Genetic polymorphisms and the effect of cigarette smoking in the comet assay

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1 Mutagenesis vol. no. pp. 9, Advance Access publication July Genetic polymorphisms and the effect of cigarette smoking in the comet assay doi:.9/mutage/gei9 Heike Hoffmann, Caroline Isner, Josef Högel and Günter Speit Universitätsklinikum Ulm, Abteilung Humangenetik, D-9 Ulm, Germany A potential genotoxic effect of cigarette smoking has repeatedly been investigated with the comet assay (alkaline single cell gel electrophoresis) and conflicting results have been reported. Besides differences in the methodology and the study design used, genetic differences between the subjects investigated might contribute to the variability of test results. Considering genetic polymorphisms of genes involved in metabolism or DNA repair has led to a better discrimination of smoking-related genotoxic effects in some cases but also led to discrepant results. We therefore evaluated our baseline comet assay effects obtained for nonsmokers and smokers in relation to selected genetic polymorphisms. Our study group comprised nonsmokers and smokers who were strictly selected to exclude potential confounding factors. We chose polymorphisms in the genes GSTM and CYPA (IleVal) because they take part in the metabolism of genotoxins contained in tobacco smoke. In a subgroup of nonsmokers and smokers we also studied polymorphisms in XPD (LysGln), XRCC (Arg99Gln) and XRCC (ThrVal) because they are part of DNA repair pathways involved in the repair of tobacco-related DNA damage. Freshly collected peripheral whole blood samples were tested in the alkaline (ph > ) comet assay. In all experiments a reference standard (untreated V9 cells) was included to correct for assay variability. An independent second evaluation was carried out for all experiments. None of these approaches revealed a significant difference between nonsmokers and smokers. Introduction The comet assay (alkaline single cell gel electrophoresis) has frequently been used to measure DNA damage related to tobacco smoking. Some studies directly tested the effect of smoking as a potential genotoxic exposure while the majority of studies considered smoking as a potential confounding factor in environmental and workplace biomonitoring. Generally, peripheral blood cells were studied and conflicting results have been reported, which have been critically reviewed (,). The reason(s) for the reported discrepancies are virtually unknown, although, various explanations (including seasonal and regional differences) have been proposed (). One critical aspect in biomonitoring is the genetic heterogeneity of humans. Genetic differences in their ability to activate and inactivate xenobiotics and differences in their capacity to repair DNA damage induced by environmental mutagens might influence the results of genotoxicity tests used in biomonitoring (,). Polymorphisms of genes encoding enzymes involved in metabolism and DNA repair might also account for inter-individual susceptibility to smoking-related biological effects (,). Specifically, tumorigenesis in tobacco-related cancer is influenced by the interaction of exposure to cigarette smoking and genetic susceptibility (). Multiple mutagens/ carcinogens have been found in cigarette smoke, with polycyclic aromatic hydrocarbons (PAHs), aromatic amines, N-nitrosamines and aldehydes representing the major classes of harmful substances (,). Many of these carcinogens need metabolic activation before they covalently bind to DNA. As tobacco smoke contains a variety of genotoxic compounds various kinds of DNA modifications are induced, which can be repaired by distinct DNA repair pathways. In particular, genes of the nucleotide excision repair (NER), the base excision repair (BER) and the DNA double strand break (DSB) repair pathways seem to play important roles in the prevention of smoking-induced mutations and cancer. Genetic polymorphisms of genes involved in metabolic activation and DNA repair are therefore of particular interest with regard to the modification of gentoxic effects and cancer risks induced by tobacco smoke and other environmental mutagens/ carcinogens (,,9). One frequently studied polymorphism is the GSTM homozygous allelic loss, GSTM-null genotype, which is present in % of Caucasians. Lack of the GSTM enzyme may result in deficient detoxification of tobacco smoke carcinogens leading to slight increase in the risk of lung cancer (). CYPA is a member of the cytochrome P family, a class of phase I enzymes that activate carcinogens