WORKSHOP REPORT
EPA/600/R-98/057
Screening Methods for Chemicals That Alter Thyroid Hormone
Action, Function and Homeostasis
June 23-25,1997
Durham, NC
Michael DeVito, Kevin Crofton and Suzanne McMaster
National Health and Environmental Effects Research Laboratory
United States Environmental Protection Agency
-------
WORKSHOP REPORT
EPA/600/R-98/057
Screening Methods for Chemicals That Alter Thyroid Hormone
Action, Function and Homeostasis
June 23-25,1997
Durham, NC
Michael DeVito, Kevin Crofton and Suzanne McMaster
National Health and Environmental Effects Research Laboratory
United States Environmental Protection Agency
-------
Screening Methods for Chemicals That Alter Thyroid Hormone Action, Function
and Homeostasis Workshop
EXECUTIVE SUMMARY 5
Background 5
Workshop Objectives 5
Assays for Thyroid System Disruption in Mammals 7
Serum Hormone Determination and Thyroid Histology; 7
Assays for Chemicals That alter Synthesis, Secretion, Transport and
Catabolism of Thyroid Hormones. 7
Screening for Chemicals That Interact with Thyroid Hormone Receptors or
Modulate Thyroid Hormone Receptor Activation 8
Developmental Assays 8
Screening for Chemicals That Alter Thyroid Action, Function, and
Homeostasis in Non-Mammalian Wildlife 9
Conclusion 9
INTRODUCTION 10
Thyroid Function and Regulation 11
ASSAYS FOR THYROID SYSTEM DISRUPTION IN MAMMALS 12
Thyroid hormone concentrations and thyroid gland histology 12
Section Summary 15
Assays for Chemicals That Alter Synthesis, Secretion, Transport and Catabolism of
Thyroid Hormones 16
Peroxidase Assay 16
Perchlorate Discharge Test 17
TRH Challenge Test 18
-------
Serum Protein Binding Assays 18
Deiodinase Assays 19
Glucuronidation Assays 21
Section Summary 21
TR Binding and Activation 22
In Vitro Binding Assays 22
Transfection and Transformation Assays 23
GH3 Cell Assay for Thyroid Hormone Action: 24
Section Summary 25
Developmental Assays 25
Neurodevelopmental 26
Morphological and Biochemical Assays in Developing Brains 26
Behavioral Testing 26
Male Reproductive System Development 27
Testes Size and Sperm Count 27
Section Summary 27
Screening for Chemicals That Alter Thyroid Function, and Homeostasis in Non-
Mammalian Wildlife 28
Tadpole Metamorphosis Assay 30
Section Summary 30
CONCLUSIONS 31
REFERENCES 32
APPENDIX 1: ABBREVIATIONS 40
APPENDIX 2: PARTICIPANTS 41
3
-------
Disclaimer
This document has been reviewed in accordance with U.S. Environmental Prote9tion
Agency policy and approved for publication. Approval does not signify that the contents
necessarily reflect the view and policies of the Agency nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
-------
Workshop On Screening Methods for Chemicals That Alter Thyroid Hormone
Action, Function and Homeostasis.
EXECUTIVE SUMMARY
Background
On June 23-25, 1997 the Workshop On Screening Methods for Chemicals That
Alter Thyroid Hormone Homeostasis, Action and Function was held at the Nicholas
School of the Environment at Duke University, Durham, North Carolina. This workshop
was attended by 21 scientists from academic, industrial and governmental laboratories.
The workshop was the third in a series of workshops sponsored by the Chemical
Manufacturers Association, World Wildlife Fund and the US EPA. The meeting was
organized by Drs. Ron Miller (CMA), Theo Colborn (WWF), Sue McMaster (EPA),
Michael DeVito (EPA), Michael McClain (Hoffman La Roche) and Peter Hauser (VA).
Workshop Objectives:
Convene a workgroup to discuss the technical merits and limitations of currently
available or under development in vivo and in vitro methods for detection of chemicals
that interfere with thyroid hormone (TH) action and homeostasis. It is not the purpose of
the meeting to recommend a screening battery nor to deal with policy issues pertaining to
the use of such screens. The product of the workshop is intended to describe the methods
which are currently available or could be developed in the near future for screening and
testing.
Workshop Summary
Chemicals that alter thyroid gland function or hormone action can act by altering
synthesis, transport, or. metabolism of thyroid hormones or by disrupting thyroid hormone
-------
receptor signaling by binding directly to these receptors as either agonists or antagonists or
through secondary mechanisms. The workshop examined more than 20 assays in
mammalian and non-mammalian systems including in vivo, ex vivo and in vitro assays.
Discussions of these assays focused on their specificity (is the response pathognomonic for
alterations in thyroid function), sensitivity (defined as the response of the method to low
doses or to weak acting chemicals), test duration, simplicity, and limitations. Confidence
in an assay is dependent upon how long the assay has been in use and how widely accepted
the method is in pharmacological and toxicological research, hence the historical use of
the assay was evaluated.
The consensus of the workshop is that there are assays available which can detect
alterations in thyroid gland function and hormone action. These assays include in vivo, ex
vivo and in vitro assays. However, only a few of these assays can be described as potential
first tier screens. Many of the assays discussed are either mechanistic in nature or are time
consuming and/or too expensive to be considered as screens. Current standard EPA test
guidelines do not include screening methods for chemicals that alter thyroid gland function
or thyroid hormone action. Several of the methods discussed at the workshop could be
added to these guidelines.
Chemicals that affect synthesis, transport or metabolism of thyroid hormones have
been well characterized and assays for the detection of these effects are well established.
Several of these assays have been used in numerous laboratories examining hundreds of
chemicals. In vivo, ex vivo and in vitro assays have been used to examine these chemicals.
Many of the assays discussed are mechanistic based and not necessarily useful as initial
screens. These assays may be useful in further testing of the chemicals that are positive in
initial screens. The workshop participants concluded that a single screen may not detect
weakly goitrogenic chemicals if the studies are not properly designed and evaluated.
-------
ASSAYS FOR THYROID SYSTEM DISRUPTION IN MAMMALS
Serum Hormone Determination and Thyroid Histology.
One possible screen for chemicals that alter thyroid function could be the
measurement of serum TH concentrations in experimental animals following treatment
with a test compound. Methods for determining serum concentrations of THs are readily
available from commercial suppliers and these assays have been in use for many years.
The determination of serum thyroid hormone concentrations in animals following
chemical exposure provides assessment of thyroid function equivalent to those used
clinically in humans. However, due to potential compensatory mechanisms, histological
assessment of thyroid gland in conjunction with the measurement of serum TH
concentrations can provide a more complete assessment of thyroid function and thyroid
hormone action (McClain, 1995; Capen, 1995). Future efforts to determine the most
appropriate time point and exposure regimen for examining serum TH concentrations are
recommended.
Assays for Chemicals That Alter Synthesis, Secretion, Transport and Catabolism of
Thyroid Hormones.
The workshop discussed the peroxidase assay, perchlorate discharge test, TRH
challenge test, serum protein binding assays, deiodinase assays, and glucuronidation
assays. The assays described in this section are specific for particular mechanisms of
action. A combination of these assays could provide predictive information on the
availability of intracellular 3,3',5-tri-iodothyronine (T3) concentrations, particularly in the
fetus. This information could be useful in assessing the potential adverse effects of
chemicals that disrupt thyroid hormone homeostasis and alter tissue TH concentrations.
These assays have been used to understand the mechanism of either altered serum
concentrations of TH and thyroid stimulating hormone (TSH) or changes in thyroid
histopathology (McClain et al., 1995; Atterwell et al., 1993; Poole et al., 1988; Olgivie
and Marsden, 1988; Brouwer 1991). These assays are useful tools in understanding the
mechanism of action of thyroid hormone disrupters. If these assays were to be used as
-------
initial screens, several of these assays should be performed in order to demonstrate that a
chemical does not alter TH concentrations.
Screening for Chemicals That Interact with Thyroid Hormone Receptors or
Modulate Thyroid Hormone Receptor Activation.
Chemicals may bind to the thyroid hormone receptors and act as either agonists,
antagonists or partial agonists. Evidence of xenobiotics which alter thyroid hormone action
by binding to the receptor is limited. While there are QSAR models which predict such
interactions, there is no direct evidence that any xenobiotic binds the thyroid hormone
receptor (TR). Chemicals can also interact with other proteins and modulate the
activation of the TR. There are a number of high throughput screens which can determine
if a test chemical binds to the TR or modulates the activation of the receptor. The most
frequently performed assays are the receptor binding assays, mammalian cell transfection
assays and yeast cell line transformation assay. Also discussed was the GH3 cell line
assay, which examines the proliferation of cells in response to TR agonists. While these
assays would have the ability to detect chemicals that bind to TR, there is limited evidence
that environmental chemicals bind to these receptors. The development and
implementation of screens should reflect known mechanisms of action of thyroid
disrupting chemicals
Developmental Assays
Hypothyroidism during development produces profound permanent changes in the
auditory system, central nervous system and the male reproductive system. A number of
assays or test systems can be used to detect chemicals that produce hypothyroidism.
However, most of these assays or tests systems are time consuming and not necessarily
specific for hypothyroidism. In addition, pronounced decreases in serum T4
concentrations are required to detect the behavioral or morphological changes. In
experimental animals,.alterations in serum THs can be detected at lower dose levels than
8
-------
those required to detect the behavioral and morphological changes in these systems.
