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.
Table 1.
Hyperfine coupling constants used for the simulation of EPR spectra for individual spin traps and for the combination of spin traps (cocktail).
Reaction with hydroxyl radical.
Table 2.
Hyperfine coupling constants used for the simulation of EPR spectra for individual spin traps and for the combination of spin traps (cocktail).
Reaction with azidyl radical.
Table 3.
Hyperfine coupling constants used for the simulation of EPR spectra for individual spin traps and for the combination of spin traps (cocktail).
Reaction with sulfite radical.
Table 4.
Hyperfine coupling constants used for the simulation of EPR spectra for individual spin traps and for the combination of spin traps (cocktail).
Reaction with methyl radical.
Table 5.
Hyperfine coupling constants used for the simulation of EPR spectra for individual spin traps and for the combination of spin traps (cocktail).
Reaction with hydroperoxyl radical.
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).