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Sarin is a highly toxic nerve agent that was developed for chemical warfare during World War II and is used in present conflicts. Immediate effects of acute sarin exposure are established; however, whether effects persist after initial signs have subsided is debated. The National Toxicology Program (NTP) conducted a systematic review to evaluate the evidence for long-term neurological effects following acute (

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl 2 , MMB4-DMS, and HLo7 DMS. The inhibition rate constants ( k i ) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8 × 10 3 to 1.4 × 10 4 M − 1 min − 1 ) compared to many other OPs. Obidoxime Cl 2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48 h) for all three species. Conclusions: (1) Obidoxime Cl 2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

Diisopropylfluorophosphate (DFP) is a potent acetylcholinesterase inhibitor commonly used in toxicological studies as an organophosphorus nerve agent surrogate. However, LD50values for DFP in the same species can differ widely even within the same laboratory, possibly due to the use of degraded DFP. The objectives here were to identify an efficient synthesis route for high purity DFP and assess the storage stability of both the in-house synthesized and commercial source of DFP at the manufacturer-recommended storage temperature of 4°C, as well as −10°C and −80°C. After 393 days, the commercial DFP stored at 4°C experienced significant degradation, while only minor degradation was observed at −10°C and none was observed at −80°C. DFP prepared using the newly identified synthesis route was significantly more stable, exhibiting only minor degradation at 4°C and none at −10°C or −80°C. The major degradation product was the monoacid derivative diisopropylphosphate, formed via hydrolysis of DFP. It was also found that storing DFP in glass containers may accelerate the degradation process by generating waterin situas hydrolytically generated hydrofluoric acid attacks the silica in the glass. Based on the results here, it is recommended that DFP be stored at or below −10°C, preferably in air-tight, nonglass containers.

Organophosphorus (OP) compounds, including pesticides and chemical warfare nerve agents (CWNA), are threats to the general population as possible weapons of terrorism or by accidental exposure whether through inadvertent release from manufacturing facilities or during transport. To mitigate the toxicities posed by these threats, a therapeutic regimen that is quick-acting and efficacious against a broad spectrum of OPs is highly desired. The work described herein sought to assess the protective ratio (PR), median effective doses (ED(50)), and therapeutic index (TI = oxime 24-h LD(50)/oxime ED(50)) of MMB4 DMS, HLö-7 DMS, and 2-PAM Cl against the OPs sarin (GB), VX, and phorate-oxon (PHO). All OPs are representative of the broader classes of G and V chemical warfare nerve agents and persistent pesticides. MMB4 DMS and HLö-7 DMS were previously identified as comparative efficacy leads warranting further evaluations. 2-PAM Cl is the U.S. FDA-approved standard-ofcare oxime therapy for OP intoxication. Briefly, PRs were determined in male guinea pigs by varying the subcutaneously (SC) delivered OP dose followed then by therapy with fixed levels of the oxime and atropine (0.4 mg/kg; administered intramuscularly [IM]). ED(50)s were determined using a similar approach except the OP dose was held constant at twice the median lethal dose (2 × LD(50)) while the oxime treatment levels were varied. The ED(50) information was then used to calculate the TI for each OP/oxime combination. Both MMB4 DMS and HLö-7 DMS provided significant protection, i.e., higher PR against GB, VX, and PHO when compared to atropine controls, but significance was not readily demonstrated across the board when compared against 2-PAM Cl. The ED(50) values of MMB4 DMS was consistently lower than that of the other oximes against all three OPs. Furthermore, based on those ED(50)s, the TI trend of the various oximes against both GB and VX was MMB4 DMS > HLö-7 DMS > 2-PAM Cl, while against PHO, MMB4 DMS > 2-PAM Cl > HLö-7 DMS.

