Use of nasal and buccal cells in human biomonitoring ... - Formacare
Use of nasal and buccal cells in human biomonitoring ... - Formacare
Use of nasal and buccal cells in human biomonitoring ... - Formacare
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<strong>Use</strong> <strong>of</strong> <strong>nasal</strong> <strong>and</strong> <strong>buccal</strong> <strong>cells</strong> <strong>in</strong> <strong>human</strong><br />
biomonitor<strong>in</strong>g studies for the detection<br />
<strong>of</strong> cytotoxic <strong>and</strong> DNA-damag<strong>in</strong>g<br />
siegfried.knasmueller@meduniwien.ac.at<br />
chemicals<br />
Siegfried Knasmüller,<br />
A. Nersesyan, M. Misik<br />
Cancer Research Institute, Innere Mediz<strong>in</strong> I<br />
Medic<strong>in</strong>e University <strong>of</strong> Vienna<br />
1
siegfried.knasmueller@meduniwien.ac.at<br />
Topics<br />
• background <strong>and</strong> historical notes<br />
• methodological aspects<br />
– MN assays with exfoliated <strong>buccal</strong> <strong>cells</strong><br />
– MN assays with <strong>nasal</strong> <strong>cells</strong><br />
– Advantages /disadvantages <strong>in</strong> comparison to<br />
MN assays with lymphocytes<br />
• <strong>Use</strong> <strong>of</strong> MN assays with exfoliated <strong>cells</strong> <strong>in</strong> <strong>human</strong><br />
biomonitor<strong>in</strong>g; the current data base<br />
• Studies on FA exposure<br />
• Conclusions<br />
2
siegfried.knasmueller@meduniwien.ac.at<br />
Background<br />
A variety <strong>of</strong> methods has been developed to detect exposure <strong>of</strong> <strong>human</strong>s<br />
to genotoxic carc<strong>in</strong>ogens<br />
<strong>in</strong> <strong>human</strong> <strong>cells</strong><br />
Adducts<br />
Chromosomal<br />
aberrrations<br />
Micronuclei<br />
Comet - Assay<br />
OH<br />
8 - Hydroxydeoxyguanos<strong>in</strong>e<br />
3
Not all endpo<strong>in</strong>ts are related to cancer risks !!!!<br />
• MN <strong>in</strong> lymphocytes <strong>and</strong> <strong>buccal</strong> <strong>cells</strong> reflect<br />
<strong>human</strong> cancer risks.<br />
• Chromosomal aberrations <strong>in</strong> lymphocytes<br />
correlate with <strong>human</strong> cancer risks.<br />
• Sister chromatid exchanges <strong>in</strong> lymphocytes: no<br />
correlation.<br />
• Comets: not known !!!!!<br />
• DNA Adducts: for certa<strong>in</strong> adducts clear<br />
associations were established.<br />
• OHdG <strong>in</strong> ur<strong>in</strong>e <strong>and</strong> lymphocytes: not known.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
4
Not all endpo<strong>in</strong>ts are related to cancer risks !!!!<br />
siegfried.knasmueller@meduniwien.ac.at<br />
5
siegfried.knasmueller@meduniwien.ac.at<br />
Micronucleus<br />
conta<strong>in</strong><strong>in</strong>g an entire<br />
chromosome<br />
Micronuclei are<br />
formed as a<br />
consequence<br />
<strong>of</strong> chromosome<br />
breaks <strong>and</strong><br />
aneuploidy<br />
6
siegfried.knasmueller@meduniwien.ac.at<br />
Historical notes<br />
• Micronuclei were discovered by Schmid <strong>in</strong> 1972.<br />
• The first experiment with exfoliated <strong>cells</strong> was conducted by<br />
Stich <strong>and</strong> Parida <strong>in</strong> 1982.<br />
• The first FA study with <strong>nasal</strong> <strong>cells</strong> was published <strong>in</strong> 1992 by<br />
Ballar<strong>in</strong> et al.<br />
• A review on the effects <strong>of</strong> FA <strong>in</strong> exfoliated <strong>cells</strong> appeared <strong>in</strong><br />
2006 (Speit et al.)<br />
• In 2004 the HUMN xl consortium was formed (M. Fenech, E.<br />
