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Thyroid Mediated CNS Dysfunction

Kevin M. Crofton, PhD US Environmental Protection Agency McKim Conference Duluth MN September 17, 2008. Thyroid Mediated CNS Dysfunction. How to use what we know about the structure and function of the thyroid system to generate data using in vitro methods that can populate QSAR models.

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Thyroid Mediated CNS Dysfunction

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  1. Kevin M. Crofton, PhD US Environmental Protection Agency McKim Conference Duluth MN September 17, 2008 Thyroid Mediated CNS Dysfunction How to use what we know about the structure and function of the thyroid system to generate data using in vitro methods that can populate QSAR models

  2. Outline • Thyroid hormones and homeostatic mechanisms • A mode-of-action for thyroid disruption and adverse outcomes on nervous system development • Targets for disruption • Targets for screening • Summary

  3. I I I I I I O O O HO HO HO O O O CH2 CH2 CH2 CH CH CH C C C H H H NH NH NH O O O I I I I I I 2 2 2 I I I I I I O O O HO HO HO O O O CH2 CH2 CH2 CH CH CH C C C H H H NH NH NH O O O I I I 2 2 2 Thyroid Hormones- Structure and Function • T3 and T4 are the principle hormones synthesized and released by the thyroid gland • Development - Critical for differentiation and growth • Transient disruption = permanent effects • Adult – Important for energy and thermoregulation • Transient disruption = transient effects Thyroxin (T4) Triiodothyronine (T3)

  4. Regulation of Thyroid Hormones TR Hypothalamus TRH TR Pit Acts as a ligand for nuclear thyroid hormone receptors (TRs) + Elimination from the body TSH Thyroid T3/T4 Catabolic Enzymes Blood T4 T3 Liver TH binding proteins 5’-deiodinases TH binding proteins Target Tissues

  5. TR TR T3 T3 T4 AAAAA Transporter DI Cellular Acton of TH Corepressors Co Activator X Zoeller, 2003

  6. Thyroid MOAs

  7. Adverse Outcome Pathways Toxicity Pathways What do we know and not know about these pathways?

  8. Major Sequelae of Thyroid Disruption • Adult Exposure • Thyroid tumors in laboratory animals • Not a relevant mechanism for human cancer • May increase incidence of cardiovascular disease • Neurodevelopment • Lack of THs result in adverse neurological development (sensory, motor, cognitive) • Species independent (fact) • Rat is appropriate animal model for neurodevelopmental effects • These are two different outcomes that can result from the same molecular targets • One is relevant for human health and one is not

  9. Neurodevelopment and Thyroid Dysfunction • FACT: without adequate TH the nervous fails to properly develop • Iodine deficiency • Congenital hypothyroidism

  10. Dose-Response and Critical WindowDioxins, Furans and PCBs - Hearing Loss Binding to PXR Hepatic Parent or Metabolite Exposure Hepatic Phase II Enzymes Binding to AhR Alter TR Mediated Proteins Loss of cochlear hair cells  Serum T4 & T3  Tissue T3 Hearing Loss

  11. CNS Protein (1) DItissue UGTs CNS malformation PXR Binding Response T4serum T3serum Functional Loss (eg. IQ) T3tissue TRactivation CNS Protein (2) Increasing Dose and/or Time

  12. PHAHs and Ototoxicity

  13. Dose-Response Perchlorate, Propylthiouracil and Hippocampal Physiology Thyroid PTU Binding to TPO Exposure  Serum T4 & T3 Thyroid Perchlorate Inhibition Of TPO Alter TR Mediated Proteins  Hippocampal T3 Synaptic Malformation Altered Synaptic Function Learning Impariment

  14. Dose-Response Perchlorate, Propylthiouracil and Hippocampal Physiology Hippocampal physiology Water maze learning

  15. What do we not know? • Dose-response relationships • Critical windows • Sensitive biomarkers (T4?) • However, we can’t get lost in the need to understand everything in the pathway • Causative and predictive is minimum • Quantitative models are the holy grail

  16. What can we do to inform QSAR models • Develop in vitro test methods for known targets

  17. In Vitro Models for Thyroid Disruptors • Iodine Symporter (NIS) • Thyroperoxidase (TPO) • Deiodinases • Transporters – Blood • Transporters – Cellular • Thyroid Receptors • Hepatic Nuclear Receptors

  18. In Vitro Models – Thyroid Receptor Beta GeneBLAzer TRb-UAS-bla HEK293 Cell Line ** Substrate ** ** ** T3 Stimulation of TRb ** ** T3 (M) ** ** Cell line contains a beta-lactamse reporter gene under the control of an UAS response element stably integrated in Hek293 cells. This line also stably expresses a fusion protein consisting of the GAL4 DNA binding domain and the TRb ligand binding domain. Courtesy of Keith Houck, NCCT

  19. T3 Levothyroxine Human TRb Reporter Gene Assay Heat Map of AC50’s1456 chemicals; 14 concentrations; agonist and antagonist modes Courtesy of Keith Houck, NCCT

  20. TH Action Assay – T-Screen (Gutleb et al. EnvToxPharm 2005) - measures TH dependent cell proliferation in GH3 cells - 96 well plate assay

  21. Summary • Thyroid pathways are known • Multiple targets involved • In vitro models are available for many of the targets • Need to begin testing chemicals

  22. Thank You

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