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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF STEM CELL AND NUCLEAR CLONING TECHNOLOGIES IN UNGULATES Title: Cytochrome P450 Expression Profile of the PICM-19H Pig Liver Cell Line: Potential Application to Rapid Liver Toxicity Assays.

Authors
item Willard, Ryan
item Shappell, Nancy
item Meekin, John - HEPALIFE
item Talbot, Neil
item Caperna, Thomas

Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 29, 2009
Publication Date: November 14, 2009
Citation: Willard, R.R., Shappell, N.W., Meekin, J.H., Talbot, N.C., Caperna, T.J. 2009. Cytochrome P450 expression erofile of the PICM-19H pig liver cell line: Potential application to rapid liver toxicity assays. In Vitro Cellular and Developmental Biology - Animals. 46(1):11-19.

Interpretive Summary: In vitro models of the liver are needed to replace animal models for the rapid assessment of drug biotransformation and toxicity. he objective of this study was to characterize metabolic functions of a PICM-19 derivative cell line, PICM-19H. The PICM-19H cell line was also compared to the tumor-derived human HepG2 C3A cell line and to primary cultures of adult porcine hepatocytes (APH). Basal levels of cytochrome P450 (CYP) enzymes (Phase I system) were low, however following induction with known inducing agents CYP-1A,-2 and-3A were determined in PICM-19H cells. Relative to APH, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. PICM19H cells also demonstrated significant ability to form water soluble conjugates with test substrates, a function required to help in the elimination of xenobiotics. Other CYP450 isozyme activities were also confirmed by the conversion of testosterone to 6ß-OH testosterone, 2a-OH testosterone and 2ß-OH testosterone, as determined by mass spectrometry in cultures of PICM-19H cells. The susceptibility of PICM-19H cells to the known toxin was determined. Toxicity and bioactivation of aflatoxin following CYP450 induction was also determined indicating that the toxin was rendered more potent due to the action of CYP450s. The results demonstrate the potential for the use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

Technical Abstract: In vitro models of the liver are needed to replace animal models for the rapid assessment of drug biotransformation and toxicity. One hepatocellular model, the PICM-19 pig liver stem cell line, may fulfill this need since these cells have many activities associated with xenobiotic phase I and phase II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and phase II metabolic functions of a PICM-19 derivative cell line, PICM-19H. The PICM-19H cell line was also compared to the tumor-derived human HepG2 C3A cell line and to primary cultures of adult porcine hepatocytes (APH). Following 48 h induction with 5uM 3-methylcholanthrene (3-MC), 50uM rifampicin or 2 mM Phenobarbital, the activities of cytochrome (CYP450) isozymes CYP-1A,-2 and-3A in PICM-19H cells. Relative to APH, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated; 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with ß-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was also confirmed by the conversion of testosterone to 6ß-OH testosterone, 2a-OH testosterone and 2ß-OH testosterone, as determined by mass spectrometry. The susceptibility of PICM-19H cells to acetaminophen toxicity determined and the LC50 was calculated to be 12.65± 075 mM. Toxicity and bioactivation of aflatoxin B1 following CYP450 induction was determined in control and 3-MC treated cultures. PICM-19H cells treated with 3-MC had a LC50 of 1.1 µM compared to controls with an LC50 of 40 µM. The results demonstrate the potential for the use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

Last Modified: 12/22/2014
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