GABAA Receptor Subtype Selectivity of the Proconvulsant Rodenticide TETS
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Arch Toxicol DOI 10.1007/s00204-017-2089-4 ORGAN TOXICITY AND MECHANISMS GABAA receptor subtype selectivity of the proconvulsant rodenticide TETS Brandon Pressly1 · Hai M. Nguyen1 · Heike Wulf1 Received: 16 June 2017 / Accepted: 5 October 2017 © The Author(s) 2017. This article is an open access publication Abstract The rodenticide tetramethylenedisulfotetramine Abbreviations (TETS) is a potent convulsant (lethal dose in humans AUC Area under the curve 7–10 mg) that is listed as a possible threat agent by the CI Confdence interval United States Department of Homeland Security. TETS has DMSO Dimethylsulfoxide previously been studied in vivo for toxicity and in vitro in EBOB 4′-Ethynyl-4-n-propylbicycloorthobenzoate binding assays, with the latter demonstrating it to be a non- EC50 Concentration producing 50% efect competitive antagonist on GABAA receptors. To determine Emax Maximal efect whether TETS exhibits subtype selectivity for a particular GABA Gamma-aminobutyric acid GABAA receptor combination, we used whole-cell patch- GFP Green fuorescent protein clamp to determine the potency of TETS on the major syn- IC50 Concentration producing 50% inhibition aptic and extrasynaptic GABAA receptors associated with nH Hill coefcient convulsant activity. The active component of picrotoxin, PTX Picrotoxinin picrotoxinin, was used as a control. While picrotoxinin did SD Standard deviation not diferentiate well between 13 GABAA receptors, TETS TETS Tetramethylenedisulfotetramine exhibited the highest activity on α2β3γ2 (IC 50 480 nM, 95% TBPS t-Butylbicyclophosphorothionate CI 320–640 nM) and α6β3γ2 (IC50 400 nM, 95% CI 290– 510 nM). Introducing β1 or β2 subunits into these receptor combinations reduced or abolished TETS sensitivity, sug- Introduction gesting that TETS preferentially afects receptors with α2/ β3 or α6/β3 composition. Since α2β3γ2 receptors make up GABAA receptors are heteropentameric ligand-gated chlo- 15–20% of the GABA A receptors in the mammalian CNS, ride channels that are activated by gamma-aminobutyric acid we suggest that α2β3γ2 is probably the most important (GABA), the main inhibitory neurotransmitter in the adult GABAA receptor for the seizure-inducing activity of TETS. CNS. GABAA receptors have a complex and often some- what promiscuous pharmacology with numerous orthosteric Keywords TETS · GABAA receptor · Electrophysiology · and allosteric sites that modulate channel function (Krall Picrotoxinin · Convulsant · Threat agent et al. 2015; Olsen 2015). While GABAA receptor agonists and positive allosteric modulators reduce neuronal excit- ability and can be used as anxiolytics and anticonvulsants, compounds that inhibit GABAA receptor functions increase * Heike Wulf neuronal fring and promote seizures. TETS (tetramethyl- [email protected] enedisulfotetramine) and picrotoxin are both potent convul- 1 sants (Haskell and Voss 1957; Zolkowska et al. 2012) that Department of Pharmacology, Genome and Biomedical can cause severe tonic–clonic seizures and are, therefore, Sciences Facility Room 3502, 451 Health Sciences Drive, School of Medicine, University of California, Davis, considered threat agents by the United States Department CA 95616, USA of Homeland Security. Both compounds are thought be to Vol.:(0123456789)1 3 Arch Toxicol work through a similar mechanism; nonselective inhibition a rodenticide in China, where several mass poisonings with of GABAA receptors, yet TETS is 30–100-fold more potent malicious intent have occurred (Whitlow et al. 2005; Zhang as a convulsant and lethal toxin in the mouse than picrotoxin et al. 2011). There have also been cases of reported TETS (Lamanna and Hart 1968; Shandra et al. 1996). poisonings in the United States with material sourced from GABAA receptors exhibit a high degree of structural China as an indoor rodenticide (Barrueto et al. 2003; Whit- heterogeneity and exist in multiple subtypes (Olsen and low et al. 2005). The frst pharmacological information about Sieghart 2009), with each subtype being a pentamer assem- TETS was generated by Alfred Haskell and Voss (1957), bled from a pool of 19 possible subunits, α1–α6, β1–β3, who tested TETS on organ preparations from frogs, rats, γ1–γ3, δ, ε, π, θ, and ρ1–ρ3. Based on the fact that TETS cats, and dogs and found that TETS acted exclusively on the and picrotoxin are structurally somewhat similar but dis- brain without exhibiting any activity on peripheral nerves tinct (Fig. 1), we wondered if the two agents would exhibit or muscle. Seizures only terminated with severance below diferent GABAA receptor subtype specifcities. Picrotoxin the medulla. The fact that sub-lethal doses of TETS could can be isolated from seeds of the moonseed family and