MECHANISM-BASED INACTIVATION OF MOUSE HEPATIC CYTOCHROME P4502B ENZYMES BY AMINE METABOLITES OF MUSK XYLENE

Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitro- musk perfume ingredient that induces and inhibits mouse cytochrome P45 028 (CYP2B) enzymes in vivo. The purpose of the present work was to de termine whether amine metabolites of MX contributed to the enzyme inhi bition and, if so, to define the nature and kinetics of this inhibitio n. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg ) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresoruf in (PROD), and [C-14]MX equivalents bound covalently to microsomal pro teins. However, when this experiment was repeated in mice pretreated w ith antibiotics to eliminate the gastrointestinal flora, no decrease i n PB-induced PROD activity and no covalent binding to microsomal prote ins were observed. Thus, the ability of antibiotic treatment to elimin ate the enzyme inhibition and covalent binding implicated amine metabo lites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were syn thesized to study enzyme inhibition directly. These metabolites were 2 -amino-4,6-dinitro-1-t-butylxylene and 4-amino-2,6-dinitro-1-t-butylxy lene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting t he position of the amine substitution relative to the t-butyl function . In the in vitro studies with PB-induced mouse liver microsomes, both amines inhibited PROD activity when preincubated in the absence of NA DPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order pro cess that displayed saturation kinetics. These results indicate that p -NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the K-j was calculated to be 10.5 mu M and the k(Ina ct) was 1.2 min(-1). As final confirmation of the inhibitory effects o f p-NH2-MX on mouse CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited es sentially all of the PB-induced PROD activity, whereas an equimolar do sage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine metabolite of MX is a m echanism-based inactivator of mouse CYP2B10. Furthermore, it is likely that the amine is responsible for the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.