TISSUE BIOMARKERS OF THYROID STATUS AS CORRELATES OF

NEURODEVELOPMENTAL IMPAIRMENT FOLLOWING GESTATIONAL AND LACTATIONAL

EXPOSURE TO THYROID DISRUPTOR PROPYLTHIOURACIL

by

MATTHEW AARON TAYLOR

(Under the direction of Duncan C. Ferguson and Jeffrey Fisher)

ABSTRACT

From its influence on mitochondrial activity and basal metabolic rate to its complex interplay

with other endocrine organs, the thyroid gland is vital for proper development and operation of the

mammalian body. Neurological development is a special concern due to the relative sensitivity of

gestating females and neonates to thyroid disruption. The studies described within investigate the effects

of developmental exposure to antithyroid compounds on the neurodevelopment of rats. As peripheral

tissues including the brain are capable of partial compensation in response to declining availability of

thyroid hormones from serum, we hypothesized that measuring thyroid-responsive parameters such as

tissue 5’-deiodinase enzyme activity thyroid hormone concentrations, and mRNA expression would

provide data which would more closely correlate to neurological endpoints than serum thyroid hormone

or thyrotropin (TSH) concentrations. We further hypothesized that younger animals would be more

sensitive to thyroid disruption than older animals, due to diminished reserve of the compensatory

mechanisms in the hypothalamic-pituitary-thyroid axis.

We used two antithyroid compounds to test these hypotheses. Propylthiouracil (PTU) at 0, 0.3, 1,

3, and 10 ppm in drinking water, and perchlorate at 150ppm in drinking water were administered from

gestational day 2 until weaning. Serum and cerebrocortical thyroid hormone concentrations were

measured at postnatal days 4, 14, and 21-32, as well as in dams and pups allowed to recover for 60 days

post-weaning. Hippocampal in vitro electrophysiological measurements were performed on pups and

adults. Open field behavioral testing was performed on adults. Overall, we found that the chosen

thyroid-responsive biomarkers were insufficient to consistently predict the presence of

electrophysiological alterations. A severe reduction in serum total T4 concentration in developing pups

was found to be a necessary condition for neurological impairment. Reductions in cortical T3

concentrations were not found to perfectly correlate with neurological outcomes.

INDEX WORDS: Rat, propylthiouracil, thyroid, hippocampus, perchlorate, neurodevelopment,

electrophysiology, deiodinase, triiodothyronine, thyroxine

TISSUE BIOMARKERS OF THYROID STATUS AS CORRELATES OF

NEURODEVELOPMENTAL IMPAIRMENT FOLLOWING GESTATIONAL AND LACTATIONAL

EXPOSURE TO THYROID DISRUPTOR PROPYLTHIOURACIL

by

MATTHEW AARON TAYLOR

BS, University of Georgia, 2003

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of

the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2008

© 2008

Matthew Taylor

All Rights Reserved

TISSUE BIOMARKERS OF THYROID STATUS AS CORRELATES OF

NEURODEVELOPMENTAL IMPAIRMENT FOLLOWING GESTATIONAL AND LACTATIONAL

EXPOSURE TO THYROID DISRUPTOR PROPYLTHIOURACIL

by

MATTHEW TAYLOR

Co-Major Professors: Duncan C. Ferguson

Jeffrey Fisher

Committee: Cham Dallas

Margarethe Hoenig

John Wagner

Electronic Version Approved:

Maureen Grasso

Dean of the Graduate School

The University of Georgia

August 2008

iv

DEDICATION

Dedicated to those for whom the mundane mysteries of the universe are wonder enough for a

lifetime without need of the supernatural.

v

ACKNOWLEDGEMENTS

I would like to thank all those who have helped me in my work. For Dr. Ferguson and my

committee, thank you for your guidance and teachings. For former labmates, especially Nadia, thank you

for your help and support. For my wife, thank you for helping me to stay on the correct side of insanity,

and for everything else you’ve done for me. And for all who have encouraged my curiosity and love for

science over the years, thank you.

vi

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS ......................................................................................................................... V

CHAPTER

1 INTRODUCTION ..................................................................................................................... 1

REFERENCES ..................................................................................................................... 2

2 LITERATURE REVIEW .......................................................................................................... 4

THYROID HORMONE PRODUCTION AND METABOLISM ....................................... 4

PLASMA THYROID HORMONE BINDING AND PHARMACOKINETICS................. 6

EFFECTS OF THYROID HORMONES ........................................................................... 13

HYPOTHALAMUS-PITUITARY-THYROID AXIS DEVELOPMENT ......................... 16

BRAIN DEVELOPMENT ................................................................................................. 17

THYROID DISRUPTION ................................................................................................. 19

ELECTROPHYSIOLOGY ................................................................................................. 27

CONCLUSION .................................................................................................................. 31

REFERENCES ................................................................................................................... 31

3 LOWER THYROID COMPENSATORY RESERVE OF RAT PUPS FOLLOWING

MATERNAL HYPOTHYROIDISM: CORRELATION OF THYROID, HEPATIC, AND

CEREBROCORTICAL BIOMARKERS WITH HIPPOCAMPAL

NEUROPHYSIOLOGY ..................................................................................................... 57

ABSTRACT ....................................................................................................................... 58

INTRODUCTION .............................................................................................................. 58

MATERIALS AND METHODS ....................................................................................... 60

RESULTS ........................................................................................................................... 65

vii

DISCUSSION .................................................................................................................... 68

REFERENCES ................................................................................................................... 71

4 DECREASED LITTER SIZE AMELIORATES THE THYROID DISRUPTIVE AND

NEURODEVELOPMENTAL EFFECTS OF GESTATIONAL AND LACTATIONAL

EXPOSURE TO PROPYLTHIOURACIL ...................................................................... 105

ABSTRACT ..................................................................................................................... 106

INTRODUCTION ............................................................................................................ 106

MATERIALS AND METHODS ..................................................................................... 109

RESULTS ......................................................................................................................... 115

DISCUSSION .................................................................................................................. 117

REFERENCES ................................................................................................................. 119

5 EXPANDING THE DOSE-RESPONSE CURVE OF PROPYLTHIOURACIL UNDER

CONDITIONS OF CONTROLLED LITTER SIZES ...................................................... 136

ABSTRACT ..................................................................................................................... 137

INTRODUCTION ............................................................................................................ 137

MATERIALS AND METHODS ..................................................................................... 139

RESULTS ......................................................................................................................... 143

DISCUSSION .................................................................................................................. 145

REFERENCES ................................................................................................................. 149

6 SUPPLEMENTAL AND COLLABORATIVE STUDIES .................................................. 177

PTU TIMECOURSE STUDY .......................................................................................... 177

DEIODINASE ACTIVITY OF PERCHLORATE TREATED RAT PUPS .................... 178

DEIODINASE ACTIVITY AND CEREBROCORTICAL T3 CONCENTRATIONS OF

PTU DOSED LONG-EVANS RATS ........................................................................ 179

7 SUMMARY AND DISCUSSION ........................................................................................ 195

REFERENCES ................................................................................................................. 203

viii

APPENDICES .......................................................................................................................................... 211

A LIST OF ABBREVIATIONS ............................................................................................... 211

B DIETARY COMPARISON .................................................................................................. 213

1

CHAPTER 1

INTRODUCTION

As our understanding of endocrine physiology advances, so does the list of chemicals which may

interfere with these regulatory systems. One endocrine organ currently of concern is the thyroid, which

impacts function in virtually every tissue of the mammalian body. Although the thyroid was first

identified about 500 years ago, new effects of thyroid hormones are still being identified, and along with

them new implications of thyroid insufficiency. While widespread inclusion of iodine into table salt has

virtually eliminated goiters and cretinism in developed nations, new environmental threats continue to

emerge.

A major concern over chemicals which impact the thyroid axis is the growing body of literature

which shows that thyroid hormones play a vital role in neural development in mammals including

humans. Hypothyroidism has been shown to have effects ranging from outright cretinism to ones as

subtle as altering the timing at which neuronal receptors switch from one isoform to another during

development. While our understanding of the effects of thyroid insufficiency during development

continues to grow, many of these experimental effects are demonstrated in the laboratory at doses that

have no known environmental relevance. Some thyroid effects have been shown at current environmental

levels of antithyroid chemicals, such as an increase in serum thyroid stimulating hormone (TSH) levels in

American women with elevated perchlorate exposure.1 [However, the degree of exposure needed to

induce adverse effects rather than merely compensatory effects is still poorly understood.

One of the biggest problems in translating exposure to antithyroid agents into detrimental effects

is the large amount of compensation possible at the central (hypothalamic-pituitary-thyroid) and

peripheral (target tissue) levels which help to ameliorate the effects of thyroid disruption caused by

exposure to such agents. While it is quite common to measure serum TSH concentrations in screening

procedures for hypothyroidism, serum hormone measurements alone do not necessarily explain what is

2

happening at the level of target tissues. Deiodinases in peripheral tissues such as the brain have cellular

adaptive mechanisms by which to avoid local conditions of hypothyroidism or hyperthyroidism.2

We sought to fill in some of these knowledge gaps and devised hypotheses linking hormonal,

biochemical, electrophysiological and behavioral assays in an effort to inform model development.

Propylthiouracil (PTU) was chosen as the anti-thyroid agent for this project due to its prior use in several

neurologically-focused studies. Specifically, it was hypothesized that

1) Thyroid hormone concentrations in serum do not provide sufficient information to predict the

availability and activity of thyroid hormones to all tissues in the body, as alterations in peripheral

metabolism help to counterbalance the effects of systemic hypothyroidism in critical tissues such as the

brain.

2) Measurement of brain-specific biomarkers such as thyroid responsive gene expression, deiodinase

activity and cerebrocortical T3 concentrations will be more reliable predictors of neurodevelopmental

impairment than serum hormone concentrations due to their improved proximity to the developmental

endpoint compared to serum effects.

3) The chosen biomarkers: cerebrocortical D2 activity, cerebrocortical T3 concentration, and expression

of thyroid-responsive genes in the brain will provide data which will aid efforts to predict both temporary

and permanent neurological dysfunction caused by any thyroid disruptive compound.

4) Younger animals are more sensitive to thyroid disruption than older animals due to their immature

thyroid axis and lesser compensatory capabilities compared to adults.

5) The neurological impact of maternal hypothyroidism is modified by the nutritional

status of developing pups, as impacted by the number of pups nursed by a single dam, as well as

alterations in dietary contents such as soy isoflavones.

References

1. Blount BC, Pirkle JL, Osterloh JD, Valentin-Blasini L, Caldwell KL. Urinary perchlorate and thyroid

hormone levels in adolescent and adult men and women living in the United States. Environmental health

perspectives. 2006 December;114(12):1865-71.

3

2. Köhrle J. The deiodinase family: selenoenzymes regulating thyroid hormone availability and action.

Cellular and molecular life sciences. 2000 December;57(13-14):1853-63.

4

CHAPTER 2

LITERATURE REVIEW

Thyroid hormone production and metabolism

Basic Thyroid Physiology

The earliest recorded references to the thyroid date back to around 2700 BCE, with a reference to

the use of seaweed to treat goiter.1 The name was associated with the gland in 1656 CE by Thomas

Wharton, due to the shield-shaped cartilage associated with the gland in mammals. Understanding of the

thyroid's purpose was hindered by the medical thinking of the era, as it did not fit into the traditional view

of the balance of humors in the body. The importance of iodine was not elucidated until about 1820.

Today, iodine's sole known role is as a micronutrient for synthesis of thyroid hormones (Figure 2.1),

which have a variety of effects on most organs in the body.2

The functional unit of the thyroid is the follicle, which consists of epithelial cells surrounding a

compartment of viscous fluid known as colloid. The follicle contains all of the machinery necessary to

take iodine from the serum, store it, produce hormones, and export the hormones back into the

bloodstream. The follicular cells take up iodine from the blood via the sodium-iodine symporter (NIS).

This iodine is then exported into the colloid, along with thyroglobulin (Tg), and a number of other

compounds. Iodine is attached to Tg, and resorbed into the epithelial cells via endocytosis, where the Tg

is digested and thyroid hormones released into the bloodstream.2

Iodine Metabolism

The thyroid gland displays a remarkable ability to concentrate iodine from the blood, thanks to

the actions of the sodium iodide symporter (NIS), located within the basolateral membrane of thyroid

follicular cells. The gene for NIS was first cloned in 1996, and both the human and rat NIS genes were

cloned that year.3,4

An 84% identity and 93% similarity was found between the NIS genes in these two

5

species.5 NIS contains 13 transmembrane domains, and several identified glycosylation sites.

6 TSH

stimulation is critical for proper targeting and function of NIS.7 TSH stimulates NIS activity through the

cAMP pathway, as well as through upregulation of NIS mRNA.8,9

Under steady-state conditions, a 20-40

fold difference between thyroid free iodine concentrations and serum iodine concentrations can be

maintained.10

NIS functions by utilizing the sodium gradient formed by the sodium-potassium ATPase.

Two sodium ions are allowed through NIS to provide the energy needed to transport one iodine ion

against its gradient. While NIS is essential to thyroid function, it should be noted that NIS is not

exclusively localized to the thyroid, but exists elsewhere in the body including salivary and mammary

glands.5

After iodine is brought into the follicular cells by NIS, it is transported into the colloid. The

colloid contains large amounts of iodine and protein, especially Tg. Colloid has been reported to contain

protein concentrations as high as 100-400 mg/ml2. The colloid is the location in which iodine is attached

to Tg to create thyroid hormones. As such, it serves as a major site for hormone storage, and is

responsible for maintaining constant circulating hormone levels despite periodic and erratic dietary

consumption of iodine.

Organification of Iodide and Production of Thyroid Hormones

Mature Tg is a dimeric protein of approximately 660 kilodaltons (kD), and a sedimentation value

of 19. The molecular weight of Tg depends in part upon the degree of iodination present. Iodination of

Tg occurs only on tyrosine residues, and each tyrosine residue can form half of a completed thyroid

hormone. Iodine is attached to Tg through a process involving thyroid peroxidase (TPO). TPO is a large

heme-containing glycoprotein enzyme of approximately 105kD. The gene for TPO is located on human

chromosome 2, and appears to have evolved separately from several other prominent human peroxidases,

myeloperoxidase, eosinophil peroxidase, and lactoperoxidase.11

Iodide must be oxidized before it can act as an effective iodinating agent. Only two known

biological oxidizing agents, H2O2 and O2, are potent enough to oxidize iodide.12

Thyroid peroxidase

reacts with H2O2 to give an unstable oxoferryl porphryin p-cation radical that reacts with protein tyrosine

6

or tryptophan and isomerizes to an oxoferryl protein radical. The porphyrin p-cation radical I catalyses a

two-electron oxidation of iodide to hypoiodite, which iodinates thyroglobulin tyrosine to diiodotyrosine.

The cation radical I then catalyses a one-electron oxidation of thyroglobulin diiodotyrosine to form

phenoxyl radicals that undergo coupling to form thyroglobulin thyroxine.13

In states of iodine sufficiency, the majority of thyroid hormones produced during iodination are

T4, which is formed from the joining of two doubly-iodinated tyrosine residues. Tg is phagocytosed and

returned to the follicular cells under TSH stimulation.2 It is thought that this process results in “first in-

first out” mechanics, as diffusion determines the position of a Tg molecule within the thyroid. As a

result, the most recently iodinated Tg molecules are generally reabsorbed first. This phenomenon is of

particular note when dealing with radioiodine, as it suggests ways to “flush” the radioactive material out

of the thyroid through application of anti-thyroid agents such as methimazole(MMI), perchlorate, or

several other compounds.

When Tg is reabsorbed by the follicular cells, the vesicles fuse with lysosomes. The lysosomal

enzymes break down Tg and release mature thyroid hormones. The vesicles targeted for excretion from

the cell send the mature hormones back into the bloodstream. A fraction of the T4 released undergoes

deiodination prior to release, resulting in the T3/T4 ration of the secreted iodothyronines being higher

than the ratio in the colloid. Once in the bloodstream, only a small fraction of the released hormones will

circulate freely. Most will be bound to a number of serum proteins such as transthyretin (TTR), albumin,

and thyroxine-binding globulin (TBG). The exact percentages usually bound to each protein vary from

species to species. However, in most cases, albumin makes up the majority of the bound serum TH pool.2

It should be noted that each iodothyroinine type has different binding affinity for serum binding proteins

which directly impacts their pharmacokinetic behavior.14

Plasma Thyroid Hormone Binding and Pharmacokinetics

As previously mentioned, three major thyroid hormone binding proteins exist in mammalian

species. The relative concentrations and binding capacity of TBG, TTR)and albumin vary by species. As

each of these three proteins have varying affinities for each thyroid hormone (affinities which again vary

7

by species), different species can have greatly differing serum clearance rates. Accordingly, the degree to

which a species may tolerate insults to the thyroid gland is partially moderated by serum binding capacity

and kinetics.

TTR was first identified from cerebrospinal fluid(CSF).15,16

TTR was originally known as

prealbumin, and is also involved in Vitamin A binding by association with retinol binding protein.17

TTR

is a 127-residue monomer which has been shown in human and chicken to function as tetramer of four

TTR monomers.14

One TTR tetramer contains two identical hormone binding sites within the central

channel. TTR is expressed in the liver and choroid plexus of eutherian mammals, marsupials, and birds,

but only in the choroid plexus in reptiles.14

This has been interpreted as implying that the extrahepatic

synthesis of TTR evolved first. However, TTR was found in the liver but not the choroid plexus of an

amphibian and fish model.14

In humans, TTR is present in serum at approximately 300 mg/liter, with a

binding capacity of 2300 µg/liter. TTR serves as the primary thyroid hormone binding protein in the

CSF.18

TTR has different binding affinities between species, such as the preferential binding of T3 in

birds and fish as opposed to T4 in human and rat. The preferential binding of TTR to thyroxine in

eutherian mammals and its abundance in CSF combine to favor transport of T4 from blood to brain.18

There is an 82% identity and 92% similarity between human and rat TTR.14

In rats, TTR is the major T4

binding protein. The Kd for T3 and T4 are similar between rats and humans. In rats, the Kd for T3 is

67.2nM, and 8.0nM for T4. In humans, the Kd's are 56.6nM and 13.6nM respectively.14

The affinity and

capacity would suggest that TTR would serve as a regulatory protein for free T4 fraction whenever

thyroxine binding globulin is not present or present only in low quantities such as in rodents, or in the

CSF of fish and mammals.

In humans, TBG binds approximately 75% of plasma T4.14

TBG is present in human serum at

approximately 15 mg/liter, with a Kd of 0.1 nM.14,19

This small amount of protein binds approximately

600 nmol/l (200 µg/l) T4 in euthyroid adults.20

TBG is a member of the serpin family of protease

inhibitors, but does not itself have inhibitory action. However, TBG retains a reactive site loop, which is

8

cleaved during sepsis.19

TBG mutations have been linked to several human thyroid disorders, with a

prevalence of around 0.01% in newborn Caucasians.21

The remaining major thyroid hormone binding protein is albumin. The gene encoding human

albumin is located on Chromosome 4, mapped to regions q11-q13. The gene contains 15 exons, spanning

over 16000 nucleotides on the chromosome. Albumin is a non-glycosylated globular protein, consisting

primarily of alpha-helix structures. It makes up a significant portion of the total serum proteins, and is

active in binding many classes of bioactive molecules, including steroids and thyroid hormones. Albumin

has multiple distinct sites that bind TH, with varying binding kinetics. There is one primary binding site,

and between 4-6 secondary binding sites. The primary binding site for T4 is shared by several other

molecules, including L-tryptophan, oleate, and linoleate. In humans, the association constant for the

primary site is 7.0x105 M

-1, and for the remaining sites, the constant is 4.8x10

4 M

-1. Many technical

difficulties have been encountered while trying to determine the dissociation rate of TH from albumin.2,22

In an average human (70kg), T3 secretion is approximately 33% that of T4, resulting from

deiodination within the thyroid gland before secretion into the serum. However, the ratio in circulating

serum pools is significantly different, with average values in humans around 1.35 ng/ml and 86 ng/ml T3

and T4 respectively.2 This is due to the much greater clearance rate for T3 than T4. These rates do not

hold for all species, including rat. The turnover rate in rats is greater than that of humans, and the

thyroidal reserve much smaller in proportion to the rat's needs.

In euthyroid rats, the average plasma T3 concentration is about 0.47 ng/ml and T4 about 36.4

ng/ml, about 40% of the concentrations in euthyroid humans. Rats have an average distribution volume

of about 17 ml/100 g BW for T4 and 236 ml/100g BW for T3. Plasma clearance rates in rat are about

0.94 ml/h/100g BW for T4 and 21.3 ml/h/100g BW for T3. The T4 secretion rate is 38.3 ng/h/100g BW.

The total body pool sizes for T4 and T3 are 702 and 100 ng/100g BW respectively. The rate of T3

production from T4 is about 6.77 ng/h/100 g BW.23

The tissue distribution of thyroid hormones varies

between T3 and T4. 33.1% of T3 is found in the intestines, 2.3% in the kidneys, 8.7% in the liver, and

9

3.6% in the blood. 18.1% of T4 is found in the intestines, 1% in the kidneys, 8% in the liver, and 31.2%

in the blood.24

Deiodinases

While the majority of secreted thyroid hormones are in the form of T4, T4 has approximately

10% of the nuclear receptor-mediated activity of T3. Deiodination to T3 is required for full bioactivity

through this receptor-mediated mechanism. Recent studies have begun to show important roles for

3,5,3’-triiodothyronine (rT3) in neural development, such as in control of neuronal outgrowth and

migration, as well as actin polymerization.25

Deiodination is accomplished by three enzymes, known as

type 1, 2, and 3 deiodinase. Each of the three deiodinases have different affinities for the various thyroid

hormones, but all have the same basic action: removal of a single iodine molecule from a thyroid

hormone molecule. Thyroid hormone molecules contain two phenolic rings. Each ring contains up to

two iodine atoms. Type 1 deiodinase (D1) is primarily a 5'-deiodinase, but also has 5-deiodinase

potential. Type 2 deiodinase (D2) is exclusively a 5'-deiodinase. Type 3 deiodinase (D3) is exclusively a

5-deiodinase (Figure 2.2).

D1

Until the 1950's, the only identified thyroid hormone was T4.26

After the identification of T3 by

Gross and Pitt-Rivers, it became clear that some process was able to remove iodine from T4, and that this

process was in a large part responsible for the circulating T3 observed.27,28

This process was soon found

to be inhibited by administration of the thionamide antithyroid drug propylthiouracil (PTU).29

Soon

thereafter, the enzyme was found to be localized to endoplasmic reticulum and plasma membrane

fractions.30

D1 has been determined to have primarily 5'-deiodinase activity, but also to have 5-

deiodinase activity under certain conditions.31,32

D1 has been localized to many tissues in the body,

including the liver, kidney, thyroid, placenta, and intestine. D1 has also been identified in the rat central

nervous system, but not that of the human.26

It should be noted that the deiodinases are selenoenzymes,

relying on a selenocysteine residue for much of their activity.26

D1 deiodination proceeds by ping-pong

kinetics involving the iodothyronine and a thiol cofactor.26

“Ping-pong” kinetics means that the

10

iodothyronine and thiol cofactor bind independently to D1, and each undergoes a half reaction. It is

theorized that the first half-reaction deiodinates the iodothyronine and forms a selenoleyl iodide

intermediate, which is subsequently reduced by the thiol cofactor.26

D1 has the greatest affinity for rT3,

followed by 3', 5'-T2; 3, 3'-T2; 3'-T1; T4; and finally T3. At pH 7.2, the Km's for rT3 and 3', 5' – T2 are

0.1 and 0.77 µM respectively.32

D1 is regulated by a variety of factors, including nutrition and thyroid hormones, such as fasting

and the ratio of glucagon to insulin.33-39

D1 activity is increased at a transcriptional level by factors such

as T3, retinoic acid, cyclic AMP, and TSH.26

D1 transcription is decreased by IL-1β, diabetes, and

TNFα.26

In general, hypothyroidism reduces D1 activity whereas hyperthyroidism increases it. At a

translational level, selenium deficiency reduces D1 activity due to the necessity of selenocysteine for

proper functionality.26

D2

The continued presence of 5'-deiodinase activity despite PTU inhibition in some brain

preparations led to the identification of the Type 2- iodothyronine deiodinase. Like D1, D2 is a

selenoenzyme, relying on selenocysteine for proper functionality of its active sites. D2 is a membrane-

bound protein, attached to the endoplasmic reticulum, rather than the plasma membrane.40

Unlike D1, D2

has only been shown to have 5'-activity. D2 also contrasts with D1 in its relative affinities for the

iodothyronines. D2 has a greater affinity for T4 than for rT3 with Km's of 0.5-3nmol/l and 1-10 nmol/l

respectively.2 As with D1, D2 is found in a number of organs, including the CNS, pituitary, and brown

adipose tissue.41,42

In the brain, D2 is found in astrocytes and tanycytes.43-45

Interestingly, D2 also shows

differences in expression patterns between rats and humans, with humans showing D2 activity in adult

skeletal muscle, which was not expected after adult rat studies.26

D2 is especially important in the brain,

where studies have shown it to be responsible for over 75% of the nuclear T3 in rat cerebral cortex46

. D2

activity peaks around PND15-20 in rats.47

In adult humans, D2 is the only remaining 5'-deiodinase

present in the brain, again unlike the rat where both D1 and D2 remain expressed and active.48

11

D2 has a relatively low Km for T4 of about 2nM, putting it several orders of magnitude lower

than that of D1.42

D2 has a similar affinity for rT3 as for T4, though the overall kinetics favor T4

slightly.49

D2 also requires a thiol cofactor for activity. D2 deiodinase activity follows sequential

reaction kinetics, which suggests that the TH substrate and thiol cofactor must be simultaneously bound

to D2.42

D2 is known to be regulated by thyroid status through both pre- and post-translational

mechanisms, as well as by stress and other physiological alterations.50-56

D2 mRNA is upregulated under

hypothyroid conditions, responding primarily to T3 mediated by nuclear receptors.57,54,45,58

However,

photoperiod and other stress factors can also play a role in determining mRNA expression.59-61,52,62

On the

posttranslational side, T4 is the driving force, though rT3 also plays a role.53

Posttranslational regulation

includes targeting for ubiquitination, a process driven by the availability of T4 for D2.63,64,25

D2 is rapidly

cycled under normal conditions, with destruction of D2 accelerated by availability of substrate.65-67

The posttranslational regulation of D2 is essential to the rapid response of this enzyme to

changing availability of thyroid hormones. Both in vivo and in vitro tests blocking gene transcription or

protein synthesis with actinomycin D or cyclohexamide, respectively, have demonstrated rapid decreases

in D2 activity when challenged with thyroid hormones.65,68,69

T4 and rT3 are far more potent than T3 in

inactivating D2 in hypothyroid animals, further reinforcing the importance of the non-nuclear aspect of its

regulation.68

One study looking at D2 mRNA and activity in rat cerebral cortex demonstrated an

approximately 50% increase in D2 mRNA at the same time that the activity was increased by 500%.54

Studies by Steinsapir et. al. of transiently expressed D2 in HEK-293 cells have helped to further

elucidate the mechanisms underlying the posttranslational regulation of D2. When cyclohexamide and

50nM rT3 was added to cells transiently expressing D2, D2 activity dropped by 50% in four hours.

However, in the presence of MG132, a specific inhibitor of the ubiquitin-proteasome pathway, this

reduction was completely blocked. Further experimentation with 75-Se labeled D2 demonstrated that this

reduction was in fact due to a 50% reduction in D2 protein, a reduction still blocked by administration of

MG132. This data indicates that posttranslational regulation of D2 by rT3 occurs solely through

12

increased targeting for ubiquitin mediated proteolysis. A further experiment with a mutant D2 which

changes one active site selenocysteine to alanine eliminates the deiodinative activity of D2, as well as its

substrate-induced degradation. Overall, this data indicates that it is in fact the interaction with substrate

which leads D2 to be targeted for ubiquitination and subsequent degradation.63

D3

D3 is the primary 5-deiodinase, and is thought of primarily as an inactivating enzyme, though

recent evidence is suggesting that rT3 has important physiological roles in neural development.70

D3 was

first identified in a monkey hepatocarcinoma cell line.71

D3 is present in many organs, including the

CNS, placenta, skin, and liver.72-76

It has been shown to be induced by TGFβ in a variety of human cell

types, including fetal and adult fibroblasts from several tissues, hemangioma cells, fetal epithelia, and

skeletal muscle myoblasts.77

The exact subcellular location of D3 is still uncertain, though it appears to

join D1 and D2 as an integral membrane protein.78

D3 catalyzes the 5-deiodination of T4, T3, and 3,3'-T2, though not the sulfoconjugates of these

hormones.79

Similarly to D2, D3 requires an unknown thiol cofactor (though DTT works in vitro), and is

insensitive to PTU inhibition.80

Another similarity to D2 is in the sequential kinetic pattern, as opposed to

the ping-pong kinetics of D1, again suggesting coordinated and contemporaneous binding of the

iodothyronine and thiol cofactor.81

D3 is upregulated by thyroid hormones, consistent with its perceived

role as an inactivating enzyme.80,26,82

As with the other deiodinase enzymes, regulation is complicated

and goes beyond only thyroid hormone stimuli.83-85

Like the other deiodinases, D3 is differentially

regulated in different tissues.86,87

Conjugation and Other Modifications

In addition to deiodination, peripheral metabolism of thyroid hormones also includes conjugation

of the phenolic hydroxyl group of the outer ring with sulfate or glucuronic acid. This type of conjugation

occurs primarily in the liver and kidney. The conjugation reactions inactivate the thyroid hormones, and

prepare them for elimination.88,89

Glucuronides are stable conjugates, and are rapidly excreted in bile, and

to some extent, urine.90

Glucuronidation is catalyzed by the enzyme UDP-glucuronosyltransferase, which

13

can be induced by a variety of compounds.91

Sulfation of the phenolic hydroxyl group blocks 5'-

deiodination, but stimulates 5'-deiodination.90

Sulfates are rapidly deiodinated in the liver, and little are

normally excreted intact in the bile or found in serum.90

T3 is a substrate for at least three phenol

sulfotransferases.92

Thyroid hormones can also undergo deamination and decarboxylase reactions in the

liver. This conversion results in deaminated acetic acid analogues of thyroid hormones.88

The analogues

of T3 and T4 are known as triiodothyroacetic acid (Triac) and tetraiodothyroacetic acid (Tetrac)

respectively.93

Effects of Thyroid Hormones

Overall Clinical Effects

Clinical effects of thyroid hormones are best identified through the organismal changes observed

in conditions of hyper- and hypothyroidism. In hypothyroidism, basal metabolic rate will decrease,

meaning that overall energy production and consumption in the body are reduced. In humans, hair

becomes coarse and sparse and the skin becomes dry and yellowish. Cold is poorly tolerated due to

impaired thermogenesis. Mental function is slowed, and memory is impaired.94,95

Hyperthyroidism, in contrast, leads an increase in basal metabolic rate, and weight loss is often

seen. Nervousness results from increased sympathoadrenal output and secondary cardiovascular effects.

Tremors can be found in extremities. Additionally, an intolerance to heat is noted, as thermogenesis is

elevated even under warm conditions.94,95

Thyroid hormone receptors

The first accepted cellular effect of thyroid hormones was action through nuclear receptors.96

The

first demonstration of thyroid hormones stimulating RNA and protein synthesis came in the mid 1960's.97-

99 Specific nuclear binding sites for T3 were first recognized in 1972.

100 Further studies found a

relationship between the amount of hormone present in nuclear preparations and the response generated.

101 In the mid-1970's, it became apparent that there were more than one type of TH receptor (TR), though

all types were believed to belong to the same family, and be conserved across species.102,2

Current

understanding has evolved to recognize five current thyroid hormone receptor isoforms, belonging to two

14

primary isoforms and several variants. The genes were first identified in chick embryo and human

placenta, and each species showed a different receptor.103,104

The gene coding for the chick isoform was

designated Thra, and its product TR-α, and the human homologue was located on chromosome 17.105

The

gene for the placental isoform was located on chromosome 3, and designated Thrb, with its product

designated TR-β.104

The thyroid hormone receptors have been classified into the nuclear hormone

receptor superfamily, which includes receptors for vitamin D, retinoic acid, and several other

hormones.106

Both of these genes have been identified in the rat as well.107,108

A second α isoform was

identified in humans in 1987.109

It was further found that there were two forms of the α2 isoform, which

were denoted TR-α2vI and TR-α2vII, or alternatively c-erbAα-2.110,111,106

The α2 and α1 isoforms are

created by alternative splicing of the TRα mRNA, though the α2 form lacks the ability to bind T3 due to

an alteration in the carboxy terminus.106,112,113

Interestingly, the noncoding strand of the Thra gene itself

codes for Rev-Erb a related protein involved in adipogenesis.114,115

An additional β isoform, designated

TR-β2 was identified in a rat pituitary tumor line.116

The thyroid hormone receptors act as ligand-regulatable transcription factors because they bind to

both thyroid hormones and thyroid hormone response elements (TRE) in the genome.106

The TRs consist

of several conserved domains. The central portion of the TR is a DNA-binding domain with two zinc

finger motifs to intercalate with the major and minor grooves of the TRE.106

The TRs must dimerize to

bind DNA, and have been shown to form heterodimers with related receptors, such as the retinoic acid

receptor.117

In addition to the dimerization, co-activators and co-repressors can also bind to the TRs.118-122

The role of the various TR isoforms is not yet fully understood. In most cases, the effects seem to be

interchangeable, with the exception of the α2 isoform.106

However, recent research has suggested that

there may be some specificity for certain genes in the hypothalamus and pituitary, such as TRH, growth

hormone, and TSH.123-125

Both the TR-α1 and TR-β1 isoforms are expressed in almost all tissues, though

the representation in rats skews towards the β1 isoform in the brain, liver and kidney, and towards the α1

isoform in skeletal muscle and brown fat.126

Recent research has shown that unliganded TRs may play a

15

role in repression of basal expression of several genes, in conjunction with several corepressor

proteins.127-129

Nongenomic actions of thyroid hormones

In recent years, mechanisms of thyroid hormone action that do not involve transcriptional

regulation have come to light. While these actions are still postulated to rely on receptors for mediation,

the location and identity of these receptors varies from that of the genomic actions.96

Many of these

actions have been localized to specific tissues such as heart, brain, muscle, liver, and circulatory

vessels.130,25,131-138

It should be noted that the class of nongenomic actions does not preclude actions

which may eventually have a genomic effect, such as the initiation of phosphorylation events which affect

DNA-binding proteins.139,140

Recent studies have even suggested the existence of thyroid hormone

receptors among extracellular matrix proteins.96

One of the first organ systems to earn consideration for nongenomic thyroid hormone effects was

the cardiovascular system.130

In the mid-1990's, it was shown that thyroid hormone could mobilize

mitochondrial magnesium stores in the heart and liver of rat through interaction with the adenine

nucleotide translocase.137

T3 has been further shown to impact phosphorylation potential in sheep heart,

in a mechanism involving a decrease in ADP concentrations.141

T3 has been shown to have an acute

effect on the inward rectifier potassium channel in guinea pig ventricular myocytes, decreasing the

duration of action potentials.142

This type of effect has also been shown in the rat, with effects including

prolonged action potentials under hypothyroid conditions.143

T3 has also been shown to increase the

throughput of the Na+ channel in ventricular myocytes by increasing the fraction of channels in the open

state at a given time.144

T3 and T4 have been shown to have direct, rapid effects on arterial vasodilation

in rat coronary artery.138

Additionally, it has recently been shown that T3 can acutely increase the blood

flow to some organs, such as the kidneys, through a vasodilative process.145

Several mechanisms of nongenomic action have also been identified in the brain. Already

mentioned is the effect of thyroid hormone on the degradation of D2.25,65,146,147

Recently, another major

mechanism has been elucidated regarding polymerization of actin, a mechanism which calls to light the

16

potential importance of rT3 in the brain.70

In hypothyroid PND14 rats, administration of rT3 or T4 served

to rapidly decrease the proportion of G-actin in favor of F-actin, which may contribute to thyroid

hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.

Notably, T3 did not have a similar effect. At the same time as the actin polymerization patterns were

changing, D2 levels were rapidly returned to euthyroid levels within the span of only a few hours.25

It has

been speculated that the cytoskeletal effects may help to regulate the degradation of D2 by attaching to

the protein and removing it from its active location in the cell.147

A further nongenomic neurological

effect of the thyroid hormones has also been identified in the hippocampus, where T4 and T3 have been

shown to rapidly decrease the activity of NMDA receptors in hippocampal cultures.136

Additional effects

have been found in the cerebral cortex, where T3 and T4 have been shown to have biphasic effects on the

phosphorylation of several proteins in synaptosomal homogenates.148

Hypothalamus-Pituitary-Thyroid axis development

It is important to consider not only the mature thyroid and its associated organs, but also to

consider the development of the hypothalamic-pituitary-thyroid (HPT) axis. Due to the difference in

developmental timescales between the rat and human, the absolute timing of events is far different

between these species.149

The rat gestational period is approximately 21 days, and weaning traditionally

occurs 21 days after birth. Thyroid hormones have been detected in rat embryos as early as GD9, refuting

the old theory that thyroid hormones will not cross the placenta.150

Several studies have shown that the

embryonic rat is highly sensitive to maternal thyroid disruption.151,152

Pituitary development begins

around GD9.5, and secretory granules can be seen by GD16.153

The pituitary portal system is well

developed by GD17.149

TSH mRNA can be found in the pituitary starting on GD15, and TSH protein can

be isolated at GD17.154-156

TSH levels peak around PND11, and slowly decrease to adult levels between

PND14 and PND40.153

,157

The development of the hypothalamus takes place at a slower pace than that of

the pituitary. The hypothalamus first becomes visible on GD12.5, but does not reach maturity, especially

with regard to the vasculature, until about six weeks after birth.153,149

The rat thyroid is first visible on

17

GD9. In vitro cultures of rat thyroid cells are about to synthesize thyroid hormones at least as early as

GD20.149

These developmental events are summarized in figure 2.3.

The human HPT axis develops at a much slower absolute rate than that of the rat. However, there

are substantial parallels between the species, with events generally occurring at the same or similar

relative developmental timepoints. The anterior pituitary begins to form from Rathke's pouch, which first

appears in the fourth week of gestation.149

By the fifth week, the anterior pituitary connects to a

derivative of the cerebrum which will become the posterior pituitary. Basophils, including thyrotropes

and gonadotropes are detectable in the pituitary by the eighth week of gestation, and large numbers of

thyrotropes are present by 12-13 weeks.153,158

The hypophyseal portal system develops around the same

time, with capillaries detectable as early as the seventh week of gestation.149

TSH production is

detectable by the twelfth week of gestation.149

Levels are relatively low until weeks 16-20, then peak

around weeks 22-30, and decrease slightly until birth.153,158

The hypothalamus is visible by the fifth week

of gestation. The hypothalamic nuclei are detectable at 12-14 weeks, and continue to mature through

about week 35.149

The vasculature connecting the hypothalamus and pituitary is visible as early as 11.5

weeks.159

The thyroid gland begins to develop in the third week of gestation, visible around GD16.153,149

The thyroid gland tissues coalesce and migrate to their final location by GD45-50, and the gland is well-

developed by the tenth week of gestation, by which time the gland accumulates iodine and produces

thyroid hormones.160

These developmental events are summarized in figure 2.4.

Brain Development

Many aspects of neurological development rely at least in part upon thyroid hormone. During

human pregnancy, there are several shifts in maternal thyroid regulation. Peripheral metabolism of

thyroid hormones increase during pregnancy, and the thyroid compensates by increasing production.161,162

In most cases this results in increased total serum T3 and T4, but no alterations in serum free T4.163,164

During the early phases of development, the thyroid hormone requirement must be met from maternal

hormone stores, with several negative consequences to maternal hypothyroidism.165-168

All of the thyroid

hormone receptors have been identified in the human brain between 10 and 16 weeks of gestation, and

18

TRα1 and TRβ1 mRNA's have been detected in the eighth week.169-171

In the rat, the TRβ1 and TRβ2

receptors have been detected at GD 12.5, and the alpha subtypes at GD14.172-174

After birth, receptor

mRNA and protein levels increase until peaking at PND10 and PND15 respectively.175,176

This timing

matches the time of eye opening in the rat, and is the time of full maturation of the HPT axis feedback

loop.

The complexities of neural development are far from fully understood. However, certain portions

of the developmental processes are better understood than others. This review will focus upon the basic

development of the hippocampus. Much of the current knowledge regarding cell formation in the rodent

hippocampus dates back to work in the 1960's and 1970's.177-179

Stem cells for both the pyramidal neurons and granule cells originate from the ventricular

germinal layers located below the ventricular wall along the CA1 area.178

Multiplying neurons directly

migrate from the ventricular zone to their final target region.180

Pyramidal neurons in the rat are

generated between day 16 of gestation and birth.181

CA3 pyramidal cell generation peaks at day 17, while

CA1 generation peaks a day or two later.181

At birth, the pyramidal layer in the rat hippocampus is

composed of 6-10 rows of neuronal somata, though in the larger adult hippocampus, this layer has

thinned to two or three rows.178

In humans, the pyramidal layer is formed in the first half of pregnancy,

with the CA1, CA2, and CA3 regions differentiated as early as gestational week 16.182

The formation of

the granule cell layer of the dentate gyrus occurs later and lasts longer than the development of the

pyramidal layer.178

In the rat dentate gyrus, the first granule cells appear a day after the first pyramidal

neurons, and only 15% of the granule cells have developed by birth.181,178

This lengthened time frame

also translates to human development. In humans, granule cell formation lasts more than 30 weeks,

starting around the 13th week, and some evidence shows continued neurogenesis in the adult dentate

gyrus.183-185

Disruption of the thyroid axis during any point of this developmental timeline will lead to

abnormal development.

19

Thyroid Disruption

The hypothalamic-pituitary-thyroid axis is vulnerable to disruption at many points. A large

number and variety of stress factors and exogenous compounds can disrupt the production, activity, and

metabolism of thyroid hormones. Within the thyroid gland itself, there are several targets for disruption.

Compounds such as perchlorate and thiocyanate directly inhibit the uptake of iodine into the thyroid.149

Amino acid deficiency or protein synthesis inhibitors can impair the production of Tg, thyroid peroxidase,

or other vital proteins. Compounds like PTU, methylmercaptoimidazole (MMI), and iopanoic acid inhibit

the incorporation of iodine into Tg. Disruption of the cytoskeleton can impair the phagocytotic processes

involved in reuptake of colloid, digestion of Tg, and export into the serum. Mutations in serum proteins

can alter the binding kinetics of thyroid hormones, disrupting the normal equilibrium between bound and

free hormone, altering the availability of hormones to peripheral tissues. Several other compounds will

also compete with thyroid hormones for binding sites, such as lipids, polychlorinated biphenyls (PCB's),

DDT, polybrominated biphenyls (PBB's), and many more. Disruption can also occur at the level of

peripheral tissue uptake. Transporter proteins such as organic anion transporting polypeptide 1C1

(OATP1C1) and monocarboxylic acid transporter 8 (MCT8) are vital for proper uptake of thyroid

hormones into the brain but transport more than just thyroid hormones. Even within the target tissues,

interference with the thyroid hormone receptors will inhibit the activity of the produced hormones.

Several agonists have been identified, including 3,5-dimethyl-4-(4-hydroxy-3-isopropylbenzyl)phenoxy

acetic acid (GC-1), tetrac, triac, PCB105, PCB118, and many more.186,187

Several antagonists have also

been identified or synthesized, including O-[4-hydroxy-3,5-diisopropylphenyl]-L-tyrosine, 3,5-dibromo-

4-(3,5-diisopropylphenoxy)benzoic acid, {4-[4-hydroxy-3-isopropyl-5-(4-nitrophenyl)- benzyl]-3,5-

dimethylphenoxy}acetic acid, and {4-[4-hydroxy- 3-isopropyl-5-(4-nitrophenylethynyl)benzyl]-3,5-

dimethylphenoxy}acetic acid, amiodarone, and bisphenol-A.188,189

Another form of disruption focuses on

the metabolism of thyroid hormones. Compounds like propylthiouracil (PTU) and iopanoic acid will

inhibit the action of some or all of the deiodinases. Several compounds including the PCB mixture

Arochlor 1254, DDT, hexachlorobenzene, phenobarbital, and thiazopyrcan induce secondary metabolism

20

in the liver through mechanisms including interactions with the retinoic acid receptors, impairing

sulfation and glucuronidation. 149

For most forms of disruption, the HPT axis has compensation mechanisms. The primary

adjustment is the negative feedback pathway between thyroid hormones and the hypothalamus and the

pituitary. A reduction in hormone availability to the hypothalamus and pituitary resulting in decreased

negative feedback will result in increased synthesis and secretion of TRH, which will in turn trigger

increased secretion of TSH, which will stimulate increased hormone production and export from the

thyroid gland. The thyroid itself has been shown to respond to disruptions such as acute iodine excess.

When plasma iodide concentrations are abnormally high, organification is reduced in the thyroid.190

This

phenomenon, known as the Wolff-Chaikoff effect, lasts for about two days before the thyroid escapes the

effect through the downregulation of NIS mRNA and protein.191

In conditions of prolonged

hypothyroidism, the ratio of T3 to T4 produced by the thyroid will shift towards T3 as a result of both

changes in the ratio in which these two hormones are produced as well as changes in deiodinase activity

within the gland.192

Different results are observed between various causes of hypothyroidism, such as

thyroidectomy and iodine deficiency. In the peripheral tissues, deiodinase levels can be adjusted within

only a few hours to compensate for increased or decreased availability of hormone.54

There are limits to

the amount of compensation possible by the thyroid and peripheral tissues. The thyroid is very efficient

at sequestering iodine, even going so far as to drastically increase the size of the gland to the point of

goiter, thus giving the original term for a thyroid disrupting compound, “goitrogen”. Even with glandular

hypertrophy, the absolute iodine availability can still become insurmountable in extreme deficient

situations.193-196

Hypothyroidism and neural development

Proper functionality of the thyroid axis is essential for proper neural development. Iodine

deficiency is recognized as being the most common preventable cause of mental retardation in the world.

While the role of iodine was obscured for a time due to the inability of thyroid hormone supplementation

after birth to reverse these changes, and the belief that thyroid hormones did not cross the placenta, it was

21

eventually understood that iodine's role came simply as a component of thyroid hormones.197

Developmental hypothyroidism caused by iodine deficiency has been shown to cause mental and growth

retardation, rigid spastic motor disorders, and deaf mutism, and has affected millions of children around

the world.1 It is estimated that more than a billion people in the world are at risk for consuming

insufficient iodine.198

Thyroid Disruption and the Brain

A substantial body of literature has developed around the investigation of the effects of

hypothyroidism on the hippocampus and the rest of the brain. The hippocampus has received special

focus because of its critical role in learning and memory.178

These investigations are made far more

challenging by the varying regional responses to hypothyroidism, both in the impact on the concentrations

of thyroid hormones, and the response to those hormones.199

Despite the currently primitive

understanding of the complexities of neurological development, as steady stream of progress has been

made in understanding the effects of developmental thyroid disruption, mainly in hypothyroid conditions.

A wide range of effects have been observed, both transitory and permanent.

In 1979, Rabié demonstrated that the longitudinal growth, area, and volume of the hippocampus

were reduced by hypothyroidism.200

In 1983, King found that the activities of 5'-nucleotidase and of 2',3'-

cyclic nucleotide 3'-phosphohydrolase were reduced in hypothyroid rats, correlating with a delay in

myelination.201

Dozin showed in 1984 that the hippocampus, cerebral cortex, olfactory bulb, and caudate

nucleus had an increased nuclear binding capacity for T3, but a decrease in binding capacity with an

increase in binding affinity in the pituitary.202

Rami found in 1986 that the arborization of the dendritic

field of granule and pyramidal cells were impaired, and that not all of these changes could be overcome

by thyroid hormone replacement.203

That same year, Rami also found that hypothyroidism did not change

the production rate of granule cells, but that it did retard their migration, though this data was refuted in

1988, when Madeira demonstrated a reduction in granule cell numbers.204,205

Madeira further showed that

hypothyroidism eliminated the bias towards greater granule cell numbers in male than female rats,

suggesting that thyroid hormone levels may play a role in the sexual dimorphism shown in the rat

22

hippocampus.205

Madeira further found that the CA3 pyramidal cell layer was decreased in volume,

combined with an increase in density and no change in cell numbers.206

In contrast, the CA1 region

showed a decrease in pyramidal cell numbers between 13.2 and 22.5% at 30 and 180 days after birth for

animals rendered hypothyroid from birth until sacrifice, a reduction that was not reversed after 150 days

of recovery.206

Glutamatergic neurons were shown to be minimally responsive to neonatal thyroid

deficiency in 1987, reinforcing the concept that thyroid hormone actions are cell-type specific.207

Neonatal hypothyroidism caused a marked retardation of the developmental patterns of choline

acetyltransferase, a marker for cholinergic neurons, though the effect diminished with age in the

hippocampus but not in the cerebral cortex and cerebellum.208

Glial cells were investigated by Rami in

1988, where it was found that hypothyroidism delayed the maturation of the radial glial fibers, as well as

a reduction in glial fibrillary acidic protein(GFAP) labeled astrocytes later in development.209

This

observation contrasts with observations in the cerebellum, where hypothyroidism leads to glial

hypertrophy.209

Perinatal PTU administration was observed in 1988 to decrease the number of

protoplasmic processes and end-feet in fibrous astrocytes in the cortical molecular layer, a reduction

which remained even after a recovery period.210

Rami continued publishing in 1989, showing that

hypothyroidism led to a delay in the arrival of cholinergic afferences, as well as a slight alteration in the

early development of high-affinity vasopressin receptors.211

In 2003, Lavado-Autric investigated cell

migration and cytoarchitecture in the somatosensory cortex and hippocampus of PND40 rats born to

iodine deficient dams, and found a significant number of cells, including neurons, at abnormal

locations.212

Farahvar took the morphometric analysis of the hippocampus to the next level, creating 2D

foldout maps of the hippocampus.213

This analysis showed a decrease of 11% and 20% in hippocampal

surface area in P25 and P90 hypothyroid rats, with the greatest reductions in the CA1 and CA4 regions.213

Rats allowed to recover for two months after weaning showed near complete return to normal sizes.213

This recovery was further confirmed by quantification of mitochondrial cytochrome oxidase staining.214

Research into the hippocampal impact of hypothyroidism continued in the 1990's, when Raimi

demonstrated a delay of about two days in synaptogenesis in the dentate gyrus, as measured by

23

synaptophysin immunostaining.215

The region-specific effects of hypothyroidism on GFAP were further

elucidated in 1991, when Faive-Sarrailh demonstrated an increase of up to 78% in GFAP mRNA in the

cerebellum between birth and PND14, falling to 53% of control levels by PND35.216

In contrast, the

GFAP mRNA levels in the hippocampus were always reduced compared to control levels.216

Sandrini

explored the responsiveness of neuronal receptors to thyroid status in the adult rat, and found that high

doses of PTU for 35 days led to a decrease in prazosin, yohimbine, and dihydroalprenolol binding sites in

the cerebral cortex.217

Imipramine binding was reduced in the cortex and hypothalamus, but increased in

the hippocampus.217

In 1992, Savage demonstrated that 0.02% PTU dosing through drinking water from

GD18-PND31 led to a 75% reduction in mossy fiber zinc density in the dorsal and ventral hippocampal

