The primary targets of SM are the skin, cornea, and respiratory tissues. Signs of injury follow an asymptomatic period of variable length, depending on the level of exposure and the target organ. The asymptomatic period is briefest for the eye and is followed by redness and irritation which can progress to corneal damage with photophobia, blepharospasm, and temporary blindness (Papirmeister et al., 1991). Airway exposure primarily targets the nasal, laryngeal, and tracheobronchial mucosa, with early mild irritation giving rise to epistaxis, laryngeal injury, and bronchial edema and damage (Papirmeister et al., 1991). Respiratory barrier function is compromised, and at higher concentrations, the lower airway epithelium becomes involved, with corresponding impairment of gas exchange (Papirmeister et al., 1991). Initial contact with the skin is typically not associated with discomfort. Erythema, the first sign of exposure, develops after a latency of 2–24 h and may be associated with intense itching. Vesicles filled with a pale yellow fluid appear several hours later, and with time, they may coalesce to form pendulous blisters. Blisters subsequently burst, resulting in formation of a necrotic layer or eschar on the affected skin surface. Typically, the wounds resolve over the course of 10–50 days, leaving pigmentation changes that may persist for months or years (Balali-Mood and Hefazi, 2005; Chiesman, 1944). Figure 2 depicts the time course of pathophysiological responses following cutaneous SM exposure. Multiple rounds of blistering and healing in the same individual have also been reported (Balali-Mood and Hefazi, 2005).

Inflammation is thought to play a significant role in SM-induced pathology with initial exposure characterized by an accumulation of both granulocytes and macrophages at sites of injury (Dannenberg et al., 1985; Guignabert et al., 2005; McClintock et al., 2002; Millard et al., 1997; Papirmeister et al., 1991; Smith et al., 1995). Exposure to high levels of SM can also result in systemic toxicity; notably bone marrow depression, consequent immune suppression, and increased susceptibility to infection (Balali-Mood and Hefazi, 2005). Chronic conditions arising from acute or long-term low-level exposure to SM have been documented involving the skin, eyes, lung, nervous system, immune system, and gastrointestinal tract (Balali-Mood and Hefazi, 2005; Ghanei et al., 2006; Khateri et al., 2003; Sidell and Hurst, 1997). Occupational exposures to SM that have occurred during its manufacture have also been associated with various forms of cancer (Easton et al., 1988; Inada et al., 1978; Pechura and Rall, 1993; Wada et al., 1968; A. Weiss and B. Weiss, 1975; Yanagida et al., 1988). The present review surveys the current knowledge of the biological effects of SM in the skin, with a focus toward understanding mechanisms involved in cutaneous blister formation.

A mouse ear model was initially used by Brinkley et al. (1989) to assess SM-induced dermal alterations and then further characterized and developed by Casillas et al. (1997). Termed the mouse ear vesicant model (MEVM), liquid SM (0.04–0.64 mg in 5 μl) is applied to the medial surface of one ear, with the other ear serving as the vehicle control. After a characteristic latency period, a dose-dependent increase in the frequency and severity of histopathological markers including edema, epidermal necrosis, and the formation of microblisters on medial and lateral surfaces is observed. Immunostaining and electron microscopy has revealed that epidermal-dermal separation occurs at the level of the upper lamina lucida, with focal cleavage into the lower portion of the stratum basale as indicated by the detection of hemidesmosomal components on both dermal and epidermal sides of the cleavage site (Monteiro-Riviere et al., 1999). This locus of separation is attributed to the unique anatomical complexity of the ear relative to other epidermal sites.

