The Toxicologist - Society of Toxicology

TNFα-induced survival signalling routes and sensitizes cells to apoptosis and consequently
the onset of DILI. We anticipate that our work will enable us to identify
mechanism-based biomarkers that can predict idiosyncratic-like DILI in a pre-clinical
drug development setting.
410 NRF2B: A NOVEL NRF2 PARALOG IN ZEBRAFISH.
A. R. Timme-Laragy 1 , S. I. Karchner 1 , D. G. Franks 1 , M. J. Jenny 2 and M. E.
Hahn 1 . 1 Biology, Woods Hole Oceanographic Institution, Woods Hole, MA and
2 University of Alabama, Tuscaloosa, AL.
NF-E2-related factor 2 (Nrf2) regulates antioxidant defenses by activating gene expression
via antioxidant response elements (AREs). An Nrf2 ortholog was identified
previously in zebrafish (Danio rerio). We have identified and cloned a novel zebrafish
Nrf2 paralog, Nrf2b. The predicted protein sequence shares several domains
with the original Nrf2 (now Nrf2a), but lacks the neh4 and neh5 transactivation
domains. Zebrafish-human comparisons demonstrate conserved synteny between
nrf2 and hox genes. nrf2a is on chromosome 9 near the hoxda cluster and nrf2b is
on chromosome 6 near the degenerate hoxdb cluster, indicating that nrf2a and
nrf2b are co-orthologs of the human NRF2 gene. Nrf2a and Nrf2b display different
patterns of expression during embryonic development. While Nrf2a expression
increased throughout development (0-120 hours post fertilization; hpf), Nrf2b had
the highest expression pre-hatch (48-60 hpf); embryos that hatched early had low
expression at these same times. In adults, Nrf2b was most highly expressed in the
ovary, female gut, and gill; lowest expression was found in male kidney and eye. In
transient transfection assays in COS-7 cells, Nrf2b was less active than Nrf2a, but
was capable of activating an ARE-regulated reporter gene in response to tert-butylhydroquinone
(tBHQ). No deformities were noted after morpholino knockdown
of either Nrf2a or Nrf2b. Nrf2a morphant embryos were more sensitive to tertbutylhydroperoxide
(tBOOH) but not tBHQ, whereas knock-down of Nrf2b did
not affect sensitivity of embryos to either chemical. Nrf2b expression was increased
following exposure to TCDD and PCB-126 in an Ahr2-dependent manner, suggesting
crosstalk between Nrf2b and Ahr2 signaling pathways. Further examination
of zebrafish Nrf2 co-orthologs may yield new understanding about the evolution,
function, and regulation of NRF2.
[Supported by F32ES017585, R01ES016366, and R01ES006272.]
411 CRITICAL CYSTEINE RESIDUES OF KEAP1 IN
SUPPRESSION OF NRF2 BASAL ACTIVITY AND
ARSENIC-SENSING BY REGULATING THE
UBIQUITINATION-PROTEASOMAL DEGRADATION
OF NRF2 PROTEIN.
X. He 1 and Q. Ma 1, 2 . 1 Receptor Biology Lab. /TMBB/HELD, NIOSH,
Morgantown, WV and 2 Biochemistry, West Virginia University, School of Medicine,
Morgantown, WV.
Nrf2 mediates the induction of antioxidant response element (ARE)-dependent
drug metabolizing enzyme genes and antioxidative genes by a wide range of structurally
divergent inducers including carcinogenic metals such as arsenic. Activation
of Nrf2 involves modulation of Keap1/Cul3-mediated ubiquitination-proteasomal
degradation of Nrf2. Here we analyzed arsenic-Keap1 cysteine thiol interactions for
Nrf2 activation. Fluorescent arsenic FlAsH and phenylarsine oxide (PAO) were
used to probe binding of arsenic to Keap1. FlAsH emitted strong fluorescence upon
binding to purified Keap1, and arsenic, tert-butyhydroquinone (tBHQ), or the
thiol reactive 2, 3–dimercaptopropanol inhibited FlAsH binding. Purified or endogenous
Keap1 was effectively pulled down by PAO affinity beads in vitro and
from hepa1c1c7 cells, and arsenic, tBHQ, free PAO, or cadmium blocked Keap1
pull down. Furthermore arsenic and free PAO significantly reduced the free thiol
contents of purified or endogenous Keap1 in cells. Therefore, arsenic, FlAsH, PAO,
tBHQ, and cadmium bind to Keap1 cysteine thiols competitively. All domains of
Keap1 bound PAO, but the linker region exhibited highest binding activity. The
function of arsenic-Keap1 interaction was evaluated in a reconstituted system that
mimics endogenous Nrf2 regulation. Mutation of C273 or C288A in linker region
resulted in higher level expression of Nrf2 protein in the absence of inducers.
Mutation of C151A abolished Nrf2 activation by arsenic. Overexpression of
C273A, C288A, or C151A altered the basal and arsenic-induced expression of
Nqo1 consistent with regulated protein levels of Nrf2. The study demonstrates important
roles of C273 and C288 in the suppression of Nrf2 by Keap1 under basal
conditions and critical function of C151 in arsenic sensing and responsiveness. Our
findings support the notion that arsenic binds to different sets of Keap1 cysteine
residues to regulate divergent functions in Nrf2 signaling.
