1. A Systems Approach to Characterizing and Predicting Thyroid Toxicity
Michael Hornung, Kara Thoemke,
Joseph Korte, Jose Serrano, John Nichols,
Patricia Schmieder, Joseph Tietge, Sigmund Degitz
US EPA, Mid-Continent Ecology Division, Duluth, MN
McKim Conference
June 27-29, 2006
Duluth, MN

2. Thyroid Toxicity Research
Endocrine Disruptors
Thyroid hormone is important for growth and development, neurodevelopment, metabolism
To understand thyroid toxicity need to look at it in the context of the whole Hypothalamus-Pituitary-Thyroid Axis (HPT)
WHY Investigate thyroid toxicity ?

3. Thyroid Hormone Regulation
Pituitary
Thyrotropes
TRH
(CRH)
Hypothalamus
Thyroid Gland
Transthyretin
(-) T4
Iodine
TPO
DIT
TSH
Peripheral Tissue
T TR/RXR DNA mRNA
Liver T3
Deiodination
(D2)
Deiodination (D3)
Conjugation
Inactivation/
Elimination
Deiodination (D2)
NIS
MIT
Thyroglobulin
colloid
Follicular cells
To begin to study thyroid toxicity we need to understand how thyroid hormone is regulated - A Systems Approach
This figure shows a compartmental view of the thyroid hormone system and the process involved in maintaining thyroid hormone homeostasis.
Thyroid hormone is controlled by thyroid stimulating hormone from the pituitary which stimulates the production and release of T4. As the amount of T4 released increases, this provides a negative feedback regulation on the pituitary to decrease TSH release, thereby maintaining circulating T4 levels within normal physiological ranges

4. Thyroid-axis Systems Model
QSAR and
in vitro Models
Thyroid Follicular Cell
Systems Model
Organismal Outcomes
Thyroid Gland
Hypertrophy
Retarded
Development
Control
Treated
Hypothalamus
TRH
(CRH)
(-)
Pituitary
TSH
Thyroid Gland
Thyroglobulin
TPO
MIT
DIT
We can look at the regulation of thyroid hormone as a part of a systems model.
Perturbations within this system will cause effects at the whole organism level that can be readily measured including histological changes in the thyroid gland and arrested development.
To make the linkages between the chemical and the outcome we need to look at the effect of the chemical at hose points in the system that determine the ultimate outcome wihich is circulating T4. Efforts put into understanding the cellular and subcellular components and thier role in T4 homeostasis allows us to understand how perturbation of specific nodes within this axis by xenobitics can alter normal T4 control and metamorphosis.
Iodine T4
DIT
Transthyretin
Inactive TH
Deiodination
Deiodination
Deiodination
Inactive TH
T3+TR/RXR
DNA
mRNA
Conjugation
Liver
Peripheral Tissues

5. Why an amphibian model ?
Metamorphosis is controlled by thyroid hormone
Simple apical endpoint to monitor disruption in vivo
Molecular events are well characterized
Easy to raise and test in the laboratory
Xenopus laevis

6. Xenopus Metamorphosis
Prometamorphosis
Climax
As I mentioned earlier, Xenopus was selected as a model organism to screen chemicals for thyroid disruption because of specific attributes of this species. The most important of these is that metamorphosis, a thyroid hormone dependent event, provides an opportunity to measure apical effects of thyroid disruption.
This figure shows the relationship of TH, expressed as T4 and T3, to the morphological changes that occur in metamorphosis.
The bottom panel shows the plasma concentrations of T4 and T3 throughout metamorphosis, which is nominally a 40 day process.
During that interval, the larvae are completely remodeled (as shown in the top panel) into the juvenile tetrapod.
The middle panel shows two important processes of metamorphosis, de novo development of the limbs and resorption of tail tissue relative to endogenous TH levels.
Based on this remarkable biological process, it was proposed that chemicals which interfere with the normal rise in TH would affect these morphological indicators of development.

7. MED Thyroid Project Objectives
Conduct studies with known HPT disruptors
Inhibitors of thyroid hormone synthesis
Thyroid Peroxidase: Methimazole, Propylthiouracil
Sodium Iodide Symporter: Perchlorate
Develop diagnostic measures
What are the appropriate tissue level endpoints?
Histology, T4, TSH
Can gene and protein expression be used as indicators of thyroid axis disruption?
Develop assays to enable ranking and prioritization of chemicals

8. Effect of Methimazole on Development and Thyroid Histology
Proportion in stage
50 mg/L
25 mg/L
12.5 mg/L
Control
Developmental
Stage
14 d Exposure 55 56 57 58 59 60 *
day 8

9. Summary of Metamorphosis Assay
X. laevis is sensitive to model thyroid pathway modulators
Methimazole, 6-PTU, Perchlorate
Early stage tadpoles (stg 51-54) can be arrested in development by T4 synthesis inhibitors, stage 60 is not
Thyroid histology is an essential component of assay
More sensitive than developmental rate (d8)
Diagnostic

