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1.
Toxicol Appl Pharmacol ; 428: 115677, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34390737

ABSTRACT

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15-30 min later by anti-TNFα antibody (15mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.


Subject(s)
Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Antibodies, Monoclonal/therapeutic use , Mustard Gas/toxicity , Oxidative Stress/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Acute Lung Injury/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Chemical Warfare Agents/toxicity , Inhalation Exposure/adverse effects , Male , Mustard Gas/administration & dosage , Oxidative Stress/physiology , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/metabolism
3.
Toxicol Sci ; 178(2): 358-374, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33002157

ABSTRACT

Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2-0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4-0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7-16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.


Subject(s)
Chemical Warfare Agents , Lung Injury , Mustard Gas , Animals , Chemical Warfare Agents/toxicity , Fibrosis , Inflammation/pathology , Lung/drug effects , Lung Injury/pathology , Mustard Gas/toxicity , Oxidative Stress , Rats
4.
Toxicol Mech Methods ; 29(8): 604-615, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31237465

ABSTRACT

Phosgene is classified as a chemical warfare agent, yet data on its short-duration high concentration toxicity in a nose-only exposure rat model is sparse and inconsistent. Hence, an exposure system for short-term/high concentration exposure was developed and characterized. Herein, we report the median lethal concentration (LC50) for a 10-min nasal exposure of phosgene in a 24-h rat survival model. Male Wistar rats (Envigo) weighing 180-210 g on the day of exposure, were exposed to phosgene gas via nose-only inhalation using a system specifically designed to allow the simultaneous exposure and quantification of phosgene. After 24 h, the surviving rats were euthanized, the lung/body mass ratio determined, and lung tissues analyzed for histopathology. Increased terminal airway edema in the lungs located primarily at the alveoli (resulting in an increased lung/body mass ratio) coincided with the observed mortality. An LC50 value of 129.2 mg/m3 for a 10-min exposure was determined. Furthermore, in agreement with other highly toxic compounds, this study reveals a LC50 concentration value supportive of a nonlinear toxic load model, where the toxic load exponent is >1 (ne = 1.17). Thus, in line with other chemical warfare agents, phosgene toxicity is predicted to be more severe with short-duration, high-concentration exposures than long-duration, low-concentration exposures. This model is anticipated to be refined and developed to screen novel therapeutics against relevant short-term high concentration phosgene exposures expected from a terrorist attack, battlefield deployment, or industrial accident.


Subject(s)
Chemical Warfare Agents/toxicity , Inhalation Exposure/adverse effects , Lung/drug effects , Phosgene/toxicity , Pulmonary Edema/chemically induced , Animals , Dose-Response Relationship, Drug , Inhalation Exposure/analysis , Lethal Dose 50 , Lung/pathology , Male , Pulmonary Edema/pathology , Rats, Wistar , Survival Analysis , Time Factors
5.
Toxicol Appl Pharmacol ; 355: 52-59, 2018 09 15.
Article in English | MEDLINE | ID: mdl-29935281

ABSTRACT

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.


Subject(s)
Chemical Warfare Agents/toxicity , Chemokines/biosynthesis , Cytokines/biosynthesis , Mustard Gas/toxicity , Skin/drug effects , Skin/metabolism , Animals , Ear, External/drug effects , Ear, External/metabolism , Ear, External/pathology , Immunohistochemistry , Inflammation/chemically induced , Inflammation/pathology , Inflammation Mediators/metabolism , Male , Matrix Metalloproteinase 12/biosynthesis , Matrix Metalloproteinase 8/biosynthesis , Mice , RNA/biosynthesis , RNA/genetics , Skin/pathology , Skin Diseases/chemically induced , Skin Diseases/metabolism
6.
Toxicol Lett ; 293: 77-81, 2018 Sep 01.
Article in English | MEDLINE | ID: mdl-29127031

ABSTRACT

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48-61% of mast cells were degranulated. SM exposure (1.4g/m3 in air for 6min) resulted in increased numbers of degranulated mast cells 1-14days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1-14days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1-3days post SM, and from 84% to 44% 7-14days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Degranulation/drug effects , Chemical Warfare Agents/toxicity , Cholinergic Antagonists/pharmacology , Mast Cells/drug effects , Mustard Gas/toxicity , Prodrugs/pharmacology , Skin/cytology , Skin/drug effects , Animals , Choline/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Dermatitis/drug therapy , Indomethacin/pharmacology , Male , Mice , Mice, Hairless , Wound Healing/drug effects
7.
Toxicol Sci ; 160(2): 420-428, 2017 Dec 01.
Article in English | MEDLINE | ID: mdl-28973427

ABSTRACT

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.


