Search

Exposure to high levels of a cholinesterase inhibiting organophosphorus (OP) agent often results in seizures that progress to status epilepticus (SE). Survivors of OP-induced SE often display neuropathological consequences the days following SE. In the current study, the temporal profile of neuropathology after SE was investigated in a rat model of diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE for one hour. Following termination of electrographic SE with urethane (0.8 g/kg, sc), cohorts of rats were euthanized 3, 24 and 48 h later and brain tissue was processed to determine immediate early gene and inflammatory mediator expression as well as blood-brain barrier changes and neurodegeneration. After SE rats displayed a time-dependent upregulation of immediate early genes such as cFos and ΔFosB as well as pro-inflammatory mediators COX-2, IL-1β and IL-6. The profile of positive cFos staining, but not ΔFosB, coincided temporally with heightened brain activity measured by cortical electroencephalography (EEG). Neurodegeneration in limbic brain regions was absent 3 h after SE, but prominent 24 h later and continued to increase 48 h after SE. Serum albumin was detected in the cortex 3 h after SE suggesting early loss of blood brain barrier integrity. However, the blood-brain barrier appeared repaired 48 h after SE. This study demonstrates that following OP-poisoning in rats, immediate early gene expression in the brain precedes neuroinflammation followed by erosion of the blood-brain barrier and neurodegeneration. The study also demonstrates that seizure activity in brain nuclei coincides with cFos expression. Together, these studies give insight into the temporal molecular changes in the brain following organophosphate-induced status epilepticus.

Oxime reactivators of acetylcholinesterase (AChE) represent an integral part of standard antidote treatment of organophosphate poisoning. Oxime K869 is a novel bisquaternary non-symmetric pyridinium aldoxime with two pyridinium rings connected by a tetramethylene bridge where two chlorines modify the pyridinium ring bearing the oxime moiety. Based on in vitro assays, K869 is a potent AChE and butyrylcholinesterase (BChE) reactivator. For the investigation of the basic pharmacokinetic properties of K869 after its intramuscular application, new HPLC-UV and LC-MS/MS methods were developed and validated for its determination in rat body fluids and tissues. In this study, the SPE procedure for sample pretreatment was optimized as an alternative to routine protein precipitation widely used in oxime pharmacokinetics studies. K869 oxime is quickly absorbed into the central compartment reaching its maximum in plasma (39 ± 4 μg/mL) between 15 and 20 min. The majority of K869 was eliminated by kidneys via urine when compared with biliary excretion. However, only a limited amount of K869 (65 ± 4 ng/g of brain tissue) was found in the brain 30 min after oxime administration. Regarding the brain/plasma ratio calculated (less than 1%), the penetration of K869 into the brain did not exceed conventionally used oximes.

Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm’s body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism’s feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins’ mode of action.

Pulmonary delivery of protein drugs into the systemic circulation is highly desirable as the lung provides a large absorption surface area and a more favorable environment for biologics compared to other delivery routes. However, pulmonary systemic delivery of proteins presents several challenges such as poor protein stability and limited bioavailability, especially for large proteins (molecular weight > 50 kDa), which exhibit an average bioavailability of 1% to 5% when delivered via the pulmonary route. Here, we demonstrated that with the conjugation of zwitterionic poly(carboxybetaine) (pCB) polymer, the bioavailability of organophosphate hydrolase (OPH) was significantly increased from 5% to 53%. OPH conjugated with pCB delivered through intubation-assisted intratracheal instillation (IAIS) into the lung exhibited improved pharmacokinetic properties and prophylactic efficacy against organophosphate poisoning, showing its application potential. Zwitterionic polymer conjugation provides the possibility for a favorable, non-invasive delivery of biological therapeutics into the systemic circulation.

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings. Cholinergic overstimulation by co-application of soman and acetylcholine (10 μM each) did not reduce the relative inhibition of propofol (1.0 μM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy. Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.

Organophosphorus (OP) 1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE), 2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation. Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors. Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.

Summary 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.

Organophosphates and carbamates are important anticholinesterase intoxicants of humans and dogs. Intermediate syndrome (IMS) typically occurs 7-96 h following a toxicity-associated acute cholinergic crisis (ACC), and manifests clinically as weakness of the proximal limb, respiratory, and neck flexor muscles. The aim of this study was to describe the prevalence, clinical findings, and outcome of IMS in dogs. The medical records of a veterinary teaching hospital were searched for dogs diagnosed with ACC, IMS, or both, between 2017 and 2021. Case files were retrospectively reviewed. Six historical IMS cases were additionally reviewed. Thirty-two dogs were diagnosed with anticholinesterase intoxication during the search period, of which 23 (72 %) were only diagnosed with ACC, seven (22 %) progressed from ACC to IMS, and two (6 %) were only diagnosed with IMS. Duration of hospitalisation was longer in the IMS group compared to the ACC only group (P = 0.005). When all dogs with IMS (n = 15, including the six historical cases) were considered, survival was 100 %, including four (27 %) that required positive pressure mechanical ventilation following respiratory failure. Serum butyrylcholine esterase activity, a marker of cholinesterase activity, was below reference interval when first measured in 14 (93 %) of dogs; however, was not a useful as a recovery marker. IMS should be suspected in dogs demonstrating respiratory, neck, and proximal limb muscle paresis or paralysis, especially following clinical signs consistent with ACC. Absence of clinical signs consistent with ACC or butyrylcholine esterase activity within the reference interval does not exclude IMS as a differential.

Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.

