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CHRISTINE L. RUSSOM (1), CARLIE A. LALONE (2), DANIEL L. VILLENEUVE* (1), and GERALD T. ANKLEY (1)
- (1)National Health and Environmental Effects Research Laboratory, Office of Research and Development, Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA
- (2)Water Resources Center, College of Food, Agricultural, and Natural Resource Sciences, University of Minnesota, St. Paul, Minnesota, USA
- Corresponding author for wiki entry (Villeneuve.email@example.com)
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OECD Project 1.3: The Adverse Outcome Pathways for Acetylcholinesterase Inhibition.
This AOP was last modified on 12/5/2016.
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The contents of this AOP page represent a network of two separate AOPs linking the MIE of acetylcholinesterase inhibition to the AO of acute mortality. Both AOPs include the KE of acetylcholine accumulation at the synapses. The downstream consequences of this KE are dependent on the tissue context and the specific type(s) of acetylcholine receptors present in those tissues. For example, one of the AOPs focuses on the effect of excess acetylcholine at the neuromuscular junctions of skeletal muscle which is mediated through impacts on nicotinic (N2, Nm) acteylcholine receptor. The other AOP considers impacts of acetycholine at the synapses in cardiac tissue, where muscarinic acetylcholine receptors (M3) are known to be involved in regulation of heart rate. While these two AOPs are represented on the basis of their most plausible linkages to acute mortality, other known symptoms of acetylcholinesterase inhibition mediated through actions on other receptors and tissues are also known and may also play a role (see Russom et al. 2014). Overall, there is strong evidence supporting the linkage of acetylcholinesterase inhibition and acetylcholine accumulation with acute mortality, however, the evidence supporting a direct role of the intermediate organ level key events is less clear. This network of AOPs as a whole, including the indirect KERs depicted, supports the potential utility of in vitro or short-term in vivo measures of acetylcholinesterase inhibition for identifying chemicals with potential to cause systemic neurotoxicity at sub-narcotic concentrations. At present, quantitative understanding is not sufficiently complete to accurately predict apical outcomes or potency from in vitro measurements alone, and well known chemical initiators of these AOPs are known to require metabolic activation, suggesting chemical-specific ADME and toxicokinetic considerations will be strong determinant of quantitative outcomes along these AOPs.
To date, review of the available literature regarding the role of intermediate key events is incomplete. It is likely that extant literature could be used to further evaluate those relationships.
Summary of the AOP
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Molecular Initiating Event
|Molecular Initiating Event||Support for Essentiality|
|Acetylcholinesterase (AchE), Inhibition||Strong|
|Event||Support for Essentiality|
|Acetylcholine in synapses, Accumulation||Strong|
|Atrioventricular block and bradycardia, Increased||Weak|
|Respiratory distress/arrest, Increased||Weak|
|Ataxia, paralysis, or hyperactivity, Induction||Weak|
|Population trajectory, Decreased|
Relationships Among Key Events and the Adverse Outcome
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Life Stage Applicability
Overall Assessment of the AOP
Weight of Evidence Summary
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.
- There is strong evidence that the initial inhibition of the AChE enzyme is required prior to triggering key events that lead to the adverse outcome of mortality (See US EPA 2006; Grue and Shipley 1984).
- There is strong empirical evidence linking the key events, beginning with the molecular initiating event; AChE inhibition, followed by an increase in the acetylcholine at synapses of muscarinic and nicotinic receptors, and subsequent physiological and biochemical response resulting in cholinergic activity resulting in the death of the organism.
- Strong evidence based on measured AChE inhibition and statistically-derived acute endpoints (e.g., LC/LD50) demonstrate a correlation of increase in enzyme inhibition and increased lethality. The open literature has many studies reporting these effects across invertebrate and vertebrate species with examples presented below.
- The overall weight of evidence supporting the indirect relationship between AChE inhibition and mortality is very strong and there are many physiological activities associated with acetylcholine neurotransmission that are plausibly linked with organism survival. However, there remain significant gaps in the current AOP description regarding which specific intermediate events are primarily responsible for the toxicity observed. It is likely that it is a combination of these physiological responses rather than any one alone, and that the key driver is also species dependent.
Essentiality of the Key Events
- There are numerous studies which have shown the blockage or reversibility of downstream events following the administration of pharmacological agents that either bypass acetylcholinesterase activity by directly activating acetylcholine receptors or that act as direct agonists or antagonists of the various types of acetylcholine receptors. Additionally, evidence from experiments with AChR morpholino antisense oligonucleotides have provided additional evidence for an essential role of acetylcholine receptor activation in mediating some of the downstream key events.
- Based on the current information assembled for this AOP, the essentiality of the key events downstream of acetylcholine accumulation is less clear. While there are several key events that correspond with well known symptoms of acetylcholinesterase inhibition (as characterized through a number of the indirect KERs), it is presently unclear which of these are the major driver of lethality. Given the abundance of literature on acetylcholine signaling and adverse effects associated with acetylcholinesterase inhibition, this is an area of the AOP that warrants further development.
Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.
Applicability of the AOP
Life Stage Applicability,
Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.
Sex: No studies were located reporting significant differences in AChE activity between male and female organisms.
Life stages: The key molecular target is the AChE enzyme, which appears to be available in all life stages of vertebrate and invertebrate species, although studies have found that AChE activity increases as the organism develops.
- AChE in developing precocial birds were found to level off at the embryo life stage, while in altricial species the AChE activity increased as the bird developed until it reached a steady state at adulthood (Grue et al., 1981).
- A study examining the acetylcholine and cholinesterase levels in developing brains of Sprague-Dawley rats found that acetylcholine synthesis was significantly lower in neonate and juveniles when compared to the adult levels, and the neonate cholinesterase levels were significantly lower than adult levels of activity (Karanth and Pope 2003). When exposed to either parathion or chlorpyrifos, the researchers found differences in peak inhibition of cholinesterase with neonates seeing the greatest impact early in the exposure (4-24 hrs), while juveniles and adults saw the most severe impacts at 96 hrs of exposure (Karanth and Pope 2003). Exposure to pesticides did not seem to impact acetylcholine production in the rats regardless of life stage, but researchers thought the pesticides may be altering other biochemical process (e.g., choline acetyltransferase) which might ultimately impact the measures of acetylcholine (Karanth and Pope 2003).
- In Drosophila, changes in AChE activity in the developing organism were observed, with egg stages displaying the lowest activity, and maximum activity at the pupae life stage (Parkash and Kaur 1982).
- In mammals and birds, studies have determined that skeletal muscles of immature birds and mammals contain both butyrylcholinesterase and AChE, with butyrylcholinesterase decreasing and AChE increasing as the animal develops (Tsim et al. 1988; Berman et al, 1987).
- Another study found that changes in AChE within the developing pig brain were dependent on the area of the brain and life stage of the animal, with significant decreases in activity within the pons and hippocampus from birth to 36 months, and no significant change in activity in the cerebellum, where activity increased up to four months of age, leveling off thereafter (Adejumo and Egbunike, 2004).
- Evidence exists that immature life stages in mammals and birds may be more sensitive to OP pesticides (see Grue et al., 1997; Grue et al., 1983; Grue et.al; 1981), although this may be related to the amount of pesticide ingested in relation to body size (Ludke et al, 1975).
- Researchers reported that frog (Bufo arenarum Hensel) embryos were more tolerant to parathion exposure than frog larvae, and associated this with the ability of the embryo AChE to recover to baseline levels faster than the larval life stage (Anguiano et al, 1994).
Taxonomic: Although AChE enzyme is found in all vertebrate and invertebrate species, the activity within taxa varies.
- An examination of the STRING (V9.0 http://string-db.org), which is a database of known and predicted protein interactions, finds that the AChE enzyme is well conserved across vertebrate and invertebrate species.
- A pattern of species sensitivity for OPs using terrestrial species found birds to be highly sensitive, mammals moderately sensitive, and fish and amphibians of lower sensitivity (Wallace 1992).