Zeiger, N. Holl<strong>and</strong>, C. Bolognesi, S Burgaz, S. Knasmüller)<br />
<strong>and</strong> started to evaluate <strong>and</strong> st<strong>and</strong>ardize MN experiments<br />
with exfoliated <strong>buccal</strong> <strong>cells</strong>.<br />
7
Methodological aspects- experiments with<br />
<strong>buccal</strong> <strong>cells</strong><br />
Holl<strong>and</strong> et al., Mutat Res 2008<br />
• Cells migrate from the basal membrane to the surface<br />
<strong>and</strong> are replaced by new <strong>cells</strong>; dur<strong>in</strong>g this process the<br />
<strong>cells</strong> divide <strong>and</strong> form MN.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
8
Different types <strong>of</strong> nucelar aberrations can be<br />
identified <strong>in</strong> the <strong>cells</strong><br />
siegfried.knasmueller@meduniwien.ac.at<br />
9
Some endpo<strong>in</strong>ts reflect cytotoxicity others are<br />
considered to be a consequence <strong>of</strong> genetic damage<br />
• Genotoxic effects:<br />
– Micronuclei: aneugenic <strong>and</strong> clastogenic effect<br />
– B<strong>in</strong>ucleates: sp<strong>in</strong>dle disturbance<br />
– Nuclear buds: gene amplification<br />
• Cytotoxicity:<br />
– Pycnosis<br />
– Karyolysis<br />
– Condensed chromat<strong>in</strong><br />
– Karyorexis<br />
siegfried.knasmueller@meduniwien.ac.at<br />
10
Nat Protoc. 2009;4(6):825-37. 2009 Buccal micronucleus cytome assay. Thomas P, Holl<strong>and</strong> N, Bolognesi<br />
C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, Fenech M<br />
siegfried.knasmueller@meduniwien.ac.at<br />
11
Development <strong>of</strong> a st<strong>and</strong>ardized protocol……<br />
siegfried.knasmueller@meduniwien.ac.at<br />
12
• Sta<strong>in</strong><strong>in</strong>g<br />
siegfried.knasmueller@meduniwien.ac.at<br />
Important aspects<br />
• Preparation <strong>of</strong> optimal slides with a cytosp<strong>in</strong><br />
centrifuge<br />
• Number <strong>of</strong> <strong>cells</strong> that has to be scored<br />
• Number <strong>of</strong> <strong>in</strong>dividuals<br />
13
siegfried.knasmueller@meduniwien.ac.at<br />
Sta<strong>in</strong><strong>in</strong>g<br />
a b<br />
c<br />
Figures a <strong>and</strong> c depict May-Grünwald-Giemsa sta<strong>in</strong>ed <strong>cells</strong>.<br />
Figures b <strong>and</strong> d show the same <strong>cells</strong> after sta<strong>in</strong><strong>in</strong>g with Feulgen <strong>and</strong><br />
DAPI.<br />
d<br />
A large number <strong>of</strong><br />
earlier studies used<br />
DNA unspecific sta<strong>in</strong>s<br />
(e.g. Giemsa) which<br />
lead to false positive<br />
results (as they sta<strong>in</strong><br />
kerat<strong>in</strong> bodies that are<br />
formed <strong>in</strong> epithelial<br />
<strong>cells</strong> as a consequence<br />
<strong>of</strong> cytotoxicity.<br />
Nersesyan et al. Cancer Epidemiol Biomarkers Prev<br />
2006;15(10). October 2006<br />
14
siegfried.knasmueller@meduniwien.ac.at<br />
Slide preparation<br />
• Optimal slide preparation<br />
is achieved by use <strong>of</strong> a<br />
cytosp<strong>in</strong>.<br />
• Direct smears will lead to<br />
„low quality slides“ that are<br />
hard to evaluate.<br />
15
Number <strong>of</strong> <strong>cells</strong> which should be scored<br />
• Ceppi et al. (2010) suggest to score per <strong>in</strong>dividual 4000<br />
<strong>cells</strong>, this number is rarely reached <strong>in</strong> earlier studies.<br />
• However Thomas et al. 2009 suggest scor<strong>in</strong>g <strong>of</strong> 2000<br />
