Long Term Goal 1: Neurodevelopment and Thyroid Homeostasis ...

Long Term Goal 1:Neurodevelopment and Thyroid Homeostasis

Reproductive Function and EDCs

Mary E. GilbertNeurotoxicology DivisionNational Health and Environmental Effects Research LaboratoryOffice of Research and DevelopmentU.S. Environmental Protection Agency

September 14, 2007

2

Linkage and Timeline for APGs to Meet LTG 1: 2004

FY03 FY04 FY05 FY06 FY07

Characterize the effects

of exposure to multiple

EDCs in variouscombinations

includingthose with similar

& different mechanisms

of action

Evaluate at least 3 existing risk management tools to reduce exposure to EDCs

Determine the shape of the dose-response curve in a variety of species exposed to ambient levels of EDCs

Determine degree to which

EDCs with definedmechanisms/modes

of action can beextrapolated

across classes ofvertebrates

LTG 1: Provide a Better Understanding of Science Underlying the Effects, Exposure, Assessment, and Management of Endocrine Disruptors

Determine critical biological factors during development resulting in toxicities later in life

FY08 FY09 FY10 FY11 FY12

Identify key risk assessment issues and develop guidance for assessing endocrine disruptors

Develop at least 2new risk manage-

ment tools

Identify risk management EDCs research

Evaluate exposure methods, measurement protocols, and models for the assessment of risk management efficacy on EDCs

Provide at least 1 computational model for assessing endocrine disruptor compounds

Develop systems models to test &

predict vulnerabilitiesof neuroendocrine

system to contaminant-induced effects

3

2013 - Provide OPPTS, OW, the Regions and other organizations with new exposure assessment and risk management tools to characterize and reduce exposure to EDCs

2013 - Provide OPPTS, OW, the Regions and other organizations with systems and models to test and predict vulnerability of the neuroendocrine system to contaminant-induced effects

2013 - Provide OPPTS, OW, the Regions and other organizations with data from the development and application of high-through put and molecular approaches, including ‘omics , and computational tools for defining mechanisms of action, extrapolation across species and improving assessments for EDCs

2010 - Provide OPPTS, OW, the Regions and other organizations with improved data on the shape of the dose-response curve as a result of exposure to environmentally relevant levels of endocrine disruptors

2009 2010 2011 2012 2013 20152014

LTG 1: Reduction in uncertainty regarding effects, exposure, assessment, and management of EDCs so that EPA has a sound scientific foundation for environmental decision making.

Linkage & Timeline for APGs to Meet LTG 1: Redefined

4

•Low Dose Effects, Developing Appropriate Animal ModelsLow Dose Effects, Developing Appropriate Animal Models

•Evaluation of Mixtures of EDCsEvaluation of Mixtures of EDCs

•Species ExtrapolationSpecies Extrapolation

•Toxicogenomics in Risk AssessmentToxicogenomics in Risk Assessment

•Biomarkers and Screening ToolsBiomarkers and Screening Tools

Examples of Research Addressing LTG1

ORD

Many projects inform issues that cut across APMs

5

Brain Malformation Induced by Prenatal Thyroid Hormone Insufficiency

Goodman and Gilbert, Endocrinology, 2007

Only seen in TH-deficient offspring

Cells are born late gestation ~ mid-pregnancy in humans

Incidence and sizeare dose-dependent

Cells are neuronalphenotype, not injuryresponse

-

CON

PTU

•Low Dose Effects/Animal ModelsLow Dose Effects/Animal Models•Mixtures of EDCsMixtures of EDCs•Species ExtrapolationSpecies Extrapolation•Toxicogenomics in RAToxicogenomics in RA•Biomarkers and ScreeningBiomarkers and Screening

NEUN

GFAP

GD13-16

GD17-19

6

Excitatory Synaptic Transmission is Impaired in Offspring of PTU and Perchlorate-Treated Dams

Gilbert and Sui, 2006Gilbert and Sui, under review

Dose-dependent reductions in T4 induced by PTU or perchlorate impair excitatory synaptic function in hippocampus of adult offspring – an area critical for learning.Correlations between level of hormone reduction and degree of impairment.

B. Baseline Population Spike mV

Stimulus Intensity (A)

100 300 500 700 900 1200 1500

Popu

lati

on S

pike

Am

plit

ude

(mV

)

0

2

4

6

8

10

12

0 ppm 30 ppm300 ppm1000 ppm

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


7

Cognitive Deficits Assessed To Mirror Health Outcomes of Concern in Humans

Subtle changes in IQ function are the most commonly reported Subtle changes in IQ function are the most commonly reported impairments observed in children born to women with thyroid impairments observed in children born to women with thyroid hormone insufficiencies. hormone insufficiencies. Extrapolation to humans from animal Extrapolation to humans from animal models is facilitated if common endpoints are assessed in both models is facilitated if common endpoints are assessed in both species.species.

Dose-dependent impairments detected in behavioral and Dose-dependent impairments detected in behavioral and electrophysiological indicators of “learning” in response to electrophysiological indicators of “learning” in response to graded degrees of thyroid dysfunctiongraded degrees of thyroid dysfunction


8

Low Levels of TH Disruption Impact Myelination

CC OligodendrocytesCC mRNA MAG

(% o

f C

ontr

ol)

***

**

0ppm 1ppm 2ppm 3ppm0

20

40

60

80

100

120 MAG mRNA

Oli

god

end

rocy

te D

ensi

ty

(Cel

ls/m

m2 )

***

***

0ppm 1ppm 2ppm 3ppm0

200

400

600 Oligodendrocyte Number

(Zoeller et al, under review)Cell type critical for myelination is reduced by low doses of PTU


9

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

Pllp

Tspan2

Tspan2

Mbp

Plp

Cldn11

Ugt8

Mag

Mobp

Cldn11

Mal

fold

cha

nge 1-0 ppm

2-0 ppm

3-0 ppm

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

Pllp

Tspan2

Tspan2

Mbp

Plp

Cldn11

Ugt8

Mag

Mobp

Cldn11

Mal

fold

cha

nge 1-0 ppm

2-0 ppm

3-0 ppm

Dose-Dependent Reductions in MyelinAssociated Genes at Low Doses of PTU

Consistent with in situ hybridization, microarrays show changes in expression of myelin genes in hippocampus of PN15 offspring are associated with low level TH insufficiencies. This approach may provide sensitive biomarkers of effect.


(Royland and Gilbert, in preparation)

10

Persistent Long-Term Functional Consequences of Early Exposure to EDCs

Permanent cognitive impairments and altered synaptic Permanent cognitive impairments and altered synaptic function result from transient thyroid hormone function result from transient thyroid hormone insufficienciesinsufficiencies

Morphological abnormalities resulting from transient Morphological abnormalities resulting from transient exposure to AR antagonists delay puberty and reduce exposure to AR antagonists delay puberty and reduce fertilityfertility

Transgenerational effects of androgen receptor Transgenerational effects of androgen receptor antagonistsantagonists


11

Effects of in utero Exposure toEDCs Transcend Generations

Low sperm counts, reduced sperm motility, Low sperm counts, reduced sperm motility, testes dysmorphology in F3 generation testes dysmorphology in F3 generation males to dosing of F0 females to low males to dosing of F0 females to low doses of vinclozilin or methoxychlordoses of vinclozilin or methoxychlor

Incidence and persistence across Incidence and persistence across generations suggests epigenetic generations suggests epigenetic reprogramming of germ line reprogramming of germ line

CAVEAT:CAVEAT: Failure to replicate from two Failure to replicate from two independent labs; number of animals this independent labs; number of animals this study small; efforts in RTD are ongoing study small; efforts in RTD are ongoing with negative findings to datewith negative findings to date

EPA STAR Grantee Anway et al., Science, 2005


spermatazoa

spermatocyte

CON

VIN

12

Developing a “Toxicological Science” of EDC Mixtures

• Dose Additivity – Default Risk Assessment Model for Dose Additivity – Default Risk Assessment Model for mixture of chemicals with mixture of chemicals with commoncommon MOA. MOA.

• Response Additivity – Default Risk Assessment Model for Response Additivity – Default Risk Assessment Model for mixtures of chemicals with mixtures of chemicals with differentdifferent MOA MOA

• Thyroid & Reproductive Mixture Studies Demonstrate:Thyroid & Reproductive Mixture Studies Demonstrate:1. Response Additivity underestimates risk 1. Response Additivity underestimates risk 2. Dose Additivity is predictive for chemicals with 2. Dose Additivity is predictive for chemicals with

same and differentsame and different MOA MOA3. Next generation models encompass both?3. Next generation models encompass both?

