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Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need)., 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 © 2020 Elsevier B.V. All rights reserved.)

Organophosphates (OPs) are found in hundreds of valuable agricultural, industrial, and commercial compounds; however, they have also been associated with a variety of harmful effects in humans. The acute toxicity of OPs is attributed to the inhibition of the enzyme acetylcholinesterase (AChE); however, this mechanism may not account for all of the deleterious neurologic effects of OPs, especially at doses that produce no overt signs of acute toxicity. In this study, the effects of two weeks of daily subcutaneous exposure to the OP-nerve agent diisopropylfluorophosphate (DFP) in doses ranging from 0.125-0.500 mg/kg on whole brain volume, white matter, and gray matter integrity were evaluated in post mortem tissues using histology and magnetic resonance imaging (MRI) methods. The effects of DFP on axonal transport in the brains of living rats were evaluated using a manganese-enhanced MRI (MEMRI) method. DFP was associated with dose-dependent impairments in red blood cell and brain AChE (down to 29 and 18% of control, respectively at the highest dose), 24 h after the last injection. However, there were no visible signs of cholinergic toxicity noted in any portion of the study. Moreover, histological and MRI analysis of post mortem brains did not reveal any pronounced alterations of whole brain, white matter, or gray matter volumes associated with DFP. Electron microscopy did reveal a DFP-related increase in structural disruptions of myelinated axons (i.e., decompactions) in the fimbria region on the corpus callosum. MEMRI indicated that DFP was also associated with dose-dependent decreases in axonal transport in the brains of living rats, an effect that was also present after a 30-day (DFP-free) washout period, when AChE was not significantly inhibited. These results indicate that repeated exposures to the nerve agent, DFP at doses that are below the threshold for acute toxicity, can result in alterations in myelin structure and persistent decreases in axonal transport in the rodent brain. These observations could explain some of the long-term neurological deficits that have been observed in humans who have been repeatedly exposed to OPs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Organophosphate (OP)-nerve agent poisoning may lead to prolonged epileptiform seizure activity, which can result in irreversible neuronal brain damage. A timely and effective control of seizures with pharmacological agents can minimize the secondary and long-term neuropathology that may result from this damage. Diazepam, the current anticonvulsant of choice in the management of OP poisoning, is associated with unwanted effects such as sedation, amnesia, cardio-respiratory depression, anticonvulsant tolerance, and dependence liabilities. In search for an efficacious and safer anticonvulsant benzodiazepine, we studied imidazenil, a potent anticonvulsant that is devoid of sedative action and has a low intrinsic efficacy at alpha1- but is a high efficacy positive allosteric modulator at alpha5-containing GABA(A) receptors. We compared the potency of a combination of 2 mg/kg, i.p. atropine with: (a) imidazenil 0.05-0.5 mg/kg i.p. or (b) equipotent anti-bicuculline doses of diazepam (0.5-5 mg/kg, i.p.), against diisopropyl fluorophosphate (DFP; 1.5 mg/kg, s.c.)-induced status epilepticus and its associated neuronal damage. The severity and frequency of seizure activities were determined by continuous radio telemetry recordings while the extent of neuronal damage and neuronal degeneration were assessed using the TUNEL-based cleaved DNA end-labeling technique or neuron-specific nuclear protein (NeuN)-immunolabeling and Fluoro-Jade B (FJB) staining, respectively. We report here that the combination of atropine and imidazenil is at least 10-fold more potent and longer lasting than the combination with diazepam at protecting rats from DFP-induced seizures and the associated neuronal damage or ongoing degeneration in the anterior cingulate cortex, CA1 hippocampus, and dentate gyrus. While 0.5 mg/kg imidazenil effectively attenuated DFP-induced neuronal damage and the ongoing neuronal degeneration in the anterior cingulate cortex, dentate gyrus, and CA1 hippocampus, 5 mg/kg or a higher dose of diazepam is required to produce similar protective effects. These finding suggests that imidazenil, a non-sedating anticonvulsant BZ ligand, is a more potent, effective, and safer drug than diazepam in protecting rats from DFP-induced seizures and the associated neuronal damage and/or ongoing neuronal degeneration.

