Abstract

Drug-induced hepatotoxicity is an important cause for disapproval, limitations of use, or withdrawal of drugs, and there is a high need for reproducible in vitro systems that can predict such toxicity. Here we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several CYPs, UDP-Glucuronosyltransferases, transporters, transcription factors, as well as several liver specific genes, as measured by low density array. The most striking effect was seen for CYP3A4 expression. Western blot analysis revealed increased amounts of CYP3A4 together with increased levels of NADPH-cytochrome P450 reductase, cytochrome b₅, and albumin with prolonged time of confluence. By using the CYP3A4 specific substrates luciferin 6′ benzyl ether (luciferin-BE), testosterone, and midazolam, we could confirm that the increased CYP3A4 gene expression also was accompanied by a similar increase in catalytic activity, inhibitable by the CYP3A4 selective inhibitor ketoconazole. The CYP3A4 activity in confluent cells was also inducible by rifampicin. Finally, the cell system could support the CYP3A4 dependent hepatotoxic activation of aflatoxin B₁, which was effectively inhibited by ketoconazole. Our results demonstrate that Huh7 cells grown confluent differentiates into a more metabolically competent cell line, especially with regard to CYP3A4.