cology 216 (2006) 44–54www.elsevier.com/locate/ytaap

Toxicology and Applied Pharma

Steroid receptor profiling of vinclozolin and its primary metabolites

José-Manuel Molina-Molina a,b, Anne Hillenweck c, Isabelle Jouanin c, Daniel Zalko c,Jean-Pierre Cravedi c, Mariana-Fátima Fernández b, Arnaud Pillon a, Jean-Claude Nicolas a,

Nicolás Olea b, Patrick Balaguer a,⁎

a INSERM Unité 540, UMI, Endocrinologie Moléculaire et Cellulaire des Cancers, 60 rue de Navacelles, 34090 Montpellier, Franceb Laboratory of Medical Investigations, Hospital Clínico, University of Granada, 18071 Granada, Spain

c Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1089 Xénobiotiques, Toulouse, France

Received 12 December 2005; revised 7 April 2006; accepted 7 April 2006Available online 5 June 2006

Abstract

Several pesticides and fungicides commonly used to control agricultural and indoor pests are highly suspected to display endocrine-disruptingeffects in animals and humans. Endocrine disruption is mainly caused by the interference of chemicals at the level of steroid receptors: it is nowwell known that many of these chemicals can display estrogenic effects and/or anti-androgenic effects, but much less is known about theinteraction of these compounds with other steroid receptors. Vinclozolin, a dicarboximide fungicide, like its primary metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), and 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), is known to bindandrogen receptor (AR). Although vinclozolin and its metabolites were characterized as anti-androgens, relatively little is known about theireffects on the function of the progesterone (PR), glucocorticoid (GR), mineralocorticoid (MR) or estrogen receptors (ERα and ERβ). Objectives ofthe study were to determine the ability of vinclozolin and its two primary metabolites to activate AR, PR, GR, MR and ER. For this purpose, weused reporter cell lines bearing luciferase gene under the control of wild type or chimeric Gal4 fusion AR, PR, GR, MR or ERs. We confirmed thatall three were antagonists for AR, whereas only M2 was found a partial agonist. Interestingly, M2 was also a PR, GR and MR antagonist(MR≫PRNGR) while vinclozolin was an MR and PR antagonist. Vinclozolin, M1 and M2 were agonists for both ERs with a lower affinity forERβ. Although the potencies of the fungicide and its metabolites are low when compared to natural ligands, their ability to act via more than onemechanism and the potential for additive or synergistic effect must be taken into consideration in the risk assessment process.© 2006 Elsevier Inc. All rights reserved.

Keywords: Vinclozolin; Fungicide; Estrogen receptors; Androgen receptor; Progesterone receptor; Glucocorticoid receptor; Mineralocorticoid receptor; Reporter cell lines

Introduction

It has been well documented that several chemicals fromagricultural, industrial, and household sources possess endocrine-disrupting properties, which provides a potential threat to humanand wildlife reproduction (Colborn, 1995; Jensen et al., 1995).Suggested mechanisms are that these environmental contam-inants alter the normal functioning of the endocrine and repro-ductive system by mimicking or inhibiting endogenous hormoneaction, bymodulating the production of endogenous hormones, orby altering hormone receptor (Sonnenschein and Soto, 1998). Themost documented mechanism for endocrine disruption by chem-

⁎ Corresponding author. Fax: +33 467540598.E-mail address: balaguer@montp.inserm.fr (P. Balaguer).

0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.taap.2006.04.005

icals is their acting as receptor agonists or antagonists throughdirect interaction with hormone receptors. Recent reports showedindeed that several pesticides and fungicides exert estrogenic andanti-androgenic activities through interaction with estrogen andandrogen receptors (Andersen et al., 2002; Kojima et al., 2004;Lemaire et al., 2004), and it is conceivable that many otherpesticides also display these activities. In this regard, one of thefirst chemicals reported to be an anti-androgen was the dicar-boximide fungicide vinclozolin (Gray et al., 1994; Kelce et al.,1998). Several pesticides and fungicides commonly used to con-trol agricultural and indoor pests are among the chemicals mostlikely suspected to act as endocrine disruptors. The ubiquitousnature of pesticide use has resulted in the contamination of foodproducts, workplaces, as well as the environment (Baumeister etal., 2002; Haith and Rossi, 2003).

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Vinclozolin, [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-oxazo-lidine 2,4-dione], is a widely used fungicide effective againstBotrytis cinerea, Schlerotinia sclerotiorum and Moniliam spp. infruits, vegetables andwines (Spencer, 1982; Papadopoulou-Mour-kidou, 1991). It is one of several dicarboximide fungicides cur-rently registered for use in the United States of America and inEurope [U.S. Environmental Protection Agency (EPA) 2003].Vinclozolin has been characterized as a potent in vitro and in vivoandrogen antagonist (Kelce et al., 1997; Gray et al., 1999; Mo-nosson et al., 1999; Wolf et al., 2000; Nellemann et al., 2003). Inthe field, vinclozolin is degraded into its two metabolites, M1 (2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoicacid) and M2 (3′,5′-dichloro-2-hydroxy-2-methylbut-3-enani-lide). Whereas vinclozolin itself is not persistent, these two meta-bolites display half-lives ofmore than 180 days and are likely to behighly mobile in the water phase. Vinclozolin is rapidly metabo-lized in mammals into the same two metabolites (Wong et al.,1995). Vinclozolin has been shown to cause adverse effects on themale reproductive system in rats. In adult male rats, chronicexposure to vinclozolin was demonstrated to result in the devel-opment of Leydig cell tumors and atrophic ventral prostate andseminal vesicles (Yu et al., 2004). In addition, oral exposure ofdams to 100 or 200mg/kg/day fromgestational day 14 to postnatalday 3 produced demasculinized male rat pups exhibiting reducedanogenital distances, nipple retention and cleft phallus withhypospadias (Gray et al., 1994; Colbert et al., 2005). Recently,vinclozolin was shown to alter the spermatogenic capacity of malegerm cells and sperm viability via its effects on DNAmethylation.Indeed, a transient embryonic exposure to vinclozolin duringgonadal sex determination in the rat reduced fertility and spermdevelopment in the adult testis (Uzumcu et al., 2004). Remarkably,this phenotype was transmitted through the male germ line to atleast generation F4 with no further exposure (Anway et al., 2005).Interestingly, the phenotype was found to be associated withmodulation of genome-wide DNA methylation patterns in themale germ line.

