AOP ID and Title:

Authors

Kendra Conrow (a)

Dennis Sinitsyn (a)

Natalia Garcia-Reyero (c)

Karen H. Watanabe (a)

(a) Arizona State University

(b) IDAEA-CSIC

(c) US Army Corps of Engineers, Engineering Research and Development Center

Status

Abstract

The enzyme acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh) in order to eliminate it from the body.  When AChE is inhibited ACh levels increase. An excess of ACh at cholinergic synapses overstimulates both muscarinic- and nicotinic- receptors (1,2). These receptors are found in most organs in the body, thus the effects of AChE inhibition can result in multiple adverse outcomes affecting a wide variety of functions (1). This AOP focuses upon an acute outcome of neurodegeneration due to AChE inhibition specifically through calcium dysregulation as that has been identified as central to the development of the most severe phenotype caused by acute organophosphate poisoning (3).

1. United States., Environmental Protection Agency., Office of Pesticide Programs. (2000). The Use of Data on Cholinesterase Inhibition for Risk Assessments of Organophosphorous and Carbamate Pesticides. https://www.epa.gov/sites/production/files/2015-07/documents/cholin.pdf accessed Nov. 2018.

2. Quick, M. W., & Lester, R. A. J. (2002). Journal of Neurobiology, 53(4), 457-478. doi:10.1002/neu.10109.

3. Faria et al. (2015). Scientific Reports, 5. doi:10.1038/srep15591.

Background

Epidemiological studies concerning OP pesticides estimated approximately 3 million cases of acute severe poisoning, as well as 300,000 deaths annually. Most of those deaths occur in developing countries of the Asia-Pacific region (Bertolote et al., 2006). These OP compounds can also be used as chemical warfare nerve agents. The improper use of OP chemicals has tragic consequences such as neurodegeneration, brain damage, and death underscoring the need for safety measures that protect both human health and the environment.

Bertolote, J. M., Fleischmann, A., Eddleston, M. & Gunnell, D. 2006. Deaths from pesticide poisoning: A global response. British Journal of Psychiatry, 189, 201-203. DOI: 10.1192/bjp.bp.105.020834.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Overall Assessment of the AOP

Domain of Applicability

Sex: The AOP is not sex-specific

Life stages: the AOP is relevant to all life stages. Immature or developing populations may be more sensitive due to their increased susceptibility to seizures and developing cholinergic systems.

Taxonomic: given that both cholinergic and glutamatergic systems are highly conserved among vertebrates, this AOP is likely to be applicable to all vertebrates.

Essentiality of the Key Events

All KEs in AOP 281 rank high for essentiality.  The provided studies demonstrate direct evidence and include experiments involving inhibition of AChE through the application of various inhibitors, gene-knockout experiments, receptor antagonist studies, and anticonvulsant treatments which are shown to result in the reduction of neurodegeneration.

• AChE Inhibition (MIE) evidence is high. This is supported by several studies that measured increases in ACh after inhibition of AChE by a variety of inhibitors (Del Pino et al., 2017, Karanth et al., 2007, Kim et al., 2003, Kosasa et al., 1999, Ray et al., 2009). Additionally, researchers have demonstrated that pretreatment with a combination of reversible AChE inhibitors, nicotinic and mAChR receptor antagonists prior to exposure to soman resulted in a significantly higher survival rate and overall reduced brain ACh levels compared to controls (Harris et al., 1980).
• ACh accumulation in synapses (KE 1) evidence is high. Blocking the effects of ACh with atropine, an mAChR antagonist, was demonstrated to significantly reduce the pathological effects and neurodegeneration associated with soman intoxication (McDonough et al., 1989).
• Activation of mAChRs (KE 2) evidence is high. M1-mAChR deficient mice through gene-knockout studies were shown to be resistant to seizures induced by pilocarpine, an mAChR agonist (Hamilton et al., 1997).
• Occurrence of Focal Seizure (KE 3) evidence is high. Treatment with diazepam, a GABA_A receptor agonist and known anticonvulsant, both prevented seizures and resulted in significantly reduced brain pathology (McDonough et al., 1989).
• Increased Glutamate (KE 4) evidence is high. Application of 500 µM of glutamate showed in reduction in neuron survival, however if NMDA antagonist MK-801 was used in conjunction with glutamate, neuron survival returned to control levels (Michaels and Rothman, 1990). Other in vivo experiments using MK-801 or ketamine demonstrated a reduction in seizure activity and reduced neurodegeneration (Borris et al., 2000, Braitman and Sparenborg, 1989, Sparenborg et al., 1992).
• Overactivation of NMDARs (KE 5) evidence is high. Multiple experiments using ketamine and MK-801, both NMDA receptor antagonists, have been demonstrated to terminate or reduce both seizure activity and neurodegeneration (Borris et al., 2000, Braitman and Sparenborg, 1989, Sparenborg et al., 1992).
• Increased Intracellular Calcium Overload (KE 6) evidence is high. Calcium chelation in zebrafish models of organophosphate exposure significantly reduced neurodegeneration (Faria et al., 2015). Additionally, Deshpande et al. (2008) demonstrated that cell death could be significantly reduced given a low extracellular calcium solution in an in vitro model of SE in rat hippocampal neurons.
• Status Epilepticus (KE 7) evidence follows that of KE 3 and is considered high. Anticonvulsant treatment using diazepam was demonstrated to significantly reduce neurodegeneration (McDonough et al., 1989).
• Cell Injury/Death (KE 8) evidence is considered high. Cell death in the context of the brain is considered a form of neurodegeneration (Przedborski et al., 2003).  Therefore, prevention of cell death directly results in the prevention of the adverse outcome.

Weight of Evidence Summary

Biological plausibility: Biological plausibility refers to the structural or functional relationship between the key events based on our fundamental understanding of "normal biology". The evidence for biological plausibility throughout this AOP from inhibition of AChE to neurodegeneration is high. It is well understood that inhibition of AChE is followed by an accumulation of ACh, which subsequently leads to activation of muscarinic acetylcholine receptors and focal seizures. The seizures then lead to increased glutamate, which binds to and overactivates NMDARs. Following that step, we find the highest biological uncertainty in the pathway, with moderate biological plausibility (from overactivation of NMDARs leading to status epilepticus to increased glutamate). The rest of the pathway is considered of high biological plausibility all the way to neurodegeneration.

Concordance of dose-response relationships: Dose response concordance considers the degree to which upstream events are shown to occur at test concentrations equal to or lower than those that cause significant effects on downstream key events, the underlying assumption being that all KEs can be measured with equal precision. There is a significant amount of quantitative data providing dose and temporal concordance for multiple species between AChE inhibitors and the resulting percent AChE inhibition and ACh concentration (Kosasa et al., 1999). Dose-response relationships have been well stablished by showing that AChE inhibition resulted in the progressive accumulation of extracellular ACh. Furthermore, the relationship between increased intracellular calcium and cell death through dose and temporal concordance has also been demonstrated. Additionally, Faria et al. (2015) demonstrated a dose-response relationship between increasing doses of the organophosphate chlorpyrifos-oxon and the prevalence of a severe phenotype marked by measurably increased necrosis.

Temporal concordance: Temporal concordance refers to the degree to which the data support the hypothesized sequence of the key events; i.e., the effect on KE1 is observed before the effect on KE2, which is observed before the effect on KE3 and so on. Temporal concordance has been shown between seizure activity and increasing levels of glutamate (KE4 and KE7). Furthermore, temporal concordance has also been established between status epilepticus and increased intracellular calcium in rats. The relationship between increased intracellular calcium and cell death through dose and temporal concordance has also been demonstrated.

Consistency: We are not aware of cases where the whole chain of key events described was observed without also observing a significant impact on neurodegeneration. Nevertheless, the final adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to other adverse outcomes.

Uncertainties, inconsistencies, and data gaps: The current main uncertainties within this AOP are related to seizures and the location of AChE inhibition. Even though it is well known that there are two phases of seizure activity driven by cholinergic and glutamatergic mechanisms, the transition between these phases is not well understood. Additionally, the levels of increasing glutamate post-AChE inhibition appear to be dependent on the location of inhibition as well as stressor specific.

