Endocrine-Disrupting Chemicals and Their Environmental Impacts - Organic Pollutants: An Ecotoxicological Perspective - page 277

consequence, in 1996, the U.S. EPA established the Endocrine Disruptor Screening
and Testing Advisory Committee (EDSTAC), which recommended the use of a bat-
tery of tests applied in a tiered approach to identify chemicals affecting the estrogen,
androgen, or thyroid hormone systems. Similar, although less intensive, activities
were activated in Europe and in Japan. The list of recommended screens and tests
included in silico, in vitro, and in vivo approaches. In silico approaches included the
use of quantitative structure-activity relationships (QSAR), in which the specific
chemical structures are modeled to establish if chemicals have a high probability of
binding to and activating a specific receptor, or posses functional groups that exist in
other known EDCs. In an analysis of the QSAR approach as applied to EDCs, it was
shown to have a high predictive capability for chemicals interacting with a specific
hormone receptor. There were exceptions to this, however, and they include kepone,
where the structure of the chemical is not especially similar to endogenous steroid
estrogen, yet it binds effectively to the estrogen receptors and triggers an estrogenic
response. Furthermore, for chemicals active via pathways other than receptors, for
example, via affecting hormone biosynthesis, chemical structure, and thus the QSAR
approach, is far less predictive.
The list of in vitro assays for EDCs includes competitive ligand-binding assays,
which investigate binding interactions of chemicals with specific hormone recep-
tors, and hormone-dependent cell proliferation or gene expression assays. The cell-
based assays include primary cultures, for example, fish hepatocytes that express
VTG mRNA/protein when exposed to estrogen, and immortalized cell lines such
as human breast cancer MCF-7 cells (Balaguer et al . 2000), yeasts ( Saccharomyces
cerevisiae; Metzger et al . 1995; Routledge and Sumpter 1996) transfected with
plasmids carrying the estrogen receptor (E-SCREEN) or androgenic receptor
(A-SCREEN) and a reporter gene incorporating a DNA response element respon-
sive to estrogens or androgens, respectively. Other cell systems have been developed
that are responsive to chemicals that interact with progesterone receptors (Soto et
al. 1995) and responsive to thyroid hormone mimics (see Zoeller et al. 2007 for a
critical review on these).
The yeast reporter gene assays not only assess for the interaction of the chemi-
cal with the hormone receptor, but also the ability of that receptor-chemical ligand
interaction to activate the hormone DNA response element. It should be realized,
however, that most of these systems have been developed with human and mam-
malian hormone receptors and differences in ligand potencies can occur between
different animal species. A comprehensive review of in vitro assays for measur-
ing estrogenic activity, and some of the issues of comparability, is provided by
Zacharewski (1997).
A major limitation of in vitro screening systems is that endocrine modifications
can be complex, and they are not necessarily limited to a specific organ, molecular
mechanism, or exposure route. As an example, an estrogenic effect could poten-
tially come about owing to an increase in gonadal estrogen production, a decrease in
gonadal androgen production, an increase in the production of gonadotrophin from
the anterior pituitary, a decrease in hepatic enzymatic degradation of estrogen, an
increase in the concentration of serum sex-hormone-binding proteins limiting free
hormone in the serum, a decrease in cytostolic binding proteins that potentially limit