Human and Rat Liver UDP-Glucuronosyltransferases Are Targets of Ketoprofen Acylglucuronide NAD ` EGE TERRIER, ETIENNE BENOIT, CLAIRE SENAY, FRANC ¸ OISE LAPICQUE, ANNA RADOMINSKA-PANDYA, JACQUES MAGDALOU, and SYLVIE FOURNEL-GIGLEUX Unite ´ Mixte de Recherche 7561 Centre National de la Recherche Scientifique-Universite ´ Henri Poincare ´ Nancy 1, Vandoeuvre-le ` s-Nancy, France (N.T., C.S., F.L., J.M., S.F.-G.); Unite ´ Associe ´ e Institut National de la Recherche Agronomique-Direction Générale Enseignement- Recherche, Toxicologie et Me ´ tabolisme Compare ´s des Xe ´ nobiotiques, Ecole Nationale Ve ´ te ´ rinaire de Lyon, Marcy l’Etoile, France (E.B.); and Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (A.R.-P.) Received February 18, 1999; accepted April 20, 1999 This paper is available online at http://www.molpharm.org ABSTRACT Acylglucuronides formed from carboxylic acids by UDP-glucu- ronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti- inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glu- curonidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronida- tion of the drug. A decrease of about 35% in the glucuronida- tion of 2-naphthol was observed when ketoprofen acylglucu- ronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence mi- croscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunore- active protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [- 32 P]5N 3 UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver micro- somes incubated with ketoprofen glucuronide led to the isola- tion of UGT adducts recognized by both anti-UGT and antike- toprofen antibodies, providing strong evidence that UGTs are targets of this metabolite. Glucuronidation is the major metabolic pathway for car- boxylic acid containing drugs, such as nonsteroidal anti-in- flammatory drugs (NSAIDs) of the series of 2-phenylpropi- onic acid (profens), as well as diuretics and hypolipidemic agents. The reaction leads to the formation of acylglucu- ronides that are excreted in bile or urine. Unlike etherglucu- ronides formed from hydroxylated molecules, acylglucu- ronides are electrophilic species known to be intrinsically reactive both in vitro and in vivo (Spahn-Langguth and Benet, 1992). They undergo spontaneous hydrolysis to the parent drug as well as intramolecular rearrangement leading to -glucuronidase-resistant 2-, 3-, and 4-O-acyl isomers. In addition, acylglucuronides bind covalently to endogenous macromolecules. Such irreversible binding with plasma pro- teins has been reported by us and others for the acylglucu- ronides of several drugs including ketoprofen (Presle et al., 1996), tolmetin (Hyneck et al., 1988), zomepirac (Smith et al., 1990), ibuprofen and ibufenac (Castillo and Smith, 1995), and benoxaprofen (Spahn et al., 1990). It also has been doc- umented that tissue proteins may be targets for acylation by metabolites of diflunisal (King and Dickinson, 1993), diclofe- nac (Kretz-Rommel and Boelsterli, 1994; Hargus et al., 1994), and tolmetin (Ojingwa et al., 1994). It has been pos- tulated that proteins modified by the formation of adducts with drug acylglucuronides may cause immunological side effects and hepatotoxicity observed for these drugs (Olson et al., 1992). These effects have led to the withdrawal from the market of several NSAIDs, such as tolmetin, zomepirac, and benoxaprofen. UDP-glucuronosyltransferases (UGTs) are a multigenic family of membrane-bound enzymes that are responsible for the glucuronidation of various drugs and endogenous com- This work was supported by the Re ´ gion Lorraine, the Ministe ` re des Affaires Etrange `res, and the Association de Recherche sur la Polyarthrite. It is in partial fulfillment of the doctoral thesis of N.T. ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; CHAPS, 3-[(3-chloroamidopropyl)dimethylammonio]-1-propane sulfonate; FITC, fluorescein isothiocyanate; PB, phenobarbital; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; UDP-GlcUA, UDP- glucuronic acid; UGT, UDP-glucuronosyltransferase. 0026-895X/99/010226-09$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:226 –234 (1999). 226 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from