Quantitative Consideration

At present, the quantitative understanding of this AOP varies by level of biological organization. While the initial KEs leading to activation of muscarinic acetylcholine receptors have a high level of quantitative understanding, following KEs leading all the way to the Adverse Outcome (Cell Injury / Death Leading to Neurodegeneration) have a much lower quantitative understanding. The exception would be KE5 (Increased Glutamate leading to Overactivation of NMDARs), that has multiple kinetic models available to evaluate quantitative relationships. Overall, better quantitative relationships need to be developed to be able to quantitively and effectively predict the adverse outcome.

References

Borris, D. J., Bertram, E. H. & Kapur, J. 2000. Ketamine controls prolonged status epilepticus. Epilepsy Res, 42, 117-22. DOI: 10.1016/s0920-1211(00)00175-3.

Braitman, D. J. & Sparenborg, S. 1989. MK-801 protects against seizures induced by the cholinesterase inhibitor soman. Brain Research Bulletin, 23, 145-148. DOI: 10.1016/0361-9230(89)90173-1.

Del Pino, J., Moyano, P., Díaz, G. G., Anadon, M. J., Diaz, M. J., García, J. M., Lobo, M., Pelayo, A., Sola, E. & Frejo, M. T. 2017. Primary hippocampal neuronal cell death induction after acute and repeated paraquat exposures mediated by AChE variants alteration and cholinergic and glutamatergic transmission disruption. Toxicology, 390, 88-99. DOI: 10.1016/j.tox.2017.09.008.

Deshpande, L. S., Lou, J. K., Mian, A., Blair, R. E., Sombati, S., Attkisson, E. & DeLorenzo, R. J. 2008. Time course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus: role of NMDA receptor activation and NMDA dependent calcium entry. Eur J Pharmacol, 583, 73-83. DOI: 10.1016/j.ejphar.2008.01.025.

Faria, M., Garcia-Reyero, N., Padrós, F., Babin, P. J., Sebastián, D., Cachot, J., Prats, E., Arick Ii, M., Rial, E., Knoll-Gellida, A., Mathieu, G., Le Bihanic, F., Escalon, B. L., Zorzano, A., Soares, A. M. & Raldúa, D. 2015. Zebrafish Models for Human Acute Organophosphorus Poisoning. Sci Rep, 5, 15591. DOI: 10.1038/srep15591.

Hamilton, S. E., Loose, M. D., Qi, M., Levey, A. I., Hille, B., McKnight, G. S., Idzerda, R. L. & Nathanson, N. M. 1997. Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice. Proceedings of the National Academy of Sciences, 94, 13311-13316. DOI: 10.1073/pnas.94.24.13311.

Harris, L. W., Stitcher, D. L. & Heyl, W. C. 1980. The effects of pretreatments with carbamates, atropine and mecamylamine on survival and on soman-induced alterations in rat and rabbit brain acetylcholine. Life Sci, 26, 1885-91. DOI: 10.1016/0024-3205(80)90617-7.

Karanth, S., Liu, J., Ray, A. & Pope, C. 2007. Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats. Toxicology, 239, 167-179. DOI: https://doi.org/10.1016/j.tox.2007.07.004.

Kim, Y. K., Koo, B. S., Gong, D. J., Lee, Y. C., Ko, J. H. & Kim, C. H. 2003. Comparative effect of Prunus persica L. BATSCH-water extract and tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) on concentration of extracellular acetylcholine in the rat hippocampus. J Ethnopharmacol, 87, 149-54. DOI: 10.1016/s0378-8741(03)00106-5.

Kosasa, T., Kuriya, Y., Matsui, K. & Yamanishi, Y. 1999. Effect of donepezil hydrochloride (E2020) on basal concentration of extracellular acetylcholine in the hippocampus of rats. European Journal of Pharmacology, 380, 101-107. DOI: 10.1016/S0014-2999(99)00545-2.

McDonough, J. H., Jr., Jaax, N. K., Crowley, R. A., Mays, M. Z. & Modrow, H. E. 1989. Atropine and/or diazepam therapy protects against soman-induced neural and cardiac pathology. Fundam Appl Toxicol, 13, 256-76. DOI: 10.1016/0272-0590(89)90262-5.

Michaels, R. L. & Rothman, S. M. 1990. Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. J Neurosci, 10, 283-92. DOI: 10.1523/jneurosci.10-01-00283.1990.

Przedborski, S., Vila, M. & Jackson-Lewis, V. 2003. Neurodegeneration: what is it and where are we? J Clin Invest, 111, 3-10. DOI: 10.1172/jci17522.

Ray, A., Liu, J., Karanth, S., Gao, Y., Brimijoin, S. & Pope, C. 2009. Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats. Toxicology and Applied Pharmacology, 236, 341-347. DOI: 10.1016/j.taap.2009.02.022.

Sparenborg, S., Brennecke, L. H., Jaax, N. K. & Braitman, D. J. 1992. Dizocilpine (MK-801) arrests status epilepticus and prevents brain damage induced by soman. Neuropharmacology, 31, 357-68. DOI: 10.1016/0028-3908(92)90068-z.

Appendix 1

List of MIEs in this AOP

Event: 12: Acetylcholinesterase (AchE) Inhibition

Short Name: AchE Inhibition

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Overview for 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).

Organophosphates

The MIE, AChE inhibition, is triggered via electrostatic interaction at the anionic site of the enzyme and binding with the serine hydroxyl group at the esteratic site of AChE (Wilson 2010; Fukuto 1990).  Organophosphate pesticides attach to the AChE via an ‘irreversible’ phosphorylation of the enzyme. Note that the use of the term ‘irreversible’ relates to the relative rate at which the phosphorylation occurs since acetylcholine and organophosphates both form covalent bonds with the enzyme. The phosphorylated form may persist for up to a week if it has undergone an ‘aging’ process; i.e., the organophosphate has undergone a dealkylation, thereby strengthening the bond between the OP and the enzyme (Mileson et al. 1998; Kropp and Richardson 2003; Sogob and Vilanova 2002).  Certain steric and electronic requirements must be met in order for an organophosphate to inhibit AChE. For instance, organophosphates require 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 latter resulting in an ineffective AChE inhibitor (Fukuto 1999).  

Metabolism also plays an important role in the potency of organophosphates.  For instance, organophosphates in the phosphorothionate and phosphorodithioate families (i.e., P=S) must undergo metabolic activation, via cytochrome P450-based monoxygenases, to an oxon form in order to inhibit AChE effectively (Fukuto 1990). 

Evidence exists that immature life stages in mammals and birds may be more sensitive to organophosphate pesticides (see Grue et al., 1997; Grue et al., 1983; Grue et.al; 1981). It has been suggested that this may be related to the amount of pesticide ingested in relation to body size (Ludke et al, 1975), but there is direct data in rats showing that differential sensitivity to OPs is determined at least in part by inadequate detoxification in the young (Moser, 2011). OP detoxification is highly dependent on enzymes such as A-esterases (paraoxonases, PON) and carboxylesterases (e.g., Benke and Murphy, 1974; Furlong, 2007; Sterri et al., 1985; Vilanova and Sogorb, 1999), which are present at lower levels in the young (e.g., Chanda et al., 2002; Mendoza, 1976; Mortensen et al., 1996; Moser et al., 1998).

N-methyl Carbamates

Carbamates trigger AChE inhibition through electrostatic interactions at the enzyme’s anionic site and binding with the serine hydroxyl group at the esteratic site (Wilson 2010; Fukuto 1990). Carbamates, which were originally based on the plant alkaloid physostigmine, attach to the AChE via a ‘reversible’ carbamylation. Note that the use of the term ‘reversible’ relates to the relative rate at which the carbamylation occurs since acetylcholine and carbamates both form covalent bonds with the enzyme. Certain steric and electronic requirements, as well as the leaving group on the pesticide, are critical to the likelihood that the methyl-carbamate will inhibit AChE (See Figure).  

