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Chapter 3.1 - Anatomy - COMPARATIVE ANATOMY AND PHYSIOLOGY

COMPARATIVE ANATOMY AND PHYSIOLOGY

Charles C. Capen

GROSS ANATOMY

The thyroid is the largest endocrine gland in humans, weighing about 20 g in an adult. The normal weight of the thyroid often is given as being considerably greater because thyroid enlargement (""goiter"") is quite common. It is frequently stated that the thyroid is so named because of its shield-shaped configuration. However, this bilobed structure bears no resemblance to a shield but is named because of its topographic relationship to the laryngeal thyroid cartilage, which does look like a Greek shield. ⁽¹⁾

The thyroid parenchyma is mostly derived from the endoderm at the base of the tongue. It may remain attached to this region even in adult life if the embryonic thyroglossal duct persists. More often, only portions of this duct survive as the pyramidal lobe extending upward from the narrow isthmus connecting the two lobes of the thyroid. The differential growth of the neck structures occasionally results in displacement of the thyroid to a level below the larynx. The isthmus of the thyroid normally overlies the second or third cartilaginous rings of the trachea in humans. Some or all thyroid tissue occasionally remains embedded in the base of the tongue as a lingual thyroid. The thyroid also includes epithelial structures derived from the ultimobranchial bodies that are described later.

ORGANOGENESIS

The thyroid gland originates as a thickened plate of epithelium in the floor of the pharynx (Fig 3-1) . It is intimately related to the aortic sac in its development and this association leads to the frequent occurrence of accessory thyroid parenchyma in the mediastinum. A portion of the thyroglossal duct may persist postnatally and form a cyst owing to the accumulation of proteinic material secreted by the lining epithelium. Thyroglossal duct cysts develop in the anterior cervical region. Their lining epithelium may undergo neoplastic transformation and give rise to follicular cell carcinomas.

Accessory thyroid tissue is common (nearly 100%), particularly in certain animal species such as the dog, and may be located anywhere from the larynx to the diaphragm. About 50% of adult dogs have accessory thyroids embedded in the fat on the intrapericardial aorta. They are completely lacking in C- (parafollicular) cells, but their follicular structure and function are the same as those of the main thyroid lobes. Such dogs under experimental conditions cannot be made hypothyroid by surgical ablation of the two lateral thyroid lobes alone.

The postnatal human thyroid gland contains solid cell nests in the middle to upper third of the lateral lobes that are derived from the ultimobranchial body. ⁽²⁾ The solid cell nests are approximately 1 mm in diameter and composed of nonkeratinizing epidermoid cells that lack intercellular bridges. ⁽³⁾ Histochemical analysis revealed the presence of mucoid material in 73%, calcitonin (CT)-immunoreactive cells in 36%, and both carcinoembryonic antigen and high--molecular-weight cytokeratin proteins in 85.7% of epidermoid cells. A central lumen in the solid cell nests usually is surrounded by mucinous cells and may contain desquamated cells, cell debris, periodic acid--Schiff (PAS)-positive granular material, and colloid. Follicles containing Alcian blue-positive acid mucins also are present in the solid cell nests of the human thyroid. ⁽⁴⁾ These follicles were composed of or related to CT-positive cells (by immunohistochemistry) and intermixed with alcianophilic mucinous cells. These findings support the hypothesis that mucoepidermoid carcinomas of the thyroid are of ultimobranchial tissue origin. ⁽²⁾

Cysts derived from remnants of the ultimobranchial body are observed frequently in the postnatal thyroid of rats lined by squamous epithelium and containing keratin debris. ⁽⁵⁾ Occasional follicles derived from ultimobranchial primordia contain a heterogeneous material in the lumen and scattered ciliated epithelial cells in the follicular wall. In Wistar rats, CT-immunoreactive cells have been demonstrated in the wall of the

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Figure 3.1-1 Embryology of thyroid and parathyroid glands and the relationship to primordia for the ultimobranchial body.

ultimobranchial tubule as well as thyroglobulin-positive staining in adjacent follicles and solid clusters. ⁽⁶⁾ These findings plus the presence of numerous mitosis and PAS-positive microfollicles support the hypothesis that the ultimobranchial body contributes to the formation of follicles in certain areas of the thyroid lobes.

To investigate cell function and proliferation of human thyroid follicles, an experimental system has been established to culture thyrocytes in collagen gel suspended in serum-free medium. ⁽⁷⁾ The human thyrocytes were functional as determined by their time- and dose-dependent response to thyroid-stimulating hormone (TSH), which consisted of a 15-fold increase in iodide uptake and organification, triiodothyronine (T₃ ) secretion, and cyclic adenosine monophosphate (cAMP) production. The same in vitro system also permits measurement of cell proliferation as indicated by ³ H incorporation and DNA content. Normal cell polarity, which is essential for thyroid follicle function, was maintained by the use of collagen gel and serum-free medium. The development of an in vitro system using thyroid cells of human origin should be of considerable use in future studies on thyroid pathophysiology in humans. Isolated thyroid follicular cells of human and porcine origin cultured in three-dimensional collagen gel have characteristic structural polarity, respond to TSH, and produce thyroid hormones. ⁽⁸⁾ ⁽⁹⁾ Thyroid follicular cells formed follicles in collagen gel culture by (1) cell division of cavity-associated single cells, and (2) through aggregation and linkage to adjacent cells. ⁽⁹⁾

HISTOLOGY AND ULTRASTRUCTURE

The thyroid gland is the largest of the organs that function exclusively as an endocrine gland. The basic structure of the thyroid is unique for endocrine glands, consisting of follicles of varying size that contain colloid produced by the follicular cells (Fig 3-2) . During folliculogenesis an intracytoplasmic cavity develops initially in individual cells. Follicles appear to grow during development by proliferation of component cells and coalescence of adjacent colloid-containing microfollicles in individual cells. ⁽¹⁰⁾ ⁽¹¹⁾ Folliculogenesis stimulated by TSH in vitro appears to require integrity of both microfilaments and microtubules because chemicals (vinblastine and colchicine) that disorganize these organelles block follicle formation. ⁽¹²⁾ Studies have shown that protein tyrosine phosphorylation and microfilament integrity are essential for thyroid cells to spread on a substrate. ⁽¹³⁾ These are potential intracellular loci where TSH and intercellular contact may regulate adhesion of follicular cells to extracellular matrix and influence thyroid cell behavior.

Studies in Wistar rats have shown that the volumetric fractions of the different histologic components (follicular cells, C cells, colloid, and interstitial tissue) change considerably during development (birth to 120 days of age). ⁽¹⁴⁾ The fraction of follicular cells decreased from 61% at birth to 37.2% at 4 months. C cells increased from 2.9% in newborns to 4% at 15 days, with no further change at 4 months. Colloid and stroma together represented 36% at birth and increased to 59% at 120 days. During the first 4 months of life in rats, the absolute volumes occupied by follicular cells, C cells, colloid, and stroma increased 13.3, 30.8, 39, and 34 times, respectively. ⁽¹⁴⁾ Delverdier and colleagues ⁽¹⁵⁾ have shown that the limits of thyroid follicles were more clearly defined in both silver-impregnated, paraffin-embedded and resin-embedded semithin sections than in routinely stained paraffin-embedded sections, permitting the more accurate measurements of thyroid structures essential during morphometric evaluation.

