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Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.

We followed-up for mortality and cancer incidence 1088 healthy non-smokers from a population-based study, who were characterized for 22 variants in 16 genes involved in DNA repair pathways. Follow-up was 100% complete. The association between polymorphism and mortality or cancer incidence was analyzed using Cox Proportional Hazard regression models. Ninety-five subjects had died in a median follow-up time of 78 months (inter-quartile range 59-93 months). None of the genotypes was clearly associated with total mortality, except variants for two Double-Strand Break DNA repair genes, XRCC3 18067 C>T (rs#861539) and XRCC2 31479 G>A (rs#3218536). Adjusted hazard ratios were 2.25 (1.32-3.83) for the XRCC3 C/T genotype and 2.04 (1.00-4.13) for the T/T genotype (reference C/C), and 2.12 (1.14-3.97) for the XRCC2 G/A genotype (reference G/G). For total cancer mortality, the adjusted hazard ratios were 3.29 (1.23-7.82) for XRCC3 C/T, 2.84 (0.81-9.90) for XRCC3 T/T and 3.17 (1.21-8.30) for XRCC2 G/A. With combinations of three or more adverse alleles, the adjusted hazard ratio for all cause mortality was 17.29 (95% C.I. 8.13-36.74), and for all incident cancers the HR was 5.28 (95% C.I. 2.17-12.85). Observations from this prospective study suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly influence the risk of cancer and non-cancer disease, and can influence mortality.

In contrast to some extensively examined food mutagens, for example, aflatoxins, N-nitrosamines and heterocyclic amines, some other food contaminants, in particular polycyclic aromatic hydrocarbons (PAH) and other aromatic compounds, have received less attention. Therefore, exploring the relationships between dietary habits and the levels of biomarkers related to exposure to aromatic compounds is highly relevant. We have investigated in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort the association between dietary items (food groups and nutrients) and aromatic DNA adducts and 4-aminobiphenyl-Hb adducts. Both types of adducts are biomarkers of carcinogen exposure and possibly of cancer risk, and were measured, respectively, in leucocytes and erythrocytes of 1086 (DNA adducts) and 190 (Hb adducts) non-smokers. An inverse, statistically significant, association has been found between DNA adduct levels and dietary fibre intake (P = 0.02), vitamin E (P = 0.04) and alcohol (P = 0.03) but not with other nutrients or food groups. Also, an inverse association between fibre and fruit intake, and BMI and 4-aminobiphenyl-Hb adducts (P = 0.03, 0.04, and 0.03 respectively) was observed. After multivariate regression analysis these inverse correlations remained statistically significant, except for the correlation adducts v. fruit intake. The present study suggests that fibre intake in the usual range can modify the level of DNA or Hb aromatic adducts, but such role seems to be quantitatively modest. Fibres could reduce the formation of DNA adducts in different manners, by diluting potential food mutagens and carcinogens in the gastrointestinal tract, by speeding their transit through the colon and by binding carcinogenic substances.

Background: The suspect carcinogens, heterocyclic amines (HAAs), found in well-done meat require host-mediated metabolic activation before inducing DNA mutations. The role of SULT1A1 and of NAT2 on the activation of HAAs suggests that NAT2 rapid acetylator genotype and SULT1A1 allele variants can have an effect on HAA carcinogenicity., Methods: Data were collected as part of a case-control study nested within the EPIC cohort, the Gen Air investigation. EPIC is a prospective study designed to investigate the relationship between nutrition and cancer. Information was collected through a non-dietary questionnaire on lifestyle variables and through a dietary questionnaire. The subjects were restricted to non-smokers. We calculated the matched odds ratio for bladder cancer risk using logistic regression, controlling for potential confounders., Results: There were 227 bladder cases and 612 controls matched 1:3. Meat intake and NAT2 genotype were not independently associated with bladder cancer risk. A significant relationship was observed between bladder cancer risk and consumption of meat only among subjects with the rapid NAT2 genotype (odds ratios [OR] 2.9, 95% CI 1.0-7.9 for the 2nd quartile of meat intake; 3.6, 95% CI 1.3-9.7 for the 3rd quartile; and 3.5, 95% CI 1.2-9.7 for the 4th quartile), and was not present among subjects with the slow genotype. An interaction between NAT2 and meat intake was found in logistic regression (P = 0.034). No association was observed for SULT1A *1/2 genotype (1.0; 95% CI 0.7-1.5) and for SULT1A1 *2/2 genotype (0.9; 95% CI 0.5-1.7)., Conclusions: These results are suggestive of a role of meat intake and NAT2 on bladder cancer risk. They support the hypothesis that among subjects with the rapid NAT2 acetylation genotype higher levels of HAAs exposure are a bladder cancer risk factor. We did not observe an effect of SULT1A1 allele variants on this cancer. The present study adds new information on the possible long-term adverse effects of diets with high meat intake.

A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P < 0.001) in flavonoid intake. Urinary phenolic content and anti-mutagenicity did not significantly change after diet, nor was a correlation found between flavonoid intake and urinary phenolic levels or anti-mutagenicity. Phenolic levels showed a significant positive correlation with urinary anti-mutagenicity. AHR levels were significantly reduced after the diet (P = 0.038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0.038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

Background: We chose a set of candidate single nucleotide polymorphisms (SNPs) to investigate gene-environment interactions in three types of cancer that have been related to air pollution (lung, bladder and myeloid leukemia)., Patients and Methods: The study has been conducted as a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (409 cancer cases and 757 matched controls). We included never and ex-smokers. SNPs were in genes involved in oxidative stress, phase I metabolizing genes, phase II metabolizing genes and methylenetetrahydrofolate reductase (MTHFR)., Results: The most notable findings are: GSTM1 deletion and bladder cancer risk [odds ratio (OR) = 1.60; 95% confidence interval 1.00-2.56]; CYP1A1 and leukemia (2.22, 1.33-3.70; heterozygotes); CYP1B1 and leukemia (0.47, 0.27-0.84; homozygotes); MnSOD and leukemia (1.91, 1.08-3.38; homozygotes) and NQO1 and lung cancer (8.03, 1.73-37.3; homozygotes). Other statistically significant associations were found in subgroups defined by smoking habits (never or ex-smokers), environmental tobacco smoke or gender, with no obvious pattern. When gene variants were organized according to the three main pathways, the emerging picture was of a strong involvement of combined phase I enzymes in leukemia, with an OR of 5 (1.63-15.4) for those having three or more variant alleles. The association was considerably stronger for leukemias arising before the age of 55.

