Assessment of Occupational Genotoxic Risk in the

Production of Rubber Tyres

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BLANCA LAFFON1,*,
JOAO PAULO TEIXEIRA2,,
SUSANA SILVA2,
JOANA ROMA-TORRES2,
BEATRIZ PREZ-CADAHA1,
JOSEFINA MNDEZ3,
EDUARDO PSARO1 and
OLGA MAYAN2
+Author Affiliations
1. 1Toxicology Unit, University of A Corua, Edificio de Servicios Centrales de
Investigacin Campus Elvia s/n, 15071-A Corua, Spain
2. 2National Institute of Health, Environmental Health and Toxicology
Department Largo 1 Dezembro, 4000-Porto, Portugal
3. 3Department of Cell and Molecular Biology, University of A Corua, Faculty of
Sciences Campus A Zapateira s/n, 15071-A Corua, Spain
*
Author to whom correspondence should be addressed. Tel: 34 981167000; fax: 34
981167172; e-mail: blaffon@udc.es

Received January 9, 2006.

Accepted April 12, 2006.

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Abstract
A broad spectrum of substances is used in the rubber industry, many of them being genotoxic and/or
carcinogenic. Convincing evidence of an excess of certain forms of cancer among rubber workers has
been provided. The objective of this study was to determine the genotoxic effects in a group of
individuals engaged in the production of rubber tyres from a Portuguese factory. Peripheral blood
samples were collected from 32 exposed workers and 32 controls, and micronucleus (MN) test, sister
chromatid exchanges (SCE) and comet assay were performed. Urinary thioethers were measured as a
general biomarker of exposure to electrophilic compounds, and genetic polymorphisms in metabolizing
enzymes (CYP2E1 Dra I, EPHX1 codons 113 and 139, GSTP1 codon 105, and GSTM1 and GSTT1
deletion polymorphisms) were analysed as susceptibility biomarkers. Excretion of thioethers was found
significantly higher in rubber workers. Also, a non-significant increase in MN frequency related to time
of exposure and no effect in SCE were observed in the exposed. Comet assay data showed decreased
TL values in the exposed population with respect to the control group, this might indicate the induction
of crosslinks by the substances present in the workplace environment. Significant increase in MN
frequency was obtained for GSTT1 null exposed individuals with respect to positive ones, and
interaction with GSTP1 polymorphism was found. Higher levels of cytogenetic test frequencies were
observed in epoxide hydrolase expected low activity donors with respect to medium and high activity
individuals. No effect of CYP2E1 or GSTM1 variants was obtained in the biomarkers analysed.