The currently fielded pre-hospital therapeutic regimen for the treatment of organophosphorus (OP) poisoning in the United States (U.S.) is the administration of atropine in combination with an oxime antidote (2-PAM Cl) to reactivate inhibited acetylcholinesterase (AChE). Depending on clinical symptoms, an anticonvulsant, e.g., diazepam, may also be administered. Unfortunately, 2-PAM Cl does not offer sufficient protection across the range of OP threat agents, and there is some question as to whether it is the most effective oxime compound available. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection at the FDA approved human equivalent dose (HED) of 2-PAM Cl against tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX, and the pesticides paraoxon, chlorpyrifos oxon, and phorate oxon. Male Hartley guinea pigs were subcutaneously challenged with a lethal level of OP and treated at approximately 1min post challenge with atropine followed by equimolar oxime therapy (2-PAM Cl, HI-6 DMS, obidoxime Cl2, TMB-4, MMB4-DMS, HLö-7 DMS, MINA, and RS194B) or therapeutic-index (TI) level therapy (HI-6 DMS, MMB4-DMS, MINA, and RS194B). Clinical signs of toxicity were observed for 24h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. When the oxime is standardized against the HED of 2-PAM Cl for guinea pigs, the evidence from clinical observations, lethality, quality of life (QOL) scores, and cholinesterase reactivation rates across all OPs indicated that MMB4 DMS and HLö-7 DMS were the two most consistently efficacious oximes.

The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD50 (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more tolerant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcholinesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24-hr lethality data, but the differences were not observed when titers were normalized to baseline levels.

Most hospital laboratories do not measure blood cyanide concentrations, and samples must be sent to reference laboratories. A simple method is needed for measuring cyanide in hospitals. The authors previously developed a method to quantify cyanide based on the high binding affinity of the vitamin B12 analog, cobinamide, for cyanide and a major spectral change observed for cyanide-bound cobinamide. This method is now validated in human blood, and the findings include a mean inter-assay accuracy of 99.1%, precision of 8.75% and a lower limit of quantification of 3.27 µM cyanide. The method was applied to blood samples from children treated with sodium nitroprusside and it yielded measurable results in 88 of 172 samples (51%), whereas the reference laboratory yielded results in only 19 samples (11%). In all 19 samples, the cobinamide-based method also yielded measurable results. The two methods showed reasonable agreement when analyzed by linear regression, but not when analyzed by a standard error of the estimate or paired t-test. Differences in results between the two methods may be because samples were assayed at different times on different sample types. The cobinamide-based method is applicable to human blood, and can be used in hospital laboratories and emergency rooms.

Anticholinesterases, such as organophosphorus pesticides and warfare nerve agents, present a significant health threat. Onset of symptoms after exposure can be rapid, requiring quick-acting, efficacious therapy to mitigate the effects. The goal of the current study was to identify the safest antidote with the highest therapeutic index (TI = oxime 24-hr LD50/oxime ED50) from a panel of four oximes deemed most efficacious in a previous study. The oximes tested were pralidoxime chloride (2-PAM Cl), MMB4 DMS, HLo-7 DMS, and obidoxime Cl2. The 24-hr median lethal dose (LD50) for the four by intramuscular (IM) injection and the median effective dose (ED50) were determined. In the ED50 study, male guinea pigs clipped of hair received 2x LD50 topical challenges of undiluted Russian VX (VR), VX, or phorate oxon (PHO) and, at the onset of cholinergic signs, IM therapy of atropine (0.4 mg/kg) and varying levels of oxime. Survival was assessed at 3 hr after onset clinical signs. The 3-hr 90th percentile dose (ED90) for each oxime was compared to the guinea pig pre-hospital human-equivalent dose of 2-PAM Cl, 149 µmol/kg. The TI was calculated for each OP/oxime combination. Against VR, MMB4 DMS had a higher TI than HLo-7 DMS, whereas 2-PAM Cl and obidoxime Cl2 were ineffective. Against VX, MMB4 DMS > HLo-7 DMS > 2-PAM Cl > obidoxime Cl2. Against PHO, all performed better than 2-PAM Cl. MMB4 DMS was the most effective oxime as it was the only oxime with ED90 < 149 µmol/kg against all three topical OPs tested.

