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Multiendpoint Profiling of Hepatotoxicants in Vitro

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Presentation on theme: "Multiendpoint Profiling of Hepatotoxicants in Vitro"— Presentation transcript:

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

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

4 Botanical Products Associated With Hepatotoxicity (from Willet et al
Common Names Suspected Toxic Principle Crotalaria spp. Rattleweed, Sunnhemp pyrrolizidine alkaloids Heliotropium spp. White Heliotrope Symphytum spp. Comfrey Teucrium chamaedrys Germander neoclerodane diterpenes Lycopodium serratum Jin Bu Huan levo-alkaloid Piper methysticum Kava kava kavapyrones Hedeoma, Mentha Pennyroyal pulegone Larrea tridentata Chaparral nordihydroguaiaretic acid Chelidonium majus Greater celandine ? alkaloid Callilepsis laureola Impila atractyloside Atractylis gummifera Gum Thistle Scutellaria Skullcap ? Polygonum multiflorum He Shon Wu June 6, 2004 T.J. Flynn

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

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

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

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

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

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

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

15 Compounds Used for Test System Pre-Validation
Chemical Class Biological Activity Hepatotoxicity Acetaminophen Phenolic Human drug (analgesic, antipyretic) Yes Androstenedione Steroid Androgen, estrogen precursor Daidzein Isoflavone Phytoestrogen, antioxidant Estriol Estrogenic Fumonisin B1 Mycotoxin Inhibits sphingolipid synthesis Genistein Phytoestrogen, PK inhibitor, antioxidant Glycochenodeoxy-cholate Detergent (bile salt) June 6, 2004 T.J. Flynn

16 Compounds Used for Test System Pre-Validation (cont’d)
Chemical Class Biological Activity Hepatotoxicity a-Naphthoflavone Flavone (synthetic) CYP inhibitor b-Naphthoflavone CYP inducer NDGA Polyphenol Antioxidant Yes Quercetin Flavone Antioxidant, CYP3A4 inhibitor, phospho-diesterase inhibitor Testosterone Steroid Androgen, anabolic Valproic acid Short-chain carboxylic acid Human drug (anticonvulsant) June 6, 2004 T.J. Flynn

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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