ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...
ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...
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FUNCTI<strong>ON</strong>AL STUDIES <strong>ON</strong> THE EFFECTS <strong>OF</strong><br />
2,3,7,8 - TETRACHLORODIBENZO-p-DIOXIN (TCDD)<br />
<strong>ON</strong> <strong>TESTIS</strong> <strong>AND</strong> <strong>EPlDlDYMlS</strong> <strong>OF</strong> <strong>RATS</strong><br />
I HE \IS .SLIB.bf 17'J-E/) 7-0 'TI/?< f'O*VDI(. HEIIR I C ,VI l.ER.S/TI'<br />
FOR THE DEC;KEE <strong>OF</strong><br />
DOCTOR <strong>OF</strong> PHILOSOPHY<br />
IN<br />
LIFE SCIENCES<br />
BY<br />
C. LATCHOUMYC<strong>AND</strong>ANE<br />
SCHOOL <strong>OF</strong> LIFE SCIENCES<br />
P<strong>ON</strong>DICHERRY UNIVERSITY<br />
P<strong>ON</strong>DICHERRY -605 014<br />
JUNE - 2002
DECLARATI<strong>ON</strong><br />
I hereby declare that the work presented in this thesis has been carried out by me<br />
under the supervision of Dr. P.P. Mathur, Professor, School of Life Sciences, <strong>Pondicherry</strong><br />
<strong>University</strong>. To the best of my knowledge, no part of this thesis has been submitted for the<br />
award of a research degree of any other <strong>University</strong>.<br />
J<br />
c. i. Lwd-+'-<br />
(C. l,A~~CliO~lMY('ANl)ANl~)<br />
Candidate
ACKNOWLEDGEMENTS<br />
I wish to express my sincere gratitude to Dr. P. P. Mathur, Professor, School<br />
of Life Sciences, Pondicheny <strong>University</strong>, <strong>Pondicherry</strong> for his invaluable guidance,<br />
unceasing help, unerring supervision, critical suggestions, encouragement and the<br />
freedom with which I could work in his laboratory throughout the course of my<br />
research work.<br />
I express my sincere gratitude to Dr. E. Vijayan, Professor and Head, School<br />
of Life Sciences, <strong>Pondicherry</strong> <strong>University</strong>, Pondicheny for providing me laboratory<br />
facilities. My thanks are due to the other faculty members of my department Dr. B.<br />
Ka~abiran, Dr. A. Ramachandra Reddy and Dr. K. Srikumar, School of Life<br />
Sciences, <strong>Pondicherry</strong> <strong>University</strong> for their encouragement.<br />
My sincere gratitude to Dr. Stefen Safe, Department of Veterinary Anatomy<br />
and Public Health, A & M <strong>University</strong>, Texas, USA for the gift of TCDD without<br />
which I could not complete my work successfully.<br />
My sincere thanks to Dr. Dheer Singh, Scientist, National Dairy Research<br />
Institute, Karnal and Dr. R.R. Manimaran, Department of Pharmacology and<br />
Therapeutics. McGill <strong>University</strong>, Montreal. Canada, for their valuahlc suggcslions<br />
and encouragements.<br />
1 would also thank Dr. S.C. Parija, Officer in-Charge and other staff of the<br />
Central Animal House. JIPMER, <strong>Pondicherry</strong> for providing animals.<br />
I am highly thankful to Dr. P.P. Mathur, Officer in-Charge and Mr. M.<br />
Sundaramohan and Ms. V. Amoudha Staff of Bioinformatics Centre, Distributed<br />
Information Sub-centre, <strong>Pondicherry</strong> <strong>University</strong>, Pondicheny for providing me the<br />
necessary facilities for literature search and other help.<br />
I sincerely thanks to Ms. K.C. Chitra, Research Scholar, School of Life<br />
Sciences, Pondicheny <strong>University</strong> for her continuous help throughout my experiments.<br />
I also thank Ms. P. Poonkothai, Ms. R. Sujatha, Mr. M. Arul Murugan, Ms. Bindhu
Gangadharan and Ms. V. Bindhu Mol, M. Phil. Scholars, School of Life Scicnces,<br />
<strong>Pondicherry</strong> <strong>University</strong> for their cooperation in the laboratory.<br />
I would also thank Mr. V. Meibalan, Deputy Librarian, Central Library,<br />
<strong>Pondicherry</strong> <strong>University</strong> and Mr. R. Desingh, Research Scholar, School of Life<br />
Sciences, <strong>Pondicherry</strong> <strong>University</strong>, <strong>Pondicherry</strong> for their support and help in many<br />
ways.<br />
I wish to thank Mr. P. Ramalingam, Mr. K. Veeraragu and Mr. R.<br />
Jagajeevanram, Office Staff, School of Life Sciences for their cooperation and help in<br />
all aspects.<br />
The financial assistance by the Indian Council of Medical Research (ICMR),<br />
New Delhi in the form of a Senior Research fellowship is deeply acknowledged.<br />
Above all, I owe a deep sense of gratitude to my parents for their everlasting<br />
encouragement and support.<br />
c / Ck4L7<br />
(C. LATCHOUMYC<strong>AND</strong>AN :)
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS<br />
Page No<br />
ACKNOWLEDGEMENTS<br />
LIST <strong>OF</strong> TABLES<br />
LIST <strong>OF</strong> FIGURES<br />
LIST <strong>OF</strong> ABBREVIATI<strong>ON</strong>S<br />
1 INTRODUCTI<strong>ON</strong><br />
1.1 Description of male reproductive system<br />
1 .I. 1 Testis<br />
1 .1,1.1 Spermatogenesis<br />
1.1.1.2 Hormonal regulation of spermatogenesis<br />
1.1.1.3 Testicular steroidogenesis<br />
1.1.1.4 Hormonal regulation of steroidogenesis<br />
1.1.1.5 Paracrine regulation of testicular functions<br />
1.1.2 Structure and functions of the epididymis<br />
1.2 Target organ response to testosterone<br />
1.3 Effect of environmental contaminants on male reproductive<br />
system<br />
1.3.1 2.3.7.8-Tetrachlorodibenzo-p-dioxin (TCDD)<br />
1.3.1.1 Effect of TCDD on various organs<br />
1.3.1.2 Effect of TCDD on male reproduction<br />
1.3.1.3 Mechanism of TCDD action<br />
1.4 Effect of oxidative stress on male reproduction<br />
2 SCOPE <strong>OF</strong> THE PRESENT STUDY<br />
3 MATERIALS <strong>AND</strong> METHODS<br />
3.1 Animals<br />
3.2 Maintenance<br />
3.3 Chemicals<br />
i<br />
iii<br />
v<br />
viii
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS (Continud)<br />
Handling of TCDD and animals<br />
Treatments<br />
Group I : Administration of TCDD to pubertal rats<br />
Group 11: Administration of TCDD along with vitamin E<br />
Killing of animals<br />
Collection of serum<br />
Collection of tissues<br />
Organ weights<br />
Daily sperm production<br />
Collection of epididymal sperm and sperm function tests<br />
Sperm viability test<br />
Epididymal sperm motility<br />
Epididymal sperm counts<br />
Histometric studies<br />
Tubular and lumen diameter measurements<br />
Quantitative determination of serum hormone levels<br />
Quantitative determination of serum FSH<br />
Quantitative determination of serum LH<br />
Quantitative determination of serum prolactin<br />
Quantitative determination of serum testosterone<br />
Quantitative determination of serum estradiol<br />
Activities of steroidogenic enzymes<br />
Assay of 3P-hydroxysteroid dehydropnase<br />
Assay of 17P-hydroxysteroid dehydrogenase<br />
Determination of nucleic acids and protein contents in testis<br />
Extraction of nucleic acids fractions<br />
Determination of DNA<br />
Page No<br />
25<br />
25<br />
25<br />
26<br />
26<br />
26<br />
26<br />
27<br />
27<br />
27<br />
28<br />
28<br />
28<br />
29<br />
29<br />
29<br />
29<br />
3 1<br />
32<br />
34<br />
35<br />
36<br />
36<br />
37<br />
37<br />
37<br />
38
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS (Continued)<br />
Determination of RNA<br />
Determination of protein<br />
Subcellular fractionation of testis<br />
Preparation of tissue homogenates of epididymis<br />
Preparation of tissue homogenate of kidney<br />
Determination of free radicals/ reactive oxygen species in tissues<br />
Determination of superoxide anion<br />
Determination of nitric oxide<br />
Hydrogen peroxide generation assay<br />
Determination of antioxidants in tissues<br />
Estimation of reduced glutathione<br />
Estimation of a-tocopherol<br />
Estimation of ascorbic acid<br />
Determination of antioxidant enzymes in tissues<br />
Assay of superoxide dismutase<br />
Assay of catalase<br />
Assay of glutathione reductase<br />
Assay of glutathione peroxidase<br />
Estimation of lipid peroxidation<br />
Statistical analysis<br />
RESULTS<br />
Administration of TCDD to rats<br />
Effect of TCDD on body and organ weights<br />
Effect of TCDD on daily sperm production<br />
Effect of TCDD on epididymal sperm viability, motility and count<br />
Effect of TCDD on seminiferous tubular tlnd lunicn dianicters<br />
Effect of TCDD on serum hormone levels
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS (Cuntinucul)<br />
Effect of TCDD on the testicular sleroidogencsis<br />
Effect of TCDD on nucleic acids and protein contents in testis<br />
Effect of TCDD on the production of reactive oxygen species in<br />
the crude homogenate, mitochondrial and ~nicrosome-rich<br />
fractions of testis of rats<br />
Effect of TCDD on the levels of antioxidants in the crude<br />
homogenate, mitochondrial and microsome-rich fractions of testis<br />
of rats<br />
Effect of TCDD on the antioxidant enzymes and lipid peroxidation<br />
in crude homopennte. mitochondrial and microsomc-rich l'ractions<br />
of testis of rat<br />
Effect of TCDD on the epididymal sperm and epididymis<br />
Effect of TCDD on the production of reaclive oxygen species in<br />
the epididymal sperm of adult rats<br />
Effect of TCDD on the levels of antioxidants in the epididymal<br />
sperm of adult rats<br />
Effect of TCDD on the antioxidant enzymes and lipid peroxidation<br />
in the epididymal sperm of adult rats<br />
Effect of TCDD on the production of reactive oxygen spccies in<br />
the capuc. corpus and cauda epididymides of adult rats<br />
Effect of TCDD on the antioxidants in the caput, corpus and cauda<br />
epididymides of adult rats<br />
4.1 .I 1.6 Effect of TCDD on the antioxidant enzymes and lipid peroxidation<br />
in the caput. corpus and cauda epididymides of adult rats<br />
4.1.12 Effects of TCDD on the levels of reactive oxygen species<br />
production, antioxidants, antioxidant enzymes and lipid<br />
peroxidation in kidney<br />
4.2 Co-administration of TCDD and Vitamin E to Rats<br />
4.2.1 Eflbcl or co-administration oTT
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS (Continued)<br />
Effect of co-administration of TCDD and vitaniin E on<br />
seminiferous tubular and lumen diamcter<br />
Effect of co-administration of TCDD and vitamin E on the levels of<br />
serum hormones<br />
Effect of co-administration of TCDD and vitamin E on thc<br />
testicular steroidogenesis<br />
Effect of co-administration of TCDD and vitamin E on nucleic<br />
acids and protein contents of testis<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of reactive oxygen species in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of testis of rats<br />
Effect of co-administration of TCDD and vitamin E on the levels of<br />
antioxidants in the crude homogenate, mitochondria1 and<br />
microsome-rich fractions of testis of rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
antioxidant enzymes and lipid peroxidation in crude homogenate,<br />
mitochondria1 and microsome-rich fractions of testis of rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
epididymal sperm of adult rats<br />
Effect of co-administration of TCDD and vitamin I: on tlic<br />
production of reactive oxygen species in the epididymal sperm of<br />
adult rats<br />
Effect of co-administration of TCDD and vitamin E on the levels of<br />
antioxidant in the epididymal sperm of adult rat<br />
Effect of co-administration of TCDD and vitamin E on the<br />
antioxidant enzymes and lipid peroxidation in the epididymal sperm<br />
of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of reactive oxygen species in the caput, corpus and<br />
cnudn cpididyniidcs of adnlt rats<br />
EHbct of co-administration of 'fCDD and vitamin Li on thc<br />
antioxidants in the caput. corpus and cauda epididyniidcs of adult<br />
rats<br />
El'f'ect of co-administration of I'CDD and vitamin li on thc<br />
antioxidant enzymes and lipid peroxidation in the caput. corpus and<br />
cauda epididymides of adult rats<br />
vii<br />
Page No<br />
56
TABLE <strong>OF</strong> C<strong>ON</strong>TENTS (Continued)<br />
viii<br />
Effect of co-administration of TCDD and vitamin E on the levels of<br />
reactive oxygen species production, antioxidants, antioxidant<br />
enzymes and lipid peroxidation of kidney of adult rats<br />
DISCUSSI<strong>ON</strong><br />
Significance of the experimental procedures<br />
Experimental treatments<br />
Significance of the parameters studied<br />
Effect of TCDD on male rats<br />
Effect of TCDD on body and organ weights<br />
Effect of TCDD on daily sperm production<br />
Effect of TCDD on the epididymal sperm viability. motility and<br />
count<br />
Effect of TCDD on seminiferous tubular and lumen diameter<br />
Effect of TCDD on serum hormone levels and steroidogenic<br />
enzymes in testis<br />
Effect of TCDD on nucleic acid and protein contcnts in lestis<br />
Effect of TCDD on antioxidant system in testis<br />
Effect of TCDD on antioxidant systelii in the epididymal spcrm<br />
Effect of TCDD on antioxidant systems in the caput, corpus and<br />
cauda epididymides of rats<br />
Effect of TCDD on antioxidant system in kidney of rat<br />
Effect of co-administration of TCDD and vitamin E on male rats<br />
Effect of co-administration of TCDD and vitamin E on body and<br />
organ weights<br />
Effect of co-administration of TCDD and vitamin E on daily sperm<br />
productio~i, opididyniol sperm viahilily. 111olilily ;111d spcr111 COUlllh<br />
Effect of co-administration of I'CDD and vitamin C on<br />
seminiferous tubular and lumen dimcier<br />
Effect of co-administration of TCDD and vitamin E on serum<br />
hormone Levels and steroidoyenic cnzymcs in ~cstis
TAULIC <strong>OF</strong> C<strong>ON</strong>'I'ISN'I'S (C'untinued)<br />
5.3.5 Effect of co-administration of TCDD and vitamin E on nucleic acid<br />
and protein contents in testis<br />
5.3.6 Effect of co-administration of TCDD and vitamin E on antioxidant<br />
systems in testis, epididymal sperm and epididymis<br />
5.3.7 Effect of co-administration of TCDD and vitamin E on antioxidant<br />
system in Kidney<br />
5.3.8 Epilogue<br />
6 C<strong>ON</strong>CLUSI<strong>ON</strong>S<br />
7 REFERENCES<br />
APPENDIX I<br />
I'REPARATI<strong>ON</strong> <strong>OF</strong> REAGENTS<br />
APPENDIX 2<br />
LIST <strong>OF</strong> PUBLICATI<strong>ON</strong>S <strong>OF</strong> THE C<strong>AND</strong>IDATE
LIST <strong>OF</strong> TABLES<br />
Page No.<br />
Table la<br />
Table lb<br />
Table 2<br />
Tablc 3<br />
'Table 4<br />
Table 5<br />
Table 6<br />
'fablc 7a<br />
Table 7b<br />
Table 7c<br />
Table 8a.<br />
Table 8b<br />
Effect of TCDD on the body weight and weights of tlic<br />
testis. epididymis, kidney and accessory sex organs of adult<br />
male rats<br />
Effect of TCDD on the body weight and weights of the<br />
testis, epididyrnis, kidney and accessory sex organs @er<br />
100 g body weight) of adult male rats<br />
Effect of TCDD on the daily sperm production. epididymal<br />
sperm viability, sperm motility and sperm cou~its in adult<br />
male rats<br />
Effect of TCDD on the seminiferous tubular and lumcn<br />
diameter of testis of adult male rats<br />
Effect of TCDD on the serum hormone levels in adult male<br />
rats<br />
Effect of TCDD on the activities of steroidogenic enzymes<br />
in rat testis<br />
Effect of TCDD on the DNA, RNA and protein contents in<br />
testis of adult male rats<br />
Effect of TCDD on the production of superoxide anion in<br />
the crude homogenate, mitochondrial and microsome-rich<br />
fractions of testis of adult rats<br />
Effect of TCDD on the production of nitric oxide in the<br />
crude homogenate, mitochondrial and microsome-rich<br />
fractions of testis of adult rats<br />
Effect of TCDD on the hydrogen perox~de generation in<br />
the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
Effect of TCDD on the level of glulnthiolic in thc crudc<br />
homogenate, mitochondrial and microsome-rich liactiol~?;<br />
of rat testis<br />
Effect of TCDD on the level of a-tocopherol in the crude<br />
homogenate, mitochondrial and microsome-rich fractions<br />
of rat testis
xi<br />
LIST <strong>OF</strong> TABLES (Continued)<br />
I'agc No.<br />
Table 8c<br />
Table 9a<br />
Table 9b<br />
Table 9c<br />
Table 9d<br />
Table 10<br />
Effect of TCDD on the levels of ascorbic acid in the crude<br />
homogenate. mitochondrial and microsome-rich fractions<br />
of rat testis<br />
Effect of TCDD on the activity of superoxide dismutase in<br />
the cmde homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
Effect of TCDD on the activity of catalase in the crude<br />
homogenafe, mitochondrial and microsome-rich fractions<br />
of rat testis<br />
Effect of TCDD on the activity of glutathione reductase in<br />
the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
Effect of TCDD on the activity of glutathione peroxidase in<br />
the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
Effect of TCDD on the level of lipid peroxidation in the<br />
crude homogenate, mitochondrial and microsonie-rich<br />
fractions of rat testis<br />
Effect of TCDD on the production of s~lpcroxidc anion.<br />
nitric oxide and hydroycn pcl.oxide ill tlic epididym~ll<br />
sperm of adult rats<br />
Table 12<br />
Table 13<br />
Table 14<br />
Table 15<br />
Table 16a<br />
Effect of TCDD on the levels of antioxidants in the<br />
epididymal sperm of adult rats<br />
Effect of TCDD on the activities of antioxidant enzymes<br />
and the level of lipid peroxidation in the epididymal sperm<br />
of adult rats<br />
Effect of TCDD on the production of superoxide anion,<br />
nitric oxide and hydrogen peroxide in the epididymides of<br />
adult rats<br />
Effect of TCDD on the levels antioxidants in tho<br />
epididymides of adult rats<br />
Effect of TCDD on the activities of antioxidant enzymes<br />
and the level of lipid peroxidation in the caput epididyniis<br />
of adult rats
LIST <strong>OF</strong> TABLES (Continued)<br />
Table 16b<br />
Table 16c<br />
Table 17a<br />
Table 17b<br />
Table 17c<br />
Table 18a<br />
Table 18b<br />
Table 19<br />
Table 20<br />
Table 21<br />
'l'ablc 22<br />
Table 23<br />
Effect of TCDD on the activities of antioxidant enzymes<br />
and the level of lipid peroxidation in the corpus epididymis<br />
of adult rats<br />
Effect of TCDD on the activities of antioxidant enzymes<br />
and the level of lipid peroxidation in the cauda epididymis<br />
of adult rats<br />
Effect of TCDD on the production of superoxide anion,<br />
nitric oxide and hydrogen peroxide in the kidney of adult<br />
male rats<br />
Effect of TCDD on the levels of antioxidants in the kidney<br />
of adult male rats<br />
Effect of TCDD on the antioxidant enzymes and lipid<br />
peroxidation in the kidney of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
body weight and weights of the testis, epididymis, kidney<br />
and accessory sex organs of adult male rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
body weight and weights of the testis, epididymis, kidney<br />
and accessory sex organs (per 100 g body wciyht) of adult<br />
male rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
daily sperm production, epididyrnal sperm viability, sperm<br />
motility and sperm counts in adult male rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
seminiferous tubular and lumen diameter of testis of adult<br />
male rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
serum hormone levels in adult male rats<br />
Effecl of co-adniinislration of TCDD and vitamin II on thc<br />
activities of steroidogcnic enzymes of rut [tstis<br />
Effect of co-administration of TCDD and vitamin E on the<br />
DNA, RNA and protein contents in testis of adult male rats
Table 24a<br />
Table 24b<br />
Table 24c<br />
Table 25a<br />
Table 25b<br />
Table 25c<br />
Table 26a<br />
Table 26b<br />
Table 26c<br />
Table 26d<br />
Table 27<br />
LIST <strong>OF</strong> TABLES (Cuntinued)<br />
Page No.<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of superoxide anion in the crude homopenate.<br />
mitochondrial and microsome-rich liuclions ol' tcstis of<br />
adult rats 8 1<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of nitric oxide in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of testis of<br />
adult rats 82<br />
Effect of co-administration of TCDD and vitamin E on the<br />
hydrogen peroxide generation in the crude homogenate,<br />
mitochondria1 and microsome-rich fractions of rat testis 82<br />
Effect of co-administration of TCDD and vitamin E on the<br />
level of glutathione in the crude homogenate,<br />
mitochondria1 and microsome-rich fractions of rat testis 83<br />
Effect of co-administration of TCDD and vitamin E on the<br />
level of vitamin E in the crude homogenate, mitochondrial<br />
and microsome-rich fractions of rat testis 83<br />
Effect of co-administration of TCDD and vitamin E on the<br />
levels of ascorbic acid in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activity of superoxide dismutase in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activity of catalase in the crude hornogenate, mitochondrial<br />
and microsome-rich fractions of rat testis<br />
Effect of co-administration of TCDD and vitamin E on thc<br />
activity of glutathione reductase in the crude homogenate.<br />
mitochondria1 and microsome-rich fractions of rat testis 85<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activity of glutathione peroxidase in the crude homogenate,<br />
mitochondria1 and microsome-rich fractions of rat testis 86<br />
Effect of co-administration of TCDD and vitamin E on the<br />
level of lipid peroxidation in the crude homoyenatc.<br />
mitochondria1 and microsome-rich fractions of rat testis 86
Table 28<br />
Table 29<br />
Table 30<br />
Table 3 1<br />
'Table 32<br />
Table 33a<br />
Table 33b<br />
Table 33c<br />
Table 34a<br />
Table 34b<br />
Table 34c<br />
LIS'I' OY 'I'AI5LICS (Cunlinucd)<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of superoxide anion, nitric oxide and hydrogen<br />
peroxide in the epididymal sperm of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
levels of antioxidants in the epididymal sperm of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activities of antioxidant enzymes and the level of lipid<br />
petoxidation in the epididymal sperm of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of superoxide anion, nitric oxide and hydrogen<br />
peroxide in the epididymidcs of adult ruts<br />
Effect of co-administration of TCDD and vitamin E on the<br />
levels antioxidants in the epididyrnides of adult rats<br />
Effect of eo-administration of TCDD and vitamin E on the<br />
activities of antioxidant enzymes and the level of lipid<br />
peroxidation in the caput epididymis of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activities of antioxidant enzymes and the level of lipid<br />
peroxidation in the corpus epididymis of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
activities of antioxidant enzymes and the level of lipid<br />
peroxidation in the cauda epididymis of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
production of superoxide anion, nitric oxide and hydrogen<br />
peroxide in the kidney of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
levels of antioxidants in the kidney of adult rats<br />
Effect of co-administration of TCDD and vitamin E on the<br />
antioxidant enzymes and lipid peroxidation in the kidney of<br />
ndult rats<br />
i'ayc No.
Fig. 1<br />
Fig. 2<br />
Effect of TCDD on the body weight gain of rats<br />
Correlation between sperm counts and DNA contents of rat<br />
epididymal sperm<br />
Body weight changes in the TCDD administered with<br />
various doses of TCDD<br />
Fig. 4a<br />
Fig. 4b<br />
Effect of TCDD or TCDD and vitamin E on the weight of<br />
the testis of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the weight of<br />
the epididymis of adult rats<br />
Effect of 'SCDD or 'SCDD icnd vitamin E on thc wcigllt ol<br />
the seminal vesicles (intact) of adult rats<br />
Fig. 4d<br />
Fig. 4e<br />
Fig. 4f<br />
Fig. 5a<br />
Fig. 5b<br />
Fig. 6<br />
Fig. 7a<br />
Fig. 7b<br />
Fig. 7c<br />
Effect of TCDD or TCDD and vitamin E on the weight of<br />
the seminal vesicles (empty) of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the weight of<br />
the ventral prostate of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the weight of<br />
the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on daily sperm<br />
production and epididymal sperm counts of adult rats<br />
Effect of TCDD or TCDD and vitamin E on epididymal<br />
sperm viability and motility of adult rats<br />
Effect of TCDD or TCDD and vitamin E on seminiferous<br />
tubular and lumen diameter of adult rats<br />
Effect of TCDD or TCDD and vitamin E on serum FSH<br />
levels in adult male rats<br />
Effect of TCDD or TCDD and vitamin E on serum LH<br />
levels in adult male rats<br />
Effect of TCDD or TCDD and vitamin E on serum .<br />
prolactin levels in adult male rats
Fig. 7d<br />
I:iy. 7c<br />
Fig. 8<br />
Fig. 9<br />
Fig. IOa<br />
Fig. lob<br />
Fig. lla<br />
Fig. I lb<br />
Fig. I lc<br />
Fig. 12a<br />
Fig. 12b<br />
LIST <strong>OF</strong> FIGURES (Continued)<br />
Effect of TCDD or TCDD and vitamin E on serum<br />
testosterone levels in adult male rats<br />
Effect of' 'fCDD or TCDI) and vitaniin li on seru~ii<br />
estradiol levels in adult male rats<br />
Effect of TCDD or TCDD and vitamin E on the activities<br />
of testicular steroidoyenic enzymes of adult male rats<br />
Effect of TCDD or TCDD and vitamin E on DNA, RNA<br />
and protein contents in testis of adult male rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of superoxide anion in the cmde homogenate.<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of nitric oxide in the crude homogenate, mitochondrial and<br />
microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of hydrogen peroxide in the crude homogenate.<br />
rnitochondrial and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
glutathione in the crude homogenate, mitochondrial and<br />
microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the level of a-<br />
tocopherol in the crude homogenate, mitochondrial and<br />
microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
ascorbic acid in the crude homogenate, mitochondrial and<br />
microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
superoxide dismutase in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
catalase in the crude hornogenate, m~tochondrial and<br />
microsome-rich fractions of rat testis<br />
Page No.<br />
119<br />
120<br />
121<br />
122
Fig. 12c<br />
Fig. 12d<br />
Fig. 13<br />
Fig. 14a<br />
Fig. 14b<br />
LIST <strong>OF</strong> FlGURES (Continued)<br />
Effect of TCDD or TCDD and vitamin E on the activity ol'<br />
glutathione reductase in the crude homogenate.<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione peroxidase in the crude hornogenate.<br />
mitochondrial and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
lipid peroxidation in the crude homogenate, mitochondrial<br />
and microsome-rich fractions of rat testis<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of superoxide anion in the epididymal sperm of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of nitric oxide in the epididymal sperm of adult rats<br />
Page No.<br />
131<br />
Fig. 14c<br />
Fig. 15a<br />
Fig. 15b<br />
Fig. 16a<br />
I:ig. IOh<br />
Fig. 16c<br />
Fig. 16d<br />
Fig. 17<br />
Fib 18a<br />
Enict ol'l'CDD or 'l'CL)I) and vitamin I: 011 tlic ~-rrc~duclit~~i<br />
of hydrogen peroxide in the epididymal sperm ofadult rats<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
glutathione in the epididymal sperm of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of a-tocopherol in the epididyrnal sperm of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
superoxide dismutase in the epididymal sperm of adult rats<br />
EFCcct of TCDD or YCDD and vilaniin 1: on 11ic activity 01'<br />
catalase in the epididymal sperm ol'adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione reductase in the epididymal sperm of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione peroxidase in the epididymal sperm of adult<br />
rats<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
lipid peroxidation in the epididymal sperm of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of superoxide anion in the caput. corpus and cauda<br />
epididyrnides of adult rats
Fig. 18b<br />
Fig. 18c<br />
Fig. 1Ya<br />
Fig. 19b<br />
Fig. 20a<br />
Fig. 2Ob<br />
Fig. 20c<br />
Fig. 2Od<br />
Fig. 21<br />
Fig. 22a<br />
Fig. 22b<br />
Fig. 22c<br />
Fig. 23a<br />
LIST <strong>OF</strong> FIGURES (Continued)<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of nitric oxide in the caput, corpus and cauda epididymides<br />
of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of hydrogen peroxide in the caput, corpus and cauda<br />
epididymides of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
glutathione in the caput, corpus and cauda epididymides of'<br />
adult rats<br />
Effect of TCDD or TCDD and vitamin E on the level of a-<br />
tocopherol in the caput, corpus and cauda epididymides of<br />
adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
superoxide dismutasc in the caput, corpus and cauda<br />
epididymides of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
catalase in the caput, corpus and cauda epididymides of<br />
adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione reductase in the caput, corpus and cauda<br />
epididymides of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione peroxidase in the caput, corpus and cauda<br />
epididymides of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the levels of<br />
lipid peroxidation in the caput. corpus and cauda<br />
epididymides of adult rats<br />
Effect of TCDD or I'CDD and vitamin B on the production<br />
of superoxide anion in the kidney ofadult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of nitric oxide in the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the production<br />
of hydrogen peroxide in the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
glutathione in the kidney of adult rats<br />
Page No.<br />
140
LIST <strong>OF</strong> FIGURES (Continued)<br />
Fig. 23b Effect of TCDD or TCDD and vitamin E on the level of a-<br />
tocopherol in the kidney of adult rats<br />
Page No.<br />
151<br />
Fig. 24a Effect of TCDD or 'I'CDD and vitamin C on the uclivity 01'<br />
superoxide dismutase in the kidney of adult rats<br />
Fig. 24b<br />
Fig. 24c<br />
Fig. 24d<br />
Fig. 25<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
catalase in the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione reductase in the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the activity of<br />
glutathione peroxidase in the kidney of adult rats<br />
Effect of TCDD or TCDD and vitamin E on the level of<br />
lipid peroxidation in the kidney of adult rats
LIST <strong>OF</strong> ABBREVIATI<strong>ON</strong>S<br />
ABP<br />
Ah<br />
AhR<br />
ATPase<br />
BSA<br />
C<br />
CAMP<br />
CYP<br />
DDT<br />
DHT<br />
DNA<br />
DRE<br />
EDTA<br />
ELlSA<br />
ER<br />
ERKO<br />
FSH<br />
g<br />
R<br />
h<br />
GTP<br />
hCG<br />
HCH<br />
HRP<br />
IGF<br />
Ke<br />
L<br />
androgen binding protein<br />
aryl hydrocarbon<br />
aryl hydrocarbon receptor<br />
adenosine triphosphatase<br />
bovine serum albumin<br />
centigrade<br />
cyclic adenosine mono-phosphate<br />
cytochrome P450<br />
dichlorodiphenyl-trichloroethane<br />
dihydrotestosterone<br />
deoxyribonucleic acid<br />
dioxin response element<br />
ethylenediaminetetraacetic acid<br />
enzyme-linked immunosorbant assay<br />
estrogen receptor<br />
estrogen receptor knockout<br />
follicle stimulating hormone<br />
gram<br />
acceleration due to gravity<br />
hour<br />
guanosine tri-phosphate<br />
human chorianic gonadotrophin<br />
hexachlorocyclohexane<br />
horseradish peroxidasc<br />
insulin-like growth factor<br />
kilogram<br />
litre
LH<br />
M<br />
PL<br />
mg<br />
mRNA<br />
N AD<br />
NADH<br />
NADP<br />
NADPH<br />
nrn<br />
YO<br />
PCA<br />
PCB<br />
PCDD<br />
PCDF<br />
PModS<br />
I'U PA<br />
SD<br />
RNA<br />
ROS<br />
rPm<br />
TBA<br />
TCA<br />
'I'CL)l><br />
TMB<br />
XRE<br />
luteinizing hormone<br />
molar<br />
microlitre<br />
milligram<br />
messenger ribonucleic acid<br />
nicotinamide adenine dinucleotide<br />
nicotinamide adenine dinucleotide (reduced)<br />
nicotinamide adenine dinucleotide phosphate<br />
nicotinamide adenine dinucleotide phosphate<br />
(reduced form)<br />
nanometre<br />
percentage<br />
perchloric acid<br />
polychlorinated biphenyls<br />
polychlorinated dibenzo-p-dioxins<br />
polychlorinated dibenzofurans<br />
peritubular modulatory substance<br />
polyunsaturi~~ud fiit~y acid?;<br />
standard deviation<br />
ribonucleic acid<br />
reactive oxygen species<br />
revolutions per minute<br />
thiobarbituric acid<br />
trichloroacetic acid<br />
2,3.7.B-Lctrachlorodihcnzo-/~-dic1xi1i<br />
tetramethylbenzidine<br />
xenobiotic rrsponsc elenlcnl
1 INTRODUCTI<strong>ON</strong><br />
1.1 Description of male reproductive system<br />
Male reproductive system comprises a pair of testes, epididymides and<br />
accessory sex glands. Testes are encapsulated ovoid organs consisting of seminiferous<br />
tubules separated by interstitial tissue. Testis has two main functions, production of<br />
spermatozoa, which transmit male's gene to embryo and male sex hormone<br />
testosterone, which plays an important role in maintaining spcrnlatoycncsis. isccssory<br />
sex organs and secondary sexual characters.<br />
The posterior border of testis is associated with epididymis and with spermatic<br />
cord, the latter incorporating ductus deferens. together with neurovascular structures<br />
running to and from the middle and upper ends of posterior border of testis (de<br />
Kretser, 1982). Epididymis is a single highly convoluted duct, closely applied to the<br />
surface of the testis extending from the anterior to the posterior pole of testis.<br />
Epididymis acts as storage place for the spermatozoa where they acquire motility and<br />
capacity to fertilize the ova.<br />
Seminal vesicles are paired, bag-shaped glands and the internal surface<br />
consists of intricate system of folds to form irregular diverticula. Seminal vesicles<br />
secrete a viscous fluid, which is expelled along with sperm. it contains several<br />
essential nutrients which are required by the sperm for their fertilizing ability.<br />
Ventral prostate is a bilobed structure situated ventral to urethra. It has<br />
numerous small ducts through which the secretions are discharged directly into the<br />
urethra. The secretions are rich in nutrients and serve as lubricant for the semen<br />
(Setchell o ul., 1994).<br />
Testis is surrounded by a dense connectiv.: tissue. the tunica albuginea which<br />
is a tough fibrous capsule enclosing testicular parenchyma. It is composed of three
layers, an outer layer of visceral peritoneum, the tunica vaginalis, the tunica albuginea<br />
proper and on the inside the tunica vasculosa. Posteriorly funica albuginea is<br />
thickened and projects into the parenchyma of testis to form mediastenum, a<br />
honeycomb-like structure, which provides a passageway linking seminiferous tubules<br />
with efferent ductules and epididymis.<br />
Testis is made up of seminiferous tubules where the spermatozoa are formed.<br />
Senliniferous tubules owe their cylindrical shape to multiple layers of cellular and<br />
extracellular elements collectively known as the lamina propria. tunica propria, or<br />
peritubular tissue. Seminiferous tubules are two ended. convoluted loops with both<br />
ends opening into rete testis, through which sperm and fluid produced by the tubules<br />
pass on their way to excurrent duct systems. There are 30 tubules in rat testis (Dym.<br />
1976) with a diameter between 50 and 100 pm (Setchell, 1970; Wing and<br />
Christensen. 1982). Within the seminiferous tubules germ cells and somatic Sertoli<br />
cells make up the seminiferous epithelium, a complex stratified arrangement of germ<br />
cells. supported by tall columnar Sertoli cells. which rest upon the basement<br />
membrane and extend apically toward the lumen of the tubule (de Kretser er rrl.<br />
1995).<br />
Germ cells are highly synchronized in their proliferation and development to<br />
lorm a distinct cellular associations, establishing a precisely coordinated 'cycle of the<br />
seminiferous epithelium', believed to be regulated in part by Sertoli cells which do<br />
not divide in adult testis (de Kretser cr ul.. 1998; Orth. 1982). Walls of' the<br />
seminiferous tubules are composed of four layers of non-cellular materials surrounded<br />
by a layer of smooth muscle-like or myoid cells which are responsible for peristaltic<br />
movements of the tubules (Clermont, 1958). Interstitial tissue between seminiferous<br />
tubules is composed of clusters of Leydig cells, mesenchymal cells. macrophages.<br />
occasional mast cells, blood and lymph vessels (for review, see Johnson el al, 1999).<br />
The interstitial tissue is mainly composed of Leydig cells, which are the site of<br />
testicular steroidogenesis. Leydig cells have an abundance of smooth endoplasmic<br />
reticulum, many mitochondria with tubular cristae. Golgi complex, centrioles,
lysosome, peroxisome and number of lipid droplets (for review, see Johnson e! ul..<br />
1999). Leydig cells are associated with blood vessels (Fawcett et 01.. 1973) or found<br />
nearer to the walls of the seminiferous tubules (Bergh, 1983). The presences of mast<br />
cells and macrophages have been reported in the interstitial compartment (Nistal el<br />
a/., 1984; Christensen et 01.. 1985). Sertoli cells are found immediately inside the<br />
boundary tissue of the seminiferous tubular and surrounded by undeveloped germ<br />
cells before puberty. The interstitial and seminiferous tubules compartments are not<br />
only structurally distinct but are also separated physiologically by cellular barrier,<br />
which develop during puberty and limit the free exchange of water-soluble materials<br />
(Johnson and Everitt, 1995). The plasma membranes of Sertoli cells and tight<br />
junctions between adjacent cells create adluminal compartment of the blood-testis<br />
barrier. Sertoli cells share the surface of the boundary tissue with the developing<br />
spermatogonia (Fawcett, 1975). Sertoli cells have intricate cytoskeleton and<br />
numerous processes, which are responsible for positioning, movement and shaping of<br />
the spermatogenic cells. Occluding junction between adjacent Senoli cells prevent<br />
free exchange of molecules from outside of the tubule into the spermatocytes a~d<br />
spermatids (de Kretser er ol., 1995). Sertoli cells have been reported to perform large<br />
number of specific functions including secretion of fluid, phagocytosis, maturation,<br />
release of spermatozoa and synthesis of intratubular androgen binding prorein<br />
(Chemes, 1986; Clermont el al. 1987).<br />
Spermatogenesis is the process of gradual transformation of germ cells into<br />
spermatozoa over an extended period of time within the boundaries of thc<br />
seminiferous tubules of testis. This process involves cellular proliferation by repeated<br />
mitotic divisions. duplication of chromosomes. genetic recombination through<br />
crossover and reduction division by meiosis to produce haploid spermatids, and<br />
terminal differentiation of sperrnatids into spermatozoa (for review, see de Kretser r/<br />
ul.. 2000). Seminiferous tubules contain a large n~rmber of germinal epithelial cells<br />
called spermatogonia, located in two to three layers along the outer border of the
tubular epithelium and continually proliferate to replenish themselves. At the start of<br />
spermatogenesis, diploid spermatogonia proliferate producing three sub-populations<br />
of cells with markedly different destinies. One sub-population of spermatogonia are<br />
presumably identical to their progenitors and continue to function as stem cells and<br />
the majority of spermatogonia have been shown to enter a differentiative pathway to<br />
become spermatozoa (for review, see de Kretser er al., 2000). The stem<br />
spern~atogonia (type A) are located immediately adjacent to the basement membrane<br />
of the germinal epithelium. Type A spermatogonia exhibiting fine pale-staining<br />
nuclear chromatin and type B with coarse granules or more heavily stained chromatin<br />
associated with nuclear membrane and nucleolus (Clermont, 1972).<br />
'Type A<br />
spermatogonia enter a cycle and produce a chain of aligned undifferentiated<br />
spermatogonia, which differentiate into type A, spermatogonia. These cells undergo<br />
a sequence of six cell cycles and mitotic divisions resulting in the formation of A2, A>.<br />
A4 intermediates and finally into slightly more differentiated cells, the type B<br />
spermatogonia. After several divisions these cells give rise to very large primary<br />
spcrniatocytes (Steinberger and Steinberger, 1975).<br />
The primary spermatocytes undergo a long meiotic prophase and subsequently<br />
form secondary spermatocytes reducing the number of chromosomes (Steinberger and<br />
Steinberger, 1975; Weinbauer er a/., 1991). In the 2-stage process they dtvide into<br />
secondary spermatocytes and then spermatids. These spermatids undergo a precise<br />
process of morphological differentiation to form spermatozoa, which are released a1 a<br />
specific stage of development from the seminiferous epithelium into the lumen of the<br />
tubules.<br />
The temporal successions of all the spermatogenic stages called<br />
'spermatogenic cycle' have been reported in different species (Johnson rr 01.. 1999).<br />
The time taken from the division of type A spermatogonia to the release of<br />
spermatozoa has been reported to be approximately 50 days in rats and 64 days in<br />
men (Clermont and Harvey, 1965). In the mouse, the total duration of<br />
spermatogenesis from the stem cell to the mature spermatid is about 34.5 days<br />
(Monesi, 1965). The mitotic phase of spermatogenesis lasts about 8 days, meiosis 13<br />
days and spermiogenesis about 13.5 days (Monesi, 1965). The DNA synthesis in
apidly proliferating cells of testis reflects the initial phase ol' sper~iiiltoycncsis<br />
including spermatogonial proliferation by mitosis and the onset of meiotic prophase in<br />
prc-leptotcne primary spermatocylcs (Monesi, 1965). ItNA synthesis is highost<br />
during the mid pachytene phase and the end of the meiotic prophase and mRNA<br />
specific for structural protein increases during prophase of meiosis (Slaughter el 01..<br />
1989). The rate of protein synthesis has been reported to be higher in type A<br />
spermatogonia than in type B (Monesi, 1965). Toxicants that would alter mitosis or<br />
differentiation of spermatogonia would be expected to have a major influence on the<br />
efficiency and total productivity of spermatogenesis.<br />
1.1.1.2 Hormonal control of spermatogenesis<br />
There is general agreement that for quantitatively normal spermatogenesis to<br />
occur, testis requires stimulation by pituitary gonadotrophins such as follicle<br />
stimulating hormone (FSH) and luteinizing hormone (LH). The progressive rise of<br />
FSH and LH during sexual maturation has been reported in humans and in other<br />
animals (for review, see de Kretser et ul., 1998). FSH and LH have been shown to<br />
stimulate seminiferous tubules and reinstate spermatogenesis in hypophysectomized<br />
rats (Greep and Fevold, 1937). Synergistic action of FSH and testosterone for the<br />
maintenance of spermatogenesis has also been reported (Steinberger. 1971). In the<br />
prepubertal rat, FSH alone is required to maintain testicular growth (Courot el al.,<br />
1970) and synthetic activity of Senoli cells (Means et al., 1976). High intratesticular<br />
concentrations of testosterone are required for normal spermatogenesis. Testosterone<br />
has been shown to act by stimulating Senoli cells and receptors for FSH are located<br />
on the Senoli cells and spermatogonia. FSH has been shown to exert its action via<br />
CAMP. Injection of FSH is associated with a stimulation of protein kinase activity<br />
and protein synthesis. FSH has been shown to play a role in stimulating mitotic and<br />
meiotic DNA synthesis in type B spermatogonia and pre-ieptotene spermatocytes as<br />
well as in preventing apoptosis of pachytene spermatocytes and round spermatids<br />
(Henricksen el ul.. 1996: Shetty el ul.. 1996). Mc lachlnn el ol. (1995) detnonstrntod<br />
that FSH alone could partially restore spermatogenesis in GnRH-immunized rats.
lmn~unization of monkeys against FSH either actively or passively has been shown to<br />
be associated with disruption of fertility and a decline in sperm concentrations. It has<br />
been reported that the specific spermatogonial subtypes are affected by withdrawal of<br />
FSH alone (Moudgal et al., 1997). FSH has been shown to promote spermatogenesis<br />
and fertility in primates (Moudgal and Sairam, 1998).<br />
Administration of highly<br />
purified FSH to GnRH antagonist treated monkeys has been shown to niaintnin<br />
spermatogonial numbers (Weinbauer el a/., 1991). Actions of FSI-I on Sertoli cells<br />
from prepubertal animals have been reported biochemically by increased estradiol<br />
production (Fritz et al., 1976). It has been reported that FSH P-subunit gene knocked<br />
out mice. rendering them FSH deficient showed that spermatogenesis could proceed<br />
to completion albeit in the presence of smaller testes (Kumar et al., 1997). The<br />
binding of LH to Leydig cells in the interstitial compartment of testis has been<br />
reported to stimulate testosterone synthesis.<br />
Initiation of spermatogenesis by testosterone and dehydrotestosterone has been<br />
reported in mouse (Singh el al.. 1995).<br />
Testosterone alone has been shown to<br />
maintain spermatogenesis qualitatively in a dose-dependent fashion in monkeys<br />
(Weinhnuer el (11.. 1988).<br />
A high concentration of tcstosteronc within thc<br />
seminiferous tubules LH has been shown to play a role in the initiation and<br />
maintenance of sperm production in mouse (di Zerega and Sherins, 1980; Matsumota<br />
and Bremmer. 1987).<br />
In rodents and primates, by virtue of its local production.<br />
intratesticular testosterone levels have been shown to be elevated approximately 100<br />
fold above those in serum (Huhtaniemi er al., 1987).<br />
A role for estrogens in normal spermatogenesis has also been reported (Hess ef<br />
ul.. 1997) and at least two major steps have been shown to be influenced by estrogens<br />
in the seminiferous tubules, stem cell number and spermatid maturation (Hess cr ul.,<br />
1997). It has been shown that in virro multiplicntion of rat gonocytes is controlled by<br />
growth factors and estradiol, which corresponds well with the presence of ERP in<br />
these cells (Hess et a!., 2000).<br />
In estrogen receptor a knockout (ERKOa) mouse<br />
estrogens are necessary for the achievement of fertility (Hess et al.. 2000).
