Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls
Introduction
Many of the toxic effects of environmentally persistent polyhalogenated dioxins, furans and biphenyls are mediated through binding to and activation of the aromatic hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor of the Per-Arnt-Sim family, which also includes the AhR dimerization partners, ARNT and ARNT2, the AhR repressor, hypoxia inducible factor 1, and the circadian pacemaker protein, Mop3 (Bunger et al., 2000, Gu et al., 2000, Baba et al., 2001). The ligand-bound AhR/ARNT complex interacts with xenobiotic response elements in the 5′ regulatory regions of a number of genes to enhance their rates of transcription. Members of the Ah gene battery of phase I and phase II xenobiotic-metabolizing enzymes induced by AhR agonistis include cytochromes P450 (CYP) 1A1, 1A2 and 1B1, NAD(P)H quinone oxidoreductase 1, UDP-glucuronosyltransferase 1A6, aldehyde dehydrogenase 3 and glutathione S-transferase Ya (Nebert et al., 2000). In addition to genes of the Ah gene battery, the ligand-activated AhR stimulates transcription of other genes, such as that of the cyclin/cyclin-dependent kinase inhibitor, p27Kip1 (Kolluri et al., 1999), which is involved in cell-cycle regulation, and the Bax gene (Matikainen et al., 2001), which controls entry into an apoptotic pathway. The ligand-activated AhR may also inhibit rather than enhance transcriptional activation, as has been reported for the human cathepsin D gene (Wang et al., 2001). The ligand-bound AhR, but not ARNT, was recently shown to interact directly with the retinoblastoma protein and to delay cell-cycle progression (Ge and Elferink, 1998, Puga et al., 1999), indicating that the AhR has functional roles in addition to that of a transcription factor.
The ability of compounds such as polychlorinated biphenyls (PCBs) to cause persistent activation of the AhR and to stimulate AhR-mediated gene transcription both in vivo and in vitro has been identified as an indicator of their potential for dioxin-like toxicity. One of the advantages of in vitro assays for Ah responsiveness is that they more readily allow for comparisons of the relative potencies among species. Together with exposure data, these assays may be useful in evaluating potential human risk from PCB exposure. Results of recent studies indicate that there may be significant differences in the effects of AhR agonists among vertebrate species, calling into question the relevance of in vivo studies using rodents to predict human susceptibility (Garrison et al., 1996, Vamvakas et al., 1996, Van drn Berg et al., 1998, Zeiger et al., 2001).
The induction of CYP1A1 has been used extensively as a quantitative measure of Ah responsiveness in terms of relative potency (REP). The ethoxyresorufin O-deethylase activity of CYP1A1 is most commonly measured, although reporter gene constructs incorporating 5′-regulatory sequences of the CYP1A1 promoters from several species have also been employed in quantitative studies investigating AhR agonist activity and the potential for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like toxicity (Postlind et al., 1993, Anderson et al., 1995, Sanderson et al., 1996, Jones and Anderson, 1999, Seidel et al., 2000, Ziccardi et al., 2000). In addition to CYP1A1, CYP1B1 has been identified as an extrahepatic P450 also under the regulatory control of the AhR (Savas et al., 1994, Sutter et al., 1994). While studies have been focused mainly on CYP1A1 expression, CYP1B1 expression may also be relevant, and in many cell types such as mammary stromal fibroblasts (Eltom et al., 1998), and some tumors (Murray et al., 1997, Murray et al., 2001, McFadyen et al., 1999, McFadyen et al., 2001) and tumor-derived cell lines (Döhr et al., 1995, Kress and Greenlee,1 997, Spink et al., 1997, Spink et al., 1998b, Sanderson et al., 2001), CYP1B1 expression exceeds that of CYP1A1. The ratio of CYP1A1 to CYP1B1 induced by xenobiotics may be altered in MCF-7 human breast cancer cells by exposure to different combinations of xenobiotics (Coumoul et al., 2001) or exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (Spink et al., 1998a). The ratio of CYP1A1 to CYP1B1 induced by xenobiotics may affect carcinogenesis, since the 4-hydroxylation pathway of 17β-estradiol (E2) metabolism, which in humans is catalyzed by CYP1B1 (Spink et al., 1994a, Hayes et al., 1996), has been associated with carcinogenesis, whereas E2 2-hydroxylation, which is catalyzed by CYP1A1 and CYP1A2 (Spink et al., 1992), appears to be protective against carcinogenesis (Liehr and Ricci, 1996, Yager and Liehr, 1996).