More recently, a newer method for assessing SM-induced skin injury using nude mice grafted with human skin has been described (Greenberg et al., 2006). Previous variations of this model used biopsied partial thickness human skin as graft tissue (Papirmeister et al., 1984; Vogt et al., 1984). In these systems, SM was found to induce edema and microvesication, but only at relatively high doses (Papirmeister et al., 1991). The newer method utilizes engineered human skin formed by growing keratinocytes at an air-liquid interface on a fibroblast-populated collagen matrix. Though devoid of adnexal structures, these grafts appear to retain many of the morphological and functional features of normal human skin (Kolodka et al., 1998; Smith et al., 1997a). Using this model, two distinct stages of SM-induced epithelial damage have been identified by Greenberg et al. (2006). The first, a prevesication stage, observed 6-h postexposure, is largely independent of dose. It is characterized by the appearance of discrete clusters of pyknotic epithelial cells located exclusively in the stratum basale. The second stage, observed at 24 h, is dose dependent and involves significantly greater number of cells in the basal and suprabasal layers of the epidermis. Typical of this stage is the formation of microblisters through separation along the basement membrane zone. At moderate doses of SM, a polymorphonuclear cellular infiltrate is also evident in the dermis at 6 h, with focal infiltration into the epidermis at 24 h. Moreover, while the distribution of type VII collagen and laminin-332 immunoreactivity is unchanged during prevesication, a discontinuous pattern is noted after 24 h, a time coincident with microvesication.

An influx of neutrophils into the forearm of skin of human subjects has been observed as early as 30 min after exposure to SM (Warthin and Weller, 1919). Similarly, in the rabbit model, increases in granulocytes and mononuclear cells have been reported within 2 h of SM administration, persisting for 24 h (Dannenberg et al., 1985). Leukocyte emigration into the papillary dermis and epidermis has also been reported in mice and in human skin explants after SM exposure (Lindsay and Rice, 1996; Wormser et al., 2005). Myeloperoxidase activity, an indicator of neutrophil influx, increases within 9 h, preceding macrophage migration into the skin of hairless mice after CEES exposure (Tewari-Singh et al., 2009). In the MEVM, pretreatment of mice with neutrophil-depleting monoclonal antibodies significantly reduces late-stage necrosis induced by SM, suggesting a role for these cells in skin toxicity (Levitt et al., 2004). In contrast, while there is evidence of dermal mast cell degranulation in SM-treated tissues and increases in dermal mast cell number after CEES treatment has been reported (Levitt et al., 2004; Rikimaru et al., 1991; Tewari-Singh et al., 2009), treatment of wild-type and mast cell–deficient (W/W^v) mice yielded similar changes in ear weight and extravasation, suggesting a lack of involvement of mast cells in the edematous stage of SM-induced skin pathology (Levitt et al., 2004).

Several in vivo studies have documented increased expression of proinflammatory cytokines in the skin following SM exposure. Using in situ hybridization techniques, increases in interleukin (IL)-1β, IL-8, monocyte chemoattractant protein (MCP)-1, and growth related gene mRNA were noted as early as 2 h after application of liquid SM to rabbit skin (Tsuruta et al., 1996). In mouse ear, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and granulocyte monocyte-colony stimulating factor have been reported to be elevated within 6 h (Ricketts et al., 2000; Sabourin et al., 2000; Wormser et al., 2005). Increases in IL-1α protein and IL-1β, TNF-α, macrophage inflammatory protein, MIP-2, and MCP-1 mRNA have also been detected in the dorsal skin of hairless mice after exposure to SM vapor (Ricketts et al., 2000; Sabourin et al., 2003). Similarly, following vapor cup exposure of weanling pigs to SM, increases in relative mRNA levels of IL-1β, IL-6, IL-8, and TNF-α were noted (Sabourin et al., 2002). In cultured human keratinocytes, SM stimulates the release of IL-1β, IL-6, IL-8, and TNF-α at 100–300μM, doses relevant to in vivo exposure (Arroyo et al., 2000). Cultured skin fibroblasts have also been shown to express IL-6 in response to SM (Arroyo et al., 2001). These cytokines are thought to be key to inflammatory cell recruitment and activation at sites of injury, initiating a second phase of soluble mediator release. Cytokine expression is controlled by several signaling molecules, including the transcription factors nuclear factor-kappaB (NF-κB) (Ghosh et al., 1998) and activator protein-1 (AP-1) (Zenz et al., 2008). NF-κB has been reported to be activated after SM exposure (Atkins et al., 2000; Minsavage and Dillman, 2007; Rebholz et al., 2008) and both AP-1 and NF-κB after CEES exposure (Pal et al., 2009).

