1. Dose-Response for Reproductive Effects of ER Binders P. Schmieder R. Johnson

2. Acknowledgements: In Vitro- J. Denny; R. Kolanczyk; Barb Sheedy; Mark Tapper Students: R. Maciewski; W. Backe; M. Dybvig; M. Mereness Post-Doc: H. Aladjov In Vivo- K. Flynn; D. Hammermeister; D. Lothenbach; F. Whiteman; Students: J. Nagel; W. Backe Post-Doc: M. Haasch

3. QSAR Inert/Antimicrobial Chemical ER Binding Altered Protein Expression Altered proteins, hormones; Ova-testis Sex reversal; Altered behavior; Repro. Estrogen Receptor (ER) Toxicity Pathway Chemical interacts with Receptor at Molecular Level, Initiates a Series of events, and leads to Adverse Outcome Toxicological Understanding Risk Assessment Relevance In vivo Assays MOLECULAR Initiating Event CELLULAR Response TISSUE/ORGAN INDIVIDUAL Skewed Sex Ratios; Yr Class POPULATION In vitro Assays

4. ER Prioritization System – Establishing Chemical Categories Test chemicals within FI and AM inventories for potential to bind ER to prioritize which chemicals, of hundreds EPA is required to evaluate, should go on for further testing. Focus in on the chemical likely to have the activity. Predictions are needed for chemicals with little of no data EPA problem is low affinity chemicals

6. Chemical Universe Contains Cycle Yes Contains 2 OH, or OH and =0, at Spec. Dist Possible High Affinity, “A-B”; “A-C”; or “A-B-C” type binder Contains some attenuating feature steric?; other? High Binding Affinity “A-B”;“A-C” or “A-B-C” type No Non binder Ex: Progesterone Corticossterone (RBA<0.00001) Special Rule Classes Log Kow <1.3 Low Affinity Binder “A-B”,“A-C” or “A-B-C” type Assess strength of attenuation steric?; other? Some Complete Contains at least one possible H- bonding site Type “A” Contains Phenol Fragment Alkyl Anilines Phthalates Branched Phenones Cyclo Phenones p-subst Cyclohexanols p-subst Cyclohexanones ring subst (o-CH3, tri-CH3) Benzoates V Possible Low Affinity Binder Type “B” Contains identif. Type B Fragment N-alkyl Phenones Non ring subst Benzoates Alkyl Phenols Alkoxy Phenols Parabens Salicylates Belongs to known Active class Potency Rules / Equations No Potency Rules / Equations No Yes No II Needs testing Yes No Yes Belongs to class with possible Type B low affinity under investigation Belongs to known Inactive class III Belongs to known Inactive class Fully-hindered Alkyl Phenols Belong to untested class with possible Type A low affinity ring subst p-CH3 Benzoates Thiophosphate Esters Mixed Organics Cyclic Alcohols (not p-subst hexanols) Cyclic Pentanones/Others Br, I halobenzenes No Yes Belongs to known Active class Belong to untested class Needs testing Non binder (RBA<0.00001) Mixed Phenols No Yes III Non binder (RBA<0.00001) No Belongs to known Inactive class Non binder (RBA<0.00001) Alkylbenzsulfonic Acids Sulfonic Acid Dyes p-Alkyl Fluorobenzenes Bis–anilines Alkoxy Anilines Imidazolidines Isothiazolines Alkyl Benzthiols Pyrrolidiones IV Yes Oxazoles Benzamide Furans Sorbitans Triazines Yes No Yes DDT-Like Tamoxifen-Like Multi-Cyclohydrocarbons Alkyl Chlorobenzenes

9. Will a chemical with rtER binding affinity 0.001 in our assay have ER-mediated effects in vivo? How do you compare dosimetry and effects across in vitro and in vivo assay systems? Dosimetry: Chemical concentration – How much? Where? Total Chemical Conc; Bound vs Free – internal bioavailability Chemical Solubility Effects: Biological Response; Toxicity How do you compare data across levels of biological organization?

