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Michael Hornung, Kara Thoemke, Joseph Korte, Jose Serrano, John Nichols, Patricia Schmieder, Joseph Tietge, Sigmund Degitz US EPA, Mid-Continent Ecology.

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Presentation on theme: "Michael Hornung, Kara Thoemke, Joseph Korte, Jose Serrano, John Nichols, Patricia Schmieder, Joseph Tietge, Sigmund Degitz US EPA, Mid-Continent Ecology."— Presentation transcript:

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

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

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 Xenopus Metamorphosis Prometamorphosis Climax

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 Effect of Methimazole on Development and Thyroid Histology Proportion in stage 50 mg/L 25 mg/L 12.5 mg/L Control Developmental Stage 14 d Exposure 555657585960 * * day 8

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 ExpressionChemical Exposure In vivo Pituitary Gene Expression: Thyroid Stimulating Hormone

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

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  Negative feedback mechanism is functional throughout development although the setpoint changes  sensitivity to T4 decreases * * * * TSH mRNA is repressed by T4

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

20 HPT-Axis QSAR Development 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 Comparison of Endpoints of T4 Synthesis Inhibition

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

22 Thyroid Peroxidase Inhibition Literature Data MM1 PTU

23  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 HPT-Axis QSAR Development

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

25 MED Thyroid Project Team S. DegitzM. Hornung J. TietgeK. Thoemke J. NicholsJ. Chowdhury G. HolcombeJ. Serrano P. KosianH. Kerr D. HammermeisterL. Korte J. KorteM. Bugge S. BattermanJ. Olson B. ButterworthJ. Haselman

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