Annals of Occupational Hygiene Advance Access originally published online on June 23, 2006
Annals of Occupational Hygiene 2006 50(6):583-592; doi:10.1093/annhyg/mel036
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Assessment of Occupational Genotoxic Risk in the Production of Rubber Tyres
1 Toxicology Unit, University of A Coruña, Edificio de Servicios Centrales de Investigación Campus Elviña s/n, 15071-A Coruña, Spain
2 National Institute of Health, Environmental Health and Toxicology Department Largo 1 Dezembro, 4000-Porto, Portugal
3 Department of Cell and Molecular Biology, University of A Coruña, Faculty of Sciences Campus A Zapateira s/n, 15071-A Coruña, Spain
*Author to whom correspondence should be addressed. Tel: 34 981167000; fax: 34 981167172; e-mail: blaffon{at}udc.es
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ABSTRACT |
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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.
Keywords: comet assay • metabolic polymorphisms • micronucleus • rubber tyres • sister chromatid exchanges
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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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 ß-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, alkali–labile 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 (Srám and Binková, 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 |
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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 –20°C 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 VacutainerTM CPTTM 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 107 cells ml–1, and frozen at –80°C in a Nalgene® Cryo 1°C Freezing Container (Nalgene Nunc International). At the time of analysis (<2 weeks), cells were quickly thawed at 37°C 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 x400. 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 PuregeneTM 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 reaction–restriction fragment length polymorphism (PCR–RFLP) assay following the method of Lin et al. (1998).
For EPHX1, two PCR–RFLP 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 µM 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 MgCl2 and 30 ng genomic DNA in a total volume of 30 µl. PCR conditions were 35 cycles of 30 s at 94°C, 30 s at 68°C and 1 min at 72°C, preceded by an initial denaturation of 90 s at 94°C and followed by a final extension of 10 min at 72°C. 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 (Sarmanová et al., 2000).
A multiplex PCR method was used to detect the presence or absence of the GSTM1 and GSTT1 genes, using ß-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 PCR–RFLP technique, following Saarikoski 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 (Kolmogorov–Smirnov 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 |
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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, GSTM1 and GSTT1 deletion polymorphisms and GSTP1 codon 105. The distribution of CYP2E1, EPHX1 and GSTP1 analysed genotypes were in Hardy–Weinberg 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; Sarmanová et al., 2000; Laffon et al., 2003; Teixeira et al., 2004).
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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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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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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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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 for GSTT1 null exposed individuals with respect to positive ones. No effect of CYP2E1, GSTM1 or GSTP1 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 of GSTP1 105Val variant allele showed significantly higher MN frequencies than GSTP1 105Ile homozygotes (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 105Ile homozygous carriers have shown significantly lower MN frequencies than both GSTT1 null and GSTP1 105Ile homozygotes or carriers of 105Val allele.
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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 |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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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 DNA–protein crosslinks would be expected (Zhu et al., 2000). In fact, it has been reported the induction of DNA–protein 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 DNA–protein crosslinks, which would retard the migration of DNA fragments. However, Somorovská 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 Wojewódzka 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 (Catalán 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 (Thier et 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 in CYP2E1.
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 exposure–genotype 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 of GSTP1 produces an enzyme with different thermal stability and substrate affinity (Sarmanová et al., 2000). In this study, effect of this polymorphism has been detected in association with GSTT1 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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ACKNOWLEDGEMENTS |
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This work was partially supported by the Comissão de Fomento à Investigação em Cuidados de Saúde 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 Coruña.
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FOOTNOTES |
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The first two authors contributed equally to this work. 
Received January 9, 2006; in final form April 12, 2006
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