Event:12

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Event Title

Acetylcholinesterase (AchE), Inhibition
Short name: Acetylcholinesterase (AchE), Inhibition

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Acetylcholinesterase inhibition leading to acute mortality MIE Strong

Chemical Initiators

The following are chemical initiators that operate directly through this Event:

  1. Organophosphates
  2. Carbamates

Taxonomic Applicability

Name Scientific Name Evidence Links

Level of Biological Organization

Biological Organization

How this Key Event works

"Acetylcholinesterase is found primarily in blood, brain, and muscle, and regulates the level of the neurotransmitter ACh at cholinergic synapses of muscarinic and nicotinic receptors. Acetylcholinesterase features an anionic site (glutamate residue), and an esteratic site (serine hydroxyl group) [20,21]. In response to a stimulus, ACh is released into the synaptic cleft and binds to the receptor protein, resulting in changes to the flow of ions across the cell, thereby signaling nerve and muscle activity. The signal is stopped when the amine of ACh binds at the anionic site of AChE, and aligns the ester of ACh to the serine hydroxyl group of the enzyme. Acetylcholine is subsequently hydrolyzed, resulting in a covalent bond with the serine hydroxyl group and the subsequent release of choline, followed by a rapid hydrolysis of the enzyme to form free AChE and acetic acid [20,21]." [Russom et al. 2014. Environ. Toxicol. Chem. 33: 2157-2169]

Molecular target gene symbol: ACHE
KEGG enzyme: EC 3.1.1.7

How it is Measured or Detected

  • Direct measures of AChE activity levels can be made using the modified Ellman method, although selective inhibitors that remove other cholinesterases not directly related to cholinergic responses (e.g., butyrylcholinesterase) are required [45,46].
  • Radiometric methods have been identified as better for measuring inhibition because of carbamylation (carbamate exposure) [20,46,47].
[Russom et al. 2014. Environ. Toxicol. Chem. 33: 2157-2169] 
  • TOXCAST: NVS_ENZ_hAChE
  • A direct measure of cholinesterase activity levels can be made within the relevant tissues after in vivo exposure, specifically the brain as well as red blood cells in mammals. Some analytical methods used to measure cholinesterase activity may not distinguish between butyrylcholinesterase, which is found with AChE in plasma and some skeletal and muscle tissues. Although the structure of butyrylcholinesterase is very similar to AChE, its biological function is not clear, and its activity is not associated with cholinergic response covered under this AOP (Lushington et al., 2006). Therefore experimental procedures used to measure cholinesterase as well as the tissue analyzed should be considered when evaluating studies reporting AChE inhibition (Wilson 2010; Wilson and Henderson 2007). For measuring AChE levels, the Ellman method is recommended with some modifications (Ellman et al., 1961; Wilson et al., 1996) while radiometric methods have been identified as better for measuring inhibition due to carbamylation (carbamate exposure) (see Wilson 2010; Wilson et al., 1996; Johnson and Russell 1975).
  • In order to effectively bind to the AChE enzyme, thion forms of OPs (i.e., RO)3P=S) must first undergo a metabolic activation via mixed function oxidases to yield the active, oxon form (Fukuto 1990). Estimating the potential toxicity in whole organisms based on in vitro data may be problematic since metabolic activation may be required (e.g., phosphorothionates) and may not be reflected in the in vitro test result (Guo et al. 2006; Lushington et al. 2006).
  • Typically, carbamates do not require metabolic activation in order to bind to the enzyme, although some procarbamates (e.g., carbosulfan) have been developed that are not direct inhibitors of AChE, but take advantage of metabolic distinctions between taxa, resulting in a toxic form in invertebrates (e.g., carbofuran) but not vertebrate species (Stenersen 2004). Therefore in vitro assays measuring AChE inhibition for procarbamates in invertebrate species will not account for metabolic activation and therefore may not represent the actual enzyme activity.

Evidence Supporting Taxonomic Applicability

  • AChE is present in all life stages of both vertebrate and invertebrate species [27].
  • Acetylcholinesterase associated with cholinergic responses in most insects is coded by the ace1 gene and in vertebrates by the ace gene [27,28].
  • Plants have AChE but it is most likely involved in regulation of membrane permeability and the ability of a leaf to unroll (Tretyn and Kendrick 1991).
  • The primary amino acid sequence of the AChE enzyme is relatively well conserved across vertebrate and invertebrate species, suggesting that chemicals are likely to interact with the enzyme in a similar manner across a wide range of animals. From the sequence similarity analyses, the taxonomic domain of applicability of this MIE likely includes species belonging to many lineages, including branchiopoda (crustaceans, e.g., daphnids), insecta (insects), arachnida (arachnids, e.g., spiders, ticks, scorpions), cephalopoda (molluscans, e.g., octopods, squids), lepidosauria (reptiles, e.g., snakes, lizards), chondrichthyes (cartilaginous fishes, e.g., sharks), amphibia (amphibians), mammalian (mammals), aves (birds), actinopterygii (bony fish), ascidiacea (sac-like marine invertebrates), trematoda (platyhelminthes, e.g., flatworms), and gastropoda (gastropods, e.g., snails and slugs) Species within these taxonomic lineages and others are predicted to be intrinsically susceptible to

chemicals that target functional orthologs of the daphnid AChE.