CA3 regions at PND31, and a continued 33-45% reduction after three months of recovery.218

Kulikov

investigated the effect of hypothyroidism on the 5-HT1A and 5-HT2A receptors, as well at the serotonin

transporter protein under an iodine deficient diet and with thyroxine replacement.219

Significant decreases

were found in the binding of ketanserun to 5-HT2A receptors in the frontal cortex in the iodine deficient

group, and the decrease was not present in the hormone replacement group.219

Vaidya studied the same

receptors with an eye towards regulation of BDNF mRNA expression, and did not find any differences in

basal expression under either hypothyroidism or chronic excess T3 conditions.220

However, acute T3

dosing did decrease dentate gyrus BDNF mRNA levels in a mechanism mediated by the 5-HT1A

receptor.220

In 1993, Rodriguez-Peña investigated the expression of myelin-associated glycoprotein (MAG)

mRNA and protein.221

Expression of this protein is a biomarker of myelination of nerves. Hypothyroid

rats demonstrated a delay of several days in the accumulation of MAG that was more severe in rostral

regions of the brain, and recovered in later stages in more caudal regions.221

No differences in

transcription of the MAG gene were observed, indicating that the mRNA differences found are a result of

thyroid hormones affecting the stability of MAG mRNA.221

Iñiguez investigated RC3/neurogranin in

1993, finding that the normal accumulation of RC3 mRNA between PND5-12 was decreased by about

50-70% in hypothyroid rats.222

This decrease could be quickly reversed by an administration of 10

24

micrograms of T4 on PND12, returning to normal levels or higher in 48 hours.222

Guadaño-Ferraz later

showed that the differential sensitivity of some cells with respect to RC3 gene expression is not correlated

with the distribution of thyroid hormone receptors in the brain.223

Navarro-Yubero reported on

neuroserpin, a thyroid-responsive serine protease inhibitor shown to have a role in the regulation of

anxiety in genetically modified mice. Neuroserpin mRNA levels were posttranslationally downregulated

in hypothyroidism in the cortical II/III and Via layers, the hippocampus, the retrospenal cortex, and the

medial hemenular nucleus, but not in cortical layer V or other areas of the brain.224

Gilbert found that the

PND21 offspring of dams treated from GD6-PND30 had diminished immunoreactivity of parvalbumin, a

calcium binding protein in the hippocampus, which could be reversed by T4 administration.225

Additionally, a decrease in GABA-mediated inhibition of the perforant path-dentate gyrus synapse was

found in adult offspring.225

Alvarez-Dolado found in 1994 that hypothyroidism reduced nerve growth factor (NGF) mRNA

by 20-50% in the cortex, hippocampus, and cerebellum at PND15 in neonatally hypothyroid rats, as well

as in adult-onset hypothyroidism.226

Martínez-Galán investigated severe iodine deficiency and MMI

administration with normal dietary iodine.227

Both treatments significantly decreased the proportion of

glial cell fibers expressing GFAP, indicating impairment of maturation in cells involved in neuronal

migration in the hippocampus.227

Vega-Núñez investigated the impact of hypothyroidism on

mitochondria in the brain of neonatal rats, finding that there was a significant decrease in mitochondrial

transmembrane potential in hypothyroid animals.228

This decrease could be reversed within 48 hours of

administration of thyroid hormones.228

Electron microscopy revealed a decrease in the area of the inner

membrane plus cristae in all areas studied, with the exception of the hippocampal CA1 neurons and

nonneuronal cell types.228

Further investigations by Martinez showed a decrease in mitochondrial

oxidative phosphorylation rate and mitochondrial transcription in the cerebral cortex and striatum, but not

in the hippocampus, cerebellum, thalamus, mid brain, and brain stem.229

Sawin investigated PTU doses from 5-25 ppm in drinking water from GD18- PND21 on the

cholinergic neuronal system by measuring the activity of choline acetyltransferase (ChAT) and

25

hemicholinium-3 binding to the high-affinity choline transmitter in the prefrontal cortex and

hippocampus.230

ChAT activity was decreased in a dose-dependence manner, but hemicholinium-3

binding was elevated, though both returned to normal after a recovery period.230

In the same year,

Alvarez-Dolado found that tenascin-C, an extracellular matrix glycoprotein involved in cell adhesion,

migration, and neurite outgrowth, was upregulated in neonatal hypothyroid rats in several areas including

the hippocampus, though downregulated in other areas such as the occipital and retrosplenal cortex.231

At

the same time, the group investigated the reelin and dab1 genes, which are involved in neuronal migration

and lamination.232

Hypothyroid rats showed decreased levels of reelin mRNA and protein between GD18

and birth.232

Reelin expression was increased in several areas by PND5, and returned to normal by

PND15, though cells cultured from PND0 animals continued to demonstrate reduced reelin expression.232

In contrast, dab1 expression was increased at birth and decreased at PND5 in hypothyroid rats.232

Alvarez-Dolado investigated TAG-1, another cell adhesion molecule, and found an upregulation of TAG-

1 mRNA and protein levels from GD20-PND15 in regions including the cerebral cortex, hippocampus,

and olfactory bulb, and adding the cerebellum after PND15.233

Calloni investigated congenital neonatal

hypothyroid rats, and found a 50% increase in ERK 1 and 2 phosphorylation, and a 50% decrease in p38

phosphorylation, showing significant impacts on the MAP kinase pathways.234

In 2002, Vara reported on an investigation into short-term synaptic plasticity and neurotransmitter

release in hippocampal slices.235

Hypothyroid rats displayed altered plasticity, and an increase in Ca2+

-

dependent neurotransmitter release.235

Expression of synapsin I, synaptotagmin I, syntaxin, and alpha-

Ca2+/

/calmodulin kinase II was investigated in an attempt to elucidate the mechanism behind this

alteration.235

Increased expression was found for synapsin I and synaptotagmin I in hypothyroid rats.235

In adult thyroidectomized rats, Lee found a decrease in the NR1 receptor mRNA in the hippocampus, as

well as the NR2B receptor mRNA in the dorsal hippocampus.236

In 2004, Hoffman reported data on the

effect of hypothyroidism on the Na+ current densities in cultured hippocampal and cortical cells.

237

Treatment of these cells with thyroid hormones increases the rates of rise, amplitudes, and firing

frequencies of action potentials.237

Kobayashi investigated synaptic gene expression and behavior in

26

hypothyroid rats with perinatal exposure to PTU, finding increased motor activity, decreased maze

performance, and altered expression of GAP-43 and M1 mRNA during neuronal network formation.238

Oh-Nishi looked at dopamine D2l-like receptor function in hippocampal slices, finding that the normal

inhibitory effect upon glutamatergic neurons becomes an excitatory effect under hypothyroid

conditions.239

In normal rats, this excitatory effect was seen in early ages, but transitioned to an inhibitory

effect by adulthood.239

Kobayashi has shown a similar effect of hypothyroidism in delaying the switch

from NR2B to NR2A subunits of the NMDA receptor in the rat cerebellum.240

While many of these effects are transitory in nature, even an alteration in the timing of

neurodevelopmental events can have devastating consequences. Even if no permanent alterations persist

to adulthood, impaired neural function during the neonatal and juvenile phases pose significant problems

to the ability to thrive in adverse conditions. Our understanding of neural development is still primitive,

and the full consequences resulting from any minor alterations to this process are beyond our

understanding. It is thus vital to focus even greater efforts on preventing exposure to thyroid disrupting

compounds during the critical stages of neurological development.

Persistent Effects of Developmental Hypothyroidism

The impact of hypothyroidism continues to affect neurogenesis even in the adult rat. Ambrogini

reports that the mitotic activity of the neural precursors is not decreased in adult-onset hypothyroidism,

but that the survival of spawned cells was dramatically decreased, leading to a lower number of immature

neurons being added to the granule cell layer, as well as a delay in differentiation of these new neurons.241

Desouza reinforces these observations by demonstrating that adult hippocampal progenitors expressed

thyroid hormone receptors.242

Pacheco-Rosado demonstrated that adult-onset hypothyroidism generated

with MMI decreased the activity of the Na+/K

+ ATPase in the cortex, amygdala, and hippocampus, but

not in cerebellum.243

Carageorgiou expanded upon this work with the inclusion of the Mg2+

ATPase and

acetylcholinesterase(AChE).244

In hyperthyroid adult rats, AChE activity was increased 22% in the

hippocampus, while Na+/K

+ ATPase activity was decreased by 47%.

244 In hypothyroidism, AChE

27

activity was reduced by 23% in frontal cortex, and increased 21% in the hippocampus, while Na+/K

+

ATPase activity was decreased by 35% and 43% respectively.244

Bruno investigated the effects of adult-onset hyper- and hypothyroidism on ATP, ADP, and AMP

hydrolysis in rat hippocampal and cortical slices, finding that hyperthyroidism inhibited hydrolysis of all

three molecules in hippocampal slices, but only of AMP in cortical slices.245

Hypothyroidism increased

hydrolysis of all three molecules in both hippocampal and cortical slices, and these effects could be

reversed by T4 replacement.245

Braganhol expands upon this work in a cell culture system in an attempt

to determine the mechanism.246

Hippocampal, cortical, and cerebellar astrocyte cultures from neonatally

hypothyroid rats were investigated.246

ATP and AMP hydrolysis are increased by 52 and 210%

respectively in cerebellar astrocytes, though T3 replacement reverses the increase in AMP hydrolysis.246

Studies have shown that ATP is directly involved in many neurological processes, including calcium

wave propagation, promotion of association between neurons and glial cells, and induction of

morphological differentiation and maturation of astrocytes.247,248

Constantinou investigated the effects of

adult onset hypothyroidism on thyroid receptor isoform mRNA expression in the hippocampus, cerebral

cortex, and cerebellum of the adult rat brain, finding a decrease of TRα2 mRNA in all regions, an increase

of TRα1 mRNA in cortex and hippocampus, and no changes in the TRβ isoforms.249

A wide variety of neurological effects have thus been observed in gestational, neonatal, and adult-

onset hypothyroidism. Whether the impact is on the structure of the brain, such as altered migration

patterns; metabolism, such as ATP and AMP hydrolysis; or any other factor, it is clear that maintenance

of normal thyroid hormone concentrations is vital to the proper development and function of the brain.

Many impacts of hypothyroidism have been shown to have permanent effects, lending even greater

urgency to the need to protect gestating females and their offspring from exposure to thyroid disrupting

compounds.

Electrophysiology

The electrical nature of nerve signaling has been studied for the better part of a century.250

With

the development of modern electronics, the electrical potentials generated by nerve cells can be monitored

28

in any scale from fractions of a cell to the entire brain. This capability has led to the use of

electrophysiological recording to analyze the function of neurons. On a basic level, electrophysiology

monitors the change in electrical potential either within a cell, across a membrane, or in the area

surrounding a cell resulting from the opening of ion channels in membrane polarization or depolarization.

When recording extracellular potential in multicellular samples, it is possible to determine the relative

strength of responses to given stimuli. It has been shown that neural pathways can alter their responses

within the course of an experiment, both in the positive and negative direction. These phenomena are

known as long term potentiation (LTP) and long term depression (LTD) respectively. The term “synaptic

plasticity” was coined in 1948, to describe the putative changes in synaptic efficacy thought by many to

be the basis of information storage in the brain.178

A formal postulate of this putative phenomenon was

advanced the following year by Donald Hebb.178

Before the discovery of LTP, a prior model for learning known as post-tetanic potentiation (PTP)

was discovered in 1947.251

PTP consisted of enhanced synaptic responses following a period of high

frequency tetanization of afferent fibers.178

The main weakness of PTP was that it lasted only about seven

minutes, making it far too short for permanent memory storage, though this was later increased to thirty

minutes in 1966.252,253

That same year also saw the publication of data showing that the potentiation

could be increased by increasing the stimulation frequency and using repeated tetani events.254

The

discovery of true LTP, with a potential duration of weeks, occurred at the beginning of the 1970's.255,256

Originally called “long-lasting potentiation”, LTP gained its current name in 1975.254,257

Electrophysiology in the Hippocampus

Two areas of the hippocampus are most commonly studied for electrophysiology. In vivo studies

usually use the dentate gyrus, while slice studies most often use the CA1 region.178

Each type of

experimentation allows testing of different parameters. Whole-brain studies have the distinct advantage

of retaining all natural neural pathways for experimentation. In contrast, hippocampal slices isolate the

regions of interest and remove external stimuli to the region of interest. Slice preparations also have the

advantage of allowing rapid application of exogenous compounds to the slice during experimentation.178

29

The usual pathways for studying LTP are the perforant path projection to granular cells of the dentate

gyrus, and the Schaffer-commissural afferents to the CA1 area.178

LTP induced in the rat dentate gyrus in

vivo has been observed to last for more than a year.258

Quite a few prior studies have looked at alterations in LTP resulting from hypothyroidism

induced in various ways and times, such as PTU, PCB's, MMI, and thyroidectomy. The results are

somewhat confusing, and even contradictory at first glance. Pavlides studied adult rats that were made

hypothyroid as neonates, and found that the previously hypothyroid animals had impaired LTP in the

dentate gyrus, as well as impairment in spatial learning.259

Altman exposed rats prenatally to 3,3',3,3'-

tetrachlorobiphenyl(TCB), and measured LTP in the visual cortex and hippocampus.260

LTP impairment

was seen in the visual cortex, but not in the hippocampus.260

These observations were reinforced by

further study with PCB-77 (coplanar) and PCB-47 (non-coplanar).261

PCB-77 replicated the impact of

TCB, but PCB-47 had minimal effects.261

Gilbert examined Arochlor 1254, dosing dams with 6

mg/kg/day from GD6-PND21, and looking at in vivo electrophysiology in the dentate gyrus of 3-6 month

old offspring, where a 50% reduction in LTP was seen in the dosed group.262

Further study was performed

via behavioral assessment of the rats, with no obvious alterations.263

A later report also showed enhanced

population spike and postsynaptic potential amplitudes in adult offspring.264

Altmann dosed rat dams

with Arochlor 1254 starting 50 days prior to breeding.265

LTP was examined in cortical and hippocampal

slices from PND10-20 pups, where a reduction in LTP was seen in the cortex, but not the hippocampus.265

Gerges looked at a combination of stress and thyroidectomy to alter in vivo early LTP (e-LTP) in adult

rats.266

In the CA1 region, hypothyroidism and stress each impaired e-LTP, but the combination was

sufficient to eliminate it, as opposed to the lack of effect seen in the dentate gyrus.266

Further study of

thyroidectomized rats showed an impairment of LTP in the CA1, but not the dentate gyrus in in vivo

electrophysiology.267

Still further study on these rats showed impaired performance in spatial memory

paradigms.268

Niemi looked at PTU dosage of rat pups starting at PND7, and continuing until sacrifice between

the second and sixth week after birth.269

Serum T3 levels were reduced by over 80% after three weeks,

30

and LTP in the CA1 region of hippocampal slices was significantly reduced after two weeks.269

Vara

used 0.02% MMI in drinking water from GD9 until weaning, finding a significant reduction in paired

pulse facilitation, but no effect on LTP or LTD.235

,270

Sui and Gilbert looked at 3 and 10ppm PTU from

GD6-PND30, and found a decrease in paired pulse facilitation and LTP in CA1 slices.271

Another report

under this paradigm showed enhanced LTP, increased baseline synaptic transmission, but reduced paired

pulse facilitation, as well as increased phosphorylation of ERK1 and ERK2.272

The same dosing protocol

led to impaired performance by adult offspring on the Morris water maze, as well as reduced LTP of the

EPSP, but interestingly increased LTP of the population spike.273

A further study using 15ppm PTU from

GD18-weaning found a decrease in in vivo fEPSP slope and population spike amplitudes, as well as

impaired LTP.274

Additionally, there was a drastic decrease in circulating thyroid hormones and in pup

growth.275

Dong used a gestational and neonatal iodine deficiency model, and found impaired in vivo

population spike, fEPSP, and LTP, as well as decreased expression of c-fos and c-jun.276

Sui

demonstrated that a 4-week dosing regimen of PTU in adult rats was sufficient to impair LTP and paired

pulse facilitation of the dorsal hippocampo-medial prefrontal cortex pathway in in-vivo

electrophysiological study.277

Goodman and Gilbert have found a cellular heterotopia in the corpus

callosum of PND 23 rats from dams dosed with PTU from GD6, which persists to adulthood.278

The cells

in this heterotopia were determined to have primarily originated as neurons between GD17-19.278

Several studies have also looked at the acute application of thyroid active compounds to brain

slices. Wong looked at acute slice application of PCB95 in adult rat, and found a decrease in CA1, as

well as a decrease in population spike and EPSP potentials.279

Niemi followed suit in 1998, showing a

decrease in LTP upon administration of Arochlor 1016, Arochlor 1254, and two specific congeners (the

mono-ortho PCB congener 2,4,4,8-trichlorobiphenyl and the coplanar congener 3,3,8,4,4,8-

tetrachlorobiphenyl, as well as a reduction in synaptic transmission at high doses of 1254.280

Gilbert also

looked at acute slice exposure to Arochlor 1254, finding an increase in population spike amplitude in the

CA1, and transient increase in EPSP amplitudes.275

Tang demonstrated the probable involvement of

31

thyroid hormone responsive protein(THRP) in LTP through the administration of a T3 injection into the

dentate gyrus, which increased LTP and THRP mRNA.281

Conclusion

A wide range of detrimental neurological effects resulting from developmental hypothyroidism

have been described. These effects range from severe physical and mental retardation to mild

hyperactivity. It is clear that developmental hypothyroidism can cause permanent alterations in the

functionality of the brain. At some dose/time combinations, clear alterations can be seen in the physical

makeup of the brain, whereas in other combinations, subtle effects can be seen in biochemical analysis

with no gross alterations. There is still no clear answer as to just how much of an insult can be tolerated

by the developing thyroid axis before the onset of permanent damage. Further research is needed to probe

the limits of compensation protecting the developing nervous system in order to understand the dangers of

subtle changes in thyroid hormone metabolism and distribution, especially in the young and other

sensitive subpopulations.

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53

3', 5' – I 3, 5 – I

Figure 2.1 – Chemical Structures of T4, T3, and rT3

T4 (Thyroxine)

Some Nuclear

Activity

T3 (3,5,3’-

triiodothyronine)

Great Nuclear

Activity

rT3 (3,3’,5’-

triiodothyronine)

Negligible

Nuclear Activity

54

Figure 2.2 - Deiodination Pathways of T4, T3, and rT3

D1- Type 1 5’-Deiodinase

D2- Type 2 5’-Deiodinase

D3- Type 3 5-Deiodinase

T4- Thyroxine

T3- (3, 3’, 5’-triiodothyronine)

rT3- (3, 5, 3’-triiodothyronine)

3, 3’-T2- (3, 3’-diiodothyronine)

T4

rT3 T3

3, 3' - T2

D1

D3

D1

D2

D1

D3

D1

D2

55

Figure 2.3 – Timeline of Rat Neurological and Thyroid Axis Development

Reprinted with permission from Howdeshell 2002149

56

Figure 2.4 – Timeline of Human Neurological and Thyroid Axis Development

Reprinted with permission from Howdeshell 2002149

57

CHAPTER 3

LOWER THYROID COMPENSATORY RESERVE OF RAT PUPS FOLLOWING MATERNAL

HYPOTHYROIDISM: CORRELATION OF THYROID, HEPATIC, AND CEREBROCORTICAL

BIOMARKERS WITH HIPPOCAMPAL NEUROPHYSIOLOGY1

__________________________

1Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. Published in Endocrinology 2008

Jul;149(7):3521-30

Copyright 2008, The Endocrine Society

58

Abstract

The developing central nervous system of the fetus and neonate are recognized as very sensitive

to maternal or gestational hypothyroidism. Despite this recognition, there is still a lack of data concerning

the relationship between thyroid-related biomarkers and neurological outcomes. We used

propylthiouracil (PTU) administered at 0, 3, or 10 ppm in drinking water from gestational day 2 until

weaning to create hypothyroid conditions to study the relationship between HPT axis compensation and

impaired neurodevelopment. In addition to serum T3, T4, free T4, and TSH concentrations,

cerebrocortical T3 concentration (cT3), hepatic Type I (D1) and cerebrocortical Type II (D2) 5'-

deiodinase activity, and thyroidal mRNA for thyroglobulin (Tg) and sodium iodide symporter (NIS) were

measured. Extracellular recordings from the CA1 region in hippocampal slices were obtained from both

PND 21-32 (pups) and PND90-100 (adults) rats to assess neurophysiological effects. Thyroidal mRNA

for Tg and NIS were increased in pups, but not in dams. Both PTU doses increased cerebrocortical D2

activity ~5-fold in pups, but only 10ppm increased D2 activity in dams. In dams, cT3 concentrations

were maintained at 3ppm, but fell 75% at 10ppm. cT3 concentration in pups fell 50% at 3ppm, and over

90% at 10ppm. In both 3 and 10ppm pups, hippocampal baseline synaptic activity correlated negatively

with cerebrocortical D2 activity. In 3ppm adults, impaired long-term potentiation was evident. In

summary, during depletion of serum T4, D2 activity served as a sensitive marker of tissue thyroid status,

an indicator of the brain's compensatory response to maintain cT3, and correlated with a

neurophysiological outcome.

Introduction

The thyroid gland influences the function and development of many organ systems, including

cardiovascular, skeletal, and nervous systems.1-5

Thyroid hormone receptors have been documented in

the brain during the first trimester.6 Because of its many roles, maintenance of thyroid hormone levels

during pregnancy is essential to the proper development of the offspring. A link between impaired

maternal thyroid status and IQ decrement in human babies has been documented.7, 8

Additionally, the

recent increase in neurological disorders in the human population, such as autism and ADHD, has been

59

theorized to correlate with the increasing prevalence of endocrine (including thyroid) disrupting

chemicals in the environment.3,7,9

Other well-documented outcomes of developmental hypothyroidism are

hearing loss, altered migration of brain layers, delayed eye opening in rats, poor performance on maze

tests, and impaired motor development.9-17

As the full range of detrimental neurodevelopmental effects of

exposure to goitrogens is not yet understood, further investigation into the effects of well-characterized

anti-thyroid compounds may assist the understanding of the impact of environmental goitrogens.

With respect to measures of impact on brain function, it is well established that disruption in the

level of thyroid hormone during development can alter synaptic transmission in regions such as the

hippocampal formation. Significant effects on long-term potentiation (LTP) have also been reported.18-27

LTP has been denoted as a cellular model for learning and memory processes; therefore, we and others

have theorized that changes in LTP could provide a functional link between thyroid hormone deficits with

deficiencies in cognitive function.28

In addition, prior studies have also demonstrated that inhibitory and

excitatory pathways may be differentially impacted by thyroid insufficiency.29,30

Such complexities may

contribute to the wide range of outcomes reported concerning the assessment of thyroid hormone

deficiency on electrophysiological parameters measured in the hippocampus.15,19,24

While significant animal research has been performed on thyroid toxicants, including studies of

neurodevelopmental toxicity, thyroid status is most commonly determined by serum hormone

concentrations such as total T4 and TSH.31-35

Many factors influence the relationship between serum and

tissue levels of free and bound thyroid hormones, and the relationships may be tissue- and region-

specific.36-38

PTU will have both dam-mediated and direct effects on the gestating pups, as it will cross

the placental barrier.39

While PTU has been shown to have effects on the liver and immune systems,

many of these effects have been demonstrated at doses well beyond those employed in this study. PTU

has been routinely used to induce developmental hypothyroidism to study neurological impact.

The hypothalamic-pituitary-thyroid axis has a variety of mechanisms which would allow

potential adaptation to a toxicological insult, including alteration of pituitary TSH secretion, alteration of

intrathyroidal sodium-iodide symporter and thyroglobulin synthesis, change in deiodinase enzyme

60

activity, and altered thyroid receptor number or affinity. However, little is known about the threshold for

triggering these changes nor about their compensatory limit. These issues become particularly critical to

understand in subpopulations at greater risk from thyroid disruption, the developing fetus and neonate.

The primary goal of the present study was to use gestational and lactational exposure to the

mechanistically well described antithyroid drug, propylthiouracil (PTU) in the rat, to study serum,

thyroid, and brain hormone markers of thyroid status in both offspring and dams. These parameters were

then correlated with electrophysiology results measured in the CA1 region of hippocampal slices obtained

from the offspring. This study identifies the biomarkers most tightly linking gestational and neonatal

thyroid insufficiency with the altered neurophysiological outcomes measured in the hippocampus.

Materials and Methods

Animal acquisition, care, and dosing

Timed pregnant CD® IGS rats were obtained from Charles River Labs (Raleigh, NC). Rats were

bred two days before arrival. Upon arrival, dams were housed individually and provided free access to

Purina 5001 chow and tap water. Animals were maintained on a 12h/12h light/dark cycle. At

approximately 1700h on the day of arrival, the water was replaced with either deionized water purified by

Millipore RiOS 8 (Millipore, Milford, MA), or deionized water containing 3 or 10ppm PTU (Sigma, St.

Louis, MO). Doses of 0, 3, and 10ppm PTU in DI drinking water were chosen to allow comparison of

electrophysiological results with prior published studies (24). PTU dosing took place from GD2 until

weaning, when pups selected for recovery were placed in standard housing with tap water and Purina

5001 chow.

Animals were weighed every other day, and water intake recorded. Water bottles were refilled

every 2-4 days, depending on the amount remaining in the bottle. New PTU solutions were made at least

every two weeks. Average water intake during gestation was approximately 200 ml/kg/day for the dams.

Pups in the control and 3ppm groups began to supplement their diet with food and water prior to weaning

at varying times after PND14, but pups in the 10ppm group were unable to do so due to reduced size and

coordination. Pups were sacrificed beginning on PND21, with two males per litter sacrificed over the

61

next eleven days. Data from these animals is presented as “PND25” pups. Additionally, all female pups

were culled at PND23. Remaining males were weaned at the end of this sacrifice period (on PND32) and

followed to PND90-110 referred to hereafter as “adults.” In this study, with this weaning protocol, the

weaned 10ppm pups were so physically and neurodevelopmentally delayed that it influenced their ability

to independently seek adequate food and water. Despite attempted supportive measures, it was not

possible to consistently maintain these animals for study in the PND90-110 adult grouping.

Animal studies were approved by the University of Georgia Institutional Animal Care and Use

Committee and were in accordance with procedures outlined in the National Institutes of Health

Guidelines for Care and Use of Laboratory Animals.

Sacrifice protocol

Male pups were weighed, anesthetized with halothane and sacrificed via decapitation. Blood,

thyroid, and liver were collected. The brain was dissected for tissue collection and slices prepared for

electrophysiology experiments (see below). In all pup and adult samples, the majority of the

hippocampus was used for slice preparation. Cortex samples were taken from areas of the brain not

impacted by dissection of the hippocampus, generally including prelimbic, motor, sensory, and cingulate

regions, and brain tissue samples were divided equally between deiodinase and hormone assays. Liver

and cortex samples intended for deiodinase assays were homogenized on the day of sacrifice in a 250mM

sucrose, 20mM KH2PO4, 1mM EDTA, 20mM DTT buffer, then stored in aliquots at -80oC until assayed.

Blood was allowed to clot and serum collected after centrifugation. All other tissues were flash-frozen

and stored at -80oC until analysis.

Biochemical Assays:

Serum Thyroid Hormone and TSH Radioimmunoassays:

T3 and T4 radioimmunoassays were validated for rat serum as previously described, with CV’s of

approximately 12% and 5% respectively.40

TSH was measured in dams with a commercial immunoassay

kit (MP Biomedicals, Solon, OH). Free T4 concentrations were measured by use of a two-step direct

dialysis commercial radioimmunoassay kit (Nichols Institute, San Juan Capistrano, CA) with CV

62

approximately 5%. All samples for serum hormone quantification were run in a single assay for each

hormone.

Thyroid RNA extraction and analysis:

Thyroglobulin (Tg) and the Sodium Iodide Symporter (NIS) were selected for mRNA analysis

because they are, at least partly, TSH-dependent. Thyroids were extracted by use of the Stratagene

Absolutely RNA kit (Stratagene, La Jolla, CA). After extraction, real-time RT-PCR was performed for

NIS and Tg at the UGA Functional Genomics Resource Facility using Taq-Man primers and probes (see

Table 3.1).

Protein assays:

All deiodinase samples were homogenized on the day of collection in 250mM sucrose, 20mM

KH2PO4, 1mM EDTA, pH 7, and frozen in aliquots at -80C until assay. Protein concentrations were

determined by the Bio-Rad protein assay reagent (Hercules, CA) using BSA as a standard.

Cerebrocortical Type II 5’-Deiodinase Activity:

This protocol was based on a modification of that of Leonard.41

Cerebrocortical Type II 5’D

activity was assayed using 2nM rT3, including 1.9MBq [125

I]-3’,5’-3-triiodothyronine (reverse T3) in

100mM KH2PO4, 1mM EDTA, 20mM DTT, pH 7 with and without 1mM PTU. Triplicate tubes were

incubated at 37oC for one hour with 50g protein for pups, or 200g for dams and adults, in a total

volume of 320l, along with tissue-free blanks. The reaction was stopped by addition of 150l ice cold

10% BSA. Tubes were then incubated 30 minutes at 4oC before addition of 500l of 20%TCA. The tubes

are spun at 2600xg at 4oC for 45 minutes. 400l of supernatant is then added to a 1.5ml column of

50WX2-200 resin (Sigma, St. Louis, MO) and eluted with 2ml 70% acetic acid. Tubes were then counted

on a gamma counter (Wallac Wizard 1470) along with tubes for total counts. The percentage of free

iodide was calculated by comparison of the elution fractions to the total counts. The radioactivity in the

eluted fractions minus that of the blanks was used to calculate the total deiodinase activity. The percent

free iodine generated in the samples containing PTU was multiplied by 2 to account for the equal

63

possibility of liberating a radioactive or nonradioactive iodide, then divided by the time and protein

amount and then multiplied by the total rT3 concentration in the assay to arrive at the final activity

measurements. The D2 activity was shown to be inhibited by iopanoic acid and excess of rT3 (data not

shown).

Hepatic Type I 5’-Deiodinase Activity:

Liver type 1 deiodinase (D1) assays were carried out using ~2nM rT3 in 200mM KH2PO4, 1mM

EDTA, 2mM DTT, pH 7 with and without 1mM PTU. Triplicate tubes were incubated at 37oC for 2 and

12 or 62 minutes (PTU-treated animals) with 2-3 g protein in a total volume of 120l. The reaction was

stopped by addition of 500l ice cold 10% calf serum and then incubated for 30 minutes on ice. 500l of

ice cold 10% TCA was then added to precipitate the reaction tubes. The tubes were centrifuged at

2590xg 4oC for 10 minutes. 500l of supernatant was placed into a separate tube and counted alongside

the pellet. The percent free iodine was calculated by multiplying by a factor of 2 to account for liberation

of nonradioactive iodine, dividing the supernatant activity by that of the pellet and multiplying by 2.24 to

account for volume. The percentage of free iodine above that of the 2-minute point was used to calculate

the total deiodinase activity.40

Cerebrocortical T3 extraction:

Frozen cerebrocortical tissue was homogenized in cold 100% methanol containing 1mM PTU

with ~0.001 Ci [125

I]3,5,3’-T3 tracer. The homogenate was centrifuged for 15min at 2000xg and the

supernatant fluid was collected into another tube and kept on ice. The pellet was resuspended in methanol

and centrifuged again. The supernatants were pooled and passed over an AG 1-X2 column (Bio-Rad,

Hercules, CA). The columns were washed and then eluted with 3ml 70% acetic acid.23

The elution

fractions were lyophilized and resuspended in 50mM KH2PO4, 0.25% bovine serum albumin before

counting in a gamma counter to determine recovery. Average recovery for this procedure was 57%. T3

concentrations in the extracts were determined by specific RIA as with the serum T3 assay. The

64

determined concentration was divided by the product of the extraction efficiency, the proportion of the

extract tested, and the initial mass of the tissue to give the final tissue concentration.

Electrophysiology:

In general, the electrophysiological procedures and analysis were conducted as previously

reported, except as noted. 42

Hippocampal Slice Preparation:

Freshly prepared hippocampal slices (500µm) were obtained following anesthesia and

decapitation. Horizontally cut slices were dissected in ice cold, oxygenated (95% O2/5% CO2) dissection

artificial cerebrospinal fluid (aCSF) containing (mM): NaCl (120), KCl (3), MgCl2 (4), NaH2PO4 (1),

NaHCO3 (26), and glucose (10). Slices recovered for one hour in an oxygenated interface holding

chamber with standard aCSF containing (mM): NaCl (120), KCl (3), MgCl2 (1.5), NaH2PO4 (1), CaCl2

(2.5), NaHCO3 (26), and glucose (10)). Slices were then transferred to a submerged recording chamber

and recovered for an additional hour at 30° C with continuously perfused standard aCSF saturated with

95% O2/5% CO2 at approximately 1ml/min before experiments were begun.

Extracellular recording:

Extracellular recording electrodes were placed in the stratum radiatum of CA1. Field excitatory

post-synaptic potential (fEPSP) responses were evoked with a bipolar stimulating electrode placed on

either the CA3 or the subicular side of the recording electrode in the stratum radiatum. Stimulus pulses

consisted of a single square wave of 270 µsec duration delivered at 60-160 µA.

Data acquisition and analysis:

Data were digitized at 10 kHz, low pass filtered at 1 kHz, and analyzed with pCLAMP9.2

software (Molecular Devices, Sunnyvale, CA). The initial slope of the population fEPSP was measured

by fitting a straight line to the first millisecond of the fEPSP immediately following the fiber volley.

Stimulus response curves were performed at the beginning of each experiment. Baseline stimulation

pulses of an intensity that gave 40-60% of the maximum response were given at a frequency of .05 Hz for

65

the entire length of the experiment. Synaptic responses were normalized by dividing all slopes by the

average of the 15 fEPSP slopes 5 minutes pre-tetanus.

Long Term Potentiation induction:

The LTP high frequency stimulation protocol consisted of a single 100 Hz train of 1 second

duration.

Statistics:

All statistical analysis was performed using SAS (The SAS group, Cary, NC). All group

comparisons were performed using ANOVA, followed by Duncan’s multiple range test. p<0.05 was

taken as the level of significance.

Results

1) Serum hormones (Table 3.2)

Dams

Serum total T4 concentrations were reduced by 60% at 3ppm and over 90% at the 10ppm dose in

the dams (p=0.001). Free T4 concentrations were also reduced by a similar amount, and were

undetectable at the 10ppm dose (p=0.001). Serum T3 concentrations were unchanged at 3ppm, but

dropped to about 25% of control values at 10ppm (p=0.03).TSH concentrations were greatly increased at

both 3 and 10ppm doses (p=0.001).

PND25 pups

Serum total T4 concentrations were reduced by 75% at 3ppm and over 90% at the 10ppm dose in

the pups, with most of the 10ppm samples below the limit of detection (p<0.0001). Serum T3 levels were

reduced by 20% at 3ppm and by 50% at 10ppm (p=0.005). Individual free T4 concentrations were not

obtained due to limited serum volumes available from pups, but pooled samples indicated a dose-

dependent decline in free T4 concentration, with undetectable levels at 10ppm. This change paralleled

that of total T4 concentrations.

66

PND100 adults

In 3 ppm dosed pups allowed to recover for approximately two months after weaning, all serum

concentrations of T3 and T4 returned to control levels.

2) Body weight (Figure 3.1)

Almost from birth, the mean body weights of the 10ppm pups were significantly reduced in

comparison to the undosed pups. In addition to the low body weights, a delay in eye opening and

decrease in overall physical coordination was observed (data not shown). At weaning, the 10ppm pups

were unable to reach food and water in standard rat cages, so special housing arrangements were

provided. While the 3ppm pups were not as affected as the 10ppm pups, they also weighed less than

controls, with the difference becoming significant around PND18, and expanding as the pups aged until

weaning.

3) Liver Type 1 5’-deiodinase activity (Figure 3.2)

Hepatic Type 1 5’-deiodinase activity was evaluated to confirm relative bioavailability and

activity of orally administered PTU. In both the dams and pups, liver D1 activity was significantly

reduced at both 3 and 10ppm. In the pups, D1 inhibition was more significant at the lower dose with 90%

reduction at 3ppm, and a near-complete reduction at 10ppm (p<0.0001). The dams were less affected,

with a 60% reduction at 3ppm, and a 95% reduction at 10ppm (p=0.0001).

4) Thyroid mRNA (Figure 3.3)

Sodium Iodide symporter (NIS)

In the pups, thyroid NIS mRNA levels were increased to about 250% of control at both the 3 and

10ppm groups (p=0.0001). No significant differences were noted in the dams, though there was a trend

towards an elevation at 3ppm (p=0.09).

Thyroglobulin (Tg)

In the pups, thyroid Tg mRNA levels were increased significantly at both the 3 and 10 ppm doses

(p=.014). The levels in the 3ppm dosed animals were about 300% of controls, whereas the levels in the

67

10ppm dose group rose to almost 1400% of control values. No significant alterations in Tg levels were

detected in dams.

5) Cerebrocortical thyroid parameters (Figure 3.4)

D2 activity

In the pups, cerebrocortical D2 activity was significantly increased at both 3 and 10ppm,

increasing to about six-fold that of control values (p<0.0001) with no significant difference between

doses. In the dams, a slightly different pattern was observed. There was a tripling in D2 activity at 3ppm,

and a further increase to 10 times control values at 10ppm (p<0.0001). However, the observed baseline

and maximal activity in the dams was about 10% of the activity in the pups. D2 activity was normalized

in 3ppm adults.

Cerebrocortical T3 concentration

In the pups, cerebrocortical T3 (cT3) levels were significantly reduced at both 3 and 10ppm

(p=.0004). At 3ppm, the levels dropped by about 50%. At 10ppm, cerebrocortical T3 levels were about

12% of control levels. As with D2, the dams were less affected by 3ppm PTU, with no significant

changes in T3 levels. However, at 10ppm, the cT3 of the dams was reduced to about 20% of control

levels. Cortical T3 concentrations were normalized in 3ppm adults.

6) Baseline synaptic transmission is impaired in hypothyroid pups (Figure 3.5)

Extracellular recordings of the field excitatory postsynaptic potential (fEPSP) response were

evoked from the stratum radiatum layer of the CA1 region of the hippocampus. The magnitude of the

baseline synaptic transmission was significantly reduced (p=0.0002) in hippocampal slices obtained from

pups (Fig. 5A&D). The maximal slope of the fEPSP was reduced by approximately 50% in both the 3

and 10 ppm dose groups, with no significant difference between these two conditions.

A normalized graph of the stimulus-response data shows no significant shift in the sensitivity of

the synaptic response at either dose group (Fig. 5B). Paired-pulse analysis of the fEPSP indicated that

there was no effect of PTU exposure on presynaptic function, as the paired-pulse facilitation ratios were

not affected. The combined results from Figs 5A and 5B suggest that while the absolute magnitude of the

68

synaptic response was decreased by PTU treatment, the sensitivity of this response was not impacted, nor

was the function of the presynaptic nerve terminals (Fig. 5C).

7) Long-term potentiation is impaired in recovered adults (Figure 3.6)

Unlike the results observed in PND21-32 animals, hippocampal slices prepared from littermates

allowed to mature in the absence of PTU from weaning until PND90-110 did not exhibit any persisting

change in baseline synaptic transmission (Fig. 6A). However, a significant impairment in the magnitude

of long-term potentiation was observed in the 3ppm adult group (Fig. 6B), suggesting that there may be

some consequences of PTU exposure during development that persist following recovery to the euthyroid

state.

8) Correlation between cerebrocortical D2 activity and serum total T4 concentration (Figure 3.7)

In both the dams and pups, a highly significant exponential correlation was observed between

cerebrocortical D2 activity and serum total T4 concentrations. As there was no evidence for an alteration

in the fractional free T4 percentage with this dosing paradigm, the correlation of cerebrocortical D2 is

most likely to be with serum free rather than total T4 concentrations. As D2 is known to be both pre- and

post-translationally regulated by thyroid hormones, this correlation was not unexpected.

9) Correlation of synaptic response to cerebrocortical D2 activity, cerebrocortical T3

concentrations, and serum total T4 (Figure 3.8)

Several prior studies have shown links between thyroid status and electrophysiological activity.

We identified correlations between the maximal synaptic response in pups and three thyroid-related

parameters. Linear correlations were observed between synaptic response and cortical T3, as well as

serum total T4 concentrations. Interestingly, and never before reported, a significant correlation was

observed between cerebrocortical D2 enzymatic activity and maximal synaptic response, supporting the

use of D2 activity as a marker of tissue thyroid status in the brain.

Discussion

Developmental thyroid disruption with PTU was initiated at gestational day 2 and maintained

until weaning. This protocol was designed to ensure that maternal thyroid hormone production was

69

compromised prior to the development of fetal thyroid function around GD17.2 This window of dosing

began earlier than that of most prior developmental studies performed in the rat with this compound.

16,17,19,21,24,29,30,45

It is noteworthy that cerebrocortical D2 activity increased in PND25 pups to maximal levels (~6-

fold) even at the 3ppm dose, a point at which cerebrocortical T3 concentrations had fallen by 50%. No

further increase was seen in D2 at the 10ppm dose, yet cerebrocortical T3 concentration fell further to

12% of control levels. In the dams, a tripling of cerebrocortical D2 appeared to allow maintenance of

cerebrocortical T3 concentrations at the 3ppm dose, but despite a ten-fold increase in D2 at the 10ppm

dose, the cerebrocortical T3 concentrations fell to 20% of control. Therefore, based upon a maximal

stimulation of D2 activity at 3ppm PTU, and reduction of cerebrocortical T3 at this dose, the brains of

pups were more severely impacted than those of the dams which were not impacted until the 10 ppm

dose. Recordings of the fEPSP response in the CA1 region of hippocampal slices revealed significantly

decreased basal synaptic transmission in both the 3 and 10ppm pups, indicating the presence of a

functional impairment in neuronal signaling as compared with 0ppm control animals. Although we

cannot point to a specific deficit based on extracellular recording results alone, the lack of effect on either

the sensitivity of the fEPSP or on paired-pulse facilitation suggests that neither postsynaptic

responsiveness nor presynaptic release were primary underlying factors. As the fEPSP recorded in the

stratum radiatum reflects a “population synaptic response”, one remaining possibility is that the synaptic

density was decreased in these PND25 pups. A prior report consistent with this speculation describes a

decrease in CA1 pyramidal cell density in hypothyroid rats.43

Cerebrocortical D2 activity was found to be a sensitive respondent to reductions in serum T4

concentrations. While this study focused on total T4, showing parallel changes in free T4 concentration

in pooled samples from pups, the true correlation is undoubtedly to the unbound free T4 concentration.

Both the dams and pups had a strong exponential correlation between declining T4 and increasing

cerebrocortical D2 activity (Figure 3.7). D2 is known to be regulated both pre- and post-translationally

70

by T3 and T4 respectively. One recognized method of post-translational regulation by T4 is control of the

rate of ubiquitination, and thus degradation of D2.44

In addition to a correlation with serum T4 concentrations, D2 activity was also found to correlate

with an important functional endpoint. A strong correlation between cerebrocortical D2 activity and the

maximal synaptic response in pups was observed (Figure 3.8). Synaptic response also correlated

positively with serum T4 and cerebrocortical T3 concentrations, although neither of these relationships

were as strong as the correlation with D2 activity (Figure 3.8). Whether representative of a mechanistic

link or reflective of a co-dependence, the correlation between D2 activity and synaptic potential is not

fully understood at this time, but D2 and cT3 concentrations did recover in 3 ppm pups as they returned to

euthyroidism at PND 90-110, along with the baseline fEPSP response. Nonetheless, D2 activity did

reflect the degree of availability of T4 from serum to cerebrocortical tissue at the time of sacrifice.37

Analysis of thyroid mRNA levels for Tg and NIS, chosen to be markers of serum TSH

bioactivity, further demonstrated the greater sensitivity to PTU in pups compared to their dams. The

upregulation of these genes reflect an attempt to compensate for diminished hormone production. The

lack of significant upregulation in the dams, compared to the pronounced upregulation in pups at both

doses, further reinforces the hypothesis that the developing rat is more sensitive to thyroid disruption than

is the adult rat. If this observation is paralleled in other mammalian systems, it reinforces the need to

consider juveniles as a hypersensitive subpopulation with regards to thyroid disruption.

Although the serum and tissue thyroid hormone concentrations returned to normal after weaning,

the LTP data obtained from littermates approximately 60 days after weaning demonstrated that some of

the effects of treatment could not be reversed by the removal of PTU at weaning. This observation

supports the conclusion that persisting changes can occur in the brain following a developmental insult,

possibly involving alterations in myelination and migration patterns of neurons.14,23,25-27

An interesting

aspect of our results was that the deficit in baseline synaptic transmission recovered concomitantly with

the hormone levels, as expected from the relationships illustrated in Figure 3.8. Despite this, our LTP

data suggest that the PTU-treated rats retained a neuronal deficiency from which they did not recover over

71

the time frame assessed in our studies. Thus in contrast to LTP of the population spike response recorded

from the dentate gyrus, an impaired capacity for synaptic plasticity of the fEPSP in the CA1 region may

be predictive of the well established cognitive impairments which persist following recover to the

euthyroid state.15

While PTU exposure is not a major environmental concern, we have attempted to use this drug as

a model goitrogenic compound. PTU targets thyroid secretion and tissue Type I 5’-deiodination, with

mechanistic effects common to dietary and environmental goitrogens, including apigenin and luteolin,

and mimics the T4 lowering effects of some PCBs, PBDEs and atrazine .46

Therefore, the results from this

study should be applicable to other compounds which disrupt the thyroid axis. Data gathered from this

study provide a correlative if not causative link between serum (free) T4, cerebrocortical D2 activity and

a neurophysiological consequence in hypothyroid rats. Such relationships could become the basis for a

predictive model of developmental thyroid disruption with regard to neurological outcomes.

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Table 3.1: Primers and Probes for Real Time-Reverse Transcriptase PCR analysis of thyroidal mRNA

Forward Reverse Probe

Tg

NIS

TGAGTGGTGCC

AGATTGGATAT

CAGCCTCGCT

CAGAACCATT

GCAATAGAACGTA

GGAGTCCAGAGT

ACCGGCTCC

GAGGATCA

TCCTGCCCACCCA

GAATCAAGGAAC

CCGGATCAACCTG

ATGGACTTTGACC

76

Table 3.2: Serum Hormone Concentrations

Serum total T4 was significantly decreased at all doses in both the dams and pups. All but one total T4

concentration in the 10ppm pups were below the limit of detection of 0.2 g/dl. The limit of detection for

the free T4 assay was 0.25 ng/dl. Serum total T3 concentrations were reduced in 3 and 10ppm pups, and

in 10ppm dams. Serum Free T4 concentrations paralleled those in total T4, leaving open the possibility

that free T4 concentration may be the true underlying serum biomarker. Serum TSH concentrations rose

significantly (p<0.05 in both doses) in the dams. No statistics are available on free T4 concentrations in

the pups, which were measured in pooled samples of serum from each male pup in a dose group.

(*p<0.05, ND = Not Determined)

Control 3 ppm 10 ppm

Dams T3 (ng/dl) 51 ± 14 52 ± 15 12 ± 6.7*

T4(µg/dl) 4.1 ± 0.8 1.6 ± 0.7*

0.30 ± 0.05*

Free T4(ng/dl) 2.4 ± 0.5 0.33 ± 0.1*

<0.25*

TSH(ng/ml) 3.5 ± 1.1 19 ± 10*

46 ± 8*

Pups T3(ng/dl) 63 ± 15 51 ± 15*

31 ± 25*

T4(µg/dl) 3.2 ± 0.7 0.8 ± 0.5*

0.3 ± 0.1*

Free T4 (pooled) (ng/dl) 1.6 0.41 <0.25

Adults T3(ng/dl) 36 ± 10 39 ± 10 ND

T4(µg/dl) 4 ± 0. 4.1 ± 1.0 ND

Free T4(ng/dl) 2 ± 0.3 1.9 ± 0.5 ND

77

Figure 3.1: Mean body weight of A) offspring and B) dams

Almost from birth, the mean body weights of the 10ppm pups were significantly reduced in

comparison to the 0ppm controls. In addition to the low body weights, a delay in eye opening and

decrease in overall physical coordination was observed (data not shown). At weaning, the 10ppm pups

were unable to reach food and water in standard rat cages, so special housing arrangements were

provided. While the 3ppm pups were not as affected as the 10ppm pups, they also weighed less than

controls, with the difference becoming significant around PND18, and expanding as the pups aged until

weaning. There was no significant difference between the weights of the 0 and 3 ppm dams at any age,

but the body weight of the 10ppm dams averaged significantly less than control on days 30-36 and 40

(p<0.05).

78

Offspring Weight Gain

Age (Post Natal Days)

0 5 10 15 20 25 30

Avera

ge p

up w

eig

ht (g

)

0

10

20

30

40

50

60

70

Control

3ppm

10ppm

79

Dam Weight Gain

Days since mating

0 10 20 30 40 50 60

Ave

rag

e d

am

we

igh

t (g

)

200

250

300

350

400

450

Control

3ppm

10ppm

80

Figure 3.2: Liver D1 activity in dams and pups

Liver D1 activity in dams and pups decreases with PTU dosage. D1 activity is directly inhibited

by PTU administration. Significant reductions in liver D1 activity are noted at both doses in both dams

and pups. Error bars represent standard deviation of the means.

*p<0.05 relative to control.

81

Liver D1 Activity

PTU Dose (ppm)

0 3 10

D1 A

cti

vit

y (

fmo

l I-

/min

/mg

)

0

500

1000

1500

2000

2500

3000

3500

PND25

Dams

* ** *

82

Figure 3.3: Thyroid Tg and NIS mRNA levels in dams and pups

A) Tg and B) NIS have different response patterns to PTU administration in pups, with NIS

increasing at 3ppm and further increasing at 10ppm, as opposed to the maximal increase of Tg at 3ppm

with no further increase at 10ppm. No significant changes in mRNA levels for these two genes were

detected in dams. Error bars represent standard deviation of the means.

* p<0.05 relative to control.

83

Thyroid Tg mRNA

Dose (ppm PTU)

0 3 10

Rela

tive a

mo

un

t

0

5

10

15

20

25

PND25

Dam

*

*

84

Thyroid NIS mRNA

Dose (ppm PTU)

0 3 10

Rela

tive a

mo

un

t

0

1

2

3

4

5

PND25

Dams*

*

85

Figure 3.4: Cerebrocortical T3 concentration and D2 activity

A,B) Cerebrocortical T3 concentrations and C) cerebrocortical D2 activity levels are affected by

both PTU doses in the pups. Dams are significantly affected at the 10ppm dose level. Error bars

represent standard deviation of the means. * p<0.05 relative to control for that age.

86

Pup Cortical D2 Activity

Dose (ppm PTU)

0 3 10

D2 a

cti

vit

y (

fmo

l I-

/hr/

mg

)

0

200

400

600

800

1000

1200

1400

1600

**

87

Dam Cortical D2 Activity

Dose (ppm PTU)

0 3 10

D2 a

cti

vit

y (

fmo

l I-

/hr/

mg

)

0

20

40

60

80

100

120

140

160

180

*

88

Cortical T3 Concentration

Dose (ppm PTU)

0 3 10

Co

rtic

al

T3

(p

g/g

)

0

1000

2000

3000

4000

5000

PND25

Dams

*

* *

89

Figure 3.5A

90

Figure 3.5: Basal synaptic transmission in hypothyroid pups

A) EPSP stimulus-response curves for the indicated treatment groups. B) Normalized stimulus-

response curves for the indicated treatment groups. C) Paired-pulse fEPSP sweeps for the indicated

treatment groups. No significant difference in the ratio of paired-pulse facilitation was observed. D) The

mean fEPSP slope values (error bars indicate SEM) at the maximal stimulus intensity of 140 mA. Results

from 2-3 slices/animal were averaged, yielding n values (animals(litters)) of 9(5); 9(5); and 8(4) for the 0

ppm, 3 ppm, and 10 ppm, treatment groups, respectively. *p<0.05 compared to controls.

91

92

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120 140

stimulus intensity (A)

norm

aliz

ed fE

PS

P s

lope

0 ppm

3 ppm

10 ppm

B

93

0 ppm:

PPF ratio=1.15

C

3 ppm:

PPF ratio=1.20

10 ppm:

PPF ratio=1.17

0 ppm:

PPF Ratio=1.15

3 ppm:

PPF Ratio=1.20

10 ppm:

PPF Ratio=1.17

94

95

Figure 3.6: Basal synaptic transmission and LTP in euthyroid adult littermates

A) fEPSP stimulus-response curves for the indicated treatment groups in adult (PND 90-100)

animals. No significant differences were noted in the weaned adults. B) Long-Term Potentiation was

induced via a single, 100 Hz/1 sec stimulus train delivered at the test pulse intensity at time=30 minutes.

The relative increase in the fEPSP response at 30, 60, and 90 minutes post tetanus are indicated. Results

from 2-3 slices/animal were averaged, yielding n values (animals(litters)) of 9(5) and 10(5) for the 0 ppm

and 3 ppm treatment groups, respectively (error bars indicate SEM). *p<0.05 compared to controls.

96

97

0.5

1.0

1.5

2.0

2.5

15 25 35 45 55 65 75 85 95 105 115

Time (minutes)

norm

aliz

ed fE

PS

P s

lope

(1.35)

B

(1.24)

(1.18)

(1.32)

(1.44) (1.41)

98

Figure 3.7: Correlation between cerebrocortical D2 activity and serum total T4 concentration

Cerebrocortical D2 activity was found to have a significant exponential correlation with serum

total T4. This correlation is consistent with the prior knowledge that cerebrocortical D2 activity directly

responds to serum T4 concentrations.

A – Dams: y=15.1+161.8*exp(-1.22*x) R2=0.92

B – Pups: y =-34.8+1355.8*exp(-0.60*x) R2=0.89

99

Serum T4 (ug/dl)

0 1 2 3 4 5 6

D2 A

cti

vit

y (

fmo

l I/m

g/h

)

0

20

40

60

80

100

120

140

160

100

Serum T4 (ug/dl)

0 1 2 3 4 5

D2 a

cti

vit

y (

fmo

l I-

/mg

/h)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

101

Figure 3.8: Correlation analysis of synaptic response vs. A) cerebrocortical D2 B) cerebrocortical

T3, and C) serum T4

A linear correlation was observed between cerebrocortical D2, cerebrocortical T3, and serum T4

with maximal synaptic response, suggesting a possible mechanistic link between these parameters. A

more negative synaptic response is indicative of stronger neural activity. Lines shown are linear

regression with 95% confidence intervals.

A – Vs. D2: y = -2.5-(1.295*10-3

)*x R2=0.578

B – Vs. T3: y = 1.269+(2.756*10-3

)*x ; R2=0.188

C – Vs. T4: y = 1.109+0.351*x ; R2=0.362

102

Cortical D2 activity (fmol I-/min/mg)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Synaptic R

esponse (

-mV

/msec)

-1

0

1

2

3

4

5

103

Cortical T3 (pg/g)

0 1000 2000 3000 4000 5000 6000 7000

Synaptic R

esponse (

-mV

/msec)

-1

0

1

2

3

4

5

104

Total T4 (ug/dl)

0 1 2 3 4 5

Synaptic R

esponse (

-mV

/msec)

-1

0

1

2

3

4

5

105

CHAPTER 4

DECREASED LITTER SIZE AMELIORATES THE THYROID DISRUPTIVE AND

NEURODEVELOPMENTAL EFFECTS OF GESTATIONAL AND LACTATIONAL EXPOSURE TO

PROPYLTHIOURACIL

__________________________

1Taylor MA, Stramiello M, Lelutiu N, Wagner JJ, Sharlin D, Zoeller T, Ferguson DC. In Preparation

106

Abstract

The developing central nervous system of the fetus and neonate are recognized as very sensitive

to maternal or gestational hypothyroidism. A previous study from this laboratory identified of 0, 3 and 10

ppm propylthiouracil resulted in significant maternal hypothyroidism, as well as hypothyroidism and

impaired electrophysiological performance in the brains of pups. Type II 5’-deiodinase (D2) activity was

elevated at both PTU doses and correlated tightly with the diminution in synaptic potential in

hippocampus of weanling rats. In the current study, lower degrees of thyroid insufficiency were sought to

explore the threshold for compensation, as well as to evaluate the effect of a relatively high dose of

perchlorate as thyroid disruptor with a different mechanism of action. We used propylthiouracil (PTU)

administered at 0, 0.3, 1, or 3 ppm or perchlorate at 150ppm in drinking water from gestational day 2 until

weaning to induce graded levels of maternal hypothyroidism. In addition to serum T3, T4, free T4, and

TSH concentrations, cerebrocortical T3 concentration (cT3), cerebrocortical Type II (D2) 5'-deiodinase

activity, and thyroidal mRNA for thyroglobulin (Tg) and sodium iodide symporter (NIS) were measured.

All assays were performed on serum or tissues from pups sacrificed on postnatal day 4 (PND4), 14, and

21-25, as well as from dams and weaned pups allowed to mature to adulthood. Extracellular recordings

from the CA1 region in hippocampal slices were obtained from both PND 21-32 (pups) and PND90-100

(adults) rats to assess neurophysiological effects, and adults were observed in open-field chambers to

investigate basic behavioral changes such as habituation and activity. Younger pups were shown to be

more sensitive to maternal thyroid insufficiency than older pups, but the overall impairment seen in this

study was less severe than that seen in the first study. While serum free and total T4, and T3

concentrations were reduced in the pups, there was no alteration of cortical T3 concentrations in the

PND21-25 pups, and no electrophysiological or behavioral changes were noted. Based on the findings in

our first study, the lack of cortical T3 impairment at PND21-25 may help to explain the lack of

electrophysiological alterations.

107

Introduction

The thyroid gland influences the function and development of many organ systems, including

cardiovascular, skeletal, and nervous systems.1-5

Thyroid hormone receptors have been documented in

the brain during the first trimester in humans, and gestational day 12.5 in rats.6 Because of its many roles,

maintenance of thyroid hormone levels during pregnancy is essential to the proper development of the

offspring. A link between impaired maternal thyroid status and IQ decrement in human babies has been

documented.7, 8

Other well-documented outcomes of developmental hypothyroidism are hearing loss,

altered migration of brain layers, delayed eye opening in rats, poor performance on maze tests, and

impaired motor development.9-17

As the full range of detrimental neurodevelopmental effects of exposure

to goitrogens is not yet understood, further investigation into the effects of well-characterized antithyroid

compounds may assist the understanding of the impact of environmental goitrogens.

With respect to measures of impact on brain function, it is well established that disruption in the

level of thyroid hormone during development can alter synaptic transmission in regions such as the

hippocampal formation. Significant effects on long-term potentiation (LTP) have also been reported.18-27

LTP has been denoted as a cellular model for learning and memory processes; therefore, we and others

have theorized that changes in LTP could provide a functional link between thyroid hormone deficits with

deficiencies in cognitive function.28

A prior study in our lab showed impaired synaptic function in

PND25 rats, and impairments in LTP persisting into adulthood even after normalization of serum thyroid

hormone concentrations.29

In addition, prior studies have also demonstrated that inhibitory and excitatory

pathways may be differentially impacted by thyroid insufficiency.30, 31

Such complexities may contribute

to the wide range of outcomes reported concerning the assessment of thyroid hormone deficiency on

electrophysiological parameters measured in the hippocampus. 15,19,24

While significant animal research has been performed on thyroid toxicants, including studies of

neurodevelopmental toxicity, thyroid status is most commonly determined by serum hormone

concentrations of T4 and TSH.32-36

Many factors influence the relationship between serum and tissue

levels of free and bound thyroid hormones, and the relationships may be tissue- and region-specific.37-39

108

The antithyroid drug propylthiouracil (PTU) was chosen for this and a prior study because this antithyroid

drug, by crossing the placental barrier, will impact thyroid hormone production and deiodinative

metabolism of both the dam and the gestating pups.40

PTU has been routinely used to induce

developmental hypothyroidism to study neurological impact. Perchlorate was also used in an attempt to

expand prior PTU studies to include an environmental antithyroid chemical. Perchlorate acts by blocking

iodine uptake into the thyroid through competitive inhibition of the sodium-iodide symporter (NIS).

The present study was designed to expand upon a prior study in our laboratories which evaluated

maternal exposure to 0, 3, and 10ppm PTU in the drinking water 29

. Since the neurological effects at 3

and 10ppm in the prior study were virtually identical at postnatal day 21, we sought to produce a graded

response in this study by expanding the PTU dose range to include 0.3 and 1ppm PTU doses, as well as

150ppm perchlorate, and to evaluate serum and tissue parameters in pups from a wider range of

developmental ages. Measurement of serum and tissue thyroid markers at postnatal days 4 and 14 were

added to evaluate pups just after birth, and during the crucial period for establishment of hypothalamic-

pituitary negative feedback, eye opening, and thermogenic autonomy. The primary goal of the present

study was to expand the dose range while continuing to evaluate serum, thyroid, and brain hormone

markers of thyroid status in both offspring and dams. As with the prior study, these parameters were then

correlated with electrophysiology results measured in the CA1 region of hippocampal slices obtained

from the offspring. We expanded our evaluation to include brain in situ hybridization studies for known

thyroid-responsive genes including RC3/Neurogranin. Additionally, we included basic habituation and

activity observations in an open field chamber at postnatal day 90-100 to assess for behavioral impacts of

persistent neurological effects after serum thyroid parameters had normalized post weaning.

Potential adaptation to a thyroid disrupting compound may occur by a variety of mechanisms

which include alteration of pituitary TSH secretion, alteration of intrathyroidal sodium-iodide symporter

and thyroglobulin synthesis, change in deiodinase enzyme activity, and altered thyroid receptor number or

affinity. However, little is known about the threshold for triggering these changes nor about their

compensatory limit. These issues become particularly critical to understand in subpopulations at greater

109

risk from thyroid disruption, the developing fetus and neonate. Our prior study quantified compensatory

mechanisms in the thyroid with adaptation of NIS and Tg mRNA levels, and in the brain by alteration of

D2 activity .29

Materials and Methods

Animal acquisition, care, and dosing

Timed pregnant CD® IGS rats were obtained from Charles River Labs (Raleigh, NC). Rats were

bred two days before arrival. Upon arrival, dams were housed individually and provided free access to

Purina 5008 chow and tap water. Animals were maintained on a 12h/12h light/dark cycle. At

approximately 1700h on the day of arrival, the water was replaced with either deionized water purified by

Millipore RiOS 8 (Millipore, Milford, MA), or deionized water containing PTU or sodium perchlorate

(Sigma, St. Louis, MO). Doses of 0, 0.3, 1, and 3ppm PTU, as well as 150ppm sodium perchlorate in DI

drinking water were chosen to expand the dose-response curve from our first study. Litters were culled to

8 pups on PND4, and to 6 pups on PND 14, each time retaining the maximal number of surviving males.

Dosing took place from GD2 until weaning, when pups selected for recovery were placed in standard

housing with tap water and Purina 5001 chow. Five dams were received per week, and each dose was

replicated once per week. Sacrifices were scheduled through an extended Latin square design to

eliminate bias in sacrifice day selection.

Animals were weighed three times per week, and water intake recorded. Water bottles were

refilled every 2-4 days, depending on the amount remaining in the bottle. New PTU dosing solutions

were made at least every two weeks from a stock solution, which was validated by analysis by HPLC.41

Perchlorate solutions were made in a similar timeframe. Average water intake during gestation was

approximately 200 ml/kg/day for the dams. Pups in the control and 3ppm groups began to supplement

their diet with food and water prior to weaning at varying times after PND14, but pups in the 10ppm

group were unable to do so due to reduced size and coordination. Pups were sacrificed on PND4,

PND14, and PND21-PND25. On PND4, only pups in litters larger than 8 were sacrificed. On PND14,

two pups per litter were sacrificed. Starting at PND21, pups were sacrificed for electrophysiological

110

analysis, with one male per litter sacrificed over a period of five days. A randomized block design was

used to ensure a lack of bias in sacrifice day selection. Data from these animals is presented as “PND25”

pups. Remaining males were weaned at the end of this sacrifice period (on PND25) and followed to

PND90-110 and are referred to hereafter as “adults.”

Animal studies were approved by the University of Georgia Institutional Animal Care and Use

Committee and were in accordance with procedures outlined in the National Institutes of Health

Guidelines for Care and Use of Laboratory Animals.

Sacrifice protocol

In an effort to better understand the impact of hypothyroidism on the development of pups, tissue

was collected at PND4, PND14, PND25, and around PND90. Data from the PND4 and PND14

timepoints should help to explain the observed effects on their older littermates.

PND4

On PND4, litters were culled to 8 remaining pups. Pups selected for culling were euthanized by

CO2 administration followed by decapitation. After decapitation, trunk blood was collected and the brain

was removed. The brain was assigned to one of four procedures. Brains selected for histological analysis

were removed from the skull and placed in a paraformaldehyde solution. Brains selected for in-situ

hybridization were placed in a container and snap frozen as whole heads in liquid nitrogen. Brains

selected for deiodinase assays were removed from the skull, and cerebral cortex subdisected, and kept on

ice until homogenization. Brains selected for thyroid hormone quantification were removed from the

skull, placed in a container, and snap frozen in liquid nitrogen.

PND14

On PND14, two additional pups were sacrificed. Pups were anesthetized with halothane or

isofluorane and sacrificed by decapitation. After decapitation, trunk blood was collected and the brain

was removed. One brain was subdisected to provide cerebral cortex tissue for deiodinase assays and

thyroid hormone quantification. These were processed as above. The remaining brain was assigned

111

either to histological analysis or in-situ hybridization. These were processed as above, except that the

brain was removed from the skull in both cases.

PND21-25

Male pups were weighed, anesthetized with halothane or isofluorane and sacrificed via

decapitation. Blood, thyroid, and liver were collected. The brain was dissected for tissue collection and

slices prepared for electrophysiology experiments (see below). In all pup and adult samples, the majority

of the hippocampus was used for slice preparation. Cortex samples were taken from areas of the brain not

impacted by dissection of the hippocampus, generally including prelimbic, motor, sensory, and cingulate

regions, and brain tissue samples were divided equally between deiodinase and hormone assays. Liver

and cortex samples intended for deiodinase assays were homogenized on the day of sacrifice in a 250mM

sucrose, 20mM KH2PO4, 1mM EDTA, 20mM DTT buffer, then stored in aliquots at -80oC until assayed.

Blood was allowed to clot and serum collected after centrifugation. All other tissues were flash-frozen

and stored at -80oC until analysis.

Biochemical assays

Serum Thyroid Hormone and TSH Radioimmunoassays:

Serum T3 was measured by radioimmunoassay based on the method of Galton.42

This assay

entailed a five day process. On the first day, 20 l of serum is added to 180 l of GAB buffer (0.2M

Glycine, 0.13M Sodium acetate trihydrate, 0.02% bovine serum albumin BSA, pH 8.6.) Next, 100 l of

GAB is added containing 2mg/ml of 8-anilino-1-napthalene-sulfonic acid (ANS). Finally, 100 l of a

1:32,000 dilution of a polyclonal rabbit anti-T3 antibody(Cat#20-TR45, Fitzgerald Industries

International, Concord, MA) was added in GAB/EDTA (0.1M EDTA in GAB). Tubes were incubated

for two days at 4C, before addition of 100 l of GAB containing approximately 14 000 dpm (~0.006 Ci)

of 125-I T3. The tubes were then incubated at 4C for an additional two days. After the final 48 hour

incubation, 100 l of a donkey anti-rabbit immunoglobulin secondary antibody coated to magnetic beads

was added (Donkey anti-rabbit magnetic separation reagent for immunoassay, Amersham, Piscataway,

112

NJ) and tubes were incubated at room temperature for 1 hour. 1 ml of phosphate buffered saline (0.01 M

NaCl, 0.01 M NaH2PO4, pH 7.5) (PBS) was then added to each tube, and tubes were centrifuged for 15

minutes at 2600xg at 4C. The supernatant fluid was then aspirated and pellets were counted in a gamma

counter (Wallac Wizard 1470). Each rat age group was always run in a single assay. This assay had a

limit of detection of 5 ng/dl with 20l of serum, or 25 ng/tube. Intraassay coefficients of variation (CV)

averaged 23.2 ± 20%(n=3) at 50ng/dl and 16.8 ± 6%(n=3) at 60 ng/dl.

Serum T4 was measured by radioimmunoassay based on the methods of Galton. The process was

similar to the T3 assay with the following modifications. 5l of serum was assayed per tube and added to

200l of GAB. The primary antibody used was a polyclonal rabbit anti-T4 antibody (Cat#20-TR40,

Fitzgerald Industries International, Concord, MA). Approximately0.006Ci of [125

I]-T4 was added on the

third day. On the fifth day, 50 l of 200g/ml (10g) solution of rabbit immunoglobulin was added,

followed by 100 l of a GAR secondary antibody solution (Cat#R0881, Sigma) prepared at 60% of the

manufacturers recommended volume for a final dilution of approximately 1:8. Tubes were incubated at

room temperature for 30 minutes before addition of 1 ml of a 25% wt/vol solution of PBS/PEG. Tubes

were then centrifuged, aspirated, and counted as in the T3 assay. Each age group was always run in a

single assay. The serum T4 assay had a limit of detection of 0.08 g/dl with 10l of serum, or 0.8g per

tube. The average intraassay CV at 3.6 g/dl (35 g/tube) was 15%.

The cortical T3 assay was also performed similarly to the serum T3 assay with the following

modifications. On the first day, 50l of cortex extract was added to 150l GAB. On the final day, the

assay was terminated as with the serum T4 assay. Each age group was always run in a single assay. The

limit of detection was about 3.5 pg T3/tube, corresponding to 70 pg per extract, or 1 g/g for a 100 mg

sample at 70% extraction efficiency. The average intraassay CV at 7 pg/tube was about 11%. The T4

radioimmunoassay for cerebrocortical tissue extracts was also performed similarly to the serum T4 assay

with the following modifications. On the first day, 100 µl of extract was added to 100ul of GAB. On the

final day, the assay was terminated as with the serum T3 assay. Each age group was always run in a

113

single assay. The limit of detection was about 1.9 pg T4/tube, corresponding to 19 pg T4 per extract, or

about 500 pg/g for a 100 mg sample at 40% extraction efficiency. The average intraassay CV at 60

pg/tube averaged 9%.

The serum TSH assay was performed using reagents provided by Dr. A. F. Parlow (National

Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) 100 l of serum was added

to a tube along with 100 l of a PBS/BSA solution. Horse serum was used as the blank serum for the

standard curve. About 14,000 dpm (0.006 Ci) of 125-I TSH was then added to the tubes in PBS,

followed by 100 l of a 1:1 500,000 dilution of the antibody. Tubes were incubated overnight at room

temperature. The next day, 100 l of Donkey Anti-Rabbit bead solution was added to the tubes and tubes

were then incubated for an hour at room temperature. 900 l of PBS was then added, and tubes were

centrifuged for 15 minutes at 2400xg. The supernatant was aspirated, and tubes were counted on a

gamma counter. Each age group was always run in a single assay. The limit of detection was about 0.6

ng/ml, and the average CV at 1.2 ng/ml and 13 ng/ml were about 18% and 3% respectively.

Free T4 fraction by Equilibrium Dialysis:

Serum free T4 fractions were determined by tracer equilibrium dialysis. The protocol was a

modification of that of Sterling to allow for smaller sample volumes.43

Briefly, 65ul of serum was spiked

with approximately 1.4 x 106 dpm (0.64 Ci) [

125I]T4. 50l of serum was transferred into one side of a

microdialysis chamber (Harvard Apparatus, Holliston, MA) with two 100 l chambers separated by a

5,000 molecular weight cut off semipermeable cellulose membrane(The Nest Group, Inc., Southborough,

MA). 100l of dialysis buffer (0.15M NaH2PO4, 20 mM HEPES pH 7.4) was added to the other side.

After an overnight dialysis at 37C, the serum and dialysate were removed, with care to precoat all

surfaces of pipettes with a T4 carrier solution. The dialysate was precipitated with a magnesium solution

(500mM MgCl2*6H2O, 100mM NaCl, 50mM Trizma base, 0.27mM NaI, pH 9.3) and washed as

previously described but with volumes adjusted proportionally (80 µl precipitation, 200µl wash) . The

interassay coefficient of variation (CV) using a pooled sample was approximately 27%.

114

Thyroid RNA extraction and analysis:

Thyroglobulin (Tg) and the Sodium Iodide Symporter (NIS) were selected for mRNA analysis as

markers which, in part, reflect TSH bioactivity. RNA extraction and analysis were extracted and analyzed

as previously reported.29

Protein assays:

All deiodinase samples were homogenized on the day of collection in 250mM sucrose, 20mM

KH2PO4 , 1mM EDTA, pH 7, and frozen in aliquots at -80C until assay. Protein concentrations were

determined by the Bio-Rad protein assay reagent (Hercules, CA) using BSA as a standard.

Cerebrocortical Type II 5’-Deiodinase Activity:

This assay was performed as previously described, but with the following alteration. Triplicate

tubes were incubated at 37oC for one hour with 50g protein for PND25 pups, 100g for PND 4 and 14

pups, or 200g for dams, in a total volume of 320l, along with tissue-free blanks. 29

Cerebrocortical Thyroid Hormone extraction:

The extraction of thyroid hormones from the brain was performed as previously described.29

Electrophysiology:

The electrophysiological procedures and analysis were conducted as previously reported.29

Habituation and Activity Behavioral Tests:

Two months following termination of PTU dosing at weaning, rats underwent behavioral tests.

This data was designed to complement hippocampal electrophysiology as a test of functional neurological

alterations resulting from developmental hypothyroidism. Rats were handled individually for

approximately two minutes each weekday preceding the week they were scheduled for analysis.

Behavior was observed using an open field chamber (ENV-515 Test Environment, Med Associates, St.

Albans, Vermont) into which rats were placed and monitored for thirty minutes per day over a period of

five days. Infrared beams in the cage recorded position and motion of the rats, and this data was analyzed

for habituation and activity levels using the Activity Monitor software package.

115

In-situ hybridization:

In-situ hybridization of brain sections for RC3 Neurogranin, a thyroid hormone-responsive gene,

was performed and analyzed as previously described.43

Statistics:

All statistical analysis was performed using SAS (The SAS group, Cary, NC). All group

comparisons were performed using ANOVA, followed by Duncan’s multiple range test. p<0.05 was

taken as the level of significance.

Results

1) Serum hormones (Table 2)

Dams

Serum total T3 and T4 concentrations were unchanged in any dose. TSH concentrations were

tripled at the 3ppm dose level (p= 0.03). No significant alterations were seen in the perchlorate dosed

animals.

PND25 pups

Serum total T4 concentrations were reduced by 30% at 1ppm and 70% at the 3ppm dose in the

pups (p<0.0001). No significant changes were noted in serum T3 concentrations. No changes were

observed in the free T4 percentage which averaged 0.15% ± 0.1% across all dose groups . Serum TSH

concentrations were about 3-fold at 1ppm, and increased to 9-fold of control at 3ppm (p<0.0001). Again,

no changes were seen in the perchlorate dosed animals.

PND14 pups

Serum total T4 concentrations were reduced by 60% at 1ppm, and 95% at 3ppm compared to

controls (p<0.0001). Again, no changes were seen in the perchlorate-dosed animals.

PND4 pups

Serum total T4 concentrations were reduced by 50% at 1ppm, and over 90% at 3ppm compared to

controls. (p<0.0001).

116

PND100 adults

In 3 ppm dosed pups allowed to recover for approximately two months after weaning, all serum

concentrations of T3 and T4 returned to those of the 0 ppm group.

2) Body weight (Figure 4.1)

The weight of all pups track closely together until the end of the pre-weaning period. On PND14,

the body weight of 1ppm pups was slightly greater than the control pups, and on PND23, the average

body weight of 3ppm pups was less than all other pups (p<0.01). At all other times, there was no

statistical difference from control values. Additionally, no significant delay in eye opening was detected.

At all times, the body weights of PTU-treated dams were similar to control values.

3) Thyroid mRNA (Figure 4.2)

(2A ) Sodium Iodide symporter (NIS)

In the PND25 pups, NIS levels were unchanged after PTU dosing, but increased by about 1000%

in the perchlorate dosed animals. The dams demonstrated a similar response pattern, with a 5-fold

increase in NIS in the perchlorate dosed animals, and no change in the PTU dosed animals.

(2B)Thyroglobulin (Tg)

In the PND25 pups, Tg levels were doubled at the 3ppm PTU dose, with no change in the

perchlorate dose. In the dams, no Tg changes were noted.

4) Cerebrocortical thyroid parameters (Figure 4.3)

(3A)D2 activity

In the dams, cerebrocortical D2 activity was increased by 50% at 3ppm (p=0.0087). In the

PND25 pups, cerebrocortical D2 activity was tripled at 3ppm (p<0.0001). The PND14 pups

demonstrated even greater sensitivity, with a 5-fold increase at 1ppm, and a 10-fold increase at 3ppm

(p<0.0001). The PND4 pups showed a 5-fold increase in D2 activity at 3ppm but not at lesser doses

(p<0.0001). No changes in D2 activity were observed in the perchlorate animals at any age.

117

(3B)Cerebrocortical thyroid hormone concentrations

No significant change in cerebrocortical T3 concentrations were noted in the dams, PND25 pups,

or adults. In the PND14 pups, cortical T3 concentrations were reduced by 60% at 3ppm. No significant

changes in T3 were detected in PND4 animals. However, significant variability was seen in all age

groups, reducing the resolution of the assay.

5) Hippocampal Electrophysiology

No significant alterations of basal synaptic activity were observed in either PND25 pups or

recovered adults. Additionally, no LTP alterations were noted.

6) Behavioral studies

No significant differences in adult behavior in the open field tests were noted.

7) In-situ (Figure 4.4)

In-situ hybridization analysis of the PND25 pups indicated an approximately 10% decline in RC3

mRNA concentration in the cerebral cortex at 3ppm PTU (p<0.05). A potential decline in RC3 mRNA in

the lower dentate gyrus was noted, though this fell short of statistical significance (p=0.064). No

alterations were noted in the upper dentate gyrus.

Discussion

Developmental thyroid disruption with PTU and perchlorate was initiated at gestational day 2 and

maintained until weaning on PND25. This protocol was designed to ensure that maternal thyroid

hormone production was compromised prior to the development of fetal thyroid function around GD17 .2

Based on our studies with adult rats, we projected that hypothyroidism was induced by GD9, prior to the

first appearance of thyroid hormone receptors in the rat brain at GD12.2 This window of dosing began

earlier than that of most prior developmental studies performed in the rat with PTU. 16, 17, 19, 21, 24, 30, 31, 45

Analysis of thyroid mRNA levels for Tg and NIS, chosen in part to be markers of serum TSH

bioactivity on the thyroid gland, once again demonstrated the greater sensitivity to PTU in pups compared

to their dams. Additionally, the striking upregulation of NIS mRNA in the perchlorate dosed animals

118

despite the lack of effect on serum thyroid hormone levels, including TSH, suggests a powerful role for

thyroid iodide availability and autoregulation in response to perchlorate exposure.46

We note that the serum and tissue hormonal, biomarker and electrophysiological changes in dams

and post-weaning pups (PND25) from this study at the dose of 3ppm PTU were essentially nonexistent

and inconsistent with our findings in our prior work. In our prior study, both serum and cortical thyroid

hormone levels were decreased in pups exposed to 3ppm PTU, and the synaptic potential in the

hippocampus was decreased.29

It is notable, however, that the chosen biomarkers largely predicted the

reduced neurological consequence to animals at this dose in this study. The alterations made in the current

experimental protocol involved in this change are as follows. First, litters were culled to 8 pups at PND4,

with a further decrease to six pups at PND14 which represent a significant decrease in litter size from the

first study, which had average litter sizes around 12 pups. Prior studies have demonstrated an alteration

in the makeup and nutritional value of maternal lactation dependent on litter size, leading us to believe

that the nutritional status of the pups in this study was superior to that of the first study .47

Secondly, the

diet for the dams was changed to the Purina 5008 chow, with a higher percentage of fat than the 5001

diet, with a corresponding 10% increase in calories per gram of food. However, this diet contains slightly

less iodine than the 5001 diet.

This study provides a glimpse at the effect of PTU dosing on pups younger than those used for

electrophysiology at PND21-25. In almost all measured parameters, pups at PND4 and PND14 were

more affected by a given dose of PTU than their PND25 littermates, and were affected at lower doses as

well. Of special note in this regard were the relative activities of cortical D2 observed at each age point.

PND4 pups had a reduced D2 activity compared to their elder littermates, further demonstrating the

heightened sensitivity to thyroid disruption at this age. While no electrophysiological or behavioral

changes were noted in this study, the data gained from the younger pups should help to understand any

such changes in future studies. Given the postulated relationship between serum and cortical thyroid

hormone concentrations and electrophysiological functionality from our prior work, it is not unexpected

that a lesser neurological effect is observed in this study, corresponding to the decreased dose effect on

119

these concentrations.28

However, the RC3 decline in cortex without a corresponding decline in the

dentate gyrus could indicate that the impact of hypothyroidism is less severe in the hippocampus than the

cortex.

It should be noted that the prior relationship observed between serum T4 and cerebrocortical D2

activity held true in this study, though the exact regression curve was slightly different. However, the

prior relationship between D2 activity and synaptic potential in the pups was no longer observed, possibly

because the severity of serum T4 depression from the lowest to the highest PTU dose did not produce the

same spread in data. It seems likely that the lack of electrophysiological changes reflected the reduced

effectiveness of 3ppm PTU in this study to diminish thyroid hormone production as compared to the first,

as indicated by the lack of reduction in cortical T4 concentrations in the PND21-25 pups, with no

detectable decline in serum T3 concentrations in PND21 pups from this study. However, the noted

decline in serum T4 concentrations was similar, with approximately a 70% reduction in this study

compared to a 75% reduction in the first at 3ppm in PND25 pups. However, the addition of TSH data in

this study indicates a pituitary perception of hypothyroidism, indicating that some regions of the brain

may still lack adequate supplies of thyroid hormones despite the lack of reduction in serum T3

concentrations. The results of this second study prompted the following third study which would span all

PTU doses used in the first and second studies, along with a culling protocol intermediate to that of the

first and second studies. The goal for the third study was clarification of the dose-response curve ranging

from no effect to marked developmental retardation, with a graded response between these extremes.

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124

Table 4.1: Serum Hormone Concentrations

Serum total T4 is reduced at both 1 and 3 ppm PTU in all three pup age groups. No significant changes in

serum total T4 are seen in the dams. No alterations in serum T3 concentrations are seen at any dose in

any age group. Serum TSH concentrations are elevated at 1 and 3 ppm PTU in PND25 pups, but only at

3ppm in the dams.

* =p<0.05

Control 0.3 ppm 1 ppm 3ppm Perchlorate

Dams T3 (ng/dl) 46.4 ± 8.8 48.3 ± 8.6 43.5 ± 5.6 47 ± 9.7 52.2 ± 17

T4(µg/dl) 6.5 ± 1.7 7.4 ± 1.7 8.7 ± 4.2 4.2 ± 1.34 4.6 ± 1.2

TSH(ng/ml) 1.8 ± 1 1.8 ± 0.8 2.8 ± 0.5 5.2 ± 4.4* 2.3 ± 0.8

PND25 T3(ng/dl) 53.6 ± 7 49 ± 8.7 58.4 ± 7.3 52.5 ± 5.2 59.8 ± 12.1

T4(µg/dl) 6.4 ± 1.8 5.9 ±0.3 4.3 ± 0.5* 1.88 ± 0.3* 5.4 ± 0.8

TSH(ng/ml) 0.6 ± 0.4 0.9 ± 0.4 1.6 ± 0.8* 4.7 ± 1* 0.3 ± 0.3

%Free T4 0.12 ± 0.1 0.17 ± 0.12 0.18 ± 0.11 0.18 ± 0.12 0.21 ± 0.14

PND14 T3(ng/dl) 75.1 ± 13.6 82 ± 3.7 89.1 ± 17.2 60.2 ± 32.5 76.4 ± 6.9

T4(µg/dl) 7.1 ± 2.7 6 ± 2.2 2.5 ± 0.9* 0.5 ± 0.4* 8 ± 2.6

PND4 T4(µg/dl) 2.1 ± 0.5 1.8 ± 0.3 1 ± 0.2* 0.2 ± 0.1* 2.4 ± 0.5

125

Figure 4.1: Mean body weight of A) offspring and B) dams

Minimal weight differences were observed in the pups prior to weaning, with the 3ppm animals

lighter only at PND23. This minor weight gap closed during the post-weaning period. No differences in

mean body weights between dose groups were seen in the dams.

126

Pup weights

PND

0 5 10 15 20 25

Weig

ht (g

ram

s)

0

10

20

30

40

50

60

70

80

Control

0.3ppm

1ppm

3ppm

Perchlorate

127

Dam Weight

Days since arrival

0 10 20 30 40 50

Weig

ht (g

ram

s)

180

200

220

240

260

280

300

320

340

360

380

Control

0.3ppm

1ppm

3ppm

Perchlorate

128

Figure 4.2: Thyroid Tg and NIS mRNA levels in dams and pups

A) Tg and B) NIS mRNA levels. Error bars represent standard deviation of the means. NIS was

increased in the dams and pups under perchlorate dosing, but Tg was increased only in 3ppm pups.

* p<0.05 relative to control.

129

Thyroid TG mRNA

Dose

Control 0.3ppm 1ppm 3ppm Perchlorate

Rela

tive Q

uantity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

PND25

Dam*

130

Thyroid NIS mRNA

Dose

Control 0.3ppm 1ppm 3ppm Perchlorate

Rela

tive Q

uantity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

PND25

Dam *

*

131

Figure 4.3: Cerebrocortical T3 concentration and D2 activity

A) Cerebrocortical D2 activity levels were increased at 3ppm in all pups and dams, as well as at

1ppm in PND14 pups. The greatest relative increase occurred in the PND14 pups, with an approximately

9-fold increase in activity at 3ppm.

B) No changes in cerebrocortical T3 concentrations were seen in dams, PND25 pups, PND4 pups,

or adults. A Cerebrocortical T3 reduction of approximately 50% was seen at 3ppm in PND14 pups.

* p<0.05 relative to control for that age.

132

PND4 PND14 PND25

D2 A

ctivity (

fmol I-

/h/m

g)

0

200

400

600

800

1000

1200

1400

1600

Control

0.3 ppm PTU

1 ppm PTU

3 ppm PTU

150 ppm perchlorate

*

*

*

*

Dam

D2

Activity (

fmo

l I-

/h/m

g)

0

10

20

30

40

50

60

70

133

PND4 PND14 PND25

Cort

ical T

3 (

pg/g

)

0

1000

2000

3000

4000

5000

Control

0.3 ppm PTU

1 ppm PTU

3 ppm PTU

150 ppm perchlorate

*

134

Figure 4.4: in-situ data

In-situ hybridization analysis of the PND25 pups indicated a decline in RC3 mRNA concentration

in the cerebral cortex at 3ppm PTU. No changes in RC3 expression levels were detected in the dentate

gyrus at any dose.

* p<0.05 relative to control

135

RC3 mRNA

Dose

Control 0.3ppm 1ppm 3ppm Perchlorate

Arb

itra

ry U

nits

0

50

100

150

200 Dentate Gyrus

Cortex

*

136

CHAPTER 5

GESTATIONAL AND NEONATAL HYPOTHYROIDISM LEADS TO HYPERACTIVITY IN

ADULT RATS: BIOCHEMICAL AND NEUROLOGICAL OUTCOMES FROM GRADED THYROID

INSUFFICIENCY

______________________________

Taylor MA, Kelamangalath L, Lelutiu N, Wagner JJ, Fisher JW, Ferguson DC. In preparation

137

Abstract

The developing nervous system is highly sensitive to thyroid disruption. To better understand the

observed outcomes in our two prior studies of the effect of graded doses of propylthiouracil (PTU)

provided to pregnant rat dams, we initiated a third study spanning the entire PTU dose range of the two

prior studies. PTU administration occurred from GD2 until weaning, and litter sizes were culled to 10

pups at PND4, and 8 at PND14. Serum thyroid hormone concentrations were measured along with

cerebrocortical T3 concentrations and D2 activity at PND4, 14, 21-25, 90, and in dams.

Electrophysiological analysis of hippocampal function was performed at PND21-25 and PND90.

Behavior in an open field test chamber was observed in adult pups. Thyroid NIS and Tg mRNA were

measured in PND21-25 pups and adults. In most measurements, younger pups were found to be more

sensitive to disruption than older pups or their dams, with decreases in serum T4 seen at 10ppm in dams

and PND21-25, 3 and 10ppm in PND14, and 1, 3, and 10ppm at PND4. Cerebrocortical D2 activity was

increased at 10ppm in dams and PND21-25, and 1, 3, and 10ppm in PND14. Maximal synaptic potential

was impaired at 3 and 10ppm at PND21-25, but not in adults. LTP alterations in PND21-25 pups were

noted at all doses except 10ppm, but not in adults. Adults from litters dosed with 10ppm PTU were found

to be hyperactive in open field testing. In summary, dose effects were found at lower doses in younger

animals, and despite the lack of persistent electrophysiological alteration, hyperactivity was seen in adults

previously exposed to 10ppm PTU.

Introduction

The thyroid gland influences the function and development of many organ systems, including

cardiovascular, skeletal, and nervous systems.1-5

Thyroid hormone receptors have been documented in

the brain during the first trimester.6 Because of its many roles, maintenance of thyroid hormone levels

during pregnancy is essential to proper development of the offspring. A link between impaired maternal

thyroid status and IQ deficit has been documented in infants.7,8

Additionally, the recent increase in

neurological disorders in the human population, such as autism and attention deficit hyperactivity disorder

(ADHD), has been theorized to correlate with the increasing prevalence of endocrine (including thyroid)

138

disrupting chemicals in the environment.3,7,9

Other well-documented outcomes of developmental

hypothyroidism are hearing loss, altered migration of layers of the cerebral cortex, delayed eye opening in

rats, poor performance on maze tests, and impaired motor development.9-17

As the full range of

detrimental neurodevelopmental effects of goitrogen exposure is not yet understood, further investigation

into the effects of well-characterized anti-thyroid compounds may assist the understanding of the impact

of environmental goitrogens.

With respect to measures of impact on brain function, it is well established that disruption in the

level of thyroid hormone during development can alter synaptic transmission in regions such as the

hippocampal formation. Significant effects on long-term potentiation (LTP) have also been reported.18-28

LTP has been denoted as a cellular model for learning and memory processes; therefore, we and others

have theorized that changes in LTP could provide a functional link between thyroid hormone deficits with

deficiencies in cognitive function.29

In addition, prior studies have also demonstrated that inhibitory and

excitatory pathways may be differentially impacted by thyroid insufficiency.30,31

Such complexities may

contribute to the wide range of outcomes reported concerning the assessment of thyroid hormone

deficiency on electrophysiological parameters measured in the hippocampus.15,19,24

While significant animal research has been performed on thyroid toxicants, including studies of

neurodevelopmental toxicity, thyroid status is most commonly determined by serum hormone

concentrations of total T4 and TSH.32-36

Many factors influence the relationship between serum and

tissue levels of free and bound thyroid hormones, and the relationships may be tissue- and region-

specific.37-39

PTU has been routinely used to induce developmental hypothyroidism to study neurological

impact. PTU, the chosen thyroid disruptor in this study, has both maternal and direct effects on the

gestating pups, as it will cross the placental barrier .40

The hypothalamic-pituitary-thyroid axis has a variety of mechanisms which would allow

potential adaptation to thyroid disruption, including alteration of pituitary TSH secretion, alteration of

intrathyroidal sodium-iodide symporter and thyroglobulin synthesis, change in deiodinase enzyme

activity, and altered thyroid receptor number or affinity. However, little is known about the threshold for

139

triggering these changes nor about their compensatory limit. These issues become particularly critical to

understand in subpopulations at greater risk from thyroid disruption, the developing fetus and neonate.

The investigators have conducted two prior studies of gestational and lactational propylthiouracil

exposure on behavioral and electrophysiological outcomes in the rat. 28,Chapter 4

At the highest common

dosage of 3 ppm PTU in the drinking water, study 1 demonstrated significant reduction of hippocampal

synaptic potential in PND21 animals with impaired LTP in the adults. In the second study, no such

detriment was seen at this dose, which was the highest examined in that study. The present study was

designed to replicate the dose ranges used in both our first and second studies to better understand the

entirety of the observed dose-response curve. All assays performed in the second study were repeated in

this third study, though slight modifications were made to the behavioral testing protocol.

Materials and Methods

Animal acquisition, care, and dosing

Timed pregnant CD® IGS rats were obtained from Charles River Labs (Raleigh, NC). Rats were

bred two days before arrival. Upon arrival, dams were housed individually and provided free access to

Purina 5008 chow and tap water. Animals were maintained on a 12h/12h light/dark cycle. At

approximately 1700h on the day of arrival, the water was replaced with either deionized water purified by

Millipore RiOS 8 (Millipore, Milford, MA), or deionized water containing 3 or 10ppm PTU (Sigma, St.

Louis, MO). Doses of 0, 0.3, 1, 3, and 10ppm PTU in DI drinking water were chosen to complete the

dose-response curve from our first and second studies. Litters were culled to 10 pups on PND4, and to 8

pups on PND 14, each time retaining the maximal number of surviving males. Dosing took place from

GD2 until weaning, when pups selected for recovery were placed in standard housing with tap water and

Purina 5001 chow.

Animals were weighed 3 times per week, and water intake recorded. Water bottles were refilled

every 2-4 days, depending on the amount remaining in the bottle. New PTU dosing solutions were made

at least every two weeks. Average water intake during gestation was approximately 200 ml/kg/day for the

dams. Pups in the control and 3ppm groups began to supplement their diet with food and water prior to

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weaning at varying times after PND14, but pups in the 10ppm group were unable to do so due to reduced

size and coordination. Pups were sacrificed for electrophysiological analysis beginning on PND21, with

one male per litter sacrificed over a period of five days. A randomized block design was used to ensure a

lack of bias in sacrifice day selection. Data from these animals is presented as “PND25” pups.

Remaining males were weaned on PND25 and allowed to recover until approximately PND90, and are

referred to hereafter as “adults.”

Animal studies were approved by the University of Georgia Institutional Animal Care and Use

Committee and were in accordance with procedures outlined in the National Institutes of Health

Guidelines for Care and Use of Laboratory Animals.

Sacrifice protocol

In an effort to better understand the impact of hypothyroidism on the development of pups, tissue

was collected at PND4, PND14, PND25, and around PND90. Data from the PND4 and PND14

timepoints should help to explain the observed effects on their older littermates. The study design was

nearly identical to that described in chapter 4, with the exception of the dose range and two extra pups

retained in each litter.

PND4

On PND4, litters were culled to 10 remaining pups. Pups selected for culling were euthanatized

by CO2 administration followed by decapitation. After decapitation, trunk blood was collected and the

brain was removed. The brain was assigned to one of four procedures. Brains selected for histological

analysis were removed from the skull and placed in a paraformaldehyde solution as previously

described.28

Brains selected for in-situ hybridization were placed in a container and snap frozen as whole

heads in liquid nitrogen. Brains selected for deiodinase assays or thyroid hormone quantification were

removed from the skull, placed in a container, and snap frozen in liquid nitrogen for later processing.

PND14

On PND14, two additional pups were sacrificed. Pups were anesthetized with halothane or

isofluorane and sacrificed by decapitation. After decapitation, trunk blood was collected and the brain

141

was removed. One brain was dissected to provide cerebral cortex tissue for deiodinase assays and thyroid

hormone quantification. These were processed as above. The brain from the second animal was assigned

either to histological analysis or in-situ hybridization. These were processed as above, except that the

brain was removed from the skull in both cases.

PND21-25

Male pups were weighed, anesthetized with halothane or isoflurane and sacrificed via

decapitation. Blood, thyroid, and liver were collected. The brain was dissected for tissue collection and

slices prepared for electrophysiology experiments (see below). In all pup and adult samples, the majority

of the hippocampus was used for slice preparation. Cerebral cortex samples were taken from areas of the

brain not impacted by dissection of the hippocampus, generally including prelimbic, motor, sensory, and

cingulate regions, and brain tissue samples were divided equally between deiodinase and hormone assays

and immediately snap-frozen. Blood was allowed to clot and serum collected after centrifugation. All

other tissues were flash-frozen and stored at -80oC until analysis.

Biochemical assays

Serum Thyroid Hormone Radioimmunoassays:

Serum thyroid hormone assays were performed as described in chapter 4.

Thyroid RNA extraction and analysis:

Extraction and analysis of thyroid mRNA was performed as previously described. 28

Protein assays:

Protein concentrations of homogenates for deiodinase activity assays were determined by the Bio-

Rad protein assay reagent (Hercules, CA) using BSA as a standard.

Thyroid iodine quantification:

Thyroid glands were collected from pups sacrificed at weaning, flash frozen, and submitted to Dr.

Braverman for analysis by a modification of the previously reported ashing technique of Wilson and Van

Zyl.41

Cerebrocortical Type II 5’-Deiodinase Activity:

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Analysis of cerebrocortical D2 activity was performed as described in chapter 4.

Cerebrocortical T3 extraction:

Extraction of thyroid hormones from cerebrocortical tissue was performed as previously

described.28

The intraassay coefficient of variation was approximately 20% at 23.8 pg T3 per tube. All

samples were run in a single assay.

Electrophysiology

Electrophysiological analysis of PND21 pups and adults was performed as previously

described.28

Behavior assay:

Rats allowed to recover from gestational and lactational PTU exposure for two months after

weaning were used for behavioral analysis. Each day for three days prior to analysis, rats were taken in

their home cage into a new room, placed in an empty cage cleaned between each animal with diluted

Windex™, a commercial multisurface cleaning agent, left in the cage for approximately two minutes,

then taken back to their home room in their home cage. Behavior was observed using an open field

behavior chamber (Med Associates, St. Albans, VT) into which rats were placed and monitored for

twenty minutes per day over a period of three days. Infrared beams in the cage recorded position and

motion of the rats, and this data was analyzed for habituation and activity levels using the associated

Activity Monitor software.

Serum isoflavone analysis:

Serum samples from PND14 pups were submitted to Dr. Daniel Doerge for analysis according to

a previously established protocol using LC/MS.42

Concentrations of genistein, diadzein, and the genistein

metabolite equol were determined in the serum samples.

Statistics:

All statistical analysis was performed using SAS (The SAS group, Cary, NC). All group

comparisons except electrophysiology were performed using ANOVA, followed by Duncan’s multiple

143

range test. Electrophysiology results were analyzed as pairwise comparisons with the control group.

p<0.05 was taken as the level of significance.

Results

1) Serum hormones (Table 5.1)

Dams

Serum total and free T4 concentrations were reduced by 80% at 10ppm (p=0.007). Serum total

T3 concentrations were reduced by 30% at 10ppm (p=0.033).

PND25 pups

Serum total and free T4 concentrations were reduced by approximately 95% at 10ppm

(p<0.0001). Serum total T3 concentrations were reduced by 50% at 10ppm (p<0.0001).

PND14 pups

Serum total T3 concentration was increased at 1ppm and decreased at 10ppm (p<0.0001). Serum

total T4 concentration was reduced at 3 and 10ppm (p=0.007).

PND4 pups

Serum total T4 concentration was decreased by 30% at 1ppm, 75% at 3ppm, and over 90% at

10ppm (p<0.0001).

PND100 adults

Serum total T4 concentration was increased by 0.5-1.2 µg/dl in adults previously exposed to 0.3,

1, and 3, and 10ppm PTU (p=0.0031)No changes were seen in any TH concentrations in adults.

2) Body weight (Figure 5.1)

On all days starting at the first recorded weight at PND4, the body weight of the 10ppm pups

were significantly decreased compared to all other dose levels. On PND16 and PND18, the body weights

of 1ppm pups were higher than control pups. No significant differences were found in the weights of the

dams.

3) Thyroid mRNA (Figure 5.2)

Sodium Iodide symporter (NIS)

144

NIS mRNA levels in PND25 pups were significantly increased at 3 and 10ppm (p<0.0001). No

changes were observed in adults.

Thyroglobulin (Tg)

Tg mRNA levels in PND25 pups are significantly increased at 10ppm (p=0.0028). No changes

were observed in adults.

4) Cerebrocortical thyroid parameters (Figure 5.3)

D2 activity

In the dams, cerebrocortical D2 activity was increased approximately 300% at 10ppm

(p=0.0013). In the PND25 pups, cerebrocortical D2 activity also increased approximately 300% at

10ppm (p<0.0001). The PND14 pups demonstrated even greater sensitivity, with an approximate tripling

at 1ppm, 800% increase at 3ppm, and 1200% increase at 10ppm (p<0.0001).

Cerebrocortical T3 concentrations

No change in cerebrocortical T3 concentrations were seen in the dams or adults. In the PND25

pups, cortical T3 was reduced by about 40% at 10ppm (p=0.026). In the PND 14 pups, cerebrocortical

T3 concentrations were reduced by 60% at 10ppm (p= 0.003). No changes were seen in cortical T3

concentrations at PND4, and high variability was observed in this age group.

5) Electrophysiology (Figure 5.4)

In the PND25 pups, several electrophysiological alterations were noted. Maximal synaptic

potential was reduced by approximately 25% at 3 and 10ppm. 30 minute LTP (weak LTP) was impaired

by approximately 10% at 1 and 3ppm (though 10ppm was nearly significant), while 90 minute LTP

(strong LTP) was impaired by approximately 10% at 0.3 and 1ppm PTU. None of these changes were

observed to persist in adults.

6) PND25 Thyroid iodine content (Figure 5.5)

The average iodine content of thyroids from the control group averaged 1.48 µg per gland. No

significant change was observed at the 0.3ppm dose. Concentrations in the 1ppm dose were reduced

about 35%. Concentrations in the 3ppm dose were reduced about 65% .Concentrations in the 10ppm

145

dose were reduced nearly 90%. 3(control) or 4(dosed) pups per litter for one litter of each dose group

were studied.

