INDUCTION AND INHIBITION OF MOUSE CYTOCHROME-P-450 2B ENZYMES BY MUSKXYLENE

Musk xylene (MX) (1,3,5-trinitro-2-t-butylxylene) is a nitromusk perfu me ingredient that although uniformly negative in a battery of genotox icity tests, produces a high incidence of liver tumors in mice. The pu rpose of this work was to characterize the profile and dose-response r elationship of microsomal enzyme induction following exposure to MX. M X was dosed by gavage to male B6C3F1 mice for 7 days at 0, 1, 5, 10, 2 0, 50, 100, and 200 mg/ kg after which microsomes were prepared. At 20 0 mg/kg, MX increased liver weight by about 65% and increased microsom al cytochrome P-450 content 2-fold over control. MX increased microsom al activity for O-dealkylation of 7-ethoxy and 7-methoxyresorufin 4- a nd 2-fold, respectively, and increased the N-demethylation of erythrom ycin approximately 2-fold. These results were generally consistent wit h increased CYP1A1, 1A2, and 3A protein levels determined by Western b lotting. In contrast, whereas no increase in O-dealkylation of 7-pento xyresorufin (PROD) was observed, MX treatment increased CYP2B protein levels about 25-fold over control at 200 mg/kg. Furthermore, a single dosage of MX (200 mg/kg) increased Cyp2b-10 mRNA to a maximal level an d with a time course similar to phenobarbital (PB). To study inhibitio n of CYP2B enzymes in vivo, mice were treated with PB (0.05% in drinki ng water for 5 days), then given a single dosage of corn oil or MX (20 0 mg/kg) at 2 or 18 hr before necropsy. PB treatment increased PROD ac tivity 25-fold, and at 2 hr after MX treatment (associated with peak p lasma levels of MX), there was no change in the PB-induced PROD activi ty. However, at 18 hr, MX treatment decreased PROD activity by 90%. De spite the in vivo inhibition, in vitro studies indicated that MX did n ot cause mechanism-based inactivation of CYP2B enzymes. The potential for nitroreduction of MX (catalyzed by anaerobic intestinal bacteria) to contribute to the inhibition of CYP2B enzyme activity was evaluated in a separate group of PB-induced mice that were dosed orally with a regimen of broad spectrum antibiotics (neomycin, tetracyline, and baci tracin) to reduce gut flora prior to administration of MX. In these an imals, MX (200 mg/kg) did not inhibit PB-induced PROD activity. In sum mary, MX treatment produced general hepatic changes consistent with in duction of CYP2B enzymes in mice and caused a large increase in CYP2B protein and mRNA levels. These data indicate that MX is a PB-like indu cer of cytochrome P-450 enzymes and may cause liver tumors in a manner analogous to PB. However, no increase in CYP2B enzyme activity was ob served, suggesting that MX or metabolites of MX also inhibit this enzy me. When the intestinal flora was eliminated by antibiotic treatment, MX no longer inhibited the CYP2B enzyme, indicating that anaerobic bac teria are capable of metabolizing MX, and suggesting that amine metabo lites formed by nitroreduction are involved in the inhibition of mouse CYP2B enzymes. (C) 1997 Academic Press