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Chlorpyrifos (CPF; 0,0-diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate), a cholinesterase inhibitor, compromised the integrity of the blood-brain barrier (BBB) when used at low concentrations during our previous experiments in vitro. To determine if BBB leakage would also occur in vivo, we used FITC-dextrans to evaluate BBB permeability in CPF-dosed mice. Results indicated BBB leakages that were evident at 2 h after treatment with 70 mg/kg CPF ip. Since vascular endothelial growth factor (VEGF), a potent vasopermeability factor, is a signaling protein that promotes the growth of new blood vessels, we investigated the possible involvement of VEGF in BBB disruption by CPF. We found that VEGF serum concentration was significantly increased at 24 h after CPF exposure. To further explore VEGF involving BBB disruption by CPF treatment, the receptor antagonist for VEGF (sFlt-1) was used for pretreatment before CPF exposure. After sFlt-1 pretreatment, gene expressions of the tight junction (TJ) proteins claudin5 and occludin were significantly downregulated at 1, 2, and 3 h, but returned to control levels at 24 h after CPF treatment. These results suggest that VEGF is involved in BBB disruption by CPF through BBB-TJs regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The well-known toxicity of chlorpyrifos (CPF) occurs via inhibition of cholinesterase (ChE), but in recent years the detrimental effects of low-dose CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We previously showed that CPF can alter gene expression of transient receptor potential canonical (TRPC) channels in vitro. In this study, we analyzed the gene expression of TRPCs at various time points after CPF treatment in vivo. The results showed that TRPC1 mRNA expression in mouse brain was significantly reduced 2-8 h after CPF treatment, but the TRPC4 mRNA expression was not significantly changed. To investigate the possible involvement of Transforming Growth Factor-beta1 (TGF-β1) in contributing to TRPCs gene alteration by CPF, we used TGF-beta receptor inhibitor (LY2109761) as a pretreatment prior to CPF treatment. The serum TGF-β1 concentration was significantly increased 24 h after CPF treatment. After pretreatment with LY2109761, both TRPC1 and TRPC5 mRNAs were significantly downregulated 1 and 2 h after CPF treatment, but were significantly upregulated 3 and 24 h after CPF treatment. TRPC4 mRNA was also significantly downregulated at 1 h. These results suggest that interference with ion channels, a non-cholinergic mechanism of CPF, may contribute to the cellular neurotoxicity of CPF., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The blood-brain barrier (BBB) is formed by specialized endothelial cells lining capillaries in the central nervous system (CNS). We previously demonstrated that exposure to very low concentrations of the organophosphorus insecticide chlorpyrifos (CPF) decreased electrical resistance across the BBB in vitro, indicating a loss of BBB integrity. The present study examined the transient effects of CPF on expression of genes contributing to tight junctions of the BBB. Rat brain endothelial cells (RBE4) were co-cultured with rat astrocytes on membrane inserts to form an in vitro BBB. The RBE4 cells in the BBB were then exposed to CPF for 2, 4 and 12 h. Total RNA was extracted from RBE4 cells and quantitative real-time PCR (qRT-PCR) was used to quantify levels of gene expression of tight junction proteins claudin5, scaffold proteins zona occludens (ZO1) and transient receptor potential (canonical) channels (TRPC4). Gene expression decreased 2 h after exposure to CPF, especially TRPC4, but the effects were reversed 12 h later. CPF exposure for only 15 min caused less effect than longer exposures, with TRPC4 gene expression above the control at 4 h. These results suggest that altering gene expression for claudin5, TRPC4 and ZO1 by CPF may directly contribute to BBB disruption, and that the alteration is reversible upon removal of CPF., (Copyright © 2012 John Wiley & Sons, Ltd.)

A study was conducted to determine if multiple exposures to several stress paradigms might affect the anticholinesterase effect of subsequently administered organophosphate insecticide chlorpyrifos. Male Sprague-Dawley rats were subject to daily periods of restraint, swimming, a combination of the two, or neither of the two (controls) (n=8/group) for 5 days per week over a six-week period. The most profound stress, as measured by reduced body weight gain and elevated levels of plasma corticosterone, was swimming. On day 39 of the study, shortly after the daily stress episode, one half of the rats in each group was dosed with 60 mg/kg chlorpyrifos subcutaneously. This had no effect on subsequent levels of plasma corticosterone. There were no stress-related differences in the degree of chlorpyrifos-induced inhibition of brain acetylcholinesterase in animals sacrificed on day 43.