10. Comparing Biological Response Comparing Dosimetry

11. ER Toxicity Pathway In vitro Assay Endpts ER Binding Altered Protein Expression Chg 2ndry Sex Char Altered Repro. Skewed Sex Ratios, Altered Repro. MOLECULAR Initiating Event CELLULAR Respopse TISSUE/ORGAN Response INDIVIDUAL Response POPULATION Response Altered proteins (VTG), Ova-testis In vivo Assay Endpts

12. ER Pathway ER Binding Altered Protein Expression Chg 2ndry Sex Char Altered Repro. Skewed Sex Ratios, Altered Repro. MOLECULAR Initiating Event CELLULAR Respopse TISSUE/ORGAN Response INDIVIDUAL Response POPULATION Response In vitro Assay Endpts In vivo Assay Endpts Trout cyto rtER Binding MaleTrout Liver Slice Vtg Induction Altered proteins (VTG), Ova-testis

13. The in vitro Assays rtER Binding – 3 H-E2 displacement cytosolic fraction from M, F trout livers cytosol diluted in TEDG buffer (Tris, EDTA, dithiothreitol, glycerol); avg protein = 4.6 mg/ml, ± 1.2, (range 3.4 - 8.8) N=97 rtER Liver Slice Vtg Induction precision cut trout liver slices incubated in 1.4ml of L-15 buffer (10% FBS); ~ 3 mg/ml protein

14. Alkyl Anilines rtER Binding

15. Alkyl Anilines rtER Gene Expression

16. Comparing Biological Response

17. 4-n-amyl aniline Comparison are made across the assay systems using one chemical RBA = 0.001

18. rtER cytosolic receptor binding - blue rtER liver slice VTG - red 4-n-amylaniline

19. Comparing Dosimetry

20. MED Analytical rt cytosol 4-n-amylaniline (AAN) [33228443]

21. -3.92 -4.69 LogM

22. Total vs. free concentration of octylphenol in an estrogenicity reporter gene assay using SPME to measure “free” chemical Heringa M.B. et al. Environ. Sci. Technol. 2004, 38, 6263-6270 Picture from J. Hermens, Utrecht University Nominal / total concentration Free aqueous concentration Dose

23. [Effect]protein “free” Solubility Limit (LogMolar)(mg/ml)fraction(LogMolar) Water0.97-3.43 Cyto rtER EC50-3.770.27-2.88 Slice Vtg LOEC-4.33.10.20-2.76 MaxEC-4.03.10.20-2.76 4- n amyl aniline in: in vitro Assay Media

24. rtER cytosolic receptor binding - blue rtER liver slice VTG - red 4-n-amylaniline Binding EC50 [free]= -3.7 Vtg maxEC [free]= -4.7

25. Comparing Biological Response In Vivo Endpoints

26. ER Pathway ER Binding Altered Protein Expression Chg 2ndry Sex Char Altered Repro. Skewed Sex Ratios, Altered Repro. MOLECULAR Initiating Event CELLULAR Respopse TISSUE/ORGAN Response INDIVIDUAL Response POPULATION Response In vitro Assay Endpts In vivo Assay Endpts Trout cyto rtER Binding MaleTrout Liver Slice Vtg Induction Altered proteins (VTG), Ova-testis

27. Medaka Bioassay with p-n-amylaniline (AAN) Generation Weeks total 12345678910 123 F0 12345678 F1 Development Reproduction Adults (F0) exposed for 21 d; Offspring (F1) exposed for 8 wks