Thyroid follicular cells are cuboidal to columnar and their secretory polarity is directed toward the lumen of the follicles. Polarity of follicular cells is important for iodine uptake, but the follicle structure is required for the synthesis of thyroid hormones. ⁽¹⁶⁾ The luminal surfaces of follicular cells protrude into the follicular lumen and have numerous microvillar projections that greatly increase the surface area in contact with colloid (see Fig 3-2) . An extensive network of interfollicular and intrafollicular capillaries provides the follicular cells with an abundant blood supply.

Follicular cells have long profiles of rough endoplasmic reticulum and a large Golgi apparatus in their cytoplasm for synthesis and packaging of substantial amounts of protein that are then transported into the follicular lumen (Fig 3-3) . Numerous electron-dense lysosomal bodies are present in the cytoplasm, which are important in the secretion of thyroid hormones. The interface between the luminal side of follicular cells and the colloid is modified by numerous microvilli (see Fig 3-3) .

The biosynthesis of thyroid hormones is also unique among endocrine glands because the final assembly of the hormones occurs extracellularly within the follicular lumen. Essential raw materials, such as iodide, are trapped efficiently

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Figure 3.1-2 Scanning electron micrograph of thyroid gland of a dog with two opened follicles (F). The luminal aspect of individual follicular cells protrudes into the follicular lumen ( arrowhead). Interfollicular space (I) with connective tissue and capillaries is present.

at the basilar aspect of follicular cells from interfollicular capillaries, transported rapidly against a concentration gradient to the lumen, and oxidized by a thyroid peroxidase in microvillar membranes to reactive iodine (I₂ ) (Fig 3-4) (Figure Not Available) . The assembly of thyroid hormones within the follicular lumen is made possible by a unique protein (thyroglobulin) synthesized on the rough endoplasmic reticulum and packaged in the Golgi apparatus of follicular cells.

Thyroglobulin is a high--molecular-weight glycoprotein synthesized in successive subunits on the ribosomes in follicular cells. The constituent amino acids (tyrosine and others) and carbohydrates come from the circulation. Recently synthesized thyroglobulin (17S) leaving the Golgi apparatus is packaged in apical vesicles and extruded into the follicular lumen. Human thyroglobulin contains complex carbohydrate units with up to four sulfate groups and units with both sulfate and sialic acid. ⁽¹⁷⁾ The amino acid tyrosine is incorporated within

Figure 3.1-3 Electron micrograph of normal thyroid follicular cells with long microvilli (V) that extend into the luminal colloid (C). Pseudopods from the apical plasma membrane surround a portion of the colloid to form an intracellular colloid droplet (CD). Numerous lysosomes (L) are present in the apical cytoplasm in proximity to the colloid droplets. An intrafollicular capillary is visible in the lower left.

the molecular structure of thyroglobulin. Iodine is bound to tyrosyl residues in thyroglobulin at the apical surface of follicular cells to form, successively, monoiodotyrosine (MIT) and diiodotyrosine (DIT). The resulting MIT and DIT combine to form the two biologically active iodothyronines (thyroxine (T₄ ) and T₃ ) secreted by the thyroid gland.

The extracellular storage of thyroglobulin in the follicle lumen is essential for maintaining constant blood levels of thyroid hormones in vertebrates under conditions of varied intake of and varying requirements for T₄ and T₃ . Storage of large amounts of thyroglobulin is made possible by compaction or the tight packing of thyroglobulin molecules in the follicular lumen. ⁽¹⁸⁾ Protein concentrations as high as 100 to 400 mg/mL have been reported in colloid collected by micropuncture techniques from the lumens of single thyroid follicles. The luminal content of follicles consists of discrete globules (20--120 mum in diameter) that, by scanning electron microscopy, show a unique cobblestone-like surface pattern from impressions of microvilli of the apical plasma membranes of thyrocytes. Thyroglobulin in isolated globules was highly iodinated (~ 55 iodine atoms per 12S subunit), suggesting that covalent nondisulfide cross-linking occurs during iodination of thyroglobulin and that this process involves the formation of intermolecular dityrosine bridges. ⁽¹⁸⁾

Most of the epithelial cells and the functionally most important cells of the thyroid are the follicular cells (Fig 3-5) . They vary in height, depending on the intensity of stimulation by pituitary TSH, between low cuboidal and tall columnar. Follicular size and shape are quite variable in the human thyroid and there is no discernible pattern in the distribution of small and large follicles within the gland. Peripherally situated follicles in rats tend to be large and central ones small. Uchiyama and coworkers ⁽¹⁹⁾ ⁽²⁰⁾ reported that distinct variations occur morphometrically in volume and numerical densities of follicles during a 24-hour period in rats and reflect changes in subcellular organelles of follicular cells. Follicular cells in the human thyroid are relatively flat compared with those of the rat, reflecting the different plasma half-life of thyroid hormones in rat (short) compared to humans (long).

The histologic appearance of the thyroid is dramatically influenced by the level of circulating TSH from the adenohypophysis.

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Figure 3.1-4 (Figure Not Available) Thyroid follicular cells illustrating two-way traffic of materials from capillaries into the follicular lumen. Raw materials, such as iodide ion (I^- ), are concentrated by follicular cells and rapidly transported into the lumen ( left side of the drawing). Amino acids (tyrosine and others) and sugars are assembled by follicular cells into thyroglobulin (Thg), packaged into apical vesicles (av), and released into the lumen. The iodination of tyrosyl residues occurs within the thyroglobulin molecule to form thyroid hormones in the follicular lumen. Elongation of microvilli and endocytosis of colloid by follicular cells occurs in response to TSH stimulation ( right side of drawing). The intracellular colloid droplets (Co) fuse with lysosomal bodies (Ly); active thyroid hormone is enzymatically cleaved from thyroglobulin; and free T₄ and T₃ are released into the cytosol and eventually into the circulation. Mt, microtubules; M, mitochondria; mf, microfilaments. (Bastenie PA, Ermans AM, Bonnyns M, Neve P, Delespese G: Molecular pathology. Springfield, IL: Charles C Thomas, 1975:243)

⁽¹⁰⁾ Thyrotropin binds to the basilar aspect of thyroid follicular cells, activates adenylate cyclase with accumulation of cAMP, and increases the rate of biochemical reactions concerned with biosynthesis and secretion of thyroid hormones. ⁽²¹⁾ One of the initial structural responses by follicular cells to TSH

Figure 3.1-5 Normal rat thyroid gland illustrating basic histologic structure of colloid-filled (C) follicles of varying size lined by cuboidal follicular cells. An extensive network of capillaries is present between the thyroid follicles. Periodic acid-Schiff reaction.

is the formation of numerous cytoplasmic pseudopods, resulting in increased endocytosis of colloid and release of preformed thyroid hormone stored within the follicular lumen (Fig 3-6) (Figure Not Available) . Nilsson and colleagues ⁽²²⁾ reported that follicular cells do not respond in an all-or-none mode to acute TSH stimulation; rather, the response (i.e., numbers of pseudopods formed after 20 minutes) was graded depending on the level of TSH.