Because of the greater sensitivity and simplicity, determination of serum TH
concentrations is recommended instead of these developmental assays.
Screening for Chemicals That Alter Thyroid Action, Function, and Homeostasis in
Non-Mammalian Wildlife.
Thyroid hormones are critical in development for non-mammalian wildlife. There
are examples of chemicals that alter thyroid hormones in non-mammalian wildlife and
produce developmental toxicities. While there are similarities between mammals and other
wildlife species there are some differences. For example, evaluating peripheral processes
controlling T3 production and tissue T3 seem more important than serum hormone
measurements. Many of the assays discussed for mammalian system can be used for non-
mammals provided these assays are sufficiently modified to examine the non-mammalian
species of interest. The tadpole metamorphosis assay is potentially useful as a screen but
requires further standardization.
CONCLUSION
The workshop participants attempted to address the merits and limitations of
numerous assays available as potential screening methods for chemicals that alter thyroid
hormone action, function or homeostasis. Not every existing assay was examined due to
limitations of time and expertise. Some of the assays evaluated may be useful as screens
but most of the assays are more appropriate for mechanistic studies.
-------
INTRODUCTION
Endocrine disruption has emerged as an environmental issue based on the
hypothesis that exposure to certain environmental chemicals alters the endocrine system,
and increases the incidence of endocrine diseases and disorders in both humans and
wildlife (Adams, 1992; Colborn et al., 1992; Kavlock et al., 1996). While research
evaluating this hypothesis is ongoing, there are thousands of synthetic and naturally
occurring chemicals that must be considered. The development of screening methodology
for endocrine disrupting chemicals (EDCs) would enable researchers to narrow the focus
of their research efforts (Kavlock et al., 1996). In the United States, screening for EDCs
was recently mandated by congressional legislation in the Food Quality Protection Act of
1996 (Public Law.104-170) and the Safe Drinking Water Act of 1996 (Public Law 104-
182). A series of workshops sponsored by the Chemical Manufacturers Association, the
United States Environmental Protection Agency and the World Wildlife Fund focused on
the development of screens for endocrine disrupting chemicals for both humans and
wildlife. The following report is a consensus from the workshop entitled Screening
Methods for Chemicals That Alter Thyroid Hormone Action, Function and Homeostasis.
The workshop focused on over 20 assays or test systems that have been used to
examine chemicals that alter synthesis, storage, transport, and catabolism of thyroxine
(T4) and 3,5,3'-triiodothyronine (T3), assays which examine ligand binding and activation
of the thyroid hormone receptor, and in vivo assays that examine the effects of antithyroid
agents and thyromimetics in mammalian and non-mammalian wildlife models. The
meeting focused on chemicals that alter thyroid gland function through pharmacodynamic
means and did not include chemicals that were directly cytotoxic to the thyroid gland. It
was not the purpose of the meeting to recommend a screening battery nor to deal with
policy issues pertaining to the use of such screens. The product of the workshop is
intended to describe the methods which are currently available or could be developed in
the near future for screening and testing.
10
-------
Thyroid Function and Regulation.
The thyroid gland produces T4 and T3. The thyroid hormones (THs) have two
predominant functions. The first is a critical role in growth and development. One of the
best examples of the importance of thyroid hormones in growth and development is the
metamorphosis of amphibians, in particular the metamorphosis of tadpoles into frogs
(Kaltenbach, 1996; Dodd et al., 1976; Kollros, 1961). Other examples of the importance
of THs in development are the transformation of salmon from freshwater dwelling par to
seawater dwelling smolts (Dickhoff and Sullivan, 1987; Specker, 1988), flounder
metamorphosis (Inui and Miwa, 1985), and development of the central nervous system in
humans and other mammals. In humans, severe hypothyroidism during development
results in cretinism (Legrand, 1979; Porterfield, 1994). The second major function of
thyroid hormones is to maintain metabolic homeostasis in mammals (Farrell and
Braverman, 1995).
The synthesis and storage of TH predominately occurs in the thyroid gland and the
synthesis is regulated by the pituitary hormone, thyroid stimulating hormone (TSH). Most
of the TH in the thyroid is present as T4. While a small proportion of thyroid localized
TH is T3, most T3 comes from the deiodination of T4 by tissue specific deiodinases. The
processes involved in the synthesis, storage, release, transport and metabolism of THs are
complex and consist of the following: (1) uptake of iodide ion by the thyroid gland, (2)
oxidation of iodide and the iodination of tyrosine, (3) coupling of iodotyrosine residues to
produce iodothyronines, (4) proteolysis of thyroglobulin and release of T4 and T3 into the
blood, (5) binding to serum transport proteins, (6) target tissue synthesis of T3 from T4,
(7) catabolism of T4 and T3 in peripheral tissues, (8) catabolism and biliary elimination of
THs in the liver. There are many examples of pharmaceutical, environmental and
naturally occurring chemicals which alter one or more of these processes in mammals,
these have been reviewed by Hill et al. (1989) and Atterwell and Aylward. (1995).
The actions of thyroid hormones are mediated by their interaction with nuclear
thyroid hormone receptors (TR). These receptors are part of the steroid receptor super
11
-------
family (Evans, 1988) and are the cellular homologs of the oncogene c-erb-A. There are
four known isoforms of the thyroid receptor which are derived from two genes c-erb-A
beta (TR beta, 2) and c-erb A alpha (TR alpha, 2) (Lazar, 1993). These TRs bind to
specific sites on DNA and also form heterodimers with other nuclear receptors such as the
retinoid X receptor (RXR). In addition, TRs form complexes with a number of additional
modulating and accessory proteins involved in gene transcription. The affinity of the
nuclear TRs is 10-20 times greater for T3 than for T4 (Oppenhiemer et al., 1987; Cody,
1991). Unlike the estrogen receptors, there is little evidence of environmental chemicals
binding the TR. However, the hypothesis that some environmental chemicals may bind to
TR resulting in toxicological responses has not been adequately tested.
There are several sites in the synthesis, transport and metabolism of THs that can
be altered by xenobiotics. In addition, it is possible that xenobiotics can alter TH signaling
through the TR either by directly binding to TR or indirectly by altering phosphorylation
of TR or through interactions with other accessory proteins. Due to the complexity of TH
function and regulation, it is unlikely that a single assay will be available to detect
chemicals that act on any or all of these pathways. The utility of a screen depends on its
specificity (is the response pathognomonic for alterations in thyroid function), sensitivity
(defined as the response of the method to low doses or to weak acting chemicals), test
duration, simplicity, and limitations. A number of assays or experimental systems were
evaluated for their potential use as screens to detect chemicals that disrupt thyroid
hormone catabolism and signaling. The workshop participants acknowledged that several
of these methods could be used as screening tools.
ASSAYS FOR THYROID SYSTEM DISRUPTION IN MAMMALS
Thyroid hormone concentrations and thyroid gland histology
In humans, alterations in thyroid function are initially diagnosed by either physical
examination, for enlarged thyroid gland, or by measuring serum hormone concentrations.
12
-------
Serum hormone concentrations are such good indicators of thyroid function in humans that
in the U.S., newborn infants are required to have blood samples collected for TSH and/or
T4 determinations prior to leaving the hospital. The American Thyroid Association has
recommended determination of serum TSH and free T4 concentrations (Surks et al., 1990)
as the standard measure of thyroid function. Total T4 was not recommended as a measure
because false positives can be caused by disease states and pharmaceutical agents which
alter thyroxine binding globulin (TBG), the main serum binding protein in humans.
Changes in TBG alters total serum T4 concentrations, but may not necessarily alter free T4
concentrations. It is thought that free T4 is available to enter the cell and that the
concentrations of free T4 are proportional to the tissue concentrations of T3 and T4. The
American Thyroid Association considers a diagnosis of primary hypothyroidism
confirmed if the patient has decreased free T4 serum concentrations accompanied by
increased serum TSH concentrations (Surks et al., 1990). Hyperthyroidism in humans is
confirmed if the patient has increased free T4 serum concentrations accompanied by
decreases in serum TSH concentrations (Surks et al., 1990).
The synthesis of THs is tightly regulated. Decreases in serum THs as a result of
chemical inhibition of TH synthesis or transport or induction of catabolism increases TSH
release from the pituitary. The increased TSH causes hypertrophy and hyperplasia of the
follicular cells of the thyroid resulting in an increase in thyroid gland weight with a
concomitant increase in synthesis and release of thyroid hormones. The stimulation of
thyroid hormone synthesis and release by TSH can result in the normalization of serum T4
and T3 concentrations. The importance of these relationships is that there is a
compensatory mechanism by which serum TH concentrations are regulated. Early on in
chemical exposure, the serum concentration of THs will decrease. The decrease in serum
THs results in increased secretion of TSH by the pituitary which may eventually restore
normal T4 and T3 serum concentrations. When examining chemical effects on serum TH
concentrations at later time points in exposures, serum concentrations may not be affected
due to the compensatory mechanisms (McClain et al., 1989; McClain, 1995). In designing
13
-------
experiments to examine the effects on serum thyroid hormones, this compensatory
mechanism must be considered and appropriate temporal relationships examined.
Another difficulty in determining serum concentrations of THs and TSH is their
responsiveness to stress and time of day of sampling. For example, transporting animals
from one room to another, will increase TSH and T3 by approximately 2 fold over a 1
hour period with initial increases occurring within 5 minutes (Dohler et al., 1979).
Circadian rhythms of THs occur in rats with peak serum concentrations occuring at
approximately noon (Dohler et al., 1979). In addition, there is some evidence of
alterations in TH concentrations associated with stages of the estrous cycle (Dohler et al.,
1979). THs also change with age and in male rats increase strikingly from postnatal day
33 to 50 (Dohler et al., 1979). These and several other confounding factors are reviewed
by Dohler et al., (1979). The determination of serum concentrations of THs requires
careful consideration of these factors particularly for weakly goitrogenic chemicals.
Finally, detecting small changes in serum TSH and TH concentrations can be problematic
due to the large inter-animal variability and small changes in TSH (20-30%) can have
significant impact on thyroid gland function (McClain, 1995).
In humans, free T4 and TSH serum concentrations are used to assess thyroid
function. In experimental animals, researchers have measured both free and total T4 and
T3 as well as TSH serum concentrations (Bastomsky, 1976; Barter and Klassen, 1992;
Brouwer et al., 1991;Brouweret al., 1998; McClain et al., 1989). Determination of both
free and total THs can provide complementary information that would guide further
testing of a chemical. For example, free T4 is an indicator of the amount of hormone
available for tissue uptake and for fetal transfer.
An area which lacks adequate experimental data is the exact time course of the
compensatory mechanism for the different classes of chemicals that alter serum TH
concentrations. Time course data for inducers of undine diphosphate
glucuronlytransferase (UDP-GT) indicates that continued dosing with these chemicals for
7, 14, and 21 days produces alterations in serum TH concentrations (Barter and Klaassen,
14
-------
1992; McClain et al., 1989; Christensen et al., 1994). Histological changes in the thyroid
or changes in thyroid weight occurred following 14 days of dosing (Barter and Klassen,
1992; McClain et al., 1989; Christensen et al., 1994). There has been no systematic
attempt to determine the time course of the compensatory response to decreases in serum
TH concentrations following exposure to different classes of chemicals which alter serum
TH concentrations through different mechanisms.
As described above, the compensatory increases in TSH result in proliferation of
the follicular cells in the thyroid gland and these changes can be detected histologically as
increased follicular cell numbers and as increases in thyroid gland weight (Capen, 1995).
These histological changes appear less sensitive to confounders described above and may
provide a better assessment of thyroid function than serum hormone concentrations.
Similar dose-response relationships between decreases in serum hormone concentrations,
histological changes in the thyroid and increases in thyroid weight were observed in rats
administered sulfamethazine for 4 weeks (McClain, 1995). Furthermore, the use of
thyroid weights and histology may allow for screening chemicals previously tested in
subchronic studies. Caution is required for studies examined during the 1970's however,
since follicular cell hypertrophy indicative of TSH stimulation were not considered
pathological changes and may not have been reported.
Section Summary: One possible screen for chemicals that alter thyroid function
could be the measurement of serum TH concentrations in experimental animals following
treatment with a test compound. Methods for determining serum concentrations of THs
are readily available from commercial suppliers and these assays have been in use for
many years. Using determination of serum thyroid hormone concentrations in animals
following chemical exposure provides assessment of thyroid function equivalent to those
used clinically in humans. However, due to potential compensatory mechanisms,
histological assessment of thyroid gland in conjunction with the measurement of serum
TH concentrations is desirable in order to provide a more complete assessment of thyroid
function and thyroid hormone action (McClain, 1995; Capen, 1995). Future efforts to
15
-------
determine the most appropriate time point and exposure regimen for examining serum TH
concentrations are recommended.
Assays for Chemicals That Alter Synthesis, Secretion, Transport and Catabolism of
Thyroid Hormones
Serum concentrations of thyroid hormones can be caused by chemicals that inhibit
thyroid hormone synthesis, release and transport and by chemicals that increase
metabolism of thyroid hormones. If a chemical decreases serum thyroid hormone
concentrations, specific assays can be used to determine the mechanism by which these
hormone concentrations are decreased. These assays may be of value in screening for
chemicals which act through specific mechanisms. The assays described address steps 1-
8 in the synthesis and regulation of serum concentrations of thyroid hormones as described
in the introduction.
Peroxidase Assav: Thyroid peroxidases are the key enzymes in the synthesis of thyroid
hormones. There are a number of classes of synthetic chemicals that inhibit thyroid
peroxidase, e.g. thionamides, such as propylthiouracil, aromatic amines, such as
sulfathiazole, and polyhydric phenols such as resorcinol (Hill et al., 1989). In addition,
there are a number of naturally occurring chemicals which inhibit thyroid peroxidase such
as goitrin found in turnips and other cruciferous vegetables (Capen, 1995) and flavonoids
found in other plant products (Divi and Doerge, 1996). Thyroid peroxidases (TPO) have
two functions. The first is the iodination of tyrosine residues on thyroglobulin. The
second reaction is coupling of specific di- and triiodotyrosyl residues on thyroglobulin.
The iodination reaction can be readily determined using bovine serum albumin or tyrosine
as substrates (Divi and Doerge, 1996). In addition, the oxidation of guaiacol can be used
as an indicator of thyroid peroxidase activity (Divi and Doerge, 1994). All chemicals that
inhibit the iodination reaction also inhibit the coupling reaction (Doerge et al., 1993). The
coupling reaction can be assayed using either human low iodine thyroglobulin, pre-
16
-------
iodinated casein or guaiacol as substrates.
A disadvantage of the TPO assay is that purified hog TPO is the only form of TPO
commercially available. Purified human TPO is not commercially available, however,
there are efforts to develop a recombinant human TPO. Purified lactoperoxidase (LPO) is
commercially available. There is a good concordance between inhibitors of TPO and LPO
(Divi and Doerge, 1994) and LPO has been used as a model for TPO actions (Divi and
Doerge 1994). While TPO can be purified from experimental animals, the size of the
gland in rodents is extremely small, and purification of rodent TPO would be impractical
as a source of enzymes for a widely used screen.
One of the advantages of the TPO assay is that the sensitivity to chemical
inhibition of thyroid peroxidase from human and experimental animals can be directly
examined. In vitro studies have shown that TPO from monkeys is more resistant to
inhibition by PTU and sulfamethazine than is TPO from rodents (Takayama et al., 1986).
Comparisons of the relative sensitivity of TPO across species would assist in risk
assessment for chemicals that inhibit TPO activity. The iodination and coupling assays are
specific for chemicals that inhibit thyroid hormone synthesis and are unlikely to produce
false positives. However, these assays examine a specific mechanism of thyroid hormone
disruption and chemicals that affect other aspects of thyroid function are not detected.
Used alone as a screen, these assays have potential for false negatives (i.e.) chemicals that
alter TH concentrations through other mechanisms would not be detected). These assays
have been performed for many years, are well established in the scientific literature, and
numerous chemicals have been tested using these assays. While there are no published
methodologies that can be defined as high throughput screens, modification of this assay
into a high through put screen is under development in several laboratories.
Perchlorate Discharee Test: This assay examines the ability of the thyroid gland to
organify iodide into thyroid hormones (Baschieri, 1963; Atterwell et al., 1987; Christenson
et al 1996). This assay has been used for decades, in both animals and humans (Baschieri
17
-------
et al., 1983; Morgan and Trotter, 1957; Atterwell et al., 1987; Christenson et al 1996). In
this assay, animals are exposed to a test chemical and then administered NaI25I followed
later with perchlorate. Accumulation of 125I in the thyroid is then determined. When
administered after a dose of radioactive iodine, perchorlate promotes release of iodine that
has not been incorporated into thyroglobulin. If a chemical inhibits or deactivates thyroid
peroxidase or blocks iodide uptake into the thyroid, there would be a decreased
accumulation of 125I in the thyroid gland. This assay is specific for chemicals which
inhibit or deactivate thyroid peroxidase or block iodine uptake. This assay has potential for
providing mechanistic information on the actions of chemicals that alter thyroid function
but does not necessarily meet the requirements of a screen.
TRH Challenge Test: This assay examines the functional integrity of the hypothalamus-
pituitary-thyroid axis (Christensen et al. 1996). Briefly, this assay measures TSH
concentrations before and after challenge with thyrotropin releasing hormone (TRH).
Challenge with TRH should increase serum concentrations of TSH. Differences in TSH
concentrations in animals treated with test compounds compared to controls may suggest a
pituitary site of action for the chemical. This assay has been used both clinically (Same
and Refetoff, 1995) and experimentally (Christensen et al., 1996). The TRH challenge can
be used to distinguish between pituitary and hypothalamic causes of hypothyroidism
(Same and Refetoff, 1995). However, the underlying assumption prior to performing this
test is that the chemical of interest produces hypothyroidism. While the TRH challenge
has potential for providing mechanistic information on the actions of chemicals that alter
thyroid function the assay may not be a useful screen due to the limited number of
chemicals which may act through this mechanism.
