Comparison of Protective Effects of Various Antioxidants Against Hydrogen Peroxide Induced RPE Death in vitro

Abstract

Purpose: : To compare the protective effects of various flavonoids, ascorbic acid, alpha-tocopherol and glutathione against hydrogen peroxide (H₂O₂) induced retinal pigment epithelial (RPE) cells death in vitro.

Methods: : Human RPE cells were seeded into 96 cell wells. Fisetin, apigenin, naringin, ascorbic acid, alpha-tocopherol and glutathione were added to the culture medium at various concentrations. Cells cultured with medium without antioxidants were used as the controls. Two hours later, H₂O₂ was added (0.3 mM). All tests were run in triplicate. After 24 hours, quantity of viable cells was measured using the MTT assay.

Results: : All tested antioxidants were not toxic at tested concentration. H₂O₂ at 0.3 mM decreased the viable cells to 8.1% the controls. Fisetin, ascorbic acid and alpha-tocopherol showed dose-dependent protective effects on H₂O₂ induced cell death from 10 to300 µM. Viable cells in H₂O₂-treated RPE cultured with fisetin, apigenin, naringin, ascorbic acid, alpha-tocopherol and glutathione at 100 µM were 56.2%, 7.4%, 9.8%, 17.6%, 24.2% and 10.6% of the controls (without H₂O₂). The difference of quantity of viable cells between various antioxidants and the control were very significant (P < 0.01) in fisetin, ascorbic acid and alpha-tocopherol treated cells, indicating that these agents protected the RPE cells against the H₂O₂ cytotoxicity. There was not significant difference in quantity of viable cells between cells cultured with apigenin, naringin and glutathione and the controls, indicating that these agents at 100 µM level could not protect the RPE against H₂O₂ induced cell death.

Conclusions: : In three flavonoids tested, only fisetin (a flavonol) shows protective effects on H₂O₂ induced RPE death. The other two flavonoids, apigenin (a flavone) and naringin (a flavanone) do not have significant protective effects against oxidative stress. Of three classic antioxidants tested, ascorbic acid and alpha-tocopherol showed protective effects against oxidative stress; glutathione does not, possibly related to the poor passage of glutathione through the cell membrane and enter the cell.

Keywords: retinal pigment epithelium • antioxidants • oxidation/oxidative or free radical damage