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.
Andrés
D
D
Instituto de Bioquímica (CSIC-UCM)Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Sanz
N
N
Zaragoza
A
A
Alvarez
A M
AM
Cascales
M
M
D000367:Age Factors; D000704:Analysis of Variance; D000818:Animals; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008297:Male; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
D017382:Reactive Oxygen Species; D016572:Cyclosporine; D010088:Oxidoreductases; D002374:Catalase; D013482:Superoxide Dismutase