No 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.

Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems.

We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels. Here, the mechanisms behind were investigated, and a transcriptome analysis revealed significant up-regulation of liver-specific functions, whereas pathways related to proliferation and cell cycle were downregulated in the confluent cells. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR). PXR expression was increased, and PXR protein accumulated in the nuclei during confluent growth. The PXR ligand rifampicin further increased the expression of CYP3A4, and siRNAmediated knock-down of PXR in confluent cells resulted in decreased CYP3A4 expression. Cyclin-dependent kinase 2 (CDK2), a known modulator of the cell cycle and a negative regulator of PXR, was more highly expressed in proliferating control cells. Trypsinization of the confluent cells and replating them subconfluent resulted in a decrease in CYP3A4 and PXR expression back to levels observed in subconfluent control cells, whereas the CDK2 levels increased. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. A phosphorylationdeficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. In conclusion, the data strongly suggest that the increased CYP3A4 expression in confluent Huh7 cells is caused by the endogenous induction of PXR as a result of cell-cell contact inhibited proliferation and subsequent decreased CDK2 activities, indicating an endogenous, non-ligand-dependent regulation of PXR and CYP3A4, possibly of physiologic and pharmacological significance.