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Created at: 2017-05-31 12:42

AOP ID and Title:


AOP 10: Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures
Short Title: Blocking iGABA receptor ion channel leading to seizures

Authors


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


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite EAGMST Under Review 1.15 Included in OECD Work Plan

Abstract


This AOP begins with a molecular initiating event (MIE) where chloride conductance through the ion channel is blocked due to chemical binding at or near the central pore of the receptor complex (i.e., the picrotoxin site). 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 sequent 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 is 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 brain.


Background


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


Stressors


Name Evidence
Picrotoxin Strong
Lindane Strong
Dieldrin Strong

Picrotoxin

Picrotoxin seizures are well defined mechanistically. They arise from GABAA receptor chloride channel blockade. (See Page 131 in "Models of Seizures and Epilepsy", edited by A. Pitkanen, P.A. Schwartzkroin, S.L. Moshe. Elsevier Academic Press. 2006)

As picrotoxin effectively inhibits chloride influx in GABA-A and other ionotropic receptors, it represents a universal "reference" channel blocker with whom other ligands may be compared. (A.V. Kalueff. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a prposed model for channel binding sites. Neurochem Int. 50(1): 61-8.)

Lindane

Neurotoxic pesticides, such as lindane, alpha-endosulphan and dieldrin, share structural similiarity (and compete for the binding site) with picrotoxin, inhibit TBPS binding, induce seizures and block Cl-currents through ionophore. (A.V. Kalueff. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a prposed model for channel binding sites. Neurochem Int. 50(1): 61-8.)

Dieldrin

See evidence text for picrotoxin and lindane.

Molecular Initiating Event

Title Short name
Binding at picrotoxin site, iGABAR chloride channel Binding at picrotoxin site, iGABAR chloride channel

667: Binding at picrotoxin site, iGABAR chloride channel

Short Name: Binding at picrotoxin site, iGABAR chloride channel

AOPs Including This Key Event

Stressors

Name
Picrotoxin
Lindane
Fipronil
RDX
Alpha-endosulfan
Penicillin

Biological Organization

Level of Biological Organization
Molecular

Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

Chemicals non-competitively bind at or near the central pore of the receptor complex (e.g., the picrotoxin site) and directly block chloride conductance through the ion channel (Kalueff 2007). It has been postulated that they fit a single binding site in the chloride channel lumen lined by five TM2 segments. This hypothesis was examined with the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al 2006). Results suggest that they fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. More computational evidence can be found in Sander et al. (2011), Carpenter et al. (2013) and Zheng et al. (2014).



Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
fruit fly Drosophila melanogaster Strong NCBI
mouse Mus musculus Strong NCBI
dogs Canis lupus familiaris Strong NCBI
Life Stage Applicability
Life Stage Evidence
Adult Strong
Sex Applicability
Sex Evidence
Unspecific Strong

Theoretically, this MIE is applicable to any organisms that possess ionotropic GABA receptors (iGABARs) in their central and/or peripheral nervous systems. Many reviews (e.g., Hoisie et al. 1997; Buckingham et al. 2005; Michels and Moss 2007; Olsen and Sieghart 2009) have summarized evidence of ubiquitous existence of iGABARs (GABAA-R in vertebrates including the humans) in species spanning from invertebrates to human. For instance, an ionotropic GABA receptor gene (GABA-receptor subunit-encoding Rdl gene) was isolated from a naturally occurring dieldrin-resistant strain of D. melanogaster (Ffrench-Constant et al., 1991,1993; Ffrench-Constant and Rocheleau, 1993). Nineteen GABAA receptor genes have been identified in the human genome (Simon et al. 2004). Direct evidence is mostly derived from in silico molecular modeling that docks ligands to the binding pockets of iGABARs in human (Carpenter et al. 2013; Chen et al. 2006; Sander et al. 2011), fruitfly and zebrafish (Zheng et al. 2014).


