Factors Associated with Personal Protective Equipment Usage Rates in the Central States: Generalized Multilevel Multinomial Models.

OBJECTIVES: This study examined the factors affecting the use of personal protective equipment (PPE) among US agriculture producers, specifically focusing on chemical, respiratory, and hearing protection. The primary goals were to identify demographic and farm-related factors linked to lower PPE usage and to explore the associations between PPE use and self-reported injuries or diseases in this occupational context. METHODS: We developed generalized multilevel multinomial logistic regressions for the agricultural safety and health survey data from 2018 and 2020. Our models aimed to explore associations between PPE use, covariates, and their impact on skin diseases, hearing loss, and respiratory diseases. The hierarchical structure of the data was accommodated by designating the survey year as the level-3 variable and the state of residence as the level-2 variable, further delineating the nested structure of the respondents. We proposed using Adaptive Quadrature methods to approximate marginal maximum likelihood function, along with Gauss-Hermite quadrature weights when estimating fixed-effects and random effects in the proposed models. Missing data in this hierarchical structure were addressed through a multiple imputation method. RESULTS: Our findings revealed older age is associated with lower PPE usage across all types. Males exhibited higher PPE use, particularly for chemical (OR: 1.26, 95% CI: 1.08, 1.47) and respiratory protection (OR: 1.33, 95% CI: 1.18, 1.52). Producers on grain-only farms showed increased chemical PPE use (OR: 1.26, 95% CI: 1.10, 1.43) compared to those on livestock-only farms. Additionally, individuals spending 0%-24% of their worktime on the farm/ranch demonstrated lower PPE use than those who spent more time engaged in farm-related activities. CONCLUSION: The study underscores the importance of addressing low PPE usage among agriculture producers. Interventions tailored to specific groups, such as female producers, livestock-only farms, older-aged producers, and part-time producers, can effectively improve PPE use. By identifying these high-risk groups, interventions can be better adapted and targeted to enhance the adoption of PPE, subsequently reducing the risk of health hazards in the agriculture sector.

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.

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.

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.

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.

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.

Assessment of Tribal Bison Worker Hazards Using Trusted Research Facilitators.

OBJECTIVES: Agriculture is one of the most hazardous industries in the United States. Within agriculture, livestock handling is particularly dangerous. While injury and fatality rates for bison handlers have not been reported, workers in many of the newly established tribal bison herds have limited safety training and animal handling experience, making this a vulnerable workforce. Veterinarians and herd managers, working with tribal bison herds, recognized the need for improvement in the working environment and for worker safety training. In response, partnerships were established and a pilot project was developed in order to characterize risks and hazards associated with bison handling under contemporary reservation field conditions. Individuals and organizations working as change agents included veterinarians at the University of Nebraska - Lincoln School of Veterinary Medicine, a tribal advocacy organization, the Intertribal Buffalo Council and researchers at the Central States Center for Agricultural Safety and Health at the University of Nebraska Medical Center. METHODS: This is a mixed-methods study and data were gathered through closed and open-ended questions pertaining to bison worker safety hazards. A veterinarian gathered data through observational safety audits at bison herding locations. American Indian bison herd managers completed surveys using a convenience sampling method. RESULTS: Findings indicate that the most common worker safety risks are associated with the use of high-stress handling methods and substandard facilities and equipment. Adverse environmental conditions also contribute to worker health risks. Most common causes of injuries included those caused by equipment and tools, adverse weather, and direct contact with animals. CONCLUSION: This collaborative research study contributes to a better understanding of hazards faced by tribal bison workers. Findings from this research influenced the ITBC in their decision to add worker safety and health training to the agenda of their yearly conference and promote tailgate trainings for their workers. UNL veterinarians have taken the lessons learned from this research and provided safety and health information to mangers of other non-tribal bison herds. This research partnership will continue with a 5-year research study focusing on best management practices and establishing training to improve the health and safety bison workers.

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.

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.

Social marketing campaign promoting the use of respiratory protection devices among farmers.

This article describes the formal use of marketing concepts in a systematic approach to influence farmers to voluntarily increase respiratory protective device (RPD) use. The planning process for the project incorporated six key decision or action points, each informed by formative research or health behavior theory. The planning process included developing behavior change strategies based on a 4P model (product, price, place, and promotion). The resulting campaign elements included print and e-mail messages that leveraged motivators related to family and health in order to increase farmers' knowledge about the risks of exposure to dusty environments, four instructional videos related to the primary barriers identified in using RPDs, and a brightly colored storage bag to address barriers to using RPDs related to mask storage. Campaign implementation included a series of e-mails using a bulk e-mail subscription service, use of social media in the form of posting instructional videos on a YouTube channel, and in-person interactions with members of the target audience at farm shows throughout the Central States Center for Agricultural Safety and Health seven-state region. Evaluation of the e-mail campaigns indicated increased knowledge about RPD use and intent to use RPDs in dusty conditions. YouTube analytic data indicated continuing exposure of the instructional videos beyond the life of the campaign. The project demonstrates the efficacy of a planning process that incorporates formative research and clear decision points throughout. This process could be used to plan health behavior change interventions to address other agriculture-related health and safety issues.

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.

Mice treated with a nontoxic dose of chlorpyrifos oxon have diethoxyphosphotyrosine labeled proteins in blood up to 4 days post exposure, detected by mass spectrometry.

Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is an established biomarker of exposure to organophosphorus poisons (OP). Inhibition of activity is due to covalent binding of the OP to the active site serine. Mass spectrometry has made it possible to monitor OP exposure by analyzing OP adducts on tyrosine in proteins that have no active site serine. Our goal was to test the hypothesis that OP-tyrosine may serve as a biomarker of OP exposure in mice. A MALDI-TOF mass spectrometry strategy to analyze diethoxyphosphate-tyrosine of m/z 318 was developed. The adduct was synthesized by incubating l-tyrosine with chlorpyrifos oxon at pH 8.1. The adduct eluted from a reverse phase HPLC column with 22-23% acetonitrile. The fragmentation spectrum of the m/z 318 precursor ion confirmed its identity as diethoxyphosphate-tyrosine. Diethoxyphosphate-tyrosine was isolated from chlorpyrifos oxon treated mouse albumin after digesting the protein with pronase. Mice (n=3 per group) were treated with a nontoxic dose of chlorpyrifos oxon (3 mg/kg) and a toxic dose (10 mg/kg transdermally). The pronase digested plasma yielded diethoxyphosphate-tyrosine up to 120 h after treatment with 3 mg/kg chlorpyrifos oxon and up to 144 h after 10 mg/kg. In contrast plasma AChE activity returned to normal after 24-72 h. In conclusion MALDI-TOF mass spectrometry can be used to diagnose exposure to chlorpyrifos oxon days after AChE inhibition assays are uninformative.

Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran.

Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit 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.

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.

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.

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.

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.