1.1.1.3 Tcsticular steroidogcnesiv<br />
Testis secretes a variety of steroids which are synthesized from cholesterol<br />
which itself can be synthesized from acetatc. Thc principal sccrctory stcn)id product<br />
in male is testosterone of which 95% is synthesized in the Leydig cells and the<br />
remainder being produced by the adrenal glands. Cholesterol is derived from two<br />
sources, consisting of an uptake mechanism by which circulating low-density<br />
lipoprotein bind to specific receptors on Leydig cells or by internalization. that<br />
provide n ready source ol'cholcstclol. It1 rat I.cydig cells. receptors lir I~igli-ilc~lsily<br />
lipoprotein have also been reported (Chen el al., 1980). Cholesterol has been shown<br />
to get converted into pregnenolone in the testicular tissues by successive oxidations<br />
under the influence of C20-C22 desmolase complex (Cytochrome P-450) in<br />
mitochondria in the presence ofNADPH and oxygen (Burstein and Gut, 1971).<br />
Tcsticular homogenates of rat has been shown to convert pregnenolone more<br />
to progesterone than that of 17-hydroxypregnenolone. Incubation of labeled 17-<br />
hydroxypregnenolone has been shown to produce dehydroepiandrosterone instead of<br />
17-hydroxyprogesterone (for review, see Hall, 1988). Existence of an alternative<br />
pathway of conversion of 17-hydroxypregnenolone to androstenediol<br />
withor~t<br />
involving dehydroepiandrosterone or androstenedione has also been reported<br />
(Slaunwhite and Burgen, 1965). Formation of A~') ketosteroids such as progesterone.<br />
17-hydroxyprogesterone, androstenedione and testosterone from A'" P-<br />
hydroxysteroids in the presence of 3P-hydroxysteroid dehydrogenasel A~~ isomerase<br />
has been reported (Hall, 1970; Eik-Nes, 1975). Progesterone has been shown to get<br />
convened into 17-hydroxyprogesterone, androstenedione and testosterone by 17-<br />
hydroxylase, 17-20 lyase and 17P-hydroxysteroid dehydrogenase respectively (Hall.<br />
1970, Eik-Nes, 1975). Progesterone has also been shown to get converted into<br />
17-hydroxyprogesterone, androstenedione and testosterone in the incubation of slices<br />
or homogenates of testis of hypophysectomized or immature rats treated with human<br />
chorionic gonadotrophin (hCG) to increase the volume of the interstitial cells<br />
(Slaunwhite and Samuels, 1956). Testis has the capacity to produce 17p-estradiol,
and this reaction is catalyzed by the enzyme cytochrome P450 aromatase (for review,<br />
see Hall, 1988; Carreau et al., 1999). This conversion involves a series of reactions<br />
resulting in hydroxylation at C3. Testis has also the capacity to secrete small amounts<br />
of dihydrotestosterone and the enzyme catalyzing the conversion of testosterone to<br />
dihydrotestosterone is 5 a-reductase (Carreau, 2000).<br />
1.1.1.4 Hormonal regulation of steroidogenesis<br />
Testosterone production in testis is controlled by LH through the specific<br />
receptors on the surface of the Leydig cells. The LH receptor has been isolated and<br />
characterized (de Kretser et al., 1971). Considerable evidence indicates that<br />
interactions of LH with its receptor activate the CAMP pathway through a GTP<br />
binding protein (Hall, 1988; Dufau et al.. 1978). Testosterone production has been<br />
shown to get stimulated when decapsulated testis of adult rats is incubated with LH or<br />
hCG (Rommerts er al.. 1972). Stimulation of androgen production has been reported<br />
when isolated Leydig cells of rats are incubated either with LH or hCG (Qazi er al..<br />
1974). Increase in the production of testosterone and de novo protein synthesis in<br />
Leydig cells of adult rats revealed a strong correlation with the dosage of LH (Janszen<br />
et al., 1978).<br />
Activity of A' 3P-hydroxysteroid dehydrogenase has been shown to respond to<br />
hCG in neonatal interstitial cells in culture (Meidan cr a/. 1985). Stimulation of 36-<br />
hydroxysteroid dehydrogenase activities in testes of i~lirnaturc hypophyscctol~liscd<br />
rats has been shown by the administration of hCG in vivo (Murono and Payne, 1979).<br />
Suppression of the activities of 7-hydroxylase and 17P-hydroxysteroid dehydrogenase<br />
has been reported in the rat testis after administration of hCG or testosterone (Inano el<br />
rtl.. 1973). lmplanta$ion of testosterone-estradiol filled silastic capsules in rats caused<br />
a reduction in the volume of smooth endoplasmic reticulum in the Leydig cells and<br />
the ability of the testis to secrete testosterone (Ewing er al.. 1983).<br />
Ascorbic acid has been shown to play an important role in the biosynthesis of<br />
testicular steroids (Luck et a/., 1995). Testosterone could regulate the rnorphogenic
and steroidogenic processes as Leydig and precursor cells have been shown to contain<br />
receptors for androgen (Verhoeven, 1980). Testosterone secretion in the prepubertal<br />
rats reduced following treatment with an androgen receptor antagonist (Puwis and<br />
Hansson, 1978). Androgen receptor mRNA and protein levels are reported to be<br />
lower in adult rather than immature Leydig cells, which might result from down<br />
regulation exerted by the pubertal rise in androgen production (Shan and Hardy,<br />
1992). Prolactin has been shown to increase the number of LH receptors and could<br />
potentiate steroidogenic effect of LH on Leydig cells and testicular prolactin receptors<br />
have been shown to be confined to the interstitial tissue of the testis (Johnson and<br />
Everitt, 1995). Prolactin also increases the uptake of androgen and increases<br />
Sa-reductase activity (Johnson and Everitt, 1995).<br />
1.1.1.5 Paracrine regulation of testicular functions<br />
Leydig cells, Sertoli cells, germ cells and peritubular myoid cells have been<br />
shown to interact with each other in order to maintain testicular functions.<br />
Cryptocrine communications are reported between germ cells and Sertoli cells in the<br />
seminiferous tubules (Funder, 1990). It has been reported that stimulatory effect of<br />
Sertoli cells on germ cells mediated by diffusible factors and to obtain full expression<br />
of these stimulation germ cells must be in contact with Sertoli cells (Rivarola er ul.,<br />
1985). Seminiferous tubules have been shown to secrete a meiosis inducing<br />
substance and its secretion is maximal at stage VIIc-d of the spermatogenic cycle, just<br />
before the onset of premeiotic DNA synthesis in preleptotene spermatocytes<br />
(Parvinen, 1982). Regulation of Sertoli cell function by germ cells has been shown in<br />
in vivo and in virro studies (Saez ef al., 1991). Regulation of FSH and testosterone on<br />
spermatogenesis is reported to be mediated by Sertoli cells, which contain FSH and<br />
androgen receptors (Tindall er al., 1977). Sertoli cells synthesize and secrete<br />
transport proteins, growth factors that are regulated by FSH and also modulate by<br />
local mechanisms related to the spermatogenic cycles. The nutrient and growth<br />
factors required for spermatogenesis was shown to be transported from the
extratubular environment or synthesized by the Sertoli cells and delivered to the<br />
developing germinal cells within the tubule (Skinner, 1987).<br />
Germ cells require lactate for swival but they are unable to metabolize<br />
glucose (Jutte el al., 1982), this energy substrate can be supplied by Sertoli cells<br />
which are able to transform glucose to lactate, a process stimulated by FSH (June er<br />
al., 1983) and insulin-like growth factor I (IGF-I) (Mita and Hall, 1982). Lactate and<br />
pyruvate secretions by Sertoli cells have been reported to regulate and maintain<br />
spermatogenesis (Grootegoed and de Boer, 1989). Degeneration of germ cells has<br />
been reported due to the absence of proper substrate required for their metabolic<br />
activiries (June el al.. 1981). It has been reported that human Sertoli cells cultured<br />
with FSH and testosterone have been reported to produce lactate, estradiol and<br />
transfemn (Foucault el a[., 1992).<br />
Sertoli cells have been shown to synthesize and secrete transport proteins and<br />
growth factors (Bardin el d.. 1988; Ritzen el al.. 1989). which are regulated by FSH.<br />
Several Sertoli cell secretory products have now been identified which include<br />
transferrin, androgen binding protein, ceruioplasmin, plasminogen activator, clusterin<br />
(sulfate glycoprotein-2). a2 macroglobulin, testin sulfated glycoprotein-I, testiburnin<br />
and others (for review, see Mruk and Cheng, 2000a). The stimulatory effects of<br />
Sertoli cells on germ cells have been reported which are mediated either by cell-cell<br />
contacts (Russell, 1980) and! or by diffusible factors (Rivarola er a!.. 1985). Sertoli<br />
cells have been reported to secrete several growth factors including IGF-I (Cailleau el<br />
01.. 1990), TF-a (Skinner et al.. 1989). TGF-P (Skinner and Moses, 1989). FGF<br />
(Smith el al., 1989) and seminiferous growth factor (Bellve and Zheng, 1989). Germ<br />
cells have been shown to regulate biosynthesis of rat Sertoli cell clusterin. a:-<br />
macroglobulin and testin (Grima et al., 1992). Peritubular cells have been shown to<br />
enhance the production of a Leydig cell stimulatory factor when co-cultured with<br />
Senoli cells (Verhoeven and Cailleau. 1986). Peritubular cells have been reportcd 10<br />
secrete a factor PMod-S, which stimulated secretion of Sertoli cell proteins (Skinner<br />
el al., 1988). PMod-S production was reported to be under androgen regulation
(Skinner and Fritz. 1985) and provided a potential mode of action for androgens to<br />
regulate Sertoli cell function indirectly (Skinner, 1987). Supprcssion of scrum 131 I<br />
concentration has been achieved following inhibin administration to castrated rats<br />
suggesting the feedback regulation of FSH secretion by inhibin (Robertson el ul..<br />
1991). Activin has been shown to stimulate LH release and circulating FSll<br />
concentration in macaques and rats in vivo (Schwall er al, 1989; McLachlan el al.,<br />
1989). Leydig cells have been shown to secrete oxytocin-like immunoreactive<br />
material to influence the contractibility of the seminiferous tubules by acting on<br />
peritubular cells (Worley et al.. 1984).<br />
Rat germ cells co-cultured with rat Sertoli cells produce an inhibited RNA and<br />
DNA synthesis of Sertoli cells (Rivarola el al.. 1985) as well as an increased secretion<br />
of androgen binding protein (ABP). transferrin and inhihin and an inhihition 01' Scrtoli<br />
cell aromatase activity (Le Magueresse and Jegou, 1988). The number of factors<br />
implicated in paracrine regulation of testicular functions has been steadily increasing<br />
and many factors have been shown to be of potential or proven physiological<br />
significance (for review, see Spiteri-Grech and Nieschlag, 1993; Griswold, 1993).<br />
1.1.2 Structure and functions of epididymis<br />
Iipididymis is composed of a single, long. highly coiled duct closcly applied to<br />
the surface of the testis and embedded in a variahle amount of adiposc tissue of<br />
epididymal fat pad. It has been reported that mammalian epididymis. histologically<br />
and functionally, can be divided into initial and middle segments where sperm<br />
maturation takes place and a terminal segment, which subserves the function of sperm<br />
storage (for review. see Robaire er 01.. 2000). Most abundant cell type found in the<br />
epididymal epithelium is the principal cell, which has prominent stereocilia that<br />
extend into the lumen. In rats, principal cells constitute 80% of the total epithelial cell<br />
population in the initial segment and this number gradually decreases to 65% in the<br />
cauda epididymis (Robairr and Hermo. 1988; Jones. 1999). Large numbers of<br />
proteins arc secreted in the epididymis and the anterior par? of it has been shown to be
the most active in protein secretion in rats and boer (Brooks, 1981; Syntin cl ul.,<br />
1996). High levels of several proteins that mediate antioxidant action are found in<br />
epithelial cells and in epididymal fluid, which may protect epithelial and/ or sperm<br />
cells against oxidative stress from exposure to endogenous reactive oxygen species or<br />
exogenous toxic substances (Hinton er al., 1995; Jones, 1999). Expression of<br />
secretory antioxidant factors has been shown to be region specific, indicating that the<br />
need for antioxidant enzymes may vary along the epididymis. Some of the other<br />
major proteins secreted by the epididymal epithelium possess carrier functions for thc<br />
transport of retinol (Porter er ul., 1985) or of cholesterol (Baker el ul., 1993; KirchhofS<br />
cr 01.. 1998).<br />
Epididymis has been shown to be dependent on androgens secreted into the<br />
peripheral circulation as well as other factors that are secreted directly into the<br />
epididymal lumen from the seminiferous tubules. rete testis and efferent ducts (for<br />
review. see Robaire er 01.. 2000). There is a time dependent dramatic weight loss of<br />
the epididymis after androgen withdrawal and has been attributed to the loss of<br />
spermatozoa and fluid from the lumen of the epididymis and in part due to changes in<br />
the epithelium of the tissue (Klinefelter and Hess, 1998). Apoptotic cell death after<br />
orchidectomy has been shown to appear first in the epithelium of the initial segment<br />
ol' the rpididymis and subsequently in more distal segments (for review, src Robaire<br />
rr ul.. 2000). The main androgen responsible for maintaining epididymal structure<br />
and functions is the 5 a-reduced metabolites of testosterone. dihydrotestostcronc<br />
(DHT). The rate-limiting enzyme in the pathway leading from testosterone to its 5 a-<br />
reduced metabolites is 4-ene steroid 5 a-reductase (Hermo er a/., 1988). In the rat.<br />
the levels of dihydrotestosterone have been shown to be approximately 10 fold higher<br />
than those of testosterone in the caput and corpus epididymis, but the levels of these<br />
androgens are similar in the cauda epididymis (Turner cr at.. 1984). l'estosteronc<br />
entering the epididymis via the lumen is associated with transport of androgen binding<br />
protein (ABP) from seminiferous tubule to the epididymis (Danzo, 1995). In rat.<br />
epithelium in cauda epididymis is characterized by the presence of numerous clear<br />
cells. These cells have been shown to be responsible for the phagocytosis of
cytoplasmic droplets shed from sperm dwing epididymal transit (Henno er 01.. 1988).<br />
The journey from the testis to the cauda epididymis has been shown to take<br />
approximately 4 days in rats (Klinefelter and Hess, 1998).<br />
Estrogen has also been shown to play an important role in fluid reabsorption in<br />
efferent ductulcs and epididymis of rats (Hess et a[., 1997; Hess et al., 2000). Effect<br />
of estrogen on the epididymis can be viewed either as indirect or direct. since<br />
estradiol is a potent suppressor of LH secretion. Administration of estradiol results in<br />
a decrease in gonadotrophins and consequently in a shutdown of Leydig cell function,<br />
and hence androgen production. The presence of aromatase activity has been reported<br />
in spermatozoa entering the epididymis (Janulis et al., 1998) and of estrogen receptors<br />
in epididymal principal cells (Hess et al., 1997). Estrogen has been shown to cause<br />
reduction in epididymal sperm number and motility in adult male rats (Kaneto er al.,<br />
1999).<br />
1.2 Target organ response to testosterone<br />
Accessory sex organs in male have been reported to be androgen dependent<br />
and thus reflect the availability of androgens (Hunt el al., 1978). Weights of the<br />
accessory sex organs in castrated male rats have been widely used as a bioassay for<br />
androgenic and antiandrogenic compounds (Neumann and Steinbeck, 1974).<br />
Weights. sizes, cytological structures and mitotic activity of the seminal vesicles of<br />
mouse were established as indicators of bioassay for androgenic hormones (Deanesly<br />
and Parkes, 1933). Measurement of weights of the accessory sex organs in intact rats<br />
has been shown to reflect the estimation of cumulative effect of biologically active<br />
testosterone over a period (Mathw and Chattopadhyny. 1982).<br />
The sensitivity of the accessory sex organs varied from organ lo orgun 21s<br />
shown by the requirement of androgen for seminal vesicles being 3 times more than<br />
that of prostate (Moore and Gallagher. 1930; Burkhart. 1942). The maintenance of<br />
structural and functional integrity of prostate gland has ken reported to be dependent<br />
upon the amount of circulating androgen pr$nccd by the testis (Butler and Schode,
1958). A rapid fall in serum testosterone levels following bilateral orchidectomy has<br />
been shown to caw prostate involution (Bruchovsky cl ul., 1975; Lee, 1981 ). Whilc<br />
replacement of androgen exogenously has been to stimulate the prostate growth<br />
(Bruchovsky er 01.. 1975).<br />
A remarkable increase in total weight, contents of water, inorganic ions and<br />
metabolic activity of seminal vesicles was induced by subcutaneous injection of<br />
testosterone propionate to castrate rats (Rudolph and Samuel, 1949). Kidneys were<br />
shown to have potential to respond to androgen stimulation for growth and metabolic<br />
activities similar to accessory sex organs (Kochakian, 1977). Kidneys of the castrated<br />
mice decreased in size by 30 to 50% in comparison to that of the intact animals.<br />
Treatment with testosterone propionate restored the weights of the kidney to normal<br />
range in the rats treated with cisplatin (Malarvizhi and Mathur, 1996). Testosterone<br />
has been shown to stimulate compensatory hypertrophy of the kidney and ameliorate<br />
renal dysfunction (de Ruiter, 1981). Androgen has been shown to produce a two to<br />
five fold increase in the specific activity of the enzyme in mouse kidney (Bardin el<br />
01. 1978).<br />
1.3 Effect of environmental contaminants on male reproductive system<br />
A large number of environmental contaminants have been shown to induce<br />
reproductive abnormalities both in wildlife and humans (Carlsen el al., 1992; Shape,<br />
1993). Mean sperm counts in men have been shown to decline in the past fifty years<br />
(Carlsen el 01.. 1992). It has been hypothesized that environmental exposure to<br />
synthetic estrogenic chem~cals nnd related endocrine octivc compounds may bc<br />
rcsponsible for a global decrease in spcrnm counts and decreased male rcproduclivc<br />
capacity (Fisher el at., 1999; Safe. 2000). The organochlorine compounds comprise<br />
dichlorodiphenyl-trichloroethane (DD'T) and metabolites. hcxacl~lorocyclol~exill~c<br />
(HCH). polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs)<br />
and polychlorinated dibcnzo-p-dioxins (PCDDs). Due to their lipophilicity they<br />
preferentially bio-accumulate in the adipose. tissue. The presence of' DI)T and
metabolites. HCH, PCBs and PCDDs has been reported in the fluids of human<br />
reproductive tract, such as seminal fluid, cervical niucus and I'olliculi~r fluid (li~r<br />
review, see Pflieger-Bruss and Schill, 2000).<br />
Changes in male reproduction of<br />
wildlife by environmental contaminants involve feminization, demasculinization.<br />
reduced fertility, reduced hatchability, reduced viability of the offspring, impaired<br />
hormone secretion or activity and altered sexual behavior (for review, see Colborn et<br />
al.. 1993).<br />
A large number of chemicals, mainly pesticides, currently being used and<br />
industrial waste have been implicated as environmental contaminants possessing<br />
estrogenic andl or antiandrogenic properties (for review, see Colborn et al., 1993;<br />
Gray er 01.. 2001).<br />
Long-term exposure to small amounts of organochlorine<br />
contaminants leads to accumulation of considerable burdens in animal and human<br />
tissues (Calabrese, 1982). Neonatal exposure of rats to methoxychlor or DDf has not<br />
been shown to affect male reproductive organ weights in adulthood (for review, see<br />
Gray el a/., 2001). Male mice exposed to methoxychlor or DDT neither induced<br />
epididymal cysts which are found frequently in mice exposed to synthetic estrogen<br />
diethylstilbestrol (McLachlan, 1989). However, exposure throughout gestation and<br />
lactat~on to methoxychlor in rodents resulted in slightly smaller testes and<br />
epididymides and in lowered sperm counts in male offspring than in controls (Gray el<br />
01.. 1989; Gray er ol., 1992). Chlordane has been shown to disturb spermatogenesis<br />
and caused dose-related damage to the testes of mice. A fungicide vinclozolin has<br />
been shown to disrupt sexual differentiation of male rats (Gray rr ul., 1994) while ye1<br />
another fungicide benomyl caused a reduction in the number of spermatocytes and<br />
produced multinucleated germ cells (Hcss cr ul.. 1991). Endosul'nn at thc dosc 01' I<br />
mg/ Kg body weighcl day and lindane at the dose of 5 mL?/ Kg body weight have been<br />
shown lo alter the weights of the testis and accessory sex organs in malo rats (('hitrn<br />
er 01.. 1999; Sujatha el a/., 2001; Chitra er a/., 2001). Administration of methoxychlor<br />
in adult rats has been shown to decrease weights of the accessory sex organs.<br />
epididyn~sl sperm counts and motility (Latchoumycandane el ul.. 2002a). Induction<br />
of oxidative stress in testis. epididymal sperm ind various regions of the epididymis
have also been reported in the rats administered with estrogenic organochlori~lc<br />
pesticides, lindane and methoxychlor (Sujatha e! a[., 2001: Chitra el al., 2001;<br />
Latchoumycandane et al., 2002a. b). Methoxychlor has been reported to induce<br />
oxidative stress in mitochondrial and microsome-rich fractions of rat testis<br />
(Latchoumycandane and Mathur, 2002).<br />
1.3.1 23,7,8-Tctmchlorodibenzo-pain (TCDD)<br />
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), prototypical member of<br />
halogenated aromatic hydrocarbons, is one of the polychlorinated dibenzo-p-dioxins<br />
which arc formed as by-products in a number of industrial processes and during<br />
combustion of indusmal waste. TCDD has been identified as an environmental<br />
contaminant in the production of chlorinated phenols and their derived products as<br />
well as other chlorinated industrial compounds. These compounds have also been<br />
detected as by-products in sewage sludge, pulp and paper mill effluents. in diverse<br />
high-temperature industrial processes, and by-products of incineration of diverse<br />
organic materials including wood and coal. TCDD is stable to both chemical and<br />
thermal degradation and this stability contributes to its persistence in the environment.<br />
This compound is also lipophilic and environmental residues preferentially<br />
bioaccumulate in the food chain and is routinely detected in fish, wildlife. human<br />
adipose tissue, serum and milk (Rappe, 1991). The widespread human exposure and<br />
environmental contamination of TCDD has spurred research on the biochemical and<br />
toxic responses and mechanism of action elicited in animal models, mammalian cells<br />
in culture and in exposed human populations.<br />
TCDD is one of the most toxic members of polychlorinated dibenzo-p-dioxins<br />
and has been extensively used as a prototype to investigate various activities of thesc<br />
compounds and their m~rhanism 01' action (1.ucic.r o (11.. 1093; Sak. IOVS). I'hc<br />
diverse spectmm of biochemical and toxic responses elicited by TCDD and related<br />
compounds include a wasting syndrome. immunosuppression. hepatotoxicity and<br />
porphyria, carcinogenic and anticarcinoge-dc activities, hypo- and hyperplastic
clrects. reproductive and developmental toxicity, tumour production i~ctivity.<br />
neurotoxicity, chloracne and other dermal effects and the disruption of several<br />
endocrine-response pathways (Safe, 2001). TCDD has been shown to inhibit<br />
estradiol induced responses (Safe, 1995). uterine wet weight increase, peroxidase<br />
activity, progesterone receptor binding, epidermal growth factor receptor binding<br />
(Safe c.1 01.. 1998). TCDD has also been reported to induce drug-metabolizing<br />
enzymes, modulate expression of many other genest gene products (Safe, 2001).<br />
TCDD has been shown to increase the respiratory tract cancer in female but not in<br />
male rats (Kociba er al.. 1976).<br />
1.3.1.1 Effect of TCDD on various organs<br />
TCDD has been reported to alter a variety of organ systems in different<br />
species. Loss of body weight is a characteristic sign observed in most animals given a<br />
sub-lethal dose of TCDD. The weight loss usually manifests itself within a few days<br />
after exposure and results in a substantial reduction of the adipose (Peterson el ol.,<br />
1984) and muscle tissues (Max and Silbergeld, 1987). With sub-lethal doses of<br />
TCDD, a dose-dependent decrease in body weight gain occurs (Kociba el ul.. 1976).<br />
The greatest species-specific differences in toxicity concern pathological<br />
alterations in the liver of the TCDD treated rats. Lethal doses to guinea pigs do not<br />
result in liver damage, which is comparable to the liver lesions described in rabbits<br />
and rats or to liver changes observed in mice (McCOM~~I el a/.. 1978; Moore rr ul..<br />
1989; Turner and Collins, 1983). Thymic atrophy has been found in all animal<br />
species given lethal doses of TCDD. Treatment of animals with TCDD inhihits the<br />
bone marrow hemopoiesis in mice, both in vrvo and in virro, by directly altering the<br />
colony growth efficiency of stem cells (Chastain and Pazdernik. 1985; I.ustcr cr 111..<br />
I V85). Among other signs and symptoms hepitic porphyrio. licmorrliagcs in vurious<br />
organs. lesticular atrophy, reduced prostate weight, reduced uterine weight. increased<br />
thyroid weight. lesions of the adreliul glands. inhibited bonc marrow hc~ni~topoi~sis.<br />
decreased serum albumin and increased serum triglycerides and free €any acids.
Effect of TCDD on heart muscle has also been reported in guinea pigs and rats<br />
(Brewster et al., 1987; Canga et al., 1988).<br />
1.3.1.2 Effect of TCDD on male reproduction<br />
TCDD decreases testis and accessory sex organ weights, causes abnormal<br />
testicular morphology, decreases spennatogenesis and reduces fertility when given to<br />
adult animals in doses sufficient to reduce feed intake andl or body weight. Some of<br />
the effects have been reported in chicken, rhesus monkeys, rats, guinea pigs, and mice<br />
treated with overtly toxic doses of TCDD (Allen and Lalich, 1962; Khera and<br />
Ruddick, 1973; Kociba er al., 1976; Chahoud er al., 1989; Moore et al., 1989).<br />
Impaired division and/ or increased attrition of germ cells during the conversion of<br />
leptotene spermatocytes to spermatowa and/ or by a concomitant reduction in Sertoli<br />
cell number have been reported in the rats treated with TCDD (Mably et al., 1992).<br />
Effects of TCDD on spermatogenesis are characterized by loss of germ cells, the<br />
appearance of degenerating spermatocytes and immature spermatozoa within the<br />
lumen of the seminiferous tubules and a reduction in the number of tubules containing<br />
mature spermatozoa in various species (Allen and Lalich, 1962; Chahoud rr a/.,<br />
1989).<br />
Prenatal exposure to TCDD has been shown to interfere with fetal<br />
development at doses lower than those causing overt toxicity in adult animals.<br />
Exposure to TCDD during development produces alterations in the reproductive<br />
systems of the developing pups, delayed puberty and reduced sperm counts in males<br />
and malformations in the external genitalia of females (Hurst rr al.. 2000). In urero<br />
and lactational exposure of male rats to TCDD profoundly altered ontoyeny of the<br />
reproductive system and caused reduction in the anogenital distance, delayed testis<br />
descent and reduction in the weights of the testis, epididymis and accessory sex<br />
organs throughout sexual development. The number of sperm in the epididymis gets<br />
reduced in the TCDD treated rats (Faqi er al., 1997). Effects of TCDD on the male<br />
reproductive system are believed to be due to an androgenic deficiency (Gray el ul.,
1997). This deficiency is characterized in adult rats by decreased plasma testostcronc<br />
and DHT concentrations, unaltered plasma LH concentrations and unchanged plasn~a<br />
clearance of androgens and LH (Moore er al., 1985; Moore el ul., 1989). The doserelated<br />
reduction in plasma testosterone and DHT concentrations along with the<br />
weights of the seminal vesicles and ventral prostate weights in rats has been reported<br />
(Moore er a/., 1985). In contrast, TCDD did not affect accessory sex organ weights in<br />
castrated adult rats implanted with either testosterone or DHT containing capsules<br />
(Bookstaff er al., 1990). Trophic responsiveness of the seminal vesicles and ventral<br />
prostate to testosterone and DHT has been shown to remain unaffected by<br />
postpubertal TCDD treatment TCDD could increase responsiveness of the pituitary to<br />
these androgens without affecting the responsiveness of the accessory sex organs<br />
(Gray er al., 1997). TCDD has been shown to increase epididyrnal transit rate of<br />
sperm through the cauda epididymis of rat primarily due to decreased transit rate ol'<br />
sperm through the caput and corpus epididymis (Wilker er al., 1996).<br />
1.3.1.3 Mechanism of TCDD action<br />
TCDD has been shown to act through aryl hydrocarbon receptor (AhR).<br />
AhR has been shown to modulate the expression of a number of phase I and phase I1<br />
enzymes responsible for detoxification1 bioactivation of a variety of lipophilic<br />
compounds (Hankinson, 1995). This modulation has been shown to be accomplished<br />
through ligand AhR complex interactions with specific DNA response elements called<br />
dioxin-response elements or xenobiotic-response elements (Safe, 1995). A number of<br />
genes, which are involved in the oxidative metabolism, havc been shown to be<br />
induced by the AhR including CYPIA1, CYPl A2 and CYPl Bl (Spink el 01.. 1990;<br />
Spink ef al., 1994; Safe. 1995; Hankinson. 1995). TCDD has been shown to induce<br />
the production of reactive oxygen species thereby causing oxidative stress in multiple<br />
tissucs through AhR activation or AhR mediated xanthine oxidascl santhinc<br />
dehydrogenase activity (Stohs, 1990: Safe, 2001; Sugihara o 01.. 2001). Thc<br />
mechanism of TCDD mediated reactive oxygen species production has been proposed<br />
to involve cytochrome P450s (Park el ol., 1996;.
1.4 Effect of oxidative stress on mvlc reproduction<br />
Reactive oxygen species (ROS) have been shown as an important part of thc<br />
defense mechanisms against infection, but excessive generation of free radicals have<br />
been shown to damage tissues (Garg and Bansal, 2000). Reactive oxygen and<br />
reactive nitrogen species have been shown to play a significant role in male<br />
reproduction (for review, see Sikka, 2001). ROS have been shown to be formed in<br />
both physiological and pathological conditions in human spermatozoa due to their<br />
high reactivity they may interact with biomolecules inducing oxidative stress<br />
(Ichikawa el 01.. 1999). which severely impair male reproduction as indicated by<br />
reduction in fertility (for review, see Sikka, 2001). In the male genital tract,<br />
spermatozoa and leukocytes including neutrophils and macrophages have been shown<br />
to generate ROS which involved in the regulation of sperm function. Excessive<br />
production of ROS has been shown to cause oxidative stress in sperm (Ochsendorf,<br />
1999).<br />
Induction of the activities of intracellular oxidases in the testicular tissue or<br />
spermatozoa have been shown to result in the production of nanomolar levels of<br />
superoxide anion resulting in direct interaction with signalling mechanisms (Aitken<br />
er 01.. 1989). Semen samples from infertile patients have been shown to contain high<br />
superoxide anion generation. Prolonged inhibition of sperm mitochondria1 function<br />
by the ROS have been suggested as the cause of inhibition of sperm motility (Aitken<br />
er 01.. 1991; Kessopoulou er ol., 1992). Sperm viability could be improved by<br />
addition of catalase to the medium, implying that in their steady state these cells<br />
generate hydrogen peroxide, which can be deleterious to their survival (Mac Leod.<br />
1943).<br />
t-lydropen peroxide has been shown to k n stnhlr rcnctlvc oxygen sprcics with<br />
high biological diffusion properties and has been reported to transvrrsc thc plasma<br />
and nuclear membranes causing DNA adduct formation (for review. see Sikka. 2001).<br />
Peroxide derived ROS has been shown to react with transition metals such as iron<br />
present in biological systems and affect signaling or tissue injury processes (Peltola el
01.. 1996). Expression of extracellular superoxidc dismutasc has bccn rcportcd in the<br />
Sertoli cells of rat testis (Mruk and Cheng, 2000b). Mitochondria1 respiration has<br />
been shown to be the main biological source of superoxide anion radicals under<br />
physiological conditions (for review, see Chance er a/. 1979).<br />
Antioxidants have been described as substances that either directly or<br />
indirectly protect cells against adverse effects of xenobiotics, drugs, carcinogens and<br />
toxic radicals reactions (Halliwell, 1995). Vitamin C (ascorbic acid), vitamin E (a<br />
tocopherol), vitamin A, p-carotene, glutathione, superoxide dismutases, catalase,<br />
glutathione peroxidasel reductase, nitric oxide synthase have been classified as<br />
antioxidants (Krishna er a/.. 1996; Halliwell, 1999). The antioxidant properties of<br />
ascorbic acid have been shown to enable it to protect tissues from reactive oxygen<br />
species (Luck ef a/., 1995). Ascorbic acid has been shown to scavenge free radicals<br />
caused as a result of environmental pollution and cellular metabolism (Dawson er 01..<br />
1990). Vitamin E has been shown to function as a chain breaking antioxidant in the<br />
cell membrane and plasma lipoproteins (Halliwell. 1999). Lipid peroxidation has<br />
been shown to be initiated by hydroxy- (OH). alkoxy- (RO), and peroxy- (ROO)<br />
radicals (Tramer el al., 1998). Cell membranes, which are structurally made up of<br />
large amounts of PUFA has been shown to be highly susceptible to oxidative attack<br />
and consequently changes in membrane fluidity, permeability. and cellular metabolic<br />
functions (Lenzi el 01.. 1994). Co-administration of vitamin E with mercuric chloride<br />
has been shown to prevent the reduction of epididymal sperm counts, motility and<br />
morphology as well as succinic dehydrogenase, ATPase, sialic acid. ascorbic acid and<br />
glutathione (Rao and Sharrna 2001).
2 SCOPE <strong>OF</strong> THE PRESENT STUDY<br />
There has been increasing awareness of the possible effects of environmental<br />
contaminants on male reproduction.<br />
Several studies have shown that male<br />
reproductive system is getting deteriorated during the last Tew decades resulting in<br />
reduced sperm counts and increased testicular and prostate cancer in men. In the<br />
present studies the effects of one of the most potent environmental contaminants,<br />
2,3,7,8-tetrachlorodibe11~0-p-dioxin (TCDD), were studied on male reproductive<br />
functions using rat as an experimental model. TCDD is formed as an unwanted by-<br />
product in the manufacture of chlorinated hydrocarbons and has also been shown to<br />
be formed in incineration processes, paper and pulp bleachings, emission from steel<br />
foundries and motor vehicles. The lipophilicity and low rate of metabolism leads to<br />
its accumulation and persistence in adipose tissues. Involvement of aryl hydrocarbon<br />
receptor has been proposed to explain for the toxicity of TCDD and its congeners.<br />
However. the nature and mechanism of action of TCDD on male reproduction<br />
remains unclear. Recently many environmental contaminants have been reported lo<br />
disturb the prooxidant and antioxidant balance of the cells by generating reactive<br />
oxygen species and oxygen free radicals thereby inducing oxidative stress. which has<br />
been shown to impair male reproduction. The present studies were undertaken to<br />
evaluate the effect of TCDD on rhe antioxidant system of testis and epididymis of<br />
rats. Funher. w-administration of antioxidant such as vitamin E was evaluated for its<br />
protective effect against TCDD induced toxicity.<br />
In the present studies the body weight of the rats were recorded to monitor the<br />
general health status and weight of the testis along with daily sperm production.<br />
dianleters of seminiferous tubules iu~d lumen werc taken to assess maintenance ot'<br />
testicular functions of rats. The weights of epididymis, seminal vesicles and ventral<br />
prostate along with kidney were taken to assess the bioavailability and/ or production
of androgen and the cumulative effect of androgenic activity. Epididymal sperm<br />
count, viability and motility were studied in order to assess the epididymal function.<br />
Serum levels of testosterone and estradiol were measured along with the<br />
activities of 3P-hydroxysteroid dehydro~enasc and 17P-hydroxystcroid<br />
dehydrogenax in testis in order to assess the status of testicular steroidogensis.<br />
Serum levels of FSH, LH and prolactin were determined to evaluate the hormonal<br />
status after TCDD exposure.<br />
Production of superoxide anion, nitric oxide and hydrogen peroxide were<br />
determined in testis as well as in the epididymal sperm and epididymis in order to<br />
assess the levels of reactive oxygen species in tissues. Determination of the levels of<br />
glutathione, a-tocopherol, ascorbic acid and the activities of superoxide dismutase,<br />
catalase, glutathione rcductase and glutathione peroxidase in testis, epididymal sperm<br />
and epididymis were determined to assess the antioxidant system. Co-administration<br />
of TCDD and vitamin E was done in order to assess if vitamin E provides any<br />
protective effects against TCDD-induced toxicity.
3 MATERIALS <strong>AND</strong> METHODS<br />
3.1 Animals<br />
Male rats of Wistar strain obtained from the Central Animal House, Jawaharlal<br />
Institute of Postgraduate Medical Education and Research (JIPMER), <strong>Pondicherry</strong><br />
were used in the study.<br />
3.2 Maintenance<br />
Animals were housed in polypropylene cages ( l8x 10"x 8") lined with paddy<br />
husk. under a well regulated light and dark (l2h: 12h) scheduled. Rats were fed with<br />
pelleted food (Kamedhenu Agencies. Bangalore, India) and water ad libitum. Growth<br />
of'the animals was monitored regularly and animals displaying poor growth rate were<br />
discarded from the experiments.<br />
3.3 Chemicals<br />
2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. Stefen Safe,<br />
Department of Veterinary Anatomy and Public Health. A & M <strong>University</strong>, Texas.<br />
USA. Trichloroacetic acid, dehydrocpiandrosterone and androsterone 3, 17-dione<br />
were purchased from Sigma Chemical Company, MO, USA. Thiobarbituric acid and<br />
malondialdehyde were obtained from E-Merck, Germany.<br />
For hormone assays.<br />
ELSA kits were obtained from Diagnostic Systems Laboratories, Inc. Webster,<br />
Texas, USA. Ham's F12 medium, vitamin E (DL-tocopherol, 99%), horseradish<br />
peroxidase (HRP), niwtinamide adenine dinucleotide (NAD), nicotinamide adenine<br />
dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate<br />
WADP), bovine =rum albumin (BSA), deoxyribonucleic acid (DNA). ribonucleic<br />
acid (RNA) and 2,6dichlorophenol indophenol (DCPIP) were obtained from Himedia<br />
Laboratory Pvt. Ltd., Mumbai, India. Nicotinamide adenine dinucleotide phosphate<br />
reduced (NADPH) and N-(I-naphthyl) ethylene diamine di-hydrochloride were
obtained from Sisco Research Laboratories Pvt. Ltd.. Mumbai, India. Glutathione<br />
reduced and glutathione oxidized were obtained from Sd. fine Chem Ltd. Mumbai.<br />
All other chemicals were of analytical grade and obtained from local commercial<br />
sources.<br />
3.4 Handling of TCDD and animals<br />
All materials contaminated with TCDD were treated as hazardous waste.<br />
Gloves, goggles. mask and laboratory coats were used while handling the powdered<br />
material and subsequent solutions. The animals were maintained and handled<br />
according to the guidelines from the Indian Council of Medical Research, New Delhi.<br />
3.5 Treatments<br />
The experiments were carried out in pubertal (postnatal days 45) male rats. In<br />
each experiments, corresponding groups of control animals, treated with vehicle alone<br />
were maintained and killed along with the treated rats. The treatments to various<br />
groups of rats are described below.<br />
3.5.1 Group I: Admhlatration of TCDD to pubertal rats<br />
TCDD was dissolved in acetone and olive oil (see Appendix 1.1) in the ratio<br />
of 1 :I9 and administered orally for 45 days. For the administration of TCDD, the<br />
pipette tip was gently placed just inside the mouth and the dosing solution was then<br />
carefully expelled from the tip allowing the animal to lick the compound from the tip.<br />
Corresponding groups of animals were administered with equal volume of vehicle<br />
alone and served as control.<br />
Subgroup I : Administration of TCDD at the dose of 1 nd Kg body weight/<br />
day.<br />
Subgroup I1 : Adminiseation of TCDD at the dose of 10 ng/ Kg body weight/<br />
by.
Subgroup III: Administration of TCDD at the dose of 100 ng/ Kg body<br />
weight1 day.<br />
3.5.2 Group 11: Administration of TCDD along with vitamin E<br />
Vitamin E was dissolved in olive oil (see Appendix 1 .I) at the dose level of 20<br />
mg/ Kg body weight and administered orally to different groups of rats along with<br />
various doses of TCDD. Co-administration of vitamin E and TCDD was done by the<br />
methods as described previously (see Section 3.4.1). Corresponding groups of control<br />
animals were administered with equal volume of vitamin E.<br />
Subgroup I : AdminisIration of vitamin E along with TCDD at the dose of 1<br />
ng/ Kg body weight/ day.<br />
Subgroup I1 : Administration of vitamin E along with TCDD at the dose of<br />
10 ng/ Kg body weight/ day.<br />
Subgroup 111: Administration of vilamin E along with TCDD at the dosc of<br />
100 ng/ Kg body weight/ day.<br />
3.6 Killing of animals<br />
AfIer 24 h of the last treatment the rats were weighed and killed using overdosage<br />
of anesthetic ether.<br />
3.7 Collection of serum<br />
Blood samples were collected in glass centrifuge tubes either by cardiac<br />
puncture method or decapitation and were kept at 4'C for 12 h and then centrifuged at<br />
ZOO0 g for 15 min. Serum samples werc collected and stored at - 20°C i111til IISC~.<br />
3.8 Collection of tissues<br />
Kidney. testes and accessory sex organs (seminal vesicles and ventral prostate)<br />
werc dissated out and washed in cold normal saline. The epididymides were<br />
dissected out and washed in normal saline at mom temperaturn.
3.9 Organ weights<br />
Kidney, testes, epididymides and accessory sex organs wcre clcarcd from thc<br />
adhering tissues and weighed to the nearest milligram on Ohaus Precision Standard<br />
electronic balance. Testes and epididymides were processed for various experiments<br />
and the details are given in the following sections.<br />
3.10 Daily sperm production<br />
Daily sperm production was determined in testis of rats by the method of<br />
Blazak rr a]. (1993). Testis was weighed, decapsulated and homogenized in 50 mL of<br />
ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100 using<br />
polytron homogenizer (Polytron PT 3000, Kinematica AG) with a speed of 5 for 60<br />
sec as was used by Sharpe er al. (1995). The homogenate was allowed to settle for 1<br />
min and then gently mixed and a 10 mL aliquot was collected into a glass vial and<br />
stored on ice. AAer thorough mixing of each sample, number of sperni heads were<br />
counted in four chambers of an improved Neubauer type hernocytometer. Number of<br />
sperm produced per gram of tenicular tissue per day were calculated according to the<br />
following formula: average count of sperm heads from four chambers x square factor<br />
(which is 5) x hemocytometer factor (which is 10') x dilution factor (which is 50)<br />
divided by testis weight (g) and the time (days) during spermatogenesis that these<br />
cells are resistant to homogenization (which is 4.61 days for rats according to Sharpe<br />
el a/. (1995). The daily sperm production was done in duplicate and the data were<br />
expressed as mean * SD for four d~fferent rats in each group.<br />
3.1 1 Collection of epididymal sperm and sperm function tests<br />
Epididymal sperm were collected by the method of Gray er al. (1989). The<br />
sperm collected from left epididymis were used for the determination of sperni<br />
viability. sperm motility and sperm counts. The sperm collected from right<br />
epididymis were used for biochemical assays. The epididyrnides collected from<br />
experimental and control rats were washed in normal saline. Epididyrnis was
choppcd with the help of sharp razor blude in 5 mL ol' Ilam's 1:12 medium and<br />
incubated for 5 min at 35'C. After several washings in the Ham's F12 medium.<br />
sperm collected in the medium were used for various experiments.<br />
3.11.1 Sperm viability test<br />
Sperm viability test was done by the method as described in the WHO<br />
Laboratory Manual (1999). An aliquot of 100 pL of epididymal sperm was mixed<br />
with I00 pL of 0.5% eosin solution. A drop ofthe ubovc mixturc wils put on a n1icl.oslide<br />
covered with a cover slip and examined after 30 sec at 200 x with a help of light<br />
microscope. Randomly two hundred spermatozoa were counted. The livc<br />
spermatozoa were unstained and dead cells were stained. The sperm viability was<br />
expressed in percentage as the number of viable sperm of the total sperm counted.<br />
3.11.2 Epididymal sperm motility<br />
Motility of epididymal sperm was evaluated by the method as described by<br />
Linder et a/. (1986) and Cooke el al. (1991). A small incision was made in the cauda<br />
epididymis using a sharp razor. The fluid, which oozed out from the cauda<br />
epididymis was taken by using a pipene tip and diiuted to 2 mL with Ham's F12<br />
medium at 3S°C. An aliquot of this solution was placed in a hemocytometer and<br />
counted for motile and non-motile sperm. First non-motile sperm were counted<br />
followed by motile sperm. Epididymal sperm motility was expressed as the<br />
percentage of motile sperm of the total sperm counted.<br />
3.1 1.3 Epididymal sperm counts<br />
Epididymal sperm were counted by the method as described in the WHO<br />
Laboratory Manual (1999). An aliquot of 5 111. of epididyn~al sperm was diluted with<br />
95 pL of diluents (see Appendix 1.2). A coverslip was placed on the counting<br />
chambers of the improved Neubauer type hemocytometer.
