The Toxicologist - Society of Toxicology

promoter activity was seen in the presence of DEHP and MEHP, but no significant
changes were observed due to other tested oxidative metabolites. As expected, the
known AR antagonists vinclozalin and flutamide reduced DHT induced promoter
activity. In studies using CV1 cells co-transfected with PSA-Luciferase and hAR,
MEHP and its oxidative metabolites reduced promoter activity in the presence of
DHT, as did vinclozalin and flutamide. Our findings indicate DEHP or its
metabolites can impact AR signaling in prostate and kidney cell lines. Surprisingly,
both agonist and antagonist actions on AR signaling were observed, which suggests
a cell line or tissue dependent effect of DEHP or its metabolites on AR function.
1471 ASSESSMENT OF AH RECEPTOR ACTIVITY IN THE
ABSENCE OF AN EXOGENOUS LIGAND.
T. A. Harper and C. Elferink. Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, TX.
The aryl hydrocarbon receptor (AhR) is traditionally recognized for its role in the
adaptive metabolism of xenobiotics, but has more recently been associated with important
physiological processes such as cell cycle regulation and apoptosis. Our lab
has recently followed up on observations suggesting that the AhR is able to promote
cell survival or death in response to intrinsic or extrinsic apoptotic stimuli, respectively.
Using primary hepatocytes isolated from the AhRfx/fx mouse, we generated
AhR null hepatocytes through infection with an adenovirus expressing the Cre recombinase.
Complete loss of AhR expression occurred within 24 hours. AhR expression
was unaltered in control cultures infected with a control adenovirus.
Susceptibility to UV-irradiation induced apoptosis was assayed by measuring caspase-3
activity, and monitoring chromatin condensation and cell blebbing. Since
Akt activity was recently implicated in promoting AhR-mediated Hepa-1 cell survival
upon intrinsic stimulation, we examined Akt expression and activity in the
primary hepatocytes immunologically. Our findings confirmed that the AhR indeed
promotes survival, but does so involving a mechanism independent of Akt activity.
In order to ascertain how the AhR functions to protect primary hepatocytes
from intrinsic cell death, we performed a detailed DNA microarray analysis linking
changes in the transcriptome directly to the loss of AhR expression—in the absence
of an exogenous receptor agonist. This screen represents a unique strategy for assessing
the receptor’s physiological role in regulating gene expression, and identified
a distinct set of genes with altered expression not previously associated with AhR
function. As a result, we are now in position to characterize new AhR-dependent
target genes and mechanisms, including protection from apoptosis, consistent with
normal AhR physiology. Supported by ES012018 and T32ES07254.
1472 FEEDBACK LOOP BETWEEN RETINOIC ACID
RECEPTORS AND NOVEL COREGULATOR TNIP1.
I. Gurevich, C. C. Zhang, C. P. Struzynski and B. J. Aneskievich. University of
Connecticut, Storrs, CT.
Transcriptional control by nuclear receptors (NRs) is mediated not only through
their ligands, but also through coregulator proteins which act either as coactivators
or corepressors of NR activity. In a yeast two-hybrid screen seeking novel NR coregulators,
we isolated TNIP1, a protein previously reported to interact with two different
HIV proteins and also to repress NF-κB activity. TNIP1 amino acid sequence
revealed the presence of two LXXLL NR box motifs suggesting it may be a
coregulator. TNIP1 does not interact with retinoid X receptor α (RXRα) but associates
with retinoic acid receptors (RAR) α and γ in a ligand dependent fashion and
exhibits other interaction hallmarks of a coactivator. However, TNIP1 represses
RAR activity in the presence of ligand. This makes TNIP1 an unusual coregulator
– a corepressor of agonist bound NRs. The repression is partially relieved by coactivator
SRC1, suggesting interference with coactivator recruitment as a mechanism
of TNIP1 action. TNIP1 does not associate with histone deacetylase (HDAC) enzymes,
suggesting the TNIP1 repression is HDAC independent. With this atypical
function, we sought to determine what might contribute to control of TNIP1 expression.
We carried out in silico analysis of its promoter to identify putative transcription
factor binding sites which predicted several retinoic acid response elements.
Transcriptional activation studies revealed that TNIP1 promoter is
positively regulated by RARs. EMSA showed RAR binding at distinct sites in the
distal portion of the TNIP1 promoter. Promoter-luciferase reporter studies confirmed
these as response elements. Our findings reveal a potential regulatory feedback
loop where TNIP1 expression is increased by RARs which, in turn, attenuates
their activity. Such regulatory feedback loops between coregulators and their target
NRs may serve to buffer cells against extremes of hormone-regulated signaling,
such as the presence of toxic ligand levels, or cells being exposed to ligand at inappropriate
times.
