Received for publication November 29, 2007.
Revised March 13, 2008.
Accepted for publication March 13, 2008.

Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies

Abstract

The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4 and 2E1, several phase II and antioxidant enzymes, membrane transporters, including OCT1 and BSEP, the nuclear receptors CAR and PXR and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B1, a potent hepatotoxicant and carcinogen, induced a dose–dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1µM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every two days exposure. Moreover, using the comet assay a dose-dependent DNA damage was observed after a 3h treatment of differentiated HepaRG cells with 1 to 5µM aflatoxin B1, in the absence of any cell damage and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver.

Key words: CAR, chronic toxicity, CYP expression, genotoxicity, hepatocytes, hepatotoxicity, in vitro toxicity assays, liver toxicity, PXR, toxicology