Free Radical Production and Oxidative Stress in Lung Tissue of

Review Cite This: Chem. Res. Toxicol. 2018, 31, 211−222

pubs.acs.org/crt

Free Radical Production and Oxidative Stress in Lung Tissue of Patients Exposed to Sulfur Mustard: An Overview of Cellular and Molecular Mechanisms Asghar Beigi Harchegani,† Eisa Tahmasbpour,*,‡ Hojat Borna,† Ali Imamy,† Mostafa Ghanei,† and Alireza Shahriary*,† †

Chemical Injuries Research Center, System Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, 19945-581 Tehran, Iran ‡ Laboratory of Regenerative Medicine & Biomedical Innovations, Pasteur Institute of Iran, Tehran, Iran ABSTRACT: Sulfur mustard (SM) is a chemical alkylating compound that primary targets lung tissue. It causes a wide variety of pathological effects in respiratory system such as chronic bronchitis, bronchiolitis obliterans, necrosis of the mucosa and inflammation, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. However, molecular and cellular mechanisms for these pathologies are still unclear. Oxidative stress (OS) induced by reactive oxygen species (ROS) is likely a significant mechanism by which SM leads to cell death and tissues injury. SM can trigger various molecular and cellular pathways that are linked to ROS generation, OS, and inflammation. Hypoxia-induced oxidative stress, reduced activity of enzymatic antioxidants, depletion of intercellular glutathione (GSH), decreased productivity of GSH-dependent antioxidants, mitochondrial dysfunction, accumulation of leukocytes and proinflammatory cytokines, and increased expression of ROS producing-related enzymes and inflammatory mediators are the major events in which SM leads to massive production of ROS and OS in pulmonary system. Therefore, understanding of these molecules and signaling pathways gives us valuable information about toxicological effects of SM on injured tissues and the way for developing a suitable clinical treatment. In this review, we aim to discuss the possible mechanisms by which SM induces excessive production of ROS, OS, and antioxidants depletion in lung tissue of exposed patients.

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CONTENTS

Introduction Mitochondrial Dysfunction Decreased Activity of Antioxidant Enzymes Glutathione Depletion ROS Producing-Related Enzymes Accumulation of Leukocytes and Inflammation Deficiency of Nitric Oxide Synthase Hypoxia Conclusion Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments Abbreviations References

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SM analogues: (i) monochloro derivative, 2-chloroethyl ethyl sulfide (CEES), (ii) an oxygen analogue, bis(β-chloroethyl) ether (BCEE), and (iii) nitrogen analogues based on the 2,2′-dichlorodiethylamine framework (e.g., HN1, HN2, and HN3)1 (Figure 1). Since SM is an oily lipophilic compound, it can

211 212 212 215 215 216 216 216 217 217 217 217 217 217 218 218 218

Figure 1. Analogues of SM. SM, sulfur mustard; CEES, 2-chloroethyl ethyl sulfide; BCEE, bis(β-chloroethyl) ether.

easily penetrate into the cell membrane of most tissues and cause cytotoxicity.2 Upon absorption, it undergoes a process of

INTRODUCTION

Sulfur mustard (SM) is a vesicant and cytotoxic agent that has been used as a chemical warfare agent. There are three basic © 2018 American Chemical Society

Received: November 19, 2017 Published: March 23, 2018 211

DOI: 10.1021/acs.chemrestox.7b00315 Chem. Res. Toxicol. 2018, 31, 211−222

Chemical Research in Toxicology

Review

antioxidant capacities.26 The resulted OS may damage DNA, leading to chromosome instability, altered expression of some genes, and genetic mutations that are associated with cell death and lung damage.27,28 SM may also induce protein and lipid oxidation, which can modify the functional activity of various enzymes, structural proteins, and cell membranes.29 Recent evidence has revealed that SM can induce overproduction of ROS and subsequently OS in lung of victims via several mechanisms including reduced activity of antioxidants, enhanced expression of ROS producing-related enzymes, accumulation of leukocytes and inflammation, mitochondrial dysfunction, depletion of glutathione (GSH), and productivity of GSH-dependent antioxidant enzymes as well as change in activity of inducible nitric oxide synthase (iNOS).22 In the following sections, we will discuss the possible mechanisms and events by which SM triggers overproduction of ROS, oxidative stress and antioxidants depletion in lung tissue of exposed patients.

