Korean J Ophthalmol > Volume 17(1); 2003 > Article
Korean Journal of Ophthalmology 2003;17(1):19-28.
DOI: https://doi.org/10.3341/kjo.2003.17.1.19    Published online June 30, 2003.
Hydrogen Peroxide-Induced Cell Death in a Human Retinal Pigment Epithelial Cell Line, ARPE-19.
Min Ho Kim, Jin Chung, Ji wook Yang, Sang Moon Chung, No Hoon Kwag, Jin Seong Yoo
1St. Mary s Eye Clinic, Seoul, Korea.
2Uijeongbu St. Mary s Eye Clinic, Kyeonggi-Do, Korea.
3Department of Ophthalmology, Uijeongbu St. Mary s Hospital, Kyeonggi-Do, Korea.
4EOS Eye Clinic, Seoul, Korea.
5Yeolin St. Mary s Eye Clinic, Seoul, Korea.
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.
Key Words: age related macular degeneration;apoptosis;hydrogen peroxide;retinal pigment epithelium

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