The Toxicologist - Society of Toxicology

tivity, specifically peroxisome proliferator activated receptors and retinoic acid receptors.
To better understand increased TNIP1 possibly contributing to such disease
states, we over-expressed recombinant TNIP1 protein in HaCaT keratinocytes
and performed a microarray analysis to determine pathways and possible target
genes. At a cutoff of a 2-fold change or greater, elevated TNIP1 resulted in 94 significantly
down-regulated genes and 3 up-regulated genes. Pathway analysis determined
inflammatory response, cell death, and cellular growth/proliferation as the
top network functions associated with the dataset. Increased TNIP1 significantly
decreased mRNA levels of several genes encoding for two heat shock protein (HSP)
families, HSPA (HSP70) and DNAJ (HSP40). The top ten down-regulated genes
included HSPA6 (HSP70B’) and DNAJB1 (HSP40), being downregulated 20-fold
and 5-fold, respectively. Heat shock proteins play integral roles not only protecting
cells in times of stress, but are also vital in tissue homeostasis. These results indicate
a role for TNIP1 as a novel regulator of heat shock protein expression; therefore
TNIP1 may play a role in regulation of cellular stress and the pathogenesis of inflammatory
diseases.
396 SEXUALLY DIMORPHIC ROLE FOR THE CIRCADIAN
CLOCK IN MODULATING XENOBIOTIC
METABOLISM.
L. A. Hooven, K. Sherman, E. Chow, L. Beaver and J. Giebultowicz. Oregon
State University, Corvallis, OR.
The circadian clock synchronizes physiological and molecular rhythms with predictable
daily oscillations in the environment such as light and temperature. There
are multiple examples of sexual dimorphism in circadian output. Although many
xenobiotic metabolizing genes are expressed in a coordinated daily rhythm, sexual
dimorphism in clock modulation of these genes has been little investigated. Here
we examine how the circadian clock differentially modulates gene expression and
response to pesticides in males and females, using both wild type flies and flies deficient
in the core clock genes CYC and PER. Flies were maintained in 12h:12h
light/dark and collected for qRT-PCR or enzyme activity, or acutely exposed to a
series of doses of propoxur or fipronil for one hour, every four hours for 24 hours.
In wild type flies, cytochrome P450 enzyme activity exhibited dissimilar daily
rhythms in males and females, as did 24h gene expression profiles of Cyp6g1,
Cyp6a8, Cyp4e2, and the nuclear receptor HNF4. The significant daily rhythm in
LC50 we previously reported in male flies in response to propoxur was absent in females,
which exhibited a much greater LC50 across the day. The response to
fipronil showed a similar rhythm in male and females. A mutation in CYC results
in increased resistance to propoxur in male flies, but not females. This mutation
also results in increased female resistance to fipronil, with no change in male response.
Conversely, a mutation in PER, part of the negative arm of the clock, results
in decreased resistance to propoxur in males and in females. The same mutation
causes no change in the male response to fipronil, but decreased resistance in
females. We are currently comparing daily expression profiles in nuclear receptors
and xenobiotic metabolizing genes in CYC and PER mutant flies in order to examine
sex-specific clock regulation of these genes. Importantly, this work reveals that
disruption of the circadian clock, an important environmental sensor, differentially
affects the ability of males and females to respond to toxicants.
397 CHARACTERIZATION OF THE PROMOTER-
PROXIMAL REGION OF HUMAN MULTIDRUG
RESISTANCE-ASSOCIATED PROTEIN 4 GENE.
J. E. Manautou and X. Gu. Pharmaceutical Sciences, University of Connecticut,
Storrs, CT.
Multidrug resistance-associated protein 4 (MRP4, ABCC4) is an efflux transporter
localized in the basolateral membrane of hepatocytes. Hepatic MRP4 expression is
very low and highly variable. MRP4/Mrp4 is inducible in mice and human by toxic
APAP exposure. Activation of transcription factors Nfe2l2, PPARα and CAR mediate
Mrp4 induction under a variety of conditions. However, knowledge on
MRP4/Mrp4 promoter structure and function is very limited. Our previous work
showed that transcription activity of human MRP4 promoter region is constitutively
active and that the activity of the promoter-proximal region is controlled by
many activating (e.g., NRF1, SP2, STAT1, TFAP2A) and repressive (e.g., HES1,
KLF15, ZFP161) transcription factors. In this study, we further characterized the
promoter-proximal region of MRP4 gene. Alignment of human and mouse 5’ regulatory
sequence shows 60%-70% homology in the promoter-proximal region and
much less homology in the distal region. Increased transcription activity of reporter
gene is only detected when the upstream promoter DNA sequence extends beyond
152 bp relative to MRP4 coding sequence starting site. Furthermore, over-expression
of E2F transcription factor 1(E2F1) in HepG2 cells up-regulates, while transcription
factors POZ (BTB) and AT hook containing zinc finger 1(PATZ1) and
ELK1 (member of ETS oncogene family) down-regulates MRP4 reporter gene ex-
pression for constructs containing upstream DNA fragments from 5 bp to 212bp
relative to MRP4 coding sequence starting site. Over-expression of E2F1, which
plays a crucial role in controlling cell cycle and as a tumor suppressor protein, resulted
in much higher induction of MRP4 promoter reporter activity (>10 fold)
than other transcription factors tested. These results suggested that MRP4 gene expression
might be coupled and tightly regulated by cell cycle. The core promoter for
MRP4 gene and physical interaction between these cis-elements and transcription
factors in liver will be further identified or characterized. This work was supported
by The National Institutes of Health Grant DK069557.
398 HIGH CONTENT ANALYSIS USING
REDISTRIBUTION® TECHNOLOGY.
A. M. Peters, D. Miller, B. Samson and Y. Fedorov. Life Science Research -
Cellomics, Thermo Fisher Scientific, Pittsburgh, PA. Sponsor: J. Haskins.
Understanding transcriptional activity and how it interacts with various stimuli can
gain insight to cellular processes and instabilities that cause toxic outcomes such as
cell stress or death in a range of conditions. In this study, four different regulators of
transcriptional activity (HIF-1α, ATF6, Rad51 and p53) were monitored using the
Redistribution technology. With this technology, proteins are GFP-tagged and observed
for accumulation and translocation of GFP chimera within the cell after
treatment. This approach enables visualization of cellular processes in a more natural
state, without having to add multiple stains, permeabilization steps, and correct
antibodies. Frozen cells were plated into 96-well plates, treated, fixed, Hoechststained,
and run on the Cellomics® ToxInsight IVT platform. Information
about individual cell morphology, cellular toxicity, and various GFP intensity outputs
were observed. For Hypoxia Inducing Factor 1 alpha (HIF-1α), unstimulated
cells exhibit a low protein level and thus show little GFP; however, upon treatment
with a compound that mimics hypoxia (2,2’-dipyridyl), HIF-1α accumulates in the
cell, producing larger levels of GFP. Activator of Transcription factor 6 (ATF6) was
observed to translocate from the cytoplasm/ER to the nucleus after treatment with
tunicamycin. Treatment with compounds inducing DNA double-stranded breaks
lead to accumulation of the Rad51-GFP chimera in the nuclear foci. Both interaction
of p53 and Hdm2 and p53 translocation can be visually observed when treated
with various compounds: Nutlin-3 caused increased fluorescence in the nucleus
(blocking p53-Hdm2 interaction), while forskolin caused non-specific localization
of GFP chimera in the cytoplasm. By using Redistribution technology and highcontent
imaging analysis, we were able to not only look at overall intensity accumulation
(which can be done using other methods), but could, on a cell-by-cell
basis, focus on localization, translocation between sites, as well as morphological
changes within the cell at one time.
399 ASSESSMENT OF NUCLEAR FACTOR KAPPA B (NFκB)
SIGNALING IN THE HIPPOCAMPUS DURING
KANIC ACID EXPOSURE USING TRANSGENIC
REPORTER MICE.
J. A. Miller 1 , R. A. Bialecki 2 and R. B. Tjalkens 1 . 1 Center for Environmental
Medicine, Colorado State University, Fort Collins, CO and 2 Safety Assessment U.S.,
AstraZeneca, Wilmington, DE.
NF-κB is a key transcriptional regulator of numerous genes including those involved
in regulating cell death / survival pathways and cellular plasticity. Chemical
induced kindling of seizure activity results from sustained alterations in neuronal
excitability, indicative of neuronal plasticity resulting in hyper-excitability. In the
present study, we employed a unique transgenic reporter mouse expressing NF-κB
dependent GFP, in order to investigate the role of NF-κB signaling in rendering
hippocampal neurons hyper-excitable. Mice were treated with kainic acid (KA, 2 x
10 mg/Kg, ip) over 48 hr and NF-κB activation was assessed 24 hours following
treatment. Animals displayed mild behavioral changes consistent with the early
stages of seizure induction including generalized immobility and facial clonus
which progressed to include mild head nodding after the second dose of KA.
Assessment of reporter expression showed that under basal conditions GFP is absent
in the hippocampus except for a pronounced expression in the CA3 region pyramidal
layer. After KA treatment, increased expression of GFP in the CA3 region and
marked expression in the stratum moleculare, dentate gyrus molecular layer and in
the dentate hilus were observed. These preliminary data show selective regional effects
of KA on NF-κB activation which mimic the sensitivity of these regions to KA
induced excitoxicity. This demonstrates the potential utility of this reporter model
in investigating NF-κB activation in seizure induction and maintenance and may
also prove useful in detecting chemically-induced effects on the hippocampus that
may underly seizuregenic activity of pharmaceutical compounds.
SOT 2011 ANNUAL MEETING 85