1.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
Korean Journal of Ophthalmology 2003;17(1):19-28
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.
Apoptosis
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Caspases/metabolism
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Catalase/pharmacology
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Cell Line
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Cell Survival/drug effects
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Enzyme Activation
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Human
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Hydrogen Peroxide/*pharmacology
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Necrosis
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Pigment Epithelium of Eye/*drug effects/enzymology/pathology/*physiology
2.Delay of Photoreceptor Cell Degeneration in rd Mice by Systemically Administered Phenyl-N-tert-butylnitrone.
Jin Hyoung KIM ; Jeong Hun KIM ; Young Suk YU ; Seon Mi JEONG ; Kyu Won KIM
Korean Journal of Ophthalmology 2005;19(4):288-292
PURPOSE: To study the effect of systemic administration of phenyl-N-tert-butylnitrone (PBN) on the degeneration of photoreceptor cells in rd mice. METHODS: PBN was injected intraperitoneally into FVB/rd mice on postnatal days (P) 5 to 14 (group A), and P10 to 18 (group B). At days P14, 16, 18, 20 and 27, morphological changes and apoptosis were analyzed by staining with hematoxylin and eosin or DAPI. The effect of PBN on apoptosis was analyzed in retinal pigment epithelial (RPE) cells by the measurement of caspase-3 activity. RESULTS: In control and group B mice, the outer nuclear layer (ONL) of the retina was composed of 8-10 rows at P12, and rapidly decreased to one row at P18. In group A mice, the ONL was preserved with 5-7 rows at P18, and decreased to one row at P22. PBN inhibited caspase-3 activity in cultured RPE cells. CONCLUSIONS: PBN delayed, but did not block, the degeneration of photoreceptor cells in rd mice. PBN may exert its inhibitory effect during the early phase of photoreceptor cell degeneration.
Retinal Degeneration/*drug therapy/metabolism/pathology
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Pigment Epithelium of Eye/drug effects/metabolism/pathology
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Photoreceptors, Vertebrate/drug effects/metabolism/*pathology
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Nitrogen Oxides/*administration & dosage/pharmacokinetics/therapeutic use
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Neuroprotective Agents/*administration & dosage/pharmacokinetics/therapeutic use
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Mice
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Male
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Injections, Intraperitoneal
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Free Radical Scavengers/*administration & dosage/pharmacokinetics/therapeutic use
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Follow-Up Studies
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Female
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Enzyme Precursors/metabolism
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Disease Models, Animal
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Cells, Cultured
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Caspases/metabolism
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Caspase 3
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Apoptosis/drug effects
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Animals