The Toxicologist - Society of Toxicology

149 HISTONE MODIFICATION IN ARSENITE-MEDIATED
ANTIOXIDANT GENE REGULATION.
P. D. Ray and Y. Tsuji. Environmental and Molecular Toxicology, North Carolina
State University, Raleigh, NC. Sponsor: J. Ninomiya-Tsuji.
Oxidative stress is implicated in various disease states including neurodegeneration,
cancer, and even aging; therefore, understanding how antioxidant enzymes are regulated
is important in the investigation of the pathogenesis of oxidative stress related
disease. Several antioxidant enzymes are transcriptionally regulated by a conserved
enhancer element, the antioxidant responsive element (ARE). This element
is found in the 5’ flanking region of such antioxidant genes as NAD(P)H quinone
oxidoreductase-1 (NQO1), heme oxygenase-1 (HO1), and the iron binding protein
ferritin, which binds excess iron, thus preventing formation of ROS via the
Fenton reaction. We hypothesized that the metalloid arsenite, a potent inducer of
histone H3 serine 10 phosphorylation (H3S10P), would stimulate ferritin H transcription
through activation of ARE and involve H3S10P. We observed that in
human keratinocytes arsenite treatment activated transcription of the ferritin H
gene through the ARE via increased NF-E2-related factor 2 (Nrf2) nuclear accumulation,
which is the primary ARE transcription factor. The antioxidant N-
acetyl-L-cysteine (NAC) reduced Nrf2 nuclear accumulation and to varying degrees,
reduced arsenite upregulation of ferritin H, NQO1, and HO-1 genes,
suggesting that oxidative stress is involved; NAC also blocked arsenite induced
H3Ser10P. In ARE ChIP assays, arsenite induced H3S10P, decreased acetylation of
H3K9, and increased methylation of H3K9; this is in stark contrast to increased
acetylation of H3K9 during ferritin H promoter activation in response to other
ARE activators we tested. These results suggest that histone modifications at
H3S10 in conjunction with the change in H3K9 status contributes to arsenite-mediated
transcriptional activation of the ARE.
150 REACTIVATION OF L1 RETROTRANSPOSON BY
BENZO(A)PYRENE INVOLVES EPIGENETIC
MECHANISMS.
D. E. Montoya-Durango 1, 2 , I. Teneng 1, 2 , J. L. Quertermous 1, 2 , V. Stribinskis 1, 2
and K. S. Ramos 1, 2 . 1 Biochemistry and Molecular Biology, University of Louisville,
Louisville, KY and 2 Center for Genetics and Molecular Medicine, University of
Louisville, Louisville, KY.
Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic
silencing during early embryonic development and remain inactive in most
differentiated cells and tissues via methylation of DNA CpG islands present on
their promoter. Conversely, both embryonic stem cells and tumor cells show
marked DNA hypomethylation and elevated expression of L1 elements. Although
L1 methylation is regulated by DNA methyltransferases, little is known about the
transcription factors and mechanisms that regulate L1 expression in the course of
development, differentiation, and disease. Recent findings in this laboratory have
shown that Rb family members play a role in L1 epigenetic regulation, suggesting
that L1 activity is mediated by mechanisms beyond DNA methylation. We sought
to study the epigenetic changes that occur at the L1 5’UTR element in HeLa cells
exposed to B(a)P, an environmental carcinogen and L1 activator. ChIP assays on
the L1 5’UTR demonstrated that (i) short-term exposure to B(a)P increases histone
H3 transcriptional activation marks K9-acetyl and K4-trimethyl, (ii) long-term exposure
to B(a)P reduces the recruitment of both DNMT1 and MBD2, but not
MBD3a proteins, indicating L1 activation by B(a)P. Bisulphite analysis and
DNMT siRNA knockdown studies showed that long-term, but not short-term exposure
to B(a)P decreases the methylation levels on the L1 promoter at two different
B(a)P-targeted CpG loci.
Thus, the initial control of L1 expression upon B(a)P exposure is epigenetically regulated
via histone changes, while the secondary response is mediated by changes in
recruitment of both DNMT1 and MBD2 as well as decreased CpG methylation
levels. Overall, L1 is subject to complex regulatory mechanisms involving covalent
histone and DNA modifications and recruitment of transcription factors to the L1
promoter.
151 BISPHENOL A UNLOCKS THE RAT PDE4D4
PROMOTER VIA EPIGENETICS.
W. Tang and S. Ho. Environmental Health, University of Cincinnati College of
Medicine, Cincinnati, OH.
