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Abstract

The in vitro enzymatic metabolism of carcinogenic 2-nitroanisole was investigated using peroxidases (horseradish peroxidase and prostaglandin H synthase) and xanthine oxidase catalyzing oxidative and reductive reactions, respectively. The oxidation of 2-nitroanisole catalyzed by horseradish peroxidase exhibits the Michaelis-Menten kinetics. The Michaelis constant (K_m) and the maximal velocity (V_max) values for this substrate were determined at pH 5.0, 7.0, 7.6 and 8.0. At optimal pH (7.6), the K_m and V_max values are 0.219 μmol/l and 34.45 pmol/min per nmol peroxidase, respectively. The oxidation of 2-nitroanisole is inhibited by radical trapping agents (NADH, ascorbate, glutathione and nitrosobenzene). This indicates that the peroxidase-mediated oxidation of 2-nitroanisole proceeds via a radical mechanism. Active oxygen species are formed during the horseradish peroxidase-catalyzed reactions in the presence of NADH, hydrogen peroxide and 2-nitroanisole. 2-Nitroanisole is also oxidized by mammalian prostaglandin H synthase. Using the nuclease P1-enhanced variation of the ³²P-postlabelling assay, the formation of DNA adducts was detected in DNA treated with 2-nitroanisole and xanthine oxidase. No DNA binding was detected after oxidation of 2-nitroanisole with horseradish peroxidase and prostaglandin H synthase. The results presented (the formation of DNA adducts after 2-nitroanisole activation by xanthine oxidase and that of radicals and/or superoxide radicals during the reactions with peroxidases) strongly suggest the participation of 2-nitroanisole both in the initiation and in the promotion phases of carcinogenesis.

Keywords: Carcinogens; Nitro compounds; Oxidation; Reduction; Radicals; DNA adducts; Initiation and promotion phases of carcinogenesis.