A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka

Elise Pesce; Marion Garde; Muriel Rigolet; Andrew J. Tindall; Gregory F. Lemkine; Lisa A. Baumann; Laurent M. Sachs; David Du Pasquier

doi:10.1021/acs.est.3c05515

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka

Elise Pesce, Marion Garde, Muriel Rigolet, Andrew J. Tindall, Gregory F. Lemkine, Lisa A. Baumann, Laurent M. Sachs, David Du Pasquier^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T₃) and thyroxine (T₄)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T₃, T₄, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Original language	English
Pages (from-to)	99-109
Number of pages	11
Journal	Environmental Science and Technology
Volume	58
Issue number	1
Early online date	20 Dec 2023
DOIs	https://doi.org/10.1021/acs.est.3c05515
Publication status	Published - 9 Jan 2024

Bibliographical note

Funding Information:
We thank Gad-Styven Boukou, Thomas Lejeune, Nicolas Loire, Nathalie Pavageau, and Mathilde Jacob for the care of the medaka facilities. We thank Romain Girard and Marine Rousseau for capturing images of transgenic fish. We thank Nicolas Buisine for running GeNorm and Flavie Vignier for her help with RT-qPCR. We thank Jérome Polentes and Céline Leteur for inviting us to perform immunofluorescence in toto at I-STEM, France. We are grateful to the “Imaging and Cytometry Core Facility” of Genethon, France, for technical support. We thank Thomas Braunbeck for inviting us to perform histology in the Centre for Organismal Studies at the University of Heidelberg, Germany. This research has been carried out with the financial support of ANRT (Association Nationale de la Recherche et de la Technologie) and is partially funded by the European Union’s Horizon 2020 research and innovation program, under grant agreement No. 825753 (ERGO).

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society

Keywords

fish
new approach methodology
Oryzias latipes
screening test
thyroid follicles
Thyroid hormone disruption

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title = "A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka",

abstract = "Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.",

keywords = "fish, new approach methodology, Oryzias latipes, screening test, thyroid follicles, Thyroid hormone disruption",

author = "Elise Pesce and Marion Garde and Muriel Rigolet and Tindall, {Andrew J.} and Lemkine, {Gregory F.} and Baumann, {Lisa A.} and Sachs, {Laurent M.} and {Du Pasquier}, David",

note = "Funding Information: We thank Gad-Styven Boukou, Thomas Lejeune, Nicolas Loire, Nathalie Pavageau, and Mathilde Jacob for the care of the medaka facilities. We thank Romain Girard and Marine Rousseau for capturing images of transgenic fish. We thank Nicolas Buisine for running GeNorm and Flavie Vignier for her help with RT-qPCR. We thank J{\'e}rome Polentes and C{\'e}line Leteur for inviting us to perform immunofluorescence in toto at I-STEM, France. We are grateful to the “Imaging and Cytometry Core Facility” of Genethon, France, for technical support. We thank Thomas Braunbeck for inviting us to perform histology in the Centre for Organismal Studies at the University of Heidelberg, Germany. This research has been carried out with the financial support of ANRT (Association Nationale de la Recherche et de la Technologie) and is partially funded by the European Union{\textquoteright}s Horizon 2020 research and innovation program, under grant agreement No. 825753 (ERGO). Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society",

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AU - Rigolet, Muriel

AU - Tindall, Andrew J.

AU - Lemkine, Gregory F.

AU - Baumann, Lisa A.

AU - Sachs, Laurent M.

AU - Du Pasquier, David

N1 - Funding Information: We thank Gad-Styven Boukou, Thomas Lejeune, Nicolas Loire, Nathalie Pavageau, and Mathilde Jacob for the care of the medaka facilities. We thank Romain Girard and Marine Rousseau for capturing images of transgenic fish. We thank Nicolas Buisine for running GeNorm and Flavie Vignier for her help with RT-qPCR. We thank Jérome Polentes and Céline Leteur for inviting us to perform immunofluorescence in toto at I-STEM, France. We are grateful to the “Imaging and Cytometry Core Facility” of Genethon, France, for technical support. We thank Thomas Braunbeck for inviting us to perform histology in the Centre for Organismal Studies at the University of Heidelberg, Germany. This research has been carried out with the financial support of ANRT (Association Nationale de la Recherche et de la Technologie) and is partially funded by the European Union’s Horizon 2020 research and innovation program, under grant agreement No. 825753 (ERGO). Publisher Copyright: © 2023 The Authors. Published by American Chemical Society

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N2 - Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

AB - Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

KW - fish

KW - new approach methodology

KW - Oryzias latipes

KW - screening test

KW - thyroid follicles

KW - Thyroid hormone disruption

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A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka

Abstract

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Keywords

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