Cocaine cytotoxicity in hepatocyte cultures from phenobarbital-induced rats: Involvement of reactive oxygen species and expression of antioxidant defense systems

The present study was designed to investigate whether cocaine modifies the production of reactive oxygen species, affects cellular enzyme-mediated ant ioxidant defense systems and, subsequently, promotes apoptosis and/or necro sis of hepatocytes. Primary cultures of hepatocytes isolated from phenobarb ital-induced rats were exposed to cocaine (0-1000 mu M) for 24 hr, and cell death (apoptosis or necrosis), antioxidant enzyme activities and mRNA leve ls, and peroxide generation were determined. Cocaine cytotoxicity by apopto sis was observed by detecting apoptotic nuclei using optic microscopy and b y measurement of the hypodiploid peak (<2C) in DNA histograms obtained by f low cytometry. Necrosis was evidenced by lactate dehydrogenase (LDH) leakag e, and peroxide production was quantified with 2',7'-dichlorodihydrofluores cein diacetate. Low concentrations of cocaine (less than 100 mu M) resulted in an increase in dichlorofluorescein fluorescence, associated with an enh ancement in apoptotic cell death and sharp decreases in the enzyme activiti es and RNAs of catalase and manganese-superoxide dismutase (Mn-SOD). The pr ogressive decrease in peroxide production in cell cultures detected in the range of 250-1000 mu M cocaine was associated with increases in LDH leakage and decreases in the percentage of apoptotic cells, accompanied by low lev els in catalase and Mn-SOD enzyme activities and mRNAs, without apparent ch anges in apoptosis. These data indicate that oxygen radicals may contribute directly or indirectly to cocaine-induced apoptosis in cultured hepatocyte s. We conclude that, in primary hepatocyte cultures, cocaine induced cell d eath by necrosis was dependent on cocaine concentration, while cell death b y apoptosis was parallel to peroxide concentration. The down-regulation of the gene expression of antioxidant enzyme systems should be one of the mech anisms involved in cocaine toxicity. (C) 1999 Elsevier Science Inc.