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Kavitha Ravi, Ravi Keshavamurthy, S Balaiya, K V. Chalam; Dose Response Study Of Cytoprotective Effect By Lutein And Zeaxanthin On Retinal Pigment Epithelium From Oxidative Stress Induced Cytotoxicity. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4755.
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© ARVO (1962-2015); The Authors (2016-present)
Lutein (LUT) and zeaxanthin (ZEA), naturally occurring antioxidants and major components in macular pigment, are currently under investigation in clinical trials as prophylactic nutritional agents for age related macular degeneration (AMD). However, dose used in these trials is empirical and not been investigated in in vitro studies. In this study, we investigated the dose response effect of LUT and ZEA in protecting retinal pigment epithelium (RPE) from oxidative stress, a common underlying pathology in AMD
Different concentrations of hydrogen peroxide (H2O2), a common oxidant, were applied to the cultured human retinal pigment epithelial cells (ARPE-19) cells for 24 hours to generate a dose response curve. 3000 cultured human retinal pigment epithelial cells (ARPE-19) were plated in 72-well plate and after 24hrs, cells were exposed to four different concentrations of LUT (4,2,1 and 0.5 µg/ml) and ZEA (0.8,0.4,0.2 and 0.1µg/ml ). After 24 hours incubation, cells were subjected to oxidative stress induced with hydrogen peroxide. Cultures containing saline solution and trichloromethane served as controls. Cell viability was assessed using the WST assay and cell counts were measured using Vi-cell counter. Caspase-3 levels were measured using quantitative Western blot analysis.
Using the WST assay, a dose dependent cytoprotective effect was observed in ARPE-19 cells exposed to hydrogen peroxide after pretreatment with both LUT and ZEA. (Cell viability as a percentage of control was 81.3, 81.1, and 88.8 % at 4, 2, and 1 µg/ml, respectively of Lutein, p<0.001). LUT at 2 µg/ml and ZEA at 0.1 µg/ml were optimum concentrations at which protective effect was observed. At higher doses, there was a reversal of cytoprotective effect. Results were validated by Vi-cell counting. Mitochondrial mediated apoptosis was confirmed by increased expression and activity of caspase-3.
Lutein at 2 µg/ml and Zeaxanthin at 0.1 µg/ml provide optimum cytoprotective effect in retinal pigment epithelium from oxidative stress induced cytotoxicity.
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