Introduction

1. Comparison of cellular responses to sulfur mustard, nitrogen mustard, and 2-chloroethyl ethyl sulfide: questioning the use of simulants in sulfur mustard research A great Student Mentored by Dr. Science Introduction Materials and Methods (cont.) Results (cont.) I really like my project.Sulfur mustard [bis-(2-chloroethyl)-sulfide] is a highly reactive chemical warfare agent that has been in use since World War I and most recently in the Iran-Iraqi conflict. Sulfur mustard (SM)is a potent vesicant that has many adverse effects on the human body. The main target organs of SM exposure are the skin, eyes, and respiratory tract. Sulfur mustard induces blistering, ulceration, inflammation, and necrosis, the premature death of cells (Dillman et al, 2004). SM infiltrates the skin without any immediate warning signs, such as burning or itching. Due to the threat sulfur mustard poses as a chemical weapon, research to find potential therapeutics that can reduce the effects of SM injury is of high interest. Nitrogen mustard (NM) and 2-chloroethyl ethyl sulfide (CEES) are related analogs of sulfur mustard that are used as simulants in some research of SM (Black et al., 2010). Like SM, both CEES and NM are alkylating agents that are highly toxic (Kisby, Springer, & Spencer, 2004; Qui, Paromov, Yang, Smith, & Stone, 2006). The suitability of NM and CEES as simulants for sulfur mustard in chemical injury has recently come into question. In this research, cell death, inflammatory marker, and selected critical signaling pathway (NFκB, p38, and p53) responses induced by these three agents were compared in HaCaT cells, a human keratinocyte cell line. • Western Blot: • I followed the EZQ assay protocol prior to performing the western blot. • Standard western blot procedures were followed. • The blot was imaged using the Typhoon™ 9400 Scanner. 3A 3B 3C Results Results illustrate that the inflammatory cytokine analyses demonstrate varied trends of SM, NM and CEES in cytokine expression of IL-1β, IL-8, IL-6 and TNF-a (Graph 1A-1D). All four cytokines were expressed with peaks at the concentration of 400 μM in SM exposed cells. Graph 2 represents the percentage cell viability after exposure to the agents, SM, NM and CEES based on the MTT assay. The western blot densitometry (Graph 3A-3C). showed that the signaling pathways were activated early in SM exposed cells, especially p38 (Graph 3A). Graph 2: Western blot densitometry of post agent exposure time in hours versus fold increase for activation of p38 (Graph 3A), p53 (Graph 3B), and IKB-α (Graph 3C) Conclusions The slope for CEES showed a more linear decrease compared to the exponential decrease of cell viability in SM and NM. In this study, cell death, inflammatory markers, and selected critical signaling pathways (p38, p53 and NFκB) induced by SM, NM and CEES were compared in HaCaT cells. The beadlyte assay demonstrated that the cytokines were expressed in dissimilar patterns in all three of the agents, SM, NM and CEES (Graph 1A-1D). The cell viability measured through the MTT assay showed that SM and NM has a comparable pattern of cell viability (Graph 1). The slope for SM and NM appeared to have an exponential decrease. The cell viability in CEES was quite irrregular and did not follow the trend represented by the SM and NM exposed cells (Graph 2). This study suggests that NM and CEES may not be suitable analogs of SM according to the results demonstrated by the cell death assays and the comparison of cellular responses to SM, NM and CEES. Materials and Methods Graph 1A Graph 1B • Cell culture: • HaCaTs were acquired from the Wright Patterson Air Force Base and seeded at two densities, 2.5 X 103 and 5.0 X 103 into T-75 flasks. • Cells were grown in DMEM low glucose (1x) keratinocyte growth medium and Fetal Bovine Serum (FBS). • The HaCaTs were reseeded into 96-well plates and 6-well plates at 80-90% confluency, and then used for exposures and experimentations. •  Exposures: • A 4 mM stock of SM in Keratinocyte Growth Medium (KGM) was made and was exposed to the HaCaTs within 15 minutes. • A 1M stock of NM in Dimethyl sulfoxide (DMSO) was prepared and added to the HaCaTs. • A 2000 μM stock of CEES in 10 mL KGM was made directly prior to use. • All exposures were performed in a certified chemical fume hood at the U. S. Army Medical Research Institute of Chemical Defense. • Beadlyte Assay: • Beadlyte assay was performed according to the manufacturer’s protocol. • Read on Luminex™ 100 platereader. • Cell death assay-Methylthiazol Tetrazolium (MTT) Assay: • MTT assay was performed according to the manufacturer’s protocol. • Absorbance was measured using the Platereader at wavelength 570 nm, the background absorbance of multiwell plates was measured at 690 nm • and subtracted from the 570 nm measurement. References Black, A.T., Joseph, L. B., Casillas, R. P., Heck, D. E., Gerecke, D. R., Sinko, P. J., & Laskin, D. L. (2010). Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide. ToxicolApplPharmacol, 15, 352-360. Dillman, J. F. III, McGary, K. L., & Schlager J. J. (2004). An inhibitor of p38 MAP kinase down regulates cytokine release induced by sulfur mustard exposure in human epidermal keratinocytes. Toxicology In Vitro, 18(5), 593–599. Kisby, G. E., Springer, N., & Spencer, P. S. (2000). In vitro neurotoxic and DNA-damaging properties of nitrogen mustard. Journal of Applied Toxicology, 20, S35–S41. Qui, M., Paromov, V. M., Yang, H., Smith, M., & Stone, W. L. (2006). Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages. BMC Cell Biology, 7-39. Graph 1C Graph 1D Graph 1: Beadlyte assay agent concentration versus pathway in pg/ml. All graphs have two axes; one for the SM and NM concentration and the other for the CEES concentration. Acknowledgments Lindsay Marron Sarah Beach Who Knows Graph 3: Percentage cell viability after exposure to the agents, SM, NM and CEES. The cell viability was measured by utilizing theMTT Assay.