Subject(s)
Arsenicals/adverse effects , Chemical Warfare Agents/adverse effects , Cornea/drug effects , Animals , Blister/chemically induced , Blister/metabolism , Blister/pathology , Blood Vessels/drug effects , Blood Vessels/metabolism , Blood Vessels/pathology , Cornea/blood supply , Cornea/metabolism , Cornea/pathology , Corneal Keratocytes/drug effects , Corneal Keratocytes/metabolism , Corneal Keratocytes/pathology , Corneal Neovascularization/chemically induced , Corneal Neovascularization/metabolism , Corneal Neovascularization/pathology , Corneal Pachymetry , Corneal Stroma/drug effects , Corneal Stroma/metabolism , Corneal Stroma/pathology , Cyclooxygenase 2/metabolism , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Epithelium, Corneal/pathology , Interleukin-8/metabolism , Keratitis/chemically induced , Keratitis/metabolism , Keratitis/pathology , Matrix Metalloproteinase 9/metabolism , Rabbits , Risk Assessment , Time Factors , Vascular Endothelial Growth Factor A/metabolism
8.
Toxicol Appl Pharmacol ; 317: 25-32, 2017 02 15.
Article in English | MEDLINE | ID: mdl-28087322

ABSTRACT

Phosgene Oxime (CX), an urticant or nettle agent categorized as a vesicant, is a potential chemical warfare and terrorist weapon. Its exposure can result in widespread and devastating effects including high mortality due to its fast penetration and ability to cause immediate severe cutaneous injury. It is one of the least studied chemical warfare agents with no effective therapy available. Thus, our goal was to examine the acute effects of CX following its cutaneous exposure in SKH-1 hairless mice to help establish a relevant injury model. Results from our study show that topical cutaneous exposure to CX vapor causes blanching of exposed skin with an erythematous ring, necrosis, edema, mild urticaria and erythema within minutes after exposure out to 8h post-exposure. These clinical skin manifestations were accompanied with increases in skin thickness, apoptotic cell death, mast cell degranulation, myeloperoxidase activity indicating neutrophil infiltration, p53 phosphorylation and accumulation, and an increase in COX-2 and TNFα levels. Topical CX-exposure also resulted in the dilatation of the peripheral vessels with a robust increase in RBCs in vessels of the liver, spleen, kidney, lungs and heart tissues. These events could cause a drop in blood pressure leading to shock, hypoxia and death. Together, this is the first report on effects of CX cutaneous exposure, which could help design further comprehensive studies evaluating the acute and chronic skin injuries from CX topical exposure and elucidate the related mechanism of action to aid in the identification of therapeutic targets and mitigation of injury.


Subject(s)
Irritants/toxicity , Oximes/toxicity , Phosgene/toxicity , Skin Diseases/chemically induced , Skin Diseases/pathology , Administration, Cutaneous , Animals , Edema/chemically induced , Edema/pathology , Erythema/chemically induced , Erythema/pathology , Male , Mice , Mice, Hairless , Skin/drug effects , Skin/pathology
9.
Exp Mol Pathol ; 100(3): 522-31, 2016 06.
Article in English | MEDLINE | ID: mdl-27189522

ABSTRACT

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20µmol) or vehicle control was applied onto 6mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6min. This resulted in absorption of approximately 4µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2-3 d, followed by wound healing after 4-5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Indomethacin/analogs & derivatives , Mechlorethamine/toxicity , Prodrugs/pharmacology , Skin/drug effects , Alkylating Agents/toxicity , Animals , Anti-Inflammatory Agents/chemistry , Cholinergic Antagonists/chemistry , Cholinergic Antagonists/pharmacology , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/chemistry , Cyclooxygenase Inhibitors/pharmacology , DNA Damage , Female , Histones/metabolism , Immunohistochemistry , Indomethacin/chemistry , Indomethacin/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Nitric Oxide Synthase Type II/metabolism , Prodrugs/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Skin/injuries , Skin/pathology , Time Factors , Wound Healing/drug effects
10.
Toxicol Appl Pharmacol ; 303: 30-44, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27125198

