Multiendpoint Profiling of Hepatotoxicants in Vitro

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Multiendpoint Profiling of Hepatotoxicants in
Vitro

• Thomas J. Flynn, Ph.D.
• FDA, Center for Food Safety and Applied Nutrition

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Disclaimer

• The views presented are those of the speaker and
  not necessarily of the FDA/CFSAN.

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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

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Botanical Products Associated With
Hepatotoxicity(from Willet et al., 2004)
Botanical Common Names Suspected Toxic Principle
Crotalaria spp. Rattleweed, Sunnhemp pyrrolizidine alkaloids
Heliotropium spp. White Heliotrope pyrrolizidine alkaloids
Symphytum spp. Comfrey pyrrolizidine alkaloids
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 atractyloside
Scutellaria Skullcap ?
Polygonum multiflorum He Shon Wu ?
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• How do you develop a relevant in vitro model for
  hepatotoxicity?

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Mechanisms of Hepatotoxicity

• Cell Death (necrosis, apoptosis)
• Cholestasis
• Steatosis
• Phospholipidosis
• Oxidative stress
• Mitochondrial dysfunction
• Modulation of CYP activities

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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

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Apoptosis

• ApoStrand
• Caspase-3
• (Maximum sensitivity at 4 hr post-treatment)

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Steatosis Phospholipidosis

• Nile red uptake
• (McMillian et al., In Vitro Mol. Toxicol. 14
  177-190, 2001)

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Oxidative Stress

• Dichlorofluorescin diacetate oxidation
• (Yerushalmi et al., Hepatology 33 616-626,
  2001).
• Glutathione depletion
• DNA strand breaks
• Lipid peroxides (TBARS)

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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?)

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Modulation of CYP450 Activities

• EROD (CYP1A), BOROD (CYP3A)
• (Donato et al., Anal. Biochem. 213 29-33, 1993)
• Testosterone hydroxylation (multiple CYP)

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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)

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Cell Lines Evaluated

• HepG2 (human hepatocarcinoma)
• HepG2/C3A (human hepatocarcinoma)
• WRL68 (heteroploid human fetal liver)
• Clone-9 (normal (?) rat liver)

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Compounds Used for Test System Pre-Validation
Compound Chemical Class Biological Activity Hepatotoxicity
Acetaminophen Phenolic Human drug (analgesic, antipyretic) Yes
Androstenedione Steroid Androgen, estrogen precursor
Daidzein Isoflavone Phytoestrogen, antioxidant
Estriol Steroid Estrogenic
Fumonisin B1 Mycotoxin Inhibits sphingolipid synthesis Yes
Genistein Isoflavone Phytoestrogen, PK inhibitor, antioxidant
Glycochenodeoxy-cholate Steroid Detergent (bile salt) Yes
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Compounds Used for Test System Pre-Validation
(contd)
Compound Chemical Class Biological Activity Hepatotoxicity
a-Naphthoflavone Flavone (synthetic) CYP inhibitor
b-Naphthoflavone Flavone (synthetic) CYP inducer
NDGA Polyphenol Antioxidant Yes
Quercetin Flavone Antioxidant, CYP3A4 inhibitor, phospho-diesterase inhibitor
Testosterone Steroid Androgen, anabolic Yes
Valproic acid Short-chain carboxylic acid Human drug (anticonvulsant) Yes
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96-Well Plate Template
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Assay Protocol - 1
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Assay Protocol - 2
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Typical Assay Results
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Log-Log Regression
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DCFDA Assay
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Rhodamine 123 Assay
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Nile Red Assay
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EROD - 1
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EROD - 2
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BOROD - 1
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BOROD - 2
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DNA Assay
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Species Comparisons?
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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.