Title Page UDP-Glucuronosyltransferases 1A6 and 1A9 are the Major Isozymes Responsible for the 7-O-Glucuronidation of Esculetin and 4-Methylesculetin in Human Liver Microsomes

Running title: UGT metabolism of esculetin and 4-methylesculetin in vitro. Abstract Esculetin (6,7-dihydroxycoumarin, ET) and 4-methylesculetin (6,7-dihydroxy-4-methylcoumarin, 4-ME) are typical coumarin derivatives that are attracting considerable attention because of their wide spectrum of biological activities, but their metabolism remains unknown. This study aimed to elucidate the in vitro UDP-glucuronosyltransferase (UGT) metabolism characteristics of ET and 4-ME. 7-O-monoglucuronide esculetin (ET-G) and 7-O-monoglucuronide 4-methylesuletin (4-ME-G) were identified by liquid chromatography–mass spectrometry (LC-MS) and 1 H-nuclear magnetic resonance (1 HNMR) when ET or 4-ME was incubated with human liver (HLM) in the presence of UDP-glucuronic acid. Screening assays with 12 human expressed UGTs demonstrated that the formations of ET-G and 4-ME-G were almost exclusively catalyzed by UGT1A6 and UGT1A9. Phenylbutazone and carvacrol (UGT1A6 and UGT1A9 chemical inhibitors, respectively) at different concentrations (50, 100, and 200 μ M) significantly inhibited the formation of glucuronidates of ET and 4-ME in HLM, UGT1A6, and UGT1A9 when the concentrations of ET and 4-ME ranged from 10 to 300 μ M (P < 0.05). 7-O-monoglucuronidation by UGT1A6 and UGT1A9 was the predominatnt UGT metabolic pathway for both ET and 4-ME in vitro. The liver is probably the major contributor to the glucuronidation metabolism of ET and 4-ME. ET showed more This article has not been copyedited and formatted. The final version may differ from this version.