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Mutagenesis Advance Access originally published online on October 18, 2005
Mutagenesis 2005 20(6):425-432; doi:10.1093/mutage/gei058
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Risk assessment of welders using analysis of eight metals by ICP-MS in blood and urine and DNA damage evaluation by the comet and micronucleus assays; influence of XRCC1 and XRCC3 polymorphisms

G. Iarmarcovai1,2,*, I. Sari-Minodier1,2, F. Chaspoul1, C. Botta1,2, M. De Méo1, T. Orsière1, J.L. Bergé-Lefranc1, P. Gallice1 and A. Botta1,2

1Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA 1784; IFR PMSE 112), Facultés de Médecine et de Pharmacie, Université de la Méditerranée, Marseille, France and 2Laboratoire de Biotoxicologie (UF 0756), Assistance Publique des Hôpitaux de Marseille, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aims of the present study were to assess the occupational risk of welders using analysis of metals in biological fluids, DNA damage evaluation by complementary genotoxic endpoints and the incidence of polymorphisms in DNA repair genes. A biomonitoring study was conducted that included biometrology (blood and urinary concentrations of aluminium, cadmium, chromium, cobalt, lead, manganese, nickel, zinc by ICP-MS), comet and cytokinesis-block micronucleus assays in peripheral lymphocytes and genetic polymorphisms of XRCC1 (p.Arg399Gln) and XRCC3 (p.Thr241Met). This study included 60 male welders divided into two groups: group 1 working without any collective protection device and group 2 equipped with smoke extraction systems. A control group (n = 30) was also included in the study. Higher chromium, lead and nickel blood and urinary concentrations were detected in the two groups of welders compared to controls. Statistically differences between welders of group 1 and group 2 were found for blood concentration of cobalt and urinary concentrations of aluminium, chromium, lead and nickel. The alkaline comet assay revealed that welders had a significant increase of OTM{chi}2 distribution at the end of a work week compared to the beginning; a significant induction of DNA strand breaks at the end of the week was observed in 20 welders out of 30. The cytokinesis-block micronucleus assay showed that welders of group 1 had a higher frequency of chromosomal damage than controls. The XRCC1 variant allele coding Gln amino acid at position 399 was found to be associated with a higher number of DNA breaks as revealed by the comet assay. Increased metal concentrations in biological fluids, DNA breaks and chromosomal damage in lymphocytes emphasized the need to develop safety programmes for welders.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Occupational exposure to welding fumes (a complex mixture of gases and oxides or salts of metals) has been associated with an increased risk of lung cancer (1Go). However, the causal relationship is impaired by exposure to other agents and the lack of a clear dose–response. Welding fumes are classified, according to the International Agency for Research on Cancer (IARC), into group 2B (possibly carcinogenic to humans) (2Go). An effective biological surveillance of welders should take into consideration the understanding of the mechanisms involved in the carcinogenic process and should be based on the genotoxicity of metals from welding fumes. Several studies have reported the induction of DNA damage and chromosome/genome mutations by heavy metals or welding fumes using the comet and the cytokinesis-block micronucleus (CBMN) assays (3GoGoGoGo–7Go).

Metals could interact directly with DNA and DNA replication, thus causing DNA damage (DNA base modifications, depurination, inter- and intra-molecular crosslinking of DNA and proteins, DNA strand breaks, chromosome rearrangements). Moreover, particle-induced inflammation in the lungs has been shown to cause the release of reactive oxygen species (ROS) by macrophages. Transition metals on the particle surface could also generate ROS through the Fenton reaction and lead to the formation of oxidative DNA damage (8Go). Other possible underlying mechanisms involve various kinds of active oxygen and several free radical species arising from metal-catalysed redox reactions. General metal-mediated pathogenic effects (increase of inflammation response, inhibition of cellular antioxidant defenses, lipid peroxidation, inhibition of DNA repair) might also contribute to mutations, changes in gene expression and modification of the cell cycle. Under conditions of sustained occupational exposure, these mechanisms contribute to carcinogenic processes. It has been reported that two of the most powerful human metal carcinogens chromium and nickel were were related to their strong oxidative capacities (7Go,9GoGo–11Go). Similar capacities have also been suggested for aluminium, cadmium, cobalt, manganese and lead (12GoGoGoGo–16Go). Nevertheless, DNA damage caused by welding fumes has been shown to differ from single metal exposure due to either the additive or synergistic effects of multiple genotoxic compounds (17Go).

