This study was conducted to investigate the effect of ascorbic acid on oxidative injury of cultured porcine retinal pigment epithelial (RPE) cells induced by t-butylhydroperoxide. The porcine RPE cells were cultured in Dulbecco's modified Eagle's medium and the culture medium was replaced with one containing 0.01 mM to 5 mM ascorbic acid and/or 0.2 mM t-butylhydroperoxide. After 2 hours incubation, the test medium was replaced with the control medium. The number of cells was counted with a Coulter counter after a 2-day incubation period. The medium was pretreated with 900 U/ml and the previous procedure was repeated to eliminate the toxic effects of hydrogen peroxide induced by ascorbic acid. Not only t-butylhydroperoxide (p < 0.01) but also ascorbic acid (p < 0.01) were found to have dose-dependent cytotoxicity on RPE cells. The cytotoxicity was more significant when both agents were added to the culture media. In the presence of catalase, the cytotoxicity of ascorbic acid became insignificant (p > 0.05). The cytotoxicity of t-butylhydroperoxide decreased when 1 mM and 5 mM of ascorbic acid was added to the culture media with catalase pretreatment (p = 0.0277). These results indicate that ascorbic acid was toxic to RPE cells in our culture model but this cytotoxicity was not detected in the presence of catalase. With catalase pretreatment, ascorbic acid in relatively high concentration provided protection against oxidative injury of t-butylhydroperoxide.
Animals ; Ascorbic Acid/*pharmacology ; Cell Count ; Cell Survival/drug effects ; Cells, Cultured ; Culture Media ; Dose-Response Relationship, Drug ; Free Radicals ; Oxidative Stress/*drug effects ; Peroxides/antagonists & inhibitors/toxicity ; Pigment Epithelium of Eye/cytology/*drug effects ; Reactive Oxygen Species/toxicity ; Swine ; tert-Butylhydroperoxide