Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies.
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 B(1), a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1 microM, 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 microM aflatoxin B(1) 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.
Jossé
Rozenn
R
Institut National de la Santé et de la Recherche Médicale U620, Université de Rennes 1, Rennes, France.
Aninat
Caroline
C
Glaise
Denise
D
Dumont
Julie
J
Fessard
Valérie
V
Morel
Fabrice
F
Poul
Jean-Michel
JM
Guguen-Guillouzo
Christiane
C
Guillouzo
André
A
D000328:Adult; D016604:Aflatoxin B1 / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D005260:Female; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D010615:Phenacetin / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D002273:Carcinogens; D012333:RNA, Messenger; D013739:Testosterone; D016604:Aflatoxin B1; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010615:Phenacetin