Determination of metal concentrations in body fluids is usually used as a biomarker of exposure. Inductively coupled plasma-mass spectrometry (ICP-MS) is a suitable technique to monitor metals during occupational exposure with a sensitive and rapid screening method (18Go).

Complementing biometrology, the assessment of early biological effects (e.g. oxidized bases, DNA strand breaks, chromosome loss) induced by metal compounds from welding fumes can be performed using genotoxic endpoints. The comet assay, also known as the alkaline single cell gel electrophoresis assay, quantifies initial DNA breakage and can detect in alkaline conditions DNA single strand breaks (SSBs), alkaline labile sites and excision repair processes in individual cells. Chromosome breakage (double strand breaks, DSBs) and chromosome loss (chromosome lagging during anaphase separation) can be assessed by the CBMN assay. Thus, micronuclei induction reflects clastogenic and/or aneugenic events. The major pathway eliminating DNA base damage and helix distortion is the excision repair pathway, subdivided into nucleotide excision repair (NER) and base excision repair (BER). If the mutagenic potentials of metal compounds are rather weak, in contrast, they exert pronounced comutagenic effects, which may be explained by the inhibition of different DNA repair systems (19Go).

Genetic polymorphism might be involved in inter-individual variations of DNA repair processes. Indeed, DNA damage measured by the comet and CBMN assays could be influenced by genetic polymorphism. The choice of two DNA repair genes, X-ray repair cross-complementing gene 1 (XRCC1) and X-ray repair cross-complementing gene 3 (XRCC3), was dictated by the type of DNA damage expected from exposure to welding fumes. XRCC1 is a key factor in the BER pathway (20Go). XRCC3 is involved in the homologous DSB repair pathway, it directly interacts with and stabilizes Rad51 (21Go). The XRCC1 variant allele coding Gln amino acid at position 399 is associated with cancers of the head and neck, breast, lung and colon/rectum; a positive association between the XRCC3 variant allele coding Met amino acid at position 241 is shown in bladder cancer, melanoma skin cancer, gastric cancer (22Go).

The aims of the present study were to assess the occupational risk of welders using analysis of metals in blood and urine, DNA damage evaluation by genotoxic endpoints and the incidence of genetic polymorphism. We evaluated occupational exposure of welders by assessing blood and urinary concentrations of eight metals including aluminium, cadmium, chromium, cobalt, lead, manganese, nickel, zinc using the ICP-MS. Additionally, we aimed to investigate the potential early biological effects of welding fumes. To this end, we performed two complementary genotoxicity tests on peripheral lymphocytes; DNA breakage was measured with the alkaline comet assay and induction of chromosome/genome mutations was monitored with the CBMN assay. We also determined the influence of sequence variation in DNA repair genes such as XRCC1 (p.Arg399Gln) and XRCC3 (p.Thr241Met) on these genotoxicity biomarkers.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects
The study was carried out during the period October 2004–January 2005. Male welders (n = 60) were recruited from 36 workshops in the building trade in the south of France and divided into two groups: group 1 (n = 27) working in areas without any collective protection device and group 2 (n = 33) working in places equipped with smoke extraction systems. The control group (n = 30) was selected from the working population with no history of occupational exposure to welding fumes or any known physical or chemical agent in the workplace. Controls were recruited from general or administration services (office workers). Controls did not differ from welders of group 1 in gender, age and smoking habits.

For each subject selected for the study, relevant data on personal or familial medical history, smoking habits, alcohol consumption, drug intake, recent X-ray diagnostic examination, excessive physical exercise or fever during the previous week end were elicited via a standard questionnaire prior to blood and urinary samples. We also ensured that all the welders had been working the month before sampling. Subjects classified as non-smokers were never smokers or former smokers who had stopped to smoke for at least 1 year.

Exclusion criteria for both exposed and control subjects were history of radiotherapy and/or chemotherapy, use of therapeutic drugs known to be mutagenic or toxic for the reproduction.

All subjects were informed of the objective of the study and gave their written informed consent. The local ethics committee approved the research procedures used in this study.

