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Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center
Email: email@example.com or firstname.lastname@example.org
Point of contact for this AOP entry: Dr. Ping Gong
Point of Contact
Ping Gong (email point of contact)
- Ping Gong
- Edward Perkins
|Author status||OECD status||OECD project||SAAOP status|
|Open for comment. Do not cite||EAGMST Approved||1.15||Included in OECD Work Plan|
This AOP was last modified on January 28, 2019 10:52
|Reduction, Ionotropic GABA receptor chloride channel conductance||February 06, 2018 18:35|
|Occurrence, A paroxysmal depolarizing shift||May 16, 2018 18:24|
|Occurrence, Epileptic seizure||June 06, 2018 12:55|
|Binding at picrotoxin site, iGABAR chloride channel||May 18, 2018 17:35|
|Reduction, Neuronal synaptic inhibition||May 16, 2018 18:42|
|Generation, Amplified excitatory postsynaptic potential (EPSP)||May 18, 2018 19:18|
|Binding at picrotoxin site, iGABAR chloride channel leads to Reduction, Ionotropic GABA receptor chloride channel conductance||May 16, 2018 18:51|
|Reduction, Ionotropic GABA receptor chloride channel conductance leads to Reduction, Neuronal synaptic inhibition||May 16, 2018 18:54|
|Reduction, Neuronal synaptic inhibition leads to Generation, Amplified excitatory postsynaptic potential (EPSP)||May 15, 2018 18:36|
|Generation, Amplified excitatory postsynaptic potential (EPSP) leads to Occurrence, A paroxysmal depolarizing shift||May 15, 2018 18:26|
|Occurrence, A paroxysmal depolarizing shift leads to Occurrence, Epileptic seizure||May 22, 2018 15:38|
|Picrotoxin||November 29, 2016 18:42|
|Lindane||November 29, 2016 18:42|
|Dieldrin||November 29, 2016 18:42|
|Heptachlor||February 11, 2018 00:24|
|Endosulfan||February 11, 2018 00:30|
|RDX||November 29, 2016 18:42|
|Fipronil||November 29, 2016 18:42|
This AOP begins with a molecular initiating event (MIE) where a chemicla binds to the picrotoxin binding site at or near the central pore of the ionotropic GABA receptor complex causing blockage of the ion channel. As a result, the first key event (KE) is a decrease in inward chloride conductance through the ligand-gated ion channel. This leads to the second KE, a reduction in postsynaptic inhibition, reflected as reduced frequency and amplitude of spontaneous inhibitory postsynaptic current (sIPSC) or abolishment of GABA-induced firing action in GABAergic neuronal membranes. Consequently, the resistance of excitatory neurons to fire is decreased, resulting in the generation of a large excitatory postsynaptic potential (EPSP), i.e., the third KE. The large EPSP is reflected as a spike (rise) of intracellular Ca2+ observed in the affected region, where a large group of excitatory neurons begin firing in an abnormal, excessive, and synchronized manner. Such a giant Ca2+-mediated excitatory firing (depolarization) causes voltage-gated Na+ to open, which results in action potentials. The depolarization is followed by a period of hyper-polarization mediated by Ca2+-dependent K+ channels or GABA-activated Cl− influx. During seizure development, the post-depolarlization hyperpolarization becomes smaller, gradually disappears, and is replaced by a depolarization. This characteristic depolarization-shrinking hyperpolarization sequence of events represents the fourth KE known as “paroxysmal depolarizing shift” (PDS), which forms a “seizure focus”. A PDS is, essentially, an indication of epilepsy at the cellular level, which serves as the foci to initiate the adverse outcome at the organismal level of epileptic seizure. The severity of symptoms is often dose- and duration- dependent, while the toxicological symptoms are associated with the type and location of affected iGABARs. Mortality can occur if the individual sustains a prolonged or pronounced convulsion or seizure. Neurotoxicity, of which seizures are an end point, is a regulated outcome for chemicals. This AOP allows for screening chemicals for the potential to cause neurotoxicity through the use of in vitro assays that demonstrate binding to the picrotoxin site, electrophysiological assays demonstrating depolarization of neuronal membranes, or electroencephalography that records electrical activity of the adult brain.
