Elsevier

Biochemical Pharmacology

Volume 66, Issue 5, 1 September 2003, Pages 841-847
Biochemical Pharmacology

Contribution of the Ah receptor to the phenolic antioxidant-mediated expression of human and rat UDP-glucuronosyltransferase UGT1A6 in Caco-2 and rat hepatoma 5L cells

https://doi.org/10.1016/S0006-2952(03)00389-7Get rights and content

Abstract

UDP-glucuronosyltransferases (UGTs) represent major phase II enzymes of drug metabolism which are regulated in a tissue-specific manner by endogenous and environmental factors. Among the latter, aryl hydrocarbon receptor (AhR) agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and phenolic antioxidants such as tert-butylhydroquinone (tBHQ) are known to induce the expression of human UGT1A6 in Caco-2 cells. While binding of the TCDD-activated AhR to one xenobiotic response element (XRE) in the 5′-flanking regulatory region of UGT1A6 was characterised previously, the mechanism responsible for tBHQ induction is unknown. Therefore, it was investigated whether antioxidant response elements (AREs) are involved in tBHQ induction of UGT1A6. Transfectants of 3 kb of its regulatory region and its deletion mutants were treated with tBHQ. These studies suggested a region with approximately 2-fold induction, including an ARE-like motif, 15 bp downstream of the previously characterised XRE. Transfectants of the point-mutated ARE-like motif showed marginally reduced response to tBHQ, but surprisingly, loss of response to TCDD, suggesting interference of flanking proteins with the AhR/Arnt complex. Coordinate responses of UGT activity after treatment with TCDD or tBHQ were also observed in rat hepatoma 5L cells, mutants without the AhR and with recomplemented AhR. The results suggest a contribution of the AhR pathway and of proteins binding to the XRE flanking region to the induction of human UGT1A6 by both AhR agonists and phenolic antioxidants.

Introduction

Mammalian microsomal UGTs are major phase II enzymes of drug metabolism. They convert hundreds of lipophilic endobiotics and xenobiotics (drugs, dietary plant constituents, carcinogens, etc.) into hydrophilic and excretable conjugates [1]. Based on evolutionary divergence human UGTs have been grouped into two families consisting of nine family 1 members encoded on chromosome 2q37 and greater than six family 2 members encoded on chromosome 4q13 [2]. UGTs are regulated in a tissue-specific manner by endogenous and environmental factors [3], [4]. Among the latter, AhR agonists such as TCDD and phenolic antioxidants such as tBHQ have been found to induce rat and human UGT1A6 [5], [6], [7], [8]. Induction by TCDD has been shown to be mediated by binding of the activated AhR/Arnt complex to one consensus xenobiotic response element (XRE; GCGTG) in the 5′-regulatory region of rat [9] and human UGT1A6 [10].

tBHQ has been found to be a prototype inducer of a novel, non-receptor signalling pathway triggered by oxidative/electrophile stress. This signalling pathway selectively induces phase II enzymes and therefore has been termed monofunctional induction, in contrast to AhR-mediated bifunctional induction in which both phase I and II enzymes are transcriptionally activated [11]. In the case of rodent GSTs and rat and human NAD(P)H quinone oxidoreductase-1 (NQO1), AREs have been characterised in their 5′-regulatory regions [12], [13], [14], [15]. Studies with NR-E2-related factor (Nrf2) knockout mice suggested that this basic leucine zipper protein is a key factor of the protein complex binding to AREs [16], [17], [18]. Evidence has been obtained that Nrf2 is normally present in the cytosol in a latent complex with the chaperone keap-1 [19]. Oxidative/electrophile stress has been proposed to activate protein kinases, i.e. MAP kinases, disrupting the Nrf2/keap-1 complex after which Nrf2 is translocated to the nucleus where it binds to AREs [20]. Moreover, studies with Nrf2 knockout mice suggested that Nrf2 may be involved in the regulation of mouse UGT1A6 induction [21], [22], [23].

UGT1A6 has been shown to efficiently conjugate benzo[a]pyrene diphenols, thereby preventing toxic quinone/quinol redox cycles [24], [25]. Hence, UGTs such as UGT1A6 may play important roles in preventing electrophilic stress. Dietary monofunctional inducers of phase II enzymes, including phenolic antioxidants, currently receive a lot of interest in the efforts of cancer chemoprotection [23]. However, mechanisms responsible for UGT1A6 induction by phenolic antioxidants are still unclear. To investigate whether a putative ARE-like motif is functional, it was mutated by site-directed mutagenesis. The results suggest a contribution of the AhR pathway and of proteins binding to the XRE flanking region to the induction of UGT1A6 by both TCDD and phenolic antioxidants.

Section snippets

Cell culture, treatment and preparation of cell homogenates

Human Caco-2 cell, clone TC7, was maintained as described [6]. Briefly, cells were cultured on 100 mm Falcon dishes in DMEM supplemented with 20% foetal calf serum and non-essential amino acids. Cells were treated with 80 μM tBHQ, 50 μM β-naphthoflavone (BNF) or 10 nM TCDD when they reached confluency and were harvested after 42 hr, the optimal time for measuring UGT1A6 mRNA. Solvent controls contained 0.1% DMSO. Before harvest, cells were washed with PBS and stored at −80°. UGT activity was

Induction of human UGT1A6 expression by treatment with tBHQ, TCDD and BNF

Treatment of Caco-2 cells with tBHQ or BNF clearly increased UGT1A6 expression (Fig. 1A and B). The effect of BNF was stronger possibly due to the fact that BNF is both an AhR agonist and a monofunctional inducer, after efficient metabolism by BNF-induced CYP1A1 to electrophilic metabolites. The results were supported by UGT activity data using 4-methylumbelliferone or 1-naphthol (not shown) as substrates which are, however, overlapping substrates of several UGT isoforms (Fig. 1C). BNF was not

Acknowledgements

The authors thank Dr. Alain Zweibaum (INSERM U-178, Villejuif, France) for providing Caco-2/TC7 cells and Dr. Martin Göttlicher and Dr. Friedrich Wiebel (Research Center Karlsruhe and GSF-Research Center, Institute of Toxicology, Oberschleißheim, Germany) for providing 5L and BP8 cells. We are grateful to Birgit Kaltschmitt and Ingrid Voith for expert technical assistance and the Deutsche Forschungsgemeinschaft for financial support.

References (30)

  • K.W. Bock et al.

    Ah receptor-controlled transcriptional regulation and function of rat and human glucuronosyltransferase isoforms

    Adv. Enzyme Regul.

    (1998)
  • L. Poellinger et al.

    The dioxin and peroxisome proliferator-activated receptors: nuclear receptors in search of endogenous ligands

    Trends Pharmacol. Sci.

    (1992)
  • Dutton GJ. Glucuronidation of drugs and other compounds. Boca Raton: CRC Press;...
  • P.I. Mackenzie et al.

    The UDP-glucuronosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence

    Pharmacogenetics

    (1997)
  • R.H. Tukey et al.

    Genetic multiplicity of the human UDP-glucuronosyltransferases and the regulation in the gastrointestinal tract

    Mol. Pharmacol.

    (2001)
  • Cited by (77)

    • UDP-Glycosyltransferases

      2018, Comprehensive Toxicology: Third Edition
    • Recovery of redox homeostasis altered by CuNPs in H4IIE liver cells does not reduce the cytotoxic effects of these NPs: An investigation using aryl hydrocarbon receptor (AhR) dependent antioxidant activity

      2015, Chemico-Biological Interactions
      Citation Excerpt :

      CYP1A can be evidenced at the enzymatic level by measuring the associated ethoxyresorufin-O-deethylase (EROD) activity. Recent data provide evidence for a cross-talk between the Nrf2 pathway and the pathway leading to the induction of XRE-driven genes by the AhR [14,16–18]. Unlike the AhR, the Nrf2 transcription factor is not ligand activated, rather it responds to xenobiotics and endogenous compounds that are thiol reactive, such as reactive oxygen species or electrophilic insults.

    • Coffee and Gastrointestinal Glucuronosyltransferases

      2015, Coffee in Health and Disease Prevention
    View all citing articles on Scopus
    View full text