Endocrine Disrupting Chemicals: New Tools for Assessing ......Jan 27, 2009 · A Short-Term in vivo...
Transcript of Endocrine Disrupting Chemicals: New Tools for Assessing ......Jan 27, 2009 · A Short-Term in vivo...
February 25, 2009Office of Research and DevelopmentNHEERL, Mid-Continent Ecology Division, Duluth, MN
G. Ankley
Endocrine Disrupting Chemicals: New Tools for Assessing Ecological Risk
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What are Endocrine-Disrupting Chemicals (EDCs)?
• “Exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental process (Kavlock et al. 2007)”
• Emphasis, to date, on mechanisms affecting reproduction and development via the hypothalamic-pituitary-gonadal (HPG) and HPT (thyroidal) axes
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11βHSD
P45011β.
GnRHNeuronal System
GABA Dopamine
Brain
Gonadotroph
Pituitary
D1 R
D2 R
?
?
Y2 R
NPY
Y1 R
GnRH R
?
Activin
Inhibin
GABAA
R
GABAB
RY2 R
D2 R
GnRH
Circulating Sex Steroids / Steroid Hormone Binding Globlulin
PACAP
Follistatin
Activin PAC1 R
Activin R
FSHβ LHβ
GPα
Circulating LH, FSH
FSH RLH R
Circulating LDL, HDL
LDL R
HDL R
Outer mitochondrial membrane
Inner mitochondrial membrane
StAR
P450scc
pregnenolone 3βHSD
P450c17
androstenedione
testosterone
Cholesterol
estradiol
Gonad(Generalized, gonadal,
steroidogenic cell)
Androgen / Estrogen Responsive Tissues ARER
Blood
Blood
Compartment
17βHSD
P450arom
11-ketotestosterone
progesterone17α-hydroxyprogesterone
17α,20β-P (MIS)
20βHSD
GeneralizedVertebrate HPG Axis
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Postulated EDC Effects: Humans
• Cervical cancer (DES daughters)• Breast Cancer• Prostate/testicular cancers• Learning/neurological deficits• Decreases in sperm quality• Precocious maturation
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Postulated EDC Effects: Wildlife
• Hermaphrodism in gastropods• Developmental abnormalities in GL fish and birds• Malformations in amphibians• Feminization of reptiles• Behavioral changes in birds• Reproductive abnormalities in fish-eating mammals• Feminization/masculinization of fish
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Assessing Ecological Risks of EDCs
• Emphasis has been on predicting possible effects of new chemicals rather than diagnosing impacts in field, where several very challenging questions exist–-Analytical (What to measure at what detection?)–-Evaluation of mixtures (Endocrine MOA of concern?
Interactive effects?)–-Biological endpoints (Biomarker vs. apical?)–-Population-level responses (How to discern?)
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Tools Needed for Assessing Risk of EDCs in the Field
• Short-term in vitro and in vivo assays suitable for complex mixtures
• Analytical/fractionation approaches to identify specific chemicals causing biological effects
• Diagnostic endpoints (biomarkers) indicative of chemical MOA
• Approaches to link diagnostic responses to alterations in individuals and populations
7 Buckeye Plant, Fenhalloway River, FL
Assessing EDC Mixtures: A Pulp Mill Case Study
Collaborators: G. Ankley, L. Durhan (EPA, MED); E. Gray, P. Hartig, C. Lambright, L. Parks, V. Wilson (EPA, RTD); L. Guillette (Univ. Florida)
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PME Masculinizes Fish in the Field
Exposed
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FieldSample
Media made with Field Sample
1 2 3
CV-1 cellshAR
MMTV-luciferase
Cells Exposedto Field Sample
I.
