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20

ETIOPATHOLOGY OF FELINE
TOXIC NODULAR GOITER

Hans Gerber, MD, Hansjakob Peter, MD,

Duncan C. Ferguson, VMD, PhD, and Mark E. Peterson, DVM

Since the first clinical reports in 1979 and 1980/9• 54• 57 overt hyperthy-
roidism in cats has been recognized with increasing frequency,* perhaps
because of the increased awareness and publicity of the disease but also
owing to a real increase of the disease in the cat population, as well as a
longer average lifespan for cats. 15 In clinical practice, hyperthyroidism is
now the most common endocrine disorder of cats, and cats represent the
only nonhuman species in which spontaneous thyrotoxicosis develops
frequently enough to allow a systematic investigation of its pathogenesis.
Because feline hyperthyroidism results from the spontaneous develop-
ment of hyperfunctional thyroid nodules, this disease offers a unique
experimental model for the study of toxic nodular goiter.
In this article, the authors discuss the etiopathology of feline toxic
nodular goiter in the context of the corresponding human disease, which
over many years has been at the focus of the experimental interest of one
of our groups.Is, 51, 53, 67-n
*References 5, 15, 28, 29, 55, 58, 62, 63.

From the Department of Clinical Chemistry, University of Bern School of Medicine (HG),
Inselspital, Bern, Switzerland; University Hospital Administration (HP), Inselspital,
Bern, Switzerland; Department of Physiology and Pharmacology, The University of
Georgia College of Veterinary Medicine (DCF), Athens, Georgia; and Department of
Medicine, The Animal Medical Center and Center for Research Animal Resources
(MEP), Cornell University Medical College, New York, New York

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE

VOLUME 24 • NUMBER 3 • MAY 1994 541

542 GERBER et al

PATHOGENESIS OF HUMAN NODULAR GOITER: A

BRIEF REVIEW

Thyroid Heterogeneity

The follicular cells of one thyroid gland, and even individual folli-
cles, do not represent a homogeneous population of thyrocytes but rather
differ from each other in many respects such as their proliferative poten-
tial, as well as all functional characteristics studied so far: iodination,
peroxidase activity, thyroglobulin synthesis, and endocytosis.18· 24· 51· 53·
67-72 Iodine organification was the first functional trait that was identified
many years ago to display marked heterogeneity within human and
animal goiters labeled with radioiodine.* Iodinating-capacity heteroge-
neity of individual cells becomes most clearly apparent when thyrotropin
(TSH) secretion is suppressed, thereby leaving only the autonomous
function of each cell.18· 19· 51 · 53
More recently, intercellular heterogeneity has been shown for thy-
roglobulin synthesis by in situ hybridization3 in thyroid sections and
between individual cells in vitro with a plaque-forming assay or
immunohistochemistry.30• 71
The macropinocytotic activity of individual cells assessed on thyroid
sections stained with PAS (periodic acid-Schiff reagent) shows a low
number of droplet-containing cells as well as a low droplet count per
individual cell in the absence of TSH; with increasing TSH stimulation,
the fraction of responsive cells steadily enlarges and the number of
droplets per cell increases.21 Obviously, subpopulations of cells within
the follicular epithelium displaying different sensitivities to TSH are in
parallel to the increasing intensity of the TSH stimulus and are gradually
recruited to participate in endocytosis.21 The stepwise recruitment of new
sets of cells upon increasing stimulation enables the thyroid to adapt its
activity in a very economical way to changing demands.21· 71 Follicular
cell recruitment thus may represent a crucial regulatory principle for the
fine tuning of thyroid hormone synthesis and secretion. During the last
few years, it has been increasingly recognized that functional heteroge-
neity and recruitment also exist in other endocrine and nonendocrine
organs.z1, 71

Thyroid Growth

A goiter arises from the multiplication of follicular epithelial cells

forming new follicles. In the course of the transformation over years and
decades of a normal thyroid into a goiter, new daughter follicles are
being generated by the epithelial cells of mother follicles. Daughter folli-
cles arise from follicular-cell subsets endowed with an inherited propen-
sity to replicate at higher than average rates.t Replication of these cells

*References 17-19, 40, 51, 53, 67-72, 80.

tReferences 18, 26, 51, 53, 65, 67, 68, 70, 71 .
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 543

