Dissociation of covalent protein adduct formation from oxidative injury in cultured hepatocytes exposed to cocaine.
1. The relationship between the oxidative and the alkylating properties of cocaine was investigated in primary cultures of hepatocytes derived from phenobarbital-pretreated rats. 2. The cytotoxic effects (LDH release) of 300 microM cocaine were preceded by depletion of intracellular glutathione (GSH) and concomitant increases of oxidized glutathione (GSSG). Furthermore, exposure to [3H]-cocaine was associated with the formation of covalent protein adducts which plateaued between 2 and 7 h and which remained stable for at least 24 h. 3. The addition of the thiol-reducing agent dithiothreitol (DTT, 0.5 mM) protected against cocaine-induced LDH release without altering the time course and extent of cocaine covalent protein adduct formation. Similarly, when DTT was added after short-term exposure to cocaine in Krebs-Henseleit buffer, the loss of viability could be prevented, indicating that alterations in the thiol redox equilibrium, and not covalent protein adduct formation per se, may be crucial for the development of hepatocyte injury. In contrast, high concentrations (2.5-5.0 mM) of DTT inhibited both cocaine bioactivation and covalent binding and thus protected through prealkylative mechanisms. 4. Data demonstrate that cocaine-induced acute lethal hepatocyte injury was mediated by non-alkylative mechanisms, and that covalent adduct formation could be clearly dissociated from the consequences of oxidative stress that lead to cell killing.
Göldlin
C
C
Institute of Toxicology, ETH, Schwerzenbach, Switzerland.
Boelsterli
U A
UA
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003042:Cocaine / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D003042:Cocaine; D004229:Dithiothreitol