Delineating Thyroid-Mediated Toxicity Pathways in an Amphibian Model System Michael W. Hornung, Sigmund J. Degitz, Joseph E. Tietge US Environmental Protection

Delineating Thyroid-Mediated Toxicity Delineating Thyroid-Mediated Toxicity Pathways in an Amphibian Model SystemPathways in an Amphibian Model System

Michael W. Hornung, Sigmund J. Degitz, Joseph E. TietgeMichael W. Hornung, Sigmund J. Degitz, Joseph E. Tietge

US Environmental Protection AgencyOffice of Research and Development

National Health and Environmental Effects Research LaboratoryMid-Continent Ecology Division

Duluth, MN, USA

McKim Conference on Predictive ToxicologySeptember 16-18, 2008

Duluth, MN

Outline

Brief background on MED thyroid research Approach to adverse outcome pathway research for

amphibian-based thyroid model Investigating endpoints at steps in the pathway Understanding toxicity in the context of pathway

information

Background on EDC AssaysBackground on EDC Assays

FQPA and SDWA directed U.S. EPA to evaluate chemicals for endocrine disruption

Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) established

Screening and testing methods recommended by EDSTAC included:• Short-term fish reproduction assay to detect HPG effects• Short-term amphibian metamorphosis assay to detect HPT

effects• Longer-term fish life-cycle assay for reproductive effects• Longer-term amphibian reproductive/developmental effects

Test OverviewTest Overview

Initiated with tadpoles at onset of thyroid function

14-21 days exposure Apical Endpoints

• Developmental stage• Thyroid histology

0

100

200

300

400

500

600

700

Con

cen

trat

ion

(n

g/d

L)

51 54 60 66

Prometamorphosis Climax

Developmental StageThyroid function

T4T3

Amphibian Metamorphosis AssayAmphibian Metamorphosis Assay

Perchlorate

0

5 0

1 0 0

0

5 0

1 0 0

55 56 57 58 59 60 61 62

Methimazole

0

5 0

1 0 0

0

5 0

1 0 0

55 56 57 58 59 60

In Vivo Gene Expression Following Exposure

NIS Gene Response

Day 0.5 Day 1 Day 1.5 Day 2 Day 4 Day 6

Cop

ies

NIS

/Cop

ies

AC

T

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Control Methimazole Perchlorate Propylthiouracil

Exposure ----------------------------------------> response

0

The Need for Predictive ToolsThe Need for Predictive Tools

Apical endpoints established with the amphibian metamorphosis assay.

However we can not test all the chemicals for which little is known about thyroid activity

Need to develop tools to aid in selecting chemicals that need to be tested and inform which chemicals are likely not active and do not need to be tested or can be assigned low priority for testing

Potential Endpoints for Thyroid Hormone DisruptionPotential Endpoints for Thyroid Hormone Disruption

TRH/CRH

Thyroid Gland

T4

IodineTPO

DIT

MIT

DIT

DIT

TSH

Peripheral Tissue

T3 + TR T3-TR:RXR DNA mRNA

T4

Deiodination

NIS

Receptor and Protein Binding

T4 (-)

Iodine UptakeSodium-Iodide Symporter

Tyrosine Iodinationand Hormone Production

Thyroid Peroxidase

Hypothalamus

Pituitary

LiverT4

elimination

metabolism/conjugation

TH-gluc

Metabolizing Enzyme Induction / Activity

I + Tyr

Adverse Outcome Pathways Adverse Outcome Pathways for Thyroid Disruptionfor Thyroid Disruption

Perchlorate

Methimazole

NIS Inhibition

TPO Enzyme Inhibition

ThyroidFollicular Cells T4 Synthesis

Serum T4 Systemic T4 Insufficiency

ArrestedAmphibian

Metamorphosis

ChemicalChemicalSubcellularSubcellular

TargetTargetCells Cells

EffectedEffectedAdverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ



ArrestedAmphibian

Metamorphosis

Toxicity PathwayToxicity Pathway

Adverse Outcomes PathwayAdverse Outcomes Pathway

Amphibian Metamorphosis Assay

Selection of a PathwaySelection of a Pathway

Methimazole TPO Enzyme Inhibition

ThyroidFollicular Cells

T4 Synthesis





ArrestedAmphibian

Metamorphosis



Serum T4Increased

Serum TSH? Thyroid Tumor ? Goiter

TPO-inhibition has potentially more types of chemicalsthat can affect it than NIS. Assays available.

