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CHAPTER 21
TOXIC RESPONSES OF THE
ENDOCRINE SYSTEM
Charles C. Capen
INTRODUCTION
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Morphologic Alterations and Proliferative Lesions of
Thyroid C Cells
PITUITARY GLAND
Normal Structure and Function
Mechanisms of Toxicity
Morphologic Alterations and Proliferative Lesions of
Pituitary Cells
PARATHYROID GLAND
Introduction
Normal Structure and Function of Chief Cells
Biosynthesis of Parathyroid Hormone
Control of Parathyroid Hormone Secretion
Xenobiotic Chemical-Induced Toxic Injury of
Parathyroids
Ozone
Aluminum
L-Asparaginase
Proliferative Lesions of Parathyroid Chief Cells
Chief Cell Adenoma
Age-Related Changes in Parathyroid Function
ADRENAL CORTEX
Introduction
Normal Structure and Function
Mechanisms of Toxicity
Pathologic Alterations and Proliferative Lesions in
Cortical Cells
ADRENAL MEDULLA
Normal Structure and Function
Mechanisms of Toxicity
Pathologic Alterations and Proliferative Lesions in
Medullary Cells
TESTIS
Introduction
Structure and Endocrinologic Regulation of Leydig
(Interstitial) Cells
Pathology of Leydig (Interstitial) Cell Tumors
Mechanisms of Leydig (Interstitial) Cell Tumor
Development
THYROID GLAND (FOLLICULAR CELLS)
Species Differences in Thyroid Hormone Economy
Mechanisms of Thyroid Tumorigenesis
Chemicals that Directly Inhibit Thyroid Hormone
Synthesis
Blockage of Iodine Uptake
Inhibition of Thyroid Peroxidase Resulting in an
Organification Defect
Chemicals that Disrupt Thyroid Hormone Secretion
Blockage of Thyroid Hormone Release by Excess
Iodide and Lithium
Xenobiotic-Induced Thyroid Pigmentation or
Alterations in Colloid
Hepatic Microsomal Enzyme Induction
Chemical Inhibition of 5-Monodeiodinase
Secondary Mechanisms of Thyroid Tumorigenesis
and Risk Assessment
OVARY
Introduction
Mechanisms of Ovarian Tumorigenesis in Rodents
Case Study: Ovarian Tumors Associated with
Xenobiotic Chemicals
Nitrofurantoin
Selective Estrogen Receptor Modulators
Ovarian Tumors in Mutant Strains of Mice
Wx/Wv Strain with Genetic Deletion of Germ Cells
in Ovarian Cortex
Hypogonadal (hpg/hpg) Mice Unable to Synthesize
Hypothalamic Gonadotrophin-Releasing Hormone
(GnRH)
Genetically Engineered Mouse Models of Ovarian
Tumors
Summary: Ovarian Tumorigenesis in Rodents
THYROID C CELLS
Normal Structure and Function
Mechanisms of Toxicity
711
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UNIT 4 TARGET ORGAN TOXICITY
INTRODUCTION
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Endocrine glands are collections of specialized cells that synthesize, store, and release their secretions directly into the bloodstream. They are sensing and signaling devices located in the extracellular fluid compartment and are capable of responding to
changes in the internal and external environments to coordinate a
multiplicity of activities that maintain homeostasis.
Endocrine cells that produce polypeptide hormones have a
well-developed rough endoplasmic reticulum that assembles hormone and a prominent Golgi apparatus for packaging hormone into
granules for intracellular storage and transport. Secretory granules
are unique to polypeptide hormone- and catecholamine-secreting
endocrine cells and provide a mechanism for intracellular storage
of substantial amounts of preformed active hormone. When the cell
receives a signal for hormone secretion, secretory granules are directed to the periphery of the endocrine cell, probably by the contraction of microfilaments.
Steroid hormone-secreting cells are characterized by prominent cytoplasmic lipid bodies that contain cholesterol and other
precursor molecules. The lipid bodies are in close proximity to an
extensive tubular network of smooth endoplasmic reticulum and
large mitochondria which contain hydroxylase and dehydrogenase
enzyme systems. These enzyme systems function to attach various
side chains to the basic steroid nucleus. Steroid hormone-producing
cells lack secretory granules and do not store significant amounts
of preformed hormone. They are dependent on continued biosynthesis to maintain the normal secretory rate for a particular
hormone.
Many diseases of the endocrine system are characterized by
dramatic functional disturbances and characteristic clinicopathologic alterations affecting one or several body systems. The affected animal or human patient may have clinical signs that primarily involve the skin (hair loss caused by hypothyroidism),
nervous system (seizures caused by hyperinsulinism), urinary system (polyuria caused by diabetes mellitus, diabetes insipidus, and
hyperadrenocorticism), or skeletal system (fractures induced by
hyperparathyroidism).
The literature suggests that chemically induced lesions of the
endocrine organs are most commonly encountered in the adrenal
glands, followed in descending order by the thyroid, pancreas, pituitary, and parathyroid glands. In the adrenal glands, chemically
induced lesions are most frequently found in the zona fasciculata/zona reticularis and to a lesser extent in either the zona
glomerulosa or medulla. In a recent survey, conducted by the Pharmaceutical Manufacturers Association, of tumor types developing
in carcinogenicity studies, endocrine tumors developed frequently
in rats, with the thyroid gland third in frequency (behind the liver
and mammary gland), followed by the pituitary gland (fourth), and
adrenal gland (fifth). Selected examples of commonly encountered
toxic endpoints involving endocrine organs in laboratory animals
are discussed in this chapter. Mechanistic data is included whenever possible to aid in the interpretation of findings in animal
toxicology studies and to determine their significance in risk
assessment.
the pars distalis, pars tuberalis, and pars intermedia, and (2) the
neurohypophyseal system, which includes the pars nervosa
(posterior lobe), infundibular stalk, and hypothalamic nuclei
(supraoptic and paraventricular) containing the neurosecretory neurons, which synthesize and package the neurohypophyseal hormones into secretory granules. The pars intermedia forms the thin
cellular zone between the adenohypophysis and neurohypophysis.
The pituitary gland lies within the sella turcica of the sphenoid
bone. The gland receives its blood supply via the posterior and anterior hypophyseal arteries, which originate from the internal
carotid arteries. Arteriolar branches penetrate the pituitary stalk
near the median eminence, lose their muscular coat, and form a
capillary plexus. These vessels drain into the hypophyseal portal
veins, which supply the adenohypophysis. The hypothalamichypophyseal portal system functionally is important as it transports
the hypothalamic releasing- and release-inhibiting hormones directly to the adenohypophysis, where they interact with their specific populations of trophic hormone-producing cells.
The adenohypophysis in many animal species completely surrounds the pars nervosa of the neurohypophyseal system, in contrast to its position in human beings, where it is situated on the anterior surface. The pars distalis is the largest portion and is
composed of the multiple populations of endocrine cells that secrete the pituitary trophic hormones. The secretory cells are surrounded by abundant capillaries derived from the hypothalamichypophyseal portal system. The pars tuberalis consists of dorsal
projections of supportive cells along the infundibular stalk. It functions primarily as a scaffold for the capillary network of the hypophyseal portal system during its course from the median eminence to the pars distalis. The pars intermedia is located between
the pars distalis and pars nervosa and lines the residual lumen of
Rathke’s pouch. It contains two populations of endocrine cells in
certain species. One of these cell types (B cells) in the dog synthesizes and secretes adrenocorticotropic hormone (ACTH).
A specific population of endocrine cells is present in the pars
distalis (and in the pars intermedia of dogs for ACTH) that synthesizes, processes, and secretes each of the pituitary trophic hormones. Secretory cells in the adenohypophysis formerly were classified either as acidophils, basophils, or chromophobes based on
the reactions of their secretory granules with pH-dependent histochemical stains. Based upon contemporary specific immunocytochemical procedures, acidophils can be further subclassified functionally into somatotrophs that secrete growth hormone (GH;
somatotrophin) and luteotrophs that secrete luteotropic hormone
(LTH; prolactin). Their granules contain simple protein hormones.
Basophils include both gonadotrophs, which secrete luteinizing
hormone (LH) and follicle-stimulating hormone (FSH), and thyrotrophs, which secrete thyrotropic hormone [thyroid-stimulating
hormone (TSH)]. Chromophobes are pituitary cells that, by light
microscopy, do not have stainable cytoplasmic secretory granules.
They include the pituitary cells involved with the synthesis of
ACTH and melanocyte-stimulating hormone (MSH) in some
species, nonsecretory follicular (stellate) cells, degranulated chromophils (acidophils and basophils) in the actively synthesizing
phase of the secretory cycle, and undifferentiated stem cells of the
adenohypophysis.
Each type of endocrine cell in the adenohypophysis is under
the control of a specific releasing hormone from the hypothalamus
(Fig. 21-1). These releasing hormones are small peptides that are
synthesized and secreted by neurons of the hypothalamus. They
are transported by short axonal processes to the median eminence,
where they are released into capillaries and are conveyed by the
PITUITARY GLAND
Normal Structure and Function
The pituitary gland (hypophysis) is divided into two major compartments: (1) the adenohypophysis (anterior lobe), composed of
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The neurohypophysis is subdivided into three anatomic parts.
The pars nervosa (posterior lobe of the human pituitary) represents
the distal component of the neurohypophyseal system. The infundibular stalk joins the pars nervosa to the overlying hypothalamus and is composed of long axonal processes from neurosecretory neurons in the hypothalamus. It is made up of numerous
capillaries, supported by modified glial cells (pituicytes), which are
termination sites for the nonmyelinated axonal processes of neurosecretory neurons. The neurohypophyseal hormones (i.e., oxytocin and antidiuretic hormone) are synthesized in the cell body of
hypothalamic neurons, packaged into secretory granules, transported by long axonal processes, and released into the bloodstream
in the pars nervosa.
Antidiuretic hormone (ADH or vasopressin) and oxytocin are
nonapeptides synthesized by neurons situated either in the supraoptic (primarily ADH) or paraventricular (primarily oxytocin) nuclei
of the hypothalamus. ADH and its corresponding neurophysin are
synthesized as part of a common larger biosynthetic precursor molecule, termed propressophysin. The hormones are packaged with
a corresponding binding protein (i.e., neurophysin) into membranelimited neurosecretory granules and transported by axons to the
pars nervosa for release into the circulation. As the biosynthetic
precursor molecules travel along the axons in secretion granules
from the neurosecretory neurons, the precursors are cleaved into
the active hormones and their respective neurophysins. These secretory products can be detected immunocytochemically. In Brattleboro rats with hereditary hypothalamic diabetes insipidus, nerve
cells in the hypothalamus that normally produce ADH and neurophysin-I are negative immunocytochemically for both proteins
whereas neurosecretory stain positive for cells that produce vasopressin and neurophysin-II are positive.
In addition to the specific trophic hormone-secreting cells, a
population of supporting cells is also present in the adenohypophysis. These cells are referred to as stellate (follicular) cells and can
be stained selectively with antibodies to S-100 protein. The stellate cells typically have elongate processes and prominent cytoplasmic filaments. These cells appear to provide a phagocytic or
supportive function in addition to producing a colloid-like material that accumulates in follicles.
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Figure 21-1. Control of trophic hormone secretion from the adenohypophysis by hypothalamic releasing hormones (RH) and releaseinhibiting hormones (RIH).
The releasing and release-inhibiting hormones are synthesized by neurons
in the hypothalamus, transported by axonal processes, and released into
capillary plexus in the median eminence. They are transported to the adenohypophysis by the hypothalamic-hypophyseal portal system, where they
interact with specific populations of trophic hormone–secreting cells to
govern the rate of release of preformed hormones, such as growth hormone
(GH), somatotropic hormone (STH), luteotropic hormone (LTH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropic hormone (TTH), adrenocorticotropic hormone (ACTH), and melanocytestimulating hormone (MSH). There are RIHs for those trophic hormones
(e.g., prolactin and growth hormone) that do not directly influence the activity of target cells and result in production of a final endocrine product
(hormone) that could exert negative feedback control.
hypophyseal portal system to specific trophic hormone-secreting
cells in the adenohypophysis. Each hormone stimulates the rapid
release of preformed secretory granules containing a specific
trophic hormone. Specific releasing hormones have been identified
for TSH, FSH and LH, ACTH, and GH. Prolactin (PRL) secretion
is stimulated by a number of factors, the most important of which
appears to be thyrotropin-releasing hormone (TRH). Dopamine
serves as the major prolactin-inhibitory factor and suppresses prolactin secretion and also inhibits cell division and DNA synthesis
of luteotrophs. Dopamine also suppresses ACTH production by
corticotrophs in the pars intermedia of some species. Another hypothalamic release-inhibiting hormone is somatostatin (somatotropin-release inhibiting hormone, SRIH). This tetradecapeptide
inhibits the secretion of both growth hormone and TSH. Control
of pituitary trophic hormone secretion also is affected by negative
feedback by the circulating concentration of target organ (thyroid,
adrenal cortex, and gonad) hormones.
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Mechanisms of Toxicity
Pituitary tumors can be induced readily by sustained uncompensated hormonal derangements leading to increased synthesis and
secretion of pituitary hormones. The absence of negative feedback
inhibition of pituitary cells leads to unrestrained proliferation (hyperplasia initially, neoplasia later). This effect can be potentiated
by the concurrent administration of ionizing radiation or chemical
carcinogens.
In the pituitary-thyroid axis, thyroxine (T4) and triiodothyronine (T3) normally regulate the pituitary secretion of TSH by a
classic negative feedback control system. Surgical removal or
radiation-induced ablation of the thyroid or interference with the
production of thyroid hormones by the use of specific chemical inhibitors of thyroid hormone synthesis leads to a stimulation of TSH
synthesis and secretion with elevated blood levels. The thyrotrophic
cells in the adenohypophysis undergo prominent hypertrophy. Subsequently, hyperplasia of the thyrotrophs occurs concurrently with
hypertrophy as a consequence of the lack of normal negative feedback control. In rodents foci of hyperplasia may progress to the
formation of adenomas in the pituitary gland. The role of gonadectomy in pituitary tumor induction has been studied most in-
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administration of calcitonin resulted in pituitary tumors that produced the common -subunit of the glycoprotein hormones
[luteinizing hormone (LH), follicle-stimulating hormone (FSH)
and thyroid-stimulating hormones (TSH)], a type of tumor that has
been reported to compose a significant fraction of pituitary tumors
in humans. Immunohistochemistry and in situ hybridization
demonstrated that most pituitary tumors associated with the chronic
administration of high doses of calcitonin expressed a glycoprotein hormone -subunit, whereas expression of the -subunit was
identified infrequently in hyperplastic lesions of control rats.
Serum levels of each of the major pituitary hormones were
measured in both sexes of Sprague-Dawley and Fisher rats administered calcitonin. There were no significant alterations in the
circulating levels of growth hormone, prolactin, or ACTH and the
tumors were negative by immunohistochemical and in situ hybridization assessment for these hormones. Serum LH and FSH
levels were unaffected by the treatment with calcitonin; however,
TSH levels were elevated 2.1-fold after calcitonin treatment in
Sprague-Dawley but not Fischer rats of both sexes (Fig. 21-2). Interestingly, thyroid weights were decreased by 43 percent in
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tensively in mice. The pituitary tumors induced by gonadectomy
in mice are markedly strain-dependent and may contain FSH, LH,
or both.
The administration of estrogens is a reproducible method for
inducing pituitary tumors in certain experimental animals. The effect of exogenous estrogen on the rat pituitary includes stimulation of prolactin secretion and the induction of prolactin-secreting
tumors. The administration of estrogens in susceptible strains results in elevated serum prolactin levels, increased numbers of prolactin cells within the pituitary, enhanced incorporation of tritiated
thymidine within the gland, and increased mitotic activity. The pituitary of the ovariectomized F344 female rat is more responsive
to the tumorigenic effect of diethylstilbestrol than the intact female;
however, there is considerable variation in the induction of pituitary
tumors by estrogens in different rat strains. For example, F344 and
Holtzman rats respond to an initial estrogen stimulus by increasing
the rate of DNA synthesis in the pituitary within 2 to 4 days. The
rate of DNA synthesis declines after 7 to 10 days of treatment to
unstimulated levels in the Holtzman strain but remains elevated in
F344 rats.
Sarkar and coworkers (1982) have reported that estrogeninduced pituitary adenomas derived from prolactin-secreting cells
are associated with loss of hypothalamic dopaminergic neurons,
which normally inhibit the function of prolactin-secreting cells.
Prolactin-producing tumors, when transplanted subcutaneously,
also were associated with degenerative changes in hypothalamic
dopaminergic neurons. The tumorigenic action of estrogen may not
be due exclusively to its effect on the hypothalamus, since estrogen can produce prolactinomas in pituitaries grafted beneath the
renal capsule. The effects of estrogens on prolactin cells have been
studied in hypophysectomized rats bearing transplanted pituitaries
beneath the kidney capsule. The studies of E1 Etreby and coworkers (1988) using this model have shown that dopamine agonists,
including lisuride and bromocriptine, antagonize the direct stimulatory effects of estrogens on the prolactin cells. These dopamine
agonists may act directly on dopaminergic receptors within the
transplanted pituitaries.
Other agents, including caffeine, have been implicated in the
development of pituitary adenomas in rats. The administration of
N-methylnitrosourea also is associated with the development of pituitary adenomas in Wistar rats. The neuroleptic agent sulpiride has
been reported to cause the release of prolactin from the anterior
pituitary in the rat and to stimulate DNA replication. The administration of clomiphene prevents the stimulation of DNA synthesis
produced by sulpiride, but does not affect prolactin release from
the gland. These findings suggest that the intracellular prolactin
content of the pituitary plays a role in the regulation of DNA synthesis through a mechanism mediated by estrogens.
Morphologic Alterations and
Proliferative Lesions of Pituitary Cells
Jameson et al. (1992) reported that the administration of salmon
calcitonin for 1 year to Sprague-Dawley and Fisher 344 rats was
associated with an increased incidence of focal hyperplasia and
adenomas in the pituitary. The association of calcitonin treatment
and pituitary tumors was dose-dependent and was more pronounced with salmon calcitonin than with porcine calcitonin. Using both immunohistochemical analysis and measurements of
serum hormone levels, they provided evidence that prolonged
Figure 21-2. Serum glycoprotein hormone levels in rats treated chronically with salmon calcitonin.
Sprague-Dawley (left panels) or Fischer rats (right panels) were treated
for 52 weeks with vehicle (dark blue bars) or calcitonin (80 IU/kg/day)
(light blue bars). Results are the mean SD; *p 0.05. (From Jameson
et al., 1992, with permission.)
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CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
subunit by immunohistochemistry and in situ hybridization for expression of -subunit mRNA.
Serum levels of -subunit were elevated in male SpragueDawley rats after 2, 5, 8, 16, 24, 40, and 52 weeks to determine
the time course for hormone elevation. Elevated levels of -subunit
were detected as early as 24 weeks in rats treated with calcitonin
and the majority of animals had increased -subunit levels by 40
weeks of treatment (Fig. 21-4), suggesting that pituitary tumors developed only after several months of exposure to calcitonin. Levels of -subunit in vehicle-treated rats were below the detection
limits of the assay at each time point.
The studies reported by Jameson et al. (1992) did not determine whether the effects of calcitonin on the pituitary were direct
or indirect. Calcitonin is known to be produced in large amounts
in the posterior hypothalamus and median eminence where it may
normally exert an effect on the hypothalamus-pituitary axis. Calcitonin receptors have been identified in the hypothalamus and
lower numbers of receptors are found in the pituitary gland. A striking feature of the calcitonin-induced pituitary tumors and elevated
serum -subunit levels was the predilection for male compared
with female rats. The basis for the sex- and species-specific effects
of calcitonin was not determined. The relevance of the effects of
calcitonin in the rat pituitary gland to human pathophysiology is
uncertain at present. However, neither the treatment of patients with
calcitonin nor patients with the multiple endocrine neoplasia syndrome II with medullary thyroid cancer and elevated serum calcitonin levels have resulted in the development of pituitary tumors.
The doses of calcitonin used in rats were from 25- to 50-fold greater
on a per-weight basis than doses administered to patients. In addition, several strains of rats are known to be highly predisposed
to develop pituitary tumors compared to humans.
The high frequency of spontaneous pituitary adenomas in laboratory rats is a well-recognized phenomenon which must be considered in any long-term toxicological study. The incidence of pituitary tumors is determined by many factors including strain, age,
sex, reproductive status, and diet. Studies from the National Toxicology Program (NTP) historical database of 2-year-old F344 rats
have shown that the incidence of pituitary adenomas was 21.7 and
44 percent for males and females, respectively. Corresponding figures for carcinomas arising in the adenohypophysis were 2.4 and
3.5 percent for males and females, respectively. Numerous hypotheses have been invoked to explain the high incidence of pituitary adenomas in certain inbred rat strains. Both hereditary factors
and the levels of circulating sex steroids have been suggested as
important etiological mechanisms. The hypothalamus has also been
incriminated in the development of these tumors. Age-related hypothalamic changes may result in diminished activity of dopamine,
the major prolactin-inhibitory factor.
Numerous studies have demonstrated the striking degree of
strain variation in the incidence of pituitary tumors in rats, which
has been reported to range from 10 to more than 90 percent. Particularly high incidences of pituitary adenomas have been reported
in Wistar, WAG/Rij, Osborne-Mendel, Long-Evans, Amsterdam,
and Columbia-Sherman rats. In the BN/Bi strain, pituitary adenomas have been found in 26 percent of females and 14 percent of
males. Adenomas were identified in 95 percent of females and 96
percent of males in the WAG/Rij strain while (WAG/Rij 3 BN) F1
rats had incidences of 83 percent for females and 64 percent for
males. Rapid body growth rates and high levels of conversion of
feed to body mass in early life or high protein intake in early adult
life predispose any strain of rat to the development of pituitary ade-
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Figure 21-3. Serum -subunit levels in individual male rats treated
chronically with calcitonin.
The serum levels for individual animals are denoted. vehicle;
calcitonin-treated. (From Jameson et al., 1992, with permission.)
˚
calcitonin-treated male rats and there was atrophy of thyroid follicular cells in some treated rats, suggesting that the immunoreactivity detected by the TSH assay was not biologically active. After
treatment with calcitonin, serum -subunit levels were increased
by at least 20-fold in Sprague-Dawley males and 4-fold in male
Fischer rats (Figs. 21-3 and 21-4). There was a good correlation
between histopathologic evidence of -subunit–producing pituitary tumors and elevated serum levels. In each of the calcitonintreated rats that had adenomas, the tumors were positive for -
Calcitonin
Figure 21-4. Time course for the increase in serum -subunit levels in
male Sprague-Dawley rats.
Symbols in the undetectable range represent values for more than one
animal. The number of animals in each group is shown in parenthesis.
control;
calcitonin-treated. (From Jameson et al., 1992, with
permission.)
˚
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cent to some adenomas. The development of tumors and hyperplasia of prolactin-secreting cells often is accompanied by increasing serum levels of prolactin. Occasionally, prolactin cells
within adenomas may be admixed with FSH/LH-, TSH-, or ACTHpositive cells in the rat pituitary.
ADRENAL CORTEX
Introduction
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nomas. In rats fed a low protein diet (less than 12.7 percent crude
protein) the overall tumor incidence, the numbers of multifocal tumors, and the degree of cellular atypia within tumors are significantly lower than in rats fed a standard diet.
“Cystoid degeneration” has been used to describe foci of
parenchymal cell loss in the adenohypophysis. Foci of cystoid
degeneration (50 to 150 mm in diameter) have margins composed
of normal secretory cells of the pars distalis. Cystoid degeneration
also may occur in hyperplastic foci and in neoplasms of the
pituitary. The frequency of cystoid degeneration is increased by
feeding diets containing diethylstilbestrol to female C3H HeN
(MTV) mice.
The separation between focal hyperplasia, adenoma, and carcinoma utilizing histopathologic techniques is difficult in the pituitary gland. However, criteria for their separation have been established and should be applied in a consistent manner in the
evaluation of proliferative lesions of the pituitary gland. For the
specific trophic hormone–secreting cells of the adenohypophysis,
there appears to be a continuous spectrum of proliferative lesions
between diffuse or focal hyperplasia and adenomas derived from
a specific population of secretory cells. It appears to be a common
feature of endocrine glands that prolonged stimulation of a population of secretory cells predisposes to the subsequent development
of a higher than expected incidence of focal hyperplasia and tumors. Long-continued stimulation may lead to the development of
clones of cells within the hyperplastic foci that grow more rapidly
than the rest and are more susceptible to neoplastic transformation
when exposed to the right combination of promoting carcinogens.
Focal (“nodular”) hyperplasia in the adenohypophysis appears
as multiple small areas that are well demarcated but not encapsulated from adjacent normal cells. Cells in areas of focal hyperplasia closely resemble the cells of origin; however, the cytoplasmic
area may be slightly enlarged and the nucleus more hyperchromatic
than in the normal cells. Adenomas usually are solitary nodules
that are larger than the often multiple areas of focal hyperplasia.
They are sharply demarcated from the adjacent normal pituitary
glandular parenchyma and there often is a thin, partial to complete,
fibrous capsule. The adjacent parenchyma is compressed to varying degrees depending on the size of the adenoma. Cells composing an adenoma closely resemble the cells of origin morphologically and in their architectural pattern of arrangement.
Carcinomas usually are larger than adenomas in the pituitary
and usually result in a macroscopically detectable enlargement.
Histopathologic features that are suggestive of malignancy include
extensive intraglandular invasion, invasion into adjacent structures
(e.g., dura mater, sphenoid bone), formation of tumor cell thrombi
within vessels, and particularly the establishment of metastases at
distant sites. The growth of neoplastic cells subendothelially in
highly vascular benign tumors of the pituitary should not be mistaken for vascular invasion. Malignant cells often are more pleomorphic than normal, but nuclear and cellular pleomorphism are
not a consistent criterion to distinguish adenoma from carcinoma
in the adenohypophysis of rodents.
The vast majority of pituitary adenomas in humans and rodents have been described as chromophobic in type by light
microscopy; however, many of these tumors have been found to
stain for prolactin by immunohistochemistry. Of the prolactinproducing tumors, most are sparsely granulated with low levels of
prolactin immunoreactivity by immunohistochemistry and having
small numbers of secretory granules by electron microscopy.
Diffuse hyperplasia of the prolactin cells has been observed adja-
The adrenal cortex of animals is prone to develop degenerative and
proliferative lesions, the etiology of which may be either spontaneous in nature or experimentally induced. Therefore, testing of
xenobiotic chemicals using various laboratory animal species is a
valid means of assessing the toxic potential for humans exposed
to various xenobiotic chemicals. The choice of test animal species
also is critical, as a number of studies have demonstrated that there
often is a variable species susceptibility to chemical toxicity. This
suggests that interspecies differences in metabolism plays a role in
the development of adrenal cortical toxicity and in the inhibition
of steroidogenesis. The age of the test animal, to a lesser degree,
can be a factor in the development of chemically induced adrenal
cortical lesions.
Normal Structure and Function
The adrenal (suprarenal) glands in mammals are flattened bilobed
organs located in close proximity to the kidneys. The adrenal glands
receive arterial blood from branches of the aorta or from the
phrenic, renal, and lumbar arteries resulting in a subcapsular sinusoidal vascular plexus that drains through the cortex into the
medulla. The ratio of cortex:medulla is approximately 2:1 in
healthy laboratory-reared animals.
The cortex is histologically characterized by defined regions
or zones. The cortical zones consist of the zona glomerulosa (multiformis), zona fasciculata, and zona reticularis. The zones are not
always clearly delineated, as in the normal rat adrenal cortex. The
mineralocorticoid-producing zona glomerulosa (multiformis) (approximately 15 percent of the cortex) contains cells aligned in a
sigmoid pattern in relationship to the capsule. Degeneration of this
zone or an interference in the ability to produce mineralocorticoids
(namely, aldosterone) results in a life-threatening retention of
potassium and hypovolemic shock associated with the excessive
urinary loss of sodium, chloride, and water. The largest part of the
cortex is the zona fasciculata comprising 70 percent of the cortical width. Cells in this zone are arranged in long anastomosing
columns separated by vascular sinusoids and are responsible for
the secretion of glucocorticoid hormones (e.g., corticosterone or
cortisol). The innermost portion of the cortex is the zona reticularis (15 percent of the cortex), which secretes minute quantities
of adrenal sex hormones.
The adrenal cortical cells contain large cytoplasmic lipid
droplets consisting of cholesterol and other steroid hormone precursors. The lipid droplets are in close proximity to the smooth endoplasmic reticulum and large mitochondria, which contain the
specific hydroxylase and dehydrogenase enzyme systems required to synthesize the different steroid hormones. Unlike
polypeptidehormone-secreting cells, there are no secretory granules in the cytoplasm, since there is direct secretion without significant storage of preformed steroid hormones.
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trophic hormone to stimulate the synthesis and secretion of aldosterone. Under normal conditions negative feedback control to inhibit further renin release is exerted by the elevated levels of angiotensin (principally angiotensin II) as well as the expanded
extracellular fluid volume resulting from the increased electrolyte
(sodium and chloride) and water reabsorption by the kidney.
The principal control for the production of glucocorticoids by
the zona fasciculata and zona reticularis is exerted by adrenocorticotropin (ACTH), a polypeptide hormone produced by corticotrophs in the adenohypophysis of the pituitary gland. ACTH release is largely controlled by the hypothalamus through the
secretion of corticotropin-releasing hormone (CRH). An increase
in ACTH production results in an increase in circulating levels of
glucocorticoids and under certain conditions also can result in weak
stimulation of aldosterone secretion. Negative feedback control
normally occurs when the elevated blood levels of cortisol act either on the hypothalamus, anterior pituitary, or both to cause a suppression of ACTH secretion.
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Steroid hormone-producing cells of the adrenal cortex synthesizes a major parent steroid with one to four additional carbon
atoms added to the basic 17-carbon steroid nucleus. Since steroid
hormones are not stored in any significant amount, a continued rate
of synthesis is required to maintain a normal secretory rate. Once
in the circulation, cortisol or corticosterone are bound reversibly
to plasma proteins (such as transcortin, albumin). Under normal
conditions 10 percent of the glucocorticoids are in a free unbound
state.
Adrenal steroids are synthesized from cholesterol by specific
enzyme-catalyzed reactions and involve a complex shuttling of
steroid intermediates between mitochondria and endoplasmic reticulum. The specificity of mitochondrial hydroxylation reactions in
terms of precursor acted upon and the position of the substrate
which is hydroxylated is confined to a specific cytochrome P450.
The common biosynthetic pathway from cholesterol is the formation of pregnenolone, the basic precursor for the three major classes
of adrenal steroids. Pregnenolone is formed after two hydroxylation reactions at the carbon 20 and 22 positions of cholesterol and
a subsequent cleavage between these two carbon atoms. In the zona
fasciculata, pregnenolone is first converted to progesterone by two
microsomal enzymes. Three subsequent hydroxylation reactions
occur involving, in order, carbon atoms at the 17, 21, and 11 positions. The resulting steroid is cortisol, which is the major glucocorticoid in teleosts, hamsters, dogs, nonhuman primates, and humans. Corticosterone is the major glucocorticoid produced in
amphibians, reptiles, birds, rats, mice, and rabbits. It is produced
in a manner similar to the production of cortisol, except that progesterone does not undergo 17-hydroxylation and proceeds directly to 21-hydroxylation and 11
-hydroxylation.
In the zona glomerulosa, pregnenolone is converted to
aldosterone by a series of enzyme-catalyzed reactions similar to
those involved in cortisol formation; however, the cells of this zone
lack the 17-hydroxylase and thus cannot produce 17-hydroxyprogesterone, which is required to produce cortisol. Therefore,
the initial hydroxylation product is corticosterone. Some of the
corticosterone is acted on by 18-hydroxylase to form 18-hydroxycorticosterone, which in turn interacts with 18-hydroxysteroid dehydrogenase to form aldosterone. Since 18-hydroxysteroid dehydrogenase is found only in the zona glomerulosa, it is not surprising
that only this zone has the capacity to produce aldosterone. In addition to the aforementioned steroid hormones, cells in the zona
reticularis also produces small amounts of sex steroids including
progesterone, estrogens, and androgens.
The mineralocorticoids (e.g., aldosterone) are the major
steroids secreted from the zona glomerulosa under the control of
the renin–angiotensin II system. The mineralocorticoids have their
effects on ion transport by epithelial cells, particularly renal cells,
resulting in conservation of sodium (chloride and water) and loss
of potassium. In the distal convoluted tubule of the mammalian
nephron, a cation exchange exists that promotes the resorption of
sodium from the glomerular filtrate and the secretion of potassium
into the lumen.
Under conditions of decreased blood flow or volume, the enzyme renin is released into the circulation at an increased rate by
cells of the juxtaglomerular apparatus of the kidney. Renin release
has also been associated with potassium loading or sodium depletion. Renin in the peripheral circulation acts to cleave a plasma
globulin precursor (angiotensinogen produced by the liver) to
angiotensin I. An angiotensin converting enzyme (ACE), subsequently hydrolyzes angiotensin I to angiotensin II, which acts as a
717
Mechanisms of Toxicity
The reason the adrenal cortex is predisposed to the toxic effects of
xenobiotic chemicals appears to be related to at least two factors.
First, adrenal cortical cells of most animal species contain large
stores of lipids used primarily as substrate for steroidogenesis.
Many adrenal cortical toxic compounds are lipophilic and therefore can accumulate in these lipid-rich cells. Second, adrenal cortical cells have enzymes capable of metabolizing of xenobiotic
chemicals, including enzymes of the cytochrome P450 family.
Many of these enzymes function in the biosynthesis of endogenous
steroids and are localized in membranes of the endoplasmic reticulum or mitochondria. A number of toxic xenobiotic chemicals
serve as pseudosubstrates for these enzymes and can be metabolized to reactive toxic compounds. These reactive compounds result in direct toxic effects by covalent interactions with cellular
macromolecules or through oxygen activation with the generation
of free radicals.
Classes of chemicals known to be toxic for the adrenal cortex include short-chain (three- or four-carbon) aliphatic compounds, lipidosis-inducers, and amphiphilic compounds. A variety
of other compounds also may affect the medulla. The most potent
aliphatic compounds are of three-carbon length with electronegative groups at both ends. These compounds frequently produce
necrosis, particularly in the zonae fasciculata and reticularis. Examples include acrylonitrile, 3-aminopropionitrile, 3-bromopropionitrile, 1-butanethiol, and 1,4-butanedithiol. By comparison,
lipidosis-inducers can cause the accumulations, often coalescing,
of neutral fats, which may be of sufficient quantity to cause a reduction or loss of organellar function and eventual cell destruction.
Cholesterol is the precursor substrate required to synthesize steroid
hormones. Steroidogenic cells obtain cholesterol exogenously from
serum lipoproteins and endogenously from de novo synthesis via
the acetyl coenzyme A pathway (Fig. 21-5). The adrenal cortical
cells and OI cells in the rat preferentially utilize serum high-density
lipoproteins (HDLs) for their primary source of cholesterol and resort to de novo synthesis if HDL does not meet the demand of
steroidogenesis. This is in contrast to Leydig cells of the testis,
which preferentially utilizes de novo synthesis of cholesterol and
uses an exogenous source only when intracellular synthesis does
not meet the demand and the cholesterol pool has been depleted
(Payne et al., 1985).
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Lysosome
Endocytosis
SER
Acetyl CoA
Steroidogenesis
nCEH
Mitochondrion
Cholesterol
ACAT
nCEH
Lipid
Droplet
CE
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Nucleus
Figure 21-5. Cholesterol metabolism and steroid biosynthesis in adrenocortical and ovarian interstitial cells.
Cholesterol is the substrate for steroid biosynthesis. Conversion of cholesterol to pregnenolone occurs in the mitochondria, and oxidative reactions
catalyzed by P450 enzymes occur in the smooth endoplasmic reticulum
and mitochondria. Sources of cholesterol include lipoprotein uptake from
serum (LDL and HDL), de novo synthesis from acetate via the acetyl coenzyme A pathway, and hydrolysis of cholesteryl ester (CE) by neutral CE
hydrolase (nCEH). The storage pool in the form of lipid droplets is derived
principally from the conversion of free cholesterol to CE catalyzed by acyl
coenzyme A: cholesterol acyltransferase (ACAT). Direct uptake of CE from
serum to the storage pool is minimal in the rat. (From Latendresse et al.,
1993, with permission.)
The zonae reticularis and fasciculata appear to be the principal
targets of xenobiotic chemicals in the adrenal cortex. Examples of
the compounds causing lipidosis include aminoglutethimide, amphenone, and anilines. Tricresyl phosphate (TCP) and other triaryl
phosphates cause a defect in cholesterol metabolism by blocking
both the uptake from serum and storage pathways. An inhibition
of cytosolic neutral cholesteryl ester hydroxylase (nCEH) by triaryl
phosphate (97 percent inhibition compared to controls) results in
the progressive accumulation of cholesteryl ester in the form of
lipid droplets in the cytoplasm of adrenal cortical and ovarian interstitial (OI) cells (Fig. 21-6) but not in testicular Leydig cells of
rats (Latendresse et al., 1993). Acyl coenzyme A: cholesterol acyl
Lysosome
Endocytosis
SER
Acetyl CoA
Steroidogenesis
nCEH
Mitochondrion
Cholesterol
ACAT
nCEH
CE
Nucleus
Lipid
Droplet
CE
CE
Figure 21-6. Pathogenesis of cholesteryl lipidosis in adrenocortical cells
and ovarian interstitial cells.
The defect in cholesterol metabolism occurs in the uptake from serum and
storage pathways. An inhibition of neutral cholesteryl ester hydrolase
(nCEH) by a xenobiotic chemical results in the accumulation of CE in the
form of lipid droplets in the cytoplasm. Acyl coenzyme A:cholesterol acyltransferase (ACAT) (catalyzes the formation of CE from cholesterol) activity remains near normal levels. (From Latendresse et al., 1993, with
permission.)
Figure 21-7. The effects of tricresyl phosphate (TCP) and butylated triphenyl phosphate (BTH) on concentration of cholesteryl ester (CE) and
cholesterol in adrenal gland and ovary of rats.
Mean SEM (n 8–9)mg/g wet weight of adrenal gland and ovary.
***Different (p 0.01, p 0.05, respectively) from control. (From
Latendresse et al., 1993, with permission.)
transferase (ACAT), an enzyme that esterifies cholesterol to make
cholesteryl ester, was depressed only 27 percent (compared to controls) resulting in elevated intracellular storage of cholesterol in the
form of lipid droplets (Fig. 21-7).
Biologically active cationic amphiphilic compounds produce
a generalized phospholipidosis that involves primarily the zonae
reticularis and fasciculata and produce microscopic phospholipidrich inclusions. These compounds affect the functional integrity of
lysosomes, which appear ultrastructurally to be enlarged and filled
with membranous lamellae or myelin figures. Examples of compounds known to induce phospholipidosis include chloroquine, triparanol, and chlorphentermine.
Another class of compounds that affect the adrenal cortex are
hormones, particularly natural and synthetic steroids. The administration of exogenous steroid hormones such as corticosteroids
may cause functional inactivity and trophic atrophy following prolonged use. Other steroid hormones such as natural and synthetic
estrogens and androgens have been reported to cause proliferative
lesions in the adrenal cortex of laboratory animals.
In addition, there is a miscellaneous group of chemicals that
affect hydroxylation and other functions of mitochondrial and
microsomal fractions (e.g., smooth endoplasmic reticulum) in the
adrenal cortex. Examples of these compounds include o,p’-DDD
and -(1,4-dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone
(DMNM). Other compounds in this miscellaneous category cause
their effects by means of cytochrome P450 metabolism and the
production of toxic metabolites. A classic example is the activation of carbon tetrachloride, resulting in lipid peroxidation and covalent binding to cellular macromolecules of the adrenal cortex.
Many of the chemicals that cause morphologic changes in the
adrenal glands also affect cortical function. Chemically induced
changes in adrenal function result either from blockage of the action of adrenocorticoids at peripheral sites or by inhibition of synthesis and/or secretion of hormone. In the first mechanism, many
antisteroidal compounds (antagonists) act by competing with or
binding to steroid hormone–receptor sites; thereby, either reducing the number of available receptor sites or by altering their binding affinity. Cortexolone (11-deoxycortisol) an antiglucocorticoid
and spironolactone, an antimineralocorticoid, are two examples of
peripherally acting adrenal cortical hormone antagonists.
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Ultrastructural alterations of adrenal cortical cells associated
with chemical injury are quite diverse in nature. The zonae reticularis and fasciculata typically are most severely affected, although
eventually the lesions involve the zona glomerulosa. These lesions
may be classified as follows: endothelial damage (e.g., acrylonitrile), mitochondrial damage (e.g., DMNM, o,p’-DDD, amphenone), endoplasmic reticulum disruption (e.g., triparanol), lipid aggregation (e.g., aniline), lysosomal phospholipid aggregation (e.g.,
chlorophentermine), and secondary effects due to embolization by
medullary cells (e.g., acrylonitrile). Mitochondrial damage with
vacuolization and accompanying changes in the endoplasmic
reticulum and autophagocytic responses appear to be among the
most common ultrastructural changes observed following chemical injury in the adrenal cortex. Since mitochondria and smooth
endoplasmic reticulum form an intimate subcellular organellar network in cortical cells with important hydroxylases and dehydrogenase enzymes, it is not surprising that many chemical agents altering the ultrastructural morphology of cortical cells inhibit
steroidogenesis.
Chemically induced proliferative lesions of the adrenal cortex are less frequently reported and include hyperplasia, adenoma,
and carcinoma. Unlike the diffuse cortical hyperplasia/hypertrophy
associated with excess ACTH stimulation, chemically induced hyperplasia usually is nodular in type, often multiple in distribution,
and composed of increased numbers of normal or vacuolated
cortical cells.
A variety of different chemicals are associated with an increased incidence of adrenal cortical neoplasia. Most of the reported tumors tend to be benign (adenomas) although an occasional
tumor may be malignant (carcinomas). The zonae reticularis and
fasciculata are more prone to develop tumors following chemical
injury whereas the zona glomerulosa is spared unless invaded by
an expanding tumor in the adjacent zones of the cortex. The tumorigenic agents of the adrenal cortex have a diverse chemical
nature and use.
Spontaneous proliferative lesions may be found in all zones
of the adrenal cortex but in adult rats are found most frequently in
the zona fasciculata. Spontaneous nodular hyperplasia of the adrenal cortex is common in the rabbit, golden hamster, rat, mouse,
dog, cat, horse, and baboon. Naturally occurring adrenal cortical
tumors are found infrequently in domestic animals except adult
dogs and castrated male goats. However, cortical adenomas and (to
a lesser extent) cortical carcinomas have been reported in moderately high incidence in certain strains of laboratory hamsters (e.g.,
BIO 4.24 and BIO 45.5 strains) and rat (e.g., Osborne Mendel,
WAG/Rij, BUF, and BN/Bi strains). The incidence often increases
markedly in rats over 18 months of age. Adrenal cortical neoplasms
in mice are uncommon but the incidence may be increased by
gonadectomy.
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Xenobiotic chemicals affecting adrenal function often do so
by altering steroidogenesis and result in histologic and ultrastructural changes in adrenal cortical cells. For example, chemicals
causing increased lipid droplets often inhibit the utilization of
steroid precursors, including the conversion of cholesterol to
pregnenolone. Chemicals that affect the fine structure of mitochondria and smooth endoplasmic reticulum often impair the
activity of 11-, 17-, and 21-hydroxylases, respectively, and are
associated with lesions primarily in the zonae reticularis and fasciculata. Atrophy of the zona glomerulosa may reflect specific inhibition of aldosterone synthesis or secretion, either directly (e.g.,
inhibition of 18-hydroxylation) or indirectly (e.g., suppression of
the renin–angiotensin system II) by chemicals such as spironolactone and captopril.
It is well documented that synthetic and naturally occurring
corticosteroids are potent teratogens in laboratory animals. The
principal induced defect is cleft lip or palate; however, there is a
paucity of information on the direct effect of xenobiotic chemicals
on the development of the adrenal cortex. For example, adrenal
aplasia occurred in 7.6 of 9.8 percent of white Danish rabbits when
thalidomide was given to their dams.
719
Pathologic Alterations and Proliferative
Lesions in Cortical Cells
Macroscopic lesions of chemically affected adrenal glands are
characterized either by enlargement or reduction in size that often
is bilateral. Cortical hypertrophy due to impaired steroidogenesis
or hyperplasia due to long-term stimulation often is present when
the adrenal cortex is increased in size. Small adrenal glands often
are indicative of degenerative changes or trophic atrophy of the adrenal cortex. Midsagittal longitudinal sections of adrenal glands
under the above conditions will reveal either a disproportionately
wider cortex relative to the medulla or vice versa, resulting in an
abnormal cortical:medullary ratio. Nodular lesions that distort and
enlarge one or both adrenal glands suggest that a neoplasm is present. A single well-demarcated nodular lesion suggests a cortical
adenoma, whereas widespread incorporation of the entire adrenal
gland by a proliferative mass is suggestive of cortical carcinoma,
especially if there is evidence of local invasion into periadrenal
connective tissues or into adjacent blood vessels and the kidney.
Nonneoplastic lesions of the adrenal cortex induced by
xenobiotic chemicals are characterized by changes ranging from
acute progressive degeneration to reparative processes such as
multifocal hyperplasia. Early degenerative lesions characterized by
enlarged cortical cells filled with cytoplasmic vacuoles (often lipid)
may result in a diffuse hypertrophy of the cortex. A lesion of this
type has been observed in rats treated with an antibacterial compound -(1,4-dioxido-3-methylquinoxalin-2-yl)-N-methylntrone
(DMNM). This type of vacuolar degeneration is a reflection of impaired steroidogenesis resulting in an accumulation of steriod precursors. More destructive lesions such as hemorrhage and/or necrosis are associated with an inflammatory response in the cortex. If
the zona glomerulosa remains functional there will be no lifethreatening electrolyte disturbances and no signs of hypoadrenocorticism (Addison’s disease). While many chemical agents that
affect the adrenal cortex initially involve the zona reticularis and
inner zona fasciculata, certain chemicals such as DMNM can cause
a progressive degeneration of the entire adrenal cortex. Occasionally, a chemical’s effect is limited to a specific zone of the adrenal
cortex and may be species-specific.
ADRENAL MEDULLA
Normal Structure and Function
The medulla constitutes approximately 10 percent of the volume
of the adrenal gland. In the normal rodent adrenal gland and in
most other laboratory animal species, the medulla is sharply demarcated from the surrounding cortex. The bulk of the medulla is
composed of chromaffin cells, which are the sites of synthesis and
storage of the catecholamine hormones. In the rat and mouse, norepinephrine and epinephrine are stored in separate cell types that
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pituitary tumors, which occur commonly in many rat strains, also
play a role in the development of proliferative medullary lesions.
In addition, several neuroleptic compounds that increase prolactin
secretion by inhibiting dopamine production have been associated
with an increased incidence of proliferative lesions of medullary
cells in chronic toxicity studies in rats.
Both nicotine and reserpine have been implicated in the development of adrenal medullary proliferative lesions in rats. Both
agents act by a shared mechanism, since nicotine directly stimulates nicotinic acetylcholine receptors whereas reserpine causes a
reflex increase in the activity of cholinergic nerve endings in the
adrenal. A short dosing regimen of reserpine administration in vivo
stimulates proliferation of chromaffin cells in the adult rat, and the
mechanism may involve a reflex increase in neurogenic stimulation via the splanchnic nerve. Several other drugs have been reported to increase the incidence of adrenal medullary proliferative
lesions. These include zomepirac sodium (a nonsteroidal antiinflammatory drug), isoretinoin (a retinoid), and nafarelin (LHRH
analog), atenolol (beta-adrenergic blocker), terazosin (alphaadrenergic blocker), ribavirin (antiviral), and pamidronate (bisphosphonate) (Davies and Monro, 1995).
Lynch et al. (1996) have reported that nutritional factors have
an important modulating effect on the spontaneous incidence of
adrenal medullary proliferative lesions in rats. Several sugars and
sugar alcohols have produced adrenal medullary tumors at high
dosages (concentrations of 10 to 20 percent in the diet), including
xylitol, sorbitol, lacitol, and lactose. Although the exact mechanism involved is not completely understood, an important role for
calcium has been suggested. High doses of slowly absorbed sugars and starches increase the intestinal absorption and urinary excretion of calcium. Hypercalcemia is known to increase catecholamine synthesis in response to stress, and low-calcium diets
will reduce the incidence of adrenal medullary tumors in xylitoltreated rats. Other compounds that may act by a similar mechanism of altered calcium homeostasis include the retinoids (which
will produce hypercalcemia) and conditions such as progressive
nephrocalcinosis in aging male rats treated with nonsteroidal antiinflammatory agents.
Roe and Bar (1985) have suggested that environmental and
dietary factors may be more important than genetic factors as determinants of the incidence of adrenal medullary proliferative
lesions in rats. The incidence of adrenal medullary lesions can be
reduced by lowering the carbohydrate content of the diet. Several
of the agents that increase the incidence of adrenal medullary lesions, including sugar alcohols, increase absorption of calcium
from the gut. Calcium ions as well as cyclic nucleotides and
prostaglandins may act as mediators capable of stimulating both
hormonal secretion and cellular proliferation.
Tischler et al. (1999) recently presented data that vitamin D
is the most potent in vivo stimulus yet identified for chromaffin
cell proliferation in the adrenal medulla. Vitamin D3 (5000; 10,000;
or 20,000 IU/kg/day in corn oil) resulted in a four- to fivefold increase in bromodeoxyuridine (BrdU) labeling at week 4 that diminished to a twofold increase by week 26 (Fig. 21-8). An initial
preponderance of epinephrine-labeled (PNMT-positive cells) subsequently gave way to norepinephrine-labeled cells. By week 26,
a total of 89 percent of rats receiving the two highest doses of vitamin D3 had focal medullary proliferative lesions (BrdU-labeled
focal hyperplasia, or “hot spots”) and pheochromocytomas in contrast to absence of proliferative lesions in controls. This increase
in medullary cell proliferation was associated with a significant increase in circulating levels of both calcium and phosphorus after
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can be distinguished ultrastructurally after fixation in glutaraldehyde and postfixation in osmium tetroxide. The norepinephrinecontaining core of the secretory granules appears electron-dense
and is surrounded by a wide submembranous space, whereas
epinephrine-containing granules are less dense; they have a finely
granular core and a narrow space beneath the limiting membrane.
Granules of varying densities may be found in the same cell types
in the adrenal medulla of immature rats. Human adrenal medullary
cells may contain both norepinephrine and epinephrine within a
single chromaffin cell.
The adrenal medulla contains variable numbers of ganglion
cells in addition to chromaffin cells. A third cell type has been described in the medulla and designated the small granule–containing
(SGC) cell or small intensely fluorescent (SIF) cell. These cells
morphologically appear intermediate between chromaffin cells and
ganglion cells, and may function as interneurons. The adrenal
medullary cells also contain serotonin and histamine, but it has not
been determined if these products are synthesized in situ or taken
up from the circulation. A number of neuropeptides also are present in rat chromaffin cells, including enkephalins, neurotensin, and
neuropeptide Y.
In the catecholamine biosynthetic pathway, tyrosine is acted
on by tyrosine hydroxylase to produce dopa, which is converted to
dopamine by dopa decarboxylase. Dopamine in turn, is acted on
by dopamine beta-hydroxylase to form norepinephrine, which is
converted to epinephrine by phenylethanolamine-N-methyltransferase (PNMT). Tyrosine hydroxylase and PNMT are the principal rate-limiting steps in catecholamine synthesis. The conversion
of tyrosine into dopa and dopamine occurs within the cytosol of
chromaffin cells. Dopamine then enters the chromaffin granule,
where it is converted to norepinephrine. Norepinephrine leaves the
granule and is converted into epinephrine in the cytosol, and epinephrine reenters and is stored in the chromaffin granule. In contrast to the synthesis of catecholamines, which occurs in the cytosol, neuropeptides and chromogranin-A proteins are synthesized
in the granular endoplasmic reticulum and are packaged into granules in the Golgi apparatus.
Innervation plays an important role in regulating the functions
of chromaffin cells. During adult life, stresses such as insulininduced hypoglycemia or reserpine-induced depletion of catecholamines produces a reflex increase in splanchnic nerve discharge, resulting both in catecholamine secretion and transsynaptic
induction of catecholamine biosynthetic enzymes, including tyrosine hydroxylase. These effects become apparent during the first
week of life, following an increase in the number of nerve terminals in the adrenal medulla. Other environmental influences including growth factors, extracellular matrix, and a variety of hormonal signals that generate cyclic AMP also may regulate the
function of chromaffin cells.
Mechanisms of Toxicity
Proliferative lesions of the medulla, particularly in the rat, have
been reported to develop as a result of a variety of different mechanisms. Warren and coworkers (1966) studied over 700 pairs of
rats with parabiosis and found that more than 50 percent of male
irradiated rats developed adrenal medullary tumors. A relationship
exists between adenohypophyseal (anterior pituitary) hormones
and the development of adrenal medullary proliferative lesions. For
example, the long-term administration of growth hormone is associated with an increased incidence of pheochromocytomas as well
as the development of tumors at other sites. Prolactin-secreting
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721
Figure 21-8. Effects of vitamin D3 on percent of chromaffin cells labeled with BrdU during weeks 4, 8, 12,
and 26 of dietary supplementation.
Asterisks indicate statistically significant increases over corn oil controls. Numbers in parentheses indicate numbers of rats scored for each point. Premature deaths or euthanasia caused loss of three animals from the group
receiving 20,000 IU/kg/day, two from the group receiving 10,000 IU/kg/day, and one control. Histologic examination of these animals’ adrenal glands showed no detectable abnormalities. One extra animal at the start of
the experiment was assigned to the control group at week 1. At least 2500 chromaffin cells were scored for each
rat. (From Tischler et al., 1999, with permission.)
vitamin D administration (Fig. 21-9). The nuclei of hyperplastic
chromaffin cells labeled by BrdU but were phenylethanolamine-Nmethyl transferase-negative indicating they most likely were norepinephrine-producing cells of the rat medulla (Fig. 21-10). The
proliferative lesions usually were multicentric, bilateral, and peripheral in location in the medulla; nearly all were PNMT negative, and they appeared to represent a morphologic continuum
rather than separate entities. Earlier studies reported by the same
research group had demonstrated that the vitamin D3 (20,000;
40,000 IU/kg/day) increase in chromaffin cell proliferation was
observed as early as 1 week and had declined by 4 weeks. These
findings support the hypothesis that altered calcium homeostasis
is involved in the pathogenesis of pheochromocytomas in rodents,
most likely via effects on increasing chromaffin cell proliferation
(Fig. 21-11). Vitamin D3, calcitriol (active metabolite of D3), lactose, and xylitol all failed to stimulate directly the proliferation of
rat chromaffin cells in vitro.
In summary, three dietary factors have been suggested to lead
to an increased incidence of adrenal medullary proliferative lesions
in chronic toxicity studies in rats (Roe and Bar, 1985). These are
(1) excessive intake of food associated with feeding ad libitum; (2)
excessive intake of calcium and phosphorus, since commercial diets contain two to three times more calcium and phosphorus than
needed by young rats; and (3) excessive intake of other food components (e.g., vitamin D and poorly absorbable carbohydrates),
which increase calcium absorption.
Pathologic Alterations and Proliferative
Lesions in Medullary Cells
The adrenal medulla undergoes a series of proliferative changes
ranging from diffuse hyperplasia to benign and malignant neoplasia. The latter neoplasms have the capacity to invade locally and
to metastasize to distant sites. Diffuse hyperplasia is characterized
by symmetrical expansion of the medulla with maintenance of the
usual sharp demarcation between the cortex and the medulla. The
medullary cell cords often are widened, but the ratio of norepinephrine to epinephrine cells is similar to that of normal glands.
Focal hyperplastic lesions are often juxtacortical but may occur
within any area of the medulla. The small nodules of hyperplasia
in general are not associated with compression of the adjacent
medulla; however, the larger foci may be associated with limited
medullary compression. The foci of adrenal medullary hyperplasia are typically composed of small cells with round to ovoid nuclei and scanty cytoplasm. At the ultrastructural level, the cells
composing these focal areas of hyperplasia contain small numbers
of dense core secretory granules resembling the granules of SIF or
SGC cells.
Larger benign adrenal medullary proliferative lesions are designated as pheochromocytomas. These lesions may be composed
of relatively small cells similar to those found in smaller hyperplastic foci or larger chromaffin cells or a mixture of small and
large cells. According to some authors, the lack of a positive chromaffin reaction in these focal proliferative lesions precludes the diagnosis of pheochromocytoma; however, the chromaffin reaction
is quite insensitive and catecholamines (particularly norepinephrine) can be demonstrated in these proliferative lesions by biochemical extraction studies and by the formaldehyde- or glyoxylicacid-induced fluorescence methods. Even in some of the larger
medullary lesions, the chromaffin reaction is equivocal but catecholamines can be demonstrated both biochemically and histochemically. Malignant pheochromocytomas invade the adrenal capsule and often grow in the periadrenal connective tissues with or
without distant metastases.
Proliferative lesions occur with high frequency in many strains
of laboratory rats. The incidence of these lesions varies with strain,
age, sex, diet, and exposure to drugs, and a variety of environmental
agents. Studies from the NTP historical data base of 2-year-old
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Figure 21-10. Photomicrographs of adrenal medullary sections stained
for BrdU (dark nuclei) and PNMT (dark cytoplasm) to compare BrdU
labeling of epinephrine (E) and norepinephrine (NE) cells.
Vitamin D3 (20,000 IU/kg/day) caused a dramatic increase in BrdU labeling of predominantly E cells at week 4. The response was greatly reduced
at week 26 and there was no longer an E-cell predominance. A representative hyperplasic nodule in the vitamin D3 –stimulated adrenal medulla at
week 26 (top left) is densely labeled with BrdU and is PNMT-negative
(bar 100 m). (From Tischler et al., 1999, with permission.)
Figure 21-9. Effects of vitamin D3 on serum calcium and phosphorus
levels at weeks 4, 8, 12, and 26.
Sustained perturbation of both Ca2 and PO4 concentrations persisted
throughout the time course. (From Tischler et al., 1999, with permission.)
F344 rats have reported that the incidence of pheochromocytomas
was 17.0 percent and 3.1 percent for males and females, respectively. Malignant pheochromocytomas were detected in 1 percent
of males and 0.5 percent of females. In addition to F344 rats, other
strains with a high incidence of pheochromocytomas include
Wistar, NEDH (New England Deaconess Hospital), Long-Evans,
and Sprague-Dawley. Pheochromocytomas are considerably less
common in Osborne-Mendel, Charles River, Holtzman, and
WAG/Rij rats. Most studies have revealed a higher incidence in
males than in females (Figs. 21-12 and 21-13). Crossbreeding of
animals with high and low frequencies of adrenal medullary proliferative lesions results in F1 animals with an intermediate tumor
frequency. Pheochromocytomas are less common in the mouse than
in most strains of rats.
There is a conspicuous relationship between increasing age
and the frequency, size, and bilateral occurrence of adrenal
medullary proliferative lesions in the rat. In the Long-Evans strain,
medullary nodules have been found in less than 1 percent of animals under twelve months of age. The frequency increases to almost 20 percent in 2-year-old animals and to 40 percent in animals
Figure 21-11. Pathogenesis of adrenal medullary proliferative lesions associated with ingestion of polyols resulting in elevation of the blood calcium concentration. (Modified from Lynch et al., 1996, with permission.)
Figure 21-12. Rat strains with a high incidence of pheochromocytomas.
(Modified from Lynch et al., 1996, with permission.)
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Figure 21-13. Rat strains with a low incidence of pheochromocytomas.
(Modified from Lynch et al., 1996, with permission.)
Figure 21-15. Species differences in mitogenic responses of chromaffin
cells in vitro from adult rat, human, bovine, and mouse adrenals. (From
Tischler and Riseberg, 1993, with permission.)
between 2 and 3 years of age. The mean tumor size increases progressively with age, as does the frequency of bilateral and multicentric occurrence.
