A Systems Approach to Characterizing and Predicting Thyroid Toxicity 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

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) WHY Investigate thyroid toxicity ?

Thyroid Hormone Regulation Pituitary Thyrotropes TRH (CRH) Hypothalamus Thyroid Gland Transthyretin (-) T4 Iodine TPO DIT TSH Peripheral Tissue T3 + TR/RXR DNA mRNA Liver T3 Deiodination (D2) Deiodination (D3) Conjugation Inactivation/ Elimination Deiodination (D2) NIS MIT Thyroglobulin colloid Follicular cells To begin to study thyroid toxicity we need to understand how thyroid hormone is regulated - A Systems Approach This figure shows a compartmental view of the thyroid hormone system and the process involved in maintaining thyroid hormone homeostasis. Thyroid hormone is controlled by thyroid stimulating hormone from the pituitary which stimulates the production and release of T4. As the amount of T4 released increases, this provides a negative feedback regulation on the pituitary to decrease TSH release, thereby maintaining circulating T4 levels within normal physiological ranges

Thyroid-axis Systems Model QSAR and in vitro Models Thyroid Follicular Cell Systems Model Organismal Outcomes Thyroid Gland Hypertrophy Retarded Development Control Treated Hypothalamus TRH (CRH) (-) Pituitary TSH Thyroid Gland Thyroglobulin TPO MIT DIT We can look at the regulation of thyroid hormone as a part of a systems model. Perturbations within this system will cause effects at the whole organism level that can be readily measured including histological changes in the thyroid gland and arrested development. To make the linkages between the chemical and the outcome we need to look at the effect of the chemical at hose points in the system that determine the ultimate outcome wihich is circulating T4. Efforts put into understanding the cellular and subcellular components and thier role in T4 homeostasis allows us to understand how perturbation of specific nodes within this axis by xenobitics can alter normal T4 control and metamorphosis. Iodine T4 DIT Transthyretin Inactive TH Deiodination Deiodination Deiodination Inactive TH T3+TR/RXR DNA mRNA Conjugation Liver Peripheral Tissues

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

Xenopus Metamorphosis Prometamorphosis Climax As I mentioned earlier, Xenopus was selected as a model organism to screen chemicals for thyroid disruption because of specific attributes of this species. The most important of these is that metamorphosis, a thyroid hormone dependent event, provides an opportunity to measure apical effects of thyroid disruption. This figure shows the relationship of TH, expressed as T4 and T3, to the morphological changes that occur in metamorphosis. The bottom panel shows the plasma concentrations of T4 and T3 throughout metamorphosis, which is nominally a 40 day process. During that interval, the larvae are completely remodeled (as shown in the top panel) into the juvenile tetrapod. The middle panel shows two important processes of metamorphosis, de novo development of the limbs and resorption of tail tissue relative to endogenous TH levels. Based on this remarkable biological process, it was proposed that chemicals which interfere with the normal rise in TH would affect these morphological indicators of development.

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

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 55 56 57 58 59 60 * day 8

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

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

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

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

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

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

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

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

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

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

Potential Endpoints for HPT-Axis QSAR Development Hypothalamus 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 T4 Peripheral Tissue metabolism/ conjugation Deiodination TH-gluc T3 + TR  T3-TR:RXR  DNA  mRNA elimination

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

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

Thyroid Peroxidase Inhibition Literature Data PTU PTU MM1

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

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 Regulatory Pathways T4 synthesis and release Feedback mechanisms Adverse Effect & Compensatory Response QSAR Ranking & Prioritization of Chemicals EPA Chemical Lists Selection for Screening

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