Approximately 10 FL of thoroughly mixed diluted specimen transferred to<br />
each of the counting chambers of the hemocytometer and allowed to stand for 5 min<br />
in a humid chamber to prevent drying out. Sperm cells sedimented during this time<br />
and were then counted with the help of light microscope at 200 x. Complete<br />
spermatowa, head with tail. were counted.<br />
3.1 2 Histometric studies<br />
The testicular tissue was fixed in Bouin's fixative immediately after isolation.<br />
After dehydration in alcoholic series and cleaning in xylol, the tissue were embedded<br />
in paraffin wax. Sectiow were made with 5 pn thickness. The sections were stained<br />
with hematoxylin-eosin and examined under a light microscope.<br />
3.12.1 Tubular nod lumen diameter measurements<br />
The diameter of the seminiferous tubule and lumen were measured with<br />
eyepiece graticules that have been calibrated with a stage micrometer. Only tubules<br />
that appeared round were considered for tubular and lumen diameter measurements.<br />
3.13 Quantitative determination of serum hormone levels<br />
Serum levels of follicle stimulating hormone (FSH), luteiniziny hormone<br />
(LH), prolactin. testosterone and esrradiol were determined by enzyme linked<br />
imrnunosorbant assay (ELISA) using kits from Diagnostic Systems Laboratories, Inc.<br />
Webstcr, Texas, USA. The assays were done strictly according to the procedure<br />
given along with the kits<br />
3.13.1 Quantitative determination of serum FSH<br />
Serum levels of FSH were determined strictly according to the procedure<br />
given along with kit. In principle FSH ELlSA kit is an enzymatically amplified 'twostep'<br />
sandwich-type immunoassay. In the assay, standards. controls and experimental<br />
serum samples are incubated in microtitration wells, which have been coated with
anti-FSH antibody. After incubation and washing, the wells are treated with another<br />
anti-FSH detection antibody labeled with the enzyme horseradish peroxidase (HRP).<br />
After a second incubation and washing step, the wells are incubated with the substrate<br />
tetramcthylbenzidine (TMB).<br />
An acidic stopping solution is then added and the<br />
degree of enzymatic turnover of the substrate is determined by absorbance<br />
measurement at 450 nm. The absorbance measured is directly proportional to the<br />
concentration of FSH present in the serum samples. A set of FSH standards is used to<br />
plot a standard curve of absorbance versus FSH concentration from which FSH<br />
concentrations in the experimental serum samples can be calculated.<br />
Before starting the assay all the reagents and serum samples were brought to<br />
room temperature and were thoroughly mixed.<br />
The assay consisted of standards<br />
containing concentrations of approximately 0, 1.5, 4.5, 15.45 and 150 mIU/ mL FSH<br />
(see Appendix 1.3), and controls containing low (Level 1) and high (Level 11)<br />
concentrations of FSH and experimental serum samples.<br />
All the assays were<br />
performed in duplicate and internal and external quality controls were maintained.<br />
The microtitration strips were marked and 100 pL of the standard. controls<br />
and experimental serum samples were added to the appropriate wells and incubated<br />
while shaking at a fast speed (500 to 700 rpm) on an orbital microplate shaker (P-<br />
Multi~ope ELISA Reader) for 60 min at room temperature.<br />
Antibody-Enzyme<br />
Conjugate solution (100 pL) was added to each well and incubated while shaking at a<br />
fast speed on an orbital microplate shaker for 30 min at room temperature. The wells<br />
were aspirated and washed five times with the wash solution using an automatic<br />
microplate washer (Labsystems Well Wash 4). The microtitration plates were dried<br />
by invening on absorbent material. TMB chromogen solution (100 pL) was added to<br />
each well and incubated while shaking at a fast speed (500 - 700 rpm) on an orbital<br />
microplate shaker for 10 min at room temperature. Stopping solution, 0.2 M sulfuric<br />
acid, (100 a) was added to each well and absorbance of the solution in the wells was<br />
read within 30 min using a microplate reader (P-Multiscope ELISA Reader) set at 450<br />
nm. Mean absorbances for standard, control and experimental serum samples were
calculated. Using a linear-linear gtaph paper mean absorbance readings were plotted<br />
Tor each of the standards along the y-axis versus the FSH concentrations in mllJ1 ml,<br />
along the x-axis. The best fitting curve through the mean of the duplicate points was<br />
drawn. The FSH concentrations of the controls and experimental serum samples were<br />
determined 'om the standard curve by matching their mean absorbance readings with<br />
the corresponding FSH concentrations.<br />
3.13.2 Quantitative determination of serum LH<br />
Serum levels of LH were determined strictly according to the procedure given<br />
along with kit.<br />
sandwich-type immunoassay.<br />
In principle LH ELISA is an enzymatically amplified 'one step'<br />
In the assay, standards, controls and experimental<br />
serum samples are incubated with anti-LH antibody in microtitration wells, which<br />
have been coated with another anti-LH antibody. After incubation and washing, the<br />
wells are incubated with the substrate tetramethylbenzidine (TMB).<br />
An acidic<br />
stopping solution is then added and the degree of enzymatic turnover of the substrate<br />
is determined by wavelength absorbance measurement at 450 nm. The absorbance<br />
measured is directly proportional to the concentration of LH present in the serum. A<br />
set of LH standards is used to plot a standard curve of absorbance versus LH<br />
concentration from which the LH concentrations in the experimental serum sanlples<br />
can be calculated.<br />
Before starting the assay all the reagents and experimental serum samples<br />
were brought to the mom temperature and were thoroughly mixed.<br />
The assay<br />
consisted of standards containing LH concentrations of approximately 1, 3, 10, 30 and<br />
100 mlU1 mL [see Appendix 1.4) and controls containing low (Level 1) and high<br />
(Level 11) concentrations of LH and experimental serum samples. All the assays were<br />
pcrlbr~i~cd in duplicutc and inlcrnal and cx~crn:~l quality controls wcrc mai~ilai~iuil<br />
The microtitration strips were marked and 50 pL of the standard, controls and<br />
experimental serum samples were added to the appropriate wells. Antibody-Enzyme<br />
Conjugate solution (100 pL.) was then added to each well and incubated. shaking at a
lust sped (500 to 700 rpm) on an orbital microplate shakcr Ibr LJO min at room<br />
temperature. The wells were aspirated and washed five times with the wash solution<br />
using an automatic microplate washer.<br />
The microtitration plates were dried by<br />
inverting plate on absorbent material. TMB chromogen solution (100 pL) was added<br />
to each well and incubated. shaking at a fast speed on an orbital microplate shaker, for<br />
10 min at room temperature. Stopping solution, 0.2 M sulfuric acid, (100 pL) was<br />
added to each well and absorbance of the solution in the wells was read within 30 min<br />
using a microplate reader set at 450 nm.<br />
Mean absorbances for standard, control and experimental serum samples were<br />
calculated. Using a linear-linear graph paper mean absorbance readings were plotted<br />
for each of the LH concentrations in mIU/ rnL in x-axis versus along the LH standards<br />
in y-axis. The best fitting curve through the mean of the duplicate points were drawn.<br />
The LH concentrations of the controls and experimental serum samples were<br />
determined from the standard curve by matching their mean absorbance readings with<br />
the corresponding LH concentrations.<br />
3.13.3 Quantitative determination of serum prolactin<br />
Serum levels of prolactin were determined strictly according to the procedure<br />
given along with kit. In principle prolactin ELlSA is an enzymatically amplified<br />
'two-step' sandwich-type immunoassay. In the assay, standards, controls and<br />
experimental serum samples are incubated in microtitration wells, which have been<br />
coated with anti-Prolactin antibody. AAer incubation and washing, the wells are<br />
treated with another anti-Prolactin detection antibody labeled with the enzyme<br />
hotseradish peroxide (HRP). After a second incubation and washing step, the wells<br />
are incubated with the substrate tetrarnethylbcnzidine (TMB). An acidic stopping<br />
solution is then added and degree of enzymatic turnover of the substrate is determined<br />
by the wavelength absorbance measurement at 450 nm. The absorbance measured is<br />
directly proportional to the concentration of prolactin present in the serum. A set of<br />
prolactin standards is used to plot a standard curve of absorbance versus prolactin
concentration from which the prolactin concentrations in the expcrinicntal serum<br />
samples can be calculated. Before starting the assay all the rcagents and cxpcrinicntnl<br />
serum samples were brought to room temperature and were thoroughly mixed. The<br />
assay consists of standards containing 0, 2, 6. 20. 60 and 180 ng prolactin1 mL (see<br />
Appendix 1.5) and controls containing low (Level I) and high (Level 11)<br />
concentrations of prolactin and experimental serum samples. All the assays were<br />
performed in duplicate and internal and external quality controls were maintained.<br />
The microtitmtion strips were marked and 25 pL of the standard, controls and<br />
unknowns were added to the appropriate wells. Added 100 pL of assay buffer and<br />
the wells were incubated while shaking at a fast speed (500 to 700 rpm) on an orbital<br />
microplate shaker for 60 min at room temperature. The wells were aspirated and<br />
washed five times with the wash solution using an automatic microplate washer. The<br />
microtitre plates were dried by inverting the plate on absorbent material. Antibody-<br />
Enzyme Conjugate solution (100 pL) was added to each well. The wells were<br />
incubated while shaking at a fast speed on an orbital microplate shaker for 60 min at<br />
room temperature. The wells were aspirated and washed five times with the wash<br />
solution using an automatic microplate washer and then dried on absorbent material.<br />
TMB chromogcn solution (100 pL) was added to each well. The wells were<br />
~ncubated while shaking at a fast speed on an orbital microplate shaker, for 10 min at<br />
room temperature. Stopping solution, 0.2 M sulfuric acid. (100 pL) was added to<br />
each well and absorbance of the solution in the wells was read within 30 niin using a<br />
microplate reader set at 450 nm. Mean absorbances for standard, control and<br />
experimental xrum samples were calculated. Using a linear-linear graph paper. nican<br />
absorbance read~ngs were plotted Ibr each ofthc standards along the y-axis versus the<br />
prolactin concentrations in ng/ mL along the x-axis. The best-fitting curve through<br />
the mean of the duplicate poinu was drawn. The prolactin concentrations of the<br />
controls and experimental serum samples were determined from the standard curve by<br />
matching their mean absorbance readings with the corresponding prolactin<br />
concentrations.
3.13.4 Quantitative determinntiun uf serum testustcrunc<br />
The serum levels of testosterone were determined strictly according to the<br />
procedure given along with kit. In principle testosterone ELlSA follows the basic<br />
principle of enzyme immunoassay where there is competition between an unlabeled<br />
antigen and enzyme-labeled antigen bound to the antibody binding sites. The amount<br />
of enzyme-labeled antigen bound to the antibody is inversely proportional to the<br />
concentration of the unlabeled analyte present. Unbound materials are removed by<br />
decanting and washing the wells. The absorbance measured is inversely proportional<br />
to the concentration of testosterone present in the serwn. A set of testosterone<br />
standards is used to plot a standard curve absorbance versus testosterone<br />
concentration from which the testosterone concentrations in the experimental serum<br />
samples can be calculated.<br />
Before starting the assay all the reagents and experimental serum samples<br />
were brought to room temperature and were thoroughly mixed. The assay consists of<br />
standards containing 0, 0.1, 0.5, 2.5, 5, 10 and 25 ng testosterone1 mL (see Appendix<br />
1.6) and controls containing low (Level I) and high (Level 11) concentrations of<br />
testosterone and experimental serum samples. All the assays were performed in<br />
duplicate and internal and external quality controls were maintained. The<br />
microtitration strips were marked and 50 pL of the standard, controls and<br />
experimental serum samples were added to the appropriate wells. Enzyme Conjugate<br />
solution (100 pL) and Testosterone-Antiserum (100 bL) were added to each well.<br />
The wells were covered and incubated while shaking at a fast speed on an orbital<br />
microplate shaker for 60 min at room temperature. The wells were aspirated and<br />
washed five times with the wash solution using an automatic microplate washer. The<br />
microtitre plates were dried by inverting on absorbent material. TMB chromogen<br />
solution (100 &) was added to each well. The wells were incubated while shaking at<br />
a fast speed on an orbital microplate shaker for 10 min at room temperature. Stopping<br />
solution. 0.2 M sulfuric acid. (100 pL) was added to each well and absorhance of the<br />
solution in the wells was read within 30 min using a microplate reader set at 450 nm.
Mean absorbances for each standard, control and experimental serum samples<br />
were calculated. Using a linear-linear graph paper mean absorbance readings were<br />
plottcd fnr each of the standards along the y-axis versus the testosterone<br />
concentrations in ny/ mL along the x-axis. The best-fitting curve through the mean of<br />
the duplicate points was drawn. The testosterone concentrations of the controls and<br />
experimental serum samples were determined from the standard curve by matching<br />
their mean absorbance readings with the corresponding testosterone concentrations.<br />
3.13.5 Quantitative determination of serum estradiol<br />
The serum levels of estradiol were determined strictly according to the<br />
procedure given along with kit.<br />
binding enzyme immunoassay format.<br />
In principle estradiol ELISA is the competitive<br />
In the assay, standards, controls and<br />
cspcr~~ncntal serum samples are incubated with biotin-labeled estradiol and rabbit<br />
ant]-estradlol antiserum in microtitration wells where the labeled and biotin-labeled<br />
antigens compete for a limited number of anti-estradiol binding sites.<br />
After<br />
~ncubation and washing, the wells are incubated with streptavidin-Horseradish<br />
perosidase (HRPO). which binds to the biotinylated estradiol.<br />
The unbound<br />
atreptavidin-HRPO is washed. followed by incubation with the substrate<br />
tetramethylbenzidine (TMB). An acidic stopping solution is then added and degree of<br />
enzymatic turnover of the substrate is determined by the wavelength absorbance<br />
measurement at 450 nm. Before starting the assay all the reagents and experimental<br />
serum samples were brought to room temperature and were thoroughly mixed. The<br />
assay consists of standards containing 0, 20. 50. 250. 750, 2000 and 6000 pg/ mL (see<br />
Appendix 1.7). controls containing low (level I) and high (Level 11) concentrations of<br />
estradiol. All the assays were performed in duplicate. The microtitration strips were<br />
marked and 50 pL of each standard, controls and experimental serum samples were<br />
addcd to the appropriate wells.<br />
Estradiol-Biotin Conjugate solution (100 PL) was<br />
added to each well. The wells were incubated while shaking at a fast speed on an<br />
orbital microplate shaker for 60 min at room temperatun. The wells were aspkred<br />
and washed five times with the wash solution using an automatic microplate washer.
The plates were dried by inverting on absorbent material. Streptavidin-Enzyme<br />
Conjugate Solution (200 pL) was added to each well and incubated while shaking at a<br />
fast speed on an orbital microplate shaker, for 30 min at room temperature. The wells<br />
were aspirated and washed five times with the wash solution using an automatic<br />
microplate washer. The plates were dried by inverting on the absorbent material.<br />
TMB chromogen solution (100 pL) was added to each well. The wells were<br />
incubated while shaking at a fast speed on an orbital microplate shaker, for 10 min at<br />
room temperature. Stopping solution, 0.2 M sulfuric acid, (100 pL) was added to<br />
each well and absorbance of the solution in the wells was read within 30 min using a<br />
microplate reader set at 450 mn. The mean absorbance for each standard, control and<br />
experimental senun samples were calculated. Using a linear-linear graph paper mean<br />
absorbance readings were plotted for each of the standards along the y-axis versus the<br />
estradiol concentrations in pg/ mL along the x-axis. The best-fitting curvc through the<br />
mean of the duplicate points was drawn. The estradiol concentrations of the controls<br />
and cxpcrimcntal serum samples wcrc determined Ikon1 tho standard cilrvc hy<br />
matching their mean absorbance readings with the corresponding estradiol<br />
concentrations.<br />
3.14 Activities of steroidogenic enzymes<br />
The activities of 3P-hydroxysteroid dehydrogenase and I7P-hydroxysteroid<br />
dehydrogenase were assayed in testis after necessary standardization experiments. A<br />
5% testicular homogenate was prepared in cold normal saline and centrifuged at 800 g<br />
for 20 min at 4°C. The supernatant was used for assaying steroidogenic enzymes in<br />
testis.<br />
3.14.1 Assay of 3p-bydroxysteroid dehydrogenase<br />
The activity of 3p-hydroxysteroid dehydrogenase (EC 1.1.1.5 1) was<br />
determined by the method as described by Bergmeyer (I 974). The reaction mixture<br />
contained 600 pL of 100 pM pyrophosphate buffer @H 9.0). 200 pL of 0.5 ph4
NAD. 100 pM dehydroepiandrosterone (see Appendix 1.8) and 2 rnL of distilled<br />
water.<br />
An aliquot of 100 pL enzyme extract was added and vortexed. Blank was<br />
prepared simultaneously by adding all the reagents except the enzyme extract.<br />
Absorbance was measured at 340 nm against blank at 20 sec intervals for 5 min on a<br />
Systronics Spectrophotometer (Systronics UV Spectrophotometer 119). The activity<br />
of enzyme was expressed as mole of NAD converted to NADHI mini rng protein.<br />
3.14.2 Assay of 17p-hydroxysteroid dehydrogenase<br />
The activity of I7p-hydroxysteroid dehydrogenase (EC 1.1.1.51) was<br />
determined by the method of Bergrneyer (1974). The reaction mixture contained 600<br />
pL of 100 pM pyrophosphate buffer (pH 9.0). 200 pL of 0.5 pM NADPH, 100 pL 0.8<br />
pM 4-androstene 3.17-dione (see Appendix 1.9) and 2 mL of distilled water.<br />
An aliquot of 100 pL enzyme extract was added and vortexed. Blank was<br />
prepared simultaneously by adding all the reagents except the enzyme extract.<br />
Absorbance was measured immediately at 340 nm against blank at 20 sec intervals for<br />
5 min on a Systronics Spectrophotometer. The activity of enzyme was expressed as<br />
nnlolc of NADPH converted to NADPI minl mg protein.<br />
3.15 Determination of nucleic acids and protein contents in testis<br />
I~c~crriiinntio~is of deoxyrihonuclcic acid (DNA). ribonucleic acid (RNA) and<br />
pr\>iclll \\CI.C curnerl uul using eslablishcd nrc\hods as described below.<br />
3.15.1 Extraction of nucleic acids fractions<br />
Extraction of nucleic acids from testicular homogenate was carried out by a<br />
modified method of Schneider (1957). Approximately. I00 mg of decapsulated testis<br />
W;IS<br />
wcighud and homogunized in normal saline with the help of glass tenon<br />
I1o1iioycnizcr. An illiquot of 4 ml. homoyenate was mixed with 2.5 ml. of 10%<br />
chilled Iricliloro;~cctic acid ('I'CA) and allowed to stand for 4OC for 60 min and wns
centrifuged at 3,500 g for I5 min on a Remi R8C Centrifuge. The supernatant was<br />
discarded and the pellet was resuspended in 2.5 mL of 10% TCA. It was then<br />
centrifuged at 3,500 g for 15 min. The supernatant was discarded and the pellet was<br />
resuspended in 5 mL of 95% ethanol. Extraction of lipids was completed with<br />
vortexing in 3:l ethanol ether mixture for 5 min and then centrifugation at 3,500 g for<br />
I5 min.<br />
The pellet was re-extracted with 5 mL of 95% ethanol. The supernatants were<br />
discarded. The pellet was resuspended in 3 mL of 5% perchloric acid (PCA) and was<br />
heated for I5 min in a hot water bath at 90°C. After cooling the tubes were<br />
centrifuged at 3.500 g for 15 min. The extraction of nucleic acids was completed by<br />
resuspending the pellet in 5% PCA and centrifugation at 3,500 g for 10 min. The<br />
supcrnalant fluids wcre pooled and were used for DNA and RNA determinations.<br />
The residual pellet was dissolved in 0.1 N sodium hydroxide after suitable dilution.<br />
3.15.2 Determination of DNA<br />
DNA was determined by diphenylamine colour reaction following the method<br />
of Burton (1956). The tubes containing 1 mL nucleic acid extract, 2 mL of IN<br />
perchloric acid and 2 mL of diphenylamine reagent (see Appendix 1 .lo) were kept in<br />
a boiling water bath for 20 min. The samples were cooled and the colour developed<br />
was read at 600 nm on a Systronics Spectrophotometer. A standard curve was<br />
prepared by using known concentrations of calf thymus DNA.<br />
3.15.3 Determination of RNA<br />
RNA was determined by orcinol reaction method (Schneider, 1957). The<br />
tubes containing 2 mL nucieic acid extract and 3 mL of orcinol reagent (see Appendix<br />
I. I I) wcre kept in a boiling water bath for 20 nlin. The samples were cooled and the<br />
colour developed was read at 665 nm on a Sysuonics Spectrophotometer. A standard<br />
curvc was prcparcd by using known concentrations of yeast RNA.
3.15.4 Determination of protein<br />
Protein contents were determined according to the method of Lowry el ul.<br />
(1951). The pellet obtained after the extraction of nucieic acid was dissolved in 10<br />
mL of 0.1 N sodium hydroxide solution (see Appendix 1.12). An aliquot of 0.1 mL of<br />
the extract was taken and was made up to I mL with distilled water. Alkaline copper<br />
reagent, 5 rnL, was added to the tubes containing protein extract and then vortexed,<br />
they were allowed to stand for 10 min at room temperature.<br />
Folin-Ciocalteau reagent IN was added to the above samples, vortexed and<br />
allowed to stand for 20 min in dark. The optical density was read at 610 nm in a<br />
Systronics Spectrophotometer. A standard calibration cwe was prepared using<br />
different concentrations of bovine serum albumin.<br />
3.16 Subcellular fractionation of testis<br />
Mitochondrial and microsome-rich fractions of testis were obtained by the<br />
method of differential centrifugation as described by Chainy e/ a/. (1997). Briefly, a<br />
20% (w/ V) homogenate was prepared in ice-cold 0.25 M sucrose solution (see<br />
Appendix 1.13) with the help of a motor-driven glass teflon homogenizer (Potter<br />
Elvehjem type, Rerni). In order to obtain nuclear pellet the homogenate was<br />
centrifuged at 1000 g for 10 min at 4°C.<br />
Mitochondrinl pellets was oblaincd by ccntril'uging post-nuclcar supcrn:it;lnt at<br />
10.000 g for 10 min at 4'C. Microsome-rich fraction was prepared by calcium<br />
chloride (CaCIz) sedimentation method (Kamath and Narayan. 1972). Postmitochondria1<br />
supernatant was diluted with ice-cold CaC12 (I M) so that the final<br />
concentration of CaC12 was 0.8 M. Incubated at 4°C was done for 10 min with<br />
occasional stirring. The sample was then centrifuged at 10,000 g for 10 rnin at 4°C<br />
and the microsome-rich pellet was obtained and dissolved in 0.25 M sucrose solution<br />
(I mg protein1 0. l mL).
3.17 Preparation of tiasue humogenates of cpididymia<br />
Epididymis was washed several times in normal saline in order to remove<br />
maximum number of sperm attached to the epididymal parenchyma. The caput,<br />
corpus and cauda regions of the epididymides were separated and homogenized<br />
separately in cold normal saline with the help of glass teflon homogenizer. The<br />
homogenates were centrifuged at 800 g for 20 min at 4°C. The supernatants were<br />
collected and used for various biochemical analyses.<br />
3.18 Preparation of tissue homogenate of kidney<br />
Kidney was homogenized in normal saline with the help of glass teflon<br />
homogenizer. The homogenate was centrifuged in a refrigerated centrifuge at the<br />
speed of 800 g for 20 min at 4'C. The supernatant was used for the biochemical<br />
analyses.<br />
3.19 Determination of free radicals/ reactive oxygen species in tissues<br />
Production of free radicals such as superoxide anion, nitric oxide and<br />
hydrogen peroxide were determined by the standard methods as described below.<br />
3.19.1 Determination ofsuperoxidc anion<br />
Superoxide anion production was measured by the method of Prodczasy and<br />
Wei (1988) which is based on the rrduction of iodonitrotetrazolium violet (INT). The<br />
reaction mixtures contained 750 pL of the tissue homogenate and equal volumes of<br />
4.9 mM INT, 0.3 mM EDTA and 0.92 mM sodium carbonate (see Appendix 1.14).<br />
The pH of the mixture was adjusted to 10.2. Blank was prepared simultaneously by<br />
adding all the reagents except the tissue extract. The reaction mixture was incubated<br />
for 15 min at room temperature. The reaction was terminated by placing the tubes in<br />
a boiling water bath for 1 min and then cooled to room temperature. After cooling<br />
absorbance was measured at 505 nrn against blank.
3.19.2 Determination of nitric oxide<br />
Production of nitric oxide was assayed by measuring the accumulation of<br />
nitrite/ nitrate in tissue using Griess reagent as described by Green el a/. (1982). The<br />
assay mixture contained I mL of tissue supernatant and I mL of Griess reagent (see<br />
Appcndix 1.15). After a I0 min incubation at room temperature absorbance was read<br />
at 550 nm in Systronics Spectropbotometer. A standard curve was prepared by using<br />
known concentrations of sodium nitrite.<br />
3.19.3 Hydrogen peroxide generation assay<br />
Hydrogen peroxide generation was assayed by the method of Pick and Keisari<br />
(198 1). The reaction mixture contained 1.641 mL phosphate buffer (50 mM, pH 7.6),<br />
54 pL of horseradish peroxidase (8.5 units/ mL), 30 pL of 0.28 nM phenol red, 5.5<br />
nM of 165 LLL of dextrose (see Appendix 1.16) and 600 pL of enzyme source. Blank<br />
was prepared simultaneously by adding all the reagents except the tissue extract. The<br />
reaction mixture was ~ncubated at 35OC for 30 rnin. The reaction was terminated by<br />
addition of 60 pL of I0 N sodium hydroxide. Absorbance was read at 610 m against<br />
a reagent blank on a Systronics Spectrophotometer. For preparation of standard<br />
curve, known amounts of hydrogen peroxide and all the above reagents except<br />
enzyme source were incubated for 30 min at 35°C and then 60 pL of 10 N sodium<br />
hydroxide was added and optical density was read at 610 nm.<br />
3.20 Determination of antioxidants in tissues<br />
Estimations of antioxidants such as reduced glutathione, a-tocopherol and<br />
ascorbic acid were done by the methods as described below.<br />
3.20.1 Estimation of reduced glutathione<br />
Total reduced glutathione content was measured by the method of Moron ef<br />
a/. (1979). To 0.5 mL of tissue homogenate 2 mL of 5% trichloro acetic acid waa<br />
added and mixed well. It was centrifuged at 800 g for 10 min and 2 mL of the
supcrnutant was taken. Hlunk was prcpurod simultaneously by addilly ull tho rcuycnls<br />
except the tissue extract. To this 1 mL of Ellman's reagent and 4 mL of 0.3 M<br />
disodium hydrogen phosphate (see Appendix 1.17) were added. l'hc yellow colour<br />
developed was read at 412 nm on a Systronics Spectrophotometer against blank. A<br />
series of standards, reduced glutathione were treated in a similar manner along with a<br />
blank.<br />
3.20.2 Estimation of a-tocopherol<br />
Tissue a-tocopherol was estimated by the method of Desai (1984):The assay<br />
mixture contained 0.5 mL of tissue homogenate, 2 rnL of petroleum ether and 1.6 mL<br />
of ethanol. Blank was prepared simultaneously by adding all the reagents except the<br />
enzyme extract. The assay mixture was mixed and centrifuged at 800 g for 10 min.<br />
To the supernatant, 0.2 mL of 2.2'-dipyridyl solution (see Appendix 1.18) was added,<br />
mixed well and kept in the dark for 5 min. An intense red colour was developed and<br />
read at 520 nm on a Systronics Spectrophotometer against blank. Various<br />
concentrations of a-tocopherol were taken and treated similarly along with a blank.<br />
The colour in the aqueous layer was read at 520 nrn and used for standard.<br />
3.20.3 Estimation of ascorbic acid<br />
Ascorbic acid was assayed by the method of Fisher (1962). Approximately,<br />
100 mg of testis was taken in 10 mL of cold 2.5% metaphosphoric acid and<br />
homogenized with the help of glass teflon homogenizr. The homogenate was<br />
centrifuged at 800 g for 20 min at 4OC. The reaction mixture contained 1 mL of<br />
supernatant (diluted with 2.5% metaphosphoric acid) and I mL of 2.6-dichlorophenol<br />
indophenol acetate solution (see Appendix 1.19). Blank was prepared sin~ultaneously<br />
by adding all the reagents except the tissue extract. The optical density was measured<br />
in a within 30 xc at 520 nm in a Systronics Spectrophotometer. A standard<br />
calibration curve was prepared using different concentrations of standard ascorbic<br />
acid.
3.21 Determination of antioxidant enzymes in tissues<br />
Activities of antioxidant enzymes such as superoxide dismutase, catalase,<br />
glutathione reductase and glutathione peroxidase were assayed by the methods as<br />
described below.<br />
3.21.1 Assay of superoxide dismutase<br />
Superoxide dismutase (EC 1. IS. 1.1) was assayed by the method of Marklund<br />
and Marklund (1974). The assay mixture contained 2.4 mL of SO mM tris HCI buffer<br />
cont;iininy I mM EDTA (pH 7.6). 300 pL of 0.2 mM pyrogallol (see Appendix 1.20)<br />
and 100 ILL enzyme source. A blank solution was prepared similarly by adding all the<br />
reagents except the enzyme source. Increase in absorbance was measured at 420 nm<br />
against blank at 10 sec intervals for 3 min on Systronics Spectrophotometer.<br />
3.21.2 Assay of catalase<br />
Catalase (EC. 1.11.1.6) was assayed by the method of Claibome (1985). The<br />
assay mixture contained 2.40 mL of phosphate buffer (50 mM, pH 7.0). 10 pL of 19<br />
mM hydrogen peroxide (see Appendix 1.21) and 50 pL enzyme source. Blank was<br />
prepared simultaneously by adding all the reagents except the enzyme extract.<br />
Decrease in absorbance was measured at 240 nm against blank at 10 sec intervals for<br />
3 min on a Systronics Spectrophotometer.<br />
3.21.3 Assay of glutathione reductnse<br />
The activity of glutathione reductase (EC. 1 h.4.2) was assayed by the method<br />
of Carlberg and Mannervik (1975). The assay mixture contained 1.75 mL of<br />
phosphate buffer (100 mM, pH 7.6). 100 pL of 200 mM NADPH, 100 pL of 10 mM<br />
EDTA (see Appendix 1.22). SO pL of 20 mM glutathione oxidized and SO pL enzyme<br />
source. Blank was prepared simultaneously by adding all the reagents except the<br />
enzyme extract. The extinction coefficient of NADPH was measured at 340 nm<br />
against blank at 10 sec intervals for 3 min on a Systronics Specwphotomet~.
3.21.4 Assay of glutathione peroxidase<br />
Glutathione peroxidase (EC. 1.1 1.1.9) was assayed by the method of<br />
Mohandas rt al. (1984). The assay mixture contained 1.59 mL of phosphate buffer<br />
(100 mM, pH 7.6). 100 pL of 10 mM EDTA, 100 pL of sodium azidc, 50 pL of<br />
glutathione reductase, 100 pL of glutathione reduced, I00 pL of 200 mM NADPH, I0<br />
pL of hydrogen peroxide (see Appendix 1.23) and 10 pL enzyme source. Blank was<br />
prepared simultanwusly by adding all the reagents except the enzyme extract. The<br />
extinction coefficient of NADPH was measured at 340 nm against blank containing<br />
all the components except the enzyme at 10 sec intervals for 3 min on a Systronics<br />
Spectrophotometer.<br />
3.22 Estimation of lipid peroxidation<br />
A break-down product of lipid peroxidation thiobarbituric acid reactive<br />
substance (TBARS) was measured by the method of Buege and Aust (1976). Briefly,<br />
the stock solution contained equal volumes of trichloroacetic acid 15% (w/ v) in 0.25<br />
N hydrochloric acid and 2-thiobarbituric acid 0.37% (wl v) in 0.25 N hydrochloric<br />
acid (see Appendix 1.24). 1 mL of the tissue test samples and 2 mL of stock reagent<br />
were mixed in a screw-capped centrifuge tube, vortexed and heated for 15 niin in a<br />
boiling water bath. Blank was prepared simultaneously by adding all the reagents<br />
except the enzyme exrract. After cooling on ice the precipitate was removed by<br />
centrifugation at 1000 g for 15 min and absorbance of the supernatant was measured<br />
at 532 nm against blank. A standard curve was constructed extrapolating the amount<br />
of malondialdchyde to the measured absorbance<br />
3.23 Statistical analysis<br />
The data in the experimental groups of rats were obtained from the individual<br />
rat, which received identical treatments. All other biochemical estimations were<br />
carried out in duplicate. The significance of the results has been analyzed using one-
way nnalysis of variance (ANOVA) followed by Duncan's Multiple Ranyc 'I'est. 'I'hc<br />
P values of less than 0.05 were considered as significant against control.
4 RESULTS<br />
4.1 Administration of TCDD to rats<br />
TCDD was administered orally at the doses of 1, 10 and 100 ng/ Kg body<br />
weight/ day to pubertal rats for 45 days. Corresponding groups of control animals<br />
were maintained and received equal volumes of vehicle only. The animals were<br />
killed after 24 h of the last treatment.<br />
4.1.1 Effect of TCDD on body and organ weights<br />
Administration of TCDD did not show any significant change in the body<br />
weight of the rats at the end of the treatments in all the experimental groups as<br />
compared to the corr'esponding groups of control animals (Fig. 1; Table la).<br />
The weight of the testis decreased significantly In rats administered with<br />
TCDD as compared to the corresponding groups of control animals (Table la). The<br />
wciyht of the testis when expressed relative to the body weight also decreased<br />
significantly in the rats treated with TCDD as compared to the corresponding groups<br />
of control animals (Table 1 b).<br />
The weight of the epididymis decreased significantly in rats treated with<br />
TCDD as compared to the corresponding groups of control rats (Table la). The<br />
weight of the epididymis when expressed relative to the body weight also decreased<br />
significantly in the rats treated with TCDD with respect to the corresponding groups<br />
of control nnininls (Table I b).<br />
Administration of TCDD decreased significantly in thc weight of the scminal<br />
vesicles with respect to the cornspanding groups of convol animals (Table la). The<br />
weight of the seminal vesicles when expressed relative to the body weight also<br />
decreased significantly in the rats treated with TCDD as compared to the<br />
Corresponding groups of control animals (Table I b).
The weight of the venlral proshte showcd a siynilicunt reduction in the 'I'C1)U<br />
treated rats as compared to the corresponding groups of control animals (Table la).<br />
The weight of the ventral prostate when expressed relative to the body weight also<br />
decreased significantly in the rats treated with TCDD as compared to the<br />
corresponding groups of control animals (Table I b).<br />
The weight of the kidney did not show any significant change in the TCDDtreated<br />
rats as compared to the corresponding groups of control animals (Table la).<br />
The weight of the kidney when expressed relative to the body weight did not decrease<br />
in the rats treated with TCDD as compared to the corresponding groups of control<br />
animals (Table I b).<br />
4.1.2 Effect of TCDD on daily sperm production<br />
Administration of TCDD decreased the daily sperm production in a dosedependent<br />
manner with respect to the corresponding group of control animals<br />
(Table 2).<br />
4.1.3 Effect of TCDD on epididymal sperm viability, motility and counts<br />
The epididymal sperm viability did not show any significant change in the<br />
'I'CDD treated rats as compared to the corresponding groups of control animals<br />
(Table 2). Administration of TCDD decreased epididymal sperm motility in a dosedependent<br />
manner as compared to the corresponding group of control animals<br />
(Table 2). The epididymal sperm counts decreased significantly in a dose dependent<br />
manner in the TCDD treated rats as compared to the corresponding group of control<br />
animals (Table 2).<br />
4.1.4 Effect of TCDD on seminiferous tubular and lumen diameter<br />
The diameter of the seminiferous tubules was noted to be decreased<br />
significantly in the rats after administration of TCDD with respect to the<br />
corresponding group of control animals (Table 3). The lumen diameter of the
seminiferous tubules was found to be decreased significantly in rats allcr 'I'CDL)<br />
treatment as compared to the corresponding group of control animals (Table 3).<br />
4.1.5 Effect of TCDD on serum hormone levels<br />
Administration of TCDD did not show any significant change in the level of<br />
serum FSH in the rats as compared to the corresponding group of control rats (Table<br />
4). The levels of serum LH remained unchanged in rats administered with TCDD<br />
with respect to the corresponding group of control animals (Table 4). The levels of<br />
serum prolactin decreased significantly in rats administered with TCDD as compared<br />
to the corresponding group of control animals (Table 4).<br />
The levels of serum testosterone decreased significantly in rats administered<br />
with TCDD as compared to the corresponding groups of control animals ('l'ablc 4).<br />
The levels of serum estradiol decreased significantly in rats administered with TCDD<br />
as compared to the corresponding group of control animals (Table 4).<br />
4.1.6 Effect of TCDD on the testicular steroidogenic enzymes<br />
The activity of 3p-hydroxysteroid dehydrogenase decreased in a dose-<br />
dependent manner in the rats treated with TCDD with respect to the corresponding<br />
groups of control animals (Tahlc 5).<br />
l'hc activity of 17P-hydroxystcroid<br />
dehydrogenase decreased in a dose-dependent manner in the rats treated with TCDD<br />
as compared to the corresponding g~oups of control animals (Table 5).<br />
4.1.7 Effect of TCDD on nucleic acids and protein contents of testis<br />
The DNA content of testis decreased significantly in a dose-dependent manner<br />
in the rats after administration of TCDD as compared to the corresponding group of<br />
control animals (Table 6). Administration of TCDD also decreased the RNA content<br />
of testis in a dose-dependent manner as compared to the corresponding group of<br />
control animals (Table 6). The protein content of the testis decreased in the rats after
administration of TCDD in a dosc-dcpcndcnt manner as comparcd to 111~.<br />
corresponding group of control animals (Table 6).<br />
4.1.8 Effect of TCDD on the production of reactive oxygen species in the crude<br />
homogenate, mitochondrial and microsome-rich fractions of testis<br />
Administration of TCDD caused a significant increase in the production of<br />
superoxide anion in crude homogenate as well as in mitochondrial and microsomerich<br />
fractions of rat testis as compared to the corresponding groups of control animals<br />
(Table 7a).<br />
Administration of TCDD caused a significant increase in the levels of nitric<br />
oxide in crude homogenate as well as in mitochondrial and microsome-rich fractions<br />
of rat testis as compared to the corresponding groups of control animals (Table 7b).<br />
Administration of TCDD caused a significant increase in the generation of<br />
hydrogen peroxide in crude homogenate as well as in mitochondrial and microsomerich<br />
fractions of rat testis as compared to the corresponding groups of control animals<br />
(Table 7c).<br />
4.1.9 Effect of TCDD on the levels of antioxidants in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of testis<br />
Administration of TCDD caused a significant decrease in the level of reduced<br />
glutathione in the crude homogenate as well as in the mitochondrial and microsome-<br />
r~ch fractions of rat testis as compared to the corresponding groups of control animals<br />
(Table 8a).<br />
Administration of TCDD caused a significant decrease in the level of a-<br />
tocopherol in the crude homogenate as well as in the mitochondrial and microsome-<br />
rich fractions of rat testis as compared to the corresponding groups of control animals<br />
(Table 8b).<br />
Administration of TCDD caused a significant increase in the level of ascorbic<br />
acid in crude homogenate as well as in the mitochondrial and microsome-rich
fractions of rat testis as compared to the corresponding groups of control animals<br />
(Table 812).<br />
4.1.10 Effect of TCDD on the antioxidant enzymes and lipid pcroxidation in<br />
crude homogenate, mitochondrial and microsome-rich fractions of testis<br />
The activity of superoxide dismutase decreased significantly in the crude<br />
homogenate as well as in the mitochondrial and microsome-rich fractions of rat testis<br />
as compared to the corresponding group of control animals (Table 9a).<br />
The activity of catalase decreased significantly in the crude homogenate as<br />
well as in the mitochondrial and microsome-rich fractions of rat testis as compared to<br />
the corresponding group of control animals (Table 9b).<br />
The activity of glutathione reductase decreased significantly in the crude<br />
homogenate as well as in the mitochondrial and microsome-rich fractions of rat testis<br />
as compared to the corresponding group of control animals (Table 9c).<br />
The activity of glutathione peroxidase decreased significantly in the crude<br />
homogenate as well as in the mitochondrial and microsome-rich fractions of rat testis<br />
as compared to the corresponding group of control animals (Table 9d).<br />
The level of lipid peroxidation increased significantly in the crude homogenate<br />
as well as in the mitochondria1 and microsome-rich fractions of rat testis as compared<br />
to the corresponding group of control animals (Table 10).<br />
4.1.1 1 Effcct of TCDD on the cpididymnl spcrm and cpididymis<br />
002661.<br />
A positive correlation (r = 0.95; n = 24) was observed between sperm counts<br />
and DNA content in the epididymal sperm (Fig. 2). The DNA contents were routinely<br />
used as an indicator of sperm counts. The production of reactive oxygen species.<br />
levels of antioxidants, activities of antioxidant enzymes and lipid peroxidation were<br />
expressed both in terms of protein and DNA.
Fig. 2. Correiation between sperm counts and DNA contents of rat epididymal<br />
sperm (14.95; n= 24)<br />
Sperm counts (x 10')
4.1.11.1 Effect of TCDD on the production of rcactivc oxygen spccics in the<br />
epididymal sperm<br />
Administration of TCDD caused a significant increase in the production of<br />
superoxide anion nitric oxide and hydrogen peroxide in the epididymal sperm of<br />
adult rats as compared to the corresponding groups of control animals (Table 1 1 ).<br />
4.1.11.2 Effect of TCDD on the levels of antioxidants in the epididymal sperm<br />
Administration of TCDD caused a significant decrease in the level of<br />
reduced glutathione and a-tocopherol in the epididymal sperm of adult rats with<br />
respect to the corresponding groups of control animals (Table 12).<br />
4.1.11.3 Effect of TCDD on the antioxidant enzymes and lipid peroxidation in<br />
the epididymal sperm<br />
The activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase decreased significantly in the epididymal sperm of the treated<br />
animals as cornpared to the corresponding group of control animals (Table 13).<br />
The level of lipid peroxidation increased significantly in the epididymal<br />
sperm of treated rats as compared to the corresponding group of control animals<br />
(Table 13).<br />
4.1.11.4 Effect of TCDD on the production of reactive oxygen species in the<br />
caput, corpus and cauda epididymides<br />
Administration of TCDD caused significant increase in the production of<br />
superoxide anion. nitric oxide and hydrogen peroxide in the caput. corpus and cauda<br />
epididynlides of the treated rats as compared to thc corresponding groups of control<br />
animals (Table 14).
4.1.11.5 Effect of TCDD on the antioxidants in the caput, corpus and cauda<br />
epididymides<br />
Administration of TCDD caused significant decrease in the levels of<br />
reduced glutathione and a-tocopherol in the caput, corpus and cauda regions of the<br />
epididymis of the treated rats with respect to the corresponding groups of control<br />
animals (Table 15).<br />
4.1.11.6 Effect of TCDD on the antioxidant enzymes and lipid peroxidation in<br />
the cnput, corpus and caudaepididymides<br />
The activity of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase decreased significantly in the caput, corpus and cauda regions<br />
of the epididymis of the treated rats as compared to the corresponding groups of<br />
control animals (Table 16a, 16b & 16c). The level of lipid peroxidation increased<br />
significantly in all the regions of the epididymis of the treated rats as compared to the<br />
corresponding groups of control animals (Table 16a. 16b & 16c).<br />
4.1.12 Effect of TCDD on the levels of reactive oxygen species production,<br />
antioxidants, antioxidant enzymes and lipid peroxidation of kidney<br />
Administration of TCDD at the dose of 1 and 10 ngl Kg body weight did not<br />
cause any significant change in the levels of superoxide anion, nitric oxide. hydrogen<br />
peroxide, glutathione, a-tocopherol, superoxide dismutase. catalase. glutathione<br />
reductase, glutathione peroxidase and lipid peroxidation as compared to the<br />
corresponding groups of control animals (Table 17a to 17c) while administration of<br />
TCDD at the dose of 100 ngl Kg body we~ght caused significant increase in the<br />
production of superoxide anion. nitric oxide. hydrogen peroxidu and<br />
lipid<br />
peroxidation and decreased in the levels of' antioxidant system in the kidney as<br />
compared to the corresponding groups of co~itrol animals (Table 17a to 17c).
4.2 Co-administration of TCDD and vitamin E to rats<br />
TCDD (1, 10, and 100 ng/ Kg body weight1 days) and vitamin E (20 mg/ Kg<br />
body weight/ day) were administered orally to pubertal rats for 45 days.<br />
Corresponding groups of animals received equal volumes of vehicle only (vitamin E<br />
dissolved in olive oil) and served as control. The animals were killed after 24 h of the<br />
last treatment.<br />
4.2.1 EfYect of co-administration of TCDD and vitamin E on the body and<br />
organ weights<br />
Administration of TCDD and vitamin E did not show any significant changes<br />
in the body weight of rats in all the experimental groups as compared to the<br />
corresponding groups of control animals (Table 18a).<br />
The weight of the testis, epididymis, seminal vesicles and ventral prostate did<br />
not alter significantly in rats administered with TCDD along with vitamin E as<br />
compared to the corresponding groups of control rats (Table 18a). The weights of the<br />
testis. epididymis and accessory sex organs when expressed relative to the body<br />
weights also showed no significant changes in the rats treated with TCDD and<br />
vitamin E as compared to the corresponding groups of control animals (Table 18b).<br />
The weight of the kidney did not show any significant changes in the TCDD<br />
and vitamin E treated rats as compared to the corresponding groups of control animals<br />
(Table 18a). The weight of the kidney when expressed relative to the body weight did<br />
not decrease in the rats treated with TCDD and vitamin E as compared to the<br />
corresponding groups of control animals (Table 18b).<br />
4.2.2 Effect of co-adminiaimtion of TCDD and vitamin E on the daily sperm<br />
production<br />
Administration of TCDD decreased daily sperm production in a dose<br />
dependent manner as compared to the corresponding group of control animals (Table<br />
19).
4.23 Effect of co-administration of TCDD and vitamin E on the epididymal<br />
sperm viability. motility and counts<br />
The epididymal sperm viability, motility and counts did not change<br />
significantly in the rats treated with TCDD and vitamin E as compared to the<br />
corresponding groups of control animals (Table 19).<br />
4.2.4 Effect of co-administration of TCDD and vitamin E on seminiferous<br />
tubular and lumen diameter<br />
The seminiferous tubules and lumen diameters remained unchanged in the rats<br />
after co-administration of TCDD and vitamin E as compared to the corresponding<br />
groups of control animals (Table 20).<br />
4.2.5 Effect of co-administration of TCDD and vitamin E on the levels of serum<br />
hormones<br />
Co-administration of TCDD and vitamin E did not significantly change in the<br />
levels of serum FSH. LH, prolactin, testosterone and estradiol in the rats as compared<br />
ro [he corresponding groups of control animals (Table 21 ).<br />
4.2.6 Effect of co-administration of TCDD and vitamin E on the testicular<br />
steroidogcnic emmes<br />
The activities of 3P-hydroxysteroid dehydrogenase and 17P-hydroxysteroid<br />
dehydrogenase did not show any significant change in the rats treated with TCDD and<br />
vitamin E as compared to the corresponding groups of control animals (Table 22).<br />
4.2.7 Effect of co-administration of TCDD and vitamin E on nueleic acids sncl<br />
protein contents of testis<br />
Co-ndministr~tion ol' I'CDI) and vilanii~~ I: did 1101 show any siynilica~i~<br />
change in DNA. RNA and protein contents of testis as compared to the corresponding<br />
groups of control animals (Table 23).
4.2.8 Effect of co-administration of TCDD and vitamin E on the production of<br />
reactive oxygen species in the crude homogenate, mitochondrial and<br />
microsome-rich fractiom of testis<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the production of superoxide anion, nitric oxide and hydrogen peroxide in<br />
crude testicular homogenate as well as in mitochondrial and microsome-rich fractions<br />
of rat testis as compared to the corresponding groups of control animals (Table 24a;<br />
24b & 24~).<br />
4.2.9 Effect of co-administration of TCDD and vitamin E on the levels of<br />
antioxidant in the crude homogenate, mitochondrial and microsome-rich<br />
fractions of testis<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the levels of reduced glutathione, a-tocopherol and ascorbic acid in the<br />
crude testicular homogenate as well as in mitochondrial and niicrosome-rich fractions<br />
of rat testis as compared to the corresponding groups ot' control animals (Table 25a;<br />
25b & 25c).<br />
4.2.10 Effect of co-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in crude homogenate, mitochondrial and<br />
microsome-rich fractions of testis<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase as well as the levels of lipid peroxidation in the crude<br />
t~sticular homoycnatc. mitochondrial and microso~nc-riel1 lii~ctil~ns 01' r;11 tcsti?, as<br />
comparcd to the corresponding groups vl'contrul animals ('lhblc 26a (o 20d LY: 27).<br />
4.2.11 Effect of co-administration of TCDD and vitamin E on epididymal sperm<br />
Rats were treated with TCDD and vitamin E in order to see the effect of co-<br />
administration on the epididymal sperm of adult rats.
4.2.11.1 Effect of co-administration of TCDD on the production of rcvctive<br />
oxygen rpeciea In the epididymal sperm<br />
Co-administration of TCDD along with vitamin E did not show any<br />
significant change in the production of superoxide anion, nitric oxide and hydrogen<br />
peroxide in the epididymal sperm of adult rats as compared to the corresponding<br />
groups of control animals (Table 28).<br />
4.2.11.2 Effect of co-administration of TCDD and vitamin E on the levels of<br />
antioxidants in the epididymal sperm<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the levels of reduced glutathione and a-tocopherol in the epididymal sperm<br />
of adult rats as compared to the corresponding groups of control animals (Table 29).<br />
4.2.11.3 Effeet of eo-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in the epididymal sperm<br />
The activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase remained unchanged in the epididymal sperm of rats treated<br />
with TCDD and vitamin E as compared to the corresponding groups of control<br />
animals (Table 30). The levels of lipid peroxidation also remained unchanged in the<br />
rpididymal sperm of the animals administered with TCDD and vitamin E as<br />
compared to the corresponding groups of control animals (Table 30).<br />
4.2.11.4 EfTect of co-administration of TCDD and vitamin E on the production<br />
of reactive oxygen spccics in the euput, corpus and eaudn cpididyntides<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the production of superoxide anion, nitric oxide and hydrogen peroxide in<br />
all the regions of the epididymis of adult rats as compared to the corresponding<br />
groups of control animals (Table 31).
4.2.11.5 Effect of co-administration of TCDD and vitamin E on the antioxidants<br />
in the caput. corpus and cauda epididyrnideo<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the levels of reduced glutathione and a-tocopherol in all regions of the<br />
epididymis of adult rats with respect to the corresponding groups of control animals<br />
(Table 32).<br />
4.2.1 1.6 Effect of co-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in the caput, corpus and cauda<br />
epididymides<br />
The activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase as well as the levels of lipid peroxidation did not alter<br />
significantly in the caput, corpus and cauda regions of the epididymis of rats treated<br />
with TCDD and vitamin E when compared to the corresponding group of control<br />
animals (Table 33a; 33b & 33c).<br />
4.2.12 Effect of co-administration of TCDD and vitamin E on the levels of<br />
reactive oxygen species production, antioxidants, antioxidant enzymes<br />
and lipid peroxidation in Kidney<br />
Co-administration of TCDD and vitamin E did not show any significant<br />
change in the production of superoxide anion, nitric oxide and hydrogen peroxide in<br />
the kidney of adult rats as compared to the corresponding groups of control animals<br />
(Table 34a). Administration of TCDD along with vitamin E did not cause any<br />
significant changc in the levcls of roduccd gluti~thic)nc and cr-Iocoplicrol in Ihc Lidncy<br />
ofadult rats with respect to the corresponding groups of control animals (.!'able 34b).<br />
The activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase and the level of lipid peroxidation of the kidney did not show<br />
any significant change in the rats administered with TCDD and vitamin E as<br />
compared to the comsponding group of control animals (Table 34c).
Table la. Effect of TCDD on the body weight and weights of the testis, epididylnis,<br />
kidney and accessory sex organs of adult male rats<br />
TCDDI Kg body weight<br />
Control I nS I0 ng I00 ng<br />
Body weight 116i 2.82 115i 1.36 1141 1.60 114+ 1.86<br />
Testis 964 + 3.86 936 f 9.97' 922 + 2.89' 92 1 5 7.08*<br />
Epididyln~s 414 f 2.63 393 * 5.20. 388 f 4.23' 386+ 4.00'<br />
Seminal vesicles<br />
Intact<br />
Empty<br />
Ventral prostate 1935 2.66 180f 1.83. 179i 2.50' 175 t 3.55.<br />
Kidney weight 578 i 8.2 1 590 i 7.26 586 f 9.38 540 f 6.40'<br />
Body weights are expressed in gram and tissue weights are expressed in mg<br />
The data are expressed as meanfSD for six animals per group<br />
'pc0.05 against the control group
Table I b. Effect of TCDD on the weights ofthe testis, epididymis, kidney and accessory<br />
sex organs of adult male rats<br />
TCDDl Kg body weight<br />
Control I nfi I0 ng 100 11g<br />
Testis 828 f 8.69 809 f 5.45. 807 i 8.93. 805 f 2.28.<br />
Epididymis 355 f 7.14 339i 6.92' 339 t 3.31. 337 + 6.87.<br />
Seminal vesicles<br />
Intact<br />
Emptr<br />
Ventral prostate 165f 2.78 155t3.31' 156i3.20* 1521 2.85'<br />
Kidney 489 i 6.1 1 498 t 8.62 480 f 6.32 443 * 4.81 '<br />
Tissue weights are expressed in mg/ loop body weight<br />
The data arc expressed as mean f SD for six aninield dose<br />
*p
Table 2. Effcct of TCDD on the daily sperm production, epididymal sperm viability,<br />
sperm motility and sperm counts in adult male rats<br />
TCDDi Kg body weight<br />
Control I ng I0 ng 100 ng<br />
Testis<br />
Daily sperm<br />
Production (x 10" 22.19 + 2.67 15.67 i 2.65. 13.65 f 2.19. 13.10' 3.16*<br />
Epididymul Sperm<br />
Sperm viability (%) 95.50 + 1.51 94.16 f 2.56 95.83 i 2.27 92.50 * 2.16<br />
Sperm motility (%) 93.66 * 2.16 74.83 i 3.54. 57.83 i 3.48' 46.16 * 3.41.<br />
Sperm counts (x 10') 8.20 i 0.14 7.51 * 0.15* 6.36 f 0. I2* 5.3 1 f 0.15*<br />
The data are expressed as mean i SD for six animals1 dose<br />
'p
Table 4. Effect of TCDD on the serum hormone levels in adult cnulc rals<br />
TCDDI Kg body weight<br />
Control 1 ng I0 ng I00 ng<br />
FSH' 14.40f 2.10 13.62 f 2.92 14.50i 1.73 13.001 1.41<br />
'ny/ mL; bPd mL<br />
The data arc expressed as mean f SD for six animals/ dose<br />
*p
Table 6. Effect of TCDD on the DNA, RNA and protein contents in testis<br />
TCDDI Kg body weight<br />
Control I ng I0 II~ I00 ng<br />
DNA<br />
RNA<br />
Protein<br />
The values are expressed in mgl g tissue wet weight<br />
The data are expressed as mean t SD for six animals1 dose<br />
*p
Table 7b. Effect of TCDD on the production of nitric oxide in the crude ho~noyenute,<br />
mitochondrial and microsome-rich fractions of' rat testis<br />
TCDDl Kg body weight<br />
Control I ng 10 ng I00 ng<br />
Crude homogenate 2.38 f 0.10 3.61 * 0.08* 3.72 rt 0.04' 4.77 f 0.06.<br />
Mitochondrial fractions 1.43 f 0.1 1 2.33 f 0.108 2.79 f 0.14' 3.10 1 0.42'<br />
Microsome-rich<br />
fractions 2.41 f 0.77 2.81 1 0.20* 3.01 10.14' 3.16* 0.72*<br />
The values are expressed in mmole/ min/ mg protein<br />
The data are expressed as mean i SD for six animals1 dose<br />
'pcO.05 against the control<br />
Table 7c. Effect of TCDD on the hydrogen peroxide generation in crude Iiomogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
TCDDI Kg body weight<br />
Control I ng 10 ng I00 ng<br />
Crudc Iromoyenatc 18.70 f 2.78 22.16 r 1.26. 74.1 7 + 2.88' 76.4 1 i 1 .St)*<br />
Mitochondrial fractions 15.73 i 2.12 18.08 f 1.21' 19.87 i 1.14' 21.76 r 1.93'<br />
Microsome-rich<br />
fractions 12.68 f 2.01 16.31 22.41' 17.37 f 1.98. 19.45 i 1.89*<br />
The values are expressed in nmold minl mg protein<br />
The data are expressed as mean * SD for six animals1 dose<br />
*p
Table 8a. Effect of TCDD on the levels of glutathione in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
TCDDI Kg body weight<br />
Control I ng I0 ng 100 ng<br />
Crude homogenate 336 5 7.66 297 + 8.33* 275 i 6.88. 267 i 7.57'<br />
Mitochondrial fractions 197 f 5.57 169 f 9.29' 159 i 8.01* 153 5 9.06*<br />
Microsome-rich<br />
fractions 158 * 4.26 135 f 9.87' 13Oi 3.01' 126 f 5.75.<br />
The values arc expressed in nmolel nig protein<br />
The data arc expressed as mean i SD for six animals! dose<br />
*pc0.05 against the control<br />
Table 8b. Effect of TCDD on the levels of a-tocopherol in the crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
TCDDI Kg body weight<br />
Control I ng I0 ng I00 ng<br />
Crude homogenate 1.32 f 0.08 0.95 i 0.08. 0.78 f 0.04. 0.76 r 0.02'<br />
Mitochondrial fractions 0.90 f 0.02 0.74 t 0.03' 0.66 f 0.06. 0.56 k 0.03'<br />
Microsome-rich<br />
fractions 0.66 f 0.04 0.48 f 0.05' 0.38 f 0.02' 0.35 * 0.03'<br />
The data arc expressed in pg/ mg protein<br />
The data an expressed as inmean f SD for six animals1 dose<br />
*p
Table 8c. Effect of TCDD on the levels of ascorbic acid in the crude homogenate,<br />
mitochondria1 and microsome-rich fractions of rat testis<br />
TCDDI Kg body weight<br />
Control I ng I0 ng I00 ng<br />
Crude homogenate 3.55 f 0.16 2.71 f 0.1 I* 2.18 + 0.13* 1.63 + 0.10'<br />
Mitochondrial fractions 2.15 f 0.15 1.92 f 0.17* 1.74 f 0.14' 1.62 f 0.15.<br />
Microsome-rich<br />
fractions 1.96f 0.17 1.54f 0.11* 1.37f 0.10' 1.80k 0.18.<br />
The data are expressed in mg/ g tissue wet weight<br />
The data are cxpressed as mean * SD for six animals/ dose<br />
*p
Table 9b.<br />
Effect of TCDD on the activities of catalase in the crude homogenate.<br />
mitochondrial and microsome-rich fractions of rat testis<br />
TCDDl Kg body weight<br />
Control 1 ng 10 ng 100 ng<br />
Crude homogenate 0.871 * 0.06 0.696 + 0.05' 0.670 + 0.05' 0.630 3. 0.05'<br />
Mitochondr~sl fractions 0.718 + 0.04 0.628 f 0.05' 0.567 i 0.04* 0.509 * 0.03-<br />
Microsome-rich<br />
fractions 0.401 * 0.04 0.356 1 0.08' 0.321 + 0.01. 0.309 k 0.01'<br />
The values are expressed in pmole/ mini mg protein<br />
The data arc expressed as mean * SD for six animals/ dose<br />
*p
Table 9d.<br />
Effect of TCDD on the activities of glutathione peroxidase in crude<br />
homogenate, mitochondrial and microsome-rich fractions of rat testis<br />
- -<br />
TCDDI Kg body weight<br />
Control I ng I0 ng I00 ng<br />
Crude homogenate 46.35 f 3.57 41.45 f 3.13' 41.57 f 2.93' 40.55 f 4.77'<br />
Mitochondrial fractions 3 1.3 1 f 2.48 25.46 i 4.1 1 24.68 f 3.26* 2 1.14 + 3.98'<br />
Microsome-rich<br />
fractions 26.11 f 4.68 20.13 *3.41* 19.64k 1.96. 19.01 i 2.01;<br />
The values are expressed in nmolel min/ mg protein<br />
The data arc expressed as mean f SD for six animals1 dose<br />
*pc0.05 against the control group<br />
Table<br />
10. Effect of TCDD on the lipid peroxidation in crude homogenate,<br />
mitochondrial and microsome-rich fractions of rat testis<br />
TCDD/ Kg body weight<br />
Crude homogenate 2.47 f 0.16 3.40 f 0.29' 3.60 f 0.37. 4.10 f 0.15*<br />
Mitochondrial fractions 2.01 f 0.78 2.97 f 0.29' 3.41 * 0.5 I* 3.67 f 0.561<br />
Microsome-rich<br />
fractions 1.20 f 0.22 1.79 f 0.27. 2.07 * 0.23' 2.1 1 f 0.41<br />
The values arc expressed in pnoleJ mg protein<br />
The data arc expressed as mean f SD for six animals/ dose<br />
'pX0.05 against the control group
Table I I. Effect of TCDD on the production of superoxide anion, nitric oxide and<br />
hydrogen peroxide in the epididyrnal spenn of adult rats.<br />
TCDDI Kg body weight<br />
Control I ng I0 ng 100 ng<br />
Superoxide aniona<br />
(mg protein) 1.93 f 0.18 2.78 f 0.47. 2.91 + 0.14' 3.14 f 0.24"<br />
(rng DNA) 1.41 f 0.04 2.07f O.lOC 2.47f 0.16. 2.91 f 0.31.<br />
Nitric oxideb<br />
(~ng protein) 1.46i0.22 2.01 i 0.16" 2.6910.1 I* ?.99f0.06*<br />
(mg DNA) 1.14 f 0.1 l 1.90 f 0.17' 2.40 k 0.27* 2.68 ~t 0.13'<br />
H202 generationC<br />
(mg protein) 20.80f 1.96 40.70 + 3.1 I* 46.10f 3.82. 55.30 f 2.32'<br />
(rng DNA) 16.81fl.18 25.42f 1.33; 31.16+1.60* 36.87i2.88'<br />
' nmoll min/ rng protein or DNA; mmol/ min/ mg protein or DNA; nmol H202<br />
generatedl min/ rng protein or DNA; The data are expressed as mean f SD for six<br />
animals! dose; *p
Table 13. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the<br />
epididymal sperm of adult rats<br />
TCDDI Kg body weight<br />
Control I ng 10 ng I00 ng<br />
Superoxide dismutase'<br />
(mg protein) 22.405 1.43 20.30i2.85' 18.50i 1.51. 16.90k 1.57'<br />
(mg DNA) 18.08+ 0.61 13.64+ 1.14' 12.48i 0.72* 11.385 1.22*<br />
catalaseb<br />
(ing protein) 2.49f 0.13 2.33 i 0.15' 2.305 0.14' 2.03 i0.13'<br />
(mg DNA) 2.01 i 0.05 1.45 i 0.06. 1.44 + 0.08* 1.35 + 0.05'<br />
Glutathione reductase'<br />
(mg protein) 40.00* 2.17 31.20f 2.41. 28.20i 2.49* 27.10f 1.57'<br />
(mg DNA) 32.41 f 0.86 19.46i 0.53. 20.l8i 0.83' 18.07i 0.76*<br />
Glutathione peroxidase'<br />
(mg protein) 71.20i 3.87 64.50i 4.88' 56.605 4.41. 48,OOi 3.97.<br />
(mg DNA) 57.58 i 1.52 40.17 i 1.05' 38.24 i 0.88. 31.94 + 1.79.<br />
Lipid peroxidatton'<br />
(trig protein)<br />
2.17 i 0.20 4.24 i0.418 4.80f 0.38' 6.08+ 0.20'<br />
(mg DNA) 1.75 + 0.82 2.W+ 0.13. 3.24i 0.08' 4.05i 0.12.<br />
nlnol/ tninl mg protein or DNA<br />
pmoll minl rng protein or DNA<br />
moll mg protein or DNA<br />
The data are expressed as meaniSD for six animals! dose<br />
*p
Table 14. Effect of TCDD on the production of superoxide anion, nitric oxide and<br />
hydrogen peroxide in the caput, corpus and cauda epididyrnides of adult rats.<br />
TCDDl Kg body weight<br />
Control I ng 10 ng I00 ng<br />
Cuput cl~ididyrrri.~<br />
Superoxide anion' 2.01 i 0.41 2.1 1 1 0.1 I* 2.91 f 0.17. 3.14 * 0.31-<br />
Nitric oxideb 1.46-t 0.13 2.66-t 0.41. 2.71 f 0.34' 2.93 * 0.23*<br />
Hydrogen peroxideb 11.52 * 1.66 19.98 f 3.31' 36.25 + 2.05' 75.42 k 5.7IL<br />
Superoxide anion0 1.31 i 0.30 2.49* 0.10' 2.71 f 0.19' 3.01 i 0.17.<br />
Nitric oxideb 1.36* 0.21 2.01 f 0.18. 2.46-t 0.41' 2..91 i 0.14'<br />
Hydrogen peroxideb 20.50 f 1.68 37.70 13.87' 58.25 2 6.17: 109.6 i 19.3'<br />
Superoxide anionn 2.07 + 0.2 I 2.71 + 0.26. 2.97 i 0.40. 3.29 i 0.14.<br />
Nirric oxideb 1.91 -t 0.33 2.61 + 0.40. 2.93 + 0.17' 3.40* 0.16.<br />
tlydrogen peroxideb 15.76 f 1.40 20.11 i 1.42, 31.86 i 3.43. 52.8 + 5.28*<br />
nrnoll mid rng protein; mmoll mid mg protein<br />
nrnol Hz02 generatedl mid mp protein<br />
The number of animals per group is six<br />
'pc0.05 against the control group
Table 15. Effect of TCDD on the levels of antioxidants in the caput, corpus and cauda<br />
epididymides of adult rats<br />
TCDDJ Kg body weight<br />
Control I ng 10 ng 100 ng<br />
Capur epididymis<br />
Glutathione* 143 f 6.83 127 r 8.11. 121 t 4.21. 116 r 3.98.<br />
a-Tocopherolb 0.84i 0.06 0.71 f 0.12: 0.70+ 0.16. 0.69i 0.09.<br />
Corpus epidrdymb<br />
Glutathione' 140 i 7.23 120 t 6.26* 117 c 6.93' 113 i 4.90'<br />
a-Tocopherolb 0.82 i 0.1 1 0.74 + 0.06* 0.72 f 0.08' 0.70 t 0.06'<br />
" nmoll min/ mg protein; pmol/ min/ mg protein<br />
'I'he data are expressed as nieanfSD for six aninlalsl dosc<br />
*p
Tablel6a. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the<br />
caput cpididymis<br />
TCDD/ Kg body weight<br />
Control 1 ng 10 ny 100 ng<br />
Supcroxide disrnutase' 31.98 f 2.18 26.00 f 1.85' 20.13 f 1.57' 17.97 i 2.29'<br />
catalaseb 5.18f0.61 4.07+0.21* 3.78f 0.22- 3.65+0.27'<br />
Glutathionc reductasen 39.91 f 2.99 28.34 + 1.68' 23.22 + 1.93* 21.33 f 4.41'<br />
Glutathione pcroxidase'61.43 f 3.61 40.05 f 2.38. 30.87 + 1 .45* 27.07 2 3.40*<br />
Lipid peroxidation' 2.45 i 0.13 3.05 2 0.27. 4.02 * 0.25. 7.56 + 0.76*<br />
" nmoll mid mg protein; pmoll rnin / mg protein; ' bmol/ mg protein<br />
The data arc expressed as meaniSD for six animals/ dose<br />
*pcO.05 against the control group<br />
Table 16b. Effect of TCDD on the antioxidant enzymes and lipid peroxidation in the<br />
corpus cpididy~nis<br />
TCDDi Kg body weight<br />
Supcroxide disrnutase' 41.06 i 2.44 33.14 + 2.44' 30.39 f 1.8 1 * 24.23k 3.68'<br />
Catalaseb 4.1 7 f 0.31 3.80 f 0.29' 3.43 i 0.35. 3.21f 0.34*<br />
Glutathione rcductasea 37.48 i 2.40 30.08 + 2.17' 27.50 f 2.63. 23.88f 5.13'<br />
Glutathione pcroxidase'73.22 f 5.82 52.12 f 4.08. 44.94 + 4.66. 36.47i 6.86*<br />
Lipid peroxidation' 1.91 * 0.18 2.82 f 0.34. 3.34 i 0.37. 4.88~ 0.52'<br />
nmoll mid mg protein; pol/ min I mg protein; ' pmol/ rng protein<br />
Thc data arc expressed as rncanfSD for six animals/ dose<br />
*p
Table 16c. Effcct of TCDD on the antioxidant enzymes and lipid peroxidation in the<br />
cauda epididyrnis<br />
TCDD! Kg body weight<br />
Control 1 ng 10 ng 100 ng<br />
Superoxide dismutase' 45.78 i 2.64 36.57 * 2.53' 30.22 i 3.26. 26.56 f 2.36'<br />
Catalaseb 3.17i0.16 2.76f 0.22' 2.72i0.23' 1.80+0.12*<br />
Glutathione reduce' 42.69 * 2.62 32.67 + 2.59' 28.35 f 2.53' 15.36 f 1.86.<br />
Glutathione peroxidase'45.44 f 2.34 40.71 i 2.1 I* 34.81 f 2.53* 29.17 1t 2.52'<br />
Lipid peroxidationC 2.21 5 0.19 3.20 2 0.34* 4.05 + 0.39' 5.94 f 0.42*<br />
a nmol/ rnin.1 mg protein; pmol/ min 1 rng protein; pmolf mg protein<br />
The data are expressed as meaniSD for six animals! dose<br />
*p
Table 17b. Effect of TCDD on the levels of antioxidants in the kidney of adult rats<br />
TCDDl Kg body weight<br />
Control 1 ng 10 ng 100 ng<br />
Glutathione' 161 i 8.11 158 i 6.23 155 i 3.87 133 f 4.18*<br />
a-~oco~herol~ 0.90* 0.12 0.86i 0.17 0.80* 0.11 0.59i 0.10'<br />
nmoll mg protein; pgl mg protein<br />
The data are expressed as mcan*SD for six animals/ dose<br />
'pc0.05 against the control group<br />
Table 17c. Effect of TCDD on the antioxidant enzyliirs and lipid pcrosidation in lllc<br />
kidney of adult rats<br />
TCDDI Kg body weight<br />
Control 1 ng 10 ng 100 ng<br />
Superoxide dismutase' 34.56 I 3.10 35.21 i 2.93 33.27 2 2.24 28.13 '-2.16'<br />
Catalaseb 4.21 i0.70 4.19i0.38 4.15 k0.48 4.01 t0.18*<br />
Gli~tathione redmtase" 37.22 * 3.12 36 53 i 2.08 35.18 * 2.65 30.17 t 2.05'<br />
Cilulatliionc pcroxidoscd56.67 * 4.81 54. I? i 3.10 53.86 + 3.1 1 40.07 i 2.13.<br />
L~pid peroxidation' 2.1 1 * 0.17 2.16 f. 0.28 2.22 f. 0.30 2.46 i_ 0.25*<br />
* n~rioll mi"/ mg pmtein; wnoll min 1 m& protein;' bmoll nlg pmtein<br />
The data arc expressed as mcarutSD for six animals1 dose<br />
*P
Table 18a. Effect of co-administration of TCDD and vitamin E on the body weight and<br />
weights of the kidney, testis, epididymis and accessory ssx organs of adult<br />
male rats<br />
TCDD and vitamin E<br />
Control<br />
Body weight 116 f 2.04<br />
Testis 991 i 7.32<br />
Epididymis 413 t 4.56<br />
Seminal vesicles<br />
Ventral prostate 198 i 2.63<br />
Kidney 56814.13<br />
Body weight is expressed in gram and tissue weights are expressed in mg<br />
The data are expressed as mean + SD for six animalsl dose
Table 18b. Effect of oo-administration of TCDD and vitamin E on the weights of the<br />
kidney, testis, epididymis and accessory sex organs of adult male rats<br />
TCDD and vitamin E<br />
Control lng+20mg 10ng+20mg lOOng+20mg<br />
Testis 847 f 18.45 845 + 21.67 850+ 16.72 845 + 13.22<br />
Epididymis 353 * 6.49 348 + 10.14 353 f 7.17 350 f 6.94<br />
Seminal vesicles<br />
Intact 829+ 16.52 822 t21.99 838 i 18.74 827i 17.23<br />
Empty 704 + 16.65 696 i 16.96 705 + 15.56 700 f 13.26<br />
Ventral prostate 169 + 3.97 165 * 6.99 169 f 5.98 :67 f 5.81<br />
Kidney 467 i 4.87 480 i 3.47 478 ?; 3.12 470 i 2.87<br />
Tissue weights are expressed in rngl lOOg body weight.<br />
The data are expi-esd as meant SD for six ansmald dose
Table 21. Effect of co-administration of TCDD and vitamin E on the serum hormone<br />
lcvels in adult male rats<br />
--<br />
TCDD and vitamin E<br />
Control 1ngs20mg 10ng+20mg 100ng+20mg<br />
Testosterone' 2.51 i 0.13 2.48 f 0.08 2.35 f 0.05 2.34 _+ 0.06<br />
FSH' 15.66f 2.08 14.00k 0.40 15.25 i 1.84 14.63 r 0.30<br />
Prolactin' 18.33 f 1.52 17.50i 0.57 18.25 i 1.25 19.75 zt 1.21<br />
'ngl mL; bpg/ mL<br />
The data are expressed as mean<br />
SD for six animals/ dose<br />
Table 22. Effect of co-administration of TCDD and vitamin E on the activities of<br />
stemidogenic enzymes in testis.<br />
TCDD and vitamin E<br />
Cotitrol Ing+20mg lOng+201ng 100ng+20mg<br />
3-P hydroxysreroid 54.31 2 2.68 56.14 k 3.96 55.63 f 3.01 53.44 + 3.46<br />
dehydrogenax<br />
The data arc expressed in nmolel mild mg protein<br />
The data are expressed and rneaniSD for six animalsi dose
Table 23. Effect of co-administration of TCDD and vitamin E on the DNA, RNA and<br />
protein contents in testis<br />
TCDD and vitamin E<br />
Control Ing+20mg 10ng+20mg 100ng+20mg<br />
DNA 10.62 t 2.1 1 11.31 f 2.48 10.48 * 1.96 12.1 1 * 2.08<br />
RNA 5.46 i 1.91 6.07 + 1.34 6.12 f 1.20 5.69 i 1.07<br />
Protein 8.76 i 0.13 9.1 1 i 1.23 9.41 t 1.40 8.97 i 0.10<br />
Nucleic acids and protein contents are expressed in mg/ g tissue wet weight<br />
The data are expressed as mean * SD for six animals! dose<br />
Table 24a. Effect of co-administration of TCDD and viranlir~ E on the productio~~ ot'<br />
superoxide anion in the c ~de homogenate, mitochondria1 and microsomerich<br />
fractions of rat testis<br />
TCDD and vitamin E<br />
Control lng+20mg lOng+20mg 100ng+20mg<br />
Crude homogenate 3.76 f 0.12 3.79 i 0.18 3.80 f 0.40 3.79 k 0.27<br />
Mitochondria1 fractions 2.13 i 0.19 2.21 i 0.12 2.19 f 0.21 2.20 i 0.17<br />
Microsome-rich<br />
fractions 1.61 i 0.31 1.62 t 0.27 1.68 i 0.12 1.67 i 0.13<br />
'l'hc data are cxpresscd in nniold cninl~ng pn)tcin<br />
The data are expressed as mean f SD for six animals! dose
Table 24b. Effect of co-administration of TCDD and vitamin E on the production of<br />
nitric oxide in the crude hornogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
-<br />
TCDD and vitamin E<br />
Control 1ng+2Omg IOng+20rng 100ng+20mg<br />
Crude homogenate 1.7850.ll 1.83f0.16 1.88fO.l2 1.87i0.21<br />
Mitochondrial fractions 1.37 i 0.16 1.47 i 0.13 1.42 f 0.1 1 1.40 * 0.17<br />
Microsome-rich<br />
fractions 1.465 0.42 1.495 0.14 1.53 * 0.13 1.51 f 0.13<br />
The data are expressed in mmold mint mg protein<br />
The data are expressed as mean * SD for six animalsi dose<br />
Table 24c. Effect of co-administration of TCDD and vitamin Eon the hydrogen peroxide<br />
generation in the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
TCDD and vitamin E<br />
Control I ng+20mg IOng+ZOmg 100ng+2Omg<br />
Cmdc homogenate 16.16 f 2.17 17.23 5 2.62 17.68 5 2.30 17.00 k 1.96<br />
Mitochondrial fractions 14.26f 2.41 15.37 f 1.96 14.46 f 1.87 13.19 f 1.91<br />
Microsome-rich<br />
fractions 10.62f 1.30 10.78 f 0.96 11.21 f 1.40 12.01 * 1.81<br />
The data are expressed in nmold minl rng protein<br />
The data ate cxpmsed as rneanfSD for six animalsi dose
Table 25a.<br />
Effect of co-administration of TCDD and vitamin E on the level of<br />
glutathione in the crude homogenate, mitochondria1 and microsome-rich<br />
fractions of rat testis<br />
TCDD and vitamin E<br />
Control 1 ng+20mg 10ng+20mg 100ng+20mg<br />
Crude homogenate 346 i 8.21 340 -+ 7.21 343 * 6.26 349 f 7.62<br />
Mitochondrial fractions 174 f 2.81 180 i 9.31 176 f 6.14 179 f 5.62<br />
Microsome-rich<br />
fractions 149f 3.13 156i 4.87 15Oi 4.91 1572 5.13<br />
The data arc expressed in nmolel mg protein<br />
The data are expressed as mcanfSD for six animals/ dose<br />
Table 2Sb. Effect of co-administration of TCDD and vitamin E on the level of a-<br />
tocopherol in the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
TCDD and vitam~n E<br />
Control 1 ng+2Omg IOng+20mg 100ng+20mg<br />
Crude homogenate 1.48 * 0.03 1.36 ;t 0.08 1.44 i 0.06 1.40 i 0.04<br />
Mitochondrial fractions 0.91 i 0.10 0.92 i 0.09 0.97 i 0.13 0.90 k 0.09<br />
Microsame-rich<br />
fractions 0.62 * 0.16 0.63 i 0.10 0.63 i 0.06 0.62 + 0.07<br />
The data are expressed in pgl mmy protein<br />
The data are expressed as msanf SD for six animals/ dose
Table 25c. Effect of co-administration of TCDD and vitamin Eon the lcvcls of ascorbic<br />
acid in the crude homogenate, mitochondria1 and ~nicrosome-rich fractions<br />
of rat testis<br />
TCDD and vitamin E<br />
Control I ng 10 ng 100 ng<br />
Crude homogenate 3.68 f 0.1 1 3.60 t 0.15 3.63 f 0.16 3.58 r 0.20<br />
Mitochondrial fractions 2.36 * 0.26 2.23 i 0.19 2.28 f 0.20 2.20 * 0.23<br />
Microsome-rich<br />
fractions 1.85f 0.18 1.79f 0.16 1.90k 0.20 1.87i 0.19<br />
The data are expressed in md g tissue wet weight<br />
The data are expressed as mean f SD for six animals/ dose<br />
Table 26a.<br />
Effect of co-administration of TCDD and vitamin E on the activity of<br />
superoxide dismumse in the crude hornogenate, mitochondria1 and<br />
microsome-rich fractions of rat testis<br />
TCDD and vitamin E<br />
Control I ng+20mg IOng+20mg 100ng+20mg<br />
Crude hornogenatc 33.14 f 1.68 32.66 f 1.43 34.1 1 i 1.21 33.16 + 1.04<br />
Mitochondrial fractions 26.98 f 2.75 25.16 2 1.92 24.48 k 1.68 25.13 + 1.88<br />
Micmsonic-rich<br />
fractions 21.21 * 1.34 20.68i2.01 21.37i 1.98 20.45 t 1.89<br />
The data are expressed in nmolc/ mid mg protein<br />
The data arc expressed as meaniSD for six animals/ dose
Table 26b. Effect of co-administration of TCDD and vitanlin E on thc activity ol'catalasc<br />
in the crude homogcnate, mitochondrial and microsome-rich fractions of rat<br />
testis<br />
TCDD and vitamin E<br />
Control 1 ng+20mg IOng+20mg 100ng+20mg<br />
Crude homogenate 0.880 f 0.04 0.879 f 0.01 0.882 f 0.03 0.881 1 0.04<br />
Mitochondrial fractions 0.630 f 0.1 1 0.627 i 0.1 0 0.63 1 i 0.09 0.63 1 + 0.1 1<br />
Microsomc-rich<br />
fractions 0.398 f 0.10 0.395 i 0.02 0.396 + 0.06 0.396 + 0.02<br />
The data are expressed in pmole/ mid mg protein<br />
The data are expressed as mcaniSD for six a~~iniald dose<br />
Table 26c. Effect of co-administration of TCDD and vitamin E on the activity of<br />
glutathione ductase in the crude homogenate, mitochondria1 and<br />
microsome-rich fractions of rat testis<br />
TCDD and vitamin E<br />
Control I ng+20mg 10ng+20mg 100ng+20mg<br />
Crude homogcnate 45.01 i 1.3 1 44.24 1 1.68 45.43 f 2.01 44.62 1 2.1 1<br />
Mitochondrial fractions 32.44 * 1.98 33.16 i 1.60 32.64 i 1.01 33.14 i 2.07<br />
The data are expressed in nmolc/ m id mg protein<br />
The data are expressed as mcanfSD for six animals1 dose
Table 26d. Effect of co-administration of TCDD and vitamin E on the activity ol'<br />
glutathiom peroxidase in the c~de homogenate, mitochondrial and<br />
microsome-rich fractions of rat testis<br />
- - - -<br />
TCDD and v~tamin E<br />
Control Ing+20mg 10ng+20mg 100ng+20m6<br />
Crude homogenate 43.53 * 2.57 44.16 i 1.88 44.67 i 1.61 43.26 i 2.10<br />
Mitochondrial fractions 30.66 * 2.10 32.14 * 1.8 1 32.66 f 1.66 3 1.26 i 1.07<br />
Microsome-rich<br />
fractions 25.11 k3.17 24.42i2.92 26.6212.01 24.141 2.93<br />
The data am expressed in nmold mid mg protein<br />
The data am expressed as mean iSD for six animalsl dose<br />
Table 27. Effect of co-administration of TCDD and vitamin E on the level of lipid<br />
peroxidation in the crude homogenate, mitochondrial and microsome-rich<br />
fractions of rat testis<br />
TCDD and vitamin E<br />
Control I ng+20mg IOng+20mg lOOng+2Omg<br />
Crude homogenate 2.17 0.16 2.21 * 0.23 2.20 f 1.04 2.18 * 1.21<br />
Mitochondrial fractions 2.1 1 i 0.21 2.14 i 0.27 2.10 * 0.17 2.1 1 + 0.19<br />
The data arc expressed in pnole/ minl mg protein<br />
The data arc expressed as mean* SD for six animals1 dose
'I'able 28.<br />
Eff'ecl ol' co-admi~listratiott ol' 'I'CVD ettd vitamill I2 otl lhc pi.oductio~t ol'<br />
suproxide anion, nitric oxide and hydrogen peroxide in the epididymal<br />
sperm of adult rats<br />
TCDD and vitamin E<br />
Control<br />
Superoxide anion'<br />
(mg protein)<br />
(mg DNA)<br />
Nitric oxideb<br />
(mg protein)<br />
(mg DNA)<br />
H102 generationc<br />
(mg protein)<br />
(mg DNA)<br />
' nmoV mid mg protein or DNA; mmoV minl mg protein or DNA; nmol H202<br />
generated1 mint mg protein or DNA; The data are expressed as meanfSD for six<br />
animals1 dose<br />
Table 29. Effect of co-administration of TCDD and vitamiti E on the levels of<br />
antioxidants in the epididymal sperm of adult rats<br />
TCDD and vitamin E<br />
Control Ing+20mg 10ng+20mg 100ng+20mg<br />
Glutathione'<br />
(mg protein) 1622 2.18 170 f 2.16 178f 4.10 176f 4.01<br />
(mg DNA) 146f2.11 153f2.11 154i2.74 145f2.24<br />
a-tocopherolb<br />
(mg protein) 0.461 f 0.04 0.477 + 0.12 0.476f 0.14 0.471 i 0.24<br />
(mg DNA) 0.354 f 0.06 0.366 f 0.28 0.367 f 0.21 0.361 f 0.26<br />
' nmoV minl mg protein or DNA; Clp/ mg protein or DNA<br />
The data arc expressed as meaniSD for six mimalsl dose
Table 30. Effect of co-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in the epididylnal sperm oradult rats<br />
TCDD and vitamin E<br />
Control 1ng+20mg 10ng+20mg 100ng+20mg<br />
Superoxide disrnutase'<br />
(mg protein) 19.84 f 0.89 19.93 f 1.36 20.16 f 1.57 19.81 -t 0.42<br />
(mg DNA) 16.84 5 0.64 17.01 f 0.76 17.01 f 0.77 17.04 & 0.89<br />
catalaseb<br />
(mg protein) 2.12i 0.08 2.13* 0.10 2.151 0.12 2.12f 0.12<br />
(mg DNA) 1.80 f 0.03 1.82 i 0.03 1.81 * 0.03 1-82 2 0.04<br />
Glutathione ductase'<br />
(mg protein) 34.03 i 1.45 33.80f 1.61 34.36 * 1.94 33.54 +- 2.38<br />
(mg DNA) 28.87* 0.51 28.85 f 0.67 29.01 f 0.45 28.76& 0.96<br />
Glutathione peroxidase'<br />
(mg protein) 58.40 f 2.58 58.70 * 2.85 58.96 i 3.19 58.06 + 3.48<br />
(mg DNA) 49.53i 0.85 50.12* 1.24 49.80f 1.13 49.92i 0.98<br />
Lipid peroxidation'<br />
(mg protein) 2.22* 0.16 2.19i 0.16 2.20f 0.19 2.19* 0.18<br />
(mg DNA) 1.88iO.09 1.87f0.07 1.8620.12 1.87+0.06<br />
nmold mid mg protein or DNA<br />
moV mid mg protein or DNA<br />
pnol rnalondialdehyde produced/ mg protein or DNA<br />
The data are expressed as mean* SD for six animals1 dose
Table 31. Effect of TCDD on the production of superoxidc anion. nitric oxide and<br />
hydrogen peroxide in the caput, corpus and cauda epididyunides of adult rats<br />
TCDD and vitamin E<br />
Control Ing+20mg IOng+20mg 100ng+20mg<br />
Capur epididymh<br />
Superoxide anion' 1.69tt 0.09 1.76i 0.12 1.79k 0.18 1.76 + 0.17<br />
Nitric oxideb 1.39i 0.19 1.43* 0.09 1.47i 0.16 1.49 i 0.11<br />
Hydrogen peroxidec 13.43 i 1.40 11.75 i 1.07 11.73 * 1.39 11.47 2 0.73<br />
Corpus epididymb<br />
Superoxide aniona 1.71 + 0.11 1.76+ 0.21 1.792 0.13 1.81 + 0.12<br />
N~tric oxideb 1.372 0.13 1.37+ 0.09 139i 0.08 1.442 0.30<br />
Hydrogen peroxidec 17.88 + 0.93 15.84 1.41 14.66 + 1.16 13.18 + 1.32<br />
Coudo epidi+mis<br />
Superoxide anion' 1.82 + 0.09 1.92 * 0.13 1.87 1 0.36 1.84 i 0.14<br />
Nitric oxideb 1.29f 0.21 1.37+ 0.09 1.33 1- 0.16 1.35+ 0.13<br />
Hydrogen peroxidec 15.85 i 0.49 15.22 i 1.08 14.89 i 0.86 14.89 + 0.74<br />
a nmole/ mini rng protein; mrnolel rnin/ mg protein<br />
nmol Hz02 generated1 minl mg protein<br />
The data are expressed as rneanYSD for six animals/ dose
Table 32.<br />
Effect of caadrninistration of TCDD and vitamin E on the levels of<br />
antioxidants in the caput, corpus and cauda epididymides of adult rats<br />
TCDD and vitamin E<br />
Control 1 ng+20mg 10ng+20mg 100ng+2Omg<br />
Glutathione' 1675399 15654.90 15826.31 160i4.11<br />
a-~ocopherol~ 0.83i 0.1 1 0.84i 0.09 0.84t 0.40 0.84i 0.17<br />
Corpus epididymis<br />
Glutathione' 149 f 6.14 152 i 2.62 150 i 3.21 151 i 4.26<br />
a-Tocopherolb 0.84 i 0.16 0.844 2 0.21 0.84 2 0.20 0.82 i 0 I l<br />
Glutathione' 160 * 5.26 163 i 6.16 166 i 7.11 162 5 7.36<br />
a-~oco~herol~ 0 85 f 0.1 l 0.86 i 0.26 0.85 + 0.21 0.85 + 0.32<br />
nmoldmg protein; pg mg protein<br />
The data are expressed as rneanfSD for six animals/ dose
Table 33a. Effect of co-administration of TCDD and vitamin E on tlic antioxidant<br />
cnzymcs and lipid pcroxidation in the caput cpididy~iiis<br />
TCDD and vitamin E<br />
Superoxide dismutasea 33.82 i 2.35 33.47 f 1.58 33.66 f 2.39 33.84 * 1.78<br />
catalaseb 2.56i0.17 2.51 i 0.10 2.52f 0.18 2.545 0.15<br />
Glutathione reductase' 43.46 f 2.82 42.84 f 1.68 43.23 * 2.96 43.49 12.84<br />
Glutathione peroxidase'64.49 f 4.32 63.53 i 3.09 63.91 k 4.25 64.1 1 f 4.22<br />
Lipid peroxidatione 2.50 i 0.1 l 2.35 * 0.20 2.30 i 0.14 2.35 f 0.21<br />
'nmold min/ mg protein; pnold minl rng protein<br />
' pmole malondialdehyde producedl mg protein<br />
The data arc expressed as me.aniSD for six animals/ dose<br />
Table 33b. Effect of co-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in the corpus epididymis<br />
TCDD and vitamin E<br />
Control 1ng+20rng IOng+20mg 100ng+20mg<br />
Superoxide dismutasc' 36.39 * 1.49 37.1 1 f 2.48 36.91 i 3.48 37.03 _+ 2.32<br />
Catalascb 3.49f 0.18 3.51 i0.17 3.47i0.27 3.45i 0.15<br />
Glutathione reductasec 32.80 * 1.53 33.20 f 1.66 32.78 It 2.73 23.63 5 1.49<br />
Glutathione peroxidasec62.36 f 3.74 62.85 f 2.98 62.24 i 4.20 62.14 f 3.52<br />
Lipid peroxidation' 2.46 f 0.30 2.49 i 0.12 2.60 i 0.29 2.55 + 0.23<br />
'nmold mint rng protein; wold minl mg protein<br />
'wnole malondialdehydc produced1 mg protein<br />
The data arc expressed as rneaniSD for six animals/ dose
Table 33c. Effcct of co-administrution or 'I'CnD and vil;~~nin I:, OII tlw :~~~tiosidi~~~t<br />
enzymes and lipid peroxidation in the cauda epididymis<br />
TCDD and vitamin E<br />
Control Ing+20mg IOng+20mg 100ng+20mg<br />
Superoxide dismutase' 43.80 * 1.88 44.71 f 2.05 44.64 r 1.57 44.79 i 2.18<br />
catalaseb 2.99i0.13 3.03 f 0.15 3.01 i0.08 3.02t0.12<br />
Glutathione reductase' 40.18 * 1.49 40.89 2 2.20 40.6 1 * 1.46 40.75 f 1.68<br />
Glutathione peroxidasec41.85 * 1.87 42.53 t 2.05 42.45 f 1.39 42.42 f 1.56<br />
Lipid peroxidationd 1.91 * 0.14 1.85 + 0.19 1.80* 0.15 1.78 i 0.13<br />
'nmolel m~nl mg protein; pnolel mid mg protein<br />
' pmole malondialdehyde produced/ mg protein<br />
The data are expressed as mcaniSD for six animals1 dose<br />
Table 34a. Effect of co-administration of TCDD and vitamin E on the production of<br />
supzmxide anion, nitxic oxide and hydrogen peroxide in the kidney of adult<br />
rats<br />
TCDD and vitamin E<br />
Control I ng + 20 mg 10 ng + 20 nig 100 11g + 20 nlg<br />
Superoxide anion' 2.1 1 i 0.18 2.16 i 0.22 2.09 i 0.1 1 2.13 i 0.21<br />
Nitric oxideb l.5lO.ll 1.53f 0.15 1.60f 0.17 1.59 f 0.16<br />
Hydrogenperoxidd 11.12 t2.01 12.76i 1.97 13.70k 2.11 13.51 _+ 1.67<br />
nmolel mid mg protein; mmoW mint mg protein<br />
' nmol H201 generated1 mint mg protein<br />
The data arc expressed as moanfSD for six animals/ dose
Table 34b. Effect of co-administration of TCDD and vitamin E on the levels of<br />
antioxidants in the kidney of adult rats<br />
-<br />
TCDD and vitamin E<br />
Control I ng+20mg 10ng+20mg 100ng+20mg<br />
Glutathione' 178 f 5.21 170 i 9.29 188 * 7.63 172 i 8.33<br />
a-~ocopherol~ 0.93 f 0.16 0.891 0.07 0.80-f 0.15 0.83 ?C 0.20<br />
nmolelmg protein; & mg protein<br />
The data arc expressed as mean* SD for six animals/ dose<br />
Table 34c. Effect of co-administration of TCDD and vitamin E on the antioxidant<br />
enzymes and lipid peroxidation in the kidney of adult rats<br />
TCDD and vitamin E<br />
Control Ing+20rng 10ng+20mg 100ng+20mg<br />
Superoxide dismutasc' 37.1 1 i 3.18 36.98 t 2.67 37.38 f 3.10 35.13 f 2.23<br />
Catalascb 2.61 0.23 2.58 i 0.20 2.56k 0.16 2.61 * 0.26<br />
Glutathione reductare' 42.16 f 3.3 1 40.01 12.56 42.76 i 3.10 40.54 i 3.91<br />
Glutathione peroxidax'63.18 i 3.12 60.8914.32 60.41 13.88 59.15 3.10<br />
Lipid peroxidationC<br />
2.22 i 0.16 2.28 f 0.26 2.31 t 0.19 2.28 ~ 0.18<br />
'nmole! mid mg protein; tunold minl mg protein<br />
' wlnolc malondialdehyde pmduccdl I I I protein<br />
~<br />
The data am expressed as mean* SD for six animals1 dose
5 DISCUSSI<strong>ON</strong><br />
5.1 Significance of the experimental procedures<br />
Rats were treated orally with graded doses of TCDD and the effects were seen<br />
on various parameters of male reproductive functions. In order to see the effects of<br />
vitamin E on TCDD-induced alterations in the testis and epididymis the rats were<br />
administered with vitamin E along with TCDD.<br />
5.1.1 Experimental treatments<br />
TCDD is one of the most potent environmental contaminants, which has been<br />
shown to alter male reproduction of rats causing reduced fertility. In the present study<br />
TCDD (1, 10 and 100 ngi Kg body weight) day) was administered orally to 45 days<br />
old male rats for 45 days in order to see the effects of subchronic exposure of TCDD<br />
on functional aspects of male reproduction. In the subchronic toxicity test with rats<br />
exposure usually begins at 6 to 8 weeks of age (US Environmental Protection Agency,<br />
1998). Rats at postnatal day 45 have been reported to be at the threshold of puberty<br />
where the first spermatozoa are seen in the lumen of the seminiferous tubules<br />
(Clermont and Perey, 1957). The duration of the whole process of spermatogenic<br />
cycle in male rats is approximately 45 days (Clermont and Perey, 1957). In the<br />
present studies rats were treated from puberty to adulthood through a complete<br />
spermatogenic cycle. TCDD was administered orally because most of the<br />
environmental contaminants enter into the body through this route.<br />
For standardization of doses, rats were treated with seven different doses of<br />
TCDD for 45 days. The doses ranged from 0 to 1000 ng/ Kg body weight/ day and 3<br />
animals were used for each dose. The body weight gains were recorded for 45-days.<br />
Administration of TCDD at the dose levels of 0 to 100 ng/ Kg body weighti day for<br />
45 days did not show any significant changes in the body weight (Fig. 2 & 3).<br />
However, adminiseation of TCDD at higher doses decreased body weight of the
animals (Fig. 2) thereby causing systemic toxicity. Thus, we selected doses ranging<br />
between 1 to 100 ng/ Kg body weight/ days for 45 days.<br />
Vitamin E is a lipophilic compound most commonly used as an antioxidant.<br />
Administration of vitamin E at the dose level of 20 rngl Kg body weight/ day is<br />
considend to be a therapeutic dose. In the present study TCDD (1, 10 and 100 ng/<br />
Kg body weight1 day) was administered orally along with vitamin E (20 mgl Kg body<br />
weight/ day) to the pubertal rats for 45 days in order to see the protective effects. if<br />
any. exerted by vitamin E supplementation on the TCDD-induced toxicity in male<br />
reproductive system of rats. The animals were killed after 24 h of the last treatment in<br />
order to see the effects of TCDD, with or without supplementation of vitamin E on the<br />
male reproductive system of rats.<br />
5.1.2 Significance of the parameters studied<br />
Monitoring body weight of the animals during treatment provides an index of<br />
the general health status of the animals and such information is important for the<br />
interpretation of reproductive effects. Depression in body weight or reduction in<br />
weight gain may reflect a variety of responses. including rejection of feed and water,<br />
treatment induced anorexia or systemic toxicity. In the present study the weights of<br />
the animals were recorded to monitor such changes. Weight of the testis is largely<br />
dependent on the mass of the differentiated spermatogenic cells and it has been used<br />
as a crude measure of the damage to spermatogenesis (Schlappack el 01.. 1988). A<br />
strong correlation exists between weight of the testis and number of germ cells (Sinha<br />
Hikim el 01.. 1989). Reduction in the weight of testis has been shown to occur due to<br />
the loss of germ cells (Setchell and Galil, 1983). In the present study weight of the<br />
testis was taken to assess the maintenance of various testicular functions.<br />
Weights of the accessory sex organs are dependent on the availability of<br />
androgens, as castration has been shown to reduce the weights due to absence of<br />
androgens. Incrcase in the availability of testosterone has teen shown to cause<br />
bpertmphy md consequent increase in the weights of the accessory sex organs.
