CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

Abstract

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 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.

Induced CYP3A4 Expression in Confluent Huh7 Hepatoma Cells as a Result of Decreased Cell Proliferation and Subsequent Pregnane X Receptor Activation

“…Previously, we have shown that Huh7 cells grown confluent up to 4 weeks achieve a more differentiated phenotype more similar to primary hepatocytes (Sivertsson et al, 2010). To investigate the change in gene expression during this phenotypical switch, a global gene expression analysis was performed.…”

“…Four-weeks confluent and subconfluent control cells, grown on glass cover slips, were fixed and stained as previously described (Sivertsson et al, 2010). The slides were incubated with goat anti-PXR (sc-9690; Santa Cruz Biotechnology) or rabbit anti-CDK2 (sc-163; Santa Cruz Biotechnology) antibodies, followed by anti-goat Cy3 (Jackson Immunoresearch, West Grove, PA) or anti-rabbit Alexa Fluor 488 (Invitrogen) conjugated antibodies.…”

“…The microsomal fraction from control and confluent Huh7 cells was isolated as previously described (Sivertsson et al, 2010). Equal amounts of protein (microsomal fractions, total-cell lysate, cytoplasmic extracts, or nuclear extracts) were mixed with Laemmli sample buffer and subjected to SDS polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose membranes as previously described (Sivertsson et al, 2010).…”

“…The microsomal fraction from control and confluent Huh7 cells was isolated as previously described (Sivertsson et al, 2010). Equal amounts of protein (microsomal fractions, total-cell lysate, cytoplasmic extracts, or nuclear extracts) were mixed with Laemmli sample buffer and subjected to SDS polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose membranes as previously described (Sivertsson et al, 2010). The membranes were incubated with goat anti-CYP3A4 (Wang et al, 2009), mouse anti-CDK2 (610145; BD Biosciences, San Jose, California), rabbit-CDK2 (sc-163; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-PXR (ab85451; ABcam, Cambridge, UK), followed by conjugated horseradish peroxidase secondary antibodies (Dako Denmark A/S, Glostrup, Denmark).…”

Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria

“…Western Blot Analysis-Subcellular fractions isolated from rat liver and cell lysates from adipocytes and preadipocytes were subjected to Western blot analysis as described previously (22).…”