There are little data available on human exposure to vin-clozolin and its possible effects on reproductive health. However,in an occupational study, where the morbidity of the personnelinvolved in the synthesis and formulation of this chemical wasthoroughly investigated (Zober et al., 1995), an increase in testi-cular abnormalities was reported in the study group comparedwith controls. The exposed men had also significantly higherfollicle stimulating hormone (FSH) levels than the controls, sug-gesting testicular damage. In addition, vinclozolin and other anti-androgenic fungicides used in agriculture may be responsible forthe recently noted link between pesticide exposure and erectiledysfunction in otherwise healthy men (Oliva et al., 2002). Themolecular mechanism proposed for the anti-androgenic effects ofvinclozolin was that its two primary metabolites –M1 and M2 –compete for AR binding and inhibit DHT-induced transcriptionalactivation by blocking AR binding to androgen response elementDNA (Kelce et al., 1994).

Although vinclozolin has been characterized as an anti-an-drogen, relatively little is known about its possible effects on thefunction of the estrogen (ERα and ERβ), progesterone (PR),glucocorticoid (GR) or mineralocorticoid (MR) receptors, clas-

sified as members of the subfamily 3 within the nuclear receptor(NR) superfamily (Nuclear Receptors Nomenclature Commit-tee, 1999). Steroid receptors generally function as ligand-de-pendent transcriptional regulators (Tsai and O'Malley, 1994).Vinclozolin and its metabolites might indeed be disruptive toother steroid receptors, as it was demonstrated for other envi-ronmental toxicants (Laws et al., 1995; Scippo et al., 2004) andin particular, to receptors belonging to the same subfamily be-cause of the large degree of homology between receptors interms of primary structure and ligand specificity (Landers andSpelsberg, 1991; Fu et al., 2003).

The aim of the present work was to obtain additional infor-mation by providing a comprehensive steroid receptor profile ofvinclozolin and its primary metabolites. For this purpose,reporter gene assays were carried out to investigate the effects ofvinclozolin, M1 and M2, on AR, PR, GR, MR and ER-mediatedinduction of transcription. Using bioluminescent reporter celllines, we determined their agonistic/antagonistic effect on ste-roid receptors. In addition, competitive binding assays wereperformed for AR, MR and ERα. These data may help us topredict the alterations induced by vinclozolin, M1 and M2 andto more fully understand their mechanism of action.

Materials and methods

Chemicals and materials. Culture medium and fetal calf serum (FCS) wereobtained from Life Technologies Inc (Cergy Pontoise, France). R1881 waspurchased from NEN Life Science Products (Paris, France). R5020 (promeges-tone) was a gift from Sanofis-Aventis (Romainville, France). Geneticin andluciferin were purchased from Promega (Charbonnieres, France). 17β-Estradiol(E2), aldosterone (ALDO), dexamethasone (DEX) and puromycin were obtainedfrom Sigma (St Louis, MO). Chemicals were dissolved in ethanol at 1 mM.

Vinclozolin was purified from the fungicide formulation Ronilan DF (BASF,Levallois, France) by liquid extraction followed by crystallization. In brief,300 g of Ronilan DF was extracted three times with 1 l of dichloromethane(Scharlau Chemie S.A., Barcelona, Spain) and then evaporated to dryness undervacuum. Residue was dissolved in 3 l of methanol and solution was stored for48 h at −20 °C. At the end of the crystallization period, methanol phase wasdiscarded, and residue was washed with methanol then solubilized indichloromethane. Solvent was evaporated using a vacuum evaporator and dryresidue was pulverized. Recovery of vinclozolin was 32.2%. Vinclozolin puritywas checked by melting point measurement on a Büchi 510 apparatus (108 °Ccorresponding to the value given in the literature) and was analyzed by HPLCcoupled to a diode array detector (Spectra, Thermo Electron, Courtaboeuf,France) and its chemical purity was found to be greater than 97%.

For M1 and M2 synthesis, vinclozolin hydrolysis was carried out accordingto Szeto et al. (1989). In brief, 56 mg of vinclozolin was mixed with 1 l ofphosphate buffer (pH 7.08), stirred and heated for 2 h at 60–70 °C then for 2days at 35–40 °C (slow solubilization). M1 and M2 recovery: aqueous solutionwas extracted three times with methylene chloride, and the organic layer,containing M2, was dried with magnesium sulphate. A column containingFlorisil deactivated by 2% of water and sodium sulfate was conditioned withmethylene chloride/petroleum ether 2/3 (v/v). M2 was eluted with acetone/methylene chloride 1/9 (v/v), then concentrated to dryness. The white solid wasrecrystallized with toluene/petroleum ether 5/95 (v/v) leading to M2 (3.7 mg).The aqueous layer, containing M1, was acidified to pH 1 with concentrated HCl,then extracted three times with methylene chloride. The organic layer was driedwith acidified sodium sulfate and evaporated to dryness. The white solid wasrecrystallized with methylene chloride leading to the obtention of M1 (31 mg).M1 and M2 analyses: HPLC-UV showed 99.5% purity for M1 (0.5%vinclozolin) and 97.5% purity for M2 (2.5% M1). HPLC analyses wereperformed with a Thermo Separation P1000XR pump and a UV3000 detectoroperating at 240 nm. The C18 semi-preparative column (5μ-Kromasil, 250 mm)

46 J.-M. Molina-Molina et al. / Toxicology and Applied Pharmacology 216 (2006) 44–54

was operated at a flow rate of 2 ml/min with the following gradient: 0–20 min(100% A); 20–25 min linear gradient from A: 100% to B: 100%; 25–35 min(100% B). Mobile phases: A = 0.2% acetic acid solution/acetonitrile 50/50 (v/v);B = 0.2% acetic acid solution/acetonitrile 35/65 (v/v). M1 and M2 structureswere confirmed by NI-ESI-MS (M1: m/z = 302; M2: m/z = 258).

