February 25, 2009Office of Research and DevelopmentNHEERL, Mid-Continent Ecology Division, Duluth, MN

G. Ankley

Endocrine Disrupting Chemicals: New Tools for Assessing Ecological Risk

1

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

2

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

3

Postulated EDC Effects: Humans

• Cervical cancer (DES daughters)• Breast Cancer• Prostate/testicular cancers• Learning/neurological deficits• Decreases in sperm quality• Precocious maturation

4

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

5

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?)

6

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)

8

PME Masculinizes Fish in the Field

Exposed

9

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

10

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

11

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

13

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

14

15

Effects of an Estrogen on Male Effects of an Estrogen on Male Secondary Sex CharacteristicsSecondary Sex Characteristics

Miles-Richardson et al. (1999) Aquat. Toxicol.

16

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)

***

*

17

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.)

18

• 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

19

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

21

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 (+)

7

8

9

10

11

12

2

1

Chemical ProbesChemical Probes

22

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

23

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)

***

24

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

25

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

26

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

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

27

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%

28

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