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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) 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.)

Sulfur mustard (SM) is a threat to both civilian and military populations. Human skin is highly sensitive to SM, causing delayed erythema, edema, and inflammatory cell infiltration, followed by the appearance of large fluid-filled blisters. Skin wound repair is prolonged following blistering, which can result in impaired barrier function. Key to understanding the action of SM in the skin is the development of animal models that have a pathophysiology comparable to humans such that quantitative assessments of therapeutic drugs efficacy can be assessed. Two animal models, hairless guinea pigs and swine, are preferred to evaluate dermal products because their skin is morphologically similar to human skin. In these animal models, SM induces degradation of epidermal and dermal tissues but does not induce overt blistering, only microblistering. Mechanisms of wound healing are distinct in these animal models. Whereas a guinea pig heals by contraction, swine skin, like humans, heals by re-epithelialization. Mice, rats, and rabbits are also used for SM mechanistic studies. However, healing is also mediated by contraction; moreover, only microblistering is observed. Improvements in animal models are essential for the development of therapeutics to mitigate toxicity resulting from dermal exposure to SM.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45 + leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14-28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest for the subject matter of this paper., (Copyright © 2022. Published by Elsevier Inc.)

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.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5 ac producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We investigated the plasma toxicokinetic behavior of free (parent) and total (parent and conjugated forms) of bisphenol S (BPS) and bisphenol AF (BPAF) in plasma of adult male rats and mice following exposure via feed for 7 days to BPS (338, 1125, and 3375 ppm) or BPAF (338, 1125, and 3750 ppm). In rats, the exposure concentration-normalized maximum concentration [C max /D (ng/mL)/(ppm)] and area under the concentration time curve [AUC/D (h × ng/mL)/(ppm)] for free was higher for BPS (C max /D: 0.476-1.02; AUC/D: 3.58-8.26) than for BPAF (C max /D: 0.017-0.037; AUC/D:0.196-0.436). In mice, the difference in systemic exposure parameters between free BPS (C max /D: 0.376-0.459; AUC/D: 1.52-2.54) and free BPAF (C max /D: 0.111-0.165; AUC/D:0.846-1.09) was marginal. Elimination half-lives for free analytes (4.41-10.4 h) were comparable between species and analogues. When systemic exposure to free analyte was compared between species, in rats, BPS exposure was slightly higher but BPAF exposure was much lower than in mice. BPS and BPAF were highly conjugated; total BPS AUC values (rats ≥18-fold, mice ≥17-fold) and BPAF (rats ≥127-fold, mice ≥16-fold) were higher than corresponding free values. Data demonstrated that there are analogue and species differences in the kinetics of BPS and BPAF.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Methyl isocyanate (MIC, "Bhopal agent") is a highly reactive, toxic industrial chemical. Inhalation of high levels (500-1000 ppm) of MIC vapor is almost uniformly fatal. No therapeutic interventions other than supportive care have been described that can delay the onset of illness or death due to MIC. Recently, we found that inhalation of MIC caused the appearance of activated tissue factor in circulation with subsequent activation of the coagulation cascade. Herein, we report that MIC exposure (500 ppm for 30 min, nose-only) caused deposition of fibrin-rich casts in the conducting airways resulting in respiratory failure and death within 24 h in a rat model (LC 90-100 ). We thus investigated the effect of airway delivery of the fibrinolytic agent tissue plasminogen activator (tPA) on mortality and morbidity in this model. Intratracheal administration of tPA was initiated 11 h post MIC exposure and repeated every 4 h for the duration of the study. Treatment with tPA afforded nearly 60% survival at 24 h post MIC exposure and was associated with decreased airway fibrin casts, stabilization of hypoxemia and respiratory distress, and improved acidosis. This work supports the potential of airway-delivered tPA therapy as a useful countermeasure in stabilizing victims of high-level MIC exposure., (© 2020 New York Academy of Sciences.)

Sulfur mustard (SM), a dermal vesicant that has been used in chemical warfare, causes inflammation, edema and epidermal erosions depending on the dose and time following exposure. Herein, a minipig model was used to characterize wound healing following dermal exposure to SM. Saturated SM vapor caps were placed on the dorsal flanks of 3-month-old male Gottingen minipigs for 30 min. After 48 h the control and SM wounded sites were debrided daily for 7 days with wet to wet saline gauze soaks. Animals were then euthanized, and full thickness skin biopsies prepared for histology and immunohistochemistry. Control skin contained a well differentiated epidermis with a prominent stratum corneum. A well-developed eschar covered the skin of SM treated animals, however, the epidermis beneath the eschar displayed significant wound healing with a hyperplastic epidermis. Stratum corneum shedding and a multilayered basal epithelium consisting of cuboidal and columnar cells were also evident in the neoepidermis. Nuclear expression of proliferating cell nuclear antigen (PCNA) was contiguous in cells along the basal epidermal layer of control and SM exposed skin; SM caused a significant increase in PCNA expression in basal and suprabasal cells. SM exposure was also associated with marked changes in expression of markers of wound healing including increases in keratin 10, keratin 17 and loricrin and decreases in E-cadherin. Trichrome staining of control skin showed a well-developed collagen network with no delineation between the papillary and reticular dermis. Conversely, a major delineation was observed in SM-exposed skin including a web-like papillary dermis composed of filamentous extracellular matrix, and compact collagen fibrils in the lower reticular dermis. Although the dermis below the wound site was disrupted, there was substantive epidermal regeneration following SM-induced injury. Further studies analyzing the wound healing process in minipig skin will be important to provide a model to evaluate potential vesicant countermeasures., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest for the subject matter of this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Silver ion has strong antimicrobial properties and is used in a number of wound dressings. In burn models, silver-nylon dressings produce elevated silver levels in the wound along with minimal systemic effect. We evaluated systemic toxicity in a non-burn wound model to see if a similar pattern of silver ion distribution would occur., Methods: Eight deep partial-thickness wounds each were created on the dorsum of 40 Gottingen minipigs using a Er-YAG Laser. Half were treated with a 21-day course of silver-nylon dressings (Silverlon®) and half were treated with moist gauze dressings. Wound, blood, liver and kidney silver levels, along with blood chemistry and hematology data were obtained at appropriate intervals., Results: All wounds healed well with healing enhanced by silver-nylon dressings. Silver ion was demonstrable in all wounds treated with silver-nylon at day 21 and after 14 days of no further treatment. Silver ion was not detected in blood, liver or kidney of any animal treated with silver-nylon or control dressings. Liver and kidney function remained normal in all animals., Conclusion: A 21-day application of silver-nylon dressings to a non-burn dermal wound produces no systemic or local toxicity in Gottingen minipigs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sulfur mustard burns are characterized by delayed symptoms, slow healing, and recurrence after closure. Incomplete debridement at the level of the basement membrane is the postulated cause. Graham pioneered laser debridement of mustard burns. For field or mass-casualty use, saline wet-to-wet or antibiotic-soak debridement is more practical. In this study, we compared laser, saline, and antibiotic debridement in a porcine model of deep partial-thickness injury. Deep-dermal sulfur mustard burns were produced in 18 anesthetized Gottingen minipigs using 10-μl saturated vapor cap exposure time of 90 minutes. Debridement was started 48 hours postinjury and consisted of a single laser treatment; 5 days of 5% aqueous mafenide acetate wet-to-wet dressings; or 7 to 12 days of saline wet-to-wet dressings. Wounds were treated with daily silver sulfadiazine for 30 days and, then, assessed by histopathology, silver-ion analysis, colorimetry, and evaporimetry. All wounds healed well with no sign of infection. Antibiotic debridement showed no advantage over saline debridement. There were no significant differences between groups for colorimetry or evaporimetry. Histopathology was graded on a mustard-specific scale of 1 to 15 where higher values indicate better healing. Mean histology scores were 13.6 for laser, 13.9 for mafenide, and 14.3 for saline. Saline debridement statistically outperformed laser (P < .05) but required the longest debridement time. Laser debridement had the benefit of requiring a single treatment rather than daily dressing changes, significantly decreasing need for wound care and personnel resources. Development of a ruggedized laser for field use is a countermeasures priority., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125-500 ppm) by nose-only vapor inhalation for 30 min. Arterial O 2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO 2 ) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.

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/m 3 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Methyl isocyanate (MIC) is an important precursor for industrial synthesis, but it is highly toxic. MIC causes irritation and damage to the eyes, respiratory tract, and skin. While current treatment is limited to supportive care and counteracting symptoms, promising countermeasures are being evaluated. Our work focuses on understanding the inhalation toxicity of MIC to develop effective therapeutic interventions. However, in-vivo inhalation exposure studies are limited by challenges in estimating the actual respiratory dose, due to animal-to-animal variability in breathing rate, depth, etc. Therefore, a method was developed to estimate the inhaled MIC dose based on analysis of an N-terminal valine hemoglobin adduct. The method features a simple sample preparation scheme, including rapid isolation of hemoglobin, hydrolysis of the hemoglobin adduct with immediate conversion to methyl isopropyl hydantoin (MIH), rapid liquid-liquid extraction, and gas-chromatography mass-spectrometry analysis. The method produced a limit of detection of 0.05 mg MIH/kg RBC precipitate with a dynamic range from 0.05-25 mg MIH/kg. The precision, as measured by percent relative standard deviation, was <8.5%, and the accuracy was within 8% of the nominal concentration. The method was used to evaluate a potential correlation between MIH and MIC internal dose and proved promising. If successful, this method may be used to quantify the true internal dose of MIC from inhalation studies to help determine the effectiveness of MIC therapeutics., (Copyright © 2018 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.)

