I present a simulation model that should be a useful tool for risk assessment of the impact of insecticide inhibitors of cholinesterase (ChE) applied in irrigated agricultural fields on non-target wildlife. I developed the model as a compartment model based on difference equations (Δt = 1 hour) and programmed with Stella® VII software. Conceptually the model is compartmentalized into six submodels describing the dynamics of (1) insecticide application, (2) insecticide movement into floodable soil, (3) irrigation and rain, (4) insecticide dissolution in water, (5) foraging and insecticide intake from water, and (6) ChE inhibition and recovery. To demonstrate application of the model, I simulate historical, current, and “worst-case” scenarios, that examined the impact of ChE-inhibiting insecticides on white-winged doves (WWDO - Zenaida asiatica) in the Lower Rio Grande Valley of Texas (LRGV), USA. To my knowledge, there are no field data verifying that the cause of ChE deprivation in WWDO is due to the ingestion of ChE-inhibiting insecticide residues dissolved in drinking water. I parameterized the model to represent a system composed of fields of cotton, sorghum, corn, citrus, and brushland that encompasses the activity range of a WWDO in the LRGV. I simulated situations representing the typical scenario of WWDO using irrigated crop fields in the absence and in the presence of rain. I also simulated “worst case” scenarios in which methyl parathion was applied at high rates and high frequency. Based on results of the simulations, I conclude that it is unlikely that WWDO are seriously exposed to ChE-inhibiting insecticides by drinking contaminated water. Only in rare cases, for example, when a rain event occurs just after the application of insecticides, are levels of ChE inhibition likely to approach diagnostic levels (20 %). The present simulation model should be a useful tool to predict the effect of ChE-inhibiting insecticides on the ChE activity of different species that drink contaminated water from irrigated agricultural fields. It should be particularly useful in identifying specific situations in which the juxtaposition of environmental conditions and management schemes could result in a high risk to non-target wildlife.

Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage. Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low. Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. / Canada Research Chair Program, OMAFRA,

Chemical warfare agents including organophosphorus nerve agents (NA) continue to be a significant threat to both military and civilian populations. The current Canadian Armed Forces (CAF) treatment of NA poisoning includes administration of the oxime HI-6 (used to reactivate inhibited acetylcholinesterase) in combination with atropine contained in an autoinjector, with a benzodiazepine also being administered. Two salts of HI-6 are currently available: HI-6 2Cl (1-[[[4-(Aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyiminio)methyl] pyridinium dichloride (MW 376.22 g/mol) and HI-6 DMS (1-[[[4-(Aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyiminio)methyl] pyridinium dimethanesulfonate (MW 477.49 g/mol). Currently HI-6 is available to the Canadian Armed Forces under a special access program. In order to attain licensure of HI-6 numerous studies must be carried out in animal models to ensure its safety (tolerability and toxicity), efficacy and pharmacokinetics prior to human clinical trials. The present experiment aimed to determine and compare the pharmacokinetic parameters of HI-6 in two animal models under various conditions including: direct comparison of salts (HI-6 2Cl compared to HI-6 DMS), comparison of routes of administration (intramuscular compared to intravenous), comparison of effect of anaesthetic, comparison of different concentrations of HI-6, determination of the effect of atropine sulphate co-administration and evaluation of calculated pharmacokinetic parameters when infusing HI-6. Serial plasma samples were collected and HI-6 levels were quantified using a HPLC method. In all studies a significant difference was reported for absorption/distribution parameters when comparing salts. Additionally the absorption/distribution parameters when comparing routes of administration were significantly different however all other parameters were similar. Significant differences in calculated parameters were reported when examining the effect of anaesthetic on the pharmacokinetics of HI-6. Similar to previous ascending dose studies, differences were reported for the absorption/distribution kinetics. Co-administration of HI-6 with atropine sulphate did not have significant effect on the pharmacokinetics of HI-6. The determined pharmacokinetic values for both salts were accurate for the determination of an infusion rate to reach and maintain a target plasma concentration. Finally the calculated animal model pharmacokinetic data was compared to previously published human clinical trial data and the calculated pharmacokinetic values were found to be similar.