Ionotropic GABA receptors (iGABARs) are ligand-gated ion channels which play important functional roles in the nervous system. As the major player in inhibitory neurotransmission, iGABARs are widely distributed in both vertebrates and invertebrates (McGonigle and Lummis 2010; Garcia-Reyero et al. 2011). In vertebrates, the iGABAR includes two subclasses of fast-responding ion channels, GABAA receptor (GABAA-R) and GABAC receptor (GABAC-R). Invertebrate iGABARs do not readily fit the vertebrate GABAA/GABAC receptor categories (Sieghart 1995). The majority of insect iGABARs are distinguished from vertebrate GABAA receptors by their insensitivity to bicuculline and differ from GABAC-Rs in that they are subject to allosteric modulation, albeit weakly, by benzodiazepines and barbiturates (Hosie et al. 1997).
Chemical interactions with iGABARs can cause a variety of pharmacological and neurotoxicological effects depending on the location of the active or allosteric site affected. Three distinct types of interactions at binding sites on iGABARs can antagonize the postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. These three types of interactions correspond to three AOPs (Gong et al. 2015). One of the three types of interaction is non-competitive channel blocking at the picrotoxin convulsant site located inside of the iGABAR pore that spans neuronal cell membranes (this MIE). The other two types of interactions are negative modulation at allosteric sites and competitive binding at the active orthosteric sites (MIEs to be developed in the future).
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||667||Binding at picrotoxin site, iGABAR chloride channel||Binding at picrotoxin site, iGABAR chloride channel|
|2||KE||64||Reduction, Ionotropic GABA receptor chloride channel conductance||Reduction, Ionotropic GABA receptor chloride channel conductance|
|3||KE||669||Reduction, Neuronal synaptic inhibition||Reduction, Neuronal synaptic inhibition|
|4||KE||682||Generation, Amplified excitatory postsynaptic potential (EPSP)||Generation, Amplified excitatory postsynaptic potential (EPSP)|
|5||KE||616||Occurrence, A paroxysmal depolarizing shift||Occurrence, A paroxysmal depolarizing shift|
|6||AO||613||Occurrence, Epileptic seizure||Occurrence, Epileptic seizure|
Relationships Between Two Key Events
(Including MIEs and AOs)
Life Stage Applicability
|bobwhite quail||Colinus virginianus||High||NCBI|
Overall Assessment of the AOP
The biological mechanisms underlying epilepsy (defined as a disorder of the central nervous system characterized by recurrent seizures unprovoked by an acute systemic or neurologic insult) have been investigated for more than six decades and are well understood except for a few intermediate details (Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). As one of the cellular mechanisms of action, blocking postsynaptic GABA-mediated inhibition can lead to epileptic seizure (Dichter and Ayala 1987; Gong et al. 2015). It has been extensively documented that non-competitive ion channel blockers such as picrotoxin, lindane, α-endosulfan and fipronil act through binding to iGABARs (Chen et al. 2006; Casida and Durkin 2015). Despite large structural diversity, it has been postulated that these blockers fit a single binding site in the chloride channel lumen lined by five TM2 (TransMembrane domain 2) segments, which was supported in the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al. 2006; Casida and Durkin 2015). The downstream cascading key events of this AOP have also been reviewed in multiple publications (e.g., Dichter and Ayala 1987; Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). Based on the extensive evidence supporting the MIE, KEs and the AO, there is a high likelihood and certainty that such GABA antagonists as non-competitive channel blockers produce seizures in both invertebrates and vertebrates that possess GABAergic inhibitory neurotransmission in central nervous systems (Treiman 2001; Raymond-Delpech et al. 2005).
