Activation of 8-methoxypsoralen by cytochrome P-450: Enzyme kinetics of covalent binding and influence of inhibitors and inducers of drug metabolism

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 β-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 K m were 26, 24 and 13 μM and for V max 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 K m of 4.7 and 117 μM and for V max 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, α-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.