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Several methods have been developed to study the genotoxicity of endogenous or exogenous stresses. To determine if such stresses could alter DNA (genotoxicology) the methods consist in the measurement of DNA lesions or their consequences (cell cycle arrest, micronuclei, mutations, chromosomal aberrations,). In our laboratory we have developed several approaches to determine the genotoxicity of ionizing (radiobiology) and non-ionizing (photobiology) radiations, nanoparticles (nanotoxicology), and HAPs (environmental toxicology). Our data and those from the abundant literature are difficult to rationalize, but all these stresses induce very low levels of DNA lesions. Thus DNA lesions or alterations could not be used as a marker of exposure to these stresses. Intriguingly, the effects of stresses on the nucleotides pool imbalance has been poorly studied. Actually, an imbalance of the nucleotide pool is known to significantly affect cell division and survival, as confirmed by the use of 5-fluorouracil (a thymine analog) in chemotherapy, due to its ability to inhibit thymine synthase and thus to induce a nucleotides pool imbalance.
The objective of that project is to develop a highly specific and quantitative method to measure at the cellular level the amounts of the different nucleotides, and to study the variations of their concentrations following exposure of isolated cells to various conditions of stress.
For such a purpose, first an HPLC coupled through electrospray ionization to tandem mass spectrometry (HPLC-MS/MS) method (routinely used in the laboratory to quantify DNA lesions) will be develop to quantify natural nucleotides including mono-, di- and tri-phosphate derivatives, for both ribo- and desoxy-ribonucleotides. In a second step, the analytical method will be also applied to the measurement of less abundant nucleotide derivatives, including natural compounds such as AMPc, GMPc, FAD, SAM, Acyl-CoA, as well as modified nucleotides including for example 8-oxodGTP (arising from the oxidation of dGTP). Using such an approach, the concentrations of the above mentioned nucleotides will be determined in cells (in vitro) treated under several conditions of stress, for different doses and concentrations. The final objective is to determine if the nucleotides pool imbalance could be used as a good biomarker of stress.