It is becoming increasingly evident that single-locus effects cannot explain complex multifactorial human diseases like cancer. We applied the multi-factor dimensionality reduction (MDR) method to a large cohort study on gene-environment and gene-gene interactions. The study (case-control nested in the EPIC cohort) was established to investigate molecular changes and genetic susceptibility in relation to air pollution and environmental tobacco smoke (ETS) in non-smokers. We have analyzed 757 controls and 409 cases with bladder cancer (n=124), lung cancer (n=116) and myeloid leukemia (n=169). Thirty-six gene variants (DNA repair and metabolic genes) and three environmental exposure variables (measures of air pollution and ETS at home and at work) were analyzed. Interactions were assessed by prediction error percentage and cross-validation consistency (CVC) frequency. For lung cancer, the best model was given by a significant gene-environment association between the base excision repair (BER) XRCC1-Arg399Gln polymorphism, the double-strand break repair (DSBR) BRCA2-Asn372His polymorphism and the exposure variable 'distance from heavy traffic road', an indirect and robust indicator of air pollution (mean prediction error of 26%, P<0.001, mean CVC of 6.60, P=0.02). For bladder cancer, we found a significant 4-loci association between the BER APE1-Asp148Glu polymorphism, the DSBR RAD52-3'-untranslated region (3'-UTR) polymorphism and the metabolic gene polymorphisms COMT-Val158Met and MTHFR-677C>T (mean prediction error of 22%, P<0.001, mean CVC consistency of 7.40, P<0.037). For leukemia, a 3-loci model including RAD52-2259C>T, MnSOD-Ala9Val and CYP1A1-Ile462Val had a minimum prediction error of 31% (P<0.001) and a maximum CVC of 4.40 (P=0.086). The MDR method seems promising, because it provides a limited number of statistically stable interactions; however, the biological interpretation remains to be understood.

To estimate the relationship between air pollution and lung cancer, a nested case-control study was set up within EPIC (European Prospective Investigation on Cancer and Nutrition). Cases had newly diagnosed lung cancer, accrued after a median follow-up of 7 years among the EPIC ex-smokers (since at least 10 years) and never smokers. Three controls per case were matched. Matching criteria were gender, age (+/-5 years), smoking status, country of recruitment and time elapsed between recruitment and diagnosis. We studied residence in proximity of heavy traffic roads as an indicator of exposure to air pollution. In addition, exposure to air pollutants (NO(2), PM10, SO(2)) was assessed using concentration data from monitoring stations in routine air quality monitoring networks. Cotinine was measured in plasma. We found a nonsignificant association between lung cancer and residence nearby heavy traffic roads (odds ratio = 1.46, 95% confidence interval, CI, 0.89-2.40). Exposure data for single pollutants were available for 197 cases and 556 matched controls. For NO(2) we found an odds ratio of 1.14 (95% CI, 0.78-1.67) for each increment of 10 microg/m(3), and an odds ratio of 1.30 (1.02-1.66) for concentrations greater than 30 microg/m(3). The association with NO(2) did not change after adjustment by cotinine and additional potential confounders, including occupational exposures. No clear association was found with other pollutants.

In cancer patients, plasma often contains mutant DNA released by cancer cells. We have assessed the significance of plasma DNA mutations for subsequent cancer development in healthy subjects in a large longitudinal prospective study. The European Prospective Investigation into Cancer and Nutrition study was analyzed with a nested case-control design. Cases were nonsmokers or ex-smokers for >10 years and newly diagnosed with lung, bladder, or upper aerodigestive tract cancers or leukemia accrued after a median follow-up of 6.3 years. Controls were matched 2:1 for follow-up, age, sex, area of recruitment, and smoking status. KRAS2 mutations were detected by mutant-enriched PCR and sequencing (n = 1,098). TP53 mutations were detected by denaturing high-performance liquid chromatography, temporal temperature gradient electrophoresis, and sequencing (n = 550). KRAS2 or TP53 mutations were detected in 13 of 1,098 (1.2%) and 20 of 550 (3.6%) subjects, respectively, 16 of whom developed cancer on average after 18.3 months of follow-up. Among 137 subjects who developed bladder cancer, 5 had KRAS2 mutations [odds ratio (OR), 4.25; 95% confidence interval (95% CI), 1.27-14.15] and 7 had TP53 mutations (OR, 1.81; 95% CI, 0.66-4.97). There was a nonsignificant trend for association between TP53 mutations and bulky adducts in lymphocyte DNA (OR, 2.78; 95% CI, 0.64-12.17). This is the first report of TP53 or KRAS2 mutations in the plasma of healthy subjects in a prospective study, suggesting that KRAS2 mutation is detectable ahead of bladder cancer diagnosis. TP53 mutation may be associated with environmental exposures. These observations have implications for monitoring early steps of carcinogenesis.

We have conducted a randomized trial which investigated the ability of dietary changes (in particular diets rich in cruciferous vegetables and flavonoids), to increase urinary antimutagenicity and inhibit DNA damage in smokers. Ninety heavy smokers were recruited and randomly assigned to three groups, and were given three different diets. The first diet was based on flavonoid-rich foods, particularly cruciferous vegetables, but not based on supplementation; the second was a normal isocaloric diet (with an adequate administration of fruits and vegetables); and the third was based on supplementation of flavonoids in the form of green tea and soy products. DNA adducts were measured by (32)P-postlabelling in exfoliated bladder cells at different times since the start of the trial. In spite of randomization, subjects in the control group smoked more than those in the experimental groups, and this can explain the higher adduct levels at baseline. A slight decrease in bulky DNA adducts in exfoliated bladder cells was observed after 1 year since the end in the supplementation group and after 1 month in white blood cells. The only statistically significant association was found in a regression model that adjusted for smoking, in which the increase in flavonoid intake was associated with a decrease in adducts after 1 year (P = 0.02). These data suggest that adherence to a diet rich in cruciferous vegetables and flavonoids might reduce genotoxicity in the human urinary bladder of smokers, but they should be interpreted with caution owing to small numbers and the uneven distribution of smoking habits in the experimental groups. Smoking is the most important single preventable cause of cancer; at the present stage of knowledge it is totally unlikely that certain dietary habits can seriously counteract the effects of tobacco smoking.

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.

Objectives were to investigate prospectively the ability of DNA adducts to predict cancer and to study the determinants of adducts, especially air pollutants. DNA adducts were measured in a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) investigation. Cases included newly diagnosed lung cancer (n = 115), upper respiratory cancers (pharynx and larynx; n = 82), bladder cancer (n = 124), leukemia (n = 166), and chronic obstructive pulmonary disease or emphysema deaths (n = 77) accrued after a median follow-up of 7 years among the EPIC former smokers and never-smokers. Three controls per case were matched for questionnaire analyses and two controls per case for laboratory analyses. Matching criteria were gender, age, smoking status, country of recruitment, and follow-up time. Individual exposure to air pollution was assessed using concentration data from monitoring stations in routine air quality monitoring networks. Leukocyte DNA adducts were analyzed blindly using 32P postlabeling technique. Adducts were associated with the subsequent risk of lung cancer, with an odds ratio (OR) of 1.86 [95% confidence interval (95% CI), 0.88-3.93] when comparing detectable versus nondetectable adducts. The association with lung cancer was stronger in never-smokers (OR, 4.04; 95% CI, 1.06-15.42) and among the younger age groups. After exclusion of the cancers occurring in the first 36 months of follow-up, the OR was 4.16 (95% CI, 1.24-13.88). A positive association was found between DNA adducts and ozone (O3) concentration. Our prospective study suggests that leukocyte DNA adducts may predict lung cancer risk of never-smokers. Besides, the association of DNA adduct levels with O3 indicates a possible role for photochemical smog in determining DNA damage.

