HYDROXYLATION OF BENZO[a]PYRENE AND BINDING OF (-)truns-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,&BENZOFLAVONE, BUTYLATED HYDROXYTOLUENE AND ASCORBIC ACID*

The catalytic activities of hepatic microsomes from untreated, phenobarbital-treated and 3methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (-)truns-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[( -)~ans-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 phenobarbital led to induction of P-450LMz and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (-)rrans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM2 exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (-)rrun.s-7,8-diol. Conversely, P-4SOLM4 was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or pnaphthoflavone, and this was associated in microsomes with an increase in the metabolism of (-)trans7,8-diol but not of benzo[a]pyrene. Homogeneous P-450LM4 preferentially catalyzed the oxygenation of (-)tran.s-7,&diol, but was largely ineffective with benzo[n]pyrene. Partially purified P-450LM7 lacked substrate specificity, for it metabolized both benzo[a]pyrene and (-)trans-7.8-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM4 and phenobarbital-induced microsomes, as well as (-)trans-7,8-diol metabolism by P-450LM4 and 3-methylcholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM4 and LM2 with benzo[a]pyrene and (-)trans-7,8-dial, 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[n]pyrene and its 7,8_dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbitaland 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-450LMz and LM4, respectively. The hepatic microsomal cytochrome P-450-containing mixed-function oxidases and related membranebound enzymes catalyze the oxidation of metaboli* This research was supported by Grant AM-103339 from the United States Public Health Service and Grant PCM7614947 from the National Science Foundation (to M. J. C.). t Present address: Istituto di Ricerche Farmacologiche “Mario Negri”, Via Critrea 62, Milano, Italy. 11 Present address: Department of Pharmacology, Northwestern University Medical and Dental Schools, 303 East Chicago Ave., Chicago, IL 60611, U.S.A. tally important substances as well as a host of xenobiotics [ 1,2]. Many foreign compounds are detoxified by these versatile catalysts, while in other instances metabolism results in activation to cytotoxic or carcinogenic species [3-S]. Following the resolution of the mixed-function oxidase system of liver microsomal membranes and reconstitution of an active hydroxylation system from the components, namely cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidylcholine [6,7], investigations in this laboratory focused on the separation and characterization of