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.

Figure 1. Structural changes in skin following exposure of hairless mice to SM

Histological sections prepared 1 and 14 days (d) following exposure to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with H&E. Stratum corneum (SC), epidermis (E), hair follicle (HF), dermis (D), and sebaceous gland (S). One representative section from 3 mice/treatment group is shown (original magnification, × 400).

Figure 2. Trichrome staining of hairless mouse skin following SM exposure

Histological sections prepared 1 and 14 days (d) following exposure to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with Gomori’s trichrome followed by aniline blue counterstaining. Nuclei, blue/black staining; keratin and cytoplasm, red staining; collagen I/III, royal blue staining. One representative section from 3 mice/treatment group is shown (original magnification, × 400).

Figure 3. Alterations in hairless mouse skin following SM exposure

Histological sections prepared 1, 3, 7, and 14 days post SM-exposure, were stained with Gomori’s trichrome followed by aniline blue counterstaining. One representative section from 3 mice/treatment group is shown. Panel A. One day post SM exposure. Note the karyolytic (asterisk) and pyknotic (arrow) nuclei within the basal keratinocytes (original magnification × 600). Panel B. Three days post SM exposure. Arrowhead shows areas of parakeratosis (original magnification × 600). Panel C. Seven days post SM exposure. Neoepidermis (N) is shown migrating beneath the eschar (original magnification × 400). Panel D. Fourteen days post SM exposure. Hyperplastic sebaceous glands and engorged follicular cysts are shown beneath new epidermis (original magnification × 200).

Figure 4. Effects of SM exposure on myeloperoxidase expression in the skin

Sections were prepared 3 days (panel A) and 7 days (panel B) post SM-exposure and stained with anti-myeloperoxidase antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown.

Figure 5. Effects of SM on K10 expression in the skin

Sections prepared 1 and 14 days after exposure of mice to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with anti-K10 antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from × mice/treatment group is shown.

Figure 6. Effects of SM on mast cell degranulation

Sections prepared 14 days after exposure of mice to air control (CTL) or SM were stained for mast cells using toluidine blue (upper panels). One representative section from 4 mice/treatment group is shown (original magnification, x 1000). ND, no degranulation; SD, slight degranulation; MD, major degranulation. Lower panels show quantification of mast cell degranulation in control and SM-treated skin at the dermal/epidermal junction. Each bar is the mean ± S.E. (n = 4) of 15 oil immersion fields, * = p<0.05.

Figure 7. Effects of SM on COX-2 expression in the skin

Sections prepared 1 and 14 days after exposure of mice to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with anti-COX-2 antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. One day post SM exposure, COX-2 was expressed in basal keratinocytes (arrow). By 3 days, increased COX-2 expression was evident in inflammatory cells at the dermal/epidermal boundary (arrow) and in the epidermal appendages (arrowhead). Seven days post exposure there was increased expression of COX-2 at the dermal/epidermal boundary (arrow).

Figure 8. Effects of SM on phospho-H2A.X expression in the skin

Sections prepared 1 and 14 days after exposure of mice to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with anti-phospho-H2A.X antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. At days 1 and 3 post SM-exposure, nuclear phospho-H2A.X expression was evident in the basal epithelium (arrow) and in the epidermal appendages (arrowhead). By 7 days, phospho-H2A.X expressing basal keratinocytes had taken on a flattened morphology (arrow), whereas after 14 days, low levels of phospho-H2A.X expression were only visible in the granular layer of the hyperplastic epidermis (arrow).

Figure 9. Effects of SM on cleaved caspase-3 expression in the skin

Sections prepared 1 and 14 days after exposure of mice to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with anti-cleaved caspase-3 antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. At one day post SM-exposure, basal keratinocytes (arrow) and inflammatory cells in the dermis (asterisk) expressed cleaved caspase-3. By day 3, cleaved caspase-3 was visible in flattened interfollicular keratinocytes (arrow) and epidermal appendages (arrowhead). After 7 days, cleaved caspase-3 staining was restricted to basophilic cells situated below the eschar (arrow).

Figure 10. Effects of SM on PCNA expression in the skin

Sections prepared 1 and 14 days after exposure of mice to air control (CTL) or 1, 3, 7, and 14 days post SM-exposure, were stained with anti-PCNA antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. Control skin constitutively expressed PCNA in basal keratinocytes (arrow) and in keratinocytes surrounding hair follicles and sebaceous glands (arrowheads). One day after SM exposure, only sporadic PCNA expression was evident in the dermis (arrowheads) while no PCNA was evident in the epidermis 3 and 7 days post-SM exposure. By 14 days, strong PCNA expression was evident in basal cells in the hyperplastic epidermis (arrow).