The global use of organophosphorus compounds (OP) for pest control and nerve agents being used in military conflicts and for assassinations renders intoxications by these agents a public health concern. OP-poisoned patients often suffer from dysrhythmias which may ultimately result in death. In this study, human-induced pluripotent stem cells derived cardiomyocytes were exposed to OP compounds in a microelectrode array system (MEA). The MEA system is widely accepted to assess the proarrhythmic properties of (candidate) drugs. The directly acting cholinergic compounds acetylcholine and carbachol and the irreversible acetylcholinesterase inhibitor cyclosarin - a highly toxic nerve agent - were assessed. All three compounds induced a dose-dependent (up to 600 nmol/L) corrected field potential duration (FPDc) prolongation of 9.7 ± 0.6% for carbachol, for 9.7 ± 1.2% acetylcholine and 9.4 ± 0.5% for cyclosarin. Additionally, the electrophysiological alterations of the clinically approved oxime reactivators obidoxime, pralidoxime and the oximes in development HI-6 and MMB-4 were investigated in the absence of OP. Neither of these oximes (up to a concentration of 300 μmol/L) caused dysrhythmia nor beat arrest. The competitive muscarinic receptor antagonist atropine as a cornerstone in the treatment of OP poisoning was also analyzed. Interestingly, atropine caused a drop in the beat rate which might result from a non-receptor action of this substance in the absence of OP. Atropine in combination with the OP nerve agent cyclosarin and the direct cholinergics acetylcholine or carabachol completely reversed the induced FPDc prolongation. However, the oxime HI-6 as potent reactivator of cyclosarin-inhibited AChE was not able to prevent the FPDc prolongation in this model. In conclusion, the current model allows the assessment of FPDc prolongation by the nerve agent cyclosarin, the cholinergic compounds carbachol, acetylcholine and the block of this effect by atropine.

In previous experiments, human and rat small bowel samples have been successfully used to study the spasmolytic effect of (potential) therapeutics in carbamate-constricted bowel specimens. Additionally, transferability from rat to human data was shown in the previous study. In the present study, the effects of atropine, scopolamine, MB327, HI-6 as well as obidoxime were examined in organophosphorus-poisoned human small bowel specimens. All substances were tested with at least seven concentrations in samples previously exposed to the nerve agent sarin. Furthermore, the cholinesterase reactivation potential of all substances was investigated. The test substances displayed a spasmolytic effect allowing the calculation of dose-response curves and EC50s. The parasympatholytic compound scopolamine had the strongest relaxing effect (EC50 = 0.05 μM) followed by atropine (EC50 = 0.07 μM). HI-6 and obidoxime were capable to reactivate the sarin-inhibited cholinesterase activity in small bowel samples. Both substances restored AChE activity in a dose-dependent way, with HI-6 being more potent (HI-6 EC50 = 3.8 μM vs obidoxime EC50 = 197.8 μM). Summarizing, our isolated human small bowel setup is a suitable tool to investigate the smooth muscle relaxing effect of (candidate) therapeutics for organophosphorus compound poisoning i.e. sarin exposure in a complex 3D tissue model.

Organophosphates (OPs) are a class of chemicals commonly used in agriculture as pesticides, that can often lead to severe toxicity in humans. Paraoxonase-1 (PON1) belongs to a family of A-esterases and hydrolyses several OPs while also serving other biological roles. Two main genetic polymorphisms have been shown to affect enzymatic ability; an A > G transition in the 192nd position (192 Q/R, rs662), and an A > T at codon 55 (55 M/L, rs854560). In this review, we searched PubMed for relevant articles published from its inception till June 2018 and included publications from 1996 to 2018. We aimed to address the distribution of the polymorphisms in various populations, the way they affect enzymatic activity and the possible use of PON1 as a biomarker. The polymorphisms present great heterogeneity between populations, with the data being clearer over 192 Q/R, and this heterogeneity is related to the phylogenetic origins of each population. Concerning enzymatic activity, the different genotypes react better or worse to different OP substrates, with studies presenting a variety of findings. Detecting the “paraoxonase status” of an individual -referring to PON1 function- seems to be important in predicting OP toxicity, as studies have shown that some specific-genotype individuals present symptoms of toxicity in higher rates than others. We are strongly convinced that in order for the scientific community to reach a consensus over which polymorphisms confer susceptibility to toxicity and whether PON1 can eventually be used as a biomarker, more studies need to be carried out, since the data thus far does not seem to reach a universal conclusion.