•Low Dose Effects/Animal ModelsLow Dose Effects/Animal Models

•Mixtures of EDCsMixtures of EDCs

•Species ExtrapolationSpecies Extrapolation

•Toxicogenomics in RAToxicogenomics in RA

•Biomarkers and ScreeningBiomarkers and Screening

13

Binary Mixtures of Phthalates Induce Dose-Additive Effects

Howdeshell et al., Toxicological Sciences, 2007

Several androgen-dependent endpoints of male reproductive tract development interact in dose-additive manner as predicted by common mechanism of toxicity during sexual differentiation.

Ano-genital Distance is Reduced in Dose-Additive Manner

Con DBP DEHP DBP+DEHP

Both phthalates reduce testicular hormone production and expressionof genes critical for steroidogenesis.

Fetal Testicular Insl3 mRNA

Con DBP DEHP DBP+DEHP

Fetal Testicular Testosterone

• Low Dose Effects/Animal ModelsLow Dose Effects/Animal Models

• Mixtures of EDCsMixtures of EDCs

• Species ExtrapolationSpecies Extrapolation

• Toxicogenomics in RAToxicogenomics in RA

• Biomarkers and ScreeningBiomarkers and Screening

14

0 0 0 0 0 0 3.3

95.8

56

25

48.856

Control

BBP

DBP

Procymidone

Vinclozolin

Linuron

DEHP

VIN+PRO

BBP+DBP

DEHP+DBP

DBP+PRO

BBP+LIN

0

20

40

60

80

100

Perc

ent

of

male

s a

aff

ecte

d

Default: Response-Additive ModelObserved: Dose-Additive Responses• Androgen Antagonists – Androgen Antagonists – vinclozolinvinclozolin, ,

procymidone,procymidone, phthalatesphthalates

• Estrogens – Estrogens – methoxychlor,methoxychlor,

bisphenol Abisphenol A

• Androgens- Androgens- trenbolonetrenbolone

• Fetal androgen synthesis Fetal androgen synthesis inhibitorsinhibitors - - linuronlinuron

• Fetal Germ Cell Toxicants- Fetal Germ Cell Toxicants- busulfanbusulfan

• Steroidogenesis InhibitorsSteroidogenesis Inhibitors - - prochlorazprochloraz

Reproductive Toxicants: Multiple Mechanisms, Different Fetal Targets

CONCLUDE: Extensive dose-response information, especially low dose levels, is needed to appropriately design and interpret Mixture Studies

• Low Dose EffectsLow Dose Effects





15

Thyroid Disrupting Chemicals: Structurally Diverse, Multiple Sites, Multiple Mechanisms

T4 -Gluc

Biliary Excretion

Free -TH

Bound -TH

T4 TTR/TBG

Hypothalamus

Pituitary

Thyroid Gland

TRH

TSH

+

+

T3 & T4

T3 & T4

Deiodinase

T4 > T3

Ah -Receptor

T4

UDPGTs

CAR/PXR

Iodine

Perchlorate

Thiocyanate

T4 T3Thyroperoxidase

I + tyrosine

T3 & T4

HO -PCBs

Dioxins

PBDEs

_

_

Transporters

Tra

nspo

rter

s

Liver

T4 -Gluc

Biliary Excretion

Plasma/BloodFree -TH

Bound -TH

T4 TTR/TBG

Hypothalamus

Pituitary

Thyroid Gland

TRH

TSH

+

+

T3 & T4

T3 & T4

T4 > T3

Ah -Receptor

T4

UDPGTs

CAR/PXR

Iodine

Perchlorate

Thiocyanate

T4 T3Thyroperoxidase

I + tyrosine

T3 & T4

PTUMMI MancozebPronamide

Thiram

HO -PCBs

Dioxins

PBDEsPCBs

Peripheral Tissues

_

_

Transporters

Tra

nspo

rter

s

Hepatic Target

Thyroid Target

• Low Dose EffectsLow Dose Effects





16 Default Dose Additivity Model predicts effects on T4 at environmental levels of exposure.

Additivity Model

Empirical ModelT4

, %

Co

ntr

ol (m

ea

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

, %

Co

ntr

ol (m

ea

n±SD

)

20

40

60

80

100

120

140

Total Mixture Dose (μg/kg/day)

0 300 600 900 1200 1500 1800 2100

Dose (ug/kg/day)10-4 10-2 100 101 102 103

T4,

% C

on

tro

l (m

ean

±SE

)

0

20

40

60

80

100

120

TCDD PCB 126 PCB 118 PCB 153 DE-71 PCB 105

Dose (ug/kg/day)10-4 10-2 100 101 102 103

T4,

% C

on

tro

l (m

ean

±SE

)

