Long-term Respiratory Effects of Mustard Vesicants

Abstract

Sulfur mustard and related vesicants are cytotoxic alkylating agents that cause severe damage to the respiratory tract. Injury is progressive leading, over time, to asthma, bronchitis, bronchiectasis, airway stenosis, and pulmonary fibrosis. As there are no specific therapeutics available for victims of mustard gas poisoning, current clinical treatments mostly provide only symptomatic relief. In this article, the long-term effects of mustards on the respiratory tract are described in humans and experimental animal models in an effort to define cellular and molecular mechanisms contributing to lung injury and disease pathogenesis. A better understanding of mechanisms underlying pulmonary toxicity induced by mustards may help in identifying potential targets for the development of effective clinical therapeutics aimed at mitigating their adverse effects.

1. Introduction

Mustard vesicants (e.g., sulfur mustard [SM] and nitrogen mustard [NM]), are cytotoxic blistering agents that cause incapacitating injury to the skin, the eyes, and the respiratory tract, as well as the gastrointestinal and nervous systems (Somani and Babu, 1989; Smith et al., 1995). Many incidents of intentional, accidental and occupational exposure to SM have been reported (Dacre and Goldman, 1996). SM continues to be a viable threat agent owing to the ease and low cost of its manufacture and remaining stockpiles (Kristinsson and Johannesson, 2009). In this regard, it has reportedly been used in the Middle East in 2017 and 2018 (Kilic et al., 2018; Kumar et al., 2018; Malaviya et al., 2018b). Although only SM has been used in chemical warfare, injuries induced by NM are comparable to SM in human and rodents (Weinberger et al., 2016; Wang and Xia, 2007). As a consequence, NM has also been classified as a high priority chemical threat agent. The severity of injury induced by mustards varies with the route of exposure, proximity to the source, environmental conditions, and use of protective gear (Graham and Schoneboom, 2013). The lipophilic nature of SM results in rapid penetration of tissues and cells, and reactions with many moieties including sulfides, sulfhydryl, carboxyl, and amino groups, heterocyclic nitrogen atoms, and organic and inorganic phosphates present in lipids, proteins, and/or nucleic acids (Giuliani et al., 1994; Smith et al., 1998).

Pulmonary toxicity of mustard is the most critical and is responsible for most morbidity and mortality in exposed victims (Balali-Mood and Hefazi, 2005; Wang and Xia, 2007; Weinberger et al., 2011; Razavi et al., 2013; Keyser et al., 2014). Both conducting and respiratory airways are affected by mustards (Ghabili et al., 2010; Graham and Schoneboom, 2013). Early symptoms include cough, sneezing, hoarseness, sore throat, mucus discharge, loss of smell and taste, and irritation of the nasal mucosa (Kehe et al., 2008, 2009; Graham and Schoneboom, 2013; Sezigen et al., 2019). Pulmonary edema and damage to the pharynx induced by acute mustard inhalation results in an inability to speak, moist rales, tachypnea and tachycardia (Sohrabpour, 1984; Kehe and Szinicz, 2005). Necrosis of the respiratory epithelium causes epithelial sloughing, pseudo membrane formation, and lung lobe collapse (Wang and Xia, 2007; Kehe et al., 2008; Ghabili et al., 2010; Rancourt et al., 2013). A characteristic feature of mustard lung toxicity is an accumulation of large numbers of inflammatory cells at sites of injury, which produce an array of bioactive mediators including reactive oxygen and nitrogen species, cytokines, chemokines, proteases and growth factors. These mediators contribute to oxidative stress, protein degradation, DNA damage, cytotoxicity and aberrant cell proliferation (Anderson et al., 2009; Wigenstam et al., 2009; Malaviya et al., 2010, 2012; Tang and Loke, 2012; Sawale et al., 2013; Venosa et al., 2016; Weinberger et al., 2016). The resolution of acute injury is critical to restoration of homeostasis and normal lung functioning. However, following mustard exposure, there is a loss of effective antiinflammatory and wound healing capabilities resulting in persistence of acute injury and the development of chronic pathologies and diseases (Laskin et al., 2019). In this article, we describe the long-term consequences of acute mustard exposure in humans and experimental animals, and current treatment approaches.