Metabolism also plays a role in the potency of some carbamates.  Select procarbamates require metabolism to form an active AChE inhibitor (e.g., carbosulfan must be metabolized to carbofuran), or are made more potent via metabolism (e.g., aldicarb oxidation to the more toxic sulfoxide form) (Sogob and Vilanova 2002; Stenersen 2004). 

Domain of Applicability

AChE is present in all life stages of both vertebrate and invertebrate species (Lu et al 2012).

• Acetylcholinesterase associated with cholinergic responses in most insects is coded by the ace1 gene and in vertebrates by the ace gene (Lu et al 2012; Taylor 2011.

• 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, 2014).

• 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).

Studies have found that AChE activity increases as the organism develops.

• Prakesh and Kaur 1982 looked at AChE inhibition across three insect species; controls and those exposed to DDVP. They saw little difference in the larval stages but did see increased inhibition in pupal and adult stages (greatest inhibition). 

• Karanth and Pope 2003 looked at AChE and acetylcholine synthesis in rat striatum in controls and animals exposed to 0.3 and 1 times the maximum tolerated dose. Although these doses are below the lethal concentrations and they mention that not observed cholinergic responses were observed, they do provide differences related to life stages of the rodents. 

• Grue et al 1981 present baseline (no toxicity exposure) in wild starlings (both sexes) of brain cholinesterase and found activity increased as birds aged from 1-20 days until it reached a steady state at adulthood.

• A study with Red Flour Beetle found that the gene associated with cholinergic functions (Ace1) was expressed at all life-stages, with increases as the organism developed from egg to larva to pupa to adult. (Lu et al., 2012 cited in Russom et al 2014.)

• 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).

Key Event Description

"Acetylcholinesterase is found primarily in blood, brain, and muscle, and regulates the level of the neurotransmitter ACh [acetylcholine] at cholinergic synapses of muscarinic and nicotinic receptors. Acetylcholinesterase features an anionic site (glutamate residue), and an esteratic site (serine hydroxyl group) (Wilson, 2010; Soreq, 2001). 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 (Wilson, 2010; Soreq, 2001)." [From 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].
• 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.

References

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• 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.

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• Kropp, T.J., and Richardson, R.J. 2003. Relative inhibitory potencies of chlorpyrifos oxon, chlorpyrifos methyl oxon, and mipafox for acetylcholinesterase versus neuropathy target esterase. J. Toxicol. Environ.l Health, Part A, 66:1145–1157.

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• Ludke JL, Hill EF, Dieter MP. 1975. Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch Environ ContamToxicol 3:1–21.

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• 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.

• Moser, Virginia C. 2011. “Age-Related Differences in Acute Neurotoxicity Produced by Mevinphos, Monocrotophos, Dicrotophos, and Phosphamidon.” Neurotoxicology and Teratology 33 (4): 451–57. https://doi.org/10.1016/j.ntt.2011.05.012.

• 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.

• Russom, Christine L., Carlie A. LaLone, Daniel L. Villeneuve, and Gerald T. Ankley. 2014. “Development of an Adverse Outcome Pathway for Acetylcholinesterase Inhibition Leading to Acute Mortality.” Environmental Toxicology and Chemistry 33 (10): 2157–69. https://doi.org/10.1002/etc.2662.

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

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

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

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

• 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.

• 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.

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

List of Key Events in the AOP

Event: 10: Acetylcholine accumulation in synapses

Short Name: ACh Synaptic Accumulation

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

• 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).

Key Event Description

• 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

• 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).

References

• 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.

Event: 1602: Activation, Muscarinic Acetylcholine Receptors

Short Name: Activation, Muscarinic Acetylcholine Receptors

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxa:

           mAChRs are found in most vertebrates, many of the studies cited are conducted using zebrafish and mice. Zebrafish are frequently used for high-throughput assays as they have well-conserved neurotransmitter structures, including acetylcholine transmitters (Garcia et al., 2016). This can provide valuable data regarding the activation of mAChRs in mammalian systems. Knockout mice also help to elucidate the functions of specific mAChR subtypes (Gainetdinov and Caron, 1999).

Life stage:

           mAChRs signal neurons throughout all life stages (Miller and Yeh, 2016). They do not only affect individuals during developmental stages, but there have been some studies conducted specifically on the developmental effects of chemicals that affect acetylcholine signaling (Burke et al., 2017). Most of the whole animal experimental data are from younger specimens, but there have also been experiments on adult individuals (Fitzgerald and Costa, 1993).

Sex:

           mAChRs are found in both males and females, with similar functions (Burke et al., 2017).

Key Event Description

           Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors (GPCRs) with five different subtypes (M1, M2, M3, M4, and M5). GPCRs are transmembrane receptors that detect extracellular signals and activate internal pathways which modulate a variety of processes such as locomotion, learning and memory, thermoregulation and epileptic seizures (Gainetdinov and Caron, 1999).  Subtypes M1, M3, and M5 are Gq- coupled receptors that activate phospholipase C enzyme resulting in two secondary messengers, inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). Subtypes M2 and M4 are inhibitory and signal using the G_i pathway (Haga, 2013). G_i protein activation inhibits adenylyl cyclase, and reduces the conversion of ATP to cAMP (Jett and Lein, 2011).

           In its resting state, the mAChR G-protein subunits (alpha, beta and gamma) are clustered together and the alpha subunit is bound to GDP.  Once a ligand binds to an mAChR, the receptor undergoes a conformation change that allows the alpha subunit to exchange its bound GDP with GTP, then the alpha subunit dissociates from the beta and gamma subunits. Once the alpha subunit is free of the beta and gamma subunits, it moves along the cell membrane to affect its target enzyme, which typically sends out secondary messenger signals (Kandel et al., 2013)

How it is Measured or Detected

           Most studies investigating the function of mAChRs involve blocking signaling from these receptors through use of selective antagonists like atropine or scopolamine, or the use of gene targeted knockout specimens (Bymaster et al. 2003; Faria et al. 2017). The distribution and density of mAChRs can be measured using radiolabeled agonists that bind to the mAChR binding site. The receptor activity can be measured by detecting secondary-messengers regulated by the G-protein.

• Use mAChR agonist [³H] quinuclidinyl benzilate (QNB) to label mAChRs (all subtypes; see Fonnum and Sterri (2011) and measure binding levels as described by Fitzgerald and Costa (1993) and Gazit et al. (1979)
• Determination of the relative levels of specific mAChR subtypes in tissues has been found through the use of subtype-specific antisera as described by Dörje et al. (1991)
• Kinetic measurements of DAG production and IP3 release can be obtained through fluorescent reporters as in Falkenburger et al. (2013) and Dickson et al. (2013).
• Changes in the activity and quantity of cAMP and the cAMP-dependent protein kinases can serve as an indicator of the activity of mAChRs bound to Gi-proteins (M2 and M4). cAMP content can be determined using a radioimmunoassay (RIA) kit (Heikkilä et al., 1991).
• Adenylyl cyclase activity can be determined through an assay as described by Salomon et al. (1974) and used by Raheja and Dip Gill (2007).

References

Burke, R. D., S. W. Todd, E. Lumsden, R. J. Mullins, J. Mamczarz, W. P. Fawcett, R. P. Gullapalli, W. R. Randall, E. F. R. Pereira and E. X. Albuquerque (2017), "Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: from clinical findings to preclinical models and potential mechanisms”, Journal of Neurochemistry 142: 162-177. DOI: 10.1111/jnc.14077.

Dickson, E. J., B. H. Falkenburger and B. Hille (2013), "Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling”, Journal of General Physiology 141(5): 521-535. DOI: 10.1085/jgp.201210886.

Dörje, F., A. I. Levey and M. R. Brann (1991), "Immunological detection of muscarinic receptor subtype proteins (m1-m5) in rabbit peripheral tissues”, Molecular Pharmacology 40(4): 459-462.