ABSTRACT

Vesicants including sulfur mustard (SM) and nitrogen mustard (NM) are bifunctional alkylating agents that cause skin inflammation, edema and blistering. This is associated with alterations in keratinocyte growth and differentiation. Endogenous cannabinoids, including N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are important in regulating inflammation, keratinocyte proliferation and wound healing. Their activity is mediated by binding to cannabinoid receptors 1 and 2 (CB1 and CB2), as well as peroxisome proliferator-activated receptor alpha (PPARα). Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH). We found that CB1, CB2, PPARα and FAAH were all constitutively expressed in mouse epidermis and dermal appendages. Topical administration of NM or SM, at concentrations that induce tissue injury, resulted in upregulation of FAAH, CB1, CB2 and PPARα, a response that persisted throughout the wound healing process. Inhibitors of FAAH including a novel class of vanillyl alcohol carbamates were found to be highly effective in suppressing vesicant-induced inflammation in mouse skin. Taken together, these data indicate that the endocannabinoid system is important in regulating skin homeostasis and that inhibitors of FAAH may be useful as medical countermeasures against vesicants.


Subject(s)
Alkylating Agents/toxicity , Chemical Warfare Agents/toxicity , Irritants/toxicity , Mechlorethamine/toxicity , Mustard Gas/toxicity , Skin/drug effects , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/metabolism , Animals , Female , Male , Mice , Mice, Hairless , PPAR alpha/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Skin/metabolism
11.
Cutan Ocul Toxicol ; 35(4): 319-28, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27002633

ABSTRACT

Ocular injury by lewisite (LEW), a potential chemical warfare and terrorist agent, results in edema of eyelids, inflammation, massive corneal necrosis and blindness. To enable screening of effective therapeutics to treat ocular injury from LEW, useful clinically-relevant endpoints are essential. Hence, we designed an efficient exposure system capable of exposing up to six New-Zealand white rabbits at one time, and assessed LEW vapor-induced progression of clinical ocular lesions mainly in the cornea. The right eye of each rabbit was exposed to LEW (0.2 mg/L) vapor for 2.5, 5.0, 7.5 and 10.0 min and clinical progression of injury was observed for 28 days post-exposure (dose-response study), or exposed to same LEW dose for 2.5 and 7.5 min and clinical progression of injury was observed for up to 56 days post-exposure (time-response study); left eye served as an unexposed control. Increasing LEW exposure caused corneal opacity within 6 h post-exposure, which increased up to 3 days, slightly reduced thereafter till 3 weeks, and again increased thereafter. LEW-induced corneal ulceration peaked at 1 day post-exposure and its increase thereafter was observed in phases. LEW exposure induced neovascularization starting at 7 days which peaked at 22-35 days post-exposure, and remained persistent thereafter. In addition, LEW exposure caused corneal thickness, iris redness, and redness and swelling of the conjunctiva. Together, these findings provide clinical sequelae of ocular injury following LEW exposure and for the first time establish clinically-relevant quantitative endpoints, to enable the further identification of histopathological and molecular events involved in LEW-induced ocular injury.


Subject(s)
Arsenicals/adverse effects , Chemical Warfare Agents/toxicity , Corneal Neovascularization/chemically induced , Eye Injuries/chemically induced , Animals , Corneal Neovascularization/pathology , Corneal Opacity/chemically induced , Corneal Opacity/pathology , Eye/drug effects , Eye/pathology , Eye Injuries/pathology , Rabbits
12.
Chem Res Toxicol ; 28(11): 2091-103, 2015 Nov 16.
Article in English | MEDLINE | ID: mdl-26451472

ABSTRACT

The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent inhibition of purified Trx and Trx in A549 lung epithelial cells. Three Trx cross-linked protein complexes were identified in both cytosolic and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes identified both Trx and TrxR, indicating that HN2 cross-linked TrxR and Trx. This is supported by our findings of a significant decrease of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2 treatment. Using purified recombinant enzymes, the formation of protein cross-links and enzyme inhibition were found to be redox status-dependent; reduced Trx was more sensitive to HN2 inactivation than the oxidized enzyme, and Trx/TrxR cross-links were only observed using reduced enzyme. These data suggest that HN2 directly targets catalytic cysteine residues in Trx resulting in enzyme inactivation and protein complex formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine residues on Trx, including Cys32 and Cys35 in the redox center of the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular thiol-disulfide balance, contributing to vesicant-induced lung toxicity.


