Quantitative Assessment of Oxidative Damage in Cultured RPE Cells by Carbonyl ELISA

Abstract

Abstract: : Purpose:Oxidative damage has been implicated in the pathogenesis of several diseases. In order to explore the role of oxidative damage and therapies to prevent it in animal models, quantitative measures of oxidative damage are needed. In this study, we evaluated the value of ELISA for carbonyl adducts on proteins for quantifying oxidative damage in cultured RPE cells. Methods:Cultured RPE cells were exposed to various concentrations of two chemical oxidants, paraquat or hydrogen peroxide, or hyperoxia. At several time points, cells were harvested and ELISA for carbonyl adducts was done on cell lysates. Cell viability was measured byMTT method. Results:Paraquat and H₂O₂ caused increased carbonyl content in cellular proteins in dose–dependent and incubation time–dependent manners. Exposure of cells to hyperoxia for one day caused increased carbonyl content, followed by a slight decrease at 2 days, and then increases for further exposure up to 72 hours. There was an inverse correlation between carbonyl content and RPE cell viability. Preincubation with vitamin E or docosahexaenoic acid (DHA) alone caused no significant change in paraquat–induced increase in carbonyl content or reduction in viability, but not that treated with H₂O₂. Combined incubation with vitamin E, C and DHA caused a significant reduction in carbonyl content and an increase in viability in paraquat and H₂O₂ treatment. Conclusions: These data suggest that ELISA for carbonyl content provides reproducible assessment of the magnitude of oxidative damage in cultured RPE cells that correlates with cell viability. This suggests that it is also likely to be valuable to quantify oxidative damage in animal models.

Keywords: retinal pigment epithelium • drug toxicity/drug effects • cell death/apoptosis