Abstract
One of the most prominent age-related changes of retinal pigment epithelium (RPE) is the accumulation of melanolipofuscin granules, which could contribute to oxidative stress in the retina. The purpose of this study was to determine the ability of melanolipofuscin granules from younger and older donors to photogenerate reactive oxygen species, and to examine if natural antioxidants could modify the phototoxic potential of this age pigment. Electron paramagnetic resonance (EPR) oximetry, EPR-spin trapping, and time-resolved detection of near-infrared phosphorescence were employed for measuring photogeneration of superoxide anion and singlet oxygen by melanolipofuscin isolated from younger and older human donors. Phototoxicity mediated by internalized melanolipofuscin granules with and without supplementation with zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells by determining cell survival, oxidation of cellular proteins, organization of the cell cytoskeleton, and the cell specific phagocytic activity. Supplementation with antioxidants reduced aerobic photoreactivity and phototoxicity of melanolipofuscin granules. The effect was particularly noticeable for melanolipofuscin mediated inhibition of the cell phagocytic activity. Antioxidants decreased the extent of melanolipofuscin-dependent oxidation of cellular proteins and disruption of the cell cytoskeleton. Although melanolipofuscin might be involved in chronic phototoxicity of the aging RPE, natural antioxidants could partially ameliorate these harmful effects.