
survival (wild-typel to a 26-fokl increase at 6O/o sunival. The influence of UmuC function was also<br />
apparent at the DNA sequence level . In the wild-type background t3 :C - > A:T transitions accounted for<br />
74% of the mutations, which were alan characterised by a significant contribution of additional<br />
mutatiomd events including Ai r=- (hC' trnnsitkms, all classes of transverskms <strong>and</strong> framoshifu . In<br />
eontrast, after DMS treatntent in a UmuC strain 82% of all mutational events were (t :C .• > A:T<br />
transitions with a different site specificity from that recovered in the wild-type background. The other<br />
claases of events found consisted of A :T => T :A <strong>and</strong> C; :C - > T:A tramversions, frameshUts <strong>and</strong> a<br />
duplication. We conclude that mutagenesis by EMS Is independent of the UmuC function in contrast to<br />
mutagenesis by 1)MS which shrn.s a marked Umu inhtence .<br />
676<br />
DETERMINATION OF SPONTANEOUS AND ENU-INDUCED MUTANT FREQUENCIES IN<br />
CYNOMOLOGOUS MONKEYS . D .M .Zimmer, C .S.Aaron, R .J .Albertini, <strong>and</strong> J .P.O'NeiII. The<br />
Upjohn Co. <strong>and</strong> University of Vermont, USA .<br />
We have undertaken a study of mutagenesis In primates (cynomologous monkeys) to<br />
determine whether such an assay offers better means of risk assessment than currently<br />
used in vitro <strong>and</strong> rodent tests. Using methods similar to those described by Albertini<br />
(Mutat . Res. 150 (1985), 411-422J, mutation at the HPRT locus was monitored by<br />
determining the ability of PHA stimulated peripheral T- lymphocytes to form clones In the<br />
50869 3746
1989 EMS Abstracts 233<br />
presence of 6-TG . Cells were grown in round bottom 96 well microtiter plates <strong>and</strong> scored Notes<br />
visually (microscopicall*l- .for evidence of cell growth . Spontaneous mutant frequencies<br />
(MF) for a colony of 9 animals (using duplicate samples taken aprox . 2 weeks apart) gave a<br />
mean of 4 .4 t 5.3/106 cells . The appearance of mutants in peripheral blood after an IF dose<br />
of 100 mg/kg ENU was followed in a single animal by sampling weekly for 9 weeks . MFs<br />
for weeks 2-9 were (respectively) 15 .3, 5 .0,16 .5, 9 .9, 20 .6,12 .9,19 .6, <strong>and</strong> 26.9/106 ;day 0 MF<br />
was 7 .1/106 cells . In general, both MF <strong>and</strong> cloning efficiency for given animals were<br />
reproducible from sample to sam le . TG resistant clones from all experiments are<br />
currently undergoing molecular analysis to determine the nature of the mutations giving<br />
rise to TG resistant clones in-cynomoiogoas monkeys .<br />
677<br />
AN INSIGHT INTO THE MECHANISMS DETERMINING THE INDUCTION OF GENETIC EFFECTS BY NTA IN<br />
THE MOUSE AND DROSOPHILA SOMATIC AND GERM CELLS .<br />
M . Zordanl, A . Russo', R . Costal, C . Beltra .el, F . Pacchierotti=, M . Ostil <strong>and</strong> A .G .<br />
Levis' . 1 Dept . of Biology, University of Padova (ITALY) . 2 Lab . Toxicology, ENEA,<br />
CRE Casaccia ROME (ITALY) .<br />
Recently we reported the ability of nitrilotriacetic acid (NTA) to induce<br />
aneuploidy in the germ cells of both Drosophila <strong>and</strong> the souse . These results prompted<br />
further research in order to evaluate the response to treatment with NTA of somatic<br />
cells in the same organisms . The experimental systems adopted consisted in : a)<br />
chromosomal counting in souse bone sarrow cells after i .p treatment with 138 or 275<br />
eg/Kg NTA ; b) evaluation of single <strong>and</strong> twin spots in the somatic recor,bint .tion <strong>and</strong><br />
mutation test (SMART) in Drosophila selanosaster employing the &w_b e.nd (j=3 wing<br />
∎arkers, after feeding different doses of NTA (Sx10-3, 2 .5x10-2 , Sx10-2 K) for 42<br />
hrs . to 2nd stage larvae . In the latter case effectively absorbed doses of NTA were<br />
also evaluated by a .ethod employing 3H-leucine additioned to the treatr.ent sedts .<br />
The results indicate that NTA does not induce aneuploidy or sister chromatid<br />
exchanges in souse somatic cells . Positive results were obtained ir .utead :n the SMART<br />
test (statistically significant dose-dependent increase in the frepuen .:y ui small<br />
single p_wZi spots) . Extension of the wing spot analysis to include phenotypically AU<br />
individuals (inversion heterozygotes) revealed that small single spots are virtually<br />
absent in these flies following treatment with NTA, suggestiug that in the normal<br />
"w , flrs trans heterozygotes tbese spots originate mainly (90% or more) from<br />
recosbinational events .<br />
SUPPORTED BY C .N .R . p .f . "ONCOLOGIA" .<br />
A<br />
678 Due to late receipt, abstracts 678-697 are presented out of alphabetical order .<br />
CURRENT STATUS OF THE GENE-TOX PROGRAM, Angela Auletta, Office of<br />
Toxic Substance, U .S . EPA, Washington, D .C . 20460 .<br />
The EPA's Gene-Tox Program is a multi-phased effort to review <strong>and</strong><br />
evaluate published literature in genetic toxicology . Phase I was a<br />
review by expert work groups of literature published from 1969-1980<br />
for 23 days . Each group published an evaluation of chemicals tested,<br />
correlation of results with carcinogenicity, recommended protocols <strong>and</strong><br />
techniques for data analysis, interpretation <strong>and</strong> presentation . In<br />
1980-81, a computerized data base of over 2,600 chemicals was<br />
established ; this data base was analyzed by individuals concerned with<br />
chemical classification, carcinogenicity, <strong>and</strong> heritable mutation . At<br />
present, work groups are evaluating literature published since 1980<br />
for selected systems . As the update proceeds, the existing data base<br />
is reanalyzed . Analysis of the data base is affected by several<br />
factors . The preponderance of results are either positive or<br />
inconclusive . The high percentage of inconclusive results largely<br />
reflects the quality of the available literature . Most agents have<br />
been tested in only one system making cross system comparisons<br />
difficult . Correlations with carcinogenicity are hampered by the<br />
nature of the chemicals evaluated <strong>and</strong> the limited number of validly<br />
tested noncarcinogens in the data base . The data base is publicly<br />
available through the NLM TOXNET system . Public availability should<br />
increase its utility, exp<strong>and</strong> the analysis <strong>and</strong> affect the manner <strong>and</strong><br />
speed with which it is updated .<br />
~<br />
679<br />
OD ~<br />
DIET AS A SOURCE OF MUTAGENESIS t0<br />
P .S . Chauhan, <strong>Molecular</strong> Biology i Agriculture Division, Bhabha Atomic Research Centre,<br />
Trombay, Bombay-400 085 (INDIA) w<br />
Diet is a complex mixture of a large number of chemicals of diverse nature <strong>and</strong> ~<br />
continues to remain a major source of human ex posure to exogenous chemicals . In ad- v<br />
dition to life support constituents, human diet contains a large number of chemicals<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Y
234 1989 EMS Abstracts<br />
- . . . .r j- . .F0= a}r'-<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes which have been i'Nntified as toxicants <strong>and</strong> those whose biological effects are not<br />
yet known . While, literAfare is replete with reports showing a number of dietary components<br />
to be mutag'enit;^-=tirrent information on inhibitors of mutagenesis has been<br />
recently reviewed (DsFlora, ed ., Mutation Res . 202, No .2, 1988) . However, majority<br />
of the investigations on antimutagenesis vis-a-vis mutagenesis have emerged from bioassays<br />
using prokaryotic systems . While, this information provides a basis for further research<br />
<strong>and</strong> these assays are suitable for mechanistic studies, the results cannot be directly<br />
extrapolated to human system . In our laboratory, inhibitory effects of dietary factors<br />
including vitamins <strong>and</strong> trace elements, on chemical <strong>and</strong> radiation mutagenesis have been<br />
investigated in somatic <strong>and</strong> germinal cells of mice, These whole animal studies, though .<br />
support some of the observations emanated from prokaryotes, divergences are apparent<br />
between the two systems . It is pointed out that the ability of a putative inhibitor<br />
to survive the physiology of the mammalian gut, its absorption <strong>and</strong> availability in<br />
biophase would greatly contribute to its effectiveness in vivo . It is also evident that<br />
any programme on dietary prevention of genotoxicity wou~t-embark upon, removal of<br />
mutagens from the diet <strong>and</strong> incorporation of inhibitors of mutagenesis . Such a dietary<br />
reciepe, if ever formulated will have the advantage of allowing intervention at the<br />
community <strong>and</strong> public health level .<br />
ENVIRONMENTAL MUTAGENESIS IN DEVELOPING COUNTRIES<br />
C . cortinas de Nava, instituto de Investigaciones siomidicas, U .N .A .M .<br />
Ap . Postal 70228, Mexieo 20 D .B ., 04510 Mexico .<br />
The term developing countries encompasses a wide variety of situations<br />
: geographical, economical, politioal, soeial, eultural, ete . . A<br />
common denominator of those countries is the need to optimise efforts<br />
to stop environmental deterioration <strong>and</strong> diminish the impact of chemical<br />
pollution on human health <strong>and</strong> the eoosystems . This mplies the nesd of<br />
programs for integral evaluation of environmental pioblems, a~iequate -<br />
technologies for their control, a rapid multiplication ef 1ooa1 experts<br />
in the field, integration of multidisciplinary research groups <strong>and</strong> the<br />
community participation in activities of environmental protection <strong>and</strong><br />
prevention of health risks . Genetic Toxicology in developing countries<br />
can only contribute sustantielly to these goals if it is incorporated<br />
to the global actions intended to set priorities,to characterize <strong>and</strong> -<br />
manage the environmental risks . Collaboration of scientists from develoM+d<br />
countries 1a needed to speed theet, aot•lons, 'in partieul+ir, thnna<br />
concerning training of personnel, development of technol0q ies <strong>and</strong> information<br />
exchange . In addition, the establishment of collaborative -<br />
research projects to evaluate <strong>and</strong> control environmental problems of -<br />
