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Focusing on the Adverse Outcomes of ER-mediated Pathways

Focusing on the Adverse Outcomes of ER-mediated Pathways. Rodney Johnson ORD/MED McKim Conference September 16-18, 2008. Female (XX). Male (XY). Medaka. Complete life cycle ~ 10 weeks Small (adults ~0.3 to 0.5 grams) Sexually dimorphic (fins and body shape)

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Focusing on the Adverse Outcomes of ER-mediated Pathways

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  1. Focusing on the Adverse Outcomes of ER-mediated Pathways Rodney Johnson ORD/MED McKim Conference September 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. 12 12 12 13 13 13 14 14 14 15 15 15 16 16 16 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 Development Development Reproduction Reproduction F0 Multigeneration Bioassay DesignOctylphenol Generation Reproduction F1 F2 Exp. Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 General Notes 5 treatments & control 6 replicates / treatment Flow-thru exposure 1.8 L tank, flow = 20 ml/min • Developmental Endpoints Embryo F1 & F2 • Hatch • Mortality • 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

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

  11. Phenotype: male (testis) Genotype: male (XY) Phenotype: female (ovary) Genotype: female (XX) Endpoint: Sex reversal Octylphenol 50 ppb Control Phenotype: female (ovary) Genotype: male (XY) Phenotype: female (ovary) Genotype: male (XY) Breeding Adult F1 Octylphenol 100 ppb Juvenile F2 Octylphenol 50 ppb

  12. Octylphenol Effects on Fecundity Eggs/day Concentration (ppb)

  13. Octylphenol effects on genotypic males

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

  15. 11 12 13 14 15 Generation F0 Reproduction F1 1 2 3 4 5 6 7 Development Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 General Notes 5 treatments, control 6 replicates / treatment Flow-thru exposure 1.8 L tank, flow = 15 ml/min • Reproductive Endpoints F0 Adult • Fecundity • Fertility • Growth • 2° Sex character • Vitellogenin • Histopathology • Developmental Endpoints F1 • Embryo • Mortality • Hatch • Sub-adult • Growth • Genotypic sex (XX, XY) • Phenotypic sex • 2° Sex Character • Vitellogenin • Histological gonadal sex • Histopathology Bioassay Design 4-n-amylaniline (AAN)

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

  17. Endpoints: Liver Vitellogenin Concentration (ppb)

  18. AAN Effects on Growth Concentration (ppb)

  19. Endpoint: Fecundity

  20. Amylaniline effects across generations

  21. In-vitro pathway Schmieder et.al. Cellular Organ Individual Population Molecular • AAN bindingto ER • Altered reproduction • Altered development • Liver sliceVtg (mRNA) • Liver slice toxicity • ER transcription factor Decreased numbers of animals ER-mediated Adverse-outcome Pathway Amylaniline (AAN) In-vivo pathway Multigen assay Individual Molecular Cellular Organ Population ♂ Liver Vtg (mRNA) dose: Sex reversal (altered gamete ratios) AAN bindingto ER ER transcriptionfactors Altered sex-ratios ? Anal fin papillae ? dose: Mixed-sex gonad ? Gonadal morphology Population reduction ? ? dose: Reduced fecundity AAN bindingto Hbg ? ? Splenic/head-kidney pathology ? 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 Student Services Contractors • Jessica Nagel • Maicie Sykes • Chad Blanksma • Hillery Waterhouse • Megyn Mereness Wilson Contract • Kevin Lott EPA • Douglas Lothenbach • Frank Whiteman • Kevin Flynn • Dean Hammermeister NRC • Mary Haasch

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