Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Divinyl sulfone, an oxidative metabolite of sulfur mustard, induces caspase-independent pyroptosis in hepatocytes.

Sulfur mustard (SM) is a highly toxic blister agent which has been used many times in several wars and conflicts and caused heavy casualties. Ease of production and lack of effective therapies make SM a potential threat to public health. SM intoxication causes severe damage on various target organs, such as the skin, eyes, and lungs. In addition, SM exposure can also lead to hepatotoxicity and severe liver injuries. However, despite decades of research, the molecular mechanism underlying SM-induced liver damage remains obscure. SM can be converted into various products via complex hepatic metabolism in vivo. There are some pieces of evidence that one of the oxidation products of SM, divinyl sulfone (DVS), exhibits even more significant toxicity than SM. Nevertheless, the molecular toxicology of DVS is still hardly known. In the present study, we confirmed that DVS is even more toxic than SM in the human hepatocellular carcinoma cell line HepG2. Further mechanistic study revealed that DVS exposure (200 µM) promotes pyroptosis in HepG2 cells, while SM (400 µM) mainly induces apoptosis. DVS induces gasdermin D (GSDMD) mediated pyroptosis, which is independent of caspases activation but depends on the large amounts of reactive oxygen species (ROS) and severe oxidative stress produced during DVS exposure. Our findings may provide novel insights for understanding the mechanism of SM poisoning and may be helpful to discover promising therapeutic strategies for SM intoxication.

Skin decontamination of Carfentanil in vitro.

Skin decontamination of Chemical Biological Radioactive and Nuclear (CBRN) materials involves the timely and effective removal of the contaminants from the skin surface. The current work evaluated Fuller's Earth & The Reactive Skin Decontaminant Lotion Kit (RSDL®) to investigate whether they were as efficacious against free base Carfentanil skin contamination as they are against chemical warfare agents. The in vitro methodology used allowed for evaluation of decontamination regimens as specified by the decontaminant manufacturer rather than as an application of a bolus dose left in situ for the study duration. A selection of novel decontaminants, including Dermal Decontamination Gel (DDGel), Trivorex®, itaconic acid (IA), N,N'-methylenebisacrylamide (MBA), 2-trifluoromethylacrylic acid (TFMAA) and NanoSan Sorb were also tested for efficacy. All the evaluated decontaminants were successful at removing the majority of the Carfentanil skin surface contamination. The current work has shown that the Fuller's Earth decontamination kit, removes as much (or more) free base carfentanil from the skin surface in comparison to other products tested in this study series.

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.

A method to assess dermal absorption dynamics of chemical warfare agents: Finite doses of volatile compounds.

Chemical warfare agents are absorbed into the body from various entry routes and may have detrimental effects on human health. As many chemical compounds in this group are lipophilic, the outer layer of the skin is at an elevated risk. This contribution explores the dynamics of skin penetration for risk assessment. A previously validated model was applied to describe how an agent is transported across the stratum corneum following dermal exposure to a finite dose of a chemical. A mathematical construct was implemented for estimating the time constants and the cumulative amount of permeant entering the bloodstream or being released into the environment. Empirical equations were selected to determine the ratio of the steady-state evaporation rate to the steady-state dermal absorption rate and the physicochemical properties of the chemical warfare agents. Wolfram Mathematica was employed to run the simulations. The results from the newly derived expressions for the time constants matched those directly obtained from the validated model. For example, sarin gas had steady-state evaporation to an absorption rate of 991.25, and a total fractional absorption and evaporation of 5.1% and 94.9%, respectively. Combined with occupational exposure limits, the findings can help researchers assess an individual's risk level and develop protection programs.

Role of hair follicles in the pathogenesis of arsenical-induced cutaneous damage.

Arsenical vesicants cause skin inflammation, blistering, and pain. The lack of appropriate animal models causes difficulty in defining their molecular pathogenesis. Here, Ptch1+/- /C57BL/6 mice were employed to investigate the pathobiology of the arsenicals lewisite and phenylarsine oxide (PAO). Following lewisite or PAO challenge (24 h), the skin of animals becomes grayish-white, thick, leathery, and wrinkled with increased bi-fold thickness, Draize score, and necrotic patches. In histopathology, infiltrating leukocytes (macrophages and neutrophils), epidermal-dermal separation, edema, apoptotic cells, and disruption of tight and adherens junction proteins can be visualized. PCR arrays and nanoString analyses showed significant increases in cytokines/chemokines and other proinflammatory mediators. As hair follicles (HFs), which provide an immune-privileged environment, may affect immune cell trafficking and consequent inflammatory responses, we compared the pathogenesis of these chemicals in this model to that in Ptch1+/- /SKH-1 hairless mice. Ptch1+/- /SKH-1 mice have rudimentary, whereas Ptch1+/- /C57BL/6 mice have well-developed HFs. Although no significant differences were observed in qualitative inflammatory responses between the two strains, levels of cytokines/chemokines differed. Importantly, the mechanism of inflammation was identical; both reactive oxygen species induction and consequent activation of unfolded protein response signaling were similar. These data reveal that the acute molecular pathogenesis of arsenicals in these two murine models is similar.

QM/MM and MM MD simulations on decontamination of the V-type nerve agent VX by phosphotriesterase: toward a comprehensive understanding of steroselectivity and activity.

Due to deadly toxicity and high environmental stability of the nerve agent VX, an efficient decontamination approach is desperately needed in tackling its severe threat to human security. The enzymatic destruction of nerve agents has been generally considered as one of the most effective ways, and here the hydrolysis of VX by phosphotriesterase (PTE) was investigated by extensive QM/MM and MM MD simulations. The hydrolytic cleavage of P-S by PTE is a two-step process with the free energy spans of 15.8 and 26.0 kcal mol-1 for the RP- and SP-enantiomer VX, respectively, and such remarkable stereospecificity of VX enantiomers in the enzymatic degradation is attributed to their conformational compatibility with the active pocket. The structurally less adaptive SP-enantiomer allows one additional water molecule to enter the binuclear zinc center and remarkably facilitates the release of the degraded product. Overall, the rate-limiting steps in the enzymatic degradation of VX by PTE involve the degraded product release of the RP-enantiomer and the enzymatic P-S cleavage of the SP-enantiomer. Further computational analysis on the mutation of selected residues also revealed that H257Y, H257D, H254Q-H257F, and L7ep-3a variants allow more water molecules to enter the active site, which improves the catalytic efficiency of PTE, as observed experimentally. The present work provides mechanistic insights into the stereoselective hydrolysis of VX by PTE and the activity manipulation through the active-site accessibility of water molecules, which can be used for the enzyme engineering to defeat chemical warfare agents.

Extended retrospective detection of regenerated sarin (GB) in rabbit blood and the IMPA metabolite in urine: a pharmacokinetics study.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70 days for 0.1 LD50 and 0.5 LD50 and 77 days for 0.9 LD50, where rapid elimination occurred in the first 8 days with an initial average half-life of 1.2 days, followed by a second, slower elimination, with a terminal average half-life of 8.4 days. The time frame for monitoring IMPA in urine was 7, 15 and 16 days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8 days on days 2-6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4 days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

In vitro human skin decontamination efficacy of MOF-808 in decontamination lotion following exposure to the nerve agent VX.

Metal-organic frameworks (MOFs) have shown promising properties for removal of chemical warfare agents, in particular for material decontamination and functionalized fabrics. The MOF-properties could also be beneficial for skin decontamination, especially when exposed to highly toxic and low volatile nerve agents. In such exposures, efficient decontamination is crucial for adequate medical management. In the present study, seven zirconium-based MOFs were evaluated for their ability to degrade VX and subsequently tested in vitro for decontamination of VX on human dermatomed skin. Of the MOFs evaluated, MOF-808 showed the greatest ability to degrade VX in an alkaline buffer with complete degradation of VX within 5 min. PCN-777, Zr-NDC and NU-1000 displayed degradation half-lives of approximately 10 min. When including MOF-808 in a skin friendly carrier with slightly acidic pH, a decreased agent degradation rate was observed, requiring over 24 h to reach complete degradation. In skin decontamination experiments, MOF-808 enhanced the efficacy compared to the carrier alone, essentially by improved agent absorption. Adding MOF-808 to Reactive Skin Decontamination Lotion (RSDL) did not improve the high effectiveness of RSDL alone. The present study showed that including MOF in skin decontamination lotions could be beneficial. Further studies should include optimizing the particulates and formulations.

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.

No promising antidote 25 years after the Tokyo subway sarin attack: A review.

On the battlefields of Syria, many innocent civilians have been killed or injured by sarin poisoning. In Malaysia in February 2017, a North Korean man was assassinated with VX at Kuala Lumpur International Airport. In the face of such threats, a more effective antidote against organophosphonate acetylcholinesterase (AChE) inhibitors is needed, one that can freely penetrate into the central nervous system (CNS) through the blood-brain barrier (BBB). In the 1995 Tokyo subway sarin attack, which produced more than 6,000 victims, 2-pyridinealdoxime methiodide was the most commonly used antidote in hospitals, but it was unable to prevent CNS damage and no other oximes have been approved for use in Japan. Ultimately, 12 people died, and many victims had severe neurological injuries or sequelae. Although more than 25 years have passed since the incident, progress has been slow in the development of a new antidote that can penetrate the BBB, restore AChE activity in the CNS, and definitely prevent brain injury. From the perspectives of countering terrorism and protecting innocent people from nerve agent attacks, the search for nerve agent antidotes should be accelerated with the goals of improving both survival and quality of life. This review gives an overview of a series of our studies on the development of a new antidote since the Tokyo subway sarin attack and emphasizes that there is unfortunately still no promising antidote for saving the CNS in Japan.

