Chromosome aberrations in workers exposed to organic solvents: Influence of polymorphisms in xenobiotic-metabolism and DNA repair genes

Mutation Research 666 (2009) 8–15

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Chromosome aberrations in workers exposed to organic solvents: Influence of polymorphisms in xenobiotic-metabolism and DNA repair genes Luz Stella Hoyos-Giraldo a,∗ , Silvio Carvajal a , Nohelia Cajas-Salazar a , Martín Ruíz b , Adalberto Sánchez-Gómez c a b c

Department of Biology, Research Group Genetic Toxicology and Cytogenetics, Faculty of Natural Sciences and Education, Universidad del Cauca, Popayán, Cauca, Colombia Department of Morphology, Research Group Health and Work, Faculty of Health Sciences, Universidad del Cauca, Popayán, Cauca, Colombia Department of Physiologic Sciences, Faculty of Health, Universidad del Valle, Cali, Valle del Cauca, Colombia

a r t i c l e

i n f o

Article history: Received 10 September 2008 Received in revised form 5 March 2009 Accepted 11 March 2009 Available online 24 March 2009 Keywords: Organic solvents Chromosome aberrations Genetic polymorphisms Xenobiotic-metabolism and DNA repair genes Susceptibility Occupational exposure

a b s t r a c t Organic solvents are widely used as diluents or thinners for oil-paints, gasoline and other organic mixtures. We evaluated chromosome aberrations (CAs) in lymphocytes of 200 workers exposed to organic solvents and 200 referents and the influence of polymorphisms in xenobiotic-metabolism (CYP2E1, GSTM1 and GSTT1) and in DNA repair genes (XRCC1194 Arg/Trp, XRCC1280 Arg/His, XRCC1399 Arg/Gln and XRCC3241 Thr/Met). Polymorphisms were determined by PCR–RFLP. Poisson regression analysis indicates a significant CA frequency increase in exposed workers, representing a higher risk in relation to the matched referent (RR 2.15, 95% CI 1.21–1.53, p < 0.001). The CA frequency in exposed workers was influenced by the polymorphic genotypes: GSTM1 null (RR 1.33, 95% CI 1.31–1.69, p < 0.001), XRCC1194 Arg/Trp, Trp/Trp (RR 1.23, 95% CI 1.08–1.40, p < 0.001) and by the wild genotypes CYP2E1 C1/C1 (RR 1.20, 95% CI 1.05–1.37, p < 0.001), GSTT1 positive (RR 1.49, 95% CI 1.31–1.69, p < 0.001), XRCC1280 Arg/Arg (RR 1.44, 95% CI 1.26–1.64, p < 0.001) and XRCC1241 Thr/Thr (RR 1.54, 95% CI 1.34–1.76, p = 0.001). We contribute to the follow-up predictive value of individual susceptibility biomarkers and their CA frequency influence during occupational organic solvent exposure. We provide tools for surveillance and prevention strategies to reduce potential health risks in countries with a large population of car painters not using protection devices and limited organic solvents use control. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The IARC reported that “there is sufficient evidence of carcinogenicity by occupational exposure as painters” and classified painting as an occupation that increases certain cancers risk [1,2]. The production of oil-paints and the use of paint diluents or “thinners” are the main sources of organic solvents exposure; 50% of these synthesized organic solvents are employed by car painters

Abbreviations: CA, chromosome aberrations; CTA, chromatid-type aberration; CSA, chromosome-type aberration; SCE, sister chromatid exchanges; MN, micronuclei; CYP2E1, cytochrome P-450 2E1 gene; ROS, reactive oxygen species; BER, base excision repair; GSTM1, GSTT1, glutathione-S-transferase M1, T1; XRCC1, X-ray repair cross-complementing group 1; XRCC3, X-ray repair cross-complementing group 3; DSB, double strand breaks; SSB, single strand breaks; HR, homologous recombination repair; PARP, polynucleotide kinase/phosphatase-poly ADP-ribose polymerase; APE1, apurinic/apyridinic endonuclease 1; PBMC, peripheral blood mononuclear cells. ∗ Corresponding author at: Vicerrectoría de Investigaciones-Universidad del Cauca, Carrera 2da N◦ 1 A-25 Barrio Caldas, Popayán, Cauca, Colombia. Tel.: +57 2 820 9800x2615; fax: +57 2 820 9860. E-mail addresses: [email protected], [email protected] (L.S. Hoyos-Giraldo). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.03.003

and therefore represent an occupational health problem. Thinners are complex commercial organic solvents mixtures that contain: benzene, toluene, xylene, hexane, some alcohols and more than 50 different organic compounds with masses <1%. Benzene is one of principal component of thinners and is a well-known clastogen and carcinogen [3]. The Pollution Prevention Act in 1990, the U.S. Environmental Protection Agency, EPA reduced the use of benzene and toluene; however, some thinners still contain regulated, low or above the permissible levels of organic solvents. By biomonitoring studies Aksoy et al. [4] reported CA frequency, SCE and MN increase in offset painting workers and Bogadi-Sare et al. [3] indicated CA increase in shoe workers, exposed to benzene and toluene. The inclusion of healthy subjects’ peripheral lymphocytes to identify harmful occupational/environmental exposures in relation to age, time of exposure and genetic factors may reflect similar cumulative damages of carcinogenic processes in target tissues. CA is the most extensively used and best validated cytogenetic biomarker in epidemiologic cohort-prospective studies, since a higher CA frequency predicts human cancer risk [5,6]. Results of these studies may be used as regulatory tools for improving heath surveillance safety and for law regulation programs. Molecular epidemiology studies have been viewed as a means to acquire a better