II. Measure Luciferase Activity
CellCell--Based Assays for Based Assays for Detecting Detecting EDCsEDCs in Mixturesin Mixtures
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PME Androgenic Activity
ConPME 1PME 2PME 2Up F
enEco
nfina
DHT
0.0
0.5
1.0
1.5
Log1
0 Fo
ld in
duct
ion
over
Med
ia C
on
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Fractionation (TIE) Analysis to Identify EDCs in Complex PME
C18SPE
25% 75%50% 80% 90% 95% 100%85%
% Methanol / Water Elutions
PME
12
0
1
2
3
4
5
6
7
DHT 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
HPLC Fraction Number
Andr
ogen
ic A
ctiv
ity
Media
Linking EDC Activity in Cells to Fractionation of PME
Androstenedione
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A Short-Term in vivo Test for Detecting Reproductive Toxicity of EDCs
• Conducted with fathead minnow, a model species used for research and regulatory work in labs throughout the world
• Three-week test includes endpoints reflective of specific classes of EDCs and apical responses useful to risk assessment –-Vitellogenin, steroids, SSC, histology–-Egg production, fertility, hatch
• Developed for EPA’s EDSP (Endocrine Disruptor Screening and Testing Program), but applicable to assessment of complex mixtures as well
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Effects of an Estrogen on Male Effects of an Estrogen on Male Secondary Sex CharacteristicsSecondary Sex Characteristics
Miles-Richardson et al. (1999) Aquat. Toxicol.
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Effects of 17β-Trenbolone on Fathead Minnow Fecundity
0 2 4 6 8 10 12 14 16 18 20
Exposure (d)
0
800
1600
2400
3200
4000
Cum
ulat
ive
Num
ber o
f Egg
s S
paw
ned Control
0.0050.050.55.050
Trenbolone (µg/L)
***
*
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Example Applications to Diagnostic Assessments/Monitoring
• Monitoring Nebraska CAFO (Concentrated Animal Feeding Operation) samples for effects on reproduction and endocrine function (Kolok, Sellin et al.)
• Evaluating UK municipal effluents for estrogenicity (Thorpe, Tyler et al.)
• Monitoring Canadian pulp/paper mill effluents for reproductive and endocrine impacts (Kovacs, Parrott et al.)
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• USEPA – Cincinnati, OH• D. Bencic, M. Kostich, D. Lattier, J. Lazorchak, G. Toth, R. Wang,
• USEPA – Duluth, MN, and Grosse Isle, MI• G. Ankley, E Durhan, M Kahl, K Jensen, E Makynen, D. Martinovic,
D. Miller, D. Villeneuve• USEPA – Athens, GA
• T. Collette, D. Ekman, M. Henderson, Q. Teng• USEPA-RTP, NC
• M. Breen, R. Conolly• USEPA STAR Program
• N. Denslow (Univ. of Florida), E. Orlando, (Florida Atlantic University), K. Watanabe (Oregon Health Sciences Univ.), M. Sepulveda (Purdue Univ.)
• USACE – Vicksburg, MS• E. Perkins, N. Garcia-Reyero
• Other partners• Joint Genome Institute, DOE (Walnut Creek, CA)• Sandia, DOE (Albuquerque, NM)• Pacific Northwest National Laboratory (Richland, WA)• O. Mekenyan (University of Bourgas)
Linkage of Exposure and Effects Using Genomics, Linkage of Exposure and Effects Using Genomics, Proteomics, and Metabolomics in Small Fish Models Proteomics, and Metabolomics in Small Fish Models
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Investigate effects of EDCs with different MOA using the fathead minnow 21-d test to establish toxicity pathways and ecologically- relevant responses
Identify the genomic responses to the same chemicals using shorter-term exposures to support development of specific biomarkers indicative both toxic MOA and adverse outcome
Concurrently integrate data in a systems and network modeling contexts, as well as relevant population modeling, as a basis for prediction
Project ObjectivesProject Objectives
2/25/2009Office of Research and DevelopmentNHEERL, Mid-Continent Ecology Division, Duluth, MN
Molecular Cellular Organ Individual Population
• Gene/Protein Expression
• Metabolite Profiles
Alterations in production of
signaling molecules
• Functional changes
• Structural changes (pathology)
Altered reproduction or development
Decreased numbers of
animals
Conceptual Toxicity (Adverse Outcome) Pathway
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11βHSD
P45011β.
