title: Construction and application of double-transfected cells expressing the human transporter P-glycoprotein and cytochrome P450 3A4.
abstract: Intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP) enzymes are known to influence oral bioavailabilities of drugs. Recombinant plasmids pcDNA3.1/Hypgro/CYP3A4 were transfected into MDCK and MDCK-MDR1 cells to construct the single-transfected cell line MDCK-CYP3A4 and double-transfected cell line MDCK-MDR1/CYP3A4. The expression of CYP3A4 in the double-transfected cell line was determined by Western blot and its activity was detected by the metabolism assays of three substrates of CYP3A4, which were 7-benzyloxy-4-trifluoro-methylcoumarin (BFC), testosterone and midazolam. In addition, the selection of monoclones with high CYP3A4 activities in the single-tranfected cell line was performed by the P450 Glo CYP3A4 assay. Through MTT assay, the interaction between P-gp and CYP3A4 was preliminarily determined based on the changes of IC50 values. The results showed that paclitaxel detoxified in the single-transfected MDCK-MDR1 cell because of P-gp efflux. And it was also less toxic in the single-transfected CYP3A4 cell line due to the metabolism by CYP3A4. In the double-transfected MDCK-MDR1/CYP3A4 cell line, the toxicity decreased dramatically because of the interplay between P-gp and CYP3A4. Therefore, the cell model could be applied to study the toxicity and detoxification of chemicals due to the metabolism by CYP3A4 and the efflux through P-gp.
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University, Zhejiang, China.
authors.forename: H H
authors.initials: HH
authors.lastname: Hu
authors.forename: C
authors.initials: C
authors.lastname: Su
authors.forename: Y
authors.initials: Y
authors.lastname: Jiang
authors.forename: L S
authors.initials: LS
authors.lastname: Yu
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Tian
authors.forename: S Y
authors.initials: SY
authors.lastname: Xu
authors.forename: H
authors.initials: H
authors.lastname: Zhou
authors.forename: X
authors.initials: X
authors.lastname: He
authors.forename: H D
authors.initials: HD
authors.lastname: Jiang
authors.forename: S
authors.initials: S
authors.lastname: Zeng
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D002999:Clone Cells; D004396:Coloring Agents; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000235:genetics; D004285:Dogs; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C022616:thiazolyl blue