Hydroxylation of benzo[ a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450 : Effect of 7,8-benzoflavone, butylated hydroxytoluene and ascorbic acid

Abstract

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (−)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene[(−) trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM 2 and a concomitant 3-fold enhancement in microsomal benzo[ a]pyrene hydroxylase activity, whereas the conversion of (−) trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM 2 exhibited the highest activity and specificity toward benzo[ a]pyrene and the lowest activity toward (−) trans-7,8-diol. Conversely, P-450LM 4 was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (−) trans-7, 8-diol but not of benzo[ a]pyrene. Homogeneous P-450LM 4 preferentially Catalyzed the oxygénation of (−) trans-7,8-diol, but was largely ineffective with benzo[ a]pyrene. Partially purified P-450LM 7 lacked substrate specificity, for it metabolized both benzo[a]pyrene and (−) trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[ a]pyrene hydroxylation by P-450LM 4 and phenobarbital-induced microsomes, as well as (−) trans-7,8-diol metabolism by P-450LM 4 and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM 4 and LM 2 with benzo[ a]pyrene and (−)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[ a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[ a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM 2 and LM 4, respectively.