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Research ArticleArticle

Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation

Hiroko Hosomi, Tatsuki Fukami, Atsushi Iwamura, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition August 2011, 39 (8) 1388-1395; DOI: https://doi.org/10.1124/dmd.110.037077
Hiroko Hosomi
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Tatsuki Fukami
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Atsushi Iwamura
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Miki Nakajima
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Tsuyoshi Yokoi
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Abstract

Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6β-hydroxylase activity (670 pmol · min−1 · mg−1). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the drug-induced cytotoxicity associated with CYP3A4.

Footnotes

  • This work was supported by Health and Labor Science Research Grants from the Ministry of Health, Labor, and Welfare of Japan [Grant H20-BIO-G001].

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.037077.

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    Nrf2
    nuclear factor erythroid 2-related factor 2
    GCLC
    glutamate-cysteine ligase, catalytic subunit
    GCLM
    glutamate-cysteine ligase, modifier subunit
    3-HAA
    3-hydroxyacetanilide
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    GAPDH
    glyceraldehyde-3-phosphate dehydrogenase
    MOI
    multiplicity of infection
    WST-8
    2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt
    TBF-A
    7,7-dimethylhept-2-ene-4-ynal.

  • Received November 4, 2010.
  • Accepted May 3, 2011.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (8)
Drug Metabolism and Disposition
Vol. 39, Issue 8
1 Aug 2011
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Research ArticleArticle

Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation

Hiroko Hosomi, Tatsuki Fukami, Atsushi Iwamura, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition August 1, 2011, 39 (8) 1388-1395; DOI: https://doi.org/10.1124/dmd.110.037077

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Research ArticleArticle

Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation

Hiroko Hosomi, Tatsuki Fukami, Atsushi Iwamura, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition August 1, 2011, 39 (8) 1388-1395; DOI: https://doi.org/10.1124/dmd.110.037077
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