Organophosphorus Anticholinesterase Research Articles

Dose- and time-related effects of acute diisopropylfluorophosphate intoxication on forced swim behavior and sucrose preference in rats

Acute intoxication by organophosphorus anticholinesterases (OPs) has been associated with depression and other neuropsychiatric disorders. We previously reported that adult male rats treated with diisopropylfluorophosphate (2.5 mg/kg, sc) showed acute cholinergic signs followed by changes (increased immobility/decreased swimming) in the forced swim test (a measure of behavioral despair) for at least one month. This study was conducted to evaluate the further persistence of changes in the forced swim test out to 4 months and to compare responses in a sucrose preference test, a measure of anhedonia. Adult male rats were treated with vehicle (peanut oil, 1 mL/kg, sc) or DFP (2.0, 2.25 or 2.5 mg/kg) followed by sacrifice 4 h later for measurement of OP-sensitive serine hydrolases (cholinesterase [ChE], fatty acid amide hydrolase [FAAH], and monoacylglycerol lipase [MAGL]) in hippocampus. Additional rats were treated similarly and evaluated for functional signs of acute toxicity from 30 min to 6 days, and then motor activity, forced swim behavior and sucrose preference at approximately 1 week, 1 month and 4 months after dosing. All dosages of DFP elicited serine hydrolase inhibition (ChE, 92–96 %; FAAH, 46–63 %; MAGL, 26–33 %). Body weight was reduced in all DFP-treated groups during the first two weeks, and lethality was noted with the higher dosages. Involuntary movements were elicited in all DFP treatment groups during the first week, but both time of onset and rate of recovery were dose-related. There was a significant reduction in ambulation at one week after the highest dosage (2.5 mg/kg), but no other significant locomotor changes were noted. Immobility was increased and swimming was decreased in the forced swim test at all three time-points by 2.25 mg/kg DFP, and at 2 of 3 time-points by the other dosages. While length of water deprivation and time after DFP dosing affected sucrose preference, DFP treatment had no main effect. We conclude that the forced swim test (a measure of behavioral despair/coping mechanism for inescapable stress) is a robust and persistent neurobehavioral consequence of acute DFP intoxication while sucrose preference, a measure of anhedonia and a common symptom of major clinical depression, is not affected.

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

The cannabinoid receptor antagonist AM251 increases paraoxon and chlorpyrifos oxon toxicity in rats

Organophosphorus anticholinesterases (OPs) elicit acute toxicity by inhibiting acetylcholinesterase (AChE), leading to acetylcholine accumulation and overstimulation of cholinergic receptors. Endocannabinoids (eCBs, e.g., arachidonoyl ethanolamide [AEA] and 2-arachidonoyl glycerol [2-AG]) are neuromodulators that regulate neurotransmission by reducing neurotransmitter release. The eCBs are degraded by the enzymes fatty acid amide hydrolase (FAAH, primarily involved in hydrolysis of AEA) and monoacylglycerol lipase (MAGL, primarily responsible for metabolism of 2-AG). We previously reported that the cannabinoid receptor agonist WIN 55,212-2 reduced cholinergic toxicity after paraoxon exposure. This study compared the effects of the cannabinoid receptor antagonist AM251 on acute toxicity following either paraoxon (PO) or chlorpyrifos oxon (CPO). CPO was more potent in vitro than PO at inhibiting AChE (≈2 fold), FAAH (≈8 fold), and MAGL (≈19 fold). Rats were treated with vehicle, PO (0.3 and 0.6mg/kg, sc) or CPO (6 and 12mg/kg, sc) and subsets treated with AM251 (3mg/kg, ip; 30min after OP). Signs of toxicity were recorded for 4h and rats were then sacrificed. OP-treated rats showed dose-related involuntary movements, with AM251 increasing signs of toxicity with the lower dosages. PO and CPO elicited excessive secretions, but AM251 had no apparent effect with either OP. Lethality was increased by AM251 with the higher dosage of PO, but no lethality was noted with either dosage of CPO, with or without AM251. Both OPs caused extensive inhibition of hippocampal AChE and FAAH (>80–90%), but only CPO inhibited MAGL (37–50%). These results provide further evidence that eCB signaling can influence acute OP toxicity. The selective in vivo inhibition of MAGL by CPO may be important in the differential lethality noted between PO and CPO with AM251 co-administration.