Background: Children exposed to pesticides are susceptible for neurodevelopmental disruption. Data from developing countries are scarce. Aim: Assessing long-term and recent pesticide exposure in Nicaraguan children in relation to parental pesticide use and examining potential associated neurobehavioral effects. Methods: In the first study, pre- and post-spraying urinary residues of the chlorpyrifos metabolite TCPY and diazinon metabolite IMPY were measured among 7 subsistence farmers and 10 plantation workers, and in one child per worker. In the second study, for 110 children in an agricultural village and 22 in a non-agricultural village, aged 7-9, parental pesticide use was assessed by hours of spraying and kilograms of active ingredients during pre-and-postnatal time windows, as proxies for children’s long term pesticide exposures. Urinary TCPY, 3-PBA (pyrethroid metabolite), and 2,4-D were determined in 211 samples of 74 children of the agricultural village. IQ components and total IQ (WISC-IV) were evaluated in all agricultural village children. Behavior was evaluated with the Conners’ Teacher Rating Scale-Revised: Short. Multivariate linear regression models assessed associations between long-term and recent exposure to organophosphates and pyrethroids and cognitive and behavioral scales. Results: In study 1, post-spraying urinary levels of pesticide metabolites of subsistence farmers and their children were highly correlated (r=0.85), but not those of plantation workers and their children. In study 2, a wide range of exposures was reported by parents for all pesticides and time windows. The median urinary TCPY (3.7 μg/g creatinine), 3-PBA (2.8), and 2,4-D (0.9) were comparable to other studies for TCPY and 3-PBA but high for 2,4-D. Maximum levels were the highest reported for all compounds. Prenatal use of organophosphates affected working memory, and methamidophos also verbal comprehension and total IQ. Urinary TCPY was associated with poorer working memory. Organophosphate exposures were not associated with children’s behavior. Pyrethroid exposure during the first year of life associated with poorer perceptual reasoning and behavior, and urinary 3-PBA with a number of cognitive functions and ADHD in girls but not in boys. Conclusion: Nicaraguan children in poor agricultural areas are highly exposed to pesticides, which is influenced by parental pesticide use in subsistence farms. Organophosphate and pyrethroid exposures adversely affect their neurobehavioral development.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ação inibitória dos organofosforados sobre as esterases, por ser específica, pode ser empregada como um eficiente biomarcador da exposição de seres vivos aos organofosforados. A inibição da acetilcolinesterase (AChE; EC 3.1.1.7) provoca acúmulo do neurotransmissor acetilcolina nas fendas sinápticas colinérgicas, o que pode resultar na morte do indivíduo. Outra atividade também afetada por organofosforados é a da enzima carboxilesterase (CarbE; EC 3.1.1.1). CarbE estão envolvidas na fase I da biotransformação de xenobióticos e atuam como captadoras (scavengers) de organofosfatos, incluindo os formados pela biotransformação dos organofosforados. As CarbE estudadas até hoje se ligam com maior velocidade aos organofosfatos do que as colinesterases. Por isto se admite que CarbE possam diminuir, por captação estequiométrica, a ligação tóxica de moléculas de organofosfatos às acetilcolinesterases das sinapses colinérgicas e das placas motoras dos músculos. Experimentos realizados em nosso laboratório mostraram que a atividade da CarbE está aproximadamente 50% menor no soro e no fígado de pacus submetidos à hipoxia. Por causa disso, em razão de uma possível diminuição da capacidade captadora da CarbE, decidimos verificar se o pacu em hipoxia seria mais sensível aos agrotóxicos organofosforados. Para este propósito foram colocados seis pacus divididos em dois tanques. No primeiro tanque, os animais foram submetidos a 24 horas de hipoxia seguidos por mais 4 horas de exposição ao organofosforado metilparation em duas concentrações diferentes (0,02 ou 0,01 mg / L). No segundo tanque os animais permaneceram em normoxia durante o mesmo período