How this Key Event Works

Figure1.png

Figure 1. Structure of ionotropic GABA receptors based on the consensus in multiple literature reviews (Source: Gong et al. 2015). Shown is a common subtype α1β2γ2 of GABAA receptors found in the mammalian CNS. (A) Five subunits from three subunit subfamilies assemble to form a heteropentameric chloride permeable channel. (B) Stoichiometry and subunit arrangement of the GABAA receptor. Also shown are the binding sites for GABA and BZ. (C) Receptor subunits consist of four hydrophobic transmembrane domains (TM1-4), where TM2 is believed to line the pore of the channel. The large extracellular N-terminus is the site for ligand binding as well as the site of action of various drugs. Each receptor subunit also contains a large intracellular domain between TM3 and TM4, which is the site for various protein–protein interactions as well as the site for post-translational modifications that modulate receptor activity. BZ: Benzodiazepines; CNS: Central nervous system; TM: Transmembrane.


As shown in Figure 1, non-competitive channel blockers (e.g., fipronil, lindane, picrotoxin and alpha-endosulfan) indirectly modulate the iGABAR activity (i.e., alter the response of the receptor to agonist) by noncompetitively binding at or near the central pore of the receptor complex (e.g., the picrotoxin site), an allosteric site distinct from that of the orthosteric agonist binding site, and inducing a conformational change within the receptor (Ernst et al. 2005; Johnston 2005).


How it is Measured or Detected

Binding to a specific site on iGABAR can be determined using a variety of methods including mutagenesis, pore structure studies, ligand binding, and molecular modeling (more details on methods can be found in Chen et al. 2006). One should choose a method in accordance with specific goal and also on the basis of available laboratory facilities. For example, Atack et al. (2007) chose the radioligand [35S]TBPS binding assay to determine the binding properties (i.e., inhibition by TBPS, picrotoxin, loreclezole and pentobarbital and modulation by GABA) at the convulsant binding site.


References

Atack JR, Ohashi Y, McKernan RM. 2007. Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain. Br J Pharmacol. 150(8):1066-74.

Buckingham SD, Biggin PC, Sattelle BM, Brown LA, Sattelle DB. 2005. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol Pharmacol 68(4):942-951.

Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary picrotoxin-binding site on the GABAA receptor. Chem Res Toxicol. 26(10):1444-54.

Chen L, Durkin KA, Casida JE. 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.

Ernst M, Bruckner S, Boresch S, Sieghart W. 2005. Comparative models of GABAA receptor extracellular and transmembrane domains: important insights in pharmacology and function. Mol Pharmacol 68(5):1291-1300.

Ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT. 1991. Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus. Proc Natl Acad Sci USA 88:7209–7213.

Ffrench-Constant RH and Rocheleau TA. 1993 Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing. J Neurochem 60:2323–2326.

Ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, and Roush RT. 1993. A single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations. Proc Natl Acad Sci USA 90:1957–1961.

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.

Johnston GA. 2005. GABA(A) receptor channel pharmacology. Curr Pharm Des 11(15):1867-1885.

Kalueff AV. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int 50(1): 61-68.

Michels G, Moss SJ. 2007. GABAA receptors: properties and trafficking. Crit Rev Biochem Mol Biol 42(1):3-14.

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

Sander T, Frolund B, Bruun AT, Ivanov I, McCammon JA, Balle T. 2011. New insights into the GABAA receptor structure and orthosteric ligand binding: receptor modeling guided by experimental data. Proteins. 79(5):1458-77.

Simon J, Wakimoto H, Fujita N, Lalande M, Barnard EA. 2004. Analysis of the set of GABA(A) receptor genes in the human genome. J. Biol. Chem. 279(40), 41422–41435.

Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference of fipronil with GABAARs in fruitfly and zebrafish: insights from homology modeling, docking, and molecular dynamics simulation studies. J Agric Food Chem 62(44):10646-53.

