Thyroid Gland

10/13/2020

STP Virtual Modular Course

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)

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Thyroid Gland

• Evolution: Conservation of T4 and T3 (mammals, birds, and teleosts) • Structure: Similar • Physiology and metamorphosis: Similarities and dissimilarities • Cancer: Dissimilarities

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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

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

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Relationship of TSH to Free T4

TSH (ng/ml)

Free T4 (pM)

T4 dose and serum TSH in hypothyroid dog

Ferguson DC, 2009, Vet Pharm & Ther

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Mouse: TSH Cell Hypertrophy & Hyperplasia (Inhibition of thyroxine synthesis)

NIS IHC: 2-month-old Mouse

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NIS IHC: 9-month-old Mouse

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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)

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

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Thyroxine Pharmacokinetics Dog and Human

(0.22 L/hr)

(0.05 L/hr)

Kaptein, Am. J. Physiol., 1993

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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

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

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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

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)

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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

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)

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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

Species Sensitivity to TPO inhibition by Sulfonamides

Sensitive Species Resistant Species

•Rat • Humans • Mouse •Primates •Dog • Guinea pig •Pig • Chicken

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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

Thyroid Gland Tumorigenesis Cytotoxicity

• Direct cytotoxicity with secondary increased proliferation – Pyrazole • Pigmentation – Minocycline • Inhibits TPO, degraded to black pigment – 2,4-diamoanisole – Synthetic vincamines

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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

TH Transport into Target Cells Inhibition

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TH Transport into Target Cells Inhibition • Plasma membrane – Monocarboxylate transporter superfamily • MCT8 – most specific for T4 •MCT10

– Organic anion transporting polypeptide superfamily • OATP1C1

– SLC17A4

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

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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

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

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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)

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

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Inducers of UDP-GT Examples

• Phenobarbital (PB)

• Pregnenolone-16-carbonitrile (PCN)

• 3-methylcholanthrene (3MC)

• Arochlor 1254 (PCB)

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

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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

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

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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

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

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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

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

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Human Relevance Framework Thyroid Follicular Tumors: MOA & Key Events

Hyperplasia Adenoma Carcinoma (death) Metastasis (death)

Response of Follicular Cells To Increased TSH Secretion

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Goiter Nature’s Experiment • Major health problem • Iodine deficiency • Over 200 million people • Iodine deficient regions • Environmental & genetic factors • No or questionable increase in thyroid cancer

Species with grossly enlarged thyroids due to TSH • Humans • Ruminants •Horses •Birds • Guinea pigs •Fish

• Not: Rats (up to 4x), mice, dogs

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Goat: Congenital Goiter

6th ESTP International Expert Workshop, Berlin, 2018

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Workshop Conclusions

1. A wide variety of factors influence the response of a thyroid gland in a nonclinical toxicity study and careful comparison to concurrent controls is essential. 2. FCHH in adult rats without other morphological changes such as focal hyperplasia or neoplasia should not be considered intrinsically adverse at the level of an isolated animal toxicity study. 3. Qualitative severity scores alone should not be used to determine whether a thyroid histological finding is adverse. 4. Other hormonal and mechanistic data are necessary to fully determine the adversity of a thyroid finding at the level of an isolated animal toxicity study. 5. Thyroid endpoints are best evaluated within the context of the entire data set for a given study, including knowledge of the compound class and MOA. 6. Threshold or benchmark responses (or simple statistical significance cut-offs) should be avoided to determine whether a change is test article-related. 7. Where possible, additional experimental assays/models/data to substantiate equivocal or marginal results are recommended.

Thyroid Follicular Neoplasia in Animals •Rats – Hyperplasia, adenoma, carcinoma (~0-6%), esp. Males •Mice – Hyperplasia, adenoma, carcinoma (~0-10%) – Transgenic mice: RET/PTC rearrangement; BRAFV600E • Papillary cancer • Guinea pigs – Adenomas and adenocarcinomas, uncommon •Cats – Multinodular goiter (hyperplasia and adenomas) • Dogs – Follicular carcinomas

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Follicular Adenoma: Rat

Follicular Adenocarcinoma: Rat

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Thyroid Follicular Neoplasia Humans • Thyroid cancer: Most common endocrine malignancy – Incidence has risen in past 4 decades – Uncommon deaths • Thyroid nodules are common – Palpable: 4-7% of adults – Ultrasound: up to 67%, usually women – Most are benign – 5-15% are malignant

Thyroid Cancer Humans

Follicular Medullary (C cell) 2% 10% Anaplastic 1%

Papillary Follicular Medullary Anaplastic Papillary 87%

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Thyroid Cancer: Humans 10-year Survival

