10/13/2020 STP Virtual Modular Course 1 Thyroid/Parathyroid: Normal, Background, Induced; Rodent/Nonrodent Terminology; Rodent-Specific Effects I and II Thomas J. Rosol, DVM, PhD, MBA, DACVP, Ohio University Heritage College of Medicine See INHAND: Endocrine (rodents, dog) 2 ©Rosol 2020 1 10/13/2020 Thyroid Gland • Evolution: Conservation of T4 and T3 (mammals, birds, and teleosts) • Structure: Similar • Physiology and metamorphosis: Similarities and dissimilarities • Cancer: Dissimilarities 3 4 ©Rosol 2020 2 10/13/2020 Thyroid Stimulating Hormone (TSH) • Glycoprotein: Alpha & Beta (novel) subunits • Short (~15 minute) half-life • Circulates free (unbound) in blood • Highly species-specific – Human, primate, rat, & dog assays – Little cross reactivity – Male rats greater than females 5 Negative Feedback of the Pituitary by Free T4 • Feedback is dependent on the free hormone – Hypothalamus – Pituitary •Pituitary – 5’ Deiodinase (D2) (intracellular on ER) – Converts T4 to T3 – Serum free T4 correlates with TSH secretion 6 ©Rosol 2020 3 10/13/2020 Relationship of TSH to Free T4 TSH (ng/ml) Free T4 (pM) 7 T4 dose and serum TSH in hypothyroid dog Ferguson DC, 2009, Vet Pharm & Ther 8 ©Rosol 2020 4 10/13/2020 Mouse: TSH Cell Hypertrophy & Hyperplasia (Inhibition of thyroxine synthesis) 9 NIS IHC: 2-month-old Mouse 10 ©Rosol 2020 5 10/13/2020 NIS IHC: 9-month-old Mouse 11 12 ©Rosol 2020 6 10/13/2020 Thyroid Hormone Deiodination • Circulating T3 is not useful to measure thyroid function –Source: liver & kidney plasma membrane deiodinase 1 (D1) • T3 is largely regulated at the cellular level in a tissue- dependent manner (by 5’ deiodinase, D2) 13 Free Serum T4 Between Species TT4 FT4 FT4 Half-life Species (g/dL) (%) (ng/dl) (hours) Rat 4.1 0.05 2.0 13 Dog 2.8 0.10 2.8 15 Cat 1.7 0.10 1.7 11 Human 6.8 0.03 2.0 120 TT4: Total T4, FT4: Free T4 14 ©Rosol 2020 7 10/13/2020 15 Thyroxine Pharmacokinetics Dog and Human (0.22 L/hr) (0.05 L/hr) Kaptein, Am. J. Physiol., 1993 16 ©Rosol 2020 8 10/13/2020 Serum Protein Binding for Thyroxine Species TBG Albumin Transthyretin Human ++ ++ + Monkey ++ ++ + Dog ++ ++ + Pig + ++ + Ruminants ++ ++ + Feline - ++ + Mouse -* ++ + Rat -* ++ + Bird - ++ + Fish - ++ + *Expressed during pregnancy; increased by estrogen 17 Thyroid Hormone Deiodination Activation and Metabolism outer ring inner ring 3’ 3 * 5’ 5 3,5,3’,5’-T4 3,5,3’-T3 3,3’,5’-rT3 3,3’T2 18 ©Rosol 2020 9 10/13/2020 Thyroid Hormone Deiodination Deiodinase 2 and 3 3’ 3 5’D2* D2** 5’ 5 5 D3 3,5,3’,5’-T4 3,5,3’-T3 3,3’,5’-rT3 5 D3* 5’D2 D2 3,3’T2 19 Thyroid Gland Toxicology • Environmental: – Goitrogens: Natural and artificial • Water: Tadpole metamorphosis as sentinel species • Radiation: I131, I125 – Fallout, therapy, diagnostic – Carcinogen: Especially children – Rats, Dogs • Drugs and Chemicals – Thyroid disruption (many in rats) – Cytotoxicity/Mutagenicity (few) 20 ©Rosol 2020 10 10/13/2020 Chemical Disruption of Thyroid Hormone Economy 1. Inhibition of iodide uptake 2. Inhibition of thyroperoxidase 3. Inhibition of thyroid hormone secretion/processing 4. Cytotoxicity 5. Altered serum protein binding 6. Inhibition of thyroid hormone transport 7. Inhibition of