Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA

Pavel Vodicka1,11, Rajiv Kumar2,3, Rudolf Stetina4, Somali Sanyal2, Pavel Soucek5, Vincent Haufroid6, Maria Dusinska7, Miroslava Kuricova7, Maria Zamecnikova8, Ludovit Musak9, Jana Buchancova9, Hannu Norppa10, Ari Hirvonen10, Ludmila Vodickova5, Alessio Naccarati1, Zora Matousu1 and Kari Hemminki2,3

1 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 14200 Prague 4, Czech Republic, 2 Department of Bioscience at Novum, Karolinska Institute, Huddinge, Sweden, 3 German Cancer Institute, Heidelberg, Germany, 4 Purkynje Military Medical Academy, Hradec Kralove, Czech Republic, 5 National Institute of Public Health, Prague, Czech Republic, 6 Catholic University of Louvain, Louvain, Belgium, 7 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 8 National Institute of Public Health, Bratislava, Slovak Republic, 9 Department of Occupational Medicine, Medical Faculty in Martin, Martin, Slovak Republic and 10 Finnish Institute of Occupational Health, Helsinki, Finland

11 To whom correspondence should be addressed. Tel: +420 2 241062694; Email: pvodicka{at}biomed.cas.cz


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We analysed the associations between genetic polymorphisms in genes coding for DNA repair enzymes XPD (exon 23 A -> C, K751Q), XPG (exon 15 G -> C, D1104H), XPC (exon 15 A -> C, K939Q), XRCC1 (exon 10 G -> A, R399Q) and XRCC3 (exon 7 C -> T, T241 M) and the levels of chromosomal aberrations (CAs) and single-strand breaks (SSBs) in peripheral lymphocytes in a central European population. We also measured the irradiation-specific DNA repair rates and the repair rates of 8-oxoguanines in these individuals. An elevated frequency of CAs was observed in individuals with the XPD exon 23 A allele (AA and AC) genotypes (F = 3.6, P = 0.028, ANOVA). In multifactorial analysis of variance, the XPD exon 23 polymorphism appeared as a major factor influencing CAs (F = 4.2, P = 0.017). SSBs in DNA, on the other hand, were modulated by XPD (F = 4.3, P = 0.023), XPG (F = 4.3, P = 0.024) and XRCC1 genotypes (F = 3.0, P = 0.064). Irradiation-specific DNA repair rates (reflecting mainly base excision repair activity) were affected by XRCC1 (F = 5.9, P = 0.010) and XPC polymorphisms (F = 4.2, P = 0.046, MANOVA). Our results from this study suggest that markers of genotoxicity are associated with polymorphisms in genes encoding DNA repair enzymes.

Abbreviations: BER, base excision repair; BPDE, benzo[a]pyrene diol epoxide; CAs, chromosomal aberrations; NER, nucleotide excision repair; SCEs, sister chromatid exchanges; SSBs, single-strand breaks; SSBs–EndoIII sites, SSBs–endonuclease III sites


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumourigenesis represents a complex, multistage process that seems to be influenced by genetic polymorphisms in a number of genes. Several studies have shown the existence of a large inter-individual variation in DNA repair capacity and individuals with repair capacity below the population mean can be at an increased risk of developing various kinds of cancer. It is likely and has been shown in recent studies that single nucleotide polymorphisms in coding and regulatory sequences may result in subtle structural alterations in DNA repair enzymes modulating cancer susceptibility (17).

Recently, polymorphisms in DNA repair genes, such as the Xeroderma pigmentosum genes XPD and XPG, have been studied for association with lung cancer (1,2) and breast cancer (3). Polymorphism in exon 7 of the XRCC3 (X-ray cross-complementation group 3) gene has been implicated in an increased risk of melanoma (4) and several types of cancer have been related to polymorphisms in the XRCC1 gene (2,5,6). Although the links between genetic polymorphisms of DNA repair genes and their phenotypic consequences were recently addressed (8), this important topic deserves additional investigation.

Associations between various genetic polymorphisms and intermediary molecular markers involved in the cascade of genotoxic/carcinogenic events may provide useful information on the modulating effects of genetic polymorphisms, on individual susceptibility towards environmental and occupational carcinogens and on the possible links between DNA repair polymorphisms and individual DNA repair rates.

