United States Office of Research and EPA/630/R-97
Environmental Protection Development March 1998
Agency Washington, DC 20460
&EPA Assessment of Thyroid
Follicular Cell Tumors
RISK ASSESSMENT FORUM
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EPA/630/R-97/002
March 1998
Assessment of Thyroid
Follicular Cell Tumors
Technical Panel
Richard N. Hill, M.D., Ph.D., Chair Thomas M. Crisp, Ph.D.
Pamela M. Hurley, Ph.D. Sheila L. Rosenthal, Ph.D.
Office of Prevention, Pesticides, Dharm V. Singh, D.V.M., Ph.D.
and Toxic Substances Office of Research and Development
Consultant
Gordon C. Hard, B.V.Sc., Ph.D., D.Sc.
American Health Foundation
Valhalla, NY
Risk Assessment Forum Staff
William P. Wood, Ph.D., Executive Director
Imogene Sevin Rodgers, Ph.D., Science Coordinator
up to February 1993
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC 20460
Printed on Recycled Paper
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Disclaimer
This document has been reviewed in accordance with U.S. Environ-
mental Protection Agency policy and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
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Abstract
The U.S. Environmental Protection Agency (U.S. EPA) conducts risk
assessments on chemicals tor carcinogenicity under the guidance provided
in its cancer risk assessment guidelines (U.S. EPA, 1986a; 1996). From
time to time scientific developments prompt the Agency to reexamine pro-
cedures that are generally applied. That is the case with the review of some
chemicals that have produced thyroid follicular cell tumors in experimental
animals. The purpose of the Risk Assessment Forum report Assessment
of Thyroid Follicular Cell Tumors (EPA/630/R-97/002) is to describe the
procedures U.S. EPA will use to evaluate these tumors and that the data
that are needed to make these judgments.
The Report presents science policy guidance that describes the pro-
cedures the Agency will use in the evaluation of potential human cancer
hazard and dose-response assessments from chemicals that are animal
thyroid carcinogens. The Forum Report describes when, under clearly speci-
fied conditions, chemical carcinogenesis in thyroid follicular cells can be
analyzed as a nonlinear phenomenon, rather than assuming low dose lin-
earity as EPA customarily does for carcinogenic compounds. Four
hypothetical cases are summarized which illustrate how to evaluate toxico-
logical data and make hazard and dose-response estimation choices. The
procedures and considerations developed in the Forum Report embody
current scientific knowledge of thyroid carcinogenesis and evolving sci-
ence policy. Should significant new information become available, the
Agency will update its guidance accordingly.
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Contents
Lists of Tables and Figures vii
Peer Reviewers viii
Preface xiii
Executive Summary 1
1. Summary Overview of Thyroid Carcinogenesis 5
1.1. Role of Tsh In Rodent Carcinogenesis 7
1.2. Genetic Influences 7
1.3. Possible Mechanistic Steps 8
1.4. Human Thyroid Carcinogenesis 11
2. Science Policy Guidance 15
2.1. Science Policy Statements 15
2.2. Evidence For Antithyroid Activity 18
2.2.1. Data Needs 18
2.2.2. Increases in Cellular Growth 19
2.2.3. Hormone Changes 19
2.2.4. Site of Action 19
2.2.4.1. Intrathyroidal 20
2.2.4.2. Extrathyroidal 21
2.2.5. Dose Correlations 22
2.2.6. Reversibility 22
2.2.7. Lesion Progression 22
2.2.8. Structure-Activity Analysis 22
2.2.9. Other Studies 22
2.3. Other Modes of Carcinogenic Action 23
2.4. Dose-response Considerations 23
2.4.1. Thyroid Tumor High-to-low Dose Extrapolation 24
2.4.1.1. Biologically Based 24
2.4.1.2. Nonlinear 24
2.4.1.3. Low-Dose Linear 26
2.4.1.4. Low-Dose Linear and Nonlinear 27
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Contents (continued)
2.4.2. Endpoints to be Employed 27
2.4.3. Estimation of Points of Departure 27
2.4.4. Interspecies Extrapolation 28
2.4.5. Human Intraspecies Evaluations 29
3. Highlights of Case Studies 32
3.1. Compound 1: A Thionamide that Affects the Synthesis of
ActiveThyroid Hormone; Thyroid Peroxidase and 5'-
monodeiodinase Inhibition 32
3.2. Compound 2: A Chlorinated Cyclic Hydrocarbon that May
Influence the Thyroid Through Effects on the Liver;
Significant Data Gaps 34
3.3. Compound 3: A Bis-benzenamine that Produces Thyroid
and Liver Tumors; Antithyroid and Mutagenic Effects 34
3.4. Compound 4: A Nitrosamine that is Mutagenic and Has
No Antithyroid Effects 35
References 35
Appendices:
A. Case Studies of Compounds
B. Synopsis of Agents Affecting the Thyroid
C. Scientific Findings from 1988 EPA Review Document
D. A Review of Recent Work on Thyroid Regulation and Thyroid Car-
cinogenesis By Gordon Hard
VI
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List of Tables
Table 1. Carcinogenic influences on the rodent thyroid 9
Table 2. Possible molecular events in human thyroid (follicular)
carcinogenesis 10
Table 3. Carcinogenic influences on the human thyroid 12
Table 4. Inter- and intraspecies differences 13
Table 5. Default dose-response procedures for thyroid carcinogens ... 17
Table 6. Data demonstrating antithyroid activity 19
Table 7. Case study summaries: dose-response assessments 33
List of Figures
Figure 1. Hypothalamic-Pituitary-Thyroid Axis 6
Figure 2. Antithyroid Effects Influencing Thyroid Carcinogenesis 20
VII
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Peer Reviewers
1988 Document
A. Independent Reviewers
Gary Boorman, D.V.M., Ph.D.
Michael Elwell, D.V.M., Ph.D.
Scott Eustis, D.V.M., Ph.D.
Robert Maronpot, D.V.M.
National Institute of Environmental Health Sciences
Research Triangle Park, NC
Gerard Burrow, M.D.
Department of Medicine
University of Toronto
Toronto, Ontario, Canada
W. Gary Flamm, Ph.D.
Ronald Lorentzen, Ph.D.
Center for Food Safety and Applied Nutrition
Food and Drug Administration
Washington, DC
Sidney Ingbar, M.D., D.Sc.
Department of Medicine
Harvard Medical School/Beth Israel Hospital
Boston, MA
R. Michael McClain, Ph.D.
Department of Toxicology and Pathology
Hoffmann-LaRoche, Inc.
Nutley, NJ
VIII
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Peer Reviewers (continued)
Jack Oppenheimer, M.D.
Departments of Medicine and Physiology
University of Minnesota-Health Sciences
Minneapolis, MN
David Schottenfeld, M.D.
School of Public Health
University of Michigan
Ann Arbor, Ml
Jerrold Ward, Ph.D.
Laboratory of Comparative Carcinogenesis
National Cancer Institute
Frederick, MD
E. Dillwyn Williams, M.D.
Department of Pathology
University of Wales College of Medicine
Cardiff, Wales
B. EPA Science Advisory Board
Charles C. Capen, D.V.M.
Department of Veterinary Pathobiology
College of Medicine
Ohio State University
Columbus, OH
(representing FIFRA Scientific Advisory Panel)
Nancy Kim, Ph.D.
Division of Environmental Health
New York Department of Health
Albany, NY
E. Chester Ridgway, M.D.
Division of Endocrinology
University of Colorado Health Sciences Center
Denver, CO
Robert A. Scala, Ph.D.
Exxon Biomedical Sciences, Inc.
East Millstone, NJ
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Peer Reviewers (continued)
Ellen Silbergeld, Ph.D.
Environmental Defense Fund
Washington, DC
James A. Swenberg, Ph.D.
Chemical Industry Institute of Toxicology
Research Triangle Park, NC
(representing FIFRA Scientific Advisory Panel)
Robert Tardiff, Ph.D.
1423 Trapline Court
Vienna, VA
1996 DOCUMENT
A. Independent Reviewers
Gordon C. Hard, B.V.Sc., Ph.D., D.Sc.
American Health Foundation
Valhalla, NY
David Hattan, Ph.D.
Center for Food Safety and Applied Nutrition
Food and Drug Administration
Washington, DC
R. Michael McClain, Ph.D.
Department of Toxicology
Hoffmann-LaRoche, Inc.
Nutley, NJ
Margaret Ann Miller, Ph.D.
Center for Veterinary Medicine
Food and Drug Administration
Rockville, MD
Christopher Portier, Ph.D.
National Institute of Environmental Health Sciences
Research Triangle Park, NC
Imogene Sevin Rodgers, Ph.D.
Silver Spring, MD
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Peer Reviewers (continued)
RobertJ. Scheuplein, Ph.D.
Center for Food Safety and Applied Nutrition
Food and Drug Administration
Washington, DC
(now with the Weinberg Group, Washington, DC)
B. EPA Science Advisory Board
Charles C. Capen, D.V.M.
Department of Veterinary Pathobiology
College of Medicine
Ohio State University
Columbus, OH
(representing the FIFRA Scientific Advisory Panel)
Adolfo Correa, Ph.D.
Johns Hopkins University School of Hygiene and
Public Health
Baltimore, Maryland
Michael Gallo, Ph.D.
Department of Environmental and
Community Medicine
UMDNJ-Robert Wood Johnson Medical School
Piscataway, NJ
David Gaylor, Ph.D.
Food and Drug Administration
National Center for Toxicological Research
Jefferson, AR
(Federal liaison)
Eugene McConnell, D.V.M.
Raleigh, NC
(representing the FIFRA Scientific Advisory Panel)
Emil Pfitzer, Ph.D.
Research Institute for Fragrance Materials, Inc.
Hackensack, NJ
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Peer Reviewers (continued)
MarkJ. Utell, M.D.
Department of Medicine
University of Rochester Medical Center
Rochester, NY
Bernard Weiss, Ph.D.
Department of Environmental Medicine
University of Rochester Medical Center
Rochester, NY
Lauren Zeise, Ph.D.
Office of Environmental Health Hazard Assessment
California Environmental Protection Agency
Berkeley, CA
XII
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Preface
The U.S. Environmental Protection Agency (U.S. EPA) conducts risk
assessments on chemicals for carcinogenicity under the guidance provided
in its cancer risk assessment guidelines (U.S. EPA, 1986a; 1996). From
time to time scientific developments prompt the Agency to reexamine pro-
cedures that are generally applied. That is the case with the review of some
chemicals that have produced thyroid follicularcell tumors in experimental
animals. The purpose of this document is to describe the procedures U.S.
EPA will use to evaluate these tumors and the data that are needed to
make these judgments.
The current 1986 EPA cancer assessment guidelines provide direction
for performing hazard and dose-response assessments for carcinogenic
substances. The guidelines generally operate on the premise that findings
of chemically induced cancer in laboratory animals signal potential haz-
ards in humans. Likewise, for dose-response analyses, the guidelines first
call for use of the most biologically appropriate means for dose extrapola-
tion. In the absence of such knowledge, assessors are directed toward the
use of a default science policy position, a low-dose linear procedure. The
National Research Council in their report Science and Judgment in Risk
Assessment (NBC, 1994) emphasized that well designed guidelines should
permit acceptance of new evidence that differs from what was previously
perceived as the general case, when scientifically justifiable. In keeping
with this principle, the NRC recommended that EPA be more precise in
describing the kind and strength of evidence that it will require to depart
from a default option and which procedures will be applied in such situa-
tions.
The scientific analysis and science policy statement in this report ap-
ply only to tumors involving follicular cells of the thyroid gland. This report
does not analyze or address comparable issues for other endocrine or-
gans. Each one will generally need to be evaluated on its own merits
(latropoulos, 1993/94; Capen et al., 1995).
The present report responds to the EPA policy concerning risks to in-
fants and children (U.S. EPA, 1995). It finds that children are more
XIII
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susceptible than adults to the thyroid carcinogenic effects of ionizing radia-
tion, and mutagenic thyroid carcinogenic chemicals should be reviewed to
determine whether children may be more sensitive than adults. There is no
indication that disruption of thyroid-pituitary status may lead to differential
age sensitivity.
In 1988, the Agency developed a review of the existing science on
thyroid follicular cell carcinogenesis and a draft science policy position cov-
ering the evaluation of chemicals that have induced thyroid tumors in
experimental animals. The EPA Science Advisory Board approved the sci-
ence review and tentatively embraced the policy position that some thyroid
tumors could be assessed using nonlinear considerations. However, they
recommended that the Agency (a) articulate more clearly the steps that
lead to the use of nonlinear considerations in assessments and (b) illus-
trate, using case studies, the ways EPA would evaluate data on animal
thyroid carcinogens and make projections of anticipated human risk and
dose-response assessments from chemicals that are animal thyroid car-
cinogens.
The present document responds to the comments of the Science Advi-
sory Board. After an abbreviated overview of thyroid carcinogenesis, the
paper presents science policy guidance that describes the procedures the
Agency will use in the evaluation of potential human cancer hazard and
dose-response assessments from chemicals that are animal thyroid car-
cinogens. Four hypothetical case studies are summarized which illustrate
how to evaluate toxicological data and make hazard and risk estimation
choices; the case studies are presented in full in Appendix A. Some of the
chemical and functional classes and individual chemicals that have pro-
duced effects on the thyroid are included in Appendix B. The overview
highlights the knowledge about thyroid carcinogenesis, but it does not con-
tain an in depth scientific review of the literature. As support for the overview
and as background for the science policy, the reader is referred to the sci-
entific findings laid out in the 1988 EPA review document and since published
(Appendix C) and to an update of some of the significant findings that have
been published since the 1988 EPA review (Appendix D). The recent sci-
entific literature further supports this science policy.
XIV
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Executive Summary
Default assumptions that in the absence of relevant data help guide
EPA in the use of animal data in evaluating potential human hazards and
risks include the following: (a) tumors in animals are indicators of potential
carcinogenic hazards in humans; (b) humans are generally as sensitive or
more sensitive to effects of chemicals than are test animals; and (c) for
carcinogens, dose and response maintain a linear relationship from high
dose to zero dose, based on an assumption that chemicals act by affecting
DNA directly to cause mutations. This paper reviews the evidence con-
cerning these presumptions for thyroid follicular cell tumors and indicates
that they do not entirely hold for some thyroid carcinogens. Data require-
ments for substantiating and describing thyroid effects of a chemical are
elaborated.
Tumors of thyroid gland follicular cells are fairly common in chronic
studies of chemicals in rodents. Experimental evidence indicates that the
mode of action for these rodent thyroid tumors involves (a) changes in the
DNA of thyroid cells with the generation of mutations, (b) disruption of thy-
roid-pituitary functioning, or (c) a combination of the two. The only verified
cause of human thyroid cancer is ionizing radiation, a mutagenic insult to
which children are more sensitive than adults.
Thyroid hormone from the thyroid gland helps to set the metabolic rate
of cells throughout the body. Too little or too much thyroid hormone is asso-
ciated with disease, hypothyroidism and hyperthyroidism, respectively.
Thyroid hormone production and thyroid cell division are regulated by a
negative feedback loop from the pituitary gland. Whenever the pituitary
detects too much circulating levels of thyroid hormone, it reduces output of
thyroid-stimulating hormone (TSH). When the pituitary perceives too little
circulating thyroid hormone—whether due to decreased synthesis or in-
creased metabolism and excretion of thyroid hormone from the body—the
pituitary increases the output of TSH. TSH goes to the thyroid and stimu-
lates the production of more thyroid hormone by existing cells or increases
cell division in the thyroid to help meet the additional demands for thyroid
hormone production.
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Treatments of rodents that cause thyroid-pituitary disruption result in
chronic reduction in circulating thyroid hormone levels, increase in TSH
levels and the development of increased cell division, increased size and
numbers of thyroid cells, increased thyroid gland weight and, finally tu-
mors of the thyroid. In some cases, there is also an increase in tumors of
the pituitary cells that produce TSH. Cessation of treatment early in the
process before tumor development results in reversal of processes back
towards normal.
Qualitatively, it is not known whether humans are susceptible, as are
rodents, to the development of thyroid cancer from thyroid-pituitary'disrup-
tion. Those human conditions that are known—namely iodide deficiency
congenital inability to synthesize thyroid hormone, and Graves' disease-^
are difficult to interpret regarding the influence of thyroid growth on thyroid
cancer. The first two conditions result in situations like those found in ro-
dents treated with chemicals that lead to thyroid-pituitary disruption. Like
rodents, these individuals develop decreased thyroid hormone and in-
creased TSH levels, increased cell division and thyroid gland enlargement,
but cancer does not necessarily follow. Some studies of persons in iodide
deficient areas show an association with thyroid cancer; other equally well
conducted studies do not, and there have only been a few reported cancer
cases in persons with an inability to synthesize thyroid hormone. Persons
with Graves' disease, an autoimmune disorder, demonstrate antibodies
against the TSH receptor which stimulate thyroid cell growth as does TSH
itself. It has not been resolved whether persons with Graves' disease dem-
onstrate an increased incidence of thyroid cancer or the clinical course is
more aggressive when they develop cancer. Quantitatively, if humans de-
velop cancer through thyroid-pituitary disruption, it appears that humans
are less sensitive to the carcinogenic effects than are rodents. Rodents
show significant increases in cancer with thyroid-pituitary disruption; hu-
mans show little, if any. However, given the data at hand and the questions
that still remain unanswered, it seems that the finding of thyroid tumors in
experimental animals cannot be totally dismissed as a hazard indicator for
humans.
Using the current understanding of thyroid carcinogenesis, the EPA
adopts the following science policy tor interpreting data on this process in
experimental animals:
1. It is presumed that chemicals that produce rodent thyroid tumors may
pose a carcinogenic hazard for the human thyroid.
2. In the absence of chemical-specific data, humans and rodents are
presumed to be equally sensitive to thyroid cancer due to thyroid-
pituitary disruption. This is a conservative position when
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thyroid-pituitary disruption is the sole mode of action, because ro-
dents appear to be more sensitive to this carcinogenic mode of action
than humans. When the thyroid carcinogen is a mutagenic chemical,
the possibility that children may be more sensitive than adults needs
to be evaluated on a case by case basis.
3. Based on data and mode of action information on a chemical that has
produced thyroid tumors, a judgment will be made concerning the
applicability of the generic EPA presumption that dose and response
maintain linearity from high dose to zero dose as follows:
a. A linear dose-response procedure should be assumed when
needed experimental data to understand the cause of thyroid tu-
mors are absent and the mode of action is unknown.
b. A linear dose-response procedure should be assumed when the
mode of action underlying thyroid tumors is judged to involve mu-
tagenicity alone.
c. A margin of exposure dose-response procedure based on
nonlinearity of effects should be used when thyroid-pituitary dis-
ruption is judged to be the sole mode of action of the observed
thyroid and related pituitary tumors. Thyroid-pituitary perturbation
is not likely to have carcinogenic potential in short-term or highly
infrequent exposure conditions. The margin of exposure proce-
dure generally should be based on thyroid-pituitary disruptive effects
themselves, in lieu of tumor effects, when data permit. Such analy-
ses will aid in the development of combined noncancer and cancer
assessments of toxicity. Results of the margin of exposure proce-
dure will be presented in a way that supports risk management
decisions for exposure scenarios of differing types (e.g., infrequent
exposure, short durations).
d. Consistent with EPA risk characterization principles, both linear
and margin of exposure considerations should be assumed when
both mutagenic and thyroid-pituitary disruption modes of action
are judged to be potentially at work. The weight of evidence for
choosing one over the other should also be presented. The appli-
cability of each to different exposure scenarios should be developed
for risk management consideration.
e. When supported by available ^data, biologically based dose-re-
sponse modeling may be conducted. This is the preferred approach
when detailed data are available to construct such a model.
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4. Adverse rodent noncancer thyroid effects (e.g., thyroid gland enlarge-
ments) following short- and long-term reductions in thyroid hormone
levels are presumed to pose human noncancer health hazards.
5. Dose-response relationships for neoplasms other than the thyroid (or
pituitary) will be evaluated using mode of action information bearing
on their induction and principles laid out in current EPA cancer risk
assessment guidelines.
Application of the science policy guidance for chemicals that have pro-
duced thyroid tumors in rodents is dependent on the generation of mode of
action information regarding potential mutagenic influences on the DNA
and whether there are'changes in thyroid-pituitary functioning. Mutagenic
influences are evaluated by short-term tests for gene and structural chro-
mosome mutations and other tests.
Influences on thyroid-pituitary functioning are evaluated in eight differ-
ent areas: data on five lines of evidence are required to evaluate this mode
of action; three are desirable. Required is information on increases in folli-
cular cell size and number, changes in thyroid and pituitary hormones,
knowledge of where the chemical affects thyroid functioning, correlations'
between doses producing thyroid effects and cancer, and reversibility of
effects when chemical dosing ceases. Desirable information consists of
knowledge of progression of lesions overtime, chemical structure-activity
relationships, and various other investigations (e.g., initiation-promotion
studies).
Dose-response relationships will incorporate biologically based mod-
els when sufficient data are available. In their absence, linear, margin of
exposure or both procedures will be employed, using extant mode of ac-
tion information. The first step is to model existing data down to the point
where information is generally no longer reliable, the dose point of depar-
ture. These include estimates of a dose producing 10% thyroid tumor
incidence and values using noncancer effects like TSH levels, thyroid weight
and other parameters. Concern for human exposure ("risk") using a margin
of exposure, involves calculation of the ratio of a dose point of departure to
an anticipated human exposure level. The larger the margin, the lower the
concern for exposure. In cases where linear considerations are applied, a
straight-line extrapolation of tumor incidence is made from the dose point
of departure (e.g., 10% tumor incidence level) to the origin.
The procedures and considerations developed in this report embody
current scientific knowledge of thyroid carcinogenesis and evolving sci-
ence policy. Should significant new information become available, the
Agency will update its guidance accordingly.
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1. Summary Overview of Thyroid Carcinogenesis
Circulating thyroid hormone determines the level of operation of most
cells of the body (Brent, 1994); too much or too little hormone results in
disease. Control of the concentration of this endocrine hormone in the blood
is regulated mainly by a negative feedback involving three organs: the thy-
roid gland, which produces thyroid hormone, and the pituitary gland and
hypothalamus, which respond to and help maintain optimal levels of thy-
roid hormone (Figure 1).
The hypothalamus stimulates the pituitary through thyrotropin-releas-
ing hormone (TRH) to produce TSH, which then prompts the thyroid to
produce thyroid hormone. The stimulated thyroid actively transports inor-
ganic iodide into the cell; it converts it to an organic form and then into
thyroid hormone molecules that can influence target organs throughout the
body. Thyroid hormone, in tissues peripheral to the thyroid, can be con-
verted from a less active thyroxine (T4) to a more active triiodothyronine
(T3) form. Thyroid hormone also is metabolized by the liver, largely by con-
jugation reactions, and excreted into the bile.
Cells in the hypothalamus and pituitary gland respond to levels of cir-
culating thyroid hormone, such that when thyroid hormone levels are high
there is a signal to reduce the output of TRH and TSH. Similarly, when
thyroid hormone levels are reduced, the pituitary is prompted to deliver
more TSH to the thyroid gland to increase the output of thyroid hormone.
This negative feedback loop helps the body to respond to varying demands
for thyroid hormone and to maintain hormone homeostasis. Circulating T4,
T3, and TSH can readily be monitored in experimental animals and hu-
mans and serve as biomarkers of exposure and effect of agents that disrupt
thyroid-pituitary status.
In higher organisms, when demands for more thyroid hormone are
small, existing thyroid follicular cells can meet the demand. With increased
need, as a result of certain chemical exposures or iodide deficiency, the
thyroid responds by increasing the size (hypertrophy) and number (hyper-
plasia) of thyroid follicular cells to enhance hormone output. With continued
TSH stimulation, there is actual enlargement of the thyroid gland (goiter)
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Hypothalmus
(paraventricular nuclei)
„
Pituitary
(thyrotroph)
w
Certain Tissues
T _ ^.y
' ^ '
i
Target Tissue
(nuclear receptor)
Liver
Conjunction
Excretion
Figure 1. Hypothalamic-Pituitary-Thyroid Axis.
and, at least in rodents, eventually neoplasia of the thyroid follicular cells.
Because TSH-producing pituitary cells also are stimulated, they too some-
times undergo hyperplasia and neoplasia.
For details about thyroid follicular cell carcinogenesis, the reader should
consult Appendix C (Hill et al, 1989) and Appendix D, an update of the
science since 1988 (Hard, 1996); only a limited number of references are
in this text. A number of recent papers and reviews are also valuable infor-
mation sources (Ward and Ohshima, 1986; Paynter et al., 1988; Capen
and Martin, 1989;Gaitan, 1989; McClain, 1989,1992,1995; Wynford-Tho-
mas and Williams, 1989; Curran and De Groot, 1991; Thomas and Williams,
1991; Capen, 1992, 1994; Williams, 1992, 1995; Farid et al., 1994). The
long history and extensive database on thyroid neoplasia also are demon-
strated in many older reviews (Bielschowsky, 1955; Morris, 1955; Furth,
1969; Doniach, 1970a, b; Christov and Raichev, 1972; Berenblum, 1974;
Jull, 1976).
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1.1. Role of TSH in Rodent Carcinogenesis
Several experimental findings in rodents that perturb thyroid-pituitary
homeostasis and lead to elevated TSH levels indicate the central role of
TSH in inducing thyroid carcinogenic effects: (1) loss of thyroid cells through
partial thyroidectomy leads to a sustained inability of the thyroid to meet
the demands for thyroid hormone, (2) iodide deficiency decreases the
thyroid's ability to synthesize adequate supplies of thyroid hormones, and
(3) transplantation of pituitary tumors that autonomously secrete TSH adds
more of the trophic hormone to graft recipients (Hill et al., 1989). Note that
these experimental manipulations are done in the absence of any exog-
enous chemical treatment but demonstrate the seminal qualitative role that
TSH plays in thyroid carcinogenesis. Quantitatively, its significance is dem-
onstrated by the correlation between the TSH level and number of tumors/
gland in an initiation-promotion study (McClain, 1989).
If a goitrogenic stimulus that would lead to thyroid tumor formation in
rodents is removed early in the process, effects reverse towards normal
(Todd, 1986). Likewise, if a goitrogenic stimulus is given (1) in conjunction
with adequate amounts of exogenous thyroid hormone (McClain et al., 1988)
or (2) after hypophysectomy to remove TSH-secreting cells (Jemec, 1980),
then hypertrophy, hyperplasia, and tumors of the thyroid do not develop. It
follows from these observations that if TSH levels are chronically elevated,
there will be thyroid cell hypertrophy, hyperplasia, and some potential for
neoplasia, but under conditions where thyroid-pituitary homeostasis is main-
tained, the steps leading to tumor formation are not expected to develop,
and the chances of tumor development are negligible.
1.2. Genetic Influences
Rodent studies indicate an interplay between genetic and nongenetic
events in the development of thyroid tumors. Evidence indicates that car-
cinogenesis often proceeds through a number of operational steps termed
initiation, promotion, and progression (Pilot and Dragan, 1991). Initiation
seems to be linked to genetic events with the induction of DNA mutations,
whereas promotion includes at least nongenetic events that lead to the
expansion of a clone of initiated cells via repeated cell division. Progres-
sion is associated with the accumulation of cell behaviors (like enzymatic
destruction of basement membranes and increased mobility) that allow
cells to invade locally and metastasize distally, probably in part due to still
other mutations. Some genetic influences do not result in mutations but in
changes in gene expression that can affect the carcinogenic process.
Treatment regimens that produce thyroid tumors in rodents can be con-
ceptualized in regard to initiation and promotion; four examples are cited. It
is recognized that these steps do not in themselves describe carcinogenic
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mechanisms but, instead, present a framework for viewing experimental
findings. (1) In two-stage experiments where a mutagenic agent such as
radioactive iodide is followed by treatment with a nonmutagenic goitrogen
(e.g., a chemical inhibitor of thyroid hormone synthesis), the first agent
acts like an initiator, while the second behaves as a promoter (Doniach,
1953). (2) When treatment with a goitrogen alone leads to tumor formation,
TSH increases cell division among normal cells, which leads to increases
in the overall chance of a spontaneous initiating mutation and then pro-
motes the altered cells that retain responsiveness to TSH; carcinogenesis
in these cases would be free of chemically induced mutagenic effects (Owen
et al., 1973). (3) Some chemicals appear to have both initiating and pro-
moting activity because they are mutagenic in many test systems and have
significant antithyroid activity (e.g., 4,4'-oxydianiline) (Murthy et al., 1985).
(4) Still other agents, such as x-irradiation (NRC, 1990) and certain chemi-
cals (e.g., some nitrosamines) (Hiasa et al., 1991), are definitely mutagenic
but lack intrinsic goitrogenic activity. These agents can easily initiate the
carcinogenic process, but tumor formation would be independent of strong
promotional activity from antithyroid effects. TSH still may play a permis-
sive role because these agents can induce cell injury and cell death, which
lead to reductions in the output of thyroid hormone and increases in TSH-
induced cell division of initiated thyroid cells.
It thus appears that thyroid cancer in experimental animals may be
due to either mutagenic influences that lead to DNA changes; to hormone
perturbations that lead to growth stimulation, which directly increases the
number of thyroid cells and indirectly leads to mutations; or to a combina-
tion of the two (Table 1). In those cases where increases in cell number are
dominant, the inciting agent or procedure may be seen as the carcinogenic
stimulus, but the proximate carcinogenic influence is TSH. For those cases
with a dominant mutagenic influence, TSH may play an enhancing role in
the carcinogenic process.
1.3. Possible Mechanistic Steps
The precise molecular steps in the carcinogenic process leading to
thyroid follicularcell cancer have not been elucidated totally, although sig-
nificant insights into the problem have been described (Farid et al., 1994;
Said et al., 1994). Normal cell division in the thyroid seems to be affected
by an interplay among several mitogenic factors, namely, TSH, insulinlike
growth factor 1 (IGF-1), insulin, epidermal growth factor (EGF), and possi-
bly fibroblast growth factor (FGF). Still other factors, such as transforming
growth factor (3 (TGF3), certain interferons, and interleukin 1, may inhibit
growth.
TSH communicates with the cell's interior by activating adenylate cy-
clase to raise levels of cyclic AMP. It also functions through the
-------
Table 1. Carcinogenic Influences on the Rodent Thyroid
DNA directed:
X rays
Mutagenic chemicals
Indirect:
Partial thyroidectomy
Transplantation of TSH-secreting pituitary tumors
Iodide deficiency
Chemicals inhibiting uptake of iodide into the thyroid
Chemicals inhibiting thyroid peroxidase
Chemicals inhibiting release of thyroid hormone from the thyroid gland
Chemicals damaging thyroid follicular cells
Chemicals inhibiting conversion of T4 to T3
Chemicals increasing hepatic thyroid hormone metabolism and excretion
phosphatidyl-inositol/Ca2+ signal transduction cascade that activates phos-
pholipase C. This latter system expresses itself through two pathways:
inositol triphosphate, which releases calcium from cellular stores, and 1,2-
diacylglycerol, which activates protein kinase C. Signal transduction
continues following protein kinase C activation through several steps, in-
cluding the ras protooncogene and various kinases, culminating in the
activation of nuclear transcription factor genes (e.g., c-fos), which leads to
cellular proliferation. The diacylglycerol pathway may account for the fact
that the phorbol ester tumor promoters, which increase protein kinase C,
also stimulate thyroid cell division.
EGF, insulin, and IGF-1 act through tyrosine kinase receptors. TSH
increases EGF binding to its receptor and enhances cell division. IGF-1
and high doses of insulin may influence the TSH receptor. Iodide decreases
thyroid cell adenylate cyclase and calcium levels, whereas reduced iodide
enhances TSH effectiveness. In sum, the actual control of normal cell divi-
sion in follicular cells may, in fact, represent some interaction of all these
factors and possibly other growth regulatory substances.
Under conditions of thyroid-pituitary imbalance, there is no question
that TSH plays a significant role in stimulating DNA synthesis and cell pro-
liferation. However, there is somewhat of a controversy concerning the extent
that normal follicular cells can proliferate. One research group claims that
cells have limited capacity to respond to the growth-inducing effects of TSH
(Wynford-Thomas et al., 1982). In this case, tumor formation would entail
mutational steps that free cells from their growth-limiting potential. Another
group of investigators thinks that follicular cells are innately heterogeneous,
-------
with some of them having stem cell-like proliferation potential while others
are more restricted (Studer and Derwahl, 1995). The stem cell-like follicu-
lar cells would continue to respond to TSH stimulation and eventually give
rise to tumors. It seems possible that both situations might actually apply.
Neoplastic transformation appears to occur in single cells that then
expand clonally. Under TSH stimulation, the yield of mutations that may
influence transformation increases, even in the absence of an increase in
the mutation rate per cell. This is because the repeated cell divisions lead
to an increased number of cells at risk for mutation or because rapid cell
turnover leaves some spontaneous DNA damage unrepaired.
The precise genetic alterations that accumulate in thyroid follicular cells
have not been clearly established in humans or in experimental systems,
although mutations involving the ras protooncogene, the p53 tumor sup-
pressor gene, and various chromosome aberrations have been reported in
the follicular variety of epithelial tumors. These changes in gene expres-
sion could lead to uncontrolled cellular growth and allow cells to attain the
ability to invade adjacent tissues and metastasize (Table 2). For the papil-
lary variety of thyroid epithelial tumors, changes in expression of other factors
have been noted, namely, PTC/ret, trk, and met (Farid et al., 1994; Said et
al., 1994).
Transformed rodent cells that are stimulated to proliferate under the
influence of continuing antithyroid stimulation retain their responsiveness
to TSH. Interestingly, human thyroid cancer cells often retain TSH recep-
tors and the ability to respond to TSH, although their receptors dissipate as
tumors become more anaplastic. For tumor cells to attain their maximal
malignant potential, they need to lose their dependence on TSH. Although
interesting observations concerning growth regulation associated with thy-
roid carcinogenesis have been made, clearly more work is needed.
Table 2. Possible Molecular Events in Human Thyroid (Follicular) Carcinogen-
esis
Thyroid follicular cells
U TSH, insulin, IGF-1, EGF, FGF
Nodular hyperplasia
ii ras, gsp, chromosome aberrations of 5. 7, and 12
Follicular adenoma
J) loss of heterozygosity at 3p
Follicular carcinoma
U p53
Anaplastic carcinoma
10
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1.4. Human Thyroid Carcinogenesis
Clinically manifest thyroid cancer in humans in the United States is
uncommon and largely nonfatal: only about 13,000 new cases occur each
year (an incidence rate of approximately 3/100,000 persons) and about
1,000 deaths annually (>90% 5-year survival, which constitutes only about
0.5% of all cancer deaths) (Boring et al., 1994). In contrast to clinically
apparent disease, small occult thyroid cancers are noted at autopsy in a
small percentage of persons in a number of surveys and up to about 50%
in other investigations (Bondeson and Ljungberg, 1984; Mortenson et al.,
1955). The incidence in autopsy studies is more like that noted in rats in
the National Toxicology Program, where about 1% of control rats are diag-
nosed with thyroid cancers at 2 years of age. However, this comparison is
somewhat misleading. Detailed histologic examinations of human and ro-
dent thyroids are not routinely performed. Histologic tumor criteria differ
over time and across reviewers. In addition, thyroid follicular cell cancer
most often is diagnosed histqlogically as papillary in humans and follicular
in rodents. The aggressiveness of tumors varies: rodent thyroid neoplasms
rarely metastasize; human cancers frequently metastasize. These differ-
ences regarding histology, along with the shortcomings of information from
descriptive and analytical epidemiologic investigations, help to emphasize
the difficulty in comparing human and rodent cancer incidence data.
For years, the only known human thyroid carcinogen was x-irradiation,
causing an increase in papillary tumors, with children being about two-fold
or more sensitive than adults (Table 3) (NRC, 1990; Ron et al., 1989).
There was a question as to whether ionizing radiation from diagnostic or
therapeutic use of radioiodine (131I) was carcinogenic in humans (Holm et
al., 1988,1991), although more recently, children exposed to 131I following
the Chernobyl reactor accident in the Ukraine have developed thyroid can-
cer; iodide deficiency is also common in the region and may augment the
response to 131I (IAEA, 1996). To date, no chemical has been identified as
being carcinogenic to the human thyroid. Most of the human chemical car-
cinogens appear to be mutagenic and cause tumors in more than one site;
some are steroid hormones. Whether mutagenic chemicals might be more
carcinogenic to the thyroids of children is not known.
Humans respond as do experimental animals in regard to short- and
mid-term disturbances in thyroid functioning from various antithyroid stimuli
such as iodide deficiency, partial thyroidectomy (surgically or 131I induced),
and goitrogenic chemicals (e.g., thionamides): when circulating thyroid
hormone levels go down, the TSH level rises and induces thyroid hypertro-
phy and hyperplasia.
However, the long-term consequences of antithyroid action are harder
to interpret, and controversy exists regarding whether the enlarged human
11
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Table 3. Carcinogenic Influences on the Human Thyroid
DNA directed:
X-rays
Indirect factors may include:
Iodide deficiency
Inborn errors of thyroid hormone metabolism
Graves' disease
thyroid gland undergoes conversion to cancer. Thyroid enlargements and
nodules have been implicated as possible antecedents to thyroid cancer in
humans, but direct evidence of conversion of these lesions to malignancy
is lacking (Table 3). Three examples of indirect effects follow. (1) Persons
who live in iodide-deficient areas of the world are unable to synthesize
adequate levels of thyroid hormones; they develop elevated TSH levels,
very enlarged thyroid glands, and lesions typified as adenomatous hyper-
plasia. There is conflicting evidence whether thyroid cancer is increased in
these people (Galanti et al., 1995; Waterhouse et al., 1982). In contrast to
these observations, it seems that domestic animals but not wild animals in
iodide-deficient areas developed elevated incidences of thyroid tumors
(Wegelin, 1928), and tumor incidence disappeared in dogs following the
advent of using iodized salt (Ivy, 1947). Iodide may have some influence as
to the histologic type of thyroid cancer, with follicular being more common
in iodide-deficient areas and papillary being more common in iodide-rich
areas (Williams, 1985). (2) People with various inborn errors of metabo-
lism who are unable to synthesize enough effective thyroid hormone develop
very enlarged thyroids, but few cases of cancer have been reported. There
are no reports of thyroid tumors among persons with resistance to thyroid
hormone (Vickery, 1981; Refetoff et al., 1993). (3) In persons with the au-
toimmune disorder Graves' disease, there are often immunoglobulins that
stimulate thyroid cells in ways analogous to TSH, even though TSH levels
per se are very low. It is not settled as to whether there is also an increase
in thyroid cancer among these patients; some studies seem to indicate
either that cancer incidence may be increased or that thyroid tumors in
these patients may be more aggressive (Belfiore et al., 1990; Mazzaferri,
1990). Overall, this qualitative information suggests that prolonged stimu-
lation of the human thyroid under certain circumstances may lead to cancer,
as in the presence of inherited metabolic conditions or long-term immuno-
logic abnormalities, but there is uncertainty in this conclusion.
In epidemiologic studies, goiter and thyroid nodules have been shown
to be risk factors for thyroid cancer. The specific causes of these enlarge-
ments are not known but, where studied, do not appear to be due to
hypothyroidism (McTiernan et al., 1984; Ron et al., 1987). Some research-
12
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ers believe that part of the association may be due to the close medical
scrutiny given to persons with suspicious thyroid enlargements (Ridgway,
1992; Mazzaferri, 1993).
In spite of the potential qualitative similarities, there is evidence that
humans may not be as sensitive quantitatively to thyroid cancer develop-
ment from thyroid-pituitary disruption as rodents. Rodents readily respond
to reduced iodide intake with the development of cancer; humans develop
profound hyperplasia with "adenomatous" changes with only suggestive
evidence of malignancy. Even with congenital goiters due to inherited blocks
in thyroid hormone production, only a few malignancies have been found
in humans.
The reasons for differences in perceived interspecies sensitivity are
not really known. However, one factor that may play a role in interspecies
quantitative sensitivity to thyroid stimulation deals with the influence of pro-
tein carriers of thyroid hormones in the blood (Table 4). Both humans and
rodents have nonspecific low-affinity protein carriers of thyroid hormones
(e.g., albumin). However, in humans, other primates, and dogs there is a
high-affinity binding protein, thyroxine-binding globulin, which binds T4 (and
T to a lesser degree); this protein is missing in rodents and lower verte-
brates. As a result, more T4 remains bound to proteins with lower affinity in
the rodent and is more susceptible to removal from the blood, metabolism,
and excretion from the body. In keeping with this finding, the serum half-life
of T4 is much shorter in rats (less than 1 day) than in humans (5 to 9 days);
this difference in T4 half-life results in a 10-fold greater requirement for
exogenous T4 in the rat with a nonfunctioning thyroid than in the adult hu-
man (Dohler et al., 1979). Serum T3 levels also show a species difference;
the half-life in rats is about 6 hr while that in humans is about 24 hr
(Oppenheimer, 1979; Larsen, 1982). There is a morphological consequence
Table 4. Inter- and Intraspecies Differences
Parameter Human Rat
Thyroxine-binding globulin
T, 'Half-life
T., Half-life
T4 Production rate/kg b.w.
TSH
Follicular cell morphology
Sex differences
Serum TSH
Cancer sensitivity
present
5-9 days
1 day
1 X
1 X
low cuboidal
sexes equal
F = 2.5xM
essentially absent
0.5-1 day
0.25 day
10 x that in humans
6-60 x that in humans
cuboidal
M<2xFa
M>F
aM = male; F = female.
13
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to these hormone differences. High thyroid hormone synthetic activity is
demonstrated in follicles in rodents: they are relatively small, surrounded
often by cuboidal epithelium. Follicles in primates demonstrate less activity
and are large with abundant colloid, and follicular cells are relatively flat-
tened (low cuboidal) (McClain, 1992).
The accelerated production of thyroid hormones in the rat is driven by
serum TSH levels that are probably about 6- to 60-fold higher than in hu-
mans. This assumes a basal TSH level in rats and humans of 200 ng/ml
and 5 uU/ml, respectively, and a potency of human TSH of 1.5 to 15 U/mg
of hormone (NIDDK, 1994). Thus, it appears that the rodent thyroid gland
is chronically stimulated by TSH levels to compensate for the increased
turnover of thyroid hormones. It follows that increases in TSH levels above
basal levels in rats could more readily move the gland towards increased
growth and potential neoplastic change than in humans. Interestingly, adult
male -ats have higher serum TSH levels than females (Chen, 1984), and
they are often more sensitive to goitrogenic stimulation and thyroid car-
cinogenesis. In humans, there is no sex difference in hormone levels, but
females more frequently develop thyroid cancer (Boring et al., 1994).
In addition to considerations about the influence of serum thyroid hor-
mone carrier proteins, there are differences between humans and animals
in size, lifespan, and pharmacokinetics and pharmacodynamics of endog-
enous and exogenous chemicals. Any comparison of thyroid carcinogenic
responses across species should be cognizant of all these factors.
The guidance given here on thyroid tumors is not unique. Other au-
thorities have recognized and incorporated advances in the understanding
about carcinogenic mechanisms into their assessments of cancer risks
(JMPR, 1990; IARC, 1991; JECFA, 1991; Vainioetal.. 1992; Poulsen, 1993;
Strauss etal., 1994).
14
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2. Science Policy Guidance
2.1. Science Policy Statements
Rodents and humans share a common physiology in regard to the
thyroid-pituitary feedback system. Short-term perturbation in this system
often leads to similar effects in both species resulting in increases and
decreases in circulating thyroid and pituitary hormones. It is well estab-
lished in rodents that disruption of thyroid-pituitary status with elevation of
TSH levels is associated with thyroid tumor and sometimes related pitu-
itary tumor development. This is true whether it is due to deficiency in iodide,
reduction in thyroid mass, presence of TSH-producing pituitary tumors, or
administration of goitrogenic chemicals. An increase in TSH stimulation of
the thyroid is a final common pathway. Likewise, administration of exog-
enous thyroid hormone or removal of a TSH-increasing stimulus reduces
the effects in the thyroid.
The role of thyroid-pituitary disruption in cancer development in hu-
mans is much less convincing than in animals. Iodide deficiency is
associated with increases in thyroid cancer in some studies but not others.
Similarly, an association between either inborn errors of metabolism affect-
ing thyroid hormone output or autoimmune-related Graves' disease and
cancer is suggested but not proved. It seems that TSH may at least play
some permissive role in carcinogenesis in humans. Accordingly, one can-
not qualitatively reject the animal model; it seems reasonable that it may
serve as an indicator of a potential human thyroid cancer hazard. How-
ever, to the extent that humans are susceptible to the tumor-inducing effects
of thyroid-pituitary disruption and given that definitive human data are not
available, it would appear that quantitatively humans are less sensitive than
rodents in regard to developing cancer from perturbations in thyroid-pitu-
itary status.
Rodents develop thyroid cancer from ionizing radiation, working by a
mutagenic mode of action, Ionizing' radiation is the only verified human
thyroid carcinogen, and children are more sensitive than adults. The role of
mutagenic chemicals in human thyroid carcinogenesis and in children is
unknown. Recognizing mode of action information linking thyroid-pituitary
15
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disruption and mutagenesis to thyroid carcinogenesis, the Agency adopts
the following science policy:
1. It is presumed that chemicals that produce rodent thyroid tumors may
pose a carcinogenic hazard for the human thyroid.
2. In the absence of chemical-specific data, humans and rodents are
presumed to be equally sensitive to thyroid cancer due to thyroid-
pituitary disruption. This is a conservative position when
thyroid-pituitary disruption is the sole mode of action, because ro-
dents appear to be more sensitive to this carcinogenic mode of action
than humans. When the thyroid carcinogen is a mutagenic chemical,
the possibility that children may be more sensitive than adults needs'
to be evaluated on a case by case basis.
3. Adverse rodent noncancer thyroid effects (e.g., thyroid gland enlarge-
ments) following short- and long-term reductions in thyroid hormone
levels are presumed to pose human noncancer health hazards.
Some chemicals that have produced thyroid follicular cell tumors in
laboratory rodents appear to work by producing a derangement in thyroid-
pituitary homeostasis; others appear to act primarily through a mutagenic
mode of action; and still others seem to show a combination of both modes
of action. The question then becomes how to evaluate the risks of thyroid
tumors for humans given exposure to any of these chemicals. If the animal
tumors are due to chemical doses that produce imbalances in thyroid-pitu-
itary functioning, it is anticipated that the chance of cancer is minimal under
conditions of hormonal homeostasis. Tumors seeming to arise from rel-
evant mutagenic influences (e.g., gene mutations and structural
chromosome aberrations) without perturbation in thyroid-pituitary status
may pose some chance of cancer across a broader range of doses. Con-
sequently, until such time that biologically based models and data become
available, EPA adopts the following science policy for conducting dose-
response assessments of chemical substances that have produced thyroid
follicular cell (and related pituitary) tumors in experimental animals:
1. A linear dose-response procedure should be assumed when needed
experimental data to understand the cause of thyroid tumors are ab-
sent and the mode of action is unknown (Table 5, example 1) (See
case study for compound 2 in Appendix A).
2. A linear dose-response procedure should be assumed when the mode
of action underlying thyroid tumors is judged to involve mutagenicity
alone.
16
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Table 5. Default Dose-Response Procedures for Thyroid Carcinogens
Array of effects
Example Mutagenic Antithyroid Dose-response methodology
1
2
3
4
either or both
yes
no
yes
unknown
no
yes
yes
linear
linear
margin of exposure
linear and margin of exposure
3. A margin of exposure dose-response procedure based on nonlinearity
of effects should be used when thyroid-pituitary disruption is judged
to be the sole mode of action of the observed thyroid and related
pituitary tumors (Table 5, example 3) (See case study for compound 1
in Appendix A). Thyroid-pituitary perturbation is not likely to have car-
cinogenic potential in short-term or highly infrequent exposure
conditions. The margin of exposure procedure generally should be
based on thyroid-pituitary disruptive effects themselves, in lieu of tu-
mor effects, when data permit. Such analyses will aid in the
development of combined noncancer and cancer assessments of tox-
icity. Results of the margin of exposure procedure will be presented in
a way that supports risk management decisions for exposure sce-
narios of differing types (e.g., infrequent exposure, short durations).
