The tertiary oxime monoisonitrosoacetone penetrates the brain, reactivates inhibited acetylcholinesterase, and reduces mortality and morbidity following lethal sarin intoxication in guinea pigs.

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.

Oral Pretreatment with Galantamine Effectively Mitigates the Acute Toxicity of a Supralethal Dose of Soman in Cynomolgus Monkeys Posttreated with Conventional Antidotes.

Earlier reports suggested that galantamine, a drug approved to treat mild-to-moderate Alzheimer's disease (AD), and other centrally acting reversible acetylcholinesterase (AChE) inhibitors can serve as adjunct pretreatments against poisoning by organophosphorus compounds, including the nerve agent soman. The present study was designed to determine whether pretreatment with a clinically relevant oral dose of galantamine HBr mitigates the acute toxicity of 4.0×LD50 soman (15.08 µg/kg) in Macaca fascicularis posttreated intramuscularly with the conventional antidotes atropine (0.4 mg/kg), 2-pyridine aldoxime methyl chloride (30 mg/kg), and midazolam (0.32 mg/kg). The pharmacokinetic profile and maximal degree of blood AChE inhibition (∼25%-40%) revealed that the oral doses of 1.5 and 3.0 mg/kg galantamine HBr in these nonhuman primates (NHPs) translate to human-equivalent doses that are within the range used for AD treatment. Subsequent experiments demonstrated that 100% of NHPs pretreated with either dose of galantamine, challenged with soman, and posttreated with conventional antidotes survived 24 hours. By contrast, given the same posttreatments, 0% and 40% of the NHPs pretreated, respectively, with vehicle and pyridostigmine bromide (1.2 mg/kg, oral), a peripherally acting reversible AChE inhibitor approved as pretreatment for military personnel at risk of exposure to soman, survived 24 hours after the challenge. In addition, soman caused extensive neurodegeneration in the hippocampi of saline- or pyridostigmine-pretreated NHPs, but not in the hippocampi of galantamine-pretreated animals. To our knowledge, this is the first study to demonstrate the effectiveness of clinically relevant oral doses of galantamine to prevent the acute toxicity of supralethal doses of soman in NHPs. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a clinically relevant oral dose of galantamine effectively prevents lethality and neuropathology induced by a supralethal dose of the nerve agent soman in Cynomolgus monkeys posttreated with conventional antidotes. These findings are of major significance for the continued development of galantamine as an adjunct pretreatment against nerve agent poisoning.

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

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.

Assessment of false transmitters as treatments for nerve agent poisoning.

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.

Evaluation of high-affinity phenyltetrahydroisoquinoline aldoximes, linked through anti-triazoles, as reactivators of phosphylated cholinesterases.

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

A Simple 3D-Printed Enzyme Reactor Paper Spray Mass Spectrometry Platform for Detecting BuChE Activity in Human Serum.

To achieve personalized healthcare, a quick, accurate, and high-throughput method to detect disease biomarkers is essential. In the traditional practice, mass spectrometry is one of the most powerful tools and is widely studied. However, the test of human serum usually requires complicated sample pretreatment, tedious operations, and precise condition control, especially for the detection of enzymes as biomarkers. As butyrylcholinesterase (BuChE) has an indicative significance in detecting degenerative disease, liver injury, and organophosphate poisoning, the quick quantification of BuChE is of vital importance to the clinic. In this paper, we report the design and fabrication of a portable 3D-printed enzyme reactor paper spray cartridge (3D ER-PS) with integrated functions: temperature control, enzyme reaction, analyte transfer, and paper spray ionization. Coupled with mass spectrometry, quantitative testing of BuChE activity in human serum was realized conveniently and accurately. While it only requires very simple sample preparation, the results from current 3D ER-PS approach are well consistent with those obtained using Ellman's method. This 3D ER-PS platform not only provides a novel solution for the liquid biopsy of BuChE in clinics but also contributes to the development of quick and targeted medical approaches for analyzing other types of serum biomarker molecules in the field of disease diagnosis.