To explore the tentative modulating effect of DNA repair polymorphisms we studied markers of genotoxicity assumed to be relevant to the onset of cancer. In the present study, we investigated the potential links between genetic polymorphisms in genes coding DNA repair enzymes and the levels of chromosomal aberrations (CAs) and single-strand breaks (SSBs) in DNA in a central European general population. We studied polymorphisms of the DNA repair genes XPD, XPG and XPC involved in nucleotide excision repair (NER), XRCC1 involved in base excision repair (BER), and XRCC3 involved in recombination repair and in maintaining chromosomal stability (911). Since clear mechanistic links between the various genetic polymorphisms of DNA repair genes and their phenotypic consequences are not known, relationships between transient markers of genotoxic effects (like SSBs and CAs) and DNA repair polymorphisms are of particular interest. SSBs in DNA are considered as transient promutagenic lesions, representing direct effects of damaging agents. They may also be related to apurinic/apyrimidinic sites (alkali-labile sites appearing as breaks) and also represent intermediates in cellular repair, since both NER and BER cut out the damage and replace it with undamaged nucleotides (12). CAs positively correlated with the onset of cancer in recent prospective studies on human populations (13,14). The molecular background for CAs is represented by double-strand breaks (DSBs), the main sources of which are ionizing irradiation, certain antibiotics and endonucleases. Additional mechanisms leading to DSBs accumulation are DNA replication and DNA excision repair due to accumulated SSBs, mitotic recombination errors and oxidative damage (15).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Population characteristics
The study population consisted of 337 healthy individuals (204 men and 129 women, mean age 41.6 ± 10.7 years) employed in local administration as clerks, employees of regional hygienic stations and research institutes (171 individuals) and in various branches of the plastics industry of central Europe (166 individuals) (central and eastern Bohemia, western Slovakia). Genotypic distribution for various polymorphisms in DNA repair genes were determined in all individuals. CAs were assayed for 253 individuals, SSBs for 158, SSBs–endonuclease III (SSBs–EndoIII) sites for 157 g irradiation-specific DNA repair rates for 96 and the rate of DNA repair of oxidative DNA damage for 84 individuals. Lower numbers for certain parameters are due to methodological limitations (i.e. immediate processing of samples without storage, as applies for SSBs and, particularly, for DNA repair rates). Confounding factors, like X-rays, medical drug treatments, dietary (vitamins, particular diets) and lifestyle habits (alcohol and coffee consumptions) and possible exposure-related effects were recorded in detailed questionnaires and considered in the statistical analyses. No significant differences in biomarkers between individuals employed in the plastics industry and those of other professions were recorded. The data on smoking habit were available for 255 subjects (108 smokers and 147 non-smokers). Those individuals who never smoked or who had quit smoking at least 5 years prior to the sampling were considered as non-smokers. Smoking was assessed as cigarettes/day as well as number of cigarettes in a lifetime.

Additionally, individuals exposed to potentially genotoxic compounds and those unexposed exhibited similar allelic distributions in DNA repair genes and were of similar age (data not shown). Our cohort is most likely a representative, ethnically homogeneous population and therefore is suitable for the assessment of possible relations between markers of genotoxicity and polymorphisms in genes coding for DNA repair enzymes.

The study design was approved by the local Ethical Committee and the participants provided their informed consent to be included in the study. The samplings of biological material were carried out according to the Helsinki Declaration over the past 5 years.

DNA repair polymorphisms
Single nucleotide polymorphisms in genes encoding various DNA repair enzymes were determined by a PCR–RFLP based method. PCR products were generated using 10 ng of genomic DNA in 10 µl volume reactions containing 20 mM Tris–HCl, 50 mM KCl, 2.0 mM MgCl2, 0.11 mM each dNTP, 0.3 µM each primer (Table I) and 0.3 U Taq DNA polymerase. The temperature conditions for PCR were set as denaturation at 94°C for 30 s, annealing (temperatures given in Table I) for 30 s, elongation at 72°C for 30 s and final extension at 72°C for 5 min. The amplified fragments were digested with appropriate restriction endonucleases and analysed (Table I). The digested PCR products were resolved on 10% polyacrylamide gels and visualized under UV light after staining with ethidium bromide. The genotype results were regularly confirmed by direct DNA sequencing of the amplified fragments.


