Establishment Human UGT1A9 Transgenic Cell Line And Its Application In Drug Metabolism

Human hepatic UDP-glucuronosyltransferases (UGT) is a family of microsomal enzymes that catalyze the glucuronidation of many important drugs, xenobiotics and endogenous compounds.Invidividual UGTs isozymes tend to exhibit un-strict and overlapping patterns of substrate specificity.Liver microsomes is the enzyme source frequently used in vitro for study of glucuronidation.An alternative that was made possible only recently is the heterologous expression of UGT encoding cDNAs that allows the production of single enzyme isoforms for metabolism studies.The enzyme expressed in cell lines helps to realize the function of a specific gene and will provide direct information related to the isozyme interested.The availability of recombinant enzymes makes it possible to obtain information on individual isoenzyme as well as on their role in metabolism of a drug compound of interest.Furthermore,they can be used for production and analysis of various metabolites of the particular enzyme.Information obtained from these assays provide further valuable knowledge about the substrate structure-activity relationships of individual isoforms of DME.In order to study the drug metabolisms by UGTs,recombinant expression plasmid pcDNA3.1 ( + ) -UGT1A9 in eukaryotic cell was constructed,then we established CHL-UGT1A9 and V79-UGT1A9 transgenic cell lines which will stably express human UGT1A9 protein and will be used to study drug glucuronidation. l.Construction of expressional combinant of human UGT1A9Mammalian expression plasmid vector pcDNA3.1(+) and recombinant pREP9-UGTlA9 were digested by restriction endonucleases Hind and Not to produce DNA segments with compatible sticky ends.The DNA segments were pruified by gel electrophoresis and dialysis and cloned directly into a unique Hind Not site of the vector pcDNA3.1 (+) ligating by T4 ligase to form the recombinant pcDNA3.1(+)-UGTlA9 The recombinant PcDNA 3.1(+) -UGT1A9 constructed was finally used to transfect CHL cells. 2.Establishment of transgenic cell linesRecombinant expression plasmid pcDNA3.1(+) -UGT1A9 was transfected into Chinese hamster lung (CHL) cells by a modified calcium phosphate mediated transfection procedure.The cells were selected in MEM and then using the appropriate G418 concentrations(400mg L-1) as screen reagent for the screening of CHL cells and generate a stable cell line expressing human UGTlA9.After one month,surviving clonies were harvested as a pool and propagated in medium containing G418,and the cell line termed CHL-UGT1A9 was established.CHL-UGTlA9 cells grown in the culture medium containing G418(200mg L-1) were collected.The total protein concentration in S9 was measured according to Lowry's. 3.1dentification of transgenic cell linesThe mRNA detection by PCR with cell total RNA extracted from CHL-UGT1A9 and CHL cells indicated that UGT1A9 was only transcripted in transgenic cells.The activity and kinetic parameters of expressed UGT1A9 were measured using kaempferol as substrate by HPLC.It was proved that CHL-UGT1A9 transgenic cell line could lead stable metabolic activation for kaempferol whereas the native UGT1A9 activity was not detectable in no infected control CHL cells. With the same way, the V79-UGT1A9 was identificated successfully. 4,Drug metabolism with protein expressed in V79-UGT1A9 cell linesThe enzyme activity of CHL-UGT1A9 and V79-UGT1A9 transgenic cell line were measured using kaempferol as substrate by HPLC.With the result the activity of V79 - UGT1A9 is better than that of CHL-UGTlA9.Some drugs including quercetin,isorhamnetin,propofol,p-nitrophenol,propranolol were tested as substratesfor glucuronidation catalyzed with V79-UGT1A9 transgenic cell line.4.1 Flavones(kaempferol,quercetin,isorhamnetin): The glucuronidation reaction were carried out in an incubation mixture of total volume in which flavone were added.After per-incubation,the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of methanol and internal standard (morin).The mixtures were stirred thoroughly and centri...