title: Activation of 8-methoxypsoralen by cytochrome P-450. Enzyme kinetics of covalent binding and influence of inhibitors and inducers of drug metabolism.
abstract: The kinetics of covalent binding of reactive metabolites of 8-methoxypsoralen (8-MOP) to protein were measured in incubations of liver microsomes of rats pretreated for 3 days with i.p. injections of 80 mg/kg/day of beta-naphthoflavone (BNF), phenobarbital (PB), 8-MOP, or vehicle. Covalent binding of radioactivity derived from [14C]8-MOP (labeled at the metabolically stable 4-position in the coumarin ring) required NADPH, obeyed classical Michaelis-Menten kinetics, and was inducible by both PB and BNF. Plots of V versus V/[S] were linear in liver microsomes of rats pretreated with vehicle, PB, or 8-MOP; respective values for Km were 26, 24 and 13 microM and for Vmax were 0.61, 1.70 and 0.50 nmol bound/min/mg protein. In microsomes of rats pretreated with BNF, high- and low-affinity components of covalent binding were observed with respective values for Km of 4.7 and 117 microM and for Vmax of 0.77 and 1.71 nmol bound/min/mg protein. Addition of glutathione and cysteine to the incubations decreased covalent binding by 33 and 67%, respectively, presumably by trapping reactive electrophilic metabolites. Inhibition of epoxide hydrolase with 1,1,1-trichloropropene-2,3-oxide did not affect covalent binding of reactive metabolites of 8-MOP. SKF-525A was a potent inhibitor of both the metabolism of 8-MOP and covalent binding in microsomes from rats pretreated with PB, but had only a slight effect in microsomes from rats pretreated with BNF. In contrast, alpha-naphthoflavone almost completely inhibited metabolism of 8-MOP and covalent binding in BNF-induced microsomes but had no effect in PB-induced microsomes. Apparent covalent binding was reduced by 39% in incubations with 8-MOP labeled with tritium in the metabolically labile methoxy group. Collectively, these results indicate that 8-MOP is biotransformed by two or more isozymes of cytochrome P-450 to reactive electrophiles capable of binding to tissue macromolecules.
authors.affiliation: Department of Family Medicine, College of Medicine, Ohio State University, Columbus 43210.
authors.forename: D C
authors.initials: DC
authors.lastname: Mays
authors.forename: J B
authors.initials: JB
authors.lastname: Hilliard
authors.forename: D D
authors.initials: DD
authors.lastname: Wong
authors.forename: N
authors.initials: N
authors.lastname: Gerber
mesh_terms: D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D007700:Kinetics; D008730:Methoxsalen / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D019324:beta-Naphthoflavone
chemical_list: D001571:Benzoflavones; D011725:Pyridines; C013109:metapyrone; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D008730:Methoxsalen; D010634:Phenobarbital