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.

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.

Evaluation of common protein biomarkers involved in the pathogenesis of respiratory diseases with proteomic methods: A systematic review.

AIM: Respiratory disease (RD) is one of the most common diseases characterized by lung dysfunction. Many diagnostic mechanisms have been used to identify the pathogenic agents of responsible for RD. Among these, proteomics emerges as a valuable diagnostic method for pinpointing the specific proteins involved in RD pathogenesis. Therefore, in this study, for the first time, we examined the protein markers involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, bronchiolitis obliterans (BO), and chemical warfare victims exposed to mustard gas, using the proteomics method as a systematic study. MATERIALS AND METHODS: A systematic search was performed up to September 2023 on several databases, including PubMed, Scopus, ISI Web of Science, and Cochrane. In total, selected 4246 articles were for evaluation according to the criteria. Finally, 119 studies were selected for this systematic review. RESULTS: A total of 13,806 proteins were identified, 6471 in COPD, 1603 in Asthma, 5638 in IPF, three in BO, and 91 in mustard gas exposed victims. Alterations in the expression of these proteins were observed in the respective diseases. After evaluation, the results showed that 31 proteins were found to be shared among all five diseases. CONCLUSION: Although these 31 proteins regulate different factors and molecular pathways in all five diseases, they ultimately lead to the regulation of inflammatory pathways. In other words, the expression of some proteins in COPD and mustard-exposed patients increases inflammatory reactions, while in IPF, they cause lung fibrosis. Asthma, causes allergic reactions due to T-cell differentiation toward Th2.

HMSCs exosome-derived miR-199a-5p attenuates sulfur mustard-associated oxidative stress via the CAV1/NRF2 signalling pathway.

Sulfur mustard (SM) is a blister-producing chemical warfare agent which could lead to a cascade of systemic damage, especially severe acute lung injury. Oxidative stress is considered to be vital processes for the SM toxicity mechanism. We previously proved the therapeutic effect of exosomes derived from bone marrow mesenchymal stromal cells in promoting the repair of alveolar epithelial barrier and inhibiting apoptosis. However, the key functional components in exosomes and the underlying mechanisms have not been fully elaborated. This research shed light on the function of the key components of human umbilical cord mesenchymal stem cell-derived exosomes (HMSCs-Ex). We noted that HMSCs-Ex-derived miR-199a-5p played a vital role in reducing pneumonocyte oxidative stress and apoptosis by reducing reactive oxygen species, lipid peroxidation products and increasing the activities of antioxidant enzymes in BEAS-2B cells and mouse models after exposure to SM for 24 h. Furthermore, we demonstrated that the overexpression of miR-199a-5p in HMSCs-Ex treatment induced a further decrease of Caveolin1 and the activation of the mRNA and protein level of NRF2, HO1 and NQO1, compared with HMSCs-Ex administration. In summary, miR-199a-5p was one of the key molecules in HMSCs-Ex that attenuated SM-associated oxidative stress via regulating CAV1/NRF2 signalling pathway.

Mustard Gas Exposure Actuates SMAD2/3 Signaling to Promote Myofibroblast Generation in the Cornea.

Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ɑSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFß, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ɑSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure.

Detoxification of the Toxic Sulfur Mustard Simulant by a Supramolecular Antidote in Vitro and in Vivo.

Although great potential hazards and threats still occur from sulfur mustard, there are no specific medicine or therapy for the intoxication of sulfur mustard. Herein, we have demonstrated a supramolecular approach for the detoxification of the sulfur mustard simulant CEES (4) in vitro and in vivo by carboxylatopillar[5]arene potassium salts (CP[5]AK 1) efficiently based on host-guest interactions. The encapsulation of CEES (4) by the cavity of the pillar[5]arene 2 is driven by C-H···π interactions between CEES (4) and the electron-rich cavity of pillar[5]arene 2, which was investigated by 1H NMR titration, density functional theory studies, and the independent gradient model studies. CEES (4) is degradated to the reactive sulfonium salts quickly in aqueous media, resulting in the alkylation of DNA and proteins. The sulfonium salts can be encapsulated by CP[5]AK 1 efficiently, which accelerates the degradation of the sulfonium salts about 14 times. The cell and animal experiments indicated that the bioactivities of the sulfonium salts are inhibited with the formation of stable host-guest complexes, and CP[5]AK 1 has a good therapeutic effect on the damages caused by CEES (4) at either pre- or post-treatments. Due to the low cytotoxicity and good therapeutic effect, the anionic pillar[5]arenes are expected to be developed as specific antidotes against sulfur mustard (HD).