Key words

comet assay

metabolic polymorphisms

micronucleus

rubber tyres

sister chromatid exchanges

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INTRODUCTION
Rubber industry uses a broad spectrum of substances (vulcanization agents, accelerators, activators,
colorants, solvents, etc.), belonging to many chemical categories (polycyclic aromatic hydrocarbons
[PAH], N-nitrosamines, 1,3-butadiene, acetonitrile, styrene, vinyl chloride, ethylene oxide, mineral oils,
other volatile organic compounds, etc.) (Fishbein, 1991;Sturaro et al., 1993; Oury et al., 1997), some of
which have been shown to be genotoxic and/or carcinogenic. In the environment of tyre producing
plants, presence of several organic solvent vapours and airborne particulate matter has been
characterized (Kromhout et al., 1994; Dost et al., 2000). In addition, elevated mutagenicity and
genotoxicity have been observed in air and particulate samples from rubber manufacturing plants
(Baranski et al., 1989; Fracasso et al., 1999; Vermeulen et al., 2000; Monarca et al., 2001), although
the substantial differences found between companies have been attributed to differences in rubber
chemicals used and the overall level of control measures (Vermeulen et al., 2000).
Convincing evidence of an excess of certain forms of cancer among rubber industry workers has been
provided by a large number of industry-based epidemiological studies (IARC, 1982,1987). A
subsequent updating of those data confirmed increased risk of bladder, laryngeal and lung cancers
and leukaemia (Kogevinas et al., 1998). Later, exposures to 1,3-butadiene and
dimethyldithiocarbamate in the synthetic rubber industry were positively associated with leukaemia in
multivariable analyses (Delzell et al., 2001), and elevated risk of bladder cancer was reported to be
associated with exposure to -naphthylamine that reversed when the substance was removed from the
rubber manufacturing process (Veys, 2004),
Assays measuring micronucleus (MN) and sister chromatid exchanges (SCE) in peripheral blood
lymphocytes are well-established cytogenetic techniques that have been used extensively for
assessing DNA damage at the chromosomal level in human biomonitoring (Carrano and Natarajan,
1988; Fenech, 1993; Lando et al., 1998). These cytogenetic biomarkers constituted valuable tool for
studying the most important occupational and environmental hazards to public health occurring in the
past few decades (Bonassi et al., 2005) and allow a reasonable epidemiological evaluation of cancer
predicitivity (Tucker and Preston, 1996). In recent years, single cell gel electrophoresis or comet assay
has been proven to be a very sensitive method to investigate the level of DNA damage, and an useful
tool for the detection of genetic damage at the individual cell level, and in human biomonitoring
(Kassie et al., 2000;Moller et al., 2000). This assay has been widely used in order to detect strand
breaks, alkalilabile sites, DNA crosslinking and incomplete excision repair sites (Fairbairn et al.,
1995;Collins et al., 1997).
Biomonitoring studies commonly describe variation in the level of genotoxicity biomarkers among
healthy individuals exposed to similar concentrations of contaminants. Polymorphic genes of low
penetrance but high allele frequency involved in the metabolism of xenobiotics may modulate the
levels of biomarkers arising from environmental and/or occupational exposure to genotoxicants
(Pavanello and Clonfero, 2000). Many studies have shown an elevated cancer proneness for individuals
carrying the potential at-risk alleles of metabolic genes, but a number of controversial results have
also been obtained (reviewed in Hirvonen, 1999). Reasons for disagreeing data and recommendations
to design studies aimed to measure the effect of polymorphic metabolic genes on cancer susceptibility
have been suggested (Garte, 2001; Imyanitov et al., 2004). Knowledge of the real impact of genetic
polymorphisms as biomarkers of susceptibility is of key significance in understanding the processes of
genetic damage involved in mutagenesis and carcinogenesis (Srm and Binkov, 2000) and could help
to minimize risks for susceptible subjects.
The objective of this study was to determine the genotoxic risk in workers engaged in the production of
rubber tyres from a Portuguese factory. Urinary thioethers were measured as a general biomarker of
exposure to electrophilic compounds. Effect biomarkers applied were MN test, SCE and comet assay. In
addition several genetic polymorphisms in xenobiotic metabolizing enzymes from phase I (CYP2E1
Dra I, and EPHX1 codons 113 and 139) and phase II (GSTP1 codon 105, and GSTM1 and GSTT1 deletion
polymorphisms) were analysed as susceptibility biomarkers.
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METHODS
Studied population
The exposed group consisted of 32 Caucasian male individuals engaged in the production of rubber
tyres in a Portuguese factory located in Oporto. Also, 32 Caucasian males were included in the control
population, matched by age and smoking habits. A complete questionnaire on lifestyle, consumption

habits such as smoking, alcohol intake, medication, recent viral infections, vaccinations, diagnostic
tests or previous occupational exposures to chemicals was filled out by all donors. Written informed
consent was obtained from each subject. The study was approved by the National Institute of Health
Research Ethics Committee.

Sample collection
Urine samples were collected post-shift. Blood samples were collected by venipuncture in heparinized
sterile tubes for genotoxicity tests, and in sterile tubes containing EDTA for DNA extraction and
genotyping. Tubes were coded and sent immediately to the laboratory, where they were processed.

Analysis of thioethers in urine samples

Urinary thioethers were determined in urine collected after the workshift and stored at 20C until
required (not longer than 15 days). Thioethers were quantified by the method described by Vainio et
al. (1978).

