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Ocular tissue, especially the cornea, is overly sensitive to chemical exposures. The availability and adoption of chemical threat agent chloropicrin (CP) is growing in the United States as a pesticide and fumigant; thereby increasing the risk of its use in warfare, terrorist attacks and non-intentional exposure. Exposure to CP results in immediate ocular, respiratory, and dermal injury; however, we lack knowledge on its mechanism of toxicity as well as of its breakdown products like chlorine and phosgene, and effective therapies are elusive. Herein, we have reviewed the recent findings on exposure route, toxicity and likely mechanisms of CP induced ocular toxicity based on other vesicating chemical warfare agents that cause ocular injury. We have focused on the implication of their toxicity and mechanistic outcomes in the ocular tissue, especially the cornea, which could be useful in the development of broad-spectrum effective therapeutic options. We have discussed on the potential countermeasures, overall hallmarks and challenges involved in studying ocular injuries from chemical threat agent exposures. Finally, we reviewed useful available technologies and methods that can assist in the identification of effective medical countermeasures for chemical threat agents related ocular injuries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phosgene oxime (CX), categorized as a vesicating chemical threat agent, causes effects that resemble an urticant or nettle agent. CX is an emerging potential threat agent that can be deployed alone or with other chemical threat agents to enhance their toxic effects. Studies on CX-induced skin toxicity, injury progression, and related biomarkers are largely unknown. To study the physiologic changes, skin clinical lesions and their progression, skin exposure of SKH-1 and C57BL/6 mice was carried out with vapor from 10 μ l CX for 0.5-minute or 1.0-minute durations using a designed exposure system for consistent CX vapor exposure. One-minute exposure caused sharp (SKH-1) or sustained (C57BL/6) decrease in respiratory and heart rate, leading to mortality in both mouse strains. Both exposures caused immediate blanching, erythema with erythematous ring (wheel) and edema, and an increase in skin bifold thickness. Necrosis was also observed in the 0.5-minute CX exposure group. Both mouse strains showed comparative skin clinical lesions upon CX exposure; however, skin bifold thickness and erythema remained elevated up to 14 days postexposure in SKH-1 mice but not in C57BL/6 mice. Our data suggest that CX causes immediate changes in the physiologic parameters and gross skin lesions resembling urticaria, which could involve mast cell activation and intense systemic toxicity. This novel study recorded and compared the progression of skin injury to establish clinical biomarkers of CX dermal exposure in both the sexes of two murine strains relevant for skin and systemic injury studies and therapeutic target identification. SIGNIFICANCE STATEMENT: Phosgene oxime (CX), categorized as a vesicating agent, is considered as a potent chemical weapon and is of high military and terrorist threat interest since it produces rapid onset of severe injury as an urticant. However, biomarkers of clinical relevance related to its toxicity and injury progression are not studied. Data from this study provide useful clinical markers of CX skin toxicity in mouse models using a reliable CX exposure system for future mechanistic and efficacy studies., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [∼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7)., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers., (U.S. Government work not protected by U.S. copyright.)

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-β-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ocular tissue is highly sensitive to chemical exposures. Chloropicrin (CP), a choking agent employed during World War I and currently a popular pesticide and fumigating agent, is a potential chemical threat agent. Accidental, occupational, or intentional exposure to CP results in severe ocular injury, especially to the cornea; however, studies on ocular injury progression and underlying mechanisms in a relevant in vivo animal model are lacking. This has impaired the development of effective therapies to treat the acute and long-term ocular toxicity of CP. To study the in vivo clinical and biological effects of CP ocular exposure, we tested different CP exposure doses and durations in mice. These exposures will aid in the study of acute ocular injury and its progression as well as identify a moderate dose to develop a relevant rodent ocular injury model with CP. The left eyes of male BALB/c mice were exposed to CP (20% CP for 0.5 or 1 min or 10% CP for 1 min) using a vapor cap, with the right eyes serving as controls. Injury progression was evaluated for 25 days post-exposure. CP-exposure caused a significant corneal ulceration and eyelid swelling which resolved by day 14 post exposure. In addition, CP-exposure caused significant corneal opacity and neovascularization. Development of hydrops (severe corneal edema with corneal bullae) and hyphema (blood accumulation in the anterior chamber) was observed as advanced CP effects. Mice were euthanized at day 25 post-CP-exposure, and the eyes were harvested to further study the corneal injury. Histopathological analyses showed a significant CP-induced decrease in corneal epithelial thickness and increased stromal thickness with more pronounced damage, including stromal fibrosis, edema, neovascularization, trapped epithelial cells, anterior and posterior synechiae, and infiltration of inflammatory cells. Loss of the corneal endothelial cells and Descemet's membrane could be associated with the CP-induced corneal edema and hydrops which could lead to long term term pathological conditions. Although exposure to 20% CP for 1 min caused more eyelid swelling, ulceration, and hyphema, similar effects were observed with all CP exposures. These novel findings following CP ocular exposure in a mouse model outline the corneal histopathologic changes that associate