Rats poisoned with sarin enter into ahyper-cholinergic crisis characterized by excessive salivation, respiratory distress, tremors, seizures, and death. Through the use of rescue medications and an anticonvulsant, death can be avoided in many animals, with the long-term consequences of poisoning partly ameliorated, especially when countermeasures are made available immediately after exposure. However, when anticonvulsant measures are delayed by as little as 30 min, clinical, neurological, cognitive, and psychiatric abnormalities may persist long after the initial exposure. This study sought to determine if the addition of the NMDA receptor antagonist Ketamine to human standard-of-care countermeasures consisting of two rescue medications (2-PAM and atropine) and an anti-convulsant (Midazolam), would afford protection against persistent neurobiological compromise. Rats were exposed to sarin (105 μg/kg via subcutaneous injection), and treated 1 min later with 2-PAM and Atropine Methyl Nitrate (IM) to minimize mortality. One of four anti-convulsant protocols was then initiated at 50 min postsarin:Midazolam alone (MDZ, a single injection (IM) at 0.66 mg/kg); Ketamine alone (KET, a series of five injections (IM) of Ketamine at 7.5 mg/kg, 90 min apart); Midazolam + low dose Ketamine (MDZ + lowKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential doses of ketamine (IM) at 2.5 mg/kg, starting at the time of Midazolam dosing and then 90 min apart); Midazolam + high dose Ketamine (MDZ + highKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential injections of 7.5 mg/kg Ketamine (IM), starting at the time of Midazolam dosing and then 90 min apart). Animals were preassigned to groups culled at post-exposure Days 1, 7 or 30, for histopathology. For all surviving animals, EEG activity was monitored through skull electrodes for 24-h beginning immediately after sarin exposure. Surviving animals also underwent 24-h EEG monitoring on Days 6, 13, and/or 29, post-sarin. Memory assessment using the Morris Water Maze was performed on Days 1, 4, 7, 14 and 30. Following sarin exposure, 85% of surviving animals demonstrated status epilepticus within 20 min. Each of the anti-convulsant protocols was sufficient to stop convulsions within 1 h of anti-convulsant administration, but all of the animals still showed signs of electrographic status for an additional 2-12 h, without substantial differentiation between treatment groups. However, for post-sarin hours 13-24, the MDZ + highKET group showed significantly less severe EEG abnormalities than the MDZ and KET groups (Mood's Median Test, p 0.005). At one month post-exposure, 90% of animals that had received Midazolam alone still showed evidence of some epileptiform activity. In contrast, 90% of animals that had received Midazolam + high dose Ketamine combination therapy had EEG profiles that were within normal limits. This difference in EEG outcomes was highly significant (Mood's Median Test, p 0.001). Likewise, on the water maze, the majority of animals that had received Midazolam combined with either high or low dose Ketamine therapy returned to near baseline levels of mnemonic performance within 2 weeks, whereas the majority of the animals that had received midazolam alone or ketamine alone demonstrated persistent and significant memory impairments even at one month postexposure (Mood's Median Test, p 0.005). With respect to neuronal necrosis, animals in the MDZ + highKET group showed significantly less overall damage than animals in other treatment groups (Mood's Median Test, p 0.001). Of special note were findings in the hippocampus, where only 12% of animals in the MDZ + highKET group showed evidence of necrosis on HE staining, whereas 100% of animals in the KET group, 70% of animals in the MDZ group, and 40% of animals in the MDZ + lowKET group showed evidence of hippocampal necrosis. Overall, the data demonstrate that Ketamine augmentation of an atropine, 2PAM, and Midazolam standard-ofcare for sarin exposure provides clinically-relevant additional protection against the negative neurobiological consequences of sarin, even when initiation of the anti-convulsant countermeasures is delayed by 50 min.

In this article the neurotoxic disorders appearing in patients exposed to organophosphorus pesticides and known mechanisms involved are reviewed. Organophosphorus compounds cause four main neurotoxic effects in humans: the cholinergic syndrome, the intermediate syndrome, organophosphate-induced delayed polyneuropathy and chronic organophosphate-induced neuropsychiatric disorder. Compared to the cholinergic syndrome, that causes millions of cases of poisoning with fatality of more than 15% each year, other disorders involve much smaller number of patients. Possible link of exposure to organophosphorus pesticides with neurodegenerative diseases, dementia, attention deficit hyperactivity disorder and Parkinson's disease in man is also approached. This article is focused on neurotoxic disorders appearing after acute and chronic exposure to organophosphates with emphasis on molecular mechanisms, clinical presentation, pathogenesis, and possibilities for prevention/medical treatment.

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as “resensitizer”. In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [3H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.

Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the γ-α and β-δ subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function.

Nerve agents and some pesticides such as diisopropylfluorophosphate (DFP) cause neurotoxic manifestations that include seizures and status epilepticus (SE), which are potentially lethal and carry long-term neurological morbidity. Current antidotes for organophosphate (OP) intoxication include atropine, 2-PAM and diazepam (a benzodiazepine for treating seizures and SE). There is some evidence for partial or complete loss of diazepam anticonvulsant efficacy when given 30 min or later after exposure to an OP; this condition is known as refractory SE. Effective therapies for OP-induced SE are lacking and it is unclear why current therapies do not work. In this study, we investigated the time-dependent efficacy of diazepam in the nerve agent surrogate DFP model of OP intoxication on seizure suppression and neuroprotection in rats, following an early and late therapy. Diazepam (5 mg/kg, IM) controlled seizures when given 10 min after DFP exposure (“early”), but it was completely ineffective at 60 or 120 min (“late”) after DFP. DFP-induced neuronal injury, neuroinflammation, and neurodegeneration of principal cells and GABAergic interneurons were significantly reduced by early but not late therapy. These findings demonstrate that diazepam failed to control seizures, SE and neuronal injury when given 60 min or later after DFP exposure, confirming the benzodiazepine-refractory SE and brain damage after OP intoxication. In addition, this study indicates that degeneration of inhibitory interneurons and inflammatory glial activation are potential mechanisms underlying these morbid outcomes of OP intoxication. Therefore, novel anticonvulsant and neuroprotectant antidotes, superior to benzodiazepines, are desperately needed for controlling nerve agent-induced SE and brain injury.

