Establishment And Application Of Cell Model Stably Expressing HOAT1and HCYP1A2

The MDCK cell models with stable expressing human organic anion transporter1(OAT1) and/or cytochrome P4501A2(CYP1A2) were established in present study. The MDCK-hOATl cell model has been applied to screen potential inhibitors/substrates of OAT1from Chinese herbs. The toxicity of aristolochic acid I was evaluated on single (MDCK-OAT1,MDCK-CYP1A2) and double-transfected (MDCK-OAT1/CYP1A2) MDCK cells.Objective:To establish MDCK cell models with stable expressing human organic anion transporter1(OAT1) and/or cytochrome P4501A2(CYP1A2), and use them as screening models.Methods:Transfected MDCK cells with recombinant plasmid pcDNA3.1(+)-hOAT1, after G418screening, monoclones were obtained by limiting dilution. Functional monoclones were selected and evaluated by uptake of the fluorescent substrate,6-carboxyfluorescein (6-CFL). The Real-Time PCR Analysis (RT-PCR) was employed to detect the expression of hOATl at transcription level. The function of monoclones was evaluated by measuring the accumulation of p-aminohippurate (PAH), an hOAT1classical substrate. And recombinant plasmids pcDNA3.1/Hypgro-CYP1A2were transfected into MDCK and MDCK-OAT1cells to construct the single-transfected cell line MDCK-CYP1A2and double-transfected cell line MDCK-OAT1/CYP1A2. The selection of monoclones with high CYP1A2activities was performed by the P450-Glo CYP1A2assay. The expression of CYP1A2in the transfected cell line was determined by RT-PCR. Then the MDCK-OAT1cell model was used to screen potential inhibitors from ingredients from traditional Chinese medicines. The toxicity of aristolochic acid I was evaluated in single (MDCK-OAT1,MDCK-CYP1A2) and double-transfected (MDCK-OAT1/CYP1A2) MDCK cells.Results:Human OAT1was highly expressed in transfected cells comparing with controls by RT-PCR, and Western blot. The results from accumulation of PAH and6-CFL also indicated the high OAT1function of the MDCK-OAT1cells. The selection of monoclones with high CYP1A2activities in the single/double transfected cell line was performed by the P450-Glo CYP1A2assay. The cells with high CYP1A2activities were characterized at mRNA level. Several active ingredients of traditional Chinese medicines such as aristolochic acid I inhibit6-CFL uptake in a concentration-dependent manner. The toxicity of aristolochic acid I increased in the MDCK-OAT1cells and MDCK-CYP1A2cells through MTT assay. In the double-transfected MDCK-OAT11/CYP1A2cell model, the toxicity increased dramatically because of the interplay between OAT1and CYP1A2.Conclusions:Cell models with stable-expressed hOAT1and/or CYP1A2are successfully established. These models can be employed to screen inhibitors/substrates of hOAT1or CYP1A2. The cell models could be also applied to study the toxicity and detoxification of chemicals due to the metabolism by CYP1A2and the uptake through OAT1.