-Sulfur mustard (HD), a very potent alkylating agent and lipopolysacchride (LPS), are both well characterized inflammatory factors. We have found that concomitant exposure of murine macrophage cells (RAW264.7) to LPS and HD induced protection against HD induced cytotoxicity. Both HD and LPS induce release of inflammatory markers in RAW264.7 cells. However, there are marked differences in the repertoire of inflammatory factors released by the two toxins: While exposure to HD, induced a dose-dependant death of these cells, no significant change in survival rate was observed following LPS (1-100 ng/ml) exposure. Additionally, LPS elicited a robust nitric oxide (NO) and TNF-α secretion whereas HD was practically ineffective. Both toxins increased PGE 2 secretion in a concentration dependent manner. Treatment of HD-exposed RAW264.7 cells with anti-inflammatory drugs such as dexamethazone (5 μM), voltaren (diclofenac) (8 μM) or doxycycline (5 μM), decreased the release of cytokines but had no effect on cell viability. Simultaneous application of LPS (100 ng/ml) and HD (20-100 μM) resulted in an amelioration of HD cytotoxicity. Adding the NO generator S-nitrosoglutathione (GSNO) or inhibiting NO production using L-N G -monomethyl Arginine, had no effect on cell viability. Moreover, addition of PGE 2 (20 ng/ml) failed to induce any changes in cell viability under basal or HD-induced toxicity. In contrast, TNF-α (20 ng/ml) provided remarkable protection against HD-induced cell death. These findings strongly suggest that LPS exerts its protective action against HD toxicity through the generation of TNF-α and may provide better understanding of the mechanism of cytoprotection.Key words: Sulfur mustard, RAW 264.7, LPS, TNF-α, NO, Cytotoxicity Correspondence: Nahum Allon (E-mail: nahuma@iibr,gov.il) Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.3, 345-355, 2010 Vol. 35 No. 3 345 Similarly, cutaneous application of HD is also associated with an acute inflammatory response accompanied by blister formation and epithelial necrosis (Dannenberg et al., 1985;Casillas et al., 2000;Dachir et al., 2002). Notably, early studies using isolated skin explants treated with HD have shown a rapid production of cytokines and prostaglandins (Rikimaru et al., 1991).In vitro studies, where the effects of adjacent tissue are absent, have indicated that HD exposure can directly induce cytokine secretion from macrophages as shown in various cells in culture including in human monocytes (Rikimaru et al., 1991;Arroyo et al., 1995). A number of cell lines such as macrophages (Amir et al., 1998, monocytes (Arroyo et al., 1995), lymphocytes and keratinocytes (Arroyo et al., 2000), show acute reaction to HD, similar to that reported in in-vivo studies, and thus, can serve as a model. These studies showed that HD can induce the release of TNFα and other inflammatory mediators by direct action on epithelial cells, suggesting that an inflammatory response can be initiated directly by HD in a single cell (Dan...