A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

Abstract

The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.

Contaminant and Environmental Influences on Thyroid Hormone Action in Amphibian Metamorphosis

“…However, sulfated TBBPA conjugates lack this T 3 displacement capacity (77). Independent studies in X. laevis recapitulated similar finding of TBBPA inhibition of T 3 -mediated reductions in head area, hindlimb length and decreased apoptosis and epithelial folds within intestines (78–80). Additionally, various groups described the restricted activation of a thibz response element-luciferase reporter assay and the reduction of tissue-specific gene expression of thrb, st3, klf9, cebpd, mmp2, dio2 , and dio3 transcripts upon TBBPA inhibition of T 3 -induced metamorphosis (Table 3) (78–80).…”

Thyroid-disrupting chemicals and brain development: an update

“…In the latter study, TBBPA was further found to alter expression of TH target genes implicated in neural stem cell function and differentiation. Whether such effects extend to other proliferative brain regions during development remains to be investigated ( 115 ).…”

Thyroid Disrupting Chemicals

“…Xenopus laevis is another animal model used to evaluate morphological developmental problem due to different signal alterations. Very recently, Mendelin et al studied the potential action of thyroid disruptors tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol-A (TBBPA) in a tadpole model from a transgenic line of Xenopus Laevis [ 51 ]. BDE-47 didn’t show a direct thyroid impact, but a rather general toxicity, while TBBPA might have a disrupting action.…”

“…BDE-47 didn’t show a direct thyroid impact, but a rather general toxicity, while TBBPA might have a disrupting action. Overall, the transgenic model resulted to be an efficient essay [ 51 ]. In vitro studies have also been conducted to evaluate the potential pathophysiology of the endocrine disruption [ 52 ].…”