such as PAHs. Several polymorphisms are known in CYPA, and the variant allele (IleVal) in exon was associated with a significantly increased CYPA inducibility (). Many polymorphisms in DNA repair genes have been studied in the context of tobaccorelated genotoxic effects and cancer (,). We investigated three of them [XPD (LysGln), XRCC (Arg99Gln) and XRCC (ThrVal)], which had been studied previously in the context of mutagen sensitivity and genotoxic effects related to smoking and exposure to PAHs ( ). XPD (or ERCC) is a major component of the transcription factor complex TFIIH, which mediates strand separation in the course of NER at the site of a DNA lesion. XRCC plays an important role in BER. It acts as a scaffold for other DNA repair proteins. XRCC is involved in the homologous recombination pathway of DNA DSB repair. It directly interacts with and stabilizes Rad. The role of these genetic variants in smoking-induced DNA damage and the usefulness of including such single polymorphisms in biomonitoring studies still need to be elucidated. With regard to DNA adducts, numerous studies showed associations of tobacco smoke exposure with the induction of DNA adducts To whom correspondence should be addressed. Tel: 9 9; Fax: 9 ; guenter.speit@medizin.uni-ulm.de Ó The Author. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please journals.permissions@oupjournals.org 9

2 H.Hoffmann et al. in human subjects (). However, the role of gene polymorphisms in modifying tobacco-related DNA adduct levels seems to be complex and sometimes inconsistent with epidemiologic findings on cancer risks (). A recent study in a central European population indicated an association between comet assay effects and genetic polymorphisms but no difference between nonsmokers and smokers in the studied population (). In our previous studies we also did not find a difference in comet assay effects between nonsmokers and smokers (,). Here we show that in our study group a difference in the level of DNA damage is also not seen when five genetic polymorphisms are considered. Materials and methods Blood samples Heparinized blood samples were obtained by venepuncture from healthy male nonsmokers and healthy male smokers. The average age of the smokers and nonsmokers was.. and..9 years, respectively. Out of them smokers smoked cigarettes per day and smoked cigarettes and more per day. Subjects were excluded from this study when there was a history of cancer, previous radiotherapy or chemotherapy, use of therapeutic drugs, exposure to diagnostic X-rays during the previous months, severe infections during the previous months, high alcohol consumption, intake of high-dose vitamins or intensive sportive activities during the previous week. All blood donors gave informed consent to participate in this study and the study was approved by the University Human Subjects Committee. Comet assay Aliquots of ml heparinized freshly collected whole blood were mixed with ml low melting agarose (.% in PBS) and added to microscope slides (with frosted ends), which had been covered with a bottom layer of.% agarose. Slides were lysed (ph ; C) and processed as described earlier (), using a time of alkali denaturation of min and electrophoresis (. V/cm) of min at a ph >. Images of randomly selected cells stained with ethidium bromide were analysed from each coded slide by image analysis (Comet Assay II, Perceptive Instruments). For all experiments, we evaluated three image analysis parameters: tail migration, tail intensity and tail moment. In none of the experiments there was a significant difference between these parameters. Therefore, in accordance with our previous studies, we chose one parameter (tail moment) for the presentation of the results. At one reviewer s request we added the results for the parameter tail intensity because this parameter is easier to understand and to compare between different laboratories. Measures of quality assurance For a reference standard ( internal standard ), a culture of exponentially growing V9 Chinese hamster cells was trypsinized, resuspended in cell culture medium with.% DMSO and immediately frozen at C in aliquots of cells in ml each. For each electrophoretic run one sample was carefully thawed, mixed with ml low melting point agarose and immediately layered onto a precoated slide. The comet assay was performed as described above, cells per slide were analysed and the mean tail moment was determined. Then the mean tail moment of all reference standard slides and the standard deviation from the mean were calculated. Reference standard slides with mean tail moment values outside SDs from the group mean were defined as outliers. These comet assay experiments (i.e. subjects tested in these electrophoretic runs) were excluded from the final evaluation. On the basis of this criterion, only three experiments (data from three subjects) had to be excluded from the evaluation. Furthermore, a second independent evaluation of slides was performed by a second investigator using another microscope and another Comet Assay II image analysis unit. DNA isolation Genomic DNA was extracted from whole blood using NucleoSpin Ò Blood Quick Pure from Macherey-Nagel (Düren). Genetic polymorphisms GSTM genotyping. A multiplex polymerase chain reaction (PCR) method was performed to detect the presence or absence of the GSTM gene. A second primer pair for b-actin was used in the same amplification mixture to confirm the presence of amplifiable DNA in the sample. For all analyses, positive and negative control reactions were run in parallel. The PCR assay detects the presence or the absence of the intact gene, but does not differentiate between heterozygous and homozygous carriers. For determination of the polymorphism GSTM, ng genomic DNA were amplified in a total volume of ml containing pmol of the following primer pairs: forward GSTM, -GTT GGG CTC AAA TAT ACG GTC G- ; reverse GSTM, -GAA CTC CCT GAA AAG CTA AAG C- ; forward b-actin, -TCA CCA ACT GGG ACG ACA TG- and reverse b-actin, -TCA TGA GGT AGT CAG TCA GGT-, and PCR buffer ( mm KCl,. mm MgCl, mm Tris HCl, ph 9.), mm of each dntp and. U Taq polymerase. The PCR amplification condition consisted of an initial denaturation step at 9 C for min, followed by cycles of 9 C for s, C for s, C for s and a final extension step at C for min. The GSTM ( bp) and b-actin (9 bp) amplification products were resolved by electrophoresis in a.% agarose gel. The subjects genotypes were categorized as either GSTM positive or null (i.e. homozygous deletion). CYPA genotyping. For determination of the polymorphism CYPA (Ile- Val) in exon, ng of genomic DNA were amplified in a total volume of ml containing pmol of the primer pair (forward CYPA, -GGC TGA ACC TTA GAC CAC ATA- and reverse CYPA, -GAA CTG CCA CTT CAG CTG TCT- ), PCR buffer, mm of each dntp and. U Taq polymerase. The PCR amplification condition consisted of an initial denaturation step at 9 C for min, followed by cycles of 9 C for min, C for min, C for min and a final extension step at C for min. The CYPA amplification product ( bp) was digested overnight at C with the restriction enzyme BseMI (MBI Fermentas, St Leon-Roth). The restriction products of CYPA codon Ile/Ile, Ile/Val and Val/Val genotypes had band sizes of / bp, // bp and bp, respectively. XPD genotyping. The XPD (LysGln) polymorphism in exon was determined as described by Sturgis et al. (9) using the restriction enzyme PstI (MBI Fermentas, St Leon-Roth). The wildtype allele has a single PstI restriction site (, bp), whereas the variant allele results in three fragments (, and bp). XRCC genotyping. The PCR for genotyping of the polymorphisms in the XRCC gene (Arg99Gln) was performed as described by Au et al. (). The amplification product ( bp) was digested with the restriction enzyme BcnI (MBI Fermentas, St Leon-Roth). The wildtype allele was cut in two fragments (9 and 9 bp), whereas the variant Gln allele could not be digested. XRCC genotyping. Genotyping of the polymorphism (ThrVal) in exon was done according to Au et al. (). The bp amplification product was digested with NcoI. The restricted products of the XRCC codon Thr/Thr, Thr/Val and Val/Val genotypes had band sizes of bp, /9/9 bp and 9/9 bp, respectively. Statistical analysis Distributions of the values for the tail moment and the tail intensity in various groups are described using mean, standard deviation, median and quartiles, and are graphically displayed using box-and-whisker plots. For statistical testing, fixed effects ANOVA (analysis of variance) models were used. In each analysis, baseline tail moment was the dependent variable. The model factors were smoking habit (yes/no) and genotype, together with a term for interaction. Genotype had two (GSTM: positive and null) or three categories (others: homozygous wildtype allele, homozygous variant allele and heterozygous). For each gene, an ANOVA was also performed using a dichotomized genotype (homozygous wildtype versus at least one variant allele). Prior to evaluation, tail moment was transformed using a Box Cox transformation (with parameter. in all analyses) in order to establish approximate normality of the model