Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

Abstract

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 μM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione-S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A4 (LTA4) hydrolase and leukotriene C4 (LTC4) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA4 hydrolase and LTC4 synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

Efficacy of Glutathione in Ameliorating Sulfur Mustard Analog-Induced Toxicity in Cultured Skin Epidermal Cells and in SKH-1 Mouse Skin In Vivo

“…HD and CEES are alkylating agents that deplete GSH, which enhances lipid peroxidation and ROS generation, causing damage to lipids, proteins, and nucleic acids and eventually leading to toxic responses (Kehe and Szinicz, 2005;Paromov et al, 2007;Ruff and Dillman, 2007;Black et al, 2010). CEES-caused mitochondrial oxidative damage and altered mitochondrial membrane potential are also reported in lung, liver, and other tissues, further indicating a role of oxidative stress in CEES-caused toxicity (Jafari, 2007;Gould et al, 2009;Black et al, 2010).…”

“…CEES-caused mitochondrial oxidative damage and altered mitochondrial membrane potential are also reported in lung, liver, and other tissues, further indicating a role of oxidative stress in CEES-caused toxicity (Jafari, 2007;Gould et al, 2009;Black et al, 2010). Therefore, supplementing GSH or its precursors, including NAC, could help minimize this oxidative stress and reduce HD/CEES-caused toxicity, although its associated mechanism and defined biological systems are not well known (Amir et al, 1998;Atkins et al, 2000;Han et al, 2004;Arfsten et al, 2007;Paromov et al, 2007Paromov et al, , 2008.…”

“…The role of oxidative and/or nitrative stress in HD/CEEScaused skin inflammation and injury is reported in various animal models (Paromov et al, 2007;Ruff and Dillman, 2007;Kehe et al, 2009;Pal et al, 2009;Black et al, 2010;Shakarjian et al, 2010). Our recent study in SKH-1 hairless mice demonstrated that induction of oxidative stress by CEES possibly leads to lipid, protein, and DNA oxidation together with the activation of mitogen-activated protein kinases and Akt pathways and activator protein-1 and nuclear factor-B transcription factors ), followed by inflammatory responses in mouse skin .…”

“…E, mice were treated topically with either acetone or CEES (2 mg), given GSH (300 mg/kg) alone via oral gavage or given GSH (300 mg/kg) via oral gavage 1 h before CEES topical application. Mice were sacrificed, and dorsal skin tissue samples were collected 24 h after the desired treatments and processed for the determination of total GSH levels in the skin as described under Paromov et al, 2007;Black et al, 2010). In our present study, treatment of skin epidermal cells with GSH or its precursor NAC attenuated CEES-caused cytotoxic responses in cell culture and skin injury in mouse skin; moreover, CEES exposure decreased GSH levels in cell culture as well as in mouse skin systems.…”

“…HD/CEES-caused oxidative stress results in the 8-oxo-2-deoxyguanosine DNA adduct as well as lipid and protein oxidation, which can cause inflammation and other toxic responses in skin Black et al, 2010). The exogenous addition of antioxidants such as GSH, N-acetyl cysteine (NAC), vitamin E, superoxide dismutase, catalase, sulforaphane, quercetin, and catalytic antioxidants such as manganese (III) meso-tetrakis (di-N-ethylmidazole)porphyrin (AEOL 10150) has been reported to attenuate lung and skin injury by HD/CEES (Gross et al, 1993;Smith et al, 1997;Amir et al, 1998;Han et al, 2004;Arfsten et al, 2007;Paromov et al, 2007Paromov et al, , 2008Gould et al, 2009;Black et al, 2010;O'Neill et al, 2010). Whereas most of these efficacy studies with antioxidants indicate that they have beneficial effects in attenuating HD/ CEES-caused lung injury, there are few, if any, reports of efficacy studies with GSH and its associated mechanism of action in relevant skin epidermal cells and skin toxicity models of HD/CEES (Paromov et al, 2007).…”