Melanosomes isolated from retinal pigment epithelial (RPE) cells support photochemical oxidation of cellular components when excited by visible light. These reactions have an action spectrum peaking between 450 and 500 nm. We now report that similar, wavelength-dependent reactions occur within intact RPE cells. The chemical probes, 2',7'-dichlorofluorescein and dihydrorhodamine 123, are non-fluorescent when reduced and fluorescent when oxidized. Cultured bovine and baboon RPE cells were labeled with these probes, and then exposed to quantum-equivalent, 488, 514.5 or 647.1 nm emissions from Argon and Krypton ion CW lasers. The probes were isolated from the cells by solid phase extraction, and the amount of oxidized probe quantified by HPLC with fluorescence detection. Alternatively, cells were imaged with a fluorescence microscope. Images were acquired at various intervals after the cells were exposed to blue (lambda(max) = 490 nm) and yellow (lambda(max) = 582 nm) wavelengths derived from the microscope exciter lamp. The kinetics and amplitude of the fluorescence change in the cells were quantified with image processing software. Both types of experiments yielded the conclusion that blue-green wavelengths, on a quantal basis, most efficiently induced photooxidative stress in the pigmented cells. The microscopy also indicated that fluorescence was restricted to the cytoplasm. These findings are consistent with the involvement of melanosomes in photooxidative reactions.

The pigments of the retinal pigment epithelium, i.e. the intracellular granules of melanin, lipofuscin, and melanolipofuscin, have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. An important question is whether these reactions are sufficient to produce oxidative damage in the eye. To address this question, the relative photoreactivity of isolated RPE pigment granules towards polyunsaturated fatty acids has been determined, including the dark as well as the light-stimulated reactions. Hydroperoxide derivatives of docosahexaenoic acid were produced by irradiation with short wavelength (< 550 nm) visible light when RPE pigments were present. Although melanosomes exhibited the greatest light-induced activity in these reactions, lipofuscin granules induced peroxidation of fatty acids in the dark. In intact, cultured RPE cells, the existence of pigment-medicated photo-reactions were demonstrated with a fluorescent indicator probe of oxidation, 2',7'- dichlorofluorescein, that was photooxidized in probe-labeled cells in a wavelength dependence fashion with peak activity in the 450 to 500 nm region. This behavior resembled the action spectrum for melanin reactivity. These findings support the hypotheses that the RPE pigments contribute to general photooxidative stress in ocular tissue, and that accumulation of lipofuscin pigment contributes to age-related oxidative stress in the RPE.