The aim of this study was to evaluate whether biomarkers of environmental tobacco smoke exposure [i.e., 4-aminobiphenyl-hemoglobin (4-ABP-Hb) adducts] were predictive of the risk of tobacco-related cancers and diseases. We did a case-control study nested within the European Prospective Investigation into Cancer and Nutrition, involving 190 controls and 149 cases (incident cancer of the lung, bladder, pharynx, larynx, oral cavity, leukemias, and chronic obstructive pulmonary disease or emphysema deaths). All individuals were never smokers or ex smokers for >10 years. 4-ABP-Hb adducts were analyzed in peripheral blood collected before the onset of the disease (median, 7 years). Overall, 4-ABP-Hb adducts were higher, although not statistically significantly so, in cases (as a whole) than controls. In the control population, high fruit and vegetable consumption significantly lowered the frequency of detectable adducts (Fisher's exact test, P = 0.025). Restricting the analysis to women, 4-ABP-Hb adducts were higher in cases than controls (Mann-Whitney P = 0.036) and the odds ratio (OR) for the presence/absence of adducts was 2.42 [95% confidence interval (95% CI), 1.18-4.98]. Moreover, the association of adducts with the individual cancer types was stronger in women than in the whole study population, although statistically significant only for leukemias (OR, 2.77; 95% CI, 1.06-7.20). The results provide some evidence that women may be more susceptible to environmental tobacco smoke, as suggested by their higher adduct levels. The most important finding of this prospective study is that, at least in women, 4-ABP-Hb adducts may help identify subjects at high risk of cancers related to environmental tobacco smoke exposure.

Several large prospective investigations are under way or are planned in different parts of the world, aiming at the investigation of gene-environment interactions for chronic diseases. Technical, practical and ethical issues are raised by such large investigations. Here we describe how such issues were approached within a case-control study nested in EPIC, a large European cohort, and the kind of validation studies that have been set up. The GenAir investigation aimed at measuring the effects of air pollution and environmental tobacco smoke on human health in EPIC with a nested design and with biological measures. Validation studies included (a) comparisons between cotinine measurements, hemoglobin adducts and questionnaire data; (b) an analysis of the determinants of DNA adduct concentration; (c) comparison among different genotyping methods; (d) an analysis of the determinants of plasma DNA amounts. We also describe how the ethical issues were dealt with in our investigation.

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.

Levels of plasma DNA concentrations in cancer patients have been shown to be higher than the plasma DNA concentrations found in healthy subjects. The value of plasma DNA levels for development of neoplastic or pulmonary disease was evaluated in a large prospective study. Plasma samples (n = 1,184) were analyzed from 776 controls, 359 cases of cancer (lung, bladder, oral cavity, pharynx, larynx, leukemia) and 49 deaths from chronic obstructive pulmonary disease (COPD), including never smokers and ex-smokers, from 9 countries across Europe. The amount of plasma DNA was variable across the European Prospective Investigation into Cancer and Nutrition (EPIC) centers. High DNA concentrations in some centers might be due to the type of population recruited and/or the treatment of the samples. An elevated and statistically significant odds ratio (OR) was found for COPD deaths (OR = 2.53; 95% CI = 1.06-6.02), while nonsignificant increased ORs were present for oral cancers, cancers of the pharynx and larynx and leukemia. When the analyses were stratified by time since recruitment (below or above 36 months), the increased ORs were limited to the more recent period of recruitment, i.e., a time elapsed between blood drawing and disease onset lower than 36 months. This was particularly true for COPD deaths (OR = 12.7; 95% CI = 1.57-103) and leukemia (OR = 2.37; 95% CI = 1.20-4.67)., (Copyright 2004 Wiley-Liss, Inc.)

We have undertaken a randomized trial to confirm the ability of a class of phenolics, flavonoids, to increase urinary anti-mutagenicity in smokers. Ninety heavy smokers were recruited and randomly assigned to three groups, who were given three different diets. One diet was rich in flavonoids, but not based on supplementation ('flavonoid'), one was a normal iso-caloric diet with an adequate administration of fruit and vegetables ('normal'), and one was based on supplementation of flavonoids in the form of green tea and soy products ('supplement'). The urinary anti-mutagenicity-as inhibiting effect of the urinary extracts on the mutations induced by MeIQx-was measured in Salmonella typhimurium YG1024 in the presence of liver S9 from male Sprague-Dawley rats treated with Aroclor 1254. The amount of total phenolics in the urinary extracts was measured by use of spectrometric analysis. We found that important dietary modifications can be achieved through special recipes and instructions given by a cook during an intensive course. The intervention was focused on increasing the flavonoid intake, and it was successful in that respect. In fact, differences in flavonoid intake were appreciated mainly between the first group (normal diet) and the other two (flavonoid-rich and supplemented diet), suggesting that dietary modification can be as effective as supplementation. However, both urinary anti-mutagenicity and the amounts of urinary phenolics did not change as a consequence of the trial. These results suggest that only a small fraction of urinary phenolics is influenced by dietary changes in the intake of flavonoids, and that most urinary anti-mutagens and phenolics are metabolites of dietary flavonoids, whose formation is more affected by the activity and diversity of bacterial flora in the colon than by the quantity and type of intake. A strong correlation was found between urinary phenolics and anti-mutagenicity in all the groups involved in the trial. Such correlation was not explained by dietary variables.

Tobacco smoking and occupation are major risk factors of bladder cancer via exposure to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. Glutathione S-transferase (GST) M1, T1 and P1 are involved in the detoxification of PAH reactive metabolites. Two N-acetyltransferase isozymes, NAT2 and NAT1, have major roles in catalyzing the N-acetylation and O-acetylation of aromatic amines. Cytochrome p450 1B1 (CYP1B1) and sulfotransferase 1A1 (SULT1A1) are also involved in the metabolism of PAHs and aromatic amines. It is hypothesized that the genetic polymorphisms of these metabolic enzymes have an effect on the individual susceptibility to bladder cancer in particular by interacting with relevant environmental exposures. A hospital-based case-control study among men in Brescia, Northern Italy recruited 201 incidence cases and 214 controls from 1997-2000. Occupational exposures were blindly coded by occupational physicians. Genotyping of polymorphisms were carried out with PCR-RFLP method. Unconditional multivariate logistic regression was applied to model the association between genetic polymorphisms and bladder cancer risk. Effect modifications by age of onset, smoking and occupational exposures to PAHs and aromatic amines were evaluated. We also conducted an analysis of interaction between genetic factors. GSTM1 and GSTT1 null genotype were associated with an increased risk of bladder cancer with an odds ratio (OR) of 1.69 (95% confidence interval [CI] = 1.11-2.56) and 1.74 (95% CI = 1.02-2.95), respectively. The effect of GSTM1 null was seen particularly in heavy smokers, and there was a combined effect with occupational exposure of aromatic amines (OR = 2.77, 95% CI = 1.08-7.10). We observed a trend (p-value < 0.01) of increasing cancer risk comparing subjects with normal GSTM1 and T1 activity to subjects with one (OR = 1.82, 95% CI = 1.16-2.85) or both null genotypes (OR = 2.58, 95% CI = 1.27-5.23). NAT2 slow acetylator was associated with marginally increased risk of bladder cancer (OR = 1.50, 95% CI = 0.99-2.27), and the OR for the joint effect with occupational exposure of aromatic amines was 3.26 (95% CI = 1.06-9.95). SULT1A1 Arg213His polymorphism showed a marginal protective effect. These findings suggest that individual susceptibility to bladder cancer may be modulated by GSTM1, GSTT1 and NAT2 polymorphisms., (Copyright 2004 Wiley-Liss, Inc.)

