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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 Kevin M. Crofton, PhD US Environmental Protection Agency McKim Conference Duluth MN September 17, 2008 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 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 I I Thyroxin (T4) HO I I Triiodothyronine (T3) I I HO CH2 CH C O H NH2 O O O I CH2 CH C O H NH2 O Regulation of Thyroid Hormones TR Hypothalamus TRH TR Pit + Elimination from the body Acts as a ligand for nuclear thyroid hormone receptors (TRs) TSH Thyroid Catabolic Enzymes T3/ Blood Liver TH binding proteins T4 T4 T3 5’-deiodinases Target Tissues Cellular Acton of TH Corepressors Co Activator TR Transporter T4 TR DI T3 X AAAAA T3 Zoeller, 2003 Thyroid MOAs Targets Thyroidal Effects & Non-Cancer Cancer Early Biological Effect Tissue Specific Effect Altered Structure/ Function Clinical Disease Thyroperoxidase Iodine Symporter Extra-Thyroidal Hepatic UDPGTs Exposure Deiodinases Cellular Transporters T4–TTR Binding Thyroid Receptors TSH Thyroid Hyperplasia Thyroid Tumors Tissue T3 Changes Altered Development Birth Defects Serum T3 & T4 Changes Adverse Outcome Pathways Toxicity Pathways Targets Thyroidal Effects & Non-Cancer Cancer Early Biological Effect Tissue Specific Effect Altered Structure/ Function Clinical Disease Thyroperoxidase Iodine Symporter Extra-Thyroidal Hepatic UDPGTs Exposure TSH Thyroid Hyperplasia Thyroid Tumors Serum T3 & T4 Changes Tissue Altered Birth Deiodinases What do we know and not knowT3about these pathways? Changes Development Defects Cellular Transporters T4–TTR Binding Thyroid Receptors 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 Neurodevelopment and Thyroid Dysfunction • FACT: without adequate TH the nervous fails to properly develop Iodine deficiency Congenital hypothyroidism Dose-Response and Critical Window Dioxins, Furans and PCBs - Hearing Loss Exposure Hepatic Parent or Metabolite Serum T4 & T3 Binding to PXR Hepatic Phase II Enzymes Binding to AhR Tissue T3 Alter TR Mediated Proteins Loss of cochlear hair cells Hearing Loss Response DItissue PXR Binding CNS Protein (1) UGTs T4serum CNS malformation T3serum Functional Loss (eg. IQ) T3tissue TRactivation CNS Protein (2) Increasing Dose and/or Time Low-frequency Hearing loss High -- Frequency -- Low Cochlear Development Conception Birth Weaning 60 50 40 Hearing Loss, dB SPL (difference from control) Thyroid Hormone Fetal/Postnatal PCB Exposure Critical-Period Model for Chemical-Induced Postnatal Hypothyroxinemia and Ototoxicity Hearing Loss, dB SPL (difference from control) PHAHs and Ototoxicity 40 30 20 10 0 100.0 60.0 30.0 10.0 Log Thyroxine Concentration, % Control 30 20 10 y = -33.68*logX + 68.72 r ² = 0.8519 0 100 60 30 10 3 Log Thyroxine Concentration, % Control Dose-Response Perchlorate, Propylthiouracil and Hippocampal Physiology Thyroid PTU Binding to TPO Exposure Thyroid Perchlorate Hippocampal T3 Inhibition Of TPO Alter TR Mediated Proteins Serum T4 & T3 Synaptic Malformation Altered Synaptic Function Learning Impariment Dose-Response Perchlorate, Propylthiouracil and Hippocampal Physiology Hippocampal physiology 100 Normalized Spike vs EPSP 80 0 ppm 3 ppm 10 ppm 70 60 120 50 Dam T4 vs BL EPSP Max - % Control 110 30 20 10 0 0 10 20 30 40 50 60 70 80 90 Normalized EPSP Water maze learning 75 0ppm (n=11) 3ppm (n=15) 10ppm (n=10)* 70 65 100 BL EPSP Max % Control 40 100 90 80 70 60 M0805 PTU M0703 PERC M0102 PTU 50 40 60 r2=0.77 30 55 Latency in Seconds Normalized Population Spike 90 0 50 10 20 30 40 50 60 70 80 Dam T4 Percent of Control 45 40 35 30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Day Cue 90 100 110 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 What can we do to inform QSAR models • Develop in vitro test methods for known targets In Vitro Models for Thyroid Disruptors • • • • • • • Iodine Symporter (NIS) Thyroperoxidase (TPO) Deiodinases Transporters – Blood Transporters – Cellular Thyroid Receptors Hepatic Nuclear Receptors In Vitro Models – Thyroid Receptor Beta GeneBLAzer TR-UAS-bla HEK293 Cell Line Ligand ** ** ** Substrate ** ** T3 Stimulation of TR ** 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 TR ligand binding domain. Courtesy of Keith Houck, NCCT Human TR Reporter Gene Assay Heat Map of AC50’s 1456 chemicals; 14 concentrations; agonist and antagonist modes T3 Levothyroxine ACTIVES SELECTIVE 65 2 ACTIVES SELECTIVE 62 0 Courtesy of Keith Houck, NCCT TH Action Assay – T-Screen (Gutleb et al. EnvToxPharm 2005) - measures TH dependent cell proliferation in GH3 cells - 96 well plate assay Summary • Thyroid pathways are known • Multiple targets involved • In vitro models are available for many of the targets • Need to begin testing chemicals Thank You