1) The study investigated the binding affinity of groundwater extracts from a wastewater treatment site to the three estrogen receptor (ER) subtypes of the Atlantic croaker fish. 2) The groundwater extracts showed the highest relative binding affinity for ER alpha and a lower but still significant binding affinity for ER beta a. ER beta b did not significantly bind to the extracts. 3) This differential binding pattern between the receptor subtypes matches what is known about the binding of specific compounds like bisphenol A, a common endocrine disruptor found in water sources.

1. Phase 1
Phase 2
Investigate the role of amino acid substitutions in differential ER binding.
Differential binding of groundwater extracts from a land-applied wastewater site to the three
estrogen receptor subtypes of the Atlantic croaker, Micropogonias undulatus
Kyrie Hooton1 Mary Beth Hawkins2
1Department of Forestry and Environmental Resources, 2Department of Biology, North Carolina State University, Raleigh, NC
Abstract:
Environmental estrogens are chemical compounds that can enter aquatic ecosystems and
cause deleterious effects in organisms through binding to estrogen receptors (ERs) and
disrupting associated endocrine functions. Teleost fish express three ER subtypes with different
ligand binding affinities for some estrogens and estrogenic compounds, but little is known about
how the structure of teleost ERs influences their ligand binding affinities to most environmental
estrogens. In this study, we characterized the relative binding affinity of groundwater from a
wastewater treatment facility to the three ER subtypes of a teleost fish, the Atlantic croaker.
Groundwater SPE samples had the highest relative binding affinity (RBA) for ER alpha and a
lower, yet still significant, binding affinity for ERβa. ERβb did not significantly bind to
groundwater extracts at any concentration tested. These results for whole water extracts agree
with previous findings from competitive binding assays with pure compounds, where ERβa has
binding affinities unique from ER alpha and ERβ-b. These differences have been attributed in
part to the amino acid substitution of a methionine in the ER alpha ligand binding domain to a
phenylalanine in the ERβa-s. We are creating reciprocal mutant receptors to investigate the role
of this position in the differential binding of wastewater extracts to fish ERs. We will compare
our results for whole water extracts to those found for individual compounds suspected to be
present in this water system. This will help us evaluate the estrogenic potential of contaminated
water resources and predict their specific effects on ER-subtype mediated physiological
processes.
Hypothesis
Groundwater extracts will bind to the three Atlantic Croaker
estrogen receptors with different binding affinities that reflect
the binding profiles of specific estrogenic compounds.
Methods
Figure 3 shows the relative binding affinity of estrogenic compounds to alpha at 10x, 5x, 2.5x, and 1x
concentrations, with 0x as the baseline.
Figure 4 compares the 1x concentrations of Beta A, Beta B, and Alpha.
* P<0.05 **P<0.01 ***P<0.001 compared to 0x concentration n=2 assays with 4-6 replicates for each
concentration.
Acknowledgements
This work was funded by an NCSU Office of Undergraduate Research Award to Kyrie Hooton
and a 2010 HHMI Undergraduate Education Core Grant to NCSU Biology.
Sources:
1. Hawkins and Thomas 2004. Endocrinology145:2968-77.
2. Katsu, Y., et.al. (2013), J. Appl. Toxicol. 33: 41–49.
3. Thriveni and Hawkins 2013. CSETAC Annual Meeting. poster presentation.
Results
• RBAs for 1X Well 3 extract were: ERa:123; ERba123;
ERbb123 compared to 0 control.
• RBA values do not increase in a concentration
dependent manner, suggesting assay inhibition at
higher concentrations.
• The RBA for ERba was not significantly different
from 0 controls.
Estrogenic Compounds Alpha Beta-a Beta-b Agreement with Well 3
E2 100 100 100 inconsistent
E1 10 2.9 3.5 Partial
EE2* 179 940 80 Partial
BPA 2.6 0.74 0.07 Consistent
Genistein 2.4 9 18 Opposite
W3 High med low Reference
PCB 2.22 0.14 2.7 inconsistentRBAs of Candidate Compounds
The water was then filtered and partially purified via solid phase
extraction (SPE).
Detect ER ligands in the water extracts using a competitive
binding assay for teleost ER fusion proteins.
Conclusions
Ranked order of binding affinities to Well 3 water:
ERα> ERβa>Erβb
This binding pattern is consistent with BPA, a
ubiquitous endocrine disruptor in water sources.
(Figure x)
The differential binding to the fish ERb subtypes may
be due to amino acid changes in their ligand binding
domains.
.
Background
Future directions.
Explore the role of specigic
amino acid substtuions in
the LBD of teleost Ers via
site-directed mutagenesis
and subsequent ligand
binding assays.