GnRHNeuronal System
GABA Dopamine
Brain
Gonadotroph
Pituitary
D1 R
D2 R
?
?
Y2 R
NPY
Y1 R
GnRH R
?
Activin
Inhibin
GABAA
R
GABAB
RY2 R
D2 R
GnRH
Circulating Sex Steroids / Steroid Hormone Binding Globlulin
PACAP
Follistatin
Activin PAC1 R
Activin R
FSHβ LHβ
GPα
Circulating LH, FSH
FSH RLH R
Circulating LDL, HDL
LDL R
HDL R
Outer mitochondrial membrane
Inner mitochondrial membrane
StAR
P450scc
pregnenolone 3βHSD
P450c17
androstenedione
testosterone
Cholesterol
estradiol
Gonad(Generalized, gonadal,
steroidogenic cell)
Androgen / Estrogen Responsive Tissues
(e.g. liver, fatpad, gonads)ARER
3
4
5
6
Blood
Blood
Compartment
17βHSD
P450arom
11-ketotestosterone
progesterone17α-hydroxyprogesterone
17α,20β-P (MIS)
20βHSD
Fipronil (-)
Muscimol (+)
Apomorphine (+)
Haloperidol (-)
Trilostane (-)
Ketoconazole (-)
Fadrozole (-)
Prochloraz (-,-)
Vinclozolin (-)
Flutamide (-)
β-Trenbolone (+)
Ethynyl estradiol (+)
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2
1
Chemical ProbesChemical Probes
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Types of Genomic DataTypes of Genomic Data
Transcriptomics Proteomics
Metabolomics
Fathead Minnow Microarray
Fathead Minnow Liver NMR Scan
Data from EPA/ EcoArray© CRADA
Representative protein expression profile in testes of control zebrafish
1091.620
1799.879
1347.669
1615.722
2143.156
890.612
1978.039 2460.281
1214.658
1504.667
2801.340
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
1000 1500 2000 2500 3000m/z
Peptide Mass Fingerprinting
Data from EPA-Cincinnati
Fathead Minnow (male)
Data from EPA-Athens
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Effects of Effects of AromataseAromatase Inhibition on Inhibition on Reproduction in the Fathead MinnowReproduction in the Fathead Minnow
CN
NN
Fadrozole
Fadrozole (µg / L)0 50
Aro
mat
ase
Act
ivity
(fmol
/mg-1
hr-1
)
0
75
150
Male Female
a
b
cc
Fadrozole (µg / L)0 50
Aro
mat
ase
Act
ivity
(fmol
/mg-1
hr-1
)
0
75
150
Male Female
a
b
cc
8
0
2
4
6
E2
(ng/
ml)
*
*
0
10
20
Vtg
(mg/
ml)
*
* *Control 2 10 50
Fadrozole (µg/l)
8
0
2
4
6
E2
(ng/
ml)
*
*
0
10
20
Vtg
(mg/
ml)
*
* *Control 2 10 50
Fadrozole (µg/l)
-20 -18 -16 -14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20Exposure (d)
0
2
4
6
8
10
(Tho
usan
ds)
Cum
ulat
ive
Num
ber o
f Egg
s
Control21050
Fadrozole (ug/L)
***
-20 -18 -16 -14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20Exposure (d)
0
2
4
6
8
10
(Tho
usan
ds)
Cum
ulat
ive
Num
ber o
f Egg
s
Control21050
Fadrozole (ug/L)
***
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Key Nodes in Toxicity Pathways:Key Nodes in Toxicity Pathways: Illustration from the HPG Axis in FishIllustration from the HPG Axis in Fish
• Vitellogenein (vtg), egg yolk protein, is produced normally by oviparous female vertebrates in response to stimulation of the ER by 17β-estradiol
• Commonly used exposure biomarker in males for exposure to exogenous estrogens
• Effective production of vtg in females critical to successful egg production
• Vtg production in females can hypothetically be decreased via several discreet mechanisms within the HPG axis
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11βHSD
P45011β
GnRHNeuronal System
GABA Dopamine
Brain
Gonadotroph
Pituitary
D1 R
D2 R
?
