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Although organophosphorus agents are used worldwide as pesticides, there have been many reports of pesticide poisoning. Nerve agents are organophosphorus agents that interfere with neurotransmission and have been used as chemical weapons in wars. These agents mainly irreversibly inhibit the action of acetylcholinesterase, an enzyme that breaks down acetylcholine, a neurotransmitter, and are believed to cause acute symptoms of poisoning. However, in recent years, the presence of subacute, delayed toxicity independent of acetylcholinesterase inhibition has been reported for some organophosphorus agents. We analyzed the subacute and delayed toxicity of bis(isopropylmethyl)phosphonate (BIMP), which has the same phosphonate group as sarin. BIMP rounded out the morphology of the cells and reduced the proportion of cells in the G1 phase of the cell cycle over time. No DNA damage was observed, suggesting that BIMP may affect cell division., (Copyright © 2021. Published by Elsevier B.V.)

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, the available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). The current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine sulfate and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. An in vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatography-tandem mass spectrometry method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication., (Published by Elsevier Inc.)

Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments., (Copyright © 2019 Elsevier B.V. All rights reserved.)

G-series nerve agents, such as sarin, tabun, and soman, would cause tremendous harm in military and terrorist attacks, so it is necessary to develop a simple method for the rapid and efficient detection of these hazardous substances. We have developed a tunable acetylcholinesterase (AChE)-functionalized two-dimensional photonic crystal (2D PhC) for the detection of a real nerve agent, sarin. In accordance with the 2D PhC previously prepared by our group, the AChE-functionalized 2D PhC was optimized by adjustment of the amount of monomer in the hydrogel, which not only increased the sensitivity of the 2D PhC, with the detection limit decreasing by two orders of magnitude, but also ensured the structural color spanned the whole visible region in the detection range. A linear relationship between the logarithm of the sarin concentration and the particle spacing of the AChE-functionalized 2D PhC was observed from 7.1 × 10 -17 to 7.1 × 10 -4 mol/L. The AChE-functionalized 2D PhC also responded to mimics of G-series nerve agents, including dimethyl methylphosphonate, diisopropyl methylphosphonate, and isodipropyl methylphosphonate, to various degrees. The proposed 2D-PhC hydrogel has potential for low-cost, trace-level, and on-site monitoring of other G-series nerve agents. Graphical abstract.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

The ability of a new bisquaternary oxime, K027 (1-[4-hydroxyiminomethylpyridinium]-3-[carbamoylpyridinium] propane dibromide), to reactivate the enzyme acetylcholinesterase (AChE) inhibited by the nerve agents Tabun, sarin and VX was evaluated. Its reactivation potency was compared to the AChE reactivators pralidoxime (2-PAM), obidoxime and HI-6; K027 seems a good reactivator of organophosphates-inhibited AChE. Its reactivation potency is lower compared to the other oximes for reactivation of sarin-inhibited AChE, but it is sufficient to significantly increase the activity of sarin-inhibited AChE. Its reactivation ability is comparable to obidoxime for reactivation of VX- and tabun-inhibited AChE and is higher than the reactivation potency of HI-6, for tabun-inhibited AChE. HI-6 is currently regarded the most promising reactivator of organophosphates-inhibited AChE.

This study addresses a comprehensive assessment of the interaction between chemical warfare agents (CWA) and acetylcholinesterase (AChE) systems, focus on the intriguing pnictogen‐bond interaction (PnB). Utilizing the crystallographic data from the Protein Data Bank pertaining to the AChE‐CWA complex involving Sarin (GB), Cyclosarin (GF), 2‐[fluoro(methyl)phosphoryl]oxy‐1,1‐dimethylcyclopentane (GP) and venomous agent X (VX) agents, the CWA is systematically displaced by increments of 0.1 Å along the PO bond axis, extending its distance by 4 Å from the original position. The AIM analysis was carried out and consistently revealed the presence of a significant interaction along the PO bond. Investigating the intrinsic nature of the PnB, the NBO and the EDA analysis unearthed the contribution of orbital factors to the overall energy of the system. Strikingly, this observation challenges the conventional σ‐hole explanation commonly associated with such interactions. This finding adds a layer of complexity to understanding of PnB, encouraging further exploration into the underlying mechanisms governing these intriguing chemical phenomena. [ABSTRACT FROM AUTHOR]