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Table I. Primers and restriction enzymes used for genotyping various single nucleotide polymorphisms in DNA repair genes and allele frequencies in the studied population

 
The determination of SSBs and SSBs–EndoIII sites
SSBs, considered as transient promutagenic lesions reflecting DNA damage induced by alkylation or apurinic/apyrimidinic sites as well as intermediates in cellular repair (Figure 1A and B), were detected in lymphocytes by single cell gel electrophoresis (Comet assay) as described earlier (16). Calibration of the method with X-rays enables quantitative expression of the DNA damage as SSBs/109 Da (16). SSBs–EndoIII sites, reflecting abasic sites and oxopyrimidines, were evaluated by a modified version of the Comet assay with and without endonuclease III treatment (17).



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Fig. 1. Human lymphocytes processed by the Comet assay for the assessment of DNA damage and DNA repair rates. (A) Lymphocyte nucleus with intact, undamaged DNA (left) and nucleus with a high degree of DNA damage exhibiting the typical ‘comet tail’ appearance (right). (B) Lymphocyte nuclei from selected individuals showing the damaged DNA. (C) Human lymphocyte nuclei after 5 Gy {gamma}-irradiation for 10 min showing initial induced DNA damage (DNA repair test). (D) Human irradiated lymphocyte nuclei after 40 min incubation showing a reduction in DNA damage, reflecting DNA repair activity (DNA repair test). Scale bar 50 µm.

 
The determination of CAs
For the determination of CAs, whole blood lymphocyte cultures were established from heparinized blood (two parallel tubes independently) according to methods described earlier (18,19). Slides were coded and 100 metaphases per sample were scored for the presence of various types of CAs. For the statistical analyses frequencies of CAs without gaps (total CAs) and chromatid type CAs with and without gaps were used.

DNA repair rates
Irradiation-specific DNA repair rates were assessed in lymphocytes isolated using a Ficol gradient (20). Briefly, cells embedded in agarose on slides were irradiated with {gamma}-rays (5 Gy for 10 min) and either lysed immediately or incubated at 37°C for 40 min prior to lysis. The induced DNA breaks are repaired during incubation according to the individual repair rate (21). The results show the number of SSBs repaired (Figure 1C and D). The number of SSBs was estimated by alkaline Comet assay calibrated for the number of SSBs (16).

Cell extracts prepared from lymphocytes of each subject were tested for the repair of 8-oxoguanines, known to be removed from DNA by the specific glycosylase OGG1 (17). Briefly, aliquots of cell extracts prepared from 107 lymphocytes were pipetted onto microscope slides with HeLa cells mounted in agarose for the Comet assay. The HeLa cells were pretreated with the photosensitizer Ro 19-8022 (Hoffmann-La Roche) and irradiated with a fluorescent lamp to induce 8-oxoguanines in DNA and subsequently incubated with cell extracts, resulting in the introduction of SSBs at sites of 8-oxoguanine. The above functional DNA repair assays have recently been employed by us to evaluate genotoxic effects of potential chemical carcinogens (21).

Statistical calculations
Statistical calculations were performed using Statgraphics, version 7 (Manugistics Inc., Cambridge, MA). For testing significant differences between groups, the non-parametrical Mann–Whitney U-test was applied. Simple linear regression and correlation analyses were used to estimate the correlation between the parameters. Analysis of variance (ANOVA) was employed to test associations between biomarkers and the various genotypes. Associations between the combined genotypes and the biomarkers were tested by multifactorial regression analysis, stepwise selection and multifactorial analysis of variance. For the statistical calculations, wild-type genotypes were assigned a value of ‘0’, heterozygous genotypes ‘1’ and homozygous genotypes ‘2’. For statistical calculations only those cases were included for which relevant corresponding data were available.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The levels of biomarkers in the population and genotype frequencies in DNA repair genes
Generally, we did not observe any statistically significant difference in the mean levels of CAs, SSBs in DNA, SSBs–EndoIII sites, irradiation-specific DNA repair rates and repair of 8-oxoguanine lesions between men and women or between smokers and non-smokers (Table II). Simple regression analysis did not reveal any significant influence of age on the frequency of CAs or on the levels of SSBs and SSBs–EndoIII sites. Irradiation-specific DNA repair rate was moderately decreased with age (R = –0.202, P = 0.059) and inversely associated with CA frequencies, particularly in males (R = –0.244, P = 0.050). Table II shows the levels of studied markers stratified for age (younger individuals <=41.6 years and individuals >41.6 years). According to this classification all parameters did not significantly differ, except for irradiation-specific DNA repair rates (moderately higher among younger subjects, P = 0.048). Results on genotype screening for the DNA repair genes are given in Table I. The mean age of individuals with particular DNA repair genotypes was remarkably uniform, ranging from a minimum of 38.5 ± 13.4 years (XPG, exon 15 CC genotype) to a maximum of 41.9 ± 10.1 years (XRCC3, exon 7 CT genotype).


