| Abstract |
Endocrine disrupting chemicals (EDCs) include a wide variety of
structurally diverse compounds that affect normal signaling pathways in
most all animal species by multiple mechanisms. Given the global
distribution of EDCs in environmental, biological and food samples and
commercial products, detection, relative quantitation, and assessment of
their biological/toxicological effects is of paramount importance. While
instrumental methods allow detection and quantitation of specific (known)
EDCs in sample extracts, these methods do not allow identification and
characterization of new EDCs. Accordingly, mechanism-based bioanalytical
methods have been developed to assist in the identification and
characterization of matrices and extracts containing EDCs. The so-called
chemically-activated luciferase expression (CALUX) bioassays and related
CALUX-type bioassays represent a collection of nuclear receptor-based
recombinant cell bioassays for detection of EDCs, including estrogenic-,
androgenic-and dioxin-like chemicals. Although CALUX assays are much
cheaper and quicker than instrumental analysis and whole animal assays,
there is a critical need to make these assays as
biologically/toxicologically relevant and consistent with our current
understanding of endocrine disruption in order to provide optimal data for
selection of chemicals (or mixtures) which warrant in vivo assessment.
This includes improved assay characteristics (i.e., minimal detection
limits (MDLs)) and response to EDCs, inclusion of more receptors (i.e.
multiple subtypes of a given nuclear receptor) and consideration of
receptor cross-talk and related mechanisms. In this dissertation, I
describe two significantly improved CALUX bioassay systems for EDCs. The
first is a set of species-specific Ah receptor (AhR)-responsive CALUX cell
lines that exhibit dramatically increased sensitivity (10-100-fold lower
MDL (to sub pM concentrations)) and responsiveness to 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like
compounds (DLCs), compared to current dioxin-responsive CALUX cell lines.
I demonstrate the utility of these novel AhR-responsive CALUX bioassays in
analysis of sediment extracts. The second CALUX cell line (estrogen
receptor (ER)-CALUX) is a modification of our existing estrogen-responsive
human cell line (BG1Luc4E2). Although this cell line has received official
regulatory approval from the USEPA and OECD (to the point of being
included in the USEPA Endocrine Disruptor Screening Program) and is
currently being utilized by the Tox21 program for chemical screening
purposes, this cell line only expresses one of the two known human ER
subtypes (ERalpha). Since both ER subtypes (ERalpha and ERbeta) can differ
in their ligand-specificity, cross-talk with other signaling and
transcription factors and gene induction profiles, optimal cell bioassay
assays should be able to detect the full spectrum of ER-active chemicals
(i.e., activators/repressors of ERalpha and/or ERbeta). Accordingly, we
stably transfected the ERalpha-containing BG1LucE2 CALUX cell line with a
human ERbeta expression vector and screening of the resulting BG1LucERbeta
clones with the ERbeta-selective ligands genistein and Br-ERbeta-041
allowed identification and isolation of a clonal cell line
(BG1LucERbetac9) expressing ERbeta and highly responsive to ERbeta-
selective ligands. Comparison of the response of BG1Luc4E2 and
BG1LucERbetac9 cell lines to a chemical library of 176 compounds revealed
that the ERalpha/ERbeta-containing BG1LucERbetac9 cell line can identify a
greater number of potential estrogenic chemicals than the ERalpha-
containing BG1Luc4E2 cell line. Since cross-talk between the AhR and ER
has been identified as a potential mechanism for endocrine disruption in
animals exposed to DLCs (which act to repress ER gene signaling), in the
final chapter I explored cross-talk between AhR and ER in a human breast
carcinoma cell line (SKBR3). I specifically focused on the effect of
ERalpha/ERbeta on AhR gene signaling in transiently transfected cells. In
SKBR3 cells (which lack functional ER), we observed an ERalpha subtype-
specific enhancement of AhR-dependent reporter gene expression (from the
transfected AhR-responsive plasmid pGudLuc6.1) in the absence of added E2
and a repression of the ER-dependent enhancement of AhR signaling by
stimulating transfected ERalpha with E2, PPT, or fulvestrant (no
repression was observed with ERbeta). The repressive effect of E2 could be
abolished by co-incubation of E2-activated ERalpha-transfected SKBR3 cells
with the ER antagonist 4-hydroxytamoxifen, demonstrating the requirement
for functionally active ERalpha. Our ER-dependent repressive results in
SKBR3 cells indicate that AhR has selective interactions with ERa versus
ERbeta. The results presented in this dissertation demonstrate that the
CALUX reporter vectors and stable cell lines not only have application for
highly sensitive high throughput screening for EDCs systems, but for
in-depth mechanistic studies examining crosstalk between receptor signaling
pathways which can play a critical role in endocrine disruption.
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