Abstract
It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a “hypothesis-driven” approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a “data-driven” approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.
Highlights
Reactive oxygen species (ROS), including free radicals, are considered to be harmful agents involved in the genesis of various pathologies, but are important mediators in a range of biological and physiological processes [1]
In the optimization procedure for the choice of the cocktail components, we discarded possible candidate spin traps, namely dimethyl-5,5 -pyrroline-N-oxide (DMPO), for which alternative reactions have been reported to lead to radical adduct artifacts in some cases [12, 13], and whose–OOHadduct stability was lower than DEPMPO-OOH [14, 15]; 2,2,4-trimethyl-2Himidazole-1-oxide (TMIO) for which we found interfering background signals without free radical sources was excluded
We first tested the ability of the cocktail of spin traps to give an Electron Paramagnetic Resonance (EPR) signature for biologically relevant free radicals produced by well-defined enzymatic or chemical reactions
Summary
Reactive oxygen species (ROS), including free radicals, are considered to be harmful agents involved in the genesis of various pathologies, but are important mediators in a range of biological and physiological processes [1]. To unequivocally identify the presence of a free radical, the method of choice is Electron Paramagnetic Resonance (EPR) spectroscopy using spin-trapping experiments. The identity of free radicals can be inferred from the particular EPR spectrum using EPR constants, such as g values and hyperfine splitting constants This method has been extended to simple sensitive immunoassays for detecting large radical molecules, such as DNA- or protein-radicals [9,10,11], EPR remains the gold standard for identifying the presence of radicals in small molecules. Experimental conditions are generally tuned to fit these a priori assumptions This renders traditional spin-trapping experiments unpopular for rapid screening for possible free radical involvement in biological or toxicological processes because multiple experimental spin traps and conditions have to be performed sequentially (Fig 1). Rapid screening assays are more and more needed to assist in the detection of radical-mediated oxidative stress without a priori knowledge of a specific mechanism
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