residuals. Each analysis yielded three P-values (association of smoking habits and genotypes with tail moments as well as effect modification). Further, nonsmokers and smokers were also compared without consideration of genotypes, using a one-factor ANOVA. Results Figures and summarize the baseline DNA effects (Figure : tail moment; Figure : tail intensity) in nonsmokers and smokers (A), GSTM-null and GSTM-positive subjects (B), and nonsmokers and smokers according to their GSTM-genotype (C). No statistically significant difference was measured between any of the groups. Subjects positive or negative for GSTM were equally distributed among nonsmokers and smokers but an influence of the genotype of the effect in the comet

3 Comet assay and smoking A B C nonsmoker smoker null positive nonsmoker, null nonsmoker, positive smoker, null smoker, positive Fig.. Distribution of baseline tail moment values in blood samples of smokers (n ) and nonsmokers (n ) in the alkaline comet assay in relationship to the GSTM-genotype. (A) Comparison between nonsmokers and smokers. (B) Comparison between GSTM-null and GSTM-positive subjects. (C) Comparison between nonsmokers and smokers according to their GSTM-genotypes. Each box represents the interquartile range of values with the horizontal line indicating the median value and the vertical line indicating the range of values that fall between the th and 9th percentile. Outliers are marked as circles. assay was not seen (C). This negative result was confirmed in a second independent evaluation (data not shown). Figures and show the effect of smoking in the comet assay in relation to the CYPA polymorphism. It has to be emphasized that there are only seven subjects (two nonsmokers and five smokers) heterozygous for the CYPA polymorphism in our study group. These seven subjects showed a high degree of variability in the comet assay (A) but the mean value was not significantly different from the mean of the subjects homozygous for the wildtype genotype. Although the differentiation between nonsmokers and smokers and the CYPA genotype is of limited value owing to the small numbers, Figure B suggests that smokers heterozygous for the CYPA gene do not reveal obvious differences in the comet assay. In the independently performed second evaluation the highest mean tail moment was measured for this subgroup but the difference was not statistically significant. In the course of the study, we decided to include genetic polymorphisms of DNA repair genes. Therefore, data are only available for nonsmokers and smokers. Table I lists the baseline comet assay effects (tail moment and tail intensity) of nonsmokers and smokers according to genetic polymorphisms in three DNA repair genes. Homozygous carriers of the XPD variant allele (LysGln) exhibited higher tail moment values than heterozygous or homozygous for the wildtype allele. A. B A B 9 C 9 nonsmoker smoker null positive nonsmoker, null nonsmoker, positive smoker, null smoker, positive Fig.. Distribution of baseline tail intensity values in blood samples of smokers (n ) and nonsmokers (n ) in the alkaline comet assay in relation to the GSTM-genotype. (A) Comparison between nonsmokers and smokers. (B) Comparison between GSTM-null and GSTM-positive subjects. (C) Comparison between nonsmokers and smokers according to their GSTM-genotypes. heterozygous homozygous WT heterozygous, nonsmoker heterozygous, smoker homozygous WT, nonsmoker homozygous WT, smoker Fig.. Distribution of baseline tail moment values in blood samples of smokers (n ) and nonsmokers (n ) in the alkaline comet assay in relation to the CYPA-genotype. (A) Comparison between CYPA-heterozygous and CYPA-homozygous subjects. (B) Comparison between nonsmokers and smokers according to their CYPA-genotypes. Only without Box Cox transformation a P-value of. was observed when comparing subjects homozygous for the variant allele with the other subjects. However, after transformation this value was P.. The level of DNA damage was higher