4. Consistent with EPA risk characterization principles, both linear and
margin of exposure considerations should be assumed when both
mutagenic and thyroid-pituitary disruption modes of action are judged
to be potentially at work (Table 5, example 4) (See case study for
compound 3 in Appendix A). The weight of evidence for emphasizing
one over the other should also be presented. The applicability of each
to different exposure scenarios should be developed for risk manage-
ment consideration.
5. Dose-response relationships for neoplasms other than the thyroid (or
pituitary) should be evaluated using mode of action information bear-
ing on their induction and principles laid out in current EPA cancer risk
assessment guidelines. There is an association between thyroid and
liver tumors in rodent cancer studies (McConnell, 1992; Haseman and
Lockhart, 1993). The reason(s) for this relationship has not been ge-
nerically established but should be carefully assessed for chemicals
on a case-by-case basis. Some may be due to induction of hepatic
microsomal enzymes.
Most of the focus in implementing this policy is devoted to answering
the following questions: (1) Does an agent that shows thyroid carcinogenic
effects have antithyroid activity? (2) Can modes of action other than thy-
17
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roid-pituitary disruption account for thyroid tumor formation by this chemi-
cal?, and (3) How can one express thyroid dose-response relationships?
Adequately answering these questions is dependent on a data-rich infor-
mation base for the chemical under review. To the extent practicable, an
effort should be made to review such information before deciding on the
possible mode of chemical action underlying the thyroid tumors and their
consequences for risk assessment. The procedures and considerations
developed in this report embody current scientific knowledge of thyroid
carcinogenesis and evolving science policy. Should significant new infor-
mation become available, the Agency will update its guidance accordingly.
2.2. Evidence for Antithyroid Activity
Different types of information on a chemical may be obtainable indicat-
ing that it has antithyroid activity, that is, whether it works via disruption of
thyroid-pituitary status. These include effects manifest in the thyroid gland
per se, various tissues peripheral to the thyroid, and the liver. All available
factors are assembled into an overall evaluation of the likelihood the chemi-
cal is acting via disruption of the thyroid-pituitary axis.
2.2.1. Data Needs
Special mechanistic studies are needed to demonstrate chemically in-
duced perturbations in thyroid-pituitary functioning. Repeat dose (e.g., 2 to
4 week and 13 week) studies that have simultaneously evaluated a num-
ber of endpoints (as discussed below) often can provide critical information
for evaluating qualitatively whether antithyroid activity exists and what it is
due to and quantitatively what dose-response relationships may pertain.
Such studies should be designed carefully to encompass multiple doses
ranging from above those clearly associated with tumors in chronic studies
down to those below which there is no indication of disturbance in critical
thyroid-pituitary parameters, so that dose-response relationships can be
defined. Special attention should be given to the time of sampling of thy-
roid and pituitary hormones because of the compensatory action of
homeostatic mechanisms and the difficulty in discerning changes after com-
pensation occurs. Hormone sampling also should be conducted at the same
time during the course of a day, and efforts should minimize stress in han-
dling animals. Effects measured only at the end of chronic rodent studies
are often difficult to evaluate and, alone, seldom provide adequate infor-
mation.
Of the eight listed areas of inquiry shown in Table 6, the determination
of the antithyroid activity of a chemical requires empirical demonstration of
the following five items. Demonstration of (1) increases in thyroid growth
and (2) changes in thyroid and pituitary hormones are considered to be the
most important. (3) Location of the site(s) of antithyroid action documents
where in the body the chemical under assessment leads to perturbations
18
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Table 6. Data Demonstrating Antithyroid Activity
Required Desirable
1. Increases in cellular growth 6. Lesion progression
2. Hormone changes 7. Structure-activity relationships
3. Site of action 8. Other studies
4, Dose correlations
5. Reversibility
in thyroid-pituitary functioning. Next is the demonstration of (4) dose corre-
lations among various effects so as to determine where the growth curve
for the thyroid gland deviates from the normal pattern of cell replacement
and how this relates to doses producing tumors. (5) Reversibility of effects
following treatment cessation during the early stages of disruption of the
thyroid-pituitary axis shows that permanent, self-perpetuating processes
have not been set into motion. The remaining three listed items are desir-
able: (6) lesion progression, (7) structure-activity analysis, and (8) other
studies. Each provides supporting information that can add profoundly to
the assessment of an agent's ability to produce antithyroid effects.
2.2.2. Increases in Cellular Growth (evidence required)
Agents that affect thyroid-pituitary functioning stimulate thyroid enlarge-
ment. Commonly measured parameters include but are not limited to
increases in absolute or relative thyroid gland weight or to histologic indi-
cations of cellular hypertrophy and hyperplasia, morphometric
documentation of alteration in thyroid cellular components, and changes in
the proliferation of follicular cells detected by DNA labeling or mitotic indi-
ces.
2.2.3. Hormone Changes (evidence required)
With a disruption in thyroid-pituitary functioning, there is typically a re-
duction in both circulating serum T4 and T3 concentrations and an increase
in TSH levels within days or a few weeks of chemical administration. In
some cases, T4 levels may be lowered while T3 levels are maintained within
normal limits. In addition, sometimes hormone levels may return to normal
over time for mild goitrogenic agents because of the homeostatic compen-
satory increase in thyroid activity and mass. Statistical tests can help
evaluate the significance of hormone perturbations, but it is the constella-
tion of changes in both thyroid and pituitary hormones that indicate whether
the negative feedback loop between the thyroid and pituitary has been
perturbed.
2.2.4. Site of Action (evidence required)
Chemicals that produce thyroid tumors alone or after administration of
a mutagenic initiator produce interference with thyroid-pituitary function by
19
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a variety of specific means (Figure 2; also see Appendix B). Effects have
been found at one or more of the following anatomical locations:
intrathyroidal and various extrathyroidal sites, including the liver and pos-
sible other sites. Sometimes clues as to the site of action can be deduced
by analysis of structurally related compounds (also see section 2.2.7).
Generally, enough information on a chemical should be given to be able to
identify the sites that contribute the major effect on thyroid-pituitary func-
tion. Given experience to date, it appears that most often the liver is the
site of action, followed by the thyroid, where thyroid peroxidase is affected;
other sites of action seem to be less common.
2.2.4.1. Intrathyroidal
Several different effects in the thyroid gland have been associated with
the development of antithyroid activity and the formation of thyroid tumors
in rodents. Iodide pump inhibition by chemicals like thiocyanate and per-
chlorate ions leads to a decrease in uptake of inorganic iodide into the
thyroid gland. Thyroid peroxidase inhibition blocks the incorporation of ac-
tive iodide into iodotyrosines and their coupling to form the nascent thyroid
hormones. Agents that are known to reversibly or irreversibly inactivate
this enzyme include various thionamides such as 6-propylthiouracil and
ethylene thiourea, certain aromatic amines such as some of the sulfona-
mides, and miscellaneous compounds such as amitrole. Toxicityto thyroid
cells, as has been seen with polychlorinated biphenyls, may affect the
gland's ability to manufacture and secrete thyroid hormones. Inhibition of
thyroid hormone release, with agents such as lithium and excess iodide,
Thyroid gland
1. Partial thyroidectomy
2. Iodide deficiency
3. Inhibition of iodide pump
4. Inhibition of thyroid peroxidase
5. Toxicity to cells
6. Inhibition of TH* release
— TH*
Peripheral tissue
7. Inhibition of 5' - mono-
deiodinase
TH*
TSH
Pituitary gland
9. Transplantation of
TSH-secreting tumor
Liver
Enhance TH* conjunction
and excretion
Figure 2. Antithyroid effects influencing thyroid carcinogenesis.
20
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results in a retention of hormones within the colloid and a paucity released
into the circulation (Green, 1978).
2.2.4.2. Extrathyroidal
2.2.4.2,1. Peripheral tissues
Several tissues and organs of the body, including skeletal muscle, kid-
neys, and liver, contain different deiodinases that remove iodine atoms
from thyroid hormones. Inhibition of 5'-monodeiodinase, the enzyme that
normally converts T4 to T3, leads to a reduction in circulating T3 and an
increase in the rT3 level via 3'-deiodination. Compounds such as FD&C
Red No. 3 (erythrosine), iopanoic acid, and 6-propylthiouracil act by com-
petitive inhibition of this enzyme or interaction with its sulfhydryl cofactor.
The deiodinase system in the pituitary is somewhat different from that in
the periphery and may respond differently to certain chemicals (Chanoine
et al., 1993).
2.2.4.2.2. Liver
A significant amount of thyroid hormone normally is metabolized by the
liver. Certain chemicals induce liver microsomal enzymes and enhance
thyroid hormone metabolism and removal. T4 conjugation with glucuronic
acid is enhanced by those agents that induce hepatic glucuronosyl trans-
ferase (Curran and De Groot, 1991). In these cases, thyroid hormone also
may show increased binding to hepatocytes, increased biliary excretion,
and increased plasma clearance. Other common manifestations of microso-
mal induction include such things as enlargement of hepatocytes in the
centrolobular region, increase in hepatic cell smooth endoplasmic reticu-
lum, increase in P-450-associated metabolism of various chemical
substrates, and increase in biliary flow.
Disparate chemical and functional classes such as polyhalogenated
hydrocarbons (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and bar-
biturates (e.g., phenobarbital) as well as various individual compounds (e.g.,
the pesticide clofentezine and the drugs spironolactone and the histamine
[H2] antagonist SK&F 934790) are known to enhance thyroid hormone
excretion via effects on microsomal enzymes. Conjugation also may occur
with sulfate, usually associated with deiodination; deamination, oxidative
decarboxylation, and ether-link cleavage are minor degradative pathways.
Interestingly, phenobarbital has been shown to be a promoter in the rodent
thyroid (McClain et al., 1988), but there is no indication it produces thyroid
cancer in humans (Olsen et al., 1989).
2.2.4.2.3. Other potential sites
Chemicals might bind thyroid hormone receptors and produce certain
effects. For instance, agents (e.g., salicylates) could displace thyroid hor-
21
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mone from plasma carrier proteins and result in reductions in effective thy-
roid hormone (Oppenheimer and Tavernetti, 1962; Hershman, 1963). They
could bind receptors in target organs (e.g., pituitary) but result in inactive
complexes. These possibilities as well as other potential sites of action
(e.g., affecting thyroid-releasing hormone, thyroid hormone-responsive el-
ements in the DNA) might be conceived as influencing thyroid-pituitary
functioning.
2.2.5. Dose Correlations (evidence required)
Confidence in an antithyroid mode of action is enhanced by evidence
of a correlation between doses of a chemical that do and do not jointly
perturb thyroid-pituitary hormone levels, produce various histologic changes
in the thyroid, and/or produce other effects, including thyroid cancer. These
are important steps in evaluating the significance of thyroid-pituitary dis-
ruption in thyroid carcinogenesis and in evaluating dose-response
relationships (see section 2.4).
2.2.6. Reversibility (evidence required)
Chemicals working through an antithyroid mode of action induce
changes in thyroid cell morphology and number and in thyroid-pituitary
hormones that are reversible upon cessation of chemical dosing.
2.2.7. Lesion Progression (evidence desirable)
Evidence for a progression of histologic lesions over time following
exposure to an agent, including cellular hypertrophy and hyperplasia, focal
hyperplasia, and neoplasia (benign and possibly malignant tumors), is com-
monly noted.
2.2.8. Structure-Activity Analysis (evidence desirable)
Analysis of chemical structure may show that an agent belongs to a
class of compounds that induces thyroid tumors via thyroid-pituitary imbal-
ance (e.g., agents that inhibit thyroid peroxidase, liver microsomal enzyme
inducers) (see Appendix B). This allows for the scientific inference that the
chemical under review may act similarly. In addition, generic information
developed on a group of analogues can be used to support the assess-
ment of the agent under review.
2.2.9. Other Studies (evidence desirable)
Many other studies bearing on thyroid-pituitary imbalance can provide
a range of findings from strong ancillary information to only suggestive
indications. A few of these studies are noted here, for example, suppres-
sion of induced effects by concurrent administration of thyroid hormone;
absence of initiating activity but presence of promoting activity in two-stage
carcinogenicity tests; localization of certain chemicals in the thyroid (e.g.,
thionamides); influences on hypothalamic responsiveness to thyroid hor-
22
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mone levels and output of thyrotropin- releasing factor; changes in TSH
mRNA transcripts in the pituitary; and alteration of thyroid hormone nuclear
receptor number or synthesis.
2.3. Other Modes of Carcinogenic Action
Another critical element in the evaluation of a thyroid carcinogen is a
determination of whether mutagenicity may account for the observed tu-
mors. Primary emphasis should be placed on those endpoints that have
mechanistic relevance to carcinogenicity. DNA reactivity is a prime predic-
tor of potential mutagenic carcinogenicity. Many of these compounds belong
to particular chemical classes (e.g., aromatic amines, nitrosamines,
polyaromatic hydrocarbons). These chemicals or their metabolites bind to
DNA, and they often induce gene mutations and structural chromosome
aberrations. In recognition that organs and tissues may have unique meta-
bolic activity, it is helpful to know in addition to traditional short-term test
results whether there is evidence of DNA reactivity in target tissues (e.g.,
DNA adducts, unscheduled DNA synthesis, single-strand breaks).
Mutagenic effects other than those associated with direct DNA reactiv-
ity need to be carefully evaluated in regard to their mechanistic implications;
some may have different cancer dose-response considerations than do
directly DNA reactive agents. Included here are items such as the ability of
the chemical under review to induce indirect effects on DNA, such as through
influence on the cell division spindle or production of reactive oxygen. Agents
also should be evaluated for the presence of structural alerts that are often
predictive of chemical reactivity or potential carcinogenicity. All these find-
ings are then melded into an overall appraisal of an agent's ability to influence
genetic processes relevant to carcinogenesis in the thyroid or other sites.
It is possible that information on carcinogenic modes of action other
than mutagenicity or thyroid-pituitary derangement will become available.
If so, this information also needs to be incorporated on a case-by-case
basis into the evaluation of a chemical's ability to produce tumors of the
thyroid (and of other sites).
2.4. Dose-Response Considerations
Evaluation of potential dose-response relationships for thyroid tumors
depends on an evaluation of the chemical's expected mode of carcino-
genic action. Major determinations include whether the thyroid tumors
appear to be due at least in part to thyroid-pituitary imbalance and whether
other modes of action (e.g., relevant mutagenicity) may be pertinent to
their formation. Other case-specific factors may provide crucial informa-
tion, such as the extent to which data gaps and uncertainties prevail. A
rationale should accompany the selection of any dose-response method.
23
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Guidance is provided below in the subparts of this section, and the case
studies presented in Appendix A help to illustrate ways one might evaluate
data and make judgments about potential thyroid cancer dose-response
relationships.
When tumor types other than thyroid follicular cell (and related pitu-
itary) pertain, mode- of-action and other considerations should help guide
the selection of the appropriate dose-response extrapolation method(s).
Separate dose-response extrapolations may apply for the different tumor
types, depending on the specifics of the case.
2.4.1. Thyroid Tumor High-to-Low Dose Extrapolation
If antithyroid influences are operative in the formation of thyroid tu-
mors, attention should be directed to biologically based procedures that
embody the mechanistic influences, if they are available. In their absence,
default procedures should be employed that incorporate nonlinear or linear
considerations. When thyroid-pituitary imbalance is not operative, other
mode of action or default considerations should be utilized; generally low-
dose linear extrapolations are appropriate. All extrapolation procedures
should be consistent with the guidance given in the EPA cancer risk as-
sessment guidelines (U.S. EPA, 1986a, 1996). Finally, when thyroid tumors
seem to arise from both a chemical's antithyroid activity and its mutagenic
potential, dose-response relationships might be projected in ways that ex-
press concerns for both possible modes of action.
2.4.1.1. Biologically Based
Optimally, mechanistic considerations that underlie thyroid tumor for-
mation would be incorporated into biologically based extrapolation models.
They should include physiologically based pharmacokinetic considerations
for the chemical and its interactions with and effects on cells. The trouble is
that generic biologically based dose-response extrapolation models have
yet to be developed and validated for the thyroid. Fortunately, work in this
area is commencing, and a model has been developed to explain effects
associated with TCDD (Kohn et al., 1996). Until mechanistic models be-
come generally available and chemical-specific data have been produced,
the Agency assessments will employ, as discussed below, one of three
default procedures in its evaluation of thyroid cancer risks: nonlinear, lin-
ear, or both nonlinear and linear.
2.4.1.2. Nonlinear
For those cases where thyroid tumors arise from chemically induced
disturbances in thyroid-pituitary functioning, tumors are considered to be
secondary to the adverse effects on thyroid gland function that precede
them. As exposures to such agents decrease, the likelihood of cancer de-
creases; risks may be seen as minimal at doses where there is no effect on
24
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thyroid-pituitary homeostasis. Generally, homeostasis is considered to ap-
ply when serum T4, T3> and TSH levels and thyroid and pituitary morphology
and growth are within their normal limits (see case study for compound 1 in
Appendix A). Risk assessments on agents should contain case-specific
and generic information that support the contention that nonlinear dose-
response relationships apply.
Empirically, there is some support for thyroid follicular cell tumors hav-
ing a dose-response curve that is less than linear (curvilinear upward).
Using the Weibull model to give some indication of the shape of the dose-
response curve and the rodent tumor incidence database of the National
Cancer Institute/National Toxicology Program, slope functions were calcu-
lated. Incidence for tumors in general was more consistent with a quadratic
than a linear dose-response curve shape (Meier et al., 1993; Hoel and
Portier, 1994). However, when slopes for thyroid follicular cell tumors were
compared with those of thyroid C cell tumors and all other tumor sites,
those from the thyroid follicular cells were even more curvilinear than the
others (Portier, personal communication, Septembers, 1994).
One argument for assuming low-dose linearity in dose-response as-
sessments is the concept of additivity to background. If a given chemical
acts in the same way or augments an endogenous or exogenous back-
ground factor that contributes to tumor development, then the effect of the
chemical will add to that of the background factor. The result is low-dose
linearity up to at least a doubling of the background rate (Crump et al.,
1976). The concept may not be applicable to certain processes subject to
hormonal regulation, such as with the thyroid gland. Normally the level of
circulating thyroid hormone is adjusted carefully by the negative feedback
with the pituitary. Elevations and reductions in thyroid hormone are met
with adjustments in the amount of TSH released from the pituitary so as to
bring thyroid hormone values back into the normal range. These excur-
sions are not part of an ongoing carcinogenic process; instead, they
represent the body's means of maintaining thyroid hormone homeostasis.
Small doses of a potentially antithyroid chemical may not result in any per-
turbation in hormone levels or stimulation of thyroid follicular cell growth
simply because homeostatic mechanisms will drive thyroid hormone levels
back into the normal range.
The Agency acknowledges that it may be difficult to establish a precise
dose where there is negligible response for a specific toxicological effect
given the sensitivity of methods to evaluate various parameters and the
variability in measurement of endpoints. In recognition of this, it is incum-
bent on the scientific community to help in the transfer of various molecular
techniques from the research laboratory to testing facilities (e.g., measure-
ment of hormone receptor mRNA production and receptor content,
25
-------
occupancy, and turnover) that may be more sensitive indicators of thyroid-
pituitary malfunction.
The way the Agency has dealt with nonlinear phenomena is to express
concern for human exposure ("risk") as a margin of exposure (MOE), the
ratio between a dose point of departure for the critical effect and the rel-
evant estimate of anticipated human exposure (incorporating dose,
frequency, and time). Large MOEs are attended with less concern than are
small ones. Traditionally, the point of departure is expressed as a no-ob-
served-adverse-effect level (NOAEL), and the critical effect is the relevant
toxicological endpoint occurring at the lowest doses in a toxicological study
(see sections 2.4.2 and 2.4.3). More recently, alternatives to the NOAEL
have been proposed to serve as points of departure for MOE calculation.1
Procedures should be employed for thyroid tumors that are consistent with
EPA cancer risk assessment guidelines and practices that are applicable
at the time.
Assessments should include adequate information to aid in interpret-
ing the significance of MOEs, such as taking into consideration the variability
in sensitivity among individuals within a species, the sensitivity of humans
relative to experimental animals, and other strengths, weaknesses, and
uncertainties that are part of the assessment. Decision makers must then
judge the adequacy of the MOE for their risk management purposes.
2.4.1.3. Low-Dose Linear
For those assessment cases where thyroid tumors do not seem to be
due to thyroid-pituitary imbalance, existing case-specific mode of action
information and default considerations should be used to develop dose-
response relationships. In other cases, there may be an absence of mode
of action information for an agent. Generally, a low-dose linear default for
the thyroid tumors may be contemplated in these two circumstances in
accordance with current EPA procedures (see case studies for compounds
2 and 4 in Appendix A). Recent cancer risk assessment guideline propos-
als suggest that linear extrapolation would involve calculation of a dose
point of departure with a straight line extrapolation from there to the origin.1
11n April 1996, EPA proposed a revision of its existing cancer risk assessment guidelines (U.S. EPA, 1986a).
A dose point of departure is determined by extrapolating effects in the observed part of the dose-response
curve. It is used, depending on the expected mode(s) of action, as the starting point for either inear
extrapolation to the origin or calculation of an MOE in the case of nonlinear extrapolation. Generally, tumor
or nontumor (e.g., hyperplasia) endpint incidence is extrapolated to the 10% effect level. The lower 95%
confidence limit on that dose may be used as the point of departure. Possibly, the point estimate at the
10% effect level may be used in lieu of the lower-bound estimate given in the proposal. Other means of
determining departure points are also proposed (U.S. EPA, 1996). The final cancer guidelines will clarify
these matters.
26
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2.4.1.4. Low-Dose Linear and Nonlinear
Finally, careful review is warranted when both antithyroid and other
determinants seem to apply to the observed thyroid tumors, such as when
there are certain mutagenic influences (e.g., structural chromosome aber-
rations). Judgments with accompanying scientific reasoning should be
presented on the most appropriate way(s) to evaluate thyroid risk: either
linear or nonlinear or, when the two procedures are about equally tenable,
both. When both procedures are presented, assessors should state the
relative merits of each procedure. In some cases, one of the two methods
may be preferable and should be given more weight; the rationale for con-
clusions should be expressly presented. Projected risks using linear
extrapolation often give rise to concerns at doses lower than those when
nonlinear techniques are applied. Thus, these two techniques usually can
be seen as putting lower and upper bounds on exposures of concern (see
case study for compound 3 in Appendix A). Assessments should include
guidance for decision makers in interpreting concerns for exposure when
both extrapolation techniques are presented.
2.4.2. Endpoints to be Employed
One optimally would have access to data on various preneoplastic
endpoints that would be evaluated (following short-term [e.g., 2 to 4 week]
and subchronic [e.g., 13 week] studies) and compared with the doses that
have produced tumors in chronic studies. Endpoints that should regularly
be evaluated and presented in dose-response analyses include (1) changes
in levels of T4 and T3, (2) increases in TSH, (3) the incidence of thyroid
follicular cell hypertrophy, hyperplasia, and neoplasia, (4) increases in cell
proliferation and thyroid weight, and (5) specific endpoints associated with
thyroid-pituitary disturbance at the site(s) of chemical action (e.g., inhibi-
tion of thyroid peroxidase, increased metabolism, and clearance of thyroid
hormone). A host of other effects as discussed above could be monitored
in the thyroid, pituitary, or thyroid hormone-responsive organs and included
on a case-by-case basis. Care needs to be taken to ensure that studies to
evaluate these parameters have been conducted (1) for adequate periods
of time and (2) at doses that clearly define dose-response relationships.
Attention also needs to be given to procedures that help reduce the vari-
ability in responses among animals (e.g., time and means of animal sacrifice
and tissue sampling).
2.4.3. Estimation of Points of Departure
For the important toxicity studies, a point of departure (e.g., NOAEL) is
determined for each thyroid toxicity endpoint and exposure duration. Doses
associated with tumors also should be noted. The departure point may be
a study dose or an estimated dose. For instance, when data permit, the
departure point can be estimated by extrapolation of doses associated with
27
-------
observed responses (e.g., TSH levels) to those attended with no signifi-
cant deviation from the control range (see case study for compound 1 in
Appendix A). In other cases, an appropriate observed study NOAEL may
be selected (see case study for compound 3 in Appendix A) or other proce-
dures in accordance with EPA guidance may be used.
Considerations for the selection of the critical endpoint to be used to
project thyroid cancer risk (i.e., calculation of the MOE) include (1) the
nature of the endpoint and its relationship to the perturbations in endocrine
balance and carcinogenicity, (2) the presence of good dose-response or
dose-severity of effect relationships, (3) the sensitivity of the endpoint vis-
a-vis other potential endpoints, and (4) the length of the dosing period and
its relevance to making judgments about the consequences of potential
chronic exposures.
2.4.4. Interspecies Extrapolation
Many considerations are relevant in attempting to extrapolate thyroid
carcinogenic effects in experimental animals to humans. The relative sen-
sitivity of humans and rodents to the carcinogenic effects of elevated TSH
are not firmly established, but important observations have been made.
Given that the rodent is a sensitive model for measuring the carcinogenic
influences of TSH and that humans appear to be less responsive (as de-
veloped in section 1.4), one would expect that projections of potential risk
for rodents would serve as conservative potential indicators of risks for
humans.
Rodent cancer studies typically include doses that lead to toxicity, in-
cluding perturbation in thyroid-pituitary functioning, over a lifetime. The
relevance of such experimental conditions to anticipated human exposure
scenarios (i.e., dose, frequency, and time) should be considered and pre-
sented in the final characterization of risk. This is especially true because
thyroidal effects are not necessarily expected at all doses. In addition, chemi-
cally induced effects that are produced by short-term disruption in
thyroid-pituitary functioning appear to be reversible when the stimulus is
removed.
Although it appears that humans are less sensitive to the carcinogenic
perturbations of thyroid-pituitary status than rodents (e.g., iodide deficiency),
such determinations should be made on a case-by-case basis. This would
depend on a host of factors involving the agent, including the depth and
breadth of the database, the congruence of the information supporting a
given mode of action, and the existence of information on humans. Deci-
sion makers should be apprised of risk assessment judgments and their
rationales. In the absence of chemical-specific information, the default as-
sumption is that humans should be considered to be as sensitive to
28
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carcinogenic effects as rodents. That is to say, a factor of one would be
used when extrapolating effects in rodents to those in humans.
2.4.5. Human Intraspecies Evaluations
Thyroid hormones are regulated within rather narrow ranges, with nor-
mal adult human serum values often being given as T4--4 to 11 ug/dL and
T3--80 to 180 ng/dL. TSH levels extend over a broader range--0.4 to 8 uU/
ml, due to the incorporation in recent years of more sensitive laboratory
methods that have extended the normal range to lower values (Ingbar &
Woeber, 1981; Surks et al., 1990). The upper bound on normal TSH has
not changed, and it is the one of import to considerations of antithyroid
effects of chemicals. During development somewhat higher levels for each
of the hormones are noted, with adult hormone values being reached be-
yond about 10 years of age (Nicholson and Pesce, 1992). Growth of the
thyroid gland continues for the first 15 years of life, going from about 1
gram at birth to an adult size of about 17 grams (Fisher and Klein, 1981;
Larsen, 1982). The control of normal thyroid growth during development is
not totally known, although the increase in gland size may be independent
of TSH stimulation (Logothetopoulus, 1963).
Extended deviations in human thyroid hormone levels either above or
below the normal range are associated with the disease states, hyperthy-
roidism and hypothyroidism, respectively. Worldwide, iodide deficiency is
the most prominent cause of thyroid disease generally, and hypothyroid-
ism specifically. In these areas, children quickly manifest characteristic
symptoms and signs which persist throughout life (Bachtarzi and Benmiloud,
1963). Early developmental inability to synthesize adequate thyroid hor-
mone leads to altered physical and mental development (cretinism)
(DiGeorge, 1992; Goldey et al., 1995).
In the U.S., most cases of hypothyroidism are associated with some
autoimmune problem. Symptoms and signs of hypothyroidism readily
prompt patients to seek medical attention. The goal of therapy is to bring
persons back into normal thyroid-pituitary balance which, secondarily, greatly
minimizes any potential for carcinogenic effects. Overt hypothyroidism, with
reduced thyroid hormone and increased TSH levels, requires treatment; it
has an incidence of about 0.2% in women, less in men. Subclinical hy-
pothyroidism may have an incidence of 5% among women; men are affected
less often; and incidence increases significantly with age. It is not agreed
as to whether these people need treatment (Tunbridge & Caldwell, 1991).
Because of the nonspecific nature of certain symptoms of hypothyroidism,
some persons may go for a length of time before diagnosis and treatment.
Seemingly, the thyroid status of persons living in an iodide-deficient
area or those who are hypothyroid due to other causes might be made
29
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worse from significant exposure to naturally occurring chemicals or
xenobiotics that can further disrupt thyroid-pituitary functioning (Eltom et
al., 1985; Hasegawaet al,, 1991). The possible consequences of chemical
exposure on this subpopulation of individuals may warrant consideration
on a case-by-case basis. To the extent that data exist in humans as to their
thyroid-pituitary status, one should carefully review central value param-
eters as well as the distribution of values between chemically exposed and
unexposed groups. Analyses may include: (a) whether the chemical or
chemicals affect the same or different sites of antithyroid action; (b) the
ways various antithyroidal and other effects might combine to influence
potential cancer risks by the same and different routes of exposure, using
the guidance in the EPA mixtures assessment guidelines (U.S. EPA, 1986b);
(c) whether there may be modes of action other than some antithyroid
means; (d) the composition of human populations; (e) the numbers and
nature of sensitive individuals; (f) the magnitude and pattern of chemical
exposure; and (g) estimates of risk to the general population and to sub-
populations with some potential increase in sensitivity.
It is recognized that the human thyroid is susceptible to ionizing radia-
tion, the only verified human thyroid carcinogen. Children are known to be
more sensitive than adults to the carcinogenic effects of radiation (NRC,
1990; IAEA, 1996). The major effect of ionizing radiation on the thyroid is
thought to be due to mutation. Antithyroid effects can also be induced at
elevated radiation doses due to cytotoxicity of follicular cells with resulting
reduction in thyroid hormone and elevation of TSH. Mutagenic chemicals,
however, do not act totally like radiation:
(a) X rays penetrate the body and target organs without having to be
absorbed. Chemicals must be absorbed and distributed to target or-
gans.
(b) Unlike most organic chemicals, radioiodine is actively transported
and concentrated in the thyroid gland, and it becomes incorporated
into nascent thyroglobulin.
(c) Given that the size of the thyroid gland is smaller in children than in
adults, for a given blood level of radioiodine, the internal dose to the
thyroid of a child is greater than that for an adult.
(d) Radioiodine in the Chernobyl accident was picked up by cattle and
incorporated into milk. Due to differences in milk consumption, the
external dose presented to children was greater than to adults. Thy-
roid cancer inducing chemicals may or may not be found in foods
differentially consumed by children.
30
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(e) Single quanta of radiation result in a series of ionizations within bio-
logical material, each of which can react with DNA to induce mutations
and affect the carcinogenic process. Chemicals are much less effi-
cient: they frequently need to be metabolized to active intermediates,
with each molecule interacting singly with DNA, usually by forming
adducts which can be converted to mutations.
(f) The spectrum of mutagenic effects vary with the source. Ionizing
radiation often results in deletions and other structural chromosomal
aberrations, while chemicals not uncommonly produce more gene
mutations.
(g)The thyroid of children is more sensitive to carcinogenic effects of
external radiation on a per unit dose basis than in adults, especially
for children less than 5 years of age. Sensitivity decreases with ad-
vancing age and seems to disappear in adulthood. It is estimated
that, overall, children may be two or more times more sensitive to
carcinogenic effects of external emitters than are adults (NRC, 1990).
The relative sensitivity of children to mutagenic chemicals that pro-
duce thyroid tumors in rodents is totally unknown. Certainly ionizing radiation
is a special risk factor for children. Recognizing that direct acting chemi-
cals can produce mutations, it seems possible that they may have enhanced
carcinogenic effects in children. To the extent possible, the role of mu-
tagenic agents in general as well as agent specific information on thyroid
carcinogenesis should be evaluated from qualitative as well as quantitative
standpoints in risk assessments. This information should be conveyed to
risk managers so that they can consider it in decision making. It would
seem possible that chemicals producing mutagenic effects like radiation
may pose some accentuated risk to children. More research is needed in
this area.
31
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3. Highlights of Case Studies
To illustrate the risk assessment guidance for thyroid tumors that is
developed in this science policy, four detailed case studies of hypothetical
chemicals are presented in Appendix A. Each case study indicates the
types of data that might be available on chemicals and the process that
can be employed in assessing their significance. Attention is placed on
determination of whether the observed thyroid tumors may be the conse-
quence of thyroid-pituitary imbalance and of ways to project potential
dose-response relationships. A summary only of those cases is presented
here (Table 7)
3.1. Compound 1: A Thionamide that Affects the Synthesis of
Active Thyroid Hormone: Thyroid Peroxidase And 5'-
Monodeiodinase Inhibition
Compound 1 is a thionamide that causes thyroid tumors in both sexes
of two rodent species and pituitary tumors in one species; no other tumors
are increased in animals, and no tumor effects have been noted in hu-
mans. Mutagenic activity is not expected to play a role in its carcinogenicity
given its chemical structure and the results of short-term testing.
The thyroid and pituitary tumors produced are thought to be the result
of alterations in thyroid-pituitary functioning because the compound inhib-
its (1) thyroid hormone synthesis in the thyroid and (2) conversion to the
active form in the periphery. As a result, circulating thyroid hormone levels
decrease and TSH levels increase, which stimulate thyroid cells to prolifer-
ate and eventually develop tumors. The process is reversible at least early
in its course.
The compound acts as a promoter in a thyroid initiation-promotion study.
Increases in thyroid cells do not develop following chemical dosing when
thyroid-pituitary balance is maintained by coadministration of exogenous
thyroid hormone. The pituitary tumors synthesize TSH, as would be ex-
pected given the negative thyroid-pituitary feedback loop.
Dose-response information was available for multiple endpoints span-
ning from 28 days to chronic administration in rats; consistent findings were
noted, which increases the confidence in the data set. In the absence of a
32
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mechanistic model, dose-response relationships were evaluated using the
most sensitive indicator, TSH levels, from a 28-day rat study to estimate an
NOAEL. A simple linear extrapolation was made from observed TSH levels
associated with doses of compound 1 down to those within the control
group range. This generates a point of departure (NOAEL estimate) for
calculation of margins of exposure. No information is available to evaluate
directly the carcinogenic effects of compound 1 in humans. In regard to
thyroid-pituitary status, exposed workers show no indication of imbalance,
and monkeys appear less sensitive on a mg/kg basis than rats.
3.2. Compound 2: A Chlorinated Cyclic Hydrocarbon that May
Influence Thyroid through Effects on the Liver;
Significant Data Gaps
Compound 2 produces a significant dose-related increase in thyroid
tumors but not tumors at other sites in rats. The agent has not been tested
for carcinogenicity in a second rodent species. Some structural analogues
have produced mouse liver tumors but not thyroid tumors.
The agent is nonmutagenic for Salmonella gene mutations; it has not
been tested in any other short-term tests. Some analogues of the chemical
produce structural chromosome aberrations. Short-term administration of
compound 2 to rats leads to enlargement of the thyroid gland and reduc-
tions in T3 and T4; TSH levels have not been measured. The chemical
induces liver microsomal enzymes.
Major data gaps preclude a definitive evaluation of the carcinogenic
potential of compound 2 and the cause of the thyroid tumors. Further re-
search is encouraged to discern the possible cause of the tumors to see if
they may be due to interference of thyroid-pituitary functioning or mutagenic
properties. Because of the data gaps and uncertainties in the case, a low-
dose linear extrapolation should be used until a more complete database
is developed.
3.3. Compound 3: A Bis-benzenamine that Produces Thyroid
and Liver Tumors; Antithyroid and Mutagenic Effects
Compound 3, a bis-benzenamine, produces thyroid follicular cell tu-
mors in rats and mice and liver tumors in mice. The thyroid tumors are
associated with thyroid-pituitary derangement; there is thyroid cell and gland
enlargement presumably due to inhibition of thyroid peroxidase activity,
and there is indication that thyroid hormone levels are reduced and TSH
levels are increased. Such effects could account for the thyroid tumors.
However, compound 3 also is DNA reactive and produces gene mutations
and structural chromosome aberrations in short-term test systems. These
DNA effects could condition tumor development in both the thyroid and
liver. Dose-response relationships should include consideration of the DNA-
34
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modulated effects for the liver tumors. For the thyroid gland, the DNA-as-
sociated effects could be used to place an upper bound on the risks, whereas
nonlinear considerations might be used to project a lower bound on "can-
cer risk."
3.4. Compound 4: A Nitrosamine that is Mutagenic and has
No Antithyroid Effects
Compound 4, a nitrosamine, produces thyroid foliicular cell tumors in
rats after a very short latency period. It also produces lung, liver, and kid-
ney tumors in rats after a short latency period and pancreatic, liver, and
lung tumors in Syrian hamsters. Compound 4 is mutagenic in various short-
term tests. Because compound 4 is mutagenic, causes both thyroid and
other tumors with a short latency, and does not cause antithyroid effects,
the thyroid foliicular cell tumors appear to be caused by a mutagenic mode
of action. Dose-response relationships for the thyroid tumors should be
evaluated using a low-dose linear default procedure.
References
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43
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Appendix A
Case Studies of Compounds
-------
Contents
List of Tables and Figures A-5
Compound 1: A Thionamide that Affects the Synthesis of
Active Thyroid Hormone; Thyroid Peroxidase and
5'-mono-Deiodinase Inhibition A-7
Executive Summary A-7
Detailed Data A-8
1. Cancer Findings A-8
2. Mechanistic Considerations A-9
a. Mutagenicity A-9
b. Thyroid Growth A-9
c. Hormones A-11
d. Site of Action A-12
e. Dose Correlations A-13
f. Ancillary Data A-13
g. Structure-Activity Relationships A-16
h. Metabolic and Pharmacokinetic Properties A-17
3. Human Observations A-17
Hazard and Dose-Response Characterization A-17
Bibliography A-21
Compound 2: A Chlorinated Cyclic Hydrocarbon that May
Influence the Thyroid through Effects on the Liver;
Significant Data Gaps A-23
Executive Summary A-23
Detailed Data A-23
1. Cancer Findings A-23
2. Mechanistic Considerations A-24
a. Mutagenicity A-24
b. Thyroid Growth A-24
c. Hormones A-24
d. Site of Action A-25
A-2
-------
Contents (continued)
e. Dose Correlations [[[ A"26
f. Metabolism [[[ A'26
g, Structure-Activity Relationships ....................................... A-27
3. Human Observations [[[ A~27
Hazard and Dose-response Characterization ................................... A-27
A OQ
Bibliography [[[ M"^a
Compound 3: A Bis-benzenamine that Produces Thyroid and
Liver Tumors; Antithyroid and Mutagenic Effects ...... A-30
Executive Summary [[[ A"30
Detailed Data [[[ A'30
1. Cancer Findings [[[ A~3^
2. Mechanistic Considerations [[[ A'31
a. Mutagenicity [[[ A"31
b. Thyroid Growth [[[ A'32
c. Hormone Levels [[[ A~32
d. Site of Action [[[ A"33
e. Dose Correlations [[[ A'34
f. Metabolic and Pharmacokinetic Properties ....................... A-34
g. Structure-Activity Relationships ....................................... A-35
3. Human Observations [[[ A"35
Hazard and Dose-Response Characterization .................................. A-35
Bibliography [[[ A"37
Compound 4: A Nitrosamine that is Mutagenic and has No
Antithyroid Effects [[[ A"38
-------
Contents (continued)
Hazard And Dose-response Characterization A-43
Bibliography ' ^-43
A-4
-------
List of Tables
A-1. F344 Rats with Thyroid Follicular Cell Tumors in a
2-Year Study of Compound 1 A-8
A-2. B6c3f1 Mice with Thyroid Follicular Cell Tumors in a
2-Year Study of Compound 1 A-9
A-3. Mean Thyroid Weights (mg) in Male F344 Rats after
Treatment with Compound 1 for 28 Days A-10
A-4. Mean Thyroid Weights (mg) in F344 Rats Treated with
Compound 1 and Sacrificed at 12 Months A-11
A-5. Incidence of Thyroid Follicular Cell Hyperplasia in Rats at
12 Months A-11
A-6. Mean Thyroid Weights (gm) in Rhesus Monkeys after
Treatment with Compound 1 for 13 Weeks A-12
A-7. Serum T3, T4, and Tsh Levels in Male F344 Rats after
Treatment with Compound 1 for 28 Days A-13
A-8. Effect of Compound 1 on Hepatic S'-monodeiodinase in
Rats A-13
A-9. Effect of T3 And T4 Treatment on Thyroid Weights and
Serum T4, T3, and Tsh Levels in Male Rats given Compound
1 over a 28-Day Period A-16
A-10. Numbers (Percent) of Male Rats with Thyroid Tumors at
20 Weeks after Treatment with DHPN followed by
Compound 1 A-16
A-11. Numbers (Percent) of Rats with Thyroid Tumors
(Adenomas/Carcinomas Combined) A-24
A-12. Mean Thyroid Weights (mg) in Male Sprague-Dawley Rats
Treated for 90 Days with Compound 2 A-25
A-13. T3 and T4 Levels in Male Sprague-Dawley Rats Treated
with Compound 2 for 14 Days A-25
A-14. Hepatic Microsomal Enzyme Activities in Male
Sprague-Dawley Rats Treated for 14 Days with
Compound 2 A-26
A-15. Summary of Effects Relevant to Evaluating the
Significance of Thyroid Tumors in Rats A-26
A-5
-------
List of Tables (continued)
A-16. Rodents with Liver and Thyroid Tumors in 2-Year
Bioassays of Compound 3 A-31
A-17. Rats and Mice with Diffuse Thyroid Hyperplasia after 2
Years of Exposure to Compound 3 A-33
A-18. Serum T4, T3> and Tsh Levels in Male Wistar Rats
Exposed to 400 ppm of Compound 3 in Water for 20
Weeks A_33
A-19. Thyroid Effects Noted in the Toxicity Studies in Rats and
Mice on Compound 3 A-34
A-20. Wistar Rats with Tumors by Site in a 26-Week Study of
Compound 4 A-39
A-21. Thyroid Follicular Cell Tumor Incidence at 30 Weeks in
Male and Female Wistar Rats Treated with Compound 4
by i.p. Injection A-40
A-22. Mean Thyroid Weights (mg) at 30 Weeks in Male and
Female Wistar Rats Treated with Compound 4 by i.p.
Injection A-40
A-23. Incidence of Focal Hyperplasia Involving Thyroid Follicular
Cells at 30 Weeks in Male and Female Wistar Rats Treated
i.p. with Compound 4 A-41
A-24. Serum T4 and Tsh Levels at 30 Weeks in Male and Female
Wistar Rats Treated i.p. with Compound 4 A-42
A-25. Comparison of Serum TSH Levels in Rats With and Without
Thyroid Tumors Induced by i.p. Treatment with a Cumulative
Dose of 600 mg/kg of Compound 4 A-42
A-26. Male Rats with Thyroid Tumors at 20 Weeks after a Single
Treatment with Compound 4 Followed by PTU A-43
List of Figures
A-1. Thyroid weight and tumor responses to compound 1 A-14
A-2. Hormone responses to compound 1 A-15
A-6
-------
Appendix A: Case Studies Of Compounds
Compound 1: A Thionamide that Affects the Synthesis
of Active Thyroid Hormone; Thyroid Peroxidase
and S'-mono-Deiodinase Inhibition
Executive Summary
Compound 1 may pose a human thyroid carcinogenic hazard at high
exposures that might be expected to produce disruptions in the thyroid-
pituitary feedback loop. Given the extensive mode-of-action information
showing that the thyroid follicular cell tumors are produced by stimulation
of the thyroid by thyroid-stimulating hormone (TSH) and not due to mu-
tagenic action, default nonlinear considerations will be employed to estimate
concerns for human exposure.
Compound 1 causes thyroid tumors in two rodent species and pituitary
tumors in one species, as do other members of this chemical class; no
other tumor incidences are increased. There are no tumor findings in hu-
mans. Mutagenicity is not expected to play a role in its carcinogenicity; it
acts as a promoter of thyroid tumors in an initiation-promotion protocol.
The tumors are thought to be the result of alterations in the thyroid-pituitary
feedback: the chemical inhibits thyroid hormone synthesis in the thyroid
and conversion to the active form in the periphery. Lowered thyroid hor-
mone levels induce the pituitary to increase TSH levels (and to enlarge),
which stimulate thyroid cells to enlarge in size, to increase in number, and
finally to develop tumors. The process is reversible at least early in its course,
and thyroid hyperplasia does not develop following chemical dosing when
thyroid-pituitary balance is maintained by exogenous thyroid hormone ad-
ministration. The pituitary tumors synthesize TSH, as would be expected
given the negative thyroid-pituitary feedback loop.
Dose-response relationships are evaluated using the most sensitive
indicator from repeat dose, subchronic, and chronic studies, that is, TSH
levels from a 28-day rat study. A simple interpolation is made from ob-
served TSH levels associated with doses of compound 1 down to the
midpoint of the control group. This generates a point of departure for cal-
culation of margins of exposure. No information is available to evaluate
A-7
-------
directly the carcinogenic effects of compound 1 in humans. In regard to
thyroid-pituitary status, exposed humans show no thyroid imbalance, and
monkeys appear less sensitive on a mg/kg basis than rats. Thus, the esti-
mated point of departure from the rat study is probably a conservative
estimate for compound 1.
Detailed Data
1. Cancer Findings
In chronic rodent toxicity tests, groups of F344 rats and B6C3F1 mice
were administered compound 1 in the feed for 2 years to produce doses of
120, 240, and 480 mg/kg/day in rats and 240, 480, and 960 mg/kg/day in
mice. The study design was adequate, and survival of the animals was
sufficient at all doses. Results are summarized below and in Tables A-1
and A-2.
Table A-1. F344 Rats with Thyroid Follicular Cell Tumors in a 2-year Study
of Compound 1
Incidence (percent) in males and females according to dose (mg/kg/day)
Dose
Sex
Follicular
adenoma
Follicular
carcinoma
Adenoma+
carcinoma
0
M
1/49
(2)
1/49
(2)
2/49
(2)
120
F M F
0/50 1/49 0/50
(2)
0/50 0/49 0/50
0/50 1/49 0/50
(2)
240
M F
10/47*
(21)
4/47*
(9)
14/47*
(30)
1 4/48*
(29)
3/48*
(6)
1 7/48*
(35)
480
M F
34/45*
(76)
8/45*
(18)
42/45*
(93)
28/46*
(61)
7/46*
(15)
35/46*
(76)
*p<.05 compared to control value.
Thyroid tumor frequency in rats and mice was elevated by compound
1. In both male and female rats, the incidences of thyroid follicular cell
adenomas, follicular cell carcinomas, and both tumors combined were sta-
tistically significantly increased at the middle and high doses (Table A-1).
Thyroid follicular cell adenomas but not carcinomas were increased signifi-
cantly in mice at the highest dose (Table A-2). Adenomas of the anterior
lobe of the pituitary gland were significantly increased in high-dose male
rats (19/45 vs. 10/49 in control) and female rats (27/46 vs. 18/50 in control)
and in high-dose male mice (8/47 vs. 1/48 in control). Tumor incidence was
not elevated at any other organ site in any species-sex combination.
