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

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Abstract

The estrogenic potential of four pesticides (endosulfan, prochloraz, tolchlofos-methyl and propamocarb) was compared in parallel with 17β-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.

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, AMAP, 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, 2003, Bonefeld-Jorgensen, 2004). Other pesticides are more easily excreted from the body, like organophosphates (tolchlofos-methyl) 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, Bonefeld-Jorgensen, 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 aromatase activity (Andersen et al., 2002). In addition, several of the pesticides tested also showed the potential to affect the cellular level of ERα/β mRNA and ERα protein (Grunfeld and Bonefeld-Jorgensen, 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, EDSTAC, 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)-promoter-luciferase 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 integrated 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.

Section snippets

Chemicals

The pesticides endosulfan, prochloraz, tolchlofos-methyl and propamocarb were purchased from Dr. Ehrenstorfer (Ausburg, Germany) (Table 1). 17β-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 μM and the MVLN cells were exposed to the pesticide alone and co-exposed with either 25 pM E2 (EC40; Fig. 1) or

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 μM. In MVLN cells, endosulfan, prochloraz, tolchlofos-methyl and propamocarb caused cytotoxic responses at concentrations higher than 10, 25, 25 and 100 μM, respectively (Table 1). Similar results were obtained in MCF-7BUS cells but endosulfan was toxic at concentration higher than 25 μM (Table 1).

Comparison of E2 dose–response

The E2 dose–response of MVLN and MCF-7BUS cells was determined in the

Discussion

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 transfected cell systems (Andersen et al., 1999, Andersen et al., 2002, Bonefeld-Jorgensen et al., 2001, Bonefeld-Jorgensen et al., 1997, Mueller, 2004) and ER expression for assessment of

Acknowledgements

We thank the technical assistants Birgitte Sloth Andersen and Inger Sørensen for excellent technical assistance.

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