Serum Protein Binding Assays: In mammalian systems, the serum binding proteins
for thyroid hormones are thyroid binding globulin (TBG), transthyretin (TTR), and
albumin. TBG and TTR are specific for THs and T4 has a greater affinity for these serum
18
-------
binding proteins than T3 (Dohler et al., 1979). TBG is present in humans and primates and
appears responsible for the much longer half-life of T4 and T3 in humans compared to
other species (Dohler et al., 1979). TTR is present in humans, rodents and non-human
primates. In humans, TBG is the predominant binding protein while in rodents TTR is the
predominant carrier of THs. In humans, the main function of TTR appears to be the
transport of T4 into the cerebral spinal fluid (Hebert et al., 1986). In addition, TTR
transports T4 into the fetus. There are a number of reports of chemicals which displace T4
from TTR and TBG. The research on environmentally relevant chemicals has focused
mainly on the polyhalogenated dibenzo-p-dioxins, biphenyls and diphenylethers (Brouwer,
et al., 1990; Brouwer, 1991; Brouwer et al.< 1998; McKinney et al., 1985; McKinney et
al., 1987; McKinney and Waller, 1994). The displacement of T4 from these serum
binding proteins is hypothesized to increase the clearance of T4 and decrease serum T4
concentrations. It has also been suggested that TTR binding is predictive of interactions
with other TH binding proteins such as the deiodinases and sulfotransferases as well as
chemicals with high fetal accumulation (Brouwer, et al., 1998).
These assays have been performed in several laboratories examining xenobiotics
for several decades (Brouwer, et al., 1990; Ogilvie and Ramsden, 1988). While these
assays can be modified for high through-put screening they are specific for chemicals that
compete with thyroid hormones for serum binding proteins and will not detect chemicals
that act through other mechanisms. In addition, the use of either TBG or TTR may not be
relevant for non-mammalian species such as teleosts. However, one of the strengths of
this assay is that it may be predictive of chemicals that alter fetal concentrations of TH and
may provide for a useful screen.
Deiodinase Assays: In mammals, approximately 80% of the T4 secreted by the thyroid
gland is deiodinated in target tissues into either T3, the most active form of the THs or into
reverse T3, an inactive iodothyronine (Engler and Burger, 1984). There are several
enzymes involved in the deiodination of T4, T3 and their metabolites and the expression
19
-------
of these proteins is tissue specific. Type I deiodinase catalyzes the 5-deiodination of T4,
rT3 and the sulfated metabolites of T4 and T3 (Visser, 1988). Type I deiodinase is
sensitive to PTU inhibition and is found in liver, lung, kidney, pituitary and thyroid
(Chopra, 1977; Green, 1978). Type n deiodinase is present in the CNS, brown adipose
tissue, anterior pituitary and the placenta (Silva et al., 1982; Visser et al., 1983; de Ona et
al., 1991). Type n deiodinase is insensitive to PTU. In the brain type U deiodinase
converts T4 into T3 and ensures adequate brain concentrations of T3 during critical
periods of development (de Ona et al., 1991; Calvo et al., 1990; Obregon, et al., 1991) and
during hypothyroidism (Escobar-Morreale et al., 1997). Type IQ deiodinase is resistant to
PTU and catalyzes the conversion of T3 and T4 into 3,3'diiodothyronine and rT3,
respectively in brain, skin, placenta and fetal tissues (Kaplan et al., 1983; Huang et al.,
1985; Huang etal., 1988).
The deiodinases are critical in regulation of serum and tissue concentrations of
THs. Decreases in serum concentrations of T4 alters expression of the different tissue
deiodinases. For example, prenatal exposure to Aroclor 1254 increases brain type n
deiodinase in rats with decreased serum T4 (Morse et al., 1996). There are also tissue-
specific and isoform-specific changes in deiodinases following thyroidectomy and T4 and
T3 replacement in rats (Escobar-Morreale, 1997). Some chemicals, such as PTU, also
inhibit deiodinase activity. Deiodinase assays have been used for decades to understand
the metabolism of THs. Because the activity of these enzymes are dependent upon serum
concentrations of these hormones, these assays would be sensitive towards chemicals that
alter serum TH concentrations. However, alterations in deiodinase activity also alters
serum TH concentrations. This dose response relationship decreases the utility of this
assay as a screen. If serum TH concentrations are changed by deiodinase inhibitors, it may
be easier to measure serum TH concentrations than it is to determine deiodinase activity.
Similar to many of the assays described above, these assays have greater utility in
understanding the mechanism of action of a chemical rather than as an initial screen.
20
-------
Glucuronidation Assays: Glucuronidation followed by biliary elimination of T4 is one
of the major pathways of deactivation of T4. In humans there is evidence of sulfation of
T4 as well. In mammals, there are at least 4 isoforms of uridine diphosphate
glucuronosyltransferases (UDP-GT) which glucuronidate T4. Several classes of chemicals
induce UDP-GTs responsible for the glucuronidation of T4 (Barter and Kla&sen, 1992;
McClain, 1995; Capen and Martin, 1989; Atterwill and Aylward 1995; Brouwer, 1991).
Induction of thyroxine glucuronidation increases clearance and decreases serum
concentrations of T4. Induction of T4 glucuronidation is typically determined in hepatic
microsomes from animals treated with test chemicals. These assays have been performed
for decades in numerous laboratories throughout the world. These ex vivo assays require
several days of dosing of the test chemical. The advantage of this type of assay is that it is
responsive to metabolic activation of the test chemical because exposure occurs in vivo.
The activity of hepatic microsomal thyroxine glucuronidation is not as sensitive to stress
and circadian rhythms as is measurements of serum TH concentrations. The disadvantage
is that these assays are not developed for high throughput screening and at present are
laborious.
Section Summary: The assays described in this section are specific for particular
mechanisms of action. A combination of these assays could provide predictive
information on the availability of intracellular T3 concentrations, particularly in the fetus.
This information could be useful in assessing the potential adverse effects of chemicals
that disrupt thyroid hormone homeostasis and tissue concentrations. These assays have
been used to understand the mechanism of either altered serum concentrations of TH and
TSH or changes in thyroid histopathology (McClain et al., 1995; Atterwell and Aylward,
1995; Poole et al., 1988; Olgivie and Marsden, 1988; Brouwer 1991). If these assays
were to be used as initial screens, all of them would have to be performed in order to
demonstrate that a chemical does not alter TH concentrations.
21
-------
TR Binding and Activation.
Chemicals can alter thyroid hormone action by binding to the T3 specific nuclear
receptors. These receptors are part of the steroid receptor superfamily (Evans, 1988) and
are the cellular homologs of the c-erbA oncogenes. There are several isoforms of the
receptors which have tissue specific localization (Lazar, 1993). The structure-activity
relationships for binding to the nuclear thyroid hormone receptor have been determined
using crude nuclear homogenates (Oppenheimer et al., 1987; Cody, 1992) as well as
various TR isoforms expressed in E. Coli. or translated in vitro (Cheng et al., 1994;
Schueler et al., 1990). These binding studies have focused on T3 analogs and not on
environmentally relevant chemicals (Oppenheimer et al., 1987; Cody, 1992; Cheng et al.,
1994; Schueler et al., 1990). Several environmentally relevant classes of chemicals have
been proposed to bind to the nuclear T3 receptors, such as the polyhalogenated dibenzo-p-
dioxins, dibenzofurans, biphenyls and diphenyl ethers (McKinney, et al., 1987; McKinney
and Waller, 1994). However, this hypothesis has not been adequately tested. At present
there is a lack of evidence that environmentally relevant chemicals bind to TRs, which
should not be confused with the presence of negative evidence. It should be noted that the
chemical that have been proposed to bind to TRs also decreases serum TH concentrations
in experimental animals and are known thyroid hormone disrupters (Bastomsky, 1976;
Barter and Klaassen, 1992; Brouwer, 1991; van Birgelen et al., 1994).
In Vitro Binding Assays. In vitro binding assays can be used as potential screens for
chemicals that bind to TRs. The classical binding assays have used nuclear extracts from a
variety of tissues and cell lines expressing TRs (Cheng et al., 1994). More recent studies
have used various TR isoforms expressed in E. Coli or translated in vitro (Cheng et al.,
1994; Schueler et al., 1990). These assays require separating bound from free hormone
using either filtering or chromatographic methods. Either separation method is
cumbersome and timejconsuming. More recent advances have employed solid-state
22
-------
binding assays using specific isoforms of TRs. The solid-state binding assays developed
allow for high throughput screening. In the solid state binding assays, the thyroid hormone
receptor is coupled to either a multiwell plate or to beads. Coupling of the receptors to
plates or beads readily enables the separation of free and bound ligand without the use of
either filtering or chromatographic methods. Only 3 of the 4 TR isoforms have ligand
binding capability and of two of these (TR beta, and TR beta^ have identical ligand
binding domains. Binding assays are expected to have a very low rate of false positives.
False negatives can occur if the chemical requires metabolic activation or if solubility
problems are encountered.
