A. Hautefeuille

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.

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.

Skin human papillomavirus type 38 alters p53 functions by accumulation of ΔNp73

The E6 and E7 of the cutaneous human papillomavirus (HPV) type 38 immortalize primary human keratinocytes, an event normally associated with the inactivation of pathways controlled by the tumour suppressor p53. Here, we show for the first time that HPV38 alters p53 functions. Expression of HPV38 E6 and E7 in human keratinocytes or in the skin of transgenic mice induces stabilization of wild‐type p53. This selectively activates the transcription of ΔNp73, an isoform of the p53‐related protein p73, which in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression and apoptosis. ΔNp73 downregulation by an antisense oligonucleotide leads to transcriptional re‐activation of p53‐regulated genes and apoptosis. Our findings illustrate a novel mechanism of the alteration of p53 function that is mediated by a cutaneous HPV type and support the role of HPV38 and ΔNp73 in human carcinogenesis.

Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: Identification of simple phenolic precursors and phenyldiazonium ions as reactive products☆

Epidemiological studies have associated the consumption of smoked fish and meat products with an increased risk of stomach cancer. Therefore, the reaction of such smoked foods with nitrite under acidic conditions was investigated and was shown to produce potent direct-acting genotoxic substances as detected by the SOS Chromotest. Similar genotoxic activity was observed in nitrosated samples of wood-smoke condensates. Simple phenolic compounds such as phenol, 3-methoxycatechol, catechol and vanillin were identified as the precursors of the genotoxic substances. These phenolic compounds also exhibited direct-acting genotoxicity after nitrosation. The major genotoxic substances formed after nitrosation of phenol were isolated and identified as 4- and 2-hydroxyphenyldiazonium ions. Nitrosation of various wood-smoke condensates was found to generate the same type of diazonium compounds, which in part account for the genotoxicity of nitrosated smoked foods.

Interactions between genetic polymorphism of cytochrome P450-1B1, sulfotransferase 1A1, catechol-o-methyltransferase and tobacco exposure in breast cancer risk

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.

Lack of mutagenicity of synthetic pyrethroids in Salmonella typhimurium strains and in V79 Chinese hamster cells

Seven pyrethroids, i.e., cypermethrin, permethrin, deltamethrin, bioresmethrin, resmethrin, cismethrin and fenvalerate, were not found to be mutagenic in (a) Salmonella typhimurium strains TA100 or TA98 in the presence or absence of a rat liver activation system using the plate incorporation assay and fluctuation tests, or (b) V79 Chinese hamster cells in the presence or absence of hepatocytes.

Identification, occurrence and mutagenicity in Salmonella typhimurium of two synthetic nitroarenes, musk ambrette and musk xylene, in Indian chewing tobacco and betel quid

During N-nitrosamine analysis of extracts of betel quid with tobacco and of the saliva of chewers of betel quid with tobacco for N-nitrosamines using a Thermal Energy Analyzer, two unknown compounds were detected. They were identified as synthetic nitro musks, musk ambrette (5-tert-butyl-1,3-dinitro-4-methoxy-2-methylbenzene, CAS No. 83-66-9) and musk xylene, (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene, CAS No. 81-15-2), by gas chromatography-mass spectrometry and Fourier transform nuclear magnetic resonance spectroscopy. These compounds were detected in several samples of betel quid with tobacco and in perfumed tobacco used for chewing in India in amounts ranging from 0.45-23.5 mg/g wet weight. Musk ambrette was found to be mutagenic in Salmonella typhimurium TA100 requiring metabolic activation by rat-liver postmitochondrial supernatant but musk xylene lacked mutagenicity.

Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.

Characterization and identification of 6 mutagens in opium pyrolysates implicated in oesophagel cancer in Iran

Abstract Previous epidemiological studies have indicated an association between the ingestion of opium pyrolysates, dietary deficiencies, and a high incidence of oesophageal cancer in subjects in north-east Iran. Laboratory studies have shown that pyrolysates of opium and particularly of morphine, a major opium alkaloid, are highly mutagenic in bacteria and induce sister-chromatid exchanges in mammalian cells after metabolic activation. We now report the ability of these pyrolysates to transform Syrian hamster embryo cells in culture and present some evidence for their carcinogenicity in mice and hamsters following topical, subcutaneous, intratracheal and intragastric administration. 6 of the most abundant mutagenic compounds present in morphine pyrolysate were isolated and purified by high-performance liquid chromatography and characterized by gas chromatography/mass spectrometry and 1 H-Fourier transform nuclear magnetic resonance spectroscopy. These hitherto unknown compounds, all containing a hydroxy-phenanthrene moiety, were identified as: 3-methyl-3 H -naphth[1,2- e ]indol-10-ol; 1,2-dihydro-3-methyl-3 H -naphth[1,2- e ]indol-10-ol; 6-methylaminophenanthren-3-ol; 2-methylphenanthro[3,4- d ][1,3]oxazol-10-ol; 2,3-dimethyl-3 H -phenanthro[3,4- d ]imidazol-10-ol and 2-methyl-3H-phenanthro[3,4- d ]imidazol-10-ol. Mutagenicity in Salmonella typhimurium TA98 of these compounds increased in the order listed, the last compound being 35 times more active than benzo[ a ]pyrene. The mechanisms, by which these mutagens are formed and metabolically activated are discussed.

Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein.

Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC.