Effect of pesticides on estrogen receptor transactivation in vitro: A comparison of stable transfected MVLN and transient transfected MCF-7 cells

Molecular and Cellular Endocrinology 244 (2005) 20–30

Effect of pesticides on estrogen receptor transactivation in vitro: A comparison of stable transfected MVLN and transient transfected MCF-7 cells Eva C. Bonefeld-Jorgensen ∗ , Heidi T. Gr¨unfeld, Irene M. Gjermandsen Unit of Environmental Biotechnology, Department of Environmental and Occupational Medicine, University of Aarhus, Vennelyst Boulevard 6, DK-8000 Aarhus, Denmark Received 30 November 2004; accepted 20 January 2005

Abstract The estrogenic potential of four pesticides (endosulfan, prochloraz, tolchlofos-methyl and propamocarb) was compared in parallel with 17␤estradiol (E2) by reporter constructs in transient transfected MCF-7BUS and in stable transfected MVLN cells. Similar detection limit and half maximum effect concentration was determined for E2, whereas the maximum effect concentration of E2 was much higher in MCF-7BUS (10 nM) than in MVLN (150 pM), with the induced response being approximately six times the level in MVLN cells. Alone the four pesticides elicited the same relative response in the two bioassays, and similar data was obtained upon co-exposure with E2 for endosulfan and propamocarb. In contrast to the transient MCF-7BUS system, endosulfan further increased the E2 induced response in MVLN cells, whereas propamocarb did not induce the E2 response in MVLN cells as observed in MCF-7BUS cells. In conclusion, high agreement between the two reporter assays was observed, although some performance characteristics have to be considered. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: MVLN; MCF-7; Transient and stable transfection; Pesticides; Estrogenicity

1. Introduction Many pesticides and their residues are found ubiquitous in the environment and in food items as a result of use in pest control in, e.g. farming, greenhouses and combating typhus and malaria. Some pesticides, such as the organochlorine DDT/DDE and related compounds, e.g. endosulfan, dieldrin, toxaphenes and prochloraz highly bio-accumulate in the body fat and milk of animal and human (AMAP, 1998, 2003; Krieger, 2001). Since they freely transverse cellular lipid membranes, they are suspected to cause long-time irreversible effects in offspring development including the reproductive-, immune- and neurological-system and increase the risk of cancer (Bonefeld-Jorgensen and Ayotte,

Abbreviations: ␤-gal, ␤-galactosidase; E2, 17␤-estradiol; EC100 , effect concentration giving 100% (maximum) response; EC50 , effect concentration giving 40% of maximum response; ER, estrogen receptor; IC50 , 50% inhibitory concentration; LOEC, lowest observed effect concentration; Luc, luciferase; MOEC, maximum observed effect concentration; REP, relative effect potencies ∗ Corresponding author. Tel.: +45 8942 6162; fax: +45 8942 6199. E-mail address: [email protected] (E.C. Bonefeld-Jorgensen). 0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.01.017

2003; Bonefeld-Jorgensen, 2004). Other pesticides are more easily excreted from the body, like organophosphates (tolchlofosmethyl) and carbamates (propamocarb), but they have a higher acute toxicity (Krieger, 2001). Environmental chemicals, which interfere with the function of the endocrine system, are called endocrine disrupting chemicals (EDCs) referring their potential adverse effects to the health of humans and wildlife. In addition to pesticides, some chemicals belonging to the group of dioxins, furans, polychlorinated biphenyls (PCBs), plastic components like bisphenol-A and phthalates and surfactants, such as alkylphenols, have been demonstrated to have EDCs potentials (Bonefeld-Jorgensen and Ayotte, 2003; BonefeldJorgensen, 2004). Many biological approaches have been used to identify EDCs, but no exact testing guideline is given so far. Few chemicals affect only a single cellular target instead they act in different cell types often at multiple targets within the same cell type (Andersen et al., 2002; Mueller, 2004). Co-workers and we demonstrated, that of 24 in vitro tested pesticides, seven possessed the ability to disturb the sex hormone functions in more than one way, including the activities of the estrogen receptor (ER), the androgen receptor (AR) and the aro-

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

matase activity (Andersen et al., 2002). In addition, several of the pesticides tested also showed the potential to affect the cellular level of ER␣/␤ mRNA and ER␣ protein (Grunfeld and BonefeldJorgensen, 2004; Hofmeister and Bonefeld-Jorgensen, 2004). Moreover, eight of the pesticides transactivated the aryl hydrocarbon receptor (AhR) in human TV101L and/or rat H4IIE hepatoma cells (Long et al., 2003). In most studies, only a single assay has been used to assess estrogenicity of chemicals, but some inter-laboratory studies have been reported (Andersen et al., 1999; Fang et al., 2000). Studying the transactivation of the ER in different cell types originating from either different organs or species by different methods complicate the comparison of data for classification of a compound as having estrogenic potential. It is of high importance to have biologically realistic and powerful screening tools to assess potential ECDs. Several advisory committees Endocrine Disrupters Screening and Testing Advisory Committee (EDSTAC) (EDSTAC, 1998, 2000) and Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) have recently recommended development of stable estrogen-dependent gene expression assays for screening chemicals for estrogenic activity because of the high specificity and the high through-put capability (Wilson et al., 2004). Transient transfection of a reporter gene construct into cells can provide similar information, but is more time consuming and the data may be variable compared to stable transfected cells due to differences in the ratio of receptor and reporter gene (Andersen et al., 1999; Vinggaard et al., 1999; Wilson et al., 2004). Besides the importance of an effective screening tool, it should be noted that a screening method does not examine the biological mechanisms underlying the estrogenic effect of the chemical. There are numerous examples of stable transfected cell lines competent for the evaluation of the estrogenicity of a chemical, e.g. the recombinant human breast cancer cell lines MVLN (Pons et al., 1990), T47D-KBluc (Wilson et al., 2004) and T47D.Luc (Legler et al., 1999), which carry an estrogen receptor responsive element (ERE)-promoterluciferase gene reporter construct. The MVLN cell line was derived from MCF-7 cells, upon stable transfection with the p-Vit-tk-Luc-Neo reporter gene construct containing an estrogen regulated luciferase gene driven by the vitellogenin ERE in front of the tyrosin-kinase-promoter (Demirpence et al., 1993; Pons et al., 1990). The MVLN cell line has been employed in several studies to elucidate, e.g. the antiestrogenic effect of retinoic acid (Demirpence et al., 1993), the interaction between ER and flavonoids (Le Bail et al., 1998), the estrogenic relative potencies (REPs) of polycyclic aromatic hydrocarbons (PAHs) (Villeneuve et al., 2002b), the estrogenic potentials of phenols (e.g. nonylphenol and bisphenol) (Rivas et al., 2002; Van den Belt et al., 2004), phytoestrogens (e.g. genistein) (Dees et al., 1997b; Gutendorf and Westendorf, 2001), polybrominated diphenyl ethers (PBDEs) (Villeneuve et al., 2002a), hydroxylated polychlorinated biphenyls (OH-PCBs) (Machala et al., 2004), pesticides (e.g. DDT) (Dees et al., 1997a) and the antiestrogenic properties of tamoxifen (Badia et al., 1994; Pons et al., 1990). As outlined, MVLN cells have many applications, but a systematic comparison to the estrogenic response in the original transient transfected MCF-7 cells is needed for an inte-

