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Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1.

ABSTRACT: Identifying uridine 5'-diphospho-(UDP)-glucuronosyltransferase (UGT)-selective probes (substrates that are primarily glucuronidated by a single isoform) is complicated by the enzymes' large overlapping substrate specificity. Here, regioselective glucuronidation of two flavonoids, 3,3',4'-trihydroxyflavone (3,3',4'-THF) and 3,6,4'-trihydroxyflavone (3,6,4'-THF), is used to probe the activity of hepatic UGT1A1. The glucuronidation kinetics of 3,3',4'-THF and 3,6,4'-THF was determined using 12 recombinant human UGT isoforms and pooled human liver microsomes (pHLM). The individual contribution of main UGT isoforms to the metabolism of the two flavonoids in pHLM was estimated using the relative activity factor approach. UGT1A1 activity correlation analyses using flavonoids-4'-O-glucuronidation vs ?-estradiol-3-glucuronidation (a well-recognized marker for UGT1A1) or vs SN-38 glucuronidation were performed using a bank of HLMs (n = 12) including three UGT1A1-genotyped HLMs (i.e., UGT1A1*1*1, UGT1A1*1*28, and UGT1A1*28*28). The results showed that UGT1A1 and 1A9, followed by 1A7, were the main isoforms for glucuronidating the two flavonoids, where UGT1A1 accounted for 92 ± 7% and 91 ± 10% of 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF, respectively, and UGT1A9 accounted for most of the 3-O-glucuronidation. Highly significant correlations (R(2) > 0.944, p < 0.0001) between the rates of flavonoids 4'-O-glucuronidation and that of estradiol-3-glucuronidation or SN-38 glucuronidation were observed across 12 HLMs. In conclusion, UGT1A1-mediated 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF was highly correlated with the glucuronidation of estradiol (3-OH) and SN-38. This study demonstrated for the first time that regioselective glucuronidation of flavonoids can be applied to probe hepatic UGT1A1 activity in vitro.

SUBMITTER: Wu B

PROVIDER: S-EPMC3404744 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1.

Wu Baojian B Zhang Shuxing S Hu Ming M

Molecular pharmaceutics 20111021 6

Identifying uridine 5'-diphospho-(UDP)-glucuronosyltransferase (UGT)-selective probes (substrates that are primarily glucuronidated by a single isoform) is complicated by the enzymes' large overlapping substrate specificity. Here, regioselective glucuronidation of two flavonoids, 3,3',4'-trihydroxyflavone (3,3',4'-THF) and 3,6,4'-trihydroxyflavone (3,6,4'-THF), is used to probe the activity of hepatic UGT1A1. The glucuronidation kinetics of 3,3',4'-THF and 3,6,4'-THF was determined using 12 reco ...[more]

PMID: 21985641

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Project description:Bupropion is a widely used antidepressant and smoking cessation aid in addition to being one of two US Food and Drug Administration-recommended probe substrates for evaluation of cytochrome P450 2B6 activity. Racemic bupropion undergoes oxidative and reductive metabolism, producing a complex profile of pharmacologically active metabolites with relatively little known about the mechanisms underlying their elimination. A liquid chromatography-tandem mass spectrometry assay was developed to simultaneously separate and detect glucuronide metabolites of (R,R)- and (S,S)-hydroxybupropion, (R,R)- and (S,S)-hydrobupropion (threo) and (S,R)- and (R,S)-hydrobupropion (erythro), in human urine and liver subcellular fractions to begin exploring mechanisms underlying enantioselective metabolism and elimination of bupropion metabolites. Human liver microsomal data revealed marked glucuronidation stereoselectivity [Cl(int), 11.4 versus 4.3 µl/min per milligram for the formation of (R,R)- and (S,S)-hydroxybupropion glucuronide; and Cl(max), 7.7 versus 1.1 µl/min per milligram for the formation of (R,R)- and (S,S)-hydrobupropion glucuronide], in concurrence with observed enantioselective urinary elimination of bupropion glucuronide conjugates. Approximately 10% of the administered bupropion dose was recovered in the urine as metabolites with glucuronide metabolites, accounting for approximately 40%, 15%, and 7% of the total excreted hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively. Elimination pathways were further characterized using an expressed UDP-glucuronosyl transferase (UGT) panel with bupropion enantiomers (both individual and racemic) as substrates. UGT2B7 catalyzed the stereoselective formation of glucuronides of hydroxybupropion, (S,S)-hydrobupropion, (S,R)- and (R,S)-hydrobupropion; UGT1A9 catalyzed the formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the metabolic pathways underlying bupropion metabolite disposition and significantly expand our knowledge of potential contributors to the interindividual and intraindividual variability in therapeutic and toxic effects of bupropion in humans.

Dataset Information

Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1.

Publications

Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1.

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