with the continuing ocular clinical effects. The data are useful in designing further studies to identify and correlate the clinical and biological markers of CP ocular injury progression with acute and long-term toxic effects on cornea and other ocular tissues. We take a crucial step towards CP ocular injury model development and in pathophysiological studies to identify molecular targets for therapeutic interventions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Over 40% of veterans from the Persian Gulf War (GW) (1990-1991) suffer from Gulf War Illness (GWI). Thirty years since the GW, the exposure and mechanism contributing to GWI remain unclear. One possible exposure that has been attributed to GWI are chemical warfare agents (CWAs). While there are treatments for isolated symptoms of GWI, the number of respiratory and cognitive/neurological issues continues to rise with minimum treatment options. This issue does not only affect veterans of the GW, importantly these chronic multisymptom illnesses (CMIs) are also growing amongst veterans who have served in the Afghanistan-Iraq war. What both wars have in common are their regions and inhaled exposures. In this review, we will describe the CWA exposures, such as sarin, cyclosarin, and mustard gas in both wars and discuss the various respiratory and neurocognitive issues experienced by veterans. We will bridge the respiratory and neurological symptoms experienced to the various potential mechanisms described for each CWA provided with the most up-to-date models and hypotheses.

Sulfur mustard (SM) has been widely used as a chemical warfare agent including most recently in Syria. Mice exposed to SM exhibit an increase in pro-inflammatory cytokines followed by immune cell infiltration in the lung, however, the mechanisms leading to these inflammatory responses has not been completely elucidated. Mast cells are one of the first responding innate immune cells found at the mucosal surfaces of the lung and have been reported to be activated by SM in the skin. Therefore, we hypothesized that nitrogen mustard (NM: a surrogate for SM) exposure promotes activation of mast cells causing chronic respiratory inflammation. To assess the role of mast cells in NM-mediated pulmonary toxicity, we compared the effects of NM exposure between C57BL/6 and B6.Cg-KitW-sh/HNihrJaeBsmJ (KitW-sh; mast cell deficient) mice. Lung injury was observed in C57BL/6J mice following NM exposure (0.125 mg/kg) at 72 h, which was significantly abrogated in KitW-sh mice. Although both strains exhibited damage from NM, C57BL/6J mice had higher inflammatory cell infiltration and more elevated prostaglandin D2 (PGD2) present in bronchoalveolar lavage fluid compared with KitW-sh mice. Additionally, we utilized murine bone marrow-derived mast cells to assess NM-induced early and late activation. Although NM exposure did not result in mast cell degranulation, we observed an upregulation in PGD2 and IL-6 levels following exposure to NM. Results suggest that mast cells play a prominent role in lung injury induced by NM and may contribute to the acute and potentially long-term lung injury observed caused by SM., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions., Competing Interests: The authors have declared that no competing interests exist.

With a possibility for the use of chemical weapons in battlefield or in terrorist activities, effective therapies against the devastating ocular injuries, from their exposure, are needed. Oxygen plays a vital role in ocular tissue preservation and wound repair. We tested the efficacy of supersaturated oxygen emulsion (SSOE) in reducing ex vivo corneal and keratocyte injury from chloropicrin (CP). CP, currently used as a pesticide, is a chemical threat agent like the vesicating mustard agents and causes severe corneal injury. Since our previous study in human corneal epithelial cells showed the treatment potential of SSOE (55 %), we further tested its efficacy in an ex vivo CP-induced rabbit corneal injury model. Corneas were exposed to CP (700 nmol) for 2 h, washed and cultured with or without SSOE for 24 h or 96 h. At 96 h post CP exposure, SSOE treatment presented a healing tendency of the corneal epithelial layer, and abrogated the CP-induced epithelial apoptotic cell death. SSOE treatment also reduced the CP induced DNA damage (H2A.X phosphorylation) and inflammatory markers (e.g. MMP9, IL-21, MIP-1β, TNFα). Further examination of the treatment efficacy of SSOE alone or in combination with other therapies in in vivo cornea injury models for CP and vesicants, is warranted., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Highly toxic industrial chemicals that are widely accessible, and hazardous chemicals like phosgene oxime (CX) that can be easily synthesized, pose a serious threat as potential chemical weapons. In addition, their accidental release can lead to chemical emergencies and mass casualties. CX, an urticant, or nettle agent, grouped with vesicating agents, causes instant pain, injury and systemic effects, which can lead to mortality. With faster cutaneous penetration, corrosive properties, and more potent toxicity compared to other vesicating agents, CX causes instantaneous and severe tissue damage. CX, a potential chemical terrorism threat agent, could therefore be weaponized with other chemical warfare agents to enhance their harmful effects. CX is the least studied vesicant and its acute and long-term toxic effects as well as its mechanism of action are largely unknown. This has hampered the identification of therapeutic targets and the development of effective medical countermeasures. There are only protective measures, decontamination, and supportive treatments available for reducing the toxic effects from CX exposure. This review summarizes CX toxicity, its known mechanism of action, and our current studies exploring the role of mast cell activation and associated signaling pathways in CX cutaneous exposure under the National Institutes of Health Countermeasures Against Chemical Threats program. Potential treatment options and the development of effective targeted countermeasures against CX-induced morbidity and mortality is also discussed.

