title: Identification of a genotoxic mechanism for the carcinogenicity of the environmental pollutant and suspected human carcinogen o-anisidine.
abstract: 2-methoxyaniline (o-anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, 32P-postlabeling and 14C-labeled o-anisidine, to show that o-anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o-anisidine to form DNA adducts in vivo. Rats were treated i.p. with o-anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by 32P-postlabeling. Two o-anisidine-DNA adducts, identical to those found in DNA incubated with o-anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 10(7) nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o-anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o-anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o-anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o-anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o-anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic. stiborov@natur.cuni.cz
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.forename: Markéta
authors.initials: M
authors.lastname: Miksanová
authors.forename: Miroslav
authors.initials: M
authors.lastname: Sulc
authors.forename: Helena
authors.initials: H
authors.lastname: Rýdlová
authors.forename: Heinz H
authors.initials: HH
authors.lastname: Schmeiser
authors.forename: Eva
authors.initials: E
authors.lastname: Frei
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001711:Biotransformation; D002274:Carcinogens, Environmental / Q000633:toxicity*; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology; D018736:DNA Adducts / Q000032:analysis / Q000378:metabolism; D004785:Environmental Pollutants / Q000633:toxicity*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000814:Aniline Compounds; D002274:Carcinogens, Environmental; D018736:DNA Adducts; D004785:Environmental Pollutants; D003577:Cytochrome P-450 Enzyme System; C003898:2-anisidine