All cell culture plastics were obtained from Falcon (Merck Eurolab,Strasbourg, France) except 96-well Cellstar plates, which were obtained fromGreiner Labortechnic (Poitiers, France). A MicroBeta Trilux luminometer (EGGWallac, Turku, Finland) was used to detect luciferase activity in intact cells.

Plasmids: vector constructs. pSG5ERα-puro, pSG5ERβ-puro, pSG5AR-puro and pGal4RE-ERE-βGlobin-Luc-SV-Neo were already described (Bala-guer et al., 2001; Paris et al., 2002). p(Gal4RE)5-βGlobin-Luc-SV-Neo andpGal4-GR-puro were gifts of H. Gronemeyer (Stasbourg, France). In pGal4-GR-puro, the encoded chimeric Gal4-GR protein consists of 1 to 147 Gal4 aminoacids, a short linker region encoding amino acids IPRA, followed by glucorticoidreceptor (GR) LBD (Ile 500 to Lys 777). GR LBDwas introduced between Xho Iand BamH I restriction sites of pGal4-puro plasmid (Webster et al., 1998). Toconstruct Gal4-MR and -PR chimeras, LBDs ofMR (Gly 671 to Lys 984) and PR(Val 633 to Lys 933) were amplified by PCR with primers containing Xho I andBamH I restriction sites and substituted to GR LBD in pGal4-GR-puro.

Reporter cell lines. The stably transfected luciferase reporter MELN cell linewas obtained as already described (Balaguer et al., 2001). Briefly, to obtainMELN cells, ERα-positive breast cancer MCF-7 cells were transfected with theestrogen-responsive gene ERE-βGlob-Luc-SV-Neo (Balaguer et al., 1999).Selection of resistant clones by geneticin was performed at 1 mg/ml. MELN cellswere cultured in DMEM F12 with phenol red, supplemented with 10% FCS and1 mg/ml G418. Basal MELN cell activity was around 15% of maximal activity(100% for10 nM E2).

Generation of HELN ERα and HELN ERβ reporter cell lines was performedin two steps (Balaguer et al., 1999). The estrogen-responsive reporter gene wasfirst stably transfected into HeLa cells, generating HELN cell line and, in asecond step, these HELN cells were transfected with ERα or ERβ plasmidconstructs to obtain the HELN ERα or ERβ cell lines, respectively. Selection bygeneticin and puromycin was done at 1 mg/ml and 0.5 mg/ml, respectively.HELN cells were cultured in Dulbecco's modified Eagle medium (DMEM),supplemented with 5% FCS, 1 mg/ml G418. HELN ERs cells were cultured inDMEM without phenol red, supplemented with 6% dextran-coated, charcoal-treated FCS, 1 mg/ml G418 and 0.5 μg/ml puromycin. Basal HELN derivates(ERα and ERβ) cell activity was around 10% of maximal activity (100% for10 nM E2).

PALM cells were obtained as already described (Terouanne et al., 2000).Briefly PC3 cells were cotransfected with an androgen-responsive gene,MMTV-Luc-SV-Neo, and an androgen receptor expressing plasmid, pSG5-hAR-puro. Selection by geneticin and puromycin was done at 1 mg/ml and 1 μg/ml, respectively. PALM cells were cultured in Ham's F12, supplemented with5% FCS, 1 mg/ml G418 and 1 μg/ml puromycin. Basal PALM cell activity wasaround 10% of maximal activity (100% for 10 nM R1881).

Generation of the progestative, glucocorticoid and mineralocorticoidreporter cell lines was done in two steps as already made for PPARα, δ and γreporter cell lines (Seimandi et al., 2005). The Gal4-responsive reporter genewas first stably transfected into HeLa cells, generating HG5LN cell line and, in asecond step, these HG5LN cells were transfected with Gal4-PR-puro, Gal4-GR-puro and Gal4-MR-puro plasmid constructs to obtain the HG5LN Gal4-PR, -GRand -MR cell lines, respectively. Selection by geneticin and puromycin was doneat 1 mg/ml and 0.5 μg/ml, respectively. HG5LN cells were cultured in DMEMsupplemented with 5% FCS and 1 mg/ml G418. HG5LN Gal4-NR cells werecultured in the same medium supplemented with 0.5 μg/ml puromycin. BasalHG5LN Gal4-NR cell activity was around 10% (10, 5 and 10% for PR, GR andMR, respectively) of maximal activity (100%) (100 nM R5020, 100 nMdexamethasone and 10 nM aldosterone for PR, GR and MR, respectively).

Luminescent and inducible clones were identified using photon countingcameras (Argus 100 from Hamamatsu or NightOWL from Berthold Technol-ogies) and the most responsive clones were isolated.

Luciferase assay: stable gene expression assay. Reporter cells were seeded ata density of 20,000 cells per well, in 96-well white opaque tissue culture plates

and maintained in DMEM without phenol red, supplemented with 6% dextran-coated, charcoal-treated FCS (excepted for PALM cells which were maintainedin Ham's F12, supplemented with 6% dextran-coated, charcoal-treated FCS).Test compounds (prepared 4× concentrated in the same medium) were added 8 hlater and cells were incubated for 16 h with the compounds (excepted for PALMwhich were incubated for 40 h). Experiments were performed in quadruplicate.At the end of incubation, the medium containing test compounds was removedand replaced by culture medium (DMEM without phenol red or Ham's F12,supplemented with 6% dextran-coated, charcoal-treated FCS) containing0.3 mM luciferin. At this concentration, luciferin diffuses into the cell andproduces a stable luminescent signal 5 min later. This signal is approximately 10-fold less intense than the signal obtained after cell lysis but it is perfectly stable forseveral hours. The 96-well plate was then introduced in a Microbeta Wallacluminometer and luminescence was measured in intact living cells for 2 s.

