Evaluation of Induction of CYP3A mRNA Using the HepG2 Cell Line and Reverse Transcription-PCR.

Sumida, Akihiko; Yamamoto, Ichiro; Zhou, Qian; Morisaki, Tomoko; Azuma, Jun-ichi

doi:10.1248/bpb.22.61

Cited by 15 publications

(16 citation statements)

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“…5). These results were consistent with those reported previously by Sumida et al 25) and Usui et al 35) Hewitt and Hewitt 36) reported that HepG2 cells have phase I enzymes, i.e., CYP1A, CYP2C9, CYP2D6, CYP2B6, CYP2E1, and CYP3A, but their activities and levels are dependent on the source and culture conditions. This discrepancy may have been due to differences in source and/or culture conditions.…”

Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wasupporting

confidence: 93%

“…The induction of CYP in HepG2 cells has been studied as a model of human liver. 15,25) In the present study, however, it was demonstrated that character of HepG2 cells with regard to CYP expression was different from that of HFL cells, although HepG2 cell lives has features of hepatoblast-like cells. 16,17) The maintenance of HFL cells is easier than that of the adult liver cells as described above.…”

Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wacontrasting

confidence: 61%

“…On the other hand, HepG2 cells that express fetal-specific proteins, such as CYP3A7 7) and AFP, 20) have been used in a number of studies of drug metabolism. 15,25) However, there have been no comparative studies of gene expression between HepG2 cells and cultured HFL cells, and only limited data are available regarding the comparative cellular responses of these two systems to inducers. The present study was performed to further characterize the utility of HFL cells as an in vitro system in which to assess the effects of drugs and chemicals on CYP expression in the fetal liver.…”

Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wamentioning

confidence: 99%

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Comparison of Basal Gene Expression and Induction of CYP3As in HepG2 and Human Fetal Liver Cells

Maruyama

Matsunaga

Harada

et al. 2007

Biological & Pharmaceutical Bulletin

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Cytochrome P450 (CYP) is a gene superfamily of hemoproteins that catalyze the oxidation of lipophilic substrates to more water-soluble products. Among them, the human CYP3A subfamily contains mainly three isoforms, CYP3A4, CYP3A5, and CYP3A7. 1) CYP3A4 is the predominant isoform expressed in adult human liver and intestine, 2,3) and plays a significant role in the metabolism of endogenous and exogenous compounds. 4) CYP3A5 is expressed polymorphically throughout development in the liver.5,6) CYP3A7 is a major CYP isoform in the human fetal and newborn liver. 7)The levels of expression of CYP3A isoforms are enhanced by treatment with various agents, such as rifampicin (RIF), phenobarbital, clotrimazole, and dexamethasone (DEX). [8][9][10] Unlike the fetal liver in most experimental animals, the human fetus supports the oxidative metabolism of a wide range of drugs and other xenobiotics. 11) Toxicological and pharmacological studies designed to assess the safety and efficacy of candidate drugs and chemicals in the human fetus depend greatly on extrapolation from animal models. However, it has been difficult to estimate alterations of drug metabolism by inducers of drug-metabolizing enzymes in the human fetus due to the problem of differences among species.12) Species differences in the inducibility of drug-metabolizing enzymes 13,14) result in important differences in the metabolism of drugs and potential carcinogens. The induction of CYP3A in human-derived cell lines has been studied as a model of human tissues.15) As research with human subjects, especially when pregnant, should be restricted due to ethical considerations, human fetal in vitro models have been evaluated with increasing frequency to find those that are most suitable for predicting human responses in vivo.The HepG2 cell line was established from a liver tumor biopsy specimen from a 15-year-old Caucasian male subject.16) This cell line has features of hepatoblast-like cells, because the cells express CYP3A7 17) and secrete a-fetoprotein (AFP) 16) into the cell culture medium, both proteins that are found predominantly in human fetal hepatocytes.7) However, it is unknown whether HepG2 is a useful cell line as a human fetal liver (HFL) model for experiments regarding drug metabolism, because there have been no comparisons of gene expression between HFL and HepG2 cells, and limited data are available regarding comparison of the responses of HepG2 and HFL cells to inducers.In the present study, to characterize the expression and induction of CYP in HFL cells, the basal gene expression patterns and inducibility of CYP, especially CYP3A isoforms, were compared to those in HepG2 cells. MATERIALS AND METHODS ChemicalsDulbecco's phosphate-buffered saline (PBS) was from Dainippon Pharmaceutical Co. (Osaka, Japan). Dulbecco's modified Eagle's medium and Williams' medium E were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Penicillin-streptomycin-neomycin antibiotic mixture and SuperScript First-Strand Synthesis System for reverse transcription-po...

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Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wasupporting

confidence: 93%

Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wacontrasting

confidence: 61%

Section: Expression Of Pxr and Gra A In Hepg2 And Hfl Cells Rt-pcr Wamentioning

confidence: 99%

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Comparison of Basal Gene Expression and Induction of CYP3As in HepG2 and Human Fetal Liver Cells

Maruyama

Matsunaga

Harada

et al. 2007

Biological & Pharmaceutical Bulletin

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“…1) and transiently transfected into HepG2 cells. Induction of endogenous CYP3A genes in rifampicintreated HepG2 cells has been previously demonstrated (Schuetz et al, 1993;Sumida et al, 1999). In line with observations by others (Hashimoto et al, 1993), the basal activity of all CYP3A4-luciferase reporter gene constructs was low (data not shown).…”

supporting

confidence: 88%

The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation ofCYP3A4by Rifampicin through a Distal Enhancer Module

1999

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Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases -7836 to -7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to approximately 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region and cis-acting elements in the proximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction of CYP3A4 by activators of hPXR.

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“…20) HepG2 cells have been suggested to be a useful in vitro model for investigation of the metabolism and toxicity of drugs compared with animal cells, since HepG2 cells retain many of the specialized functions which are characteristic of normal human hepatocytes, including the synthesis and secretion of plasma proteins. 21) In order to clarify the efficacy of soyasapogenol B and sophoradiol glucuronides and their aglycones on human-liver-derived cell lines, we investigated their hepatoprotective effects against the cytotoxicity of tBuOOH in HepG2 cells.…”

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confidence: 99%

Hepatoprotective Constituents in Plants 14. Effects of Soyasapogenol B, Sophoradiol, and Their Glucuronides on the Cytotoxicity of tert-Butyl Hydroperoxide to HepG2 Cells1)

Kinjo

Hirakawa

Tsuchihashi

et al. 2003

Biological & Pharmaceutical Bulletin

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Evaluation of Induction of CYP3A mRNA Using the HepG2 Cell Line and Reverse Transcription-PCR.

Cited by 15 publications

References 0 publications

Comparison of Basal Gene Expression and Induction of CYP3As in HepG2 and Human Fetal Liver Cells

Comparison of Basal Gene Expression and Induction of CYP3As in HepG2 and Human Fetal Liver Cells

The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation ofCYP3A4by Rifampicin through a Distal Enhancer Module

Hepatoprotective Constituents in Plants 14. Effects of Soyasapogenol B, Sophoradiol, and Their Glucuronides on the Cytotoxicity of tert-Butyl Hydroperoxide to HepG2 Cells1)

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