Tobacco smoking and occupational exposure are major risk factors of bladder cancer via exposure to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines, which lead to oxidative stress and DNA damage. Several enzymes, which play key roles in oxidative stress are polymorphic in humans. Myeloperoxidase (MPO) produces a strong oxidant for microbicidal activity, and activates carcinogens in tobacco smoke. Catechol-O-methyltransferase (COMT) catalyzes the methylation of endo- and xenobiotics and prevents redox cycling. NAD(P)H:quinone oxidoreductase (NQO1) catalyzes the two-electron reduction of quinoid compounds, which also protects cells from redox cycling. Manganese superoxide dismutase (MnSOD) protects cells from free radical injury. To test the hypothesis that the risk of bladder cancer can be influenced by polymorphisms in the genes that modulate oxidative stress, in particular by interacting with environmental carcinogens, we conducted a hospital-based case-control study among men in Brescia, Northern Italy. We recruited and interviewed 201 incident cases and 214 controls from 1997 to 2000. Occupational exposures to PAHs and aromatic amines were coded blindly by occupational physicians. Unconditional multivariate logistic regression was applied to model the association between genetic polymorphisms and bladder cancer risk and the effect of modifications of smoking and occupational exposures were evaluated. MPO G-463A homozygous variant was associated with a reduced risk of bladder cancer with an OR of 0.31 (95% CI = 0.12-0.80). MnSOD Val/Val genotype increased the risk of bladder cancer with OR of 1.91 (95% CI = 1.20-3.04), and there was a combined effect with smoking (OR = 7.20, 95% CI = 3.23-16.1) and PAH (OR = 3.02, 95% CI = 1.35-6.74). We did not observe an effect of COMT Val108Met polymorphism. These findings suggest that individual susceptibility of bladder cancer may be modulated by MPO and MnSOD polymorphisms, and that the combination of genetic factors involved in oxidative stress response with environmental carcinogens may play an important role in bladder carcinogenesis.

Background: Genetic association studies are generating much information, usually in the form of single nucleotide polymorphisms in candidate genes. Analyzing such data is challenging, and raises issues of multiple comparisons and potential false-positive associations. Using data from a case-control study of bladder cancer, we showed how to use hierarchical modeling in genetic epidemiologic studies with multiple markers to control overestimation of effects and potential false-positive associations., Methods: The data were first analyzed with the conventional approach of estimating each main effect individually. We subsequently employed hierarchical modeling by adding a second stage (prior) model that incorporated information on the potential function of the genes. We used an empirical-Bayes approach, estimating the residual effects of the genes from the data. When the residual effect was set to zero, we instead used a semi-Bayes approach, in which they were pre-specified. We also explored the impact of using different second-stage design matrices. Finally, we used two approaches for assessing gene-environment interactions. The first approach added product terms into the first-stage model. The second approach used three indicators for subjects exposed to gene-only, environment-only, and both genetic and environmental factors., Results: By pre-specifying the prior second-stage covariates, the estimates were shrunk to the mean of each pathway. The conventional model detected a number of positive associations, which were reduced with the hierarchical model. For example, the odds ratio for myeloperoxidase (G/G, G/A) genotype changed from 3.17 [95% confidence interval (CI), 1.32-7.59] to 1.64 (95% CI, 0.81-3.34). A similar phenomenon was observed for the gene-environment interactions. The odds ratio for the gene-environment interaction between tobacco smoking and N-acetyltransferase 1 fast genotype was 2.74 (95% CI, 0.68-11.0) from the conventional analysis and 1.24 (95% CI, 0.80-1.93) from the hierarchical model., Conclusion: Adding a second-stage hierarchical modeling can reduce the likelihood of false positive via shrinkage toward the prior mean, improve the risk estimation by increasing the precision, and, therefore, represents an alternative to conventional methods for genetic association studies.

Cytochrome P450 1B1 (CYP1B1) is implicated in the activation of potentially carcinogenic xenobiotics and oestrogens. The polymorphism of the CYP1B1 gene at codon 432 (Val-->Leu) is associated with change in catalytic function. In a case-series study of breast cancer patients, we investigated the interaction between this polymorphism and environmental exposure. The women carrying the Val CYP1B1 allele and who had lived near to a waste incinerator for more than 10 years had a higher risk of breast cancer than those never exposed with the Leu/Leu genotype (odds ratio of interactions (ORi)=3.26, 95% confidence interval (CI) 1.20-8.84). Also, the Val CYP1B1 allele increased the susceptibility to breast cancer for women exposed during their life to agricultural products used in farming (ORi = 2.18, 95% CI 1.10-4.32). These xenobiotics, mainly organochlorine hydrocarbons, are known to bind to the aromatic hydrocarbon receptor (AhR), and to induce the expression of CYP1B1 enzyme. The excess risk for exposed women with a Val CYP1B1 homo/heterozygous genotype could result from a higher exposure to activated metabolites of pesticides or dioxin-like substances. Also, a higher induction of CYP1B1 enzyme by xenobiotics could increase the formation of genotoxic catechol-oestrogens among exposed women carrying the Val CYP1B1 allele. Our results suggested that the Val CYP1B1 allele increases the susceptibility to breast cancer in women exposed to waste incinerator or agricultural pollutants.

Genetic polymorphisms of enzymes involved in the metabolism of xenobiotics and estrogens might play a role in breast carcinogenesis related to environmental exposures. In a case-only study on 282 women with breast cancer, we studied the interaction effects (ORi) between smoking habits and the gene polymorphisms of Cytochrome P450 1B1 (Val432Leu CYP1B1), Phenol-sulfotransferase 1A1 (Arg213His SULT1A1) and Catechol-O-methyltransferase (Val158Met COMT). The smokers carrying the Val CYP1B1 allele associated with a high hydroxylation activity had a higher risk of breast cancer than never smokers with the Leu/Leu genotype (ORi=2.32, 95%CI: 1.00-5.38). Also, the smokers carrying the His SULT1A1 allele associated with a low sulfation activity had a 2-fold excess risk compared to never smokers carrying Arg/Arg SULT1A1 common genotype (ORi= 2.55, 95%CI: 1.21-5.36). The His SULT1A1 allele increased the risk only in premenopausal patients. The Met COMT allele with a lower methylation activity than Val COMT did not modify the risk among smokers. The excess risk due to joint effect could result from a higher exposure to activated tobacco-compounds for women homo/heterozygous for the Val CYP1B1 allele. Also, a lower sulfation of the tobacco carcinogens among women with His SULT1A1 could increase exposure to genotoxic compounds. Alternatively, the Val CYP1B1 or His SULT1A1 allele with modified ability to metabolize estrogens could increase the level of genotoxic catechol estrogen (i.e., 4-hydroxy-estradiol) among smokers. Our study showed that gene polymorphisms of CYP1B1 and SULT1A1 induce an individual susceptibility to breast cancer among current smokers., (Copyright 2003 Wiley-Liss, Inc.)