may be triggered by TSH, growth-stimulating immunoglobulins or other

growth factors already at low concentration.*

Growth Factors
Our current knowledge on the role of thyroid growth modulators
and their mechanisms of action is largely based on in vitro studies with
primary cultures of human, rat, sheep, dog, and pig thyroid glands and
with thyroid cell lines (most notably the rat thyroid-derived FRTL-5 cell
line).t Despite the large number of factors that have been shown to
stimulate or interfere with follicular-cell proliferation in vitro, their phys-
iological role in thyroid growth control in vivo and in goitrogenesis
remains to be fully elucidated.
Thyrotropin is a growth stimulator for normal human thyroid cells,
acting both by directly inducing follicular-cell proliferation through the
cyclic adenosine monophosphate (cAMP) pathway,4· 13· 38 and by indi-
rectly increasing, even in low concentration, the sensitivity of the thyro-
cytes for other growth factors such as insulin-like growth factor I (IGF-
I).38 Whereas iodine deficiency and many dietary or environmental goi-
trogens interfere with thyroid hormone synthesis, thus increasing TSH
secretion and thereby stimulating thyroid growth, the role of TSH in
many sporadic and endemic goiters is not fully established.67· 68
Other growth factors, such as IGF-I, epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), and the still debated growth-
stimulating immunoglobulins, may well be involved in goitrogenesis.:f:
A positive growth response of thyroid cells to EGF has been observed in
most species including the cat.U· 13• 22· 23· 25 EGF inhibits differentiation in
thyroid cells, including TSH-mediated differentiated functions such as
cAMP accumulation and iodine uptake, and organification in many sys-
tems.4· 13· 38 The effects of transforming growth factor (TGF)-a are similar
to those of EGF and may replace EGF in transformed cells. TGF-13, an
extremely ubiquitous growth modulator being present in most tissues
and cells, acts on both mesenchymal and epithelial cells. In cultured
FRTL-5 cells, TGF-13 inhibits growth and stimulates differentiated func-
tions.42 Furthermore, these cells actually secrete TGF-13 themselves sug-
gesting that this factor may be part of an autocrine or paracrine regula-
tory system.42 IGF-I is a mitogen for many cell types of both epithelial
and mesenchymal origin. Evidence for an autocrine role of IGF-I in sheep
thyroid cells is provided by its production by ovine thyroid glands and
human thyroid adenomas.78 Insulin and IGF-1 stimulate thyroid cell
growthY· 13 Interleukin-1 and basic fibroblast growth factor (bFGF) also
have been implicated in modulation of thyroid growth. 4• 12· 13
Loss or decrease of inhibitory effects of growth modulators have
also been discussed as possible mechanisms involved in the pathogenesis
of adenomatous goiter. For example, the development of follicular-cell

*References 4, 12, 13, 25, 38, 67, 68, 70, 71.

tReferences 4, 12, 13, 20, 25, 38, 67, 68, 70, 71.
tReferences 4, 12, 13, 25, 37, 38, 67, 68, 72, 76, 78.
544 GERBER et al

adenomas may be the result of the preferential growth of cell clones with
reduced sensitivity to TGF-13, a factor inhibitory to thyroid growth.42 To
date, the research findings are consistent with the concept that any goi-
trogen, be it TSH; a growth-stimulating immunoglobulin; an environ-
mental factor; or any other hormonal, paracrine, or autocrine factor,
induces preferential proliferation of thyrocytes with reduced sensitivity
to inhibiting factors or with high intrinsic (perhaps autonomous) growth
potential.*

Thyroid Autonomy
What is autonomy? Most of us will agree that in clinical thyroidol-
ogy, autonomy means "autonomy from TSH," that is growth or function
of the thyroid gland, or part of it, in the absence of TSH. We should keep
in mind, however, that in pathophysiological terms, particularly for in
vitro studies, the term "autonomy" could also be used in respect to other
growth factors. We should also realize that autonomous growth or func-
tion in the absence of TSH does not preclude that growth or function
cannot be further stimulated by TSH.51• 67• 68• 71 Autonomy of function and
autonomy of growth are hallmarks in the evolution of a goiter that may
or may not occur concomitantly in the same follicle or nodule. 5 1• 53• 67• 68• 71
Unfortunately, the tWo events are often taken as synonymous in clinical
thyroidology.
Autonomy of Growth. As discussed previously, normal thyroids
contain subpopulations of follicular cells with a constitutively high
growth potential. In a thyroid destined to become a goiter, a fraction of
these cells may replicate autonomously, that is, by constitutive activation
of the steps that lead the cells to enter the mitotic cycle. Once autono-
mously and rapidly dividing cells are present in large enough numbers,
the process of autonomous growth may continue in the absence of any
further extra thyroidal stimulator.67• 68• 71
Autonomy of Function. Some degree of residual autonomous iodine
turnover is a constitutive property of the follicles in the normal gland.t
In human goiter tissue, large variations in the degree of autonomous
iodine turnover can usually be found. 18• 40• 51• 53• 67- 71 The only difference
between scintigraphically hot and cold goiter areas may be the higher
number of autonomously functioning follicles in the hot area. The un-
even distribution of follicles with high or low functional autonomy rep-
resents the substrate of the patchy pattern found in scintiscans of human
toxic multinodular goiter.

Thyroid Nodules and Tumors

Mechanisms of Nodule and Tumor Formation
The common observation that nearly every long-standing goiter and
even most normal thyroids of aged individuals become nodular70 can be
explained by three mechanisms.
*References 12, 13, 18, 51, 67, 68, 71, 72, 78.
tReferences 18, 19, 40, 51, 53, 67- 71.
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 545

The presence of thyrocyte subpopulations and their tendency to

remain clustered causes uneven proliferation within the thyroid gland
and leads to focal hyperplasia or nodular transformation.67• 68• 70• 71 In
human goiters, we have described recently a marked heterogeneity in
the expression of the ras proto-oncogene product p21.72 Although the
activation of a whole set of growth factors appears to be required to
trigger proliferation of a cell, however, and p21 expression alone is not
synonymous with proliferation, foci of thyrocytes with intense ras-p21
expression may represent the earliest yet observed stages of thyroid
nodules. 72
A second mechanism of nodule formation is probably that mecha-
nism operating in clonal tumor formation thought to be the result of a
somatic mutation conferring a heritable growth advantage to a single
cell.1 6• 25• 67• 68• 71• 72• 81 These first two mechanisms might even be linked
occasionally. Considering the increased vulnerability of fast-growing
cells to acquire any kind of genetic defect (e.g., growth regulation), we
have suggested that adenomas and perhaps even malignant thyroid tu-
mors may be but the late consequence of the existence of cell subpopu-
lations with high intrinsic proliferation rate. 5 1• 67- 72 Recently, this concept
has found further support by Groch and Clifton/ 6 and very similar ideas
on the origin and development of liver cancer have been presented by
Faber and Rubin. 14
A third mechanism is the nodular growth pattern caused by the
network of fibrous strands resulting from scarring necrosis and hemor-
rhages which occur ih most growing goiters?0

