Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression

doi:10.1016/S0300-483X(01)00570-4

Toxicology

Volume 171, Issues 2–3, 28 February 2002, Pages 137-146

https://doi.org/10.1016/S0300-483X(01)00570-4 Get rights and content

Abstract

In the present study, we investigated the inducibility of the drug conjugate transporter genes MRP1 and MRP2 by redox-active compounds such as tertiary butylated hydroquinone (tBHQ) and quercetin and by chemicals known to activate the pregnane X receptor (PXR) such as rifampicin and clotrimazol and by the metalloid compound arsenite. The human MRP2 gene was found to be inducible in HepG2 cells by rifampicin, clotrimazol, arsenite and tBHQ. As MRP1 expression is extremely low in HepG2 cells, its inducibility was studied in MCF-7 cells. However, only tBHQ and quercetin acted as inducers, but not the other compounds investigated. Reporter gene assays demonstrated that proximal promoter regions of the genes contribute to the induction by tBHQ, quercetin (MRP1) and clotrimazol (MRP2). However, the deletion of binding sites supposed to mediate the induction process (a PXR-binding element-like sequence for the clotrimazol effect and an ARE (antioxidative response element) for the tBHQ/quercetin effect) did not result in a significant decrease in the induction factor indicating that other parts of the promoter are probably involved in the induction process. In summary, expression of both genes can be up-regulated by redox-active compounds, while the other compounds tested induced only MRP2 but not MRP1 expression.

Introduction

The subfamily C of the ATP-binding cassette (ABC) transporter superfamily comprises seven multidrug resistance proteins (MRP1–MRP7), among which MRP1 and MRP2 are best characterized (Hipfner et al., 1999, König et al., 1999). Human MRP1 (ABCC1) is able to mediate multidrug resistance of tumor cells, and to protect cells from toxic insults by eliminating conjugates of toxic compounds like S-glutathionyl aflatoxin B1 (Loe et al., 1997). MRP2 (ABCC2) is the most important biliary efflux pump known so far. Both, xenobiotic drugs like methotrexate and endogenous compounds such as leukotriene C₄ or bilirubin glucuronide are eliminated via MRP2. In addition, MRP2 is able to confer multidrug resistance (Cui et al., 1999) in tumor cells, probably by eliminating drugs like cisplatin or vincristine in association with glutathione. Furthermore, it was suggested to be involved in the biliary elimination of carcinogenic heterocyclic amines like PhIP (Dietrich et al., 2000).

Changes in the expression of drug transporters might influence hepatic detoxification, drug distribution, and multidrug resistance of tumor cells. Therefore, it was the aim of the present study to investigate the influence of selected drugs and toxins on both, human MRP1 and MRP2 gene expression.

In this study, we focussed first on compounds (clotrimazol, rifampicin) described as activators of the pregnane X receptor (PXR) inducing human cytochrome P450 (CYP) 3A4 via this pathway. Human PXR, also named steroid and xenobiotic receptor (SXR) is an orphan receptor cloned 3 years ago (Blumberg et al., 1998, Lehmann et al., 1998) for which various xenobiotic and steroid ligands are described (Moore et al., 2000). PXR mediates the induction of several human CYP 3A genes. Furthermore, gene expression of transmembrane transporters like Oatp2 or MDR1 is regulated by PXR/SXR (Staudinger et al., 2001, Synold et al., 2001). Interestingly, PXR/SXR is able to mediate resistance to lithocholic acid induced liver damage (Staudinger et al., 2001, Xie et al., 2001). Bile acid conjugates such as 3α-sulfatolithocholyltaurine are described as MRP-substrates (König et al., 1999). Therefore, it is of interest if PXR is also involved in MRP regulation.

As many redox-active compounds induce phase II enzymes of drug metabolism like UDP-glucuronosyl-transferases (Münzel et al., 1999, Bock et al., 2000) thus generating MRP substrates, the study includes compounds such as tert-butylated hydroquinone (tBHQ) and the flavonoid quercetin. Finally, arsenite was included in the study, as MRP1 confers resistance to arsenite and because it was previously described as MRP1 inducer in a resistant leukemia cell line (Cole et al., 1994, Ishikawa et al., 1996). In the second part of the study, we aimed at elucidating the relevance of the proximal promoter regions of both genes for the induction process.

