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. In this study, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several cytochromes P450 (P450s), UDP-glucuronosyltransferases, transporters, transcription factors, and 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-P450 reductase, cytochrome b5, and albumin with prolonged time of confluence. By using the CYP3A4-specific substrates luciferin 6′ benzyl ether, 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 show that Huh7 cells grown confluent differentiate into a more metabolically competent cell line, especially with regard to CYP3A4.
Footnotes
This work was supported by The Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning; AstraZeneca; and The Swedish Research Council.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.032367.
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ABBREVIATIONS:
- P450
- cytochrome P450
- DMSO
- dimethyl sulfoxide
- HL
- human liver
- PXR
- pregnane X receptor
- LDA
- low-density array
- TBP
- TATA-box binding protein
- UGT
- UDP-glucuronosyltransferase
- KCZ
- ketoconazole
- POR
- NADPH-cytochrome P450 reductase
- LDH
- lactate dehydrogenase
- PCR
- polymerase chain reaction
- CYB5A
- cytochrome b5 type A
- HNF
- hepatic nuclear factor.
- Received January 22, 2010.
- Accepted March 16, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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