Oncogenes and Thyroid Tumors

Considerable evidence that alterations of cellular proto-oncogenes
might be involved in the genesis of benign and malignant tumors has
been accumulated?· 64 Proto-oncogenes have been shown to code for
growth factors, growth-factor receptors, signal transducers, protein ki-
nases, and transciptional activators.2• 64 For the thyroid gland, the subject
has been reviewed by Melmed.39 It has been shown in porcine thyroid
follicles, that EGF rapidly stimulates the expression of the proto-onco-
genes c-fos and c-myc, whereas TSH alone had no effect on these proto-
oncogenes.27 These findings are consistent with the inability of TSH to
stimulate proliferation in porcine thyrocytes. 27 In FRTL-5 cells, which are
of rat origin, TSH and cAMP do stimulate the c-myc, e-ras, and c-fos
proto-oncogene expressions. 10 Neoplastic thyroid lesions, including thy-
roid adenomas, have been shown to display increased c-myc expression.82
Screening of human thyroid tumors for gene rearrangements of the
proto-oncogenes c-myc, c-myb, c-fos, c-erb-Bl, c-erb-B2, c-erb-A, N-ras, K-
ras, and H-ras performed in one study has revealed only mutations of H-
ras.46 It has been proposed that point mutations in the ras oncogene are
an early event in thyroid tumorigenesis.45 Another study indicated that
alterations in the nuclear oncogene family, including c-myc, N-myc, L-
myc, fos, myb, and p53, might play a role in the development of thyroid
tumors.81 A study of 21 follicular adenomas and 20 follicular carcinomas
546 GERBER et al

in humans by immunocytochemistry utilizing specific monoclonal anti-

bodies against her-2/neu and c-myc oncogenes showed no expression of
the her-2/neu oncogene in the specimens studied. Cytoplasmic staining
for c-myc was observed in 3 of 21 adenomas (14%) and 9 of 20 (45%)
carcinomas.1 Mutations in the gene for the alpha polypeptide chain of
the G stimulatory protein Gs associated with adenylate cyclase was
found in 38% of the functional but none of the nonfunctional thyroid
adenomas.47 Immunohistochemical analysis of the ras oncogene p21
product showed that elevated ras expression may be important in the
development of some adenomas and the conversion to carcinomas as
well. 48• 72 In human goiters, we have recently identified a marked hetero-
geneity in the expression of the ras proto-oncogene product p21. 72 A
whole set of cellular events appears to be required to trigger prolifera-
tion, and even though enhanced expression of p21 alone is not synony-
mous with proliferation, foci of thyrocytes with intense ras-p21 expres-
sion may represent the earliest yet observed stages of thyroid nodules.
Immunocytochemical studies of feline thyroid tissue indicate that ras-p21
expression is also expressed in both proliferating normal and nodular
goiter tissue. 72
In addition to proto-oncogene activation, the loss of genetic control
mechanisms, that is, the loss of tumor suppressor genes, may be involved
with the progression of a normal cell to an adenoma or a malignancy. 60• 66
The cloning of the RB-1 retinoblastoma gene and the p53 protein has led
to the understanding that tumors may be the result of removing an
inhibiting influence upon cell-cycle functions. 66

Excessive Hormone Production: Toxic Nodular Goiter

in Humans (Plummer's Disease)
From the mechanisms underlying thyroid growth, heterogeneity,
autonomy, and nodularity discussed previously, the evolution of exces-
sive hormone production and thyrotoxicosis can easily be understood.69
The basic process of goitrogenesis is the generation of new follicles, some
of which have a high level of autonomous function whereas others may
be entirely cold. While the total number of hot, autonomously function-
ing follicles slowly increases in the course of years and decades, their
joint hormone production insidiously rises and finally exceeds the need
of the organism.69 At this point, endogenous TSH secretion is shut off
and hormone production in normal follicles is thereby set at its lowest
possible level, which is always somewhat above zero. Thus, simple goiter
has become toxic goiter.

FELINE TOXIC NODULAR GOITER: KEY

OBSERVATIONS
History
Until very recently, few references to pathologic abnormalities of the
feline thyroid gland had been reported.61 In a 1913 review of thyroid
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 547

hyperplasia in dogs and cats, Carlson9 described only the congenital

goiter associated with endemic iodine deficiency in the Great Lakes re-
gion. In Switzerland, Bourgeois7 reported in 1933 that the great majority
of cats from Bern (an area with endemic iodine-deficiency goiter in hu-
mans) had goiters in which adenomas were found in 2.2%, 3.6%, and
8.8% of animals below the age of 8 months, above 8 months, and above
8 years, respeCtively. In a 1964 study by Lucke36 and a 1976 study by
Leav and coworkers/5 gross enlargement of the thyroid gland had been
found at necropsy in cats, and nodules were observed during histopatho-
logical examinations, but these abnormalities were relatively rare and
had not been associated with the clinical condition described later.5• 28• 29•
54• 57• 58 In a review of approximately 500 cats per year necropsied at The

Animal Medical Center from 1970 to 1984, an average of fewer than 2

cats per year were found to have gross evidence of thyroid enlargement
of any origin in the period before 1977 when the first cat with hyperthy-
roidism was diagnosed at that institution. Since 1977, both the prevalence
of thyroid pathologic abnormalities and the associated clinical state of
hyperthyroidism have been detected at a markedly increasing frequency,
with the present incidence being about 1 out of 300 cats examined.55
Functional thyroid adenomatous hyperplasia, involving one or both thy-
roid lobes, is the pathologic abnormality associated with 97% to 99% of
the cases of feline hyperthyroidism.28• 29• 50• 55• 75 A striking feature of feline
hyperthyroidism is that bilateral thyroid enlargement is observed in ap-
proximately 70% of cases. 15
Thyroid carcinoma, the primary cause of canine hyperthyroidism,
rarely causes hyperthyroidism in the cat with a prevalence of approxi-
mately 1% to 2%.75 As a result, etiological studies must focus upon
stimuli that would cause thyroid cells to proliferate while maintaining
an otherwise normally differentiated phenotype.

