1. 0 Focusing on the Adverse Outcomes of ER-mediated Pathways Rodney Johnson ORD/MED McKim Conference September 16-18, 2008

2. 1 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. 2 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. 3 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. 4 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. 5 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. 6 Medaka exposure system

8. 7 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. 8 Multigeneration Bioassay Design Octylphenol General Notes 5 treatments & control 6 replicates / treatment Flow-thru exposure 1.8 L tank, flow = 20 ml/min Reproductive Endpoints Adult: F0,F1, F2 Fecundity Fertility Growth 2° Sex character Vitellogenin Histopathology Sub-adult F1 & F2 Growth Genotypic sex (XX, XY) Phenotypic sex 2° Sex Character Vitellogenin Histological gonadal sex Histopathology Developmental Endpoints Embryo F1 & F2 Hatch Mortality 2425262728293031 Exp. Weeks F0 F2 F1 Reproduction 121516131412345678910 ReproductionDevelopment 121516131411 1234567891011121314151617181920212223 12345678910 ReproductionDevelopment 121516131411 Generation

10. 9 Control male male phenotype Estrogen-exposed male female phenotype Endpoint: Anal fin papillae

11. 10 Breeding Adult F1 Octylphenol 100 ppb Juvenile F2 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. 11 Octylphenol Effects on Fecundity Eggs/day 06132550100 Concentration (ppb)

13. 12 Octylphenol effects on genotypic males Vitellogenin (copies x 10 3 mRNA / total RNA) 0 ppb 6 ppb 13 ppb 25 ppb 50 ppb 100 ppb F1 2.41.01.77.0101.0169.0 F2 3.70.50.320.0370.0 no samples Anal Fin Papillae (number) 0 ppb 6 ppb 13 ppb 25 ppb 50 ppb 100 ppb F1 222738950 F2 362813126 no samples Gonadal Sex Reversal 0 ppb 6 ppb 13 ppb 25 ppb 50 ppb 100 ppb F1 0/130/120/51/124/125/5 F2 0/12 2/142/125/12 no samples increased no change decreased Fecundity (fertile eggs per pair/day) 0 ppb 6 ppb 13 ppb 25 ppb 50 ppb 100 ppb F1 1321 14130 F2 152192241

14. 13 ER-mediated Adverse-outcome Pathway Octylphenol Molecular CellularOrganIndividualPopulation Octylphenol binding to ER Liver slice Vtg (mRNA) Altered reproduction Altered development Decreased numbers of animals In-vitro pathway Schmieder et.al. ER transcription factor In-vivo pathway Multigen assay -dose: sex reversal (altered gamete ratios) -dose: reduced fecundity Population reduction Octylphenol- ER binding ER transcription factors ♂ Liver Vtg (mRNA) Anal fin papillae? Gonadal morphology? ? -dose: mixed-sex gonad MolecularPopulationCellular Individual Organ Altered sex-ratios ? ?

15. 14 Bioassay Design 4-n-amylaniline (AAN) Generation Weeks 123456789101112131415161718192021 F0 1234567 F1 Development Reproduction 1114151213 General Notes 5 treatments, control 6 replicates / treatment Flow-thru exposure 1.8 L tank, flow = 15 ml/min Developmental Endpoints F1 Embryo Mortality Hatch Reproductive Endpoints F0 Adult Fecundity Fertility Growth 2° Sex character Vitellogenin Histopathology Sub-adult Growth Genotypic sex (XX, XY) Phenotypic sex 2° Sex Character Vitellogenin Histological gonadal sex Histopathology

16. 15 Endpoints: Anal fin papillae Effects of amylaniline on anal fin papillae 011461124031430 Concentration (ppb)

17. 16 Endpoints: Liver Vitellogenin 011461124031430 Concentration (ppb)

18. 17 011461124031430 Concentration (ppb) AAN Effects on Growth

19. 18 Endpoint: Fecundity

20. 19 Amylaniline effects across generations increased no change decreased Vitellogenin (copies per ng total RNA) 0 ppb 11 ppb 46 ppb 112 ppb 403 ppb 1430 ppb F0 7900550025008000204000737000 F1 32006800140003000029000 no samples Anal Fin Papillae 0 ppb 11 ppb 46 ppb 112 ppb 403 ppb 1430 ppb F0 12310011410211197 F1 98100103820 no samples Body Weight (mg) 0 ppb 11 ppb 46 ppb 112 ppb 403 ppb 1430 ppb F0 371354366332368270 F1 21019822520798 no samples Gonadal Sex Reversal 0 ppb 11 ppb 46 ppb 112 ppb 403 ppb 1430 ppb F0 0/4 no samples F1 gen 0/240/12 1/121/7 no samples Fecundity (daily number of eggs/pair) 0 ppb 11 ppb 46 ppb 112 ppb 403 ppb 1430 ppb F0 23212320132

21. 20 ER-mediated Adverse-outcome Pathway Amylaniline (AAN) Molecular CellularOrganIndividualPopulation AAN binding to ER Liver slice Vtg (mRNA) Liver slice toxicity Altered reproduction Altered development Decreased numbers of animals In-vitro pathway Schmieder et.al. ER transcription factor In-vivo pathway Multigen assay dose: Sex reversal (altered gamete ratios) Population reduction AAN binding to ER ER transcription factors ♂ Liver Vtg (mRNA) Anal fin papillae? Gonadal morphology? ? dose: Mixed-sex gonad MolecularPopulationCellular Individual Organ Altered sex-ratios ? AAN binding to Hbg ? Splenic/head- kidney pathology ? dose: Reduced fecundity dose: Reduced growth? ?

22. 21 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. 22 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