Induction of drug-metabolizing enzymes by phenobarbital in layered co-culture of a human liver cell line and endothelial cells.
ABSTRACT Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various CYP genes were significantly increased compared with the monolayer cultured HepG2 cells. Here, we examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Moreover, the induction of hepatic drug transporters by phenobarbital was detected. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism.
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ABSTRACT: Early in vitro toxicity screening might improve the success rate of new chemical entities in pharmaceutical development. In previous studies, the advantage of cytotoxicity screening with the HepG2 cell line was shown. Cytotoxicity could be identified for 70% of the compounds in these assays as compared with known toxicity in either in vitro assays in primary hepatocytes, in in vivo assays in rats, or in (pre-)clinical development in humans. The low Phase I and II enzyme levels in HepG2 cells might have been responsible for the fact that 30% of the compounds scored negative. Therefore, we performed two follow-up studies in which Cytochrome P450 (CYP) enzymes and Phase II metabolism were examined. In the present study, the transcript levels of CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 were measured with quantitative PCR. Results showed that transcripts of all CYPs were present in HepG2 cells, however, mRNA levels of most CYPs were dramatically lower than in primary human hepatocytes. These results were confirmed with luminometric assays which were used to measure the enzyme activities of CYP1A1, 1A2, 2C9, and 3A4. Regulation of CYP1A1, 1A2, 2B6, 2C8, 2D6, 2E1, and 3A4 by the aryl hydrocarbon receptor, pregnane X receptor and constitutive androstane receptor was studied in HepG2 cells at the mRNA and/or enzyme level. Regulation of CYP1A1, 1A2, 2B6, and 3A4 mRNA levels was similar to the regulation in primary human hepatocytes. In contrast, CYP2C8 mRNA levels are inducible in primary human hepatocytes, but not in HepG2 cells, after treatment with PXR/CAR activators. Consistent with other studies, CYP2D6 and 2E1 transcript levels were not changed after treatment with AhR, PXR, and CAR activators. Moreover, CYP1A1 and 1A2 enzyme levels could be induced by AhR agonists and CYP3A4 by PXR agonists. As a consequence of the low levels of CYPs in HepG2 cells, cytotoxicity of several compounds might have been missed or underestimated as compared with cytotoxicity in primary human hepatocytes. Inducing HepG2 cells with particular receptor stimulators might lead to higher toxicity for several of the tested compounds. Compared to primary human hepatocytes, HepG2 cells are a relatively easy-to-handle tool to study the up-regulation of CYP1A1, 1A2, 2B6, and 3A4.Toxicology in Vitro 01/2008; 21(8):1581-91. · 2.65 Impact Factor
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ABSTRACT: To mimic native tissue function, coculture systems are an extremely useful model. In many cases, differentiated functions can be maintained only through the interactions of various cell types. Therefore, methods for examining the interactions between cocultured cells are necessary. The assessment of cell-to-cell cross-talk at the level of gene expression is one such method to examine interactions between different cell types. However, it is generally difficult to determine the gene expression of specific cell types in coculture without first separating cell populations. To overcome these obstacles, we have established a novel method to determine gene expression levels of a targeted cell population in coculture, using species-specific primers. With this approach, we were able to determine hepatocyte-specific gene expression of Fao cells (a rat hepatocyte cell line) in culture with human umbilical vein endothelial cells (HUVECs). Expression of both albumin and apolipoprotein A-I (apoA-I) increased time dependently for 10 days and maintained significantly higher expression in the coculture system as compared with isolated Fao cells. This indicates that hepatocyte function increased gradually in our coculture system and could be maintained long-term, suggesting that the construction of mature cell-to-cell communication between the two cell lines required a considerable amount of time. The expression of HNF-4 and HNF-1alpha, which are liver-enriched transcription factors, did not differ between the monolayer and cocultured Fao cells, suggesting that expression of HNF-4 and HNF-1alpha was not responsible for the increased expression albumin and apoA-I. Our findings suggest that this novel method for the detection of gene expression of targeted cell populations can be a useful tool in determining the molecular mechanisms that regulate communication between different cell types.Tissue Engineering 01/2005; 11(11-12):1650-7. · 4.07 Impact Factor
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ABSTRACT: Although CYP2C8, CYP2C9, and CYP2C19 play an important role in drug biotransformation, factors influencing the expression and activity of these CYP2C P450s in human liver remain largely undefined. We used primary cultures of human hepatocytes from 15 subjects to assess the inducibility of CYP2C enzyme expression by prototypical inducer agents, including rifampicin, dexamethasone, and phenobarbital. After culture for 72 h in serum-free medium on collagen, Western blotting revealed that CYP2C9 was the only CYP2C enzyme expressed at appreciable levels in untreated hepatocytes. Subsequent treatment with 25 microMrifampicin for 48 h elicited marked increases in CYP2C8 (700 +/- 761%), CYP2C19 (854%), and CYP2C9 (209 +/- 176%) protein content versus a 550 +/- 170% enhancement of CYP3A4 enzyme levels. Parallel increases in CYP2C mRNAs, measured by Northern blotting and/or RNase protection, were found in rifampicin-treated hepatocytes, with CYP2C8, CYP2C9, and CYP2C19 transcripts exhibiting increases of 688 +/- 635, 207 +/- 49, and 230 +/- 60%, respectively, versus an 8.8-fold enhancement of CYP3A4 mRNA levels. Dexamethasone (10 microM) treatment enhanced CYP2C8 mRNA (360 +/- 100%) and protein (274%) content, although this steroid had less effect on CYP2C9 and CYP2C19 transcripts (23 +/- 21% and 21 +/- 36%, respectively) and enzyme levels (55 and 143%, respectively). Phenobarbital (100 microM) was a powerful inducer of CYP2C9 (850%) and CYP2C19 (735%) mRNA content, and also increased CYP2C8 (610%) and CYP3A4 (205%) transcripts. Our results show that CYP2C enzyme expression in human hepatocytes is highly inducible by rifampicin, dexamethasone, and phenobarbital. Because these xenobiotics are ligands and/or activators of the pregnane X receptor and/or constitutive androstane receptor, such orphan nuclear receptors and their response elements may partake in regulating CYP2C gene expression in humans.Journal of Pharmacology and Experimental Therapeutics 09/2002; 302(2):475-82. · 3.89 Impact Factor