2 Thus, we have decided to extend our analysis to the individuals included in a lung cancer case-control study. 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. 2. Materials and Methods 2.1. Study Population. Peripheral blood samples were col- lected, 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 pneu- moconiosis. 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. 2.2. DNA Adduct and Polymorphism Analyses. 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 [13]. DNA samples were stored at −80◦C until laboratory analysis. PBL DNA adducts were analysed using the nuclease P1 modiﬁcation of the 32P- postlabelling technique [13]. DNA samples (1–5 μg) were digested with micrococcal nuclease (32.17 mU) and spleen phosphodiesterase (21.6 mU). Hydrolized DNA was treated with nuclease P1 (110 mU) for 30 . The nuclease P1 resis- tant DNA samples were then labelled by incubation with 25 μCi of carrier-free [γ-32P]ATP (3000 Ci/mM) and T4- polynucleotide kinase (112.5 mU). The obtained 32P-labelled samples were analysed using 1.0 M sodium phosphate, pH 6.8. DNA adduct resolution was achieved using 4.0 M lithium formate, 7.5 M urea, pH 3.5 and 0.65 M lithium chloride, 0.45 M Tris base, 7.7 M urea, pH 8.0. Chromatograms were Journal of Nucleic Acids ﬁnally developed using 1.7 M sodium phosphate pH 5.0. Detection and quantiﬁcation of PBL DNA adducts and normal nucleotides (nn) were obtained by storage phosphor imaging techniques employing intensifying screens [15]. After background subtraction, the levels of DNA adducts were expressed such as relative adduct labelling (RAL) = screen pixel in adducted nucleotides/screen pixel in nn. Polymerase Chain Reaction followed by enzymatic diges- tion was used for the genotyping of XRCC1 Arg194Trp and Arg399Gln, and XRCC3 Thr241Met [12]. 2.3. Statistical Analysis. Logistic regression analysis was car- ried 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 108 nn). A multiple regression analysis has also been performed grouping individuals according to the number of at risk alleles. A P-value less than or equal to 5% was considered signiﬁcant. All the analyses were performed by the statistical package SPSS. 3. Results and Discussion Genotype and allele frequencies were calculated by counting, and genotype distributions were in Hardy-Weinberg equi- librium. 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 [9]. Characteristics of the study population are summarized in Table 1. PBL DNA adducts were increased in former and current smokers in respect to nonsmokers. The highest levels of DNA adducts were detected in individuals that reported to smoke for more than 40 years. A slightly increment of DNA damage was observed in benign lung disease and lung cancer patients in respect to controls. Multiple regression analysis shows an increased fre- quency of PBL DNA adducts in smokers for more than 40 years (OR = 5.28, 95% conﬁdence interval (C.I.) 1.00–27.72, P = .049). A signiﬁcant trend with increasing number of smoked cigarettes was found (P for trend <.05). After the previous cited adjustments, no diﬀerences were observed comparing controls with benign lung diseases and lung cancer patients. When the associations of DNA adducts with DNA polymorphisms were considered, a association with XRCC1 Arg194Trp and Arg388Gln polymorphisms was found (OR = 4.08, 95% C.I. 0.77–21.48, P = .098 and OR = 1.32, 95% C.I. 0.34–5.18; P = .689, resp.). Conversely, a statistically signiﬁcant inverse eﬀect was observed with XRCC3 Thr241Met polymorphism (OR = 0.17, 95% C.I. 0.05–0.61, P = .006). Then, we investigated the combination of the variant allele/s of XRCC1 Arg194Trp and Arg399Gln polymor- phisms with the wild type allele of XRCC3 Thr241Met.