kevin m. crofton, phd us environmental protection agency mckim conference duluth mn september 17,...
TRANSCRIPT
Kevin M. Crofton, PhD US Environmental Protection Agency
McKim Conference Duluth MN
September 17, 2008
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
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
Triiodothyronine (T3)
Thyroxin (T4) OH
O
I
I
I
HO CH2
NH2 O
CH C
I
OH
O
I
I
I
HO CH2
NH2 O
CH C
I
OH
O
I
I
I
HO CH2
NH2 O
CH C
I
OH
O
I
I
I
HO CH2
NH2 O
CH C OH
O
I
I
I
HO CH2
NH2 O
CH C OH
O
I
I
I
HO CH2
NH2 O
CH C
Catabolic Enzymes
Hypothalamus
Pit
TRH
Thyroid
TSH
Blood
T3/T4
Target Tissues
Liver
Elimination from the body
+
TH binding proteins
TH binding proteins
5’-deiodinases
T4 T3
Acts as a ligand for nuclear thyroid hormone receptors (TRs)
Regulation of Thyroid Hormones
TR
TR
Cellular Acton of TH
TR XCorepressors
TR
Co Activator AAAAA
Transporter
DI
T3
T3T4
Zoeller, 2003
ThyroperoxidaseIodine Symporter
Exposure
Hepatic UDPGTs
Thyroidal
Extra-Thyroidal
Deiodinases
Thyroid Receptors
T4–TTR Binding
Targets
Cellular Transporters
EarlyBiological
Effect
TissueSpecificEffect
Altered Structure/Function
ClinicalDisease
SerumT3 & T4
Changes
TSH
TissueT3 Changes
AlteredDevelopment
ThyroidHyperplasia
ThyroidTumors
BirthDefects
EffectsCancer & Non-Cancer
Thyroid MOAs
ThyroperoxidaseIodine Symporter
Exposure
Hepatic UDPGTs
Thyroidal
Extra-Thyroidal
Deiodinases
Thyroid Receptors
T4–TTR Binding
Targets
Cellular Transporters
EarlyBiological
Effect
TissueSpecificEffect
Altered Structure/Function
ClinicalDisease
SerumT3 & T4
Changes
TSH
TissueT3 Changes
AlteredDevelopment
ThyroidHyperplasia
ThyroidTumors
BirthDefects
EffectsCancer & Non-Cancer
Toxicity Pathways
Adverse Outcome Pathways
What do we know and not know about these pathways?
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 WindowDioxins, Furans and PCBs - Hearing Loss
Exposure Hepatic Phase II Enzymes
Hepatic Parent or Metabolite
SerumT4 & T3
TissueT3
Alter TR Mediated Proteins
Loss of cochlear hair cells
HearingLoss
Binding to PXR
Binding to AhR
UGTs
T4serumT3serum
DItissue
T3tissue
TRactivation
CNS Protein (1)
CNS Protein (2)
CNS malformation
Functional Loss (eg. IQ)
Increasing Dose and/or Time
Res
pon
se
PXR Binding
PHAHs and Ototoxicity
BirthConception Weaning
Th
yroid
Ho
rmo
ne
Fe
tal/P
os
tnat
al P
CB
Ex
po
su
re
Cochlear Development
Critical-Period Model for Chemical-Induced Postnatal Hypothyroxinemia and Ototoxicity
High -- Frequency -- Low
Low-frequencyHearing loss
BirthConception Weaning
Th
yroid
Ho
rmo
ne
Fe
tal/P
os
tnat
al P
CB
Ex
po
su
re
Cochlear Development
Critical-Period Model for Chemical-Induced Postnatal Hypothyroxinemia and Ototoxicity
High -- Frequency -- Low
Low-frequencyHearing loss
Log Thyroxine Concentration, % Control
3103060100
Hea
ring
Loss
, dB
SP
L (
diffe
ren
ce fr
om
con
trol
)
0
10
20
30
40
50
60
y = -33.68*logX + 68.72 r ² = 0.8519
Log Thyroxine Concentration, % Control
10.030.060.0100.0
He
ari
ng
Lo
ss
, d
B S
PL
(d
iffe
ren
ce
fro
m c
on
tro
l)
0
10
20
30
40
Dose-Response Perchlorate, Propylthiouracil and Hippocampal
Physiology
Exposure SerumT4 & T3Thyroid
Perchlorate
HippocampalT3
Alter TR Mediated Proteins
Synaptic Malformation
LearningImpariment
Binding to TPO
Inhibition Of TPO
ThyroidPTU
Altered SynapticFunction
Dam T4 vs BL EPSP Max - % Control
Dam T4 Percent of Control0 10 20 30 40 50 60 70 80 90 100 110
BL
EPS
P M
ax %
Con
trol
30
40
50
60
70
80
90
100
110
120
M0805 PTUM0703 PERCM0102 PTU
r2=0.77
Normalized Spike vs EPSP
Normalized EPSP
0 10 20 30 40 50 60 70 80 90 100
Nor
mal
ized
Pop
ulat
ion
Spik
e
0
10
20
30
40
50
60
70
80
90
100
0 ppm3 ppm10 ppm
Hippocampal physiology
Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cue
Lat
ency
in S
econ
ds
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
0ppm (n=11)3ppm (n=15)10ppm (n=10)*
Water maze learning
Dose-Response Perchlorate, Propylthiouracil and Hippocampal
Physiology
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
GeneBLAzer TR-UAS-bla HEK293 Cell Line
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.
LigandLigand
T3 Stimulation of TR
T3 (M)
**
****
**
****
**** Substrate
Courtesy of Keith Houck, NCCT
In Vitro Models – Thyroid Receptor Beta
Human TR Reporter Gene Assay Heat Map of AC50’s
1456 chemicals; 14 concentrations; agonist and antagonist modes
Levothyroxine
T3
ACTIVES 62
SELECTIVE 0
ACTIVES 65
SELECTIVE 2
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