Delineating Thyroid-Mediated Toxicity Pathways in an Amphibian Model System Michael W. Hornung, Sigmund J. Degitz, Joseph E. Tietge US Environmental Protection Agency Office of Research and Development National Health and Environmental Effects Research Laboratory Mid-Continent Ecology Division Duluth, MN, USA McKim Conference on Predictive Toxicology September 16-18, 2008 Duluth, MN

Outline Brief background on MED thyroid research Approach to adverse outcome pathway research for amphibian-based thyroid model Investigating endpoints at steps in the pathway Understanding toxicity in the context of pathway information

Background on EDC Assays FQPA and SDWA directed U.S. EPA to evaluate chemicals for endocrine disruption Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) established Screening and testing methods recommended by EDSTAC included: Short-term fish reproduction assay to detect HPG effects Short-term amphibian metamorphosis assay to detect HPT effects Longer-term fish life-cycle assay for reproductive effects Longer-term amphibian reproductive/developmental effects

Test Overview Initiated with tadpoles at onset of thyroid function 14-21 days exposure Apical Endpoints Developmental stage Thyroid histology 100 200 300 400 500 600 700 Concentration (ng/dL) 51 54 60 66 Prometamorphosis Climax Developmental Stage Thyroid function T4 T3

Amphibian Metamorphosis Assay Methimazole 55 56 57 58 59 60 Perchlorate 55 56 57 58 59 60 61 62

In Vivo Gene Expression Following Exposure NIS Gene Response Exposure ----------------------------------------> response

The Need for Predictive Tools Apical endpoints established with the amphibian metamorphosis assay. However we can not test all the chemicals for which little is known about thyroid activity Need to develop tools to aid in selecting chemicals that need to be tested and inform which chemicals are likely not active and do not need to be tested or can be assigned low priority for testing

Potential Endpoints for Thyroid Hormone Disruption Hypothalamus TRH/CRH Tyrosine Iodination and Hormone Production Thyroid Peroxidase Pituitary TSH T4 (-) Thyroid Gland Iodine Uptake Sodium-Iodide Symporter 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

Adverse Outcome Pathways for Thyroid Disruption Subcellular Target Cells Effected Adverse Outcome Chemical Tissue/Organ Methimazole TPO Enzyme Inhibition Thyroid Follicular Cells  T4 Synthesis  Serum T4 Systemic T4 Insufficiency Arrested Amphibian Metamorphosis Perchlorate NIS Inhibition Thyroid Follicular Cells  T4 Synthesis  Serum T4 Systemic T4 Insufficiency Arrested Amphibian Metamorphosis Toxicity Pathway Adverse Outcomes Pathway Amphibian Metamorphosis Assay

Selection of a Pathway Subcellular Target Cells Effected Adverse Outcome Chemical Tissue/Organ Thyroid Follicular Cells  T4 Synthesis Methimazole TPO Enzyme Inhibition Systemic T4 Insufficiency Arrested Amphibian Metamorphosis  Serum T4 Methimazole TPO Enzyme Inhibition Thyroid Follicular Cells  T4 Synthesis ? Thyroid Tumor ?  Serum T4 Increased Serum TSH Goiter TPO-inhibition has potentially more types of chemicals that can affect it than NIS. Assays available. Single chemical with potential for multiple adverse outcome pathways related to thyroid

Thyroid Explant Cultures and In Vitro Assays to Understand Responses Outside of the Compensatory Mechanisms of the Whole Animal

Thyroid Gland Explant Cultures Ex Vivo: Thyroid Gland Explant Cultures Dissect thyroid glands from pro-metamorphic tadpoles Culture in 96-well plates in L-15 media Stimulate with TSH and treat with graded concentrations of test chemical Measure T4 released to media by RIA Measure TSH responsive gene expression by QPCR. TSH Chemical X T4 0.5 mm Paired thyroid glands in NF stage 59 X. laevis tadpole

Adverse Outcomes Pathway for Thyroid Toxicity Subcellular Target Cells Effected Tissue/Organ Chemical Thyroid Follicular Cells  T4 Synthesis Methimazole TPO Enzyme Inhibition Systemic T4 Insufficiency Arrested Amphibian Metamorphosis  Serum T4 Toxicity Pathway Adverse Outcomes Pathway Verification of chemical activity at organ level Thyroid Follicular Cells  T4 Synthesis Inhibition of T4 released from glands Methimazole TPO Enzyme Inhibition T4 TSH X Thyroid Explant Culture Assay

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

Gene Expression in Thyroid Gland Explant Cultures

Thyroid Gene Expression: Response to Inhibitors Methimazole, PTU, and Perchlorate Treatment NIS TPO Fresh 0h (-) TSH 24h (+) TSH Freshly Dissected No Inhibitor Methimazole PTU Perchlorate