Subject(s)
Cross-Linking Reagents/toxicity , Mechlorethamine/toxicity , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/metabolism , Cell Line, Tumor , Disulfides/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Insulin/metabolism , Lung/cytology , Models, Molecular , Oxidation-Reduction , Thioredoxins/chemistry , Thioredoxins/genetics
13.
Toxicol Appl Pharmacol ; 280(2): 236-44, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25127551

ABSTRACT

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal-epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Cholinergic Antagonists/therapeutic use , Mustard Gas/toxicity , Skin Diseases/drug therapy , Animals , Cyclooxygenase 2 , Ki-67 Antigen/analysis , Male , Matrix Metalloproteinase 9 , Mice , Mice, Hairless , Skin/pathology , Skin Diseases/chemically induced , Skin Diseases/pathology , Wound Healing/drug effects
14.
Exp Mol Pathol ; 96(3): 316-27, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24662110

ABSTRACT

Sulfur mustard (SM) is a bifunctional alkylating agent causing skin inflammation, edema and blistering. A hallmark of SM-induced toxicity is follicular and interfollicular epithelial damage. In the present studies we determined if SM-induced structural alterations in hair follicles and sebaceous glands were correlated with cell damage, inflammation and wound healing. The dorsal skin of hairless mice was treated with saturated SM vapor. One to seven days later, epithelial cell karyolysis within the hair root sheath, infundibulum and isthmus was apparent, along with reduced numbers of sebocytes. Increased numbers of utriculi, some with connections to the skin surface, and engorged dermal cysts were also evident. This was associated with marked changes in expression of markers of DNA damage (phospho-H2A.X), apoptosis (cleaved caspase-3), and wound healing (FGFR2 and galectin-3) throughout pilosebaceous units. Conversely, fatty acid synthase and galectin-3 were down-regulated in sebocytes after SM. Decreased numbers of hair follicles and increased numbers of inflammatory cells surrounding the utriculi and follicular cysts were noted within the wound 3-7 days post-SM exposure. Expression of phospho-H2A.X, cleaved caspase-3, FGFR2 and galectin-3 was decreased in dysplastic follicular epidermis. Fourteen days after SM, engorged follicular cysts which expressed galectin-3 were noted within hyperplastic epidermis. Galectin-3 was also expressed in basal keratinocytes and in the first few layers of suprabasal keratinocytes in neoepidermis formed during wound healing indicating that this lectin is important in the early stages of keratinocyte differentiation. These data indicate that hair follicles and sebaceous glands are targets for SM in the skin.


Subject(s)
Hair Follicle/drug effects , Mustard Gas/toxicity , Sebaceous Glands/drug effects , Animals , Apoptosis/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , DNA Damage/drug effects , Disease Models, Animal , Down-Regulation , Epithelial Cells/drug effects , Galectin 3/genetics , Galectin 3/metabolism , Hair Follicle/pathology , Histones/genetics , Histones/metabolism , Keratinocytes/drug effects , Male , Mice , Mice, Hairless , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Sebaceous Glands/pathology , Skin/drug effects , Skin/pathology , Wound Healing/drug effects
15.
Chem Res Toxicol ; 27(1): 61-75, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24274902

ABSTRACT

Oxidative stress plays a key role in mechlorethamine (methylbis(2-chloroethyl)amine, HN2) toxicity. The thioredoxin system, consisting of thioredoxin reductase (TrxR), thioredoxin, and NADPH, is important in redox regulation and protection against oxidative stress. HN2 contains two electrophilic side chains that can react with nucleophilic sites in proteins, leading to changes in their structure and function. We report that HN2 inhibits the cytosolic (TrxR1) and mitochondrial (TrxR2) forms of TrxR in A549 lung epithelial cells. TrxR exists as homodimers under native conditions; monomers can be detected by denaturing and reducing SDS-PAGE followed by western blotting. HN2 treatment caused marked decreases in TrxR1 and TrxR2 monomers along with increases in dimers and oligomers under reducing conditions, indicating that HN2 cross-links TrxR. Cross-links were also observed in rat lung after HN2 treatment. Using purified TrxR1, NADPH reduced, but not oxidized, enzyme was inhibited and cross-linked by HN2. LC-MS/MS analysis of TrxR1 demonstrated that HN2 adducted cysteine- and selenocysteine-containing redox centers forming monoadducts, intramolecule and intermolecule cross-links, resulting in enzyme inhibition. HN2 cross-links two dimeric subunits through intermolecular binding to cysteine 59 in one subunit of the dimer and selenocysteine 498 in the other subunit, confirming the close proximity of the N- and C-terminal redox centers of adjacent subunits. Despite cross-linking and inhibition of TrxR activity by HN2, TrxR continued to mediate menadione redox cycling and generated reactive oxygen species. These data suggest that disruption of the thioredoxin system contributes to oxidative stress and tissue injury induced by HN2.