If the secretion of TSH is sustained (hours or days), thyroid follicular cells become more columnar and follicular lumens become smaller and appear as slit-like spaces because of increased endocytosis of colloid ⁽¹⁰⁾ ⁽²³⁾ ⁽²⁴⁾ (Fig 3-7) . Numerous PAS-positive colloid droplets are present in the luminal aspect of the hypertrophied follicular cells. TSH stimulation not only elicits a highly individual macropinocytotic response among different follicular cells but, in addition, the fraction of TSH-responsive cells is also a function of dose. ⁽²⁵⁾

Iodine deficiency in the diet resulting in diffuse thyroid hyperplasia was common in animals and humans in many goitrogenic areas throughout the world before the widespread addition of iodized salt to the diet (Fig 3-8) . Marginal iodine-deficient diets containing certain goitrogenic compounds may result in thyroid follicular cell hypertrophy and hyperplasia with clinical evidence of goiter with hypothyroidism. These goitrogenic substances include thiouracil, sulfonamides, anions of the Hofmeister series, and a number of plants from the genus Brassica, among others.

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Figure 3.1-6 (Figure Not Available) Scanning electron micrograph of apical surface of hypertrophied thyroid follicular cell 4 hours after TSH stimulation. Numerous elongated microvilli and cytoplasmic projections ( arrows) extend into the follicular lumen to engulf colloid as part of the initial stages of thyroid hormone secretion in response to TSH. (Collins WT, Capen CC. Ultrastructure and functional alterations of the rat thyroid gland produced by polychlorinated biphenyls compared with iodide excess and deficiency, and thyrotropin and thyroxine administration. Virchows Arch (B) 1980;33:213)

In response to long-term stimulation of follicular cells by TSH, as occurs with chronic iodine deficiency, both lateral lobes of the thyroid are uniformly enlarged (see Fig 3-8) . The enlargements may be extensive and result in prominent swelling in the cranial cervical area. The affected lobes are firm and dark red because an extensive interfollicular capillary network develops under the influence of long-term TSH stimulation. The thyroid enlargements are the result of intense hypertrophy and hyperplasia of follicular cells, often with the formation of papillary projections into the lumens of follicles or multiple layers of cells lining follicles (Fig 3-9) . Endocytosis of colloid usually proceeds at a rate greater than synthesis, resulting in progressive depletion of colloid. Thyroid follicles become smaller than normal and there may be a partial collapse of follicles owing to the lack of colloid (see Fig 3-9) . The hypertrophic lining follicular cells are columnar with a deeply eosinophilic cytoplasm and small hyperchromatic nuclei that often are situated in the basilar part of the cell. The follicles are lined by either single or multiple layers of hyperplastic follicular cells that in some follicles form papillary projections into the lumen (see Fig 3-9) .

The converse of what has just been described occurs in follicular cells as a response to an increase in circulating thyroid hormones and a corresponding decrease in circulating pituitary TSH (e.g., after exogenous thyroxine therapy), or in patients with a large space- occupying pituitary lesion that

Figure 3.1-7 Response of thyroid follicular cells 8 hours after TSH stimulation. The follicular cells are hypertrophic and columnar. Many follicles are nearly depleted of colloid and are partially collapsed ( arrow).

markedly decreases the ability to secrete TSH. ⁽¹⁰⁾ ⁽²⁶⁾ Thyroid follicles become greatly enlarged and distended with densely staining colloid as a result of decreased TSH-mediated endocytosis of colloid. Follicular cells lining the involuted follicles are low cuboidal and there are few endocytotic vacuoles at the interface between the colloid and follicular cells (Fig 3-10) . The luminal surface of follicular cells is flattened. Microvilli extending into the colloid are widely separated and short in response to a long-standing decreased secretion of TSH (Fig 3-11) (Figure Not Available) .

The thyroid stroma is exceptionally rich in blood vessels that form extensive interfollicular capillary plexuses lying close to the follicular basement membranes. There is also a network of lymphatics in the gland. The stroma encloses a number of nerve fibers, some of which are parasympathetic, but most are sympathetic. These nerves terminate on blood vessels or in apposition to follicular cells.

Much less numerous, especially in the human thyroid, are cells concerned with the secretion of the peptide hormone of the mammalian thyroid. CT has been shown to be secreted by a second endocrine cell population in the mammalian thyroid gland. C cells (parafollicular or light cells) are distinct from follicular cells in the thyroid that secrete T₄ and T₃ . ⁽²⁷⁾ They are situated either within the follicular wall immediately beneath the basement membrane or between follicular cells (Fig 3-12) and as small groups of cells between thyroid follicles. C cells do not border the follicular

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Figure 3.1-8 Diffuse hyperplastic goiter in a pup, resulting in prominent symmetric enlargements of both thyroid lobes (T). The hyperplastic thyroids were freely movable from the trachea ( arrow) in the cervical region. H, heart.

colloid directly and their secretory polarity is oriented toward the interfollicular capillaries (see Fig 3-12) . The distinctive feature of C cells, compared to thyroid follicular cells, is the presence of numerous, small membrane-limited secretory granules in the cytoplasm. Immunocytochemical techniques have localized the CT activity of C cells to these secretory granules. ⁽²⁸⁾

Calcitonin-secreting thyroid C cells have been shown to be derived embryologically from cells of the neural crest. Primordial cells from the neural crest migrate ventrally and become incorporated within the last (ultimobranchial) pharyngeal pouch (Fig 3-13) (Figure Not Available) . They move caudally with the ultimobranchial body to the point of fusion with the midline primordia that gives rise to the thyroid gland (see Fig 3-13) (Figure Not Available) . The ultimobranchial body fuses with and is incorporated into the thyroid near the hilus in mammals, and C cells subsequently are distributed throughout the gland. Although C cells are present throughout the thyroid gland of humans and most other mammals in postnatal life, they often remain more numerous near the hilus and point of fusion with the ultimobranchial body. Under certain conditions, colloid-containing follicles lined by follicular cells also can differentiate from cells of ultimobranchial origin. ⁽²⁹⁾

In submammalian species C cells and CT activity remain segregated in the ultimobranchial gland, which is anatomically distinct from both the thyroid and the parathyroid glands (Fig 3-14) . In the avian ultimobranchial gland a network of stellate cells with long cytoplasmic processes supports the C-cells. ⁽³⁰⁾

Figure 3.1-9 Diffuse hyperplastic goiter (see Fig 3-8) illustrating papillary projections of thyroid follicular cells ( arrows) into follicular lumens in response to long-term TSH stimulation. Partial collapse of follicles is due to increased endocytosis of colloid.

