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. 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 Triiodothyronine (T3) Thyroxin (T4) O H O I I I HO CH2 NH 2 O CH C I O H O I I I HO CH2 NH 2 O CH C I O H O I I I HO CH2 NH 2 O CH C I O H O I I I HO CH2 NH 2 O CH C O H O I I I HO CH2 NH 2 O CH C O H O I I I HO CH2 NH 2 O CH C

4. Catabolic Enzymes Hypothalamus Pit TRH Thyroid TSH Blood T3 / T4 Target Tissues Liver Elimination from the body + TH binding proteins 5’-deiodinases T 4 T 3 Acts as a ligand for nuclear thyroid hormone receptors (TRs) Regulation of Thyroid Hormones TR 

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

6. Thyroid MOAs

7. Toxicity Pathways Adverse Outcome 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 Window Dioxins, Furans and PCBs - Hearing Loss Exposure Hepatic Phase II Enzymes Hepatic Parent or Metabolite  Serum T4 & T3  Tissue T3 Alter TR Mediated Proteins Loss of cochlear hair cells Hearing Loss Binding to PXR Binding to AhR

11. UGTs T4 serum T3 serum DI tissue T3 tissue TR activation CNS Protein (1) CNS Protein (2) CNS malformation Functional Loss (eg. IQ) Increasing Dose and/or Time Response PXR Binding

12. PHAHs and Ototoxicity

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

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

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

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

19. GeneBLAzer TR  -UAS-bla HEK293 Cell Line 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. T3 Stimulation of TR  T3 (M) ** Substrate Courtesy of Keith Houck, NCCT In Vitro Models – Thyroid Receptor Beta

20. Human TR  Reporter Gene Assay Heat Map of AC50’s 1456 chemicals; 14 concentrations; agonist and antagonist modes Levothyroxine T3 Courtesy of Keith Houck, NCCT

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

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

23. Thank You