4 H.Hoffmann et al. Table I. Comet assay effects in nonsmokers and smokers stratified by polymorphisms in DNA repair genes XPD (LysGln) XRCC (Arg99Gln) XRCC (ThrVal) Number moment SD intensity SD Number moment SD intensity SD Number moment SD intensity SD Homozygous wildtype All NS S Heterozygous All NS S Homozygous polymorphism All NS S A 9 B 9 heterozygous homozygous WT heterozygous, nonsmoker heterozygous, smoker homozygous WT, nonsmoker homozygous WT, smoker Fig.. Distribution of baseline tail moment values in blood samples of smokers (n ) and nonsmokers (n ) in the alkaline comet assay in relation to the CYPA-genotype. (A) Comparison between CYPA-heterozygous and CYPA-homozygous subjects. (B) Comparison between nonsmokers and smokers according to their CYPA-genotypes. in nonsmokers than in smokers in homozygous carriers of the wildtype allele but lower in heterozygous or homozygous carriers of the polymorphic allele. But these differences also did not reach statistical significance. No difference was seen within the study group or between nonsmokers and smokers when considering the polymorphisms of XRCC (Arg99Gln) and XRCC (ThrMet). With regard to the statistical analysis, transformation to normality worked sufficiently well. None of the statistical tests performed yielded a significant result, all P-values were >. after Box Cox transformation. The results do not provide evidence for any impact of smoking habit (P. without consideration of genotypes) and genotypes on DNA effects in the comet assay. To determine the statistical power of our approach for a comparison between nonsmokers and smokers, we assumed a sample size of each and a standard deviation of. for the tail moment under normal distribution. A two-sided t-test with a level of significance of % then has a power of 9% to yield a significant test result, even if the underlying mean difference between the groups is as small as.. In a two-factor design with probands for each combination of smoking habit and binary genotype, a difference between smoking habits or genotypes, as well as the presence of interaction, can be detected with a power of more than %, e.g. if nonsmokers have mean tail moment of., smokers homozygous for the wildtype allele will also have mean tail moment of., whereas smokers with at least one variant allele will have a mean of.9. Hence, even under moderate deviations from the normality assumption, the study has sufficient power when biologically relevant effects exist. Discussion Our previous studies (,) as well as several other published human biomonitoring studies failed to show an effect of smoking on DNA migration in the comet assay, while some studies indicated such an effect (,). Tobacco smoke is known to contain numerous genotoxic chemicals and thus represents a relevant model for exposure of humans to genotoxins (,). The conflicting results obtained for smoking-related DNA effects in the comet assay thus cast doubt on the general suitability of the comet assay with human peripheral blood cells in biomonitoring. Various modifications of the standard comet assay protocol have been suggested to improve its sensitivity, including the use of DNA repair inhibitors (,) and lesion-specific enzymes (,,,). However, these modifications have the disadvantage that they may increase assay variability and up to now they have not enabled an unequivocal detection of smoking-induced DNA damage in the comet assay. Comet assay results as well as results from other genotoxicity tests used in human biomonitoring might be influenced by the genetic heterogeneity of the study groups. Theoretically, polymorphisms of genes encoding for proteins involved in the metabolism of xenobiotics and repair of induced DNA damage may account for inter-individual variability in handling mutagens/carcinogens present in tobacco smoke. We studied a potential link between genetic polymorphisms in five genes (GSTM, CYPA, XPD, XRCC and XRCC) and the level of DNA strand breaks in nonsmokers and smokers, but did not find a clear effect on the level of DNA damage for any of them. Previous studies reported conflicting results with regard to the contribution of the GSTM genotype to genotoxic effects in human biomonitoring. Peluso et al. () found increased levels of DNA adducts in lymphocytes of smokers but no significant

5 Comet assay and smoking influence of the GSTM polymorphism. In contrast, higher levels of DNA adducts were measured in lymphocytes of coke-oven workers with the GSTM-null genotype (). The evaluation of DNA damage by the comet assay in workers exposed to organic solvents did neither reveal increased levels of DNA damage nor an influence of GSTM genotype on the DNA effects in nonsmokers and smokers (). In a study comparing nonsmokers and smokers the levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III modification of the comet assay were not significantly different from those of nonsmokers. The GSTM polymorphism did not affect the amount of DNA base damage (). In contrast, higher levels of endonuclease III sensitive sites were measured by Dusinska et al. (), but the amount of comet assay effects did not depend on the GSTM genotype. In this