H J Hyun1, J H Sohn, D W Ha, Y H Ahn, J Y Koh, Y H Yoon. 1. National Creative Research Initiative Center for the Study of CNS Zinc, University of Ulsan College of Medicine, Seoul, Korea.
Abstract
PURPOSE: Although zinc deficiency may contribute to the pathogenesis of age-related macular degeneration, how it leads to retinal pigment epithelium (RPE) degeneration is unknown. To investigate this, cultured human RPE cells were rendered zinc depleted with a membrane-permeant metal chelator, N,N,N',N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), and the resultant cytopathic changes were examined. METHODS: RPE cell degeneration was examined with light microscopy, TdT-mediated dUTP nick end labeling (TUNEL) staining, Hoechst dye staining, and electron microscopy and quantified with cell counting or lactate dehydrogenase release assay. The effect of sublethal zinc depletion on the vulnerability of RPE cells to UV irradiation or hydrogen peroxide (H(2)O(2)) exposure, was studied in cultures without or with pretreatment with low-concentration TPEN. RESULTS: Exposure to 1 to 4 microM TPEN for 48 hours induced RPE cell death in a concentration-dependent manner. Features of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation, and caspase-3 activation, accompanied the TPEN-induced cell death. Addition of equimolar zinc or copper completely reversed TPEN-induced apoptosis, whereas addition of iron had no effect. As in apoptosis of several other cell types including neurons, a protein synthesis inhibitor as well as caspase inhibitors blocked TPEN-induced apoptosis. On the contrary, at sublethal concentrations, TPEN increased the vulnerability of RPE cells to subsequent UV irradiation but not to H(2)O(2) exposure. CONCLUSIONS: The present results suggest that depletion of intracellular zinc and copper, but not copper alone, may be harmful to RPE cells, directly inducing apoptosis or indirectly increasing vulnerability of RPE cells to UV injury. The present culture model may be useful for gaining insights into the mechanisms of zinc depletion-associated RPE cell degeneration.
PURPOSE: Although zinc deficiency may contribute to the pathogenesis of age-related macular degeneration, how it leads to retinal pigment epithelium (RPE) degeneration is unknown. To investigate this, cultured human RPE cells were rendered zinc depleted with a membrane-permeant metal chelator, N,N,N',N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), and the resultant cytopathic changes were examined. METHODS: RPE cell degeneration was examined with light microscopy, TdT-mediated dUTP nick end labeling (TUNEL) staining, Hoechst dye staining, and electron microscopy and quantified with cell counting or lactate dehydrogenase release assay. The effect of sublethal zinc depletion on the vulnerability of RPE cells to UV irradiation or hydrogen peroxide (H(2)O(2)) exposure, was studied in cultures without or with pretreatment with low-concentration TPEN. RESULTS: Exposure to 1 to 4 microM TPEN for 48 hours induced RPE cell death in a concentration-dependent manner. Features of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation, and caspase-3 activation, accompanied the TPEN-induced cell death. Addition of equimolar zinc or copper completely reversed TPEN-induced apoptosis, whereas addition of iron had no effect. As in apoptosis of several other cell types including neurons, a protein synthesis inhibitor as well as caspase inhibitors blocked TPEN-induced apoptosis. On the contrary, at sublethal concentrations, TPEN increased the vulnerability of RPE cells to subsequent UV irradiation but not to H(2)O(2) exposure. CONCLUSIONS: The present results suggest that depletion of intracellular zinc and copper, but not copper alone, may be harmful to RPE cells, directly inducing apoptosis or indirectly increasing vulnerability of RPE cells to UV injury. The present culture model may be useful for gaining insights into the mechanisms of zinc depletion-associated RPE cell degeneration.
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