Work description
The work processes of selected welders consisted in welding, torch cutting, grinding, scraping and painting on mild steel (containing iron, carbon, cadmium, manganese) and galvanized steel (covered with a layer of zinc metal). Stainless steel (with higher concentrations of chromium and nickel) or aluminium represented <5% of the welded metals. Welders used at least one of the following processes: manual arc welding with covered electrode (MMA), semi-automatic gas-shielded bare wire metal arc welding (MIG-MAG), manual arc welding with a non-consumable tungsten electrode under inert gas shielding (TIG).

Blood and urinary samples
Blood samples were collected between 7 and 8 a.m. before work shifts and outside the working facilities. Blood samples were obtained by venipuncture on Monday (at the beginning of a work week) from all subjects for metal analysis. The comet and CBMN assays were carried out only on welders of group 1 and controls. Blood samples were also collected on Friday (at the end of a work week) from group 1 and group 2 welders for metal analysis and only from group 1 welders for comet assay. Blood for genotyping was collected on Monday from all subjects.

Spot urine samples were obtained from all subjects in the morning on Monday (pre-shift) and in the evening on Thursday (post-shift) only from group 1 and group 2 welders for metal analysis. Urine creatinine analyses were performed by the standardized Jaffe reaction (23Go).

All samples were kept on ice and in the dark and were processed within 6 h.

ICP-MS
Following homogenization, aliquots of venous blood (0.5 ml) and of urine (1 ml) were diluted one-twentieth and one-tenth, respectively, with a nitric acid (Suprapur, 65%, Merck, Darmstadt, Germany) solution (1% v/v), containing internal standards (scandium, germanium, yttrium, terbium) (Agilent, DE) at a final concentration of 25 µg/l. For determining metal content in whole blood and urine, an HP 4500 ICP-MS was used (Yokogawa Analytical Systems Inc.). Metal analysis by ICP-MS was performed as previously described (24Go,25Go). For each analysis, mass of interest (m/z) was: aluminium, 27; cadmium, 111; chromium, 50/52/53; cobalt, 59; lead, 206/207/208; manganese, 55; nickel, 58/60; zinc, 64/66/68. Height point calibration curves were created by dilution of multielement solution (SCP Sciences, Baie d'Urfé, Canada) in the nitric acid solution with internal standards. Quantization was performed using software Hewlett Packard Chemstation (Palo Alto, CA).

Alkaline comet assay
The alkaline comet assay was performed on isolated lymphocytes under alkaline conditions, as previously described by Singh et al. with slight modifications (26Go,27Go). The assay was carried out under yellow light to prevent any additional damage that could be induced by natural light. Briefly, an aliquot 20 µl of blood was mixed with 1 ml of RPMI (Eurobio, Paris, France) supplemented with 10% foetal calf serum (FCS; Eurobio, Paris, France) and L-glutamate. Then, 100 µl of lymphocytes separation medium (LSM; Eurobio, Paris, France) was added and lymphocytes were separated by centrifugation at 3000 g, for 5 min. Afterwards, 100 µl of the interface RPMI/LSM, containing lymphocytes, was removed and suspended again in 1 ml RPMI/FCS/glutamate. The suspension was centrifuged again at 3000 g for 5 min and the cells were mixed with 75 µl volume of 0.5% Low Melting Point agarose (Promega, Charbonnières, France) in phosphate-buffered saline (PBS) without Ca2+ and Mg2+ at 37°C. The cell suspension was placed on microscope slides previously coated with 2% agarose in Ca2+ and Mg2+ free PBS and layered with 85 µl of solidified agarose gel (0.8% in PBS without Ca2+ and Mg2+). A third layer of 75 µl of 0.5% Low Melting Point agarose was poured onto the slides and allowed to solidify. Then, the slides were immersed in cold lysis (Sigma Chemicals, Saint Quentin Fallavier, France) solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% N-lauryl-sarcosinate, pH 10–12, 1% Triton X-100 and 10% dimethyl sulphoxide) for 90 min at 4°C. After lysis, the slides were placed in a horizontal gel electrophoresis tank (Fisher) and the DNA was allowed to unwind in freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA, pH > 13) for 20 min at room temperature. Electrophoresis was conducted for 20 min at 25 V adjusted to 300 mA. The slides were then washed three times with a cold neutralizing buffer (0.4 M Tris–HCl, pH 7.5) for 5 min and dehydrated in pure methanol. The slides were kept in a dry atmosphere chamber until measurement. To visualize and analyse DNA damage, slides were stained with ethidium bromide (2 µg/ml in MilliQ water).

