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Evaluation of in vitro assays for determination of estrogenic activity in the environment

3 Description of the used methods and cell cultures

In vitro assays are useful techniques for the determination of estrogenic activity in environmental samples containing complex mixtures of contaminants. They enable estimation of total biological activity of all compounds that act through the same mode of action present in extracts of any environmental media.
The molecular mechanisms of estrogen action are the basis for the development of in vitro test systems. Therefore, a short description of these mechanisms is given here. The effects of estrogens are mediated by the estrogen receptor (ER), a member of the nuclear receptor superfamily (Ing and O’Malley, 1995). Inactive ERs exist in large complexes associated with heat shock proteins. Upon binding of an estrogenic compound to the ER, the heat shock proteins disassociate, inducing a conformational change that activates the receptor, and causes dimerization. The resulting homodimer complex exhibits high affinity for specific DNA sequences referred to as estrogen response elements (EREs) positioned in the regulatory region of estrogen-inducible genes in the nucleus. After binding to the ERE, the homodimer complex recruits transcription factors to the target gene promoter, which leads to gene activation and transcription. Following transcription, mRNA is then translated into proteins that are the ultimate effectors of the observed responses. By inducing the synthesis of new proteins that alter cellular functions, estrogens can have profound effects on cell function and physiology. Xenoestrogens can act as ER ligands that bind to the receptor, thus modulating endocrine pathways via a receptor-mediated process. Several in vitro assays have been developed to assess the estrogenic activity of individual compounds or complex mixtures (Zacharewski, 1997). Most of these assays fall into one of three categories: 1) estrogen receptor (ER) competitive ligand binding assays that measure the binding affinity of a chemical for ER; 2) reporter gene assays that measure ER binding-dependent transcriptional and translational activity; and 3) cell proliferation assays that measure the increase in cell number of target cells during the exponential phase of proliferation. In the following, the in vitro assays most widely used to assess estrogenic activity in wastewater and surface water are described.

3.1 ER binding assays

Competitive ligand binding assays are based on the primary mode of action of (xeno)estrogens, which is binding to the ER. In vitro competitive binding assays for the ER are well established and have been extensively used to investigate ER-ligand interactions. ER binding assays can be performed with receptors obtained from cytosolic or nuclear extracts of various mammalian and other vertebrate tissues (Ankley et al., 1998). Most ER binding assays quantifies the ability of a test compound to compete with radiolabelled 17b-estradiol for binding to the ER. In a typical competitive hormone binding assay, a high-speed centrifugal fraction of rat uterine cytosol or cell extract is incubated with excess radiolabelled 17b-estradiol ([³H]E2) and various concentrations of unlabelled test compounds. If the compounds compete with the [³H]E2 for receptor binding they will displace a fraction of the [³H]E2 from the receptor in a concentration dependent manner. The greater the concentration of the unlabelled competitor, the more [³H]E2 is displaced from the ER, and the less bound activity. The free [³H]E2 is separated from the bound [³H]E2 by filtration, hydroxyapatite extraction, or other methods and quantified by liquid scintillation counting (Gray et al., 1997). Non-specific binding is measured by addition of excesses of radioinert diethylstilbestrol (DES) or 17b-estradiol (E2). Total specific binding of [H³]E2 to the ER is calculated by subtracting the amount of [H³]E2 bound in the presence of DES or E2 from the amount of [H³]E2 bound in the absence of a competitor. Decreased specific binding of the [H³]E2 in the presence of a test sample suggests that the sample contains compounds, which can competitively bind to the ER ligand-binding site. In this assay, the compounds can reach the ER without having to pass a cell membrane.
Non-radioactive methods employing fluorescent polarization (Bolger et al., 1998) or enzyme-linked receptor assays (Seifert et al., 1999) have also been reported. However, these methods have not been widely used for environmental samples.
The concentration at which the tested compound results in a 50% decrease of the binding of [H³]E2 to the receptor is denoted as the IC50. Results are expressed as IC50 or as a relative binding affinity, which is the ratio between the IC50 of the test compound and that of unlabelled E2 (Soto et al., 1998).

ER binding assays are essential for the characterization of a compound as a ligand for the ER. However, ER binding determinations do not classify the ligand as agonist or antagonist. Moreover, the ability of a substance to initiate the molecular cascade of events implicated in gene transcription and protein synthesis associated with adverse effects is not determined in this assay. Furthermore, high concentrations of competitor ligand may result in non-competitive displacement (Zacharewski, 1997; Jobling, 1998). Finally, the cell-free nature of ER binding assays may lead to positive results for compounds, which have physical characteristics that would make it unlikely that they would normally enter the cell.

3.2 Reporter gene assays

The ER functions by modulating the rate of transcription of its target cell genes. Reporter gene assays are based on the ability of a compound to stimulate ER-dependent transcriptional activity. Thus, reporter gene expression is a result of the molecular cascade of events implicated in receptor activation, and as such provides a more integral indication of the estrogenic activity of a compound.

