International Journal of Toxicology http://ijt.sagepub.com/
Mustard Gas Inhalation Injury: Therapeutic Strategy Brian M. Keyser, Devon K. Andres, Wesley W. Holmes, Danielle Paradiso, Ashley Appell, Valerie A. Letukas, Betty Benton, Offie E. Clark, Xiugong Gao, Prabhati Ray, Dana R. Anderson and Radharaman Ray International Journal of Toxicology published online 6 May 2014 DOI: 10.1177/1091581814532959 The online version of this article can be found at: http://ijt.sagepub.com/content/early/2014/04/28/1091581814532959
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Article International Journal of Toxicology 1-11 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581814532959 ijt.sagepub.com
Mustard Gas Inhalation Injury: Therapeutic Strategy Brian M. Keyser1, Devon K. Andres1, Wesley W. Holmes2, Danielle Paradiso2, Ashley Appell1, Valerie A. Letukas1, Betty Benton1, Offie E. Clark1, Xiugong Gao3, Prabhati Ray3, Dana R. Anderson2, and Radharaman Ray1
Abstract Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds. Keywords sulfur mustard, inhalation, apoptosis, antioxidants, anti-inflammatory, protease
Sulfur mustard (SM), a blistering chemical warfare agent, was used first in World War I (WWI) and as recently as the 1980s in the Iran–Iraq war. It is easy to make, store, and deploy this chemical agent additionally, SM has been the most widely used chemical weapon during WWI.1 Because of these reasons, a recent concern is that SM could also be used as a terrorism agent. Human casualties during past wars (100 000þ in the IranIraq war alone) have shown that whole-body SM exposure causes debilitating skin blisters, respiratory distress, and ocular damage2; these effects are attributed to SM-induced epithelial damage, mainly basal cell apoptosis.1,3 Most mortality following SM exposure, however, has been attributed to respiratory tract lesions and pulmonary damage.4 Common postexposure complications in surviving victims are the development of a variety of chronic forms of pulmonary disease, including laryngitis, tracheobronchitis, bronchiolitis, bronchopneumonia, an unique form of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, large airway narrowing, pulmonary fibrosis, chronic bronchitis, bronchiolitis obliterans, and acute respiratory distress syndrome (ARDS).5-13 The multiplicity of the different cellular components (DNA, proteins, thiol compounds, etc) and the pathways affected by SM have complicated the efforts to establish a clearly defined mechanism-based therapeutic approach for SM exposure. As schematically indicated in Figure 1, some of the toxic effects of
SM and the half mustard (2-chloroethyl sulfide, CEES) include oxidative stress,14-20 proinflammatory pathways,21,22 apoptosis,19,23-25 and DNA damage.26,27 In addition to these toxic effects, low-level exposure to SM causes weight loss and sensory irritation.28 Associated with these different toxic effects are the cellular pathways that are affected, for example, intracellular free Ca2þconcentration increase due to either reduced glutathione (GSH) depletion or oxidative stress; phospholipase-mediated membrane effects and inflammation; and activation of caspases and apoptosis. Moreover, there could be cross-talks between these pathways. As such, these events provide some potential targets for intervention against SM injury. The majority of efforts to discover a therapeutic for 1
Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA 2 Analytical Toxicology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA 3 Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD, USA Corresponding Author: Radharaman Ray, Cellular and Molecular Biology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD 21010-5400, USA. Email: [email protected]
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Figure 1. Sulfur mustard toxicity. Points of intervention discussed in this article are highlighted in red.
SM exposure have historically explored one or more of the 4 different intervention points shown in Figure 1 (red bubbles). Therefore, the 4 categories of potential medical countermeasures are (1) anti-inflammatory compounds, (2) antioxidants, (3) protease inhibitors, and (4) antiapoptotic drugs or measures. This review summarizes the progress in each category, particularly the in vivo results.
Anti-inflammatory Drug Sulfur mustard acts as an electrophile that alkylates cellular and extracellular components of living tissue. As a result, some complex cellular events are initiated including cell cycle arrest, synthesis and release of inflammatory mediators, and cytotoxicity. Following these cellular effects, there are tissue responses such as inflammation and tissue damage.29 The cause of the acute injury appears to be the premature, sudden, and massive release of destructive enzymes and mediators of inflammation such as proinflammatory cytokines, which include interleukin (IL) 1b, IL-6, IL-8, and tumor necrosis factor a (TNF-a), and inducible nitric oxide synthase (iNOS).30,31 Based on what has been described earlier, various antiinflammatory or related drugs (eg, doxycycline and macrolide antibiotics) have been tested both in vitro and in vivo for their benefits in preventing the injuries caused by SM. Macrolides are a group of antibiotics that were initially studied and used
because of their antibacterial properties. The name ‘‘macrolide’’ is derived from their structure, which is characterized by a macrocyclic lactone ring with various amino sugars attached. In the recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases through mechanisms distinct from their antibacterial activity.32 Although the mechanisms underlying this effect are still unclear, macrolides have been shown to affect several pathways of the inflammatory process, including the migration of neutrophils, the oxidative burst in phagocytes, and the production of proinflammatory cytokines.33 The use of macrolide antibiotics has been previously shown to reduce SM-induced expression of iNOS and proinflammatory cytokines in monocytes and human airway epithelial cells (AECs) in vitro.34-36 In experiments using SM inhalation in rats, macrolides were able to cause a modest improvement in tracheal pathology scores at 3 days but not at 1 or 7 days after SM exposure.37 These results were only from a pilot study; a more detailed study would probably provide more insight into the possible benefits of macrolides in SM inhalation injury. However, the dose of antibiotic used was twice the normal dose used in clinical settings, increasing the chances of possible toxic side effects of this treatment. Another class of anti-inflammatory drugs that has been examined is glucocorticoid steroids. Once glucocorticoid
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steroids bind to their receptors (glucocorticoid receptor [GR]) on the cell membrane, the activated GR translocates to the nucleus and associates with DNA at specific binding domains. Activated GR binding to DNA causes the repression of many proinflammatory gene products, for example, cytokines, chemokines, adhesion molecules, and inflammatory enzymes.38 The 2 glucocorticoid steroids that have been used as SM antidotes in vivo are betamethasone and dexamethasone and both of these compounds are nonselective anti-inflammatory drugs. The administration of betamethasone for 7 days following SM inhalation in guinea pigs abolished the airway muscle hyperresponsiveness and partially restored tracheal epithelium neutral endopeptidase (a signaling peptide involved in inflammation and pain) activity.39,40 However, betamethasone was not able to completely reduce the increased tracheal epithelium height and cell density resulting from SM exposure.39 A single injection of dexamethasone, when administered to mice 1 hour after exposure to nitrogen mustard (NM, which is structurally similar to SM and produces similar injuries), was able to reduce inflammation and release cytokines, IL-1, and IL-6 in bronchoalveolar lavage fluid (BALF) that was examined at 6 and 18 hours postexposure.41 This effect of acute dexamethasone treatment also reduced the long-term lymphocytic response by decreased collagen deposition in the lungs when examined 2 to 4 weeks after NM exposure. 41 Other studies have shown that dexamethasone administered to guinea pigs was able to reduce the number of eosinophils and improve tracheal responsiveness when measured 14 days after SM exposure; however, the levels of IL-4 were higher than in SM-only-exposed controls.42 Two studies performed in Iran–Iraq war casualties have shown that both a short-term high dose and a long-term low dose of prednisolone (a commonly prescribed corticosteroid) were able to significantly (P < 0.05) improve lung function.43,44 However, some of the patients in these studies did not respond to this therapy. Additionally, there are many possible side effects with long-term administration of a corticosteroid such as weight gain, osteoporosis, lowered immunity, myopathy, avascular necrosis, changes in mood, memory deficit, psychosis, hyperglycemia, peptic ulceration, gastritis, and gastrointestinal hemorrhage (for a complete review see 45). These serious adverse effects are cause for concern for using glucocorticoids in the treatment of people who have been exposed to SM. Other compounds with anti-inflammatory properties in addition to their primary mechanism of action have been examined in SM- or CEES-induced lung injury. These compounds include salbutamol,46 tissue plasminogen activator,47 and the dual acting antioxidant/anti-inflammatory AEOL 10150. All of these compounds have shown improvement in SM- and CEESinduced lung injury.48-51 These results indicate not only the complex toxic mechanisms of action of these vesicating agents but also the need for future research in this area in order to identify more selective anti-inflammatory agents as well as possible combinations of pharmacologic antagonists targeting other affected pathways.