Concordance of dose-response relationships
Numerous pharmacological studies have reported quantitative dose-response relationships between the dose of non-competitive antagonists and the recorded electrophysirological esponse of epileptic seizures. See examples for picrotoxin (Newland and Cull-Candy 1992; Ikeda 1998; Stilwell et al. 2006), RDX (Williams et al. 2011) and dieldrin (Babot et al. 2007; Ikeda 1998).
Temporal concordance among the key events and the adverse outcome
Given that the basic mechanism of neuronal excitability is the action potential, a hyperexcitable state can result from many causes including decreased inhibitory neurotransmission (KE2). Moreover, action potentials occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal. The action potential occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. Membrane potential thus varies with the activation of ligand-gated channels, whose conductance is affected by binding to neurotransmitters. For instance, the conductance is decreased (KE1) due to the binding at allosteric sites in the chloride channel of iGABAR by non-competitive blockers (MIE).
Seizure initiation: The hypersynchronous discharges that occur during a seizure may begin in a very discrete region of the cortex and then spread to neighboring regions. Seizure initiation is characterized by two concurrent events: 1) high-frequency bursts of action potentials, and 2) hypersynchronization of a neuronal population. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on the EEG (electroencephalogram), i.e., amplified excitatory postsynaptic potential (KE3). At the level of single neurons, epileptiform activity consists of sustained neuronal depolarization resulting in a burst of action potentials, a plateau-like depolarization associated with completion of the action potential burst, and then a rapid repolarization followed by hyperpolarization. This sequence is called the paroxysmal depolarizing shift (KE4).
Seizure propagation (AO), the process by which a partial seizure spreads within the brain, occurs when there is sufficient activation to recruit surrounding neurons. This leads to a loss of surround inhibition and spread of seizure activity into contiguous areas via local cortical connections, and to more distant areas via long association pathways such as the corpus callosum. The propagation of bursting activity is normally prevented by intact hyperpolarization and a region of surrounding inhibition created by inhibitory neurons. With sufficient activation there is a recruitment of surrounding neurons via a number of mechanisms. The above description is excerpted and summarized from Bromfield et al. (2006).
Strength, consistency, and specificity of association of adverse effect and initiating event
Drug- or chemical-induced focal or generalized seizures are not limited to any specific group of chemical structures, neuroreceptors or taxonomy. This AOP addresses a specific group of chemicals that are capable of binding to the picrotoxin convulsant site of iGABARs, leading to epileptic seizures. Literature evidence strongly and consistently supports such a forward association, i.e., binding to the picrotoxin site leads to epileptic seizures (see reviews Gong et al. 2015; Bromfield et al. 2006; Raymond-Delpech et al. 2005; Treiman 2001; Dichter and Ayala 1987). For instance, dose- and time-dependent correlations between picrotoxin site binding by chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986), whereas poisoning with the organochlorine insecticide endosulfan caused seizure, status epilepticus, or refractory status epilepticus in humans (Durukan et al. 2009; Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009; Roberts et al. 2004), and eventually led to the death of a farmer (Roberts et al. 2004) and a toddler (Parbhu et al. 2009).
Uncertainties, inconsistencies, and data gaps
No inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, the process by which seizures typically end, usually after seconds or minutes, and what underlies the failure of this spontaneous seizure termination in the life-threatening condition known as status epilepticus are less well understood (Bromfield et al. 2006). The spread of epileptic activity throughout the brain, the development of primary generalized epilepsy, the existence of “gating", mechanisms in specific anatomic locations, and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain more complex forms of epilepsies observed in human and other animals, all are also not yet fully understood (Dichter and Ayala 1987). The remarkable plasticity of GABAergic neurons and iGABARs (e.g., iGABAR regulation by phosphorylation, and expression of potassium chloride cotransporters (KCCs) or sodium dependent anion exchangers (NDAE)) in response to insults and injury constitutes additional complexity and creates another layer of uncertainties in the emergence of epileptic seizures (Ben-Ari 2006; Galanopoulou 2008; Scharfman and Brooks-Kayal 2014).
