Effect of pesticides on estrogen receptor transactivation in vitro: A comparison of stable transfected MVLN and transient transfected MCF-7 cells
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.
References (48)
The human health effect programme in Greenland, a review
Sci. Total Environ.
(2004)- et al.
Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity
Toxicology
(2001) - et al.
MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity
J. Steroid Biochem. Mol. Biol.
(1993) - et al.
Interaction of xenobiotics with estrogen receptors alpha and beta and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus)
Gen. Comp. Endocrinol.
(2004) - et al.
Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds?
Sci. Total Environ.
(1999) - et al.
Effect of in vitro estrogenic pesticides on human oestrogen receptor alpha and beta mRNA levels
Toxicol. Lett.
(2004) - et al.
Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens
Toxicology
(2001) - et al.
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
(2004) - et al.
Estrogen action in target cells: selective requirements for activation of different hormone response elements
Mol. Cell. Endocrinol.
(1995) - et al.
Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz)
Toxicology
(1989)
Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids
Cancer Lett.
Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line
Toxicology
Estrogenic effect of a series of bisphenol analogues on gene and protein expression in MCF-7 breast cancer cells
J. Steroid Biochem. Mol. Biol.
Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells
J. Steroid Biochem. Mol. Biol.
Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol
Aquat. Toxicol.
Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals
Toxicol. Appl. Pharmacol.
Interactions between endosulfan and dieldrin on estrogen-mediated processes in vitro and in vivo
Reprod. Toxicol.
Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals
Environ. Health Perspect.
Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro
Toxicol. Appl. Pharmacol.
Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF-7-derived cell line
Cancer Res.
Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands
Luminescence
Effect of toxaphene on estrogen receptor functions in human breast cancer cells
Carcinogenesis
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