Section snippets

Cell culture

HepG2 cells were purchased from ATCC (Manassas, VA, USA), MCF-7 cells were kindly provided by Dr T.W. Gant (MRC Toxicology Unit, Leicester, UK). Cell culture was performed on 94 mm petri dishes (treated with collagen) in DMEM containing 1% penicillin/streptomycin and 20% FCS. Substances tested as inducers were purchased from Sigma (Deisenhofen, Germany). All test compounds were dissolved in DMSO, controls were treated with DMSO only.

RNA isolation, Northern blot analysis and RT-PCR

Total RNA from HepG2 and MCF-7 cells was isolated by a

Results

In the first part of this study, the inducibility of MRP1 and MRP2 by selected types of compounds as mentioned above was tested. MRP1 mRNA and protein expressions were not changed markedly after treatment with rifampicin or arsenite. In contrast, the redox-active compound tBHQ increased MRP1 mRNA expression up to 1.8-fold and also a slight increase in the MRP1 protein level was noted. Therefore, additional pro-/antioxidants were tested. In these experiments quercetin treatment resulted in the

Discussion

The results of this study demonstrate that both, MRP1 and MRP2 are inducible by tBHQ. Moreover, we observed that beside tBHQ, other redox-active compounds such as quercetin are able to induce MRP1 expression. Reporter gene assays suggest that the inducing effect of quercetin and tBHQ on MRP1 expression is at least partially mediated by sequences in the 5′-flanking region of the gene. A recent study demonstrates that MAPK (mitogen-activated protein kinase) pathways may link signals to the

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany. The authors thank Dr J. Carlstedt-Duke (Department of Medical Nutrition, Karolinska Institutet, Huddinge, Sweden) for fruitful discussions, and Dr T.W. Gant (MRC Toxicology Unit, University of Leicester, UK) for the provision of MCF-7 cells.

References (33)

K.W. Bock et al.
Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells
Biochem. Pharmacol.
(2000)
P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987)
M.F. Fromm et al.
The effect of rifampin treatment on intestinal expression of human MRP transporters
Am. J. Pathol.
(2000)
A. Geick et al.
Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin
J. Biol. Chem.
(2001)
D.R. Hipfner et al.
Structural, mechanistic and clinical aspects of MRP1
Biochim. Biophys. Acta
(1999)
T. Ishikawa et al.
Coordinated induction of MRP/GS-X pump and γ-glutamylcysteine synthetase by heavy metals in human leukemia cells
J. Biol. Chem.
(1996)
H.-M. Kauffmann et al.
Sequence analysis and functional characterization of the 5′-flanking region of the rat ‘multidrug resistance protein 2’ (mrp2) gene
Biochem. Biophys. Res. Commun.
(1998)
H.-M. Kauffmann et al.
Induction of cmrp/cmoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in hepatocytes
Hepatology
(1997)
S.A. Kliewer et al.
An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway
Cell
(1998)
J. König et al.
Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated resistance
Biochim. Biophys. Acta
(1999)

L.B. Moore et al.

Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands

J. Biol. Chem.

(2000)

J.M. Pascussi et al.

Evidence for the presence of a functional pregnane X receptor response element in the CYP 3A7 promoter gene

Biochem. Biophys. Res. Commun.

(1999)

T. Xie et al.

ARE- and TRE-mediated regulation of gene expression. Response to xenobiotics and antioxidants

J. Biol. Chem.

(1995)

Y. Yamane et al.

Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress

J. Biol. Chem.

(1998)

R. Yu et al.

Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via Nrf2-dependent mechanism

J. Biol. Chem.

(2000)

L. Bernstam et al.

Molecular aspects of arsenic stress

J. Toxicol. Environ. Health B Crit. Rev.

(2000)

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Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression

Abstract

Introduction

Section snippets

Cell culture

RNA isolation, Northern blot analysis and RT-PCR

Results

Discussion

Acknowledgements

Biochem. Pharmacol.

Anal. Biochem.

Am. J. Pathol.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Hepatology

Cell

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Molecular aspects of arsenic stress

J. Toxicol. Environ. Health B Crit. Rev.