In Vitro Approaches for Testing Neurotoxicity Through

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In vitro approaches for testing neurotoxicity through the GABAA receptor - Summary Neurotoxicity The effect of compounds (enhancement or inhibition) on GABA –mediated chloride flux may be predictive of their potential to produce inhibitory or excitatory transmission neurotoxicity through the GABAA receptor.

Objective & Application

TYPE OF TESTING : Part of a test battery, Screening LEVEL OF ASSESSMENT : Toxic potency, Toxic potential PURPOSE OF TESTING : Classification/labelling, Mechanistic studies, Ranking

The GABAA receptor is the molecular target of a wide type of compounds, including pesticides, industrial compounds and pharmaceuticals. Several in vitro approaches based on the determination of the function of the GABAA receptor are being developed for the screening of compounds that may produce neurotoxicity by impairing the physiological inhibitory neural transmission.

Basis of the Method

Gamma-aminobutyric acid (GABA) operates primarily as an inhibitory neurotransmitter in the mature brain and exerts a crucial role in regulating brain excitability. GABA mediates these processes by the activation of ionotropic (GABA A) and metabotropic (GABAB) receptors. The binding of GABA to its recognition site on the GABAA receptor leads to the opening of a channel permeable to chloride ions resulting, in most neurons, in an inhibitory signal. Blockage of the GABA-gated chloride channel by both competitive or non-competitive GABA antagonists leads to hyper-excitation of the central nervous system, convulsions and death, whereas excessive activation of the channel by GABA agonists or activators results in ataxia, paralysis and death.

The GABAA receptor is the molecular target of several organochlorine insecticides, convulsant compounds, the active component of absinthe a-thujone, the anthelmintic avermectine, and the Amanita muscaria mushroom poison, muscimol. The GABAA receptor is also the molecular target of important classes of therapeutic drugs, such as benzodiazepines, barbiturates and anaesthetics. The different binding sites for GABA, benzodiazepines, barbiturates, anaesthetic steroids and the convulsant picrotoxinin are allosterically coupled. There are comprehensive updated reviews of the GABAA receptor (Kardos, 1999; Mehta and Ticku, 1999; Sieghart et al., 1999; Rudolph et al., 2001; Korpi et al., 2002).

In vitro testing of neurotoxicity through the GABAA receptor is performed by measuring ion flux by means of electrophysiological and transmembrane 36Cl- flux techniques and by radioreceptor binding assays by means of radioligand binding at the different recognition sites of the GABAA receptor. Guidances for neurotoxicity testing states that neurochemical endpoints are valuable where they are related to the mechanism of toxicity and where they are expected to have neurophysiological, neuropathological, or neurobehavioral correlates.(US EPA, 1998; OECD 2000).

Experimental Description Biological and Endpoint Measurement:

RECEPTOR BINDING: by measuring radioligand binding

GABAA RECEPTOR FUNCTION (Cl- FLUX): by means of transmembrane 36Cl- ion flux or electrophysiological measurements

Endpoint Value:

IC50

Experimental System:

Brain tissue preparations and primary cultured neurons from rat and mice, engineered cell lines and Xenopus oocytes expressing GABAA receptors

In vitroassays for determining Cl- flux through the GABAA receptor. Electrophysiological (patch-clamp) or radiotracer-based (36Cl- uptake) techniques are used to determine https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/method-summaries page 1 / 8 © ECVAM DB-ALM: Method Summary

GABAA receptor function. Primary neuronal cultures, engineered cell lines and Xenopus oocytes expressing native or recombinant GABAA receptor subunits, are used in patch-clamp electrophysiology experiments (Nagata and Narahashi 1994, 1995; Nagata et al., 1994, 1996; Belelli et al., 1996; Aspinwall et al., 1997; Maskell et al., 2001). The assay determines the amplitude of the ionic current induced by GABA and the modifications (inhibition or enhancement) produced by the addition of the test agents. Intact primary cultured neurons and microsacs or synaptoneurosomes from brain tissue are used in the 36Cl- uptake assay (Obata et al., 1988; Bloomquist et al., 1986; Gant et al., 1987; Pomés et al., 1994a, b; Huang and Casida, 1996; Vale et al., 1999, 2003). The neuronal preparation is incubated with 36Cl- and GABA in the presence of test agents and 36Cl- uptake is measured by liquid scintillation counting.

