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T. J. Sarna, A. Zadlo, J. M. Burke; The Effects of Photobleached and Untreated Bovine RPE Melanosomes on Photoinduced Peroxidation of Lipids. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5941.
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We recently showed that untreated porcine RPE melanosomes had no effect on photic injury to ARPE-19 cells or on the photosensitized oxidation of melanosome-associated proteins in cell-free systems. However, after melanosomes were experimentally photobleached to simulate age-related melanin photo-oxidation, the melanosomes increased both light-induced cytotoxicity and protein oxidation. Here we analyzed untreated and photobleached bovine RPE melanosomes for their ability to modify the photoinduced peroxidation of lipids in model membranes.
Melanosomes from bovine RPE were purified by ultracentrifugation in a discontinuous sucrose gradient and subjected to controlled photobleaching using visible light from a solar simulator. Multilamellar vesicles, prepared from lipids extracted from bovine retinas by the Folch method, were incubated with or without melanosomes or control black latex beads and irradiated with intense visible light or by selective photoexcitation of the photosensitizing dyes Rose Bengal (RB) and methylene blue (MB). Progress of the photoinduced peroxidation of lipids was monitored by electron spin resonance oximetry and iodometric assay of lipid hydroperoxides.
Untreated melanosome moderately inhibited the peroxidation of lipids induced by intense visible light. Melanosomes that were photobleached not only lost the ability to inhibit peroxidation, they acquired photosensitizing activity. Untreated melanosomes also moderately inhibited the peroxidation of lipids photosensitized by the anionic dye RB; photobleached melanosomes had only a minor effect, similar to that of controlled particles. In contrast, when the cationic dye MB was used as a sensitizer, melanosomes almost completely inhibited the photosensitized peroxidation of lipids.
In heterogeneous model systems, RPE melanin is an inefficient photoprotective and antioxidant agent. The limited photoprotective properties of RPE melanosomes are further compromised when melanin is photobleached, a change that is believed to occur in the long-lived melanosomes of the aging eye. After photobleaching melanosomes exhibit photosensitizing properties. The very efficient protection against photoinduced peroxidation of lipids in the presence of the cationic photosensitizer can be explained by the ability of melanin to quench excited states of the bound-to-melanin dye molecule
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