Subject(s)
Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Disulfides/antagonists & inhibitors , Epithelial Cells/drug effects , Lung/cytology , Lung/drug effects , Mechlorethamine/chemistry , Mechlorethamine/pharmacology , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Thioredoxin-Disulfide Reductase/chemistry , Animals , Cells, Cultured , Cross-Linking Reagents/metabolism , Disulfides/chemistry , Disulfides/metabolism , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Humans , Lung/enzymology , Lung/metabolism , Male , Mechlorethamine/metabolism , Models, Molecular , Oxidation-Reduction/drug effects , Rats , Rats, Wistar , Thioredoxin-Disulfide Reductase/metabolism
16.
Toxicol Appl Pharmacol ; 268(2): 178-87, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23357548

ABSTRACT

The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24h to 168h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury.


Subject(s)
Chemical Warfare Agents/toxicity , Endoplasmic Reticulum Stress/drug effects , Mustard Gas/toxicity , Skin/drug effects , Animals , Apoptosis/drug effects , Ear , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/analysis , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/pathology , Laminin/biosynthesis , Male , Mice , Models, Animal , Skin/pathology , Transcription Factor CHOP/analysis , Wound Healing
17.
Exp Mol Pathol ; 91(2): 515-27, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21672537

ABSTRACT

Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1-14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.


Subject(s)
DNA Damage , Inflammation/pathology , Mustard Gas/toxicity , Skin/drug effects , Skin/pathology , Animals , Apoptosis/drug effects , Biomarkers/metabolism , Caspase 3/metabolism , Cell Degranulation/drug effects , Cell Differentiation/drug effects , Cyclooxygenase 2/metabolism , Histones/metabolism , Keratinocytes/drug effects , Keratinocytes/pathology , Keratins/metabolism , Male , Mast Cells/drug effects , Mast Cells/pathology , Mast Cells/physiology , Mice , Mice, Hairless , Peroxidase/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Skin/enzymology , Staining and Labeling , Wound Healing/drug effects
18.
Toxicol Appl Pharmacol ; 253(2): 112-20, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21457723

ABSTRACT

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT™). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000µM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-ß-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.


Subject(s)
Caveolae/physiology , HSP27 Heat-Shock Proteins/analysis , HSP70 Heat-Shock Proteins/analysis , Mustard Gas/analogs & derivatives , Skin/drug effects , Animals , Caveolin 1/analysis , Heat-Shock Proteins , Humans , MAP Kinase Signaling System , Mice , Molecular Chaperones , Mustard Gas/toxicity , Skin/chemistry
19.
Toxicol Appl Pharmacol ; 249(2): 178-87, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20840853

ABSTRACT

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 µM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 µM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE2 synthases, leukotriene (LT) A4 hydrolase and LTC4 synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.


Subject(s)
Irritants/toxicity , Mustard Gas/analogs & derivatives , Skin/drug effects , Biomarkers/metabolism , Blotting, Western , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Eicosanoids/biosynthesis , Histones/biosynthesis , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Mustard Gas/toxicity , Poly Adenosine Diphosphate Ribose/biosynthesis , Proliferating Cell Nuclear Antigen/biosynthesis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Time Factors
20.
Toxicol Appl Pharmacol ; 245(3): 352-60, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20382172

ABSTRACT

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 microM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A(4) (LTA(4)) hydrolase and leukotriene C(4) (LTC(4)) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA(4) hydrolase and LTC(4) synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.


Subject(s)
Antioxidants/metabolism , Chemical Warfare Agents/toxicity , Inflammation Mediators/metabolism , Inflammation/chemically induced , Irritants/toxicity , Keratinocytes/drug effects , Mitogen-Activated Protein Kinases/metabolism , Mustard Gas/analogs & derivatives , Oxidative Stress/drug effects , Animals , Cell Line , Dose-Response Relationship, Drug , Eicosanoids/metabolism , Enzyme Activation , Gene Expression Regulation, Enzymologic/drug effects , Inflammation/enzymology , Inflammation/immunology , Keratinocytes/enzymology , Keratinocytes/immunology , Mice , Mustard Gas/toxicity , Protein Carbonylation/drug effects
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