Sulfur mustard analog, 2-chloroethyl ethyl sulfide-induced skin injury involves DNA damage and induction of inflammatory mediators, in part via oxidative stress, in SKH-1 hairless mouse skin

Abstract

Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9–48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1 h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM.

Introduction

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a powerful vesicating (blistering) and bifunctional alkylating warfare agent, which readily penetrates the skin causing histopathological changes, related to inflammation, and delayed blistering (Wormser, 1991, Dacre and Goldman, 1996, Pal et al., 2009). The first target of SM is the basal epidermal keratinocytes; it causes apoptotic cell death followed by protease digestion of anchoring filaments at epidermal–dermal junctions leading to a skin blistering response via epidermal–dermal separation (Kan et al., 2003, Greenberg et al., 2006, Hayden et al., 2009). The mechanism/s of SM-caused responses has been linked mainly to its alkylating property, and its effect on nucleophilic antioxidant, and glutathione (GSH) depletion. GSH depletion could lead to oxidative stress and macromolecular damage, including that of DNA that possibly induces the activation of signaling pathways (Rice, 2003, Kehe and Szinicz, 2005, Paromov et al., 2007, Brimfield et al., 2009, Jowsey et al., 2009, Laskin et al., 2010, Shakarjian et al., 2010, Tewari-Singh et al., 2010). The cellular GSH depletion could be due to its conjugation with SM, and protective effects of exogenous GSH and its precursor, NAC, in reducing SM or its analog-induced skin toxicity have been reported (Gross et al., 1993, Smith et al., 1997, Amir et al., 1998, Han et al., 2004, Arfsten et al., 2007, Paromov et al., 2007).

Several reported studies including a study in cylooxygenase-2 (COX-2) null mice suggest that COX-2, and COX-derived prostaglandins are important inflammatory mediators in SM-induced skin toxicity (Casillas et al., 2000, Nyska et al., 2001, Wormser et al., 2004). Nitric oxide synthases (NOSs), mainly endothelial NOS (eNOS) and inducible NOS (iNOS), predominantly found in mononuclear phagocytes and neutrophils, are the proposed important inflammatory mediators in SM-induced skin toxicity (Nyska et al., 2001, Gao et al., 2007). SM induced up-regulation of iNOS expression is shown to lead to the production of nitric oxide (NO), a potent oxidizing agent. However, at lower concentrations, NO acts as an antioxidant and plays a role in would healing (Gao et al., 2007). After SM exposure, matrix metalloproteinase (MMP) family proteases are reported to be up-regulated causing the degradation of extracellular proteins, which are the major component of basement membrane and separate epidermis from dermis causing vesication (Shakarjian et al., 2010). Constitutively expressed matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) (induced by cytokines, chemokines) are the most studied MMPs in SM-induced skin toxicity. Additionally, MMP-9 has been shown to play an important role in SM-induced skin toxicity using the mouse ear vesicant model (Johansson et al., 1997, Van den Steen et al., 2002, Shakarjian et al., 2006).

Because the molecular mechanism/s involved in SM-induced blistering and skin inflammation are not completely known, the identification of effective medical countermeasures for SM-caused skin injuries has not been possible. Consequently, to further understand the mechanism of SM-induced skin injury, we conducted studies in the SKH-1 hairless mouse using the monofunctional sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES). Due to the requirement of containment facilities for SM, CEES has been widely used as a valid experimental alternative to SM for studies in laboratory settings (Han et al., 2004, Jowsey et al., 2009, Tewari-Singh et al., 2009). This research demonstrated the induction of oxidative stress by CEES leading to the activation of transcription factors AP-1 and nuclear factor-κB (NF-κB) via upstream signaling pathways including mitogen activated protein kinases (MAPKs) and Akt, which were identified as possible mechanisms of the inflammatory responses observed in this skin injury model (Pal et al., 2009, Tewari-Singh et al., 2009). To further establish a link between CEES-induced signaling pathways and inflammatory responses, our objective in the present study was to analyze the involvement of DNA damage and inflammatory mediators in CEES-induced inflammatory and microvesication responses in SKH-1 hairless mice. Furthermore, because our earlier studies showed the involvement of GSH and oxidative stress in CEES-induced molecular and biological responses (Pal et al., 2009, Tewari-Singh et al., 2009, Tewari-Singh et al., 2011), we also assessed their impact on DNA damage and the expression of inflammatory mediators in the present study. This study is a valuable addition in understanding of the mechanism of action of SM analog. Additionally, the study outcomes could help in the selection of countermeasures, which alone or in combination can target the identified pathways to attenuate SM and other vesicating agents’ related skin injuries in humans.