Precision-cut lung slices (PCLS) are a suitable model for analyzing the acetylcholinesterase (AChE) activity and subsequent effects after exposure to organophosphorus (OP) compounds. In this study, the AChE activity was determined in intact PCLS for the first time. Since the current standard therapy for OP poisoning (atropine + oxime + benzodiazepine) lacks efficiency, reliable models to study novel therapeutic substances are needed. Models should depict pathophysiological mechanisms and help to evaluate the beneficial effects of new therapeutics. Here PCLS were exposed to three organophosphorus nerve agents (OPNAs): sarin (GB), cyclosarin (GF), and VX. They were then treated with three reactivators: HI-6, obidoxime (OBI), and a non-oxime (NOX-6). The endpoints investigated in this study were the AChE activity and the airway area (AA) change. OPNA exposure led to very low residual AChE activities. Depending on the reactivator properties different AChE reactivation results were measured. GB-inhibited PCLS-AChE was reactivated best, followed by VX and GF. To substantiate these findings and to understand the connection between the molecular and the functional levels in a more profound way the results were correlated to the AA changes. These investigations underline the importance of reactivator use and point to the possibilities for future improvements in the treatment of OPNA-exposed victims. • Acetylcholinesterase (AChE) activity determination in precision-cut lung slices (PCLS): Use of PCLS as an ex vivo model. • Comprehensive OPNA exposure and reactivation study: Exposure of PCLS to GB, GF, and VX, followed by reactivation with HI-6, OBI, and aNOX-6. • AChE reactivation results: Revealing varying reactivation efficiencies for different nerve agents: GB > VX > GF. • Correlation of molecular and functional levels: Correlating AChE activities with airway area (AA) changes. [ABSTRACT FROM AUTHOR]

The Iran-Iraq war (1980–1988) was the first modern war in which chemical weapons were used. The purpose of this work is to summarize the experience of treating victims, affected by nerve agents. The publications of Iranian authors in different scientific journals were used for the preparation of the article. The first use of chemical weapons by the Iraqi army occurred in August 1983 in Kurdistan (sulfur mustard). Nerve agent (tabun) was first used on February 29, 1984 in the battles for the Majnun Islands. In total, 140 tons of tabun and about 600 tons of sarin / cyclosarin were used against Iranian troops during the war. Around 5.5 thousand servicemen died directly from the nerve agents. For one dead from tabun, there were at least 7 poisoned people who needed qualified toxicological assistance; lethal outcomes among servicemen affected by the herd amounted to at least 15%. Military personnel without protective equipment, who received large doses of nerve agents, lost consciousness within 30 seconds and died in positions within a few minutes. The most sensitive to such agents were women, children and the elderly, who accidentally fell under a chemical attack. The use of nerve agents by the enemy was accompanied by panic in the troops and the arrival of a huge number of military personnel in critical condition at medical stations. Sweating, fasciculations and miosis were the first clinical signs by which those affected by such toxic agents were recognized. The effectiveness of antidote therapy (atropine + oxime + diazepam) depended on the time of medical care. The most effective was the use of atropine by titration (fractional intravenous drip) to achieve ease of breathing, absence of bronchospasm, disappearance of secretions from the respiratory tract and heart rate >80 beats/min. The oximes turned out to be not effective in the treatment of those poisoned by tabun. The most effective was degassing by physical removal of toxic chemical. Degassing of the affected had to be carried out by washing with sea or river water, using solutions of chlorine-containing household chemicals, and sprinkling with dry earth.

Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [18F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.

Introduction Sarin is a highly toxic organophosphorus nerve agent that irreversibly inhibits neuronal enzyme acetylcholinesterase. In the prevailing scenario, it is of paramount importance to develop early diagnosis and medical countermeasures for sarin exposure. A deeper understanding of the molecular mechanism of sarin intoxication and perturbations in the associated cellular processes is likely to provide valuable clues for the elucidation of diagnostic markers and therapeutic targets for sarin exposure. Methods Present study, uncovered the changes in phosphorylation patterns of multiple proteins in different rat brain regions after sarin intoxication using 2-DE/MS approach. It provided a holistic view of the phosphorylation-mediated changes in the cellular proteome and highlighted various signaling and response pathways affected at an early time point of sarin intoxication. Results We found total 22 proteins in the cortex, 25 proteins in the corpus striatum, and 17 proteins in the hippocampus, showed ≥1.5 fold changes (hyper- or hypo- phosphorylated) with respect to control, either at 2.5 h or 1 d after sarin exposure. These results indicated the differential expression of phosphoproteins involved in protein folding in the endoplasmic reticulum, carbon metabolism, metabolic function, and energy metabolism. Conclusion Four candidates (protein disulfide-isomerase A3, heat shock cognate 71 kDa protein, alpha-enolase, and creatine kinase B-type), hyperphosphorylated in all three brain regions, can be further studied to understand the molecular mechanism behind neurodegenerative changes mediated by sarin exposure. The study sheds light on major pathogenic processes initiated during sarin intoxication and provides putative diagnostic markers/therapeutic targets for further validation. [ABSTRACT FROM AUTHOR]