A variety of techniques may be used for the demonstration of
catecholamines in tissue sections. The chromaffin reaction is the
oxidation of catecholamines by potassium dichromate solutions
and results in the formation of a brown-to-yellow pigment in
medullary tissue. The chromaffin reaction as traditionally performed possesses a low level of sensitivity and should not be used
for the definitive demonstration of the presence of catecholamines
in tissues. Similarly, both the argentaffin and argyrophil reactions,
which have been used extensively in the past for the demonstration of chromaffin cells, also possess low sensitivity and specificity.
Fluorescence techniques using formaldehyde or glyoxylic acid represent the methods of choice for the demonstration of catecholamines at the cellular level. These aldehydes form highly fluorescent derivatives with catecholamines, which can be visualized
by ultraviolet microscopy. Immunohistochemistry provides an alternative approach for the localization of catecholamines in chromaffin cells and other cell types. Antibodies are available that permit epinephrine- and norepinephrine-containing cells to be
distinguished in routinely fixed and embedded tissue samples. Several of the important enzymes involved in the biosynthesis of catecholamine hormones also may be demonstrated by immunohistochemical procedures. Antibodies to chromogranin-A can be used
for the demonstration of this unique protein in chromaffin cells.
Pheochromocytomas in rats and human beings are both composed of chromaffin cells with variable numbers of hormonecontaining secretory granules (Fig. 21-14). The incidence is high
in many strains of rats by comparison to human patients, where
pheochromocytomas are uncommon except in patients with inherited clinical syndromes of multiple endocrine neoplasia (MEN).
These tumors in rats usually do not secrete excess amounts of catecholamines, whereas human pheochromocytomas episodically secrete increased amounts of catecholamines leading to hypertension
and other clinical disturbances. There appears to be a striking
species difference in the response of medullary chromaffin cells to
mitogenic stimuli with rats being very sensitive compared to humans (Fig. 21-14).
Tischler and Riseberg (1993) reported that adult rat chromaffin cells had a marked increase (from not more than 10 to 40 percent) in bromodeoxyuridine (BrdU)–labeled nuclei in vitro
following the addition of the following mitogens: nerve growth
factor (NGF), fibroblast growth factor (FGF), forskolin, and phorbol myristate (PMA), whereas human chromaffin cells had a minimal (0.1 percent) response to the same mitogens (Fig. 21-15).
This striking difference in sensitivity to mitogenic stimuli may
explain the lower frequency of adrenal medullary proliferative
lesions in humans compared to many rat strains (Tischler and
Riseberg, 1993). The mouse adrenal medulla, which, as in humans,
has a low spontaneous incidence of proliferative lesions of chromaffin cells, also failed to respond to a variety of mitogenic stimuli (Fig. 21-15). These findings and others suggest that chromaffin cells of the rat represent an inappropriate model to assess the
potential effects of xenobiotic chemicals on chromaffin cells of the
human adrenal medulla (Lynch et al., 1996).
THYROID GLAND (FOLLICULAR
CELLS)
Species Differences in Thyroid
Hormone Economy
Figure 21-14. Characteristics of pheochromocytomas in rats compared
to humans. (Modified from Lynch et al., 1996, with permission.)
Long-term perturbations of the pituitary-thyroid axis by various
xenobiotics or physiologic alterations (e.g., iodine deficiency, partial thyroidectomy, and natural goitrogens in food) are more likely
to predispose the laboratory rat to a higher incidence of proliferative lesions [e.g., hyperplasia and benign tumors (adenomas) of fol-
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Table 21-1
Thyroxine (T4) Binding to Serum Proteins in Selected Vertebrate Species
T4 BINDING
GLOBULIN
POSTALBUMIN
ALBUMIN
PREALBUMIN
Human being
Monkey
Dog
Mouse
Rat
Chicken
*
*
—*
—
—
—
—
—
—
—
—
—
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SPECIES
KEY:
, , Degree of T4 binding to serum proteins; —, absence of binding of T4 to serum proteins.
Döhler et al., 1979, with permission.
SOURCE:
licular cells] in response to chronic TSH stimulation than in the
human thyroid (Capen and Martin, 1989; Curran and DeGroot,
1991). This is particularly true in the male rat which has 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 halflife of thyroxine T4 in rats than in humans due to the considerable
differences in the transport proteins for thyroid hormones between
these species (Döhler et al., 1979).
The plasma T4 half-life in rats is considerably shorter (12 to
24 h) than in humans (5 to 9 days). In human beings and monkeys
circulating T4 is bound primarily to thyroxine-binding globulin
(TBG), but this high-affinity binding protein is not present in rodents, birds, amphibians, or fish (Table 21-1).
The binding affinity of TBG for T4 is approximately a thousand times higher than for prealbumin. The percent 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 prealbumin. Therefore, a rat without a functional thyroid requires about 10 times more
T4 (20 g/kg body weight) for full substitution than an adult human (2.2 g/kg body weight). Triiodothyronine (T3) is transported
bound to TBG and albumin and transthyretin in the human being,
monkey, and dog but only to albumin in the mouse, rat, and chicken
(Table 21-2). In general, T3 is bound less avidly to transport proteins than T4, resulting in a faster turnover and shorter plasma halflife in most species. These differences in plasma half-life of thyroid hormones and binding to transport proteins between rats and
human beings may be one factor in the greater sensitivity of the
rat thyroid to developing hyperplastic and/or neoplastic nodules in
response to chronic TSH stimulation.
Thyroid-stimulating hormone levels are higher in male than
female rats and castration decreases both the baseline serum TSH
and response to thyrotropin-releasing hormone (TRH) injection.
Follicular cell height often is higher in male than in female rats in
response to the greater circulating TSH levels. The administration
of exogenous testosterone to castrated male rats restores the TSH
level to that of intact rats. Malignant thyroid tumors (carcinomas
or “cancer”) develop at a higher incidence following irradiation in
males than females (2:1) and castration of irradiated male rats decreases the incidence to that of intact irradiated female rats. Testosterone replacement to castrated male rats restores the incidence of
irradiation-induced thyroid carcinomas in proportion to the dose of
testosterone and, similarly, serum TSH levels increase proportionally to the dose of replacement hormone. Likewise, higher incidence of follicular cell hyperplasia and neoplasia has been reported
in males compared to female rats following the administration of
a wide variety of drugs and chemicals in chronic toxicity/carcinogenicity testing.
There also are marked species differences in the sensitivity of
the functionally important peroxidase enzyme to inhibition by
xenobiotics. Thioamides (e.g., sulfonamides) and other chemicals
can selectively inhibit the thyroperoxidase and significantly interfere with the iodination of tyrosyl residues incorporated in the thyroglobulin molecule, thereby disrupting the orderly synthesis of T4
Table 21-2
Triiodothyronine (T3) Binding to Serum Proteins in Selected Vertebrate Species
T4 BINDING
SPECIES
GLOBULIN
POSTALBUMIN
ALBUMIN
PREALBUMIN
Human being
Monkey
Dog
Mouse
Rat
Chicken
—
—
—
—
—
—
—
—
—
—
—
—
—
—
KEY:
, T3 binding to serum proteins; —, absence of binding of T3 to serum proteins.
Döhler et al., 1979, with permission.
SOURCE:
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and T3. A number of studies have shown that the long-term administration of sulfonamides results in the development of thyroid
nodules frequently in the sensitive species (such as the rat, dog,
and mouse) but not in species resistant (e.g., monkey, guinea pig,
chicken, and human beings) to the inhibition of peroxidase in follicular cells.
Mechanisms of Thyroid Tumorigenesis
secreting pituitary tumor transplants (Furth, 1954). The pathogenetic mechanism of thyroid follicular cell tumor development in
rodents involves a sustained excessive stimulation of the thyroid
gland by TSH. In addition, iodine deficiency is a potent promoter
of the development of thyroid tumors in rodents induced by intravenous injection of N-methyl-N-nitrosurea (Fig. 21-16) (Ohshima
and Ward, 1984). The subsequent parts of thyroid section discuss
specific mechanisms by which xenobiotic chemicals disrupt thyroid hormone synthesis and secretion, induce hepatic microsomal
enzymes that enhance thyroid hormone catabolism or inhibit enzymes involved in monodeiodination in peripheral tissues that result in perturbations of thyroid hormone economy which in rodents
predisposes to the development of follicular cell tumors in chronic
studies.
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Numerous studies have reported that chronic treatment of rodents
with goitrogenic compounds results in the development of follicular cell adenomas. Thiouracil and its derivatives have this effect
in rats (Napolkov, 1976) and mice (Morris, 1955). This phenomenon also has been observed in rats that consumed brassica seeds
(Kennedy and Purves, 1941), erythrosine (FD&C Red No. 3)
(Capen and Martin, 1989; Borzelleca et al., 1987), sulfonamides
(Swarm et al., 1973), and many other compounds (Hill et al., 1989;
Paynter et al., 1988). The pathogenetic mechanism of this phenomenon has been understood for some time (Furth, 1954) and are
widely accepted by the scientific community. These goitrogenic
agents either directly interfere with thyroid hormone synthesis or
secretion in the thyroid gland, increase thyroid hormone catabolism and subsequent excretion into the bile, or disrupt the peripheral conversion of thyroxine (T4) to triiodothyronine (T3). The
ensuing decrease in circulating thyroid hormone levels results in a
compensatory increased secretion of pituitary thyroid stimulating
hormone (TSH). The receptor-mediated TSH stimulation of the
thyroid gland leads to proliferative changes of follicular cells that
include hypertrophy, hyperplasia, and ultimately, neoplasia in
rodents.
Excessive secretion of TSH alone (i.e., in the absence of any
chemical exposure) also has been reported to produce a high incidence of thyroid tumors in rodents (Ohshima and Ward, 1984,
1986). This has been observed in rats fed an iodine-deficient diet
(Axelrod and Leblond, 1955) and in mice that received TSH-
725
Chemicals that Directly Inhibit Thyroid
Hormone Synthesis
Blockage of Iodine Uptake The biosynthesis of thyroid hormones is 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 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. 21-17). The
mechanism of active transport of iodide has been shown to be
associated with a sodium-iodide (Na-I) symporter (NIS)
present in the basolateral membrane of thyroid follicular cells.
Transport of iodide ion across the thyroid cell membrane is linked
to the transport of Na. The ion gradient generated by the Na,
K-ATPase appears to be the driving force for the active cotransport of iodide. The transporter protein is present in the basolateral membrane of thyroid follicular cells (thyrocytes) and is a
Figure 21-16. Data demonstrating the potent promoting effects of iodine deficiency (ID) in rats administered
a single intravenous dose of a known initiator [N nitrosomethylurea (NMU)] of thyroid neoplasms. (From
Ohshima and Ward, 1984, with permission.)
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Blockage of the iodide trapping mechanism has a disruptive effect
on the thyroid-pituitary axis, similar to iodine deficiency. The blood
levels of T4 and T3 decrease, resulting in a compensatory increase
in the secretion of TSH by the pituitary gland. The hypertrophy
and hyperplasia of follicular cells following sustained exposure results in an increased thyroid weight and the development of goiter.
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Inhibition of Thyroid Peroxidase Resulting in an Organification Defect A wide variety of chemicals, drugs, and other
xenobiotics affect the second step in thyroid hormone biosynthesis (Fig. 21-17). The stepwise binding of iodide to the tyrosyl
residues in thyroglobulin requires oxidation of inorganic iodide (I2)
to molecular (reactive) iodine (I2) by the thyroid peroxidase present in the luminal aspect (microvillar membranes) of follicular cells
and adjacent colloid. Classes of chemicals that inhibit the organification of thyroglobulin include (1) the thionamides (such as
thiourea, thiouracil, propylthiouracil, methimazole, carbimazole,
and goitrin); (2) aniline derivatives and related compounds (e.g.,
sulfonamides, paraaminobenzoic acid, paraaminosalicylic acid, and
amphenone); (3) substituted phenols (such as resorcinol, phloroglucinol, and 2,4-dihydroxybenzoic acid); and (4) miscellaneous inhibitors [e.g., aminotriazole, tricyanoaminopropene, antipyrine,
and its iodinated derivative (iodopyrine)] (Fig. 21-18).
Many of these chemicals exert their action by inhibiting the
thyroid peroxidase, which results in a disruption both of the
iodination of tyrosyl residues in thyroglobulin and also the
coupling reaction of iodotyrosines [e.g., monoiodothyronine (MIT)
and di-iodothyronine (DIT)] to form iodothyronines (T3 and T4)
(Fig. 21-19). Propylthiouracil (PTU) has been shown to affect each
step in thyroid hormone synthesis beyond iodide transport in rats.
The order of susceptibility to the inhibition by PTU is the coupling
reaction (most susceptible), iodination of MIT to form DIT, and
iodination of tyrosyl residues to form MIT (least susceptible).
Thiourea differs from PTU and other thioamides in that it neither
inhibits guaiacol oxidation (the standard assay for peroxidase) nor
inactivates the thyroid peroxidase in the absence of iodine. Its ability to inhibit organic iodinations is due primarily to the reversible
reduction of active I2 to 2I.
The goitrogenic effects of sulfonamides have been known for
more than fifty years, since the reports of the action of sulfaguanidine on the rat thyroid. Sulfamethoxazole and trimethroprim exert
a potent goitrogenic effect in rats, resulting in marked decreases in
circulating T3 and T4, a substantial compensatory increase in TSH,
and increased thyroid weights due to follicular cell hyperplasia.
Figure 21-17. Mechanism of action of goitrogenic chemicals on thyroid
hormone synthesis and secretion. (From Capen and Martin, 1989, with
permission.)
large protein containing 643 amino acids with 13 transmembrane
domains.
Immunohistochemical staining using a polyclonal antibody
against the human NIS fusion protein (hNIS) revealed that expression of the protein is heterogenous in the normal human thyroid and detected only in occasional thyrocytes of a follicle (Jhiang
et al., 1998). The hNIS-positive thyrocytes usually were detected
in small follicles composed of cuboidal to columnar cells but
rarely were detected in large follicles composed of flattened thyrocytes. The heterogeneity of hNIS expression among thyroid follicles is consistent with the finding that iodide concentrating ability also varies between follicles in the thyroid gland (Spitzweg et
al., 2000).
Other tissues such as the salivary gland, gastric mucosa,
choroid plexus, ciliary body of the eye, and lactating mammary
gland also have the capacity to actively transport iodide, albeit at
a much lower level than the thyroid. In the salivary glands the hNIS
protein was detected in ductal cells but not in acinar cells. Only
the thyroid follicular cells accumulate iodide in a TSH-dependent
manner. The NIS gene is complex (15 exons, 14 introns) and its
expression in the thyroid is up-regulated by TSH. The functionally
active iodine transport system in the thyroid gland has important
clinical applications in the evaluation, diagnosis, and treatment of
several thyroid disorders, including cancer. The NIS and active
transport of iodide can be selectively inhibited by competitive anion inhibitors, thereby effectively blocking the ability of the gland
to iodinate tyrosine residues in thyroglobulin and synthesize thyroid hormones.
The initial step in the biosynthesis of thyroid hormones is the
uptake of iodide from the circulation and transport against a gradient across follicular cells to the lumen of the follicle. A number
of anions act as competitive inhibitors of iodide transport in the
thyroid, including perchlorate (ClO4), thiocyanate (SCN), and
pertechnetate (Fig. 21-17). Thiocyanate is a potent inhibitor of iodide transport and is a competitive substrate for the thyroid peroxidase, but it does not appear to be concentrated in the thyroid.
Figure 21-18. Chemicals disrupting thyroid function (decreased synthesis of thyroid hormones) by inhibiting thyroperoxidase.
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fluorescent dye] and a twofold increase in apoptosis in both treated groups. Therefore, inhibition of GJIC by PTU or a low-iodine
diet may result in increased thyroid follicular cell proliferation,
similar to other tissues, possibly by disrupting the passage of regulatory substance(s) through these highly permeable intercellular channels.
A contemporary example of a chemical acting as a thyroperoxidase inhibitor is sulfamethazine. This is a widely used antibacterial compound in food-producing animals with a current permissible tissue residue level of 100 ppb. Recently completed
carcinogenicity studies at NCTR reported a significant increase of
thyroid tumors in male Fischer 344 rats administered the high dose
(2400 ppm) of sulfamethazine (McClain, 1995). The incidence of
thyroid tumors was increased in both male and female B6C3F1 mice
after two years in the high-dose (4800 ppm) group but not in the
lower-dose groups. Quantitative risk assessment based upon these
new carcinogenicity findings, using low-dose linear extrapolation,
yielded a 1 106 lifetime risk of 90 ppb in female rats and 40
ppb in male rats. A consideration of the ratio of intact drug to
metabolites further reduced the tissue residue level to 0.4 ppb,
which would be unachievable in practice (McClain, 1995).
A number of mechanistic studies have been performed in collaboration with Dr. McClain and others with the objective of developing a database that would support the hypothesis that the thyroid tumors observed in rats and mice from chronic studies were
secondary to hormonal imbalances following the administration of
high doses of sulfamethazine. In a 4-week mechanistic study, the
effects of 10 dose levels (0 12,000 ppm) of sulfamethazine, spanning the range that induced thyroid tumors in rodents, were evaluated on thyroid hormone economy in Sprague-Dawley rats. There
was a characteristic log-dose response relationship in all parameters of thyroid function evaluated. There were no significant
changes at the six lower doses (20 800 ppm) of sulfamethazine,
followed by sharp relatively linear changes at the four higher dose
levels (1600 12,000 ppm) in percent decrease of serum T3 and
T4, increase in serum TSH, and increase in thyroid weight. A
similar, nonlinear dose response was present in the morphologic
changes of thyroid follicular cells following the feeding of varying
levels of sulfamethazine. Follicular cell hypertrophy was observed
at lower doses of sulfamethazine than hyperplasia, which was increased only at dose levels of 3300 ppm and above (Fig. 21-21).
Other mode-of-action studies have demonstrated sulfamethazine to be a potent inhibitor of thyroperoxidase in rodents with a
IC50 of 1.2 106 M. The morphologic effects on the thyroid
Figure 21-19. Mechanisms by which xenobiotic chemicals decrease thyroid hormone synthesis by inhibiting thyroperoxidase (TPO) in follicular cells.
The dog also is a species sensitive to the effects of sulfonamides,
resulting in markedly decreased serum T4 and T3 levels, hyperplasia of thyrotrophic basophils in the pituitary gland, and increased
thyroid weights.
By comparison, the thyroids of monkeys and human beings
are resistant to the development of changes that sulfonamides
produce in rodents (rats and mice) and the dog. Rhesus monkeys
treated for 52 weeks with sulfamethoxazole (doses up to
300 mg/kg/day) with and without trimethroprim had no changes
in thyroid weights and the thyroid histology was normal. Takayama
and coworkers (1986) compared the effects of PTU and a goitrogenic sulfonamide (sulfamonomethoxine) on the activity of thyroid
peroxidase in the rat and monkey using the guaiacol peroxidation
assay. The concentration required for a 50 percent inhibition of the
peroxidase enzyme was designated as the inhibition constant50
(IC50). When the IC50 for PTU was set at 1 for rats it took 50 times
the concentration of PTU to produce a comparable inhibition in
the monkey. Sulfamonomethoxine was almost as potent as PTU in
inhibiting the peroxidase in rats. However, it required about 500
times the concentration of sulfonamide to inhibit the peroxidase in
the monkey compared to the rat. Studies such as these with sulfonamides demonstrate distinct species differences between rodents and primates in the response of the thyroid to chemical
inhibition of hormone synthesis. It is not surprising that the sensitive species (e.g., rats, mice, and dogs) are much more likely to
develop follicular cell hyperplasia and thyroid nodules after longterm exposure to sulfonamides than the resistant species (e.g.,
subhuman primates, human beings, guinea pigs, and chickens)
(Fig. 21-20).
Recent evidence suggests that propylthiouracil (PTU) or feeding a low-iodine diet markedly increase thyroid follicular cell proliferation in rats by disrupting the movement of low-molecularweight ions and molecules through gap junctions (Kolaja et al.,
2000). Inhibition of gap-junction intercellular communication
(GJIC) prior to induction of cell proliferation has been reported
with several tumor promoters and in proliferative diseases. After
14 days of either PTU or a low iodine diet (plus 1 percent KClO4
in water) serum T3 and T4 were decreased to undetectable levels,
serum TSH was increased significantly and thyroid follicular cell
proliferation was increased nearly threefold. This was accompanied by a 30 to 35 percent decrease in GJIC [determined by an ex
vivo method with Lucifer yellow—a low-molecular-weight (457)
Figure 21-20. Variable species sensitivity of thyroperoxidase (TPO) inhibition by sulfonamides. (From Takayama et al., 1986, with permission.)
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728
Figure 21-21. Nonlinear dose-response in morphologic changes in thyroid follicular cells in response to 10
dose levels of sulfamethazine administered in the feed to male Sprague-Dawley rats. (From Capen, 1997, with
permission.)
were reversible after withdrawal of compound and addition of supplemental T4 to the diet inhibited the development of the functional
and morphologic changes in thyroid follicular cells (McClain,
1995). Hypophysectomized rats (with no TSH) administered sulfamethazine did not develop morphologic changes in the thyroid.
Sulfamethazine did not increase thyroid cell proliferation in vitro
in the absence of TSH and there was no effect on thyroid structure/function in cynomolgus monkeys administered sulfamethazine. Nonhuman primates and human beings are known to be more
resistant than rodents to the inhibition of thyroperoxidase.
Chemicals that Disrupt Thyroid
Hormone Secretion
Blockage of Thyroid Hormone Release by Excess Iodide and
Lithium Relatively few chemicals selectively inhibit the secretion of thyroid hormone from the thyroid gland (Fig. 21-17). An
excess of iodine has been known for years to inhibit secretion of
thyroid hormone and occasionally can result in goiter and subnormal function (hypothyroidism) in animals and human patients.
High doses of iodide have been used therapeutically in the treatment of patients with Graves’ disease and hyperthyroidism to lower
circulating levels of thyroid hormones. Several mechanisms have
been suggested for this effect of high iodide levels on thyroid hormone secretion, including a decrease in lysosomal protease activity (in human glands), inhibition of colloid droplet formation (in
mice and rats), and inhibition of TSH-mediated increase in cAMP
(in dog thyroid slices). Studies in my laboratory demonstrated that
rats fed an iodide-excessive diet had a hypertrophy of the cytoplasmic area of follicular cells with an accumulation of numerous
colloid droplets and lysosomal bodies (Collins and Capen, 1980).
However, there was limited evidence ultrastructurally of the fusion
of the membranes of these organelles and of the degradation of the
colloid necessary for the release of active thyroid hormones (T4
and T3) from the thyroglobulin. Circulating levels of T4, T3, and
rT3 all would be decreased by an iodide-excess in rats.
Lithium also has been reported to have a striking inhibitory
effect on thyroid hormone release (Fig. 21-17). The widespread use
of lithium carbonate in the treatment of manic states occasionally
results in the development of goiter with either euthyroidism or occasionally hypothyroidism in human patients. Lithium inhibits colloid droplet formation stimulated by cAMP in vitro and inhibits
the release of thyroid hormones.
Xenobiotic-Induced Thyroid Pigmentation or Alterations in
Colloid The antibiotic minocycline produces a striking black discoloration of the thyroid lobes in laboratory animals and humans
with the formation of brown pigment granules within follicular
cells. The pigment granules stain similarly to melanin and are best
visualized on thyroid sections stained with the Fontana-Masson
procedure. Electron-dense material first accumulates in lysosomelike granules and in the rough endoplasmic reticulum. The pigment
appears to be a metabolic derivative of minocycline and the administration of the antibiotic at high dose to rats for extended periods may result in a disruption of thyroid function and the development of goiter. The release of T4 from perfused thyroids of
minocycline-treated rats was significantly decreased but the follicular cells retained the ability to phagocytose colloid in response to
TSH and had numerous colloid droplets in their cytoplasm.
Other xenobiotics [or metabolite(s)] selectively localize in the
thyroid colloid of rodents resulting in abnormal clumping and increased basophilia to the colloid. Brown to black pigment granules
may be present in follicular cells, colloid, and macrophages in the
interthyroidal tissues resulting in a macroscopic darkening of both
thyroid lobes. The physiochemically altered colloid in the lumen
of thyroid follicles appears to be less able than normal colloid either of reacting with organic iodine in a stepwise manner to result
in the orderly synthesis of iodothyronines or being phagocytized
by follicular cells and enzymatically processed to release active
thyroid hormones into the circulation. Serum T4 and T3 are decreased, serum TSH levels are increased by an expanded population of pituitary thyrotrophs, and thyroid follicular cells undergo
hypertrophy and hyperplasia. As would be expected, the incidence
of thyroid follicular cell tumors in 2-year carcinogenicity studies
is significantly increased at the higher dose levels, usually with a
greater effect in males than females. Autoradiographic studies of-
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Xenobiotics that induce liver microsomal enzymes and disrupt thyroid function in rats include CNS-acting drugs (e.g., phenobarbital, benzodiazepines); calcium channel blockers (e.g.,
nicardipine, bepridil); steroids (spironolactone); retinoids; chlorinated hydrocarbons (e.g., chlordane, DDT, TCDD), polyhalogenated biphenyls (PCB, PBB), among others. Most of the hepatic
microsomal enzyme inducers have no apparent intrinsic carcinogenic activity and produce little or no mutagenicity or DNA damage. Their promoting effect on thyroid tumors usually is greater in
rats than in mice, with males more often developing a higher incidence of tumors than females. In certain strains of mice these
compounds alter liver cell turnover and promote the development
of hepatic tumors from spontaneously initiated hepatocytes.
Phenobarbital has been studied extensively as the prototype
for hepatic microsomal inducers that increase a spectrum of cytochrome P450 isoenzymes (McClain et al., 1988). McClain and
associates (1989) reported that the activity of uridine diphosphate
glucuronyltransferase (UDP-GT), the rate limiting enzyme in T4
metabolism, is increased in purified hepatic microsomes of male
rats when expressed as picomoles/min/mg microsomal protein (1.3fold) or as total hepatic activity (threefold). This resulted in a significantly higher cumulative (4-h) biliary excretion of 125I-labeled
T4 and bile flow than in controls.
Phenobarbital-treated rats develop a characteristic pattern of
changes in circulating thyroid hormone levels (McClain et al.,
1988, 1989). Plasma T3 and T4 are markedly decreased after 1 week
and remain decreased for 4 weeks. By 8 weeks T3 levels return to
near normal due to compensation by the hypothalamic-pituitarythyroid axis. Serum TSH values are elevated significantly throughout the first month but often decline after a new steady state is attained. Thyroid weights increase significantly after 2 to 4 weeks
of phenobarbital, reach a maximum increase of 40 to 50 percent
by 8 weeks, and remain elevated throughout the period of treatment.
McClain and coworkers (1988) in a series of experiments have
shown that supplemental administration of thyroxine (at doses that
returned the plasma level of TSH to the normal range) blocked the
thyroid tumor-promoting effects of phenobarbital and that the promoting effects were directly proportional to the level of plasma
TSH in rats. The sustained increase in circulating TSH levels results initially in hypertrophy of follicular cells, followed by hyperplasia, and ultimately places the rat thyroid at greater risk to
develop an increased incidence of benign tumors.
Phenobarbital has been reported to be a thyroid gland tumor
promoter in a rat initiation-promotion model. Treatment with a nitrosamine followed by phenobarbital has been shown to increase
serum TSH concentrations, thyroid gland weights, and the incidence of follicular cell tumors in the thyroid gland (McClain 1988;
McClain et al., 1989). These effects could be decreased in a doserelated manner by simultaneous treatment with increasing doses of
exogenous thyroxine. McClain et al. (1989) have demonstrated that
rats treated with phenobarbital have a significantly higher
cumulative biliary excretion of 125I-labeled thyroxine than controls
(Fig. 21-23). Most of the increase in biliary excretion was accounted for by an increase in T4-glucuronide due to an increased
metabolism of thyroxine in phenobarbital-treated rats. This is consistent with enzymatic activity measurements which result in increased hepatic T4-UDP-glucuronyl transferase activity in phenobarbital-treated rats (Fig. 21-24). Results from these experiments
are consistent with the hypothesis that the promotion of thyroid tumors in rats was not a direct effect of phenobarbital on the thyroid
gland but rather an indirect effect mediated by TSH secretion from
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ten demonstrate tritiated material to be preferentially localized in
the colloid and not within follicular cells. Tissue distribution studies with 14C-labeled compound may reveal preferential uptake and
persistence in the thyroid gland compared to other tissues. However, thyroperoxidase activity is normal and the thyroid’s ability to
take up radioactive iodine often is increased compared to controls
in response to the greater circulating levels of TSH. Similar thyroid changes and/or functional alterations usually do not occur in
dogs, monkeys, or humans.
Hepatic Microsomal Enzyme Induction
Hepatic microsomal enzymes play an important role in thyroid hormone economy because glucuronidation is the rate-limiting step in
the biliary excretion of T4 and sulfation primarily by phenol sulfotransferase for the excretion of T3. Long-term exposure of rats
to a wide variety of different chemicals may induce these enzyme
pathways and result in chronic stimulation of the thyroid by
disrupting the hypothalamic-pituitary-thyroid axis (Curran and
DeGroot, 1991). The resulting chronic stimulation of the thyroid
by increased circulating levels of TSH often results in a greater
risk of developing tumors derived from follicular cells in 2-year or
lifetime chronic toxicity/carcinogenicity studies with these compounds in rats (Fig. 21-22). Recent studies have suggested that glucuronidation and enhanced biliary excretion of T3 may be the reason why serum TSH is increased in short-term (7 days) studies
with some microsomal enzyme inducing chemicals (e.g., phenobarbital, pregnenolone-16-carbonitrile) but is less affected with
others (3-methylcholanthrene, PCB) (Hood and Klaassen, 2000).
However, microsomal enzyme inducers are more effective in
reducing serum T4 than serum T3 (Hood and Klaassen, 2000).
Outer-ring deiodinase (ORD) activity, an enzyme involved in the
peripheral conversion of T4 (major secretory product of the thyroid) to T3, was reduced (not increased as would be expected if
this was the mechanism) following the administration of four wellcharacterized enzyme inducers in rats. Type I ORD was measured
in thyroid, kidney, and liver whereas type II ORD was quantified
in brown adipose tissue, pituitary gland, and brain.
Figure 21-22. Hepatic microsomal enzyme induction by the chronic administration of xenobiotic chemicals, leading to thyroid follicular cell hyperplasia and neoplasia.
