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
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.
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