O-(1-Fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate is a reactive organophosphate ester (OP) developed as a surrogate of the chemical warfare agent sarin that forms a similar covalent adduct at the active site serine of acetylcholinesterase. The radiolabeled O-(1-[18 F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate ([18 F] fluorosarin surrogate) has not been previously prepared. In this paper, we report the first radiosynthesis of this tracer from the reaction of bis-(4-nitrophenyl) methylphosphonate with 1-[18 F]fluoro-2-propanol in the presence of DBU. The 1-[18 F]fluoro-2-propanol was prepared by reaction of propylene sulfite with Kryptofix 2.2.2 and [18 F] fluoride ion. The desired tracer O-(1-[18 F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate was obtained in a >98% radiochemical purity with a 2.4% ± 0.6% yield (n = 5, 65 minutes from start of synthesis) based on starting [18 F] fluoride ion and a molar activity of 49.9 GBq/μmol (1.349 ± 0.329 Ci/μmol, n = 3). This new facile radiosynthesis routinely affords sufficient quantities of [18 F] fluorosarin surrogate in high radiochemical purity, which will further enable the tracer development as a novel radiolabeled OP acetylcholinesterase inhibitor for assessment of OP modes of action with PET imaging in vivo.

Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 μg/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule.

Past assassinations and terrorist attacks demonstrate the need for a more effective antidote against nerve agents and other organophosphates (OP) that cause brain damage through inhibition of acetylcholinesterase (AChE). Our lab has invented a platform of phenoxyalkyl pyridinium oximes (US patent 9,277,937) that demonstrate the ability to cross the blood–brain barrier in in vivo rat tests with a sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) and provide evidence of brain penetration by reducing cessation time of seizure‐like behaviors, accumulation of glial fibrillary acidic protein (GFAP), and hippocampal neuropathology, as opposed to the currently approved oxime, 2‐pyridine aldoxime methyl chloride (2‐PAM). Using two of the novel oximes (Oximes 1 and 20), this project examined whether gene expression changes might help explain this protection. Expression changes in the piriform cortex were examined using polymerase chain reaction arrays for inflammatory cytokines and receptors. The hippocampus was examined via quantitative polymerase chain reaction for the expression of immediate‐early genes involved in brain repair (Bdnf), increasing neurotoxicity (Fos), and apoptosis control (Jdp2, Bcl2l1, Bcl2l11). In the piriform cortex, NIMP significantly stimulated expression for the macrophage inflammatory proteins CCL4, IL‐1A, and IL‐1B. Oxime 20 by itself elicited the most changes. When it was given therapeutically post‐NIMP, the largest change occurred: a 310‐fold repression of the inflammatory cytokine, CCL12. In the hippocampus, NIMP increased the expression of the neurotoxicity marker Fos and decreased the expression of neuroprotective Bdnf and antiapoptotic Bcl2l1. Compared with 2‐PAM, Oxime 20 stimulated Bcl2l1 expression more and returned expression closer to the vehicle control values. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Organophosphorus (OP) nerve agent poisoning made the headlines in 2018 with the nerve agent 'Novichok' poisonings in Salisbury, England. This event highlighted a gap in the knowledge of most clinicians in the UK. In response, this special article aims to enlighten and signpost anaesthetists and intensivists towards the general management of OP nerve agent poisoned patients. Drawing on a broad range of sources, we will discuss what OP nerve agents are, how they work, and how to recognise and treat OP nerve agent poisoning. OP nerve agents primarily act by inhibiting the enzyme acetylcholinesterase, causing an acute cholinergic crisis; death usually occurs through respiratory failure. The antimuscarinic agent atropine, oximes (to reactivate acetylcholinesterase), neuroprotective drugs, and critical care remain the mainstays of treatment. The risk to medical staff from OP poisoned patients appears low, especially if there is a thorough decontamination of the poisoned patient and staff wear appropriate personal protective equipment. The events in Salisbury in the past year were shocking, and the staff at Salisbury District General Hospital performed admirably in treating those affected by Novichok nerve agent poisoning. We eagerly anticipate their future clinical publications so that the medical community might learn from their valuable experiences. [ABSTRACT FROM AUTHOR]