de 24 horas e depois foram expostos ao metilparation como no primeiro tanque. As atividades da AChE ensaiada com acetiltiocolina, a da butirilcolinesterase (BChE) ensaiada com butiriltiocolina e a da CarbE ensaiada com p-nitrofenilacetato foram avaliadas no soro, fígado, cérebro, músculo e coração dos pacus. Houve redução de aproximadamente 35% da atividade de CarbE no soro dos pacus submetidos a 24 horas de hipoxia. Uma queda de 85% na atividade de CarbE do soro foi observada nos animais que sofreram hipoxia e subsequente exposição a 0,02 mg de metilparation por litro. Com metilparation a 0,01 mg/L a diminuição observada foi de 48,2%. No músculo dos pacus expostos a 0,02 mg/L, as atividades de AChE e BChE cairam pela metade quando os mesmos foram submetidos à hipoxia quando comparados a animais que permaneceram em normoxia. Nos diversos tecidos dos pacus expostos a 0,01 mg/L de metilparation não observamos diferenças significativas nas atividades de AChE, BChE ou CarbE. Concluímos que a duplicação da concentração de metilparation de 0,01 para 0,02 mg/L levou à atividade residual de CarbE do soro de 51,8% para 15%. A ausência de mudanças nas atividades das esterases dos tecidos de animais expostos a 0,01 mg/L entre os grupos hipoxia e normoxia deve ter ocorrido porque a concentração de organofosforado não foi suficiente para superar a primeira barreira de proteção das esterases séricas e atingir os tecidos. Mas, no experimento com 0,02 mg/L de metilparation, as inibições de AChE e de BChE no músculo dos animais em hipoxia podem ser explicadas pela diminuição da atividade de CarbE do soro dos pacus. / The inhibitory action of organophosphates on cholinesterases is a specific phenomenon, and it is used as a biomarker of exposure to organophosphate in live animals. Inhibition of cholinesterases results in the accumulation of acetylcholine in the synaptic clefts of cholinergic synapses, which can lead to death. Another enzyme which is affected by organophosphates is the carboxylesterase (CarbE). CarbE are involved in phase I of xenobiotic biotransformation and can act as a "scavenger" of organophosphates. The CarbE studied to the present date can bond to organophosphate with greater speed than other cholinesterases. Therefore, CarbE might decrease by stoichiometric uptake the binding of organophosphate at the acetylcholinesterase at cholinergic synapses and motor end plates of muscles. Experiments conducted in our laboratory showed that the CarbE activity is about 50% lower in serum and liver of pacu subjected to hypoxia. Due to the detoxifying role of CarbE we verified whether pacu, under conditions of oxidative stress, would be more sensitive to organophosphate pesticides. Nine pacus were divided into two tanks. In the first tank the animals underwent 24 hours of hypoxia followed by a further 4 hours of exposure to methylparathion in two different concentrations (0,02 or 0,01 mg/L). Thus, in the second tank the animals remained in normoxia for 24 hours and were subsequently exposed to methylparathion for 4 hours. The activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and CarbE were tested in serum, liver, brain, muscle and heart. We found a decrease of approximately 35% of CarbE activity in serum of the animals subjected to 24 hours of hypoxia. A fall of 85% in CarbE activity was detected in the serum of animals that suffered hypoxia and subsequent exposure to 0,02 mg/L, yet in animals exposed to 0,01 mg/L there is a decrease of 48,2%. In muscle the activities of AChE and BChE declined by half in animals submitted to hypoxia compared with animals in normoxia when exposed to 0,02 mg/L. In the tissues of pacus exposed to 0,01 mg/L of methylparathion we did not observe significant differences in the activities of AChE, BChE or CarbE. We concluded that doubling the concentration of methylparathion from 0,01 to 0,02 mg/L causes CarbE activity in the serum to decrease from 51,8% to 15%. The absence of changes in activities in the tissues of animals exposed to 0,01 mg/L between the groups hypoxia and normoxia may have occurred because the concentration of methylparathion was not sufficient to break the first enzymes barrier of protection in serum. In the experiment with 0,02 mg/L of methylparathion the activities of AChE and BChE in the muscle were most inhibited in the hypoxia group than the normoxia group. This phenomenon can be explained by the decrease in the CarbE activity in the serum of animals under hypoxia.