Abstract
Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Aconitate Hydratase / antagonists & inhibitors
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Animals
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Antibodies, Monoclonal / pharmacology
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Apoptosis / drug effects
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Caspase 3
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Caspases / metabolism
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Cattle
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Cells, Cultured
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Endocytosis / drug effects
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Endothelium, Vascular / cytology
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Endothelium, Vascular / drug effects
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Endothelium, Vascular / metabolism*
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Enzyme Activation / drug effects
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Fluoresceins / metabolism
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Fluoresceins / pharmacokinetics
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Fluorescent Dyes / metabolism
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Fluorescent Dyes / pharmacokinetics
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Glutathione / pharmacology
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Hydrogen Peroxide / pharmacology*
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Iron / metabolism*
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Iron / pharmacokinetics
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Iron Chelating Agents / pharmacology
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Iron Regulatory Protein 1 / metabolism
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Lipid Peroxides / pharmacology
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Metalloporphyrins / pharmacology
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Oxidants / pharmacology
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Oxidation-Reduction / drug effects
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Oxidative Stress / drug effects
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Oxidative Stress / physiology*
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Receptors, Transferrin / antagonists & inhibitors
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Receptors, Transferrin / metabolism*
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Signal Transduction / drug effects
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Signal Transduction / physiology
Substances
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Antibodies, Monoclonal
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Fluoresceins
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Fluorescent Dyes
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Iron Chelating Agents
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Lipid Peroxides
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Metalloporphyrins
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Oxidants
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Receptors, Transferrin
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2',7'-dichlorodihydrofluorescein
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Hydrogen Peroxide
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Iron
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Caspase 3
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Caspases
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Aconitate Hydratase
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Iron Regulatory Protein 1
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Glutathione