Measurement of weights of the accessory sex organs in intact male rats has been<br />
demonstrated to reflect the cumulative effect of biologically active testosterone over a<br />
period of time (Mathur and Chattopadhyay, 1982). In the present studies weights of<br />
epididyrnis, seminal vesicles and ventral prostate were taken to assess the<br />
bioavailability andl or production of androgen and the cumulative effect of<br />
androgenic activity. Weight of kidneys of the castrated mice decreased by 30 - 50%<br />
in comparison to that of intact animals and has been reported to be androgen<br />
dependent. The toxicity threshold varies in different species as well as varies in<br />
different organs in the same animals. In the present study weight of kidney was taken<br />
to assess the toxicity threshold between reproductive and non-reproductive tissue.<br />
Daily sperm production in rats reflects the status of spermatogenesis in control<br />
and experimental animals. Epididymal sperm viability. epididymal sperm motility<br />
and sperm count alter when exposed to environmental toxicants. In the present study<br />
daily sperm production, epididymal sperm viability, motility and count were done in<br />
order to assess the status of spermatogenesis.<br />
Histometric studies on testis provide strongest evidence to see the difference<br />
between control and experimental tissues (Russell rl a/., 1990).<br />
A positive<br />
relationship exists between tubular diameter and spermatogenic activity of testis<br />
(Sinha Hikim ct a/.. 1989). Measurements of tubular diameter have bren reponed to<br />
discriminate between varying levels of sp.rnmatogcnic damage (Russcll ct
of testosterone, estradiol, FSH, LH and prolactin were determined in extracellular<br />
fluid to assess any alteration in their availability to target sites.<br />
The two key enzymes involved in the biosynthetic pathway of testosterone are<br />
3p-hydroxysteroid dehydrogenase and I7P-hydroxysteroid dehydrogenase. The<br />
activities of 3P-hydroxysteroid dehydrogenase and 17P-hydroxysteroid<br />
dehydrogenase have been used to study testicular steroidogenesis of rats in different<br />
experimental conditions (Ghosh e! a/., 1991; 1995: Sujatha er u/., 2001). It has been<br />
reported that increased production of reactive oxygen species decreased the activities<br />
of steroidogenic enzymes in testis of rats (Sujatha el a/.. 2001). Determination of<br />
activities of 30-hydroxysteroid dehydrogenase and 17P-hydroxysteroid<br />
dehydrogcnase in testis reflected the status of steroidogenesis.<br />
Nucleic acid contents in testis vary in different stages of spermatogenesis and<br />
the amount of DNA in the primary spermatocyte, secondary spermatocyte and<br />
spermatid has been shown to be present in the ratio of 4: 2: 1 (Davson and Segal,<br />
1975). Protein and RNA synthesis occur both in somatic cells and in germ cells. In<br />
the present study quantitative determination of DNA in testis indicated the rate of cell<br />
division and subsequent maturation. Determination of the levels of FWA and protein<br />
showed thc resultant effect of intracellular synthetic and breakdown processes.<br />
The superoxide anion. nitric oxide and hydrogen peroxide are reactive oxygen<br />
species produced from the cell under normal cellular metabolism. Increased<br />
production of superoxide anion and hydrogen peroxide under oxidative stress has<br />
been reported in the reproductive and and non-reproductive tissues of the animals<br />
(Hassoun el 01.. 2001; Sujatha er al., 2001). Productions of superoxide anion, nitric<br />
oxide and hydrogen peroxide were determined in testis as well as in the epididyrnal<br />
sperm and epididymis in order to assess the levels of reactive oxygen species in<br />
tissues.<br />
The levels of antioxidants such as glutathione, a-tocopherol and ascorbic acid<br />
play an imponant role in the maintenance of tissue against reactive oxygen species.<br />
These antioxidants have been reported to be distributed in the subcellular
compartment of the cells (Chainy el at., 1997; Reddy er at., 1999). Determination of<br />
the levels of glutathione, a-tocopherol and ascorbic acid reflect the status of<br />
antioxidants in tissues.<br />
Superoxide dismutase, catalase, glutathione reductase and glutathione<br />
peroxidase are antioxidant enzymes distributed in different subcellular fractions of<br />
testis as well as in the epididymal sperm and epididymis (Sujatha et al., 2001; Chitra<br />
er a/., 2001; Latchoumycandane et at., 2002a, b, c). Superoxide dismutase is<br />
considered to be the first line of defense against deleterious effects of oxyradicals in<br />
cell by catalyzing dismutation of superoxide anion to hydrogen peroxide and<br />
molecular oxygen. Catalase, glutathione reductase and glutathione peroxidase protect<br />
cells from highly toxic hydrogen peroxide by catalyzing hydrogen peroxide into water<br />
and oxygen. In the present studies the activities of superoxide dismutase, catalase,<br />
glutathione reductase and glutathione peroxidase in testis. epididymal sperm and<br />
epididymis were determined to assess the activities of the antioxidant enzymes in the<br />
TCDD-treated rats.<br />
5.2 Effect of TCDD on male rats<br />
Effect of TCDD on the body weight, organ weights and reproductive<br />
parameters of male rats are discussed in the following sections.<br />
5.2.1 Effect of TCDD on body and organ weights<br />
Administration ol"l'CDD did not cause any signilicant cha~~gc in h)d) weight<br />
of rats as compared to the corresponding groups of control animals (Fig. 3; Table la)<br />
~ndicating that the general me~aboliconditions of the animals wcrc within the nonnal<br />
range and TCDD at these doses did not cause systemic toxicity.<br />
Administration of TCDD caused a significant reduction in the weight of the<br />
testis as compared to the corresponding groups of control animals (Fig. 4a; Table la<br />
& I b). In the male rats TCDD exposure during adulthood has been shown to decrease<br />
weight of the testis (Johnson er at., 1992). It has been reported that TCDD induces a
eduction in Leydig cell volume per testis (Wilker, 1996). Reduction in the weight ol'<br />
the testis could be due to the Loss of germ cells by direct inhibition of TCDD on<br />
spermatogenesis or an indirect effect through inhibition of testosterone production.<br />
The present findings are consistent with previous report in which reduction in weight<br />
of the testis may be due to the loss of germ cells and Sertoli cells in the TCDD-treated<br />
rats (Mably el al., 1992; Johnson el ul., 1992).<br />
The weights of epididymis, seminal vesicles and ventral prostate decreased<br />
significantly in adult rats while weight of the kidney remained unchanged following<br />
administration of TCDD (Fig. 4b to 4f; Table la & I b). Epididyrnis and accessory<br />
sex organs require a continuous androgenic stimulation for their normal growth and<br />
functions (Klinefelter and Hess, 1998). It has been reported that accessory sex organs<br />
are androgen dependent and thus reflect the availability of androgen over a period of<br />
time (Mathw and Chanopadhyay. 1982). TCDD has also been reported to reduce<br />
plasma testosterone and dihydrotestosterone in the rats (Mably er ul., 1992).<br />
Inhibition on the weights of the epididymis, seminal vesicles and ventral prostate may<br />
be either due to the reduction in the levels of serum testosterone or a direct action of<br />
TCDD on the epididymis and accessory sex organs. 'She observed reduction in thu<br />
weight of accessory sex organs also reflected reduced bioavailability andl or<br />
production of androgen in the TCDD-treated rats.<br />
In the present studies<br />
administration of TCDD at these doses did not affect the weights of the extra-<br />
reproductive tissues like kidney though the activity of P-gluco~nidase in kidney has<br />
been demonstrated as testosterone dependent enzyme (Malarvizhi and Mathur, 1996).<br />
5.2.2 Effect oCTCDD oo daily spcrm production<br />
Adn>inistntion of TCDD dccrcascd siynilicunlly thc doily spcrm production<br />
as compared to the corresponding groups of control animals (Fig. 5a: Table 2). Thc<br />
observed reduction in the daily sperm production also rellects reduction in<br />
spermatogenesis. The present findings are consistent with the earlier reports in which
the reduction in daily sperm production has been shown in TCDI>-treated rats<br />
(Sommer el ul., 1996; Faqi el ul., 1997; 'l'hcohald and I'ctorson, 1997).<br />
5.2.3 Effect of TCDD on epididymal sperm viability, motility and count<br />
The epididymal sperm viability remained unchanged while the epididymal<br />
sperm motility decreased significantly in a dose-dependent manner in the animals<br />
administered with TCDD (Fig. 5a & 5b; Table 2).<br />
Administration of TCDD decreased epididymal sperm count significantly<br />
when compared to the corresponding groups of control animals (Fig. Sa; Table 2).<br />
The present findings are consistent with the earlier report in which the reduction in<br />
daily sperm production and epididymal sperm count in the rats treated with TCDD<br />
(Sommer el al.. 1996; Faqi el al., 1997; Theobald and Peterson, 1997).<br />
5.2.4 Effect of TCDD on seminiferous tubular and lumen diameter<br />
Diameter of the seminiferous tubular and lumen decrcased signilicantly alicr<br />
administration of TCDD as compared to the corresponding groups of control animals<br />
(Fig. 6; Table 3). The reduction in the tubular and lumen diameter of' thc<br />
seminiferous tubules may be due to a direct action of TCDD on the seminiferous<br />
tubules or an indirect effect through inhibition of testosterone production. The<br />
reduction in the tubular diameter has been correlated with decreased spermatogenic<br />
activity of the testis (Sinha Hikim cl a/., 1989).<br />
5.2.5 EfTcct of TCDD on the levels of serum horlnoi~cs and stcrc~idogei~ic<br />
enzymes in testis<br />
The levels of serum testosterone and rstradiol were found lo be decreased in<br />
the TCDD-treated rats along with reduction in the activities of 3P-hydroxysteroid<br />
dehydrogenase and I7p-hydroxysteroid dehydrogenase in testis (Fig. 7a to 7e & 8:<br />
Table 4 & 5). A decrease in the intratesticular and serum testosterone concentration<br />
has been reported in rats following administration of TCDD (Wilker. 1996). It has
een reported that TCDD decreases volume of the smooth e~idoplasmic reticulum and<br />
mitochondria per testis and has been shown to alter testicular steroidogenesis in rats<br />
and thereby reduce the production of testosterone and estradiol in rats (Wilker, 1996).<br />
The decrease in the serum testosterone levels in adult rats could be due to the<br />
diminished responsiveness of Leydig cells to LH and or the direct inhibition of<br />
testicular steroidogenesis. It has been reported that two of the steroidogenic enzymes<br />
involved in the conversion of cholesterol to testosterone in Leydig cells are<br />
cytochrome P450 enzymes (P450scc and P450c17). Molecular oxygen and electrons<br />
donated from NADPH were used for hydroxylation of the substrate. During normal<br />
steroidogenesis, ROS (superoxide anion and or hydroxy radical) can be produced by<br />
electron leakage outside the electron transfer chains (Homsby and Crivello, 1983;<br />
Hanukoglu el al., 1993), and these free radicals can initiate lipid peroxidation, which<br />
can inactivate P450 enzymes (Homsby, 1980). The inability of the pseudosubsuate to<br />
be oxygenated promotes the release of reactive oxygen species (Peltola et al., 1996).<br />
It has been reported that increased production of reactive oxygen species following<br />
lindane treatment decreased the activities of steroidogenic enzymes in testis of rats<br />
(Sujatha el a/.. 2001).<br />
The levels of serum FSH and LH remained unchanged while the level of<br />
prolactin decreased significantly in the animals administered with TCDD (Fig. 7c to<br />
7e; Table 4). This is supported by previous reports that TCDD does not alter serum<br />
FSH and LH levels in rats (Moore er 01..<br />
1989) thereby suggesting that pituitary<br />
hypofunction may not be a major cause of the initial stages of subchronic TCDD<br />
toxicity. It has also been suggested that growth retardation in TCDD-treated rats may<br />
not be the result of a deficiency of growth hormone (GH), alterations in plasma<br />
conicosterone concentrations may due to altered responsiveness of the adrenal to<br />
ACTH stimulation rather than to changes in plasma ACTH concentrations. and that<br />
impaired spermatogenesis may not be associated with a decrease in plasma FSH<br />
concentrations (Moore el ul., 1989).<br />
l'hc levels of scruni prolnctili dccrcucd<br />
significantly in the animals wated with TCDD. Similar changes have also been
eported in the TCDD-treated rats (Moore rt al.. 1989). It has been rcportcd that<br />
prolactin increases the number of LH receptors and potentiate steroidogenic effect of<br />
LH on Leydig cells while testicular prolactin receptors have been shown to be<br />
confined to the interstitial tissue of the testis (Johnson and Everitt, 1995). Similarly<br />
prolactin has been shown to increase the uptake of androgen and increases 5-a<br />
reductas activity in the prostate, while testosterone maintains the number of prolactin<br />
receptors in the prostate. Prolactin has also been shown to potentiate the effect of<br />
testosterone on accessory sex organs (Mathur and Chattopadhyay, 1986). The<br />
reduction in the levels of serum testosterone may also be due to decreased serum<br />
prolactin level in the TCDD-treated rats.<br />
5.2.6 Effect of TCDD on nucleic acid and protein contents in testis<br />
Administration of TCDD decreased in DNA, RNA and protein contents of the<br />
testis as compared to the corresponding groups of control animals (Fig. 9; Table 6).<br />
The observed reduction in the levels of DNA. RNA and protein in the TCDD-treated<br />
rats reflects the reduced synthetic and metabolic activities of the testis.<br />
5.2.7 Effect of TCDD on antioxidant system in testis<br />
Administration of TCDD increased the production of superoxide anion, nitric<br />
oxide and hydrogen peroxide in the crude homogenate, mitochondrial and<br />
microsome-rich fractions of testis of rats when compared to the corresponding groups<br />
of control animals (Fig. 10a to IOc; Table 7a to 7c). Treatments of rats with TCDD<br />
decreased the level of antioxidants such as glutathione, a-tocopherol and ascorbic<br />
acid in the crude homogenate, mitochondrial and microsonle-rich fractions of testis of'<br />
rats (Fig. 1 la to I Ic; Table 8a to 8c).<br />
Administration of TCDD decreased the activities of superoxide dismutase.<br />
camlase, glutathione rcductase and glutathione peroxidase (Fig. 12a to 12d; Table 9a<br />
to 9d) while the level of lipid peroxidation (Fig. 13; Table 10) increased significantly<br />
in the crude homogenate, mitochondria1 and microsome-rich fractions of testis of rats
as compared to the corresponding groups of control animals. Increased production of'<br />
reactive oxygen species. decreased levels of antioxidants as well as the activities of'<br />
antioxidant enzymes and increased levels of lipid peroxidation reflected that testis are<br />
In oxidative stress.<br />
Increased production of superoxide anion in the hepatic and brain tissues of<br />
rats after exposure to TCDD has been reponed (Hassoun r! ul., 2001). TCDD<br />
increased the production of reactive oxygen species in mitochondria1 and microsomal<br />
fractions of rat testis (Latchoumycandane er ul., 2002 c). Nitric oxide has been shown<br />
to react with superoxide anion to produce peroxynitrite, which actively react with<br />
glutathione, cysteine, deoxyribose and thiolsl thioethers (Sikka, 2001). It has been<br />
reported that acute exposure of TCDD in mice decreased the levels of glutathione and<br />
a-tocopherol in hepatic tissues (Slezack el a[.. 1999). Glutathione is important in the<br />
regulation of the cellular redox state and a decline in its cellular level in TCDDtreated<br />
rats is considered to be indicative of increased oxidative stress. The reduction<br />
in testicular glutathione levels in TCDD-treated rats might be in part attributed to the<br />
inhibition of glutathione reductase activity, which is responsible for regcneration of<br />
glutathione from its oxidised form Sikka (2001) showed that glutathione reductase is<br />
inactivated by superoxide anion. Therefore, reduced superoxide dismutase activity in<br />
testis might have enhanced flux of superoxide radicals that potentially could damage<br />
glutathione rcductase and decrease glutathione content in testis. In addition, decrease<br />
in glutathione level might reflect a direct reaction between glutathione and free<br />
radicals generated by TCDD. This is consistent with function of glutathione of<br />
scavenging oxidants by binding to them covalently. Furthermore. a regression of thc<br />
antioxidant recycling mechanism duc to a decrease in ascorbic acid level in 'l'CI)Vtreated<br />
rats might contributed to a decline in glutathione level in testis. This is in<br />
accordance with the position of glutathione at the end of the cycling mechanism.<br />
which includes vitamin E and vitamin C (Shindo er ul.. 1994).<br />
Ascorbic acid is the most important free radical scavenger within membranes<br />
and lipoproteins. Alpha-tocopherol is a well-known chain breaking antioxidant; its
lilnction is to ililcrccpt lipid pcroxyl rudiouls and thcrchy tcr~lii~l~tc lipid pc~.oxid;~livo<br />
chain reactions.<br />
The free radical clearing ability of u-tocopherol is due to<br />
delocalisation of an unpaired electron in its conjugated double bond system. The<br />
observed reduction in the levels of antioxidants in the testis of rats treated with TCDD<br />
might be due reduced recycling of antioxidants in tissues.<br />
Oxidative stress induced by TCDD has been shown to occur primarily in the<br />
mitochondria followed by microsome-rich fractions (Latchoumycandane el a/.,<br />
2002~). Decreased activities of antioxidant enzymes and increased level of lipid<br />
peroxidation in the mitochondrial and rnicrosomal fractions have been reported in the<br />
testis of rats following TCDD treatments (Latchoumycandane el a[., 2002~).<br />
5.2.8 Effect of TCDD on antioxidant system in the epididymal sperm<br />
Administration of TCDD increased the production of superoxide anion, nitric<br />
oxide and hydrogen peroxide in the epididymal sperm of rats when compared to the<br />
corresponding groups of control animals (Fig. 14a to 14c; Table 11). Administration<br />
of TCDD decreased the level of antioxidants such as glutathione and a-tocopherol in<br />
the epididyrnal sperm of rats (Fig. 1 Sa & 15b; Table 12). Administration of TCDD<br />
decreased the activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidax (Fig. 16a to 16d: Table 13) while the level of lipid<br />
peroxidation increased significantly in the epididymal sperm of rats as compared to<br />
the corresponding groups of control animals (Fig. 17; Table 13).<br />
TCDD has been shown to increase the production of reactive oxygen species<br />
in the epididymal sperm of rats (Latcho~~~iiycand;~nc ct 111.. 2002h). Incrcilscd<br />
production of supcroxide anion nnd hydrogen pcroxidc in the cpididymul hpcrtn ct~uld<br />
reflect the adverse effect of TCDD 011 the generation of reactive oxygen species. l'he<br />
observed reduction in the level of glutathione and u-tocopherol reflect adverse effect<br />
of TCDD on the antioxidants in the epididymal sperm of nts. Reduction in the<br />
activities of antioxidant enzymes and increased levels of lipid peroxidation in the<br />
epididyrnal sperm has already been reported following TCDD treatment
(Latchuumycandane el ul.. 2002 b). Uccrcasod activitics of antioxidant cnzymcs and<br />
increased levels of lipid peroxidation in the epididymal sperm could rcveal the<br />
adverse effect of TCDD on the antioxidant system of rats.<br />
5.2.9 Effect of TCDD on antioxidant system in the caput, corpus and cauda<br />
epididymides<br />
Administration of TCDD increased the production of superoxide anion, nitric<br />
oxide and hydrogen peroxide in the caput, corpus and cauda epididymides of rats<br />
when compared to the corresponding groups of control animals (Fig. 18a to 18c;<br />
'Table 14). Administration of TCDD decreased the levels of antioxidants such as<br />
glutathione and a-tocopherol in the caput, corpus and cauda epididymides of rats<br />
(Fig. l9a & 19b; Table 15). Administration of TCDD decreased the activities of<br />
superoxide dismutase. catalase, glutathione reductase and glutathione peroxidase (Fig.<br />
20a to 2Od; Table 16a to 16c), while the level of lipid peroxidation incroascd<br />
s~gnificantly in the caput, corpus and cauda epididymides of rats (Fig. 21: Table 16a<br />
to 16c). Similar observation has also been reported in caput, corpus and cauda<br />
epididymides of rats foIlowing treatment with lindane (Chitra eta/.. 2001).<br />
5.2.10 Effect of TCDD on antioxidant system in kidney<br />
Administration of TCDD at the dose level of 100 ngl Kg body weight! day<br />
increased the production of superoxide anion, nitric oxide and hydrogen peroxide in<br />
the kidney of rats when compared to the corresponding groups of control animals<br />
(Fig. 22a to 22c; Table 17a). TCDD decreased the levels of antioxidants such as<br />
glutathione and a-tocopherol in the kidney of rats at 100 ng dose level (Fig.23a L<br />
23b; Table 17b) when compared to the corresponding groups of control animals.<br />
Administration of TCDD decreased the activities of superoxide dismutase. catalase.<br />
glutathione reductase and glutathione peroxidase at 100 ng dose level (Fig. 24a to<br />
24d; Table 17c) while the level of lipid petoxidation increased significantly in the<br />
kidney of rats (Fig. 25: Table 17c). However, no significant changes were observed
in the animals administered with TCDD at I and 10 ng dose Icvcls. Thc lcvcls of<br />
some of the antioxidant enzymes were higher in kidney us compared to testis and<br />
epididymis. Thus the levels of antioxidant mechanisms could be higher in kidney<br />
which could prevent it from TCDD-induced oxidative stress at low doses. It is also<br />
possible that toxicity response threshold in terms of oxidative stress is lower in testis<br />
and epididymis than in kidney since low doses of TCDD could not induce oxidative<br />
stress in the kidney of rat.<br />
5.3 Effect of co-administration of TCDD end vitamin E on male rats<br />
Effects of co-administration of TCDD and vitamin E on various parameters of<br />
male reproduction were studied and the results are discussed below.<br />
5.3.1 Effect of co-administration of TCDD and vitamin E on body and organ<br />
weights<br />
Co-administration of TCDI) and vitamin E did not show any significant<br />
change in the body weight of rats as compared to the corresponding groups of control<br />
an~mals (Table 18a) indicating that the general metabolic conditions of the animals<br />
were within the normal range and that the administration oT'TCDD along with vitamin<br />
E did not cause systemic toxicity.<br />
Administration of TCDD along with vitamin E did not cause any reduction in<br />
the weight of the testis when compared to the corresponding groups of control<br />
animals (Fig. 4 a; Table 18a & 18b). It is thus suggested that co-administration of<br />
vitamin E along with TCDD could impart protective effect against TCDD on the<br />
weight of the testis. The weight of the epididymis, seminal vesicles and ventral<br />
prostate did not show any significant dinermces in the rats administered with 'TCDI)<br />
along with vitamin E when compared to the corresponding groups of control animals<br />
(Fig. 4b to 4e; Table 18a & lab). The results suggested that co-administration of<br />
vitamin E along with TCDD could give protection to the epididymis and accessory
sex organs against TCDD. Weight of the kidney remained unchanged in the animals<br />
co-administered with TCDD and vitamin E (Fig. 4f; Table 18a & 18b).<br />
5.3.2 Effect of co-administration of TCDD and vitamin E on daily sperm<br />
production, epididymal sperm viability, motility and sperm count<br />
The daily sperm production and epididymal sperm viability, motility as well as<br />
epididymal sperm count remained unchanged in the animals administered with TCDD<br />
and vitamin E as compared to the corresponding groups of control animals (Fig. Sa &<br />
Sb; Table 19). It is thus suggested that co-administration of vitamin E along with<br />
TCDD could impart protective effect against TCDD on the epididymal spenn<br />
viability and motility as well as daily sperm production and epididymal sperm count.<br />
5.3.3 Effect of co-administration of TCDD and vitamin E on seminiferous<br />
tubular and lumen diameter<br />
Diameter of the seminiferous tubular and lumen did not show any significant<br />
change in the rats co-administrated with TCDD and vitamin E as compared to the<br />
corresponding groups of control animals (Fig. 6; Table 20). Thus co-administration<br />
of vitamin E along with TCDD could impart protective effect against TCDD to<br />
seminiferous tubules of rat testis.<br />
5.3.4 Effect of co-administration of TCDD and vitamin E on serum hormone<br />
levels and steroidogenic enzymes in testis<br />
The levels of serum testosterone and estradiol did not show any significant<br />
change in the rats co-administered with TCDD and vitamin E as were found to be<br />
decreased in the TCDD-treated rats (Fig. 7a to 7e; Table 21). Co-administration of<br />
TCDD and vitamin E did not cause any significant change the activities of' 3Phydroxysteroid<br />
dehydrogenase and 17P-hydroxysteroid dchydrogenasc in tcslis as<br />
compared to the corresponding groups of control animals (Fig. 8; Table 22). It is thus<br />
suggested that co-administration of vitamin E along with TCDD could impart
protective effects on serum testostcronc and cstradiol lcvcls by prcvcnting tho<br />
decreased activities of steroidogenic enzymes in testis of TCDD-treated rats.<br />
The levels of serum FSH and LH remained unchanged in the animals<br />
administered with TCDD and vitamin E which is similar to TCDD-treated groups.<br />
However, the levels of serum prolactin did not decrease significantly in the animals<br />
treated with TCDD and vitamin E as was shown to decrease in the rats treated with<br />
TCDD alone (Fig. 7a to 7e; Table 21). It is thus suggested that co-administration of<br />
vitamin E along with TCDD could impart protective effect on serum prolactin.<br />
53.5 Effect of co-administration of TCDD and vitamin E on nucleic acid and<br />
protein contents in testis<br />
Administration of TCDD along with vitamin E did not show any significant<br />
chanye in the DNA. RNA and protein contents of the testis as was shown to decrease<br />
~n tlx TCDD-treated rats (Fig. 9; l'ablc 23). It is thus suggcstcd that coadministration<br />
of vitamin 1:. along with TCIID could inipart protective efl'cct on<br />
synthetic and metabolic activities of the testis<br />
5.3.6 Effect of co-administration of TCDD and vitamin E on antioxidant system<br />
in testis, epididymal sperm and epididymis<br />
Administration of TCDD along with vitamin E did not show any significant<br />
chanye in the production of superoxide anion, nitric oxide and hydrogen peroxide in<br />
the crude homogenate, mitochondrial and microsome-rich fractions of testis of rats as<br />
well as epididymal sperm and various regions of the epididymis when compared to<br />
the corresponding groups of control animals (Fig. !Oa to IOc; 'l'ahlc 24a lo 245; I;ig.<br />
14a to 14c; Table 28 and Fig. 18a to 18c; 'l'ablc 3 I ).<br />
Treatments of rats with TCDD along with vitamin E did not alter the level of<br />
antioxidants such as glutathione, a-tocopherol and ascorbic acid in the crude<br />
homogenate, rnitochondrial and microsome-rich fractions of testis as well as<br />
epididymal sperm and epididymis of rats (Fig. 1 la to 1 Ic; Table 2Sa to 25c; Fig. 15a
to Fig. 15b; Table 29 and Fig. 19a lo I'if I9b; 'l'ablc 32). Administralion of'1'CL)I)<br />
along with vitamin E did not show any significant change in the activities of<br />
superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase (Fig<br />
12a to IZd, Table 26a to 26d; Fig. 16a to Fig. 16d; Table 30 and Fig. 20a to Fig. 2Od;<br />
Table 33a to 33c) as well as the level of lipid peroxidation (Fig.13; Table 27; Fig. 17;<br />
Table 30; and Fig. 21; Table 33a to 33c) in the crude homogenate, mitochondria1 and<br />
microsome-rich fractions of testis. epididymal sperm and various regions of the<br />
epididymis of rats as compared to the corresponding groups of control animals. The<br />
results suggested that vitamin E could protect testis and epididymis against TCDDinduced<br />
reactive oxygen species mediated toxicity.<br />
5.3.7 Effect of co-administration of TCDD and vitamin E on antioxidant system<br />
in kidney<br />
Administration of TCDD along with vitamin E did not cause any significant<br />
change In the production of superoxide anion. nitric oxide and hydrogen peroxide in<br />
the kidney of rats as was shown to decrease in the TCDD-treated rats at 100 ng dose<br />
level (Fig. 22a to 22c; Table 34a). The levels of glutathione and a-tocopherol in the<br />
kldney of rats treated with TCDD and vitamin E remained unchanged (Fig.23a to 23b;<br />
'Table 34b). There were, however, no changes in the levels of antioxidants in TCDD<br />
alone treated groups at lower doses such as I and I0 ng levels.<br />
Co-administration of TCDD and vitamin E did not cause any significant<br />
change in the activities of superoxide dismutase, catalase, glutathione reductase and<br />
glutathione peroxidase (Fig. 24a to 24d; Table 34c) as well as the level of lipid<br />
peroxidation in the kidney of rats as compared to the corresponding groups of control<br />
animals (Fig. 25; Table 34c). However, no significant changes were observed in the<br />
animals administered with TCDD at 1 and 10 ng dose levels. Low dose of TCDD<br />
were nor able to induce oxidative stress in kidney of rat thereby suggesting that thc<br />
antioxidant mechanism is higher in kidney as well as the toxicity threshold is lower in<br />
testis and epididymis than in kidney in terms of oxidative stress. The results
suygcstccl that vivamin 1: conld imp;lrt p~.otcclivc cI'(Lc~s i~gi~itlst 'I'('I>Il lo 11o11-<br />
reproductive tissue as well.<br />
5.4 Epilogue<br />
On the basis of the foregoing discussion it can be summarized that exposure to<br />
TCDD increases oxidative stress in rats as evidenced by increase in reactive oxygen<br />
species, lipid peroxidation and suppression of antioxidant system in testis. epididymis,<br />
sperm and kidney. The generation of oxidative stress is triggered by low doses of<br />
TCDD in the male reproductive system as compared to kidney where it is generated<br />
only at high doses. Thus male reproductive system appears to be more susceptible to<br />
the damage caused by environmental contaminants like TCDD. Co-administration of<br />
TCDD along with vitamin E imparts protective effects against TCDD-induced<br />
reactive oxygen species mediated toxicity in the reproductive and non-reproductive<br />
tissues. Thus vitamin E could be uscd as a protective measure against TCDD-induced<br />
toxicity.
Fig.4a. Effect of TCDD or TCDD and vitamin E on the weight of the testis of<br />
adult rats (mean* SD; *P
Fig.4~. ENect of TCDD or TCDD and vitamin E on the weight of the sen~inal<br />
vesicles (Intact) of adult rats (meanfSD; *P
Fig.4~. Effect of TCDD or TCDD and vitamin E on the weight of the ventral<br />
prostate of adult rats (meaniSD; *P
tE<br />
W<br />
d<br />
In<br />
w<br />
it<br />
I<br />
I<br />
(,OCX) 3s PUB (,OCX) dsa<br />
i
Fig. 'la. Effect of TCDD or TCDD and vitamin E on the rerum FSH levds in<br />
adult male rats (meanfSD; *PcO.05)<br />
OOng olng ml0ng EelOOng<br />
TCDD<br />
TCDD +Vitamin E<br />
Fig. 7b. Effect oTTCDD or TCDD and vitamin E on the serum LH levels of<br />
adult rats (meaniSD; *P-=0.05)<br />
Dong Blng BlOng 01100ng<br />
TCDD<br />
TCDD + Vitamin E
Fig. 7c.<br />
Effect of TCDD or TCDD and vitamin E oa the serum pmlrctin levels<br />
of adult rats (meanfSD; *P
Fig. 7c. ElTect of TCDD or TCDD and vitamin E 011 the serum estradiol levels of<br />
adult rats (meanfSD; *PcO.OS)<br />
TCDD<br />
TCDD +Vitamin E
u 3 f ~ = m w * N O<br />
.-<br />
~Slew aew enssg 6 1 6 ~
Fig.lOa. EBut of TCDD or TCDD and vitamin E on the production of superoxide anion in crude homogenate, mitochondrial and<br />
microsome-rich fractions of rat tcstis (mcmf SD: *P
Fig.lOb. ENmt of TCDD or TCDD and vitamin Eon the production of nitric oxide in crude homogenate, mitochondrial and<br />
microsomcrich fractions of rat testis (mcantSD; *P
Filk Effect of TCDD or TCDD and vitamin E on the hydrogen peroxide generation in crude homogenatc, mitocbondrhl m d<br />
microsome-rich fnctiow of rat tatis (munfSD; *P
Fig. 1Ir Effect of TCDD or TCDD md vitamin E on the 1 4 of ascorbic acid in crude homogenatq mitochondria1 md micmsomc<br />
rich fractions of rat tcctir (mean+SD; *P
Fig. 14a. Effect of TCDD or TCDD and vitamin E on the production of superoxide<br />
anion in the epididymal sperm of adult rats (mean *SD; *P
Fig. 14c. Effect of TCDD or TCDD and vitamin E on the hydrogen peroxide<br />
generation in the epididymai sperm of adult rats<br />
(mean kSD; *P
Fig. 1Sb. Effect of TCDD or TCDD and vitamin E on the level of a-tocopherol in<br />
the epididymal sperm of adult rnts (mean *SD; *P
Fig. 16b. Effect of TCDD or TCDD and vitamin E on the activity ofcatalase in tlte<br />
epididymal sperm of adult rats (mean iSD: *P
Fig.16d. Effect of TCDD or TCDD and vitnn~in E 011 the activity of glulell~iune<br />
peroxidase in the epididymal sperm of adult rats<br />
(mean MD; *Pc0.05)<br />
mg protein mg DNA mg protein mg DNA<br />
TCDD<br />
TCDD + Vitamin E<br />
Fig.17. Effect of TCDD or TCDD and vita~nin E 011 level of lipid geruxid:~liu~~ ill<br />
the epididymal sperm of adult rats (mean fSD; *P
Fig.18~. EKect of TCDD or TCDD and vitamin Eon the production of hydrogen peroxide in the caput, corpus and<br />
cauda epididymides of adult rats (mean +SD; *P
Fig. 19a. Effect of TCDD or TCDD and vitamin Eon the lcvd of glutathione in thc caput, corpus md<br />
cauda cpididymis of adult rats (me~nfSD;*PQ.05)<br />
TCDD TCDD +Vitamin E TCDD TCDD +Vitamin E TCDD TCDD +Vitamin E<br />
Caput epididymis Corpus epididymis Cauda epididymis<br />
142
1 1.2 l4<br />
Fig. 19b. ENect of TCDD or TCDD and vitamin E on the level ofa-tocopherol in the caput, corpus and<br />
cauda epididymides of adult rats (mean fSD: *P
60<br />
Fig. 20a. Effect of TCDD or TCDD and vitamin E on the activity of superoxide dismutasc in the caput, corpus and<br />
cauda epididymides oladult rats (mean +SD; 'P
Fig. ZOb. Elkt of TCDD or TCDD and vitamin Eon the activity of catalrse in the eaput, corpus md<br />
uuda cpididymis of adult rats (mean f SD; *P
Fig. 2k. Ekt of TCDD or TCDD and vitamin Eon the activity of glutathione reductase in the caput, corpus and<br />
cauda epididymids of adult rats (mean fSD: *P
Fig. 21.<br />
Effect olTCDD or TCDD and vitamin Eon the level of lipid peroxidation in the caput, corpus md audr<br />
epididymidu of adult rab (mean fSD; 'P
Fig. 22a. EKect of TCDD or TCDD and vitamin E on the production of<br />
superoxide anion in the kidney of adult rats (meanfSD; *P
Fig. 22c. Effect of TCDD or TCDD and vitamin E on the production of<br />
hydrogen peroxide in the kidney of adult rats (meanfSD; *P
Fig. 23b. Effect of TCDD or TCDD and vitamin E on the level of a-tocopherol in<br />
the kidney of adult rats (mean*SD; *P
Fig. 24b. Eflect ofTCDD or TCDD and vitamin E on the activity of catalase in<br />
the kidney ofadult rats (meanfSD; *P
Fig. 24d. Effect of TCDD or TCDD and vitamin E on the activity of glutathione<br />
peroxidase in the kidney of adult rats (meanfSD; .P
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1.1 Preparation of dosing solution<br />
APPENDIX 1<br />
PREPARATI<strong>ON</strong> <strong>OF</strong> REAGENTS<br />
1.1.1 2.3.7.8-Tetrachlorodibem-p-dioxin (TCDD)<br />
100 pg of TCDD was dissolved in acetone and olive oil (1:19) and used as<br />
stock solution. The stock solution was serially diluted and used for treatments.<br />
1.1.2 Vitamin E<br />
200 mg of vitamin E was made up to 10 mL with olive oil and stored in amber<br />
coloured bottle at 4OC. This solution contained 20 mg vitamin E/ mL.<br />
1.2 Epididymal sperm counts<br />
1.2.1 Diluents<br />
The diluents for sperm counts were prepared by the addition of 50 g sodium<br />
bicarbonate, 10 mL 35% formalin and 0.25 g trypan blue in a volumetric flask<br />
and made up to a final volume of 1 L with distilled water.<br />
1.3 Serum FSH<br />
1.3.1 Anti-FSH-Coated microtitration strips<br />
One strip holder contained 96 microtitration wells coated with anti-FSH<br />
antibody. Stored at 2-8'C in the resealable pouch with a desiccant to protect<br />
from moisture.<br />
1.3.2 FSH standards<br />
One vial, 2 mL, labeled A contained 0 mlU1 mL and five vials. 1 mL each,<br />
labeled B-F, contained concentrations of approximately 1.5, 4.5, 15, 45 and<br />
150 mIU/ mL FSH (from human pituitary gland) in porcine serum with a nonmercury<br />
preservative.<br />
1.3.3 FSH controls<br />
Two vials, I rnL each, Levels I and 11. contained low and high concentrations<br />
of FSH (from human pituitary gland) in porcine serum with a non-mercury<br />
preservative.
1.3.4 Antibody-Enzyme Conjugate solution<br />
One amber vial, 0.3 mL, contained anti-FSH antibody conjugated to the<br />
enzyme horse+adish peroxidase in a protein-based buffer with a non-mercury<br />
preservative. The Antibody-Enzyme Conjugate concentrate was diluted at a<br />
ratio of 1 part into 50 parts of the assay buffer.<br />
1.3.5 Tctramethylbenzidine Chromogen solution (TMB)<br />
One amber bottle, 11 mL contained a solution of TNIB in citrate buffer with<br />
hydrogen peroxide.<br />
1.3.6 Wash concentrate<br />
One bottle, 105 mL contained buffered saline with a nonionic detergent.<br />
Diluted 10-fold with deionized water prior to use.<br />
1.4.1 Anti-LH-Coated Microtitration strips<br />
One strip holder contained 96 microtitration wells coated with anti-LH<br />
antibody. Stored at 2-8°C in the resealable pouch with a desiccant to protect<br />
from moisture.<br />
1.4.2 LH standards<br />
One vial, 1 mL, labeled A contained 0 mlU/ mL and five vials, 0.5 mL each,<br />
labeled B-F, contained concentrations of approximately I, 3, 10, 30 and 100<br />
mlU/ mL LH equine serum with a non-mercury preservative.<br />
1.4.3 LH controls<br />
Two vials, 0.5 mL each. Levels I and 11, contained low and high<br />
concentrations of LH in equine serum matrix with a non-preservative.<br />
1.4.4 Antibody-Enzyme Conjugate solution<br />
One amber vial, 0.3 mL, contained anti-LH antibody conjugated to the enzyme<br />
horseradish peroxidase in a protein-based buffer with a non-mercury<br />
preservative. The Antibody-Enzyme Conjugate concentrate was diluted at a<br />
ratio of 1 part into 50 parts of the assay buffer.<br />
1.4.5 Ternethylbemidine Chromogen solution (TMB)<br />
One amber bottle, 11 mL contained a solution of TMB in citrate buffer with<br />
hydrogen peroxide.
