STAR Review WorkshopUS EPA Endocrine Disruptors Program
Thyroid Toxicants: Developmental Outcomes and Chemical MixturesMary Gilbert, PhD
Office of Research and Development MultiYear Plan for Endocrine Disruptors
•Focus Area ADevelopment/standardization of protocols to identify endocrine disrupting chemicals in the environment
•Focus Area BLong-term consequences of developmental exposure to endocrine disrupting chemicals - gonadal steroids and thyroid hormones
•Focus Area CCumulative risk/mixtures of endocrine disrupting chemicals Extrapolation across species
NHEERL and STAR Thyroid ProjectsComplex Mixtures of Thyroid Hormone Disruptors: Mechanisms and Predictive Modeling. Intramural Research in NTD and ETD
Low Dose Thyroid Hormone Insufficiencies and Neurological Outcomes in Rodent Models. Intramural Research in NTD
Endocrine Disrupting Chemicals and Thyroid Outcomes – Exposure-Effect Studies in Humans. STAR Grant to Dr. Henry Anderson, Wisconsin Dept Health and Family Services
Low Dose Effects of Thyroid Toxicants on Neurodevelopment –Mechanistic Studies. STAR COOP between NHEERL and Dr. Thomas Zoeller, U Massachusetts
Development of BBPK Model for the Thyroid Axis in Pregnant Rat and Fetus for Dose Response Analysis of Developmental Neurotoxicity. STAR COOP between NHEERL and Dr. Jeffrey Fisher, U Georgia
Thyroid Hormone Disruption & Neurological Dysfunction
•TH critical for brain development - Iodine Deficiency, Congenital Hypothyroidism
•Treatment CH still leads to subtle cognitive deficits(Rovet, 2000)
•Hypothyroxinemia – pregnant women with low T4, childrenincidence attention deficit disorder
lower global IQ
(Haddow et al., 1999; Allen et al., 2000)
Research Challenges• Interaction in brain development is complex: Thyroid
hormone serves different roles in different cells at different times
• Many structurally diverse chemicals affect thyroid axis by interaction with a variety of targets
• Unclear how much change is adverse. We know little at the low end of the dose-response function
• Unclear which profiles of TH disturbance are predictive of adversity
• Extrapolation from animal models to humans
• Real world exposures are to complex mixtures of thyroid disrupting chemicals
Induced THCatabolism
TH ReceptorActivation
TH Synthesis
Exposure Early BiologicalEffect
TH-Regulated Protein Expression
HearingLoss
Administered Dose
Induced TH Catabolism
Learning Deficits
MODE-OF-ACTION MODEL
SerumTransport
TH Receptor Activation
Thyroid Liver Blood Brain TH
TH Synthesis
Exposure TargetDose
Early BiologicalEffect
Altered Structure/Function
Clinical Disease
TH-Regulated Protein Expression
Synaptic Dysfunction
Hearing Loss
Structural Anomalies Biochemical Imbalances
Induced THCatabolism
TH ReceptorActivation
TH Synthesis
Exposure Early BiologicalEffect
TH-Regulated Protein Expression
HearingLoss
Administered Dose
Induced TH Catabolism
TH-dependent Signaling, Gene Transcription
Learning Deficits
BIOLOGICALLY-BASED DOSE-RESPONSE MODEL
SerumTransport
TH Receptor Activation
Thyroid Liver Blood Brain TH
TH Synthesis
Exposure TargetDose
Early BiologicalEffect
Altered Structure/Function
Clinical Disease
TH-Regulated Protein Expression
Synaptic Dysfunction
Hearing Loss
Structural Anomalies Biochemical Imbalances
Liver
T4-Gluc
Biliary Excretion
Plasma/BloodFree-TH
Bound-TH
T4 TTR/TBG
Hypothalamus
Pituitary
Thyroid Gland
TRH
TSH
+
+T3 & T4
T3 & T4
Ah-ReceptorT4
UDPGTs
CAR/PXR
Iodine
PerchlorateThiocyanate
T4T3
Thyroperoxidase
I + tyrosineT3 & T4
PTUMancozeb
HO-PCBs Dioxins
PCBsDeiodinase
T4 > T3
Thiram
Peripheral tissues
_
_
T4
CNS tissues
Deiodinase
Xenobiotics?