Abstract Diisopropyl fluorophosphate (DFP), a surrogate of nerve agent sarin, is an organophosphorus (OP) compound which inhibits neuronal enzyme acetylcholinesterase (AChE). Exposure of this compound leads to a wide range of toxic symptoms and survivors may exhibit long term neurotoxicity related to cognitive and memory defects. Due to ease of availability and similar mechanism of action to other highly toxic nerve agent, DFP is widely used as model compound to trace changes associated with nerve agent exposures. Proximal fluids are widely used for the elucidation of biomarkers for exposure to toxic substances and to study the mechanism of toxicity. Using a rat model of OP intoxication, the present study was carried out to elucidate proteomic changes in plasma associated with DFP intoxication. Rats were exposed to a single dose (0.5 LD 50) of DFP and their plasma proteome was studied, one day post exposure by two dimensional gel electrophoresis - mass spectrometry (2DE-MS). Some of the milestone changes were validated by Western blot analysis. A total 15 proteins showed significant fold changes in expression with respect to control after 1 day of DFP intoxication. Most of the proteins showing changes in expression at initial stages were related to immunogenic function, acute phase response, blood coagulation, and stress response. Experiments reported here demonstrate that 0.5 LD 50 DFP intoxication leads to AChE inhibition, modulation of immunogenic function, and generation of stress at an early stage. Although, some proteins and their putative functional ramifications indicated similarity with those observed in our previous plasma proteome study, neurodegenerative changes were not observed in plasma of 0.5 LD 50 DFP treated animals. Highlights • Plasma proteome was evaluated after 0.5 LD 50 DFP intoxication. • Comparative evaluation was carried out with sarin plasma proteome study. • DFP exposure influences the stress and immunogenic responses. • DFP exposure leads to changes in the proteins belonging to various processes. • Comparative toxicities are likely to provide new therapeutic avenues. [ABSTRACT FROM AUTHOR]

O‐(1‐Fluoropropan‐2‐yl)‐O‐(4‐nitrophenyl) methylphosphonate is a reactive organophosphate ester (OP) developed as a surrogate of the chemical warfare agent sarin that forms a similar covalent adduct at the active site serine of acetylcholinesterase. The radiolabeled O‐(1‐[18F]fluoropropan‐2‐yl)‐O‐(4‐nitrophenyl) methylphosphonate ([18F] fluorosarin surrogate) has not been previously prepared. In this paper, we report the first radiosynthesis of this tracer from the reaction of bis‐(4‐nitrophenyl) methylphosphonate with 1‐[18F]fluoro‐2‐propanol in the presence of DBU. The 1‐[18F]fluoro‐2‐propanol was prepared by reaction of propylene sulfite with Kryptofix 2.2.2 and [18F] fluoride ion. The desired tracer O‐(1‐[18F]fluoropropan‐2‐yl)‐O‐(4‐nitrophenyl) methylphosphonate was obtained in a >98% radiochemical purity with a 2.4% ± 0.6% yield (n = 5, 65 minutes from start of synthesis) based on starting [18F] fluoride ion and a molar activity of 49.9 GBq/μmol (1.349 ± 0.329 Ci/μmol, n = 3). This new facile radiosynthesis routinely affords sufficient quantities of [18F] fluorosarin surrogate in high radiochemical purity, which will further enable the tracer development as a novel radiolabeled OP acetylcholinesterase inhibitor for assessment of OP modes of action with PET imaging in vivo. Synthesis of a fluorine‐18‐labeled PET tracer surrogate of sarin to evaluate the exposure mechanism and reactivation of acetylcholinesterase. [ABSTRACT FROM AUTHOR]

Abstract Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure. [ABSTRACT FROM AUTHOR]

