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W Valesky, JS Armstrong, DP Jones, P Sternberg; Redox Regulation of Apoptosis in Retinal Pigment Epithelial Cells: Role of Endogenous Reactive Oxygen Intermediate (ROI) Production . Invest. Ophthalmol. Vis. Sci. 2002;43(13):672.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether endogenous generation of reactive oxygen intermediates ROI) in cultured human retinal pigment epithelial (hRPE) cells is sufficient to induce apoptosis when the GSH-dependent antioxidant system is impaired. Methods: Primary cultured hRPE cells were incubated with L-buthionine S,R sulfoximine (0-96 h) which inhibits gamma-glutamyl cysteine synthetase and blocks synthesis of glutathione (GSH). Cell viability was assessed using trypan blue exclusion. Apoptosis was determined by cell cycle analysis where the fraction of apoptotic cells were estimated from propidium iodide (PI) stained cells in the subG1 fraction. ROI formation was measured by using the peroxide sensitive dye dichlorofluorescein diacetate (H2DCF-DA) with flow cytometry. Mitochondrial membrane potential (Dym) was determined using the potentiometric dye tetra-methylrhodamine methylester (TMRM) in combination with flow cytometry. GSH and GSH disulfide (GSSG) levels were determined by HPLC. Results: BSO treatment resulted in loss of GSH and caused a time-dependent loss over a 96 h-time course. There was both a loss of cell viability as determined by trypan blue staining and an increase in apoptotic fraction as determined by flow cytometry. Increased DCF fluorescence (indicative of ROI formation) was detectable at 24 h when mitochondrial Dym was intact, indicating that ROI generation was an early event in activation of the apoptotic pathway. The thiol donor dithiothreitol (DTT) prevented ROI formation and apoptotic cell death. Conclusions: Endogenous generation of ROI is sufficient to activate apoptosis in hRPE cells. These results suggest that conditions which impair GSH synthesis in vivo, such as low cysteine diet or fasting/starvation, could contribute to RPE cell loss as occurs in aging and age-related macular degeneration.
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