Purpose: Aldicarb and methomyl are carbamate pesticides commonly implicated in human poisonings. The primary toxic mechanism of action for carbamate poisoning is cholinesterase (ChE) inhibition. As such, it is logical to assume that the currently accepted therapies for organophosphate poisoning (muscarinic antagonist atropine and the oxime acetylcholinesterase reactivator pralidoxime chloride [2-PAM Cl]) could afford therapeutic protection. However, oximes have been shown to be contraindicated for poisoning by some carbamates. Methods: A protective ratio study was conducted in guinea pigs to evaluate the efficacy of atropine and 2-PAM Cl. The ChE activity was determined in both the blood and the cerebral cortex. Results: Coadministration of atropine free base (0.4 mg/kg) and 2-PAM Cl (25.7 mg/kg) demonstrated protective ratios of 2 and 3 against aldicarb and methomyl, respectively, relative to saline. The data reported here show that this protection was primarily mediated by the action of atropine. The reactivator 2-PAM Cl had neither positive nor negative effects on survival. Both blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were significantly reduced at 15 minutes postchallenge but gradually returned to normal within 24 hours. Analysis of cerebral cortex showed that BChE, but not AChE, activity was reduced in animals that succumbed prior to 24 hours after challenge. Conclusion: The results suggest that coadministration of atropine and 2-PAM Cl at the currently recommended human equivalent doses for use in the prehospital setting to treat organophosphorus nerve agent and pesticide poisoning would likely also be effective against aldicarb or methomyl poisoning.

It has been suggested that pesticide exposure may be a contributing factor underlying the increased incidence of asthma in the United States and other industrialized nations. To test this hypothesis, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used organophosphate pesticide. Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 h or 7 days after a single subcutaneous injection of either 390 mg/kg or 70 mg/kg of chlorpyrifos, respectively. Mechanisms by which chlorpyrifos may cause airway hyperreactivity include inhibition of acetylcholinesterase (AChE) or dysfunction of M3 muscarinic receptors on airway smooth muscle or of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 h after treatment with 390 mg/kg of chlorpyrifos, but not 7 days after injection of 70 mg/kg of chlorpyrifos. Acute exposure to eserine (250 μg/ml) also significantly inhibited lung AChE but did not potentiate vagally induced bronchoconstriction. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifos-treated animals, pilocarpine dose-response curves were shifted significantly to the right, demonstrating decreased responsiveness of neuronal M2 receptors. In contrast, chlorpyrifos treatment did not alter methacholine-induced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway smooth muscle. These data demonstrate that organophosphate insecticides can cause airway hyperreactivity in the absence of AChE inhibition by decreasing neuronal M2 receptor function.

This study tested the hypothesis that the inhibition of acetylcholinesterase is greater when the insecticide chlorpyrifos (CPF) is in the presence of several polycyclic aromatic hydrocarbons (PAHs) found in house dust. CPF-oxon (CPFO) inhibition curves of purified AChE (electric eel) were generated in the presence or absence of different concentrations of the PAHs pyrene, benzo(a)pyrene, anthracene, and fluoranthene. Without CPF-oxon, all four PAHs themselves inhibited AChE activity with IC50 values in the range 8.2-17 microM. The IC50 for benzo(a)pyrene with human recombinant AChE was 1.5 microM. When AChE was incubated with CPF-oxon together with the PAHs, the inhibitory effect on AChE was additive. This was exemplified by large (60-80%) and significant (P

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemicals pose a threat to the United States military and civilian populations because they are relatively inexpensive and easy to obtain, and have the potential to cause mass casualties if released by accident, natural disaster, or by a deliberate act. Chemical threat agents include nerve agents, vesicating or blister agents, pulmonary agents, and blood agents or metabolic poisons. The traditional chemical warfare agents include the organophosphorus nerve agents such as sarin and VX, and the mustard vesicating agents. These remain in several stockpiles around the world. Toxic industrial chemicals (TICs) manufactured, stored, and transported across the nation are not regulated as strictly as traditional warfare agents, and many are stored in large amounts and are readily available. Unfortunately and unlike biological and radiation threats, there are many examples of human suffering and death after deliberate releases of chemical welfare agents (CWAs) (sarin, sulfur mustard) and TICs (cyanide, chlorine). Requirements for an effective response to a civilian chemical event include (i) post exposure treatments that are effective within an often short therapeutic time window, (ii) drugs and devices that can be used by medical personnel at the scene of the event or in a pre hospital setting to treat many victims, (iii) drugs and devices that are appropriate for a diverse population including pediatric and elderly victims, and individuals with preexisting medical conditions, and (iv) rapid and effective diagnostic technologies to determine the chemical agent and pathophysiology. The National Institutes of Health (NIH) has developed a research program called Countermeasures Against Chemical Threats (CounterACT) to develop new and improved diagnostic technologies and therapies. This program has identified several new opportunities to develop better medical intervention strategies that treat the most severe symptoms, and broad spectrum drugs that target common physiological mechanisms of injury that could be used to treat victims exposed to many different kinds of chemicals. CounterACT and other programs at Department of Health and Humman Services (DHHS) charged with this important goal are essential to national security and will require strong support to meet the challenges ahead. It will require a commitment that is consistent with the cost and time required for drug discovery and development, and should support research at the basic, translational, and clinical levels. Keywords: chemical threats; medical countermeasures; research; Counter ACT; NIH; sarin; VX; cyanide; sulfur mustard; chlorine; phosgene

