Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA.
2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [14C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The 32P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to 32P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the 32P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3'-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the 32P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.
Stiborová
Marie
M
Department of Biochemistry, Faculty of Natural Sciences, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic. stiborov@prfdec.natur.cuni.cz
Miksanová
Markéta
M
Havlícek
Vladimír
V
Schmeiser
Heinz H
HH
Frei
Eva
E
D000814:Aniline Compounds / Q000493:pharmacokinetics / Q000633:toxicity*; D000818:Animals; D001665:Binding Sites; D002273:Carcinogens / Q000493:pharmacokinetics / Q000633:toxicity*; D004247:DNA / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007213:Indomethacin / Q000494:pharmacology; D007784:Lactoperoxidase / Q000378:metabolism; D008297:Male; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D012669:Seminal Vesicles / Q000187:drug effects / Q000378:metabolism; D012756:Sheep; D021241:Spectrometry, Mass, Electrospray Ionization; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced*
D000814:Aniline Compounds; D002273:Carcinogens; D009243:NAD; D004247:DNA; D006735:Horseradish Peroxidase; D007784:Lactoperoxidase; D005978:Glutathione; C003898:2-anisidine; D007213:Indomethacin