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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.

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.

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.

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.

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.

Paraoxonase 1 (PON1) is a type of aromatic esterase widely existing in mammals. It can hydrolyze various kinds of compounds effectively in vivo and in vitro. Previous studies have confirmed that PON1 can be used as antidote against organophosphorus poisonings (OPs). In this study, we obtained two subtype isozymes (i.e. rhPON1R192 and rhPON1Q192) by gene recombination and compared their detoxification effects against different OPs in rats. The rhPON1R192 demonstrated better detoxification effect against chlorpyrifos poisoning than the rhPON1Q192, whose detoxification effect against diazinon poisoning was prior to the former. Both of them showed poor detoxification effect against trithion. Therefore, we concluded that, to different OPs, better detoxification effect may be achieved by selecting the PON1 subtype isozyme with higher specific hydrolytic activity.

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.

A method is described allowing forensic analysis of plasma samples to prove human poisoning with the organophosphorus pesticides omethoate (OM) and dimethoate (DIM). Upon incubation of human serum albumin (HSA) with both pesticides tyrosine residues were phosphorylated. In addition, a novel disulfide-adduct between the identical thiol-containing leaving group of OM and DIM (2-mercapto-N-methylacetamide, MNMA) and the only free cysteine residue in HSA (Cys34) was formed. Following pronase-catalyzed proteolysis either O,O-dimethyl phosphotyrosine (Tyr-dmp) or O,O-dimethyl thiophosphotyrosine (Tyr-dmsp) as well as the cysteine-proline dipeptide disulfide-adduct (MNMA-CysPro) were produced. All biomarkers were simultaneously detected using modern microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). Corresponding limits of identification (LOI) for tyrosine-adducts (LOIOM: 30 μM, LOIDIM: 120 μM) and disulfide-adducts (LOIOM: 1.2 μM, LOIDIM: 30 μM) demonstrated that MNMA-CysPro allowed a considerably more sensitive detection. Finally, this novel method was applied to a plasma sample of an 87-year-old man, who had unintentionally ingested the pesticide Roxion® containing DIM as active ingredient. Unambiguous proof of poisoning demonstrated suitability of the novel biomarkers for sensitive verification analysis.

We herein report on the forensic analysis of plasma samples to prove human poisoning with oxydemeton-S-methyl (ODM), S-(2-(ethylsulfinyl)ethyl)-O,O-dimethyl phosphorothioate. This organophosphorus pesticide is the active ingredient of Metasystox®, that was swallowed by a 77-year-old woman to commit suicide. ODM belongs to the class of dimethyl phosphoryl (DMP) pesticides, contains a 2-(ethylsulfinyl)ethanethiol (ESOET) leaving group and undergoes adduct formation with endogenous molecules as elaborated herein with human serum exposed to pesticides in vitro. A novel bioanalytical micro liquid-chromatography-electrospray ionization tandem high-resolution mass spectrometry method (μLC-ESI MS/HR MS) was developed to target multiple biomarkers of exposure. Following pronase-catalyzed proteolysis of patient plasma and subsequent ultrafiltration, the filtrate was analyzed. Diverse reaction products of ODM as well as of its oxidized biotransformation product demeton-S-methyl sulfone (DSMS), that possesses a 2-(ethylsulfonyl)ethanethiol (ESO2ET) leaving group, were simultaneously detected. Phosphorylated tyrosine residues (Tyr-DMP) derived from human serum albumin (HSA) as well as novel dipeptide-adducts containing the Cys34 residue of HSA coupled to ESOET and ESO2ET via a disulfide bond (ESOET-CysPro and ESO2ET-CysPro) were found. In addition, a related disulfide-product was detected comprising the single amino acid cysteine and ESOET (ESOET-Cys). Whereas Tyr-DMP only proved the intake of any DMP pesticide in general, its simultaneous detection with ESOET-CysPro, ESO2ET-CysPro and ESOET-Cys allowed unambiguous identification of the ingested pesticide. Therefore, the novel biomarkers and the method developed expand the possibilities of forensic investigations of ODM poisoning.

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.