1.4.6 Wash concentrate<br />
One bottle, 105 mL contained buffered saline with a nonionic detergent.<br />
Diluted 10-fold with deionized water prior to use.<br />
1.5.1 Anti-Prolactin-Coated microtitration strips<br />
One strip holder contained 96 microtitration wells coated with anti-prolactin<br />
antibody. Stored at 2-8°C in the resealable pouch with a desiccant to protect<br />
from moisture.<br />
1.5.2 Prolactin standards<br />
One vial, labeled A contained 0 ngl rnL and five vials, labeled B-F, contained<br />
concentrations of approximately 2, 6, 20, 60 and 180 ngl mL prolactin in<br />
porcine serum with a non-mercury preservative.<br />
1.5.3 Prolactin serum controls<br />
Two vials, Levels I and 11, contained low and high concentrations of prolactin<br />
in porcine serum with a non-mercury preservative.<br />
1.5.4 Antibody-Enzyme Conjugate solution<br />
One vial, 0.3 mL, contained anti-prolactin antibody conjugated to the enzyme<br />
horseradish peroxidase in a protein-based (BSA) buffer with a non-mercury<br />
preservative. The Antibody-Enzyme Conjugate concentrate was diluted at a<br />
ratio of 1 part into SO parts of the assay buffer.<br />
1.5.5 Tetramethylbenzidine Chromogen solution (TMB)<br />
One amber bottle, 11 mL contained a solution of TMB in citrate buffer with<br />
hydrogen peroxide.<br />
1.5.6 Wash concentrate<br />
One bottle. 60 mL contained buffered saline with a nonionic detergent. Diluted<br />
25-fold with deionized water prior to use.<br />
1.6 Serum testosterone<br />
1.6.1 GARG-Coated microtitmtion strips<br />
One strip holder contained 96 polystyrene microtitre wells coated with goat<br />
anti-rabbit 1gG immobilized to the inside wall of each well. Stored at 2-S°C in<br />
the resealable pouch with a desiccant to protect from moisture.
1.6.2 Testosterone standards<br />
One vial, 0.5 mL, labeled A contained 0 ng/ mL testosterone and six vials, 0.5<br />
mL each, labeled B-G, contained concentrations of approximately 0.1. 0.5.<br />
2.5, 5.0, 10 and 25 ngl mL testosterone in human serum with a non-mercury<br />
preservative.<br />
1.6.3 Testosterone controls<br />
Two viats, 0.5 mL each, Levels I and II, contained low and high<br />
concentrations of testosterone in human serum with a non-mercury<br />
preservative.<br />
1.6.4 Testosterone-Enzyme Conjugate concentrate<br />
One vial, 0.3 mL, contained testosterone conjugated to horseradish peroxidase<br />
in a protein-based (BSA) buffer with a non-mercury preservative. The<br />
Antibody-Enzyme Conjugate concentrate was diluted at a ratio of 1 part into<br />
50 parts of the assay buffer.<br />
1.6.5 Testosterone antiserum<br />
One vial, I I mL, contained rabbit anti-testosterone (polyclonal) serum in<br />
protein based (BSA) buffer with a non-mercury preservative.<br />
1.6.6 Tetramethylbenzidine chromogen solution (TMB)<br />
One amber bottle. I I mL contained a solution of TMB in citrate buffer with<br />
hydrogen peroxide.<br />
1.6.7 Wash concentrate<br />
One bottle, 60 mL contained buffered saline with a nonionic detergent. Diluted<br />
25-fold with deionized water prior to use.<br />
1.7 Serum estmdiol<br />
1.7.1 Anti-Esuadiol-Coated microtitration strips<br />
One strip holder contained 96 polystyrene microtitre wells coated with rabbit<br />
anti-estradiol IgG immobilized to the inside wall of each well. Stored at 2-8°C<br />
in the resalable pouch with a desiccant to protect from moisture.<br />
1.7.2 Esuadiol standards<br />
One vial, 3 mL, labeled A contained 0 pgl mL and six vials, 1 rnL each,<br />
labeled B-G, contained concentrations of approximately 20, 50, 250, 750.<br />
2000 and 6000 pgl mL estradiol in human serum with a non-mercury<br />
preservative.
1.7.3 Estradiol controls<br />
Two vials. 1 mL each, Levels I and 11, contained low and high conccntrulions<br />
of esuadiol in human serum with a non-mercury preservative.<br />
1.7.4 Estradiol-Biotin Conjugate concentrate<br />
One vial, 0.3 mL, contained biotinylated estradiol in a protein-based (BSA)<br />
buffer with a non-mercury preservative. Diluted prior to use with the<br />
estradiol -biotin conjugate diluent.<br />
1.7.5 Tetramethylbenzidine Chromogen solution (TMB)<br />
One amber bonle, 11 mL contained a solution of TMB in citrate buffer with<br />
hydrogen peroxide.<br />
1.7.6 Wash concentrate<br />
One bottle, 60 mL contained buffered saline with a nonionic detergent. Diluted<br />
25-fold with deionized water prior to use.<br />
1.7.7 Stopping solution<br />
One vial, 11 mL contained 0.2 M sulfuric acid<br />
1.8 3P-Hydroxystcroid dchydrugcnasc<br />
1.36 g of tris was dissolved in 100 mL of distilled water (Solution A)<br />
0.9 mL of concentrated hydrochloric acid was made up to 100 rnL with<br />
distilled water (Solution B).<br />
62.5 mL of solution A and 55.25 mL of solution B were mixed and made up to<br />
250 mL with distilled water. The pH was adjusted to 7.2 with 1 N sodium<br />
hydroxide.<br />
1.8.2 Sodium pyrophosphate buffer (100 pM; pH 9.0)<br />
13.3 mg of tetrasodium pyrophosphntc was dissolvcd and made up to 500 1111~<br />
with distilled water and pH was adjusted to 9.0.<br />
1.8.3 NAD (0.5 pM)<br />
1.66 mg of NAD was dissolved in 5 mL of distilled water.
22.91 pg of 4-androstcne 3,17-dione was dissolved in 100 mL of distilled<br />
water.<br />
1.9 17p-Hydroxysteroid dehydrogenase<br />
1.9.1 Sodium pyrophosphate buffer (1 00 pM; pH 9.0)<br />
13.3 rng of tetrasodium pyrophosphate was dissolved and made up to 500 mL<br />
with distilled water and pH was adjusted to 9.0.<br />
1.9.2 NADPH (0.5 mM)<br />
2.08 mg of NADPH was dissolved in 5 mL of distilled water<br />
2.86 mg of androstene 3,17-dione was dissolved in I0 mL of 50% ethanol<br />
1.10 Estimation of deoryribonucleic acids (DNA)<br />
1.10.1 Perchloric acid (PCA)<br />
Perchloric acid 16.66 mL (60%) was made upto 100 mL with distilled water.<br />
1.10.2 Diphenylamine reagent<br />
1.5 g of diphenylamine reagent was dissolved in 100 rnL of glacial acetic acid.<br />
To this 1.5 mL of concentrated sulphuric acid was added and stored at 4°C in<br />
amber coloured bottle.<br />
To every 20 mL of this reagent, 0.1 mL of 1.6% aqueous acetaldehyde was<br />
added to potentiate the colour development.<br />
1.10.3 DNA standard<br />
2 mg of calf thymus DNA was dissolved in 10 mL of hot 5% trichloroacetic<br />
acid to give a concentration of 200 pg/ mL and stored at 4°C.<br />
1.1 1 Estimation of ribonudeic acid (RNA)<br />
1.1 1 .I Orcinol reagent<br />
I g of ferric chloride was dissolved in 1 L of concentrated hydrochloric acid<br />
and 35 mL of 6% orcinol was added.
1.1 1.2 Orcinol(6%)<br />
6 g of orcinol was dissolved in I00 mL of ethyl alcohol.<br />
1.1 1.3 RNA standard<br />
2 mg of RNA was dissolved in 10 mL of distilled water to give a concentration<br />
of 200 pg/ mL and stored at 4'C.<br />
1.12 Estimation of protein<br />
1.12.1 Alkaline copper reagent<br />
50 mL of reagent A and I mL of reagent B were mixed fresh at the time of<br />
use.<br />
Reagent A<br />
2 g of sodium carbonate was dissolved in and made up to 100 mL with 0.1 N<br />
sodium hydroxide solution.<br />
Reagent B<br />
5 mg of copper sulfate in 1 mL of 4% sodium potassium tartarate.<br />
1 .I 2.2 Sodium hydroxide solution (0.1 N)<br />
0.4 g of sodium hydroxide was dissolved in and made up to 100 mL with<br />
distilled water.<br />
1.12.3 Sodium potassium tamirate (4%)<br />
4 g sodium potassium tartarate was dissolved in and made up to 100 mL with<br />
distilled water.<br />
1.12.4 Bovine serum albumin (BSA) standard<br />
100 mg of bovine serum albumin was dissolved in and made up to 100 mL<br />
with distilled water. This solution was diluted 10 times to obtain a<br />
concentration of 0.1 mg/ mL.<br />
1.12.5 Folin Ciocalteau reagent (IN)<br />
Commercial Folin Ciocalteau reagent was diluted with equal volume of<br />
distilled water.
1.13 Subcellulnr frnctionation of testis<br />
1.13.1 Sucrose solution (0.25 M)<br />
Sucrose 8.55 g was dissolved in 100 mL distilled water.<br />
1.13.2 Calcium chloride (1 M)<br />
14.70 g of calcium chloride was dissolved in 100 mL distilled water.<br />
1.14 Superoxide anion<br />
1.14.1 Iodonitrotetrazolium (INT; 4.9 mM)<br />
230.91 mg of INT was dissolved in I00 mL of distilled water.<br />
1.14.2 Ethylene diarnine tetraacetic acid (EDTA; 0.3 mM)<br />
11.16 mg of EDTA was dissolved in 100 mL of distilled water.<br />
1.14.3 Sodium carbonate (0.92 mM)<br />
9.74 mg of sodium carbonate was dissolved in 100 mL of distilled water.<br />
1.15 Nitric oxide<br />
1.1 5.1 Griess reagent<br />
10% Sulfanilamide was added to 40% phosphoric acid (Solution A).<br />
1 g of N-[-naphthyllethylenediamine dichloride was diluted to 100 mL with<br />
distilled water (Solution B).<br />
Mix equal volume of solution A and B at the time of use.<br />
1.16 Hydrogen peroxide generation<br />
I. 16.1 Phosphate buffer (0.05 M; pH 7.6)<br />
0.89025 g of disodium hydrogen phosphate dihydrate was dissolved in 100<br />
mL distilled water (Solution A).<br />
0.69175 g of sodium dihydrogen phosphate monohydrate was dissolved in 100<br />
mL distilled water.<br />
Mixed 13 mL of solution A with 87 mL of solution B and made up to 200 mL<br />
with distilled water and pH was adjusted to 7.6.
1.16.2 Horseradish peroxidase (8 Units1 mg)<br />
1 mg of 80 units horseradish peroxidase was dissolved in 10 mL of distilled<br />
water to make 8 units/ ml.<br />
1.16.3 Phenol red (28 nM)<br />
354.4 mg of phenol red was dissolved in IrnL of distilled water to give a<br />
concentration of 1 M. This solution was made up to 1 L to get the<br />
concentration of lmM, and this solution was made up to 1 L to get the<br />
concentration of 1 pM. 28 mL of 1pM phenol red was made up to 1L with<br />
distilled water to get the concentration of 28 nM.<br />
1.16.4 Dextrose (100 nM)<br />
180.1 1 mg of dextrose was dissolved in I mL of distilled water and made up<br />
to 1 L to get the concentration of 1 mM. 1 mL of ImM dextrose solution was<br />
made up to 1 L with distilled water to get the concentration of 1 pM. 1 mL of<br />
IpM dextrose solution was dissolved and made up to 100 mL with distilled<br />
water to get the concentration of 100 nM.<br />
1.16.5 Sodium hydroxide (10 N)<br />
40 g of sodium hydroxide pellet was dissolved in 100 mL of distilled water.<br />
1.17 Glutathione reduced<br />
1.17.1 Phosphate buffer (0.1 M; pH 7.0)<br />
1.78 g of disodium hydrogen phosphate dihydrate was dissolved in 100 mL of<br />
distilled water (Solution A).<br />
1.38 g of sodium dihydrogen phosphate dihydrate was dissolved in 100 mL of<br />
distilled water (Solution B).<br />
Mixed 39 mL of solution A and 61 mL of solution B and made up to 200 rnL<br />
with distilled water and pH was adjusted to 7.0.<br />
I. 17.2 Trichloroacetic acid (5%)<br />
5 mL of 100% TCA was diluted to 100 mL with distilled water.<br />
1.17.3 Ellman's reagent<br />
34 mg of dithionitrobis-benzoic acid was mixed in 10 mL of 0.1% sodium<br />
citrate.
1.17.4 Disodium hydrogen phosphate (0.3 M)<br />
5.34 g of disodium hydrogen phosphate was dissolved in I00 tnL of distilled<br />
water.<br />
1.17.5 Glutathione standard<br />
100 mg of glutathione reduced in 100 mL distilled water and stored as<br />
working standard. Stock was diluted to get a concentration of 100 pd mL.<br />
1 .I 8.1 2,Z'-Dipyridyl solution (0.2%)<br />
0.2 g of dipyridyl was dissolved in I00 mL of ethanol.<br />
1 .I 8.2 Ferric chloride solution (0.5%)<br />
0.5 g of ferric chloride was dissolved in 100 mL of ethanol.<br />
1.18.3 a-Tocopherol (standard)<br />
100 mg of DL-tocopherol was dissolved in 100 mL of distilled ethanol. Stock<br />
solution was diluted to get a concentration of 10 pgl mL.<br />
1.19 Ascorbic acid<br />
1.19.1 Metaphosphoric acid (2.5%)<br />
2.5 g of mctaphosphoric acid was dissolved in 100 mL of distilled water.<br />
1.19.2 Dichlorophenol indophenol acetate<br />
7.5 mg of dichlorophenol indophenol was dissolved in 100 mL of distilled<br />
water at 85OC, filtered hot, cooled and diluted to 250 mL (Solution A).<br />
4.5309 g of sodium acetate was dissolved in 100 mL of distilled water and pH<br />
was adjusted to 7.0 with 0.5 M acetic acid (solution B).<br />
Mixed equal volume of solution A and Solution B at the time of use<br />
1.19.3 Ascorbic acid (standard)<br />
100 mg of ascorbic acid was dissolved in 100 mL distilled water and stored as<br />
working standard. Stock was diluted to get a concentration of 100 pgl mL.
1.20 Superoxide dismutase<br />
1.20.1 Tris HCI buffer (50 mM; pH 8.2)<br />
605.7 mg of Tris HCI was dissolved in I00 mL of distilled water (50 mM). To<br />
this solution 0.0372 g of EDTA was added and pH was adjusted to 8.2.<br />
12.6 mg of pyrogallol was dissolved in 50 mL of I0 mM HCI.<br />
1.20.3 10 mM HCI<br />
0.0416 mL or 41.6 pL of coocentrated HCI was made up to 50 mL in a<br />
standard flask.<br />
1.21 Catalase<br />
1.2 1.1 Phophate buffer (0.0SM; pH 7.0)<br />
0.89025 g of disodium hydrogen phosphate dihydrate was dissolved in 100<br />
mL of distilled water (Solution A).<br />
0.691 75 g of sodium dihydrogen phosphate monohydrate was dissolved in 100<br />
mL of distilled water (Solution B).<br />
Mixed 39 mL of solution A with 61 mL of solution B and made upto 200 mL<br />
with distilled water and then pH was adjusted to 7.0.<br />
1.21.2 Hydrogen peroxide (0.019M)<br />
58pL of 30% hydrogen peroxide was made up to 100 mL with distilled water<br />
and stored in cool and dark place.<br />
1.22 Glutathione reductnse<br />
1.22.1 Phosphate buffer (0.1 M, pH 7.6)<br />
1.78 g of disodium hydrogen phosphate was dissolved in 100 mL of distilled<br />
water (Solution A).<br />
13.835 g of sodium dihydrogen phosphate was dissolved in 100 mL of<br />
distilled water (Solution B).<br />
13 mL of solution A and 87 mL of solution B was taken and diluted to 200 mL<br />
and pH was adjusted to 7.6.
1.22.2 NADPH (0.2 pM)<br />
16.6672 mg of NADPH was dissolved in 10 mL of distilled water<br />
1.22.3 Glutathione oxidized (2 mM)<br />
Glutathione oxidized of 12.2526 my was dissolved in 10 mL of distilled water<br />
(2 mM).<br />
1.22.4 Ethylenediaminetetraacetic acid (EDTA, 0.01 M)<br />
37.224 mg of EDTA was dissolved in I0 mL of distilled water.<br />
1.23 Glutathione peroxidase<br />
1.23.1 Phosphate buffer (0.05 M. pH 7.0)<br />
0.89 g of disodium hydrogen phosphate was dissolved in 100 mL of distilled<br />
water (Solution A).<br />
0.78005 g of sodium dihydrogen phosphate was dissolved in 100 mL of<br />
distilled water (Solution B).<br />
Take 39 mL of solution A and 61 ml of solution B was taken and diluted to<br />
200 ml and pH was adjusted to 7.6<br />
1.23.2 Glutathione reductase<br />
3 1.25 mg of glutathione reductase was dissolved in 5 mL of distilled water.<br />
1.23.3 EDTA (0.01 M)<br />
37.224 mg of EDTA was dissolved in 10 mL of distilled water.<br />
1.23.4 Sodium azide<br />
6.5 rng of sodium azide was dissolved in 10 mL of distilled water.<br />
1.23.5 Glutathione (reduced)<br />
30.733 mg of glutathione (reduced) was dissolved in 10 mL of distilled water.<br />
1.23.6 NADPH (0.2 pM)<br />
16.6672 mg of NADPH was dissolved in I0 mL of distilled water.<br />
1.24 Lipid peroxidation<br />
1.24.1 15% v/ v trichloro acetic acid (TCA)<br />
15 mL of 100 % TCA was made up to 100 mL with distilled water.
1.24.2 Thiobarbituric acid (TBA; 0.37% w/ v)<br />
0.375 mg of TBA was dissolved in 100 mL of distilled water.<br />
1.24.3 Hydrochoric acid (HCI; 0.25 N)<br />
1.08 mL of HCI is made upto 50 mL with distilled water.<br />
1.24.4 TCA: TBA: HCI (1:I:I)<br />
Equal volumes of the above mentioned 3 solutions were mixed to make a 1 : 1 :<br />
1 ratio of TCA:TBA:HCI.
APPENDIX 2<br />
LIST <strong>OF</strong> PUBLICATI<strong>ON</strong>S <strong>OF</strong> THE C<strong>AND</strong>IDATE<br />
During the course of the study the candidate has published following papers and<br />
the available reprints/ preprints are being enclosed.<br />
1 K.C. Chitra, C. Latchoumycandane and P.P. Mathur (1999). Effect of<br />
endosulfan on the testicular functions of rat. Asian Journal of Andrology 1: 203-<br />
206.<br />
2 R. Sujatha, K.C. Chitra C. Latchoumycandane and P.P. Mathur (2001). Effect<br />
of lindane on the testicular antioxidant system and steroidogenic enzymes in adult<br />
rats. Asian Journal of Andrology. 3: 135-1 38.<br />
3 K.C. Chitra, R. Sujatha., C. Latchoumycandane and P.P. Mathur (2001). Effect<br />
of lindane on the epididymal antioxidant enzymes in adult rats. Asian Journal of<br />
Andrology. 3: 205-208.<br />
4 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). The effect of<br />
2,3,7,8-tetrachlorodibenzo-p-dioxin on the antioxidant system of rat testis<br />
mitochondrial and microsomal fraction. Toxicology, 171 : 127-35.<br />
5 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). Induction of<br />
oxidative stress on the epididymal sperm of rats after exposure to 2,3,7,8-<br />
tetrachlorodibenzo-dioxin. Archives of Toxicology, 76: 1 13-1 18.<br />
6 C. Latchoumycandane, K.C. Chitra and P.P. Mathur (2002). The effect of<br />
methoxychlor on the epididymal antioxidant system of adult male rats.<br />
Reproductive Toxicology, 16: 16 1-1 72.<br />
7 C. Latchoumycandane, and P.P. Mathur (2002). Effect of methoxychlor on the<br />
antioxidant system of mitochondrial and microsome-rich fractions of rat testis.<br />
Toxicology, in press.<br />
8 C. Latchoumycandane, and P.P. Mathur (2002). lnduction of oxidative stress in<br />
testis of rat after short-term exposure to organochlorine pesticide methoxychlor.<br />
Archives of Toxicology, in press.<br />
9 K.C. Chitra, C. Latchoumycandane. P.P. Mathur (2002). Effect of nonylphenol<br />
on the antioxidant system in epididymal sperm of rats. Archives of Toxicologv, in<br />
press.<br />
10 C. Latchoumycandane, and P.P. Mathur (2002). Effects of vitamin E on<br />
reactive oxygen species mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in<br />
rat testis. Journal of Applied Toxicology, in press.
Chronic effect: of enclosulfan on the testiculalfi~nctions<br />
of rat<br />
:lillt: 'To lind out Llic toxic vlicct of endoililiu~ ol! Ulc tusuc~~lnr ionctiw~ 01 puknill rrb. MilJloti~m\j~ns. I~esu11s: In ~rldt)s~~liiu~.lwaI~tl<br />
;:it&, UICW WR. n IV~UC~~OII ill LhC lWdy wig111 und tlr \wi@~ts ui Ic\ti\lia ;~nd:tccu:.u,ry rcr urgrns, a rl?.<br />
CIUW in lllc I~~IICUIU i~~ulc and pymvillc wtivitiu, u;d ut d~c lt~liculitr DNA m~d IINA C~III~.CIIIT.I~~O~I\I i~hc~a\ ih~<br />
~L.SUCU~Y pmuUI CO~CC~LTJUO~ was slighlly i t m d ; dw spsific xliviiy of leslicu!iu ~t~~iduglnif CIIBIIIC, :i$-Otis~ciuid<br />
&hyJ~c)gL?n.m ad IIC iluorbic cad level WPC decrciaul, wllich wlr corrtlated wih a dwrcsc in rtciuidoge-<br />
~tcsis. 1 1 lywucu~ul ~ emynu? ncid phosplwtw rind brwh-LmrJcr cuyntc nlhlillins pho~phnww ~cliviriu wotr; also Jcc:cwd<br />
in rhc tcrtis of uwtcd mu. Conclusion: In puberl;ii mLs, endosullm (rca.mtt inl~ibik !he wticukd fu~icttuns.<br />
(Ailinr~ J Arulru! 1YB Dec; 1 : ?a3 - 206)<br />
llrm is gro\vutg couccnt lhxt cn!'iroilll~rni;ll clii,llli.<br />
cJr. tdh n~lurdl md IIUII-ntnde, h?vi!lg csux-gmic<br />
propeny #nay he ~iusii~g n vvicty of rcpmluctive disor-<br />
4rs in u,ildlifc d IIWMI ~~plaliUIUj. Most of the<br />
rl;s~~iicd~. \vl;icll u.4 a p-sticides ;ul: noi !tifill)<br />
rlccti\%-, but av gcirdly toxic lo ltlimj tloll-lwgcl<br />
spies, incluili$ tnnn &ad oo(her winds. Endosulfm,<br />
;u~ inusucidc ol cyclcdiur group is cxluwivcly urul irq<br />
UI in..;ricide ntdnly in ~g~iculm and in some cpunlricr<br />
ii~ public 114d1['~. Ar a ruulr of ik wide s p d luc, it<br />
rial lr a p~sntid cnvimtd unlill~u~uif ud may<br />
mmlu: a pUic lu.dlh l*wrd('l. hKbPulf;m 11s b t l<br />
-----<br />
Cura&lrr to I*. P. P. hlYlur<br />
c.nml: ~plulluOydrn.mnn<br />
'1~1: +91.41:4.
~lLllc mu ~LYIC~L~ II*: ~[IL'IIII WIIIS ill UC u~UJ..II cpi.<br />
didyt~lis :eul d,c izi~nacs~icul;~r slrniuilid rw11u, ilrur;i.<br />
.ILI iviilr uii ck~rrIio~i tii UIC xlivili~ of sl~xific IG\I~CU.<br />
rru~gc~ri~J using Ralli R~llu-Uvd!jcni Inx~n~gcnircr.<br />
I0 5, I ~ul~t~wlC Wuv prclmi~d ill IUWIEII s:ilittt: ~1 11<br />
si~p,~l;runi ru.ul lw Iiicrlu~rio~l &'d:1y. 'llr cxrr*cii,<br />
1;v ~ii;utcr CII~IIW. s so~lrilol dcl~yhwga~w, IWLIC u~rl ~klmniwtion of M A mlJ RNA m wnie~<br />
~kliydmgcir:~~!, ~.glur.u:~yl muqrpl~d:~~<br />
uul gluc.cru C. !ull~,wi~rg Ur l
enbw1h1.W mu IUY Wcab unpiliml~t at &ticactivity<br />
ilrw with llic dcvclojr~~~e~ti of sr~ntr;,lo<br />
Ub. piluilalY. Cf ~1yp~IPliY1lic lcwl, 'nu ~pididyi~ii~,<br />
C~ICK~"'~. Acliviliu; ot truc ly~u,~~ul UIL~IU !wvr iull<br />
:cini~ud wick wid vc~~utll INWUI~: an: tl11 ul&rwl. dwWl\ In nu, dm enlicuhr slcruicbgc~uiis il; ill.<br />
~&lI orpimr, dying On ICstoslM~ ,fur kir ucuu~I'*'~. A &acmc in UIC %id ohosDhahr ill lrer<br />
grwnh nnd fUnclion[lol. A rcducljon in (heir wcidlk stlr ww~d bus rcthl d d lesicu~pr smido8em-<br />
11uy r c b u ~ Jcuwred bianvi~ilability wd pralunion of sis in Ur W - m u ad UI my ba cmdnkd will1 1110<br />
aI~enr("'.<br />
ducal rasrerion of g ~ p h i A ~ deacare . in tk<br />
nl!dinc phosphaurw ocuvity in cndosulfan.mlcd n$ in-<br />
J i W lhnl emlobulf~~~ unLrw111 prduccd s sue of deurarcd<br />
stMidogwcris whuc Ihc inrcr- and invrcllular<br />
Wnrporr Was &ccd as rhc melabolic d m lo channnlize<br />
the MU~Y~ input8 for slemidogurais slowed<br />
do*n("l.<br />
In conclusion, 'he own1 studics indicate hat en-<br />
Pmeid IO..%tz.r) 13.12+1.lrF<br />
IINA' ll.l:l*l.:L~~ ?,i9*(1,#~><br />
WIA' ;1.1')tU.W 0.93t0.2ib<br />
AX.5.W acid' 2.5+0.81 I.M+O.IP<br />
LYw' 4.5.08+7. 32.64+Y.L?<br />
M-2 31.nts.m 1r.utz.w<br />
3 POH.uarxd 3.WtI.W l.e8*0.31"<br />
JC~>*IWJ<br />
Aca -y' 10L.07+25.1 79.%~10,&5~<br />
Wim phy.p"ouel W.50r.X.42 55.61*15.2UL<br />
' III~L wet wcidll 01 1 ~~1~s<br />
ln~~~V~og 11tu1ein<br />
' ruwl of NAD conuuled lo NADIUmidnrp pmlcin<br />
'l,nwl rt.~~~~~~~l~lw~~~l<br />
IIIC~;~I~I~:I~ T/::lI III~II/IIXI (21;<br />
I8,Ulflll<br />
PPM sc*norvlcgcs the rscipl of financial support<br />
horn Uc Popllation Council, New York. USA.<br />
References<br />
1 WllO kpn. Inlau!hm! yropmmr m c l a d olsly.<br />
In, Lv(mwW IWL Cri~iu-W. Gx!dlu~ L WllD.<br />
Gus"") 1w: I.<br />
2 Siwl, SK, lbrky US 'rvricilg or ctuknilr.lll ax! LLlirg ,,I<br />
llule NY LI .IYIIUI le chg nrohiiul~s c~u)%sa ;yul ltucm.<br />
rarrrl lipicl pwrilluim. llwlisll J Lp llid I'm): 27: 725 -<br />
:I l\>l.b" VII, l\$d
Effect of lindane on testicular antioxidant system and<br />
steroidogenic enzymes in adult rats<br />
K. Sujeh. K.C. ChiVd, C. L utchoumyct, P.P. Mahur<br />
*hd MLp 5clcnw, Pndrclvny Un,uniIy, Pmdichny 6% ill4 . Ida<br />
Abreact<br />
&: To fird wl hc efiozl of lib<br />
on Ic~iiculw arlioxihl system nnd lesiiculer rlcmidupmsis in dull male<br />
r.B. Methods: Adult male rats wus orally dminislued wilh iindan at a dm of 5.0 rng/kg body wighl per day<br />
fiu RO duyr. Twenty-[our hours nRCr LhC 13Y Mllmenl Ulc mLs w a kilkd ~ using n d i c &r. Testes, cpididymis.<br />
wnlinttl v~%iclc% ed wtml pmtake wcrr mwd nnd wigM. A 10% (alicuk lrgenute wa prcpDlsd ud mn-<br />
1lirug4 nl ,IT. Ihc wpcmacun~ w 4 tor biwlmn~wl eslimntions. Results: 'Ihe buly wiyhl MII he<br />
weights of ~sler, qdddymis, reminal &la end vend p w ware red& in limhw-Mlllcd mls. Then was a<br />
rignikm klinc in rhc Daivilia of Mlioxhnt cmyms mpuoxidc dismutasc (SOD). nnd glubthiar Rduc-<br />
L= dlik cut it- in hfi- pcmxide (YO,) gencmlion wa* churned. 7he spwiiic activilie% or Lerlicular<br />
urnihqar av.ria:+hrJRUwd ddlyil~~$uwp nnd llp-hydmxyslemid dehydquwsc wn 4324. Tk<br />
kuslr o( DNA. RNA anl pmdn wcrr alsn dsnased in lindmr-vmtcd ram. Conclusion: Lindme indum oxi&-<br />
liw.mxrMddoourstaPntiO*dPIIf~~~~in<malcm~. (kiv1JAnlml2WI: 3: 135-138)<br />
V+<br />
on rqxodunive functions in wildlife ad hunw~"~. Lin-<br />
1 InmJdvction<br />
dam has ban pviwly shown to muse loxic cfiozls on<br />
The orpwchlolinc insmid& lih ( 7-hu- tcrres of m. In fanulc rats li& exem anlialm-<br />
Irhlorocyc~) is widcly 4 m a pacicidc in genic sctivity by diwping ulmus cycle with a reduction<br />
m y muwria. In India it b widcly used in agriculhln in rhc ulaine vhi$~[~). Lindsm hss been rcponod to al-<br />
Md puhlic MUI 7.<br />
Lindm WUWx lcci rhc dcvelymeru d MLW crnbv in vifm in e<br />
lip*bilk: ClPMQ MJ enm Inlo Ur food chin mull- dosbdcpndcnl rmnd').<br />
ing in bbmmdmim in Iho kdy Iixue6, Mood rind L i h when given orally lo mia during wrly,<br />
brrwtmiikofhuwrMdrvi~~ilc"~. L i h d o k mid Md lalo ppmm& hna been repfled lo muse IMal<br />
nlxac of imlnnmtion aim, locnl rcfiaption of fHusC..<br />
d low binh'weighc of plp. wilh M i-v in number<br />
n*'adoaitm &n ad my cut dvslst<br />
of dad faw, rrspclinly[", Thc m1m arc highly<br />
nnaplib* D lindm r It the blaod-ds bsrricr<br />
Cl%-al a. P.P. k(l.h.. SkddWa%m=. ud with s n d c Rduction .<br />
IWdq WW. RwwImYaw. ha.<br />
Td, + P14~2lZ Rr, +9lW912<br />
Ed. ~ O p . p a . d c . h o ~ O ~ . u a \<br />
YIWW SILI-17<br />
rntllwl
cbniuis 11~11 b"nci:~lc ~wlive oxygen<br />
01 5 .(I mvkg buJy wdght/JPy ra 3) drys. The control<br />
IROS)(~~.<br />
animals &ved a dmilor volw or lk whiele h.<br />
ROS nto M impumnt p;m or hu & r e m- ~wcnty-r~ hours slkr ~hc lat -I ~hc<br />
nisn iyninq inrmum. bt excnive gwwlion ol rm u+hd and killed ming mahdc akr. T*, opi.<br />
Oxygm dials #my ILunogwfc lisw. ROS arc fwmal in dkJymis, anid rrsickx and vcmd wee n.<br />
b h phys'rdogiul d pahlogicol mndilhs in mm- mod. c!mdofIhc.dhaingriaus.nlusi@. A<br />
mlim ti-. ROS inclula supmxidc mion. hydmryl 10% Igcia*rhmagar*.gnawinmmulsalin<br />
r;rlicll, hydncm pmai(k and r,aypcn ion, nll or which wing IWW hampim ml [he hmmwge~le WT<br />
;I= u~w~ahk i(ld rnllly wwliw It1 9111~2 LJIv1m b Ik ~t111ri1ubpI 01 HIXI x 8 fir X) min at .I?. The .wp<br />
-<br />
huly mlting ill WOS mwmh lo subk sun* end IIuMl wa5 lgcd lor vnria. biachcmi mays. Raein<br />
~10luuks~'~. When hu balm bawepl ROs and m- war eslimted by the mthd of lawyI1". Euimstions<br />
tioxibnt .spyrtar is losl ' oxidalive m' multsiq). Re- of auprmidc dLmdd8l. ~.ld'~).<br />
gluliuhim m.<br />
wliw ox&! wies has tun dawn to be inn44 in d~xnd"~ end hhydmBan -ids mian rcpylzlJ<br />
inlatilily due lo drreuive f ~ ~ l ' n and ~ " in ~ wac daa. 'he aivicies d -ic mzymcs 3p.<br />
ClyFlorchiistn a upm upmum! lo toxic &mi- hydmxyW<br />
rd 17phydmaysbroid<br />
Culs"','~J. I1 lu7 ha\n shown Ih hu (hc m dchdlqmm vsc eaimld irmrding 10 &gmyeozymrr<br />
01 hu Semi&&' pathway ux mdccular oxy- dP1, The cxaCan md d.caminnciar or DNA end<br />
gen Jnd ekama mfu fmm NADR1 lo hydmxylnre RNA nwe CdWW M fdkwing lk lshique d<br />
the mh~nld"'. In chis pmxxs, rupmxide mion or ~chnidd"'<br />
Mher oxygen fm dials wre pmmad a e rcrult ci<br />
elecuon leakage in normal wlim or hr lo inlcracfion<br />
o f s t a v i d ~ ~ o r a with h hum- a ~ thelhcdmpmmprrnavdntts. Stalirialmlyzymt<br />
1%) &r pviuur vmk ha% dawn rhol cndaulrvl sit wn &amd wing Stu(a('a ' I' test ~nsdomr["l.<br />
oltus tcui~ular furujons of pubcror mtst"' Md Signi~mnccordiff~uwse~~ pc0.05.<br />
mhxychla i- restive oxym ud &-<br />
u r n g,idiily~~ul<br />
~ ~ m*i~,xkliull eri~yma in dull IWIO rab<br />
~ ~ ~ i r W d a L 1 ) . T l r ~ 1 ~ ~ 1 ~ r o<br />
evalusreIhecllslofLindYronlk~~idru lh bqdy wigha or !inbae-lwcd rarq did rm<br />
spm and it? inn- on geroidoprsis in Ihc helaun. h a y n@fi dugesung cflk<br />
mamen. Ho*crrr. lhs bady &gku uar algniWy<br />
2 Mamink and m e W<br />
~.rlkendo~lkuamanammpndtoIhc<br />
~ i n g ~ d m o(Ftgurr v I). d the ~<br />
mi*ofdIbc,enb,,pllinl~)and<br />
vaunl pawc werr M dpifdy han thmc<br />
ofhumnPd~mupdninuh(T.MeI).<br />
Lildac (7-huphlaocyslohaae) was b giR fmn<br />
layakridm Rslicidg, Sah. Tmil Ndu, India. Andmsmmc<br />
3, 17 dim, p-niuophcnyl -. p-niv<br />
o p h e n d M d d e l l 7 w -<br />
fmnSigmaUankaI6. (Sl.Louis, Mo.. USA). All<br />
thc drmblr ud wn. d mlylial gdc obW [nwn<br />
kwl m m b l aurEs.<br />
2.2 ~Lrandrdrrrmmmr<br />
Wirvrmkmu(10-12wr+ktdgc)nweobwizd<br />
Cmn Ihc CaurJl Anid Fsiiily d Um lsWdllPl<br />
llrrll~lc or PBcgndujc WiI Eduoslm nd bmdl<br />
(JIPMER). Wichary. I*. Ths m .me mdnwlwd<br />
undn a wll-roguQtal li@ uJ darL (1%: IZh)<br />
ululukrZ4tR't~~~~~lr.aa~M<br />
crsiuy dm* and mp Wa. Lindrme M dial4 in<br />
o~~vc oil iu a amx~mtmirni or 5.0 464. ml Ike tau<br />
~nwp W" giw> by crd iauhuritnl u( S i u a hap
ILXlll hnl, .I 121; 1.0 -/.a<br />
. Ill<br />
CinvrJ<br />
. - -.- -- ...-<br />
uuly wigla (8) nl * 16 1at9<br />
Tcxw<br />
(111g) llXP t 50 &%$id<br />
I b .W5t10 SI~~,@<br />
t$*I,"i~<br />
(#I+) .ULltttB :2u + ah<br />
0 I b . w l WJtW %*&<br />
Sonin*l &kd<br />
< 11%) (YL3*70 575 * Sb<br />
-<br />
hlal<br />
Wwwim Cuvol Twcd<br />
Slrpau.* Jlxnuslc' 23.41 t5.68 11.2ltZ.W<br />
ChILJ;lr' e.05+0.~5 1.n*0.ul1<br />
Oldur~ n&w Ill .let 3.P U~.Iulf 2.7dk<br />
HPhiP w L5.16+0.05 ZI.GStZ.@<br />
d<br />
-<br />
n my'<br />
Rucin' &5.25+0.3 38,16tO.&<br />
DNA' Z.PiO.11 O.B7oO.Lt?<br />
WAS 4.Wt0.01 3.46+0.l@<br />
W W W ~ &).J)t8.(U 61.%3*2.YP<br />
M~y*lrln.l~ur*<br />
17l~hyruwnul s,.a+d.a U.W*~.~JF<br />
',,ml( mwnl~ wmaWmin pa nu pusin d 32F<br />
5nd ycb, m-~.rvnlin p.r s. w#&n 6 IT.<br />
bs.4 NAWW ~xaa.V#9rn pr a&! ~ ld X?t. n<br />
Crrol~~,awna~n~,ponlspadn~~~.<br />
*hy( a &$I dm.<br />
'brtml NAD 011tnnal 10 NMmin pa F a n d 32%.<br />
1111;l1ion (WHO Repon) under lhc wagoly or whnchnlcal<br />
pmluCls a% being moJMlely hplnrdous Md it is still<br />
uscd arnpslicidcinmywwnuiw.. Adoseof5rngof<br />
liWkg body weight ha? been consi- by WHO LV<br />
No Chscrved Elk4 Level (NOEL). Bul in the present<br />
study the testis might8 of lindane-treated rat8 were significanlly<br />
dsueascd. Thc tcrlis hat been shown to be<br />
highly susceptible to lin& a8 it C- bid-testis<br />
h e r ilnd deprrsse ynmwlogemis["l. The d~cnose in<br />
the lcaticulor weight ul lindam-lrenled me my be due lo<br />
d u d Iubule sizc, spmatogenic m$t and inhib~lion<br />
01 steroid biosynbis af Lcydig<br />
I l l g h . . Tiftt.fl 4W+&<br />
The mighe of cpididymis. wninnl vaicles md<br />
VLI*~~ Inuiuc.<br />
venml pwle ill lindnne-1mLed me wen decreased.<br />
Oll6) lUU*Pl IzutIP<br />
(m~ttllyb.~.) Ill taO 9)oP<br />
Sevual stdi have sham chat !he epididymis and ac-<br />
=\wry KX orgnns quire n wntinuws Yld~genic slimuintion<br />
fw vwntion ol their noml smcturnl and<br />
7hc qxcific oelivilics of supemxi& dimuIn%. functid int~~ril$~'. l%us Lhe slight rtduction in the<br />
uwlw sul gluulhicne dubwc domd while the<br />
levels or hydmgm peroxide was found lo be elcvald in<br />
mlcd rats when mpand to the corrapcnding grwp of<br />
mlrol animals. 7hC wific oetivities of ~Midocenic<br />
weight ol che cpididymis and ncctasoly sex orgm in be<br />
vcated rats may be due 10 low bioavdlabilicy of andm-<br />
The mtioxidnnt sysm plays an errective role in<br />
arg~~xrr. :I&hyJruxywuid Jchydmgcw nnd I7khy. pmwcting Lcrm onl Mher biologicul tissu*; below a cricdruxyslwid<br />
kl~ydm~m~~w uue deumd signiliwrlly id lhrrxhvld or wdve oxygen specie? thus prrventilrg<br />
(P cO.a5). 'Iherr ms u sipiftam dswEpsc in the lev.<br />
Icrticulor dysl~nctionl''~. Anlioxidmt ciuymc$ constiUte<br />
cis of lcviculv DNA. RNA md win in mtal goup<br />
a rnudly suppoRive ltam or defence ngninsl Mctive<br />
W~II urn@ lo Ilr mud mim11I6 (ToMe 2).<br />
oxygen npcicr (RChS). In ~hc present study IhC acdviticq<br />
T.h*Z.<br />
of antioxidant wym, wpemride di.unulpsc, malase<br />
E i T a d l i n l v n m ~ ~ i n W e ~ o l<br />
~nuknu, ,,=!a. A*S. bP< 1l.a vrlhccmrulp~up.<br />
iuvl glutnlhiw redwin.. were (!ecreuced in limlune.wl.<br />
ed me. Thc levels of hydmgen pmxide gencmlion were<br />
loud (o be elevaed which ma ar a h e r of in.<br />
crud ROS. Thus in he p m l study m increaw In<br />
hydrogen peroxide wilh a duction in snrioxidnnt cnzym<br />
idiw LhC.oxi&live Ues. induced hy lindime.<br />
Ihc dsreDsnl aivities of slemidogenic enrymer 3&hydmrysWuiJ<br />
dchydmpa~ and ll~hydmxystemid dehydrogenas<br />
were indicative of the duced Isticulx<br />
slemidopunsis, Lidme hns been shown to inhibit<br />
h lo thc puthqu'wis or li&r inducal rrpmlwtive<br />
dysfundionl". 7he Wiun 01 supruxide dint~ulw<br />
suggestis that it is involved in antioxidant defmce and it<br />
h;r$ ken Nwm~ to WI ns an aitcwa ngulalury swikb in<br />
lnticulilr nwidogelursidZ']. Ilr cyld~nulc PC41 alzymcs<br />
of Lhe sluuicbgenic plhway M known to produce<br />
rtte radiEals. Thesc free mdids M pmluad s s resull<br />
of elecm kSP.p due to Ik inlem~iun ol steroid pmJ-<br />
WL$ or & pwwbubxlmlc? wilh thc cnayllrcq. lhc inability<br />
of LhC ~ ~ lo be orygwtd l phmola e<br />
the relmse or wtive oxygen spie~(~"I. In lildururuld<br />
1x1s IhC (!ecm in IhC teSli~vlx NnIelllS of
DNA, RNA ;uxl Wcin wcrr abarval, whkh show<br />
Ihac wlive oxygcn rpccies WI atmk vllnl mnparnt~<br />
or the all-like 11w1cic uci& and pieim.<br />
In oMlflusicm, che pant s!wh indiik bl )ind;ur<br />
sllrrs lwiUUlilr fuwtiam PhTiMy by i&ig rroctin:<br />
~u).yl~ qxim md &ansing LI\C a~tiw.ii tnxyilcs<br />
lhuchy disrupting tntk rrpNJuEtion.<br />
Ilw: ;w~lxm hi~t cb SUIT o( Binbmwicn Centre,<br />
Wickny Univmicy, Rmdichary lor help. PPM<br />
&nnvlalw (he rtipl or iimisl rmn he I'M8 4: 2m - 6.<br />
Itl(ul;~~iis Cinl~il. New Y$11,. USA (CSUII Nth. WJJ. 17 LWIY OH, k- NJ. Ih AL. -1 K). !+*in<br />
M7P-I)/ lCMC onl t3YJ.W KM;).<br />
maurcmerr~U1PoBrr~reaa.IBblorm<br />
IR1; 198: %-15.<br />
I8 hlskhxi S. hbkhd 0. hwdVemn d -a nim<br />
References<br />
nbdh.liolldrbndm.dlm*ibn*ml<br />
I WH0lkp.m. l-prgrracm~rd~.<br />
lawcYnur. %I&mtem1974:.yU-l4<br />
In: WIh ad Sufny M- 5( Unic (W.-)<br />
19 CUbm A. C*.lr mivky. Ln: Onan*lld R. edlw<br />
IVJI: p 14,<br />
GKlmtu&dnrhoc(lfammrrknlmrch. Bax<br />
2 aAnm7. n.nsdR.Su"Au.yurard RM. M; CRC Mi IW. pan-81.<br />
mlrrinc-diming ~ 4 ~ in ~ viMrlc 1 % rrl hmn. Enw- XI ~I.MnnsvU81.W~dcmEahnsnd<br />
nn HwXh IWJi 101: 378-8(.<br />
hRvmz,m~-fmrmIher. JBi<br />
:I Caxq.~ Kt.. Clsch&+ RW. RJrhab OL. GrUm IM. aa Irn, 261: M74-a,.<br />
Ik.111 KC. Ihn Jl,. M by. t!X~%i 41(l*cn hmn- El lk'tB,kbiY.Syuu&~nrrll~l.A,jmJaivlhy<br />
UI ~xlnll
Effect of lindane on antioxidant enzymes in<br />
epididymis and epididymal sperm of adult rats<br />
K.C. Chitre, R. Sujah, C. Latchoumycandanc, P.P. Malhur<br />
M#(lk-, Pm&myUn(wniy. W " y M Z O l 4 , Idk<br />
-: 1-1 Y -I epididymir: Mcioxidulai mUvc oxygen species<br />
Ab.tnct<br />
d lbr- W. h: h llerhllbd mis, hum we& ignitka& duaiona inin& qklkiy'<br />
nrlwdqhc.rpieQrml~~~~oDuadmocUiryrrmpndwlBth?~. SigniTmcdaMlerinchewpmjd.<br />
(80D),-, #atidam nhrPr d @uUthiare pm*duc dvitia ud signitmt inmaws in thc<br />
~plarbodtlpld~~r*o~inUlscpididymirudepididyrmltprmoflindan~ucPted<br />
a. Lip*D dPElOIDl the bYOL of mdoddmf CN- in lk epididymia and epididyd ~gcrm of<br />
dor~WS~lndushp&wM. ( ~ ~ ~ ~ J A I ~ ~ ~ ~ W I S ~ ~ I S : M S - ~ I % O B )<br />
ud d v e oxygen spkkr (ROS) thereby inducing ox-<br />
Mvc we&". ROS have bkn shorn a damage 81-<br />
most all chs rmcmmksvlcs of che dl incluing mm-<br />
LXUIO~O~~~~~~IWRWI f.lty~c/ds (WFA). CBUSiw<br />
hpnimm or plldor fudons[" ). Thc cpididyznis<br />
ir highly rW in RlFA aal lhusfoa suPcqxible IO (la<br />
&vs MO. &pcsm lo wvimnmenlal conlominpnl<br />
lindur bu tea Ibom lo enhsna oxidalive urrrr in liver<br />
and d"". Ihe -1 6lUdy WM wdcnnken I0 C-<br />
vducdc PL U T d ~ linJMs on che pnciaxht cmym<br />
d cpididymL ad epLWytrlnl yrnn ie &I1 NU.<br />
Iiadna (99%) w a gin fmn Jayhrishn. &tic&,<br />
Srbm. %nil Nadu. India. All Mhcr drmicnls
Mt~~m(10-lZsu&)vacob-<br />
Wfmn Ihehml ~ n i m l H a a o l h ~ ~ & ~<br />
InUilUc d Rnlpdue Medi 6Judion nd Rkirch<br />
(JIPMER). hdiehey, Ida. Mmb *as mintnincd<br />
in pMic cya u*la 8 dl-qiwd S i d<br />
drlr(lZh:lZh)~k*(#t5)~.Sadrd<br />
m m r r d a l ~ d n w a d l Q ~ * c n r ~ e d<br />
l k . Undnc (5.0 m&hL, h o h dl) wm @vcn<br />
dlyMa~of~.Om&!xdyrdgpr&y14<br />
ad. fhcmmmllnimhnoind8rbnibrrdvrrol<br />
chevehick. BodywighQmt~oncnyIlcanale<br />
dny mil IC ad d Ihe qahml. 'Ibo mhmb<br />
ux killal by Uhcr 24 h llla OD Y -. Bpld<br />
i vim mval, dcnsd dl Ihe ldahy ligucr<br />
md wished. Thc epidi qona a- ad d h y<br />
weir. dm bmrd*cty.nd mddq qam vac<br />
cdkc4alMduscdfn~m)n.
