The Effect of A2–E and Its Photo–Derivatives to RPE–Cells

Abstract

Purpose: : A2–E, a major component of the retinal pigment epithelium (RPE) lipofuscin, is known as a compound which can neither be degraded by nor eliminated from the cells and which is toxic as well as phototoxic to the cells. Illumination of A2–E with short wavelength light results in isomerisation, photo–oxidation, as well as photolysis. Cytotoxic intermediates (free oxygen radicals) and reaction products (peroxides) are involved in this process. Here, we examined the mechanism of A2–E photobleaching as well as its effect on cultured RPE cells.

Methods: : A2–E solutions (1.28 mM in ethanol or 10 µM cell culture medium) were kept in dark, exposed to blue light (450 – 490 nm, 0.2 mW/mm²) for 15 minutes, or to white light (8.9mW/mm²) for one hour respectively and supplemented to the culture medium of primary porcine RPE cells in 10 µM concentration for 24 hours. Damaged cells were determined by staining with propidium iodide. Absorption as well as fluorescence spectra (UV–excitation: 340–380 nm, blue light excitation: 450–490 nm) were measured by micro–spectrophotometry from A2–E and its photo–derivatives generated as described above as well as in an oxygen–depleted environment.

Results: : Exposure of A2–E to blue as well as white light results in a remarkable, irreversible photo–bleaching of absorption and fluorescence. The peaks of absorption and fluorescence were shifted towards shorter wavelengths. The bleaching by blue light was retarded under oxygen depletion. Primary culture of porcine RPE provided confluent mono–layers of morphologically and functionally intact, slightly pigmented cells. In the control cells, grown without A2–E, a damage rate of 5.3% was found. This increased to 28.1% after A2–E supplementation. The incubation of RPE with 0.2 mW/mm² blue light–irradiated A2–E increased the damage rate to 91.5% whereas irradiation of A2–E with high dosage white light reduced the damage rate to 15.2%.

Conclusions: : Spectral shifts by bleaching with blue light as well as its retardation under oxygen depletion support the theory of photo–epoxidation of A2–E to oxiranes. The cytotoxicity of the oxiranes is demonstrated by the increased damage rate found in cells supplemented with blue light–irradiated A2–E. The reduction of the toxicity of A2–E exposed to a high light dosage, taken together with the spectral data, indicate a breakdown of the molecule into relatively harmless lower weight components.

Keywords: retinal pigment epithelium • cell survival • oxidation/oxidative or free radical damage