Silver-based dressings are commonly used in burn care. Silver sulfadiazine use is associated with elevated blood, urine, and tissue levels of silver ion. We examined wound and tissue levels of silver ion in a two-species model of sulfur mustard chemical burn injury treated with two different silver-based dressings. Superficial dermal and moderate thickness dermal chemical burns were induced in 16 hairless guinea pigs and in 16 Gottingen minipigs by exposure to sulfur mustard vapor. After debridement, silver-nylon burn dressings or silver-calcium alginate dressings were applied and changed every 7 days until wound healing or a maximum of 60 days post exposure. At autopsy, liver, spleen, and wound samples were harvested. Silver ion was measured using inductively coupled plasma-mass spectrography with a lower level of detection of 0.02 parts per billion. Negligible silver ion levels were found in the liver (mean < 0.003 μg/g of tissue) and spleen (mean < 0.05 μg/g) of all 32 animals. Wound biopsies showed silver ion levels ranging from 0.07 to 19.5 μg/g of tissue. Wound levels were higher in minipigs than in hairless guinea pigs and were higher in animals treated with silver-nylon burn wound dressings than with silver-calcium alginate dressings. Silver ion could be detected in some wounds 40 days after dressings were removed. In a chemical burn model, application of silver-nylon or silver-calcium alginate dressings is associated with elevated wound levels but negligible tissue levels of silver ion.

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.

Lewisite is a potent arsenic-based chemical warfare agent known to induce painful cutaneous inflammation and blistering. Only a few modestly effective antidotes have so far been described in the literature. However, the discovery of effective antidotes for lewisite was hampered by the paucity of the exact molecular mechanism underlying its cutaneous pathogenesis. We investigated the molecular mechanism underlying lewisite-induced cutaneous blistering and inflammation and describe its novel antidotes. On the basis of our initial screening, we used a highly sensitive murine model that recapitulates the known human pathogenesis of arsenicals-induced cutaneous inflammation and blistering. Topically administered lewisite induced potent acute inflammation and microvesication in the skin of Ptch1(+/-)/SKH-1 mice. Even at a very low dose, lewisite up-regulates unfolded protein response signaling, inflammatory response, and apoptosis. These cutaneous lesions were associated with production of reactive oxygen species and extensive apoptosis of the epidermal keratinocytes. We confirmed that activation of reactive oxygen species-dependent unfolded protein response signaling is the underlying molecular mechanism of skin damage. Similar alterations were noticed in lewisite-treated cultured human skin keratinocytes. We discovered that chemical chaperone 4-phenyl butyric acid and antioxidant N-acetylcysteine, which significantly attenuate lewisite-mediated skin injury, can serve as potent antidotes. These data reveal a novel molecular mechanism underlying the cutaneous pathogenesis of lewisite-induced lesions. We also identified novel potential therapeutic targets for lewisite-mediated cutaneous injury., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Time courses of MMB4 concentrations in plasma were characterized following 7-day repeated intramuscular (IM) administrations of MMB4 DMS to male and female Sprague-Dawley rats, New Zealand White rabbits, beagle dogs (single dose only), and rhesus monkeys at drug dose levels used in earlier toxicology studies. In general, there were no significant differences in MMB4 toxicokinetic (TK) parameters between males and females for all the species tested in these studies. After a single IM administration to rats, rabbits, dogs, and monkeys, MMB4 DMS was rapidly absorbed, resulting in average T max values ranging from 5 to 30 minutes. Although C max values did not increase dose proportionally, the overall exposure to MMB4 in these preclinical species, as indicated by area under the curve (AUC) extrapolated to the infinity (AUC∞) values, increased in an approximately dose-proportional manner. The MMB4 DMS was extensively absorbed into the systemic circulation after IM administration as demonstrated by greater than 80% absolute bioavailability values for rats, rabbits, and dogs. Repeated administrations of MMB4 DMS for 7 days did not overtly alter TK parameters for MMB4 in rats, rabbits, and monkeys (150 and 300 mg/kg/d dose groups only). However, C max and AUC values decreased in monkeys given 450 and 600 mg/kg IM doses of MMB4 DMS following repeated administrations for 7 days. Based on the TK results obtained from the current study and published investigations, it was found that the apparent volume of distribution and clearance values were similar among various preclinical species, except for the rat.