Transfection and Transformation Assays. One of the problems with TR binding assays
is that they cannot differentiate between agonists and antagonists. Alternative assays
which would examine receptor binding and differentiate between agonists and antagonists
are systems in which a specific TR is transfected into a mammalian cell line along with a
reporter gene, typically coding for either luciferase or beta-galactosidase (Allegreto and
Hayman, 1992). Transformed yeast cell lines containing TR gene constructs have also
been developed. In these systems T3, or other ligands to TR, bind and activate the
receptor which then interacts with specific response elements upstream from the reporter
gene and enhance its transcription. The increased transcription is determined by increased
enzymatic activity of the reporter gene product, e.g. luciferase. Chemicals can be tested
alone or in combination with T3 to determine agonist or antagonist properties. Similar
systems have been used to examine the interactions of TR with different response
elements (Lazar et al., 1994), different cofactors (Katz et al., 1993) and with
phosphorylation of TR (Boat, et al., 1994). While these systems have not been used for
screening for environmental chemicals which are TR ligands, similar screens have been
developed for estrogens and androgen agonists and antagonists (Allegreto and Heyman,
1996).
Transformed yeast cell and transfected mammalian cell lines have been used to
23
-------
study several of the steroid hormone receptor super family members. There are
differences between the assays used for estrogen, androgen and thyroid hormone receptors.
There are only two estrogen receptors and a single androgen receptor in contrast to the four
isoforms of TR. TRs form heterodimers with RXR (Berrodin et al., 1992; Reginato, et
al., 1996; Meier et al., 1993) while the estrogen and androgen receptors form homodimers.
Both TR and peroxisome proliferator-activated receptor (PPAR) form heterodimers with
RXR and agonists of PPAR can alter TR mediated gene expression by binding competing
for RXR (Bhat et al., 1996). Hence, chemicals can alter TR activation by altering RXR or
PPAR pathways. TR activation is also regulated by phosphorylation (Bhat et al., 1994),
similar to the estrogen and androgen receptors. In designing a screen for TR ligands,
chemicals may have different effects depending upon the TR transfected, the response
element used and their interactions with PPAR and RXR. Because of the complexity of
this system, several different screens would have to be incorporated to account for the
multiplicity of interactions of the different TR isoforms. An advantage of the transfection
assays is that chemicals that alter TR activation through mechanisms not involving direct
binding to TR would be detected in these assays. Another advantage of these assays is
that they are readily adapted to high through put screens.
A major disadvantage of these in vitro screens is the lack of metabolic capability of
the cells or assays. It is possible that the metabolites of some chemicals would produce
these effects and not the parent compound. The cell lines typically used in these assays
have limited ability to metabolize the test compounds, particularly persistent organic
pollutants such as the PCBs and the dioxins. The transformation assays in the yeast have
additional drawbacks in that for many chemicals entry into the yeast is limited due to the
cell wall.
GH} Cell Assay for Thyroid Hormone Action: An in vitro bioassay has been
designed which can detect TR agonists (Hohenwarter et al., 1996). This assay uses the rat
pituitary tumor cell line GH3. The growth of these cells are dependent upon thyroid
hormones when plated at low-density in serum-free medium (Hohenwarter et al., 1996).
24
-------
In addition, the morphology of these cells is also altered by THs in a dose-dependent
manner. One form of the assay measures cell proliferation in response to TR agonists by
the determination of the transformation of MTT tetraxolium salt into MTT fromazan by
mitochondrial enzymes (Hohenwarter et al., 1996). This assay is performed on micro-well
plates and can be considered a high through-put screen. Although this assay is relatively
new, it has the potential to provide valuable information as a screen for chemicals that
activate TR.
Section Summary: There are clear examples of environmental estrogens, both
synthetic and naturally occurring, that bind to the estrogen receptor and act as agonists,
antagonists or partial agonists (Gray et al., 1997). In addition, there are several chemicals
found in the environment that act as anti-androgens (Gray and Kelce, 1996). There are no
know environmental chemicals that act as either TR agonists or antagonists. While the
hypothesis that environmental chemicals bind TR has been proposed, this hypothesis has
not been adequately tested. Recent methodological developments resulting in high
through-put screens could be performed on a limited number of chemicals to test this
hypothesis. However, broad-based screening should reflect known biological
mechanisms.
Developmental Assays.
The role of thyroid hormones in developing humans and other animals is well
documented. Hypothyroidism during development leads to permanent alterations in a
number of organ systems including the central nervous system and the male reproductive
system. The sensitivity of developing animals may provide models for testing and
screening chemicals that alter thyroid hormone catabolism or interfere with thyroid
hormone signaling.
25
-------
Neurodevelopmental Assays
The development of the central nervous system is dependent upon thyroid
hormones for control of neuronal proliferation, initiation of neuronal differentiation,
formation and development of neuronal processes and timely myelinization of the neurons
(Porterfield, 1994). In humans, hypothyroidism induced by iodine deficiency results in
neurologic endemic cretinism. This disorder is characterized by a high incidence of severe
mental retardation, deaf-mutism, and problems with gross and fine motor coordination.
Maternal hypothyroidism during pregnancy results in a increased incidence of neurologic
and behavioral disorders in the offspring. In rodents maternal hypothyroidism produces a
variety of behavioral and morphological changes in the brain similar to those observed in
humans.
Morphological and Biochemical Assays in Developing Brains: Morphological and
biochemical changes in the developing brain have been observed in animals exposed to
agents that decrease thyroid hormone concentrations, such as PTU. For example,
decreased brain weight occurs in rodents with marked decreases in serum THs during
perinatal development (Balaza et al., 1968; Behnam-Rassoli et al., 1991). Perinatal
hypothyroidism also results in morphological abnormalities in the organ of Corti (Deol,
1973; Uziel et al., 1981, 1983). Biochemical changes observed in hypothyroid animals
include decreases in myelin basic protein and alterations in neurotransmitter
concentrations among others (Porterfield, 1994). The morphological and biochemical
changes induced by hypothyroidism are detectable when maternal, fetal or neonatal serum
T4 concentrations are significantly decreased.
Behavioral Testing: Numerous behavioral assays have examined the effects of
goitrogens or iodine deficiency in developing mammals. Hypothyroidism during
development delays eye opening (Coner and Norton, 1982), reflex development (Coner
and Norton, 1982) and weaning (Blake and Henning, 1985) in rodents. Depressed motor
activity has also been demonstrated following developmental hypothyroidism (Rastogi et
al., 1976). Exposure to PTU in the drinking water from gestational day 18 to postnatal day
26
-------
21 produces delays in eye opening, reduced body weights, decreased or delayed
preweaning motor activity, and increased postweaning motor activity (Goldey et al., 1995).
Similar to humans, developmental hypothyroidism in rodents permanently alters auditory
function (Goldey et al., 1995a; Goldey et al., 1995b). These behavioral assays can be used
to detect hypothyroidism, however, most of these behavioral changes may not be specific
to hypothyroidism and have potential for a high rate of false negatives. More importantly,
these behavioral changes occur only when there are significant decreases in serum T4
concentrations (Goldey et al., 1995a; Goldey et al., 1995b).
Male Reproductive System Development
Testes Size and Sperm Counts: Thyroid hormones are critical for developing humans and
animals. The effects of hypothyroidism in infants can be severe and are permanent.
Hypothyroidism in humans during the juvenile stage in boys is associated with
megalotestis and high sperm counts. Maternal iodine deficiency or repeated exposure to
goitrogens, such as PTU or PCBs during lactation increases testes size and sperm counts in
rats when the animals reach maturity (Kirby et al., 1992; Cooke et al., 1993; Cooke et al.,
1996). Similar findings have been reported in mice, hamsters and roosters. Conversely,
neonatal hyperthyroidism results in the decreased testis size and lower sperm counts (van
Haaster et al., 1993). Hence, testis weight and sperm counts can be used as measures of
thyroid status in developing animals. An advantage of these measurements is their ease.
Testes weights are simple to determine and methods to measure sperm counts have been
developed over decades and are readily performed. A disadvantage of this assay is that it
requires repeated dosing of the animals during lactation and a waiting period of several
weeks prior to measuring the endpoints. In addition, these responses are observed only
when there are significant decreases in serum thyroid hormones concentrations (Kirby et
al. 1992; Cooke et al., 1993).
Section Summary: Hypothyroidism during development produces profound
permanent change in the 'auditory system, central nervous system and the male
reproductive system. A number of assays or test systems can be used to detect chemicals
27
-------
that produce hypothyroidism. However, most of these assays or tests systems are time
consuming and not necessarily specific for hypothyroidism. In addition, pronounced
decreases in serum T4 concentrations are required to detect the behavioral or
morphological changes. Alterations in serum THs can be detected at lower dose levels
than those required to detect the behavioral and morphological changes in these systems.
Because of the greater sensitivity and simplicity, determination of serum TH
concentrations is recommended instead of these developmental assays. It should be
remembered that using adult, pubescent or prepubescent animals may be qualitatively
predictive of fetal response, it may not be quantitatively predictive of dose or response in
fetal tissue.
Screening for Chemicals That Alter Thyroid Function, and Homeostasis in Non-
Mammalian Wildlife.
Similar to mammalian systems, the thyroid and THs are critical in the development
of amphibians, birds, fish and reptiles (Kaltenbach, 1996;McNabb, 1992; Bales and
Brown, 1993). While there are similarities in the basic structure and function of the
thyroid system among vertebrate species (Gorbman et al., 1993; Bales and Brown, 1993),
there are also differences that must be considered when recommending tests of thyroid
function. TRs have been cloned in one species of teleosts (Yamano and Inui, 1995), in
two species of frogs (Tata, 1996), and in chickens (Lazar, 1993). TRs from all species
examined show similar structure-binding activity relationships with regard to T4, T3 and
their analogs (Gallon, 1980; Darling et al., 1982; Bres and Bales, 1986; Sullivan, 1987).