regional interest will make the efforts of scientists from devaloping<br />
countries, working on the field, more relevant .<br />
680<br />
681<br />
Transgenic mice as a model system for studying gene mutations in vivo .<br />
Jan A . Gossen, W .F .J . de Leeuw, C .H .T . Tan <strong>and</strong> Jan Vijg, TNO Institute<br />
for Experimental Gerontology, P .O . Box 5815, 2280 HV Rijswijk, The<br />
Netherl<strong>and</strong>s .<br />
In order to study gene mutations in different organs <strong>and</strong> tissues of an<br />
experimental aniT .al, we constructed transgenic nice harbouring<br />
bacteriophage 1a^:bda shuttle vectors integrated in the qeno^ :e in a headto-tail<br />
arrangement . As a*.arazt fcr rnutagenesis, the selectable<br />
bac*.erial LacZ aene was cloned in the vector . The integrated vectors were<br />
rescued fror total genonic DN1, with high efficiency by in vitro packaging<br />
<strong>and</strong> propagation of the phages in an E .cc C Lac2- strain . This systex<br />
allowed the detection of :-.utation frequencies down to about 5 .10-6, the<br />
background frequency in different organs . Treatment of adult fenale<br />
transgenic :rice with fi- ..thyl-N-nitrosourea (ENU) resulted in a dosedependent<br />
increase of the mutation frequency in the vectors isolated fro^t<br />
brain D!tA, up to 10-1 at 250 :^g E1N per kg bodyweight . At this dose, in<br />
liver <strong>and</strong> bone -arrow Dtd:A of the sa :^e :r:ice, mutation freyu_ncies were 2 .9<br />
}: 10-' ar.d 8 .5 . . 10-°, respectively . Restriction-enzyme analysis<br />
indicated that the mutations observed in the LacZ target gene were point<br />
mutations (
682<br />
: 1989 EMS Abstracts 235<br />
MODULATORY EFFECTS OF WHEAT SEEDLINC,S HGMOjF39ATE (S14) ON (,ENOTOXICITY<br />
CF SOME SYSPEMIC PE4TICIDES . I .S . GRGVER <strong>and</strong> S•S .LacYiar, School of Life<br />
Sciences .Guru Nangk-Dev University, Amritsar-143005, INDIA<br />
Modulatory effects of wheat seedlings homogenate (S14) on genotoxicity<br />
of some systemic pesticides viz . benomyl, Ekatin, . . FernoXone,<br />
<strong>and</strong> monocrotophos have been assessed employing histidine ceversibn<br />
assay in Salrtonella yj~urium (Ames assay) <strong>and</strong> d;romosoiosl aberrations<br />
ih root meristems inlium =p-a . All these pesticides induced mitotic<br />
depression <strong>and</strong> a broad spectrum of physiological (a-mitosis, stickiness,<br />
vagrant chromosomes, laggards eta .) <strong>and</strong> .clastogenic (chromosome breaks,<br />
ring chromosomes <strong>and</strong> micronuclei) aberrations . However, the frequency<br />
of induced physiological aberratior.s remain unaltered by S14 homogenate<br />
but the percentage of cells with clastogenic aberrations was reduced<br />
significantly with the supplementation of 514 . None of these pesticides<br />
enhanced significantly the histidine revertants in TA98> TA1V2 <strong>and</strong><br />
TA1535 of .5 . tvnhimurlum . No appreciable effect was observed with the<br />
supplementation of rat liver S9 homogenate or wheat seedllno S14<br />
homogenate .<br />
683<br />
GENEl`GX EVALUATION OF METASYSTUX IN CHRGMC-SOMAL ABERRATIONS AND CHLORC :-<br />
YHYLL DEFICIENT MUTATI(~N ASSAY IN HQRDEUM VULGARE, I .S .GFGVER, Vinlta<br />
<strong>and</strong>'H .Grover, School of Life Sciences, Guru Nanak Dev University,<br />
Amritsai an`i3 +S .R.Govt .College for Women, Amritsar . INDIA .<br />
Metasystox-an organophosphorus pesticide is one of the mo4t wic3ely<br />
used pesticides whose genotoxic nature is uncertain . The present report<br />
describes its genotoxic effect employing chr'omosoeal aberrations assay<br />
in root meristems <strong>and</strong> pollen mother cells <strong>and</strong> chlorophyll deficient<br />
mutation assay in }jordeum yylqAy .'It induced a significarit increase in<br />
chromosomal aberrations in root meristems . The spectrum of chromosomal<br />
aberrations included chromosome breaks, c-mitotic effects, leading to<br />
polyploid cells, chromatin'bridgesn laggards .tri- <strong>and</strong> tetraPolar cells<br />
<strong>and</strong> micioruclei . The effect was found to be "dose-dependent• Po'llen mother<br />
cells with univalents was the moet common aberration encountered at<br />
metaphase-I .Rarely a quadrivalent attributable to exchanges was also<br />
noticed . Unequal distr'ibution, chro.matin bridges <strong>and</strong> laggards were also<br />
recd rded . M2 analysis at the seedling stage revealed chlorophyll<br />
deficient mutants<br />
. Xa ntha, tigrina, maculata, albovirldis <strong>and</strong> virido-<br />
chlorophyll deficient mutantsuwarrantTitsifuc~herrstudies~iccaebatteryaof<br />
assays<br />
684<br />
EVOLUTIONARY SIGNIFICANCE OF MUTATION AND REPAIR . Robert H . Haynes, Department of<br />
Biology, York University, Toronto, Canada, H3J 1P9 .<br />
Heredity is a manifestation of the stability of genes, chromosomes <strong>and</strong> genomee<br />
from one generation of cells <strong>and</strong> organisms to the next . Heritable variation is a<br />
manifestation of various types of change in the genetic material of c .lls . In the<br />
absence of mechanisms to promote an extremely high level of replicational fidelity,<br />
the long genetic messages of contemporary organisms could not have evolved . In addition,<br />
were it not for the existence of processes which effect the repair or bypass of<br />
damage to DNA which arises from many ever-present natural sources, cellular activity<br />
would collapse from what might be called 'genetic meltdown' . On the other h<strong>and</strong>, if<br />
the various mechanisms which promote genetic stability were capable of functioning<br />
with perfect accuracy, genetic variation, <strong>and</strong> hence evolution, also would not occur .<br />
Many different genetic loci are involved in coding <strong>and</strong> control of the complex array<br />
of biochemical processes which maintain genetic stability . This is consistent with<br />
the notion that if very great fidelity is to be achieved with equipmmnt of poor precision,<br />
extensive checking procedures must be built into the system . For optimum<br />
economy, the 'cost' of such procedures should be just sufficient to reduce the errorrate<br />
to a tolerable level . Thus, the genetic machinery of cells can be viewed as a<br />
remarkable example of a highly reliable, dynamically stable system built from vulnerable<br />
<strong>and</strong> unreliable parts . Recent work on the genetic consequences of nucleotide pool<br />
imbalance suggests that natural selection has fashioned all major aspects of DNA<br />
metabolism to minimise mortality <strong>and</strong> mutability . However, the existence of inducible<br />
error-prone processing of DNA damage indicates that the maintenance of cellular viability<br />
takes precedence over genetic fidelity .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes
236 1989 EMS Abstracts . :__ __<br />
~ 000. ..~<br />
685<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes tSMROMiNfAI . MJfXMM1S IN GHINA<br />
J . L . HSUF}I <strong>and</strong> C . C . TAN~<br />
Institute of Cenetics~ Fl~iverstty, Shanghai 200433, China<br />
China is a developing country, having a population of irarly 1 .2 billion people. Along with the progress<br />
of modernization, the caaxry is also facing the problen of enviranmental pollution through the release of<br />
industrial aastes into the air <strong>and</strong> keterr.mys . This, together with population pressure <strong>and</strong> agricultural problems,<br />
has already received the wide attention of the Chinese public . China is prepared to exercise quality<br />
control <strong>and</strong> envirarnental monitoring, <strong>and</strong> a series of regulations have been established . The Chinese <strong>Environmental</strong><br />
lLtagen Society (CElfi) ws found in 1983, it is essential to the development of environnental nutagenesis<br />
. In the year of 1983, 1985, 1987 <strong>and</strong> 1988, -the CE2S hdld his annual meetings in Shenghai, Wuhan,Slanghai "<br />
<strong>and</strong> Beijing. Over than 100 independent qualified laboratories for identifying autagens are now operating in<br />
Chira . They are distributed on more than 20 provinces . lbst of these assays are used to help identify nutagens,<br />
sore can also identify carcinogens . Of these laboratories, t .u-thrids are using nutagenlc assays <strong>and</strong><br />
the other one-third is doing either carcinogenetic or teratogenetic assays . 'kre in vitro assays use a variety<br />
of cell types ranging form bacterial to huren, fram somatic cells to genn cells ; other tests can be done<br />
directly on Drosophila, rodents <strong>and</strong> plants . Recently, the recc.rbinant DNA tecFnique has been introduted into<br />
the study of environrental nutagenesis . We have developed a shuttle vector system for studying mutational<br />
specificity, the shuttle vector contains the EB virus origin <strong>and</strong> E . colt XyIE gene, then introduced into human<br />
cultured cells by transfection <strong>and</strong> allowed to replicated autorously in the cell nucleus . During the replication<br />
period of the vector, the cells are exposed to a nutagen, an increased in nutation frequency above the<br />
spontaneous background is readily obtained . Induced mutants can be rapidly isolated <strong>and</strong> characterized by color<br />
change . Nowadays, all new medtcines, pesticides, food additives, contraceptions <strong>and</strong> Chinese medicinal<br />
herbs are subjected to the kres test, micronucleus test, chronosane aberrations <strong>and</strong> SCE analysis, U06 <strong>and</strong><br />
SOS et al routine screening procedures .<br />
686<br />
"THE POTENTIAL ROLE OF CELL DIFFERENTIATION IN TUMOR PROMOTION," E . Nuberman,<br />
Biological, <strong>Environmental</strong>, <strong>and</strong> Medical Research Division, Argonne National Laboratory,<br />
Argonne, Illinois 60439<br />
Chemicals that promote tumor formation, including phorbol 12-myristate 13-acetate<br />
(PMA), in general do not bind to DNA <strong>and</strong> are devoid of mutagenic activity ;<br />
nevertheless, many of these promoters induce differentiation in various cell types<br />
including the human promyelocytic RL-60 leukemia cells . Thus, tumor promotion may<br />
involve the expression of growth-facilitating genes, which, as a result of prior<br />
genetic alterations (during tumor initiation), have been placed under the control of<br />