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insight in to the cancer risk associated with genotoxic damage influenced by individual susceptibility, thus making them relevant for hazard exposures identification and risk assessment in order to prevent future cancers. Organic solvents metabolism induces oxidation by CYP2E1 enzyme during phase I [7]. One of the identified CYP2E1 polymorphisms includes a base substitution in the 5 -flanking region that has been associated with an altered transcription regulation and with an increase of gene expression [8]. CYP2E1 polymorphisms have been associated with CA frequency increase and cancer susceptibility [9]. Benzene is primary oxidized to phenols, hydroquinones and then re-oxidized to benzoquinones, which are the most potent genotoxic metabolites produced by organic solvents [10]. Also organic solvents during redox processes generate ROS [11]. ROS and benzoquinones induce oxidative damage such as: oxidized bases, SSB, DSB and cross-linkings, that can be expressed in CA and MN formation [12,13]. Glutathione-S-transferases are a super family of enzymes involved in phase II, transforming of oxidated-reactive intermediates, to less-reactive metabolites for easier excretion. The GSTM1 and GSTT1 enzymes play a crucial role in organic solvents reactive intermediates detoxification [14]. The deleted polymorphism in GSTM1 and GSTT1 results in the total lack of enzyme activity by homozygous null genotype [15]. The null genotypes have been associated with CA frequency increase [13,16]. The oxidative damage caused by organic solvents metabolites that escape detoxification is repaired by BER [17] and by HR [18], that if is not repaired, lead to mutagenesis, tumor promotion and progression. The scaffolding protein XRCC1 plays an important part in BER [19] by promoting an efficient SSB repair; after removal of damaged bases by DNA glycosylases. XRCC1 mediates protein–protein interactions with DNA ligase III, PARP and DNA polymerase ␤ [20]. The removal of damaged bases by APE1 involves an intermediary step, where one DNA strand is nicked and may promote indirect DSB formation by DNA replication [21], which lead to CTA formation and chromosome instability [22]. Three major coding polymorphisms, XRCC1194 Arg/Trp, XRCC1280 Arg/His, XRCC1399 Arg/Gln, were studied for XRCC1 gene [23]. Protein XRCC3 is one of the RAD51-like proteins, required for efficient HR repair of DSB induced either directly or indirectly [18]. Only one coding polymorphism has been reported (XRCC3241 Thr/Met), but little is known about the functional consequences of this amino acid change. Given the importance of CYP2E1, GSTM1 and GSTT1 in organic solvents metabolism and the crucial role of DNA ROS damage repair by XRCC1 gene in BER and XRCC3 gene in HR, we hypothesized that their genetic polymorphisms may influence individual differences in activation and detoxification during metabolism [24] and decreased DNA repair capacity, that may result in CA frequency increase in lymphocytes [25] and thereby higher cancer risk. The aim of this cross-sectional study was to evaluate the CA frequency in workers exposed to organic solvents and the influence of polymorphisms in xenobiotic-metabolism (CYP2E1, GSTM1 and GSTT1) and in DNA repair genes (XRCC1194 Arg/Trp, XRCC1280 Arg/His, XRCC1399 Arg/Gln and XRCC3241 Thr/Met). 2. Materials and methods 2.1. Subjects The study population included a total of 400 healthy males. 200 car-painters, recruited from several workshops in south-west Colombia, exposed to the same commercial thinner 0.14, to dilute oil-paints, a complex mixture which contains: toluene 21.58%, isobutane 17.45%, m-xylene 15.77, hexane 11.45%, novene 8.58, 2,3-dimetylhexane 6.76%, ethylbenzene 8.01, p-xylene 5.79,octane 3.55%, o-xylene 0.48, ∼50 compounds mass <1% (gas chromatography-PIANO analysis hydrocarbon analysis to determine the amount of paraffin (P), isoparaffins (I), aromatics (A), naphthalene (N), and olefins (O), in the mixture). They all worked 8 h average per day in their occupation setting, without use of protective devices such as masks

