Lack of antioxidative properties of vitamin C and pyruvate in cultured retinal pigment epithelial cells

Abstract

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. 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 microM 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. 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. 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.