genes that regulate normal cell differentiation . Consequently, we are studying the<br />
ability of PHA <strong>and</strong> related agents to initiate early biochemical events that cause<br />
alterations in gene expression leading to modulation of differentiation processes .<br />
These phenomena are analyzed in NL-60 cells that are either susceptible or resistant to<br />
the induction of cell differentiation by PMA . Differentiation is characterized by<br />
changes in functional proteins, enzymatic markers, <strong>and</strong> reactivity with monoclonal<br />
antibodies generated against maturation-specific cell-surface <strong>and</strong> nuclear proteins .<br />
Our results indicate that a number of early events (2-30 m1n) such as subcellular<br />
translocation <strong>and</strong> activation of protein kinase C, phosphorylation of a number of<br />
proteins including nuclear proteins, <strong>and</strong> modulation of topoisomarase II activity may be<br />
early steps in the signal transduction that results in the induction of cell<br />
differentiation <strong>and</strong> perhaps tumor promotion by PNA <strong>and</strong> related agents . Work supported<br />
by the U .S . Department of Energy under Contract No . W-31-109-ENG-38 .<br />
687<br />
DETECTION OF THE MUTAGENS IN URINE OF CIVILIANS EXPOSED TO MUSTARD GAS<br />
M .A .Jabbar ; F .Pourismnili ; G .H .Riazi <strong>and</strong> M . Nouri Dalawi<br />
Biochemistry <strong>and</strong> Biophysic Institute, Tehran University, Box 6479-11365<br />
Tehran , IRAN .<br />
Thous<strong>and</strong>s of miliatry <strong>and</strong> civilian people in Iran were exposed to sublethal<br />
doses of mustard gas during the Iraqi-Iran war . Therefore, a rapid<br />
prediction for the hazardous impact of this agent on man <strong>and</strong> his environment<br />
is urgently needed to impliment any possible preventive measure .<br />
Ames <strong>and</strong> the fluctuation assays were used to detect the mutagenic metabolites<br />
of mustard gas in urine samples of 20 non-smoker patients who<br />
were wounded in Sardasht area( west of Iran )in May, 1986 . Salmonellae<br />
_t~~himurium TA98, TAIOO, <strong>and</strong> TA202 were used in the screening proce3ures .<br />
Tfie resul-ts showed that the fluctuation test was more sensitive than<br />
Ames test in detection the mutagenic agents of mustard gas metabolites .<br />
More than 701 of the urine samples were mutagenic for TAI02 strain when<br />
fluctuation assay was implimented . These results reveal that most of the<br />
mutagenic metabolites in the urine samples of the wounded patients were<br />
of the oxidative type . Some patients were given garlic Juce to see its<br />
antimutagenicity in vivo but there was no significant difference in the<br />
mutagenic activity of their uring concentrates befor or after treatment .<br />
In conclu sion, more work is needed to spe ify the mutagenic metabolites<br />
of mustarsd gas <strong>and</strong> the actual hazard of wh~ch on man or his offspring .<br />
50869 3750
688<br />
CYfOGENETIC EFFECTS OF PHORBOL ESTER TUMOR PROMOTERS : POSSIBLE ROLE IN IIULTISTEP TUKORI-<br />
GENESIS. V . Kinzel . B. Farber, R .T. Petrusevska . N . E . Fusenle . Institute of Exp. Pathology,<br />
Institute of Biochemistry', German Cancer Research Center . D-8900 Heldelber¢ . PRG<br />
Tumoriyenesis in mouse skin can be effected in a number of steps : by initiation with DMBA, by<br />
conversion with one or two applications of TPA (12-0-tetradecanoylphorbol-13-acetate) <strong>and</strong> by reprated<br />
treatment with RPA (12-0-retinoylphorbol-13-acetate) . The conversion step effected by TPA<br />
(but not by RPA) Is characterized by a half-life of 10-12 weeks (in NMRI aice) : it is Sndependent<br />
from initiation (Fiirstenberger . G . et al ., 1985, Science, L3-0. 76) . '1'his specific effect of TPA<br />
may be explained best by the clastogenic activity of TPA shown In mouse keratinocytes In culture<br />
(Petrusevska, R.T. at al . 198fi, Carcirto¢enesis $, 1207) as well as in ex vivo studies (Fiirstenbereer,<br />
G . et al ., 1988, Carcino¢eneals . In press). Details of the olastoYenic action of TPA were studied in<br />
HeLa cells which exhibit a rodiuaimetic cell cycle response to TPA (Klnzel, V . at al ., 1980, Science<br />
210 . 429) . Only TPA exerted a significant elastogenic activity at non-eytotoxic concentrations (10-8<br />
to 10-6 M) measured after 24 <strong>and</strong> 48 hrs . Values obtained with RPA were close to those obtained in<br />
the presence of solvent (acetone 0.2k). The response to TPA was only to some extent correlated with<br />
the dose. Chromosome aberrations including gaps <strong>and</strong> breaks were predominantly of the chromatid type ;<br />
they are earliest measurable after 6-8 hrs, I .e. as the cells recover from TPA-induced 02-inhibition<br />
(Kinzel . V . et al., 1988. Cancer Res . 40 . 1759). A 30 ∎in exposure to TPA (10-7 N) is sufficient to<br />
induce aberrations . The data point to an indirect, possibly receptor-mediated action of TPA . If it<br />
is supposed that TPA-iuduced chromosome lesions represent a key event required for conversion it<br />
might be further speculated that the absolute requirement for DNA replication In the conversion step<br />
(Kinsel, V . et a1 ., 1984 . PNAS $1. 5858) is necessary to "fix" a certain degree of chromosome damaQe<br />
(supported by the DFG) .<br />
689<br />
ASSESSMENT OF EXPOSURE AND POTENTIAL RISK FROM MUTAGENIC AIR POLLUTANTS . Joellen<br />
Levtas, U .S . <strong>Environmental</strong> Protection Agency, Research Triangle Park, N .C . 27711<br />
(U .S .A .) .<br />
Genetic bioassays have been used to identify airborne mutagens <strong>and</strong> tlieir sources,<br />
to measure human exposure <strong>and</strong> to provide comparative assessment of potential cancer<br />
risk from air pollutants . Recent air pollution studies have integrated the use of<br />
genetic bioassays into sampling <strong>and</strong> analysis strategies to seet these goals . Complex<br />
mixtures of urban air pollutants from vehicles <strong>and</strong>aresidential heating sources have<br />
been studied in field investigations in both Western <strong>and</strong> Eastern cities in the U .S .<br />
as part of EPA's Integrated Air Cancer Project . Genetic bioassays were applied to<br />
source emissions <strong>and</strong> ambient outdoor <strong>and</strong> indoor air to characterize the exposure <strong>and</strong><br />
potential risk from the gaseous, semi-volatile <strong>and</strong> particle-bound organic species .<br />
Source apportionment of the mutagens in these airsheds has been accomplished through<br />
receptor-modeling of mutagens using multiple linear regression analysis .<br />
Mutagenesia, tumorigenesis <strong>and</strong> DNA adduct dosimetry studies of these air pollution<br />
mixtures have been used in further developing a comparative potency method for<br />
assessing cancer risk from complex mixtures . This is an abstract of a proposed<br />
presentation <strong>and</strong> does not necessarily reflect EPA policy .<br />
690<br />
THE GENETIC TOXICITY OF THE HUMAN CARCINOGENS BENZIDINE AND BENZIDINE-<br />
BASED DYES : CHROMOSOMAL ANALYSIS IN EXPOSED WORKERS<br />
E .Mirkova <strong>and</strong> S .Lalchev,The Medical Academy,Sofia (Bulgaria)<br />
The activities of the human carcinogens benzidine (BENZ) <strong>and</strong> BENZ-based<br />
dye Direct Black 38 in the rodent bone marrow micronucleus assays ( BM<br />
MNA) were established unequivocally (Ashby <strong>and</strong> Mirkova,1988 ;Beije 1987) .<br />
The lack of data on their genetic effects in humans acted as the stimulus<br />
for the present cytogenetic study .Chromosomal analysis was performed<br />
in the lymphocytes of 23 BENZ-based dyes manufacturing workers <strong>and</strong> 30<br />
matched control .individuals . The period of exposure was 7-31 yr . BENZ<br />
was detecSed in the workplace air at concentration levels of 0 .42 -<br />
0 .86 mg/m <strong>and</strong> in the urine of exposed workers respectively at mean level<br />
of 1 .78 ± 1 .4 µg/1 . The total airborne particulate levels of BENZ -<br />
based dyes (mainly Direct Black 38) ranged from 7 .8 to 32 .3 mg/m3 . i<br />
significant increase in the X of aberrant cells (-gaps) was found in the<br />
exposed group (7 .9 ± 5 .9) as compared with the control incidence of<br />
0 .87 ± 0 .26X aberrant cells (-gaps) . Aberrations were mainly chromatid<br />
breaks . The X frequency of polyploid cells in the exposed grou was<br />
found to be 1 .1 ± 0 .1 <strong>and</strong> the control incidence was 0 .17 * 0 .5~ . The<br />
present data "establish that the rodent BM MNA has predicted correctly<br />
the mutagenicity to humans of the IARC carcinogens BENZ <strong>and</strong> BENZ-based<br />
dyes .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
1989 EMS Abstracts 237<br />
Notes
238 1989 EMS Abstracts _ e _ -- - - 691<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
NO LE S REVERSION OF CANCER PR~ENOTYPES BY ALTERATIONS IN GENOTYPE . Minako NAGAO, National<br />
Cancer Center Research nstitute, Tokyo (Japan)<br />
NIH3T3 cells Zian`e
694<br />
: 1989 EMS Abstracts 239<br />
Notes<br />
COMPARISON OF HUMAN AND RODENT RESPONSE TO GENOTOEICANTS<br />
Schm X hl, D ., Institute of Tozicology <strong>and</strong> Chemotherapy, German Cancer<br />
Research Center, Im=-Neuenheimer Feld 280, D-6900 Heidelberg, Federal<br />
Republic of Germany<br />
On the basis of results found in genetic toxicology, animal experi-<br />
ments with rodents <strong>and</strong> human data, two groups of compounds are discussed,<br />
namely anticancer drugs <strong>and</strong> N-nitroso compounds . Regarding anticancer<br />
drugs, there is a good agreement between resulte obtained in<br />
vitro in tests on genotoxioity <strong>and</strong> in carcinogenicity ezperimente in<br />
rodents as well ae in -human-carcinogenesie . Investigations with Nnitroso<br />
compounds ehowed that many N-nitroso compounde such as die-<br />
thylnitrosamine are carcinogenic in many laboratory animals, but the<br />
organotropism of the carcinogenic activity varies considerably . Epide-<br />
miological studies on people monitoring concerning the extrapolation<br />
of results obtained in animal experiments to the human situation have<br />
to take into consideration that the organotropism of the carcinogenic<br />
activity is often hard to predict . Possible reasons are epeculatively<br />
discussed .<br />
695<br />
