1. Introduction to the Toxicology of the Follicular
Thyroid Gland
Dr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT
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2. Learning Objectives
• To understand the key basic functional aspects of the thyroid;
• To understand how thyroid dysfunction is detected and
measures;
• To understand and accurately interpret changes in thyroid
parameters;
• To understand and accurately interpret the human relevance
of changes to thyroid parameters in rodent toxicology studies;
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3. Basic Terms
• Triiodothyronine = T3 = 3, 5, 3’ triiodothyronine; the most
active of the follicular thyroid hormones
• Reverse triiodothyronine = rT3 = 3,3’5’-triiodothyronine; a
biologically inactive isomer of T3
• Thyroxine = T4 = 3,5,3’,5’ tetraiiodothyronine; has some
activity in the periphery but mostly functions in vivo as a pro-
hormone (converted to T3 in the periphery)
• Thyroglobulin = Tg; a 660 kDa dimeric protein produced by the
follicular cells of the thyroid and used to produce T3 and T4.
Tg forms the colloid in the follicular thyroid gland
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4. Basic Terms
• MIT = monoiodotyrosine; forms part of the thyroid colloid.
Colloid thryoperoxidase enzymatically binds iodine to tyrosine
residues in Tg in the thyroid colloid
• DIT = diiodotyrosine; synthesis is similar to that of MIT
• MIT + DIT combine to form T3 or r-T3
• DIT + DIT combine to form T4
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5. Basic Thyroid Histology
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6. Basic Concepts: Synthesis of Thyroid Hormones
• Tg is synthesized in the RER and
follows the secretory pathway to
enter the colloid in the lumen of the
thyroid follicle by exocytosis.
• - Meanwhile, a sodium-iodide (Na/I)
symporter pumps iodide (I-) actively
into the cell
• - I- enters the follicular lumen from
the cytoplasm by the transporter
pendrin
• - In the colloid, I- is oxidized to iodine
(I0) by an enzyme called
thyroperoxidase.
• - I0 is very reactive and iodinates the
Tg at tyroine residues in its protein
chain.
• During conjugation, adjacent tyrosine
residues are paired together.
• The entire complex re-enters the
follicular cell by endocytosis.
• Proteolysis by various proteases
liberates T3, rT3 and T4 which enter
the blood by largely unknown
mechanisms.
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7. Important Species Differences in Thyroperoxidases
• Thyroperoxidase in rats, mice and dogs are sensitive to
inhibition by xenobiotics
• Result of inhibition is disruption of T4 synthesis  increased
thyroid load (see thyroid economy below)  increased
sensitivity to thyroid follicular hyperplasia/neoplasia
• Thioamides (sulfonamides) are notably inhibitors of
thyroperoxidase
• Other notable inhibitors: propylthiouracil
(PTU), methimazole, aminotriazole, acetoacetamide
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8. T3 and T4 Transport in the Blood
• Normal ratio of T4 to T3 in blood is about 20:1
• In most species the majority of T3 and T4 in plasma is bound
to proteins
• TBG is the critical protein – affinity for T4 is about 1000 times
higher than that of other proteins
• Bound T3 and T4 are not biologically active; only the unbound
T3 and T4 are biologically active (important when
measuring/interpreting plasma or serum T3 and T4 levels)
• Protein binding of T3 and T4 is very important – increases the
circulating T½ of the hormones, which in turn protects the
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follicular thyroid gland

9. T3 and T4 Transport in the Blood
• There is a critical difference with big biological consequences
in rodents (notably rats):
– The plasma T½ for T4 in rats is short (12-24 hours compared with 5 – 9
days in humans)
– Plasma T½ for T3 is about 1 day in most species (what are the
implications?)
– Rats have low plasma TBG – T4 binding is primarily limited to albumin
and prealalbumin which have much lower binding affinities
– What are the implications of all of this?
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10. T3 and T4 Transport in the Blood
• There is a critical difference with big biological consequences
in rodents (notably rats):
– Net result is that rats have to produce about 10 times more T4 per unit
body weight than humans
– T3 in humans is transported in the plasma mostly bound to
TBG, however in rats and mice, T3 does not bind to TBG  faster T3
turnover in rats versus humans  greater thyroid demand
– Overall result is that rats and mice have a substantially lower thyroid
economy compared with humans  much greater sensitivity to
developing thyroid hyperplasia and neoplasia than humans
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11. Thyroid Hormones in the Periphery
• T4 is converted to the active T3 within cells by deiodinases (5'-
iodinase)
– All three isoforms of the deiodinases are selenium-
containing enzymes, thus dietary selenium is essential for
T3 production.
• These are further processed by decarboxylation and
deiodination to produce inactive iodothyronamine (T1a) and
thyronamine (T0a).
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12. Actions of T3 in the Periphery
• Increases cardiac output
• Increases heart rate
• Increases ventilation rate
• Increases basal metabolic rate
• Potentiates the effects of catecholamines (i.e. increases
sympathetic activity)
• Potentiates brain development
• Thickens endometrium in females
• Increases metabolism of proteins and carbohydrates
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13. Metabolism and Excretion of T3 and T4
UGT = UDP-glucuronosyltransferases
SULT = Sulfotransferases
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14. Regulation of Thyroid Hormones:
The Hypothalamic-Pituitary-Thyroid Axis
• TSH is released from the anterior pituitary gland in
response to thyroid releasing hormone from the
hypothalamus and causes the synthesis and
secretion of T3 and T4 by the thyroid gland. This is
acheived by stimulation of the thyroid follicular cells
by binding of TSH to the receptor on the basal
surface of the cell and activation of acetylate
cyclase. This leads to increased iodide uptake.
• T3 and T4 exert negative feedback on both the
pituitary production of TSH and the hypothalamic
production of TRH.
• Other factors affecting release of TRH from the
hypothalamus include blood levels glucose and the
body's metabolic rate.
• Somatostatin inhibits TSH secretion and estrogen
has been shown, in rats, to reverse the negative
feedback affect of T3 and T4 on the TSH response to
TRH.
• SS is produced by the GI, pancreas and
hypothalamus – important in regulation of body
size and in obesity
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15. Sex Differences in TSH Drive
• There is a critical difference with big biological consequences
in male rats:
– TSH levels are higher in males than in females
– Effect is related to testosterone levels
– Higher TSH levels in males  greater demand on the
follicular thyroid  increased sensitivity to thyroid
neoplasia compared with female rats
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16. Measurement of Thyroid Status: Thyroid Function Tests
• Typically a combination of measures are required – single
tests are frequently insufficient to determine thyroid status!
• Available measures are:
– Serum TSH
– Total Serum T4
– Free Serum T4
– Total Serum T3
– Free Serum T3
– Serum T3:T4 ratio
– Serum TBG = T3 resin uptake test (T3U)
– Serum Tg
• 123I uptake
• Imaging
• Histopathology/fine needle aspirate (really a human thing)
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17. Thyroid Function Tests: Serum TSH
• TSH levels peak in the evening and are lowest in the afternoon, with
marked variations due to physiologic conditions such as illness, and low
energy intake.
• A variety of drugs can result in artifactual or biological changes to TSH
levels
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18. Thyroid Function Tests: Serum TSH
• Increases in TSH are associated with loss of negative feedback on the
hypothalamic-pituitary-thyroid axis due to:
– Primary hypothyroidism due to disruption of T4 or T3 synthesis (iodine
deficiency, disruption of I- uptake, inhibition of
thyroperoxidase, inhibition of Tg synthesis, inhibition of T3/T4 release)
– Increased utilization/destruction of T4 or T3 in the periphery (e.g.
induction of 5’ iodinases)
– TSH producing pituitary tumor (rare)
– Pituitary resistance to T3 and T4 (disruption of negative feedback)
– Increased demand for T3 and T4 e.g. hypermetabolic states
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19. Thyroid Function Tests: Serum TSH
• Decreases in TSH are associated with excessive negative
feedback on the hypothalamic-pituitary-thyroid axis due to:
– Hyperthyroidism
– Pituitary (secondary) hypothyroidism (decreased TSH
production; rare)
– Some non-thyroid related diseases (rare)
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20. Thyroid Function Tests: Total Serum T4 (Thyroxine)
• Increased total T4 occurs in hyperthyroid states or if there is an increased
TBG (can be acquired) or if the animals are treated with thyroxine
• Decreased total T4 occurs in hypothyroid states or with TBG deficiency
– Heritable TBG deficiency occurs in humans and is non-harmful;
heritable gene difference
– Acquired TBG deficiency can be caused by protein
malnutrition, chronic liver and kidney disease, glucocorticoids and
androgenic steroids
• Remember that total T4 is a relatively insensitive measure of the T4
biological activity (T4 has to be non-protein bound to be biologically
available)
– In some senses it is a measure of the readily available reserve of free
(biologically active) T4 and T3
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21. Thyroid Function Tests: Serum Free T4 (Thyroxine)
and Free T4 Index
• Despite the names, these are an indirect estimate of unbound
T4 in the blood
• The advantage is that the biologically available form is
measured, correcting for variations in TBG levels
• The disadvantage is that no currently available test accounts
for all the currently known T4 protein binding abnormalities
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22. Thyroid Function Tests: Serum Total T3
• Advantage: measuring the ultimate hormone rather than a
pro-hormone
• Disadvantage: high inter-laboratory variability in
measurement and normal ranges
• Again, the measurement reflects the circulating storage pool
of T3 rather than the small fraction of unbound T3 that is
biologically active
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23. Thyroid Function Tests: Serum Free T3
• Rarely performed because of variation in the normal range
across different populations and inter-laboratory variation
• Has the advantage that you are measuring the biologically
available levels of the ultimate-hormone
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24. Thyroid Function Tests: Serum T3:T4 ratio
• The T3/T4 ratio is a simply calculated index reflecting thyroid
function and the action of hormones on the tissues
• In normal subjects the T3/T4 ratio is influenced neither by the
body weight nor by the physical activity level, sex, or the
blood sampling conditions
• The nutritional status can influence the ratio if there is
inadequate iodine intake or if there is weight loss
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25. Thyroid Function Tests: Serum T3:T4 ratio
• T3/T4 is increase in hyperthyroid states because of increased
T3 secretion relative to T4 secretion by the thyroid
• T3/T4 is increased in hypothyroid states because of increased
T3 secretion relative to T4 secretion + higher relative decline
in blood T4 levels (increased tissue 5’-deiodinase activity)
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26. Thyroid Function Tests:
Serum TBG = T3 resin uptake test (T3U)
• This test does not measure any of the thyroid hormones, it
simply provides an indication of the amount TBG present
• It is called "T3" resin uptake because T3 is used in the test
procedure.
• The normal range varies considerable among laboratories
• The test is designed so that the higher the value, the lower
the amount of binding globulin (TBG) which is present.
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27. Thyroid Function Tests:
Serum rT3
• Serum rT3 is increased if 5’-deiodinases are inhibited
• Not routinely available
• rT3 increases if 5’-deiodinases are blocked because:
– T4 accumulates which is subsequently converted to rT3 in the
periphery
– T3 accumulates which is subsequently converted to rT3 in the
periphery
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28. Thyroid Function Tests:
Radioactive Iodine Uptake Test (RAIU) + Thyroid Scan
• Determines the uptake by the thyroid of 123I by the thyroid
• Extremely sensitive
• Critical test if the xenobiotic is suspected of disrupting I
uptake by the thyroid
• Generally low uptake is an indicator of hypothyroidism (either
primary or secondary)
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29. Thyroid Function Tests:
Radioactive Iodine Uptake Test (RAIU) + Thyroid Scan
• A high uptake of tracer spread evenly in the thyroid gland is
an indicator of general hyperthyroidism
• An uneven spread of tracer in the thyroid gland (with either
low or high areas of uptake) may mean that hyperthyroidism
is caused by a nodular hyperplasia or nodular thyroid tumors.
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31. Nodular goiter
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32. Thyroid tumor
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33. Practical Use of the Thyroid Function Tests
• General screening for thyroid dysfunction: at the
minimum, total serum T4 + serum TSH should be performed
– Both experimental + historical controls (normal ranges) are critical for
interpretation
– Normal ranges must be for the strain being used and the age of
animals being used (and preferably developed specifically for the
testing lab)
• Additional tests should be included if there is any indications
that the xenobiotic may result in thyroid disruption
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34. Mechanisms of Thyroid Toxicity
• Inhibition of thyroid hormone synthesis
(primary hypothyroidism)
– Inhibition of iodine uptake
– Inhibition of thyroperoxidase and other organification defects
• Blockage of T3 and T4 secretion (primary hypothyroidism)
• Thyroid pigmentation and other changes in colloid
• Increased T4 clearance associated with hepatic microsomal enzyme
induction
• Inhibition of 5’ deiodenases
• Thyroid carcinogenesis
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35. Inhibition of Iodine Uptake and Iodine Trapping
• Involves blockage of or competition for the Na I symporter
(NIS)
– Note: NIS is also found in the salivary gland, gastric mucosa, choroid
plexus, ciliary body of the eye, lactating mammary gland
• Common NIS competitive inhibitors
– Perchlorate (ClO4-)
– Thiocyanates (SCN-) e.g. brassica-based foods, cassava etc
– Pertechnetate
• Sodium chlorate (NaClO3) is a common NIS inhibitor
• Net result is hypothyroidism (decreased T3, T4 + increased
TSH) + goiter (follicular cell hypertrophy and hyperplasia due
to increased TSH and excessive thyroid stimulation)
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36. Inhibition of thyroperoxidase
and other organification defects
• Thionamides
– Propylthiouracil used to treat hyperthyroidism is the classical example
– Sulfonamide antibiotics
• Anilines
• Substituted phenols
• Can act either by inhibiting thyroperoxidase or the coupling
reactions (i.e. reaction of MIT + DIT to form T3 and T4)
• Humans are relatively resistant to these effects compared
with rodents and dogs
• Net result is hypothyroidism (primary)
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37. Blockage of T3 and T4 Secretion
• High doses of iodine – mode of action is uncertain
• Lithium – classical side effect of the use of lithium to treat
manic states is hypothyroidism and goiter
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38. Thyroid Pigmentation and Colloid Abnormalities
• Many xenobiotics localize and concentrate in colloid
• May result in pigmentation or clumping of colloid or increased
basophilic staining of colloid
• Accumulation of xenobiotics in colloid may or may not disrupt
thyroid function
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39. Increased T3 and T4 Excretion:
Hepatic Microsomal Enzyme Induction
• Hepatic glucuronidation is the rate-limiting step for biliary
excretion of T4
• Hepatic sulfation (primarily phenol sulfotransferase) is the
rate-limiting step for biliary excretion of T3
• Induction of these enzymes results in increased T3 and T4
clearance in rats  this combined with the lower thyroid
economy in rats results in reduced negative feedback on the
hypothalamic-pituitary-thyroid axis  increased TSH 
increased thyroid stimulation + hypothyroidism  increased
incidence of thyroid hyperplasia and thyroid follicular cell
neoplasia
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40. Increased T3 and T4 Excretion:
Hepatic Microsomal Enzyme Induction
• Classical examples are phenobarbital and Ah receptor agonists
(e.g. arochlors/co-planar PCBs) and pyrethrins  induce
UDP-GT  increased glucuronidation  increased rate of T4
excretion
• Can also occur via activation of the AhR, CAR, PXR and PPARα
xenosensor systems
• Hepatic microsomal enzyme inducers also increase the uptake
of T4 into the liver and biliary excretion of unconjugated T4
(presumably by upregulating membrane transporters)
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41. Increased T3 and T4 Excretion:
Hepatic Microsomal Enzyme Induction
• Increased T4 excretion as a mechanism of disruption of the hypothalamic-
pituitary thyroid axis (hypothyroidism) and thyroid follicular
carcinogenesis appears to be rat specific and is regarded as not relevant to
humans
– Epidemiology of patients treated with barbituates for seizure disorders
indicate no increased risk
– Increases of serum TSH are not seen in humans with microsomal
induction
– Sustained abnormally high TSH levels do not appear to increase the
risk of thyroid cancer in humans
– Thyroid economy is much greater in humans than in rodents
– The only verified cause of thyroid cancer in humans is ionizing
radiation exposure (123I and similar)
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42. Increased T3 and T4 Excretion:
Hepatic Microsomal Enzyme Induction
• Disruption of the hypothalamic-pituitary-thyroid axis and thyroid follicular
tumors secondary to increased thyroid demand is an extremely common
finding in rodent toxicology studies
