A Systems Approach to Characterizing and Predicting Thyroid Toxicity

1. Michael Hornung, Kara Thoemke, Joseph Korte, Jose Serrano, John Nichols, Patricia Schmieder, Joseph Tietge, Sigmund Degitz US EPA, Mid-Continent Ecology Division, Duluth, MN McKim Conference June 27-29, 2006 Duluth, MN A Systems Approach to Characterizing and Predicting Thyroid Toxicity

2. Thyroid Toxicity Research • Endocrine Disruptors • Thyroid hormone is important for growth and development, neurodevelopment, metabolism • To understand thyroid toxicity need to look at it in the context of the whole Hypothalamus-Pituitary-Thyroid Axis (HPT)

3. Hypothalamus (CRH) TRH (-) Pituitary Thyrotropes T4 TSH Thyroid Gland Thyroglobulin TPO DIT MIT NIS Iodine DIT DIT T4 T4 Transthyretin Deiodination (D2) T4 Inactivation/ Elimination T3 Deiodination (D2) Deiodination (D3) Conjugation T3 + TR/RXR DNA mRNA Liver Peripheral Tissue Thyroid Hormone Regulation colloid Follicular cells

4. QSAR and in vitro Models Organismal Outcomes Systems Model Retarded Development Thyroid Gland Hypertrophy Thyroid Follicular Cell Treated Control Development Thyroid-axis Systems Model Hypothalamus TRH (CRH) (-) Pituitary TSH Thyroid Gland Thyroglobulin TPO MIT DIT Iodine DIT T4 Transthyretin Inactive TH Deiodination Deiodination Deiodination Inactive TH T3+TR/RXR DNA mRNA Conjugation Peripheral Tissues Liver

5. Why an amphibian model ? • Metamorphosis is controlled by thyroid hormone • Simple apical endpoint to monitor disruption in vivo • Molecular events are well characterized • Easy to raise and test in the laboratory • Xenopus laevis

6. Climax Prometamorphosis Xenopus Metamorphosis

7. MED Thyroid Project Objectives • Conduct studies with known HPT disruptors • Inhibitors of thyroid hormone synthesis • Thyroid Peroxidase: Methimazole, Propylthiouracil • Sodium Iodide Symporter: Perchlorate • Develop diagnostic measures • What are the appropriate tissue level endpoints? • Histology, T4, TSH • Can gene and protein expression be used as indicators of thyroid axis disruption? • Develop assays to enable ranking and prioritization of chemicals

8. 14 d Exposure * 50 mg/L * 25 mg/L Proportion in stage 12.5 mg/L Control day 8 55 56 57 58 59 60 Developmental Stage Effect of Methimazole on Development and Thyroid Histology

9. Summary of Metamorphosis Assay • X. laevis is sensitive to model thyroid pathway modulators • Methimazole, 6-PTU, Perchlorate • Early stage tadpoles (stg 51-54) can be arrested in development by T4 synthesis inhibitors, stage 60 is not • Thyroid histology is an essential component of assay • More sensitive than developmental rate (d8) • Diagnostic

10. Diagnostic Research Approach • Link Chemical-Biomolecular Interaction to Organism Response • Examine gene expression during normal metamorphosis and following chemical exposure • Examine protein changes • Circulating T4 and TSH • Responses of tissues isolated from compensatory mechanisms • Pituitary explant culture: TSH – T4 feedback • Thyroid explant culture: TSH stimulation, chemical inhibition of T4 release • Develop computational – predictive approaches

11. Developmental Expression Chemical Exposure In vivo Pituitary Gene Expression: Thyroid Stimulating Hormone

12. Developmental Expression Chemical Exposure In Vivo Thyroid Gland Gene Expression Sodium/Iodide Symporter

13. Pituitary Explant Culture Objective: Characterize function of the pituitary during development and the relationship between T4 and TSH Method: Culture pituitaries from tadpoles at multiple stages of development Measure TSH expression in the pituitaries Gene expression or T4 release in thyroid glands treated with media conditioned by pituitary culture

