Abstract

Induction of cytochrome P-450-dependent monooxygenases with phenobarbital (PB) or other hepatic drug-metabolizing enzyme inducers in the rat is associated with enhanced cocaine hepatotoxicity both in vivo and in cultured rat hepatocytes. To demonstrate whether the major PB-inducible P-450 subfamily (P-450IIB) could be involved in the metabolic activation of cocaine, rates of cocaine N-demethylation (the first step of cocaine bioactivation) and the rate of irreversible (covalent) binding of tritiated cocaine to hepatic microsomal proteins (a measure for the overall bioactivation) were determined in microsomes from saline or PB-pretreated rats. PB pretreatment augmented Vmax (6-fold), but not KM, of cocaine N-demethylation. Similarly, the rate of irreversible protein binding was 3-fold increased in microsomes from PB-pretreated rats as compared with those from saline controls. Addition of benzphetamine, a substrate of P-450IIB, markedly inhibited cocaine irreversible binding. In addition, various concentrations of cocaine inhibited microsomal pentoxyresorufin O-depentylase activity in a competitive-type pattern. A polyclonal antibody raised against purified rat P-450IIB1 markedly inhibited cocaine N-demethylation as compared with control incubations with preimmune IgG. Finally, pretreatment of rats with PB potentiated cocaine-induced cytotoxicity in primary, short-term cultured hepatocytes, assessed as lactate dehydrogenase release into the culture medium. This enhancing effect of PB became even more evident in glutathione-depleted cells. These results suggest that cocaine is metabolized and bioactivated by P-450IIB1 in the rat liver, and that induction of this isoform with various agents may be associated with enhanced lethal hepatocyte injury in the rat.