Journal of Steroid Biochemistry & Molecular Biology 82 (2002) 45–53

Estrogenic effect of a series of bisphenol analogues on gene and protein expression in MCF-7 breast cancer cells Ana Rivas a , Marc Lacroix b , Fátima Olea-Serrano a , Ioanna La¨ıos b , Guy Leclercq b , Nicolás Olea a,∗ a b

Laboratory of Medical Investigations, School of Medicine, University of Granada, 18071 Granada, Spain Laboratoire J.-C. Heuson de Cancérologie Mammaire, Service de Médecine Interne, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium Received 30 January 2002; accepted 3 July 2002

Abstract Bisphenols constitute a family of compounds, which includes many substances that have as a common chemical structure two phenolic rings joined together through a bridging carbon. In the present study, we aimed to determine whether several events triggered by 17 ␤-estradiol (E2 ) in MCF-7 breast cancer cells were also observed in response to various bisphenol-A (BPA) analogues. We studied the expression of estrogen controlled genes by measuring the induction of pS2 (mRNA and protein) and progesterone receptor (PgR) as well as the expression of a luciferase reporter gene transfected into MVLN cells. These data were compared to the cell proliferation potency and effectiveness as the latest expression of estrogen controlled functions. Bisphenols showed an agonistic effect in all our assays, suggesting that these compounds may act through all the response pathways triggered by the natural hormone. We found differences between the assays in the potency of bisphenols, defined as the minimum concentration required to produce a maximal effect. In the cell proliferation assay, all tested compounds needed a lower concentration than in the other assays to give maximal response. Our results suggest that the polarity and nature of the substituent in the central carbon determines the estrogenic potency. Presence of two propyl chains at the central carbon appears to confer the greatest potency in both gene and protein expression assays. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bisphenols; MCF-7; Gene expression

1. Introduction Bisphenols is a broad term that includes many substances which have as a common chemical structure two phenolic rings joined together through a bridging carbon (Fig. 1). Bisphenol-A (BPA) is a well-known estrogenic molecule [1] used extensively as a monomer in the plastics industry [2]. Apart from BPA, very little is known about the hormonal activity of bisphenols. This study appears mandatory, since they form a part of innumerable manufactured products such as epoxy, polycarbonate and polyester resins, are included in pesticides, certain emplastrums and rubber bridging materials [3]. The estrogenicity of bisphenols was reported for the first time in 1936 by Dodds and Lawson [4], who looked for synthetic estrogens devoid of the phenantrenic nucleus. These authors classified stilbenes and bisphenols by their ability to mimic 17 ␤-estradiol (E2 ) in increasing the uter∗

Corresponding author. Tel.: +34-958-242864; fax: +34-958-249953. E-mail address: [email protected] (N. Olea).

ine weight of ovariectomized rats. Stilbenes were found to be much more potent than bisphenols and one of them, diethylstilbestrol (DES), was selected for pharmaceutical use. Bisphenols were subsequently discarded for pharmaceutical purposes. In 1944, Reid and Wilson [5] again studied the relationship of the structure of some bisphenols to estrogenic activity in vivo compared with stilbene derivatives. Interestingly, some bisphenols were already used at this time in the plastic industry. For instance, bisphenol-F was part of Bakelite plastic, invented in 1909. The early reported estrogenicity of bisphenols was not considered a toxicological problem, and new bisphenols were synthesised for use in many industrial applications. Fifty years later, Gilbert et al. [6] studied the relationship between correspondence factor analysis and structure–activity in bisphenols by testing the effect of these compounds on proliferation of MCF-7 breast cancer cells, and by testing their binding specificity to the estrogen receptor. They proposed that no single structural feature defines estrogenic activity and that hydrophobic volume, together with hydroxy groups and conjugation with basic groups in bisphenol

0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 0 7 6 0 ( 0 2 ) 0 0 1 4 6 - 2

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A. Rivas et al. / Journal of Steroid Biochemistry & Molecular Biology 82 (2002) 45–53

Fig. 1. Chemical structures of test compounds.

