In the absence of appropriate medical care, exposure to organophosphorus nerve agents, such as VX, can lead to respiratory failure, and potentially death by asphyxiation. Despite the critical role of respiratory disturbances in organophosphorus-induced toxicity, the nature and underlying mechanisms of respiratory failure remain poorly understood. This study aimed to characterize respiratory alterations by determining their type and duration in mice exposed to a subcutaneous sublethal dose of VX. Respiratory ventilation in Swiss mice was monitored using dual-chamber plethysmography for up to 7 days post-exposure. Cholinesterase activity was assessed via spectrophotometry, and levels of inflammatory biomarkers were quantified using Luminex technology in blood and tissues involved in respiration (diaphragm, lung, and medulla oblongata). Additionally, a histological study was conducted on these tissues to ensure their structural integrity. Ventilatory alterations appeared 20-25 minutes after the injection of 0.9 LD50 VX and increased until the end of the recording, i.e., 40 minutes after intoxication. Concurrent with the occurrence of apnea, increased inspiratory and expiratory times resulted in a significant decrease in respiratory rate in exposed mice compared to controls. Ventilatory amplitude and, consequently, minute volume were reduced, while specific airway resistance significantly increased, indicating bronchoconstriction. These ventilatory effects persisted up to 24 or even 72 hours post-intoxication, resolving on the 7th day. They were correlated with a decrease in acetylcholinesterase activity in the diaphragm, which persisted for up to 72 hours, and with the triggering of an inflammatory reaction in the same tissue. No significant histologic lesions were observed in the examined tissues. The ventilatory alterations observed up to 72 hours post-VX exposure appear to result from a functional failure of the respiratory system rather than tissue damage. This comprehensive characterization contributes to a better understanding of the respiratory effects induced by VX exposure, which is crucial for developing specific medical countermeasures.

Protein adducts are vital targets for exploring organophosphorus nerve agents (OPNAs) exposure and identification, that can be used to characterize the chemical burden and initiate chemical safety measures. However, the use of protein adducts as biomarkers of OPNA exposure has developed slowly. To further promote the development of biomarkers in chemical forensics, it is crucial to expand the range of modified peptides and active sites, and describe the characteristics of OPNA adducts at specific reaction sites. This study utilized multi-species and multi-source albumins as the protein targets. We identified 56 peptides in albumins from various species (including human, horse, rat and pig), that were modified by at least two OPNAs. Diverse modification characteristics were observed in response to certain agents: including (1) multiple sites on the same peptide modified by one or more agents, (2) different reactivities at the same site in homologous albumins, and (3) different preferences at the same active sites associated with differences in the biological matrix during exposure. Our studies provided an empirical reference with rationalized underpinnings supported by estimated conformation energetics through molecular modeling. We employed different peptide markers for detection of protein adducts, as (one would do) in forensic screening for identification and quantification of chemical damage. Three characteristic peptides were screened and analyzed in human albumin, including Y287ICENQDSISSK, K438VPQVS443TPTLVEVSR, and Y162LY164EIAR. Stable fragment ions with neutral loss were found from their tandem MS/MS spectra, which were used as characteristic ions for identification and extraction of modified peptides in enzymatic digestion mixtures. Coupling these observations with computer simulations, we found that the structural stability of albumin and albumin-adduct complexes (as well as the effective force that promotes stability of different adducts) changes in the interval before and after adduct formation. In pig albumin, five active peptides existed stably in vivo and in vitro. Most of them can be detected within 30 min after OPNA exposure, and the detection window can persist about half a month. These early findings provided the foundation and rationale for utilizing pig albumin as a sampling target for rapid analysis in future forensic work.

