Oxidative Stress–Induced Iron Signaling Is Responsible for Peroxide-Dependent Oxidation of Dichlorodihydrofluorescein in Endothelial Cells

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Abstract
Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H 2 O 2 -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. H 2 O 2 -induced DCF fluorescence was inhibited by the monoclonal IgA–class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H 2 O 2 -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. H 2 O 2 -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.