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Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies

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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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Abbreviations

ACN:

acetonitrile

AhR:

aryl hydrocarbon receptor

BNF:

β-naphthoflavone

CAR:

constitutive androstane receptor

CV:

coefficient of variation

CYP:

cytochrome P450

DMSO:

dimethyl sulfoxide

GLP:

good laboratory practice

HLM:

human liver microsomes

HPLC:

high-performance liquid chromatography

INDG:

indigo

IND:

indirubin

3-MC:

3-methylcholanthrene

LLOQ:

lower limit of quantification

MRM:

multiple reaction monitoring

MS:

mass spectrometry

MS/MS:

tandem mass spectrometry

MTT:

3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide

OME:

omeprazole

PB:

phenobarbital

PXR:

pregnane X receptor

QC:

quality control

RIF:

rifampicin

RME:

relative error of measurement

TCDD:

2,3,7,8-tetrachlorodibenzeno-p-dioxin

UDPGA:

UDP-glucuronic acid

UGT:

UDP-glucuronosyltransferase

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Acknowledgements

This work was supported by an EEC contract (LINTOP-037499) and the Spanish Ministry of Science and Innovation/Instituto de Salud Carlos III through a Miguel Server contract (CP08/00125) (to A.L.).

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Authors and Affiliations

  1. Unidad de Hepatología Experimental, Centro de Investigación Hospital Universitario “La Fe”, Avda. Campanar, 46009, Valencia, Spain

    M. Teresa Donato, Sandra Montero, José V. Castell, M. José Gómez-Lechón & Agustín Lahoz

  2. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Avda. Blasco Ibáñez, 46010, Valencia, Spain

    M. Teresa Donato & José V. Castell

Authors
  1. M. Teresa Donato
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  2. Sandra Montero
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  3. José V. Castell
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  4. M. José Gómez-Lechón
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  5. Agustín Lahoz
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Corresponding author

Correspondence to Agustín Lahoz.

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Figure S1

Glu-Standards Stability. QC samples at a concentration of 2, 5 μM were stored in the autosampler at 10 °C and quantified at 0, 10, 17, 24 and 31 h. (PDF 466 kb)

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Donato, M.T., Montero, S., Castell, J.V. et al. Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies. Anal Bioanal Chem 396, 2251–2263 (2010). https://doi.org/10.1007/s00216-009-3441-1

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  • DOI: https://doi.org/10.1007/s00216-009-3441-1

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