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 mechanism of action for the isozyme-selective cytochrome P450 (P450) inhibitors N-benzyl-1-aminobenzotriazole (BBT), N-(alpha-methylbenzyl)-1-aminobenzotriazole (alpha MB), and N-(alpha-ethylbenzyl)-1-aminobenzotriazole (alpha EB) was investigated in hepatic microsomes from untreated, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs. Similar to other complex forming amines, MI complex formation was observed as an absorbance maximum at approximately 455 nm by optical-difference spectroscopy, was dependent upon incubation with NADP(H), and was dissociable by the addition of 50 microM 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 limiting concentrations of NADP(H). Maximal complexation with the three compounds was observed in microsomes from phenobarbital-induced guinea pigs where the initial rate of complex formation was dependent upon inhibitor concentration and apparent Km values of 108 +/- 44, 338 +/- 96, and 84 +/- 15 microM for BBT, alpha MB, and alpha EB, respectively, were found. Inclusion of 1 mM glutathione in the incubation mixtures had a significant attenuating effect upon complex formation, suggesting the involvement of an electrophilic, reactive intermediate. Complex formation was not observed with the three inhibitors in pulmonary microsomes from either guinea pigs or rabbits. MI complexation is not likely to contribute to the mechanism-based inactivation of guinea pig hepatic 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 of formed MI complexes, and the absence of MI complex formation with guinea pig or rabbit pulmonary P450.