<strong>cells</strong>.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
16
Number <strong>of</strong> participants<br />
requested<br />
siegfried.knasmueller@meduniwien.ac.at<br />
• The number <strong>of</strong><br />
participants which<br />
should be evaluated<br />
depends on the<br />
magnitude <strong>of</strong> the<br />
effect.<br />
• Ceppi et al. (2010)<br />
state that 120<br />
<strong>in</strong>dividuals (60<br />
controls <strong>and</strong> 60<br />
exposed) have to be<br />
analyzed <strong>in</strong> order to<br />
detect a 50% <strong>in</strong>crease<br />
<strong>of</strong> MN over the<br />
background.<br />
Mutat Res. 2010 Jul-Sep;705(1):11-9. Ceppi M, Biasotti B, Fenech M, Bonassi S.<br />
17
Average background frequencies <strong>of</strong> different anomalies<br />
Nuclear anomaly Number <strong>of</strong> subjects Mean±SE Ref.<br />
MNed <strong>cells</strong> 703 0.83±0.04<br />
Nuclear buds (broken<br />
egg)<br />
siegfried.knasmueller@meduniwien.ac.at<br />
789 1.36±0.08<br />
B<strong>in</strong>ucleated <strong>cells</strong> 852 3.04±0.20<br />
Karyorrhectic <strong>cells</strong> 408 2.23±0.25<br />
Pycnotic <strong>cells</strong> 210 4.38±0.52<br />
MNed <strong>cells</strong> Metanalysis <strong>of</strong> 63<br />
studies<br />
Bonassi et al., 2011<br />
1.10±0.55 Ceppi et al., 2010<br />
18
Methodological aspects: experiments with<br />
<strong>nasal</strong> <strong>cells</strong><br />
• The morphological features <strong>of</strong> the anomalies are similar as<br />
those found <strong>in</strong> <strong>buccal</strong> <strong>cells</strong>; however, the nuclei <strong>of</strong> the <strong>cells</strong><br />
are larger, many ciliated <strong>cells</strong> are found <strong>in</strong> the smears <strong>and</strong><br />
also goblet <strong>cells</strong> as well as lymphocytes <strong>and</strong> granulocytes.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
19
Numbers <strong>in</strong> the picture A <strong>in</strong>dicate:<br />
1 - ciliated cell with 3 MNi<br />
2 - nuclear bud<br />
3 - normal ciliated cell<br />
4 - karyorrhexis<br />
5 - karyolysis<br />
6 - condensed chromation<br />
Picture B - 2 normal ciliated <strong>cells</strong><br />
A<br />
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B<br />
20
siegfried.knasmueller@meduniwien.ac.at<br />
Methodological aspects<br />
• The collection <strong>of</strong> <strong>cells</strong> with a cytobrush is more<br />
tricky as the collection <strong>of</strong> <strong>cells</strong> from the oral<br />
cavity.<br />
• The site <strong>of</strong> collection was shown to have an<br />
impact on the results. Apart from nuclear<br />
anomalies also metaplastic <strong>and</strong> dysplastic <strong>cells</strong><br />
can be scored.<br />
21
Figure 1. Nasal cytopathologic<br />
f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong> nonsmok<strong>in</strong>g customs<br />
<strong>of</strong>ficers exposed to DEE<br />
throughout the<br />
year (Papanicolaou sta<strong>in</strong>). (A)<br />
Squamous cell metaplasia. (B)<br />
Cyl<strong>in</strong>der cell dysplasia. (C) Squamous<br />
cell<br />
dysplasia with mitotic figure (arrow).<br />
The scale bar <strong>in</strong> (B) also applies to (A)<br />
<strong>and</strong> (C).<br />
Glück et al. 2003 Environ<br />
Health Perspectives<br />
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22
siegfried.knasmueller@meduniwien.ac.at<br />
The current data base<br />
23
MN studies with exfoliated <strong>buccal</strong> <strong>cells</strong><br />
• In total ca. 300 studies have been published so far.<br />
• They concern the effects <strong>of</strong> lifestyle factors (betel<br />