In contrast to the iodothyronines (T₄ and T₃ ) produced by follicular cells, CT is a polypeptide hormone composed of 32 amino acid residues arranged in a straight chain. ⁽³¹⁾ The concentration of calcium ion in plasma and extracellular fluids is the principal physiologic stimulus for the secretion of CT by C cells. CT is secreted continuously under conditions of normocalcemia, but the rate of secretion of CT is increased greatly in response to elevations in blood calcium.

C cells store substantial amounts of CT in their cytoplasm in the form of membrane-limited secretory granules (see Fig 3-12) . In response to hypercalcemia there is a rapid discharge of stored hormone from C cells into interfollicular capillaries. The hypercalcemic stimulus, if sustained, is followed by hypertrophy of C cells and an increased development of cytoplasmic organelles concerned with the synthesis and secretion of CT. C-cell hyperplasia occurs in response to long-term hypercalcemia. When the blood calcium is lowered, the stimulus for CT secretion is diminished and numerous secretory granules accumulate in the cytoplasm of C cells. The storage of large amounts of preformed hormone in C cells and its rapid release in response to moderate elevations in blood calcium probably reflect the physiologic role of CT as an ""emergency"" hormone to protect against the development of hypercalcemia.

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Figure 3.1-10 Response of thyroid follicular cells to long-term decreased levels of TSH. The follicular cells ( arrows) are low-cuboidal, and thyroid follicles are distended with dense colloid (C) in response to decreased TSH secretion.

HISTOCHEMISTRY AND HISTOPHYSIOLOGY

Follicular cells show striking polarity orientated toward the follicular lumen (Fig 3-15) (Figure Not Available) . Varying numbers of lysosomes, histochemically stainable for enzymes such as acid phosphatase, are found in the apical portion of the cell. ⁽³²⁾ ⁽³³⁾ Soon after stimulation by TSH in follicular cells, intracellular droplets (phagosomes), corresponding to those demonstrated light microscopically by the PAS reaction and representing ingested colloid, are more numerous than in the resting state. ⁽³³⁾ Some of these form phagolysosomes in follicular cells by fusion with lysosomes.

The apical portion of the follicular cell develops prominent elongations of microvilli shortly after stimulation by TSH that form cytoplasmic processes (pseudopods) that surround portions of the follicular colloid (see Fig 3-15) (Figure Not Available) . Pseudopods appear to collect thyroglobulin located at some distance from the apical surface and may provide a mechanism of selective macropinocytosis by which newly synthesized thyroglobulin recently delivered to the follicle lumen is prevented from undergoing immediate reuptake ⁽³⁴⁾ (Fig 3-16) (Figure Not Available) . This process, termed ""endocytosis,"" results in the formation of colloid droplets in the cytoplasm of follicular cells. ⁽³³⁾ ⁽³⁵⁾ ⁽³⁶⁾ In addition, small, clathrin-containing coated vesicles appear to be involved in the uptake and transport of iodinated thyroglobulin from the follicular lumen to the lysosomal compartment of thyroid follicular cells. ⁽³⁷⁾ This process of receptor-mediated endocytosis of colloid (""micropinocytosis"") may be a major pathway of thyroglobulin uptake in the normal thyroid gland

Figure 3.1-11 (Figure Not Available) Scanning electron micrograph of luminal surface of thyroid follicular cells from a rat that was given 100 mug of T₄ daily for 4 weeks. In response to decreased TSH levels, microvilli ( arrows) are widely separated and short. There is no evidence of formation of cytoplasmic pseudopodia into the luminal colloid, as occurs in actively secreting thyroid follicles (contrast with Fig 3-6) (Figure Not Available) . (Collins WT, Capen CC. Ultrastructural and functional alterations of the rat thyroid gland produced by polychlorinated biphenyls compared with iodide excess and deficiency, and thyrotropin and thyroxine administration. Virchows Arch (B) 1980;33:213)

under conditions in which the demands for thyroid hormone secretion are low. During the vesicular transport of thyroglobulin through the cytoplasm of follicular cells (""transcytosis""), the molecule does not undergo cleavage and its electrophoretic mobility remains unchanged. ⁽³⁸⁾ Thyroglobulin may be released as an intact molecule into the circulation in small quantities by this TSH-regulated transepithelial vesicular transport. Clearance of thyroglobulin from the circulation with release of thyroid hormones occurs in the liver by macrophages (Kupffer cells). ⁽³⁹⁾

Microtubules and microfilaments in the cytoplasm of follicular cells beneath the apical plasma membrane are important in moving colloid droplets into close proximity to lysosomal bodies. ⁽⁴⁰⁾ The membranes of these two organelles fuse, resulting in the local release of enzymes that break down the colloid and release T₄ and T₃ into the cytosol.

The active thyroid hormones subsequently diffuse out of the cell and enter the abundant interfollicular capillaries, which have a fenestrated endothelial lining. The iodinated tyrosines (MIT and DIT) released from the colloid droplets are deiodinated enzymatically and the iodide generated either is recycled to the lumen to iodinate new tyrosyl residues or released into the circulation. These unique structural and functional characteristics of the phylogenetically oldest endocrine gland suggest that the thyroid may have evolved toward a more ""ideal"" structure to perform its vital metabolic functions. ⁽⁴¹⁾

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Figure 3.1-12 Electron micrograph of thyroid, illustrating a C cell in the wall of a follicle, wedged between several follicular cells (F). Follicular cells line the follicle directly and extend microvilli ( arrow) into the colloid (C). The cytoplasm of C cells has many calcitonin-containing secretion granules (S) and a prominent Golgi apparatus (G). The secretory polarity of C cells is directed toward interfollicular capillaries (E), rather than toward the follicle lumen, as with follicular cells.

The functionally most important enzyme in the thyroid hormone synthetic pathway is present in the apical plasma membrane and microvilli as well as in other structures of the follicular cells ⁽⁴²⁾ ⁽⁴³⁾ (Fig 3-17) (Figure Not Available) . Thyroperoxidase in the human thyroid is a membrane-bound, heme-containing glycoprotein composed of 933 amino acids with a transmembrane domain. ⁽⁴⁴⁾ This important enzyme oxidizes iodide ion (I^- ) taken up by follicular cells into reactive iodine, which binds to the tyrosine residues in the thyroglobulin. Iodine is incorporated not

Figure 3.1-13 (Figure Not Available) Schematic representation of neural crest origin of calcitonin-secreting C cells. Primordial cells arising from neural crest migrate ventrally during embryonic life to become incorporated in the last (ultimobranchial) pharyngeal pouch. The ultimobranchial body fuses with primordia of the thyroid and distributes C cells throughout the mammalian thyroid gland. (Foster GV, Byfield PGH, Gudmundsson TV. Calciton. Clin Endocrinol Metab 1972;1:93)

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Figure 3.1-14 Calcitonin-secreting C cells in submammalian vertebrates remain in an anatomically distinct endocrine organ separate from the thyroid gland. The ultimobranchial gland in chickens is caudal to the two pairs of parathyroids and thyroid gland along the carotid artery.

only into newly synthesized thyroglobulin recently delivered to the follicular lumen but into molecules already stored in the lumen. ⁽⁴⁵⁾ Thyroperoxidase also functions as a ""coupling"" enzyme to combine MIT and DIT to form T₃ or two DITs to form T₄ .

