Increased Expression of the Endocannabinoid System in Mouse Skin Following Exposure to Sulfur Mustard and Nitrogen Mustard (Mechlorethamine)
Authors: Heck Diane E , Wohlman Irene M , Composto Gabriella M , Heindel Ned D , Casillas Robert P , Gerecke Donald R , Laskin Debra L , Joseph Laurie B , Laskin Jeffrey D 
Published: November 2023

Vesicants including sulfur mustard (SM, bis(2‐chloroethyl) sulfide) and nitrogen mustard (NM, bis(2‐chloroethyl)methylamine) are highly reactive bifunctional alkylating agents that target the skin. In humans and rodents, vesicants initially induce skin injury which is associated with an early inflammatory response; depending on the dose and time following exposure, tissue damage and blistering can ensue. This is associated with alterations in keratinocyte growth and differentiation. The precise mechanisms by which vesicants induce tissue injury and blistering are poorly understood. Both SM and NM have been reported to modify many cellular components including proteins, lipids and nucleic acids; they can also stimulate tissue production of cytokines, growth factors and proinflammatory mediators. Endocannabinoids including N‐arachidonoylethanolamine (anandamide, AEA) and 2‐arachidonoyl glycerol (2‐AG), along with the N‐acylethanolamines palmitoylethanolamine (PEA) and oleyolethanolamine (OEA), are important endogenous fatty acid signaling molecules involved in regulating skin homeostasis and inflammation. They are known to regulate keratinocyte proliferation and wound healing. Their activity is mediated by binding to classical cannabinoid receptors, CB1 and CB2, which have been identified in the skin. Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH); inhibitors of this enzyme are known to suppress inflammation. Endocannabinoid signaling through CB1 and CB2 upregulates genes for lipid synthesis, immune cell signaling and migration, and dermal inflammation. We found that treatment of mouse skin with SM or NM caused epidermal damage including stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Markers of apoptosis and DNA damage including expression of cleaved caspase‐3 and phosphorylated histone 2A.X (phospho‐H2A.X), respectively, were increased in epidermal cells, along with enzymes generating pro‐inflammatory mediators including myeloperoxidase, inducible nitric oxide synthase and cyclooxygenase‐2. Control mouse skin was found to express the endocannabinoid proteins FAAH, CB1 and CB2. This was evident in epidermis and dermal appendages including sebaceous glands and hair follicles. SM or NM, at concentrations that induce tissue injury, markedly upregulated FAAH, CB1 and CB2; increased expression of these proteins persisted in the tissue during the wound healing process and was associated with areas of extensive keratinocyte proliferation, as measured by cells expressing proliferating cell nuclear antigen. To determine if the endocannabinoid system is regulated by vesicants, mouse skin was treated topically with vanillyl alcohol carbamate (VAC), a potent inhibitor of FAAH. VAC was found to be highly effective in suppressing vesicant‐induced inflammation. Our findings that NM and SM upregulate FAAH, CB1 and CB2, and that FAAH inhibitors have the capacity to reduce skin injury support the idea the endocannabinoids contribute to the pathogenic responses to vesicants. They further support the idea that FAAH inhibitors may be useful in mitigating tissue injury induced by SM and NM.Support or Funding InformationSupported by NIH grants U54AR055073, U01NS079249, ES004738 and ES005022.