However, there are some differences in the regulation of THs by non-mammalian wildlife.
In teleosts negligible amounts of T3 is synthesized and secreted (Bales and Brown, 1993)
from the thyroid gland. The plasma proteins involved in transport of THs in teleosts bind
T3 preferentially in contrast to the mammalian plasma proteins which bind T4
preferentially (Bales and Brown, 1993). The serum TH binding proteins in teleosts does
not appear to be structurally related to transthyretin. While the serum binding protein
28
-------
found in the bullfrog tadpole is a homolog of transthyretin, it preferentially binds T3
(Yamauchi et al., 1993) In addition, there are seasonal changes in thyroid hormones in
teleosts that are not apparent in mammalian systems.
One important difference between mammals and fish and amphibians is the
hypothalamic control of TSH from the pituitary. In teleosts, the hypothalamus negatively
controls release of TSH, while in mammals it is positively controlled. In developing
tadpoles, the hypothalamus positively controls TSH release via corticotropin releasing
factor rather than TRH. TRH in tadpoles and adult frogs appear to play a role in
osmoregulation by regulating prolactin release from the pituitary (Denver, 1996). This
suggests that tests routinely used in rodents, such as the TRH challenge and the TTR
binding assays, may not be uniformly applicable in non-mammalian species. Some of the
assays used to assess thyroid function in rodents must be viewed cautiously when applied
to non-mammalian systems.
Despite some of the species differences in TH regulation, there is a concordance
between mammals and fish in response to many chemicals that alter thyroid hormone
function or homeostasis (Bales and Brown, 1993). Chemicals which demonstrate
significant differences in species sensitivity are the mono-ortho substituted PCBs which
are extremely efficacious in decreasing plasma or serum T4 in rodents but have little
effects on plasma TH in fish. Also several metals such as Hg and Cd alter TH
concentrations in fish but not in mammals (Bleau et al., 1996; Hontella et al., 1996;
Kirubagaran and Joy, 1995). Many of the assays described in previous sections could be
used to examine chemical effects on TH function and homeostasis in fish and other
wildlife if appropriately adapted for the species of interest. Alterations in thyroid function
can be examined histologically in teleosts (Bales and Brown, 1993), similar to the
mammalian system. However, it should be noted that the thyroid gland in teleosts is not
encapsulated and consists of diffuse, scattered follicles making metrics like thyroid
weights difficult to obtain. Because the thyroid consists of diffuse and scattered follicles,
histological evaluation can be difficult, particularly for weak goitrogens. In fish, there
29
-------
appear to be considerable control of the thyroid system via the mechanisms controlling
peripheral T3 production (Bales and Brown, 1993). Consequently measures of deiodinase
activities in conjunction with peripheral T4 assessments are required to thoroughly
evaluate T3 availability to target tissues.
Tadpole Metamorphosis Assay: The development of tadpoles into frogs occurs in
multiple stages with different organ systems developing at different times. Thyroid
hormones are required for metamorphosis (Kaltenbach, 1996; Dodd et al., 1976; Kollros,
1961) but TH action is modulated by other hormones (Wright et al., 1994; Iwamuro and
Tata, 1995). In conjunction with T3, corticosterone accelerates metamorphosis at later
stages of development (Gallon, 1990).. Circulating prolactin concentrations increase
toward the end of metamorphosis (Kaltenbach, 1996) and prolactin down regulates TR
expression, apparently modulating the stimulatory action of T3 (Tata, 1996). Chemicals
that alter tadpole development may not interact directly with TRs or directly alter TH
concentrations, but may act indirectly by altering other endocrine pathways. In addition,
chemicals that alter calcium homeostasis such as calmodulin antagonists also alter
metamorphosis (Kumar et al., 1993) The tadpole metamorphosis assay may be a valuable
tool for screening chemicals that alter TH signaling pathways either directly or indirectly.
However, this assay requires further validation and standardization prior to use as a screen.
Section Summary: Thyroid hormones are critical in development for non-mammalian
wildlife and there are examples of chemicals that alter thyroid hormones and produce
alterations in non-mammalian wildlife. Many of the assays discussed for mammalian
system can be used for non-mammals provided these assays are sufficiently modified to
examine the non-mammalian species of interest. The tadpole metamorphosis assay is
potentially useful as a screen but requires further validation and standardization.
30
-------
Conclusions:
The workshop participants attempted to address the merits and limitations of
numerous assays available as potential screening methods for chemicals that alter thyroid
hormone action, function or homeostasis. Not every existing assay was examined due to
limitations of time and expertise. Some combinations of the assays evaluated may be
useful as screens. Chemicals appear to alter thyroid hormone action by either inhibiting
synthesis of THs, altering serum binding to transport proteins or by increasing TH
metabolism. Few if any environmentally relevant chemicals have been demonstrated to
act as either TR agonists or antagonists. The development and implementation of screens
should reflect the known mechanism of action of thyrotoxic chemicals.
Screening for chemicals using either thyroid histology or serum TH concentrations
in mammals should provide tests that would produce few false negatives or false positives
Subchronic studies in mammals examining thyroid histology provide the most useful
measure of a chemicals thyrotoxic potency and efficacy. However, these assays are not
necessarily screens, and require dosing animals for at least 2-6 weeks to observe consistent
responses. Determination of serum TH concentrations in short-term tests may provide an
adequate initial screen for these chemical in mammals. The exact dosing regimen and time
course for these responses have not been adequately examined in the published literature.
Determination of serum TH concentrations and thyroid histology may also be of value in
teleosts however, indices of peripheral T3 production are better markers of thyroid status
and should be included when determining the effects of chemicals on teleosts.
31
-------
REFERENCES
Adams, NR. (1995). Organizational and activational effects of phytoestrogens on the
reproductive tract of the ewe. Proc. Soc. Exper. Biol. Med. 208:87-91.
Atterwill, CK, and Aylward, SP (1995). Endocrine toxicology of the thyroid for industrial
compounds. In "Toxicology of Industrial Compounds. H Thomas, R. Hess and F.
Waechter eds. Taylor and Francis Publishers, pp257-280.
Atterwill, CK, Collins, P, Brown, CG, and Harland, RF (1987). The perchlorate discharge
test for examining thyroid function in rats. J. Pharmacol. Methods. 18:199-203.
Barter, R and Klassen, C. (1992). UDP-glucuronosyltransferase inducers reduce thyroid
hormone levels in rats by an extra thyroidal mechanism. Toxicol. Appl. Pharmacol.
111:36-42.
Bastomsky, CH (1977). Enhanced thyroxine metabolism and high uptake goiters in rats
after a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology 101:292-296.
Berrodin, T. J., M. S. Marks, et al. (1992). Heterodimerization among thyroid hormone
receptor, retinoic acid receptor, retinoid X receptor, chicken ovalbumin upstream promoter
transcription factor, and an endogenous liver protein. Mol Endocrinol 6(9): 1468-78.
Balaza, R, Kovacs, S, Teichgraber, P, Cocks, WA, and Eayrs, JT (1968). Biochemical
effects of thyroid deficiency on the developing brain. J. Neurochem. 15:1335-1349.
Behnam-Rassoli, M, Hebert, LC, Howard, V, Pharoah POD, and Stanisstreet, M (1991).
Effect of propylthiouracil treatment during prenatal and early postnatal development on
the neocortex of rat pups. Neuorendocrinology 53:321-327.
Blake, HH, and Hennings, SJ (1985). Effect of propylthiouracil dose on serum thyroxine,
growth and weaning in young rats. Am J Physiol. 248:R524-R530.
Bres, O., and Bales, J.G. (1986). Thyroid hormone binding to isolated trout (Salmo
gairdneri) liver nuclei in vitro; binding affinity, capacity and chemical specificity. Gen
Comp. Endocrinol. 69:71-79.
Brouwer, A. (1991). The role of biotransformation in PCB-induced alterations in vitamin
A and thyroid hormone metabolism in laboratory and wildlife species. Biochem. Soc.
Trans. 19:731-7.
32
-------
Brouwer, A, Klasson-Wehler, E, Bokdam, M, Morse, DC, and Traag, WA (1990).
Competitive inhibition of thyroxine binding to transthyretin by monohydroxy metabolites
of 3,4,3',4'-tetrachlorobiphenyl. Chemosphere 20:1257-1262.
Brouwer, A., Morse, DC, Lans, MC, Gerlienke Schuur, A., Murk, AJ, Klasson-Wehler, E.,
Bergman, A., and Visser, TJ (1998). Interactions of persistent environmental
organohalogens with the thyroid hormone system: Mechanisms and possible consequences
for animal and human health. Toxicol. Indust. Health 14:59-84.
Calvo. R, Obergon, MJ,. Ruiz de Ona, C, Escobar del Ray, F, Morreale de Escobar, G
(1990). Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine
but not of 3,5,3'-triiodothyronine in the protection of the fetal brain. J. Clin. Invest.
86:889-899.
Chopra, IJ (1977). A study of extra thyroidal conversion of thyroxine to 3,3',5-
triiodothyronine in vitro. Endocrinology 101:453-463.
