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