The Toxicologist - Society of Toxicology

effects of bisphenol A. Two factors contributing to such differing results include
highly variable estrogen-like contamination and the exquisite sensitivity of endocrine
systems. In rodent studies possible environmental factors affecting background
EDC levels includ: types of caging, bedding, and sanitation materials,
drinking water and diet. Laboratory rodent diet remains a significant uncontrolled
variable in many experiments assessing the effects of EDCs. Previously, Thigpen et
al. demonstrated that dietary formulation and mill dates of rodent chow impact
sensitivity of end-points of estrogen-like activity. The purpose of the current study
was to determine the optimal “reduced EDC” diet for consistent detection of the
effects of estrogen-like EDC exposure. Treatment-related differences in reproductive
and developmental endpoints were observed among groups fed four diets having
reduced phytoestrogen content. Animals were continuously fed from 2 weeks
before copulation (F0 treatment) until sacrifice with offspring (F1) treated
throughout life. A positive control diet with 17α-ethinylestradiol was included.
Changes were observed in onset of puberty, organ weights, and adipose deposition.
These findings support the proposal by Thigpen et al. that caloric content of the
diet is a confounder in EDC exposure studies and reveal that a reduction of diet
caloric content and elimination of EDC phytochemicals can reduce diet-induced
study variability to yield increased reproducibility of animal studies investigating
actions of EDCs.
2363 DEVELOPMENTAL TOXICITY OF O-METHYLATED
METABOLITES OF BISPHENOL A.
K. C. Pangallo, E. Kirichenko, K. Cooper and L. A. White. Biochemistry and
Microbiology, Rutgers University, New Brunswick, NJ.
Bisphenol A (BPA), an estrogen agonist and known endocrine disruptor, is a common
component of plastics and epoxy resins. Migration of BPA out of commercial
products results in environmental contamination, and provides opportunity for
human exposure to BPA and biotransformation to BPA metabolites. Recent work
in our lab has demonstrated O-methylation of BPA to monomethyl and dimethyl
ether metabolites (BPA-MME and BPA-DME, respectively) under aerobic conditions
by soil-dwelling Mycobacterium species. This transformation results in increased
toxicity, as BPA-MME and BPA-DME have lower LC50s than their parent
compound to developing zebrafish (Danio rerio) embryos. Here we investigate the
mechanism of this increased toxicity, and show that the O-methylated metabolites
are stronger inducers of matrix metalloproteinases (MMP-2, -9 and -13) than the
parent compound during D. rerio development. The increased toxicity of Omethylated
metabolites, and the ubiquity of organisms capable of O-methylation,
suggests that this transformation pathway must be considered when making a risk
assessment of BPA exposure.
2364 SERINE PROTEASE INHIBITOR, AEBSF, STRONGLY
IMPAIRS EXPRESSION OF PROINFLAMMATORY IFNγ
AND RELATED BIOMOLECULES FROM SPLENOCYTES
FROM ESTROGEN-TREATED C57BL/6 MICE.
E. Karpuzoglu 1 , R. M. Gogal 1 and A. Ahmed 2 . 1 Department of Anatomy and
Radiology, University of Georgia, Athens, GA and 2 Department of Biomedical Sciences
and Pathobiology, Virginia Maryland Regional College of Veterinary Medicine,
Blacksburg, VA.
Estrogen is shown to alter immune responses and associated with enhanced susceptibility
to autoimmunity in females. We have demonstrated that estrogen enhances
proinflammatory IL-12, IFNγ, iNOS, and MCP1, that has a critical role in Th1
profile as well as female predominant autoimmune diseases. However, the immune
modulation of estrogen mediated inflammatory responses remains to be elucidated.
In this study, the immunomodulatory effects of a serine protease inhibitor, 4-(2aminoethyl)-benzenesulfonyl
fluoride (AEBSF), on estrogen-mediated induction of
inflammatory cytokines (IFNγ, IL-6, MCP1, iNOS, NO) and related transcription
factors (STAT1) in ConA or rIL-12-treated splenocytes are explored. AEBSF was
found to downregulate the estrogen-induced proinflammatory IFNγ, IL-17, IL-6,
MCP1, iNOS expressions from ConA-activated splenocytes. The AEBSF-derived
reduction of IFNγ and MCP1 were also observed upon IL-12receptor specific stimulation
with Th1-inducing rIL-12 after 24 h from splenocytes from estrogentreated
mice. The cell cycle analysis showed no marked change in phases of cell
cycle (G1/G0, S, or G2/M) due to rIL-12 and/or AEBSF treatment in splenocytes
from estrogen and placebo-treated mice after 24h of culture. Furthermore, AEBSF
alters the estrogen-enhanced expression IFNγ and STAT1 expression but did not
markedly affect IRF1 protein levels at 3h compared to placebo controls. These
novel findings indicate that AESBF could alter estrogen-mediated proinflammatory
events in the immune system, and thus might lead to new avenues in regulation of
autoimmune diseases.
