Benzo(a)pyrene activation to 7,8-dihydrodiol 9,10-oxide by rat liver microsomes. Control by selective product inhibition.

ABSTRACT

Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.