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or/and gloves. The 200 referents (non-exposed) were selected from the University administration services, with no occupational exposure history to organic solvents or any known physical or chemical agent in the workplace. Exposed workers were matched to referent by age (±2 years) and similar social-economic status. Confounding and exclusion factors were collected from all participants who responded to an interviewer-administered, detailed, standard questionnaire. Data of health status, cancer history, other chronic diseases, lifestyle, nutrition, smoking habits, alcohol and medication intake, occupational and time exposure, protective measures, and previous exposure to medical X-rays or treatment with known carcinogens. Exclusion criteria for exposed and referent groups were current and ex-smoking habits, medical treatment up to 3 months or X-ray up to 1 year before sampling and therapeutic drugs intake, known to be mutagenic. Exclusion criterion for exposed workers was an organic solvents exposure <5 years. All data was organized and recorded in databases. No major differences social-economic status and dietary habits were recorded. Written informed consent was obtained from all participants who were informed of the study’s aim, methodology, risks and possible benefits from study participation. All could cancel their participation at any time during the study according to the Helsinki II declaration. The study design and the informed consent format were approved by the institutional Ethical Committee for Scientific Investigation, Universidad del Cauca, Colombia based on the WHO guidelines [26]. 2.2. Blood samples After informed consent was obtained, peripheral blood samples from all 400 participants were collected in each shop or office at the end of the working shift. By venipuncture we collected 5 mL of blood in heparin tubes (Becton Dickenson, vacutainer) for the CA assay and 5 mL in EDTA coated tubes for DNA isolation and genotypification. The blood sampling was done by a nurse and the material was carried out in the frame of regular preventive examinations of employees occupationally exposed to potentially harmful chemicals and performed according to the Helsinki Declaration [27]. All blood samples tubes were coded and transported in dark in less than 2 h to the laboratory and processed for analysis immediately upon arrival. Blood sapling was done from June 2006 to January 2008, with 10–20 subjects being sampled per week. 2.3. Chromosome aberration assay and analysis Heparinized whole blood (1 mL) was added to 5 mL of RPMI 1640 medium (Sigma R8758, USA) supplemented with 2 mM l-glutamine (Sigma A5955, USA), 10% fetal bovine serum (Gibco/Invitrogen 15000-044, Brazil), 100 ␮L/mL antibiotic–antimycotic (Sigma A5955, USA) and 2% phytohemagglutinin (Sigma L8754, USA) to stimulate the lymphocytes. Cultures were incubated at 37 ◦ C in the dark for 46 h in a 5% CO2 atmosphere. Two parallel cultures were set in tubes (Falcon 3033) for each sample. Cultures were treated with Demecolcine (Sigma D6165, USA), during the last 2 h of culture. The cells were harvested by centrifugation, resuspended in hypotonic solution (0.075 M KCl at 37 ◦ C), for 20 min and fixed three times with Carnoy Fixer (glacial acetic acid: methanol 1:3, v/v) according to standard procedure [28]. Lymphocyte suspension from last fixation per culture was dropped onto two slides, air-dried and stained with 10% Giemsa (pH 6.8). The slides from each culture were randomly numbered and scored as “blind” in numerical order. A total of 100 well spread metaphases (46 centromers) were scored per subject. Scoring was done by two microscopists with harmonized standard scoring criteria [29]. The following CA categories were registered: CTA, CSA, dicentric chromosomes and chromatid exchanges, were considered as two CTA. The results are presented as CA percentage. 2.4. DNA isolation For each 5 mL of blood sample collected in EDTA coated tube, buffy coat containing PBMC was isolated by Ficoll–Paque. PBMC were lyzed by proteinase-K digestion. Genomic DNA was isolated from PBMC by means of commercially available DNeasy Blood and Tissue® extraction kit (Qiagen, USA), according to the manufacturer’s instructions. Each DNA sample was stored at −20 ◦ C until analysis. 2.5. Genetic polymorphisms in xenobiotic-metabolism and DNA repair genes PCR and RFLP techniques were used for the genotype analysis. GSTM1 and GSTT1 genotyping detects homozygous deleted genes and was carried out by allele-specific multiplex PCR as described earlier, with the CYP1A1 gene as an internal control [30]. The GSTM1 and GSTT1 genotypes were classified as null or positive (at least one undeleted allele). The CYP2E1 polymorphism was genotyped as described by Anwar et al. [31]. XRCC1194 and XRCC1399 genotypes were assessed as previously described by Lunn et al. [32], with a slight modification in the 3 end of 194F primer, where three bases were added to obtain uniform annealing temperature in the multiplex PCR–RFLP [33]. XRCC1280 and XRCC3241 genotypes were determined according to Tuimala et al. [33]. All PCRs were performed in a total volume of 30 ␮L, 200 ␮M of each dNTP (Invitogen, Brazil), 10× PCR buffer (Promega), 3 mM MgCl2 , 10 pmol of each primer, 100–400 ng of genomic DNA, and 0.5 U Taq polymerase (Promega, USA). For XRCC1194 and XRCC1399 PCR we added as well the Platinum® Taq DNA polymerase, antibody (Invitrogen, Brazil). Positive and negative controls were included

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Table 1 Characteristics of the study population and the mean chromosome aberration frequency for referent and exposed workers. No. of subjects (%) Referent Age (mean ± S.D., years) Time of exposure (mean ± S.D., years) Frequency of chromatid-type aberration/100 cells (mean ± S.E.) Frequency of chromosome-type aberration/100 cells (mean ± S.E.) Frequency of chromosome aberration/100 cells (mean ± S.E.)

