Oxidants formed from oxidative and nitrative metabolism include reactive oxygen species (ROS) such as superoxide, hydrogen peroxide/lipid hydroperoxides and reactive nitrogen species (RNS) (e.g., peroxynitrite [ONOO-] and nitrogen dioxide), and reactive halogenated species (e.g., hypochlorous acid [HOCl]). Increasingly, ROS and RNS are implicated in tumorigenesis as well as tumor growth, progression, and metastasis. Recently, ROS were implicated in drug resistance, metabolic reprogramming, and T-cell metabolism in immunotherapy. Mostly, fluorescent probes have been used in cell culture systems. The identity of species is obtained by LC-MS analyses of diagnostic marker products. However, extrapolation of these assays to cancer xenografts is difficult if not impossible. Thus, development of a probe-free assay for monitoring and assessing oxidant formation in tumor cells and tumor xenografts is critical and timely. Here, we describe the use of ex vivo electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures as a uniquely useful probe-free technique for assessing intracellular oxidation and oxidants via EPR signals from redox centers, particularly iron-sulfur clusters, in mitochondrial and cytosolic redox proteins. Examples of cancer cells subjected to inhibition of mitochondrial oxidative phosphorylation are presented. This ex vivo methodology can be readily extended to monitor oxidant formation in tumor tissues isolated from mice and humans.
Keywords: Electron paramagnetic resonance; Mitochondrial targeting; Oxidative phosphorylation; Reactive oxygen species.