Histologic and Autoradiographic Findings

Histology of Feline Toxic Nodular Goiter

Histologic studies have revealed that the thyroids of hyperthyroid
cats contain single or multiple hyperplastic nodules ranging in size from
less than 1 mm to 3 em (Fig. 1).29• 50 The size of the cells as well as the
volume of the nuclei were invariably much larger within the hyperplastic
tissue than in the surrounding, apparently normal paranodular tissue.
The hyperplastic follicles were lined by a columnar epithelium and con-
tained only faintly PAS-stained colloid.5° Follicular size varied consider-
ably between different nodules from an almost solid growth pattern in
some nodules to a macrofollicular architecture in others. As a rule the
nodular tissue was clearly delimited from the surrounding normal tissue,
however, zones of "smooth transition" between normal and hyperplastic
tissue in which it became difficult to distinguish the two types of tissue
were also seen.50
The extranodular tissti~ was largely built up by colloid-rich follicles
548 GERBER et a!

Figure 1. Histologic section across a nodular goiter of a 13-year·old thyrotoxic cat. Four
nodules consisting of follicles lined by cuboidal epithelial cells with large nuclei (see inset)
and filled with pale, barely stained colloid are shown. Some areas within the nodules are
almost solid structures with only tiny follicular lumina (upper left quadrant) (A). Other nodules
may contain some large follicles separated by densely cellular parenchyma (right upper
quadrant) (8). Occasionally, even a giant follicle may form within the nodules (middle,
below). The inset illustra)es the large cuboidal cells of a hyperplastic focus lying back to
back with normal follicles. PAS stain. Magnification 35x. (From Peter HJ, Gerber H, Studer
H, et al: Autonomy of growth and iodine metabolism in hyperthyroid feline goiters trans-
planted onto nude mice. J Clin Invest 80:491-498, 1987; with permission.)

lined by a flat epithelium.50 The histologic appearance of this paranodu-

lar tissue resembles that of thyroid glands obtained from animals treated
with T4, 19 and therefore, it reflects the suppression of TSH in the thyro-
toxic cats.

Autoradiographic Findings
Autoradiographs obtained from hyperthyroid cats injected with 1251
before surgery have shown that the hyperplastic nodules have a high
radioiodine incorporation (Fig. 2), whereas little 1251 is taken up by the
paranodular tissue, which obviously is gradually suppressed as the hor-
mone production of the growing hyperplastic nodules increases.50 In-
creased radioiodine incorporation in the nodules was not observed in all
follicles, but it varied from nil to very intense in individual follicles,S 0
thus resembling the impressive interfollicular heterogeneity of iodine
metabolism known from normal, and moreso from goitrous, human and
animal thyroid glands.* Marked heterogeneity of radioiodine incorpora-
tion was also observed in normal cat thyroids. 5° Fewer than 4 hours after
1251-administration, that is, before the newly iodinated thyroglobulin mol-

ecules had moved away from the colloid-thyrocyte border and mixed

*References 17-19, 40, 51, 53, 67-71, 80.

 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 549

Figure 2. Autoradiograph of a hyperthyroid cat goiter labeled with 20 f.LCi of 1251for 4 hours
prior to surgery showing intense iodine organification within a hyperplastic nodule and very
little iodine uptake within the adjacent paranodular tissue. In some hyperplastic nodules
there was considerable heterogeneity of radioiodine organification among individual follicles
even within the same nodule. Nuclear fast red stain, exposure time 65 days. Magnification
350x. (From Peter HJ, Gerber H, Studer H, et al: Autonomy of growth and iodine metabo-
lism in hyperthyroid feline goiters transplanted onto nude mice. J Clin Invest 80:491-498,
1987; with permission.)

with the rest of the colloid/9 clearcut differences in the amount of

radioiodine metabolism even among the cells of individual follicles
were observed.50 In this aspect, feline thyroids are no exception to
other species.

Xenotransplantation Studies

A key study demonstrated that feline toxic nodular goiter tissue

transplanted into dysthymic nude mice resulted in growth of the tissue
in its original histologic pattern within the host.5° Proliferation and iodine
incorporation by the transplanted thyroid tissue was studied by injecting
mice with 1251, or 1311 and tritium-thymidine, respectively. The mice were
treated either with thyroxine to suppress or with methimazole to raise
TSH levels.5°· 51

Iodine Metabolism (Evaluated by Iodine 125 or Iodine 131

Incorporation)
As shown by autoradiography, suppression of the mouse's TSH
subsequently decreased radioiodine uptake in the mouse thyroid,
whereas uptake and organification of radioiodine was very intense in the
hyperplastic nodules of the xenotransplanted tissue and much lower in
550 GERBER et a!

the extranodular follicles (Fig. 3).50 In contrast to bovine TSH, hyperthy-

roid cat serum did not increase the radioiodine uptake of normal or
adenomatous cat thyroid xenografts (see Fig. 3). 50 These findings
show that, regardless of the initiating cause, at the time of diagnosis,
xenotransplanted toxic feline thyroid tissue remains h yperfunctional
in the host mouse and thus is independent from circulating stimulatory
factors.