10. Diagnostic Research Approach
Link Chemical-Biomolecular Interaction to Organism Response
Examine gene expression during normal metamorphosis and following chemical exposure
Examine protein changes
Circulating T4 and TSH
Responses of tissues isolated from compensatory mechanisms
Pituitary explant culture: TSH – T4 feedback
Thyroid explant culture: TSH stimulation, chemical inhibition of T4 release
Develop computational – predictive approaches

11. In vivo Pituitary Gene Expression: Thyroid Stimulating Hormone
Developmental Expression
Chemical Exposure

12. In Vivo Thyroid Gland Gene Expression Sodium/Iodide Symporter
Developmental Expression
Chemical Exposure

13. Pituitary Explant Culture
Objective:
Characterize function of the pituitary during development and the relationship between T4 and TSH
Method:
Culture pituitaries from tadpoles at multiple stages of development
Measure TSH expression in the pituitaries
Gene expression or T4 release in thyroid glands treated with media conditioned by pituitary culture

14. Pituitary Explant Culture
TSH mRNA is repressed by T4 *
Negative feedback mechanism is functional throughout development although the setpoint changes
sensitivity to T4 decreases

15. Thyroid Gland Explant Culture
Objective:
Define thyroid-specific outputs in response to TSH and xenobiotics in the absence of whole organism compensatory response
Method:
Culture thyroid glands from prometamorphic tadpoles and treat with TSH and T4 synthesis inhibitors
Measure T4 release and gene expression

16. Thyroid Gland Explant Culture: Time relationship of T4 release inhibition
1000 ng TSH/ml
1000 ng TSH/ml + MM1
2000 ng TSH/ml
2000 ng TSH/ml + MM1 

17. Pituitary Explant Culture
Feedback mechanisms in the pituitary
Negative feedback by T4 on the pituitary is present in metamorphosis
Sensitivity of the pituitary to this inhibition decreases over time
- in early metamorphosis prevent excess T4
- allow more T4 later to complete metamorphosis

18. Thyroid Explant Culture Interpretation of compensatory and direct effects
In vitro…
Release T4 in response to TSH is dose related
T4 reserves must be depleted before synthesis inhibition significantly affects T4 release
In vivo…
Early stages are more sensitive to arrested metamorphosis by T4 inhibitors than late stages
At late prometamorphosis, thyroid glands are larger and reserve T4 is sufficient to complete metamorphosis
Exposure time 0 does not equal effect time 0 for circulating T4
Need to measure circulating hormone levels to interpret gene expression and protein responses in vivo

19. Potential Endpoints for HPT-Axis QSAR Development
Hypothalamus
TRH/CRH
Tyrosine Iodination
and Hormone Production
Pituitary
TSH
T4 (-)
Thyroid Gland
Iodine Uptake
MIT
I + Tyr
DIT
NIS
TPO
Iodine
DIT
DIT
Receptor and Protein
Binding T4
Metabolizing Enzyme
Induction / Activity T4
Liver T4
Peripheral Tissue
metabolism/
conjugation
Deiodination
TH-gluc
T3 + TR  T3-TR:RXR  DNA  mRNA
elimination

20. HPT-Axis QSAR Development
Comparison of Endpoints of T4 Synthesis Inhibition
NIS activity
Membrane protein transports iodine into the follicular cell
Limited data on chemical inhibitors of NIS - mostly monovalent anions of similar size as iodide
Lack of data makes it difficult to make informed chemical selection
Difficult assay to transform to high throughput format
TPO activity
TPO iodinates tyrosine and couples iodo-tyrosines to produce thyroid hormone
TPO inhibition data available for more chemicals & classes of chemicals
Methimazole – PTU
Flavonoids
Resorcinols
More data aids chemical selection process and QSAR model development
Spectrophotometric determination of iodination of tyrosine to MIT
Potential for conversion to high throughput assay

21. HPT-Axis QSAR Development
TPO Inhibitors
Methimazole
Plant Flavonoids
flavone
myricetin
Resorcinol & Derivatives
recorcinol
Propylthiouracil

22. Thyroid Peroxidase Inhibition Literature Data
PTU
PTU
MM1

23. HPT-Axis QSAR Development
Develop Xenopus-based in vitro assay to begin to test known inhibitors of TPO activity
Expand the range of chemicals and classes
Select from EPA Chemical Lists
Predictive Linkages
in vitro → ex vivo (explant culture) → in vivo

24. Systems Approach to Predicting Thyroid Toxicity
Molecular
Effects
Biological Responses
Tissue Organism
Chemical
Gene Expression
Enzyme Activities
TPO
UDPGT
Protein Binding TR
Transthyretin
Serum Albumin
Regulatory Pathways
T4 synthesis and release
Feedback mechanisms
Adverse Effect
&
Compensatory
Response
QSAR
Ranking & Prioritization
of Chemicals
EPA Chemical
Lists
Selection for
Screening

25. MED Thyroid Project Team
S. Degitz M. Hornung
J. Tietge K. Thoemke
J. Nichols J. Chowdhury
G. Holcombe J. Serrano
P. Kosian H. Kerr
D. Hammermeister L. Korte
J. Korte M. Bugge
S. Batterman J. Olson
B. Butterworth J. Haselman