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WF Obritsch, SJ Coloso, H Van Remmen, J Yang, DS Gregerson, DA Ferrington; Increased Apoptosis in Retinal Pigment Epithelial Cells With Reduced Antioxidant Enzymes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):674.
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Purpose: Apoptosis in retinal pigment epithelial (RPE) cells has been proposed to be a mechanism for age-related macular degeneration, and some supportive evidence has been reported. Oxidative stress has been shown to induce apoptosis in cultured RPE cells. We postulated that decreased protection by antioxidant enzymes could place the RPE at greater risk for apoptotic cell death. Methods: RPE cells were isolated from wild type and transgenic knock-out (KO) mice expressing decreased levels of either copper-zinc superoxide dismutase (SOD1 +/-) or glutathione peroxidase (GPX1 -/-). Cells were transformed, grown to confluence, then exposed to varying levels of hydrogen peroxide (1-4 mM) for one hour. The relative concentration of intracellular hydrogen peroxide was monitored using dichlorodihydrofluorescein (DCHF). Apoptosis was determined by quantification of DNA fragmentation by FACS analysis of TUNEL positive cells. Cell membrane permeability was evaluated from propidium iodide (PI) fluorescence. Results: Following exposure of RPE cells to increasing concentrations of hydrogen peroxide, we observed a peroxide dose-dependent response in DCHF fluorescence, confirming an intracellular increase in hydrogen peroxide. Four hours after peroxide stress, there was a two-fold increase in TUNEL positive cells for RPE from KO mice compared with RPE from wild type mice. There was a peroxide dose-dependent increase in PI fluorescence in all cell lines, with the greatest increase exhibited in RPE from GPX1 mice. Conclusion: RPE cells with decreased levels of copper-zinc SOD and GPX are more susceptible to peroxide-induced apoptotic cell death. Decreased GPX activity may place RPE cells at a greater risk for membrane damage during oxidative stress.
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