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ação inibitória dos organofosforados sobre as esterases, por ser específica, pode ser empregada como um eficiente biomarcador da exposição de seres vivos aos organofosforados. A inibição da acetilcolinesterase (AChE; EC 3.1.1.7) provoca acúmulo do neurotransmissor acetilcolina nas fendas sinápticas colinérgicas, o que pode resultar na morte do indivíduo. Outra atividade também afetada por organofosforados é a da enzima carboxilesterase (CarbE; EC 3.1.1.1). CarbE estão envolvidas na fase I da biotransformação de xenobióticos e atuam como captadoras (scavengers) de organofosfatos, incluindo os formados pela biotransformação dos organofosforados. As CarbE estudadas até hoje se ligam com maior velocidade aos organofosfatos do que as colinesterases. Por isto se admite que CarbE possam diminuir, por captação estequiométrica, a ligação tóxica de moléculas de organofosfatos às acetilcolinesterases das sinapses colinérgicas e das placas motoras dos músculos. Experimentos realizados em nosso laboratório mostraram que a atividade da CarbE está aproximadamente 50% menor no soro e no fígado de pacus submetidos à hipoxia. Por causa disso, em razão de uma possível diminuição da capacidade captadora da CarbE, decidimos verificar se o pacu em hipoxia seria mais sensível aos agrotóxicos organofosforados. Para este propósito foram colocados seis pacus divididos em dois tanques. No primeiro tanque, os animais foram submetidos a 24 horas de hipoxia seguidos por mais 4 horas de exposição ao organofosforado metilparation em duas concentrações diferentes (0,02 ou 0,01 mg / L). No segundo tanque os animais permaneceram em normoxia durante o mesmo período de 24 horas e depois foram expostos ao metilparation como no primeiro tanque. As atividades da AChE ensaiada com acetiltiocolina, a da butirilcolinesterase (BChE) ensaiada com butiriltiocolina e a da CarbE ensaiada com p-nitrofenilacetato foram avaliadas no soro, fígado, cérebro, músculo e coração dos pacus. Houve redução de aproximadamente 35% da atividade de CarbE no soro dos pacus submetidos a 24 horas de hipoxia. Uma queda de 85% na atividade de CarbE do soro foi observada nos animais que sofreram hipoxia e subsequente exposição a 0,02 mg de metilparation por litro. Com metilparation a 0,01 mg/L a diminuição observada foi de 48,2%. No músculo dos pacus expostos a 0,02 mg/L, as atividades de AChE e BChE cairam pela metade quando os mesmos foram submetidos à hipoxia quando comparados a animais que permaneceram em normoxia. Nos diversos tecidos dos pacus expostos a 0,01 mg/L de metilparation não observamos diferenças significativas nas atividades de AChE, BChE ou CarbE. Concluímos que a duplicação da concentração de metilparation de 0,01 para 0,02 mg/L levou à atividade residual de CarbE do soro de 51,8% para 15%. A ausência de mudanças nas atividades das esterases dos tecidos de animais expostos a 0,01 mg/L entre os grupos hipoxia e normoxia deve ter ocorrido porque a concentração de organofosforado não foi suficiente para superar a primeira barreira de proteção das esterases séricas e atingir os tecidos. Mas, no experimento com 0,02 mg/L de metilparation, as inibições de AChE e de BChE no músculo dos animais em hipoxia podem ser explicadas pela diminuição da atividade de CarbE do soro dos pacus. / The inhibitory action of organophosphates on cholinesterases is a specific phenomenon, and it is used as a biomarker of exposure to organophosphate in live animals. Inhibition of cholinesterases results in the accumulation of acetylcholine in the synaptic clefts of cholinergic synapses, which can lead to death. Another enzyme which is affected by organophosphates is the carboxylesterase (CarbE). CarbE are involved in phase I of xenobiotic biotransformation and can act as a "scavenger" of organophosphates. The CarbE studied to the present date can bond to organophosphate with greater speed than other cholinesterases. Therefore, CarbE