2-chloroethyl ethyl sulfide (CEES) is a vesicant agent, commonly referred to half mustard due to its ability to form monofunctional adducts with DNA. In this study, we evaluated the chemoprotective potential of 13 compounds and their mixtures with sodium 2-mercaptoethanesulfonate (MESNA) against CEES-induced geno- and cytotoxicity in human lung cell line A-549. MESNA, L-glutathione (GSH), thiourea, sodium thiosulfate, hexamethylenetetramine, 4-acetamidophenol, asoxime dichloride (HI-6), N-acetyl-L-cysteine (NAC), sodium pyruvate, myo-inositol, 3-aminobenzamide (3-AB), nicotinamide, and Nω-nitro-L-arginine methyl ester hydrochloride and combinations of these compounds with MESNA were applied 30 min before CEES. DNA alkylation was measured using modified comet assay 1 and 24 h after the exposure. Cell viability was determined using MTT assay at 24 and 72 h. The mono-therapeutical approach identified MESNA and GSH to provide significant chemoprotection. NAC and 3-AB supported DNA damage repair, while cell viability remained unaffected. Mixtures of GSH or NAC with MESNA showed protective synergism against DNA damage. Other compounds or their combinations with MESNA failed due to the potentiation of CEES-induced cytotoxicity. The chemoprotection against CEES remains limited; however, the combination of substances can provide protective synergy and may represent a promising strategy in the treatment of accidental exposure to monoalkylating agents.

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 Organisation 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 (UHPLC-MS/MS) approach to the conjugate of CEES to glutathione and two of its metabolites: the cysteine and the N-acetylcysteine conjugates. The N7-guanine 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-acetylcysteine 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 similar to SM, the results are promising for application to this toxic of interest.

Mustard gas (sulfur mustard, SM), a highly vesicating chemical warfare agent, was first deployed in warfare in 1917 and recently during the Iraq-Iran war (1980s) and Syrian conflicts (2000s); however, the threat of exposure from stockpiles and old artillery shells still looms large. Whereas research has been long ongoing on SM-induced toxicity, delineating the precise molecular pathways is still an ongoing area of investigation; thus, it is important to attempt novel approaches to decipher these mechanisms and develop a detailed network of pathways associated with SM-induced toxicity. One such avenue is exploring the role of microRNAs (miRNAs) in SM-induced toxicity. Recent research on the regulatory role of miRNAs provides important results to fill in the gaps in SM toxicity-associated mechanisms. In addition, differentially expressed miRNAs can also be used as diagnostic markers to determine the extent of toxicity in exposed individuals. Thus, in our review, we have summarized the studies conducted so far in cellular and animal models, including human subjects, on the expression profiles and roles of miRNAs in SM- and/or SM analog-induced toxicity. Further detailed research in this area will guide us in devising preventive strategies, diagnostic tools, and therapeutic interventions against SM-induced toxicity.

Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/-). Exposure to NM (3.2mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/- mice at 24h. However, by 72h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/- mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24h persisting through 72h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72h after exposure in p53+/- mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/- mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury.