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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

A Cell-Based Reporter Gene Assay for Determining Induction of CYP3A4 in a High-Volume System

Judy Raucy, Lyndon Warfe, Mei-Fei Yueh and Scott W. Allen
Journal of Pharmacology and Experimental Therapeutics October 2002, 303 (1) 412-423; DOI: https://doi.org/10.1124/jpet.102.038653
Judy Raucy
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Lyndon Warfe
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Mei-Fei Yueh
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Scott W. Allen
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Abstract

Assessing the inducibility of CYP3A4 by various xenobiotics can predict potential drug interactions. In the present investigation, human hepatoma cells were stably integrated with either the CYP3A4 enhancer region and a luciferase reporter gene or the CYP3A4-luciferase construct and the human pregnane X receptor (PXR). Several colonies containing one to three copies of luciferase per cell were identified by Southern blot analysis. Those transformants producing high luciferase activity in response to rifampicin were used to standardize a 96-well plate screening system with minimal inter- and intraplate variability. Standardization also consisted of assessing viability of cells cultured in medium containing various serum concentrations. In cells maintained for 48 h in medium with less than 5% serum, a significant (p < 0.01) decline was observed in viability accompanied by altered induction. A defined serum-free medium also produced less viable cells but did not alter the inductive response. Treatment of transformants with various concentrations of rifampicin produced a dose-response curve with maximal induction at 10 μM (5.6 ± 0.18- and 2.1 ± 0.3-fold above dimethyl sulfoxide (DMSO)-treated cells in transformants with and without PXR, respectively). Of additional agents examined for their ability to induce CYP3A4, omeprazole (200 μM) was the most potent inducer (12.8 ± 1.9- and 2.4 ± 0.2-fold above DMSO-treated cells in transformants with and without PXR, respectively). Mifepristone and mevastatin produced modest induction (∼3-fold) in the cell line containing exogenous PXR, but produced less than 1.2-fold increases in cells lacking PXR. Thus, only potent inducers can be identified in the cell line without PXR. In contrast, cells containing the receptor can be used to rank CYP3A4 induction. Because a high volume of chemicals can be readily and accurately screened for their ability to induce CYP3A4 with this format, such a system could be valuable in the initial stages of preclinical drug development.

Footnotes

  • This research was supported by National Institutes of Health Grant GM58287 (to M.-F.Y.).

  • DOI: 10.1124/jpet.102.038653

  • Abbreviations:
    P450
    cytochrome P450
    PXR
    pregnane X receptor
    PCN
    pregnenolone 16α-carbonitrile
    bp
    base pair(s)
    hPXR
    human pregnane X receptor
    DMSO
    dimethyl sulfoxide
    PXRE
    pregnane X response element
    DMEM
    Dulbecco's modified Eagle's medium
    FBS
    fetal bovine serum
    HMM
    hepatocyte maintenance media
    TCDD
    2,3,7,8-tetrachlorodibenzo-p-dioxin
    SF
    serum-free media
    RLU
    relative light unit
    OMP
    omeprazole
    HTS
    high throughput screening
    CAR
    constitutive androstane receptor
    • Received May 9, 2002.
    • Accepted June 20, 2002.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 303 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 303, Issue 1
1 Oct 2002
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

A Cell-Based Reporter Gene Assay for Determining Induction of CYP3A4 in a High-Volume System

Judy Raucy, Lyndon Warfe, Mei-Fei Yueh and Scott W. Allen
Journal of Pharmacology and Experimental Therapeutics October 1, 2002, 303 (1) 412-423; DOI: https://doi.org/10.1124/jpet.102.038653

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

A Cell-Based Reporter Gene Assay for Determining Induction of CYP3A4 in a High-Volume System

Judy Raucy, Lyndon Warfe, Mei-Fei Yueh and Scott W. Allen
Journal of Pharmacology and Experimental Therapeutics October 1, 2002, 303 (1) 412-423; DOI: https://doi.org/10.1124/jpet.102.038653
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