A-8
-------
Table A-2. B6C3F, Mice with Thyroid Follicular Cell Tumors in a 2-year Study
of Compound 1
Incidence (percent) in males and females according to dose (mg/kg/day)
Dose
Sex
Follicular
adenoma
0
M
1/48
(2)
F
2/50
(4)
M
1/46
(2)
120
F
1/47
(2)
240
M
2/44
(5)
F
1/47
(2)
M
16/47
(34)
480
F
* 11/46*
(24)
Follicular 0/48 0/50 0/46 0/47 0/44 0/47 0/47* 1/46*
Adenomas-
carcinoma
1/48
(2)
2/50
(4)
1/46
(2)
1/47
(2)
2/44
(5)
1/47
(2)
16/47*
(34)
11/46*
(26)
*p<,05 compared to control value.
In conclusion, the tumor data provide solid evidence of a tumorigenic
effect of compound 1 for the thyroid in both sexes of rat and mouse as well
as a weak effect on the pituitary in rats and male mice.
2. Mechanistic Considerations
a. Mutagenicity
This compound did not demonstrate mutagenic effects in the Salmo-
nella mutation assay in a set of tester strains both for frameshift and
base-pair interactions, with and without metabolic activation. Point muta-
tion tests in Saccharomyces were negative. In mammalian cells in culture,
the mouse lymphoma test produced negative results, but there were weakly
positive results for the frequency of sister chromatid exchanges in CHO
cells. In in vivo tests, compound 1 was negative for chromosomal aberra-
tions in the rat bone marrow and mouse dominant lethal tests. The
compound was investigated for DNA reactivity in rat liver cells in vivo using
14C- and 35S- labeled compound. There was no evidence of DNA binding in
these cells at the limit of sensitivity. Saccharomyces was studied for mitotic
gene conversion and gave a weak positive indication of effect. Even though
there is some genetic activity in a fungal test system and a mammalian cell
test, these test systems are not indicators of mutagenicity and are difficult
to interpret as to their significance for a cancer mode of action. In addition,
given the lack of DNA reactivity and gene and structural chromosomal mu-
tations, it is concluded that there is an overall lack of mutagenic activity for
compound 1 relevant to the evaluation of cancer.
b. Thyroid Growth
Following the results in the chronic toxicity studies, the company de-
signed an extensive 28-day study in male rats that included the collection
A-9
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of information on thyroid weight and hormone levels at seven different
doses (15 rats/dose). The mean thyroid weights were significantly increased
at the three highest doses (Table A-3).
Table A-3. Mean Thyroid Weights (mg) in Male F344 Rats after Treatment with
Compound 1 for 28 Days
Dose (mg/kg/day)
0 15 30 60 120 240 480 960
21.4
±0.6
22.0
+0.7
22.1
±0.8
22.7
±0.7
23.9
±0.9
29.9*
±1.9
48.0*
+2.6
48.6*
±3.9
*p<.05 compared to control value.
Thyroid weight data were also available from male and female rats in
the 2-year study (including its 12-month interim sacrifice). The observa-
tions from these studies were consistent with the information derived from
the 28-day study in male rats. As an example, thyroid weights were signifi-
cantly increased at the 240 and 480 mg/kg/day dose levels in male and
female rats sacrificed at the interim period of 12 months in the cancer bio-
assay (Table A-4). In all of these studies on thyroid weight, expressing the
thyroid weight data as a percentage of body weight (relative thyroid weight)
produced exactly the same results, indicating that the thyroid weight gain
was chemically induced and not just a normal growth change.
The increase in thyroid weight at the different times correlated with a
progressive increase in hypertrophy and hyperplasia of the thyroid follicu-
lar cells, noted histologically. These changes were diffuse throughout the
gland, as opposed to focal. Hypertrophy was characterized by an increase
in size of cells lining the follicles from the normal flattened or low cuboidal
shape to columnar. Hyperplasia involved a generalized increase in the
number of follicular cells and the number of follicles. Increased cellularity
was accompanied by a reduction in the size of the follicles and in colloid
content. Hyperplastic follicles were often irregular in shape with a narrowed,
slitlike lumen. The follicular epithelium was often convoluted, sometimes
projecting as papillary formations into the follicular lumen. These changes
were more pronounced centrally within the gland than at the periphery.
Nodular hyperplasia was reported at one or more sites in animals in the top
two doses. The incidence of hyperplasia at 12 months in male and female
F344 rats is summarized in Table A-5.
In the 28-day study, the mitotic index for follicular cells was determined
in the male rats receiving doses of 480 mg/kg/day by manually counting
mitotic figures (metaphases) in standard unit areas of thyroid tissue. The
mitotic index, expressed as the number of cells in metaphase per 10,000
A-10
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Table A-4. Mean Thyroid Weights (mg) in F344 Rats Treated with Compound 1
and Sacrificed at 12 Months
Dose (mg/kg/day)
0 120 240 480
Males 29.3±2.1 29.4+2.7 44.2+3.8* 52,613.2*
Females 25.313.2 25.1±2.1 34.3±2.3* 44.7+2.0*
*p<.05 compared to control value.
Table A-5. Incidence of Thyroid Follicular Cell Hyperpiasia in Rats at 12
Months
Dose (mg/kg/day)
0 120 240 480
Males 0/15 0/15 10/15* 12/15*
Females 0/15 0/15 8/15* 11/15*
*p<.05 compared to control value.
nuclei, was increased fivefold over the untreated control rats (control value
1.5+0.3 vs. treated group value 7.8±0.2), providing further evidence of thy-
roid follicular cell proliferative activity in response to compound 1. Data on
thyroid weights and morphology from a 13-week oral study with rhesus
monkeys were provided (Table A-6). Males (four per group) were adminis-
tered a dose of compound 1 (300 mg/kg/day) that was above those
producing thyroid tumors and weight changes in rats on a mg/kg basis.
There were no treatment-related effects on thyroid weights, nor were there
any differences in gross and histologic observations of the thyroid between
treated and control monkeys. Specifically, follicular cell hyperplasia, either
diffuse or focal, was not observed at either dose of compound 1. Addition-
ally, all clinical chemistry determinations in the monkeys were within the
normal range.
In conclusion, the data on thyroid weights and morphology show that
compound 1 has a specific effect on the thyroid in rats, increasing thyroid
size through stimulation of cellular hypertrophy and diffuse follicular cell
hyperplasia. Limited data from monkeys suggest that primates may be less
sensitive to these thyroid effects than rats. The sensitivity of mice requires
more study.
c. Hormones
As part of the mechanistic studies on compound 1, T3 (triiodothyro-
nine), T4 (thyroxine), and TSH levels were measured in male rats from the
A-11
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Table A-6. Mean Thyroid Weights (gm) in Rhesus Monkeys after Treatment
with Compound 1 for 13 Weeks
Dose (mg/kg/day)
0 300
Males 0.57 0.45
+0.3 +0.2
28-day study at all seven dose levels. Table A-7 presents mean levels (15
animals per group). Serum T4 and T3 levels were significantly lower than
controls in the three higher doses tested but not the four lower doses. TSH
levels were significantly elevated in a dose-related manner at doses of 120
mg/kg/day and above; no such increases were noted in the two lowest
doses, which were comparable to the control. Comparable changes in se-
rum hormone levels were noted at the same doses in the interim sacrifice
of the 2-year rat study. These hormone changes along with the alterations
in thyroid histology noted above provide unequivocal evidence that com-
pound 1 influences thyroid- pituitary functioning.
d. Site of Action
(1) Thyroid.
To provide data on the site of perturbation of the thyroid-pituitary axis,
information was obtained on the effect of compound 1 on thyroid peroxi-
dase. Hog thyroid peroxidase activity was inhibited in vitro at several dose
levels through a reversible interaction not involving covalent binding and
suicide inactivation, as has been reported for some of the other thionamides.
(2) Other.
Another study showed that compound 1 is also capable of inhibiting 5'-
monodeiodinase activity. The effect of compound 1 on the enzymatic
conversion of T4 to the active form, T3, was investigated by incubating the
supernatant fractions (containing 5'-deiodinase) from perfused livers of
control rats and rats treated with three i.p. doses of compound 1. The amount
of T3 generated in the incubation mixture was a measure of enzyme activ-
ity; inhibition of the enzyme results in decreased generation of T3 and
increased production of the inactive form, reverse T3 (rT3). The results (Table
A-8) indicate a dose-dependent inhibition of 5'-deiodinase activity by com-
pound 1 and provide evidence of a mechanism for reducing effective thyroid
hormone levels and enhancing TSH production. Collectively, these mecha-
nistic data confirm the position that compound 1 exerts actions centrally on
the thyroid and peripherally (e.g., liver) to reduce thyroid hormone forma-
tion.
A-12
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Table A-7, Serum T3, T4, and TSH Levels in Male F344 Rats after Treatment
with Compound 1 for 28 Days
Dose (mg/kg/day)
0 15 30 60 120 240 480 960
T.
(ng/dl)
T4
(9/dL)
TSH
(ng/ml)
56.2
±3.3
3.4
±0.2
3.3
±0.4
58.1
±2.4
3.3
±0.1
3.0
±0.5
61.2
±3.1
3.6
±0.2
3.6
±0.4
63.5
±2.4
3.4
±0.2
3.9
±0.6
59.7
±3.3
3.1
±0.1
5.9*
±0.7
44.4*
±2.5
2.5*
+0.3
7.8*
±0.9
26.4*
±2.6
0.6*
±0.1
12.2*
±0.7
23.5*
±2.0
0.2*
±0.1
13.8*
±1.0
*p<.05 compared to control value.
Table A-8. Effect of Compound 1 on Hepatic 5'-monodeiodinase in Rats
Dose Hepatic T3 generation from T4
(mg/kg/day) (% of control value)
0 100
125 97.5
250 61.0*
500 39.5*
*p<.05 compared to control value.
e. Dose Correlations
The plethora of data on compound 1 lead to some important correla-
tions between dose and various neoplastic and preneoplastic lesions as
well as other effects. They are discussed later in the Hazard and Dose-
Response Characterization section of this Appendix; specific correlations
are illustrated in Figures A-1 and A-2.
f. Ancillary Data
Data were provided on reversibility of induced effects, pituitary changes
and initiation-promotion aspects for compound 1 in support of the position
that the thyroid tumors occurred as the result of thyroid-pituitary disruption.
(1) Reversibility studies.
Male rats were administered 240 mg/kg/day of compound 1 for 4 weeks
to induce increases in thyroid weight and hormone changes. Animals were
then allowed to recover for 4 additional weeks, by which time thyroid weights
and serum T3 and T4 and TSH had returned to the control range. This study
demonstrates that cells are not irreversibly committed to progress to neo-
A-13
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100
o
£
"CD
Thyroid tumors (2 years)
Thyroid weight (28 days)
— - Thyroid Weight (1 year)
200
400 600
Dose (mg/kg/day)
800
1000
Figure A-1. Thyroid weight and tumor responses to compound 1.
plasia. Instead, weight and thyroid hormone changes are completely re-
versible to preexposure values upon cessation of treatment.
(2) Effect of exogenous thyroid hormone.
In conjunction with the study described in d(1) above, additional groups
of male rats were administered compound 1 (240 mg/kg/day) for 4 weeks
in combination with equal amounts of T4 and T3 at three different dose
levels. Thyroid weights and serum T4, T3, and TSH levels were measured
at the end of the 4-week period. The results, shown in Table A-9, are con-
sistent with an interpretation that T4/T3 treatment blocks the antithyroid
effects of compound 1.
These two studies emphasize the effect of compound 1 on circulating
TSH levels as an ultimate mechanism that can be manipulated by a recov-
A-14
-------
- 3
Serum TSH (ng/ml)
Serum T4 (ug/dL)
— 2
200
—I 1 1
400 600
Dose (mg/kg/day)
T3
O)
-1
800
1000
Figure A-2. Hormone responses to compound 1.
ery phase or replacement therapy back to the normal range of thyroid-
pituitary functioning.
(3) Initiation-promotion data.
The carcinogen N-bis(2-hydroxypropyl) nitrosamine (DHPN) is used
frequently as a model initiator in rodent studies involving the thyroid as a
target organ. In one of these studies, male F344 rats, 6 weeks old, were
administered a single i.p. injection of DHPN and fed compound 1 in the diet
(240 mg/kg/day), beginning 1 week after injection, for 19 weeks. The inci-
dence of thyroid follicular cell tumors in the group receiving DHPN and
compound 1 was 95% compared with 5% in the DHPN only group and 0%
in the compound 1 only group. The results, summarized in Table A-10,
A-15
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Table A-9. Effect Of T3 and T4 Treatment on Thyroid Weights and Serum T4, T3
and Tsh Levels in Male Rats given Compound 1 over a 28-day
Period
Doses
Compound 1
(mg/kg/day) 0 240 240 240 240
(&g/kg/day each) 0 0 10 30 50
Thyroid weight
(mg)
(ng/dL)
(9/dL)
TSH
(ng/ml)
18.4
±0.9
60.2
+2.8
3.9
±0.1
3.01
±0.6
27.3*
±0.9
45.5*
±2.8
2.5*
±0.4
6.36*
±0.7
22.2
±1.7
50.7*
±2.1
2.9
±0.5
4.84*
±0.7
27.6
±0.9
66.3
±3.1
4.3
±0.1
3.08
±0.6
17.5
±0.8
63.1
±1.7
4.5
±0.3
2.62
±0.4
*p<.05 compared to control value.
Table A-10. Numbers (Percent) of Male Rats with Thyroid Tumors at 20 Weeks
after Treatment with DHPN Followed by Compound 1
Treatments
None DHPN Compound 1 DHPN + Compound 1
Thyroid 0/20 1/20 0/20 19/20
tumors (0) (5) (0) (95)
provide convincing evidence that compound 1 can act as a promoting agent
for the development of thyroid tumors in rats. Alone, the compound did not
induce tumors at the end of the observation period, but following a mu-
tagenic initiator a significant proportion of animals developed tumors.
(4) Pituitary changes.
Because of the finding of pituitary adenomas in the 2-year rat and mouse
studies, male rats in the expanded 28-day study were investigated as to
the histologic origin of the pituitary lesions. At weekly intervals, groups of
animals were sacrificed, and sections of pituitary gland were stained with
the aldehyde-thionine-PAS stain or immunohistochemically with an antirat
TSH antibody using the peroxidase staining method. Microscopic evalua-
tion of aldehyde-thionine-PAS stained sections showed hypertrophy and
A-16
-------
hyperplasia of basophilic cells (thyrotrophs) that secrete TSH after 1 week.
Immunohistochemical staining of pituitary sections showed a quantifiable
increase in TSH immunoreactivity by 7 days. An increase in the number of
thyrotrophs induced by compound 1 correlates with an expected stimula-
tion of the pituitary under conditions of reduced thyroid hormone levels and
with increased production of TSH; it is a further indication of an effect on
the thyroid-pituitary axis. Accordingly, it is reasonable to conclude that the
pituitary adenomas arose from basophilic thyrotrophs.
g. Structure-Activity Relationships
Chemically, compound 1 is a thionamide, a class of compounds that
has produced thyroid tumors in rodents and sometimes liver tumors in mice.
The chemicals frequently are inhibitors of thyroid peroxidase, and some
are also inhibitors of hepatic 5'-monodeiodinase activity. The biological and
tumor findings with compound 1 are consistent with these structural at-
tributes.
h. Metabolic and Pharmacokinetic Properties
Metabolism and excretion studies in the rat were conducted with 14C-
labeled compound 1 administered orally or intravenously. No comparative
studies have been done in the mouse or the monkey. The results, reported
in the literature, were in agreement regardless of route of administration.
Radioactivity in rat blood was identified as unchanged chemical. Less than
5% of radioactivity was detected in the feces, but 80% to 85% of the ad-
ministered dose was excreted via the kidneys within 24 hours, all radioactivity
clearing by 48 hours. About 15% of the administered dose was excreted in
the urine as the unchanged compound, while the major urinary metabolite
was the chemical conjugated with glucuronide. Approximately 15% of the
compound was excreted into the bile as a glucuronide conjugate (different
from the major urinary metabolite), indicating enterohepatic circulation. No
oxidative metabolites could be demonstrated in urine or plasma.
Accumulation of compound 1 in the thyroid was investigated by admin-
istering the chemical radiolabeled with 35S i.p. to rats and comparing the
radioactivity in the thyroid to the serum. The results of this study showed
that the intact parent compound preferentially accumulated in the thyroid
gland, as has been found for some of the other thiol-containing chemicals
and which is consistent with some direct antithyroidal action for compound
1.
3. Human Observations
The manufacturer of compound 1 has produced the chemical, using
batch processing, for many years. The medical department monitored thy-
roid function of about 150 employees directly involved in the synthetic
process every 2 years over the preceding 10 years. Overall, the results
A-17
-------
showed no significant differences between exposed workers and normal
values reported in the literature for serum T4 and TSH. T3, measured in the
most recent surveillance, was also within normal limits. No worker exposed
to compound 1 reported symptoms or showed signs consistent with hy-
pothyroidism, and medical examination did not reveal any enlarged thyroid
glands. Occupational exposure levels never reached the level of quantifi-
cation using a rather insensitive air monitoring system.
Hazard and Dose-Response Characterization
Compound 1 may pose a carcinogenic hazard to the human thyroid if
doses perturb thyroid-pituitary homeostasis. It produces thyroid tumors
(males/females) and related pituitary tumors (males) in rats and mice. It is
readily absorbed following oral exposure, and because of its small size
and physical properties, it also should be readily absorbed through the
lung and to a lesser extent the skin. Extensive mode-of-action information
demonstrates that compound 1 inhibits both the synthesis of thyroid hor-
mone in the thyroid gland and the conversion of thyroid hormone (T4) to its
more active form (T3) in organs outside the thyroid gland. Given this infor-
mation and the absence of relevant mutagenicity, it would appear that thyroid
risk would be minimal under conditions of thyroid-pituitary homeostasis.
Therefore, in the absence of a mathematical model that incorporates mode-
of-action data on a chemical like this, a dose- response relationship that
involves nonlinear default extrapolation will be projected.
There is strong evidence that compound 1 has carcinogenic activity in
laboratory animals. The agent is a thionamide, a group of chemicals that
often produces thyroid (and sometimes other) tumors in rodents. Both male
and female rats and mice receiving compound 1 in feed showed significant
increases in thyroid follicular cell tumor (benign and malignant tumors in
rats, benign only in mice) incidence. Rats and male mice also showed an
increase in benign pituitary tumors. No other tumors were increased in
dosed animals. There is no information on the carcinogenicity of compound
1 in humans.
Overall, mutagenicity studies fail to demonstrate relevant effects. There
is no indication from those endpoints that there is some direct mechanistic
relationship to carcinogenic processes: gene mutations (Salmonella and
cultured mammalian cells), structural chromosome aberrations (rat bone
marrow), DNA reactivity using radiolabeled compound 1, and analysis of
structural analogues. Although other studies (yeast mitotic gene conver-
sion and crossing over and mammalian cell sister chromatid exchange)
show at least some indication of a positive response, the implication of
chemically induced increases in these effects to carcinogenicity is not well
understood. There are no studies of the potential genetic effects of com-
pound 1 in the thyroid per se; such information may be useful.
A-18
-------
An extensive range of testing demonstrated that the thyroid tumors
may be due to disruption of thyroid-pituitary functioning. The chemical
causes a dose-related enlargement of the thyroid gland (goitrogenic effect)
and a progression of lesions over time. After 28 days of dosing, individual
thyroid cells show enlargement in size (hypertrophy), and the proportion of
cells in cell division (mitotic index) increases. In addition, there is an in-
crease in the number of thyroid cells (hyperplasia). These factors combined
lead to increased thyroid weights of dosed rats. These effects persist in
rats sacrificed at 1 year, and after 2 years of treatment, thyroid tumors are
noted.
Studies also indicate that compound 1 results in altered thyroid-pitu-
itary hormone levels. By 28 days of treatment, there are profound
dose-related reductions in serum T4 and T3 and increases in TSH. There is
a dose correlation between those that influence both hormone levels and
thyroid histology. Hormone and histologic effects are reversible when treated
animals are returned to a diet without compound 1. All histologic changes
in the above studies occur at doses of 240 mg/kg and higher; all changes
in hormones occur at the same doses, except TSH at 28 days, which is
significantly increased at 120 mg/kg and higher.
The site of action of compound 1's influence on thyroid-pituitary status
seems to be centered on the thyroid gland and extrathyroidal sites. The
compound is concentrated in the thyroid gland like some other thionamides
and produces reversible reduction in thyroid peroxidase activity (in vitro),
the enzyme that results in iodination of tyrosyl moieties and their coupling
into thyroid hormones. In addition, there is reduction in liver 5'-
monodeiodinase activity (>250 mg/kg), which reduces the normal conversion
of circulating T4 to the cellular active form, T3. Studies of other potential
sites of antithyroid action of compound 1 (e.g., interference of thyroid up-
take of inorganic iodide and induction of liver enzymes) have not been
conducted. However, the identified influence of compound 1 on thyroid
hormone synthesis and conversion can adequately explain the increases
in TSH levels and the resulting growth of the thyroid gland.
Other studies indicate that thyroid-pituitary imbalance leads to tumor
formation. Rat pituitary adenoma cells contain TSH, which is consistent
with reductions in thyroid hormone levels with corresponding increased
stimulation of the pituitary to produce TSH. Compound 1 promotes thyroid
tumors after treatment with an initiator. In addition, thyroid gland and hor-
monal changes are blocked by coadministration of compound 1 and amounts
of thyroid hormones that maintain normal thyroid-pituitary status.
All mechanistic work is consistent with this chemical producing thyroid
and pituitary tumors in rodents by a nonmutagenic mode of action that
involves (1) inhibition of the synthesis of active thyroid hormone, (2) feed-
A-19
-------
back stimulation of the pituitary to enlarge and synthesize TSH, and (3)
TSH stimulation of growth of the thyroid gland. Although the precise events
accounting for transformation of hyperplastic cells into neoplastic cells are
unknown, they seem to occur under conditions of continued TSH stimula-
tion. If exposures to compound 1 can be kept at levels that do not lead to
thyroid-pituitary disruption, there would be no stimulus for tumor develop-
ment in either the thyroid or the pituitary.
Relevant dose-response data demonstrate a consistent alteration in
test parameters in rats at doses of at least 120 mg/kg. Few data are avail-
able in the mouse other than the finding of tumors at a dose (900 mg/kg/
day) higher than in rats; therefore, information in rats is used. Figure A-1
shows dose-response plots of thyroid weight after 28 days, thyroid weight
after 1 year, and thyroid tumors after 2 years of dosing. Figure A-2 shows
similar dose-response plots for T4 and TSH levels after 28 days of dosing.
Other endpoints could have been depicted, but they add nothing signifi-
cant to what is depicted here; besides, they show similar dose-response
relationships. Tumor incidences are increased at 240 mg/kg, a value con-
sistent with increases in thyroid weight (after both 28 days and 1 year of
dosing) and decreases in thyroid hormone levels at 28 days. TSH, how-
ever, is significantly elevated at doses of 120 mg/kg and above.
Since TSH plays a pivotal role in thyroid stimulation and there is a
progressing doubling of doses to help illustrate the effects of dose and
effect, the TSH level is used to estimate a dose of compound 1 that would
not be expected to increase the hormone level in rats above that of normal,
untreated animals. The TSH response appears to be essentially flat from
the control through the 15, 30, and 60 mg/kg/day groups, with a profound
upward change in slope from 60 mg/kg/day up to the highest dose tested,
960 mg/kg/day.
The question is how far to extrapolate downward from high doses of
compound 1 associated with pronounced increases in TSH to reach a value
that probably is without significant effect on the thyroid. Given the promi-
nence of TSH in priming the carcinogenic process in the rodent thyroid,
one would not want chemically induced additions to the normal homeo-
static levels of TSH. In addition, the information at hand is quite revealing;
it seems that some doses of compound 1 are without effect on circulating
TSH levels. For instance, doses up to about 60 mg/kg/day do not seem to
add to the underlying TSH level in any significant way. It appears that some
level of compound 1 is required before it can interfere with thyroid homeo-
stasis, that is, to inhibit both thyroid peroxidase and T4 deiodination to T3,
to decrease circulating thyroid hormone levels, and to increase TSH. In
recognition of these findings, the 60 mg/kg/day no-observed-adverse-ef-
fect level (NOAEL) from the 28-day rat study is a reasonable estimate of a
A-20
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point of departure for projecting concern for human exposures and calcu-
lating a margin of exposure.
There is limited information on compound 1 concerning the ability to
extrapolate the finding of thyroid tumors in rodents to humans. Workers
exposed to undisclosed levels of compound 1 have no indication of pertur-
bation of thyroid-pituitary status; all exposed individuals are without
significant physical findings and have relevant hormone values with the
same mean and distribution as in the general population. A limited number
of monkeys exposed to the compound at 300 mg/kg failed to show any
thyroid effects; these findings suggest that primates (and possibly humans)
may be less sensitive than rats (possibly, at least fivefold on a mg/kg basis)
to the antithyroid effects of the chemical. Therefore, using the midpoint of
the control TSH level as a point of departure may be a conservative esti-
mate of the potency of compound 1. This information should be considered
in the evaluation of the significance of the calculated margin of exposure
for humans.
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Ithiouracil analogues: comparison of 5'-deiodinase inhibition and anti-
thyroid activity. Endocrinology 118:1598-1605.
Owen, NV; Worth, HM; Kiplinger, GF. (1973) The effects of long-term in-
gestion of methimazole on the thyroids of rats. Fd Cosmet Toxicol
11:649-653.
Sitar, DS; Thornhill, DP. (1972) Propylthiouracil: absorption, metabolism,
and excretion in the albino rat. J Pharmacol Exp Therap 183:440-448.
Steinhoff, D; Weber, H; Mohr, U; et al. (1983) Evaluation of amitrole
(aminotriazole) for potential carcinogenicity in orally dosed rats, mice,
and golden hamsters. Toxicol Appl Pharmacol 69:161-169.
Swarm, RL; Roberts, GKS; Levy, AC; et al. (1973) Observations on the
thyroid gland in rats following the administration of sulfamethoxazole
and trimethoprim. Toxicol Appl Pharmacol 24:351-363.
Thomas GA; Williams, ED. (1991) Evidence for and possible mechanisms
of non-genotoxic carcinogenesis in the rodent thyroid. Mutat Res
248:357-370.
Wynford-Thomas, D; Stringer, BMJ; Williams, ED. (1982) Goitrogen-induced
thyroid growth in the rat: quantitative morphometric study. J Endocrinol
94:131-140.
A-22
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Compound 2: A Chlorinated Cyclic Hydrocarbon that
May Influence the Thyroid through Effects on the
Liver; Significant Data Gaps
Executive Summary
Compound 2 is a chlorinated cyclic hydrocarbon that produces thyroid
but no other tumors following chronic dosing in male and female rats. Some
structural analogues produce liver tumors in mice; none has produced thy-
roid tumors.
In studies on the mode of carcinogenic action, compound 2 produces
enlargement of the thyroid gland and a decrease in serum T4 but not T3.
There is an increase in liver weight in rats in the chronic study. Induction of
liver microsomal enzymes is reported in one acute study using a very high
dose. Compound 2 is nonmutagenic in the only test in which it has been
evaluated (Salmonella gene mutation); however, some analogues produce
structural chromosome aberrations.
Many uncertainties are associated with the assessment of compound
2. Major data gaps include a cancer study in a second species, further
mutagenicity testing, demonstration of the chemical's influence on TSH
levels, and a delineation as to whether enhanced thyroid metabolism and
excretion by the liver may account for the observed thyroid tumors.
Given the data at hand, a low-dose linear approach is used for dose-
response purposes until such time that further data are developed and a
mode of action is determined.
Detailed Data
1. Cancer Findings
The animal cancer bioassay provides convincing evidence of a posi-
tive tumorigenic effect by compound 2 for the thyroid in rats. This study
was conducted in male and female rats, 50 animals per group, using three
dose levels of compound 2 (15, 50, and 150 mg/kg) and a control, admin-
istered by gavage each day, seven times weekly. Over the 2-year study
period, survival of the animals was excellent, with the high-dose males
exhibiting the highest mortality at 10%.
A-23
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Thyroid tumor incidences with respect to dose are summarized in Table
A-11. A dose-related increase in thyroid tumors was statistically significant
in the mid- and high-dose males and females.1 A significant increase in
tumors was not detected for any other organ. In particular, out of a total of
389 rats in this carcinogenicity bioassay, only two liver tumors, both hepa-
tocellular adenomas, were observed, one in a mid-dose male and one in a
control female.
Table A-11. Numbers (Percent) of Rats with Thyroid Tumors (Adenomas/
Carcinomas Combined)
Dose (mg/kg/day by gavage)
Sex 0 15 50 150
Male 2/50 3/50 7/48* 7/45*
(4) (6) (15) (16)
Female 1/50 1/50 7/49* 9/47*
(2) (2) (14) (19)
*p<.05 compared to control value.
2. Mechanistic Considerations
a. Mutagenicity
Compound 2 was nonmutagenic in Salmonella assays for frameshift
(TA-98) and base-pair (TA-100) mutations. No other endpoints have been
assayed.
b. Thyroid Growth
Information on thyroid weight and histology is available from a 90-day
study in which compound 2 was administered daily by gavage to male rats,
10 per group. Thyroid weights were increased in a dose-related manner at
all doses tested (Table A-12). Effects were significant at each dose as com-
pared with the control. Microscopic examination of the animals showed
that the increase in thyroid weight was due to diffuse follicular cell hyper-
plasia, characterized by more numerous but smaller follicles. There was
no statement as to whether cells were hypertrophied.
c. Hormones
In a 14-day study using male rats (10 per dosage group), the chemical
was administered daily by gavage, and serum T3 and T4 concentrations
were then measured (Table A-13). T4 concentrations showed dose-related
decreases and were statistically significant at the two highest doses. T3
1The investigators did not report separate incidences for thyroid adenomas and carcinomas; see Table A-
11.
A-24
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Table A-12. Mean Thyroid Weights (Mg) in Male Sprague-Dawley Rats Treated
for 90 Days with Compound 2
Dose (mg/kg/day by gavage)
0 15 50 150
16.412.5 23.814.1* 24.1+5.8* 32.614.6*
*p<.05 compared to control value.
Table A-1 3. T3 And T4 Levels in Male Sprague-Dawley Rats Treated with
Compound 2 for 14 Days
Dose (mg/kg/day by gavage)
Hormone 0 5 10 50 150
T (ng/dl) 94.4±19.1 89.3115.5 94.5+11.2 90.7+14.8 91.3+10.6
4.6+1.2 4.510.9 4.0+0.6 3.510.7* 3.310.6*
*p<.05 compared to control value.
levels were not affected by dosing. There was no measurement of TSH
levels.
d. Site of Action
(1) Thyroid.
No studies investigated the effects of compound 2 on the thyroid gland
per se.
(2) Other.
There is a lack of information concerning the effects of compound 2 on
5'- monodeiodinase activity.
(3) Liver.
In the 2-year rat study, the livers in low-, mid-, and high-dose animals
were heavier on an absolute and relative-to-body-weight basis than those
in controls; increases were statistically significant in the mid- and high-
dose groups. There was no mention of histologic changes in the livers.
Compound 2's mixed-function oxidase (MFO)-inducing properties have
been demonstrated in one acute study in Sprague-Dawley rats. Animals
received a single dose of compound 2 that was equivalent to one-half the
acute LD50 (1,000 mg/kg). Liver microsomal enzyme activities were mea-
sured 3 days later and included cytochrome P-450 content,
benzphetamine-N-demethylase, 7-ethoxycoumarin O-deethylase, and
A-25
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NADPH-cytochrome c-reductase. In all cases, the values were significantly
greater in treated animals than in controls (Table A-14).
These data provide evidence that compound 2 induces liver enzyme
activity in the rat at a high acute dose. No studies have been conducted
following repeat dosing and at chemical doses around those that are asso-
ciated with the development of thyroid tumors. Similarly, there is an absence
of data on the activity of uridine diphosphate glucuronosyl-transferase in
the liver and of T4 clearance from serum or excretion in the bile.
Table A-14. Hepatic Microsomal Enzyme Activities in Male Sprague-Dawley
Rats Treated for 14 Days with Compound 2
Cytochrome Benzphetamine- 7-Ethoxycoumarin
NADPH-cytochrome P-450 N-demethylase O-deethylase
Dose
(mg/kg)
0
1,000
c-reductase
(nmol reduced/mg)
1.4810.10
2.09A0.21*
(nmol/mg
protein/min)
6.54±0.93
9.27A0.84*
(nmol/mg
protein/min)
2.14+0.30
3.84A0.20*
(nmol/mg
protein/min)
253.3A70.7
370.8A51.4*
*p<.05 compared to control value.
e. Dose Correlations
Given the data gaps, only preliminary correlations can be developed
between dose and specific endpoints. These are discussed later (see Table
A-15).
f. Metabolism
In the rat, compound 2 is freely absorbed through the gastrointestinal
tract and extensively metabolized in the liver; individual metabolites have
not been characterized. Little else is known about the handling of the chemi-
cal, and no comparative studies in different species are available.
Table A-15. Summary of Effects Relevant to Evaluating the Significance of
Thyroid Tumors in Rats
Dose (mg/kg/day)
Study Effect
5 10 15 50 150 1,000
2-Year study: tumors - + +
liver weight + + +
90-Day study: thyroid weight + + +
14-Day study: T, decrease - ± + +
Acute study: MFO induction +
A-26
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g. Structure-Activity Relationships
Some structural analogues produce liver tumors in mice, but most have
not been studied at all for carcinogenicity. None has induced thyroid tu-
mors. Some analogues produce structural chromosome aberrations in
cultured mammalian cells; one close analogue also induces these aberra-
tions in mammals in vivo.
3. Human Observations
No relevant data are available on thyroid-related function or carcino-
genicity of this chemical in humans.
Hazard and Dose-Response Characterization
Compound 2, a chlorinated cyclic hydrocarbon, produces a small but
significant increase in thyroid tumors in gavaged male and female rats. No
other tumor increases are noted. The chemical has not been tested for
carcinogenicity in a second laboratory species, and nothing is known about
its potential carcinogenic effects in humans. Data on the carcinogenicity of
structural analogues do not indicate carcinogenic effects in the thyroid,
although some produce liver tumors in mice; most analogues have not
been studied for carcinogenicity. It is likely that compound 2 may pose a
carcinogenic hazard to humans.
The existing mutagenicity information indicates negative effects for gene
mutations in Salmonella. Some structural analogues produce structural
chromosome aberrations in mammalian cells that could influence carcino-
genicity.
There are data indicating that compound 2 affects thyroid-pituitary func-
tioning. Within 90 days of dosing, there is thyroid enlargement associated
with diffuse hyperplasia and an increase in thyroid weight. It is not known
whether there is cellular hypertrophy in the thyroid. Hormone changes were
investigated after dosing for only 14 days. Serum T4 decreased with dos-
ing while serum T3 remained unchanged; TSH levels were not measured.
There are no studies measuring hormones at times beyond 14 days.
Concerning the site of action, compound 2 produced liver enlargement
in the chronic rat study, and consistent with this, it induced microsomal
enzymes in the rat liver (e.g., increased P-450 content and benzphetamine-
N-demethylase activity) following a single, high acute dose. No studies
have been conducted following subchronic dosing or at doses around those
that produced thyroid tumors in rats.
A mode of action that accounts for production by compound 2 of thy-
roid tumors in rats can be proposed. It seems to be acting by nonmutagenic
processes that involve chemically induced antithyroid activity. The com-
A-27
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pound may enhance metabolism and excretion of thyroid hormone by stimu-
lating hepatic microsomal enzymes. These enzymes would be expected to
enhance clearance of thyroid hormone from the body and result in a lower-
ing of serum thyroid hormone levels. These decreases would result in reflex
increases in TSH levels with stimulation and enlargement of the thyroid
gland and, eventually, neoplasia. These observations and presumptions
support the use of a default dose-response analysis using threshold con-
siderations (use of an NOAEL and computation of the margins of exposure).
Significant uncertainties exist, which bring into question the proposed
mode of action and the merit in using the margin of exposure method.
Compound 2 has not been tested for cancer in a second species, and
some related chemicals are known to induce liver tumors in mice. In addi-
tion, the agent has not been adequately tested for gene mutations, structural
chromosome aberrations, and other such endpoints. The site of antithyroid
action has not been adequately investigated; studies should investigate
the thyroid and periphery as well as the liver where preliminary evidence
suggests an effect. Mode-of-action studies are needed following subchronic
dosing and at dosages adequate to explain thyroid tumor formation.
Because of the uncertainties and until such time that more information
is available that explains a mode of action for compound 2, dose-response
relationships will be projected by the low-dose linear approach.
There is not an extensive or totally appropriate database to evaluate
the antithyroid effects of compound 2, but the existing dose correlations
are summarized in Table A-15, assuming that the liver may play some role
in tumor development. Preneoplastic effects are noted at 15 mg/kg/day
(thyroid weight) and 10 mg/kg/day (T4 decrease), which are lower than the
dose producing thyroid tumors (and increased liver weight) in the chronic
study (50 mg/kg/day). An estimate of the NOAEL can be derived from the
14-day study where a dose of 5 mg/kg/day had no significant effect on the
T4 level. Further work is needed.
Depending on the outcome of future testing, a linear default may be
maintained if the agent is mutagenic, if more than one tumor site is noted,
or if adequate mode of action information is not developed to understand
thyroid tumor production. A nonlinear default may be used if no other tu-
mors are found in a second species, the compound is not mutagenic, and
an antithyroid mode of action is found to describe the formation of thyroid
tumors. Both methods may be retained when more than one mode of ac-
tion leads to the conclusion that both linear and nonlinear projections are
about equally tenable.
A-28
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Bibliography
The following articles were consulted in developing the data used in
this case study of the hypothetical chemical.
Comer, CP; Chengelis, CP; Levin, S; et al. (1985) Changes in thyroidal
function and liver UDP-glucuronosyl-transferase activity in rats follow-
ing administration of novel imidazole (SC-37211). Toxicol Appl
Pharmacol 80:427-436.
Lumb, G; Newberne, P; Rust, JH; et al. (1978) Effects in animals of chronic
administration of spironolactone—a review. J Environ Pathol Toxicol
1:641-660.
Sanders, JE; Eigenberg, DA; Bracht, LJ; et al. (1988) Thyroid and liver
trophic changes in rats secondary to liver microsomal enzyme induc-
tion caused by an experimental leukotriene antagonist (L-649,923).
Toxicol Appl Pharmacol 95:378-387.
Semler, DE; Chengelis, CP; Radzialowski, FM. (1989) The effects of chronic
ingestion of spironolactone on serum thyrotropin and thyroid hormones
in the male rat. Toxicol Appl Pharmacol 98:263-268.
A-29
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Compound 3: A Bis-benzenamine that Produces Thyroid
and Liver Tumors; Antithyroid and Mutagenic Effects
Executive Summary
Compound 3 produces thyroid follicular cell and liver tumors in two
rodent species. The thyroid tumors are associated with interference in thy-
roid-pituitary functioning, presumably due to inhibition of thyroid peroxidase
activity. There is no mechanistic information concerning the development
of liver tumors. Compound 3 is also DNA reactive and mutagenic, and these
effects could influence tumor development in both the thyroid and the liver.
Accordingly, it is likely that compound 3 may pose a human cancer hazard,
and a low-dose linear procedure should be used for the quantification of
both cancer dose-response relationships, although a lower bound on the
thyroid cancer "risk" should also include nonlinear considerations.
Detailed Data
1. Cancer Findings
Chronic laboratory animal studies provide strong evidence of a tumori-
genic effect of compound 3 for both the thyroid and liver in both sexes of rat
and for the female mouse. F344 rats and B6C3F1 mice were administered
compound 3 at three doses of 200, 400, and 500 (20, 40, and 50 mg/kg/
day) and 150, 300, and 800 ppm (30, 60, and 160 mg/kg/day) in tap water,
respectively. There were 50 animals per sex/species/dose, with the control
groups receiving plain tap water. No animal died before 52 weeks of dos-
ing. Survival was lowest in the control male rats (50%) and the high-dose
female rats (26%) at 104 weeks. In all other groups (rats and mice), sur-
vival ranged from 60% to 82%. The tumor incidence results are summarized
in Table 16 for both rats and mice.
The incidences of liver tumors (adenomas, carcinomas) were signifi-
cantly increased in the low-, mid-, and high-dose male rats, in the mid- and
high-dose female rats, and in the high-dose female mice. Male mice showed
no increase in liver tumors. Although foci and areas of hepatocellular alter-
ation were present, consistent with the preneoplastic phases of liver
carcinogenesis, there was no indication of hepatocellular hypertrophy char-
acteristic of MFO-inducing compounds.
A-30
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Table A-16. Rodents with Liver and Thyroid Tumors in 2-Year Bioassays of
Compound 3
Rats
Tumor incidence (percent) according to dose (ppm in water)
(N=50 animals/group)
Sex
Liver (hepatocellular)
Adenoma
Carcinoma
Thyroid (follicular cell)
Adenoma
Carcinoma
0
M
1
(2)
0
1
(2)
0
200
F
3
(6)
0
0
0
M
9*
(18)
4*
(8)
1
(2)
2
(4)
F
0
0
2
(4)
2
(4)
400
M
18*
(36)
23*
(46)
8*
(16)
9*
(18)
F
20*
(40)
4*
(8)
17*
(34)
12*
(24)
500
M
17*
(34)
22*
(44)
13*
(26)
15*
(30)
F
11*
(22)
6*
(12)
16*
(32)
7*
(14)
Mice
Sex
Liver (hepatocellular)
Adenoma
Carcinoma
Thyroid (follicular cell)
Adenoma
0
M
11
(22)
18
(36)
0
150
F
4
(8)
4
(8)
1
(2)
M
13
(26)
27
(54)
0
F
6
(12)
7
(14)
0
M
11
(22)
23
(46)
2
(4)
300
F
9
(18)
6
(12)
0
800
M
10
(20)
26
(52)
2
(4)
F
14*
(28)
15*
(30)
7*
(14)
*p<.05 compared to control value.
The incidences of benign and malignant thyroid follicular cell neoplasms
were significantly increased in the mid- and high-dose male and female
rats and in the high-dose female mice (benign only). Male mice showed no
increase in thyroid tumors. Tumors occurring at sites other than the liver
and thyroid in rats and mice were not statistically related to treatment.
2. Mechanistic Considerations
a. Mutagenicity
Compound 3 was studied in both in vitro and in vivo short-term tests
for mutagenicity. In the Salmonella mutation assay, the compound was
A-31
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positive in the presence of an 39-activating system using strains TA-98
and TA-100. The compound induced transformation in BHK cells, DNA-
strand breaks, and point mutations in V79 cells and DNA repair in rat
hepatocytes. There was an equivocal response for sister chromatid ex-
changes in mouse bone marrow but positive results for chromosomal
aberrations in the same system. Because compound 3 is DNA reactive
and produces point and structural chromosomal mutations, it is regarded
as having mutagenic activity relevant to carcinogenicity,
b. Thyroid Growth
To date, the only data generated for compound 3 specifically on thyroid
weights were from a published account of its administration to male Wistar
rats at 400 ppm in tap water for 20 weeks. At cessation of treatment, the
mean values for thyroid weight were statistically significantly different in
treated (30+6 mg) as compared with control (18+8 mg) groups (21 rats per
group), respectively.
In the 13-week study, male and female Fischer rats were given 200,
400, and 700 ppm of compound 3 in tap water, while male and female mice
received 100, 300, and 700 ppm. Thyroid follicular cell hyperplasia was
found in 5/10 and 7/10 mid-dose male and female rats, respectively, but
only in 1/9 high-dose males. Adenomatous goiters were observed in 3/10
mid-dose males and 1/10 mid-dose females, while more severe goiters
were found at the high dose in 8/9 males and 10/10 females. In mice,
adenomatous goiters (less severe than those found in rats) were observed
in 1/10 of high-dose males and 1/10 high-dose females. None of the low-
dose rats or mice showed thyroid lesions.
The incidence of diffuse hyperplasia of the thyroid gland in the 2-year
bioassay was significantly increased in the mid- and high-dose rats of both
sexes and the high-dose mice of both sexes, as summarized in Table A-17.
In sum, many of these data indicate that compound 3 has an enhanc-
ing effect on thyroid growth by inducing diffuse thyroid follicular cell
hyperplasia and adenomatous goiter and increased thyroid gland weight
on repeated administration.
c. Hormone Levels
The studies completed so far provide limited data on the effect of com-
pound 3 on serum levels of T4, T3, and TSH. Serum levels for these three
hormones were measured in a group of five male rats after exposure to
400 ppm of compound 3 for 20 weeks and compared with those from a
control group (also five male rats), as illustrated in Table A-18. Circulating
levels of both T3 and T4 were significantly reduced and TSH was signifi-
cantly elevated, indicating an effect of the compound on thyroid-pituitary
functioning.
A-32
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Table A-17. Rats and Mice with Diffuse Thyroid Hyperplasia after 2 Years of
Exposure to Compound 3
Incidence (percent) according to dose (ppm in water)
(N=50 animals/group)
Sex
Rats
Sex
Mice
0 200
M F M
0 0 1
(2)
0 200
M F M
000
F
1
(2)
F
0
400
M
11*
(22)
400
M
0
500
F
16*
(32)
M
13*
(26)
F
22
(44)
500
F
0
M
26*
(52)
F
25*
(50)
*p<.05 compared to control value.
Table A-18. Serum T4, T3, and TSH Levels in Male Wistar Rats Exposed to 400
ppm of Compound 3 in Water for 20 Weeks
Control Treated
T., 54.6+1.8 40.2+2.2*
(ng/dL)
T 5.2+1.1 3.0+0.3*
(94/dL)
TSH 4.1+0.8 6.1+0.7*
(ng/mL)
*p<.05 compared to control value.
d. Site of Action
No studies have yet been conducted to investigate the action of com-
pound 3 on components of the iodide pump, thyroid peroxidase, peripheral
deiodination of T4, or MFO induction. However, some close analogues are
known to reduce radioactive iodine accumulation in the thyroid; presum-
ably this is due to an inhibition of thyroid peroxidase activity and not a
function of affecting the iodide pump.
In the animal bioassays, the liver shows sinusoidal dilatation in male
rats, fatty metamorphosis and focal cellular change in male and female
rats, and degeneration in male mice. It has been speculated that the meta-
A-33
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bolic activation of compound 3 to a reactive electrophile may be respon-
sible for these nonneoplastic lesions. This is consistent with the Salmonella
mutation data, where effects are noted only after metabolic activation. No
other information is available concerning the biological processes leading
to liver tumor development.
e. Dose Correlations
Important correlations can be made between the dose of compound 3
and various effects. These are discussed later in this Appendix (see Table
A-19).
f. Metabolic and Pharmacokinetic Properties
No information is available on the tissue distribution of compound 3. It
has been estimated that 90% is absorbed through the gastrointestinal and
respiratory tracts in rats. The compound is rapidly excreted in the urine
with two major metabolites. No comparative metabolic information is avail-
able in mice or humans.
Table A-19. Thyroid Effects Noted in the Toxicity Studies in Rats and Mice on
Compound 3
Dose in drinking water (ppm)
Rats 200 400 500 700
2-Year study
Tumors - + +
Hyperplasia - + +
13-Week study
Hyperplasia/goiter - + +
20-Week study
Thyroid/pituitary
hormones and thyroid
weight +
Mice 100 150 300 700 800
2-Year study
Tumors - - +
Hyperplasia - - +
13-Week study
Goiter - - +
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g. Structure-Activity Relationships
Compound 3 is a member of the bis-benzenamine class of chemicals,
being an alkyl-substituted derivative of 4,4'-methylene-dianiline. Several
dianilines with methylene, oxygen and sulfur bridges, and their alkyl-sub-
stituted derivatives have been shown to (1) inhibit iodine accumulation in
the thyroid, (2) possess goitrogenic activity following repeated administra-
tion, and (3) produce thyroid tumors and sometimes liver tumors in rats
and/or mice. These aromatic amines are mutagenic in a number of short-
term tests relevant for evaluation of carcinogenicity, namely, DNA reactivity
and gene and structural chromosome mutations.
