Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats.

The in vitro effect of cyclosporine A (CsA) was studied in reference to the production of reactive oxygen species (peroxides and superoxide anion) and to cell enzyme-mediated antioxidant defence in hepatocytes isolated from rats aged 2 and 12 months. Primary cultures of hepatocytes were incubated in the presence of concentrations of cyclosporine in the range of 0 to 50 microM for 24 hr, and the release of lactate dehydrogenase into the culture medium was evaluated as a parameter of cytotoxicity and membrane lysis. Peroxides were quantified by using 2',7'-dichlorodihydrofluorescein diacetate, and superoxide anion levels were evaluated by the fluorescence of dihydroethidium. Enzyme activity and gene expression of catalase and Mn- and Cu,Zn-superoxide dismutase were also assayed. CsA cytotoxicity was significantly higher in hepatocytes from rats aged 12 months when compared to those aged 2 months. Intracellular peroxide content resulted in a dose-dependent increase, while the anion superoxide intracellular level slightly decreased as CsA increased from 0-50 microM. The progressive increase in intracellular peroxides in cell cultures in the range from 0-50 microM CsA was associated with the loss of cell viability and accompanied by significantly higher levels of Mn- and Cu, Zn-superoxide dismutase enzyme activities and mRNAs, and slight increases in catalase activity and mRNA. We conclude that, in primary hepatocyte cultures, the cytotoxicity of CsA was dose-dependent in both age groups and significantly higher in cultures from 12-month-old rats when compared to those from 2-month-old animals. The non-coordinated regulation of the gene expression of antioxidant enzyme systems, i.e. catalase and Mn- and Cu,Zn-superoxide dismutases, evidenced to a greater extent in hepatocytes from the older group of rats, could be one of the mechanisms involved in CsA toxicity.