729
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Figure 21-23. Cumulative biliary excretion of 125I-thyroxine (percentage of administered dose) in control
and phenobarbital treated rats (100 mg/kg/day in the diet for 4 to 6 weeks).
Phenobarbital treatment resulted in an increase in the cumulative excretion of thyroxine over a 4-h period. Thinlayer chromatography of bile samples indicated that most of the increase in biliary excretion was accounted for
by an increase in the fraction corresponding to thyroxine-glucuronide. (From McClain, 1989, with permission.)
the pituitary secondary to the hepatic microsomal enzyme-induced
increase of T4 excretion in the bile.
The activation of the thyroid gland during the treatment of
rodents with substances that stimulate thyroxine catabolism is a
well-known phenomenon and has been investigated extensively
with phenobarbital and many other compounds (Curran and
DeGroot, 1991). It occurs particularly with rodents, first because
UDP-glucuronyl transferase can easily be induced in rodent
species, and second because thyroxine metabolism takes place very
rapidly in rats in the absence of thyroxine-binding globulin. In humans a lowering of the circulating T4 level but no change in TSH
and T3 concentrations has been observed only with high doses of
very powerful enzyme-inducing compounds, such as rifampicin
with and without antipyrine.
Although phenobarbital is the only UDP-glucuronyl transferase (UDP-GT) inducer that has been investigated in detail to act
as a thyroid tumor promoter, the effects of other well-known UDPGT inducers on the disruption of serum T4 TSH and thyroid gland
have been investigated. For example, pregnenolone-16-carbonitrile (PCN), 3-methylcholanthrene (3MC) and aroclor 1254 (PCB)
induce hepatic microsomal UDP-GT activity towards T4 (Barter
and Klaassen, 1992a). These UDP-GT inducers reduce serum T4
levels in both control as well as in thyroidectomized rats that are
infused with T4, indicating that reductions in serum T4 levels are
not due to a direct effect of the inducers on the thyroid gland (Barter
and Klaassen, 1992b, 1994). However, serum TSH levels and the
thyroid response to reductions in serum T4 levels by UDP-GT inducers is not always predictable. While PCN increased serum TSH
and resulted in thyroid follicular cell hyperplasia similar to that
observed with phenobarbital, 3MC and PCB in these short-duration
experiments and at the dose levels used did not increase serum TSH
levels or produce thyroid follicular cell hyperplasia (Liu et al.,
1995; Hood et al., 1995). These findings support the overall hypothesis that UDP-GT inducers can adversely affect the thyroid
gland by a secondary mechanism, but this applies only to those
UDP-GT inducers that increase serum TSH in addition to reducing serum T4.
Additional investigations by this research group demonstrated
that hepatic microsomal enzyme inducing xenobiotic chemicals
[e.g., phenobarbital and pregnenolone-16-carbontrile (PCN)] increased serum TSH (0 75 percent) much less than the thyroperoxidase inhibitor propylthiouracil (PTU) (830 percent) (Hood et
al., 1999). Phenobarbital and PCN administration increased thyroid weight approximately 80 percent compared to a 500 percent
increase in PTU-treated rats. Thyroid follicular cell proliferation
(determined by BrdU labeling) was increased 260, 330, and 850
percent in rats treated with phenobarbital, PCN, and PTU, respectively, for 7 days but the labeling index had returned to control levels by the 45th day of treatment. These findings demonstrate that
certain hepatic microsomal enzyme-inducing chemicals that result
in mild to modest elevations in serum TSH lead to dramatic increases in thyroid follicular cell proliferation that peak after 7 days
of treatment and then rapidly return to control values. These
findings are similar to those of Wynford-Thomas et al. (1982), who
reported a maximal proliferative response (evaluated by mitotic index) after 7 days of treatment with aminotriazole (a thyroperoxidase inhibitor). The decline in thyroid follicular cell proliferation
was suggested to be due to desensitization of the cells to the mitogenic actions of TSH (Wynford-Thomas et al., 1982a).
Hood et al. (1999) reported that moderate increases in serum
TSH of between 10 and 20 ng/ml increased the number of proliferating thyroid follicular cells but had no effect on thyroid weight,
emphasizing that small increases in serum TSH can be sufficient
to stimulate proliferation. These important findings suggest that
quantitation of follicular cell proliferation may be more useful than
thyroid weights for assessing alterations in thyroid growth in rats
administered xenobiotic chemicals that produce only small to moderate increases in serum TSH.
There is no convincing evidence that humans treated with
drugs or exposed to chemicals that induce hepatic microsomal
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731
Figure 21-24. Hepatic thyroxine glucuronyltransferase activity in control and phenobarbital-treated rats (100
mg/kg/day in the diet for 4 weeks).
Glucuronyltransferase activity was measured in hepatic microsomes using thyroxine as a substrate. Phenobarbital treatment induced thyroxine-glucuronyltransferase in male and female rats; however, the effect in male rats
was quantitatively larger. (From McClain, 1989, with permission.)
enzymes are at increased risk for the development of thyroid cancer (Curran and DeGroot, 1991). In a study on the effects of microsomal enzyme–inducing compounds on thyroid hormone
metabolism in normal healthy adults, phenobarbital (100 mg daily
for 14 days) did not affect the serum T4, T3, or TSH levels
(Ohnhaus et al., 1981). A decrease in serum T4 levels was observed
after treatment with either a combination of phenobarbital
plus rifampicin or a combination of phenobarbital plus antipyrine;
however, these treatments had no effect on serum T3 or TSH
levels (Ohnhaus and Studer, 1983). Epidemiologic studies of patients treated with therapeutic doses of phenobarbital have reported no increase in risk for the development of thyroid neoplasia
(Clemmesen et al., 1974; Clemmesen and Hualgrim-Jensen, 1977,
1978, 1981; White et al., 1979; Friedman, 1981; Shirts et al., 1986;
Olsen et al., 1989). Highly sensitive assays for thyroid and pituitary hormones are readily available in a clinical setting to monitor circulating hormone levels in patients exposed to chemicals potentially disruptive of pituitary-thyroid axis homeostasis.
Likewise, there is no substantive evidence that humans treated
with drugs or exposed to chemicals that induce hepatic microsomal enzymes are at increased risk for the development of liver cancer. This is best exemplified by the extensive epidemiologic information on the clinical use of phenobarbital. Phenobarbital has been
used clinically as an anticonvulsant for more than eighty years.
Relatively high microsomal enzyme-inducing doses have been used
chronically, sometimes for lifetime exposures, to control seizure
activity in human beings. A study of over 8000 patients admitted
to a Danish epilepsy center from 1933 to 1962 revealed no evidence
for an increased incidence of hepatic tumors in phenobarbitaltreated humans when patients receiving thorotrast, a known human
liver carcinogen, were excluded (Clemmesen and Hjalgrim-Jensen,
1978). A follow-up report on this patient population confirmed and
extended this observation (Clemmesen and Hjalgrim-Jensen, 1981;
Olsen et al., 1989). The results of two other smaller studies (2099
epileptics and 959 epileptics) also revealed no hepatic tumors in
patients treated with phenobarbital (White et al., 1979).
Chemical Inhibition of
5-Monodeiodinase
FD&C Red No. 3 (erythrosine) is an example of a well-characterized xenobiotic that results in perturbations of thyroid function in
rodents and in long-term studies is associated with an increased
incidence of benign thyroid tumors. Red No. 3 is a widely used
color additive in foods, cosmetics, and pharmaceuticals. A chronic
toxicity/carcinogenicity study revealed that male Sprague-Dawley
rats fed a 4 percent dietary concentration of Red No. 3 beginning
in utero and extending over their lifetime (30 months) developed
a 22 percent incidence of thyroid adenomas derived from follicular cells compared to 1.5 percent in control rats and a historical incidence of 1.8 percent for this strain (Borzelleca et al., 1987; Capen,
1989) (Fig. 21-25). There was no significant increase in follicular
cell adenomas in the lower-dose groups of male rats or an increase
in malignant thyroid follicular cell tumors. Female rats fed similar amounts of the color did not develop a significant increase in
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to determine the effects of the color on thyroid hormone economy.
The experimental design of the study was to sacrifice groups of
rats (n 20 rats/interval and dose) fed Red No. 3 and their control groups after 0, 3, 7, 10, 14, 21, 30, and 60 days.
A consistent effect of Red No. 3 on thyroid hormone economy was the striking increase in serum reverse T3 (Fig. 21-26). In
the rats fed high doses, reverse T3 was increased at all intervals
compared to controls and this also held for rats killed at 10, 14,
and 21 days in the low-dose group. The mechanisms responsible
for the increased serum reverse T3 appear to be, first, substrate (T4)
accumulation due to 5-monodeiodinase inhibition with subsequent
conversion to reverse T3 rather than active T3; and, second, reverse
T3 accumulation due to 5-monodeiodinase inhibition resulting in
an inability to degrade reverse T3 further to diiodothyronine (T2).
Serum triiodothyronine (T3) was decreased significantly at all intervals in rats of the high-dose group compared to interval controls
(Fig. 21-27). The mechanism responsible for the reduced serum T3
following feeding of Red No. 3 was decreased monodeiodination
of T4 due to an inhibition of the 5-monodeiodinase by the color.
Serum TSH was increased significantly at all intervals in rats
of the high-dose (4 percent) group compared to controls. Rats fed
0.25 percent Red No. 3 had increased serum TSH only at days 21,
30, and 60 (Fig. 21-28). The mechanism responsible for the increased serum TSH following ingestion of Red No. 3 was a compensatory response by the pituitary gland to the low circulating levels of T3 that resulted from an inhibition of the 5-monodeiodinase.
Serum T4 also was increased significantly at all intervals in rats
fed 4 percent Red No. 3 compared to controls (Fig. 21-29). The
mechanism responsible for the increased serum T4 was, first, accumulation due to an inability to monodeiodinate T4 to T3 in the
liver and kidney from the inhibition of 5-monodeiodinase by the
color; and, second, TSH stimulation of increased T4 production by
the thyroid gland.
125
I-labeled T4 metabolism was significantly altered in liver
homogenates prepared from rats fed 4 percent FD&C Red No. 3.
Degradation of labeled T4 was decreased to approximately 40 percent of the values in control homogenates (Fig. 21-30). This was
associated with a 75 percent decrease in percent generation of 125I
and an approximately 80 percent decrease in percent generation of
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Figure 21-25. Thyroid lesions in male Sprague-Dawley rats fed varying
doses of FD&C Red No. 3 beginning in utero and for a lifetime of 30
months. (From Borzelleca et al., 1987, with permission.)
either benign or malignant thyroid tumors. Feeding of the color at
the high dose (4 percent) level provided male rats with 2464 mg/kg
of Red No. 3 daily; by comparison human consumption in the
United States is estimated to be 0.023 mg/kg/day.
The results of mechanistic studies with FD&C Red No. 3 have
suggested that a primary (direct) action on the thyroid is unlikely
to result from (1) failure of the color (14C-labeled) to accumulate
in the gland, (2) negative genotoxicity and mutagenicity assays, (3)
lack of an oncogenic response in mice and gerbils, (4) a failure to
promote thyroid tumor development at dietary concentrations of
1.0 percent or less in male and female rats (Capen, 1989), and (5)
no increased tumor development in other organs. Investigations
with radiolabeled compound have demonstrated that the color does
not accumulate in the thyroid glands of rats following the feeding
of either 0.5 percent or 4.0 percent FD&C Red No. 3 for 1 week
prior to the oral dose of 14C-labeled material.
Mechanistic investigations with FD&C Red No. 3 included,
among others, a 60-day study of male Sprague-Dawley rats fed either 4 percent (high dose) or 0.25 percent (low dose) Red No. 3
compared to controls, whose food was without the color, in order
Figure 21-26. Rapid and significant increase in serum reverse triiodothyronine (rT3) levels in Sprague-Dawley rats (N 20 per group and
interval) administered a high (4 percent) and low (0.25 percent) dose of
FD&C Red No. 3.
The significant increase in rT3 was detected at the initial interval of 3 days
and persisted during the 60-day experiment in the high-dose group. (Courtesy of the Certified Color Manufacturers Association, Inc., and Dr. L.E.
Braverman and Dr. W.J. DeVito, University of Massachusetts Medical
School.)
Figure 21-27. Changes in serum triiodthyronine (T3) following administration of a high (4 percent) and low (0.25 percent) dose of FD&C Red
No. 3 in the diet to Sprague-Dawley rats. (Courtesy of the Certified Color
Manufacturers Association, Inc., and Dr. L.E. Braverman and Dr. W.J.
DeVito, University of Massachusetts Medical School.)
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CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
Figure 21-28. Changes in serum thyroid stimulating hormone (TSH) following administration of a high (4 percent) and low (0.25 percent) dose
of FD&C Red No. 3 in the diet to Sprague-Dawley rats. (Courtesy of the
Certified Color Manufacturers Association, Inc., and Dr. L.E. Braverman and Dr. W.J. DeVito, University of Massachusetts Medical School.)
125
I-labeled T3 from radiolabeled T4 substrate. These mechanistic
investigations suggested that the color results in a perturbation of
thyroid hormone economy in rodents by inhibiting the 5’-monodeiodinase in the liver, resulting in long-term stimulation of follicular cells by TSH, which over their lifetime predisposed to an
increased incidence of thyroid tumors (Capen and Martin, 1989;
Borzelleca et al., 1987). The color tested negative in standard genotoxic and mutagenic assays, and it did not increase the incidence
of tumors in other organs.
Morphometric evaluation was performed on thyroid glands
from all rats at each interval during the 60-day study. Four levels
of exposure of rat thyroid to Red No. 3 were evaluated, with 25
measurements from each rat. The direct measurements included
the diameter of thyroid follicles, area of follicular colloid, and
height of follicular cells. Thyroid follicular diameter was decreased
significantly in both low- and high-dose groups at 3, 7, 10, and
Figure 21-30. Effects of dietary FD&C Red No. 3 on the hepatic metabolism of 125I-labeled thyroxine in male Sprague-Dawley rats fed diets
containing 0.5 and 4.0 percent color compared to controls. (Courtesy of
the Certified Color Manufacturers Association, Inc., and the late Sidney
H. Ingbar, M.D.)
14 days compared to interval controls. The area of follicular colloid generally reflected the decrease in thyroid follicular diameter
and was decreased significantly at days 3 and 10 in high-dose rats
and days 7 and 10 in the low-dose group compared to interval controls. These reductions in thyroid follicular diameter and colloid
area were consistent with morphologic changes expected in response to an increased serum TSH concentration.
Thyroid follicular height was increased significantly only after feeding FD&C Red No. 3 for 60 days in both the high- and
low-dose groups compared to interval controls. The absence of
morphometric evidence of follicular cell hypertrophy at the shorter
intervals was consistent with the modest increase (15.8 percent) in
thyroid gland:body weight ratio after this relatively short exposure
to the color. The lack of follicular cell hypertrophy at the shorter
intervals of feeding Red No. 3 in rats with severalfold elevations
in serum TSH levels may be related, in part, to the high iodine
content (58 percent of molecular weight) interfering with the
receptor-mediated response of thyroid follicular cells to TSH. The
thyroid responsiveness to TSH is known to vary inversely with iodine content (Ingbar, 1972; Lamas and Ingbar, 1978). Thyroid
glands of rats fed FD&C Red No. 3 would be exposed to an increased iodine concentration primarily from sodium iodide contamination of the color and, to a lesser extent, from metabolism of
the compound and release of iodide.
Secondary Mechanisms of Thyroid
Tumorigenesis and Risk Assessment
Figure 21-29. Changes in serum thyroxine (T4) following administration
of a high (4 percent) and low (0.25 percent) dose of FD&C Red No. 3 in
the diet to Sprague-Dawley rats. (Courtesy of the Certified Color Manufacturers Association, Inc., and Dr. L.E. Braverman and Dr. W.J. DeVito,
University of Massachusetts Medical School.)
Understanding the mechanism of action of xenobiotics on the thyroid gland provides a rational basis for extrapolation findings from
long-term rodent studies to the assessment of a particular compound’s safety for humans. Many chemicals and drugs disrupt one
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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
“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 human patients who have marked changes in thyroid function and elevated TSH levels, as is common 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 (Doniach,
1970; Curran and DeGroot, 1991). 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 (McClain, 1989).
Human patients with congenital defects in thyroid hormone
synthesis (dyshormonogenetic goiter) and markedly increased circulating TSH levels have been reported to have an increased incidence of thyroid carcinomas (Cooper et al., 1981; McGirr et al.,
1959). Likewise, thyrotoxic patients with Graves’ disease, in which
follicular cells are autonomously stimulated by an immunoglobulin (long-acting thyroid stimulator, or LATS) also appear to be at
greater risk of developing thyroid tumors (Pendergrast et al., 1961;
Clements, 1954). Therefore, the literature suggests that prolonged
stimulation of the human thyroid by TSH will induce neoplasia
only in exceptional circumstances, possibly by acting together with
some other metabolic or immunologic abnormality (Curran and
DeGroot, 1991).
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2996R_ch21_711-759
Figure 21-31. Multiple sites of disruption of the hypothalamic-pituitarythyroid axis by xenobiotic chemicals.
Chemicals can exert direct effects by disrupting thyroid hormone synthesis or secretion and indirectly influence the thyroid through an inhibition
of 5’-deiodinase or by inducing hepatic microsomal enzymes (e.g., T4-UDP
glucuronyltransferase). All of these mechanisms can lower circulating levels of thyroid hormones (T4 and/or T3), resulting in a release from negativefeedback inhibition and increased secretion of thyroid stimulating hormone
(TSH) by the pituitary gland. The chronic hypersecretion of TSH predisposes the sensitive rodent thyroid gland to develop an increased incidence
of focal hyperplastic and neoplastic lesions (adenomas) by a secondary
(epigenetic) mechanism.
or more steps in the synthesis and secretion of thyroid hormones,
resulting in subnormal levels of T4 and T3, associated with a compensatory increased secretion of pituitary TSH (Fig. 21-31). When
tested in highly sensitive species, such as rats and mice, early on
these compounds resulted in follicular cell hypertrophy/hyperplasia and increased thyroid weights, and in long-term studies they
produced an increased incidence of thyroid tumors by a secondary
(indirect) mechanism associated with hormonal inbalances.
Review of the Physicians’ Desk Reference (PDR) reveals a
number of marketed drugs that result in a thyroid tumorigenic
response when tested at high concentrations in rodents, primarily
in rats. A broad spectrum of product classes are represented including antibiotics, calcium-channel blockers, antidepressants,
hypolipidemic agents, among others (Fig. 21-32). Amiodarone (an
antiarrhythmic drug) and iodinated glycerol (an expectorant) are
highly iodinated molecules that disrupt thyroid hormone economy by mechanisms similar to the food color FD&C Red No. 3
(Fig. 21-33).
In the secondary mechanism of thyroid oncogenesis in rodents, the specific xenobiotic chemical or physiologic perturbation
THYROID C CELLS
Normal Structure and Function
Calcitonin (CT) has been shown to be secreted by a second population of endocrine cells in the mammalian thyroid gland that are
much less numerous than follicular cells. C cells (parafollicular or
light cells) are distinct from follicular cells in the thyroid that
secrete T4 and T3 (Kalina and Pearse, 1971). They are situated either within the follicular wall immediately beneath the basement
membrane or between follicular cells and as small groups of cells
between thyroid follicles. C cells do not border the follicular colloid directly and their secretory polarity is oriented toward the in-
Figure 21-32. Examples of marketed drugs with a tumorigenic response in the thyroid gland of rats. (Modified from Davis and Monro, 1995, with permission.)
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735
Figure 21-33. Marketed drugs with a thyroid tumorigenic response. (Modified from Davis and Monro, 1995,
with permission.)
terfollicular capillaries. 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 calcitonin activity of
C cells to these secretory granules (DeGrandi et al., 1971).
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. 21-34). They move caudally with the ultimobranchial body
to the point of fusion with the midline thyroglossal duct primordia that gives rise to the thyroid gland. The ultimobranchial body
fuses with and is incorporated into the thyroid near the hilum in
mammals, and C cells subsequently are distributed throughout the
Figure 21-34. Schematic representation of neural crest origin of calcitonin-secreting C cells.
Primordial cells arising from the 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 to varying degrees throughout the mammalian thyroid gland. (From Foster, 1972, with
permission.)
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cell) thyroid tumors in Wistar rats but that high dietary calcium
intake (2,000 mg/100 g) did not further incease the incidence of
C cell tumors in irradiated rats. Further studies by Thurston and
Williams (1982) found that irradiated rats receiving diets high in
vitamin D that developed hypercalcemia had a higher incidence of
C-cell tumors than rats fed diets adequate or deficient in vitamin
D. Stoll et al. (1978) reported that the antithyroid drug thiamazole
can result in proliferative lesions (hyperplasia and adenoma) in thyroid C cells as well as in follicular cells in rats.
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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 hilum 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 (Takayama et al., 1986).
In contrast to the iodothyronines (T4 and T3) produced by
follicular cells, calcitonin is a polypeptide hormone composed of
32 amino acid residues arranged in a straight chain (Copp, 1970).
The concentration of calcium ion in plasma and extracellular fluids is the principal physiological 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. 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.
Hyperplasia of C cells 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.
Calcitonin exerts its function by interacting with target cells,
primarily in bone and kidney. The action of calcitonin is antagonistic to that of parathyroid hormone on mobilizing calcium from
bone but synergistic on decreasing the renal tubular reabsorption
of phosphorus. The storage of large amounts of preformed hormone in C cells and rapid release in response to moderate elevations of blood calcium are a reflection of the physiologic role of
calcitonin as an “emergency” hormone to protect against the development of hypercalcemia. Calcitonin and parathyroid hormone,
acting in concert, provide a dual negative feedback control mechanism to maintain the life-sustaining concentration of calcium ion
in extracellular fluids within narrow limits. Calcitonin secretion is
increased in response to a high-calcium meal often before a significant rise in plasma calcium can be detected. Gastrin, pancreozymin, and glucagon are examples of gastrointestinal hormones
whose secretion is stimulated by an oral calcium load which, in
turn, act as secretagogues for calcitonin release from the thyroid
gland.
Morphologic Alterations and
Proliferative Lesions of Thyroid C Cells
C-cell proliferative lesions occur commonly in many rat strains but
are uncommon in the mouse. Rat strains with high incidences of
these lesions include WAG/Rij, Sprague-Dawley, Fisher, Wistar,
Buffalo, and Osborne-Mendel. The incidence varies from 19 to
33 percent with no obvious sex differences. Burek (1978) reported
that cross-breeding of high- (WAG/Rij) and low- (BN/Bi) incidence
strains results in F1 animals with intermediate tumor incidences.
As with proliferative lesions of other endocrine organs, there is a
correlation between the age of the animal and the presence of the
entire spectrum of C-cell proliferative lesions. For example, LongEvans rats under 1 year of age rarely have C-cell neoplasms in the
thyroid. Rats that were between 12 and 24 months of age had a
10 percent incidence, whereas approximately 20 percent of 2- to
3-year-old rats had C-cell tumors. Similarly, the frequency of focal and diffuse C-cell hyperplasia increased progressively with
age in this strain of rat. According to the NTP historical database
of 2-year-old F344 rats, the incidence of C-cell neoplasms was 8.9
percent in males and 8.5 percent in females.
There are two types of C-cell hyperplasia: diffuse and focal
(nodular) (Fig. 21-35). In diffuse hyperplasia, the numbers of
C cells are increased throughout the thyroid lobe to a point where
Mechanisms of Toxicity
Nodular and/or diffuse hyperplasia of C cells occurs with advancing age in many strains of laboratory rats and in response to longterm hypercalcemia in certain animal species and human beings.
Focal aggregation of C cells near the thyroid hilum are a normal
anatomic finding in the thyroids of dogs and should not be overinterpreted as areas of C-cell hyperplasia. There is suggestive evidence that focal or diffuse hyperplasia precedes the development
of C-cell neoplasms (DeLellis et al., 1977) (Fig. 21-35). Other
studies (Triggs and Williams, 1977) have demonstrated significantly elevated circulating levels of immunoreactive calcitonin in
rats with C-cell neoplasms compared to either young or old rats
without C-cell tumors. Neither consistent changes in total blood
calcium and phosphorus nor bone lesions have been reported in
rats with calcitonin-secreting C-cell neoplasms.
Triggs and Williams (1977) reported that radiation (5 or 10
Ci 131I) increased the incidence of C-cell (as well as follicular
Figure 21-35. Focal and nodular hyperplasia of C cells in the thyroid
often precedes the development of C-cell neoplasms. (From DeLellis et
al., 1977, with permission.)
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more intense in diffuse or nodular hyperplasia, whereas in adenomas and carcinomas calcitonin immunoreactivity is much more
variable between tumors and in different regions of a tumor. Hyperplastic C cells adjacent to adenomas and carcinomas usually are
intensely positive for calcitonin. In addition to calcitonin, some of
the tumor cells and adjacent hyperplastic C cells have positive
staining for somatostatin or bombesin.
PARATHYROID GLAND
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they may be more numerous than follicular cells. In contrast to the
predominantly central distribution of C cells in thyroids from young
rats, C cells in the more severe forms of hyperplasia extend to the
extreme upper and lower poles as well as the peripheral regions of
the lobes. Focal (nodular) hyperplasia of C cells often occurs concurrently with diffuse hyperplasia in the thyroid glands of rats.
Follicular cells adjacent to the proliferating C cells often are compressed and atrophic with prominent supranuclear accumulations
of lipofuscin. In the later stages, follicles with intense C-cell hyperplasia often assume irregular, twisted, and elongated configurations. Occasional colloid-filled thyroid follicles are entrapped
among the proliferating C cells.
The histologic distinction between focal hyperplasia and adenoma of C cells is indistinct and somewhat arbitrary. The diagnosis of C-cell hyperplasia refers to a focal or diffuse increase of
C cells between thyroid follicles and/or within the follicular basement membrane. The C cells appear normal with an abundant,
lightly eosinophilic, granular cytoplasm and a round-to-oval nucleus with finely stippled chromatin. In focal C-cell hyperplasia
the accumulations of proliferating C cells are of a lesser diameter
than five average colloid-containing thyroid follicles, with minimal evidence of compression of adjacent follicles. Hyperplastic
C cells within the follicular basement membrane may compress individual thyroid follicles.
The ultimobranchial body (last, usually fifth, pharyngeal
pouch) that delivers the neural crest–derived C cells to the postnatal thyroid gland fuses with each thyroid lobe at the hilum during embryonic development and distributes C cells throughout each
lobe to varying degrees in different species (Fig. 21-34). In the dog
nodular aggregations of C cells frequently persist along the course
of the major vessels to the thyroid; therefore, C-cell hyperplasia in
dogs should be diagnosed only when there is a definite increase in
C cells throughout each thyroid lobe compared to age-matched controls. Both thyroid lobes should be sectioned longitudinally in a
consistent manner for microscopic evaluation. This will minimize
the prominent regional differences of C cells in the thyroid glands
of normal dogs that can result in the overinterpretation of these focal aggregations of C cells as a significant lesion. There are occasional ultimobranchial-derived, colloid-containing follicles within
the focal accumulations of microscopically normal C cells along
the course of vessels within the thyroid lobe or in the connective
tissues of the thyroid hilum in dogs.
By comparison, C-cell adenomas are discrete, expansive
masses of C cells larger than five average colloid-containing thyroid follicles. Adenomas either are well circumscribed or partially
encapsulated from adjacent thyroid follicles that often are compressed to varying degrees. C cells composing an adenoma may be
subdivided by fine connective tissue septae and capillaries into
small neuroendocrine packets. Some C-cell adenomas are composed of larger pleomorphic cells with amphophilic cytoplasm,
large nuclei with coarsely clumped chromatin, and prominent nucleoli; histologically, they resemble ganglion cells. Occasional
amyloid deposits may be found both in nodular hyperplasia and in
adenomas.
C-cell carcinomas often result in macroscopic enlargement of
one or both thyroid lobes due to the extensive proliferation and infiltration of C cells. There is evidence of intrathyroidal and/or capsular invasion by the malignant C cells, often with areas of hemorrhage and necrosis within the neoplasm. The malignant C cells
are more pleomorphic than those making up benign proliferative
lesions. Immunoperoxidase reactions for calcitonin generally are
737
Introduction
Calcium plays a key role as an essential structural component of
the skeleton and also in many fundamental biological processes.
These processes include neuromuscular excitability, membrane
permeability, muscle contraction, enzyme activity, hormone release, and blood coagulation, among others. The precise control of
calcium in extracellular fluids is vital to health. To maintain a constant concentration of calcium, despite marked variations in intake
and excretion, endocrine control mechanisms have evolved that primarily consist of the interactions of three major hormones —
parathyroid hormone (PTH), calcitonin (CT), and cholecalciferol
(vitamin D) (Fig. 21-36).
Normal Structure and Function of
Chief Cells
Biosynthesis of Parathyroid Hormone Parathyroid chief cells
in humans and many animal species store relatively small amounts
of preformed hormone, but they respond quickly to variations in
the need for hormone by changing the rate of hormone synthesis.
Parathyroid hormone, like many peptide hormones, is first synthesized as a larger biosynthetic precursor molecule that undergoes
posttranslational processing in chief cells. Preproparathyroid hormone (preproPTH) is the initial translation product synthesized on
ribosomes of the rough endoplasmic reticulum in chief cells. It is
composed of 115 amino acids and contains a hydrophobic signal
or leader sequence of 25 amino acid residues that facilitates the
penetration and subsequent vectorial discharge of the nascent peptide into the cisternal space of the rough endoplasmic reticulum
(Kronenberg et al., 1986) (Fig. 21-37). PreproPTH is rapidly
converted within 1 min or less of its synthesis to proparathyroid
hormone (ProPTH) by the proteolytic cleavage of 25 amino acids
from the NH2-terminal end of the molecule (Habener, 1981). The
intermediate precursor, proPTH, is composed of 90 amino acids
and moves within membranous channels of the rough endoplasmic
reticulum to the Golgi apparatus (Fig. 21-37). Enzymes within
membranes of the Golgi apparatus cleave a hexapeptide from the
NH2-terminal (biologically active) end of the molecule, forming
active PTH. Active PTH is packaged into membrane-limited,
macromolecular aggregates in the Golgi apparatus for subsequent
storage in chief cells. Under certain conditions of increased demand
(e.g., a low calcium ion concentration in the extracellular fluid compartment), PTH may be released directly from chief cells without
being packaged into secretion granules by a process termed bypass
secretion.