Comparison of the reactivation rates of acetylcholinesterase modified by structurally different organophosphates using novel pyridinium oximes.

A novel panel of oximes were synthesized, which have displayed varying degree of reactivation ability towards different organophosphorus (OP) modified cholinesterases. 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 OPs. 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).

[Organophosphorus poisoning: Towards enzymatic treatments]. / Intoxication aux organophosphorés : vers des traitements enzymatiques.

Organophosphorus compounds (OP) are toxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). Most OP are neurotoxic and act as nervous system disruptors by blocking cholinergic transmission. They are therefore responsible for many poisonings worldwide. OP toxicity may result either from acute or chronic exposure, and their poisoning effect were evaluated using several animal models. These latter were also used for evaluating the efficacy of antidotes. Strategies based on enzymes that can trap (stoichiometric bioscavengers) or degrade (catalytic bioscavengers) OP, were particularly studied since they allow effective decontamination, without toxicity or environmental impact. This review summarizes the results obtained in vivo with enzymes through three levels: prophylaxis, treatment and external decontamination. The efficiency of enzymatic treatments in different animal models is presented and the relevance of these models is also discussed for a better extrapolation to humans.

Acetylcholinesterase activity in the brain of wild birds in Korea-2014 to 2016.

Acetylcholinesterase (AChE) activity level can be used as a diagnostic marker for anticholinesterase pesticide poisoning. In this study, we aimed to establish a baseline level of normal brain AChE activity in wild birds. AChE activity was measured in the brains of 87dead wild birds (26 species). The level of AChE activity ranged from 6.40 to 15.9 µmol/min/g of brain tissue in normal wild birds. However, the brain tissue AChE activity level in wild birds exposed to organophosphate (OP) pesticide was 48.0%-96.3% of that in the normal birds. These results may serve as reference values to facilitate routine diagnosis and monitoring of OP-poisoned wild birds.

Pretreatment with pyridostigmine bromide has no effect on seizure behavior or 24 hour survival in the rat model of acute diisopropylfluorophosphate intoxication.

Acute intoxication with organophosphate cholinesterase inhibitors (OPs) is a significant human health threat, and current medical countermeasures for OP poisoning are of limited therapeutic efficacy. The rat model of acute intoxication with diisopropylfluorophosphate (DFP) is increasingly being used to test candidate compounds for efficacy in protecting against the immediate and long-term consequences of acute OP toxicity. In this model, rats are typically pretreated with pyridostigmine bromide (PB), a reversible cholinesterase inhibitor, to enhance survival. However, PB pretreatment is not likely in most scenarios of civilian exposure to acutely neurotoxic levels of OPs. Therefore, the goal of this study was to determine whether PB pretreatment significantly increases survival in DFP-intoxicated rats. Adult male Sprague Dawley rats were injected with DFP (4 mg/kg, s.c.) or vehicle (VEH) followed 1 min later by combined i.m. injection of atropine sulfate (2 mg/kg) and 2-pralidoxime (25 mg/kg). Animals were pretreated 30 min prior to these injections with PB (0.1 mg/kg, i.m.) or an equal volume of saline. DFP triggered rapid and sustained seizure behavior irrespective of PB pretreatment, and there was no significant difference in average seizure behavior score during the first 4 h following injection between DFP animals pretreated with PB or not. PB pretreatment also had no significant effect on survival or brain AChE activity at 24 h post-DFP exposure. In summary, PB pretreatment is not necessary to ensure survival of rats acutely intoxicated with DFP, and eliminating PB pretreatment in the rat model of acute DFP intoxication would increase its relevance to acute OP intoxication in civilians.

A novel fluorescence based assay for the detection of organophosphorus pesticide exposed cholinesterase activity using 1-naphthyl acetate.

