PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides
Introduction
OCP are produced by industrial processes and used during agricultural procedures. They contaminate the food chains and display toxic effects in animals and human populations. For example, dieldrin, a commonly used insecticide, is suspected to increase breast cancer risks [1]. Indeed, most OCP display estrogenic actions because they bind and activate ER [2]. Moreover, estrogen exposure is known to increase breast cancer risk [3]. As a consequence, these xenobiotics are called xenoestrogens.
Activation of ERα leads to an increase of the transcription of a plethora of genes and in the repression of other genes [4]. We and other groups have previously shown that E2 inhibits CYP1A1-induced transcription [5]. CYP1A1 is an enzyme involved in xenobiotic metabolism and belongs to the CYP superfamily best known as major phase I xenobiotic metabolizing enzymes (XMEs). Expression of the CYP1 (1A1, 1A2, 1B1) subfamily is induced by dioxins, furans and polychlorinated biphenyls and this effect depends on the activation of the aryl hydrocarbon receptor (AhR) transduction pathway [6]. Inhibition of dioxin-mediated CYP1A1 activation in mammary carcinoma cells depends on ERα. As a consequence, we have studied the effects of xenoestrogens on this gene and shown that these xenobiotics display the same actions as natural estrogens [5]. However, in the hepatoma cell line, HepG2, which does not contain functional ERα, OCP but not E2 repress CYP1A1 gene expression [5]. This result shows that other transduction pathways could be involved in xenobiotic effects.
Recent studies have shown that some OCP could activate the PXR, a new member of the nuclear hormone receptor superfamily [7], [8], which is involved in CYP3A transcription in the liver and the gut [9], [10]. These studies have shown an activation of rodent PXRs (rat and mouse) and subsequent CYP3A23 (mouse) and CYP3A1 (rat) induction [9], [10]. Binding of pesticides to rodent PXR required the ligand binding domain (LBD) which is poorly conserved in the human gene [11], [12], [13], [14]. As a consequence, some ligands (rifampicin) of the human PXR do not bind rodent PXR [15]. Here, we show that three OCP, α-endosulfan, chlordane and dieldrin (Fig. 1), activate human PXR and CYP3A4 transcription and could thus alter the metabolism of a large number of endogenous as well as xenobiotic compounds.
Section snippets
Chemicals
Chlordane, dieldrin and α-endosulfan were obtained from Promochem and dissolved in ethanol (10 mM, volume of treatment 0.1%). The certified value of purity of these pesticides is 64.78, >99 and >99%, respectively. Other chemicals were obtained from Sigma (unless otherwise stated) and oligonucleotides were from Genset. DMEM culture medium was purchased from Invitrogen.
Cell culture
The human hepatoma cell line HepG2 (ATCC) and the human mammary tumor cell line MCF-7 (ATCC) were maintained in Dulbecco’s
Results
Human PXR is most expressed in liver and intestine, two major detoxication organs. We used human hepatocytes to evaluate the effect of pesticides (Fig. 1) and 17β-estradiol (E2) on CYP3A4 expression. Fig. 2 shows that treatment of cultured human hepatocytes with α-endosulfan (10 μM) for 24 hr increases CYP3A4 mRNA levels. In contrast, 17β-estradiol (E2) (10 nM) had no effect. This observation shows that although E2 and α-endosulfan display similar effects on CYP1A1 regulation, they exert a
Discussion
Our data provide novel evidence for the activation of human PXR by organochlorine pesticides and subsequent activation of CYP3A4 gene expression. These observations show that CYP1A1 and CYP3A4 are regulated in opposite ways by pesticides. Indeed, CYP1A1 is down-regulated by these compounds in the MCF-7 and the HepG2 cell lines whereas CYP3A4 gene expression is up-regulated in the HepG2 cell line as well as in human hepatocytes. We have demonstrated that the human PXR but not the ER is required
Acknowledgements
This work was supported by INSERM, Université Paris V—René Descartes, Fondation pour la Recherche Médicale (Grant No. 1000031401), Ministère de l’environnement, ARC, La Ligue contre la Cancer (X. Coumoul is a grant recipient from L.L.C) and Région Ile de France. We thank P. Maurel (from INSERM U128, IFR24, Campus CNRS, 1919 Route de Mende, 34293 Montpellier, France) for providing the human hepatocytes and R. Evans (Howard Hughes Medical Institute, Gene Expression Laboratory. The Salk Institute
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