36.75 ± 0 2.34 ± 0.18 ± 2.52 ±

p-Value Exposed

9.13 0.132 0.033 0.137

37.49 16.24 3.17 0.26 3.43

± ± ± ± ±

9.35 9.27 0.173 0.056 0.188

0.073a 0.001a , * 0.392a <0.001a , *

S.D.: standard deviation; S.E.: standard error. * Significant difference, p < 0.05. a Determinated by Mann–Whitney U-test.

in each run. The amplified fragments and digested PCR products were resolved on 2% agarose gel, except for the digested products from XRCC1194 and XRCC1399 PCR–RFLP which were resolved on 3% high-resolution MetaPhor agarose gel (Bioproducts, Rockland, ME). All PCR–RFLP products were visualized under UV light (Transilluminator, FBTIV-88, Fisher Scientific) after staining with ethidium bromide (Sigma, USA). All genotypes were evaluated and agreed on by two persons independently. The genotype results were confirmed by random re-genotyping more than 10% of the samples, for each analyzed polymorphism. 2.6. Statistical analysis Statistical analysis was performed using SPSS software for Windows version 13.0 (SPSS Inc., Chicago, IL, USA). Differences for qualitative variables and CA frequency differences between exposed workers and referent were calculated using 2 -test and the non-parametric Mann–Whitney rank sum U-test, respectively. The association between time of exposure to organic solvents and CA frequency was examined using the Kruskal–Wallis test. Hardy–Weinberg equilibrium was tested for each polymorphism, the observed genotype frequencies were compared to the expected frequencies using the goodness-of-fit 2 -test. The association between ageing and the CA frequency, in the whole study population, exposed and referent groups was assessed by Spearman’s rank correlation test. The relative risk (RR) and 95% confidence intervals (95% CI) were obtained from the Poisson regression analysis in order to evaluate the independent association between the CA frequency and the xenobiotic-metabolism (CYP2E1, GSTM1 and GSTT1) and in DNA repair genes (XRCC1194 Arg/Trp, XRCC1399 Arg/Gln, XRCC1280 Arg/His and XRCC3241 Thr/Met) polymorphisms. For all genes, the homozygous and heterozygous carriers of the polymorphism were classified as polymorphic genotypes and were combined in the statistical analysis owing to the small numbers of homozygote variants. Statistical significance was set at p < 0.05 (˛) with power (ˇ) at ≥0.8.

3. Results The general characteristics of the study population and the mean CA frequency for referent and exposed workers are shown in Table 1. There was no significant difference in health problems, medication intake and family common health problems between exposed workers and referent (not shown). The mean age ± standard deviation (S.D.) was 36.75 ± 9.13 years in referent and 37.49 ± 9.35 years in exposed workers. The mean time exposure ± S.D. was 16.24 ± 9.27 years. Mann–Whitney U-test revealed that exposure to organic solvents significantly influenced the CA frequency increase (Mann–Whitney U-test p < 0.001) between exposed workers and referent (3.43 ± 0.18% vs 2.52 ± 0.13%, respectively), in both cases independent of alcohol intake. The CTA was the most common CA in the study population and significantly higher in exposed workers compared to referent (Mann–Whitney U-test p = 0.001). No statistical difference was detected in CSA between referent and exposed workers (Table 1). The CA frequency increased significantly from referent to exposed workers with a minimum time of exposure of 5 years (Kruskal–Wallis test p = 0.011, Fig. 1). CA frequency did not increase with aging in the study population (Spearmen’s rank correlation test p > 0.05). Table 2 presents the distribution of CYP2E1, GSTM1, GSTT1, XRCC1194 , XRCC1280 , XRCC1399 and XRCC3241 genotypes and alleles frequencies in the study population, with exposure stratification. All the genotypes were in concordance with Hardy–Weinberg equilibrium making selection bias less likely, since no statistically significant differences were observed between genotype distributions in referent and exposed

Fig. 1. Association of time of exposure to organic solvents with frequency of chromosome aberration. a Referent CA baseline. Error bars represent the standard deviation of the mean. Asterisk indicates significant CA frequency increase in association with time of exposure according to Kuskall–Wallis test, p < 0.05.

workers. All variant allele frequencies are in agreement with literature values for Caucasian and Brazilian population [34,35]. Table 3 shows the mean CA frequencies and the Poisson regression analysis. The RR was calculated using as reference the wild genotype referent as the lowest risk genotype. There was a significant CA frequency increase in exposed workers, representing a higher risk in relation to the matched referent (RR 2.15, 95% CI 1.21–1.53, p < 0.001). The CA frequency in exposed workers was influenced by the polymorphic genotypes: GSTM1 null (RR 1.33, 95% CI 1.31–1.69, p < 0.001), XRCC1194 Arg/Trp, Trp/Trp (RR 1.23, 95% CI 1.08–1.40, p < 0.001) and by the wild genotypes CYP2E1 C1/C1 (RR 1.20, 95% CI 1.05–1.37, p < 0.001), GSTT1 positive (RR 1.49, 95% CI 1.31–1.69, p < 0.001), XRCC1280 Arg/Arg (RR 1.44, 95% CI 1.26–1.64, p < 0.001) and XRCC1241 Thr/Thr (RR 1.54, 95% CI 1.34–1.76, p = 0.001). Even though, CYP2E1 C1/C2, C2/C2 in referent and in exposed were all statistically significant, it is evident that the RR, for the CYP2E1 C1/C1 in exposed workers, is higher than the sum of the RRs for CYP2E1 C1/C2, C2/C2 in referent and exposed workers. This same relation occurs for the RR bold values of the genotypes with significant effects on CA frequency, since their values are higher than the sum of the other two remaining RR values, for the same gene, as stated in Table 3. 4. Discussion During the last 30 years, several non-positive [36], positive in vitro [37] and biomonitoring studies using a single [38] or combined organic solvent exposures [39,40] have been published, that