cyclization to form a sulphonium ion, which in turn alkylates DNA, proteins, and lipids.3,4 A large number of studies have reported pathological effects of SM on different organs and systems of the victims.5 Dermatologic, hematologic, psychological, neurological, ocular, gastrointestinal, and immunological complications, as well as reproductive and sleep disorders, have been frequently reported in victims.2 Respiratory system is one of the main targets of SM toxicity that occurs in a dose-dependent manner from the nasal mucosa to the terminal bronchioles.6 These adverse effects are often lethal in acute phase, while they can be associated with clinical symptoms and disabilities in chronic phase.7 Bloody sputum, pain and disturbance in the nose or sinuses, bronchiolitis obliterans, feeling of tightness and pain in the chest, shortness of breath over nights, hoarseness, sore throat, generalized wheezing, decreased lung sounds, necrosis of the mucosa, and inflammation are the major respiratory complications in patients exposed to SM.8−10 SM has been also involved in parenchymal tissue destruction and airway obstruction, which can lead to asthma or chronic obstructive pulmonary disease (COPD).11 Decrease in pulmonary function tests (PFTs) values can be observed in these patients.11 Balali-Mood et al. reported the presence of pulmonary fibrosis in 7.7% of the patients.12 A 10 year follow-up study illustrated chronic bronchitis (58%), COPD (12%), asthma (10%), large airway narrowing (9%), and bronchiectasis (8%) as the most common pulmonary complications among SM exposed patients.13 Several cellular and molecular mechanisms have been recommended for toxicological effects of SM.14 Recent evidence have suggested that SM toxicity is mainly because of its direct interaction with DNA, proteins, and lipids.15 SM can form adducts with these macromolecules and inhibit nucleic acid and protein biosynthesis as well as ATP generation.14 DNA alkylation seems to be the primary initiator of SM toxicity that is associated with proteins or genome modifications as well as DNA replication and transcription defects,.15−17 DNA damages can also induce poly(ADP-ribose) polymerase (PARP) activation, which in turn associated with ATP depletion, proteases activation, cell death, and injury.18 Upregulation of Fas and Fas ligand (FasL), which is associated with Caspases activation, proteins degradation, and apoptosis, is another mechanism of SM toxicity and cellular injuries.19 SM also increases contents of NF-κ B, calcium, and calmodulin that enhance cell death, inflammation, and tissue injury.20,21 Oxidative stress (OS) induced by reactive oxygen species (ROS) and as the result antioxidants depletion is now considered as the major mechanism of SM toxicity on lung injuries.22 Many studies showed overproduction of ROS and increased oxidative stress biomarkers following exposure to SM (Table 1). It has been shown to be involved in deficiency of extracellular matrix remodeling, pulmonary cells apoptosis, impaired mitochondrial respiration, cell proliferation failure, defects in maintenance of surfactant and protease activity, as well as deficiency of alveolar repair responses and immunity modulation, which are associated with inflammation and lung tissue damage.23,24 Therefore, there is a tight link between SM and oxidative stress. Oxidative stress is caused by an imbalance between the production of ROS and normal antioxidant capacity, which in turn induces critical failure of biological functions and cell death.25 ROS are highly reactive and short half-life compounds that target DNA, proteins, and lipids to compensate their unpaired electrons. It is now elucidated that SM can accelerate OS through induction of ROS generation and decrease of

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MITOCHONDRIAL DYSFUNCTION There is a tight link between oxidative stress and mitochondrial dysfunction because it is both a generator and target for ROS.30 Mitochondrial dysfunction can be associated with overproduction of endogenous ROS and oxidative damages. A great number of experimental studies demonstrated that SM can result in severe mitochondria abnormalities, which are subsequently associated with increased production of ROS, DNA damage, membrane lipid peroxidation, and decrease in intracellular antioxidants.1,23,31−33 SM exposure has been shown to be associated with fuzzy mitochondrial cristae and morphological abnormalities in type I alveolar epithelial cells and sever lung injury.34−36 Ray et al. demonstrated that SM can mediate Caspases-induced apoptosis by mitochondrial dysfunction in cultured human airway epithelial cells.37 Gould et al. showed that CEES exposure is associated with inhibition of mitochondrial respiratory chain, electron transfer dysfunction, overproduction of O2•− from the mitochondria, and as the result of increased cytotoxicity.30 These data indicate that SM may function as an uncoupler in mitochondrial respiratory chain and increase ROS generation in damaged mitochondria (Figure 2). However, there is no study that considered mitochondrial dysfunction in lung biopsies of patients long-term after SM exposure.

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DECREASED ACTIVITY OF ANTIOXIDANT ENZYMES Reduced activity of antioxidant enzymes is another mechanism by which SM can increase endogenous production of ROS and oxidative damages. A large number of studies have considered the effect of SM on expression and activity of various antioxidant enzymes at chronic phase of injury.38 Although some studies have shown overexpression of different enzymatic antioxidants at the mRNA level, the activity of these enzymes or their expression at the protein level has been reported to be decreased long years after exposure to SM.39 Superoxide dismutase (SOD) is one of the most important antioxidants because it converts anionic superoxide (O2•) to hydrogen peroxide (H2O2) and O2.40 SOD has been reported to be a primary target for SM.41 Numerous studies have reported decreased activity of this enzyme long-term after exposure to SM.42,43 In a study by Mirbagheri et al., they revealed that SOD mRNAs were upregulated in lung biopsies of SM-exposed patients at chronic phase of injury, while SOD proteins were 212



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Table 1. Change in Biomarkers of Oxidative Stress and Inflammation after SM Exposure in Different Study Models study models Human lung biopsies lung biopsies lung biopsies lung biopsies lung biopsies lung biopsies lung biopsies BAL fluids serum BAL and plasma serum serum In Vivo lung of male mice lung of rats liver and brain of rats serum BAL and lung of mice lung of mouse BAL, serum, and alveolar septum lung of rats lung of rats lung of rats serum of rats muscles, livers, and kidneys of rats serum of rats brain of rats In Vitro human skin fibroblast HepG2-derived cells A549 lung epithelial cells A549 lung epithelial cells human epidermal keratinocyte human keratinocyte human skin

