Occupational stress among farm and ranch operators in the midwestern United States.

BACKGROUND: This study used surveillance data from 2018 and 2020 to test the stability of work-related strain symptoms (high stress, sleep deprivation, exhaustion) with demographic factors, work characteristics, and musculoskeletal symptoms among farm and ranch operators in seven midwestern states of the United States. METHODS: Cross-sectional surveys were conducted among farm and ranch operators in 2018 (n = 4423) and 2020 (n = 3492). Operators were asked whether, in the past 12 months, they experienced extended work periods that resulted in high stress levels, sleep deprivation, exhaustion/fatigue, or other work-related strain symptoms. Covariates included personal and demographic factors, work characteristics, number of injuries, work-related health conditions, and exposures on the operation. Summary statistics were tabulated for explanatory and outcome variables. The classification (decision) tree approach was used to assess what variables would best separate operators with and without reported strain symptoms, based on a set of explanatory variables. Regularized regression was used to generate effect estimates between the work strain variables and explanatory variables. RESULTS: High stress level, sleep deprivation, and exhaustion were reported more frequently in 2018 than 2020. The classification tree reproduced the 2018 model using 2020 data with approximately 80% accuracy. The mean number of reported MSD symptoms increased slightly from 1.23 in 2018 to 1.41 in 2020. Older age, more time spent in farm work, higher gross farm income (GFI), and MSD symptoms in six body regions (ankles/feet, knees, lower back, neck, shoulders, wrists/hands) were associated with all three work strain symptoms. CONCLUSIONS: Musculoskeletal pain and discomfort was a strong predictor for stress, sleep deprivation, and exhaustion among farmers and ranchers. This finding indicates that reducing MSD pain and discomfort is beneficial for both physical and mental health.

Polyclonal antibody to soman-tyrosine.

Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin but not nonphosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10(-11) M. The limit of detection on Western blots was 0.01 µg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity.

Gene-delivered butyrylcholinesterase is prophylactic against the toxicity of chemical warfare nerve agents and organophosphorus compounds.

Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD(50) dose of echothiophate and 5× LD(50) dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.

Prolonged toxic effects after cocaine challenge in butyrylcholinesterase/plasma carboxylesterase double knockout mice: a model for butyrylcholinesterase-deficient humans.

Death and toxicity after cocaine use do not correlate with cocaine blood levels. One explanation for this observation is that cocaine abusers may posses one or more of the 58 possible known mutations in the butyrylcholinesterase gene (BCHE). Butyrylcholinesterase (BChE) serves as the primary cocaine hydrolase producing a nontoxic product ecgonine methyl ester. A reduction in endogenous levels of BChE may result in increased metabolism by hepatic carboxylesterase to produce norcocaine, a toxic product. Humans have carboxylesterase in tissues but not in plasma, whereas wild-type mice have significant amounts of carboxylesterase in tissues and plasma. Knockout mice with no plasma carboxylesterase were created to eliminate the contribution of plasma carboxylesterase in cocaine hydrolysis, thereby simulating human enzyme levels. This study tested the hypothesis that reductions in BChE such as those in humans with BChE mutations contribute to increased toxicity after cocaine use. Carboxylesterase and BChE double knockout mice, models for humans with BChE deficiency, were challenged with a nonlethal dose of 100 mg/kg (-)-cocaine. Carboxylesterase/BChE double knockout mice demonstrated toxic signs significantly longer than did wild-type and carboxylesterase knockout mice. The carboxylesterase/BChE-deficient mice took approximately 2.5 times as long to recover from cocaine toxicities, including the following: hypothermia, hyperactivity, stereotypical behavior, ocular effects, and dorsiflexion of the tail. The carboxylesterase/BChE double knockout mouse model demonstrates the importance of endogenous BChE for protection against cocaine toxicity and provides an in vivo system for studying drug sensitivity of humans who carry a BChE mutation.

Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons.

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase.

Production of ES1 plasma carboxylesterase knockout mice for toxicity studies.

