Low Concentrations of the Organophosphate VX Affect Spontaneous and Evoked Transmitter Release from Hippocampal Neurons: Toxicological Relevance of Cholinesterase-Independent Actions

doi:10.1006/taap.1999.8733

Toxicology and Applied Pharmacology

Volume 159, Issue 1, 15 August 1999, Pages 31-40

https://doi.org/10.1006/taap.1999.8733 Get rights and content

Abstract

In the present study, the patch–clamp technique was applied to cultured hippocampal neurons to evaluate the effects of the nerve agent VX on evoked and spontaneous postsynaptic currents mediated by γ-aminobutyric acid (GABA) and glutamate. At 0.01 nM, VX reduced the amplitude of evoked GABAergic currents, and only at concentrations >1 nM did it decrease the amplitude of evoked glutamatergic currents. The effect of VX on GABAergic currents, which was partially reversible upon washing of the neurons with VX-free external solution, could be prevented by the muscarinic antagonist atropine. In contrast, the effect of VX on glutamatergic currents, which was not reversible upon washing, appears to be related to the VX-induced reduction of the amplitude and frequency of repetitively firing by action potentials. In the presence of the Na⁺-channel blocker tetrodotoxin (TTX), VX (≥10 nM) increased the frequency of GABA- and glutamate-mediated miniature postsynaptic currents (MPSCs). This effect of VX was unrelated to cholinesterase inhibition and was Ca²⁺ dependent. The lack of effect of VX on MPSC kinetics indicates that VX-induced alterations of evoked and spontaneous currents are exclusively due to alterations of the transmitter release processes. The ability of VX to affect transmitter release in the brain may underlie some of its neurotoxic effects and may provide the basis for the development of therapeutic countermeasures to treat and/or prevent VX-induced neurotoxicity.

