title: Characterization of chrysin glucuronidation in UGT1A1-overexpressing HeLa cells: elucidating the transporters responsible for efflux of glucuronide.
abstract: Active transport of glucuronide out of cells is a critical process in elimination of drugs via the glucuronidation pathway. Here, HeLa cells were stably transfected with UGT1A1 and the contributions of BCRP and MRP family transporters to the cellular efflux of chrysin glucuronide (CG) were determined. The cDNA of UGT1A1 was introduced into HeLa cells using the lentiviral transfection method. The modified cells were functional in generation of the glucuronide from chrysin. Ko143 at 10-20 μM (a dual inhibitor of BCRP and UGT1A1) caused a marked decrease (51.3%-59.7%, P < 0.01) in the excretion rate and efflux clearance of CG. Likewise, MK-571 at 5-20 μM (an inhibitor of MRPs but an activator of UGT1A1) resulted in a significant reduction in the excretion rate (18.2%-64.0%, P < 0.01) and efflux clearance (37.0%-90.2%, P < 0.001). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on CG excretion. The chemical inhibition indicated that excretion of CG was contributed by the MRP family transporters, whereas the role of BCRP was unclear. Furthermore, short hairpin RNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of CG (38.6% for BCRP, 39.3% for MRP1, 36.4% for MRP3, and 28.7% for MRP4; P < 0.01). Transporter silencing also led to substantial decreases in the efflux clearance (44.7% for BCRP, 60.4% for MRP1, 36.7% for MRP3, and 28.7% for MRP4; P < 0.01). The gene silencing results suggested that BCRP, MRP1, MRP3, and MRP4 were significant contributors to excretion of CG.
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, China (E.Q., X.Z., B.W.); and Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, Guangzhou, China (H.W., D.D.).
authors.forename: Enxi
authors.initials: E
authors.lastname: Quan
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, China (E.Q., X.Z., B.W.); and Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, Guangzhou, China (H.W., D.D.).
authors.forename: Huailing
authors.initials: H
authors.lastname: Wang
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, China (E.Q., X.Z., B.W.); and Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, Guangzhou, China (H.W., D.D.).
authors.forename: Dong
authors.initials: D
authors.lastname: Dong
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, China (E.Q., X.Z., B.W.); and Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, Guangzhou, China (H.W., D.D.).
authors.forename: Xingwang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, China (E.Q., X.Z., B.W.); and Guangzhou Jinan Biomedicine Research and Development Center, Jinan University, Guangzhou, China (H.W., D.D.) bj.wu@hotmail.com.
authors.forename: Baojian
authors.initials: B
authors.lastname: Wu
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D005419:Flavonoids / Q000378:metabolism; D020868:Gene Silencing; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins; D013379:Substrate Specificity; D014162:Transfection; D000099148:UGT1A1 Enzyme
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D005419:Flavonoids; D020719:Glucuronides; D014453:Glucuronosyltransferase; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme; C073492:ABCC4 protein, human; C116614:ABCG2 protein, human; C114462:multidrug resistance-associated protein 3; C043561:chrysin; C421109:multidrug resistance-associated protein 1