The LD(50) for soman is 10-20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1-/- mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1-/- mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes but was lethal only to ES1-/- mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1-/- mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.

Role of acetylcholinesterase on the structure and function of cholinergic synapses: insights gained from studies on knockout mice.

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.

Adenovirus-transduced human butyrylcholinesterase in mouse blood functions as a bioscavenger of chemical warfare nerve agents.

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.

Mass spectrometry identifies multiple organophosphorylated sites on tubulin.

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.

Effects of rivastigmine and donepezil on brain acetylcholine levels in acetylcholinesterase-deficient mice.

PURPOSE: Alzheimer s disease is characterized by a dysfunction of central cholinergic systems and is treated by inhibitors of acetylcholinesterase (AChE). This study tests the effect of two AChE inhibitors in therapeutic use, rivastigmine and donepezil, in mice that are devoid of AChE (AChE-/- mice). Rivastigmine is an inhibitor of both AChE and butyrylcholinesterase (BChE) whereas donepezil is a selective inhibitor of AChE. METHODS: We have used in vivo microdialysis to investigate the effects of the two drugs on the extracellular concentration of acetylcholine (ACh) in the hippocampus of AChE-/- mice. RESULTS: Extracellular ACh levels in the hippocampus were 30-fold elevated in AChE-/- mice compared to wild-type (AChE+/+) animals. Infusion of rivastigmine (1 and 10 microM) caused a further doubling of ACh levels in AChE-/- mice within 90-120 min. In contrast, infusion of donepezil (1 microM) did not affect hippocampal ACh levels in AChE-/- mice although it increased ACh levels more than twofold in wild-type mice. CONCLUSIONS: In the absence of AChE, rivastigmine enhances the levels of extracellular ACh by inhibiting BChE. This finding may be of therapeutic relevance because BChE activity is preserved, but AChE activity is strongly decreased, in late-stage Alzheimer s disease.

Comparison of agricultural injuries reported in the media and census of fatal occupational injuries.

The Bureau of Labor Statistics (BLS) publishes annual statistics on occupational injuries and fatalities in the United States. The BLS fatality data include all agricultural workers while the non-fatal injury data only cover hired employees on large farms. In 2012, the Central States Center for Agricultural Safety and Health (CS-CASH) began collecting regional media monitoring data of agricultural injury incidents to augment national statistics. The aims of this report were: a) to compare CS-CASH injury and fatality data collected via print and online sources to data reported in previous studies, and b) to compare fatality data from media monitoring to BLS Census of Fatal Occupational Injuries (CFOI) data. CS-CASH media monitoring data were collected from a news clipping service and an internet detection and notification system. These data covered years 2012-2017 in seven Midwestern states (Iowa, Kansas, Minnesota, Missouri, Nebraska, North Dakota, and South Dakota). CS-CASH occupational fatality data were compared with aggregate CFOI data for the region during 2012-2015. Media monitoring captured 1048 injury cases; 586 (56%) were non-fatal and 462 (44%) were fatal. The numbers of occupational fatality cases from media monitoring and CFOI were nearly identical (280 vs. 282, respectively), and the distributions by type of injury were similar. Findings suggest that media monitoring can capture equal numbers of fatalities compared to CFOI. Non-fatal injuries, not captured by national surveillance systems, can be collected and tracked using print and electronic media. Risk factors, identified in media sources, such as gender, age, time, and source of the incident are consistent with previously reported data. Media monitoring can provide timely access to detailed information on individual cases, which is important for detecting unique and emerging hazards, designing interventions and for setting policy and guiding national strategies.

The butyrylcholinesterase knockout mouse as a model for human butyrylcholinesterase deficiency.

