SPIN BioRad

In the last 20 years, the study of Reactive Oxygen and Nitrogen Species (RONS) has appeared as an increasing field of research. This interest is related to their roles as critical mediators in various physiological processes (second messengers and regulators of signal transduction) and pathologies (cancers, neurodegenerative diseases, ischemia / reperfusion damages, diabetes, atherosclerosis). Among these species, the oxygen-centered free radicals are of particular biomedical importance. An accurate technique for the detection of these species is critical for a better understanding of the physiological and pathophysiological processes in which they are implicated. Many methods have been developed to assay O2'- in biochemical, cellular, and in vivo systems such as: cytochrome c reduction, chemiluminescence, fluorescent dyes, hydroethidine oxidation, and nitroblue tetrazolium reduction. Unfortunately, using these techniques it is difficult to discriminate between biological oxidants and O2'-. Beside to above-mentioned techniques, the technique of Electron Paramagnetic Resonance (EPR) spectroscopy allows the detection of free radicals and an extensive characterization of their generation, kinetics, and reactions. Thus, EPR spectroscopy is one of the most powerful techniques for the study of free radicals. However, combinations of rapid decays and low steady-state concentration prevent the direct EPR detection of most biological relevant radicals (e.g., O2'-, NO', HO') under physiological conditions. To circumvent these difficulties, the implication of free radicals can be inferred using the spin trapping technique in which short-lived free radicals specifically react with a nitrone spin trap to form a persistent radical adduct that is conveniently detected by EPR spectroscopy. In 1999, Swartz et al. evaluated DEPMPO, a new spin trap, as spin trapping agent in biological systems and the authors concluded that DEPMPO is a potentially good candidate for trapping radical in biological systems and represents an improvement over DMPO. And more recently, Swartz et al. evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on CHO cells and the stability of the radical adducts in the presence of cells. As a conclusion, the authors indicated that with appropriate controls and selection of spin traps, the spin trapping of reactive free radicals in biological systems is likely to be effective using the newly available spin traps. Since this work, few experiments have been performed successfully in biological systems. The longest half-lives for radical adducts in vivo have been estimated to be around 1 or 2 min with DEPMPO. For the study of free radicals in biological systems, the technique is still limited by the following main drawbacks: - the short lifetime of the superoxide spin adducts, - the readily reduction of nitroxide spin adducts to EPR silent compounds - the low rate constants observed for the trapping of superoxide radical ' the very low steady-state concentration of superoxide radical - the low concentration of the spin trap on the spot of the radical event As a consequence, application of spin trapping technique to in vivo systems is still challenging. There is still a growing need in tools that can provide information on free radical processes occurring in biological systems, and this is illustrated by the incorporation of organic chemists in leading groups involved in free radical biology and in grant proposals focused on this topic. We propose to study the spin trapping of reactive oxygen species in biological systems at the level of theoretical calculations, in buffer solution, in subcellular fractions, in cells and in small animals. These combined studies should allow us to gain insight into the way nitrone spin traps distribute in cells, in the intrinsic mechanism of degradation of the superoxide adduct, on the metabolism of the spin adducts, on the trapping rate of superoxide radical and on the valuable strategies useful to protect the spin adducts in biological systems. We hope to get the following major results: - to develop new spin traps with improved superoxide trapping rate, - to develop a theoretical model to correlate the hyperfine coupling constants to the structure and reactivity of the spin adducts, - to highlight the metabolic pathways of spin adducts, - to enhance the lifetime of the paramagnetic adducts in biological systems, - to determine the important molecular features governing the spin trapping efficiency, - to prepare efficient spin traps to investigate free radical processes in functional biological systems. A the end of the program, we expect that we will be able to offer new improved spin traps for biological systems to the scientific community. The tools and methods that will be developed during the project will be useful also in other fields such as: imaging techniques (PEDRI, OMRI, DNP), therapeutic studies, and immuno-spin trapping technique.