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0-100 μM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Introduction: Roughly 13% of all battlefield injuries include some form of ocular trauma. Ocular tissue preservation is critical for wound healing for warfighters with ocular injuries. Our team hypothesized that oxygen plays a vital role in ocular tissue preservation and wound healing and has developed a supersaturated oxygen emulsion (SOE) for the topical treatment of ocular trauma., Materials and Methods: The partial pressure of oxygen (PO2) was measured in the SOE. Safety and efficacy studies were carried out in primary human corneal epithelial (HCE) cells, as the outermost layer is the first barrier to chemical and mechanical injury. Western blot, scratch assay, and MTT assays were conducted to determine the effect of the SOE on various molecular markers, the rate of scratch closure, and cellular viability, respectively., Results: Data indicate that the SOE releases oxygen in a time-dependent manner, reaching a partial pressure within the emulsion over four times atmospheric levels. Studies in HCE cells indicate that application of the SOE does not lead to DNA damage, promote cell death, or hinder the rate of scratch closure and enhances cellular viability. Preliminary studies were carried out with chloropicrin (CP; developed as a chemical warfare agent and now a commonly used pesticide) as a chemical agent to induce ocular injury in HCE cells. CP exposures showed that SOE treatment reverses CP-induced DNA damage, apoptotic cell death, and oxidative stress markers., Conclusions: Maintaining adequate tissue oxygenation is critical for tissue preservation and wound repair, especially in avascular tissues like the cornea. Further studies examining the application of the SOE in corneal injury models are warranted., (© Association of Military Surgeons of the United States 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metalloproteinase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phosgene Oxime (CX, Cl 2 CNOH), a halogenated oxime, is a potent chemical weapon that causes immediate acute injury and systemic effects. CX, grouped together with vesicating agents, is an urticant or nettle agent with highly volatile, reactive, corrosive, and irritating vapor, and has considerably different chemical properties and toxicity compared to other vesicants. CX is absorbed quickly through clothing with faster cutaneous penetration compared to other vesicating agents causing instantaneous and severe damage. For this reason, it could be produced as a weaponized mixture with other chemical warfare agents to enhance their deleterious effects. The immediate devastating effects of CX and easy synthesis makes it a dangerous chemical with both military and terrorist potentials. Although CX is the most potent vesicating agent, it is one of the least studied chemical warfare agents and the pathophysiology as well as long term effects are largely unknown. CX exposure results in immediate pain and inflammation, and it mainly affects skin, eye and respiratory system. There are no antidotes available against CX-induced injury and the treatment is only supportive. This review summarizes existing knowledge regarding exposure, toxicity and the probable underlying mechanisms of CX compared to other important vesicants' exposure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vesicating agent, Sulfur mustard (SM), causes devastating eye injury; however, there are no effective antidotes available. Using nitrogen mustard (NM), a bi-functional analog of SM, we have earlier reported that NM-induced corneal injury in ex vivo rabbit cornea organ culture model parallels corneal injury reported with SM. Using this model, we have demonstrated the therapeutic efficacy of dexamethasone (DEX), doxycycline (DOX) and silibinin (SB) in reversing NM (2h exposure)-induced corneal injuries when added immediately after washing NM. In the present study, we further examined the efficacy of similar/higher doses of these agents when added immediately, 2, or 4h after washing NM following its 2h exposure. All three treatment agents caused a reversal in established NM-induced injury biomarkers when added immediately or 2h after washing NM following its 2h exposure; however, when treatments were carried out 4h after washing NM, there was no significant effect. Together, our results further show the beneficial effect of these agents in reversing NM-induced corneal injury and indicate the time window for effective treatment. This could be useful towards future development of targeted therapeutics against vesicant-induced ocular injury., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Exposure to the vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) causes severe skin injury with delayed blistering. Depending upon the dose and time of their exposure, edema and erythema develop into blisters, ulceration, necrosis, desquamation, and pigmentation changes, which persist weeks and even years after exposure. Research advances have generated data that have started to explain the probable mechanism of action of vesicant-induced skin toxicity; however, despite these advances, effective and targeted therapies are still deficient. This review highlights studies on two SM analogs, 2-chloroethyl ethyl sulfide (CEES) and NM, and CEES- and NM-induced skin injury mouse models that have substantially added to the knowledge on the complex pathways involved in mustard vesicating agent-induced skin injury. Furthermore, employing these mouse models, studies under the National Institutes of Health Countermeasures Against Chemical Threats program have identified the flavanone silibinin as a novel therapeutic intervention with the potential to be developed as an effective countermeasure against skin injury following exposure to mustard vesicating agents., (© 2016 New York Academy of Sciences.)