Agonist and antagonist assays. To study AR, PR, GR, MR and ERs (ERα andERβ) agonistic activities, PALM, HG5LN Gal4-PR, HG5LN Gal4-GR, HG5LNGal4-MR and HELN ERs cells, respectively, were tested in the presence ofincreasing concentrations (0.01–10 μM) of vinclozolin, M1 and M2. Antagonisticassays (for each receptor) were performed using at a concentration of agonistyielding between 60 and 90% of maximal luciferase activity. The antagonisticactivities of these compounds (tested at 0.01–10 μM) were determined by coin-cubation with R1881 (0.2 nM), R5020 (7 nM), dexamethasone (7 nM), aldosterone(1 nM) or E2 (0.1 nM) agonists, for AR, PR,GR,MR and ERs, respectively. At theseconcentrations, activities reach approximately 75, 60, 60, 75, 90 and 70% ofmaximal luciferase activity (for AR, PR, GR, MR, ERα and ERβ, respectively).

AR, MR and ERα competitive binding assays. Briefly PALM, Gal4-MR andMELN cells were seeded at a density of 200,000 cells per well, in 24-well tissueculture plates and grown in DMEM without phenol red, supplemented with 6%dextran-coated, charcoal-treated FCS. Twenty-four hours later, PALM cellswere labeled with 0.3 nM [3H]-R1881 (72.6 Ci/mmol specific activity) at 37 °Cfor 3 h in the absence or presence of vinclozolin, M1, M2 (1, 3 and 10 μM) orunlabeled R1881 (100 nM). Gal4-MR cells were labeled with 0.3 nM [3H]-aldosterone (39 Ci/mmol specific activity) at 37 °C for 3 h in the absence orpresence of vinclozolin, M1, M2 (1, 3 and 10 μM) or unlabeled aldosterone(100 nM). MELN cells were labeled with 0.1 nM [3H]-E2 (41.3 Ci/mmolspecific activity) at 37 °C for 3 h in the absence or presence of vinclozolin, M1,M2 (1, 3 and 10 μM) or unlabeled E2 (100 nM). The final incubation volumewas 500 μl, and each point was performed in duplicate. After incubation,unbound material was aspirated and cells washed three times with 500 μl of coldPBS. Then 250 μl lysis buffer (400 mM NaCl, 25 mM Tris phosphate pH 7.8,2 mMDTT, 2 mMEDTA, 10% glycerol, 1% triton X-100) was added, and plateswere shook for 5 min. Total cell lysate (200 μl) was mixed with 4 ml of LSC-cocktail (Emulsifier-Safe, Packard BioScience), and [3H] bound radioactivitywas liquid scintillation counted (LS-6000-SC, Beckman-Coulter, Roissy,France). Protein concentration was determined by Bio-Rad protein assay.Non-specific binding was determined in presence of 100 nM unlabeled E2,R1881 or aldosterone. Specific binding was calculated by subtractingnonspecific binding from total binding. Bound radioactivity values wereexpressed in dpm (disintegrations per minute).

Each compound was tested at least three times. In absence of competitor,specific bound radioactivity was 750–1000 dpm for MELN, 13,300–14,500dpm for PALM and 1150–1300 dpm for Gal4-MR. Results were plotted as dpmversus concentration of tested compounds.

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) toxicityassay. The effect of vinclozolin, M1 and M2 on cell viability was assessedwith the MTT test using Denizot and Lang's modified technique (1986). Briefly,PALM, HELN ERs and HG5LN-derived cells were cultured in DMEM withoutphenol red or in Ham's F12, supplemented with 6% dextran-coated, charcoal-treated FCS and were seeded as 20,000 cells per well in 96-well tissue culturegrade, multiwell plates and left to attach overnight. Twenty-four hours afterchemical addition, 0.5 mg/mlMTTwas dissolved in medium, and 100 μl of MTTsolution was added to each well, and the plate was incubated for 2 h. Viable cellscleaved the MTT tetrazolium ring into a dark blue formazan reaction product,whereas dead cells remained colorless. The MTT-containing medium was gentlyremoved and DMSO was added to each well. After shaking, the plates were read

Fig. 1. Transcriptional activity of AR in response to vinclozolin and its metabolitesM1 andM2. PALMcells were treatedwith vinclozolin (triangle),M1 (square), andM2 (circle) for 40 h at the indicated concentrations. Maximal luciferase activity(100%) was that obtained with 10 nM R1881. Values were the mean ± SD fromthree separate experiments. *P b 0.05 and **P b 0.01 (versus control).

Fig. 3. Transcriptional activity of AR in response to vinclozolin and itsmetabolites. PALM cells were treated with 0.2 nM R1881, in the presence ofincreasing concentrations of vinclozolin (triangle), M1 (square) and M2 (circle)for 40 h. Maximal luciferase activity (100%) was that obtained with 10 nMR1881. Values were the mean ± SD from three separate experiments. *P b 0.05and **P b 0.01 (versus R1881 0.2 nM).

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in absorbance at 540 nm. All results are expressed as the average of three wells.Additional control consisted of medium alone with no cells.

Data analysis. Each compoundwas tested at various concentrations in at leastthree independent experiments. For each experiment, tests were performed inquadruplicate for each concentration and data were expressed asmean ± SD.Datawere analyzed for significant differences using one-way ANOVA followed byDunnett's postcomparison test (versus control). Differences were consideredstatistically significant when P b 0.05. Individual dose–response curves, in theabsence and in the presence of agonist, were fitted using the sigmoidal dose–response function of a graphics and statistics software (Graph-Pad Prism, version4.0, 2003, Graph-Pad Software Incorporated, San Diego. CA). Transactivationdata are presented as EC50 values, effective concentration for half-maximalluciferase activity and as IC50 values, half-maximal inhibitory concentration foreach compound tested.

Results

Gene expression modulation via the androgen receptor (AR)

To study AR agonistic activity, PALM cells were tested inthe presence of increasing concentrations (0.01–10 μM) of

Fig. 2. Dose–response curves of E2, R1881, R5020, DEX and ALDOwith HELN (ERcells, respectively. Cells were treated with indicated steroids compounds for 16 h (exceMaximal luciferase activity (100%) was that obtained with 10, 10, 100, 100 and 10 nMthe mean ± SD from three separate experiments.

vinclozolin, M1 and M2. At 10 μM M2 showed partialagonistic activity (20% of maximal activity) while vinclozolinand M1 did not (Fig. 1). The EC50 value of R1881 was0.1 nM (Fig. 2). The antagonistic activity of the compoundswas tested against the synthetic androgen R1881 (0.2 nM). Atthis R1881 concentration, vinclozolin and its metabolites wereclearly potent antagonists for AR (Fig. 3, Table 1). At highestconcentration (10 μM), M1 decreased the R1881-induced ARtranscriptional activity (24.4%), although less efficiently thanthat observed with vinclozolin and M2 at the same con-centration (75 and 78%, respectively). Despite its partialagonistic activity, M2 was a better antagonist (IC50 =0.17 μM) than vinclozolin and M1 (IC50 = 0.3 and 53 μM,respectively).

