Dermal-Epidermal Junction Disruptors: Toxicodynamic Mechanisms

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Project Summary/Abstract Dermal-epidermal junction (DEJ) disruptors are cutaneous poisons that cause the epidermis to detach from the dermis, an effect that results in skin blistering (vesication). The prototype DEJ disruptor is mechlorethamine (MEC), a nitrogen mustard; however, the toxicodynamic mechanisms involved in vesication by MEC and other DEJ disruptors remains mostly undescribed in the scientific literature. The overall goal of the work proposed here is to investigate the toxicodynamic mechanisms associated with DEJ disruption. This will be achieved by studying the role of mast cell derived mediators (histamine and the serine protease tryptase), neutrophil-derived mediators (myeloperoxidase, MPO and matrix metalloproteinase-9, MMP-9) and keratinocyte-specific collagens (collagen IV, COL4 and collagen XVII, COL17) in the response of skin to MEC. In Aim 1 of the proposal, the hypothesis that mast cell-derived mediators influence DEJ disruption will be evaluated. To this end, mouse ear skin will be topically exposed to saline (naïve), dimethylsulfoxide (DMSO, vehicle) or MEC. Additional groups of mice will be treated 20 min prior to exposure to saline, vehicle or MEC with the antihistamine doxepin (5% cream administered topically) or the tryptase inhibitor leupeptin (at test doses of 10 or 30 mg/kg, ip). All ear tissues will be harvested at 2, 8 or 24 hr and either fixed for histopathologic analysis or homogenized and assayed for the presence of histamine via ELISA and tryptase via western blotting. In Aim 2 of the proposal, we will test the hypothesis that neutrophil-derived MMP-9 contributes to DEJ disruption by MEC. To test this hypothesis, we will analyze naïve, vehicle and MEC-treated mouse ear biopsies from wildtype and Mmp9 -/- knockout mice obtained at 2, 8 or 24 hr after exposure for the presence of DEJ disruption using light microscopy and for the presence of myeloperoxidase, a neutrophil-selective marker, using immunohistochemistry (IHC). In Aim 3 of the proposal, we will test the hypothesis that MEC promotes blistering by interfering at the level of epidermal keratinocytes and affecting the expression of key keratinocyte-expressed collagens involved in maintaining the DEJ, namely COL4 and COL17. To test this hypothesis, we will analyze mouse ear biopsies from the time points and samples described above for Aim 1 but here carry out IHC on the formalin-fixed paraffin embedded tissues to evaluate how the expression of these two proteins, COL4 and COL17, changes over time after following exposure to MEC. In addition, experiments in Aim 3 will utilize human 3D skin cultures exposed to MEC for 2, 8 or 24 hr to investigate effects on COL4 and COL17 protein expression in human tissues. All in all, the proposal seeks to investigate three related yet independent hypotheses that will shed new light on the toxicodynamic mechanisms used by DEJ disruptors to cause blisters and will uncover novel targets for the purpose of reducing chemically induced cutaneous blistering. The work proposed here will provide a broad, “whole tissue” toxicodynamic response to injury by MEC at the levels of the cutaneous mast cells, the infiltrating neutrophils and the epidermal keratinocytes and will be complemented by the 3D human skin study. Most importantly, students involved in this project will learn a wide range of skills including experimental design, animal care, animal dosing, histology techniques, IHC, western blotting and how to use ImageJ software to assess tissue protein expression.