Publisher Summary The chapter discusses the role of acetylcholinesterase (AChE) inhibition in the toxicity of organophosphorus (OP) and carbamate (CM) pesticides, with particular emphasis on lower doses that are sublethal, but induce changes in cholinergic receptors, either by indirect modulation of ACh levels via AChE inhibition or by direct interaction with the receptor. Case studies are presented that suggest that some of the toxicities associated with OP insecticides may not involve AChE or cholinergic receptors. Inhibition of AChE and subsequent cholinergic dysfunction represent a common mechanism of toxicity after acute exposure to high levels of OP and CM pesticides. However, the range of toxicities among different compounds is broad, and the potency of a compound for the inhibition of AChE is not a good predictor of its toxicity. Furthermore, toxicities associated with repeated exposures to lower levels of OPs do not correlate well with AChE inhibition. These observations suggest that significant interaction of OPs and CMs with other critical molecules within the cell may be important in explaining some of the toxicity of these compounds not attributable to AChE inhibition. Examples are presented with AChE null mice, direct interactions with muscarinic receptors and other molecules, impairment of cognitive development in weaning rats, OP-induced airway hyperreactivity mediated by M2 receptors, and direct effects on axonal growth in cell culture. The case studies provide strong evidence that noncholinesterase mechanisms are involved in the toxicity of OPs, and they have identified important gaps in the understanding of the mechanism of action of these important classes of insecticides.

Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations > or =0.001 microM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (> or =1 microM) and CPFO (> or =1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system.

Previous studies have shown that moderate to high levels of chlorpyrifos (CPF) alter cognitive function in adult and immature rats. In the present study, we tested the hypothesis that lower-level exposure to CPF before or immediately after weaning causes deficits in cognitive function. A total of 78 Long–Evans rats were injected subcutaneously with 0, 0.3 or 7.0 mg/kg CPF every 4 days before or after weaning and were tested with the Morris swim task from postnatal day 24 through 28. Exposure to CPF before weaning did not cause signs of overt cholinergic intoxication or impaired growth nor did the exposures cause significant inhibition of regional brain cholinesterase (ChE) activity or reduction in muscarinic receptors 24 h after the last injection. However, spatial learning was impaired after 5 days of training in the group of weanling rats administered 7.0 mg/kg CPF. Rats administered 0.3 or 7.0 mg/kg CPF after weaning were also impaired in the task, without significant changes in brain ChE activity. These data indicate that low-level exposure to CPF caused deficits in cognitive function in weanling rats, and these effects did not appear to be mediated by the inhibition of brain ChE. It is suggested that the alteration of cognitive function in juvenile rats is an important functional correlate of the cellular and molecular effects of CPF in the immature brain. The mechanisms for CPF-induced cognitive dysfunction are unknown.

Brain acetylcholinesterase activity in field-caught meadow voles (Microtus pennsylvanicus) was depressed after a field-spray of Orthene® (acephate: acetylphosphoramidothioic acid O,S-dimethyl ester) by as much as 32% in 1982 and 38% in 1983. Short-term recovery was demonstrated and occurred in a time-dependent fashion in 1982. Plasma Cholinesterase levels were more variable but also were depressed. Residues were detected in vegetation samples and in the gastrointestinal tracts of exposed voles. Residues in vegetation were diluted or absent 7 to 8 d following the treatment.