Protein adducts are important biological targets for traceability of organophosphorus nerve agents (OPNAs). Currently, the recognized biomarkers that can be used in actual samples in the field of chemical forensics only include Y411 in albumin and the active nonapeptide in butyrylcholinesterase (BChE). To explore stable and reliable protein adducts and increase the accuracy of OPNAs traceability further, we gradually expanded OPNAs-albumin adducts based on single and group adduct collection. Several stable peptides were found via LC-MS/MS analysis in human serum albumin (HSA) exposed to OPNAs in a large exposure range. These adducts were present in HSA samples exposed to OPNAs of each concentration, which provided data support for the reliability and stability of using adducts to trace OPNAs. Meanwhile, the formation mechanism of OPNAs-cysteine adduct was clarified via computer simulations. Then, these active sites found and modified peptides were used as raw materials for progressive expansion of albumin adducts. We constructed an OPNAs-HSA adducts group, in which a specific agent is the exposure source, and three or more active peptides constitute data sets for OPNAs traceability. Compared with single or scattered protein adducts, the OPNAs-HSA adduct group improves OPNAs identification by mutual verification using active peptides or by narrowing the identity range of the exposure source. We also determined the minimum detectable concentration of OPNAs for the adduct group. Two or more peptides can be detected when there is an exposure of 50 times the molar excess of OPNAs in relation to HSA. This improved the accuracy of OPNAs exposure and identity confirmation. A collection of OPNAs-albumin adducts was also examined. The collection was established by collecting, classifying, and integrating the existing albumin adducts according to the species to which each albumin belongs, the types of agents, and protease. This method can serve as a reference for discovering new albumin adducts, characteristic phosphonylated peptides, and potential biomarkers. In addition, to avoid a false negative for OPNAs traceability using albumin adducts, we explored OPNAs-cholinesterase adducts because cholinesterase is more reactive with OPNAs than albumin. Seven active peptides in red blood cell acetylcholinesterase (RBC AChE) and serum BChE can assist in OPNAs exposure and identity confirmation.

Chemical attribution is a vital tool to attribute chemicals or related materials to their origins in chemical forensics via various chemometric methods. Current progress related to organophosphorus nerve agents (OPNAs) has mainly focused on the attribution of chemical sources and synthetic pathways. It has not yet been applied in matching exposed biological samples to their sources. This work used chemical attribution to explore organic impurity profiles in biological samples exposed to various OPNAs. Chemical attribution was first used to identify the exposure source of biological samples based on the full-scan data via comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometer (GC × GC-TOFMS). Taking peak area as the only variable, it can quickly match exposed samples to their sources by applying unsupervised or supervised models, screen difference compounds via one-way ANOVA or t-tests, and then identify valuable impurities that can distinguish different types of exposed samples. To further obtain the impurity profile only applicable to a certain weapon\' samples, the irrelevant components were removed via conventional methods. The findings showed there were 53 impurities that can promote distinguishing six groups of OPNA exposed samples, as well as 42 components that can be used as valuable impurities to distinguish class G and class V samples. These were all unique impurities that appear in a certain weapon\' samples. The outcomes can be a reference for tracing the source for OPNA-exposed samples, which was beneficial to the further development in source matching of forensic samples. Moreover, the chemical attribution for impurity profiles in biological samples after weapons exposure may inspire research into the characteristics of impurity profile in biological samples as well as practical applications of chemical attribution for OPNA-exposed samples, that may expand potential biomarkers and break the limits of existing markers in the future.

Zirconium-based metal-organic frameworks (Zr-MOFs) have been demonstrated as potent catalysts for the hydrolytic detoxification of organophosphorus nerve agents and their simulants. However, the practical implementation of these Zr-MOFs is limited by the poor processability of their powdered form and the necessity of water media buffered by a volatile liquid base in the catalytic reaction. Herein, we demonstrate the efficient solid-state hydrolysis of a nerve agent simulant (dimethyl-4-nitrophenyl phosphate, DMNP) catalyzed by Zr-MOF-based mixed matrix membranes. The mixed matrix membranes were fabricated by incorporating MOF-808 into the blending matrix of poly(vinylidene fluoride) (PVDF), poly(vinylpyrrolidone) (PVP), and imidazole (Im), in which MOF-808 provides highly active catalytic sites, the hydrophilic PVP helps to retain water for promoting the hydrolytic reaction, and Im serves as a base for catalytic site regeneration. Impressively, the mixed matrix membranes displayed excellent catalytic performance for the solid-state hydrolysis of DMNP under high humidity, representing a significant step toward the practical application of Zr-MOFs in chemical protective layers against nerve agents.

