Developmental Toxicology II

1. Developmental Toxicology II Sid Hunter 6 November 2007 Hunter.Sid@EPA.GOV

2. Techniques and Models in TeratologyIssue?

5. Whole Embryo Culture System Trophoblast 3-5 Somite Embryo Amnionic Cavity Parietal Yolk Sac Allantois Reichert's Membrane Visceral Yolk Sac Visceral Yolk Sac Extraembryonic Cavity Ectoplacental Cone Ectoplacental Cone Fig. B Fig. A 30 rpm 30 ml Flask Fig. C Fig. D Schematic representation of the technique of embryo culture. The conceptus is removed from the uterus (Fig.A), dissected free of maternal tissues, trophoblasts, parietal yolk sac and Reichert's membrane (Fig.B), placed into a culture flask of medium (Fig.C), and rotated in an incubator at 30 rpm at 38C (Fig.D).

6. Day 8 Mouse Embryo

7. Embryos after 24 hours in Culture

9. Developmental Effects of Ethylene Glycol Metabolites in Mouse Whole Embryo Culture

10. Physiological changes induced by Ethylene glycol administration Bicarbonate pH Osmolality

11. Administration of sodium bicarbonate decreased the incidence of ethylene glycol- induced skeletal effects

12. Ethylene Glycol Example: • Administration of ethylene glycol produced species dependent induction of malformations • Ethylene Glycol did not alter rat embryonic development when conceptuses were directly exposed in vitro • Ethylene glycol metabolites do induce malformations in rat/mouse embryos grown in vitro • Metabolites have different potencies • Administration of Ethylene glycol produces physiological changes in the mother • Amelioration of maternal physiological changes decreases (BUT DOES NOT ELIMINATE) the teratogenic effects of Ethylene glycol administration

13. HYPOTHESIS Physiological changes induced by ethylene glycol administration are an important contributor to developmental effects AND Likely Proximate Teratogen?

14. Does administration of glycolic acid produce malformation and how do they compare to those produced by ethylene glycol

17. Ethylene Glycol Developmental Toxicity The developmental effects produced by ethylene glycol administration are may be due to glycolic acid and maternal acidosis Administration of glycolic acid recapitulates most but not all of the effects of ethylene glycol. (Follow-up study to evaluate relationship between AUC and defects / resorptions) Toxicity enhanced by or produced by maternal acidosis?

18. PBPK Model for Ethylene Glycol and Glycolic Acid

19. Effects of a 6-hour exposure to the HAA-metabolites

20. Administration of drug (D) results in distribution of the drug and its metabolites (M) The placenta is NOT a barrier to D or M distribution

21. You can not use disposition information from male or non-pregnant females to determine the distribution during pregnancy!!!!!

22. How do chemicals cross the Placenta?? Carrier-mediated transport facilitated diffusion: not energy dependent high substrate specificity follows [ ] gradient D-glucose active transport: energy dependent high substrate specificity can function against [ ] gradient amino acids; creatine; calcium receptor-mediated endocytosis: Immunogloblins (IgG) Iron-transferrin simple diffusion Not energy dependent Follows [ ] gradient lipophilic > hydrophilic size, structure, pKa depends on placental structure sheep = multilayer = no lactate transfer human = 3 layers = lactate transfer

23. Control of Rates of Xenobiotic Transfer across the Placenta Xenobiotics - primarily Passive Diffusion Embryo Embryo Some xenobiotics limit embryonic exposure by decreasing uterine blood flow

24. Control of Rates of Xenobiotic Transfer across the Placenta Xenobiotics - primarily Passive Diffusion pHi 7.64 (day 9) Mouse 7.61 (day 11) Rat 7.05 (day 14) Rat Plasma pH 7.26 Mouse 7.44 Rat 7.47 Rat

25. Sheep, Cow, Horse, Pig Epitheliochorial Dog, Cat, ferret Endotheliochorial Mouse, Rat, Rabbit, Human Haemochorial

27. Ratio of Chemicals in Fetus to Maternal Serum Concentrations

28. The rate of elimination may be as important as the rate of product formation for accumulation of a toxic intermediate

29. Techniques and Models in Developmental Toxicologyin vivo veritas !!

33. Techniques and Models in Developmental Toxicologyin vitro veritas !!

34. In vitro Developmental Toxicity Test Systems Cultured Mammalian Embryos / Primorida Non-Eutherian Embryos Primary Cell Culture Established Cell Lines

35. In vitro Developmental Toxicity Test Systems Established Cell Lines Human Embryonic Palate Mesenchyme cells Mouse ovarian tumour cells Neuroblastoma Cells Mouse/Human Embryonic Stem Cells

36. In vitro Developmental Toxicity Test Systems Non-Eutherian Embryos Medaka Zebrafish Xenopus laevis Frog Embryo Teratogenesis Assay Xenopus (FETAX) Drosophilia Chick Chick Embryotoxicity Screening Test (CHEST) Crickets Hydra

37. In vitro Developmental Toxicity Test Systems Cultured Mammalian Embryos / Primorida Whole Embryos - postimplantation Mouse Rat Rabbit Opossum Palatal Shelves / Head Limbs Mandibles It has been VERY difficult to successfully culture preimplantation-staged embryos to post-implantation stage. Chen LT, Hsu YC.Development of mouse embryos in vitro: preimplantation to the limb bud stage.Science. 1982 Oct 1;218(4567):66-8. PMID: 7123220

38. Whole Embryo Culture System Trophoblast 3-5 Somite Embryo Amnionic Cavity Parietal Yolk Sac Allantois Reichert's Membrane Visceral Yolk Sac Visceral Yolk Sac Extraembryonic Cavity Ectoplacental Cone Ectoplacental Cone Fig. B Fig. A 30 rpm 30 ml Flask Fig. C Fig. D Schematic representation of the technique of embryo culture. The conceptus is removed from the uterus (Fig.A), dissected free of maternal tissues, trophoblasts, parietal yolk sac and Reichert's membrane (Fig.B), placed into a culture flask of medium (Fig.C), and rotated in an incubator at 30 rpm at 38C (Fig.D).

39. Mouse Embryos 24H Culture PreCulture

40. Effects of LY294002 on embryonic development 10µM 75µM 50µM

41. Log (1/BD) = a(Pka) - b(Elumo) + c; r2 = 0.94

42. Morphological effects of HAAs and 3 metabolites in Mouse WEC

43. Short Term Exposure to xenobiotics Control Medium Begin Culture Period Morphological Evaluation Exposure to toxicant

44. Toxicity depends upon both concentration and length of exposure

45. Time Dependent induction of dysmorphogenesis by DCA, DBA and BCA in Whole Embryo Culture

46. Whole Embryo Culture Advantages No Maternal Metabolism Know what embryo is exposed to No Maternal Disposition Know how long embryo is exposed Select Embryos at All embryos have same # somites beginning of culture All embryos have same sensitivity Ability to manipulate Embryo Overexpress genes Knock-down expression Compare sensitivity across strains and species Mechanistic Studies Disadvantages No Maternal Metabolism No Maternal Disposition Select Embryos at beginning of culture No ability to assess effects of compound on the mom

47. Embryonic Stem Cell Test (EST) Embryonic stem cells are a permanent cell line established from the ICM of embryos The basis of the EST is to determine 3 endpoints IC50 for inhibition of ESC differentiation to beating heart cells IC50 for induction of cell death in ESC IC50 for induction of cell death in 3T3 cells