Then, the slides were observed at 250x magnification using a BH2-RFL fluorescence microscope (Olympus Optical Co., Tokyo, Japan) equipped with a 20BG-W2 dichroic mirror (band-pass filter, 515–560 nm; long-pass filter, 590 nm) and a D plan-Apo 20xUV objective (oil immersion). Image analysis was performed using the Fenestra Komet software (Kinetics Imaging, Liverpool, UK, version 3.1) on 100 randomly selected cells from duplicate slides per individual. DNA damage was quantified by the increase of Olive tail moment (OTM). Normalized frequencies of distribution of OTM were calculated using 40 OTM classes between minimal and maximal values for each set of data. A non-linear regression analysis was performed with the Table Curve 2D software (Jandel Scientific Software, version 5.0) on the OTM distribution frequencies by using a {chi}2 function. The calculated degree of freedom (n) of the function has been previously shown to be a quantitative parameter of the level of DNA damage in samples and was named OTM{chi}2 (28Go,29Go). OTM{chi}2 was expressed in arbitrary units and used as the main parameter to assess levels of DNA damage throughout this study.

CBMN assay
The CBMN assay was performed as previously described by Sari-Minodier et al. with slight modifications (30Go). Cultures were carried out by adding 0.7 ml of whole blood to 9.3 ml of X Vivo 10TM (serum-free medium with gentamicine, Bio WhittakerTM, Walkersville, MD) supplemented with heparin (50 U/ml) and 1% PHA (Life technologiesTM, Grand Island, NY) for 72 h in a humidified CO2 incubator at 37°C. Cytochalasin B (Sigma Chemicals Company, St Louis, MO) was added to the culture (6 µg/ml) 44 h after PHA stimulation. At 72 h, the cells were subjected to a mild hypotonic treatment (1 min in KCl 0.075 M), fixed twice with methanol/acetic acid (3/1), then smeared on precleaned microscope slides and air dried. Staining was performed with 5% Giemsa in milli-Q water for 10 min. For the scoring of micronuclei, stained slides were then coded and scored by light microscopy at 1000x magnification. For each slide, 1000 Giemsa-stained binucleated lymphocytes with a well-preserved cytoplasm were scored for the presence of micronuclei according to criteria previously described (31Go). Micronuclei were expressed in terms of binucleated micronucleated cell rate (BMCR) per 1000 cells.

Genetic polymorphism
The DNA polymorphisms of XRCC1 and XRCC3 gene were detected by sequencing amplified PCR fragments using PCR and sequencing primers described in http://snp500cancer.nci.nih.gov. In the present report, DNA sequence variation was named according to the recommendations of den Dunnen and Antonarakis (32Go,33Go). We used the terms p.Arg399Gln of XRCC1 and p.Thr241Met of XRCC3 because an amino acid change is always based on protein sequence, thus the ‘p.’ prefix is implied. The guanosine to adenine substitution in XRCC1 gene leading to a Arg399Gln amino acid change and the cytidine to thymidine substitution in XRCC3 gene leading to a Thr241Met amino acid change were monitored. Sequence analysis was performed on a CEQ 8000 Beckman sequencing device.

Statistical analysis
Differences between donor groups were calculated with the non-parametric Mann–Whitney U-test and the {chi}2 test for quantitative and qualitative variables, respectively.

Differences of metal concentrations and OTM{chi}2 distribution between the beginning and the end of a work week were assessed by the non-parametric Wilcoxon W-rank sum test (median comparison). The association between quantitative variables was tested using nonparametric Spearman's rank correlation analysis. The statistical analysis included multiple regression analyses (backward multivariate regression) to examine the possible influence of several independent variables (smoking and drinking habits, age, exposure) on the genotoxic endpoints (OTM{chi}2, BMCR). Statistical significance was set at P < 0.05. Statistical analysis was performed with SPSS 10.1 program for Windows (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
General characteristics of the subjects
Welders were exposed from 0.5 to 45 years. They were welding on average 14.6 ± 6.9 h a week for welders of group 1 and 13.5 ± 6.1 h a week for group 2. The three groups studied had similar general characteristics, such as age (group 1: 43.9 ± 12.6 years; group 2: 44.4 ± 7.8 years; controls: 43.1 ± 11.0 years), percentage of smokers (group 1: 37%, group 2: 33%, controls: 53%).