1473 PROGRESSIVE EXPOSURE TO AMBIENT FINE
PARTICLES IS ASSOCIATED WITH CHANGES IN
PULMONARY AND SYSTEMIC INFLAMMATION IN
MICE.
L. E. Plummer and K. E. Pinkerton. Center for Health and the Environment,
University of California Davis, Davis, CA.
Growing evidence suggests exposure to particulate matter is associated with increases
in morbidity and mortality due to cardiopulmonary complications. To examine
for the presence of early markers of pulmonary and systemic inflammation,
including changes in hematologic parameters, male Balb/C mice were progressively
exposed to concentrated ambient particles (CAPS) in the fine/ultrafine size range or
to filtered air in Fresno, CA during the summer of 2009 for 6 hours a day for 3, 6,
9, or 12 days. Total lung cells recovered by bronchoalveolar lavage (BAL) were significantly
increased at 6 days, while significantly reduced at 12 days, compared with
filtered air control values. Lung neutrophils were found to be increased with progressive
CAPS exposure, attaining statistical significance at 12 days. Hematologic
assessment demonstrated significantly increased white blood cells in the circulation
at 6 days. These pulmonary and systemic findings provide further confirmation for
a role of progressive exposure to concentrated inhaled fine/ultrafine particles to enhance
the pro-inflammatory activity in the lungs along with secondary markers of
cellular change in the systemic circulation of mice. Research funded by U.S. EPA
RD 832414.
1474 PARTIAL CHARACTERIZATION OF AMBIENT PM2.5
EXTRACT FROM SAHARAN DUST EVENTS.
R. Rodríguez 1 , L. B. Méndez-Torres 2 and B. D. Jiménez-Vélez 1 . 1 Biochemistry,
University of Puerto Rico-Medical Sciences Campus, San Juan and 2 University of
Puerto Rico-Medical Sciences Campus, San Juan.
Every year million of tons of African dust (Sahara) are transported into the
Caribbean. As much as 8 million tons of African dust was estimated as reaching the
Puerto Rican coast in one month alone (July 2000). This input of dust coupled
with other natural and anthropogenic sources of particulate matter could impact air
quality and intensify respiratory problems. The expression of interleukins in human
bronchial epithelial cells (BEAS-2B) exposed to PM2.5 enriched with Saharan dust
and its relation with oxidative stress was evaluated. PM2.5 Teflon filters were obtained
from the PR Environmental Quality Board (EQB). Satellite information
Total Ozone Mapping Spectrometer (TOMS) aerosol index, in conjunction with
EQB’s data, was combined to determine Saharan dust events (SDE). Filters were
extracted for 24 hours in 175mL hexane/acetone (1:1). BEAS-2B cells were seeded
onto a 96-well plate and incubated 48hrs prior exposure to the organic extracts.
Attached cells were exposed for 24hrs at different PM extract concentrations (25,
50, 75 and 100 μg/ml) before being analyzed for biological response. Cell viability
was assessed using the Neutral Red bioassay (Sigma). A direct relationship between
extract concentration and cell viability was identified. Non-SDE extracts were not
cytotoxic at concentrations less than 100 ug/ml. A direct dose relationship was also
found between PM2.5 extract and IL-8 expression (Luminex, Millipore) with both
SDE and Non-SDE organic extracts. However the magnitude of response was significantly
higher for SDE extracts. Similar findings were observed with IL-6 expression
but not highly significant. Preliminary results pointed to a decrease in glutathione
levels of SDE extract, suggesting that components in Sahara dust might
induce oxidative stress in BEAS-2B. Metals analyses on both extracts are currently
being performed.
1475 PULMONARY BIOASSAY STUDY WITH POTASSIUM
TITANATE NONFIBROUS PARTICULATES (TERRACESS
JS) IN RATS.
D. B. Warheit 1 , K. L. Reed 1 and S. Sakai 2 . 1 DuPont Haskell Laboratory, Newark,
DE and 2 Otsuka Chemical Company, Osaka, Japan.
Terracess JS particles are composed of nonfibrous potassium titanate (K2Ti6O13)-
particle-types and have been proposed for friction and filling applications. The aim
of this study was to assess lung toxicity in rats exposed to potassium titanate particle-types
relative to positive and negative controls. Groups of male rats were intratracheally
instilled with doses of 1 or 5 mg/kg of Terracess JS particles or α-quartz
particles. Phosphate-buffered saline (PBS) solution instilled rats served as vehicle
controls. Following exposures, the lungs of PBS and particle-exposed rats were evaluated
for bronchoalveolar lavage (BAL) fluid inflammatory biomarkers at post-instillation
time points of 1 week, 1 month, and 3 months or lung histopathology at
1 month or 3 months. Pulmonary exposures to quartz particles produced sustained
lung inflammation and significant cytotoxic effects. In contrast, exposures to potassium
titanate particle-types produced no significant lung inflammatory or cell in-
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