Studies were conducted in Sprague-Dawley rats, New Zealand White (NZW) rabbits, and rhesus monkeys to characterize the toxicity of 1,1'-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate (MMB4 DMS) following intramuscular administration. Rats received MMB4 DMS once daily for 7 days at 100, 200, 400, and 800 mg/kg/d; rabbits received a range of dose levels in 3 separate 7-day studies from 3 to 800 mg/kg/d and in a single-dose study from 50 to 200 mg/kg; and monkeys received MMB4 DMS at 150 to 600 mg/kg/d. Mortality was noted in rats and rabbits administered ≥ 200 mg/kg. All monkeys survived until scheduled termination. Adverse clinical observations were noted in the rats at ≥ 400 mg/kg/d and in rabbits administered ≥ 200 mg/kg; no adverse findings were noted in the monkeys. Clinical pathology changes were noted in the rabbit related to cardiac and renal function. In the rabbit and monkey, elevations in myoglobin, alanine aminotransferase/aspartate aminotransferase, platelets, creatine kinase, and coagulation factors were related to local inflammation at the intramuscular administration site. Light microscopic examination at the injection sites revealed acute skeletal muscle necrosis in vehicle control and treated groups. Target tissues in the rabbit studies were identified as kidney, heart, and lungs at ≥ 100 mg/kg/d. All changes noted in all the species demonstrated partial to complete recovery comparable to control values or to a clinically irrelevant level of effect. The NZW rabbit was the most sensitive species, and the no observed adverse effect level (NOAEL) was determined as 50 mg/kg/d; the NOAEL in the rat was 100 mg/kg/d; and the NOAEL in rhesus monkeys was >600 mg/kg/d.

Recent events have heightened the need for the rapid development of vaccines directed against pandemic influenza H1N1 viruses circulating during 2009 to 2010. The current study was conducted to establish a virus challenge dose for a subsequent CA/04 vaccine efficacy study in 3-mo-old domesticated ferrets. An additional consideration in using CA/04 in ferrets is the selection of endpoints on which to base the challenge dose, given the potential nonlethality of this particular model. Four doses ranging from 10(4) to 10(7) TCID(50) units of CA/04 per animal were administered by intranasal instillation to groups of male and female ferrets, and virus titers in nasal washes obtained 1, 3, and 5 d thereafter were determined in MDCK cells. Dosed ferrets developed clinically mild infections. Peak virus titers occurred on day 3 after instillation regardless of dose. Virus-treated ferrets had less weight gain than did untreated ferrets. In conclusion, 3-mo-old ferrets can be infected with doses as low as 10(4) TCID(50) units of CA/04, and virus titers in nasal washes and decreased body weight gain can be used to assess the course of nonlethal infection of 3-mo-old ferrets by CA/04.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces aberrant release of gonadotropins, FSH, and LH and blocks ovulation during induced ovarian follicular development in rats by an unknown mechanism. In the current study, TCDD (0, 8, or 32 microg/kg orally) was administered to immature female Sprague-Dawley rats, and synchronous follicular development was induced 24 h later with equine chorionic gonadotropin (eCG, 5 IU s.c.). Both doses of TCDD induced a significant premature increase in serum FSH and LH (P < 0.05) at 12 h post-eCG. This premature gonadotropin surge was facilitated by the administration of a long-acting estradiol (estradiol cypionate, 0.01, 0.1, and 0.5 mg/kg s.c.), whereas the progesterone and cortisol receptor antagonist RU486 (0, 1, and 10 mg/kg s.c.) potentiated the premature release of FSH and LH following TCDD as well. Pentobarbital (32 mg/kg i.p.) administered at 6 or 9 h, but not 0 h, post-eCG ablated the ability of TCDD to stimulate the release of FSH and LH in vivo. TCDD had no significant effect on GnRH accumulation in vitro from immortalized GnRH neuronal (GT1-7) cells and failed to alter the cell number. Transfection of these cells with a rat GnRH promoter-reporter construct revealed no significant acute effect of TCDD on GnRH promoter activity. Aryl hydrocarbon receptor mRNA was not detected in the GT1-7 cells by reverse transcription polymerase chain reaction. TCDD appears to stimulate premature gonadotropin release in the gonadotropin-primed immature rat by interacting with an estradiol- and pentobarbital-sensitive neural signal for GnRH release but not by acting upon the GnRH neuron directly.