Cocaine induces acute lethal cell injury in rat hepatocytes following N-oxidative metabolic activation by cytochrome P450-dependent and flavin-dependent monooxygenases. Beside this oxidative bioactivation pathway, hepatic carboxylesterases may cleave the carboxymethylester or the benzoylester linkage which leads to molecules found to be non-toxic in vivo. To elucidate the structural requirements of the cocaine molecule for its bioactivation and inactivation, the cytotoxic potential of the natural (-)-cocaine relative to two isomeric forms, (+)-cocaine* (the unnatural enantiomer) and (-)-psi-cocaine (the C2 epimer of the unnatural cocaine) were investigated. Primary short-term cultures of rat hepatocytes obtained from phenobarbital (PB)-pretreated rats were exposed to the drugs for up to 24 h. (-)-Cocaine produced marked time- and concentration-dependent release of lactate dehydrogenase (LDH) into the extracellular medium, whereas the other forms were not cytotoxic (0-1 mM). Furthermore, depletion of cellular glutathione (GSH) with diethylmaleate enhanced LDH release in (-)-cocaine-treated cells and caused marginal cytotoxicity in hepatocytes exposed to the other isomers. To investigate the mechanisms that could be responsible for these isomer-specific effects, the time-dependent metabolic degradation was determined both in cultured hepatocytes and in hepatic microsomes in the presence or absence of the serine carboxylesterase inhibitors, phenylmethylsulfonylfluoride (PMSF) or NaF. All three cocaine analogs were enzymatically degraded, but the rates of ester cleavage greatly varied among the stereoisomers. (-)-Cocaine was primarily N-oxidized via SKF-525A-sensitive pathways, whereas (+)-cocaine was predominantly hydrolyzed by PMSF-sensitive carboxylesterases. In contrast, (-)-psi-cocaine, which is very stable in the absence of cells at 37 degrees C and pH 7.4, was subject to extremely fast enzymatic ester cleavage. In conclusion, these results indicate that the isomer-specific differential cytotoxicity of (-)-cocaine, (+)-cocaine and (-)-psi-cocaine in hepatocytes may be related to stereoselective differences in the rates of hydrolytic inactivation by hepatic carboxylesterases and that the N-oxidative pathway, resulting in hepatocyte injury, may thus be relevant only for (-)-cocaine.