2. Long-term effects of mustard exposure in humans

Chronic clinical and pathological manifestations of mustard exposure have been observed in survivors of chemical attacks and in manufacturing facility workers. The relative risk of pulmonary complications from mustard exposure increases with the age of the victim at the time of exposure, and with advancing time after the exposure (Zarchi et al., 2004). Thus, when compared to young victims (≤ 25 years), individuals over the age of 36 years are two times more susceptible to SM-induced lung toxicity. The annual incidence of recorded pulmonary complications also increases rapidly several years after mustard exposure, relative to those recorded one-year post exposure. The most common symptoms in long-term survivors of mustard gas exposure are chronic cough, dyspnea, increases in sputum and hemoptysis (airway bleeding) (Bijani and Moghadamnia, 2002; Ghanei et al., 2004b). High-resolution CT, bronchoscopy, bronchoalveolar lavage and lung biopsies of victims 15 - 20 years following SM exposure showed progressive airway deterioration, hyperreactivity and stenosis of the conducting airways, which were associated with a decline in pulmonary function, (Rowell et al., 2009; Weinberger et al., 2011; Graham and Schoneboom, 2013). Twenty-five years after mustard exposure, pathologies included dysphonia (79%), chronic sinusitis (55%), post-nasal discharge (42%), impaired vocal cord activity (26%), mucosal inflammation of the larynx (15%), wheezing (45%), ronchi (45%) and crackle (24%) (Balali-Mood et al., 2011). Lung function analyses in victims showed evidence of obstructive (55%), restrictive (6.8%) and mixed (6.8%) lung disease. More recently, Darchini-Maragheh et al. (2018) reported mostly restrictive (42%) and mixed (12%) patterns of disease in victims exposed 25-30 years earlier, which was correlated with a greater degree of disability.

Common pathologies in victims of the Iran-Iraq war 10 years after exposure to a single high-dose of SM included asthma (11%), bronchitis (59%), bronchiectasis (9%), airway narrowing (10%), and pulmonary fibrosis (12%) (Emad and Rezaian, 1997; Weinberger et al., 2011). Twenty five years post exposure to SM, a higher incidence of COPD (84%) and bronchiectasis (44%) was observed indicating that the pathologies are progressive (Balali-Mood et al., 2011). Frequent respiratory tract infections and bronchopneumonia early after mustard exposure are thought to contribute to bronchiectasis (Balali-Mood et al., 2011). Air trapping (50%), pulmonary fibrosis (25%), and ground glass opacity (17%) were also evident in patients 25 years post-SM exposure (Darchini-Maragheh et al., 2018). Mustard gas exposure is also associated with relatively early onset of lung cancer in non-smokers (Hosseini-khalili et al., 2009). Emphysema (22%), bronchiectasis (19%), centrilobular nodules (13%), bronchial wall thickening (8%), reticular opacity (10%), ground glass opacity (7%), consolidation (5%), honeycombing (2%) and other respiratory pathologies have similarly been reported in survivors of mustard gas exposure in a manufacturing factory (Easton et al., 1988; Nishimura et al., 2016). A relatively higher incidence of mortality from lung cancer and respiratory diseases was also noted in mustard gas factory workers which was related to the duration of employment (Wada et al., 1968; Easton et al., 1988; Doi et al., 2011; Mukaida et al., 2017).