Metal concentrations
Metal concentrations in blood (Table I) and urine (Table II) were determined.


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  		Table I.. Metal concentrations in blood (µg/l)

 

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  		Table II.. Metal concentrations in urine (µg/g creatinine)

 
Blood concentrations of aluminium and zinc and urinary concentrations of cadmium and manganese were not significantly different within controls, group 1 or group 2. Blood and urinary concentrations of chromium, lead and nickel were significantly higher in the two groups of welders compared to controls. Blood concentrations of cadmium, cobalt and manganese and urinary concentrations of aluminium, cobalt and zinc were significantly higher in welders of group 1 than controls. Blood concentrations of cadmium, cobalt and urinary concentrations of zinc were significantly higher in welders of group 2 than controls.

Blood concentrations of cobalt at the beginning of the week were found to be higher in group 1 than in group 2. Aluminium and lead urinary concentrations at the beginning of the week and chromium and nickel urinary concentrations at the end of the week were higher in group 1 than group 2.

No significant difference was observed in blood metal concentrations between the beginning and the end of the week. Manganese urinary concentrations appeared to increase between the beginning and the end of the week in group 1 (P = 0.002) and in group 2 (P = 0.006). Chromium urinary concentrations increased between the beginning and the end of the week only in group 2 (P = 0.031).

Mean metal concentrations did not differ between smokers and non-smokers in exposed or control populations, but cadmium was significantly higher in smokers (data not shown).

Alkaline comet assay
DNA damage was studied in a total of 30 welders (26 welders of group 1 and 4 welders of group 2) at the beginning and at the end of a work week, and in 22 controls only at the beginning of the week. The results are given in Figure 1A and B. No significant difference was observed in OTM{chi}2 means between controls and welders at the beginning of the week. Welders had a significant increase of OTM{chi}2 distribution at the end of the week when compared to the beginning (4.54 ± 1.68 versus 2.84 ± 0.75; P < 0.001). The alkaline comet assay revealed an induction of DNA strand breaks between the beginning and the end of the week in 20 welders out of 30.



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  		Fig. 1.. Distribution of OTM medians (A) and OTM{chi}2 (B) of the control group (BWControl) and the exposed group at the beginning (BWExposed) and at the end of a work week (EWExposed). Each box represents the inter-quartile range of values, with the horizontal line indicating the median value and the vertical line indicating the range of values that fall between the 10th and 90th percentile. Outliers are marked as crosses, means as squares. Difference between control group and exposed group at the beginning of the week was not statistically significant (Mann–Whitney U- test). Difference between exposed group at the beginning of the week and exposed group at the end of the week was statistically significant (Wilcoxon W test; ***P < 0.001).

 
OTM{chi}2 was not correlated with time of welding during the week. No significant difference in OTM{chi}2 distribution was observed between smokers and non-smokers or between alcohol and no-alcohol drinkers (Table III).


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  		Table III.. Multiple linear regression analysis of genotoxic endpoints

 
CBMN assay
The CBMN assay was performed in 27 welders (group 1) and in 30 matched-controls. The average BMCR was significantly higher in the welders than in the controls as shown in Figure 2 (6.3{per thousand} ± 2.9 versus 4.7{per thousand} ± 1.8; P = 0.03). Smoking or drinking habits did not have a significant effect on BMCR (Table III). BCMR was not correlated with age or duration of occupational exposure to welding fumes.



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  		Fig. 2.. Distribution of BMCR in the control group and the exposed group. For details, see caption of Figure 1. Significant statistical difference between exposed and control groups by the Mann–Whitney U- test; *P < 0.05.

