Use of Glucuronidation Fingerprinting To Describe and Predict Mono- and Dihydroxyflavone Metabolism by Recombinant UGT Isoforms and Human Intestinal and Liver Microsomes

Abstract

The present study aims to predict the regiospecific glucuronidation of three dihydroxyflavones and seven mono-hydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven mono-hydroxyflavones (or HFs), 2′-, 3′-, 4′-, 3-, 5-, 6-, and 7-hydroxyflavone, and three di-hydroxyflavones (or diHFs), 3,7-dihydroxyflavone (3,7-diHF), 3,5-dihydroxyflavone (3,5-diHF) and 3,4′-dihydroxyflavone (3,4′-diHF) were chosen and rates were measured at 2.5, 10 and 35 μM. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 are the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used, 3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoformspecific metabolism rates generally correlated well with the rates of glucuronidation in human intestinal and liver microsomes at each of the three tested concentrations. In conclusion, the glucuronidation "fingerprint" of seven selected mono-hydroxyflavones was affected by UGT isoforms used, positions of the −OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.

UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP

“…The method was modified on the basis of a previously published article (Tang et al, 2010). In brief, we mixed cell lysates (final protein concentration from 0.053 to 0.21 mg/ml) or human UGT1A9 Supersomes (final protein concentration from 0.013 to 0.053 mg/ml), magnesium chloride (0.88 mM), saccharolactone (4.4 mM), and alamethicin (0.022 mg/ml) and different concentrations of genistein (0.5-50 M) or apigenin (0.5-40 M) in a 50 mM potassium phosphate solution (pH 7.4) diluted from 100ϫ concentrated stock solutions in organic solvent (DMSO-methanol; 1:4).…”

“…The conditions for UPLC analysis of genistein, apigenin, and their glucuronides were modified on the basis of a previously published method (Tang et al, 2010;Zhu et al, 2010). The conditions were as follows: system, Acquity with a binary pump and a 2996 DPA diode array detector (Waters, Milford, MA); column, Acquity UPLC BEH C18 column (50 ϫ 2.1 mm, i.d.…”

“…The conversion factors, representing the molar extinction coefficient ratio of glucuronides to aglycones, were used to quantify the amounts of genistein and apigenin glucuronides as described previously (Liu and Hu, 2002;Tang et al, 2010).…”

Three-Dimensional Quantitative Structure-Activity Relationship Studies on UGT1A9-Mediated 3-O-Glucuronidation of Natural Flavonols Using a Pharmacophore-Based Comparative Molecular Field Analysis Model

“…Enzyme kinetics parameters of glucuronidation by UGT1A9 were determined by measuring initial gluc-uronidation rates of flavonols at a series of concentrations. The experimental procedures of UGT assays were exactly the same as those described previously (Joseph et al, 2007;Tang et al, 2009Tang et al, , 2010Singh et al, 2010). Glucuronidation rates were calculated as the amount of glucuronides formed per protein quantity per reaction time (or nmol/mg/min).…”

“…Therefore, the objective of this article is to construct a position (3-OH)-specific CoMFA model to predict 3-O-glucuronidation. 3-O-glucuronidation was chosen because it is the most active position in UGT1A9-mediated glucuronidation (Otake et al, 2002;Tang et al, 2010). Kinetics parameters (K m , V max , CL int ) of UGT1A9-mediated 3-O-glucuronidation with 30 flavonols were determined.…”