Bio-assays provide an attractive alternative towards traditional analytical mass based assays. Bio-assays may provide useful information regarding the mode of action of specific contaminants (Geisy et al., 2002) as well as the integrated possible action of various contaminants from different environmental matrices. Bio-assays include various in vitro and in vivo assays based on a number of mechanisms including cell proliferation, ligand binding, vitellogenin induction, luciferase induction, or antigen-antibody interaction. Bio-assays may provide either qualitative or quantitative responses. Bio-assays, which include both in vitro and in vivo assays, may use whole organisms, whole cells, or biological materials like antibodies or estrogen receptors.
2.7.2.1 In Vitro bio-assays
A large number of in vitro tests have been developed for the screening of environmental EDCs (ECETOC, 1996; Holmes et al., 1998). In general, in vitro assays are designed to be sensitive and rapid to facilitate the screening of large numbers of chemicals. The rapid response and lower equipment requirements, resulted in in vitro bio-assays being widely used as alternative to conventional analytical techniques for environmental monitoring, particularly when measuring relative increases in total estrogenic activity is the monitoring objective. The most widely used in vitro bio-assays for the prediction of estrogenic endocrine disruption are listed in table 2.6.
2.7.2.2 Receptor Binding Assays
Steroid hormones, such as 17β-estradiol act on their specific target cells by binding to a high affinity hormone receptor within the cell nucleus (Fig. 2.4). Receptor binding assays have been developed to assess the ability of a substance, or environmental chemical mixture, to bind directly to the hormone receptor (Jensen and Jacobson, 1960; Ireland et al., 1980; Baker et al., 1999 a, b, 2000).
Table 2.6. Main types of in vitro test systems for the detection of endocrine disrupters
Assay Type Endpoint Measured
Receptor Binding Assays Binding affinity of a substance to a hormone receptor
Cell proliferation assays Ability of a substance to stimulate growth of hormone responsive cells (e.g. MCF-7, E-screen)
Reporter gene Assays Ability of a substance to activate transcription of a reporter gene construct in cells (mammalian/yeast)
Analysis of hormone- sensitive gene expression
Ability of a substance to induce expression of hormone-sensitive genes (e.g. pS2)
Non-cellular bio-assays A range of assays not requiring viable cells i.e. ELISA, RIA
Receptor binding assays have been widely used because it is rapid, easy to use and also relatively cheap. These assays are very appropriate to use when screening a large number of samples. However, these assays have some limitations. They cannot distinguish between agonistic and antagonistic effects, as binding to the receptor do not necessarily result in transcriptional activation. They are also only suitable for the detection of hormone-receptor mediated effects and cannot detect pro-estrogens (absence of metabolism in cell-free systems).
2.7.2.3 Cell proliferation assays.
Cell proliferation assays to monitor estrogenicity are well known and used by several research groups. These assays are based on the ability of a test substance to stimulate growth of an estrogen dependent cell line (Soto et al., 1992, 1995). The most commonly used cancer cell lines for cell proliferation assays are MCF-7 and T47D cells. In these assays breast cancer cells are exposed to both positive (17β-estradiol) and negative controls (vehicle control) as well as to the environmental sample being analysed. The cell proliferation of the environmental sample compared to the controls provides the basis for demonstrating estrogenic responses. Cell proliferation assays have the advantage over ER binding assays in that they take cellular, biological and metabolic responses into account. These assays are therefore useful to determine both synergistic and antagonistic estrogenic characteristics of complicated biological mixtures. Cell proliferation assays also have some disadvantages. Various studies have reported inter laboratory variation of the MCF-7 proliferation assay. This is mainly due to differences in culture conditions, as well as different MCF-7 strains (Osborne et al., 1987; Villalobos et al., 1995; Jones et al., 1997, 1998; Odum et al., 1998). Moreover, some studies have reported that MCF-7 cells can be stimulated to proliferate by a
range of non-oestrogenic substances including epidermal growth factor (EGF), progesterone, dihydrotestosterone, insulin-like growth factors, lithium, chloride and ethanol (Hackenburg et al., 1988; Osborne et al., 1990; Savouret et al., 1990; Jones et al., 1998).
2.7.2.4 Reporter gene assays
Reporter gene assays measure the ability of a substance to activate the transcription of a hormone (estrogen)-sensitive promoter in eukaryotic cells (e.g. yeast and mammalian cells). These assays are established using eukaryotic cells that are transfected with an expression vector encoding the human estrogen receptor, as well as estrogen response elements, which is in reading frame with a reporter gene (e.g. luciferase and β-galactosidase that can be readily detected and quantified). When single chemicals or complex environmental mixtures that contain e-EDCs are exposed to these cells, it will activate the receptor and response elements to stimulate expression of the reporter gene. A large number of reporter gene assays have been developed for a range of steroid hormone receptors using both yeast and mammalian cells. The use of yeast cells in these assays (Routledge et al., 1998; Gaido et al., 1997a,b) offer a number of advantages. Yeast cells are fairly easy to culture, ideal for genetic manipulation and also contain some degree of metabolic competence. The yeast reporter gene screens also have some limitations as reported by Joyeux et al (1997). The sensitivity of the system can depend on the number of receptor protein being expressed, the response element and also the reporter gene used. Yeast cell membranes have also been reported to be impermeable to some test substances and may result in false negatives (Gray et al., 1997).
2.7.2.5 Non-cellular bio-assays
Non-cellular bio-assays do not require viable cells in order to test for environmental endocrine disruptors. These assays have the advantage of not having difficulties with membrane permeability, cell function, and organism life stage, as well as toxicity responses to a given sample. Many of these assays are quantitative and provide reasonable detection limits for measurement of e-EDCs. Enzyme linked Immunosorbent Assay (ELISA) and Radio Immuno Assay (RIA) kits have been developed for many environmentally relevant surfactants and estrogenic compounds, as well as for some pesticides, antibiotics, and other personal care products (Gascón et al., 1997; Neogen Corp Lexington, KY; ALPCO Diagnostics Salem, NH; Assay Designs Inc. Ann Arbor, MI; Bio-Quant Inc., San Diego, CA; BioSource Internacional, Camarillo, CA; Cayman Chemical Company, Ann Arbor, MI; Immuno-Biological Laboratories, Inc., Minneapolis, MN; Envirologix. Portland, Me: York Nutritional
Laboratory, Osbaldwick, York, UK). In addition to these immunosorbent assays, advances have been made in electrochemical sensors, fluorescent indicators, and microarray relative binding assays (Zhihong et al., 1999; Murata et al., 2001; Awais et al., 2004).