GEi:OTOXIC ASSAY OF TI(0 DIF:TARY FURP2dS BY SOW IN VIVO CYTOGENETIC PARAI+R9TERS<br />
S . Subramanyam, D . SailaJa <strong>and</strong>D . Rathnaprabha<br />
Department of Genetics, Osmania University,<br />
Hyderabad 500007, India<br />
Furans found in vegetarian <strong>and</strong> nonvegetarian foods <strong>and</strong> used to prepare resins,<br />
varnisiies, pesticides <strong>and</strong> germicides are suspected to oause respiratory tract<br />
infections, liver <strong>and</strong> lung oezicers . In the absence of proper information, the<br />
genotozic potentials of two furans, Furfural <strong>and</strong> 2-methyl furan, were evaluated by<br />
the st<strong>and</strong>ard in vivo cytogre netic protocols by employing somatic <strong>and</strong> molotio tissues<br />
<strong>and</strong> nultiple paemeters on 8 to 10 week old Swiss albino mice as test system . In<br />
somatic system o1L omosome mutatio : .s were scored from bone marrox oells, 24,48 <strong>and</strong><br />
72 hr . after oral feeding at 1000,2000 <strong>and</strong> 4000 p}m concentrations at 24 hr . intervals<br />
for five dqyc . Furitiral induced chromosome matatious only with the highest<br />
dose after 24 <strong>and</strong> 48 hr . 2-methyl furan did not do so . Both did not inhibit<br />
synthesis of snindle proteins . There was no retardatiosi of oell division . In<br />
neiotic test system, the test was . carried out for 24 hr . <strong>and</strong> from I to V weeks<br />
at xeeYly intervals to cover one spermatogenetic cycle . Both compounds did not<br />
induce Genotozio effects . They did not also cause sperm head abnormalities which<br />
indicete point mutations induced in sex end autosomal genes governing their mospholof•y<br />
. This analysis illustrates thF .t cumulative doses of the two flurane do not<br />
cause any genotoxic hazards to the mammalian teet system .<br />
696<br />
GENETIC TOXICOLOGY OF COMPLEX MIXTURES--DVERVIEW AND SUMMARY OF THE WASHINGTON<br />
SATELLITE MEETING . Waters, M ., U .S . Environ . Prot . Agency, Res . Tri . Park, NC (USA) .<br />
Complex mixture research in genetic toxicology has advanced rapidly since the first<br />
EPA-sponsored Symposium on "Short-Term Bioassays in the Analysis of Complex<br />
<strong>Environmental</strong> Mixtures" held in 1978 . The purpose of this biennial symposium is to<br />
present state-of-the-art techniques in bioassay <strong>and</strong> chemical analyses applied to<br />
complex mixtures <strong>and</strong> to foster continued advancement of the field . The Washington<br />
ICEM Satellite Meeting will begin with a presentation on the latest techniques in<br />
molecular biology applicable to complex mixture research <strong>and</strong> then will separately<br />
address complex mixtures in air <strong>and</strong> in water, <strong>and</strong> exposure/effects assessment . The<br />
session on air focuses on the identification <strong>and</strong> characterization of component<br />
chemicals <strong>and</strong> chemical classes found in combustion sources <strong>and</strong> in ambient <strong>and</strong> indoor<br />
air . The thrust of research in this medium is to separately characterize the<br />
contributions of various combustion sources as volatile <strong>and</strong> particulate components as<br />
well as atmospheric transformation products to the observed genotoxicity of the air<br />
mixture reaching the human receptor . In the water medium, the main emphasis of<br />
research is on the characterization of mutagenic compounds formed during disinfection<br />
of drinking water <strong>and</strong> on the development of new methods to detect genotoxicants in<br />
various wastewaters . Exposures/effects assessment research deals with the use of DNA<br />
<strong>and</strong> protein adduct dosimetry in conjunction with low dose extrapolation models used to<br />
estimate cancer risk from environmental genotoxicants . Presentations will consider<br />
cigarette smoking, occupational <strong>and</strong> industrial exposures as well as environmental<br />
exposures . A final presentation will address future avenues of research in the field .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
M<br />
I<br />
I<br />
~
240 1989 EMS Abstracts _ ._ -- - .<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Notes "~CHEMOPREVENTI*fOFTMTOXICITY IN VIVO . Wattenber~, L . W . Dept . of Laboratory<br />
Medicine <strong>and</strong> Pathology . Univers~iLy' o-Minnesota, Minneapolis, MN 55455, USA .<br />
Cnemopreventiqp gi56he effects of genotoxic compounds in vivo can be brought about<br />
by compounds ("bloc~c'Tng agents") that prevent the genotoxi~c agent from reaching or<br />
reacting with critical cellular targets or by compounds ("suppressing agents") that<br />
prevent manifestations of genotoxicity in cells in which the target molecules have<br />
already been hit . Using carcinogens as an example of one type of genotoxic compound,<br />
blocking agents can be shown to act via three mechanisms : (1) by inhibiting activation<br />
reactions of carcinogens requiring metabolic activation in order to form reactive<br />
electrophiles, (2) by enhancing carcinogen detoxification <strong>and</strong> (3) by trapping<br />
reactive carcinogenic species . Examples of all three will be discussed . Particular<br />
emphasis will be on naturally-occurring organosulfur compounds <strong>and</strong> aromatic<br />
isothiocyanates that can both induce increased activity of detoxification systems <strong>and</strong><br />
can inhibit carcinogen activation . Trapping agents to be discussed will include aromatic<br />
thiols <strong>and</strong> sodium thiosulfate . Data relating to suppressive effects of cruciferous<br />
vegetables <strong>and</strong> citrus fruit oils will be presented . Current evidence indicates<br />
that there are surprisingly large numbers of pure chemicals <strong>and</strong> natural products<br />
that can inhibit genotoxicity in vivo . Supported by Grant SIG 5A from the<br />
American Cancer Society .<br />
. 697
a-<br />
Author Index to Abstracts<br />
Numbers refer to abstract numbers, not page numbers<br />
AL-Allak, B .M .A ., 539<br />
Aardema, M .J ., 1, 350<br />
Aaron, C ., 657<br />
Aaron, C .S ., 2, 3, 235, 676<br />
Aaron, S ., 588<br />
Abarca M ., 133<br />
Abbond<strong>and</strong>olo, A ., 66, 371 483<br />
Abbott, M .G ., 4<br />
Abdalla, N .A ., 153<br />
Abe, T ., 420<br />
Abu-Shakra, A ., 5<br />
Adair, G, 87<br />
Adhikari, N ., 6<br />
Adhvaryu, S .G ., 7<br />
Adler, L-D ., 8<br />
Aeschbacher, H .U ., 9<br />
Afzal, V ., 639<br />
Agarwal, K ., 526<br />
Agostinelli, D .A ., 49<br />
Agostini, J .M .S ., 83<br />
Ahnstrom, G ., 332<br />
Ahuja, Y .R ., 10, 35, 463<br />
Aibara, K ., 490<br />
Aidoo, A ., 11<br />
Akintownwa, D .A .A ., 12<br />
Akiyama, N ., 13<br />
Al-Ghaith, L .K ., 154<br />
AlaLeviE, M ., 174<br />
Albertini, R .J ., 14, 412, 471, 545, 676<br />
Albertini, S ., 15, 16<br />
Alc .Intara-Diaz, D ., 17<br />
Alex<strong>and</strong>er, J ., 28, 616, 617<br />
Allen, J .W ., 36, 259, 630<br />
Allen, K .L ., 123<br />
Alonso, C ., 494<br />
Alvi, N .K ., 19<br />
Ammenheuser, M .M ., 20, 623<br />
An, Y .m . 562<br />
Anaya, P., 26<br />
Anderson, D ., 21<br />
Andrews, A .F ., 246<br />
Andrews, P .W ., 22<br />
Antilla, S ., 228<br />
Anzick, S .L ., 422<br />
Aranez, A .T ., 23, 24<br />
Aravindakshan, M ., 25<br />
Arce, G .T., 49<br />
Arena, G ., 483<br />
Arenaz, P ., 26<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Arimoto, S ., 27<br />
Armstrong, J .D :, 28, 310, 311<br />
Arras, C .A ., 534<br />
Arreola, G .G ., 336<br />
Arrimanoglou, I .I ., 29<br />
Arumikkili, N ., 528<br />
Ashby, J ., 30, 81<br />
Athwal, R .S ., 223<br />
Atsumi, G ., 31<br />
Au, W .W ., 20, 32<br />
Auletta, A ., 678<br />
Ault, K .T., 241<br />
Austin, S .J ., 320<br />
Autrup, H ., 33<br />
Awogi, T., 565<br />
Azian, A ., 63<br />
Baan, R .A ., 34<br />
.<br />
Babu, P.P., 35<br />
Backer, L .C ., 36, 259<br />
Bai, C .J ., 643, 647<br />
~Bailey, G ., 122<br />
Bakale, G ., 38<br />
Baker, R .S .U ., 245<br />
Bakke, J .P. 230<br />
Bala, S ., 221<br />
Balakrishnan, S ., 53<br />
Balcgzar, M ., 574<br />
Balczon, R .D ., 74<br />
Baldini, M ., 483<br />
Bali, D ., 541<br />
Ball, J .C ., 39<br />
Ball, S .T., 441<br />
Balwierz, P.S ., 428<br />
Banerjee, A ., 526<br />
Banga, S .S ., 70<br />
Bansal, M .P., 408<br />
Bansal, M .R ., 40<br />
Barbosa, H .S., 475<br />
Bardwell, L ., 176<br />
Barnes, W.s ., 112<br />
Barnett, L .B ., 41<br />
Barrett, J.C., 637<br />
Baruthio, FL . 157<br />
Bassani, B ., 431<br />
Basu, A .K . 337<br />
Battista, J .R ., 42<br />
Battula, N ., 43<br />
Bautista, A .R .P.L., 493<br />
241<br />
Bayley, S .T ., 37<br />
Bayuley, B .C ., 169<br />
Becher, G . 18, 616, 617<br />
Beeman, D .K ., 320<br />
Beije, B ., 379<br />
Bel<strong>and</strong>, F .A ., 44<br />
Bel<strong>and</strong>, F .A ., 581<br />
Bell, D .A ., 45<br />
Belli, J .A ., 20<br />
Beltrame, C ., 677<br />
Bempong, M .A ., 46<br />
Benasutti, M ., 337<br />
Benigni, R ., 47, 106<br />
Benova, D.K ., 48<br />
Bentley, K .S .<br />
Berger, N .A . 98<br />
Berger, S .J ., 98<br />
Bergtold, D .S ., 50<br />
Bernini, L .F ., 51<br />
Bertr<strong>and</strong>, F., 128<br />
Bessho, T., 31, 52<br />
Bewsey, B ., 419<br />
Beyers, J ., 290, 439<br />
Beyersmann, D ., 242<br />
Bhatia, K, 548<br />
Bhatt, B ., 53<br />
Bhatt, J ., 602<br />
Bhattacharjee, S .B ., 194, 518<br />
Bhattacharya, N .P., 54<br />
Bhattacharya, R .K, . 54<br />
Bhaumik, G ., 194<br />
Bhilwade, H .N., 56<br />
Bi, D .Q ., 263<br />
Bieszczad, M .J ., 556, 557<br />
Bigbee, W .L ., 271, 319<br />
Bin, Y ., 233, 460<br />
Bineva, M ., 48<br />
Bishop, J .B ., 57<br />
Bisi, J .E ., 637<br />
Blackburn, G .R ., 58<br />
Blaich, G ., 59<br />
Blake, B .W., 60, 61<br />
Blakey, D .H ., 62<br />
Blevins, R .D ., 63<br />
Bloom, S .E., 343, 375<br />
Bochkov, N .P., 64<br />
Bodell, W .J ., 65, 634<br />
Boehm, R .M ., 161<br />
Bonatti, S ., 66, 371<br />
I
242 Author Index to Abstracts<br />
Boobis, A .R ., 206<br />
Boothman, D .A ., 67<br />
Boreiko, C .J ., 352<br />
Borgstedt, H .H ., 60, 61<br />
Boroffice ; R .A ., 68<br />
BOrrsen, A .-L., 69<br />
Bourre, F ., 498<br />
Boyd, J .L ., 70<br />
Boyes, B .G ., 71<br />
Boyley, J .M ., 62<br />
Brady, A ., 627<br />
Br<strong>and</strong>riff, B .F ., 72<br />
Branstetter, D ., 2<br />
Brefia-Valle, M ., 17<br />
Brendler, S .Y ., 449, 450<br />
Bridges, B ., 111<br />
Bridges, B .A ., 73<br />
._Brillinger, R .L ., 410<br />
Brinkley, B .R ., 74<br />
Brinson, E .C ., 75<br />
