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1.
Anal Biochem ; 685: 115388, 2024 01 15.
Article in English | MEDLINE | ID: mdl-37967783

ABSTRACT

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.


Subject(s)
Soman , Animals , Humans , Rabbits , Soman/metabolism , Europium , Microfluidics , Retrospective Studies , Serum Albumin, Human , Fluorescent Antibody Technique
2.
Biol Pharm Bull ; 46(4): 563-573, 2023.
Article in English | MEDLINE | ID: mdl-37005300

ABSTRACT

This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague-Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I-IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I + II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I-IV, I-III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.


Subject(s)
Soman , Transcription Factors , Rats , Male , Animals , Transcription Factors/metabolism , Reactive Oxygen Species/metabolism , Soman/metabolism , Acetylcholinesterase/metabolism , Electron Transport , Rats, Sprague-Dawley , Liver/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism
3.
Chem Biol Interact ; 309: 108714, 2019 Aug 25.
Article in English | MEDLINE | ID: mdl-31228470

ABSTRACT

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.


Subject(s)
Chemical Warfare Agents/chemistry , Cholinesterase Inhibitors/chemistry , Cholinesterases/metabolism , Protective Agents/chemistry , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Binding Sites , Catalytic Domain , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/metabolism , Cholinesterases/chemistry , Humans , Kinetics , Molecular Dynamics Simulation , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Protective Agents/metabolism , Quantitative Structure-Activity Relationship , Soman/chemistry , Soman/metabolism
4.
Arch Toxicol ; 93(7): 1853-1863, 2019 07.
Article in English | MEDLINE | ID: mdl-31161358

ABSTRACT

A major challenge in organophosphate compound (OP) and OP nerve agent (OPNA) research has been in the identification and utilization of reliable biomarkers for rapid, sensitive, and efficient detection of OP exposure. Albumin has been widely studied as a biomarker for retrospective verification of exposure to OPNAs, including soman (GD), by detecting the phosphonylation of specific amino acid residues. The aim of the present study was to identify binding sites between GD and rabbit serum albumin in vitro and in vivo. A nano-liquid chromatography coupled with a quadrupole-orbitrap mass spectrometry (nLC-Q-Orbitrap-MS) was used to examine the GD-modified adducts of rabbit albumin. A total of 11 GD-modified sites were found in rabbit serum albumin across three experimental models. The following five GD-modified rabbit albumin sites, which were all lysine residues, were established in vivo: K188, K329, K162, K233, and K525. Two of these five lysine residues, K188 in peptide EK*ALISAAQER and K162 in peptide YK*AILTECCEAADK, were stable for at least 7 days in vivo. Molecular simulation of the GD-albumin interaction provided theoretical evidence for reactivity of the identified lysine residues. The findings suggest that these modifiable lysine residues are potential biomarkers of GD exposure for retrospective analysis by Q-Orbitrap-MS.


Subject(s)
Biomarkers/metabolism , Nerve Agents/metabolism , Serum Albumin/metabolism , Soman/metabolism , Animals , Binding Sites , Biomarkers/analysis , Chromatography, Liquid , Lysine/metabolism , Mass Spectrometry , Rabbits
5.
Chem Biol Interact ; 308: 170-178, 2019 Aug 01.
Article in English | MEDLINE | ID: mdl-31129133

ABSTRACT

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (HuBChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of HuBChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. HuBChE purified under current Good Manufacturing Practices (CGMP) was injected intramuscularly at 13.1 mg/kg, producing an average peak plasma value (Cmax) of over 27 Units/ml. The apparent time to maximum concentration (Tmax) approximated 7 h, the elimination half-life approximated 102 h, and plasma levels returned to pre-administration (baseline) levels by 14 days. No behavioral disruptions following HuBChE administration were observed on either neurobehavioral test, even in monkeys injected 24 h later with an otherwise lethal dose of soman. Thus, HuBChE provided complete neurobehavioral protection from soman challenge. The present data replicate and extend previous results from our laboratory that had used a different route of administration (intravenous), a different species (rhesus macaque), and a different BChE product (non-CGMP material). The addition of two sensitive neurobehavioral tests coupled with the PK/PD results convincingly demonstrates the neurobehavioral safety of plasma-derived HuBChE at therapeutic levels. Protection against an otherwise-lethal dose of soman by a pre-exposure treatment dose that is devoid of side effects establishes a foundation for additional testing using other exposure routes and treatment times, other challenge agents/routes, or other classes of organophosphate scavengers.


Subject(s)
Behavior, Animal/drug effects , Butyrylcholinesterase/administration & dosage , Chemical Warfare Agents/metabolism , Soman/metabolism , Animals , Butyrylcholinesterase/blood , Butyrylcholinesterase/pharmacokinetics , Chemical Warfare Agents/toxicity , Half-Life , Humans , Injections, Intramuscular , Macaca fascicularis , Male , Soman/toxicity
6.
Anal Chim Acta ; 1033: 100-107, 2018 Nov 29.
Article in English | MEDLINE | ID: mdl-30172315

ABSTRACT

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL-1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events.


Subject(s)
Dried Blood Spot Testing , Nerve Agents/analysis , Organophosphorus Compounds/blood , Organothiophosphorus Compounds/blood , Sarin/blood , Soman/blood , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Nerve Agents/metabolism , Organophosphorus Compounds/metabolism , Organothiophosphorus Compounds/metabolism , Sarin/metabolism , Soman/metabolism , Tandem Mass Spectrometry
7.
Anal Chem ; 90(14): 8495-8500, 2018 07 17.
Article in English | MEDLINE | ID: mdl-29888904

ABSTRACT

The NMR-observable nuclei of the acidic and basic compounds experience pH dependence in chemical shift. This phenomenon can be exploited in NMR titrations to determine p Ka values of compounds, or in pH measurement of solutions using dedicated pH reference compounds. On the other hand, this sensitivity can also cause problems in, for example, metabolomics, where slight changes in pH result in significant difficulties for peak alignment between spectra of set of samples for comparative analysis. In worst case, the pH sensitivity of chemical shifts can prevent unambiguous identification of compounds. Here, we propose an alternative approach for NMR identification of pH-sensitive analytes. The 1H and X (13C, 15N, 31P, ...) chemical shifts in close proximity to the acidic or basic functional group should, when presented as ordered pairs, express piecewise linear correlation with distinct slope, intercept, and range. We have studied the pH dependence of 1H and 31P chemical shifts of the CH3-P moiety in urinary metabolites of nerve agents sarin, soman and VX using 2D 1H-31P fast-HMQC spectroscopy. The 1H and 31P chemical shifts of these chemicals appear in very narrow range, and due to subtle changes in sample pH the identification on either 1H or 31P chemical shift alone is uncertain. However, if the observed 1H and 31P chemical shifts of the CH3-P moiety of individual compounds are presented as ordered pairs, they fall into distinct linear spaces, thus, facilitating identification with high confidence.


Subject(s)
Chemical Warfare Agents/pharmacokinetics , Magnetic Resonance Spectroscopy/methods , Nerve Agents/pharmacokinetics , Sarin/urine , Soman/urine , Chemical Warfare Agents/metabolism , Humans , Hydrogen/metabolism , Hydrogen/urine , Hydrogen-Ion Concentration , Nerve Agents/metabolism , Phosphorus Isotopes/metabolism , Phosphorus Isotopes/urine , Sarin/metabolism , Soman/metabolism
8.
Comput Biol Chem ; 75: 74-81, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29747078

ABSTRACT

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 A1 receptor agonist N6-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.


Subject(s)
Acetylcholinesterase/metabolism , Adenosine A1 Receptor Agonists/metabolism , Adenosine/analogs & derivatives , Nerve Agents/metabolism , Organophosphorus Compounds/metabolism , Sarin/metabolism , Soman/metabolism , Adenosine/chemistry , Adenosine/metabolism , Adenosine/pharmacology , Adenosine A1 Receptor Agonists/chemistry , Adenosine A1 Receptor Agonists/pharmacology , Animals , Molecular Structure , Nerve Agents/chemistry , Nerve Agents/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Quantum Theory , Rats , Sarin/chemistry , Sarin/pharmacology , Soman/chemistry , Soman/pharmacology , Thermodynamics
9.
Int J Mol Sci ; 19(4)2018 Apr 23.
Article in English | MEDLINE | ID: mdl-29690585

ABSTRACT

Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways.


Subject(s)
Chemical Warfare Agents/metabolism , Phosphoric Triester Hydrolases/metabolism , Biodegradation, Environmental , Organophosphates/metabolism , Organophosphorus Compounds/metabolism , Principal Component Analysis , Soman/metabolism
10.
Enzyme Microb Technol ; 112: 65-71, 2018 May.
Article in English | MEDLINE | ID: mdl-29499783

ABSTRACT

The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry.


Subject(s)
Aryldialkylphosphatase/genetics , Aryldialkylphosphatase/metabolism , Nerve Agents/metabolism , Amino Acid Sequence , Amino Acid Substitution , Biocatalysis , Kinetics , Mutagenesis, Site-Directed , Organophosphorus Compounds/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarin/metabolism , Soman/metabolism , Stereoisomerism , Substrate Specificity
11.
Anal Bioanal Chem ; 410(3): 1039-1051, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28971225

ABSTRACT

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL-1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.


Subject(s)
Butyrylcholinesterase/metabolism , Chemical Warfare Agents/pharmacokinetics , Cholinesterase Inhibitors/blood , Sarin/blood , Soman/blood , Butyrylcholinesterase/blood , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/metabolism , Chromatography, Affinity/methods , Chromatography, Liquid/methods , Humans , Sarin/analogs & derivatives , Sarin/metabolism , Soman/analogs & derivatives , Soman/metabolism , Tandem Mass Spectrometry/methods
12.
Article in English | MEDLINE | ID: mdl-28531843

ABSTRACT

A direct approach for the determination of a specific hydrolysis product of organophosphorus nerve agents such as methylphosphonic acid (MPA) in urine by ion chromatography and tandem mass spectrometry (IC-MS/MS) has been developed. The first advantage of the proposed approach is a rapid and simple sample preparation, which does not require a large sample volume, complicated and laborious preconcentration and derivatization steps, and takes less than 7min per sample. The second advantage is the fast and selective IC determination of MPA carried out on a noncommercial anion exchanger based on a poly(styrene-co-divinylbenzene) (PS-DVB) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer, which enables complete resolution of MPA from major urine matrix components and allows one to overcome matrix effects. Hyphenation of IC with tandem mass spectrometry results in highly sensitive and reliable MPA determination with the lowest detection limit (4ngmL-1) reported so far for HPLC determination of MPA in urine. The proposed approach is successfully applied for the analysis of urine from rats exposed to nonlethal doses of organophosphorus nerve agents such as sarin, soman, and VR in up to 13days after initial exposure, which shows the possibility to verify the nerve agent exposure after a long period of time.


Subject(s)
Nerve Agents/metabolism , Organophosphorus Compounds/urine , Tandem Mass Spectrometry/methods , Animals , Limit of Detection , Linear Models , Organophosphorus Compounds/metabolism , Organothiophosphorus Compounds/administration & dosage , Organothiophosphorus Compounds/metabolism , Rats , Reproducibility of Results , Sarin/administration & dosage , Sarin/metabolism , Soman/administration & dosage , Soman/metabolism
13.
Chem Res Toxicol ; 29(9): 1439-57, 2016 09 19.
Article in English | MEDLINE | ID: mdl-27509164

ABSTRACT

A mechanistic investigation has been carried out to explore all possible gas phase unimolecular isomerization as well as decomposition pathways of toxic organophosphorus compounds (OPCs), namely, sarin (GB) and soman (GD), which are better known as nerve agents. We have identified a total of 13 detoxication pathways for sarin, where the α-H, ß-H, and γ-H take part in the H-transfer process. However, for soman, due to the presence of ω-H, three additional detoxication pathways are obtained, where the ω-H is involved in the H-transfer process. Among all the pathways, the D3 decomposition pathway, where the phosphorus oxoacid derivative and alkene are generated via the formation of a six-membered ring in the transition state, is identified as the most feasible pathway from the perspective of both activation barrier and reaction enthalpy values. Moreover, we have studied the feasibility of the isomerization and decomposition pathways by performing the reaction kinetics in the temperature range of 300 K-1000 K using the one-dimensional Rice-Ramsperger-Kassel-Marcus (RRKM) master equation. From the RRKM calculation also, D3 pathway is confirmed as the most feasible pathway for both OPCs. The rate constant values associated with the D3 pathway within the temperature range of 600 K-700 K imply that the degradation of the OPCs is possible within this temperature range via the D3 pathway, which is in good agreement with the earlier reported experimental result. It is also observed that at higher temperature range (∼900 K), the increased rate constant values of other detoxication pathways indicate that along with D3, all other pathways become more or less equally feasible. Therefore, the entire work provides a widespread idea about the kinetic as well as thermodynamic feasibility of the explored detoxication pathways of the titled OPCs.


Subject(s)
Sarin/metabolism , Soman/metabolism , Thermodynamics , Gases , Kinetics , Molecular Structure , Phase Transition , Sarin/chemistry , Sarin/toxicity , Soman/chemistry , Soman/toxicity
14.
Anal Chem ; 88(12): 6523-30, 2016 06 21.
Article in English | MEDLINE | ID: mdl-27161086

ABSTRACT

Several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure. However, parent nerve agents and known metabolites are generally rapidly excreted from biological matrixes typically used for analysis (i.e., blood, urine, and tissues), limiting the amount of time after an exposure that verification is feasible. In this study, hair was evaluated as a long-term repository of nerve agent hydrolysis products. Pinacolyl methylphosphonic acid (PMPA; hydrolysis product of soman) and isopropyl methylphosphonic acid (IMPA; hydrolysis product of sarin) were extracted from hair samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.15 µg/kg and 7.5 µg/kg and linear ranges were 0.3-150 µg/kg and 7.5-750 µg/kg, respectively. To evaluate the applicability of the method to verify nerve agent exposure well after the exposure event, rats were exposed to soman, hair was collected after approximately 30 days, and stored for up to 3.5 years prior to initial analysis. PMPA was positively identified in 100% of the soman-exposed rats (N = 8) and was not detected in any of the saline treated animals (N = 6). The hair was reanalyzed 5.5 years after exposure and PMPA was detected in 6 of the 7 (one of the soman-exposed hair samples was completely consumed in the analysis at 3.5 years) rat hair samples (with no PMPA detected in the saline exposed animals). Although analysis of CWA metabolites from hair via this technique is not appropriate as a universal method to determine exposure (i.e., it takes time for the hair to grow above the surface of the skin and typical analysis times are >24 h), it complements existing methods and could become the preferred method for verification of exposure if 10 or more days have elapsed after a suspected exposure.


Subject(s)
Chemical Warfare Agents/analysis , Hair/chemistry , Nerve Agents/analysis , Organophosphorus Compounds/analysis , Soman/analogs & derivatives , Chemical Warfare Agents/metabolism , Chromatography, High Pressure Liquid/methods , Hair/metabolism , Humans , Limit of Detection , Nerve Agents/metabolism , Organophosphorus Compounds/metabolism , Sarin/analysis , Sarin/metabolism , Soman/analysis , Soman/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods
15.
Chem Biol Interact ; 259(Pt B): 133-141, 2016 Nov 25.
Article in English | MEDLINE | ID: mdl-27062893

ABSTRACT

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators.


Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Reactivators/metabolism , Nerve Agents/metabolism , Oximes/chemistry , Phenols/metabolism , Pralidoxime Compounds/metabolism , Acetylcholinesterase/chemistry , Acetylcholinesterase/genetics , Animals , Cholinesterase Reactivators/chemistry , Cholinesterase Reactivators/therapeutic use , Erythrocytes/enzymology , Guinea Pigs , Half-Life , Humans , Kinetics , Male , Nerve Agents/chemistry , Nerve Agents/poisoning , Organophosphate Poisoning/drug therapy , Organophosphates/chemistry , Organophosphates/metabolism , Phenols/chemistry , Phenols/therapeutic use , Pralidoxime Compounds/chemistry , Pralidoxime Compounds/therapeutic use , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sarin/chemistry , Sarin/metabolism , Soman/chemistry , Soman/metabolism , Structure-Activity Relationship
16.
Comb Chem High Throughput Screen ; 19(4): 334-44, 2016.
Article in English | MEDLINE | ID: mdl-27012528

ABSTRACT

During World War II, organophosphorus compounds with neurotoxic action were developed and used as the basis for the development of structures currently used as pesticides in the agricultural industry. Among the nerve agents, Tabun, Sarin, Soman and VX are the most important. The factor responsible for the high toxicity of organophosphorus (OP) is the acetylcholinesterase inhibition. However, one of the characterized enzymes capable of degrading OP is Phosphotriesterase (PTE). This enzyme has generated considerable interest for applications of rapid and complete detoxification. Due to the importance of bioremediation methods for the poisoning caused by OP, this work aims to study the interaction mode between the PTE enzyme and organophosphorus compounds, in this case, Sarin, Soman, Tabun and VX have been used, which are potent acetylcholinesterase inhibitors, taking into account the enantiomers "Rp" and " Sp" of each compound, with the Sp-enantiomers presenting the higher toxicity. With that, we were able to demonstrate the existence of the stereochemical preference by PTE in these compounds. With the purpose of increasing the speed of the hydrolysis mechanism, we have proposed a modification in the enzyme active site structure, where Zn(2+) ions were substituted by Al(3+) ions. To analyze the stability of Al(3+) ions in the wild-type PTE active site, MD simulations were also performed. This mutation brought relevant results; in this case, there was a reduction of the reaction energy barrier for all the compounds, mainly for VX in which the reaction presented lower activation energy values, and consequently, a faster hydrolysis process.