findings

ref

SM SM SM SM SM SM SM SM SM SM SM SM

↑SOD mRNA; ↓SOD protein ↑SOD mRNA; ↑CAT mRNA; ↓GSR mRNA; ↑GPX mRNA; ↑GSS mRNA; ↓MT3 mRNA; ↑GST mRNA ↑GST mRNAs; ↓GST protein ↑Metallothionein-1A mRNAs; ↓MT-1A protein ↑PRDX mRNAs; ↑SRXN1 mRNA ↑DOUX1 mRNA; ↑DOUX2 mRNA; ↑TPO mRNA; ↑EPO mRNA; ↑MPO mRNA ↑SOD mRNA; ↑GST mRNA; ↑Heme oxygenase-1 mRNA ↑PC; ↑MDA; ↓Total antioxidant capacity (TAC) ↓SOD activity; ↓CAT activity; ↓GSH; ↑MDA↓IL-2; ↓INF-γ; ↑TNF-α; ↑IL-4; ↑IL-6; ↑IL-10; ↑MMP-9; ↑TGF-β ↓SOD activity; ↓CAT activity; ↓GPX; ↓GSH ↓GSH; ↑MDA; ↓SOD activity; ↓CAT activity ↓Paraoxonase-1; ↓Albumin

7 46 39 52 64 67 113 64, 67 41 42 10, 54 114

CEES SM SM SM SM CEES SM

92 79 43 44 76 115 116

SM CEES CEES SM SM

↑iNOS; ↑COX-2; ↑SOD; ↑TNF-α ↑COX-2; ↑TNF-α; ↑iNOS; ↑MMP-9↓HOX-1; ↓SF-D; ↓Anti-inflammatory collectin ↓SOD activity; ↓CAT activity; ↓GPX activity; ↓GST activity; ↓GSH; ↑MDA ↑ROS; ↑8-OHdG; ↑MPO activity; ↑iNOS activity;↓GSH ↑Activated macrophages; ↑MPO; ↑LDH; ↑MMP-9; ↑MMP-2; ↑MDA; ↓GSH ↓GSH; ↓total protein; ↑DNA oxidation; ↑MDA; ↓SOD activity; ↓CAT activity; ↓GST activity ↓GPX activity; ↓LDH activity; ↓Heme oxygenase-1;↑Activated macrophages; ↑TNF-α; ↑IL-1β; ↑IL-6; ↑CRP; ↑ MDA; ↑8-OHdG ↓GPX; ↓total protein; ↓albumin; ↓LDH ↑IL-1β, ↑TNF-α, ↑IL-2, ↑IL-6, ↑TGF-β, ↑IL-17 ↓TrxR activity ↑Apoptotic cells; ↑TNF-α; ↑IL-1β; ↑IL-6; ↓LDH; ↓GPX activity; ↑MDA ↑MDA; ↓GSH; ↓Metallothionein; ↓GSR activity; ↓GPX activity; ↓GST activity; ↑Caspase-3

SM SM

↓Ferric reducing antioxidant power (FRAP); ↓GSH; ↑MDA; ↓GST; ↓GSR; ↑Caspase-3 SM-DNA adducts

62 118

SM SM SM HN2 SM SM CEES

↓Cells viability; ↓GSH; ↓CAT activity ↑Metallothionein 2A promoter ↑DNA cross-links; ↑DNA breaks ↓TrxR1, 2 activity; ↑HN2-Trx2 cross-links ↓iNOS ↑Arachidonic acid release ↑COX-2,5; ↑LT-A4; ↑LT-C4; ↑Microsomal PGE(2) synthases; ↓GSTs

119 120 26 47 97 94 93

agent

down-expressed.7 Catalase (CAT) is another enzymatic antioxidant that has been reported to be modified long years after SM exposure.41,44 It is a cytosolic antioxidant enzyme that protects cells against toxic effects of H2O2 by catalyzing its decomposition into O2 and H2O.45 Panahi et al. reported a significant reduction in activity of SOD and CAT enzymes in serum of patients suffering from chronic SM-induced complications.41 Recent investigations have indicated that SM also reduces the glutathione peroxidase (GPX) activity in lung tissue either at chronic or acute phases.36,42 It is a selenocysteine-containing enzyme that converts lipid and hydrogen peroxides to their corresponding alcohols and catalyzes the reducing of H2O2 to H2O by utilizing GSH.46 Decreased activity of glutathione-stransferase (GST) and glutathione reductase (GSR) enzymes has been also reported after SM exposure.24,39,42 In a more recent study, Tahmasbpour et al.38 have revealed higher expression of SOD, CAT, GST, GSS, and GPX mRNAs in lung biopsies of patients several years after SM exposure. However, they did not consider the expression of these antioxidants at the protein levels. The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is critical in regulation

117 87 50 36 24

and protection of all living cells against oxidative stress.47 TrxR is a selenocysteine-containing flavoprotein that specifically catalyzes the reduction of oxidized Trx as well as other redox-active proteins and small molecules such as oxidized glutathione (GSSG) and H2O2 using NADPH as a source of reducing equivalents.48 Reduced Trx can serve as a reductase for various enzymes, many of which are essential for controlling of oxidative stress, antioxidant defense system, DNA synthesis, signal transduction, and protein folding through thiol redox control.14,49 Recent evidence have revealed that Trx system can be a main target for SM or its analogues. SM alkylates catalytic residues in both the N- and C-terminal redox motif of the TrxR and terminates enzyme cross-linking.50 Jan et al. reported that mechlorethamine (HN2), a SM analogue, cross-links with TrxR and inhibits the activity of cytosolic (TrxR1) and mitochondrial (TrxR2) forms of TrxR in cultured lung epithelial cells.47 In another study, Jan et al. suggested that inhibitory effect of SM on TrxR activity may be an important mechanism mediating oxidative stress and lung tissue injury.50 Since the Trx system serves as electron donors in redox regulation, Trx system deficiency can inhibit these processes and cause overgeneration of ROS, oxidative stress, and toxicity (Figure 3). Nevertheless, 213



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Figure 2. SM triggers massive production of ROS through its cytotoxic effects, leukocytes recruitments at the site of injury, overexpression of ROS producing-related enzymes such as, AOX1, DUOXs, iNOS, and TPO. Mitochondrial deficiency is another source of ROS overproduction after SM exposure. SM not only increases arachidonic acid release from cell membrane, but also it enhances overexpression of COX-2 and 12-LO enzymes that convert arachidonic acid to PG and 12-HETE, respectively. These proinflammatory intermediates recruit leukocytes at the site of injury, which cause to increased secretion of MPO and EPO enzymes into the cells. Both enzymes are responsible for the production of ROS. Increased contents of ROS cause oxidative damage to cellular DNA, protein, and lipids, which may be closely related to cell death, inflammation, and tissue damage in respiratory organs. SM, sulfur mustard; OS, oxidative stress; ROS, reactive oxygen species; COX-2, cyclooxygenase-2; PG, prostaglandin; 12-LO, 12-Lipoxygenase; 12-HETE, 12-hydroxyeicosatetra-enoic acid; iNOS, inducible nitric oxide synthase; AOX1, aldehyde oxidase-1; DUOXs, dual oxidases; MPO, myeloperoxidase; EPO, eosinophil peroxidase; TPO, thyroid peroxidase.

Figure 3. Mechanisms by which SM causes to GSH depletion and reduced activity of GSH-dependent antioxidant enzymes. SM can induce intracellular GSH depletion though downregulation of GSR. GSH depletion is then associated with decreased productivity of GSTs, GPXs, Srx1, Prdx enzymes, and as the result massive production of ROS and OS. SM, sulfur mustard; GSR, glutathione reductase; GPX, glutathione peroxidase; GST, glutathione-s-transferase; TrxR, Thioredoxin reductase; Srx1, Sulfiredoxin-1; Prdx, Peroxiredoxins; ROS, reactive oxygen species; OS, oxidative stress; GSH, reduced glutathione; GSSG, oxidized glutathione. 214



Review

lung tissue either at chronic or acute phases,36,42 which may be related to depletion of GSH. Sawale et al. found that exposure to CEES depletes intracellular GSH level and activity of GSR, GPX, and GST enzymes, which play a major role in preventing ROS production and oxidative stress.63 Therefore, there is a tight link between GSH contents and activity of these antioxidants in oxidatively damaged cells. Peroxiredoxins (Prdx) are a new class of thiol-specific antioxidants that protect cells from the action of H2O2 in their reduced form.64 Prdx antioxidative activity is dependent on cellular GSH content. Sulfiredoxin-1, which is a crucial oxidoreductase enzyme, catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins using ATP and GSH.65 Recent studies propose that the level of Prdx is increased in numerous pathological states accompanied by oxidative stress.64,66 Tahmasbpour et al. revealed overexpression of Prdx and Srx-1 mRNAs in lungs of SM exposed patients at chronic phase of injury, indicating the existence of oxidative stress in mustard lungs.64 Since Srx1 catalyzes the reduction of oxidized Prxs using GSH, intracellular depletion of GSH may reduce the effectiveness of these enzymes in the lungs of SM-patients (Figure 3). Therefore, decreased intracellular GSH level induced by SM may affect activity of these antioxidant enzymes and cause to accumulation of ROS and oxidative damages to lung tissue.

there is no study that investigated thioredoxin system in lung biopsies of patients several years after SM exposure. Recent investigations have also found reduced expression of metallothionein-3 (MT3) mRNA in lung biopsies of SM-injured patients at chronic phase of injury.38 MT3 is a low molecular weight and sulfhydryl-rich intracellular antioxidant that protects lung against inflammation through the regulation of pulmonary endothelial and epithelial integrity and its antioxidative property.51 Pohanka et al. indicated that SM exposure causes to depletion of metallothionein in the livers, kidneys, and muscles of the Wistar rats.24 A study by Nourani et al. found up-regulation of metallothionein-1A (MT-1A) mRNAs in endobronchial biopsy samples of SM-injured patients several years after exposure, but MT-1A protein was significantly down-regulated.52 This may be because of creation of mutations in the genome sequence, phosphorylation, or other post-translational alterations of proteins upon SM exposure that can lead to structural and functional changes in protein. Furthermore, alterations in some miRNAs responsible for regulating post-translation events may inhibit the expression of the antioxidant proteins in the poisoned cells at translational level.

