Intracellular photochemical reactions in the RPE cell exhibit a wavelength dependence that resembles the action spectrum of melanin

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 (λ _max = 490 nm) and yellow (λ _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.