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Barry S. Winkler, Shu-Chu Chen, Maureen Thomas, Nasihah Barlaskar; Organic Hydroperoxide-Induced Damage to Human Retinal Pigment Epithelial Cells: Dependence on Mitochondrial Electron Transport. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4782.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether the generation of oxygen free radicals from mitochondrial electron transport influences peroxide-induced damage in cultured human retinal pigment epithelial cells (hRPE).
Monolayer cultures of hRPE were grown to confluence in serum-containing MEM media. Cells were subsequently incubated in serum-free MEM for 4 hrs under the following four conditions: control; mitochondrial electron transport inhibited (+ 0.01 mM Antimycin A); +1 mM tertiary butyl hydroperoxide (tBHP); +1 mM hydrogen peroxide (H2O2); each peroxide together with Antimycin A. Measurements were made of lactic acid production, ATP content, morphology, and numbers of cells.
Control cells produced lactate at a rate of 0.85 µmoles/hr/106 cells, had 10.8 nmoles ATP/106 cells and were of normal cytological appearance. Cells incubated in media containing Antimycin A demonstrated a 2-fold increase in lactate production (Pasteur Effect), while maintaining ATP content and cytological appearance similar to that observed in control cells. Thus, hRPE cells are very resistant to chemically-induced inhibition of mitochondrial activity. Cells exposed to 1 mM H2O2 (no added Antimycin A) showed a near-normal production of lactic acid aerobically and only a small decrease in ATP content, and maintained normal cytological features. When H2O2 was added to Antimycin A-containing media, the Pasteur Effect was decreased by 75% relative to the control value, ATP content declined by 40% but after 4 hrs cellular cytological appearance was good. Most interesting, addition of 1 mM tBHP to control MEM caused only a small decrease in aerobic lactic acid production, but ATP content was reduced by 90% and the cells exhibited clear signs of morphological damage and a 30-40% reduction in cell numbers. In contrast, cells incubated in media containing Antimycin A and 1 mM tBHP were protected, i.e., ATP content was reduced by only 25% and there was little change in morphology or cell numbers relative to the control condition.
These results demonstrate that inhibition of mitochondrial electron transport in hRPE cells protects against the short-term damaging effects of tBHP but not those of H2O2, an effect that likely depends on the lipophilic nature of tBHP and differences in mechanisms that detoxify H2O2 and tBHP. Our hypothesis is that mitochondrial electron transport generates free radicals that influence the redox state of transition metals (iron and copper) that react with the lipophilic tBHP in mitochondria to initiate membrane damage.
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