The term organophosphate (OP) refers to a diverse group of chemicals that are found in hundreds of products worldwide. As pesticides, their most common use, OPs are clearly beneficial for agricultural productivity and the control of deadly vector-borne illnesses. However, as a consequence of their widespread use, OPs are now among the most common synthetic chemicals detected in the environment as well as in animal and human tissues. This is an increasing environmental concern because many OPs are highly toxic and both accidental and intentional exposures to OPs resulting in deleterious health effects have been documented for decades. Some of these deleterious health effects include a variety of long-term neurological and psychiatric disturbances including impairments in attention, memory, and other domains of cognition. Moreover, some chronic illnesses that manifest these symptoms such as Gulf War Illness and Aerotoxic Syndrome have (at least in part) been attributed to OP exposure. In addition to acute acetylcholinesterase inhibition, OPs may affect a number of additional targets that lead to oxidative stress, axonal transport deficits, neuroinflammation, and autoimmunity. Some of these targets could be exploited for therapeutic purposes. The purpose of this review is thus to: 1) describe the important uses of organophosphate (OP)-based compounds worldwide, 2) provide an overview of the various risks and toxicology associated with OP exposure, particularly long-term neurologic and psychiatric symptoms, 3) discuss mechanisms of OP toxicity beyond cholinesterase inhibition, 4) review potential therapeutic strategies to reverse the acute toxicity and long term deleterious effects of OPs.

Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY*411TKKVPQVSTPTL (pepsin) and as the tripeptide Y*411TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (μLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS). Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and −30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 μM and 4.0 μM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis.

Pets can act as sentinels for human health and thus surveillance of pet dogs has the potential to improve awareness of emerging risks for animal and public health. The aim of this study was to investigate factors associated with the risk of canine poisoning. In a large population-based epidemiological investigation in Italy performed from January 2015 to January 2016 and April 2016 to April 2017, descriptive statistics were acquired and analysed to determine variables associated with poisoning events in pet dogs. Results were validated in a test population and forecast analysis of risk was performed. The cumulative incidence of poisoning events was low (10.2/1000 dogs/year). Anticoagulant rodenticides, organophosphate pesticides, metaldehyde and strychnine were the most frequent causes of intoxications. Territory characteristics significantly modulated both the frequency and the nature of the involved substances. The seashore area was associated with poisoning by rodenticides (odds ratio, OR, 1.81, 95% confidence interval, CI, 1.54–2.13) and metaldehyde (OR 1.61, 95% CI 1.16–2.28). The hill country area was associated with poisoning by organophosphate pesticides (OR 1.73, 95% CI 1.38–2.15), metaldehyde (OR 2.26, 95% CI 1.53–3.25) and strychnine (OR 1.86, 95% CI, 1.34–2.57). The mountain area was associated with strychnine poisoning (OR 3.79, 95% CI 2.84–5.06). The prospective cumulative incidence of poisoning over 10 years was 9.74% (95% CI 9.57–9.91). These results may be useful for predicting the risk of poisoning and for estimating the risk index related to specific toxic compounds in specific territories. This study suggests that poisoning events in dogs may represent a problem of public health with the potential to affect wildlife and human beings.

BackgroundCognitive impairment is a major health concern among older Mexican Americans, associated with significant morbidity and mortality, and may be influenced by environmental exposures.ObjectivesTo investigate whether agricultural based ambient organophosphorus (OP) exposure influences 1) the rate of cognitive decline and mortality and 2) whether these associations are mediated through metabolic or inflammatory biomarkers.MethodsIn a subset of older Mexican Americans from the Sacramento Area Latino Study on Aging (n = 430), who completed modified mini-mental state exams (3MSE) up to 7 times (1998-2007), we examined the relationship between estimated ambient OP exposures and cognitive decline (linear repeated measures model) and time to dementia or being cognitively impaired but not demented (CIND) and time to mortality (cox proportional hazards model). We then explored metabolic and inflammatory biomarkers as potential mediators of these relationships (additive hazards mediation). OP exposures at residential addresses were estimated with a geographic information system (GIS) based exposure assessment tool.ResultsParticipants with high OP exposure in the five years prior to baseline experienced faster cognitive decline (β = 0.038, p = 0.02) and higher mortality over follow-up (HR = 1.91, 95% CI = 1.12, 3.26). The direct effect of OP exposure was estimated at 241 (95% CI = 27-455) additional deaths per 100,000 person-years, and the proportion mediated through the metabolic hormone adiponectin was estimated to be 4% 1.5-19.2). No other biomarkers were associated with OP exposure.ConclusionsOur study provides support for the involvement of OP pesticides in cognitive decline and mortality among older Mexican Americans, possibly through biologic pathways involving adiponectin.

Soman, an organophosphorus (OP) compound, disrupts nervous system function through inactivation of acetylcholinesterase (AChE), the enzyme that breaks down acetylcholine at synapses. Left untreated, a state of prolonged seizure activity (status epilepticus, SE) is induced, causing widespread neuronal damage and associated cognitive and behavioral impairments. Previous research demonstrated that therapeutic stimulation of A1 adenosine receptors (A1ARs) can prevent or terminate soman-induced seizure. This study examined the ability of three potent A1AR agonists to provide neuroprotection and, ultimately, prevent observable cognitive and behavioral deficits following exposure to soman. Sprague Dawley rats were challenged with a seizure-inducing dose of soman (1.2 x LD50) and treated 1 min later with one of the following A1AR agonists: (6)-Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine (CCPA) or N-bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (cdENBA). An active avoidance shuttle box task was used to evaluate locomotor responses to aversive stimuli at 3, 7 and 14 days post-exposure. Animals treated with CPA, CCPA or cdENBA demonstrated a higher number of avoidance responses and a faster reaction to the aversive stimulus than the soman/saline control group across all three sessions. Findings suggest that A1AR agonism is a promising neuroprotective countermeasure, capable of preventing the long-term deficits in learning and memory that are characteristic of soman intoxication.