Acetylcholinesterase (AChE) is the primary target of organophosphorus pesticides (OPs). Ellman's method using Acetylthiocholine (ATCh) is the standard approach for the detection of AChE activity. Though ATCh is a popular substrate, it has certain drawbacks as well. Because of these limitations, there is a need for the development of reliable and rapid assays for determination of AChE activity in cases of OP poisoning. In the present work, we have used 1-Naphthyl acetate (1-NA) as a fluorogenic substrate for the estimation of AChE activity of human erythrocytes. It is well known that due to inter-individual variation in AChE activity, the baseline value cannot be correctly predicted. Therefore, using 1-NA, we have developed a rapid, sensitive and baseline free assay for the estimation of AChE activity. The assay is based on reactivation and fluorescence quenching using a cocktail of oximes for the determination of cholinesterase activity in a post-exposure sample. Moreover, it is free from interference due to oximolysis which is an established limitation of ATCh. We feel that such an assay using 1-NA has the potential to be explored at the point of care for rapid detection of OP poisoning.

Prophylactic potential of memantine against soman poisoning in rats.

BACKGROUND: Carbamates physostigmine and pyridostigmine have been used as a pretreatment against poisoning with nerve agents in order to reversibly inhibit and thus protect from irreversible inhibition a portion of acetylcholinesterase (AChE) in brain and respiratory muscles that is crucial for survival. Memantine, an adamantine derivative, has emerged as a promising alternative to carbamates, since it prevented the fasciculations and skeletal muscle necrosis induced by carbamates and organophosphates, including nerve agents. AIM: This experimental study was undertaken in order to investigate and compare the protective and behavioural effects of memantine and standard carbamates physostigmine and pyridostigmine in rats poisoned with soman and treated with atropine, oxime HI-6 and diazepam. Another goal was to elucidate the mechanisms of the antidotal effect of memantine and its potential synergism with standard antidotes against nerve agents. MATERIALS AND METHODS: Male Wistar rats were used throughout the experiments. In dose-finding experiments memantine was administered at dose interval 0-72 mg/kg sc 60 min before sc injection of soman. In time-finding experiments memantine was injected 18 mg/kg sc 0-1440 min before soman. Standard treatment antidotes - atropine 10 mg/kg, HI-6 50 mg/kg and diazepam 2.5 mg/kg - were administered im within 15 s post-exposure. Soman 0.75 LD50 was used to study its inhibitions of neuromuscular transmission on the phrenic nerve-diaphragm preparation in situ and of tissue AChE activity. Behavioural effects of the prophylactic antidotes were investigated by means of the rotarod test. Based on these data therapeutic index and therapeutic width was calculated for all three prophylactic agents. RESULTS: Memantine pretreatment (18 mg/kg sc) produced in rats poisoned with soman significantly better protective ratios (PRs) than the two carbamates - 1.25 when administered alone and 2.3 when combined with atropine pretreatment and 6.33 and 7.23 with atropine/HI-6 and atropine/HI-6/diazepam post-exposure therapy, respectively. The highest PR of 10.11 obtained in Atr/HI-6-treated rats was achieved after pretreatment with memantine 36 mg/kg. This additional protection lasted for 8 h. All three prophylactic regimens antagonised the soman-induced neuromuscular blockade, but the effect of memantine was fastest. Pretreatment with memantine assured higher AChE activity in brain and diaphragm than in unpretreated rats (46% vs 28% and 68% vs. 38%, respectively). All three prophylactic regimens affected the rotarod performance in rats, but the effect of memantine was relatively strongest. Memantine and pyridostigmine had lowest and highest therapeutic index and therapeutic width, respectively. CONCLUSIONS: Although memantine assures better and longer-lasting protection against soman poisoning in rats than the two carbamates, its small therapeutic index and narrow therapeutic width seriously limit its potential as a pretreatment agent. Despite its behavioural effects, memantine seems to be beneficial antidote when administered after soman, along with atropine/HI-6/diazepam therapy. Mechanism of the antidotal effect of memantine against soman poisoning appears to be a combination of AChE-protecting and NMDA receptor-blocking action.

