Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Abstract

Here we report that ferricytochrome c (cyt c 3+ ) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX ™ Red) forming highly characteristic homo-and heterodimeric products. Using a HPLC-electrochemical (EC) method, several products were detected from cyt c 3+ -catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E + -E + ; Mito-HE-Mito-HE, Mito-E + -Mito-E + ) and heterodimers (HE-E + and Mito-HE-Mito-E + ), as well as the monomeric ethidium (E + ) and mito-ethidium (Mito-E + ). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c 3+ were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E + or Mito-E + , the dimeric analogs (E + -E + and Mito-E + -Mito-E + ) were not fluorescent. Superoxide ( ) or Fremy's salt react with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E + ) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c 3+ -dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

“…Although E ϩ and HE-derived dimeric products are not formed in the presence of O 2 . alone, other 1-electron oxidants (perferryl iron or ONOO Ϫ -derived oxidants) induce their formation, and consequently, decrease intracellular HE levels (13). This can lead to a decrease in 2-OH-E ϩ formation.…”

“…Other oxidants (ONOO Ϫ -derived radicals, hydroxyl radical, perferryl iron) react with HE and MitoSOX TM to form the corresponding oxidation/dimeric products (E ϩ , Mito-E ϩ , and dimeric products) but do not yield the same O 2 . -mediated hydroxylated products (13). Understanding of the oxidation mechanisms of HE and MitoSOX TM has made it possible to use these probes with discretion for detecting intracellular O 2 .…”

Iron Chelators with Topoisomerase-Inhibitory Activity and Their Anticancer Applications

“…2¢,7¢-Dichlorfluorescein-diacetate (DCFH-DA) is another fluorescent probe that is used for measuring intracellular H 2 O 2 and oxidative stress (109). However, this probe may not be specific for H 2 O 2 and could give misleading results due to oxidation by lipid hydroperoxides and H 2 O 2 , as mediated by cellular iron-sensing molecules (304,305). Oxidant signaling by redox-active iron, heme proteins, or cytosolic, enzymatically active cytochrome C had been implicated in the changes observed in DCFH-DA (305).…”

Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium

“…For much of the last 20 years, the red fluorescence of ethidium (E ϩ ), an oxidation product of HE, has been used as a measure of O 2 . in biological systems (4 (9). In addition, HE is oxidized to currently unidentified products upon reaction with hydroxyl radical, ONOO Ϫ and HOCl (4).…”