Aop: 10

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures in adult brain

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Blocking iGABA receptor ion channel leading to seizures

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center

Email: ping.gong@usace.army.mil or edward.j.perkins@usace.army.mil

Point of contact for this AOP entry: Dr. Ping Gong

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Ping Gong   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Ping Gong
  • Edward Perkins

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment WPHA/WNT Endorsed 1.15 Included in OECD Work Plan
This AOP was last modified on June 04, 2021 10:13
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Reduction, Ionotropic GABA receptor chloride channel conductance February 06, 2018 18:35
Occurrence, A paroxysmal depolarizing shift April 29, 2019 12:32
Occurrence, Epileptic seizure April 29, 2019 12:33
Binding at picrotoxin site, iGABAR chloride channel April 29, 2019 12:27
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 April 29, 2019 12:14
Reduction, Ionotropic GABA receptor chloride channel conductance leads to Reduction, Neuronal synaptic inhibition April 29, 2019 12:40
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

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

This AOP begins with a molecular initiating event (MIE) where a chemical 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-depolarization 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.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

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, which is the MIE described in the current AOP. 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).

It is worth noting that there exist another class of GABA receptors called metabotropic G-protein-coupled receptors (mGABAR) or GABAB receptors. This AOP is not applicable to GABAB receptors because they mediate slow and sustained inhibitory responses to GABA and are involved in absence epilepsy (Han et al. 2012).

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
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)

This table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help
Name Evidence Term
Picrotoxin High
Lindane High
Dieldrin High
Heptachlor High
Endosulfan High
RDX High
Fipronil High

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Adults High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
bobwhite quail Colinus virginianus High NCBI
zebrafish Danio rerio Moderate NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Male High
Female High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

Biological plausibility

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 electrophysiological response 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

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

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

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

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).

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

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).

ScoreTable.jpg

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

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)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

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).

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

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.

Han HA, Cortez MA, Snead OC III. 2012. GABAB Receptor and Absence Epilepsy. In: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012. Available from: https://www.ncbi.nlm.nih.gov/books/NBK98192/.

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.

Lomen-Hoerth C, Messing RO. (2010) Chapter 7: Nervous system disorders. In: Stephen J. McPhee, and Gary D. Hammer (Eds), Pathophysiology of disease: an introduction to clinical medicine (6th Edition). New York: McGraw-Hill Medical. ISBN 9780071621670.

McGonigle I, Lummis SC. (2010) Molecular characterization of agonists that bind to an insect GABA receptor. Biochemistry. 49(13):2897-902.

Moon JM, Chun BJ, Lee SD. (2017) In-hospital outcomes and delayed neurologic sequelae of seizure-related endosulfan poisoning. Seizure. 51:43-49.

Moses V, Peter JV. (2010) Acute intentional toxicity: endosulfan and other organochlorines. Clin Toxicol (Phila). 48(6):539-44.

Newland CF, Cull-Candy SG. (1992) On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol, 447: 191–213.

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.

Olsen RW, Sieghart W. (2009) GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56(1):141-8.

Parbhu B, Rodgers G, Sullivan JE. (2009) Death in a toddler following endosulfan ingestion. Clin Toxicol (Phila). 47(9):899-901.

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.

Sieghart W.(1995) Structure and pharmacology of gamma-aminobutyric acid A receptor subtypes. Pharmacol.Rev. 47(2):181-234

Stilwell GE, Saraswati S, Littleton JT, Chouinard SW. (2006) Development of a Drosophila seizure model for in vivo high-throughput drug screening. Eur J Neurosci, 24(8):2211-22.

Taketo M, Yoshioka T (2000) Developmental change of GABA(A) receptor-mediated current in rat hippocampus. Neuroscience 96(3):507-514.

Treiman DM. (2001) GABAergic mechanisms in epilepsy. Epilepsia, 42(Suppl. 3):8–12.

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.