WnCr, inyminn~nt of spmri motility, nducsd futiliza- dicnlg (hsl lLdan inducts oxidative s w in epididymis<br />
licm ability, lbnamal ~pum in mcn and and cpididymal sprm. ?hs significant incresrc in Lhs<br />
wildlifd"' . Our @OUS Uudia on onc of be avimn- lipid puoxidation of cpididymal spem in linjarr.tmled<br />
maurl mnminanta adosulfan<br />
-<br />
have shaMl to esusc im- rnts has dm brm ascciaLed with the dsnsscd sprm<br />
primant of tentkulur furrcionr Pnd k t the Bndrogmic- mocilily. 'Ihc gcnascion of ROS has bm mrnlated with<br />
ityinrsul". AQr&kanlinIhcpcsmtuudy (5.0 f&alogicaI cprm ~ o r as i oligosprmis ~ tanned as<br />
~oflindac/~~bodywdght)h~becnrmridacdas defeaivc spmns' with Rduced cprm motility, futility<br />
No Obaaved meu Led (~05)['). Lindsrr treat- Md span-mcyfc<br />
In the prrrcnt atudy, tke he.<br />
muH haa dmvn a tignirmnt in thc body wight pididymd spam mts and spm motility wn &-<br />
ud thc 4ghU of mtia. vcnlnl pmsfate and 6aninal crraccd in IindPnc-treaud rats as cwparod to the mnml<br />
~eoickr('~. Ln thc -I .udicr Ibc epididw wight anhla ilxlbiq thst epididymia was undsrgoing oxidaof<br />
lindnne-W IUIO was deacsrd eignifu.nntly and liw rmss.<br />
Illis my be due to lk drrravd biavaihbility and produccim<br />
of albgas(~~.<br />
Ro-oxidon1 onl ~ hiQIidPI1( bdum k viml r~ nornvllbidoplcPlrunaioningorlk~.<br />
lrsnyofthc<br />
can&, ampmu arh rc cllvirmnccltll motsminonta<br />
nff~Urhl.nacanpmvukeuecssiwpoduc(ion<br />
of ROS that is effaivcly wawgcd by ~~ antioxidant<br />
defapt apbn"). In 7, sewrnl an-<br />
(w paein)<br />
(mg mu)<br />
SuprmkCdim?Smb<br />
(ma padn)<br />
(mg DNA)<br />
Glu*hions-c<br />
(ma W)<br />
(ma DNA)<br />
aunhDncpm*duce<br />
(nlg ~m(cin)<br />
(w DNA)<br />
&or w -9<br />
(ms Imuin)<br />
(mg mu)<br />
wpaojdsaon'<br />
(W &) 5.28tO.!a 5.61tO.M'<br />
(mo DNA) 4.Pr0.37 5.74ro.39'<br />
PC0.05 W m a d u a m<br />
liOxi&"l *lg P6 glutahhe pauxidasI"', supmai*<br />
ciismu~py[al catadm) u. kmwn to w.<br />
Cytqbn of spmnmm is uuanly liud in volume In mnclusicm. fhe pent studies mflcct that linand<br />
WWi, a the polpmauwd fmty acids that dune indm oliiduiw smas in rat epididym* Md epibound<br />
in Ihe spurn p l w mmbnrr are wy sumbk<br />
lo RQS d~Y'.<br />
els or an~ioxidmt sraym~l with a pdbie raductiMl in e-<br />
didw spm, by inacasing ROS snd dsming levkubxidut<br />
auym rmrrliurce a mutually support- pididymal sprm mnD Md qididyd sperm molility.<br />
in ICM of defence against ROS. In Ua pocnt sudy<br />
thc rpoific rtivitic. of atnlar, rupaoxidc dimtase,<br />
#lulnthnrr tuk@nsE onl gluwi pcrmk WM<br />
lurd lo hc k& whik nn inumff in thc hydmgen<br />
m ilulhas h1k UT SW~T of Bioinfmtics CenpaoxWcpaxmcionMdlipid~wuc~<br />
UP., Pmlichcrry <strong>University</strong>. P~dichury lw pviding<br />
in~Md@did~rpnnoflklindPno~- -a. fdtia. KCC vluawicdges My Tata MMOed<br />
n(r. Tlnm in Ua pwuu thc incrasc in lipid rhl Twt. W, India fw the Junior Sehol~6hip.<br />
-Mi with a rrduction in .n(iori atym in- a Pcxmwkdgu Mian Cauril of Medical Ra+arrh,
New Delhi, llrliil Ib SCI~KK Rcm& Miavship. PPM<br />
nckmwkdgm Ihe receipt or fi~ndnl auppn fmn Ropl.<br />
inlim Council. New Yak, USA (Grant Nm. 699.<br />
(H7P79/ HMC md B93.M W IQrlC).<br />
Ref-<br />
: RmW Kt%. Mia. P. *a* P. Ddta KK, D**rilh 75.<br />
Wcal rwn~~tk<br />
~ilwy I* I k n B r in rar. J ljrvinn<br />
m h 11111; r,: 4ln -Ln.<br />
2 Oudvid; RW, Cmpa RL, 0-q J. Rdnbsl a. M9-<br />
myW. Rmjbk-r(iviyd1irdncinfanJe<br />
ma. I Wichcln Tm'd I'AU; 3: 147-XI.<br />
-<br />
n m. w e.<br />
4 Imh D. OIyp midy ad aygn<br />
~nhnp<br />
W* I. ~sts HI. M I.<br />
RqnI*ni~ luxbly a] ti- mrarmbs d iingr in<br />
irWl nmk mt. Hun Toriml 1%: 15: U6- 10.<br />
-ips<br />
inmmmmls, liaRrlBidMad1989: 7r 67-1OB.<br />
pl4.<br />
mi mioxib aNm in implnn*kn R1 m: m-<br />
A h H, T- U. Tam H. ldvarc d mpc&m+rn<br />
qrra m III~&UI*: .-. Tol- 15%: 50: 2.<br />
~ o n ~ d r m a c m b r p r * . i l m Rqmd .<br />
6 -A, ti-&4~cdFlrJhna-<br />
T& 1%: 10: al-s.<br />
~ r ~ s c i d . ( m ~ ) : ~ ~ ~ a d pmmKC. o l i-C.<br />
-<br />
MhrPP. --el-<br />
Mcsmmprr-. FmuBiacWr 5: 1-15.<br />
~mdm+mlfmmtk~~ffvllirmdn. kimJ<br />
7 Vukh IA, hna Am. Iem, AP. K d OR. JWn Amhd IWt I: m-6.<br />
VW. I*+& Haubrbiuly .taws Ur arly<br />
mWrcswm~inbra(inUrhiu~rUeIm.<br />
dncimkam. Toxd MLtam 115: 45-51.<br />
ti r*lpul R. a"- KC. ~ilcmun)cn*rr C. ~ d u<br />
w.<br />
U i e d l i n L u c m C I i c v l r ~ ~ d ~ MaebMRF, kn R. RnprCYl d*+rmamJ npmrgarmzpminorhhm~.AdmJAnW~l;3:<br />
-<br />
135- i a d . M n d ~ d ~ d ~ k<br />
R<br />
m.ca+dl46dpid&hb,ema. BiahrmJ<br />
9 tie RE, be& I.?. MFmy WK. Marrmcn d cpi. l5Uq 1919 289-97.<br />
~slqmnmilrlys.mnvtCinUrU. BJIPnvi. Jdin C, .%z% IC. Weka P, Mmln DL. C.l**rr R.<br />
mCm;lmTmml LW; ?&: 317-24.<br />
uJ+ah*ykhmm~ndadnlC*m<br />
lo adxme A. mi*iy In: 0,eawdd R,aiIa. OmsrRa15u';I, Is-%.<br />
CXC Hanhxt oTmIhxh fa m m &-.<br />
Rm- MlienRS.WD.WD. UedaIl.)irrm-<br />
'h. UuHncn: a\CRa: l'B. pm-4.<br />
~kndd&.yanmimwehcFpaol*<br />
II M ~ S . -0. Imdmmnd-aim<br />
a7wdrrrh*awenprdsanh."mprmrrm. J<br />
n a b t ~ ~ o l a m d ~ n d * m r m a a m y<br />
lusmmz'&dnm*k. WJBiDdan19741 47: 4W-<br />
5086-91.<br />
w ; R . K a b a i Y . ~ r i o n d Y 0 , ~ 4 .<br />
chsnbllydkklprW--inbadbrmhi.<br />
p* -*ldi. Qll tmnml lulli Wl XI1 - Iu.<br />
-H.<strong>ON</strong>NN, YadK.kay(aUpldpcd&h<br />
ln~auabymkbrbl&~rcrtim.~Bioda<br />
l!691%: ll-8.<br />
Lo*rl ai. lldnQl@ NJ, b7 AL. Prim a. Rein<br />
~VimhDfolinphaDlrrqpr. JBidOan<br />
IS11 1% 2s-75.<br />
BuwK. AO.*dUrmnetand-dtk<br />
~~tukcdabrra*ahrdmd&-<br />
oxyWm&k dd. Biahcrn J lP561.52: 316-a).<br />
m c g ~ l c a n a i a l a s m r m ~ l l aIn:<br />
y .<br />
HmK, d SJay OuiQLin*nr. Oarn: WHO; IW1.<br />
uHer lo, sis.q BT. Rde d $udim pxwnm? in<br />
pmse&ynnnrf*rlanrua*hanladmtilly<br />
dbyqarmaalipidrumbnbl. OmeCRa198);<br />
23, n-50.<br />
Rz+dRnillPPJi Wr441-I.<br />
-T.WT. M H . Sdlill WB. RadlUmYp+dmbllarrberrmm-hrmaia~llvlnhnn-.<br />
InJAnWIP)9;P:S7-4.
The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin 011 the<br />
antioxidant system in mitochondrial and microsomal<br />
fractions of rat testis<br />
C. Lalchoumycandane, K.C. Chitra, P.P. Mathur *<br />
Scllwl oJ UJr Srmtrrr. Porsbrlirrry U#zri.rr.rrly, l'un,rllerrj, - WS 014 hrao<br />
Rc-cc~vcJ 21 Auyun ZMlI: rutivcd in rvvircd lirrtll I S Scplclnhr IKII: acceplctl i Norcmhr !lull<br />
The abilitv 01 2.3.7.8~tetrachlorodiben~~~~n-diorio (TCDD) to inducc oxidntive alrerr in heoutic and some<br />
extmhepatic iirrua of animals hu ban reporied. The precise nature and mhanism of action ~ ~TCDD on the male<br />
rc~roductive rwsm is not dcar. In the orucnl sludv, we hnve invcstiaaled the induction of oaidalivc slrcss in tllc<br />
leilic of ral dncr expoaurr to low do& 01 TCDD. TCDD (I. 10,-and 100 nukg body weipht pcr dey) wus<br />
udministercd orally lo the rat lor 45 dam. Aner 24 h of the lust treatment the ruts were killed using uncslhctic e~hcr.<br />
The weight8 ofthc -ti&, cpididymu, remind verrlo and renlral prostate dccrekvd uh~le the bod; uclghl rcmalneJ<br />
uncksn@ tn the mu adm~n~stercd wllh TCDD. M~lochondr~ul and micrmmal lrsct~ons of the lesl!s %ere oblnlncd<br />
by thc niclhd or dilTcrcnlivl antrilugulion. Thc ~lclivicy of u~rtioxidulrr cnzymcs such ax snperaxi~tc d~smnv~~sc.<br />
C I~IM, glululhionc rcducluse, und gluluthio~~c puroaid;~se dc%redml sig~~ilic~~~lly in llic LII~~II~IIIS trci~lc~l wilh TCIIU<br />
in u doae4cw11dcnt tliunncr in the milocho~idriirl UIJ ~I~C~OWIIIUI<br />
huelions 01 rut tc6lis. Tlic 1cvcI1 of ihydr(1~C11<br />
peroxde pencntlon (HIOI) and llpd prox~datton Increased In m~~ochondr~al, and m~crosomrl Iracl~onr of 111e lest s<br />
Thc ruolrr su9~crttd that the bw dovr of TCDD cllc~l dcp~el~on of ant~oatdant enzymes and concomltdnl tncrerw<br />
in the lcvclr O~H,O, and lipid peroxidation difkrentially in mitochondrial and microsomsl fraclions of rat testis. In<br />
conclusion the adverse erst oTTCDD on mslc reproduction could bc due to induction oloxidslive arcs. 0 202<br />
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Kqmrw~lr: 2.3.7.B.Te1mchiomlibc1~o.pdw1in: Tutir Anliuxkdnn~ mzymn: Lipid prroxidstion: Mi~ocllondriul Irucltons: MIcroro.<br />
mu1 fnniollr Rul<br />
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tlic possible clTects of environmental contanli-<br />
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4136>3211.<br />
E.,~WII .ilu*h.~~~: p p ~ t h ~ ~ ~ ppmrthur~hol.<br />
~ . ~ ~ . ~ i ZOOU). ~ . i We ~ have reporlcd the effect ofenvironate~imuiI.com<br />
(P.P. Mulhurl.<br />
lal co~ilarninnnls endosulrun ultd li~idil~te 011 tile<br />
Il.r(URJXnI26 . n: Iron1 nmtler 0 M2 Elrvier Scicmr lrclilnd LJ. All rich11 mrvd<br />
PII: S0300-4I3XfOI 100563-1
tiii~lc rcprmluction or rut (Cllitru ct HI., 1999:<br />
Suj:ltl181 ct ill.. 2(K)I: Cliilnl Ct el.. 2001). Onc of<br />
lllc mtist ptcnt cnvironmcnlnl contaminants is<br />
2..1.7.R-tctr;1chlo1~dibenzo-p-tlioxi11 (TCDD).<br />
wl~iclis Ihrnicd ;IS ii by.ptoducl in the manuracrllrc<br />
ol'chlorini~lcd Iiydrmrbons. This compound<br />
is :!lso Fcirnicd in the incineration p m of niu.<br />
~~iciliill ri3i~slc, pilpcr and pulp hlcilching, cmissio~~<br />
Ijt)lll \Iccl li~~~~iilrics i~nd lnotor vcliiclcs (Skc~ic ct<br />
ill.. 1'189). Tlie lipphilicily and low rate or<br />
rncli~h~ilism of TCDD leads to its accumuhtion<br />
;lncI ~~rsistc~icc in udipor tissues (Enan et HI..<br />
I0'18) It II;IS lrcl~ rcptirtd 1Iii11 niillc rills cxpml<br />
to TCI)D displ;~y rcditccd fcrtility, dcliiyed pebcrly<br />
;lnJ altcrcd rcprductivc organ weights<br />
(Cri~y ct ;~l.. 1997). Millc MIS cxpoxd to TCDD<br />
sllt~wcil dwrc;~snl spcrln cou~irs (Gray ct ill..<br />
1997) ~IIIJ IIII ~I~L~ICCJ numbcr or ubnonnsl<br />
svrlli (I::~qi ct ill.. 1997). Scvec~l niwhi~nis~ns<br />
llilvc Iwcn prop~rcd to cxplilin llic toxicity or<br />
TCI)I) i111c1 irs coIigcncn (SIR. 1990). Thc most<br />
co~isis~cnt biwhcmic;~l cnht or TCDD hi~r been<br />
sliown lo be the induction of cytcchrome P450<br />
iasax~tll~tl nnlonnoxygcnas ilnd rcletcd protcin<br />
(Pal:~~~il i~nil Kimbrough. 1984). Reantly nryl<br />
I~ydrwi~rbon (Ah) rwplor mdinled xnnrliinc ox.<br />
idi~x and xunthine dchydrogcnasc system has<br />
been rclated lo oxidative stma in vivo in the<br />
TCDD.tra11cd rats (Sugiharn et nl.. 2001). Acule<br />
highdore exposure to TCDD results in oxidative<br />
strcss in mulriplc tissua and species (Mohammnd.<br />
pur CI al.. 1988). TCDD has ken nporled to<br />
indacc suproxidc anion, lipid peroaidution und<br />
DNA damage in the hepai~ and brain lhua of<br />
rat (Hassoun et al.. 2001). Mitochondrial and<br />
niicros
I kl<br />
wciglit (b.wt.) per day for 45 days. For administration<br />
of TCDD, the pipclte tip was aently<br />
placed just inside the mouth and the dosing solution<br />
was then slowly explkd from UK tip al.<br />
lowing the animal to lick the compound.<br />
Corresponding groups of control animals were<br />
niai~ltni~ied und admi~~istered with vehicle nlone.<br />
'I'Ac inrinrals wcn? I'asicd overnight, wcighcd<br />
and killed by using anesthetic ether on the day<br />
following the lasl dosing. Testis. cpididymir,<br />
seminal vesicles and vend prostate wre rc-<br />
~iiovcd und clcnred from the adherin8 tissues.<br />
The weights of the tissues wrr recorded in mg<br />
as well as mgj100 g b.wt. Tatis wu used for<br />
subccllulur fratlion~lion and bialiemiw~l nud.<br />
ics.<br />
Mitocl~ondri&l and microsomal Iructions of<br />
Ihc tutis wen obtained by the dilTutntial an.<br />
trifugation method as duaibd by Chainy et al.<br />
(1997) afler rtandardizution. Briefly. n 2P/. (wlv)<br />
homopnutc was preprcd in &sold 0.25 M sucrose<br />
wlulion with the help of r motor-driven<br />
glass leflon homogcnh. Tbc hmogcnatc was<br />
antrifugcd at lOW x 8 for \O min nt J *C to<br />
obtain the nuclear pllet. Mitochondrial pllet<br />
was obtained by anvifuging tbe port-nuclear<br />
ruprnnlnnt at 10000 x 1 for 10 min at 4 *C.<br />
The microsomnl plkt wu prepad by the calcium<br />
chloride (CaCIJ Pedimcnlrtion method of<br />
Kamath and Nmym (1972). BMy. the plmilochondriel<br />
superrutant vu diluted with icecold<br />
CnCI, (I M) w that the final caranIralion<br />
of CaCI, was 0.8 M. 11 w bblted at 4 T<br />
for I0 min with occasional stirria& Tbm th<br />
snmpk wan cmlrifu@d at 10000 x 8 br. 10 min<br />
at 4 *C and UIC microtorml pellet wu obtnined.<br />
All the frnctiom wn wuhed UIk with iced<br />
d 1.1 5% polouium chloride solution md dirrolvnl<br />
in tlu 0.25 M sucnuc wlution (I mg<br />
prolcin/O.l ml). Thc mitochondrial, and microwmal<br />
fraclioru wn uud for biochuniarl dUdis.<br />
Superoxide ditmulu~ (EC 1.15.1.1) was<br />
sryed by the method of Morklu~ld ,<br />
Mnrklund (1974). Briefly. the uswy ~nixtuve c<br />
tained 2.4 ml of 5U mM Tris-HCI buffer<br />
wining I mM EDTA (pH 7.6). 300 p1 of<br />
mM pyrogallol und 300 111 elizylnc source.<br />
ilwrcux in clbaorh~nce was mci~sund irli~n<br />
rctely ut 420 nm ugtlinst blllnk co~ilnining HII<br />
cwmponenb cxctpt tl~c enzyme and pyrogullo<br />
I0 s intervals for 3 min on a Hitschi U-2<br />
Spsctrophotometer. The activity of enzyme<br />
expcard in pmol pyrogslbl oxidizcd/min<br />
mg protein.<br />
Catnlaw (EC 1.11.1.6) was arrayed by !<br />
method of Clviborne (1985). BrieRy. the ass<br />
mixture conbitled 2.40 ml of phosphate bur<br />
(SO mM. pH 7.0). 10 pl of 19 mM hydr<br />
peroxide and 50 pl enzyme souru. The dcc<br />
in nbwrbmcc wsr mwud immediately at<br />
nm against bhk conmining all the compon<br />
exccpt the enzyme at 10 s inarvals for 3 mil?<br />
a Hilachi U.2000 Spctrophotomem. The ac<br />
icy of enzyme WM expW in pmol of hyd<br />
gcn peroxide consumed/min per mg protei~,.<br />
The oclivity ot gluUthionc reductax<br />
1.6.4.2) was utlyd by the method of Carl<br />
and Mannnik (1975). Briefly. the 1ury<br />
lure conlrinal t.75 ml of phaphntc bulTcr (1<br />
mM, pH 7.6). 100 MI of 200 mM NADPH.<br />
pl of 10 mM EDTA. 50 1 of 20 mM<br />
lathione oxidii and M pI enzyme wurcc.<br />
appevrancc of NADPH ww mws<br />
immediutcly at 340 nm rgainrl blvnk contvinl<br />
all the cornponenu exapl the enzyme a1<br />
intcrualt for 3 min on a Hiinchi U-IOM) S<br />
[rophotomcter. Thc ucrivity d enzyme wus<br />
pmwl in nmol 01 NADPH oxidi&/min<br />
mg prolein.
C'. I~~I~~~~IIIII~~O~,~~~~~~<br />
R6+ 4,lNl'<br />
'Tlic sctivi~y orcutulnr Jecreuud signifiantly in a<br />
Joscdepcndcnl manner in the mitochondriul and<br />
microrotnul Iractionc when compared to the cor-<br />
rapondig group of control ~nimalr (Fig. 2). The<br />
levels of hydrogcn pemride &nention and lipid<br />
~roxidntion wcrc incmscd rignificantly (P <<br />
(1.05) in rnitcchondrial and mimsomal fractions<br />
of thc tatis when cornpnred lo the wrmponding<br />
prnup of c~>orm\ animds (Figr. 3 and 4).<br />
(with and wi~hout secretions) nnd ventral prost;tte<br />
in a Josc-dcpcndent manner. In the mi~le rats<br />
TCDD exposurc during adulthood lius been<br />
shown to dccrcnsc the weights of the leslis, sctninal<br />
vesicles and ventrnl prostate us well as altcred<br />
1csticular und epididymal morphology Oobnson et<br />
al.. 1992). These eTTKts may be due to reduced<br />
TCDD one of the envimnmntul mnominunts.<br />
hut been &own to induce reproductive abnormaLilia<br />
in both wildlie and humma with reduction<br />
in futility (U-Sakuwy ct 11.. 1998). In the<br />
present dudy, the animals mrc administered with<br />
TCDD at low daa (I. 10 .ad 100 nag b.wt.<br />
pr day) lor 45 day& Sin@ muMIl expsurc lo<br />
low Jwcr ol TCDD (50 nL) haa been reported to<br />
rsduathcwdJIuoFthetcltirand~ryrr;<br />
orpm of nu (Otuako sc .I., 2001). Thc body<br />
weight of MI& lrutod ,with TCDD did not<br />
chuyo indiating that l s pml metabolic conditiona<br />
of the mi& wom within noml nnge.<br />
Administration of TCDD ad duaion in the<br />
wi#hu of lhc mllr, rpididymk minrl wicks<br />
FI. I. TCDD on lhe anivitin 01 ruprcrxide din~~ulaw.<br />
Ju~thionr ~~JWIIL 8rd $luelhinlx pcrnxaur ill Ik mil*<br />
chodrid (Mill m d mieraeml lractiont (MLI 01 1111 Icblir.<br />
The silvilia crpd in nmollmin per 111s prolcill al<br />
35 .C. Tb nlw *re upraxd u man f SD (01 six rnnltal<br />
and a iruporimaul mimuh pr pup. 'P
0.1 00 01<br />
" 0.8 I . .lo Dl*<br />
rcnn IW *c wl ~*rbtl<br />
I:#* ? lin'iw or rrDo on I& nnimty d mlah in ~hc<br />
~llll~el~nJri;~l and micmaml fmh nf nt lalb. Thc<br />
;rlirilh.i .In cxpn%ud tn pmllmin pr mp pmKin 11 35 .C.<br />
I Ihr V~IIIM IR. PIF%~(R( ns mn f SD Inr six mntd anl is<br />
cr~riltrnl~~l iu~ltn~ill. rn #mu*. 'Pz0.05 hmnridcltd lo h<br />
.~pmik.!nl npilm.il mnlml<br />
numbcr or Lcydip dl. germ cell and<br />
steroiilogcne enzyme activity. as well na impaired<br />
Lcydip ell LH mponsivencs (Mably el al., 1992:<br />
Joli~tw~n ct 211.. 1992). fhc weight or the epi.<br />
didynlo und u~crssory sex orgsns rquirc r continuous<br />
rlndrogcnic stimulation Tor Ihcir normal<br />
gro~vll~ and l'u~~u~io~~s (Kli~ulcltcr und Ha.<br />
I'FlUt. 7'hc wcigllts or u-ry sex organs have<br />
hrn uwd its b~wswy or andrugens (Milrkur and<br />
I lc I I IILY~ or 1'c'1)1) Im ~ kwh d hydm(m Fox&<br />
.... f,.,., ,,, IIIC IIIII~I.~IIY~~~~:#~ awl oranwwlu!l rt.aliutr<br />
,,,,,.,,, IIK ,.l~lr. rtr srl~r\w+~l ~knwl lwn I-r nbp tn8*r?8l<br />
.,I I* '1 .Is I ~ !\I) K ~ rlv $1, ~ conlrd ~ ~ and us crpmlrltlsl<br />
,1,1. ~r Fvq,p .I'rllll5 n ~~,n~nlmrl In k stfnikionl<br />
,1. l,,,.l 1111111111<br />
4.4. Elrm dTCDDon lk*wkdUpidpraiiblbnin<br />
milochondri~l and miaolavl tnnia d N W. The<br />
nlua mn apnrd in Whh w mt pmccin nl I5 T as<br />
rmn f SO Icr ua mnlml and Sia riprirrnUl anirmls pl<br />
mp. 'P < 0.05 nl ranidnal lo be sbiiknt .pin.<br />
mnrml.<br />
Chattopadhyay. 1982). TCDD has ken reported<br />
to reduce plasma tcatwlerom. Sadihydrotcatosterone<br />
(DHT) and LH (Mably el al.. 1992). The<br />
observed reduction in the weights of ~k amnory<br />
su orpm rdkts reduad bioavailability and/or<br />
production of andmgm in the TCDD-lrci~tcd<br />
MIS.<br />
TCDD has bccn shown to ad through Ah<br />
mptor. a lipnd aninled nuckar lrnnrriplion<br />
raclor (Safe. 1990). The nuclear Ah wptor conrplea<br />
is a heterodimcr conlnining the Ah receptor<br />
und Ah naptor nuclear lrnnsloetltor prolcinr and<br />
the molcculur mechanism or Ah +or sction<br />
has bem shown to be assodated with bindinp or<br />
\he Merodimcr lo dioxin mponsin ekrmts in<br />
rcgulr~ry regionn of Ah-mponsive gene (Sure.<br />
H)OI). It hu bacn repod that TCDD and its<br />
mngnm bind to the Ah mqlor, which mulls<br />
in the induction of m m I enzyme rysremr that<br />
may be mpomiMe Tor the production or rcaftive<br />
intmndnta such 1s Tree rndbh IKimbmu~h.<br />
1974: Neben a 01. 1981: Slohr. I WO: Slohs el ul..<br />
1991). The mechanism of TCDD-mediatnl rcuctlve<br />
oxypn spdn production has k n p r o w<br />
to involve the cytmhmme 1'450 IAl and lA2<br />
(I)iliherlc~ CI ul.. 1997). 1'1% Ah nwptor mdiolnl<br />
lndwtion of runthine oridnx'nnd xnntllinc Jchydropnnr<br />
activity by TCDI) ~IIS ~llro bccn rc-<br />
~I,,CI~I 1,) k n.*p~\~ihk. lor !In' pr~uhlni~~!~ 111'
'lstrr,tb~xr 171 i?lYJ?I 127 /.IS Ill<br />
rcuctiw oxygen rwim in liver (Sugihilrs ct ill.,<br />
2OUI). In the pracnl aludy adminiutrution ol'<br />
TCDI) dccwd tla ttctivitim of ci~t~ilitw, xc~pr.<br />
oxide diuilutuw, &IuIathio~ic rcducttt~, itlid 8111-<br />
tutliiolw proxiduw. wl~ile the lcvcls of bydrop11<br />
peroxide and lipid peroxidntion incrwscd in tlrc<br />
mitochondrial and microrom~l fnctlonr of the<br />
testis. Mitochondrial rapintion is the main biological<br />
wurm of tuproxide anion radicals undcr<br />
physiological conditions. The mtioxidanl enzyme<br />
supemaide dimutasc hps bmn rhown to aculerute<br />
the dimutation of the superoxide anion into<br />
hydrogen peroxide (Nirscn and Knyscl. 1983).<br />
Cntulase allows degmdation of hydrogen peroxide<br />
into water und oxygcn (Jeulin el al., 1989) and llic<br />
glutathione pcroxidue/reductue system ha^ been<br />
rhown to calalysc the degradation of hydrogen<br />
peroxide and lipid hydroperoxide by using re.<br />
dud glutathione (Alvam and Storey. 1989).<br />
The reduction in the activitin of antioxidant enzymes<br />
and incruuc in hydrogen peroxide and<br />
lipid peroxidation could reflect the adverse effect<br />
of TCDD on tho untioxidunt system in testis<br />
subcellular frnctions. Oxidative stress has been<br />
shown to occur primarily in the mitochondria<br />
fol\owed by microsomn. Orievau and Lunnou<br />
11097) rcprtnl that ROS such as HIOt uppca,rs<br />
to k u key agent in the cytotoxic effects In<br />
spemlowe and in udJition to their dim effect<br />
on cellular constituent produce oxidative stress by<br />
dwrc~~sil~y the c11zymatic JeRm of the testis.<br />
Un& high ROS mnrrntrntions pre-dumuged<br />
rpcnnutowu an exposed to lipid peroxidatioli by<br />
polyunwturuted fatty acids (Ichikawrr ct al..<br />
1999). The mitochondriul and microsomal membrann<br />
have ken reported to undergo permurbility<br />
changes following enhanad Lipid peroxidation<br />
and Jutathione depletion (Chancc ct al.. 1979).<br />
One of the important upatl of anlioxidwt en.<br />
rymer ii their nature of synergistic functioning: a<br />
decnrsc in superoxide dirmulav clctivity hm been<br />
shown to increase the level of supcroride anion,<br />
which is known to inactivate catlrlrro llctivily<br />
(KOIO and Fridovich. 1982). Similarly. when<br />
wtahuc or gluuthbne psmxiduc faib to climinate<br />
H,O1 from the all, the accumulated H,O,<br />
hru been ahown to caw inactivation of suproxide<br />
dismuw (Sine1 md Garber. 1981). Reactive<br />
oxygcli spccics Iisr bceci sliown 111 d;~tiii~yc ;llliioal<br />
ill1 ~llscromolcculcr. viz. prolein, lipids. ccc.. i111d<br />
lllc cliiinpr in llic rcprtnh~ctivc orpiill wciphts<br />
111:ly slso Iw duc lo iIi~~l~ii$c ~IIIIIIIC~~I hy IUIICI~VC<br />
Uxygc11 rwiw (&l~~ultdt%rl: IDYO). 'I'llc ~.acllla<br />
sugycst thut the low doses or TCDV clicil dcplc.<br />
lion in antioxidunt defense system diflerentislly in<br />
mitmhondrial and microsonial rrilctions indici~ling<br />
TCDD-induced oxidative strcsr ill tculis suhccllular<br />
fractions. This may lend to disruption of<br />
functional integrity of cell organelles. In conclusion<br />
the adverse long. term effects or low doscs or<br />
TCDD on male reproduction of mts may be due<br />
lo the induction of oxidative stress in testis.<br />
Acknowledgments<br />
Tlie aulhors ock~~owledge Dr Slcplien Safc. TX.<br />
USA, for the generous girt of TCDD and thc still'l'<br />
or the Bioinrormutia Center. <strong>Pondicherry</strong> Unlversity.<br />
for various incilitics. C.L. acknowledges<br />
the Indian Council of Medicnl Rcseilrch. New<br />
Delhi. for Senior Research Fellowship. K.C. ilck~iowledges<br />
the Lady Tstu Meniorial Trust.<br />
Munibai for u Junior Scholursliip ~IIIJ P.I1.M.<br />
ucknowledges Ihe Population Council. New Yolk<br />
for financial assistance (Grant Nos. B99.047P-91<br />
ICMC md B99.048RIlCMC).<br />
Ra. 23. 77-90.<br />
Bury. I.A.. Ausl. S.D.. 1976. Lrclo~roridlw cill;tlyml Ilpal<br />
proriblion of micrororn~l and ~~rlilicrrl n!nn~brnnn.<br />
Biochim. Biophy+ Acts 444. 192-201.<br />
Curlbrre. I., Mmnmik. 8.. 1975. Purifimlion und chlmtctcrl.<br />
ulion d Ur Ilrvanzym plulrlhiom rrJurlax from nil<br />
livcr. J. Bbl. M. 250. Yl4-5480.<br />
Chainy, O.B.N.. S.nunwrry. 6.. Salnanla. L. 1991. Tnlov<br />
temne-induced sbnp in taticulur nnlioxiJilnl ~~(ICIII<br />
Androlopiu 29, 341-MY.<br />
Chuncc. 0.. Sie* H.. Bonrit. A,. 1979, llydrapcro~de<br />
nlslubolirm in murnmulwn orylnl. PII).riol, Kcv 59. $27-<br />
514.<br />
Chw. S.E.. 2WO. Endwrinr disruploi. innd nlalr rrpruducltsc<br />
futction - a &an ncuka. tat. 1. Andrul. 21. 45-46.
Chilr:~. K.C.. L!olnn!myci!~dum. C.. Milhur. P.P.. IWP. Kimbroufi. R.D.. 1914. The loaklty d the p+l~Io+.~cd<br />
('l~io~tk cN~yl ol'e~dtaulhn un Ik Inlicukr funstions or imlmciic m d s m d chffabla. CRC Crit.<br />
ra A'iiiln J Adrol 1. 203-206<br />
Rn. Toriwl. L 443-498.<br />
Chtlr;~. K.C.. Sul;!lha. R.. Lulchoun~yclnJan. C.. Mathur. Klirrkksr. 0.1, Heo, I.&, 1998, To* 01 lhc male<br />
P 1'. XKll, EGXI rf lindme nn entinxid.nl ctuymn in uarmnt d m ad r4 *.dl. In: Kod. K.s.<br />
cpal*ly~nir >~tmI r~t'dym~l sprm of ntlull mu. Asinn J (Ed.). R*nodustk and R*okmul Toikdqy, pp.<br />
Andt,-l J. ?(I5 XIR.<br />
-((1. -- 0- . 1<br />
,<br />
('littlwrn~.,\.. lVX5. 11,. C~RVIIWI~!~. R. (W.1. IlmJhr,k Kono. V. FtlJa!ch. I.. IPU. Supmxidc ndbl inhibits<br />
Mrlln
S.tlc. S.. ?IXII. Mubuli~r bioluyy ol 1 l ,411 ~ rwqvlut iwd it* IOIC Sl1n11h.S l .Alrlt.~c~l.N.J .Sh;tr;!. M,A..Al~ll,~y:tl~./. A,. Wi!ltl?.t.<br />
in ath'iaop~xsis. 'Turir'ol. Lull. 120. 1-7.<br />
Z.Z., IWI. Bvidmwr. tor IIIC it~duclioa ol ill! ox3;clive sllcrr<br />
Slliarp. R.M.. 1993. Oxlining rprlll ~ounlr in mcn - i: ll>elu is rut hcp;nlcmilwh~ndria by 2.3.1.8~MIrucl~l~~rodibE1~~~~1~.<br />
"a cdwrinc ruur'? I, lidwrinui. 116. 317. Jbll.<br />
dtoxin fTCI)I)). Ad", lirp. MIYI. Ricll. 2x1, &?I R3l<br />
Silwl. I1.M.. tirrlrr. I'.. I1)XI. i~~wliv~~lk~~~<br />
a%( 11111111111 ('11 /.!I SIIFIII.II:I. K.. Kilt~~l,l~ti~. S . Yiu~tl~d:~. I .. l )Itlit. S.. )liw1:~\liil,8,<br />
r~~~ur~~r~Jiaa~~luwduri~\lsr~~u~Llt~O~~~~JII~O,~<br />
Arch. K., YI~su~I~, M.~ lri~,i~i.Kt~riy:,~tt;t, Y,, 21811 Aryl lhycl~twa~.<br />
II~x.ln~t. Ilu!pl~y. 212. 41-1 4Ib.<br />
Skme, S.A.. Lkwltunl. IS.. (imcnhry. M.. 1989. I'oiyel!lu!i.<br />
IIUIL~ dibnus.(#-dit)ai8ih atd polychbrinul~d rlikwh,.<br />
lur~l rvruelar IAIIW I.,t!etli;llcd l~alt!eli~n~t,l xi~~~ll~i~lc ~ull~l:)~.f<br />
x;~t~lllilludcl~ydru&~~!i~~-;~~livily by !.~.~.R.IcI~~~cI~~~~II~II~II<br />
,<br />
I'untn.: Ilr rill 10 bulnna hullll, A Rcv. Ilunl. 'l'ari~r,l 8. llrr).<br />
177 ?lIJ.<br />
huj;ellt;t. H..Cltllr;t. LC.. I.i~lul~~nt~~~yvi~lId:~l,c.C.. M,llll\rr, I'.I' .<br />
S~ohx. SJ.. IW. O~idulive atw induced by 2.3.7.8-lclrd.<br />
?WI. E ~ of ltndsm I on Icsicubr rnl~o~iJa,lr syrlcln und<br />
chloralibmw.pdwxi~~ ITCDU). Fra Rud. Biol. Mcd. 9.<br />
79-W.<br />
aerotdoscnic cnzymn in adult ruts. Astun J. Androl. 3.<br />
135-138.