Antioxidant Drug Exposure to SM can also cause oxidative stress due to the depletion of cellular antioxidants, mainly GSH, a tripeptide and rapidly oxidizes to oxidized glutathione, which may lead to the production of reactive oxygen species (ROS) and membrane lipid peroxidation. 52 This may result in an imbalance between the production of ROS and the level of antioxidants in the lung and respiratory tract. This imbalance is believed to be the primary event triggering the inflammatory cascade and tissue injury.53 The specific oxidants that are involved in SM-induced cytotoxicity are still not known. A growing body of evidence is pointing toward reactive nitrogen species being one of the main oxidant classes in SM-induced lung injury.53 Nitric oxide, produced by iNOS, reacts with superoxide anion, forming peroxynitrite. Nitric oxide and peroxynitrite modify cellular proteins and DNA and, thereby, induce cytotoxicity and inflammation. Expression of iNOS is upregulated in lung macrophages and epithelial cells following exposure to SM.31 Use of ebselen (2-phenyl-1, 2-benzisoselenazol-3(2H)-one), a peroxynitrite scavenger, and 3 analogs have been shown to reduce CEES toxicity in vitro.54 Also, an NM exposure model has been shown to reduce histopathology but not NM-induced elevation in lung iNOS levels.55 Melatonin, a scavenger of both reactive nitrogen and oxygen species, when given to NM-exposed rats, was able to reduce NM-induced increase in iNOS, glutathione peroxidase activity, and reduced inflammation in the lung.56 Transgenic mice lacking a functional iNOS were found to have reduced expression of proinflammatory genes and markers of oxidative stress when exposed to CEES.57 Use of an iNOS inhibitor, silibinin,58 in vitro has shown protection against SM; however, further investigations are needed to further establish treatment conditions.59 The use of other antioxidants to treat SM-induced inhalation toxicity showed some limited success. Two antioxidants, Trolox (a water soluble analog of vitamin E), vitamin E, and quercetin (bioflavonoid) have been shown to enhance survival time and offer protective effects from oxidative damage in animals exposed to SM via inhalation or percutaneously.60-62 These antioxidants have been shown to reduce oxidative markers after SM exposure as well as increased survival. In one of these studies, the GSH used was not found to have the same protective effect as Trolox or quercetin.60 One reason for this result could be that GSH is not cell permeable and is easily degraded by peptidases in vivo. Another antioxidant, N-acetyl cysteine (NAC), which is a precursor for glutathione synthesis, has been explored as a potential therapeutic for SM-induced lung injury.63 When it was administered intraperitoneally in the beginning of SM exposure, it was shown to reduce the levels of lactate dehydrogenase, albumin, total protein, and the number of neutrophils in BALF fluid 24 hours postexposure.64 In our recent studies using the same rat exposure model, NAC, given as a postexposure treatment via inhalation (nebulized and given every 2 hours for 12 hours postexposure), was effective in reducing the pulmonary flow obstruction (expiratory flow at 50% [EF50]) and improving the oxygen saturation in SM inhaled rats at 24 hours after SM exposure
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Figure 2. Oxygen saturation and mid-expiratory flow at 50% (EF50) measures at 24 hours postinhalation of sulfur mustard (SM) vapor. Sulfur mustard caused a decrease in O2 saturation and an increase in EF50, a measure of airway obstruction, in comparison to control animals. Treatment with N-acetyl cysteine (NAC), administered immediately following SM exposure then every 2 hours of 12 hours postexposure, improved tissue oxygenation and airway flow in comparison to SM exposed animals. *P < 0.05 versus control; þ P < 0.05 versus SM. Data were analyzed using 1-way analysis of variance (ANOVA) with Tukey post hoc test.
(Figure 2). The efficacy of NAC as a postexposure therapeutic for SM inhalation injury was also demonstrated in a large animal model such as the swine where it showed improvements in arterial blood oxygenation, arterial blood bicarbonate, and shunt fraction.65 In these experiments, NAC was given via inhalation and was given 0.5, 2, 4, 6, 8, and 10 hours postexposure. N-Acetyl cysteine was also tested by administering orally, intravenously, and intratracheally (encapsulated in liposomes) after CEES exposure.66-68 In these studies, NAC was able to protect either as a pretreatment (3 days prior to CEES, oral) or as a therapeutic (90 minutes after CEES, intratracheal) by reducing *70% the CEES-induced lung injury. N-Acetyl cysteine has been reported to reduce the infiltration of inflammatory cells into the lung via the downregulation of the transcription factor activator protein 1,69 but the exact mechanism of protection due to NAC is not known. These observations and others have suggested that antioxidants could serve as prospective therapeutics for pulmonary damage due to SM.70
Protease Inhibitor Drug Both serine proteases and matrix metalloproteinases (MMPs) have been implicated in SM effects, particularly its vesication mechanism. Matrix metalloproteinases are responsible for the degradation of extracellular matrix proteins, contributing to inflammatory cell recruitment, tissue injury, and fibrosis.71 Matrix metalloproteinases have been shown to be upregulated as early as 6 hours postexposure to SM.31 This upregulation is dose dependent and persists for 24 hours after SM exposure.72 Following SM exposure, MMP activity is found in BALF and at
sites of decreased alveolar epithelial integrity and increased BAL albumin content (an indicator of airway damage).31 It is possible that MMP-9 plays a role in SM-induced detachment of epithelium and epithelial cell sloughing.31 When aprotinin (a serine-protease inhibitor) or ilomastat (a nonselective MMP inhibitor) was administrated just prior to SM, a marked improvement in pulmonary function (PF) was observed when measured 24 hours following SM exposure in rats.73 Aprotinin reduced SM-induced increases in IL-1a, IL-13, total BAL protein, and lung histopathology including inflammation. The use of epigallocatechin gallate, MMP antanogist, and antioxidant was shown not be highly effective as a prophylactic against SM.74 Doxycycline, a tetracycline derivative, has been shown to be a nonselective MMP inhibitor. Using doxycycline as a pretreatment (3 hours prior to SM), the results were similar to those obtained using aprotinin and ilomastat. In these experiments, doxycycline was able to inhibit MMP-2 and MMP-9, which decreased lung inflammation and injury.75 However, in addition to inhibiting MMPs, doxycycline has been shown to inhibit iNOS and consequently nitric oxide production.76 Hence, the beneficial effects of all of these drugs (aprotinin, ilomastat, and doxycycline) could be due to the nonselective effects and not wholly the MMP inhibition. Taken together, protease inhibitors also seem to be promising candidates as therapeutics for SM injury.