T4 > T3
CNS tissues
Deiodinase
Xenobiotics?
T4 > T3
Target Sites and Early Biological IndicatorsStructurally Diverse chemicals - Multiple Sites & Mechanisms
Induction of Liver Enzymes Reduces T4
What is the most appropriate model for extrapolation to humans?
Glucuronidation of T4 is differentially induced in rats and mice following exposure chemicals
Species ExtrapolationA number of environmental contaminants induce liverglucuronidation of T4. These chemicals display distinctdose-response profiles
Does additivity theory predict the effects of complex mixtures that induce metabolism of T4?
Mixtures
Additivity Model predicted effects on T4 of mixtures of chemicals with common MOA at low doses, but underestimated effects at high doses.
Additivity Model
Empirical ModelT4, %
Con
trol
(mea
n±SD
)
20
40
60
80
100
120
140
Total Mixture Dose (µg/kg/day)
0 300 600 900 1200 1500 1800 2100
Additivity Model
Empirical ModelT4, %
Con
trol
(mea
n±SD
)
20
40
60
80
100
120
140
Total Mixture Dose (µg/kg/day)
0 300 600 900 1200 1500 1800 2100
What of mixtures with different MOA?
How do these effects extrapolate to humans?
Induction of Liver Enzymes Reduces T4
A number of environmental contaminants induce liverglucuronidation of T4. These chemicals display distinctdose-response profiles
Does additivity theory predict the effects of complex mixtures that induce metabolism of T4?
Mixtures
What is the most appropriate model for extrapolation to humans? Develop reporter gene assays in hepatocytes from mouse, rat, human
Glucuronidation of T4 is differentially induced in rats and mice following exposure chemicals
Species Extrapolation
Induced THCatabolism
TH ReceptorActivation
TH Synthesis
Exposure Early BiologicalEffect
TH-Regulated Protein Expression
HearingLoss
Administered Dose
Induced TH Catabolism
Learning Deficits
MODE-OF-ACTION MODEL
SerumTransport
TH Receptor Activation
Thyroid Liver Blood Brain TH
TH Synthesis
Exposure TargetDose
Early BiologicalEffect
Altered Structure/Function
Clinical Disease
TH-Regulated Protein Expression
Synaptic Dysfunction
Hearing Loss
Structural Anomalies Biochemical Imbalances
Induced THCatabolism
TH ReceptorActivation
TH Synthesis
Exposure
TH-Regulated Protein Expression
HearingLoss
Administered Dose
Induced TH Catabolism
TH-dependent Signaling, Gene Transcription
Learning Deficits
BIOLOGICALLY-BASED DOSE-RESPONSE MODEL
SerumTransport
TH Receptor Activation
Thyroid Liver Blood Brain TH
TH Synthesis
Exposure TargetDose
Early BiologicalEffect
Altered Structure/Function
Clinical Disease
TH-Regulated Protein Expression
Synaptic Dysfunction
Hearing Loss
Structural Anomalies Biochemical Imbalances
Low Level Thyroid Disruption and Neurodevelopment
•Subclinical hormone reductions in pregnant women leads to IQ deficits in offspring
•Developing fetus and newborn are populations of concern
•Cognitive function is endpoint of concern
•Evaluation at low levels of hormone disruption is neededT4
Toxicant
Gestational Age in Months Postnatal Age in Months
Critical Factors Impacting Outcome:Magnitude, Timing and Duration of TH Insufficiency
Gestational Age in Months Postnatal Age in Months
Mature Brain
Neuronal MaturationDendritic Arborization, Synapse Formation
Oligodendrocyte DevelopmentProliferation Migration Maturation Myelination
Cerebellar & HippocampalGranule Cell
Proliferation Migration Maturation
0 10 15 20 20 25 305 15105Rat
0 63 6 30Human
Gestational Age in Days Postnatal Age in Days
Neuronal Histogenesis
Neuronal Migration
Neuronal MaturationDendritic Arborization, Synapse Formation
Oligodendrocyte DevelopmentProliferation Migration Maturation Myelination
Cerebellar & HippocampalGranule Cell
Proliferation Migration Maturation
Neuronal MaturationDendritic Arborization, Synapse Formation
Oligodendrocyte DevelopmentProliferation Migration Maturation Myelination
Cerebellar & HippocampalGranule Cell
Proliferation Migration Maturation
Oligodendrocyte DevelopmentProliferation Migration Maturation Myelination
Cerebellar & HippocampalGranule Cell
Proliferation Migration Maturation
0 10 15 20 20 25 305 15105Rat
0 63 6 30Human
Gestational Age in Days Postnatal Age in Days
Neuronal Histogenesis
Neuronal Migration
B
B
Adapted from Anderson et al. (2003) Thyroid, 13:1039-1056
Timing and Duration are Important Elements for Consideration
• Prenatal hypothyroidism – errors in migration?Functional Implications?
