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Research ArticleArticle

CYP2C9-Mediated Metabolic Activation of Losartan Detected by a Highly Sensitive Cell-Based Screening Assay

Atsushi Iwamura, Tatsuki Fukami, Hiroko Hosomi, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition May 2011, 39 (5) 838-846; DOI: https://doi.org/10.1124/dmd.110.037259
Atsushi Iwamura
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Tatsuki Fukami
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Hiroko Hosomi
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Miki Nakajima
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Tsuyoshi Yokoi
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Abstract

Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4′-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatography-tandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9.

Footnotes

  • This work was supported in part by Research on Advanced Medical Technology, Health and Labor Science Research 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.110.037259.

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

  • ABBREVIATIONS:

    P450
    cytochrome P450
    Nrf2
    nuclear factor-E2 p-45-related factor
    GFP
    green fluorescent protein
    GAPDH
    glyceraldehyde-3-phosphate dehydrogenase
    siRNA
    small interfering RNA
    HPLC
    high-performance liquid chromatography
    WST-8
    2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium monosodium salt
    LC
    liquid chromatography
    MS/MS
    tandem mass spectrometry
    LCMS-IT-TOF
    liquid chromatography ion trap time-of-flight mass spectrometry
    MOI
    multiplicity of infection
    BSO
    buthionine sulfoximine
    ALT
    alanine aminotransferase
    FLU-1
    4-nitro-3-(trifluoromethyl)phenylamine.

  • Received November 14, 2010.
  • Accepted February 14, 2011.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (5)
Drug Metabolism and Disposition
Vol. 39, Issue 5
1 May 2011
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Research ArticleArticle

CYP2C9-Mediated Metabolic Activation of Losartan Detected by a Highly Sensitive Cell-Based Screening Assay

Atsushi Iwamura, Tatsuki Fukami, Hiroko Hosomi, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition May 1, 2011, 39 (5) 838-846; DOI: https://doi.org/10.1124/dmd.110.037259

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Research ArticleArticle

CYP2C9-Mediated Metabolic Activation of Losartan Detected by a Highly Sensitive Cell-Based Screening Assay

Atsushi Iwamura, Tatsuki Fukami, Hiroko Hosomi, Miki Nakajima and Tsuyoshi Yokoi
Drug Metabolism and Disposition May 1, 2011, 39 (5) 838-846; DOI: https://doi.org/10.1124/dmd.110.037259
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