Single chemical with potential for multipleadverse outcome pathways related to thyroid

Thyroid Explant Cultures and In Vitro Assays to Understand Responses Outside of the

Compensatory Mechanisms of the Whole Animal

Ex Vivo:

Thyroid Gland Explant CulturesDissect thyroid glands from pro-metamorphic tadpoles• Culture in 96-well plates in L-15 media • Stimulate with TSH and treat with graded concentrations of test chemical• Measure T4 released to media by RIA• Measure TSH responsive gene expression by QPCR.

TSH

Chemical XT4

Paired thyroid glands in NF stage 59 X. laevis tadpole

Thyroid Gland Explant CulturesThyroid Gland Explant Cultures

0.5 mm

Adverse Outcomes Pathway for Thyroid ToxicityAdverse Outcomes Pathway for Thyroid Toxicity



T4 Synthesis

Serum T4Systemic T4 Insufficiency



EffectedEffected

Adverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ

Thyroid Explant Culture Assay



T4 Synthesis

Inhibition of T4 releasedfrom glands

ArrestedAmphibian

Metamorphosis


Adverse Outcomes PathwayAdverse Outcomes PathwayVerification of chemical activity

at organ level

T4

TSH

X

Day

1 2 3 4 5 6 8 10 12

T4

Rel

ease

d (

ng

T4

/ mm

3 g

lan

d /

24h

)

0

100

200

300

400

1000 ng TSH/ml

1000 ng TSH/ml + MM1

2000 ng TSH/ml

2000 ng TSH/ml + MM1

Thyroid Gland Explant CultureThyroid Gland Explant Culture: Time & dose relationship of T4 release inhibition

Gene Expression in Thyroid Gland Gene Expression in Thyroid Gland Explant CulturesExplant Cultures

Gene Expression in Response to TSHTTF, NIS, and TPO

TTF NIS TPO

Co

pie

s /

ng

RN

A

0

5000

10000

15000

20000

25000

30000

35000

Fresh 0h Control 24h No TSH 24h TSH

Methimazole, PTU, and Perchlorate Treatment

Freshly Dissected

No Inhibitor

Methimazole

PTU

Perchlorate

NIS TPO

Fresh0h

(-) TSH24h

(-) TSH24h

(+) TSHFresh

0h(-) TSH

24h(-) TSH

24h(+) TSH

Cop

ies

of N

IS p

er R

PL32

0.00

0.02

0.04

0.06

0.08

0.10

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Thyroid Gene Expression: Response to InhibitorsThyroid Gene Expression: Response to Inhibitors

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 UNDERSTAND DOSIMETRY / PK & PD

• Need to measure circulating hormone levels (T4, TSH) to interpret gene expression and protein responses in vivo

Thyroid Explant CultureThyroid Explant CultureInterpretation of compensatory and direct effects

Inhibition of TSH-Stimulated T4 Release Inhibition of TSH-Stimulated T4 Release by Cultured Thyroid Glandsby Cultured Thyroid Glands

Perchlorate – Iodine uptake inhibitionPTU & Methimazole – TPO inhibition

Understand results in the context of the assay

Thyroid glands from X. laevis tadpoles (NF stage 59) were cultured in L-15 media in the

presence of 2000 ng TSH/ml alone or TSH and graded concentrations of chemical.

Media was collected and analyzed by RIA for T4.

Methimazole

Concentration (µM)

1 10 1000

25

50

75

100

125

150

175

200IC50 = 1 µM IC50 = 7 µM IC50 = 11 µM

Propylthiouracil

Concentration (µM)

1 10 1000

25

50

75

100

125

150

175

200

Perchlorate

Concentration (µM)

0.001 0.1 1 10 100

T4

Rel

ease

d

(ng

T4/

mm

3 gla

nd

/ 48

h)

0

25

50

75

100

125

150

175

200

Assess Pathway for Thyroid Disruption Assess Pathway for Thyroid Disruption vs Thyroid Follicular Cell Toxicityvs Thyroid Follicular Cell Toxicity



EffectedEffected


Thyroid Explant Culture Assay

Chemical TPO Enzyme Inhibition


T4 Synthesis

Inhibition of T4 releasedfrom glands

T4

TSH

X

Chemical ? ? ?mitochondria


MTT Assayfor mito function

Cell Death

Thyroid Disrupting Chemical

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

20

40

60

80

100

MTT

T4 Release

Concentration (log M)

Res

po

nse

(%

of

Co

ntr

ol)

Thyroid Toxic Chemical

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

20

40

60

80

100

MTT

T4 Release


Res

po

nse

(%

of

Co

ntr

ol)

Chemical Testing Chemical Testing and Predictive Model Developmentand Predictive Model Development

