2025

Juvonen, Risto O.; Laitinen, Tuomo; David, Joana; Ferreira, Margarida; Etemadi, Jasmin; Mekhalia, Oussama; Onatsu, Ina; Pätsi, Emma; Huuskonen, Juhani; Finel, Moshe; Raunio, Hannu; Lassheikki, Veera; Heikkinen, Aki T.; Auriola, Seppo

Introduction/Objective: Glucuronidation and sulfonation enzymes conjugate compounds containing a hydroxyl group, while paraoxonase enzymes hydrolyze lactonecontaining compounds. 3-Hydroxy-6H,7H-chromeno [3,4-c]chromene-6,7-dione (3- hydroxy-V-coumarin) contains both a hydroxyl substituent and two lactone substructures and is strongly fluorescent. This study evaluates glucuronidation, sulfonation and hydrolysis of 3-hydroxy-V-coumarin reactions, which abolish its fluorescence. Methods: Glucuronide, sulfate, and hydrolyzed product formation were confirmed using accurate LC-MS. Fluorescence-based multi-well plate assays were established to determine rates of glucuronidation, sulfonation and hydrolysis of 3-hydroxy-V-coumarin. Results: A decrease in fluorescence correlated with the formation of conjugation or hydrolyzed metabolites of the reactions was observed. 3-Hydroxy-V-coumarin was glucuronidated by human UGT1A1, 1A3, 1A4, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4 and 2B7 and by dog UGT1A1, 1A2 and 1A11, and sulfonated by human SULT1A1, 1A2 and 1E1. The results indicated that paraoxonase 2 hydrolyzed lactone of 3-hydroxy-V-coumarin. 3-Hydroxy-Vcoumarin interacted via a hydrogen bond with serine 221 and via hydrophobic interaction with phenyls 71 and 291 of paraoxonase 2. The hydrolysis rate of 3-hydroxy-V-coumarin was determined in ten rat tissues. In human liver cytosol, the hydrolysis was slower than in rat, mouse, dog, pig, rabbit, and sheep liver cytosol. Conclusion: 3-hydroxy-V-coumarin is a new model substrate for studying glucuronidation, sulfonation and lactone hydrolysis reactions.