Subject(s)
Metals/chemistry , Organophosphorus Compounds/metabolism , Phosphoric Triester Hydrolases/metabolism , Aluminum , Catalytic Domain , Cholinesterase Inhibitors/metabolism , Cholinesterase Inhibitors/toxicity , Hydrolysis , Kinetics , Molecular Docking Simulation , Organophosphates/metabolism , Organophosphates/toxicity , Organophosphorus Compounds/toxicity , Organothiophosphorus Compounds/metabolism , Organothiophosphorus Compounds/toxicity , Sarin/metabolism , Sarin/toxicity , Soman/metabolism , Soman/toxicity , Stereoisomerism
17.
Nanoscale ; 8(18): 9537-47, 2016 May 05.
Article in English | MEDLINE | ID: mdl-26730700

ABSTRACT

The toxic nerve agent (NA) soman is the most toxic artificially synthesized compound that can rapidly penetrate into the brain and irreversibly inhibit acetylcholinesterase (AChE) activity, leading to immediate death. However, there are currently few brain-targeted nanodrugs that can treat acute chemical brain poisoning owing to the limited drug-releasing speed. The present study investigated the effectiveness of a nanodrug against NA toxicity that has high blood-brain barrier penetration and is capable of rapid drug release. Transferrin-modified mesoporous silica nanoparticles (TF-MSNs) were conjugated with the known AChE reactivator HI-6. This nanodrug rapidly penetrated the blood-brain barrier in zebrafish and mice and restored cerebral AChE activity via the released HI-6, preventing the brain damage caused by soman poisoning and increasing the survival rate in mice. Furthermore, there was no toxicity associated with the MSNs in mice or rats. These results demonstrate that TF-MSNs loaded with HI-6 represent the most effective antidote against NA poisoning by soman reported to date, and suggest that MSNs are a safe alternative to conventional drugs and an optimal nanocarrier for treating brain poisoning, which requires acute pulse cerebral administration.


Subject(s)
Brain/drug effects , Nanoparticles , Nerve Agents/metabolism , Oximes/administration & dosage , Pyridinium Compounds/administration & dosage , Silicon Dioxide , Soman/metabolism , Animals , Drug Liberation , Mice , Rats , Zebrafish
18.
Chem Commun (Camb) ; 51(13): 2601-4, 2015 Feb 14.
Article in English | MEDLINE | ID: mdl-25572650

ABSTRACT

Novel 2,3-heterodisubstituted ß-cyclodextrin derivatives were designed as artificial enzymes to degrade chemical warfare agents. One of them reduced the acetylcholinesterase inhibitory potential by soman faster than its monosubstituted analog.


Subject(s)
Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/metabolism , beta-Cyclodextrins/chemistry , beta-Cyclodextrins/metabolism , Acetylcholinesterase/metabolism , Chemical Warfare Agents/chemistry , Chemical Warfare Agents/pharmacology , Cholinesterase Inhibitors/pharmacology , Molecular Structure , Soman/chemistry , Soman/metabolism , Soman/pharmacology
19.
Anal Bioanal Chem ; 406(21): 5231-4, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24481624

ABSTRACT

A derivatization procedure for the qualitative gas chromatography-mass spectrometry (GC-MS) analysis of pinacolyl alcohol (PA) that employs phenyldimethylchlorosilane (PhDMClS) and the promoter N-methylimidazole is described. While PA, underivatized, can be detected using conventional gas chromatographic methods, its polarity and low boiling point make its detection in complex matrices challenging. The silylation procedure described herein generates a PA-derivative exhibiting an increased on-column retention time, thus shifting its GC-MS signal away from commonly encountered, volatile, interfering analytes. Derivatized PA could be distinguished from other PhDMClS-derivatized isomeric alcohols by its unique retention time and mass spectrum. The derivatization was demonstrated to perform well in the GC-MS analysis and identification of PA in samples from Proficiency Tests administered by the Organisation for the Prohibition of Chemical Weapons (OPCW).


Subject(s)
Chemical Warfare Agents/analysis , Gas Chromatography-Mass Spectrometry/methods , Hexanols/analysis , Silanes/chemistry , Biotransformation , Hexanols/chemistry , Humans , In Vitro Techniques , Laboratory Proficiency Testing , Limit of Detection , Soman/metabolism
20.
Rapid Commun Mass Spectrom ; 27(15): 1697-704, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23821563

ABSTRACT

RATIONALE: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.


Subject(s)
Chemical Warfare Agents/analysis , Chemical Warfare Agents/metabolism , Tandem Mass Spectrometry/methods , Humans , Organophosphorus Compounds/metabolism , Organophosphorus Compounds/urine , Organothiophosphorus Compounds/metabolism , Organothiophosphorus Compounds/urine , Sarin/metabolism , Sarin/urine , Soman/metabolism , Soman/urine
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