 


Key Events

Title Short name
Reduction, Ionotropic GABA receptor chloride channel conductance Reduction, Ionotropic GABA receptor chloride channel conductance
Occurrence, A paroxysmal depolarizing shift Occurrence, A paroxysmal depolarizing shift
Reduction, Neuronal synaptic inhibition Reduction, Neuronal synaptic inhibition
Generation, Amplified excitatory postsynaptic potential (EPSP) Generation, Amplified excitatory postsynaptic potential (EPSP)

64: Reduction, Ionotropic GABA receptor chloride channel conductance

Short Name: Reduction, Ionotropic GABA receptor chloride channel conductance

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Cellular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rats Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI
Drosophila melanogaster Drosophila melanogaster Strong NCBI

Banerjee et al. (1999) reported functional modulation of GABAA receptors by Zn2+, pentobarbital, neuroactive steroid alphaxalone, and flunitrazepam in the cerebral cortex and cerebellum of rats undergoing status epilepticus induced by pilocarpine.

Babot et al. (2007) measured the reduction in mouse GABAA receptor function by 3 μM dieldrin using the GABA-induced 36Cl- uptake method.

Bromfield et al. (2006) reviewed evidence for GABA-A receptors in human and mammalian brains.

Grolleau and Sattelle (2000) reported a complete blocking of inward current by 100 μM picrotoxin in the wild-type RDL (iGABAR) of Drosophila melanogaster.


How this Key Event Works

This key event occurs at the cellular level and is characterized by a dose-dependent post-synaptic inhibition of membrane currents in iGABAR-containing cells, especially neuronal cells (Dichter and Ayala 1987; Bromfield et al. 2006), leading to the reduction of iGABAR chloride channel conductance.


How it is Measured or Detected

The change in membrane conductance can be measured by determining the alteration (i.e., inhibition) in muscimol-stimulated (Banerjee et al. 1999) or GABA-induced uptake (Babot et al. 2007) of 36Cl- in cortical and cerebellar membranes or primary cerebellar granule cell cultures, prior to and after exposure to a GABA antagonist.


References

Babot Z, Vilaro MT, 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.

Banerjee PK, Olsen RW, Snead OC, III. (1999) Zinc inhibition of gamma-aminobutyric acid(A) receptor function is decreased in the cerebral cortex during pilocarpine-induced status epilepticus. J Pharmacol Exp Ther 1999; 291(1):361-366.

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

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: a status report. Science 237(4811), 157-164.

Gong P. Hong HH, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomark. Med. 9(11):1225-39.

Grolleau F, Sattelle DB. (2000) Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line. Br J Pharmacol. 130(8):1833-42.

 


616: Occurrence, A paroxysmal depolarizing shift

Short Name: Occurrence, A paroxysmal depolarizing shift

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Tissue

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI

Most of the supporting evidence come from studies on human and rodents. See the reviews of Bromfield (2006) and Lomen-Hoerth and Messing (2010) for examples.


How this Key Event Works

A paroxysmal depolarizing shift (PDS) or depolarizing shift is a cellular manifestation of epilepsy. As summarized by Lomen-Hoerth and Messing (2010), brain electrical activity is non synchronous under normal conditions. In epileptic seizures, a large group of neurons begin firing in an abnormal, excessive, and synchronized manner, which results in a wave of depolarization known as a paroxysmal depolarizing shift (Somjen, 2004). Normally after an excitatory neuron fires it becomes more resistant to firing for a period of time, owing in part to the effect of inhibitory neurons, electrical changes within the excitatory neuron, and the negative effects of adenosine. However, in epilepsy the resistance of excitatory neurons to fire during this period is decreased, likely due to changes in ion channels or inhibitory neurons not functioning properly. This then results in a specific area from which seizures may develop, known as a "seizure focus".

Increased, abnormal neuron firing causes a wave of depolarization throughout the brain/neuronal tissue. 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 (PDS). The bursting activity resulting from the relatively prolonged depolarization of the neuronal membrane is due to influx of extracellular Ca2+, which leads to the opening of voltage-dependent Na+ channels, influx of Na+, and generation of repetitive action potentials. The subsequent hyperpolarizing afterpotential is mediated by iGABA receptors and Cl- influx, or by K+ efflux, depending on the cell type (Bromfield et al 2006).