The vesicating agents sulfur mustard (SM) and lewisite (LEW) are potent chemical warfare agents that primarily cause damage to the ocular, skin, and respiratory systems. However, ocular tissue is the most sensitive organ, and vesicant exposure results in a biphasic injury response, including photophobia, corneal lesions, corneal edema, ulceration, and neovascularization, and may cause loss of vision. There are several reports on ocular injury from exposure to SM, which has been frequently used in warfare. However, there are very few reports on ocular injury by LEW, which indicate that injury symptoms appear instantly after exposure and faster than SM. In spite of extensive research efforts, effective therapies for vesicant-induced ocular injuries, mainly to the most affected corneal tissue, are not available. Hence, we have established primary human corneal epithelial cells and rabbit corneal organ culture models with the SM analog nitrogen mustard, which have helped to test the efficacy of potential therapeutic agents. These agents will then be further evaluated against in vivo SM- and LEW-induced corneal injury models, which will assist in the development of potential broad-spectrum therapies against vesicant-induced ocular injuries., (© 2016 New York Academy of Sciences.)

Purpose: To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to the vesicating agent, nitrogen mustard (NM), a bifunctional alkylating analog of the chemical warfare agent sulfur mustard., Methods: Toxic effects and associated mechanisms were examined in maximally affected corneal tissue using corneal cultures and human corneal epithelial (HCE) cells exposed to NM., Results: Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation, and levels of vascular endothelial growth factor, cyclooxygenase 2, and matrix metalloproteinase-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53, and H2A.X ser139 levels. NM exposure also induced caspase-3 and poly ADP ribose polymerase cleavage, suggesting their involvement in NM-induced apoptotic death in the rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in cyclooxygenase 2, matrix metalloproteinase-9, and vascular endothelial growth factor levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation, and neovascularization. NM exposure also induced activation of activator protein 1 transcription factor proteins and upstream signaling pathways including mitogen-activated protein kinases and Akt protein kinase, suggesting that these could be key factors involved in NM-induced corneal injury., Conclusions: Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant-induced ocular injuries, which could be helpful in the development of targeted therapies.