 
Genetic polymorphism
Genotypes of XRCC1 and XRCC3 were analysed to study a possible association between the distribution of allele (Table IV) and genotoxic endpoints. Allele frequencies were similar to those observed in other Caucasian population (22Go). When comparing workers homozygous for the variant XRCC3 sequence (Met/Met p.Thr241Met) and heterozygous or homozygous for the wild-type sequence, the mean values of OTM{chi}2 and BMCR were not significantly different within controls, exposed or total populations. However, by compiling data on control and exposed populations, with regard to XRCC1 polymorphism, OTM{chi}2 distribution of subjects bearing Gln/Gln p.Arg399Gln sequence was found higher than OTM{chi}2 distribution of subjects Gln/Arg or Arg/Arg (3.08 ± 0.77 versus 2.62 ± 0.57; P = 0.047). These results suggest a protective effect of Arg/Gln or Arg/Arg phenotype. No other significant difference between the XRCC1 genotypes was observed.


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  		Table IV.. Distribution of XRCC1 and XRCC3 genotypes in the study population

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The strengths of the present study were (i) the similar characteristics of welders working in areas with or without protective devices, (ii) the recruitment of welders from several workshops making the study population more representative of different exposure conditions and (iii) the assessment of DNA damage induction during the work week. Furthermore, a very strict collection protocol for the alkaline comet assay was implemented with sampling in the morning before work shifts to avoid oxidative stress that could be linked to intense physical activities (34Go,35Go).

To evaluate the exposure to metals, concentrations of eight metals in blood and urine of the welders were determined. Of the elements requested, chromium was the most difficult to measure using ICP-MS because of spectral interferences in blood as previously reported (24Go). The present study revealed an occupational exposure particularly to chromium, lead and nickel. The mean levels of these metals in exposed groups were higher than in controls as reported in several studies on welders (5Go,7Go,36GoGo–38Go). Blood and urinary concentrations of chromium, cobalt, lead and nickel were found to be higher in the welders working in areas without any protection device than those working with smoke extraction systems. Nevertheless, the differences were not all statistically significant. A possible explanation may be the relatively limited number of subjects, the recent equipment of workplaces with smoke extraction systems (mean of 2 years), the delayed excretion metabolism of metals (39Go). Our data indicated that urinary manganese and chromium concentrations appeared to increase between the beginning and the end of the week and/or during the work process. This underlines the importance of sampling time and the number of samples per individual.

Workers without any collective protection device were investigated for genotoxic effects of welding using complementary endpoints (comet and CBMN assays); results obtained from both assays highlighted DNA lesions in the peripheral lymphocytes of the exposed subjects. DNA lesions and cytogenetic aberrations in lymphocytes are surrogate endpoints in surrogate cells, presumed to represent genetic alterations involved in carcinogenesis of target tissues. Indeed, results of genotoxic endpoints might also be explained by individual susceptibility factors such as genetic polymorphism affecting genomic stability (DNA repair, folate metabolism) and carcinogen metabolism (40Go). Smoking did not have a significant effect on DNA damage in the present study. According to Faust et al., only nine human biomonitoring studies with the alkaline comet assay have found a significant relationship between DNA damage and smoking habits (41Go). The analysis from the Human MicroNucleus project has shown that smokers do not experience an overall increase in micronuclei frequency, although when the interaction with occupational exposure is taken into account, heavy smokers were the only group showing a significant increase in genotoxic damage as measured by the CBMN assay in lymphocytes (42Go). In our study, the subgroup of heavy smokers (>30 cigarettes per day) could not be specifically evaluated because it was not large enough to satisfy statistical requirements. Micronutrients (vitamins and minerals) are required as cofactors for enzymes or as part of the structure of proteins (metallo-enzymes) involved in DNA synthesis and repair, prevention of oxidative damage as well as maintenance methylation of DNA. Thus micronutrient deficiency (e.g. antioxidant such as zinc, vitamins C and E or folate, vitamine B12) can also cause genome damage (43Go,44Go). In our study, due to the relatively small sample size, we did not determine the impact of diet and vitamins status on the MN index (44Go).

