Acute Organophosphate Exposure Research Articles

Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

Developing a Prenatal Acute Exposure and Medical Countermeasure Evaluation Model for Organophosphate Compounds in a Genetically Modified Mouse

Organophosphate (OPs) compounds make up the most common class of pesticides as well as chemical warfare nerve agents (CWNA), all of which covalently inhibit the active site of acetylcholinesterase (AChE), resulting in intoxication via a cholinergic crisis. While the biochemical mechanism of action resulting in acute intoxication of an exposed individual is well understood, less is known about the mechanism of OP intoxication via other exposure routes, particularly prenatal exposure. The clinical effects of non‐acute prenatal OP exposure have been previously studied primarily in cases involving diet and agricultural labor, while case studies documenting the effects on conception of acute exposure during chemical warfare attacks (Japan 1995, Syria 2013 and 2017) have also been published. Such exposure events were linked to a range of serious neurodevelopmental anomalies in children, but the underlying mechanism of action as well as the potential benefits of medical countermeasures have not been clearly elucidated. A robust human‐relevant animal model to test both is therefore essential for evaluating current and future OP countermeasures. The present study used a novel humanized mouse strain expressing human AChE and an inactivated native serum carboxylesterase to create a prenatal acute exposure and emergency medical countermeasure evaluation model. Pregnant mice were acutely exposed to an OP at a critical point in cholinergic development, generally equivalent to the first trimester in humans. Dams were challenged with either sarin or the pesticide paraoxon, followed by either a saline vehicle control or 2‐PAM and an atropine/midazolam cocktail, the currently fielded emergency treatment for OP intoxication. Mice were allowed to carry pups to term, and upon reaching maturity, cortex, cerebellum, bone marrow and gonads were collected from offspring in each exposure group. Phenotypical and histopathological evaluation of tissues and other factors in dams and offspring were evaluated. Gene expression analyses conducted for each tissue generated a detailed profile associated with each OP exposure/treatment group, providing robust criteria for model development. The resulting data demonstrated a significant beneficial impact of medical countermeasures for both sarin and paraoxon exposures, significantly reducing the amount and the scope of gene expression changes induced by OP exposure. This model provides insight into the developmental pathologies resulting from prenatal acute OP exposure as well as serves as an evaluation model for future treatments.

The effect of acute organophosphate intoxication on female rat hippocampus cornu ammonis region pyramidal neuron numbers, biochemistry and morphology

BackgroundThe most commonly used insecticides and pesticides worldwide are organophosphate compounds, chemicals that irreversibly inhibit the cholinesterase enzyme. Acute intoxication with cholinesterase inhibitors is known to cause permanent effects on both the human and rat brains. AimTo investigate the effect of acute organophosphate intoxication on hippocampus morphology, biochemistry, and pyramidal neuron numbers in female rats. MethodsTwenty-one rats were randomly divided into three groups. The control group received normal nutrition and underwent no procedures. The sham group received intraperitoneal physiological serum, while the experimental group received intraperitoneal 0.8 g/kg fenthion. Rats were sacrificed 24 h after these procedures. The brains were removed and divided in two halves medially, with one side being kept in 10% neutral formalin. After fixation procedures, tissues were embedded in blocks, sliced, and stained. A neuron count was then performed for the hippocampus. The other hippocampus was homogenized and used for biochemical procedures. ResultsHippocampus sections from rats in the experimental group exhibited swelling and loss of shape in pyramidal cells, while no changes were observed in the control or sham groups. The number of neurons in the experimental group was lower than in the control and sham groups. Biochemical analysis revealed higher MDA and GSH values in the experimental group compared to the control and sham groups. ConclusionOur results show increased apoptotic neurodegeneration of cells in the cornu ammonis region of the hippocampus and changes in biochemical values in rats with acute organophosphate exposure.

Dichlorvos exposure to the Kölliker-fuse nuclei is sufficient but not necessary for OP induced apnea