Domain of Applicability
This AOP is applicable to all vertebrates and invertebrates possessing iGABARs, without restrictions pertaining to sex and taxonomy. This AOP may not be applicable to young animals during their embryonic and early developmental stages when GABA acts as an excitatory neurotransmitter due to increased intracellular Clˉ concentration in immature or developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006). A key feature of the immature type function of GABAA receptors is the depolarizing signaling, attributed to the inability of young neurons to maintain low intracellular chloride. The regulation of GABAergic switch is different in neurons with depolarizing vs hyperpolarizing GABAergic signaling. In mature neurons, recurrent and prolonged seizures may trigger a pathological reemergence of immature features of GABAA receptors, which compromises the efficacy of GABA-mediated inhibition. In immature neurons with depolarizing GABAergic signaling, the physiological and pathological regulation of this system is completely different, possibly contributing to the different outcomes of early life seizures (Galanopoulou 2008).
Essentiality of the Key Events
The MIE, four key events and resulted adverse outcome listed for this AOP are all essential based on current knowledge and understanding of the structure, pharmacology, localization, classification of ionotropic GABA receptors (e.g., GABAA receptors) (Olsen 2015; Olsen and Sieghart 2009), the basic neurophysiology, neurochemistry and cellular mechanisms underlying epilepsies (Dichter and Ayala 1987; Bromfield et al. 2006), and the pathophysiology of seizures (Lomen-Hoerth and Messing 2010).
A novel subject matter expertise driven approach was developed for weight of evidence (WoE) assessment (Collier et al. 2016). This approach, tailored toward the needs of AOPs and in compliance with the AOP Users' Handbook (OECD 2017), was based on criteria and metrics related to data quality and causality (i.e., the strength of causal linkage between key events). The methodology consists of three main steps: (1) assembling evidence (preparing the AOP), (2) weighting evidence (criteria weighting and scoring), and (3) weighting the body of evidence (aggregating lines of evidence). We adopted the General Assessment Factors (GAF) established by the US EPA as the criteria for data quality evaluation, and a set of five criteria known as Bradford Hill criteria to measure the strength of causal linkages (see Table below). The numerical scoring scale corresponds to the descriptive scoring scale recommended in the Users' Handbook as follows: 4 or 5 is equivalent to High, 3 is equivalent to Moderate, and 1 or 2 is equivalent to Low. Detailed description on, supporting evidence for and quantitative understanding of each KE/KER can be found by clicking the link on each KE/KER above.
The authors of Collier et al. (2016), who served as the developers for several AOPs (including this one), represented subject matter experts, and they applied their expertise and best professional judgment to assign weights to the criteria and scores to each line of evidence. Final criteria scoring represented the consensus scores agreed upon after debates among the authors. For example, the MIE has been intensively reviewed where numerous documented studies provided supporting evidence. Hence, the MIE received high scores for all five GAF criteria. However, the Bradford Hill criteria connecting KE2-->KE3 and KE3-->KE4 received relatively lower scores because there still exist knowledge gaps in the spread of epileptic activity throughout the normal CNS and the mechanism underlying the generalized epilepsies. The following table shows the results of our WoE assessment (note that scores may be inexact due to rounding).
Many studies have reported quantitative relationships between chemicals such as drugs and pesticides and electrophysiological response. For instance, long-term exposure of primary cerebellar granule cell cultures to 3 µM dieldrin reduced the GABAA receptor function to 55% of control, as measured by the GABA-induced 36Cl- uptake (Babot et al. 2007). Juarez et al. (2013) observed that picrotoxin exerted concentration-dependent and reversible inhibition of GABA-induced membrane currents in primary cultured neurons obtained from the guinea-pig small intestine. The stepwise qualitative relationships between consecutive events (MIE, KEs and AO) are well established but quantitative ones are rarely documented.