Analysis of lung tissue, bronchoalveolar lavage fluid (BAL) and serum from SM victims 27 years after exposure showed alterations in the airway epithelium, as well as increases in markers of oxidative stress and inflammation (Tahmasbpour et al., 2016; Weinberger et al., 2016; Layali et al., 2018). Histopathological changes in the airway included thickened epithelium and basement membrane, and structural alterations and/or damage of epithelial cells. Oxidative stress related genes including dual oxidases, aldehyde oxidase 1, thyroid peroxidase, myeloperoxidase and eosinophil peroxidase were also upregulated in the lung (Table 1) (Tahmasbpour Marzony et al., 2016b). Additionally, an accumulation of protein carbonyls and lipid peroxidation products such as malondialdehyde (MDA) have been observed, along with decreases in antioxidants like glutathione, glutathione reductase and total antioxidant capacity in lungs and/or blood from survivors 20 - 27 years post exposure (Shohrati et al., 2010; Tahmasbpour et al., 2016; Tahmasbpour Marzony et al., 2016a; Layali et al., 2018). Expression of metallothionein-3 and glutathione reductase was down regulated, while the thiol-specific antioxidants, peroxiredoxin and oxidoreductase, sulfiredoxin, superoxide dismutase (SOD)2 and SOD3 were upregulated, further supporting prolonged oxidative stress following mustard exposure (Tahmasbpour et al., 2016; Tahmasbpour Marzony et al., 2016a, 2016b).

Table 1.

Inflammatory Mediators Implicated in Long-term Pathologies Induced by Mustard Vesicant

Species	Increase	Decrease	References
Human
Serum	Selectin E, CCL2, soluble Fas ligand, MDA, CX3CL1, elastase	GSH, Selectin L, selectin P, TNFα, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8, CCL-5	Ghazanfari et al., 2009a, 2009b; Pourfarzam et al., 2009; Yaraee et al., 2009a, 2009b; Shohrati et al., 2010; Pourfarzam et al., 2013)
Sputum	TNFα, IL-1β, MDA	GSH, IL-1Ra, fibrinogen, CX3CL1	Pourfarzam et al., 2013; Yaraee et al., 2013; Nejad-Moghaddam et al., 2016)
BAL	MDA, IL1β, IL-5, IL-6, IL-8, IL-12, TNFα, EGF, IGF-1, TGFβ, CCl2, CCL3, CCL4, CCL5, CCL11	GSH, TAC	Aghanouri et al., 2004; Emad and Emad, 2007a, 2007b, 2007c; Tahmasbpour Marzony et al., 2016b; Layali et al., 2018
Lung	IL-17A, PTGS-2, SOD2, SOD3, GPXs, GSTs, TGFβ1, TGFβ3, GSS,DUOXs, CAT, AOX1, TPO, MPO, EPO, PRDXNs, SRXN1, OXSR1, FOXM1, SMAD3, SMAD4; APOE; HO-1, 12-LO	PTGS-1, iNOS, SOD-1, MT-3, GSR, SFTPD, TXNRD2, IL-10	Zarin et al., 2010; Adelipour et al., 2011; Imani et al., 2016b; Tahmasbpour et al., 2016; Tahmasbpour Marzony et al., 2016a, 2016b; Layali et al., 2018
Guinea pig
Serum	IL-4, IFNγ		Boskabady et al., 2011
BAL	LDH, GSH		Allon et al., 2009
Lung	MDA, hydroxyproline, IL-1β, IL-6, SOD1, C/EBPβ, ICAM-1		Mukhopadhyay et al., 2006; Mukherjee et al., 2009; Mukhopadhyay et al., 2010; Roy et al., 2017
Rat
Plasma	PAI		McGraw et al., 2018
BAL	TGFβ1, PDGF, PAI, CCSP		McGraw et al., 2017, 2018
Lung	PCNA, HO-1, SOD-2, TNFα, TGFβ, PDGF, iNOS, COX-2, Ym-1, Gal-3, CCR2, CX3CR1, collagen, CCSP, α-SMA, CTGF, PDGFRα, IL-17, iNOS, COX-2, IL-12α, MMP-9, IL-10, PTX-2, CTGF, APOE	NOTCH1, NOTCH3, HES1, CCR2, CCL2, CCR5, CCL5, CX3CR1, CX3CL1, CCSP	Malaviya et al., 2012; Mishra et al., 2012; Gustafsson et al., 2014; Malaviya et al., 2015b; Venosa et al., 2016; McGraw et al., 2017, 2018
Mouse
Plasma	IL-1α, IL-1β, IL-10, IFNγ, TNFα	IL-4, IL-10	Sawale et al., 2013
BAL	LDH, IL-23, MPO, MMP-2, MMP-9, β-glucuronidase	GSH	Ekstrand-Hammarstrom et al., 2011; Kannan et al., 2016
Lung	MDA, 8-OHdG, PCO, OSI, GSSG, collagen, MPO	CAT, SOD, GST, GPX, GSR, GSH	Wigenstam et al., 2009; Ekstrand-Hammarstrom et al., 2011; Wigenstam et al., 2012; Sawale et al., 2013; Kannan et al., 2016; Varmazyar et al., 2019

