Involvement of oxidative mechanisms in blue-light-induced damage to A2E-laden RPE.

Purpose: The lipofuscin fluorophore A2E is known to be an initiator of blue-light-induced apoptosis in retinal pigment epithelial cells (RPE). The purpose of this study was to evaluate the role of oxidative mechanisms in mediating the cellular damage.

Methods: Human RPE (ARPE-19) cells that had accumulated A2E were exposed to blue light in the presence and absence of oxygen, and nonviable cells were quantified. Potential suppressors (histidine, azide, 1,4-diazabicyclooctane [DABCO], and 1,3-dimethyl-2-thiourea [DMTU]) and enhancers (deuterium oxide [D(2)O] and 3-aminotriazole [3-AT]) of oxidative damage, were also screened for their ability to modulate the frequency of nonviable cells. A2E in PBS, with and without an oxygen-depleter or singlet-oxygen quencher and A2E-laden RPE, were exposed to 430-nm light and examined by reversed-phase high performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry (FAB-MS).

Results: The death of blue-light-illuminated A2E-laden RPE was blocked in oxygen-depleted media. When A2E-laden RPE were transferred to D(2)O-based media and then irradiated (480 nm), the number of nonviable cells was increased, whereas the latter was decreased in the presence of histidine, DABCO, and azide. Conversely, no affect was observed with 3-AT and DMTU. When A2E, in either acellular or cellular environments, was irradiated at 430 nm, FAB-MS revealed the generation of a series of higher molecular mass derivatives of A2E. The sizes of these species increased by increments of mass 16. The generation of these photo-products was accompanied by the consumption of A2E, the latter being diminished, however, when illumination was performed after oxygen depletion and in the presence of a singlet-oxygen quencher.

Conclusions: The augmentation of cell death in the presence of D(2)O and the protection afforded by quenchers and scavengers of singlet oxygen, indicates that the generation of singlet oxygen may be involved in the mechanisms leading to the death of A2E-containing RPE cells after blue light illumination. The finding that irradiation also produces oxygen-dependent photochemical changes in A2E, indicates that the effects of singlet oxygen may be mediated either directly or through the generation of reactive photo-products of A2E.