Poster #M14 USE OF FOLATE TO PREVENT SCHIZOPHRENIA IN ANIMAL MODEL OF KETAMINE INDUCE SCHIZOPHRENIA IN RATS

Acetylcholine (ACh) release is modulated pre-synaptically by both muscarinic and nicotinic receptor-mediated processes. While muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release and thus disruption of these processes could potentially affect cholinergic toxicity following exposure to anticholinesterases. Marked age-related differences in sensitivity to some organophosphorus (OP) anticholinesterases have been reported. We compared nicotinic autoreceptor function (NAF) during maturation and aging and evaluated its potential modulation by the common OP insecticide, chlorpyrifos (CPF). Cortical synaptosomes were pre-loaded with [3H]choline, superfused (0.6 ml/min) with physiological buffer and [3H]ACh release was evoked with potassium (KCl, 9 mM), with or without co-addition of exogenous ACh to stimulate nicotinic autoreceptors. Fractions of perfusate were subsequently collected and area under the curve (AUC) for [3H] was analyzed by scintillation counting. The difference in evoked release due to co-addition of exogenous ACh was defined as NAF. Under these conditions, atropine (ATR, 0.1 μM) appeared requisite for NAF; thus this muscarinic antagonist was subsequently added to all perfusion buffers. In synaptosomes from adult tissues, exogenous ACh (3–100 μM) significantly increased release in a concentration-dependent manner. The nicotinic antagonist mecamylamine (MEC, 100 μM) substantially reduced the potassium-evoked release elicited by co-addition of ACh (10 μM). Interestingly, the nicotinic agonists nicotine (NIC) and dimethylphenylpiperazinium (DMPP; 0.1–10 μM) had no effect on release. The active metabolite of CPF (i.e. chlorpyrifos oxon (CPO), 1–10 μM) inhibited NAF in vitro. Maturation-related expression of NAF was noted (AUC with co-addition of 10 μM ACh: 7-day rats, 7±6; 21-day rats, 44±6; 90-day rats, 196±37; 24-month rats, 173±52). NAF was substantially reduced (67–91%) 96 h after maximum tolerated dosages of CPF in adult and aged rats (279 mg/kg, sc) but not in juveniles (127 mg/kg, sc), even though AChE inhibition was similar among the age groups (>80%). Together these data suggest that NAF is differentially expressed during maturation and that this neuromodulatory process may be selectively altered by some OP insecticides, potentially contributing to age-related differences in response to AChE inhibitors. As NAF has been postulated to be activated under conditions of ‘impaired’ cholinergic function, selective alteration of this pre-synaptic process by OP anticholinesterases may be also important in age-related conditions associated with cholinergic hypofunction.

Effects of occupational exposure to chlorpyrifos on neuropsychological function: A prospective longitudinal study

BackgroundExposure to chlorpyrifos (CPF), an organophosphorus (OP) anticholinesterase insecticide, occurs typically in settings where multiple agents are present (e.g., agriculture) and quantitative dose measures may be absent (e.g., pesticide application). Such exposures allow few opportunities to study potential neurobehavioral effects of CPF alone. We studied the relationship between CPF exposure and behavioral function among CPF manufacturing workers, which allowed identification, measurement, and estimation of exposure and important non-exposure variables that potentially could affect study findings. MethodsA prospective longitudinal study design was used to compare neurobehavioral function over a one-year period among 53 CPF workers and 60 referent workers. Quantitative and qualitative measures were used, and potential confounders were identified and tested for possible inclusion in our statistical models. Neurobehavioral function was assessed by neuropsychological tests covering various behavioral domains that may be adversely affected by exposure to CPF in sufficient amount. ResultsCPF workers had significantly greater CPF exposures during the study period than did referents at levels where physiologic effects on plasma butyrylcholinesterase (BuChE) activity were apparent and with higher 3,5,6-trichloro-2-pyridinol (TCPy/Cr) urinary excretion (p<0.0001) and lower average BuChE activity (p<0.01). No evidence for impaired neurobehavioral domains by either group of workers was observed at baseline, on repeat examination, or between examinations. CPF workers scored higher than referent workers on the verbal memory domain score (p=0.03) at baseline, but there were no significant changes in verbal memory over time and no significant group-by-time interactions. ConclusionsThe study provides important information about CPF exposure in the workplace by not supporting our working hypothesis that CPF exposure associated with various aspects of the manufacturing process would be accompanied by adverse neurobehavioral effects detectable by quantitative neurobehavioral testing. Some aspects making this workplace site attractive for study and also present limitations for the generalization of results to other situations that might have exposures that vary widely between and within different facilities and locations. For example, these results might not apply to occupations such as applicators with higher exposure or to workers with low educational levels.