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Table II. Mean values (±SD) for various parameters of genotoxicity and repair rates of irradiation-specific and oxidative DNA damage in the population stratified by main confounders

 
Based on the data from questionnaires we have analysed possible associations between biomarkers and different dietary and lifestyle factors. It appeared that irregular exposure to a broad variety of chemicals, used in the workplace as well as in the household, was linked with the level of SSBs (R = 0.239, P = 0.009, F = 7.1, P = 0.009 ANOVA) and with the rates of irradiation-specific DNA repair (R = 0.295, P = 0.004, F = 8.8, P = 0.004 ANOVA). In addition, the consumption of coffee positively affected irradiation-specific DNA repair rates (R = 0.204, P = 0.039, F = 2.6, P = 0.029 ANOVA), but decreased the capacity to repair oxidative damage (R = –0.218, P = 0.022, F = 2.6, P = 0.029 ANOVA). Other possible influencing factors, such as medical treatment, X-ray investigations, lifestyle and dietary habits, did not significantly affect any of the biomarkers studied. It should be noted that the above significant associations may emerge by chance and it is not possible to draw any firm conclusions at the moment, since the data are based only on questionnaires without any biochemical confirmations (e.g. caffeine and vitamins in blood, urinary cotinine, etc.).

Links between genotypes and chromosomal aberrations
As shown in Table III, CA frequencies were decreased in individuals with homozygous genotypes of the XPD exon 23 variant C allele in comparison with those with the wild-type AA and heterozygous AC genotypes (P = 0.012). ANOVA showed a modulation of CA frequencies by XPD exon 23 genotype (F = 3.6, P = 0.028). On the other hand, MANOVA, employed to discern the effect of genotypes on CA frequency in combination, confirmed that XPD exon 23 polymorphism is a major factor influencing the level of CAs (F = 4.2, P = 0.017). Similar tendencies were found for chromatid-type CAs with and without gaps, when tested separately. The effect of XPD genotype on CAs was particularly apparent in smokers, where wild-type AA homozygotes had a CA frequency of 2.6 ± 1.6 (n = 32), while CC homozygotes had a CA frequency of 1.3 ± 1.4 (n = 16) (R = –0.227, P = 0.030 by simple regression analysis and F = 5.6, P = 0.006 by ANOVA).


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Table III. Mean values (±SD) for various parameters of genotoxicity and repair rates of irradiation-specific and oxidative DNA damage in the population stratified by individual DNA repair genotypes

 
Genetic polymorphism in the XPG gene was associated with marginally elevated CA frequencies in wild-type GG homozygotes (2.4 ± 1.6) as compared with CC homozygotes (2.0 ± 1.8), while in XPC gene the lowest CA frequency was observed in AA homozygotes (2.1 ± 1.5) in comparison with CC homozygotes (2.4 ± 1.5); these differences were not statistically significant. No significant modulating effects on CA frequencies were observed for the other DNA repair polymorphisms (XRCC1 exon 10 G -> A and XRCC3 exon 7 C -> T) (Table III).

Links between SSBs, SSBs–EndIII sites and genotypes
Significantly lower levels (P = 0.033) of SSBs were observed for individuals bearing a homozygous (CC) genotype compared with those with the XPD exon 23 wild-type alleles (AA) and heterozygous (AC) genotype. A 2-fold difference, although not statistically significant, was also found between individuals with the homozygous XPG exon 15 wild-type GG genotype (0.7 ± 0.5 SSB/109 Da) and those with the homozygous CC genotype (0.3 ± 0.2 SSB/109 Da, P = 0.066). In contrast, no clear relationships were found between XPC, XRCC1, XRCC3 genotypes and SSB levels (Table III).

