Chloropicrin induced ocular injury: Biomarkers, potential mechanisms, and treatments.

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.

Identification of early events in nitrogen mustard pulmonary toxicity that are independent of infiltrating inflammatory cells using precision cut lung slices.

Nitrogen mustard (NM; mechlorethamine) is a cytotoxic vesicant known to cause acute lung injury which can progress to chronic disease. Due to the complex nature of NM injury, it has been difficult to analyze early responses of resident lung cells that initiate inflammation and disease progression. To investigate this, we developed a model of acute NM toxicity using murine precision cut lung slices (PCLS), which contain all resident lung cell populations. PCLS were exposed to NM (1-100 µM) for 0.5-3 h and analyzed 1 and 3 d later. NM caused a dose-dependent increase in cytotoxicity and a reduction in metabolic activity, as measured by LDH release and WST-1 activity, respectively. Optimal responses were observed with 50 µM NM after 1 h incubation and these conditions were used in further experiments. Analysis of PCLS bioenergetics using an Agilent Seahorse showed that NM impaired both glycolytic activity and mitochondrial respiration. This was associated with injury to the bronchial epithelium and a reduction in methacholine-induced airway contraction. NM was also found to cause DNA damage in bronchial epithelial cells in PCLS, as measured by expression of γ-H2AX, and to induce oxidative stress, which was evident by a reduction in glutathione levels and upregulation of the antioxidant enzyme catalase. Cleaved caspase-3 was also upregulated in airway smooth muscle cells indicating apoptotic cell death. Characterizing early events in NM toxicity is key in identifying therapeutic targets for the development of efficacious countermeasures.

Effective skin decontamination with RSDL® (reactive skin decontamination lotion kit) following dermal exposure to a Novichok class nerve agent.

In recent years, various poisoning incidents have been reported, involving the alleged use of the so-called Novichok agents, resulting in their addition to the Schedule I list of the Organisation for the Prohibition of Chemical Warfare (OPCW). As the physicochemical properties of these agents are different from the 'classical' nerve agents, such as VX, research is needed to evaluate whether and to what extent existing countermeasures are effective. Here, we evaluated the therapeutic potential of RSDL® (Reactive Skin Decontamination Lotion Kit) for the neutralization of percutaneous toxicity caused by Novichok agents, both in vitro and in vivo. Experiments showed the three selected Novichok agents (A230, A232, A234) could be degraded by RSDL lotion, but at a different rate. The half-life of A234, in the presence of an excess of RSDL lotion, was 36 min, as compared to A230 (<5 min) and A232 (18 min). Following dermal exposure of guinea pigs to A234, application of the RSDL kit was highly effective in preventing intoxication, even when applied up until 30 min following exposure. Delayed use of the RSDL kit until the appearance of clinical signs of intoxication (3-4 h) was not able to prevent intoxication progression and deaths. This study determines RSDL decontamination as an effective treatment strategy for dermal exposure to the Novichok agent A234 and underscores the importance of early, forward use of skin decontamination, as rapidly as possible.

Dexamethasone sodium phosphate loaded nanoparticles for prevention of nitrogen mustard induced corneal injury.

Nitrogen mustard (NM) is a potent vesicating chemical warfare agent that is primarily absorbed through skin, inhalation, or ocular surface. Ocular exposure of NM can cause acute to chronic keratopathy which can eventually lead to blindness. There is a current lack of effective countermeasures against ocular exposure of NM despite their imperative need. Herein, we aim to explore the sustained effect of Dexamethasone sodium phosphate (DSP)-loaded polymeric nanoparticles (PLGA-DSP-NP) following a single subconjunctival injection in the management and prevention of corneal injury progression upon exposure to NM. DSP is an FDA approved corticosteroid with proven anti-inflammatory properties. We formulated PLGA-DSP-NP with zinc chelation ion bridging method using PLGA polymer, with particles of approximately 250 nm and a drug loading of 6.5 wt%. Under in vitro sink conditions, PLGA-DSP-NP exhibited a sustained drug release for two weeks. Notably, in NM injured cornea, a single subconjunctival (SCT) injection of PLGA-DSP-NP outperformed DSP eyedrops (0.1%), DSP solution, placebo NP, and saline, significantly mitigating corneal neovascularization, ulceration, and opacity for the two weeks study period. Through PLGA-DSP-NP injection, sustained DSP release hindered inflammatory cytokine recruitment, angiogenic factors, and endothelial cell proliferation in the cornea. This strategy presents a promising localized corticosteroid delivery system to effectively combat NM-induced corneal injury, offering insights into managing vesicant exposure.

The risk associated with organophosphorus nerve agents: from their discovery to their unavoidable threat, current medical countermeasures and perspectives.

The first organophosphorus nerve agent was discovered accidently during the development of pesticides, shortly after the first use of chemical weapons (chlorine, phosgene) on the battlefield during World War I. Despite the Chemical Weapons Convention banning these substances, they have still been employed in wars, terrorist attacks or political assassinations. Characterised by their high lethality, they target the nervous system by inhibiting the acetylcholinesterase (AChE) enzyme, preventing neurotransmission, which, if not treated rapidly, inevitably leads to serious injury or the death of the person intoxicated. The limited efficacy of current antidotes, known as AChE reactivators, pushes research towards new treatments. Numerous paths have been explored, from modifying the original pyridinium oximes to developing hybrid reactivators seeking a better affinity for the inhibited AChE. Another crucial approach resides in molecules more prone to cross the blood-brain barrier: uncharged compounds, bio-conjugated reactivators or innovative formulations. Our aim is to raise awareness on the threat and toxicity of organophosphorus nerve agents and to present the main synthetic efforts deployed since the first AChE reactivator, to tackle the task of efficiently treating victims of these chemical warfare agents.

Galectin-3 inhibition ameliorates alveolar epithelial cell pyroptosis in phosgene-induced acute lung injury.

Phosgene is a type of poisonous gas that can cause acute lung injury (ALI) upon accidental exposure. Casualties still occur due to phosgene-induced acute lung injury (P-ALI) from accidents resulting from improper operations. The pathological mechanisms of P-ALI are still understudied. Thus, we performed scRNA-seq on cells isolated from all subpopulations of the BALF in P-ALI and found that Gal3 expression was significantly higher in the gas group than in the control group. Further analysis revealed a ligand-receptor correspondence between alveolar macrophages (AMs) and alveolar epithelial cells (AEC), with Gal3 playing a key role in this interaction. To confirm and elaborate on this discovery, we selected four time points during the previous week: sham (day 0), day 1, day 3, and day 7 in the P-ALI mouse model and found that Gal3 expression was significantly elevated in P-ALI, most abundantly expressed in AM cells. This was further confirmed with the use of a Gal3 inhibitor. The inhibition of Gal3 and elimination of AMs in mice both attenuated epithelial cell pyroptosis, as confirmed in in vitro experiments, and revealed the Gal3/caspase-8/GSDMD signaling pathway. These findings suggest that Galectin-3 inhibition can ameliorate AEC pyroptosis by inhibiting the Gal3/caspase-8/GSDMD signaling pathway, thus reducing alveolar damage in mice with P-ALI. This finding provides novel insights for improving treatment efficacy for P-ALI.

The estimation of acute oral toxicity (LD50) of G-series organophosphorus-based chemical warfare agents using quantitative and qualitative toxicology in silico methods.

The idea of this study was the estimation of the theoretical acute toxicity (t-LD50, rat, oral dose) of organophosphorus-based chemical warfare agents from the G-series (n = 12) using different in silico methods. Initially identified in Germany, the G-type nerve agents include potent compounds such as tabun, sarin, and soman. Despite their historical significance, there is a noticeable gap in acute toxicity data for these agents. This study employs qualitative (STopTox and AdmetSAR) and quantitative (TEST; CATMoS; ProTox-II and QSAR Toolbox) in silico methods to predict LD50 values, offering an ethical alternative to animal testing. Additionally, we conducted quantitative extrapolation from animals, and the results of qualitative tests confirmed the acute toxicity potential of these substances and enabled the identification of toxicophoric groups. According to our estimations, the most lethal agents within this category were GV, soman (GD), sarin (GB), thiosarin (GBS), and chlorosarin (GC), with t-LD50 values (oral administration, extrapolated from rat to human) of 0.05 mg/kg bw, 0.08 mg/kg bw, 0.12 mg/kg bw, 0.15 mg/kg bw, and 0.17 mg/kg bw, respectively. On the contrary, compounds with a cycloalkane attached to the phospho-oxygen linkage, specifically methyl cyclosarin and cyclosarin, were found to be the least toxic, with values of 2.28 mg/kg bw and 3.03 mg/kg bw. The findings aim to fill the knowledge gap regarding the acute toxicity of these agents, highlighting the need for modern toxicological methods that align with ethical considerations, next-generation risk assessment (NGRA) and the 3Rs (replacement, reduction and refinement) principles.

Role of macrophage bioenergetics in N-acetylcysteine-mediated mitigation of lung injury and oxidative stress induced by nitrogen mustard.

Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.

Effects of the nerve agent VX on hiPSC-derived motor neurons.

Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.

The prediction of hydrolysis and biodegradation of organophosphorus-based chemical warfare agents (G-series and V-series) using toxicology in silico methods.