Butyrylcholinesterase (BChE) is an important enzyme for metabolism of ester drugs. Many humans have partial or complete BChE deficiency due to genetic variation. Our goal was to create a mouse model of BChE deficiency to allow testing of drug toxicity. For this purpose, we created the BChE knockout mouse by gene-targeted deletion of a portion of the BCHE gene (accession number M99492). The BChE(-/-) mouse had no BChE activity in plasma, but it had low residual butyrylthiocholine hydrolase activity in all other tissues attributed to carboxylesterase ES-10. The BChE(-/-) mouse had a normal phenotype except when challenged with drugs. Nicotinic receptor function as indicated by response to nicotine seemed to be normal in BChE(-/-) mice, but muscarinic receptor function as measured by response to oxotremorine and pilocarpine was altered. Heart rate, blood pressure, and respiration, measured in a Vevo imager, were similar in BChE(+/+) and BChE(-/-) mice. Like BChE(-/-) humans, the BChE(-/-) mouse responded to succinylcholine with prolonged respiratory arrest. Bambuterol was not toxic to BChE(-/-) mice, suggesting it is safe in BChE(-/-) humans. Challenge with 150 mg/kg pilocarpine i.p., a muscarinic agonist, or with 50 mg/kg butyrylcholine i.p., induced tonicclonic convulsions and death in BChE(-/-) mice. This suggests that butyrylcholine, like pilocarpine, binds to muscarinic receptors. In conclusion, the BChE(-/-) mouse is a suitable model for human BChE deficiency.

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice.

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 microM in AChE-deficient mice and 4.36 microM in wild-type mice. Addition of choline (10 microM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 microM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.

The butyrylcholinesterase knockout mouse is obese on a high-fat diet.

Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE-/- mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE-/- mice fed a standard 5% fat diet had normal body weight. However, BChE-/- mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.

Whole body and tissue imaging of the butyrylcholinesterase knockout mouse injected with near infrared dye labeled butyrylcholinesterase.

Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and organophosphates. Phase I safety trials of human BChE are currently being conducted and large-scale production of recombinant BChE is underway. Information on the real-time distribution of BChE from the injection site has not been well characterized. This study utilized the BChE nullizygote (BChE-/-) mouse and tetrameric equine BChE labeled with LI-COR fluorescent IRDye 800CW to track, quantify and determine the retention time of BChE in vivo following intramuscular injection. In vivo images were acquired with Xenogen's IVIS 200 imager and the LI-COR Odyssey Imaging System fitted with the MousePOD. Plasma and tissues were tested for BChE activity. The 2 mg of BChE spread from the injection site to heart, liver, intestine, kidneys, lungs, salivary glands, and muscle, but did not enter the brain or the skin. Fluorescence intensity in organs and BChE activity in plasma peaked on day 1. BChE activity in plasma was undetectable by day 16, at a time when there was still significant fluorescent signal and BChE activity in the liver (0.32 units/g), injected quadriceps (0.13 units/g) and in most of the organs analyzed. It is concluded that the tetrameric BChE glycoprotein of 340 kDa diffuses from the muscle injection site to blood and peripheral organs and has a longer residence time in the organs than in blood.

Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo.

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.

Neuropathological and immunochemical studies of brain parenchyma in acetylcholinesterase knockout mice: implications in Alzheimer's disease.

The 'cholinergic hypothesis', based on the correlation of the reduction of cholinergic activity in Alzheimer's disease (AD) with cognition and memory, is currently the most widely-held view for AD. Drug treatments for AD focus mainly on inhibition of acetylcholinesterase (AChE), and to some extent butyrylcholinesterase (BChE). In addition to changes in AChE in AD, there is a rise in the level of the sister enzyme BChE. However, the role of the two cholinesterases is poorly understood in vivo. We characterized several proteins immunohistochemically in brain sections from AChE nullizygote (AChE-/-) and wild type AChE+/+ mice. Previous studies had shown that AChE-/- mouse tissues are devoid of AChE activity and that the overall cholinesterase activity is significantly decreased in the knockout group [16]. Despite the differences of cholinesterase activity, we found no significant structural alterations between the experimental groups. Immunohistochemical examination revealed no neuronal, dendritic, astrocytic, synaptic, microglial, and endothelial differences between AChE-/- and AChE+/+ mice. Similarly, the histochemical examination showed no morphologic alterations between AChE-/- and AChE+/+ mice. Our studies show that neither the absence of AChE nor the presence exclusively of BChE is associated with neuroglial and vascular pathology.

