Gleich Alexander, Kaiser Bastian, Honscha Walther, Fuhrmann Herbert, Schoeniger Axel
Institute of Biochemistry, University of Leipzig, An den Tierkliniken 1, 04103, Leipzig, Germany.
Institute of Veterinary Physiology, University of Leipzig, An den Tierkliniken 7, 04103, Leipzig, Germany.
Cytotechnology. 2019 Feb;71(1):231-244. doi: 10.1007/s10616-018-0279-4. Epub 2019 Jan 8.
The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux transporters ABCG2 and ABCC1. The EROD assay revealed a dose-dependent CYP1A induction after treatment with benzo[a]pyrene (BP). LC-MS/MS analysis of cell culture supernatants showed the production of bile acids including taurocholic acid, tauro-chenodeoxycholic acid, taurodeoxycholic acid and taurolithocholic acid. The results strongly suggest the applicability of the cell line BFH12 for subsequent experiments in the emerging field of bovine biotransformation.
关于牛体内药物代谢酶（DMEs）的知识相当有限。评估了源自牛胎儿肝细胞的细胞系BFH12作为生物转化模型的能力。通过逆转录聚合酶链反应（RT-PCR）对DMEs进行基因表达分析。通过免疫细胞化学观察外排转运蛋白的存在，并通过乙氧异吩恶唑酮-O-脱乙基酶（EROD）试验评估细胞色素P450（CYP）1A的功能诱导。通过液相色谱-串联质谱法（LC-MS/MS）测量胆汁酸的产生。RT-PCR显示细胞色素1A1、1A2、3A4以及II相酶UGT1A1、UGT1A6和GSTM1的表达。免疫荧光显示外排转运蛋白ABCG2和ABCC1。EROD试验显示在用苯并[a]芘（BP）处理后CYP1A呈剂量依赖性诱导。对细胞培养上清液的LC-MS/MS分析显示产生了包括牛磺胆酸、牛磺鹅去氧胆酸、牛磺脱氧胆酸和牛磺石胆酸在内的胆汁酸。结果强烈表明细胞系BFH12适用于牛生物转化这一新兴领域的后续实验。
