DNA repair genes have an important role in protecting individuals from cancer-causing agents. Polymorphisms in several DNA repair genes have been identified and individuals with non-dramatic reductions in the capacity to repair DNA damage are observed in the population, but the impact of specific genetic variants on repair phenotype and cancer risk has not yet been clarified. In 308 healthy Italian individuals belonging to the prospective European project EPIC, we have investigated the relationship between DNA damage, as measured by ³² P-DNA adduct levels, and three genetic polymorphisms in different repair genes: XRCC1 -Arg399Gln (exon 10), XRCC3 -Thr241Met (exon 7) and XPD -Lys751Gln (exon 23). DNA adduct levels were measured as relative adduct level (RAL) per 10 ⁹ normal nucleotides by DNA ³² P-post-labelling assay in white blood cells from peripheral blood. Genotyping was performed by PCR–RFLP analysis. The XRCC3 -241Met variant was significantly associated with higher DNA adduct levels, whereas XRCC1 -399Gln and XPD -751Gln were associated with higher DNA adduct levels only in never-smokers. XRCC3 -241Met homozygotes had an average DNA adduct level of 11.44 ± 1.48 (±SE) compared with 7.69 ± 0.88 in Thr/Met heterozygotes and 6.94 ± 1.11 in Thr/Thr homozygotes ( F = 3.206, P = 0.042). Never-smoking XRCC1 -399Gln homozygotes had an average DNA adduct level of 15.60 ± 5.42 compared with 6.16 ± 0.97 in Gln/Arg heterozygotes and 6.78 ± 1.10 in Arg/Arg homozygotes ( F = 5.237, P = 0.007). A significant odds ratio (3.81, 95% CI 1.02–14.16) to have DNA adduct levels above median value was observed for XPD -751Gln versus XPD -751Lys never-smoking homozygotes after adjustment for several confounders. These data show that all the analysed polymorphisms could result in deficient DNA repair and suggest a need for further investigation into the possible interactions between these polymorphisms, smoking and other risk factors.

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M(1)dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M(1)dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

Objectives To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death. Design Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC). Participants 303 020 people from the EPIC cohort (total 500 000) who had never smoked or who had stopped smoking for at least 10 years, 123 479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment. Main outcome measures Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people. Results Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours). Conclusions This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.

Individuals differ widely in their ability to repair DNA damage, and DNA‐repair deficiency may be involved in modulating cancer risk. In a case‐control study of 124 bladder‐cancer patients and 85 hospital controls (urological and non‐urological), 3 DNA polymorphisms localized in 3 genes of different repair pathways (XRCC1‐Arg399Gln, exon 10; XRCC3‐Thr241Met, exon 7; XPD‐Lys751Gln, exon 23) have been analyzed. Results were correlated with DNA damage measured as ³²P‐post‐labeling bulky DNA adducts in white blood cells from peripheral blood. Genotyping was performed by PCR‐RFLP analysis, and allele frequencies in cases/controls were as follows: XRCC1‐399Gln = 0.34/0.39, XRCC3‐241Met = 0.48/0.35 and XPD‐751Gln = 0.42/0.42. Odds ratios (ORs) were significantly greater than 1 only for the XRCC3 (exon 7) variant, and they were consistent across the 2 control groups. XPD and XRCC1 appear to have no impact on the risk of bladder cancer. Indeed, the effect of XRCC3 was more evident in non‐smokers [OR = 4.8, 95% confidence interval (CI) 1.1–21.2]. XRCC3 apparently interacted with the N‐acetyltransferase type 2 (NAT‐2) genotype. The effect of XRCC3 was limited to the NAT‐2 slow genotype (OR = 3.4, 95% CI 1.5–7.9), suggesting that XRCC3 might be involved in a common repair pathway of bulky DNA adducts. In addition, the risk of having DNA adduct levels above the median was higher in NAT‐2 slow acetylators, homozygotes for the XRCC3‐241Met variant allele (OR = 14.6, 95% CI 1.5–138). However, any discussion of interactions should be considered preliminary because of the small numbers involved. Our results suggest that bladder‐cancer risk can be genetically modulated by XRCC3, which may repair DNA cross‐link lesions produced by aromatic amines and other environmental chemicals.

Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N -nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic mutations and cancer. Our study investigated the relationships between 22 polymorphisms (and their haplotypes) in 16 DNA repair genes belonging to different repair pathways in 1094 controls and 567 cancer cases (bladder cancer, 131; lung cancer, 134; oral–pharyngeal cancer, 41; laryngeal cancer, 47; leukaemia, 179; death from emphysema and chronic obstructive pulmonary disease, 84). The design was a case–control study nested within a prospective investigation. Among the many comparisons, few polymorphisms were associated with the diseases at the univariate analysis: XRCC1 -399 Gln/Gln variant homozygotes [odds ratios (OR) = 2.20, 95% confidence intervals (CI) = 1.16–4.17] and XRCC3 -241 Met/Met homozygotes (OR = 0.51, 95% CI = 0.27–0.96) and leukaemia. The recessive model in the stepwise multivariate analysis revealed a possible protective effect of XRCC1 -399Gln/Gln in lung cancer (OR = 0.22, 95% CI = 0.05–0.98), and confirmed an opposite effect (OR = 2.47, 95% CI = 1.02–6.02) in the leukaemia group. Our results also suggest that the XPD/ERCC1- GAT haplotype may modulate leukaemia (OR = 1.28, 95% CI = 1.02–1.61), bladder cancer (OR = 1.38, 95% CI = 1.06–1.79) and possibly other cancer risks. Further investigations of the combined effects of polymorphisms within these DNA repair genes, smoking and other risk factors may help to clarify the influence of genetic variation in the carcinogenic process.

The aim of this project was to devise and test improved protocols of the ³²P-postlabelling assay for the detection of carcinogen–DNA adducts. The intention was to reverse the drift of different investigators using increasingly divergent experimental conditions. This would lead to a more standardized assay that can be used in future applications by different investigators for the monitoring of human exposure to genotoxic agents, permitting more meaningful comparisons between different studies or between different participants in the same study. As part of this process, there was perceived to be a need for carcinogen-modified DNA standards of known levels of adducts for use as positive controls, as standards for normalization of results with unknown samples and to assist interlaboratory comparisons. The preparation of characterized DNA standards modified by benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), 4-aminobiphenyl (ABP), an aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine, and N-methyl-N-nitrosourea (MNU), a methylating agent yielding DNA containing O⁶-methylguanine, was carried out. A critical appraisal of all aspects of the ³²P-postlabelling procedure and investigations to examine the influence of a number of key variations on the assay were conducted. There followed testing of a consensus protocol in a first interlaboratory trial involving 25 participants in Europe and the USA, conducted on the prepared synthetic DNA standards, the assessment of interlaboratory variability and the reasons for it. Revision of the protocols was followed by further testing in a second interlaboratory trial in which liver DNA from mice treated with BaP or ABP were assayed together with the synthetic DNA standards. Adduct levels were found to be significantly lower by ³²P-postlabelling than by ³H incorporation. A recommended set of procedures has been developed for the detection and quantitation of DNA adducts formed by PAHs, aromatic amines and methylating agents. These trials have led to a much clearer idea as to what are the critical features and procedures of the ³²P-postlabelling assay and there is a set of standard DNA samples for use in quality control and against which biological samples can be normalized. Use of these standards and procedures has reduced interlaboratory variability in quantitation of DNA adducts.