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Title: Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway

Abstract

Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100 years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by {approx} 3-fold following exposure to CEES. Our data also suggested that CDDO-Me may actmore » additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin. - Highlights: > CDDO-Me treatment increased intracellular GSH in human keratinocytes. > CDDO-Me increased cell viability following exposure to the half-mustard, CEES. > The cytoprotective effect of CDDO-Me was likely due to scavenging with endogenous GSH.« less

Authors:
; ; ; ;  [1];  [2];  [1];  [1]
  1. Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX 78957 (United States)
  2. Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX 77030 (United States)
Publication Date:
OSTI Identifier:
21587827
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 255; Journal Issue: 2; Other Information: DOI: 10.1016/j.taap.2011.06.012; PII: S0041-008X(11)00234-1; Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BRASSICA; CHEMICAL WARFARE AGENTS; CYSTEINE; DETOXIFICATION; DMSO; EDTA; EPIDERMIS; GLUTATHIONE; LIGASES; LUNGS; NAPHTHALENE; SULFIDES; SYNTHESIS; TOXICITY; VIABILITY; AMINO ACIDS; ANIMAL TISSUES; AROMATICS; BODY; CARBOXYLIC ACIDS; CHALCOGENIDES; CHELATING AGENTS; CONDENSED AROMATICS; DRUGS; ENZYMES; EPITHELIUM; FOOD; HYDROCARBONS; MAGNOLIOPHYTA; MAGNOLIOPSIDA; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; ORGANS; PEPTIDES; PLANTS; POLYPEPTIDES; PROTEINS; RADIOPROTECTIVE SUBSTANCES; RESPIRATORY SYSTEM; RESPONSE MODIFYING FACTORS; SKIN; SULFOXIDES; SULFUR COMPOUNDS; THIOLS; VEGETABLES; WEAPONS

Citation Formats

Abel, Erika L., Bubel, Jennifer D., Simper, Melissa S., Powell, Leslie, McClellan, S. Alex, Andreeff, Michael, MacLeod, Michael C., and DiGiovanni, John, E-mail: john.digiovanni@austin.utexas.edu. Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway. United States: N. p., 2011. Web. doi:10.1016/j.taap.2011.06.012.
Abel, Erika L., Bubel, Jennifer D., Simper, Melissa S., Powell, Leslie, McClellan, S. Alex, Andreeff, Michael, MacLeod, Michael C., & DiGiovanni, John, E-mail: john.digiovanni@austin.utexas.edu. Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway. United States. doi:10.1016/j.taap.2011.06.012.
Abel, Erika L., Bubel, Jennifer D., Simper, Melissa S., Powell, Leslie, McClellan, S. Alex, Andreeff, Michael, MacLeod, Michael C., and DiGiovanni, John, E-mail: john.digiovanni@austin.utexas.edu. Thu . "Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway". United States. doi:10.1016/j.taap.2011.06.012.
@article{osti_21587827,
title = {Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway},
author = {Abel, Erika L. and Bubel, Jennifer D. and Simper, Melissa S. and Powell, Leslie and McClellan, S. Alex and Andreeff, Michael and MacLeod, Michael C. and DiGiovanni, John, E-mail: john.digiovanni@austin.utexas.edu},
abstractNote = {Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100 years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by {approx} 3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin. - Highlights: > CDDO-Me treatment increased intracellular GSH in human keratinocytes. > CDDO-Me increased cell viability following exposure to the half-mustard, CEES. > The cytoprotective effect of CDDO-Me was likely due to scavenging with endogenous GSH.},
doi = {10.1016/j.taap.2011.06.012},
journal = {Toxicology and Applied Pharmacology},
number = 2,
volume = 255,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 2011},
month = {Thu Sep 01 00:00:00 EDT 2011}
}
  • 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 {mu}M) 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,more » 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{sub 4} (LTA{sub 4}) hydrolase and leukotriene C{sub 4} (LTC{sub 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{sub 4} hydrolase and LTC{sub 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.« less
  • Sulfur mustard [bis(2-chloroethyl)sulfide, SM] is a well-known DNA-damaging agent that has been used in chemical warfare since World War I, and is a weapon that could potentially be used in a terrorist attack on a civilian population. Dermal exposure to high concentrations of SM produces severe, long-lasting burns. Topical exposure to high concentrations of 2-(chloroethyl) ethyl sulfide (CEES), a monofunctional analog of SM, also produces severe skin lesions in mice. Utilizing a genetically engineered mouse strain, Big Blue, that allows measurement of mutation frequencies in mouse tissues, we now show that topical treatment with much lower concentrations of CEES inducesmore » significant dose- and time-dependent increases in mutation frequency in mouse skin; the mutagenic exposures produce minimal toxicity as determined by standard histopathology and immunohistochemical analysis for cytokeratin 6 and the DNA-damage induced phosphorylation of histone H2AX (γ-H2AX). We attempted to develop a therapeutic that would inhibit the CEES-induced increase in mutation frequency in the skin. We observe that multi-dose, topical treatment with 2,6-dithiopurine (DTP), a known chemical scavenger of CEES, beginning 1 h post-exposure to CEES, completely abolishes the CEES-induced increase in mutation frequency. These findings suggest the possibility that DTP, previously shown to be non-toxic in mice, may be useful as a therapeutic agent in accidental or malicious human exposures to SM. -- Highlights: ► 200 mM 2-(chloroethyl) ethyl sulfide (CEES) induces mutations in mouse skin. ► This dose of CEES is not overtly toxic, as assayed by histopathology. ► 2,6-Dithiopurine (DTP), applied after CEES-treatment, abolishes CEES-mutagenesis. ► This supports the idea that sulfur mustards exhibit long biological half-lives.« less
  • The formation and removal of DNA cross-links caused by treatment with 1-(4-amino-2-methylpyrimidin-5-yl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) were assayed by the technique of alkaline elution for DNA in comparison with the cytotoxicities in ACNU-sensitive rat 9L cells or ACNU-resistant subclones of 9L cells (9L/R cells). The ACNU-resistant 9L/R cells appeared to be about 16 times more resistant against ACNU than were the 9L cells. The DNA cross-links immediately after the treatment with ACNU were not detectable in 9L or 9L/R cells. Although the level of cross-links for 9L cells had reached a maximum at 6 hours and then persisted at almost the same levelmore » as that at 24 hours after the treatment with ACNU, the level for 9L/R cells was very low at 6 hours and then gradually decreased at 24 hours after treatment with ACNU. Inhibition of the formation of DNA interstrand cross-links caused by the treatment with ACNU might be a factor for the ACNU resistance in 9L/R cells. Also, the capacity for the repair of DNA interstrand cross-links might participate in the mechanisms of the ACNU resistance in 9L/R cells.« less
  • Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT{sup TM}, a commercially available full-thickness human skin equivalent. CEES (100-1000 {mu}M) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 {mu}M), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histonemore » H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE{sub 2} synthases, leukotriene (LT) A{sub 4} hydrolase and LTC{sub 4} synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.« less
  • Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT{sup TM}). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000 {mu}M) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealedmore » that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-{beta}-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.« less