Nitrogen mustard exposure of murine skin induces DNA damage, oxidative stress and activation of MAPK/Akt-AP1 pathway leading to induction of inflammatory and proteolytic mediators

Abstract

Our recent studies in SKH-1 hairless mice have demonstrated that topical exposure to nitrogen mustard (NM), an analog of sulfur mustard (SM), triggers the inflammatory response, microvesication and apoptotic cell death. Here, we sought to identify the mechanism/s involved in these NM-induced injury responses. Results obtained show that NM exposure of SKH-1 hairless mouse skin caused H2A.X and p53 phosphorylation and increased p53 accumulation, indicating DNA damage. In addition, NM also induced the activation of MAPKs/ERK1/2, JNK1/2 and p38 as well as that of Akt together with the activation of transcription factor AP1. Also, NM exposure induced robust expression of pro-inflammatory mediators namely cyclooxygenase 2 and inducible nitric oxide synthase and cytokine tumor necrosis factor alpha, and increased the levels of proteolytic mediator matrix metalloproteinase 9. NM exposure of skin also increased lipid peroxidation, 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation, protein and DNA oxidation indicating an elevated oxidative stress. We also found NM-induced increase in the homologous recombinant repair pathway, suggesting its involvement in the repair of NM-induced DNA damage. Collectively, these results indicate that NM induces oxidative stress, mainly a bi-phasic response in DNA damage and activation of MAPK and Akt pathways, which activate transcription factor AP1 and induce the expression of inflammatory and proteolytic mediators, contributing to the skin injury response by NM. In conclusion, this study for the first time links NM-induced mechanistic changes with our earlier reported murine skin injury lesions with NM, which could be valuable to identify potential therapeutic targets and rescue agents.

Nitrogen Mustard-Induced Corneal Injury Involves DNA Damage and Pathways Related to Inflammation, Epithelial-Stromal Separation, and Neovascularization

“…Hence, this study further corroborates these biomarkers as important endpoints following NM exposure in corneal organ culture. COX-2 and iNOS are reported as key mediators in SM- and NM-induced skin inflammation and injury 20, 21 , and results from present study also show their involvement in NM-induced corneal injury. Activation of proteolytic enzymes, especially MMPs, which degrade the components of extracellular matrix has been shown to result in blister formation upon vesicant exposure 40, 41 .…”

“…In addition, results from this study also show that continuous or 2 h NM exposure in HCE cells led to the activation of similar pathways. The molecules and related pathways (DNA damage, MAPKs, COX-2, MMP-9, VEGF and oxidative stress) in NM-induced ocular injury, identified in present study, are comparable to those associated with SM and NM-induced skin injury 20, 34 .…”

Mustard vesicating agent–induced toxicity in the skin tissue and silibinin as a potential countermeasure

“…Results from additional studies on pathways in SKH‐1 hairless mice also show that p53 paucity caused a delay in NM‐induced early apoptosis and inflammatory response, suggesting a crucial role of p53‐related pathways in mustard vesicating agent–induced skin injury. Induction of the homologous recombination repair (HRR) pathway following NM exposure in mice and skin keratinocytes suggests that HRR is the key pathway involved in the repair of NM‐induced DNA damage . Using Mpo knockout mice, our studies show that MPO, which produces microbicidal oxidants and is indicative of neutrophil infiltration, plays a significant role in NM‐induced skin injuries …”

Antioxidants as potential medical countermeasures for chemical warfare agents and toxic industrial chemicals

“…NM mechanism of action produces DNA alkylation that is mediated with an immonium ion instead of a sulphonium ion[22]. NM is believed to directly cause oxidative stress in affected tissues, which then mediates activation of signaling pathways leading to cell microvesication and death[24]. …”

“…Silibinin has been studied for its utility against oxidative stress, inflammation, and cancer, and has demonstrated potential medical utility in several models of toxicity. Its exact mechanism of action is poorly understood, but it has shown protection in models of vesicant toxicity including nitrogen mustard, CEES and SM[10,24,97–99]. Silibinin might also reduce the nitrosative stress observed following in-vitro SM exposures through its ability to inhibit iNOS[99].…”