21

Fig. 1. E2 dose–response in MVLN cells. The MVLN cells were seeded in 96-well plates at a density at 3 × 104 cells/well and exposed to E2 in the concentration range 0.05–500 pM for 24 h. Solvent control (EtOH) was set to 100%. Mean values ± standard deviations are shown, n = 10. RLU; relative luciferase unit per ␮g cell protein as described in Section 2.

grated assessment of estrogenic chemicals. In this study, we compare the estrogenic potential of four pesticides using the transient transfected MCF-7BUS and stable-transfected MVLN cells. 2. Materials and methods 2.1. Chemicals The pesticides endosulfan, prochloraz, tolchlofos-methyl and propamocarb were purchased from Dr. Ehrenstorfer (Ausburg, Germany) (Table 1). 17␤Estradiol (E2) was obtained from Sigma, Denmark. A stock solution of 10 nM was prepared for each pesticide and for E2 in 96% ethanol (extra pure) from Merck (Darmstadt, Germany). The final concentration range of the tested pesticides was 0.5–50 ␮M and the MVLN cells were exposed to the pesticide alone and co-exposed with either 25 pM E2 (EC40 ; Fig. 1) or with 50 nM ICI 182,780 (strong ER antagonist). MCF-7BUS cells were exposed to pesticide ±10 nM E2 (EC100 ; Fig. 2) as described by Andersen et al. (2002). Each concentration

Fig. 2. E2 dose–response in MCF-7BUS cells. The MCF-7BUS cells were seeded in 24-well plates at a density at 2 × 105 cells/well and exposed to E2 in the concentration range 1 pM to 0.1 ␮M for 24 h, n = 3. () Data used for extrapolation, response at EtOH (solvent control) exposure. See legend Fig. 1.

22

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

Table 1 Cytotoxicity of the tested pesticides in MVLN and MCF-7BUS cells Test compound

Code/chemical classesa

Cas no.

Cytotoxicity in MVLN cells (␮M)

Cytotoxicity in MCF-7BUS cells (␮M)b

Endosulfan Prochloraz Tolchlofos-methyl Propamocarb

i/OC fu/HC,OC fu/OP fu/CB

115-29-7 67747-09-5 1394594 24579-73-5

>10 >25 >25 >100

>25 >25 >25 >100

a As used in rapports on pesticides evaluation from WHO, Internal Programme on Chemical Safety (1999). Codes for use; in, insecticide; fu, fungicide. Codes for chemical class: CB, carbamate; HC, heterocyclic; OC, organochlorine; OP, organophosphorous. b Andersen et al. (2002).

was run in heptaplicates. Negative control (media alone), solvent control (0.1% EtOH) and E2 positive control (EC100 ) was included in each assay.

2.2. Cell culture The stable transfected MVLN cell line (Demirpence et al., 1993; Pons et al., 1990) and MCF-7BUS cells (Andersen et al., 2002) were cultured at 37 ◦ C in humidified atmosphere of 5% CO2 in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM) (Biochrome, Berlin, Germany) supplemented with 5% fetal calf serum (FCS) (HyClone, Belgium), 4 mM glutamine (Sigma), 6 ␮g/l insulin (Sigma), 64 mg/l garamycin (Schering-Plough, Brussel, Belgium), 1 nM sodium pyruvate (Invitrogen, Denmark) and 20 mM HEPES (Invitrogen). Cells were maintained according to Bonefeld-Jorgensen et al. (2001).

2.3. Cytotoxicity The cells were seeded in 96-well plates for 24 h. MVLN cells at a density at 3 × 104 cells/well in 1% charcoal/dextran treated FCS (DC-FCS) (HyClone) DMEM, and MCF-7BUS cells at a density of 1 × 104 cells/well in 5% DCFCS (Andersen et al., 2002). Serial dilutions of test pesticide in 0.5 or 5% DC-FCS DMEM were added to MVLN or MCF-7BUS cells, respectively, and incubated for 24 h. Cytotoxicity tests were performed according to CellTiter 96 Cell Proliferation assay from Promega (Madison WI, USA) as described by Andersen et al. (2002).

2.4. Estrogen receptor chemically activated luciferase expression (ER-CALUX) assay MVLN cells were seeded in 96-well plates with cell density at 3 × 104 cells/well and cultured in media containing 1% DC-FCS for 24 h, and then exposed to the test compound in 0.5% DC-FCS DMEM. After incubation for another 24 h, cells were lysed in 50 ␮l lysis buffer (Roche, Basel, Switzerland). Luciferase (luc) activity was measured in a LUMIstar luminometer (RAMCON, Denmark) in the 96-well plates according to a standard curve of recombinant luciferase (Bie & Berntsen, Denmark) as described (Andersen et al., 2002). Protein (internal standard) content was determined by adding 50 ␮l fluorescamine (0.5 g/l) diluted in acetonitrile to each well and fluorometric measurements in the WALLAC VICTOR2 (Perkin-Elmer, USA) at 355/460 nM wavelength according to a standard curve of bovine serum albumin (BSA) (Promega). The measured luciferase data were then corrected for cell density using the protein measurements. Each pesticide was tested as heptaplicates in at least three independent assays, except for endosulfan co-exposed with ICI, which was tested in two independent assays. The MCF-7BUS cells were seeded in 24-well plates at a density of 2 × 105 cells/well in 5% DC-FCS DMEM for 24 h and the test compounds were added in 5% DC-FCS DMEM as described (Andersen et al., 2002). Then the MCF-7BUS cells were transfected with 0.35 ␮g pERE-sv-Luc (vitellogenin ERE cloned in the pGL3-promoter vector; Promega) and 0.1 ␮g pON249 (␤-galactosidase (␤-gal) expression vector as internal standard) using 1.25 ␮l FuGene transfection reagent (Roche, Basel, Switzerland). After another 24 h of incubation, the cells were lysed in 110 ␮l lysis buffer (Roche) and luciferase activity was measured using the “luciferase reporter gene assay” from Roche.

␤-Gal was measured using the “␤-gal reporter gene assay” from Roche. The measured luciferase activities were then normalized to the ␤-gal activity and cell protein as described (Andersen et al., 2002).

2.5. E2 and ICI dose–response analysis MVLN cells were seeded in 96-well plates at a density at 3 × 104 cells/well in 1% DC-FCS DMEM for 24 h. Serial dilutions of E2 or ICI 182,780 plus 50 pM E2 in 0.5% DC-FCS DM were added and the cells incubated for 24 h. The luciferase activity and cell protein were measured as described in Section 2.4. In the MVLN cells, the E2 dose–response analysis was performed in 10 independent assays as triplicates or quadruplicates and the ICI dose–response analysis was performed in two independent assays as heptaplicates. MCF-7BUS cells were cultured, transfected and induced as described in Section 2.4 with serial dilution of E2 and incubated for 24 h. In the MCF-7BUS cells, the E2 dose–response analysis was performed in three independent assays as quadruplicates.

