Inactivation of Cytochromes P450 2B Protects Against Cocaine-Mediated Toxicity in Rat Liver Slices

Abstract

Mechanism-based inactivators of rat liver cytochrome P450 2B1 and 2B2 were used to evaluate the role of these enzymes in the hepatotoxicity of cocaine. Loss of liver microsomal androstenedione 16β-hydroxylation was monitored to determine the extent of P450 2B1/2 inactivation by chloramphenicol (CAP) or its 2B-selective analogue, N-(2- p-nitrophenethyl)chlorofluoroacetamide ( pNO 2C1FA). The effect of P450 2B1/2 inactivation on cocaine-mediated hepatotoxicity was assessed in rat liver slices. Exposure of slices from phenobarbital-induced Lewis rats to CAP concentrations ranging from 100 to 500 μM resulted in a concentration-dependent decrease in P450 2B activity and a corresponding decrease in cytotoxicity as measured by K + loss following exposure to 1 mM cocaine. Treating slices from PR-induced rats with 250 μM pNO 2C1FA protected slices against cocaine-mediated cytotoxicity after exposure to 500 μM cocaine. In vivo administration of 300 mg/kg CAP or 200 mg/kg pNO 2C1FA to phenobarbital-induced Lewis rats decreased androstenedione 16β-hydroxylation to 30 or 39% of control, respectively, and blocked cocaine-mediated K + loss in rat liver slices. Rat liver microsomes from animals treated with either CAP or pNO 2C1FA displayed approximately 40% of the control rate of cocaine N-demethylation. Experiments with phenobarbital-treated Munich Wistar (WM) rats, which lack 2B2, revealed similar rates of microsomal N-demethylation and comparable in vitro hepatotoxicity to Lewis rats. The capacity of a specific P450 2B1/2 inactivator to protect against cocaine-mediated hepatotoxicity both in vivo and in vitro and the results with the WM rats support the identification of P450 2B1 as a major cocaine bioactivating form.