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Table 2 Distribution of CYP2E1, GSTM1, GSTT1, XRCC1194 , XRCC1280 , XRCC1399 and XRCC3241 genotypes and allele frequencies in referent and organic solvents exposed workers. Gene

Genotype

Referent (%) (n = 200)

Exposed (%) (n = 200)

Total (%) (n = 400)

Allele frequency

p-Valuea

C1/C1 C1/C2 C2/C2

149 (74.5) 49 (24.5) 2 (1.0)

138 (69.0) 61 (30.5) 1 (0.5)

287 (71.8) 110 (27.5) 3 (0.8)

C1: 0.85 C2: 0.15

0.353

Positive Null

116 (58.0) 84 (42.0)

117 (58.5) 83 (41.5)

233 (58.3) 167 (41.8)

Positive: 0.65 Null: 0.35

0.919

Positive Null

160 (80.0) 40 (20.0)

173 (86.5) 27 (13.5)

333 (83.3) 67 (16.8)

Positive: 0.59 Null: 0.41

0.082

Arg/Arg Arg/Trp Trp/Trp

172 (86.0) 27 (13.5) 1 (0.5)

158 (79.0) 42 (21.0) 0 (0.0)

330 (82.5) 69 (17.3) 1 (0.3)

Arg: 0.91 Trp: 0.09

0.088

Arg/Arg Arg/His His/His

158 (79.0) 37 (18.5) 5 (2.5)

160 (80.0) 37 (18.5) 3 (1.5)

318 (79.5) 74 (18.5) 8 (2.0)

Arg: 0.89 His: 0.11

0.774

Arg/Arg Arg/Gln Gln/Gln

89 (44.5) 90 (45.0) 21 (10.5)

89 (44.5) 83 (41.5) 28 (14.0)

178 (44.5) 173 (43.3) 49 (12.3)

Arg: 0.66 Gln: 0.34

0.526

Thr/Thr Thr/Met Met/Met

131 (65.5) 59 (29.5) 10 (5.0)

145 (72.5) 50 (25.0) 5 (2.5)

276 (69.0) 109 (27.3) 15 (3.8)

Thr: 0.83 Met: 0.17

0.210

CYP 2E1

GSTM1

GSTT1

XRCC1194

XRCC1280

XRCC1399

XRCC3241

a

Determinated by 2 -test.

included non-smokers and smokers, in relatively small numbers of subjects [4,41–44]. We evaluated an organic solvents complex mixture exposure, that resembles real car painters exposure conditions, since most diluents and thinners are mixtures containing more than 55% of organic solvents, at low doses and sometimes at higher levels than the regulated [45]. In spite of these low doses, our results demonstrate a significant CA frequency increase in exposed workers compared to referent. This result can be attributed to the additive, potentiality and synergistic properties of organic solvents in mixtures which can enhance the clastogenic effect as well [46,47]. The sensitivity of our study relies on an adequate population sample, enabling us to establish the genotoxic effect at presumably low dose exposure. CA in lymphocytes are an intermediate end point in carcinogenic progress and is the best validated cytogenetic biomarker to predict cancer risk [5,6,25,29]. Therefore our study gives a better cancer risk insight associated to organic solvents exposure in order to avoid future cancers. Our CA frequency increase results are consistent with previous cross-sectional studies, in out-door and in-door painters occupationally exposed to organic solvents [13,43,48,49]. We obtained a significant CTA increase but not a CSA increase. If we evaluate the frequency ratio between CTA and CSA in exposed and referents is 1.35 and 1.44, respectively; even though the absolute difference between the study groups is larger for CSA, only CTA was significantly different between exposed and referents. The rare occurrence of CSA caused by organic solvents may explain the difference. CTA arise predominantly during S-phase of the cultured lymphocytes, in vitro, in response to base oxidations and SSB induced by S-phase dependent clastogens in vivo, as organic solvents metabolites [5,50–52]. We also evidenced a significant CA frequency increase in workers with a minimum time of exposure (5 years) compared to referent CA baseline. Hazardous occupational exposure comes with DNA oxidative damage threat, as well it is possible that a reduction or deficient DNA repair can be evidenced with increased time of exposure, augmenting CA frequency [5]. We did not evidence a CA frequency

increase by ageing or alcohol intake on the whole study population. All our CA frequency results are comparable with other organic solvents studies that evaluated genotoxic effects [3,10,44,48,53]. Even though previous studies describe genotoxic effects by occupational organic solvents exposure, the non-inclusion of individual susceptibility biomarkers, as genetic polymorphisms, has limited the elucidation of individual differences, since these biomarkers influence the genotoxic outcomes [54]. Our study, reports a significant influence of CYP2E1 C1/C1 genotype on CA frequency increase in exposed workers. CYP2E1 binds to organic solvents macromolecules and forms electrophilic intermediates that can induce DNA damage. The non-base substitution in the 5 -flanking region could be maintaining the normal or up-regulating the gene expression. There are limited and controvertible reports of the functional consequences of CYP2E1 C1/C2 polymorphism, as individual susceptible biomarker, during organic solvents exposure; however, the present result disagrees with previous studies [54,55]. Garte et al. [56] reported that there are significant differences in the frequencies and transcription rates of metabolic genes, like CYP2E1, according to population stratification. We did not include molecular markers to determine this ancestry factor. However, it is possible that CYP2E1 polymorphisms have significant differences between ethnic sub-groups in our study population [57]. Boccia et al. [58] reported no association between CYP2E1 C1/C2 polymorphism and increased head–neck cancer risk. Exposed workers with GSTM1 null genotype had a statistical significant CA frequency increase compared to referent and exposed workers with GSTM1 positive genotype. As indicated by Norppa [25] the induced damage in subjects by organic solvents metabolites may increase in lymphocytes with GSTM1 null genotype that totally lack detoxification activity and may influence the amount of DNA damage. Previously, the GSTM1 null genotype has also been reported to increase cytogenetic biomarkers in epidemiologic studies evaluating exposure to organic solvents [13,59,60]. Carlsten et al. [61] reported an increased lung cancer risk