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The contact poison VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate) is a chemical warfare agent that is one of the most toxic organophosphorus compounds known. Its primary mechanism of toxic action is through the inhibition of acetylcholinesterase and resultant respiratory paralysis. The majority of work on VX has thus concentrated on its potent anticholinesterase activity and acute toxicity, with few studies investigating potential long-term effects. In this report we describe the effects of VX in aggregating rat brain cell cultures out to 28 days post-exposure. Cholinesterase activity was rapidly inhibited (60 min IC₅₀ = 0.73 +/− 0.27 nM), but recovered towards baseline values over the next four weeks. Apoptotic cell death, as measured using caspase-3 activity was evident only at 100 μM concentrations. Cell type specific enzymatic markers (glutamine synthase, choline acetyltransferase and 2′,3′-cyclic nucleotide 3′-phosphodiesterase) showed no significant changes. Total Akt levels were unchanged, while an increased phosphorylation of this protein was noted only at the highest VX concentration on the first day post-exposure. In contrast, significant and delayed (28 days post-exposure) decreases were noted in vascular endothelial growth factor (VEGF) levels, a protein whose reduced levels are known to contribute to neurodegenerative disorders. These observations may indicate that the long-term effects noted in some survivors of nerve agent intoxication may be due to VX-induced declines in brain VEGF levels.
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The widespread use of chemicals in everyday human life has a constant threat of toxic chemicals exposure from different sources. The use of toxic chemicals with goals of assault, demoralization, homicide, and lethality has been documented as early as in Stone Age and on the battlefields. The misuse of chemical warfare agents (CWAs) on civilians in Syria, Malaysia, and the UK demonstrates the constant threat of CWAs to the global population. CWAs, such as nerve and blister agents, are generally called weapons of mass destruction due to their high mortality and rapid toxicity, even in small quantities. The current chapter focuses on nerve and blister agents due to space constraints. Blister agents cause DNA alkylation, poly (ADP-ribose) polymerase (PARP) activation, severe oxidative stress, inflammation, energy depletion, and cell death. Nerve agents have profound effects on the human body mediated via cholinergic and other neurotransmitters linked to ligand, voltage-gated ion channels, and GPCR receptors leading to subsequent activation of multiple downstream signaling pathways through second messengers causing an array of toxic effects, including death.
Neurotoxicity of organophosphate nerve agents
2020, Advances in Neurotoxicology
Citation Excerpt :
Lallement et al. (1991a,b) suggested that soman-induced overstimulation of glutamatergic receptors contributed to maintenance of seizure. In another study, VX increased the frequency of GABA- and glutamate-mediated miniature postsynaptic currents- a Ca2 +-dependent effect reported to be unrelated to AChE inhibition (Rocha et al., 1999). Chabebo et al. (1999) reported that 0.3–1 nM of sarin reduced the amplitude of GABA-mediated postsynaptic currents but had no effect on the amplitude of glutamatergic-mediated postsynaptic currents.
Organophosphate nerve agents of the G-series (tabun, GA; sarin, GB; cyclosarin, GF, and soman, GD) and V-series (VX, RVX, Novichok, etc.) were developed prior to, during and after World War II. These are all toxic ester derivatives of phosphonic acid containing a cyanide (GA), fluoride (GD or GF), or sulfur (VX) substituent and are commonly termed “nerve” agents as a consequence of their anticholinesterase properties as well as their effects on both the central nervous system (CNS) and the peripheral nervous system (PNS). These nerve agents produce toxicity primarily in the CNS and skeletal muscles by cholinergic and noncholinergic mechanisms involving various neurotransmitters, receptors, enzymes, and other molecules. Various biomarkers of exposure, effects and susceptibility to nerve agents have been identified. Treatment of OP poisoning rests with specific antidotes (atropine sulfate and oximes). Since no single oxime is effective against all nerve agents, a specific oxime can be indicated only against a particular nerve agent(s). Various decontaminating products are available, which are found to be very effective in removing or minimizing nerve agents' residue, thereby avoiding further exposure to victims and emergency responders.
Behavioral toxicity of nerve agents
2020, Handbook of Toxicology of Chemical Warfare Agents
The behavioral changes belong to the most important and frequent symptoms of acute and delayed toxicity of organophosphorus nerve agents. The incidence of behavioral effects is higher in individuals who have been severely exposed to nerve agents, but they may also occur in individuals who have received a low-level exposure below those producing convulsions and other severe clinical signs of toxicity. The behavioral effects usually start within a few hours and last from several days to several weeks or months. The most frequent symptoms include disruption of cognitive functions, feelings of uneasiness, tenseness, and fatigue. Persistent severe alteration in behavior and cognitive functions, especially impairment of learning and memory, was observed after exposure to high doses of nerve agents as a result of severe brain neuropathology that involves neuronal degeneration and necrosis of various brain regions including the hippocampus that is connected with the alteration of cognitive functions. Nevertheless, repeated or long-term exposure to low levels of nerve agents can also cause behavioral alterations due to downregulation of muscarinic receptors in the hippocampus as a reaction on acetylcholine accumulation at muscarinic receptor sites due to acetylcholinesterase inhibition.
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2020, Handbook of Toxicology of Chemical Warfare Agents
Chemical warfare nerve agents are potent anticholinesterase compounds deliberately formulated to induce debilitating effects or death during wartime hostilities. A key need for both community emergency preparedness, and restoration of military installations where agents have been processed and/or stored, is access to concise and timely information on agent characteristics and treatment, as well as health-based exposure guidelines derived in a clear manner by contemporary methods of data analysis. These parameters are summarized for the nerve agents GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), and VX, as well as for agent GE. Toxicological analysis and logic incorporated into estimation of vapor exposure and ingestion guidelines for these agents is also summarized. Recent experimental data characterizing low-level nerve agent vapor exposures for multiple agents and species have placed existing guidelines in context and further substantiates their protective nature.
Retrograde activation of CB1R by muscarinic receptors protects against central organophosphorus toxicity
2019, Neuropharmacology
Citation Excerpt :
OP exposure was also shown to elevate eCB levels in brain, which was initially attributed to direct inhibition of eCB hydrolases by OPs (see below) (Liu et al., 2013; Nomura et al., 2008). Decreased excitatory signaling in hippocampal circuits has been demonstrated in rat and guinea pig brain slices at delayed times after in vivo soman challenge (Alexandrova et al., 2014; Munirathinam and Bahr, 2004); in cultured hippocampal neurons exposed to VX (Rocha et al., 1999); in hippocampal slices exposed to paraoxon (Narimatsu et al., 2010); and at rat SC synapses exposed to the reversible AChE antagonist physostigmine (an effect that was originally attributed to tonic activation of presynaptic mAChRs) (Mans et al., 2014). Separately it was found that physostigmine-induced presynaptic depression could be blocked by treatment with a CB1R antagonist, but not a M2 antagonist (Colgin et al., 2003).
The acute toxicity of organophosphorus-based compounds is primarily a result of acetylcholinesterase inhibition in the central and peripheral nervous systems. The resulting cholinergic crisis manifests as seizure, paralysis, respiratory failure and neurotoxicity. Though overstimulation of muscarinic receptors is the mechanistic basis of central organophosphorus (OP) toxicities, short-term changes in synapse physiology that precede OP-induced seizures have not been investigated in detail. To study acute effects of OP exposure on synaptic function, field excitatory postsynaptic potentials (fEPSPs) were recorded from Schaffer collateral synapses in the mouse hippocampus CA1 stratum radiatum during perfusion with various OP compounds. Administration of the OPs paraoxon, soman or VX rapidly and stably depressed fEPSPs via a presynaptic mechanism, while the non-OP proconvulsant tetramethylenedisulfotetramine had no effect on fEPSP amplitudes. OP-induced presynaptic long-term depression manifested prior to interictal spiking, occurred independent of recurrent firing, and did not require NMDA receptor currents, suggesting that it was not mediated by activity-dependent calcium uptake. Pharmacological dissection revealed that the presynaptic endocannabinoid type 1 receptor (CB1R) as well as postsynaptic M₁ and M₃ muscarinic acetylcholine receptors were necessary for OP-LTD. Administration of CB1R antagonists significantly reduced survival in mice after a soman challenge, revealing an acute protective role for endogenous CB1R signaling during OP exposure. Collectively these data demonstrate that the endocannabinoid system alters glutamatergic synaptic function during the acute response to OP acetylcholinesterase inhibitors.