Proliferation (Evaluated by Tritium-Thymidine

Incorporation)
Autoradiographic assessment of follicular cell proliferation in tissue
from toxic feline goiters grafted to T4 -treated nude mice using labeling
with tritium-thymidine showed autonomously proliferating follicular
cells within the hyperplastic tissue but not within the paranodular tissue
(Fig. 4).50

serum

NaCI

40 000
TSH

NaCI serum
~rzm
0 n=5 3
m 5
grafts from anormal cat grafts from a
hyperthyroid cat

Figure 3. Radioiodine uptake into xenotransplanted normal and toxic goiter tissue growing
in T4 -treated host mice labeled with 20 j.LCi of 131 1 for 3 hours before sacrifice. 13 1 1-uptake
(mean ± SEM) is low in the absence of TSH and is not enhanced by administration of
hyperthyroid cat serum, whereas it is readily stimulated by TSH. Toxic goiter tissue shows
an unabated high autonomous iodine uptake that is not significantly altered by administration
of serum from the hyperthyroid donor cat. (From Peter HJ, Gerber H, Studer H, et al:
Autonomy of growth and iodine metabolism in hyperthyroid feline goiters transplanted onto
nude mice. J Clin Invest 80:491 - 498, 1987; with permission.)
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 551

·. '·
...
.. : I.

....·.,~·~·
. . ..
• !) ••

:.c
.. ~;;, ...
.....
. ,. 4 .. ··,.:"' .
. ..... '-• ,;t ~ ·{
•.'\
-~
·~...
I
• ' ·' •t
·:: :•.
·~· ·.·· ,- ~ ...~

Figure 4. Xenograft of a toxic goiter of an 18-year-old cat grown in a T 4 ·treated host mouse
injected with 3 H·thymidine for 3 days and with 131 1 for 3 hours before sacrifice. A, Autoradi·
ograph taken from a hyperplastic area and exposed 3 months after radioiodine labeling
shows that despite the absence of TSH a considerable fraction of the follicular cells has
incorporated the thymidine label into their nuclei. The grains within the follicular lumina result
from residual' 31 1activity. No incorporation of 3 H-thymidine into follicular cell nuclei is found
in 8, autoradiograph showing a paranodular area exposed under identical conditions. Nu-
clear fast red stain, exposure time 4 days. Magnification 1000x. (From Peter HJ, Gerber H,
Studer H, et al: Autonomy of growth and iodine metabolism in hyperthyroid feline goiters
transplanted onto nude mice. J Clin Invest 80:491-498, 1987; with permission.)

In Vitro Studies

Primary Cultures of Follicles and Thyrocytes from Normal

and Toxic Adenomatous Cat Thyroids
Primary cultures of enzymatically dissociated follicles from hyper-
thyroid and normal cats were either embedded in collagen (Fig. 5) or
552 GERBER et al

75J,Jm
I I

Figure 5. A, 8, Clusters of follicles obtained from a normal cat thyroid cultured in collagen.
In the absence of TSH (A) the follicles contain abundant strongly PAS-positive colloid. 8, In
the presence of TSH, 10 mU/mL, the follicles are lined by hyperplastic cuboidal or columnar
follicular cells and contain thin, weakly PAS-positive colloid. C, D, Follicles from a hyperthy-
roid cat goiter cultured in collagen in the absence of TSH.
Illustration continued on opposite page

grown as monolayers.6' n, 22' 23' 33 In vitro studies were in line with the
findings obtained in the xenotransplantation experiments: even in vitro,
collagen-embedded follicles from the hyperplastic nodules grown in the
absence of TSH exhibited intense radioiodine incorporation (Fig. 6) and
higher thymidine incorporation (Fig. 7) than follicles from normal tis-
sue.49 Obviously, follicles derived from hyperplastic nodules retain a
high degree of functional and growth autonomy, and therefore, differ
from the behavior of follicles from the extranodular tissue or normal
glands.
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 553

Figure 5 Continued. The architecture of follicles in C closely resembles that found in hyper-
plastic nodules in vivo whereas that of the follicles in D is very similar to paranodular tissue.
Staining with PAS and hematoxylin.

Moreover, the growth pattern and the effects of various growth

factors on the proliferation of monolayers growing out from isolated
follicles of normal and toxic feline thyroids have been investigated re-
cently to identify alterations of the control mechanisms and particularly
those of the adenylate-cyclase system regulating thyroid follicular cell
proliferation and function. Phase-contrast microscopy has revealed defi-
nite differences between cultured cells from hyperthyroid cats and nor-
mal cats. Adenomatous cells form three-dimensional structures (pseudo-
follicles) consisting of a few layers of cells. On the contrary, cells from
normal cats formed a monolayer with more homogeneity. 15 Normal fe-
line thyroid cells can be subcultured only once or twice, whereas cells
from thyrotoxic glands can be passaged at least three or four times, and
554 GERBER et al

B c
···~

75 J.JmI
I

Figure 6. Semiquantitative autoradiographic assessment of radioiodine organificatiori in

follicles embedded in collagen gel. In normal follicles cultured without TSH, the density of
silver grains within the lumina is low (A), whereas intense ' "'1-organificatiori occurs in the
presence of TSH (B). In contrast, follicles from hyperthyroid goitrous tissue display a.hyper-
plastic appearance (C) and intensely accumulate the iodine label despite the absence of
TSH. All cultures were labeled under identical conditions and slides were exposed for 5
days. Nuclear fast red staining. (From Peter HJ, Gerber H, Studer H, et al: Autonomous
growth and function of cultured thyroid follicles from cats with spontaneous hyperthyroidism.
Thyroid 1:331-338,1991 ; with permission.)
 ETIOPATHOLOGY OF FEUNE TOXIC NODULAR GOITER 555