binding to TH receptors 8. Decreased activation by 5’-deiodinase 9. Increased metabolism and excretion 10.Central inhibition of TSH or TRH secretion 11.Mutagenicity/Genotoxicity 21 Inhibition of Hormone Synthesis Lack of Iodide Uptake by NIS • Sodium-Iodide Symporter • Iodide Deficiency – Mountaineous/inland regions of the world • Inhibition of NIS – Thiocyanate – Perchlorate (rocket fuel, environmental contamination) 22 ©Rosol 2020 11 10/13/2020 Inhibition of Hormone Synthesis Inhibition of Thyroperoxidase (TPO) • Organification of I2 to tyrosine and coupling of iodotyrosines - – Thiourea: reduces I2 to I • Inhibition of TPO – Thioamides • Propylthiouracil, Mercaptoimidazole • Methimazole, carbimazole, aminotriazole • Sulfonamides, such as sulfamethazine – Sulfonylureas (antidiabetic drugs) –1st generation: acetohexamide, chlorpropamide, tolbutamide, tolazamide – Substituted phenols • Resorcinol, salicylamide • Species specificity 23 Species Sensitivity to TPO inhibition by Sulfonamides Sensitive Species Resistant Species •Rat • Humans • Mouse •Primates •Dog • Guinea pig •Pig • Chicken 24 ©Rosol 2020 12 10/13/2020 Inhibition of Hormone Secretion Excess of Iodide, Lithium • Excess of iodide – Decreased lysosomal proteases (humans) – Inhibition of colloid droplet formation (rats, mice) – Inhibition of TSH-mediated cAMP (dogs) – Excessive maternal intake of iodine • Goiter in neonate • Lithium – Inhibits colloid droplet formation by cAMP – Inhibits hormone release 25 Thyroid Gland Tumorigenesis Cytotoxicity • Direct cytotoxicity with secondary increased proliferation – Pyrazole • Pigmentation – Minocycline • Inhibits TPO, degraded to black pigment – 2,4-diamoanisole – Synthetic vincamines 26 ©Rosol 2020 13 10/13/2020 Serum Thyroid Hormone Binding Proteins Competition • Less important in species with TBG • Binding to prealbumin (transthyretin, TTR) – Chlorophenols, chlorophenoxy acids, nitrophenols • Decreased T4 in rats – Pentachlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), dinoseb, bromoxynil, polychlorinated biphenyls (PCBs) • Decreased T3 in rats – Bromoxynil • Decreased T4 and T3 in rats –2,4-D 27 TH Transport into Target Cells Inhibition 28 ©Rosol 2020 14 10/13/2020 TH Transport into Target Cells Inhibition • Plasma membrane – Monocarboxylate transporter superfamily • MCT8 – most specific for T4 •MCT10 – Organic anion transporting polypeptide superfamily • OATP1C1 – SLC17A4 29 Thyroid Hormone Receptor (TR) Inhibition • Synthetic agonists – Triac, Tetrac, NH-3 • Receptor antagonists – Bromine, brominated flame retardants – Bisphenol A (BPA): Reduces TR • May be specific for TR or TR 30 ©Rosol 2020 15 10/13/2020 Thyroxine (T4) Deiodination Inhibition • Selenium deficiency – Se: cofactor for 5’-deoidinase – Lack of Se leads to decreased T3 and increased T4 • FD&C Red No. 3 – Inhibits 5’-deiodinase – Rats: Increased T4, decreased T3, increased reverse T3, increased TSH • Lipid peroxidation – 5’-deiodinase 31 Thyroxine (T4) Deiodination Inhibition • Inhibitors of Deoidinases – Iopanoic acid (iodinated, x-ray contrast) • Inhibits 5’-deiodinase 2 and 3 – Pyrethroid insecticides • Decreased 5’-deiodinase 2 32 ©Rosol 2020 16 10/13/2020 