context it is interesting to note that coke-oven workers with the GSTM-null genotype had significantly higher levels of BPDE-DNA adducts in peripheral white blood cells (). However, an earlier study () did not find an induction of DNA adducts or comet assay effects in coke-oven workers occupationally exposed to PAH and no significant influence of the GSTM genotype on one of these biomarkers. No association between the amount of DNA damage in PAH-exposed subjects measured by the comet assay and the GSTM polymorphism was also reported in a study with traffic policemen (), a study with coke-oven and graphiteelectrode-producing plant workers (9), and in a study with workers employed in tire plants (). In some of these studies, the study population was also genotyped for the CYPA polymorphism but no association with the effects in the comet assay was detected (,,,9). In addition, a recently published study with pesticide sprayers indicated increased comet assay effects in the exposed group but no association between the CYPA polymorphism and the higher level of DNA damage measured by the comet assay (). In a recent study, various CYPA polymorphisms revealed different effects on DNA adduct levels in lymphocytes after exposure of nonsmokers to environmental tobacco smoke (). DNA effects in the comet assay do not only measure DNA damage but also indicate the DNA repair capacity of the subjects studied (,). DNA strand breaks measured by the comet assay are non-specific indicators of transient DNA damage, reflecting an equilibrium between damage formation and removal at the particular sampling time. Thus genetic differences in DNA repair genes, which modify the DNA repair capacity, may directly influence the level of DNA damage in subjects exposed to genotoxins. However, the three polymorphisms tested in our study did not indicate an association between these genetic differences and comet assay effects in nonsmokers and smokers. Vodicka et al. () recently reported results from a study in a central European population where they found an association between the level of strand breaks in the comet assay and the XPD exon polymorphism (LysGln). In contrast, in other studies, no association was determined between this XPD polymorphism and the frequency of chromosome aberrations or DNA adducts in smokers (,). Vodicka et al. () did not find an association between comet assay effects and the polymorphisms in XRCC (Arg99Gln) and XRCC (ThrVal). In another study of the same group, workers in tire plants exposed to,-butadiene and PAHs did neither show increased DNA effects in the comet assay in relation to the workplace exposure nor differences between nonsmokers and smokers. Furthermore, no significant association was found between genetic polymorphisms (among them XPD, XRCC and XRCC) and the amount of strand breaks in the comet assay (). It is interesting to note that in the same studies positive associations between repair genotypes and the frequency of chromosome aberrations were reported (,). Possibly, chromosomal aberrations that directly reflect inappropriate repair of (induced) DNA damage are better suited than comet assay effects for the determination of repair capacities. On the other hand, the comet assay offers further possibilities for measuring repair capacity, e.g. the use of repair inhibitors (,) or in vitro challenge assays (,,). An association between the irradiation-specific DNA repair rate and the polymorphism in XRCC (Arg99Gln) has been reported (). Results concerning different repair rates between nonsmokers and smokers are inconsistent (,) and an influence of polymorphisms in repair gene has not been established yet. However, the database is limited and further studies are needed to elucidate the usefulness of the comet assay to determine individual repair rates and susceptibility towards environmental mutagens. It should also be kept in mind that due to the vast number of genetic polymorphisms the combined effect of multiple variant alleles may be decisive and the investigation of single polymorphisms might not be sufficient to detect functional differences and cancer risks (,). Acknowledgements The authors gratefully acknowledge the co-operation of all the blood donors. The authors would like to thank Petra Schütz for the excellent technical assistance. The study was financially supported by the project BWPLUS at the Forschungszentrum Karlsruhe with funds from the Department for Environment, Baden-Württemberg, Germany. References. Moller,P., Knudsen,L.E., Loft,S. and Wallin,H. 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