chew<strong>in</strong>g, smok<strong>in</strong>g), health status, dietary factors<br />
<strong>and</strong> occupational exposure.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
24
siegfried.knasmueller@meduniwien.ac.at<br />
25
MN studies with <strong>nasal</strong> <strong>cells</strong><br />
• In total only 19 studies were published, <strong>in</strong> most <strong>of</strong> them only<br />
MN were scored (three studies <strong>in</strong>cluded also other nuclear<br />
anomalies).<br />
• The <strong>in</strong>vestigations concerned the effects <strong>of</strong> occupational<br />
exposure (n=17/ 8 FA exposure), 2 <strong>in</strong>vestigations concerned<br />
air pollution <strong>in</strong> cities.<br />
siegfried.knasmueller@meduniwien.ac.at<br />
26
Chicken manure<br />
B: ─ / N ─<br />
Studies conducted <strong>in</strong> Vienna (ICR)<br />
Welders<br />
B: + (2 studies)<br />
siegfried.knasmueller@meduniwien.ac.at<br />
Khat chewers Coca chewers<br />
B: ─ / N + B: +<br />
B: ─<br />
Smok<strong>in</strong>g<br />
r<strong>in</strong>g trial (<strong>in</strong> progress)<br />
27
Fold <strong>in</strong>crease over background<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Which endpo<strong>in</strong>ts are the most sensitive<br />
ones???<br />
Nersesyan et al. 2006 -<br />
Smokers <strong>of</strong> unfiltered cigarets<br />
MN BE KR<br />
siegfried.knasmueller@meduniwien.ac.at<br />
Fold <strong>in</strong>crease over background<br />
8<br />
6<br />
4<br />
2<br />
0<br />
Celik et al. 2004 -<br />
pa<strong>in</strong>ters (n=60 <strong>in</strong> each group)<br />
MN BE<br />
Rekhadevi et al. 2010 -<br />
fuel fill<strong>in</strong>g station attendants (n=100 <strong>in</strong> each group)<br />
Fold <strong>in</strong>crease over background<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
MN BE KR<br />
28
Which cell types are more sensitive, <strong>nasal</strong> or <strong>buccal</strong> <strong>cells</strong>?<br />
MN/1000 <strong>cells</strong><br />
MN/1000 <strong>cells</strong><br />
Gonsebatt et al. 2000 -<br />
Air pollution <strong>in</strong> Mexico City (n=20/group)<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0.0<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Nasal Buccal Nasal Buccal<br />
Polluted area Control area<br />
Titenko-Holl<strong>and</strong> et al. 1996 -<br />
formadehyde exposure (n=19/group)<br />
Nasal Buccal Nasal Buccal<br />
Exposed Controls<br />
siegfried.knasmueller@meduniwien.ac.at<br />
MN/1000 <strong>cells</strong><br />
MN/1000 <strong>cells</strong><br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
3<br />
2<br />
1<br />
0<br />
Burgaz et al. 1998 -<br />
Welders I (n=32/group)<br />
Nasal Buccal Nasal Buccal<br />
Exposed Controls<br />
Wultsch et al. unpublished -<br />
Welders II (n=25/group)<br />
Nasal Buccal Nasal Buccal<br />
Exposed Controls<br />
29
Why experiments with exfoliated <strong>cells</strong>?<br />
(comparison <strong>of</strong> advantages <strong>and</strong> disadvantages <strong>in</strong><br />
relation <strong>of</strong> experiment with lymphocytes)<br />
Lymphocytes Buccal/Nasal <strong>cells</strong><br />
Large database (ca 2100 studies) only around 300 studies<br />
Automated evaluation available automated evaluation <strong>in</strong> preparation<br />
sampl<strong>in</strong>g <strong>in</strong>vasive sampl<strong>in</strong>g not <strong>in</strong>vasive<br />
long turnover time <strong>of</strong> lymphocytes short turnover time (2-3-weeks)<br />
Efficient DNA repair low repair capacity <strong>of</strong> the <strong>cells</strong><br />
(higher sensitivity)<br />
Indirect relation to cancer ≥90% <strong>of</strong> cancers are <strong>of</strong> epithelial orig<strong>in</strong><br />