2.6. Statistics The data of the E2 dose–response analyses were related to the solvent control (100%), and evaluated in SigmaPlot 8.0 using the “Chapman, 4 parameter” sigmoidal equation. Detection limit, EC25 , EC50 , EC75 and EC100 were determined for both cell lines. The ICI dose–response data were related to the solvent control (100%), and evaluated in SigmaPlot 8.0 using the “Hill, 4 parameter” sigmoidal equation. In Figs. 4–7, the ER-CALUX data in MVLN cells were related to the negative control sample (media), which were set to 100%. In Figs. 8–10 and in Tables 3 and 4, the normalized luciferase data were related to E2 maximum response (EC100 ; Figs. 1 and 2), which were set to 100%. The dose–response analysis of endosulfan, prochloraz + E2 and tolchlofos-methyl were evaluated in SigmaPlot 8.0 using “Sigmoid, 4 parameter” for endosulfan and “Chapman, 4 parameter” for prochloraz + E2 and tolchlofos-methyl. The statistical analysis was performed on the mean values from each independent assay. Due to inequality of variance and relatively few data per concentration, non-parametric statistics were employed. Using SPSS, the Kruskal–Wallis test was used to compare differences between the different concentrations and the Jonckheere–Terpstra test (two tailed) was employed to analyse the linear trend between concentration and response. If one or both test showed significant difference (p < 0.05), the Mann–Whitney test was used to compare each concentration group with the control group.

3. Results 3.1. Cytotoxicity of tested pesticides The cytotoxicity tests of the four pesticides were performed in the MVLN and MCF-7BUS cells in the concentration range of 0.5–100 ␮M. In MVLN cells, endosulfan, prochloraz, tolchlofos-methyl and propamocarb caused cytotoxic responses at concentrations higher than 10, 25, 25 and 100 ␮M, respectively (Table 1). Similar results were obtained in MCF-7BUS

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

23

Table 2 Comparison of half maximum (E2–EC50 ) and maximum induction (E2–EC100 ) normalized to cell number Cell line

MVLN MCF-7BUS

Plate type (no. wells)

Cells/well

3 × 104 2 × 105

96 24

At LOEC

At EC50

E2 (pM)

nmol E2 per

10 10

3.3 2.5

105

cells

At EC100

E2 (pM)

nmol E2 per

33 43

11.0 10.8

105

cells

E2 (pM)

nmol E2 per105 cells

150 10000

50 2500

cells but endosulfan was toxic at concentration higher than 25 ␮M (Table 1). 3.2. Comparison of E2 dose–response The E2 dose–response of MVLN and MCF-7BUS cells was determined in the concentration range of 0.05–500 pM and 1–0.1 ␮M, respectively. Using SigmaPlot 8.0 the detection limit, EC25 , EC50 , EC75 and EC100 was determined to be 0.5, 16, 33, 60 and 150 pM, respectively, for MVLN cells (Fig. 1), and for the MCF-7BUS cells, to 0.1, 13, 43, 110 and 10.000 pM, respectively (Fig. 2). The maximum response (EC100 ) above background in MCF-7BUS cells was approximately six times the response at EC100 obtained in the MVLN cells, but at an E2 concentration approximately 60 times higher the EC100 of MVLN cells. Accordingly, the E2 concentration per cell number was similar at LOEC and at EC50 (Table 2), but at EC100 , the E2 concentration was 50 times higher for MCF-7BUS than for MVLN cells. 3.3. ICI dose–response of MVLN The antagonistic ICI dose–response of the E2 induced luciferase activity in MVLN cells was determined in the concentration range of 50 pM to 5 ␮M ICI. At 50 nM ICI exerted a maximum antagonistic response to background level of the 50 pM E2 (EC70 ) induced luciferase activity (Fig. 3). In MCF7 cells, it was earlier determined that 100 nM ICI antagonised EC100 –E2 (10 mM) induced transactivation, returning it to background levels (Bonefeld-Jorgensen et al., 2001). 3.4. Effect of the pesticides on ER transactivity The MVLN cells were exposed to endosulfan, prochloraz, tolchlofos-methyl and propamocarb in the concentration range

Fig. 3. ICI dose–response in MVLN cells. The MVLN cells were seeded in 96well plates and exposed to 50 pM E2 (EC70 ) and ICI 182,780 in the concentration range 50 pM to 5 ␮M for 24 h, n = 2. ( ) Data used for extrapolation, response at 50 pM E2 exposure. See legend Fig. 1.

of 0.5–50 ␮M alone, or co-exposed with the pure anti-estrogen ICI (50 nM) or with 25 pM E2 (EC40 ; see Fig. 1). The comparison of data for ER transactivation in the stable ERE-CALUX transfected MVLN cells, the ER transactivation in transient transfected ERE-CALUX MCF-7BUS cells, and cell proliferation in MCF-7BUS is presented in Table 3 (exposed to pesticide alone) and Table 4 (co-exposed with E2). 3.4.1. Comparison of the effects of pesticides on ER transactivation in MVLN and MCF-7BUS cells For endosulfan, prochloraz and tolchlofos-methyl similar results were obtained in both test systems. In MVLN and MCF7BUS cells, endosulfan alone significantly increased the ER transactivation in the range of 1–10 ␮M and 1–25 ␮M, reaching

Table 3 Summary of pesticide-induced agonistic response in the ER transactivation assay in MVLN and MCF-7BUS cells and in cell proliferation in MCF-7BUS cells Test compound

EtOH (control) 17␤-Estradiol Endosulfan Prochloraz Tolchlofos-methyl Propamocarb a

ER transactivation in MVLN cells

ER transient transactivation in MCF-7BUS cellsa

Cell proliferationa

LOEC (␮M)

MOEC (␮M)

Percent of 17␤-E2 max at MOEC

LOEC (␮M)

MOEC (␮M)

Percent of 17␤-E2 max at MOEC

LOEC (␮M)

MOEC (␮M)

Percent of 17␤-E2 max at MOEC

– 5 × 10−6 1 – 1 –

– 1.5 × 10−4 10 – 25 –

27 100 55 – 45 –

– 1 × 10−5 1 – 5 –

– 1 × 10−2 25 – 10 –

9 100 67 – 26 –

– 1 × 10−6 1 – 10 –

– 1 × 10−4 25 – 25 –

– 100 68 – 33 –

Andersen et al. (2002), n ≥ 3.