Apoptosis Inhibitors Apoptosis can proceed by 2 different pathways (Figure 3), referred to as the intrinsic (mitochondrial) and the extrinsic (death receptor) pathways. The intrinsic pathway is activated via the release of proapoptotic molecules from the mitochondria into the cytosol, which leads to the activation of caspase-9 and ultimately caspase-3. The extrinsic pathway is induced by cell surface death receptors (eg, Fas). Stimulation of these receptors facilitates recruitment of the adaptor Fas-associated protein with death domain (FADD) and tumor necrosis factor receptor superfamily member 1A-associated via death domain (TRADD), and these, in turn, associate with procaspase-8 and procaspase-10 (initiator caspases). The assembly of FADD/TRADD and procaspase-8/10 is called the death-inducing signaling complex (DISC). The DISC promotes self-processing and activation of the initiator procaspases to active caspase-8 and caspase-10 and their subsequent dissociation from the protein complex. Active initiator caspase-8 and/or caspase-10 directly activates downstream caspases (caspase-3 and caspase-7) and causes cell death. Apoptosis plays an important role in lung pathologies after injury, such as SM exposure, in 2 different ways. First, failure to clear unwanted cells by apoptosis will prolong the inflammation because the release of their toxic elements delays the repair processes77; second, excessive apoptosis may cause diseases (eg, adult respiratory distress syndrome [ARDS] and pulmonary fibrosis).78 The survival and recovery of epithelial and endothelial cells and the resolution of inflammatory cells appear to be the keys in normal repair.77 Matute-Bello et al in 1999 demonstrated that lung injury and the sequential inflammation indicated a dual role for the Fas/FasL system as a proapoptotic/
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Figure 3. Apoptosis pathways. Adapted from Keyser et al.24
proinflammatory system. It appears that controlling the degree of apoptosis after an injury to the lung (ie, SM exposure) has a direct correlation with the patient’s prognosis.77 Upregulation of the Fas/FasL pathway in epithelial cells has been shown to contribute to the severe epithelial damage that occurs in ARDS, one of the postexposure complications of SM. 79 Recently, FasL molecules were reported to be expressed in a-smooth muscle actin positive cells in mice with bleomycin-induced pulmonary fibrosis and in humans with idiopathic pulmonary fibrosis (IPF).80 The BALF from patients with ARDS or IPF could induce apoptosis in cultured small AECs through the Fas pathway.78,80 Further evidence of the Fas pathway in pulmonary fibrosis is that neutralization of FasL antibody by Fas–Ig fusion protein or neutralizing antiFasL antibody was able to prevent the development of bleomycin-induced pulmonary fibrosis in vivo.81 Blocking the Fas/FasL system reduced the development of endotoxininduced acute lung injury (ALI).82 Fas and FasL mutant animals exhibit less pulmonary epithelial cell apoptosis in ALI and have higher survival rates.78,83 Based on these facts, the Fas pathway may play a critical and important role in the development of the lung diseases that are observed after exposure to SM. After SM exposure, apoptosis may occur via both the intrinsic mitochondrial pathway and the extrinsic death receptor (Fas) pathway.84,85 It has been reported that blocking the Ca2þ, calmodulin-mediated mitochondrial pathway or the Fasmediated pathway antagonizes SM-induced apoptosis in cultured primary human epidermal keratinocytes.85,86 In in vivo, it
has been demonstrated that blocking the SM-induced Fas response by overexpression of dominant-negative FADD (FADD-DN) in human skin grafted onto nude mice blocks apoptosis in the basal epidermis and, therefore, microvesication induced by vapor cup SM exposure.85 In cultured human AECs, SM induces apoptosis predominantly via a caspase-8 or Fas-mediated pathway.3 This SM-induced and Fas-mediated apoptosis in AEC is prevented by (1) a Fas antagonistic antibody (ZB4; B. M. Keyser, unpublished results), (2) through the suppression of the Fas/FADD response by small interfering RNAs (B. M. Keyser, unpublished results), or (3) simply by the general caspase inhibitor Z-VAD-FMK.87 A recent report has also demonstrated that the use of RNA interference against the Fas receptor, applied up to 8 hours postexposure in vitro, was able to significantly reduce apoptosis 24 hours after SM exposure.24 This use of a highly selective antagonist of the Fas-mediated pathway of apoptosis added further evidence for an important role of this pathway in SMinduced apoptosis. Other proteins in the extrinsic apoptosis pathways (eg, cFLIP, DR-4, and DR-5) could play a role in SM-induced apoptosis. To date, there have been no studies involving these proteins in the lung. We have shown previously that dominant-negative FADD skin did not show any evidence of microblistering whereas this was not seen with TNF-a antagonism.85 If this is the case in the airway remains to be seen. Hence, further development of an effective pharmacological antagonism or a molecular suppression of the Fas-mediated pathway of apoptosis may provide a highly prospective therapeutic approach for SM inhalation injury.
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Figure 4. Airway cast formation at 24 hours after intratracheal sulfur mustard (SM) inhalation in rats. A, In general, a cast or plug is composed of a sparsely to moderately cellular eosinophilic homogenous material surrounding a less dense central core, hematoxylin and eosin. B, When present, fibrin stains dark blue (arrows), affects up to 15% of the section only, and is observed only at the periphery of the cast, phosphotungstic acid hematoxylin (PTAH). C, Bright red fibrin staining (arrows) mirrors the fibrin staining observed with PTAH; light green mucinous ground substance (*) is seen mostly in the less dense central areas; the yellowish-material is suggestive of collagen and/or reticular fibers, pentachrome. D, Light pink mucin (arrows) is observed with mucicarmine in the less dense central areas, mucicarmine. All images taken at 2 magnification.
Sulfur Mustard Inhalation Exposure and Drug Administration Inhalation exposure to SM vapor was performed via tracheal intubation in rats as described previously.88 Briefly, male Sprague Dawley rats (*200 g, 8 per group) were anesthetized with a combination of ketamine (80 mg/kg, intraperitoneally [ip]) and xylazine (10 mg/kg, ip). After induction of anesthesia, a diagnostic otoscope was used to facilitate tracheal intubation. Sulfur mustard (diluted in absolute ethanol at 1.0 mg/kg) was placed in a heated (37 C) water-jacketed glass vapor generator, and the rats were connected to this device by an endotracheal tube and exposed to SM vapor or ethanol (vehicle control) for 50 minutes. This exposure model has been shown to produce a consistent pathology at 24 hours after exposure.88 At the conclusion of the exposure (SM or ethanol), rats were placed on a heated water blanket (37 C) in a cage until they recovered from
anesthesia. N-Acetyl cysteine (0.35 mg/mL) was administered via nose-only nebulization using an Aeroneb (Aerogen Inc., Marietta, Georgia) nebulizer system. Immediately following exposure (SM or ethanol) and then every 2 hours of 12 hours postexposure, conscious rats were placed into nose-only restraint tubes (Buxco Research Systems, Wilmington, North Carolina) and positioned directly in the effluent of the nebulizer. Air flow to the nebulizer was metered through a mass flow controller (Brooks Instrument, Hatfield, Pennsylvania) at a rate of 0.5 LPM. N-Acetyl cysteine of 1 mL was placed in the Aeroneb and was nebulized at a rate of 0.3 mL/min with a particle size of 4.0 to 6.0 mm.
Lung Cast Staining Lung casts were fixed with 4% neutral-buffered paraformaldehyde, trimmed, paraffin embedded, and cut on a microtome
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Figure 5. Airway cast formation at 24 hours after intratracheal sulfur mustard (SM) inhalation in rats. A. In general, a cast or plug is composed of a sparsely to moderately cellular eosinophilic homogenous material surrounding a less dense central core, hematoxylin and eosin. B, When present, fibrin stains dark blue (arrows), affects up to 15% of the section only, and is observed only at the periphery of the cast, phosphotungstic acid hematoxylin. C, Bright red fibrin staining (arrows) mirrors the fibrin staining observed with PTAH; light green mucinous ground substance (*) is seen mostly in the less dense central areas; the yellowish material is suggestive of collagen and/or reticular fibers, pentachrome. D, Light pink mucin (arrows) is observed with mucicarmine in the less dense central areas, mucicarmine. Note that all images shown here are the same casts as in Figure 4, but at a magnification of 20 to reveal additional details.
into 5-mm thick sections. Slides of the lungs were stained with hematoxylin and eosin (H&E) for routine histopathology. Phosphotungstic acid hematoxylin staining was done as reported previously.89 Pentachrome staining was performed following.90 Mucicarmine staining protocol was in accordance as reported previously.91
Whole Body Plethysmography A Buxco unrestrained whole-body plethysmography (WBP) system with Biosystems XA software (Buxco Research Systems) was used to monitor PF parameters in conscious rats. This included expiratory flow, from which the EF50 value could be derived. Prior to SM or ethanol exposure, rats were placed in the WBP chambers for a total of 2 hours (1 hour for acclimation and 1 hour for data collections) to establish a baseline for PF parameters. At the conclusion of each exposure (SM or ethanol), the rats were placed in their original cages to recover from anesthesia. Twenty-three hours postexposure, the rats were placed in the WBP chambers again and allowed to be acclimated for 1 hour; PF data were recorded through the 24-hour
time point. Data were averaged by group for each 1-hour data collection period.
Blood Oxygen Saturation (PO2) Analysis At 24 hours after SM exposure, rats were deeply anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). A 0.5 mL blood sample was then obtained from the descending aorta. The blood sample was quickly injected into the TruPoint CC (IRMA, Edison, NJ) cartridge and analyzed per manufacturer’s instructions.