might decrease by stoichiometric uptake the binding of organophosphate at the acetylcholinesterase at cholinergic synapses and motor end plates of muscles. Experiments conducted in our laboratory showed that the CarbE activity is about 50% lower in serum and liver of pacu subjected to hypoxia. Due to the detoxifying role of CarbE we verified whether pacu, under conditions of oxidative stress, would be more sensitive to organophosphate pesticides. Nine pacus were divided into two tanks. In the first tank the animals underwent 24 hours of hypoxia followed by a further 4 hours of exposure to methylparathion in two different concentrations (0,02 or 0,01 mg/L). Thus, in the second tank the animals remained in normoxia for 24 hours and were subsequently exposed to methylparathion for 4 hours. The activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and CarbE were tested in serum, liver, brain, muscle and heart. We found a decrease of approximately 35% of CarbE activity in serum of the animals subjected to 24 hours of hypoxia. A fall of 85% in CarbE activity was detected in the serum of animals that suffered hypoxia and subsequent exposure to 0,02 mg/L, yet in animals exposed to 0,01 mg/L there is a decrease of 48,2%. In muscle the activities of AChE and BChE declined by half in animals submitted to hypoxia compared with animals in normoxia when exposed to 0,02 mg/L. In the tissues of pacus exposed to 0,01 mg/L of methylparathion we did not observe significant differences in the activities of AChE, BChE or CarbE. We concluded that doubling the concentration of methylparathion from 0,01 to 0,02 mg/L causes CarbE activity in the serum to decrease from 51,8% to 15%. The absence of changes in activities in the tissues of animals exposed to 0,01 mg/L between the groups hypoxia and normoxia may have occurred because the concentration of methylparathion was not sufficient to break the first enzymes barrier of protection in serum. In the experiment with 0,02 mg/L of methylparathion the activities of AChE and BChE in the muscle were most inhibited in the hypoxia group than the normoxia group. This phenomenon can be explained by the decrease in the CarbE activity in the serum of animals under hypoxia.

No description available.

Organophosphorus compounds (OPs), both nerve agents and insecticides, may induce damage leading to long-term neurological deficits. Oxime 20, the lead compound from a platform of substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937), is an oxime that reactivates OP-inhibited acetylcholinesterase (AChE) and has displayed neuroprotection within the brain of rats in in vivo tests where the currently approved treatment, pralidoxime (2-PAM), has not. This research investigated the neuroprotective effect of novel oxime therapy. Gene expression in rat piriform cortex was analyzed to determine if neurotoxicity, stress, and synaptic plasticity-associated genes were down-regulated by Oxime 20 following a sublethal dose of sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). Significant therapeutic changes from the NIMP+Oxime20 group relate to cell viability and attenuation of neuroinflammation, while networks formed by Ingenuity Pathway Analysis (IPA) emphasize neuroinflammatory and cell death pathways. Neuropathology cell biomarkers glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN), ionized calcium-binding adapter molecule 1 (IBA-1), and cyclooxygenase-2 (COX-2) were measured immunohistochemically in rat CA1 region of the hippocampus following a lethal dose of NIMP plus Oxime 20 or 2-PAM therapy. For GFAP and IBA-1, an expected trend of NIMP activation and Oxime 20 recovery was seen and was more pronounced in the apex CA1 region. Loss of neurons were observed most often in the NIMP experimental group. A platform of novel oximes was selected based on percent brain AChE reactivation (24–35% and 10–17%) following exposure of rats to NIMP to determine if interactions between ATP-binding cassette efflux transporters of the blood-brain barrier, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), contribute to oxime in vivo efficacy. In vitro human P-gp and BCRP membrane assays determined activation and inhibition measurements of total ATPase activity. Human P-gp transport was variable among oximes. Oxime 55 was the only oxime transported by BCRP. Therefore, the lower efficacy of 10-17% in the reactivation oxime group was not due to greater P-gp or BCRP efflux. The explanation might lie with different transporter or mechanism. These tests support our laboratory testing OP inhibited AChE reactivators to understand their efficacy and evaluate neuroprotection from damage caused by OP exposure

