The threat of warfare mustard vesicants' exploitation also as potential terrorist agents poses challenges to the research community to develop effective therapies against vesicant-related injuries 
. Skin injuries by HD can take several months to heal and require long-term medical management. However, the best remedy currently available is decontamination along with supportive treatment to relieve pain, prevent infection and promote healing 
. Exposure to HD triggers an array of complex signal transduction pathways, suggesting the need for multifunctional/pleiotropic agents or combination therapies to treat skin injuries by this vesicant 
. Findings in the present study demonstrated the strong therapeutic efficacy of natural flavanone silibinin in attenuating HD analog CEES-induced cutaneous toxic responses. These studies further give insight into the antioxidant and potential pleiotropic mechanisms of silibinin in reversing toxic effects of CEES exposure.
The current study was carried out using skin toxicity models of CEES, a less toxic monofunctional alkylating analog of HD that only has the ability to form adducts instead of cross-links with biological molecules 
. However, since CEES has similar chemical properties and toxic effects as HD, this study supports earlier reports of its use as a practical substitute to study molecular effects of injury to screen treatment agents in the laboratory settings 
. Once identified, therapeutic agents need to be confirmed for efficacy in bifunctional alkylating vesicant skin injury model. Constantly dividing basal epidermal keratinocytes are reported to be the primary targets of the vesicating agents. However, due to the presence of dermal injury and infiltration of inflammatory molecules, toxic effects on fibroblasts are also evident, which contribute to vesication by HD 
. Therefore, efficacy studies herein were carried out in both skin epidermal cells and dermal fibroblasts employing biomarkers established in our earlier reported studies 
. The observed therapeutic efficacy of silibinin in cell culture studies was then advanced in our in vivo
Vesicating agents are strong alkylating agents, and their interaction with cellular thiols, especially GSH, leads to accumulation of endogenous ROS, resulting in lipid peroxidation, protein adduct formation and DNA damage which can subsequently activate an array of signaling pathways 
. Knowledge of these complex mechanisms of action of vesicating agents is important to develop effective therapies. Accordingly, our recent studies in skin cells and mouse skin tissue have revealed that HD analog CEES induces oxidative stress, DNA damage-related cell cycle checkpoint signaling, activation of signaling pathways including MAPKs and Akt, and subsequent activation of transcription factors AP-1 and NF-κB, resulting in the induction of inflammatory (COX-2, iNOS) and vesicating mediators (MMP-9) 
. The consequences of CEES exposure on skin observed in our studies and reported by others fairly resemble those of ultraviolet B (UVB) radiation, where silibinin is reported to be an effective chemopreventive agent 
. Silibinin is reported to target p53, Cip1/p21 and other cell cycle-regulatory molecules to prevent UVB-induced skin carcinogenesis, in part by controlling apoptotic cell death and DNA damage repair 
. Upon exposure to vesicating agents, p53 is phosphorylated leading to its stabilization and accumulation. Ultimately, there is an increase in the nuclear level of p53 with the formation of a homotetrameric complex acting as a transcriptional suppressor or activator 
. In the current study, the ability of silibinin to attenuate CEES-induced apoptotic cell death, p53 accumulation and ser15 phosphorylation demonstrates its mode of action on p53-dependent apoptosis, cell cycle arrest and DNA damage repair. Silibinin treatment 30 min after CEES-exposure also reversed CEES-induced H2A.X ser139 phosphorylation, which is an indicator of the cellular response to DNA damage, mainly the DSB's. Similarly, silibinin treatment also reversed CEES-induced cellular and mitochondrial O2−
levels which are reported to be important events leading to oxidative DNA damage 
. Therefore, oxidative stress could be a target of silibinin in the regulation of CEES-induced DNA damage. However, silibinin could also directly influence p53 and H2A.X in cell cycle regulation, apoptosis and DNA repair, suggesting their importance as mediators of therapeutic efficacy by silibinin against CEES-induced skin injuries ().
Schematic of possible therapeutic targets for silibinin in CEES-induced skin injury pathways identified in our studies.
Silibinin is also known to target an array of cellular signaling pathways and molecules such as MAPKs, Akt, AP-1, NF-κB, COX-2, iNOS and MMP-9, which are known to play a key role in skin tissue damage and inflammatory responses by both physical and chemical carcinogens 
. Consistent with these, in the present study, silibinin treatment 30 min after CEES-exposure significantly reversed CEES-induced a) inflammatory responses including increase in skin bi-fold and epidermal thickness, apoptotic cell death, neutrophil infiltration and MPO activity; b) induction of biochemical mediators: iNOS, COX-2 and MMP-9; c) activation of NF-κB and AP-1; and d) oxidative stress response: lipid peroxidation, protein oxidation and oxidative DNA damage in mouse skin. Diverse cell-injury stimuli, including oxidative stress, activate MAPKs and Akt pathways, which then activate transcription factors AP-1 and NF-κB are central players in regulating inflammatory and vesicant responses by facilitating the transcription of COX-2, iNOS and MMPs 
. Our recent studies have reported CEES-induced oxidative stress-mediated activation of these pathways, leading to skin inflammatory and injury responses 
. Therefore, the results shown here indicate that oxidative stress could be a target of therapeutic efficacy of silibinin against CEES-induced skin injury (). Since an increase in MPO activity, indicating neutrophil infiltration, can also result in further increase of ROS generation, reversal in CEES-induced MPO activity by silibinin further suggests its strong antioxidant effect by scavenging ROS or its potential to up regulate cells' antioxidant potential ().
Although oxidative stress, inflammation and activation of proteases in vesicant-related skin injury are reported as major consequences of vesicant exposure; antioxidants, scavengers, anti-inflammatory drugs and protease inhibitors alone do not exhibit strong therapeutic effects 
. This suggests that agents are needed with diverse activity towards multiple pathways including targeting the oxidative stress. Natural flavanone silibinin is reported to be a strong antioxidant, and it also possesses strong anti-inflammatory, anti-angiogenic, anti-metastatic, and DNA repair properties 
.Whereas targeting induction of oxidative stress has been a major focus for development of effective rescue therapies for vesicants 
, current study shows that reversal of the toxic effects of CEES by silibinin could be due to its antioxidant as well as other pleiotropic properties which target multiple signaling pathways related to DNA damage, inflammation and vesication (). This study also supports further optimization of silibinin as a rescue agent in HD-skin injury models to allow its development as an effective novel therapeutic agent against vesicant-induced skin injury.