Effect of cholinergic crisis on the potency of different emergency anaesthesia protocols in soman-poisoned rats.

BACKGROUND: In a military or terrorist scenario, combination of organophosphorus compounds (OP) poisoning with physical trauma requiring surgical treatment and thus general anaesthesia are possible. Previous in vitro studies showed an altered potency of relevant anaesthetics during cholinergic crisis. Hence, it is not clear, which anaesthetics are suitable to achieve the necessary stage of surgical anaesthesia in OP poisoning. METHODS: In the present study, different anaesthetic regimens (ketamine-midazolam, propofol-fentanyl, thiopental-fentanyl), relevant in military emergency medicine, were examined in soman-poisoned rats. Clinical signs and cardiovascular variables were recorded continuously. Blood samples for acetylcholinesterase (AChE) activity were drawn. After euthanasia or death of the animals, brain and diaphragm were collected for cholinesterase assays. RESULTS: Propofol-fentanyl and thiopental-fentanyl resulted in surgical anaesthesia throughout the experiments. With ketamine-midazolam, surgical anaesthesia without respiratory impairment could not be achieved in pilot experiments (no soman challenge) and was therefore not included in the study. Soman-poisoned and control animals required a comparable amount of propofol-fentanyl or thiopental-fentanyl. In combination with atropine, significantly less propofol was needed. Survival rate was higher with thiopental compared to propofol. Atropine improved survival in both groups. Blood and tissue AChE activities were strongly inhibited after soman administration with and without atropine treatment. DISCUSSION: The current in vivo study did not confirm concerns of altered potency of existing anaesthetic protocols for the application of propofol or thiopental with fentanyl due to soman poisoning. Despite severe cholinergic crisis, sufficient anaesthetic depth could be achieved in all animals. CONCLUSION: Further experiments in in vivo models closer to human pharmaco- and toxicokinetics (e.g., swine) are required for confirmation of the initial findings and for improving extrapolation to humans.

Effects of magnesium chloride on in vitro cholinesterase and ATPase poisoning by organophosphate (chlorpyrifos).

The present study investigated possible benefits of magnesium ion (as MgCl2) in organophosphorus poisoning targeting its ability to interact with substrates and membrane enzymes. Blood samples collected from volunteered healthy adult by venepuncture into anticoagulant test tubes containing EDTA were separated into plasma and red blood cell and divided into three groups namely: normal, pesticide only (0.25-2.0 mmol/L chlorpyrifos) and pesticide (0.25-2.0 mmol/L chlorpyrifos) + 0.1 mol/L MgCl2. Acetylcholinesterase, Na+/K+ ATPase and Ca2+ ATPase activities were evaluated. Results showed that Chlorpyrifos significantly (P < .05) reduced the levels of cholinesterase both in plasma and on red blood cells. Red blood cells Na+/K+ ATPase and Ca2+ ATPase were also significantly (P < .05) reduced by chlorpyrifos while MgCl2 counteracted effects of chlorpyrifos with significant (P < .05) increase in the levels of cholinesterase, Na+/K+ ATPase and Ca2+ ATPase. We concluded that MgCl2 neutralized effects of chlorpyrifos by promoting normal ATPase activities and inhibiting release of acetylcholine from cell.

Fatal poisoning by terbufos following occupational exposure.

CONTEXT: Terbufos (TBF) is a class Ia (extremely hazardous) organophosphate pesticide (OP) and its distribution in industrialized countries has been severely restricted. Thus, acute occupational poisoning is rather uncommon. However, it still occurs in rural areas of some developing countries, where the sale of TBF is not controlled and its use is thus not properly regulated. We report a case of a 43-year-old female farmer who died after applying TBF granules. CASE: The patient died within 3 h after applying 20 bags of 5% TBF granules (900 g per bag). Investigation showed that her personal protective equipment (PPE) did not provide effective protection against dermal and inhalational exposure. Postmortem analysis revealed extremely low red blood cell acetylcholinesterase activity. Toxicological analysis of TBF showed 1.45 × 10-2 µg/ml in the heart blood and 0.17 µg/g in the liver. DISCUSSIONS: This patient died as a result of toxicity from dermal and inhalational exposure to TBF. Over-application, improper equipment, inadequate and defective PPE, and lack of hygienic precautions were all contributing factors. CONCLUSIONS: TBF is a highly toxic OP. Inadequate regulatory control, improper environmental application, and ineffective PPE resulted in a fatal human exposure.