[Russom et al. 2014. Environ. Toxicol. Chem. 33: 2157-2169]


  • Advanced computational approaches such as crystal structures of the enzyme and transcriptomics have provided empirical evidence of the enzyme structure, relevant binding sites, and function across species (Lushington et al., 2006; Lu et al., 2012; Wallace 1992).

Evidence for Chemical Initiation of this Molecular Initiating Event

  • Organophosphate and carbamate insecticides are prototypical AChE inhibitors. The OP and carbamate pesticides were synthesized specifically to act as inhibitors of AChE, with OPs developed from early nerve agents (e.g., sarin) and carbamate pesticides based on the natural plant alkaloid physostigmine (Ecobichon 2001).
  • A positive and significant correlation between the log of the Eserine IC50 (in vitro) for AChE inhibition and the log Km value for the AChE in the fish and crustacea species has been reported, explaining 92% of the variation in enzyme inhibition (Monserrat and Bianchini, 2001). Similar success was found in relating the rate constants for inhibition of AChE in housefly and the pseudo first-order hydrolysis rate constant for active forms of OPs (Fukuto 1990).
  • The open literature includes many studies on vertebrate and invertebrate species that demonstrate a clear dependence of AChE activity on the dose or concentration of the substance with increased concentrations leading to an increase in the inhibition of AChE (e.g., fish ( Karen et al., 2001), birds (Hudson et al., 1984 (see dimethoate and disulfoton), Grue and Shipley 1984; and Al-Zubaidy et al., 2011); cladocera (Barata et al., 2004); nematodes (Rajini et al., 2008); rodents (Roberts et al., 1988; and mollusk (Bianco et al., 2011)).
  • The open literature includes many studies on vertebrate and invertebrate species that demonstrate a clear relationship between increasing AChE inhibition as duration of exposure increases (e.g., amphibians ( Venturino et al., 2001); fish (Rao 2008; Ferrari et al., 2004); insects (Rose and Sparks 1984); birds (Ludke 1985; Grue and Shipley 1984); annelids (Reddy and Rao 2008); cladocera (Barata et al., 2004)).
  • Rao et al. 2008 exposed the estuarine fish Oreochromis mossambicus to a 24 h LC50 concentration of chlorpyrifos and reported that it took 6 hr to reach >40% AChE inhibition and 24 hr to reach 90% AChE inhibition. It took >100 days to recover to normal AChE levels when fish were placed in clean water.
  • A time course study of earthworms (Eisenis foetida) exposed to the 48 hr LC50 of profenofos found a significant relationship (between increases in percent inhibition of AChE and increase in time of exposure from 8-48 hrs (Chakra Reddy and Rao 2008).

References

[20] Wilson BW. 2010. Cholinesterases. Hayes’ Handbook of Pesticide Toxicology, 3rd ed, Vol 2. Elsevier, Amsterdam, The Netherlands, pp 1457-1478.

[21] Soreq H, Seidman S. 2001. Acetylcholinesterase -- New roles for an old actor. Nature Reviews Neurosci 2:294-302.

[27] Lu Y, Park Y, Gao X, Zhang X, Yoo J, Pang X-P, Jiang H, Zhu KY. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci Rep 2:1-7.

[28] Taylor P. 2011. Anticholinesterase agents. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 12th ed, McGraw Hill, New York, NY, USA, pp 255-276.

[29] Tretyn A, Kendrick RE. 1991. Acetylcholine in plants: Metabolism and mechanism of action. Bot Rev 57:33-73.

[45] Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. 1961. A new and rapid colormetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88-95.

[46] Wilson BW, Padilla S, Henderson JD, Brimijoin S, Dass PD, Elliot G, Jaeger B, Lanz D, Pearson R, Spies R. 1996. Factors in standardizing automated cholinesterase assays. J Toxicol Environ Health 48:187-195.

[47] Johnson CD, Russell RL. 1975. A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations. Anal Biochem 64:229-238.

[48] Augustinsson KB. 1957. Assay methods for cholinesterases. Methods of Biochemical Analysis, Vol 5, Interscience Publishers, Inc., New York, NY, USA, pp 1-63.

Lushington, G.H., J-X. Guo, and M.M. Hurley. 2006. Acetylcholinesterase: Molecular modeling with the whole toolkit. Curr. Topics Medic. Chem. 6: 57-73.

Wilson, B.W. and J.D. Henderson. 2007. Determination of cholinesterase in blood and tissue. Current Protocols in Toxicology 12.13.1-12.13.16.

Guo, J.-X., J.J.-Q. Wu, J.B. Wright, and G.H. Lushington. 2006. Mechanistic insight into acetylcholinesterase inhibition and acute toxicity of organophosphorus compounds: A molecular modeling study. Chem. Res. Toxicol. 19: 209-216.

Stenersen, J. 2004. Specific enzyme inhibitors. In: Chemical Pesticides: Mode of action and toxicology. (41 p). CRC Press, Boca Raton, FL.

Ecobichon, D.J. 2001. Toxic effects of pesticides. In: C.D. Klaassen (Ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons; Sixth Edition. (pp. 763-810). McGraw-Hill, New York, NY.

Monserrat, J.M. and A. Bianchini. 2001. Anticholinesterase effect of eserine (physostigmine) in fish and crustacean species. Braz. Arch. Biol. Technol. 44(1): 63-68.

Fukuto, TR. 1990. Mechanism of action of organophosphorus and carbamate insecticides. Environ Health Perspect. 87:245-254.