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

24

Table 4 Summary of pesticide-induced effects of 17␤-estadiol-induced ER transactivation assay in MVLN and MCF-7BUS cells and in cell proliferation in MCF-7BUS cells Test compound

EtOH (control) 17␤-Estradiol Endosulfan Prochloraz Tolchlofos-methyl Propamocarb a b c

ER transactivation in MVLN cellsa

ER transient transactivation in MCF-7BUS cellsb

Cell proliferationc

LOEC (␮M)

MOEC (␮M)

LOEC (␮M)

MOEC (␮M)

Percent of 10 nM (EC100 ) 17␤-E2 response

LOEC (␮M)

MOEC (␮M)

Percent of 0.01 nM (EC50 ) 17␤-E2 response

– 5 × 10−6 10 25 – –

– 28 1.5 × 10−4 100 10 130 25 70 – – – –

– 1 × 10−5 – 10 – 0.5

– 0.01 – 25 – 50

9 100 – 51 – 198

– 1 × 10−6 7.5 1 – –

– 1 × 10−4 10 25 – –

– 100 175 47 – –

Percent of 25 pM (EC40 ) 17␤-E2 response

Pesticides added together with 25 pM 17␤-E2 ∼50% of 17␤-E2 max response. Pesticides added together with 10 nM 17␤-E2 ∼100% of 17␤-E2 max response; Andersen et al. (2002). Pesticides added together with 0.01 nM 17␤-E2 ∼50% of 17␤-E2 max response, n ≥ 3.

55 and 67% of E2 maximum induction, respectively (Fig. 4A and Table 3). In MVLN cells, the observed dose–response effect of endosulfan was counteracted by 50 nM ICI to background level (Fig. 4B). Co-treatment of MVLN cells with endosulfan (0.5–10 ␮M) and 25 pM E2 elicited a dose–response CALUX activity being significantly increased at 10 ␮M being 130% of the response of 25 pM E2 alone (Fig. 4C). In MCF-7BUS cells, no effect was observed upon co-treatment with E2 (Table 4). Cell treatment with prochloraz in the range 0.5–25 ␮M exerted no effect in MVLN neither in MCF-7BUS cells (Fig. 5A and Table 3). Treatment of MVLN cells with 50 nM ICI alone or in combination with prochloraz (0.5–25 ␮M) antagonised the relative luciferase units (RLU) to a level below the constitutive background activity (Fig. 5B). Upon co-treatment of MVLN cells with 25 pM E2 (EC40 ), 25 ␮M prochloraz elicited a significantly decreased E2 response to a level being 70% of E2 alone (Fig. 5C and Table 4). In MCF-7BUS cells co-exposure with 10 nM E2 (EC100 ) and 10–25 ␮M prochloraz an antagonistic response was observed at 25 ␮M being 51% of the E2 level alone (Table 4). A clear dose–response CALUX activity was observed upon MVLN treatment with tolchlofos-methyl in the range of 1–25 ␮M reaching 80 and 45% of E2–EC40 and E2–EC100 , respectively (Fig. 6A and Table 3). Similarly, 5–10 ␮M tolchlofos-methyl induced the luciferase activity reaching 26% of EC100 (10 nM) in MCF-7BUS cells (Table 3). In MVLN cells, 50 nM ICI antagonised the luciferase activity induced by tolchlofos-methyl to a level below the constitutive background level (Fig. 6B). Tolchlofos-methyl had no effect together with E2 in either of the cell lines (Fig. 6C and Table 4). Alone propamocarb elicited no response in either of the two cells lines (Fig. 7A and Table 3). Parallel exposure of MVLN cells to 50 nM ICI alone or with propamocarb or 25 pM E2 significantly decreased the RLU to a level below the constitutive background activity (Fig. 7B). Upon co-exposure with 25 pM E2, no effect was observed for propamocarb in the MVLN cells (Fig. 7C), whereas in MCF-7BUS cells, an increased luciferase activity being 198% of the response of 10 nM E2 (EC100 ) was determined (Table 4).

Fig. 4. Effect of endosulfan on ER transactivation in MVLN cells. The compound was tested in the concentration range 0.5–50 ␮M alone (A), or +50 nM ICI (B) or +25 pM E2 (EC40 ) (C), for 24 h in independent assays. The blank control (cells only; not shown), the solvent control (EtOH) and the positive controls, 25 pM E2 (EC40 ) and 150 pM E2 (EC100 ; not shown) were run in parallel. Endosulfan was cytotoxic at concentrations >10 ␮M. Asterisk (*) significant (p ≤ 0.05) different from the solvent control response; (**) significant (p ≤ 0.05) different from the response of 25 pM E2; ( ) significant (p ≤ 0.05) different from the effect of test compound alone at the same concentration. Mean values ± standard deviations are shown, n = 3; endosulfan + ICI, n = 2. For RLU, see legend Fig. 1.

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

Fig. 5. Effect of prochloraz on ER transactivation in MVLN cells. Prochloraz was cytotoxic at concentrations >25 ␮M. Asterisk (*) significant (p ≤ 0.05) different from the solvent control response; (**) significantly (p ≤ 0.05) different from the response of 25 pM E2; ( ) significant (p ≤ 0.05) different from the effect of test compound alone at the same concentration, n = 3. See legend Fig. 4.

For comparison of the stable (MVLN) and transient (MCF7BUS) ER reporter assays, we introduced the obtained dose–response data to Sigma Plot Version 8. For endosulfan and tolchlofos-methyl, the E2–EC100 was set to 100% and for prochloraz co-exposed with E2, the reference E2 concentrations were set to 100%. The dose–response of endosulfan, prochloraz + E2 and tolchlofos-methyl in the two bioassays for estrogenicity are shown in Figs. 8–10, respectively. Generally,

25

Fig. 6. Effect of tolchlofos-methyl on ER transactivation in MVLN cells. Asterisk (*) significant (p ≤ 0.05) different from the solvent control response; (**) significant (p ≤ 0.05) different from the response of 25 pM E2; ( ) significant (p ≤ 0.05) different from the effect of test compound alone at the same concentration, n = 3. See legend Fig. 4.

compared to the response in MCF-7BUS cells, the background level was highest in the MVLN cells and the maximum response was reached at lower concentrations in MVLN cells. However, similar EC50 values were observed for the three pesticides in both bioassays (Figs. 8–10 and Table 5). For endosulfan the EC50 concentrations were calculated to 5.5 and 4.8 ␮M in MVLN and MCF-7BUS cells, respectively. In contrast to MCF-7BUS

Table 5 EC50 and relative estrogenic potencies (REPs) of the tested compounds Test compound

17␤-Estradiol Endosulfan Prochloraza Tolchlofos-methyl Propamocarb

MCF-7BUS cells (transient transfection)

MVLN cells (stable transfected)

EC50 (M)

REP

EC50 (M)

REP

1 9.0 × 10−6 2.0 × 10−6 1.4 × 10−5 –

33 × 10−12

1 6.0 × 10−6 1.4 × 10−6 7.7 × 10−6 –

43 × 10−12

4.8 × 10−6 22 × 10−6a 3.1 × 10−6 –

REP = EC50 E2/EC50 test compound. a IC , half maximum concentration of prochloraz to inhibit the E2–EC induced effect, which is set to 1. 50 40

5.5 × 10−6 24 × 10−6a 4.3 × 10−6 –

26

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

Fig. 8. Comparison of dose–response analysis of endosulfan in MVLN and MCF-7BUS cells. Cells were exposed to endosulfan in the concentration range 0.5–25 ␮M. The EC100 RLU value for E2 was set to 100%. MVLN, EC100 = 150 pM and MCF-7BUS, EC100 = 10 nM. Endosulfan was cytotoxic for the MVLN cells at 25 ␮M. ( ) and ( ) reference points for EC50 values for MVLN and MCF-7BUS cells, respectively. Mean values ± standard deviations are shown, n = 3. For RLU, see legend Fig. 1.