3. Human Observations
No data are available concerning the effects on humans of exposure to
compound 3. However, it is known from accidental and certain high-dose
industrial incidents that exposure to structurally related chemicals in the
bis-benzenamine class have the potential to produce toxic hepatitis.
Hazard and Dose-Response Characterization
Compound 3 is likely to pose a carcinogenic hazard to humans given
that it induces both thyroid and liver tumors in rodents and has gene mu-
tagenic activity in microbes and cultured mammalian cells. Mutagenic
properties may account for the tumor responses; in addition, the thyroid
tumors may be due to perturbation in thyroid-pituitary functioning. There-
fore, cancer dose-response relationships should be projected using a
low-dose linear procedure; a lower bound on the thyroid cancer risks may
be characterized using nonlinear considerations.
Cancer studies on the bis-benzenamine, compound 3, show signifi-
cant increases in thyroid follicular cell carcinomas and adenomas in male
and female rats and adenomas alone in female mice; male and female rats
and female mice also show significant increases in hepatocellular carcino-
mas and adenomas. Male mice do not show increases in either of these
tumor types. Structurally related compounds often produce thyroid tumors
in rats and mice and liver tumors in mice. Just as compound 3 shows DNA
reactivity, gene mutations, and possible structural chromosome mutations,
structural analogues also produce mutagenic effects relevant to carcino-
genicity.
Several lines of investigation indicate that compound 3 affects thyroid-
pituitary functioning. (1) Significant dose-related goitrogenic effects (e.g.,
increase in thyroid weight, diffuse hyperplasia) are noted after subchronic
and chronic dosing in rats and mice. There is no mention whether follicular
cell hypertrophy was noted in any of the studies. (2) Following 20 weeks of
treatment, hormone levels are altered in rats, including decreases in both
T4 and T3 and increases in TSH. (3) No studies have been done on com-
A-35
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pound 3 to discern its site of action on thyroid-pituitary functioning, although
several structural analogues inhibit the accumulation of iodide in the rat
thyroid, probably the result of thyroid peroxidase inhibition. (4) Some indi-
cation of progression of histologic lesions is noted: thyroid hyperplasia and/
or adenomatous goiter develop after subchronic dosing in rats and mice,
while neoplasms form after chronic dosing. In mice, seemingly the more
resistant species (on a mg/kg/day basis), the only thyroid neoplasms are
adenomas, while in rats, both adenomas and carcinomas are found in
roughly comparable frequencies. (5) Dose correlations are presented in
Table A-19 and are discussed later. In sum, these observations constitute
an adequate database to evaluate antithyroid effects, but there is a lack of
specific information on the site of action.
The above influences of compound 3 on thyroid-pituitary status could
account for thyroid tumor formation following chronic exposure. However,
there is also mutagenic activity relevant to carcinogenicity for both com-
pound 3 and structural analogues that include DNA reactivity, gene
mutations, and structural chromosome aberrations. The compound is seem-
ingly metabolized to produce reactive products that are mutagenic. Only
minimal metabolic information is available in rats, and none exists in mice
and humans. These mutagenic effects could account for both the thyroid
and liver tumors.
Characterization of cancer dose-response relationships should prima-
rily rely on mutagenic considerations for the thyroid and liver tumors using
a low-dose linear procedure. However, the thyroid tumor responses may
be due to both its mutagenic and antithyroid properties. Other chemicals
with both mutagenic and antithyroid effects also have led to high thyroid
tumor incidences, as have combinations of mutagenic and antithyroid stimuli.
Because it is not possible to totally discern the relative impacts of these
influences for compound 3, threshold considerations should be used in
addition to a linear extrapolation so as to estimate the lower bound on the
thyroid cancer risk.
In making a decision about potential thyroid cancer risk due to antithy-
roid activity, toxicity dose correlations on compound 3 are important
considerations (Table A-19). Thyroid effects are noted in rats at doses of
400 ppm and higher in studies spanning the subchronic and chronic dos-
ing periods, but not at 200 ppm. In mice, effects are noted at 700 and 800
ppm, but not at 300 ppm and below. It is recognized that there are thyroid-
pituitary hormone levels only in rats after dosing with 400 ppm and no data
in mice; more extensive testing in mice would help to ensure identification
of doses where hormone alterations are not noted. In their absence, em-
phasis can be placed on the above identified no-observed-effect-levels
(NOEL), recognizing that there is some degree of uncertainty in relying on
A-36
-------
the values. Rats appear to be as sensitive or more sensitive to the antithy-
roid effects of compound 3 as mice on a mg/kg/day basis. With the existing
database, the estimated NOEL for rats is 200 ppm (20 mg/kg/day), while
that for mice is 300 ppm (60 mg/kg/day). The 20 mg/kg/day value in rats
can be used in calculating margins of exposure. Linear risks for thyroid
tumors might be estimated by drawing a straight line from the 10% esti-
mated tumor incidence level to the origin or the lower 95% bound on that
incidence. Risks for other tumors would depend upon the mode of action
information available on those sites.
Bibliography
The following articles were consulted in developing the data used in
the case study of this hypothetical chemical.
McQueen, CA; Williams, GM. (1990) Review of the genotoxicity and carci-
nogenicity of 4,4'-methylene-dianiline and 4,4'-methylene-bis-2-
chloroaniline. Mutat Res 239:133-142.
Weisberger, EK; Murthy, ASK; Lilja, HS; et al. (1984) Neoplastic response
of F344 rats and B6C3F1 mice to the polymer and dyestuff intermedi-
ates 4,4'-methylenebis(N,N- dimethyl)benzenamine, 4,4'-oxydianiline,
and 4,4'-methylenedianiline. J Natl Cancer Inst 72:1457-1463.
A-37
-------
Compound 4: A Nitrosamine that is Mutagenic and has
No Antithyroid Effects
Executive Summary
Compound 4, a nitrosamine, produces thyroid follicular cell tumors in
rats after a very short latency period. It also produces lung, liver, and kid-
ney tumors in rats after a short latency period and pancreatic, liver, and
lung tumors in Syrian golden hamsters. Compound 4 is mutagenic in vari-
ous short-term tests for DNA alkylation, DNA damage, and gene and
structural chromosome mutations. Because compound 4 is mutagenic,
causes tumors with short latencies at multiple anatomical sites including
the thyroid, and does not appear to interrupt the thyroid endocrine axis, the
thyroid follicular cell tumors appear to be caused by a mutagenic mecha-
nism, indicating that linear risk assessment procedures for low-dose
extrapolation apply in this case.
Detailed Data
1. Cancer Findings
Although compound 4 has not been tested in a conventional 2-year
rodent bioassay, tumor incidence data for the chemical are available from
various shorter term biomedical research studies. The results from one of
these studies using male Wistar rats are summarized in Table A-20. The
doses administered to groups of 20 rats were 0, 50, 100, and 200 ppm
(corresponding to 0, 5, 10, and 20 mg/kg/day or 0, 910, 1,820, and 3,640
mg/kg total dose) in drinking water that was provided ad libitum. The study
was terminated after 26 weeks of exposure. Although only one death (in
the high-dose group) had occurred at that time, adenomas and carcino-
mas of the lung, liver, kidneys, and thyroid were found. Lung tumors, induced
at all dose levels tested, were found in 100% of the mid- and high-dose
animals. Tumors in the liver, kidney, and thyroid occurred with lower inci-
dences at the mid- and high-dose levels, but showed dose responsiveness.
Separate studies have shown that compound 4 is effective in produc-
ing tumors in a similar range of organ sites by other routes of administration,
for example, by weekly subcutaneous injection in both male and female
Sprague-Dawley rats or by i.p. injection to Wistar rats. Another study, using
oral administration, demonstrated compound 4's ability to produce pancre-
A-38
-------
Table A-20. Wistar Rats with Tumors by Site in a 26-Week Study of Compound
4
Tumor incidence (percent) according to dose
Organ site 0 50 100 200
Lung
Liver
Kidney
Thyroid
0/20
0/20
0/20
0/20
5/20
(25)
0/20
0/20
0/20
20/20
(100)
5/20
(25)
3/20
(15)
0/20
(10)
19/19
(100)
8/19
(42)
4/19
(21)
5/19
(26)
atic cancer, as well as lung and liver tumors, in the Syrian golden hamster.
In the experiment using the i.p. route of administration, compound 4 was
administered to male and female rats at three dose levels once weekly for
4 weeks. The experiment was terminated at 30 weeks, at which time thy-
roid tumors were diagnosed at the mid and high doses in both male and
female rats (Table A-21). There was a particularly high incidence (80%) in
the high-dose males. Since the cumulative doses in the i.p. study were well
below those in the drinking water study, the i.p. route appears to be a more
effective mode for inducing thyroid tumors in the rat with compound 4.
These data in both sexes of multiple species, multiple organs, and by
different routes of administration indicate that compound 4 is a potent car-
cinogen in laboratory animals. The chemical's potency is further emphasized
by the very short latency period of induction, the low total dose of the chemi-
cal, and the high tumor incidences.
2. Mechanistic Considerations
a. Mutagenicity
A review of the literature indicates that compound 4 is consistently posi-
tive in short-term tests for mutagenicity in the presence of a metabolic
activation system. It has tested positively in various Salmonella assays for
frameshift and base-pair mutations with strains TA-1537 and TA-1535, re-
spectively, and produces sister chromatid exchange and chromosomal
aberrations in CHO cells and transformation of BHK cells. DNA reactivity
has been shown by positive results for, DNA single-strand breakage in cul-
tured mouse epithelial cells, for alkylation both in vitro and in vivo, and for
inducing DNA-repair replication in human lymphocytes.
A-39
-------
b. Thyroid Growth
The effects of compound 4 on the thyroid, both grossly and histologi-
cally, were different from the effects of chemicals acting on the
thyroid-pituitary axis. Thyroid weights were recorded at 30 weeks in the i.p.
injection cancer study of compound 4. These are summarized in Table A-
22.
Although thyroid follicular cell tumors were present in the mid- and
high-dose males and in the high-dose females, thyroid weights overall
showed no statistically significant difference among groups and therefore
no correlation with the incidences of thyroid tumors. This lack of statistical
significance remained-when the thyroid weights were calculated relative to
body weights. At the histologic level, the thyroids from treated groups showed
a dose-response relationship for the incidence of follicular cell hyperplasia
(Table A-23). The hyperplasia, however, was not of the diffuse form typical
of antithyroid compounds, but manifested as small solitary foci, presum-
ably representing the first stage in the continuum of hyperplasia to adenoma
to carcinoma. Thus, focal hyperplasias were described as small aggre-
Table A-21. Thyroid Follicular Cell Tumor Incidence at 30 Weeks in Male and
Female Wistar Rats Treated with Compound 4 by i.p. Injection
Total cumulative dose
_ (mg/kg)
0 200 400 600
0 1/25 6/25 20/25
(4) (24) (80)
Female 0 0 1/24 5/24
(4) (21)
Table A-22. Mean Thyroid Weights (mg) at 30 Weeks in Male and Female
Wistar Rats Treated with Compound 4 by i.p. Injection
Total cumulative dose
(mg/kg)
0 200 400 600
Male 15.6+2.2 15.2+2.2 14.8+1.4 299+355*
Female 11.1+2.6 10.6+1.3 10.8+1.3 11.9+3.5
*The large variation in thyroid weights in the high-dose males was due to the
presence of several very large tumors. The high standard deviation for this
group resulted in a lack of significance from controls.
A-40
-------
Table A-23. Incidence of Focal Hyperplasia Involving Thyroid Follicular Cells at
30 Weeks in Male and Female Wistar Rats Treated i.p. with
Compound 4
Total cumulative dose
(mg/kg)
Male
Female
0
0
0
200
4/25
(16)
0
400
9/25
(36)
1/24
(4)
600
18/25*
(72)
3/24
(12)
*p<.05 compared with control values.
gates of basophilic epithelial cells involving part of a follicle or a few fol-
licles, but without fibrous encapsulation. Uninvolved tissue lacked cellular
hypertrophy, and the follicles contained amounts of colloid within the nor-
mal range.
c. Hormone Levels
In the i.p. cancer study, serum T4 and TSH levels (but not T3) were also
measured (summarized in Table A-24). There were no differences in se-
rum T4 or TSH levels in treated groups when compared with levels in control
animals. In addition, comparison of serum TSH concentrations in high-dose
rats with and without thyroid tumors demonstrated no significant differences
(Table A-25).
A separate study provided limited information on serum T3 levels at 20
weeks posttreatment in male Wistar rats receiving a single i.p. injection of
compound 4 at a dose of 1,000 mg/kg. Compared to the mean value of
80.8+3.8 ng/ml for 20 control animals, the mean value for serum T3 in the
treated group (n=20) was not significantly different at 90.7+11.5 ng/ml.
These various data, showing serum T3, T4, and TSH levels within the
control ranges after treatment with compound 4, provide further evidence
that the chemical has no effect on the function of the thyroid/pituitary axis.
d. Site of Action
No studies were available that have investigated the effects of com-
pound 4 on thyroid peroxidase, the deiodination pathway, or effects on
thyroid metabolism and excretion in the liver.
However, the chemical is without goitrogenic or thyroid-pituitary hor-
mone effects, indicating that such studies are not needed. Obviously, there
are no significant dose correlations to consider.
A-41
-------
Table A-24. Serum T4 and TSH Levels at 30 Weeks in Male and Female Wistar
Rats Treated i.p. with Compound 4
Total cumulative dose
(mg/kg)
0 200 400 600
T4
(ug/dl)
TSH
(ng/ml
M
3.4
+0.5
4.5
+ 1.1
F
2.9
+0.7
3.2
+0.5
M
3.8
+0.4
5.8
+2.5
F
3.3
+0.4
3.9
+1.4
M
3.0
+0.5
4.0
+1.0
F
2.3
+0.7
4.4
+ 1.5
M
3.3
+0.4
4.4
+1.1
F
3.0
+0.6
4.0
+1.2
Table A-25. Comparison of Serum TSH Levels in Rats with and without Thyroid
Tumors Induced by i.p. Treatment with a Cumulative Dose of 600
mg/kg of Compound 4
Without tumors
With tumors
No.
M
9
11
of rats
F
19
3
TSH concentration (ng/ml)
M
4.9+1.4
4.2+1.0
F
4.0+1.1
4.0+0.5
e. Ancillary Data
The initiating effect of compound 4 on two-stage thyroid carcinogen-
esis was investigated using the antithyroid agent propylthiouracil (PTU) as
a promoter. Male Wistar rats, 6 weeks old, were administered a single 1,000
mg/kg i.p. dose of compound 4 and fed PTU in the basal diet for 19 weeks
(beginning of week 2 to week 20) at a level of 0.15%. The experiment was
terminated at 20 weeks, and the thyroids were examined histologically for
neoplastic lesions. The results are tabulated in Table A-26. The incidence
of thyroid follicular tumors in the group receiving compound 4 followed by
administration of PTU was 95% compared to 5% in the compound 4 only
group and 0% in the PTU only group. The results provide evidence that
compound 4 acts as an initiating agent for the development of thyroid tu-
mors in rats that can be promoted with PTU.
f. Structure-Activity Relationships
Compound 4, a nitrosamine, belongs to a notorious class of chemicals
recognized to be potent mutagenic carcinogens, producing tumors in many
anatomical sites in both sexes of multiple species. Many other members of
this class are also used in research studies involving mechanisms of car-
cinogenesis. Enzymic hydroxylation of these chemicals is required for
A-42
-------
Table A-26. Male Rats with Thyroid Tumors at 20 Weeks after a Single Treat-
ment with Compound 4 Followed by PTU
Treatments*
None Compound 4 PTU Compound 4 + PTU
No. (%) of rats with 0/20 1/20 0/20 19/20
thyroid tumors (0) (5) (0) (95)
"Compound 4: single i.p., 1,000 mg/kg; PTU: 0.15% in diet for 19 weeks.
generation of the proximate carcinogens/mutagens, which are strong alky-
lating agents.
g. Metabolism and Pharmacokinetic Properties
It is known from mechanistic studies that compound 4 is rapidly and
evenly distributed throughout the body water. Although approximately 60%
of the administered dose is excreted unchanged in the urine, the chemical
is also oxidized in the liver to a ketone that has been shown to be a methy-
lating agent. The enzyme system responsible for oxidation has not been
determined.
3. Human Data
No direct information exists on the acute or chronic effects of this chemi-
cal in humans.
Hazard and Dose-Response Characterization
Compound 4 produces tumors at several sites, including the thyroid.
The thyroid tumors do not result from disruption of thyroid-pituitary status.
The chemical does not produce follicular cell hypertrophy or diffuse hyper-
plasiaand does not alter the levels of circulating thyroid hormones orTSH.
Although there are no specific studies on the chemical's effect on thyroid
hormone synthesis or disposition, there is no indication that it has such
effects. Instead, it appears to act, as do many other members of the nitro-
samine group, as a clear-cut mutagenic carcinogen. It possesses
demonstrable gene and chromosome-breaking mutagenic activity and an
ability to alkylate DNA both in vitro and in vivo. It also causes DMA damage,
has initiating capacity in a thyroid two-stage carcinogenesis test, acts as a
complete carcinogen producing tumors at multiple sites in addition to the
thyroid, is effective as a carcinogen by more than one route of administra-
tion and in more than one species, and exhibits a very short tumor latency.
For these reasons, compound 4 has the potential to be a human carcino-
gen, functioning as a mutagenic carcinogen rather than by disruption of
thyroid-pituitary status. Accordingly, thyroid dose-response assessments
for this substance should be conducted using a low-dose linear procedure.
A-43
-------
Risk estimation at other sites would depend on the mode of action informa-
tion.
Bibliography
The following articles were consulted in developing the data used in
this case study of this hypothetical chemical.
Hiasa, Y; Kitahori, Y; Kato, Y; et al. (1987) Potassium perchlorate, potas-
sium 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.
Hiasa, Y; Kitahori, Y; Kitamura, M; et al. (1991) Relationships between se-
rum thyroid stimulating hormone levels and development of thyroid tu-
mors in rats treated with N-bis(2-hydroxypropyl)nitrosamine. Carcino-
genesis 12:873-877.
Kitahori, Y; Hiasa, Y; Konishi, N; et al. (1984) Effect of propylthiouracil on
the thyroid tumorigenesis induced by N-bis(2-hydroxypropyl)nitrosamine
in rats. Carcinogenesis 5:657-660.
A-44
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Appendix B
Synopsis of Agents Affecting the Thyroid
-------
Appendix B
Synopsis Of Agents Affecting The Thyroid
Extensive investigation of physical and chemical factors have identi-
fied many treatments that influence the thyroid gland. Some directly affect
thyroid gland functioning (Table B-1), such as those that damage the ge-
netic material and those that in some way affect the synthesis or release of
thyroid hormone from the gland. Other agents influence the thyroid indi-
rectly (Table B-2), as by reducing the formation of circulating T3 from T4
due to inhibition of 5'- monodeiodinase. Chemicals from many different
structural and functional classes also indirectly affect the thyroid by induc-
ing liver microsomal enzymes and increasing the metabolism and excretion
of thyroid hormones.
Still other agents can affect the thyroid in more than one way; several
examples are applicable. Thiocyanate ion inhibits both iodide uptake into
the thyroid and thyroid peroxidase activity, while propylthiouracil inhibits
both thyroid peroxidase and 5'-monodeiodinase activities. These agents
doubly reduce effective thyroid hormone levels. Other compounds may have
both thyroid cancer initiation and promotion activity. For instance, both 3-
methylcholanthrene and 4,4'-methylenedianiline are mutagenic and may
affect thyroid cell DMA, while the former is also a liver microsomal enzyme
inducer that increases thyroid hormone metabolism and excretion and the
latter also reduces thyroid hormone synthesis by probably inhibiting thy-
roid peroxidase.
Not all agents that have some direct or indirect effect on the thyroid
have demonstrated carcinogenic effects. In humans, for instance, only x-
irradiation has been demonstrated to produce thyroid cancer. In rodents,
many compounds that have produced some effect on the thyroid have never
been tested in chronic studies. Even those that have been tested do not
always produce thyroid tumors. For instance, phenytoin produces many
different effects on the thyroid, but it was negative for thyroid tumors in
rodent studies conducted by the National Toxicology Program (NTP). The
same is true for certain polyhydroxy aromatic compounds that are inhibi-
tors of thyroid peroxidase, in that neither resorcinol nor 4-hexylresorcinol
produced thyroid tumors in NTP rodent studies. This is interesting, given
that resorcinol has been shown to be goitrogenic in short-term animal stud-
ies and in humans.
B-2
-------
Table B-1. Illustrative Treatment Regimens Directly Affecting the Thyroid Gland
Iodide deficiency Thyroid peroxidase inhibition
thionamides
Iodide pump inhibition goitrin
perchlorate ion propylthiouracil
pertechnetate ion thiocyanate ion
thiocyanate ion thiouracil
aniline derivatives
Thyroid hormone release inhibition 4,4'-methylenedianiline
excess iodide p-aminobenzoic acid
lithium sulfamethazine
sulfathiazole
Thyroid gland damage polyhydroxy aromatics
polybrominated biphenyls 4-hexylresorcinol
polychlorinated biphenyls phloroglucinol
resorcinol
miscellaneous
2-aminothiazole
amitrole
Mutagenic
x-irradiation
,31,
glycidol
3-methylcholanthrene
4,4'-methylenedianiline
N-bis (2-hydroxypropyl) nitrosamine
tribromomethane
Liver microsomal enzyme inducers are very heterogeneous: they span
many different structural and functional classes of chemicals, they differ in
regard to the specific microsomal enzymes that are induced, and they vary
greatly in their potency for enzyme induction.
Accordingly, only some enzyme inducers have been shown to influ-
ence thyroid-pituitary functioning following short-term administration. Among
those with antithyroid effects, thyroid-pituitary status may return to homeo-
stasis following continued dosing. Thus, in long-term dosing studies in
rodents, only a proportion of these agents go on to induce thyroid neopla-
sia. Generally, thyroid carcinogenic responses are noted in a relatively low
percentage of animals with the enzyme inducers, in contrast to those agents
that inhibit thyroid peroxidase that often produce much more pronounced
carcinogenic effects.
As of February 1996, the experience from the NTP indicates that 33 of
approximately 460 chemicals (about 7%) (Table B-3) tested in chronic stud-
ies show thyroid follicular tumors in rats and/or mice. A number of others
show some preneoplastic lesion (usually hyperplasia but also hypertro-
B-3
-------
Table B-2. Illustrative Agents Indirectly Influencing the Thyroid Gland
Liver microsomal enzyme induction 5'-Monodeiodinase inhibition
channel blocker amiodarone
nicardipine FD&C Red No. 3
CNS active iopanoic acid
phenobarbital propranolol
phenytoin propylthiouracil
histamine (H2) antagonist
SK&F 93479
CoA reductase inhibitor
simvastatin
imidazole antibiotic
SC-37211
leukotriene antagonist
L-649,923
pesticide
clofentezine
thiazopyr
polyaromatic hydrocarbon
3-methylcholanthrene
polyhalogenated hydrocarbon
C12 chlorinated paraffins
dieldrin
polychlorinated biphenyls
2,3,7,8-tetrachlorodibenzo-p-dioxin
toxaphene
retinoid
etretinate
steroid
spironolactone
Table B-3. Chemicals Producing Thyroid Follicular Cell Tumors in NTP Studies
aldrin isobutyl nitrite
3-amino-4-ethoxyacetanilide malonaldehyde, sodium salt
o-anisidine hydrochloride manganese sulfate monohydrate
azinphosmethyl mercuric chloride
2,2-bis (bromomethyl)-1,3-propanediol 4,4'-methylenebis (N,N-dimethyl)
tertiary butyl alcohol benzenamine
chlorinated paraffins: C12, 60% chlorine 4,4'-methylenedianiline dihydrochloride
C.I. Basic Red 9 monohydrochloride 1,5-naphthalenediamine
C.I. Pigment Red 3 oxazepam
decabromodiphenyl oxide 4,4'-oxydianiline
2,4-diaminoanisole sulfate 2,3,7,8-tetrchlorodibenzo-p-dioxin
N,N'-diethylthiourea p,p-tetrachlorodiphenylethane (ODD)
ethylene thiourea (ETU) 1-trans-delta-9-tetrahydrocannabinol
glycidol 4,4'-thiodianiline
HC Blue No. 1 toxaphene
heptachlor trimethylthiourea
iodinated glycerol tris (2-chloroethyl) phosphate
B-4
-------
phy) without corresponding neoplasia. Most of the tested chemicals have
not been studied as to their antithyroid potential. However, it appears that a
number of these chemicals inhibit thyroid peroxidase or are microsomal
enzyme inducers; still others have mutagenic activity that might account
for the thyroid neoplasms. For instance, short-term toxicity studies have
been completed on the thionamide 2-mercaptobenzimidazole that show
both significant thyroid hyperplasia (Gaworski et al., 1991) and antithyroid
activity; thyroid tumors would be expected in chronic studies. Interestingly,
2-mercaptobenzothiazole, a closely related compound, failed to show anti-
thyroid effects (Bywater et al., 1945) and likewise failed to induce thyroid
cancer in NTP chronic testing.
References
Bywater, WG; McGinty, DA; Jenesel, ND. (1945) Antithyroid studies. II.
The goitrogenic activity of some imidazoles and benzimidazoles. J
Pharmacol Exp Therap 85:14-22.
Gaworski, CL; Aranyi, C; Vana, S; et al. (1991) Prechronic inhalation toxic-
ity studies of 2-mercaptobenzimidazole (2-MB) in F344/N rats. Fundam
ApplToxicol 16:161-171.
B-5
-------
-------
Appendix C
Thyroid Follicular Cell Carcinogenesis
-------
-------
FUNDAMENTAL AND APPLIED TOXICOLOGY 12, 629-697 (1989)
REVIEW
Thyroid Follicular Cell Carcinogenesis1
RICHARD N, HILL,* LINDA S. ERDREiCH.f'2 ORVILLE E. PAYNTER,*
PATRICIA A. ROBERTS,^ SHEILA L. RosENTHAL,f'3 AND CRISTOPHER F. WILKINSON*-*
^Office of Pesticides and Toxic Substances, fOffice of Research and Development, ^Office of General Counsel, Risk
Assessment Forum, U.S. Environmental Protection Agency, Washington, D.C. 20460
Received November 10, 1988 .-accepted December 2, 1988
Thyroid Follicular Cell Carcinogenesis. HILL, R. N., ERDREICH, L. S., PAYNTER, O. E., ROB-
ERTS, P. A., ROSENTHAL, S. L., AND WILKINSON, C. F. (1989). Fundam. Appl. Toxicol. 12,629-
697. Ample information in experimental animals indicates a relationship between inhibition of
thyroid-pituitary homeostasis and the developmental thyroid follicular cell neoplasms. This is
generally the case when there are long-term reductions in circulating thyroid hormones which
have triggered increases in circulating thyroid stimulating hormone. Such hormonal derange-
ments leading to neoplasms have been produced by different regimens, including dietary iodide
deficiency, subtotal thyroidectomy, and administration of natural and xenobiotic chemical sub-
stances. The carcinogenic process proceeds through a number of stages, including follicular cell
hypertrophy, hyperplasia, and benign and sometimes malignant neoplasms. Given the interrela-
tionship between the thyroid and pituitary glands, conditions that result in stimulation of the
thyroid can also result in stimulation of the pituitary, with the development of hyperplastic and
neoplastic changes. The progression of events leading to thyroid (and pituitary) neoplasms can
be reversed under certain circumstances be reestablishing thyroid-pituitary homeostasis. Most
chemicals that have induced follicular cell tumors seem to operate through inhibition of the
synthesis of thyroid hormone or an increase in their degradation and removal. For some of these
• compounds, it appears that genotoxic reactions may not be playing a dominant role in the
carcinogenic process. A seemingly small group of thyroid carcinogens seems to lack influence
on thyroid-pituitary status and may in part be operating via their genotoxic potential. In con-
trast with the weU-established relationship between thyroid-pituitary derangement and follicu-
lar cell neoplasms in animals, the state of information in humans is much less certain. At this
time, ionizing radiation is the only acknowledged human thyroid carcinogen, a finding well
established in experimental systems as well. Although humans respond to goitrogenic stimuli
as do animals, with the development of cellular hypertrophy, hyperplasia, and under certain
circumstances nodular lesions, disagreement exists as to whether malignant transformation oc-
curs in any predictable manner. It would seem that if humans develop thyroid tumors following
long-term derangement in thyroid-pituitary status, they may be less sensitive than the com-
monly used animal models.
' This document has been reviewed in accordance constitute endorsement or recommendation for
with the U.S. Environmental Protection Agency pro- use.
cedures and has been approved for publication. Ap- 2 Present address: Clement Associates, Inc., Edison, NJ.
proval does not signify that the contents necessarily 3 Present address: U.S. Environmental Protection
reflect the views and policies of the Agency nor does Agency, Region IX, San Francisco, CA.
the mention of trade names or commercial products > Present address: Versar, Inc., Springfield, VA.
629 0272-0590/89 $3.00
-------
630
HILL ET AL.
FOLLtCLE CELL
FOUJCULAR LUMEN
FIG. 1. Schematic representation of thyroid hormone biosynthesis and secretion. The protein portion of
thyroglobulin is synthesized on rough endoplasmic reticulum, and carbohydrate moieties are added by the
Golgi apparatus. Thyroglobulin proceeds to the apical surface in secretory vesicles which fuse with the cell
membrane and discharge their contents into the lumen. Iodide enters the cell by active transport, is oxi-
dized by a peroxidase at the apical border, and is incorporated into tyrosine residues in peptide linkage in
thyroglobulin. Two iodinated tyrosyl groups couple in ether linkage to form thyroxine, which is still
trapped in thyroglobulin. For the secretory process, thyroglobulin is engulfed by pseudopods at the apical
border of the follicular lumen and resolved into vesicles that fuse with lysosomes. Lysosomal protease
breaks down thyroglobulin to amino acids, T4, T3, diiodotyrosine (DIT), and monoiodotyrosine (MIT).
T4 and T3 are secreted by the cell into the blood. DIT and MIT are deiodinated to free tyrosine and iodide,
both of which are recycled back into iodinated thyroglobulin. Source: Goodman and van Middlesworth
(1980).
I. THYROID-PITUITARY
PHYSIOLOGY AND BIOCHEMISTRY
In order to examine the possible role of pitu-
itary, thyroid, and related hormones in thy-
roid carcinogenesis, it is important to first un-
derstand the physiology and biochemistry of
the thyroid-pituitary hormonal system. Ac-
cordingly, this section summarizes the na-
ture, formation, and secretion of the thyroid
hormones and discusses the mechanisms by
which circulating levels of the hormones are
regulated. References are mainly to recent re-
views (see Paynter et al, 1986; 1988) rather
than to the original scientific literature.
A. Synthesis of Thyroid Hormones
The thyroid hormones are synthesized in
the thyroid gland and are stored as amino
acid residues of thyroglobulin, a protein con-
stituting most of the colloid in the thyroid fol-
licles (Goodman and van Middlesworth,
1980; Taurog, 1979; Haynes and Murad,
1985). Thyroglobulin is a complex glycopro-
tein made up of two identical subunits each
with a molecular weight of 330,000 D.
The first stage in the synthesis of the thy-
roid hormones is the uptake of iodide from
the blood by the thyroid gland (Fig. 1). Up-
take is active in nature (requires energy) and
is effected by the so-called "iodide pump."
Under normal conditions the thyroid may
concentrate iodide up to about 50-fold its
concentration in blood, and this ratio may be
considerably higher when the thyroid is ac-
tive. Iodide uptake may be blocked by several
anions (e.g., thiocyanate and perchlorate)
and, since iodide uptake involves concurrent
uptake of potassium, it can be also blocked
by cardiac glycosides that inhibit potassium
accumulation.
The next step in the process is a concerted
reaction in which iodide is oxidized to an ac-
tive iodine species that in turn iodinates the
-------
THYROID CARCINOGENESIS REVIEW
631
HO-/0\CH2-CH-COOH
3 NH2
tyros i ne
Monofodotyroslne {HID) = 3-1odotyrosine
Oiiodotyrosfne (010) « 3,5-dilodotyroslne
5'
HO-( 0 )-0-( 0 VCHj-CH-COOH
3' 3 HH2
thyronine
Thyroxlne (14) " 3,5,3',5'-tetraiodothyronine
Triiodothyronine (Tj) - 3,5,3'-tri1odothyron1ne
Reverse tr1iodothyrontne {rTj) * 3,3',5'-trifodothyron(ne
FIG. 2. lodinated compounds of the thyroid gland.
tyrosyl residues of thyroglobulin. The reac-
tion is effected by a heme-containing peroxi-
dase in the presence of hydrogen peroxide.
While diiodotyrosyl (DIT) residues constitute
the major products, some monoiodotyrosyl
(MIT) peptides are also produced (Fig. 2).
Additional reactions involving the coupling
of two DIT residues or of one DIT with one
MIT residue (each with the net loss of ala-
nine) lead to peptides containing residues of
the two major thyroid hormones, thyroxine
(T4) and triiodothyronine (T3), respectively
(Fig. 1). It is thought that these reactions are
catalyzed by the same peroxidase effecting
the iodination reaction, and it seems that
both peroxidase steps are blocked by certain
compounds such as thiourea and some sul-
fonamides.
The release of T4 and T3 from thyroglobu-
lin or smaller peptides is effected by endocy-
tosis of colloid droplets into the follicular epi-
thelial cells and subsequent action of lyso-
somal proteases. The free hormones are
subsequently released into the circulation. It
is not known whether thyroglobulin must be
hydrolyzed completely to permit release of T4
and T3.
Although T4 is by far the major thyroid
hormone secreted by the thyroid (normally
about 8 to 10 times the rate of T3, although it
varies as a function of the iodine intake), it is
usually considered to be a prohormone.
Thus, T3 is about fourfold more potent than
T4, and about 33% of the T4 secreted under-
goes 5'-deiodination to T3 in the peripheral
tissues; another 40% undergoes deiodination
of the inner ring to yield the inactive material,
reverse triiodothyronine (rT3) (Fig. 2).
B, Transport of Thyroid Hormones in Blood
On entering the circulation, both T4 and T3
are transported in strong, but not covalent,
association with plasma proteins (Fig. 3). The
major carrier protein in humans is thyroxine-
binding globulin, a glycoprotein (MW
63,000) that forms a 1:1 complex with the
thyroid hormones. Thyroxine-binding globu-
lin has a very high affinity for T4 (Ka about
10'° M) and a lower affinity for T3. (This spe-
cific carrier protein is absent in rodents, cats,
HYPOTHALAMUS
FIG. 3. Hypothalmic-pituitary-thyroid-peripheral or-
gan relationships. TRH, thyrotropin-releasing hormone;
TSH, thyroid-stimulating hormone; TH, thyroid hor-
mones; TBG, thyroxine-binding globulin; TBP, thyrox-
ine-binding prealbumin.
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632
HILL ET AL.
and rabbits (Dohler el ai, 1979),) Thyroxine-
binding prealbumin and albumin also trans-
port thyroid hormones in the blood; the pre-
albumin has Ka values of about 107 and 106
M forT4 and T3, respectively. Normally, only
about 0.03% of the T4 in the circulation is free
and available for cell membrane penetration
and thus hormone action, metabolism, or ex-
cretion. The levels of free thyroid hormones
in the circulation may be changed through
competitive binding interactions of certain
drugs and other foreign compounds (Haynes
andMurad, 1985).
C. Metabolism and Excretion
As previously discussed, T4, the major hor-
mone secreted from the thyroid, is considered
to be a prohormone and is converted to the
more active T3 by a 5'-monodeiodinase in a
variety of peripheral tissues, including the pi-
tuitary. T4 is also metabolized to rT3 which is
hormonally inactive and has no known func-
tion, except perhaps as an inhibitor of the
conversion of T4 to T3. The 5'-monodeiodi-
nase also reacts with rT3 and coverts it to a
diiodo-derivative (Larson, 1982b). Under
normal conditions the half-life of T4 is 6 to 7
days in humans (see Thomas and Bell, 1982).
Degradative metabolism of the thyroid
hormones occurs primarily in the liver and
involves conjugation with either glucuronic
acid (mainly T4) or sulfate (mainly T3)
through the phenolic hydroxyl group. The re-
sulting conjugates are excreted in the bile into
the intestine. A portion of the conjugated ma-
terial is hydrolyzed in the intestine, and the
free hormones thus released are reabsorbed
into the blood (enterohepatic circulation).
The remaining portion of the conjugated ma-
terial (20*to 40% in humans) is excreted in the
feces.
As stated previously, most thyroid hor-
mone is carried in the blood of humans by
thyroxine-binding globulin and thyroxine-
binding prealbumin. In the absence of thy-
roxine-binding globulin, as in the rat and
mouse, more thyroid hormone is free of pro-
tein binding and is subject to metabolism and
removal from the body. As a consequence,
the half-life of T4 in the rat is only about 12-
24 hr in contrast to 6-7 days in humans (see
Thomas and Bell, 1982). To compensate for
the increased turnover of thyroid hormone,
the rat pituitary secretes more TSH. Baseline
serum TSH levels in humans are on the order
of 2.5 ^LJ/ml, while in rats it ranges from 55.5
to 65 /iU/ml in males and 36.5 to 41 /iU/ml
in females, (about a 2-fold sex difference). It
has been suggested that rats require a 10-fold
higher T4 production rate per kilogram body
weight than do humans to maintain physio-
logical levels (about 15- to 20-fold higher se-
rum levels) (Dohler et al., 1979; Peer Review
Panel, 1987).
D. Physiologic Actions
of Thyroid Hormones
While not of direct relevance to this discus-
sion, the thyroid hormones play numerous
and profound roles in regulating metabolism,
growth, and development and in the mainte-
nance of homeostasis. It is generally believed
that these actions result from effects of the
thyroid hormones on protein synthesis.
There is considerable evidence to suggest
that many of the various biological effects of
the thyroid hormones are initiated by the in-
teraction of T3 with specific nuclear receptors
in target cells, presumably proteins (Oppen-
heimer, 1979). Recent evidence points to
these receptors being the products of the c-
erb-A oncogene (Weinberger et ai, 1986; Sap
et al., 1986). Such interactions can lead, di-
rectly or indirectly, to the formation of a di-
versity of mRNA sequences and ultimately to
the synthesis of a host of different enzyme
proteins. Qualitative and quantitative differ-
ences in the responses resulting from forma-
tion of T3-receptor complexes may occur in
different target tissues. Such differences may
be controlled at a local cellular level and may
be mediated through metabolic or hormonal
factors.
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THYROID CARCINOGENESIS REVIEW
633
E. Regulation of Thyroid Hormone
Synthesis/Secretion
Homeostatic control of thyroid hormone
synthesis and secretion in the thyroid gland is
effected by a sensitive feedback mechanism
that responds to changes in circulating levels
of the thyroid hormones T4 and T3. The
mechanism involves the hypothalamus and
anterior pituitary of the brain (Fig. 3) (Pay-
nter, et al, 1986; Larsen, 1982a; Houk,
1980).
Of central importance in the feedback
mechanism is the thyroid-stimulating hor-
mone (TSH, thyrotropin), which is secreted
by the anterior pituitary gland and causes the
thyroid to initiate new thyroid hormone syn-
thesis. Increases in iodide uptake, the iodin-
ation of thyroglobulin, and endocytosis and
proteolysis of colloid are all observed in re-
sponse to TSH stimulation. The effects of
TSH on the thyroid appear to be the conse-
quence of binding to cell-surface receptors
and activation of adenyl cyclase and protein
kinase with subsequent phosphorylation of
cellular proteins. Cyclic adenosine mono-
phosphate (cAMP) can itself mimic most of
the actions of TSH on thyroid cells (Van-
Sande et al., 1983; Roger and Dumont,
1984). Further details of the molecular biol-
ogy of TSH action on the thyroid are dis-
cussed elsewhere in this document (Sec-
tion II.C).
The rate of release of TSH from the pitu-
itary is delicately controlled by the amount of
thyrotropin-releasing hormone (TRH) se-
creted by the hypothalamus and by the circu-
lating levels of T4 and T3. If for any reason
there is a decrease in circulating levels of thy-
roid hormones, TSH is secreted and thyroid
function is increased; if exogenous thyroid
hormone is administered, TRH secretion is
suppressed and eventually the thyroid gland
becomes inactive and regresses. It appears
that the plasma concentrations of both T4
and T3 (and possibly intracellular formation
of T3 from T4 in the pituitary) are important
factors in the release of TSH; they may also
modulate the interaction of TRH with its re-
ceptors in the pituitary (Goodman and van
Middlesworth, 1980;HinkleandGoh, 1982;
Larsen, 1982a; Ross et al., 1986). Lastly, in
the pituitary T4 undergoes 5'-mono-deiodina-
tion to T3. In the rat about 50% of T3 within
pituitary cells arises from this means. When
serum T4 is reduced but T3 is normal, pitu-
itary intracellular T3 is reduced and cells are
able to respond to the decreased serum T4
and increase TSH secretion (Larsen, 1982a).
Thyroid hormone responsive tissues con-
tain a variable number of nuclear receptors
for thyroid hormones (mainly T3), usually in
excess of several thousand per cell (Oppen-
heimer, 1979). Under euthyroid conditions
in the rat, usually about 30 to 50% of the sites
are occupied by T3, although in the pituitary
more like 80% of the sites are filled under
physiological conditions. The T3-receptor
complex is quite labile with a half-life for dis-
sociation of about 15 min; the released T3 re-
enters the exchangeable cellular pool where it
can complex with another receptor or exit the
cell. The half-life for T3 clearance from the
plasma in experimental animals is variable,
being about 6 hr in the rat (Oppenheimer,
1979).
Studies on the regulation of TSH output
from the pituitary have indicated a link be-
tween T3 nuclear receptor occupancy and the
mRNA levels for the TSH subunit chains.
Administration of exogenous T3 resulted in
decreases in TSH mRNA levels in the pitu-
itaries and in transplanted pituitary tumors
of thyroidectomized mice within 1 day of ad-
ministration (Chin et al., 1985). Subunit mes-
senger RNA elongation in nuclei isolated
from pituitary tumors of mice treated in vivo
with T3 decreased within 0.5 hr after hor-
mone administration, and mRNA levels
were reduced within 1 hr (Shupnik et al.,
1985). It appears that the decrease in mRNA
is due either to decreased transcription or to
decreased stability of the mRNA transcripts.
A straight-line relationship existed between
the proportion of nuclear T3 receptors occu-
pied and the proportional reduction in TSH
subunit transcripts in transplanted pituitary
tumors (Shupnik et al., 1986). A 50% reduc-
-------
634
HILL ET AL.
tion in mRNA transcripts occurred when
about 45% of the receptors were occupied;
this occurred at plasma T3 levels of about 1
ng/ml(1.5x 1(T9M).
Other studies have investigated the effects
of withdrawal of T3 on TSH mRNA levels in
thyroidectomized mice bearing transplanted
pituitary tumors (Ross el ai, 1986). Plasma
T3 levels dropped precipitously within 1 day
after withdrawal; plasma TSH concentra-
tions rose fourfold between 1 and 2 days; and
tumor TSH subunit mRNA levels increased
markedly between Days 1 and 2.
These experiments demonstrate the rapid
response of the pituitary gland to increases
and decreases in plasma T3 levels. It seems
that pituitary cells modulate the levels in
TSH subunit mRNAs as a function of the
proportional occupancy of the numerous
nuclear receptors for T3.
II. THYROID AND PITUITARY
GLAND NEOPLASIA
As described in the previous section, the pi-
tuitary exerts a delicate control over the mor-
phological and functional status of the thy-
roid, and thyroid hormones are in turn im-
portant regulators of pituitary function. It is
perhaps not surprising, therefore, that the pi-
tuitary may be affected profoundly by factors
causing thyroid gland dysfunction. Because
of this close dependency, it is appropriate to
discuss thyroid and pituitary neoplasia in the
same section.
A. Thyroid Neoplasia
While, statistically, clinical thyroid cancer
is not a serious human health problem in the
United States, occult thyroid cancer discov-
ered at autopsy (Sampson et al., 1974) is
much more common (average about 2% of
autopsies). The American Cancer Society es-
timates there will be 11,000 new cases of thy-
roid cancer in 1988, which represents about
1% of the total expected cancer cases. In the
same period it is expected there will be 1100
deaths from thyroid cancer, which is only
0.2% of the projected cancer deaths (Silver-
berg and Lubera, 1988). Overall, thyroid can-
cer 5-year relative survival rates are in excess
of 90%. Although the trends in the average
annual percentage change for thyroid cancer
incidence has been increasing over the last 13
years (0.3%/year), the trend is not statistically
significant (NCI, 1988). Other thyroid le-
sions, like "nodules" noted upon palpation of
the thyroid, occur in about 4 to 7% of adults
and are of concern to physicians because they
may be or develop into thyroid malignancies
(Payntere/a/., 1986; DeGroot, 1979; Samp-
sons al.. 1974;RojeskiandGharib, 1985).
1. Induction
Thyroid neoplasia may be induced by ex-
posure of experimental animals to a variety
of treatment regimens, exogenous chemicals,
or physical agents. Some of these are dis-
cussed in more detail later. It has been recog-
nized for some time that neoplasms induced
in experimental animals by a number of these
treatments result from thyroid gland dys-
function, in particular, hypothyroidism.
Among the thyroid cancer-causing factors
inducing a hypothyroid state are iodine defi-
ciency (Bielschowsky, 1953; Axelrod and
Leblond, 1955; Schaller and Stevenson,
1966) and subtotal thyroidectomy (Dent et
al., 1956). In addition, thyroid tumors can re-
sult from the transplantation of TSH-secret-
ing pituitary tumors (Dent et ai, 1956; Har-
an-Guerae/a/., 1960;Sinhaera/., 1965). The
one factor common to each of these condi-
tions is that they all lead to increased produc-
tion of TSH and prolonged stimulation of the
thyroid gland by "excess" TSH. In the first
two conditions, elevated TSH results from
chronic stimulation of the pituitary in re-
sponse to a deficiency in the circulating levels
of thyroid hormone. Also note that nothing
has been given to these animals: instead the
tumors developed in the absence of some-
thing that is normally present (i.e., iodine and
thyroid gland mass). It should rightfully be
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THYROID CARCINOGENESIS REVIEW
635
pointed out, however, that the animals are
under chronic stress due to deficiency of thy-
roid hormone. In the third case, excess TSH
comes from the transplanted pituitary tumor.
Thus, irrespective of the cause, it appears that
prolonged stimulation of the thyroid-pitu-
itary feedback mechanism that results in re-
lease of elevated levels of TSH by the pitu-
itary may lead to thyroid gland neoplasia.
Support for the role of TSH in thyroid car-
cinogenesis also comes from irradiation stud-
ies. X-irradiation is the only demonstrated
human thyroid carcinogen. High doses of ir-
radiation commonly associated with thyroid
tumor development are associated with thy-
roid parer.chymal cell killing and compensat-
ing increase in TSH. The types of tumors pro-
duced by irradiation are the same as those
noted following purposeful manipulation of
TSH levels (e.g., by iodine deficiency). In ad-
dition, treatments which raise TSH levels co-
operate with irradiation in increasing the fre-
quency of thyroid tumors, while ablation of
TSH stimulation (e.g., hypophysectomy) un-
der these experimental conditions blocks tu-
mor development (Doniach, 1970a,b, 1974;
Nadler el ai, 1970; NAS, 1980). Thus, part
of the irradiation-induced carcinogenicity
appears to be due to or responsive to in-
creases in TSH levels.