Brockman, H .E ., 456, 627<br />
BrOgger, A ., 69<br />
Brognoli, I ., 83<br />
Broit, M ., 582<br />
Bronzetti, G ., 76, 182<br />
Brookman, K .W ., 579<br />
Brooks, A .L ., 77, 476<br />
Brunborg, G ., 78<br />
Brunny, J ., 79<br />
Brusick, D .J ., 80, 81<br />
Bruzzone, E ., 483<br />
Bryant, M .F ., 82<br />
Bueno, A .M .S ., 83<br />
Bulich, A .A ., 84<br />
Burger, G .T., 144, 322<br />
Burgess, W .M ., 366<br />
Burkart, W., 609<br />
Burkhart-Schultz, K ., 75, 85<br />
Burlingame, S .F., 65<br />
Burlinson, B ., 86, 192<br />
Burrell, M ., 286<br />
Busch, D . B ., 87<br />
Busk, L ., 88, 607<br />
Butler, M .A ., 89<br />
Butterworth, B .E ., 49, 635<br />
Cai, G ., 327<br />
Cai, Y ., 90<br />
Cain, K .T., 516<br />
Campbell, J .A ., 259, 630<br />
Campfield, A ., 583<br />
Cantelli Forti, G ., 76<br />
Cao, J ., 91<br />
Cao, L .F ., 646<br />
Cao, N ., 621<br />
Cao, S .-y ., 92<br />
Caprathe, B.W ., 307<br />
Carere, A ., 115<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Cariello,_ N', ~=" - -<br />
Carr, J ., 1~38<br />
Carr, R., 446 ~<br />
Carrano, A.V ., 72<br />
Carty, M ., 138<br />
Carvajal de Gil L .A ., 218<br />
Casciano, D .A ., 93, 388, 406<br />
Caskey, C .T., 484, 649<br />
Caspary, W.J ., 123<br />
Castagnero, M ., 126<br />
Caterson, C ., 530, 531<br />
Cattley, R .C ., 451<br />
Cebula, T .A ., 94<br />
Cebulska-Wasilewska, A . 95, 96<br />
Cederberg, H ., 607<br />
Cercignanni, G ., 66<br />
Cerqueira, E .M .M ., 475<br />
Chakraborty, P.K ., 199<br />
Ch<strong>and</strong>orkar, M ., 209<br />
Ch<strong>and</strong>rika, K .V., 467<br />
Chang, C .C ., 277<br />
Chang, F ., 459, 672<br />
Chang, S .C ., 117<br />
Chang, X .-p ., 168<br />
Chang, Y .-y ., 168<br />
Chang, Z .-y ., 92<br />
Channarayappa, J .N ., 97<br />
Chattetjee, S ., 98<br />
Chaubey, R .C ., 56<br />
Chaudhry, M .A ., 99<br />
Chauhan, P .S ., 56, 679<br />
Chen, A .T .L ., 472<br />
Chen, D .s, 132<br />
Chen, H .-H ., 147<br />
Chen, J .-K ., 640, 648<br />
Chen, K .-Z ., 100<br />
Chen, R ., 101<br />
Chen, S ., 262<br />
Chen, S .M ., 263<br />
Chen, T .D ., 347<br />
Chen, X .R ., 658, 659<br />
Chen, Y ., 102, 655, 665<br />
Chen, Z., 273<br />
Cheng, M .F., 98<br />
Cherney, B ., 548<br />
Chionglo, D ., 568<br />
Chiu, S .M, 103<br />
Choi, I .S ., 104<br />
Chouroulinkov, I ., 425<br />
Chowdhury, J .B ., 183<br />
Chu, J .-X ., 168<br />
Chung, H .W ., 383, 636<br />
Ciaravino, V ., 575<br />
Cifone, M .A ., 105, 173<br />
Citro, G ., 106<br />
Clapp, D .W ., 148<br />
Claxton, L .D ., 107<br />
Cliet, I ., 108<br />
Clive, D ., 109, 201, 303<br />
Clonfero, E ., 110<br />
Cobb, R .R ., 430<br />
Cochrane, J ., 470, 471<br />
Coelho, M .C .L .S ., 494<br />
Cohen, M .D., 549<br />
Cohen, M .M ., 508, 509<br />
Coimbriio, C .A ., 494<br />
Cole, J ., 111<br />
Coles, B ., 112<br />
Colyer, S .P., 275, 333<br />
Combes, R .D ., 521, 522<br />
Conley, E.C., 114<br />
Conner, M .K ., 377<br />
Conti, G ., 115<br />
Cooper, A .J ., 176<br />
Cordier, A ., 108<br />
Corey, L .A ., 369<br />
Corrie, M ., 379<br />
Corsale, G ., 162, 434<br />
Cortinas De Nava, C ., 680<br />
Costa, R ., 677<br />
Crebelli, R ., 106, 115<br />
Crespi, C .L ., 116<br />
Cross, F .T., 276<br />
Cui, Y .Q ., 117<br />
Cunningham, M .L ., 118<br />
Curren, R .D., 173<br />
Custer, L ., 105<br />
Czeizel, A., 119<br />
de Leeuw, W .F .J ., 681<br />
de la Rosa, M .E ., 574<br />
de S . Cblus, I .M ., 113<br />
de Serres, F.J ., 81, 127, 430<br />
D'Souza, S .J ., 405<br />
Dai, Y .F ., 658<br />
Dalawi, M .N ., 687<br />
Dallaire, L ., 545<br />
Damodaran, T.V., 120<br />
Daniel, F .B ., 367<br />
Daniels, C .B ., 121<br />
DaniBre, M .C ., 157<br />
Danzl, T ., 186<br />
Das, T., 526<br />
Dashwood, R ., 122<br />
Daston, D .S ., 123<br />
Dave, B .J ., 7<br />
Davies, D .S ., 206<br />
Davison, A .J ., 485<br />
Day, J ., 165<br />
Daya-Grosjean, L ., 124<br />
Dayrit, F., 610<br />
De Carli, L ., 594<br />
De Ferrari, M ., 66<br />
De Flora, S ., 125<br />
De Marini, D.M ., 45, 259<br />
De MEo, M .P., 126
De Sario, A ., 594 ~<br />
De Wals, P ., 128<br />
De, M., 526 r<br />
DeAngelo, A .B ., 367<br />
Dearfield, K .L ., 130, 131<br />
Deitch, R .A ., 58<br />
Del Carratore, R ., 76, 182<br />
Delclos, K .B ., 93<br />
Della Croce, C ., 182 -<br />
Demkowicz-Dobrzafiski, K .K ., 248<br />
Demple, B ., 132<br />
Deng, D .J ., 665<br />
Denham, J ., 167<br />
Descailleauz, J ., 135<br />
Dewi, D ., 437<br />
Dhillon, H .S ., 134<br />
Dhir, H ., 135<br />
Diggle, 136<br />
DineshKumar, B ., 10<br />
Dipple, A ., 137<br />
Dixon, K ., 138<br />
Dobbs, R .A . 140<br />
Dock, L ., 248<br />
Doehmer, J ., 139<br />
Doerger, J .U ., 140<br />
Doerr, C .L ., 130<br />
Doess, C .L ., 141<br />
Dolara, P., 142, 143<br />
Dolk, H ., 128<br />
Domon, O .E ., 388<br />
Donahoe, R . M ., 520<br />
Dong, Z . W . , 117<br />
Donnelly, C .E ., 42<br />
Donovan, C ., 148<br />
Doolittle, D .H ., 144<br />
Doolittle, D .J ., 322<br />
Dou, G ., 562<br />
Douglas, G .R ., 62<br />
Dragan, Y ., 445<br />
Drake, J .W ., 145<br />
Dresp, J .H ., 146<br />
Driscoll, S ., 283<br />
Drougard, C ., 124<br />
Du, Y .-X ., 147<br />
Dubins, J .S ., 430<br />
Duker, M .J ., 184<br />
Dumenco, L .L ., 148<br />
Dum6nil, G ., 126<br />
Dunbar, V .G ., 520<br />
Dunphy, E ., 149<br />
Dybing, E ., 78 •<br />
Eastmond, D.A ., 150, 546, 590<br />
Ebata, J ., 151<br />
Ehling, U .H . 152<br />
Eiche, A ., 458<br />
El-Seehi, M ., 247<br />
EI-Tarras, A ., 153<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
El-Zawahri, M .M ., 154<br />
El-Zyat, H ., 247<br />
ElKoweidy, A.H ., 389<br />
Elespuru, R .E ., 155, 156<br />
Elias, Z ., 157<br />
Elmore, E ., 158<br />
Endo, 0 ., 285, 355, 356<br />
Ennever, F .K ., 159<br />
Enslein, K ., 60, 61, 204<br />
Epe, B ., 160<br />
Erexson, G .L., 82, 161, 294, 630<br />
Esaki, S ., 288<br />
Esancy, J ., 107<br />
Eskelinen, A ., 344<br />
Esposito, A ., 162, 434<br />
Essigmann J .M ., 337<br />
Etoh, H., 436<br />
Fahrig, R ., 163<br />
Falek, A ., 520<br />
Falta, M .T., 545<br />
Fan, Q ., 102<br />
Fan, X ., 264<br />
Fang, F .D ., 646<br />
Fang, L., 168<br />
Fang, M ., 658<br />
F3irrber, B ., 688<br />
Favor, J ., 164<br />
Fedorwicz, G ., 165<br />
Felton, J .~ 600<br />
Felton, J .S ., 166, 366, 592<br />
Fenech, M ., 167<br />
Feng, P.-C ., 168<br />
Fengming, G ., 185<br />
Ferguson, L .R., 169<br />
Ferguson, R .J ., 246<br />
Ficsor, G ., 170<br />
Fielder, 136<br />
Finch, P., 610<br />
Fiskesjo, G ., 171<br />
Fleck, E .W ., 276<br />
Foiles, P.G ., 19<br />
Fong, A ., 122<br />
Fornace, A .J ., Jr, 172<br />
Forster, R ., 512<br />
Fort, F.L ., 173<br />
Fournier, E ., 108<br />
Fox, M ., 99, 484<br />
Francis, W., 167<br />
FranekiE, J ., 174<br />
Freeman, H ., 107<br />
Frei, H ., 175<br />
French, J .E ., 57<br />
Friedberg, E .C ., 176<br />
Friedman, L.R ., 103<br />
Froes, N .C ., 177<br />
Frohberg, H ., 301<br />
Frome, E .L ., 275, 333<br />
.<br />
Author Index to Abstracts 243<br />
Fronza, G ., 371, 483<br />
Frosina, G ., 483<br />
Fu, J ., 102<br />
Fu, S ., 562, 673<br />
Fujika, Y ., 252<br />
Fukui, S ., 252<br />
Fullerton, N .F ., 44<br />
Furihata, C., 178<br />
Furukawa, A ., 179<br />
Furukawa, H ., 180<br />
Fusenig, N .E ., 688<br />
Gad, S .C ., 428<br />
Gaidzinsk, K ., 83<br />
Gairola, C .G ., 578<br />
Galas, D .J ., 181<br />
Galati, R ., 106<br />
Galli, A ., 182<br />
G<strong>and</strong>hi, G ., 183<br />
Ganguly, T., 184<br />
Gao, H .L., 646<br />
Gao, M ., 656<br />
Gao, N ., 1I<br />
Gao, Q ., 646<br />
Garcia, M .V., 177<br />
Garin, K .E ., 230<br />
Garriott, M ., 79, 290, 439<br />
Garry, V., 186<br />
Gasper, K .P. 276<br />
Gatehouse, D .G ., 86, 187, 192, 291, 593<br />
Geard, C .R ., 189, 309<br />
Geddes, A .D ., 190<br />
Geethanjali, D ., 404<br />
Gelboin, H .V ., 343<br />
Gemmell, M .A ., 197<br />
Generoso, W .M ., 191, 516<br />
Gentile, G .J ., 165, 492<br />
Gentile, J .M ., 165, 301, 492<br />
Gentile, S .L ., 483<br />
George, E ., 192<br />
Gerson, S .L ., 148<br />
Getman, S .M ., 424<br />
Ghalib, M .A ., 193<br />
Ghosh, A ., 135<br />
Ghosh, A .K ., 135<br />
Ghosh, B .B ., 571<br />
Ghosh, R ., 194<br />
Ghosh, S ., 135<br />
Gibbs, R .A ., 649<br />
Gibson, D .P., I<br />
Gibson, E .S ., 585<br />
Giesi, R .A ., 276<br />
Gilbert, A .M ., 482<br />
Gill, B .S ., 195<br />
Gille, J .J .,P., 196<br />
Gilman, J.P.W ., 410<br />
Gilot-Delhalle, J ., 390<br />
Ginsberg, L .C ., 170<br />
e
244 Author Index to Abstracts<br />
Giometti, C .S ., 197<br />
Giordano, G .G ., 162, 434, 553<br />
Giordano, P.C ., 51<br />
Giri, A .K ., 198, 199, 542<br />
Giromini, L ., 182<br />
Glatt, H .R ., 139<br />
Glattke, M ., 28, 310, 311<br />
Glickman, B.W ., 200, 208, 219, 315,<br />
510, 654, 675<br />
Glover, P., 201, 237<br />
Gluecksohn-Waelsch, S ., 197<br />
Goldstein, B .D ., 428<br />
Gollapudi, B .B ., 202, 203<br />
Gombar, V.K ., 60, 61, 204<br />
Gomes, M ., 205<br />
Gooderham, N .J ., 206<br />
Gopalan, H .N .B ., 207<br />
Gopinath, P.M ., 351, 468<br />
6ordon, A .J .E ., 208, 315<br />
Gordon, L .A ., 72<br />
Gorodetzkaya, N ., 148<br />
Gossen, J .A ., 681<br />
Goswami, H .K ., 209<br />
Goswami, R .P., 209<br />
Goto, S ., 210, 285<br />
Goudreau, B ., 335<br />
Gowri, R ., 528<br />
Goyle, S ., 211<br />
Graf, U ., 212<br />
Grant, W .F ., 213<br />
Grassi, P ., 142<br />
Gray, J ., 443<br />
Green, C .L . 337<br />
Green, M .R ., 214, 215<br />
Greenberg, J .T, 132<br />
Greer, G ., 216<br />
Griffith, J ., 186<br />
Grilli, S ., 76<br />
Grimmer, G ., 450<br />
Grollman, A .P., 217<br />
Groot de Restrepo, H ., 218<br />
Grosovsky, A .J ., 219<br />
Grover, H ., 683<br />
Grover, I .S ., 6, 183, 220, 221, 222,<br />
682, 683, 692<br />
Grummt, T ., 561<br />
Grusick, D . J ., 80, 81<br />
Gryseels, B .J .A.M ., 301<br />
Grzesiuk, E ., 270<br />
Gudi, R ., 223<br />
Guengerich, F .P., 89, 224<br />
Guevara, M .L ., 133<br />
Guida, M ., 434<br />
Guirong, D ., 233<br />
Gulati, D ., 161, 578<br />
Gulati, D .K ., 638<br />
Gupta, R .L ., 225<br />
Gustaffson, J .A ., 293, 379<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Guttenplan„I .$„r226<br />
Guzzie; RJ2!r'E<br />
Hachiya, S .IN6541-5<br />
Hackman, P., 228<br />
Hall, B .G . 229<br />
Halliday, H .A ., 208<br />
Halperin, E .C ., 294<br />
Hamamy, H .A., 539 _<br />
Hameil'a, M ., 418<br />
Hamilton, C .M . 230<br />
Han, J ., 231<br />
Han, Y ., 452<br />
Hanawalt, P.C ., 232<br />
Hanekamp, J ., 577<br />
Hanmantha, P., 466<br />
Hanson, R .W ., 148<br />
Hanxiao, S ., 233<br />
Hanying, J ., 185<br />
Haque, J ., 548<br />
Hara, M ., 234<br />
Hara, T ., 537<br />
