Abstract
Purpose: :
We have previously demonstrated that lipofuscin is phototoxic to retinal pigmented epithelial (RPE) cells in vitro (Davies et al. 2001). The goal of this study was to correlate lipofuscin-induced acute phototoxicity in cultured RPE cells with lipofuscin density, intensity of light, and wavelength of the light.
Methods: :
ARPE-19 cells were cultured to confluence in 24-well plates. Cells were fed with varying numbers of granules of lipofuscin (0-700 granules per cell) isolated from human RPE (donor age 50-70 years) or maintained without lipofuscin. Granule uptake was determined by FACS analysis. Immediately before illumination, culture medium was replaced with photosensitizer-free medium. Cells were exposed to three different light conditions: short wavelength light (400-500 nm, 2.8 mW/cm2) or full visible spectrum light (390-720 nm) at either 2.8 or 13 mW/cm2 for up to 48 hours at 37°C. After illumination, cell viability was assessed by microscopy and the MTT assay.
Results: :
The density of lipofuscin in RPE cells correlated directly with the number of granules to which the cells were exposed. Under each of the three lighting conditions RPE cell viability decreased as the lipofuscin granule concentration per cell increased (p<0.05). Interestingly, the dose response differed between cells exposed to short wavelength and those exposed to full visible spectrum light. Following exposure to light between 400-500nm, RPE cell viability showed a linear decrease to 10% at 700 lipofuscin granules per cell. Exposure to the 390-720 nm full visible spectrum light at the higher intensity (13 mW/cm2) resulted in RPE cell viability decreasing rapidly to 25% at 100 lipofuscin granules per cell and then decreasing slowly to 10% at 700 lipofuscin granules per cell. However, cell viability was much less affected by exposure of cells to lower intensity full visible spectrum light (2.8 mW/cm2).
Conclusions: :
The phototoxicity of lipofuscin on RPE cells is dependent on the lipofuscin granule concentration per cell and the intensity and wavelength of light. The results demonstrate that this is an excellent model for investigating acute phototoxicity on RPE cells in vitro, as was clearly demonstrated by the response to different intensities of full visible spectrum light.
Keywords: ipofuscin • oxidation/oxidative or free radical damage • aging