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
ThyroidFollicular Cells T4 Synthesis
Serum T4 Systemic T4 Insufficiency
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
Serum T4 Systemic T4 Insufficiency
ChemicalChemicalSubcellularSubcellular
TargetTargetCells Cells
EffectedEffectedAdverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ
ArrestedAmphibian
Metamorphosis
Methimazole TPO Enzyme Inhibition
ThyroidFollicular Cells T4 Synthesis
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
Methimazole TPO Enzyme Inhibition
ThyroidFollicular Cells
T4 Synthesis
Serum T4Systemic T4 Insufficiency
ChemicalChemicalSubcellularSubcellular
TargetTargetCells Cells
EffectedEffected
Adverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ
Thyroid Explant Culture Assay
Methimazole TPO Enzyme Inhibition
ThyroidFollicular Cells
T4 Synthesis
Inhibition of T4 releasedfrom glands
ArrestedAmphibian
Metamorphosis
Toxicity PathwayToxicity Pathway
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
ChemicalChemicalSubcellularSubcellular
TargetTargetCells Cells
EffectedEffected
Adverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ
Thyroid Explant Culture Assay
Chemical TPO Enzyme Inhibition
ThyroidFollicular Cells
T4 Synthesis
Inhibition of T4 releasedfrom glands
T4
TSH
X
Chemical ? ? ?mitochondria
ThyroidFollicular Cells
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
Concentration (log M)
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
Methimazole TPO Enzyme Inhibition
ThyroidFollicular Cells
T4 Synthesis
Serum T4 Serum TSH
Systemic T4 Insufficiency
ChemicalChemical SubcellularSubcellularTargetTarget
Cells Cells EffectedEffected
Adverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ
In Vitro TPO Inhibition AssayQSAR = Chemical + Cellular Target
Methimazole TPO Enzyme Inhibition
ArrestedAmphibian
Metamorphosis
Toxicity PathwayToxicity Pathway
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
Concentration (log M)
-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
Concentration (log M)
-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
Toxicity PathwayToxicity Pathway
Adverse Outcomes PathwayAdverse Outcomes Pathway
Methimazole TPO Enzyme Inhibition
ThyroidFollicular Cells
T4 Synthesis
Serum T4 Systemic T4 Insufficiency
ArrestedAmphibian
Metamorphosis
ChemicalChemicalSubcellularSubcellular
TargetTargetCells Cells
EffectedEffectedAdverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ
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