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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


Synaptic Dysfunction

Hearing Loss

Structural Anomalies Biochemical Imbalances



TH Synthesis



HearingLoss

Administered Dose


TH-dependent Signaling, Gene Transcription

Learning Deficits

BIOLOGICALLY-BASED DOSE-RESPONSE MODEL

SerumTransport



TH Synthesis

Exposure TargetDose



Clinical Disease



Hearing Loss


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



TH Synthesis



HearingLoss

Administered Dose


Learning Deficits

MODE-OF-ACTION MODEL

SerumTransport



TH Synthesis

Exposure TargetDose



Clinical Disease



Hearing Loss




TH Synthesis

Exposure


HearingLoss

Administered Dose


TH-dependent Signaling, Gene Transcription

Learning Deficits

BIOLOGICALLY-BASED DOSE-RESPONSE MODEL

SerumTransport



TH Synthesis

Exposure TargetDose



Clinical Disease



Hearing Loss


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












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

star review workshopus epa endocrine disruptors program5 10 15 20 25 30 35 40 45 50 55 60 65 70 75...

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