has reverse pentobarbital-induced decreases in blood pressure an extremely bitter taste. It is an equimolar mixture of two and respiration in dogs pointed towards a mechanism poten- tricyclic sesquiterpenes: picrotin and the active component tially involving GABAA receptors. TETS was later shown to picrotoxinin (Slater and Wilson 1951). TETS has similar displace [35S]t-butylbicyclophosphorothionate ([35S]TBPS) physicochemical properties, but is easy to synthesize, taste- binding to rat brain membranes with an IC 50 of 1 μM (Esser less and odorless, and stable in drinking water for months et al. 1991; Squires et al. 1983), and to prevent 36Cl− uptake (Knaack et al. 2014). These characteristics make TETS a (Ratra et al. 2001) or to inhibit chloride currents (Barnych tangible threat. et al. 2017) through α1β2γ2 GABAA receptors expressed in TETS was frst synthesized in 1933 from sulfamide and HEK293 cells with IC 50 values of 1.3 or 8 μM, respectively. formaldehyde (Wood and Battye 1933) and then used as Interestingly, recent work from the Casida laboratory (Zhao an anti-mold agent for upholstery. Its severe toxicity frst et al. 2014) showed that “cold”-TETS displaces [14C]TETS became apparent in a German furniture factory where work- from rat brain membranes with a much lower IC 50 of 80 nM ers accidentally exposed to TETS-impregnated wool suf- than it displaces another radiolabeled caged convulsant, fered from disorientation and seizures (Hagen 1950). TETS 4′-ethynyl-4-n-[3H]propylbicycloorthobenzoate (EBOB), was later resynthesized under laboratory conditions and suggesting that TETS is binding to a site that is only partially found to be exceptionally poisonous with an extremely low overlapping with the EBOB or TBPS site on various GABA A parenteral LD50 of 0.1–0.3 mg/kg in rodents (Casida et al. receptors or that it might exhibit a specifc GABAA recep- 1976; Haskell and Voss 1957). The United States Forestry tor subtype selectivity. We here tested the later possibility Department explored TETS as a rodenticide but eventually using patch-clamp electrophysiology in an attempt to fnd abandoned it due to its extreme toxicity, the lethal dose in an explanation for the discrepancy between the relatively adult humans being 7–10 mg (Guan et al. 1993), the lack of low potency of TETS in inhibiting GABA A receptors and its a viable rescue agent, and its persistence in the environment high in vivo toxicity (Lamanna and Hart 1968; Zolkowska (Whitlow et al. 2005). TETS is currently banned world- et al. 2012). wide, but continues to be easily obtainable and popular as Materials and methods H O O Chemicals O O O O Picrotoxinin (PTX), fpronil, bicuculline, propofol, sali- N OH S cylidene salicylhydrazide, zinc chloride, GABA, dexameth- H O asone, zeocin, and geneticin were purchased from Sigma N N Aldrich (St. Louis, MO, United States). Diazepam, allopreg- O N N S nanolone, and DS2 (4-chloro- -[2-(2-thienyl)imidazo[1,2-a] pyridin-3-yl]benzamide) were purchased from Tocris Bio- O science (Bristol, United Kingdom). TETS was synthesized in the laboratory of Dr. Bruce Hammock, University of Califor- nia, Davis, CA (Zhao et al. 2014). 10 mM stocks of GABA TETS Picrotoxinin (PTX) were made fresh daily using Ringer solution (see below for composition). 10 mM stocks of PTX and TETS were Fig. 1 Chemical structures of TETS and picrotoxinin prepared in DMSO and diluted down into Ringer solution 1 3 Arch Toxicol immediately before application onto the cell. Both TETS dexamethasone was added to the media to induce α4 and β3 and PTX waste were treated with nitric acid and disposed of expressions (Mortensen et al. 2010). L929 or COS-7 cells using the waste accumulation program at UC Davis. were transfected using FuGENE 6 (ThermoFisher, Grand Island, NY, United States) transfection reagent in Opti- ® Preparation of cells expressing the GABAA receptors MEM reduced serum medium (Life Technologies, Benicia, CA, United States) with an equal amount of each of the sub- The human GABAA receptors α1, α2, α6, β1, β3, γ2L, and units (1:1:1) in combination with green fuorescent protein δ and the rat GABAA receptor β2 cloned into pcDNA3.1 (GFP) expressed from the pEGFP-C1 vector (Invitrogen). expression vectors were a gift from Dr. Robert L. Mac- The ratio of total cDNA to transfection reagent was 2:1. 48 h donald, Vanderbilt University, Nashville, TN. The human post-transfection, and cells were detached by trypsinization, GABAA receptor α4 cloned into a pcDNA3.1 expression washed, and plated onto poly-L-lysine-coated glass cover- vector was a gift from Dr. Richard Olson, University of slips. Transfected cells were identifed as GFP-expressing California, Los Angeles, CA. The human GABA A receptor cells, using an epifuorescence microscope for electrophysi- γ1 cloned into a pcDNA3.1 expression vector and a Lt-K