Transcript Document

Delineating Thyroid-Mediated Toxicity
Pathways in an Amphibian Model System
Michael W. Hornung, Sigmund J. Degitz, Joseph E. Tietge
US Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Mid-Continent Ecology Division
Duluth, MN, USA
McKim Conference on Predictive Toxicology
September 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 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 Overview
Prometamorphosis
Climax
700
Concentration (ng/dL)
 Initiated with tadpoles at
onset of thyroid function
 14-21 days exposure
 Apical Endpoints
• Developmental stage
• Thyroid histology
T4
T3
600
500
400
300
200
100
0
51
54
Thyroid function
60
Developmental Stage
66
Amphibian Metamorphosis Assay
Methimazole
Perchlorate
100
100
50
50
0
0
100
100
50
50
0
0
55
56
57
58
59
60
55 56
57 58 59
60 61
62
In Vivo Gene Expression Following Exposure
NIS Gene Response
0.18
Copies NIS/Copies ACT
0.16
Control
Methimazole
Perchlorate
Propylthiouracil
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
Day 0.5
Day 1
Day 1.5
Day 2
Day 4
Day 6
Exposure ----------------------------------------> response
The 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 Disruption
Hypothalamus
TRH/CRH
Tyrosine Iodination
and Hormone Production
Pituitary
Thyroid Peroxidase
TSH
T4 (-)
Thyroid Gland
Iodine Uptake
Sodium-Iodide Symporter
MIT
I + Tyr
NIS
Iodine
TPO
DIT
DIT
DIT
T4
Metabolizing Enzyme
Induction / Activity
T4
Liver
metabolism/
conjugation
TH-gluc
elimination
T4
Receptor and Protein
Binding
Peripheral Tissue
Deiodination
T3 + TR  T3-TR:RXR  DNA  mRNA
Adverse Outcome Pathways
for Thyroid Disruption
Subcellular
Target
Cells
Effected
Methimazole
TPO Enzyme
Inhibition
Thyroid
Follicular Cells
 T4 Synthesis
Perchlorate
NIS
Inhibition
Thyroid
Follicular Cells
 T4 Synthesis
Chemical
Tissue/Organ
Adverse
Outcome
 Serum T4
Systemic T4
Insufficiency
Arrested
Amphibian
Metamorphosis
 Serum T4
Systemic T4
Insufficiency
Arrested
Amphibian
Metamorphosis
Toxicity Pathway
Adverse Outcomes Pathway
Amphibian Metamorphosis Assay
Selection of a Pathway
Chemical
Methimazole
Methimazole
Subcellular
Target
TPO Enzyme
Inhibition
TPO Enzyme
Inhibition
Cells
Effected
Thyroid
Follicular Cells
Adverse
Outcome
Tissue/Organ
 Serum T4
Arrested
Amphibian
Metamorphosis
Systemic T4
Insufficiency
 T4 Synthesis
Thyroid
Follicular Cells
 T4 Synthesis
 Serum T4
Increased
Serum TSH
TPO-inhibition has potentially more types of chemicals
that can affect it than NIS. Assays available.
Single chemical with potential for multiple
adverse outcome pathways related to thyroid
Goiter
? Thyroid Tumor ?
Thyroid Explant Cultures and In Vitro Assays
to Understand Responses Outside of the
Compensatory Mechanisms of the Whole Animal
Thyroid Gland Explant Cultures
Ex Vivo:
Thyroid Gland Explant Cultures
Dissect 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 X
T4
0.5 mm
Paired thyroid glands in
NF stage 59 X. laevis tadpole
Adverse Outcomes Pathway for Thyroid Toxicity
Chemical
Methimazole
Subcellular
Target
Cells
Effected
Thyroid
Follicular Cells
TPO Enzyme
Inhibition
Adverse
Outcome
Tissue/Organ
 Serum T4
Systemic T4
Insufficiency
 T4 Synthesis
Arrested
Amphibian
Metamorphosis
Toxicity Pathway
Adverse Outcomes Pathway
Methimazole
TPO Enzyme
Inhibition
Thyroid
Follicular Cells
 T4 Synthesis
Inhibition of
T4 released
from glands
Verification of chemical activity
at organ level
TSH
T4
Thyroid Explant Culture Assay
X
Thyroid Gland Explant Culture:
Time & dose relationship of T4 release inhibition
T4 Released (ng T4 / mm3 gland / 24h)
400
1000 ng TSH/ml
1000 ng TSH/ml + MM1
2000 ng TSH/ml
2000 ng TSH/ml + MM1
300
200
100










