Assessing Risks of Endocrine- Disrupting Chemicals: A Scientific ...

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Assessing Risks of Endocrine- Disrupting Chemicals: A Scientific ...

Assessing Risks of Endocrine-

Disrupting Chemicals: A Scientific

Odyssey

Gary Ankley

USEPA, Duluth, MN

0


What are EDCs?

• Substantial attention and research on

chemicals that cause effects by mimicking

estrogens

• Restrictive definition in terms of endocrine

functions/targets that could be affected by

chemicals

• Efforts are expanding past just estrogens to

other points of disruption (e.g., androgen

receptor, steroid production, etc.)

• Greatly increases universe of potential EDCs


EDCs: Not a New Phenomenon

• Reproductive problems in female cattle,

sheep exposed to phytoestrogens in clover

(hundreds of years)

• Use of synthetic steroids (e.g., ethinyl

estradiol; EE2, diethylstilbestrol; DES) in

humans (1930s)

• Utilization of synthetic steroids to modulate

reproductive cycles and enhance livestock

growth (1940s)

2


EDC Impacts: Humans

• Increases in breast cancer

• Increases in prostate/testicular cancers

• Learning/neurological deficits

• Metabolic syndrome (obesogens)

• DES-induced health effects

• Decreases in sperm quality


DES: An Established EDC in Humans

• Prescribed to prevent miscarriage and

premature births (through 1971)

• Estimated 5-10 million Americans exposed

directly or in utero

• Range of adverse health effects

– Vaginal cancer (daughters)

– Reproductive tract abnormalities (daughters, sons)

• Hallmark EDC ―properties‖

– Sensitive window(s) of exposure

– Latent effects

5


Decreased Sperm Quality:

An EDC Effect in Humans?

• Analysis published by Danish researchers documenting

large temporal decreases in sperm quality world-wide

has been major ―poster child‖ for EDC issue

• Subsequent follow-up studies by others produced mixed

results, with some showing declines and others no

change

• Recent, more robust study by same Danish group

reportedly failed to produce same results as initial

analysis

• Highlights complex interplay between public perception

and scientific progress-a common theme for EDC issue

6


EDC Impacts: Wildlife

• Hermaphrodism in gastrapods

• Feminization of alligators

• Developmental lethality/abnormalities in GL

fish and birds

• Reproductive abnormalities in fish-eating

mammals

• Feminization of fish

• Malformations in amphibians


Malformed Frogs: An EDC Effect?

-Extensive occurrence at several North

American locations of anurans with

multiple limb/digit malformations

-Malformations suggestive of chemical

effects on retinoid signaling pathways

- Intense public concern/interest

-Exhaustive studies initiated to

identify causative chemicals

-Subsequent monitoring/experimental

work revealed little support for chemical

etiology, suggesting instead a biological

basis for effects (trematodes)


Feminization of Fish: UK Story

-Fishermen and biologists in the early 90’s

noted increased prevalence of intersex

condition described as ova-testis (OT)

-Often seen in effluent-dominated

streams-estrogens from WWTP suspects

-Male feminization (OT; VTG induction)

has received substantial public attention

From Jobling et al. (1996)

11/15/2011 9


Work by UK team provided first direct evidence for contribution

of synthetic and natural steroidal estrogens (E1, E2, EE2) to

estrogenicity of waste water, and to feminizing effects in fish

Direct (whole lake) and indirect studies have shown that

levels of estrogenic steroids in wastewater can be sufficient to

cause population-level impacts in fish

Subsequent work world-wide has shown WWTP effluent

estrogenicity to be widespread, and often (but not always)

due to steroids

10


EEQ (ng/L)

Estrogenic WWTP Effluents in Minnesota

St. Paul WWTP Effluent Effects on

Male Fathead Minnows*

Statewide Survey of Estrogenic Potential of

Effluents and Receiving Waters**

100

90

80

70

60

50

40

30

20

10

0

WLSSD (Duluth) Effluent

Temporal Estrogenicity*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Day

rtERA

EIA

*Martinovic et al. (2007; 2008) Environ. Toxicol. Chem. & Environ. Sci. Technol.

**Lee, K.E. et al. (2011) Endocrine active chemicals, pharmaceuticals, and other chemicals of concern in surface water,

wastewater-treatment plant effluent, and bed sediment, and biological characteristics in selected streams, Minnesota—

design, methods, and data, 2009: U.S. Geological Survey Data Series 575


Responses to EDCs as ―Novel‖ Risks

• Increase in monitoring programs focused on

non-traditional chemicals that could affect

endocrine function (e.g., phthalates, BPA,

steroids, etc.)