The cytotoxicity of the fungicide vinclozolin and its primarymetabolites (M1 and M2) was assessed using the MTT test.Vinclozolin as well as its metabolites were devoid of any cyto-toxicity (cell survival ranging from 95 to 100%) in the 0.01–10 μM range (data not shown).

α and ERβ), PALM, HG5LN Gal4-PR, HG5LN Gal4-GR and HG5LN Gal4-MRpted for PALM cells which were treated for 40 h) at the indicated concentrations.for E2, R1881, R5020, dexamethasone and aldosterone, respectively. Values were

Table 1Inhibitory concentrations (IC50) of vinclozolin and its metabolites ontranscriptional activation through AR, PR, GR and MR

Ligands AR (0.2 nM) PR (7 nM) GR (7 nM) MR (1 nM)

IC50 (μM)

Vinclozolin 0.30 ± 0.04 18.3 ± 5.2 – 3.26 ± 0.25M1 53.4 ± 11.2 – – –M2 0.17 ± 0.03 3.3 ± 1.4 41.2 ± 20.3 1.46 ± 0.21

Fig. 5. Transcriptional activity of GR in response to vinclozolin and itsmetabolites. Gal4-GR cells were treated with 7 nM dexamethasone, in thepresence of increasing concentrations vinclozolin (triangle), M1 (square) andM2 (circle) for 16 h. Maximal luciferase activity (100%) was that obtained with100 nM dexamethasone. Values were the mean ± SD from three separateexperiments. *P b 0.05 and **P b 0.01 (versus dexamethasone 7 nM).

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Gene expression modulation via the progesterone receptor (PR)

Firstly, vinclozolin and its metabolites were first tested on theHG5LN constitutive cell line, which expresses only the Gal4-driven reporter gene. None of the compounds activated (non-specific activation) or inhibited (toxicity) the luciferase reportergene in HG5LN cells (data not shown). When HG5LN Gal4-PRcells were used to determine PR agonistic/antagonistic activityof vinclozolin, M1 and M2, no agonistic activity was observed(data not shown). On the contrary, antagonist activity wasobserved. The EC50 value of the synthetic progesterone R5020was 5 nM. Vinclozolin and M2 were full antagonists for PR buthad a very weak affinity (Fig. 4, Table 1). M2 was the mosteffective antagonists at high concentration (IC50 = 3.3 μM),while vinclozolin was slightly less effective (IC50 = 18 μM).

Gene expression modulation via the glucocorticoid receptor(GR)

The potential activity of vinclozolin and its metabolites viaGR was investigated using HG5LN Gal4-GR cells, in whichnone of the above-mentioned compounds show agonisticactivity (data not shown). The EC50 value of dexamethasonewas 5 nM. Antagonist activity was determined by coincubationwith 7 nM dexamethasone. The IC50 value for M2 was 41 μM(Fig. 5, Table 1). At 10 μM,M2 slightly decreased the luciferaseexpression. This antagonistic effect was greater when dexa-

Fig. 4. Transcriptional activity of PR in response to vinclozolin and itsmetabolites. Gal4-PR cells were treated with 7 nM R5020, in the presence ofincreasing concentrations of vinclozolin (triangle), M1 (square) and M2 (circle)for 16 h. Maximal luciferase activity (100%) was that obtained with 100 nMR5020. Values were the mean ± SD from three separate experiments. *P b 0.05and **P b 0.01 (versus R5020 7 nM).

methasone concentration was lower (data not shown), indicat-ing that M2 could be a full antagonist but with very low affinityfor GR. In contrast, vinclozolin and M1 did not show anyantagonistic activity toward GR.

Gene expression modulation via the mineralocorticoid receptor(MR)

Mineralocorticoid and anti-mineralocorticoid activity ofvinclozolin and its metabolites was investigated in HG5LNGal4-MR cells. The EC50 value of aldosterone was 0.5 nM.Antagonistic activity was determined by coincubation with1 nM aldosterone. Vinclozolin and M2 were potent antagonistsfor MR (Fig. 6, Table 1). For the two compounds, the bestinhibitory effect was found at 10 μM (70 and 84% for

Fig. 6. Transcriptional activity of MR in response to vinclozolin and itsmetabolites. Gal4-MR cells were treated with 1 nM aldosterone, in the presenceof increasing concentrations of vinclozolin (triangle), M1 (square) and M2 (circle)for 16 h at the indicated concentrations. Maximal luciferase activity (100%) wasthat obtained with 10 nM aldosterone. Values were the mean ± SD from threeseparate experiments. *P b 0.05 and **P b 0.01 (versus aldosterone 1 nM).

Fig. 8. Transcriptional activity of ERβ in response to vinclozolin and itsmetabolites. HELNERβ cells were treatedwith vinclozolin (triangle),M1 (square)andM2 (circle) for 16 h at the indicated concentrations.Maximal luciferase activity(100%) was that obtained with 10 nM E2. Values were the mean ± SD from threeseparate experiments. *P b 0.05 and **P b 0.01 (versus control).

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vinclozolin and M2, respectively). Interestingly, again, M2 wasthe best antagonist (IC50 = 1.4 μM) followed by vinclozolin(IC50 = 3.2 μM). However, vinclozolin and its metabolites didnot show agonistic activity toward MR (data not shown).

