Musk xylene (MX) (1,3,5-trinitro-2-t-butylxylene) is a nitromusk
perfume ingredient that although uniformly negative in a battery of genotoxicity tests, produces a high incidence of liver
tumors in mice. The purpose of this work was to characterize the profile and dose-response relationship of microsomal enzyme induction following exposure to MX. MX was dosed by gavage to male B6C3F1 mice for 7 days at 0, 1, 5, 10, 20, 50, 100, and 200 mg/kg after which microsomes were prepared. At 200 mg/kg, MX increased liver weight by about 65% and increased microsomal
cytochrome P-450 content 2-fold over control. MX increased microsomal activity for O-dealkylation of 7-ethoxy and
7-methoxyresorufin 4- and 2-fold, respectively, and increased the N-demethylation of
erythromycin approximately 2-fold. These results were generally consistent with increased
CYP1A1, 1A2, and 3A
protein levels determined by Western blotting. In contrast, whereas no increase in O-dealkylation of 7-pentoxyresorufin (
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 and with a time course similar to
phenobarbital (PB). To study inhibition of CYP2B
enzymes in vivo, mice were treated with PB (0.05% in
drinking water for 5 days), then given a single dosage of
corn oil or MX (200 mg/kg) at 2 or 18 hr before necropsy. PB treatment increased
PROD activity 25-fold, and at 2 hr after MX treatment (associated with peak plasma levels of MX), there was no change in the PB-induced
PROD activity. However, at 18 hr, MX treatment decreased
PROD activity by 90%. Despite the in vivo inhibition, in vitro studies indicated that MX did not 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
bacitracin) to reduce gut flora prior to administration of MX. In these animals, MX (200 mg/kg) did not inhibit PB-induced
PROD activity. In summary, MX treatment produced general hepatic changes consistent with induction 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 inducer of
cytochrome P-450 enzymes and may cause liver
tumors in a manner analogous to PB. However, no increase in CYP2B
enzyme activity was observed, suggesting that MX or metabolites of MX also inhibit this
enzyme. When the intestinal flora was eliminated by
antibiotic treatment, MX no longer inhibited the CYP2B
enzyme, indicating that anaerobic bacteria are capable of metabolizing MX, and suggesting that
amine metabolites formed by nitroreduction are involved in the inhibition of mouse CYP2B
enzymes.