Our recent studies in SKH-1 hairless mice have demonstrated that topical exposure to nitrogen mustard (NM), an analog of sulfur mustard (SM), triggers the inflammatory response, microvesication and apoptotic cell death. Here, we sought to identify the mechanism/s involved in these NM-induced injury responses. Results obtained show that NM exposure of SKH-1 hairless mouse skin caused H2A.X and p53 phosphorylation and increased p53 accumulation, indicating DNA damage. In addition, NM also induced the activation of MAPKs/ERK1/2, JNK1/2 and p38 as well as that of Akt together with the activation of transcription factor AP1. Also, NM exposure induced robust expression of pro-inflammatory mediators namely cyclooxygenase 2 and inducible nitric oxide synthase and cytokine tumor necrosis factor alpha, and increased the levels of proteolytic mediator matrix metalloproteinase 9. NM exposure of skin also increased lipid peroxidation, 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation, protein and DNA oxidation indicating an elevated oxidative stress. We also found NM-induced increase in the homologous recombinant repair pathway, suggesting its involvement in the repair of NM-induced DNA damage. Collectively, these results indicate that NM induces oxidative stress, mainly a bi-phasic response in DNA damage and activation of MAPK and Akt pathways, which activate transcription factor AP1 and induce the expression of inflammatory and proteolytic mediators, contributing to the skin injury response by NM. In conclusion, this study for the first time links NM-induced mechanistic changes with our earlier reported murine skin injury lesions with NM, which could be valuable to identify potential therapeutic targets and rescue agents., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Currently, there is no effective antidote to prevent skin injuries by sulfur mustard (SM) and nitrogen mustard (NM), which are vesicating agents with potential relevance to chemical warfare, terrorist attacks, or industrial/laboratory accidents. Our earlier report has demonstrated the therapeutic efficacy of silibinin, a natural flavanone, in reversing monofunctional alkylating SM analog 2-chloroethyl ethyl sulfide-induced toxic effects in mouse skin. To translate this effect to a bifunctional alkylating vesicant, herein, efficacy studies were carried out with NM. Topical application of silibinin (1 or 2mg) 30 min after NM exposure on the dorsal skin of male SKH-1 hairless mice significantly decreased NM-induced toxic lesions at 24, 72 or 120 h post-exposure. Specifically, silibinin treatment resulted in dose-dependent reduction of NM-induced increase in epidermal thickness, dead and denuded epidermis, parakeratosis and microvesication. Higher silibinin dose also caused a 79% and 51%reversal in NM-induced increases in myeloperoxidase activity and COX-2 levels, respectively. Furthermore, silibinin completely prevented NM-induced H2A.X phosphorylation, indicating reversal of DNA damage which could be an oxidative DNA damage as evidenced by high levels of 8-oxodG in NM-exposed mouse skin that was significantly reversed by silibinin. Together, these findings suggest that attenuation of NM-induced skin injury by silibinin is due to its effects on the pathways associated with DNA damage, inflammation, vesication and oxidative stress. In conclusion, results presented here support the optimization of silibinin as an effective treatment of skin injury by vesicants., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) are reported to be easily absorbed by skin upon exposure causing severe cutaneous injury and blistering. Our studies show that topical exposure of NM (3.2mg) onto SKH-1 hairless mouse skin, not only caused skin injury, but also led to significant body weight loss and 40-80% mortality (120 h post-exposure), suggesting its systemic effects. Accordingly, further studies herein show that NM exposure initiated an increase in circulating white blood cells by 24h (neutrophils, eosinophils and basophils) and thereafter a decrease (neutrophils, lymphocytes and monocytes). NM exposure also reduced both white and red pulp areas of the spleen. In the small intestine, NM exposure caused loss of membrane integrity of the surface epithelium, abnormal structure of glands and degeneration of villi. NM exposure also resulted in the dilation of glomerular capillaries of kidneys, and an increase in blood urea nitrogen/creatinine ratio. Our results here with NM are consistent with earlier reports that exposure to higher SM levels can cause damage to the hematopoietic system, and kidney, spleen and gastrointestinal tract toxicity. These outcomes will add to our understanding of the toxic effects of topical vesicant exposure, which might be helpful towards developing effective countermeasures against injuries from acute topical exposures., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Our previous studies and other published reports on the chemical warfare agent sulfur mustard (SM) and its analog 2-chloroethyl ethyl sulfide (CEES) have indicated a role of oxidative stress in skin injuries caused by these vesicating agents. We examined the effects of the catalytic antioxidant AEOL 10150 in the attenuation of CEES-induced toxicity using our established skin injury models (skin epidermal cells and SKH-1 hairless mice) to validate the role of oxidative stress in the pathophysiology of mustard vesicating agents. Treatment of mouse epidermal JB6 and human HaCaT cells with AEOL 10150 (50μM) 1h post-CEES exposure resulted in significant (p < 0.05) reversal of CEES-induced decreases in both cell viability and DNA synthesis. Similarly, AEOL 10150 treatment 1h after CEES exposure attenuated CEES-induced DNA damage in these cells. Similar AEOL 10150 treatments also caused significant (p < 0.05) reversal of CEES-induced decreases in cell viability in normal human epidermal keratinocytes. Cytoplasmic and mitochondrial reactive oxygen