- Taxonomic differences as they relate to key events within the AOP may be due to a number of factors. For instance, the organism’s habitat (e.g., sediment, water, and grass), diet, body size, behavior, mobility, and skin/exoskeleton permeability could all affect the level of exposure and the ability of the chemical to reach the molecular target. A complicating factor when identifying relative species sensitivities are differences in routes of exposure which may impact the time it takes for the substance to reach the molecular target, and impacts to adsorption, distribution, metabolism, and elimination (ADME) within the organism.
- Phase I metabolism of phosphorothioates and phosphorodithioates by mixed function oxidases results in the more toxic oxon, which is the form that binds to AChE. Differences in species sensitivity may be impacted by the rate at which this reaction occurs, as would any mechanism related to detoxification of the oxon form. For instance, in mammals the oxon form undergoes an ester detoxification pathway which is not present in insect species, resulting in insects having a higher susceptibility to OPs than mammals (Ecobichon 2001). Similarly for procarbamates, invertebrates are able to convert the substance to the active form, while vertebrate species lack this metabolic mechanism (Stenersen 2004).
- Studies have provided evidence that differences in the AChE enzyme across taxa may better explain differences in species sensitivity. Evidence suggests that the relative activity of AChE is linked to the hydrophobic and electronic configuration of the enzyme, which directly impacts the speed and/or efficiency that a substance can bind to the enzyme, and could impact the ability/speed of the enzyme to reactivate to its normal state (Wallace, 1992; Wallace and Kemp 1991). For instance, taxonomic differences in the electronic and steric properties of the esteratic site, nucleophilic strength of the enzyme center, the distance between the anionic and esteratic sites, and the electronic/steric properties of the anionic site may all impact the relative binding efficiency of the enzyme to the pesticide (O’Brien 1963; Monserrat and Bianchini 2001; Wallace, 1992; Wallace and Kemp 1991).
- Baseline levels of the enzyme can significantly vary depending on species, strains, age class, sex, season, reproductive and nutritional status, and disease state (Cowman and Mazanti, 2000; Hill 1988; Rattner and Fairbrother 1991).
- There are specific differences in the genes that code for the cholinergic AChE enzyme. In vertebrate species, AChE is encoded by a single gene (Ace) resulting in a conserved enzyme across the taxonomic group (Lu et al., 2012; Taylor 2011). AChE is encoded in the Diptera suborder Brachycera (e.g., Drosophila, common house fly) by the gene Ace2, while in other insects both an Ace1 and Ace2 gene encode AChE (Lu et al., 2012). The Ace1 gene produces an AChE with a cysteine residue, which is not found in vertebrate AChE, or in the AChE from the Ace2 gene form. Acetylcholinesterase from Ace1 is associated with neurotransmissions within the insect, while AChE from the Ace2 gene is responsible for non-cholinergic activity such as embryonic development, growth, and reproduction (Lu et al., 2012).
- Comparisons of susceptibility of Xenopus laevis and human forms of AChE to OP and carbamate pesticides found that the enzyme in frog embryos has a much higher resistance to these pesticides than the human form of the enzyme (Shapira et al., 1998).
- Acetylcholinesterase is found in presynaptic membranes of motor neurons in the spinal cord, cranial nerves within skeletal muscle, and preganglionic sympathetic and postganglionic parasympathetic neurons of vertebrates (Mileson et al., 1998). In invertebrates, AChE appears to be associated with sensory, brain, and other muscle activity (Fulton and Key, 2001; Habig and Di Giulio 1991; Mileson et al. 1998; Ware and Whitacre 2004).
- In plants, the function of AChE is not well understood, but levels of acetylcholine appear to be involved in the regulation of membrane permeability, and the ability of a leaf to unroll (Tretyn and Kendrick 1991).
Chemical Domain: In order to trigger the AChE inhibition AOP (Figure2), the substance must meet certain electronic and steric constraints, and for OPs, have a leaving group sufficiently electronegative to ensure the formation of a reactive electrophile (Fukuto 1990; Sogob and Vilanova 2002; Schűűrmann 1992). Substances with subtle structural differences can result in major changes in AChE inhibition capabilities. For example, OPs having identical R and R1 alkyl groups, display decreasing AChE inhibition as the R / R1 carbon chain increases from a single carbon to a propyl moiety, with the later resulting in an ineffective inhibition of AChE (Fukuto 1990). Metabolism also plays an important part in the potency of OPs and to some extent carbamates. For instance, OPs in the phosphorothionate and phosphorodithioate families (i.e., P=S) must undergo metabolic activation to the oxon form in order to inhibit AChE effectively (Fukuto 1990). Similarly, procarbamates require metabolism to form an active AChE inhibitor (e.g., carbosulfan must be metabolized to carbofuran), and others are made more potent via metabolism (e.g., aldicarb oxidation to the more toxic sulfoxide form) (Sogob and Vilanova 2002; Stenersen 2004).
Considerations for Potential Applications of the AOP (optional)
- U.S. Environmental Protection Agency (U.S. EPA). 2006. Organophosphorus Cumulative Risk Assessment – 2006. Update. U.S. EPA, Office of Pesticide Programs, Washington, DC. 522 p.
- Grue, C.E. and B.K. Shipley. 1984. Sensitivity of nestling and adult starling to dicrotophos, and organophosphate pesticide. Environ. Res. 35:454-465.
- Grue, C.E., G.V.N. Powell, and N.L. Gladson. 1981. Brain cholinesterase (ChE) activity in nestling starlings: Implications for monitoring exposure of nestling songbirds to ChE inhibitors. Bull. Environ. Contam. Toxicol. 26: 544-547.
- Karanth, S. and Pope, C. 2003. Age-Related Effects of Chlorpyrifos and Parathion on Acetylcholine Synthesis in Rat Striatum. Neurotoxicol.Teratol. 25, 599-606.
- Parkash, R. and K. Kaur. 1982. Ontogeny of acetylcholinesterases in hybridizing Drosophila species. Proc. Indian Nat. Sci. Acad. B48(5): 659-666.
- Tsim, K.W.K., W.R. Randall, and E.A. Barnard. 1988. Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogenous enzyme. EMBO J 7(8): 2451-2456.
- Berman, H.A., M.M. Decker, and J. Sangmee. 1987. Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle. Dev. Biol. 120(1): 154-161.
- Adejumo, D.O. and G.N. Egbunike. 2004. Changes in acetylcholinesterase activities in the developing and aging pig brain and hypophyses. Int. J. Agric. Rural. Dev. 5: 46-53.
- Grue, C.E., P.L. Gibert, and M.E. Seeley. 1997. Neurophysiological and behavioral changes in non-target wildlife exposed to organophosphate and carbamate pesticides: Thermoregulation, food consumption, and reproduction. Amer. Zool. 37: 369-388.
- Grue, C.E., W.J. Fleming, D.G. Busby, and E.F. Hill. 1983. Assessing hazards of organophosphate pesticides to wildlife. In: Transactions of the 48th North American Wildlife and Natural Resources Conference, The Wildlife Management Institute, Washington, DC. pp. 200-220.
- Ludke, J.L., E.F. Hill, and M.P. Dieter. 1975. Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch. Environ. Contam. Toxicol. 3(1): 1-21.
- Anguiano, O.L., C.M. Montagna, M. Chifflet de Llamas, L. Gauna, and A.M. Pechen de D'Angelo. 1994. Comparative toxicity of parathion in early embryos and larvae of the toad, Bufo arenarum Hensel. Bull. Environ. Contam. Toxicol. 52(5): 649-655.
- Wallace, K.B. 1992. Species-selective toxicity of organophosphorus insecticides: A pharmacodynamics phenomenon. In: J. E. Chambers and P. E. Levi, (Eds.), Organophosphates— Chemistry, Fate, and Effects. pp. 79-105. Academic Press, San Diego.