Reporter gene assays are carried out with genetically engineered mammalian cells or strains of yeast, with cells transformed (tranfected) by introducing vectors containing DNA sequences for the receptor, along with EREs linked to a reporter gene, and the reporter gene itself. A number of assays are available using cell lines with an endogenous ER (T47D cells or MCF-7 cells) or cell lines without an endogenous ER (e.g. yeast cells or HeLa cells). The reporter gene used in human cancer cells usually codes for luciferase and the reporter gene used in yeast cells usually codes for b-galactosidase.Reporter genes can be introduced into cells for the duration of the experiment only (transient transfection) or permanently, generating a genetically altered subline (stable transfection). Regardless of whether transient or stably transfected cells are utilized in the assays, test substances that enter the cells interact with the ER, which becomes activated by a change in its conformation. The activated ER then binds with soluble cell factors, and the resulting complex binds to the ERE on the reporter plasmid. This biding initiates the expression of the reporter gene and thereby the production of the enzyme. An appropriate substrate in the incubation mixture is metabolized by the newly synthesized enzyme, resulting in the production of an easily detected product.

In agonism studies, the cells are treated with a test substance and the induction of the reporter gene product is utilized to measure the response. For an assessment of relative potency, the induction can be compared to the induction by a reference estrogen. Alternatively, when dose-response data are generated, the EC50 for the test substance can be determined and compared with that for the reference estrogen.
For antagonism studies, the cells are exposed simultaneously to the reference estrogen and the test substance, while control cells are exposed to the reference estrogen only. The difference in induction of the reporter gene product in the presence and absence of the test substance is used as a measure of estrogen antagonism.

3.2.1 Mammalian-based reporter gene assays

3.2.1.1 ER-mediated chemical activated luciferase gene expression (ER-CALUX) assay

The ER-CALUX assay is a relatively new method developed in the Netherlands and is not yet widely used. The assay uses T47D human breast adenocarcinoma cells expressing endogenous ER and stably transfected with an estrogen-responsive luciferase reporter gene containing three EREs. In the ER-CALUX assay, exposure of cells to xenoestrogens results in binding to endogenous ER, activation of the receptor, and consequently, binding of the ligand-receptor complex to the EREs present in the promoter region of the stably integrated luciferase gene. This leads to expression of the luciferase gene, which is assayed by lysing cells, adding the substrate luciferin and measuring light output in a luminometer (Legler et al., 1999, 2003).

3.2.1.2 MVLN cell assay

The principles of this assay are similar to those of the ER-CALUX assay. However, the MVLN cell assay utilizes a derivate of the MCF-7 breast cancer cell line (MVLN) expressing endogenous ER and stably transfected with an estrogen-responsive luciferase reporter gene (Pons et al., 1990; Demirpence et al., 1993). Like in the ER-CALUX assay, the estrogen specific transcription activity of a test compound is directly related to the luciferase activity measured in the lysate of treated MVLN cells.

3.2.1.3 Chimeric receptor/reporter gene assays

Chimeric receptor/reporter gene constructs have also been proven to have utility in screening compounds for estrogenic activity. For example, the E2 Bioassay (Zacharewski et al., 1995) consists of a chimeric receptor (with the ligand binding domain of the ER and the DNA binding domain of the yeast transcription factor Gal4) and a Gal4-regulated reporter gene consisting of a luciferase gene regulated by a basal promoter and five tandem Gal4 response elements. Both of these constructs have been transiently or stably tranfected into recipient MCF-7 cells or HeLa cells (human cervical cancer cells). HGELN cells are stably transfected HeLa cells (Gutendorf and Westendorf, 2001). The transfected cells are treated with the test compounds. Estrogenic compounds will bind to the ER ligand-binding domain of the chimeric receptor and transform the construct into an activated high affinity DNA binding receptor complex, which binds to the Gal4 response element on the luciferase reporter gene. Binding of this activated complex will then initiate expression of the luciferase gene, which results in the induction of luciferase activity. Thus, luciferase activity is a direct measure of estrogenic activity.

3.2.2 Yeast-based reporter gene assay

3.2.2.1 Yeast estrogen screen (YES)

Yeast cells do not contain endogenous steroid hormone receptors. However, Metzger et al. (1988) showed that the human ER functions in yeast. The yeast strain Saccharomyces cerevisiae has been extensively used to investigate receptor structure and function as well as the activity of selected ligands (Zacharewski, 1997). The recombinant yeast estrogen screen (YES) developed by Glaxo, U.K. and first published by Routledge and Sumpter (1996) has been widely used to rapidly screen various estrogenic compounds. In this assay, yeast cells Saccharomyces cerevisiae have been stably transfected with the gene for the human ER (which has essentially the same specificity as the trout estrogen receptor (Le Dréan et al., 1995)) and a plasmid containing EREs and the lac-Z gene as a reporter gene coding for the enzyme b-galactosidase. The stably transfected yeast is incubated with the test compound for about 3 days. Activation of the receptor, by binding of a ligand, causes expression of lacZ, which produces b-galactosidase. This enzyme is secreted into the culture medium where it metabolizes the chromogenic substrate chlorophenol red-b-d-galactopyranoside, thus inducing a change in colour from yellow to red. The intensity of the red colour can be readily measured spectrophotometrically (Routledge and Sumpter, 1996). A dilution series of E2 as an estrogenic reference is assayed alongside the samples. The estrogenic activity for each sample is then compared to the E2 standard.
To determine whether compounds possess anti-estrogenic activity, E2 is added to the medium at a concentration that produces a sub-maximal response. The ability of the compounds to inhibit the colour change induced by E2 is then determined (Routledge and Sumpter, 1997; Sohoni and Sumpter, 1998).

