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

Pharmacology and Toxicology, Rutgers University, Piscataway, NJ, USA.
Toxicology and Applied Pharmacology (Impact Factor: 3.71). 04/2010; 245(3):352-60. DOI: 10.1016/j.taap.2010.04.001
Source: PubMed


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 microM) 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 A(4) (LTA(4)) hydrolase and leukotriene C(4) (LTC(4)) 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, LTA(4) hydrolase and LTC(4) 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.

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Available from: Laurie B Joseph
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    • "Hence, this study builds a foundation where our in vivo model with NM could be used to further understand the role of DNA damage and related p53 activation, and Rad51 and HRR pathway in vesicant-induced DNA repair, which would help develop novel agents for the therapeutic intervention of vesicants-induced skin injuries. NM exposure resulting in an increase in the phosphorylation of MAPKs ERK1/2, JNK1/2 and p38 in mouse skin, as observed in the present study, supports their role in vesicant-induced skin injury, as reported in earlier studies with SM in keratinocytes and CEES in keratinocytes and mouse skin (Black et al., 2010b; Dillman et al., 2004; Pal et al., 2009; Rebholz et al., 2008). Since many of the downstream targets of MAPKs activation lead to the production and action on inflammatory mediators, our results suggest that activation of MAPKs by NM in mouse skin plausibly plays a major role in NM-induced increase in COX2, iNOS, TNFa and MMP9 levels, resulting in inflammatory and vesicating responses similar to those by SM (Rosenthal et al., 1998). "
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    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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    Full-text · Article · Apr 2015 · Toxicology Letters
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    • "Vesicating agents with strong alkylating properties can cause either direct DNA damage or interact with cellular thiols leads to the accumulation of ROS causing mainly lipid peroxidation, protein oxidation and DNA damage [10] [12] [20] [33]. CEES-induced DNA damage could lead to activation of DNA-damage related signaling pathways that cause cellular toxic responses including cell death as reported earlier [10] [17] [18] [28] [34]. In this study, 50 μM AEOL 10150 treatment applied 1 h after CEES exposure caused a reversal in CEES-induced loss of cell viability in mouse epidermal JB6 cells, human HaCaT, and NHEK (primary epidermal) cells. "
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    ABSTRACT: Our previous studies and other published reports with the chemical warfare agent sulfur mustard (SM) and its analog 2-chloroethyl ethyl sulfide (CEES) have indicated a role of oxidative stress in skin injuries caused by these vesicating agents. We examined the effects of the catalytic antioxidant AEOL 10150 in attenuation of CEES-induced toxicity in our established skin injury models (skin epidermal cells and SKH-1 hairless mice) to validate the role of oxidative stress in the pathophysiology of mustard vesicating agents. Treatment of mouse epidermal JB6 and human HaCaT cells with AEOL 10150 (50μM) 1h post CEES exposure resulted in significant (p<0.05) reversal of CEES-induced decreases in both cell viability and DNA synthesis. Similarly, AEOL 10150 treatment 1h after CEES exposure attenuated CEES-induced DNA damage in these cells. Similar AEOL 10150 treatments also caused significant (p<0.05) reversal of CEES-induced decreases in cell viability in normal human epidermal keratinocytes. Cytoplasmic and mitochondrial reactive oxygen species measurements showed that AEOL 10150 treatment drastically ameliorated the CEES-induced oxidative stress in both JB6 and HaCaT cells. Based on AEOL 10150 pharmacokinetic studies in SKH-1 mouse skin, mice were treated with topical formulation plus subcutaneous (injection; 5mg/kg) AEOL 10150, 1h after CEES (4mg/mouse) exposure and every 4h thereafter for 12h. This AEOL 10150 treatment regimen resulted in over 50% (p<0.05) reversal in CEES-induced skin bi-fold and epidermal thickness, myeloperoxidase activity, and DNA oxidation in mouse skin. Results from this study demonstrate potential therapeutic efficacy of AEOL 10150 against CEES-mediated cutaneous lesions supporting AEOL 10150 as a medical countermeasure against SM-induced skin injuries.
    Full-text · Article · May 2014 · Free Radical Biology and Medicine
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    • "Driving this process is activation of signaling molecules and transcription factors that control expression of these genes. In keratinocytes, these include mitogen activated protein (MAP) kinases and phospho-inositide-3- kinase (PI3K)/Akt, as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and NF-κB (Black et al., 2010; Dhar et al., 2002; Haarmann- Stemmann et al., 2012; Keum et al., 2006; Marrot et al., 2008). Molecules thought to be involved in mediating stress-induced alterations in adaptive response genes include lipid peroxidation products such as malondialdehyde, acrolein and 4-HNE (Pizzimenti et al., 2013). "
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    ABSTRACT: 4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86-98 fold within 6h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6h with 30μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2-/- mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress.
    Full-text · Article · Jan 2014 · Toxicology and Applied Pharmacology
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