Role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene in microsomes from rats treated with phenobarbital or Sudan III.

Abstract

The rates of formation of diols of dimethylbenz[a]anthracene (DMBA-3,4-diol, DMBA-5,6-diol and DMBA-8,9-diol) have been determined in hepatic microsomes prepared from untreated rats or from animals treated with phenobarbital (PB) or Sudan III. PB treatment enhanced the formation of the proximate carcinogen, DMBA-3,4-diol, and of the 5,6-diol while treatment with Sudan III suppressed the formation of DMBA-3,4-diol but greatly increased the rates of formation of the other two diols. Metyrapone, a reagent which is specific for members of the major PB-induced cytochrome P-450 subfamily (P-450-PB3), did not alter the rate of formation of the diols other than in microsomes prepared from PB-treated animals in which formation of the 5,6-diol was inhibited. Incubation of DMBA with microsomes resulted in the formation of covalent, DMBA-microsome adducts. Treatment of the animals with PB and Sudan III increased the rate of formation of DMBA-microsome adducts to a similar extent (approximately 5-fold). The formation of adducts could be inhibited by metyrapone in microsomes from untreated and PB-treated animals but the reagent had no effect on adduct formation in microsomes from Sudan III-treated animals. These observations may indicate that adduct formation in microsomes from Sudan-treated animals involves primary epoxide metabolites while in microsomes from PB-treated animals secondary metabolites are involved and these may be formed by a P-450-PB3 isoenzyme. Specific P-450 isoenzymes involved in the regioselective formation of DMBA-diols have been identified by the use of antibodies directed against specific isoenzymes. An antibody to P-450-MC1b inhibited the formation of DMBA-5,6-diol and 8,9-diol in microsomes from Sudan III-treated animals. Western blot analysis demonstrated that P-450-MC1b was induced in microsomes of animals treated with Sudan III but was not present in the other two microsomal preparations. In accord with the observations with metyrapone, anti-P-450-PB3 inhibited formation of DMBA-5,6-diol in microsomes from PB-treated animals but was without effect on the formation of other diols. Anti-P450-PB1 inhibited the formation of DMBA-3,4-diol in microsomes from PB-treated animals. Western blot analysis of microsomes from animals treated with several xenobiotics indicated a qualitative correlation between the content of P-450-PB1 and the rate at which DMBA-3,4-diol was formed.