Christenson, WR, Becker, BD, Wahle, BS, Moore, KD, Das, PD, Lake, SG, Van Boethem,
DL, Stuart, BP, Sangha, GK, and Thyssen, JH. (1996). Evidence of chemical stimulation
of hepatic metabolism by an experimental acetanilide (FOE 5043) indirectly mediating
reductions in circulating thyroid hormone levels in the male rat. Toxicol. Appl.
Pharmacol. 29:251-259.
Cody, V. (1991). Thyroid hormone structure-function relationships. In, Werner and
Ingbar's The Thyroid (Braverman, LE and Utiger, RD, eds) JB Lippincott Co.,
Philidelphia, pp. 887-919.
Colborn, T and Clement, C (eds). Chemically induced alterations in sexual and functional
development: the wildlife/human connection. Advances in Modem environmental
Toxicology, Vol XXI. Princeton, NJ:Princeton Scientific Publishing, 1992;401pp.
Coner, CP, and Norton, S. (1982). Effects of perinatal methimazole exposure on a
developmental test battery for neurobehavioral toxicity in rats. Toxicol. Appl. Pharmacol.
63:133-141.
Cooke, PS, Kirby, JD, and Porcelli, J. (1993). Increased testis growth and sperm
production in adult rats following transient neonatal goitrogen treatment: Optimization of
the propylthiouracil dose and effects of methimazole. J. Reprod. Fertil. 97:493-499.
Cooke, PS, Zhao, Y-D, and Hansen, LD (1996). Neonatal polychlorinated biphenyl (PCB)
treatement increases adult testis size and sperm production in the rat. Toxicol. Appl.
Pharmacol. 136:112-117.
33
-------
Darling, D.S., Dickhoff, W.W., and Borbman, A. (1982). Comparisons of thyroid
hormone binding to hepatic nuclei of the rat and a teleost (Oncorhynchus kisutch).
Endocrinology 111: 1936-42.
Davis, K. D. and M. A. Lazar (1992). Selective antagonism of thyroid hormone action by
retinoic acid. J Biol Chem 267(5): 3185-9.
Denver, RJ. (1996). Neuroendocrine control of amphibian metamorphosis. In L.I. Gilbert,
J.R. Tata, and E.G. Atkinson, eds., Metamorphosis: Postembryonic reprogramming of
gene expression in amphibian and insect cells. Academic Press, New York, New York
USA, pp 433-464.
Dickhoff, W.W. and Sullivan, C.V. (1987). Involvement of the thyroid gland in
smoltification , with special reference to metabolic and developmental processes.
American Fisheries Society Symposium, 1:197-210.
Divi, R. L. and D. R. Doerge (1994). Mechanism-based inactivation of lactoperoxidase
and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32): 9668-74.
Divi, R. L. and D. R. Doerge (1996). Inhibition of thyroid peroxidase by dietary
flavonoids. Chem Res Toxicol 9(1): 16-23.
Dodd, MHL, and Dodd, JM (1976). The biology of metamorphosis. In: Lofts B (ed)
Physiology of the Amphibia. Academic Press, New York, pp 467-599.
Doel, MS (1973). An experimental approach to understanding and treatment of hereditary
syndromes with congenital deafness and hypothyroidism. J. Med. Genet. 10:2039-2045.
Doerge, D. R., C. J. Decker, et al. (1993). Chemical and enzymatic oxidation of
benzimidazoline-2-thiones: a dichotomy in the mechanism of peroxidase inhibition.
Biochemistry 32(1): 58-65.
Dohler, K-D, Wrong, CC, and von Ur. Muhlen, A (1979). The rat as model for the study
of drug effects on thyroid function: consideration of methodological problems. Pharmac.
Ther. 5:305-318.
Bales, JG, and Brown, SB (1993). Measurement and regulation of thyroidal status in
teleost fish. Rev Fish Biol. Fisheries. 3:299-347.
Engler, D, and Burger, AG (1984). The deiodination of the iodothyronines and their
derivatives in man. Endocr. Rev. 5:151 -184.
34
-------
Gallon, V.A., (1980). Binding of thyroid hormones in vivo by hepatic nuclei of Rana
catesbeiana tadpoles. Endocrinology, 106:859-870.
Gallon, V. A. (1990). Mechanisms underlying the acceleration of thyroid hormone-
induced tadpole metamorphosis by corticosterone. Endocrinology 127(6): 2997-3002.
Goldey, E.S., Rehnberg, GL, and Crofton, KM (1995a). Effects of Developmental
hypothyroidism on auditory and motor function in the rat. Toxicol and Appl Pharmacol
135: 67-77.
Goldey, ES., Lau, C, Rehnberg, GL, and Crofton, KM (1995b). Developmental exposure
to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone
concentrations and causes hearing deficits in rats. Toxicol and Appl Pharmacol 135: 77-
88.
Gorbaman, A., Dickhoff, W.W, Vigna, S.R., Clark, C.L., and Ralph, C.L. (1983).
Comparative endocrinology. John Wiley and Sons, New York, New York.
Gray LE and Kelce, WR (1996). Latent effects of pesticides and toxic substances on
sexual differentiation of rodents. Toxicol. Indust. Health. 12:515-531.
Gray LE et al., (1997). Endocrine Screening methods workshop report: Detection of
estrogen and androgenic hormonal and antihormonal activity for chemicals that act via
receptor or steroidogenic enzyme mechanisms. Reproductive Toxicology 5:719-750.
Green, WL (1978). Metabolism of thyroid hormones by the rat thyroid tissue in vitro.
Endocrinology, 103:826-837.
Hill, RC, Erdreich, LS, Paynter, OE, Roberts, PA, Rosenthal, SL, and Wilkinson, CF
(1989). Thyroid follicular cell carcinogenesis. Fund. Appl. Toxicol. 12:629-697.
Hohenwarter, O., Waltenberger, A., and Katinger, H. (1996). An in vitro system for
thyroid hormone action. Anal. Biochem. 234:56-59.
Huang, T., Chopra, IJ, Beredo, A., Solomon, DH, ChuaTeco, GN (1985). Skin is an
active site of inner ring monodeiodination of thyroxine to 3,3',5-triiodothyronine.
Endocrinology, 117:20106-2113.
Huang, T., Chopra, IJ, Boado, R., Solomon, DH, Chua Teco, GN (1988). Thyroxine inner
ring monodeiodinating activity in fetal rat tissues. Pediatr. Res. 23:196-199.
35
-------
Iwamuro, S. and J. R. Tata (1995). Contrasting patterns of expression of thyroid hormone
and retinoid X receptor genes during hormonal manipulation of Xenopus tadpole tail
regression in culture. Mol Cell Endocrinol 113(2): 235-43.
Juge Aubry, C. E., A. Gorla Bajszczak, et al. (1995). Peroxisome proliferator-activated
receptor mediates cross-talk with thyroid hormone receptor by competition for retinoid X
receptor. Possible role of a leucine zipper-like heptad repeat. J Biol Chem 270(30): 18117-
22.
Kaltenbach, J.C. (1996). Endocrinology of amphibian metamorphosis. In L.I. Gilbert,
J.R. Tata, and B.G. Atkinson, eds., Metamorphosis: Postembryonic reprogramming of
gene expression inamphibian and insect cells. Academic Press, New York, New York
US A, pp 403-431.
Kaplan, MM, Visser, TJ, Yaskoski, KA, and Leonard, JL (1983). Characteristics of
iodothyronine tyrosil ring deiodination by rat cerebral cortical nicrosomes. Endocrinology
112:35-42.
Katz, D., M. J. Reginato, et al. (1995). Functional regulation of thyroid hormone receptor
variant TR alpha 2 by phosphorylation. Mol Cell Biol 15(5): 2341-8.
Kavlock, RJ, Daston, GP, DeRosa, C., Fenner-Crisp, P (1996). Research needs for the
risk assessment of health and environmental effects of endocrine disrupters: A report of
the U.S. EPA-sponsored Workshop. Environ. Health Persp. 104 (4):715-740
Kirby, J. D., A. E. Jetton, et al. (1992). Developmental hormonal profiles accompanying
the neonatal hypothyroidism-induced increase in adult testicular size and sperm production
in the rat. Endocrinology 131(2): 559-65.
Kollros, JJ (1961). Mechanisms of amphibian metamorphosis: Hormones. Am. Zol.
1:107-114.
Kumar, B. A., K. R. Vinod, et al. (1993). Effect of calcium and calmodulin antagonists on
metamorphosis in the anuran tadpole, Rana tigerina. Funct Dev Morphol 3(4): 237-42.
Lazar, M. A. (1993). Thyroid hormone receptors: multiple forms, multiple possibilities.
Endocr Rev 14(2): 184-93.
Legrand, J (1979). Morphogenic actions of thyroid hormones. Trends Neurosci. 2:234-
236.
36
-------
McClain, R.M., Levin, A.A., Posch, R., and Downing, J.C. (1989). The effect of
phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl.
Pharmacol. 99:216-228.
McClain, R. M. (1995). Mechanistic considerations for the relevance of animal data on
thyroid neoplasia to human risk assessment. Mutat Res 333(1-2): 131-42.
McKinney, JD, Fannin, R., Jordan, S, Chae, K., Rickenbacher, U., and Pedersen L (1987).
Polychlorinated biphenyls and related compound interactions with specific binding sites
for thyroxine in rat liver nuclear extracts. N. Med. Chem. 30:79-86.