Falkenburger, B. H., E. J. Dickson and B. Hille (2013), "Quantitative properties and receptor reserve of the DAG and PKC branch of G_q-coupled receptor signaling”, The Journal of General Physiology 141(5): 537-555. DOI: 10.1085/jgp.201210887.

Faria, M., Prats, E., Padrós, F., Soares, A. M., & Raldúa, D. (2017). Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication. Archives of toxicology, 91(4), 1891-1901.

Fitzgerald, B. B. and L. G. Costa (1993), "Modulation of Muscarinic Receptors and Acetylcholinesterase Activity in Lymphocytes and in Brain Areas Following Repeated Organophosphate Exposure in Rats”, Fundamental and Applied Toxicology 20(2): 210-216. DOI: 10.1006/faat.1993.1028.

Fonnum, F. and S. H. Sterri (2011), “Tolerance Development to Toxicity of Cholinesterase Inhibitors”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 257-267.

Gainetdinov, R. R. and M. G. Caron (1999), "Delineating muscarinic receptor functions”, Proceedings of the National Academy of Sciences of the United States of America 96(22): 12222-12223. DOI: 10.1073/pnas.96.22.12222.

Garcia, G. R., P. D. Noyes and R. L. Tanguay (2016), "Advancements in zebrafish applications for 21st century toxicology”, Pharmacology and Therapeutics 161: 11-21. DOI: 10.1016/j.pharmthera.2016.03.009.

Gazit, H., I. Silman and Y. Dudai (1979), "Administration of an organophosphate causes a decrease in muscarinic receptor levels in rat brain”, Brain Research 174(2): 351-356. DOI: 10.1016/0006-8993(79)90861-8.

Haga, T. (2013), "Molecular properties of muscarinic acetylcholine receptors”, Proceedings of the Japan Academy Series B: Physical and Biological Sciences 89(6): 226-256. DOI: 10.2183/pjab.89.226.

Heikkilä, J., C. Jansson and K. E. O. Åkerman (1991), "Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells”, European Journal of Pharmacology: Molecular Pharmacology 208(1): 9-15. DOI: 10.1016/0922-4106(91)90045-J.

Jett, D. A. and P. J. Lein (2011), “Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 233-245.

Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), “Modulation of Synaptic Transmission: Second Messengers”, in Principles of Neural Science, Fifth Edition, Blacklick, United States, McGraw-Hill Publishing: 236-259.

Miller, S. L. and H. H. Yeh (2016), “Neurotransmitters and Neurotransmission in the Developing and Adult Nervous System”, in Conn's Translational Neuroscience: 49-84.

Raheja, G. and K. Dip Gill (2007), "Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain”, Toxicology and Industrial Health 23(1): 25-37. DOI: 10.1177/0748233707072490.

Salomon, Y., C. Londos and M. Rodbell (1974), "A highly sensitive adenylate cyclase assay”, Anal Biochem 58(2): 541-548. DOI: 10.1016/0003-2697(74)90222-x.

Event: 1623: Occurrence, Focal Seizure

Short Name: Occurrence, Focal Seizure

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxa

           Seizures have been observed and studied in many different species including vertebrate and invertebrates. Listed species above are specifically referenced in the cited sources.

Age

           There is evidence indicating that in developing rat brains GABAergic activity might be excitatory, not inhibitory (Li and Xu, 2008). Increased sensitivity shown by younger individuals to some substances that induce seizures may possibly be affected by this phenomenon (Miller, 2015).

Sex

           Both males and females can develop focal seizures, with some possible differences in sensitivity to certain forms of epileptic activity (Belelli et al., 1990). Despite some differences the effect of the key event is conserved for both sexes.

Key Event Description

           This key event is characterized as the start of synchronized neural signaling in a specific group of neurons. It is possible that when the ratio between excitatory (glutamatergic) over inhibitory (GABAergic) currents in brain tissue increases past the threshold of the network , seizure starts to occur (Miller, 2015). The initial occurrence of epileptiform activity, in specific regions of the brain, can begin a signaling cascade leading to seizure spread throughout the brain (i.e., secondary generalization leading to status epilepticus) (Kandel et al., 2013).

 Acetylcholinesterase inhibition induced seizure

           For the signaling cascade caused by acetylcholinesterase inhibition to continue to propagate, some studies suggest that stimulation specifically in the basolateral amygdala plays a key role in the development of seizure activity (McDonough Jr and Shih, 1997). Other studies indicate that the piriform cortex as well as the hippocampus also play a role in seizure development caused by nerve agents (Myhrer, 2007).

How it is Measured or Detected

• An electrocorticogram record can be used to measure brain activity to monitor seizure development (Braitman and Sparenborg, 1989).
• Brain electroencephalographic (EEG) activity can also record the development of the seizure (Acon-Chen et al., 2016; Kandel et al., 2013).
• Whole cell recordings of spontaneous inhibitory postsynaptic currents and excitatory postsynaptic currents have also been used to study the initial seizures occurring from exposure to organophosphates (Miller, 2015).

References

Acon-Chen, C., J. A. Koenig, G. R. Smith, A. R. Truitt, T. P. Thomas and T. M. Shih (2016), "Evaluation of acetylcholine, seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats”, Toxicology Mechanisms and Methods 26(5): 378-388. DOI: 10.1080/15376516.2016.1197992.

Belelli, D., N. C. Lan and K. W. Gee (1990), "Anticonvulsant steroids and the GABA/benzodiazepine receptor-chloride ionophore complex”, Neuroscience & Biobehavioral Reviews 14(3): 315-322. DOI: https://doi.org/10.1016/S0149-7634(05)80041-7.

Braitman, D. J. and S. Sparenborg (1989), "MK-801 protects against seizures induced by the cholinesterase inhibitor soman”, Brain Research Bulletin 23(1-2): 145-148. DOI: 10.1016/0361-9230(89)90173-1.

Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), “Seizures and Epilepsy”, in Principles of Neural Science, Fifth Edition, Blacklick, United States, McGraw-Hill Publishing: 1116-1139.

Li, K. and E. Xu (2008), "The role and the mechanism of gamma-aminobutyric acid during central nervous system development”, Neuroscience bulletin 24(3): 195-200. DOI: 10.1007/s12264-008-0109-3.

McDonough Jr, J. H. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology”, Neuroscience and Biobehavioral Reviews 21(5): 559-579. DOI: 10.1016/S0149-7634(96)00050-4.

Miller, S. L. (2015), The Efficacy of LY293558 in Blocking Seizures and Associated Morphological, and Behavioral Alterations Induced by Soman in Immature Male Rats and the Role of the M1 Muscarinic Acetylcholine Receptor in Organophosphate Induced Seizures. Doctor of philosophy in the neuroscience graduate program Doctoral dissertation, Uniformed Services University.

Myhrer, T. (2007), "Neuronal structures involved in the induction and propagation of seizures caused by nerve agents: Implications for medical treatment”, Toxicology 239(1-2): 1-14. DOI: 10.1016/j.tox.2007.06.099.

Event: 1350: Increased, glutamate

Short Name: Increased, glutamate

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxa:

Zebrafish neurotransmitter systems, including glutamate, are being used more for investigating chemical toxicity (Horzmann and Freeman 2016). Some cited sources above have data from rat experiments.

Life Stage:

Glutamate is functional throughout all life stages. Liu et al. (1996) suggests that immature rat brains show less glutamate-induced neurotoxicity than adult brains.

Sex:

Glutamate and glutamate receptors have been studied in both males and females, with similar functionality (Jafarian et al. 2019).

Key Event Description

Glutamate (Glu) release into the synaptic cleft is primarily caused by excitatory glutamatergic neurons, however there is evidence showing astrocytes releasing glutamate through a calcium-dependent process. A mechanism explaining how astrocytes release glutamate is not well defined, but it could be released through exocytosis(Nedergaard et al. 2002). Glutamate is the main excitatory transmitter in the brain and spinal cord, where it activates both ionotropic and metabotropic receptors. There are 3 main ionotropic receptor classifications, AMPA, Kainate, and NMDA receptors, which are always excitatory (Kandel et al. 2013: 213). Excessive extracellular glutamate release overactivates these signaling pathways, and propagates the excitotoxicity caused by some nerve agents (McDonough and Shih 1997).