Cytokinesis-blocked MN assay
Aliquots of 0.5 ml of heparinized whole blood were used to establish duplicate lymphocyte cultures for
cytokinesis-blocked MN test, as described in Laffon et al. (2005). A total of 1000 binucleated
lymphocytes with well-preserved cytoplasm were scored blindly for each subject (500 from each
duplicate culture) by the same reader to determine the number of MN. Criteria from Kirsch-Volders et
al. (2000) for identifying binucleated cytokinesis-blocked cells and MN were followed.

Sister chromatid exchanges

Lymphocyte cultures for SCE were established in duplicate as described previously (Teixeira et al.,
2004) from 0.5 ml of heparinized whole blood. Differential chromatid staining was performed with the
fluorescence-plus-Giemsa procedure (Perry and Wolff, 1974). A single observer scored 50 second
division metaphases for each donor (25 from each duplicate culture) on coded slides to determine the
number of SCE/cell.

Comet assay
BD Vacutainer CPT Cell Preparation Tubes with sodium heparin (Becton Dickinson) were used for
the isolation of mononuclear leukocytes, following manufacturer's instructions. Cells were suspended
in freezing medium (50% foetal calf serum, 40% RPMI 1640 and 10% DMSO) to obtain 10 7 cells ml1,
and frozen at 80C in a Nalgene Cryo 1C Freezing Container (Nalgene Nunc International). At the
time of analysis (<2 weeks), cells were quickly thawed at 37C and trypan blue exclusion technique
was used to check viability, being >85% in all cases.
The alkaline version of the comet assay, basically as described by Singh et al. (1988), was applied with
minor modifications (Laffon et al., 2002). Two slides were prepared for each donor and a blind scorer
examined 50 randomly selected cells from each slide (100 cells/donor) using a magnification of 400.
QWIN Comet software (Leica Imaging Systems, Cambridge, UK) was used for image capture and
analysis. Comet tail length (TL), measured from the estimated centre of the cell, was evaluated as DNA
damage parameter.

Genotyping
Genomic DNA was isolated by means of Puregene DNA isolation kit (Gentra Systems, Minneapolis,
MI, USA). All genotype analyses were performed at least in duplicate to confirm the study results.
CYP2E1 Dra I site polymorphism (T7632A, intron 6) was analysed by means a polymerase chain
reactionrestriction fragment length polymorphism (PCRRFLP) assay following the method of Lin et al.
(1998).
For EPHX1, two PCRRFLP assays were used to detect the T to C mutation in exon 3 (Tyr113His) and
the A to G transition in exon 4 (His139Arg). Codon 113 polymorphism was analysed as described
in Laffon et al. (2003). For determination of codon 139 polymorphism, a 540 bp fragment was
amplified using 0.75 U Taq polymerase, 0.2 M of each primer (5-CCA GCT GTC AGG GGG CAC C-3 and
5-TGG CGA GGA CGG GGC AGT-3), 0.2 mM deoxynucleoside triphosphates, 1.5 mM MgCl 2 and 30 ng
genomic DNA in a total volume of 30 l. PCR conditions were 35 cycles of 30 s at 94C, 30 s at 68C
and 1 min at 72C, preceded by an initial denaturation of 90 s at 94C and followed by a final
extension of 10 min at 72C. The PCR product was digested with Rsa I, generating fragments of 499
and 41 bp in the case of wild-type allele and 322, 177 and 41 bp in the case of variant allele. Once the

individuals were genotyped for codons 113 and 139, they were classified according to the expected
epoxide hydrolase enzyme activity (Sarmanov et al., 2000).
A multiplex PCR method was used to detect the presence or absence of the GSTM1 and GSTT1genes,
using -globin as internal control, as described in Laffon et al. (2003). Genotypes were classified as
positive (at least one undeleted allele) or null (both alleles deleted). GSTP1 exon 5 (Ile105Val)
polymorphism analysis was performed using a PCRRFLP technique, followingSaarikoski et al. (1998).