Mechanistic Studies Mode of Action • Hormone assays – Free T4 and TSH most important • Liver histopathology and weights • Pituitary histopathology (+/- IHC) • Large rat group sizes (15 or greater) • Enzyme activity assays (frozen tissues) – May be optional

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Conclusions

• Mode of Action (rats only) – Secondary carcinogenesis of TSH in rats • Mode of Action (multiple species) – Target site(s) and mechanisms • Mechanistic experiments (in vivo and in vitro) – Human relevance – Human biomarker (Free T4, TSH, other) – Post registration monitoring

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Thyroid C-cells

• Neuroectoderm • Migration from neural crest • Separate glands in fishes & birds (Ultimobranchial glands)

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Calcitonin Content in Endocrine Glands Calcitonin Activity Gland (MRC units/mg) • Utimobranchial 4000-6000 • Salmon • Chicken • Thyroid 100-200 • Pig • Sheep • Human

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Role of Calcitonin Mammals

• No clinical conditions associated with calcitonin excess or deficiency • Prevents postprandial hypercalcemia and preserves the maternal skeleton by inhibiting osteoclasts • Exogenous CT: hypocalcemia

Pathophysiology of Calcitonin Mammals

• No clinical conditions associated with calcitonin excess or deficiency

•Biomarker: C-cell hyperplasia, tumors

• Drug Tx: Hypercalcemia, osteoporosis

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Location of C-Cells in Thyroid

•Rat – Central – Single cells to clusters; clusters increase with age •Dog – Central, hilus (C-cell complexes) • Non-Human Primate – Greater variability • Ungulates – Dispersed, single cells

C-Cell Complexes in Dogs

• Clusters in central gland and near the hilus – C cells – Small undifferentiated cells – Small, colloid-containing follicles – Cysts • Colloid containing; TG+ • Do not accumulate iodide • Ultimobranchial remnants

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C-Cell Complexes in Dogs

C-Cell Numbers

• Humans – < 1% of thyroid cells – May decrease with age •Rats – 5% of thyroid cells – Increase to 10% after 120 days – Individual cells (intrafollicular and parafollicular) – Interfollicular clusters • increase in number and size with age

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C-Cell Distribution: Rat Thyroid Gland

Cranial 1 2

2 3 3

4 4 5

6 5

7 Caudal 6

Martín-Lacave, Cell Tissue Res, 1992 71

Calcitonin Immunohistochemistry

• Human antiserum – Monkey, rat, mouse hamster, rabbit, guinea pigs • Porcine antiserum – Dogs, cats, cattle, pigs

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Calcitonin Secretogogues

• Calcium (Ca2+), Mg2+, calcimemetics • GI Hormones –Gastrin – Cholecystokinin – Secretin – Glucagon – Enteroglucagon (glicentin or oxyntomodulin) – GLP-1 (rodents)

Thyroid C-Cell Tumors Species Occurrence

• Rat (F344, SD, WAG/Rij ) – Higher incidence in females (Wistar) • OVX decreases CT synthesis and secretion • Bull (ultimobranchial) • Dog • Mouse • Horse • Ferret (with islet cell tumors) • Sheep (Mouflon) ++ • Zebrafish (ultimobranchial) (Ca in H20)

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Factors That Influence C-cell Proliferative Lesions

• Irradiation

• Vitamin D

• High Dietary Calcium – PTG adenoma with C-cell carcinoma (Dog)

Diffuse C-Cell Hyperplasia

• Response to chronic stimulation – Hypercalcemia • Increase in number of follicles with C-cells – Intrafollicular and Parafollicular • Increase in number of C-cells per follicle • Increase in number of interfollicular C-cells

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Diffuse C-Cell Hyperplasia - Dog

C-Cell Focal Hyperplasia

• Focal accumulation of C-cells

• Well delineated; Lack of compression

• Absence of a fibrous capsule

• Diameter < area of 5 average colloid-filled thyroid follicles (rats)

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Focal C-Cell Hyperplasia - Rat

C-Cell Adenoma

• Discrete, expansive nodule of C-cells • Well circumscribed, partially encapsulated • Variable compression (depending on size) of adjacent thyroid follicles • Neuroendocrine packeting – Abundant cytoplasmic area – Amphophilic cytoplasm • Wide size range (> 5 follicles to majority of the gland)

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Amyloid in C-Cell Tumors

• Produced by neoplastic cells • Usually confined to the tumor • Protein unrelated to immune amyloid • Similarity of amyloid and calcitonin – Shared amino acid sequences – Additional amino acid sequences of calcitonin gene product