Organophosphosphates (OPs) are highly effective acetylcholinesterase (AChE) inhibitors that are used worldwide as cheap, multi-purpose insecticides. OPs are also used as chemical weapons forming the active core of G-series and V-series chemical agents including tabun, sarin, soman, cyclosarin, VX, and their chemical analogs. Human exposure to any of these compounds leads to neurotoxic accumulation of the neurotransmitter acetylcholine, resulting in abnormal nerve function and multiple secondary health complications. Suicide from deliberate exposure to OPs is particularly prevalent in developing countries across the world and constitutes a major global health crisis. The prevalence and accessible nature of OP compounds within modern agricultural spheres and concern over their potential use in biochemical weapon attacks have incentivized both government agencies and medical researchers to enact stricter regulatory policies over their usage and to begin developing more proactive medical treatments in cases of OP poisoning. This review will discuss the research undertaken in recent years that has investigated new supplementary drug options for OP treatment and support therapy, including progress in the development of enzymatic prophylaxis.

Isolated organs proofed to be a robust tool to study effects of (potential) therapeutics in organophosphate poisoning. Small bowel samples have been successfully used to reveal smooth muscle relaxing effects. In the present study, the effects of obidoxime, TMB-4, HI-6 and MB 327 were investigated on human small bowel tissue and compared with rat data. Hereby, the substances were tested in at least seven different concentrations in the jejunum or ileum both pre-contracted with carbamoylcholine. Additionally, the cholinesterase activity of native tissue was determined. Human small intestine specimens showed classical dose response-curves, similar to rat tissue, with MB 327 exerting the most potent smooth muscle relaxant effect in both species (human EC50=0.7×10-5M and rat EC50=0.7×10-5M). The AChE activity for human and rat samples did not differ significantly (rat jejunum=1351±166 mU/mg wet weight; rat ileum=1078±123 mU/mg wet weight; human jejunum=1030±258 mU/mg wet weight; human ileum=1293±243 mU/mg wet weight). Summarizing, our isolated small bowel setup seems to be a solid tool to investigate the effects of (potential) therapeutics on pre-contracted smooth muscle, with data being transferable between rat and humans.

Medical countermeasures for acute poisoning by organophosphorus nerve agents are generally assessed over 24 h following poisoning and a single administration of treatment. At 24 h, the antinicotinic bispyridinium compound MB327 (1,10-(propane-1,3-diyl)bis(4-tert-butylpyridinium)) dimethanesulfonate is as effective as the oxime HI-6 against poisoning by soman, when used as part of a treatment containing atropine and avizafone. In this study, we hypothesised that an earlier endpoint, at 6 h, would be more appropriate for the pharmacokinetics and mechanism of action of MB327 and would therefore result in improved protection. MB327 diiodide (33.8 mg/kg) or the oxime HI-6 DMS (30 mg/kg), in combination with atropine and avizafone (each at 3 mg/kg) was administered intramuscularly to guinea pigs 1 min following subcutaneous soman and the LD50 of the nerve agent was determined at 6 h after poisoning for each treatment. The treatment containing HI-6 gave a similar level of protection at 6 h as previously determined at 24 h (protection ratios 3.9 and 2.9, respectively). In contrast, the protection achieved by treatment containing MB327 showed a striking increase at 6 h (protection ratio >15.4) compared to the 24 h end point (protection ratio 2.8). The treatment gave full protection for at least 5 h against doses of soman up to 525 μg/kg; in contrast, mortality began in animals treated with HI-6 after 1 h. This study demonstrates the importance of using an appropriate end point and has shown that treatment including MB327 was far superior to oxime-based treatment for poisoning by soman, when assessed over a pharmacologically-relevant duration. The improved outcome was seen following a single dose of treatment: it is possible that additional doses to maintain therapeutic plasma concentrations would further increase survival time. Antinicotinic compounds therefore offer a promising addition to treatment, particularly for rapidly aging or oxime-insensitive nerve agents.