• Legislated mandates (FQP, SDW Acts) issued

for development of screening and testing

programs for certain classes of EDCs

– USEPA Endocrine Disruptor Screening and Testing

Program (EDSP)

12


EDSP Overview

• Tiered approach comprised first of screening (hazard id)

followed by focused testing to support risk assessment

• Considers human and ecological effects mediated

through HPG/T axes

• Goal of screening TSCA inventory (>80,000 chemicals)

• First set of Tier 1 (screening) test orders recently issued

for 60+ pesticides/pesticide inerts

• Validated, SAP-approved Tier 1 methods

– In vitro assays (e.g., receptor binding, steroid synthesis)

– Mammalian (rat) in vivo tests (Uterotrophic, Hershberger and

pubertal assays)

– Non-mammalian (amphibian, fish) in vivo assays

13


EDSP Tier 1 Fish Screen

• Initiated with mature, spawning fathead minnows

• 14-21 day pre-exposure followed by < 21 day

chemical exposure

- Behavior

- Fecundity

- Fertility

- Hatch

- Secondary sex characteristics

- Gonadal status (GSI, histology)

- Plasma vitellogenin

- Plasma steroids (E2, T, KT)


Responses in Tier 1 Fish Assay

b-Estradiol ER Agonist ND -- + ND ND

Methoxychlor ER Agonist + -- + + +

Methyltestosterone AR Agonist + + + + +

α- and β-Trenbolone AR Agonist + + + + +

Vinclozolin AR Antagonist + + + + +

Flutamide AR Antagonist + -- + + +

Fadrozole Steroidogenesis Inhibitor + -- + + +

Ketoconazole Steroidogenesis Inhibitor + -- -- -- +

Trilostane Steroidogenesis Inhibitor + -- + -- +

Prochloraz

Fenarimol

ND, not determined

Steroidogenesis

Inhibitor/AR Antagonist

Steroidogenesis

Inhibitor/ER-AR Antagonist

+ ND + + +

+ ND + + +


EDSP Tier 1 Resource Use

Screening Assays

Animals

Used* Median Cost **

In vitro

ER Binding – rat cytosol ? $17,250

ER α Transcriptional Activation $15,000

AR Binding - rat cytosol ? $17,250

Steroidogenesis H295R $13,750

Aromatase Recombinant $25,000

In vivo

Uterotrophic 18 $43,050

Hershberger 18 - 36 $47,400

Pubertal Male 45 $93,500

Pubertal Female 45 $87,100

Fish Short-term Reproduction 72 $92,500

Amphibian Metamorphosis 320 $80,597

Analytical chemistry $12,000

Total 518-536 $544,397

*Willitt et al. 2011. Toxicol. Sci. 123:15-25

**Draft Guidance Document (GD) on the Assessment of Chemicals for Endocrine Disruption. 2010.

Organization for Economic Cooperation and Development (OECD).


Predictive Toxicology

• Predicting potential toxicity of chemicals with limited

empirical data

• Identification of organizing principles and pathways that

underlie biological responses to chemicals

• Determination and measurement of key physicochemical

and/or biological properties of chemicals that

enable them to perturb these pathways

– In silico (computational) approaches (e.g., QSAR)

– In vitro (e.g., HTP) toxicity pathway assays

– Short-term in vivo tests with pathway/mechanism-specific

endpoints (molecular, biochemical alterations)

18


• USEPA (NERL) – Cincinnati, OH

• D. Bencic, M. Kostich, D. Lattier, J. Lazorchak, G. Toth, R.-L.

Wang,

• USEPA (NHEERL)– Duluth, MN, and Grosse Isle, MI

• G. Ankley, E. Durhan, K. Jensen, M. Kahl, C. LaLone, E.

Makynen, D. Miller, D. Villeneuve

• USEPA (NERL)– Athens, GA

• T. Collette, D. Ekman, K. Ralston-Hooper, M. Henderson, Q.

Teng , D. Skelton

• USEPA-RTP, NC

• M.&M. Breen, R. Conolly (NCCT/NHEERL)

• S. Edwards (NHEERL)

• L. Burgoon (NCEA)

• USEPA (NCER) STAR Program

• N. Denslow (Univ. of Florida), E. Orlando, (Univ. of Maryland), K.

