Smoke constituents can induce DNA adducts that cause mutations and lead to lung cancer. We have analyzed DNA adducts and polymorphisms in two DNA repair genes, for example, XRCC1 Arg194Trp and Arg399Gln genes and XRCC3 Thr241Met gene, in 34 lung cancer cases in respect to 30 subjects with benign lung cancer disease and 40 healthy controls.
When the study population was categorized in base to the number of risk alleles, adducts were significantly increased in individuals bearing 3-4 risk alleles (
Lung cancer is a leading cause of cancer death in the world [
The role of tobacco smoking in the aetiology of lung cancer has been widely evaluated [
It has been hypothesized that interindividual difference in lung cancer risk may be due to differences in DNA repair. In support of this hypothesis, different studies have indicated that DNA variation in DNA repair genes may influence cancer susceptibility [
In this study, we have analyzed the levels of DNA adducts and DNA polymorphisms in two DNA repair genes, for example, XRCC1 Arg194Trp and XRCC1 Arg399Gln, and XRCC3 Thr241Met polymorphisms, representing the base excision repair (BER) and the double-strand breaks repair (DSB) pathways, in lung cancer cases in respect to individuals with benign lung disease and to healthy controls. Then, we decided to investigate the combination of the variant allele/s of XRCC1 Arg194Trp and Arg399Gln polymorphisms with the wild type allele of XRCC3 Thr241Met. The analysis of the effects of different combinations of DNA repair polymorphisms on DNA adducts has been done under the assumption that the combination of polymorphisms can have additive or more than additive effects on DNA adduct formation.
Peripheral blood samples were collected, after written informed consent to participate in the present study, from 34 nonsmall cell lung cancer patients (26 males and 8 females, mean age 63.4 years) and from 30 (22 males and 8 females, mean age 63.5 years) subjects with benign lung diseases admitted to the National Cancer Institute and San Martino Hospital, Genoa, Italy. 40 controls (25 males and 15 females, mean age 63.4 years) were recruited from a group of blood donors. Lung cancer cases were asked to participate in the study after the diagnosis, but before radio and chemotherapy. The group of benign lung diseases was formed by subjects affected by Chronic Obstructive Pulmonary Disease (COPD), asthma, and pneumoconiosis. A standard questionnaire was administered to all volunteers by personal interview at the time of blood collection. Smoking status was defined as smoker, within the last year, former smoker, at least one year before diagnosis, and nonsmoker.
Peripheral blood lymphocytes (PBLs) were separated from 5 mL freshly collected whole blood by centrifugation on a Ficoll gradient. PBL DNA was extracted and purified using a method that requires RNA and protein digestion and extraction with organic solvents [
Polymerase Chain Reaction followed by enzymatic digestion was used for the genotyping of XRCC1 Arg194Trp and Arg399Gln, and XRCC3 Thr241Met [
Logistic regression analysis was carried out to calculate Odds Ratios (ORs) adjusted for different covariates (i.e., age, sex, smoking, and DNA polymorphisms, as appropriate) categorizing DNA adduct levels by RAL median value (above/below 0.1 DNA adducts per
Genotype and allele frequencies were calculated by counting, and genotype distributions were in Hardy-Weinberg equilibrium. Genotype frequencies were, respectively: XRCC1 Arg194Arg = 88.8%, Arg194Trp = 11.2%, XRCC1 Arg399Arg = 38.5%, Arg399Gln = 49%, Gln399Gln = 12.5%, XRCC3 Thr241Thr = 40%, Thr241Met = 41%, and Met241Met = 19%, in keeping with those reported previously [
Characteristics of the study population are summarized in Table
Means of DNA adducts ± standard error (SE) for different variables considered in the study plus the parameter estimates of the multivariate regression model.