Repeated stress has been reported to cause reversible impairment in the central nervous system (CNS). It was proposed that alterations in glutamatergic, cholinergic, and monoamine neurotransmitter systems after exposure to stress are initial CNS events contributing to this impairment and that exacerbation could occur with concurrent exposure to cholinesterase inhibitors. Effects of concurrent exposure to repeated stress and chlorpyrifos on activities of acetylcholinesterase (AChE), carboxylesterase, and choline acetyltransferase (ChAT); concentrations of excitatory amino acids, monoamines, and their metabolites; and maximum binding densities (B(max)) and equilibrium dissociation rate constants (K(d)) of glutamatergic N-methyl-d-aspartate (NMDA) and total muscarinic cholinergic receptors were studied in the blood, hippocampus, cerebral cortex, or hypothalamus of adult Long-Evans rats. Stress treatments extended over 28 days included (1) control rats handled 5 days/week; (2) rats restrained 1 h/day for 5 days/week; (3) rats swum 30 min for 1 day/week; or (4) rats restrained 4 days/week and swum for 1 day/week. On day 24, each stress treatment group was randomly divided and injected either with corn oil or chlorpyrifos, 160 mg/kg subcutaneously (sc) (60% of the maximum tolerated dose), 4 h after restraint. Blood and brain tisssues were collected on day 28. Rats restrained and swum had a statistical trend toward increasing concentrations of glutamate in the hippocampus when compared to rats only swum (p = .064). Chlorpyrifos administration decreased restraint-induced elevated aspartate in the hippocampus, and decreased B(max) of total muscarinic receptors in the cerebral cortex. In addition, chlorpyrifos decreased B(max) and K(d) of total muscarinic receptors in the cerebral cortex of swum rats. Results demonstrated that chlorpyrifos inhibited AChE activity in blood, cerebral cortex, and hippocampus, but stress did not affect AChE activity. Carboxylesterase activity was inhibited by chlorpyrifos and by repeated restraint with swim. Swim stress decreased concentrations of norepinephrine in the hippocampus and hypothalamus, and increased concentrations of dopamine and its metabolite, DOPAC, in the hypothalamus. Both stress and chlorpyrifos altered serotonin concentrations, and the interactions of repeated stress and chlorpyrifos on serotonin approached significance in the hippocampus (p = .06) and hypothalamus (p = .08). Therefore, stress models were demonstrated to alter glutamatergic and monoamine responses, whereas chlorpyrifos alone had effects on cholinergic and monoamine systems in the rat CNS. However, the interactions between stress and chlorpyrifos significant at p < 0.05 were restricted to attenuation of elevated aspartate in the hippocampus of restrained with swim rats and decreased K(d) of acetylcholine receptors in the cerebral cortex of swum rats and restrained rats.

The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.

Adult male Long-Evans rats were exposed to 2 neurotoxic organophosphates in a setting of chronic stress, over a 63-day period. The organophosphates were tri-ortho-tolyl phosphate (TOTP) administered in 14 gavage doses of 75, 150 or 300 mg/kg, and chlorpyrifos, given in two 60 mg/kg subcutaneous exposures. Corticosterone was added to the drinking water at 400 microg/ml, to model aspects of chronic stress. These compounds/dosages were administered individually and in combination, with appropriate controls, giving rise to 16 experimental groups. The major neuropathologic change was the presence of axonal degeneration progressing to myelinated fiber degeneration, mainly in distal regions of selected fiber tracts and peripheral nerve, seen in animals sacrificed on experimental day 63. The cervical spinal cord and medullary levels of the sensory gracile fasciculus were most prominently affected. This axonopathy/fiber degeneration was TOTP dose-related at the 300 and 150 mg/kg levels. There was association of this lesion with inhibition of the enzyme neurotoxic esterase in hippocampal tissue from TOTP-treated rats. Such an association categorizes this disease process as organophosphate ester-induced delayed neuropathy. Neither chlorpyrifos nor corticosterone appeared to contribute to the neuropathic events or the enzyme inhibition. A cohort of rats was maintained on the corticosterone dosing, but without additional exposure to TOTP or chlorpyrifos, for an additional 27 days. When these rats were examined on day 90, the nerve fiber degeneration had progressed in all experimental groups administered the 300 mg/kg dose of TOTP (lower doses were not studied at the 90-day interval), although hippocampal neurotoxic esterase had returned to control values.

In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests. [ABSTRACT FROM PUBLISHER]

It has been proposed that exposure to environmental toxicants plays a role in the development of neurodegenerative diseases. Mechanisms of action underlying this process, however, have not yet been clearly established. The present study was designed to address potential mechanisms that contribute to brain injury after exposure to the organophosphorus insecticide chlorpyrifos. C57BL/6 mice were administered a single intraperitoneal injection of chlorpyrifos in increasing dosages (20, 70, and 140 mg/kg). At 4, 8, 16, and 24 h of exposure, four discrete regions of the brain (hippocampus, cerebral cortex, striatum, and cerebellum) were isolated, and expression levels of pro-inflammatory mediators were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). A significant increase in expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), MCP-1, and E-selectin was observed in all brain regions in a time- and dose-dependent manner. These data suggest that chlorpyrifos-induced brain injury may be mediated through pro-inflammatory pathways. It also offers the potential to contribute to the development of new therapeutic strategies specifically targeted against pro-inflammatory pathways of environmental agent-induced neurotoxicity. [ABSTRACT FROM AUTHOR]