Human and Rat Liver UDP-Glucuronosyltransferases Are Targets of Ketoprofen Acylglucuronide

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal antiinflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation 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 glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide 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 microscopy 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 immunoreactive 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, [b-P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite. Glucuronidation is the major metabolic pathway for carboxylic acid containing drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) of the series of 2-phenylpropionic acid (profens), as well as diuretics and hypolipidemic agents. The reaction leads to the formation of acylglucuronides that are excreted in bile or urine. Unlike etherglucuronides formed from hydroxylated molecules, acylglucuronides 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 b-glucuronidase-resistant 2-, 3-, and 4-O-acyl isomers. In addition, acylglucuronides bind covalently to endogenous macromolecules. Such irreversible binding with plasma proteins has been reported by us and others for the acylglucuronides 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 documented that tissue proteins may be targets for acylation by metabolites of diflunisal (King and Dickinson, 1993), diclofenac (Kretz-Rommel and Boelsterli, 1994; Hargus et al., 1994), and tolmetin (Ojingwa et al., 1994). It has been postulated 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 comThis work was supported by the Région Lorraine, the Ministère des Affaires Etrangères, 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, UDPglucuronic 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 A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from pounds containing hydroxyl, carboxyl, amino, or sulfhydryl groups (Mackenzie et al., 1997). From the 50 UGT cDNA that have been isolated and characterized in rat and human until now, only two isoforms (UGT2B1 and UGT2B7) have been identified to glucuronidate NSAIDs chemically related to 2-phenylpropionic acid to an appreciable extent. The human UGT2B7 isoform, which has been expressed in HK293 cells, also glucuronidates catechol estrogens and androgens (Coffman et al., 1998). We recently stably expressed the cDNA encoding UGT2B1 in V79 fibroblasts (Pritchard et al., 1994). Analysis of the substrate specificity of this isoform revealed that carboxylic substances such as NSAIDs (ketoprofen, ibuprofen, and carprofen), hypolipidemic agents (clofibric acid), and short-chain fatty acids were the major substrates of this enzyme, whereas hydroxylated substances, such as 2-naphthol, were also glucuronidated but at a lower rate. Because of its potency in catalyzing the formation of acylglucuronides, UGT2B1 is a model enzyme for studying the formation and reactivity of the acylglucuronides. Ketoprofen, a widely used NSAID, is mainly glucuronidated in the liver as an acylglucuronide (Upton et al., 1980). Taking into account the reactivity of ketoprofen acylglucuronide and its main source of formation in the liver by the UGTs, it is likely that it could also bind to intracellular proteins, including UGTs themselves. In the present study, we investigated the reactivity of the glucuronide of racemic ketoprofen toward microsomal and recombinant UGT isoforms, particularly UGT2B1, responsible for the acylglucuronide formation. The results clearly show, for the first time, that ketoprofen glucuronide covalently binds to UGTs, which are irreversibly inactivated as a result of adduct formation. Materials and Methods Chemicals and Reagents Ketoprofen [R,S-2-(3-benzoylphenyl)propionic acid], 1-naphthol, 2-naphthol, 1-naphthyl-b-D-glucuronide, D-saccharic acid 1,4-lactone, sodium cyanide, dimethyl sulfoxide, 3-[(3-chloroamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), paraformaldehyde, gelatin from bovine skin, and rabbit anti-goat and goat antirabbit alkaline phosphatase-conjugated IgG were obtained from Sigma (L’Isle d’Abeau, St. Quentin Fallavier, France). Saponin was provided by Aldrich (L’Isle d’Abeau, St. Quentin Fallavier, France). UDP-glucuronic acid (UDP-GlcUA) (sodium salt) was obtained from Boehringer (Mannheim, Germany). Dulbecco’s modified Eagle’s medium was obtained from Gibco-BRL (Eragny, France). Fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat and goat antirabbit IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Affi-Gel Protein A mitogen-activated protein II was purchased from Bio-Rad (Ivry-sur-Seine, France). Diethylaminoethyl (DEAE)-Sephacel (DEAE-cellulose anion exchanger) was purchased from Sigma. Blue Sepharose CL-6B (Cibacron Blue 36-A covalently attached to Sepharose CL-6B by the triazine coupling method) was purchased from Pharmacia Biotech (St. Quentin-Yvelines, France). The UGT inhibitor, 7,7,7-triphenylheptanoic acid, was synthesized according to Fournel-Gigleux et al. (1989). The radiolabeled photoaffinity probe, [b-P]5N3UDP-GlcUA (specific activity 2–5 mCi/mmol), was synthesized as previously described (Drake et al., 1992).