Gene expression modulation via the estrogen receptor (ER)

In HELN ERα and HELN ERβ cells the EC50 values of E2

were approximately 0.019 nM for ERα and 0.07 nM for ERβ.Vinclozolin and its metabolites were first tested on the HELNparental cell line and luciferase expression was not induced(data not shown). Vinclozolin and its metabolites were thenapplied on HELN ERα and HELN ERβ (Figs. 7, 8).Vinclozolin, M1 and M2 increased luciferase expression(M2≫M1NVinclozolin), but their potency was very lowcompared to E2 (Fig. 2, Table 2). M1 and M2 displayed higherERα transactivation activity (EC50 = 21 and 1 μM, respectively)than vinclozolin (EC50 = 34 μM). Vinclozolin and itsmetabolites were less active on ERβ. M2 presented the bestpotency to activate ERβ (EC50 = 18 μM) compared to othercompounds (EC50 = 69 and 53 μM for vinclozolin and M1,respectively). Vinclozolin and its metabolites were also testedfor their antagonistic potential on ERα and ERβ, and none ofthese compounds exhibited ERα or ERβ antagonistic activity(data not shown).

Effect of vinclozolin, M1 and M2 on R1881, aldosterone and E2

binding to AR, MR and ERα

To determine whether the fungicide inhibitory effects ob-served in transactivation assays reflected the abilities of vinclo-zolin, M1 and M2 to inhibit the binding of R1881, aldosteroneand E2 to AR, MR and ERα, respectively, “whole-cell”competition binding assays with PALM, Gal4-MR and MELNcells were performed. The binding affinity of vinclozolin and itsmetabolites for AR was assessed using 0.3 nM [3H]-R1881 astracer. Vinclozolin andM2 inhibited the binding of R1881 to AR

Fig. 7. Transcriptional activity of ERα in response to vinclozolin and its meta-bolites. HELN ERα cells were treated with vinclozolin (triangle), M1 (square)and M2 (circle) for 16 h at the indicated concentrations. Maximal luciferaseactivity (100%) was that obtained with 10 nM E2. Values were the mean ± SDfrom three separate experiments. *P b 0.05 and **P b 0.01 (versus control).

in a dose-dependent manner, and complete inhibition byM2wasachieved at 10 μM concentration (Fig. 9). The least effectivecompound was M1, which showed low binding affinity for AR.The effect of vinclozolin and its metabolites on the binding ofaldosterone to MR was assessed using 0.3 nM [3H]-aldosteroneas tracer. Vinclozolin and M2 were able to displace aldoste-rone from MR (36 and 70%, respectively), while M1 was not(Fig. 10). Finally, the binding affinity of these compounds forERα was assessed using 0.1 nM [3H]-E2 as tracer. M2 was themost effective compound, inhibiting [3H]-E2 binding byapproximately 50% at the highest concentration (10 μM). Onthe contrary, the binding affinity of vinclozolin andM1 was verylow but were significantly able to displace E2 from ERα at10 μM concentration (Fig. 11).

Discussion

Vinclozolin as well as its two main metabolites exhibited apotent anti-androgenic activity in PALM cells – a stable pros-tatic cell line – used to evaluate androgenic activities. M2metabolite was the most potent antagonist followed byvinclozolin itself and metabolite M1 (IC50 = 0.17, 0.3 and53 μM for 0.2 nM R1881, respectively). These results are inagreement with those previously obtained by our group(Balaguer et al., 2000; Sultan et al., 2001) and with thoseobtained byWong et al. (1995), using a reporter gene assay, whofound that vinclozolin and its metabolites clearly show potent

Table 2Effective concentrations (EC50) of vinclozolin and its metabolites ontranscriptional activation through ERs and AR

Ligands ERα ERβ AR

EC50 (μM)

Vinclozolin 34.3 ± 14.2 69.9 ± 27.5 120.3 ± 50.5M1 21.1 ± 1.3 53.2 ± 13.2 400.4 ± 182.3M2 1.1 ± 0.2 18.6 ± 2.1 84.3 ± 12.5

Fig. 9. Competition inhibition of [3H]-R1881 binding to AR by vinclozolin and its metabolites. PALM cells were incubated with different concentrations – 1 μM(black columns), 3 μM (grey columns), and 10 μM (white columns) – of vinclozolin, M1 and M2 in presence of 0.3 nM [3H]-R1881. Values were the mean ± SD fromthree separate experiments. *P b 0.05 and **P b 0.01 (versus 0.3 nM [3H]-R1881).

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antagonist effects (M2Nvinclozolin≫M1). However, slightlydifferent results – generally higher IC50 –were recently reportedby Körner et al. (2004) for the binding of vinclozolin to AR in acell-based androgen reporter assay to detect androgen agonistsand antagonists in an international interlaboratory study.Moreover, Kelce et al. (1994) reported that metabolites ofvinclozolin, in receptor competition assays, exhibited a potentbinding ability to AR, whereas the parent compound showedlittle activity (vinclozolin and M1 were about 100- and 10-foldless potent, respectively, than M2). This result differs from thefinding in the current study. However, in agreement with ourstudy, it was reported that vinclozolin itself showed potent anti-androgenic activity in the hAR assay, but it showed poor bindingability to hAR (Kojima et al., 2004). These results suggested thatCHOK1 cells used in the last study possess biotransformationcapacity, producing vinclozolin metabolites, as Vinggaard et al.(1999) pointed out. In this context, the intermediate effective-ness of vinclozolin, in our study, relative to M1 and M2 ininhibiting R1881-induced transcription, would be consistent

Fig. 10. Competition inhibition of [3H]-aldosterone binding to MR by vinclozoliconcentrations – 1 μM (black columns), 3 μM (grey columns), and 10 μM (white cValues were the mean ± SD from three separate experiments. *P b 0.05 and **P b

with its metabolization to M1 and M2 in PALM cells. The factthat vinclozolin produced a potent anti-androgenic response inour assay could indicate that PALM cells possess at least somebiotransformation capacity, as the active anti-androgeniccompounds were identified as metabolites M1 and M2 (Wonget al., 1995).

Interestingly, we also found that M2 showed moderateandrogenic activity at the highest concentrations tested, whereasM1, and vinclozolin, showed a slight androgenic activity. This isin agreement with several previous reports (Wong et al., 1995;Wilson et al., 2002; Körner et al., 2004), which found that M2 isan agonist at 10 μM, and with Nellemann et al. (2003) andSonneveld et al. (2005), which observed that vinclozolin is anagonist at concentrations higher than 3 μM in an AR reportergene assay using CHO cells and CALUX cells, respectively. Asreported by others (Wong et al., 1995; Kemppainen and Wilson,1996; Terouanne et al., 2002; Wilson et al., 2002), somecompounds – cyproterone actate, chlormadinone acetate andhydroxyflutamide –may display a mixed agonistic/antagonistic

n and its metabolites. HG5LN Gal4-MR cells were incubated with differentolumns) – of vinclozolin, M1 and M2 in presence of 0.3 nM [3H]-aldosterone.0.01 (versus 0.3 nM [3H]-aldosterone).