Considerations for Potential Applications of the AOP (optional)
This AOP can be used to establish the mode of neurotoxicological actions for chemicals capable of binding to the picrotoxin convulsant site of iGABARs. It can also be applied to risk assessment where AOP can assist in predictive modeling of chemical toxicity. Chemicals acting through this AOP can be distinguished from neurotoxicants acting on other types of iGABAR sites (e.g., orthosteric or allosteric binding sites) or other types of neuroreceptors (e.g., ardrenergic, dopaminergic, glutaminergic, cholinergic and serotonergic receptors). More information relevant to this topic can be found in Gong et al. (2015).
Babot Z, Vilaro M T, Sunol C. (2007) Long-term exposure to dieldrin reduces gamma-aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures of mouse cerebellar granule cells. J Neurosci Res, 85(16):3687-3695.
Ben-Ari Y. (2006) Seizures beget seizures: the quest for GABA as a key player. Crit Rev Neurobiol. 18(1-2):135-44.
Bromfield EB, Cavazos JE, Sirven JI. (2006) Chapter 1, Basic Mechanisms Underlying Seizures and Epilepsy. In: An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society; Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/.
Casida JE, Durkin KA. (2015) Novel GABA receptor pesticide targets. Pesticide Biochem Physiol. 121:22-30.
Chen L, Durkin KA, Casida J. (2006) Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site. Proc Natl Acad Sci USA. 103(13):5185-5190.
Cole LM, Casida JE. (1986) Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore. Life Sci. 39 (20):1855-62.
Collier ZA, Gust KA, Gonzalez-Morales B, Gong P, Wilbanks MS, Linkov I, Perkins EJ. (2016) A weight of evidence assessment approach for adverse outcome pathways. Regul Toxicol Pharmacol. 75:46-57.
Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237: 157-164.
Durukan P, Ozdemir C, Coskun R, Ikizceli I, Esmaoglu A, Kurtoglu S, Guven M. (2009) Experiences with endosulfan mass poisoning in rural areas. Eur J Emerg Med. 16(1):53-6.
Galanopoulou AS. (2008) GABAA Receptors in Normal Development and Seizures: Friends or Foes? Curr Neuropharmacol. 6(1): 1–20.
Garcia-Reyero N, Habib T, Pirooznia M, Gust KA, Gong P, Warner C, Wilbanks M, Perkins E. (2011) Conserved toxic responses across divergent phylogenetic lineages: a meta-analysis of the neurotoxic effects of RDX among multiple species using toxicogenomics. Ecotoxicology. 20(3):580-94.
Gong P, Hong H, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine 9(11):1225-39.
Hosie AM, Aronstein K, Sattelle DB, ffrench-Constant RH. (1997) Molecular biology of insect neuronal GABA receptors. Trends Neurosci. 20(12): 578-583.
Ikeda T, Nagata K, Shono T, Narahashi T. (1998) Dieldrin and picrotoxinin modulation of GABA(A) receptor single channels. Neuroreport 9(14):3189-3195.
Juarez EH, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M, Barajas-Lopez C. (2013) Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol, 34(1-2):1-8.
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OECD. (2017) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs. OECD Environment Directorate, Environment, Health and Safety Division, Series on Testing & Assessment No. 233, Series on Adverse Outcome Pathways No. 1, ENV/JM/MONO(2016)12, Paris, France.
Olsen RW. (2015) Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.
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Raymond-Delpech V, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB. (2005) Ion channels: molecular targets of neuroactive insecticides. Invert Neurosci, 5(3-4):119-133.
Roberts DM, Dissanayake W, Sheriff MHR, Eddleston M. (2004) Refractory status epilepticus following self-poisoning with the organochlorine pesticide endosulfan. J Clinical Neurosci. 11(7): 760-2.
Scharfman HE, Brooks-Kayal AR. (2014) Is Plasticity of GABAergic Mechanisms Relevant to Epileptogenesis? In: Scharfman HE and Buckmaster PS (eds.), Issues in Clinical Epileptology: A View from the Bench, Advances in Experimental Medicine and Biology 813, pp.133-150.
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Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. (2011) RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ Health Perspect, 119(3):357-363.