1

Focusing on the Adverse Outcomes of ER-mediated Pathways

Rodney JohnsonORD/MED

McKim ConferenceSeptember 16-18, 2008

2

Female (XX)

Male (XY)

Medaka

• Complete life cycle ~ 10 weeks• Small (adults ~0.3 to 0.5 grams)• Sexually dimorphic (fins and body shape)•Male sex-determination gene (DMY) identified and sequenced• Spawns daily (~25 to 35 eggs per spawn)• Genome sequenced• Gene arrays available• Large historical literature database

3

Positive Attributes of Medaka for Short-term and Multigeneration Tests

• Simple ID of genetic sex of individuals• Small aquaria and pair spawning

–optimize replication– improve statistical power

• Rapid life cycle reduces test duration

4

Purpose of Tier II Fish Tests

• Evaluate nature and extent of adverse effects for chemicals implicated in Tier I tests• Evaluate population-level responses to potential EDCs• Establish dose-response parameters for chemicals implicated as EDCs in Tier I tests

5

Purpose of Multigeneration Fish Tests

Test Development• Develop datasets for developing efficient Tier II test protocols.

Risk Assessment• Evaluate transgenerational effects of EDCs

–determine if present– if so, which MOAs (i.e. estrogens, androgens, etc)– if so, magnitude of effect

6

Transgenerational Effects

• Definition: a between-generation increase or decrease in sensitivity of the test organism to the test agent–Requirements for evaluation• Same test conditions for each generation

- chemical concentration

- life-stage

- endpoint

7

Medaka exposure system

8

Test protocol and data analysis based on genotypic sex (DMY)

• Set up breeding pairs: 1 male (DMY) and 1 female• Endpoints analyzed by sex genotype (XX) or (XY)• Gonad histology phenotype (ovary or testis)• Liver vitellogenin mRNA phenotype (high female or low male) • Secondary Sex: anal fin papillae phenotype

9

Multigeneration Bioassay DesignOctylphenol

General Notes5 treatments & control6 replicates / treatmentFlow-thru exposure1.8 L tank, flow = 20 ml/min

Reproductive Endpoints Adult: F0,F1, F2

FecundityFertilityGrowth2° Sex characterVitellogeninHistopathology

Sub-adult F1 & F2GrowthGenotypic sex (XX, XY)Phenotypic sex

2° Sex CharacterVitellogeninHistological gonadal sex

Histopathology

Developmental Endpoints Embryo F1 & F2

HatchMortality

24 25 26 27 28 29 30 31Exp. Weeks

F0

F2

F1

Reproduction

12 15 1613 14

1 2 3 4 5 6 7 8 9 10

ReproductionDevelopment

12 15 1613 1411

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 3 4 5 6 7 8 9 10

ReproductionDevelopment

12 15 1613 1411

Gen

eration

10

Control male• male phenotype

Estrogen-exposed male• female phenotype

Endpoint: Anal fin papillae

11

Breeding Adult F1Octylphenol 100 ppb

Juvenile F2Octylphenol 50 ppb

Octylphenol 50 ppb

Endpoint: Sex reversal

Phenotype: female (ovary)Genotype: female (XX)

Phenotype: male (testis)Genotype: male (XY)

Phenotype: female (ovary)Genotype: male (XY)

Phenotype: female (ovary)Genotype: male (XY)

Control

12

Octylphenol Effects on Fecundity

0

5

10

15

20

25

30

1 2 3 4 5 6

F0

F1

F2Eggs/day

0 6 13 25 50 100

Concentration (ppb)

13

Octylphenol effects on genotypic males

Vitellogenin (copies x 103 mRNA / total RNA)

0ppb

6 ppb

13 ppb

25 ppb

50 ppb

100 ppb

F1 2.4 1.0 1.7 7.0 101.0 169.0

F2 3.7 0.5 0.3 20.0 370.0no

samples

Anal Fin Papillae (number)

0ppb

6 ppb

13 ppb

25 ppb

50 ppb

100 ppb

F1 22 27 38 9 5 0

F2 36 28 13 12 6no

samples

Gonadal Sex Reversal

0ppb

6 ppb

13 ppb

25 ppb

50 ppb

100 ppb

F1 0/13 0/12 0/5 1/12 4/12 5/5

F2 0/12 0/12 2/14 2/12 5/12no

samples

 increased

 no change

 decreased

Fecundity (fertile eggs per pair/day)

0ppb

6 ppb

13 ppb

25 ppb

50 ppb

100 ppb

F1 13 21 21 14 13 0

F2 15 21 9 22 4 1

14

ER-mediated Adverse-outcome PathwayOctylphenol

Molecular Cellular Organ Individual Population• Octylphenol

binding to ER• Liver slice

Vtg (mRNA)

•Altered reproduction

•Altered developmentDecreased numbers of animals

In-vitro pathwaySchmieder et.al.