In vitroradioreceptor binding assays at the GABAA receptor. Squires et al. (1983) first described the use of the radioligand [35S]-t-butyl bicyclophosphorothionate ([35S]TBPS) to label the picrotoxinin recognition site (also called the convulsant site) at the GABAA receptor in brain membranes. Membranes obtained from brain tissue or neuronal cultures as well as intact cultured neuronal cells are incubated with [35S]TBPS in the presence of test agents followed by the determination of bound radioactivity by using liquid scintillation counting (Squires et al., 1983, 1990; Llorens et al., 1990; Pomés et al., 1993; 1994a; Vale et al., 1997; Elster et al., 2000). The addition of competitive GABAA receptor antagonists to the binding assay allows to differentiate compounds that directly inhibit [35S]TBPS binding from those that allosterically inhibit [35S]TBPS binding (Squires et al., 1983, 1984; Vale et al., 1997). [3H]flunitrazepam binding to the benzodiazepine recognition site at the GABAA receptor is performed in membranes obtained from brain tissue or cultured neurons as well as in intact cultured neuronal cells (Mehta and Ticku 1987, 1988, 1992; Squires et al., 1990; Harris et al., 1993; Vale et al., 1997; 1998; 1999; Elster et al., 2000; Ali and Olsen 2001; Fonfría et al., 2001). [3H]muscimol or [3H]GABA binding to the GABA recognition site at the GABAA receptor is performed in membranes obtained from brain tissue or cultured neurons (Horng and Wong 1979; Maksay 1988; Vale et al., 1997; Elster et al., 2000; Fonfría et al., 2001).

Data Analysis/Prediction Model

So far, no prediction model for neurotoxicity testing has been described yet. However, data found in the literature reveal that, in general, the relative potency of convulsants in inhibiting [ 35S]TBPS binding and GABA-stimulated chloride flux is in good agreement with their mammalian toxicity (Lawrence and Casida, 1984; Squires et al., 1984; Cole and Casida, 1986; Gant et al., 1987; Abalis et al., 1985; Bloomquist et al., 1986; Obata et al., 1988), establishing the toxicological relevance of this binding site for closely related compounds (Casida, 1993).

Test Compounds

All chemicals; agrochemicals.

Modifications

Patch clamp electrophysiology methods yield detailed information concerning mechanisms of action, but suffer with regard throughput screening. High throughput electrophysiology may be a reality in the future with the introduction of automated patch clamp instruments (Xu et al., 2001). Gross et al. (1997) and Egert et al. (1998, 2002) developed a method to assess neuronal activity in cultures grown on substrate-integrated microelectrode arrays: higher throughput but lower mechanistic specificity is obtained (Gramowski et al., 2000; Keefer et al., 2001; Potter et al., 2001). Fluorescent assays are being developed as alternative assays for measuring Cl- flux (Inglefield and Schwartz-Bloom, 1997; Chistina Grobin et al., 1999; Inglefield and Shafer, 2000; Kuner and Agustine, 2000; Adkins et al., 2001; Sah et al., 2002). At present, these assays are not fully developed and have not yet been used in neurotoxicity testing. Lawrence and Casida (1984) performed [35S]TBPS binding in the presence of a metabolic system. Compounds that undergo in vivo activation/detoxification showed enhanced/reduced potencies in the coupled in vitro TBPS assay. Tritiated radioligands have been developed for the analysis of the picrotoxinin site of the GABA-gated chloride channel (Huang and Casida, 1996, 1997; Rauh et al., 1997; Hamon et al., 1998). Among them, [3H]ethynylbicycloorthobenzoate ([3H]EBOB) has higher affinity and longer radioisotopic half-life than [ 35S]TBPS. https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/method-summaries page 2 / 8 © ECVAM DB-ALM: Method Summary

Ozoe and Akamatsu (2001) reported 3D-QSAR analyses of non-competitive GABA antagonists by using [35S]TBPS binding data of about 50 compounds.