Recent events have shown that organophosphorus nerve agents (OPNAs) are a serious threat. Cholinesterase inhibition by OPNAs results in acetylcholine accumulation, a cholinergic crisis leading to death if untreated. Efficacy assessment of new medical countermeasures against OPNAs relies on translational animal models. We developed a swine model of percutaneous VX intoxication and a simple plate reader-based enzymatic method to quantify plasmatic VX over time. Juvenile pigs anesthetized with sevoflurane were poisoned with a single supralethal (n = 5; 1200 μg/kg) or sublethal (n = 6; 320 μg/kg) percutaneous dose of VX. These intoxicated animals were compared to 7 control animals. Repeated blood sampling was performed up to 6 h post-intoxication. Blood cholinesterase activities were measured using the Ellman assay. Nanomolar plasma concentrations of VX were measured by exogenous butyrylcholinesterase added to an aliquot of plasma. As expected, we observed a steady increase in plasma concentration of VX over time concomitant to a decrease in blood cholinesterase activities for all intoxicated pigs. Despite the simplicity of the enzymatic method, the results obtained are in good agreement with those of the liquid chromatography-mass spectrometry method. This method is also applicable to other OPNAs such as novichoks with minor adaptations.

Organophosphorus neurotoxic agents (OPNAs) seriously damage the nervous system, inhibiting AChE activity and threatening human health and life. Timely and accurate detection of biomarkers in biomedical samples is an important means for identifying OPNA exposure, helping to recognize and clarify its characteristics and providing unambiguous forensic evidence for retrospective research. It is therefore necessary to summarize the varieties of biomarkers, recognize their various characteristics, and understand the principal research methods for these biomarkers in the retrospective detection of OPNA exposure. Common biomarkers include mainly intact agents, degradation products and protein adducts. Direct agent identification in basic experimental research was successfully applied to the detection of free OPNAs, however, this method is not applicable to actual biomedical samples because the high reactivity of OPNAs promotes rapid metabolism. Stepwise degradation products are important targets for retrospective research and are usually analyzed using a GC-MS, or an LC-MS system after derivatization. The smaller window of detection time requires that sampling be accomplished within 48 h, increasing the obstacles to determining OPNA exposure. For this reason, the focus of retrospective identification of OPNA exposure has shifted to protein adducts with a longer lifetime. Compared to the fluoride-induced reactivation method, which cannot be used for aged adducts, digestive peptide analysis is the more elegant method for detecting various adducts, identifying more active sites, exploring potential biomarkers and excavating characteristic ions. Retrospective identification of biomarkers after OPNA poisoning is of primary importance, providing unambiguous evidence for forensic analysis in actual cases and judgment of chemical accidents. At present, degradation products, the nonapeptide from BChE adducts and Y411 from human serum adducts are used successfully in actual cases of OPNA exposure. However, more potential biomarkers are still in the discovery stage, which may prove inconclusive. Therefore, there is an urgent need for research that screens biomarker candidates with high reactivity and good reliability from the potential candidates. In addition, mass spectrometry detection with high resolution and reactivity and an accurate data processing system in the scanning mode must also be further improved for the retrospective identification of unknown agents.

In this work, blow flies were investigated as environmental chemical sample collectors following a chemical warfare attack (CWA). Blow flies sample the environment as they search for water and food sources and can be trapped from kilometers away using baited traps. Three species of blow flies were exposed to CWA simulants to determine the persistence and detectability of these compounds under varying environmental conditions. A liquid chromatography mass spectrometry (LC-MS/MS) method was developed to detect CWA simulants and hydrolysis products from fly guts. Flies were exposed to the CWA simulants dimethyl methylphosphonate and diethyl phosphoramidate as well as the pesticide dichlorvos, followed by treatment-dependent temperature and humidity conditions. Flies were sacrificed at intervals within a 14 day postexposure period. Fly guts were extracted and analyzed with the LC-MS/MS method. The amount of CWA simulant in fly guts decreased with time following exposure but were detectable 14 days following exposure, giving a long window of detectability. In addition to the analysis of CWA simulants, isopropyl methylphosphonic acid, the hydrolysis product of sarin, was also detected in blow flies 14 days post exposure. This work demonstrates the potential to obtain valuable samples from remote or access-restricted areas without risking lives.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.