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard.

Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL-1) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL-1). Five new alkylation sites were accurately identified on the protein (α-His72, α-His87, α-His89, ß-His2 and ß-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide α-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 µg.mL-1. The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 µg.mL-1). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin ß. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL-1. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

Monitoring of hydrolysis products of mustard gas, some sesqui- and oxy-mustards and other chemical warfare agents in a plant material by HPLC-MS/MS.

At present, there is a real threat of chemical warfare agents being used in terrorist acts and military clashes. Sulfur and nitrogen mustards are blister agents with high lethality and rapid disruption of armed forces. These highly poisonous substances are hydrolyzed to the characteristic marker compounds when released into the environment. Analysis of environmental objects allows to establish the fact of alleged use of chemical warfare agents and to reveal their type. However, water and soil samples are not always reliable for retrospective analysis. The resulting chemical warfare agent markers may be washed out from the application site over time by groundwaters or atmospheric condensations. This study shows the potential for using plants as a convenient material for retrospective analysis. Garden cress (Lepidium sativum) was chosen as a model plant for this purpose, since it can be easily and quickly grown hydroponically. The plants were cultivated in the environment of the selected markers to study an accumulation of these compounds by the plants. An effective and fast method of homogenization with subsequent ultrasonic extraction was applied. The extracts were analyzed using a specially developed and validated HPLC-MS/MS approach. Separation of the hydrophilic markers was carried out on a reversed-phase column with a polar endcapping. Sensitive mass spectrometric detection was performed in the multiple reaction monitoring mode. Achieved limits of detection for most markers were in the range of 2-40 ng mL-1. It was discovered from the research that after the removal of markers from the growing medium the plants are able to store and concentrate these markers for at least 5 weeks, ensuring a high retrospectivity of the analysis. The obtained results indicate the perspective of using plants as additional objects of analysis during the investigation of incidents related to the use of chemical warfare agents. However, more complex plants and models should be studied in the future.

Comparison of the toxicity of sulfur mustard and its oxidation products in vitro.

The molecular toxicology of the chemical warfare agent sulfur mustard (SM) is still not completely understood. It has been suggested that in addition to SM itself also biotransformation products thereof mediate cytotoxicity. In the current study, we assessed this aspect by exposing a human hepatocyte cell line (HepG2) to SM or to its oxidation products sulfur mustard sulfoxide (SMO), sulfur mustard sulfone (SMO2), and divinyl sulfone (DVS). Cytotoxicity, determined with the XTT assay, revealed a significant higher toxicity of SMO2 and DVS compared to SM while SMO had no effect at any concentration. The exact biotransformation of SM leading to SMO, SMO2 and finally DVS is unknown so far. Involvement of the CYP450 system is discussed and was also investigated in the presented study. Modulation of CYP1A2 activity, taken as a model enzyme for CYP450, affected cytotoxicity of SM, SMO2 or DVS significantly. Induction of CYP1A2 with omeprazole led to decreased cytotoxicity for all compounds whereas inhibition with cimetidine resulted in an increased cytotoxicity for SM, but not for SMO2 and DVS. Our results indicate a distinctive role of the CYP450 system in SM poisoning. Future studies should address the metabolic conversion of SM in more detail. Our data may suggest the well-tolerated drug omeprazole as a potential co-treatment after contact to SM.

PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner.

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H2O2, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H2O2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.

Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA.

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.

Optimized Gas Chromatography-Tandem Mass Spectrometry for 1,1'-sulfonylbis[2-(methylthio) ethane] Quantification in Human Urine.

Sulfur mustard (SM) which is a bifunctional alkylating vesicant is one of the mostly used chemical warfare agent in First World War and the Iran-Iraq War. ß-Lyase metabolites of SM especially 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) is an unequivocal biomarker of the exposure. An optimized gas chromatography-tandem mass spectrometry method was developed and validated for the retrospective detection of SBMTE in human urine. Urine samples were treated with acidic titanium trichloride to reduce ß-lyase metabolites to the single analyte SBMTE. After neutralization and precipitation, SBMTE was extracted from urine by C8 solid-phase extraction cartridge and analyzed in the multiple-reaction monitoring mode. The lower limit of quantification was 1 ng/mL with relative standard deviation of <10%. Acceptable intra-day and inter-day precisions and accuracies were obtained. The developed method was successfully measured various levels of SBMTE which could be used as the forensic evidence of such a chemical attack.