REP values in MVLN cells. The linear correlation of logarithmic transformed REP for MVLN and MCF-7BUS (Fig. 11) demonstrates a highly significant correlation with the R2 and regression equation coefficient being equal to 1. 4. Discussion

Fig. 7. Effect of propamocarb on ER transactivation in MVLN cells. Asterisk (*) significant (p ≤ 0.05) different from the solvent control response; (**) significant (p ≤ 0.05) different from the response of 25 pM E2; ( ) significant (p ≤ 0.05) different from the effect of test compound alone at the same concentration, n = 3. See legend Fig. 4.

Several bioassay systems based on the ER response mechanism have been developed including stable transfected cell lines (Balaguer et al., 2001; Fang et al., 2000; Gollapudi and Oblinger, 1999; Hyder et al., 1995; Legler et al., 1999; Pons et al., 1990; Tonetti et al., 2003; Wilson et al., 2004), transient

cells endosulfan was cytotoxic for MVLN cells at 25 ␮M, but at 10 ␮M similar relative luciferase activity was obtained in the two cell assays (Tables 1 and 3 and Fig. 8). Also the antagonistic effect of prochloraz was very similar in the two cell systems with IC50 concentrations being 24 and 22 ␮M in MVLN and MCF-7BUS cells, respectively (Fig. 9 and Table 4). Comparison of the tolchlofos-methyl dose–response in the two cell systems showed similar curves with same fold increase but being parallel displaced with the MVLN luciferases dose–responses at a higher level than MCF-7BUS (Fig. 10). The calculated EC50 of tolchlofos-methyl was 4.3 and 3.1 ␮M in MVLN and MCF-7BUS cells, respectively. 3.4.2. Relative potency In Table 5, is summarized the EC50 and the calculated relative potencies for the tested pesticides. As presented, the EC50 of E2 was lower in MVLN (33 pM) than in MCF-7BUS cells (43 pM). In contrast, although very similar, the EC50 of the endosulfan, prochloraz and tolchlofos were slightly higher in the MVLN CALUX system, which result in general little lower

Fig. 9. Comparison of dose–response analysis of prochloraz in MVLN and MCF-7BUS cells. MVLN cells, prochloraz + 25 pM E2 (EC40 ) vs. MCF-7BUS cells, prochloraz + 10 nM E2 (EC100 ) with prochloraz in the concentration range 0.5–50 ␮M. The E2 reference concentration (25 pM for MVLN and 10 nM for MCF-7BUS) was set to 100%. ( ) and ( ) reference points for IC50 values for MVLN and MCF-7BUS cells, respectively. () and (
) data used for extrapolation, corresponds the solvent control (EtOH) response. ( ) response at 25 pM E2; data used for extrapolation, n = 3. See legend Fig. 8.

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

Fig. 10. Dose–response analysis of tolchlofos-methyl in MVLN and MCF7BUS cells. MVLN cells, EC100 = 150 pM and in MCF-7BUS, EC100 = 10 nM. Cells were exposed to tolchlofos-methyl in the concentration range 0.5–50 ␮M. Tolchlofos-methyl was cytotoxic for both cell line at 50 ␮M. ( ) and ( ) reference points for EC50 values for MVLN and MCF-7BUS cells, respectively, n = 3. See legend Fig. 8.

transfected cell systems (Andersen et al., 1999, 2002; BonefeldJorgensen et al., 2001; Bonefeld-Jorgensen et al., 1997; Mueller, 2004) and ER expression for assessment of estrogenic chemicals (Grunfeld and Bonefeld-Jorgensen, 2004; Hofmeister and Bonefeld-Jorgensen, 2004). For an integrated assessment and comparison of the estrogenic potential of various chemicals in the different bioassays systems a quantitative comparison must be carried out. The aim of the present study was to compare the estrogenic potential of four pesticides, endosulfan, prochloraz, tolchlofosmethyl and propamocarb in parallel with 17␤-estradiol in transient pERE-sv-Luc transfected MCF-7BUS cells and the MVLN cell line derived from MCF-7 stably transfected with pVit-tkLuc (Pons et al., 1990). In both bioassays, the ER response element of Xenopus laevis vitellogenin A2 gene is involved. Time course studies of E2 did show similar relative luciferase units after 24 and 48 h of treatment in both cell systems (not shown). The LOEC and EC50 of E2 was similar in the two bioassays, although lower in MVLN compared to MCF-7BUS cells, LOEC being 5 and 10 pM and EC50 33 and 43 pM,

Fig. 11. The correlation of log REP for MCF-7BUS against log REP for MVLN. The R2 = 1 and the regression equation is y = x.