0

20

40

60

80

100

120

TCDD PCB 126 PCB 118 PCB 153 DE-71 PCB 105

Mixtures Of Thyroid Hormone Disruptors:Evolving Statistical Models to Predict Outcome

(Flippin et al., under review)

R mixture = [UGT inducers]

Dose Addition

Modeled and Observed Results of 4 Day PHAH and Pesticide Exposure

Dose of PHAH and Pesticide (% of Stock Solns)

0 10 20 30 40 50 60 70

T4

(%

of

Co

ntr

ol)

20

40

60

80

100

120

140

Effect AdditionEffect Addition

Rmixture=[Thyroid peroxidase inhibitors] + [UGT inducers]

Dose Addition Dose Addition

Hepatic Target – Similar MOA Hepatic + Thyroid Targets – Different MOA

Combined Mixture Model accurately predicts effects of mixtures with components with different MOA.

Predicted

Empirical

(Crofton et al., 2005)

Default Dose Additivity Model Combined Dose + Effect Additivity Model

18 PHAHs18 PHAHs + 3 Pesticides

17

Mixtures of chemicals that alter gonadal or thyroid hormones via a Mixtures of chemicals that alter gonadal or thyroid hormones via a common MOA behave in a Dose-Additive mannercommon MOA behave in a Dose-Additive manner

Regardless of the molecular MOARegardless of the molecular MOA, c, chemicals that disrupt sexual hemicals that disrupt sexual differentiation produce dose-additive responses,– differentiation produce dose-additive responses,– i.e., need to thinki.e., need to think of of common pathways of toxicitycommon pathways of toxicity

Unlike carcinogenesis risk assessments, Unlike carcinogenesis risk assessments, Response-Additivity Response-Additivity Models do not fit the dataModels do not fit the data, , combined mixture models may prove to combined mixture models may prove to be the most predictivebe the most predictive

Knowledge about the precise mechanism of toxicity is Knowledge about the precise mechanism of toxicity is not necessary necessary to predict the interactions, to predict the interactions, but extensive dose-response data are needed for each chemical

Dose-Additivity Model Best Fits the Data and Most Consistent with Biology of Hormonal Action

18

Studies to Examine Extrapolation Across Species and Inform Screening Efforts

Estrogen and Androgen Receptors Across SpeciesEstrogen and Androgen Receptors Across Species

Aromatase Activity – MOA for EDCs in Rats and Fish?Aromatase Activity – MOA for EDCs in Rats and Fish?






19

Species Extrapolation: Invertebrate and Vertebrate

SPECIES STATUS

Wilson et al., 200?

Chimp AR

?

Japanese Quail

Northern Leopard

Frog

Daphnia Magna

?

Mud Snail

Rainbow Trout

AR and ER

Obtain Animal Tissues

Prepare cDNA library

Isolate ER or AR

Sequence ER or AR

Express ER or AR

ER or AR Function

Compare Function Across Species

Fathead Minnow

AR and ER

Red belly turtle

giant salamander

American Alligator

SPECIES STATUS

Wilson et al., 200?

Chimp AR

?

Japanese Quail

Northern Leopard

Frog

Daphnia Magna

?

Mud Snail

Rainbow Trout

AR and ER



Isolate ER or AR

Sequence ER or AR

Express ER or AR

ER or AR Function


Fathead Minnow

AR and ER

Red belly turtle

giant salamander

American Alligator

Wilson et al., 200?

Chimp AR

?

Japanese Quail

Northern Leopard

Frog

Daphnia Magna

?

Mud Snail

Rainbow Trout

AR and ER



Isolate ER or AR

Sequence ER or AR

Express ER or AR

ER or AR Function


Fathead Minnow

AR and ER

Red belly turtle

giant salamander

American Alligator

Hartig et al, in press

Wilson et al., 2007 *

* *

*

20

Aromatase Activity – MOA for EDCs in Rats and Fish? A Case Study with Atrazine

Goal: Improve species extrapolation by understanding cellular events leading to altered aromatase activity/gene expression in different target tissues & species.

Increased serum estrogens are not caused byactivation of aromatase or increased cyp19 geneexpression in brain or testes. Major metabolites present in plasma.

Reduced reproductive success with increase inbrain aromatase at the lowest dose. Atrazine is major component in plasma.