Nerve agents (G- and V-series) are a group of extremely toxic organophosphorus chemical warfare agents that we have had the opportunity to encounter many times on a massive scale (Matsumoto City, Tokyo subway and Gulf War). The threat of using nerve agents in terrorist attacks or military operations is still present, even with establishing the Chemical Weapons Convention as the legal framework. Understanding their environmental sustainability and health risks is critical to social security. Due to the risk of contact with dangerous nerve agents and animal welfare considerations, in silico methods were used to assess hydrolysis and biodegradation safely. The environmental fate of the examined nerve agents was elucidated using QSAR models. The results indicate that the investigated compounds released into the environment hydrolyse at a different rate, from extremely fast (<1 day) to very slow (over a year); V-agents undergo slower hydrolysis compared to G-agents. V-agents turned out to be relatively challenging to biodegrade, the ultimate biodegradation time frame of which was predicted as weeks to months, while for G-agents, the overwhelming majority was classified as weeks. In silico methods for predicting various parameters are critical to preparing for the forthcoming application of nerve agents.

No-observed-adverse-effect-level (NOAEL) assessment as an optimized dose of cholinesterase reactivators for the treatment of exposure to warfare nerve agents in mice.

Despite the international convention on the prohibition of chemical weapons ratified in 1997, the threat of conflicts and terrorist attacks involving such weapons still exists. Among these, organophosphorus-nerve agents (OPs) inhibit cholinesterases (ChE) causing cholinergic syndrome. The reactivation of these enzymes is therefore essential to protect the poisoned people. However, these reactivating molecules, mainly named oximes, have major drawbacks with limited efficacy against some OPs and a non-negligible ChE inhibitor potential if administered at an inadequate dose, an effect that they are precisely supposed to mitigate. As a result, this project focused on assessing therapeutic efficacy, in mice, up to the NOAEL dose, the maximum dose of oxime that does not induce any observable toxic effect. NOAEL doses of HI-6 DMS, a reference oxime, and JDS364. HCl, a candidate reactivator, were assessed using dual-chamber plethysmography, with respiratory ventilation impairment as a toxicity criterion. Time-course modeling parameters and pharmacodynamic profiles, reflecting the interaction between the oxime and circulating ChE, were evaluated for treatments at their NOAEL and higher doses. Finally, the therapeutic potential against OPs poisoning was determined through the assessment of protective indices. For JDS364. HCl, the NOAEL dose corresponds to the smallest dose inducing the most significant therapeutic effect without causing any abnormality in ChE activity. In contrast, for HI-6 DMS, its therapeutic benefit was observed at doses higher than its NOAEL, leading to alterations in respiratory function. These alterations could not be directly correlated with ChE inhibition and had no adverse effects on survival. They are potentially attributed to the stimulation of non-enzymatic cholinergic targets by HI-6 DMS. Thus, the NOAEL appears to be an optimal dose for evaluating the efficacy of oximes, particularly when it can be linked to respiratory alterations effectively resulting from ChE inhibition.

The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity.

We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser198) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography-electrospray ionisation tandem-mass spectrometry (µLC-ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (-)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (-)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.

Dexamethasone targets actin cytoskeleton signaling and inflammatory mediators to reverse sulfur mustard-induced toxicity in rabbit corneas.

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.

Establishing a Dexamethasone Treatment Regimen To Alleviate Sulfur Mustard-Induced Corneal Injuries in a Rabbit Model.

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.

Mesna Improves Outcomes of Sulfur Mustard Inhalation Toxicity in an Acute Rat Model.

Inhalation of high levels of sulfur mustard (SM), a potent vesicating and alkylating agent used in chemical warfare, results in acutely lethal pulmonary damage. Sodium 2-mercaptoethane sulfonate (mesna) is an organosulfur compound that is currently Food and Drug Administration (FDA)-approved for decreasing the toxicity of mustard-derived chemotherapeutic alkylating agents like ifosfamide and cyclophosphamide. The nucleophilic thiol of mesna is a suitable reactant for the neutralization of the electrophilic group of toxic mustard intermediates. In a rat model of SM inhalation, treatment with mesna (three doses: 300 mg/kg intraperitoneally 20 minutes, 4 hours, and 8 hours postexposure) afforded 74% survival at 48 hours, compared with 0% survival at less than 17 hours in the untreated and vehicle-treated control groups. Protection from cardiopulmonary failure by mesna was demonstrated by improved peripheral oxygen saturation and increased heart rate through 48 hours. Additionally, mesna normalized arterial pH and pACO2 Airway fibrin cast formation was decreased by more than 66% in the mesna-treated group at 9 hour after exposure compared with the vehicle group. Finally, analysis of mixtures of a mustard agent and mesna by a 5,5'-dithiobis(2-nitrobenzoic acid) assay and high performance liquid chromatography tandem mass spectrometry demonstrate a direct reaction between the compounds. This study provides evidence that mesna is an efficacious, inexpensive, FDA-approved candidate antidote for SM exposure. SIGNIFICANCE STATEMENT: Despite the use of sulfur mustard (SM) as a chemical weapon for over 100 years, an ideal drug candidate for treatment after real-world exposure situations has not yet been identified. Utilizing a uniformly lethal animal model, the results of the present study demonstrate that sodium 2-mercaptoethane sulfonate is a promising candidate for repurposing as an antidote, decreasing airway obstruction and improving pulmonary gas exchange, tissue oxygen delivery, and survival following high level SM inhalation exposure, and warrants further consideration.