27

respectively. However, the MOEC of E2 was 150 pM and 10 nM in MVLN and MCF-7BUS, respectively, and the maximum fold induced luciferase activity in MCF-7BUS cells was approximately six times the level in MVLN cells. Accordingly, Pons et al. (1990) reported for MVLN cells an E2–EC50 and E2–EC100 at 20–30 and 100–1000 pM, respectively. Thus, the range between LOEC and MOEC is much wider in the transient MCF-7BUS (10 pM–10 nM) than that of the stable MVLN (5–150 pM) ERE reporter system. The cause of the higher ER transactivation level in MCF-7BUS cells compared to MVLN cells is not known, but may be one of several possible, e.g. the use of two different ERE-reporter constructs; effects of flanking sequences at the pVit-tk-Luc integration site in MVLN; and/or different ratios between endogenous expressed ERs and the number of ERE-reporter-genes. The ERE-reporter construct, pERE-sv-luc, used for transient transfection of MCF-7BUS cells, is carrying the Xenopus laevis vitellogenin ERE only upstream the SV40 promotor and the coding region of the firefly luciferase gene. In contrast, the pVit-tk-Luc reporter vector stable integrated in the MVLN cell line carries the 5 flanking region of the Xenopus laevis vitellogenin A2 gene, which contains an ERE, in front of the herpes simplex virus promoter for thymidine kinase (tk) and the luciferase coding region (Demirpence et al., 1993; Pons et al., 1990). In the primary established MVLN cells, 100–1000 pM E2 induced a maximum response 4–5 times the basal level (Pons et al., 1990). Demirpence et al. (1993) reported that 10 nM E2 induced a similar fold luciferase units above background level for MVLN and pVit-tk-Luc transient transfected MCF-7 cells being 5.4 and 5.2, respectively. However, they did not report whether there are differences in MOEC between the MVLN and MCF-7 cells at higher concentrations. In our hands, the maximum induced fold above background was 3.5 and 20 times in MVLN and pERE-sv-Luc transfected MCF-7BUS cells, respectively. In accordance, we calculated similar level of nmol E2 per 105 cells at LOEC and EC50 for the two cells systems, but 20 times higher level of nmol E2 per 105 cells at MOEC in MCF-7 cells. Considering similar levels of ERs in the two reporter bioassays, which are based on the same cell line, the ratio between endogenous expressed ERs and number of EREreporter-genes may differ between the MVLN and MCF-7BUS system. Possibly, in the transient transfected MCF-7BUS system more copies of reporter vectors per cells and thus per ERs are found, causing a higher capacity E2 dose–response ER transactivation compared to MVLN. In summary, similar sensitivity can be expected for the two reporter assays at the concentration range below 150 pM for agonistic compounds with ER affinities like E2, but at higher concentrations a dose–response effect might only be obtained in the transient transfected MCF-7BUS ERE-reporter system. We did observe a dose–response antagonistic ER-response of ICI 182.780 in MVLN cells with maximum inhibition at 50 nM of the relative luciferase units induced by 50 pM E2. An antagonistic effect of ICI 182.780 in MVLN cells was also reported by (Dees et al., 1997b) and the inhibition of ER transactivation by tamoxifen has been reported as well (Gutendorf and Westendorf, 2001; Le Bail et al., 1998). Earlier we showed antagonistic activity of ICI 182.780 (0.1 and 2.5 ␮M) and tamoxifen (2.5 ␮M) in

28

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

MCF-7 cells transiently transfected with a pERE-tk-cat reporter vector (Andersen et al., 1999; Bonefeld-Jorgensen et al., 2001, 1997). It is known that the antagonistic effect of ICI is mediated through binding to ER␣ followed by a rapid degradation of the receptor (Havrilesky et al., 2001; Howell et al., 2002; Vladusic et al., 2000), which is believed to reduce the steady state level of ER in MCF-7 cells (Dauvois et al., 1992, 1993). In a recent study, we showed by Western Blot that ICI 182.780 totally can degrade measurable ER␣ within 3 h, and that ICI abolished the E2 downregulation of ER␣ and ER␤ mRNA within 48 h (Hofmeister and Bonefeld-Jorgensen, 2004). The organochlor insecticide endosulfan was earlier assessed as an estrogenic compound in several in vitro biosystems (Andersen et al., 1999, 2002; Fang et al., 2000; Graumann et al., 1999; Hodges et al., 2000; Hunter et al., 1999; Legler et al., 1999; Soto et al., 1995; Wade et al., 1997). Endosulfan binds to ER with low affinity (Andersen et al., 1999) but binds to the human progesterone receptor (hPR) with higher affinity than the hER␣ (Scippo et al., 2004) and competes for the 3 H-E2 binding to steroid hormone binding globulin (SHBG) at high concentrations (Gale et al., 2004). In the stable transfected T47D.luc ER-CALUX transactivation system, the LOEC and MOEC of endosulfan were reported to be 1 and 10 ␮M, respectively (Legler et al., 1999). For endosulfan we determined the same LOEC (1 ␮M) in both ER transactivation systems and identical to the LOEC reported for the MCF-7BUS E-screen proliferation test (Andersen et al., 2002), but the MOEC was lower in MVLN cells (10 ␮M) than for the MCF-7BUS ER transactivation and E-screen (25 ␮M), possibly explained by the toxic effect of the compound on MVLN cells at 25 ␮M. Similar relative response was observed in both ER transactivation and E-screen assays being 55–68% of E2 MOEC level. Also the EC50 of ensdosulfan was similar for the two ER transactivation assays, MVLN (5.5 ␮M) and MCF-7BUS (4.8 ␮M), a concentration close to the EC50 (5.9 ␮M) determined for the T47D.luc cells (Legler et al., 1999). Co-exposure of MVLN cells with 10 ␮M endosulfan and E2–EC40 induced a response being 130% of the level induced by E2 alone, which is similar to the level observed upon co-response observed in E-screen analyses reaching 175% of E2–EC50 level. In contrast, endosulfan did not elicit any effect upon co-exposure with E2 on the transiently expressed ERE-luc in MCF-7BUS cells. Whether that can be explained by a different ratio of ER and reporter construct between MVLN and MCF-7BUS cells and/or the less sensitivity of the transient MCF-7BUS system at lower concentrations is not known. The organochloride fungicide prochloraz alone did not exert any estrogenic effect in either of the two ER transactivation assays or on MCF-7BUS cell proliferation. However, the compound significantly antagonized the E2-induced luciferase response in both ER transactivation assays and MCF-7BUS cell proliferation, with an antagonistic sensitivity of prochloraz in the assays decreasing in the order: E-screen > MCF7BUS > MVLN, where the inhibited effect of E2-induced response in the three assays were 53, 49 and 30%, respectively. The IC50 of prochloraz was determined to 24 and 22 ␮M for MVLN and MCF-7BUS cells, respectively. In a recent study, we did show that prochloraz also inhibit androgen receptor

transactivity and aromatase activity (Andersen et al., 2002), and significantly decreased ER␣ and ER␤ mRNA levels upon 48 h exposure, which could be reversed by co-treatment with ICI 182.780 (Hofmeister and Bonefeld-Jorgensen, 2004). In addition, as determined by Western Blot we showed that prochloraz could induce a degradation of ER␣ within 3 h at the level observed for ICI (Hofmeister and Bonefeld-Jorgensen, 2004). Prochloraz has been demonstrated to inhibit some cytochrome P450 and induce others (Laignelet et al., 1989). Whether the P450s are involved in the ER degradation and inhibition of E2 induced responses by prochloraz requires further investigations. The LOEC of the organophosphorus fungicide tolchlofosmethyl in the MVLN and MCF-7BUS ER transactivation assays where 1 and 5 ␮M, respectively, whereas 10 ␮M was determined for the E-screen assay (Andersen et al., 2002). The MOEC was 25 ␮M for MVLN and the E-screen assays and 10 ␮M in MCF7BUS cells. At the MOEC, the estrogenic response of tolchlofosmethyl was 45% (at 25 ␮M), 26% (at 10 ␮M) and 33% (at 25 ␮M) of the maximum RLU at E2-MOEC in the MVLN, MCF-7BUS and E-screen assays, respectively. Also for this compound similar EC50 was determined for MVLN and MCF7BUS cells being 4.3 and 3.1 ␮M, respectively. Tolchlofosmethyl seems to be most sensitive in the MVLN test having the lowest LOEC and the highest MOEC response of the estrogenic response. In none of the three estrogenic assays (MVLN, MCF-7BUS and E-screen) was tolchlofos-methyl able to further increase the E2 induced response. Recently, we demonstrated that tolchlofos-methyl antagonized the E2 induced decrease of ER␣ and ER␤ mRNA (Grunfeld and Bonefeld-Jorgensen, 2004). The carbamate fungicide propamocarb did not exert estrogenic responses on its own in neither of the ER transactivation assays nor MCF-7BUS cell proliferation analyses. In MVLN cells we did not observe any effect of propamocarb upon coexposure with E2, which is in accordance with the results observed in the E-screen analyses (Andersen et al., 2002). However, for the transient expression of the ER-regulated luciferase in the MCF-7BUS transient bioassay a further increase of the E2induced luciferase activity up to 198% of the E2–EC100 response was obtained. We do not know the reason for this difference between the compared estrogenic response assays. Although not obvious from the data, the reason might be that propamocarb affects the co-transfected reporter vector (pERE-sv-luc) and the vector used as internal standard (the ␤-galactosidase expression vector pON249) in different ways e.g. lowering the ␤-galactosidase expression level causing a higher ratio between luciferase and ␤-galactosidase upon normalization. A comparison of endosulfan, prochloraz and tolchlofosmethyl in the two ER transactivation systems is best performed using the determined EC50 for expression of the REPs compared to E2. Similar REPs were obtained for the four pesticides in the decreasing order tolchlofos-methyl > endosulfan > prochloraz but in general a slightly higher REP was observed for the transient MCF-7BUS system. However, the linear correlation of the logarithmic transformed REPs showed high identity between the two cell-based ER transactivation systems with a R2 equal to 1.

