Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems

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.

Fig 1. Pathway to demonstrate the involvement of free radicals in a toxicological process.

Top: Hypothesis that a compound may lead to the production of free radicals in a biological medium. This process may involve various free radicals that can be produced extracellularly or in different cellular compartments. Middle: Traditional spin trapping pathway: the design of the experiment requires an a priori assumption regarding the nature of the free radical. Because the choice of spin-trap depends on its ability to react with the expected free radical or the expected localization of the site of production (intracellular or extracellular), this leads to a long iterative process. The hook needs to be adapted to each type of fish and its localization. Bottom: Radicalomics, using a cocktail of spin traps, reveals the presence of free radicals in one step, whatever the chemical nature of the free radical or the site of production. The EPR signature obtained can be deconvolved to characterize the likely radical involved. Comparison: Whatever the fish and its localization, it will be hooked after a single cast.

Fig 2. Chemical structures of the individual spin traps used in the proposed cocktail.

Fig 3. EPR spectra from the cocktail in the presence of different free radical generating systems.

In Black, experimental spectra. In red, simulated spectra (using parameters described in Table 1). In grey, control experiments without generating system. A: methyl radical, B: hydroxyl radical, C: azidyl radical, D: sulfite radical, E: superoxide anion radical.

Fig 4. EPR spectra of K562 cells exposed to selected toxic agents.

A: Tert-butylhydroperoxide, B: Menadione, C: Hydrogen peroxide, D: Phenylhydrazine. E: control experiment (K562 cells in the presence of cocktail without toxic agent).

Fig 5. EPR spectra of K562 cells after exposure to menadione using individual spin trapping agents.

A: PBN, B: POBN, C: DEPMPO, D: EMPO, E: Cocktail of spin traps (black) which is actually the sum of EPR spectra recorded using DEPMPO and EMPO (red dots).

Fig 6. Influence of superoxide dismutase on the EPR spectra recorded after exposure to menadione.

EPR spectra of a preparation of K562 cells (2x10⁶cells/ml) exposed to menadione (1 mM): A. in presence of DEPMPO (50 mM) (gray) or of DEPMPO (50 mM) + PEG-SOD (100U/ml) (black); B. in presence of EMPO (50 mM) (gray) or of EMPO (50 mM) + PEG-SOD (100U/ml) (black); C. in presence of cocktail (50 mM/spin trap) (gray) or of cocktail(50 mM) + PEG-SOD (100U/ml) (black).