Data Analysis Data are shown as mean + standard deviation. Group comparisons were conducted using 1-way analysis of variance followed by Tukey post hoc multiple comparison test. Significant results were identified when P < .05 or smaller.
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Concluding Remarks Sulfur mustard exposure of the respiratory system causes immediate and long-term health effects. In severe exposure, it is the inhalation injury that has been known to result in most fatalities compared to the skin or ocular injury. Sulfur mustard inhalation effects include chronic forms of pulmonary diseases, including laryngitis, tracheobronchitis, bronchiolitis, bronchopneumonia, COPD, bronchiectasis, asthma, large airway narrowing, pulmonary fibrosis, chronic bronchitis, and ARDS.5-10 Thus, it is essential to discover potential therapeutic compounds to combat these effects. As has been explained earlier in this article, the major target for the tissue or organ damage due to SM is the epithelial layer. This is particularly important for the injury to the upper airway where the epithelial cells die and detach from the substratum. The mechanism of this epithelial detachment, although not fully established, might involve protease stimulation that could degrade the proteins responsible for the epithelial cell attachment to the substratum. The detached cells contribute to cast formation along with inflammatory cells, fibrin, and mucus which is released from goblet cells. In experimental animals such as the rat SM inhalation model,88 cast formation has been found to be responsible for acute mortality due to the airway obstructive effect following the inhalation exposure (Figures 4 and 5). Based on their mechanisms of action, all of the proposed therapeutic approaches, that is, using (1) antioxidants, (2) protease inhibitors, (3) antiapoptotic therapeutics, and (4) antiinflammatory compounds should be able to counteract this cast formation; moreover, measures to degrade such casts once those are formed might also be helpful at least to reduce the acute mortality. From what has just been described, the major factors attributable to the cellular and/or tissue toxicity due to SM appear to be (1) genotoxicity and cytotoxicity, (2) protease stimulation, (3) cell death, and (4) inflammation. Based on the earlier discussions, it is apparent that all of the 4 different categories of drugs, that is, antioxidants, protease inhibitors, apoptosis inhibitors, and anti-inflammatory compounds are potential medical countermeasures against SM toxicity. All of these different types of drugs when tested singly have been found to have some benefit against SM inhalation injury, but whether their combinations and which combinations would be more beneficial remain to be studied. One important consideration is the temporal sequence of events following SM exposure, which is lacking. Under such circumstances, a reasonable approach to treat SM airway injury seems to be preventing the cytotoxicity (cell death) and at the same time minimizing the other associated toxic events, that is, inflammation, protease stimulation, and oxidative damage. Acknowledgments This work was supported by the Defense Threat Reduction Agency— Joint Science and Technology Office, Medical S&T Division [Grant CBM.RESP.01.10.RC.015]. Additionally, this research was supported
in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC. We acknowledge LTC Derron A. Alves for his expertise in the lung cast staining.
Authors’ Note The views expressed in this article are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the US Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89-544), as amended.
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International Journal of Toxicology tracheal muscle responsiveness and lung inflammation in sulfur mustard exposed guinea pigs. J Ethnopharmacol. 2011;137(2): 1028-1034. Ghanei M, Panahi Y, Mojtahedzadeh M, Khalili AR, Aslani J. Effect of gamma interferon on lung function of mustard gas exposed patients, after 15 years. Pulm Pharmacol Ther. 2006; 19(2):148-153. Ghanei M, Khalili AR, Arab MJ, et al. Diagnostic and therapeutic value of short-term corticosteroid therapy in exacerbation of mustard gas-induced chronic bronchitis. Basic Clin Pharmacol Toxicol. 2005;97(5):302-305. Moghadam-Kia S, Werth VP. Prevention and treatment of systemic glucocorticoid side effects. Int J Dermatol. 2010;49(3): 239-248. Uzkeser H, Cadirci E, Halici Z, et al. Anti-inflammatory and antinociceptive effects of salbutamol on acute and chronic models of inflammation in rats: involvement of an antioxidant mechanism. Mediators Inflamm. 2012;2012:438912. Del Rosso M, Fibbi G, Pucci M, Margheri F, Serrati S. The plasminogen activation system in inflammation. Front Biosci. 2008;13:4667-4686. O’Neill HC, White CW, Veress LA, et al. Treatment with the catalytic metalloporphyrin AEOL 10150 reduces inflammation and oxidative stress due to inhalation of the sulfur mustard analog 2-chloroethyl ethyl sulfide. Free Radic Biol Med. 2010;48(9): 1188-1196. van Helden HP, Kuijpers WC, Diemel RV. Asthmalike symptoms following intratracheal exposure of Guinea pigs to sulfur mustard aerosol: therapeutic efficacy of exogenous lung surfactant curosurf and salbutamol. Inhal Toxicol. 2004;16(8): 537-548. Boskabady M, Boskabady MH, Amel Zabihi N, Boskabady M. The effect of chemical warfare on respiratory symptoms, pulmonary function tests and their reversibility 23-25 years after exposure [published online December 20, 2012]. Toxicol Ind Health. 2012. Veress LA, Hendry-Hofer TB, Loader JE, Rioux JS, Garlick RB, White CW. Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am J Respir Cell Mol Biol. 2013;48(4):439-447. Tewari-Singh N, Agarwal C, Huang J, Day BJ, White CW, Agarwal R. Efficacy of glutathione in ameliorating sulfur mustard analoginduced toxicity in cultured skin epidermal cells and in SKH-1 mouse skin in vivo. J Pharmacol Exp Ther. 2011;336(2):450-459. Weinberger B, Laskin JD, Sunil VR, Sinko PJ, Heck DE, Laskin DL. Sulfur mustard-induced pulmonary injury: therapeutic approaches to mitigating toxicity. Pulm Pharmacol Ther. 2011; 24(1):92-99. Pino MA, Pietka-Ottlik M, Billack B. Ebselen analogues reduce 2-chloroethyl ethyl sulphide toxicity in A-431 cells. Arh Hig Rada Toksikol. 2013;64(1):77-86. Yaren H, Mollaoglu H, Kurt B, et al. Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats. Res Vet Sci. 2007;83(1):116-122. Ucar M, Korkmaz A, Reiter RJ, et al. Melatonin alleviates lung damage induced by the chemical warfare agent nitrogen mustard. Toxicol Lett. 2007;173(2):124-131.
57. Sunil VR, Shen J, Patel-Vayas K, Gow AJ, Laskin JD, Laskin DL. Role of reactive nitrogen species generated via inducible nitric oxide synthase in vesicant-induced lung injury, inflammation and altered lung functioning. Toxicol Appl Pharmacol. 2012;261(1): 22-30. 58. Ramasamy K, Dwyer-Nield LD, Serkova NJ, et al. Silibinin prevents lung tumorigenesis in wild-type but not in iNOS-/- mice: potential of real-time micro-CT in lung cancer chemoprevention studies. Clin Cancer Res. 2011;17(4):753-761. 59. Balszuweit F, John H, Schmidt A, Kehe K, Thiermann H, Steinritz D. Silibinin as a potential therapeutic for sulfur mustard injuries. Chem Biol Interact. 2013;206(3):496-504. 60. Kumar O, Sugendran K, Vijayaraghavan R. Protective effect of various antioxidants on the toxicity of sulphur mustard administered to mice by inhalation or percutaneous routes. Chem Biol Interact. 2001;134(1):1-12. 61. Vijayaraghavan R, Sugendran K, Pant SC, Husain K, Malhotra RC. Dermal intoxication of mice with bis(2-chloroethyl)sulphide and the protective effect of flavonoids. Toxicology. 1991;69(1): 35-42. 62. Vijayaraghavan R, Gautam A, Sharma M, Satish HT, Pant SC, Ganesan K. Comparative evaluation of some flavonoids and tocopherol acetate against the systemic toxicity induced by sulphur mustard. Indian J Pharmacol. 2008;40(3): 114-120. 63. Bobb AJ, Arfsten DP, Jederberg WW. N-acetyl-L-Cysteine as prophylaxis against sulfur mustard. Mil Med. 2005;170(1): 52-56. 64. Anderson DR, Byers SL, Vesely KR. Treatment of sulfur mustard (HD)-induced lung injury. J Appl Toxicol. 2000;20(suppl 1): S129-S132. 65. Jugg B, Fairhall S, Smith A, et al. N-acetyl-L-cysteine protects against inhaled sulfur mustard poisoning in the large swine. Clin Toxicol. 2013;51(4):216-224. 66. McClintock SD, Hoesel LM, Das SK, et al. Attenuation of half sulfur mustard gas-induced acute lung injury in rats. J Appl Toxicol. 2006;26(2):126-131. 67. Das SK, Mukherjee S, Smith MG, Chatterjee D. Prophylactic protection by N-acetylcysteine against the pulmonary injury induced by 2-chloroethyl ethyl sulfide, a mustard analogue. J Biochem Mol Toxicol. 2003;17(3):177-184. 68. McClintock SD, Till GO, Smith MG, Ward PA. Protection from half-mustard-gas-induced acute lung injury in the rat. J Appl Toxicol. 2002;22(4):257-262. 69. Mukhopadhyay S, Mukherjee S, Stone WL, Smith M, Das SK. Role of MAPK/AP-1 signaling pathway in the protection of CEES-induced lung injury by antioxidant liposome. Toxicology. 2009;261(3):143-151. 70. Pohanka M. Antioxidants countermeasures against sulfur mustard. Mini Rev Med Chem. 2012;12(8):742-748. 71. Chakrabarti S, Patel KD. Matrix metalloproteinase-2 (MMP-2) and MMP-9 in pulmonary pathology. Exp Lung Res. 2005; 31(6):599-621. 72. Calvet JH, Planus E, Rouet P, et al. Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs. Am J Physiol. 1999;276(5 pt 1):L754-L762.
