Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells

PURPOSE. To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS. Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t- butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtΔΨ) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS. t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL- positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtΔΨ was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS. Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.