• Postnatal PV-Immunohistochemistry – altered neuronal phenotype?
Functional Implications?
• Postnatal Cochlear Development – hair cell lossFunctional Implications?
Prenatal : Cortical Malformations
B
3ppm PTU
10ppm PTU
NEUN
GFAP
* *
CCH
CTX
* *
PN86
TH Replacement
CorticalMalformation
Dose-Dependent Neuronal PersistentTH-Dependent
Postnatal: Parvalbumin Expression0 ppm 3 ppm 10 ppm
Hippo
Cortex
A
E FD
B C
0 ppm 3 ppm 10 ppm
Hippo
Cortex
A
E FD
B C
20% Max
Interpulse Interval (msec)
10 20 30 70 250
Pulse
2/P
ulse
1 X
100
50
100
150
200
250
*
**
*
*
Density PV-IR Neurons in Hippocampus
DG CA1 CA3
Mea
n +/
- SE
Cel
l Den
sity
X 1
0-6
0
2
4
6
8
10
120 ppm (n=5)3 ppm (n=5)10 ppm (n=6)
*
*
*
*
*
*
Postnatal: Cochlear Damage
Postnatal PCB exposure reduces T4, induces hair cell loss in the basal turn of the cochlea
Low frequency hearing loss is evidenced by behavioral measures of acoustic startle reflex
Dose-Response Relationships:Thyroid Hormones and Hearing Loss
Log Thyroxine Concentration, % Control3103060100
Hea
ring
Loss
, dB
SPL
(diff
eren
ce fr
om c
ontro
l)
0
10
20
30
40
50
60 A1254 T4 Rep PropylthiouracilDioxin #1A1254 #2
A1254#1
Dioxin#2PCB 126 A1254 SubchronicDE-71 (PBDE)
A1254 CrossFoster
A1254 T4 Rep A1254 T4 Rep PropylthiouracilPropylthiouracilDioxin #1Dioxin #1A1254 #2A1254 #2
A1254#1A1254#1
Dioxin#2Dioxin#2PCB 126 PCB 126 A1254 SubchronicA1254 SubchronicDE-71 (PBDE)DE-71 (PBDE)
A1254 CrossFosterA1254 CrossFoster
Developmentally-induced reductions in T4 on PN15 are predictive of hearing loss.
Sensitivity of Cognitive Endpoints?
Cellular Models Behavioral Models
Hippocampal Synaptic Transmission is Impaired
Normalized Spike vs EPSP
Normalized EPSP0 10 20 30 40 50 60 70 80 90 100
Nor
mal
ized
Pop
ulat
ion
Spik
e0
10
20
30
40
50
60
70
80
90
100
0 ppm3 ppm10 ppm
Synaptic Plasticity is Impaired
•Long term potentiation, a cellular model of learning and memory, is reduced in a dose-dependent manner
StimulusArtifact
Population Spike
EPSP Slope
*
EPSP Slope High Intensity Series
Minutes PostTrain1 15Pe
rcen
t of P
reTr
ain
EPSP
Slo
pe-20
-10
0
10
20
30
0 ppm3 ppm10 ppm
Hippocampal Learning is Impaired
Trace fear conditioning is impaired in adult offspring.
