ISOZYME-SELECTIVE METABOLIC INTERMEDIATE COMPLEX-FORMATION OF GUINEA-PIG HEPATIC CYTOCHROME-P450 BY N-ARALKYLATED DERIVATIVES OF 1-AMINOBENZOTRIAZOLE

The capacity for metabolic intermediate (MI) complex formation as a me chanism of action for the isozyme-selective cytochrome P450 (P450) inh ibitors N-benzyl-1-aminobenzotriazole (BBT), N-(alpha-methylbenzyl)-1- aminobenzotriazole (alpha MB), and N-(alpha-ethylbenzyl)-1-aminobenzot riazole (alpha EB) was investigated in hepatic microsomes from untreat ed, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs . Similar to other complex forming amines, MI complex formation was ob served as an absorbance maximum at approximately 455 nm by optical-dif ference spectroscopy, was dependent upon incubation with NADP(H), and was dissociable by the addition of 50 mu M potassium ferricyanide. MI complexes formed by BBT, alpha MB, and alpha EB were also dissociable by sedimentation and resuspension, as well as in the presence of limit ing concentrations of NADP(H). Maximal complexation with the three com pounds. was observed in microsomes from phenobarbital-induced guinea p igs where the initial rate of complex formation was dependent upon inh ibitor concentration and apparent K-m values of 108 +/- 44, 338 +/- 96 , and 84 +/- 15 mu M for BBT, alpha MB, and alpha EB, respectively, we re found. Inclusion of 1 mM glutathione in the incubation mixtures had a significant attenuating effect upon complex formation, suggesting t he involvement of an electrophilic, reactive intermediate. Complex for mation was not observed with the three inhibitors in pulmonary microso mes from either guinea pigs or rabbits. MI complexation is not likely to contribute to the mechanism-based inactivation of guinea pig hepati c P450 2Bx, the homologue of rabbit P450 2B4, due to the irreversible inactivation of this isoform at very low inhibitor concentrations, the lack of glutathione attenuation of this destruction the instability o f formed MI complexes, and the absence of MI complex formation with gu inea pig or rabbit pulmonary P450.