Arachidonic acid and its cyclooxygenase and lipooxygenase products are important inflammatory mediators that have also been observed in the skin after SM exposure (Blaha et al., 2000; Dachir et al., 2004; Lefkowitz and Smith, 2002; Rikimaru et al., 1991; Tanaka et al., 1997). Several of these mediators increase capillary permeability facilitating the influx of additional inflammatory substances including complement components, kinins, and fibrin into the dermal interstitium (Rikimaru et al., 1991). Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin biosynthesis, has also been identified in the epidermis of SM-treated mice (Nyska et al., 2001). Findings that nonsteroidal anti-inflammatory agents (NSAIDs) reduce skin injury suggest that these mediators are important in SM toxicity (Casillas et al., 2000). That COX-2 is involved in toxicity is also supported by studies showing that the extent of ear swelling and histopathological signs of lesion severity are markedly reduced in COX-2 null mice treated with SM or in wild-type mice treated with celecoxib, a COX-2–specific inhibitor (Wormser et al., 2004). In contrast, loss of COX-1, the constitutive isoform of the enzyme, has no effect on cutaneous injury induced by SM. Taken together, these studies suggest an involvement of inflammatory mediators in SM cutaneous pathology. However, it remains to be determined which of these are important in the vesication process.

Much has been theorized regarding the potential for SM and its analogs to produce oxidative and electrophilic stress, processes often associated with inflammation, and the role that this plays in toxicity (Dacre and Goldman, 1996; Paromov et al., 2007). Under homeostatic conditions, a net reducing environment is maintained in tissues by the presence of glutathione (GSH), which serves as a buffer against cytotoxic electrophiles and reactive oxygen species (ROS). The propensity of SM to react with sulfhydryls is thought to lead to a concentration-dependent depletion of reducing equivalents within cells. Recent findings also suggest that SM and related vesicants can interact with key intracellular reductases to generate mustard-free radicals (Brimfield et al., 2009). In addition, inflammatory cells, which infiltrate into the skin in response to SM-induced injury, generate additional ROS that contribute to oxidative stress (Dröge, 2002). This raises the possibility that the toxicity of mustard alkylating agents involves oxidative stress. In this regard, lipid peroxides formed by the reaction of ROS with membrane lipids, have been reported to be elevated systemically after percutaneous intoxication of rats with SM (Vijayaraghavan et al., 1991). There is also evidence of increases in lipid peroxidation in A431 cells after HN2 or CEES (Pino et al., 2007), in cultured human keratinocytes after SM (Steinritz et al., 2009), and on the dorsal skin of hairless mice after CEES exposure (Pal et al., 2009). Support for a role of superoxide anion in SM-induced injury comes from findings that administration of superoxide dismutase reduces cutaneous toxicity in a guinea pig model (Eldad et al., 1998). Human skin cell lines pretreated with the GSH-depleting agent, buthionine sulfoximine, display enhanced toxicity to SM (Simpson and Lindsay, 2005). Conversely, the cytotoxic actions of SM are reduced in primary keratinocytes pretreated with sulforaphane, a cytoprotective agent which increases intracellular GSH levels by activating the transcription nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Gross et al., 2006). Direct evidence for the GSH-depleting action of chloroethyl alkylating agents has been described in lymphocytes treated with CEES (Han et al., 2004). It has been suggested that GSH depletion by SM can lead to the production of quinone-generated free radicals in melanocytes (Smith, 1999). Findings that pharmacological inhibition of these quinone radicals protects G361 melanocytes against SM-induced toxicity provides support for this concept (Smith and Lindsay, 2001). Based on these findings, it is tempting to speculate that unregulated oxidative/electrophilic stress contributes significantly to the cutaneous vesicating action SM; however, this remains to be determined.

The most extensively investigated molecular target of SM alkylation is DNA. Its significance was initially established in bacteria where inhibition of DNA replication was noted at doses relevant to in vivo mammalian toxicity (Papirmeister et al., 1991). Subsequent use of ³⁵S-labeled SM led to the identification of alkylation sites on DNA. Approximately 65% and 17% of the DNA alkylation products are monofunctional adducts on the N⁷ of guanine and the N³ of adenine, respectively, and approximately 17%, N⁷ guanine bifunctional cross-links. In mammalian cells, it has been estimated that 25% of the DNA cross-links occur between complementary DNA strands, while the remainder are intrastrand (Walker, 1971). The fact that monofunctional analogs such as CEES exhibit reduced potency relative to SM suggests that DNA cross-linking is important in toxicity. However, the observation that CEES is a biologically active vesicant suggests that bifunctional cross-links are not required for skin injury.