Fig. 11. Competition inhibition of [3H]-E2 binding to ERα by vinclozolin and its metabolites. MELN cells were incubated with different concentrations – 1 μM (blackcolumns), 3 μM (grey columns), and 10 μM (white columns) – of vinclozolin, M1 and M2 in presence of 0.1 nM [3H]-E2. Values were the mean ± SD from threeseparate experiments. *P b 0.05 and **P b 0.01 (versus 0.1 nM [3H]-E2).

51J.-M. Molina-Molina et al. / Toxicology and Applied Pharmacology 216 (2006) 44–54

activity. At high concentrations these ligands apparently inducea receptor conformation compatible with AR DNA binding andtranscriptional activation.

Despite the existing information regarding AR interactionof vinclozolin and its metabolites, much less is known aboutthe interaction of theses compound with other steroid receptorslike PR, GR and MR. We used three established reporter celllines to investigate the progestagenic, glucocorticoid andmineralocorticoid properties. In parallel, vinclozolin and itsmetabolites were tested on the HG5LN constitutive cell line,which expresses only the Gal4-driven reporter gene, andallowed us to characterize non-specific responses. Interesting-ly, none of the compounds activated or inhibited the luciferasereporter in HG5LN cells.

Vinclozolin andM2 induced a low, but clearly detectable, fullantagonistic effect towards PR, withM2 being the most effectiveanti-progestagen (IC50 = 3.3 μM in presence of 7 nM R5020).The anti-progestagenic response observed with these chemicalsis in accordance with several other studies. For instance, Laws etal. (1996) observed a low but detectable affinity of M1 and M2for the cytosolic progesterone receptor. Recently, Scippo et al.(2004), using receptor binding assays, also showed thatvinclozolin displays a low affinity for hPR with an IC50 valueof 233 μM. In this work, we showed that M2 could be a fullantagonist for GR, although the binding affinity for GR appearedto be very low. At 10 μM concentration, luciferase expressionwas slightly but significantly decreased. However, we concludethat vinclozolin and M1 are not antagonists.

Our results also showed that vinclozolin and M2 had a verystrong full antagonistic activity for MR, at the highestconcentrations (1–10 μM), M2 acted as a full MR antagonist(IC50 = 1.4 μM) in competition with 1 nM aldosterone.Vinclozolin, also significantly reduced aldosterone-mediatedtranscription, but less dramatically than M2. To our knowledge,there are no data available on the – possible – anti-minera-locorticoid effect of vinclozolin and its metaboliteM2. Thus, thisis the first work demonstrating the anti-mineralocorticoid effectof these chemicals.

The intermediate effectiveness of vinclozolin compared toM1 and M2, for AR, PR, GR and MR receptors tested in thiswork, could be consistent with its biotransformation intoM1 andM2 in cells – M1 reaches 40–75 times higher concentrationsthan M2 (Wong et al., 1995). Hence, the higher concentration ofthe less potent metabolite M1 suggests that it contributes, withM2, to the anti-androgenic, anti-progestagenic, and anti-mine-ralocorticoid effects of vinclozolin.

Since several environmental chemicals with anti-androgenicactivity have been shown to possess estrogenic activity as well(Sohoni and Sumpter, 1998; Paris et al., 2002; Willemsen et al.,2004; Kojima et al., 2004; Sonneveld et al., 2005), we decidedto investigate the possible estrogenic activity of vinclozolin andits metabolites. We carried out a reporter gene assay using ERnegative, ERα and ERβ cell lines (Balaguer et al., 1999). Theparallel use of HELN parental cells allowed the characterizationof non-specific responses, while HELN ERα and HELN ERβwere used to identify chemicals that interact specifically withERα and ERβ.

Our results demonstrated that vinclozolin and its metaboliteswere agonists towards ERα and ERβ although with a loweraffinity for ERβ. M2 displayed a higher ERα and ERβtransactivation activity (EC50 = 1 and 18 μM, respectively) thanM1 and vinclozolin itself. For both ER homologues, therefore,M2 was the most effective metabolite (95 and 42% of maximalactivity, for ERα and ERβ, respectively). Results obtained withHELN ERα cells were confirmed with MCF-7 cells stablytransfected with an estrogen-responsive luciferase reporter –MELN cell line (Balaguer et al., 2001) – and also used toevaluate the estrogenicity of vinclozolin and its metabolites(data not shown). Only a limited number of studies (Scippo etal., 2004) have previously shown a measurable estrogenicactivity – via ERα – for vinclozolin. In addition, we found thatvinclozolin and its metabolites, especially M2, possessed anERβ agonistic activity. It is in contrast with other in vitro studies(Willemsen et al., 2004; Kojima et al., 2004; Sonneveld et al.,2005) which found no estrogenic activity for vinclozolin inreporter gene assay and in vitro yeast screen system (Sohoni and

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Sumpter, 1998). It is necessary to emphasize that few studieshave investigated the estrogenic effects of M1 and M2vinclozolin metabolites via ER, despite the abundant informa-tion on the parent compound.

Using the competitive receptor binding assays, we identifiedthat the (anti)androgenic, anti-mineralocorticoid and estrogenicactivities of vinclozolin and/or its metabolites, detected by ourAR, MR and ERα assay systems, were mediated by way of thebinding of these compounds to AR, MR and ERα, respec-tively. The binding abilities of vinclozolin, M1 and M2 for ARand MR were consistent with the (anti)androgenic and anti-mineralocorticoid activities defined in our reporter gene assaysystem. However, the binding abilities of these compounds forERα were somewhat low and did not correlate well with theERα-transcriptional activation. The latter discrepancy may rep-resent a difference in sensitivity between the reporter gene assayand receptor binding assay.