}_jj
B
'
• ...•
+-
~

~
~

50 j..lmI
I

Figure 7. Autoradiograph showing follicular cell proliferation in follicles from normal and
hyperfunctioning thyroid tissue cultured in the absence of TSH. A, In normal follicles, only
an occasional cell has incorporated the thymidine label. 8, In contrast, some clusters of
follicles contain up to 12% of 3H-thymidine labeled cells, in cultures from hyperthyroid goiters.
Nuclear fast red stain. (From Peter HJ, Gerber H, Studer H, et al: Autonomous growth and
function of cultured thyroid follicles from cats with spontaneous hyperthyroidism. Thyroid
1:331-338, 1991; with permission.)

in approximately 20% of the cases can be maintained as cell strains that

can be passaged many times or even may give rise to immortal cell
lines. 20• 22• 49• 52 Primary cultures of follicles from toxic nodules retain con-
siderably higher proliferation rates (as measured by the fraction of trit-
ium-thymidine labeled cells) and iodine metabolism in a fully defined
TSH-free medium, as compared to follicles from paranodular tissue or
normal feline thyroids. It is felt that the high degree of activity in respect
to function and proliferation in the absence of TSH found in the adeno-
matous tissue is likely due to graded alterations at the follicular-cell level
556 GERBER et al

rather than due to exogenous-stimulating factors. Another possibility is

that these cells might produce growth factors themselves resulting in
continued proliferative responses in vitro.

Cell Lines Established from Feline Toxic Nodular Goiters

Until now, a total of five continuous cell lines called PETCAT-1, 2,

3, 4, and ROMCAT-1 have been established from feline toxic goiters in
two laboratories.6• 11 • 20• 22• 23• 49• 52 The cell lines widely differ in morphologic,
functional, and growth parameters. Two of them (PETCAT-1 and ROM-
CAT-I) are able to form follicle-like lumina in vitro (Fig. 8).20• 49• 52

Proliferation Studies in Vitro

A number of studies have focused upon the proliferation of primary
and continuously cultured feline thyrocytes in response to known
'growth factors and on the adenylate-cyclase system in these cells. TSH
does not stimulate proliferation of normal or abnormal feline thyroid
cells. TSH stimulates cAMP in normal cells but has a smaller relative
effect in adenoma cells and the cell lines.33 As forskolin, a direct potent
stimulator of adenylate cyclase, increases cAMP levels in all cells, the
results are consistent with an abnormality of the TSH receptor or G
proteins in the adenomatous cells.
Further studies have shown that normal and adenomatous cells
proliferate in response to EGF, while only adenomatous cells respond to
bFGF, and only cells from the cell strains respond to insulin and IGF-
1.22· 23 Furthermore, each of the cell lines tested displayed an individual
pattern of growth-factor effects.20• 22• 23 The results available so far suggest
a heterogeneous pattern of intrinsic cellular alterations interfering at
different sites of the growth regulating machinery.
Epidermal Growth Factor has been identified as the most potent
factor stimulating proliferation of feline thyrocytes (primary cultures of
normal and goitrous thyroids; celllines).22• 23 Under no circumstance have
we found that TSH, by itself, is a proliferative stimulant for cat thyroid
cell lines. Iodine inhibited proliferation of three out of five of the feline
thyroid cell lines and retinoic acid inhibited four of five cell lines,22
Therefore, if Brown's8 findings of growth-stimulating immunoglobulins
binding at the TSH receptor are to explain the feline thyroid cell abnor-
mality, then TSH would need to be at least a weak thyroid proliferative
factor. The possibility of cooperative or potentiating effects of multiple
growth factors in vivo has not been entirely ruled out to date. The
pattern of growth factor stimulation would indicate that each of the
tumors and cell lines is heterogeneous with respect to growth factor
response, Furthermore, the possibility that growth factors are being se-
creted by the cells themselves needs also to be explored.
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 557

II
I

Figure 8. PETCAT-1 cells embedded as single cells into collagen gel and cultured without
TSH for 28 days. During the last 24 h the cells were labeled with 3 H-thymidine 1 11Ci/ml. A,
Single cells have developed into follicle-like structures containing cells with 3 H-thymidine-
labeled nuclei. 8, The cells are clearly polarized with an apical membrane forming microvilli
protruding towards the lumen and the basal region facing the side of the collagen. C, Near
the apical region a well-defined junctional complex consisting of tight and intermediate
junction as well as desmosomes seal the lumen from the intercellular space. (From Peter
HJ, Gerber H, Studer H, et al: Autonomous growth and function of cultured thyroid follicles
from cats with spontaneous hyperthyroidism. Thyroid 1:331-338, 1991 ; with permission.)

Evidence For and Against Circulating Thyroid

Stimulators

The two most important forms of thyrotoxicosis in humans are

Graves' disease and Plummer's disease. In Graves' disease, excessive
hormone production of the thyroid gland is caused by thyroid-stimulat-
ing autoantibodies (TSAb), which bind to the TSH receptor and thereby
mimic the effect of TSH. 37• 77 This results in an intense and uniform
overactivity of all follicles while the thyroid is usually only modestly
558 GERBER et al