Background: The relationships between both obesity and diabetes and the exposure to insecticides, specifically organophosphates and pyrethroids, in the adult Canadian population are not well-understood. Methods: Urinary concentrations of 4 organophosphate metabolites (DEP, DEPT, DMP, and DMPT) and of 4 pyrethroid metabolites (cis-DBCA, cis-DCCA, 3-PBA, and trans-DCCA) were measured for 1,147 adult Canadians aged between 18-79 years old. The geometric means and medians of both creatinine-adjusted and unadjusted urinary insecticide metabolites were estimated. Multiple linear regression and logistic regression analyses were employed to examine the associations between the insecticide metabolite concentrations and obesity and diabetes measures. Results: Both insecticides had detectable levels in over 70% of CHMS respondents. Most metabolites demonstrated a negative significant relationship between their urinary concentrations and BMI as well as waist circumference. No significant relationship was found in regard to HbA1c levels or for diabetes. Conclusion: Organophosphate and pyrethroid metabolites were detected in more than 70% of Canadian adults. Our data showed no evidence that organophosphate and pyrethroid exposures increase the risks of obesity and diabetes in adults. These results should be interpreted with caution as diet may play a large confounding role in the relationships of study.

Chlorpyrifos (CPF) is one of the most widely used organophosphorus insecticides (OPs). The developmental exposure to low levels of CPF results in the inhibition of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) and in altered emotional behavior (increased social play) without affecting the acetylcholinesterase, the canonical target of OPs. However, the molecular mechanisms responsible for this increased social play are not known. In this study, male rat pups were exposed orally to either corn oil, 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845 (PF; a specific inhibitor of FAAH) daily from postnatal day 10 (PND10) - PND16. This dosage of CPF does not alter brain cholinergic activity but inhibits FAAH. Once these rats reached adolescence (PND38), they were divided into two cohorts and each cohort contained all treatments. One cohort underwent social behavior testing and the other cohort remained naïve to behavioral testing. Following testing, the amygdala was collected from each cohort and protein expression was determined using a labelree shotgun proteomic approach. The obtained differentially expressed proteins from the different cohorts were analyzed by DAVID and Ingenuity Pathway Analysis software. Comparison of control non-behavior and control behavior rats suggests that social play altered the systems involved in the regulation of reward such as the opioid, dopaminergic, and serotonergic systems. These data also suggest that synaptic levels of GABA and glutamate increased during play. Comparison of non-behavior control and treated rats suggests that FAAH inhibition resulting from developmental exposure to CPF and PF persistently affects glutamatergic and GABAergic signaling. These data also suggest that there is a similar pattern of protein expression between CPF and PF. Comparison of the data from the behavioral groups of rats suggests that alterations in glutamatergic and GABAergic signaling and improper activation of opioid signaling could be responsible for the increased social play behavior. These alterations in the neurotransmitter signaling were observed in both CPF and PF treated rats. Overall, the results suggest that FAAH inhibition by either CPF or PF leads to alterations in opioid, glutamatergic, and GABAergic signaling that could be responsible for increased levels of social play.