In Vitro TPO Inhibition

ChemicalSelection

EPA Chemical Lists

Model T4 Synthesis InhibitorsQSAR

Ex Vivo T4 Release Inhibition

Literature – Thyroid Toxicity

Chemical Structure Similarities

In Vivo Amphibian

Metamorphosis Assay

Adverse Outcomes Pathway for Thyroid ToxicityAdverse Outcomes Pathway for Thyroid Toxicity



T4 Synthesis

Serum T4 Serum TSH

Systemic T4 Insufficiency

ChemicalChemical SubcellularSubcellularTargetTarget

Cells Cells EffectedEffected


In Vitro TPO Inhibition AssayQSAR = Chemical + Cellular Target


ArrestedAmphibian

Metamorphosis


Thyroid Peroxidase InhibitionThyroid Peroxidase Inhibition

In Vitro:

Prepare microsomes containing thyroid peroxidase activity from pig thyroid glands

Test chemicals for potency for inhibiting two TPO-mediated reactions

1. Tyrosine Iodination: Conversion of Tyrosine to MIT/DIT

2. Guaiacol Oxidation: Surrogate coupling reaction (DIT + DIT = T4)

Guaiacol Oxidation Assay

OH

OCH3

OO

OO

CH3CH3

TPO

H2O2

Abs (470 nm)

Relative Potency of Model T4 Synthesis Relative Potency of Model T4 Synthesis Inhibitors for TPO InhibitionInhibitors for TPO Inhibition


-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0

Ch

ang

e in

ab

s / m

in /

mg

pro

tein

0

1

2

3

4

5

6

Methimazole is the most potent of the model T4 synthesis inhibitors in the TPO inhibition assay. Perchlorate had very low potency for TPO inhibition, but was the most potent of the three inhibitors in the ex vivo and in vivo assays. The primary mechanism of action for perchlorate inhibition of T4 synthesis is by inhibiting iodide uptake into the follicular cells.

IC50 (µM)Methimazole 2.7 PTU 8.8 Perchlorate 13,400

TPO Inhibition: Alkylphenol Series


-7 -6 -5 -4 -3 -2

Ch

ang

e in

ab

s/m

in/m

g p

rote

in

0

1

2

3

4

5

6

Phenol MethylphenolEthylphenoln-Propylphenol n-Butylphenol Methimazole

0

All alkylphenols tested exhibited no inhibition of TPO activity

Screening Chemicals for Higher Tier Testing

24 chemicals tested in vitro in TPO Inhibition Assay

7 Inhibit TPO In Vitro

NH

S

SH

NH

N

SH

CH3

N

N

CH3

CH3

OH

Positive:Inhibits T4 Release

Model TPO Inhibitors

NH

NH

S

O

CH3

Thyroid Gland Explant Culture: T4 Release

Cl

O

O

O

O-

Na+

O

OH

CH3

NH2

I

I

I

NH

S

SH

Cytotoxic / Negative

N

NH

NClCl

O-

O

O

N

N

CH3

CH3

OH

N

NH

NClCl

O-

O

O

17 Did Not Inhibit TPO

• Alkylphenol series• Phthalates• Isothiazoline• Mixed Iodo Phenyl• OH-PCDE• Triazole• Triazines• Conazole

Test In VivoAmphibian

Metamorphosis Assay

Active Inactive

TPO Assay SummaryTPO Assay Summary

The TPO assay can be used to rapidly screen chemicals for further

testing in the higher level thyroid toxicity assays, and can be used to

begin to develop predictive models incorporating structure activity

relationships between chemical structure and T4 synthesis inhibition

This suite of assays can be an effective tool to determine the capacity

of previously untested or unsuspected classes of chemicals to disrupt

normal thyroid hormone production

Adverse Outcomes Pathways Adverse Outcomes Pathways for Thyroid Disruptionfor Thyroid Disruption


Adverse Outcomes PathwayAdverse Outcomes Pathway



T4 Synthesis


ArrestedAmphibian

Metamorphosis




In Vitro - QSAR

Ex Vivo Gland/Tissue Culture

Amphibian Metamorphosis Assay

Mike HornungSig DegitzJoe TietgeJohn NicholsJose SerranoJoe KorteGary HolcombePat KosianDean HammermeisterJon HaselmanBrian ButterworthSherri Batterman

Post Docs Kara Thoemke Jasim Chowdhury Robin Sternberg

Student Contractors Hollie Kerr Megan Bugge Lisa Korte Jessica Olson Emily Burgess Scott Moen

MED Thyroid Project TeamMED Thyroid Project Team

Documents

Delineating Thyroid-Mediated Toxicity Pathways in an Amphibian Model System Michael W. Hornung, Sigmund J. Degitz, Joseph E. Tietge US Environmental Protection