Although the principal form of active PTH secreted from chief
cells is a straight-chain peptide of 84 amino acids (molecular
weight 9500), the molecule is rapidly cleaved into amino- and
carboxy-terminal fragments in the peripheral circulation and especially in the liver. The purpose of this fragmentation is uncertain
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Figure 21-36. Interrelationship of parathyroid hormone (PTH), calcitonin (CT), and 1,25-dihydroxycholecalciferol (1,25(OH)2VD3) in the regulation of calcium (Ca) and phosphorus in extracellular fluids.
Receptors for PTH are on osteoblasts and for CT on osteoclasts in bone. PTH and CT are antagonistic in their
action on bone but synergistic in stimulating the renal excretion of phosphorous. Vitamin D exerts its action
primarily on the intestine to enhance the absorption of both calcium and phosphorus.
because the biologically active amino-terminal fragment is no more
active than the entire PTH molecule (amino acids 1 to 84). The
plasma half-life of the N-terminal fragment is considerably shorter
than that of the biologically inactive carboxy-terminal fragment of
parathyroid hormone. The C terminal and other portions of the PTH
molecule are degraded primarily in the kidney and tend to accumulate with chronic renal disease.
Control of Parathyroid Hormone Secretion Secretory cells in
the parathyroid gland store small amounts of preformed hormone
Figure 21-37. Biosynthesis of parathyroid hormone (PTH) and parathyroid secretory protein (PSP) by parathyroid chief cells.
Active PTH is synthesized as a larger biosynthetic precursor molecule (preproPTH) that undergoes rapid posttranslational processing to proPTH prior
to secretion from chief cells as active PTH (amino acids 1 to 84).
but are capable of responding to minor fluctuations in calcium concentration by rapidly altering the rate of hormonal secretion and
more slowly by altering the rate of hormonal synthesis. In contrast
to most endocrine organs that are under complex controls involving both long and short feedback loops, the parathyroids have a
unique feedback controlled by the concentration of calcium (and
to a lesser extent magnesium) ion in serum. The concentration of
blood phosphorus has no direct regulatory influence on the synthesis and secretion of PTH; however, several disease conditions
with hyperphosphatemia in both animals and humans are associated clinically with secondary hyperparathyroidism. An elevated
blood phosphorus level may lead indirectly to parathyroid stimulation by virtue of its ability to lower blood calcium, primarily by
suppressing the 1-hydroxylase in the kidney and decreasing the
production of the active form of vitamin D [1,25-(OH)2-cholecalciferol] thereby diminishing the rate of intestinal calcium absorption. Magnesium ion has an effect on parathyroid secretion rate
similar to that of calcium, but its effect is not equipotent to that of
calcium.
Serum Ca2 binds to a recently identified Ca receptor on the
chief cell, which permits the serum Ca2 to regulate chief cell function (Pollak et al., 1993). The receptor is present on the plasma
membranes of parathyroid chief cells, renal epithelial cells, and
other cells that respond to extracellular Ca2. Mutations in one or
both of the Ca2-sensing receptor genes in humans results in familial hypocalciuric hypercalcemia or neonatal severe hypercalcemia, respectively. Interaction of serum Ca2 with its receptor on
chief cells results in the formation of an inverse sigmoidal relationship between serum Ca2 and PTH concentrations (Cloutier et
al., 1993; Silver, 1992).
The serum [Ca2] that results in half maximal PTH secretion
is defined as the serum calcium “set point” and is stable for an
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cells had prominent Golgi complexes and endoplasmic reticulum,
aggregations of free ribosomes, and swelling of mitochondria
(Atwal and Pemsingh, 1981).
Inactive chief cells with few secretory granules predominate
in the parathyroids in the later stages of exposure to ozone. There
was evidence of parathyroid atrophy from 12 to 20 days after ozone
exposure, with mononuclear cell infiltration and necrosis of chief
cells. The reduced cytoplasmic area contained vacuolated endoplasmic reticulum, a small Golgi apparatus, and numerous lysosomal bodies. Plasma membranes of adjacent chief cells were disrupted, resulting in coalescence of the cytoplasmic area. Fibroblasts
with associated collagen bundles were prominent in the interstitium, and the basal lamina of the numerous capillaries often was
duplicated.
The parathyroid lesions in ozone-exposed animals are similar
to isoimmune parathyroiditis in other species (Lupulescu et al.,
1968). Antibody against parathyroid tissue was localized near the
periphery of chief cells by indirect immunofluorescence, especially
14 days following ozone injury (Atwal et al., 1975).
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individual animal. The sigmoidal relationship between serum
[Ca2] and PTH secretion permits the chief cells to respond rapidly to a reduction in serum Ca2. The major inhibitors of PTH
synthesis and secretion are increased serum [Ca2] and 1,25dihydroxyvitamin D. Inhibition of PTH synthesis by 1,25-dihydroxyvitamin D completes an important endocrine feedback loop
between the parathyroid chief cells and the renal epithelial cells
since PTH stimulates renal production of 1,25-dihydroxyvitamin D.
Chief cells synthesize and secrete another major protein
termed parathyroid secretory protein (I), or chromogranin A. It is
a higher-molecular-weight molecule (70 kDa) composed of from
430 to 448 amino acids that is stored and secreted together with
PTH. A similar molecule has been found in secretory granules of
a wide variety of peptide hormone–secreting cells and in neurotransmitter secretory vesicles. An internal region of the parathyroid secretory protein or chromogranin A molecule is identical in
sequence to pancreastatin, a C terminal amidated peptide, which
inhibits glucose-stimulated insulin secretion. This 49–amino acid
proteolytic cleavage product (amino acids number 240 to 280) of
parathyroid secretory protein has been reported to inhibit low
calcium-stimulated secretion of parathyroid hormone and chromogranin A from parathyroid cells. These findings suggest that
chromogranin A–derived peptides may act locally in an autocrine
manner to inhibit the secretion of active hormone by endocrine
cells, such as chief cells of the parathyroid gland (Barbosa et al.,
1991; Cohn et al., 1993; Fasciotto et al., 1990) (Fig. 21-38).
739
Xenobiotic Chemical-Induced Toxic
Injury of Parathyroids
Ozone Inhalation of a single dose of ozone (0.75 ppm) for 4 to
8 h has been reported to produce light and electron microscopic
changes in parathyroid glands (Atwal and Wilson, 1974). Subsequent studies have utilized longer (48-h) exposure to ozone in order to define the pathogenesis of the parathyroid lesions (Atwal et
al., 1975; Atwal, 1979). Initially (1 to 5 days after ozone exposure), many chief cells undergo compensatory hypertrophy and hyperplasia with areas of capillary endothelial cell proliferation, interstitial edema, degeneration of vascular endothelium, formation
of platelet thrombi, leukocyte infiltration of the walls of larger vessels in the gland, and disruption of basement membranes. Chief
Aluminum Evidence for a direct effect of aluminum on the
parathyroid was suggested from studies of patients with chronic
renal failure treated by hemodialysis with aluminum-containing
fluids or orally administered drugs containing aluminum. These
patients often had normal or minimal elevations of immunoreactive parathyroid hormone (iPTH), little histologic evidence of osteitis fibrosa in bone, and a depressed response by the parathyroid
gland to acute hypocalcemia (Bourdeau et al., 1987). Studies by
Morrissey and coworkers (1983) have reported that an increase in
aluminum concentration in vitro over a range of 0.5 to 2.0 mM in
a low-calcium medium (0.5 mM) progressively inhibited the secretion of PTH. At 2.0 mM aluminum, PTH secretion was inhibited by 68 percent, while a high-calcium medium (2.0 mM) without aluminum maximally inhibited PTH secretion by only 39
percent. The inhibition of PTH secretion by aluminum does not
appear to be related to an irreversible toxic effect because normal
secretion was restored when parathyroid cells were returned to the
0.5 mM calcium medium without aluminum. The incorporation of
[3H] leucine into total cell protein, parathyroid secretory protein,
proparathyroid hormone, or PTH was not affected by aluminum;
however, the secretion of radiolabeled protein by dispersed
parathyroid cells was inhibited by aluminum (Morrissey et al.,
1983).
The molecular mechanism by which aluminum inhibits PTH
secretion, reducing diglyceride levels in chief cells (Morrissey and
Slatopolsky, 1986), appears to be similar to that of the calcium ion.
Aluminum appears to decrease diglyceride synthesis, which is reflected in a corresponding decrease in synthesis of phosphatidylcholine and possible triglyceride; however, phosphatidylinositol
synthesis was not affected by aluminum. The mechanism by which
aluminum decreases diglycerides and maintains phosphatidylinositol synthesis in parathyroid cells is not known.
L-Asparaginase
Figure 21-38. Autocrine/paracrine action of chromogranin A (CgA)derived peptides. PTH and CgA are coreleased from chief cells in response to a low calcium ion signal.
Pancreastatin (PST) is a 49–amino acid peptide derived from CgA that exerts local negative feedback on chief cells and decreases PTH secretion
from chief cells. (Modified from Cohn et al., 1993, with permission.)
Tettenborn and colleagues (1970) and Chisari et
al. (1972) reported that rabbits administered L-asparaginase develop severe hypocalcemia and tetany characterized by muscle
tremors, opisthotonos, carpopedal spasms, paralysis, and coma.
This drug was of interest in cancer chemotherapy because of the
beneficial effects of guinea pig serum against lymphosarcoma in
mice.
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incorporated by the adenoma. Chief cells in this rim often are compressed and atrophic due to pressure and the persistent hypercalcemia. Peripherally situated follicles in the adjacent thyroid lobe
may be compressed to a limited extent by larger parathyroid adenomas. The parathyroid glands that do not contain a functional adenoma also undergo trophic atrophy in response to the hypercalcemia and become smaller.
Influence of Age on Development of Parathyroid Tumors There
are relatively few chemicals or experimental manipulations reported in the literature that significantly increase the incidence of
parathyroid tumors. Long-standing renal failure with intense diffuse hyperplasia does not appear to increase the development of
chief cell tumors in rats. The historical incidence of parathyroid
adenomas in untreated control male F344 rats in studies conducted
by the NTP was 4/1315 (0.3 percent); for female F344 rats, it was
2/1330 (0.15 percent). However, parathyroid adenomas are an example of a neoplasm in F344 rats whose incidence increases dramatically when life-span data are compared to 2-year studies.
Solleveld and coworkers (1984) reported that the incidence of
parathyroid adenomas increased in males from 0.1 percent at 2
years to 3.1 percent in lifetime studies. Corresponding data for female F344 rats was 0.1 percent at 2 years and 0.6 percent in lifetime studies. Hexachlorobenzene (40ppm) fed to Sprague-Dawley
rats (F0 rats for 3 months and until weaning of F1 rats that subsequently were fed for the rest of their life of 130 weeks) has been
reported to increase the incidence of parathyroid adenomas in the
male rats (24.5 percent; 12 of 49 rats) (Arnold et al., 1985).
Influence of Gonadectomy Oslapas and colleagues (1982) reported an increased incidence of parathyroid adenomas in female
(34 percent) and male (27 percent) rats of the Long-Evans strain
administered 40 Ci sodium 131I and saline at 8 weeks of age.
There were no significant changes in serum calcium, phosphorus,
and parathyroid hormone compared to controls. Gonadectomy performed at 7 weeks of age decreased the incidence of parathyroid
adenomas in irradiated rats (7.4 percent in gonadectomy versus 27
percent in intact controls), but there was little change in the incidence of parathyroid adenomas in irradiated females. X-irradiation
of the thyroid-parathyroid region also increased the incidence of
parathyroid adenomas. When female Sprague-Dawley rats received
a single absorbed dose of x-rays at 4 weeks of age, they subsequently developed a 24 percent incidence of parathyroid adenomas
after 14 months (Oslapas et al., 1981).
Influence of Xenobiotic Chemicals Parathyroid adenomas have
been encountered infrequently following the administration of a
variety of chemicals in 2-year bioassay studies in Fischer rats. In
a study with the pesticide rotenone in F344 rats, there appeared to
be an increased incidence of parathyroid adenomas in high-dose
(75 ppm) males (4 of 44 rats) compared to low-dose (38 ppm)
males, control males (1 of 44 rats), or NTP historical controls (0.3
percent) (Abdo et al., 1988). It was uncertain whether the increased
incidence of this uncommon tumor was a direct effect of rotenone
feeding or the increased survival in high-dose males. Chief cell hyperplasia was not present in parathyroids that developed adenomas.
Influence of Irradiation and Hypercalcemia Induced by Vitamin
D Wynford-Thomas and associates (1982) reported that irradiation significantly increases the incidence of parathyroid adenomas
in inbred Wistar albino rats and that the incidence could be modified by feeding diets with variable amounts of vitamin D. Neonatal Wistar rats were given either 5 or 10 Ci radioiodine (131I)
within 24 h of birth. In rats 12 months of age and older, parathy-
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Parathyroid chief cells appeared to be selectively destroyed
by L-asparaginase (Young et al., 1973). Chief cells were predominately inactive and degranulated, with large autophagic vacuoles
present in the cytoplasm of degenerating cells. Cytoplasmic organelles concerned with synthesis and packaging of secretory products were poorly developed in chief cells. Rabbits developed hyperphosphatemia, hypomagnesemia, hyperkalemia, and azotemia
in addition to acute hypocalcemia. Rabbits with clinical hypocalcemic tetany did not recover spontaneously; however, administration of parathyroid extract prior to or during treatment with
L-asparaginase decreased the incidence of hypocalcemic tetany.
The development of hypocalcemia and tetany has not been observed in other experimental animals administered L-asparaginase
(Oettgen et al., 1970). However, this response may not be limited
to the rabbit, because some human patients receiving the drug
also have developed hypocalcemia (Jaffe et al., 1972). The Lasparaginase-induced hypoparathyroidism in rabbits is a valuable
model for investigating drug–endocrine cell interactions, somewhat analogous to the selective destruction of pancreatic beta cells
by alloxan with production of experimental diabetes mellitus.
Proliferative Lesions of Parathyroid
Chief Cells
Chief Cell Adenoma Introduction Parathyroid adenomas in
adult-aged rats vary in size from microscopic to unilateral nodules
several millimeters in diameter; they are located in the cervical
region by the thyroids or infrequently in the thoracic cavity near
the base of the heart. Parathyroid neoplasms in the precardiac mediastinum are derived from ectopic parathyroid tissue displaced
into the thorax with the expanding thymus during embryonic development. Tumors of parathyroid chief cells do not appear to be
a sequela of long-standing secondary hyperparathyroidism of either renal or nutritional origin. The unaffected parathyroid glands
may be atrophic if the adenoma is functional, normal if the adenoma is nonfunctional, or enlarged if there is concomitant hyperplasia. In functional adenomas, the normal mechanism by which
PTH secretion is regulated—changes in the concentration of blood
calcium ion—is lost and hormone secretion is excessive in spite
of an increased level of blood calcium.
Adenomas are solitary nodules that are sharply demarcated
from adjacent parathyroid parenchyma. Because the adenoma compresses the rim of surrounding parathyroid to varying degrees depending upon its size, there may be a partial fibrous capsule, resulting either from compression of existing stroma or from
proliferation of fibrous connective tissue.
Adenomas are usually nonfunctional (endocrinologically inactive) in adult-aged rats from chronic toxicity/carcinogenicity
studies. Chief cells in nonfunctional adenomas are cuboidal or
polyhedral and arranged either in a diffuse sheet, in lobules, or in
acini with or without lumens. Chief cells from functional adenomas often are closely packed into small groups by fine connective
tissue septa. The cytoplasmic area varies from normal size to an
expanded area. There is a much lower density of cells in functional
parathyroid adenoma compared to the adjacent rim with atrophic
chief cells.
Larger parathyroid adenomas, such as those that are detected
macroscopically, often nearly incorporate the entire affected gland.
A narrow rim of compressed parenchyma may be detected at one
side of the gland, or the affected parathyroid may be completely
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roid adenomas were found in 33 percent of rats administered 5 Ci
131
I and in 37 percent of rats given 10 Ci 131I compared to 0 percent in unirradiated controls. The incidence of parathyroid adenomas was highest (55 percent) in normocalcemic rats fed a low vitamin D diet and lowest (20 percent) in irradiated rats fed a diet
high in vitamin D (40,000 IU/kg) that had a significant elevation
in plasma calcium.
nexal structures or serous membranes, and, last, a group of tumors
derived from the supporting connective tissues and vessels of the
testis.
Neoplasms of the gonadal stroma include benign and malignant tumors derived from Leydig (interstitial) cells, Sertoli cells of
the seminiferous tubules, as well as a rare mixed tumor with an
admixture of both cell types. The Leydig cell tumor is the most
common tumor developing in the rodent testis and frequently presents a problem in separating between focal hyperplasia and early
neoplastic growth (i.e., adenoma formation).
The incidence of Leydig cell tumors in old rats varies considerably depending upon the strain. In general, Sprague-Dawley,
Osborne-Mendel, and Brown-Norway strains have a much lower
incidence than other strains frequently used in chronic toxicity/carcinogenicity studies, including the Fischer 344 and Wistar strains.
The spontaneous incidence of Leydig cell tumors in three different strains of rats is illustrated in Fig. 21-39 (Bär, 1992). The actual incidence of Leydig cell tumors in old rats is lowest in SpragueDawley, highly variable in Wistar rats, and highest in Fischer rats
(in which the incidence at 2 years of age often approaches 100 percent). The specific numerical incidence of benign Leydig cell tumors in rats also will vary considerably depending upon the histologic criteria used by the pathologist in the separation of focal
hyperplasia from adenomas.
The incidence of Leydig cell tumors in human patients, in
comparison to rodents, is extremely rare, something on the order
of 1 in 5 million, with age peaks at approximately 30 and 60 years.
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Age-Related Changes in
Parathyroid Function
741
Serum immunoreactive parathyroid hormone (iPTH) [as well as
calcitonin (iCT)] has been reported to be different in young compared to aged Fischer 344 (F344) rats; however, the serum calcium
concentration does not change with age (Wongsurawat and
Armbrecht, 1987). This suggests that the regulation of iPTH (and
iCT) secretion may be affected by the process of aging. The decreased responsiveness of chief cells to calcium may be due to agerelated changes in the regulation of the secretory pathway. This
could include age-related changes in the effect of calcium on release of stored PTH, intracellular degradation of PTH, or modification of adenylate cyclase activity as observed in other tissues that
utilize calcium as an intermediary signal (Brown, 1982). Therefore, the sensitivity of parathyroid chief cells to circulating calcium
ion concentration appears not to be fixed but may change during
development, aging, and in response to certain disease processes.
The increased secretion of iPTH with advancing age in rats
could be due to several factors: first, an increased number of
parathyroid secretory cells with age; or, second, an altered regulation of chief cells in response to calcium ion associated with the
process of aging. For example, a decreased sensitivity of chief cells
to negative feedback by calcium ion could result in the higher blood
levels of iPTH in aged F344 rats. Wada et al. (1992) reported that
the early age-related rise in plasma PTH in F344 rats was neither
a consequence of low plasma calcium nor of renal insufficiency.
Age-related changes in the responsiveness of chief cells to circulating levels of other factors that modulate iPTH secretion,
particularly 1,25-dihydroxycholecalciferol and alpha- and betaadrenergic catecholamines, also could contribute to the variations
in blood levels of iPTH in rats of different ages. In addition, target cell responsiveness to PTH also decreases with advancing age
in rats. PTH does not increase the renal production of 1,25-dihydroxyvitamin D in adult (13-month-old) male F344 rats compared
to young (2-month-old) rats, where its production was increased
61 percent (Armbrecht et al., 1982). Older rats have a decreased
calcemic response and decreased renal production of 1,25-dihydroxycholecalciferol compared to young rats (Armbrecht et al.,
1982; Kalu et al., 1982).
TESTIS
Introduction
Leydig (interstitial) cell tumors are among the more frequently
occurring endocrine tumors in rodents in chronic toxicity/carcinogenicity studies, and a great deal of research has been published
investigating their pathogenesis and implications for safety assessment. Rodent testicular tumors are classified into five general
categories, including tumors derived from cells of the gonadal
stroma, neoplasms of germ cell origin, tumors derived from ad-
Figure 21-39. Spontaneous incidence of Leydig cell tumors of the testis
in Sprague-Dawley, Wistar, and Fischer rats. (From Bär, 1992, with
permission.)
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Ninety or more percent of Leydig cell tumors in humans are benign and some appear to be endocrinologically active and associated clinically with gynecomastia.
Structure and Endocrinologic
Regulation of Leydig (Interstitial) Cells
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Although the numbers of Leydig cells vary somewhat among different animal species and humans, the basic structural arrangement
is similar. In the rat, there are small groups of Leydig cells clustered around blood vessels in the interstitium, between seminiferous tubules with an incomplete layer of endothelial cells around
the groups of Leydig cells. In humans, the Leydig cells are present as small groups in the interstitium near blood vessels or in loose
connective tissue but without the surrounding layer of endothelial
cells. Leydig cells are much more numerous in some animal
species, such as the domestic pig. Microscopic evaluation of the
normal rat testis reveals the inconspicuous clusters of Leydig cells
in the interstitium between the much larger seminiferous tubules
composed of spermatogonia and Sertoli cells. The close anatomic
association of Leydig cells and interstitial blood vessels permits
the rapid exchange of materials between this endocrine cell population and the systemic circulation.
The endocrinologic regulation of Leydig cells involves the coordinated activity of the hypothalamus and adenohypophysis (anterior pituitary) with negative feedback control exerted by the blood
concentration of gonadal steroids (Fig. 21-40). Hypothalamic
gonadotrophin-releasing hormone (GnRH) stimulates the pulsatile
release of both luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) from gonadotrophs in the adenohypopthysis.
Luteinizing hormone is the major trophic factor controlling the activity of Leydig cells and the synthesis of testosterone. The blood
levels of testosterone exert negative feedback on the hypothalamus
and, to a lesser extent, on the adenohypophysis. Follicle-stimulating
hormone binds to receptors on Sertoli cells in the seminiferous
tubules and, along with the local concentration of testosterone,
plays a critical role in spermatogenesis. Testosterone, by controlling GnRH release, is one important regulator of FSH secretion by
the pituitary gland. The seminiferous tubules also produce a gly-
copeptide, designated as inhibin, which exerts negative feedback
on the release of FSH by the gonadotrophs.
Leydig cells have a similar ultrastructural appearance as other
endocrine cells that synthesize and release steroid hormones.
The abundant cytoplasmic area contains numerous mitochondria,
abundant profiles of smooth endoplasmic reticulum, prominent
Golgi apparatuses associated with lysosomal bodies, and occasional lipofucsin inclusions. However, they lack the hormone-containing secretory granules that are found characteristically in peptide hormone–secreting endocrine cells.
The hormonal control of testicular function is largely the result of the coordinated activities of LH and FSH from the pituitary
gland. LH binds to high-affinity, low-capacity receptors on the surface of Leydig cells and activates adenylate cyclase in the plasma
membrane resulting in the generation of an intracellular messenger, cyclic AMP (Fig. 21-41). The cyclic AMP binds to a protein
kinase, resulting in the phosphorylation of a specific set of proteins in the cytosol, which increases the conversion of cholesterol
to pregnenolone by making more substrate available and increasing the activity of an enzyme that cleaves the side chain of cholesterol. The pregnenolone in Leydig cells is rapidly converted to
testosterone, which is released into interstitial blood vessels or
taken up by adjacent Sertoli cells. Testosterone in the Sertoli cells
binds to nuclear receptors, where it increases genomic expression
and transcription of mRNAs that direct the synthesis of proteins
(e.g., androgen-binding protein and others) involved in spermatogenesis. In the rat, the mitotic phase of gametogenesis can occur without hormonal stimulation but testosterone is necessary for
meiosis of spermatocytes to spermatids. FSH is required for the later stages for spermatid maturation to spermatazoa (Fig. 21-41).
Once FSH and testosterone initiate spermatogenesis at puberty in
the rat, testosterone alone is sufficient to maintain sperm production.
Pathology of Leydig (Interstitial)
Cell Tumors
Before discussing the pathology of focal proliferative lesions of
Leydig cells, a few points should be made about the importance of
Figure 21-40. Hypothalamus–anterior pituitary gland–gonad axis in the endocrine control of Leydig and
Sertoli cells by luteinizing hormone (LH) and follicle stimulating hormone (FSH). (From Hedge et al. 1987,
with permission.)
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CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
Figure 21-41. Hormonal control of testicular function.
Luteinizing hormone (LH) stimulates testosterone (T) release by binding to its receptor (R) and increasing the
conversion of cholesterol (chol) to pregnenolone (preg) via a cAMP-protein kinase (PK) cascade. Spermatogenesis (bold arrows) is controlled by both FSH and testosterone acting via Sertoli cells. (From Hedge et al., 1987,
with permission.)
standardized sectioning methods for the complete evaluation of the
rodent testis. It is not unusual to have less than optimal sections to
evaluate, due either to a lack of a consistent plane of section or to
inadequate fixation. The goal should be to include the largest testicular area containing all anatomic features on a mid-sagittal section along the long axis of the testis, including spermatic vessels,
attachment sites of the epididymis, the tubulus rectus, and the intratesticular rete testis. It is also important to emphasize the need
to cut the thick outer covering (tunica albuginea) of the testis at
several points prior to immersion in the fixative in order to permit
more rapid penetration of the formalin or other fixing solution.
The major issue in the interpretation of focal proliferative lesions of Leydig cells in the rodent testis is the accurate and consistent separation of focal hyperplasia from benign tumors (adenomas) that possess autonomous growth. The separation of focal
hyperplasia from adenomas derived from Leydig cells is arbitrary
based upon current methods of evaluation and often is based primarily on the size of the focal lesion, since cytologic features usually are similar between focal hyperplastic and benign neoplastic
lesions derived from Leydig cells.
In the multistage model of carcinogenesis, proliferative lesions are designated as beginning with hyperplasia, often progressing to benign tumors (adenomas); infrequently, a few assume
malignant potential and form carcinomas (“cancer”) (Fig. 21-42).
Although this terminology often is applied to focal proliferative lesions in rodent endocrine tissues for convenience and standardization, it is essential to understand that the separation, especially between focal hyperplasia and adenoma, is based primarily on size
and morphologic changes in the proliferating Leydig cells. It is important to emphasize that focal proliferative lesions associated with
hormonal imbalances in rodent endocrine tissues—also including
Leydig cells, adrenal medullary cells, thyroid follicular cells, and
C cells, among others—represent a morphologic continuum that
begins with hyperplasia and progresses often but not always to the
formation of adenomas that grow autonomously and only occasionally undergo a malignant transformation to form carcinomas
(“cancer”) (Fig. 21-43).
The National Toxicology Program (NTP), in an attempt to
standardize the classification of focal proliferative lesions of Leydig cells between studies with different xenobiotic chemicals and
different testing laboratories, established the following diagnostic
criteria (Boorman, 1987): (1) Hyperplasia was defined as a focal
collection of Leydig cells with little atypia and a diameter of less
than 1 seminiferous tubule. (2) An adenoma was defined as a mass
of Leydig cells larger in diameter than 1 seminiferous tubule with
some cellular atypia and compression of adjacent tubules. (3) It
Figure 21-42. Multistage model of carcinogenesis of proliferative lesions
in endocrine tissues of rodents.
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Table 21-3
Changes in Rodent Endocrine Sensitivity over Time
(1970s–1990s)
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1. Animal husbandry practices
Specific pathogen-free (SPF) conditions
Greater survival to 2 years
High body weight (obesity)
Immobility
2. Genetic selection process
High productivity
Large litters
High lactation yield
Rapid growth
Figure 21-43. Morphologic continuum of proliferative lesions in endocrine tissues of rodents.
was emphasized that the separation was arbitrary, since at that time
little was known about the biological behavior of these lesions in
rodents.
A more contemporary set of diagnostic criteria for focal proliferative lesions of Leydig cells has been published recently by
the Society of Toxicologic Pathologists (STP) (McConnell et al.,
1992). Recognizing that many small focal proliferative lesions of
Leydig cells (i.e., between one and three tubule diameters) will
regress following removal of the inciting stimulus, they recommend
the diagnosis of Leydig cell adenoma be used for a mass of interstitial cells equal to or greater than the diameter of three adjacent
seminiferous tubules plus one or more of the following criteria:
symmetrical peripheral compression of adjacent tubules, evidence
of cellular pleomorphism or an increase in the nuclear:cytoplasmic
ratio, an endocrine sinusoidal vascular network, increased mitotic
activity, or coalescence of adjacent cell masses.
Leydig cell neoplasms in laboratory rats associated with hormonal imbalances rarely undergo malignant transformation with
progression to the development of carcinomas (“cancer”). Histologic features of malignancy include invasion into the epididymis,
spermatic cord, or tunica albuginea. The most definitive criteria of
malignancy is the demonstration of metastases in extratesticular
sites. Leydig cell carinomas are large and often distort the overall
contour of the affected testis, with extensive areas of both hemorrhage and necrosis. The cytology of Leydig cell carcinomas usually is more pleomorphic than with adenomas consisting of an
admixture of poorly differentiated cells with an increased nuclear:cytoplasmic ratio and larger, more differentiated cells with an
abundant vacuolated eosinophilic cytoplasmic area. The frequency
of mitotic figures may be increased either in focal areas or throughout the Leydig cell carcinomas. The most convincing evidence of
malignancy in carcinomas is the establishment of foci of growth
outside of the testis, such as multiple foci of tumor cell emboli
growing within and distending vessels of the lung.
several factors, including (1) animal husbandry practices, such as
specific pathogen-free conditions, that result in a greater survival
for 2 years, high body weight related to over feeding, and immobility; and (2) the genetic selection process for high productivity
and rapid growth.
Pathogenic mechanisms reported in the literature to be
important in the development of proliferative lesions of Leydig
cells include irradiation, the species and strain differences mentioned previously, and exposure to certain chemicals such as cadmium salts and 2-acetoaminofluorene (Prentice and Meikle, 1995)
(Table 21-4). Other pathogenic mechanisms include physiologic
perturbations such as cryptorchidism, a compromised blood supply to the testis, or heterotransplantation into the spleen. Hormonal
imbalances also are important factors in the development of focal
proliferative lesions of Leydig cells, including increased estrogenic
steroids in mice and hamsters and elevated pituitary gonadotrophins
resulting from the chronic administration of androgen receptor antagonists, 5-reductase inhibitors, testosterone biosynthesis inhibitors, GnRH agonists, and aromatase inhibitors (Fig. 21-44)
(Clegg et al., 1997; Cook et al., 1999). Many xenobiotic chemicals administered chronically to rats disrupt the hypothalamicpituitary-testis axis at one of several possible sites, interfering with
negative feedback control and resulting in an overproduction of
luteinizing hormone (LH), which causes the proliferative changes
Table 21-4
Pathogenic Mechanisms for Development of Leydig
(Interstitial) Cell Proliferative Lesions in Rodents
Mechanisms of Leydig (Interstitial) Cell
Tumor Development
Leydig (interstitial) cells of the testis frequently undergo proliferative changes with advancing age and following chronic exposure
to large doses of xenobiotic chemicals. In addition, it should be
emphasized that the “sensitivity” of rodent endocrine tissues, such
as Leydig cells of the testis and other populations of endocrine
cells, appears to be increasing over time particularly if one compares data generated in the 1970s to that gathered in the 1990s for
the same compound (Table 21-3). This appears to be the result of
Copyright © 2001 by The McGraw-Hill Companies
Physiologic perturbations
Cryptorchidism
Compromised blood supply
Heterotransplantation (spleen)
Hormonal imbalances
Decreased testosterone
Increased estrogens (mice, hamsters)
Increased pituitary gonadotropins (e.g., LH)
Irradiation
Species/strain differences
Chemicals
Cadmium salts
2-Acetoaminofluorene
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CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
creasing the incidence of Leydig cell adenomas compared to the
ad libitum–fed group (Table 21-7). For example, at the 30-month
sacrifice, only 17 percent of feed-restricted F344 rats had developed Leydig cell adenomas, compared to 100 percent in the ad libitum group.
Investigations reported by Chatani and associated (1990)
documented the importance of hormonal imbalances on the incidence of Leydig cell adenomas and hyperplasia in Fischer rats
(Table 21-8). The incidence of adenomas was 70 percent and of
hyperplasia 100 percent in control rats killed at 70 weeks of age.
In rats administered testosterone for 28 weeks (by silastic tubes
implanted subcutaneously at 42 weeks of age), the incidence of
Leydig cell adenomas and hyperplasias was decreased to 0 percent
for both at 70 weeks of age. This dramatic reduction in the incidence of focal proliferative lesions of Leydig cells was associated
with a significant lowering in circulating levels of LH, through negative feedback exerted by testosterone on the pituitary gland.
The important studies reported by Chatani and coworkers
(1990) also demonstrated that hormones other than testosterone
could markedly decrease the development of Leydig cell adenomas in F344 rats (Table 21-9). The administration of estradiol-17
for 28 weeks (by silastic tubes implanted subcutaneously) decreased the incidence of Leydig cell adenomas to 0 percent (compared to 100 percent in controls) and significantly reduced serum
LH, due to negative feedback control on the pituitary gland. An
LH-releasing hormone agonist administered continuously for 28
weeks (injection of microcapsules every 4 weeks at a dose of
5 mg/2 mL/kg) also decreased the incidence of Leydig cell adenomas to 0 percent and significantly decreased circulating LH levels, most likely a result of the known-down regulation of LH-RH
receptors on pituitary gonadotrophs (Table 21-9).
The studies of Bartke and colleagues (1985) demonstrated that
hyperprolactinemia also markedly decreased the incidence of Leydig cell adenomas in Fischer rats (Table 21-10). Pituitaries transplanted beneath the renal capsule (four per rat) resulted in a chronic
elevation of circulating prolactin levels, owing, most likely, to the
lack of dopamine inhibition of prolactin secretion, which occurs
when the pituitary gland is in its normal anatomic location in close
proximity to the hypothalamus. In this interesting experiment, 83
percent of sham-operated rats developed Leydig cell adenomas at
21 to 24 months of age, whereas 0 percent of rats developed tumors in animals with ectopic pituitaries and elevated serum prolactin levels.
The administration of a calcium-channel blocker (SVZ 200110) at high doses (62.5 mg/kg/day for 2 years) significantly increased the incidence of Leydig cell adenomas in Sprague-Dawley
rats. Endocrinologic studies demonstrated that increased serum levels of LH and FSH were present only after 52 and 66 weeks, respectively, and persisted to week 104 for LH. This compound is
unusual in that most xenobiotic chemicals that cause hormonal imbalances result in earlier significant changes in circulating hormone
levels.
Another important mechanism by which xenobiotics increase
the incidence of Leydig cell tumors in rats is by inhibition of testosterone synthesis by cells in the testis. For example, lansoprazole is
a substituted benzimidazole, which inhibits the hydrogenpotassium ATPase (proton pump) responsible for acid secretion by
the parietal cells in the fundic mucosa of the stomach (Fort et al.,
1995). The presence of the imidazole moiety in lansoprazole was
suggestive of an effect on testosterone synthesis, since several imidazole compounds (e.g., ketoconazole and miconazole) are known
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Figure 21-44. Model of action of nongenotoxic compounds that produce
Leydig cell hyperplasia/adenoma in rodents.
(hyperplasia, adenoma) in Leydig cells (Fig. 21-45). For example,
chronic exposure to chemicals with antiandrogenic activity, such
as procymidone due to binding to the androgen receptor, increases
circulating levels of LH and results in stimulation of Leydig cells
leading to an increased incidence of hyperplasia and adenomas in
rats (Murakami et al., 1995).
Data from several studies in the recent literature emphasize
the importance of several of these pathogenetic factors in the frequent development of Leydig cell tumors in rats. Thurman and colleagues (1994) reported the effects of food restriction on the development of Leydig cell adenomas in the high incidence strain of
Fischer 344 rats (Table 21-5). Beginning at 13 weeks of age, rats
were either continued on an ad libitum feeding or were foodrestricted 40 percent (NIH-31 diet with 1.673 fat-soluble and B
vitamins) over their lifetime until they died or became moribund
due to spontaneous disease. The incidence of Leydig cell adenomas was decreased to 19 percent in food-restricted rats compared
to 49 percent in the ad libitum–fed group (Table 21-5). In another
group from the food restriction study reported by Thurman and
coworkers (1994), rats were periodically removed for serial sacrifice at 6-month intervals. Food restriction resulted in a similar
marked reduction in Leydig cell adenomas (23 percent compared
to 60 percent in ad libitum–fed rats) (Table 21-6). Examination of
the serially sacrificed F344 rats in this study also demonstrated that
feed restriction delayed the onset of development as well as de-
Figure 21-45. Regulation of the hypothalamic-pituitary-testis (HPT) axis
and control points for potential disruption by xenobiotic chemicals. Symbols: () feedback stimulation; () feedback inhibition; receptor stimulation; enzyme or receptor inhibition. (Modified from Cook et al.,
1999, with permission.)
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Table 21-5
Effect of Food Restriction on the Development of Interstitial (Leydig) Cell Adenomas
in F344 Rats*
Interstitial Cell Adenomas
FEEDING
NO. OF RATS
NUMBER
PERCENT
Ad libitum
Food-restricted (40%)
49
52
24
10
49
19
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*Lifetime study without periodic sacrifice removals (died from spontaneous disease).
SOURCE: Thurman et al., 1994, with permission.
to inhibit testosterone synthesis. Lansoprazole resulted in decreased
circulating levels of testosterone, increased levels of LH, and an
increased incidence of Leydig cell hyperplasia and benign neoplasia (adenomas) in chronic studies in rats (Fort et al., 1995). The
most sensitive site for inhibition of testosterone synthesis by lansoprazole was the transport of cholesterol to the cholesterol side
chain cleavage enzyme.
Although several hormonal imbalances result in an increased
incidence of Leydig cell tumors in rodents, several disease conditions associated with chronic elevations in serum LH (including
Klinefelter’s syndrome and gonadotroph adenomas of the pituitary
gland) in human patients have not been associated with an increased
development of this type of rare testicular tumor.
There are a number of reports in the literature of xenobiotic
chemicals (many of which are marketed drugs) that increase the
incidence of proliferative lesions of Leydig cells in chronic toxicology/carcinogenicity in rats. These include indomethacin, lactitol, muselergine, cimetidine, gemfibrozil, and flutamide, among
many others (Table 21-11).
Flutamide is a potent nonsteroidal, antiandrogen compound
that displaces testosterone from specific receptors in target cells
and decreases negative feedback on the hypothalamus-pituitary
gland, resulting in elevated circulating levels of LH and FSH. The
chronic administration of flutamide is known to result in a striking increase in the incidence of Leydig cell adenomas in rats. The
Schering-Plough Research Institute’s Department of Drug Safety
and Metabolism completed an important reversibility study in
which Sprague-Dawley rats were administered flutamide daily either for 1 year, 1 year followed by a 1-year recovery period, or
continuously for 2 years (Table 21-12). This important study emphasizes the lack of autonomy of many focal proliferative lesions
of Leydig cells in rats and their continued dependence upon com-
pound administration for stimulation of growth. There was a reduction in the incidence of Leydig cell adenomas (using the
conservative NTP criteria of greater than 1 tubule diameter) in rats
administered three dose levels of flutamide daily for 1 year
followed by a 1-year recovery prior to termination compared to
rats given flutamide for 1 year and immediately evaluated (Table
21-12). Conversely, in rats administered flutamide for 2 years the
numbers of adenomas continued to increase until 95 percent of animals in the mid- and high-dose groups (30 and 50 g/kg, respectively) had developed Leydig cell tumors. There also was a marked
reduction in the incidence of focal hyperplasia (focus less than 1
tubule diameter) after 1 year of recovery, compared to rats terminated immediately following 1 year of flutamide administration, a
finding that emphasizes the frequent reversibility of these small
proliferative lesions of Leydig cells.
Although a number of xenobiotics have been reported to increase the incidence of Leydig cell adenomas in chronic studies in
rats, similar compounds such as cimetidine, ketoconazole, and certain calcium channel blocking agents have not resulted in an increased incidence of Leydig cell neoplasia in humans. In summary,
Leydig cell tumors are a frequently occurring tumor in rats, often
associated mechanistically with hormonal imbalances; however,
they are not an appropriate model for assessing the potential risk
to human males of developing this rare testicular tumor.
OVARY
Introduction
Ovarian tumors in rodents can be subdivided into five broad categories, including epithelial tumors, sex cord–stromal tumors, germ
cell tumors, tumors derived from nonspecialized soft tissues of the
Table 21-6
Effect of Food Restriction on the Development of Interstitial (Leydig) Cell Adenomas
in F344 Rats*
Interstitial Cell Adenomas
FEEDING
NO. OF RATS
NUMBER
PERCENT
Ad libitum
Food-restricted (40%)
98
112
59
26
60
23
*Lifetime study with periodic removal of serially sacrificed rats (died from spontaneous disease).
SOURCE: Thurman et al., 1994, with permission.
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Table 21-7
Effects of Food Restriction on the Development of Interstitial
(Leydig) Cell Adenoma (ICA) in F344 Rats at Different Ages
ICA [Tumors/No. Rats (%)]
FEED-
AGE AT
SACRIFICE (MONTHS)
SOURCE:
RESTRICTED
0/12 (0)
5/12 (42)
10/12 (83)
9/9 (100)
—
(40%)
0/12 (0)
0/12 (0)
1/12 (8)
2/12 (17)
4/9 (44)
ithelium and sex cord–derived ovarian interstitium rather than being two distinct types of ovarian tumors. The histogenic origin of
this unique ovarian tumor in mice has been a controversial topic
in the literature, but most investigators currently agree that it is derived from the ovarian surface epithelium, with varying contributions from stromal cells of the ovarian interstitium. However, some
early reports suggested an origin from the rete ovarii or thecal/granulosal cells of the ovary.
Another important group of ovarian tumors are those derived
from the sex cords and/or ovarian stroma. These include the
granulosal cell tumors, luteoma, thecoma, Sertoli cell tumor, tubular adenoma (with contributions from ovarian stroma), and undifferentiated sex cord–stromal tumors. The granulosal cell tumor
is the most common of this group which, according to Alison and
Morgan (1987), accounts for 27 percent of naturally occurring ovarian tumors in mice. Granulosal cell tumors may develop within
certain tubular or tubulostromal adenomas following a long-term
perturbation of endocrine function associated with genic deletion,
irradiation, oocytotoxic chemicals, and neonatal thymectomy (Frith
et al., 1981; Li and Gardner, 1949; Hummel, 1954).
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12
18
24
30
36
AD LIBITUM
Thurman et al., 1994, with permission.
ovary, and tumors metastatic to the ovary from distant sites. The
epithelial tumors of the ovary include cystadenomas and cystadenocarcinomas, tubulostromal adenomas, and mesothelioma. The
tubular (or tubulostromal) adenomas are the most important of the
ovarian tumors in mice, and they are the tumors whose incidence
often is increased by various endocrine perturbations associated
with exposure to xenobiotics, senescence, or inherited genic deletion (Murphy and Beamer, 1973). Tubular adenomas are a unique
lesion that develops frequently in the mouse ovary, accounting for
approximately 25 percent of naturally occurring ovarian tumors in
this species (Alison and Morgan, 1987; Rehm et al., 1984). They
are uncommon in rats, rare in other animal species, and not recognized in the ovaries of women. In some ovarian tumors of this
type in mice, there is an intense proliferation of stromal (interstitial) cells of sex cord origin. These tumors often are designated
tubulostromal adenomas or carcinomas to reflect the bimorphic appearance.
The tubulostromal adenomas in mice are composed of numerous tubular profiles derived from the surface epithelium, plus
abundant large luteinized stromal cells from the ovarian interstitium. The differences in histologic appearance of this type of
unique ovarian tumor in mice are interpreted to represent a morphologic spectrum with variable contributions from the surface ep-
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Mechanisms of Ovarian Tumorigenesis
in Rodents
Five model systems of ovarian tumorigenesis in mice have been
reported in the literature. The first model system identified was the
production of ovarian neoplasms by radiation (Furth and Boon,
1947; Gardner, 1950). After acute radiation exposure, the initial
change was a rapid loss of oocytes and a destruction of graafian
follicles. There was proliferation and down-growth of the ovarian
epithelium into the stroma within 10 weeks after radiation exposure. The first ovarian tumors developed approximately 1 year after exposure. The tubular adenomas that develop following radiation often were bilateral, endocrinologically inactive, and not lethal
unless they reached a very large size. Some irradiated mice also
developed endocrinologically active granulosal cell tumors, which
transplantation experiments have shown to be different from the
tubular adenomas. Granulosal cell tumors were transplantable into
the spleen and often grew rapidly, whereas tubular adenomas grew
slowly after transplantation, most successfully in castrated animals.
Table 21-8
Effect of Aging and Testosterone on the Incidence of Interstitial Cell Adenomas (ICA) and
Hyperplasia (ICH) in Fischer 344 Rats*
SERUM LH
TESTES
ICH†
(NODULES/
TESTES)
10
95
90
100
0‡
0.3 0.8
5.0 2.3
8.3 7.0
11.1 6.2
0.0 0.0‡
20.9 19.5
N.D.
48.7 16.7
22.1 8.4
8.4 8.8‡
TERMINAL
ICA
ICH
TREATMENT
AGE
%
(23 WEEKS)
(WEEKS)
%(NO./NO.
TESTES)
0
0
0
0
Testosterone§
42
50
60
70
70
0 (0/20)
0 (0/20)
0 (0/10)
70 (14/20)
0 (0/10)‡
(ng/mL)†
*NTP criteria: ICA greater and ICH less than one normal seminiferous tubule diameter.
†Mean SD.
‡p 0.05 compared to controls.
§Silastic tubes implanted subcutaneously at 42 weeks.
SOURCE: Chatani et al., 1990, with permission.
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Table 21-9
Effect of Testosterone, Estradiol, and LH-RH Agonist on Incidence of Interstitial Cell
Adenoma (ICA) in F344 Rats*
TERMINAL
ICA
SERUM LH
AGE (WEEKS)
% (NO./NO. TESTES)
(ng/mL)†
Control
Testosterone‡
Estradiol-17‡
LH-RH Agonist¶
88
88
88
88
100 (18/18)
0 (0/18)§
0 (0/18)§
0 (0/16)§
12.9 11.7
1.7 1.6§
4.7 2.4§
4.9 3.5§
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TREATMENT
(28 WEEKS)
*NTP criteria: ICA greater than one normal seminiferous tubule diameter.
†Mean SD.
‡Silastic tubes implanted subcutaneously.
§p 0.05 compared to controls.
¶Injected subcutaneously at 60 weeks of age.
SOURCE: Chatani et al., 1990, with permission.
Table 21-10
Effect of Hyperprolactinemia from Ectopic Pituitary Transplants on the Incidence of
Interstitial Cell Adenoma (ICA) and Testicular/Seminal Vesicle Weights in Fischer 344 Rats
WEIGHT OF
%
(NO./NO. RATS)
ICA
RAT GROUP
Sham-operated
With tumors
Without tumors
Pituitary-grafted†
With tumors
Without tumors
WEIGHT OF
TESTES
SEMINAL
(g)*
VESICLES
(g)†
83 (20/24)
17 (4/24)
4.88 0.22‡
2.66 0.48
0.55 0.08‡
1.35 0.14
0 (0/0)
100 (24/24)
—
2.79 0.05
—
1.26 0.07
*Pituitary transplants (four per rat) beneath renal capsule or sham-operated at 2 to 5 months of age; terminated at 21 to 24
months of age.
†Mean SE.
‡p 0.05 compared to other two groups.
SOURCE: Bartke et al., 1985, with permission.
Table 21-11
Selected Examples of Drugs that Increase the Incidence of Proliferative Lesions of Leydig
Cells in Chronic Exposure Studies in Rats or Mice
NAME
SPECIES
Indomethacin
Lactitol
Metronidazole
Muselergine
Buserelin
R
R
R
R
R
Cimetidine
R
Flutamide
Gemfibrozil
Spironolactone
Nararelin
Tamoxifen
Vidarabine
Clofibrate
Finasteride
R
R
R
R
M
R
R
M
CLINICAL INDICATION
Anti-inflammatory
Laxative
Antibacterial
Parkinson’s disease
Prostatic and breast
carcinoma, endometriosis
Reduction of gastric acid
secretion
Prostatic carcinoma
Hypolipidemia
Diuretic
LH-RH analog
Antiestrogen
Antiviral
Hypolipidemia
Prostatic hyperplasia
Copyright © 2001 by The McGraw-Hill Companies
REFERENCE
Roberts et al., 1989
Bär, 1992
Rustia and Shubik, 1979
Prentice et al., 1992
Donaubauer et al., 1989
PDR, 1992
PDR, 1992
Fitzgerald et al., 1981
PDR, 1994
PDR, 1994
PDR, 1994
PDR, 1994
PDR, 1994
Prahalada et al., 1994
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749
Table 21-12
Flutamide: Incidence of Interstitial Cell (IC) Adenoma in Sprague-Dawley Rats after Various Dosing Intervals
1-Year Dosing
1-Year Recovery
1-Year Dosing
DOSE
SOURCE:
0
10
30
50
0
10
30
50
0
10
30
50
58
0
57
28
57
43
57
40
52
6
53
25
53
23
53
25
55
6
55
50
55
52
55
52
6
39
44
48
8
7
10
17
5
12
9
12
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Number/group
IC adenoma
(1 tubule)
IC hyperplasia
(1 tubule)
2-Year Dosing
Courtesy of Schering Plough Research Institute, Department of Drug Safety and Metabolism, Lafayette, NJ.
The second model of ovarian tumorigenesis arose out of the
work published by Biskind and Biskind (1944). They transplanted
ovaries into the spleen of castrated rats to prevent negative feedback by circulating sex hormones on the hypothalamus and pituitary gland because estrogen is degraded as it circulates through
the liver. Transplantation resulted in a rapid loss of ovarian follicles as well as an interference with estrogen feedback on the hypothalamus. Following the loss of graafian follicles, the epithelial
covering of the ovary began to proliferate and invaginate into the
ovary, with an accompanying increase in stromal tissue, which ultimately resulted in the formation of tubular adenomas and, occasionally, granulosal cell tumors (Guthrie, 1957). The presence of
a single functioning gonad prevented the development of the proliferative lesions in the ovary, suggesting that the lack of negative
feedback from estrogen was necessary for the changes to develop.
Administration of exogenous estrogen or testosterone after transplantation completely suppressed development of the proliferative
changes in the ovarian cortex.
The third model of ovarian tumorigenesis was described by
Marchant (1957, 1960), who reported that ovarian tumors developed in mice exposed to dimethylbenzanthracene. This chemical
is a reproductive toxicant that is cytotoxic to oocytes, resulting in
the loss of graafian follicles from the ovary. This was followed by
a proliferation of the interstitial (stromal) tissue, invaginations of
the surface epithelium, and subsequent development of tubular
adenomas and occasionally granulosal cell tumors of the ovary
(Taguchi et al., 1988). Support for an endocrinologic mechanism
of hormonal imbalance included the observation that the xenobiotic chemical first must cause sterility, because the presence of a
single normal gonad prevented the development of the hyperplastic
lesions and tumors of the ovary. The administration of estrogen
prevented tumor formation even in sterile mice, and hypophysectomy also prevented the development of ovarian tumors.
The fourth model of ovarian tumorigenesis was described by
Nishizuki and associates (1979). They reported that removal of the
thymus from neonatal mice resulted in ovarian dysgenesis and the
development of ovarian tumors. Thymectomy prior to 7 days of
age resulted in an immune-mediated destruction of follicles in the
ovary. Because estrogen was not produced by the follicles, these
mice also developed hormone-mediated proliferative lesions and
ovarian tumors identical to those in the previously described models. After the immune-mediated destruction of follicles, there was
a proliferation of the interstitial (stromal) and surface epithelial
cells of the ovary, resulting in the formation of tubular adenomas.
If the mice survived for longer periods, some animals developed
granulosal cell tumors and luteomas in the ovary. Because this
model also did not involve exposure to any carcinogen, it is another indication that the prerequisite for ovarian tumor response in
mice is the production of sterility, which results in hormonal imbalances that lead to stimulation of the sensitive populations of target cells (Michael et al., 1981).
Case Study: Ovarian Tumors Associated
with Xenobiotic Chemicals
Nitrofurantoin Nitrofurantoin is an example of a chemical that,
when fed at high doses to mice for 2 years in a NTP study, increased the incidence of ovarian tumors of the tubular or tubulostromal type (Table 21-13). Nitrofurantoin fed at both low (1300
ppm) and high (2500 ppm) doses to B6C3F1 mice caused sterility
due to the destruction of ovarian follicles, leading to hormonal imbalances, which resulted in the development of an increased incidence of this unique type of ovarian tumor.
Mice administered nitrofurantoin had a consistent change in
the ovarian cortex, termed ovarian atrophy. This lesion was characterized by an absence of graafian follicles, developing ova, and
corpora lutea; by focal or diffuse hyperplasia with localized or diffuse down-growth of surface epithelium into the ovary; and by
varying numbers of polygonal, often vacuolated, sex cord–derived
stromal (interstitial) cells between the tubular profiles. The ovaries
were small, had irregular surfaces due to the tubular down-growths
into the cortex, and had scattered eosinophilic stromal cells between tubular profiles. In addition, there was a lack of graafian follicles and corpora lutea throughout the ovarian cortex.
The benign ovarian tumors in this study were classified either
as tubular adenomas (5 of 50 mice) or as tubulostromal tumors
(4 of 50 mice) (Table 21-13). In tubulostromal adenomas, the proliferating stromal (interstitial) cells between the tubules were considered to represent a significant component of the lesion. However, the separation between these two types of proliferating
ovarian lesions in mice was not distinct and both appeared to be
part of a continuous morphologic spectrum. Because all treated
mice in the NTP nitrofurantoin feeding study were sterile due to
ovarian atrophy, an indirect mechanism secondary to a disruption
of endocrine function leading to hormonal imbalances was
suggested to explain the development of the ovarian tubular
adenomas.
The results of an investigative study demonstrated that nitrofurantoin had an effect on graafian follicles in the ovary of B6C3F1
mice. Female mice of the same strain were fed 350 or 500
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Table 21-13
NTP Nitrofurantoin Study of Treatment-Related Ovarian Lesions in B6C3F1 Mice
(50/Group Exposed for 2 Years in Feed)
CONTROL
Tubular adenoma
Tubulostromal adenoma
Benign GCT
Malignant GCT
Cystadenoma
Cysts
Abscess
Ovarian atrophy
Survival at 2 Years
0
0
0
0
2
14
18
0
19
LOW DOSE
HIGH DOSE
(0.13%)
(0.25%)
0
0
3
0
1
15
0
49
37
5*
4†
2
1
1
10
0
48
37
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LESION
KEY:
NTP, National Toxicology Program; GCT, granulosa cell tumor.
*Trend test positive (p 0.05).
†Fischer exact test positive (p 0.05).
mg/kg/day of nitrofurantoin beginning at 7 weeks of age. These
levels approximate the low (1300 ppm) and high (2500 ppm) doses
used in the NTP study with nitrofurantoin. Ten female mice per
group were sacrificed at 4, 8, 13, 17, 43, and 64 weeks of feeding
nitrofurantoin. The numbers of follicles were quantified from
nitrofurantoin-treated and control mice on serial sections of the
ovary. The morphometric data revealed that the numbers of small,
medium, and large follicles in nitrofurantoin-treated mice were numerically decreased at 17 weeks compared with controls and were
significantly decreased in rats fed 350 and 500 mg/kg/day nitrofurantoin after 43 and 64 weeks (Fig. 21-46). All mice in the treated
groups were sterile by 43 weeks of feeding nitrofurantoin.
Figure 21-46. Morphometric evaluation of ovaries of mice following the
administration of 350 and 500 mg/kg/day of nitrofurantoin.
The numbers of small, medium, and large ovarian follicles were decreased
after 17 weeks and significantly decreased after 43 and 64 weeks due to a
direct action of the nitrofurantoin.
Selective Estrogen Receptor Modulators Selective estrogen receptor modulators (SERMs) are compounds that have estrogen agonist effects on some tissues and estrogen antagonist actions on
other tissues (Cohen et al., 2000). The triphenylethylene SERMs
tamoxifen and toremifene have estrogen antagonist effects in the
breast and currently are used in the medical management of breast
cancer. The benzothiophene SERM raloxifene has estrogen agonist effects on bone and serum lipids but estrogen antagonist actions on the uterus and breast. This is in contrast to tamoxifen,
which has an estrogen agonist effect on bone and also may stimulate the uterine endometrium. These SERMs (e.g., tamoxifen,
toremifene, and raloxifene) all have been reported to increase the
incidence of ovarian tumors when administered chronically to
mice. For example, CD1 mice administered raloxifene (9, 50,
225 mg/kg) per day for 21 months developed an increased incidence of granulosa/theca cell tumors (benign and malignant) and
tubular/papillary adenomas of the ovary. However, there is no evidence of an increased risk for ovarian cancer in women administered SERMs, since tamoxifen has been used clinically since 1978
(Fisher et al., 1994; Cook et al., 1995).
Raloxifene binds to the estrogen receptor (Yang et al., 1996)
and appears to block the negative feedback of circulating levels of
estrogen on the hypothalamus, resulting in a sustained increase in
circulating levels of LH. Mice (CD1) administered raloxifene (233
or 236 mg/kg) daily for 2 and 4 weeks had a dose-dependent
significant elevation of serum LH levels (4- to 7-fold and 4.4-fold
compared to controls, respectively) (Cohen et al., 2000).
Raloxifene-treated mice had sustained elevations in serum LH over
a 24-h period and did not have the preovulatory LH surge present
in many control mice. Histomorphologic changes in the ovary were
indicative of arrested follicular maturation including anovulatory
hemorrhagic follicles, some developing follicles, and few corpora
lutea. Following a recovery period of 3 weeks during which no
raloxifene was administered, serum LH concentrations were indistinguishable from controls and follicular maturation and corpora
lutea distribution were normal (Long et al., 2001). Raloxifene binding to the estrogen receptor resulted in an elevation of serum LH
and ovarian tumor development similar to those in estrogen receptor- knockout mice with genetic deletion of the estrogen re-
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ceptor (Korach, 1994), suggesting that the tumors in both instances
develop secondary to the hormonal imbalances.
Ovarian Tumors in Mutant
Strains of Mice
tologically to the larger tubular adenomas in the high-dose (2500
ppm) mice of the NTP study. Several of the larger ovarian
neoplasms of the 20-month-old mutant mice had evidence of malignancy with invasion of tumor cells through the ovarian capsule
into the periovarian tissues, often accompanied by a localized
desmoplastic response. Histopathologic evidence of malignancy
was not observed in the ovarian tubular adenomas from the
high-dose (2500 ppm) female mice in the NTP study. An occasional mutant mouse at 20 months of age also had developed focal
areas of hyperplasia of granulosal cells or small granulosa cell
tumors.
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Wx/Wv Strain with Genetic Deletion of Germ Cells in Ovarian
Cortex In an attempt to arrive at further mechanistic explanations
for the development of tubular adenomas in B6C3F1 mice fed high
doses of nitrofurantoin, ovaries were evaluated from several mutant mouse strains not exposed to any xenobiotic chemicals but
known to develop ovarian tumors. In this mutant mouse strain, referred to as Wx/Wv, few germ cells migrate into the ovary during
development. Murphy (1972) reported that less than 1 percent of
the normal complement of oocytes were present in the ovary at 1
day of age and the numbers of graafian follicles decrease progressively until none were present at 13 weeks of age. In this mutant
mouse strain (Wx/Wv), a mutant allele at the C-kit locus encodes
for a defective protein kinase receptor, resulting in an inability to
respond to stem cell growth factor encoded by the Steel locus
(Witte, 1990; Majumder et al., 1988). A failure of proliferation of
primordial germ cells during gonadogenesis leads to the marked
reduction of graafian follicles in the ovarian cortex.
