title: The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species.
abstract: The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002703:Chloranil / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006873:Hydroquinones / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011817:Rabbits; D051381:Rats
chemical_list: D006873:Hydroquinones; D002703:Chloranil; D009243:NAD; D009249:NADP; C044578:2,3,5,6-tetrachlorohydroquinone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D005978:Glutathione; D001205:Ascorbic Acid