Two human neuronal differentiated cell lines (IMR-32 and NT2-N) that express different GABAA receptors subunits have been reported. Their pharmacology may depend on their degree of differentiation and heterogeneous expression of GABAA receptor isoforms (Anderson et al., 1993; Noble et al., 1993; Neelands et al., 1998, 1999; Sapp and Yeh, 2000).

Discussion

Measurements of GABA-induced Cl- flux and radioligand binding have demonstrated that the organochlorine pesticides lindane and cyclodiene-related compounds block the GABAA receptor, in agreement with their convulsant properties (Bloomquist and Soderlund, 1985; Bloomquist et al., 1986; Cole and Casida, 1986; Llorens et al., 1990; Pomés et al., 1993, 1994a,b; Ogata et al., 1988, Nagata et al., 1994, 1996; Nagata and Narahashi 1994, 1995). Mutagenesis and pharmacophoric studies of the GABAA receptor support a mechanism of action in which these convulsants bind within the chloride channel lumen (Zhang et al., 1994; Jursky et al., 2000; Vale et al. 2003). Furthermore, there is a quantitative correlation between the potency to inhibit [35S]TBPS or [3H]EBOB binding and the potency to block GABA-stimulated chloride flux for a large variety of convulsant compounds (Gant et al., 1987; Bloomquist et al., 1986; Pomés et al., 1994b; Huang et al., 1996). Likewise, depressant compounds, including pharmaceuticals (benzodiazepines, barbiturates, anesthetics) and toxic agents, enhance GABA-stimulated chloride flux; in addition, some of them allosterically increase [3H]flunitrazepam binding in neuronal preparations (Mehta and Ticku, 1987,1992, Moody et al., 1993; Pomés et al., 1993, 1994b; Mihic et al., 1994; Belelli et al., 1996; Aspinwall et al., 1997; Vale et al., 1997; Maskell et al., 2001; Ratra et al., 2001)

GABAA receptors are composed of five subunits that can belong to eight different subunit classes. Subunit composition of GABAA receptors affects response to GABA agonists and modulators (Alsbo et al., 2001; Hansen et al., 2001; Korpi et al., 2002). In general, convulsants interacting at the picrotoxinin site show similar potency on inhibiting [35S]TBPS, [3H]EBOB binding or Cl- flux regardless of GABAA receptor subunities with the exception of the human b3 subunit (Nagata et al., 1994, 1996; Llorens et al., 1990; Aspinwall et al. 1997; Huang and Casida, 1997; Maskell et al., 2001; Ratra and Casida 2001; Ratra et al., 2001). However, specificity among compounds and mammal selective toxicity are better predicted by using the native GABAA receptor (Ratra et al., 2001). Contrarily, the allosteric effects of GABA mimetics on the picrotoxinin recognition site may depend on receptor subunit composition (Huang and Casida, 1997).

Advantages: Binding and electrophysiology assays provide data that are linked to the mechanism of action of the tested agents. Therefore, the data obtained are relevant for the prediction of toxicity. Binding assays are easy to be performed. Patch-clamp electrophysiologic techniques allow the measurement of any receptor and ion channel in practically any type of cells with a high degree of precision and time resolution. Determination of the GABAA receptor function in the presence of tested agents may identify convulsant and depressant compounds.

Limitations: Electrophysiology assays require high expertise and specific equipment. Performance of radiochemical assays requires that the laboratory have a radioactive installation. The 36Cl- uptake assay may not be suitable for screening purposes, due to the high radiation energy and long half-life of the radionuclide. The use of bioactivation systems has not been described so far. Only compounds that act on the GABAA receptor are identified.

Status

The herein described techniques still need significant basic research before they will be useful in tiered screening systems. Further development is needed to establish the adequate/s radioligand and fluorescent probes, as well as for the choice of the most adequate in vitro system(s) for the testing of neurotoxicity through the GABAA receptor.

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Last update: January 2003

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