structure, are involved in the triggering of cell proliferation. In 1998, we studied the estrogenic potency of some diphenylalkanes with bisphenol structure and demonstrated their ability to stimulate MCF-7 cell proliferation in vitro and to induce specific E2 -responsive proteins [7]. We proposed that both the length and the nature of the substituent groups at the bridging carbon of BPA analogues affected the estrogenic potency of these compounds. Good correlation was found between the relative binding affinity and the proliferative potency of each compound, suggesting that the proliferative effects of bisphenols are mediated through the binding to the estrogen receptor. Concern about the estrogenicity of bisphenols in plastics and the consequences of human exposure to them was

raised by Krishnan, who suggested that BPA was responsible for the estrogenicity of water sterilised in polycarbonate flasks [8]. We later demonstrated the leaching of various bisphenols (BPA and methyl-BPA from food cans [9] and dental sealants (BPA and BPA dimethacrylate) [10]. More recently, Hashimoto and Nakamura [11] showed the estrogenicity of a range of bisphenols used as dental materials. We demonstrated, for the first time, the estrogenicity of chlorinated BPA [12]. It has also been shown that polybrominated BPA, together with pure polybrominated diphenyl ethers (PBDEs) and hydroxylated PBDEs, are estrogen mimics in in vitro assays [12]. Chlorinated and brominated BPA with the lowest degree of halogenation showed the highest estrogenic potencies and the latter were agonists of both ER-␣ and ER-␤ receptors [13]. Other hormone-like actions, such as thyroid hormone-like

A. Rivas et al. / Journal of Steroid Biochemistry & Molecular Biology 82 (2002) 45–53

action, have been described for brominated bisphenols [14]. In the present study, we aimed to determine whether several events triggered by E2 in MCF-7 cells were also observed in response to various BPA analogues. The proliferative effect of these agents was also investigated. We explored the expression of the estrogen controlled genes by measuring the mRNA of the pS2 protein and the related protein released to the culture medium, the induction of the progesterone receptor (PgR), and the expression of a luciferase reporter gene transfected in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-Luc reporter-containing plasmid [15]). These data were compared to the cell proliferation potency and effectiveness as the latest expression of estrogen controlled functions.

2. Materials and methods 2.1. Chemicals E2 was purchased from Sigma (St. Louis, MO). RU 58 668 was provided by Dr. P. Van de Velde (Roussel UCLAF, Romainville, France). 1,1-bis(4-hydroxyphenyl)ethane (BP-1); 1,1-bis(4-hydroxyphenyl)propane (BP-2); 2,2-bis(4-hydroxyphenyl)butane (BP-3); 3,3-bis(4-hydroxyphenyl)pentane (BP-4); 2,2-bis(4-hydroxyphenyl)heptane (BP-5); 2,2-bis(4hydroxy-3-methylphenyl)propane (BP-6); 2,2-bis(4-hydroxyphenyl)perfluoropropane (BP-7); bis(4-hydroxyphenyl) ketone (BP-8) and 2,2-bis(4-hydroxyphenyl)propanol (BP-9) were synthesised by Dr. M. Metzler. Purity of these chemicals was at least 97%. Chemicals were prepared in ethanol to a final concentration of 1 mM and stored at 20 ◦ C, until use. 2.2. Cell culture The MCF-7 breast cancer cell line (clone BUS) was a gift of Dr. C. Sonnenschein (Tufts University, Boston, USA), and clone BB was obtained in 1977 from the Michigan Cancer Foundation. MVLN cells were obtained from Dr. M. Pons, INSERM U 58, Montpellier, France. The stable MVLN transfectant expressing the luciferase reporter gene under estrogen control has been described elsewhere [15]. These MCF-7-derived cells are stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene [15]. For routine maintenance, BUS cells were grown in Dulbecco’s modification of Eagle’s medium (DME) supplemented with 5% fetal bovine serum (FBS) (Biowhittaker, Boehringer Ingelheim Bioproducts Partnership, Verviers, Belgium). BB cells and MVLN cells were grown in minimal essential medium (MEM) with phenol red, supplemented with 10% fetal calf serum (FCS), l-glutamine, penicillin, streptomycin and gentamycin (Gibco BRL, Life Technologies;