Tobacco smoking and occupational exposures are the main known risk factors for bladder cancer, causing direct and indirect damage to DNA. Repair of DNA damage is under genetic control, and DNA repair genes may play a key role in maintaining genome integrity and preventing cancer development. Polymorphisms in DNA repair genes resulting in variation of DNA repair efficiency may therefore be associated with bladder cancer risk. A hospital-based case-control study was conducted in Brescia, Italy, to assess the relationship between polymorphisms in DNA repair genes XRCC1 (Arg(399)Gln), XRCC3 (Thr(241)Met), and XPD (Lys(751)Gln) and bladder cancer risk. A total of 201 male incident bladder cancer cases and 214 male controls with urological nonneoplastic diseases were recruited and frequency-matched on age, period, and hospital of recruitment. Detailed information was collected using a semistructured questionnaire on demographic, dietary, environmental, and occupational factors. Genotypes were determined by PCR-RFLP analysis. The XRCC3 codon 241 variant genotype exhibited a protective effect against bladder cancer [odds ratio (OR), 0.63; 95% confidence interval (CI), 0.42-0.93], which was prominent among heavy smokers (OR, 0.49; 95% CI, 0.28-0.88) but not among never and light smokers. No overall impact of the XRCC1 codon 399 polymorphism was found (OR, 0.86; 95% CI, 0.59-1.28), but a protective influence of the homozygous variant was suggested among heavy smokers (OR, 0.38; 95% CI, 0.14-1.02). XPD polymorphisms did not show an association with bladder cancer (OR, 0.92; 95% CI, 0.62-1.37). There was no statistical evidence of an interaction between these three genetic polymorphisms and either tobacco smoking or occupational exposure to polycyclic aromatic hydrocarbons and aromatic amines. The XRCC3 codon 241 polymorphism had an overall protective effect against bladder cancer that was most apparent among heavy smokers. Similarly, the XRCC1 codon 399 polymorphism also had a protective effect on bladder cancer among heavy smokers. The XPD polymorphism was not, however, associated with bladder cancer risk.

Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables.

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 (32)P-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.

We have analyzed the tumor biopsies of 45 patients with bladder cancer for p53 mutations by direct sequencing. In addition to N-acetyltransferase-2 (NAT2) and GSTM1 allelisms, which were examined previously, we have analyzed the genetic polymorphisms of GSTT1, GSTP1, COMT, NQO1, TS-SULT and MPO in buffy coat DNA using PCR-based methods. All subjects were interviewed through a questionnaire on smoking, dietary habits and other risk factors. No specific pattern was evident for p53 mutations. Eight out of ten mutations occurred in grade 3 tumors. All p53 mutations occurred in subjects with the COMT mutated allele (p=0.03). The prevalence of cases with p53 mutations was 3.5-fold higher in subjects with wild type than in those with variant GSTP1 alleles (p=0.03). The other polymorphisms investigated were not associated with p53 mutations.

The 'Mediterranean diet', a diet rich in cereals, fruit and vegetables, has been associated with lowering the risk of a variety of cancers of the digestive tract and the bladder. In a previous study, we showed that the high phenolic content these dietary components produce in the urine could be associated with higher antimutagenic properties of the urine and lower arylamine-DNA adducts in exfoliated bladder cells. We have conducted a case-control study on 162 bladder cancer patients and 104 hospital controls. Total aromatic DNA adducts were measured in white blood cells (WBC) of all subjects by (32)P-post-labelling. Genetically based metabolic polymorphisms were analysed by PCR-RFLP (NAT2, GSTM1, GSTT1, GSTP1, COMT and NQO1). All subjects were interviewed about their tobacco use, dietary habits and other risk factors. The odds ratio (OR) for the risk of bladder cancer according to the presence/absence of WBC DNA adducts (detection limit 0.1 RALx10(8)) was 3.7 [95% confidence interval (CI) 2.2-6.3] and a dose-response relationship with levels of adducts was apparent. The association between case/control status and the presence of WBC DNA adducts was significantly stronger in the subjects who consumed fewer portions of fruit or vegetables per day (OR 7.80, 95% CI 3.0-20.30 for 0-1 portions of vegetables) than in the heavy consumers (OR 4.98 for consumers of 2 portions daily, OR 1.97 for consumers of > or =3 portions; similar but lower estimates were found for the intake of fruit). No association was noticed between tobacco smoking and WBC DNA adducts. Only NAT-2, among the several genotypes considered, was associated in a statistically significant way with the risk of bladder cancer (OR 1.72, 95% CI 1.03-2.87) and with the levels of WBC DNA adducts. Our report suggests that fruit and vegetables could protect against bladder cancer by inhibiting the formation of DNA adducts.

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by (32)P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.

Chronic inflammation induced by Helicobacter pylori infection has been associated with an increased risk of stomach cancer. We have analysed 167 stomach biopsies from 99 patients for H. pylori infection and immunohistochemically for the expression of inducible nitric oxide synthase (iNOS), catalase and superoxide dismutases (SODs) as markers of oxidative stress. Biopsies were graded as follows on the basis of histology: normal, superficial gastritis, variable severity of atrophic gastritis with or without intestinal metaplasia, and dysplasia. iNOS was detected in inflammatory cells in all types of gastritis with or without H. pylori infection and independently of its severity. In foveolar cells, iNOS was observed in approximately 25% of all biopsies showing any type of gastritis, but in a markedly higher proportion of dysplastic samples. Catalase and Mn-type SOD in inflammatory cells and catalase in foveolar cells were more frequently observed in marked atrophic gastritis biopsies than in less severe gastritis. Individual differences were found in the expression of these enzymes within groups with the same severity of gastritis. Prolonged oxidative stress in severe gastritis and dysplasia may play an important role in gastric carcinogenesis, through increased damage of DNA and tissue by reactive oxygen and nitrogen species.

Glutathione S-transferases (GSTs) are known to take part in detoxification of many potentially carcinogenic compounds. Therefore, polymorphisms of the GST genes have been considered as potentially important modifiers of individual risk of environmentally induced cancers. The association between lack of glutathione S-transferase M1 gene (GSTM1 null genotype) and susceptibility to smoking-related lung cancer has been actively studied, with contradictory results. In contrast, little is known about the more recently found polymorphisms in GSTM3, GSTP1 and GSTT1 genes with respect to individual responses to environmental exposures. In this study, we determined the genotype distribution of all these genes, and their combinations, among 208 Finnish lung cancer patients and 294 population controls. None of the genotypes studied had a statistically significant effect on lung cancer risk, when studied separately. However, a significant association was observed for concurrent lack of the GSTM1 and GSTT1 genes and susceptibility to squamous cell carcinoma. For that cell type, the risk was more than 2-fold when compared with that of individuals having other genotype combinations (OR = 2.3; 95% CI = 1.0-5.3; p = 0.05). Moreover, the risk was mostly attributable to patients with smoking history of 40 pack-years or less (OR = 2.9; 95% CI = 1.1-7.7; p = 0.03). In contrast, this genotype combination did not affect the risk for other histological types of lung cancer, and the other genotype combinations had no effects on individual susceptibility to this malignancy. The overall role of GST polymorphisms in modifying the lung cancer risk may therefore be more limited than has been so far anticipated.