How it is Measured or Detected

Paroxysmal depolarizing shifts can be measured in vitro using patch clamp methods or in vivo using electroencephalography techniques (Niedermeyer and da Silva 2005).


References

Bromfield EB, Cavazos JE, Sirven JI. 2006. An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society; Chapter 1, Basic Mechanisms Underlying Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/.

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

Niedermeyer E, da Silva FL. 2005. Electroencephalography: basic principles, clinical applications, and related fields. Lippincott Williams & Wilkins.

Somjen GG. 2004. Ions in the Brain Normal Function, Seizures, and Stroke. New York: Oxford University Press. p. 167.

 


669: Reduction, Neuronal synaptic inhibition

Short Name: Reduction, Neuronal synaptic inhibition

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Cellular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus Strong NCBI
guinea pig Cavia porcellus Strong NCBI
human Homo sapiens Strong NCBI
Japanese quail Coturnix japonica Strong NCBI

See Juarez et al. (2013) for supporting evidence for Guinea pig; For rat, whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of GABAA receptor mediated sIPSCs and the amplitude of GABA-evoked postsynaptic currents, whereas in extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges (Williams et al, 2011).


How this Key Event Works

A reduction in GABA-mediated inhibition of neuronal synaptic signalling is reflected as decreased frequency and amplitude of iGABAR-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) or abolishment of GABA-induced firing action (Newland and Cull-Candy 1992).


How it is Measured or Detected

Juarez et al. (2013) used primary cultured neurons obtained from the guinea-pig small intestine to detect picrotoxin concentration-dependent (and reversible) inhibition of GABA-induced membrane currents. Williams et al. (2011) used whole-cell in vitro recordings in the rat basolateral amygdala (BLA) to detect the reduced frequency and amplitude of GABAA receptor mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and the amplitude of GABA-evoked postsynaptic currents, both of which were induced by RDX.


References

Newland C F, Cull-Candy S G. On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol 1992; 447: 191–213.

Juarez E H, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M, Barajas-Lopez C. Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol 2013; 34(1-2):1-8.

Williams L R, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. 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 2011; 119(3):357-363.

 


682: Generation, Amplified excitatory postsynaptic potential (EPSP)

Short Name: Generation, Amplified excitatory postsynaptic potential (EPSP)

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Cellular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
mouse Mus musculus Strong NCBI
rat Rattus norvegicus Strong NCBI
guinea pig Cavia porcellus Strong NCBI

Miura et al. (1997) reported supporting evidence from guinea pigs whereas Dichter and Ayala (1987) and Bromfield et al. (2006) summarized relevant studies on humans.


How this Key Event Works

In neuroscience, an excitatory postsynaptic potential (EPSP) is defined as a neurotransmitter-induced postsynaptic potential change that depolarizes the cell, and hence increases the likelihood of initiating a postsynaptic action potential (Purves et al. 2001). On the contrary, an inhibitory postsynaptic potential (IPSP) decreases this likelihood. Whether a postsynaptic response is an EPSP or an IPSP depends on the type of channel that is coupled to the receptor, and on the concentration of permeant ions inside and outside the cell. In fact, the only factor that distinguishes postsynaptic excitation from inhibition is the reversal potential of the postsynaptic potential (PSP) in relation to the threshold voltage for generating action potentials in the postsynaptic cell. When an active presynaptic cell releases neurotransmitters into the synapse, some of them bind to receptors on the postsynaptic cell. Many of these receptors contain an ion channel capable of passing positively charged ions (e.g., Na+ or K+) or negatively charged ions (e.g., Cl-) either into or out of the cell. In epileptogenesis, discharges reduced GABAA receptor-mediated hyperpolarizing IPSPs by shifting their reversal potentials in a positive direction. At the same time, the amplitudes of Schaffer collateral-evoked RS-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated EPSPs and action potential-independent miniature EPSPs were enhanced, whereas N-methyl-d-aspartate receptor-mediated EPSPs remained unchanged. Together, these changes in synaptic transmission produce a sustained increase in hippocampal excitability (Lopantsev et al. 2009).


