A Multiwell-Based Assay for Screening Thyroid Hormone Signaling Disruptors Using thibz Expression as a Sensitive Endpoint in Xenopus laevis

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.

Keywords: Xenopus laevis; multiwell plate; screening assay; thibz gene; thyroid hormone.

Figure 1

Relative expression of thyroid hormone (TH)-response genes in stage 48 Xenopus tadpoles following 24-h exposure to T3. Data are shown as mean ± SEM (n = 3). * indicates significant differences between T3 exposure and the control (p < 0.05). klf9: Kruppel-like factor 9; thibz: thyroid hormone induced bZip protein; thrb: thyroid hormone receptor beta; st3: stromelysin-3; dio3: type 3 deiodinase; mmp13: matrix metallopeptidase 13.

Figure 2

Relative expression of thyroid hormone (TH)-response genes in stage 48 Xenopus tadpoles following 24-h exposure to bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) in the absence or presence of 1 nM T3. Data are shown as mean ± SEM (n = 3). * and # indicate significant differences between BPA or TBBPA exposure and the control and between BPA or TBBPA + T3 and T3 treatment, respectively (p < 0.05). klf9: Kruppel-like factor 9; thibz: thyroid hormone induced bZip protein; thrb: thyroid hormone receptor beta; st3: stromelysin-3; dio3: type 3 deiodinase; mmp13: matrix metallopeptidase 13.

Figure 3

Relative expression of thibz (thyroid hormone induced bZip protein) in stage 48 Xenopus tadpoles following 24-h exposure to betamipron, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), triclocarban (TCC), triclosan (TCS), benzophenone (BP), and benzophenone-3 (BP-3) in the absence or presence of 1 nM T3. Data are shown as mean ± SEM (n = 3). * and # indicate significant differences between the chemical exposure and the control and between chemical + T3 and T3 treatment, respectively (p < 0.05).