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n=18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (e.g., quercetin, isorhamnetin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells., (Copyright 1998 Elsevier Science B.V.)

Carcinogens present in cigarette smoke and diet have been associated with pancreatic cancer. We hypothesized that heterocyclic and aromatic amines implicated in these exposures could be involved as causative agents and that therefore genetic variation in enzymes metabolizing these carcinogens could modify the risk of developing malignant and benign pancreatic disease. The effect of the genetic polymorphism of acetyltransferases (NAT1) and NAT2), glutathione S-transferase M1 (GSTM1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) on the risk of pancreatic diseases (cancer, pancreatitis) was examined in a case-control study. PCR-based assays were used for genotype analysis of genomic DNA from whole blood cells. Samples collected from Caucasian patients with diagnosed pancreatic cancer (n = 81), with non-alcoholic (n = 41) and alcoholic pancreatitis (n = 73) and from asymptomatic control subjects (n = 78) were analysed. The prevalence of GSTM1 null genotype and of NAT2 fast and slow acetylator genotypes and the distribution of frequencies for NQO1 genotypes did not differ in subjects with pancreatic diseases vs controls. For NAT1 slow acetylators a non-significant excess (P = 0.18) was found among pancreatic cancer cases vs controls. There was a significant over-representation of the GSTM1 AB or B genotype in all pancreatic disease cases combined (OR = 2.6; P < 0.05). When concurrent controls were pooled with literature controls (n = 1427), OR was 1.4 (P = 0.08). The results of this study, requiring confirmation, suggest that the polymorphism of GSTM1 and NAT1 enzymes may be associated with a modest increase in susceptibility to pancreatic diseases.

We conducted a case-control study on 114 bladder cancer patients and 46 hospital controls. DNA adducts were measured in WBCs by 32P postlabeling and showed no association with smoking habits and the glutathione-S-transferase M1 genotype. A strong association between adduct levels and the N-acetyltransferase (NAT2) genotype was found (P = 0.0002). The NAT2 genotype was associated in a nonstatistically significant way to the case-control status (odds ratio, 1.6; 95% confidence interval, 0.8-3.2). In a logistic regression model, the log of DNA adduct levels was associated in a highly significant way to the risk of bladder cancer (regression coefficient, 0.75; P = 0.0006), independently of smoking habits. Using the median of DNA adducts (RAL, 0.3) as a cutoff point, the odds ratio for the risk of bladder cancer was 4.1 (age-adjusted; 95% confidence interval, 1.9-9.0). Our study suggests that sources other than tobacco smoke contribute to the formation of aromatic DNA adducts in WBCs. The role of WBC-DNA adducts in predicting bladder cancer is still to be clarified.

Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms.

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified N-(deoxyguanosine-8-yl)-4-aminobiphenyl adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. Urinary anti-mutagenicity in vitro appears thus to be a good indicator of the anti-genotoxicity exerted by substances excreted in urine, that protect the bladder mucosal cells (and possibly other cells) against DNA damage. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n = 18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (quercetin, isorhamnetin and naringenin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited noncompetitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Several structural features of the flavonoids were identified as necessary for the inhibition of PhIP and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxiline mutagenicity. Fractionation by reverse-phase HPLC and subsequent analysis of two urinary extracts, showed the presence of several anti-mutagenic substances and phenolics; more lipophilic phenolics displayed the highest specific inhibitory activity. This suggests that enzymatic conversion of dietary flavonoids into their more lipophilic and anti-mutagenic O-methylcatechol derivatives, as noted for quercetin, may occur in vivo in man. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells. This protective effect of dietary phenolics against the cancer of the bladder (and possibly other sites) should be verified and explored as a part of a chemoprevention strategy.

Cytochrome P4501A2 (CYP1A2) activity may be related to bladder cancer risk through metabolic activation of aromatic amines, such as 4-aminobiphenyl (ABP), to reactive intermediates that can form DNA and hemoglobin (Hb) adducts. In the context of a study on smoking and bladder cancer risk, 97 healthy male volunteers were investigated. CYP1A2-dependent N-oxidation activity was measured using a molar ratio of urinary caffeine metabolites [(paraxanthine + 1,7-dimethyluric acid)/caffeine] obtained between the fourth and fifth h after drinking a standardized cup of coffee. N-Oxidation activity was induced by blond tobacco smoke, meat consumption the dinner before the test, or more than four cups of coffee a day. The regular use of medication appeared associated with a decrease in N-oxidation levels. Age and alcohol consumption were not related with CYP1A2 activity. A polymorphic distribution of the CYP1A2 and N-acetyltransferase-2 (determined by the caffeine metabolite ratio 5-acetylamino-6-formylamino-3-methyluracil:1-methylxanthine) phenotypes was examined in relation to susceptibility to ABP-Hb adduct formation. Rapid oxidizers and subjects with the combined slow acetylator-rapid oxidizer phenotype showed the highest ABP-Hb adduct levels at a low smoking dose. Blond tobacco smokers exhibited higher adduct levels compared with black tobacco smokers, after adjustment for the quantity of cigarettes smoked. At the highest levels of smoking exposure, no major difference in ABP-Hb adduct levels was found among the different combinations of CYP1A2 and N-acetyltransferase-2 phenotypes. In a subset of only 45 available samples, no association was seen between the ABP-Hb adduct levels and the glutathione S-transferase M1 genotype.

High red meat diets have been linked with risk of sporadic colorectal cancer; but their effects on mutations which occur in this cancer are unknown. G-->A transitions in K-ras occur in colorectal cancer and are characteristic of the effects of alkylating agents such as N-nitroso compounds (NOC). We studied th effect of red meat consumption on faecal NOC levels in eight male volunteers who consumed diets low or high in meat (60 or 600 g/day), as beef, lamb or pork, whilst living in a metabolic suite. Increased intake of red meat induced a significant (P<0.024) 3-fold increase from 40 + or - 7 to ab average of 113 + or - 25 microgram/day NOC, a range of exposure in faeces similar to that from tobacco-specific NOC in cigarette smoke. THe diets were isoenergetic and contained equal amounts of fat, but concentrations of heterocyclic amines were low. Faecal excretion of the promotor ammonia was significantly increased to 6.5 + or - 1.08 mmol/day. When the high red meat diets were supplemented with 20 g phytate-free wheat bran in six volunteers there was no reduction in NOC levels (mean 138 + or - 41 microgram/day NOC), but faecal weight increased. Higher starch and non-starch polysaccharide intakes reduced intraluminal cross-linking in microcapsules (r=-0.77) and reduced faecal pH (r=-0.64). In two volunteers there was no effect of 600 g white meat and fish o faecal NOC (mean low white meat diet 68 + or - 10 microgram/day, high white meat 56 + or -6 microgram/day nor on faecal nitrate, nitrite and iron. Faecal nitrite levels increased on changing from a white to red meat diet (mean high white meat diet 46 + or - 7 mg/day, high red meat diet mean 80 + or - 7 mg/day.) Increased endogenous production of NOC and precursors from increased red meat, but not white meat and fish, consumption may be relevant to the aetiology of colorectal cancer.