species measurements showed that AEOL 10150 treatment drastically ameliorated the CEES-induced oxidative stress in both JB6 and HaCaT cells. Based on AEOL 10150 pharmacokinetic studies in SKH-1 mouse skin, mice were treated with a topical formulation plus subcutaneous injection (5mg/kg) of AEOL 10150 1h after CEES (4mg/mouse) exposure and every 4h thereafter for 12h. This AEOL 10150 treatment regimen resulted in over 50% (p < 0.05) reversal of CEES-induced skin bi-fold and epidermal thickness, myeloperoxidase activity, and DNA oxidation in mouse skin. Results from this study demonstrate the potential therapeutic efficacy of AEOL 10150 against CEES-mediated cutaneous lesions, supporting AEOL 10150 as a medical countermeasure against SM-induced skin injuries., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The pathologic mechanisms of skin injuries, following the acute inflammatory response induced by vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) exposure, are poorly understood. Neutrophils which accumulate at the site of injury, abundantly express myeloperoxidase (MPO), a heme protein that is implicated in oxidant-related antimicrobial and cytotoxic responses. Our previous studies have shown that exposure to SM analog 2-chloroethyl ethyl sulfide (CEES) or NM results in an inflammatory response including increased neutrophilic infiltration and MPO activity. To further define the role of neutrophil-derived MPO in NM-induced skin injury, here we used a genetic approach and examined the effect of NM exposure (12h and 24h) on previously established injury endpoints in C57BL/6J wild type (WT) and B6.129X1-MPOtm1Lus/J mice (MPO KO), homozygous null for MPO gene. NM exposure caused a significant increase in skin bi-fold thickness, epidermal thickness, microvesication, DNA damage and apoptosis in WT mice compared to MPO KO mice. MPO KO mice showed relatively insignificant effect. Similarly, NM induced increases in the expression of inflammatory and proteolytic mediators, including COX-2, iNOS and MMP-9 in WT mice, while having a significantly lower effect in MPO KO mice. Collectively, these results show that MPO, which generates microbicidal oxidants, plays an important role in NM-induced skin injuries. This suggests the development of mechanism-based treatments against NM- and SM-induced skin injuries that inhibit MPO activity and attenuate MPO-derived oxidants., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sulfur mustard (SM) is a vesicant warfare agent which causes severe skin injuries. Currently, we lack effective antidotes against SM-induced skin injuries, in part due to lack of appropriate animal model(s) that can be used for efficacy studies in laboratory settings to identify effective therapies. Therefore, to develop a relevant mouse skin injury model, we examined the effects of nitrogen mustard (NM), a primary vesicant and a bifunctional alkylating agent that induces toxic effects comparable to SM. Specifically, we conducted histopathological and immunohistochemical evaluation of several applicable cutaneous pathological lesions following skin NM (3.2mg) exposure for 12-120h in SKH-1 and C57BL/6 mice. NM caused a significant increase in epidermal thickness, incidence of microvesication, cell proliferation, apoptotic cell death, inflammatory cells (neutrophils, macrophages and mast cells) and myleoperoxidase activity in the skin of both mouse strains. However, there was a more prominent NM-induced increase in epidermal thickness, and macrophages and mast cell infiltration, in SKH-1 mice relative to what was seen in C57BL/6 mice. NM also caused collagen degradation and edema at early time points (12-24h); however, at later time points (72 and 120h), dense collagen staining was observed, indicating either water loss or start of integument repair in both the mouse strains. This study provides quantitative measurement of NM-induced histopathological and immunohistochemical cutaneous lesions in both hairless and haired mouse strains that could serve as useful tools for screening and identification of effective therapies for treatment of skin injuries due to NM and SM., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

To identify effective therapies against sulfur mustard (SM)-induced skin injuries, various animals have been used to assess the cutaneous pathology and related histopathological changes of SM injuries. However, these efforts to establish relevant skin injury endpoints for efficacy studies have been limited mainly due to the restricted assess of SM. Therefore, we employed the SM analog nitrogen mustard (NM), a primary vesicating and bifunctional alkylating agent, to establish relevant endpoints for efficient efficacy studies. Our published studies show that NM (3.2 mg) exposure for 12-120 h in both the hairless SKH-1 and haired C57BL/6 mice caused clinical sequelae of toxicity similar to SM exposure in humans. The NM-induced cutaneous pathology-related structural changes were further analyzed in this study and quantified morphometrically (as percent length or area of epidermis or dermis) of skin sections in mice showing these lesions. H&E stained skin sections of both hairless and haired mice showed that NM (12-120 h) exposure caused epidermal histopathological effects such as increased epidermal thickness, epidermal-dermal separation, necrotic/dead epidermis, epidermal denuding, scab formation, parakeratosis (24-120 h), hyperkeratosis (12-120 h), and acanthosis with hyperplasia (72-120 h). Similar NM exposure in both mice caused dermal changes including necrosis, edema, increase in inflammatory cells, and red blood cell extravasation. These NM-induced cutaneous histopathological features are comparable to the reported lesions from SM exposure in humans and animal models. This study advocates the usefulness of these histopathological parameters observed due to NM exposure in screening and optimization of rescue therapies against NM and SM skin injuries.

Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/-). Exposure to NM (3.2mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/- mice at 24h. However, by 72h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/- mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24h persisting through 72h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72h after exposure in p53+/- mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/- mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A paucity of clinically applicable biomarkers to screen therapies in laboratory is a limitation in the development of countermeasures against cutaneous injuries by chemical weapon, sulfur mustard (SM), and its analog nitrogen mustard (NM). Consequently, we assessed NM-caused progression of clinical cutaneous lesions; notably, skin injury with NM is comparable to SM. Exposure of SKH-1 hairless and C57BL/6 (haired) mice to NM (3.2 mg) for 12-120 h caused clinical sequelae of toxicity, including microblister formation, edema, erythema, altered pigmentation, wounding, xerosis and scaly dry skin. These toxic effects of NM were similar in both mouse strains, except that wounding and altered pigmentation at 12-24 h and appearance of dry skin at 24 and 72 h post-NM exposure were more pronounced in C57BL/6 compared to SKH-1 mice. Conversely, edema, erythema and microblister formation were more prominent in SKH-1 than C57BL/6 mice at 24-72 h after NM exposure. In addition, 40-60% mortality was observed following 120 h of NM exposure in the both mouse strains. Overall, these toxic effects of NM are comparable to those reported in humans and other animal species with SM, and thus represent clinically-relevant cutaneous injury endpoints in screening and optimization of therapies for skin injuries by vesicating agents.

There are no effective and approved therapies against devastating ocular injuries caused by vesicating chemical agents sulfur mustard (SM) and nitrogen mustard (NM). Herein, studies were carried out in rabbit corneal cultures to establish relevant ocular injury biomarkers with NM for screening potential efficacious agents in laboratory settings. NM (100nmol) exposure of the corneas for 2h (cultured for 24h), showed increases in epithelial thickness, ulceration, apoptotic cell death, epithelial detachment microbullae formation, and the levels of VEGF, cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9). Employing these biomarkers, efficacy studies were performed with agent treatments 2h and every 4h thereafter, for 24h following NM exposure. Three agents were evaluated, including prescription drugs dexamethasone (0.1%; anti-inflammatory steroid) and doxycycline (100nmol; antibiotic and MMP inhibitor) that have been studied earlier for treating vesicant-induced eye injuries. We also examined silibinin (100μg), a non-toxic natural flavanone found to be effective in treating SM analog-induced skin injuries in our earlier studies. Treatments of doxycycline+dexamethasone, and silibinin were more effective than doxycycline or dexamethasone alone in reversing NM-induced epithelial thickening, microbullae formation, apoptotic cell death, and MMP-9 elevation. However, dexamethasone and silibinin alone were more effective in reversing NM-induced VEGF levels. Doxycycline, dexamethasone and silibinin were all effective in reversing NM-induced COX-2 levels. Apart from therapeutic efficacy of doxycycline and dexamethasone, these results show strong multifunctional efficacy of silibinin in reversing NM-induced ocular injuries, which could help develop effective and safe therapeutics against ocular injuries by vesicants., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants.