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Table 3 Poisson regression analysis of chromosome aberrations (CA): interaction of genetic factors and organic solvents exposure. Gene

Genotype

Exposure

Chromosome aberration frequency (mean ± S.D.)

Chromosome aberration frequency

Referent Exposed

2.52 ± 0.137 3.43 ± 0.188

Reference 2.15

1.21–1.53

<0.001*

C1/C1 C1/C2, C2/C2 C1/C1 C1/C2, C2/C2

Referent Referent Exposed Exposed

2.61 2.20 3.41 3.47

± ± ± ±

1.933 1.919 2.701 2.591

Reference 0.29 1.20 0.50

0.24–0.35 1.05–1.37 0.47–0.65

<0.001* <0.001* <0.001*

Positive Null Positive Null

Referent Referent Exposed Exposed

2.41 2.67 3.18 3.77

± ± ± ±

1.729 2.186 2.269 3.113

Reference 0.80 1.12 1.33

0.21–0.32 0.96–1.32 1.31–1.69

0.014* 0.163 <0.001*

Positive Null Positive Null

Referent Referent Exposed Exposed

2.51 2.55 3.45 3.30

± ± ± ±

1.978 1.768 2.559 3.291

Reference 0.26 1.49 0.22

0.21–0.32 1.31–1.69 0.18–0.28

<0.001* <0.001* <0.001*

Arg/Arg Arg/Trp, Trp/Trp Arg/Arg Arg/Trp, Trp/Trp

Referent Referent Exposed Exposed

2.53 2.43 3.39 3.57

± ± ± ±

1.992 1.550 2.713 2.481

Reference 0.16 0.35 1.23

0.12–0.21 0.29–0.42 1.08–1.40

<0.001* <0.001* <0.001*

Arg/Arg Arg/His, His/His Arg/Arg Arg/His, His/His

Referent Referent Exposed Exposed

2.47 2.67 3.52 3.05

± ± ± ±

2.002 1.663 2.671 2.621

Reference 0.29 1.44 0.31

0.23–0.35 1.26–1.64 0.26–0.38

<0.001* <0.001* <0.001*

Arg/Arg Arg/Gln, Gln/Gln Arg/Arg Arg/Gln, Gln/Gln

Referent Referent Exposed Exposed

2.49 2.53 3.44 3.41

± ± ± ±

1.804 2.040 2.709 2.634

Reference 1.26 1.37 1.71

1.06–1.51 1.16–1.64 1.44–2.01

0.009* <0.001* <0.001*

Thr/Thr Thr/Met, Met/Met Thr/Thr Thr/Met, Met/Met

Referent Referent Exposed Exposed

2.50 2.54 3.48 3.29

± ± ± ±

2.088 1.614 2.622 2.780

Reference 0.53 1.54 0.55

0.44–0.64 1.34–1.76 0.46–0.66

<0.001* <0.001* <0.001*

RR

95% CI

p-Value

CYP2E1

GSTM1

GSTT1

XRCC1194

XRCC1280

XRCC1399

XRCC3241

S.D.: standard deviation; RR: relative risk; CI: confidence intervals. Bold for significant effect of the genotype. * Significant difference, p < 0.05.

for GSTM1 null genotype. We determined a relationship between GSTT1 positive genotype and a CA frequency increase in exposed workers. Parl [62] suggested that combined conjugation activities of wild genotypes, for all glutathione-S-transferases, may lead to glutathione activity depletion and thereby become counterproductive [14]. Interestingly our results report a protective influence by GSTT1 null genotype, over CA frequency increase, without any combined effect of other glutathione-S-transferases polymorphisms in non-smokers. This protective influence corroborates Pitarque et al. [63] results during organic solvents exposure. Cajas-Salazar et al. [64] did report a CA frequency increase in subjects with combined GSTM1/GSTT1 positive genotype in a case–control study evaluating lung cancer risk. Whether exposure to relatively low levels of organic solvents could lead to glutathione pool depletion and explain CA frequency increase in subjects’ carriers of GSTM1 and GSTT1 positive genotypes needs further investigation [14]. Cancer susceptibility not only correlates with polymorphisms in xenobiotic-metabolism genes but also to polymorphisms in DNA repair genes, since they can influence DNA oxidative damage repair capacity and CA frequency increase [25,65]. In our study the exposed workers carriers of XRCC1194 Arg/Trp and Trp/Trp genotypes had statistically higher CA frequency compared to those with the XRCC1194 Arg/Arg wild genotype. The XRCC1194 Arg/Trp poly-

