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D. Amado, P. Krajacic, T. Dentchev, N. Lukinova, J. Dunaief; The Iron Chelator SIH is Effective in Protecting Retinal Pigment Epithelial Cells Against Oxidative Damage . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2081.
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© ARVO (1962-2015); The Authors (2016-present)
Iron is known to cause oxidative stress. Elevated iron levels are found in the retinas of patients with age–related macular degeneration, suggesting that iron may play a role in the pathogenesis of AMD. Previous studies have demonstrated the efficacy of certain chelators in reducing iron levels in various cell types, including RPE cells; it has also been shown that iron chelation confers cytoprotection against oxidative damage in cardiac myocytes. The aim of the present study is to identify those chelators most effective at reducing iron levels in RPE cells and their optimal doses, and to then use these to determine the protective effects of iron chelation against oxidative stress in the human retinal pigment epithelium.
ARPE–19 cells stably transfected to express a reporter for intracellular iron levels (IRE–GFP) were grown in culture. The chelators deferoxamine (DFO), zinc deferoxamine (ZnDFO), salicylaldehyde isonicotinoyl hydrazone (SIH), and deferiprone (DFP) were applied to separate wells over a wide range of doses, and reduction in green fluorescence was used as a measure of reduction in iron levels at 4h, 8h, and 24h. SIH, the most efficacious chelator, was then applied to ARPE–19 RPE cells one hour prior to the addition of concentrations of hydrogen peroxide ranging from 0 mM to 10 mM. Cell death was assayed at 4h, 8h, and 24 h and quantified by LDH assay and ethidium bromide uptake. Levels of cell death were compared between chelated and non–chelated cells.
Of the four chelators tested, SIH demonstrated optimal reduction in green fluorescence and was non–cytotoxic at 24h. DFO also demonstrated significant fluorescence reduction, but at doses that proved cytotoxic by 8h; ZnDFO was highly cytotoxic, and neither it nor DFP demonstrated effective chelation. For these reasons, SIH was selected for further experimentation. At all time points and at all H2O2 concentrations tested, those cells treated with SIH showed levels of LDH release at or below baseline, indicating protection from cell death, while non–SIH–treated cells treated with the same doses of H2O2 showed up to a 72% increase from baseline LDH release, indicating significant cytotoxicity.
These data show that SIH is an effective chelator in RPE cells and is non–cytotoxic at 24h. Furthermore, SIH is highly effective in protecting RPE cells from oxidative stress induced by hydrogen peroxide, suggesting a possible eventual therapeutic role for SIH or related compounds in patients afflicted with, or at risk of developing, AMD.
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