Increased Liver Metabolism & Excretion • Phase I (deethylases) and II enzymes (UDP-GT) • Polyaromatic hydrocarbons • Organochlorine pesticides (DDT, methoxychlor) • Polychlorinated biphenyls (PCBs) • Dioxin and dioxin-like chemicals • Polybrominated diphenyl ethers (PBPEs) • Alachlor herbicide (chloroacetanilide) • Phenobarbital • (Increased liver weight and hypertrophy) 33 UDP-GT Uridine 5’-diphospho-glucuronosyltransferase • Important Phase II conjugative enzyme – Elimination of drugs and foreign chemicals – Not present in cats – Induction sensitivity: Rats > Humans, Dogs, Mice • Transfers gluronosyl from uridine 5’- diphospho-glucuronic acid • Increases water solubility 34 ©Rosol 2020 17 10/13/2020 Inducers of UDP-GT Examples • Phenobarbital (PB) • Pregnenolone-16-carbonitrile (PCN) • 3-methylcholanthrene (3MC) • Arochlor 1254 (PCB) 35 Effects of Microsomal Enzyme Inducers in Rats PB PCN 3MC PCB T4-UDP-GT T3-UDP-GT Serum T4 Serum T3 Serum TSH Thyroid Cell Proliferation CD Klaassen, Tox. Pathol., 2001 36 ©Rosol 2020 18 10/13/2020 Central Inhibition TSH or TRH Secretion • Benzodiazepines – Clonazepam – Diazepam • Inhibit cold-stimulated TSH secretion in rats • Likely not clinically significant in humans • May also suppress CRH and increase GH secretion 37 Thyroid Gland Tumorigenesis Genotoxicity • Genotoxins/Mutagens – Ionizing radiation (only known human carcinogen, e.g., 131I) – Chemicals (rodents) • N-methyl-N-nitrosourea (MNU) – Enhanced by iodide deficiency • N-bis(2-hydroxypropyl) nitrosamine (DHPN) • Methylcholanthrene • Dichlorobenzidine • Polycyclic hydrocarbons • Acetylaminofluoride 38 ©Rosol 2020 19 10/13/2020 Endocrine Interactions Thyroid Axis • Glucocorticoids – Antagonism of the thyroid axis – Decrease thyroid binding globulin • Adrenalectomy increases TBG – Regulate deiodinase activity – Regulate pituitary gland function • Estrogens – Interfere with thyroid endpoints; false negative results – Atrazine (aromatase effects): minimal effects on thyroid axis 39 Conundrum • Why is TSH an indirect carcinogen in rats? – Gaps in knowledge – Genetics and epigenetics of thyroid follicular cells in rats – Strain differences – Human relevance • TSH in humans – Relevant in patients with thyroid cancer 40 ©Rosol 2020 20 10/13/2020 Human Relevance Framework Rat Thyroid Follicular Tumors • Fundamental differences in thyroid hormone economy in rats – Rapid half-life of T4 – Lack of thyroid binding globulin – High TSH concentrations (greater in males) – Low secretion rate of T4 (inherently less able to make T4 compared to humans) – Sensitive to the tumorigenic effects of drugs that decrease T4 or T3 – Robust TSH response decreased T4 or T3 41 Human Relevance Framework Thyroid Follicular Tumors: MOA & Key Events T4 or T3 (many mechanisms) Inhibition of thyrotropes in pituitary TSH secretion, thyrotrope hyperplasia Thyroid follicular cell hypertrophy Colloid depletion Increased cell proliferation Hyperplasia 42 ©Rosol 2020 21 10/13/2020 Human Relevance Framework Thyroid Follicular Tumors: MOA & Key Events Hyperplasia Adenoma Carcinoma (death) Metastasis (death) 43 Response of Follicular Cells To Increased TSH Secretion 44 ©Rosol 2020 22 10/13/2020