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73. Anderson DR, Taylor SL, Fetterer DP, Holmes WW. Evaluation of protease inhibitors and an antioxidant for treatment of sulfur mustard-induced toxic lung injury. Toxicology. 2009;263(1): 41-46. 74. Pohanka M, Sobotka J, Stetina R. Sulfur mustard induced oxidative stress and its alteration by epigallocatechin gallate. Toxicol Lett. 2011;201(2):105-109. 75. Guignabert C, Taysse L, Calvet JH, et al. Effect of doxycycline on sulfur mustard-induced respiratory lesions in guinea pigs. Am J Physiol Lung Cell Mol Physiol. 2005;289(1): L67-L74. 76. Wasil M, Halliwell B, Moorhouse CP. Scavenging of hypochlorous acid by tetracycline, rifampicin and some other antibiotics: a possible antioxidant action of rifampicin and tetracycline? Biochem Pharmacol. 1988;37(4):775-778. 77. Kuwano K. Involvement of epithelial cell apoptosis in interstitial lung diseases. Intern Med. 2008;47(5):345-353. 78. Matute-Bello G, Liles WC, Steinberg KP, et al. Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J Immunol. 1999;163(4):2217-2225. 79. Albertine KH, Soulier MF, Wang Z, et al. Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome. Am J Pathol. 2002;161(5):1783-1796. 80. Golan-Gerstl R, Wallach-Dayan SB, Amir G, Breuer R. Epithelial cell apoptosis by fas ligand-positive myofibroblasts in lung fibrosis. Am J Respir Cell Mol Biol. 2007;36(3):270-275. 81. Kuwano K, Hagimoto N, Kawasaki M, et al. Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis. J Clin Invest. 1999;104(1):13-19. 82. Kitamura Y, Hashimoto S, Mizuta N, et al. Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung
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injury in mice. Am J Respir Crit Care Med. 2001;163(3 pt 1): 762-769. Perl M, Chung CS, Perl U, et al. Fas-induced pulmonary apoptosis and inflammation during indirect acute lung injury. Am J Respir Crit Care Med. 2007;176(6):591-601. Rosenthal DS, Simbulan-Rosenthal CM, Iyer S, et al. Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2þ-calmodulin and caspase-dependent pathway. J Invest Dermatol. 1998;111(1):64-71. Rosenthal DS, Velena A, Chou FP, et al. Expression of dominantnegative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard. J Biol Chem. 2003;278(10):8531-8540. Simbulan-Rosenthal CM, Ray R, Benton B, et al. Calmodulin mediates sulfur mustard toxicity in human keratinocytes. Toxicology. 2006;227(1-2):21-35. Ray R, Keyser B, Andres D, et al. Human bronchial/tracheal epithelial cells (BEC) are more sensitive than small airway epithelial cells (SAEC) to sulfur mustard-induced apoptosis apparently due to a Fas (death receptor) response amplification loop. FASEB J. 2008;22(1_MeetingAbstracts):648.646. Anderson DR, Yourick JJ, Moeller RB, Petrali JP, Young GD, Byers SL. Pathologic changes in rat lungs following acute sulfur mustard inhalation. Inhal Toxicol. 1996;8(3):285-297. Lane BP, Europa DL. Differential staining of ultrathin sections of Epon-embedded tissues for light microscopy. J Histochem Cytochem. Sep-Oct 1965;13(7):579-582. Hummler E, Barker P, Gatzy J, et al. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet. 1996;12(3):325-328. Dresbach M. A modification of Mayer’s mucicarmine stain for mucin. Arch Pathol. 1947;44(4):391.
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Mustard Gas Inhalation Injury: Therapeutic Strategy Brian M. Keyser, Devon K. Andres, Wesley W. Holmes, Danielle Paradiso, Ashley Appell, Valerie A. Letukas, Betty Benton, Offie E. Clark, Xiugong Gao, Prabhati Ray, Dana R. Anderson and Radharaman Ray International Journal of Toxicology published online 6 May 2014 DOI: 10.1177/1091581814532959 The online version of this article can be found at: http://ijt.sagepub.com/content/early/2014/04/28/1091581814532959
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Article International Journal of Toxicology 1-11 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581814532959 ijt.sagepub.com
Mustard Gas Inhalation Injury: Therapeutic Strategy Brian M. Keyser1, Devon K. Andres1, Wesley W. Holmes2, Danielle Paradiso2, Ashley Appell1, Valerie A. Letukas1, Betty Benton1, Offie E. Clark1, Xiugong Gao3, Prabhati Ray3, Dana R. Anderson2, and Radharaman Ray1
Abstract Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds. Keywords sulfur mustard, inhalation, apoptosis, antioxidants, anti-inflammatory, protease
Sulfur mustard (SM), a blistering chemical warfare agent, was used first in World War I (WWI) and as recently as the 1980s in the Iran–Iraq war. It is easy to make, store, and deploy this chemical agent additionally, SM has been the most widely used chemical weapon during WWI.1 Because of these reasons, a recent concern is that SM could also be used as a terrorism agent. Human casualties during past wars (100 000þ in the IranIraq war alone) have shown that whole-body SM exposure causes debilitating skin blisters, respiratory distress, and ocular damage2; these effects are attributed to SM-induced epithelial damage, mainly basal cell apoptosis.1,3 Most mortality following SM exposure, however, has been attributed to respiratory tract lesions and pulmonary damage.4 Common postexposure complications in surviving victims are the development of a variety of chronic forms of pulmonary disease, including laryngitis, tracheobronchitis, bronchiolitis, bronchopneumonia, an unique form of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, large airway narrowing, pulmonary fibrosis, chronic bronchitis, bronchiolitis obliterans, and acute respiratory distress syndrome (ARDS).5-13 The multiplicity of the different cellular components (DNA, proteins, thiol compounds, etc) and the pathways affected by SM have complicated the efforts to establish a clearly defined mechanism-based therapeutic approach for SM exposure. As schematically indicated in Figure 1, some of the toxic effects of
SM and the half mustard (2-chloroethyl sulfide, CEES) include oxidative stress,14-20 proinflammatory pathways,21,22 apoptosis,19,23-25 and DNA damage.26,27 In addition to these toxic effects, low-level exposure to SM causes weight loss and sensory irritation.28 Associated with these different toxic effects are the cellular pathways that are affected, for example, intracellular free Ca2þconcentration increase due to either reduced glutathione (GSH) depletion or oxidative stress; phospholipase-mediated membrane effects and inflammation; and activation of caspases and apoptosis. Moreover, there could be cross-talks between these pathways. As such, these events provide some potential targets for intervention against SM injury. The majority of efforts to discover a therapeutic for 1
Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA 2 Analytical Toxicology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA 3 Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD, USA Corresponding Author: Radharaman Ray, Cellular and Molecular Biology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD 21010-5400, USA. Email: [email protected]
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Figure 1. Sulfur mustard toxicity. Points of intervention discussed in this article are highlighted in red.