Still further support for the role of TSH in
thyroid carcinogenesis comes from experi-
ments using chemicals which reduce circulat-
ing thyroid hormone levels and result in in-
creases in TSH (see Section III.B). Thyroid
hyperplasia and neoplasia in these cases can
be blocked by doses of exogenous thyroid
hormone that reestablish thyroid-pituitary
homeostasis or by hypophysectomy (for ex-
amples see Yamada and Lewis, 1968; Jemec,
1980).
In general, thyroid neoplasms that have
been induced in animals by excessive TSH
stimulation remain dependent upon ongoing
TSH stimulation, as when tissue fragments
are transplanted from the original animals to
a second host (see Doniach, 1970; for excep-
tion, note Ohshima and Ward, 1986). This is
in keeping with the observation that thyroid
tumors in animals and humans retain their
ability to respond to TSH in regard to differ-
entiated cell functions and growth (Biel-
schowsky, 1955;Larsen, 1982b).
2. Morphological Stages in Thyroid Neopla-
sia
The progressive morphological changes
that occur in thyroid tissues in response to
prolonged elevated levels of TSH have been
studied in some detail and are qualitatively
similar irrespective of the nature of the stimu-
lus causing TSH elevation (low iodine diet,
goitrogen exposure, etc.) (Gorbman, 1947;
Denefefa/., 1981; Philprta/., 1969;Santler,
1957; Wynford-Thomas el al, 1982a; Woll-
man and Breitman, 1970). Following initia-
tion of long-term TSH stimulation, changes
in the thyroid exhibit three different phases—
an initial lag phase of several days, a period
of rapid growth, and a period of declining
growth rate as a plateau is attained.
During the lag or latent period that may
last for several days, thyroid weight and DNA
content remain relatively constant. Rapid
changes occur in the morphology of the gland
during this period, however, characterized by
resorption of colloid from the follicular lu-
men and by increases in epithelial cell vol-
ume (the cells change from a cuboidal to a
more columnar form) and vascularity. Con-
sequently, the latent period is characterized
by a redistribution of thyroid tissue and com-
partment volumes and particularly by hyper-
trophy of the follicular epithelial cells.
With continued TSH stimulation, the la-
tent period is followed by a rapid and pro-
longed increase in thyroid weight and size.
Although all thyroid tissue components pro-
liferate to some extent, the major changes ob-
served are associated with follicular cell hy-
perplasia. Thus, there are dramatic increases
in both mitotic activity and in the number of
follicular cells per gland (Wynford-Thomas el
ai, 1982a). There are, however, limits to the
extent to which thyroid hyperplasia, as well
as thyroid weight and size, can continue to
-------
636
HILL ET AL,
increase. Thus, despite a sustained TSH stim-
ulus (e.g., administration of goitrogen) and
sustained increases in the circulating levels of
TSH, the mitotic activity of the follicle cells
progressively declines, and thyroid size and
weight level off to a plateau (after about 80
days of goitrogen treatment) (Wynford-
Thomas el al, 1982a,b). If the TSH stimulus
is withdrawn for 25 days and then reintro-
duced, the maximum size of the thyroid is
unchanged (Wynford-Thomas et al., 1982b).
Although far from definitive, the mechanism
of this "desensitization" to the stimulating
effects of TSH does not appear to be due to
a significant "downregulation" (decrease) of
the number of TSH receptors per cell (Witte
and McKenzie, 1981; Davies, 1985). While
subsequent studies (Wynford-Thomas et al.,
1982c; Stringer^ al., 1985) have failed to elu-
cidate the desensitization mechanism, it has
been suggested that it is mediated by an intra-
cellular change in the follicular cell either at
the receptor or at the postreceptor level.
Clearly, there exists an intracellular or inter-
cellular control mechanism that limits the
mitotic response of thyroid follicle cells to
TSH, which led Wynford-Thomas et al.
(1982c) to propose that the failure of this con-
trol mechanism might be the first step in neo-
plasia. Possibly thyroid cells undergoing re-
peated cell division become irreversibly com-
mitted to a differentiated state and are no
longer able to respond to TSH. On the other
hand, cellular responsiveness to TSH may de-
pend upon interactions with other growth
mediators. In support of this, TSH-induced
increases in cell number in vivo are closely
correlated with changes in receptor density
for another protein growth factor, soma-
tomedin A (Polychronakos et al., 1986).
Certainly, under experimental conditions
of prolonged stimulation by TSH, diffuse thy-
roid hyperplasia may progress to a nodular
proliferation of the follicular cells and even-
tually to neoplasia (Gorbman, 1947; Money
and Rawson, 1950; Griesbach et al., 1945;
Doniach and Williams, 1962). While many
of the resulting tumors are benign, prolonged
and excessive thyroid stimulation may result
in malignant tumors. The morphology of
thyroid tumors in laboratory rodents has
been discussed in several reviews (Doniach,
1970b; Boorman, 1983; Frith and Heath,
1983). Studies with humans show a similar
morphologic progression of the thyroid up
through nodular hyperplasia and "adenoma-
tous" lesions following prolonged stimula-
tion by TSH (Ingbar and Woeber, 1981; see
Section IV of this paper).
3. Reversibility of Morphological Progres-
sion to Thyroid Cancer
Several important questions arise concern-
ing the progression of the different morpho-
logical states toward thyroid cancer, particu-
larly with respect to the extent to which the
progression is reversible. Thus, it is important
to know at what point (if any) and by what
mechanism the progression through hyper-
trophy, hyperplasia, nodule formation, and
neoplasia becomes irreversibly committed to
the formation of a malignant tumor. Un-
doubtedly, the final answer to these and other
questions will have to await a more thorough
understanding of the molecular biology of the
complex events resulting in thyroid neoplasia
(see Section II.C).
There is ample experimental evidence,
however, showing that, to a significant
though unknown extent, the morphological
progression toward thyroid malignancy can
be halted and at least partially reversed by re-
moving the source of, and/or correcting for,
the excessive thyrotropic stimulation. This
may be achieved by administering adequate
amounts of thyroid hormones to hypothy-
roid animals (Purves, 1943; Bielschowsky,
1955;Furth, 1969; Paynter etal., 1986) or by
effecting surgical hypophysectomy (Astwood
et al,, 1943; MacKenzie and MacKenzie,
1943; Nadler et al., 1970). Goiters in persons
living in iodine-deficient areas tend to reverse
following introduction of iodine in persons
with hyperplasias of short duration (Ingbar
and Woeber, 1981; see Section IV of this pa-
per). In each case, these procedures counter
the effect of the source of TSH stimulation.
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THYROID CARCINOGENESIS REVIEW
637
The extent to which morphological pro-
gression in the thyroid can be reversed, how-
ever, clearly depends on the extent to which
the process has progressed, i.e., the severity
and particularly the duration of the insult
causing TSH stimulation. On cessation of
long-term goitrogen treatment or replace-
ment of a long-term, low iodine diet with a
high iodine diet, the size and weight of the
thyroid typically decreases. If the pathologi-
cal process has not progressed too far (e.g.,
hyperplastic goiter), regression may be com-
plete (Gorbman, 1947; Greer el al., 1967; In-
gbar and Woeber, 1981). There is even one
report that propylthiouracil-induced cellular
proliferation (including metastasis to the
lung) regressed to normal when goitrogen ad-
ministration to animals was stopped (Dunn,
1975). In the same study, propylthiouracil-
stimulated thyroid tissue transplanted into
other animals did not continue to proliferate
and retain its tumorigenic status unless the
animals were treated with propylthiouracil.
Others have pointed out the need for ongoing
TSH stimulation in the perpetuation of "hy-
perplastic-neoplastic" thyroid lesions either
in the animals where the lesions arose or in
hosts receiving transplants of the material
(Todd, 1986;Doniach, 1970b).
In contrast, little or no indication of mor-
phological reversibility was observed when
rats that had received up to 500 ppm ethylene
thiourea in their diets for a period of 2 years
were returned to a control diet (Graham et
al., 1973). In another study (Bielschowsky
and Goodall, 1963), methylthiouracil-in-
duced thyroid lesions in the mouse continued
to progress after goitrogen administration
was stopped and replaced by thyroid hor-
mone treatment. Most other studies indicate
varying degrees of reversibility following dis-
continuation of goitrogen administration
(Arnold et al., 1983; Wollman and Breitman,
1970; Wynford-Thomas et al., 1982c) or re-
turn of animals from a low iodine to a high
iodine diet (Greer et al., 1967).
In humans it has been common clinical
practice to use high doses of thyroid hormone
to try to suppress the growth of thyroid "nod-
ules" and help differentiate nonneoplastic
from neoplastic growths (Rojeski and
Gharib, 1985). The idea is that prenoplastic
lesions would regress upon cessation of TSH
stimulation brought about by the added hor-
mone. Although variable success in reducing
nodule size has been noted in the past, a re-
cent study failed to show any treatment-re-
lated reductions (see study and review,
Gharib et al., 1987). Thus the role of TSH in
maintaining the size of human thyroid nod-
ules and their potential for reversal upon ces-
sation of TSH stimulation requires further in-
vestigation.
Typically, reversal is marked by a reduc-
tion of thyroid gland size and weight begin-
ning a few days after removal of the TSH
stimulus and this is associated with a loss of
DNA indicating a decrease in the number of
cells present; some of this seems to be due to
a reduction in the number of follicular cells
(Wollman and Breitman, 1970; Wynford-
Thomas et al., 1982c). The mechanism by
which cells are lost from the thyroid may be
cell death or migration. Regression is associ-
ated with involution of the thyroid that in-
volves a decrease in vascular dilation, a
marked diminution of follicular cell size and
shape (from columnar to cuboidal), and a re-
turn of follicular colloid material (Gorbman,
1947). These qualitative changes in thyroid
histology almost always occur following the
removal of the TSH stimulus. However, if the
goiter has been present for several weeks, or
months, the thyroid gland continues to re-
main at least two to three times its normal
size and weight despite a return to its normal
histological appearance (Greer et al., 1967;
Wollman and Breitman, 1970; Wynford-
Thomas et al.. 1982c).
B. Pituitary Neoplasia
Following chronic iodine deficiency (Axel-
rod and Leblond, 1955), treatment with goi-
trogens (Griesbach, 1941; Griesbach et al.,
1945), or surgical or I31l-induced thyroidec-
tomy (Doniach and Williams, 1962; Carlton
-------
638
HILL ET AL
and Gries, 1983), the anterior pituitary fre-
quently exhibits a loss of acidophilic cells and
an increase in basophil cells, and develops
swollen "thyroidectomy cells," some of
which contain cytoplasmic granules. These
cells contain TSH (Osamura and Takayama,
1983) and, according to some researchers,
may progress to TSH-secreting adenomas
(Furth et al, 1973; Bielschowsky, 1955), al-
though other authors have failed to demon-
strate tumors in such treated animals (for in-
stance, see Ohshima and Ward, 1984, 1986).
Pituitary hyperplasia and neoplasia appear to
result from the same treatments causing thy-
roid neoplasia—conditions leading to pro-
longed circulating thyroid hormone decrease
and excessive secretion of TSH by the pitu-
itary gland.
C. Molecular Considerations
in Thyroid Carcinogenesis
Any hypothesis developed to explain the
mechanism for carcinogenesis must be con-
sistent with what is known about the specific
type of cancer and the physiological and bio-
chemical system in which it develops. Ani-
mal experiments have clearly shown that in-
creased levels of TSH are associated with de-
velopment of thyroid hyperplasia and, later,
with thyroid neoplasia. These endpoints, hy-
perplasia and neoplasia, manifest two pro-
cesses that are going on in the thyroid: one
is an increased commitment to cell division,
which leads to hyperplasia; the other is the
transformation of normal cells into neoplas-
tic cells. Recent work at the cellular level indi-
cates that induction of cell division (which
can lead to hyperplasia) and the transforma-
tion of normal to altered (neoplastic) cells are
the result of a complex interaction of differ-
ent cell systems. For thyroid follicular carci-
nogenesis, it appears that TSH is a compo-
nent in these interactions.
It is generally recognized that, under nor-
mal conditions, the control of cell division re-
quires the interaction of a number of endoge-
nous factors which work through a number
of common pathways; exogenously added
materials may also have profound effects on
this system. It seems there are at least two
such control steps centered in the pre-DNA
synthetic part of the cell cycle and that TSH
is one of the factors operating there in thyroid
cells. Certain protein growth factors which
operate through receptors on the cell surface
are other stimuli that influence cell division.
In a similar manner, the transformation of
normal cells into an altered state with neo-
plastic potential also seems to be dependent
upon the interaction of different factors. TSH
may also play an active role here.
This section reviews available molecular
information about the control of cell growth
in thyroid cells and their conversion to neo-
plastic cells and attempts to incorporate this
information into a plausible mechanistic
framework. Although there are gaps in the
understanding of the processes involved,
what is known about the thyroid is consistent
with the existing understanding of the com-
ponents involved with the control of mam-
malian cell division. It is also consistent with
current thinking that carcinogenesis is a mul-
tistep process and that multiple factors may
influence its course. And finally, it accords
special weight to TSH as playing a significant
role in cell proliferation and in carcinogenesis
of the thyroid gland.
1. Stimulation of Cell Division
a. Influence of TSH. TSH interaction with
its receptor on the surface of the thyroid cell
results in activation of adenyl cyclase and re-
sultant production of cAMP, the activation
of the phosphatidylinositol pathway, com-
mencement of certain thyroid-specific dif-
ferentiated functions that result in the forma-
tion of thyroid hormones, and stimulation of
cell division. Although all cultured cells do
not respond to TSH alone by increasing cell
division (murine and canine do; porcine,
ovine, and human do not [see Saji et a!.,
1987]), the following steps have been identi-
fied in those that do respond. Almost imme-
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THYROID CARCINOGENESIS REVIEW
639
diately (within 15 to 30 min) after addition
of TSH to quiescent thyroid cells in culture,
there are marked increases in the levels of the
mRNAs for the cellular protooncogene, c-fos.
A similar pattern is found for transcripts of
the protooncogene, c-myc, but the induction
is delayed somewhat, with the peak occurring
at about 1 to 2 hr after TSH addition. These
effects of TSH can be mimicked by direct ad-
dition of cAMP analogs or other factors that
increase cellular cAMP (Dere et al, 1985;
Tramontane el al, 1986a; Colletta el al.,
1986). Interestingly, human thyroid adeno-
mas and carcinomas are characterized by c-
myc expression, which is not found in the sur-
rounding normal thyroid tissue. In addition,
like normal cells in culture, adenoma cells re-
spond to TSH in a dose-related manner by
increasing the levels of c-myc transcripts (Ya-
mashita et ai, 1986). This finding in human
cells is in contrast to that cited above (Saji et
ai, 1987).
The protein products of the c-fos and c-
myc protooncogenes are thought to play a
role in the replication of cells. Both c-myc and
c-fos code for proteins that are largely re-
stricted to the cell nucleus and appear to be
functionally linked to DNA synthesis. The
latter is illustrated by experiments showing
that when monoclonal antibody to human c-
myc protein is added to isolated nuclei, there
is an inhibition of DNA synthesis and replica-
tive DNA polymerase activity; the inhibition
can be overcome by the addition of excess c-
myc protein (Studzinski et al., 1986). Further
investigation is required in this area, since
there are some questions about the original
report (Gutierrez et al., 1988; Studzinski,
1988).
There is additional evidence to indicate
that oncogene expression may be an impor-
tant factor in triggering cell division. For in-
stance, certain human cancers have been
shown to have chromosome rearrangements
involving c-myc. This relationship has been
well established for cases of Burkitt lym-
phoma (B-cell cancer) (Taub et al., 1982; ar-
Rushdi et al, 1983; Nishikura et al, 1983)
and to a lesser extent for certain T-cell leuke-
mias (Erikson et al, 1986; Finger et al,
1986). It is thought that chromosomal trans-
locations move c-myc to the regulatory units
of immune response genes in these cells and
bring about constitutive activation of the on-
cogene which then provides a continued
stimulus for cell proliferation (see review by
Croce, 1986), although recent evidence indi-
cates that a number of Burkitfs cases also
have point mutations at the binding site for a
nuclear protein (Zajac-Kaye et al, 1988).
TSH also seems to affect to some extent the
phosphatidylinositol pathway within cells
(Kasai and Field, 1982; Tanabe et al, 1984;
Bone et al, 1986), which is a major transduc-
tion system of signals across cell membranes
(see Nishizuka, 1986 and next section) as is
the cAMP system. Just how this effect of TSH
may influence thyroid cell division has not
yet been determined.
b. Other factors. Experiments in a number
of cell systems have identified control points
in the pre-DNA synthetic part of the cell cycle
which must be passed for cells to replicate
DNA and go into cell division. For instance,
mammalian cells treated with one chemical
stimulus (e.g., platelet-derived growth factor
which is known to stimulate c-myc) did not
commence DNA synthesis until other sub-
stances were added to the medium (Stiles et
al., 1979; Smeland et al, 1985). Current in-
vestigations on the interaction of various fac-
tors in the control of cell division have been
summarized by Goustin et al. (1986) and Ro-
zengurt(1986).
Work with thyroid cells also indicates that
a number of growth factors and cell systems
are operating which influence a cell's com-
mitment to cell division. For illustrative
purposes, emphasis here will be placed on
three of these: epidermal growth factor, the
protein kinase c system (see Table 1), and the
somatomedins.
Epidermal growth factor (EGF) is a natu-
rally occurring polypeptide present in a num-
ber of organs that binds to specific receptors
on sensitive cells. This binding results in acti-
vation of receptor-associated tyrosine kinase
which phosphorylates the EGF receptor and
-------
640
HILL ET AL.
TSH
EOF
TPA"
TABLE I
EFFECTS OF STIMULI ON THYROID CELLS
Stimulus
Enzyme
activity
Induces
c-fos and c-myc
Stimulates
cell
division
Effect on
differentiated
functions
Other
Adenyl cyclase
Tyrosine kinase
Protein kinase c
Enhances
Inhibits
Inhibits
Enhances EGF binding
to its receptor
Inhibits EGF binding
to its receptor and
tyrosine kinase
activity
° 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester.
other sites and helps to bring about its cellular
action. EGF is present in adult tissues; a re-
lated growth factor, transforming growth fac-
tor type a, is present in neoplasms and em-
bryonic tissues and may be an embryonic
form of EGF. It is interesting to note that one
of the viral oncogenes, v-erbB, is a mutation
of the EGF receptor gene where the binding-
site portion of the receptor has been deleted,
and that this mutation may result in constitu-
tive activation, resulting in continued cell
proliferation (Goustin el ai, 1986).
There is some work that indicates that EGF
plays a role in the regulation of cellular activ-
ity and cell division in thyroid cells in culture.
Its role i/i vivo needs to be ascertained. Unlike
TSH, EGF blocks certain differentiated func-
tions that typify thyroid action, such as for-
mation of thyroglobulin by thyroid cells in
culture (Westennark et ai, \ 983; Bachrach et
al, 1985; Roger et ai, 1986). In in vivo stud-
ies, infusion of sheep over a 24-hr period with
EGF resulted in a profound drop in serum T4
and T3 which started within 10 hr after com-
mencing administration. Part of this reduc-
tion in circulating thyroid hormones appears
to be due to their enhanced metabolism (Cor-
coran et al., 1986). These authors cite other
work which shows that thyroid hormone ad-
ministration results in increased tissue levels
and urinary excretion of EGF. It thus seems
that some feedback exists between levels of
EGF and thyroid hormones.
EGF also produces increases in cell divi-
sion in thyroid cells. By about 1 day after ad-
dition of EGF to thyroid cells in culture, there
is stimulation in DNA synthesis (Wester-
mark et ai, 1983; Roger et ai, 1986), as was
seen after administration of TSH. TSH in-
creases the binding of EGF to its receptor on
thyroid cells and, in combination with EGF,
enhances DNA synthesis above that seen
with EGF alone (Westermark et ai, 1986).
Another cell surface-related mechanism re-
sults in the activation of protein kinase c. It is
generally recognized that this system is one of
the major information-transferring mecha-
nisms from extracellular to intracellular sites
in many cells throughout the body (see re-
view by Nishizuka, 1986). Receptor binding
of a host of biologically active substances
(e.g., hormones, neurotransmitters) is fol-
lowed by hydrolysis of inositol phospholipids
along two paths: one leads to calcium mobili-
zation, the other to activation of protein ki-
nase c. The kinase transfers phosphate groups
to various proteins which results in a modula-
tion of their action. Many studies have dem-
onstrated that certain tumor promoters in the
two-stage mouse skin carcinogenesis model,
including the phorbol esters, can bind to cell
receptors and activate protein kinase c (see
Nishizuka, 1986).
Phorbol esters, like EGF, inhibit differen-
tiated thyroid cell functions and stimulate
cell division. As in other cells (Friedman et
-------
THYROID CARCINOGENESIS REVIEW
641
al, 1984), phorbol esters increase protein ki-
nase c activity and block EGF binding of its
receptor in thyroid cells (see Table 1) (Bach-
rach era/., 1985; Ginsberg and Murray, 1986;
Roger el al, 1986). It is not known if EGF
and phorbol esters stimulate expression of the
c-fos and c-myc protooncogenes in the thy-
roid, although there is some evidence for this
in mouse 3T3 cells (Kruijer et al., 1984;
Muller et al., 1984; Kaibuchi et al., 1986).
A series of polypeptide substances related
to insulin and termed somatomedins (insu-
lin-like growth factors, IGFs) are known to
exist which help to control cell growth in nu-
merous tissues (see Goustin et al., 1986).
Concentrations of somatomedins in the
blood are regulated by growth hormone.
They are produced by the liver and almost all
organs of the body, seemingly the products of
mesenchymal cells (Han et al., 1987). Al-
though they may or may not stimulate DNA
synthesis in cells when they are the only
added factor, they frequently interact with
other growth factors in bringing about cell di-
vision (Stiles et al., 1979).
In cultured rat thyroid cells, very high con-
centrations of insulin alone will induce cells
to replicate DNA (Smith et al., 1986). It was
hypothesized, then demonstrated, that this
effect was most likely due to cross-reactivity
of insulin with the somatomedin C (IGF-I)
receptor (Tramontane et al., 1986b, 1987;
Saji et al., 1987). In rat thyroid cells, TSH and
somatomedin C (or insulin) synergize in in-
ducing DNA synthesis, but are additive in re-
gard to increasing cell growth (Tramontane
et al, 1986b); such DNA replication synergy
was not noted in porcine cells (Saji et al,
1987).
Although studies on thyroid cells indicate
that TSH, EGF, phorbol esters, and soma-
tomedin C (and insulin) can each stimulate
cell division in cultured thyroid cells, it does
not mean that these factors are the only ones.
For instance, many of the culture systems
used in these studies included serum, which
is known to contain a number of growth fac-
tors. In other cases, the culture medium was
supplemented with hormones, growth fac-
tors, and other substances (e.g., somatostatin,
cortisol, transferrin) which are known to
effect cell cycle traverse (Bachrach et al,
\985; Colletta et al, 1986; Westermark et al,
1983).
c. Possible controls of thyroid cell division.
As discussed earlier, it appears that the con-
trol of cell division in certain mammalian
cells in the pre-DNA synthetic portions of the
cell cycle. By using combinations of sub-
stances, two control points have been identi-
fied; both points must be passed for cells to
commence DNA replication. Although there
are significant differences in response among
cell systems, factors that seem to affect the
first regulatory point include such things as
platelet-derived growth factor and the c-fos
and c-myc oncogenes, whereas those operat-
ing at the second control point include soma-
tomedin C, EGF, and the c-ras oncogene
(Stiles etal, 1979;Leofrta/., 1982; see Gous-
tin et al, 1986). Since TSH is also known to
activate adenyl cyclase and c-fos and c-myc
expression in thyroid cells (Dere et al, 1985;
Colletta et al, 1986; Tramontane et al,
\ 986a), it seems possible that it may act at the
first control point. This is supported by the
observation that combinations of TSH with
EGF or somatomedin C lead to enhanced
DNA synthesis in thyroid cells (EGF and so-
matomedin C are putative second control
step agents) (Westermark et al, 1986; Tra-
montane e/a/., 1986b, 1987).
The placement of the protein kinase c sys-
tem in the control of thyroid gland cell divi-
sion is uncertain, since its effect on cell prolif-
eration is not enhanced by either TSH or
EGF. As indicated previously, phorbol ester
administration to thyroid cells diminished
EGF binding to its receptor (Bachrach et al,
1985). It also appears that TSH itself may in-
crease the phosphatidylinositol pathway in
addition to affecting cAMP (Bone et al,
1986). On the other hand, the protein kinase
c and adenyl cyclase systems often play com-
plementary roles in mammalian cells to en-
hance cell division and other functions (Nis-
hizuka, 1986; Rozengurt, 1986). More infor-
mation is needed in this area.
-------
642
HILL ET AL.
TSH
platelet-derived growth factor
insulin
Induction of adeny! cyclace
and c-fos/c-myc
Pre-DNA
replication
DNA
replication
Cell
division
' Activation of
protein kinase c
somatomedin C
c-ras
insulin
FIG. 4. Possible control points for cell division in the pre-DNA synthetic portion of the cell cycle.
Insulin (and related substances) seems to
play a facilitating role in the thyroid. Alone
in high concentrations it can induce thyroid
cells in medium without serum to synthesize
DNA, and it enables TSH to enhance this
effect (Wynford-Thomas #a/., 1986). Insulin
is active at both control points in certain
mouse 3T3 cells as well (Rozengurt, 1986).
A model can be constructed for control of
cell division in the thyroid gland (Fig. 4) that
includes the two pre-DNA synthetic steps.
The model engenders the known effects of
various factors on thyroid cells and reflects
certain observations in other mammalian cell
systems. Although the model is not fully satis-
factory, due to the inconsistencies across cell
systems, it depicts certain interactions that
may exist in the thyroid gland and suggests
possible future research directions.
2. Cellular Transformation
As with the control of cell division, com-
plex interactions among different factors
seem to be operating during the transforma-
tion of normal to altered cells with neoplastic
potential. Although activation of a single on-
cogene may not be sufficient in all cases to
produce transformation, activation of two
different oncogenes is commonly sufficient to
transform cells (see reviews by Weinberg,
1985; Barbacid, 1986). Frequently the coop-
eration includes the coordinate expression of
an oncogene whose product is localized to the
nucleus (e.g., c-fos, c-myc) with one whose
product is in the cytoplasm (e.g., c-ras, c-src).
As was mentioned previously, nuclear onco-
genes can be activated by chromosomal
translocation of the oncogene to cellular reg-
ulatory sequences; other activation mecha-
nisms include the insertion of viral regulatory
segments next to the nuclear oncogene, gene
amplification (increase in the number of cop-
ies of the oncogene per cell), and stabilization
of the oncogene gene product. On the other
hand, cytoplasmic oncogenes tend to be acti-
vated by point or chromosomal mutations
which affect the structure of their gene prod-
ucts (Weinberg, 1985).
TSH enhances c-fos and c-myc expression
that may in turn interact with other factors
in bringing about cell transformation. If the
stimulus for TSH secretion from the pituitary
is long term, as in the case of continued expo-
sure to an antithyroid substance, it seems
possible there could be continued oncogene
transcription and a continued emphasis on
-------
THYROID CARCINOGENESIS REVIEW
643
cell proliferation which could result in hyper-
plasia. Still other stimuli (e.g., activation of a
second oncogene, certain point or structural
mutations, interplay with growth factors)
may aid in the transformation process and
bring about neoplasia.
This hypothesis is consistent with recent
studies which indicate that c-myc may be a
necessary component in cellular transforma-
tion, but that it is not sufficient in itself to
bring about the condition. Studies of trans-
genie mice support this conclusion (Adams et
al., 1985; Langdon et al, 1986). Combina-
tions of the DN A of c-myc and the enhancer
region of the E^-immunoglobulin locus were
constructed and injected into fertilized
mouse eggs which were transplanted into ma-
ternal hosts. The DNA became incorporated
into the cells of the body of the developing
organism (transgenic recipients). Within a
few months after birth, almost all animals de-
veloped malignant B-cell lymphomas and
died. It seems that during development there
is a constitutive expression of c-myc with a
great expansion of multiple clones of B-cell
precursors. However, only one clone devel-
ops into a tumor and this seems to occur at
variable times during development. This has
led the authors to propose that although c-
myc expression favors proliferation of B-cell
precursors, some genetic event, like activa-
tion of a second oncogene, may be required
for transformation to malignancy.
Studies on the thyroid gland are consistent
with the idea that c-myc (through TSH stimu-
lation) may interact with other stimuli in
bringing about cell transformation. For in-
stance, an enhancement of the carcinogenic
response is noted when a treatment that in-
creases TSH (e.g., iodide deficiency) follows
application of a genotoxic agent (e.g., irradia-
tion, nitrosamine) (see Section IV.B.4) which
might produce a mutation that activates a
second oncogene or some other effect.
One is still faced, however, with the obser-
vation that treatments that ensure prolonged
TSH stimulation, as have been discussed pre-
viously, lead to neoplasia. Three possibilities
exist: (1) TSH simply enhances spontane-
ously occurring events (e.g., mutations in reg-
ulatory sequences like oncogenes). The find-
ing of thyroid neoplasms in about 1 % of some
untreated laboratory animals (Haseman et
al., 1984) is in keeping with the idea that
"spontaneous mutations" might exist in con-
trol animals that might predispose animals
for development of thyroid tumors. (2)
Through its effect on cell division, TSH may
expand the thyroid cell population at risk for
a spontaneous event and then promote neo-
plasia once a spontaneous mutation occurs.
(3) TSH alone, via some yet undisclosed
mechanism, might produce cellular transfor-
mation.
III. EXOGENOUS FACTORS
INFLUENCING THYROID-
PITUITARY CARCINOGENESIS
The observations presented in the previous
section demonstrated that prolonged in-
creases in TSH output are associated with
thyroid cellular hypertrophy and hyperplasia
and, finally, with neoplasia in the absence of
exogenously added agents. This section sum-
marizes known information on thyroid carci-
nogenesis following application of exogenous
stimuli. In the main, it, too, shows the impor-
tant role of chronic TSH stimulation in thy-
roid carcinogenesis. Information on physical
and chemical agents affecting thyroid-pitu-
itary physiology and carcinogenesis is sum-
marized. Chemical classes associated with
thyroid tumors in the National Cancer Insti-
tute/National Toxicology Program (NCI/
NTP) animal studies are listed, and analyses
are conducted on the specific chemicals from
those classes as to their antithyroid activity
and genotoxicity.
A. Physical Factors
External ionizing radiation is a known thy-
roid carcinogen in humans and experimental
animals (NAS, 1980). Internal radiation, fol-
lowing administration of I3II (a (3- and a 7-
radiation emitter), produces thyroid tumors
-------
644
HILL ET AL.
in animals, but the evidence in humans from
the follow-up of treated Graves' disease pa-
tients is less firmly established (NAS, 1980;
NCRP, 1985; see Becker, 1984). A recent pa-
per purports the hypothesis that radioiodines
may account for thyroid nodules following
the detonation of a hydrogen bomb in the
Marshall Islands in the Pacific Ocean (Hamil-
ton et ai, 1987). Although irradiation can al-
ter DNA and induce mutation and, thus, in-
fluence thyroid carcinogenesis via genotoxic
mechanisms, others have speculated that the
follicular cell damage induced by irradiation
may also impair the gland's ability to produce
thyroid hormone and, thus, places the thy-
roid under conditions of long-term TSH
stimulation.
B. Chemical Factors
1. Goitrogens
Early interest in naturally occurring chemi-
cals causing thyroid enlargement arose from
observations that rabbits fed diets composed
mainly of cabbage leaves frequently devel-
oped goiters (Chesney et ai, 1928). Similar
observations were subsequently made with
two purified synthetic chemicals (sulfaguani-
dine and l-phenyl-2-thiourea) during nutri-
tional/physiological studies with rats (Mac-
kenzie et ai, 1941;RichterandClisby, 1942).
When it was realized that the primary action
of these and related compounds was to in-
hibit synthesis of the thyroid hormones, their
potential therapeutic value in hyperthyroid-
ism became evident.
a. Naturally occurring (dietary) substances.
These materials have been reviewed in detail
by Van Etten (1969). The early observations
of goiters in rabbits maintained on cabbage
leaf diets (Chesney et at., 192 8) were followed
by the discovery that the seeds of rape and
other brassica species (cabbage, brussels
sprouts, turnips, and mustard) also contained
substance(s) that were goitrogenic when in-
corporated into rat diets (Hercus and Purves,
1936; Kennedy and Purves, 1941). Prolonged
dietary exposure to rape seed led to the devel-
opment of adenomatous goiters (100% in 27
months) in rats (Griesbach et ai, 1945). L-5-
Vinyl-2-thiooxazolidone (goitrin) has been
identified as the active goitrogen in turnips
and the seed and green parts of other crucifer-
ous plants. Goitrin from these sources may
be passed to humans in the milk of cows feed-
ing on such plants. In humans, goitrin ap-
pears to be about as active as propylthiouracil
(Haynes and Murad, 1985). Peanuts are also
reported to be goitrogenic in rats (Srinivasan
et ai, 1957), the active component being the
glucoside, arachidoside.
b. Synthetic compounds. Synthetic chemi-
cals exhibiting goitrogenic activity may be di-
vided into three major structural groups: thi-
onamides, aromatic amines, and polyhydric
phenols. The synthetic goitrogens are dis-
cussed briefly below, but have been exten-
sively reviewed by Cooper (1984) and Pay-
nleretal. (1986).
(i) Thionamides: These include derivatives
of thiourea and heterocyclic compounds con-
taining the thioureylene group. The latter in-
cludes most of the compounds (e.g., propyl-
thiouracil, methimazole, and carbimazole)
used therapeutically for hyperthyroidism in
humans. Among the many chemicals in this
group, one nitrogen atom may be replaced by
oxygen or sulfur; however, the thionamide
group is common to all. Other active com-
pounds in this class are derivatives of imidaz-
ole, oxazole, thiazole, thiadiazole, uracil, and
barbituric acid. The naturally occurring goi-
trin, present in cruciferous plants, also be-
longs to this group of compounds.
(ii) Aromatic amines: Examples of com-
pounds of this type are the sulfonamides, sul-
fathiazole, and sulfadiazine (Haynes and
Murad, 1985). Optimal antithyroid activity
of this group of compounds is associated with
a para-substituted aminobenzene structure
with or without aliphatic (e.g., methyl) substi-
tution on the amino nitrogen. It is of interest
that several methylene- and oxydianilines
(and alkyl substituted derivatives) have also
been shown to possess goitrogenic activity
(Hayden et ai, 1978) and, like the sulfon-
-------
THYROID CARCINOGENESIS REVIEW
645
amides, to increase thyroid neoplasms in rats
(Weisburgere/a/., 1984).
(iii) Polyhydric phenols: The antithyroid
activity (hypothyroidism and goiter) of resor-
cinol was first observed following the use of
this material for treatment of leg ulcers in hu-
mans (Haynes and Murad, 1985). Subse-
quent studies have established that antithy-
roid activity is associated with compounds
with meta polar-substituents on the benzene
ring. Thus, hexyresorcinol, phloroglucinol,
2,4-dihydroxybenzoic acid, and meta-zmmo-
phenol are active, whereas catechol, hydro-
quinone, and pyrogallol are not (Paynter el
al., 1986).
c. Modes of action. Antithyroid agents be-
longing to structural groups (i), (ii), or (iii) all
exert at least part of their activity by direct
interference with the synthesis of thyroid hor-
mone in the thyroid gland. All appear to
block the incorporation of iodine into tyrosyl
residues of thyroglobulin and by inhibiting
the coupling of the idotyrosyl residues into
idothyronines. It was proposed by Taurog
(1976) that the antithyroid agents inhibit the
enzyme peroxidase that is responsible for the
conversion of iodide to the iodinating species
and the subsequent iodination and coupling
of the tyrosyl residues. This has been con-
firmed by subsequent studies (Davidson et
al, 1978;Englere?a/., 1982) showing that the
compounds bind to and inactivate peroxi-
dase when the heme of the enzyme is in the
oxidized state. It is likely that these com-
pounds show some inhibitory selectivity to-
ward the different peroxidase-catalyzed reac-
tions (i.e., iodination vs coupling) (Haynes
and Murad, 1985). There is also evidence that
some of the compounds (e.g., propylthioura-
cil) inhibit the peripheral deiodination of T4
and T3 (Geffner et al, 1975; Saberi et al.,
1975).
Because of their ability to inhibit thyroid
hormone synthesis, all of the above com-
pounds have the potential to reduce circulat-
ing levels of T4 and T3 and, consequently, to
induce the secretion of TSH by the pituitary.
As a result, prolonged exposure to such com-
pounds can be expected to induce thyroid
gland hypertrophy and hyperplasia and ulti-
mately may lead to neoplasia.
2. Enzyme Inducers
In addition to chemicals exerting effects di-
rectly at the thyroid, as was summarized in
the previous section, a number of others act-
ing at peripheral sites can cause equally pro-
found disturbances in thyroid function and
morphology. Of particular interest are those
compounds that induce hepatic and/or extra-
hepatic enzymes responsible for the metabo-
lism of many endogenous and exogenous
compounds. These chemicals can increase
the metabolism of thyroid hormone, can re-
sult in a reduction in circulating thyroid hor-
mone, and can stimulate an increase in TSH.
Following long-term exposure to these
agents, the thyroid gland undergoes hypertro-
phy and hyperplasia and finally, neoplasia.
a. Foreign compound metabolism and en-
zyme induction, (i) General: The enzymes re-
sponsible for the metabolism of foreign com-
pounds constitute a remarkably diverse
group of proteins that catalyze a variety of re-
actions associated with either the primary
(Phase I) metabolic attack on a chemical (oxi-
dation, reduction, hydrolysis) or with its sub-
sequent secondary (Phase II) metabolism
(e.g., conjugation with glucuronide, sulfate,
amino acids, and glutathione) (Testa and
Jenner, 1976). The enzymes are associated
with the endoplasmic reticulum or cytosol of
the liver and a number of extrahepatic tis-
sues. The enzymes serve an important func-
tional role in increasing the polarity, water
solubility, and excretability of the vast major-
ity of fat soluble foreign compounds that re-
sults in a decrease in their biological activity
or toxicity. Because of the latter, they are fre-
quently referred to as detoxication enzymes
(Wilkinson, 1984).
(ii) Induction: Enzyme induction refers to
the phenomenon whereby exposure of an an-
imal to a given foreign compound results in
the enhanced activity through de novo syn-
thesis of a spectrum of the enzymes involved
-------
646
HILL ET AL.
in Phase I and Phase II metabolism (Cooney,
1967). Induction typically results in an in-
crease in the rate at which the inducer and
other compounds are metabolized and ex-
creted.
Since the enzymes responsible for foreign-
compound metabolism are thought by many
to have evolved as a biochemical defense
against potentially harmful environmental
chemicals (Wilkinson, 1984), induction may
be viewed as a biological adaptation that can
provide important short-term benefits for
survival. On the other hand, in light of in-
creasing evidence that the enzymes detoxify-
ing one chemical may activate another
(Cummings and Prough, 1983), there has
been concern that enzyme induction may
represent a mechanism through which poten-
tially dangerous lexicological interactions
can occur following chemical exposure.
Another cause for some concern is that sev-
eral of the enzymes that participate in for-
eign-compound metabolism are also known
to play important roles in the metabolism of
physiologically important endogenous chem-
icals such as hormones. Clearly, any changes
in the levels of enzymes responsible for the
synthesis or breakdown of such compounds
could lead to physiological imbalances with
potentially serious consequences (Conney,
1967).
(Hi) Different inducer types: Inducers of the
enzymes involved in foreign-compound me-
tabolism have been divided into at least two
different categories on the basis of their char-
acteristic effects on cytochrome P450 and
monooxygenase activity (Mannering, 1971;
Lu and West, 1978, 1980; Ryan et at., 1978).
One of these, typified by phenobarbital, led
to a significant increase in liver size and
weight and caused the substantial prolifera-
tion of hepatic endoplasmic reticulum. In-
duction was associated with increases in cyto-
chrome P450 and a large number of mono-
oxygenase reactions that enhanced metabolic
(oxidative) capability toward many foreign
compounds. The spectrum of oxidative reac-
tions induced is now known to result mainly
from the induction of one major isozyme of
cytochrome P450 that, in rats, is referred to
as cytochrome P450b (Ryan et al, 1978). A
large number of drugs and other foreign com-
pounds, including the chlorinated hydrocar-
bon insecticides (DDT and its analogs and
the cyclodienes like chlordane and aldrin),
exhibit induction characteristics similar to
phenobarbital and are generally referred to as
"PB-type" inducers.
Early studies with the polycyclic hydrocar-
bon, 3-methyl cholanthrene (3MC), clearly
indicated that the induction characteristics of
this compound were quite distinct from those
of PB (Mannering, 1971). In contrast to the
latter, treatment of animals with 3MCdid not
cause large increases in liver size or in the pro-
liferation of endoplasmic reticulum; neither
did it result in large increases in cytochrome
P450. Instead, 3MC resulted in the formation
of a qualitatively different form of cyto-
chrome P450, known generally as cyto-
chrome P448 and now referred to in rats as
cytochrome P450c (Mannering, 1971; Lu
and West, 1978;Ryanrta/.. 1978). This cyto-
chrome is associated with a rather limited
number of oxidative reactions, the best
known of which is aryl hydrocarbon hydrox-
ylase (AHH) (Ryan et al., 1978; Eisen et al,
1983; Conney, 1982). AHH has received a lot
of attention in recent years because of its role
in the metabolic activation of compounds
like benzo[a]pyrene to potent carcinogens
(Eisen et al, 1983; Conney, 1982). Inducers
of the "3MC-type" include a number of poly-
cyclic aromatic hydrocarbons, naphthofla-
vone, and several halogenated dibenzo-/j-di-
oxins. 2,3,7,8-Tetrachlorodibenzo-/Miioxin
(TCDD) is the most effective inducer of this
type to be discovered (Poland and Glover,
1974). The mechanism of action of inducers
of this type involves high affinity binding to a
cytosolic receptor and subsequent migration
of the inducer-receptor complex to the nu-
cleus where the transcriptional effect leading
to enhanced protein synthesis is initiated
(Eisen et al, 1983). Induction of this type is
genetically controlled by the so-called Ah lo-
cus in rodents and, while the true identity of
the cytosolic receptor remains unknown, it is
-------
THYROID CARCINOGENESIS REVIEW
647
hypothesized to be a receptor for some hor-
mone or other physiologically important li-
gand.
While the PB-type and 3MC-type inducers
still constitute the two major categories of in-
ducers, it is now recognized that a number of
other types exists, each characterized by in-
creased levels of a distinct spectrum of iso-
zymes of cytochrome P450 and other en-
zymes. It is also apparent that a number of
compounds share some of the characteristics
of more than one group and cannot be strictly
classified. Technical mixtures of polyhaloge-
nated biphenyls (PCBs and PBBs), for exam-
ple, exhibit characteristics of both PB- and
3MC-type inducers (Alvares et ai, 1973),
probably due to the presence in the mixtures
of a number of isomers representing each
type.
In addition to inducing a characteristic
spectrum of isozymic forms of cytochrome
P450, many of the inducers also result in en-
hanced tilers and activities of other enzymes
involved in foreign-compound metabolism.
While these have not been well documented,
they include epoxide hydratases, glutathione
(GSHJ-S-transferases, and several of the
transferases (UDP-transferases, sulfo-trans-
ferases) associated with secondary conjuga-
tion reactions (Jacobsen et ai, 1975; Lucier
et ai, 1975; Ecobichon and Comeau, 1974).
It has been suggested that, like cytochrome
P450, these enzymes may also exist in multi-
ple isozymic forms and that different induc-
ers may enhance the activity of specific iso-
zymes with a characteristic range of substrate
specificities.
b. Metabolism of thyroid hormones. The
liver not only constitutes a target tissue for
the thyroid hormones, but is also an organ re-
sponsible for the metabolic inactivation of
the hormones and their elimination from the
body. About half the T4 elimination from the
body of the rat occurs via the bile, whereas in
humans only about 10 to 15% is lost in this
way (Oppenheimer, personal communica-
tion 1987). While there appear to be quanti-
tative differences in the relative rates of elimi-
nation of T4 and T3, it is probable that both
are excreted by a qualitatively similar mecha-
nism. The major pathway of elimination in-
volves conjugation of the phenolic hydroxyl
group of T4 with glucuronic acid and biliary
excretion of the resulting glucuronide (Fig. 1)
(Galton, 1968; Bastomsky, 1973); sulfate
conjugates may also be produced and ex-
creted. On entering the intestine a portion of
the conjugate may undergo hydrolysis by in-
testinal bacteria to release free thyroid hor-
mone that may be reabsorbed into the circu-
lation; this process is referred to as enterohep-
atic circulation. Unhydrolyzed conjugate
cannot be reabsorbed and is excreted in the
feces(Houk, 1980).
c. Effect of inducers on thyroid function and
morphology, (i) PB-type inducers: Initial re-
ports on the goitrogenic effects of a number
of PB-type inducers in both birds and rodents
began to appear in the mid- to late 1960s.
Modest to substantial increases in thyroid
weight were reported in rats treated with phe-
nobarbital (Japundzic, 1969; Oppenheimer
et ai, 1968) and isomers of ODD (Fregly et
ai, 1968), in pigeons treated with p,p/-DDE
(Jefferies and French, 1969), /?,//-DDE, or
dieldrin (Jefferies and French, 1972), and in
bobwhite quail exposed to p.rJ-DDT or toxa-
phene (Hurst et ai, 1974). Chlordane, an-
other chlorinated hydrocarbon, enhanced
thyroid function and caused hepatic accumu-
lation of I25I-T4 in rats (Oppenheimer et ai,
1968). Histological examination of the thy-
roids of treated animals typically showed a re-
duction in follicular colloidal material and
increased cellular basophilia and hyperplasia
(Fregly et ai, 1968; Jefferies and French,
1972), and it was noted by several workers
that these changes were similar to those oc-
curring in response to increased circulating
levels of TSH. Support for the effect being a
response to increased TSH, rather than a di-
rect effect on the thyroid, is found in studies
demonstrating that the goitrogenic response
of the thyroid to phenobarbital could be pre-
vented by hypophysectomy or the adminis-
tration of T4 (Japundzic, 1969).