- 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.
- Stenersen, J. 2004. Specific enzyme inhibitors. In: Chemical Pesticides: Mode of action and toxicology. (41 p). CRC Press, Boca Raton, FL.
- Wallace, K.B., and J.R. Kemp. 1991. Species specificity in the chemical mechanisms of organophosphorus anticholinesterase activity. Chem. Res. Toxicol. 4: 41-49.
- O’Brien, R.D. 1963. Mode of action of insecticides: Binding of organophosphate to cholinesterases. Agric. Food Chem. 11(2): 163-166.
- 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.
- Cowman, D.F. and L.E. Mazanti. 2000. Ecotoxicology of “new generation” pesticides to amphibians. In: D.W. Sparling, G. Linder, and C.A. Bishop (Eds.), Ecotoxicology of Amphibians and Reptiles, pp 233-268, SETAC Press, Pensacola, FL.
- Hill, E.F. 1988. Brain cholinesterase activity of apparently normal wild birds. J.Wild. Dis. 24(1): 51-61.
- Rattner, B.A. and A. Fairbrother. 1991. Biological variability and the influence of stress on cholinesterase activity. In: P. Mineau (Ed.), Cholinesterase-inhibiting Insecticides: Their Impact on Wildlife and the Environment. (pp. 89-107). Elsevier, Amsterdam, The Netherlands.
- Lu, Y., Y. Park, X. Gao, X. Zhang, J. Yoo, Y.-P. Pang, H. Jiang, and K.Y. Zhu. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci. Rep. 2(Article No. 288):1-7.
- Taylor, P. 2011. Anticholinesterase agents. In: L.J. Brunton (Ed.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics; 12th Edition. (pp. 255-276). McGraw Hill, New York, NY. (Accessed from the web: http://accessmedicine.com/resourceTOC.aspx?resourceID=651).
- Shapira, M., S. Seidman, N. Livni, and H. Soreq. 1998. In vivo and in vitro resistance to multiple anticholinesterases in Xenopus laevis tadpoles. Toxicol. Lett. 102-103: 205-209.
- Mileson, B.E., J.E. Chambers, W.L. Chen, W. Dettbarn, M. Ehrich, A.T. Eldefrawi, D.W. Gaylor, K. Hamernik, E. Hodgson, A.G. Karczmar, S. Padilla, C.N. Pope, R.J. Richardson, D.R. Saunders, L.P. Sheets, L.G. Sultatos, and K.B. Wallace. 1998. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41: 8-20.
- Fulton, M.H. and P.B. Key. 2001. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorous insecticide exposure and effects. Environ. Toxicol. Chem. 20(1): 37-45.
- Habig, C. and R.T. DiGiulio. 1991. Biochemical characteristics of cholinesterases in aquatic organisms. In: P. Mineau (Ed.), Cholinesterase-inhibiting Insecticides: Their Impact on Wildlife and the Environment. pp. 19-33. Elsevier, Amsterdam, The Netherlands.
- Ware, G.W. and D.M. Whitacre. 2004. An Introduction to Insecticides. In: E. B. Radcliffe,W. D. Hutchison and R. E. Cancelado [Eds.], Radcliffe's IPM World Textbook. 34 pp. University of Minnesota, St. Paul, MN. (Accessed from the web: URL: http://ipmworld.umn.edu).
- Tretyn, A. and R.E. Kendrick. 1991. Acetylcholine in plants: Metabolism and mechanism of action. Bot. Rev. 57(1): 33-73.
- Fukuto, TR. 1990. Mechanism of action of organophosphorus and carbamate insecticides. Environ Health Perspect. 87:245-254.
- Sogob MA, Vilanova E. 2002. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicol Lett 128:215-228.
- Schűűrmann G. 1992. Ecotoxicology and structure-activity studies of organophosphorus compounds. Rational Approaches to Structure, Activity, and Ecotoxicology of Agrochemicals, CRC Press, Boca Raton, FL, USA pp 485-541