Standard therapeutic options in organophosphate (OP) poisoning are limited to the administration of atropine and oximes, a regimen often lacking in efficacy and applicability. Treatment alternatives are needed, preferably covering a broad spectrum of OP intoxications. Although recent research yielded several promising compounds, e.g. bioscavengers, modulators of the muscarinic acetylcholine (ACh) receptor or bispyridinium non-oximes, these substances still need further evaluation, especially regarding effects on the potentially lethal respiratory symptoms of OP poisoning. Aim of this study was the development of an applicable and easy method to test the therapeutic efficiency of such substances. For this purpose, airway responsiveness in viable precision cut lung slices (PCLS) from rats was analysed. We showed that ACh-induced airway contractions were spontaneously reversible in non-poisoned PCLS, whereas in OP poisoned PCLS, contractions were irreversible. This effect could be antagonized by addition of the standard therapeutic atropine, thereby presenting a clear indication for treatment efficiency. Now, candidate therapeutic compounds can be evaluated, based on their ability to counteract the irreversible airway contraction in OP poisoned PCLS.

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G0/G1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells.

Background: To determine a new pralidoxime (PAM) treatment guideline based on the severity of acute organophosphate intoxication patients, APACHE II score, and dynamic changes in serum butyrylcholinesterase (BuChE) activity. Methods: This is a randomization trial. All patients received supportive care measurements and atropinization. Each enrolled patient was treated with 2 gm PAM intravenously as the loading dose. The control group was treated according to the WHO's recommended PAM regimen, and the experimental group was treated according to their APACHE II scores and dynamic changes in BuChE activity. If a patient's APACHE II score was ≧26 or there was no elevation in BuChE activity at the 12th hour when compared to the 6th, doses of 1 g/h PAM (i.e., doubled WHO's recommended PAM regimen) were given. The levels of the serum BuChE and red blood cells acetylcholinesterase and the serum PAM levels were also measured. Results: Forty-six organophosphate poisoning patients were enrolled in this study. There were 24 patients in the control group and 22 patients in the experimental group. The hazard ratio of death in the control group to that of the experimental group was 111.51 (95% CI: 1.17–1.613.45; p = 0.04). The RBC acetylcholinesterase level was elevated in the experimental group but was not in the control group. The experimental group did not exhibit a higher PAM blood level than did the control group. Conclusion: The use of PAM can be guided by patient severity. Thus, may help to improve the outcomes of organophosphate poisoning patients.

Introduction The information on patient characteristics and outcome in patients requiring invasive mechanical ventilation (IMV) is critical for better use of resources and clinical decision making in a rural ICU. Objective To understand characteristics and outcome of patients on IMV. Design This is a retrospective study in patients admitted in medical intensive care unit of a rural hospital who were on IMV during August 2013 to February 2015. Adult patients with failing respiratory drive and/or those who failed oxygen therapy or NIV (non invasive ventilation) were considered eligible for invasive ventilation. Patients exclusively on NIV were excluded (reason for exclusion was to study the outcome in an expensive intervention like IMV). Patients who were weaned and extubated and subsequently shifted to medicine ward were considered “good” outcome and “adverse” (not-extubated) if they died or sought discharge against medical advice. Outcome measure All-cause mortality during ICU stay. Results A total of 505 patients, of which 74.7% were male with mean age of 52 years (IQ range 38–65 years). Comorbidities were seen in 76.4% patients; significantly higher in not-extubated (94.85% vs 5.15%) ( p = 0.008). The ICU stay, days on ventilation and total hospital stay were 5 (3–9) days, 2 (1–5) days and 5(3–9) days respectively. Primary cause for IMV was sepsis, neurological, cardiac, renal and respiratory and others like envenomation, drug overdose, organophosphate poisoning, etc. Hypertension and diabetes were the commonest co-morbidities. Conclusion The mortality in patients requiring invasive ventilation support from low-resource setting is high.

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.

Pyridinium oximes are strong nucleophiles and many are effective reactivators of organophosphate-inhibited cholinesterase (ChE). However, the current oxime reactivators are ineffective at crossing the blood-brain barrier and reactivating brain ChE in the intact organism. Our laboratories have developed a series of substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937 B2) with the goal of identifying reactivators effective in crossing the blood-brain barrier. The first 35 of the series were found to have similar in vitro efficacy as reactivators of ChE inhibited by a sarin surrogate (phthalimidyl isopropyl methylphosphonate, PIMP) or a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP) in bovine brain preparations as previously observed in rat brain preparations. A number of these novel oximes have shown the ability to decrease the level of ChE inhibition in the brains of rats treated with a high sublethal dosage of either a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the VX surrogate NEMP. Levels of reactivation at 2 h after oxime administration were up to 35% while the currently approved therapeutic, 2-PAM, yielded no reduction in brain ChE inhibition. In addition, there was evidence of attenuation of seizure-like behavior with several of the more effective novel oximes, but not 2-PAM. Therefore these novel oximes have demonstrated an ability to reactivate inhibited ChE in brain preparations from two species and in vivo data support their ability to enter the brain and provide a therapeutic action. These novel oximes have the potential to be developed into improved antidotes for nerve agent therapy.

Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in vivo was ≤10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 × 107 M-1 min-1 and a plasma concentration of 0.4 μM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.

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.

Two polyhydroxyfullerenes, which decrease organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition in vitro, were administered by the intraperitoneal (ip) route or applied topically at doses of 0.9–24 mg/kg to protect adult male mice from enzyme-inhibiting and behavioral effects indicative of OP toxicity resulting from exposure to 1.7 - 2 mg/kg diphosphorofluoridate (DFP) ip or 2.3 – 2.7 mg paraoxon topical. Dosing paradigms included OP-fullerene simultaneous administration by the ip route, and 20 min post-OP polyhydroxyfullerene treatment topically. Benefits of OP sequestration by the polyhydroxyfullerene were noted and were dependent on the OP compound as well as timing and route of the polyhydroxyfullerene treatment.