How it is Measured or Detected

• Glutamate uptake by astrocytes and synaptic cleft concentration can be measured using liquid scintillation spectrometry and radiolabeled glutamate (H³ glutamate) (Lallement et al. 1991). Liquid scintillation spectrometry counts the activity of a radioactive sample by mixing the glutamate with a liquid scintillator (a material that fluorescens) and count photon emissions.
• Another mechanism to measure the glutamate concentration in the synaptic cleft is by microdialysis sampling. This mechanism is inexpensive and easy to use. When microdialysis is paired with other analytical methods such as High-Pressure Liquid Chromatography (HPLC), there is a higher instrumental selectivity and sensitivity (Watson et al. 2006).

References

Horzmann, K. A. and J. L. Freeman (2016), "Zebrafish get connected: investigating neurotransmission targets and alterations in chemical toxicity.” Toxics 4(3).

Jafarian, M., S. M. Modarres Mousavi, F. Alipour, H. Aligholi, F. Noorbakhsh, M. Ghadipasha, J. Gharehdaghi, C. Kellinghaus, S. Kovac, M. Khaleghi Ghadiri, S. G. Meuth, E. J. Speckmann, W. Stummer and A. Gorji (2019), "Cell injury and receptor expression in the epileptic human amygdala.” Neurobiology of Disease 124. DOI: 10.1016/j.nbd.2018.12.017.

Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), Principles of Neural Science, Fifth Edition. Blacklick, United States, McGraw-Hill Publishing.

Lallement, G., P. Carpentier, A. Collet, I. Pernot-Marino, D. Baubichon and G. Blanchet (1991), "Effects of soman-induced seizures on different extracellular amino acid levels and on glutamate uptake in rat hippocampus.” Brain Research 563(1-2). DOI: 10.1016/0006-8993(91)91539-D.

Liu, Z., C. E. Stafstrom, M. Sarkisian, P. Tandon, Y. Yang, A. Hori and G. L. Holmes (1996), "Age-dependent effects of glutamate toxicity in the hippocampus.” Brain Res Dev Brain Res 97(2).

McDonough, J. H., Jr. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology.” Neurosci Biobehav Rev 21(5).

Nedergaard, M., T. Takano and A. J. Hansen (2002), "Beyond the role of glutamate as a neurotransmitter.” Nature Reviews Neuroscience 3(9). DOI: 10.1038/nrn916.

Watson, C. J., B. J. Venton and R. T. Kennedy (2006), "In vivo measurements of neurotransmitters by microdialysis sampling.” Analytical Chemistry 78(5).

Event: 388: Overactivation, NMDARs

Short Name: Overactivation, NMDARs

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

It is important to note that in invertebrates the glutamatergic synaptic transmission has an inhibitory and not an excitatory role like in vertebrates. This type of neurotransmission is mediated by glutamate-gated chloride channels that are members of the ‘cys-loop’ ligand-gated anion channel superfamily found only in invertebrates. The subunits of glutamate-activated chloride channel have been isolated from C. elegans and from Drosophila (Blanke and VanDongen, 2009).

Key Event Description

Biological state: Please see MIE NMDARs, Binding of antagonist

Biological compartments: Please see MIE NMDARs, Binding of antagonist

General role in biology: Please see MIE NMDARs, Binding of antagonist

The above chapters belong to the AOP entitled: Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities since the general characteristic of the NMDA receptor biology is the same for both AOPs.

Additional text, specific for this AOP:

At resting membrane potentials, NMDA receptors are inactive. Depending on the specific impulse train received, the NMDA receptor activation triggers long term potentiation (LTP) or long-term depression (LTD) (Malenka and Bear, 2004; Luscher and Malenka, 2012). LTP (the opposing process to LTD) is the long-lasting increase of synaptic strength. For LTP induction both pre- and postsynaptic neurons need to be active at the same time because the postsynaptic neuron must be depolarized when glutamate is released from the presynaptic bouton to fully relieve the Mg2+ block of NMDARs that prevents ion flows through it. Sustained activation of AMPA or KA receptors by, for instance, a train of impulses arriving at a pre-synaptic terminal, depolarizes the post-synaptic cell, releasing Mg2+ inhibition and thus allowing NMDA receptor activation. Unlike GluA2-containing AMPA receptors, NMDA receptors are permeable to calcium ions as well as being permeable to other ions. Thus NMDA receptor activation leads to a calcium influx into the post-synaptic cells, a signal that is instrumental in the activation of a number of signalling cascades (Calcium-dependent processes are describe in Key Event Calcium influx, increased). Postsynaptic Ca2+ signals of different amplitudes and durations are able to induce either LPT or LTD.

Conversely to LTP, LTD is induced by repeated activation of the presynaptic neuron at low frequencies without postsynaptic activity (Luscher and Malenka, 2012). Therefore, under physiological conditions LTD is one of several processes that serves to selectively weaken specific synapses in order to make constructive use of synaptic strengthening caused by LTP. This is necessary because, if allowed to continue increasing in strength, synapses would ultimately reach a ceiling level of efficiency, which would inhibit the encoding of new information (Purves, 2008).

LTD is an activity-dependent reduction in the efficacy of neuronal synapses lasting hours or longer following a long patterned stimulus. It has also been found to occur in different types of neurons however, the most common neurotransmitter involved in LTD is L-glutamate that acts on the NMDARs, AMPAR, KARs and metabotropic glutamate receptors (mGluRs). It can result from strong synaptic stimulation (as occurs e.g. in the cerebellar Purkinje cells) or from persistent weak synaptic stimulation (as in the hippocampus) resulting mainly from a decrease in postsynaptic AMPA receptor density, although a decrease in presynaptic neurotransmitter release may also play a role. Moreover, cerebellar LTD has been hypothesized to be important for motor learning and hippocampal LTD may be important for the clearing of old memory traces (Nicholls et al., 2008; Mallere et al., 2010). The main molecular mechanism underlying-LTD is the phosphorylation of AMPA glutamate receptors and their synaptic elimination (Ogasawara et al., 2008).

It is now commonly understood in the field of spine morphology that long lasting NMDAR-dependent LTD causes dendritic spine shrinkage, reduces number of synaptic AMPA receptors (Calabrese et al., 2014), possibly leading to synaptic dysfunction, contributing to decreased neuronal network function and impairment of learning and memory processes.

Additional text, specific for the AOP “Acetylcholinesterase inhibition leading to neurodegeneration”:

              Seizures caused by cholinesterase dependent mechanisms result in an excess of glutamate release  that activates the NMDA receptors.  As a result, intracellular Ca2+ levels at the postsynaptic neuron can overload the calcium-control mechanisms, activating without control all the calcium-dependent enzymes (proteases, lipases…) (Deshpande et al., 2014; Garcia-Reyero et al., 2016). In cases of strong acetylcholinesterase inhibition of the CNS, the NMDAR overactivation initiated by cholinergic mechanisms can result, after the initial seizure activity (focal seizure), in the development of status epilepticus. This key event separates the initial toxicity, driven by cholinergic activity, from the secondary toxicity, which is cholinergic independent  (McDonough and Shih, 1997).

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

No OECD methods are available to measure the activation state of NMDA receptors.

The measurement of the activation or the inhibition of NMDA receptors is done indirectly by recording the individual ion channels that are selective to Na+, K+ and Ca2+ by the patch clamp technique. This method relies on lack of measurable ion flux when NMDA ion channel is closed, whereas constant channel specific conductance is recorded at the open state of the receptor (Blanke and VanDongen, 2009). Furthermore, this method is based on the prediction that activation or inhibition of an ion channel results from an increase in the probability of being in the open or closed state, respectively (Ogdon and Stanfield, 2009; Zhao et al., 2009).

The whole-cell patch clamp recording techniques have also been used to study synaptically-evoked NMDA receptor-mediated excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) in brain slices and neuronal cells, allowing the evaluation of the activated or inhibited state of the receptor.