A scheme summarizing current understanding of the potential mechanisms contributing to the dermal toxicity of SM is depicted in Figure 4. Because of its ability to alkylate a wide variety of small and large molecules, SM is likely to have multiple targets, including DNA, protein, and small molecules that contribute to its vesicating action. DNA damage can lead directly to cytotoxicity, or it may trigger other changes resulting in cellular dysfunction, cell death or tissue repair. For example, whereas overactivation of PARP following exposure to high concentrations of SM can trigger apoptotic or necrotic cell death, depending on the extent of ATP depletion, milder activation can trigger DNA repair mechanisms that may prove to be either beneficial or detrimental to cell survival. Although less well studied, intracellular and extracellular protein alkylation by SM might also contribute to vesication. Current data suggest that alkylation of cytoskeletal, cell anchoring–related, and ECM proteins may weaken keratinocyte-basement membrane interactions, ultimately leading to epidermal-dermal separation and anoikis. However, one cannot exclude the possibility that less abundant protein targets are also significant to the pathogenic process. Finally, intracellular GSH likely exemplifies a small molecule target and the depletion of this important reducing agent following exposure to SM can initiate oxidative stress leading to lipid peroxidation and other oxidative cellular damage.

A variety of proinflammatory proteins are induced following dermal exposure to SM including enzymes that produce RNS and ROS, chemokines, cytokines, and proteases. These most likely act in concert to promote tissue injury and amplify the inflammatory response. At present, the precise role of these different inflammatory mediators and cells in the toxicity of SM is unknown. Evidence suggests that disruption of cell-matrix interactions is key to the process, and it remains to be determined if inflammatory cells participate in this pathogenic step.

Studies on the mechanism of action of SM have suggested several potential therapeutic approaches. For example, the observation that SM induces inflammation and the release of cytotoxic/proinflammatory mediators suggests that anti-inflammatory drugs may be efficacious in mitigating the cytotoxic actions of SM. In this regard, both administrations of NSAIDs including indomethacin as well as COX-2 inhibitors have proven to be efficacious against SM-induced injury in a variety of in vivo models (Babin et al., 2000; Casillas et al., 2000; Yourick et al., 1995; Zhang et al., 1995b). Neurogenic inflammation is also thought to be an important component of tissue damage induced by SM. Nonmyelinated sensory C-fibers function as dual sensory afferents, transmitting sensory information to the central nervous system and also releasing nociceptive and inflammatory neuropeptides, such as substance P (Szallasi and Blumberg, 1999). Many of these actions are mediated by transient receptor potential V1 (TRPV1) in the skin. Capsaicin, a constituent of hot pepper, has been reported to exert its actions via TRPV1, and several capsaicin analogs significantly reduce SM-induced inflammation in the mouse ear model (Casillas et al., 2000; Sabourin et al., 2003). Targeting specific proinflammatory cytokines and chemokines may also prove clinically efficacious in treating SM poisoning. This is supported by findings that antibodies to TNF-α inhibit SM damage in the MEVM (Wormser et al., 2005).

A variety of enzyme inhibitors have also been tested as SM therapeutics. For example, recent studies have shown that PARP inhibitors abrogate SM toxicity in the MEVM (Smith, 2009). Similarly, inhibition of caspases that mediate apoptosis have been shown to suppress the toxic and vesicating actions of SM (Mol et al., 2009; Rosenthal et al., 2003). Moreover, blocking the activity of matrix-degrading enzymes reduces injury from SM exposure in human skin organ cultures (Mol et al., 2009).

Another interesting approach to treating SM toxicity is the use of iodine. Wormser and colleagues recently isolated a peptide factor from iodine-treated burns that may partially mediate iodine's actions (Brodsky et al., 2008). The peptide, an 11 amino acid fragment of histone H2A, reduces the severity of SM-induced ear swelling in the MEVM, and transfection of a plasmid encoding this factor into HaCaT cells significantly increases their resistance to the cytotoxic effects of SM.