Adaptation to excess acetylcholine by downregulation of adrenoceptors and muscarinic receptors in lungs of acetylcholinesterase knockout mice.

The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.

Sensitivity of butyrylcholinesterase knockout mice to (--)-huperzine A and donepezil suggests humans with butyrylcholinesterase deficiency may not tolerate these Alzheimer's disease drugs and indicates butyrylcholinesterase function in neurotransmission.

Butyrylcholinesterase (EC 3.1.1.8 BChE) is present in all human and mouse tissues, and is more abundant than acetylcholinesterase (EC 3.1.1.7 AChE) in all tissues except brain. People who have no BChE activity due to a genetic variation are healthy. This has led to the hypothesis that BChE has no physiological function. We tested this hypothesis by challenging BChE and AChE knockout mice, as well as wild-type mice, with the AChE specific inhibitors, (--)-huperzine A and donepezil, and with serine hydrolase inhibitors, echothiophate and chlorpyrifos oxon. (--)-Huperzine A and donepezil caused mortality and significant toxicity in the BChE-/- animals. The BChE heterozygote (BCHE+/-) mice with approximately one-half the BChE activity of the BChE wild type (BChE+/+) exhibited intermediate toxic symptoms, and survived a longer period. The BChE+/+ animals displayed comparatively minor toxic symptoms and recovered by 24h post-dosing. Plasma AChE activity was inhibited to the same extent in BChE-/-, +/-, and +/+ mice, whereas BChE activity was not inhibited. This indicated that the protective effect of BChE was not due to scavenging (--)-huperzine A. AChE-/- mice were unaffected by (--)-huperzine A and donepezil, demonstrating the specificity of these inhibitors for AChE. AChE-/- mice treated with chlorpyrifos oxon lost all BChE activity, had severe cholinergic symptoms and died of convulsions. This showed that BChE activity was essential for survival of AChE-/- mice. In conclusion, we propose that the protective effect of BChE is explained by hydrolysis of excess acetylcholine in physiologically relevant regions such as diaphragm, cardiac muscle, and brain. Thus, BChE has a function in neurotransmission. People with BChE deficiency are expected to be intolerant of standard doses of the anti-Alzheimer's drugs, (--)-huperzine A and donepezil.

Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol.

Ethanol is one of the most commonly abused psychotropic substances with deleterious effects on the central nervous system. Ethanol exposure during development results in the loss of neurons in brain regions and when exposed to ethanol cultured cells undergo apoptosis. To date no information is available on whether abnormally high AChE activity is characteristic of apoptosis in animals exposed to ethanol. The aims of the present study were to determine whether induction of AChE activity is associated with ethanol-induced apoptosis and to explore the mechanism of enhanced AChE activity induced by ethanol. For this purpose, in vitro and in vivo experiments were performed. AChE activity was quantified by spectrophotometry and apoptosis by flow cytometer in SH-SY5Y cells exposed to ethanol. The results showed that cells treated with 500mM ethanol for 24h had a 9-fold increase in apoptotic cells and a 6-fold increase in AChE activity compared with controls. Mice exposed acutely to 200µl of 20% ethanol daily on days 1-4 had elevated AChE activity in plasma on days 3-7. On day 4, plasma AChE activity was 2.4-fold higher than pretreatment activity. More apoptotic cells were found in the brains of treated mice compared to controls. Cells in brain sections that were positive in the TUNEL assay stained for AChE activity. In conclusion, AChE activity and apoptosis were induced in SH-SY5Y cells and mice treated with ethanol, which may indicate that increased AChE may related to apoptosis induced by ethanol. Unusually high AChE activity may be an effect marker of exposure to ethanol. The relationship between AChE and apoptosis might represent a novel mechanism of ethanol-associated neuronal injury.