Hydrogen peroxide-induced cell death in a human retinal pigment epithelial cell line, ARPE-19.
The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.
Kim
Min-Ho
MH
St. Mary's Eye Clinic, Seoul, Korea.
Chung
Jin
J
Yang
Ji-wook
JW
Chung
Sang-Moon
SM
Kwag
No-Hoon
NH
Yoo
Jin-Seong
JS
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009336:Necrosis; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology / Q000502:physiology*
D006861:Hydrogen Peroxide; D002374:Catalase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases