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Tadeusz Jan Sarna, Magdalena Olchawa, Grzegorz Szewczyk, Andrzej Zadlo, Michal Sarna; The efficiency of melanin to photogenerate potentially toxic singlet oxygen significantly increases after partial photobleaching of the pigment. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4495.
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A growing body of experimental evidence indicates that age-related changes of human RPE melanosomes may result from photoinduced oxidative modifications of the melanin. While an enhanced ability of in vitro “photoaged” bovine and porcine RPE melanosomes to photogenerate superoxide anion has been previously reported by us, it remains unknown whether such pigment granules can photogenerate cytotoxic singlet oxygen (SO). We addressed this issue by analyzing the efficiency of synthetic melanins and melanosomes from horse RPE, subjected to controlled photobleaching, to photogenerate SO and induce phototoxic reaction in ARPE-19 cells containing photoaged melanosomes.
Synthetic eumelanin and pheomelanin, and purified melanosomes isolated from horse RPEs were subjected to intense aerobic irradiation from a light-emitting diode (405 nm; 0.2 W/cm2). Progress of melanin photobleaching was monitored by UV-VIS absorption and electron paramagnetic resonance spectroscopies. The efficiency of control and photobleached melanins to photogenerate SO was determined by direct measurements of singlet oxygen phosphorescence at 1270 nm. Phototoxic effects of photobleached melanosomes after phagocytosis by ARPE-19 cells were measured by standard cell survival methods and the extent of cellular protein oxidation by the fluorogenic probe CBA.
Photobleaching of synthetic melanins was accompanied by a dramatic increase in their efficiency to photogenerate SO after excitation in near UV and short-wavelength visible range. The enhanced ability of melanins to photogenerate SO, coincided with their reduced efficiency to quench this reactive oxygen species. We also observed an elevated photoformation of SO by photobleached horse RPE melanosomes. When incorporated in ARPE-19 cells by phagocytosis, photobleached melanosomes photoinduced oxidation of cellular proteins and cell killing. Both effects were significantly stronger than those observed for control unbleached melanosomes.
Photobleaching of RPE melanosomes, an in vitro model for melanosome photoaging, induces profound oxidative changes in the melanin structure and facilitates the release of photomodified melanin nanoaggregates with elevated photochemical reactivity. Such changes, if they occur in situ, could increase the risk of chronic photoinduced oxidative stress in the human RPE.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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