Sulfur mustard (SM), a chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM that could be easily accessible in urine or plasma. Because investigations using SM are prohibited by the Organization for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. We developed an ultra-high-pressure liquid chromatography -tandem mass spectrometry approach (UHPLC-MS/MS) to the conjugate of CEES to glutathione and two of its metabolites, the cysteine and the N-acetyl-cysteine conjugates. The N7guanine adduct of CEES (N7Gua-CEES) was also targeted. After synthesizing the specific biomarkers, a solid phase extraction protocol and a UHPLC-MS/MS method with isotopic dilution were optimized.We were able to quantify N7Gua-CEES in the DNA of HaCaT keratinocytes and of explants of human skin exposed to CEES. N7Gua-CEES was also detected in the culture medium of these two models, together with the glutathione and the cysteine conjugates. In contrast, the N-acetyl-cysteine conjugate was not detected. The method was then applied to plasma from mice cutaneously exposed to CEES.All four markers could be detected. Our present results thus validate both the analytical technique and the biological relevance of new, easily quantifiable biomarkers of exposure to CEES. Because CEES behaves very similarly to SM, the results are promising for application to this toxic of interest.

Sulfur mustard, a chemical warfare agent known to be a vesicant of skin, readily diffuses in the blood stream and reaches internal organs. In the present study, we used the analog (2chloroethyl)-ethyl-sulfide (CEES) to provide novel data on the systemic diffusion of vesicants and on their ability to induce brain damage, which result in neurological disorders. SKH-1 hairless mice were topically exposed to CEES and sacrificed at different time until 14 days after exposure. A plasma metabolomics study showed a strong systemic impact following a self-protection mechanism to alleviate the injury of CEES exposure. This result was confirmed by the quantification of specific biomarkers in plasma. Those were the conjugates of CEES with glutathione (GSH-CEES), cysteine (Cys-CEES) and N-acetyl-cysteine (NAC-CEES), as well as the guanine adduct (N7Gua-CEES). In brain, N7Gua-CEES could be detected both in DNA and in organ extracts. Similarly, GSH-CEES, Cys-CEES and NAC-CEES were present in the extracts until day14. Altogether, these results, based on novel exposure markers, confirm the ability of vesicants to induce internal damage following dermal exposure. The observation of alkylation damage to glutathione and DNA in brain provides an additional mechanism to the neurological insult of SM.

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