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

Abstract

Hydroethidine (or dihydroethidium) (HE) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E+). In biological systems, another red fluorescent product, ethidium (E+), is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we have reviewed the methods to selectively detect the superoxide-specific product (2-OH-E+) and the factors affecting its levels in cellular and biological systems. The most important conclusion of the present review is that it is nearly impossible to assess the intracellular levels of the superoxide specific product, 2-OH-E+, using the confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E+, in order to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red ®) with superoxide is similar to the reactivity of HE with superoxide and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or Mito-SOX) as well.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

“…We have previously shown that the fluorescence spectral overlap between 2-OH-E ϩ and E ϩ makes it impossible to assign HE-derived red fluorescence to either 2-OH-E ϩ or E ϩ without guidance from HPLC measurements (22). As shown in Fig.…”

“…Under these conditions, it is likely that there is negligible formation of ONOO Ϫ or HOCl. The global profiling of HE and HE-derived oxidation products is essential for proper interpretation of results obtained under different experimental conditions (22). Measurement of intracellular superoxide via monitoring of 2-OH-E ϩ , the diagnostic marker product of O 2 .…”

“…have not yet been totally validated (22). The fluorescent probes, dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR), have been most widely used to measure intracellular oxidants (H 2 O 2 , ONOO Ϫ ).…”

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

“…Discrimination between these two is still be possible, however, due to an extra excitation band between 350 and 400 nm for 2-OH-E + (Robinson et al 2006). However, as the ratio E + /2-OH-E + is often 10 or more, contribution of E + might still be significant (Zielonka and Kalyanaraman 2010). MitoSOX is a DHE derivative coupled to a positively charged triphenylphosphonium group (TPP + ), enabling efficient targeting to the mitochondria for selective detection of mitochondrial O 2…”

“…525 nm, em. 616 nm) and is often present at a much higher concentration (Zielonka and Kalyanaraman 2010). Discrimination between these two is still be possible, however, due to an extra excitation band between 350 and 400 nm for 2-OH-E + (Robinson et al 2006).…”