The activity of human cholinesterases, erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) represents an important marker when monitoring exposure to pesticides/nerve agents, and may also be used in occupational medicine in diagnosis and prognosis of some diseases. In this study "normal/baseline" AChE and BChE activity has been investigated in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age (categories 18-25, 26-35 and 36-45 years old) and smoker status. The modified Ellman's method was used for enzyme activity assessment in 387 young and healthy individuals (201 males and 186 females aged 18-45). A significant inter-sexual difference in AChE and BChE activity was found (AChE: 351±67 for males and 377±65 for females, (μmol/min)/(μmol of hemoglobin), p0.001; BChE: 140±33 for males and 109±29 for females, μkat/l, p0.001; mean±SD). Despite the finding that mean AChE activity somewhat decreased whereas BChE activity grew within the age categories of the tested subjects, no significant effect of age on cholinesterase activity was found (p0.05). Smoking influenced cholinesterase activity - AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p0.05). Smoking was found not to have any effect on BChE activity. Reference values based on confidence intervals for AChE and BChE activity were established. The presented results might be useful in routine clinical practice where the monitoring of blood AChE and plasma BChE activity is crucial for prognosis and diagnosis of organophosphate poisoning, in occupational medicine and in relevant mass casualty scenarios.

Acute kidney injury (AKI) is common following glyphosate surfactant herbicide (GPSH) self-poisoning. Serum creatinine (sCr) is the most widely used renal biomarker for diagnosis of AKI although a recent study in rats suggested that urinary kidney injury molecule-1 predicted AKI earlier and better after GPSH-induced nephrotoxicity. We explored the utility of a panel of biomarkers to diagnose GPSH-induced nephrotoxicity in humans. In a prospective multi-centre observational study, serial urine and blood samples were collected until discharge and at follow-up. The diagnostic performance of each biomarker at various time points was assessed. AKI was diagnosed using the Acute Kidney Injury Network (AKIN) definitions. The added value of each biomarker to sCr to diagnose AKI was assessed by the integrated discrimination improvement (IDI) metric. Of 90 symptomatic patients, 51% developed AKI and 5 patients who developed AKIN≥2 died. Increased sCr at 8 and 16h predicted moderate to severe AKI and death. None of the 10 urinary biomarkers tested increased above normal range in patients who did not develop AKI or had mild AKI (AKIN1); most of these patients also had only minor clinical toxicity. Absolute concentrations of serum and urinary cystatin C, urinary interleukin-18 (IL-18), Cytochrome C (CytoC) and NGAL increased many fold within 8h in patients who developed AKIN≥2. Maximum 8 and 16h concentrations of these biomarkers showed an excellent diagnostic performance (AUC-ROC ≥0.8) to diagnose AKIN≥2. However, of these biomarkers only uCytoC added value to sCr to diagnose AKI when assessed by IDI metrics. GPSH-induced nephrotoxicity can be diagnosed within 24h by sCr. Increases in uCytoC and uIL-18 confirm GPSH-induces apoptosis and causes mitochondrial toxicity. Use of these biomarkers may help to identify mechanism specific targeted therapies for GPSH nephrotoxicity in clinical trials.

Paraoxonase-1 (PON1) is a native enzyme that is synthesized in the liver and is capable of hydrolyzing organophosphates (OPs). It is regarded as part of a promising approach for the pretreatment and therapy of OP poisoning. Previous experiments with purified rabbit serum PON1 have established that it can protect rats against many OP exposures. In the current paper, we described a preparation of active recombinant human PON1 (rHuPON1) by engineering an Escherichia coli expression system. Recombinant HuPON1 was purified by Ni-NTA affinity chromatography followed by DEAE sepharose fast-flow chromatography. After purification, rHuPON1 was chemically modified with polyethyleneglycol (PEG)-20K. Recombinant HuPON1 exhibited a mean residence time (MRT) of 8.9h, which was threefold shorter than that of native HuPON1 in rats. However, rHuPON1 chemically modified with PEG-20K displayed an MRT of 19.5h, suggesting that PEG modification can prolong the circulatory stability of rHuPON1. PEG-rHuPON1 had a catalytic efficiency sufficient in protecting rats against OP poisoning, as measured by acetylcholinesterase activity in tissues and signs after poisoning.