Oxime-type acetylcholinesterase reactivators in pregnancy: an overview

Oxime-type acetylcholinesterase reactivators (oxime-AChER) are used as an adjunct in the treatment for organophosphorus anticholinesterase poisoning. Because of the widespread usage and exposure of organophosphorus compounds (OPCs), its poisoning and fatalities is obvious in pregnant women, embryos and fetuses. OPCs irreversibly inhibit acetylcholinesterase (AChE) at nerve synapses. Furthermore, the role of AChE other than neurotransmission termination has been defined in the literature. The growing evidences show that cholinergic mechanisms are involved during growth and development of other organ systems. In contrary to the fact, the data on the use of oxime-AChER in OPC poisoning in pregnancy are scanty. The present review aimed to comprehend the status of oximes in pregnancy in lieu of the published literature. A thorough literature search was performed in January 2013, using ten popular search engines including Medline/PubMed, Google scholar, etc., using nine standard keywords. The search period was set from 1966 to present. The search did not reveal substantial data. No considerable studies were retrieved which could really demonstrate either the beneficial, harmful or even null effect of oxime-AChER usage in pregnancy. Only eighteen relevant articles were obtained for a period of about 47 years. In the literature, there is no report available to demonstrate the risk of using oxime-AChER in pregnancy for the treatment of OPC poisoning. The study reveals that the use of oxime-AChER in pregnancy is largely un-addressed, inconclusive and based on speculation albeit the incidences of OPC poisoning are quite prevalent. Well-designed studies are warranted for a tangible conclusion.

Efficacious Oxime for Organophosphorus Poisoning: A Minireview

Oximes are well known as acetylcholinesterase reactivators and are used in organophosphorus poisoning to reactivate inhibited acetylcholinesterase. Therapeutically available oximes, namely, pralidoxime (2-PAM), obidoxime, trimedoxime and Hagedorn oxime (HI-6), have no broad-spectrum activity against structurally different kinds of organophosphorus anticholinesterases. The widely used oxime, 2-PAM, is least effective. The focus of the review is to find an oxime that is broad spectrum and superior to the presently available oximes for the treatment of organophosphorus poisoning. Numerous oxime-based reactivators have been synthesized - in laboratories in Croatia, United States of America, Israel and most recently in Czech Republic. Some experimental oximes synthesized in Czech Republic and named as K-series of oximes have been found promising. Among them, K-27 and K-48 have higher or comparable efficacy to all available oximes though it is not effective against all organophosphorus (OP) nerve agents. They are also efficacious in pretreatment protocol for OP anticholinesterases. K-27 oxime is a promising candidate to replace therapeutically available oximes with respect to insecticide/pesticide organophosphorus poisoning. K27 and K48 may be candidates to replace the only approved pretreatment drug, pyridostigmine, in military combat medicine for OP nerve agent.Keywords: K-oximes, K-27 and K-48 oxime, Bispyridinium aldoxime, Acetylcholinesterase reactivators, OPC poisoning.

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (−/−) mice. Mice of both genotypes (n = 5–6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82–95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 μM) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20–23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner.

Dual mechanism of the antidotal action of atropine-like drugs in poisoning by organophosphorus anticholinesterases.

ABSTRACT Although more potent anti-acetylcholine drugs than atropine have been described, attempts to find atropine-like drugs that are significantly superior to atropine for the treatment of poisoning by anticholinesterases have been unsuccessful. Possible reasons for this have been investigated by comparing enantiomeric pairs of atropine-like drugs in a variety of tests for anti-acetylcholine activity (e.g. affinity constants on ileum, mydriasis, blockade of oxotremorine-induced tremors and salivation) and for their anticholinesterase antidotal properties. Large and consistent anti-acetylcholine enantiomeric potency ratios were observed, thereby providing an indication of a similar receptor structure in the PNS, CNS and in different species. However, the enantiomers of any compound studied showed equal antidotal properties. The conclusion reached was that atropine-like drugs have anti-convulsant as well as anti-acetylcholine properties.

An analysis of the action of drugs on the circular muscle strips from the caecum of the guinea-pig.

