Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells

Abstract

Purpose: To determine whether hydrogen peroxide (H2O2), a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells.

Methods: To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50-200 mM H2O2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-rho-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting.

Results: Caspase-3 activity significantly increased in cells exposed to H2O2. Also, the expression of bcl-2 in cells treated with 200 microM H2O2 was diminished, whereas expression of p53 and p21waf-1 was increased compared to the controls.

Conclusions: Exposure of retinal pigment epithelial cells to concentrations of H2O2 that cause in vitro mitochondrial DNA damage also promotes apoptosis.