morphism is located in XRCC1 NLS domain, vicinal to other domains which mediate polymerase ␤ and APE1 interactions [20]. Therefore, this polymorphism may disturb the XRCC1 conformation, resulting in a decreased protein affinity or decreased DNA damage binding and ineffective DNA repair. There is limited information of XRCC1194 Arg/Trp polymorphism influence in workers exposed to organic solvents. Zhu et al. [20,66] and Zhang et al. [67] reported that this polymorphism significantly decreased DNA damage repair in vinyl chloride monomer and benzene exposed workers, respectively. Ramachandran et al. [68] reported an association between XRCC1194 Arg/Trp polymorphism and increased oral cancer risk. We report an association between the genotype XRCC1280 Arg/Arg with a significant CA frequency increase in exposed workers. The XRCC1 codon 280 is located close to domains that mediate interactions with PARP and DNA polymerase ␤ in BER [20]. It may be that the XRCC1280 Arg/His polymorphism has a higher affinity for protein interactions, facilitating a better oxidative damage repair caused by organic solvents [33]. Therefore, it is suggested that XRCC1280 Arg/Arg wild genotype may decrease repair capacity of SSB and influence an unstable CA formation. This present finding may not yet be clearly interpreted; unless further biochemical, population and clinical studies are carried out to confirm this finding. Tuimala et al. [69] encountered lower CA frequency for

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the XRCC1280 Arg/His polymorphism carriers during a case-control study in healthy Caucasians. There is limited knowledge about the functional consequences of XRCC3241 Thr/Met polymorphism [70]. In our study we obtained a significant CA frequency increase influenced by XRCC3241 Thr/Thr wild genotype. It might be that this wild position does represent a significant deficiency in the Rsd51 stabilization or DSB affinity decrease, during HR; however, more studies are needed to confirm this observation. On the contrary, other results do report a significant CA frequency increase influenced by XRCC3241 Thr/Met polymorphism during benzene exposure [67,71]. Our exposed workers carriers of XRCC1399 Arg/Gln polymorphism did not have a significant CA frequency increase compared to those exposed workers carriers of the wild XRCC1399 Arg/Arg genotype. It may be that XRCC1399 Arg/Gln polymorphism does not alter the affinity of XRCC1for PARP. Our finding is in disagreement with those of Zhang et al. [67], Chanvaivit et al. [72] and Kim et al. [49] who reported a significant CA frequency increase, by XRCC1399 Arg/Gln polymorphism, in benzene exposed workers. In conclusion, our results report a statistically significant CA frequency increase influenced by the polymorphic genotypes: GSTM1 null, XRCC1194 Arg/Trp, Trp/Trp and by the wild genotypes CYP2E1 C1/C1, GSTT1 positive, XRCC1280 Arg/Arg and XRCC3241 Thr/Thr in exposed workers. These unfavorable genotypes are individual susceptibility factors that have a functional impact and influence on potential cancer risk in organic solvents exposed workers. From these results it should be considered that if DNA repair pathways are able to correct induced damage, caused by ROS that escape detoxification systems, then the consequences of high-risk metabolic genotypes are less significant, since finally successful DNA repair pathways maintain chromosome integrity. This molecular epidemiology, cross-sectional type study, is the most extensive investigating genotoxic effects of occupational exposure to organic solvents mixtures in low levels, influenced by individual susceptibility biomarkers and analyzed in an adequate population sample. The present study suggests that occupational exposure to organic solvents is associated with chromosome damage in surrogated and possible target tissues and higher cancer risk that may be influenced by dosage, time of exposure and susceptible genotypes of xenobiotic-metabolism and DNA repair genes. Further studies, evaluating occupational exposure to organic solvents, should include all our factors and additional ones, such as: studied and unstudied polymorphisms that are still not well understood, population stratification and exposure biomarkers, in order to better characterize exposure response in inter- and intra-individual susceptible levels [73]. Our results need to be tested by gold standard prospective large-scale studies to provide more robust results on gene–gene and gene–environment interaction to obtain a better understanding of risk assessment influenced by genetic polymorphisms [74]. Our study will provide tools for direct intervention to prevent genetic damage risk in exposed organic solvents workers and will contribute to future follow-up of the predictive value of these susceptibility biomarkers as individual cancer risk factors [75]. Also our results could be incorporated in establishing surveillance, prevention strategies and policies, especially in countries with limited organic solvent regulations, with large car painters population paying special attention to exposed workers carrying “unfavorable” genotypes simultaneously [76]. As suggested by Krajinovic et al. [77] only one polymorphism may affect organic solvents toxicity, but the combined influence of multiple polymorphisms of xenobiotic-metabolism and DNA repair genes may increase, in a higher proportion, CA frequency, cancer risk and other diseases. The present study informed to all exposed participants about the global study results and emphasized the need to use protection devices and develop safety programs.