Harbach, P.R ., 235<br />
Harbell, J ., 530,531<br />
Harnois, M .C ., 236, 268<br />
Harosh, I ., 176<br />
Harrington-Brock, K ., 130, 237, 381<br />
Harris, C .H ., 238<br />
Harris, K ., 298<br />
Harris, S .B ., 239<br />
Harrison, D .y ., 369<br />
Hartman, P .E ., 240, 241<br />
Hartman, Z ., 241<br />
Harttig, U .H ., 160<br />
Hartwig, A ., 242<br />
Hashimoto, N ., 417<br />
Hauser, J ., 138<br />
Hay, D .B ., 557<br />
Hayashi, M ., 243, 565, 615<br />
Hayatsu, H ., 27, 31, 52, 244, 269, 407,<br />
489<br />
Hayes, A .W ., 144, 322<br />
Haynes, R .H ., 684<br />
He, S ., 245<br />
He, Y ., 90<br />
Heflich, R .H ., 11, 581<br />
Heikkila, L ., 228<br />
Heikkila, P., 418<br />
Hein, D .W., 246<br />
Helmi, S ., 247<br />
Hemminki, K ., 399<br />
Hemminki, N .F .I ., 504<br />
Hendricks, J ., 122<br />
Hennig, E .E ., 248<br />
Hennig, U .G .G ., 249, 619<br />
Henry, K .Y ., 324<br />
Henschler, D ., 289<br />
Herrera, L .A ., 250<br />
Hertner, Th ., 457<br />
Hesso, A ., 418<br />
Hewer, A ., 504<br />
Hilliard, C ., 251<br />
Hinson, W.G ., 388<br />
Hirayama, T ., 252<br />
Hisamatsu, Y ., 253<br />
Hitotsumachi, S ., 536<br />
Hittelman, W.N ., 254<br />
Hiyama, K ., 502<br />
Hofnung, M ., 461, 587<br />
Holme, J .A ., 18, 78, 616<br />
Holmes, R .M ., 230<br />
Hong, H ., 641<br />
Hong, X .,k, 255<br />
Hong, Z., 473<br />
Honorb, G ., 294<br />
Hook, G .J ., 256, 455<br />
Horesovsky . G .J ., 257, 455<br />
Horikawa, K ., 258<br />
Horiya, N ., 537<br />
Horsfall, M .J ., 208<br />
Horvath, J ., 112<br />
Hosoi, J ., 637<br />
Hou, E .W ., 637<br />
Housey, G .M ., 302<br />
Hovig, E ., 69<br />
Howard, D .R ., 259<br />
Howard, P.C ., 260, 361<br />
Hsiao, W.W .-1 ., 302<br />
Hsie, A.W ., 649<br />
Hsu, G .S ., 335<br />
Hsueh, J .L ., 255, 685<br />
Hu, Y .-C ., 261<br />
Huang, J ., 327<br />
Huang, M ., 666, 668<br />
Huang, N ., 262<br />
Huang, S .L ., 367<br />
Huang, X ., 264<br />
Huberman, E ., 686<br />
Hulina, G ., 174<br />
Hunsicker, P.R ., 488<br />
Huppi, C ., 548<br />
Huse, W .D., 300<br />
Husgafvel-Pursiainen, K ., 228, 330<br />
Huston, J .L., 161<br />
Hwang, M ., 600<br />
Igras, V., 42<br />
Ikushima, T., 266<br />
Imanishi, H ., 267<br />
Inouye, T., 151, 267<br />
Ipata, P.L ., 66<br />
Irwin, S .E ., 58<br />
Ishidate, M ., Jr ., 236, 243, 268, 291,<br />
355, 550, 624, 625, 626<br />
Issa, T., 437<br />
Issac, G .S ., 193, 278
Itoh, S ., 538 - =<br />
0<br />
Ivett, 1 .L ., 531<br />
Iwado, H ., 269<br />
Iwahara, S ., 490<br />
Iwasaki, M ., 89, 224<br />
Iwata, S ., 384<br />
Jabbar, M .A ., 687<br />
Jacobs, A ., 190<br />
Jacobson-Kram, D ., 645<br />
Jaen, J .C ., 307<br />
James, S ., 438<br />
Janion, C ., 270<br />
Jantunen, K ., 330<br />
Jarventaus, H ., 418<br />
Jayaraman, G ., 468<br />
Jenkinson, G ., 424<br />
Jensen, R .H ., 271, 319<br />
Jenssen, D ., 664<br />
Ji, X .Y ., 117<br />
Jia, S .-Z ., 671<br />
Jia, X ., 90<br />
liang, Z ., 272, 273<br />
Jin, C ., 274<br />
Jin, S ., 665<br />
Joardar, M ., 526<br />
Joe, C ., 323<br />
Joenje, H ., 196<br />
Johnson, E ., 531<br />
Johnson, M .D ., 302<br />
Joiner, E .E ., 275, 333<br />
Jones, I .M ., 75, 85<br />
Jones, N .J ., 579<br />
Jones, R .C ., 629<br />
Jones, R .L ., 534<br />
Jong, X ., 346<br />
Jordan, P., 301<br />
Joshi, V .P., 376<br />
Jostes, R .F., 276<br />
1ou, Y .S ., 277<br />
Juneja, T.R ., 225<br />
Jung, K .-Y ., 542<br />
Jurs, P.C ., 391<br />
Jyothy, A ., 193, 278, 465<br />
Kadhim, M ., 437<br />
Kadlubar, F .F., 89, 559<br />
Kafer, E ., 279<br />
Kalinowski, D ., 280<br />
Kalliomaki, P.-L ., 228<br />
Kamat, J .P., 405<br />
Kamiya, A ., 281<br />
Kamiya, S ., 288<br />
Kanazashi, K ., 490<br />
Kang, V ., 548<br />
Kangsadalumpaui, K ., 265<br />
Kaniguchi, Y ., 372<br />
Kappas, A ., 282<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Kari, F., 283, 630<br />
Kashyap, K ., 10, 463<br />
Kato, T ., 267, 287, 624<br />
Katoh, M ., 284, 537, 554, 596<br />
Katz, N ., 301<br />
Kaur, I .P . 225<br />
Kaur, R ., 408<br />
Kaur, S ., 65, 122<br />
Kauranen, P ., 344<br />
Kawai, A ., 285<br />
Kawai, K ., 180, 384<br />
Kawakishi, S ., 427<br />
Kawamoto, M ., 234<br />
Kawatani, N ., 377<br />
Kelsey, K .T ., 286, 633<br />
Kemin, B ., 342<br />
Keohavong, P., 577<br />
Kerckaert, G .A ., 321<br />
Kessen, S ., 148<br />
Khanna, D ., 40<br />
Kier, L .D ., 411<br />
Kikugawa, K ., 287<br />
Kim, J .B ., 104<br />
Kinae, M ., 288<br />
Kind, R ., 289<br />
Kindig, D ., 79<br />
Kinding, D ., 290<br />
Kingston, D .G .I ., 598<br />
.<br />
Kinzel, V ., 688<br />
Kirkl<strong>and</strong>, D .J ., 291<br />
Kirlin, W .10., 246<br />
Kito, H ., 501<br />
Klein, C .B ., 292<br />
Klein, P., 450<br />
Klein, W.J ., 506<br />
Kleman, M ., 293<br />
Klepetka, J .F., 170<br />
Kligerman, A .D ., 294, 630<br />
Klopman, G ., 295, 481<br />
Knasmuller, S ., 296, 359<br />
Kniewald, J ., 174<br />
Knize, M .G ., 166, 366, 600<br />
Knudsen, I ., 297<br />
Koch, W .H ., 94<br />
Kochhar, T .S ., 298<br />
Kodaira, M ., 502<br />
Kodell, R .L., 388<br />
Kogiso, S ., 234<br />
Kohalmi, L ., 28<br />
Kohalmi, S .E., 299, 310, 311<br />
Kohler, S.W., 300<br />
Koop, P.C ., 361<br />
Kopfler, F .C ., 505<br />
Koreeda, M ., 542<br />
Korzeniowski, R ., 96<br />
Kramers, P.G .N ., 301<br />
Krauss, R .S ., 302<br />
Krehl, R ., 201, 303<br />
Author Index to Abstracts 245<br />
Kretz, P.L., 300<br />
Krishna, G ., 304, 575, 576<br />
Krishnamoorthy, R ., 405<br />
Krishnaswamy, K ., 10<br />
Krishrakumar, A ., 305<br />
KrOkje, A ., 306<br />
Kropko, G ., 575<br />
Kropko, M .L ., 304, 307, 576<br />
Kryptopoulos, S ., 29<br />
Kumar, A ., 446<br />
Kumar, D ., 308<br />
Kumari, C .K ., 278, 465<br />
Kumaroo, V ., 2, 3<br />
Kung, J .S ., 309<br />
Kunkel, T.A ., 693<br />
Kunz, B .A ., 28, 299, 310, 311, 374<br />
Kuo, B ., 443<br />
Kuo, S, 312<br />
Kwanyuen, P., 82<br />
Ladhar, S .S ., 682<br />
Lafi, A ., 313<br />
Lafuente, N .M ., 554, 596<br />
Lagenbach, R ., 318<br />
Laget, M ., 126<br />
Lake, R .S ., 314<br />
Lalchev, S ., 690<br />
Lambert, I .B ., 315<br />
Lamela, R ., 633<br />
Lampelo, S ., 317<br />
Langenbach, R ., 283<br />
Langlois, R .G ., 271, 319<br />
Larimer, F.W ., 280<br />
Lasher, L ., 508, 509<br />
Lasko, D .D ., 586<br />
Lasley, J .A ., 320<br />
Lasne, C ., 425<br />
Laufer, C ., 445<br />
Lavappa, K .S ., 534<br />
Lave, L .B ., 159<br />
Le Boeuf, R .A ., 1, 321<br />
Lechat, M .F ., 128<br />
Lecona, S .U ., 486<br />
Lee, C .K ., 144, 322<br />
Lee, H .G ., 213<br />
Lee, H .O . 323<br />
Lee, J .E ., 45<br />
Lee, J .K ., 534<br />
Lee, J .S ., 436<br />
Lee, M .A ., 323<br />
Lee, P .S ., 324<br />
Legator, M .S ., 32, 623<br />
Leon<strong>and</strong>, B ., 298<br />
Leopardi, P., 106<br />
Levin, J .D ., 132<br />
Levine, A .S ., 138<br />
Levis, A .G ., 110, 677<br />
Lewis, P., 166
246 Author Index to Abstracts<br />
Lewis, S .E ., 41, 325<br />
Lewtas, J ., 210, 689<br />
Li, A .P., 326<br />
Li, D .S ., 646<br />
Li, H ., 327<br />
Li, J .-H ., 328<br />
Li, J .J ., 675<br />
Li, X .-y ., 622<br />
Li, Z ., 90<br />
Li, Z .-Q ., 669<br />
Lia, C .-e ., 671<br />
Lian, X .-g ., 671<br />
Liang, T ., 90<br />
Liang, W .-Z ., 147<br />
Liang, X .-R ., 147<br />
Liao, M .-y ., 622<br />
Liber, H ., 470<br />
Lichtenberger, A ., 498<br />
Liegibel, U .L ., 506<br />
Lihua, S ., 342<br />
Lim, I . K . , 148<br />
Lim-Sylianco, C .Y . 329<br />
Lin, F ., 262<br />
Lin, G ., 346<br />
Lin, Y ., 668<br />
Lin, Y .-x ., 92<br />
Lindahl, T., 586<br />
Lindeskog, P ., 293<br />
Linnaimaa, K .I ., 330, 418<br />
Linscombe, V .A ., 202, 203<br />
Little, J .B ., 286<br />
Littlefield, L .G ., 275, 333<br />
Liu, B ., 102<br />
Liu, D ., 334<br />
Liu, P.K ., 335<br />
Liu, S .B ., 117<br />
Liu, S .F ., 65<br />
Liu, Y ., 331<br />
Liu, Y .P., 665<br />
Ljungman, M ., 332<br />
Loarca, F .P., 336<br />
Lodovici, M ., 142<br />
Loechler, E .L ., 337<br />
Lofroth, G ., 338<br />
Logan, D .M ., 462<br />
Lohman, P., 81<br />
London, F ., 390<br />
Longan, D .M ., 213<br />
Longid, C .S ., 399, 340, 341<br />
Longzhan, Y ., 342<br />
Lon:, N .A ., 343<br />
Lotjonen, S ., 317, 344<br />
Lovel<strong>and</strong>, R ., 122<br />
Lowe, K .W ., 363<br />
Lu, Y ., 641<br />
Lucas, J ., 443<br />
Luks, H .J ., 629<br />
Lynch, A ., 438<br />
Lynch, D .W ., 286<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
--<br />
•-->Ma, G .-Y ., 669 w--`<br />
Ma, G .J ., 65T<br />
Ma, T.-H ., 336, 3ft-346, 347, 486<br />
MacGregor, J .T., 348, 583<br />
MacPhee, D .G ., 532<br />
Mackerer, C .R ., 58<br />
MacInnes, M .A ., 392<br />
Madden, J .J ., 520<br />
Maddux, S .C ., 488<br />
Magnusson, J ., 607<br />
Mah, M .C .-M ., 653<br />
Maher, V.M ., 55, 360, 349, 653<br />
Mahran, H ., 424<br />
Mailhesi, J .B ., 350<br />
Maity, S ., 526<br />
Majeeth, M .A ., 351<br />
Maki-Paakkanen, J ., 415<br />
Maness, S .C . 352<br />
Mara de S . Colus, L, 113<br />
Marchetti, F ., 431<br />
Margolin, B .H ., 353<br />
Marimuthu, K .M ., 120, 351<br />
Marques, M .M ., 44<br />
Marr, K ., 548<br />
Marsman, D .S ., 451<br />
Martin, M .V ., 224<br />
Martin, R .H ., 354<br />
Mason, J ., 57<br />
Mass, M .H ., 320<br />
Mass, M .J ., 320<br />
Matsshita, H ., 210<br />
Matsuda, H ., 537<br />
Matsumoto, K ., 31, 267<br />
Matsuoka, A ., 550<br />
Matsushima, T., 81, 178, 611<br />
Matsushita, H ., 253, 285, 355, 356<br />
Matsushita, H ., Jr ., 355, 356<br />
Matter, B ., 81<br />
Matthews, E .J ., 105, 239, 357, 358<br />
Mattila, S ., 228<br />
Mattson, K ., 330<br />
Matula, T.I ., 71<br />
Mayne, L ., 395<br />
Mayo, J ., 2, 588<br />
Mazurek, J ., 2, 3<br />
McCalla, D.R ., 315, 585<br />
McCartney, M .A ., 359<br />
McClintock, M .L ., 203<br />
McCormick, J .J ., 55, 203, 349, 360, 653<br />
McCoy, E .C ., 260, 359<br />
McCoy, G .D ., 361<br />
McCreary, R .D . 38<br />
McCune, S .L ., 74<br />
McDaniels, A .E ., 362<br />
McDowell, M ., 634<br />
McFee, A .F ., 4, 477<br />
McGarrity, L .J ., 388<br />
McGee, A .F ., 363<br />
McGregor, D ., 364<br />
McKelvey, V .J ., 365<br />
McKenna, P.G ., 365<br />
McKenzie, W .H ., 591<br />
McKinley, T .W ., Jr ., 516<br />
McLaren, K .F., 556, 557<br />
McLean, J .R ., 62<br />
McMahon, T .F ., 118<br />
McManus, M .E ., 366<br />
McManus, T .P., 170<br />
McMillin, D ., 121<br />
McSparrin, L ., 112 ,<br />
Means, J .C ., 121<br />
Mecca, D .J ., 557<br />
Meenakshi, N .D ., 528<br />
Meier, J .R ., 140, 367, 368, 505<br />
Meiyue, R ., 473<br />
Melancon, S .B ., 545<br />
Melchior, W .B ., Jr ., 44<br />
Melcion, C ., 108<br />
Meli, C ., 512<br />
Melluso, G ., 434<br />
Mendelsohn, M ., 81<br />
Mendrala, A .L ., 203<br />