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C-Cell Carcinoma - Rat

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C-Cell Carcinoma - Rat

Rat C-Cell Immunohistochemistry

Calcitonin CGRP Chromogranin Somatostatin Normal +++ ++ ++ Rare Diffuse Rare, small +++ ++ ++ Hyperplasia clusters Focal ++ + + Few + cells Hyperplasia Adenoma ++ ++ + -/++ (Well Diff.) (homogeneous) (homogeneous) (homogeneous) (variable) Adenoma + + + -/++ (Pleiomorphic) (heterogeneous) (heterogeneous) (heterogeneous) (variable)

Carcinoma ++/+++ +/+++ +/+++ -/+++ (heterogeneous) (heterogeneous) (heterogeneous) (most +)

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C-Cell Histopathology: Mice

• Use immunohistochemistry – Normal C-cells – Diffuse hyperplasia – Early focal hyperplasia

• Can only see focal hyperplasia and tumors in routine histopathology

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Mouse: Normal Diffuse Hyperplasia

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Development of C-Cell Tumors

• Drugs and chemicals – Direct or Indirect – Mode of Action usually unknown – Examples: Aledronate, Arformoterol, Atenolol, Colesevelam, Naratriptan, Palonosetron – GLP-1 agonists (rats and mice only)

• Multiple Endocrine Neoplasia – Humans, Dogs, other species

Glucose Homeostasis Fasting vs. Postprandial

• Fasting glucose level – Insulin – Glucagon • Postprandial glucose level – Incretin (GI) hormones • GLP-1 (glucagon-like peptide-1) • GIP (gastric inhibitory polypeptide; glucose-dependent insulinotropic polypeptide) • Only GLP-1 increases insulin in diabetics

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GLP-1 Very Short Half-Life in Blood

• Half-life: 1-5 minutes

• GLP-1 (7-36) amide and 7-37 forms

• Degraded by plasma DPP-4 (dipeptidyl peptidase-4) – DPP-4 inhibitors: Limited ability to increase GLP-1

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Exenatide (Byetta®)

• First GLP-1 agonist (2005) • Synthetic form of exendin-4 – Isolated from Gila monster salivary glands – 50% homology to human GLP-1 – Longer half-life in humans • Adjunctive therapy for DM • Twice daily injections

C-cells and GLP-1 agonists Rats and Mice • C-cell hyperplasia – Diffuse – Focal

• C-cell adenomas

• C-cell carcinomas

• Rats are more sensitive than mice

GLP-1 Agonist-Induced C-cell Proliferation in Rodents

• Rodent-specific effect • Receptor expression greatest in rodents • GLP-1R KO mice confirm role of receptor – Physiologic role in rodents • No proliferation in dogs and NHP • No increase in calcitonin in dogs and NHP • Equivocal or no increase in calcitonin in humans

• Long-term effect in humans need to be monitored

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Parathyroid Gland

‘In the Beginning was Calcium and Calcium was in the Sea’

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PTH Set Point 30

25 Ca2+ vs PTH Logistic Regression 20

15 a-d y  b +d x 10 1+c PTH (pM)

0 3456789101112 Ca2+(mg/dL)

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Oxyphil (Mitochondria-Rich) Cells

• Puberty: First appear • Species: – Dog, cat, cattle, horses, humans, NHP • Derivation: – Metabolically altered chief cells • Increase with age • Can form diffuse or nodular hyperplasia

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Structural Response of Chief Cells

Chronic Hypocalcemia

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‘Water-Clear’ Cells

Parathyroid Chief Cells

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Water Clear Cell Hyperplasia: Dog

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Kursteiner’s Cysts

Cats, Rodents, other species

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Ectopic Thymus

Rat, Mouse

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Fibrosis

Rat, Mouse

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Multinucleated ‘Syncytial’ Chief Cells Rat, Dog

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Parathyroid Gland

Proliferative Lesions

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Parathyroid Chief Cell Neoplasms

• Endocrinologically Active – ‘Functional’

• Endocrinologically Inactive – ‘Nonfunctional’

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Chief Cell Adenoma

Non-Functional

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Diffuse Chief Cell Hyperplasia

Chronic Renal Disease Nutritional Imbalances

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Primary Multinodular Chief Cell Hyperplasia

Dog, Humans Sprague-Dawley Rat

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Parathyroid Gland

Carcinoma

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Factors Influencing Development of Parathyroid Proliferative Lesions

•Age • Irradiation • Gonadectomy • Hypocalcemia • Calcitriol – suppresses chief cells

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PTH: Treatment for Osteoporosis

• PTH (1-34)

• Daily injection – Intermittent vs. continuous action

• Rat toxicology (lifetime) studies – High incidence of osteosarcomas – Tumors dependent on treatment duration and dose • ‘No Effect’ dose for tumors exists, while still inducing bone formation

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