Abstract The taenia-free circular muscle strips of the caecum of the guinea-pig contracted to acetylcholine or histamine. 5-Hydroxytryptamine produced small contractile responses but high concentrations (40 μg/ml or more) induced relaxation. The contractions to 5-hydroxytryptamine were enhanced by the organophosphorus anticholinesterase drug mipafox. The responses to nicotine were always relaxations but contractions were obtained after incubating the preparation with mipafox. Hence circular muscle strips treated with mipafox were used to investigate the mechanism of contractions to all the drugs. The site of action of acetylcholine or histamine was located on the smooth muscle cells because the responses were not blocked by pentolinium, dimethylphenylpiperazinium, procaine or cocaine. Hyoscine blocked the responses to acetylcholine but left those to histamine unchanged. The contractions to nicotine were blocked by hyoscine, local anaesthetics or ganglion blocking drugs. Nicotine is thought to activate cholinergic ganglion cells. 5-Hydroxytryptamine produced contractions which were potentiated by mipafox, blocked by hyoscine and almost abolished by local anaesthetics. The responses were not modified by pentolinium but were reduced by dimethylphenylpiperazinium at a concentration which did not block the responses to acetylcholine or histamine. It is concluded that the action of 5-hydroxytryptamine was wholly indirect, on nervous tissues, part of which was located on cholinergic ganglion cells.

Effect of aldicarb insecticide formulation on its detection in postmortem human specimen

A growing body of evidence indicates that young animals exhibit an increased susceptibility to the lethal effects of cholinesterase (ChE)-inhibiting insecticides. Our laboratory is engaged in defining factors which may explain this age-related sensitivity. This report includes results from experiments designed to compare the developmental profiles, kinetic parameters and intrinsic (i.e. in vitro) sensitivity of developing male rat brain acetylcholinesterase (AChE) activity to carbamate and organophosphorus anticholinesterases. Total ChE activity in whole brain for each age was composed of about 90% AChE and 10% butyrylcholinesterase (BuChE) activity for the six ages examined. Brain AChE activity showed an age-related increase in Vmax until postnatal day 17 with no change in Km (average of all six ages ∼72 μM). Optimal substrate (acetylthiocholine) concentration for each age was 1 mM, and there was substantial (≈10%) substrate inhibition at 2.5 mM. IC50s (the concentration of compound that inhibits 50% of the AChE activity in 30 min at 26°C) defined concomitantly for postnatal day 4 and adult brain AChE using either aldicarb, carbaryl, chlorpyrifos-oxon or malaoxon were virtually identical at both ages with average IC50 values being: aldicarb=2.4 μM, carbaryl=1.7 μM, chlorpyrifos-oxon=4.9 nM and malaoxon=140 nM. In summary, AChE in young and adult brain differs mostly in specific activity while the Kms, substrate profiles, and in vitro sensitivity to selected anticholinesterase insecticides are not different. Therefore, these data support and extend the hypothesis that the greater sensitivity of the young animals to anticholinesterase pesticides is not due to the greater sensitivity of the target molecule AChE to these inhibitors.

Non-accidental chlorpyrifos poisoning—an unusual cause of profound unconsciousness

Chlorpyrifos is an organophosphorus anticholinesterase insecticide, and organophosphate intoxication can induce symptoms such as miosis, urination, diarrhea, diaphoresis, lacrimation, excitation of central nervous system, salivation, and consciousness disturbance (MUDDLES). Although accidental poisoning of children with drugs and chemicals is a common cause for consciousness disturbance in children, the possibility of deliberate poisoning is rarely considered. We report on a healthy 5-year 6-month-old boy with recurrent organophosphate intoxication. Reports of chlorpyrifos intoxication in children are quite rare. This case report demonstrates decision-making process and how to disclose deliberate chlorpyrifos poisoning of the toddler by the stepmother, another example of Munchausen syndrome by proxy.

Quantum chemical calculations for understanding and predicting toxicity. II. The phosphorylation step in the inhibition of ache by organophosphorus anticholinesterases

The first step in the inhibition of acetylcholinesterase (AChE) by organophosphorus anticholinesterases is formation of the organophosphorous molecule-AChE complex. In the first paper of this series it was shown that the first step can be modeled well by the use of three-dimensional electrostatic molecular potential contour maps around the P compound. The second phosphorylation step of the mechanism by which organophosphorous anticholinesterases of the general formula inhibit AChE is related to the acidity of X and to the bond strength of the P—X bond. For a series of compounds, aryl N-methyl methyl phosphoramidates, in which the P—X bond is constant, this paper shows that the quantum chemically calculated P-O total overlap populations (TOPs) correlate the experimental log I50 for inhibition of AChE over four orders of magnitude with correlation coefficients of 0.96 [r2: TOP 0.9581; (TOP)1/2 0.9614].