SM exposure have historically explored one or more of the 4 different intervention points shown in Figure 1 (red bubbles). Therefore, the 4 categories of potential medical countermeasures are (1) anti-inflammatory compounds, (2) antioxidants, (3) protease inhibitors, and (4) antiapoptotic drugs or measures. This review summarizes the progress in each category, particularly the in vivo results.
Anti-inflammatory Drug Sulfur mustard acts as an electrophile that alkylates cellular and extracellular components of living tissue. As a result, some complex cellular events are initiated including cell cycle arrest, synthesis and release of inflammatory mediators, and cytotoxicity. Following these cellular effects, there are tissue responses such as inflammation and tissue damage.29 The cause of the acute injury appears to be the premature, sudden, and massive release of destructive enzymes and mediators of inflammation such as proinflammatory cytokines, which include interleukin (IL) 1b, IL-6, IL-8, and tumor necrosis factor a (TNF-a), and inducible nitric oxide synthase (iNOS).30,31 Based on what has been described earlier, various antiinflammatory or related drugs (eg, doxycycline and macrolide antibiotics) have been tested both in vitro and in vivo for their benefits in preventing the injuries caused by SM. Macrolides are a group of antibiotics that were initially studied and used
because of their antibacterial properties. The name ‘‘macrolide’’ is derived from their structure, which is characterized by a macrocyclic lactone ring with various amino sugars attached. In the recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases through mechanisms distinct from their antibacterial activity.32 Although the mechanisms underlying this effect are still unclear, macrolides have been shown to affect several pathways of the inflammatory process, including the migration of neutrophils, the oxidative burst in phagocytes, and the production of proinflammatory cytokines.33 The use of macrolide antibiotics has been previously shown to reduce SM-induced expression of iNOS and proinflammatory cytokines in monocytes and human airway epithelial cells (AECs) in vitro.34-36 In experiments using SM inhalation in rats, macrolides were able to cause a modest improvement in tracheal pathology scores at 3 days but not at 1 or 7 days after SM exposure.37 These results were only from a pilot study; a more detailed study would probably provide more insight into the possible benefits of macrolides in SM inhalation injury. However, the dose of antibiotic used was twice the normal dose used in clinical settings, increasing the chances of possible toxic side effects of this treatment. Another class of anti-inflammatory drugs that has been examined is glucocorticoid steroids. Once glucocorticoid
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steroids bind to their receptors (glucocorticoid receptor [GR]) on the cell membrane, the activated GR translocates to the nucleus and associates with DNA at specific binding domains. Activated GR binding to DNA causes the repression of many proinflammatory gene products, for example, cytokines, chemokines, adhesion molecules, and inflammatory enzymes.38 The 2 glucocorticoid steroids that have been used as SM antidotes in vivo are betamethasone and dexamethasone and both of these compounds are nonselective anti-inflammatory drugs. The administration of betamethasone for 7 days following SM inhalation in guinea pigs abolished the airway muscle hyperresponsiveness and partially restored tracheal epithelium neutral endopeptidase (a signaling peptide involved in inflammation and pain) activity.39,40 However, betamethasone was not able to completely reduce the increased tracheal epithelium height and cell density resulting from SM exposure.39 A single injection of dexamethasone, when administered to mice 1 hour after exposure to nitrogen mustard (NM, which is structurally similar to SM and produces similar injuries), was able to reduce inflammation and release cytokines, IL-1, and IL-6 in bronchoalveolar lavage fluid (BALF) that was examined at 6 and 18 hours postexposure.41 This effect of acute dexamethasone treatment also reduced the long-term lymphocytic response by decreased collagen deposition in the lungs when examined 2 to 4 weeks after NM exposure. 41 Other studies have shown that dexamethasone administered to guinea pigs was able to reduce the number of eosinophils and improve tracheal responsiveness when measured 14 days after SM exposure; however, the levels of IL-4 were higher than in SM-only-exposed controls.42 Two studies performed in Iran–Iraq war casualties have shown that both a short-term high dose and a long-term low dose of prednisolone (a commonly prescribed corticosteroid) were able to significantly (P < 0.05) improve lung function.43,44 However, some of the patients in these studies did not respond to this therapy. Additionally, there are many possible side effects with long-term administration of a corticosteroid such as weight gain, osteoporosis, lowered immunity, myopathy, avascular necrosis, changes in mood, memory deficit, psychosis, hyperglycemia, peptic ulceration, gastritis, and gastrointestinal hemorrhage (for a complete review see 45). These serious adverse effects are cause for concern for using glucocorticoids in the treatment of people who have been exposed to SM. Other compounds with anti-inflammatory properties in addition to their primary mechanism of action have been examined in SM- or CEES-induced lung injury. These compounds include salbutamol,46 tissue plasminogen activator,47 and the dual acting antioxidant/anti-inflammatory AEOL 10150. All of these compounds have shown improvement in SM- and CEESinduced lung injury.48-51 These results indicate not only the complex toxic mechanisms of action of these vesicating agents but also the need for future research in this area in order to identify more selective anti-inflammatory agents as well as possible combinations of pharmacologic antagonists targeting other affected pathways.
Antioxidant Drug Exposure to SM can also cause oxidative stress due to the depletion of cellular antioxidants, mainly GSH, a tripeptide and rapidly oxidizes to oxidized glutathione, which may lead to the production of reactive oxygen species (ROS) and membrane lipid peroxidation. 52 This may result in an imbalance between the production of ROS and the level of antioxidants in the lung and respiratory tract. This imbalance is believed to be the primary event triggering the inflammatory cascade and tissue injury.53 The specific oxidants that are involved in SM-induced cytotoxicity are still not known. A growing body of evidence is pointing toward reactive nitrogen species being one of the main oxidant classes in SM-induced lung injury.53 Nitric oxide, produced by iNOS, reacts with superoxide anion, forming peroxynitrite. Nitric oxide and peroxynitrite modify cellular proteins and DNA and, thereby, induce cytotoxicity and inflammation. Expression of iNOS is upregulated in lung macrophages and epithelial cells following exposure to SM.31 Use of ebselen (2-phenyl-1, 2-benzisoselenazol-3(2H)-one), a peroxynitrite scavenger, and 3 analogs have been shown to reduce CEES toxicity in vitro.54 Also, an NM exposure model has been shown to reduce histopathology but not NM-induced elevation in lung iNOS levels.55 Melatonin, a scavenger of both reactive nitrogen and oxygen species, when given to NM-exposed rats, was able to reduce NM-induced increase in iNOS, glutathione peroxidase activity, and reduced inflammation in the lung.56 Transgenic mice lacking a functional iNOS were found to have reduced expression of proinflammatory genes and markers of oxidative stress when exposed to CEES.57 Use of an iNOS inhibitor, silibinin,58 in vitro has shown protection against SM; however, further investigations are needed to further establish treatment conditions.59 The use of other antioxidants to treat SM-induced inhalation toxicity showed some limited success. Two antioxidants, Trolox (a water soluble analog of vitamin E), vitamin E, and quercetin (bioflavonoid) have been shown to enhance survival time and offer protective effects from oxidative damage in animals exposed to SM via inhalation or percutaneously.60-62 These antioxidants have been shown to reduce oxidative markers after SM exposure as well as increased survival. In one of these studies, the GSH used was not found to have the same protective effect as Trolox or quercetin.60 One reason for this result could be that GSH is not cell permeable and is easily degraded by peptidases in vivo. Another antioxidant, N-acetyl cysteine (NAC), which is a precursor for glutathione synthesis, has been explored as a potential therapeutic for SM-induced lung injury.63 When it was administered intraperitoneally in the beginning of SM exposure, it was shown to reduce the levels of lactate dehydrogenase, albumin, total protein, and the number of neutrophils in BALF fluid 24 hours postexposure.64 In our recent studies using the same rat exposure model, NAC, given as a postexposure treatment via inhalation (nebulized and given every 2 hours for 12 hours postexposure), was effective in reducing the pulmonary flow obstruction (expiratory flow at 50% [EF50]) and improving the oxygen saturation in SM inhaled rats at 24 hours after SM exposure
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Figure 2. Oxygen saturation and mid-expiratory flow at 50% (EF50) measures at 24 hours postinhalation of sulfur mustard (SM) vapor. Sulfur mustard caused a decrease in O2 saturation and an increase in EF50, a measure of airway obstruction, in comparison to control animals. Treatment with N-acetyl cysteine (NAC), administered immediately following SM exposure then every 2 hours of 12 hours postexposure, improved tissue oxygenation and airway flow in comparison to SM exposed animals. *P < 0.05 versus control; þ P < 0.05 versus SM. Data were analyzed using 1-way analysis of variance (ANOVA) with Tukey post hoc test.