Watanabe (Oregon Health Sciences Univ.), M. Sepulveda

(Purdue Univ.)

• USACE – Vicksburg, MS

• E. Perkins, N. Garcia-Reyero, T. Habib, M. Mayo

• Other partners

EDCs in fish: Developing Exposure Indicators

and Predictive Models of Effects Based on MOA

• D. Martinovic (St. Thomas, MN)

• UC-SB, J. Shoemaker, K. Gayen (Santa Barbara, CA)

• Joint Genome Institute, DOE (Walnut Creek, CA)

11/15/2011 19

• Sandia, DOE (Albuquerque, NM)

• Pacific Northwest National Laboratory (Richland, WA)


Project Objectives

Identify molecular markers of effects of

chemicals representing different HPG

pathways in small fish models

―Anchor‖ these markers to a response

(reproduction) relevant to ecological risk

assessments using the adverse outcome

pathway (AOP) framework

Use as basis for providing more efficient,

mechanism-based approaches for

screening and monitoring EDCs

20


Compartment

Pathway-Specific “Probes”

Brain

GABA

?

GnRH

Neuronal

System

GnRH

D1 R

Y 2 R

NPY

?

Dopamine

1

2

Fipronil (-)

Muscimol (+)

?

GABA A

R

D2 R

PACAP

GABA B

R

Y 2 R

Pituitary

Follistatin

Activin

Activin R

PAC 1 R

GnRH

R

Gonadotroph

GPa

Y 1 R

D2 R

3

4

Apomorphine (+)

Haloperidol (-)

FSHb

LHb

Blood

Circulating LDL, HDL

LDL R

HDL R

Outer mitochondrial

membrane

Circulating LH, FSH

LH R FSH R

Cholesterol

StAR

5

6

Trilostane (-)

Ketoconazole (-)

Gonad

(Generalized, gonadal,

steroidogenic cell)

Activin

Inhibin

Inner mitochondrial

membrane

pregnenolone

17 α -hydroxyprogesterone

P450scc

P450c17

3bHSD

progesterone

7

8

Fadrozole (-)

Prochloraz (-,-)

20βHSD

androstenedione

17α,20β-P (MIS)

17βHSD

testosterone

P45011β.

P450arom

9

Vinclozolin (-)

11βHSD

11-ketotestosterone

estradiol

10

Flutamide (-)

Blood

Circulating Sex Steroids / Steroid

Hormone Binding Globlulin

11

17β-Trenbolone (+)

ER

AR

12

17 α -Ethynylestradiol (+)

21


Assessing Reproduction

22


Intens. [a.u.]

1.5

1.0

0.5

0.0

1000 1500 2000 2500 3000

Types of Molecular Data Collected

Transcriptomics

Fathead Minnow Microarray

Proteomics

Peptide Mass Fingerprinting

x10 4

1091.620

1799.879

1347.669

2143.156

Representative protein expression

profile in testes of control zebrafish

890.612

1214.658

1504.667

1615.722

1978.039 2460.281

2801.340

Data from EPA/ EcoArray© CRADA

Metabolomics

Fathead Minnow Liver NMR Scan

Data from EPA-Cincinnati

m/z

Fathead Minnow (male)

Data from EPA-Athens


An Adverse Outcome Pathway (AOP) is a conceptual framework that portrays existing knowledge

concerning the linkage between a direct molecular initiating event and an adverse outcome, at a

level of biological organization relevant to risk assessment.

(Ankley et al. 2010, Environ. Toxicol. Chem., 29: 730-741.)

Toxicant

Macro-

Molecular

Interactions

Cellular

Responses

Organ

Responses

Organism

Responses

Population

Responses

Chemical

Properties

Receptor/Ligand

Interaction

DNA Binding

Protein Oxidation

Gene

activation

Protein

production

Altered

signaling

Altered

physiology

Disrupted

homeostasis

Altered tissue

development/

function

Lethality

Impaired

Development

Impaired

Reproduction

Structure

Recruitment

Extinction

Provides transparent basis for communication among scientists

and between scientists and risk assessors/managers relative to

translation of mechanistic data into risk assessment


AOP Derivation and Application: An Example

for Aromatase Inhibition in Fish

Vtg (mg/ml)

E2 (ng/ml)

Cumulative Number of Eggs

(Thousands)