Reactivation of acetylcholinesterase (AChE), an essential enzyme in neurotransmission, is a key point in the treatment of acute poisoning by nerve agents and pesticides, which structurally belong to organophosphorus compounds (OP). Due to the high diversity of substituents on the phosphorous atom, there is a variety of OP-AChE conjugates deriving from AChE inhibition, and therefore not only is there no universal reactivator efficient enough for the most toxic OPs, but for some nerve agents there is still a lack of any reactivator at all. The endeavor of many chemists to find more efficient reactivators resulted in thousands of newly-designed and synthesized oximes−potential reactivators of AChE. For an evaluation of the oximés reactivation efficiency, many research groups employ a simple spectrophotometric Ellman method. Since parameters that describe reactivator efficiency are often incomparable among laboratories, we tried to emphasize the critical steps in the determination of reactivation parameters as well as in the experimental design of a reactivation assay. We highlighted the important points in evaluation of reactivation kinetic parameters with an aim to achieve better agreement and comparability between the results obtained by different laboratories and overall, a more efficient evaluation of in vitro reactivation potency. [ABSTRACT FROM AUTHOR]

Isopropyl methylphosphonoselenofluoridate (sarin-Se) was synthesized in order to determine its analytical data and to compare them as well as its reactivity parameters with those of sarin. After synthesis, sarin-Se was characterized using 1H, 13 C and 31P NMR spectroscopy and high resolution mass spectrometry (HRMS). 1H and 13 C NMR chemical shifts were also calculated using density functional theory (DFT) and compared with experimental data, showing a close agreement. Electrophilicity indices of sarin and sarin-Se were calculated at B3LYP/6-31++G (2d, 2p) level of theory. HOMO-LUMO analysis provides a basis to explain electrophilic nature of sarin-Se and sarin, which are almost similar. Based on calculated data, 31P NMR chemical shift and the reactivity of sarin-Se towards some enzymes such as acetylcholinesterase and albumin were explained. [ABSTRACT FROM AUTHOR]

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A 1 receptor agonist N 6 -cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development. [ABSTRACT FROM AUTHOR]

Efficacy of two oximes treatments evaluated during inhalation of sarin vapor (LCt50, 755.9 mg/min/m3) in simulated real scenario in vivo. Majority of mice either became moribund or died within 1-2 min during exposure to multifold-lethal concentrations of sarin vapor. Protection indices were determined by exposing to sarin vapor in two sessions, 1 min exposure followed by treatments with or without HNK-102 (56.56 mg/kg, im) or 2-PAM (30 mg/kg, im) and atropine (10 mg/kg, ip), and again exposed for remaining 14 min. Protection offered by HNK-102 was found to be four folds higher compared to 2-PAM in the same toxic environment. Secondly, sub-lethal concentration of sarin vapor (0.8 × LCt50 or 605 mg/min/m3), 24 h post investigations revealed that the oximes could not reactivate brain and serum acetylcholinesterase (AChE) activity. The treatments prevented increase in protein concentration (p < .05) and macrophages infiltration compared to sarin alone group in broncho-alveolar lavage fluid. Lung histopathology showed intense peribronchial infiltration and edema with desquamating epithelial lining and mild to moderate alveolar septal infiltration in sarin and atropine groups, respectively. Noticeable peeling-off observed in epithelial lining and sporadic mild infiltration of epithelial cells at bronchiolar region in 2-PAM and HNK-102 groups, respectively. The oximes failed to reactivate AChE activity; however, the mice survived up to 6.0 × LCt50, proved involvement of non-AChE targets in sarin toxicity. Atropine alone treatment was found to be either ineffective or increased the toxicity. HNK-102, exhibited better survivability with lung protection, can be considered as a better replacement for 2-PAM to treat sarin inhalation induced poisoning. [ABSTRACT FROM AUTHOR]

Sarin is an organophosphorus (OP) chemical warfare agent which irreversibly inhibits acetylcholinesterase. Acute toxicity after sarin exposure is because of hyper activation of the nicotinic and muscarinic receptor. Survivors of sarin exposure often develop long-term neuropathology referred as OP ester-induced chronic neurotoxicity. However, the exact mechanism of chronic neurotoxicity is yet unknown. We studied proteomic changes in rat brain regions after 0.5 LD50 dose of sarin and investigated some milestone changes associated with long-term CNS injury.We used two-dimensional gel electrophoresis/mass spectrometry approach to identify early proteomic changes and traced expression of selected proteins for longer time points. This study shows changes in chaperone function, endoplasmic reticulumstress, and defect in cytoskeleton functions at earlier stages. Predictive interaction analysis demonstrated putative role of Parkinson's disease-related proteins after sarin exposure. Our results clearly indicated neurodegenerative changes which started after 2.5h and showed prominence after 3-month postexposure. The study also unmasks changes in proteins related to movement and cognitive function. The markers for astrocytosis (GFAP) and neurodegenerative changes (alpha-synuclein and amyloid precursor protein) exhibited altered expression in brain. This is the first proteomic study among survivors of sarin exposure in animal model. Some of the early changes, including those involved in neurodegeneration, movement, and cognitive function, defects in chaperone function and cytoskeleton, were shown to persist for a longer period. The study provides a preliminary framework for further validation of major mechanisms of sarin toxicity is suggested here and opens new avenues for elucidation of therapeutic intervention. [ABSTRACT FROM AUTHOR]

