Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future

• Published in: The Journal of biological chemistry 2018-06, Vol.293 (26), p.10363-10380
• Main Authors: Cheng, Gang, Zielonka, Monika, Dranka, Brian, Kumar, Suresh N., Myers, Charles R., Bennett, Brian, Garces, Alexander M., Dias Duarte Machado, Luiz Gabriel, Thiebaut, David, Ouari, Olivier, Hardy, Micael, Zielonka, Jacek, Kalyanaraman, Balaraman
• Format: Article
• Language: English
• Subjects: Aconitate Hydratase - metabolism; bioenergetics; Cell Line; Chemical Sciences; electron paramagnetic resonance (EPR); Electron Transport Complex I - antagonists & inhibitors; Electron Transport Complex III - antagonists & inhibitors; Energy Metabolism - drug effects; Enzyme Inhibitors - pharmacology; Humans; low-temperature EPR; Methods and Resources; Mitochondria - drug effects; Mitochondria - metabolism; mitochondrial oxidants; Molecular Probe Techniques; Oxidation-Reduction - drug effects; Oxidative Stress - drug effects; peroxiredoxin; radical scavengers; reactive oxygen species (ROS); Reactive Oxygen Species - metabolism; redox probes; superoxide ion; Superoxides - metabolism; thiol-specific antioxidant enzymes, bioenergetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.003044

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.