Microelectrode array (MEA) recordings are used to measure mainly spontaneous network activity of cultured neurons (Keefer et al., 2001, Gramowski et al., 2000 and Gopal, 2003; Johnstone et al., 2010). However, using specific agonists and antagonists of a receptor, including NMDAR, MEA technology can be used to measure evoked activity, including glutamatergic receptor function (Lantz et al., 2014). For example it has been shown that MEA-coupled neuronal cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission (Frega et al., 2012). MEAs can also be applied in higher throughput platforms to facilitate screening of numerous chemical compounds (McConnell et al., 2012).

Excessive excitability can be also measured directly by evaluating the level of the extracellular glutamate using the enzyme-based microelectrode arrays. This technology is capable of detecting glutamate in vivo, to assess the effectiveness of hyperexcitability modulators on glutamate release in brain slices. Using glutamate oxidase coated ceramic MEAs coupled with constant voltage amperometry, it is possible to measure resting glutamate levels and synaptic overflow of glutamate after K(+) stimulation in brain slices (Quintero et al., 2011).

Neuronal network function can be also measured using optical detection of neuronal spikes both in vivo and in vitro (Wilt et al., 2013).

Drebrin immunocytochemistry: drebrin, a major actin-filament-binding protein localized in mature dendritic spines is a target of calpain mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. The NMDA-induced degradation of drebrin in mature neurons occurres concomitantly with a loss of f-actin. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. These findings suggest that calpain-mediated degradation of drebrin is mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin (A and E) regulates the synaptic clustering of NMDARs. Therefore, degradation of drebrin can be used as a readout for excitotoxicity induced by NMDAR overactivation. Degradation of drebrin can be evaluated quantitatively by Western blot analysis (mRNA evel) or by immunocytochemistry (at protein level) (Chimura et al., 2015: Sekino et al., 20069.

NMDAR overactivation-induced long lasting LTD can be measured by the dendritic spine shrinkage by quantification of cofilin and phospho-cofilin in neurons expressing eGFP and combined with immunocytochemical techniques (Calabrese et al., 2014).

References

Blanke ML, VanDongen AMJ., Activation Mechanisms of the NMDA Receptor. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; 2009, Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5274/.

Calabrese B, Saffin JM, Halpain S. Activity-dependent dendritic spine shrinkage and growth involve downregulation of cofilin via distinct mechanisms. PLoS One., 2014, 16;9(4):e94787.

Chimura T., Launey T., Yoshida N.,Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo PLOS ONE, 2015, |DOI:10.1371/journal.pone.0125119.

Deshpande, L. S., D. S. Carter, K. F. Phillips, R. E. Blair and R. J. DeLorenzo (2014), "Development of status epilepticus, sustained calcium elevations and neuronal injury in a rat survival model of lethal paraoxon intoxication”, NeuroToxicology 44: 17-26. DOI: 10.1016/j.neuro.2014.04.006.

Frega M, Pasquale V, Tedesco M, Marcoli M, Contestabile A, Nanni M, Bonzano L, Maura G, Chiappalone M., Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing. Neurotoxicol Teratol. 2012: 34(1):116-27.

Garcia-Reyero, N., L. Escalon, E. Prats, M. Faria, A. M. V. M. Soares and D. Raldúa (2016), "Targeted Gene Expression in Zebrafish Exposed to Chlorpyrifos-Oxon Confirms Phenotype-Specific Mechanisms Leading to Adverse Outcomes”, Bulletin of Environmental Contamination and Toxicology 96(6): 707-713. DOI: 10.1007/s00128-016-1798-3.

Gopal K., Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis. Neurotoxicol Teratol., 2003, 25: 69-76.

Gramowski A, Schiffmann D, Gross GW., Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultures on microelectrode arrays. Neurotoxicology, 2000, 21: 331-342.

Johnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ.,Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology, 2000, 31: 331-350.

Keefer E, Norton S, Boyle N, Talesa V, Gross G., Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays. Neurotoxicology, 2001, 22: 3-12.

Lantz SR, Mack CM, Wallace K, Key EF, Shafer TJ, Casida JE. Glufosinate binds N-methyl-D-aspartate receptors and increases neuronal network activity in vitro. Neurotoxicology. 2014, 45:38-47.

Luscher C. and Malenka R.C., NMDA Receptor-Dependent Long-Term Potentiation and Long-Term Depression (LTP/LTD). Cold Spring Harb Perspect Biol., 2012, 4:a005710.

Malenka RC, Bear MF., LTP and LTD: An embarrassment of riches. Neuron, 2004, 44: 5–21.

Malleret G, Alarcon JM, Martel G, Takizawa S, Vronskaya S, Yin D, Chen IZ, Kandel ER, Shumyatsky GP., Bidirectional regulation of hippocampal long-term synaptic plasticity and its influence on opposing forms of memory". J Neurosci., 2010, 30 (10): 3813–25.

McConnell ER, McClain MA, Ross J, LeFew WR, Shafer TJ., Evaluation of multi-well microelectrode arrays for neurotoxicity screening using a chemical training set Neurotoxicology, 2012, 33: 1048-1057.

McDonough, J. H., Jr. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology”, Neurosci Biobehav Rev 21(5): 559-579.

Nicholls RE, Alarcon JM, Malleret G, Carroll RC, Grody M, Vronskaya S, Kandel ER., Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility". Neuron, 2008, 58 (1): 104–17.

Ogasawara H, Doi T, Kawato M. Systems biology perspectives on cerebellar long-term depression. Neurosignals, 2008, 16 (4): 300–17.

Ogdon D, Stanfield P., Patch clamp techniques for single channel and whole-cell recording. Chapter 4, pages 53-78, (http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch04.pdf).

Paradiso MA, Bear MF, Connors BW., Neuroscience: exploring the brain. 2007, Hagerstwon, MD: Lippincott Williams & Wilkins. p. 718. ISBN 0-7817-6003-8.

Purves D., Neuroscience (4th ed.). Sunderland, Mass: Sinauer., 2008, pp. 197–200. ISBN 0-87893-697-1.

Sekino Y, Tanaka S, Hanamura K, Yamazaki H, Sasagawa Y, Xue Y, Hayashi K, Shirao T., Activation of N-methyl-D-aspartate receptor induces a shift of drebrin distribution: disappearance from dendritic spines and appearance in dendritic shafts. Mol Cell Neurosci. 2006, 31(3):493-504.

Quintero JE, Pomerleau F, Huettl P, Johnson KW, Offord J, Gerhardt GA. 2011. Methodology for rapid measures of glutamate release in rat brain slices using ceramic-based microelectrode arrays: basic characterization and drug pharmacology. Brain Res.2011, 1401:1-9.

Wilt BA, Fitzgerald JE, Schnitzer MJ., Photon shot noise limits on optical detection of neuronal spikes and estimation of spike timing. Biophys J. 2013, 8; 104(1):51-62.

Zhao Y, Inayat S, Dikin DA, Singer JH, Ruoff RS, Troy JB., Patch clamp techniques: review of the current state of art and potential contributions from nanoengineering. Proc. IMechE 222, Part N: J. Nanoengineering and Nanosystems, 2009, 149. DOI: 10.1243/17403499JNN149.

Event: 1788: Status epilepticus

Short Name: Status epilepticus

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

See KE Occurrence, Focal Seizure.

Key Event Description

Focal seizures occur when a small group of neurons start synchronized neural signaling (See KE Occurrence, Focal Seizure). Once started, focal seizures can spread to the entire brain through various axonal pathways. GABA-ergic interneurons help inhibit seizure spread from the seizure focus forming an inhibitory region. If the activity in the focus is intense enough that inhibitory region breaks down and the seizure spreads (Kandel et al., 2013). Once the epileptiform activity has expanded to other areas in the brain, i.e., once both hemispheres of the brain are involved for approximately 5 minutes, the focal seizure has been secondarily generalized (status epilepticus) (Lowenstein and Alldredge, 1998).

Acetylcholinesterase inhibition induced seizure

In the case of acetylcholinesterase inhibition, status epilepticus has been seen to be regulated through NMDAR activation and increasing intracellular Ca2+, which is distinct from the initial focal seizure through mAChRs (Acon-Chen et al., 2016).  Anticholinergic drugs (atropine, 2-PAM…) are ineffective if administrated after seizure generalization, whereas NMDAR antagonists (memantine, MK-801…) can still be effective 35 minutes after exposure (Lallement et al., 1999; McDonough and Shih, 1997). 

How it is Measured or Detected

See KE Occurrence, Focal Seizure.

References

Acon-Chen, C., J. A. Koenig, G. R. Smith, A. R. Truitt, T. P. Thomas and T. M. Shih (2016), "Evaluation of acetylcholine, seizure activity and neuropathology following high-dose nerve agent exposure and delayed neuroprotective treatment drugs in freely moving rats”, Toxicology Mechanisms and Methods 26(5): 378-388. DOI: 10.1080/15376516.2016.1197992.

Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), “Seizures and Epilepsy”, in Principles of Neural Science, Fifth Edition, Blacklick, United States, McGraw-Hill Publishing: 1116-1139.

Lallement, G., D. Clarencon, M. Galonnier, D. Baubichon, M. F. Burckhart and M. Peoc'h (1999), "Acute soman poisoning in primates neither pretreated nor receiving immediate therapy: value of gacyclidine (GK-11) in delayed medical support”, Arch Toxicol 73(2): 115-122. DOI: 10.1007/s002040050595.

Lowenstein, D. H. and B. K. Alldredge (1998), "Status Epilepticus”, New England Journal of Medicine 338(14): 970-976. DOI: 10.1056/nejm199804023381407.

McDonough, J. H., Jr. and T. M. Shih (1997), "Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology”, Neurosci Biobehav Rev 21(5): 559-579.

Event: 389: Increased, Intracellular Calcium overload

Short Name: Increased, Intracellular Calcium overload

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Please see KE Calcium influx, Decreasedin the AOP entitled Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

Additional text, specific for the AOP “Acetylcholinesterase Inhibition leading to Neurodegeneration”:

Zebrafish have shown dysregulation in intracellular calcium ion levels following exposure to organophosphate compounds through similar mechanisms demonstrated in mammals (Faria et al. 2015).

Key Event Description

NMDAR agonist binding results in increased intracellular calcium, whereas NMDAR antagonist binding results in decreased intracellular calcium levels. For the relevant paragraphs below please see AOP entitled Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

Biological state: KE Calcium influx, Decreased

Biological compartments: KE Calcium influx, Decreased

General role in biology: KE Calcium influx, Decreased

The text specific for the AOP "ionotropic glutamatergic receptors and cognition” and “Acetylcholinesterase inhibition leading to neurodegeneration”:

It is now well accepted that modest activation of NMDARs leading to modest increases in postsynaptic calcium are optimal for triggering LTD (Lledo et al. 1998; Bloodgood and Sabatin, 2007; Bloodgood et al. 2009), whereas much stronger activation of NMDARs leading to much larger increases in postsynaptic calcium are required to trigger LTP (Luscher and Malenka, 2012; Malenka 1994). Indeed, high-frequency stimulation causes a strong temporal summation of the excitatory postsynaptic potentials (EPSPs), and depolarization of the postsynaptic cell is sufficient to relieve the Mg2+ block of the NMDAR and allow a large amount of calcium to enter into the postsynaptic cells. Therefore, intra-cellular calcium is measured as a readout for evaluation NMDAR stimulation.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Please see KE Calcium influx, Decreasedin the AOP entitled: Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities.

References

Bloodgood BL, Sabatini BL., Nonlinear regulation of unitary synaptic signals by CaV2.3 voltage-sensitive calcium channels located in dendritic spines. Neuron, 2007, 53:249–260.

Bloodgood BL, Giessel AJ, Sabatini BL., Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines. PLoS Biol., 2009, 7: e1000190.

Faria, M., N. Garcia-Reyero, F. Padrós, P. J. Babin, D. Sebastián, J. Cachot, E. Prats, M. Arick Ii, E. Rial, A. Knoll-Gellida, G. Mathieu, F. Le Bihanic, B. L. Escalon, A. Zorzano, A. M. Soares and D. Raldúa (2015), "Zebrafish Models for Human Acute Organophosphorus Poisoning.” Sci Rep 5. DOI: 10.1038/srep15591.

Lledo PM, Zhang X, Sudhof TC, Malenka RC, Nicoll RA., Postsynaptic membrane fusion and long-term potentiation. Science, 1998, 279: 399–403.

Malenka RC. Synaptic plasticity in the hippocampus: LTP and LTD. Cell, 1994, 78: 535–538.

Luscher C. and Robert C. Malenka. NMDA Receptor-Dependent Long-Term Potentiation and Long-Term Depression (LTP/LTD). Cold Spring Harb Perspect Biol., 2012, 4: a005710.

Event: 55: Cell injury/death

Short Name: Cell injury/death

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).

Key Event Description

Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased intracellular Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.

DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining (see explanation below). Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.

Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell (Fujikawa, 2015). Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process (Fujikawa, 2015; Malhi et al., 2010). Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA (Fujikawa, 2015; Malhi et al., 2010; Kaplowitz, 2002; Kroemer et al., 2009).  

How it is Measured or Detected

Necrosis:

Lactate dehydrogenase (LDH) is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (Chan et al., 2013). 

The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes include XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light (Berridgeet al.,2005).

Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm (Moore et al.,1998)

Alamar Blue (resazurin) is a fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12).

Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13). Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega).

ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega).

Apoptosis:

TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.

Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence (Li, Peng et al., 2004).

Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis (Loo, 2002; Kubbies and Rabinovitch, 1983). 

Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.

References

• Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.
• Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.
• Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2, http://www.medscape.com/viewarticle/433631 (accessed on 20 January 2016).
• Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.
• Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.
• Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.
• Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.
• Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.
• Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.
• Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.
• Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.
• O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.
• Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.

List of Adverse Outcomes in this AOP

Event: 352: N/A, Neurodegeneration

Short Name: N/A, Neurodegeneration

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Domain of Applicability

The necrotic and apoptotic cell death pathways are quite well conserved throughout taxa (Blackstone and Green, 1999, Aravind et al., 2001). It has been widely suggested that apoptosis is also conserved in metazoans, although despite conservation of Bcl-2 proteins, APAF-1, and caspases there is no biochemical evidence of the existence of the mitochondrial pathway in either C. elegans or Drosophila apoptosis (Baum et al., 2007; Blackstone and Green, 1999).

Key Event Description

The term neurodegeneration is a combination of two words - "neuro," referring to nerve cells and "degeneration," referring to progressive damage. The term "neurodegeneration" can be applied to several conditions that result in the loss of nerve structure and function, and neuronal loss by necrosis and/or apoptosis

Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of “neurodegenerative diseases" including Huntington's, Alzheimer’s and Parkinson’s disease. All of these conditions lead to progressive brain damage and neurodegeneration.

Alzheimer's disease is characterised by loss of neurons and synapses in the cerebral cortex and certain subcortical regions, with gross atrophy of the affected regions; symptoms include memory loss.

Parkinson's disease (PD) results from the death of dopaminergic neurons in the midbrain substantia nigra pars compacta; symptoms include bradykinesia, rigidity, and resting tremor.

Several observations suggest correlative links between environmental exposure and neurodegenerative diseases, but only few suggest causative links:

Only an extremely small proportion (less than 5%) of neurodegenerative diseases are caused by genetic mutations (Narayan and Dragounov, 2017). The remainders are thought to be caused by the following:

·      A build up of toxic proteins in the brain (Evin et al., 2006)

·      A loss of mitochondrial function that leads to the oxidative stress and creation of neurotoxic molecules that trigger cell death (apoptotic, necrotic or autophagy) (Cobley et al., 2018)

·      Changes in the levels and activities of neurotrophic factors (Kazim and Iqbal, 2016; Machado et al., 2016; Rodriguez et al., 2014)

·      Variations in the activity of neural networks (Greicius and Kimmel, 2012)

Protein aggregation: the correlation between neurodegenerative disease and protein aggregation in the brain has long been recognised, but a causal relationship has not been unequivocally established (Lansbury et al., 2006; Kumar et al., 2016). The dynamic nature of protein aggregation mean that, despite progress in understanding its mechanisms, its relationship to disease is difficult to determine in the laboratory.

Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered pre-symptomatically, drastically reduce the incidence of these diseases.

Loss of mitochondrial function: many lines of evidence suggest that mitochondria have a central role in neurodegenerative diseases (Lin and Beal, 2006). Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Dysfunction of mitochondria induces oxidative stress, production of free radicals, calcium overload, and mutations in mitochondrial DNA that contribute to neurodegenerative diseases. In all major examples of these diseases there is strong evidence that mitochondrial dysfunction occurs early and acts causally in disease pathogenesis. Moreover, an impressive number of disease- specific proteins interact with mitochondria. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, or specific interactions of disease-related proteins with mitochondria, hold great promise.

Decreased level of neurotrophic factors: decreased levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer disease and Parkinson disease (Zuccato and Cattaneo, 2009). These studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. Therefore BDNF treatment is being considered as a beneficial and feasible therapeutic approach in the clinic.

Variations in the activity of neural networks: Patients with various neurodegenerative disorders show remarkable fluctuations in neurological functions, even during the same day (Palop et al., 2006). These fluctuations cannot be caused by sudden loss or gain of nerve cells. Instead, it is likely that they reflect variations in the activity of neural networks and, perhaps, chronic intoxication by abnormal proteins that the brain is only temporarily able to overcome.

Neurodegeneration in relation to COVID19

SARS-CoV-2 patients present elevated plasma levels of neurofilament light chain protein (NfL), which is a well-known biochemical indicator of neuronal injury (Kanberg et al., 2020). Postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination (Archie and Cucullo 2020; Heneka et al. 2020).

Autopsy results of patients with SARS showed ischemic neuronal damage and demyelination; viral RNA was detected in brain tissue, particularly accumulating in and around the hippocampus (Gu et al. 2005).

Brain magnetic resonance imaging (MRI) investigations in SARS-CoV-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (FLAIR), along with intracerebral hemorrhagic and microhemorrhagic lesions, and leptomeningeal enhancement (Kandemirli et al. 2020; Kremer et al. 2020; Mohammadi et al., 2020).

Moreover, eight COVID-19 patients with signs of encephalopathy had anti–SARS-CoV-2 antibodies in their CSF, and 4 patients had CSF positive for 14-3-3-protein suggesting ongoing neurodegeneration (Alexopoulos et al. 2020).

How it is Measured or Detected

The assays for measurements of necrotic or apoptotic cell death are described in the Key Event: Cell injury/Cell death

Recent neuropathological studies have shown that Fluoro-Jade, an anionic fluorescent dye, is a good marker of degenerating neurons. Fluoro-Jade and Fluoro-Jade B were found to stain all degenerating neurons, regardless of specific insult or mechanism of cell death (Schmued et al., 2005). More recently, Fluoro-Jade C was shown to be highly resistant to fading and compatible with virtually all histological processing and staining protocols (Schmued et al., 2005). In addition, Fluoro-Jade C is a good tool for detecting acutely and chronically degenerating neurons (Ehara and Ueda, 2009).

Regulatory Significance of the AO

Currently the four available OECD Test Guidelines (TGs) for neurotoxicity testing are entirely based on in vivo neurotoxicity studies: (1)Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (TG 418); (2) Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (TG 419); (3) Neurotoxicity Study in Rodents (TG 424) involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer); (4) Developmental Neurotoxicity (DNT) Study (TG 426) evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. One of the endpoints required by all four of these OECD TGs is evaluation of neurodegeneration that, so far, is performed through in vivo neuropathological and histological studies. Therefore, neurodegeneration described in this AOP as a key event, has a regulatory relevance and could be performed using in vitro assays that allow a reliable evaluation of neurodegeneration using a large range of existing assays, specific for apoptosis, necrosis and autophagy ( see also KE Cell injury/Cell death).

References

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Baum, J. S., Arama, E., Steller, H., and McCall, K. (2007). The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis. Cell Death Differ 14, 1508-1517.

Blackstone, N. W., and Green, D. R. (1999). The evolution of a mechanism of cell suicide. Bioessays 21, 84-88.

Cobley JN, Fiorello ML, Bailey DM (2018) 13 reasons why the brain is susceptible to oxidative stress. Redox Biol 15: 490-503

Ehara A, Ueda S. 2009. Application of Fluoro-Jade C in acute and chronic neurodegeneration models: utilities and staining differences. Acta histochemica et cytochemica 42(6): 171-179.

Evin G, Sernee MF, Masters CL (2006) Inhibition of gamma-secretase as a therapeutic intervention for Alzheimer's disease: prospects, limitations and strategies. CNS Drugs 20: 351-72

Greicius MD, Kimmel DL (2012) Neuroimaging insights into network-based neurodegeneration. Curr Opin Neurol 25: 727-34

Kazim SF, Iqbal K (2016) Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease. Mol Neurodegener 11: 50

Kumar V, Sami N, Kashav T, Islam A, Ahmad F, Hassan MI (2016) Protein aggregation and neurodegenerative diseases: From theory to therapy. Eur J Med Chem 124: 1105-1120

Lansbury1 PT & Lashuel HA (2006) A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 443, 774-779.

Lin1 MT & Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787-795

Machado V, Zoller T, Attaai A, Spittau B (2016) Microglia-Mediated Neuroinflammation and Neurotrophic Factor-Induced Protection in the MPTP Mouse Model of Parkinson's Disease-Lessons from Transgenic Mice. Int J Mol Sci 17

Narayan P, Dragunow M (2017) Alzheimer's Disease and Histone Code Alterations. Adv Exp Med Biol 978: 321-336

Palop JJ, Chin1 J & Mucke L, Review Article A network dysfunction perspective on neurodegenerative diseases. 2006, Nature 443, 768-773

Rodrigues TM, Jeronimo-Santos A, Outeiro TF, Sebastiao AM, Diogenes MJ (2014) Challenges and promises in the development of neurotrophic factor-based therapies for Parkinson's disease. Drugs Aging 31: 239-61

Schmued LC, Stowers CC, Scallet AC, Xu L. 2005. Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035(1): 24-31.

Zuccato C & Cattaneo E, Brain-derived neurotrophic factor in neurodegenerative diseases.2009, Nature Reviews Neurology 5, 311-3

COVID19-related references relevant to KE Neurodegeneration:

Alexopoulos et al. Anti-SARS-CoV-2 antibodies in the CSF, blood-brain barrier dysfunction, and neurological outcome: Studies in 8 stuporous and comatose patients. Neurol Neuroimmunol Neuroinflamm. 2020 Sep 25;7(6):e893.

Archie SR, Cucullo L. Cerebrovascular and neurological dysfunction under the threat of COVID-19: is there a comorbid role for smoking and vaping? Int J Mol Sci. 2020 21(11):3916 12.

Gu J et al. Multiple organ infection and the pathogenesis of SARS. J Exp Med. 2005;202:415–424.

Heneka MT, et al. Immediate and long-term consequences of COVID-19 infections for the development of neurological disease. Alzheimers Res Ther. 2020 12(1):1–3.

Kandemirli SG, et al. Brain MRI findings in patients in the intensive care unit with COVID-19 infection. Radiology. 2020 Oct;297(1):E232-E235.

Kanberg N, et al. Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19. Neurology. 2020 Sep 22;95(12):e1754-e1759.

Kremer S, et al. Brain MRI findings in severe COVID-19: a retrospective observational study. Radiology. 2020 Nov;297(2):E242-E251.

Mohammadi S. et al. Understanding the Immunologic Characteristics of Neurologic Manifestations of SARS-CoV-2 and Potential Immunological Mechanisms. Mol Neurobiol. 2020 Dec;57(12):5263-5275.

Appendix 2

List of Key Event Relationships in the AOP

10 µM