How it is Measured or Detected

EPSPs are usually recorded using intracellular electrodes. See Miura et al. (1997) and Bromfield et al. (2006) for details.


References

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/.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237:157-64.

Lopantsev V, Both M, Draguhn A. 2009. Rapid Plasticity at Inhibitory and Excitatory Synapses in the Hippocampus Induced by Ictal Epileptiform Discharges. Eur J Neurosci 29(6):1153–64.

Miura M, Yoshioka M, Miyakawa H, Kato H, Ito KI. (1997) Properties of calcium spikes revealed during GABAA receptor antagonism in hippocampal CA1 neurons from guinea pigs. J Neurophysiol. 78(5):2269-79.

Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO, Williams SM (Eds). 2001. Neuroscience. 2nd edition. Chapter 7. Neurotransmitter Receptors and Their Effects. Sunderland (MA): Sinauer Associates. Available from: http://www.ncbi.nlm.nih.gov/books/NBK10799/.


Adverse Outcomes

Title Short name
Occurrence, Epileptic seizure Occurrence, Epileptic seizure

613: Occurrence, Epileptic seizure

Short Name: Occurrence, Epileptic seizure

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Individual

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI
honeybee Apis mellifera Strong NCBI
eisenia fetida eisenia fetida Strong NCBI

A wide range of species including invertebrates and vertebrates have been documented (see Tingle et al. (2003) and Gunasekara et al. 2007 for reviews on the list of aquatic and terrestrial species affected by fipronil). For instance, fipronil can induce seizures in fruit flies (Stilwell et al. (2006)) and house flies (Gao et al. 2007).


How this Key Event Works

Blockage of the GABA-gated chloride channel reduces neuronal inhibition and induces focal seizure. This may further lead to generalized seizure, convulsions and death (Bloomquist 2003; De Deyn et al. 1990; Werner and Covenas 2011). For instance, exposure to fipronil produces hyperexicitation at low doses and convulsion or tonic-clonic seizure and seizure-related death at high doses (Gunasekara et al. 2007; Tingle et al. 2003; Jackson et al. 2009).

Seizure propagation, 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. Repetitive discharges lead to: 1) an increase in extracellular K+, which blunts the extent of hyperpolarizing outward K+ currents, tending to depolarize neighboring neurons; 2) accumulation of Ca2+ in presynaptic terminals, leading to enhanced neurotransmitter release; and 3) depolarization-induced activation of the NMDA subtype of the excitatory amino acid receptor, which causes more Ca2+ influx and neuronal activation. Of equal interest, but less well understood, is 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 (Bromfield et al. 2006).


How it is Measured or Detected

Electrophysiological measurements and physical (visual) observation (for mortality) are the methods often used to detect epileptic seizure-related effects (Ulate-Campos et al. 2016). One may also visit http://www.mayoclinic.org/diseases-conditions/epilepsy/diagnosis-treatment/diagnosis/dxc-20117234 for more information on how medical doctors diagnose epilepsy in patients.


Regulatory Examples Using This Adverse Outcome

As a neurotoxicity endpoint, information with regard to the seizure or epilepsy is often used by regulators such as EPA, FDA and DHS for human and environmental health assessment and regulation of chemicals, drugs and other materials. For instance, the Office of Pesticide Programs (OPP) in US EPA regulates, monitors and investigates the use of all pesticides in accordance with the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (https://www.epa.gov/laws-regulations/summary-federal-insecticide-fungicide-and-rodenticide-act). Many pesticides like fipronil target the iGABAR causing seizure and mortality. Another example is the regulatory actions of US FDA to ensure drug saety (see https://www.fda.gov/Drugs/DrugSafety/ucm436494.htm).


References

Bloomquist JR. 2003. Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol 54(4), 145-156.

Bromfield EB, Cavazos JE, Sirven JI, editors. 2006. An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society. Chapter 1 Basic Mechanisms Underlying Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/

De Deyn PP, Marescau B, Macdonald RL. 1990. Epilepsy and the GABA-hypothesis a brief review and some examples. Acta Neurol. Belg. 90(2), 65-81.

