EVIDENCE FOR THE STABILITY AND CYTOCHROME-P450 SPECIFICITY OF THE PHENOBARBITAL-INDUCED REDUCTIVE HALOTHANE-CYTOCHROME-P450 COMPLEX FORMED IN RAT HEPATIC MICROSOMES

The hypothesis that the reduced spectral halothane-cytochrome P450 complex formed in rat hepatic microsomes is a stable cytochrome P450 specific species was examined. Comparisons of the cytochrome P450 inducers, phenobarbital (PB), pregnenolone-16-alpha-carbonitrile (PCN) and beta-naphthoflavone (beta-NF) showed that PB was the most effective inducer of the halothane-cytochrome P450 complex and the cytochrome P450 which liberates the halothane metabolites, 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). However, the ratio of CDE produced to quantity of complex was found to be reduced 70-77% in these microsomes. A large portion of total microsomal cytochrome P450 was destroyed upon halothane reduction (up to 39%), yet the complexed cytochrome P450, particularly in microsomes from PB-treated animals, was resistant to the irreversible inactivation mechanisms of halothane reduction. The effects of reductive halothane metabolism on subsequent warfarin metabolism showed that 7-hydroxywarfarin formation from either (R)- or (S)-warfarin in microsomes from PCN-treated, PB-treated or untreated rats was highly susceptible to irreversible inhibition. In microsomes from PB-treated, but not PCN or untreated rats, the formation of one warfarin metabolite, 4'-hydroxywarfarin from (R)-warfarin, could be shown to be increased when complex was eliminated by photodissociation. These results suggest that PB-B is preferentially bound as complex and resistant to inactivation because of complex stability, and that halothane reduction readily destroys the cytochrome P450 form, PB-C.