The ability to overcome cellular barriers in the body is crucial for efficient delivery of drugs to the target where intervention is needed. For drugs acting in the brain it is essential to overcome the blood-brain barrier (BBB). Such drugs include antidotes for the treatment of organophosphate poisoning, a current warfare and terroristic threat. Being lipophilic compounds, organophosphates readily penetrate the brain and block the enzyme acetylcholinesterase (AChE). They cause severe symptoms which may have fatal consequences. A major drawback of currently available oxime reactivators is their inability to reactivate AChE in the central nervous system (CNS) as they are unable to cross the blood-brain barrier. An important obstacle preventing many drugs from reaching their therapeutic target in the brain is the efflux transporter P-glycoprotein (P-gp), whose function is to prevent the penetration of potentially harmful substances. The aim of this study was to evaluate the effect of P-gp on the permeation of oximes into the brain. The study of this interaction was carried out on the CACO-2 cell line, stably expressing P-gp. As it turned out, P-gp has no essential influence on the central availability of clinically used oxime reactivators within this study.

Certain ligands slowly bind to acetylcholinesterase. As a result, there is a slow establishment of enzyme-inhibitor equilibrium characterized by a slow onset of inhibition prior reaching steady state. Three mechanisms account for slow-binding inhibition: a) slow binding rate constant kon, b) slow ligand induced-fit following a fast binding step, c) slow conformational selection of an enzyme form. The slow equilibrium may be followed by a chemical step. This later that can be irreversible has been observed with certain alkylating agents and substrate transition state analogs. Slow-binding inhibitors present long residence times on target. This results in prolonged pharmacological or toxicological action. Through several well-known molecules (e.g. huperzine) and new examples (tocopherol, trifluoroacetophenone and a 6-methyluracil alkylammonium derivative), we show that slow-binding inhibitors of acetylcholinesterase are promising drugs for treatment of neurological diseases such as Alzheimer disease and myasthenia gravis. Moreover, they may be of interest for neuroprotection (prophylaxis) against organophosphorus poisoning. This article is part of the special issue entitled ‘Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield’.

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63μmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37μmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.

Background Paraoxonase-1 (PON1) activity toward organophosphorus(OP) compounds shows inter-individual variations, rendering the identification of individuals’ PON1 allozymes valuable in treating patients suffering from organophosphorus intoxication. One of the most important cytochrome P450 monooxygenases (CYPs) is CYP2D6. The CYP2D6 G1934A polymorphism leads to good, poor or no enzyme activity. Genetic testing helps identification of high risk individuals as well as management of chronic intoxicated patients. Objective to investigate a possible association between genetic polymorphisms of PON1 Q192R, and CYP2D6 G1934A as well as PON1 and pseudo-cholinesterase (PChE) enzyme activity levels and chronic organophosphate exposed patients, and hence, susceptibility for organophosphorus chronic poisoning. Design and methods Thirty chronic organophosphate exposed farm workers were compared to 29 healthy controls as regards PON1 Q192R and CYP2D6 G1934A polymorphisms using PCR-RFLP technique. Also serum PON1 and PChE activities were determined spectrophotometrically. Results Serum PChE was significantly reduced in chronic intoxicated patients compared to the control group ( p = 0.02), while PON1 activity was increased, but just failed to reach significance ( p = 0.06). PON1 192 RR genotype and R allele were significantly increased in chronic OP intoxicated patients ( p = 0.005 & p = 0.002 respectively). CYP2D6 1934 A allele was significantly increased in chronic OP patients ( p = 0.045). combining the two SNPs showed a significant statistical difference between the two groups with PON1QQ and CYP2D6 GG genotypes being more represented in the healthy controls ( p = 0.001). Fatigue and motor weakness were the most prevalent neurological symptoms seen in chronic cases (56.7%), followed by headache and lacrimation (30% each), depression (23%), tingling and sensory symptoms (20%), sleep disorders and limb pain (13%). The mean duration of environmental exposure to organophosphates was 7.7 ± 5.2 years and no association was found between chronic symptoms of intoxication and duration of exposure, provided that all workers were exposed for at least 3 years. Conclusion PON1 192RR genotype and CYP2D6 1934 A allele were found to be related to the susceptibility to organophosphate chronic toxicity in Egyptians. Larger scale gene-environmental interaction studies are recommended to confirm results and Genotyping is recommended during selection of agricultural pesticide workers to exclude high risk group.

Organophosphate pesticide exposure and neurodegeneration DOI: 10.1016/j.cortex.2015.10.003 URL: http://www.sciencedirect.com/science/article/pii/S0010945215003500 FIliació URV: SI Organophosphate pesticides (OPs) are used extensively throughout the world. The main sources of contamination for humans are dietary ingestion and occupational exposures. The major concerns related to OP exposure are delayed effects following high level exposures as well as the impact of low level exposures during the lifespan which are suggested to be a risk factor for nervous system chronic diseases. Both high and low level exposures may have a particularly high impact in population subgroups such as aged or genetically vulnerable populations. Apart from the principle action of OPs which involves inhibition of the acetylcholinesterase (AChE) enzyme, several molecular targets, such as hormones; neurotransmitters; neurotrophic factors; enzymes related to the metabolism of beta amyloid protein as well as inflammatory changes have been identified for OP compounds. Here we review the main neurological and/or cognitive deficits described and the experimental and epidemiological relationships found between pesticide exposure and Alzheimer's, Parkinson's, and Amyotrophic Lateral Sclerosis (ALS) diseases. This report also focuses on possible individual differences making groups resilient or vulnerable to these toxicants. A critical discussion of the evidence obtained from experimental models and possible sources of bias in epidemiological studies is included. In particular this review aims to discuss common targets and pathways identified which may underlie the functional deficits associated with both pesticide exposure and neurodegeneration.