(Figure 2). The efficacy of NAC as a postexposure therapeutic for SM inhalation injury was also demonstrated in a large animal model such as the swine where it showed improvements in arterial blood oxygenation, arterial blood bicarbonate, and shunt fraction.65 In these experiments, NAC was given via inhalation and was given 0.5, 2, 4, 6, 8, and 10 hours postexposure. N-Acetyl cysteine was also tested by administering orally, intravenously, and intratracheally (encapsulated in liposomes) after CEES exposure.66-68 In these studies, NAC was able to protect either as a pretreatment (3 days prior to CEES, oral) or as a therapeutic (90 minutes after CEES, intratracheal) by reducing *70% the CEES-induced lung injury. N-Acetyl cysteine has been reported to reduce the infiltration of inflammatory cells into the lung via the downregulation of the transcription factor activator protein 1,69 but the exact mechanism of protection due to NAC is not known. These observations and others have suggested that antioxidants could serve as prospective therapeutics for pulmonary damage due to SM.70
Protease Inhibitor Drug Both serine proteases and matrix metalloproteinases (MMPs) have been implicated in SM effects, particularly its vesication mechanism. Matrix metalloproteinases are responsible for the degradation of extracellular matrix proteins, contributing to inflammatory cell recruitment, tissue injury, and fibrosis.71 Matrix metalloproteinases have been shown to be upregulated as early as 6 hours postexposure to SM.31 This upregulation is dose dependent and persists for 24 hours after SM exposure.72 Following SM exposure, MMP activity is found in BALF and at
sites of decreased alveolar epithelial integrity and increased BAL albumin content (an indicator of airway damage).31 It is possible that MMP-9 plays a role in SM-induced detachment of epithelium and epithelial cell sloughing.31 When aprotinin (a serine-protease inhibitor) or ilomastat (a nonselective MMP inhibitor) was administrated just prior to SM, a marked improvement in pulmonary function (PF) was observed when measured 24 hours following SM exposure in rats.73 Aprotinin reduced SM-induced increases in IL-1a, IL-13, total BAL protein, and lung histopathology including inflammation. The use of epigallocatechin gallate, MMP antanogist, and antioxidant was shown not be highly effective as a prophylactic against SM.74 Doxycycline, a tetracycline derivative, has been shown to be a nonselective MMP inhibitor. Using doxycycline as a pretreatment (3 hours prior to SM), the results were similar to those obtained using aprotinin and ilomastat. In these experiments, doxycycline was able to inhibit MMP-2 and MMP-9, which decreased lung inflammation and injury.75 However, in addition to inhibiting MMPs, doxycycline has been shown to inhibit iNOS and consequently nitric oxide production.76 Hence, the beneficial effects of all of these drugs (aprotinin, ilomastat, and doxycycline) could be due to the nonselective effects and not wholly the MMP inhibition. Taken together, protease inhibitors also seem to be promising candidates as therapeutics for SM injury.
Apoptosis Inhibitors Apoptosis can proceed by 2 different pathways (Figure 3), referred to as the intrinsic (mitochondrial) and the extrinsic (death receptor) pathways. The intrinsic pathway is activated via the release of proapoptotic molecules from the mitochondria into the cytosol, which leads to the activation of caspase-9 and ultimately caspase-3. The extrinsic pathway is induced by cell surface death receptors (eg, Fas). Stimulation of these receptors facilitates recruitment of the adaptor Fas-associated protein with death domain (FADD) and tumor necrosis factor receptor superfamily member 1A-associated via death domain (TRADD), and these, in turn, associate with procaspase-8 and procaspase-10 (initiator caspases). The assembly of FADD/TRADD and procaspase-8/10 is called the death-inducing signaling complex (DISC). The DISC promotes self-processing and activation of the initiator procaspases to active caspase-8 and caspase-10 and their subsequent dissociation from the protein complex. Active initiator caspase-8 and/or caspase-10 directly activates downstream caspases (caspase-3 and caspase-7) and causes cell death. Apoptosis plays an important role in lung pathologies after injury, such as SM exposure, in 2 different ways. First, failure to clear unwanted cells by apoptosis will prolong the inflammation because the release of their toxic elements delays the repair processes77; second, excessive apoptosis may cause diseases (eg, adult respiratory distress syndrome [ARDS] and pulmonary fibrosis).78 The survival and recovery of epithelial and endothelial cells and the resolution of inflammatory cells appear to be the keys in normal repair.77 Matute-Bello et al in 1999 demonstrated that lung injury and the sequential inflammation indicated a dual role for the Fas/FasL system as a proapoptotic/
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Figure 3. Apoptosis pathways. Adapted from Keyser et al.24
proinflammatory system. It appears that controlling the degree of apoptosis after an injury to the lung (ie, SM exposure) has a direct correlation with the patient’s prognosis.77 Upregulation of the Fas/FasL pathway in epithelial cells has been shown to contribute to the severe epithelial damage that occurs in ARDS, one of the postexposure complications of SM. 79 Recently, FasL molecules were reported to be expressed in a-smooth muscle actin positive cells in mice with bleomycin-induced pulmonary fibrosis and in humans with idiopathic pulmonary fibrosis (IPF).80 The BALF from patients with ARDS or IPF could induce apoptosis in cultured small AECs through the Fas pathway.78,80 Further evidence of the Fas pathway in pulmonary fibrosis is that neutralization of FasL antibody by Fas–Ig fusion protein or neutralizing antiFasL antibody was able to prevent the development of bleomycin-induced pulmonary fibrosis in vivo.81 Blocking the Fas/FasL system reduced the development of endotoxininduced acute lung injury (ALI).82 Fas and FasL mutant animals exhibit less pulmonary epithelial cell apoptosis in ALI and have higher survival rates.78,83 Based on these facts, the Fas pathway may play a critical and important role in the development of the lung diseases that are observed after exposure to SM. After SM exposure, apoptosis may occur via both the intrinsic mitochondrial pathway and the extrinsic death receptor (Fas) pathway.84,85 It has been reported that blocking the Ca2þ, calmodulin-mediated mitochondrial pathway or the Fasmediated pathway antagonizes SM-induced apoptosis in cultured primary human epidermal keratinocytes.85,86 In in vivo, it
has been demonstrated that blocking the SM-induced Fas response by overexpression of dominant-negative FADD (FADD-DN) in human skin grafted onto nude mice blocks apoptosis in the basal epidermis and, therefore, microvesication induced by vapor cup SM exposure.85 In cultured human AECs, SM induces apoptosis predominantly via a caspase-8 or Fas-mediated pathway.3 This SM-induced and Fas-mediated apoptosis in AEC is prevented by (1) a Fas antagonistic antibody (ZB4; B. M. Keyser, unpublished results), (2) through the suppression of the Fas/FADD response by small interfering RNAs (B. M. Keyser, unpublished results), or (3) simply by the general caspase inhibitor Z-VAD-FMK.87 A recent report has also demonstrated that the use of RNA interference against the Fas receptor, applied up to 8 hours postexposure in vitro, was able to significantly reduce apoptosis 24 hours after SM exposure.24 This use of a highly selective antagonist of the Fas-mediated pathway of apoptosis added further evidence for an important role of this pathway in SMinduced apoptosis. Other proteins in the extrinsic apoptosis pathways (eg, cFLIP, DR-4, and DR-5) could play a role in SM-induced apoptosis. To date, there have been no studies involving these proteins in the lung. We have shown previously that dominant-negative FADD skin did not show any evidence of microblistering whereas this was not seen with TNF-a antagonism.85 If this is the case in the airway remains to be seen. Hence, further development of an effective pharmacological antagonism or a molecular suppression of the Fas-mediated pathway of apoptosis may provide a highly prospective therapeutic approach for SM inhalation injury.