• Thyroid follicular tumors induced by this MOA in rodents is a classical
example of non-genotoxic (non-mutagenic) carcinogenesis
• Provided the necessary criteria are met (see below), this MOA is generally
not regarded as relevant to humans
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43. Human Relevance of Thyroid Follicular Cell Tumors and
Disruption of the Hypothalamic-Pituitary-Thyroid Axis in
Rodents
• US EPA guidance documents:
http://www.epa.gov/raf/publications/thyroid-follicular-cell-
tumor.htm
• IPCS guidance document:
http://www.inchem.org/documents/harmproj/harmproj/harmpr
oj4.pdf
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44. Human Relevance of Thyroid Follicular Cell Tumors and
Disruption of the Hypothalamic-Pituitary-Thyroid Axis in
Rodents
– US EPA/IPCS takes the extremely conservative stance that rodents and
humans are equally sensitive to thyroid effects due to disruption of
the hypothalamic-pituitary-thyroid axis unless there is chemical-
specific data
– However, if the MOA is clearly due to hepatic enzyme induction, the
effects are regarded as not relevant to humans
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45. Human Relevance of Thyroid Follicular Cell Tumors and
Disruption of the Hypothalamic-Pituitary-Thyroid Axis in
Rodents
• US EPA/IPCS guidance documents:
– Must establish the following to demonstrate the MOA:
• NOT MUTAGENIC or CLASTENOGENIC or CLASSICALLY GENOTOXIC
• Induction of hepatic UGT activity
• increase in hepatic metabolism and biliary excretion of T4
• decrease in serum T4 half-life and concentration
• increase in circulating TSH concentration
• Resulting cellular thyroid hypertrophy and follicular cell
hyperplasia
• Appropriate temporal relationships i.e. UGT induction occurs
before decreased serum T4 T½ which occurs before increases in
TSH which occurs before follicular hypertrophy and hyperplasia
which occurs before the tumors
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46. Human Relevance of Thyroid Follicular Cell Tumors and
Disruption of the Hypothalamic-Pituitary-Thyroid Axis in
Rodents
• US EPA/IPCS guidance documents:
– KEY THINGS TO CHECK AND THINK ABOUT WHEN
INTERPRETING THIS TYPE OF DATA:
• Is the weight of evidence sufficient to establish a mode
of action (MOA) in animals?
• Can human relevance of the MOA be reasonably
excluded on the basis of fundamental, qualitative
differences in key events between experimental
animals and humans?
• Can human relevance of the MOA be reasonably
excluded on the basis of quantitative differences in
either kinetic or dynamic factors between experimental
animals and humans?
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47. Inhibition of 5’ Monodeiodinase
• Classical example is FD&C Red No. 3 (eythrosine) food and
cosmetic dye
• The effects include:
– Functional hypothyroidism and increased thyroid demand since T4
cannot be converted into the more active T3
– Increases TSH due to decreased negative feedback on the
hypothalamic-pituitary-thyroid axis
– Long-term stimulation of the thyroid due to increased TSH  thyroid
follicular hypertrophy and hyperplasia  thyroid follicular neoplasia
• MOA is not regarded as human-relevant
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48. Case Study 1
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49. Instructions and Objectives
• From the data provided below:
– Determine if there is evidence of thyroid abnormalities associated
with the administration of this xenobiotic
– Propose a MOA for any putative effects
– Compose a table that provides a comparison of key events in rats
versus humans for the effects you have observed in the study
– Can human relevance of the MOA be reasonably excluded on the basis
of fundamental, qualitative differences in key events between
experimental animals and humans? Please provide a detailed
justification for your answer
– Can human relevance of the MOA be reasonably excluded on the basis
of quantitative differences in either kinetic or dynamic factors
between experimental animals and humans? Please provide a detailed
justification for your answer
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50. Results of Genotoxicity Studies
• Ames test with and without metabolic activation – clearly
negative all strains
• In vitro miconucleus test - clearly negative
• Chromatid exchange test – clearly negative
• In vitro chromosome aberration assay – clearly negative
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54. Case Study 2
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55. New Data
• New studies for the xenobiotic examined in Case Study 1 have become
available
• The new data indicates:
– The chemical concentrates in the thyroid colloid
– The chemical substance is a substrate for thyroperoxidase
– A highly reactive intermediate is generated in the reaction catalyzed
by thyroperoxidase
– The reactive intermediate has the potential to produce DNA adducts in
vitro
• Would you change your conclusions regarding the human relevance of the
MOA of the thyroid neoplastic effects? If so, why?
• What additional studies would you perform to examine the human
relevance of the MOA of the thyroid neoplastic effects?
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