Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

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.

Meat intake and bladder cancer in a prospective study: a role for heterocyclic aromatic amines?

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.

Genetic susceptibility according to three metabolic pathways in cancers of the lung and bladder and in myeloid leukemias in nonsmokers.

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.

Multi-factor dimensionality reduction applied to a large prospective investigation on gene-gene and gene-environment interactions.

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.

DNA repair polymorphisms and cancer risk in non-smokers in a cohort study.

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.

Methodology of laboratory measurements in prospective studies on gene-environment interactions: the experience of GenAir.

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.

Environmental tobacco smoke and risk of respiratory cancer and chronic obstructive pulmonary disease in former smokers and never smokers in the EPIC prospective study.

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.

Interaction between genetic polymorphism of cytochrome P450-1B1 and environmental pollutants in breast cancer risk.

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.

DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study.

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.

Impact of polymorphisms in xeno(endo)biotic metabolism on pattern and frequency of p53 mutations in bladder cancer.

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.

White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer.

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 controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms.

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.

Genetic polymorphism of N-acetyltransferases, glutathione S-transferase M1 and NAD(P)H:quinone oxidoreductase in relation to malignant and benign pancreatic disease risk. The International Pancreatic Disease Study Group.

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.

Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers.

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.

Combined effect of polymorphic GST genes on individual susceptibility to lung cancer.

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.

White blood cell DNA adducts, smoking, and NAT2 and GSTM1 genotypes in bladder cancer: a case-control study.

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.

4-Aminobiphenyl-DNA adducts and p53 mutations in bladder cancer.

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.

Inducible nitric oxide synthase, anti-oxidant enzymes and Helicobacter pylori infection in gastritis and gastric precancerous lesions in humans.

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.

Dietary phenolics as anti-mutagens and inhibitors of tobacco-related DNA adduction in the urothelium of smokers.

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: enzyme induction and genetic control in determining 4-aminobiphenyl-hemoglobin adduct levels.

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.