The Toxicologist - Society of Toxicology

2344 A NOVEL SENSITIVE AND SELECTIVE REAL-TIME
CELLULAR ASSAY FOR DETECTION OF ENDOCRINE
DISRUPTORS USING NATIVE ENDOCRINE
SIGNALING PATHWAYS.
C. Jin 1 , Y. Abassi 1 , X. Xu 1 , X. Wang 1 , W. Zhang 2 and S. Gabos 2 . 1 Acea
Biosciences, San Diego, CA and 2 Alberta Health and Wellness, Edmonton, AB,
Canada. Sponsor: M. Lo.
A cell based assay for detection of endocrine disruptors was developed using real
time impedance monitoring of native endocrine signaling pathways in T47D breast
cancer cell line. T47D cells express estrogen receptor (ER) as well as some other
steroid hormone receptors such as progesterone receptor (PR) and glucocorticoid
receptors (GR). Stimulation of T47D cells with respective endocrine agonists leads
to an increase in cell number which can be detected by gold microelectrodes embedded
in the bottom of the well of specialized microelectronic plates. Addition of
estrogen agonists including 17beta-estradiol, DPN and PPT to T47D cells induce
a signature biphasic and dose-dependent increase in impedance readout. The observed
EC50 values are comparable or better than existing in vitro cell based reporter
assay systems. The specificity of the assay for ER activity was established
using well characterized ER antagonists, such as ICI 182780, Raloxifene and
ZK164015. Using this assay system, several environmental hazardous compounds
known to disrupt endocrine signaling pathways were tested, including bisphenol A,
daidzein, nonylphenol, and PBDE-47. All of these compounds induced a dose-dependent
impedance-based response profile similar to ER agonists and were inhibited
by ER antagonists. The calculated EC50 values were comparable to lowest
published EC50 values generated in different assay systems. Moreover, the T47D
system was also used to detect response to PR agonist, progesterone and GR agonist,
dexamethasone. These two compounds also produced impedance-based
biphasic responses although with different kinetic profile compared to ER activation.
The data suggests the T47D assay system has the capacity to sensitively, selectively
and quantitatively detect three different nuclear hormone responses.
2345 THE IMPACT OF EPIDERMAL GROWTH FACTOR
RECEPTOR INHIBITION ON ENERGY HOMEOSTASIS.
M. Biggs 1 , D. Threadgill 2 , T. Ghashghaei 3 and T. Lee 4 . 1 Toxicology, University of
North Carolina, Chapel Hill, NC, 2 Genetics, North Carolina State University,
Raleigh, NC, 3 Department of Molecular and Biomedical Sciences, North Carolina
State University, Raleigh, NC and 4 Genetics, University of North Carolina, Chapel
Hill, NC.
As a result of the worldwide rise in obesity and obesity-related complications, such
as diabetes, stroke, and cardiovascular disease, understanding the mechanisms associated
with this disease is necessary. Obesity results from taking in more calories
than required for energy homeostasis. Body weight is regulated by this balance between
food intake and energy expenditure, through a highly integrated, multiorgan
system. Accumulating evidence suggests signaling through the epidermal
growth factor receptor (EGFR) is required for normal adipocyte development and,
therefore, this signaling may be important in obesity development. Our lab has previously
shown that EGFR inhibition retards adipose deposition in diet-induced
obesity models, either pharmacologically with a small molecule inhibitor of the
EGFR tyrosine kinase, AG1478, or genetically in a constitutionally impaired
EGFR tyrosine kinase model (Egfr wa2 ). However, the mechanisms behind this phenotype
are unknown. Here we show that central EGFR signaling is important in
appetite and metabolism regulation in diet-induced obesity. We found significantly
less body weight and fat mass in mice with a conditional deletion of EGFR in the
central nervous system fed a high-fat diet. Contributing to this decrease in fat mass
is an increase in energy expenditure due to hyperphagia, increased oxygen consumption,
diet-induced thermogenesis, and lipolysis in the white adipose tissue of
these animals compared to control littermates. These findings suggest that altered
signaling between the CNS and periphery, due to this increase in energy expenditure,
accounts for this resistance to diet-induced obesity in mice with central EGFR
inhibition.
2346 SCIENTIFICALLY RELEVANT INFORMATION TO
EVALUATE THE ENDOCRINE DISRUPTING
POTENTIAL OF ISOPHORONE.
N. M. Berdasco and D. R. Geter. Toxicology and Environmental Research and
Consulting, The Dow Chemical Company, Midland, MI.
In 1996, Congress directed the U.S. EPA to develop a prioritization and screening
program to evaluate potential endocrine disrupting compounds. This program is
now referred to as the Endocrine Disruptor Screening Program (EDSP). Tier 1
EDSP assays are composed of 5 in-vitro, 4 mammalian in vivo, and 2 eco-toxicity
504 SOT 2011 ANNUAL MEETING
screening assays designed to identify substances with the potential to interact with
the estrogen, androgen, or thyroid (EAT) hormone systems. Isophorone, a solvent
and crop protecting compound, was selected in the initial round of EDSP testing.