Markers of exposure to polycyclic aromatic hydrocarbons (urinary 1-hydroxypyrene-glucuronide) and aromatic amines (4-aminobiphenyl-hemoglobin adducts), as well as urinary mutagenicity, were measured in 47 healthy smokers and 50 nonsmokers. DNA adducts were determined by P32-postlabeling in the exfoliated bladder cells of 39 healthy subjects. Both 1-hydroxypyrene-glucuronide (1-OHPG) and 4-aminobiphenyl adducts (4-ABP-Hb) were associated with smoking habits, but only 4-ABP-Hb adducts were associated with consumption of black, air-cured tobacco. The levels of 2 DNA adducts (numbers 2 and 4) in urothelial cells were clearly associated with 4-ABP-Hb adducts, in all subjects and in smokers. Levels of one of these DNA adducts (number 2) were also associated with 1-hydroxypyrene-glucuronide in urines, but in smokers the association was not statistically significant. Overall, these observations constitute further evidence of a role of arylamines in tobacco-induced bladder cancer.

A case-control study on lung cancer patients demonstrated the pronounced effect of tobacco smoke on pulmonary carcinogen metabolism and suggested the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer. Lung cancer patients who were recent smokers showed in their lungs (i) significantly induced CYP1A1-related enzyme activity vs smoking non-lung cancer patients; (ii) increased benzo(a)pyrene (BP) tetrol formation from BP 7,8-diol by lung microsomes; and (iii) high levels of cytochrome P4501a1 by immunohistochemical staining. Levels of bulky aromatic DNA adducts (by 32P-postlabelling) and of BP-diol-epoxide (BPDE) adducts (by HPC/fluorometry) were quantified in lung parenchyma. Aryl hydrocarbon hydroxylase activity and the level of BPDE-DNA adducts (r = 0.91; p < 0.001) and to a lesser degree bulky DNA adducts were correlated. Thus pulmonary CYP1A1 expression (inducibility) controls in part polycyclic aromatic hydrocarbon-DNA adduct formation in tobacco smokers and, therefore, appears to be associated with lung cancer risk. High risk subjects for lung cancer among smokers may be identifiable through genotyping for polymorphic drug metabolizing enzymes in combination with molecular dosimetry of carcinogen-DNA adducts and mutation analysis in target (surrogate) cells. Such studies in a Finnish cohort of lung cancer patients and controls are in progress. Interim results of the effect of metabolic polymorphism on the level of PAH-DNA adducts and on the excretion of mutagens in urine are summarized.

Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 micrograms/ml) were spiked with an internal standard, d9-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130 degrees C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [3H]N-hydroxy-4-ABP modified DNA standards, we observed 59 +/- 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 10(8) to 1 in 10(4) nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the 32P-postlabeling method. There was excellent agreement (r = 0.994, p < 0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the 32P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be < 0.32 to 49.5 adducts per 10(8) nucleotides in the lungs and < 0.32 to 3.94 adducts per 10(8) nucleotides in the bladder samples.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic activation or inactivation of carcinogens varies considerably in human populations, and is partly genetically determined. Inter-individual variability in the susceptibility to carcinogens may be particularly important at low degrees of environmental exposure. Examples of probable human carcinogens that present widespread low-dose exposures are environmental tobacco smoke and diesel exhaust. We have determined levels of DNA adducts in bladder cells and of 4-aminobiphenyl-haemoglobin adducts in 97 volunteers, together with the N-acetylation non-inducible phenotype, the corresponding genotype, and the levels of nicotine-cotinine in the urine. We find that among the slow acetylators, 4-aminobiphenyl adducts were higher than in rapid acetylators at low or null nicotine-cotinine levels, whereas the difference between slow and rapid acetylators was less evident at increasing nicotine-cotinine levels. The N-acetyltransferase genotype is highly predictive of the acetylation phenotype. Our results indicate that the clearance of low-dose carcinogens is decreased in the genetically based slow-acetylator phenotype. Such genetic modulation of low-dose environmental risks is relevant to 'risk assessment' procedures.

Ochratoxin A (OTA), its major metabolite in rodents, ochratoxin alpha, and seven structurally related substances were assayed for SOS DNA repair inducing activity in Escherichia coli strain PQ37. At concentrations of 0.1-4 mM, OTA, chloroxine, 5-chloro-8-quinolinol, 4-chloro-meta-cresol and chloroxylenol induced SOS DNA repair in the absence of an exogenous metabolic activation system. Ochratoxin B, ochratoxin alpha, 5-chlorosalicylic acid and citrinin were inactive, but all except ochratoxin alpha were cytotoxic. Thus, the presence of chlorine at C-5 appears to be one determinant of genotoxicity in these substances. Amino oxyacetic acid, an inhibitor of the cysteine conjugate beta-lyase, decreased the cytotoxicity of OTA but did not alter its genotoxic activity, suggesting the formation of a cytotoxic thiol-containing derivative. The mechanisms by which OTA and some of its active analogues induce SOS DNA repair activity was further investigated in E. coli PQ37 and in three derived strains (PQ300, OG100 and OG400), containing deletions within the oxy R regulon. The response in strain PQ37 was measured in the absence and presence of Trolox C, a water-soluble form of vitamin E. Trolox C completely quenched the genotoxicity of OTA, and the effect was similar in the mutant and wild-type strains. These results implicate an OTA-derived free radical rather than reduced oxygen species as genotoxic intermediate(s) in bacteria.