N

N

Aromatase

inhibition

Reduced E2,

Vtg synthesis

8

6

4

2

0

*

*

Impaired

vitellogenesis

10

8

6

4

Reduced fecundity

Fadrozole (ug/L)

Control

2

10

50

*

*

*

CN

20

2

10

0

*

*

*

Control 2 10 50

Fadrozole (µg/l)

0

-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20

Exposure (d)

Practical Implications

- Identification of meaningful biochemical/histological endpoints for in vivo

tests focused on EDC screening (e.g., E2, lack of VTG deposition)

- Provides basis for developing/interpreting in vitro assays suitable for

high-throughput screening and testing (aromatase inhibition; AI)

- Facilitates transparent derivation of predictive AI QSAR models

- Supports translation of mechanistic effects observed in field-collected

animals (e.g., VTG depression) to population-level responses

25


Microarray-Based

Biomarker Discovery

and Pathway

Definition

11/15/2011 26


Development of ―Generation 2‖

EDC Screening Tools

N

N

CN

Small Battery of Molecular

Responses

•Established linkage both to specific HPG

pathways and impaired reproduction in

fish

•Generated from short-term exposures

(e.g., 4 d) with small numbers of fish

•Analyses amenable to HTS automation

•Enhances testing of greater number of

chemicals more quickly

27


Summary and Prospectus

• EDCs have been—and will continue to be—a

high visibility human and ecological health issue

• Variety of mechanism-based concepts and

state-of-the-art tools are starting to be employed

effectively both for prospective (screening) and

retrospective (monitoring) assessments of EDCs

• Pathway/mechanism-based approaches being

developed for EDCs are providing prototypes for

dealing with other classes of chemicals in a

more efficient, robust manner

28


Contributors

• USEPA, Duluth: J. Cavallin, E. Durhan, K. Jensen, M. Kahl, C.

LaLone, E. Makyen, S. Skolness, L. Thomas, J. Tietge, D.

Villenueve (and many others)

• USEPA, Athens: T. Collette, D. Ekman, D. Skelton, Q. Teng

• USEPA, Cincinnati: D. Bencic, J. Lazorchak, R.L. Wang

• USEPA, Chicago: T. Smith

• USEPA, RTP: M. Breen, L. Burgoon, R. Conolly, S. Edwards, E.

Gray, P. Hartig, V. Wilson

• USCOE, Vicksburg (and partners): N. Garcia-Reyero, T. Habib, M.

Mayo, E. Perkins

• USFWS, St. Paul: D., DeVault, A. Trowbridge

• Others: N. Denslow (Univ. Florida); B. Hoke (DuPont); K. Lee

(USGS); D. Martinovic (Univ. St. Thomas); K. Ralston-Hooper/L.

Ferguson (Duke); K. Watanabe (Oregon Health Sciences Univ.)

29


11/15/2011 30


Development of Pathway-Based Effects

Monitoring Approaches for the Great Lakes:

A GLRI-Supported Multi-Agency Effort

11/15/2011 31


Background and Project Overview

• Past approaches for monitoring of contaminants in the

GL have relied mostly on analytical techniques

• Although providing critical information, augmentation

with biological monitoring can lend substantial insights

(e.g., unknown/undetected chemicals, mixtures)

• Multi-agency (USGS, USFWS, USEPA, Environment

Canada) project focused on CECs in several GL AOCs

• Integrated suite of chemical and biological measures

including molecular, biochemical and histological

responses

• Endpoint selection and data interpretation based on AOP

concept

32


Pilot Study Sites in

Duluth-Superior Harbor


t[2]

Impact of WLSSD Discharge on Female

Liver Metabolite Profiles

t[2]

t[2]

WLSSD1 (~10 m from discharge)

WLSSD2 (~250 m from discharge)

LabCon-F

WLSSD1-F

LabCon-F

WLSSD1-F

LabCon-F

WLSSD2-F

LabCon-F

WLSSD2-F

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8

-0.9

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

R2X[1] = 0.354721 R2X[2] = 0.17155 Ellipse: Hotelling T2 (0.95)

t[1]

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

-0.3

-0.4

SIMCA-P+ 11 - 3/9/2011 11:02:07 AM

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

t[1]

R2X[1] = 0.439394 R2X[2] = 0.127986 Ellipse: Hotelling T2 (0.95)

SIMCA-P+ 11 - 3/9/2011 10:58:09 AM

-0.5

-0.6

-0.7

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