siegfried.knasmueller@meduniwien.ac.at<br />
30
FA studies with exfoliated <strong>cells</strong><br />
• S<strong>in</strong>ce 2006 four further studies were<br />
published<br />
siegfried.knasmueller@meduniwien.ac.at<br />
31
Micronucleus frequencies <strong>in</strong> exfoliated <strong>buccal</strong> <strong>cells</strong> <strong>of</strong> <strong>human</strong> subjects after<br />
formaldehyde exposure<br />
Study size (subjects/<strong>cells</strong> scored) Exposur<br />
e (ppm)<br />
29 Pre- <strong>and</strong> post exposure<br />
1500 <strong>cells</strong>/subject<br />
28 Pre- <strong>and</strong> post exposure<br />
500–4000 <strong>cells</strong>/per subject<br />
28 Exposed/18 controls<br />
3000 <strong>cells</strong>/subject<br />
80 Exposed/85<br />
Controls<br />
2000 <strong>cells</strong>/subject<br />
16 Exposed/23 controls<br />
????? <strong>cells</strong>/subject<br />
56 Exposed/85 controls<br />
2000 <strong>cells</strong>/subject<br />
21 Pre- <strong>and</strong> post exposure 1000 -<br />
2000 <strong>cells</strong>/subject<br />
1.4<br />
(peak:<br />
6.6)<br />
1.4<br />
(peak:<br />
6.6)<br />
siegfried.knasmueller@meduniwien.ac.at<br />
Sta<strong>in</strong><strong>in</strong>g Result: <strong>buccal</strong> <strong>cells</strong><br />
(exposed<br />
(‰)/controls (‰))<br />
Feulgen/Fast green 0.6/0.046<br />
↑ 1.6<br />
FISH/propidium<br />
iodide<br />
2.0/0.6<br />
↑ 3.3<br />
2–4 Feulgen /fast green 7.1/3.3<br />
↑ 2.2<br />
0.25 Feulgen/no<br />
counter sta<strong>in</strong><br />
1.27/0.13<br />
↑ 9.8<br />
0.64/0.13<br />
↑ 4.9<br />
0.1 Wright's sta<strong>in</strong> 0.86/0.57<br />
↔<br />
0.16 Feulgen/no<br />
counter sta<strong>in</strong><br />
0.96/0.16<br />
↑ 6.0<br />
13.5 DAPI 1.33/0.90<br />
↔<br />
Ref. Notes<br />
Suruda et al.,<br />
1993<br />
Titenko-<br />
Holl<strong>and</strong> et al.,<br />
1996<br />
Burgaz et al.,<br />
2002<br />
Viegas et al.,<br />
2010<br />
Y<strong>in</strong>g et al.,<br />
1997<br />
Ladeira et al.,<br />
2011<br />
Speit et al.,<br />
2007<br />
0 MN <strong>in</strong> 22 males,<br />
but 0.19‰ <strong>in</strong><br />
females<br />
First study <strong>of</strong> <strong>buccal</strong><br />
<strong>cells</strong> with FISH<br />
technique<br />
-<br />
-<br />
Not reliable results<br />
because <strong>of</strong> sta<strong>in</strong><br />
♀↑ 10.4/♂↑ 2.8<br />
Exposure under<br />
controlled<br />
conditions<br />
32
Micronucleus frequencies <strong>in</strong> exfoliated <strong>nasal</strong> <strong>cells</strong> <strong>of</strong> <strong>human</strong> subjects after<br />
formaldehyde exposure<br />
Study size (subjects/<strong>cells</strong><br />
scored)<br />
15 Exposed/15 controls<br />
6000 <strong>cells</strong>/subject<br />
29 Pre- <strong>and</strong> post exposure<br />
1500 <strong>cells</strong>/subject<br />
28 Pre- <strong>and</strong> post exposure<br />
500–4000 <strong>cells</strong>/per subject<br />
23 Exposed/25 controls<br />
3000 <strong>cells</strong>/subject<br />
18 Exposed/23 controls<br />
3000 <strong>cells</strong>/subject<br />
16 Exposed/23 controls<br />
????? <strong>cells</strong>/subject<br />
41 Exposed/the same subject were<br />
controls 1000-2000 cell subject<br />
Exposure<br />
(ppm)<br />
siegfried.knasmueller@meduniwien.ac.at<br />
Sta<strong>in</strong><strong>in</strong>g<br />
0.1–0.39 Feulgen/Fast green<br />
1.4 (peak:<br />
6.6)<br />
1.4 (peak:<br />
6.6)<br />
Feulgen/Fast green<br />
FISH/propidium<br />
iodide<br />
2–4 Feulgen/Fast green<br />
1 Wright's sta<strong>in</strong><br />
0.1 Wright's sta<strong>in</strong><br />
0 to 0.7<br />
