Abstract
The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74–94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4–8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19–59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (fmet) in the presence of Ko143 was observed. In contrast, leukotriene C4 and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM70737].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- BCRP
- breast cancer resistance protein
- MRP
- multidrug resistance protein
- SN-38
- 7-ethyl-10-hydroxycamptothecin
- siRNA
- small interfering RNA
- LTC4
- leukotriene C4
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- DMSO
- dimethyl sulfoxide
- HPLC
- high-performance liquid chromatography
- UPLC
- ultraperformance liquid chromatography
- HBSS
- Hanks' balanced salt solution
- MS/MS
- tandem mass spectrometry.
- Received June 30, 2011.
- Accepted November 9, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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