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GLUTATHIONE DEPLETION Depletion of cellular GSH is now considered as one the main mechanisms of SM toxicity. GSH is a key nonenzymatic antioxidant that plays critical roles in free radical scavenging. In addition to actions as an oxyradical scavenger, GSH also functions as a cofactor or hydrogen donor for a subset of antioxidant enzymes such as GPX, GSTs, and sulfiredoxin-1 (Srxn1).53 Therefore, decreasing GSH contents mediated by SM can reduce the productivity of these antioxidant enzymes and exacerbate OS and toxicity in lung endothelial cells22 (Figure 3). Reduced level of GSH has been frequently reported in serum and bronchoalveolar lavage (BAL) fluids of patients several years after SM exposure.42,54,55 Several clinical trial studies on SM-exposed patients with chronic lung injury revealed that treatment with GSH precursors, especially with N-acetylcysteine (NAC), compensates GSH depletion and consequently decrease markers of OS and toxicity induced by SM.56,57 Tewari-Singh et al. demonstrated that NAC therapy has protective effects against CEES-caused cytotoxicity in rats.58 These findings point out the importance function of cellular GSH pools as protective mechanism against oxidative stress and lung injuries. The actual mechanism by which SM decreases cellular GSH contents is not clear. Recent evidence indicates that SM can interact with GSH to produce SM-GSH metabolites, which deplete cellular GSH and increase intracellular ROS as well as oxidative stress.42 Glutathione reductase and TrxR deficiency are now considered as the major mechanisms of GSH depletion after SM exposure (Figure 3). GSR and TrxR are tightly related oxidoreductases with a particularly similar structure and reaction mechanism.59 GSR is a key regulatory antioxidant enzyme that converts GSSG to GSH using NADPH.60 Several studies reported GSR deficiency in patients previously exposed to SM.38,61,62 A more recent study has found that GSR is severely down-regulated in lung tissue of patients who previously exposed to SM.46 Impairment of GSR system can be associated with deficiencies in the regeneration of GSH and its depletion as well as declined productivity of GPXs and GSTs enzymes, which are an initial leading cause of OS and lung injury22 (Figure 3). Several lines of studies have demonstrated that SM declines the activity of GPXs and GSTs enzymes in the human

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ROS PRODUCING-RELATED ENZYMES Overexpression of ROS producing enzymes is another significant mechanism in which SM induces OS and lung injury. In a more recent study, we have shown higher expression of aldehyde oxidase-1 (AOX1), dual oxidases 1, 2 (DUOX1 and DUOX2), myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO) enzymes in chronic mustard lungs.67 Overexpression of these enzymes can be associated with higher production and secretion of free radicals and subsequently oxidative damages (Figure 2). Dual oxidases are the major sources of H2O2 production that plays a pivotal role in host defense in human airways.68,69 Thyroid peroxidase can produce reactive nitrogen radical (NO2•) through interaction between H2O2 and nitrite.69 In airways epithelium of SM-exposed patients, where high levels of H2O2 exist, TPO can couple with H2O2-producing oxidases such as DUOXs and increases NO2• production.69 Aldehyde oxidase-1 generates cellular ROS through the oxidation of NADH.70 Myeloperoxidase is a major component of neutrophil cytoplasmic granules and monocyte lysosomes.71 Increased activity of MPO is considered as a direct indicator of neutrophil presence and lung injury.72 Upon neutrophil activation, primary granules fuse with the plasma membrane and cause MPO secretion into extracellular milieu. MPO converts H2O2 to highly reactive hypochlorous acid (HClO), which attacks thiols, nucleotides, proteins, and fatty acids to generate protein adducts and genetic mutations, and affects signaling pathways.73,74 Several lines of studies showed increased activity of MPO after SM exposure.63,75 Tahmasbpour et al. have revealed increased expression of MPO in lung biopsies of SM-exposed patients at chronic phase of injury.38 Zhang et al. have indicated that subcutaneous injection of SM into mice decreases GSH level and increases the activity of serum MPO at acute phase of injury.44 In another study, Kannan et al. demonstrated that SM exposure enhanced the number of activated macrophages, degranulation of neutrophils, and MPO activity in BAL fluids of SM-exposed mice at chronic phase of injury.76 O’Neill et al. 215



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long-term after SM exposed.38 Similarly, experimental studies reported overexpression of COX-2 in lungs of rats exposed to either SM or CEES.79,92 Another pervious study observed increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE-2 synthases, leukotriene-A4 hydrolase, and leukotriene-C4 synthase in CEES-treated skin.93 Lefkowitz et al. demonstrated that exposure to SM causes a 5−8-fold increase in arachidonic acid release from human keratinocytes.94 The increased metabolites of 5-lipoxygenase pathway were also reported in human lung parenchyma long-term after SM exposure.78 An in vitro study showed SM increases arachidonic acid release from cell membrane in rat glioma hybrid NG108−15 clonal cell line.33 Therefore, increased expression and activity of 12-LO and COX-2 may be responsible for inflammatory responses and overproduction of ROS in lung tissue of SM-injured patients (Figure 2).

revealed that lung MPO activity was increased at 18 h postCEES exposure.72 SM-induced injuries may recruit macrophages and neutrophils with a subsequent release of inflammatory mediators that recruit and activate other leukocytes in lung.77−79 Therefore, increased expression or activity of MPO suggests degranulation of neutrophils due to SM toxicity in the lungs (Figure 2). Eosinophil peroxidase is a key enzyme involved in generation of reactive oxidants by eosinophils.80 Eosinophils are a member of inflammatory response that are recruited to the damaged tissue and secret high levels of free radicals.80 Boskabady et al. observed an increased number of eosinophils in lungs and BAL fluids of guinea pigs 14 days postexposure to SM.81,82 Similarly, we have found overexpression of EPO in lungs of individuals who previously exposed to SM.67 However, the mechanism in which eosinophils are increased after SM exposure is unclear. Increased number of eosinophils in lung of SM-exposed patients can be associated with higher EPO activity, overproduction of free radicals, and marked peroxidatic pulmonary damage (Figure 2).

