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.

Knowledge, Attitudes, and Practices for Respiratory and Hearing Health among Midwestern Farmers.

OBJECTIVE: The purpose of this study was to assess knowledge, attitudes, and practices for hearing and respiratory health/safety among farmers in seven Midwestern states served by a federally funded Agricultural Center. Findings provided a baseline to longitudinally track the Agricultural Center's program outcomes and to design community education to improve safety and health among farmers. DESIGN AND SAMPLE: This was a cross-sectional study using a 30 item mailed survey to describe farmers' operations, demographics, health conditions, related information sources, and knowledge/attitude/practices for personal protective equipment (PPE) (i.e., ear plugs/muffs and dust masks/respirators). MEASURES: Frequencies and percentages were calculated for each item and according to responses from younger versus older farmers. The unit of study was farm operators (N = 280) randomly selected from a publicly available database of corn/soybean and hog farmers in seven Midwestern states. RESULTS: Findings revealed important knowledge gaps among respondents regarding (1) hazardous exposure sources; (2) long-term health consequences of noise/dust exposure; (3) proper selection/fitting of PPE. CONCLUSIONS: Public health nurses and primary care providers in rural communities should address specific knowledge gaps in order to enhance farmers' perceived understanding of their susceptibility to hazardous exposures. Increasing farmers' knowledge through preferred venues may help to improve PPE effectiveness.

Rural Natural Disaster Stress: A Survey of Community Resource Use and Effect.

OBJECTIVE: This study identified rural community experiences and preferences related to available resources and their effects on stress following tornadoes and floods. METHODS: The Rural Natural Disaster Stress and Recovery survey was distributed from December 2021 to February 2022 in rural disaster-affected communities. Within the analysis sample (N = 159) of self-selected participants representing both agricultural and non-agricultural occupations, 125 responded to the Resource Use and Effect survey component that evaluated the effects of 22 resources on post-disaster stress. Additional qualitative questions provided further data. RESULTS: The most frequently used resources post-disaster were found to be talking about the event (98.3%), friends and neighbors (97.6%) and family (97.6%). Using a derived Impact per Use score, groups from the neighboring community and personal faith activities most often reduced stress. Resources that increased stress were identified as FEMA or other government organizations; repairing, replacing, or rebuilding property; and following news or social media. Participants reported help from their community (35.8%) or things they did for themselves (31.2%) most effectively decreased disaster stress. Family, friends, faith, neighbors, and community were top choices to decrease stress in a future disaster. CONCLUSION: Rural residents identified available resources for reducing disaster stress, but some common post-disaster activities were found to increase stress. Rural emergency management should adapt response and recovery plans and activities to leverage readily accessible people, groups, and activities to mitigate negative mental and emotional health effects.

The Use of Injury and Fatality Narratives to Convey Agricultural Safety and Health Messages and to Develop Effective Resources Through Collaborative, Multi-Disciplinary Approaches (Tell a Story, Save a Life).

OBJECTIVE: Storytelling engages audiences, passes down traditions and history, educates, and helps people understand and interpret their environment. Many of those who work in agriculture have been part of the storytelling tradition since childhood. Research has demonstrated the emotional impact of personal stories and how prevention information is conveyed effectively "farmer to farmer" through this method of communication. METHODS: Since 2016, the Telling the Story Project has provided a space for those directly or indirectly involved in an agricultural incident to share their story and unique perspectives on how similar incidents can be avoided. RESULTS: This collaborative project, developed between the National Institute for Occupational Safety and Health (NIOSH) Agriculture Safety Centers, has resulted in 11 stories on a dedicated website, safety and health resources, and educational guides. The stories and educational guidelines have been marketed extensively through traditional and social media sources, employed in safety training, and embraced by educators in agricultural programs. The website has provided a national and international reach with more than 35,000 visits. CONCLUSION: Qualitative thematic analysis of the stories provided data on the circumstances leading up to each incident, valuable information on how the storytellers interpreted the aftermath, and a novel perspective on how safety professionals can create messaging that will resonate with the farming community.

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.

Rural and Agricultural Natural Disaster Stress and Recovery: A Comparison.