Rapid analysis of sulfur mustard oxide in plasma using gas chromatography-chemical ionization-mass spectrometry for diagnosis of sulfur mustard exposure.

Sulfur mustard (SM) is the most utilized chemical warfare agent in modern history and has caused more casualties than all other chemical weapons combined. SM still poses a threat to civilians globally because of existing stockpiles and ease of production. Exposure to SM causes irritation to the eyes and blistering of skin and respiratory tract. These clinical signs of exposure to SM can take 6-24 h to appear. Therefore, analyzing biomarkers of SM from biological specimens collected from suspected victims is necessary for diagnosis during this latent period. Here, we report a rapid, simple, and direct quantitative analytical method for an important and early SM biomarker, sulfur mustard oxide (SMO). The method includes addition of a stable isotope labeled internal standard, SMO extraction directly into dichloromethane (DCM), rapid drying and reconstitution of the extract, and direct analysis of SMO using gas chromatography-chemical ionization-mass spectrometry. The limit of detection of the method was 0.1 µM, with a linear range from 0.5 to 100 µM. Method selectivity, matrix effect, recovery, and short-term stability were also evaluated. Furthermore, the applicability of the method was tested by analyzing samples from inhalation exposure studies performed in swine. The method was able to detect SMO from 100% of the exposed swine (N = 9), with no interferences present in the plasma of the same swine prior to exposure. The method presented here is the first of its kind to allow for easy and rapid diagnosis of SM poisoning (sample analysis <15 min), especially important during the asymptomatic latency period.

Characterization of FM2382 from Fulvimarina manganoxydans sp. Nov. 8047 with potential enzymatic decontamination of sulfur mustard.

Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min. The catalytic activity of FM2382 was activated by Co2+ and Mg2+, and inhibited by Zn2+, Cu2+ and Fe3+. Furthermore, site-specific mutagenesis proved that D34, K207 D232, D237 were amino acid residues related to the catalytic activity of SM. In conclusion, we found a thermostable haloacid dehalogenases (HAD) family dehalogenase showing SM-degradation activity, which may be useful for practical application in the future.

Synthesis, Characterization, and Identification of New in Vitro Covalent DNA Adducts of Divinyl Sulfone, an Oxidative Metabolite of Sulfur Mustard.

Divinyl sulfone (DVS) is an important oxidative metabolic product of sulfur mustard (SM) in vitro and in vivo. Although DVS is not a classical blister agent, its high reactivity and toxicity induced by vinyl groups can also cause blisters like SM upon contact with the skin, eyes, and respiratory organs. The purpose of this paper was to identify whether DVS could covalently bind to DNA bases to form new DNA adducts in cells in vitro. A series of adducts were synthesized and characterized using purine, nucleoside, or DNA, separately, as starting materials. The covalent site, pattern, and relative reactivity of adduct formation were identified and discussed in detail. The results showed that five high abundance site-specific DNA adducts, including two monoadducts and three cross-linked adducts, were obtained when DNA was used as a substrate. When HaCaT cells were exposed to 30 µM of DVS, four new DNA adducts containing monoadducts and cross-linked adducts were found and identified in cells, including N3-A monoadduct, N7-G monoadduct, N7G-N7G bis-adduct, and N3A-N7G cross-linked adduct. Among them, the abundance of N3-A monoadduct was 10 times higher than that of the other three adducts. DNA adduct formation with DVS showed significant differences from that observed with SM. The observation of these new DNA adduct in vitro cells revealed that DNA damage could be also induced by DVS.

High throughput quantitative analysis of the ß-lyase sulfur mustard metabolite, 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] in urine via high performance liquid chromatography tandem mass spectrometry.

Sulfur Mustard (HD) has a 100year history of use as a chemical warfare agent and recent events in the Middle East are causing it to once again be a potential concern. We report a new high-throughput method for the determination of HD exposure by the analysis of the ß-lyase metabolite 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) in human urine. This method features a hydrogen peroxide (H2O2) oxidative conversion of the ß-lyase metabolites to SBMSE, followed by sample extraction and concentration using solid phase extraction in 96-well plate format. Subsequent high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis gave linear quantitation over a calibration range of 0.1-100ng/mL, with a method detection limit of 0.03ng/mL. Liquid chromatographic separation was achieved using a hydrophilic interaction liquid chromatography (HILIC) column with an analyte retention time of 0.9min and method time of 1.5min (cycle time=2.0min). Users of this method could prepare and analyze approximately 650 samples in 24h which would be important for an emergency response.

