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Research ArticleArticle

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Louise Sivertsson, Monica Ek, Malin Darnell, Irene Edebert, Magnus Ingelman-Sundberg and Etienne P. A. Neve
Drug Metabolism and Disposition June 2010, 38 (6) 995-1002; DOI: https://doi.org/10.1124/dmd.110.032367
Louise Sivertsson
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Monica Ek
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Malin Darnell
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Irene Edebert
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Magnus Ingelman-Sundberg
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Etienne P. A. Neve
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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.

  • 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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Drug Metabolism and Disposition: 38 (6)
Drug Metabolism and Disposition
Vol. 38, Issue 6
1 Jun 2010
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Research ArticleArticle

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Louise Sivertsson, Monica Ek, Malin Darnell, Irene Edebert, Magnus Ingelman-Sundberg and Etienne P. A. Neve
Drug Metabolism and Disposition June 1, 2010, 38 (6) 995-1002; DOI: https://doi.org/10.1124/dmd.110.032367

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Research ArticleArticle

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Louise Sivertsson, Monica Ek, Malin Darnell, Irene Edebert, Magnus Ingelman-Sundberg and Etienne P. A. Neve
Drug Metabolism and Disposition June 1, 2010, 38 (6) 995-1002; DOI: https://doi.org/10.1124/dmd.110.032367
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