Result: <strong>nasal</strong> <strong>cells</strong><br />
(exposed (‰)/controls<br />
(‰))<br />
0.90/0.25<br />
↑ 3.6<br />
0.5/0.41<br />
↔<br />
2.5/2.0<br />
↔<br />
1.0/0.61<br />
↑ 1.6<br />
2.70/1.25<br />
↑ 2.2<br />
3.9/1.2<br />
↑ 3.3<br />
DAPI 0.21/0.17<br />
↔<br />
Ref. Notes<br />
Ballar<strong>in</strong> et al.,<br />
1992<br />
Suruda et al.,<br />
1993<br />
Titenko-Holl<strong>and</strong><br />
et al., 1996<br />
Burgaz et al.,<br />
2001<br />
Ye et al., 2005<br />
Y<strong>in</strong>g et al., 1997<br />
Zeller et al.,<br />
2011<br />
-<br />
13% <strong>in</strong>crease <strong>in</strong><br />
males, 37% <strong>in</strong><br />
females<br />
First study <strong>of</strong> <strong>nasal</strong><br />
<strong>cells</strong> with FISH<br />
technique<br />
-<br />
Not reliable results<br />
because <strong>of</strong> sta<strong>in</strong><br />
Not reliable results<br />
because <strong>of</strong> sta<strong>in</strong><br />
Exposure under<br />
controlled<br />
conditions 33
siegfried.knasmueller@meduniwien.ac.at<br />
Comments I<br />
• Two studies used Wright's sta<strong>in</strong> (a modified Giemsa<br />
sta<strong>in</strong><strong>in</strong>g procedure), their results are irrelevant as<br />
unspecific sta<strong>in</strong>s give mislead<strong>in</strong>g results.<br />
• In one study (Suruda et al. 1993) no MN were found <strong>in</strong><br />
samples from males (controls), this result is out <strong>of</strong> the<br />
acceptable range calculated by Bonassi et al. (2011) <strong>and</strong><br />
is therefore, irrelevant.<br />
34
siegfried.knasmueller@meduniwien.ac.at<br />
Comments II<br />
• The results <strong>of</strong> the rema<strong>in</strong><strong>in</strong>g studies are controversial; none <strong>of</strong><br />
them fulfills the criteria for optimal <strong>in</strong>vestigations<br />
• Ladeira et al (2011) <strong>and</strong> also Viegas et al (2010) conducted<br />
relatively large studies; they evaluated 2000 <strong>cells</strong> <strong>and</strong> used<br />
Feulgen without counter sta<strong>in</strong>.<br />
• All other studies were smaller as suggested <strong>and</strong> <strong>in</strong> some <strong>of</strong><br />
them the number <strong>of</strong> evaluated <strong>cells</strong> was below 2000 (Holl<strong>and</strong><br />
et al, 1996, Zeller et al, 2011).<br />
• In none <strong>of</strong> the FA studies published so far nuclear anomalies<br />
other than MN were evaluated.<br />
35
siegfried.knasmueller@meduniwien.ac.at<br />
Overall conclusions<br />
The methods for MN studies with exfoliated <strong>cells</strong> have been<br />
substantially improved <strong>in</strong> the last years by the jo<strong>in</strong>t efforts <strong>of</strong> the<br />
HUMN XL consortium. It was shown that the MN frequencies <strong>of</strong><br />
exfoliated <strong>cells</strong> <strong>of</strong> the oral cavity correlate with <strong>human</strong> cancer<br />
risks <strong>and</strong> are therefore valuable biomarkers for the detection <strong>of</strong><br />
exposure to genotoxic carc<strong>in</strong>ogens. Nasal <strong>cells</strong> have been less<br />
frequently used as oral <strong>cells</strong> but the morphological<br />
characteristics <strong>of</strong> their nuclear anomalies are similar as those<br />
seen <strong>in</strong> <strong>buccal</strong> <strong>cells</strong>. Results obta<strong>in</strong>ed so far with FA exposed<br />
<strong>in</strong>dividuals yielded controversial results; three studies <strong>in</strong> which<br />
positive results were obta<strong>in</strong>ed are <strong>in</strong>adequate due to<br />
methodological shortcom<strong>in</strong>gs.<br />
36