The MDA-MB-231 line of estrogen receptor-negative cells was shown to respond to exposure to AhR agonists with highly preferential induction of CYP1B1 as opposed to CYP1A1 (Döhr et al., 1995, Spink et al., 1998b, Angus et al., 1999). In this study we evaluated the effects of exposure to non-ortho-substituted PCBs on CYP1B1 expression in MDA-MB-231 cells by using three independent, quantitative assays. We measured (1) the effects of PCB exposure on induction of estrogen metabolism, (2) the levels of CYP1B1 and CYP1A1 mRNAs as determined by competitive reverse-transcriptase polymerase chain reaction (RT-PCR), and (3) the ability of the PCBs to activate the human CYP1B1 promoter in a luciferase reporter gene assay. The PCB congeners chosen for study (Table 1) include the four non-ortho-substituted PCBs for which the World Health Organization has assigned toxic equivalency factors, PCBs 77, 81, 126 and 169, and eight others for the purpose of comparing structure–activity relationships for human CYP1B1 induction.
Section snippets
Chemicals
TCDD was purchased from Cambridge Isotope Laboratories (Woburn, MA, USA). The PCB congeners (99+% purity) listed in Table 1 were obtained from AccuStandard (New Haven, CT, USA). N,O-bis(trimethylsilyl)trifluoroacetamide was from Pierce (Rockford, IL, USA). Type H-2 β-glucuronidase/aryl sulfatase, dimethyl sulfoxide (DMSO), and resveratrol were from Sigma (St Louis, MO, USA). 3′,4′-Dimethoxyflavone was from Lancaster Synthesis (Windham, NH, USA).
Culture of MDA-MB-231 and MCF-7 cells
MDA-MB-231 cells were cultured in Dulbecco's
Effects of PCB exposure on viability of MDA-MB-231 cells
Exposure to 1 μm of each of the PCB congeners, or to 10 nm TCDD in medium, for 72 h had no significant effect on cell viability of MDA-MB-231 cells, as evaluated by Trypan blue dye exclusion. After 72 h of exposure to the solvent vehicle, 0.1% (v/v) DMSO in medium, 80.0±5.0% (mean±SD; n=6 determinations) of MDA-MB-231 cells totally excluded the dye. Dye exclusion by MDA-MB-231 cultures after 72 h of exposure to 10 nm TCDD was 82.7±1.9% (n=3 determinations), and after 72 h of exposure to 1 μm of
Discussion
CYP1A1 is known to be weakly inducible in MDA-MB-231 cells (Vickers et al., 1989). Later studies showed that CYP1B1, rather than CYP1A1, is preferentially expressed in these cells (Döhr et al., 1995, Spink et al., 1998b, Angus et al., 1999). Our results show that TCDD- and PCB-induced E2 metabolism in MDA-MB-231 cells occurs primarily through the 4-hydroxylation pathway. The catechol metabolites of E2, 2-hydroxyestradiol and 4-hydroxyestradiol, are converted to 2-MeOE2 and 4-MeOE2 by the action
Acknowledgements
This research was supported by US Public Health Service Grants CA81243 and ES04913. The authors gratefully acknowledge use of the Wadsworth Center's Tissue Culture, Biochemistry, and Molecular Genetics Core Facilities.
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Present address: Purdue Pharma, Ardsley, NY, USA