Abbreviations: 8-OHdG, 8-hydroxy-2'-deoxyguanosine; AOX, aldehyde oxidase; APOE, Apolipoprotein E; BAL, bronchoalveolar lavage; CAT, catalase, CCL, chemokine ligand; CCR, chemokine receptor; CCSP, clara cell secretory protein; C/EBP, CCAAT-enhancer binding protein; COX, cyclooxygenase; CTGF, connective tissue growth factor; CX3CL, CX3C chemokine ligand; CX3CR, CX3C chemokine receptor; DUOX, dual oxidase: EGF, epidermal growth factor; EPO, eosinophil peroxidase; FOXM, forkhead box M; Gal, galactin, GPXs, glutathione peroxidases; GSH, glutathione; GSR, glutathione reductase; GSS, glutathione synthetase; GSSG, glutathione disulfide; GSTs, glutathione-s-transferases; HES, a transcription suppressor; HO, heme oxygenase; ICAM, intracellular adhesion molecule; IFN, interferon: IGF, insulin growth factor; IL, interleukin; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LO, lipoxygenase; MDA, malondialdehyde; MMP, matrix metalloproteinase; MPO, myeloperoxidase; MT, metallothionein; NOTCH, a signaling receptor; OSI, oxidative stress index; OXSR, oxidative stress response gene; PAI, plasminogen activator inhibitor; PCNA, proliferating cell nuclear antigen; PCO, protein carbonyls; PDGF, platelet-derived growth factor; PRDXN, peroxiredoxin; PTGS, prostaglandin endoperoxide synthase; PTX, pentraxin; SFTPD, surfactant protein D, SMA, smooth muscle actin; Smad, transcription factor for TGFβ signaling; SOD, superoxide dismutase; SRXN, sulfiredoxin; TAC, total antioxidant capacity; TGF, transforming growth factor; TNF, tumor necrosis factor; TPO, thyroid peroxidase; TXNRD, thioredoxin reductase; Ym, chitinase-like3 protein.

There is also evidence of systemic and pulmonary inflammation in survivors including alterations in serum levels of adhesion molecules, growth factors, cytokines and chemokines (Amiri et al., 2009; Ghazanfari et al., 2009b; Pourfarzam et al., 2009; Yaraee et al., 2009a, 2009b). Thus, circulating levels of the macrophage chemokine, CCL2 and selectin E were elevated in mustard gas victims 20 years after exposure, whereas levels of L- and P-selectin, tumor necrosis factor (TNF)α, interleukin (IL)-1α, IL-1β, IL-1Ra, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, CCL5, and CX3CL1 were reduced, when compared to controls. In contrast, serum levels of IL-6 and C-reactive protein (markers of systemic inflammation) were increased in mustard victims with COPD, which correlated with disease severity (Attaran et al., 2009, 2010). Similarly, serum and/or BAL levels of the pro-apoptotic protein, soluble Fas ligand, were elevated in long-term survivors of SM poisoning, and this correlated with altered pulmonary function (Ghazanfari et al., 2009a; Pirzad et al., 2010). Increased levels of TNFα, IL-1β and MDA in sputum have also been described in mustard exposed individuals with moderate to severe pulmonary complications (Yaraee et al., 2013). Pulmonary fibrosis following SM exposure is associated with increased numbers of eosinophils, neutrophils and lymphocytes, and inflammatory cytokines and chemokines in the lung, including, IL-1α, IL-1β, IL-5, IL-6, IL-8, IL-12, IL-13, TNFα, transforming growth factor (TGF)β, insulin growth factor-1, epidermal growth factor, CCL2, CCL3, CCL4, CCL5, and CCL11 (Emad and Emad, 2007a, 2007b, 2007c); moreover, levels of IL-8 and TGFβ directly correlated with the degree of fibrosis. Upregulation of Smad3 and Smad4 transcription factors in the lung of mustard victims suggests TGFβ-mediated signaling and fibrosis (Adelipour et al., 2011). A higher Th17/Treg ratio and increased IL-17A levels in the blood and lung of exposed victims were associated with chronic Th17-mediated inflammatory response and fibrosis (Imani et al., 2016a; Iman et al., 2017; Panahi et al., 2018).