After more than 70 years of considerable efforts, research on medical defense against nerve agents has come to a standstill. Major progress in medical countermeasures was achieved between the 50s and 70s with the development of anticholinergic drugs and carbamate-based pretreatment, the introduction of pyridinium oximes as antidotes, and benzodiazepines in emergency treatments. These drugs ensure good protection of the peripheral nervous system and mitigate the acute effects of exposure to lethal doses of nerve agents. However, pyridostigmine and cholinesterase reactivators currently used in the armed forces do not protect/reactivate central acetylcholinesterases. Moreover, other drugs used are not sufficiently effective in protecting the central nervous system against seizures, irreversible brain damages and long-term sequelae of nerve agent poisoning.New developments of medical counter-measures focus on: (a) detoxification of organophosphorus molecules before they react with acetylcholinesterase and other physiological targets by administration of stoichiometric or catalytic scavengers; (b) protection and reactivation of central acetylcholinesterases, and (c) improvement of neuroprotection following delayed therapy.Future developments will aim at treatment of acute and long-term effects of low level exposure to nerve agents, research on alternative routes for optimizing drug delivery, and therapies. Though gene therapy for in situ generation of bioscavengers, and cell therapy based on neural progenitor engraftment for neuronal regeneration have been successfully explored, more studies are needed before practical medical applications can be made of these new approaches.

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and an oxime has a limited efficacy. An alternative approach is the development of stoichiometric or catalytic (bio-)scavengers which should be able to prevent systemic toxicity. Recently, a β-cyclodextrin derivative, 6-OxP-CD, bearing a pyridinium oximate in 6-position of one glucose unit was synthetized and shown to possess a promising detoxification potential against a variety of alkyl methylfluorophosphonates in vitro. In order to investigate the suitability of 6-OxP-CD as a small molecule scavenger an in vivo guinea pig model was established to determine the protective effect of 6-OxP-CD against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of 6-OxP-CD (100mg/kg) prevented systemic toxicity in cyclosarin (∼2LD50) poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity but did not protect erythrocyte AChE activity. A lower 6-OxP-CD dose (50mg/kg) reduced systemic toxicity and prevented mortality in all animals. Thus, the results of this proof of concept study indicate that 6-OxP-CD may be considered as a potential small molecule scavenger to protect against the toxic effects of a range of highly toxic OP nerve agents.

In order to investigate the mechanism of muscular weakness in the intermediate myasthenia syndrome (IMS) following acute organophosphate poisoning, the effect of dimethoate on the neuromuscular transmission was studied in rats by using the electrical stimulation single fiber electromyography (SSFEMG) and repetitive nerve stimulation (RNS). The results showed that there was a prolongation of mean consecutive difference (MCD) of the latencies of single fiber potential shown by SSFEMG in dimethoate intoxicated rats during the presence of muscle weakness when the stimuli were given at 10 or 20 Hz, and there was a remarkable decrement of compound muscle action potential (CMAP) of gastrocnemius muscle evoked by RNS on the sciatic nerve at 20 Hz in some rats with myasthenia. The frequency of neuromuscular transmission abnormalities detected by SSFEMG was significantly higher than those detected by RNS. This study demonstrates that the SSFEMG is a more sensitive electrophysiological method in the detection of neuromuscular transmission block occurred in rats of acute organophosphate poisoning with muscle weakness.

Although the importance of atropine in therapy of organophosphate (OP) poisoning is generally recognized, its dosing is a matter of debate. A retrospective analysis of atropine dosing was undertaken in 34 patients who had been enrolled in a clinical study assessing obidoxime effectiveness in OP-poisoning. All patients were severely intoxicated (suicidal attempts) and required artificial ventilation. Atropine was administered routinely by intensive care physicians for life-threatening muscarinic symptoms, with the recommendation to favor low dosage. The pharmacological active enantiomere S-hyoscyamine was determined by a radioreceptor assay. When RBC-AChE activity ranged between 10% and 30%, S-hyoscyamine plasma concentrations of approx. 5 nmol L⁻¹ were sufficient. This concentration could be maintained with about 0.005 mg h⁻¹ kg⁻¹ atropine. Only when RBC-AChE was completely inhibited, therapy of cholinergic crisis required atropine doses up to 0.06 mg h⁻¹ kg⁻¹. Elimination half-life of S-hyoscyamine was 1.5 h, showing occasionally a second slow elimination phase with t(½)=12 h. Malignant arrhythmias were observed in some 10% of our cases, which occurred late and often in the absence of relevant glandular cholinergic signs, when the S-hyoscyamine concentration was below 2.5 nmol L⁻¹. Arrhythmias mostly resolved on reinstitution of atropine.

Objectives Reactivation of inhibited acetylcholinesterase (AChE) with oximes is a causal therapy of intoxication with organophosphorus compounds (OPs). Maximal oxime effects are expected when effective doses are administered as soon as possible and as long as reactivation can be anticipated. An obidoxime plasma level in the range of 10–20 μM was estimated as appropriate. The achievement of this target was assessed in 34 severely OP-poisoned patients. Methods After admission to the intensive care unit (ICU) the obidoxime regimen (250 mg i.v. as bolus, followed by 750 mg/24 h) was started and maintained as long as reactivation was possible. Plasma concentrations of obidoxime were determined by HPLC. Results A total amount of 2269 ± 1726 mg obidoxime was infused over 65 h ± 55 h resulting in a steady state plasma concentration of 14.5 ± 7.3 μM. Obidoxime was eliminated with t 1/2(1) 2.2 and t 1/2(2) 14 h. The volumes of distribution amounted to 0.32 ± 0.1 L/kg ( V (1) ) and 0.28 ± 0.12 ( V (2) ) L/kg. Postmortem examination of tissue in one patient showed obidoxime accumulation in cartilage, kidney and liver and pointed to brain concentrations similar to plasma concentration. Conclusions Using the suggested obidoxime regimen, the targeted plasma concentration could be achieved. Obidoxime was eliminated biphasically and was well tolerated. This result allows the recommendation of using this definite regimen for adults also in case of mass casualties.

Thirty-four adult patients with severe organophosphorus compounds (OP) poisoning requiring artificial ventilation were enrolled in a clinical study and received atropine and obidoxime (250 mg i.v., followed by 750 mg/24 h) as antidotal treatment. Here, we re-analyzed the cholinesterase status (red blood cell acetylcholinesterase (RBC-AChE) activity, reactivatability of RBC-AChE, and plasma butyrylcholinesterase (Pl-BChE) activity) in relation to the neuromuscular transmission (NMT) data. When RBC-AChE activity ranged between 100% and 30% NMT was unimpaired after tetanic stimulation with frequencies up to 50 Hz. A further decrease in RBC-AChE activity was accompanied by a marked disturbance of NMT, being strongly impaired at AChE activities5% of normal. Higher stimulation frequencies (30 Hz) facilitated the discrimination of the types of impairment. The neuromuscular transmission was the best quantified by using the ratio of the ninth to the first amplitude, while the standard method was less discriminative. At RBC-AChE levels higher than 40% of normal weaning from the ventilator may be considered. Completely aged RBC-AChE as indicated by loss of reactivatability loses its guidance function. Then, steadily increasing Pl-BChE activity suggests lack of circulating poison. One-week later, neuromuscular transmission may be largely normal and patients could be weaned from the respirator if other complications are not withstanding.

Organophosphorus compounds (OPs) are used as pesticides and developed as warfare nerve agents such as tabun, soman, sarin, VX and others. Exposure to even small amounts of an OP can be fatal and death is usually caused by respiratory failure. The mechanism of OP poisoning involves inhibition of acetylcholinesterase (AChE) leading to inactivation of the enzyme which has an important role in neurotransmission. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the nervous systems. During more than five decades, pyridinium oximes have been developed as therapeutic agents used in the medical treatment of poisoning with OP. They act by reactivation of AChE inhibited by OP. However, they differ in their activity in poisoning with pesticides and warfare nerve agents and there is still no universal broad-spectrum oxime capable of protecting against all known OP. In spite of enormous efforts devoted to development of new pyridinium oximes as potential antidotes against poisoning with OP only four compounds so far have found its application in human medicine. Presently, a combination of an antimuscarinic agent, e.g. atropine, AChE reactivator such as one of the recommended pyridinium oximes (pralidoxime, trimedoxime, obidoxime and HI-6) and diazepam are used for the treatment of OP poisoning in humans. In this article the available data related to medical treatment of poisoning with OP pesticides are reviewed and the current recommendations are presented.

