CYP3A4 catalytic activity is induced in confluent Huh7 hepatoma cells.
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 b(5), 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 B(1), 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.
Sivertsson
Louise
L
Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Nanna Svartz väg 2, SE-171 77 Stockholm, Sweden. Louise.Sivertsson@ki.se
Ek
Monica
M
Darnell
Malin
M
Edebert
Irene
I
Ingelman-Sundberg
Magnus
M
Neve
Etienne P A
EP
D016604:Aflatoxin B1; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D004347:Drug Interactions; D015870:Gene Expression; D006801:Humans; D007654:Ketoconazole / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D008874:Midazolam; D012293:Rifampin; D013739:Testosterone; D014157:Transcription Factors
D014157:Transcription Factors; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam; D007654:Ketoconazole; D012293:Rifampin