Background Organophosphates are the basis of many insecticides, herbicides, and nerve agents. They were listed as highly acutely toxic agents. Findings in knockout mice suggest that paraoxonase 1 may modulate the toxicity resulting from exposure to organophosphorus compounds. In human, there is no enough data about genetic modulation of acute organophosphorus intoxication. CYP2D6 is involved in the metabolism of about 30% of xenobiotics. Prompt accurate management of OP acute intoxication can promote patient's survival. Design and methods Forty acute organophosphorus intoxicated patients were divided according to presence of clinical toxicity manifestations and serum level of pseudo-cholinesterase into two groups of acute symptomatic and acute asymptomatic patients. A third group of 29 healthy volunteers served as control. Paraoxonase 1 Q192R and CYP2D6 G1934A polymorphisms, (QQ, QR, and RR for PON1) and (GG, GA, and AA for CYP2D6), were studied using polymerase chain reaction-restriction fragment length polymorphism technique. Serum paraoxonase 1 and pseudo-cholinesterase activities were measured spectrophotometrically. Results Serum pseudo-cholinesterase was significantly reduced in both acute intoxication groups compared to the controls (p = 0.000). Paraoxonase 1 was significantly reduced in the symptomatic acute intoxication patients in comparison to the asymptomatic group (p = 0.002). There was a significant increase in paraoxonase 1 192 RR genotype and R allele in the symptomatic patients in comparison to the controls and asymptomatic patients (p = 0.006 and p = 0.01, respectively). For CYP2D6 G1934A genotypes and alleles, no significant difference was found between groups (p = 0.3 and p = 0.18, respectively). However, one case of the two recorded fatalities was for a symptomatic female patient with the only traced AA genotype. The combination of both single nucleotide polymorphisms revealed a significant distribution difference between groups, with QQ + GG genotypes being more represented in the controls, while RR + GA genotypes were exclusively present in the group of symptomatic patients (p = 0.04), none of the participants was found to have RR + AA genotypes. Some nicotinic (fasciculation and weakness), and muscarinic symptoms (bronchospasm, salivation, lacrimation, and diarrhea), increased with high significance in the symptomatic group compared to the asymptomatic one (p Conclusion Paraoxonase 1 Q192R modulates patient's response, and CYP2D6 may be related to the acute organophosphorus intoxication in the context of other genetic-environmental factors. Paraoxonase 1 enzyme level is related to symptom severity in acute OP poisoning, while pseudo-cholinesterase level indicates exposure to OP rather than severity of clinical manifestations.

This manuscript describes the development and clinical testing of a paper-based, metabolic assay designed for rapid, semi-quantitative measurement of organophosphate poisoning. Paper-based platforms, including point-of-care devices and 96-well plates, provided semi-quantitative information regarding the concentration of AchE (a biomarker for organophosphate poisoning). The paper-based 96-well-plate developed and implemented in this study was used to measure the level of organophosphate poisoning in three different clinical patients. Results were comparable to those obtained using conventional hospital methods currently considered the "gold standard". This diagnostic device offers several advantages over conventional methods, including short operating time (twice as fast as conventional methods), procedure simplicity, and reduced fabrication cost. With further commercialization efforts, the methods described in this manuscript could be applied to a wide range of potential diagnostic applications in the field.

The current standard of care for treatment of organophosphate (OP) poisoning includes pretreatment with the weak reversible acetylcholinesterase (AChE) inhibitor pyridostigmine bromide. Because this drug is an AChE inhibitor, similar side effects exist as with OP poisoning. In an attempt to provide a therapeutic capable of mitigating AChE inhibition without such side effects, high-throughput screening was performed to identify a compound capable of increasing the catalytic activity of AChE. Herein, two such novel positive allosteric modulators (PAMs) of AChE are presented. These PAMs increase AChE activity threefold, but they fail to upshift the apparent IC 50 of a variety of OPs. Further development and optimization of these compounds may lead to pre- and/or postexposure therapeutics with broad-spectrum efficacy against pesticide and nerve agent poisoning. In addition, they could be used to complement the current therapeutic standard of care to increase the activity of uninhibited AChE, potentially increasing the efficacy of current therapeutics in addition to altering the therapeutic window.

Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.

In a previous work we showed that the organophosphate pesticide (OP) chlorpyrifos (CPF) reduces the protective chemoreflex and baroreflex responses in rats. However, whether the antidotes atropine (ATR) and pralidoxime (2-PAM) are capable of restoring these reflex functions remains unexplored. Rats were poisoned with CPF (30 mg.kg−1, i.p.) and one hour after the intoxication, ATR (10 mg.kg−1, i.p.) and 2-PAM (40 mg.kg−1, i.p.) were administrated separately or in combination. Cardiorespiratory parameters were recorded in awake rats 24 h after CPF. Systolic blood pressure (SBP) and heart rate (HR) variability and spontaneous baroreflex sensitivity (sBRS) were derived from undisturbed recordings (30 min), while chemoreflex was assessed through potassium cyanide (KCN) i.v. injections (10, 20, 40, 80 μg/rat). CPF poisoning increased SBP variability and low frequency/high frequency (LF/HF) ratio of the HR variability spectrum, indicating autonomic imbalance with increased cardiac sympathetic tone. sBRS was not changed. Treatment with 2-PAM restored SBP variability, whilst both antidotes increased LF/HF ratio. CPF poisoning reduced the hypertensive, bradycardic and tachypneic chemoreflex responses. Chemoreflex-induced hypertensive response was restored by 2-PAM treatment, while ATR recovered the bradycardic response. Both antidotes restored the chemoreflex tachypneic response. Our data show distinct effects of ATR and 2-PAM on cardiorespiratory parameters affected by OP poisoning. While 2-PAM rescued the chemoreflex hypertensive response, ATR reversed chemoreflex bradycardic dysfunction. Although 2-PAM clinical use is questioned in some countries, our data indicate that summation of effects of both antidotes appears beneficial on the cardiorespiratory system and peripheral chemoreflex function.

