Studies on covalent binding of (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to cytochromes P-450 LM₂ and LM₄ and NADPH-cytochrome P-450 reductase

Abstract

1. Metabolism of ¹⁴C-labelled benzo[α]pyrene (-)trans-7,8-dihydrodiol to protein and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM₄ than with LM₂.

2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM₂, but only 50% of those formed by LM₄. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone.

3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM₄ represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system.

4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM₄ than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM₂ is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM₄ as opposed to LM₂, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM₄ and LM₂ towards (-)trans-7,8-diol.

5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM₄ with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[α]pyrene.