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Multiendpoint Profiling of Hepatotoxicants in Vitro

Multiendpoint Profiling of Hepatotoxicants in Vitro. Thomas J. Flynn, Ph.D. FDA, Center for Food Safety and Applied Nutrition. Disclaimer . The views presented are those of the speaker and not necessarily of the FDA/CFSAN. Hepatotoxicity Team. Chung Kim, Ph.D. - Pharmacokinetics

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Multiendpoint Profiling of Hepatotoxicants in Vitro

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  1. Multiendpoint Profiling of Hepatotoxicants in Vitro Thomas J. Flynn, Ph.D. FDA, Center for Food Safety and Applied Nutrition T.J. Flynn

  2. Disclaimer • The views presented are those of the speaker and not necessarily of the FDA/CFSAN. T.J. Flynn

  3. Hepatotoxicity Team • Chung Kim, Ph.D. - Pharmacokinetics • Paddy Wiesenfeld, Ph.D. – Apoptosis, Lipid metabolome • Saura Sahu, Ph.D. – Oxidative damage • Phil Sapienza, M.S. – Research chemist • Ivan Ross, M.S. – Research biologist • Widmark Johnson – Technical assistance T.J. Flynn

  4. Botanical Products Associated With Hepatotoxicity(from Willet et al., 2004) T.J. Flynn

  5. How do you develop a relevant in vitro model for hepatotoxicity? T.J. Flynn

  6. Mechanisms of Hepatotoxicity • Cell Death (necrosis, apoptosis) • Cholestasis • Steatosis • Phospholipidosis • Oxidative stress • Mitochondrial dysfunction • Modulation of CYP activities T.J. Flynn

  7. Cell Death (Necrosis) • Total double-stranded DNA (H33258) • (Rago et al., Anal. Biochem. 191: 31-34, 1990) • Resazurin reduction (“Alamar blue”) • LDH, ALT, AST, ALP release • Total ATP T.J. Flynn

  8. Apoptosis • ApoStrand® • Caspase-3 • (Maximum sensitivity at 4 hr post-treatment) T.J. Flynn

  9. Steatosis & Phospholipidosis • Nile red uptake • (McMillian et al., In Vitro Mol. Toxicol. 14: 177-190, 2001) T.J. Flynn

  10. Oxidative Stress • Dichlorofluorescin diacetate oxidation • (Yerushalmi et al., Hepatology 33: 616-626, 2001). • Glutathione depletion • DNA strand breaks • Lipid peroxides (TBARS) T.J. Flynn

  11. Mitochondrial Dysfunction • Rhodamine 123 uptake and retention • (Rat et al., Cell Biol. Toxicol. 10: 329-337, 1994) • (Measure 3 hr post exposure) • (Also measures P-glycoprotein?) T.J. Flynn

  12. Modulation of CYP450 Activities • EROD (CYP1A), BOROD (CYP3A) • (Donato et al., Anal. Biochem. 213: 29-33, 1993) • Testosterone hydroxylation (multiple CYP) T.J. Flynn

  13. Desirable Properties of an In Vitro Model • Display as many liver-specific functions as possible • Use post-mitotic cells (closer to “reality”) • Primary cells or cell lines at confluence • Low glucose medium (closer to “reality”) • “Reasonable” maximum dose (1000 mg/mL or limit of solubility) • Dose response – should not disregard usable data (e.g., EC50) T.J. Flynn

  14. Cell Lines Evaluated • HepG2 (human hepatocarcinoma) • HepG2/C3A (human hepatocarcinoma) • WRL68 (heteroploid human fetal liver) • Clone-9 (normal (?) rat liver) T.J. Flynn

  15. Compounds Used for Test System Pre-Validation T.J. Flynn

  16. Compounds Used for Test System Pre-Validation (cont’d) T.J. Flynn

  17. 96-Well Plate Template T.J. Flynn

  18. Assay Protocol - 1 T.J. Flynn

  19. Assay Protocol - 2 T.J. Flynn

  20. Typical Assay Results T.J. Flynn

  21. Log-Log Regression T.J. Flynn

  22. DCFDA Assay T.J. Flynn

  23. Rhodamine 123 Assay T.J. Flynn

  24. Nile Red Assay T.J. Flynn

  25. EROD - 1 T.J. Flynn

  26. EROD - 2 T.J. Flynn

  27. BOROD - 1 T.J. Flynn

  28. BOROD - 2 T.J. Flynn

  29. DNA Assay T.J. Flynn

  30. Species Comparisons? T.J. Flynn

  31. Conclusions • Each model compound generated a unique response pattern among the six endpoints evaluated. • The response pattern discriminated between the following pairs of closely related compounds: • Androstenedione - Testosterone • -Naphthoflavone - -Naphthoflavone • Daidzein - Genistein • The response pattern discriminated between multiple biological mechanisms of action. • For model compounds that are human drugs with known hepatotoxicity (acetaminophen and valproic acid), some endpoints responded at medium concentrations comparable to known human blood levels. T.J. Flynn

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