The follicular cell of the thyroid is involved concurrently in luminally directed processes of thyroglobulin synthesis and exocytosis as well as basally directed processes of colloid endocytosis with breakdown and eventual release of thyroid hormones into the interfollicular capillaries. Radioautographs prepared at increasing time intervals after pulse labeling of thyroids with a radioactive amino acid such as ³ H-leucine (Fig 3-18) (Figure Not Available) have shown that its incorporation into peptides occurs in the rough endoplasmic reticulum. Labeled material subsequently appears in the Golgi region, then over vesicles between the Golgi apparatus and the lumen, and finally in the lumen. ⁽⁴⁶⁾ With the use of tritiated monosaccharides, it can be shown that the synthesis of the carbohydrate chains of thyroglobulin starts in the endoplasmic reticulum and is completed in the Golgi apparatus. ⁽⁴⁷⁾

The thyroid takes up iodine in the form of iodide ion. Although the active transport of iodide occurs at the base of the follicular cells near the interfollicular capillaries, iodide that has entered the thyroid cell is transported rapidly to the follicular lumen ⁽⁴⁸⁾ (Fig 3-19) (Figure Not Available) . Iodide in the thyroid is oxidized to a higher valence state by the thyroperoxidase in microvilli. This oxidized form of iodine becomes rapidly attached to the tyrosyl residues in thyroglobulin in proximity to the apical microvilli. ⁽⁴⁹⁾ ⁽⁵⁰⁾

COMPARATIVE ASPECTS OF THYROID

Comparative studies of thyroid structure and function have contributed, to an important and often not fully appreciated degree, to mammalian and clinical thyroidology. The first iodoproteins and their incorporated iodotyrosines were discovered in invertebrate organisms long before their association was known with the thyroid gland. The relationship between iodine lack and thyroid hypertrophy was first worked out in hatchery trout and was quickly applied to clinical situations in human patients. ⁽⁵¹⁾

Morphology

The thyroid in all adult vertebrates has a basic follicular pattern and it would be difficult, with few exceptions, to differentiate among species solely on the basis of thyroid histology. Similarity in thyroid structure has been found between lower vertebrates and mammals at the ultrastructural level as well ⁽⁵²⁾ (Fig 3-20) (Figure Not Available) .

The macroscopic shape of the thyroid is formed by amalgamation of the numerous histologic units (i.e., follicles) and can vary quite considerably among the different vertebrate species (Fig 3-21) . However, the function of the thyroid gland as a whole is not influenced by its macroscopic shape, which suggests this anatomic variation among species is not of fundamental evolutionary significance. ⁽⁵¹⁾

There is not an organized thyroid in the adult cyclostomes (lampreys, hagfish) and teleost fish. However, follicles occur scattered in the subpharyngeal connective tissue in a pattern roughly approximating the ventral aorta and its principal branches into the gills. In a few species of teleosts (parrotfish, swordfish), most thyroid follicles may be gathered into an organized gland. An interesting finding in fish is the occurrence of thyroid follicles in nonpharyngeal areas. The most frequent location of heterotopic follicles in teleosts is in the kidney; other, less common sites include the eye, brain, heart, esophagus, and spleen.

The thyroid in elasmobranch fishes usually is aggregated into a single encapsulated organ near the tip of the lower jaw (see Fig 3-21) . In amphibians there are two rounded thyroid lobes, often quite widely separated and associated with branches of the hyoid cartilage. Thyroid shape in reptiles is variable, with turtles having a large disc of thyroid tissue immediately in front of the heart at the branching of the two systemic aortae. Lizards have a bilobed gland connected by an isthmus that crosses the trachea. Birds have two widely separated, rounded thyroid lobes, one on each side of the trachea at the level of the clavicles (see Figs. 3-14 and 3-21) . Mammals are fairly consistent in the well known pattern that consists of two lobes connected by an isthmus.

Physiology

Marine algae are efficient concentrators of iodine, indicative of some kind of iodide or halide pump. ⁽⁵³⁾ Most groups of invertebrates are able to form iodoproteins, usually in skeletal or fibrous scleral layers. As a rule, iodination of such rigid proteins does not go beyond formation of diiodotyrosine, ⁽⁵⁴⁾ ⁽⁵⁵⁾ presumably because the iodotyrosines are not free to couple. There

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Figure 3.1-15 (Figure Not Available) Electron micrograph of the apical portion of a follicular cell stained for acid phosphatase. The enzyme is present in the darkly stained lysosomes, some of which are attached to colloid droplets (phagosomes, arrows). The large droplets with irregular black material are phagolysosomes, whose colloid has become intermingled with lysosomal contents. (Wetzel BK, Spicer CC, Wollman SH. Changes in fine structure and acid phosphatase localization in rat thyroid cells following thyrotropin administration. J Cell Biol 1965;25:593)

are reports of formation of significant proportions of T₄ in certain invertebrate genera, such as in Musculium (small freshwater clams). This cannot be taken as a significant finding because in vitro iodination of pure proteins under oxidative conditions yields a certain amount of T₄ .

Although there has been no conclusive evidence of a function for thyroid hormones in invertebrates despite the general occurrence of iodotyrosines and T₄ , there is evidence from coelenterates of the metabolism of iodine to T₄ . ⁽⁵⁶⁾ Data from one of the most primitive invertebrate groups support the conclusion that T₄ formation is an ancient biochemical phenomenon in animals and that T₄ may have a function in some invertebrates. However, T₄ in many of the primitive forms may be merely an accidental product of the oxidative iodination of proteins exposed to high levels of environmental iodine.

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Figure 3.1-16 (Figure Not Available) Colloid droplet (cd) enclosed within a thin pseudopod on apical surface of a follicular cell 30 minutes after TSH injections. Note dense granules (dg), mitochondria (m), endoplasmic reticulum (er), and centriole (c). Vesicles (v) of moderate size with contents similar to those of the luminal colloid (LC) are clustered about the Golgi zones (Gz) and beneath the apical membrane. (MV, microvilli; JC, junctional complex; n, nucleus; uv, microvesicles). (Wetzel; BK, Spicer SS, Wollman SH. Changes in fine structure and acid phosphatase localization in rat thyroid cells following thyrotropin administration. J Cell Biol 1965;25:593)

THYROID IODOPROTEINS IN LOWER VERTEBRATES

Molecular variations have not been reported in the structure of T₄ and T₃ among animals. All vertebrates that metabolize iodine into an organic form, whether in a thyroid or in any other pharyngeal epithelial structure, form T₄ and variable proportions of a triiodinated form of the thyronine molecule.