Growing concern on the problem of mycotoxins in the alimentary chain underlines the need to investigate the mechanisms explaining the cholinergic effects of aflatoxin B(1) (AFB(1)). We examined the effect of AFB(1), a mycotoxin produced by Aspergillus flavus, on mouse brain acetylcholinesterase (AChE) and specifically on its molecular isoforms (G(1) and G(4)) after in vitro exposure. AFB(1) (from 10(-9) to 10(-4)M), inhibited mouse brain AChE activity (IC(50) = 31.6 x 10(-6)M) and its G(1) and G(4) molecular isoforms in a dose-dependent manner. Michaelis-Menten parameters indicate that the K(m) value increased from 55.2 to 232.2% whereas V(max) decreased by 46.2-75.1%. The direct, the Lineweaver-Burk and the secondary plots indicated a non-competitive-mixed type antagonism, induced when the inhibitor binds to the free enzyme and to the enzyme-substrate complex. AFB(1)-inhibited AChE was partially reactivated by pyridine 2-aldoxime (2-PAM) (10(-4)M) but the AChE-inhibiting time courses of AFB(1) (10(-4)M) and diisopropylfluorophosphate (DFP) (2 x 10(-7)M) differed. Overall these data suggest that AFB(1) non-competitively inhibits mouse brain AChE by blocking access of the substrate to the active site or by inducing a defective conformational change in the enzyme through non-covalent binding interacting with the AChE peripheral binding site, or through both mechanisms.

Such organophosphorus (OP) compounds as diisopropylfluorophosphate (DFP), sarin and soman are potent inhibitors of acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs). The acute toxicity of OPs is the result of their irreversible binding with AChEs in the central nervous system (CNS), which elevates acetylcholine (ACh) levels. The protective action of subcutaneously (SC) administered antidotes or their combinations in DFP (2.0 mg/kg BW) intoxication was studied in 9-10-weeks-old Han-Wistar male rats. The rats received AChE reactivator pralidoxime-2-chloride (2PAM) (30.0 mg/kg BW), anticonvulsant diazepam (2.0 mg/kg BW), A(1)-adenosine receptor agonist N(6)-cyclopentyl adenosine (CPA) (2.0 mg/kg BW), NMDA-receptor antagonist dizocilpine maleate (+-MK801 hydrogen maleate) (2.0 mg/kg BW) or their combinations with cholinolytic drug atropine sulfate (50.0 mg/kg BW) immediately or 30 min after the single SC injection of DFP. The control rats received atropine sulfate, but also saline and olive oil instead of other antidotes and DFP, respectively. All rats were terminated either 24 h or 3 weeks after the DFP injection. The rats treated with DFP-atropine showed severe typical OP-induced toxicity signs. When CPA, diazepam or 2PAM was given immediately after DFP-atropine, these treatments prevented, delayed or shortened the occurrence of serious signs of poisoning. Atropine-MK801 did not offer any additional protection against DFP toxicity. In conclusion, CPA, diazepam and 2PAM in combination with atropine prevented the occurrence of serious signs of poisoning and thus reduced the toxicity of DFP in rat.

The neuropathic potential of acute and repeated exposures of the phosphoramidates tabun (GA) and isofenphos (IFP), of diisopropyl fluorophosphate (DFP) and paraoxon (PO) were examined in the hen with treatments for up to 90 days via intramuscular injections of the highest tolerated doses with atropine protection. Plasma acetylcholinesterase (AChE), non-specific butyrylcholinesterase (BChE) and creatine kinase (CK) activities were measured in order to monitor whether the compounds were present at biologically active concentrations. Locomotor behavior was observed and tissues from the peripheral and central nervous systems were examined for signs of organophosphate-induced delayed neuropathy (OPIDN). No behavioral or histological evidence of OPIDN was observed after treatments with GA, IFP, PO, saline or atropine sulfate. DFP-treated birds displayed locomotor and neuropathological signs of OPIDN with a no effect level (NOEL) between 25 and 50 micrograms/kg.

Experiments were designed to assess the mechanisms of diisopropyl fluorophosphate (DFP)-induced changes in thermoregulation of the rat. In one study, male rats of the Long-Evans strain were injected with DFP (s.c.) at doses ranging from 0 to 2.0 mg/kg while maintained at an ambient temperature (Ta) of 20--24 degrees C. Body (Tb) and tail skin (Tt) temperatures were recorded for 5 h post-injection. DFP doses of greater than or equal to 1.0 mg/kg resulted in significant decreases in Tb lasting up to 5 h and increases in Tt lasting up to 1 h post-injection. In a second study, metabolic rate (MR), evaporative water loss (EWL), motor activity (MA), Tb, and Tt were measured at 2 h post-injection of 0, 0.5, 1.0, and 1.5 mg/kg DFP (s.c.) at Ta values of 10, 20, and 30 degrees C. DFP treatment resulted in hypothermia at all three Ta values, but the effect was attenuated at 30 degrees C. MR was significantly reduced at a Ta of 20 degrees C following 1.5 mg/kg, unaffected by DFP at a Ta of 30 degrees C, and stimulated at 10 degrees C following 0.5 mg/kg DFP. EWL was significantly elevated at 30 degrees C following 1.5 mg/kg DFP. MA was significantly reduced following greater than or equal to 1.0 mg/kg DFP at 20 and 30 degrees C and 1.5 mg/kg at 10 degrees C. Tt was elevated and reduced by DFP at Ta values of 30 and 10 degrees C, respectively. In a third study, rats were injected with DFP and placed in a temperature gradient for 1 to 2 h post-injection while selected Ta and Tb were monitored. While both control and DFP-treated rats remained in the cool end of the gradient, rats administered DFP at doses of 1.0 and 1.5 mg/kg were significantly hypothermic. It was also found that Ta values of 10, 20, and 30 degrees C had no effect on DFP-induced inhibition of cholinesterase activity of plasma and erythrocyte fractions of whole blood. Overall, these data support the hypothesis that acute DFP may lower the set-point for the control of body temperature in the rat and demonstrates that the toxicity of DFP is modified by changes in Ta.

