Structural changes in hair follicles and sebaceous glands of hairless mice following exposure to sulfur mustard

Abstract

Sulfur mustard (SM) is a bifunctional alkylating agent causing skin inflammation, edema and blistering. A hallmark of SM-induced toxicity is follicular and interfollicular epithelial damage. In the present studies we determined if SM-induced structural alterations in hair follicles and sebaceous glands were correlated with cell damage, inflammation and wound healing. The dorsal skin of hairless mice was treated with saturated SM vapor. One to seven days later, epithelial cell karyolysis within the hair root sheath, infundibulum and isthmus was apparent, along with reduced numbers of sebocytes. Increased numbers of utriculi, some with connections to the skin surface, and engorged dermal cysts were also evident. This was associated with marked changes in expression of markers of DNA damage (phospho-H2A.X), apoptosis (cleaved caspase-3), and wound healing (FGFR2 and galectin-3) throughout pilosebaceous units. Conversely, fatty acid synthase and galectin-3 were down-regulated in sebocytes after SM. Decreased numbers of hair follicles and increased numbers of inflammatory cells surrounding the utriculi and follicular cysts were noted within the wound 3-7 days post-SM exposure. Expression of phospho-H2A.X, cleaved caspase-3, FGFR2 and galectin-3 was decreased in dysplastic follicular epidermis. Fourteen days after SM, engorged follicular cysts which expressed galectin-3 were noted within hyperplastic epidermis. Galectin-3 was also expressed in basal keratinocytes and in the first few layers of suprabasal keratinocytes in neoepidermis formed during wound healing indicating that this lectin is important in the early stages of keratinocyte differentiation. These data indicate that hair follicles and sebaceous glands are targets for SM in the skin.

Keywords: Hair follicles; Phosphorylated histone H2A.X; Sebaceous glands; Sulfur mustard.

Figure 1. Structural changes in hairless mouse skin following SM

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

Figure 2. Trichrome staining of hairless mouse skin following SM

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with Gomori's trichrome containing hematoxylin which stains nuclei blue/black, eosin which stains keratin and cytoplasm red, and aniline blue which stains collagen I/III royal blue. One representative section from 3 mice/treatment group is shown (original magnification, × 400). Hair follicle (HF).

Figure 3. Effects of SM on expression of fatty acid synthase

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to fatty acid synthase. Antibody binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. Arrows indicate sebaceous glands.

Figure 4. Effects of SM on sebaceous glands

Histological sections prepared 1 day and 3 days after SM exposure were stained with Gomori’s trichrome followed by aniline blue counterstaining (upper panels) or with anti-fatty acid synthase antibody (lower panels). One representative section from 3 mice/treatment group is shown (original magnification, × 400).

Figure 5. Effects of SM on phospho-H2A.X expression

Histological sections were prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to 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. Arrows indicates sebaceous glands, asterisk indicates inflammatory cells at the dermal/epidermal junction, and arrowhead indicates interfollicular and outer root sheath basal keratinocytes.

Figure 6. Effects of SM on cleaved caspase-3 expression

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to 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. Arrow indicates interfollicular and outer root sheath basal keratinocytes and arrowhead indicates basophilic cells in the dermis.

Figure 7. Effects of SM on PCNA expression

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to PCNA. Antibody binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown. Arrows indicate interfollicular and outer root sheath basal keratinocytes, arrowheads indicate sebocytes and asterisks indicate inflammatory cells at the dermal/epidermal junction.

Figure 8. Effects of SM on FGFR2 expression

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to FGFR2. Antibody binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown.

Figure 9. Effects of SM on galectin-3 expression

Histological sections prepared 1 and 14 days after air exposure (CTL), and 1, 3, 7 and 14 days after SM exposure were stained with an antibody to galectin-3. Antibody binding was visualized using a Vectastain Elite ABC kit (original magnification, × 400). One representative section from 3 mice/treatment group is shown.