Jacek Zielonka

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

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.

Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine

Various detection methods of the specific product of reaction of superoxide (O2•−) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E+), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E+, the unique product of HE/O2•− in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E+ and E+ in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E+ and E+, we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E+ needs to be separated from other HE-derived products for unequivocal quantification.

Direct oxidation of boronates by peroxynitrite: Mechanism and implications in fluorescence imaging of peroxynitrite

In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO(-)) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO(-), hypochlorous acid (HOCl), and hydrogen peroxide (H(2)O(2)) and found that ONOO(-) reacts with 4-acetylphenylboronic acid nearly a million times (k=1.6x10(6) M(-1) s(-1)) faster than does H(2)O(2) (k=2.2 M(-1) s(-1)) and over 200 times faster than does HOCl (k=6.2x10(3) M(-1) s(-1)). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO(-) formed in cellular and cell-free systems.

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy.

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatins antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.

Mitochondria targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death

Cancer cells are long known to exhibit increased aerobic glycolysis, but glycolytic inhibition has not offered a viable chemotherapeutic strategy in part because of the systemic toxicity of antiglycolytic agents. However, recent studies suggest that a combined inhibition of glycolysis and mitochondrial function may help overcome this issue. In this study, we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTD) in combination with 2-deoxy-d-glucose (2-DG), a compound that inhibits glycolysis. Using the MTDs, termed Mito-CP and Mito-Q, we evaluated relative cytotoxic effects and mitochondrial bioenergetic changes in vitro. Interestingly, both Mito-CP and Mito-Q synergized with 2-DG to decrease ATP levels in two cell lines. However, with time, the cellular bioenergetic function and clonogenic survival were largely restored in some cells. In a xenograft model of human breast cancer, combined treatment of Mito-CP and 2-DG led to significant tumor regression in the absence of significant morphologic changes in kidney, liver, or heart. Collectively, our findings suggest that dual targeting of mitochondrial bioenergetic metabolism with MTDs and glycolytic inhibitors such as 2-DG may offer a promising chemotherapeutic strategy.

Peroxynitrite is the major species formed from different flux ratios of co-generated nitric oxide and superoxide: direct reaction with boronate-based fluorescent probe

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O2̇̄) and nitric oxide (•NO). It is well known that O2̇̄ and •NO rapidly react to form a potent oxidant, peroxynitrite anion (ONOO−). However, indirect measurements with the existing probes (e.g. dihydrorhodamine) previously revealed a bell-shaped response to co-generated •NO and O2̇̄ fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO− per se at various fluxes of •NO and O2̇̄. Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO− (k = 1.1 × 106 m−1s−1), we report that ONOO− formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated •NO and O2̇̄ fluxes. We conclude that ONOO− is formed as the primary intermediate during the reaction between •NO and O2̇̄ co-generated at different fluxes.

HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements.

Methods for the detection and quantitation of hydroethidine (HE) and its oxidation products by HPLC analysis are described. Synthetic methods for preparation of authentic standards (2-hydroxyethidium and diethidium) are provided. Potential applications of the HPLC methods to chemical and biological systems are discussed. Specific examples of chromatograms obtained using UV-Vis absorption, fluorescence, electrochemical, and mass spectrometry detectors are provided. The development of a dual electrochemical and fluorescence detection methodology and its applications are described. The HPLC-based method enables analyses of HE and its oxidation products such as ethidium and the dimeric products of HE. The ramifications of HPLC measurement of HE and its oxidation products in the detection and quantitation of 2-hydroxyethidium, the diagnostic marker product of superoxide and HE, in the intracellular milieu are discussed. Similarly, mitochondria-targeted HE conjugated to a triphenylphosphonium group (Mito-HE or Mito-SOX) also forms oxidation products (dimers of Mito-HE and Mito-E+) that can affect the detection and quantitation of 2-hydroxy-mito-ethidium, the diagnostic marker product of Mito-HE and superoxide in mitochondria.

Doxorubicin Inactivates Myocardial Cytochrome c Oxidase in Rats: Cardioprotection by Mito-Q

Doxorubicin (DOX) is used for treating various cancers. Its clinical use is, however, limited by its dose-limiting cardiomyopathy. The exact mechanism of DOX-induced cardiomyopathy still remains unknown. The goals were to investigate the molecular mechanism of DOX-induced cardiomyopathy and cardioprotection by mitoquinone (Mito-Q), a triphenylphosphonium-conjugated analog of coenzyme Q, using a rat model. Rats were treated with DOX, Mito-Q, and DOX plus Mito-Q for 12 weeks. The left ventricular function as measured by two-dimensional echocardiography decreased in DOX-treated rats but was preserved during Mito-Q plus DOX treatment. Using low-temperature ex vivo electron paramagnetic resonance (EPR), a time-dependent decrease in heme signal was detected in heart tissues isolated from rats administered with a cumulative dose of DOX. DOX attenuated the EPR signals characteristic of the exchange interaction between cytochrome c oxidase (CcO)-Fe(III) heme a3 and CuB. DOX and Mito-Q together restored these EPR signals and the CcO activity in heart tissues. DOX strongly downregulated the stable expression of the CcO subunits II and Va and had a slight inhibitory effect on CcO subunit I gene expression. Mito-Q restored CcO subunit II and Va expressions in DOX-treated rats. These results suggest a novel cardioprotection mechanism by Mito-Q during DOX-induced cardiomyopathy involving CcO.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems HIGH-THROUGHPUT REAL-TIME ANALYSES

Background: Recently, new “targeted” fluorescent probes that react selectively with reactive oxygen and nitrogen species to yield specific products have been discovered. Results: High-throughput fluorescence and HPLC-based methodology for global profiling of ROS/RNS is described. Conclusion: This methodology enables real-time monitoring of multiple oxidants in cellular systems. Significance: The global profiling approach using different ROS/RNS-specific fluorescent probes will help establish the identity of oxidants in redox regulation and signaling. Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.

Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides

Boronates, a group of organic compounds, are emerging as one of the most effective probes for detecting and quantifying peroxynitrite, hypochlorous acid, and hydrogen peroxide. Boronates react with peroxynitrite nearly a million times faster than with hydrogen peroxide. Boronate-containing fluorogenic compounds have been used to monitor real time generation of peroxynitrite in cells and for imaging hydrogen peroxide in living animals. This perspective highlights potential applications of boronates and other fluorescent probes to high-throughput analyses of peroxynitrite and hydroperoxides in toxicological studies.