The effects of PB-type inducers on thyroid
function are now known to be quite complex
-------
648
HILL ET AL.
and to involve a number of factors relating to
the distribution, tissue binding, metabolism,
and excretion of thyroid hormones. Animals
treated with phenobarbital show increased
hepatocellular binding of T4 combined with
enhanced biliary excretion of the hormone
(Oppenheimer et ai, 1968, 1971). In intact
rats, these changes simply result from an in-
creased rate of turnover of T4 that is compen-
sated by release of TSH and enhanced thyroi-
dal secretion of new hormone. As a result, no
change in serum protein-bound iodine (FBI)
is observed following treatment with pheno-
barbital (Oppenheimer et ai, 1968). In thy-
roidectomized rats, however, phenobarbital
reduces serum FBI and also reduces the hor-
monal effects of administered T4 (Oppenhei-
meretal., 1968, 1971). The ability of pheno-
barbital to reduce circulating levels of exoge-
nously supplied T4 in a human hypothyroid
patient has been reported. The major factors
leading to enhanced turnover of T4 in ani-
mals treated with PB-type inducers seem to
be increased hepatocellular binding due
mainly to proliferation of the endoplasmic re-
ticulum (Schwartz et ai, 1969) and a modest
increase in bile flow that enhances the overall
rate of biliary clearance (Oppenheimer el al.,
1968). Phenobarbital (Oppenheimer et al.,
1968) and DDT (Bastomsky, 1974) cause
only minimal increase in biliary T4 excretion,
and in rats treated with DDD isomers, fecal
excretion of I3II-T4 was not observed until 24
hr after hormone treatment (Fregly et al.,
1968). While DDT slightly enhanced the pro-
portion of biliary 125I present at T4-glucuro-
nide, neither PB nor DDT (Bastomsky, 1974)
are reported to have significant effects on the
rate of glucuronidation of T4
Several studies have been conducted on the
effects of PB-type inducers on thyroid hor-
mone status in healthy human volunteers or
in patients on different drug regimens. Drugs
studied include phenobarbital, carbamazep-
ine, rifampicin, and phenytoin (diphenylhy-
dantoin). Most of the studies report de-
creased serum levels of T4 (both protein-
bound and free) (Rootwelt et al., 1978; Faber
etaL, 1985;OhnhausandStuder, 1983), but
reports vary on the changes observed in se-
rum levels of T, and rT3 depending on the
type and concentration of the inducer em-
ployed. Ohnhaus and Studer (1983) observed
a relationship between increasing levels of
microsomal enzyme induction and decreas-
ing serum levels of T4 and rT3 in healthy vol-
unteers treated with combinations of antipy-
rine and rifampicin. An effect was only ob-
served, however, at induction levels that
decreased the half-life of antipyrine by more
than 60%. Induction of hepatic enzymes is
apparently only one of several mechanisms
through which diphenylhydantoin can re-
duce circulating levels of T4 (Smith and
Surks, 1984). Other possible mechanisms by
which diphenylhydantoin might act include
serum protein displacement of the thyroid
hormones, effects on the binding and biologi-
cal activity of T3, and even effects on hypo-
thalamic and pituitary regulation of TSH.
Despite significantly decreased serum levels
of T4, there seem to be a few reports of hu-
mans being placed in a hypothyroid condi-
tion as a result of treatment with drugs that
induce liver microsomal enzyme activity. An
exception is the observation that persons be-
ing maintained on exogenously supplied thy-
roid hormone become hypothyroid when
given diphenylhydantoin or phenobarbital
unless their thyroid hormone doses are
changed (Oppenheimer, personal communi-
cation 1987). Furthermore, TSH levels never
change significantly from those observed in
the controls.
(ii) 3MC-type inducers: The effects on the
thyroid of 3MC-type hepatic enzyme induc-
ers (polycyclic aromatic hydrocarbons,
TCDD, etc.) are perhaps the best understood
of the compounds under discussion. A major
mechanism involved seems to be the induc-
tion of the T4-UDP-glucuronyl transferase
that constitutes the rate-limiting step in the
biliary excretion of T4 (Bastomsky, 1973).
The effect is particularly well illustrated with
reference to a variety of thyroid hormone pa-
rameters 9 days after treatment of rats with a
single dose of 25 Mg/kg TCDD (Bastomsky,
1977a). Biliary excretion of I25I (during the
-------
THYROID CARCINOGENESIS REVIEW
649
first hour after injection of I25I-T4) and the
biliary clearance rate of plasma I25I-T4 were
increased about 10-fold. Somewhat unex-
pectedly, the biliary excretion of T3 was un-
affected by TCDD. As a direct consequence
of these changes in metabolism and excre-
tion, serum T4 concentrations (but not those
of T3) were reduced to half those in controls.
Other workers have reported decreased se-
rum T4 concentrations following TCDD
treatment of rats (Potter et al., 1983; Pazder-
nik and Rozman, 1985; Rozman et al.,
1985). TCDD treatment elevated serum con-
centrations of TSH and, as a resuh, produced
thyroid goiters (measured by elevated thyroid
weight) and enhanced 13II uptake by the thy-
roid. There are conflicting reports as to
whether TCDD enhances bile flow (Bastom-
sky, 1977a; Hwang, 1973), but this does not
seem to be a major factor in its goitrogenic
action. Interestingly, in hamsters, a species
resistant to the acute toxic effects of TCDD,
administration of the chemical raised T4 and
T3 levels (Henry and Gasiewicz, 1987).
While TCDD is an unusually potent in-
ducer of UDP-glucuronyl transferases, it ap-
pears to be at least somewhat similar to com-
pounds such as 3MC (Bastomsky and Papa-
petrou, 1973; Newman et al., 1971), 3,4-
benzo[a]pyrene (Goldstein and Taurog,
1968), and the polychlorinated and poly-
brominated biphenyls (PCBs and PBBs) (see
below), all of which have been shown to en-
hance the biliary excretion of T4 at least
partly by increasing the formation of T4-gluc-
uronide. TCDD did not uniformly increase
hepatic UDP-glucuronyl transferase activity
toward all substrates; it enhanced activity to-
ward p-nitrophenol about fivefold but not to-
ward testosterone or estrone. At the single
dose of TCDD which produces maximal in-
duction of mixed function oxidase activity in
the livers of rats and hamsters there is about
a 25-30% increase in transferase activity to-
ward T4 (Henry and Gasiewicz, 1987).
Recently, some investigators have sug-
gested that the explanation for the interac-
tions of TCDD with thyroid hormone levels
is that T4 and TCDD have common molecu-
lar reactivity properties that might allow
them to react with the same receptors (Mc-
Kinney et al., 1985a,b). Indeed, McKinney
and his co-workers consider that many of the
toxic effects of TCDD result directly from its
action as a thyroxine agonist. This theory
contrasts with the views of Poland's group
(Poland and Knutsen, 1982) that TCDD tox-
icity segregates with the Ah locus and in-
volves TCDD binding to the cytosolic recep-
tor. Moreover, McKinney's views are not
consistent with recent experimental results
(Potter et al., 1986), and the entire area re-
quires more research attention.
(iii) Mixed-type: Perhaps as a result of their
widespread contamination of the environ-
ment and their well-documented occurrence
in human foods, the toxicological properties
of PCBs and PBBs have received consider-
able attention (Kimbrough, 1974).
Daily feeding of commercial mixtures of
PCB (Arochlors) or PBB (Firemaster) to rats
(5, 50, and 500 ppm) led to striking dose- and
time-dependent histological changes in thy-
roid follicular cells (Collins et al., 1977; Kasza
et al., 1978). These changes included in-
creased vacuolization and accumulation of
colloid droplets and abnormal lysosomes
with strong acid phosphatase activity in folli-
cle cells. Microvilli on the lumen surface be-
came fewer in number, shortened and irregu-
larly branched, and Golgi bodies were
smaller; at higher exposures mitochondria
were swollen with disrupted cristae. It has
been suggested that the combined presence of
an abnormally large number of colloid drop-
lets and lysosomes in the follicle cells might
indicate interference with the normal synthe-
sis and/or secretion of thyroid hormones
(e.g., cleavage of active thyroxine from thyro-
globulin). PBB has been found to accumulate
preferentially in the thyroid following 20 days
of treatment and was still present 5 months
after administration (Allen-Rowlands et al.,
1981). Sequestration of PBB in the thyroid
might indicate binding to thyroidal macro-
molecules, and it has been suggested that
PBB might interfere with the organification
-------
650
HILL ET AL.
of iodide by peroxidase. More work in this
area is needed.
Instead of comprising a single layer of cu-
boidal or low columnar epithelium, the follic-
ular cells of PCB-treated animals became
more columnar with multiple layers and hy-
perplastic papillary extensions into the col-
loid. Similar follicular cell hyperplasia has
been reported in other chronic (Norris el al.,
1975) and subchronic studies (Sleight el al,
1978) with PBBs. The histological changes,
which are similar to those observed in ani-
mals treated with TSH (Seljeld el al, 1971),
were accompanied by substantially decreased
(> threefold) serum thyroxine levels in PCB-
treated rats (Collins el al. 1977). Residual
effects were observed 12 weeks after termina-
tion of exposure, probably reflecting the per-
sistent nature of the PCBs. However, it is im-
portant to note that, even in animals exposed
to the highest doses of PCBs, both the histo-
logical and functional abnormalities were re-
versible and were minimal 35 weeks after ces-
sation of treatment.
The search for a mechanistic explanation
of PCB- or PBB-induced thyroid hyperplasia
has focused on the biochemical events occur-
ring on exposure to these compounds. Direct
effects on the thyroid cannot be discounted,
and recent evidence suggests that distur-
bances in thyroid hormone synthesis and dis-
tribution may occur following long-term ad-
ministration (Byrne el al, 1987). More work
is needed in this area. However, most atten-
tion has been given to peripheral effects that
modify the distribution, metabolism, and ex-
cretion of thyroid hormones and as a conse-
quence may indirectly cause thyroid hyper-
plasia through activation of the normal feed-
back mechanism involving TSH. Thyroid
parameters changed following short-term
oral or cutaneous administration of PCBs to
rats have been extensively studied by Bas-
tomsky and co-workers (Bastomsky, 1974,
1977b; Bastomsky and Murthy, 1976; Bas-
tomsky el al, 1976) and include:
(a) Increased biliary excretion (about 5-
fold) and bile:plasma ratio (about 12-fold)
following injection of I25I-T4.
(b) Increased biliary clearance rate of
plasma 125I-T4 (more than 20-fold).
(c) Modest increase in bile flow (less than
2-fold).
(d) Decreased total serum and free T4 con-
centrations.
(e) Increased I3II uptake by thyroid.
It is apparent from these data that PCBs
have effects that are similar to both PB-type
and 3MC-type inducers. PCBs are reported
to be potent inducers of liver T4-UDP-gluc-
uronyl transferase (Bastomsky and Murthy,
1976) and, as with the 3MC-type inducers
such as TCDD, this undoubtedly accounts, at
least partially, for the increased biliary excre-
tion of T4. On the other hand, PCB also dis-
placed the thyroid hormones from their bind-
ing proteins in the serum (Bastomsky, 1974;
Bastomsky el al, 1976), an effect usually as-
sociated more with PB-type compounds. Be-
cause of its PB-like activity, it is also possible
that PCB enhances hepatic binding of T4. It
may be a combination of the induction of T4-
UDP-glucuronyl transferase and the dis-
placement from serum-binding proteins that
lead to such high bileiplasma ratios of T4 fol-
lowing PCB treatment; much smaller T4 bile:
plasma ratios are observed with compounds
like salicylate that effect displacement but not
enzyme induction (Osorio and Myant, 1963).
Conversely, the effects of changes in binding
proteins on metabolism of thyroid hormone
under steady-state conditions do not seem to
have been studied, and at least some argu-
ments can be mounted that would suggest
that no change in metabolism would occur
under those conditions.
PCBs are reportedly quite specific in their
ability to selectively induce different iso-
zymes of UDP-glucuronyl transferase. Thus,
in addition to inducing the glucuronidation
of T4, the PCB-induced isozyme(s) will also
enhance activity toward />-nitrophenol
(Ecobichon and Comeau, 1974) and 4-meth-
ylumbelliferone (Grote el al, 1975); PCB did
not enhance the glucuronidation of bilirubin,
however (Bastomsky el al, 1975).
-------
THYROID CARCINOGENESIS REVIEW
651
The effects of PCB treatment on circulating
levels of T3 are clearly different from those of
T4. It has been suggested that since T3 is more
active than T4 and because it is generated pe-
ripherally by 5'-monodeiodination of T4, T4
may be serving simply as a prohormone. It is
now generally accepted, however, that T4
does have intrinsic hormonal activity. It is of
considerable interest to note that, in constrast
to the case with T4, treatment of rats with
PCB does not result in any marked change
in total serum or free concentrations of T3.
While this may result from a number of
different factors (Bastomsky et ai, 1976), no
completely satisfactory explanation has yet
been proposed. There is some suggestion that
the relatively constant circulating levels of T3
might be due to enhanced thyroidal secretion
and enhanced peripheral conversion of T4
and T3 in response to the PCB-induced hypo-
thyroidism.
In summary, in addition to possible direct
effects on the thyroid, mixed-type inducers
such as the PCBs and PBBs have several
effects that, either alone or in combination,
reduce circulating levels of the thyroid hor-
mones and cause the pituitary to release
TSH. These are (a) an induction of T4-UDP-
glucuronyl transferase, (b) a displacement of
T4 from' serum proteins, and (c) an increase
in bile flow.
3. Inhibitors ofS'-Monodeiodinase
Certain thionamides, in addition to their
known inhibition of iodination and coupling
of tyrosine moieties into thyroid hormone,
have the ability to inhibit the peripheral con-
version of T4 to T3. This is due to effects on 5'-
iodothyronine deiodinase, a monodeidinase
which specifically removes the 5'-iodine from
substituted thyronines. The enzyme requires
a sulfhydryl-containing cofactor for activity,
and it appears that some of the thionamides
interfere with the cofactor to affect enzyme
activity (Larson, 1982b). Compounds like
thiouracil, propylthiouracil, and methylthi-
curaci! inhibit the monodeiodination of T4 to
T3 and as a result reduce urinary iodide excre-
tion, raise serum T4 levels, and reduce the
hormone effectiveness of T4 by reducing con-
version to T3. Other thionamides, like thio-
urea and methimazole, and the thiocyanate
ion do not result in reduced thyroid hormone
effectiveness (Green, 1978).
The activity of 5'-monodeiodinase can also
be reduced by competitive inhibition of the
enzyme by certain iodinated compounds like
the radiocontrast agents, iopanoic acid and
sodium ipodate, and the antiarrhythmic,
amiodarone (Borowski et ai, 1985; Larsen,
1982b). The color additive, FD&C, Red No.
3 (Peer Review Panel, 1987), may also fall
into this category.
FD&C Red No. 3 has been shown to pro-
duce thyroid tumors in dosed rats. With inhi-
bition of the 5'-monodeiodinase, treated ani-
mals under certain conditions showed ele-
vated T4, lowered or normal T3, and elevated
TSH serum levels. Also, since the 5'-mono-
deiodinase seems to metabolize rT3 to a diio-
do-derivative, inhibition of the enzyme by
Red No. 3 leads to elevated rT3 levels too
(Larsen, 1982b; Peer Review Panel, 1987).
4. Direct-Acting Chemicals and Treatment
Combinations
In addition to those chemicals that act di-
rectly upon the thyroid gland to inhibit the
synthesis of thyroid hormone or act distal to
that site to enhance thyroid hormone metab-
olism and removal from the body (see Sec-
tion IV.B for some other agents active in hu-
mans), there is a small group of compounds
that have produced thyroid tumors in experi-
mental animals that do not share these char-
acteristics. Also, several investigations have
indicated that combined treatment regimens
are associated with thyroid carcinogenic re-
sponses in excess of that produced by either
single treatment alone.
a. Direct-acting chemicals. A few com-
pounds have been identified that produce
thyroid tumors that are not known to influ-
ence thyroid-pituitary status (see Hiasa et ai,
-------
652
HILL ET AL.
1982), two of which are A-nitroso com-
pounds. Rats given eight injections of N-
methyl-A-nitrosourea (NMU) over a 4-week
period developed thyroid tumors by Week 36
without any development of goiter (Tsuda el
ai, 1983). Likewise, there was no evidence of
diffuse follicular hyperplasia in rats given a
single dose of NMU and observed at 33
weeks, even though some animals had thy-
roid neoplasms (Ohshima and Ward, 1984).
In a similar way, Ar-bis(2-hydroxypropyl)ni-
trosamine (DHPN) administration for 8
weeks led to thyroid tumors by 20 weeks
without any increase in thyroid weight (Hiasa
el ai, 1982); this observation was confirmed
in a second laboratory (Kitahori el a/., 1984).
Both nitrosamines produce tumors at sites
other than the thyroid.
The nitrosamines are a notorious group of
compounds as to their potential to produce
carcinogenic effects in multiple species fol-
lowing metabolism to reactive intermediates.
Many are genotoxic in multiple test systems
for different end effects.
b. Combined treatment studies. Although
goitrogenic stimuli that increase TSH levels
(e.g., amitrole, iodine deficiency) are known
to induce thyroid hyperplasia and neoplasia
alone, many experiments have demonstrated
an enhancement of the neoplastic response
when these treatments are combined with
other exposures. Thus, when animals are first
exposed to genotoxic physical agents (i.e., I3II
or X-rays) or chemical substances (e.g., cer-
tain nitroso compounds, 2-acetyl-aminoflu-
orene) followed by a goitrogenic stimulus,
carcinogenic responses (e.g., incidence of tu-
mor-bearing animals, multiplicity of tumors
per animal, incidence of malignancies, and
tumor latency) are greater than following sin-
gle treatments alone (see Appendix A).
Some have likened this response in the thy-
roid to the initiation-promotion (two-step)
phenomena originally described for mouse
skin. In that case, treatment with the first
agent (initiator) confers a permanent change
in cells, such that exposure (usually pro-
longed) to the second agent (promoter) re-
sults in neoplasms; reversal of treatments is
ineffective as to tumor production. Over time
it has become generally recognized that carci-
nogenesis is a multistep process that usually
includes an initiation step as well as a promo-
tional phase (OSTP, 1985).
The thyroid combined treatment studies
are consistent with the concepts of initiation-
promotion. The genotoxic agent might per-
manently alter the thyroid cell so that its ac-
centuated growth under a goitrogenic stimu-
lus would result in neoplasms. Also consis-
tent with this notion is the finding that the
effect of the initial treatment in the thyroid is
long lived. Rats can be treated with 4-methyl-
2-thiouracil (promoter) after intervals of time
at least up to 18 weeks after exposure to 2-
acetyl-aminofluorene (initiator) and still go
on to show an enhanced neoplastic response
(Hall, 1948). On the other hand, protocols
employing treatment with the "promoter"
before the "initiator" have not been con-
ducted for the thyroid. Thus, the correspon-
dence of effects in the thyroid to those in the
classical two-stage model is not established
(although they are testable).
c. Summary. Both physical and chemical
agents have been implicated in thyroid carci-
nogenesis. Ionizing radiation remains the
only confirmed carcinogenic agent for the
human thyroid, an observation corroborated
in experimental animals. Laboratory re-
search has demonstrated that many sub-
stances can directly interfere with the synthe-
sis of thyroid hormone (e.g., certain inorganic
substances, thionamides, aromatic amines).
Under conditions of reduced thyroid hor-
mone levels, the pituitary increases TSH
stimulation of the thyroid, which leads to a
predictable set of responses, including cellu-
lar hypertrophy and hyperplasia, nodular
hyperplasia and, finally, neoplasia. Pitui-
tary tumors are also sometimes increased,
seemingly due to the increased pituitary
stimulation resulting from lowered circulat-
ing thyroid hormone levels.
Direct thyroidal effect is not the only way
chemicals produce reductions in circulating
thyroid hormone. Enzyme inducers increase
the removal of thyroid hormone from the
-------
THYROID CARCINOGENESIS REVIEW
653
blood while inhibitors of 5'-monodeiodinase
block the formation of T3 from T4; in turn,
both of these result in stimulation of the pitu-
itary gland to secrete more TSH. The result,
again, of long-term exposure is hypertrophy,
hyperplasia, and eventually neoplasia. Only a
limited number of chemicals have produced
thyroid follicular tumors in animals in the ab-
sence of some antithyroid effect.
C. Structure-Activity Relationships
1. Chemicals Producing Thyroid Neoplasms
in Animals
One means of testing hypotheses concern-
ing the mechanism of follicular cell thyroid
carcinogenesis is to review those chemicals
known to produce such neoplasms in experi-
mental animals. The NCI/NTP data base is
a valuable source of information because it
consists of about 300 chemicals that have
been subject to a somewhat standard proto-
col in certain strains of rats and mice. Al-
though about half of the chemicals tested
have shown neoplastic effects at one or more
anatomical sites, only 21 chemicals have
been associated with the development of fol-
licular cell neoplasms of the thyroid (Ta-
ble 2).
These 21 compounds are not representa-
tive of the spectrum of classes of chemicals
that were tested in the bioassays. Instead
there is an overabundance of chemicals in
structural classes that are known to influence
thyroid hormone status. Over half of them
(13 of 21) are either thionamides (3) or aro-
matic amines (10), two chemical classes that
have often been linked with antithyroid activ-
ity primarily due to peroxidase inhibition.
The bulk of the remaining chemicals (7 of 21)
are complex halogenated hydrocarbons;
members of this class are often inducers of
microsomal enzymes, and at least some are
known to increase the clearance of thyroid
hormone from the blood. The remaining
chemical, an organophosphorous com-
pound. is not from a group typically linked
TABLE 2
CHEMICALS IN THE NCI/NTP BIOASSAY PROGRAM
SHOWING AT LEAST SOME EVIDENCE OF THYROID FOL-
LICULAR CELL NEOPLASIA
I Thionamides
,W,A"-Dicyclohexylthiourea
A^W-Diethylthiourea
Trimethylthiourea
2. Aromatic amines
a. Single ring
3-Amin0-4-ethoxyacetanilide
oAmsidine
hydrochlonde
2,4-DiaminoanisoIe
sulfate
HC Blue No. 1
b. Bridged double rings
4,4'-MethyIenebis(Af,A'-dimethyl)benzenamine
4,4'-Methylenedianilinedihydrochloride
4,4'-Oxydianiline
4,4'-Thiodianiline
c. Miscellaneous
C.I. Basic Red 9 monochloride
1,5-Naphthalenediamine
3. Complex halogenated hydrocarbons
Aldnn
Chlordane
Chlorinated paraffins (C,2, 60% chlorine)
Decabromodiphenyl oxide
2,3J,8-Tetrachlorodibenzo-p-dioxin
Tetrachlorodiphenylethane (p,p '-ODD)
Toxaphene
4. Organophosphous Compounds
Azinphosmethyl
to effects on the thyroid. Thus, in 20 of 21
instances, there is some basis to think that
thyroid neoplasms may be related to a reduc-
tion in thyroid hormone with a concomitant
increase in pituitary stimulation of the thy-
roid through TSH.
Although most compounds producing thy-
roid neoplasms are members of specific
chemical classes, not all members of those
groups have been shown to produce such tu-
mors. For instance, among the thionamides
tested by NCI/NTP, jV.jV'-dicyclothiourea,
A^W-diethylthiourea, and trimethylthiourea
yielded positive thyroid effects whereas sev-
eral others did not (see Table 3).
It, therefore, seems reasonable to postulate
that while a thionamide structure increases
-------
654
HILL ET AL.
TABLE 3
THIONAMIDES NEGATIVE FOR THYROID NEOPLASIA FN NCI/NTP STUDIES
1. 2,5-Dithiobiurea
S = C
\
NH
I
NH
NH2
2. Lead dimethyldithiocarbamate
N(CH3)
s = c'
S~
3. l-Phenyl-2-thiourea
'\
NIL
4. Sodium diethyldithiocarbamate
N(C2H5)2
'\
Na*
5. SulfaUate
N(C2H5),
S = C
\
6. Tetraethylthiuram disulfide
/N(C2H5)2
S = CN
S — C
"N(C2H5)2
the chance that a chemical will produce thy-
roid tumors in long-term animal tests, struc-
ture alone is not sufficient in itself to generate
such activity. The same is true for certain aro-
matic amines (see Section HI.C.2.b).
2. Antithyroid Activity and Thyroid Carcino-
genesis
Given that many of the chemicals produc-
ing thyroid tumors in the NCI/NTP series
come from chemical classes known to pro-
duce antithyroid effects by inhibition of thy-
roid peroxidase, a review was made of specific
thionamides and aromatic amines to see if
antithyroid activity was a prerequisite for thy-
roid carcinogenic activity. The hypothesis
was borne out for the thionamides and at
least some of the aromatic amines.
Generally, the criteria for selecting the spe-
cific chemicals required that they had been
(1) tested for animal carcinogenicity (NCI/
NTP or IARC review) and (2) evaluated for
antithyroid activity. However, in some cases
a chemical had been studied for carcinogenic-
ity, but not antithyroid activity. In those
cases, structurally related compounds that
had been tested for antithyroid activity were
chosen to act as surrogate indicators of a
compound's antithyroid potential.
Antithyroid activity has been measured for
a number of chemicals in rats and, to some
extent, in humans. For rats, chemicals were
administered orally at different doses for 10
days. Iodine concentrations in the thyroid
were measured, and from the dose-response
curve the dose that reduced the iodine con-
centration to a standard level was estimated
(EDc). For comparison, the dose of thiouracil
(a well-studied antithyroid agent) that re-
duced iodine concentration to the same level
was also estimated (EDt). Antithyroid activ-
ity was expressed as the ratio of the estimated
dose of thiouracil relative to that for the
chemical (EDt/EDc), where thiouracil (in
this review) is given a value of 100 (Astwood
etal., 1945;McGintyandBywater, 1945a,b).
For humans, antithyroid activity for a
chemical was again measured against the
-------
THYROID CARCINOGENESFS REVIEW
655
TABLE 4A
THIONAMIDES: RELATIONSHIP BETWEEN ANTITHYROID ACTIVITY AND THYROID CARCINOGENICITY
Heterocyclic compounds
Relative
antithyroid activity
(thiouracil = 100)
Neoplasms'*
Thyroid
Rat*
Human'
Rat
Mouse
Other sites
1 . 2-Thiouracil
NH— CH
CH
100
100
Mouse-liver
2. 6-Methylthiouracil
.Nil— C
CH3
NH-C
O
3. 6-n-Propylthiouracil
C,H7
NH-C
S-C CH
O
4. Ethylene thiourea
S = C
/NH —CH2
\
NH—CH,
100
100
1100
75
40
50
Mouse-liver and pituitary
Mouse-pituitary
Mouse-liver
° From IARC reviews.
* Astwood « a/. (1945).
' Stanley and Astwood (1947).
d Mouse study did not examine thyroid.
' McGinty and Bywater (1945a).
/Not tested = n.
effects of thiouracil (value = 100 for this re-
view) (Stanley and Astwood, 1947). Subjects
were given 13II by mouth, and iodine in the
thyroid was monitored externally by Geiger-
Muller measurement. After 1 to 2 hr, the
chemical was given orally, and the influence
of the agent on the further time course uptake
of radioactivity into the gland was evaluated.
The degree to which accumulation was
affected was graded depending upon the com-
pleteness and duration of inhibition. Usually
chemicals were studied at two or more doses.
a. Thionamides. For the heterocyclic
thionamides there is strong support for the
premise that there may be a correlation be-
tween a chemical's ability to induce thyroid
tumors and its ability to significantly inhibit
iodine localization in the thyroid of rats and
humans (Table 4A). For the thiourea-like thi-
onamides (Table 4B), namely thiourea, tri-
methylthiourea, and jV.JV-diethylthiourea,
relative antithyroid activities of about 10 or
more were associated with thyroid tumor in-
duction. In keeping with a correlation be-
-------
656 HILL ET AL.
TABLE 4B
THIONAMIDES: RELATIONSHIP BETWEEN ANTITHYROID ACTIVITY AND THYROID CARCINOGENICITY
1
2.
3.
Thiourea derivatives
. Thiourea
yNH2
NNH2
, Trimethylthiourea
XN — (CH3)2
NNH— CH3
N, N '-Diethyllhiourea
XNH-C2H5
Relative
antithyroid activity
(thiouracil= 100) Neoplasms"
Rat Thyroid
ABH MB' Human' Rat Mouse Other sites
12 9 100 + -t- Rat-liver, head, face
Mouse-skull
10 n' n +
40 47 n +
4. 2,5-Dilhiobiurea
S = C
NH
I
NH
NH,
5. Tetraethylthiuram disulfide
S = C
N(C2H5)2
s = c
6. Tetramethyllhiurarn disulfide
,N(CH3)2
S —C
s
I
s^c/S
XN(CH3)2
7. l-Phenyl-2-thiourea
s-c
s-c
14
-------
THYROID CARCINOGENESIS REVIEW
TABLE 4B—Continued
657
Relative
antithyroid activity
(thiouracil = 100)
Neoplasms"
Rat
Thyroid
Thiourea derivatives
ABH* MB' Human' Rat Mouse
Other sites
8. N,N'-Dicyclohexylthiourea
s = c:
9. l,3-Diethyl-l,3-diphenyl
thiourea
C2H5
,N-< O
S = C
N,,®
C2H5
" From NCI studies, except thiourea (IARC review).
* Astwood et al (1945).
' Stanley and Astwood (1947).
•* Mouse study did not examine thyroid.
' McGinty and Bywater(1945a).
1 Not tested = n.
tween these effects, 2,5-dithiobiurea and tet-
raethylthiuram disulfide (with its structural
analog, tetramethylthiuram disulfide) both
lacked antithyroid activity and did not pro-
duce thyroid neoplasia.
On the other hand, two other chemicals in
the series of thiourea-like compounds need
clarification. In the case of l-phenyl-2-thio-
urea, a relative antithyroid value of 14 was
found in rats, but the long-term NCI study in
rats and mice was negative for thyroid tumors
or thyroid hyperplasia. There was an absence
of any toxic manifestations in dosed rats in
the long-term study and a question whether a
maximum tolerated dose had been used. In
addition, after 78 weeks of chemical adminis-
tration, dosed animals were observed for an
additional 26 weeks in rats and 13 weeks in
mice before termination. Since thyroid hy-
perplasia is often times reversible, it is possi-
ble that any lesions produced by dosing may
have regressed during the observation period.
Other investigators have reported thyroid hy-
perplasia after 6 weeks of phenylthiourea ad-
ministration to rats (Richter and Clisby,
1942), indicating that the chemical may in-
duce thyroid neoplastic effects under certain
conditions. Further work on this compound
may bear this out.
In the second case, TV.yV-dicyclohexylthio-
urea showed increased incidences of thyroid
follicular hyperplasia in dosed rats and mice
in the NCI study, and there were some in-
creases in follicular cell carcinomas in male
rats. Although N, W-dicyclohexylthiourea has
not been tested for antithyroid activity, its
structural analog, l,3-diethyl-l,3-diphenyl-
thiourea, failed to show significant antithy-
roid effects in the rat.
b. Bridged double ring aromatic amines.
Like the tnionamides, certain aromatic amines
with double rings attached by a simple ether-
-------
658
HILL ET AL.
TABLE 5
AROMATIC AMINES: RELATIONSHIP BETWEEN ANTITHYROID ACTIVITY AND THYROID CARCINOGENESIS
Neoplasms"
Bridged double ring compounds
Relative antithyroid
activity: rat
(thiouracil = 100) Rat Mouse
Thyroid
Other sites
1. 4,4'-Methylenedianiline dihydrochloride
2. 4,4'-MethyIenebis(N,A'-dimethyl)benzenamine
(CH3)iN-/ O V-CHj-/ O \-N(CH3)2
3. 4,4'-Thiodianiline
/ \
-NH,
NH2
NH2—( O >—S-
4, 4,4'-Oxydianiline
5. 4,4'-Sulfonyldianiline
O
/ \
NH-
6. Michler's ketone
NH,—( O
O )—NH2
7. 4,4'-Diaminodiphenylsulfoxide
O
NH2—( O
O V-NH,
8. 4,4'-Methylenebis(2-chloroaniline)'
Cl
Q
2-/ O \— CHj-Y O V-
9. 4,4'-Methylenebis(2-methylaniline)/'
NH
NH2—( O V- CH2—( O
Nil,
CH,
CH,
25"
25"
15C
4'
12'
+ + Mouse-liver
Rat-liver
Mouse-liver
+ + Mouse-liver
Rat-liver
n n
Mouse-liver, harderian
gland
Rat-liver
Rat-mesenchymal
Mouse-liver
Mouse-liver, vascular
Rat-liver, lung
n Rat-liver
° NCI/NTP bioassay except for last two chemicals in table.
*Astwood eial. (1945).
' McGinty and Bywater (1945b).
d Not tested.
' McGinty and Bywater (1946a).
^IARC review of carcinogenicity.
-------
THYROID CARCINOGENESIS REVIEW
659
like bridge show a correlation between anti-
thyroid activity and thyroid carcinogenesis
(Table 5). 4,4'-Methylenedianiline, 4,4'-meth-
ylenebis (N,N' - dimethyl) benzenamine, and
4,4'-thiodianiline (chemicals No. 1 through 3,
respectively) show both attributes, and al-
though 4,4'-oxydianiline (No. 4) has not been
tested for antithyroid activity, it has close
structural similarity with the other three
chemicals and also produces thyroid neo-
plasms. In keeping with its potential for anti-
thyroid effects, chemical No. 4 produced in-
creases in the number of TSH-secreting cells
in the pituitary in rats following chronic ad-
ministration (Murthy et al., 1985), and
chemicals No. 4 and No. 1 both produced
thyroid enlargements in the NCI 90-day pre-
chronic studies. All of these observations—
antithyroid activity, thyroid enlargement in
subchronic studies, and increases in the cell
types of the pituitary that secrete TSH—are
consistent with the hypothesis that bridged
ring aromatic amines induce thyroid neo-
plasms by reducing circulating thyroid hor-
mone levels and increasing TSH.
Other compounds in this series show re-
sults that are hard to interpret. 4,4'-Sulfonyl-
dianiline (No. 5), which has an -SO2- bridge
between the rings, had a low antithyroid
value of 4 in rats and was negative for thyroid
tumors. Compound No. 6 with a -C(O)-
bridge was also negative for thyroid tumors.
Although chemical No. 7, which has an
-S(O)- bridge, was negative for thyroid neo-
plasms, it was associated with an antithyroid
value of 12 in the rat. Antithyroid values in
the 10 to 15 range have been linked with posi-
tive thyroid tumorigenic effects for chemical
No. 3 and some of the thionamides, e.g., thio-
urea. Further studies on antithyroid activity
may help to clarify this inconsistency.
It is also interesting to note that com-
pounds structurally identical to 4,4'-methy-
lenedianiline (No. 1), except for substitution
on the rings in the 2,2'-positions (chemicals
Nos. 8 and 9), are negative for thyroid tu-
mors. It would be interesting to measure their
antithyroid activity.
In summary, for both the thionamides and
bridged double ring aromatic amines there
appears to be support for concluding that
there is a good relationship between antithy-
roid activity and thyroid carcinogenesis, al-
though further work needs to be done to be
able to interpret some results. It seems possi-
ble that agents that are known to inhibit thy-
roid hormone output may be potential thy-
roid carcinogens under certain experimental
conditions.
c. Characteristics of single ring aromatic
amines. Many single ring aromatic amines
have been evaluated for carcinogenicity in
experimental systems and have shown posi-
tive effects (Clayson and Garner, 1976;
Weisburger et al., 1978; see review by La-
venhar and Maczka, 1985), but only a few of
them have produced neoplasms in the thy-
roid. Of the single ring compounds that have
been tested by NCI/NTP (Appendix B), o-an-
isidine (No. 1), 2,4-diaminoanisole (No. 2),
3-amino-4-ethoxy-acetanilide (No. 3), and
HC Blue No. 1 (No. 9) were the only ones to
produce thyroid neoplasms. Of these agents
only 2,4-diaminoanisole produced thyroid
tumors in all four species-sex categories; the
others produced such tumors in only one
group.
The single ring aromatic amines have not
been examined systematically as to their anti-
thyroid activity; therefore, these agents can-
not be analyzed as to the relationship be-
tween peroxidase inhibition and thyroid car-
cinogenesis. However, from a preliminary
review of structural analogs that have been
tested for carcinogenicity (Appendix B), there
is little indication that specific ring substitu-
tions are influencing thyroid carcinogenic po-
tential.
3. Genotoxicity and Thyroid Carcinogenesis
It has been generally accepted by the scien-
tific community that mutagenesis plays a role
in carcinogenesis. In the case of thyroid follic-
ular cell tumors, however, it has been sug-
gested that a hormonal feedback mechanism
-------
660
HILL ET AL.
TABLE 6
GENOTOXICITY DATA FOR THIONAMIDES
1.
2.
Chemicals positive for thyroid tumors
A^TV'-Dicyclohexylthiourea
A',A''-Diethylthiourea
Trimethylthiourea
Chemicals negative for thyroid tumors
1 -Phenyl-2-thiourea
2,5-Dithiobiurea
Tetraethylthiuram disulfide
Sulfallate
Lead dimethyldithiocarbamate
Sodium diethyldithiocarbamate
Chromosomal
Gene mutations effects
SA ML SLRL CA SCE
~ ~~ n — +
+
_
- u n + +
n n - +
+ n +
+ n n n n
+ u - + +
+ n
Note. SA, Salmonella reverse mutation; ML, mouse lymphoma L5178Y cell thymidine kinase locus; SLRL, sex-
linked recessive lethal in Drosophila; CA, chromosomal aberrations in CHO cells; SCE, sister chromatid exchange in
CHO cells; -, negative result; +, positive result; n, not tested; w, weak positive result; ?, equivocal result; /, results
from two or more laboratories; u, under test by NTP.
involving increased output of thyroid-stimu-
lating hormone from the pituitary gland in
response to low thyroid hormone levels may
be operating (Woo et al, \ 985; Paynter et al,
\ 986). Even though hormone imbalance may
play a role in thyroid carcinogenesis, it is also
important to evaluate the mutagenic poten-
tial of agents causing these tumors.
This section explores the relationship be-
tween the induction of thyroid neoplasms in
rodents and their outcome on several short-
term tests of genotoxicity. If the hypothesis
that TSH plays a significant role in thyroid
carcinogenesis is true, one might expect that
chemicals producing thyroid tumors in ex-
perimental animals would not show geno-
toxic potential in any predictable way. If,
instead, thyroid carcinogenesis was largely
due to chemical reactivity and not to hor-
monal derangement, then thyroid carcino-
gens might be genotoxic agents.
This review largely draws upon those com-
pounds that were tested in rats and mice for
carcinogenicity by NCI/NTP and produced
thyroid neoplasms. Structurally related com-
pounds that did not produce thyroid tumors
are included for comparison. The genotoxicity
data on these chemicals are from the NTP,
much of which has not been published in peer-
reviewed journals and at least some of which
could be considered preliminary in nature.
Chemicals are divided into structural
classes: thionamides, aromatic amines, and
halogenated hydrocarbons. The NTP short-
term test data on many compounds are lim-
ited and, therefore, are hard to interpret. In
order to get a better appreciation of the spec-
trum of genotoxic effects that may occur
among members of a chemical class, two
compounds, ethylene thiourea and 4,4'-oxy-
dianiline, were considered in detail (using the
open literature) as examples of thionamides
and aromatic amides, respectively. An exam-
ple of the halogenated hydrocarbon class was
not included, since members of this group
generally show little indication of genotoxic
potential. A third compound, amitrole, was
also included for detailed review; it does not
belong to any of the above chemical classes,
but it is recognized as being an inhibitor of
thyroid peroxidase as are certain thionamides
and aromatic amines.
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THYROID CARCINOGENESIS REVIEW
661
TABLE 7
GENOTOXICITY DATA FOR SINGLE RJNG AROMATIC AMINES
Gene mutations
SA
ML
SLRL
Chromosomal
effects
CA
SCE
1. Chemicals positive for thyroid tumors
3-Amino-4-ethyloxyacetanilide
o-Anisidine hydrochlonde
2,4-Diaminoanisole sulfate
HC Blue No. 1
2. Chemicals negative for thyroid tumors
/j-Cresidine
5-Nitro-o-anisidine
;>-Anisidine
2,4-Dimethoxyaniline hydrochloride
m-Phenylenediamine
p-Phenylenediamine hydrochloride
2-Nitro-/>-phenyIenediamine
n
n
w
Note SA, Salmonella reverse mutation; ML, mouse lymphoma L5178Y cell thymidine kinase locus; SLRL, sex-
linked recessive lethal in Drosophila; CA, chromosomal aberrations in CHO cells; SCE, sister chromatid exchange in
CHO cells; -, negative result; +, positive result; n, not tested; w, weak positive result; ?, equivocal result; /, results
from two or more laboratories; u, under test by NTP.
a. Thionamides. For the three chemicals
tested by NCI/NTP that were positive for thy-
roid tumors, the existing information gives
little indication of significant genotoxic po-
tential (Table 6). Of 14 chemical test compar-
isons on these agents for both gene mutation
and chromosomal effects, there are only two
positive responses. There appears to be
slightly more positive genotoxicity data in the
case of thionamides that tested negative for
thyroid follicular cell tumors (10 of 19 tests)
than for those that tested positive. However,
no firm conclusions can be drawn from this
limited data set.
The genotoxicity of ethylene thiourea, a
compound known to produce thyroid tu-
mors, was assessed in greater detail (see Ap-
pendix C). Although it was concluded from
the journal articles that there is evidence for
genotoxicity when ethylene thiourea is sup-
plemented with sodium nitrite (Salmonella
with metabolic activation, in vivo cytogenet-
ics, dominant lethal, micronucleus), presum-
ably via the formation of N-nitrosoethylene
thiourea, there is much less evidence for the
genotoxic potential of ethylene thiourea it-
self. The compound shows little indication of
gene mutation activity: negative to weakly
positive effects in bacteria, negative in Dro-
sophila, and conflicting information in yeast
and mammalian cells in culture (negative in
CHO cells and divergent results in mouse
lymphoma cells). Chromosomal effects are
not demonstrated in cells of higher eukary-
otes in culture or in vivo. DNA damage tests
showed conflicting results in bacteria, yeast,
and human cells in culture.
In contrast to the effects listed above, sev-
eral thionamides are positive for in vitro
transformation. Thiourea, /V.jV'-dicyclohex-
ylthiourea, and ethylene thiourea have
shown positive effects in Syrian hamster cells
(SHE and BHK), and the first two also trans-
formed rat embryo cells (Rauscher murine
leukemia virus-infected) (Heidelberger et al.,
1983; Styles, 1981; Daniel and Dehnel,
1981). However, these three chemicals and
Ar,./V'-dietnylthiourea were reported negative
in simian adenovirus-7-infected Syrian ham-
ster and rat cells (Heidelberger et al., 1983).
-------
662
HILL ET AL.
TABLE 8
GENOTOXICITY DATA FOR BRIDGED DOUBLE RING AROMATIC AMINES
Gene mutations
SA
ML
SLRL
Chromosomal
effects
CA
SCE
Chemicals positive for thyroid tumors
4,4'-Methylenedianilinedihydrochloride
4,4'-Methylenebis(A',yV-dimethyl)benzenamine
4,4'-Thiodianiline
4,4'-Oxydianiline
Chemicals negative for thyroid tumors
Michler's ketone
4,4'-Sulfonyldianiline
Note. SA, Salmonella reverse mutation; ML, mouse lymphoma L5178Y cell thymidine kinase locus; SLRL, sex-
linked recessive lethal in Drosophila; CA, chromosomal aberrations in CHO cells; SCE, sister chromatid exchange in
CHO cells; -, negative result; +, positive result; n, not tested; w, weak positive result; ?, equivocal result; /, results
from two or more laboratories; u, under test by NTP.
In sum, the lack of genotoxic effects noted
with the thionamides that produced thyroid
tumors in the NCI/NTP studies is borne out
by the detailed review of ethylene thiourea.
There is little indication of gene mutation or
chromosomal effects. There are conflicting
results with the DNA damage tests and in vi-
tro transformation.
b. Aromatic amines. Unlike thionamides,
the class of aromatic amines commonly dem-
onstrates genotoxic effects for both point mu-
tations and chromosomal effects (Tables 7,8,
and 9). This is the case for chemicals that pro-
duced thyroid tumors as well as for analogs
that did not.
The genotoxic potential of 4,4'-oxydiani-
line was evaluated in more detail using infor-
mation from the published literature (Appen-
dix D) to supplement that generated by NTP
(Table 3). It is concluded that it is a frame-
shift and perhaps base-pair substitution mu-
tagen in Salmonella that requires metabolic
activation for an effect to be noted. In keeping
with its mutagenic effects on bacteria, 4,4'-ox-
ydianiline also produced gene mutations,
chromosome aberrations, and sister chroma-
TABLE 9
GENOTOXOCITY DATA FOR MISCELLANEOUS AROMATIC AMINES
Chemicals positive for thyroid tumors
C.I. basic red 9 monochloride
1 ,4-Naphthalenediamine
Gene mutations
SA ML SLRL
+/? +/? n
+ n n
Chromosomal
effects
CA
n
SCE
+
n
Note. SA, Salmonella reverse mutation; ML, mouse lymphoma L5178Y cell thymidine kinase locus; SLRL, sex-
linked recessive lethal in Drosophila; CA, chromosomal aberrations in CHO cells; SCE, sister chromatid exchange in
CHO cells; -, negative result; +, positive result; n, not tested; w, weak positive result; ?, equivocal result; /, results
from two or more laboratories; u, under test by NTP.
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THYROID CARCINOGENESIS REVIEW
663
tid exchanges (SCE) in cultured mammalian
cells. However, SCE are not increased in vivo,
and two DN A damage assays in vivo gave dis-
cordant results. In vitro transformation stud-
ies were generally positive. Thus, the analysis
of 4,4'-oxydianiline confirms the suspicion
from Tables 7 through 9 that aromatic
amines are genotoxic agents.
c. Complex halogenaled hydrocarbons.
For the class of halogenated hydrocarbons
there are a few scattered positive genotoxicity
results (3 out of 16 chemical-test compari-
sons among the agents producing thyroid tu-
mors) (Table 1 0), although many compounds
have not been well characterized as to gene
mutations and chromosomal effects. Other
than toxaphene, all compounds are negative
in the Salmonella test. Structural analogs that
have not produced thyroid tumors also show
a paucity of genetic responses (7 positives
among 1 7 comparisons). No firm conclusion
can be drawn on these compounds because
the data are limited but, in general, it appears
that complex halogenated hydrocarbons fail
to demonstrate much genotoxic potential.
d. Amitrole. Amitrole has not been investi-
gated by the NTP concerning its carcinoge-
nicity, but from other long-term animal stud-
ies it is known to produce thyroid, pituitary,
and liver tumors (see Paynter et ai, 1986).
Like the thionamides and aromatic amines,
amitrole inhibits thyroid peroxidase. Al-
though it lacks the thiol group of thionam-
ides, it does show some structural similar-
ity ( an R grouping), as illustrated with the
— N— C— N—
comparison with thiourea.
NH2
H
N-NH
S=C
\
H2N— C
NH2
\
= CH
Thiourea Amitrole
Gene mutation testing of amitrole has
spanned prokaryotes, yeast, insects, and
mammalian cells in culture (Appendix E).
Many replications of bacterial testing in Sal-
monella and E, coli have almost uniformly
failed to demonstrate mutagenic effects,
which led a review group to declare amitrole
negative (see Bridges et ai, 1981). Point mu-
tation tests in Saccharomyces and Drosophila
were also negative (positive in one case; see
Appendix E). Test results in mammalian cells
in culture have been conflicting, with con-
firmed negative results in mouse lymphoma
cells but positive effects in one laboratory for
two different loci in Syrian hamster embryo
cells. Thus, submammalian testing indicates
little concern about point mutations, whereas
results in mammalian cells are positive in
Syrian hamster but not mouse cells.
Testing for chromosomal effects includes
evaluation of numerical aberrations, struc-
tural aberrations, and sister chromatid ex-
change. Negative results have been obtained
in yeast and insect nondisjunction systems
and in mammalian cells in culture. Two in
vivo mouse micron ucleus assays, which can
give some indication of numerical chromo-
some aberrations, were also negative.
Tests for structural chromosome aberra-
tions have been uniformly negative and in-
clude the following: human lymphocytes in
culture, mouse bone marrow cytogenetics,
and mouse micronucleus and dominant le-
thal tests.
An increase was reported in the frequency
of SCE in CHO cells in culture in two studies;
a negative response was recorded in a third
study in the same cells.
DNA damage tests have been performed
on bacteria, fungi, and mammalian cells in
culture. Of six bacterial tests, five were re-
ported as negative. Thus, there is little indica-
tion in bacteria of a DNA-interactive effect.
Two of six DNA damage tests in Saccharo-
myces were positive. One such test in Asper-
gillus gave a weak positive reaction.