A comprehensive symptomatological obervational battery in conjunction with acute toxicity testing, evaluated with the method of a hyperbolic curve relating survival time (T) to dose (D), was used to characterize quantitatively the lethal toxicities of the organophosphorus (OP) compound, sarin, in mice. The experimental data observed with sarin (0.4–4.0 mg kg s.c. ) , alone or in combination with atropine (ATR) (20 mg kg i.p.), were plotted as a graph of D/T against D, a linearizing transformation of the hyperbolic function. This linearized plot gave two straight lines, deflecting at 1.2 mg kg , in terms of latency to whole body tremor (BT) and loss of the righting reflex (LR). At lower lethal doses of sarin (0.4–1.2 mg kg ) with ATR pretreatment, the D/T vs. D curves of BT and LR were shifted in parallel to the left, while at high lethal doses (1.6–4.0 mg kg ) these curves interpolatedly converged. The sequelae and/or severity of symptoms were also comparatively different between the range of lower and high lethal doses as noted above. It has been claimed that the protective actions of ATR and clonidine (CLD) against the lethal effects of cholinesterase inhibitors are associated with different underlying mechanisms, i.e. presynaptic versus postsynaptic cholinergic inhibition. The protective effects of a single dose of ATR (20 mg kg ) and CLD (1.0 mg kg ), after 38 and 15 min of intraperitoneal pretreatment, respectively, alone or in combination, challenged with 2 ×, 4 × and 8 × LD50's of sarin were also comparatively evaluated. ATR resulted in a nonsignificant increase in latency to onset of BT and LR. CLD significantly delayed the onset of these symptoms against all 3 dose levels of sarin intoxication, whereas ATR plus CLD additively increased the latency to the onset of these symptoms. The present results indicate that at lower dosages of sarin (⩽ 4 × LD50's) its mode of action appears to be mediated mainly by a muscarinic mechanism, whereas at high doses it is mediated by some other non-specific actions superimposed on the cholinergic actions of sarin. The present study also lends support to the hypothesis of the existence of different forms of OP intoxication on the strength of lethal exposure. The possible mechanisms of both sarin lethality and ATR- and CLD-mediated protection are briefly discussed.

Pralidoxime iodide (2-PAM), an antidote approved for the reactivation of inhibited acetylcholinesterase (AChE) in organophosphate poisoning, dose-dependently hydrolyzed an acetylthiocholine iodide (ASCh). The AChE (0.3 U) activity inhibited by VX analog (ENMP, 0.1 microM) increased to approximately 200% of normal levels after a dosage of 5 mM 2-PAM (control 0.132+/-0.012 U/ml, 5 mM 0.253+/-0.026 U/ml). This result indicates that 2-PAM produced a thiocholine from the ASCh by hydrolysis. High-performance liquid chromatography (HPLC) analysis was then performed to further clarify the hydrolysis of ASCh with 2-PAM. It was clear that 2-PAM was converted to acetylated 2-PAM with acetic acid produced from ASCh by hydrolysis. Next, we tried to compare this esterase-like activity of 2-PAM with that of obidoxime, which is known as a strong reactivator of inhibited AChE, and with diacetylmonoxime, known as a weak reactivator. All of these oximes showed esterase-like activity, and their strengths were consistent with those of known reactivators of inhibited AChE. These results indicate that a great deal of the data obtained previously with ASCh relating to the effects of oximes must be rechecked. It is clear that oximes easily hydrolyze ASCh. We therefore strongly caution that the method of determining AChE activity with ASCh is not suitable for examining the effects of oximes.

Organophosphorus compounds (OPs) are being used as insecticides and warfare agents. OP insecticides represent an important problem of public health, causing around 200,000 deaths annually. The World Health Organization has pointed to the necessity to introduce new medical practices that improve the results of classical treatments. Many studies have shown that the administration of phosphotriesterases (enzymes that detoxify OPs through hydrolysis) is a promising treatment of persons poisoned with OPs. Such an enzyme-based treatment might introduce important improvements in the treatment of patients having ingested large amounts of OPs. Phosphotriesterases might also be suitable for prophylactic treatment of persons at risk to be severely exposed. The new experimental treatments do not exhibit the intrinsic neurotoxicity of the classical prophylaxis based on carbamates and antimuscarinic drugs. Experimental data suggest that might be time to initiate clinical trials in order to study the efficacy of phosphotriesterases in the therapy and prophylaxis of OP intoxication.