Nitrogen Mustard-Induced Ex Vivo Human Cornea Injury Model and Therapeutic Intervention by Dexamethasone.

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).

Dermal Exposure to Vesicating Nettle Agent Phosgene Oxime: Clinically Relevant Biomarkers and Skin Injury Progression in Murine Models.

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 µl 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.

HSP90, a Common Therapeutic Target for Suppressing Skin Injury Caused by Exposure to Chemically Diverse Classes of Blistering Agents.

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90α expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.

The Involvement of Unfolded Protein Response in the Mechanism of Nitrogen Mustard-Induced Ocular Toxicity.

Nitrogen mustard (NM) is a known surrogate of sulfur mustard, a chemical-warfare agent that causes a wide range of ocular symptoms, from a permanent reduction in visual acuity to blindness upon exposure. Although it has been proposed that the two blistering agents have a similar mechanism of toxicity, the mode of NM-induced cell death in ocular tissue has not been fully explored. Therefore, we hypothesized that direct ocular exposure to NM in mice leads to retinal tissue injury through chronic activation of the unfolded protein response (UPR) PERK arm in corneal cells and VEGF secretion, eventually causing cell death. We topically applied NM directly to mice to analyze ocular and retinal tissues at 2 weeks postexposure. A dramatic decline in retinal function, measured by scotopic and photopic electroretinogram responses, was detected in the mice. This decline was associated with enhanced TUNEL staining in both corneal and retinal tissues. In addition, exposure of corneal cells to NM revealed 228 differentially and exclusively expressed proteins primarily associated with the UPR, ferroptosis, and necroptosis. Moreover, these cells exhibited activation of the UPR PERK arm and an increase in VEGF secretion. Enhancement of VEGF staining was later observed in the corneas of the exposed mice. Therefore, our data indicated that the mechanism of NM-induced ocular toxicity should be carefully examined and that future research should identify a signaling molecule transmitted via a prodeath pathway from the cornea to the retina. SIGNIFICANCE STATEMENT: This study demonstrated that NM topical exposure in mice results in dramatic decline in retinal function associated with enhanced TUNEL staining in both corneal and retinal tissues. We also found that the NM treatment of corneal cells resulted in 228 differentially and exclusively expressed proteins primarily associated with ferroptosis. Moreover, these cells manifest the UPR PERK activation and an increase in VEGF secretion. The latter was also found in the corneas of the cexposed mice.

Efficacy of the oxime HI-6 dimethanesulphonate in the treatment of guinea-pigs exposed to the nerve agents GB and GD.

The bispyridinium oxime HI-6 DMS is in development as an improved therapy for the treatment of patients exposed to organophosphorus nerve agents. The aim of the work described in this paper was to provide non-clinical data to support regulatory approval of HI-6 DMS, by demonstrating efficacy against an oxime-sensitive agent, GB and an oxime-resistant agent, GD. We investigated the dose-dependent protection afforded by therapy including atropine, avizafone and HI-6 DMS in guinea-pigs challenged with GB or GD. We also compared the efficacy of 30 mg.kg-1 of HI-6 DMS to an equimolar dose of the current in-service oxime P2S and the dichloride salt of HI-6 (HI-6 Cl2). In the treatment of GB or GD poisoning there was no significant difference between the salt forms. The most effective dose of HI-6 DMS in preventing lethality following challenge with GB was 100 mg.kg-1; though protection ratios of at least 25 were obtained at 10 mg.kg-1. Protection against GD was lower, and there was no significant increase in effectiveness of HI-6 DMS doses of 30 or 100 mg.kg-1. For GD, the outcome was improved by the addition of pyridostigmine pre-treatment. These data demonstrate the benefits of HI-6 DMS as a component of nerve agent therapy. © Crown copyright (2023), Dstl.