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Figure 4. Airway cast formation at 24 hours after intratracheal sulfur mustard (SM) inhalation in rats. A, In general, a cast or plug is composed of a sparsely to moderately cellular eosinophilic homogenous material surrounding a less dense central core, hematoxylin and eosin. B, When present, fibrin stains dark blue (arrows), affects up to 15% of the section only, and is observed only at the periphery of the cast, phosphotungstic acid hematoxylin (PTAH). C, Bright red fibrin staining (arrows) mirrors the fibrin staining observed with PTAH; light green mucinous ground substance (*) is seen mostly in the less dense central areas; the yellowish-material is suggestive of collagen and/or reticular fibers, pentachrome. D, Light pink mucin (arrows) is observed with mucicarmine in the less dense central areas, mucicarmine. All images taken at 2 magnification.
Sulfur Mustard Inhalation Exposure and Drug Administration Inhalation exposure to SM vapor was performed via tracheal intubation in rats as described previously.88 Briefly, male Sprague Dawley rats (*200 g, 8 per group) were anesthetized with a combination of ketamine (80 mg/kg, intraperitoneally [ip]) and xylazine (10 mg/kg, ip). After induction of anesthesia, a diagnostic otoscope was used to facilitate tracheal intubation. Sulfur mustard (diluted in absolute ethanol at 1.0 mg/kg) was placed in a heated (37 C) water-jacketed glass vapor generator, and the rats were connected to this device by an endotracheal tube and exposed to SM vapor or ethanol (vehicle control) for 50 minutes. This exposure model has been shown to produce a consistent pathology at 24 hours after exposure.88 At the conclusion of the exposure (SM or ethanol), rats were placed on a heated water blanket (37 C) in a cage until they recovered from
anesthesia. N-Acetyl cysteine (0.35 mg/mL) was administered via nose-only nebulization using an Aeroneb (Aerogen Inc., Marietta, Georgia) nebulizer system. Immediately following exposure (SM or ethanol) and then every 2 hours of 12 hours postexposure, conscious rats were placed into nose-only restraint tubes (Buxco Research Systems, Wilmington, North Carolina) and positioned directly in the effluent of the nebulizer. Air flow to the nebulizer was metered through a mass flow controller (Brooks Instrument, Hatfield, Pennsylvania) at a rate of 0.5 LPM. N-Acetyl cysteine of 1 mL was placed in the Aeroneb and was nebulized at a rate of 0.3 mL/min with a particle size of 4.0 to 6.0 mm.
Lung Cast Staining Lung casts were fixed with 4% neutral-buffered paraformaldehyde, trimmed, paraffin embedded, and cut on a microtome
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Figure 5. Airway cast formation at 24 hours after intratracheal sulfur mustard (SM) inhalation in rats. A. In general, a cast or plug is composed of a sparsely to moderately cellular eosinophilic homogenous material surrounding a less dense central core, hematoxylin and eosin. B, When present, fibrin stains dark blue (arrows), affects up to 15% of the section only, and is observed only at the periphery of the cast, phosphotungstic acid hematoxylin. C, Bright red fibrin staining (arrows) mirrors the fibrin staining observed with PTAH; light green mucinous ground substance (*) is seen mostly in the less dense central areas; the yellowish material is suggestive of collagen and/or reticular fibers, pentachrome. D, Light pink mucin (arrows) is observed with mucicarmine in the less dense central areas, mucicarmine. Note that all images shown here are the same casts as in Figure 4, but at a magnification of 20 to reveal additional details.
into 5-mm thick sections. Slides of the lungs were stained with hematoxylin and eosin (H&E) for routine histopathology. Phosphotungstic acid hematoxylin staining was done as reported previously.89 Pentachrome staining was performed following.90 Mucicarmine staining protocol was in accordance as reported previously.91
Whole Body Plethysmography A Buxco unrestrained whole-body plethysmography (WBP) system with Biosystems XA software (Buxco Research Systems) was used to monitor PF parameters in conscious rats. This included expiratory flow, from which the EF50 value could be derived. Prior to SM or ethanol exposure, rats were placed in the WBP chambers for a total of 2 hours (1 hour for acclimation and 1 hour for data collections) to establish a baseline for PF parameters. At the conclusion of each exposure (SM or ethanol), the rats were placed in their original cages to recover from anesthesia. Twenty-three hours postexposure, the rats were placed in the WBP chambers again and allowed to be acclimated for 1 hour; PF data were recorded through the 24-hour
time point. Data were averaged by group for each 1-hour data collection period.
Blood Oxygen Saturation (PO2) Analysis At 24 hours after SM exposure, rats were deeply anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). A 0.5 mL blood sample was then obtained from the descending aorta. The blood sample was quickly injected into the TruPoint CC (IRMA, Edison, NJ) cartridge and analyzed per manufacturer’s instructions.
Data Analysis Data are shown as mean + standard deviation. Group comparisons were conducted using 1-way analysis of variance followed by Tukey post hoc multiple comparison test. Significant results were identified when P < .05 or smaller.
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Concluding Remarks Sulfur mustard exposure of the respiratory system causes immediate and long-term health effects. In severe exposure, it is the inhalation injury that has been known to result in most fatalities compared to the skin or ocular injury. Sulfur mustard inhalation effects include chronic forms of pulmonary diseases, including laryngitis, tracheobronchitis, bronchiolitis, bronchopneumonia, COPD, bronchiectasis, asthma, large airway narrowing, pulmonary fibrosis, chronic bronchitis, and ARDS.5-10 Thus, it is essential to discover potential therapeutic compounds to combat these effects. As has been explained earlier in this article, the major target for the tissue or organ damage due to SM is the epithelial layer. This is particularly important for the injury to the upper airway where the epithelial cells die and detach from the substratum. The mechanism of this epithelial detachment, although not fully established, might involve protease stimulation that could degrade the proteins responsible for the epithelial cell attachment to the substratum. The detached cells contribute to cast formation along with inflammatory cells, fibrin, and mucus which is released from goblet cells. In experimental animals such as the rat SM inhalation model,88 cast formation has been found to be responsible for acute mortality due to the airway obstructive effect following the inhalation exposure (Figures 4 and 5). Based on their mechanisms of action, all of the proposed therapeutic approaches, that is, using (1) antioxidants, (2) protease inhibitors, (3) antiapoptotic therapeutics, and (4) antiinflammatory compounds should be able to counteract this cast formation; moreover, measures to degrade such casts once those are formed might also be helpful at least to reduce the acute mortality. From what has just been described, the major factors attributable to the cellular and/or tissue toxicity due to SM appear to be (1) genotoxicity and cytotoxicity, (2) protease stimulation, (3) cell death, and (4) inflammation. Based on the earlier discussions, it is apparent that all of the 4 different categories of drugs, that is, antioxidants, protease inhibitors, apoptosis inhibitors, and anti-inflammatory compounds are potential medical countermeasures against SM toxicity. All of these different types of drugs when tested singly have been found to have some benefit against SM inhalation injury, but whether their combinations and which combinations would be more beneficial remain to be studied. One important consideration is the temporal sequence of events following SM exposure, which is lacking. Under such circumstances, a reasonable approach to treat SM airway injury seems to be preventing the cytotoxicity (cell death) and at the same time minimizing the other associated toxic events, that is, inflammation, protease stimulation, and oxidative damage. Acknowledgments This work was supported by the Defense Threat Reduction Agency— Joint Science and Technology Office, Medical S&T Division [Grant CBM.RESP.01.10.RC.015]. Additionally, this research was supported
in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC. We acknowledge LTC Derron A. Alves for his expertise in the lung cast staining.
Authors’ Note The views expressed in this article are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the US Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89-544), as amended.
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