Published in:
01-02-2007 | Laboratory Investigation
Lack of antioxidative properties of vitamin C and pyruvate in cultured retinal pigment epithelial cells
Published in: Graefe's Archive for Clinical and Experimental Ophthalmology | Issue 2/2007
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Background
Oxidative damage to the retinal pigment epithelium might be involved in the pathogenesis of age related macular degeneration. Thus antioxidative protection represents a rationale for a causative therapy or prophylaxis. The aim of the present study is to evaluate antioxidative properties of vitamin C and pyruvate at retinal pigment epithelial (RPE) cells exposed to oxidative stress.
Methods
The ability of vitamin C and pyruvate to quench hydroxyl radicals was tested using the di-hydro-rhodamine (DHR) assay. Cells of the human RPE cell line ARPE-19 were exposed for 8 min to hydroxyl radicals generated by the Fenton reaction from 2.25 mM H2O2 and 30 μM Fe3+-nitrilo-tri-acetate. This was done in the absence and presence of 0.3–3.0 mM pyruvate and vitamin C, respectively. Cell survival was analysed by vitality staining (life-dead-assay) and expressed as cell survival ratio. A survival ratio <1.0 indicates cell loss.
Results
At concentrations from 0.1 to 1.0 mM vitamin C and pyruvate quench hydroxyl radicals in the DHR assay in absence of living matter. In the presence of 0.1– 0.3 mM vitamin C and pyruvate, ARPE-19 showed a reduced survival ratio (0.87±0.01 to 0.89±0.02 after 6 h) which was not the case at the higher concentrations between 1 and 3 mM. The exposure of ARPE-19 cells to hydroxyl radicals reduced the survival ratio to 0.92±0.02. At concentrations at which vitamin C and pyruvate exert toxic effects, a potentiation of radical induced cell death can be observed (survival ratio 0.79±0.02 and 0.82±0.03, respectively). Higher concentrations of vitamin C or pyruvate had no explicit protective effect to the hydroxyl radical induced damage.
Discussion
Although vitamin C and pyruvate are potent hydroxyl radical quenchers in vitro they failed to protect cultured ARPE-19 cells from oxidative stress induced cell death. In contrast, when applying the scavengers at low concentrations a potentiation of cell damage was observed.