Iodination of tyrosine molecules appears to take place in a characteristic protein, which in the mammalian thyroid is thyroglobulin. However, mammals and a few species of birds, reptiles, amphibians, elasmobranchs, and teleosts that have been studied also synthesize lesser amounts of 12S and 27S iodoproteins. ⁽⁵⁷⁾ ⁽⁵⁸⁾ Smaller thyroproteins also have been identified with iodination to various degrees in different species and referred to as R5SS or R3-8SS fractions.

30

Figure 3.1-17 (Figure Not Available) Rat thyroid cytochemically stained to demonstrate the distribution of peroxidase. Precipitate corresponding to the location of the enzyme is most evident in the apical membrane, including the portion lining a microvillus ( arrows). There is also some precipitate in apical vesicles. (Tice LW, Wollman SH. Ultrastructural localization of peroxidase activity on some membranes of the typical thyroid epithelial cell. Lab Invest 1972;26:23. Courtesy of US-Canadian Division of the International Academy of Pathology)

The thyroid iodoproteins of the Agnatha are of special interest because they come from the most primitive existing vertebrates. Conflicting reports that the principal thyroid iodoprotein was 12S in one species of lamprey and 19S in another most likely are the result of procedural differences. This is suggested by the finding of 19S thyroglobulin in thyroid tissue of the Pacific lamprey ( Entosphenus tridentatus) on sucrose density gradient centrifugation, but 12S on fractional precipitation and then purification with ammonium sulfate. ⁽⁵⁹⁾ ⁽⁶⁰⁾

The findings of mean T₄ levels of 1.5 mug/dL in leopard frogs, 3.4 mug/dL in Pacific hagfish, 0.5 mug/dL in migrating adult lampreys, ⁽⁶¹⁾ and 3.7 mug/dL in a Japanese hagfish offer little opportunity for generalizations except that marine fish have higher circulating levels of T₄ than freshwater fish. Functions for T₄ have been demonstrated in teleosts, and a negative feedback for T₄ has been observed on TSH secretion. In agnathans there is no demonstrated action of T₄ and no evidence for TSH production with feedback control. ⁽⁵²⁾

Regard and colleagues ⁽⁶²⁾ and others ⁽⁶³⁾ have reported a surge in both T₃ and T₄ at the time of metamorphosis in several species of amphibians. They speculated that because their assay failed to detect measurable plasma T₃ and T₄ in postmetamorphic or adult amphibians, there may be no function for thyroid hormones in adults. ⁽⁶²⁾ However, it is known that the adult amphibian thyroid gland actively metabolizes iodine and responds to goitrogenic treatment. ⁽⁶³⁾ ⁽⁶⁴⁾ In addition, there are reported actions of thyroidectomy and thyroid hormones on skin, nervous function, and intermediary metabolism in amphibians.

In anuran amphibia, metamorphosis is accompanied by alterations in thyroid hormone receptor concentration and marked changes in the activities of the iodothyronine deiodinase systems. ⁽⁶⁵⁾ All of these changes contribute to enhancing the peripheral sensitivity to circulating T₄ . The Mexican axolotl, Ambystoma mexicanum, is a neotenous salamander that rarely undergoes anatomic metamorphosis but can be induced to undergo metamorphosis by the administration of T₄ . ⁽⁶⁵⁾ The neoteny results primarily from low levels of plasma T₄ secondary to a low secretory rate of TSH from the pituitary. Tissues of the Mexican axolotl do not undergo specific changes that enhance the physiologic response to T₄ (summarized previously), as in anuran amphibia. ⁽⁶⁵⁾

Metamorphosis in amphibians is a complex metabolic process controlled by thyroid hormones. The limbs of Xenopus laevis grow and differentiate concomitant with the formation of

31

Figure 3.1-18 (Figure Not Available) High-resolution radioautographs of rat thyroid follicular cells at various times after injection of radioactive precursors of thyroglobulin. ( A) Thyroid 10 minutes after ³ H-leucine injection. Silver gains over ribosomes studding the membranes of the rough endoplasmic reticulum indicate that the synthesis of thryoglobulin starts in association with the ribosomes. ( B) Thyroid 30 minutes after ³ H-leucine injection. Silver grains over the cisternae of the endoplasmic reticulum indicate that the newly synthesized protein portion of thyroglobulin migrates from the ribosomes to the cisternae. ( C) Thyroid 1 hour after ³ H-leucine injection. Silver grains in association with Golgi zone indicate that the newly synthesized protein molecule is transported from the endoplasmic reticulum to the Golgi apparatus. ( D) Thyroid 15 minutes after ³ H-galactose injection. Note silver grains in association with the Golgi zone. ( E) Thyroid 2 hours after ³ H-leucine injection. Silver grains over the region of apical vesicles indicate that the glycoprotein molecules emerge from the Golgi apparatus contained in vesicles that move to the apical surface of follicular cells. ( F) Thyroid 4 hours after ³ H-leucine injection. Silver grains over the colloid in the lumen indicate that thyroglobulin is secreted by the follicular cells into the colloid of the lumen ( A through C, courtesy of Dr. B.A. Young; D, courtesy of Dr. E.J.H. Nathaniel)

the thyroid gland and increasing levels of thyroid hormones. More than 120 genes are up-regulated within 24 hours after induction of metamorphosis by thyroid hormone. ⁽⁶⁶⁾ Some of the genes respond directly, but most appear to be secondary response genes judging from their delayed kinetics and cyclohexamide sensitivity. Up-regulation of nuclear thyroid hormone

32

Figure 3.1-19 (Figure Not Available) High resolution radioautograph of thyroid follicle. The thyroid gland was removed from a rat 1 minute after injection of a tracer dose of ¹²⁵ I. With few exceptions, the silver grains are located over the colloid in the follicular lumen, indicating that iodination of thyroglobulin takes place in the lumen near the apical surface. (Courtesy of Dr. Huberta van Heyningen)

receptor mRNA is one of the earliest changes in gene expression in Xenopus tadpoles in response to thyroid hormones, which correlates closely with the progress of metamorphosis. ⁽⁶⁷⁾

A 56-kd protein composed of four identical subunits has been reported in the plasma of the bullfrog ( Rana catesbeiana), the amino acid composition of which was highly homologous with the mammalian transthyretins (e.g., T₄ -binding prealbumin). ⁽⁶⁸⁾ In contrast to mammalian transthyretins, the affinity of bullfrog transthyretin for T₃ was 360 times greater than for T₄ . These findings suggest that bullfrog transthyretin plays an important role in transporting T₃ in the blood during metamorphosis. ⁽⁶⁸⁾ In turtles ( Trachemys scripta), plasma T₄ is

33

Figure 3.1-20 (Figure Not Available) Electron micrograph of thyroid epithelial cells of a hagfish ( Eptatretus stouti). The follicular lumen contains no colloid, but colloid-like secretion droplets are numerous in the cytoplasm. Other structures, including the microvilli of the apical surface, are similar to the equivalent ones in thyroid cells of higher vertebrates. (Courtesy of N.E. Henderson, University of Calgary).

bound principally to a relatively high-affinity, low-capacity T₄ -binding protein. ⁽⁶⁹⁾ Because of the low concentrations of albumin in turtle blood (~ 10 mg/mL), T₄ -binding protein appears to account for a greater proportion of T₄ binding than T₄ -binding globulin (TBG) in humans.