Quantum chemical calculations for understanding and predicting toxicity. I. Ab initioMODPOT/VRDDO calculations and electrostatic molecular potential contour maps of organophosphorus anticholinesterases

To demonstrate the feasibility of quantum chemical and other theoretical techniques in the understanding and correlation and then in the prediction of toxicity of a specific class of compounds, the organophosphorus acetylcholinesterase (AChE) inhibitors were chosen since these have relatively clear-cut one-dose acute lethal toxicity, both LD50, and ED50 for the triggering event inhibition of AChE, to serve as well-defined end points. It was possible to identify particular quantum chemical requisites which are related to and predictive of the toxic effects of these molecules. Ab initio calculations were carried out on a number of organophosphorus compounds using our own programs which incorporate a number of desirable options for ab initio calculations on large molecules, including ab initio effective core model potentials (MODPOT), which permit calculations of valence electrons only explicitly, yet accurately, and a charge-conserving integral prescreening evaluation (which we named VRDDO—variable retention of diatomic differential overlap) especially effective for spatially extended molecules. Ab initio calculations were carried out for the two optical isomers of GB: These isomers differ in their toxicities. From the wavefunctions of the optical isomers of GB (calculated relative to internal body-fixed coordinates) the electrostatic molecular potential contour maps (relative to the same absolute space-fixed reference coordinates) were generated in three dimensions around each molecule. These maps indicate vividly the differing stereoelectronic requisites for the different optical isomers and suggest the basis for the difference in their in vivo toxicities. These maps form three-dimensional stereoelectronic holograms and can even be used for reverse image holography to ascertain if new even hypothetical molecules fulfill the requisites. This is related to the first step in the inhibition of AChE, complex formation of the organophosphorus compound with AChE.

&lt;b&gt;Story of Muscarinic Receptors, Alkaloids with Muscarinic Significance and of Muscarinic Functions and Behaviors&lt;/b&gt;

This review of the studies of the muscarinic receptors, their synaptic activities and their functional and behavioral roles will begin with the history of the research of the autonomic and central nervous systems and their transmitters, the development of the notion of the receptor, and the tale of the significance of muscarine and other alkaloids as well as of organophosphorus (OP) anticholinesterases for these studies; we will then segue into the modern status of muscarinic receptors and of their functional and behavioral expression

Paraoxonase status and plasma butyrylcholinesterase activity in chlorpyrifos manufacturing workers

Chlorpyrifos is an organophosphorus (OP) anticholinesterase insecticide. Paraoxonase (PON1) is an enzyme found in liver and plasma that hydrolyzes a number of OP compounds. PON1 polymorphisms include a glutamine (Q)/arginine (R) substitution at position 192 (PON1(Q192R)) that affects hydrolysis of OP substrates, with the PON1(192Q) allotype hydrolyzing chlorpyrifos oxon less efficiently than the PON1(192R) allotype, a variation potentially important in determining susceptibility to chlorpyrifos. We studied 53 chlorpyrifos workers and 60 referents during 1 year and estimated chlorpyrifos exposure using industrial hygiene and employment records and excretion of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP). Plasma butyrylcholinesterase (BuChE) activity, which may by inhibited by chlorpyrifos exposure, was measured monthly. In addition, plasma samples were assayed for paraoxonase (PONase), diazoxonase (DZOase), and chlorpyrifosoxonase (CPOase) activity to determine PON1 status (inferred genotypes and their functional activity). Linear regression analyses modeled BuChE activity as a function of chlorpyrifos exposure and covariates. We postulated that the level of CPOase activity and the inferred PON1(192) genotype (together reflecting PON1 status) would differ between groups and that PON1 status would modify the models of chlorpyrifos exposure on BuChE activity. Chlorpyrifos workers and referents had a 100-fold difference in cumulative chlorpyrifos exposure. Contrary to our hypotheses, mean CPOase activity was similar in both groups (P=0.58) and PON1(192Q) showed a slight overrepresentation, not an underrepresentation, in the chlorpyrifos group compared with referents (PON1(192QQ), 51% chlorpyrifos, 40% referent; PON(192QR), 43% chlorpyrifos, 40% referent; PON(192RR), 6% chlorpyrifos, 20% referent, P=0.08). In our models, BuChE activity was significantly inversely associated with measures of interim chlorpyrifos exposure, but the biological effects of chlorpyrifos exposure on BuChE activity were not modified by PON1 inferred genotype or CPOase activity.

