Download T3 - McKim Conferences

Thyroid Mediated CNS
Dysfunction
How to use what we know about the structure and function of
the thyroid system to generate data using in vitro methods
that can populate QSAR models
Kevin M. Crofton, PhD
US Environmental Protection Agency
McKim Conference
Duluth MN
September 17, 2008
Outline
• Thyroid hormones and homeostatic
mechanisms
• A mode-of-action for thyroid disruption and
adverse outcomes on nervous system
development
• Targets for disruption
• Targets for screening
• Summary
Thyroid Hormones- Structure and Function
• T3 and T4 are the principle hormones synthesized and released
by the thyroid gland
• Development - Critical for differentiation and growth
• Transient disruption = permanent effects
• Adult – Important for energy and thermoregulation
•Transient disruption = transient effects
I
I
Thyroxin (T4)
HO
I
I
Triiodothyronine (T3)
I
I
HO
CH2 CH C O
H
NH2 O
O
O
I
CH2 CH C O
H
NH2 O
Regulation of Thyroid Hormones
TR
Hypothalamus
TRH
TR
Pit
+
Elimination from
the body
Acts as a
ligand for
nuclear
thyroid
hormone
receptors
(TRs)
TSH
Thyroid
Catabolic Enzymes
T3/
Blood
Liver
TH binding
proteins
T4
T4
T3
5’-deiodinases
Target Tissues
Cellular Acton of TH
Corepressors
Co Activator
TR
Transporter
T4
TR
DI
T3
X
AAAAA
T3
Zoeller, 2003
Thyroid MOAs
Targets
Thyroidal
Effects
&
Non-Cancer
Cancer
Early
Biological
Effect
Tissue
Specific
Effect
Altered
Structure/
Function
Clinical
Disease
Thyroperoxidase
Iodine Symporter
Extra-Thyroidal
Hepatic UDPGTs
Exposure
Deiodinases
Cellular Transporters
 T4–TTR Binding
Thyroid Receptors
 TSH
Thyroid
Hyperplasia
Thyroid
Tumors
Tissue
T3 Changes
Altered
Development
Birth
Defects
Serum
T3 & T4
Changes
Adverse Outcome Pathways
Toxicity
Pathways
Targets
Thyroidal
Effects
&
Non-Cancer
Cancer
Early
Biological
Effect
Tissue
Specific
Effect
Altered
Structure/
Function
Clinical
Disease
Thyroperoxidase
Iodine Symporter
Extra-Thyroidal
Hepatic UDPGTs
Exposure
 TSH
Thyroid
Hyperplasia
Thyroid
Tumors
Serum
T3 & T4
Changes
Tissue
Altered
Birth
Deiodinases
What
do we know and not knowT3about
these
pathways?
Changes
Development
Defects
Cellular Transporters
 T4–TTR Binding
Thyroid Receptors
Major Sequelae of Thyroid Disruption
• Adult Exposure

Thyroid tumors in laboratory animals
•
•
Not a relevant mechanism for human cancer
May increase incidence of cardiovascular disease
• Neurodevelopment

Lack of THs result in adverse neurological development
(sensory, motor, cognitive)



Species independent (fact)
Rat is appropriate animal model for neurodevelopmental effects
These are two different outcomes that can result
from the same molecular targets

