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Anna K Pilat, Magdalena M Olchawa, Tadeusz J Sarna; Sub-lethal photodynamic stress mediated by phagocytized lipofuscin granules induces oxidation of proteins in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2388.
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© ARVO (1962-2015); The Authors (2016-present)
Chronic oxidative stress in human RPE, induced by sub-lethal phototoxic reactions, mediated by lipofuscin, could contribute to development of age related macular degeneration. To evaluate photooxidizing capabilities of the age pigment, peroxidation of proteins in ARPE-19 cells containing phagocytized lipofuscin granules was determined by the use of a sensitive fluorescent probe.
Lipofuscin granules (LFG), isolated from human RPEs from donors of different age, were enriched with a combination of zeaxathin and alpha tocopherol (A). Control or antioxidant enriched lipofuscin granules (A-LFG) were introduced to APRE-19 cells by phagocytosis. Control cells, A-treated cells or cells with LFG and A-LFG, irradiated with blue light for selected time intervals, were analyzed for the presence of protein hydroperoxides using the coumarin boronic acid (CBA) indicator. Photoreactivity of LFG and A-LFG was tested in model systems by determining the granules ability to photooxidize albumin.
Irradiation of LFG containing cells with blue light induced peroxidation of cellular proteins with the effect being light dose dependent. The extent of protein oxidation mediated by lipofuscin was higher for lipofuscin isolated from older donors (age: 30-39) compared to younger donors (age: 20-29). Enrichment of lipofuscin granules isolated from both age groups with zeaxathin and alpha tocopherol reduced protein photoperoxidation. Consistent results were also observed in model systems, in which photoperoxidation of albumin was analyzed.
Photooxidation of proteins in ARPE-19 cells mediated by phagocytized lipofuscin granules can be reproducibly analyzed by CBA used as sensitive indicator of protein hydroperoxides. Such analyses can be carried out on a moderate number of surviving cells subjected to sub-lethal oxidative stress. Photoreactivity of lipofuscin granules in cells and in model system can be modulated by combination of antioxidants.<br />
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