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June 6, 2004 T.J. Flynn1 Multiendpoint Profiling of Hepatotoxicants in Vitro Thomas J. Flynn, Ph.D. FDA, Center for Food Safety and Applied Nutrition.

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Presentation on theme: "June 6, 2004 T.J. Flynn1 Multiendpoint Profiling of Hepatotoxicants in Vitro Thomas J. Flynn, Ph.D. FDA, Center for Food Safety and Applied Nutrition."— Presentation transcript:

1 June 6, 2004 T.J. Flynn1 Multiendpoint Profiling of Hepatotoxicants in Vitro Thomas J. Flynn, Ph.D. FDA, Center for Food Safety and Applied Nutrition

2 June 6, 2004 T.J. Flynn2 Disclaimer The views presented are those of the speaker and not necessarily of the FDA/CFSAN.

3 June 6, 2004 T.J. Flynn3 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

4 June 6, 2004 T.J. Flynn4 Botanical Products Associated With Hepatotoxicity (from Willet et al., 2004) BotanicalCommon NamesSuspected Toxic Principle Crotalaria spp.Rattleweed, Sunnhemppyrrolizidine alkaloids Heliotropium spp.White Heliotropepyrrolizidine alkaloids Symphytum spp.Comfreypyrrolizidine alkaloids Teucrium chamaedrysGermanderneoclerodane diterpenes Lycopodium serratumJin Bu Huanlevo-alkaloid Piper methysticumKava kavakavapyrones Hedeoma, MenthaPennyroyalpulegone Larrea tridentataChaparralnordihydroguaiaretic acid Chelidonium majusGreater celandine? alkaloid Callilepsis laureolaImpilaatractyloside Atractylis gummiferaGum Thistleatractyloside ScutellariaSkullcap? Polygonum multiflorumHe Shon Wu?

5 June 6, 2004 T.J. Flynn5 How do you develop a relevant in vitro model for hepatotoxicity?

6 June 6, 2004 T.J. Flynn6 Mechanisms of Hepatotoxicity Cell Death (necrosis, apoptosis) Cholestasis Steatosis Phospholipidosis Oxidative stress Mitochondrial dysfunction Modulation of CYP activities

7 June 6, 2004 T.J. Flynn7 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

8 June 6, 2004 T.J. Flynn8 Apoptosis ApoStrand® Caspase-3 (Maximum sensitivity at 4 hr post-treatment)

9 June 6, 2004 T.J. Flynn9 Steatosis & Phospholipidosis Nile red uptake (McMillian et al., In Vitro Mol. Toxicol. 14: , 2001)

10 June 6, 2004 T.J. Flynn10 Oxidative Stress Dichlorofluorescin diacetate oxidation (Yerushalmi et al., Hepatology 33: , 2001). Glutathione depletion DNA strand breaks Lipid peroxides (TBARS)

11 June 6, 2004 T.J. Flynn11 Mitochondrial Dysfunction Rhodamine 123 uptake and retention (Rat et al., Cell Biol. Toxicol. 10: , 1994) (Measure 3 hr post exposure) (Also measures P-glycoprotein?)

12 June 6, 2004 T.J. Flynn12 Modulation of CYP450 Activities EROD (CYP1A), BOROD (CYP3A) (Donato et al., Anal. Biochem. 213: 29-33, 1993) Testosterone hydroxylation (multiple CYP)

13 June 6, 2004 T.J. Flynn13 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  g/mL or limit of solubility) Dose response – should not disregard usable data (e.g., EC 50 )

14 June 6, 2004 T.J. Flynn14 Cell Lines Evaluated HepG2 (human hepatocarcinoma) HepG2/C3A (human hepatocarcinoma) WRL68 (heteroploid human fetal liver) Clone-9 (normal (?) rat liver)

15 June 6, 2004 T.J. Flynn15 Compounds Used for Test System Pre- Validation CompoundChemical ClassBiological ActivityHepatotoxicity AcetaminophenPhenolicHuman drug (analgesic, antipyretic) Yes AndrostenedioneSteroidAndrogen, estrogen precursor DaidzeinIsoflavonePhytoestrogen, antioxidant EstriolSteroidEstrogenic Fumonisin B1MycotoxinInhibits sphingolipid synthesis Yes GenisteinIsoflavonePhytoestrogen, PK inhibitor, antioxidant Glycochenodeoxy- cholate SteroidDetergent (bile salt)Yes

16 June 6, 2004 T.J. Flynn16 Compounds Used for Test System Pre- Validation (cont’d) CompoundChemical ClassBiological ActivityHepatotoxicity  -Naphthoflavone Flavone (synthetic)CYP inhibitor  -Naphthoflavone Flavone (synthetic)CYP inducer NDGAPolyphenolAntioxidantYes QuercetinFlavoneAntioxidant, CYP3A4 inhibitor, phospho- diesterase inhibitor TestosteroneSteroidAndrogen, anabolicYes Valproic acidShort-chain carboxylic acid Human drug (anticonvulsant) Yes

17 June 6, 2004 T.J. Flynn17 96-Well Plate Template

18 June 6, 2004 T.J. Flynn18 Assay Protocol - 1

19 June 6, 2004 T.J. Flynn19 Assay Protocol - 2

20 June 6, 2004 T.J. Flynn20 Typical Assay Results

21 June 6, 2004 T.J. Flynn21 Log-Log Regression

22 June 6, 2004 T.J. Flynn22 DCFDA Assay

23 June 6, 2004 T.J. Flynn23 Rhodamine 123 Assay

24 June 6, 2004 T.J. Flynn24 Nile Red Assay

25 June 6, 2004 T.J. Flynn25 EROD - 1

26 June 6, 2004 T.J. Flynn26 EROD - 2

27 June 6, 2004 T.J. Flynn27 BOROD - 1

28 June 6, 2004 T.J. Flynn28 BOROD - 2

29 June 6, 2004 T.J. Flynn29 DNA Assay

30 June 6, 2004 T.J. Flynn30 Species Comparisons?

31 June 6, 2004 T.J. Flynn31 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.


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