?
Y2 R
NPY
Y1 RGnR H R
?
Activin
Inhibin
GAB AA R
GAB AB R
Y2 R
D2 R
GnR H
Circulating Sex Steroids / Steroid Hormone Binding Globlulin
PACAP
Follistatin
Activi n
PAC1
R
Activin R
FSHβ LHβ
GP α
Circulating LH, FSH
FSH R
LH R
Circulating LDL, HDL
LDL R
HDL R
Outer mitochondrial membrane
Inner mitochondrial membrane
StAR
P450scc
pregnenolone 3βHSD
P450c17
androstenedion e
testosterone
Cholestero l
estradiol
Gonad(Generalized, gonad,
steroidogenic cells and oocytes)
Androgen / Estrogen Responsive Tissues
(e.g. liver, fatpad, gonads)
AR
ER Fenarimol
Blood
Blood
Compartment
17βHSD
P450arom
11-ketotestosterone
progesterone17α-hydroxyprogesterone
17α,20β-P (MIS)
20βHSD
Fadrozole
Prochloraz
β
trenbolone
α
trenbolone
Estradiol
+Vtg
(steroidogenic cells)
(oocytes)
Molecular Mechanisms Molecular Mechanisms of Inhibition of VTG of Inhibition of VTG ProductionProduction
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Linking Molecular Responses to Apical Effects: Linking Molecular Responses to Apical Effects: VTG and FecundityVTG and Fecundity
Chemical Exposure Concentrations
17β-trenbolone 0.005µg/l, 0.05µg/l, 0.5µg/l, 5µg/l, and 50µg/l17α-trenbolone 0.003µg/l, 0.01µg/l, 0.03µg/l, and 0.1µg/lProchloraz 0.03mg/l, 0.1mg/l, and 0.3mg/lFenarimol 0.1mg/l and 1mg/lFadrozole 2µg/l, 10µg/l, and 50µg/l
Fecundity = -0.042 + 0.95 * Vtg (R2 = 0.88)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1Relative Vitellogenin
Rel
ativ
e Fe
cund
ity
Fathead Minnow Fecundity vs Vtg
0
0.1
0.2
0.3
0.4
0.5
0.6
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0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1Relative Vitellogenin
Rel
ativ
e Fe
cund
ity
Fathead Minnow Fecundity vs Vtg
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prochlorazfenarimol
fadrozole
Vtg
17β-trenbolone17α-trenbolone
prochlorazfenarimol
fadrozole
Measurement of vtg concentrations andfecundity for female fathead minnows
Life table with age specific vital rates of survival and fecundity for the fathead minnow population
Carrying capacity for the fathead minnowpopulation
Projection of density dependentlogistic population trajectories forthe fathead minnow populationbased upon change in vtg
Fecu
ndity
Population projection for populations at carrying capacity exposed to stressors that depress vitellogenin production
Population Forecasts Based on Molecular ResponsesPopulation Forecasts Based on Molecular Responses
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Size
(Pro
port
ion
of C
arry
ing
Cap
acity
)
A
B
C
DE
Forecast Population Trajectories
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20Time (Years)
Aver
age
Popu
latio
n S
ize
(Pro
porti
on o
f Car
ryin
g C
apac
ity)
A
B
CDE
0%
25%
50%
75%>95%0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Size
(Pro
port
ion
of C
arry
ing
Cap
acity
)
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Size
(Pro
port
ion
of C
arry
ing
Cap
acity
)
A
B
C
DE
Forecast Population Trajectories
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20Time (Years)
Aver
age
Popu
latio
n S
ize
(Pro
porti
on o
f Car
ryin
g C
apac
ity)
A
B
CDE
0%
25%
50%
75%>95%
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Tools Needed for Assessing Risk of EDCs in the Field
• Short-term in vitro and in vivo assays suitable for complex mixtures
• Analytical/fractionation approaches to identify specific chemicals causing biological effects
• Diagnostic endpoints (biomarkers) indicative of chemical MOA
• Approaches to link diagnostic responses to alterations in individuals and populations