A surge in plasma T₄ or T₃ , or both, occurs in developing salmon at the stage when the ""parr"" becomes a ""smolt."" ⁽⁷⁰⁾ The exact timing of the smoltification T₄ surge appears to be keyed to the phase of the moon. ⁽⁷¹⁾ There are several

Figure 3.1-21 Distribution of thyroid follicles and their organization into thyroid glands of different characteristic shapes in vertebrates.

reports that peripheral deiodination of T₄ to T₃ appears at the time of amphibian metamorphosis, ⁽⁷²⁾ and a similar phenomenon occurs in developing salmonids. ⁽⁷³⁾ Deiodination of T₃ during parr--smolt transformation in Atlantic salmon proceeds exclusively through an inner ring deiodinase pathway, which permits regulation of T₃ degradation independently of the outer ring deiodinase pathway responsible for T₃ formation. ⁽⁷⁴⁾ By comparison, deiodination of reverse T₃ in salmon occurs primarily through an outer ring deiodinase pathway, but reverse T₃ inner ring deiodinase activity does occur in some tissues.

The actions of thyroid hormones in different animal groups and species reflect adaptiveness in the evolutionary sense. ⁽⁵¹⁾ Particular target tissues for T₃ and T₄ appear to vary in their sensitivity to these hormones at different stages of development. For example, brain and gut are sensitive to T₄ in tadpoles but are much less so in adult frogs. ⁽⁷⁵⁾ ⁽⁷⁶⁾ The characteristic action of thyroid hormone in stimulating oxygen consumption and heat production in mammals is absent or different in the cold-blooded vertebrates. Most of the actions of thyroid hormones eventually are explicable on the basis of translation of specific ribonucleic acid messages for synthesis of particular structural or enzyme proteins. It appears from the multiplicity of actions of thyroid hormones that cellular receptors are widespread and that receptor protein synthesis is relatively easily evoked (or suppressed) during evolution or ontogenetic development. ⁽⁵¹⁾

CONTROL OF THYROID ACTIVITY

Different elements in the thyroid control system have been subjected to evaluation in nonmammalian vertebrates, with some interesting differences detected. Hypophyseal thyrotropic activity has been demonstrated in most vertebrate groups. ⁽⁷⁷⁾ In the cartilaginous elasmobranch fishes, evidence indicates that TSH is synthesized primarily in the ""ventral lobe"" of the pars distalis. ⁽⁷⁸⁾ Definitive chemical identification of thyrotropin-releasing hormone (TRH) in the hypothalamus has been achieved for several mammalian species and in the salamander. ⁽⁷⁹⁾ However, TRH was found distributed through all parts of the brain and in the pituitary of rats, chickens, snake ( Thamnophis), leopard frogs and their tadpoles, and Atlantic salmon, as well as in the ""head region"" of Amphioxus. ⁽⁸⁰⁾ Frog hypothalamic extract had TRH biologic activity in rats in proportion to its immunoreactive TRH content. Recent studies have reported that TRH, corticotropin-releasing hormone (ovine), and gonadotropin-releasing hormone (mammalian) all stimulated the secretion of bioactive TSH by frog ( Rana esculenta) pituitary glands in vitro. ⁽⁸¹⁾ Preincubation with T₄ for 6 hours suppressed the TRH- and corticotropin-releasing hormone--induced secretion of TSH but did not affect the response to gonadotropin-releasing hormone, whereas preincubation with T₃ reduced both the TRH- and gonadotropin-releasing hormone--stimulated release of TSH. The results suggest that thyroid hormones exert a negative feedback control on the secretion of TSH in adult frogs by a direct action on the pituitary.

The action of T₄ on TSH secretion in amphibians indicates negative but no positive feedback. ⁽⁸²⁾ ⁽⁸³⁾ Thus, the surging high plasma levels of T₄ in metamorphosing tadpoles may reflect extreme changes in the set point for negative feedback during development, analogous to the changes in sex steroid feedback on gonadotropins during puberty in mammals. ⁽⁵¹⁾

34

In the turtle, Pseudemys scripta, hypothyroidism induced by surgical thyroidectomy or a goitrogen (methimazole) resulted in marked depression of plasma binding of T₄ , and T₄ treatment restored binding after 4 to 6 weeks. ⁽⁸⁴⁾ The T₄ -binding protein in turtles has a high degree of structural homology (68% of NH₂ terminal region) to the mammalian vitamin D-binding protein (rat, mouse, and human) rather than mammalian TBG. ⁽⁸⁵⁾ Binding studies confirmed that the turtle T₄ -binding protein likely also represents the major vitamin D-binding protein and is electrophoretically distinct from the sex hormone-binding proteins. ⁽⁸⁶⁾ T₄ tends to enhance the affinity and the capacity for transporting vitamin D₃ in this species. Therefore, turtles have a single binding protein, resembling vitamin D-binding protein, that performs two major functions that are normally served by proteins representing different multigene families in mammals. ⁽⁸⁵⁾

Pathology

Although the basic hypothalamic--pituitary--thyroid axis functions in a similar manner in animals and humans, there are important differences between species that are significant when extrapolating data from chronic toxicity and carcinogenicity studies of drugs and chemicals in animals for human risk assessment. ⁽⁴¹⁾ ⁽⁸⁷⁾ Long-term perturbations of the pituitary--thyroid axis by various xenobiotics or physiologic alterations (e.g., iodine deficiency, partial thyroidectomy) are more likely to predispose laboratory rodents (e.g., rat and mouse) to a higher incidence of proliferative lesions (e.g., hyperplasia and tumors) of follicular cells than in the human thyroid. ⁽⁸⁸⁾ This appears to be particularly true in the male rat, in which there usually are higher circulating levels of TSH than in females. The greater sensitivity of the rodent thyroid to derangement by drugs, chemicals, and physiologic perturbations also is related to the shorter plasma half-life of T₄ than in humans, which results, in part, from the considerable differences between species in the transport proteins for T₄ .