Yeast has a number of advantages over other systems, including the absence of endogenous steroid hormone receptors and consequent lack of complex interactions between the ER and other receptors (Routledge and Sumpter, 1996). In addition, since the ER is transfected into the cell there is no concern about the effect of mutant or variant receptors, which are known to be present in receptor-positive cell lines such as MCF-7 cells (Sluyser, 1992; Pfeffer et al., 1996). Furthermore, the yeast cells grow in a medium devoid of steroid hormones, thereby ensuring low background levels. A disadvantage of the yeast-based assay is the presence of a yeast cell wall and active transport mechanisms that may differ from those found in mammalian cells and may affect the activity of some test compounds (Legler et al., 2002a). Furthermore, the YES assay cannot detect all anti-estrogens (Beresford et al., 2000; Graumann and Jungbauer, 2000).

Yeast-based reporter gene assays other than the YES assay employed by Routledge and Sumpter exist. Among these are a similar assay employed by Gaido et al. (1997) and a yeast two-hybrid assay employed by Nishikawa et al. (1999). However, these assays are more sensitive to toxic effects than the YES assay (Saito et al., 2002). In a comparative study of the three yeast-based assays, the YES assay measured estrogenic activity in each of 13 samples of influent sewage and final discharge. However, the assay employed by Gaido et al. and the yeast two-hybrid assay did not detect estrogenic activity in 5 or 9 of the 13 samples, respectively, because the yeast growth was inhibited (Saito et al., 2002).

3.3 Cell proliferation assays

3.3.1 E-screen assay

The MCF-7 cell line, which was developed at the Michigan Cancer Foundation in the early 1970s, derives from a woman in the late stages of metastatic mammary carcinoma (Soule et al., 1973). The MCF-7 cell line has been widely utilized in studies of cancer, steroid hormone biochemistry and toxicology. One of the most common applications of MCF-7 cells is for the study of estrogenic compounds. The estrogen-responsive cell growth of MCF-7 cells was discovered in 1976 by Lippman et al. In the E-screen assay developed by Soto et al. (1992), proliferation of MCF-7 cells as a response to estrogen is measured. The E-screen is based on the following three premises: (i) factors in human serum inhibit the proliferation of MCF-7 cells, (ii) estrogens induce cell proliferation by negating this inhibitory effect, and (iii) non-estrogenic steroids and growth factors do not neutralize the inhibitory signal present in human serum (Soto et al., 1992, 1995; Sonnenschein et al., 1996; Zacharewski, 1997). A similar number of MCF-7 cells are seeded in each well, they are allowed to attach for 24 hours, and then the medium is changed. Cells are then allowed to proliferate for 4-6 days in the presence of medium containing serum rendered estrogenless by charcoal-dextran adsorption, along with a range of concentrations of the compound being tested. After incubation, the cells are lysed and nuclei counted on a Coulter counter. The E-screen then compares the number of cells present following incubation in the presence or absence of the test substance (Soto et al., 1992, 1998). The end point of the E-screen has been modified by Körner et al. (1998), who, rather than counting cells or nuclei, utilize a colorimetric end point.
Antagonists are identified in a two-step test by a modification of the E-screen assay. In the first step the ability of the compound to inhibit estrogen action is tested. A range of concentrations of the presumptive antagonist is added to the medium containing the minimal dose of E2 that induces maximal proliferation. If it is established that a compound inhibits estrogen action, it should be verified that this is a receptor-mediated phenomenon; that is, increasing the concentration of E2 can reverse it. In this second step, the minimal dose of the antagonist needed for maximal inhibition is tested in the presence of a range of doses of E2 (Soto et al., 1998).

One potential disadvantage of the E-screen is its lack of estrogen specificity, as studies have shown that the MCF-7 cells proliferate in response to a range of mitogens, cytokines, growth factors, nutrients and hormones other than estrogens (Osborne et al., 1990; van der Burg et al., 1992; Dickson and Lippman, 1995; Jones et al., 1998; Diel et al., 1999; Andò et al., 2002). Thus, the E-screen assay could lead to false positive determinations of estrogenic compounds. Conversely, cytotoxic substances and general growth inhibitors could lead to identification of false negatives.

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