Gao JR, Kozaki T, Leichter CA, Rinkevich FD, Shono T, Scott JG. 2007. The A302S mutation in Rdl that confers resistance to cyclodienes and limited crossresistance to fipronil is undetectable in field populations of house flies from the USA. Pestic. Biochem. Physiol. 88, 66−70.

Gunasekara AS, Truong T, Goh KS, Spurlock F, Tjeerdema RS. 2007. Environmental fate and toxicology of fipronil. J. Pestic. Sci. 32(3), 189-199.

Jackson D, Cornell CB, Luukinen B, Buhl K, Stone D. 2009. Fipronil Technical Fact Sheet. National Pesticide Information Center, Oregon State University Extension Services,

Stilwell GE, Saraswati S, J. Troy Littleton JT, Chouinard SW. 2006. Development of a Drosophila seizure model for in vivo high-throughput drug screening. European J Neurosci. 24, 2211-2222.

Tingle CC, Rother JA, Dewhurst CF, Lauer S, King WJ. 2003. Fipronil: environmental fate, ecotoxicology, and human health concerns. Rev. Environ. Contam Toxicol. 176, 1-66.

Ulate-Campos A, Coughlin F, Gaínza-Lein M, Fernández IS, Pearl PL, Loddenkemper T. 2016. Automated seizure detection systems and their effectiveness for each type of seizure. Seizure. 40:88-101.

Werner FM, Covenas R. 2011. Classical neurotransmitters and neuropeptides involved in generalized epilepsy: a focus on antiepileptic drugs. Curr. Med. Chem. 18(32), 4933-4948.

 


Scientific evidence supporting the linkages in the AOP

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Binding at picrotoxin site, iGABAR chloride channel directly leads to Reduction, Ionotropic GABA receptor chloride channel conductance Strong Strong
Reduction, Ionotropic GABA receptor chloride channel conductance directly leads to Reduction, Neuronal synaptic inhibition Strong Strong
Occurrence, A paroxysmal depolarizing shift directly leads to Occurrence, Epileptic seizure Strong Moderate
Reduction, Neuronal synaptic inhibition directly leads to Generation, Amplified excitatory postsynaptic potential (EPSP) Strong Moderate
Generation, Amplified excitatory postsynaptic potential (EPSP) directly leads to Occurrence, A paroxysmal depolarizing shift Moderate Moderate

Graphical Representation

Overall Assessment of the AOP

Biological plausibility

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). 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). 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 GABA antagonists including 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 electrophysiresponse 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). 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 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 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. Repetitive discharges lead to: 1) an increase in extracellular K+, which blunts the extent of hyperpolarizing outward K+ currents, tending to depolarize neighboring neurons; 2) accumulation of Ca2+ in presynaptic terminals, leading to enhanced neurotransmitter release; and 3) depolarization-induced activation of the NMDA subtype of the excitatory amino acid receptor, which causes more Ca2+ influx and neuronal activation. 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).

Uncertainties, inconsistencies, and data gaps

No inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, it is less well understood about 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 (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 not yet fully understood (Dichter and Ayala 1987).


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Adults Strong
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
mouse Mus musculus Strong NCBI
rat Rattus norvegicus Strong NCBI
bobwhite quail Colinus virginianus Strong NCBI
zebrafish Danio rerio Moderate NCBI
Sex Applicability
Sex Evidence
Male Strong
Female Strong

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 because GABA acts as an excitatory neurotransmitter due to increased intracellular Clˉ concentration during development of the nervous system (Taketo and Yoshioka 2000). 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).

Weight of Evidence Summary

See Collier et al. (2016) for details.

ScoreTable.jpg

Quantitative Consideration

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 possessing 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


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.

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/.

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.

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. Regulatory Toxicology and Pharmacology 75:46-57.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237: 157-164.

Galanopoulou AS. (2008) GABA(A) 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.

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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.

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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.

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.

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.

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.

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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.