Patients exposed to organophosphate (OP) compounds demonstrate a central apnea. The Kölliker-fuse nuclei (KF) are cholinergic nuclei in the brainstem involved in central respiratory control. We hypothesize that exposure of the KF is both necessary and sufficient for OP induced central apnea. We performed an animal study of acute OP exposure. Anesthetized and spontaneously breathing Wistar rats (n=24) were exposed to a lethal dose of dichlorvos using three experimental models. Experiment 1 (n=8) involved systemic OP poisoning using subcutaneous (SQ) 2,2-dichlorovinyl dimethyl phosphate (dichlorvos) at 100mg/kg or 3× LD50. Experiment 2 (n=8) involved isolated poisoning of the KF using stereotactic microinjections of dichlorvos (625μg in 50μl) into the KF. Experiment 3 (n=8) involved systemic OP poisoning with isolated protection of the KF using SQ dichlorvos (100mg/kg) and stereotactic microinjections of organophosphatase A (OpdA), an enzyme that degrades dichlorvos. Respiratory and cardiovascular parameters were recorded continuously. Animals were followed post exposure for 1h or until death. There was no difference in respiratory depression between animals with SQ dichlorvos and those with dichlorvos microinjected into the KF. Despite differences in amount of dichlorvos (100mg/kg vs. 1.8mg/kg) and method of exposure (SQ vs. CNS microinjection), 10min following dichlorvos both groups (SQ vs. microinjection respectively) demonstrated a similar percent decrease in respiratory rate (51.5 vs. 72.2), minute ventilation (49.2 vs. 68.8) and volume of expired gas (17.5 vs. 0.0). Animals with OpdA exposure to the KF during systemic OP exposure demonstrated less respiratory depression, compared to SQ dichlorvos alone (p<0.04). No animals with SQ dichlorvos survived past 25min post exposure, compared to 50% of animals with OpdA exposure to the KF. In conclusion, exposure of the KF is sufficient but not necessary for OP induced apnea. Protection of the KF during systemic OP exposure mitigates OP induced apnea.

Effect of short-term exposure to dichlorvos on rat hepatocyte: molecular and histopathological approach

Background: Organophosphate (OP) insecticides are the most widely used in both agricultural and landscape pest control. The mortality and morbidity rate of OP poisoning is high. The aim of the present study is to investigate the effect of acute organophosphate exposure on hepatocyte and to examine caspase 1 and caspase-3 gene expression, and cell apoptosis related genes as p53, Tumor Necrosis Factor-alpha, Hypoxia Inducible Factor 1-alpha expression changes in rat hepatocyte. Material and Methods: 10 adult Wistar Albino female rats weighing 250-300 g were divided into control (n=5) and experiment (n=5) groups. In experimental group, rats were treated 25 mg/kg of dichlorvos (Bayer DDVP EC 550, Bayer) in corn oil by 16 gauge oral gavage tube. In control group, rats were treated only 2.5 ml corn oil by oral gavage. After seven days, all of the rats were sacrificed by cervical dislocation under anesthesia. The liver was removed and divided into fragments. Hepatocyte density and histopathological examination were performed in fixed liver tissues. For this purpose, sections were taken and stained with hematoxylin-eosin. A part of the liver was used for gene expression analysis. Total RNA was extracted from the liver tissue using an RNA isolation reagent via manufacturer's instruction. Changes in mRNA levels, detected using semi-quantitative reverse transcription-polymerase chain reaction, were calculated as the proportion of the target gene amplification products to the amplification products of the housekeeping gene GAPDH. Results: Hepatocyte density were decreased in experimental group compared to control group (p<0.05). The histopathological changes, such as portal inflammation and picnosis were observed in liver sections of experimental group. According to molecular genetics analysis, Caspase 1, Caspase 3, and p53 gene expression were increased in liver tissue after dichlorvos treated rats compared with the control group. There were no expression changes for TNF-Alpha and Hif1-Alpha gene expression level among groups. Conclusion: Acute organophosphate exposure leads to loss in hepatocyte. Correlation with histopathological results, OP compound-induced cytotoxicity may be modulated through multiple sites including caspase1-3 pathway and also changes in the quantitative criteria of molecular markers of apoptosis in the rat hepatocytes on formation of behavioral skills were characterized by increased in caspase expressions in the hepatocyte.

Correlation of defects in regional cerebral blood flow determined by 99mTc SPECT with residual neurocognitive testing abnormalities during and 3 months post exposure in acutely poisoned patients with organophosphates

Introduction. Following acute organophosphate exposure, morphological changes in certain regions of the brain have been reported to develop within a few hours and involve neuronal degeneration. Single photon emission computed tomography (SPECT) has been used to determine changes in the regional cerebral blood flow and attempts have been made to correlate these changes with long-term neurological sequelae. Purpose of study. The aim of the study was to determine changes in the regional cerebral blood flow by 99mTc-ECD SPECT following acute organophosphate poisoning and to correlate these defects with abnormalities in neurocognitive testing carried out during admission and at 3 months post exposure, in order to determine whether any changes in the cerebral blood flow could help in predicting future development of neurocognitive deficit. Patients and methods. Twenty-eight patients with acute organophosphorous poisoning were included in the study. The inclusion criteria were a history of ingestion or accidental exposure, clinical features of cholinergic crises, and low serum acetylcholinesterase (AChE). Twenty age- and sex-matched patients from a previous study were used as controls for the neurocognitive tests. There were no controls for SPECT. Results. Of the 28 patients studied, 27 had abnormalities in the regional cerebral blood flow on SPECT with men having significantly higher abnormalities than women (p < 0.05). The right side of the brain had more defects than the left, with the occipital lobes being the most commonly involved. Of seven neurocognitive function tests carried out on patients who had regional cerebral blood flow defects during admission, abnormalities were observed in six tests. In 18 of 26 patients who could be tested at 3 months post exposure, improvement was observed in Trail B and Visual retention tests. However, others tests remained significantly abnormal. Conclusion. We conclude that a single episode of clinically significant organophosphate intoxication can lead to persistent residual neurocognitive deficits. Detection of regional cerebral blood flow defects on 99mTc-ECD SPECT can possibly help in predicting long-term deficits in neurocognitive functions in such patients.