3. Long-term effects of mustard exposure in animals

The long-term effects of mustard exposure on the lung have been defined at the cellular and molecular levels in animal models using SM, NM, and the half mustard, 2-chloroethyl ethyl sulfide (CEES) (Table 2). As mechanisms underlying the toxicity of these three agents are similar, consideration of their pathophysiologic effects in animals is useful for target identification and drug development (Weinberger et al., 2016).

Table 2.

Long-Term Histopathological Alterations in the Respiratory Tract of Experimental Animals Following Mustard Exposure.

Species	Agent (Route)	Time post exposure	Response	References
Non-human primate	SM (INH; 150 mg/m3)	14 days	Neutrophilia, stratified squamous epithelium	Mishra et al., 2012
Guinea pig	CEES (IT; 0.3 mg/kg)	14 days	Tracheal epithelial ulceration and aberrant regeneration, peribronchial edema, atelectasis, airway hyperreactivity	Calvet et al., 1994
Guinea pig	SM (INH; 40 mg/m3 for 10 min)	14 days	Tracheal epithelium desquamation, atelectasis, congestion, inflammatory cell accumulation, hemorrhage, mucus in airways, emphysema	Gholamnezhad et al., 2016
Guinea pig	CEES (IT; 0.5 mg/kg)	21 days	Bronchial constriction, mucus, leukocyte accumulation in alveoli and bronchi	Das et al., 2003
Guinea pig	CEES (IT; 2 mg/kg)	30 days	Bronchial constriction, hypertrophy of goblet cells, luminal debris, neutrophilia, eosinophilia, intraalveolar hemorrhage, edema, alveolar accumulation of leukocytes, collapsed alveoli, fibrin and collagen deposits	Mukherjee et al., 2009; Roy et al., 2017
Guinea pig	SM (INH; 0.7 – 2.2 mg min/L for 10 min)	30 days	Aberrant regeneration and differentiation of tracheal epithelium, microvesication in basal layer, edema and blood congestion in lamina propria, leukocyte accumulation in distal airways, extracellular matrix deposits, lung remodeling	Allon et al., 2009
Rat	SM (INH; 150 mg/m3 for 10 min)	14 - 39 days	Proximal airway epithelium denudation, metaplasia, squamous epithelia, bronchiolitis obliterans, myofibroblast proliferation, interstitial fibrosis, abnormal collagen deposition	Mishra et al., 2012; McGraw et al., 2017, 2018
Rat	NM (IT; 0.125 – 1.2 mg/kg)	21 - 90 days	Leukocyte accumulation, perivascular and peribronchial edema, squamous metaplasia of bronchial wall, debris in airway lumen, bronchiolization of alveolar walls, fibroplasia, emphysema, fibrin and collagen deposition, collapsed alveolar structure	Malaviya et al., 2012; Gustafsson et al., 2014; Malaviya et al., 2015b
Mouse	CEES (PC; 1068 mg/kg)	7 - 14 days	Inflammatory cell accumulation, activated macrophages, oxidative stress, MPO, inflammatory cytokines	Sawale et al., 2013; Kannan et al., 2016
Mouse	CEES (10 mg/kg, IP)	7 – 180 days	Congestion/hyperemia, perivascular and peribronchial inflammation emphysema, atelectasis, parenchymal inflammation/fibrosis, smooth muscle hypertrophy, goblet cell hyperplasia and metaplasia	Varmazyar et al., 2019
Mouse	NM (IT; 0.08 - 1.0 mg/kg)	14 – 90 days	Increases in lung leukocytes, pulmonary fibrosis, collagen deposition	Wigenstam et al., 2009, 2012; Ekstrand-Hammarstrom et al., 2011; Sunil et al., 2018, 2019)