Our results are consistent with previous studies (5Go,7Go). The alkaline comet assay revealed an induction of DNA strand breaks during the work week in 20 welders out of 30; these 20 welders did not differ from the others for smoking habits, alcohol consumption, duration of exposure to welding fumes, time of welding or physical exercise. This significant increase of DNA strand breaks during the work week may be related to their occupational exposure to welding fumes or to oxidative stress that could be linked to physical activities included in the work process of welders (34Go,35Go). In the comet assay, OTM{chi}2 represents DNA strand breaks consecutive to either direct effect of welding fumes or indirect effect linked to DNA repair processes (17Go,45Go). Hartwig has reported that some heavy metals (such as cadmium, cobalt and nickel) may not only be genotoxic, but also inhibit DNA repair systems including NER and BER at low, non-cytotoxic concentrations (46Go). Thus, DNA damage in the exposed individuals might also be a secondary event due to DNA repair inhibition, since a decreased repair capacity will enhance susceptibility to direct DNA strand breaks as previously suggested (47Go). The fact that the OTM{chi}2 means are similar between controls and welders at the beginning of the week is consistent with the reversibility of the DNA lesions. Further experiments are needed to discriminate between direct and indirect DNA breaks or to assess DNA repair activities. Welders showed a higher frequency of micronuclei (a consequence of chromosomal aberrations and/or spindle disturbances) compared to controls. Several studies have indicated the clastogenic and aneugenic ability of different metal salts (4Go,48GoGoGo–51Go). Chromosome breakage (clastogenic events) is caused by DSB induced either directly or by the conversion of a SSB into a DSB after cell replication. Alternatively a significant proportion of induced micronuclei may be the result of aneugenic effects of metals. Micronuclei result either from lesions/adducts at the level of DNA or chromosomes, or at the level of proteins directly or indirectly involved in chromosome segregation (e.g. tubulin). There are two classical processes that lead to aneuploidy: chromosome loss and chromosome non-disjunction (52Go).

This study takes into consideration the involvement of genetic polymorphisms in DNA repair genes in genotoxic effects evaluated by the comet and CBMN assays. In this study, we focused on XRCC1 and XRCC3, which play an important part in the BER pathway of DNA and in the DSB repair pathway, respectively. DNA polymorphism of other products involved in DNA repair, such as hOGG1, should be considered in further investigations (6Go). At the present time, our data indicate that, in the total population, subjects homozygous for the XRCC1 variant allele coding Gln amino acid at position 399 showed a higher number of DNA breaks as revealed by the comet assay. The influence of polymorphism in DNA damage might be due to its location in the hinge region of XRCC1 gene, which may alter the capacity of XRCC1 to interact with several acting enzymes and then decrease the efficiency of BER repair (6Go,53Go). Recently, the effect of three XRCC1 polymorphisms on DNA damage and DNA repair in EM9 cells using the CBMN assay was reported. It had been observed that only the Arg399Gln polymorphism influenced the ability of XRCC1 to repair DNA (54Go). The influence of XRCC1 polymorphism on BMCR was not statistically significant, in contrast to its contribution in the comet assay, possibly reflecting that most of the transient SSB due to DNA repair processes did not induce micronuclei as previously suggested by Mateuca et al. (6Go).

Higher chromium, lead and nickel blood and urinary concentrations have been observed in welders compared to controls. We assessed the relation between genotoxic effects and exposure to welding fumes. DNA breaks increased at the end of the work week and chromosomal damage was higher in welders than in controls in peripheral lymphocytes. These genotoxicity endpoints can be considered as intermediate biomarker endpoints in relation to cancer risk (55Go). Genotoxic endpoints in complement with determination of genetic polymorphism could be incorporated in establishing surveillance for occupational prevention. The present study emphasizes the need to inform workers exposed to known or suspected carcinogens and to develop safety programs.


   Acknowledgments
 
We acknowledge the occupational physicians from APAMETRA BTP (Dr Fassi, Dr Pittilloni, Dr Vigneron, Dr Braunstein) for their collaboration. We thank Chantal Bideau, Danielle Iniesta, Laetitia Decome and Jocelyne Pompili for technical assistance, André Lanteaume for statistical advice and the Direction Régionale du Travail, de l'Emploi et de la Formation Professionnelle (PACA, France) for financial support (contract grant number 93CA 2004-004.0).


   Notes
 
* To whom correspondence should be addressed at: EA 1784, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France. Tel: +33 04 91 32 45 71; Fax: +33 04 91 32 45 72; Email: gwenaelle.iarmarcovai{at}medecine.univ-mrs.fr


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    1. Moulin,J.J. (1997) A meta-analysis of epidemiologic studies of lung cancer in welders. Scand. J. Work Environ. Health, 23, 104–113.[ISI][Medline]

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Received on August 5, 2005; revised on September 21, 2005; accepted on September 22, 2005.


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