0

20

40

60

80

100

120

140

0 0.24 1.2 2.4 12

*

Eg

g P

rod

uc

tio

n

0 0.24 1.2 2.4 120

20

40

60

80

100

120

140

0 0.24 1.2 2.4 12

*

Eg

g P

rod

uc

tio

n

0 0.24 1.2 2.4 12

Egg Production Decreased Increase Brain Aromatase

Atrazine metabolites are primary components in rat plasma

Atrazine is primary component in fish plasma

Differential Metabolism

cyp19

Proposed MOA Atrazine

Aro

mat

ase

Act

ivity

0

2

4

6

8

10

12

14

16

*

Female Fish Brains

0 0.24 1.2 2.4 12

Atrazine (mg / kg)

16

14

12

10

8

6

4

2

0

Aro

mat

ase

Act

ivity

0

2

4

6

8

10

12

14

16

*

Female Fish Brains

0 0.24 1.2 2.4 12

Atrazine (mg / kg)

16

14

12

10

8

6

4

2

00

2

4

6

8

10

12

14

16

*

Female Fish Brains

0 0.24 1.2 2.4 12

Atrazine (mg / kg)

16

14

12

10

8

6

4

2

0

0

25

50

75

100

125

150

175

**

SerumEstrone

* *

SerumEstradiol

0 50 200 0 50 200

pg

/ml

Serum Estrogen IncreasedNo Change in Aromatase

Aromatase Activity in Testicular MicrosomesFollowing 3 Days of Exposure

0 50 2000.0

0.1

0.2

Atrazine (mg/kg)

pm

ol/

hr/

mg

Testes Aromatase Activity

CONCLUDE: Induction of aromatase is not the primary MOA for atrazine-induced toxicity in rat or fish. Differential metabolism may underlie species- & tissue- specific effects.

21

Biomarkers, Toxicogenomics, and Screening Tools

Biomarkers of Reproductive & Thyroid DysfunctionBiomarkers of Reproductive & Thyroid Dysfunction• Biomarkers of Thyroid Dysfunction • Toxicogenomics in Risk Assessment• Proteomics as Bioindicators of Reproductive Toxicity

Amphibian Model for Thyroid Hormone DisruptorAmphibian Model for Thyroid Hormone Disruptor• Development of alternative biochemical and molecular screens

for EDCs acting on thyroid axis






22

Using Toxicogenomics in Risk Assessment: A Case Study with Dibutyl Phthalate

• Increasing use of ‘omics technology:Increasing use of ‘omics technology:• How can this type of data be used in risk assessment?

• What are its limitations?

•How best can this info be interfaced with toxicity data?

• Recommendations for Toxicogenomics Studies:Recommendations for Toxicogenomics Studies:• Parallel genomic and toxicity study design characteristics (e.g.,

dose, timing of exposure, tissues)

• Time-course data over critical window of exposure for endpoints of interest

• Increase # samples and replicates to improve study power and to permit pathway analysis.

• Incorporate multiple doses and low doses to address dose-response

23

Future Work on Reproductive & Thyroid EDCs•Low Dose

•Biomarkers of TH disruption and neurodevelopmental outcomes•Quantitative BBDR for thyroid disruption in fetus and neonate

•Reproductive and thyroid toxicity to inform mixtures studies

•Mixtures•Multiple chemicals with different MOA, critical developmental periods, evaluate both genders, improved statistical models

•Extrapolation•Animal models of human neurodevelopmental outcomes

•AR/ER receptors – Expand chemicals, species, receptors• Aromatase - ADME, low dose, brain as site of action?

•Biomarkers•Identify biomarkers of TH disruption, incorporate proteomics into toxicogenomic profiles of reproductive toxicants

•Screening•AR and ER binding assays across multiple species

•Amphibian and mammalian models for thyroid hormone disruption

24

The NHEERL Players …

RTD ScientistsRTD Scientists• Ralph Cooper• Earl Gray• Phillip Hartig• Gary Klinefelter• Susan Laws• Tammy Stoker• Vickie Wilson

NTD ScientistsNTD Scientists• Kevin Crofton• Mary Gilbert

MED ScientistsMED Scientists• Sig Diegtz• Mike Hornung• Joseph Tietge

AED ScientistsAED Scientists• Lesley Mills

ETD ScientistsETD Scientists• Mike DeVito

Elaine Francis, National Program DirectorDoug Wolf, Assistant Laboratory Director

Long Term Goal 1: Neurodevelopment and Thyroid Homeostasis ...

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