Abstract

Microsomes prepared from livers of male and female rats of nine inbred and outbred strains and of male Sprague-Dawley rats pretreated with monooxygenase-inducing agents were used to study N-dealkylation of diazepam and temazepam and C3-hydroxylation of diazepam and nordazepam. Both C3-hydroxylation reactions were more rapid in male than in female liver preparations, but this gender-dependent pattern was not seen with the N-dealkylation reactions. These results indicate the lack of identity of the monooxygenases responsible for the two kinds of reaction and suggest that male-specific enzyme(s) are responsible for the C3-hydroxylations. Induction studies were undertaken to further define these enzymes. To do this, liver microsomes prepared from male Sprague-Dawley rats pretreated with a variety of agents known to have different monooxygenase induction effects were used. With triacetyloleandomycin, dexamethasone, and phenobarbital pretreatment, the specific activities of the C3-hydroxylation reactions were selectively elevated over corresponding control values. These particular xenobiotics are known to enhance the abundance of cytochrome P450IIIA family enzymes, and our results strongly suggest the involvement of these enzymes in the benzodiazepine B ring monooxygenations. Formation of temazepam was also shown to be inhibited by triacetyloleandomycin. This effect was demonstrated to be equal in both saline-treated and dexamethasone-treated male Sprague-Dawley rat liver microsomes, with the antibiotic present either with diazepam throughout the entire incubation period or initially with NADPH in a preincubation mix for 15 min, following which C3-hydroxylation was initiated by the addition of diazepam. These results confirm the uniformity of the involvement of cytochrome P450IIIA family enzymes in diazepam C3-hydroxylation in untreated and inducer-treated rat liver microsomes. Recent studies with human and rabbit liver microsomal preparations have shown that orthologues of these enzymes also catalyze an equivalent hydroxylation in the B ring of midazolam. These findings, considered with the present results showing that the adjacent methyl N-substituent (absent in nordazepam but present in diazepam) did not affect the selectivity of these enzymes for the C3-hydroxylation reaction, suggest that neither steric nor electronic factors markedly influence catalysis of this monooxygenation by these enzymes.