Abbreviations: BAL, bronchoalveolar lavage; CEES, 2-chloroethyl ethyl sulfide; INH, inhalation; IP, intraperitoneal; IT, intratracheal; MPO, myeloperoxidase; NM, nitrogen mustard; PC, percutaneous; SM, sulfur mustard.

Tracheal edema, mucosal necrosis, lung congestion, luminal accumulation of diphtheritic membranes, and edematous and hemorrhagic atelectasis have been observed in rabbits 4 - 6 days after mustard exposure (Dacre and Goldman, 1996). Neutrophilia and stratified squamous bronchial epithelium were observed in lungs of non-human primates 2 weeks after SM inhalation (Mishra et al., 2012), while guinea pigs demonstrate peribronchial edema, mucus in bronchi, emphysema and severe atelectasis (Gholamnezhad et al., 2016). The tracheal epithelium was ulcerated in guinea pigs, showing incomplete regeneration and/or abnormal differentiation 30 days after mustard exposure, whereas the mucosa continued to exhibit edema and inflammation (Calvet et al., 1994; Allon et al., 2009; Gholamnezhad et al., 2016). Airway hyperreactivity to substance P and histamine have also been noted in guinea pigs, consistent with the onset of asthma-like symptoms in SM exposed victims (Calvet et al., 1994; Emad and Rezaian, 1997; Hefazi et al., 2005). Peribronchial edema, parenchymal accumulation of inflammatory cells (eosinophils, neutrophils and monocytes), hemorrhage, fibrin exudation and emphysema developed and persisted up to 3 weeks post exposure (Das et al., 2003; Gholamnezhad et al., 2016). Thirty days after mustard exposure, increases in the transcription factor CCAAT enhancer binding protein (C/EBP)β, which is responsible for inducing intracellular adhesion molecule (ICAM)-1 and inflammatory cytokines including IL-1β and IL-6, were noted in the lung of guinea pigs (Table 1), along with septal and perivascular fibrin and collagen deposits (Roy et al., 2017). Oxidative stress, a primary event in mustard-induced lung injury, also persisted in the lung of guinea pigs for at least one month after exposure (Mukherjee et al., 2009; Mukhopadhyay et al., 2010).

Mustard inhalation is associated with proximal bronchiolar epithelial injury including denudation of the epithelium 3 weeks post exposure in rats (McGraw et al., 2017). Multifocal lesions (Table 2) characterized by perivascular and peribronchial edema, luminal accumulation of cellular debris, bronchiolization of alveolar walls, and multiple areas of fibrosis containing collagen fibers have also been described in rodent lung 3-4 weeks post exposure (Wigenstam et al., 2009; Malaviya et al., 2012; Gustafsson et al., 2014; Malaviya et al., 2015b; Varmazyar et al., 2019). In addition, fibroplasia, squamous metaplasia of the bronchial wall, and emphysema-like changes in alveolar tissue were evident. Fibrin and collagen deposition tended to increase progressively in the lung leading to a collapse of alveolar structures and the appearance of honeycombing (Malaviya et al., 2012; Mishra et al., 2012; McGraw et al., 2018). Progressive hypoxemia and respiratory distress gradually impair pulmonary function. The chronic phase of respiratory injury is also characterized by a predominance of enlarged foamy macrophages, lymphocytes, neutrophils, and IL-17⁺ cells in rodent lung, along with increases in TNFα, TGFβ, platelet derived growth factor (PDGF), PDGFRα, myofibroblast proliferation and collagen (Ekstrand-Hammarstrom et al., 2011; Mishra et al., 2012; Gustafsson et al., 2014; Malaviya et al., 2015b; Venosa et al., 2016; McGraw et al., 2018). Antiinflammatory/profibrotic macrophages expressing IL-10, pentraxin-2, Ym-1, galectin-3 and connective tissue growth factor are prominent in rat lung 28 d after mustard exposure, along with smaller numbers of proinflammatory/cytotoxic macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, TNFα, IL-12α, and matrix metalloproteinase (MMP)-9 (Malaviya et al., 2012; Mishra et al., 2012; Venosa et al., 2016). Decreases in total antioxidant capacity, glutathione, glutathione-S-transferase, glutathione reductase, SOD and catalase, coupled with increases in the antioxidant enzyme, heme oxygenase (HO)-1, 8-hydroxy-2'-deoxyguanosine in DNA, protein carbonyls and MDA (Table 1), suggest that oxidative stress persists in the lung of mustard exposed rats or mice (Malaviya et al., 2012; Sawale et al., 2013; Kannan et al., 2016; Varmazyar et al., 2019).