Taken together, our results suggest that vinclozolin and itsmetabolites could act as ERs agonists and/or AR antagonists,which may help to explain some effects observed in animalmodels, such as feminization (Gimeno et al., 1996) with anoverall anti-androgenic effect in vivo (Gray et al., 1994). Theobservation that vinclozolin (or its metabolites) can directlyinteract with AR, ER, PR, GR and MR receptors in vitrosuggests that they may exhibit multiple hormonal activities,which renders difficult the interpretation of their mechanism ofaction. An early study by Diamanti-Kandarakis et al. (1995)suggested that steroidal and non-steroidal anti-androgens mayexert a wide range of other hormonal and anti-hormonaleffects by interacting with receptors for progesterone,glucocorticoids, and mineralocorticoids. We showed thatvinclozolin and its metabolites exhibited affinities for MR,PR, GR and ER, indicating that the interaction with otherreceptors than AR could contribute to the endocrine-disruptingeffects imputed to these chemicals. We are studying theestrogenic, (anti)androgenic, anti-progestagenic and anti-min-eralocorticoid activities of vinclozolin using in vivo biosensorsconstituted of our bioluminescent cells implanted in nudemice. We already used this technique to compare in vitro andin vivo estrogenic activities of a few xenoestrogens (Pillon etal., 2005). This kind of work generalized to AR, PR and MRwill inform us whether vinclozolin could also display in vivoefficacy. Moreover, our group is also investigating vinclozolinpresence and concentrations in human fat tissues, motherblood, cord serum blood and placentas from an importantnumber of individuals (n = 200) recruited in Southern Spain.Unpublished data confirmed that vinclozolin is very frequentlyfound in all kinds of samples. For example, in serum of adultpeople, the frequency of positive samples (above detectionlimit) is 95% with mean values of 9.79 ng/ml. In placentaltissues, the frequency is 83% with mean values of 2.08 ng/g oftissue. In blood from umbilical cords of the same series ofplacentas, the frequency is 86% with mean values 8.36 ng/g oftissue (Lopez-Espinosa et al., paper in preparation). Althoughthese concentrations are low, it cannot be excluded thatsynergistic effects acting through different nuclear receptorsbound to vinclozolin alone or to other endocrine disruptors

could mediate the in vivo endocrine disrupting effects of thesecompounds.

Although disruption of the progestative, glucocorticoid andmineralocorticoid activities was rarely addressed in the past, incontrast to estrogenic and anti-androgenic activities, it could wellcontribute to trigger a wide range of hormonal and/or anti-hor-monal effects in vivo. Vinclozolin and its metabolites couldpotentially disrupt sex hormone functions via multiple pathways.Indeed, in vivo effects of vinclozolin on male fertility werealready observed in mouse (Anway et al., 2005; Gray et al.,1999), and they were attributed to its anti-androgenic activity.However, it cannot be excluded that estrogenic and anti-progesta-genic activities of vinclozolin would play a role in its in vivoeffect on male sexual differentiation. Previous studies indicatedindeed that environmental chemical, which are able to inhibit PRbinding, may also have an important impact on abnormalitiesassociatedwith the developing reproductive system (Vonier et al.,1996; Pickford and Morris, 1999). Estrogens and progestagensare two key regulators of proliferation and differentiation inreproductive tissues (Graham and Clarke, 1997), and progesta-gens are known to oppose the biological effects of estrogens inmany systems (Zhu et al., 2000; Guo et al., 2001). Suchfunctional interplay between estrogen and progesterone is fun-damental to maintain a significant physiological process (Rose,1996). Moreover, as reported by Tran et al. (1996), the estrogenicactivity of some synthetic estrogens may be enhanced by theiranti-progestagenic activity. Based on reports demonstrating thatPR can quench the activity of ER in mammalian cells (Kraus etal., 1995), a chemical functioning as both an estrogen and an anti-progestagen would be expected to produce a stronger estrogenicresponse than a chemical that exhibits estrogenic activityexclusively. Finally, although anti-mineralocorticoid activity isexpected to have beneficial effects (Oelkers, 2005; White et al.,2005), it cannot be excluded that prolonged exposure to anti-mineralocorticoids could induce negative effects. Hence, al-though the potency of this fungicide (or its metabolites) to behaveas a hormone agonist or antagonist is low compared to the naturalligands, its ability to act via more than one mechanism mightcontribute to its biological effects in the intact organism, since thefinal response will likely be determined by the interactions of allthe implicated pathways. In addition, since living organisms areexposed to a large variety of xenobiotics simultaneously(Andersen et al., 2002) potential additive and/or synergisticeffects must also be taken into consideration.

In summary, we confirm that vinclozolin, M1 and M2, whichup to now have been reported to possess only an anti-andro-genic activity, are indeed potent antagonists for AR (M2Nvinclozolin≫M1), whereas M2 is also a PR, GR and MRantagonist (MR≫PRNGR) and vinclozolin is only a MR andPR antagonist (MR≫PR). Furthermore, they are agonists forERs, especially metabolite M2, via ERα. We demonstrate forthe first time that these compounds – mainly vinclozolin andM2 – possess an anti-mineralocorticoid activity. The presentstudy also demonstrates the effectiveness of our reporter geneassays for detecting chemical interactions with AR, PR, GR,MR and ERs and for discerning receptor agonists from antag-onists. This study shows that a single compound can exert

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different effects on several hormone receptors. However, it isnot possible to predict the overall endocrine disruptive effectfrom in vitro studies. Endocrine-disrupting chemicals often actthrough multiple endocrine pathways, emphasizing the need todemonstrate that the purported mechanism of action identifiedin vitro also is operative in vivo. In this regard, the work of ourteam is now oriented towards in vivo studies. We are studyingthe estrogenic, (anti)androgenic, anti-progestagenic and anti-mineralocorticoid activities of vinclozolin in mice grafted withour reporter cell lines. This kind of experiment will indicate ifvinclozolin could have significant in vivo nuclear receptoractivities. Thus, for a detailed risk assessment of vinclozolin (orits metabolites) concerning endocrine disruption in humans andanimals, more data are needed, especially in vivo data.

Acknowledgment

This study has been partially supported by the EuropeanUnion network CASCADE (FOOD-CT-2003-506319).

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