The hypothesis that intragastric synthesis of N-nitroso compounds (NOC) in early life could play a role in gastric carcinogenesis was tested by applying the N-nitrosoproline (NPRO) test to about 50 children living in high- and low-risk areas for stomach cancer in Costa Rica. The median values of excretion of NPRO and the sum of three nitrosamino acids (micrograms/12 h urine) were 10-20% of those in adults from other geographical high-risk areas for stomach cancer. The urinary NPRO level after proline intake was higher in children from the high-risk area (P < 0.04) and markedly reduced after ingestion of ascorbic acid together with proline (P < 0.05). NPRO levels on the day of proline intake were highly correlated with levels of nitrate excretion (P < 0.001). Mean levels of total NOC in an aqueous (pH 2) extract of cooked beans from the high- and low-risk areas were similar. Acid-catalyzed nitrosation of the extract increased the total NOC concentration up to 1000-fold, but there was no difference between samples from the two areas. About 10% of bean extracts from both areas showed weak direct-acting genotoxicity in Escherichia coli; after acid-catalyzed nitrosation, all samples were genotoxic at similar levels. The diet of children in the low-risk area satisfied recommended levels of intake of energy and most nutrients except riboflavin and retinol equivalents. Diets from the high-risk area were deficient in energy intake and all nutrients except protein and vitamin C.(ABSTRACT TRUNCATED AT 250 WORDS)

In the assessment of genotoxic risk factors in the environment, the measurement of DNA adducts in aquatic organisms and in plants may have considerable implications. Using 32P-postlabelling, we have detected DNA adducts in the liver of carp (Chondrostoma nasus) from the River Rhône (France), both downstream and upstream from a polychlorinated biphenyl incineration plant. Some of the DNA adducts were specific to downstream fish, suggesting a differential pattern of exposure. We have also detected DNA damage in needles in a declining spruce forest. We found that, in the declining forest, the amounts of DNA adducts increase in relation to the degree of damage to the needles whereas, in a healthy forest, the levels of DNA adducts were low. We have also found DNA adducts in the leaves of hops grown in fields where heptachlor residues persisted.

This study examined whether elevated risk of gastric cancer is associated with high levels of total N-nitroso compounds (NOC), their precursors and nitrosation-dependent genotoxins in gastric juice (GJ). An improved method for quantifying total NOC was used and genotoxicity was assayed in E. coli. Results from patients (n = 210) with or without precancerous lesions of the stomach and living in three areas with up to 8-fold variations in gastric cancer risk (U.K., France, Colombia) were compared. The level of nitrite (range < 1-472 mumol/l) was found to increase with the pH of GJ from the three countries and was dependent on country of collection. The levels of NOC (range: < or = 0.01-8.0 mumol/l) in GJ were not affected by stomach histology and country of collection. NOC levels increased linearly with nitrite concentrations, but the slope of the regression line was greater for acidic GJ (pH < or = 4). These data together suggest that chemical nitrosation contributes at least as much as other nitrosation pathways to the intragastric formation of NOC. Acid-catalysed nitrosation of GJ in vitro increased the NOC concentration (range: 7-1332 mumol/l) up to several 1000-fold but this increase was not predictive of gastric cancer risk either by country or by stomach histology. After acid-catalysed nitrosation, direct genotoxicity (SOS-inducing potency) was significantly higher in GJ with original pH > 4 and highest in samples from Colombia. The results (a) provide no support that intragastric total NOC levels are elevated in subjects with precancerous stomach lesions or living in a high risk area for stomach cancer; (b) confirm that a high nitrite level and elevated pH in GJ are strongly associated, the level of nitrite being associated with precancerous stomach conditions only in Colombia; (c) reveal the presence of precursor compounds in GJ, that after nitrosation yield direct mutagens that probably contain NOC and other substances. As their concentrations were significantly higher in achlorhydric subjects and highest in Colombian patients, these data together provide support for a role of intragastrically formed nitrite-derived direct mutagens in gastric cancer aetiology.

Tobacco smoking causes a major fraction of male urinary bladder cancers and the relative risk of bladder cancer is reported to be two to three times higher for smoking of black (air-cured) than for smoking of blond (flue-cured) tobacco. In molecular dosimetry studies to examine the hypothesis that aromatic amines in tobacco smoke are primarily responsible for bladder cancer, the higher bladder cancer risk in smokers of black tobacco was correlated with two to five times higher exposure to carcinogenic aromatic amines present in black tobacco smoke, notably 4-aminobiphenyl (ABP). For the same amount of smoking, black tobacco smokers had levels of ABP-haemoglobin (Hb) adducts 1.5 times higher and excreted a 1.8-fold higher level of urinary mutagens. These mutagens were characterised as aromatic amines, and included the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a known mutagen and multiorgan/species carcinogen. In smoking volunteers, the ABP-Hb adduct level depended significantly on the acetylator and P-450IA2 phenotypes, being 1.3- to 1.5-fold lower in fast acetylators, slow/intermediate P-450IA2 individuals. The N-(deoxyguanosine-8-yl)-ABP adduct was a major smoking-related DNA adduct in bladder biopsies from surgical patients. It was also tentatively identified in exfoliated urothelial cells of smoking volunteers, who showed a significant and linear correlation between adduct levels of ABP with Hb and with deoxyguanosine in urothelial DNA; both were related to number of cigarettes smoked per day. Levels of several smoking-related DNA adducts in urothelial cells were 2-20 times elevated in smokers. Similar convex dose-response relationships have been found between the number of cigarettes smoked and the relative risk for bladder cancer and between the levels of ABP-Hb adducts and markers of recent smoking. A possible explanation is that fast and slow acetylators have different susceptibility to aromatic amine carcinogens. Case-control studies have consistently revealed an excess of variable magnitude of slow acetylators in subgroups exposed occupationally to carcinogenic aromatic amines. Altogether, results from these studies reinforce the association between cigarette smoking, carcinogen-DNA adducts in urothelial cells, and implicate primary aromatic and possibly heterocyclic amines as bladder carcinogens.

Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.

The formation of DNA adducts, using the (32)P-postlabelling assay, and induction of 7-ethoxyresorufin O-deethylase (EROD) were investigated in a primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene (B[a]P). Concentrations of 0.1 and 1 mum-B[a]P were shown to induce EROD whereas 10 mum was an inhibitory concentration. DNA adducts were detected for 12 hr to 72 hr after exposure to 1 mum-B[a]P whereas EROD activity was increased 36 hr after treatment. The pattern of adducts was shown to be identical to that obtained after B[a]P treatment of rainbow trout in vivo, as demonstrated by co-chromatography of the adducts. Pre-exposure of hepatocytes for 48 hr to beta-naphthoflavone (betaNF) and subsequent 24-hr exposure to 1 mum-B[a]P did not lead to increased DNA adduct formation although betaNF treatment led to a 3.4-fold induction of EROD activity at the time of B[a]P addition. This study suggests that primary culture of rainbow trout hepatocytes provides a suitable method for studying DNA adduct formation and its modulating factors in vitro.

The main polycyclic aromatic hydrocarbon-inducible cytochrome P450 was studied in lung tissue from 57 lung cancer patients by immunohistochemistry, using a monoclonal antibody (1-7-1) that recognizes P450IA1 and P450IA2 isozymes. The intensity of immunostaining was compared with the pulmonary activity of a P450IA1-dependent enzyme, aryl hydrocarbon hydroxylase (AHH), and with P450IA2-related metabolic activity estimated from the ratio of caffeine metabolites in urine. Immunostaining was not observed in peripheral lung tissue of nonsmokers or ex-smokers but was seen in the bronchiolar and alveolar epithelium of all patients who were smokers and had a peripheral carcinoma (16/16) and of 60% (10/17) of those who had a bronchial carcinoma. AHH activity was positively related to the intensity of immunostaining, and an almost 2-fold increase due to smoking was detected in the ratios of caffeine metabolites. These results demonstrate that tobacco smoke induces P450IA1 in the lung and probably P450IA2 in the liver, and suggest a role for certain metabolic phenotypes of P450IA1 in peripheral pulmonary carcinoma.