TitleSelective fluorescent imaging of superoxide in vivo using ethidium-based probes.
Publication TypeJournal Article
Year of Publication2006
AuthorsRobinson KM, Janes MS, Pehar M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS
JournalProc Natl Acad Sci U S A
Date Published2006 Oct 10
KeywordsAnimals, Animals, Newborn, Cells, Cultured, Chromatography, High Pressure Liquid, Ethidium, Fluorescent Dyes, Microscopy, Confocal, Mitochondria, Phenanthridines, Rats, Rats, Sprague-Dawley, Spectrometry, Fluorescence, Spectrometry, Mass, Electrospray Ionization, Superoxides

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID17015830
PubMed Central IDPMC1586181
Grant ListR03 TW006482 / TW / FIC NIH HHS / United States
R01 AG017141 / AG / NIA NIH HHS / United States
PAT002034-01 / / PHS HHS / United States
AT002034-02 / AT / NCCIH NIH HHS / United States
ES 00040 / ES / NIEHS NIH HHS / United States
P01 ES000040 / ES / NIEHS NIH HHS / United States
MC_U105663142 / / Medical Research Council / United Kingdom
AG17141A / AG / NIA NIH HHS / United States
TW006482-02 / TW / FIC NIH HHS / United States
ES00240 / ES / NIEHS NIH HHS / United States
P01 AT002034 / AT / NCCIH NIH HHS / United States