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DEFICIENCY OF NITRIC OXIDE SYNTHASE

Inducible nitric oxide synthase (iNOS) is responsible for generation of nitric oxide (NO) from the amino acid L-arginine.95 Nitric oxide serves as a signal molecule in many parts of the organism. At physiological concentration, it is involved in modulation of vascular tone, pulmonary neurotransmission, host defense, relaxation of vascular smooth muscle, and remodeling of pulmonary circulation.96 However, increased content of NO can be associated with inflammation, oxidative stress, and severe cytotoxicity.97 In a more recent study, Zhang et al. have demonstrated that SM exposure enhanced activity of iNOS and MPO in serum of mice, which was subsequently associated with oxidative stress, inflammation, and DNA damages.44 Although several lines of studies showed that SM exposure can be associated with up-regulation and increased activity of iNOS enzyme and consequently higher generation of NO,92,98−100 a very limited study considered expression and activity of iNOS in respiratory epithelial cells of SM-exposed patients. On the other hand, a recent study has revealed downregulation of iNOS enzyme in lung biopsies of SM exposed individuals.38 Therefore, SM can cause significant alterations in iNOS expression and its activity, which may be associated with oxidative stress, wound healing, and also deficiency in airways remodelling (Figure 2).

ACCUMULATION OF LEUKOCYTES AND INFLAMMATION SM exposure can also result in massive release and upregulation of inflammation mediators in human respiratory epithelial cells in chronic phase. It primary damages lung epithelial cells and induces inflammatory responses, causing pulmonary symptoms.75,83 Recent investigations have shown inflammatory cells accumulate with signs of necrosis in the respiratory tract of SM-exposed patients.11,84 Increased level of inflammatory cytokines such as IL-1α, IL-1β, IL-5, IL-6, IL-8, IL-12, IL-13, and TNFα was reported in serum and BAL fluids of patients several years after SM exposure.85 Experimental studies have demonstrated increased expression and secretion of different proinflammatory cytokines, chemokines, and growth factors such as TNF-α, TGF-β, IL-α, IL-β, IL-2, IL-6, IL-8, IL-10, IL-13, IL-15, INF-γ, transcription factors NF-kappaB, AP-1 macrophage chemotactic protein (MCP)-1, matrix metalloproteinases (MMPs) in serum, and lung tissue of animal models after exposure to SM.44,75,86,87 SM can also accumulate macrophages and neutrophils, as a major source of inflammatory mediators, at the site of tissue injury. Increased macrophage and neutrophil level can subsequently be associated with recruitment and activation of other leukocytes (Figure 2). This causes to overproduction of ROS that can overwhelm the antioxidant strategies, leading to oxidative stress.42 Numerous experimental studies reported the increase neutrophil and macrophage infiltration in both lung tissue and alveolar septum of rats after exposure to SM that can be associated with high level of ROS and oxidative damages.34,88 Increased activity of cyclooxygenase-2 (COX-2) and 12-lipoxygenase (12-LO) is now considered as one of the other main mechanisms by which SM recruits leukocytes and induces inflammatory reactions, ROS production, oxidative stress, and cell damage in mustard lungs (Figure 2). COX-2 is a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PG), as a key regulatory factor in the generation of the inflammatory response.89 PG is a cardinal sign of acute inflammation and its biosynthesis can be enhanced in damaged tissues.90 12-LO is responsible for oxidative metabolism of arachidonic acid and generates bioactive lipid mediators with inflammatory properties.91 A more recent study has reported overexpression of COX-2 and 12-LO in lung biopsies of patients

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HYPOXIA Hypoxia is a condition that has been shown to be associated with increased production of ROS and OS by altering the activity of cytochrome chain that is responsible for mitochondrial oxidative phosphorylation.101 This process reduces ATP synthesis and enhances ROS production.102 In a recent study, Tuleta et al. have reported that intermittent hypoxia contributes to the lung damage by massive production of ROS, OS, inflammation, and imbalance in protease/antiprotease system.103 Hypoxia-induced oxidative stress may be another mechanism by which SM mediates lung injury in chronic phase. Previous studies showed that SM induces hypoxia in damaged tissues.104,105 Recent evidence has indicated that exposure to SM or its analogue, 2-chloroethyl ethyl sulfide (CEES), is also associated with O2/CO2 problems and severe hypoxemia106,107 (Figure 4). SM exposure has also been shown to be associated with reduced level of arterial blood oxygenation (PaO2) and saturation, increased content of carbon dioxide (PaCO2), and declined value of arterial blood pH and bicarbonate 216



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Figure 4. Effect of SM on hypoxia-induced oxidative stress. (A) Normal and (B) SM-exposed lung. Toxicity effect of SM causes to lung epithelial cells damage, which in turn leads to O2/CO2 exchange problem, hypoxia condition, and OS. CYGB and MB are overexpressed in response to hypoxia. Gas exchange failure is associated with hypoxemia, decreased blood HCO3 and pH, reduced blood PO2, increased PCO2, and as the result oxidative stress. SM, sulfur mustard; ROS, reactive oxygen species; OS, oxidative stress; CYGB, cytoglobin; MB, myoglobin.