Enantioselective in-vitro elimination kinetics of nerve agents in blood monitored by derivatization and LC-MS/MS analysis.

We present a simple method for chiral separation and analysis of organophosphorus nerve agents and apply it to monitor the enantioselective blood elimination kinetics of sarin in-vitro. The method is implemented in standard reverse phase LC-MS operating conditions, relieving the user of the dedicated operating conditions frequently demanded in chiral LC-MS analysis. The method consists of formation of diastereomers by a rapid derivatization with (R)-2-(1 aminoethyl) phenol, followed by LC-MS/MS analysis. Derivatization enantioselectivity was studied by comparing the reaction of optically pure sarin and racemic sarin, proving no substantial enantiomeric preference in the reaction and demonstrating the enantiomeric discrimination abilities of the technique. Enantioselective sarin elimination pathways were probed in-vitro by following the fast elimination kinetics of the two sarin enantiomers as well as its hydrolysis metabolite (isopropyl methyl-phosphonic acid, IMPA) in whole blood and plasma compared to water. Sarin enantiomers showed the known marked differences in elimination kinetics with rapid elimination of the (+) enantiomer and slower elimination of the (-) enantiomer in whole blood and plasma as well as dose-dependent kinetics (faster elimination at lower concentrations). We found that small amounts of acetonitrile in plasma prevent the rapid elimination of the (+) enantiomer, resulting in similar, slower elimination kinetics for both enantiomers.

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent.

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in KM and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased KM by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.

Studying the metabolism of toxic chemical warfare agent-related phenylarsenic chemicals in vitro in cod liver.

Large quantities of chemical warfare agents (CWAs), such as phenylarsenic chemicals, were disposed by sea-dumping after World War II. Nowadays, the release of these toxic chemicals from munitions poses a potential threat to living organisms. This study investigates the fate of these chemicals in fish by exposing selected CWA-related phenylarsenic chemicals and their oxidation products to cod (Gadus morhua) liver S9 fraction in vitro. Clark I (DA), Adamsite (DM) and their corresponding oxidation products as well as triphenylarsine oxide (TPA[ox]) and phenylarsonic acid (PDCA[ox]) were used as chemicals in in vitro experiments. Glutathione (GSH) conjugates of DA, DM and PDCA-related chemicals were found to be the most dominant metabolites, and methylated metabolites were detected as well, suggesting that these compounds are metabolised in the presence of cod liver enzymes. TPA[ox] was the only compound tested that did not form a GSH conjugate or methylated metabolite, indicating a different biotransformation pathway for this compound. Furthermore, hydroxylated metabolites were detected for each tested chemical. Due to their reactive nature, GSH conjugates may be difficult to detect in fish samples from CWA dumpsites. In contrast, both methylated and hydroxylated metabolites of phenylarsenic chemicals are promising target chemicals for the detection of CWA-related contamination in fish.

A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Protein adduct binding properties of tabun-subtype nerve agents after exposure in vitro and in vivo.

Upon entering the body, nerve agents can bind active amino acid residues to form phosphonylated adducts. Tabun derivatives (O-alkyl-N,N-dialkyl phosphoroamidocyanidates) have strikingly different structural features from other G-series nerve agents, such as sarin and soman. Here, we investigate the binding mechanism for the phosphonylated adducts of nerve agents of tabun derivatives. Binding sites for three tabun derivatives, O-ethyl-N,N- dimethyl phosphoramidocyanidate (GA), O-ethyl-N,N-ethyl(methyl) phosphoramidocyanidate, and O-ethyl-N,N-diethylphosphoramidocyanidate were studied. Quadrupole-orbitrap mass spectrometry (Q-Orbitrap-MS) coupled to proteomics was used to screen adducts between tabun derivatives and albumin, immunoglobulin, and hemoglobin. The results reveal that all three tabun derivatives exhibit robust selectivity to lysine residues, rather than other amino acid residue types. A set of 10 lysine residues on human serum albumin are labeled by tabun derivatives in vitro, with K525 (K*QTALVELVK) and K199 (LK*CASLQK) peptides displaying the most reactivity. Tabun derivatives formed stable adducts on K525 and K414 (K*VPQVSTPTLVEVSR) for at least 7 days and on K351 (LAK*TYETTLEK) for at least 5 days in a rabbit model. Three of these peptides-K525, K414, and K351-have the highest homology with human serum albumin of all 5 lysine residues that bound to examined rabbit blood proteins in vivo. Molecular simulation of the tabun-albumin interaction using structural analysis and molecular docking provided theoretical evidence supporting lysine residue reactivity to phosphonylation by tabun derivatives. K525 has the lowest free binding energy and the strongest hydrogen bonding to human albumin. In summary, these findings identify unique binding properties for tabun derivatives to blood proteins.

