Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies |
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Authors: | M Teresa Donato Sandra Montero José V Castell M José Gómez-Lechón Agustín Lahoz |
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Institution: | 1. Unidad de Hepatología Experimental, Centro de Investigación Hospital Universitario “La Fe”, Avda. Campanar, 46009, Valencia, Spain 2. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Avda. Blasco Ibá?ez, 46010, Valencia, Spain
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Abstract: | UDP-glucuronsyltransferases (UGTs) are a family of conjugating enzymes that participate in the metabolism of many drugs. The
study of potential drug–drug interactions involving UGTs has been largely hindered by the limited availability of selective
functional assays for individual UGT enzymes. We propose a sensitive and reproducible procedure for the activity measurements
of four major human hepatic UGT forms. The assays are based on analysis and quantification by high-performance liquid chromatography–tandem
mass spectrometry of glucuronides formed from selective probe substrates, namely, β-estradiol (UGT1A1, 3-glucuronide), 1-naphthol
(UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). The analytical methods developed in the present study have been validated
under good laboratory practice compliance following FDA recommendations. The assays can be easily applied to both phenotyping
UGT reactions in liver-derived cellular and subcellular systems, and drug–drug interaction in vitro studies. Chemical inhibition
of UGTs was tested in human liver microsomes at substrate concentrations lower than the corresponding K
M values. Under these conditions, selective inhibition of UGT2B7 by fluconazole and low amitriptyline concentrations were observed,
whereas diclofenac and quinidine were shown as non-enzyme-selective inhibitors of UGTs. Induction of UGTs was studied in primary
human hepatocytes and HepG2 cells cultured in 96-well plates. Aryl hydrocarbon receptor ligands (except indirubin in hepatocytes)
increased the UGT1A1 activity in both cell models. The highest effects were observed in HepG2 cells exposed to indirubin (21-fold
over the control) and omeprazole or β-naphthoflavone (about sixfold). Although variable effects were observed in other UGT
enzymes, the degree of induction was generally lower than that for UGT1A1. |
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