Effects of different types of oxidative stress in RPE cells

Oxidative damage to retinal pigmented epithelial (RPE) cells and photoreceptors has been implicated in the pathogenesis of age-related macular degeneration (AMD). In order to develop new treatments, it is necessary to characterize the antioxidant defense system in RPE cells to better define their vulnerabilities and how they can be remedied. In this study, we sought to investigate the effects of three different types of oxidative stress on cultured RPE cells. Carbonyl content in RPE cells increased with increasing concentrations of oxidants or increasing duration of exposure with high reproducibility, validating ELISA for carbonyl content as a valuable quantitative measure of oxidative damage. Compared to other cell types, RPE eel Is were able to survive exposure to H₂O₂ quite well and exposure to paraquat extremely well. Comparison of the total amount of oxidative damage at the IC₅₀ for each type of stress showed a rank order of hyperoxia > paraquat > H₂O₂, and since these stressors primarily target different cellular compartments, it suggests that the endogenous defense system against oxidative damage in RPE cells protects well against damage to mitochondria and endoplasmic reticulum, and is less able to handle oxidative damage at the cell surface. Supplementation of media with ascorbic acid provided significant protection from H₂O ₂-induced oxidative damage, but not that induced by paraquat or hyperoxia. Supplementation with docosahexaenoic acid or α-tocopherol significantly reduced oxidative damage from H₂O₂ or hyperoxia, but not that induced by paraquat. We conclude that exposure to different types of oxidative stress results in different patterns of accrual of oxidative damage to proteins in RPE cells, different patterns of loss of viability, and is differentially countered by antioxidants. This study suggests that multiple types of oxidant stress should be used to probe the vulnerabilities of the retina and RPE in vivo, and that ELISA for carbonyl content provides a valuable tool for quantitative assessment of oxidative damage for such studies.