Construction and application of double-transfected cells expressing the human transporter P-glycoprotein and...

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.