Abstract
There is a need for rapidly screening thyroid hormone (TH) signaling disruptors in vivo considering the essential role of TH signaling in vertebrates. We aimed to establish a rapid in vivo screening assay using Xenopus laevis based on the T3-induced Xenopus metamorphosis assay we established previously, as well as the Xenopus Eleutheroembryonic Thyroid Assay (XETA). Stage 48 tadpoles were treated with a series of concentrations of T3 in 6-well plates for 24 h and the expression of six TH-response genes was analyzed for choosing a proper T3 concentration. Next, bisphenol A (BPA) and tetrabromobisphenol A (TBBPA), two known TH signaling disruptors, were tested for determining the most sensitive TH-response gene, followed by the detection of several suspected TH signaling disruptors. We determined 1 nM as the induction concentration of T3 and thibz expression as the sensitive endpoint for detecting TH signaling disruptors given its highest response to T3, BPA, and TBBPA. And we identified betamipron as a TH signaling agonist, and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) as a TH signaling antagonist. Overall, we developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis.
Highlights
Thyroid hormones (THs, T3 and T4) produced by the thyroid gland are critical for growth and development in vertebrates [1]
The present study aimed to develop a simple and rapid screening assay for detecting thyroid hormone (TH) signaling disruptors using wild-type Xenopus tadpoles, referencing the Xenopus Eleutheroembryonic Thyroid Assay (XETA) and the T3-induced Xenopus metamorphosis assay (TIXMA)
When 1 nM T3 upregulated the expression of all the test TH-response genes, 1000 and/or 100 nM both bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) antagonized T3 actions, showing that this screening assay is effective in detecting TH signaling antagonists
Summary
Thyroid hormones (THs, T3 and T4) produced by the thyroid gland are critical for growth and development in vertebrates [1]. Several in vitro binding assays [14] and in vitro transfection assays based on reporter genes [15] have been developed to detect TH signaling disruptors. In vitro TR binding assays can detect whether chemicals bind to TR, but cannot distinguish whether they are agonists or antagonists of TH signaling. In vitro transfection assays can screen TH signaling agonists or antagonists but not completely reflect what happens in vivo due to the lack of the capacity to metabolize test chemicals in cells. The reporter-enzymes-based GH3-TRE-Luciferase assay established by Freitas et al (2011) [12] has been modified into a high throughput screening assay in the Tox project, aiming to screen TH signaling agonists or antagonists. It is still necessary to develop rapid in vivo screening assays for detecting TH signaling disruptors
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