4. Therapeutic approaches and treatment options

To date, there are no medications approved for treating either the acute or long-term effects of mustard on the lung. Current management strategies have been mainly adopted from treatments for pulmonary diseases or injuries with similar pathophysiology. Supportive care after acute exposure includes oxygen supplementation, humidification, hydration, bronchodilators and/or bronchoscopy with using isotonic saline to remove luminal debris, endotracheal intubation, tracheostomy, and respiratory physical therapy (Weinberger et al., 2016). Symptomatic interventions including therapeutic bronchoscopy, laser therapy, and respiratory tract stents also provide limited help to victims suffering from chronic manifestations of mustard injury (Razavi et al., 2013; Weinberger et al., 2016). Airway obstruction from fibrin casts following acute SM inhalation has been reported to lead to respiratory failure and death in victims of mustard poisoning. Veress et al. (2015) have reported that intratracheal administration of a potent fibrinolytic agent, tissue plasminogen activator, provides relief by normalizing pulmonary oxygenation and by reducing mortality in rats following high dose mustard inhalation.

As described above, inflammation and oxidative stress are hallmarks of acute, as well as chronic mustard toxicity in both human and animals (Malaviya et al., 2015a; Beigi Harchegani et al., 2018). Inhibitors of enzymes such as iNOS, which generates cytotoxic nitric oxide and other oxidants, reduce mustard induced toxicity. For example, the iNOS inhibitor, aminoguanidine, mitigated NM-induced acute lung injury in rats (Malaviya et al., 2012). This is consistent with studies in mice deficient in iNOS, which are less sensitive to the cytotoxic effects of the half-mustard, CEES, than wild-type controls (Sunil et al., 2012). In rodents, treatment with the corticosteroid, dexamethasone, or liposome-encapsulated vitamin E, alone or in combination with the antioxidant, N-acetyl cysteine (NAC), inhibited acute inflammation, reduced lymphocyte influx and suppressed collagen deposition induced by CEES or NM (Hoesel et al., 2008; Mukherjee et al., 2009; Wigenstam et al., 2009, 2012). Similarly, systemic and/or inhaled corticosteroids and long-term β2 adrenergic agonists have been reported to reduce airway obstruction and stenosis in mustard victims (Ghanei et al., 2007). NAC administration also mitigated signs of chronic mustard lung toxicity in humans by reducing sputum, cough and dyspnea, and by improving pulmonary function (Ghanei et al., 2008; Shohrati et al., 2008; Razavi et al., 2013; Shohrati et al., 2014). NAC, together with clarithromycin, also provided some benefit in patients with SM-induced bronchiolitis obliterans (Ghanei et al., 2004a; Shohrati et al., 2014). Mesenchymal stem cell therapy has been used to treat chronic injury in mustard gas victims (Nejad-Moghaddam et al., 2016, 2017, 2018). Mesenchymal cells have been shown to downregulate oxidative stress and inflammation, and to significantly improve overall quality of life. Carvacrol, a food additive with antioxidant, antiinflammatory and immunomodulatory activity, reportedly improved pulmonary function in mustard gas victims, a response associated with increases in serum levels of antiinflammatory cytokines and decreases in inflammatory cytokines (Khazdair and Boskabady, 2019).