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

In summary, similar results were obtained when the four pesticides were tested alone in the two ER-reporter assays, the stable transfected MVLN and transiently transfected MVF-7BUS, as clarified by similar REP and a linear correlation between the logarithmic transformed REPs of the two ER-reporter systems. Also the data obtained upon co-exposure with E2 was similar but for endosulfan and propamocarb. In MVLN cells, endosulfan further intensified the E2 induced luciferase response as observed in the stable transfected T47D.luc ER-CALUX assay and the MCF-7BUS E-screen, whereas in our transient transfected MCF-7BUS system endosulfan did not affect the E2 induced response. In addition, propamocarb exerted a further increase of the E2 induced response in the transient transfected MCF-7BUS system. We do not know the reason for these differences but believe that it is easier to reproduce data in the stable MVLN cells in the range between E2 LOEC and MOEC induced RLU because no transfection, and thus normalization is required in the MVLN system. The transient transfection system may be more labile requiring co-transfection of two vectors into the cells, the ER-reporter and the ␤-gal expression vector used for normalization, where the test compound must not affect the internal standard for proper normalization. Moreover, the transient transfection system is based on the assumption that equal numbers of the two vectors are introduced into the cells, which also introduce some uncertainty into the assay system. Thus, in conclusion our analyses suggest that there is a general agreement between the two ER-reporter assays, although there are performance characteristics to be considered. Standardized estrogenic assays should be used for comparison of the estrogenic potential of chemicals, and as suggested (Gutendorf and Westendorf, 2001) the estrogenic potential of a complex mixture should in analogy to the TEQ-values for the Ah-receptor be expressed as estradiol-equivalents (EEq) expressed as the sum of relative potencies multiplied by the concentrations of a compound in the mixture. Acknowledgements We thank the technical assistants Birgitte Sloth Andersen and Inger Sørensen for excellent technical assistance. References AMAP, 1998. Assessment Report: Arctic Pollution Issues. Arctic Monitoring and Assessment Programme (AMAP), vol. xii, Oslo, Norway, 859 pp. AMAP, 2003. AMAP Assessment 2002: Human Health in the Arctic. Artic Monitoring and Assessment Programme (AMAP), vol. xiv, Oslo, Norway, 137 pp. Andersen, H.R., Andersson, A.M., Arnold, S.F., Autrup, H., Barfoed, M., Beresford, N.A., Bjerregaard, P., Christiansen, L.B., Gissel, B., Hummel, R., Jorgensen, E.B., Korsgaard, B., Le Guevel, R., Leffers, H., McLachlan, J., Moller, A., Nielsen, J.B., Olea, N., Oles-Karasko, A., Pakdel, F., Pedersen, K.L., Perez, P., Skakkeboek, N.E., Sonnenschein, C., Soto, A.M., 1999. Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals. Environ. Health Perspect. 107, 89–108. Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M., Bonefeld-Jorgensen, E.C., 2002. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl. Pharmacol. 179, 1–12.

29

Badia, E., Duchesne, M.J., Fournier-Bidoz, S., Simar-Blanchet, A.E., Terouanne, B., Nicolas, J.C., Pons, M., 1994. Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF7-derived cell line. Cancer Res. 54, 5860–5866. Balaguer, P., Boussioux, A.M., Demirpence, E., Nicolas, J.C., 2001. Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands. Luminescence 16, 153–158. Bonefeld-Jorgensen, E.C., 2004. The human health effect programme in Greenland, a review. Sci. Total Environ. 331, 215–231. Bonefeld-Jorgensen, E.C., Andersen, H.R., Rasmussen, T.H., Vinggaard, A.M., 2001. Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity. Toxicology 158, 141–153. Bonefeld-Jorgensen, E.C., Autrup, H., Hansen, J.C., 1997. Effect of toxaphene on estrogen receptor functions in human breast cancer cells. Carcinogenesis 18, 1651–1654. Bonefeld-Jorgensen, E.C., Ayotte, P., 2003. Toxicological properties of POPs and related health effects of concern for the arctic populations, in: AMAP assessment 2002: Human Health in the Arctic. Arctic Monitoring and Assessment Programme (AMAP), vol. xiv, Oslo, Norway, p. 137 (Chapter 6). Dauvois, S., Danielian, P.S., White, R., Parker, M.G., 1992. Antiestrogen ICI 164, 384 reduces cellular estrogen receptor content by increasing its turnover. Proc. Natl. Acad. Sci U.S.A. 89, 4037–4041. Dauvois, S., White, R., Parker, M.G., 1993. The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J. Cell Sci. 106, 1377–1388. Dees, C., Askari, M., Foster, J.S., Ahamed, S., Wimalasena, J., 1997a. DDT mimicks estradiol stimulation of breast cancer cells to enter the cell cycle. Mol. Carcinog. 18, 107–114. Dees, C., Foster, J.S., Ahamed, S., Wimalasena, J., 1997b. Dietary estrogens stimulate human breast cells to enter the cell cycle. Environ. Health Perspect. 105, 633–636. Demirpence, E., Duchesne, M.J., Badia, E., Gagne, D., Pons, M., 1993. MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity. J. Steroid Biochem. Mol. Biol. 46, 355–364. EDSTAC, 1998. Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). http://www.epa.gov/oscpmont/oscpendo/history/ coverv14.pdf. EDSTAC, 2000. Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). http://www.epa.gov/scipoly/oscpendo/edsow4.pdf. Fang, H., Tong, W., Perkins, R., Soto, A.M., Prechtl, N.V., Sheehan, D.M., 2000. Quantitative comparisons of in vitro assays for estrogenic activities. Environ. Health Perspect. 108, 723–729. Gale, W.L., Patino, R., Maule, A.G., 2004. Interaction of xenobiotics with estrogen receptors alpha and beta and a putative plasma sex hormonebinding globulin from channel catfish (Ictalurus punctatus). Gen. Comp. Endocrinol. 136, 338–345. Gollapudi, L., Oblinger, M.M., 1999. Stable transfection of PC12 cells with estrogen receptor (ERalpha): protective effects of estrogen on cell survival after serum deprivation. J. Neurosci. Res. 56, 99–108. Graumann, K., Breithofer, A., Jungbauer, A., 1999. Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds? Sci. Total Environ. 225, 69–79. Grunfeld, H.T., Bonefeld-Jorgensen, E.C., 2004. Effect of in vitro estrogenic pesticides on human oestrogen receptor alpha and beta mRNA levels. Toxicol. Lett. 151, 467–480. Gutendorf, B., Westendorf, J., 2001. Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology 166, 79–89. Havrilesky, L.J., McMahon, C.P., Lobenhofer, E.K., Whitaker, R., Marks, J.R., Berchuck, A., 2001. Relationship between expression of coactivators and corepressors of hormone receptors and resistance of ovarian cancers to growth regulation by steroid hormones. J. Soc. Gynecol. Invest. 8, 104–113. Hodges, L.C., Bergerson, J.S., Hunter, D.S., Walker, C.L., 2000. Estrogenic effects of organochlorine pesticides on uterine leiomyoma cells in vitro. Toxicol. Sci. 54, 355–364.