The plasma half-life of T₄ in rats is considerably shorter (12--24 hours) than in humans (5--9 days). This is related in part to differences between species in the transport proteins for T₄ and T₃ . ⁽⁸⁹⁾ In human beings and the monkey, circulating T₄ is bound primarily to TBG; however, this high-affinity binding protein for T₄ is not present in rodents, birds, amphibians, or fish. The binding affinity of TBG for T₄ is approximately 1000 times higher than for transthyretin. The percentage of unbound active T₄ is lower in species with high levels of TBG than in animals in which T₄ binding is limited to albumin and transthyretin.

Although T₄ is the principal secretory product of the thyroid, it functions primarily as a prohormone and undergoes a single deiodination of the phenolic ring in extrathyroidal tissues to form the metabolically more active T₃ . ⁽⁹⁰⁾ T₃ is transported bound to TBG and albumin in human beings, monkey, and dog but only to albumin in mouse, rat, and chicken. These differences in plasma half-life of thyroid hormones and binding to transport proteins between rats and humans may be one factor in the greater propensity of the rat thyroid to development of hyperplastic or neoplastic lesions in response to chronic TSH stimulation.

Many chemicals and drugs disrupt one or more steps in the synthesis and secretion of thyroid hormones or enhance the metabolism of thyroid hormones, especially those that increase hepatic cytochrome P450 T₄ -metabolizing enzymes. This results in subnormal levels of T₄ and T₃ associated with a compensatory increased secretion of pituitary TSH in long-term rodent studies for safety assessment of a particular chemical for humans. ⁽⁹¹⁾ ⁽⁹²⁾ ⁽⁹³⁾ ⁽⁹⁴⁾ ⁽⁹⁵⁾ ⁽⁹⁶⁾ When tested in highly sensitive species, such as rats and mice, these compounds result in early follicular cell hypertrophy/hyperplasia and increased thyroid weights, and long-term studies show an increased incidence of thyroid tumors by a secondary (indirect) mechanism associated with hormonal imbalances.

In the secondary mechanism of thyroid oncogenesis in rodents, the specific xenobiotic chemical or physiologic perturbation evokes another stimulus (e.g., chronic hypersecretion of TSH) that promotes the development of nodular proliferative lesions (initially hypertrophy, followed by hyperplasia, subsequently adenomas, infrequently carcinomas) derived from follicular cells. Thresholds for a no-effect on the thyroid gland can be established by determining the dose of xenobiotic that fails to elicit an elevation in the circulating level of TSH. Compounds acting by this indirect (secondary) mechanism with hormonal imbalances usually show little or no evidence for mutagenicity or for producing DNA damage.

In humans who have markedly altered changes in thyroid function and elevated TSH levels, as in areas with a high incidence of endemic goiter due to iodine deficiency, there is little if any increase in the incidence of thyroid cancer. ⁽⁹⁷⁾ ⁽⁹⁸⁾ The relative resistance to the development of thyroid cancer in humans with elevated plasma TSH levels is in marked contrast to the response of the thyroid gland to chronic TSH stimulation in rats and mice. The human thyroid is much less sensitive to this pathogenetic phenomenon than rodents. ⁽⁹⁹⁾

Although numerous in vitro thyroid follicular cell culture systems have been described, ⁽¹⁰⁰⁾ ⁽¹⁰¹⁾ ⁽¹⁰²⁾ ⁽¹⁰³⁾ ⁽¹⁰⁴⁾ ⁽¹⁰⁵⁾ secretion of the thyroid hormones T₃ and T₄ is seldom measured in vitro because of the reduced ability of follicular cells growing in monolayer culture (i.e., lacking follicular organization) to concentrate and efficiently iodinate thyroglobulin. Thyroid function in vitro often

Figure 3.1-22 (Figure Not Available) Fluorescence photomicrograph of a small cluster of FRTL-5 cells with numerous latex beads ( arrowheads) located within the cytoplasm. The beads are approximately 2 mum in diameter. (Ozaki A, Sagartz JE, Capen CC. Phagocytic activity of FRTL-5 rat thyroid cells as measured by ingestion of fluorescent latex beads. Exp Cell Res 1995;219:547)

35

is assessed by evaluating specific phases of thyroid hormone synthesis, including iodide trapping ⁽¹⁰⁶⁾ ; thyroglobulin expression, ⁽⁷⁾ ⁽⁸⁾ ⁽⁹⁾ ⁽¹⁰⁾ ⁽¹¹⁾ ⁽¹²⁾ secretion, ⁽¹¹³⁾ ⁽¹¹⁴⁾ and iodination ⁽¹¹⁵⁾ ⁽¹¹⁶⁾ ; thyroid peroxidase synthesis ⁽¹¹⁷⁾ ⁽¹¹⁸⁾ and activity ⁽¹¹⁹⁾ ; and TSH receptor expression. These functions are TSH-dependent and can be altered in response to various growth factors, hormones, second messengers, and xenobiotics.

The normal Fischer rat thyroid line (FRTL-5) cell line has most of the known in vivo functional responses to TSH and has thus been a useful model for the study of thyroid pathophysiology in vitro. Numerous TSH-induced functional responses have been described, including iodide influx, trapping, and efflux; thyroglobulin synthesis, secretion, and iodination; thyroid peroxidase synthesis and activity; thyroid hormone production; and morphologic differentiation. In addition to these functional responses, FRTL-5 cells have been used to measure the effects of TSH on cell growth.

Thyroid follicular cell phagocytic activity can be quantified by a sensitive nonradioactive assay using a normal rat thyroid follicular cell line (FRTL-5) with fluoresceinated latex beads and flow cytometry ⁽¹²⁰⁾ (Fig 3-22) (Figure Not Available) . This in vitro assay permits discrimination of both the number of functionally active cells as well as the ability to estimate the level of activity of these cells. Electron microscopic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes (Fig 3-23) (Figure Not Available) . Phagocytosis can be stimulated by forskolin, cholera toxin, 8-Br-cAMP, calcitriol, and transforming growth factor beta. In contrast, phagocytosis was inhibited by insulin, NaI, CaCl₂ , and aminotriazole. ⁽¹²¹⁾ The phagocytosis of latex beads was regulated in a manner similar to iodide trapping and could be altered by the addition of numerous compounds. Phagocytic activity was stimulated by both cAMP-dependent and cAMP-independent pathways. Flow cytometric evaluation of phagocytosis of fluorescent latex beads provides a simple, rapid, nonradioactive index of thyroid function in vitro after exposure to a variety of xenobiotic chemicals.

Figure 3.1-23 (Figure Not Available) Phagocytosis of fluorescent latex beads by FRTL-5 rat thyroid follicular cell grown on polycarbonate membrane. Note the apical microvilli and the presence of a latex bead (B) within a cytoplasmic vacuole and on the cell surface. (Sagartz JE, Ozaki A, Capen CC. Phagocytosis of fluorescent T beads by rat thyroid follicular cells (FRTL-5): comparison with iodide trapping as an index of functional activity of thyrocytes in vitro. Toxicol Pathol 1995;23. In press)

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