7) Serum Isoflavones

Serum from control PND14 pups from this study and the prior study were submitted to Dr. Daniel

Doerge for analysis of soy isoflavones and metabolites. Serum concentrations of genistein, daidzein and

equol were determined. Concentrations of genistein and daidzein were both unchanged between the

studies. Concentrations of equol, the primary metabolite of genistein, approximately 0.19+/-0.09 µM

(n=7) in the second study, and 0.45+/-0.19 (n=8) µM in the third (p=0.006), supportive of reduced dietary

isoflavone exposure of the dam and/or pup bioavailability of the maternal metabolite.

8) Behavior (Figure 5.6)

Adult rats monitored in the open field chamber demonstrated significant behavioral alterations at

the 10ppm dose. Rats in this dose group demonstrated a significantly greater amount of ambulatory

activity over each 20 minute session. No alterations were seen in the number of vertical counts or central

zone entries, indicating hyperactivity without a modification of anxiety.

8) Cortical D2 correlations (Figure 5.7)

Cortical D2 continues to display a significant correlation with serum total T4 in pups. (PND25:

y=597.2-223*ln(x) R^2=0.88, PND14: y=789.1-323*ln(x) R^2=0.89) where y=D2 activity (fmol/hr/mg)

and x=serum total T4 concentration (µg/dl)

Discussion

As in the prior two studies, developmental thyroid disruption with PTU was initiated at

gestational day 2 and maintained until weaning. This protocol was designed to ensure that maternal

thyroid hormone production was compromised prior to the initiation of fetal thyroid hormone secretion

around GD17.2 Serum and brain data obtained at the PND4 timepoint confirmed the effectiveness of our

dosing routine in inducing neonatal as well as gestational hypothyroidism. The doses employed in this

study demonstrated a variety of effects, including neurological changes of varying degree and nature at all

146

doses. The presence of LTP impairment in PND25 pups from the 0.3ppm dose group is confusing, as no

decline in thyroid hormone concentrations have been detected in any age group exposed to 0.3ppm PTU

except for elevated T4 concentrations in the adult. Further studies on the gestational period are warranted

at this dose, to determine if any perturbations in thyroid hormone concentrations are caused prior to our

first sacrifice point at PND4.

As was seen in our second study, serum total thyroid hormone concentrations were affected to a

greater extent in younger pups than in older pups or dams. It is worth noting that the threshold for serum

effects was only 1ppm at PND4, whereas reductions in thyroid hormones were only seen starting at 3ppm

at PND14, and at 10ppm in the PND25 pups and dams. It is clear from this data that the ability of the

pups to compensate for insults to the thyroid system improved with maturity. However, interpretation of

the potential for permanent effects requires a complete understanding of the timing of thyroid hormone-

dependent neurodevelopmental effects. As this data shows, younger animals are more likely to be

affected by a given level of insult to the thyroid system, creating the potential for neurological damage

that may persist after serum thyroid hormone concentrations normalize.

The 3ppm dose effects on most of the observed parameters in this study were less severe than

those observed in the first study, but were more severe than the second study. At the same time, the

overall increase in body weight of the pups was increased in the second and third studies compared to the

first, with control pups averaging about 40 grams at PND21 in the first study, versus about 57 grams in

the second and 53 grams in the third. This alteration in growth patterns extended even to the 10ppm dose,

averaging about 25 grams at PND21 in the first study, and 35 in the third. Considering this data, it is

likely that the basic nutritional profile of the pups varied significantly between the three studies. Between

the first and second studies, the alteration in diet from Purina 5001 to 5008 may play a part, but the same

diet was used in the second and third studies. However, serum equol levels in PND14 pups were

significantly higher in the third study than in the second. As equol is a metabolite of soy isoflavones, this

indicates a greater isoflavone content of the diet in the third study, which may have increased the insult to

the thyroid axis in this study.42-46

Soy isoflavones have been shown to impact the thyroid axis at many

147

levels, including nuclear receptors and the retinoic acid system.42-46

For example, interactions with the

retinoic acid receptor in the liver can cause hypertriglyceridemia.46

Additionally, soy proteins were

shown to regulate the expression of genes for fatty acid synthase (FAS), malic enzyme (ME), acetyl-CoA

carboxylase (ACC), hydroxymethylglutaryl- CoA reductase, LDL receptor, and CYP7A1 in humans,

animals, and cultured human hepatoma cells.45

In this light, we interpret that litter size likely played a

significant role in resulting in a difference in nutritional plane for the pups in study 1 compared with 2

and 3. In study 1, no pups were culled until at least PND21 resulting in average litter sizes around 13. The

second study had the lowest average litter size (8 pups at PND4 and 6 at PND14,) while study 3 had two

more pups than the second, but still fewer than the first (10 pups at PND4, 8 at PND14). Studies by other

investigators have shown that the nutritional content of milk varies with the size of the litters being

nursed.47

This data emphasizes the need to carefully consider liter size and dietary constituents when

comparing data across studies.

Cerebrocortical Type II 5’-deiodinase (D2) enzymatic activity was again found to be a sensitive

respondent to reductions in serum T4 concentrations. The correlation was similar at PND25 and PND14,

with differences mainly arising from the variations in normal serum T4 concentration and basal D2

activity between the two age groups (Figure 6). While the observed correlations are not exactly identical

to those observed in prior studies, a strong similarity is still observed, and normalized data from each

study overlap, suggesting that the differences in D2 alterations observed in each study relate mainly to the

relative efficacy of PTU in reducing serum T4 concentrations. D2 is known to be regulated both pre- and

post-translationally by T3 and T4 respectively. One recognized method of post-translational regulation

by T4 is control of the rate of ubiquitination, and thus degradation of D2.48

Accordingly, the observed D2

activities are highly informative as to the immediate availability of thyroid hormones to the brain at the

time of sacrifice. The correlation of D2 to serum total or fT4 remains the strongest observed correlation

of D2.

Surprisingly, serum T4 concentrations were found to be elevated by about 20% in all doses in the

mature (adult) rats having been exposed to high doses of PTU. Prior studies have shown a restoration of

148

normal serum T4 concentrations in all doses, and we are unable to explain this surprising finding.

Behavioral data obtained from littermates approximately 60 days after weaning demonstrated that some

of the effects of the 10ppm treatment could not be reversed by restoration of euthyroidism after weaning.

This observation of altered behavior supports the conclusions made previously by other researchers that

persisting changes can occur in the brain following a developmental insult, possibly involving alterations

in myelination and migration patterns of neurons.14,23,25-28

Electrophysiological alterations were not

shown to persist, to adulthood unlike the first study.28

However, even in the absence of

electrophysiological alterations, significant hyperactivity was seen in the 10ppm pups. This effect was

observed on every day of testing, and provides evidence that some neurological alterations were still

present in the adults, despite the lack of electrophysiological alterations. While the exact cause of the

hyperactivity is unclear, this finding suggests the need for expanded behavioral testing in future studies.

This behavioral outcome parallels recent studies on the relationship between thyroid disruption and

hyperactivity in human populations.49

While electrophysiological changes were again observed in this study, the magnitude of the

impacts on synaptic potential were half that of our first study.28

Impairments in either 30-minute or 90-

minute LTP were observed in all doses except 10ppm. Unlike our first study, these alterations are not

observed in adults.

While there are quantitative and qualitative differences between the results obtained in our three

studies, a coherent picture emerges when all three are considered. First, litter size can moderate the

effects of insults to the thyroid axis in neonatal pups, probably through nutritional status. Second,

younger animals are more sensitive to insults to the thyroid axis than their older littermates. Finally,

sufficient neurological hypothyroidism in developing pups leads to irreversible neurological dysfunction,

indicated by electrophysiology in the first study and behavior in the third. Unfortunately, no significant

linear correlations between any of the observed biomarkers and electrophysiological outcome have been

consistently observed across the three studies. Therefore, the chosen electrophysiological markers are

unlikely to be the most sensitive possible indicators of neurodevelopment. It is also unfortunate that

149

histological data on physical brain structure is currently unavailable, as alterations in physical structure

would provide valuable data to explain the observed electrophysiological and behavioral results. More

sensitive functional measurements of neurodevelopmental impairment would be helpful in overcoming

this hurdle in future studies. Further studies to elucidate the cause of the LTP impairment in the PND25

pups exposed to 0.3ppm PTU would be especially helpful in understanding the true nature of the observed

changes. Traditional maze testing in particular may aid in detecting subtle changes in neurological

function which were undetectable through in vitro electrophysiology in the hippocampus. With the

exception of the puzzling reduction in LTP noted in the 0.3ppm dosed pups in this study, all

electrophysiological changes were accompanied or preceded by measurable increases in cerebrocortical

D2 activity, providing support for the thyroid-dependent nature of the observed neurological alterations.

However, the increased concentrations of isoflavones detected in the serum of this third study may help to

explain some of the differences between the first and second studies, as genistein may be modulating the

effects of thyroid hormones on the brain.50

Any future studies should strongly consider the need to

control and characterize isoflavone content for consistency across studies.

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154

Table 5.1 - Serum total T4, free T4, and total T3 Concentrations

Serum total T4 concentrations in PND4 pups are reduced by approximately 30% at 1ppm, 80% at 3ppm

and over 90% at 10ppm. In PND14 pups. The reduction is approximately 85% at 3ppm, and over 95% at

10ppm. In PND25 pups, the reduction is approximately 90% at 10ppm, and 80% at 10ppm in the dams.

(p<0.05) No alterations in the fraction of free T4 were seen at any dose.

Serum total T3 concentrations in PND14 pups are increased approximately 30% at 1ppm and decreased

approximately 80% at 10ppm. In PND25, serum T3 concentrations are reduced approximately 50% at

10ppm, and approximately 30% at 10ppm in the dams. (p<0.05)

Control 0.3 ppm 1 ppm 3ppm 10ppm

Dams T3 (ng/dl) 52.8 ± 8.9 50.8 ± 8.7 52.3 ± 11.6 51 ± 3.7 36.9 ± 12.1*

T4(µg/dl) 7.2 ± 2.6 7.9 ± 2 9.7 ± 6.6 7.8 ± 2.2 1.7 ± 1.3*

%Free T4 0.07 ± 0.02 0.06 ± 0.01 0.07 ± 0.01 0.06 ± 0.01 0.12 ±0.14

Free T4

(ng/dl)

4.8 ± 1.9 4.9 ± 1.4 6.3 ± 4.7 4.6 ± 1.3 1.1 ± 0.6*

PND25 T3(ng/dl) 51.7 ± 8.4 53.2 ± 7.7 51.7 ± 8.2 50 ± 14.2 23.6 ± 12.9*

T4(µg/dl) 4.7 ± 1 5.4 ± 2 5.6 ± 2.9 3.5 ± 2.1 0.3 ± 0.2*

%Free T4 0.1 ± 0.01 0.09 ± 0.01 0.1 ± 0.04 0.1 ± 0.02 0.09 ± 0.04

Free T4

(ng/dl)

4.9 ± 1.4 4.6 ± 1.7 6.3 ± 5.5 3.6 ± 2.6 0.2 ± 0.1*

PND14 T3(ng/dl) 27.4 ± 11.3 35.1 ± 5.4 40.7 ± 11.5* 28.8 ± 17.4 5.2 ± 1.2*

T4(µg/dl) 7.8 ± 4.5 6.4 ± 2.5 3.8 ± 2.4 0.9 ± 0.8* <0.1*

PND4 T4(µg/dl) 1.0 ± 0.2 0.9 ± 0.1 0.7 ± 0.2* 0.2 ± 0.02* <0.1*

Adult T4(µg/dl) 4.2 ± 0.4 5.5 ± 0.8 5.0 ± 0.8 5.5 ± 0.8 4.7 ± 0.7

155

Figure 5.1) A) Pup and B) Dam weight gain

Pups exposed to 10ppm PTU were significantly lighter than all other doses starting from the first

recorded weights at PND4. 3ppm pups became significantly lighter than controls only at PND23. No

weight differences were noted in dams.

156

Pup Weights

Post Natal Day

0 5 10 15 20 25

Avera

ge L

itte

r W

eig

ht (g

)

0

10

20

30

40

50

60

70

80

Control

0.3ppm

1ppm

3ppm

10ppm

157

Dam Weights

Days since arrival

0 10 20 30 40 50

Dam

Weig

ht (g

)

180

200

220

240

260

280

300

320

340

360

380

Control

0.3ppm

1ppm

3ppm

10ppm

158

Figure 5.2) Thyroid RNA

NIS mRNA was slightly upregulated in PND25 pups exposed to 3 ppm PTU, and increased by

approximately 5-fold at 10 ppm. Tg mRNA concentrations were unchanged below 10 ppm, and only

slightly upregulated at 10ppm.

159

PND25 Thyroid RNA

Dose (ppm PTU)

0 0.3 1 3 10

Rela

tive Q

uantity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NIS

TG

**

*

160

Figure 5.3) Cortical A)D2 B)T3

A) Cerebrocortical D2 activity in PND14 pups was increased approximately 200% at 1ppm,

600% at 3ppm, and 1000% at 10ppm. Cerebrocortical D2 activity in PND25 pups increased

approximately 400% only at 10ppm. Cerebrocortical D2 activity was increased approximately 300% in

dams exposed to 10 ppm PTU.B) In PND14 pups, cerebrocortical T3 concentrations were reduced

approximately 50% at 10 ppm. In PND25 pups, cerebrocortical T3 concentrations were reduced

approximately 40% at 10ppm. No significant changes in cerebrocortical T3 concentrations were seen at

any dose in PND4 pups or dams. No significant alterations remained until adulthood in weaned pups.

161

162

163

Figure 5.4) PND25 Electrophysiology: A) Baseline Synaptic Response, B) Maximal Synaptic Response,

C) LTP, D) wLTP, E)sLTP

Maximal synaptic potential was decreased by approximately 20% at 3 and 10ppm. Weak LTP

was impaired by approximately 10% at 1 and 3ppm, and strong LTP was impaired by approximately 10%

at 0.3 and 1ppm PTU. (p<0.05)

164

-2

-1.5

-1

-0.5

0

40 60 80 100 120 140

fEP

SP

slo

pe (

mV

/ms)

control 0.3 ppm 1 ppm 3 ppm 10 ppm

baseline synaptic response

165

30

40

50

60

70

80

90

100

110

120

130

control 0.3 ppm 1 ppm 3 ppm 10 ppm

max f

EP

SP

(%

contr

ol)

synaptic response at 140 A

* *

166

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

norm

aliz

ed f

EP

SP

slo

pe

Long-Term Potentiation

HFS:100Hz *3

167

30

40

50

60

70

80

90

100

110

120

130

control 0.3 ppm 1 ppm 3 ppm 10 ppm

% c

ontr

ol pote

ntiation

LTP at 30 minutes post HFS

*

*

168

Control 0.3ppm 1ppm 3ppm 10ppm

90 m

inute

LT

P (

% o

f contr

ol)

0

20

40

60

80

100

120

* *

169

Figure 5.5) PND25 Thyroid iodine content

The iodine content was reduced in pups dosed with 1, 3, and 10ppm PTU by approximately 30%,

60%, and 90% respectively. Only a single litter from each dose group was analyzed, so error bars

represent standard deviation within the litter.(p<0.05)

170

Thyroid iodine content - PND25

Dose (ppm PTU)

0 0.3 1 3 10

Thyro

id iodin

e c

onte

nt (u

g/g

land)

0.0

0.5

1.0

1.5

2.0

2.5

171

Figure 5.6) Open Field Behavior

Adult rats previously exposed to 10ppm PTU were found to be hyperactive in a 20 minute open

field test session. Data shown is total activity per 20 minute session. One session was performed each

day for three days. No significant alterations were found in the proportion of this activity in the center of

the open field vs. the periphery indicating no change in the levels of anxiety.

172

Ambulatory Activity

Behavior day

1 2 3

Am

bula

tory

Counts

0

500

1000

1500

2000

2500

3000

Control

0.3ppm

1ppm

3ppm

10ppm *

*

*

173

Figure 5.7) Serum A)Free T4, B)Total T4 vs. Cortical D2 – PND25, C) Serum total T4 vs. Cortical D2 –

PND14

Cortical D2 (Y) (fmol I-/mg/h) demonstrates a tight and highly significant correlation to (x) serum

total T4 concentrations (µg/dl)

PND25: Y=597.2-223*ln(x) R^2=0.88

Cortical D2 (Y) (fmol I-/mg/h) demonstrates a tight and highly significant correlation to (x) serum

free T4 concentrations (ng/dl)

PND25: Y=571.4-212.7*ln(x) R^2=0.89

Cortical D2 (Y) (fmol I-/mg/h) demonstrates a tight and highly significant correlation to (x) serum

total T4 concentrations (µg/dl)

PND14: Y=789.1-323*ln(x) R^2=0.89

174

Cortical D2 vs. Serum Total T4 - PND 25

Serum Total T4 (ug/dl)

0 2 4 6 8 10 12

Cort

ical D

2 A

ctivity (

fmol/m

g/h

)

0

200

400

600

800

1000

1200

175

Cortical D2 vs. Serum Free T4 - PND25

Serum free T4 (ng/dl)

0 5 10 15

Cort

ical D

2 (

fmol/m

g/h

)

-200

0

200

400

600

800

1000

1200

1400

176

Cortical D2 vs. Serum Total T4 - PND14

Serum Total T4 (ug/dl)

0 5 10 15

Cort

ical D

2 (

fmol/m

g/h

)

-1000

-500

0

500

1000

1500

2000

177

CHAPTER 6

SUPPLEMENTAL AND COLLABORATIVE STUDIES

1) PTU Timecourse study

Materials and Methods:

Young adult male CDS rats (Charles River Labs, Raleigh NC) were maintained on standard diet

and water until onset of study. Animals were weighed and transferred to single cages immediately

preceding the onset of study. Weights and water intake were recorded daily. PTU doses of 0, 0.3 and

3ppm were employed in DI drinking water with 4 rats per group.

On Day 0, animals were lightly anaesthetized and bled via retroorbital sinus. Approximately

600ul of blood was collected. This blood was allowed to clot, spun down for serum collection, and frozen

until assay. After the initial bleed, the animals were given dosed water for their cage. Subsequent bleeds

occurred at 1, 2, 4, and 7 days after the onset of dosing, each of approximately 600ul. On day 10, the

animals were terminated by decapitation under anesthesia. Trunk blood was collected at this time.

Additionally, cerebral cortex was collected and used for deiodinase assays.

Serum T4 concentration and cortical D2 were measured as previously described (Chapter 3).

Results:

Serum T4 Concentration (Figure 6.1)

There were no significant changes from initial serum T4 concentrations on the first day after

dosing, but a 25% decrease in all three groups was noted on the second. This decrease was maintained in

the control and 3ppm groups on the fourth day, but the 0.3ppm rats returned to initial levels on that day.

On the seventh day, an elevation of approximately 15% was seen in the 0.3ppm group, while the control

group was still reduced by about 20%, and the 3ppm group was further reduced to 50% of initial values.

On the tenth and final day, the 0.3ppm group remained elevated, while the control group had returned to

normal and the 3ppm group remained depressed by 50%.

178

Cortical D2 (Figure 6.2)

Cortical D2 activity was measured after the terminal bleed. An 80% reduction in D2 activity was

seen in the 3ppm adults treated for 10 days.

Discussion:

Evaluating the control group, it is clear that frequent repeat bleeds on days 1, 2, and 4 were likely

associated with stress or an anesthetic drug effect which resulted in lowering serum T4. Regardless, it

seems clear from this study that a clear decrease in serum T4 levels in adult rats is achieved after no more

than seven days of dosing with 3ppm PTU in drinking water. Strangely, D2 levels are not elevated in the

dosed animals, suggesting that the repeated stress of bleeding impacted D2 regulation in these subjects, as

has been described in nonthyroidal illness.

2) Deiodinase activity of perchlorate treated rat pups

Materials and Methods:

CDS Rats were treated in Dr. Zoeller’s lab at the University of Massachusetts. Timed pregnant

rat dams were exposed to perchlorate at doses of 0, 10, 100, 250, 500, and 5000 ppb in drinking water

from GD6 until weaning. Brains from PND21 animals were collected, flash frozen, and shipped to UGA.

Cerebral cortex was removed from the frozen brains and homogenized for cortical deiodinase

measurement. D2 assays were performed as previously described (Chapter 3).

Results:

No significant changes were noted in cortical D2 activity across any of the measured doses.

Discussion:

This data indicates that continued exposure to up to 5ppm perchlorate in the drinking water of

gestational and neonatal rats provides an insufficient insult to the thyroid axis to trigger a deiodinase

response in the brain. We would predict from this data that there are no alterations in thyroid hormone

availability to the brain under these treatments, at least at PND21.

179

3) Deiodinase activity and cerebrocortical T3 concentrations of PTU dosed Long-Evans rats

Materials and Methods:

Pregnant Long-Evans rats were obtained from Charles River (Raleigh, NC) on gestational day

(GD) 2 and housed individually in standard plastic hanging cages in an Association Assessment and

Accreditation of Laboratory Animal Care-approved animal facility at the USEPA laboratories in RTP,

NC. All animal treatments were in strict accordance with the National Institutes of Health Guide for the

Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals and their

suffering. Animal rooms were maintained on a 12-h light, 12-h dark schedule, and animals were permitted

free access to food (rat chow, Purina; St. Louis, MO) and tap water.

Beginning on GD 6 and continuing until PND23, dams were rendered hypothyroid by addition of

0, 1, 2, or 3 ppm of the thyroid hormone synthesis inhibitor propylthiouracil (PTU; Sigma, St. Louis, MO)

to the drinking water. The day of birth was designated PN0 and all litters were culled to 10 pups on PN4,

retaining the maximal number of males per litter. On PND23, the offspring were weaned, transferred to

plastic hanging cages (two to four/cage) and were permitted free access to food and tap water. A subset of

animals were sacrificed for tissue collection on PND4, PND14, PND15, PND21, and PND22. Cortical

tissue from dams, PND14-15 and PND21-22 animals were frozen and shipped to UGA for analysis of

cortical D2 activity and cortical T3 concentrations. These assays were performed as previously described

(Chapter 3). Serum thyroid hormone data was provided for correlation with these data.

Results:

D2 (Figure 6.3)

In PND21 animals, cortical D2 activities were increased by about 250% and 650% at 2 and 3ppm

respectively (p<0.0001). In the PND 14 animals, this upregulation is about 600% at both 2 and

3ppm(p=0.0005). In dams, D2 activities were increased by about 250% at 3ppm, with no change at

2ppm(p<0.0001).

Cortical T3 Concentration

Cortical T3 concentrations were unchanged at PND14, PND21 and in dams.

180

Cortical T4 (Figure 6.4)

Cortical T4 concentrations were reduced by about 40% and 70% at 2 and 3ppm respectively in

PND21 animals (p=0.0043). Cortical T4 concentrations were reduced by about 55% in all three doses at

PND14 (p=0.0084). Cortical T4 concentrations were reduced by about 30% at 1 and 2ppm, and about

60% at 3ppm in dams (p=0.0005).

Correlations of D2 Activity with Serum Concentration Data (Gilbert lab)

Cortical D2 was again found to correlate tightly with serum total T4 concentration. Each age

group was found to fit a logarithmic curve. The relationship in the PND14 animals is somewhat obscured

by a larger number of serum T4 levels below the limit of detection than the other ages.

PND14 (Figure 6.5)

Y=1677-390*ln(X) R^2 = 0.58

PND21 (Figure 6.6)

Y=2159-533*ln(X) R^2 = 0.90

Dams (Figure 6.7)

Y=219-51*ln(X) R^2 = 0.76

Where x= serum T4 concentration in ng/ml and Y = cortical D2 activity in fmol I-/hr/mg

Discussion:

Despite the differences in dosing protocol and rat strain between the animals studied in Dr.

Gilbert’s lab and those in this laboratory, cortical D2 data correlates with serum T4 concentrations in a

very similar fashion. This correlation provides further evidence that the regulation of D2 in the brain is

qualitatively identical between the CDS and Long-Evans strains of rats dosed with PTU. As D2 is

regulated both pre- and post-translationally by thyroid hormones, this similarity is not unexpected, but its

similarity should aid in modeling D2 response to serum T4 across rat strains. As PTU has not been

shown to change the free T4 fraction, this relationship should hold for both free and total T4.

181

Figure 6.1 – Serum T4 Concentration in PTU Timecourse Study

A significant (20-30%) reduction in serum total T4 concentration is seen in all three dose groups at day 2

of testing. This reduction is fully reversed by day 4 in the 0.3ppm group, but the reduction remains until

day 10 in the control group. A further reduction to about 50% of control levels is seen in the 3ppm group

at day 7, and persists at termination on day 10.

*=P<0.05 compared to control for that day

182

PTU Timecourse Serum T4

Days since onset of PTU dosing

0 2 4 6 8 10 12

Seru

m T

ota

l T

4 (

% initia

l)

40

60

80

100

120

140

Control

0.3ppm

3ppm

*

* *

*

*

183

Figure 6.2 – D2 in PTU Timecourse Study

Cortical D2 activity is reduced by approximately 80% at the 3ppm dose level after 10 days of dosing.

*=p<0.05

184

PTU Timecourse D2

PTU dose (PPM)

0 0.3 3

Cort

ical D

2 a

ctivity (

fmol/m

g/h

)

0

10

20

30

40

50

60

70

*

185

Figure 6.3 – Cortical D2 in Gilbert Study

At PND14, cerebrocortical D2 concentrations are increased approximately 800% at both the 2 and 3ppm

doses. At PND21, an approximately 150% increase is seen at 2ppm, and approximately a 900% increase

at 3ppm. In the dams, an approximately 200% increase is seen at 3ppm.*=p<0.05

186

Cortical D2

Animal age

PND14 PND21 Dam

Cort

ical D

2 A

ctivity (

fmol/m

g/m

in)

0

200

400

600

800

1000

1200

1400

1600

Control

1ppm

2ppm

3ppm

*

*

*

**

187

Figure 6.4 – Cortical T4 in Gilbert Study

Cerebrocortical T4 concentrations are reduced by approximately 50% at all three doses in PND 14 pups.

At PND21, an approximately 50% reduction is seen at 2ppm, and an 80% reduction at 3ppm. In dams, a

50% reduction is seen at 2ppm, and an approximately 75% reduction at 3ppm.*=p<0.05

188

Cortical T4

Animal age

PND14 PND21 Dam

Cort

ical T

4 (

ug/m

g)

0

1000

2000

3000

4000

5000

6000

Control

1ppm

2ppm

3ppm

*

* *

(1,2,3ppm)

**

189

Figure 6.5 – Cortical D2 vs. Serum T4 at PND14 in Gilbert Study

Cortical D2 and serum T4 displayed a strong correlation.

Y=1677-390*ln(x) R^2 = 0.58

*=p<0.05

190

Serum T4 vs. Cortical D2 - PND14

Serum T4

0 20 40 60 80

Cort

ical D

2 a

ctivity (

fmol/m

in/m

g)

0

500

1000

1500

2000

2500

191

Figure 6.6 – Cortical D2 vs. Serum T4 at PND23 in Gilbert Study

Cortical D2 activity and serum total T4 concentrations displayed a strong logarithmic correlation.

Y=2159-533*ln(x) R^2 = 0.90

*=p<0.05

192

Serum T4 vs. Cortical D2 - PND21

Serum T4

0 20 40 60 80

Cort

ical D

2 a

ctivity (

fmol/m

in/m

g)

-200

0

200

400

600

800

1000

1200

1400

1600

1800

193

6.7 – Cortical D2 vs. Serum T4 in Dams in Gilbert Study

Cortical D2 activity and serum total T4 concentrations displayed a strong logarithmic correlation.

Y=219-51*ln(x) R^2 = 0.76

*=p<0.05

194

Serum T4 vs. Cortical D2 - Dams

Serum T4

0 20 40 60 80

Cort

ical D

2 a

ctivity (

fmol/m

in/m

g)

-50

0

50

100

150

200

250

195

CHAPTER 7

SUMMARY AND DISCUSSION

Review of hypotheses:

1) Thyroid hormone concentrations in serum do not provide sufficient information to predict the

availability and activity of thyroid hormones to all tissues in the body, as alterations in peripheral

metabolism help to counterbalance the effects of systemic hypothyroidism in critical tissues such as the

brain.

This hypothesis is partially supported, at least for the PTU model of hypothyroidism.

Specifically, no clear correlation was found between serum thyroid hormone concentrations and cortical

T3 concentrations. This is due in large part to the sensitive upregulation of D2 in response to declining

T4. It should be noted that the relationship between serum T4 and cortical D2 activity is very close, and

the serum T4 concentrations in an animal from each tested age group is sufficient to predict the cortical

D2 activity with a fair degree of precision. Further study may help to better bridge the gap between

serum and tissue availability and effectiveness of thyroid hormones. This study presents no data to clarify

the effects of any alterations of serum or receptor binding kinetics caused by some other antithyroid

agents. Future studies comparing the relative impact of these alternative pathways of disruption would

help to clarify the degree to which deiodinase regulation can compensate for generalized insults to the

thyroid axis.

2) Measurement of brain-specific biomarkers such as thyroid responsive gene expression, deiodinase

activity and cerebrocortical T3 concentrations will be more reliable predictors of neurodevelopmental

impairment than serum hormone concentrations due to their improved proximity to the developmental

endpoint compared to serum effects.

Again, this hypothesis is partially supported. Cortical D2 levels seem to correlate very well with

serum T4 concentrations, potentially allowing accurate modeling of cortical D2 regulation in response to

196

serum T4 perturbations. However, cortical D2 upregulation seems to be limited to a single order of

magnitude. Cortical T3 concentrations are maintained despite moderate serum T4 depletion, but will fall

under severe T4 depletion. However, this data does not address any potential changes that would result

from mechanistically dissimilar anti-thyroid agents. An additional strike against this hypothesis lies in

the lack of any consistent correlation between serum or tissue biomarkers and observed

electrophysiological results, despite the strong correlation between synaptic potential and D2 activity

noted only in the first study. This does not mean that no such correlations exist, and further investigation

of thyroid hormone availability to the brain at varying timepoints during development may help to clarify

the exact mechanisms responsible for hypothyroidism’s effect on electrophysiological parameters of

neural development.

3) The chosen biomarkers: cerebrocortical D2 activity, cerebrocortical T3 concentration, and expression

of thyroid-responsive genes in the brain will provide data which will aid efforts to predict both temporary

and permanent neurological dysfunction caused by any thyroid disruptive compound.

No biomarkers were observed in this study which consistently correlated with electrophysiological

outcomes. Expanding future studies to include new biomarkers and more sensitive neurological outcomes

may provide the missing data. It is likely that data from late prenatal and early postnatal periods may

provide the critical link, as many structures of the brain are still developing during these times.1 Further

analysis during periods of critical development in specific brain regions, combined with sensitive testing

of morphology and functionality of these regions in mature animals would help to understand which

markers of thyroid hormone availability to the target tissues are most critical for proper development.

Apart from the puzzling impairment of 90-minute LTP in the third study, a measureable decline in at least

serum T4 levels was required before any neurological alterations were noted. It is possible that an

elevation of available T4 at an inappropriate developmental period might also lead to adverse

neurophysiological events. Given the potential perturbations caused by the varying levels of soy

isoflavones from lot to lot of the same diet, even the tissue thyroid hormone concentrations may not be

providing the full picture of thyroid hormone availability in the brain.

197

4) Younger animals are more sensitive to thyroid disruption than older animals due to their immature

thyroid axis and lesser compensatory capabilities compared to adults.

This hypothesis is well supported by the data obtained in these studies. In almost every case,

thyroid hormone reductions were seen both at lower doses, and to a greater extent at equivalent doses than

their older littermates or dams. The period of greatest impact seems to be PND14, though the limited

sample size at PND4 complicates analysis of that age group. Based on this data, it is possible that doses

of antithyroid compounds which are insufficient to cause noticeable effects in mature animals may still

have detrimental consequences for younger animals. It is thus critical to investigate effects on the

gestational and neonatal periods when considering the true impact of exposure to thyroid disrupting

compounds. Measureable

5) The neurological impact of maternal hypothyroidism is modified by the nutritional status of

developing pups, as impacted by the number of pups nursed by a single dam, as well as alterations in

dietary contents such as soy isoflavones.

The data obtained by the three UGA studies supports, but does not confirm this hypothesis. The

severity of results obtained in the three studies ranks in the same order as litter sizes, but further analysis

would be necessary to confirm the nutritional status of the pups. The best direct support for this

hypothesis comes from the weight gain of control pups. The pups from the second and third studies were

significantly heavier than those from the first, and the pups in the second study were slightly heavier than

those in the third. This would be expected if litter size were truly important, as litters were largest in the

first study, smaller in the third, and smallest in the second. While significant differences in isoflavone

content were noted between the second and third study, the lack of parallel data from the first study

makes it impossible to declare how strongly the varying isoflavone content of the diet impacted the results

noted in the study.

Study 1 results (Table 7.1):

The first study employed doses of 0, 3, and 10ppm PTU to induce gestational and neonatal

hypothyroidism. The PND25 pups proved to be more sensitive to disruption than their dams.

198

Specifically, serum T3 levels were reduced at 3ppm in pups, but not in dams. Also, the pups showed an

increase in thyroidal NIS and Tg mRNA, which was not seen in the dams. Both the 3 and 10ppm doses

served to decrease the weight gain of dosed pups, and both led to impaired synaptic functionality at the

PND25 timepoint. A striking correlation was noted between synaptic potential and cortical D2 activity,

suggesting a link between immediate thyroid status and synaptic function. The 10ppm dose further

caused gross physical retardation, and left the affected pups unable to survive in standard housing after

weaning. No significant differences were found in the degree of electrophysiological impairment in the

PND25 pups between the doses, so a second study was designed to use lower doses of PTU to produce a

graded effect. Only 3ppm and control pups were maintained to adulthood, and the 3ppm pups

demonstrated a reduction in LTP at PND90, confirming a permanent effect of the gestational and neonatal

dosing.

Study 2 results (Table 7.1):

The second study employed doses of 0, 0.3, 1, and 3 ppm PTU, as well as 150ppm perchlorate.

The perchlorate dose chosen had been previously shown to induce chronic depression of serum thyroid

hormone levels. To harmonize the study design with collaborators at the University of Massachusetts and

the US EPA, culling was implemented to reduce litter sizes to eight pups at PND4. Tissue collection was

also initiated at PND4 and PND14 to explore the impact of hypothyroidism on several points in neonatal

development. Only one pup per litter was used for electrophysiological analysis, as opposed to two pups

per litter in the first study. As a result, weaning was moved to PND25 instead of PND32.

No serum thyroid hormone alterations were noted in the perchlorate dosed animals, but a striking

increase in NIS mRNA in the thyroids of PND25 pups and dams was noted. This large increase was not

accompanied by an increase in Tg mRNA, unlike the response observed to PTU in the first study.

Additionally, only a Tg response was seen in the PTU doses in the second study, with no NIS response

mirroring that seen in the first study. When combined with the lack of serum thyroid hormone alterations,

this suggests that NIS is autoregulated by the thyroid in response to iodine availability, even in the

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absence of TSH modulation. Autoregulation of genes in the thyroid in the absence of TSH stimulation

has been previously observed by other investigators.2,3

The data obtained from pups in the second study further supported our hypothesis that younger

pups would be more impacted by insults to the thyroid axis. Serum T4 concentrations were reduced at

both 1 and 3ppm dose levels in PND4, PND14, and PND25 pups, though the reduction was smaller in the

PND25 pups. Conversely, serum T4 concentrations were unchanged in dams. This differential

sensitivity translated into the brain, where cortical D2 continued to correlate nicely with serum T4. No

alterations in serum T3 were noted in this study, nor were any electrophysiological alterations observed in

either PND25 pups or adults. When considering the decline in cerebrocortical RC3 expression which is

not mirrored in the hippocampus, it is possible that the hippocampus is more highly protected against

hypothyroidism than the cortex. Analysis of adult behavior in an open field chamber over five testing

sessions of 30 minutes each also showed no significant alterations. Additionally, pups in the second

study had higher body weights than their counterparts in the first study.

Overall, the effects of the 3ppm dose in the second study were substantially reduced from the

effects in the prior study. The major differences in animal handling between the first and second studies

were culling and alteration of the pre-weaning diet from Purina 5001 to Purina 5008. Comparison of the

nutritional profiles of the two diets showed a slightly higher fat content in the 5008 diet (6.5% in 5008 vs.

4.5% in 5001), but a 20% decrease in the iodine concentration. See Appendix B for a more detailed

comparison.. Accordingly, litter sizes emerged as the likely candidate for the leftward shift of the PTU

dose-response curve. A third study was designed to verify the shift of the dose response curve, as

opposed to a qualitative alteration in the effects of PTU dosing.

Study 3 results (Table 7.1):

The third study employed PTU doses of 0, 0.3, 1, 3, and 10ppm PTU, replicating all PTU doses

used in the first two studies. The litters were culled in a similar fashion to the second study, but the litter

sizes were two pups larger. The behavioral analysis was refined to three 20-minute sessions. Overall, the

electrophysiological results obtained in the third study were intermediate between those seen in the prior

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two studies. Unfortunately, the lack of LTP data in the PND25 pups from the first study does not allow

the full comparison of electrophysiological results between all studies.

Once again in the third study, younger animals were observed to be more sensitive to thyroid

insults than their older littermates and dams. Serum T4 was reduced starting at 1ppm at PND4, 3ppm at

PND14, and 10ppm at PND25 and in dams. Serum T3 was reduced starting at 3ppm at PND14, and

10ppm at PND25 and in dams. The weights of 10ppm pups were reduced starting on the first recorded

weight at PND4, though they were still heavier than the 10ppm pups from the first study.

Measurements of thyroid hormone concentrations and deiodinase activity in the brain also

showed age-related differences. Cortical D2 activities were increased starting at 1ppm in the PND14

pups, and only at 10ppm in dams and PND25 pups. Little data of this nature is available in the literature,

and previous studies have shown lower cortical D2 activity in PND12 pups than PND22, so the greater

activity noted in PND14 pups than PND21-25 in our studies is somewhat surprising.4 Cortical T3

concentrations were reduced only at 10ppm in PND14 and PND25 pups, and were unchanged in the

dams. As in the first study, synaptic potential in the PND25 pups was reduced at 3ppm and 10ppm.

Changes in LTP were also seen in the PND25 pups, with impaired weak LTP at the 1 and 3ppm dose

levels, and impaired strong LTP at the 0.3 and 1ppm dose levels. The effects on the 0.3ppm dose level

are especially surprising, as no thyroid hormone concentrations in serum or brain were observed to be

altered at that dose in any age group. Given the generally lesser effect of dosing in the second study, it is

possible that fluctuations in thyroid hormone concentrations earlier on in development may have

impacted the 0.3ppm pups in the third study but not in the second. An additional study further expanding

the sampling of early developmental timepoints may help to explain this effect. Unlike the first study, a

change in LTP magnitude was not observed in the recovered adults in the third study. However,

behavioral observation in an open field chamber revealed that the adults previously exposed to 10ppm

PTU were hyperactive. No changes were observed in habituation patterns or activity in the central zone,

indicating no impairment in simple place recognition or anxiety.

Supplemental and collaborative results:

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Between the first and second UGA studies, Dr. Zoeller at the University of Massachusetts

conducted an experiment dosing timed pregnant rats with perchlorate doses up to 5ppm in drinking water.

Brains from these animals were sent to UGA, where cortical D2 activity levels were determined. No

changes were seen in cortical D2 activity in any of these animals. This data, with the consideration of

prior literature demonstrating persistent hypothyroidism in rats exposed to 30mg/kg/day of perchlorate

was used to arrive at the 150ppm dose used in the second UGA study, which still showed no D2 effects.

Also between the first and second UGA studies, cortex sections were sent to UGA from rats

dosed with 0, 1, 2, and 3 ppm PTU by Dr. Gilbert at the US EPA. Cortex D2 activity, as well as cortex

T3 and T4 concentrations were determined at UGA. This data was correlated with serum hormone data

obtained elsewhere. The correlation between cortical D2 activity and serum total T4 concentrations was

found to be nearly identical to that found in the UGA studies, further validating the quantitative

relationship between these two biomarkers of thyroid status as it was observed in two strains of rats

studied in 2 laboratories. Preliminary analysis of the in-vivo electrophysiology data from Gilbert’s study

does not indicate a correlation between D2 activity and synaptic potential in vivo.

Contemporaneously with the second study, a timecourse study was conducted to verify the time

at which serum thyroid hormones would be impacted by drinking water administration of 0.3 or 3ppm

PTU. This study showed a slight increase in serum T4 levels after 7 days of dosing with 0.3ppm PTU,

and a significant decrease in serum T4 under 3ppm PTU at the same time. However, this data was

somewhat clouded by the effects of frequent bleeding under anesthesia of these animals, as a slight

reduction in serum T4 levels were observed even in the controls after two days of dosing and bleeding.

Overall, this data suggests that normal adult rats will be impacted by our PTU dosing protocol after no

more than seven days of treatment. This would further imply that dams dosed beginning on day 2 of

gestation would be significantly affected well before thyroid hormone receptors are first detected in the

developing rat brain around GD12. If this is the case, then it confirms that rats born to dams dosed

starting at either GD6 (Gilbert and Zoeller) or GD2 (UGA) will develop under hypothyroid conditions at

all relevant times during neural development.

202

Overall:

The studies of this dissertation confirmed some of the hypotheses in one of the studies, while

refuting it in others. In general, the first study best fit the hypotheses, whereas the lesser effects of dosing

in the second and third study did not fit the patterns observed in the first study. While several striking

findings arose, such as the correlation between deiodinase activity and hippocampal synaptic activity

found in the first study, no linear correlations were found to exist when looking at all three datasets. Of

all the studied data, the only completely consistent finding was the relationship between cortical D2

activity and serum total T4 concentrations, with a doubling of D2 for each 30-35% reduction in serum T4

concentrations below control levels. However, given the presence of electrophysiological alterations in

doses without concomitant T4 reductions or D2 elevations at PND25, it becomes clear that perturbations

which subside before PND25 may still leave a lasting impact on neurological function. Based on our

observations of PND4 and PND14 animals in the second and third studies, it seems clear that any future

studies of hypothyroidism and neurodevelopment must focus on identifying the critical gestational and

neonatal timepoints associated with abnormalities in the weanling and/or adult. Pups in these two younger

age categories were more strongly affected by each tested PTU dose than their dams or PND25

littermates. In the thyroid, either Tg, NIS, or both of the mRNA’s were upregulated at both 3 and 10ppm

in all studies. In general, a decline in cortical T3 concentrations at PND25 was linked with neurological

impairment, though impairment was also seen in the absence of cortical T3 changes at PND25. Each of

the three studies helped to refine our understanding of the effects of thyroid insufficiency on the

hippocampal neurodevelopment of the rat, but our experiments failed to definitively link any tested

thyroid related parameters such as deiodinase activity or hormone concentrations to neurological

outcome, though with the exception of the 0.3ppm LTP impairment in the third study, measurable

declines in serum T4 in at least preweaning age group were linked to adverse neurological effects. New

endpoints capable of measuring function in other critical brain regions, such as maze testing, startle

responses, and other common developmental toxicity tests should be considered for any future work. In

addition, the prioritization of histological analysis of the brain will assist in detecting physical

203

malformations not impacting the chosen neurofunctional assays. By expanding brain measurements

beyond the hippocampus, many more changes in the brain may be detected, greatly expanding our

understanding of the neurodevelopmental impacts of hypothyroidism. Further characterization of

regional concentrations of thyroid hormones in the brain should also help to understand the true

relationship between thyroid hormone depression and adverse neurological alterations. Except for study 1

which showed a relationship between D2 and srmax and cortical T3 and srmax in PND25 pups, the other

studies did not replicate these findings, suggesting that the larger litter size and/or a reduced nutritional

plane may have been key to this relationship. The isoflavone concentrations in the pups were not

measured in study 1 so variation relative to studies 2 and 3 cannot be confirmed. Further studies would be

necessary to confirm this possibility of dietary effects.

References

1. Howdeshell KL 2002 A model of the development of the brain as a construct of the thyroid system.

Environ Health Perspect. 110 Suppl 3:337-48

2. Suzuki K, Lavaroni S, Mori A, Ohta M, Saito J, Pietrarelli M, Singer DS, Kimura S, Katoh R,

Kawaoi A, Kohn LD 1998 Autoregulation of thyroid-specific gene transcription by thyroglobulin.

PNAS 95(14):8251-6

3. Leoni SG, Galante PA, Ricarte-Filho JC, Kimura ET 2008 Differential gene expression analysis of

iodide-treated rat thyroid follicular cell line PCCI3. Genomics 91(4):356-66

4. Bates JM, St. Germain DL, Galton VA 1999 Expression profiles of the three iodothyroinine

deiodinases, D1, D2, and D3, in the developing rat. Endocrinology 140(2)844-51

204

Table 7.1-Overall findings

□ : Not Studied; ═: No Change; ▲: Increased; ▼:Decreased

Dose 0.3 1 3 10 0.3 1 3 10

Study 1/2/3 1/2/3 1/2/3 1/2/3 1/2/3 1/2/3 1/2/3 1/2/3

PND4 Dam

T4 □/ ═/ ═ □/ ▼/ ▼ □/ ▼/ ▼ □/ □/ ▼ T3 □/ ═/ ═ □/ ═/ ═ ═/ ═/ ═ ▼/ □/ ▼

D2 □/ ═/ □ □/ ═/ □ □/ ▲/ □ T4 □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ═ ▼/ □/ ▼

cT3 □/ ═/ ═ □/ ═/ ═ □/ ═/ ═ □/ □/ ═ TSH □/ ═/ □ □/ ═/ □ ▲/ ▲/ □ ▲/ □/ □

PND14 D2 □/ ═/ ═ □/ ═/ ═ ═/ ═/ ═ ▲/ □/ ▲

T3 □/ ═/ ═ □/ ═/ ▲ □/ ═/ ═ □/ □/ ▼ cT3 □/ ═/ ═ □/ ═/ ═ ═/ ═/ ═ ▼/ ═/ ═

T4 □/ ═/ ═ □/ ▼/ ═ □/ ▼/ ▼ □/ □/ ▼ NIS □/ ═/ □ □/ ═/ □ ═/ ═/ ═/ □/ □

D2 □/ ═/ ═ □/ ▲/ ▲ □/ ▲/ ▲ □/ □/ ▲ TG □/ ═/ □ □/ ═/ □ ═/ ═/ □ ═/ □/ □

cT3 □/ ═/ ═ □/ ═/ ═ □/ ▼/ ═ □/ ═/ ▼ Adult

PND25 wLTP □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ═ □/ ═/ ═

T3 □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ═ ▼/ □/ ▼ sLTP □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ═ □/ ═/ ═

T4 □/ ═/ ═ □/ ▼/ ═ ▼/ ▼/ ═ ▼/ □/ ▼ activity □/ ═/ ═ □/ ═/ ═ □/ ═/ ═ □/ ═/ ▲

TSH □/ ═/ □ □/ ▲/ □ □/ ▲/ □ □/ □/ □

D2 □/ ═/ ═ □/ ═/ ═ ▲/ ▲/ ═ ▲/ □/ ▲

cT3 □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ═ ▼/ □/ ▼

srmax □/ ═/ ═ □/ ═/ ═ ▼/ ═/ ▼ ▼/ □/ ▼

wLTP □/ ═/ ═ □/ ═/ ▼ □/ ═/ ▼ □/ □/ ═

sLTP □/ ═/ ▼ □/ ═/ ▼ □/ ═/ ═ □/ □/ ═

NIS □/ ═/ ═ □/ ═/ ═ ▲/ ═/ ▲ ▲/ □/ ▲

TG □/ ═/ ═ □/ ═/ ═ ▲/ ▲/ ═ ▲/ □/ ▲

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Figure 7.1-Serum T4 vs. Cortical D2 – Dams

When all studies were adjusted to calculate T4 and D2 alterations on a percent of control basis, all studies

clustered tightly into a common relationship between serum total T4 and cortical D2 activity. No major

differences are noted in this relationship across studies. In the following equation, Y is the percentage of

control cortical D2 activity, and x is the percentage of control serum T4 concentration

Y=94.5+880*e^(-0.05*x) R^2 = 0.7

206

D2 vs. Serum T4 - Dams

Serum T4 (% of control)

0 100 200 300

D2 (

% o

f contr

ol)

0

200

400

600

800

1000

1200

207

Figure 7.2-Serum T4 vs. Cortical D2 - PND21

When all studies were adjusted to calculate T4 and D2 alterations on a percent of control basis, all studies

clustered tightly into a common relationship between serum total T4 and cortical D2 activity. No major

differences are noted in this relationship across studies. In the following equation, Y is the percentage of

control cortical D2 activity, and x is the percentage of control serum T4 concentration

Y=-8+747*e^(-0.02*x) R^2 = 0.68

208

Serum T4 vs. Cortical D2 - PND21

Serum T4 (% control)

0 50 100 150 200 250

Cort

ical D

2 (

% c

ontr

ol)

0

200

400

600

800

1000

1200

1400

209

Figure 7.3-Serum T4 vs. Cortical D2 - PND14

When all studies were adjusted to calculate T4 and D2 alterations on a percent of control basis, all studies

clustered tightly into a common relationship between serum total T4 and cortical D2 activity. No major

differences are noted in this relationship across studies. In the following equation, Y is the percentage of

control cortical D2 activity, and x is the percentage of control serum T4 concentration

Y=93+1134*e^(-0.04*x) R^2 = 0.78

210

Serum T4 vs. Cortical D2 - PND14

Serum T4 (% control)

0 50 100 150 200 250

Cort

ical D

2 (

% c

ontr

ol)

0

200

400

600

800

1000

1200

1400

1600

1800

211

APPENDIX A

LIST OF ABBREVIATIONS

AMP Adenosine monophosphate

ATP Adenosine triphosphate

BCE Before Common Era

BW Body weight

CSF Cerebrospinal fluid

CE Common Era

D1 Type 1 deiodinase

D2 Type 2 deiodinase

D3 Type 3 deiodinase

GD Gestational day

kD Kilodalton

LTP Long term potentiation

MMI Methimazole

NIS Sodium/iodide symporter

NOAEL No Observed Adverse Effect Level

PND Postnatal day

ppm Parts per million

PTU Propylthiouracil

rT3 3,3’,5’-triiodothyroinine

T3 3,5,3’-triiodothyronine

T4 Thyroxine

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TBG Thyroxine-binding globulin

Tg Thyroglobulin

TH Thyroid hormone

TPO Thyroid peroxidase

TSH Thyroid stimulating hormone

TTR Transthyretin

213

APPENDIX B

DIETARY COMPARISON

Parameter Diet

5008 5001

Protein 23.5% 23.9%

Fat (ether extract) 6.5% 5.0%

Fat (acid hydrolysis) 7.5% 5.7%

Starch 34.9% 31.9%

Glucose 0.22% 0.22%

Fructose 0.24% 0.30%

Sucrose 2.57% 3.70%

Lactose 0.39% 2.01%

Calories from Protein 26.849% 28.507%

Calories from Fat (ether extract) 16.710% 13.496%

Calories from Carbohydrates 56.441% 57.996%

Gross Energy, kcal/gm 4.15 4.07

Metabolizable Energy, kcal/gm 3.31 3.02

Iodine (ppm) 0.8 1.0