The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells

PURPOSE. TO determine whether the lipofuscin fluorophore A2E participates i n blue light-induced damage to retinal pigmented epithelial (RPE) cells. METHODS. Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 mu M concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance l iquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of co nfluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 /- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of m embrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) o f apoptotic cells and by Annexin V labeling for phosphatidylserine exposure . RESULTS. Nonviable cells were located in blue light-exposed zones of A2E-co ntaining RPE cells, whereas cells situated outside the illuminated areas re mained viable. As shown by fluorescence labeling of the nuclei of membrane- damaged cells and by the presence of TUNEL-positive cells, the numbers of n onviable cells increased with exposure duration and as a function of the co ncentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL- positive cells also increased in advance of th e increase in labeling of membrane-compromised cells, a finding that, toget her with Annexin V labeling, indicates an apoptotic form of cell death. Con versely, blue right-exposed RPE cells that did not contain A2E remained via ble. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. CONCLUSIONS. These studies implicate A2E as an initiator of blue light-indu ced apoptosis of RPE cells.