Considering the various microscopic reactions as well as toxicokinetic and pharmacokinetic principles in therapy of organophosphate poisoning, the administration of obidoxime by an initial bolus dose followed by continuous infusion appears rational. Using this protocol, six patients each with parathion or oxydemeton methyl poisoning were treated. In parathion poisoning, reactivation was possible up to 7 days. At paraoxon concentrations0.1 microM obidoxime only partially reactivated acetylcholinesterase (AChE) of erythrocytes in vivo although reactivation could be assessed in vitro, which roughly fitted theoretical calculations. AChE-inhibitory material was detected up to 5 days. Cholinergic signs soon subsided when AChE was above 20% of normal, and atropine plasma levels could be kept below 7 ng/ml. In one patient brain damage persisted. Oxydemeton methyl poisoning responded to obidoxime therapy only when the oxime was instituted shortly after poisoning. Out of six patients one died. No intermediate syndrome and no signs of permanent hepatic dysfunction were found in the 12 patients.

This study compared the efficacy of HI6 and 2-PAM against nerve agent (soman, tabun, sarin, and VX)-induced lethality in the atropinesterase-free rabbits pretreated with vehicle (controls) or pyridostigmine. Treatment was administered at signs or 2 min after agent challenge and consisted of oxime (100 mumol/kg) + atropine (13 mg/kg) (alone or together with diazepam). Twenty-four-h LD50 values were calculated for soman- and tabun-intoxicated animals, whereas 24-h survival was noted in animals given 10 LD50s of sarin or VX. In pyridostigmine and control rabbits intoxicated with soman and treated with oxime + atropine (alone or together with diazepam), HI6 was 3-5 times more effective than 2-PAM. In contrast, HI6 was less effective than 2-PAM against tabun poisoning. In pyridostigmine-pretreated animals exposed to tabun, efficacy was increased more than 3-fold when compare to tabun-challenged animals treated with atropine + HI6 alone. Both oximes were highly effective against sarin and VX. These findings suggest that HI6 could replace 2-PAM as therapy for nerve agent poisoning, because it is superior to 2-PAM against soman, and when used in pyridostigmine-pretreated animals, it affords excellent protection against all four nerve agents when used in combination with atropine (alone or together with diazepam) therapy.

Phenyl di-n-pentylphosphinate was synthesised by interaction of phenyl phosphorodichloridate and n-pentyl magnesium bromide. The product was purified by silica chromatography (yield 25%). Although much more stable at physiological pH than its 4-nitrophenyl analogue, this ester is a good inhibitor of neuropathy target esterase (NTE): kappa a = 1.7 X 10(5) M-1 min-1. It is a very weak anticholinesterase (kappa a congruent to 10 M-1 min-1). In vivo only 5-10 mg/kg is required to inhibit hen brain and spinal cord NTE. The inhibited NTE can be reactivated fully by incubation in vitro with iso-nitrosoacetophenone (INAP) (19 mM at 37 degrees C and pH 8.5 for 60 min): this property enables study to be made of the fate of inhibited NTE in vivo.

Groups of guinea pigs were injected with a range of dosages for sarin (0, 140, 279, 557 micrograms/kg) followed by pralidoxime (2-PAM) and atropine sulfate (16 mg/kg). Poisoning by sarin in these animals elevated plasma pralidoxime content in a dose-dependent manner within 10 min of intoxication. Plasma levels after administration of 3.12 mg/kg of 2-PAM were elevated from a control mean of 6.18 micrograms/ml to a maximum of 13.78 micrograms/ml in animals given 557 micrograms/kg of sarin at 2 min after the injection of the therapeutic compounds. This suggests that pathophysiological changes following intoxication by potent inhibitors of cholinesterase result in a decrease in the rate and extent of distribution of therapeutic compounds. This effect is most likely a consequence of changes in cardiovascular functions influencing blood flow to various organs.

The specific features of working conditions in greenhouses were studied and related to the health status of greenhouse workers. A higher level of morbidity with temporary disability in female greenhpuse workers, as compared with women engaged in outdoor vegetable growing, was established. The effect of working conditions in greenhouses on the development of functional disorders in the central nervous and cardiovascular systems, on respiration, as well as the effect on blood morphology, activity of specific liver enzymes, cholinesterase (ChE) and some indices of lipid metabolism was studied in female greenhouse workers. The results showed the need for a selective use of pesticides in greenhouses, taking health considerations into account, as well as for a thorough medical surveillance of workers.