enlarged. On the contrary, excessive hormone production in toxic nodu-

lar goiters (Plummer's disease) is not caused by a circulating stimulatory
factor but by the TSH-independent overactivity of a large number of so-
ca,lled autonomous follicles.40, 51, 53, 67- 71
Increased thyroid growth and function in feline hyperthyroidism
might theoretically be due to circulating stimulatory factors such as im-
munoglobulins. However, two separate studies have failed to identify
circulating thyroid-stimulating activity in serum from affected cats as
measured by cAMP accumulation.8' 56 Furthermore, when adenomatous
thyroid tissue removed from cats with hyperthyroidism is transplanted
into nude mice, it retains a histological appearance identical to that of
the donor tissue, continues to hyperfunction and to proliferate.50 Admin-
istration of serum from hyperthyroid cats to the mice did not further
increase the radioiodine uptake of normal or adenomatous cat thyroid
xenografts.50 On the other hand, increased titers of thyroid growth-stim-
ulating immunoglobulins (TGis) have been measured in sera from thy-
rotoxic cats.8 Such autoantibodies, which act to promote thyroid growth
but not to stimulate thyroid hormone secretion, also have been reported
in human patients with toxic nodular goiter, as well as in patients with
Graves' disease, Hashimoto's thyroiditis, and euthyroid goiter.37, 67, 68
Brown and coworkers8 studied the effect of purified immunoglobulin
IgG preparations on a FRTL-5 cell line. When compared with IgG of a
normal cat, hyperthyroid cat IgG caused significantly increased tritium-
thymidine incorporation into DNA and stimulated cell proliferation 15-
fold. Stimulation of thymidine incorporation tended to be biphasic and
was completely inhibited by a potent, specific TSH-receptor blocking
antibody. Hyperthyroid cat IgG also significantly inhibited bovine TSH
binding to porcine thyroid membranes, an effect that could be repro-
duced using electrophoretically pure IgG and normal cat thyroid mem-
branes. Despite the effect on growth, hyperthyroid cat lgG did not stim-
ulate intracellular cAMP, and there was no correlation between thyroid
function in vivo and IgG growth-promoting activity in vitro. These data
suggest that elevated titers of thyroid growth IgGs, probably acting
through the TSH receptor, are present in feline hyperthyroidism and
may play a role in goiter formation. 8 However, the ultimate biological
significance of these in vitro findings for goiter formation and hyperfunc-
tion in vivo remains to be further investigated because, as mentioned
previously, administration of sera obtained from hyperthyroid cats into
host mice failed to stimulate the xenografts.50 The xenotransplantation
studies also show that in vivo growth and hyperfunction of toxic cat
goiters no longer depend on circulating extrathyroidal stimulators.49' 50
Rather, the basic lesion appears to be an excessive intrinsic growth poten-
tial of some thyroid cell subsets,50 similar to that observed in human toxic
nodular goiter.51' 53, 67- 71 Of course, a very weak stimulator of thyroid
growth (e.g., TGI) enhancing the transformation of a normal thyroid into
a nodular goiter over many years could well be at play initially. If one
day this is confirmed, then a viral agent should be considered, because
the possible involvement of different viruses has been evoked in human
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 559

and animal autoimmune disease/4• 77 for example spumaretroviruses in

Graves' disease.34

Epizootiological Studies of Feline Hyperthyroidism

Epizootiological studies of feline hyperthyroidism aimed at detect-

ing environmental and nutritional clues have been summarized by
Scarlett62• 63 and Ferguson. 15
In a study of 56 cats with hyperthyroidism and 117 matched con-
trols, Scarlett and coworkers63 identified an increased risk for hyperthy-
roidism associated with regular treatment with flea sprays or powders;
living strictly indoors; and having reported exposure to lawn herbicides,
fertilizers, and pesticides. The greater the amount of canned food in the
diet, the greater the risk. Cats fed a majority of canned food in their diet
had a 3.4 times greater risk of developing hyperthyroidism.63
Siamese cats appeared to have a 10-fold lower likelihood for devel-
oping the condition,63 an observation which may eventually provide
clues to the genetic factors associated with this disease.

Geographical Distribution
According to Scarlett and coworkers,63 feline hyperthyroidism has
least commonly been reported in the mid-section of the country and
most commonly in California. However, Ferguson15 has pointed out, that
all of the original reports emanated from the East coast (New York,
Massachusetts, Pennsylvania)15• 28• 29• 54• 57• 58 and that in the year 1979 only
a few cases had been identified at the University of California, Davis.
Most veterinary schools were reporting the disease by 1980. The number
of cats over 7 years of age presented for veterinary care increased slowly
from 1978 to 1986.63 Reports of feline hyperthyroidism now also com-
monly come from Europe,33• 74 Australia, and New Zealand.31 • 73 If some
environmental exposure or change in nutrition precipitated the onset of
this disease, it is interesting that the exposure or change occurred in a
relatively short time. Another possibility is that cats are living longer,
and owners are seeking veterinary care more intensively.

Iodine
There is ample evidence in in vivo and in vitro systems for a role
for iodide as a moderator of thyroid growth and function. 4• 13• 38• 67- 71
Iodide administration to human patients with nodular goiter can induce
hyperthyroidism, the so-called "jodbasedow" effect.59 This effect has also
been identified in iodine-replete areas (Boston) in otherwise clinically
normal individuals.5 9 A characteristic of iodine-induced hyperthyroidism
in patients with multinodular goiter is its transient course. This does not,
by itself, explain the unremittingly autonomous and progressive nature
of hyperthyroidism in cats.
560 GERBER et al

