Retinal pigment epithelium cell damage by A2-E and its photo-derivatives

PURPOSE: A2-E, a major component of the retinal pigment epithelium (RPE) lipofuscin, is a compound that can neither be degraded by nor eliminated from cells and is toxic as well as phototoxic to the cells. Illumination of A2-E with short wavelength light results in isomerization, photooxidation, as well as photolysis. Cytotoxic intermediates (free oxygen radicals) and reaction products (peroxides) are involved in this process. METHODS: A2-E solution (1.28 mM in ethanol or 10 microM phosphate-buffered saline) was kept in dark, exposed to blue light (450-490 nm, 0.2 mW/mm2) for 15 min, or to white light (8.9 mW/mm2) for 60 min, respectively and supplemented to the culture medium of primary porcine RPE cells for 24 h. Damaged cells were determined by staining with propidium iodide in 24 experiments. The photooxidation products of A2-E were analyzed by ultraviolet-visible spectroscopy and MALDI-TOF mass spectrometry. RESULTS: Supplementation of A2-E for 24 h resulted in a rate of damaged cells of 28%. Blue light exposure of A2-E before supplementation increased the rate to 91% whereas the exposure to high dosage white light reduced it to 14%. Irradiation of A2-E resulted in a dosage-dependent addition of one through four oxygen atoms. CONCLUSIONS: The increase of the cell damage rate by A2-E irradiated with low dosage light supports the hypothesis of direct DNA damage by oxidized A2-E. Furthermore, we found a reduced cell damage rate from intensively irradiated A2-E resulting in a tetraoxidized molecule which was rather stable and thus less toxic.