Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth

We have reviewed the methods to selectively detect the superoxide-specific product and the factors affecting its levels in cellular and biological systems

Jacek Zielonka; B. Kalyanaraman


Scholarcy highlights

  • The discovery of the enzyme superoxide dismutase has revolutionized our understanding of the role of reactive oxygen species in biology and medicine
  • Methods for detecting HE and its oxidation products in cells and tissues As ethidium has long been assumed to be the sole product of HE oxidation, the quantification of HE oxidation was based on the changes in the fluorescence intensity
  • There is an ever increasing number of reports on the detection of superoxide with HE and Mito-HE, its mitochondria-targeted analog. These reports often lack any information with respect to superoxide reaction with the probes. It has been nearly six years after publication of the initial report suggesting that HE/superoxide reaction forms 2-hydroxyethidium but not ethidium as a major product
  • Many publications still claim that ethidium is the major product of HE and superoxide
  • The major conclusion is that the knowledge of the whole profile of HE oxidation products together with the intracellular levels of HE is required to make any conclusion regarding the amount of intracellular superoxide and/or the effect of the specific inhibitors
  • As radicals derived from HE and Mito-HE do not react with oxygen to form additional superoxide and H2O2, disproportionation and radical recombination products derived from HE and Mito-HE yield a quantitative measurement of oxidants formed in the cytosolic and mitochondrial regions

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