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Berliner LJ, Parinandi NL, editors. Measuring Oxidants and Oxidative Stress in Biological Systems [Internet]. Cham (CH): Springer; 2020. doi: 10.1007/978-3-030-47318-1_2
Chapter 2Pitfalls of Reactive Oxygen Species (ROS) Measurements by Fluorescent Probes and Mitochondrial Superoxide Determination Using MitoSOX
Balaraman Kalyanaraman.
Author Information and Affiliations
1.Published online: August 9, 2020.
Intracellular and mitochondrial superoxide formation is detected using phenanthrene-based dyes such as hydroethidine, mitochondria-targeted hydroethidine, or MitoSOX. HE and MitoSOX are redox probes, which undergo two-electron oxidation forming ethidium (E+) and Mito-ethidium (Mito-E+). The two-electron oxidation products derived from these probes exhibit the characteristic fluorescence that aids in fluorescence microscopy or flow cytometry or related techniques that are used to detect and determine superoxide (sometimes referred to as ROS, mitochondrial ROS, or mROS). This chapter briefly addresses the pitfalls of fluorescence-based techniques for detecting the intracellular superoxide.
Keywords:
Reactive oxygen species measurement, Pitfalls of ROS detection and determination, Fluorescent probes for ROS, Mitochondrial superoxide determination, MitoSOX ROS determination pitfall, Hydroethidine mitochondrial ROSPhenanthrene-based dyes (e.g., hydroethidine, mitochondria-targeted hydroethidine, or MitoSOX) have been used to detect intracellular and mitochondrial superoxide formation [1, 2]. Both HE and MitoSOX are redox probes and undergo two-electron oxidation forming ethidium (E+) and Mito-ethidium (Mito-E+). The two-electron oxidation products derived from these probes exhibit characteristic fluorescence, and therefore, fluorescence microscopy or flow cytometry or related techniques have been used to detect and quantitate superoxide (sometimes referred to as ROS, mitochondrial ROS, or mROS). Most recently, MitoSOX-derived fluorescence was used to detect mitochondrial ROS formed in activated T cells [3]. This short commentary addresses the pitfalls of fluorescence-based techniques for detecting intracellular superoxide. Readers are referred to the previous reviews on this topic [4–8].
Many years ago, we showed that superoxide reacts with hydroethidine and other analogs including hydropropidine (HPr) and MitoSOX, forming a characteristic hydroxylated product (e.g., 2-hydroxyhydroethidium or 2-OH-E+ and Mito-2-OH-E+), but not the corresponding ethidium (E+ or Mito-E+) [9, 10]. The fluorescence parameters of 2-OH-E+ and Mito-2-OH-E+ are significantly different, and therefore, monitoring the red fluorescence of HE or MitoSOX in cells will not measure intracellular superoxide formation, and the increase in fluorescence intensity is merely indicative of increased oxidation of HE and MitoSOX. Although the exact mechanism of oxidation is not determined, it is conceivable that redox metal ions (iron, for example) and/or peroxidatic mechanism is responsible for the oxidation of HE to E+ and MitoSOX to Mito-E+. Therefore, the use of MitoSOX to measure mitochondrial superoxide formation, using the fluorescence technique, is incorrect and flawed.
Evidence for one-electron oxidant formation in extracellular and intracellular settings was obtained by determining dimeric product formation (e.g., E+-E+, Mito-E+-Mito-E+) using HPLC and LC-MS techniques [11]. The dimeric products are not fluorescent.
Another caveat is the uptake of redox dyes into cells. The intracellular uptake varies depending upon the experimental conditions (changes in oxidative profile, membrane potential, apoptosis). Measuring the intracellular concentration of the fluorescent probes is critical for interpreting the results. At the same rate of intracellular oxidant generation, an increase or decrease in probe uptake will alter the amount of product formation.
Improper use of these probes during extraction or incubation procedure can induce hydrolysis, like in other assays, and give rise to confounding results [12]. We have used isotopically labeled oxidants (e.g., O-18-labeled superoxide) and unequivocally shown that the oxygen atom in 2-OH-E+ is incorporated from molecular oxygen and not from water (unpublished results).
In conclusion, irrespective of the probes (HE, Mito-SOX or HPr) used, it is important to obtain a global profile of probe uptake and oxidation products in order to fully assess the extracellular, intracellular, and mitochondrial oxidant formation.
References
- 1.
- Zielonka J, Kalyanaraman B. Hydroethidine- and Mito-SOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth. Free Radic Biol Med. 2010;48:983–1001. Review. [PMC free article: PMC3587154] [PubMed: 20116425]
- 2.