28. Ctrl-7.18-6.55-6.16-5.61-5.06 21d-LethalityF0 MALE NOEC50% F0 FEMALE NOEC25%100% Fecundity (eggs/female)F0 FEMALE NOECLOEC:↓↓ Body WeightFO MALE LOEC:↓ F0 FEMALE NA Liver VitellogeninFO MALE LOEC F0 FEMALE Sex Reversal (M to F)FO MALE Papil. Process Body WeightF1 MALE NOECLOEC:↓ F1 FEMALE NOECLOEC:↓ Liver VitellogeninF1 MALE NOECLOEC:↑↑ F1 FEMALE NOECLOEC: ↓↓ Sex Reversal (M to F)F1 MALE NOECLOEC:↓↓ Papil. Process Adults – 21d Eggs from exposed adults hatched and raised thru 8 wk exposure – F1 ER mediated responses were observed Some genetic males had only ovarian tissue with no sign of testes -4.87 96hr LC50 Newly hatched Lethality (reduced hatch)F1 M,F NOECLOEC:↑

29. Pathology -FO MALE NOECLOEC:↑ SpleenF0 FEMALE NOECLOEC:↑ Pathology-FO MALE none kidney hematopoesisF0 FEMALE none Pathology -F1 MALE LOEC:↑ SpleenF1 FEMALE NOECLOEC:↑ Pathology-F1 MALE NOECLOEC kidney hematopoesisF1 FEMALE NOEC Additional pathology observed is indicative of aromatic amine toxicity (via metabolic activation)

30. ER Toxicity Pathway In vitro Assay Endpts Trout cyto rtER Binding MaleTrout Liver Slice Vtg Induction ER Binding Altered Protein Expression Chg 2ndry Sex Char Altered Repro. Skewed Sex Ratios, Altered Repro. MOLECULAR Initiating Event CELLULAR Respopse TISSUE/ORGAN Response INDIVIDUAL Response POPULATION Response Altered proteins (VTG), Ova-testis In vivo Assay Endpts Male Medaka Liver Vtg Induction Female Medaka Liver Vtg decr. Male Medaka Chg in 2ndary sex characteristic; behavior? Papillary Processes Male Medaka Gonad Complete conversion to ovary Female Medaka Fecundity Hatch Sex Reversal Genetic Males to Phenotypic Females Reproductive Impairment, but is it EDC in F0??

31. Comparing Dosimetry

32. Acute Toxicity – 4-n-pentyl aniline In Vivo96h LC50 Species (mg/L)LogMolarSource Fathead minnow4.3 -4.58 ASTER-calc Medaka2.2-4.87MED-msrd Trout1.7-4.99ASTER-calc Activity = 96h LC50= -4.87=0.038 Water Solubility -3.45 21d LC50 Species LogMolarSource Medaka~-5.3msrd Activity = 21d LC50= -5.3=0.014 Water Solubility -3.45 How do we factor in time component in comparing effects?

33. Chronic ER-mediated Endpoint Vitellogenin Induction In vitro – Liver slice conc where Vtg induction was observed Media = -4.0 LogM In vivo – Lowest conc where Vtg Induction was observed Water = -6.16 LogM

34. rtER cytosolic receptor binding - blue rtER liver slice VTG - red 4-n-amylaniline

35. In vivo uptake of 4-n-amylaniline in Medaka Liver (7 d) Water Conc = 0.117 ppm; -6.14 LogMolar BCF =160

36. Medaka VTG induction in Males, [Water Conc]= -6.16 Estimate Liver Conc as 160 times Water Conc = -3.95 Trout Liver Slice Media Conc for Vtg induction = -4.0 We don’t have a measured free concentration in Medaka blood (although it can be estimated from Log Kow), but we do have a measured free fraction of AAN in slice media = 0.20. Correcting Vtg Induction conc for free fraction = -4.7 Activity = Effect conc (“free”) = -4.7 = 0.056 Water Solubility -3.45

37. Common Reference Point for comparing among in vitro assays and from in vitro to in vivo?

40. Couldn’t go from binding EC50 to in vivo Vtg, But could go from in vitro Vtg to in vivo Vtg if you get on a comparable conc basis Need to compare similar biological effect as well as get dosimetry on common scale