The Toxicologist - Society of Toxicology

docrine disruptor activities of EDCs including ER / AR/ TR mediated reporter
gene assay, E-screening / A-screening / T-screening, pS2 mRNA expression assay,
ER competitive-binding assay, amphibian metamorphosis (Xenopus laevis),
steroidogenesis (H295R cell line). Combining these assays, we can detect chemicals
with (anti)estrogenic, (anti)androgenic and (anti)thyroid hormone activities effects,
and identify chemicals that affect steroidogenesis. We have screened several chemicals
including industrial chemicals and pesticides, such as alkylphenols, bisphenol A
and its halogenated derivatives, phthalates, pyrethroids, perfluorinated chemicals,
carbaryl and related metabolites. Use this screening battery, we can primarily screen
and classify EDCs into estrogenic, androgenic and thyroid hormone and steroidogenesis
disruptors. These sensitive assays are capable of high-throughput processing
and provide mechanistic insight. We also explored the potential molecular mechanisms
of TR disruption. We found that phthalates could affect the metamorphic
development of X. laevis by affecting the gene expression, enhanceing the interactions
between co-repressor Silence Mediator of Retinoic acid and Thyroid hormone
(SMRT) and TR, changing the methylation status of promoter region of TRβ gene.
Take together, our studies could provide some useful information for the assessment
of environment chemicals on human health.
2368 EFFECTS OF CHOLESTEROL-ALTERING
PHARMACEUTICALS ON CHOLESTEROL
METABOLISM, STEROIDOGENESIS, AND GENE
EXPRESSION IN THE FATHEAD MINNOW
(PIMEPHALES PROMELAS).
S. Y. Skolness 1, 2 , J. Cavallin 2 , E. Durhan 2 , K. Jensen 2 , M. Kahl 2 , C. LaLone 2 ,E.
Makynen 2 ,D. Villeneuve 2 , L. Wehmas 2 and G. Ankley 2 . 1 University of Minnesota,
Duluth, MN and 2 U.S. EPA Mid-Continent Ecology Division, Duluth, MN.
Pharmaceuticals that target cholesterol biosynthesis and uptake are among the most
widely prescribed drugs and have been detected in the aquatic environment.
Fibrates are a class of pharmaceuticals that indirectly modulate cholesterol biosynthesis
through effects on peroxisome proliferator-activated receptors. Statins lower
endogenous cholesterol production by inhibiting 3-hydroxy-3-methylglutaryl
coenzyme A reductase, the rate limiting step in cholesterol synthesis. The objective
of the present study was to assess the physiological and reproductive impacts of a fibrate,
gemfibrozil, and a statin, rosuvastatin, on adult fathead minnows (Pimephales
promelas). Fathead minnows were exposed to gemfibrozil or rosuvastatin in three
different studies. Cholesterol, triglyceride, vitellogenin, and sex steroid (testosterone
(T), 17β-estradiol (E2)) concentrations were determined in the plasma.
Expression of a number of cholesterol metabolism-related genes in liver and
steroidogenesis-related genes in the gonad was determined. Ex vivo production of T
and E2 by gonad tissue was also determined. Gemfibrozil significantly lowered
plasma cholesterol concentrations in the males exposed to 600 μg/L for 8 days.
Expression of several genes important to lipid metabolism was significantly altered,
suggesting that gemfibrozil does affect lipid metabolism in fish. There was also a
significant reduction in male ex vivo T production after 2 days of exposure to gemfibrozil.
In addition, a 21 day reproduction study with gemfibrozil was completed
to further investigate the effects observed in the male fathead minnows and their
potential implications for fish reproduction. This study adds to our knowledge of
possible effects of common pharmaceutical pollutants on fish.
2369 RETINOIC ACID MODULATES AHR-MEDIATED
EFFECTS OF TCDD IN OSTEOBLASTIC CELLS.
M. Herlin 1 , R. Heimeier 1 , M. Korkalainen 2 , M. Viluksela 2 and H. Håkansson 1 .
1 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden and
2 National Institute for Health and Welfare, Kuopio, Finland.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor
that can be activated by a range of structurally diverse chemicals, including ubiquitous
environmental contaminants such as dioxins. An increasing number of studies
show that exposure to high affinity AhR ligands, such as TCDD (2,3,7,8-
Tetrachlorodibenzo-p-dioxin), can induce alterations in bone geometry, bone mineral
density and bone mechanical strength. Exposure to such exogenous hormonally
active substances can thus have deleterious implications to human health. This
study proposed to identify critical AhR-modulated signaling pathways that may be
disrupted in bone by TCDD based on molecular analysis in vitro. Retinoic acid
(RA) signaling is one such pathway of interest as retinoid metabolism seems to be
involved in TCDD-induced toxicity. RA is a lipid soluble hormone, which exerts a
wide variety of effects on development, cellular differentiation and homeostasis in
various tissues, including bone growth and development. Currently the role of RA
in bone toxicity is unexplored. In this study, we exposed MCT3T-E1 cells, an osteoblast
precursor cell line, to TCDD in the presence or absence of all-trans RA or
9-cis RA. Both RA isoforms showed a protective effect against TCDD-induced
AhR activation. These results suggest that RA could modulate AhR-mediated effects
of TCDD in differentiating osteoblastic cells. Our finding that RA inhibits
AhR-pathways that affect bone differentiation in vitro thus provides a novel mechanism
for the toxicity effects of dioxins on bone development. Further studies are
needed to delineate the molecular mechanisms involved and potential crosstalk between
AhR and RA signaling pathways.