One is relevant for human health and one is not
Neurodevelopment and
Thyroid Dysfunction
• FACT: without adequate TH the
nervous fails to properly develop
 Iodine deficiency
 Congenital hypothyroidism
Dose-Response and Critical Window
Dioxins, Furans and PCBs - Hearing Loss
Exposure
Hepatic
Parent or
Metabolite
 Serum
T4 & T3
Binding
to PXR
Hepatic
Phase II
Enzymes
Binding
to AhR
 Tissue
T3
Alter TR
Mediated
Proteins
Loss of cochlear
hair cells
Hearing
Loss
Response
DItissue
PXR
Binding
CNS
Protein (1)
UGTs
T4serum
CNS
malformation
T3serum
Functional
Loss (eg. IQ)
T3tissue
TRactivation
CNS
Protein (2)
Increasing Dose and/or Time
Low-frequency
Hearing loss
High -- Frequency -- Low
Cochlear Development
Conception
Birth
Weaning
60
50
40
Hearing Loss, dB SPL (difference from control)
Thyroid Hormone
Fetal/Postnatal PCB Exposure
Critical-Period Model for Chemical-Induced
Postnatal Hypothyroxinemia and Ototoxicity
Hearing Loss, dB SPL (difference from control)
PHAHs and Ototoxicity
40
30
20
10
0
100.0
60.0
30.0
10.0
Log Thyroxine Concentration, % Control
30
20
10
y = -33.68*logX + 68.72
r ² = 0.8519
0
100
60
30
10
3
Log Thyroxine Concentration, % Control
Dose-Response
Perchlorate, Propylthiouracil and Hippocampal
Physiology
Thyroid
PTU
Binding
to TPO
Exposure
Thyroid
Perchlorate
 Hippocampal
T3
Inhibition
Of TPO
Alter TR
Mediated
Proteins
 Serum
T4 & T3
Synaptic
Malformation
Altered
Synaptic
Function
Learning
Impariment
Dose-Response
Perchlorate, Propylthiouracil and Hippocampal
Physiology
Hippocampal physiology
100
Normalized Spike vs EPSP
80
0 ppm
3 ppm
10 ppm
70
60
120
50
Dam T4 vs BL EPSP Max - % Control
110
30
20
10
0
0
10
20
30
40
50
60
70
80
90
Normalized EPSP
Water maze learning
75
0ppm (n=11)
3ppm (n=15)
10ppm (n=10)*
70
65
100
BL EPSP Max % Control
40
100
90
80
70
60
M0805 PTU
M0703 PERC
M0102 PTU
50
40
60
r2=0.77
30
55
Latency in Seconds
Normalized Population Spike
90
0
50
10
20
30
40
50
60
70
80
Dam T4 Percent of Control
45
40
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Day
Cue
90
100 110
What do we not know?
• Dose-response relationships
• Critical windows
• Sensitive biomarkers (T4?)
• However, we can’t get lost in the need to
understand everything in the pathway
 Causative and predictive is minimum
 Quantitative models are the holy grail
What can we do to inform
QSAR models
• Develop in vitro test methods for
known targets
In Vitro Models for Thyroid
Disruptors
•
•
•
•
•
•
•
Iodine Symporter (NIS)
Thyroperoxidase (TPO)
Deiodinases
Transporters – Blood
Transporters – Cellular
Thyroid Receptors
Hepatic Nuclear Receptors
In Vitro Models – Thyroid Receptor Beta
GeneBLAzer TR-UAS-bla HEK293 Cell Line
Ligand
**
**
** Substrate
** **
T3 Stimulation
of TR
**
T3 (M)
**
**
Cell line contains a beta-lactamse reporter gene under the control of an UAS response
element stably integrated in Hek293 cells. This line also stably expresses a fusion protein
consisting of the GAL4 DNA binding domain and the TR ligand binding domain.
Courtesy of Keith Houck, NCCT
Human TR Reporter Gene Assay
Heat Map of AC50’s
1456 chemicals; 14 concentrations; agonist and antagonist modes
T3
Levothyroxine
ACTIVES
SELECTIVE
65
2
ACTIVES
SELECTIVE
62
0
Courtesy of Keith Houck, NCCT
TH Action Assay – T-Screen (Gutleb et al. EnvToxPharm 2005)
- measures TH dependent cell proliferation in GH3 cells
- 96 well plate assay
Summary
• Thyroid pathways are known
• Multiple targets involved
• In vitro models are available for many of the
targets
• Need to begin testing chemicals
Thank You