Organophosphate (OP) chemicals include pesticides such as parathion, and nerve gases such as sarin and soman and are considered major chemical threat agents. Acute OP exposure is associated with a cholinergic crisis and status epilepticus (SE). It is also known that the survivors of OP toxicity exhibit neurobehavioral deficits such as mood changes, depression, and memory impairment, and acquired epilepsy. Our research has focused on addressing the need to develop effective therapeutic agents that could be administered even after prolonged seizures and would prevent or lessen the chronic morbidity associated with OP-SE survival. We have developed rat survival models of OP pesticide metabolite paraoxon (POX) and nerve agent sarin surrogate diisopropyl fluorophosphate (DFP) induced SE that are being used to screen for medical countermeasures against an OP attack. Our research has focused on studying neuronal calcium (Ca(2+)) homeostatic mechanisms for identifying mechanisms and therapeutics for the expression of neurological morbidities associated with OP-SE survival. We have observed development of a “Ca(2+) plateau” characterized by sustained elevations in neuronal Ca(2+) levels in OP-SE surviving rats that coincided with the appearance of OP-SE chronic morbidities. These Ca(2+) elevations had their origin in Ca(2+) release from the intracellular stores such that blockade with antagonists like dantrolene, carisbamate, and levetiracetam lowered OP-SE mediated Ca(2+) plateau and afforded significant neuroprotection. Since the Ca(2+) plateau lasts for a prolonged period, our studies suggest that blocking it after the control of SE may represent a unique target for development of novel countermeasures to prevent long term Ca(2+) mediated OP-SE neuropsychiatric comorbidities such as depression, anxiety, and acquired epilepsy (AE).

In this issue of the Biomedical Journal, we highlight new data supporting the use of pralidoxime in the treatment of cases of organophosphate poisoning, which also suggest that WHO treatment guidelines should be updated. We also learn about a modified surgical technique to repair severe spinal injuries, as well as new insight into the structure of human adenovirus that could inform vaccine development.

A novel panel of oximes were synthesized, which have displayed varying degree of reactivation ability towards different organophosphorus (OP) modified cholinesterases following insecticide exposures. In the present article, we report a comparative reactivation profile of a series of quaternary pyridinium-oximes for electric eel acetylcholinesterase (EEAChE) inhibited by the organophosphorus (OP) inhibitors methyl paraoxon (MePOX), ethyl paraoxon (POX; paraoxon) and diisopropyl fluorophosphate (DFP) that are distinguishable as dimethoxyphosphoryl, diethoxyphosphoryl and diisopropoxyphosphoryl AChE-OP-adducts. Most of the 59-oximes tested led to faster and more extensive reactivation of MePOX- and POX-inhibited EEAChE as compared to DFP-modified EEAChE. All were effective reactivators of three OP-modified EEAChE conjugates showing 18–21% reactivation for DFP-inhibited AChE and ≥45% reactivation for MePOX- and POX-inhibited EEAChE. Oximes 7 and 8 showed kr values better than pralidoxime (1) for DFP-inhibited EEAChE. Reactivation rates determined at different inhibition times showed no significant change in kr values during 0–90 min incubation with three OP’s. However, a 34–72% decrease in kr for MePOX and POX and > 95% decrease in kr for DFP-inhibited EEAChE was observed after 24 h of OP-exposure (aging).

WOS: 000486357600008 PubMed: 31152871 Background: The most commonly used insecticides and pesticides worldwide are organophosphate compounds, chemicals that irreversibly inhibit the cholinesterase enzyme. Acute intoxication with cholinesterase inhibitors is known to cause permanent effects on both the human and rat brains. Aim: To investigate the effect of acute organophosphate intoxication on hippocampus morphology, biochemistry, and pyramidal neuron numbers in female rats. Methods: Twenty-one rats were randomly divided into three groups. The control group received normal nutrition and underwent no procedures. The sham group received intraperitoneal physiological serum, while the experimental group received intraperitoneal 0.8 g/kg fenthion. Rats were sacrificed 24 h after these procedures. The brains were removed and divided in two halves medially, with one side being kept in 10% neutral formalin. After fixation procedures, tissues were embedded in blocks, sliced, and stained. A neuron count was then performed for the hippocampus. The other hippocampus was homogenized and used for biochemical procedures. Results: Hippocampus sections from rats in the experimental group exhibited swelling and loss of shape in pyramidal cells, while no changes were observed in the control or sham groups. The number of neurons in the experimental group was lower than in the control and sham groups. Biochemical analysis revealed higher MDA and GSH values in the experimental group compared to the control and sham groups. Conclusion: Our results show increased apoptotic neurodegeneration of cells in the cornu ammonis region of the hippocampus and changes in biochemical values in rats with acute organophosphate exposure. Ordu University Scientific Research Coordination Office [AP 1703] This study was supported by the Ordu University Scientific Research Coordination Office under project number AP 1703.