Mitochondrial involvement in cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine.
The cytotoxicity of cocaine (0 - 1000 microM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats. Cytotoxicity was dose-dependent and LDH leakage was significantly enhanced above 100 microM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 microM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase-3 like DEVDase activity and the level of 20 kDa subunit, a product of pro-caspase-3 cleavage. The protective effect of N-acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine-induced necrosis/apoptosis. We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase-3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC. These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage.
Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.
A
A
Zaragoza
C
C
Díez-Fernández
A M
AM
Alvarez
D
D
Andrés
M
M
Cascales
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003042:Cocaine / Q000633:toxicity*; D003574:Cytochrome c Group / Q000187:drug effects* / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology; D003676:Deferoxamine / Q000494:pharmacology; D018765:Dopamine Uptake Inhibitors / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers / Q000494:pharmacology*; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D009124:Muscle Proteins / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D002614:Chelating Agents; D003574:Cytochrome c Group; D018765:Dopamine Uptake Inhibitors; D016166:Free Radical Scavengers; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009124:Muscle Proteins; C087458:Hspb6 protein, rat; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003042:Cocaine; D003676:Deferoxamine; D000111:Acetylcysteine