Iodine appears to modulate the effects of growth factors on the

thyroid. 4· 13· 38· 67-71 Transforming growth factor-[3 has recently been shown
to be decreased in multinodular goiters; iodine induced the production
of this inhibitor of cell growth.4 The thyroidal receptors for EGF, a potent
growth promotant of thyroid cells, are reduced in the presence of io-
dine.41 Furthermore, the growth of three out of five feline thyroid cell
lines is inhibited by iodine.22
Several studies have examined the iodine content of cat foods in an
attempt to implicate iodine in the cause of feline hyperthyroidism.15 Total
iodine in cat foods ranged from 1 to 36.8 mg/kg dry weight. 15 The
recommended level of iodine is from 0.14 to 3 mg/kg dry weight.15 A
study of toxic constituents in pet food demonstrated that all cat-food
samples contained added iodine in synthetic form. 43 Red Dye #3, which
was banned from use in human food products because it caused thyroid
tumors when given in high amounts to rats, releases free iodine when
foods are processed.15 In canned foods, there has been a trend toward
more gourmet products containing more animal tissue (often marine
fish) .15 The iodine content of several American cat foods is up to ten
times the recommended level.l5· 31
Although perhaps not pertinent to the origin of hyperthyroidism,
one study in cats with preexisting hyperthyroidism showed that low-
iodine diets had little effect on the thyroid function tests in these cats.55
Another study examined the effect of variation in dietary iodine intake
on the serum free T4 concentration (and presumably total T4 concentra-
tion)?3 The researchers followed urinary iodine excretion and free T4 by
analog assay (likely proportional to total T4) in young healthy cats. As
expected, urinary iodine correlated with dietary iodine intake, which
varied by over a hundred fold, and the free T4 measurements were
inversely correlated with the urinary iodine. The results showed that the
eat's thyroid hormone homeostasis is extremely sensitive to iodine in-
take, most likely via the well-described "Wolff-Chaikoff effect"/9 a mech-
anism by which iodide uptake results in inhibition of iodine organifica-
tion and reduced hormone synthesis. The feeding study only examined
the effect of 2 weeks of feeding a fixed amount of iodine, and no study
has determined the level of iodine which might be detrimental. Further-
more, the study did not address if the inhibition seen was permanent or
only temporary as is the case in rat and humans?9 The authors of the
study suggested that the wide fluctuation in iodine intake might result
in stimulation of the thyroid gland in some cats, previously on iodine-
deficient diets, receiving a diet which provided excess iodide. 15· 73

Possibility of Dietary or Environmental Goitrogens

Aside from iodine, most cat foods contain relatively high levels of
goitrogenic compounds like phthalates.44 There are many other goitro-
genic materials (e.g., resorcinol, polyphenols, PCBs, etc.), which cats may
be exposed to, either through their diets (particularly fish-containing
diets) or in the environment, that could contribute to the development of
the thyroid adenomatous lesions in the h yperthyroid cat. Most of these
 ETIOPATHOLOGY OF FELINE TOXIC NODULAR GOITER 561

hydrocarbons are metabolized by glucuronidation, a process which is

particularly slow in the cat. 15 The same environmental factors could also
play a role in the pathogenesis of the human disease, making the cat a
monitor species for the cohabiting human population.

Conclusions and Future Directions for Research

Toxic nodular goiter of the cat may be a very useful model for
studying some aspects of human toxic nodular goiter pathogenesis or,
more generally, the growth mechanisms acting in the genesis of endo-
crine tumors.* Feline hyperthyroidism is a distinctly geriatric disease,
and its diagnosis and treatment are now relatively routine. We must now
turn our attention to the understanding of the progression from the
normal thyroid to the hyperfunctional adenomatous gland, and to docu-
menting the apparent increase in the incidence of hyperthyroidism and
uncovering an explanation for it. A major problem with identifying caus-
ative agents in food or environment relates to the delay in onset of this
disease: most cats are more than 10 years of age at onset, and the average
age is 13 years. Prospective experimental trials become prohibitively
expensive and, unless strong evidence is obtained for candidate mecha-
nisms, are unlikely to yield definitive results. It is hoped that cell biolog-
ical and molecular biological studies of the diseased cat tissue in parallel
with epidemiological studies will ultimately shed light on the initial
steps in the evolution of feline toxic goiter. Of particular concern are the
possible dietary and environmental influences, as the disease now evi-
dent in our feline companions may herald changes occurring in our
thyroid glands as well.

SUMMARY

We have discussed the etiopathology of feline toxic nodular goiter

in the context of human nodular goiter pathogenesis. We have reviewed
thyroid heterogeneity, growth regulation, functional and growth auton-
omy, nodule and tumor formation, and the evolution of toxic nodular
goiter in the human being. By addressing toxic nodular goiter of the cat,
the history, morphologic findings, xenotransplantation and cell culture
studies, evidence for and against circulating thyroid stimulators and
epizootiological studies of the feline disease have been summarized.
Due to its structure, the thyroid gland offers some unique possibili-
ties to study the mechanisms that are responsible for cellular heteroge-
neity, the emergence of autonomous nodular growth and function, and,
ultimately, the development of tumors. The demonstration of naturally
occurring clones of cells with high intrinsic proliferation potential within
the follicular epithelium of the thyroid has fostered promising new con-

*References 14, 16, 25, 39, 45-48, 50, 67- 72, 78, 81, 82.
562 GERBER et a!

cepts on the genesis of nodular growth of benign and possibly malignant

endocrine tumors.
Hyperthyroid cat goiters contain single or multiple, autonomously
(i.e., ISH-independently) functioning and growing nodules. Neither hy-
perfunction nor growth of these nodules depends on extrathyroidal cir-
culating stimulators. The basic lesion appears to be an excessive intrinsic
growth capacity of some thyroid cells. The factors enhancing the trans-
formation of a normal thyroid into a nodular hyperfunctioning goiter
over many years are still unknown. Immunological, environmental, and
nutritional factors are the focus of ongoing studies, but an infectious
agent can not yet be excluded.

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Hans Gerber, MD
Department of Clinical Chemistry
University of Bern
Inselspital, Freiburgstrasse
CH- 3010 Bern, Switzerland