Dimerization of human butyrylcholinesterase expressed in bacterium for development of a thermally stable bioscavenger of organophosphorus compounds.

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.

Multiple metabolic pathways are predictive of ricin intoxication in a rat model.

INTRODUCTION: Exposure to ricin can be lethal and treatments that are under development have short windows of opportunity for administration after exposure. It is therefore essential to achieve early detection of ricin exposure to provide the best prognosis for exposed individuals. Ricin toxin can be detected in clinical samples via several antibody-based techniques, but the efficacy of these can be limited due to the rapid processing and cellular uptake of toxin in the body and subsequent low blood ricin concentrations. Other diagnostic tools that perform, in an orthogonal manner, are therefore desirable. OBJECTIVES: To determine time-dependent metabolic changes in Sprague-Dawley rats following intravenous exposure to ricin. METHODS: Sprague-Dawley rats were intravenously exposed to ricin and multiple blood samples were collected from each animal for up to 48 h following exposure in two independent studies. Plasma samples were analysed applying HILIC and C18 reversed phase UHPLC-MS assays followed by univariate and multivariate analysis. RESULTS: In Sprague-Dawley rats we have demonstrated that metabolic changes measured in blood can distinguish between rats exposed intravenously to ricin and controls prior to the onset of behavioral signs of intoxication after 24 h. A total of 37 metabolites were significantly altered following exposure to ricin when compared to controls. The arginine/proline, bile acid and triacylglyceride metabolic pathways were highlighted as being important with two triacylglycerides at 8 h post exposure giving an AUROC score of 0.94. At 16 h and 24 h the AUROC score increased to 0.98 and 1.0 with the number of metabolites in the panel increasing to 5 and 7, respectively. CONCLUSIONS: These data demonstrate that metabolites may be a useful tool to diagnose and detect ricin exposure, thus increasing the effectiveness of supportive therapy and future ricin-specific medical treatments.

The in vitro protective effects of the three novel nanomolar reversible inhibitors of human cholinesterases against irreversible inhibition by organophosphorous chemical warfare agents.

Acetylcholinesterase (AChE) is an enzyme which terminates the cholinergic neurotransmission, by hydrolyzing acetylcholine at the nerve and nerve-muscle junctions. The reversible inhibition of AChE was suggested as the pre-treatment option of the intoxications caused by nerve agents. Based on our derived 3D-QSAR model for the reversible AChE inhibitors, we designed and synthesized three novel compounds 8-10, joining the tacrine and aroylacrylic acid phenylamide moieties, with a longer methylene chain to target two distinct, toplogically separated anionic areas on the AChE. The targeted compounds exerted low nanomolar to subnanomolar potency toward the E. eel and human AChE's as well as the human BChE and showed mixed inhibition type in kinetic studies. All compounds were able to slow down the irreversible inhibition of the human AChE by several nerve agents including tabun, soman and VX, with the estimated protective indices around 5, indicating a valuable level of protection. Putative noncovalent interactions of the selected ligand 10 with AChE active site gorge were finally explored by molecular dynamics simulation suggesting a formation of the salt bridge between the protonated linker amino group and the negatively charged Asp74 carboxylate side chain as a significant player for the successful molecular recognition in line with the design strategy. The designed compounds may represent a new class of promising leads for the development of more effective pre-treatment options.

[Analysis of Countermeasures against Chemical Terrorism].

As a countermeasure against terrorism involving highly toxic chemical warfare agents, the rapid identification of the causative toxic substances is extremely important. This symposium review describes analytical methods the author's group has developed for detecting nerve gases after either high level or low level exposure. As a method for assessing human exposure to high levels of nerve gases, a technology that detects nerve gas hydrolysis products, i.e., strong anion exchange extraction-tert-butyldimethylsilyl derivatization-selectable one-dimensional or two-dimensional GC-MS, is explained. As a method for assessing human exposure to low levels of nerve gases, two technologies that detect adducts of nerve gas with blood cholinesterase, i.e., adduct purification-enzymatic digestion-LC/MS and fluoride-mediated regeneration-solid phase extraction-large volume introduction GC-MS, are explained.