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1 Vascular Biology Laboratory, Heart Institute, University of São Paulo School of Medicine, Sao Paulo, São Paulo, Brazil
2 Vascular Biology Laboratory, Heart Institute, University of São Paulo School of Medicine, São Paulo, São Paulo, Brazil
3 Vascular Biology Laboratory, Heart Institute, University os Sao Paulo School of Medicine, Sao Paulo, Sao Paulo, Brazil
* To whom correspondence should be addressed. E-mail: expfrancisco{at}incor.usp.br.
Dihydroethidium (DHE) is a widely used sensitive superoxide (O2
-) probe. However, DHE fluorescence is due to its oxidation to at least two products, 2-hydroxyethidium (EOH), known to be more specific for O2-, and less specific ethidium (E). We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree Fenton or peroxynitrite/carbon dioxide systems led to EOH and E formation. Peroxidase/hydrogen peroxide but not hydrogen peroxide alone yielded E as the main product. In vascular smooth muscle cells (VSMC) incubated with angiotensin II (100 nM, 4h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in VSMC membrane fractions, showing that EOH was uniquely increased after angiotensin II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was ca. (1.5-2) fold increase in EOH/DHE and E/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of E product and EOH/E ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in E generation. Thus, HPLC analysis of DHE-derived oxidation products can improve assessment of O2- production or NADPH oxidase activity in many vascular experimental studies.
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