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Drug Metabolism and Disposition Fast Forward
First published on September 24, 2004; DOI: 10.1124/dmd.104.001206

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Received for publication June 30, 2004.
Revised September 22, 2004.
Accepted for publication September 22, 2004.

Identification of the rabbit liver UDP-glucuronosyltransferase catalyzing the glucuronidation of p-ethoxyphenylurea (dulcin)

Yoshihiro Uesawa 1*, Adam G Staines 2, Audrey O'Sullivan 2, Kiminori Mohri 3, Brian Burchell 2

1 Meiji pharmaceutical university 2 University of Dundee 3 Meiji Pharmaceutical University

* Address correspondence to: E-mail: uesawa{at}my-pharm.ac.jp

Abstract

Dulcin (DL), 4-ethoxyphenylurea, a synthetic chemical about 200 times as sweet as sucrose, has been proposed for use as an artificial sweetener. DL is excreted as a urinary ureido-N-glucuronide after oral administration to rabbits. The phenylurea N-glucuronide is the only ureido conjugate with glucuronic acid known at present, so DL is interesting as a probe to search for new functions of UDP-glucuronosyltransferases (UGTs). Seven UGT isoforms (UGT1A3, UGT1A4, UGT1A7, UGT2B13, UGT2B14 and UGT2B16) have been identified from rabbit liver (RabL), but these UGTs have not been investigated using DL as a substrate. In this work, the identities of UGT isoforms catalyzing the formation of DL glucuronide were investigated using rabbit liver microsomes (RabLM) and cloned/expressed as rabbit UGT isoforms. DL N-glucuronide (DNG) production was determined quantitatively in RabLM and homogenates of COS-7 cells expressing each UGT isoform by using electrospray liquid chromatography-tandem mass spectrometry (LC/MS-MS). Analysis of DNG formation using RabLM, by Eadie-Hofstee plot, gave a Vmax of 0.911 nmol/min/mg protein and the Km of 1.66 mM. DNG formation was catalyzed only by cloned expressed rabbit UGT1A7 and UGT2B16 (Vmax of 3.98 and 1.16 pmol/min/mg protein and a Km of 1.23 and 1.69 mM, respectively). Substrate inhibition of UGT1A7 by octylgallate confirmed the significant contribution of UGT1A7 to the formation of DNG. Octylgallate was further shown to competitively inhibit DNG production by RabLM (Ki=0.149 mM). These results demonstrate that UGT1A7 is the major isoform catalyzing the N-glucuronidation of DL in RabLM.


Key words: drug disposition, glucuronidation, liver microsomes, phase II drug metabolism, UDP glucuronyltransferases


This article has been cited by other articles:


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L. Xu, D. M. Krenitsky, A. M. Seacat, J. L. Butenhoff, T. R. Tephly, and M. W. Anders
N-GLUCURONIDATION OF PERFLUOROOCTANESULFONAMIDE BY HUMAN, RAT, DOG, AND MONKEY LIVER MICROSOMES AND BY EXPRESSED RAT AND HUMAN UDP-GLUCURONOSYLTRANSFERASES
Drug Metab. Dispos., August 1, 2006; 34(8): 1406 - 1410.
[Abstract] [Full Text] [PDF]


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