McKinney JD, and Waller CL (1994). Polychlorinated biphenyls as hormonally active
structural analogues. Environ. Health Persp. 102:290-297.
Meier, C. A., C. Parkison, et al. (1993). Interaction of human beta 1 thyroid hormone
receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-
dependent dominant negative potency. J Clin Invest 92(4): 1986-93.
Morse, DC, Klasson-Wehler, E, Wesseling, W, Koeman, JH, and Brouwer, A. (1996).
Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to
polychlorinated biphenyls (Aroclor 1254). Toxicol Appl. Pharmacol. 136:269-279.
Obregon, MJ, Ruiz de Ona, C., Calvo, R., Escobar del Rey, FM and Morreale de Escobar
G. (1990). Outer ring iodothyronine deiodnases and thyroid hormone economy: responses
to iodine deficiency in rat fetus and neonate. Endocrinology 129-2663-2673.
Olgivie, LM and Ramsden, DB (1988). loxynil and 3,5,3'-triiodothyronine: comparison of
binding to human plasma proteins. Toxicol. Lett. 44:281-287.
Oppenheimer, JH, Schwartz, HL, Mariash, CN, Kinlaw, WB, Wrong, NCW, and Freake,
HC. (1987). Advances in our understanding of thyroid hormone action at the cellular
level. Endocr. Rev. 8:288-308.
Poole, A., Pritchard, D., Jones, R.B., Catto, L., and Leonard, T. (1990). In vivo biliary
excretion and in vitro cellular accumulation of thyroxine by rats or cultured tat hepatocytes
treated with a novel histamine H,-receptor antagonist. Arch Toxicol. 64:474-481.
Porterfield, SP (1994). Vulnerability of developing brain to thyroid abnormalities:
Environmental insults to the thyroid system. Environ Health Persp. 102(2): 125-130.
37
-------
Rastogi, RB, Lapierre, Y, and Singhal, RL (1976). Evidence for the role of brain biogenic
amines in depressed motor activity seen in chemically thyroidectomized rats. J.
Neurochem. 26:443-449.
Reginato, M. J., J. Zhang, et al. (1996). DNA-independent and DNA-dependent
mechanisms regulate the differential heterodimerization of the isoforms of the thyroid
hormone receptor with retinoid X receptor. J Biol Chem 271(45): 28199-205.
Ruiz de Ona, C., Morreale de Escobar, G., Calvo, R., Escobar del Rey, F, and Obregon,
MJ (1991). Thyriod hormones and 5'-deiodinase in the rat fetus late in gestation: effects of
maternal hypothyroidism. Endocrinology 128:422-432.
Silva, JE, Leonard, JL, Crantz, FR, and Larsen, PR. (1982). Evidence for two tissue-
specific pathways for in vivo thyroxine 5'-deiodination in the rat. J. Clin. Invest. 69:1176-
1184.
Specker, J.L. (1988). Preadaptive role of thyroid hormones in larval and juvenile salmon:
Growth, the gut and evolutionary considerations. Am. Zool. 28:337-349.
Sullivan, CV, Darling, DS, and Dickhoff, WW (1987). Nuclear receptors for L-
triiodothyronine in trout erythrocytes. Gen. Comp. Endocrinol. 65:149-160.
Surks, I. J. C., Gary N., Mariash, John T. Nicoloff, David H. Solomon (1990). American
Thyroid Association guidelines for use of laboratory tests in thyroid disorders. Journal
American Medical Association 263(11): 1529-1532.
Tata, J. R. (1994). Hormonal regulation of programmed cell death during amphibian
metamorphosis. Biochem Cell Biol 72(11-12): 581-8.
Tata, J.R. (1996). Hormonal interplay and thyroid hormone receptor expression during
amphibian metamorphosis. In L.L Gilbert, J.R. Tata, and E.G. Atkinson, eds.,
Metamorphosis: Postembryonic reprogramming of gene expression inamphibian and
insect cells. Academic Press, New York, New York USA, pp 466-503.
Uziel, A, Gabrion, J, Ohresser, M., and Legrand, C (1981). Effects of hypothyroidism on
the structural develpoment of the organ of Corti in the rat. Acta Otolaryngol. 92:469-480.
Uziel, A., Legrand, C., Ohresser, M., and Marot, M. (1983). Maturational and
degenerative processes in the organ of Corti after neonatal hypothyroidism. Hearing Res.
11:203-218.
38
-------
Van Haaster, LH, de Jong, FH, Docter, R and de Rooji, DG (1993). High neonatal
triiodothyronine levels reduce the period of Sertoli cell proliferation and accelerate tubular
lumen formation in the rat testis, and increase serum inhibin levels. Endocrinology,
133:755-760.
Visser, TJ (1988) Metabolism of thyroid hormones. In Hormones and their action (BA
Cooke, RJB King, and HJ van der Molen, eds) Elsevier, New York, vol. 1 pp. 81-103.
Visser, TJ, Kaplan, MM, Leonard, JL and Larsen, PR (1983). Evidence for two pathways
of iodothyronine S'-deiodination in rat pituitary that differ in kinetics propylthiouracil
sensitivity and response to hypothyroidism. J. Clin. Invest. 71:992-1002.
Wright, M. L., L. J. Cykowski, et al. (1994). Anterior pituitary and adrenal cortical
hormones accelerate or inhibit tadpole hind limb growth and development depending on
stage of spontaneous development or thyroxine concentration in induced metamorphosis. J
Exp Zool 270(2): 175-88.
Yamano, K. and Inui, Y. (1995). cDNA cloning of the thyroid hormone receptor beta for
Japanese flounder. Gen. Comp. Endocrinol. 99:197-203.
Yamauchi, K., T. Kasahara, et al. (1993). Purification and characterization of a 3,5,3'-L-
triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of
mammalian transthyretin. Endocrinology 132(5): 2254-61.
39
-------
Appendix 1
LIST OF ABBREVIATIONS
EDCs - Endocrine Disrupting Chemicals
Lactoperoxidase - LPO
PPAR - Proxisomal Proliferator-Activating Receptor
PTU - Propylthiouracil
rT3 - reverse T3
RXR - Retinoid X receptor
T3 - triiodothyronine
T4 - thyroxine
TBG - Thyroid Binding Globulin
TH - thyroid hormone
TPO - thyroid peroxidase
TR - thyroid receptor
TRH - Thyrotropin Releasing Hormone
TSH - Thyroid Stimulating Hormone
TTR - Transthyretin
UDP-GT - Uridine Diphosphate Glucuronlytransferase
40
-------
Appendix 2
PARTICIPANTS
Lisa Biegel
Haskell Laboratory for Industrial Medicine
Molecular Carcinogenesis & Mutagenesis
PO Box 50 Elkton Rd.
Newark DE, 19714
Abraham Brouwer
Wageningen Agricultural University
Department of Toxicology
Tuinlaan 5
Wangeningen 6307
Netherlands
Scott Brown
National Water Resource Institute
Box 5050
867 Lake Shore Road
Burlington Ont L7R 4A6
Canada
Ann Olive Cheek
Tulane Medical Center
Center for Bioenvironmental Research
1430 Tulane Ave. SL3
New Orleans LA 70112
Francoise Brucker-Davis
World Wildlife Fund
1250 24th St,NW
6th Floor
Washington DC 20037
Russ Christensen
Bayer Corporation
Agriculture Division of Toxicology
17745 South Metcalfe
Stilwell Kansas 66085-9104
41
-------
Paul Cooke
University of Dlinois
College of Veterinary Medicine
2001 south Lincoln Ave
UrbanaDl,61802
James Crissman
The Dow Chemical Co.
Health & Environmental Research Laboratory
1803 Building
Midland, MI 48674
Kevin Crofton
USEPA
Neurotoxicology Division
NHEERL (MD74-b)
RTF, NC 27711
Michael DeVito (Chair)
USEPA
Experimental Toxicology Division
NHEERL (MD-74)
RTF, NC 27711
Dan Doerge
Dept. Of Health and Human Services
Division of Chemistry
3900 NCTR Lane
Jefferson AR 72079-9502
Earl Gray
USEPA
Reproductive Toxicology Division
NHEERL (MD-72).
RTF, NC 27711
Peter Hauser
Psychiatry Services (116A)
VA Mdical Center
10 North Green Street
Baltimore MD 21201
42
-------
Pamela Hurley
USEPA
Crystal Mall 2
1921 Jefferson Davis Highway
Arlington VA 22202
Michael Kohn
NffiHS
POBox 12233
MD-A3-06
RTP, NC 27709
Jozef Lazar
Psychiatry Services (116A)
VA Mdical Center
10 North Green Street
BaltimoreMD21201
Michael McClain
Hoffmann-LaRoche Inc
Preclinical Development Administration
340 Kingsland Street
NutleyNJ07110
Eugene McConnell
3028 Ethan Lane
Lawndale Est
Raleigh NC 27613
Suzanne McMaster
USEPA
NHEERL(MD-51A)
RTP, NC 27711
43
-------
Joseph Tietge
USEPA
NHEERL/MED
6201 Cogdon Blvd.
Duluth, MN 55804
Shelly Tyl
Research Triangle Institute
Herman Laboratory Building
Room 45
POBox 12194
RTF, NC 27711
44
------- |