508 SOT 2011 ANNUAL MEETING
2365 LOW-DOSE BPA ALTERS CA CYCLING AND PROMOTES
ARRHYTHMOGENESIS IN THE FEMALE HEART.
S. M. Belcher, S. Yan, Y. Chen, M. Dong and H. Wang. Pharmacology,
University of Cincinnati, Cincinnati, OH.
There is wide-spread human exposure to the ubiquitous estrogenic chemical
bisphenol A (BPA) and growing evidence implicating BPA as a significant concern
to human health. Epidemiologic studies have demonstrated that higher urine BPA
concentrations are associated with cardiovascular diseases in humans. However, the
nature and underlying mechanism of the cardiac effects of BPA exposure are unknown.
In this study, we examined the rapid effects of BPA and 17α-estrodial (E2)
on rodent ventricular myocytes and whole hearts and the underlying mechanisms.
Physiological low-dose (10-9 M) of BPA or E2 rapidly induced arrhythmogenic
triggered activities in isolated myocytes from female, but not male, rat hearts. The
effects of BPA were particularly pronounced with combined with E2. BPA and E2
also promoted ventricular arrhythmias, particularly malignant ventricular fibrillation
under catecholamine-induced stress in female, but not male, rat whole hearts.
The dose-response curves for both BPA and E2 have an inverted-U shape, with the
most efficacious doses at 10-9 M. The cellular mechanism of the pro-arrhythmic effects
of BPA and E2 were investigated. The estrogens rapidly enhanced sarcoplasmic
reticulum (SR) Ca2+ load and release, and markedly increased frequency of
Ca2+ sparks, or spontaneous SR Ca2+ “leak”. Pharmacological blockade of SR
Ca2+ leak suppressed estrogen-induced triggered activities. Ablation of ERβ in an
ERβ knockout mouse model completely abolished triggered activities induced by
estrogens in female myocytes. In conclusion, low-dose of BPA and E2 rapidly promote
arrhythmias in the heart in a female-specific manner; the pro-arrhythmic actions
of estrogens are mediated by ERβ-signaling through alterations of myocyte
Ca2+ cycling, particularly increases in Ca2+ leak from the SR.
2366 ARSENITE INHIBITS 3T3-L1 ADIPOGENESIS AND
SUPPRESSES INDUCTION OF NUCLEAR RECEPTORS
DURING THE EARLIEST STAGES OF
DIFFERENTIATION.
F. J. Zandbergen 1 , V. Chatikavanij 1 , C. D. Kozul-Horvath 2 and J. W. Hamilton 1 .
1 Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA and 2 Department
of Immunology, Dartmouth Medical School, Lebanon, NH.
Exposure to arsenic (As) is associated with an increased risk of serious illnesses, including
cardiovascular disease (CVD). Obesity, defined as excess adipose tissue, is
associated with elevated plasma triglyceride levels, a strong risk factor for CVD. We
have previously shown that arsenite (AsIII) can suppress the activation of steroid receptors,
which are part of the nuclear receptor family. We hypothesized that AsIII
might affect lipid metabolism through targeting other members of this family as
well. Since adipose tissue plays a major role in lipid homeostasis, we used the mouse
3T3-L1 cell line, which can be induced to differentiate into adipocytes, for our
studies. AsIII markedly suppressed mRNA expression of several markers of adipogenesis
in a concentration dependent manner. In addition, expression of nuclear receptors
that are induced very early in 3T3-L1 differentiation was significantly suppressed.
Correspondingly, AsIII treatment from the time of differentation
induction resulted in a concentration dependent decrease in lipid accumulation.
But when AsIII treatment was started several days after the cells had been induced
to differentiate, expression of adipogenic markers was increased. These results show
that depending on cell differentiation stage AsIII may have different, or even opposite
effects on adipogenesis, and that inhibition of adipogenesis may be related to
suppression of nuclear receptors very early in 3T3-L1 differentiation. We speculate
that in vivo, the effects of AsIII on adipogenesis might decrease lipid storage capacity,
which could be disadvantageous under obesity promoting conditions if the result
is an elevated level of plasma lipids. Interference of AsIII with lipid metabolism
in differentiated adipocytes might also change plasma lipid levels, likewise providing
a possible explanation for the association of AsIII with increased risk for CVD.
(NIH-NIEHS P42 ES007373)
2367 SCREENING, CLASSIFICATION, AND MECHANISM OF
ACTION OF ENDOCRINE DISRUPTING CHEMICALS.
X. Wang, G. Du, O. Shen, H. Sun, L. Xu, H. Chen, Y. Xia, A. Gu and S.
Wang. Nanjing Medical University, Institute of Toxicology, Nanjing Jiangsu, China.
The potential effects of substances with endocrine activities in humans and wildlife,
so-called endocrine-disrupting chemicals (EDCs), constitute a major concern
among the public and the scientific community. Screening of chemicals for endocrine
disrupting (ED) activity is a major goal of research groups and regulatory
agencies worldwide. Our laboratory have set up a battery of assays to detect the en-