Sulfur mustard induced mast cell degranulation in mouse skin is inhibited by a novel anti-inflammatory and anticholinergic bifunctional prodrug

Abstract

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48-61% of mast cells were degranulated. SM exposure (1.4g/m³ in air for 6min) resulted in increased numbers of degranulated mast cells 1-14days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1-14days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1-3days post SM, and from 84% to 44% 7-14days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM.

Keywords: Acetylcholinesterase; Countermeasures; Epidermis; Mast cells; Sulfur mustard.

Figure 1

Chemical structure of NDH 4338 [NDH4338 4-(((3,3-dimethylbutoxy)carbonyl)oxy) benzyl 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate] is a carbamate composed of an anti-inflammatory moiety, indomethacin, linked via an aromatic ester–carbonate to a choline bioisostere 3.3-dimethyl-1-butanol.

Figure 2

Toluidine blue staining of mast cells in hairless mouse skin following SM exposure. Histological sections prepared 1, 3, 7, and 14 days post exposure to control or SM without and with NDH4338 treatment were stained with toluidine blue (original magnification, ×400). Left Panel: Control mouse skin. Center Panel: Mouse skin treated with SM. Right Panel: Mouse skin treated with SM and NDH4338. Black arrows, mast cells. Yellow arrows, degranulated mast cells. Insets show intact and degranulated mast cells. One representative section from 4 mice/treatment group is shown.

Figure 3

Effect of SM on mast cell degranulation. Histological sections prepared 1, 3, 7, and 14 days post exposure to control or SM without and with NDH4338 treatment were stained with toluidine blue. Left Panel: Total number of mast cells. Right Panel: Percentage of degranulated mast cells. Each bar is the mean ± S.E. (n = 4) of 15 oil immersion fields, *Significantly different from control (p<0.05). ⁺Significantly different from SM exposure (p<0.05).