• ER transcription factor

In-vivo pathwayMultigen assay

-dose: sex reversal (altered gamete ratios)

-dose: reduced fecundity

Population reductionOctylphenol-ER binding

ER transcriptionfactors

♂ Liver Vtg (mRNA)

Anal fin papillae?

Gonadal morphology?

?

-dose: mixed-sex gonad

Molecular PopulationCellular IndividualOrgan

Altered sex-ratios?

?

15

Bioassay Design 4-n-amylaniline (AAN)

Generation

Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

F0

1 2 3 4 5 6 7F1

Development

Reproduction

11 14 1512 13

General Notes5 treatments, control6 replicates / treatmentFlow-thru exposure1.8 L tank, flow = 15 ml/min

Developmental Endpoints F1Embryo

MortalityHatch

Reproductive Endpoints F0 Adult

FecundityFertilityGrowth2° Sex characterVitellogeninHistopathology

Sub-adultGrowthGenotypic sex (XX, XY)Phenotypic sex

2° Sex CharacterVitellogeninHistological gonadal sex

Histopathology

16

Endpoints: Anal fin papillae

Effects of amylaniline on anal fin papillae

0 11 46 112 403 1430

Concentration (ppb)

17

Endpoints: Liver Vitellogenin

0 11 46 112 403 1430

Concentration (ppb)

18

0 11 46 112 403 1430

Concentration (ppb)

AAN Effects on Growth

19

Endpoint: Fecundity

20

Amylaniline effects across generations

 increased

 no change

 decreased

Vitellogenin (copies per ng total RNA)

0ppb

11 ppb

46 ppb

112 ppb

403 ppb

1430 ppb

F0 7900 5500 2500 8000 204000 737000

F1 3200 6800 14000 30000 29000 no samples

Anal Fin Papillae

0ppb

11 ppb

46 ppb

112 ppb

403 ppb

1430 ppb

F0 123 100 114 102 111 97

F1 98 100 103 82 0 no samples

Body Weight (mg)

0ppb

11 ppb

46 ppb

112 ppb

403 ppb

1430 ppb

F0 371 354 366 332 368 270

F1 210 198 225 207 98no

samples

Gonadal Sex Reversal

0ppb

11 ppb

46 ppb

112 ppb

403 ppb

1430 ppb

F0 0/4 0/4 0/4 0/4 0/4 no samples

F1 gen 0/24 0/12 0/12 1/12 1/7 no samples

Fecundity (daily number of eggs/pair)

0ppb

11 ppb

46 ppb

112 ppb

403 ppb

1430 ppb

F0 23 21 23 20 13 2

21

ER-mediated Adverse-outcome PathwayAmylaniline (AAN)

Molecular Cellular Organ Individual Population• AAN binding

to ER• Liver slice

Vtg (mRNA)

• Liver slice toxicity

•Altered reproduction

•Altered developmentDecreased numbers of animals

In-vitro pathwaySchmieder et.al.

• ER transcription factor

In-vivo pathwayMultigen assay

dose: Sex reversal (altered gamete ratios)

Population reduction

AAN bindingto ER

ER transcriptionfactors

♂ Liver Vtg (mRNA)

Anal fin papillae?

Gonadal morphology??

dose: Mixed-sex gonad

Molecular PopulationCellular IndividualOrgan

Altered sex-ratios?

AAN bindingto Hbg ?

Splenic/head-kidney pathology

?

dose: Reduced fecundity

dose: Reduced growth ?

?

22

Conclusions

• The in-vivo bioassays suggest that the ER- mediated pathway is more sensitive than other adverse outcome pathways for both octylphenol and AAN• The AAN data suggests that the ER-pathway is only slightly more sensitive than aromatic amine toxicity. • The Effectopedia could be very helpful for constructing and evaluating pathways.

23

Acknowledgements

EPA • Douglas Lothenbach• Frank Whiteman• Kevin Flynn• Dean Hammermeister

NRC • Mary Haasch

Student Services Contractors• Jessica Nagel• Maicie Sykes• Chad Blanksma• Hillery Waterhouse• Megyn Mereness

Wilson Contract• Kevin Lott