API

Event: 10

Key Event Title

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Acetylcholine accumulation in synapses

Short name

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ACh Synaptic Accumulation

Biological Context

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Level of Biological Organization
Cellular

Cell term

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Cell term
eukaryotic cell


Organ term

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Key Event Components

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Process Object Action
acetylcholine increased

Key Event Overview


AOPs Including This Key Event

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Stressors

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Taxonomic Applicability

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Term Scientific Term Evidence Link
zebra fish Danio rerio High NCBI

Life Stages

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Life stage Evidence
All life stages High

Sex Applicability

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Term Evidence
Unspecific High

Key Event Description

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  • Acetylcholine is a neurotransmitter that is stored in nerve endings at cholinergic synapses in the central and peripheral nervous systems (Soreq and Seidman, 2001; Lushington 2006).

  • Acetylcholine can bind multiple types of nicotinic and muscarinic receptors. The downstream consequences of those events are tissue and receptor-specific.

  • Acetylcholine is released into the synaptic cleft when stimulation of the nerve occurs, and then binds to a receptor protein; either muscarinic (metabotropic) or nicotinic (ionotropic). The binding to the receptor results in changes in the flow of ions across the cell, thereby signaling activity (Fukuto 1990; Mileson et al 1998; Soreq and Seidman 2001; Lushington 2006).  

    • Inhibition of acetylcholine binding at the serine site via AChE inhibition results in an accumulation of acetylcholine in synapses associated with muscarinic and nicotinic receptors, resulting in unregulated excitation at neuromuscular junctions of skeletal muscle; pre-ganglionic neurotransmitters and post-ganglionic nerve endings of the autonomic nervous system; and neurotransmitters in the brain or central nervous system (CNS). 


How It Is Measured or Detected

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  • Several techniques are available to measure acetylcholine levels, including the Hestrin method (Augustinsson 1957, Hestrin 1949, Stone 1955), molecular probes or assays, microdialysis techniques (Zapata, 2009, Russom, 2014) or by liquid chromatography - tandem mass spectrometer LC-MS/MS (Gómez-Canela et al., 2017).

  • Hestrin’s method involves a colorimetric measurement of esterase activity. The rate of hydrolysis of acetylcholine with hydroxylamine to form hydroxamic acid is measured to determine the amount of acetylcholine:

RCOOR’ + H2NOH -> RCONHOH + R’OH

This method is performed at alkaline pH in water and is applicable over a wide range of ester concentrations (Hestrin 1949).

  • Hydrolysis of acetylcholine by acetylcholinesterase in the synaptic cleft is fast, so concentration in the extracellular fluid is low (0.1-6 nM). Brain microdialysate studies quantify nanomolar concentrations of acetylcholine in extracellular fluid using chromatographic mass spectrometric techniques (Nirogi 2009). Choice of analytical method should provide detection limits below the lowest concentration expected in the dialysate and requiring the smallest sample volume. High-pressure liquid chromatography coupled to electrochemical detection (HPLC-EC) is based on enzymatic conversion of acetylcholine into choline and acetate by acetylcholinesterase, and subsequent oxidation of choline by choline oxidase to betaine and hydrogen peroxide, which can be oxidized on a platinum electrode. This method permits detection of dialysate acetylcholine concentrations in the 5-10 nM range (Zapata, 2009). Other microdialysis techniques for quantification of acetylcholine are liquid chromatography mass spectrometry (Nirogi 2009) and pyrolysis-gas chromatography (Szilagyi 1968).

Domain of Applicability

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  • Acetylcholine and cholinergic receptors are found in invertebrate and vertebrate species. Specific examples from the literature documenting acetylcholine accumulation include:  Penaeid prawn exposed to sublethal exposure of methylparathion and malathion showed significantly increased ACh levels, in nervous tissue (Reddy 1990).

  • Brain tissue of tadpoles exposed to single sublethal concentrations methyl parathion for 24 h showed an increase in acetylcholine levels (Nayeemunnisa and Yasmeen 1986).  

  • Acute (48h) sublethal exposure to methyl parathion resulted in increased AChE levels in brain tissue in fish (Oreochromis mossambicus) (Rao and Rao, 1984). Researchers found a significant increase in acetylcholine at all time points measured (12-48hr) with acetylcholine levels increasing from 33-83% as compared to controls over the same time span. 

  • A study of male quail (Coturnix japonica) exposed to lethal concentrations of two OP pesticides (i.e., DDVP or fenitrothion), found significant increases in total and free acetylcholine (Kobayashi et al., 1983).

  • Mice singly injected with propoxur displayed changes in cholinergic parameters in the brain: increased brain ACh content, decreased AChE activity, and high-affinity choline uptake into synaptosomes (Kobayashi 1988). 

  • AChE levels and acetylcholine synthesis in rat striatum were compared in controls and animals exposed to 0.3 and 1 times the maximum tolerated dose. Acetylcholine was present in significantly less concentrations than in the adult rats (Karanth, 2003).


References

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  • Augustinsson, K.B. 1957. In: Glick,D.(Ed.); Methods of Biochemical Analysis, Interscience Publishers, Inc., New York, NY.

  • Gómez-Canela, C., D. Tornero-Cañadas, E. Prats, B. Piña, R. Tauler and D. Raldúa (2018), "Comprehensive characterization of neurochemicals in three zebrafish chemical models of human acute organophosphorus poisoning using liquid chromatography-tandem mass spectrometry”, Analytical and Bioanalytical Chemistry 410(6): 1735-1748. DOI: 10.1007/s00216-017-0827-3.
  • Fukuto TR.  1990. Mechanism of action of organophosphorus and carbamate insecticides.  Environ Health Perspect 87:245-254.    

  • Hestrin, S. (1949). The Reaction of Acetylcholine and Other Carboxylic Acid Derivatives with Hydroxylamine, and its Analytical Application. J. Biol. Chem. 180(1): 249-61.

  • Karanth, S., Pope, C. 2003. Age-related effects of chlorpyrifos and parathion on acetylcholine synthesis in rat striatum. Neurotoixol. Teratol. 25(5): 599-606.  

  • Kobayashi H, Yuyama A, Kudo M, Matsusaka N. 1983. Effects of organophosphorus compounds, O,O‐dimethyl‐o‐(2,2‐dichlorovinyl)phosphate (DDVP) and O,O‐dimethyl‐o‐(3‐methyl 4‐nitrophenyl)phosphorothioate (fenitrothion), on brain acetylcholine content and acetylcholinesterase activity in Japanese quail. Toxicology 28:219–227.

  • Kobayashi, H., Yuyama, A., Ohkawa, T., and Kajita, T. 1988. Effect of Single or Chronic Injection with a Carbamate, Propoxur, on the Brain Cholinergic System and Behavior of Mice. Jpn.J.Pharmacol. 47[1], 21-27.

  • Lushington GH, Guo J-X, Hurley MM.  2006. Acetylcholinesterase: Molecular modeling with the whole toolkit.  Curr Topics Medic Chem 6:57-73.

  • Mileson, BE, Chambers JE, Chen WL, Dettbarn W, Ehrich M, Eldefrawi AT, Gaylor DW, Hamernik K, Hodgson E, Karczmar AG, Padilla S, Pope CN, Richardson RJ, Saunders DR, Sheets LP, Sultatos LG, Wallace KB.  1998. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol Sci 41:8-20.

  • Molecular Probes. (2004). Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Retrieved from: http://tools.thermofisher.com/content/sfs/manuals/mp12217.pdf

  • Nayeemunnisa, Yasmeen N. 1986. On the presence of calmodulin in the brain of control and methyl parathion‐exposed developing tadpoles of frog, Rana cyanophlictis. Curr Sci (Bangalore) 55:546–548.

  • Nirogi, R., Mudigonda, K., Kandikere, V. Ponnamaneni, R. (2010). Quantification of Acetylcholine, an Essential Neurotransmitter, in Brain Microdialysis Samples by Liquid Chromatography Mass Spectrometry. Biomed Chromatogr. 24(1), 39-48. 

  • Rao KSP, Rao KVR. 1984. Impact of methyl parathion toxicity and eserine inhibition on acetylcholinesterase activity in tissues of the teleost (Tilapia mossambica)—A correlative study. Toxicol Lett 22:351–356.

  • Reddy MS, Jayaprada P, Rao KVR. 1990. Impact of methyl parathion and malathion on cholinergic and non‐cholinergic enzyme systems of penaeid prawn, Metapenaeus monoceros. Biochem Int 22:769–780.

  • Sogob MA, Vilanova E.  2002. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis.  Toxicol  Lett 128:215-228.

  • Szilagyi, P.I.A., Schmidt, D.E., Green, J.P. (1968). Microanalytical determination of acetylcholine, other choline esters, and choline by pyrolysis-gas chromatography. Analytical Chemistry. 40(13), 2009-2013. 

  • Zapata, A., V.I. Chefer, T.S. Shippenberg, and L. Denoroy. 2009. Detection and quantification of neurotransmitters in dialysates. Curr. Protoc. Neurosci. Chapter 7:Unit 7.4.1-30.