The role of secondary metabolism in the metabolic activation of 7,12-dimethylbenz[a]anthracene by rat liver microsomes

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Abstract
The effect of induction by either phenobarbital (PB) or 3-methylcholanthrene (MC) on the kinetics of both primary and secondary metabolism of 7,12-dimethylbenz[a]-anthracene (DMBA) have been studied. PB and MC induction stimulate the initial rate of total DMBA metabolism 4- and 8-fold, respectively. Individual metabolites exhibited distinct time courses dependent on the inducer. With both induced and uninduced microsomes, formation of DMBA 5,6-dihydrodiol exhibited a 2–5 min time lag before reaching a maximum rate while, in contrast, formation of 7-hydroxy-methyl-12-methylbenz[a]anthracene (7HOMMBA) declined substantially from linearity at a very early point in the reaction. This was most apparent after 2 min and total levels decreased after 5 min. Regioselectivity for both DMBA and 7HOMMBA were measured based on the initial rates of mono-oxygenation derived from these kinetics. In each set of microsomes, prior 7-hydroxylation redirected metabolism towards DMBA 3,4-dihydrodiol formation and away from 5,6-dihydrodiol. Large differences were noted for the effectiveness and regioselectivity of secondary metabolism via 7HOMMBA. MC-induced liver microsomes exhibited a stronger preference for secondary metabolism of 7HOMMBA than PB-induced and control microsomes. In all cases, 7HOMMBA dihydrodiols, formed from DMBA, appeared in the same ratio as when formed directly from 7HOMMBA, suggesting formation from 7HOMMBA rather than from 7-hydroxylation of primary dihydrodiols. The extent of secondary metabolism of primary products was calculated as the difference between the calculated formation (determined from the appropriate regioselectivity and DMBA disappearance) and the quantitated level at a particular reaction time. However, for MC-induced microsomes, quantitation of 7HOMMBA mono-oxygenated metabolites accounted for <50% of the calculated 7HOMMBA secondary metabolism. This discrepancy results from selective further oxidation of 7HOMMBA phenols. For control and PB-induced liver microsomes, quantitation of identifiable 7HOMMBA mono-oxygenated metabolites agreed well with calculated secondary 7HOMMBA metabolism. Based on the results presented here, factors that favor hydroxylation of PAH prior to bay-region diol-epoxide formation are discussed.