Statistical analysis
All statistical analyses were conducted using the SPSS for Windows statistical package, version 11.5
(IL, USA). Distribution of every variable obtained in this study did not depart significantly from
normality (KolmogorovSmirnov goodness of fit test), and therefore parametric tests were considered
adequate for the statistical analysis of these data. Analysis of variance (ANOVA), followed by
Bonferroni's correction for multiple comparisons among groups when the overall F-test was significant,
was used to assess the contribution of exposure, age, smoking habits and genotypes to the variability
of genotoxicity variables studied. The associations between two variables were analysed by Pearson's
correlation. For all the polymorphisms considered (excepting GSTM1 and GSTT1), the homozygous and
heterozygous carriers of the variant alleles were combined in the statistical analyses, owing to the low
number of variant homozygotes.
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RESULTS
The present article illustrates the results of a genotoxicity study (MN test, SCE and comet assay) on
peripheral blood leukocytes obtained from 32 workers of a Portuguese rubber tyre factory and 32
control subjects. Characteristics of the study populations are gathered in Table 1. Age was very similar
in both groups, as well as smoking habits, 39% of the individuals being smokers. Exposure time
covered a wide range, with 19 individuals (59.4%) exposed for <7 years and 13 subjects (40.6%)
exposed for >14 years. To assess the possible influence of metabolic polymorphisms on the genetic
biomarkers investigated, the study subjects were genotyped for the following polymorphisms: CYP2E1
Dra I, EPHX1 codons 113 and 139, GSTM1and GSTT1 deletion polymorphisms and GSTP1 codon 105.
The distribution of CYP2E1, EPHX1and GSTP1 analysed genotypes were in HardyWeinberg equilibrium
(2-test). The frequencies of variant alleles or genotypes (in case of GSTM1 and GSTT1) were the
expected for the Caucasian population and similar to those reported previously (Pemble et al.,
1994; Sarmanovet al., 2000; Laffon et al., 2003; Teixeira et al., 2004).

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Table 1
General characteristics of study subjects
Comet assay results were evaluated by means of the three most frequently used parameters to
describe DNA damage: TL, percentage of DNA in the comet tail and tail moment (which results from
multiplying the other two variables). Statistical analysis obtained the same results for all of them (in
terms of statistical significances), thus we only show TL data. Table 2 shows results obtained in the
measurement of internal exposure dose and in genotoxicity assays, both in the control and exposed
groups and stratified by tobacco consumption. Exposed subjects had levels of urinary thioethers
significantly higher than controls. When smokers were separated from non-smokers, both groups
presented elevated values for urinary thioethers in the exposed subjects as compared to controls, but
statistical significance was only maintained in non-smokers. No significant increase was observed in
MN or SCE frequencies in the exposed population as compared with control individuals, although MN
frequencies were higher in the exposed group. Nevertheless, a significant decrease in TL was obtained
for the exposed with respect to control individuals. This decrease was also significant when comparing
DNA damage in non-smoker exposed with non-smoker controls, but not when the comparison was
made between smokers. Smoking did not significantly affect results of exposure or effect biomarkers
analysed.

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Table 2
Exposure and effect biomarkers in the populations examined (mean SE)

The influence of duration of exposure on MN, SCE and comet tests is reflected in Fig. 1. MN frequencies
increased with time of exposure, although significance was not reached. SCE results were not affected
by the duration of the employment, and TL decreased significantly in both groups as compared to
controls, but they did not differ from each other. Correlation between MN frequency and TL with
exposure time was found to be significant at the level of 0.05 (r = 0.274 and r = 0.267, respectively).

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Fig. 1
Effect of exposure time on (a) MN frequency, (b) SCE frequency and (c) TL. The number of individuals
included in each group is indicated inside each bar. **P < 0.01, *P < 0.05, significant difference with
respect to the control group.
Figure 2 shows the effect of age on genotoxicity assays performed. Higher MN and SCE frequencies
were observed in the older group, although significant differences were only obtained between the two
established groups (35 and >35 years) among controls in MN frequencies and among exposed
subjects in SCE frequencies. Significant associations between age and cytogenetic parameters were
found (r = 0.387, P < 0.01 for MN and r = 0.225, P < 0.05 for SCE). TL values were not affected by this
factor.

