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

Brenda J. Mengeling, Yuzhu Wei, Lucia N. Dobrawa, Mischa Streekstra, Jochem Louisse, Vikrant Singh, Latika Singh, Pamela J Lein, Heike Wulff, Albertinka J. Murk, John Furlow

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)1-10
Number of pages10
JournalAquatic Toxicology
Volume190
DOIs
StatePublished - Sep 1 2017

Fingerprint

thyroid hormones
Thyroid Hormones
hormone
assay
assays
tadpoles
Thyroid Hormone Receptors
Larva
luciferase
Luciferases
metamorphosis
Hormone Antagonists
Xenopus laevis
DNA
antagonists
cell proliferation
precocious metamorphosis
Cell Proliferation
Retinoid X Receptors
brain

Keywords

  • Amphibian metamorphosis
  • Endocrine disruption
  • Flame retardants
  • Thyroid hormone
  • Xenopus laevis

ASJC Scopus subject areas

  • Aquatic Science
  • Health, Toxicology and Mutagenesis

Cite this

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling. / Mengeling, Brenda J.; Wei, Yuzhu; Dobrawa, Lucia N.; Streekstra, Mischa; Louisse, Jochem; Singh, Vikrant; Singh, Latika; Lein, Pamela J; Wulff, Heike; Murk, Albertinka J.; Furlow, John.

In: Aquatic Toxicology, Vol. 190, 01.09.2017, p. 1-10.

Research output: Contribution to journalArticle

Mengeling, Brenda J. ; Wei, Yuzhu ; Dobrawa, Lucia N. ; Streekstra, Mischa ; Louisse, Jochem ; Singh, Vikrant ; Singh, Latika ; Lein, Pamela J ; Wulff, Heike ; Murk, Albertinka J. ; Furlow, John. / A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling. In: Aquatic Toxicology. 2017 ; Vol. 190. pp. 1-10.
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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.",
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AU - Wei, Yuzhu

AU - Dobrawa, Lucia N.

AU - Streekstra, Mischa

AU - Louisse, Jochem

AU - Singh, Vikrant

AU - Singh, Latika

AU - Lein, Pamela J

AU - Wulff, Heike

AU - Murk, Albertinka J.

AU - Furlow, John

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N2 - 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.

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KW - Endocrine disruption

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KW - Thyroid hormone

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