As part of the testing program, Other Scientifically Relevant Information (OSRI)
for isophorone was presented to the EPA in consideration for aiding in the understanding
of possible interactions with the EAT hormone systems. The isophorone
OSRI included summarized information on a developmental toxicity study in rats,
a limited one-generation reproduction toxicity study, 2 sub-chronic toxicity studies,
and 2 chronic toxicity/oncogenicity studies. Isophorone was also assessed in two in
vitro assays, an estrogen-receptor alpha (ERα) binding and an ERα transcriptional
activation assay. In summary, this OSRI concluded that the solvent isophorone
does not bind to either the estrogen or androgen receptor, was neither embryotoxic
nor teratogenic, and did not alter pregnancy rates or litter sizes. From these data, it
was concluded that isophorone did not demonstrate any pattern of effects consistent
with adverse endocrine-mediated effects on reproductive organs or processes,
nor were any alterations seen that support the potential for operating by an endocrine-related
mode of action.
2347 EXENATIDE AND SITAGLIPTIN: THEIR ROLE IN
AUTOPHAGOSOME REGULATED TOXICITY IN
PANCREATITIS.
R. Rouse, L. Zhang and D. A. Volpe. U.S. FDA, Silver Spring, MD.
The anti-diabetic drugs exenatide (EXN), a glucagon-like peptide 1 (GLP-1) receptor
agonist, and sitagliptin (SIT), an inhibitor of dipeptidyl peptidase-4 (GLP-1 inhibitor)
have been linked to post-marketing reports of drug-induced acute pancreatitis
(DIAP). To better understand the risk of this effect, a series of in vitro studies
were undertaken to investigate the toxicity mechanisms contributing to DIAP.
MTS viability assays were performed to evaluate the proliferation of rat pancreatic
acinar cells (AR42J), ductal cells (DSL-6A/C1) and islet cells (RIN-m5F) dosed
with EXN or SIT for 8, 24 or 48 hrs. SIT was toxic in all cell types at 1 mg/ml at 8,
24, or 48 hrs. No viability decreases were found at concentrations below 0.1
mg/ml. At 48 hrs, 1mg/ml EXN induced proliferation in the islet cell line but inhibited
ductal and acinar cell line proliferation. LC3 (autophagosome) co-localization
with intracellular compartments marked with amylase (zymogen), LAMP2 or
LAMP1 (lysosome), Rab7 (lysosome or late endosome), EEA1 (early endosome),
and cathepsin B (activate trypsinogen) were also investigated using AR42J or primary
rat acinar cells. The results obtained demonstrated effects of EXN and SIT on
co-localization of autophagosomes in lysosomes and other intracellular compartments.
Finally, we inhibited the activities or expressions of cathepsin B, trypsinogen
and protease-activated receptor-2 (PAR-2) to evaluate the roles these proteins or
pathways play in intracellular autophagosome localization. The inhibition of the
above proteins altered the colocalization between autophagosome and other intracellular
compartments induced by EXN or SIT. These results suggest that EXN and
SIT may play a role in the onset of acute pancreatitis by altering vacuole accumulation
and localization in acinar cells through several signaling pathways encountered
in pancreatic cells.
2348 IMPROVING THE IN VIVO PREDICTIVE VALUE OF
THE ER-CALUX BIOASSAY BY COMBINING IN VITRO
ESTROGENICITY RESULTS WITH KINETIC
CHARACTERISTICS OF ESTROGENIC COMPOUNDS.
W. Brand 1 , A. Punt 1, 2 , A. J. Murk 2 , M. Schriks 1 , A. P. van Wezel 1 and M. B.
Heringa 1 . 1 KWR Watercycle Research Institute, Nieuwegein, Netherlands and
2 Division of Toxicology, Wageningen University, Wageningen, Netherlands.
In vitro bioassays play an important role in effect-directed analysis, providing a
rapid way of screening samples for the presence of bioactive compounds. An example
is the ER-Calux bioassay, a reporter gene assay based on T47D cells stably transfected
with an estrogen-responsive luciferase reporter gene, which allows sensitive
detection of estrogenic (and anti-estrogenic) compounds. A limitation of many in
vitro bioassays however, is that they generally do not take the ADME characteristics
of compounds into account, which can hamper the in vivo predictive value of these
assays.
The present study compares the estrogenicity of a suite of (xeno-)estrogenic compounds,
relative to ethinyl estradiol (EE2) as determined with the ER-Calux bioassay,
to literature-derived in vivo estrogenic responses obtained with the rat uteroptrophic
assay. To correct for compound specific differences in ADME
characteristics, albumin binding constants were determined for the suite of compounds,
as well as hepatic availability (i.e. the dose that escapes hepatic metabolism),
determined by incubations with liver microsomes and cytosol. The results
demonstrate that combining the EC 50 values for estrogenic activity determined
with the ER-Calux bioassay with the compound-specific kinetic characteristics for
albumin binding and hepatic availability, improves the correlation with the in vivo