Simultaneous quantification of four metabolites of sulfur mustard in urine samples by ultra-high performance liquid chromatography-tandem mass spectrometry after solid phase extraction.

Four HD urinary metabolites including hydrolysis metabolite thiodiglycol (TDG), glutathione-derived metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] (SBSNAE), as well as the ß-lyase metabolites 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio) ethylsulfonyl]ethane (MSMTESE) are considered as important biomarkers for short-term retrospective detection of HD exposure. In this study, a single method for simultaneous quantification of the four HD metabolites in urine samples was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The four urinary metabolites were simultaneously extracted from urinary samples using a solid phase extraction (SPE) method with high extraction recoveries for all four metabolites varied in the range of 71.1-103% followed by UHPLC-MS/MS analysis. The SPE is simple and high effective only requiring 0.1mL of urinary samples and 0.5h time consuming. The problem of previous co-elution of TDG and SBSNAE in UHPLC was well solved, and complete separation of TDG, SBSNAE, SBMSE and MSMTESE from SPE-processed urine matrix was obtained to increase specificity and sensitivity. A full method validation was performed for each analyte in urine matrix. The linear range of calibration curves for the four analytes were respectively from 0.50-500ngmL-1 for TDG and SBSNAE, 0.05-500ngmL-1 for SBMSE and MSMTESE with coefficient of determination value (R2) ≥0.990. The limit of detection was 0.25ngmL-1 for TDG and SBSNAE, 0.01ngmL-1 for SBMSE and MSMTESE spiked in normal urine. The intra/inter-day precision for each analyte at three QC levels had relative standard deviation (%RSD) of ≤10.3%, and the intra/inter-day accuracy ranged between 88.0-108%. This developed method allows for simultaneous and trace measurement of four HD urinary metabolites within one single determination with the lowest usage amount of urine samples over all previous methods This study provides a useful tool for early diagnosis and monitoring of HD poisoning for medical treatment with high confidence, avoiding the need for application of several analysis methods.

Chemical Weapons Exposures in Iraq: Challenges of a Public Health Response a Decade Later.

An October 14, 2014 article in The New York Times reported that the US Department of Defense (DoD) concealed, for nearly a decade, circumstances surrounding service members' exposure to chemical warfare agents (CWA) while deployed to Iraq in support of Operations Iraqi Freedom and New Dawn from March 13, 2003, to December 31, 2011, and alleged failure of the DoD to provide expedient and adequate medical care. This report prompted the DoD to devise a public health investigation, with the Army Public Health Center (Provisional) as the lead agency to identify, evaluate, document, and track CWA casualties of the Iraq war. Further, the DoD revisited and revised clinical guidelines and health policies concerning CWA exposure based on current evidence-based guidelines and best practices.

Multi-inhibitor prodrug constructs for simultaneous delivery of anti-inflammatory agents to mustard-induced skin injury.

The molecular pathology of sulfur mustard injury is complex, with at least nine inflammation-related enzymes and receptors upregulated in the zone of the insult. A new approach wherein inhibitors of these targets have been linked by hydrolyzable bonds, either one to one or via separate preattachment to a carrier molecule, has been shown to significantly enhance the therapeutic response compared with the individual agents. This article reviews the published work of the authors in this drug development domain over the last 8 years.

Solid-Phase Extraction of Sulfur Mustard Metabolites Using an Activated Carbon Fiber Sorbent.

A novel solid-phase extraction method using activated carbon fiber (ACF) was developed and validated. ACF has a vast network of pores of varying sizes and microporous structures that result in rapid adsorption and selective extraction of sulfur mustard metabolites according to the pH of eluting solvents. ACF could not only selectively extract thiodiglycol and 1-methylsulfinyl-2-[2-(methylthio)-ethylsulfonyl]ethane eluting a 9:1 ratio of dichloromethane to acetone, and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] and 1,1'-sulfonylbis- [2-S-(N-acetylcysteinyl)ethane] eluting 3% hydrogen chloride in methanol, but could also eliminate most interference without loss of analytes during the loading and washing steps. A sample preparation method has been optimized for the extraction of sulfur mustard metabolites from human urine using an ACF sorbent. The newly developed extraction method was applied to the trace analysis of metabolites of sulfur mustard in human urine matrices in a confidence-building exercise for the analysis of biomedical samples provided by the Organisation for the Prohibition of Chemical Weapons.