0
1
2
3
4
5
Day
6
8
10
12

Gene Expression in Thyroid Gland
Explant Cultures
Gene Expression in Response to TSH
TTF, NIS, and TPO
35000
Copies / ng RNA
30000
25000
Fresh
0h Control
24h No TSH
24h TSH
20000
15000
10000
5000
0
TTF
NIS
TPO
Thyroid Gene Expression: Response to Inhibitors
Methimazole, PTU, and Perchlorate Treatment
TPO
NIS
Copies of NIS per RPL32
0.10
1.2
1.0
0.08
0.8
0.06
0.6
0.04
0.4
0.02
0.2
0.00
0.0
Fresh
0h
(-) TSH
24h
(-) TSH
24h
(+) TSH
Freshly Dissected
No Inhibitor
Fresh
0h
(-) TSH
Methimazole
PTU
Perchlorate
24h
(-) TSH
24h
(+) TSH
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
UNDERSTAND DOSIMETRY / PK & PD
•
Need to measure circulating hormone levels (T4, TSH) to interpret gene
expression and protein responses in vivo
Inhibition of TSH-Stimulated T4 Release
by Cultured Thyroid Glands
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.
T4 Released
(ng T4/ mm3 gland / 48h)
200
Methimazole
Propylthiouracil
Perchlorate
IC50 = 1 µM
200
IC50 = 7 µM
200
175
175
175
150
150
150
125
125
125
100
100
100
75
75
75
50
50
50
25
25
25
0
0.001
0
0
0.1
1
10
100
Concentration (µM)
IC50 = 11 µM
1
10
Concentration (µM)
Perchlorate – Iodine uptake inhibition
PTU & Methimazole – TPO inhibition
Understand results in the context of the assay
100
1
10
Concentration (µM)
100
Assess Pathway for Thyroid Disruption
vs Thyroid Follicular Cell Toxicity
Chemical
???
mitochondria
Adverse
Outcome
Tissue/Organ
Thyroid
Follicular Cells
Inhibition of
T4 released
from glands
 T4 Synthesis
Thyroid
Follicular Cells
MTT Assay
for mito function
Cell Death
Thyroid Disrupting Chemical
Thyroid Explant Culture Assay
TSH
T4
X
Thyroid Toxic Chemical
100
100
Response (% of Control)
Chemical
TPO Enzyme
Inhibition
Cells
Effected
Response (% of Control)
Chemical
Subcellular
Target
80
60
40
MTT
T4 Release
20
80
60
40
MTT
T4 Release
20
0
0
-12 -11 -10
-9
-8
-7
-6
Concentration (log M)
-5
-4
-3
-12 -11 -10
-9
-8
-7
-6
Concentration (log M)
-5
-4
-3
Chemical Testing
and Predictive Model Development
Ex Vivo
T4 Release Inhibition
In Vitro
TPO Inhibition
Chemical
Selection
In Vivo
Amphibian
Metamorphosis Assay
Model T4 Synthesis Inhibitors
QSAR
Literature – Thyroid Toxicity
Chemical Structure Similarities
EPA Chemical Lists
Adverse Outcomes Pathway for Thyroid Toxicity
Chemical
Methimazole
Subcellular
Target
TPO Enzyme
Inhibition
Cells
Effected
Thyroid
Follicular Cells
 T4 Synthesis
Toxicity Pathway
Methimazole
TPO Enzyme
Inhibition
In Vitro TPO Inhibition Assay
QSAR = Chemical + Cellular Target
Tissue/Organ
 Serum T4
 Serum TSH
Systemic T4
Insufficiency
Adverse
Outcome
Arrested
Amphibian
Metamorphosis
Thyroid 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
CH3
OH
O
CH3
O
O
TPO
H2O2
H3C
O
O
Abs (470 nm)
Relative Potency of Model T4 Synthesis
Inhibitors for TPO Inhibition
Change in abs / min / mg protein
6
5
Methimazole
PTU
Perchlorate
4
IC50 (µM)
2.7
8.8
13,400
3
2
1
0
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
Concentration (log M)
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.
TPO Inhibition: Alkylphenol Series
Change in abs/min/mg protein
6
5
4
3
Phenol
Methylphenol
Ethylphenol
n-Propylphenol
n-Butylphenol
Methimazole
2
1
0
0
-7
-6
-5
-4
-3
-2
Concentration (log M)
All alkylphenols tested exhibited no inhibition of TPO activity
Screening Chemicals for Higher Tier Testing
24 chemicals tested in vitro in TPO Inhibition Assay
Inactive
Active
7 Inhibit TPO In Vitro
Model TPO Inhibitors
17 Did Not Inhibit TPO
N
H3C
N
H
N
OH
SH
H
N
SH
O
S
H
N
N
CH3
O
NH
S
CH3
-
I
+
Cl
O Na
O
O
H
N
-
I
O
O
H2N
H3C
N
O
I
N
Cl
OH
CH3
Cl
O
Thyroid Gland Explant Culture: T4 Release
Cytotoxic / Negative
O
H
N
-
O
H3C
N
N
N
N
Cl
Positive:
Inhibits T4 Release
OH
H
N
SH
Cl
O
CH3
S
• Alkylphenol series
• Phthalates
• Isothiazoline
• Mixed Iodo Phenyl
• OH-PCDE
• Triazole
• Triazines
• Conazole
Test In Vivo
Amphibian
Metamorphosis
Assay
TPO 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
for Thyroid Disruption
Chemical
Methimazole
Subcellular
Target
TPO Enzyme
Inhibition
Cells
Effected
Thyroid
Follicular Cells
Tissue/Organ
 Serum T4
 T4 Synthesis
Systemic T4
Insufficiency
In Vitro - QSAR
Ex Vivo Gland/Tissue Culture
Toxicity Pathway
Amphibian Metamorphosis Assay
Adverse Outcomes Pathway
Adverse
Outcome
Arrested
Amphibian
Metamorphosis
MED Thyroid Project Team
Mike Hornung
Sig Degitz
Joe Tietge
John Nichols
Jose Serrano
Joe Korte
Gary Holcombe
Pat Kosian
Dean Hammermeister
Jon Haselman
Brian Butterworth
Sherri Batterman
Post Docs
Kara Thoemke
Jasim Chowdhury
Robin Sternberg
Student Contractors
Hollie Kerr
Megan Bugge
Lisa Korte
Jessica Olson
Emily Burgess
Scott Moen