Odds Ratio | C.I. | ||||
---|---|---|---|---|---|
Gender | |||||
Woman | 31 | 1 | |||
Male | 73 | 0.65 | 0.19–2.16 | .480 | |
Age (years) | |||||
Per unit | 104 | 1.02 | 0.97–1.06 | .516 | |
Smoking habit | |||||
Nonsmoker | 21 | 1 | |||
Former smoker | 44 | 1.60 | 0.39–6.58 | .517 | |
Smoker | 11 | 2.38 | 0.41–13.90 | .336 | |
Smoker | 27 | 5.28 | 1.00–27.72 | .049 | |
Status | |||||
Controls | 40 | 1 | |||
Benign lung disease | 30 | 0.93 | 0.28–3.03 | .898 | |
Lung cancer | 34 | 1.17 | 0.39–3.47 | .783 | |
XRCC1 Arg194Trp | |||||
Arg/Arg | 87 | 1 | |||
Arg/Trp | 11 | 4.08 | 0.77–21.48 | .098 | |
XRCC1 Arg399Gln | |||||
Arg/Arg | 37 | 1 | |||
Arg/Gln | 47 | 0.93 | 0.36–2.39 | .882 | |
Gln/Gln | 12 | 1.32 | 0.34–5.18 | .689 | |
XRCC3 Thr241Met | |||||
Thr/Thr | 40 | 1 | |||
Thr/Met | 41 | 0.38 | 0.15–0.97 | .043 | |
Met/Met | 19 | 0.17 | 0.05–0.61 | .006 | |
Number of risk alleles | |||||
0-1 | 25 | 1 | |||
2 | 31 | 0.92 | 0.31–2.69 | .877 | |
3-4 | 38 | 4.1 | 1.28–13.09 | .009 |
Means of DNA adducts ± standard error (SE) case-control status considering the number of DNA repair risk alleles plus the parameter estimates of the multivariate regression model.
Odds Ratio | C.I. | ||||
---|---|---|---|---|---|
0-1 risk alleles | |||||
Smoking habit | |||||
Nonsmoker | 6 | 1 | |||
Former smoker | 12 | 0.08 | 0.00–9.76 | .302 | |
Smoker | 2 | 0.33 | 0.00–67.26 | .686 | |
Smoker | 5 | 0.16 | 0.00–27.05 | .480 | |
Status | |||||
Controls | 13 | 1 | |||
Benign lung disease | 4 | 1.23 | 0.08–19.30 | .884 | |
Lung cancer | 8 | 0.43 | 0.05–4.00 | .458 | |
2 risk alleles | |||||
Smoking habit | |||||
Nonsmoker | 7 | 1 | |||
Former smoker | 10 | 2.46 | 0.12–50.98 | .560 | |
Smoker | 5 | 0.86 | 0.03–28.37 | .933 | |
Smoker | 9 | 20.91 | 0.62–709.04 | .091 | |
Status | |||||
Controls | 12 | 1 | |||
Benign lung disease | 7 | 1.11 | 0.12–10.00 | .928 | |
Lung cancer | 12 | 2.39 | 0.25–22.62 | .447 | |
3-4 risk alleles | |||||
Smoking habit | |||||
Nonsmoker | 5 | 1 | |||
Former smoker | 20 | 9.47 | 0.52–173.16 | .130 | |
Smoker | 9 | 36.38 | 1.17–1132.84 | .040 | |
Status | |||||
Controls | 12 | 1 | |||
Benign lung disease | 15 | 0.45 | 0.06–3.69 | .460 | |
Lung cancer | 11 | 4.54 | 0.33–62.93 | .259 |
Multiple regression analysis shows an increased frequency of PBL DNA adducts in smokers for more than 40 years (OR = 5.28, 95% confidence interval (C.I.) 1.00–27.72,
When the associations of DNA adducts with DNA polymorphisms were considered, a null association with XRCC1 Arg194Trp and Arg388Gln polymorphisms was found (OR = 4.08, 95% C.I. 0.77–21.48,
Then, we investigated the combination of the variant allele/s of XRCC1 Arg194Trp and Arg399Gln polymorphisms with the wild type allele of XRCC3 Thr241Met. The choice was based on the different association of XRCC1 and XRCC3 polymorphisms with lung cancer risk, for example, positive for XRCC1 Arg194Trp and Arg399Gln and negative for XRCC3 Met241Met [
When the study population was categorized in base to the number of risk alleles, the levels of DNA adducts were statistically significantly increased in individuals bearing three-four risk alleles (OR=4.1 95% C.I. 1.28–13.09,
Tobacco smoking is recognised as the primary preventable cause of human cancer. Therefore, many studies have explored the influence of smoking on the levels of DNA adducts in nucleated blood cells in order to identify an early and sensitive biomarker of effective intake of tobacco carcinogens [
In our study, we have analyzed PBLs as surrogate and more accessible tissues than bronchial biopsies, and we compared the levels of DNA adducts in patients with lung cancer in respect to those with benign lung diseases and controls. Our aim was to evaluate whether the levels of DNA adducts were associated with benign or malignant lung chronic diseases. The question of the utility of PBLs as a valid surrogate for a specific organ like lung, representing the events occurring in the target tissue, is still open [
