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Focusing on the Adverse Outcomes of ER-mediated Pathways
Rodney JohnsonORD/MED
McKim ConferenceSeptember 16-18, 2008
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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
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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
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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
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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
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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
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Medaka exposure system
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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
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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
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Control male• male phenotype
Estrogen-exposed male• female phenotype
Endpoint: Anal fin papillae
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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
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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)
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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
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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?
?
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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
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Endpoints: Anal fin papillae
Effects of amylaniline on anal fin papillae
0 11 46 112 403 1430
Concentration (ppb)
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Endpoints: Liver Vitellogenin
0 11 46 112 403 1430
Concentration (ppb)
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0 11 46 112 403 1430
Concentration (ppb)
AAN Effects on Growth
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Endpoint: Fecundity
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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
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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 ?
?
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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.
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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