Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole.
1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.
Kao
L R
LR
Department of Entomology, Cornell University, Ithaca, New York 14853.
Wilkinson
C F
CF
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D001564:Benzo(a)pyrene / Q000494:pharmacology; D001571:Benzoflavones / Q000494:pharmacology; D001579:Benzopyrene Hydroxylase / Q000037:antagonists & inhibitors*; D001667:Binding, Competitive; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism / Q000502:physiology*; D004149:Dioxoles / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009249:NADP / Q000502:physiology; D010078:Oxazines / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D051381:Rats; D011919:Rats, Inbred Strains; D012451:Safrole / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D004149:Dioxoles; D007093:Imidazoles; D010078:Oxazines; C035396:dihydrosafrole; D001564:Benzo(a)pyrene; D009249:NADP; C007649:ethoxyresorufin; D019324:beta-Naphthoflavone; C037738:1-phenylimidazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D001579:Benzopyrene Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D012451:Safrole