Meng, J .F ., 263<br />
Merl, T ., 369<br />
Messerly, E .A ., 199, 542<br />
Metzler, M ., 59, 160, 507<br />
Meuth, M ., 370<br />
Midtvedt, T., 379<br />
Miele, M ., 371<br />
Mikalsen, A ., 18<br />
Mikamo, M ., 372<br />
Miles, C ., 370<br />
Miller, G ., 166<br />
Miller, H ., 343<br />
Miltenburger, H .G ., 612<br />
Mir<strong>and</strong>a, A ., 124<br />
Mirkova, E ., 690<br />
Mirsalis, J .C ., 3, 230, 373<br />
Mis, J .R .A ., 310, 374<br />
Misra, R .R ., 375<br />
Mitsuoka, T., 427, 567<br />
Mittelstaedt, R .A ., 581<br />
Miyamoto, J ., 234<br />
Miyazawa, T., 180<br />
Mochizuki, M ., 27, 355, 356<br />
Mody, R ., 376<br />
Modzelewski, R .A ., 377<br />
Mohn, G .R ., 387<br />
Mohran, Z ., 548<br />
Mohrenweiser, H.W., 378<br />
Mbller, L ., 379<br />
Mollis, S ., 511<br />
Monach, P ., 132<br />
Moneti, G ., 143<br />
Montero, R ., 250, 380<br />
Montgomery, J .C ., 637<br />
Moody, W., 298<br />
Moore, D ., 81
Moore, M .M ., 130, 141,-381<br />
Moraga, A .A ., 212<br />
Morales-Ramirez, P., 382r<br />
Moreira, J ., 83<br />
Moreno, F ., 380<br />
Morgan, T .L ., 276<br />
Morgan, W .F ., 383, 636<br />
Mori, H., 384 _<br />
Morichetti, E ., 76<br />
Morita, T ., 385<br />
Morley, A .A ., 167, 386<br />
Morris, D .L ., 387, 623<br />
Morris, S .M ., 388<br />
Mostafa, M .H ., 247, 389<br />
Mott, K . E . , 301<br />
Mottus, K .M ., 280<br />
Moustacchi, E ., 435<br />
Moutschen, J ., 390<br />
Moutschen-Dahmen, M ., 390<br />
Moyer, S .R ., 391<br />
Mudgett, J .S ., 392<br />
Mukherjee, A ., 393, 394<br />
Mullenders, L.H .F ., 395, 604<br />
Mullenders, P.H .M ., 599<br />
Miiller, B ., 114<br />
Miiller, D ., 457<br />
Muller, E.W., 612<br />
Mulvihill, J .J ., 301, 396<br />
Munzy, D.M ., 484<br />
Murata, M ., 210<br />
Murli, H., 397<br />
Murota, T., 537<br />
Murray, B .P., 206<br />
Murray, S ., 206<br />
Murray, T.H ., 398<br />
Murthy, K .,<br />
Mustonen, R ., 399<br />
Nagabhushan, M ., 400, 401, 402<br />
Nagao, M ., 691<br />
Nagarajan, B ., 403<br />
Nagase, H ., 501<br />
Naito, M ., 269<br />
Nakajima, K ., 654<br />
Nakamuro, K ., 489<br />
Namiki, M ., 427<br />
Naram, R ., 404<br />
Narurkar, L.M ., 405<br />
Narurkar, M .V ., 405<br />
Natarajan, A .T., 51, 395<br />
Nauman, C .H ., 22, 583<br />
Neff, R .E ., 406<br />
Negishi, K ., 31, 52<br />
Negishi, T ., 407<br />
Nehru, B ., 408<br />
Nelson, R ., 186<br />
Nesnow, S ., 81, 409<br />
Nestmann, E .R ., 410, 411<br />
Nianjun, H ., 263<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Nicklas, 1 .A., 471, 545<br />
Nillson-Tillgren, T ., 474<br />
Nishioka, H ., 417<br />
Nitta, H ., 83<br />
Nivard, M .J .M, 413, 540<br />
Nohmi, T., 625, 626<br />
Nonaka, M ., 414<br />
Norppa, H ., 243, 415<br />
Notani, N .K ., 376, 416<br />
Nunoshiba, T ., 417<br />
Nylund, L ., 418<br />
O'Neill, J .P., 412, 471, 545, 676<br />
Oberly, T ., 419<br />
Oenick, N .L ., 103<br />
Oesch, F ., 139<br />
Ogier, H ., 545<br />
Ogolla, F ., 246<br />
Oh, K . C . , 347<br />
Ohe, T ., 420<br />
Ohshima, T ., 536<br />
Ohta, T ., 42, 267, 624<br />
Ohuchida, A ., 179, 565<br />
Ohwovoriole, A .E ., 421<br />
Okeke, G .C .E ., 421<br />
Okinaka, R .T ., 422<br />
Okmura, K ., 385<br />
Oleson, F .B ., 423, 424<br />
Oliveira, M .D.M ., 493<br />
Oliveri, G .,,,639<br />
Olivero, 0 ., 426<br />
Oliviera, M .D .M ., 475<br />
Omenn, G.S ., 159<br />
Ong, T., 97, 535, 631, 640, 648<br />
Orellana, M .M ., 554<br />
Orfila, L ., 425<br />
Osawa, T., 427<br />
Ose, Y ., 501<br />
Oshiro, Y ., 227, 428<br />
Osorio, S ., 198<br />
Osowole, O.A ., 429<br />
Osti, M ., 677<br />
Ostrosky-Wegman, P., 250, 380<br />
Otsuka, H ., 258<br />
Ottaggio, L ., 371<br />
Overton, L .K ., 127, 430<br />
Overvik, E., 293<br />
Ozasa, S ., 252<br />
Pacchierotti, F ., 431, 677<br />
Padmasani, V ., 497<br />
Pagano, D .A ., 433, 558<br />
Pagano, G ., 162, 434, 553<br />
Palin-Edlund, K ., 330<br />
Palit, S ., 135<br />
Panneerselvam, N ., 528<br />
Paolini, M ., 76<br />
Papadopulo, D ., 435<br />
Papapetropoulos, A.,479<br />
.<br />
Author Index to Abstracts 247<br />
Paradisin, W .M ., 651<br />
Pardee, A .B ., 67<br />
Pardo, K . C . , 123<br />
Paredes, M, 133<br />
Park, E.-H ., 436<br />
Park, S .D ., 104<br />
Park, S . S . , 343<br />
Parker, C ., 283<br />
Parker, L., 381<br />
Parry, E .M ., 437, 438<br />
Parry, J .M., 313, 437, 438<br />
Parton, J ., 290, 439<br />
Pastnik, A ., 413, 540<br />
Patterson, J ., 440<br />
Pawlak, A .L ., 632<br />
Pelaez, V ., 26<br />
Pellom, A ., 583<br />
Peng, G .-Y ., 100, 101<br />
Penman, B .W ., 116<br />
Perez, A ., 336<br />
Perry, B .A ., 378<br />
Peters, J ., 441<br />
Petrusevska, V., 688<br />
Petry, T., 2<br />
Phear, G ., 370<br />
Phillips, B .J ., 21<br />
Phillips, D.H ., 442, 504<br />
Phillips, J .W ., 383, 636<br />
Picardi, R ., 440<br />
Ping, L ., 261<br />
Pinkel, D ., 150, 443<br />
Piper, C .E ., 428<br />
Piperakis, S .M ., 444<br />
Plewa, M .J ., 445, 280, 446, 447, 547,<br />
613<br />
Ploem, J .S ., 51<br />
Pohl, H ., 448<br />
Poirier, M ., 44, 426<br />
Poirot, 0 ., 157<br />
Polverini, P ., 401<br />
Pongracz, K ., 65<br />
Pool, B .L ., 449, 450, 506<br />
Poorman-Allen, P., 237<br />
Popoff, S .C ., 132<br />
Popp, J . A, 451<br />
Popp, R .A ., 41<br />
Porfirio, B ., 435<br />
Porter, R ., 437<br />
Pourismaili, F, 687<br />
Povrik, L .F., 452, 560<br />
Prakhya, B ., 453<br />
Prasad, N .V., 10, 463<br />
Prasad, V.S ., 35<br />
Preston, R .H ., 216<br />
Preston, R .J ., 256, 257, 454, 455<br />
Provost, G .S ., 300<br />
Pupatwibul, K ., 456<br />
Puri, E .C ., 457<br />
Putman, K .L ., 504
248 Author Index to Abstracts<br />
Putnam, D ., 530<br />
Pyron, M ., 445<br />
Qian, B .-L ., 458<br />
Qian, L, 90<br />
Qingfan, Z ., 233, 459, 460<br />
Qingxia, Z ., 459<br />
Qiu, XE, 255<br />
Quattrochi, L .C ., 366<br />
Quillardet, P., 461, 587<br />
Rafter, J ., 379<br />
Raimondi, E ., 594<br />
Raj, A .S ., 462<br />
Rajah, T.T., 463<br />
Rajendran, M ., 528<br />
Ramanujam, V .M .S ., 623<br />
Ramel, C ., 464<br />
Ramesh, A ., 497<br />
R<strong>and</strong>erath, K ., 49, 504<br />
R<strong>and</strong>hawa, S .K ., 692<br />
Rani, A .S ., 278, 465<br />
Rao, K .P., 467, 555<br />
Rao, K .R ., 35<br />
Rao, K .V .S ., 468<br />
Raposa, T., 469<br />
Rappaport, S .M ., 65, 651<br />
Rathnaprabha, D ., 695<br />
Raychoudhury, A ., 526<br />
Raymer, G .D ., 516<br />
Recio, L ., 412, 470, 471, 545<br />
Reddy, O .S ., 193, 465<br />
Reddy, P.P., 278, 404<br />
Reidy, J .A ., 448, 472<br />
Renli, W ., 572<br />
Resnick, M .A ., 474<br />
Rexroat, M ., 419<br />
Reyes, A .L ., 362<br />
Riazi, G .H ., 687<br />
Ribiero, L .R ., 475, 493<br />
Rice, J ., 195<br />
Richard, S ., 492<br />
Richardson, C ., 291<br />
Richardson, K., 419<br />
Richmond, F., 111<br />
Richter, S . P ., 432<br />
Ringh<strong>and</strong>, H .P ., 505<br />
Rithidech, K ., 476<br />
Rizvi, T., 445<br />
Roberts, D .W ., 559<br />
Roberts, J .D ., 693<br />
Robertson, M .L ., 546<br />
Robertson, S .D ., 477<br />
Rocco, M ., 66<br />
Rodriguez, L .A . 133<br />
Rodriguez-Reyes, R ., 382<br />
Rodriguez-Arnaiz, R ., 478<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Rogers, C .Q.,4t _<br />
RohrbacheK E~428<br />
Roilides, E ., 131.<br />
Rojanapo, W .`26S<br />
Rokosh, D .A ., 492<br />
Romagna, F., 479, 564<br />
Ronen, A ., 582<br />
Rosellini, D ., 76<br />
Rosenkranz, H .S ., 159, 295, 359,f180,-<br />
481<br />
Rosin, M .P., 482, 485<br />
Ross, L .S ., 219<br />
Rossi, 0 ., 483<br />
Rossiter, 484, 649<br />
Rossl<strong>and</strong>, O .J ., 617<br />
Rossman, T .G ., 292, 328<br />
Rotenberg, S .A . 302<br />
Rotmensch, J ., 519<br />
Roupova, I ., 48<br />
Rousseau, E .J ., 485<br />
Rowl<strong>and</strong>, I .R ., 521, 522<br />
Roy, A .K ., 526<br />
Roza, L ., 34<br />
Rudd, C .J . 123, 324<br />
Rudo, K ., 283<br />
Ruiz, E .F ., 486<br />
Russell, L .B ., 487<br />
Russell, W .L ., 488<br />
Russo, A ., 677<br />
Sabharwal, P ., 578<br />
Sadler, B.M ., 127<br />
Sailaja, D ., 695<br />
Sakaguchi, K ., 551<br />
Sakamoto, H ., 489<br />
Sakamoto, K . 490<br />
Sakamoto-Hojo, E .T ., 113, 491<br />
Sakata, Y ., 420<br />
Salamone, M .F., 213<br />
Salazar, E .P., 579, 628<br />
Salmeen, I ., 39<br />
Salvadori, D .M .F ., 475, 493<br />
Salvadori, M ., 143<br />
Samtella, R .M ., 205<br />
Sancar, A ., 515<br />
Sanchez, P.S ., 494<br />
S<strong>and</strong>hu, D . 541<br />
S<strong>and</strong>hu, S .S ., 195, 223<br />
S<strong>and</strong>oval, M ., 380<br />
Sankaranarayanan, K ., 495<br />
Santella, R.M ., 496, 559<br />
Santhiya, S .T., 497<br />
Santos, S .J ., 570<br />
Sarasin, A ., 124, 498<br />
Sareen, P.K ., 183<br />
Sargent, G ., 370<br />
Sasaki, M .S ., 499, 500<br />
Sasaki, Y .F ., 267, 615<br />
Satish, S ., 139<br />
Sato, M .I .Z ., 494<br />
Sato, S . 566, 615<br />
Sato, T., 180, 501<br />
Satoh, C ., 502<br />
Savage, J .R .K ., 503<br />
Savela, K ., 504<br />
Sawada, M ., 550<br />
Sayato, Y ., 489<br />
Scariolo, S ., 594<br />
Schenck, K .M ., 367, 505<br />
Schiffmann, D ., 160, 507<br />
Schlehofer, J .R ., 450<br />
Schlepegrell, RL, 242<br />
SchmBl, D ., 694<br />
Schmezer, P., 449, 450, 506<br />
Schneider, O . , 157<br />
Schneiter, S ., 511<br />
Schnitzler, R ., 507<br />
Schoeny, R ., 440<br />
Schol, H .M ., 406<br />
Schorschinsky, N .S ., 320<br />
Schreibner, M .G ., 84<br />
Schreuder-Rotteveel, A .H.M ., 51<br />
Schut, H .A .J ., 43<br />
Schwartz, J .L ., 149, 519<br />
Schwartz, S ., 508, 509<br />
Schy, W .E., 510<br />
Scott, B .S ., 77<br />
Sedwick, W .D ., 511, 602<br />
Seeberg, A ., 512<br />
Seed, J .L ., 301<br />
Sega, G .A ., 513<br />
Sehgal, S .S ., 514<br />
Selby, C .P., 515<br />
Selby, P.B ., 516, 517<br />
Sen, M .P., 71<br />
Sen, S ., 525<br />
Sengupta, L .K ., 209<br />
Sengupta, S ., 518<br />
Sera, N ., 258<br />
Seth, R .K ., 513<br />
Shaddock, J .G ., 93<br />
Shadley, J .D ., 519<br />
Shafer, D .A ., 520<br />
Shah, A .B ., 521, 522<br />
Shahin, M .M ., 523<br />
Sh<strong>and</strong>alis, A ., 219<br />
Shane, B .S ., 524<br />
Shankel, D.M ., 312<br />
Shao, H ., 656<br />
Sharaf, A .N ., 153<br />
Sharma, A ., 135, 393, 525, 526, 571<br />
Sharma, C .B .S .R ., 527, 528, 529<br />
Sharma, R .K ., 530, 531<br />
Shaw, T., 532
Shelby, M .D .,57, 161,251, 533, 550,638<br />
Shen, J ., 647<br />
Sheu, C .W., 534<br />
Shi, X ., 535, 648<br />
Shi, Z.Z ., 659<br />
Shibuya, T ., 536, 537<br />
Shimada, H ., 27, 538, 565, 615<br />
Shimada, T ., 224<br />
Shiotani, T., 407<br />
Shirasu, Y ., 267, 624<br />
Short, J .M ., 300<br />
Shreiner, C .A ., 58<br />
Shubber, E.K ., 539<br />