C. hteboumyeradlos . KC. CMtn . P.P. Msibu~<br />
Induction of oxidative stress in rat epididymal spenn<br />
alter exposure to 2,~7,8-tetmchlorodi~~ioxin<br />
Rmclved: 23 Auewl 2001 IArrcptd: 7 Nomnkr 2WI / hbl~rhcd online: 6 February 2002<br />
0 SprinptVcrhg 2002<br />
Ahtnct The ability of 2,3,7.&tetrachlorodiknzo-p DNA) in the epididymal sperm. The results suggest that<br />
dioxin (TCDD) to induce oxidative 8tW in various graded do= of TCDD elicit depletion of antioxidant<br />
ti^^ of animala hu been reporled. The nalure and defense system in sprm, indicating TCDD-induced<br />
mecbanLmofwtionof~DDonthcnntioxidsntsyslem oxidstivc stress in the epididymal sperm. In conclusion,<br />
of spurn has no1 bccm Uudied. In tbe pnscnt study we theadvenedfccton malereproductioninTCDD-treated<br />
huvc wught to inwatigatc whether TCDD induas <strong>ON</strong>- ruts may be due to the induction of oxidativc stress in<br />
dativc mtror in the opididymal tpcrm of rau. Subchronic sperm.<br />
dose6 of TCDD !I. 10. and 100 ndkn bodv wci~ht Der<br />
day)w& pdminik& orally to m
(I't,l:~t~cl 211x1 Kin~hronglt IVX4). It has bccn'rclwrt~d 111111 Jilulr~i vllh 95 N ot dilucnlr Tbc h i p . on the wunling<br />
i~cutc hiflh-do~ cxwsurc lo TCDD results in oxidative ehrmbcn d Lh. impad Ncukaw4ypt -t 4%<br />
surd.<br />
strcss inn~ultiple iissues and species (~ohammad~ou;<br />
A ~ I<strong>ON</strong> ot rbs ~ lbomWNy ~ .M diluku Y<br />
sp&.nm *u vudnd to oh Omdq dunk. dthc hem*<br />
el 811. IVXX). TCIID h;~s hccn rcpnrlcd to indua suwr- cyIanna.TLo~x.lMW(L.ndIbr5minina<br />
;rxi
I. 1 Ilal 01 2 1.7 # I c I ~ ~ L I ~ i IlLl>l>l u I $,,t ~ WIR. ~ ~ 1, Ellbcl ~ ~ or TCUU ~ o un ~ tllc ~ uelivlly ~ ol ~uprorldo di\mulil in<br />
lls rpddynrul Ipml cww SF saunu wrc ac@rmslro ,n lhc epididymul sperm ol rum. The cnzymc uctivtly $8 exprrssed sr<br />
dupllate lmm ul upnmntsl and w; sonlrol nu The dau are ~nomolcr pcr rmnule end mlllinrtm or rolei in or m~lli~rsm al<br />
upraad am mun+SD. and PCOOJ ImIerub) u corv~drrcd 13 DNA at 35'C. The dnla ate hpd'a$ mcani~~~toi~r~a<br />
~ndrrlr I u)nthcsnl dlllmrur telrren opnmcnut and concrol cxpenmenlal and ci% conlrol groups. P
t11.3. Wal 01 TCDD on !he acttnly ouhe !n the cpoddymll<br />
.p.m, nr ,a$, llc ctv>tt>e ;oafvtly I- urn<br />
as mlcramola pm<br />
-wtttae uw twll~pc~n~ ~(PIo!c~~ ~rrnilbmmofDNA .I IrC Rr<br />
data sm ctpnxkd 08 mntSV for-w apnmcnul and mr<br />
mnlml SroLF P
Fin. L Corrclatwn bclwfcn "perm counts and DNA cootent$ or<br />
Fil. 7. Urea of TCDD on lied mlidrtion in the s~ididvrnal nl cpididymli rprm. A paaltivc corrclaten (r-0.9% n-24) war<br />
Grin or la.. 7hc m m adktyu in m~rdm.i"<br />
found between rprm wunl and DNA contcnl<br />
pr<br />
mtnulc and rnillignnn or mein Ot millisnm 01 DNA. n o data<br />
urn n W M munlSD Iw ax uprimcnUl and am control<br />
mum. PcO.05 lm~erldx) il lo indiate a rienificant activity (Kono and Fridovich 1982). Cataiasc or<br />
glutathione peroxidae rails to eliminate HZ02 from the<br />
cell, and the accumulated H202 has been shown to cause<br />
inactivation of superoxidc dismutase (Sine1 and Garber<br />
1981). Griveau and Lannou (1997) reported lhal ROS<br />
dovl of TCDD (0.15 ndkg per day on 5 days/week for<br />
13 weeks) wuld induce oxidative stmu in liver, spleen,<br />
lung and kidncy following rubchronic exposure in mice<br />
(Skwk ct ill. WOO). In tho orrscat rtudv administration<br />
of TCDD cuuml II ducll& In the sperin wunts, which<br />
1s wnrrclcnl wtth esrlter reporu (Fwt el al 1991. Gra)<br />
cl uI 1997) S~nce a wry ugn~hcrat wrnlauon (r- 0 95.<br />
18-24) was ohncrvfd bitwan cprm counb and DNA<br />
conlcnb in cpididyntnl,sperm. DNA wnlcnt wus routinely<br />
ucsd M an indicator of sprm wunt (Mukherja<br />
cl ul. 19921. Adminiatnlion of TCDD decreased the<br />
hydrogen peroxide and lipid peroddauon roo in the<br />
enididvmal soerm of mla in tams of boLb ~mlcin and<br />
~ N *LI~~I; A doso-response wsr not obscr;od wtl! re.<br />
galrd lo .any ofllr hit)~.hw~luI ptmmctcn In our rlvdx\.<br />
which is auito similar to (he mulb obrwed in studies of<br />
the ctTcct.of TCDD on the hepatic and braln tluues of<br />
nu ( Hmun el d. 2001). M~tochondnPl mplntlon tr<br />
the main biolo+icnl mum of tuamxidc anion radials<br />
in physiolopi&l conditions. 7% antioxidant snrymc<br />
superoxide dimuusc lsaknla the diimuulion of Ihc<br />
runeroxidc anion into hvdroncn mxidc. Cnlal;~se ilc-<br />
such as HIOl appear to be key agents in the cytoloxic<br />
elTects in spennalozoa, and in addition lo their direct<br />
erect on the cellular wnstimcnt, to produce oxida~ve<br />
stress by darcasing the enzymatic defenses. Thc rise in<br />
the kvels of hydrogen peroxide generation by 160-170%<br />
reflccls the induction bv TCDD of the oroduction of<br />
mctlve oxygen rpcctcs~lhat arc no1 e.ttn'~nated b\ lhr<br />
ant~oxtdanc enzyme, rush as catalase and g.u~ath~one<br />
reductase/proxidase systems, and this can induce lipid<br />
oeroxidation. The wssibilitv of eflects other than the<br />
uppnrcnt darease In unt!oa~d.lnt actl\lltcs tn caus ng 1hc<br />
tncw m peroude product.on also cdnnol bs r~lcd<br />
our. Cytoplasm of the spermatozoa is extremely limited<br />
in volume and localization. so that the ~olvunsaturated<br />
fatty aads lhsl nhound In Ihe eprm plkm'a mcmnrd.w<br />
urc vcry suscept~blc to ROS ultdck (Al.kcn et ul tYSJ,<br />
When ROS &nczntrationr are high, predamaged spcrmatozoa<br />
are exposed to lipid peroxidation by polyunsatunlcd<br />
fatty acids (Ichikawa el al. 1999). The sperm<br />
membranes have been teported to undergo permeabilily<br />
chanua followinn enhanced linid mroxidation and<br />
glut~htone deplc
IIR<br />
h r k a ~ r Ilw ~ ;njllmr~ l ~ Ilnionk Dr. Stcrhcn Sitrc. TEXIIX.<br />
USA, li,r Ihr: gctwmu\ gn uITCUU, and Ihc rlail of thc 01otnr0rm:ltin<br />
Ccnlcr. Pondichcrry Uncvcnily lot ~riow Iscilain.<br />
C L tr the Iholder oli, Setunr Rwurch Fcli~w~hip Im thc Indian<br />
~ ...., . ",<br />
('rrhg iron? tk LnJy Tala Memorial Tnul. Mumhi; w t r and bn~n ((PYOI d nu Inn eubshronic 1;<br />
ilnd P.P M hi83 refcivd Ilwndal ;,nmtlnn: rmm tk Popvialion m~nlumo~TCDD.ndlU n IApplTrmcol2l~2lI-219<br />
Tclt~ml. Nrv Yolk r$mnla nn. RW YlP.P/ICMC.nd BPP.MRRI lehok.m T . ~ o d l ~ . ~ h mWB(1999) o n ~ R- oxyen<br />
Il'M('1 'Ihc ia~*~llvn LILYI:IP tlnill IIK cx~ri~ncnln done Jurin~ won tnfluaa Ik o a m a raa.on hut nn r.ain anans.<br />
tllru. ato~lnz R)III~~Y wvlh llle cl4rrelll 181~1 or lhr co~ntry.<br />
J&,G&:fi
Iionshii<br />
nepmdvctivc mxicity ad uptm m RewrA (JlPhf6Rh Pondichay. In& llx mhnls ~nr.<br />
chaniuls Lhat geasltc ructive Oxyw apsic. (ROS) 191. nuinainodvndcra12h:IZh~of~.ad~md<br />
It has ken rtur mthoxychlw urdapm -5 pmvidad*.tad.rdsommcrd.l~Wadruer<br />
-rygcnrrc mdialld mivation ad the 8d libilurn Metbmychla ( l . l . l - u ! ~ 2 3 ~ ~ y -<br />
mulunt d v e mtrbolitar, pxsibly free ndiulr, bind P ~ Y I I C X ~ ~ ~ 95%. sima mmnw Ca) wu<br />
mnknUy m micmroml commnu [lo]. Antioxidanw diualvtd in mna ad olive 011 (1:19) md .dmlnlncred<br />
free ndiul m m and suifhydyl WnWning mm- dlyndaad50. 1 0 0 . a 2 0 0 ~ b o d y ~ d a y<br />
pOunds inhibit wvhl binding of mihorychlm in hurmn for 1.4. a 7 days. An Antiaul group of minub was<br />
liM miuaomm sumttg that the mdve inmmd(aln -ldministaed -w 600 mslLI WY VclOhv<br />
~m+ndiulr[11].IthasllsokenrrpMtrdr)uthuman<br />
day)mdritMinEZD~bdy~for7d.y~.<br />
cytochmm P4U) cnzymar respmsibk fm conversion of<br />
~&~oftkdodn~d~oll,tbepfpme~p~~<br />
methoxychla im 16 nqior mmblhea, the m o w -<br />
pcnUy pld<br />
jW inaide the mmlh ad tbe doring solution<br />
mcthylatcd dsivPive, ixluding CWlA2 play the p+e.<br />
dominant mlc in this reaction [Ill. Thc ability of the<br />
w lhsl slowly orpolled fmrn the tip dlowing the m i d m<br />
cyl*<br />
lid<br />
chmm P-450 systsn lo induce the poduction of d v e<br />
the cnmpound. A carrrpondiw Omvp of mnml ani-<br />
OXyECn swim in hepatic and other tisucr has bcen re-<br />
MIS was admini- M equal volum of vehicle alone.<br />
paned 1131.<br />
It has bcen rhom thtt mthoxychlor rndvcc oxidative 2.Z. Necm~~<br />
and eo"~lb7 oj~pidIdyn~1 rprm<br />
stress in the epididyd sprm of gost 1141. ROS are famcd<br />
on h ~ physblogr h<br />
pnlhologic euditlon~ in mumlw. The nnimlr vtrr fmed owmight, wcighd.<br />
-<br />
and killed<br />
lion tisuu: due meir high -tivity, ROS my int- with by using ancrVlctic clhcr on the &y folldnp Ihc last<br />
biomloeuia inducing oxiduiw [IS]. This oxidative bins. The wighu of the linr. kklney. -ti& apididymis.<br />
rocss i mlmd m the antioxidant defense qsem, and an ~C"ind nriCla, aad vend pmM werc romrdad. The<br />
ovaail babct bwkn prwxidano and mti0lid.n~ is ieR cduda epididymL wu weighed aad wed fa qrrm<br />
q u i d m rminhin ecllvl~ hmcmasis. Environmaral mvnu and Iprm motility. Ihe E.Ph md mud.<br />
wnumirunb have ken rcpard m dirnvb the pm-oxidant maions Of the right Wid- and rpididyrml rpcrm vm<br />
-<br />
and mtkridant bel- of sells 1161 and to -11 in en- vocd for bbckdd ny.. Thc @id@ rpmr<br />
ention of oxygen free radicals aad ROS Ilfl. A mk of ROS mllsEccd by cvuinp the wididymi, into anU w in<br />
m inf&ity due m Mmiw spcnn function has bca m-<br />
q c d 118-201. cxaruve gmaslhn of ROS has been<br />
shorn w caw pxidnive to the plasma mmbnrr.<br />
W h k.dr m irnplirrd rprm funom I2 11. Suproxide<br />
diMHUue dcptcdon ir Wght m be auocimd wilh<br />
spcrmImmotility [21l.Tofisul.rpodvEfionofhsendiulr<br />
and rtiv&an of the ~doxjdant dmnsc rym have ken<br />
rrponcd aftu expome to wxic chuniulr [I8301 and tkc<br />
epididymal pDdvction of hse ndifals md function of the<br />
anuouht dmnse synrm quire fvrtbcr sndia. Rcviova<br />
rodia 6um ow llbaroy have sham lindur idoxidative<br />
meu in the twis u well as in the cpidndymia aad<br />
cpididyd sprm of 8duit ram 123,241. In Ihc prrynt study.<br />
vs have sought to duemine the effsct of nulhoxychlor on<br />
the epididynul antioxidant system of adult ntr by nuw-<br />
~ng the antioxidant rnrymr ad lipid papridUion in epidrdyd<br />
rpcrm d in vshs regions ofthe cpldidymis. We<br />
aim erdvltcd the Mccl of mahoxychlor on cpididmf<br />
spmuumaadspammailityinordcrm.uarimnvlc<br />
qmdua(ve mrieity.<br />
Epididyual qam rmnt wu deOBmiacd by tbe mahod<br />
u dmaibcd it1 tk WHO MMlul 1261. Brkfly a S-6<br />
diquol ofapididyrml .pnn was dild wiLh 956 dil~t<br />
(50prodiumbkpboartr. IOmL35%farmll4mdO.S8<br />
ayplablucaar.Mod.adm&qmaw~umofl<br />
L with dirtlllsd e). A mw llip vu .sMd a the<br />
monungdumbarof~Ncuhtypc~.&<br />
pmatnutaly 10 I3. of the Uaovghly mirod dilulld ipscinmw~aredmsuhoftbe4np~oftk<br />
~ ~ , v h i c h w ~ . U o v m d m ~ f m 5 m t n i n s<br />
bunM hanba .w -1 wlu. Tile cab ~lmnlld<br />
ddn~~histimmd-rmnaddChaLilh(micmopc<br />
m m x.<br />
Roprrri*c.prm~Uy*.l.~lrhUdrbmd.rdmc(bodI27YIbyobpinhy~~tbe~<br />
epididymla xi* 8 pipoac Up and diktiw a 2 mL 4th<br />
H.m'sP12mrdi~.135.CAa.UQmcd~1alumn
C L m r ~ n . l l ~ T a d ~ 1 6 W J O m - O C O<br />
a. Superoxide dismutase
o. Glutathione reductage<br />
mg pmWn mgDNA mg Protein mgDNA mg protein mgDNA<br />
1 d.). 4 d.ys<br />
d. GlutaUlione peroxidase
c urbmwen&n= s a( I n.pradpradpradnu lad*<br />
I6 rm) mo-mo<br />
a. Hydrogen peroxide generation<br />
I d.Y<br />
b. Lipid peroxidation<br />
I d.)r,
IOWAME -. Tor. #558517 PME: 9 SESS: 5 OUTPUT: Tuc Jan 29 10:00:51 2002<br />
hieblW-mM1-#mOlm/msr26-02.<br />
-<br />
1- in th opididYIni1 Md a dcuura in the weight of thaxychlor into Its mujar melaboiitrr and CYPlA2 hrr<br />
Ihc 80% Orom8 my dn lea biwv&ility bccn shown lo play a predominant role bn rhlr reaction<br />
Or pmdvaiw Of Mdrapsn in wtbozychlor--mi rau. IIZI. The ability of the cytochrome P450 syslcm to in-<br />
Tho weigh1 of tho Uvu UKI Udvy dld not ihow any duce the pmdvction of ROS has ken wcll documented<br />
lloniacMI in tho MbmIr lmald~wid Moxy. 1131. In the present study, admnisuatian of methaxychla.<br />
OnY Q II. npottcd IhQ ldmiairrruion of mc- chlor dccrurrcd the activrtics of catslare, superoxids dis-<br />
IhOxyshlor Q dce4 &@ng from 200 c 400 mglkdday murase. plutath~onc reduclass, and giutslh~one pcroxifar<br />
11 nwr& dlarod tho W y wci@ htd veiphtr of the darc, and concom3untly increased the Levels of hydrogen<br />
live# Md Udmy 1361. Thc ebomr & of admininn- perondc and lipid . . proridstion in eddidymnl rwrm 85<br />
tion in out uwiy-nuy -I fcxiho I.ek of o b ~ ~ e d wcll as in the cpididymis. Suprox~dc dismutasc ia cunetion<br />
on (b..o a* wciphu.<br />
Thc acb.niim of momol;ychla-mediated oxidative<br />
rtdered the fimt line of defense against deleterious cffccrr<br />
of oxyradicsll in Ihe cell by coulynng the d~rmuiot$on<br />
~irfflc~butilhu~lbowncbomsdiaudby superoxide radicals to hydrogen peromde und molecuivr<br />
Ihc vdvuloll of nkxumnl moaoorygenue, which is<br />
involved in tho WII~!QXI of mDZboxysbior iaw iu re.<br />
vdn mmbliccr 1101. Dudly Uhir nrtioa. mtivc<br />
oxygen. The reduction in thc aclivity of catalase m;ly<br />
reflect an inability of cpididymal sperm md the epidid.<br />
ymir lo eliminate hydropcn pmxide prcduced by the<br />
M $ V & poulbly fno ndiul:, bind covllently to activation of methoxychlor and iu mclabolilcr or lo enmi-<br />
mmp~wmr 1101. ~stioxid.n~fioe r.dic~.i zyme mscuvauon caused by excess ROS production In<br />
luvenlar lad hirulfhydiyisonuini~ compounds Inhibit<br />
COV&lll Mlldiq of maborycNa in hwnrn llver microcpsdidymai<br />
sperm and the cpididymls 1371 The nnrioxldant<br />
enzymes caalase and peroiidaic prolocl ruperoude<br />
unna, ruwdng Uul UT M~VC inlediate is I. flK dirmutssc agatnst ~nactlvation by hydrogen proxidc. Rendtul<br />
1101. It hu been rhown that human cylahmme<br />
P450 my- po rolporulblc of rhc converrion of mcctpmcally.<br />
1hC rvperoxide d~rmulasc protects cntalasc<br />
and prnxidase against inhrbitian by superoxide anlon.
- aaarslu30 ,.*mra. L~jl;lr;<br />
-P4-w~~w'IPldnopd.oSd)o<br />
'wDU----w3p<br />
~ r c p o ~ ~ a ~ ~ ~ ~<br />
~ * p a ~ l a . q l = I ~ ~ ~ ! J l ~<br />
~ ~ ~ J ~ ~ ~ P ~ . C P * P F I ~ ~ ( O O J<br />
u08.p(rd PW% pcn wmd polorpKq u! muul<br />
I p p c n - ~ ~ ~ ) O . . ~ u ~ p ~ . ~ a ~ ~<br />
~ ~ ~ s n n o r n ~ I r m X p l p ! &<br />
Po. O(tP wwandnt almpylna<br />
u1 pOtllWI wm-d<br />
or en- aq Knu wu*. WIKZw rM J.3 nu.l.q '.nu
JOBNAME: Rqm. Tm. M5W7 PAOB: 11 SBS: 5 OUmJT Tuc an 29 IO:W:~I zm2<br />
MsbZla-m+m,iwasw-m<br />
cpididyrnis against lipid peraxidation mduced by me.<br />
thoxychlor by inCreasing antioxidant enzymes md de.<br />
crcains ROS geamtion. 1n addition, tosopbcml sup-<br />
PrcsEM lhe prcducdon of reactive oxidants and pmly<br />
impmvcl s pm motility (401. It has bsaa repartcd that<br />
vitamin E raises superoxide dismutasc activity in bovine<br />
sprm [411.<br />
Out results suggcst that exposure to melhoxychlor<br />
induces depletion in antioxidant defenrc system in the<br />
cpldidyrnie and opididymal sperm indisarlng methorychlor-induced<br />
oxidative smss in the epidrdymia. Thtr<br />
effect may lead to disruption in the functional integrity of<br />
cell orgmcller. We present study also suggests thal coadnunisvation<br />
of vitamin E with methorychlor prevents<br />
the advcne cffecfa of methorychlor on antioxidant status<br />
8s well ar improving sperm motility in adult rats. In<br />
conflvrion the adverse effect of mcthorychior on male<br />
reproduction of rats may be due to the induction of<br />
oxidative rmrr. These effects could be nilevistcd by the<br />
antioxidant viumin E.<br />
IM authors aclmowledgc Dr. Ufe Tiem. Germany for<br />
lhe gifl of mcthoxychlor md the sfaff of the Bioinformat>ci<br />
Ccnlr. Pondichcny <strong>University</strong> for various foeilltier. C. L.<br />
thank8 lo thc Indian Council of Medical Rwrch. Ncw<br />
Delhi for Senior Research Fcilowrtup and P.P.M, acknawledger<br />
the Populntlon Council. New Yark for finunclul it*.<br />
r,stunce (Gnni Nu~.DY~.~U~P-Y~CMC and II1)P,lUYI
(=I 23. w- C Olln (LC W W. Ubrth d-1.<br />
Mrrmiaul.sWdmd-dmke-m w.7.1.<br />
v A = b T o l * o i . b p r r .<br />
1261 W O ~ ~ ( a h r ~ d h m m s , - a d<br />
~--I999.UhBdlt(Pr~U~.<br />
-.-,---.<br />
1321 WLI + v- WN. sw*. oa guda* w gllimw<br />
- d ~ ~ p l o i ( d n I l . b Q i n<br />
Med 1967:701SB-69.<br />
P31 Fid; E K dPi Y. S-M boa ad MIL -a br<br />
. .<br />
Anr LU. 19z-ml<br />
1351 MSh. PP. Oatop.dgq. 5.190 d- .rr<br />
r-<br />
-<br />
rn n-ldrnd m- (n rr ICL<br />
8- ,.- 873-G ...-"<br />
1361 om. 5 w. I, coos. RL. r 4 lops m -k ad<br />
~--%w..*alm~llrt<br />
mdbchmarhhol*-phdt-y.)I.aLnldpDarttD<br />
in rmb DI TmM. M H..h4 IS, 37-47<br />
1371 mloar.r-.r.-~-d-ccn-<br />
M.-.D.-J.<br />
19W.01.18*1-<br />
d~M-mdElPUCIDni.dmbY-.nda-Wd
t<br />
02<br />
ELSEVIER<br />
Toricology OM) (2W1) DM-wo<br />
wwv.el~vicr.com/lo~~le~loii~)l<br />
Effect of methoxychlor on the antioxidant system in<br />
mitochondrial and microsome-rich fractions of rat testis<br />
C. Latchoumycandane, P.P. Mathur I*<br />
S'hwl v/ LII Scicnrrr. Pvldtchrrry Un~urriry. Pondrdcrry 605 014, Indra<br />
Rccctvcd 21 N~vcmbcr ZmI, rccetvcd m rcvn~d iorm 19 h m h r 2001. a~cc~~cd 28 March 2W1<br />
Abstract<br />
Mcthoaychior. an cnvironmenlal contdmlnanl, which is widely used as a pesticide in many countries and has k n<br />
shown lo ~nducc rcproductivc abnormalities ~n mule rats The precise nature and mechanism ot action of methoxy<br />
cllior on lllc lniblc rcproducllve ryslcm is no1 clwr In tho prcxnt study, we have sought to invesligille the induction<br />
of o*ldat~vc stress In thc tesl~l of rat after expoure to methorychlor Melhoiychlor (I, 10, and IW mg kg-' body<br />
wcnghl wr Jsy) was adrninisttrsd ormlly to the rat for 45days Aitcr 24 h of the last treatment the anlmals were killed<br />
wing :~ne\.thct~c clhcr. The body welght of thc anlmalr adm~n~slcrcd with methorychlor d~d not show any s~gn~flcam<br />
chungc Thc wcaghts of the testis, cpadtdymis, seminal vn!cles and ventral prostate dscreawd significantly in 100 mg<br />
dox but rcma~ncd unchsngtd In I and 10 ma do~s<br />
Miloehondrial and microsome-rich fract~ons of the test~s were<br />
oblatned by the method or d~llcrcntlal centrifugation. The activitles of anttoridant enzymes such as superoxide<br />
dlsmutasc. cslnlarc, plutath~one rcducmre and glutnthlone prox*dasc decreued s~gnificantly ,n the animals treated<br />
well8 tnc~horyci~lor In a dose-dependent manner In thc m~lochondilal and micromme-rich fraetloer of rat testis The<br />
lcvclr of hydrogcn peroiidc pncmllon (H,OI) and lip~d peroxtdatlon increased in m~~ochondriul and m~crorome-rich<br />
lracl~ons of the lcstts of the rats tmtd with melhoxychlor The rcsulu suppaled that the low to medlum dosn ol<br />
rncthuxyci,lor clcn deplet8on of antloxtdanl enzymes and concom~lant increase In the levels or HIO* and lipid<br />
prortdiltian dtllcrcnl\ally in mitochondrial and micro~omc-nch fractions of rat testis In conclusion, the adverse<br />
cflccl 01 mcthoxychlor on malc rsproducuon could be duc to lhc lnduclion of oxidal~ve stress in lestis. Q 2002<br />
Publl,hd by Elrvicr Sclsncc Ireland Ltd<br />
lcywvrdr Mc~horychior, Tuur, Anboridanl cnqmn. L~ptd pcrondalaon. Mllochondnsl fnclionr, Mncroromcrich Irlclionl: Rat<br />
The exposure of humans to cnvironmcntal contaminants<br />
that negatlvely affccl the male reproductive<br />
svstcm is increasing -(Sham, . 1993: Chia,<br />
'Cormpondlny author Tel. +91413615.211, 81. +91. 2000,, 11;~ important to notc that many cnviron.<br />
413.bJJ.lll<br />
K.n~~a1~~d~,rcu pp~~~;~~I~ut~~!l~olm~~I,com<br />
(P.P Muthur) mcnta' like organochlor'ne<br />
' B.mwl vpnnlhurgpl pon.n~.tn, cider, accumulate in fatty tissues. Therefore,
continuous exposure of humans to increasing<br />
numbcn and amounts of thns cnvtronmcntal<br />
contaminants niay have cumulative efTects that<br />
lcad to reproductive disorders (Lafuentc et al.,<br />
2000). Mctlioiychlor is onc of the cnv~ronmentnl<br />
contamlnonts wli~clt as w~dcly urd as a pesticide<br />
in many countries that was developed to replau<br />
dichloro-diphenyl-tnchloroethane (DDT), and its<br />
chemical name is 1.l.l-trichloro-2.2-his@-<br />
mcthoxy phenyl) cthanc. Mcthoxychlor displays<br />
both ntrogcnic and antrandrogcnic activities in<br />
vivo and In vitro (Maness el nl.. 1998). Methoxychlor<br />
1s wnstdcred to be a proatrogcn that 1s<br />
mctabolired into mono- and bs-hydroxy melabohtcs,<br />
wh~ch have been shown to have more estrogenic<br />
properties than the parental compounds<br />
(Bulger el al., 1978). Due to the estrogcnictty of<br />
mcthoxychlor metabolites. exposure of either<br />
neonatal or adult organlsm to high doses of<br />
mclhoxychlor may place the male reproductive<br />
system at risk. It has been reported that methoxychlor<br />
can affect malc rcprodvction of rats by<br />
Jccrcnsina spc-rm counts (Chnpin ct al.. 1997).<br />
Mctlioxychlor has also been shown to inducc<br />
ox~dat~ve strm In the ep~didymal sperm of goat<br />
(Gnligudhar~a ct al.. 2001). It has bcen rcportcd<br />
th;it mcthoxyclilor undcrgo hcpatic m~crosomal<br />
nmnooxygcnax mcdiatcd actwallon and the resultant<br />
rcactlve metabol~tes possibly free radicals<br />
bind wvalcnlly to microsomal components<br />
(Bulger et al.. 1983). Ant$ox~dants/froe radical<br />
scavengers, and sulfhydryl containing compounds<br />
inhibat covalent bindlng of methoxychlor in human<br />
liver microsomes, suggesting that the rcastive<br />
intermediate is a frec radlcal (Bulgcr and Kupfur,<br />
1989). It has also been reported that human cy.<br />
tmhrome P-450 enzymes responsible for wnversion<br />
of methoxychlor into its major metaboliter,<br />
the mono-o-dcmethylatcd dcrivatlvn and<br />
CYPIA2, havc bcen shown lo play predomrnant<br />
role tn thas rcaction (Strcrxr and Kupfcr. 1998).<br />
Thc abklity of cylmhrorne P-450 system to mduu<br />
rcacuvc oxygcn spx~es (ROS) has ban reported<br />
(Bondy and Naderi. 1994). ROS are formed in<br />
both phys~ological and patholog~cal wnditions in<br />
mammalran ttssun, duc to lhcir high reactivity<br />
thcy s,;ty tmcrilct with hiomol~zuln inducing oxidat~ve<br />
stress (Ochxndocrf, 1999). Mitochondr~al<br />
respiration is the main bioloaical source of suoeror~dc<br />
anion radicals und~r-~h~siolo~iul conditions.<br />
F m radiFalsiROS generated in tissues mnd<br />
subcellular wmpMmonts are cfficicntly rcavcngcd<br />
by the antioxidant defence system, which<br />
co~ts(itutcs antioxidant enzymes such as supcror.<br />
ide dismutaac, &lase. xlutathione rcduclnse and<br />
glutathlonc peroxidase. Under normal physiological<br />
conditions ires radiulaIROS are generated in<br />
subcellular compartments of testis which are subsequently<br />
mvcngd by the antioxidant defence<br />
system of the corresponding cellular compartments.<br />
The suixzllular membranes an more sus-<br />
ceptible lo l~p~d pcroxidat~ons, which are rich in<br />
unslaturated fatty acids and has bcen shown to<br />
contain low amount of antiox~dants. The subcellular<br />
membranes have been reported to undergo<br />
pcrmeab~lity changes following enhanced lip~d<br />
pcroxidation and glutathione depletion (Chance ct<br />
al.. 1979). The testicular production or free radicals<br />
and function of the antioxidant dcfcna systcm<br />
have ken reported upon exposure to toxlc<br />
chcm~calr (Peltola ct sl.. 1994; Sujotha ct al..<br />
2WI. Latchoumvcandanc el al.. 2W2a) Prevaous<br />
stud~es from ou; laboratory havc shown that the<br />
cnvironmcntal wntamrnants lake Ilndanc.<br />
mcthoxychlor and 2.3.7.8-tetrachlorodibenzo.pdioxin<br />
indua oxidauvc suns in the epid~dymrs<br />
and eptdidymal sperm of rats (Chttra el al.. 2WI;<br />
Latchournycdndane el al., 2002b,c). In order to<br />
explain the mechanism of action of methoxychlor<br />
at suixzlluhr levels In testis, the present studies<br />
wen undertaken to evaluate the eNm of<br />
methoxychlor an the antioxidant systcm in mitochondrial<br />
and microsome-rich frlclions of rat<br />
testlS.<br />
2. Material and arthods<br />
Methoxychlor (I.l.I-lrichloro-2.2.b~~[~<br />
mcthoxyphenyl] cthanc), Appror. 95% purc,<br />
Sigma Chemical Company, was obta~ncd as gift<br />
from Dr Ute Ticmann. Rescarch institute for the<br />
Biology of Farm An~mals. Dummcntorf, Germany.<br />
Th~obarbitunc acid and malondialdchyde
----<br />
-<br />
wcre obtu~ncd from E-Monk. Germany. All other<br />
chcmicnls were of analytical grade and obtained<br />
from local commercial sources.<br />
Alblno male rats of Wistar strain (45 davs old)<br />
werc used an thc present study and oblalncd from<br />
the Central Anhmal House. laaaharlal lnst~lutc of<br />
Portgradu~le Mcdteal Wucatlon and Re~earch.<br />
Pond~cherry. India. The animals wcre maintained<br />
undcr a wcll-regulated linht and dark schedule<br />
(12.12 hl and tcmperatureil f 3 'C and were fed<br />
wllh sltlndard comn>erclal pcllcted fced and water<br />
ad I~b>lum Mcthoaycblor was dissolved in acelonc<br />
and olwe oil ([:I91 and administered orally<br />
at the doses of 1. 10 and 100 mg kg-' body<br />
wcipl~t wr Jay Tor 45-days. Corrsaponding groups<br />
of annrnals wcrc admintstercd with vehtcle alone<br />
dnd scrved us conlrol.<br />
The animals wcrc fasted overnight, weighed and<br />
k~llcd by urxng ancslhclic cthcr on the day following<br />
the I;tst dosing Tcst~s, cptdidymts. scmtnal<br />
vcs~cles and ventral prostatc were removed and<br />
clearcd from thc adhertng tissues. The weights of<br />
thc ttssucs wcrc rccordcd in mg as well as mg<br />
kg - ' body wctght. Both thc testa of each animal<br />
wcrc u\cd Tor subccllvlar irilcl~onat~on and biochcmlcal<br />
studtcs.<br />
M~tochoodr~al and mtcrosome-rlch fractions of<br />
the tertls wcrc obtalncd by diffcrcntlal centrtfugatlon<br />
mcthod as dcscrabcd prcv~ously (Lutchoumyrn~nd;knc<br />
cl ul.. 2002a). Briefly, a 20% (w/v)<br />
tcst~cular homogcnate was prepared in ~cecald<br />
0.25 M sucrose solulnon with the help of a motordrtvcn<br />
xlifss tcflon homo~enircr, The homoaenale<br />
wa- . re8;tnfuscd -~ at 1000 ; e for 10 rnin at 4-oc to<br />
~ - - ~ -<br />
obtain the nuclear pellet. Mitochondrial pellet<br />
was obtatncd by ccntr~rugtng the post-nuclear supernatant<br />
at 1OW x I: for 10 min at 4 -C. The<br />
m~croromc-rlch fractions were prepared by calclum<br />
chloridc (CaClJ sedimentation method of<br />
Kamath and Narayan (1972). Briefly, the post-ms.<br />
tachandr~al supernatant was diluted wzth ice-cold<br />
CaCI, (I M) so that the final concentration of<br />
CaCI, was 0 8 molar. It was incubated at 4 'C for<br />
10 min with occasional stirring. The sample was<br />
then centr~iuged at IOWO x g for 10 min at 4 "C<br />
and the m~crosome-rich fractions were obtained.<br />
All Ihe lraclions werc washed three t!mes w~th<br />
~ce-cold 1.15% potassium chloride solution and<br />
d~ssolved in 0.25 M sucrose solution (1 mg protein<br />
per 0.1 ml). The mitochondr~al and mlcrosomerich<br />
iractions were used for biochemical studies.<br />
Superoxlde dlsmutase (EC 1.15 1 1) was assayed<br />
by the method of Marklund and Marklund<br />
(1974) Br~efly, the assay mlxture contained 2.4 mi<br />
of 50 mM tris-HCI buffer conlaming I mM<br />
EDTA (pH 7.6). 300 PI of 0.2 mM pyrogallol and<br />
300 PI cnzymc source. The incrcase I" absorbance<br />
was rnoasurcd immediatelv at 420 nm arainst<br />
blank contamin8 all the components except the<br />
enzyme and pyrogallol at 10 s intetvals for 3 mln<br />
on a Systronic~ Speztrophatometer. The enzyme<br />
activtty was cxpresscd in nmolc pyrogallol oridtzcd<br />
per min mg-' prolcin.<br />
Catalase (EC, 1.1 1.1.6) was assayed by the<br />
mcthod of Claiborne el al. (1985). Br~efly, the<br />
ansay rnlxture contained 240 ml of phosphate<br />
buffer (50 mM. pll 7.0). 10 $4 of 19 mM hydrogcn<br />
peroxide (H20,) and 50 gl enzyme source.<br />
The decrease in absorbance was measured Immediatcly<br />
at 240 nm against blank conlainlng all the<br />
components except the enzyme at 10 s intervals<br />
for 3 min on a Systronics Spcctrophotometcr. The<br />
enzyme actwily was expressed in lrmole oi H A<br />
consumed per mln mg-' protein.<br />
2.7. Glurorhione reducrose<br />
The activity of glutathione reductase (EC.<br />
1.6.4.2) was assayed by the method or Carlberg<br />
and Manncrv~k (1975). BneRy, the assay mixture<br />
contatned 1 75 rnl of phosphate buffer (100 mM.
pH 7.6). I00 pl of 200 mM NADPH, IW pI of 10<br />
mM EDTA. 50 p1 of 20 mM glutathione oxidized<br />
and 50 MI enzyme soum. Disappearance of<br />
NADPH was measured immcd~ately at 340 nm<br />
seainst blank containing all tho componcnts exccpt<br />
thc enzymc at 10 s mtcrvals for 3 min on a<br />
Systrotl!u S~trophotometer. The enzyme activ-<br />
~ty was cxprcsscd In nmole of NADPH oxidized<br />
pcr min mg-' protein.<br />
Ci1ut;tlhionc pcroxidasc (EC 1 .I 1.1.9) was nsr;tycJ<br />
by tine mclllod of l'ugl~;t and Valcntlac<br />
(1967). linefly. thc assay mirturc contamed 1.59<br />
ml of phosphatc bufTcr (100 mM. pH 7.6). 100 p1<br />
of 10 mM EDTA. 100 g1 of sod~um azide, 50 pl of<br />
glutathlonc rcductasc, 100 pl of glutathione reduced<br />
100 p1 of 200 mM NADPH. 10 pl of H,O,<br />
and 10 el cnzyme sourcc. Disappearance of<br />
NADPH was rncasurcd immcd~atcly at 340 nm<br />
ng.lmst blank containing all the components exccpt<br />
thc cnzymc at 10 s intcrvdls for 3 min on a<br />
Systron~cs Spatrophotomcter. The enzyme actlv-<br />
~ty was cxprcsscd in nmolc of NADPH oxidized<br />
pcr znln mp- ' prutcitl.<br />
2.9. Hydrogen pcroxrde generalton assoy<br />
11,0, pocr;trton was aswycd by thc mcthod or<br />
I'lch and Kcisr~ (1981). Drlcfly, the incubatson<br />
mtrriurc wntaincd 1.64 ml phosphate bukr (50<br />
mM. pH 7.6). 54 4 of horseradish peroxidasc (8.5<br />
Vlml). 30 pl of 0.28 nM phenol red. 5.5 nM of<br />
165 pl of dextrose and 100 pl of enzyme aource<br />
was done at 35 'C for 30 min. The reaction was<br />
tcrmlnatcd by the addition of 60 p1 of 10 N<br />
sodtum hydrondc. Thc absorbance was read at<br />
610 nM against a reagent blank on a Syrtromu<br />
Spcctrophotomctcr. The quantlty of H,O, produccd<br />
was exprcsxd as nmolc H,O, pcnentcd pr<br />
min per mg protcln at 35 'C. For standard curve.<br />
known amount or H,O, and all the above<br />
rcagcnb except cnzymc source wcrc incubated for<br />
30 mrn at 35 'C and then added 60 14 of 10 N<br />
saltum hydroxide and optical dsnsbty WM read at<br />
610 nM.<br />
2.10. Lipid pcrox~dadorion<br />
A break-down product of lipid peroxidation<br />
thiobarbituric actd reactive subslance (TEARS)<br />
was measured by the method of Bucp and Aust<br />
(1976). Ensfly. the stock solution wntained equal<br />
volumes of trichloroaoct~c actd 15% (wlv) in 0.25<br />
N hydrochloric acid and 2-thiobarbituric acid<br />
0.37% (wlv) in 0.25 N hydrmhloric add. h e<br />
volume of the test sample and two volumes of<br />
stock reagent were mixed in a screwsopped centrifuge<br />
tube. vortexcd and heated for IS min on a<br />
boiling water bath. After cooling on ia the precipilalc<br />
was rcmovcd by centrifugation at 1000 x<br />
g for I5 man and absorbanes of the supernalant<br />
was measured at 532 nM against blank conlaming<br />
all the reagents except test sample. A standard<br />
CUPS was Constructed extrapolating the amount<br />
of commercially bought product malondialdchyde<br />
to the rneasurcd absorbance. The value is cxpressed<br />
In pmole of malond~aldchydc formed pr<br />
mg protctn.<br />
Data wcrc crprcrrcd as mean *S.D, lor four<br />
anlmals per dose and analyrcd statistically using<br />
one-way analysis of varlana (ANOVA) followed<br />
by Tukey's 161. Probability valun Ins than 0.05<br />
wcrc dcternllned to be statistically rtgn!ficant<br />
agalnst control.<br />
3.1. Body weaghr and organ wctghfs<br />
Thc body weight of methoxychlor-lrcatsd rats<br />
d~d not change r~gnificantly when wmparod wlth<br />
the corresponding group of control animals<br />
(Tabk I), the weights of the tutia, cpid~dymir,<br />
=mind ves~lcr and ventral prmtate decreased<br />
significantly in the rau treated with 100 mg<br />
mcthoaychlor. However, the weighta remained<br />
unchanged in the animals treated with melhory.<br />
shlor at lower do= (Table I).
3.2. A,III~AILNI syrlo~t of ,nIrochondrml nnd<br />
,nicro~o~,te-rrch ftaertons qf Ihe terrir<br />
The iCvCl of H202 In mltochondrlal and micro.<br />
some-rlch fractions of tcalis increased slgnllicantly<br />
In the animals administered with methoxychlor as<br />
compared w~th the corresponding group of con.<br />
Thc actwty of suprox~de dismulase In mitochondrisl<br />
fractions of rat testis decrea~d s~gnlfi- fro1 animals (Table 2).<br />
wurtly III lhc ~~llinv;lls troatcd with mcthoxychlor at ThC level of lipid proxidation incrcascd slgnin.<br />
the doscs of 10 and 100 mg ss wmparcd with the<br />
in mitochondr~al fractlons of rcstts at 10<br />
correspond!ng group of control animals (Table 2). and 100 doses and all the doses in mlcraromc-<br />
Adm!ntstration of varsous doses or methoxychlor rich fractions in the animals administered w~th<br />
dccrolscd thc activtty of superoxide dirmutasc methoxychlor as compared with the correspondmlCro~on~~.ri~h<br />
fractg Broup of control animals (Tablc 2).<br />
sory sex organ weights and suppression<br />
of tcsllcu.<br />
Admtnlslralion of various doses of methoxychlor<br />
decreased the actrvlty of glutath~one reductase 25 mg kg-) body weight per day (Gray el al..<br />
s~gnlficantly ~n mlcrosomc-nch fractions as corn. 1989) Rcduc~on in fert~l~ty has also been repdrcd<br />
with the corresponding group of control ported in the methoxychlor treated rats (Gray el<br />
an~mals (Tablc 21. Adm~nistration of mcthoxy a].. 1989). It has been reported that methoxychlor<br />
chlor dccrcascd the activlty ol glutathione peroa- decreased the epidtdymal spcm counts and motil.<br />
~dusc II, rnllochondrial and m~crosome-rich ~ty In adult rats (Latchoumycandane ct al..<br />
fractbons of testis as compared w~th the corre- 2002b). Female rats gavaged with methoxychlor<br />
spo~ldtng group of control animals (Table 2), at the doses of 5, 50 and 150 mg kg-' body<br />
~ K ~ ~ ~ ~ ~ ~ $ ~ ~ ~ ~ ~ ~ ~ ~ ~ , " C ~ , , " , " ' ~ ~<br />
T"h1f I<br />
Enm, or mcthorysh~ar on the bodl vclphc md ~.,~hu or ,he ~uia. cpldtdymlr and .-row<br />
wx organa or *dull mt<br />
--<br />
Melhaxychlor pi Kg body wcighl per day<br />
cornre1 1 nig 10 mg IW ms<br />
Udy wc13n UI tl6f 366 118*513 I18* l 19 114zk116<br />
TsrllHmgl 972 t 15.W 968f 1145 977 i 5 91 9I53II89.<br />
Eptdrdymlr lmg) 412311 I7 401 3 22 60 4D4k1281 367 i 499'<br />
Scmrnnl rcrtcln (ml) 947i 1707 941 i I5 I5 94631608 873 4 12'<br />
Vcntrsl pr0rt.r (mp)<br />
110 i I8 1s 19934 11 1W*515 176 f 432'
ioavaiiability andlor production of testosterone<br />
and also thc cstrogfnic and antiandrogenic activitics<br />
of thc mcthoxychlor and its metabolits.<br />
Thc rncchiinisn~ of mclhoxychlor mcdialod 0x1-<br />
ililllvc strcss is not "cry clcar but ~t has bocn<br />
shown lo bc mcdiatcd by thc activation of microsome-nch<br />
monooxygenax, which a involved ~n<br />
the convcrsnon of methoxychior Into its reacttve<br />
mctabolitcs (Bulger el ai.. 1983). During this reaclloll<br />
tllc ~cibctlvc 1I1~l.lb0lilCs. possibly free ~di.<br />
cals. b~nd wvaicntly to microsome-rich<br />
components (Bulger et al . 1983). 11 has been<br />
shown that human cytochrome P450 enzymes.<br />
rcsponslblc for tlic conversion of mothoxychlor<br />
lnto 11s major mclaboitler and CYPIAZ, havc<br />
been shown to play e predommilnt role in'thir<br />
reaclloii (Slrcsscr and Kupfer, 1998). The ablilty<br />
of cytoclirome P-450 system lo lnduce ROS has<br />
been wcll documented (Bondy and Naderi. 1994)<br />
It has bcco reported that ROS such as H,O,<br />
appcdrs lo be a key agent In the cytotoxtc erects<br />
In alxrn?.\tozo;t iwd 111 itddltlo!, to thew dlrcot<br />
cffcct on ccllulnr constituent produce oxcdatlve<br />
stress by dccreas~ng the enzymatic defenses of the<br />
tcais (Grlv~au and Lannou. 1997). In the present<br />
nudy. ad~nlnlstration of mcthoxychlor dccrcased<br />
thc i~cliv~t~cs of ~ttulurc, superox~dc d~smutnrc.<br />
piutathionc reductax, and glulathtonc peroxidase<br />
and concomitant incrcaw In the levels of H?0,<br />
and hpld pcroxldal~on dificrentiaily in the mltochondrral<br />
and msrosomc-rich fractions of rat<br />
tcrtls Thc marlmum decllnc In the actlvltlcs of<br />
ontioxId.tnl cnrynlcs was obscrvcd In mlcrosomc<br />
rlch fracuans followcd by mltachondrtal fractions<br />
aftcr mcthoxychlor trcntment. Superoxlde d~smutarc<br />
1s conskdercd the first llne of defence against<br />
delcter~aus cKec(s of oxyradicais In cell by catalyz~ng<br />
the d~rmutat!on of superoxidc rad~cals to<br />
H,02 and molecular oxygen. The rcduct~on in the<br />
ncttvaty of catalar may rcflect ~nabilzty of tesllculilr<br />
m~tochondrta and mscrosomes to cl~mtnatc<br />
11,0, produccd by thc actwellon of mcthoxychlor<br />
and 11s n~elabol~ler. Th~s may also be due to<br />
cnzyme ~naclbvation cawed by taws ROS production<br />
in mttochondr~s and mtcrosomes (Pigco-<br />
Icl et "1.. Il99U). Thc nnt~oridant cnrymcs cnlnlusc<br />
and pcroxtdarc protect superoxide dismutasc<br />
against mactlvatlon by H,O,. Rcc~procally, the<br />
superoxide d~smutase protects the catalase and<br />
peroxidase against inhibition by superoxtdc anion.<br />
Thus. the balance of this enzyme system may be<br />
crscntlal to gct rid oTsuperoxidc anion and pcror.<br />
ides generated in subaeliular companmmts of the<br />
testis. The reduction in the activities of anttoxldant<br />
enzymes and increase in H,O, and lipld<br />
peroxidation wuld reflcct the adverse effect of<br />
mcthoxychior on the antioxidant system in testis<br />
suhccllular fruchons. The ROS cause damage to<br />
m~lochondr~al and other cytoplasmic organelle<br />
membrane structures through peroxldatlon of<br />
phospholipids, proteins and nucleotides. Undcr<br />
hlgh ROS concentrations prc-damaged spermalolo*<br />
arc exposed to hpid peroxldatlon by polyunsaturated<br />
fatty acids (ichikawa el a].. 1999) The<br />
milochondrial and m!crosome-rich membranes<br />
havc been reported to undergo permeabllily<br />
changes following enhanced lip~d peroxidation<br />
and giutath~onc dcplction (Chance et al., 1979).<br />
The results suggested that the low to medium<br />
dnscs exposure or pubcrtal rat lo mcthoxychlor<br />
~nducen dcpletlon in antioxidant defcncc systems<br />
diflerentially in milochandrial and microsome.<br />
rich rracllons ind~cating melhaxychlor-lnduced<br />
ox~daltve stress tn tcrtis rubcellular fractions. Thls<br />
crrcct may lead to dtsruplion In functtonal integrity<br />
of cell organelles in testis and thus the<br />
advcrsc effcct of methoxychlor on male rcproduction<br />
of rats may be due to the induction of<br />
oridanve stress in testls.<br />
The authors acknowledge Dr Ute Ticmann,<br />
Research lnslrtutc for the Biology of Farm Anl.<br />
rnuir. Dummerstorf, Germany for the generous<br />
gait of mclhoxychlor and the staff of the Biolnfor<br />
matlcs Center. Pondcherry <strong>University</strong> for various<br />
facillt~es C. Latchoumycandane acknowledgcs the<br />
Indian Council of Medical Research, New Delhi<br />
for Scnior Research Fellowship and P.P. Mathur<br />
acknowlcdgcs the Population Council. Ncw York<br />
for financial assistance (Grant Nos.B99.047P-91<br />
lCMC and B99.048RilCMC).
Uc,#!dy. SC. N;tdcru. S , 1% Conlrbbulton or hcpatic cy.<br />
lmhrome P4Se ryncml lo the genenuan or rractire oiy.<br />
yrn rmia U~~hsm Phsrmncol 48, 155-159<br />
nucpe. J.A . Aun. S D.. 1976 baopmildul utalyd hpd<br />
vrox8drunn or mcro.omc-rrh and arllrrul mmbrsnu<br />
Btoch~m Unphys As" 444. 192-201<br />
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~ ~<br />
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