30

E.C. Bonefeld-Jorgensen et al. / Molecular and Cellular Endocrinology 244 (2005) 20–30

Hofmeister, M.V., Bonefeld-Jorgensen, E.C., 2004. Effects of the pesticides prochloraz and methiocarb on human estrogen receptor alpha and beta mRNA levels analyzed by on-line RT-PCR. Toxicol. In Vitro 18, 427–433. Howell, A., Robertson, J.F., Quaresma Albano, J., Aschermannova, A., Mauriac, L., Kleeberg, U.R., Vergote, I., Erikstein, B., Webster, A., Morris, C., 2002. Fulvestrant, formerly ICI 182, 780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment. J. Clin. Oncol. 20, 3396–3403. Hunter, D.S., Hodges, L.C., Vonier, P.M., Fuchs-Young, R., Gottardis, M.M., Walker, C.L., 1999. Estrogen receptor activation via activation function 2 predicts agonism of xenoestrogens in normal and neoplastic cells of the uterine myometrium. Cancer Res. 59, 3090–3099. Hyder, S.M., Shipley, G.L., Stancel, G.M., 1995. Estrogen action in target cells: selective requirements for activation of different hormone response elements. Mol. Cell. Endocrinol. 112, 35–43. Krieger, R.I., 2001. Handbook of Pesticide Toxicology, 1. Academic Press, San Diego, USA. Laignelet, L., Narbonne, J.F., Lhuguenot, J.C., Riviere, J.L., 1989. Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz). Toxicology 59, 271–284. Le Bail, J.C., Varnat, F., Nicolas, J.C., Habrioux, G., 1998. Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids. Cancer Lett. 130, 209–216. Legler, J., van den Brink, C.E., Brouwer, A., Murk, A.J., van der Saag, P.T., Vethaak, A.D., van der Burg, B., 1999. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol. Sci. 48, 55–66. Long, M., Laier, P., Vinggaard, A.M., Andersen, H.R., Lynggaard, J., Bonefeld-Jorgensen, E.C., 2003. Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line. Toxicology 194, 77–93. Machala, M., Blaha, L., Lehmler, H.J., Pliskova, M., Majkova, Z., Kapplova, P., Sovadinova, I., Vondracek, J., Malmberg, T., Robertson, L.W., 2004. Toxicity of hydroxylated and quinoid PCB metabolites: inhibition of gap junctional intercellular communication and activation of aryl hydrocarbon and estrogen receptors in hepatic and mammary cells. Chem. Res. Toxicol. 17, 340–347. Mueller, S.O., 2004. Xenoestrogens: mechanisms of action and detection methods. Anal. Bioanal. Chem. 378, 582–587, Epub 2003 October 2016. Pons, M., Gagne, D., Nicolas, J.C., Mehtali, M., 1990. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules. Biotechniques 9, 450–459.

Rivas, A., Lacroix, M., Olea-Serrano, F., Laios, I., Leclercq, G., Olea, N., 2002. Estrogenic effect of a series of bisphenol analogues on gene and protein expression in MCF-7 breast cancer cells. J. Steroid Biochem. Mol. Biol. 82, 45–53. Scippo, M.L., Argiris, C., Van De Weerdt, C., Muller, M., Willemsen, P., Martial, J., Maghuin-Rogister, G., 2004. Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors. Anal. Bioanal. Chem. 378, 664–669, Epub 2003 October 2025. Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Serrano, F.O., 1995. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 103, 113–122. Tonetti, D.A., Rubenstein, R., DeLeon, M., Zhao, H., Pappas, S.G., Bentrem, D.J., Chen, B., Constantinou, A., Craig Jordan, V., 2003. Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells. J. Steroid Biochem. Mol. Biol. 87, 47–55. Van den Belt, K., Berckmans, P., Vangenechten, C., Verheyen, R., Witters, H., 2004. Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol. Aquat. Toxicol. 66, 183–195. Villeneuve, D.L., Kannan, K., Priest, B.T., Giesy, J.P., 2002a. In vitro assessment of potential mechanism-specific effects of polybrominated diphenyl ethers. Environ. Toxicol. Chem. 21, 2431–2433. Villeneuve, D.L., Khim, J.S., Kannan, K., Giesy, J.P., 2002b. Relative potencies of individual polycyclic aromatic hydrocarbons to induce dioxinlike and estrogenic responses in three cell lines. Environ. Toxicol. 17, 128–137. Vinggaard, A.M., Joergensen, E.C., Larsen, J.C., 1999. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals. Toxicol. Appl. Pharmacol. 155, 150– 160. Vladusic, E.A., Hornby, A.E., Guerra-Vladusic, F.K., Lakins, J., Lupu, R., 2000. Expression and regulation of estrogen receptor beta in human breast tumors and cell lines. Oncol. Rep. 7, 157–167. Wade, M.G., Desaulniers, D., Leingartner, K., Foster, W.G., 1997. Interactions between endosulfan and dieldrin on estrogen-mediated processes in vitro and in vivo. Reprod. Toxicol. 11, 791–798. Wilson, V.S., Bobseine, K., Gray Jr., L.E., 2004. Development and characterization of a cell line that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen receptor agonist and antagonists. Toxicol. Sci. 81, 69–77, Epub 2004 May 2027.