2370 DETERMINING STRUCTURAL DETERMINANTS OF
PHTHALATE ANTIANDROGENIC POTENCY IN VITRO
USING RAT AND MOUSE LEYDIG TUMOR CELLS.
R. A. Clewell, K. Edwards, J. Campbell, H. Clewell and M. Andersen. The
Hamner Institutes, Research Triangle Park, NC.
Some phthalate esters (PEs) disrupt sexual development in the male rat after gestational
exposure, which is at least partially due to reduced testosterone (T) synthesis
in the fetal Leydig cell. We previously developed a mouse Leydig cell (MA-10) assay
to test antiandrogenic activity of the PEs in vitro. However, several important differences
exist between this cell line and cells affected in vivo. MA-10s are derived
from the adult mouse and require luteinizing hormone (LH) receptor stimulation
for testosterone (T) production. In vivo antiandrogenic effects are only seen in the
rat, after fetal exposure, and fetal T is not regulated by LH. Thus, we explored
whether a rat tumor derived cell line (R2C) that produces T independent of LH
would provide an accurate and more relevant model for PE inhibition. A variety of
PEs were tested in both cells, with alkyl chains from 1 to 9 carbons (C1 - C9) in
length and various degrees of branching. RT-PCR was performed for 4 genes typically
down-regulated in the fetal testis after PE exposure (Cyp11a1, Cyp17a1, StAR
and Scarb1). All four genes were down-regulated in MA-10s after MEHP treatment,
while only Cyp11a1 and Cyp17a1 were reduced in the R2Cs. While R2Cs
were somewhat less sensitive to T inhibition, the trend across PEs was the same in
the two cell lines, indicating that these in vitro assays are useful for predicting PE
relative activity in vivo. In agreement with in vivo studies, T synthesis in both cell
lines was inhibited by monoethylhexyl (MEHP) and monobutyl (MBP), but not
monoethyl phthalate (MEP). EC 50 s were 4 and 16 uM (MEHP), 3 and 12 uM
(MBP) and > 100 uM (MEP) in the MA-10 and R2Cs, respectively. In both cell
lines, potency was highest in PEs with alkyl chain backbone lengths of 4-5 carbons.
In longer chain PEs, branching tended to increase potency for T inhibition.
2371 ISOLATION AND CHARACTERIZATION OF
CHEMOPREVENTATIVE ANALYTES FROM AN
ACETONE EXTRACT OF KOLA ACUMINATA.
J. Wynder 1, 2 and W. G. Gray 1, 2 . 1 Chemistry, Southern University A&M College,
Baton Rouge, LA and 2 Environmental Toxicology, Southern University Baton Rouge,
Baton Rouge, LA.
The search for effective treatment and potential eradication of prostate and breast
cancer has seen a merger between the use of synthetic chemo-preventative agents
and natural dietary products. The World Health Organization has recognized that
more than fifty percent of scientifically developed drugs are either natural products,
a derivative of natural products, or inspired by natural products. Our laboratory has
been engaged in characterizing the androgenic and “putative” chemo-preventative
properties of the common Jamaican bush tea “bizzy” nut (Kola acuminata, also
known as obi ) using the androgen responsive (LNCaP-AR+) and estrogen responsive
(MCF-7-) cell lines.. The goal of this research project is to test the hypothesis
that Biz-E3 contains a unique set of analytes that modulates cancer cell function.
The specific objective, of this study was to generate Biz-E3 and Biz-E5 specific
chemoinformatic libraries with its associated gene expression profile data. To accomplish
this objective a High Performance Liquid Chromatograph (HPLC) and
UV-Vis spectroscopy profiles for Biz-E3 and Biz-E5 were generated. We examined
the expression of key regulatory genes along the apoptotic pathway in the presence
of these extracts. Characterization of Biz-E3 by HPLC revealed the presence of
seven distinct peaks, all eluting within the first 32 minutes of the HPLC run. Biz-
E5 showed fewer peaks as compared to Biz-E3. The wavelength spectra of selected
Biz-E3 HPLC peaks detected the presence of 4 to 5 individual analytes per peak.
The crude Biz-E3 extract and selected HPLC fractionations were tested in growthinhibitory
assays in the MCF-7 cells and found to have strong anti-estrogen properties.
Our results suggest that Bizzy nut contains unique analytes may be responsible
for the “putative” chemo- preventative effects reported for this natural product.
SOT 2011 ANNUAL MEETING 509