1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes.
The preparation of 1-ethynylpyrene (EP) is described. Incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products, but the production of these products ceases after 15 min. Addition of fresh microsomes restores the original rate of EP metabolism. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in microsomes from phenobarbital- (PB) pretreated rats or from untreated, control animals. EP strongly inhibits the hydroxylation of benzo[a]pyrene (BP) by liver microsomes; after 20 min in the presence of EP, BP metabolism nearly ceases. Addition of fresh microsomes restores the original rate of BP hydroxylation. EP more effectively inhibits the oxidation of BP in liver microsomes from rats pretreated with BF than from rats pretreated with PB or from untreated, control animals. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparently irreversible. Kinetic analyses demonstrate that the observed inhibition of BP hydroxylation is due to loss of the enzymatic activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. One such first-order process, with a t 1/2 of 3.5 min and a Ks for EP of 40 microM, is observed in microsomes from BF-pretreated rats. Two such first-order processes, one with t 1/2 of 6.9 min and Ks of 46 microM and one with t 1/2 of 12.7 min and Ks of 33 microM, are observed in microsomes from PB-pretreated rats. It is proposed that a self-catalyzed inhibition (suicide inhibition) of the cytochrome P-450 dependent BP hydroxylation occurs in the presence of EP. Incubation with EP under conditions that result in loss of about 90% of the BP hydroxylase activity in microsomes from BF-pretreated rats and about 66% of the activity in microsomes from PB-pretreated rats causes the loss of only 6 and 12% of the cytochrome P-450, respectively. It is concluded that loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. The selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is also observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the suicide inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D001564:Benzo(a)pyrene / Q000378:metabolism*; D001571:Benzoflavones / Q000494:pharmacology; D001579:Benzopyrene Hydroxylase / Q000037:antagonists & inhibitors*; D003577:Cytochrome P-450 Enzyme System / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology; D011721:Pyrenes / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D011721:Pyrenes; D001564:Benzo(a)pyrene; C042881:1-ethynylpyrene; D009249:NADP; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D001579:Benzopyrene Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; D010634:Phenobarbital