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Fig. 2
Effect of age on (a) MN frequency, (b) SCE frequency and (c) TL. The number of individuals included in
each group is indicated inside each bar. **P<0.01, *P<0.05, significant difference with respect to the
minor age group.
Possible modulation of results obtained in the effect biomarkers by metabolic polymorphisms is
reflected in Table 3 for CYP2E1 and GST genes and in Fig. 3 for EPHX1 gene (expressed as expected
activity of epoxide hydrolase). Significant increase in MN frequency was observed forGSTT1 null
exposed individuals with respect to positive ones. No effect of CYP2E1, GSTM1 orGSTP1 variants was
obtained in SCE or MN test. Nevertheless, when individuals were stratified by their GSTT1 genotype,
among the exposed population those GSTT1 positive carriers ofGSTP1 105Val variant allele showed
significantly higher MN frequencies than GSTP1 105Ilehomozygotes (2.10 0.43 versus 1.00
0.30, P < 0.05, N = 10 for both groups). Combining these two polymorphisms (Fig. 4) exposed
subjects GSTT1 positive and GSTP1 105Ilehomozygous carriers have shown significantly lower MN
frequencies than both GSTT1 null andGSTP1 105Ile homozygotes or carriers of 105Val allele.

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Fig. 3
Effect of epoxide hydrolase expected activity on (a) MN frequency, (b) SCE frequency and (c) TL. The
number of individuals included in each group is indicated inside each bar. *P<0.05, significant
difference with respect to low activity genotype.

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Fig. 4
Effect of combined GSTT1 and GSTP1codon 105 polymorphisms on MN frequency in the exposed
population. The number of individuals included in each group is indicated inside each
bar. **P<0.01,*P<0.05, significant difference with respect to GSTT1 positive and GSTP1Ala/Ala
individuals.

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Table 3
Effect of genetic polymorphisms on genotoxicity tests
As for EPHX1, higher MN frequency among controls and SCE frequency among exposed were detected
for the epoxide hydrolase expected low activity donors with respect to medium and high activity
individuals, reaching statistical significance only in the later case. TL values appeared not to be
affected by any of the genetic polymorphisms analysed.
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DISCUSSION
The present report aims to evaluate the genotoxic effects associated with the exposure to xenobiotic
substances in the production of rubber tyres, and to determine the relationships between transient
markers of genotoxic/carcinogenic effect and several genetic polymorphisms in genes coding for
xenobiotic metabolizing enzymes.
Owing to the great variety of chemical compounds present in the ambient air of rubber tyre factories,
evaluation of the internal exposure dose was performed by analyzing urinary thioethers, since this
measure constitutes a general biomarker of exposure to electrophilic compounds. Significantly
increased levels of this biomarker were observed in the exposed population as compared to controls,
but when the population was divided by tobacco consumption, significance for the difference between
exposed and controls was only maintained among non-smokers. This was caused by the elevated
excretion of thioethers compounds by smokers, as has been described previously (Sinues et al., 1990),
that decreased the importance of urinary thioethers coming from the exposure.
A range of potentially carcinogenic compounds occur as workplace contaminants during rubber
manufacturing, such as PAH, N-nitrosamines, 1,3-butadiene, styrene and various alkenes. The
genotoxic effects that these agents may simultaneously induce are largely unknown, but synergistic
interactions can be expected. In this study a non-significant increase in MN frequency has been
observed in exposed individuals, but no effect of exposure was detected in SCE. Our results agree with
those from Moretti et al. (1996), who reported elevated MN frequencies but not SCE frequencies in a
rubber-exposed population, although thioethers excretion described was clearly higher than in our
study. Nevertheless, other authors found increases in chromosome aberrations and SCE in workers
from the rubber industry (Sorsa et al., 1983; Sasiadek et al., 1992), but unfortunately no indication on
the quantitative level of exposure was given in these studies.
Comet assay data have shown decreased TL values in the exposed population with respect to the
control group that might indicate the induction of crosslinks by the substances present in the
workplace environment. In contrast to other DNA alterations, crosslinks may stabilize chromosomal
DNA and inhibit DNA migration in the comet assay (Pfuhler and Wolf, 1996; Merk and Speit, 1999). As
some of the active metabolites from rubber production compounds can react with nucleophilic regions
of both protein and DNA, similar to those from alkenes and styrene (Philips and Farmer, 1994), the

