Environmental Engineering Reference
In-Depth Information
van der Linden et al., 2013; WHO, 2011, 2013; Ishihara et al., 2009; Murata and Yamauchi,
2008; Ma et al., 2005; Kusk et al., 2011), signiicant attention and investment have been
devoted to their detection. A variety of methods, including laborious chemical methods
of isolation and identiications by a combination of high-performance liquid chromatogra-
phy (HPLC), liquid or gas chromatography (GC), and/or mass spectroscopy (MS), as well
as “omic” approaches (genomics, transcriptomics, proteomics, and/or metabolomics on
ish and other affected organisms), have been described (Mansilha et al., 2010; Jordan et al.,
2011). However, high cost, lack of uniform quantiication, and uncertainty of the biological
response prevented their use for eficient detection and monitoring of EDCs in the envi-
ronment, particularly in the water.
In an attempt to streamline the detection of EDCs in water sources, a number of assays
have been introduced (Roy and Pereira, 2005). One of the irst described assays is a yeast
estrogen screen assay, which is based on ER gene-modiied yeast cells (Routledge and
Sumpter, 1996). These cells carry human ER and ER-responsive gene, which codes for
β-galactosidase. In the presence of estrogen, the activity of this artiicial gene is turned
“on,” producing the enzyme. β-Galactosidase in turn modiies the yellow chlorophenol
red-β-galactopyranoside, present in the growth medium, to a red product that is quantiied
spectrophotometrically at 540 nm. Another approach has been the use of luciferase reporter
assay. In this assay, the expression of luciferase gene is governed by regulatory elements
responsive to a speciic hormone-activated receptor, and accumulation of the protein lucif-
erase is an indication of receptor activation. Recently, several luciferase-expressing cell lines
capable of detecting EDCs that inluence the function of the AR, AhR, and TR have been
developed and implemented (Kusk et al., 2011). For example, AhR-activating contaminants
in water have been detected by the chemical-activated luciferase gene expression (CALUX)
assay (Murk et al., 1996; Long et al., 2003, 2007). TR-mediated luciferase gene expression
assay was used to detect T3-like and anti-T3 activity in water (Murata and Yamauchi, 2008;
Ishihara et al., 2009). The T-screen is another bioassay based on thyroid hormone-depen-
dent cell proliferation of a rat pituitary tumor cell line (GH3) in serum-free medium (Gutleb
et al., 2005). A cell line (MVLN) derivative of ER-positive MCF-7 breast cancer cells carrying
ER response element (ERE)-luciferase reporter vector (Bonefeld-Jorgensen et al., 2005) has
been used to test for the potency of water contamination with estrogens. Methods to detect
the rate of hormone synthesis and breakdown in response to EDCs have also been described.
Hilscherova et al. (2004) implemented a human adrenocortical carcinoma cell line (H295R)
to measure the expression of steroidogenic genes by using real-time PCR. Another inter-
esting approach is the use of the transgenic zebraish (Lee et al., 2012). This model has the
potential to assess the impact of environmental estrogens on a variety of body systems. The
transgenic zebraish is engineered to contain an estrogen-inducible promoter composed of
multiple EREs and a Gal4ff-UAS system. An estrogenic signal is detected when the chemi-
cal-ER complex binds to the ERE, which activates Gal4ff. Subsequently, Gal4ff protein binds
to UAS to induce the luorescent reporter (green luorescent protein, GFP). In spite of many
attempts of detect EDCs in the environment, most assays are manually demanding, require
considerable time for analysis and are frequently limited to detection of a single hormone.
Our laboratory has recently developed a unique high throughput assay for detection
and biological testing of EDCs in mammalian cells (Stavreva et al., 2012a). We engineered
mouse mammary cells that express GFP-tagged nuclear steroid receptors, such as glu-
cocorticoid receptor (GR) (Walker et al., 1999). This assay is based on the fact that, in the
absence of the hormone, the receptor resides in the cytoplasm bound to various heat shock
proteins and immunophilins in a large multiprotein complex (Pratt and Toft, 1997). Upon
hormone binding, GR dissociates from this complex and translocates to the cell nucleus