Ovaries from mutant mice at age 13 weeks were small, had
an irregular surface, were devoid of graafian follicles, and had numerous hyperplastic tubules growing into the cortex. These tubules
were lined by a hyperplastic cuboidal epithelium similar to that on
the surface of the ovary. Interspersed between the tubular profiles
were luteinized stromal cells of the ovarian interstitium with a
lightly eosinophilic, often vacuolated, cytoplasm. The proliferative
changes observed in the ovary of these mutant (Wx/Wv) mice at
13 weeks of age were similar morphologically to the ovarian atrophy lesions in the NTP nitrofurantoin study.
The ovaries of heterozygous controls (1/1) of this strain were
larger than in the mutant mice and had a histologic appearance similar to normal mouse ovaries. The ovarian cortex in control mice
had plentiful graafian follicles with developing ova. The surface
epithelium covering the ovary consisted of a single layer of
cuboidal cells without the down-growth of tubules into the underlying cortex.
The ovaries of mutant (Wx/Wv) mice at 22 weeks of age had
a more intense proliferation of surface epithelium, either with extensive down-growths of hyperplastic tubules into the cortex or the
formation of small tubular adenomas. The tubular adenomas in the
mutant (Wx/Wv) mice with genetic deletion of graafian follicles
but without any exposure to xenobiotic chemicals were composed
of proliferating tubules of surface epithelium that replaced much
of the ovary. They were similar microscopically to the smaller tubular adenomas in the B6C3F1 mice fed the high dose (2500 ppm)
of nitrofurantoin in the 2-year NTP feeding study. Interspersed between the hyperplastic profiles of surface epithelium in the tubular adenomas were scattered luteinized stromal cells with varying
degrees of vacuolation of the eosinophilic cytoplasm. In mutant
(Wx/Wv) mice, age 22 weeks, whose ovaries had been under longterm intense gonadotrophin stimulation, there appeared to be a
morphologic continuum between ovarian atrophy and tubular
adenomas.
At age 20 months, ovaries of the mutant (Wx/Wv) mice without any exposure to xenobiotic chemicals consistently had large tubular adenomas that incorporated all of the ovarian parenchyma
and greatly enlarged the ovary. These neoplasms were similar his-
751
Hypogonadal (hpg/hpg) Mice Unable to Synthesize Hypothalamic Gonadotrophin-Releasing Hormone (GnRH) Mutant hypogonadal mice, designated hpg/hpg, are unable to synthesize normal amounts of hypothalamic GnRH (Tennent and Beamer, 1986).
They have low circulating levels of pituitary gonadotrophins (both
FSH and LH); however, hypogonadal mice have a normal complement of ovarian follicles (Cattanach et al., 1977).
In the studies of Tennent and Beamer (1986), both genetically
normal littermates and hypogonadal (hpg/hpg) mice were irradiated at age 30 days to destroy the oocytes. The irradiated control
mice of this strain produced normal amounts of pituitary gonadotrophic hormones and developed ovarian tubular adenomas at
age 10 to 15 months. The tumors that developed in the absence of
any exposure to xenobiotic chemicals had similar histological characteristics as tubular adenomas in the high-dose (2500 ppm) females of the nitrofurantoin study. They either were small nodules
involving only a portion of the ovary or large masses that completely incorporated the affected gonad. They were composed predominantly of tubular profiles, some of which were dilated, with
interspersed stromal cells.
The irradiated hypogonadal (hpg/hpg) mice failed to develop
tubular adenomas or to have intense hyperplasia of the ovarian surface epithelium and interstitial (stromal) cells in the absence of
GnRH and with low circulating levels of pituitary gonadotrophins.
The ovaries of irradiated hypogonadal mice were small and had
single- or multiple-layered follicles, without oocytes, scattered
throughout the ovary. There also was an absence of stromal cell
hypertrophy and hyperplasia, a change frequently observed in
ovaries of the irradiated normal littermates.
The experiments reported by Tennent and Beamer (1986)
demonstrated that a normal secretion of hypothalamic GnRH and
pituitary gonadotrophins was necessary for the intense proliferation of ovarian surface epithelium and stromal cells, leading to the
formation of tubular adenomas in mice, which develop subsequent
to irradiation-induced loss of ovarian follicles and decreased ability to produce gonadal steroids (especially estradiol-17).
Genetically Engineered Mouse Models of Ovarian Tumors Transgenic mice expressing a chimeric lutenizing hormone
(LH) submit (LH) in pituitary gonadotrophs have increased pituitary expression of LH mRNA and elevated circulating levels of
LH (as well as estradiol and testosterone) but are infertile (Risma
et al. 1995 and 1997). They ovulate infrequently, maintain a prolonged luteal phase, and develop a variety of ovarian lesions including cyst (blood and fluid filled) formation and ovarian tumors.
A subset of LH transgenic mice developed ovarian granulosa and
theca–interstitial cell tumors by 4 to 8 months of age as a result
of the chronic stimulation by the elevated gonadotropin (LH) lev-
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Summary: Ovarian Tumorigenesis
in Rodents
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A review of studies on mutant mice and the NTP study of nitrofurantoin support an interpretation that the unique intense hyperplasia of ovarian surface epithelium and stromal cells, leading eventually to an increased incidence of tubular adenomas and
occasionally granulosa cell tumors, develops secondary to chronic
pituitary gonadotrophic hormone stimulation. Factors that destroy
or greatly diminish the numbers of ovarian follicles—such as
senescence, genetic deletion of follicles, x-irradiation, drugs, and
chemicals such as nitrofurantoin, and early thymectomy with the
development of autoantibodies to oocytes—are known to diminish sex steroid hormone secretion by the ovary. This results in elevated circulating levels of gonadotrophins, especially LH, due to
decreased negative feedback on the hypothalamic-pituitary axis by
estrogens and possibly other humoral factors produced by the
graafian follicles (Carson et al., 1989). The long-term stimulation
of stromal (interstitial) cells, which have receptors for LH (Beamer
and Tennent, 1986), and, indirectly, the ovarian surface epithelium
appears to place the mouse ovary at increased risk for developing
the unique tubular or tubulostromal adenomas.
The finding of similar tubular adenomas in the ovaries of the
xenobiotic-treated and genetically sterile mice not exposed to exogenous chemicals supports the concept of a secondary (hormonally mediated) mechanism of ovarian oncogenesis associated with
hormonal imbalances (Fig. 21-47). The ovarian tumors developed
only in sterile mice in which the pituitary-hypothalamic axis was
intact; administration of exogenous estrogen early in the course
will prevent ovarian tumor development. The intense proliferation
of ovarian surface epithelium and stromal (interstitial) cells with
the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of human
adult females.
Experimental ovarian carcinogenesis has been investigated in
inbred and hybrid strains of mice and induced by a diversity of
Figure 21-47. Secondary mechanisms of ovarian oncogenesis in mice.
els. The findings of granulosa and stromal cell tumors in transgenic
mice whose only genetic alteration is the addition of a gene encoding a chimeric gonadotropin suggest that abnormal gonadotropin stimulation is tumorigenic to the ovary in mice. In
addition, genetically altered mice deficient in inhibin (hormone
which suppresses the secretion of follicle stimulating hormone
[FSH]) have elevated blood concentrations of FSH (two- to threefold) and develop granulosa cell tumors and mixed or incompletely
differentiated gonadal stromal tumors of the ovary (Matzuk et
al., 1992).
Another example of hormonal dysregulation leading to the induction of ovarian tumors are the estrogen receptor knockout
(ERKO) mice that lack the alpha estrogen receptors and are unable to regulate gonadotropin secretion due to a lack of negative
feedback control by the blood estrogen level (Lubahan et al., 1993;
Korach, 1994). Female mice are infertile with hypoplastic uteri and
hyperemic ovaries with no detectable corpora lutea. There was no
uterine stimulation (uterotropic response) when these animals were
treated with tamoxifen (an estrogen agonist in mice). ERKO mice
also develop ovarian granulosa cell tumors in response to the
chronic elevations in circulating gonadotropin levels.
Figure 21-48. Multiple pathogenic mechanisms in ovarian tumorigenesis of mice resulting in decreased negative feedback by diminished levels of gonadal steroids, particularly estrogen.
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CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
Figure 21-49. Decreased circulating estrogens release the hypothalamus-pituitary gland from negative feedback inhibition.
The increased gonadotrophin levels (LH and FSH) result in the mouse ovary being at greater risk of developing tubular adenomas in chronic studies.
mechanisms, including x-irradiation, oocytotoxic xenobiotic chemicals, ovarian grafting to ectopic or orthotopic sites, neonatal
thymectomy, mutant genes reducing germ cell populations, and aging (Fig. 21-48). Disruptions in the function of graafian follicles
by a variety of mechanisms results in a spectrum of ovarian proliferative lesions, including tumors. The findings in mutant mice
support the concept of a secondary (hormonally mediated) mechanism of ovarian carcinogenesis in mice associated with sterility.
Multiple pathogenetic factors that either destroy or diminish the
numbers of graafian follicles in the ovary result in decreased sex
hormone secretion (especially estradiol-17), leading to a compensatory overproduction of pituitary gonadotrophins (particularly
LH) (Fig. 21-48), which places the mouse ovary at an increased
risk for developing tumors (Fig. 21-49). The intense proliferation
of ovarian surface epithelium and stromal (interstitial) cells with
the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of human
adult females.
REFERENCES
GENERAL
Alison RH, Capen CC, Prentice DE: Neoplastic lesions of questionable significance to humans. Toxicol Pathol 22:179–186, 1994.
Capen CC: Correlation of mechanistic data and histopathology in the evaluation of selected toxic endpoints of the endocrine system. Toxicol
Lett 102–103:405–409, 1998.
Davies TS, Monro A: Marketed human pharmaceuticals reported to be tumorigenic in rodents. J Am Coll Toxicol 14:90–107, 1995.
Doss JC, Gröne A, Capen CC, et al: Immunohistochemical localization of
chromogranin A in endocrine tissues and endocrine tumors of dogs.
Vet Pathol 35:312–315, 1998.
Frith CH, Botts S, Jokinen MP, et al: Non-proliferative lesions of the
endocrine system in rats, in Guides for Toxicologic Pathology.
Washington, DC: STP/ARP/AFIP, 2000, pp 1–22.
Haseman JK, Hailey JR, Morris RW: Spontaneous neoplasm incidences in
Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity studies: A national toxicology program update. Toxicol Pathol 26:428–
441, 1998.
Keenan KP, Smith PF, Hertzog P, et al: The effects of overfeeding and dietary restriction of Sprague-Dawley rat survival and early pathology
biomarkers of aging. Toxicol Pathol 22:300–315, 1994.
Keenan KP, Soper KA, Smith PF: Diet, overfeeding, and moderate dietary
restriction in control Sprague-Dawley rats: I. Effects on spontaneous
neoplasms. Toxicol Pathol 23:269–286, 1995.
McClain RM: Mechanistic considerations in the regulation and classification of chemical carcinogens, in Kotsonis FN, Mackey M, Hjelle J
(eds): Nutritional Toxicology. New York: Raven Press, 1994, pp 273–
304.
Roe FJC, Lee PN, Conybeare G, et al: The biosure study: Influence of composition of diet and food consumption on longevity, degenerative diseases and neoplasia in Wistar rats studied for up to 30 months post
weaning. Food Chem Toxicol 33 (suppl 1): 1S–100S, 1995.
Solleveld HA, Haseman JK, McConnell EE: National history of body
weight gain, survival and neoplasia in the F344 rat. J Natl Cancer Inst
72:929–940, 1984.
Thurman JD, Bucci TJ, Hart RW, et al: Survival, body weight, and spontaneous neoplasms in ad libitum-fed and food-restricted Fischer-344
rats. Toxicol Pathol 22:1–9, 1994.
PITUITARY GLAND
Attia MA: Neoplastic and nonneoplastic lesions in aging female rats with
special reference to the functional morphology of the hyperplastic and
neoplastic changes in the pituitary gland. Arch Toxicol 57:77–83,
1985.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
754
4/26/01
4:58 PM
Page 754
UNIT 4 TARGET ORGAN TOXICITY
itary histology and serum prolactin levels as related to aging. Mech
Ageing Dev 42:75–90, 1988.
Yamagami T, Handa H, Takeuchi J, et al: Rat pituitary adenoma and hyperplasia induced by caffeine administration. Surg Neurol 20:323–
331, 1983.
ADRENAL CORTEX
Colby HD: Adrenal gland toxicity: chemically induced dysfunction. J Am
Coll Toxicol 7:45–69, 1988.
Dominick MA, Bobrowski WA, MacDonald JR, et al: Morphogenesis of a
zone-specific adrenocortical cytotoxicity in guinea pigs administered
PD 132301-2, an inhibitor of acyl-CoA; cholesterol acyltransferase.
Toxicol Pathol 21:54–62, 1993.
Dominick MA, McGuire EJ, Reindel JF, et al: Subacute toxicity of a novel
inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs. Fundam Appl Toxicol 20:217–224, 1993.
Hart MM, Swackhamer ES, Straw JA: Studies on the site of action of
o,p’-DDD in the dog adrenal cortex: II. TPNH- and corticosteroid
precursor-stimulation of o,p’-DDD inhibited steroidogenesis. Steroids
17:575–586, 1971.
Latendresse JR, Azhar S, Brooks CL, Capen CC: Pathogenesis of cholesteryl lipidosis of adrenocortical and ovarian interstitial cells in F344
rats caused by tricresyl phosphate and butylated triphenyl phosphate.
Toxicol Appl Pharmacol 122:281–289, 1993.
Latendresse JR, Brooks CL, Capen CC: Pathologic effects of butylated
triphenyl phosphate-based hydraulic fluid and tricresyl phosphate on
the adrenal gland, ovary and testis in the F344 rat. Toxicol Pathol
22:341–352, 1994.
Latendresse JR, Brooks CL, Capen CC: Toxic effects of butylated triphenyl phosphate-based hydraulic fluid and tricresyl phosphate in female
F344 rats. Vet Pathol 32:394–402, 1995.
Latendresse JR, Brooks CL, Fleming CD, Capen CC: Reproductive toxicity of butylated triphenyl phosphate (BTP) and tricresyl phosphate
(TCP) fluids in F344 rats. Fundam Applied Toxicol 22:392–399, 1994.
Reindel JF, Dominick MA, Bocan TM, et al: Toxicologic effects of a novel
acyl-CoA: Cholesterol acyltransferase (ACAT) inhibitor in cymomolgus monkeys. Toxicol Pathol 22:510–518, 1994.
Ribelin WE: The effects of drugs and chemicals upon the structure of the
adrenal gland. Fundam Appl Toxicol 4:105–119, 1984.
Rothuizen J, Reul JM, Fijnberk A, et al: Aging and the hypothalamuspituitary-adrenocortical axis, with special reference to the dog. Acta
Endocrinol 125(suppl):73–76, 1991.
Szabo S, Huttner I, Kovacs K, et al: Pathogenesis of experimental adrenal
hemorrhagic necrosis (“apoplexy”). Ultrastructural, biochemical, neuropharmacologic, and blood coagulation studies with acrylonitrate in
the rat. Lab Invest 42:533–546, 1980.
Vilar O, Tullner WW: Effects of o,p’-DDD on histology and 17-hydroxycorticosteroid output of the dog adrenal cortex. Endocrinology 65:80–
86, 1959.
Yarrington JT, Huffman KW, Gibson JP: Adrenocortical degeneration in
dogs, monkeys, and rats treated with -(1,4-dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone. Toxicol Lett 8:229–234, 1981.
Yarrington JT, Huffman KW, Leeson GA, et al: Comparative toxicity of
the hematinic MDL 80,478 effects on the liver and adrenal cortex of
the dog, rat and monkey. Fundam Appl Toxicol 3:86–94, 1983.
Yarrington JT, Johnston JO: Aging in the adrenal cortex, in Mohr U,
Dungworth DL, Capen CC (eds): Pathobiology of the Aging Rat.
Washington, DC: ILSI Press, 1994, pp 227–244.
Yarrington JT, Loudy DE, Sprinkle DJ, et al: Degeneration of the rat and
canine adrenal cortex caused by -(1,4-dioxido-3-methylquinoxalin2-yl)-N-methylnitrone (DMNM). Fundam Appl Toxicol 5:370, 1985.
Co
py
rig
hte
dM
ate
ria
l
Azad N, Nayyar R, Tentler J, et al: Anatomical and functional effects of
estrogen-induced prolactinomas on the rat hypothalamus. J Exp Pathol
4:237–249, 1989.
Barsoum NJ, Moore JD, Gough AW, et al: Morphofunctional investigations
on spontaneous pituitary tumors in Wistar rats. Toxicol Pathol 13:200–
208, 1985.
Berkvens JM, van Nesselrooy JHJ, Kroes R, et al: Spontaneous tumours
in the pituitary gland of old Wistar rats. A morphological and immunocytochemical study. J Pathol 130:179–191, 1980.
Berry PH: Effect of diet or reproductive status on the histology of spontaneous pituitary tumors in female Wistar rats. Vet Pathol 23:606–618,
1986.
Brown WR, Fetter AD, Van Ryzin RJ, et al: Proliferative pituitary lesions
in rats treated with salmon or porcine calcitonin. Toxicol Pathol 21:81–
86, 1993.
Capen CC: Functional and pathologic interrelationships of the pituitary gland and hypothalamus in animals, in Jones TC, Capen CC,
Mohr U (eds): Endocrine System. Series II. Monographs on Pathology of Laboratory Animals, 2d ed. International Life Sciences Institute Series. Berlin, Heidelberg, New York: Springer-Verlag, 1996, pp
3–32.
El Etreby MF, Lorenz B, Habenicht UF: Immunocytochemical studies on
the pituitary gland and spontaneous pituitary tumors of SpragueDawley rats. Pathol Res Pract 183:645–650, 1988.
Furth J, Boon MC: Induction of ovarian tumors in mice by x-rays. Cancer
Res 7:241–245, 1947.
Gunnison AF, Bowers A, Nadziejko C, et al: Modulation of the inflammatory effects of inhaled ozone in rats by subcutaneous prolactinsecreting, pituitary-derived tumors. Fundam Appl Toxicol 37:88–94,
1997.
Jameson JL, Weiss J, Polak JM, et al: Glycoprotein hormone alpha-subunitproducing pituitary adenomas in rats treated for one year with calcitonin. Am J Pathol 140:75–84, 1992.
McComb DJ, Kovacs K, Beri J, et al: Pituitary gonadotroph adenomas in
old Sprague-Dawley rats. J Submicrosc Cytol 17:517–530, 1985.
McComb DJ, Kovacs K, Beri J, et al: Pituitary adenomas in old SpragueDawley rats: A histologic, ultrastructural, and immunocytochemical
study. J Natl Cancer Inst 73:1143–1166, 1984.
Osamura RY, Komatsu N, Izumi S, et al: Ultrastructural localization of prolactin in the rat anterior pituitary glands by preembedding peroxidaselabeled antibody method: Observations in normal, castrated, or
estrogen-stimulated specimen. J Histochem Cytochem 30:919–925,
1982.
Parkening TA, Collins TJ, Smith ER: A comparative study of prolactin levels in five species of aged female laboratory rodents. Biol Reprod
22:513–518, 1980.
Pickering CE, Pickering RG: The effect of repeated reproduction on the incidence of pituitary tumours in Wistar rats. Lab Anim 18:371–378,
1984.
Sandusky GE, Van Pelt CS, Todd GC, et al: An immunocytochemical study
of pituitary adenomas and focal hyperplasia in old Sprague-Dawley
and Fischer 344 rats. Toxicol Pathol 16:376–380, 1988.
Sarkar DK, Gottschall PE, Meites J: Damage to hypothalamic dopaminergic neurons is associated with development of prolactin-secreting pituitary tumors. Science 218:684–686, 1982.
Satoh H, Kajimura T, Chen C-J, et al: Invasive pituitary tumors in female
F344 rats induced by estradiol dipropionate. Toxicol Pathol 25:462–
469, 1997.
Sher SP, Jensen RD, Bokelman DL: Spontaneous tumors in control F344
and Charles River-CD rats and Charles River CD-1 and B6C3HF1 mice.
Toxicol Lett 11:103–110, 1982.
van Putten LJA, van Zwieten MJ: Studies on prolactin-secreting cells in
aging rats of different strains: II. Selected morphological and immunocytochemical features of pituitary tumors correlated with serum
prolactin levels. Mech Ageing Dev 42:115–127, 1988.
van Putten LJA, van Zwieten MJ, Mattheij JAM, et al: Studies on prolactinsecreting cells in aging rats of different strains. I. Alterations in pitu-
ADRENAL MEDULLA
Baer A: Sugars and adrenomedullary proliferative lesions: The effects of
lactose and various polyalcohols. J Am Coll Toxicol 7:71–81, 1988.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
4/26/01
4:58 PM
Page 755
CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol Appl Pharmacol 113:36–42, 1992.
Borzelleca JF, Capen CC, Hallagan JB: Lifetime toxicity carcinogenicity
study of FC&C Red No. 3 (erythrosine) in rats. Food Chem Toxicol
25:723–733, 1987.
Capen C, Sagartz J: A Ret transgenic mouse model of thyroid carcinogenesis. Lab Anim Sci 48:580–583, 1998.
Capen CC, DeLellis RA, Williams ED: Experimental thyroid carcinogenesis: Role of radiation and xenobiotic chemicals, in: Proceedings of
the First International Conference on Radiation and Thyroid Cancer.
St. John’s College, Cambridge, UK, 20-23 July 1998; and in Thomas
G, Karaoglou A, Williams ED (eds): Radiation and Thyroid Cancer.
Singapore, New Jersey, London, Hong Kong: World Scientific Publishing, 1999, pp 167–176.
Capen CC: Comparative anatomy and physiology of the thyroid, in Braverman LE, Utiger RD (eds): Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text, 8th ed. Philadelphia: Lippincott-Raven,
2000, pp 20–44.
Capen CC: Thyroid and parathyroid toxicology, in Harvey PW, Rush K,
Cockburn A (eds): Endocrine and Hormonal Toxicology. Sussex,
England: Wiley, 1999, pp 33–66.
Capen CC: Mechanistic data and risk assessment of selected toxic end
points of the thyroid gland. Toxicol Pathol 25:39–48, 1997.
Capen CC: Mechanistic considerations for thyroid gland neoplasia with
FD&C Red No. 3 (erythrosine), in The Toxicology Forum. Proceedings of the 1989 Annual Winter Meeting, Washington, DC, 1989; pp
113–130.
Capen CC, Martin SL: The effects of xenobiotics on the structure and function of thyroid follicles and C cells. Toxicol Pathol 17:266–293, 1989.
Clements FW: Relationship of thyrotoxicosis and carcinoma of thyroid to
endemic goiter. Med J Aust 2:894–899, 1954.
Clemmesen J, Fuglsang-Frederiksen V, Plum CM: Are anticonvulsants
oncogenic? Lancet 1:705–707, 1974.
Clemmesen J, Hjalgrim-Jensen S: Does phenobarbital cause intracranial tumors? A follow-up through 35 years. Ecotoxicol Environ Saf 5:255–
260, 1981.
Clemmesen J, Hjalgrim-Jensen S: Is phenobarbital carcinogenic? A followup of 8078 epileptics. Ecotoxicol Environ Saf 1:255–260, 1978.
Clemmesen J, Hjalgrim-Jensen S: On the absence of carcinogenicity to man
of phenobarbital, in Statistical Studies in the Aetiology of Malignant
Neoplasms. Acta Pathol Microb Scand 261 (suppl):38–50, 1977.
Collins WT Jr, Capen CC: Ultrastructural and functional alterations in thyroid glands of rats produced by polychlorinated biphenyls compared
with the effects of iodide excess and deficiency, and thyrotropin and
thyroxine administration. Virchows Arch B Cell Pathol 33:213–231,
1980.
Cooper DS, Axelrod L, DeGroot LJ, et al: Congenital goiter and the development of metastatic follicular carcinoma with evidence for a leak
of non-hormonal iodide: Clinical, pathological, kinetic, and biochemical studies and a review of the literature. J Clin Endocrinol Metab
52:294–306, 1981.
Copp DH: Endocrine regulation of calcium metabolism. Annu Rev Physiol
32:61–86, 1970.
Curran PG, DeGroot LJ: The effect of hepatic enzyme-inducing drugs on
thyroid hormones and the thyroid gland. Endocr Rev 12:135–150,
1991.
Döhler K-D, Wong CC, von zut Mühlen A: The rat as model for the study
of drug effects on thyroid function: consideration of methodological
problems. Pharmacol Ther 5:305–313, 1979.
Doniach I: Aetiological consideration of thyroid carcinoma, in Smithers D
(ed): Tumors of the Thyroid Gland. London: E & S Livingstone, 1970;
pp 55–72.
Friedman GD: Barbiturates and lung cancer in humans. J Natl Cancer Inst
67:291–295, 1981.
Gutshall DM, Pilcher GD, Langley AE: Effect of thyroxine supplementation on the response to perfluoro-n-decanoic acid (PFDA) in rats. J
Toxicol Environ Health 24:491–498, 1988.
Co
py
rig
hte
dM
ate
ria
l
Lynch BS, Tischler AS, Capen C, et al: Low digestible carbohydrates (polyols and lactose): Significance of adrenal medullary proliferative lesions in the rat. Regul Toxicol Pharmacol 23:256–297, 1996.
Manger WM, Hulse MC, Forsyth MS, et al: Effect of pheochromocytoma
and hypophysectomy on blood pressure and catecholamines in NEDH
rats. Hypertension 4(suppl 2):200–202, 1982.
Mosher AH, Kircher CH: Proliferative lesions of the adrenal medulla in
rats treated with Zomepirac sodium. J Am Coll Toxicol 7:83–93, 1988.
Nyska A, Haseman JK, Hailey JR, et al: The association between severe
nephropathy and pheochromocytoma in the male F344 rat—The national toxicology program experience. Toxicol Pathol 27:456–462,
1999.
Puchacz E, Stumpf WE, Stachowiak EK, et al: Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells.
Mol Brain Res 36:193–196, 1996.
Reznik G, Ward JM, Reznik-Shuller H: Ganglioneuromas in the adrenal
medulla of F344 rats. Vet Pathol 17:614–621, 1980.
Roe FJC, Bär A: Enzootic and epizootic adrenal medullary proliferative
disease of rats: Influence of dietary factors which affect calcium absorption. Hum Toxicol 4:27–52, 1985.
Tischler AS, DeLellis RA: The rat adrenal medulla: I. The normal adrenal.
J Am Coll Toxicol 7:1–21, 1988.
Tischler AS, DeLellis RA: The rat adrenal medulla: II. Proliferative lesions.
J Am Coll Toxicol 7:23–44, 1988.
Tischler AS, DeLellis RA, Nunnemacher G, et al: Acute stimulation of
chromaffin cell proliferation in the adult rat adrenal medulla. Lab
Invest 58:733–735, 1988.
Tischler AS, DeLellis RA, Perlman RL, et al: Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats: Comparison
to PC12 cells, small granule-containing cells, and human adrenal
medullary hyperplasia. Lab Invest 53:486–498, 1985.
Tischler AS, McClain RM, Childers H, et al: Neurogenic signals regulate
chromaffin cell proliferation and mediate the mitogenic effect of reserpine in the adult rat adrenal medulla. Lab Invest 65:374–376, 1991.
Tischler AS, Powers JF, Downing JC, et al: Vitamin D3, lactose, and xylitol stimulate chromaffin cell proliferation in the rat adrenal medulla.
Toxicol Appl Pharmacol 140:115–123, 1996.
Tischler AS, Powers JF, Pignatello M, et al: Vitamin D3-induced proliferative lesions in the rat adrenal medulla. Toxicol Sci 51:9–18, 1999.
Tischler AS, Riseberg J: Different responses to mitogenic agents by adult
rat and human chromaffin cells in vitro. Endocr Pathol 4:15–19, 1993.
Tischler AS, Riseberg J, Cherington V: Multiple mitogenic signaling pathways in chromaffin cells: A model for cell cycle regulation in the nervous system. Neurosci Lett 168:181–184, 1994.
Tischler AS, Ruzicka LA, Van Pelt CS, et al: Catecholamine-synthesizing
enzymes and chromogranin proteins in drug-induced proliferative lesions of the rat adrenal medulla. Lab Invest 63:44–51, 1990.
Tischler AS, Ziar J, Downing JC, et al: Sustained stimulation of rat adrenal chromaffin cell proliferation by reserpine. Toxicol Appl Pharmacol 135:254–257, 1995.
Warren S, Gruzdev L, Gates O, et al: Radiation induced adrenal medullary
tumors in the rat. Arch Pathol 82:115–118, 1966.
THYROID GLAND (FOLLICULAR CELLS)
Atterwill CK, Collins P, Brown CG, et al: The perchlorate discharge test
for examing thyroid function in rats. J Pharmacol Methods 18:199–
203, 1987.
Axelrod AA, Leblond CP: Induction of thyroid tumors in rats by low iodine diet. Cancer 8:339–367,1955.
Barter RA, Klaassen CD: Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: Characterization, induction, and form
specificity. Toxicol Appl Pharmacol 115:261–267, 1992.
Barter RA, Klaassen CD: Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol Appl Pharmacol 128:9–17, 1994.
Barter RA, Klaassen CD: UDP-glucuronosyltransferase inducers reduce
755
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
756
4/26/01
4:58 PM
Page 756
UNIT 4 TARGET ORGAN TOXICITY
Lamm SH, Doemland M: Has perchlorate in drinking water increased the
rate of congenital hypothyroidism? J Occup Environ Med 41:409–411,
1999.
Li Z, Li FX, Byrd D, et al: Neonatal thyroxine level and perchlorate in
drinking water. J Occup Environ Med 42:200–205, 2000.
Liu J, Liu Y, Barter RA, et al: Alteration of thyroid homeostasis by UDPglucuronosyltransferase inducers in rats: A dose-response study.
J Pharmacol Exp Ther 273:977–985, 1995.
Many M-C, Denef J-F, Haumont S, et al: Morphological and functional
changes during thyroid hyperplasia and involution in C3H mice:
Effects of iodine and 3,5,38-triiodothyronine during involution.
Endocrinology 116:798, 1985.
McClain RM: The use of mechanistic data in cancer risk assessment: Case
example sulfonamides, in Low-Dose Extrapolation of Cancer Risk: Issues and Perspectives. International Life Sciences Institute Series.
Washington, DC: ILSI Press, 1995, pp 163–173.