(HCO3−)108 (Figure 4). Tahmasbpour et al. have found overexpression of hypoxia-related genes, cytoglobin (Cygb) and myoglobin (Mb), in lung tissue of patients long years after SM-exposure, indicating the increased incidence of hypoxia in mustard lungs.38 Cytoglobin and myoglobin, which are responsible in oxygen and redox homeostasis, have been shown to be upregulated in cellular response to hypoxia, OS, ischemiainduced cell death, and fibrogenesis.109 Given the regulatory function of Cygb and Mb to scavenge free radicals,110 we hypothesize that hypoxia is the ultimate signal that triggers Cygb and Mb induction in mustard lungs. However, we believe Cygb and Mb expression may be a compensatory mechanism by which the lung copes with hypoxic and OS conditions and to control the metabolism of free radical production.111 Furthermore, these proteins can help lung epithelial cells to produce energy in hypoxic conditions by supplying mitochondria with oxygen.112 Therefore, SM-induced hypoxia can be considered as one of the other mechanisms for ROS overproduction and OS in lung tissue of patients at chronic phase of injury (Figure 4).

leukocytes and inflammatory reactions, and consequently imbalances between the production and detoxification of ROS in cells. Although several studies reported increased expression of different antioxidant enzymes in mustard lungs, this is likely not sufficient to overwhelm exogenous ROS generation. Massive production and accumulation of ROS suppresses the capacity of antioxidant defense systems in lung cells. GSH depletion can be considered as the major reason for cytotoxic effects of SM toxicity, which is associated with decreased activity of several GSH-dependent antioxidants such as GPXs, GSTs, Srx1, and Prxs in lung cells. Therefore, antioxidant therapy may be useful to mitigate pulmonary systems against SM-induced damages. However, successful therapy is depended on disease severity, antioxidants dosage, and their improved delivery to target tissues.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 0021-82482502. *E-mail: [email protected]. Phone: 0021-9111193051.

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ORCID

CONCLUSION Excessive production of ROS and oxidative stress is now considered as one of the significant mechanisms of SM that can be associated with DNA, lipid, and protein oxidation and subsequently cell death. SM can induce oxidative damage in airway epithelial cells via several mechanisms including hypoxia, mitochondrial deficiency, enhanced activity of ROS-producing enzymes, reduced activity of intracellular antioxidants enzymes, iNOS deficiency, GSH depletion and decreased activity of GSH-dependent enzymatic antioxidants, accumulation of

Eisa Tahmasbpour: 0000-0002-7213-1494 Notes

The authors declare no competing financial interest. Biographies Mr. Asghar Beigi is a MSc graduate in clinical biochemistry from Shahid-Beheshti University of Medical Sciences Tehran, Iran. He worked on cell death pathways in myocardial infarction in the context of his MSc thesis. At present, he is a researcher in the Genomic Research Center at Baqiyatallah University of Medical Sciences. 217



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PaCO2, blood carbon dioxide; PaO2, blood oxygenation; PARP, poly(ADP-ribose) polymerase; PG, prostaglandins; Prdx, peroxiredoxins; ROS, reactive oxygen species; SM, sulfur mustard; SOD, superoxide dismutase; Srxn1, sulfiredoxin-1; TPO, thyroid peroxidase; Trx, thioredoxin; TrxR, thioredoxin reductase; 12-LO, 12-lipoxygenase

His research is now focused on studying the consequences of oxidant and antioxidant imbalance, especially in lung diseases and male infertility. Dr. Eisa Tahmasbpour has been a postdoctoral research fellow at the Laboratory of Regenerative Medicine & Biomedical Innovations, Pasteur Institute of Iran since 2017, and does research concerning induced pluripotent stem cell generation and differentiation from somatic cells. Dr. Tahmasbpour holds a PhD in molecular medicine where he gained experience in mesenchymal stem cell therapy for patients with lung diseases. Dr. Tahmasbpour has published over 40 original and review articles. Although his research is mainly focused on studies of pathophysiology and molecular genetic of male infertility, he is also interested in stem cell biology, cell therapy, and pulmonary biology.

