Structural changes in the skin of hairless mice following exposure to sulfur mustard correlate with inflammation and DNA damage

Abstract

Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1–14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.

Time course of skin features and inflammatory biomarkers after liquid sulfur mustard exposure in SKH-1 hairless mice

“…In the present study, we demonstrate that topical SM exposure was associated with a dose-dependent degranulation of mast cells and a time-dependent change in mast cells number. Our results are consistent with previous report (Joseph et al, 2011) showing an early activation of mast cells (degranulation of mast cells) after vapour SM exposure in hairless mice associated with a decrease in mast cells number 3 days after exposure. These findings are also in agreement with studies showing a maximal decrease in mast cells number that occurs on day 3 in a burn wound healing model in mice (Nishikori et al, 1998).…”

“…Following exposure of hairless mice to different doses of liquid SM, our results show evident dose-and time-dependent histopathological changes associated with a marked inflammatory response including structural changes in epidermis and dermis which is consistent with previous reports in different skin models after liquid or vapor SM exposure (Dorandeu et al, 2011;Greenberg et al, 2006;Joseph et al, 2011;Smith et al, 1997bSmith et al, , 1998. In the epidermis a disorganization of basal cells associated with an increase in the number of pyknotic nuclei, which is concordant with the dose-dependent increase in apoptosis that we have recently observed in the same model (Batal et al, 2013), led to the formation of microvesications and the complete destruction of epidermis in a dose-dependent manner.…”

“…In the last years, the hairless mouse has become the model of choice for investigating the effects of SM or of its monofunctional analogue CEES (Batal et al, 2013;Clery-Barraud et al, 2012;Dorandeu et al, 2011,b;Jain et al, 2011a,b;Joseph et al, 2011;Tewari-Singh et al, 2009;Vallet et al, 2012). Emphasis was placed in many of these studies on the inflammatory skin response, either after liquid CEES exposure (Jain et al, 2011a,b;Tewari-Singh et al, 2009) or SM vapor exposure (Joseph et al, 2011;Vallet et al, 2012). Yet because of differences in metabolism and mechanism of action between SM and CEES (Gautam et al, 2006) and differences of inflammatory response after liquid or vapor SM exposure , a more complete picture of the skin response to topical exposure to SM remains to be drawn.…”

“…Histopathologically, SM-exposed skin is characterized by change in mast cells associated with a dermal infiltration of neutrophils, basophils and macrophages (Dannenberg et al, 1985;Joseph et al, 2011;Lomash et al, 2013;Rikimaru et al, 1991). This skin inflammation in response to SM exposure is controlled by an important regulation of a network of inflammatory mediators to recruit and activate inflammatory cells to the site of injury.…”

“…During the last years, the hairless mouse model stood out as the model of choice for studying the skin pathogenesis of SM Dorandeu et al, 2011;Joseph et al, 2011;Vallet et al, 2012) or its analogues the chloroethyl ethyl sulfide (CEES) (Jain et al, 2011a,b,b;Tewari-Singh et al, 2009) or the nitrogen mustard Jain et al, 2014;Tewari-Singh et al, 2013. Furthermore, it has been shown that the mouse ear model which is considered as the vesicant model for studying SM-induced inflammatory response and histopathological changes has altered reaction to SM-injury when compared to hairless mouse skin .…”