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

https://doi.org/10.1016/j.taap.2011.06.012Get rights and content

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 ~ 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.

Introduction

Sulfur mustard (SM) is an extremely reactive bifunctional alkylating agent capable of inducing DNA damage and necrosis of epithelial cells of the lung, cornea and skin (reviewed in Balali-Mood and Hefazi, 2005, Shakarjian et al., 2010). Following cutaneous exposure to the aerosolized liquid, cytotoxicity occurs primarily in the basal layer of the epidermis resulting in epidermal–dermal separation and blistering (Smith et al., 1998, Kehe et al., 2009). For this reason SM is categorized as a vesicant. Recovery requires weeks to months, often with permanent changes in skin pigmentation (reviewed in Balali-Mood and Hefazi, 2005, Shakarjian et al., 2010). Due to these incapacitating effects as well as effects on vision and respiratory function following exposure, mustards have been used as warfare agents since World War I. More recent exposure incidents have occurred in the Iran–Iraq war (United Nations Security Council, 1984) and among fishermen who encountered sunken stockpiles of SM at sea (Wulf et al., 1985, Aasted et al., 1987). Currently, no clinically-validated antidotes are available. Because of the potential for use of SM as a chemical terrorism agent, there is renewed interest in developing effective chemopreventive and therapeutic measures.

SM undergoes intramolecular cyclization to form an electrophilic episulfonium ion, which can adduct cellular macromolecules such as DNA (reviewed in Balali-Mood and Hefazi, 2005). Analyses of urinary metabolites in rats and humans have suggested that SM (presumably in the cyclized form) either participates in a spontaneous reaction with nucleophilic glutathione (GSH) or is a substrate for GSH S-transferase (GST)-mediated metabolism (Black et al., 1992, Black and Read, 1995). The cysteinyl sulfur atom of GSH is predicted to provide electrons for a nucleophilic attack on the episulfonium ring, rendering the compound less reactive; therefore, GSH-conjugation may represent a major detoxification pathway for SM. In previous work, a spontaneous reaction between CEES and GSH was detected only when the pH was raised above the pKa of the sulfhydryl moiety of GSH (Liu et al., 2010); however, GST-mediated conjugation of structurally-similar nitrogen mustards has been previously demonstrated and is predicted to occur for SM (Dulik et al., 1986, Bolton et al., 1991).

Exposure to SM or its monofunctional analog, CEES, has been shown to deplete GSH stores in vitro and in vivo (Vijayaraghavan et al., 1991, Gross et al., 1993, Atkins et al., 2000, Kumar et al., 2001, Han et al., 2004, Gautam and Vijayaraghavan, 2007). GSH depletion may inhibit further clearance of SM, and, importantly, elicit oxidative stress, lipid peroxidation and macromolecular damage (Pal et al., 2009, Laskin et al., 2010). In line with these ideas, supplementation with GSH or GSH analogs protected against CEES- or SM-induced cytotoxicity in a number of cell lines (Amir et al., 1998, Andrew and Lindsay, 1998, Lindsay and Hambrook, 1998, Han et al., 2004), whereas blocking GSH synthesis rendered cells more sensitive to mustard toxicity (Gross et al., 1993, Atkins et al., 2000). Recently, pre- and post-treatment with GSH was shown to increase the viability of keratinocytes exposed to CEES (Tewari-Singh et al., 2011). Taken together, these findings suggest that agents capable of elevating levels of GSH and/or GST for extended periods of time may inhibit the toxic effects of SM and serve as effective and long-lasting chemopreventive agents in skin.

GSH is a tripeptide antioxidant composed of glutamate, cysteine and glycine residues. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL) (reviewed in Franklin et al., 2009, Lu, 2009). GCL is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are encoded by separate genes in humans. GCLC catalyzes the reaction between glutamate and cysteine. GCLM regulates the activity of GCLC by lowering the Km for glutamate and raising the Ki for GSH; therefore, GSH synthesis is more efficient in the presence of GCLM (Huang et al., 1993a, Huang et al., 1993b, Chen et al., 2005). Transcriptional regulation of GSTs and GSH synthesis enzymes can occur through activation of the transcription factor, Nrf2 (reviewed in Lu, 2009). Under basal conditions, Nrf2 complexes with Keap1 in the cytosol where it is targeted for ubiquitination and proteolysis (reviewed in Kensler et al., 2007). Upon stimulation by oxidative stress signals or electrophilic agents, Nrf2 is released from the Keap-1 mediated ubiquitination allowing for newly translated Nrf2 to translocate to the nucleus and elicit transcription of its target genes via binding to antioxidant response elements. A number of cancer chemopreventive agents have been shown to activate signaling through Nrf2 (Zhang and Hannink, 2003, Iida et al., 2004, Liby et al., 2005, Yates et al., 2009).

The goal of this study was to identify agents that could induce the GSH conjugation detoxification pathway and protect against CEES-induced cytotoxicity in human keratinocytes. Six potential chemopreventive agents were screened for their ability to induce expression of GSTs and/or elevate reduced GSH content in the human keratinocyte cell line, NCTC2544. Our studies suggest that the synthetic triterpenoid, methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), can stimulate Nrf2 translocation to the nucleus, elevate GSH content, and provide protection against CEES-induced cytotoxicity.