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REFERENCES

(1) Inturi, S., Tewari-Singh, N., Gu, M., Shrotriya, S., Gomez, J., Agarwal, C., White, C. W., and Agarwal, R. (2011) Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts. Free Radical Biol. Med. 51, 2272−2280. (2) Mansour Razavi, S., Salamati, P., Saghafinia, M., and Abdollahi, M. (2012) A review on delayed toxic effects of sulfur mustard in Iranian veterans. Daru, J. Pharm. Sci. 20, 51. (3) Hefazi, M., Attaran, D., Mahmoudi, M., and Balali-Mood, M. (2005) Late respiratory complications of mustard gas poisoning in Iranian veterans. Inhalation Toxicol. 17, 587−592. (4) Rao, S., Matsumura, A., Yoon, J., and Simon, M. C. (1999) SPI-B activates transcription via a unique proline, serine, and threonine domain and exhibits DNA binding affinity differences from PU.1. J. Biol. Chem. 274, 11115−11124. (5) Hassan, Z. M., Ebtekar, M., Ghanei, M., Taghikhani, M., Noori Daloii, M. R., and Ghazanfari, T. (2006) Immunobiological consequences of sulfur mustard contamination. Iranian journal of allergy, asthma, and immunology 5, 101−108. (6) Ghanei, M., and Harandi, A. A. (2007) Long term consequences from exposure to sulfur mustard: a review. Inhalation Toxicol. 19, 451− 456. (7) Mirbagheri, L., Habibi Roudkenar, M., Imani Fooladi, A. A., Ghanei, M., and Nourani, M. R. (2013) Downregulation of super oxide dismutase level in protein might be due to sulfur mustard induced toxicity in lung. Iranian journal of allergy, asthma, and immunology 12, 153−160. (8) Ghanei, M., and Harandi, A. A. (2010) Lung carcinogenicity of sulfur mustard. Clin. Lung Cancer 11, 13−17. (9) Akhlaghpoor, S., Ahari, A. A., Shabestari, A. A., Ghanei, M., Ali, H. A., and Shakiba, M. (2011) Comparison of virtual bronchoscopy with fiberoptic bronchoscopy findings in patients exposed to sulfur mustard gas. Acta Radiol. 52, 1095−1100. (10) Shohrati, M., Ghanei, M., Shamspour, N., and Jafari, M. (2008) Activity and function in lung injuries due to sulphur mustard. Biomarkers 13, 728−733. (11) Khazdair, M. R., Boskabady, M. H., and Ghorani, V. (2015) Respiratory effects of sulfur mustard exposure, similarities and differences with asthma and COPD. Inhalation Toxicol. 27, 731−744. (12) Balali-Mood, M., Afshari, R., Zojaji, R., Kahrom, H., Kamrani, M., Attaran, D., Mousavi, S. R., and Zare, G. A. (2011) Delayed toxic effects of sulfur mustard on respiratory tract of Iranian veterans. Hum. Exp. Toxicol. 30, 1141−1149. (13) Balali-Mood, M., Mousavi, S., and Balali-Mood, B. (2008) Chronic health effects of sulphur mustard exposure with special reference to Iranian veterans. Emerging health threats journal 1, e7. (14) Tahmasbpour, E., Reza Emami, S., Ghanei, M., and Panahi, Y. (2015) Role of oxidative stress in sulfur mustard-induced pulmonary injury and antioxidant protection. Inhalation Toxicol. 27, 1−14. (15) Jowsey, P. A., Williams, F. M., and Blain, P. G. (2012) DNA damage responses in cells exposed to sulphur mustard. Toxicol. Lett. 209, 1−10. (16) Dillman, J. F., 3rd, Phillips, C. S., Dorsch, L. M., Croxton, M. D., Hege, A. I., Sylvester, A. J., Moran, T. S., and Sciuto, A. M. (2005) Genomic analysis of rodent pulmonary tissue following bis-(2chloroethyl) sulfide exposure. Chem. Res. Toxicol. 18, 28−34. (17) Ghanei, M., and Harandi, A. A. (2011) Molecular and cellular mechanism of lung injuries due to exposure to sulfur mustard: a review. Inhalation Toxicol. 23, 363−371.

Mr. Hojat Borna holds an MSc degree in biochemistry from Tehran University. He is working as researcher in the Systems Biology Institute at Baqiyatallah University of Medical Sciences. His research is focused mainly on molecular pathways, as well as genomics and proteomics of cancer and immune system cells, and their interactions. He collaborates with different national leading universities and institutes including Pasteur Institute, Shahid-Beheshti University of Medical Sciences, and Tehran University to address systems biology of cancer and immune system interactions. Dr. Ali Imamy is a general practitioner who works in hospitals in Tehran, Iran. He is also interested in biomedical sciences projects. He likes to know molecular and cellular mechanisms of different diseases such as pulmonary and cardiovascular diseases. Currently, he collaborates with the Systems Biology Institute at Baqiyatallah University of Medical Sciences. Dr. Mostafa Ghanei (MD) has been a Professor at Baqiyatallah University of Medical Sciences since 1993. His research is mainly focused on studies of pathophysiology of pulmonary disorders such as COPD, pulmonary fibrosis, and chemicaly injured patients with pulmonary problems. Dr. Ghanei has published extensively with over 200 original peer-reviewed articles, 15 book chapters, and over 50 presentations at scientific meetings. Dr. Alireza Shahriary is an assistant professor at Baqiyatallah University of Medical Sciences. His research interest is toxicology and systems biology as he has published several original and review articles and also some books in Persian. Much of his research deals with the detrimental effects of sulfur mustard on health as well as the victims of the perilous substance.

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ACKNOWLEDGMENTS We are deeply indebted to past and present collaborators. ABBREVIATIONS AOX1, aldehyde oxidase-1;; BAL, bronchoalveolar lavage; CAT, catalase; CEES, 2-chloroethyl ethyl sulfide; COPD, obstructive pulmonary disease; COX-2, cyclooxygenase-2; CYGB, cytoglobin; DUOX, dual oxidases; EPO, eosinophil peroxidase; FasL, Fas ligand; GPX, glutathione peroxidase; GSH, reduced glutathione; GSR, glutathione reductase; GSSG, oxidized glutathione; GST, glutathione-s-transferase; H2O2, hydrogen peroxide; HClO, hypochlorous acid; HCO3−, bicarbonate; HN2, mechlorethamine; iNOS, inducible nitric oxide synthase; MB, myoglobin; MCP, macrophage chemotactic protein; MMPs, matrix metalloproteinases; MPO, myeloperoxidase; MT-1A, metallothionein-1A; MT3, metallothionein-3; NAC, N-acetylcysteine; NO, nitric oxide; NO2•, nitrogen radical; O2•, anion superoxide; OS, oxidative stress; 218



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