Primary cell cultures of mouse embryo spinal cords were used to study the biological effects of exposure to organophosphate esters and neurotoxic amino acids. The effects of exposure were correlated with markers of cholinergic function. The purpose of this study was to examine the mechanisms whereby xenobiotics produce neurotoxicologic damage.

Numerous studies have suggested a role for the nervous system in renal function. Several cholinergic agents have been examined for effects in vivo and in renal slices. Effects mediated through the vascular system and also direct effects have been reported. In this study an irreversible cholinesterase inhibitor, diisopropylfluorophosphate (DFP) in doses of 1-4 mg/kg, was tested on renal function. A single dose of DFP (2, 3 or 4 mg/kg) caused an increased flow of urine of low osmolality over the 6 h after the administration of the drug with essentially a return to control status by 24 h after either of the lower doses. Twenty-four hours after 4 mg/kg, urine volume was less and urine osmolality greater than control. Renal and brain cholinesterases remained depressed 24 h after DFP. In acute experiments on anesthetized animals, inulin clearance was increased by the lower doses and decreased by the highest dose of DFP. Renal blood flow measured with an electromagnetic flow meter showed a similar dose-response relationship. However, urine flow increased at all doses. The increased urine flow associated with decreased inulin clearance (4 mg/kg) and renal blood flow (3 or 4 mg/kg) suggest a direct effect of DFP on renal tubular function. These effects do not appear to be related to inhibition of cholinesterase.

The objective of the present investigation was to assess the comparative efficacy of prophylactic treatment with d-tubocurarine (d-TC) (0.075 mg/kg), atropine sulfate (16 mg/kg), and atropine methylnitrate (16 mg/kg), employed singly or in combination against the diisopropylphosphorofluoridate (DFP)-induced myopathy in rat. DFP (1.5 mg/kg, s.c.) produced signs of cholinergic toxicity with predominantly peripheral involvement manifest as severe muscle fasciculations beginning within 5-7 min and persisting in excess of 4-6 h. Maximal muscle fiber necrosis was observed within 24 h. Rats were protected against the apparent behavioural and morphological changes as well as electrophysiological signs of neuromuscular toxicity by all pretreatment agents. Combined pretreatment with d-TC (0.075 mg/kg, s.c.) and atropine methylnitrate (16 mg/kg, s.c.) was found to be most effective in attenuating DFP-induced muscle fiber necrosis as evidenced by complete absence of lesions and the prevention of DFP-induced hyperactivity in nerve and muscle. Significant protection was afforded by all pretreatment agents when given alone. It is suggested that the pretreatment agents act presynaptically by preventing drug-induced backfiring and muscle fasciculations possibly by reducing the release of acetylcholine (ACh). The protective drugs in the concentrations used had no significant effect on the normal characteristics of conduction and transmission.

Some organophosphorus compounds (OP) induce a delayed polyneuropathy (OPIDP) which is initiated by the phosphorylation of the so-called neuropathy target esterase (NTE). In this work some aspects of hen sciatic nerve NTE are studied. The assay method is reported and modifications are discussed and a combined method proposed. Proximo-distal distribution showed a significant difference from proximal (100 +/- 10%) to distal (69 +/- 9%) fragments, in accordance with reported data. The time course of in vivo regeneration after a single TOCP dose (200 mg/kg, post oral) showed some differences when compared with hen brain NTE. Sciatic nerve NTE showed a delay of 2-3 days before regeneration but then regenerated faster (74% activity at day 7) than brain NTE (50% activity at day 7). A slower rate of regeneration of distal than proximal segments has been suggested to explain higher sensitivity of distal segments [3], however in this work no significant differences were observed in the rate of regeneration when comparing proximal and distal fragments.