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

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Abstract

Hydroethidine (HE; or dihydroethidium) 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, is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we review 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 this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E+, using 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+, 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 MitoSOX) as well.

Introduction

The discovery of the enzyme superoxide dismutase (SOD) has revolutionized our understanding of the role of reactive oxygen species (ROS) in biology and medicine [1], [2], [3], [4], [5]. Once viewed as a deleterious by-product of oxygen metabolism, superoxide radical anion (O2radical dot) is now considered to play a role in modulating signaling by reacting with radical dotNO, a second messenger, and as a precursor for H2O2, another second messenger. Despite tremendous progress in the understanding of the biological reactions and physiological signal-ing functions of superoxide, intracellular detection and quantitation of O2radical dot remain a challenge. Direct detection of intracellular superoxide is virtually impossible because of its short half-life and rapid intracellular scavenging. This has led to the development of many probes (e.g., luminol, nitroblue tetrazolium, hydroethidine) that can react with O2radical dot, producing easily detectable, relatively stable products that accumulate with time [6], [7], [8], [9], [10]. However, only a few of those probes yield products that are specific for superoxide. These probes include lucigenin (a chemiluminescent probe, which forms N-methylacridone in the excited state via an unstable dioxetane), hydroethidine (which forms a fluorescent product—2-hydroxyethidium), and cyclic nitrone spin traps (which form spin adducts exhibiting characteristic spectra of superoxide spin adducts detectable by electron paramagnetic resonance spectrometry). As discussed in previous reviews, lucigenin undergoes redox-cycling (self-generating O2radical dot) [11], [12], [13] and nitrone spin traps react slowly with superoxide, forming a relatively unstable superoxide adduct [14], [15], [16]; consequently, hydroethidine (HE; also known as dihydroethidium (DHE), Fig. 1) has become a probe of choice for the detection of intracellular superoxide. The method based on the detection of 2-hydroxyethidium was described as a “gold standard” for superoxide detection in cardiovascular research [7].

Over the past 20 years, hydroethidine has been used to detect superoxide produced in a variety of biological systems, ranging from intracellular organelles to whole cells and whole organs in live animals. Hydroethidine and its mitochondria-targeted analog (Mito-HE or MitoSOX) have been used to detect O2radical dot formed from oxidative burst in leukocytes, pro-and antioxidative actions of cellular endogenous compounds and xenobiotics, apoptosis and the anti-and proapoptotic actions of drugs, neurosignaling, neurodegeneration and neuroprotection, ischemia and reperfusion, hypertension, mechanical stress-induced oxidant production, vascular signaling and pathology, renal function and pathology, and mitochondrial and radiation-induced damage. These studies provide evidence for the involvement of superoxide in many physiological and pathophysiological conditions and contribute to the understanding of the molecular mechanisms of many diseases, potentially allowing for development of new strategies for treatment. In almost all studies, the “red fluorescence” generated from HE, attributed to DNA-bound ethidium, was used as a marker of superoxide formation. In this review, we discuss the reactions of HE with superoxide and other oxidants, some of which generate oxidative red fluorescent product (different from that of the superoxide and HE reaction product) and other oxidative nonfluorescent dimeric products. In addition, we emphasize the need to monitor the levels of HE and its oxidation products to correctly interpret the 2-hydroxyethidium data obtained from HPLC-based studies.

Section snippets

HE—product of reduction of the ethidium cation (E+)

Hydroethidine, a product of two-electron reduction of E+ (Fig. 2), was initially synthesized by reacting ethidium bromide with sodium borohydride [17]. HE does not avidly bind to DNA as it lacks a positive charge and has a nonplanar geometry of the phenanthridine moiety. Reduction of E+ to HE was proposed as a way to release E+ from DNA [17]. The ultraviolet–visible (UV–Vis) absorption spectrum of an aqueous solution of HE at neutral pH consists of several absorption bands in the range of

Reaction between HE and oxidants

From reviewing the previous literature, it is evident that despite its extensive use in biological systems, the basic chemical reactivity of HE remains poorly understood. Thus, we feel it is necessary to present a short overview of the state of the art of HE oxidation/reduction chemistry.

Acid–base properties

At neutral pH, the ethidium cation exists as a single, positively charged species. However, in acidic solution, it undergoes double protonation with pKa values of 0.4–0.8 and 2.0–2.1 for the protonation of aromatic amine groups at positions 3 and 8, respectively [115], [116]. In the case of 2-OH-E+, an additional acid–base equilibrium is expected—with the involvement of the aromatic hydroxyl group [18], [116]. In fact, the pKa value of 7.3, next to 0.5 and 2.2, has been reported for this cation

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 fluorescence intensity. With increased understanding of the oxidative chemistry of HE, it became apparent that even for qualitative analysis, the selective detection of the specific products is critical. The fluorescence settings with greater sensitivity for 2-OH-E+ than for E+ have been established to achieve more selective detection of superoxide-specific

Factors affecting the yield of 2-hydroxyethidium in cells and tissues

The question is, is HE the ideal intracellular probe for measuring superoxide? Intracellularly, HE has to compete with SOD (kSOD + O2radical dot =  2 × 109 M−1 s−1), which is present at micromolar concentrations in the cytosol. To outcompete SOD, HE levels should reach millimolar concentrations in cells, which may be cytotoxic. Thus, although 2-hydroxyethidium, a specific marker for superoxide radical anion, has been used to monitor changes in superoxide levels, the amount of 2-OH-E+ produced is determined

HE-based localization of the sources of superoxide

The HE-based fluorescence assay has been used to identify the localization of superoxide sources in tissues and cells. Those experiments are based on several assumptions:

  • 1.

    The effectiveness of scavenging of superoxide by HE is similar in different cellular compartments, i.e., HE is distributed uniformly over the whole cell or tissue.

  • 2.

    The fluorescent compound formed from HE oxidation remains stationary at the site of its formation, i.e., no intracellular or intercellular transport of the

Summary

Currently, HE is the most commonly used ROS-specific probe, especially for detecting intracellular superoxide. 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 [129]. It has been nearly 6 years since publication of the initial report suggesting that HE/superoxide reaction forms 2-hydroxyethidium but not ethidium as a

Acknowledgment

This work was supported by National Institutes of Health Grants NS40494, HL073056, and HL063119.

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