14. * * * * Pituitary Explant Culture TSH mRNA is repressed by T4 • Negative feedback mechanism is functional throughout development although the setpoint changes • sensitivity to T4 decreases

15. Thyroid Gland Explant Culture • Objective: • Define thyroid-specific outputs in response to TSH and xenobiotics in the absence of whole organism compensatory response • Method: • Culture thyroid glands from prometamorphic tadpoles and treat with TSH and T4 synthesis inhibitors • Measure T4 release and gene expression

16. 1000 ng TSH/ml 1000 ng TSH/ml + MM1 2000 ng TSH/ml 2000 ng TSH/ml + MM1            Thyroid Gland Explant Culture:Time relationship of T4 release inhibition

17. Pituitary Explant Culture • Feedback mechanisms in the pituitary • Negative feedback by T4 on the pituitary is present in metamorphosis • Sensitivity of the pituitary to this inhibition decreases over time • - in early metamorphosis prevent excess T4 • - allow more T4 later to complete metamorphosis

18. Thyroid Explant CultureInterpretation of compensatory and direct effects • In vitro… • Release T4 in response to TSH is dose related • T4 reserves must be depleted before synthesis inhibition significantly affects T4 release • In vivo… • Early stages are more sensitive to arrested metamorphosis by T4 inhibitors than late stages • At late prometamorphosis, thyroid glands are larger and reserve T4 is sufficient to complete metamorphosis • Exposure time 0 does not equal effect time 0 for circulating T4 • Need to measure circulating hormone levels to interpret gene expression and protein responses in vivo

19. Hypothalamus Potential Endpoints for HPT-Axis QSAR Development TRH/CRH Tyrosine Iodination and Hormone Production Pituitary TSH T4 (-) Thyroid Gland Iodine Uptake MIT I + Tyr DIT NIS TPO Iodine DIT DIT Receptor and Protein Binding T4 Metabolizing Enzyme Induction / Activity T4 Liver Peripheral Tissue T4 metabolism/ conjugation Deiodination T3 + TR T3-TR:RXR  DNA  mRNA TH-gluc elimination

20. HPT-Axis QSAR Development Comparison of Endpoints of T4 Synthesis Inhibition • NIS activity • Membrane protein transports iodine into the follicular cell • Limited data on chemical inhibitors of NIS - mostly monovalent anions of similar size as iodide • Lack of data makes it difficult to make informed chemical selection • Difficult assay to transform to high throughput format • TPO activity • TPO iodinates tyrosine and couples iodo-tyrosines to produce thyroid hormone • TPO inhibition data available for more chemicals & classes of chemicals • Methimazole – PTU • Flavonoids • Resorcinols • More data aids chemical selection process and QSAR model development • Spectrophotometric determination of iodination of tyrosine to MIT • Potential for conversion to high throughput assay

21. HPT-Axis QSAR Development TPO Inhibitors Methimazole Plant Flavonoids flavone myricetin Resorcinol & Derivatives recorcinol Propylthiouracil

22. Thyroid Peroxidase InhibitionLiterature Data PTU PTU MM1

23. HPT-Axis QSAR Development • Develop Xenopus-based in vitro assay to begin to test known inhibitors of TPO activity • Expand the range of chemicals and classes • Select from EPA Chemical Lists • Predictive Linkages in vitro→ex vivo (explant culture)→in vivo

24. Systems Approach to Predicting Thyroid Toxicity Molecular Effects Biological Responses Tissue ------------ Organism Chemical Gene Expression Enzyme Activities TPO UDPGT Protein Binding TR Transthyretin Serum Albumin Adverse Effect & Compensatory Response Regulatory Pathways T4 synthesis and release Feedback mechanisms QSAR Ranking & Prioritization of Chemicals Selection for Screening EPA Chemical Lists

25. MED Thyroid Project Team S. Degitz M. Hornung J. Tietge K. Thoemke J. Nichols J. Chowdhury G. Holcombe J. Serrano P. Kosian H. Kerr D. Hammermeister L. Korte J. Korte M. Bugge S. Batterman J. Olson B. Butterworth J. Haselman