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Etude de profils en adduits à l'ADN comme biomarqueurs potentiels d'exposition aux polluants aériens en milieu urbain dans une approche de type adductomique

Abstract : Many studies in the second half of the 20th century have shown that genotoxic carcinogens, either directly or after metabolic activation, react with DNA to form covalently bonded adducts that are absolutely central in the carcinogenic process. Although there is compelling evidence of the presence of multiple DNA adducts in the lungs of subjects exposed to smoking or occupational exposure to a given aldehyde, it is clear that this is an area in which further research has been necessary. The aim of this thesis is to establish exocyclic DNA adducts profiles induced by the mixture of aldehydes, which could eventually be considered as a genotoxic marker of aldehyde exposure, both endogenous environmental. For this reason, we have validated a fast, sensitive and precise method on liquid chromatography coupled to mass spectrometry in tandem mode (UHPLC-MS/MS) using isotopic dilution, for trace quantification of 9 exocyclic DNA adducts derived from 8 major exogenous and endogenous aldehydes, including formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. These adducts were synthesized and purified as well as their labeled homologues, identified and quantified through standard curves ranging from 0.25 (LLOQ) to 250 ng/mL (ULOQ) adducts in water and in DNA to describe the matrix effects. Quality control (QC) samples were prepared and analyzed to verify the accuracy and precision of the method in repeatability and intermediate fidelity situations. The absence of cross-contamination has also been demonstrated. The method is able to differentiate the 9 analytes of interest and their internal standards using for each analyte a quantification transition and a confirmation transition. This method has been validated according to the recommendations of the European Medicines Agency (EMA) concerning bioanalytical methods. It meets all the essential criteria to guarantee the acceptability of the performances and the reliability of the analysis results. This method is the very first validated and can be used in adductomics in the context of studies on the exposome. In addition, the exocyclic adducts were simultaneously measured by an in vitro approach in calf thymus DNA exposed to different concentrations of each aldehyde apart or in equimolar mixtures. This approach allowed us to establish dose-dependent relationships for all aldehydes with the exception of malondialdehyde and methylglyoxal. A dose-response relationship was also observed with equimolar mixtures of aldehydes. It made it possible to define different reactivities of aldehydes in mixture versus DNA. The profiles of these exocyclic adducts were also determined in the blood DNA of smokers and non-smokers. Cigarette smoke contains several aldehydes known to covalently bind to DNA bases, so the DNA adduct may be considered as biomarker of tobacco exposure. Significant differences in adducts levels were obtained between smokers and non-smokers DNA with the exception of malondialdehyde-induced DNA adduct. Correlations were established between each adduct and smoking-related markers without any significant correlation of all adducts with a specific marker. Furthermore, we have shown that exposure to formaldehyde, butanal and benzaldehyde had an effect on the concentrations of urinary MDA measured in Lebanese police stationed at the intersection for 7 hours a day and after 5-day exposure to road traffic. An increase in plasma MDA has been described; years of work had an impact on the concentrations of this biomarker. These results are promising and it would be interesting to validate in population the profile of 9 exocyclic adducts as biomarkers of exposure to both exogenous and endogenous aldehydes as part of an adductomic approach to understand the carcinogenic risk in relation to aldehydes exposures in urban areas.
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Helena Alamil. Etude de profils en adduits à l'ADN comme biomarqueurs potentiels d'exposition aux polluants aériens en milieu urbain dans une approche de type adductomique. Médecine humaine et pathologie. Normandie Université; Université libanaise, 2019. Français. ⟨NNT : 2019NORMC407⟩. ⟨tel-02373739⟩

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