This study used a novel survey instrument to evaluate the hypothesis that U.S. agricultural producers have significantly different stress and recovery experiences following acute-onset natural disaster compared to their non-agricultural counterparts. Participants were recruited through local organizations and targeted email and social media in communities in Arkansas and Nebraska that had experienced violent tornadoes in 2014 and/or severe flooding in 2019. The survey instrument incorporated the Brief Resilience Scale, the Revised Impact of Event Scale referencing two time points, the Posttraumatic Growth Inventory-Short Form, and original questions. Demographic, exposure, stress, and recovery measures were analyzed in SAS with Chi-square tests, t-tests, Wilcoxon tests, and multiple linear regression modeling to test for differences between agricultural and non-agricultural groups in resilience, event exposure, stress symptoms in the week after the event, stress symptoms in the month before the survey, a calculated recovery ratio, and posttraumatic growth. Analysis sample (N = 159) contained 20.8% agricultural occupation, 71.1% female, and 49.1% over age 55. No significant differences were found between agricultural and non-agricultural participants when comparing resilience, stress, or recovery ratio measures. Unadjusted posttraumatic growth score was significantly lower in the agriculture group (P = .02), and an occupation group by sex interaction was significantly associated with posttraumatic growth score (P = .02) when controlled for number of initial posttraumatic stress symptoms in the adjusted model, with agricultural women showing lower growth. Overall, there was no evidence of significant difference in disaster stress and recovery between agricultural and rural, non-agricultural groups in this study. There was some evidence that women in agriculture may have lower levels of recovery. Data indicated that rural residents continue to experience posttraumatic-type symptoms up to 8 years beyond the acute-onset natural disaster events. Communities should include strategies to support mental and emotional health in their preparedness, response, and recovery plans with intentional inclusion of agricultural populations.

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.

Development of an Educational YouTube Channel: A Collaboration between U.S. Agricultural Safety and Health Centers.

Objectives: The National Institute for Occupational Safety and Health (NIOSH) Agriculture, Forestry, and Fishing Centers (AFF Centers) collaborated to initiate a joint YouTube channel in order to raise awareness of agricultural, forestry, and fishing occupational hazards; provide information to prevent AFF injuries and illnesses; increase the visibility and influence of the AFF Centers; and establish a collaborative model that can be replicated by other organizations.Methods: The collaborators sought to produce a structured channel with high scientific standards. Policies, procedures, and a standard review process were established. Representatives from the AFF Centers coordinated the review process and the procedures by which videos were uploaded to the site. A marketing plan was created including a press release and ideas to promote new videos. Promotions are targeted to agricultural cooperative extension agents, educators, producers, owners, operators, first responders, families and community organizations. Viewership was tracked using YouTube metrics.Results: The site was launched in November 2013. Over a 6-year period, the channel grew from 48 videos to 125 videos with over 10,500 cumulative watch time hours. The channel is promoted by the AFF Centers through email, social media, conference presentations and outreach exhibits. The channel is also publicized during coordinated national outreach events.Conclusion: Each AFF Center benefitted from increased exposure of their content and the collaboration provided an opportunity to achieve labor efficiencies. YouTube metrics demonstrated that coordinated marketing increases views, watch time and subscriptions. In addition, the success of the channel communicates the benefits to collaboration among organizations with common missions.

Virtual Outreach: Using Social Media to Reach Spanish-speaking Agricultural Workers during the COVID-19 Pandemic.

Face-to-face outreach and in-person training have traditionally been key strategies in reaching agricultural producers, workers, and communities with safety and health information, but the COVID-19 pandemic has forced outreach educators to be creative and find alternative ways to reach, communicate, and share such information. In this commentary, we describe our use of social media to reach Latino/a cattle feedyard workers with COVID-19 related information. As a result of our effort, we reached over 54,000 people and demonstrated there is an audience for Spanish-language agricultural safety and health information. Social media can be a cost-effective method for virtual outreach in this new normal. We should look at this time as an opportunity to learn more about how our stakeholders obtain information and about how best we can connect with them. Although our outreach methods may be changing, our goal is not - we will continue to work to improve the safety and health of those who work in agriculture.

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.