- Kalyanaraman B. What effects do antioxidants have on tumors? Sci Trends. 2018. https:
//sciencetrends .com/what-effects-do-antioxidants-have-on-tumors/. - 3.
- De Biasi S, Gibellini L, Bianchini E, Nasi M, Pinti M, Salvioli S, Cossarizza A. Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry. Cytometry A. 2016;89(12):1106–10. [PubMed: 27575554] [CrossRef]
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- Zielonka J, Joseph J, Sikora A, Kalyanaraman B. Real-time monitoring of reactive oxygen and nitrogen species in a multiwell plate using the diagnostic marker products of specific probes. Methods Enzymol. 2013;526:145–57. [PMC free article: PMC6941666] [PubMed: 23791099] [CrossRef]
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- Zielonka J, Hardy M, Kalyanaraman B. HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements. Free Radic Biol Med. 2009;46:329–38. [PMC free article: PMC3375818] [PubMed: 19026738] [CrossRef]
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- Zielonka J, Vásquez-Vivar J, Kalyanaraman B. Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine. Nat Protoc. 2008;3:8–21. [PubMed: 18193017] [CrossRef]
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- Kalyanaraman B, Hardy M, Zielonka J. A critical review of methodologies to detect reactive oxygen and nitrogen species by NADPH oxidase enzymes: implications in pesticide toxicity. Curr Pharmacol Rep. 2016;2:193–201. [PMC free article: PMC5070669] [PubMed: 27774407] [CrossRef]
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- Kalyanaraman B, Hardy M, Podsiadly R, Cheng G, Zielonka J. Recent developments in detection of superoxide radical anion and hydrogen peroxide: opportunities, challenges, and implications in redox signaling. Arch Biochem Biophys. 2017;617:38–47. [PMC free article: PMC5318280] [PubMed: 27590268] [CrossRef]
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- Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vásquez-Vivar J, Kalyanaraman B. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med. 2003;34:1359–68. [PubMed: 12757846] [CrossRef]
- 10.
- Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vásquez-Vivar J, Kalyanaraman B. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl Acad Sci U S A. 2005;102:5727–32. [Erratum: Proc Natl Acad Sci USA. 102:9086, 2005]. [PMC free article: PMC556312] [PubMed: 15824309] [CrossRef]
- 11.
- Zielonka J, Srinivasan S, Hardy M, Ouari O, Lopez M, Vásquez-Vivar J, Avadhani NG, Kalyanaraman B. Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers. Free Radic Biol Med. 2008;44:835–46. [PMC free article: PMC2692199] [PubMed: 18155177] [CrossRef]
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- Xiao Y, Meierhofer D. Are hydroethidine-based probes reliable for reactive oxygen species detection? Antioxid Redox Signal. 2018;31(4):359–67. https://doi
.org/10.1089/ars.2018.7535. [PMC free article: PMC6602104] [PubMed: 29790367] [CrossRef]
- Review HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection.[Biochim Biophys Acta. 2014]Review HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection.Kalyanaraman B, Dranka BP, Hardy M, Michalski R, Zielonka J. Biochim Biophys Acta. 2014 Feb; 1840(2):739-44. Epub 2013 May 10.
- Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.[Methods Enzymol. 2014]Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.Polster BM, Nicholls DG, Ge SX, Roelofs BA. Methods Enzymol. 2014; 547:225-50.
- Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers.[Free Radic Biol Med. 2008]Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers.Zielonka J, Srinivasan S, Hardy M, Ouari O, Lopez M, Vasquez-Vivar J, Avadhani NG, Kalyanaraman B. Free Radic Biol Med. 2008 Mar 1; 44(5):835-46. Epub 2007 Dec 4.
- Are Hydroethidine-Based Probes Reliable for Reactive Oxygen Species Detection?[Antioxid Redox Signal. 2019]Are Hydroethidine-Based Probes Reliable for Reactive Oxygen Species Detection?Xiao Y, Meierhofer D. Antioxid Redox Signal. 2019 Aug 1; 31(4):359-367. Epub 2018 Jun 22.
- Review Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.[Free Radic Biol Med. 2010]Review Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.Zielonka J, Kalyanaraman B. Free Radic Biol Med. 2010 Apr 15; 48(8):983-1001. Epub 2010 Jan 29.
- Pitfalls of Reactive Oxygen Species (ROS) Measurements by Fluorescent Probes and...Pitfalls of Reactive Oxygen Species (ROS) Measurements by Fluorescent Probes and Mitochondrial Superoxide Determination Using MitoSOX - Measuring Oxidants and Oxidative Stress in Biological Systems
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