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 organic cation transporter 1 and bile salt export pump, the nuclear receptors constitutive androstane receptor and pregnane X receptor, 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 0.1 μM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every 2-day exposure. Moreover, using the comet assay, a dose-dependent DNA damage was observed after a 3-h treatment of differentiated HepaRG cells with 1 to 5 μM 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.
Footnotes
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This study was supported by European Economic Community Contracts LIINTOP-STREP-037499 and COMICS STREP 037575, Agence Nationale de la Recherche Contract 06SEST17, and by the Ligue 35 Contre le Cancer.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019901.
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ABBREVIATIONS: AFB1, aflatoxin B1; P450, cytochrome P450; 3-MC, 3-methylcholanthrene; DMSO, dimethylsulfoxide; MMS, methyl methane sulfonate; FCS, fetal calf serum; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; PBS, phosphate-buffered saline; OTM, Olive tail moment; UGT, UDP-glucuronosyl transferase; GST, glutathione transferase; mEH, microsomal epoxide hydrolase; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; OCT, organic cation transporter; BSEP, bile salt export pump; PXR, pregnane X receptor; CAR, constitutive androstane receptor; MnSOD, manganese superoxide dismutase.
- Received November 28, 2007.
- Accepted March 13, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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