The Toxicologist - Society of Toxicology

qHTS format at concentrations ranging from 0.59 nM to 92 μM for their ability to
induce this pathway in ARE-bla HepG2 cells transfected with a beta-lactamase reporter
gene under the control of ARE and in HepG2 cells transfected with a luciferase
reporter measuring Nrf2 specific ARE activation. Raw data were normalized
relative to beta-napththoflavone (100%) and DMSO (basal, 0%).
Concentration response curves were generated for each substance, with EC50 values
and efficacy calculated for substances classified as positive in either assay. We
identified 364 and 44 compounds that stimulated ARE in the bla and luciferase assays,
respectively, with 34 of these compounds active in both assays. Based on either
a positive response in both assays or a structure-activity relationship to an active
compound, we re-tested 63 compounds in the two ARE assays and also for their
ability to deplete glutathione, as a downstream assay. Fifty-six out of 63 compounds
were confirmed for ARE activity and several (e.g., hydroquinone) additionally depleted
glutathione levels. Our results indicate the robustness of these two different
approaches for assessing the ability of much larger compound libraries to induce
ARE. Supported by NIEHS Interagency Agreement Y2-ES-7020-01.
2502 NUCLEAR FACTOR E2-RELATED FACTOR 1 IS
INVOLVED IN ARSENIC-INDUCED ANTIOXIDANT
RESPONSE IN HUMAN KERATINOCYTES.
R. Zhao 1 , Y. Hou 1 , P. Xue 1 , C. G. Woods 1 , J. Fu 1 , M. P. Waalkes 2 , M. E.
Andersen 1 and J. Pi 1 . 1 The Hamner Institutes, Reaserach Triangle Park, NC and
2 NTP, NIEHS, NIH, Research Triangle Park, NC .
Human exposure to inorganic arsenic, a potent oxidative stressor, causes various
dermal disorders, including hyperkeratosis and skin cancer. Nuclear factor E2-related
factor 1 (NRF1) plays a critical role in regulating the expression of many antioxidant
response element (ARE)-dependent genes. The current study investigates
the role of NRF1 in arsenic-induced antioxidant response and cytotoxicity in cultured
human keratinocytes. In HaCaT cells, inorganic arsenite (iAs3+) enhanced
the protein accumulation of long isoforms (120-140 kDa) of NRF1 in a dose- and
time-dependent fashion. The long isoforms of NRF1 induced by iAs3+ were
mainly accumulated in nucleus of HaCaT cells. Selective deficiency of NRF1 by
lentiviral shRNAs in HaCaT cells (NRF1-KD) led to decreased expression of γ-glutamatecysteine
ligase catalytic subunit (GCLC) and regulatory subunit (GCLM)
and a reduced level of intracellular glutathione. In response to acute iAs3+ exposure,
induction of some ARE-dependent genes, including NAD(P)H: quinone oxidoreductase
1 (NQO1), GCLC and GCLM, was significantly attenuated in
NRF1-KD cells. However, the iAs3–induced expression of heme oxygenase 1
(HMOX-1) was unaltered by silencing of NRF1, suggesting HMOX-1 is not regulated
by NRF1. In addition, lack of NRF1 in HaCaT cells did not disturb iAs3+induced
NRF2 accumulation, but noticeably decreased KEAP1 protein levels
under basal and iAs3+-exposed conditions, suggesting a potential interaction between
NRF1 and KEAP1. Consistent with the critical role of NRF1 in the transcriptional
regulation of some ARE-bearing genes, knockdown of NRF1 significantly
increased iAs3+-induced cytotoxicity and apoptosis. The current study, for
the first time, demonstrates that long isoforms of NRF1 contribute to arsenic-induced
antioxidant response in human keratinocytes and protect the cells from acute
arsenic cytotoxicity.
2503 BENEFICIAL ROLE OF NRF2 IN REGULATING NADPH
GENERATION AND CONSUMPTION.
K. C. Wu, J. Y. Cui and C. D. Klaassen. Pharmacology, Toxicology, and
Therapeutics, University of Kansas Medical Center, Kansas City, KS.
Nrf2 (nuclear factor E2 p45-related factor 2) is a central regulator that promotes
the transcription of cytoprotective genes in response to oxidative and electrophilic
stresses. Most functions of Nrf2 were identified by studying biological models with
Nrf2 deficiency, however, little is known about the effects of graded Nrf2 activation.
In the present study, hepatic phenotypes as well as genomic gene expression
profiles by microarray analysis were characterized with a ‘gene dose-response’ model
in livers of Nrf2-null mice, wild-type mice, Keap1-knockdown (Keap1-KD) mice
with enhanced Nrf2 activation, and Keap1-hepatocyte knockout (Keap1-HKO)
mice with maximum hepatic Nrf2 activation. Hepatic nuclear Nrf2 protein, glutathione
concentrations, and known Nrf2 target genes were increased dose dependently.
In total, 115 genes were identified to be constitutively induced and 80 genes
suppressed with graded Nrf2 activation. Specifically, mRNA of genes encoding enzymes
in the pentose phosphate pathway as well as malic enzyme were low with
Nrf2 deficiency and high with Nrf2 activation, indicating that Nrf2 is important
for NADPH production. NADPH is the major reducing power in the body to
scavenge oxidative stress including regenerating glutathione and thioredoxin, and is
also used for anabolic pathways including lipid synthesis. HPLC-UV analysis confirmed
that hepatic NADPH concentration was lowest in Nrf2-null mice and highest
in Keap1-HKO mice. In addition, genes involved in fatty acid synthesis and de-
536 SOT 2011 ANNUAL MEETING
saturation were down-regulated with graded Nrf2 activation. In conclusion, the
present study suggests that Nrf2 fights against environmental insults to benefit cell
survival by promoting the generation of NADPH, and by pushing its consumption
into the direction of scavenging oxidative stress rather than fatty acid synthesis and
desaturation. (Supported by NIH grants ES-09649, ES-09716, ES-07079,
DK081461, and RR-021940)
2504 ASSESSMENT OF NANOPARTICLE EXPOSURE IN
OCCUPATIONAL SETTINGS AND IN INHALATION
TOXICOLOGY STUDIES: IS THERE A BEST
DOSEMETRIC TO USE?
D. B. Warheit 1 and G. Oberdorster 2 . 1 DuPont Haskell Global Centers, Wilmington,
DE and 2 University of Rochester, Rochester, NY.
Nanotechnology involves the design and manipulation of materials at the nanoscale
size range. The capacity for assessing hazards or quantifying exposures to engineered
nanomaterials for workers or consumers is limited, and may require new or
different methodologies to provide information for effective risk assessment and
risk management. Exposure assessments are important components for determining
health risks. However, currently used methodologies are inadequate for quantifying
nanoparticulate exposures, because most of the occupational exposure data is
represented in the gravimetric form of mass/volume of sampled air. Our goal is to
address this controversial issue with a focus on practicability of measurements
under workplace conditions. Therefore it is important to discuss the proposed
change in dose metrics for inhalation toxicity studies with engineered aerosolized
amorphous silica nanoparticles. Accordingly, rats were exposed to freshly generated
nanoparticles at two different particle size ranges. The particle aerosols were characterized
by particle numbers using SMPS technology. Our panel of experts will address
the merits of using surface area metrics based on measurements of particle
numbers and material-specific density for quantifying particle exposures and their
use for risk assessment. Our panel will present real-time instrumentation for measuring
airborne particle surface area and morphology of nanoparticle agglomerates—two
of the important metrics for toxicity evaluation. In addition, recent exposure
assessments in manufacturing facilities involving silicon nanoparticles and
carbon nanotubes will be presented. It is also important to note the effect of agglomerate
structure size on the bioactivity of nanoparticles and present current
methods used to disperse nanoparticles for in vitro and in vivo testing which we will
address. Finally, we will discuss methods to convert in vivo lung burdens to appropriate
in vitro test concentrations of nanoparticles for hazard screening.
2505 PROGRESS OF THE TOX21 CONSORTIUM IN HIGH-
THROUGHPUT BIOACTIVITY PROFILING OF
CHEMICALS.
R. Kavlock 1 and C. Austin 2 . 1 U.S. EPA, Research Triangle Park, NC and 2 NIH
Chemical Genomics Center, Gaithersburg, MD.
In 2008, the National Institute of Environmental Health Sciences/National
Toxicology Program, the NIH Chemical Genomics Center, and the U.S. EPA’s
National Center for Computational Toxicology entered into a Memorandum of
Understanding to collaborate on the research, development, validation, and translation
of new and innovative test methods to characterize key steps in toxicity pathways.
The U.S. FDA joined this consortium in 2010. A central component is the
exploration of high-throughput screening, as well as high-throughput whole
genome analytical methods, to evaluate mechanisms of toxicity. The goals of the
Tox21 Community are to investigate the use of these new tools, along with existing
chemical and biological information, to prioritize substances for further in-depth
toxicological evaluation, identify mechanisms of action for further investigation,
and develop predictive models for in vivo biological response. Success is expected to
result in test methods for toxicity testing that are more mechanistically based and
economically efficient; as a consequence, a reduction or replacement of animals in
regulatory testing is anticipated to occur in parallel with an increased ability to evaluate
the large numbers of chemicals that currently lack adequate toxicological evaluation.
In the past year, Tox21 has completed assembly of a library of ~10,000
chemicals and began screening the library against molecular targets and pathways at
the rate of one assay per week. Our panel of experts will inform the scientific community
of progress in meeting the Tox21 goals, by focusing on the strategies for
chemical and assay selection, workflows for data management and analysis, and understanding
the human significance of results. The Tox21 effort represents the
largest and most comprehensive evaluation of interaction of environmental chemicals
with toxicity pathways and is helping to pave the way for the use of highthroughput
screening tools in hazard identification, chemical prioritization, and
risk assessment.