Abstract
Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance–associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4′-O-glucuronide (R4′G)] were excreted into the extracellular compartment after incubation of resveratrol (1–100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4′G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)–mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%–46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4′G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.
Footnotes
- Received October 7, 2015.
- Accepted January 8, 2016.
↵1 S.W. and F.L. contributed equally to this work.
This work was supported by the National Natural Science Foundation of China [Grant 81573488]; the Young Scientist Special Projects in Biotechnological and Pharmaceutical Field of 863 Program [Grant 2015AA020916]; and the Outstanding Youth Fund from the Natural Science Foundation of Guangdong Province [Grant 2014A030306014].
↵This article has supplemental material available at dmd.aspetjournals.org.
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
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