Shuli, F ., 233, 460<br />
Shyr, L .-S ., 77<br />
Siciliano, M .J ., 579<br />
Siede, W ., 176<br />
Sierra, L.M ., 540<br />
Silva, A .R ., 475, 493<br />
Silvestro, C ., 483<br />
Simic, M .G ., 50<br />
Simons, J .W .I .M ., 599<br />
Simpson, D ., 412, 470, 471, 545<br />
Simpson, S ., 548<br />
Singh, H ., 541<br />
Singh, J .R ., 134, 541<br />
Singh, V .P., 541<br />
Sinha, A .K ., 202, 203<br />
Sinsheimer, J .E ., 198, 199, 542<br />
Sivaramakrishnan, V .M . 305<br />
Skelly, M .F ., 367<br />
Skipper, P .L ., 543<br />
Skopek, T.R ., 412, 470, 471, 544, 545<br />
Smillie, R .H ., 532<br />
Smith, A .L ., 235<br />
Smith, B .A ., 44, 581<br />
Smith, M .T ., 546<br />
Smith, S .J ., 472<br />
Smith, S .R ., 547<br />
Smith-Oliver, T ., 49<br />
Smollinger, J .K ., 591<br />
Smulson, M .E ., 548<br />
Smylie, J ., 675<br />
Snow, E .T., 549<br />
Snyderwine, E .G ., 43, 366<br />
Sobrero, A ., 483<br />
SOderlund, E .J ., 78<br />
Soelter, S .G ., 428<br />
Sofumin, T., 550<br />
Sofuni, T., 236, 243, 268, 565<br />
Solberg, K .E ., 617<br />
Solt, D ., 401<br />
Song, J .-M ., 176<br />
Sonop, A ., 551<br />
SOrensen, B .S ., 69<br />
Sorg, R ., 3<br />
Sorge, J ., 300<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Soriano, J .D ., 340<br />
Sorsa, M ., 418, 552<br />
Soskoline, C .L ., 553<br />
Soto, J .P., 554<br />
Souza, S .C ., 570<br />
Spierto, F .W ., 472<br />
Sridevi, K ., 467, 555<br />
Stack, F ., 627<br />
Stahlberg, M ., 607<br />
Stankowski, L ., 3<br />
Stankowski, L.F ., Jr ., 556, 557<br />
Stark, A .A ., 558<br />
Stef<strong>and</strong>is, M ., 559<br />
Steighner, R .J ., 560<br />
Steinmetz, K .L ., 3, 230<br />
Stelma, G .N ., 140<br />
Stensman, C ., 338<br />
Stewart, J .D, 631<br />
Stoltz, S .L ., 37<br />
Stowesan, G .S ., 230<br />
Strniste, G .F ., 392, 422<br />
Strobel, K ., 561<br />
Strout, C .L., 75, 85<br />
Stupar, L .L ., 156<br />
Suarez, H .G ., 124<br />
Subramanyam, S ., 695<br />
Sugie, S ., 384<br />
Sugita, K., 253<br />
Suhonen, S ., 418<br />
Sujatha, M ., 465<br />
Sun, H ., 5b2<br />
Sun, W ., 346<br />
Suryakumari, T ., 563, 595<br />
Suter, W., 564<br />
Sutou, S ., 566, 615<br />
Suttajit, M ., 611<br />
Suzuki, K., 427, 567<br />
Swartout, J ., 440<br />
Swierenga, S .H .H ., 410<br />
Sylianco, C .Y .L., 610<br />
Szegedi, M ., 569<br />
Takagi, Y . 210<br />
Takahashi, C .S ., 113, 491, 570, 606<br />
Takahashi, N ., 502<br />
Takahashi, S ., 287<br />
Takatori, K ., 490<br />
Takayama, S ., 538<br />
Takeda, K ., 385<br />
Talitha, T.R ., 10<br />
Talukder, G ., 135, 525, 526<br />
Tammilehto, K ., 330<br />
Tan, C .C ., 685<br />
Tan, C .H .T ., 681<br />
Tan, Y ., 572<br />
Tanaka, T ., 384<br />
Tang, W .D ., 589<br />
Author Index to Abstracts 249<br />
Tannenbaum, S .R ., 543<br />
Tano, S ., 572, 573<br />
Tateno, H ., 372<br />
Tates, A ., 51<br />
Tavera, L ., 574<br />
Taylor, S ., 446<br />
Temenak, J .J ., 347<br />
Terzetti, F., 157<br />
Theiss, J .C ., 157, 304, 307, 575, 576<br />
Thilagar, A ., 2, 3, 530<br />
Thilly, W .G ., 577<br />
Thomas, D .C ., 693<br />
Thomas, M .A ., 578<br />
Thomassen, D.G ., 476<br />
Thompson, L .H., 87, 149, 579, 628<br />
Thorgeirsson, S .S ., 43, 580<br />
Thorton-Manning, J .R ., 581<br />
Tiah, M ., 582<br />
Tianbao, T ., 660<br />
Tice, R .R ., 22, 251, 583<br />
Tiveron, C ., 431<br />
ToftgArd, R ., 379<br />
Tokiwa, H ., 258<br />
Tollaksen, S .L ., 197<br />
Tomer, K ., 283<br />
Tometsko, A .M ., 584<br />
Tomkins, D .J ., 585<br />
Tomkinson, A.E ., 586<br />
Tompa, A ., 449<br />
Toohill, M ., 519<br />
Tbrnquist, S ., 379<br />
Toscano, W ., 633<br />
Touati, E .,461, 587<br />
Townsend, L .B ., 198<br />
Tozzi, M .G ., 66<br />
Trask, B ., 443<br />
Traul, K .A., 530, 531<br />
Travis, C .C ., 432<br />
Tregerman, L ., 588<br />
Trevizo, S ., 26<br />
Trinidad, A ., 246<br />
Tritscher, A .M ., 59<br />
Trivedi, A .H ., 7<br />
Trosko, J .E ., 277<br />
Troungos, C ., 29<br />
Tsuda, S ., 420<br />
Tsujimura, T ., 55<br />
Tu, Z .H ., 589<br />
Tucker, J .D ., 590<br />
Tukey, R .H ., 366<br />
Tulis, D .A ., 591<br />
Tuman, W .G ., 556, 557<br />
Tung, K .-K ., 84<br />
Turner, D .R ., 386<br />
Turtletaub, K .W ., 592<br />
Tutikawa, K ., 536<br />
Tweats, D .J ., 86, 593<br />
e
250 Author Index to Abstracts<br />
Ueamworapong, C ., 265<br />
Ukawa, S ., 27<br />
Uwaifo, A .O ., 429<br />
van Berkel, C .G.M ., 196<br />
van Hoffen, A ., 395, 604<br />
van Loon, A .A .W.M ., 34<br />
van Rooijen, J ., 599<br />
van Zeel<strong>and</strong>, A .A ., 395, 599, 604<br />
van de Klundert, F .A . J . M ., 196<br />
van der Schans, G .P ., 34<br />
von Borstel, R .C ., 619<br />
Vagnarelli, P., 594<br />
Vaidyanath, K ., 563, 595<br />
Vainio, H ., 228<br />
Valdivia, R .P.A ., 284, 554, 596<br />
Valencia, D ., 380<br />
Valent, G .U ., 494<br />
-Vallarino-Kelly, T., 382<br />
Valtierra, E.R ., 486<br />
Van Benthem, J ., 597<br />
Van Schaik, M ., 212<br />
Van Tassell, R .L ., 598<br />
Van der Wulp . C .J .M ., 34<br />
V<strong>and</strong>erlan, M ., 600<br />
Varkonyi, J ., 469<br />
Vartiainen, T ., 344<br />
Vartiainen, T ., 317<br />
Vartiainen, T ., 601<br />
Vavrek, T., 629<br />
Veigl, M .L ., 511, 602<br />
Veleminsky, J ., 603<br />
Vellosi, R., 76<br />
Venema, J ., 395, 599, 604<br />
Venier, P., 110<br />
Venitt, S ., 605<br />
Verdier, M .M ., 547, 613<br />
Verdina, A ., 106<br />
Veronese, M .E ., 366<br />
Viaggi, S ., 66, 371<br />
Victorin, K ., 607<br />
Vierling, Th ., 608<br />
Vijayalaxmi, N .F.I ., 608<br />
Vijg, J ., 681<br />
Villasenor, I .M ., 610<br />
Vincenti-Dias, V .E .P., 606<br />
Vinitketkumnuen, U ., 611<br />
Vo-Dirh, T ., 205<br />
Vogel, E.W ., 413, 540<br />
Vblkner, W ., 612<br />
Von Borstel, R .C ., 249<br />
Vrieling, H ., 69, 599<br />
Vuglenov, A ., 48<br />
Wagner, E .D ., 547, 613<br />
Wagner, T., 148<br />
Wairimu, A .N ., 207<br />
Wakabayashi, K ., 614<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
Wakata, A., W6, 565, 615.<br />
Walbousn ;~Cs&!f84<br />
Walker, It, 32<br />
Walker, G:C,*L--<br />
Walker, R .P., 93<br />
Wallin, H ., 616, 617<br />
Walsh, D .B ., 107<br />
Wang, B .-S ., 669<br />
Wang, D ., 618<br />
Wang, H .-z., _<br />
Wang, J ., 655<br />
Wang, J .X ., 670<br />
Wang, L, 273<br />
Wang, L ., 674<br />
Wang, L .-h ., 92<br />
Wang, M .Y ., 589<br />
Wang, Q ., 619, 620<br />
Wang, S ., 264<br />
Wang, S ., 650<br />
Wang, X ., 327<br />
Wang, X ., 621<br />
Wang, X .L ., 255<br />
Wang, Y ., 117<br />
Wang, Y ., 55<br />
Wang, Y ., 562<br />
Wang, Z .-q ., 622<br />
Wantanabe, K ., 624<br />
Wantanabe, M ., 355<br />
Wantanabe, T ., 252<br />
Ward, J .B ., Jr ., 20, 623<br />
Warman, B ., 148<br />
Warr, T ., 438<br />
Wasserman, S .S ., 508, 509<br />
Watanabe, M ., 267, 624, 625, 626<br />
Waters, M ., 81, 627, 696<br />
Watkins, B .E., 592, 600<br />
Wattenberg, L .W ., 697<br />
Waymack, P.P., Jr., 472<br />
Weber, C .A ., 579, 628<br />
Wei, L., 666, 668<br />
Wei, Z.-W., 663<br />
Weichoelbaum, R .R ., 149<br />
Weincke, J .K ., 286, 634<br />
Weinstein, I .B ., 302<br />
Weisburger, J .H ., 629<br />
Wen, S .-z ., 622<br />
Wenqing, L ., 473<br />
West, S .C ., 114<br />
Westbrook-Collins, B ., 630<br />
Whitlock, J ., 519<br />
Whong, W .-Z ., 631, 640, 648<br />
Whorton, E., 186<br />
Wicnienski, N ., 657<br />
Wielgosz, S .M ., 632<br />
Wiencke, J ., 633<br />
Wierzba, K ., 179<br />
Wild, D ., 289<br />
Wilkins, T.D ., 598<br />
Wilkinson, D ., 62<br />
William§, G .M ., 19<br />
Williams, J .R ., 645<br />
Williams, L ., 583<br />
Willis, A .E., 586<br />
Wilmer, J.W .G.M ., 597<br />
Wilson, D .M ., 635<br />
Winegar, R .A ., 383, 636<br />
Winkfield, L ., 26<br />
Winston, G .W ., 524<br />
Wise, D ., 198<br />
Wise, L .D ., 307<br />
Wiseman, R.W ., 637<br />
Wiser, S .K ., 235, 657<br />
Witt, K .L ., 57<br />
Witz, G ., 428<br />
Wojciechowski, J .P., 578, 638<br />
Wold, S .A ., 307<br />
Wolff, S ., 639<br />
Working, P.K ., 49<br />
Wu, G ., 641<br />
Wu, H .Y ., 665<br />
Wu, Z .-L ., 640, 648<br />
Wu, Z .L ., 147<br />
Wiirgler, F .E ., 175, 642<br />
Wymer, L .J ., 362<br />
Xi Li, L ., 643<br />
Xia, W ., 346<br />
Xiao, B .Q ., 644<br />
Xiao, S ., 645<br />
Xie, D .Y ., 646<br />
Xie, W ., 90<br />
Xili, L ., 647<br />
Xing, S .G, 648<br />
Xu, E ., 643<br />
Xu, J ., 346<br />
Xu, L .S ., 549<br />
Xu, Z ., 640<br />
Xue, K .X ., 650<br />
Xue, L .Y ., 103<br />
Yager, J .W ., 546, 651<br />
Yagova, A ., 48<br />
Yamada, F ., 234<br />
Yamasaki, H ., 652<br />
Yamashita, M ., 288<br />
Yang, B ., 562, 673<br />
Yang, H .-L. 349<br />
Yang, H .F ., 646<br />
Yang, J .-L ., 653<br />
Yang, W.-l ., 92<br />
Yannan, Y ., 185<br />
Yaozhong, W ., 473<br />
Yatagai, F ., 654<br />
Yerokun, T., 246<br />
Yi, A .-K ., 436<br />
Yin, M ., 655
Yin, S .-1 ., 656<br />
Yin, X ., 334<br />
Yongjun, W ., 233<br />
Yoshimi, N ., 384<br />
Yoshitake, A ., 234<br />
Young, R .R ., 531<br />
Yu, R ., 657<br />
Yu, Y ., 649<br />
Yu, Y .-q ., 656<br />
Yu, Y .N ., 658, 659<br />
Yuanl, Z .P ., 350<br />
Yue, S ., 641<br />
Yuhui, A ., 233<br />
Yun, J ., 148<br />
Yuzhi, W ., 660<br />
Zang, S ., 661<br />
Zdzienicka, M .Z ., 599<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
~<br />
Zeiger, E ., 57, 433, 558, 630, 662<br />
Zeller, U ., 506<br />
Zempel, J .A ., 203<br />
Zernik, M ., 138<br />
Zhang, H .-J ., 663<br />
Zhang, J ., 671<br />
Zhang, L .H ., 664<br />
Zhang, Q .X ., 644<br />
__Zhang, R .F ., 665<br />
Zhang, W ., 646<br />
Zhang, X .-I ., 656<br />
Zhang, Z ., 572<br />
Zhao, Q ., 672<br />
Zhao, W ., 667<br />
Zhao, Z ., 666, 668<br />
Zheng, Lin, 342<br />
Zheng, S ., 674<br />
Zhong, B .-Z ., 669<br />
Author Index to Abstracts 251<br />
Zhou, J.W ., 670<br />
Zhou, R ., 621<br />
Zhou, X ., 347<br />
Zhou, Z ., 671<br />
Zhu, M .C., 644<br />
Zhu, Q ., 672, 673<br />
Zhu, S .X ., 562, 665, 674<br />
Zhu, Y .-Z ., 663<br />
Zhuo, Jian, B ., 91<br />
Zhuo, P ., 650<br />
Zielenska, M ., 675<br />
Zijno, A ., 106<br />
Zimmer, D .M ., 676<br />
Zimmering, S ., 478, 574<br />
Zinkowski, R .P., 74<br />
Zito, R ., 106<br />
Zordan, M ., 110, 677
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It
Environmentai <strong>and</strong><br />
<strong>Molecular</strong> <strong>Mutagenesis</strong><br />
JOURNAL OF THE ENVIRONMENTAL MUTAGEN SOCIETY<br />
Volume 14, Supplement 15 1989<br />
Abstracts of the Fifth International Conference on <strong>Environmental</strong> Mutagens . . . . . . . . . . . . . . . . 3<br />
Author Index to Volume 14, Supplement 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241<br />
http://legacy.library.ucsf.edu/tid/clb93d00/pdf<br />
A<br />
.