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9-48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Sulfur mustard (HD) is a vesicating agent that has been used as a chemical warfare agent in a number of conflicts, posing a major threat in both military conflict and chemical terrorism situations. Currently, we lack effective therapies to rescue skin injuries by HD, in part, due to the lack of appropriate animal models, which are required for conducting laboratory studies to evaluate the therapeutic efficacy of promising agents that could potentially be translated in to real HD-caused skin injury. To address this challenge, the present study was designed to assess whether microvesication could be achieved in mouse skin by an HD analog 2-chloroethyl ethyl sulfide (CEES) exposure; notably, microvesication is a key component of HD skin injury in humans. We found that skin exposure of male SKH-1 hairless mice to CEES caused epidermal-dermal separation indicating microvesication. In other studies, CEES exposure also caused an increase in skin bi-fold thickness, wet/dry weight ratio, epidermal thickness, apoptotic cell death, cell proliferation, and infiltration of macrophages, mast cells and neutrophils in male SKH-1 hairless mouse skin. Taken together, these results establish CEES-induced microvesication and inflammation-related histopathological changes in mouse skin, providing a potentially relevant laboratory model for developing effective countermeasures against HD skin injury in humans., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Exposure to chemical warfare agent sulfur mustard (HD) is reported to cause GSH depletion, which plays an important role in HD-linked oxidative stress and skin injury. Using the HD analog 2-chloroethyl ethyl sulfide (CEES), we evaluated the role of GSH and its efficacy in ameliorating CEES-caused skin injury. Using mouse JB6 and human HaCaT epidermal keratinocytes, we observed both protective and therapeutic effects of exogenous GSH (1 or 10 mM) in attenuating a CEES-caused decrease in cell viability and DNA synthesis, as well as S and G(2)M phase arrest in cell cycle progression. However, the protective effect of GSH was stronger than its ability to reverse CEES-induced cytotoxic effect. The observed effect of GSH could be associated with an increase in intracellular GSH levels after its treatment before or after CEES exposure, which strongly depleted cellular GSH levels. N-Acetyl cysteine, a GSH precursor, also showed both protective and therapeutic effects against CEES-caused cytotoxicity. Buthionine sulfoximine, which reduces cellular GSH levels, caused an increased CEES cytotoxicity in both JB6 and HaCaT cells. In further studies translating GSH effects in cell culture, pretreatment of mice with 300 mg/kg GSH via oral gavage 1 h before topical application of CEES resulted in significant protection against CEES-caused increase in skin bifold and epidermal thickness, apoptotic cell death, and myeloperoxidase activity, which could be associated with increased skin GSH levels. Together, these results highlight GSH efficacy in ameliorating CEES-caused skin injury and further support the need for effective antioxidant countermeasures against skin injury by HD exposure.

Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.

A monofunctional analog of the chemical warfare agent sulfur mustard (HD), 2-chloroethyl ethyl sulfide (CEES), induces tissue damage similar to HD. Herein we studied the molecular mechanisms associated with CEES-induced skin inflammation and toxicity in SKH-1 hairless mice. Topical CEES exposure caused an increase in oxidative stress as observed by enhanced 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation and an increase in protein oxidation. The CEES-induced increase in the formation of 8-oxo-2-deoxyguanosine indicated DNA oxidation. CEES exposure instigated an increase in the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38). After CEES exposure, a significant increase in the phosphorylation of Akt at Ser473 and Thr308 was observed as well as upregulation of its upstream effector, PDK1, in mouse skin tissue. Subsequently, CEES exposure caused activation of AP-1 family proteins and the NF-kappaB pathway, including phosphorylation and degradation of IkappaBalpha in addition to phosphorylation of the NF-kappaB essential modulator. Collectively, our results indicate that CEES induces oxidative stress and the activation of the transcription factors AP-1 and NF-kappaB via upstream signaling pathways including MAPKs and Akt in SKH-1 hairless mouse skin. These novel molecular targets could be supportive in the development of prophylactic and therapeutic interventions against HD-related skin injury.

Sulfur mustard (HD) is an alkylating and cytotoxic chemical warfare agent, which inflicts severe skin toxicity and an inflammatory response. Effective medical countermeasures against HD-caused skin toxicity are lacking due to limited knowledge of related mechanisms, which is mainly attributed to the requirement of more applicable and efficient animal skin toxicity models. Using a less toxic analog of HD, chloroethyl ethyl sulfide (CEES), we identified quantifiable inflammatory biomarkers of CEES-induced skin injury in dose- (0.05-2 mg) and time- (3-168 h) response experiments, and developed a CEES-induced skin toxicity SKH-1 hairless mouse model. Topical CEES treatment at high doses caused a significant dose-dependent increase in skin bi-fold thickness indicating edema. Histopathological evaluation of CEES-treated skin sections revealed increases in epidermal and dermal thickness, number of pyknotic basal keratinocytes, dermal capillaries, neutrophils, macrophages, mast cells, and desquamation of epidermis. CEES-induced dose-dependent increases in epidermal cell apoptosis and basal cell proliferation were demonstrated by the terminal deoxynucleotidyl transferase (tdt)-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen stainings, respectively. Following an increase in the mast cells, myeloperoxidase activity in the inflamed skin peaked at 24 h after CEES exposure coinciding with neutrophil infiltration. F4/80 staining of skin integuments revealed an increase in the number of macrophages after 24 h of CEES exposure. In conclusion, these results establish CEES-induced quantifiable inflammatory biomarkers in a more applicable and efficient SKH-1 hairless mouse model, which could be valuable for agent efficacy studies to develop potential prophylactic and therapeutic interventions for HD-induced skin toxicity.