McClain RM: The significance of hepatic microsomal enzyme induction
and altered thyroid function in rats: Implications for thyroid gland neoplasia. Toxicol Pathol 17:294–306, 1989.
McClain RM, Levin AA, Posch R, et al: The effects of phenobarbital on
the metabolism and excretion of thyroxine in rats. Toxicol Appl Pharmacol 99:216–228, 1989.
McClain RM, Posch RC, Bosakowski T, et al: Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital. Toxicol Appl Pharmacol 94:254–265, 1988.
McGirr EM, Clement WE, Currie AR, et al: Impaired dehalogenase activity as a cause of goiter with malignant changes. Scott Med J 4:232–
242, 1959.
Morris HP: The experimental development and metabolism of thyroid gland
tumors. Adv Cancer Res 3:51–115, 1955.
Napalkov NP: Tumours of the thyroid gland, in Turusov VS (ed): Pathology of Tumors in Laboratory Animals: Part 2. Vol 1. Tumors of the
Rat. IARC Scientific Publication No. 6. Lyons, France: IARC, 1976,
pp 239–272.
Nilsson M, Engström G, Ericson LE: Graded response in the individual
thyroid follicle cell to increasing doses of TSH. Mol Cell Endocrinol
44:165, 1986.
Ohnhaus EE, Burgi H, Burger A, et al: The effect of antipyrine, phenobarbital, and rifampicin on the thyroid hormone metabolism in man.
Eur J Clin Invest 11:381–387, 1981.
Ohnhaus EE, Studer H: A link between liver microsomal enzyme activity
and thyroid hormone metabolism in man. Br J Clin Pharmacol 15:71–
76, 1983.
Ohshima M, Ward JM: Dietary iodine deficiency as a tumor promoter and
carcinogen in male F344/NCr rats. Cancer Res 46:877–883, 1986.
Ohshima M, Ward JM: Promotion of N-methyl-N-nitrosourea-induced thyroid tumors by iodine deficiency in F344/NCr rats. J Natl Cancer Inst
73:289–296, 1984.
Olsen JH, Boice JD, Jensen JP, et al: Cancer among epileptic patients exposed to anticonvulsant drugs. J Natl Cancer Inst 81:803–808, 1989.
Ozaki A, Sagartz JE, Capen CC: Phagocytic activity of FRTL-5 rat thyroid
follicular cells as measured by ingestion of fluorescent latex beads.
Exp Cell Res 219:547–554, 1995.
Paynter OH, Burin GJ, Jaeger RB, et al: Goitrogens and thyroid follicular
cell neoplasmia: Evidence for a threshold process. Reg Toxicol Pharmacol 8:102–119, 1988.
Pendergrast WJ, Milmore BK, Marcus SC: Thyroid cancer and toxicosis
in the United States: Their relation to endemic goiter. J Chronic Dis
13:22–38, 1961.
Poirier LA, Doerge DR, Gaylor DW, et al: An FDA review of sulfamethazine toxicity. Regul Toxicol Pharmacol 30:217–222, 1999.
Sagartz JE, Jhiang SM, Tong Q, Capen CC: Thyroid stimulating hormone
promotes growth of thyroid carcinomas in transgenic mice with targeted expression of ret/PTC1 oncogene. Lab Invest 76:307–318, 1997.
Sagartz JE, Ozaki A, Capen CC: Phagocytosis of fluorescent beads by rat
thyroid follicular (FRTL-5) cells: Comparison with iodine uptake as
an index of functional activity of thyrocytes in vitro. Toxicol Pathol
23:635–643, 1995.
Co
py
rig
hte
dM
ate
ria
l
Hard GC: Recent developments in the investigation of thyroid regulation
and thyroid carcinogenesis. Environ Health Perspect 106:427–436,
1998.
Hiasa Y, Kitahori Y, Kato Y, et al: Potassium perchlorate, potassium iodide,
and propylthiouracil: Promoting effect on the development of thyroid
tumors in rats treated with N-Bis(2-hydroxypropyl)-nitrosamine. Jpn
J Cancer Res 78:1335–1340, 1987.
Hill RN, Crisp TM, Hurley PM, et al: Risk assessment of thyroid follicular cell tumors. Environ Health Perspect 106:447–457, 1998.
Hill RN, Erdreich LS, Paynter O, et al: Assessment of thyroid follicular
cell tumors: Risk Assessment Forum. Washington, DC: U.S. Environmental Protection Agency, March 1998, pp 1–43.
Hill RN, Erdreich LS, Paynter O, et al: Thyroid follicular cell carcinogenesis. Fundam Appl Toxicol 12:629–697, 1989.
Hood A, Harstad E, Klaassen CD: Effects of UDP-glucuronosyltransferase
(UDP-GT) inducers on thyroid cell proliferation. Toxicologist 15:229,
1995.
Hood A, Hashmi R, Klaassen CD: Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol Appl Pharmacol 160:163–170, 1999.
Hood A, Klaassen CD: Differential effects of microsomal enzyme inducers on in vitro thyroxine (T4) and triiodothyronine (T3) glucuronidation. Toxicol Sci 55:78–84, 2000.
Hood A, Klaassen CD: Effects of microsomal enzyme inducers on outerring deiodinase activity toward thyroid hormones in various rat tissues. Toxicol Appl Pharmacol 163:240–248, 2000.
Hood A, Liu YP, Gattone VH II, et al: Sensitivity of thyroid gland growth
to thyroid stimulating hormone (TSH) in rats treated with antithyroid
drugs. Toxicol Sci 49:263–271, 1999.
Hood A, Liu YP, Klaassen CD: Effects of phenobarbital, pregnenolone16-carbonitrile, and propylthiouracil on thyroid follicular cell proliferation. Toxicol Sci 50:45–53, 1999.
Hotz KJ, Wilson AGE, Thake DC, et al: Mechanism of thiazopyr-induced
effects on thyroid hormone homeostasis in male Sprague-Dawley rats.
Toxicol Appl Pharmacol 142:133–142, 1997.
Hurley PM, Hill RN, Whiting RJ: Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents. Environ Health
Perspect 106:437–445, 1998.
Ingbar SH: Autoregulation of the thyroid. Response to iodide excess and
depletion. Mayo Clin Proc 47:814, 1972.
Jhiang SM, Cho J-Y, Furminger TL, et al: Thyroid carcinomas in RET/PTC
transgenic mice, in Schwab M, Rabes H, Munk K, Hofschneider PH
(eds): Genes and Environment in Cancer. Recent Results in Cancer
Research Vol 154, Berlin: Springer-Verlag, 1998, pp 265–270.
Jhiang SM, Cho J-Y, Ryu K-Y, et al: An immunohistochemical study of
Na/I symporter in human thyroid tissues and salivary gland tissues.
Endocrinology 139:4416, 1998.
Jhiang SM, Sagartz JE, Tong Q, et al: Targeted expression of the ret/PTC1
oncogene induces papillary thyroid carcinomas. Endocrinology
137:375–378, 1996.
Kanno J, Nemoto T, Kasuga T, et al: Effects of a six-week exposure to
excess iodide on thyroid glands of growing and nongrowing male
Fischer-344 rats. Toxicol Pathol 22:23–28, 1994.
Kennedy TH, Purves HD: Studies on experimental goiter: Effect of Brassica seed diets on rats. Br J Exp Pathol 22:241–244, 1941.
Kolaja KL, Klaassen CD: Dose-response examination of UDP-glucuronosyltransferase inducers and their ability to increase both TGF- expression and thyroid follicular cell apoptosis. Toxicol Sci 46:31–37,
1998.
Kolaja KL, Petrick JS, Klaassen CD: Inhibition of gap-junctional-intercellular communication in thyroid-follicular cells by propylthiouracil and
low iodine diet. Toxicology 143:195–202, 2000.
Lamas L, Ingbar SH: The effect of varying iodine content on the susceptibility of thyroglobulin to hydrolysis by thyroid acid protease.
Endocrinology 102:188–197, 1978.
Lamm SH, Braverman LE, Li FX, et al: Thyroid health status of ammonium perchlorate workers: A cross-sectional occupational health study.
J Occup Environ Med 41:248–260.
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
4/26/01
4:58 PM
Page 757
CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
Thurston V, Williams ED: Experimental induction of C-cell tumours in thyroid by increased dietary content of vitamin D2. Acta Endocrinol
100:41–45, 1982.
Triggs SM, Williams ED: Experimental carcinogenesis in the rat thyroid
follicular and C cells. Acta Endocrinol 85:84–92, 1977.
PARATHYROID GLAND
Abdo KM, Eustis SL, Haseman J, et al: Toxicity and carcinogenicity of
rotenone given in the feed to F344/N rats and B6C3F1 mice for up to
two years. Drug Chem Toxicol 11:225–235, 1988.
Armbrecht HJ, Wongsurawat N, Zenser TV, et al: Differential effects of
parathyroid hormone on the renal 125-dihydroxyvitamin D3 and
24,25-dihydroxyvitamin D3 production of young and adult rats.
Endocrinology 111:1339–1344, 1982.
Arnold DL, Moodie CA, Charbonneau SM, et al: Long-term toxicity of
hexachlorobenzene in the rat and the effect of dietary vitamin A. Chem
Toxicol 23:779–793, 1985.
Atwal OS: Ultrastructural pathology of ozone-induced experimental
parathyroiditis: IV. Biphasic activity in the chief cells of regenerating
parathyroid glands. Am J Pathol 95:611–632, 1979.
Atwal OS, Pemsingh RS: Morphology of microvascular changes and endothelial regeneration in experimental ozone-induced parathyroiditis:
III. Some pathologic considerations. Am J Pathol 102:297–307, 1981.
Atwal OS, Samagh BS, Bhatnagar MK: A possible autoimmune parathyroiditis following ozone inhalation: II. A histopathologic, ultrastructural, and immunofluorescent study. Am J Pathol 80:53–68, 1975.
Atwal OS, Wilson T: Parathyroid gland changes following oxone inhalation. A morphologic study. Arch Environ Health 28:91–100, 1974.
Barbosa JA, Gill BM, Takiyyuddin MA, et al: Chromogranin A: Posttranslational modifications in secretory granules. Endocrinology
128:174–190, 1991.
Bourdeau AM, Plachot JJ, Cournot-Witmer G, et al: Parathyroid response
to aluminum in vitro: Ultrastructural changes and PTH release. Kidney Int 31:15–24, 1987.
Brown EM: PTH secretion in vivo and in vitro. Regulation by calcium and
other secretagogues. Miner Electrolyte Metab 8:130–150, 1982.
Capen CC: Age-related changes in structure and function of the parathyroid gland in laboratory rats, in Mohr U, Dungworth DL, Capen CC
(eds): Pathobiology of the Aging Rat. Vol. II. Washington, DC: International Life Sciences Press (ILSI), 1994, pp 199–226.
Capen CC: Pathobiology of parathyroid gland structure and function in
animals, in Jones TC, Capen CC, Mohr U (eds): Endocrine System.
Series II. Monographs on Pathology of Laboratory Animals, 2d ed.
International Life Sciences Institute Series. Berlin, Heidelberg, New
York: Springer-Verlag, 1996, pp 293–327.
Capen CC, Gröne A, Bucci TJ, et al: Changes in structure and function of
the parathyroid gland, in Mohr U, Dungworth D, Capen C, Sundberg
J (eds): Pathobiology of the Aging Mouse. Washington, DC: International Life Sciences (ILSI) Press, 1996, pp 109–124.
Chisari FV, Hochstein HD, Kirschstein RL: Parathyroid necrosis and
hypocalcemic tetany induced in rabbits by L-asparaginase. Am J Pathol
69:461–476, 1972.
Cloutier M, Rousseau L, Gascon-Barré M, et al: Immunological evidences
for post-translational control of the parathyroid function by ionized
calcium in dogs. Bone Miner 22:197–207, 1993.
Cohn DV, Fasciotto BH, Zhang J-X, et al: Chemistry and biology of chromogranin A (secretory protein-I) of the parathyroid and other endocrine glands, in Bilezikian JP, Marcus R, Levine MA (eds): The
Parathyroids: Basic and Clinical Concepts. New York: Raven Press,
1993, pp 107–119.
Fasciotto BH, Gorr S-U, Bourdeau AM, et al: Autocrine regulation of
parathyroid secretion: Inhibition of secretion by chromogranin-A
(secretory protein-I) and potentiation of secretion by chromogranin-A
and pancreastatin antibodies. Endocrinology 127:1329–1335, 1990.
Goltzman D, Bennett HPJ, Koutsilieris M, et al: Studies of the multiple
molecular forms of bioactive parathyroid hormone and parathyroid
hormone-like substances. Recent Prog Horm Res 42:665–703, 1986.
Co
py
rig
hte
dM
ate
ria
l
Shirts SB, Annegers JF, Hauser WA, et al: Cancer incidence in a cohort of
patients with seizure disorders. J Natl Cancer Inst 77:83–87, 1986.
Spitzweg C, Heufelder AE, Morris JC: Review: Thyroid iodine transport.
Thyroid 10:321–330, 2000.
Swarm RL, Roberts GKS, Levy AC: Observations on the thyroid gland in
rats following the administration of sulfamethoxazole and trimethoprim. Toxicol Appl Pharmacol 24:351–363, 1973.
Tajima K, Miyagawa J-I, Nakajima H, et al: Morphological and biochemical studies on minocycline-induced black thyroid in rats. Toxicol Appl
Pharmacol 81:393–400, 1985.
Takayama S, Aihara K, Onodera T, et al: Antithyroid effects of propylthiouracil and sulfamonomethoxine in rats and monkeys. Toxicol Appl
Pharmacol 82:191–199, 1986.
Waritz RS, Steinberg M, Kinoshita FK, et al: Thyroid function and thyroid
tumors in toxaphene-treated rats. Regul Toxicol Pharmacol 24:184–
192, 1996.
White SJ, McLean AEM, Howland C: Anticonvulsant drugs and cancer. A
cohort study in patients with severe epilepsy. Lancet 2:458–461, 1979.
Wynford-Thomas D, Smith P, Williams ED: Proliferative response to cyclic
AMP elevation of thyroid epithelium in suspension culture. Mol Cell
Endocrinol 51:163, 1987.
Wynford-Thomas D, Stringer BM, Williams ED: Desensitisation of rat thyroid to the growth-stimulating action of TSH during prolonged goitrogen administration. Persistence of refractoriness following withdrawal
of stimulation. Acta Endocrinol Copenh 101:562–569, 1982.
Wynford-Thomas D, Stringer BM, Williams ED: Dissociation of growth
and function in the rat thyroid during prolonged goitrogen administration. Acta Endocrinol Copenh 101:210–216, 1982.
Wynford-Thomas V, Wynford-Thomas D, Williams ED: Experimental induction of parathyroid adenomas in the rat. J Natl Cancer Inst 70:127–
134, 1982.
Zbinden G: Hyperplastic and neoplastic responses of the thyroid gland in
toxicological studies. Arch Toxicol Suppl 12:98–106, 1988.
THYROID C CELLS
Burek JD: Pathology of Aging Rats. West Palm Beach, FL: CRC Press,
1978.
DeGrandi PB, Kraehenbuhl JP, Campiche MA: Ultrastructural localization
of calcitonin in the parafollicular cells of the pig thyroid gland with
cytochrome c-labeled antibody fragments. J Cell Biol 50:446–456,
1971.
DeLellis RA, Dayal Y, Tischler AS, et al: Multiple endocrine neoplasia
syndromes. Cellular origins and interrelationships. Int Rev Exp Pathol
28:163–215, 1986.
DeLellis RA, Nunnemacher G, Bitman WR, et al: C cell hyperplasia and
medullary thyroid carcinoma in the rat: An immunohistochemical and
ultrastructural analysis. Lab Invest 40:140–154, 1979.
DeLellis RA, Nunnemacher G, Wolfe HJ: C cell hyperplasia, an ultrastructural analysis. Lab Invest 36:237–248, 1977.
Foster GV, Byfield PGH, Gudmundsson TV: Calcitonin, in MacIntyre I
(ed): Clinics in Endocrinology and Metabolism. Vol. 1. Philadelphia:
Saunders, 1972, pp 93–124.
Kalina M, Pearse AGE: Ultrastructural localization of calcitonin in C cells
of dog thyroid: An immunocytochemical study. Histochemie 26:1–8,
1971.
Leblanc B, Paulus G, Andreu M, et al: Immunocytochemistry of thyroid
C-cell complexes in dogs. Vet Pathol 27:445–452, 1990.
Nonidez José F: The origin of the “parafollicular” cell, A second epithelial component of the thyroid gland of the dog. Am J Anat 49:479–
505, 1931–1932.
Stoll R, Faucounau N, Maraud R: Les adenomes a cellules folliculaires
et parafolliculaires de la thyroide du rat soumis au thiamazole. [Development of follicular and parafollicular adenomas in the thyroid of
rats treated with thiamazole.] Ann Endocrinol (Paris), 39, 179–189,
1978.
Teitelbaum SL, Moore KE, Shieber W: C cell follicles in the dog thyroid:
Demonstration by in vivo perfusion. Anat Rec 168:69–78, 1970.
757
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
4/26/01
4:58 PM
Page 758
758
UNIT 4 TARGET ORGAN TOXICITY
Fort FL, Miyajima H, Ando T, et al: Mechanism for species-specific induction of Leydig cell tumors in rats by lansoprazole. Fundam Appl
Toxicol 26:191–202, 1995.
McConnell RF, Westen HH, Ulland BM, et al: Proliferative lesions of
the testes in rats with selected examples from mice, in Guides
for Toxicologic Pathology. Washington, DC: STP/ARP/AFIP, 1992, pp
1–32.
Murakami M, Hosokawa S, Yamada T, et al: Species-specific mechanism
in rat Leydig cell tumorigenesis by procymidone. Toxicol Appl Pharmacol 131:244–252, 1995.
Prahalada S, Majka JA, Soper KA, et al: Leydig cell hyperplasia and adenomas in mice treated with finasteride, a 5-reductase inhibitor: A
possible mechanism. Fundam Appl Toxicol 22:211–219, 1994.
Prentice DE, Meikle AW: A review of drug-induced Leydig cell hyperplasia and neoplasia in the rat and some comparisons with man. Hum
Exp Toxicol 14:562–572, 1995.
Waalkes MP, Rehm S, Devor DE: The effects of continuous testosterone
exposure on spontaneous and cadmium-induced tumors in the male
Fischer (F344/NCr) rat: Loss of testicular response. Toxicol Appl Pharmacol 142:40–46, 1997.
Co
py
rig
hte
dM
ate
ria
l
Habener JF: Recent advances in parathyroid hormone research. Clin
Biochem 14:223–229, 1981.
Jaffe N, Traggis D, Lakshmi D, et al: Comparison of daily and twice-weekly
schedule of L-asparaginase in childhood leukemia. Pediatrics 49:590–
595, 1972.
Kalu DN, Hardin RR, Murata I, et al: Age-dependent modulation of
parathyroid hormone action. Age 5:25–29, 1982.
Kronenberg HM, Igarashi T, Freeman MW, et al: Structure and expression
of the human parathyroid hormone gene. Recent Prog Horm Res
42:641–663, 1986.
Lupulescu A, Potorac E, Pop A: Experimental investigation on immunology of the parathyroid gland. Immunology 14:475–482, 1968.
Morrissey J, Rothstein M, Mayor G, et al: Suppression of parathyroid hormone secretion by aluminum. Kidney Int 23:699–704, 1983.
Morrissey J, Slatopolsky E: Effect of aluminum on parathyroid hormone
secretion. Kidney Int 29:S41–S44, 1986.
Oettgen HF, Old LJ, Boyse EA, et al: Toxicity of E. coli L-asparaginase in
man. Cancer 25:253–278, 1970.
Oslapas R, Prinz R, Ernst K, et al: Incidence of radiation-induced parathyroid tumors in male and female rats. Clin Res 29:734A, 1981.
Oslapas R, Shah KH, Hoffman, et al: Effect of gonadectomy on the incidence of radiation-induced parathyroid tumors in male and female rats.
Clin Res 30:401A, 1982.
Pollak MR, Brown EM, Chou YW, et al: Mutations in the human Ca2sensing receptor gene cause familial hypocalciuric hypercalcemia and
neonatal severe hypercalcemia. Cell 75:1297–1303, 1993.
Silver J: Regulation of parathyroid hormone synthesis and secretion, in Coe
FL, Favus MJ (eds): Disorders of Bone and Mineral Metabolism. New
York: Raven Press, 1992, pp 83–106.
Tettenborn D, Hobik HP, Luckhaus G: Hypoparathyroidismus beim Kaninchen nach Verabreichung von L-asparaginase. Arzneimittelforschung
20:1753–1755, 1970.
Wada L, Daly R, Kern D, et al: Kinetics of 1,25-dihydroxyvitamin D metabolism in the aging rat. Am J Physiol 262 (Endocrinol Metab, 25):
E906–E910, 1992.
Wongsurawat N, Armbrecht HJ: Comparison of calcium effect on in vitro
calcitonin and parathyroid hormone release by young and aged thyroparathyroid glands. Exp Gerontol 22:263–269, 1987.
Young DM, Olson HM, Prieur DJ, et al: Clinicopathologic and ultrastructural studies of L-asparaginase-induced hypocalcemia in rabbits. An
experimental animal model of acute hypoparathyroidism. Lab Invest
29:374–386, 1973.
TESTIS
Bär A: Significance of Leydig cell neoplasia in rats fed lactitol or lactose.
J Am Coll Toxicol 11:189–207, 1992.
Bartke A, Sweeney CA, Johnson L, et al: Hyperprolactinemia inhibits development of Leydig cell tumors in aging Fischer rats. Exp Aging Res
11:123–128, 1985.
Boorman GA, Hamlin MH, Eustis SL: Focal interstitial cell hyperplasia,
testis, rat, in Jones TC, Möhr U, Hunt RD (eds): Monographs on
Pathology of Laboratory Animals. Berlin: Springer-Verlag, 1987; pp
200–220.
Chatani F, Nonoyama T, Katsuichi S, et al: Stimulatory effect of luteinizing hormone on the development and maintenance of 5-reduced
steroid-producing testicular interstitial cell tumors in Fischer 344 rats.
Anticancer Res 10:337–342, 1990.
Clegg ED, Cook JC, Chapin RE, et al: Leydig cell hyperplasia and adenoma formation: Mechanisms and relevance to humans. Reprod
Toxicol 11:107–121, 1997.
Cook JC, Klinefelter GR, Hardisty JF, et al: Rodent Leydig cell tumorigenesis: A review of the physiology, pathology, mechanisms, and relevance to humans. Crit Rev Toxicol 29:169–261, 1999.
Cook JC, Murray SM, Frame SR, et al: Induction of Leydig cell adenomas
by ammonium perfluorooctanoate: A possible endocrine-related mechanism. Toxicol Appl Pharmacol 113:209–217, 1992.
OVARY
Alison RH, Morgan KT: Ovarian neoplasms in F344 rats and B6C3F1 mice.
Environ Health Perspect 73:91–106, 1987.
Amsterdam A, Selvaraj N: Control of differentiation, transformation, and
apoptosis in granulosa cells by oncogenes, oncoviruses, and tumor
suppressor genes. Endocr Rev 18:435–461, 1997.
Biskind MS, Biskind JR: Development of tumors in the rat ovary after
transplantation into the spleen. Proc Soc Exp Biol Med 55:176–179,
1944.
Blaakaer J, Baeksted M, Micic S, et al: Gonadotrophin-releasing hormone
agonist suppression of ovarian tumorigenesis in mice of the Wx/Wv
genotype. Biol Reprod 53:775–779, 1995.
Capen CC, Beamer WG, Tennent BJ, et al: Mechanisms of hormonemediated carcinogenesis of the ovary in mice. Mutat Res 333:143–
151, 1995.
Carson RS, Zhang Z, Hutchinson LA, et al: Growth factors in ovarian function. J Reprod Fertil 85:735–746, 1989.
Cattanach BM, Iddon A, Charlton HM, et al: Gonadotropin releasing hormone deficiency in a mutant mouse with hypogonadism. Nature
269:238–240, 1977.
Cohen IR, Sims ML, Robbins MR, et al: The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice.
Reprod Toxicol 14:37–44, 2000.
Cook LS, Weiss NS, Schwartz SM, et al: Population-based study of tamoxifen therapy and subsequent ovarian, endometrial, and breast cancers. J Natl Cancer Inst 87:1359–1364, 1995.
Davis BJ, Maronpot RR: Chemically associated toxicity and carcinogenicity of the ovary, in Huff J, Boyd J, Barrett J (eds): Cellular and Molecular Mechanisms of Hormonal Carcinogenesis: Environmental Influences, New York, Wiley-Liss, 1996, pp 285–309.
Delmas PD, Bjarnason NH, Mitlak BH, et al: Effects of raloxifene on bone
mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N Engl J Med 337:23:1641–
1647, 1997.
Evista Package Insert. Indianapolis, IN: Eli Lilly, 1997.
Fisher B, Costantino JP, Redmond CK, et al: Endometrial cancer in
tamoxifen-treated breast cancer patients: Findings from the National
Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J Natl
Cancer Inst 86:527–537, 1994.
Frith CH, Zuna RF, Morgan K: A morphologic classification and incidence
of spontaneous ovarian neoplasms in three strains of mice. J Natl Cancer Inst 67:693–702, 1981.
Furth J: Morphologic changes associated with thyrotropin-secreting pituitary tumors. Am J Pathol 30:421–463, 1954.
Gardner WU: Ovarian and lymphoid tumors in female mice subsequent to
Copyright © 2001 by The McGraw-Hill Companies
Retrieved from: www.knovel.com
2996R_ch21_711-759
4/26/01
4:58 PM
Page 759
CHAPTER 21 TOXIC RESPONSES OF THE ENDOCRINE SYSTEM
of ovarian tumorigenesis in mice of the Wx/Wv genotype. Cancer Res
33:721–723, 1973.
Murphy ED: Hyperplastic and early neoplastic changes in the ovaries of
mice after genic deletion of germ cells. J Natl Cancer Inst 48:1283–
1295, 1972.
Nishizuki Y, Sakakura T, Taguchi O: Mechanism of ovarian tumorigenesis
in mice after neonatal thymectomy. J Natl Cancer Inst Monogr 51:89–
96, 1979.
Payne AH, Quinn PG, Stalvey JRD: The stimulation of steroid biosynthesis by luteinizing hormone, in Ascoli M (ed): Luteinizing Hormone
Action and Receptors. Boca Raton, FL: CRC Press, 1985, pp 136–
173.
Rao AR: Effects of carcinogen and/or mutagen on normal and
gonadotrophin-primed ovaries of mice. Int J Cancer 28:105–110, 1981.
Rehm S, Dierksen D, Deerberg F: Spontaneous ovarian tumors in
Han:NMRI mice: Histologic classification, incidence, and influence
of food restriction. J Natl Cancer Inst 72:1383–1395, 1984.
Risma KA, Clay CM, Nett TM, et al: Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries,
and ovarian tumors. Proc Natl Acad Sci USA 92:1322–1326, 1995.
Risma KA, Hirshfield AN, Nilson JH: Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology. Endocrinology 138:3540–
3547, 1997.
Taguchi O, Michael SD, Nishizuka Y: Rapid induction of ovarian granulosa cell tumors by 7,12-dimethylbenz[a]anthracene in neonatally estrogenized mice. Cancer Res 48:425–429, 1988.
Tennent BJ, Beamer WG: Ovarian tumors not induced by irradiation and
gonadotropins in hypogonadal (hpg) mice. Biol Reprod 34:751–760,
1986.
Willemsen W, Kruitwagen R, Bastiaans B, et al: Ovarian stimulation and
granulosa-cell tumour. Lancet 341:986–988, 1993.
Witte ON: Steel locus defines new multipotent growth factor. Cell 63:5–6,
1990.
Yang NN, Venugopalan M, Hardikar S, et al: Identification of an estrogen
response element activated by metabolites of 17-estradiol and raloxifene. Science 273:1222–1225, 1996.
Co
py
rig
hte
dM
ate
ria
l
Roentgen-ray irradiation and hormone treatment. Proc Soc Exp Biol
Med 75:434–436, 1950.
Guthrie MJ: Tumorigenesis in intrasplenic ovaries in mice. Cancer 10:190–
203, 1957.
Hart JE: Endocrine pathology of estrogens: Species differences. Pharmacol Ther 47:203–218, 1990.
Hsu SY, Hsueh AJW: Hormonal regulation of apoptosis: An ovarian perspective. Trends Endocrinol Metab 8:207–213, 1997.
Hummel KP: Induced ovarian tumors. J Natl Cancer Inst 15:711–715,
1954.
Korach KS: Insights from the study of animals lacking functional estrogen
receptor. Science 266:1524–1527, 1994.
Li MH, Gardner WU: Further studies on the pathogenesis of ovarian tumors in mice. Cancer Res 9:35–44, 1949.
Long GG, Cohen IR, Gries CL, et al: Proliferative lesions of ovarian granulosa cells induced in rats by a selective estrogen-receptor modulator.
Toxicol Pathol 27, in press.
Lubahn DB, Moyer JS, Golding TS, et al: Alteration of reproductive function but not prenatal sexual development after insertional disruption
of the mouse estrogen receptor gene. Proc Natl Acad Sci USA
90:11162–11166, 1993.
Majumder S, Brown K, Qiu F-H, et al: c-kit Protein, a transmembrane kinase: Identification in tissues and characterization. Mol Cell Biol
8:4896–4903, 1988.
Marchant J: The chemical induction of ovarian tumours in mice. Br J Cancer 11:452–464, 1957.
Marchant J: The development of ovarian tumors in ovaries grafted from
mice treated with dimethylbenzanthracene. Inhibition by the presence
of normal ovarian tissue. Br J Cancer 14:514–519, 1960.
Matzuk MM, Finegold MJ, Su JG, et al: Alpha-inhibin is a tumoursuppressor gene with gonadal specificity in mice. Nature 360:313–
319, 1992.
Michael SD, Taguchi O, Nishizuka Y: Changes in hypophyseal hormones
associated with accelerated aging and tumorigenesis of the ovaries in
neonatally thymectomized mice. Endocrinology 108:2375–2380,
1981.
Murphy ED, Beamer WG: Plasma gonadotrophin levels during early stages
759
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Retrieved from: www.knovel.com