Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard

Abstract

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal-epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.

Keywords: Non-steroid bifunctional anti-inflammatory and anti-cholinergic agent; Skin; Sulfur mustard; Wound repair.

Figure 1

Chemical structure of the bifunctional anticholinergic NSAID compound NDH 4338. The drug combines two anti-inflammatory moieties (indomethacin and a choline bioisostere, 3,3-dimethyl-1-butanol), attached via an aromatic ester-carbonate linkage.

Figure 2

Structural changes in hairless mouse skin following exposure to SM. Histologic sections, prepared 4 days after exposure to air (Ctl) (panels A and C) or SM exposure (panels B and D), were stained with H&E. Panels A and B, scale bar = 200 µm; panels C and D, scale bar = 20 µm. Panels C and D are enlargements of the black dotted squares shown in panels A and B, respectively. dermis (D), epidermis (E), hair follicle (HF), pyknotic nuclei (P),sebaceous glands (S), stratum corneum (SC).

Figure 3

Effects of NDH 4338 on SM-induced skin injury. Histological sections prepared 4 days after exposure to SM, SM plus vehicle, or SM plus NDH 4338, were stained with H&E. The dotted line denotes the dermal/epidermal junction. The arrow designates the leading edge of the migrating keratinocytes. Scale bar = 20 µm.

Figure 4

Effects of SM exposure on skin thickness. Skin section, prepared 4 days after exposure to air (Ctl) or SM, were analyzed for dermal (panel A) and epidermal (panel B) thickness. Ctl (open bars), SM (black bars), SM plus vehicle (Veh)(gray bars), SM plus NDH 4338 (striped bars). Data are presented as the mean ± SE (n = 20), *p<0.001, compared to control tissues; +p<0.001, compared to SM exposed tissues.

Figure 5

Effects of NDH 4338 on SM-induced alterations in COX2 expression in mouse skin. Pooled samples from each group were analyzed by Western blotting using a COX2 antibody. Lane 1, untreated control; lane 2, vehicle alone; lane 3, NDH 4338 alone; lane 4, SM; lane 5, SM plus vehicle; lane 6, SM plus NDH 4338. GAPDH expression was used to ensure equal loading of proteins on the gels.

Figure 6

Effects of NDH 4338 on SM-induced COX2 expression in mouse skin. COX2 was visualized by confocal microscopy using a secondary antibody conjugated to Dylight 549 (red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.

Figure 7

Effects of NDH 4338 on SM-induced MMP9 expression in mouse skin. MMP9 was visualized by confocal microscopy using a secondary antibody conjugated to Dylight 549 (red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.

Figure 8

Effects of NDH 4338 on SM-induced alterations in K10 expression in mouse skin. K10 was visualized by confocal microscopy using a secondary antibody conjugated to Dylight 549 (red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.

Figure 9

Effects of NDH 4338 on SM-induced alterations in K6 expression in mouse skin. K6 was visualized by confocal microscopy using a secondary antibody conjugated to Dylight 549 (red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.

Figure 10

Effects of NDH 4338 on SM-induced alterations in Ki67 expression in mouse skin. Ki67 was visualized by confocal microscopy using a secondary antibody conjugated to Alexa Fluor 488 (shown in red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.

Figure 11

Effects of NDH 4338 on SM-induced expression of laminin 332 in mouse skin. Laminin 332 was visualized by confocal microscopy using a secondary antibody conjugated to Dylight 549 (red). Control mouse skin (panel A), SM (panel B), SM plus vehicle control (panel C), and SM plus NDH 4338 (panel D). Nuclei were counterstained with DAPI (shown in blue), scale bar = 20 µm.