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^☆: Supported by U.S. Army Medical Research and Development Command contract DAMD-17-95-C-5063. Some information contained in this paper was presented in the form of abstracts at the 26th Annual Meeting of the Society of Neuroscience in Washington, D.C. and at the Proceedings of the 1996 Medical Defense Bioscience Review.

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Regular ArticleLow Concentrations of the Organophosphate VX Affect Spontaneous and Evoked Transmitter Release from Hippocampal Neurons: Toxicological Relevance of Cholinesterase-Independent Actions☆

Abstract

Brain Res.

Eur. J. Pharmacol.

Pestic. Biochem. Physiol.

Brain Res.

Toxicol. Appl. Pharmacol.

Trends Pharmacol. Sci.

The role of carbamates and oximes in reversing toxicity of organophosphorus compounds: A perspective into mechanisms

Activation and blockade of the nicotinic and glutamatergic synapses by reversible and irreversible cholinesterase inhibitors

Multiple actions of anticholinesterase agents on chemosensitive synapses: Molecular basis for prophylaxis and treatment of organophosphate poisoning

Fundam. Appl. Toxicol.

Molecular targets and synaptic effects of carbamates, organophosphates, oximes, and pyridiniums: Implications for prophylaxis and therapy of organophosphate toxicity

Report to U.S. Army Research and Development Command

Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes

J. Pharmacol. Exp. Ther.

Neuronal nicotinic acetylcholine receptor activation modulates γ-aminobutyric acid release from CA1 neurons of rat hippocampal slices

J. Pharmacol. Exp. Ther.

Modulation of cellular excitability in neocortex: Muscarinic receptor and second messenger-mediated actions of acetylcholine

Synapse

Computer analysis of electrophysiological signals

Hippocampal synaptic transmission enhanced by low concentrations of nicotine

Nature

Regular Article
Low Concentrations of the Organophosphate VX Affect Spontaneous and Evoked Transmitter Release from Hippocampal Neurons: Toxicological Relevance of Cholinesterase-Independent Actions☆