An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos

The knowledge on environmentally relevant chemicals that may interfere with thyroid signaling is scarce. Here, we present a method for the screening of goitrogens, compounds that disrupt the thyroid gland function, based on the automatic orientation of zebrafish in a glass capillary and a subsequent imaging of reporter gene fluorescence in the thyroid gland of embryos of the transgenic zebrafish line tg(tg:mCherry). The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to a negative control compound (3,4-dichloroaniline), or a concentration series of known goitrogenic compounds (resorcinol, methimazole, potassium perchlorate, 6-propyl-2-thiouracil, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on mortality and/or solubility. Exposure to 3,4-dichloroaniline decreased the fluorescence signal. All goitrogenic compounds exhibited clear concentration-dependent inductions of reporter fluorescence 1.4 to 2.6 fold above control levels. Concentration-response modelling was used to calculate goitrogenic potencies based on EC50 values. The new automated method offers an efficient screening approach for goitrogenic activity.

[1]  Mehmet Fatih Yanik,et al.  High-throughput in vivo vertebrate screening , 2010, Nature Methods.

[2]  Xiaowei Zhang,et al.  Zebrafish embryos/larvae for rapid determination of effects on hypothalamic-pituitary-thyroid (HPT) and hypothalamic-pituitary-interrenal (HPI) axis: mRNA expression. , 2013, Chemosphere.

[3]  C. Y. Chen,et al.  Thyroid hormone receptor represses miR-17 expression to enhance tumor metastasis in human hepatoma cells , 2013, Oncogene.

[4]  G. Diamante,et al.  Influence of Temperature on the Thyroidogenic Effects of Diuron and Its Metabolite 3,4-DCA in Tadpoles of the American Bullfrog (Lithobates catesbeianus). , 2016, Environmental science & technology.

[5]  R. Nagel.  DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology. , 2002, ALTEX.

[6]  Hilda Witters,et al.  Zebrafish embryos as an alternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations. , 2012, Reproductive toxicology.

[7]  Kevin M. Crofton,et al.  Thyroid-Disrupting Chemicals: Interpreting Upstream Biomarkers of Adverse Outcomes , 2009, Environmental health perspectives.

[8]  Bruce C Allen,et al.  Benchmark dose (BMD) modeling: current practice, issues, and challenges , 2018, Critical reviews in toxicology.

[9]  Ashok Kumar,et al.  Severe hyperthyroidism induces mitochondria‐mediated apoptosis in rat liver , 2004, Hepatology.

[10]  B. Mughal,et al.  Thyroid-disrupting chemicals and brain development: an update , 2018, Endocrine connections.

[11]  A. V. van Wezel,et al.  Narcosis due to environmental pollutants in aquatic organisms: residue-based toxicity, mechanisms, and membrane burdens. , 1995, Critical reviews in toxicology.

[12]  Demetrio Raldúa,et al.  Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. , 2009, Environmental science & technology.

[13]  R. Altenburger,et al.  Development of a general baseline toxicity QSAR model for the fish embryo acute toxicity test. , 2016, Chemosphere.

[14]  U. Liebel,et al.  Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing , 2014, BMC Biotechnology.

[15]  S. Reibe,et al.  Analysis of origin and growth of the thyroid gland in zebrafish , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[16]  B. Piña,et al.  Deiodinases and thyroid metabolism disruption in teleost fish. , 2014, Environmental research.

[17]  Jan Huisken,et al.  Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development. , 2012, Developmental biology.

[18]  J. Wheeler,et al.  A review of the evidence for endocrine disrupting effects of current-use chemicals on wildlife populations , 2018, Critical reviews in toxicology.

[19]  Colin D. Brown,et al.  LogD: lipophilicity for ionisable compounds. , 2008, Chemosphere.

[20]  Daniel Zalko,et al.  An in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption. , 2007, Environmental science & technology.

[21]  Albertinka J Murk,et al.  Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[22]  É. Fetter,et al.  Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos. , 2015, Reproductive toxicology.

[23]  Demetrio Raldúa,et al.  Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. , 2011, Environmental science & technology.

[24]  Stefan Scholz,et al.  Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis. , 2012, Environmental science & technology.

[25]  Ksenia Tonyushkina,et al.  Embryonic exposure to excess thyroid hormone causes thyrotrope cell death. , 2014, The Journal of clinical investigation.

[26]  Daniel M. Rotroff,et al.  Development of a thyroperoxidase inhibition assay for high-throughput screening. , 2014, Chemical research in toxicology.

[27]  Adam Lillicrap,et al.  The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. , 2010, Aquatic toxicology.

[28]  A. Lombardi,et al.  Genetic–epigenetic dysregulation of thymic TSH receptor gene expression triggers thyroid autoimmunity , 2014, Proceedings of the National Academy of Sciences.

[29]  Mineyuki Mizuguchi,et al.  Biosensor recognition of thyroid-disrupting chemicals using transport proteins. , 2006, Analytical chemistry.

[30]  Thorsten Meinl,et al.  KNIME: The Konstanz Information Miner , 2007, GfKl.