The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7–10 mg) that is listed as a possible threat agent by the United States Department of Homeland Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a noncompetitive antagonist on GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patchclamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2β3γ2 (IC50 480 nM, 95% 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, suggesting that TETS preferentially affects receptors with α2/β3 or α6/β3 composition. Since α2β3γ2 receptors make up 15–20% of the GABAA receptors in the mammalian CNS, we suggest that α2β3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.
Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABA A R) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABA A R antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABA A R antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABA A R antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABA A R positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABA A R antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABA A R antagonism.
ABSTRACT:We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D) labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a significantly improved synthesis of NH-3. ■ INTRODUCTIONThyroid hormones regulate growth, metabolism, and homeostasis of vertebrates. They bind to thyroid hormone receptors (THRs) found in almost all cells of the body and exert their effect by altering gene expression. 1 Various developmental and metabolic disorders can result from under-or over-activity of the thyroid. Thus, both THR agonists and antagonists have immense medicinal importance. 2,3 NH-3 (Figure 1) is currently the most potent synthetic small-molecule THR antagonist. 4,5 For a study investigating disrupting effects of environmental toxicants on thyroid hormone regulated neurodevelopmental processes, NH-3 was required as a reference compound. NH-3, which is not commercially available, can only be obtained by following a long synthetic route originally reported by Nguyen et al. 6 In our hands, the synthesis of NH-3 following this procedure resulted in samples of NH-3 that contained high amounts of an inseparable benzofuran impurity (EBI). Through deuterium-labeling experiments, we have demonstrated that the presence of EBI is the result of a base-mediated annulation of an o-alkylnylphenol precursor. The mechanism of formation of benzofurans from o-alkylnylphenols by Pd-, 7a,b Cu-, 7c 7d and Pt-catalyzed 7e reactions has previously been studied. Jacubert et al. 7f have reported an acid-mediated annulation of o-alkylnylphenol methyl ethers and in one case used deuterium labeling to establish the electrophilic nature of the transformation. Our experiment reveals that NH-3 converts into EBI via base-mediated intramolecular annulation without a transition-metal catalyst. There have been speculations about EBI by other research groups, but it could never previously be identified. 8 Therefore, we decided to identify, synthesize, and study the biological activity of EBI and investigate its mechanism of formation along with developing an improved and higher-yielding synthesis of Received: November 20, 2015 Figure 1. Structures of the THR antagonist NH-3 and the benzofuran EBI. ■ RESULTS AND DISCUSSIONOur synthesis of the key intermediates 9 and 10 is depicted in Scheme 1. This sequence is similar to the one reported by Nguyen et al. 6 which includes several steps involving the use of n-BuLi, a pyrophoric substance, posing a safety hazard. The use of BuLi not only necessitates extremely careful handling but also adversely affects reaction scale-up in academic laboratory settings. Therefore, we decided to minimize or eliminate the use of n-BuLi from the sequence and made some cha...
Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABA A) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD 50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 μM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α 2 β 3 γ 2 GABA A-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.