Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

Abstract

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.

Fig. 1. Morphologic changes in full-thickness human skin equivalents following CEES treatment

EpiDerm-FT™ was treated with CEES (100–1000 μM) or vehicle control. After 24 hr, tissue samples were stained with hematoxylin and eosin (Panel A) or trichrome (Panel B). Arrows indicate abnormal trichrome staining in the stratum corneum. Original magnification, 400×.

Fig. 2. Alterations in basal keratinocytes in a full-thickness human skin equivalent following CEES treatment

EpiDerm-FT™ was treated with CEES (100–1000 μM) or vehicle control. After 24 hr, tissue samples were stained with hematoxylin and eosin. Note the prominent nuclear condensation in basal keratinocytes following exposure to 300 μM and 1000 μM CEES. Original magnification, 1000×.

Fig. 3. Effects of CEES on PCNA expression

EpiDerm-FT™ was treated with CEES (1000 μM) or control. Tissues were collected 2–72 hr later and stained with anti-PCNA antibody. Binding was visualized using a peroxidase DAB substrate kit. Original magnification, 400×.

Fig. 4. Effects of CEES on expression of markers of injury and inflammation

EpiDerm-FT™ was treated with CEES (100, 300 or 1000 μM) or control for 15 min, 6 hr, or 24 hr, as indicated. Panel A. Epidermal sheets were collected and analyzed for protein expression by Western blotting using anti-PCNA, PARP, phospho-H2AX, COX-2, 5-LOX, and LTA₄ hydrolase antibodies. β-actin was used as a control to ensure equal protein loading. Panel B. Densitometry was performed on Western blots shown in Panel A. Data is presented in arbitrary units.

Fig. 5. Effects of CEES on PARP expression

EpiDerm-FT™ was treated with CEES (1000 μM) or control. Tissues were collected 2–72 hr later and stained with anti-PARP antibody. Binding was visualized using a peroxidase DAB substrate kit. Original magnification, 400×.

Fig. 6. Effects of CEES on phospho-H2AX expression

EpiDerm-FT™ was treated with CEES (1000 μM) or control. Tissues were collected 2–72 hr later and stained with anti-phospho-H2AX antibody. Binding was visualized using a peroxidase DAB substrate kit. Original magnification, 400×.

Fig. 7. Effects of CEES on COX-2 expression

EpiDerm-FT™ was treated with CEES (1000 μM) or control. Tissues were collected 2–72 hr later and stained with anti-COX-2 antibody. Binding was visualized using a peroxidase DAB substrate kit. Original magnification, 400×.

Fig. 8. Effects of CEES on mRNA expression of eicosanoid biosynthetic enzymes

EpiDerm-FT™ was treated with CEES (100, 300 or 1000 μM) or control. After 6 or 24 hr, epidermal sheets were collected and mRNA isolated and analyzed for gene expression by real-time PCR. Data are presented as fold change in gene expression relative to untreated cells. *Significantly different from control (p < 0.05).

Fig. 9. Effects of CEES on keratinocyte mRNA expression of primary antioxidants and glutathione S-transferases

EpiDerm-FT™ was treated with CEES (100, 300 or 1000 μM) or control. After 6 or 24 hr, epidermal sheets were collected and mRNA isolated and analyzed for gene expression by real-time PCR. Data are presented as fold change in gene expression relative to untreated cells. *Significantly different from control (p < 0.05).