Uppermost, we have considered the effect of smoking on DNA adduct levels. Our findings show that the levels of DNA adducts of smokers were higher than those of former and nonsmokers. Our finding shows that PBL DNA adduct may reflect exposures to carcinogens, such as those contained in tobacco smoke better than other surrogate tissues, such as leukocyte DNA adducts. However, discrepant results have been also reported with PBLs [
Next results show that the effect of smoking on DNA damage was more marked in the subjects that reported to smoke for more than 40 years. Although detailed information on smoking history, for example, number of pack of cigarettes smoked per years was missing, our findings support the hypothesis that the formation of DNA adducts is significantly influenced by chronic carcinogen exposure. Furthermore, when study population was subgrouped for the number of risk alleles, a significant association with smoking was observed in the subjects carrying three or more risk alleles who reported to smoke for more than 40 years. Conversely, no effect of smoking was observed in smokers bearing one or less risk allele.
The contribution of duration of exposure to cigarette smoke has important implications for both research studies and prevention strategies. It has been shown that the age at first exposure and duration are associated influences to the levels of DNA adducts [
Our next results show that DNA adduct levels were comparable in individuals with benign lung disease or with lung cancer and in controls. However, when specific combinations of variant alleles were investigated, a not significant increased lung cancer risk was observed in individuals bearing the same number of risk alleles.
In a meta-analysis of cancer and bulky DNA adducts [
No increment of DNA adducts has been found in subjects with different benign bronchial pathologies, such as COPD, asthma, and pneumoconiosis, characterized by important inflammatory processes in respect to controls. However, such inflammatory phenomena can influence DNA adduct levels in lung target cells by increasing the biologically effective dose of PAH [
DNA damage primarily reflects exposures to carcinogens but is modulated by inherited and acquired susceptibilities. Age, gender, and life-style and dietary habits have been reported to influence levels of DNA adducts [
To study the effect of different combinations of DNA repair single nucleotide polymorphisms on DNA adducts, we have investigated the combination of the variant allele/s of XRCC1 Arg194Trp and Arg399Gln polymorphisms with the wild type allele of XRCC3 Thr241Met. This was based on the different association of XRCC1 and XRCC3 polymorphisms with lung cancer risk, for example, positive for XRCC1 Arg194Trp and Arg399Gln and negative for XRCC3 Thr241Met [
Furthermore, a previous report has shown that smoking is strong harmful factor that can eliminate the effect of DNA polymorphisms of DNA repair genes on lung cancer susceptibility [
The results of the present study support the utilisation of PBLs as surrogate and more accessible tissues than bronchial biopsies. In fact, we have observed a stronger effect of smoking on DNA adducts of 40 years smokers. When study population was subgrouped for number of risk alleles, the association with smoking was concentrated in carriers of 3-4 risk alleles that reported to have smoked for more than 40 years. A nonsignificant increased lung cancer risk was observed in individuals bearing the same number of risk alleles. Our results suggest that analysis of risk alleles can predict the interindividual variation in DNA adduct levels observed in smokers and lung cancer cases.
This study was partially supported from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy. The authors are grateful to Rosa Filiberti, Donatella Ugolini, Monica Neri, and Andrea Ardizzoni for their efforts in specimens collection.