Increases in unscheduled DNA synthesis
have been reported in human cells. For HeLa
cells, a positive dose-response effect for ami-
trole was noted in the presence of rat liver S9;
no such increase was noted in the absence of
-------
664 HILL ET AL.
TABLE 10
GENOTOXICITY DATA FOR COMPLEX HALOGENATED HYDROCARBONS
Chromosomal
Gene mutations effects
SA ML SLRL CA SCE
I . Chemicals positive for thyroid tumors
Aldrin
Chlordane
s
(r)
n
(t)
n n
(r)
n
(r)
— + n — +
Chlorinated paraffins (Ci2,60% chlorine) — n n n n
Decabromodiphenyl oxide - - n - —
2,3,7,8-Tetrachlorodibenzo-p-dioxin - - - — —
p,p'-Tetrachlorodiphenylethane(p,p'-DDD) - n n u u
Toxaphene + n n n n
2. Chemicals negative for thyroid tumors
Dieldrin - + n - +
Heptachlor — u n + +
Chlorinated paraffins (C23, 43% chlorine) — n n n n
PBB mixture (FiremasterFF-1) - - n _ _
p,;?'-Dichlorodiphenyldichloroethylene(/>,p'-DDE) - + +/- - vv
Note. SA, Salmonella reverse mutation; ML, mouse lymphoma L5178Y cell thymidine kinase locus; SLRL, sex-
linked recessive lethal in Drosophila; CA, chromosomal aberrations in CHO cells; SCE, sister chromatid exchange in
CHO cells; s, selected for testing by NTP; r, reagent grade; t, technical grade; -, negative result; +, positive result; n,
not tested; w, weak positive result; ?, equivocal result; /, results from two or more laboratories; u, under test by NTP.
exogenous activation (Martin and McDer- lustrates that thyroid carcinogenesis is not uni-
mid, 1981). Also, amitrole was reported in an formly tied to genotoxicity. Thionamides (and
abstract to be positive in human EUE cells; amitrole) and complex halogenated hydrocar-
the conditions of the study were not given. bons demonstrated only limited indication of a
Lastly, several positive studies have been genotoxic potential, whereas aromatic amines
reported for in vitro transformation in Syrian regularly showed positive short-term test re-
hamster and rat embryo cells, which argue for suits. Emphasis on this point is gained from re-
some type of genotoxic effect, view of structural analogs from these classes
In sum, there is limited evidence for the that did not produce thyroid tumors; their out-
genotoxicity of amitrole. This effect is proba- come on the tests was basically similar to that
bly not mediated through mutagenic mecha- of the thyroid carcinogens. Thus, thyroid car-
nisms: there is no indication of the produc- cinogens do not show a consistent response on
tion of chromosomal mutations and, at best, genotoxicity tests.
the point mutagenic evidence is inconclusive. If we look at chemical classes as to their in-
There are indications, however, that under fluence on thyroid peroxidase, we again fail
some circumstances amitrole produces to see a consistent pattern as to their genotox-
DNA-damaging effects. These results are aug- icity. Chemicals from within the thionamides
mented by confirmed positive responses in in and aromatic amines (as well as amitrole) are
vitro transformation. Thus, there is support known to inhibit thyroid peroxidase. How-
for amitrole having a weak DNA-interactive ever, the reviewed thionamides (and ami-
or genotoxic effect that probably does not in- trole) are generally not genotoxic, whereas
volve mutation per se. the amines are active. Thus, genotoxicity is
e. Conclusion. The review of three chemical not correlated with functional activity on per-
classes demonstrating thyroid carcinogenesis il- oxidase.
-------
THYROID CARCINOGENESIS REVIEW
665
It is well recognized that aromatic amines
are often carcinogenic in animals and that
many means are available within organisms
to activate these structures to reactive inter-
mediates that have genotoxic potential. To
the extent that certain aromatic amines also
inhibit thyroid peroxidase, it seems possible
that such agents may have two means to in-
fluence thyroid carcinogenesis: to induce
DNA damage and to increase the output of
TSH from the pituitary.
Although the remarks made in the previ-
ous paragraph are representative impressions
of the data on chemical classes as a whole,
they certainly do not necessarily apply to any
one chemical within a class. Many times
chemicals give a smattering of positive and
negative results. In other cases, such as with
the thionamides and amitrole, the evidence
indicates a general lack of activity for some
end points (e.g., gene mutations and chromo-
somal aberrations), but the potential pres-
ence for other effects (e.g., in vitro transfor-
mation). Each of these cases makes it difficult
to reach an all-inclusive position on genotox-
icity. Still, within the limits of the present re-
view, there does not seem to be a consistent
relationship across chemical classes that pro-
duce thyroid tumors as to their ability to pro-
duce genotoxic effects.
different approaches and merging data from
clinical observations, studies of clinical popu-
lations, and epidemiologic studies.
Currently, the only verified cause of thy-
roid cancer in humans is X-irradiation (Ron
and Modan, 1982; NCRP, 1985), and this
finding is well documented in experimental
animals. There are conflicting data in hu-
mans bearing on an association of iodine de-
ficiency and thyroid cancer, unlike the case
in animals where the association is well estab-
lished. In contrast to the situation in animal
studies, no studies follow a single human
population directly through the sequence
from exposure to chemical substances or ini-
tiation of some other treatment through hy-
perplasia and eventually to neoplasia. Conse-
quently, the information on humans must be
analyzed in separate steps, describing the role
of certain treatments on the development of
hyperplasia and then describing risk factors
or antecedent conditions for thyroid neopla-
sia. The combination of these two analyses al-
lows one to make some inferences about the
overall comparability of animal models and
humans regarding thyroid carcinogenesis.
A. Thyroid-Pituitary Function
IV. HUMAN DATA ON THYROID
HYPERPLASIA AND NEOPLASIA
The goals of this section are to compare hu-
man and animal information bearing on thy-
roid physiology, disruption of thyroid func-
tion, and development of hyperplasia (goiter)
and neoplasia. As has been related, it has
been well established by long-term experi-
ments in animals that certain chemical sub-
stances and other treatments cause thyroid
hyperplasia that will progress to neoplasia.
While evaluation of laboratory experiments
garners useful information on likely pro-
cesses in humans, verification of this for hu-
man thyroid carcinogenesis requires evaluat-
ing the weight of evidence from several
It is widely accepted that the pituitary-thy-
roid axis and the nature, body handling, and
function of thyroid hormones and TSH are
quite similar in experimental animals and
humans. For instance, in a review of thyroid
function in humans, Larsen (I982a) pre-
sented clinical data on the feedback regula-
tion of thyrotropin secretion by thyroid hor-
mones and the tissue conversion of T4 to T3
that is basically like that in experimental ani-
mals. Recent evidence, however, helps to
point out some of the differences that may ex-
ist between animals and humans. For in-
stance, in the rat there is active conversion of
T4 to T3 which then regulates TSH produc-
tion, whereas in humans circulating T3 may
play a more dominant role (Fish et ai, 1987).
-------
666
HILL ET AL.
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THYROID CARCINOGENESIS REVIEW
667
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-------
668
HILL ET AL.
B. Causes of Thyroid Hyperplasia
Animals and humans respond similarly to
a number of treatments that disrupt thyroid
function such as (1) a lack of dietary iodide,
(2) blockage of the iodide transport mecha-
nism (ionic inhibitors), (3) interference with
the synthesis of thyroid hormone (peroxidase
inhibition), (4) suppression of thyroid activ-
ity by high concentrations of iodide, (5) en-
hanced peripheral metabolism of thyroid
hormones, and (6) damage to the thyroid
gland by ionizing radiation (see also Section
III of this report; Oilman and Murad, 1975;
Green, 1978; Paynter et al., 1986; De Groot
and Stanbury, 1975; Meyers et al., 1976).
Each of these can lead to goiters in humans.
1. Chemical Inhibitors
Several examples of chemical substances
that influence thyroid status in humans are
summarized in Table 11 to illustrate the na-
ture of the effects. The agents include such
things as thyroid peroxidase inhibitors (e.g.,
ethylene thiourea, sulphonylureas, resor-
cinol), a cation (lithium), an organiodide
(amiodarone), and inducers of mixed func-
tion oxidases (phenobarbital, PBB). In each
case exposures result in reduction in circulat-
ing thyroid hormone levels and in some cases
elevated TSH levels or goiters. These re-
sponses are like those seen in animals.
Because the data base varies among the
chemicals, a summary of supporting refer-
ences, including those reported in the study,
is included in a separate column entitled
"data base." For example, the goitrogenic
effect in humans of sulfonyl ureas and of ami-
odarone has been reported in several clinical
studies. Differences in quantitative value of
the results among studies are to be expected
because of differences in health status, age,
sex, and dietary factors. In some studies these
factors are controlled (patients of similar age)
or evaluated in the analysis (sex differences).
The value of a case report in support of the
hypothesis is strengthened if cessation of
treatment with the putative goitrogen or
other agent is followed by a return of thyroid
function tests to normal. These temporal as-
sociations are important in assessing the evi-
dence for the association because subjects are
exposed to other drugs or possible confound-
ing factors. This information, which is impor-
tant in assessing the strength of the evidence,
is summarized in the table column titled
"Temporal." Prospective clinical studies pro-
vide valuable information because subjects
are euthyroid prior to exposure.
Other observations point out the compara-
bility of response in humans as in animals. In
hypothyroid animals the cells of the pituitary
enlarge and become "thyroidectomy cells"
(Baker and Yu, 1971) and, according to some
authors, may undergo hyperplasia and finally
neoplasia (see Section II.B). Indirect studies
in humans also demonstrate some of these
findings. The bony covering of the human pi-
tuitary, the sella turcica, normally enlarges
with age up to about 20 years and then re-
mains essentially constant in size. Enlarge-
ment in the sella turcica beyond normal lim-
its is noted in cases of hypothyroidism, and
there is an inverse relationship between the
blood levels of thyroid hormones and sella
size and a direct one between TSH levels and
size of the sella turcica (Yamada et al., 1976;
Bigos et al., 1978). It is interesting to note that
there are also a few clinical reports linking
chemical hypothyroidism and pituitary ade-
nomas, and at least some of them appear to
be TSH-secreting tumors (e.g., Samaan et al.,
1977; Katz et al., 1980; see review by Balsam
and Oppenheimer, 1975), although the case
is not established with any certainty.
2. Dietary Factors
Much of the human investigations of dis-
ruption in thyroid function following envi-
ronmental modifications have come from the
study of populations where there are dietary
changes, namely deficiency of iodide and the
consumption of foods containing goitrogenic
substances.
-------
THYROID CARCINOGENESIS REVIEW
669
a. Iodine deficiency. The most striking
pattern of the geographic distribution of pop-
ulations with goiter is attributed to deficiency
of iodine in the diet as a result of low environ-
mental iodine levels. Endemic goiter has oc-
curred throughout the world, particularly in
mountainous areas such as the Alps, Himala-
yas, and Andes, and in the United States in
areas around the Great Lakes. De Groot and
Stanbury (1975) cite the report of thyroid hy-
perplasia in domestic goats and in wild ro-
dents in endemic areas of iodine deficiency
in the Himalayas, which again points out the
similarity of response among mammals. Goi-
ter incidence has been virtually eliminated in
the United States and Europe by the intro-
duction of iodized salt (Williams et a/., 1977;
De Groot and Stanbury, 1975; Hedinger,
1981).
Several arguments support iodine defi-
ciency as a cause of goiter: (1) there is an in-
verse correlation between iodine content of
soil and water and the appearance of goiter in
the population; (2) metabolism of iodine and
TH and TSH status in patients with this dis-
order fits the pattern expected and is reversed
with iodine prophylaxis; and (3) there is a
sharp reduction in goiter prevalence with io-
dine prophylaxis (Williams et al., 1977; Hed-
inger, 1981).
Iodine deficiency in humans can result in
profound thyroid hyperplasia. Goiters up to
5 kg (a 100-fold increase in weight) have been
observed in iodine-deficient areas as a com-
pensatory response to inability to synthesize
thyroid hormone. Generally, the impairment
in hormone synthesis is overcome in time
and the individual becomes clinically euthy-
roid, even in the presence of some derange-
ment in T4 and TSH levels. Often in goitrous
populations repeated cycles of hyperplasia
and involution occur which can lead to mul-
tinodular goiter. In contrast to the hyperplas-
tic goiter, multinodular goiters do not regress
upon administration of iodine. Likewise, thy-
roid hormone usually has no effect on long-
standing goiters (Ingbar and Woeber, 1981).
Adenomatous hyperplasia is a less common
cause of nodularity but is significant because
it is difficult to distinguish from neoplasia,
thus complicating the assessment of the asso-
ciation between hyperplasia and neoplasia.
As will be developed later in this section, it
does not appear that thyroid cancer is a major
problem arising from iodine-deficient goiters,
in contrast to the observations in experimen-
tal animals which indicate that tumors fre-
quently arise under iodine-deficient condi-
tions.
b. Other goilrogens. Observations of goiter
distribution suggest that factors other than io-
dine deficiency could be important. The inci-
dence of goiter varies within the population
in endemic areas, and the severity is not uni-
form among all inhabitants; these suggest the
presence of risk factors in addition to iodine
deficiency. Although it is considered unlikely
that natural goitrogens in food are a primary
cause of goiter in humans, variability in re-
sponse within endemic areas has led some to
conclude (De Groot and Stanbury, 1975) that
"natural goitrogens acting in concert with io-
dine deficiency may determine the pattern
and severity of goiter."
As discussed before, the thionamide, goi-
trin, with antithyroid activity in animals and
in humans, has been isolated from certain
cruciferous foods (e.g., turnips). It exists nat-
urally as progoitrin, an inactive thioglyco-
side, which is hydrolyzed in vivo to goitrin.
Human data exist to illustrate the thyroid-
inhibiting effect of the monovalent hydrated
anion, thiocyanate (TCN), and of cyanogenic
glucosides that are hydrolyzed in the body to
thiocyanate. TCN blocks the uptake of iodide
into the thyroid. Chemicals that are metabo-
lized to thiocyanates are found in seeds of the
plants of the genus Brassica, in Cruciferae,
Compositae, and Umbelliferae. These in-
clude cabbage, kale, brussel spouts, cauli-
flower, turnips, rutabagas, mustard, and
horseradish. The effect was established in
man as a result of clinical use of potassium
thiocyanate (Oilman and Murad, 1975).
It has been assumed, therefore, that eating
foods producing the thiocyanate ion or goi-
trin contributes to endemic goiter. De Groot
and Stanbury (1975) cite studies in Australia,
-------
670
HILL ET AL.
Finland, and England that suggest cattle have
passed these goitrogens to humans through
milk. Progoitrin has been detected in com-
mercial milk in goitrous regions of Finland,
but not in nongoitrous regions. Seasonal de-
velopment of goiter in school children has
been related to milk from cows fed kale (De
Groot and Stanbury, 1975).
Several dietary items that are staples in
some cultures contain cyanogenic glucosides.
These include cassava, sorghum, maize, and
millet. In its raw form, cassava contains toxic
levels of cyanogenic glucoside, and although
much of it is removed by pounding and soak-
ing, poorly detoxified cassava is a suspected
cause of goiter in Central Africa.
Recent studies in Africa contribute more
direct evidence to support an interactive
effect of TCN (or cyanogenic glucosides) and
a diet low in iodine. In an iodine-deficient re-
gion of the Sudan where goiter prevalence
may reach 55%, the frequency of large goiters
is higher in rural than in urban areas (Eltom
et al, 1985). The predominant staple food in
rural Darfur is millet. Rural subjects with goi-
ters had statistically significantly higher levels
of TSH and T3 and lower levels of T4 and free
T4 index than urban subjects with goiters. Se-
rum TCN was significantly higher in rural
subjects, but the elevated levels of urinary
TCN did not reach statistical significance.
The urinary iodine excretion, a reflection of
quantity of iodine ingested, was not signifi-
cantly different between the two groups.
These results are consistent with the hypothe-
sis that TCN overload in conjunction with io-
dine deficiency causes more severe thyroid
dysfunction than iodine deficiency alone. Ev-
idence of a possible effect has also been re-
ported in North Zaire in Central Africa in
children with iodine deficiency (Vanderpas el
al, 1984).
C. Causes of Thyroid Cancer in Humans
Epidemiologists search for clues to causes
of disease and to factors that increase an indi-
vidual's risk of disease (risk factors) by exam-
ining descriptive data or designing analytic
studies. Descriptive data consist of morbid-
ity, mortality, or incidence rates of diseases
in population groups. Incidence rates (newly
diagnosed cases in a population over a given
time period) reveal patterns of disease by age,
race, sex, ethnic group, and geographic lo-
cale. These rates and their changes over time
and space identify high risk groups and pro-
vide indirect evidence for causes of disease.
Associations between host factors and disease
are hypothesized.
Analytical epidemiology consists of case
control, often termed retrospective, and co-
hort or prospective studies. These studies per-
mit greater control of confounding factors
and an opportunity to link exposure and re-
sponse information in individuals. Thus, evi-
dence for causes of disease is more direct.
As a result of descriptive and analytic epi-
demiologic data, radiation is a well-docu-
mented cause of thyroid cancer in humans
(Schottenfeld and Gershman, 1978; Ron and
Modan, 1982). Incidence rates for thyroid
cancer rose roughly twofold between the
1940s and the 1970s for persons under age
55. The change in pattern coincides with ad-
ministration of X-ray for various medical
treatments and is consistent with the hypoth-
esis that ionizing radiation is a cuase of thy-
roid cancer in children and young adults.
Childhood irradiation was observed more of-
ten in thyroid cancer cases than controls. Ron
and Modan (1982) summarize eight epidemi-
ologic studies of populations exposed to X-
ray therapy, atomic bomb explosions, and
fallout from nuclear weapons testing.
The epidemiologic approach to investigat-
ing whether hyperplasia (goiter) leads to thy-
roid cancer in humans is to (1) examine de-
scriptive data, (2) compare the cancer rates
between endemic goiter areas and goiter-free
areas, (3) examine time trends for thyroid
cancer after prophylactic measures (iodine
supplementation) reduce endemic goiter fre-
quency in a given area, and (4) evaluate
whether goitrous individuals have a greater
risk of thyroid cancer or whether thyroid can-
cer cases have a more frequent history of hy-
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THYROID CARCINOGENESIS REVIEW
671
perplasia and nodules than controls. These
steps are summarized in the sections below.
1. Descriptive Epidemiology
Variations in cancer incidence rates by
country and race may be studied to evaluate
the role of host and environmental factors on
disease. Despite the striking geographic pat-
terns for goiter, no similar trends are detected
for incidence of thyroid carcinomas in the ar-
eas for which cancer incidence data are avail-
able. It is one of the rarest and generally least
virulent carcinomas, and although it has in-
creased somewhat in recent decades, purport-
edly because of medical radiation exposure,
it is not considered a major public health
problem (Ron and Modan, 1982).
For several countries, thyroid cancer shows
rising age-adjusted incidence rates with age
and consistently higher rates for women than
men, particularly in young adults. Rates for
males range from 0.6 to 5 per 100,000 and for
females from 1.2 to 16 per 100,000. Varia-
tions by country are relatively small com-
pared with that for other cancer sites (about
10-fold) and are not consistently related to ge-
ography or race. The highest age-adjusted
rates in females (1967-1971) were for Hawai-
ians in Hawaii (16/100,000), Iceland (16.3/
100,000), and Israeli Jews(8.3/100,000)(Wa-
terhousee/a/., 1982).
The incidence of thyroid cancer detected
clinically shows interesting distinctions from
prevalence of occult thyroid cancer detected
at autopsy. At autopsy, thyroid carinoma is
equally frequent in men and women, and
high rates have been diagnosed in popula-
tions that have unremarkable clinical rates of
thyroid cancer (Shottenfeld and Gershman,
1978). These observations have led these au-
thors and others to hypothesize that the host
and environmental factors that enhance the
development of clinically detected thyroid
cancer are different from those that incite tu-
morigenesis.
Experimental evidence in several labora-
tory species demonstrates that iodine defi-
ciency, certain chemicals, and other causes of
prolonged TSH stimulation result in thyroid
enlargements and eventually thyroid tumors.
In the absence of such information in hu-
mans other studies need to be conducted to
get some handle on human thyroid carcino-
genesis.
Much of the work on the relationship be-
tween goiter and thyroid cancer has focused
on populations differing in iodine intake,
since iodine deficiency (endemic goiter) has
been and still remains a major health prob-
lem in various parts of the world. Numerous
reviews of the subject have been written
which conclude that past studies are conflict-
ing about the role of goiter in thyroid carcino-
genesis(e.g., Alderson, 1980;Hedinger, 1981;
Riccabona, 1982). Doniach (I970a) reviews
much of the information available to that
time and questions the link between endemic
goiter and thyroid cancer development.
In geographical epidemiologic studies, thy-
roid cancer rates are compared in geographi-
cal areas with different goiter rates. Wegelin
(1928) compared the frequency of thyroid
cancer in an autopsy series in five areas. The
largest percentage with thyroid cancer oc-
curred in Berne, Switzerland, an area where
goiter was highly endemic. The lowest per-
centage of cancer appeared in Berlin where
endemic goiter was rare. Other geographic
correlation studies have followed, yet reports
have been conflicting. For example, no corre-
lations were found in reports from Australia
and Finland (Alderson, 1980; Ron and Mo-
dan, 1982), and Pendergrast (1961) found no
associated increase in the cancer rates in goi-
ter areas in the United States compared with
nongoiter areas. Hedinger (1981) cites inci-
dence statistics that show no decline in fre-
quency of thyroid malignancies despite the
virtual elimination of goiter by iodine pro-
phylaxis. On the other hand, Wanner et al.
(1966) did show a positive correlation when
they compared the incidence of thyroid can-
cer in Cali, Colombia, an endemic goiter
area, to similar data in New York state and
Puerto Rico. Thyroid cancer rates for both
-------
672
HILL ET AL.
sexes were about three times higher in Co-
lombia than in the other two sites.
Several reasons may account for differing
study outcomes. Some of the correlations are
based on reports of high thyroid cancer rates
generated from pathology studies of surgery
cases, and are likely to suffer from a selection
bias because thyroid disease suspected of car-
cinogenicity is likely to be referred to surgery
(De Groot and Stanbury, 1979). Different
causes of cancer may result in different histo-
pathological types of thyroid cancer. In the
United States, in particular, radiation-in-
duced cancer associated with therapy in
childhood could have masked a decrease as-
sociated with iodine prophylaxis. After the
introduction of iodized table salt in Switzer-
land and the decreasing incidence of goiter,
thyroid cancer rates remained stable but an
increasing proportion of thyroid cancers were
classified as papillary (Shottenfeld and Gersh-
man, 1977). Therefore, the conflicting data
cited above are inconclusive and difficult to
interpret.
Recent geographical studies consider the
histological type of thyroid cancer. In Cali,
Colombia, an endemic goiter area, at least
90% of the follicular and anaplastic cancer
specimens showed evidence of goiter,
whereas about 50% of the papillary tumors
were associated with goiter (Wahner et al.,
1966). These results suggest some relation-
ship between goiter and the histological type
of cancer.
In Zurich, Switzerland before the advent of
iodine supplementation, few of the tumors
were papillary (7.8%), whereas after that time
the proportion of papillary cancers among
the total increased (33.4%) while the propor-
tion of follicular and anaplastic tumors de-
creased (Hedinger, 1981; Riccabona, 1982).
Since papillary cancers have the best progno-
sis and anaplastic the worst, with follicular in-
termediate, these results suggest that thyroid
cancer in endemic goiter regions may be asso-
ciated with more aggressive forms of cancer.
Further evidence of a relationship between
iodine intake (from inadequate to hypernor-
mal) and the form of thyroid cancer comes
from a review of thyroid cancer cases coming
to surgery in Northeast Scotland, a region
with average iodide intake, and Iceland, an
island with very high iodide intake (Williams
et al., 1977). Persons from Iceland have un-
usually small thyroid glands, high concentra-
tions of iodide in plasma and the thyroid
gland, and low plasma TSH levels. Papillary
cancer incidence was about fivefold higher
and the proportion of papillary cancers
among the total was greater in Iceland than
in Scotland (71% vs 54%). Offsetting the
difference in papillary cancers, the propor-
tion of follicular tumors was comparable in
the two groups, but anaplastic cancers were
more common in Scotland than Iceland (19%
vs 10%).
In contrast to the above studies suggesting
some relationship between iodide intake and
the form of thyroid cancer in humans, others
fail to support this hypothesis. For instance,
Waterhouse et al. (1982) report that the rela-
tive frequencies of the major histological
types for several countries show the highest
proportion of follicular carcinoma in Sao
Paulo, Brazil, Bombay, India, and Zaragoza,
Spain—all areas not noted for endemic goi-
ter. The highest proportion of papillary carci-
noma was reported from all North America
cancer registries and from Hawaii, Israel, and
Singapore. In addition to noting the potential
for disagreement in diagnoses among experi-
enced pathologists, the authors conclude that
the significance of these differences is unclear.
Therefore, geographic correlations with and
without histology data are inconclusive and
do not show a consistent relationship be-
tween endemic goiter areas and thyroid can-
cer rates.
Probably the most profound disruptions in
thyroid functioning occur in cases of familial
goiter where there are inherited blocks in thy-
roid hormone production (Stanbury et al.,
1979). When left untreated, these patients de-
velop profound hyperplasia and nodular (be-
nign tumor) changes, but only a very few
cases have gone on to develop thyroid carci-
noma (see review by Vickery, 1981). Like
with endemic goiter, it appears that the en-
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THYROID CARCINOGENESIS REVIEW
673
TABLE 12
EPIDEMIOLOGIC STUDIES OF THYROID CANCER AND ITS RELATIONSHIP TO GOITER AND THYROID NODULES
Odds ratio (95% confidence
limits)"
Goiter
Thyroid nodules
Comment
Ref.
4.5(1.6-12.2)* 8.7(1.6-47.5)*
10.5(2.5-44.8)'
5.6(1.0-41)'' 33 (4.5-691)*
Women aged 18-80
White women aged 15-40
Adjusted for age, sex, and prior radiation
exposure
McTiernan el al. (1984)
Preston-Martin et al. (1987)
Ron et al, (1987)
" Odds ratio estimates risk of disease with the trait (or exposure) compared to risk without the trait. Confidence
limits that overlap 1.0 are not significant.
* Data for those unexposed to radiation. The risk for all cases was goiter 6.6 (2.8-15.6) and nodules 12.0 (2.3-63.8).
c Presence of goiter or benign nodules.
•* These data are from univariate analysis. The odds ratios of a multiple logistic regression adjusted for age and sex
were thyroid nodules (28.0) and goiter (3.8) (not significant).
larged thyroids in these patients do not often
undergo malignant transformation; this con-
trasts with the findings in long-term animal
studies where blocks in thyroid production
regularly lead to thyroid cancer.
Although not much seems to have been
done concerning the follow-up of patients
with Graves' disease (hyperthyroidism) as to
thyroid cancer development, the little that
has been done (a follow-up of 30,000 pa-
tients) suggests there may not be a significant
thyroid cancer problem in these cases (Do-
byns et al., 1974; see also Doniach, 1970a).
[One very small study of Graves' patients sug-
gested a higher than expected frequency of
thyroid cancer (Shapiro et al., 1970).] The
reason Graves' patients may be at risk is the
finding that many of the persons carry immu-
noglobulms in their blood which bind to the
TSH receptor on thyroid cells and, at least in
vitro, act like TSH to stimulate DNA synthe-
sis and cell division (Valente et al., 1983; Tra-
montane et al., 1986b). Since these patients
frequently have enlarged thyroid glands, one
cannot help but think that the immunoglob-
ulins may stimulate thyroid cell division in
vivo as well. A small number of cases of thy-
roid cancer in Graves' disease have recently
been reported (Filettie? al., 1988).
The single investigation of Graves' disease
patients treated with antithyroid agents (i.e.,
thionamides) for at least I year failed to show
any thyroid cancers in over 1000 patients
(Dobyns et al., 1974). Again, this suggests
that at least circumscribed use of antithyroid
drugs is not attended with a marked thyroid
cancer risk. It should be pointed out, how-
ever, that the goal of antithyroid treatment
for Graves' disease is to bring patients into
euthyroid and not a hypothyroid status where
increases in TSH may occur. Thus, the fol-
low-up of treated case of Graves' disease dees
not provide significant evidence to impugn or
acquit antithyroid agents.
In the case of Hashimoto's thyroiditis, a
common condition considered to be an auto-
immune disorder, patients commonly have
high circulating levels of TSH (Larsen,
1982b). The clinical impression is that the
only association between this disease and thy-
roid cancer is with the thyroid lymphoma
and not follicular cell carcinoma (Woolner,
1959).
2. Analytical Epidemiology
Of all the various types of data on humans
from which causal associations can be in-
ferred, the strongest evidence is derived from
-------
674
HILL ET AL.
analytical epidemiology—cohort or case-
control studies—that evaluate data on indi-
viduals and suitable controls. Analytical epi-
demiologic studies have helped to establish
ionizing radiation as a cause of thyroid cancer
(Ron and Modan, 1982).
Three case-control studies of thyroid carci-
noma in the United States have recently been
completed which evaluated risk factors for
cancer, including preexisting thyroid disease
(Table 12). These studies were designed to
test a potential hypothesized role of endoge-
nous female hormones in thyroid cancer.
Hormonal factors are suspected as a cause of
thyroid cancer because of the consistently
higher rates in females and the peak occur-
rence in females at between ages 15 and 29
when hormonal activity is enhanced (Hen-
derson et al, 1982; Ron and Modan, 1982).
Each study showed significant increases in
thyroid nodules and goiters among thyroid
cancer patients.
McTiernan el al. (1984) studied 183
women aged 18 to 80 located from a popula-
tion-based cancer surveillance system and
394 controls. The two groups had similar
family history, weight, and smoking habits.
The most common confounding factor in the
analyses was age; therefore, relationships
were adjusted to five age groups.
History of goiter for individuals unexposed
to radiation showed a statistically significant
and high odds ratio (OR) equal to 4.5. Fur-
ther analysis of preexisting goiter by histo-
pathological type resulted in an OR = 16.4
for follicular compared with 3.3 for papillary
cancer. Radiation exposure doubled the risk
for those with papillary histology, but did not
change the risk for follicular. Thyroid nod-
ules were also a statistically significant ante-
cedent in those unexposed to radiation (OR
= 8.7) and were strongly related to papillary
or mixed papillary-follicular thyroid cancer.
There are some potential biases in the Mc-
Tiernan el al. (1984) study such as recall bias,
relatively low ascertainment rate (65%), the
lack of reevaluation of the histopathology,
and the reliance on telephone interviews
rather than medical history. However, it is
doubtful that these could be the cause of asso-
ciations of the magnitude noted.
Preston-Martin el al. (1987) conducted a
case-control study in which they questioned
110 female cases aged 15 to 40 and an equal
number of matched controls. Diagnoses of
cases were histologically confirmed, and thy-
roid disease was recorded if a physician was
consulted at least 2 years prior to the cancer
diagnosis. Statistically significant risk factors
were found for thyroid enlargement as an ad-
olescent (OR = 10) and any goiter or benign
nodules (OR = 10.5). The odds ratio of any
thyroid disease was 14.5. The small number
of cases of follicular carcinoma prevented
analysis by histological type.
Ron el al. (1987) also found increased risk
with parity as well as increased risk with goi-
ter and nodules. This case-control study in-
cluded 159 cases (109 female and 50 male)
ascertained through a cancer registry and 318
controls from the general population. A re-
view of the pathology was included. Thyroid
nodules were evaluated separately from goi-
ter and had a far greater risk (OR = 33) com-
pared with goiter (OR = 5.6); both were sta-
tistically significant. The authors offer as ca-
veats the fact that thyroid disease status was
not medically verified and the response rate
was only 62%.
In conclusion, these three recent case-con-
trol studies in the United States consistently
showed thyroid cancer strongly related to
preexisting goiter and to thyroid nodules (Ta-
ble 12). There is insufficient evidence to iden-
tify a quantitative difference in this relation-
ship between follicular or papillary tumor
types. One concern is that the association be-
tween thyroid disease and thyroid cancer
may be increased as a result of closer medical
attention; after all, there must have been
some clinical indication that the patients may
have had a thyroid neoplasm prior to the time
of surgery (like the presence of a nodule in the
gland). In addition, the criteria used to define
goiter were never defined in the studies. How-
ever, the consistency among studies, the
-------
THYROID CARC1NOGENESIS REVIEW
675
strength of the association, and the consis-
tency with established causes (e.g., in all stud-
ies, ORs were increased with radiation)
strongly support the hypothesis that thyroid
nodules and, to a lesser degree, goiter are risk
factors (potential causes) of thyroid cancer in
humans. It should be pointed out, however,
that in the two studies that were analyzed for
an association between hypothyroidism and
thyroid cancer, neither showed a relationship
(McTiernane/a/., 1984; Ron et ai, 1987).
In summary, there is considerably less sup-
port for a role for TSH in thyroid carcinogen-
esis in humans than in experimental animals.
To the extent TSH pertains, humans appear
to be less sensitive to its effects than animals.
APPENDIX A
COMBINED TREATMENT STUDIES PRODUCING THYROID TUMORS
Test animal
Treatment A
Treatment B
Results
Ref.
Wistar rat
(female)
AAF (2.5 mg MTU (0. 1 g/liter
gavage, 4-6x in drinking
for 1 week) water up to 2 1
weeks)
Combined treatment Hall (1948)
showed multiple
adenomas/gland. MTU
alone caused hyperplasia
or single tumors. AAF
stated as having no tumor
effect
Combined treatment
showed multiple
adenomas when interval
between treatments
extended for 4- 1 8 weeks.
Lister rat (male
and female)
Lister rat (male
and female)
AAF(100mg/
liter in drinking
water for 1 3
months)
mI(30MCi, ip)
MTU ( 1 g/liter in
drinking water
for 1 3 months
concurrent with
AFF)
MTU ( 1 g/liter in
in drinking
water for 1 5
months)
Wistar rat (male)
Wistar rat (male)
X-rays (300 rad to
neck)
DHPN (70 mg/
100 g body wt
given sc once/
week for 4 or 8
weeks)
MTU(1 g/liter in
drinking water
for 15-18
months)
Amitrole (2000
ppm in diet for
12 weeks)
Combined treatment
showed more adenomas/
gland than single
treatment groups.
Combined treatment
produced more
adenomas/gland and
malignancies not seen in
single treatment groups.
Combined treatment
increased incidence of
tumor-bearing animals
and malignancies that
were not seen with single
treatments.
Amitrole after 4 weeks of
DHPN-induced thyroid
adenomas at 91 % and
carcinomas at 9%. No
tumors with DHPN or
amitrole alone.
Amitrole accelerated
development of
Doniach(1950)
Doniach(1953)
Christov(1975)
Hiasa el al.
(1982a)
-------
676
HILL ET AL.
APPENDIX A.—Continued
Test animal
Treatment A
Treatment B
Results
Ref.
Wistar rat (male)
DHPN (70 mg/
100 g body wt
given sc once/
week for 4 or 6
weeks)
PB (500 ppm in
diet for 12
weeks)
BB (500 ppm in
diet for 12
weeks)
Wistar rat (male)
DHPN (single sc
dose of 280 mg/
100 g body wt)
PB (500 ppm in
diet for 6, 12, or
19 weeks)
Wistar rat (male)
DHPN (single sc
dose of 280 mg/
PTU( 1500 ppm
in diet for 19
adenomas and increased
carcinomas after 8 weeks
of DHPN (no amitrole:
58% adenomas, 18%
carcinomas; with
amitrole: 100%
adenomas, 42%
carcinomas). No tumors
with amitrole alone.
PB after 4 weeks of DHPN-
induced thyroid
adenomas at 66% and
carcinomas at 10%. No
tumors with DHPN or
PB alone.
PB after 6 weeks of DHPN-
accelerated development
of adenomas and induced
carcinomas (no PB: 23%
adenomas, no
carcinomas; with PB:
100% adenomas, 25%
carcinomas; no tumors
with PB alone).
PB after 4 weeks of DPHN-
induced thyroid
adenomas (23%) but no
carcinomas. No tumors
with BB alone.
BB after 6 weeks of DHPN-
accelerated development
of adenomas and induced
a small number of
carcinomas (no BB: 23%
adenomas, no
carcinomas; with BB:
45% adenomas, 10%
carcinomas; no tumors
with BB alone).
PB for 12 or 19 weeks after
DHPN-enhanced
development of thyroid
adenomas. PB for 19
weeks after DHPN-
induced thyroid
carcinomas at 12%. Not
seen with DHPN alone.
PB alone produced no
tumors.
PTU after DHPN-
enhanced development
Hiasa et al.
(1982b)
Hiasa et al. (1983)
Kitahori et al.
(1984)
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THYROID CARCINOGENESIS REVIEW
APPENDIX A—Continued
677
Test animal
Treatment A
Treatment B
Results
Ref.
100 g body wt)
weeks)
Wistar rat (male)
DHPN(singleip
dose of 280 mg/
lOOgbodywt)
MDA(1000ppm
in diet for 19
weeks)
F344/NCr rat
(male)
NMU (single iv
dose of 41.2
mg/kg body wt)
Iodine-deficient
diet after 2
weeks until 20
or 33 week
F344/NCr rat
(male)
NMU (single iv
dose of 41.2
mg/kg body wt)
Iodine deficiency
after 2 weeks
until 52 and 77
week
of thyroid follicular cell
adenomas and induced
carcinomas (no PTU:
19% adenomas, 0%
carcinomas; with PTU:
100% adenomas, 52%
carcinomas). PTU alone
produced no tumors.
MDA after DHPN-
enhanced development
of thyroid tumors and
induced carcinomas (no
MDA:28% tumors, 0%
carcinomas; with MDA:
90% tumors, 9.5%
carcinomas). MDA alone
produced no tumors.
Iodine deficiency after
NMU-enhanced
development of thyroid
follicular cell adenomas
and carcinomas (NMU
alone: 10% adenomas at
20 weeks and 70%
adenomas at 33 weeks,
10% carcinomas at 33
weeks; NMU with iodine
deficiency: 100%
adenomas at 20 weeks
and 100% carcinomas at
33 weeks; no tumors
following iodine
deficiency alone).
Iodine deficiency after
NMU-enhanced
development of the
thyroid follicular cell
carcinomas (NMU alone:
32% carcinomas at 52
weeks; NMU with iodine
deficiency: 90% at 52
weeks).
Iodine deficiency alone
induced mostly thyroid
adenomas and a few
carcinomas (40%
adenomas at 52 weeks,
60% adenomas at 77
weeks, and 10%
carcinomas at 77 weeks).
Hiasaera/. (1984)
Ohshima and
Ward (19861
Ohshima and
Ward (1984)
-------
678
HILL ET AL.
APPENDIX A—Continued
Test animal
Treatment A
Treatment B
Results
Ref.
Wistar rat
(female)
NMU(40mg/kg
body wt by
gavage for 3
days)
F344 rat (female)
NMU (single iv
dose of 50 mg/
kg body wt)
F344 rat (male)
NMU (20 mg/kg
ip2x/wk for 4
weeks)
MTU [ 1 g/liter in
drinking water
from 4 weeks
after NMU
until death (60
week)
PTU(3, 10, and
30 mg/literin
drinking water)
I3'I(1 and 10MCI)
PB (0.05% in diet
for 32 weeks)
Combined treatment
resulted in appearance of
thyroid follicular cell
adenomas (within 13
weeks) and carcinomas
(after 16 weeks) that
metastatized to the lung
(after 30 weeks). No
single treatment groups
were included, and the
fate of untreated controls
was not described.
PTU after NMU-induced
development of thyroid
adenomas and
carcinomas (NMU alone:
no tumors; with 3 mg/
liter PTU: 17%
adenomas, 23%
carcinomas; with 10 and
30mg/literPTU: 100%
carcinomas). No PTU
alone group was
included.
No thyroid tumors.
PB after NMU-induced
thyroid papillary
carcinomas. NMU alone
did not induce tumors.
PB was not tested alone.
Schaffer and
Muller(1980)
Milmore et al.
(1982)
Tsuda el al.
(1983)
Note. AAF, 2-acetylaminofluorene; MTU, 4-methyl-2-thiouracil; DHPN, A'-bis(2-hydroxyprooyl)nitrosamine;
amitrole, 3-amino-1,2,4-triazole; PB, phenobarbital; BB, barbital; PTU, propylthiouracil; MDA, methylenedianiline;
NMU, Ar-methyl-Ar-nitrosourea.
APPENDIX B: SINGLE RING AROMATIC AMINES
Several structurally related, single ring aro-
matic amines have been tested for carcinoge-
nicity and are illustrated in the accompany-
ing table. Of the 11 structural analogs, only
o-anisidine (No. 1), 2,4-diaminoanisole (No.
2), 3-amino-4-ethoxyacetanilide (No. 3), and
HC Blue No. 1 (No. 9) were positive for thy-
roid tumors.
Although the first three chemicals share
amino and methoxy substituents in the or-
tho position on the ring, other tested chemi-
cals with this conformation (No. 4, No. 5)
did not produce thyroid tumors. Both
chemicals, No. 2 and No. 3, have amino
groups in the meta position on the ring;
however, compound No. 8, which also has
this configuration, lacked thyroid tumor ac-
tivity. Chemicals No. 2 and No. 3 also
shared amino and methoxy groups in the
para positions; compounds No. 6 and No.
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THYROID CARCINOGENESIS REVIEW
679
7 with these constituents were negative for logs No. 10 and No. 11 failed to show this
thyroid tumors. Likewise, for HC Blue No. response. Thus, it is not readily apparent
1 (No. 9), which showed a thyroid tumor re- which, if any, substitutions on the ring may
sponse in the NTP bioassay, structural ana- impact thyroid tumor activity.
STRUCTURE-ACTIVITY RELATIONSHIPS AMONG CHEMICALS TESTED BY THE NCI/NTP
Thyroid tumors
Rat Mouse
Other tumors
Rat
M F M F M
Mouse
M
1. o-Amsidine
NH2
/ O \- OCH3
2. 2,4-DiaminoanisoIe
NH,
NH,-/ O V- OCH3
3. 3-Amino-4-ethoxyacetaniIide
NH'
,
CHj— C— NH— / O V-OCH
4. p-Cresidine
NH,
CH3— ( O V- OCH3
- Bladder Bladder
Kidney
+ + + + Skin Skin
Liver Liver
Bladder Bladder
__ ~*_ 4- —
Bladder Bladder
Nasal Nasal
Liver -
Liver
Bladder Bladder
- Liver
5. 5-Nitro-o-anisidine
NH,
NO,—/ O V- OCH,
6. p-Anisidine
NH.
O V-OCH,
- - - - Skin Skin
Zymbal Zymbal gland -
Clitoral gland Liver
-------
680
HILL ET AL.
APPENDIX B—Continued
Thyroid tumors
Rat Mouse
Other tumors
Rat
M F M F
M
Mouse
M F
7. 3,4-Dimethoxyaniline
OCH,
8. m-Diphenylenediamine
NH2
/(
NH2—< O
9. HC Blue No. 1
(HOCH2—CH2)2N
10. />-Phenylenediamine
NO,
— — + — Liver
O V-NH— CH3
-NIL
NH.
11. 2-Nitro-p-phenylenediamine
N02
NH.
O
-NH,
Lung
Liver Liver
APPENDIX C
GENOTOXOCITY: ETHYLENE THIOUREA
Reported effect
Ref.
1. Gene mutations
A. Bacteria
Salmonella (Ames)
G46
G46
W-nitrosoethylenethiourea
Multiple strains
(-NOJ)
(+NOJ)
Mouse/rat host mediated G46
(-NOJ)
(+N02)
Multiple strains
w
+
Seiler(1974)
Seiler(1977)
Shirasu«a/. (1977)
+ TA 1530only Schupbach and Hummler( 1977)
-------
THYROID CARCINOGENESIS REVIEW
681
APPENDIX C—Continued
Reported effect
Ref.
Mouse host mediated G46, TA 1530
Multiple strains
TA 1950
(-NOD
(+NOJ)
Mouse host mediated (TA 1950)
(-NOJ)
(+NOJ)
Multiple strains
Mouse host mediated (TA 1950)
(-N02)
(+N02)
Multiple strains/replications in different labs
Multiple strains/replications in different labs
E. co/i
WP2
(-NOJ)
(+N02)
WP2
B. Eukaryotic microorganisms
Saccharomyces(XV 185-14Q
Schizosaccharomyces
C. Higher eukaryotes
Mouse lymphoma cells (TK)
Mouse lymphoma cells
Chinese hamster ovary (several loci)
Drosophila XLRL
Drosophila XLRL
Dfosophila XLRL
Chromosome effects
A. Numerical aberrations
Saccharomyces mitotic aneuploidy
Mouse micronucleus (see B, below)
B. Structural aberrations
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Mouse micronucleus (B6C3F1)
Mouse micronucleus (ICR)
Mouse micronucleus (CD-1)
Mouse micronucleus
(-NaNO2)
(+NaNON2)
Mouse micronucleus
Mouse dominant lethal
Mouse dominant lethal
Mouse dominant lethal
(+NaNO2) preimplantation loss
postimplantation loss
Chinese hamster bone marrow
+ TA 15 30 only
+ in all
w
+
wTA 1535 only
wTAI535
- all others
+ requires S9
- injection
? feeding
Anderson and Styles (1978)
Autioe/a/. (1982)
Moriynetal. (1983)
Braune/a/. (1977)
Mortelmans el al. (1986)
Bridges etal. (1981)
Shirasuera/. (1977)
Mehta and von Borstel (1981)
Loprieno(1981)
Jotz and Mitchel( 1981)
NTP(1986)
Carver el al. (1981)
Valencia and Houtchens (1981)
Woodruff^ al (1985)
NTP(1986)
Parry and Sharp (1981)
Shirasu«fl/. (1977)
Nastaranjan and van Kesteren-
vanLeeu wen (1981)
NTP(1986)
Salamone
-------
682
HILL ET AL.
APPENDIX C—Continued
Reported effect
Ref.
(+NaNOz)
Rat bone marrow
Drosophila reciprocal translocation
C. Sister chromatid exchanges
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Mouse wviVo(CBA/J)
3. DNA damage
B. subtilis (rec)
E. coli (pol A)
£. coli (rec)
E. coli (rec, pol A)
E. coli (pol A)
E. coli (lambda induction)
Saccharomyces mitotic cross-over
Saccharomyces mitotic gene conversion
Saccharomyces mitotic gene conversion
Saccharomyces (JDI) mitotic gene conversion
Saccharomyes (RAD) differential growth
Unscheduled DNA synthesis WI-38 cells
Human fibroblasts
Mouse sperm morphology
Mouse sperm morphology
4. In vitro transformation
Baby hamster kidney (BHK 21)
Baby hamster kidney (BHK 21)
Syrian hamster embryo, adenovirus infected
(SHE-SA7)
w without S9
- with S9
+ with S9
w without S9
- with S9
+ without S9
Seiler(1977)
Shirasu«a/. (1977)
NTP(1986)
Evans and Mitchel (1981)
Nastaranjan and van Kesteren-
van Leeuwen(1981)
Perry and Thomson (1981)
NTP(1986)
Paika etal. (1981)
Kada(1981)
Green (1981)
Ichinotsubo « a/. (1981)
Tweats(198l)
Rosenkranz el al. (1981)
Thomson (1981)
Kassinova « a/. (1981)
Jagannath « a/. (1981)
Zimmemann and Scheel (1981)
Sharp and Perry (1981 a)
Sharp and Perry (1981 b)
Robinson and Mitchell (1981)
Agrelo and Amos (1981)
Wyrobeke/a/. (1981)
Tophan(1980)
Daniel and Dehnel (1981)
Styles (1981)
Hatch et al. (1986)
Note. +, positive; w, weak positive; ?, equivocal; -, negative.
APPENDIX D
GENOTOXICITY: 4,4'-OxvDiANiLiNE
Reported effect
Ref.
