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D. Kokkinou, T. Lamah, I. Semkova, B. Martiny, B. Kirchhof, U. Schraermeyer; Combination of Melanin and Zinc Protects from Oxidative Damage by UVA Irradiation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):462.
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Purpose: In the eye, zinc is stored in melanin pigment. Lack of zinc and melanin is present in patients with age-related macula degeneration.Both, zinc and melanin, protects from oxidative stress caused by light exposure, by several different mechanisms. We investigated the role of melanin in combination with zinc as possible protector of cellular oxidative damage. Methods: 8 experimental groups were investigated, including controls. Melanin, zinc and UV light were used alone or in different combination. Cultured mouse fibroblasts were fed with melanin (Sepia officinalis/Sigma, Deisenhofen, Germany, at a concentration of 1mg/ml DMEM for 4 hours). After 24 hours, we incubated the cell-cultures with zinc chloride (200µM for 2 hours). The treated and non treated cell cultures were loaded in 60µM of 2',7'-dichlorofluorescin Diacetate that fluoresce when oxidized by H2O2 and exposed to UVA light during loading with the dye for 15 min. Stock solution of dye was prepared in DMSO and diluted 1:1000 in phosphate saline buffer for use.The cells were incubated at room temperature. The UVA light was derived of a lamp ( Radium HRL 125W Germany:20mW/cm²). Fluorescence intensity was collected through a fluorescence microscope (Axionvert 135, Zeiss, Germany) and a CY3 filter (Analysentechnik, Feuerbacher, Tuebingen, Germany) connected to a personal computer equiped with a video camera and a CytoFluor Multi-well Plate Reader (PerSeptive Biosystems, Germany). The fluorescence intensity was measured and the statistical evaluation was based on student's t- test. Results: Cell cultures without melanin and zinc were more fluorescent after UVA radiation than the fibroblats with addition of the same combination. We confirmed that H2O2 was produced by UVA exposure by comparing the control group under the same condition and without UV irradiation. The fluorescence intensity was lowest in cells that had phagocytosed melanin and had been treated with zinc after UVA exposure. Conclusions: The results confirm the hypothesis for protective role of melanin. The protection of melanin in combination with zinc from H2O2 formation was more effective than the use of the substances alone. Probably zinc and melanin treatment in combination can enlarge catalase activity as is shown by our group in an other presentation. This mechanism probably is functional also in pigmented cells of the eye.
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