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Conflict of interest statement None. Acknowledgments This study was supported by COLCIENCIAS grant 295/1103-0418263/2005 and by Vicerrectoría de Investigaciones Universidad del Cauca, Colombia. We are grateful with the study population, nurse Elsa Betty Velasco for her valuate participation in collecting the blood samples. We really appreciate Biologists Luisa Fernanda ˜ Escobar, Ingrid Reyes, Adriana Munoz, Luis Alfonso Ruíz and Maria Belén Trujillo for their excellent and invaluable contribution and Carolyn Wenholz for her English review. References [1] IARC, Occupational exposures in paint manufacture and painting, in: IARC (Ed.), Monographs in Evaluation Carcinogenic Risk in Humans, International Agency for the Research on Cancer, Lyon, 1989. [2] E. Lynge, A. Anttila, K. Hemminki, Organic solvents and cancer, Cancer Causes Control 8 (1997) 406–419. [3] A. Bogadi-Sare, V. Brumen, R. Turk, V. Karacic, M. Zavalic, Genotoxic effects in workers exposed to benzene: with special reference to exposure biomarkers and confounding factors, Ind. Health 35 (1997) 367–373. [4] H. Aksoy, S. Yilmaz, M. Celik, D. Yuzbasioglu, F. Unal, Genotoxicity study in lymphocytes of offset printing workers, J. Appl. Toxicol. 26 (2006) 10–15. [5] S. Bonassi, L. Hagmar, U. Stromberg, A.H. Montagud, H. Tinnerberg, A. Forni, P. Heikkila, S. Wanders, P. Wilhardt, I.L. Hansteen, L.E. Knudsen, H. Norppa, Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. European Study Group on Cytogenetic Biomarkers and Health, Cancer Res. 60 (2000) 1619–1625. [6] L. Hagmar, U. Stromberg, S. Bonassi, I.L. Hansteen, L.E. Knudsen, C. Lindholm, H. Norppa, Impact of types of lymphocyte chromosomal aberrations on human cancer risk: results from Nordic and Italian cohorts, Cancer Res. 64 (2004) 2258–2263. [7] J.L. Valentine, S.S.T. Lee, M.J. Seaton, B. Asgharian, G. Farris, J.C. Corton, F.J. Gonzalez, M.A. Medinsky, Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression, Toxicol. Appl. Pharmacol. 141 (1996) 205–213. [8] S. Hayashi, J. Watanabe, K. Kawajiri, Genetic polymorphisms in the 5 -flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene, J. Biochem. (Tokyo) 110 (1991) 559–565. [9] N. Cajas-Salazar, C.H. Sierra-Torres, S.A. Salama, J.B. Zwischenberger, W.W. Au, Combined effect of MPO, GSTM1 and GSTT1 polymorphisms on chromosome aberrations and lung cancer risk, Int. J. Hyg. Environ. Health 206 (2003) 473–483. [10] A. Pandey, M. Bajpayee, D. Parmar, R. Kumar, S. Rastogi, N. Mathur, P. Thorning, M. de Matas, Q. Shao, D. Anderson, Multipronged evaluation of genotoxicity in Indian petrol-pump workers, Environ. Mol. Mutagen. 49 (2008) 000–10. [11] D. Ross, The role of metabolism and specific metabolites in benzene-induced toxicity: evidence and issues, J. Toxicol. Environ. Health A 61 (2000) 357–372. [12] L. Zhang, M.L. Robertson, P. Kolachana, A.J. Davison, M.T. Smith, Benzene metabolite, 1,2,4-benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells, Environ. Mol. Mutagen. 21 (1993) 339–348. [13] S.Y. Kim, J.K. Choi, Y.H. Cho, E.J. Chung, D. Paek, H.W. Chung, Chromosomal aberrations in workers exposed to low levels of benzene: association with genetic polymorphisms, Pharmacogenetics 14 (2004) 453–463. [14] M. Kirsch-Volders, R.A. Mateuca, M. Roelants, A. Tremp, E. Zeiger, S. Bonassi, N. Holland, W.P. Chang, P.V. Aka, M. Deboeck, L. Godderis, V. Haufroid, H. Ishikawa, B. Laffon, R. Marcos, L. Migliore, H. Norppa, J.P. Teixeira, A. Zijno, M. Fenech, The effects of GSTM1 and GSTT1 polymorphisms on micronucleus frequencies in human lymphocytes in vivo, Cancer Epidemiol. Biomark. Prev. 15 (2006) 1038–1042. [15] S.E. Pemble, K.R. Schroeder, S.R. Spencer, D.J. Meyer, E. Hallier, H.M. Bolt, B. Ketterer, J.B. Taylor, Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism, Biochem. J. 300 (Pt 1) (1994) 271–276. [16] J. da Silva, C.R. Moraes, V.D. Heuser, V.M. Andrade, F.R. Silva, K. Kvitko, V. Emmel, P. Rohr, D.L. Bordin, A.C. Andreazza, M. Salvador, J.A. Henriques, B. Erdtmann, Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes, Mutagenesis 23 (2008) 415–422. [17] D.M. Wilson III, L.H. Thompson, Life without DNA repair, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 12754–12757. [18] M.A. Brenneman, B.M. Wagener, C.A. Miller, C. Allen, J.A. Nickoloff, XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination, Mol. Cell 10 (2002) 387–395. [19] R. Brem, J. Hall, XRCC1 is required for DNA single-strand break repair in human cells, Nucleic Acids Res. 33 (2005) 2512–2520.

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