Estimation upregulate subsequent reporter gene expression (and in vitro

Estimation of total estrogenic activity is
possible through in vitro bioassays which can be a rapid, sensitive, and inexpensive
integrative screening procedure by analyzing all compounds in mixtures which act
through the same mode of action (Hilscherova et al., 2000). Simple compounds and complex mixtures can be measured for
estrogenic activity with several
in vitro bioassays. The natural processes of endocrine systems can be interfered
by chemicals in several ways such as affecting the synthesis or metabolism of
natural hormones, binding to hormone receptors, and/or disrupting hormone
receptor synthesis or metabolism (Katzenellenbogen and Muthyala, 2003;
Zacharewski, 1997). Estrogenicity detection is most frequently
performed by in vitro transactivation assays (Kinnberg, 2003) which determine the
capacity for samples to stimulate estrogen receptors and upregulate subsequent reporter
gene expression (and in vitro estrogenicity assays). Additionally, tiered
monitoring of environmental waters is currently being considered to be
evaluated by in vitro estrogenicity assays (Leusch et al., 2010). Comparing estrogenic
activity of environmental samples determined by different in vitro assays
demonstrate that the assays are effective for environmental monitoring (Leusch et al., 2010;
Murk et al., 2002).Genetically engineered yeast, fish, or mammalians
cells are normally subjected to transfection with an estrogen-responsive
element (ERE) DNA sequence connected to a reporter gene when conducting reporter
gene assays (Zacharewski, 1997; Kinnberg, 2003). After an agonist binds to the
receptor, a series of molecular events results in the receptor binding to the
ERE and activating the gene expression process (Figure 1). The reporter gene
then generates a product that can be measured appropriately (galactosidase or
luciferase genes represent most reporter genes, with protein/enzymatic products
easily quantified by spectrophotometry and luminometry). Vertebrate assays are
typically performed with fish (Ackermann et al., 2002; Rutishauser et al.,
2004) or mammalian cells (Legler et al., 1999; Vinggaard et al., 1999; Balaguer
et al., 2000; Wilson et al., 2004) of which the luciferase gene is usually
transfected with an ERE. Simple yeast-based assays (Routledge and Sumpter,
1996; Sohoni and Sumpter, 1998; Garcia-Reyero et al., 2001) reportedly
transfect yeast cells with a galactosidase reporter gene linked mammalian ERE. An
expression plasmid containing a mammalian ER is also introduced as yeast do not
possess an endogenous ER. Finally, yeast Gal4 DNA binding domains and chimeric
receptors with a mammalian ligand binding domain are utilized by chimeric yeast
reporter gene assays which harvest the natural yeast genetic machinery
(Nishikawa et al., 1999).  Field
research demonstrates the persistence of estrogenic properties in soils amended
with biosolids. Soil from plots in Australia were measured for the YES bioassay
response described above (Langdon et al., 2012). Even at low
concentrations, estrogenic activity persisted in the treat soils (with control
plots exhibiting no measurable estrogenic response) even after 4 months of
application.

 

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