Chemistry is a science that contributes to all aspects of our everyday lives and our professions. There are clear examples in law enforcement (forensics) and public health and perhaps less clear (but equally important) uses of chemicals in applications that include automobile manufacturing, electronics, packaging materials, currency printing, and even waste management (recycling and value-added products from garbage). Chemistry can also influence international diplomacy - an area that is likely to be unfamiliar to many chemistry professionals. Take for example the United Nations led investigation into the alleged use of chemical weapons in Syria in August of 2013. Environmental and biomedical samples were collected and analyzed, and they undisputedly confirmed the use of the nerve agent sarin. The results were published in a report by the United Nations Secretary-General and were one of the many influences leading to the accession of The Syrian Arab Republic to the Chemical Weapons Convention (an international treaty prohibiting chemical weapons) and the declaration and dismantlement of a chemical weapons programme. Using this investigation as an example, we highlight some of the chemistry that influenced decision making in a high visibility international event. [ABSTRACT FROM AUTHOR]

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group. [ABSTRACT FROM AUTHOR]

The high number of annual fatalities following suicidal poisoning by organophosphorus (OP) pesticides and the recent homicidal use of the chemical warfare nerve agent sarin against civilian population in Syria underlines the continuous threat by these highly toxic agents. The need for an effective treatment of OP poisoning resulted in the implementation of a combination therapy with the muscarinic receptor antagonist atropine and an oxime for the reactivation of OP-inhibited acetylcholinesterase (AChE). Since the invention of the first clinically used oxime pralidoxime (2-PAM) in the 1950s ongoing research attempted to identify more effective oximes. In fact, several thousand oximes were synthesized in the past six decades. These include charged and non-charged compounds, mono- and bispyridinium oximes, asymmetric oximes, oximes with different substitutes and more recently non-oxime reactivators. Multiple in vitro and in vivo studies investigated the potential of oximes to reactivate OP-inhibited AChE and to reverse OP-induced cholinergic signs. Depending on the experimental model, the investigated species and the tested OP largely variable results were obtained by different laboratories. These findings and the inconsistent effectiveness of oximes in the treatment of OP-pesticide poisoned patients led to a continuous discussion on the value of oximes. In order to provide a forward-looking evaluation of the significance of oximes in OP poisoning multiple aspects, including intrinsic toxicity, in vitro reactivation potency, efficacy and pharmacokinetics, as well as the impact of the causative OP have to be considered. The different influencing factors in order to define the benefit and limitations of oximes in OP poisoning will be discussed. [ABSTRACT FROM AUTHOR]

The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via (CH 2 ) n , (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3–10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro . 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (k r = 3.6 × 10 2 vs. 0.2 × 10 2 M −1 min −1 , respectively, 37 °C). These hybrids inhibited AChE reversibly, IC 50 = 16–48 μM, thereby decreasing the inhibition rates by OPs. The LD 50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 μmol/kg in rats and 144, 203 and >506 μmol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD 50 sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs. [ABSTRACT FROM AUTHOR]

Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator as an antidote. Structurally different oximes are widely studied to examine their kinetic and mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related 1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides ( 5a–5e, 9a–9c ) were synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and VX-inhibited human acetylcholinesterase ( h AChE). The observed results were compared with the reactivation efficacy of standard reactivators; 2-PAM, obidoxime and HI-6. Amongst the synthesized oximes, 5a , 9a and 9b were found to be most potent reactivators against sarin-inhibited h AChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM. Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the reactivation strength of prepared reactivator. Physicochemical properties of synthesized reactivators have also been evaluated. [ABSTRACT FROM AUTHOR]

The enzyme acetylcholinesterase (AChE) is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer), it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol /L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples.