Biological and molecular mechanisms of sulfur mustard analog-induced toxicity in JB6 and HaCaT cells: Possible role of ATM/ ATR-cell cycle checkpoint pathway

Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, owing to limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here we investigated the molecular mechanism of HD analog, 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to control, up to 1 mM CEES treatment of these cells for 2, 4 and 24 h, caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ATM at ser1981 and ATR at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide (PI) staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature and latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and PARP cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of caspase pathway. Together, our findings suggest a DNA damaging effect of CEES that activates ATM/ATR-cell cycle checkpoint signaling as well as caspase-PARP pathways leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers and epidermal cell models in this study, have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity. Our results indicate that CEES-induced decrease in cell viability and DNA synthesis, and S and G2-M phase cell cycle arrest were associated with a DNA damage followed by the activation of DNA-damage sensor kinases ATM and ATR, and modulation of S and G2-M phase regulatory molecules in both JB6 and HaCaT cells. In addition, CEES-induced apoptosis/necrosis in both cell lines was associated with cleavage of caspase-3 and PARP.