occurrence of DNAprotein crosslinks would be expected (Zhu et al., 2000). In fact, it has been
reported the induction of DNAprotein crosslinks by several PAH (Perin-Rousel et al., 1984; Park et al.,
2002). On the other hand, the comet assay also detects DNA breaks coming from incomplete excision
repair processes, so data obtained may reflect a lower DNA repair capacity of the exposed
subjects. Moretti et al. (1996) and Vodicka et al. (2004) did not find significant differences in comet
results in rubber-exposed individuals, and Zhu et al. (2000) described increase in DNA damage in
rubber workers but they used proteinase K in the comet assay to break down possible DNAprotein
crosslinks, which would retard the migration of DNA fragments. However, Somorovsk et al.
(1999) reported higher level of DNA breaks, evaluated by the comet assay, and also of chromosome
aberration and MN frequencies in workers from a rubber factory. Differences found between studies
could be attributed to the different spectra of chemical substances used (Vermeulen et al., 2000).
Tobacco consumption did not affect results obtained in cytogenetic tests, except that a non-significant
increase in SCE frequency was observed in smokers in comparison to non-smokers among control
group. Smoking is known to increase SCE (Lazutka et al., 1994; Barale et al., 1998), and therefore
smoking effect could be masked by exposure in rubber workers, although the low number of smoker
subjects included in this study may also affect our results. On the other hand, tobacco smoking did not
appear to have a clear effect on the frequency of MN (Thierens et al., 1996; Barale et al., 1998).
According to this, results from the HUman MicroNucleus Project have shown a significant increase of
MN frequency only in heavy smokers (30 cigarettes or more per day) (Bonassi et al., 2003). Smoking
habit did not either significantly increase the levels of DNA damage in control or exposed subjects, in
agreement with findings by Hellman et al. (1997, 1999) and Wojewdzka et al. (1999).
The present investigation shows increasing MN frequencies with duration of exposure, confirming the
fact that this biomarker reflects accumulated chromosomal damage (Kirsch-Volders and Fenech, 2001).
However, maybe owing to the small sample size the difference does not reach statistical significance.
In this study, significant associations were found between age and cytogenetic parameters. Age is
known to affect the frequency of MN and SCE (Thierens et al., 1996; Bolognesi et al., 1997; Barale et
al., 1998; Bonassi et al., 2003), its effect being particularly clear on MN, apparently mostly because of
the age-dependent micronucleation of sex chromosomes (Cataln et al., 1998).
Cytochrome P450 (CYP) monooxygenases are enzymes which catalyse the insertion of one atom of
molecular oxygen into a substrate in a typical reaction of activation (phase I). Of particular interest for
the industrial field is the isozyme CYP2E1, since substrate spectrum of this enzyme includes many
compounds of classical relevance for industrial toxicology (Thieret al., 2003). Genetic polymorphisms
in CYP2E1 appear to act on the transcription levels of the enzyme, increasing its activity (Pavanello
and Clonfero, 2000). We did not observe any association between biomarkers measured in the present
study and Dra I polymorphism inCYP2E1.
Microsomal epoxide hydrolase, coded by the EPHX1 gene, catalyses the addition of a molecule of H2O
to an epoxide to form a dihydrodiol, generating more soluble and less reactive metabolites that can be
readily conjugated and excreted. Replacement of Tyr to His at codon 113 reduces enzyme activity,
while substitution of His to Arg at codon 139 of exon 4 is associated with increased activity (Hasset et
al., 1994). It has been suggested that the amino acid substitutions may result in altered protein
stability, since they do not affect the specific activity of the enzyme (Raaka et al., 1998). In this work,
significantly increased SCE frequencies have been observed in exposed workers with low expected
epoxide hydrolase activity, with respect to those individuals with medium and high activity. The same
effect, although non-significant, has been also obtained in MN frequencies in the control population. In
a previous report, individuals working in a tyre plant with low epoxide hydrolase expected activity
exhibited higher CA frequencies than those with medium and high activity (Vodicka et al. 2004). In
addition, elevated HPRT lymphocyte mutant frequencies were observed in workers with low epoxide
hydrolase activity exposed to 1,3-butadiene (Abdel-Rahman et al., 2003) or to low levels of PAH
(Viezzer et al., 1999). These results confirm the role of epoxide hydrolase in the detoxification of
xenobiotic substances present in the environment of rubber tyre factories.
Glutathione S-transferases (GST) are a superfamily of polymorphic enzymes involved in the
conjugation of reactive chemical intermediates, and play an important role in the detoxification of
endogenous and exogenous compounds. The polymorphisms of GSTM1 and GSTT1 owing to gene
deletions result in null alleles, and homozygous individuals for the deletions lack enzyme activity. As in
the present study, no association between chromosomal aberrations or DNA damage
and GSTM1 deletion polymorphism was detected in rubber workers (Vodicka et al., 2004). The lack
of GSTM1 appears to be associated with increased sensitivity to genotoxicity of tobacco smoking
(Norppa 2004). Nevertheless, no significant increase in cytogenetic or DNA damage among GSTM1 null
smokers has been detected in this study in comparison with GSTM1 positive smokers, neither in
controls nor in exposed (data not shown), probably owing to the fact that the effect of smoking itself
on the mean number of SCE per cell is usually small.
Deletion of GSTT1 gene has been found to yield an increase in baseline SCE frequency (Norppa, 2004).
Also, increased MN frequency was consistently shown for GSTT1 null individuals (Bonassi et al., 2005).
The reported effect of GSTT1 genotype on baseline SCE level was quite small, which may explain why
it has not been detected in this study, in addition to the low number of individuals included in the