Cue Test Day Activity Counts 1st Min Post CS Presentation
BL CS
Num
ber o
f Cou
nts
0
20
40
60
80
100
0 ppm 3 ppm10 ppm
Morris water maze acquisition and reversal learning are impaired in adult offspring
Day1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Reversal Cue
Late
ncy
in S
econ
ds
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
750ppm (n=11)3ppm (n=10) deletes 5 nonlearners10ppm (n=6) - litter corrected
15 secTone/light CS
Trace Interval30 sec
US0.5 sec, 1mA
Footshock
Trace fear conditioning is impaired in adult offspring.
Cue Test Day Activity Counts 1st Min Post CS Presentation
BL CS
Num
ber o
f Cou
nts
0
20
40
60
80
100
0 ppm 3 ppm10 ppm
Morris water maze acquisition and reversal learning are impaired in adult offspring
Day1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Reversal Cue
Late
ncy
in S
econ
ds
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
750ppm (n=11)3ppm (n=10) deletes 5 nonlearners10ppm (n=6) - litter corrected
15 secTone/light CS
Trace Interval30 sec
US0.5 sec, 1mA
Footshock
STAR Thyroid ProjectsEndocrine Disrupting Chemicals and Thyroid Outcomes – Exposure-Effect Studies in Humans. STAR Grant to Dr. Henry Anderson, Wisconsin Dept Health and Family Services
Low Dose Effects of Thyroid Toxicants on Neurodevelopment –Mechanistic Studies. STAR COOP between NHEERL and Dr. Thomas Zoeller, U Massachusetts
Development of BBPK Model for the Thyroid Axis in Pregnant Rat and Fetus for Dose Response Analysis of Developmental Neurotoxicity. STAR COOP between NHEERL and Dr. Jeffrey Fisher, U Georgia
BBDRDevelopmentalNeurotoxicity
PBPK
Administered Dose
BBDR Thyroid axis
Describes the kinetics of the toxicant.
Describes the perturbations in the thyroid axis via MOA.
Describe alterations in CNS development due to altered hormones
Exposure-Dose-Response Model
PharmacokineticEffort
Simulate exposure-induced changes in thyroid dosimetrics associated with CNS toxicity.
PharmacodynamicEffort
U Georgia COOP
U Georgia COOP
Target Dose
Serum & Tissue
Hormones
Synthetic & Catabolic Enzymes
mRNAProteins
Structure Function
BBDRDevelopmentalNeurotoxicity
PBPK
Administered Dose
BBDR Thyroid axis
Describes the kinetics of the toxicant.
Describes the perturbations in the thyroid axis via MOA.
Describe alterations in CNS development due to altered hormones
PharmacokineticEffort
Simulate exposure-induced changes in thyroid dosimetrics associated with CNS toxicity.
PharmacodynamicEffort
U Georgia COOP
U Georgia COOP
U Mass COOPNTD
U Georgia COOPU Mass COOPNTD/ETD
Target Dose
Serum & Tissue
Hormones
Synthetic & Catabolic Enzymes
mRNAProteins
Structure Function
Endocrine Disrupting Chemicals and Thyroid Outcomes – Exposure-Effect Studies in Humans.
EPA STAR Grant Dr. Henry Anderson
Wisconsin Dept Health and Family Services
Low Dose Effects of Thyroid Toxicants on Neurodevelopment – Mechanistic Studies.
EPA STAR COOPDr. Thomas ZoellerU Massachusetts
Development of BBPK Model for the Thyroid Axisin Pregnant Rat and Fetus for Dose Response
Analysis of Developmental Neurotoxicity
EPA STAR COOPDr. Jeffrey Fisher
University of Georgia