Innovative oximes as potential blood‐brain barrier penetrating cholinesterase reactivators

Organophosphate (OP) nerve agents are extremely toxic because they inhibit acetylcholinesterase (AChE). The US Army currently utilizes pralidoxime (2‐PAM) for the reactivation of peripheral OP‐inhibited AChE. However, 2‐PAM does not cross the blood‐brain barrier (BBB) at therapeutically relevant levels; therefore acute OP exposure can lead to status epilepticus (SE) which can cause brain damage. We propose to prevent SE by reactivating CNS AChE. Our oximes will potentially be transported across the BBB because they contain a sugar moiety, hence the terminology 'sugar‐oximes'. This sugar moiety could be recognized by the facilitative transporter GLUT1. Published data has shown that glucose‐oxime conjugates can prevent the body temperature inducing properties of OPs in mice, indicating a CNS effect. We have synthesized these previously reported, but understudied, sugar‐oximes and have developed an in vitro model for evaluating their reactivation of OP inhibited RBC‐ChE. Our results show several sugar‐oximes are good candidates for further studies, including in vivo efficacy studies and in vitro assays to show that the sugar‐oxime interacts with GLUT1. This work is supported by the Defense Threat Reduction Agency (DTRA).

Mitochondrial ATP synthase inhibition and nitric oxide are involved in muscle weakness that occurs in acute exposure of rats to monocrotophos

Organophosphate poisoning in the context of self-harm is a common medical emergency in Asia. Prolonged muscle weakness is an important but poorly understood cause of morbidity and mortality of the poisoning. This study examined mitochondrial function and its modulation by nitric oxide in muscle weakness of rats exposed to an acute, oral (0.8LD50) dose of monocrotophos. Muscle mitochondrial ATP synthase activity was inhibited in the rat in acute exposure to monocrotophos while respiration per se was not affected. This was accompanied by decreased mitochondrial uptake of calcium and increased levels of nitric oxide. Reactive cysteine groups of ATP synthase subunits were reduced in number, which may contribute to decreased enzyme activity. The decrease in ATP synthase activity and reactive cysteine groups of ATP synthase subunits was prevented by treatment of animals with the nitric oxide synthase inhibitor, L-NG Nitroarginine methyl ester, at 12 mg/kg body weight for 9 days in drinking water, prior to monocrotophos exposure. This indicated a role for nitric oxide in the process. The alterations in mitochondrial calcium uptake may influence cytosolic calcium levels and contribute to muscle weakness of acute organophosphate exposure.

Evidence of necrosis in human intercostal muscle following inhalation of an organophosphate insecticide

Intercostal muscle samples obtained from autopsy of a 51-year-old male exposed to an organophosphate insecticide were analyzed for cholinesterase activity and muscle fiber integrity. Muscle cholinesterase activity, 5 days after exposure, was reduced to 53% of control values. Histological analysis indicated the presence of muscle fibers with subsarcolemmal grouped granular basophilic inclusions and scattered necrotic fibers. Results indicate that acute organophosphate exposure through inhalation can lead to skeletal muscle fiber damage in humans, similar to results obtained by ingestion. Furthermore, the pathology is comparable to the histological alterations observed in rats following acute injection of organophosphates.

Evidence of Necrosis in Human Intercostal Muscle following Inhalation of an Organophosphate Insecticide

Intercostal muscle samples obtained from autopsy of a 51 year old male, exposed to an organophosphate insecticide by inhalation, were analyzed for cholinesterase (ChE) activity and muscle fiber integrity. Muscle ChE activity, five days after exposure, was still reduced to 53% of control values. Histological analysis indicated the presence of muscle fibers with subsarcolemmal grouped granular basophilic inclusions and scattered necrotic fibers. Results indicate that acute organophosphate exposure through inhalation can lead to skeletal muscle fiber damage in humans, similar to results obtained by ingestion. Furthermore, the pathology is comparable to the histological alterations observed in rats following acute injection of organophosphates.