C. Malaveille

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

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

Abstract 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.

Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems

Results from tests on a total of 180 compounds in the Salmonella/microsome assay and its adapted procedures are summarized. The following specific problems were analyzed: the predictive value of the test; frequency distribution of chemicals (classes) according to their mutagenic activity; quantitative relationship between mutagenicity versus electrophilicity versus carcinogenicity of some selected carcinogens; chemicals that are activated into mutagens by human-liver enzymes; compounds that have been tested in the presence of rodent hepatic versus extra-hepatic tissue fractions; and some factors involved in the efficient detection of mutagens in vitro, i.e. the source and concentration of liver microsomal protein required for maximal mutagenic activity. As 34 chemicals have also been tested in microsome- or cell-mediated mutagenicity assays by using V79 Chinese hamster cells, an intercomparison is made of test results obtained in bacterial and mammalian assays.

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. Int. J. Cancer<0R> 77:516–521, 1998.

Mutagenic and alkylating metabolites of halo-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues

Mutagenicity, expressed as the number of his+ revenants per μmole of test compound per hour of exposure, was estimated in two strains of S. typhimurium in the presence of a postmitochondrial mouse-liver supernatant, following exposure to vapours of one of a series of halo-olefins. Their activity was in the following descending order: 3,4-dichlorobutene-1 > 1-chlorobutadiene (technical grade) > 2-chlorobutadiene > vinyl bromide > vinylidene chloride > vinyl chloride; marginal mutagenicity was detected in the presence of 1,1,2-trichloroethylene and 1,1-difluoroethylene, and none with tetrachloroethylene and vinyl acetate. Liver fractions from humans converted vinyl chloride, vinyl bromide, vinylidene chloride and 2-chlorobutadiene into mutagens. In the plate incorporation assay, 1,4-dichlorobutene-2 was mutagenic per se, and addition of microsomal fractions from human or mouse liver enhanced the mutagenicity; a synthetic putative metabolite, 1,4-dichloro-2,3-epoxybutane was less mutagenic than the parent olefin in strain TA100. Treatment of rats with phenobarbital or 3-methylcholanthrene caused an up to 2-fold increase in the liver microsome-mediated mutagenicities of vinyl chloride and vinylidene chloride in S. typhimurium TA1530; while treatment with pregnenolone-16α-carbonitrile, aminoacetonitrile or disulfiram decreased the mutagenic effects. Vinyl chloride, and probably vinyl bromide, were shown to be epoxidized by mouse-liver microsomes; volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine and analysed spectrally. 2-Chlorobutadiene also yielded an alkylating intermediate, but 1,1-difluoroethylene, 1,1-dichloroethyleneand 1,1,2-trichloroethylene did not. 2-Chloro- and 1-chlorobutadiene, 3,4-dichlorobutene-1, 1,4-dichlorobutene-2 and its 2,3-epoxy derivative showed alkylating activity with 4-(4-nitrobenzyl)pyridine, which was not related quantitatively to mutagenic activity in S. typhimurium TA100 in the absence of a metabolic activation system. These data support the hypothesis that an oxidation of the double bond in certain halo-olefins, which is dependent on microsomal mono-oxygenases is a common pathway in the formation of biologically reactive intermediates. The relevance of the metabolites formed during such oxidative processes to the mutagenic, toxic and carcinogenic activities in vivo of some of the parent compounds is discussed.ZusammenfassungDie mutagene Wirkung einer Reihe halogenierter Kohlenwasserstoffe wurde in Gegenwart einer postmitochondrialen Mäuseleberfraktion in zwei Stämmen von S. typhimurium gemessen. Wenn die Bakterien den gasförmigen Testsubstanzen ausgesetzt wurden, war die Mutagenität in folgender Reihenfolge: 3,4-Dichlorbuten-1 > 1-Chlorbutadien (technischer Reinheitsgrad) > 2-Chlorbutadien > Vinylbromid > Vinylidenchlorid > Vinylchlorid. 1,1,2-Trichloräthylen und 1,1-Difluoräthylen zeigten schwachen, Tetrachloräthylen und Vinylacetat keinen mutagenen Effekt. Gewebefraktionen von Menschenleber konnten Vinylchlorid, Vinylbromid, Vinylidenchlorid und 2-Chlorbutadien in mutagene Metabolite überführen. Im konventionellen Plattentest zeigte 1,4-Dichlorbuten-1 eine direkte mutagene Wirkung, die durch Zusatz von mikrosomalen Leberfraktionen von Menschen oder von der Maus erhöht wurde. Der vermutete reaktive Metabolit 1,4-Dichlor-2,3-Epoxybutan wurde synthetisiert, erwies sich aber im Stamm TA100 als ein schwächeres Mutagen als die Muttersubstanz. Die Vorbehandlung von Ratten mit Phenobarbital oder 3-Methylcholanthren verdoppelte die mikrosomenabhängige Mutagenität von Vinylchlorid und Vinylidenchlorid in S. typhimurium TA1530, Pregnenolon-16α-Carbonitril, Aminoacetonitril oder Disulfiram dagegen verminderten die mutagene Wirkung. Eine Epoxidierung von Vinylchlorid und wahrscheinlich Vinylbromid durch Mäuselebermikrosomen wurde durch die Bildung und Abfangen von flüchtigen, alkylierenden Metaboliten mit 4-(4-Nitrobenzyl)pyridin sowie spektralen Untersuchungen nachgewiesen; 2-Chlorbutadien bildete ebenfalls eine alkylierende Zwischenstufe, die in ähnlichen Experimenten mit 1,1-Difluoräthylen, 1,1-Dichloräthylen und 1,1,2-Trichloräthylen nicht nachgewiesen werden konnte. 2-Chlor- und 1-Chlorbutadien, 3,4-Dichlorbuten-1,1,4-Dichlorbuten-2 und sein 2,3-Epoxid zeigten alkylierende Wirkung in Gegenwart von 4-(4-Nitrobenzyl)pyridin, die sich quantitativ nicht mit der mutagenen Aktivität in S. typhimurium TA100 in Abwesenheit metabolischer Aktivierung korrelieren ließ. Die Ergebnisse zeigen einen gemeinsamen Bildungsweg in halogenierten Olefinen auf, der über eine mikrosomale Oxidation der Doppelbindung zu biologisch reaktiven Zwischenstufen führt. Die Bedeutung derartig gebildeter Metabolite für die mutagene, toxische und karzinogene Wirkung der Muttersubstanzen in vivo wird zusammenfassend erörtert.

Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

Abstract When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1- i ]purine.

GST, NAT, SULT1A1, CYP1B1 genetic polymorphisms, interactions with environmental exposures and bladder cancer risk in a high‐risk population

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.

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

Abstract When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his+ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.

Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of his+ rev./plate 16, 12 or 5 times over the spontaneous mutation rate. The mutagenic response for TA 1530 strain was enhanced 7, 4 or 5-fold when fortified S-9 liver fractions from humans, rats or mice were added. In TA 1530 strain, chloroacetic acid showed only toxic effects, while chloroacetaldehyde, chloroethanol and chloroethyleneoxide caused a mutagenic response. The latter compound was shown to be a strong alkylating agent.

Carcinogenic nitrosamines: Free radical aspects of their action

NDMA and other nitrosamines may be activated into DNA binding intermediates by a cytochrome P450-dependent formation of alpha-nitrosamino radicals or photochemically. Within the catalytic site of cytochrome P450, these radical intermediates either combine with HO. to form alpha-hydroxynitrosamines or decompose into nitric oxide and N-methylformaldimine. In the presence of phosphate, nutagenic alpha-phosphonooxy derivatives are formed from radicals generated chemically/photochemically. Studies on lipid peroxidation, in vivo and in vitro, have further suggested that radicals are formed as intermediates from N-nitrosodialkylamines. The level of nitrosamine-induced lipid peroxidation parallels hepatocarcinogenicity in rats. These data, although preliminary, provide further evidence that free radical damage and DNA alkylation are involved in carcinogenesis induced by nitrosamines.