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Merelbeke, Belgium) at the usual concentrations. Cell cultures were kept in an atmosphere of 5% CO2 /95% air under saturating humidity at 37 ◦ C. The cells were subcultured at weekly intervals using a mixture of 0.1% trypsin and 0.02% EDTA. 2.3. Charcoal-dextran treatment of serum Plasma-derived human serum was prepared from outdated plasma by adding calcium chloride to a final concentration of 30 mM to facilitate clot formation. Sex steroids were removed from serum by charcoal-dextran stripping [16]. Briefly, a suspension of 5% charcoal (Norit A, Sigma, St. Louis, MO) with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden) was prepared. Aliquots of the charcoal-dextran suspension of a volume similar to the serum aliquot to be processed were centrifuged at 1000 × g for 10 min. Supernatants were aspirated and serum aliquots were mixed with the charcoal pellets. This charcoal–serum mixture was maintained in suspension by rolling at six cycles per minute at 37 ◦ C for 1 h. The suspension was centrifuged at 1000 × g for 20 min, and the supernatant was then filtered through a 0.20 ␮m filter (Gelman Sciences, Ann Arbor, MI). Charcoal-dextran-treated human serum (CDHuS) was stored at −20 ◦ C until needed. Charcoal-dextran fetal bovine serum (CDFBS) was prepared following a similar protocol. 2.4. Cell proliferation experiments MCF-7 cells were used in the test of estrogenicity according to a technique slightly modified [17] from that originally described by Soto et al. [18]. Briefly, cells were trypsinized and plated in 24-well plates (Limbro, McLean, VA) at initial concentrations of 10,000 cells per well in 5% FBS in DME. Cells were allowed to attach for 24 h; then the seeding medium was replaced with 10% CDHuS-supplemented phenol red-free DME. Different concentrations of the test compounds were added, and the assay was stopped after 144 h by removing medium from wells, fixing the cells and staining them with sulforhodamine-B (SRB). The staining technique was modified from that described by Skehan et al. [19]. Briefly, cells were treated with cold 10% trichloracetic acid and incubated at 4 ◦ C for 30 min, washed five times with tap water and left to dry. Trichloroacetic-fixed cells were stained for 10 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Wells were rinsed with 1% acetic acid and air-dried. Bound dye was solubilized with 10 mM Tris base (pH 10.5) in a shaker for 20 min. Finally, aliquots were transferred to a 96-well plate and read in a Titertek Multiscan apparatus (Flow, Irvine, CA) at 492 nm. Linearity of the SRB assay with cell number was verified prior to cell growth experiments. Mean cell numbers from each experiment were normalised to the steroid-free control cultures to correct for differences in the initial seeding density.

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A. Rivas et al. / Journal of Steroid Biochemistry & Molecular Biology 82 (2002) 45–53

2.5. Progesterone receptor induction MCF-7 cells seeded in T-25 flasks (Nunc, Roskilde, Denmark) were incubated in 10% CDHuS for 72 h with 0.1–10 nM of E2 , and a parallel set of flasks was exposed to 0.1–10 ␮M of the chemicals. Controls received vehicle alone. At the end of the experiment, the medium was aspirated, and the cells were frozen in liquid N2 . To extract receptor molecules, cells were incubated with 1 ml of extraction buffer (0.5 M KCl, 10 mM potassium phosphate, 1.5 mM EDTA, and 1 mM monothioglycerol, pH 7.4) at 4 ◦ C for 30 min. PgR was measured in extracted cells by enzyme immunoassay using the Abbott progesterone receptor kit (Abbott Diagnostics, Chicago, IL) according to the manufacturer’s instructions. 2.6. Induction and secretion of pS2 protein pS2 protein was measured in the culture medium of MCF-7 cells with the ELSA-pS2 immunoradiometric assay (CIS Bio International, Gif-sur-Yvette, France). Cells were subcultured in 24-well plates for 144 h in 10% CDHuS. The culture medium was centrifuged at 1200 × g for 10 min to eliminate floating and detached cells. 2.7. Quantification of pS2 mRNA expression BB MCF-7 cells were trypsinized and plated in 100-mm dishes in phenol red-free MEM containing 10% dextran-coated charcoal fetal calf serum (DCC-FCS). After 4 days of subculture, different concentrations of the test compounds were added. The assay was stopped after 24 h by removing medium from dishes. Controls received vehicle alone. Total RNA was extracted with TriZol according to the instructions of the manufacturer (Gibco). The RNAs, dissolved in Rnase-free water, were quantified by spectrophotometry at 260–280 nm, and 15 ␮g were separated on a 0.9% agarose gel in 2.2% formaldehyde, 0.02 M 3-(N-morpholino)propane sulfonic acid (MOPS) and 1 mM EDTA before transfer onto a nylon membrane (Hybond-N, Amersham) and UV-cross linked. Prehybridization (4 h) and hybridisation (18 h) were performed at 42 ◦ C (at room temperature when using the 28S ribosomal oligonucleotide) in 50% formamide, 5× SSPE (20× SSPE = 0.2 M phosphate buffer (pH 7.4), 2.98 M NaCl, 0.02 M EDTA), 0.1% SDS, 10% 5× Denhard, 5% dextran sulphate, and 100 ␮g/ml sheared salmon sperm DNA. Hybridised membranes with pS2 cDNA probe (probe was obtained from American Type Culture Collection) were washed twice at room temperature in 2× SSC (1× SSC is 0.15 M NaCl and 15 mM sodium citrate, pH 7.0) 0.1% sodium dodecyl sulphate (SDS), followed by three washes in 0.1× SSC 0.1% SDS at 55 ◦ C. The filters hybridised with the 28S ribosomal RNA oligonucleotide probe (Clontech) were washed only at room temperature in 2× SSC 0.1% SDS. Northern blots were analysed by autoradiography in DuPont Cronex Lightning

