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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
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
•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
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
•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
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
•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
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.
•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
(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
•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
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
•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
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
• Mixtures of EDCsMixtures of EDCs
• Species ExtrapolationSpecies Extrapolation
• Toxicogenomics in RAToxicogenomics in RA
• Biomarkers and ScreeningBiomarkers and Screening
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
• Mixtures of EDCsMixtures of EDCs
• Species ExtrapolationSpecies Extrapolation
• Toxicogenomics in RAToxicogenomics in RA
• Biomarkers and ScreeningBiomarkers and Screening
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?
• 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
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
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
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
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
• 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
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