PON1 multitasks to protect health

The enzyme paraoxonase 1 (PON1) is an excellent example of a multitasking protein, displaying at least two very important functions: hydrolysis of organophosphorus (OP) anticholinesterases and prevention of oxidation of low density lipoproteins (LDLs) and high density lipoproteins (HDLs). PON1 is associated with the HDL particle and is a member of a family of calcium-dependent hydrolases (1). Interest in PON1 has escalated in recent years because of its two highly important roles, the first related to protection from OP compound poisoning from nerve agents (chemical warfare agents also used in terrorist attacks) and from agricultural insecticides, and the second related to protection against cardiovascular disease. The current study from the Furlong laboratory in this issue of PNAS (2) demonstrates the feasibility of producing in Escherichia coli recombinant human PON1 engineered to be more efficient catalytically toward several substrates than are the native human forms of PON1. This greater enzymatic efficiency offers promise of a potential catalytic therapeutic agent more effective than current therapies at saving lives and preserving health.

In vitro sensitivity of cholinesterases and [ 3H]oxotremorine-M binding in heart and brain of adult and aging rats to organophosphorus anticholinesterases

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [ 3H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [ 3H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.

Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys

Human butyrylcholinesterase (HuBuChE), purified from outdated human plasma, is being evaluated for efficacy against nerve agents in guinea pigs and cynomolgus monkeys. Previous studies in rodents and nonhuman primates demonstrated that pretreatment of animals with enzymes that can scavenge nerve agents could provide significant protection against behavioral and lethal effects of nerve agent intoxication. In preparation for evaluation of efficacy of HuBuChE prior to initiating an investigational new drug (IND) application, the pharmacokinetics of HuBuChE were evaluated in guinea pigs and in cynomolgus monkeys. HuBuChE was injected intramuscularly (i.m.) at two doses, and blood samples were taken to follow the time-course of HuBuChE in blood for up to 168 h after administration. In guinea pigs, the two doses of HuBuChE, 19.9 and 32.5 mg/kg, produced similar times of maximal blood concentration ( T max of 26.0 and 26.8 h, respectively) and similar elimination half-times ( t 1/2 of 64.6 and 75.5 h, respectively). Enzyme levels were still 10-fold over baseline at 72 h. Based on these data, guinea pigs were administered 150 mg/kg of enzyme i.m. and challenged at T max. Soman or VX doses were approximately 1.5, 2.0 and 2.0 × LD 50 administered subcutaneously (s.c.) in sequence at 90–120 min apart. None of the animals displayed signs of organophosphorus (OP) anticholinesterase intoxication at any of the challenge levels, and all survived for the 14-day duration of the experiment. Similar experiments were carried out with cynomolgus monkeys to determine the pharmacokinetics of HuBuChE and its efficacy against soman. The complete survival of nearly all animals tested to date, coupled with the maximal blood concentration and half-life elimination profile obtained for HuBuChE after i.m. injection, provides strong support for the continued development of HuBuChE as a product to protect against nerve agents.

Use of Electrochemical Biosensor and Gas Chromatography for Determination of Dichlorvos in Wheat

Two methods for the determination of dichlorvos in durum wheat by electrochemical assay and gas chromatography, respectively, have been developed. Dichlorvos, an organophosphorus anticholinesterase pesticide, was extracted from wheat with hexane, and the filtered extract was directly analyzed by gas chromatography with nitrogen-phosphorus flame detection (NPD). Recoveries of dichlorvos from milled wheat spiked at 0.25-1.5 microg/g ranged from 96.5 to 100.9%, and the limit of detection was 0.02 microg/g. The electrochemical assay was based on the detection of choline, the acetylcholinesterase product, via a choline oxidase biosensor. An aliquot of the filtered hexane extract was partitioned with phosphate buffer solution, and the organic layer was evaporated prior to electrochemical analysis. A limit of detection of 0.05 microg/g of dichlorvos was obtained with mean recoveries of 97-103% at spiking levels of 0.25-1.5 microg/g. A good correlation (r = 0.9919) was found between the results obtained with the electrochemical and those obtained with the gas chromatographic methods. The electrochemical method was peer-validated in two laboratories that analyzed 10 blind samples (5 duplicates), including a blank and 4 spiked samples with dichlorvos at levels of 0.25, 0.60, 1.00, and 1.50 microg/g. Within-laboratory repeatability (RSDr) and between-laboratory reproducibility (RSDR) ranged from 5.5 to 7.8% and from 9.9 to 17.6%, respectively.