SSB levels were inversely correlated with capacity to repair oxidative damage in DNA (R = –0.218, P = 0.047). In multiple regression analysis, SSBs were influenced by capacity to repair both irradiation-specific and oxidative DNA damage (F = 13.3 and 6.9, respectively), XPD (F = 5.3) and XPG (F = 4.9) polymorphisms (R2 for this model was 0.432). MANOVA showed that polymorphisms in XPD (F = 4.3, P = 0.023), XPG (F = 4.3, P = 0.024) and XRCC1 (F = 3.0, P = 0.064) modulated SSB levels. When the cohort was stratified for smoking habit, significantly elevated SSB levels were found in non-smokers with the XPD AA and AC polymorphisms (0.6 ± 0.4 and 0.7 ± 0.5 SSB/109 Da, respectively) as compared with the XPD CC polymorphism (0.3 ± 0.2 SSB/109 Da, P = 0.025).

SSBs–EndoIII sites did not seem to be affected by any of the genetic polymorphisms investigated (Table III). Multiple regression analysis revealed a statistically significant positive relationship between irradiation-specific DNA repair capacities and SSBs–EndoIII sites (F = 9.4).

DNA repair polymorphisms and DNA repair rates
The irradiation-specific repair rates in DNA were significantly affected by the XRCC1 polymorphism, being lower in heterozygous GA and variant AA genotypes as compared with the wild-type GG homozygotes (P = 0.018 and P = 0.011, respectively) (Table III). The correlation between XRCC1 genotypes and irradiation-specific DNA repair capacity (R = –0.27, P = 0.019) was additionally confirmed by ANOVA (F = 3.3, P = 0.042).

Irradiation-specific DNA repair capacity was found to be moderately higher in XPG exon 15 CC homozygotes (1.0 ± 0.3 SSB/109 Da) as compared with the homozygotes for the wild-type G allele (0.8 ± 0.6 SSB/109 Da) (Table III), the difference being of borderline significance (P = 0.089). An interesting although not significant tendency was also seen for the XPC exon 15 polymorphism; the mean DNA repair rate was 1.0 ± 0.5 SSB/109 Da for wild-type AA homozygotes and 0.8 ± 0.5 SSB/109 Da for the CC variant homozygotes (Table III). For XPG, XPC and XRCC1 polymorphisms, the highest irradiation-specific DNA repair rates seemed to be associated with lower levels of SSBs. While XRCC1 polymorphism significantly affected DNA repair rates in all sub-cohorts (males, females, smokers and non-smokers), XPG polymorphism was associated with the DNA repair rates particularly in women (1.1 ± 0.4 SSB/109 Da for variant CC genotype versus 0.5 ± 0.4 SSB/109 Da for the wild-type G allele) and in non-smokers (individuals with the homozygous CC genotype exhibited almost 2-fold higher DNA repair rates than those with the wild-type GG genotype; 1.0 ± 0.3 versus 0.6 ± 0.5 SSB/109 Da). MANOVA confirmed that irradiation-specific DNA repair rates are indeed affected by XRCC1 (F = 5.9, P = 0.010) and XPG polymorphisms (F = 4.2, P = 0.046), while the influence of age was marginal (F = 3.0, P = 0.081). None of the DNA repair polymorphisms studied showed a clear link to the rates of the repair of 8-oxoguanines from DNA (Table III).

An illustration of the modulating effect of genotype combinations on the level of CAs
Based on the results described above, we compared the frequencies of CAs for two groups of combined DNA repair gene genotypes; one group consisted of combinations of XPD exon 23 (AA and AC) genotype, XPG exon 15 GG genotype, XPC exon 15 CC genotype and XRCC1 exon 10 (GA and AA) genotype and the other of combinations of XPD exon 23 CC genotype, XPG exon 15 (CC and GC) genotype, XPC exon 15 (AA and AC) genotype and XRCC1 exon 10 (GG and GA) genotype. The former combination exhibited significantly higher mean frequencies of CA than the latter (Figure 2).



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Fig. 2. Modulating effect of combinations of DNA repair polymorphisms on CA frequencies. The dark bars represent mean CA frequency for an ‘adverse’ genotype combination (leading to an increase in CA frequencies), while the white bars represent genotype combination exhibiting lower CA frequencies. (A) Individuals with the XPD exon 23 AA and AC genotypes combined (n = 119) in comparison with individuals with the XPD exon 23 CC genotype (n = 36); (B) comparison between the XPD exon 23 AA and AC, XPG exon 15 GG genotype combination (n = 112) and the XPD exon 23 CC genotype, XPG exon 15 CC and CG genotype combination (n = 23); (C) comparison between the XPD exon 23 AA and AC, XPG exon 15 GG, XPC exon 15 CC genotype combination (n = 25) and the XPD exon 23 CC, XPG exon 15 CC and CG, XPC exon 15 AA and AC genotype combination (n = 18); (D) comparison between the XPD exon 23 AA and AC, XPG exon 15 GG, XPC exon 15 CC, XRCC1 exon 10 GA and AA genotype combination (n = 16) and the XPD exon 23 CC, XPG exon 15 CC and CG, XPC exon 15 AA and AC, XRCC1 exon 10, GG and GA genotype combination (n = 16). P values given were evaluated using the Mann–Whitney U-test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we investigated different transient markers of genotoxic/carcinogenic effects and a set of genetic polymorphisms in genes coding for DNA repair enzymes. Our results indicate a relationship between markers of genotoxic effects and polymorphisms in genes coding for DNA repair enzymes. In the concept of multiple comparisons for the assessment of the modulating effect exerted on different end-points by genetic polymorphisms in combination, cautious interpretation is required due to the random occurrence of some positive findings. The determination of individual DNA repair rates represents an important mechanistic aspect and a phenotypic measure. These data along with the DNA repair polymorphisms provide a novel contribution of importance for the understanding of individual response to genotoxic/carcinogenic processes.

In the present study, CA frequencies were significantly lower in individuals homozygous for the exon 23 variant allele of the XPD gene. This effect was particularly evident in smokers, suggesting that the CC genotype results in an enhanced repair capacity towards CA-forming lesions like those induced by tobacco smoke. The XPD exon 23 polymorphism did not affect the frequency of sister chromatid exchanges (SCEs), another cytogenetic end-point, in lymphocytes of smokers and non-smokers (22). Our finding is in agreement with the elevated level of benzo[a]pyrene diol epoxide (BPDE) adducts observed in mononuclear leukocyte DNA of smoking GSTM1 null lung cancer patients homozygous for the XPD exon 23 and 10 wild-type alleles (23). On the other hand, an increased level of aromatic DNA adducts in mononuclear leukocytes of lung cancer patients and control subjects was associated with XPD exon 23 and 10 variant alleles (1). Two other studies have also suggested that the exon 23 variant allele is associated with elevated DNA adduct levels. Never-smokers homozygous for the exon 23 variant allele (9) and traffic workers who carried these alleles (24) showed an increase in bulky DNA adducts in leukocytes. The repair of cyclobutane dimers induced by UV radiation in the skin of a group of melanoma patients, basalioma patients and control subjects appeared to be lowered in homozygotes for the XPD exon 23 variant allele who were 50 years of age or older (25). Additionally, moderately elevated CA levels seemed to be associated with two other polymorphisms of NER, i.e. the G allele in exon 15 of the XPG gene and the C allele in exon 15 of the XPC gene. The C allele of the exon 15 XPC polymorphism has been suggested to be associated with depressed levels of NER in vitro (26).