1. Gene mutation
A. Bacteria
Salmonella (Ames)
TA98
TA 100
TA98
TA100
TA98
4- requires S9
+ assayed only in
presence of S9
w requires S9
+ requires S9
+ requires S9
Lavoieetal. (1979)
Varo&etal. (1981)
Tanakaefa/. (1985)
-------
THYROID CARC1NOGENES1S REVIEW 683
APPENDIX D—Continued
Reported effect Ref,
TA 100
TA97
TA98
TA 100
TA 1535
TA1537
+ requires S9
+ requires S9
+ requires S9
+ with or without S9
+ requires hamster S9
+ assayed only with S9;
requires hamster S9
NTP(1987)
(personal communication
E, Zeiger)
B. Eukaryotes
Mammalian cells in culture
Mouse lymphoma + NTP (1986)
Chromosome effects
Chinese hamster ovary cells
Structural chromosome f NTP (1986)
aberrations
Sister chromatid exchanges +
Rat bone marrow
Sister chromatid exchanges - Parodi et al. (1983)
DNA damage
Unscheduled DNA synthesis
(rat hepatocytes)
In vivo - Mirsalis el al (1983)
In vitro -
In vitro transformation
Syrian hamster embryo cells ? Tuetal. (1986)
Enhancement of virus-infected + Hatch et al. (1986)
transformation of Syrian
hamster embryo cells
Note. +, positive; w, weak positive; ?, equivocal; -, negative.
APPENDIX E
GENOTOXICITY: AMITROLE
Reported effect Ref.
1. Gene mutations
A. Bacteria
Salmonella (Ames) - See multiple bacterial
tests summarized in
Bridges et al. (1981)
- McCann and Ames
(1976)
TA 1950, mouse host mediated
(-NO2~) - Braune/a/. (1977)
(+NO2) w
Dunkel(1979)
- Rosenkranz and Poirier
(1979)
Moriyaetal. (1983)
NTP (1986)
-------
684
HILL ET AL.
APPENDIX E—Continued
Reported effect
Ref.
E. coli
WP2uvrA (P)
WP2uvrA
WP2uvrA/pKM101
Streptomyces
B. Eukaryotic microorganisms
Saccharomyces (RV)
C. Higher eukaryotes
Drosophila XLRL
Mouse lymphoma L5178 Y cells (TK.)
Syrian hamster embryo cells
Ouabain
6-Thioguanine
2. Chromosome effects
A. Numerical aberrations
Saccharomyces (D6)
Aspergillus mitotic nondisj unction
Drosophila sex chromosome
nondisjunction
B. Structural aberrations
Human lymphocytes in vitro
' Mousemicronucleus(B6C3Fl)
(CD-I)
Mouse dominant lethal (Ha, 1 CR)
C. Other effects
Sister chromatid exchange
CHO
CHO
3. DNA damage
Bacillus subtilis
Rec
E. coli
Rec
Rec
Rec
Rec
PolA
feeding, ?;
injection,
/ /
Venitt and Crofton-
Sleigh(1981)
Matsushima et al.
(1981)
Matsushima et al.
(1981)
Careree/a/. (1978)
Mehta and von Borstel
(1981)
Laamanen et al. (1976)
Vogel«a/. (1980)
Vogeletal. (1981)
NTP(1986)
Woodruffs al. (1985)
NTP(1986)
Tsutsui etal. (1984)
Tsutsuie/a/. (1984)
Parry and Sharp (1981)
Bignami
-------
THYROID CARCINOGENESIS REVIEW
APPENDIX E—Continued
685
Reported effect
Ref.
Lambda prophage induction
Saccharomyces cerivisiae
(D3) mitotic crossover
(race X11) mitotic crossover
(D4) mitotic gene conversion
(D7) mitotic gene conversion
(JD1) mitotic gene conversion
(RAD) cell growth
Aspergillus mitotic crossover
Unscheduled DNA synthesis (HeLa)
MLV integration enhancement
(C3H2K)
Mouse sperm head abnormality
4. In vitro transformation
Syrian hamster embryo cells
Baby hamster kidney cells (BHK)
Rat embryo cells
Rauscher murine leukemia virus
infected
w
+
Thomson (1981)
Simmon (1979)
Kassinova elal(1981)
Jagannath « a/. (1981)
Zimmerman and Scheel
(1981)
Sharp and Perry (1981,
1981a)
Sharp and Perry
(1981b)
Bignamiera/. (1977)
Martin and McDermid
(1981)
Yoshikur and
Matsushima(1981)
Tophan(1980)
Dunkelefa/. (1981)
TsutsuieM/. (1980)
Styles (1980)
Styles (1981)
Daniel and Dehnel
(1981)
Dunkeletal. (1981)
NTP(1983)
Note. +, positive; w, weak positive; ?, equivocal; -, negative.
ACKNOWLEDGMENTS
This report was developed under the auspices of EPA's
Risk Assessment Forum. The authors acknowledge the
input of numerous staff from within the Agency, review-
ers from outside the Agency, and those assembled
through the EPA Science Advisory Board. Substantive
comment was received from Gary A. Boorman, Michael
R. Elwell, Scott L. Eustis, and Robert P. Maronpot; Ge-
rard N. Burrow; W. Gary Flamm and Ronald J. Lorent-
zen; Sidney H. Ingbar; Jack H. Oppenheimer; E. Chester
Ridgway; David Schottenfeld; Jerrold M. Ward; and E.
Dillwyn Williams. Special appreciation is given to R. Mi-
chael McClain. Karlene Thomas and especially Pamela
Bassford are commended for their clerical skills.
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Appendix D
A Review of Recent Work on Thyroid
Regulation and Thyroid Carcinogenesis
Gordon C. Hard, B.V.Sc., Ph.D., D.Sc.
American Health Foundation
Valhalla, NY
December 1996
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Notice
This report was prepared by Dr. Gordon C. Hard of the American Health
Foundation initially under subcontract to Eastern Research Group, Inc.
(ERG), Lexington, Massachusetts, for the U.S. Environmental Protection
Agency (EPA) Risk Assessment Forum. ERG assembled and produced
the final September 1992 report. Since then, the 1992 report has been
further updated by Dr. Hard to include papers into 1996. The views pre-
sented are those of the author, and not necessarily those of EPA.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
D-2
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Contents
Introduction 0-4
Thyroid Regulation D-5
Metabolism and Excretion of Thyroid Hormones D-9
Control of TSH Secretion in the Central Nervous System D-9
Mitogenic Effect of TSH on Thyroid Tissue D-10
Rodent Thyroid Cancer Studies D-11
Effects of Specific Chemicals on the Thyroid-Pituitary
Axis D-12
Epidemiology and Etiology of Human Thyroid Cancer D-15
Changes in Gene Expression in Thyroid Carcinogenesis D-16
Data Gaps and Research Needs D-18
References D-19
D-3
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Introduction
Most risk assessment issues involving the thyroid concern the role of
the prolonged elevation of circulating thyroid-stimulating hormone (TSH)
levels on the development of follicular cell neoplasia in laboratory animals
and the appropriate procedures for extrapolation of these results to hu-
mans. A Technical Panel of the U.S. Environmental Protection Agency (EPA)
Risk Assessment Forum (Forum) examined this issue and concluded in a
1988 draft report that, under certain circumstances, thyroid follicular cell
tumors develop through an ordered linkage of steps beginning with inter-
ference in thyroid-pituitary status. When there is no direct interaction of the
chemical with DNA, the Technical Panel concluded that thyroid follicular
neoplasia involves a threshold process and would not develop unless there
is prolonged interference with the thyroid-pituitary feedback mechanisms.
The mechanistic information assembled in the 1988 draft was published in
1989 (Hill et al., 1989). The Forum is presently preparing a science policy
statement for assessing risk of thyroid follicular cell neoplasia and requested
an update of the pertinent literature as part of this process.
Since the EPA report on thyroid follicular cell carcinogenesis, was pub-
lished (Hill et al., 1989), over 600 pertinent papers on thyroid function,
regulation, carcinogenesis, and epidemiology have appeared in the litera-
ture. This review of the new publications highlights selected information on
the mechanisms of normal and abnormal thyroid growth and function and
the action of chemicals thereon.
Briefly, recent studies on regulation of the thyroid gland and thyroid
follicular cell neoplasia present a broad array of new data that add depth
and complexity to the information available in 1988 on this fundamental
biological process. These studies provide information on growth factors
and messenger systems, the control of TSH secretion in the central ner-
vous system, the intrinsic heterogeneity in follicular cell populations with
regard to proliferative potential, and new data on thyroid cancer in rodents
and humans.
The studies also confirm previous suggestions regarding the impor-
tance of chemically induced thyroid peroxidase inhibition and the inhibition
D-4
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of 3,3',5,5'-tetraiodothyronine (T4, thyroxine) deiodinases on disruption of
the thyroid-pituitary axis and thyroid neoplasia. In particular, new investiga-
tions that couple mechanistic studies with information from animal cancer
bioassays (e.g., sulfamethazine studies) confirm the linkage between pro-
longed disruption of the thyroid-pituitary axis and thyroid neoplasia. Many
new initiation/promotion studies also add to previous information suggest-
ing that chronic stimulation of the thyroid induced by goitrogens can result
in thyroid tumors.
It is now known that thyroid regulation occurs through a complex inter-
active network mediated through different messenger systems. Increased
TSH levels activate the signal transduction pathways to stimulate growth
and differentiation of the follicular cell. Although oncogene activation and
tumor suppressor gene inactivation may also be factors in the develop-
ment of thyroid cancer, the important role of TSH on growth as well as
function helps to explain how disruptions in the thyroid-pituitary axis may
influence thyroid neoplasia.
Other new data from epidemiologic studies contribute to the under-
standing of thyroid neoplasia. Acute exposure to ionizing radiation, especially
in childhood, remains the only verified cause of thyroid carcinogenesis in
humans. Iodine deficiency studies as a whole remain inconclusive, even
though several new studies in humans examine the role of dietary iodine
deficiency in thyroid cancer.
EPA's analysis in the 1988 draft report focused on the use of a thresh-
old for risk assessment of thyroid follicular tumors. New studies, involving
several chemicals, provide further information that suggests there will be
no antithyroid activity until critical intracellular concentrations are reached.
Thus, for chemically induced thyroid neoplasia linked to disruptions in the
thyroid-pituitary axis, a practical threshold for thyroid cancer would be ex-
pected. More information on thyroid autoregulation, the role of oncogene
mutations and growth factors, and studies directly linking persistently high
TSH levels with the sequential cellular development of thyroid follicular cell
neoplasia would provide further confirmation.
Thyroid Regulation
Numerous recent studies point to the conclusion that the physiological
regulation of thyroid cell growth and function involves a complex interac-
tive network of trophic factors (endocrine, paracrine, and autocrine) and
that the effects of these factors are mediated through a number of different
second messenger systems. It is well established that TSH is the main
growth factor for thyroid cells, maintaining as well the differentiated state of
the thyroid and controlling thyroid hormone secretion. Other growth regula-
tors involved in the complex web include insulin/insulinlike growth factor-l
D-5
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(IGF-I), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),
transforming growth factor 6 (TGFB), as well as an endogenous iodide-
dependent mechanism (Wynford-Thomas, 1993; Farid et al, 1994).
TSH exerts its action on thyroid follicular cells via receptor sites, re-
stricted mainly to the basal membranes of follicle cells (Mizukami et al.,
1994). The advent of recombinant DNA technology has led to the cloning
of the TSH receptor of both rat (Akamizu et al., 1990) and human thyroid
(Libert et al., 1989; Nagayama et al., 1989; Parmentier et al., 1989; Misrahi
et al., 1990). The TSH receptor is a plasma membrane site able to bind G
(guanine nucleotide-binding) protein for signal transduction (Parmentier et
al., 1989). G protein activitation by the TSH receptor appears to be a highly
complex effector system involving all four G protein families (Laugwitz et
al., 1996). The gene for the TSH receptor is virtually constitutive in the
thyroid cell, occurring far along the pathway of transformation, as demon-
strated by persistent expression in normal thyroid tissue as well as in
differentiated thyroid tumors, but not undifferentiated carcinoma (Brabant
et al., 1991). Current models indicate that the human TSH receptor is a
heptahelical glycoprotein molecule with an extremely large extracellular
domain at the N-terminus, a transmembrane/intracellular region consisting
of seven intramembrane helices connected by three alternating intracellu-
lar and extracellular loops, and an intracellular tail at the carboxyl terminus
(Nagayama and Rapoport, 1992; Vassart and Dumont, 1992). The extra-
cellular domain is the ligand-binding site, conferring a high affinity for TSH
binding and distinguishing it from other G protein-coupled receptors. It is
thought that the three extracellular loops help the.ligand to fit to the tertiary
structure, while the intramembrane and intracellular segments appear to
be critical for signal transduction. The available evidence indicates that the
overall conformation of the TSH receptor in rats is probably the same as in
humans.
TSH, through activation of its receptor, has been shown to stimulate
multiple signal transduction pathways in the regulation of both growth and
differentiated function. Each pathway may be related to specific cellular
events. The main effector of TSH on proliferation and differentiation in a
variety of species, man and rat included, is the cAMP signal transduction
pathway, that is, the cascade involving activation of adenylate cyclase re-
sulting in cAMP generation (Maenhaut et al, 1990; Dumont et al., 1992).
There is increasing evidence that the physiological stimulation of thyroid
cell function by TSH is achieved as well via the phosphatidyl-inositol/Ca2+
(Pi-C) signal cascade, with activation of phospholipase C, in rat and hu-
man thyroid cells (Laurent et al., 1987; Leer et al., 199t; Dumont et al.,
1992; D'Arcangelo et al., 1995), although, as a species difference, appar-
ently not in dog thyroid cells (Mockel et al, 1991). Thus, the TSH receptor
activates both pathways, but with a different efficacy, because in contrast
D-6
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to the cAMP pathway, much higher concentrations of TSH are required to
stimulate the Pi-C phospholipase C cascade (Laurent et al., 1987). The
signalling by these diverse pathways results in a range of metabolic conse-
quences, including iodine uptake and release, thyroid peroxidase
generation, organification of residues on thyroglobulin, thyroid hormone
synthesis and release, and thyroid cell growth and division (Dumont et al.,
1992), In this complex regulatory network, the TSH-cAMP cascade is func-
tionally responsible for secretion, while the Pi-C phospholipase C cascade
controls H?02 generation and thyroid hormone synthesis (Corvilain et al.,
1994). It is~generally accepted that cAMP accounts for the mitogenic effect
of TSH (Uyttersprot et al., 1995), although higher concentrations of TSH
and more prolonged stimulation of the cAMP cascade are necessary to
induce cell proliferation than for expression of differentiated function (Roger
et al., 1988). cAMP also appears to play a central role in iodide uptake and
metabolism by the follicular cell (Filetti and Rapoport, 1983, 1984) and in
thyroglobulin and thyroid peroxidase (TPO) gene expression (Van
Heuverswyn et al., 1985; Chazenbalk et al., 1987).
In addition to the above second messenger systems, the possibility of
a nontranscriptional regulatory pathway involving a phosphorylation site
on the TSH receptor that is kinase-C sensitive has also been suggested
(Akamizu et al., 1990).
Growth factors also play a key role, along with TSH, in the complex
regulation of thyroid cell proliferation. However, there are few data yet to
explain how these trophic factors interact with the cell cycle to stimulate or
inhibit cell division in the thyroid. There is evidence that both TSH receptor
gene expression and thyroglobulin gene expression are under the control
of insulin/IGF-1 as well as TSH, at a transcriptional level in the rat cell-line
FRTL-5 (Santisteban et al., 1987; Takahashi et al., 1990; Saji et al., 1992).
Similar evidence for a complex autoregulatory feedback mechanism in-
volving insulin/IGF-1 operative at several levels of interactive signalling is
accumulating for other primary thyroid cell culture systems (Gerard et al.,
1989; Eggo et al., 1990). Collective data suggest that a complex interac-
tion between the 1,2-diacylglycerol/protein kinase C (one of the bifurcating
second messenger pathways of the Pi-C signal cascade) and the adenyl
cyclase signal transduction systems is important in the regulation of thy-
roid growth by TSH and IGF-1 (Fujimoto and Brenner-Gati, 1992). Thus, in
rat and man, insulin/IGF-1 is considered a necessary cofactor for the ac-
tion of TSH on follicle cells, synergizing with TSH to induce thyrocyte
proliferation while maintaining differentiated function (Farid et al., 1994). In
humans, it has been recorded that benign and malignant thyroid tumors
produce increased levels of IGF-1 (Minuto et al., 1989; Williams et al., 1989).
Such findings have led to the suggestion that emergent adenoma cells
lose their dependence on exogenous IGF-1, acquiring the capability for
D-7
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autocrine production of this growth factor, resulting in continued
autostimulation of ceil replication and thus allowing thyroid nodules to be-
come autonomous (Williams et al., 1988; Thomas'and Williams, 1991).
Other autocrine/paracrine regulators of thyroid growth, with potent mi-
togenic activity for thyroid cells demonstrable in vitro include EOF and bFGF.
EGF is synthesized within the thyroid gland and induces proliferation in
thyroid cells from a wide range of species at the expense of dedifferentia-
tion and loss of specialized thyroid-specific function (Asmis et al., 1995;
Nilsson, 1995). bFGF is present in human thyroid tissue (Taylor et al.,' 1993)^
and there are stores of FGF in the basement membrane of follicles in adult
normal rat thyroid (Logan et al., 1992). EGF has been shown to stimulate
the growth and invasion of differentiated human thyroid cancer cells in cul-
ture and in nude mice (Hoelting et al., 1994), whereas bFGF expression
increases during thyroid hyperplasia in the rat (Becks et al., 1994).
Transforming growth factor 6, (TGFI3,) is a putative negative regulator
of thyroid growth, as studies in all normal cell systems have shown it to
inhibit thyrocyte proliferation, including that mediated by TSH (Morris et al
1988; Colletta et al., 1989; Roger, 1996). Although the actual role of TGFB'
in the thyroid is not known, cell culture and rodent studies suggest that it is
a limiting autocrine influence on thyroid cell hyperplasia and cancer growth
(Holting et al., 1994; Logan et al., 1994). In one study, TGF1 was detected
in approximately 50% of human thyroid carcinomas, but not in adenomas,
with a striking correlation being observed between the dual presence of
TGF1 expression and arginine substitution at codon 61 of the H-ras
oncogene (Jasani et al., 1990)
There also has been additional evidence that iodine is a major media-
tor of thyroid autoregulation, involving numerous inhibitory actions (Wolff,
1989). One of these is a decrease in cAMP formation in response to TSH,'
resulting in an inhibition of all cAMP-mediated stimulatory effects of TSH
on the thyroid. Excess iodide therefore exerts a negative control on differ-
ent thyroid parameters, inhibiting iodide uptake and organification, protein
and RNA biosynthesis, hormone secretion, as well as paracrine mitogenic
activity on endothelial cells and fibroblasts (Chazenbalk et al., 1988; Pisarev
et al., 1988; Gartner et al., 1990). Most of these actions appear to be medi-
ated by an intracellular organified iodine intermediate of as yet unknown
identity. Various derivatives of arachidonic acid have been proposed as
this putative regulator(s), including the iodinated eicosanoid -iodolactone
(Gartner et al., 1990). However, this compound had no effect on TSH-me-
diated cAMP formation in porcine follicles (Dugrillon and Gartner, 1995).
Considered to be a more likely candidate is 2-iodohexadecanal (Boeynaems
et al., 1995), a major iodolipid formed in horse thyroid when incubated with
iodide (Pereira et al., 1990), but which is also detectable in the thyroid of
other species, including man and rat.
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Metabolism and Excretion of Thyroid Hormones
There has been much recent progress in understanding the enzymatic
pathways responsible for metabolism of T4, T3, and the inactive T3 analog,
reverse T3 (rT3). T4 is secreted by the thyroid but has little biological activity
unless deiodinated to T3. Two isoenzymes catalyze this 5'-deiodination re-
action: type I 5'-deiodinase abundant in liver, kidney, and thyroid, and type
II 5'-deiodinase found primarily in brain, pituitary, and brown adipose tissue
(Leonard and Visser, 1986; Chanoine et al., 1993). In man, about 80% of
circulating T3 derives from peripheral 5'-monodeiodination of T4, particu-
larly that by liver and kidney, while 20% of T3 is secreted by the thyroid
(Pekary et al., 1994). In the rat, intrathyroidal conversion of T4 to T3 pro-
vides the major source of T3 (Chanoine etal., 1993), and ratthyroidal levels
of the 5'- deiodinase are the highest so far reported for any species.
Recent studies have also confirmed that thyroid hormones in rats are
metabolized predominantly through conjugation with either glucuronic acid
or suifate (de Herder etal., 1988; Eelkman Roodaet al., 1989; Visser etal.,
1990). The enzymes responsible for glucuronidation of thyroid hormones
are UDP-glucuronysyltransferases located mainly in the endoplasmic reticu-
lum of the liver, but also of intestines and kidney. It appears that there are
at least three UDP-GT isoenyymes involved in rat liver. T4 and rT3 are
glucuronidated by types I and II isoenzyme, while T3 is glucuronidated by
the type III isoform (Visser et al., 1993). The T3 glucuronide conjugate is
excreted in bile, which may represent a reversible pathway as the conju-
gate is hydrolyzed by intestinal bacteria, creating an enterohepatic cycle
enabling reabsorption of free T3 (de Herder et al., 1988; Rutgers et al.,
1989a). The evidence also suggests that there may be a more effective
enterohepatic circulation in humans than in rats (Rutgers et al., 1989a).
Suifate conjugation of thyroid hormones is an alternative metabolic
pathway. The suifate conjugate of T3 is rapidly deiodinated by type I
deiodinase through successive deiodinations of the tyrosyl (inner) and phe-
nolic (outer) rings (Visser et al., 1988), thus releasing iodine into the
circulation for reutilization by the thyroid (Rutgers et al., 1989b). In man,
the majority of nondeiodinative disposal of T3 occurs via this pathway
(LoPresti and Nicoloff, 1994).
Control of TSH Secretion in the Central Nervous System
At the central nervous system (CNS) level, recent work has provided
additional information on the control of TSH secretion by thyroid hormones
in the anterior pituitary and via the hypothalamus. A discrete population of
neurons synthesizing thyrotropin-releasing hormone (TRH), located in the
paraventricular nucleus of the hypothalamus, is under negative feedback
regulation by circulating thyroid hormones (Kolleret al., 1987; Segerson et
al., 1987). Some results suggest that the biosynthesis of TRH is regulated
D-9
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by both T3 and T4 (Kakucska et al., 1992), although the mechanism by
which T4 plays an inhibitory role is unknown. The negative feedback of
thyroid hormones on TSH secretion caused by antithyroid compounds ap-
pears to be exerted mainly at the pituitary level. This is because the increase
in TRH release into hypophyseal portal blood produced by propylthiouracil
(PTU) is relatively small (less than 50%) compared to the pronounced in-
crease (up to 20 times at 3 weeks) in serum TSH (Rondeel et al., 1992). In
rats, the data suggest that serum T3 has a greater inhibitory action on TSH
secretion from the pituitary than does serum T4 (Emerson et al., 1989), at
least in the euthyroid or mildly hypothyroid states. In humans, new highly
sensitive immunometric assays used for measurement of TSH serum con-
centrations have underscored earlier work showing that thyroid hormone
negative feedback on pituitary TSH secretion is mediated mainly by local
generation of T3 within the pituitary from T4 by the 5'-deiodinase enzyme
system (Spencer, 1996).
Mitogenic Effect of TSH on Thyroid Tissue
Evidence concerning the mitogenic role of TSH for thyroid cells in vivo
has been further consolidated over recent years. Studies from various labo-
ratories employing tritiated thymidine labeling, metaphase-arrest techniques
for mitotic index, or immunohistochemical decoration of statin (a nonprolif-
eration-specific nuclear antigen identifying quiescent Go-phase cells) show
that TSH stimulates, in a dose- and time-dependent way, the recruitment of
noncycling Go cells into the cycling compartment, entry into S-phase, and
entry of G2 cells into mitosis (Bayer et al., 1992).
Recent work has indicated that the normal rodent (and human) thyroid
may have an intrinsic heterogeneity in the follicular cell population regard-
ing the capacity for proliferative response to TSH. One hypothesis suggests
that there are a few subsets of stemlike follicular cells having a high growth
potential compared to the majority of the population, and that this trait is
stable and heritable (Peter et al., 1985; Smeds et al., 1987; Groch and
Clifton, 1992; Studer and Derwahl, 1995). According to this model, the clones
of cells with extensive proliferative potential are the origin of the adenomas
that develop under conditions of chronic TSH stimulation (Smeds et al.,
1987; Groch and Clifton, 1992).
As suggested by Studer et al. (1989), the intrinsic stem cell clone model
reconciles earlier kinetic observations (Wynford-Thomas et al., 1982;
Christov, 1985) that hyperstimulated thyroid attains a plateau phase of
growth or state of "desensitization," with the vast majority of follicular cells
becoming refractory to the mitogenic effects of increased TSH levels, be-
fore the emergence of adenomas. In this model, chronic TSH stimulation
would select preexisting thyrocyte clones with the greatest proliferative
potential, and thus greater risk of neoplastic transformation. An alternative
D-10
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model for explaining the self-limited nature of hypothyroidism and the de-
velopment of hormone-responsive tumors in the chronically stimulated rat
thyroid proposes that clones of cells escape from the desensitization mecha-
nism through mutational events and natural selection, leading ultimately to
tumor formation (Thomas and Williams, 1991). Both models agree that
thyroid carcinogenesis involves rare subsets of cells responsive to contin-
ued TSH stimulation but differ concerning the origin of follicular cell
heterogeneity. Additional support for the intrinsic subset concept and/or
the controlling influence of TSH on the development of selected clones of
thyroid follicular cells comes from other studies on cell proliferation (Bayer
et al., 1992), or those using transplantation methodology (Watanabe et al.,
1988; Domann et al., 1990; Ossendorp et al., 1990).
Rodent Thyroid Cancer Studies
Although additional bioassays have revealed new compounds with thy-
roid tumor- inducing capability in rodents, such as malonaldehyde (NTP,
1988), or have provided stronger evidence of tumorigenicity as in the case
with sulfamethazine (Littlefieid et al., 1989,1990), the most significant stud-
ies in this area concern the promoting activity of antithyroid compounds.
These studies have employed N-bis(2-hydroxypropyl)nitrosamine (DHPN)
as the initiating agent. Promoting activity in the rat has been confirmed for
many antithyroid compounds, including thiourea and potassium thiocyan-
ate (Kanno et al., 1990), PTU and potassium perchlorate (Hiasa et al.,
1987), 4,4'-methylenebis'(N,N-dimethyl)-benzenamine (MDBA) (Kitahori et
al., 1988), 2,4-diaminoanisole sulfate (Kitahori et al., 1989), sulfadimethoxine
(Mitsumori et al., 1995), and phenobarbital (McClain et al., 1988; Kanno et
al., 1990). Where thyroid gland and hormone parameters were measured,
there were strong correlations between the tumor-promoting activity on the
one hand and increased thyroid weight, decreased serum T3 and T4 levels,
and increased circulating TSH on the other (Kitahori et al., 1988, 1989;
McClain et al., 1988). One of these studies (with MDBA) also linked the
increased circulating level of TSH with increased numbers of TSH-positive
cells in the anterior lobe of the pituitary gland (Kitahori et al., 1988). The
phenobarbital study was noteworthy in demonstrating a clear dose-depen-
dent negation of thyroid tumor promotion and plasma TSH elevation by T4
replacement therapy (McClain et al., 1988). In the investigations where cell
proliferative activity has been determined, the results strongly support an
important role for high serum levels of TSH in the early stages of thyroid
tumorigenesis (Shimo et al., 1994; Mitsumori et al., 1995).
These tumor promotion studies add further and consistent support to
the hypothesis that antithyroid compounds exert effects secondarily on the
thyroid through the chronic stimulation of persistently elevated levels of
TSH.
D-11
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Effects of Specific Chemicals on the Thyroid-Pituitary Axis
Several recent studies have provided more quantitative data on the
time- and dose-dependent effects of specific antithyroid chemicals on thy-
roid hormone status, including PTU (de Sandro et al., 1991), sulfamethazine
(McClain, 1995), and phenobarbital (McClain et al., 1988; de Sandro et al.,
1991). These studies defined the effects as early but persistent decreases
in circulating T3 and T4 levels and a substantial increase in circulating TSH.
Particularly noteworthy are observations on sulfamethazine where the dose-
responsiveness for thyroid parameters in rats was studied with 10 dose
levels spanning 3 orders of magnitude (McClain, 1995). Consistently, the
parameters of thyroid weight and plasma T3, T4, and TSH levels displayed
nonlinear dose-response curves with a major break in linearity from zero
slope at around the 1,600 ppm dose level. These data suggested that, if
coincident with tumor incidence data, a benchmark approach might con-
ceivably be applied through the most sensitive indicator to provide a scientific
basis for high- to low-dose extrapolation for secondary thyroid carcinogen-
esis.
There is also more detailed information available on the metabolic path-
ways and metabolites of such antithyroid compounds as
methylmercaptoimidazole (MMI) and PTU (Taurog and Dorris, 1988; Taurog
et al., 1989). Some of these data confirm the importance of antithyroid
drugs accumulating in the target organ and thus achieving effective intrac-
ellular concentrations at the intrathyroidal site of iodide organification as a
requirement for antithyroid activity. Accordingly, it has been calculated that
the intracellular concentration of PTU in the rat thyroid reaches approxi-
mately 20 M (Taurog and Dorris, 1989), sufficiently high for iodination
inhibition.
A particularly critical site of action representing a common intrathyroidal
mechanism shared by PTU, ethylenethiourea (ETU), MMI, and aminotriazole
(ATZ) is TPO inhibition. Antithyroid chemicals appear to be able to inacti-
vate TPO in one of two ways, either by a reversible reaction that does not
involve covalent binding, or by an irreversible interaction involving suicide
inactivation of the enzyme. The latter reaction comprises branched path-
ways that proceed concurrently, namely, inactivation of the enzyme and
turnover of the suicide substrate (Doerge, 1988). Thus, suicide inhibitors
inactivate TPO by covalent binding to the prosthetic heme group in the
presence of H2O2, resulting in H?02-dependent catalytic formation of reac-
tive intermediates (Doerge, 1988; Doerge and Niemczura, 1989). In this
case, de novo synthesis is required to restore the lost enzyme activity
(Doerge and Takazawa, 1990). The reaction between either PTU or ETU
and theTPO/H2O2 generating system is reversible, representing metabolic
detoxification of these compounds in thyroid and not suicide inactivation
D-12
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(Doerge, 1988; Taurog and Dorris, 1989; Doerge and Takazawa, 1990).
This contrasts with the effects of MMI and other thiocarbamide goitrogens,
as well as ATZ, which cause suicide inactivation of the enzyme via cova-
lent binding (Doerge, 1988; Doerge and Niemczura, 1989; Doerge and
Takazawa, 1990). The difference in action between PTU or ETU and MMI,
involving reversible inhibition of TPO on the one hand, and on the other,
irreversible inactivation requiring de novo synthesis of enzyme to restore
activity, may account for the longer duration of effect and greater clinical
potency of MMI (Doerge and Takazawa, 1990). The different mechanism
of TPO inactivation between compounds would not necessarily implicate a
different overall mechanism of rodent thyroid carcinogenesis. However, the
commonality of this intrathyroidal mechanism among antithyroid chemi-
cals that appear to induce follicular cell carcinogenesis via secondary effects
on the thyroid/pituitary axis suggests that TPO inactivation might be a use-
ful additional criterion for categorizing these chemicals. It has been
suggested further, that for risk assessment purposes, the effect on TPO
provides a biochemical basis for the existence of a no-observed-effect level
(NOEL) in chemically induced thyroid toxicity (Doerge and Takazawa, 1990).
A marked species difference may exist between primates and rodents
in the inhibition of TPO by antithyroid compounds. As examples, inhibition
of monkey TPO requires approximately 50 and 450 times the concentra-
tion of PTU and sulfamonomethoxine, respectively, than does rat TPO
(Takayama et al., 1986). This inequality might explain the greater suscep-
tibility of the rat to the antithyroid effects of such compounds compared to
the primate.
More recent data concerning the extrathyroidal action of antithyroid
compounds on the peripheral conversion of T4 and T3 involving inhibition of
T4 deiodinases primarily in liver (reviewed by Curran and De Groot, 1991)
is available for PTU (de Sandro et al., 1991), ATZ (Cartier et al., 1985), and
phenobarbital (McClain et al., 1989; de Sandro et al., 1991; Barter and
Klaassen, 1992). Unlike PTU, ATZ did not affect the outer ring 5'-deiodination
pathway but stimulated inner ring 5-deiodination of T4 with a consequent
increase in serum concentration of rT3 (Cartier et al., 1985), an inactive
form of T3. ATZ has no thiocarbonyl (or aromatic) group in its structure, and
the lack of sulfur could be a possible explanation for the difference in pe-
ripheral action from PTU. The studies with phenobarbital have confirmed
that this drug acts through an extrathyroidal mechanism by increasing both
hepatic glucuronidation of T4 as well as increasing the clearance of T4 from
serum (McClain et al., 1989; de Sandro et al., 1991; Barter and Klaassen,
1992). These effects were mediated by the substantial induction of the
enzyme responsible for T4 metabolism, as well as an increased biliary flow.
The effect of phenobarbital solely on the peripheral pathway and the less
potent consequence for thyroid hormone levels, compared to the effects of
D-13
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a thiourylene compound like PTU, accords with its role as a promoter rather
than an inducer of rodent thyroid tumorigenesis. There is still no epidemio-
logic evidence that chemicals such as phenobarbital, which affect thyroid
function through a peripheral mechanism involving thyroid hormone me-
tabolism, are associated with thyroid neoplasia in humans (McClain, 1989;
Curran and De Groot, 1991).
In keeping with an indirect action on TSH hypersecretion from the an-
terior pituitary, some antithyroid compounds have been shown to have CNS
effects. Qualitative increases in TSH-producing thyrotrophs in the rat pitu-
itary have been associated with administration of PTU (Samuels et al.,
1989) and 4,4'-oxydianiline (Murthy et al., 1985), with a rapid reversal of
morphological changes upon cessation of treatment in the case of PTU. In
the PTU study, there was also a coincident decline in growth hormone
cells, suggesting transdifferentiation of somatotrophs into thyrotrophs
(Horvath et al., 1990), thus implying changes in cell type rather than total
cell number in the hypothyroid state. At the molecular level, this accords
with the induction of increased levels of TSH mRNA in the anterior pituitary
by PTU and a concomitant fall in growth hormone mRNA (Franklyn et al.,
1986; Wood et al., 1987). The profound effect on cytoplasmic TSH levels
affected both TSH and subunits (Franklyn et al., 1986; Mirell et al., 1987;
Samuels et al., 1989), but the increases were relatively greater in TSHB
than in the subunit. T3 replacement reversed these specific subunit changes
(Samuels et al., 1989). These effects of PTU were dose and time depen-
dent, and the various studies confirmed a direct influence of "thyroid status"
on the regulation of pituitary hormones at a pretranslational level.
Importantly, there is evidence that genotoxic chemicals able to induce
thyroid cancer in rodents have different morphological and physiological
effects from those of known goitrogens. Thus, DHPN induces rat thyroid
tumors along a multistage pathway involving focal atypical hyperplasia (origi-
nating from single follicles) rather than diffuse follicular hyperplasia (Kawaoi
et al., 1991). Furthermore, DHPN and N-nitrosomethylurea (NMU) appear
not to influence the thyroid-pituitary axis during the induction of thyroid
carcinogenesis because these compounds did not increase thyroid weights
unrelated to tumor development or cause a persistent elevation of serum
TSH levels or changes in serum T4 levels (Mori et al., 1990; Hiasa et al.,
1991; Hiasa et al., 1992).
Collectively, these studies with a range of compounds strengthen the
hypothesis that antithyroid agents in rodents act by secondarily causing
sustained elevations in serum TSH levels associated with the develop-
ment of thyroid carcinogenesis. They also highlight the differences in
pertinent effects between antithyroid compounds and those rodent thyroid
carcinogens that are directly DNA reactive.
D-14
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Epidemiology and Etiology of Human Thyroid Cancer
In man, tumor histology is important to the understanding of the etiol-
ogy of thyroid cancer because different types appear to represent separate
biological entities with different clinical and epidemiologic features
(Pettersson et al., 1991, 1996). The most frequent type is papillary carci-
noma, accounting for approximately 60% of all thyroid cancers, while
follicular carcinoma represents about 20% (Goepfert and Callender, 1994).
In Sweden, there are regional differences in the incidence of papillary and
follicular types of thyroid cancer defined by iodine status, iodine-deficient
areas being associated with a higher risk of follicular cancer (Pettersson et
al., 1996). There is also some evidence that the incidence rates of these
histologic entities may be changing. The data from one study reflect an
increase for papillary thyroid cancer in Sweden since 1919 but a decline
for follicular cancer in cohorts born since 1939 (Pettersson et al., 1991).
Although residence in endemic goiter areas in Switzerland was linked to a
modestly increased probability of developing thyroid cancer (Levi et al.,
1991), overall, there remains a general view that no convincing evidence
has yet emerged to link environmental thyroid cancer with areas of iodine
deficiency. Furthermore, the long-standing program of supplementation of
food items with iodine in Sweden has not affected thyroid cancer trends in
iodine-deficient or iodine-rich areas (Pettersson et al., 1996). Vegetables
known to contain, or endogenously generate, thiocyanate have not been
found to enhance the risk of thyroid cancer, but possibly exert a protective
influence (Franceschi et al., 1993). A meta-analysis of four similarly de-
signed case-control studies conducted in high-thyroid cancer areas of
Switzerland and Italy revealed an association with diets rich in starchy foods
and fats, while raw ham and fish were protective (Franceschi et al., 1991).
The only verified cause of thyroid cancer in humans is exposure to
ionizing radiation. This association has been established for x-radiation
therapy (de Vathaire et al., 1988; Hawkins and Kingston, 1988; Ron et al.,
1989; Tucker et al., 1991) and for radioactive fallout (Kerber et al., 1993;
Nikiforov et al., 1996). Of the several events exemplifying the latter, the
Chernobyl nuclear power-plant disaster provides the most striking correla-
tion. Since 1990, a very high incidence of childhood thyroid cancer has
been recorded in the Republic of Belarus, affecting predominantly children
who were less than 1 year old at the time of the accident (Nikiforov et al.,
1996). Almost all of the cases have been papillary carcinomas with short
latency (Nikiforov and Gnepp, 1994), in keeping with the observation that
radiation-associated thyroid tumors are predominantly of the papillary type
(Goepfert and Callender, 1994). The most biologically significant isotopes
released in the fallout were radioiodines, primarily 131I, and consequently
radioiodine has been accepted as the causative factor (Williams, 1996).
This stands in marked contrast to the lack of evidence incriminating diag-
D-15
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nostic or therapeutic doses of 131I (Holm et al., 1988, 1991). As with the
Chernobyl experience, age at the time of treatment with x-radiation therapy
is also an important factor in thyroid cancer development, the 67-fold risk
at 12 years mean age declining to nonsignificance at a mean age of 29
years (Tucker et al., 1988). In contrast to the risk posed by high-level ioniz-
ing radiation, a well-designed Chinese study indicates that lifetime exposure
to low-level environmental radiation, with an estimated cumulative dose of
9cGy, is not a risk factor for human thyroid cancer (Wang et al., 1990).
Graves' disease, an autoimmune thyroid condition, is associated with
the presence of circulating antibodies stimulating the TSH receptor (TSAb)
(Rees Smith et al., 1988; Paschke et al., 1995). Whether this disease car-
ries an increased risk of thyroid carcinoma has been controversial.
Nevertheless, from the collective published reports, Mazzaferri (1990) con-
cludes that thyroid cancer incidence in surgically treated Graves' disease
patients is between 5% and 10%. It is generally agreed that thyroid cancer
occurring in Graves' disease is an aggressive form (Belfiore et al., 1990).
There is strong support for a pathophysiological role of TSAb rather than
circulating TSH in thyroid cancer development associated with Graves' dis-
ease (Filetti et al., 1988; Belfiore et al., 1990), particularly as serum TSH
levels are suppressed in hyperthyroid patients with thyroid cancer, while
TSAb's are present in most cases (Belfiore et al., 1990). Evidence that
TSAb's are circulating autoantibodies to the TSH receptor comes from the
finding that they, and monoclonal antibodies to the human TSH receptor in
thyroid tissue, mimic many of the activities of TSH on thyroid cells (Rees
Smith et al., 1988; Belfiore et al., 1990; Marion et al., 1992). The role of
TSH in mediating the growth of thyroid nodules in humans needs further
clarification, however (Ridgway, 1992).
Changes in Gene Expression in Thyroid Carcinogenesis
To date, there have been numerous studies at the molecular level ex-
ploring the changes in gene expression that might accompany human thyroid
carcinogenesis, involving a wide range of protooncogenes, oncogenes,
tumor suppressor genes, or gene protein products. Despite the technical
evolution from transfection methodology to polymerase chain reaction
amplification coupled with sequence-specific oligonucleotide hybridization
or probing, some of the findings are discordant. For example, conflicting
results have been obtained for the erb family of "nuclear" oncogenes, and
for involvement of mutations of the TSH receptor or retinoblastoma (Rb)
gene in thyroid tumor development, while other investigations have indi-
cated clear evidence of no role in thyroid cancer for mutation of the APC,
plgiNM or nm23 genes, or for abnormal expression of the central regulat-
ing genes, myc, myb, fos, or jun (Wynford-Thomas, 1993; Fagin, 1994;
Farid et al., 1994; Said et al., 1994).
D-16
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There is general agreement emerging that some of the genetic changes
observed can be correlated with tumor histotype and stage of tumor devel-
opment. Point mutations of ras are the most frequent single genetic
abnormalities found in human thyroid tumors, occurring in about 50% of
those of follicularcell type (Wynford-Thomas, 1993; Fagin, 1994; Said et
al., 1994), and these mutations are regarded as early molecular events in
the development of this tumor type (Lemoine et al., 1989; Wright et al.,
1989; Shi et al., 1991). Although all three ras family members have been
involved (that is, Ha-ras, Ki-ras, N-ras), the changes have been associated
mainly with Ha-ras mutation, the most common mutation site being codon
61 with glutamine_>arginine substitution (Wright et al., 1989; Namba et al.,
1990; Shi etal., 1991). When comparing the data for thyroid cancer asso-
ciated with areas of iodine sufficiency, one study found a higher rate of ras
mutation in thyroid tumors from iodine-deficient areas (Shi et al., 1991).
Also prevalent in follicular adenomas are gsp mutations, occurring in about
25% of cases with a possible predilection for microfollicular adenomas
(Suarezetal., 1991; O'Sullivan et al., 1991; Farid et al., 1994; Said et al.,
1994). This distribution suggests that gsp mutation is another early event
in the development of follicular thyroid cancer, although there appear to be
some discrepancies between studies concerning the involvement of G pro-
teins in thyroid neoplasia (Esapa et al., 1997).
In contrast to follicular tumors, rearrangements of ret and trk
protooncogenes are associated with the papillary type of cancer. The ret/
PTC rearrangement is specific for the thyroid and found in up to 30% of
human papillary carcinomas (Jhiang and Mazzaferri, 1994; Said et al., 1994;
Viglietto et al., 1995). As this alteration has been detected in over 40% of
occult papillary carcinomas, generally considered to be the early stage of
papillary malignancy, it is believed to represent an early event in the devel-
opment of this tumor histotype (Viglietto et al., 1995). A higher prevalence
of ret protooncogene rearrangement, in up to two-thirds of cases, has now
been recorded in papillary thyroid carcinomas from children exposed to the
Chernobyl nuclear reactor accident (Fugazzola et al., 1995; Klugbauer et
al., 1995). Unlike the situation in adult tumors where the most common ret
translocation is retfPTCI, the alteration in the childhood radiation tumors
from Belarus was preferentially ref/PTC3 rearrangement (Fugazzola et al.,
1995; Klugbauer et al., 1995). Interestingly, the only reproducible cytoge-
netic abnormality found in papillary thyroid cancer has been an inversion of
chromosome 10 at the 10q11.2 locus, which is known to involve the ret
protoocogene at that locus (Donghi et al., 1989; Jenkins et al., 1990). Acti-
vating rearrangement of the trk protoocogene has also been found only in
papillary carcinomas (Fagin, 1994; Said et al., 1994), while overexpression
of the met oncogene is another molecular aberration observed mainly in
papillary thyroid cancer (Said et al., 1994; Farid et al., 1995).
D-17
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Little is known concerning molecular changes involved in the transition
from adenoma to carcinoma, although in follicular tumors a loss of het-
erozygosity involving chromosome 3p was considered to be specific for
follicular carcinoma, appearing to correlate with the transition from the ad-
enoma to the carcinoma stage (Herrmann et al., 1991), Chromosomal
analysis of follicularthyroid tumors has also indicated the existence of three
cytogenetically distinct subsets of adenoma, with numerical changes in
chromosomes 5,7, and 12 as the most frequent cluster of anomalies (Roque
et al., 1993a). A similar cluster of alterations found in some thyroid nodular
hyperplasias has been interpreted as support for a biological continuum
between hyperplastic nodules and the most common subset of adenomas
(Roque et al., 1993b). At the histologic level, polysemies for chromosomes
7 and/or 12 have been observed only in lesions with an exclusive or pre-
dominant microfollicular component (Criado et al., 1995). There is some
evidence from several studies that mutation of the tumor suppressor gene
p53 is a late genetic event in thyroid carcinogenesis involved in the pro-
gression to a more aggressive phenotype in the form of undifferentiated or
anaplastic cancer (Nakamura et al., 1992; Ito et al., 1993; Nikiforov et al.,
1996).
Genetic alterations have also been detected in rat thyroid carcinogen-
esis, but only a few investigations have been reported. Ha-ras activation
was exclusively involved in a majority of tumors induced by the direct-act-
ing genotoxin NMD (Lemoine et al., 1988). In DHPN-induced rat thyroid
tumors, however, mutations involved the Ki-ras gene via a G to A transition
at the second base of codon 12 (Kitahori et al., 1995). The same point
mutation was detected at an early time point in preneoplastic thyroid, sug-
gesting that Ki-ras mutations may play a role in the development of
DHPN-induced rodent thyroid cancer. In about half of the cases, radiation-
induced thyroid tumors in rats were associated preferentially with Ki-ras
activation (Lemoine et al., 1988). In this respect the rat data conform with
that for radiation-induced papillary thyroid tumors in humans, which also
are associated with Ki-ras mutation (Wright et al., 1991). On the other hand,
ATZ-induced adenomas in the rat showed only a very low incidence of Ki-
ras activation (Lemoine et al., 1988).
Data Gaps and Research Needs
Much of the recent new data strengthen the hypothesis that
nongenotoxic antithyroid compounds induce rodent thyroid follicular cell
carcinogenesis by an indirect mechanism involving thyroid-pituitary feed-
back regulation, and none of the data negate that notion. Some of the new
evidence also supports the view that humans may be less sensitive to the
antithyroid process than rodents. Nevertheless, there are gaps in the avail-
able information that require further research.
D-18
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1, Despite the voluminous literature on thyroid autoregulation, more re-
search is needed before the complex interactive network is fully
elucidated.
2. More data are required to clarify the role and interaction of oncogene
mutations and growth factor alterations in thyroid carcinogenesis in
both rodents and humans, and particularly in defining differences, if
any, between rodent thyroid cancer induced by antithyroid compounds
compared to the action of genotoxic carcinogens.
3. More information is required concerning the differences that might
distinguish the thyroid-related mechanisms of action of antithyroid
compounds versus genotoxic carcinogens, such as DNA adduct analy-
sis within the thyroid, and the effects of DNA-reactive carcinogens on
TPO.
4. More information is required directly linking persistently high levels of
circulating TSH with a step-by-step sequence involved in the cellular
development of thyroid follicular cell neoplasia.
5. Well-conducted studies are needed to better define the comparative
sensitivity of humans, relative to rodents, to the long-term effects of
antithyroid factors.
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