Plus cassettes (DuPont, Germany) with intensifying screens (1–5 days exposure, Kodak XAR-5 film, Eastman Kodak, Rochester, NY), followed by quantification of signals by densitometry using a Hoefer GS300 densitometer (Hoefer Instruments, San Francisco, CA) and the GS370 Densitometry Analysis System 3.0 for Macintosh. To account for variations in RNA loading, pS2 values were normalised to 28S rRNA values. All membranes were exposed in autoradiography for various periods of time to ensure that only signals obtained in the linear range of film sensitivity were quantified. To rehybridize filters, former probes were first removed by incubating the membranes in pure water at 85 ◦ C. 2.8. Luciferase induction assay MVLN cells were cultured for 3–4 days in 35 mm ∅ plastic Falcon dishes (80,000 cells per dish) in DME containing 10% DCC-FCS. Test compounds under investigation were subsequently added to the culture medium and cultured for 24 h prior to luciferase assay. For that purpose, the medium was removed and the cell monolayer was washed twice with PBS. A minimal volume (250 ␮l) of a five-fold diluted lysis solution (Promega E153A) was then added to the dishes and maintained under mild agitation for 20 min to extract luciferase. Lysed cells were subsequently detached with a scraper (Costar, 2001) and centrifuged for 5 s at 10,000 × g to clarify the extracts. Finally, 20 ␮l of extracts were mixed at room temperature with 100 ␮l of a luciferase reaction medium (Promega E151A/E152A) prepared according to the manufacturer’s protocol. Induced light was measured with a Berthold luminometer (Lumat LB 9507); induction was expressed in arbitrary units with regard to the light measured with a blank relative light unit (RLU). To compare RLU data, the protein content of each extract was measured by the BCA assay (Pierce; Rockville, IL), and the data were expressed per mg of protein. The luciferase activity of each experiment was normalised to the steroid-free control cultures in order to correct for differences in the initial seeding density. 2.9. Analysis of the data Results from experiments were expressed as means±S.D. All experiments were repeated at least five times. The effect over control (i.e. PE, RIE) was expressed as the ratio between the highest cell yield obtained with the bisphenol tested and the hormone-free control. The relative effect (i.e. RPE, RPI) was calculated as 100× the ratio between the effect of test compounds-1 and the effect of E2 -1. The lowest observed effect concentration (LOEC) was defined as the lowest concentration of the bisphenol needed to produce maximal yield. The relative proliferation potency (RPP) and relative inductive potency (RIP) were calculated as 100× the ratio between the dose of E2 and that of bisphenol needed to produce maximal yield.

A. Rivas et al. / Journal of Steroid Biochemistry & Molecular Biology 82 (2002) 45–53

The differences between the different chemicals treatment groups were assessed by analysis of variance and then a posteriori Shaffe’s test. A P value of