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 UNDERSTAND DOSIMETRY / PK & PD Need to measure circulating hormone levels (T4, TSH) to interpret gene expression and protein responses in vivo

Inhibition of TSH-Stimulated T4 Release by Cultured Thyroid Glands Thyroid glands from X. laevis tadpoles (NF stage 59) were cultured in L-15 media in the presence of 2000 ng TSH/ml alone or TSH and graded concentrations of chemical. Media was collected and analyzed by RIA for T4. IC50 = 1 µM IC50 = 7 µM IC50 = 11 µM Perchlorate – Iodine uptake inhibition PTU & Methimazole – TPO inhibition Understand results in the context of the assay

Thyroid Explant Culture Assay Assess Pathway for Thyroid Disruption vs Thyroid Follicular Cell Toxicity Adverse Outcome Subcellular Target Cells Effected Tissue/Organ Chemical Thyroid Follicular Cells  T4 Synthesis Inhibition of T4 released from glands Chemical TPO Enzyme Inhibition Chemical ? ? ? mitochondria Thyroid Follicular Cells MTT Assay for mito function Cell Death Thyroid Explant Culture Assay T4 TSH X

Chemical Testing and Predictive Model Development In Vitro TPO Inhibition Chemical Selection EPA Chemical Lists Model T4 Synthesis Inhibitors QSAR Ex Vivo T4 Release Inhibition Literature – Thyroid Toxicity Chemical Structure Similarities In Vivo Amphibian Metamorphosis Assay

Adverse Outcomes Pathway for Thyroid Toxicity Subcellular Target Cells Effected Adverse Outcome Chemical Tissue/Organ Thyroid Follicular Cells  T4 Synthesis Methimazole TPO Enzyme Inhibition  Serum T4  Serum TSH Systemic T4 Insufficiency Arrested Amphibian Metamorphosis Toxicity Pathway Methimazole TPO Enzyme Inhibition In Vitro TPO Inhibition Assay QSAR = Chemical + Cellular Target

Thyroid Peroxidase Inhibition Guaiacol Oxidation Assay In Vitro: Prepare microsomes containing thyroid peroxidase activity from pig thyroid glands Test chemicals for potency for inhibiting two TPO-mediated reactions 1. Tyrosine Iodination: Conversion of Tyrosine to MIT/DIT 2. Guaiacol Oxidation: Surrogate coupling reaction (DIT + DIT = T4) Guaiacol Oxidation Assay TPO H2O2 Abs (470 nm)

Relative Potency of Model T4 Synthesis Inhibitors for TPO Inhibition IC50 (µM) Methimazole 2.7 PTU 8.8 Perchlorate 13,400 Methimazole is the most potent of the model T4 synthesis inhibitors in the TPO inhibition assay. Perchlorate had very low potency for TPO inhibition, but was the most potent of the three inhibitors in the ex vivo and in vivo assays. The primary mechanism of action for perchlorate inhibition of T4 synthesis is by inhibiting iodide uptake into the follicular cells.

TPO Inhibition: Alkylphenol Series All alkylphenols tested exhibited no inhibition of TPO activity

Screening Chemicals for Higher Tier Testing 24 chemicals tested in vitro in TPO Inhibition Assay Active Inactive 7 Inhibit TPO In Vitro 17 Did Not Inhibit TPO Model TPO Inhibitors Alkylphenol series Phthalates Isothiazoline Mixed Iodo Phenyl OH-PCDE Triazole Triazines Conazole Thyroid Gland Explant Culture: T4 Release Cytotoxic / Negative Positive: Inhibits T4 Release Test In Vivo Amphibian Metamorphosis Assay

TPO Assay Summary The TPO assay can be used to rapidly screen chemicals for further testing in the higher level thyroid toxicity assays, and can be used to begin to develop predictive models incorporating structure activity relationships between chemical structure and T4 synthesis inhibition This suite of assays can be an effective tool to determine the capacity of previously untested or unsuspected classes of chemicals to disrupt normal thyroid hormone production

Adverse Outcomes Pathways for Thyroid Disruption Subcellular Target Cells Effected Adverse Outcome Chemical Tissue/Organ Methimazole TPO Enzyme Inhibition Thyroid Follicular Cells  T4 Synthesis  Serum T4 Systemic T4 Insufficiency Arrested Amphibian Metamorphosis In Vitro - QSAR Ex Vivo Gland/Tissue Culture Toxicity Pathway Amphibian Metamorphosis Assay Adverse Outcomes Pathway

MED Thyroid Project Team Post Docs Kara Thoemke Jasim Chowdhury Robin Sternberg Student Contractors Hollie Kerr Megan Bugge Lisa Korte Jessica Olson Emily Burgess Scott Moen Mike Hornung Sig Degitz Joe Tietge John Nichols Jose Serrano Joe Korte Gary Holcombe Pat Kosian Dean Hammermeister Jon Haselman Brian Butterworth Sherri Batterman