group of GSTT1 null controls. However, a clear influence of this polymorphism has been obtained in MN
frequencies among rubber workers. Since this effect has been obtained only in the exposed group, it
suggests the existence of an exposuregenotype interaction (Norppa, 2003).
GSTP1 is the most abundant isoform in the lungs; thus, it has particular importance in the
detoxification of inhaled toxicants (Saarikoski et al., 1998). Polymorphism in codon 105
ofGSTP1 produces an enzyme with different thermal stability and substrate affinity (Sarmanov et al.,
2000). In this study, effect of this polymorphism has been detected in association
withGSTT1 genotype, since the lack of GSTT1 activity and the presence of GSTP1 105Val variant allele
determined increasing MN frequencies. This observation suggests an increased risk of genotoxic
effects in individuals with particular genotype combinations.
Data obtained in this study indicated that genotoxic risk of occupational exposure associated with the
production of rubber tyres cannot be excluded. In addition, a certain role of genetic polymorphisms
in EPHX1 and GSTT1 in the modulation of cytogenetic tests results has been suggested, together with
an interaction between GSTP1 and GSTT1 polymorphisms. Nevertheless, these results must be
cautiously interpreted, owing to the relatively low number of exposed and control individuals included
in this study.
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Acknowledgments
This work was partially supported by the Comisso de Fomento Investigao em Cuidados de
Sade and by a grant from the Xunta de Galicia (PGIDT04PXIB10602PR). B.L. was supported by a
postdoctoral fellowship from the Xunta de Galicia. B.P.-C. was supported by a predoctoral fellowship
from the University of A Corua.
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Footnotes

The first two authors contributed equally to this work.

The Author 2006. Published by Oxford University Press on behalf of the British Occupational
Hygiene Society
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