Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofenacylglucuronide

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferas es (UGTs) are electrophilic metabolites able to covalently bind proteins. I n 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 glucuronidatio n of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by th e recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuro nidation of P-naphthol was observed when ketoprofen acylglucuronide was pro duced in situ in cultured V79 cells expressing UGT2B1. Inhibition was alway s associated with the formation of microsomal protein-ketoprofen adducts. T he presence of these covalent adducts within the endoplasmic reticulum of c ells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibo dies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuro nide and probed with antiketoprofen antibodies revealed the presence of sev eral protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct format ion partially prevented the photoincorporation of the UDP-glucuronic acid ( UDP-GlcUA) analog, [beta-P-32]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 microsomes incubated with ketopr ofen glucuronide led to the isolation of UGT adducts recognized by both ant i-UGT and antiketoprofen antibodies, providing strong evidence that UGTs ar e targets of this metabolite.