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Development of an in vitro Hepatocyte Model to Investigate Chemical Mode of Action
Citation:
Hester, S., L. A. MURPHY, J. HARRILL, AND C. CORTON. Development of an in vitro Hepatocyte Model to Investigate Chemical Mode of Action. Presented at Society of Toxicology (SOT) Annual Meeting, Washington, DC, March 06 - 10, 2011.
Impact/Purpose:
Our goal is to use in vitro models for mode of action prediction which recapitulate critical cellular processes underlying in vivo hepatotoxicity and which express key mediators of hepatotoxicant modes-of-action, including nuclear receptors.
Description:
There is a clear need to identify and characterize the potential of liver in vitro models that can be used to replace animals for mode of action analysis. Our goal is to use in vitro models for mode of action prediction which recapitulate critical cellular processes underlying in vivo hepatotoxicity and which express key mediators of hepatotoxicant modes-of-action, including nuclear receptors. We assessed the potential of an immortalized mouse liver cell line, AML-12, as an in vitro model for detecting chemical-mediated increases in cell proliferation. Cultures were exposed to several reference compounds known to increase hepatocyte proliferation in vivo and which activate proliferation through different MOA, including: Tumor Necrosis Factor-alpha (TNF-a), WY-14,643, Epidermal Growth Factor (EGF), the fungicide propiconazole and phenobarbital. Cell proliferation was assessed by incorporation of BrdU at 4, 24, and 48 hrs using the Cellomics ArrayScan VTI high content image analysis platform. BrdU incorporation increased after phenobarbital (200, 500, and 1000 uM), WY-14,643 (25, 50 uM), EGF (5 uM) and propiconazole (10, 25, and 50 uM) exposure. In addition, more than 50 other cellular measurements were captured including number of nuclei, nuclear area, cell count per field, BrdU % positive, average intensity of BrdU positive cells on a well to well basis. Future assessments of this in vitro mouse model will include markers for cytotoxicity and apoptotic endpoints. The coupling of phenotypic data with expression of biomarker genes of nuclear receptors will allow a prediction of MOA for chemicals of interest to EPA. This abstract does not reflect EPA policy.