Studies in rodents have demonstrated a role for the inflammatory cytokine, TNFα in mustard-induced acute lung injury and fibrosis (Oikonomou et al., 2006; Malaviya et al., 2015b; Venosa et al., 2016). In mice lacking TNF receptor (TNFR)1, the receptor mediating the proinflammatory actions of TNFα, significant protection from CEES-induced injury, oxidative stress, and inflammation was observed (Sunil et al., 2011). CEES-induced expression of iNOS, COX-2 and monocyte chemotactic protein-1 mRNA was also attenuated in TNFR1^−/− mice, while upregulation of Cu-Zn-SOD and Mn-SOD was delayed or absent. Similarly, pharmacologic inhibition of TNFα using pentoxifylline reduced NM-induced acute lung injury, inflammation, and oxidative stress, as reflected by improved histopathology, and by decreases in BAL cell and protein content, and levels of HO-1 and lipocalin 2 (Sunil et al., 2014). NM-induced increases in activated proinflammatory/cytotoxic lung macrophages expressing COX-2 and MMP-9 were also decreased after pentoxifylline, while CD163⁺ and Gal-3⁺ antiinflammatory macrophages increased. Of particular importance are findings using anti-TNFα antibody. Thus, administration of monoclonal anti-TNFα antibody once every 9 days, beginning 30 min after NM was found to reduce progressive histopathologic alterations in the lung including perivascular and peribronchial edema, macrophage/monocyte infiltration, interstitial thickening, bronchiolization of alveolar walls, fibrin deposition, emphysema and fibrosis up to 28 d post exposure (Malaviya et al., 2015b). Inhibition of TNFα also reduced NM-induced damage to the alveolar-epithelial barrier, blocked HO-1 expression and suppressed the accumulation of proinflammatory/cytotoxic M1 macrophages in the lung up to 28 d after exposure, whereas antiinflammatory/wound repair M2 macrophages were increased or unchanged. Treatment of rats with anti-TNFα antibody also reduced NM-induced increases in expression of the profibrotic mediator, TGF-β (Malaviya et al., 2015a, 2015b). Monoclonal anti-TNFα antibody has also been found to be effective in mitigating SM-induced pulmonary toxicity in rats, as measured by reduced BAL cell and protein content, expression of inflammatory proteins, and markers of oxidative stress (Malaviya et al., 2018a). These findings suggest that blocking TNFα using biologics hold great promise as a countermeasure against SM and related vesicants.

Another potential approach to treating mustard-induced lung injury is increasing levels of surfactant protein (SP)-D, a pulmonary collectin known to suppress proinflammatory macrophage activity. Mice lacking SP-D have been reported to be hypersensitive to mustards, exhibiting exacerbated oxidative stress, edema, bronchiectasis and fibrosis (Sunil et al., 2018). Curosurf, a natural surfactant, has been reported to reduce SM-induced mortality in guinea pigs (van Helden et al., 2004).

5. Summary and conclusions

Mustard vesicant-induced lung injury progresses over time leading to chronic bronchitis, bronchiectasis, airway stenosis, lung fibrosis, and/or acute respiratory distress syndrome. This is associated with persistent inflammation, oxidative and nitrosative stress and increases in the levels of inflammatory cytokines, growth factors and chemokines in the lung. Animal models of mustard-induced lung injury demonstrate similar pathological patterns, and hence have been useful in mechanistic studies of mustard toxicity, and for testing potential therapeutics. Treatment with antiinflammatory agents, corticosteroids, antioxidants, or with drugs that suppress TNFα and/or TGFβ have shown some efficacy in relieving disease pathology associated with mustard poisoning. Further studies are required to more clearly elucidate mechanisms underlying mustard-induced pulmonary injury, which will help in identification and development of more efficacious clinical approaches to treating chronic mustard toxicity.