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Marie Roser-Gilardoni

Quantification of biomarkers of exposure to vesicant agents in easy to collect biological matrices

Published on 4 October 2021
Thesis presented October 04, 2021

Abstract:
Use of chemical warfare agents is a current concern shown in several recent conflicts (Iraq and Syria) and associated to terrorist threat. Availability of biomonitoring assays for the identification of toxic compounds and assessment of individual exposure is an important challenge. Sulphur mustard, a chemical warfare agents belonging to the vesicants family, and its analog CEES will be the focus of this PhD work. In the present work, we developed a sensitive non-invasive ultra-high-pressure liquid chromatography associated with tandem mass spectrometry approach (UHPLC-MS/MS) to quantify specific and persistent biomarkers. We also performed biological experiments, in vivo and in vitro, to validate their use as biomarkers. A DNA adduct, N7Guanine-CEES was detected, both in nuclear DNA and as depurinated base, in all biological experiments. In particular, in the plasma of mice, it is persistent until fourteen days. The glutathione conjugate, GSH-CEES, is short-lived and closed to the limit of detection because it is readily converted into the cysteine conjugate, Cys-CEES. The quantity of Cys-CEES is high compared to the other biomarkers but it could be tricky to analyse it because of its polar properties and the presence of interfering substances. Cys-CEES is converted into the mercapturate acid conjugate. NAC-CEES was detected in vivo in exposed mice but not in skin in vitro models. It is persistent at least for two weeks. The analytical method used for the CEES biomarkers was adapted for the sulphur mustard biomarkers and improved by an on-line Solid Phase Extraction (SPE). This validated method allows for detecting in human plasma biomarkers of sulphur mustard Cys-Yp, NAC-Yp and N7Gua-Yp in eleven minutes including the online SPE step. In summary, our work allowed us to validate new biomarkers of exposure to vesicants and provided information on the metabolism of these toxic agents.

Keywords:
Biomarkers, Mass spectrometry, Solid Phase Extraction, Mercapturic acid pathway