In silico and in vitro evaluation of two novel oximes (K378 and K727) in comparison to K-27 and pralidoxime against paraoxon-ethyl intoxication.

Organophosphate (OP) poisoning is a major global health issue; while compounds from this group have been used intensively over the last century, an effective antidote is still lacking. Oxime-type acetylcholinesterase (AChE) reactivators are used to reactivate the OP inhibited AChE. Pralidoxime is the only US Food and Drug Administration approved oxime for therapeutic use but its efficacy has been disappointing. Two novel oximes (K378 and K727) were investigated in silico and in vitro and compared with an experimental oxime (kamiloxime; K-27) and pralidoxime. In silico the molecular interactions between AChE and oximes were examined and binding energies were assessed. LogP (predicted log of the octanol/water partition coefficient) was estimated. In vitro the intrinsic ability of the oximes to inhibit AChE (IC50) and their reactivation potency (R50) when used in paraoxon inhibited human RBC-AChE was determined. Molecular docking revealed that K378 and K727 bind to the peripheral site(s) with high binding energies in contrast to the central binding of K-27 and pralidoxime. LogP values indicating that the novel compounds are significantly less hydrophilic than K-27 or pralidoxime. IC50 of K378 and K727 were comparable (0.9 and 1 µM, respectively) but orders of magnitude lower than comparators. R50 values revealed their inability to reactivate paraoxon inhibited AChE. It is concluded that the novel oximes K378 and K727 are unlikely to be clinically useful. The in silico and in vitro studies described allow avoidance of unnecessary in vivo animal work and contribute to the reduction of laboratory animal use.

In Vitro Cysteine Reactivates Organophosphate Insecticide Dichlorvos-Inhibited Human Cholinesterases.

OBJECTIVES: Organophosphate (OP) pesticides inhibit both red blood cell (RBC) and plasma cholinesterases (ChEs). Oximes, especially pralidoxime (2-PAM), are widely used as antidotes to treat OP poisoning. In addition, N-acetylcysteine (NAC) is sometimes used as an adjuvant antidote. The current study aimed to assess the feasibility of using NAC as a single therapeutic agent for OP poisoning in comparison to in vitro 2-PAM. METHODS: This study was carried out at the Razi Drug Research Center of Iran University of Medical Sciences, Tehran, Iran, between April and September 2014. A total of 22 healthy human subjects were recruited and 8 mL citrated blood samples were drawn from each subject. Dichlorvos-inhibited blood samples were separately exposed to low and high doses (final concentrations of 300 and 600 µmol.L-1, respectively) of 2-PAM, NAC and cysteine. Plasma and RBCs were then separated by centrifugation and their ChE activity was measured using spectrophotometry. RESULTS: Although cysteine-and not NAC-increased the ChE activity of both plasma and RBCs over those of dichlorvos, it did not increase them over those of a high dose of 2-PAM. CONCLUSION: These results suggest that the direct reactions of 2-PAM and cysteine with dichlorvos and the reactivation of phosphorylated ChEs occurr via an associative stepwise addition-elimination process. High therapeutic blood concentrations of cysteine are needed for the elevation of ChE activity in plasma and RBCs; however, both this agent and NAC may still be effective in the reactivation of plasma and RBC ChEs.

Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis.

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), p<0.001; BChE: 140±33 for males and 109±29 for females, µkat/l, p<0.001; 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 (p>0.05). Smoking influenced cholinesterase activity - AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p<0.05). 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.

Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity.