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 nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the present work was to determine whether amine metabolites of MX contributed to the enzyme inhibition and, if so, to define the nature and kinetics of this inhibition. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX equivalents bound covalently to microsomal proteins. However, when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PB-induced PROD activity and no covalent binding to microsomal proteins were observed. Thus, the ability of antibiotic treatment to eliminate the enzyme inhibition and covalent binding implicated amine metabolites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were synthesized to study enzyme inhibition directly. These metabolites were 2-amino-4,6-dinitro-1-t-butyl-xylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting the 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 NADPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order process that displayed saturation kinetics. These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki was calculated to be 10.5 microM and the Kinact was 1.2 min-1. As final confirmation of the inhibitory effects of 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 essentially all of the PB-induced PROD activity, whereas an equimolar dosage 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 mechanism-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.