88 SOT 2011 ANNUAL MEETING
412 THE KEAP1-NRF2 SYSTEM REGULATES
METALLOTHIONEIN EXPRESSION AND PROTECTS
VASCULAR ENDOTHELIAL CELLS FROM CADMIUM
CYTOTOXICITY.
Y. Shinkai 1 , Y. Kumagai 1 , T. Kimura 2 , C. Yamamoto 3 , M. Yamamoto 4 , H.
Jinno 5 , T. Tanaka-Kagawa 5 and T. Kaji 6 . 1 Graduate School of Comprehensive
Human Sciences, University of Tsukuba, Tsukuba, Japan, 2 Faculty of Pharmaceutical
Sciences, Setsunan University, Osaka, Japan, 3 Faculty of Pharmaceutical Sciences,
Hokuriku University, Kanazawa, Japan, 4 Graduate School of Medicine, Tohoku
University, Sendai, Japan, 5 Division of Environmental Chemistry, National Institute
of Health Sciences, Tokyo, Japan and 6 Faculty of Pharmaceutical Sciences, Tokyo
University of Science, Noda, Japan. Sponsor: A. Naganuma.
Cadmium, a ubiquitous heavy metal, is an important industrial and environmental
pollutant that can target the vascular endothelium. However, the molecular and cellular
mechanisms of protection against cadmium toxicity are poorly understood. In
the present study, we investigated role of the Keap1-Nrf2 system in cellular response
to cadmium and protection against this metal in bovine aortic endothelial
cells. Exposure of the cells to cadmium caused Nrf2 activation, resulting in the expression
of antioxidant proteins such as heme oxygenase-1, NAD(P)H: quinone
oxidoreductase-1, and glutamate cysteine ligase modulatory subunit. We found
that siRNA-mediated knockdown of Nrf2 attenuated expressions of not only these
antioxidant proteins but also metallothionein-I/II, thereby enhancing cadmium cytotoxicity,
whereas siRNA-mediated knockdown of Keap1, the negative regulator
for Nrf2, potentiated induction of metallothionein genes, leading to protection of
the cells from cadmium cytotoxicity. Moreover, chromatin immunoprecipitation
assay showed that Nrf2 was recruited to the antioxidant response element region of
the metallothionein gene promoter in the presence of cadmium. Taken together,
these results suggest that the Keap1-Nrf2 system serves as a defense mechanism
against cadmium toxicity through positive regulation of metallothionein genes in
the vascular endothelial cells.
413 MECHANISTIC INVOLVEMENT OF THE NRF2/KEAP1
ANTI-OXIDANT RESPONSE IN THE REGULATION OF
HUMAN ABCC3.
M. D. Merrell 1 , M. J. Canet 1 , F. Zhao 1 , T. Wu 1 , J. M. Maher 2 and N. J.
Cherrington 1 . 1 Pharmacology and Toxicology, University of Arizona, Tucson, AZ and
2 Department of Medical Biochemistry, Tohoku University, Sendai, Japan.
Members of the ATP-binding cassette C (ABCC) family, including ABCC3, play
an important role in toxicology and disease by actively effluxing a wide variety of
endogenous and exogenous substrates. Induction of human ABCC3 has been reported
during hepatic stress (disease, toxicant exposure) and in the progression of
some forms of cancer. Multiple investigations have implicated the transcription factor
Nrf2 in this induction, and the functional antioxidant response element (ARE)
has been characterized in the mouse ABCC3 gene. However, while recent studies in
A549 human lung cells (which over-express Nrf2) have demonstrated a clear role
for Nrf2 in the induction of human ABCC3, no functional human response element
has been defined. Results of a ChIP-sequencing experiment revealed a specific
interaction between Nrf2 and the eighth intron (-26.3kb to -27.5kb) of the human
ABCC3 gene, and not the upstream promoter. Subsequent in silico analysis of this
intron revealed several putative AREs and ARE-like elements. Additionally, while
previously identified putative response elements in the upstream promoter are not
evolutionarily conserved, sequence alignment revealed sequence conservation of an
intronic ARE across ten eutherian mammal species, including five non-primate
species. Luciferase reporter gene assays using an Nrf2 overexpressing cell line
(A549) and constructs containing 1.2kb of the eighth intron revealed increased activity
over vector-controls. Finally, rtPCR-based chromatin immunoprecipitation
using antibodies against Nrf2 confirmed the specific DNA-Nrf2 interaction identified
in the ChIP-sequencing experiment. Our findings identifying an Nrf2 response
element within an intron of the human ABCC3 gene may provide a mechanistic
understanding of the induction of human ABCC3 during the antioxidant
response.
414 ATTENUATION OF THE NRF2 SIGNALING PATHWAY
IN HEPATOCYTES LACKING SIRTUIN 1 (SIRT1).
W. Wei 1 , J. Xu 1 , S. Kulkarni 1 , X. Li 2 and A. Slitt 1 . 1 BPS, University of Rhode
Island, Kingston, RI and 2 National Institute of Environmental Health Sciences,
Research Triangle Park, NC.
Primary hepatocytes are effective tools for in vitro evaluation of biotranformation
enzyme and transporter induction. Tertiary butylhydroquinone (tBHQ) is a phenolic
antioxidant that stabilizes the protein NF-E2-related factor 2 (Nrf2, Nfe2l2)
and enhances antioxidant response element-mediated gene transcription. Sirt1 is a