Our results indicate an association between the frequencies of CAs, mainly of the chromatid type, and polymorphisms in DNA repair genes, particularly XPD, involved in NER. Chromatid-type aberrations observed in cultured lymphocytes are considered to be formed in the S phase of the cell cycle from DNA lesions originally induced in G0 stage lymphocytes in vivo by, for example, various clastogenic chemicals such as genotoxic constituents of tobacco smoke (14,22,27). NER can be assumed to affect the rate of chromatid-type CAs by repairing DNA adducts produced by such clastogens. In males, CA frequencies decreased with increasing irradiation-specific DNA repair rates, suggesting a role of BER in CA formation as well. A striking difference was found on comparing CA frequencies in two extreme combinations of DNA repair polymorphisms: XPD exon 23 (AA and AC) genotype, XPG exon 15 GG genotype, XPC exon 15 CC genotype and XRCC1 exon 10 (GA and AA) genotype versus XPD exon 23 CC genotype, XPG exon 15 (CC and GC) genotype, XPC exon 15 (AA and AC) genotype and XRCC1 exon 10 (GG and GA) genotype. Out of the various combinations these two were selected as an illustration of the modulating effect of ‘adverse’ genotypes of DNA repair polymorphisms on individual susceptibility to genotoxic response. Apparently, the XPD exon 23 polymorphism had a major influence on CA frequencies, followed by XPC exon 15 polymorphism.

SSBs were found to be lower in individuals bearing the XPD exon 23 variant CC allele and homozygous for the XPG exon 15 variant CC allele. This pattern of modulation of SSB levels was similar to that described for CAs, indirectly suggesting a relationship between SSBs and CAs, as well as a role of NER in the repair (or formation) of SSBs.

Additionally, an increase in SSBs was observed in individuals homozygous for the exon 10 variant allele of the XRCC1 gene. This association suggests that BER also participates in modulating SSB levels. The XRCC1 exon 10 variant allele has previously been observed to be associated with increased levels of SCEs in lymphocytes (28) and glycophorin A variant erythrocytes in smokers (29), polyphenol DNA adducts in mononuclear cells (30) and bulky DNA adducts in leukocytes in never-smokers (9). The XRCC1 exon 10 polymorphism did not affect the level of bulky DNA adducts in leukocytes of bladder cancer patients (31) or BPDE–DNA adducts in mononuclear leukocytes of lung cancer patients (23), as these DNA lesions are mainly repaired by NER. Apparently, a combination of ‘adverse’ genotypes (XPD exon 23 AA and AC genotypes and XPG GG genotype) may increase the risk of SSB formation, but a detailed analysis, similar to that for CAs, was not performed due to the limited data on SSBs. Additionally, cautious interpretation is required for an assessment of the modulating effect exerted by multiple genetic polymorphisms in combination on different end-points due to the random occurrence of positive findings.

Interesting results emerged by relating DNA repair polymorphisms to DNA repair rates in humans. Although the rate of 8-oxoguanines repair was not affected by any of the DNA repair genotypes tested, higher irradiation-specific DNA repair rates were detected among individuals with the XPG exon 15 variant allele and the XPC exon 15 wild-type allele. However, the most striking difference was recorded for the XRCC1 exon 10 polymorphism, the repair rate of irradiation-induced DNA damage being almost 2-fold higher in wild-type homozygote individuals as compared with variant allele homozygotes. These results are in good agreement with the SSB and CA data. The lower irradiation-specific DNA repair rates were accompanied by higher levels of SSBs and CAs, particularly in men.

The irradiation-specific DNA repair rate is believed to represent predominantly BER (20,32), in agreement with the role of the XRCC1 polymorphism. The moderate influence of XPG and XPC polymorphisms may be explained either as a chance finding, which may arise due to multiple comparisons, or by a participation of NER as well, probably in the repair of photoproducts or other DNA lesions induced by {gamma}-irradiation. In an earlier study, an association between DNA repair kinetics of UV-induced DNA damage and polymorphisms in the XPD gene had been indicated (25). The participation of NER genes may be solved by direct phenotypic determination of this repair pathway in future studies.

In conclusion, we report here an association between a set of genetic polymorphisms and important genotoxic effects end-points, related to cancer risk. Our results, in combination with individual DNA repair rates, contribute to our understanding of the role of individual susceptibility in genotoxic carcinogenesis.


    Acknowledgments
 
This study was supported by grants GACR 310/01/0802, 310/03/0437 and QLK4-CT-1999-01386, EU ASHRAM, grant QLK4-CT-2001-00264, the Swedish Council for Working Life and Social Research, grant QLK4-CT-2000-00628, the Finnish Work Environment Fund, the Academy of Finland and by grant AVOZ5039906. V.H. was the recipient of a grant from ECETOC.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 6, 2003; revised November 25, 2003; accepted December 16, 2003.