Metabolism of benzo(a)pyrene and benzo (a)pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes

Abstract

A highly purified and reconstituted hepatic microsomal monooxygenase system, completely free of epoxide hydrase and consisting of cytochrome P-448 from 3-methylcholanthrene-treated rats, NADPH-cytochrome c reductase, phosphatidylcholine, and NADPH, metabolizes benzo (a)pyrene to products highly mutagenic in strains TA 98 and TA 1538 of Salmonella typhimurium. The formation of mutagenic metabolites is completely dependent on the presence of benzo (a)pyrene, NADPH, NADPH-cytochrome c reductase, and cytochrome P-448 and is partially dependent on phosphatidylcholine. Mutation frequency in both strains is linearly related to amount of cytochrome P-448 and to time of incubation. Highly purified cytochrome P-450 from phenobarbital-treated rats is relatively poor in catalyzing the formation of mutagenic metabolites from benzo (a)pyrene. Addition of 7.5 to 75 units of highly purified epoxide hydrase to the cytochrome P-448-dependent monooxygenase system decreases the number of mutations by approximately 50% and30% in strains TA 1538 and TA 98, respectively. Additional amounts of epoxide hydrase (300 units) fail to further suppress mutations, indicating that at least some, but probably not all, of the mutagenic metabolites of benzo (a)pyrene are arene oxides. In the absence of a monooxygenase system, mutations induced by benzo (a)pyrene 4,5-oxide are readily quenched by epoxide hydrase, whereas mutations induced by a diol epoxide metabolite of benzo (a)pyrene [(+/-)-7 beta, 8alpha-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo (a)pyrene] are not. Several known and potential phenolic and dihydrodiol metabolites of benzo (a)pyrene are metabolized to products mutagenic in the Salmonella. The number of mutations induced per nmol of hemoprotein is approximately 3- to 4-fold higher when trans-7,8-dihydroxy-7,8-dihydrobenzo (a)pyrene replaces benzo (a)pyrene as a substrate for the cytochrome P-448-dependent monooxygenase system. Little or no mutagenic activity is observed with trans-dihydrodiols at positions 4,5, 9,10, or 11,12 of the hydrocarbon, either in the absence or presence of the active monooxygenase system. Of the 12 possible isomeric monophenols of benzo (a)-pyrene, only 6- and 12-hydroxybenzo (a)pyrene are moderately active bacterial mutagens; 1-, 2-, 3-, 6-, 9-, and 12-hydroxybenzo (a)pyrene are premutagens (i.e. metabolized to mutagenic products); and 4-, 5-, 7-, 8-, 10-, and 11-hydroxybenzo (a)pyrene have little or no mutagenic activity with or without further oxidative metabolism. Benzo (a)pyrene 7,8-oxide, a carcinogen on mouse skin, is weakly mutagenic but can be further metabolized to a highly active bacterial mutagen(s), presumably diol epoxide(s), by a combination of epoxide hydrase and the cytochrome P-448 monooxygenase system. This is the first example of a direct role of epoxide hydrase in the metabolic activation of a chemical to a toxic product.