title: Development of a whole-cell screening system for evaluation of the human CYP1A2-mediated metabolism.
abstract: Cytochrome P450 1A2 (CYP1A2) is an important member of cytochrome P450 involved in drug metabolism. In this study, a cell line, Huh7-1A2-I-E, with high expression level of CYP1A2 is established based on Huh7 cells. To achieve this, we constructed a recombinant lentiviral vector, pLenti-1A2-I-E, containing a single promoter encoding CYP1A2 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP1A2 and EGFP proteins would be based on a single bi-cistronic mRNA. The Huh7-1A2-I-E cells were evaluated as a cell-based model for identification of CYP1A2 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of Huh7-1A2-I-E cells and the Huh7-E control cells with aflatoxin B1 showed that cells with CYP1A2 expression are much more sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in Huh7-1A2-I-E cells could be prevented by furafylline, a CYP1A2 inhibitor. A collection of approximately 200 drugs were screened using this system and results indicate that for most drugs the metabolism by CYP1A2 is unlikely to have made a major contribution to the in vitro cytotoxicity except for thimerosal and evoxine. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study.
authors.affiliation: Department of Chemical Engineering, National Tsing-Hua University, Hsin-Chu, Taiwan, Republic of China.
authors.forename: Chih-Chieh
authors.initials: CC
authors.lastname: Chu
authors.forename: Kao-Lu
authors.initials: KL
authors.lastname: Pan
authors.forename: Hsien-Tsung
authors.initials: HT
authors.lastname: Yao
authors.forename: John Tsu-An
authors.initials: JT
authors.lastname: Hsu
mesh_terms: D001599:Berberine / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013806:Theophylline / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
chemical_list: D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D001599:Berberine; D013806:Theophylline; C050131:furafylline; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2