Section snippets

Chemicals

2-Chloroethylethyl sulfide (CEES) was obtained from Aldrich Chemicals (St. Louis, MO). Working stocks of CEES were prepared in ethanol at 200 or 600 mM and stored at − 20 °C. Stocks were verified for alkylating ability just prior to use by a spectrophotometric assay (Liu et al., 2010). CEES is a toxic, vesicating agent, which may potentially damage DNA; therefore, CEES and CEES-containing samples were handled with gloves in a chemical fume hood. CEES containing solutions were decontaminated with

Results

The goal of this study was to identify agents that could increase the rate of clearance of SM by elevating the overall GST conjugation activity in keratinocytes through induction of expression of GST isoforms or by increasing the levels of the GST cofactor, GSH. Initially, 6 agents with known cancer chemopreventive or Nrf2-activating properties [sulforaphane (Xu et al., 2006, Clarke et al., 2008), CDDO-Me (Liby et al., 2005, Yates et al., 2007), CDDO-Im (Yates et al., 2007, Petronelli et al.,

Discussion

In the current study, we have shown that treatment with CDDO-Me in the nanomolar range induced expression and nuclear localization of Nrf2, elevated the expression of GCLM, and, ultimately, increased the intracellular level of reduced GSH. Additionally, our preliminary studies revealed that pretreatment of keratinocytes with CDDO-Me prevented the loss of viability following exposure to the half-mustard, CEES. The cytoprotective effect of CDDO-Me was presumably due to scavenging of electrophilic

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

We thank Stephanie Tomlinson, Hilary Graham, and Joi Holcomb for assistance with manuscript preparation. We also thank Collin White for his helpful discussions concerning the reduced GSH assay. This work was funded by the National Institutes of Health CounterACT Program through the National Institute of Neurological Disorders and Stroke (award #U01NS058191) and by an NIEHS Center grant (P30ES007784).

References (65)

  • K. Kehe et al.

    Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering

    Toxicology

    (2009)
  • O. Kumar et al.

    Protective effect of various antioxidants on the toxicity of sulphur mustard administered to mice by inhalation or percutaneous routes

    Chem. Biol. Interact.

    (2001)
  • M.K. Kwak et al.

    Role of phase 2 enzyme induction in chemoprotection by dithiolethiones

    Mutat. Res.

    (2001)
  • S.C. Lu

    Regulation of glutathione synthesis

    Mol. Aspects Med.

    (2009)
  • A. Pal et al.

    Sulfur mustard analog induces oxidative stress and activates signaling cascades in the skin of SKH-1 hairless mice

    Free Radic. Biol. Med.

    (2009)
  • I. Samudio et al.

    2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) directly targets mitochondrial glutathione to induce apoptosis in pancreatic cancer

    J. Biol. Chem.

    (2005)
  • R.P. Singh et al.

    Mechanisms and preclinical efficacy of silibinin in preventing skin cancer

    Eur. J. Cancer

    (2005)
  • V. Tamasi et al.

    Ebselen augments its peroxidase activity by inducing nrf-2-dependent transcription

    Arch. Biochem. Biophys.

    (2004)
  • R.K. Thimmulappa et al.

    Nrf2-dependent protection from LPS induced inflammatory response and mortality by CDDO-Imidazolide

    Biochem. Biophys. Res. Commun.

    (2006)
  • R. Vijayaraghavan et al.

    Dermal intoxication of mice with bis(2-chloroethyl)sulphide and the protective effect of flavonoids

    Toxicology

    (1991)
  • C.C. White et al.

    Fluorescence-based microtiter plate assay for glutamate-cysteine ligase activity

    Anal. Biochem.

    (2003)
  • H.C. Wulf et al.

    Sister chromatid exchanges in fishermen exposed to leaking mustard gas shells

    Lancet

    (1985)
  • A. Aasted et al.

    Mustard gas: clinical, toxicological, and mutagenic aspects based on modern experience

    Ann. Plast. Surg.

    (1987)
  • E. Aktunc et al.

    N-acetyl cysteine promotes angiogenesis and clearance of free oxygen radicals, thus improving wound healing in an alloxan-induced diabetic mouse model of incisional wound

    Clin. Exp. Dermatol.

    (2010)
  • A. Amir et al.

    Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro

    Hum. Exp. Toxicol.

    (1998)
  • D.J. Andrew et al.

    Protection of human upper respiratory tract cell lines against sulphur mustard toxicity by glutathione esters

    Hum. Exp. Toxicol.

    (1998)
  • K.B. Atkins et al.

    N-acetylcysteine and endothelial cell injury by sulfur mustard

    J. Appl. Toxicol.

    (2000)
  • M. Balali-Mood et al.

    The pharmacology, toxicology, and medical treatment of sulphur mustard poisoning

    Fundam. Clin. Pharmacol.

    (2005)
  • R.M. Black et al.

    Biological fate of sulfur mustard, 1,1′-thiobis(2-chloroethane). Urinary excretion profiles of hydrolysis products and beta-lyase metabolites of sulfur mustard after cutaneous application in rats

    J. Anal. Toxicol.

    (1992)
  • R.M. Black et al.

    Biological fate of sulphur mustard, 1,1′-thiobis(2-chloroethane): identification of beta-lyase metabolites and hydrolysis products in human urine

    Xenobiotica

    (1995)
  • M.G. Bolton et al.

    Specificity of isozymes of murine hepatic glutathione S-transferase for the conjugation of glutathione with l-phenylalanine mustard

    Cancer Res.

    (1991)
  • G. Drasch et al.

    Concentrations of mustard gas [bis(2-chloroethyl)sulfide] in the tissues of a victim of a vesicant exposure

    J. Forensic Sci.

    (1987)
  • Cited by (0)

    View full text