The Effect of Antioxidants on Photoreactivity and Phototoxic Potential of RPE Melanolipofuscin Granules from Human Donors of Different Age

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.

Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Retinal Pigment Epithelial Cells

“…These findings underline the importance of co-localisation of antioxidants with potential sources of reactive oxygen species. Some previous studies also indicate that zeaxanthin and/or lutein can exert protection in cultured ARPE-19 cells against photooxidative damage, and that protection is more efficient if carotenoids are co-localized with photosensitizers [92][93][94][95]. Wrona et al have shown that zeaxanthin supplemented to ARPE-19 cells exposed to visible light and merocyanine 540, a well-characterized photosensitizer which associates with cell membranes, can substantially diminish oxidation of cholesterol but does not offer a significant protection against cytotoxicity unless it is given in combination with vitamin C or vitamin E and the exposure to photooxidation is short [92].…”

“…In those studies zeaxanthin was administered to ARPE-19 cells 24 hours prior to exposure to the photosensitizer and light. In other studies, where the photosensitizers were present inside the cells, such as phagocytosed lipofuscin, melanolipofuscin, or melanosomes, a combination of zeaxanthin with vitamin E were, at least in part, preserving cell viability [93][94][95]. In these experiments, pigment granules were enriched with antioxidants prior to administration to cultured cells.…”

Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Cultured Retinal Pigment Epithelial Cells

“…These findings underline the importance of co-localization of antioxidants with potential sources of reactive oxygen species. Some previous studies also indicate that zeaxanthin and/or lutein can exert protection in cultured ARPE-19 cells against photo-oxidative damage, and that protection is more efficient if carotenoids are co-localized with photosensitizers [ 47 , 115 , 116 , 117 ]. Wrona et al have shown that zeaxanthin supplemented by ARPE-19 cells exposed to visible light and merocyanine 540, a well-characterized photosensitizer which associates with cell membranes, can substantially diminish oxidation of cholesterol but does not offer a significant protection against cytotoxicity unless it is given in combination with vitamin C or vitamin E and the exposure to photo-oxidation is short [ 47 ].…”

“…In those studies, zeaxanthin was administered to ARPE-19 cells 24 h prior to exposure to the photosensitizer and light. In other studies, where the photosensitizers were present inside the cells, such as phagocytosed lipofuscin, melanolipofuscin, or melanosomes, a combination of zeaxanthin with vitamin E were, at least partly, preserving cell viability [ 115 , 116 , 117 ]. In these experiments, pigment granules were enriched with antioxidants prior to administering to cultured cells.…”

Photoreactivity of Hair Melanin from Different Skin Phototypes—Contribution of Melanin Subunits to the Pigments Photoreactive Properties

“…EPR spin trapping of photogenerated radicals were performed using 100 mM DMPO (5,5-Dimethyl-1-Pyrroline N -oxide) (Sigma-Aldrich, Saint Louis, MO, USA) as a spin trap, according to the procedure described elsewhere [ 45 ]. In brief, samples containing 0.061 mg/ml melanin in 70% DMSO (Sigma-Aldrich, Saint Louis, MO, USA) were irradiated employing the same light source as described for oxygen photoconsumption.…”