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
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Abstract |
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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; SSBsEndoIII sites, SSBsendonuclease III sites
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Introduction |
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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).
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Materials and methods |
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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 PCRRFLP based method. PCR products were generated using 10 ng of genomic DNA in 10 µl volume reactions containing 20 mM TrisHCl, 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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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 -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 MannWhitney 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.
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Results |
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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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Links between SSBs, SSBsEndIII 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).
SSBsEndoIII 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 SSBsEndoIII 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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Discussion |
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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 BPDEDNA 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 -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.
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Acknowledgments |
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References |
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