Bisphenol A (BPA), a mimic of estrogen, is now used in the manufacture of polycarbonate
plastics and epoxy resins in a variety of consumer products. The leakage
of BPA from these products makes the xenoestrogen found ubiquitously in the environment
and in human bodily fluids and tissues. Its estrogenic effects suggest it
can reprogram developing human and animal tissues, particularly those sensitive to
estrogens, like the prostate. We previously demonstrated that a CpG island in the
phosphodiesterase type 4 variant 4 (PDE4D4) promoter was demethylated in rat
prostates neonatally exposed to environmental low dose of BPA or estradiol-17beta
(E2) when compared to ones in oil-treated controls. Here, we investigate the underpinning
mechanism of BPA- or E2-induced hypomethylation of PDE4D4 promoter
coupled with aberrant over-expression of the gene in an immortalized normal
prostatic epithelial cell line (NbE-1). Results illustrated notable difference
between the epigenetic regulation of PDE4D4 by E2 versus by BPA. While E2 primarily
signaled via a nuclear receptor mechanism, BPA induced PDE4D4 transcription
and promoter demethylation via multiple pathways. Thus, its action was
blocked by ICI182780, Erk1/2 inhibitor, U0126, and DNA methylation substrate,
SAM. Furthermore, the impact of BPA was found to associate with the up-regulation
of MBD2 and down-regulation of DNMT3A. Reversal of promoter demethylation
and gene transcription of PDE4D4 occurred if antisense MBD2 DNA
oligonucleotides or DNMT3A siRNA were applied. Collectively, these findings
support our hypothesis that estrogen mimics epigenetically reprogrammed key prostatic
growth regulatory genes in a manner different from E2 through the involvement
of genomic and non-genomic estrogen action and recruitment of DNA
methyltransferases and methyl-CpG binding domains.
Research support: ES016817, ES006096, ES015584, ES018758, and ES018789.
152 REACTIVATION OF THE EPIGENETICALLY SILENCED
TUMOR SUPPRESSOR GENE - TISSUE FACTOR
PATHWAY INHIBITOR-2 (TFPI-2) BY CURCUMIN
CAUSES CELL DEATH IN HEPATOCELLULAR
CARCINOMA CELLS.
A. Moghe 1 , A. S. Barve 3 , S. Ghare 2 , L. Gobejishvili 2 , S. Joshi-Barve 2 , C. J.
McClain 2, 1 and S. Barve 2, 1 . 1 Pharmacology and Toxicology, University of Louisville,
Louisville, KY, 2 Internal Medicine, University of Louisville, Louisville, KY and
3
University of Louisville, Louisville, KY.
Hepatocellular carcinoma (HCC) is considered the fifth most common cancer
worldwide and the third most fatal, with a rising incidence in the US as the result
of an increase in alcoholic liver disease and obesity. Curcumin, a phenolic compound
from the rhizome of the plant Curcuma longa has been shown to inhibit
growth and induce cell death in various types of cancer cells including HCC.
However, the anti-HCC mode of action of curcumin has not yet been elucidated.
In HCC, aberrant promoter methylation and histone deacetylation are implicated
in the inactivation of tumor suppressor genes which has a significant impact on carcinogenesis.
Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease
inhibitor, is a tumor suppressor gene that is frequently epigenetically silenced
in human HCC and HCC cell lines. Restoration of TFPI-2 expression in tumor tissue
has been shown to not only inhibit invasion, tumor growth, metastasis and angiogenesis
but also induce apoptosis. Hence, the goal of this study was to examine
the ability of curcumin to reverse the epigenetic silencing of TFPI-2 expression in
the HCC cell line HepG2. HepG2 cells were treated with demethylating agent 5-
Azacytidine (5-Aza), histone deacetylase inhibitor trichostatin A (TSA) and curcumin.
Similar to 5-Aza and TSA, curcumin was able to significantly reactivate
TFPI-2 gene expression in a dose dependent (10 to 50 μM) manner. Further, curcumin
in combination with either 5-Aza or TSA was able to robustly re-establish
TFPI-2 expression. These data strongly suggest that curcumin can have a therapeutic
role in the treatment of HCC by reversing the epigenetic alterations involving
promoter hypermethylation and histone deacetylation and reactivating the expression
of the silenced tumor suppressor gene -TFPI-2.
153 HYPOXIA INDUCES TRI-METHYLATED H3 LYSINE 4
BY INHIBITION OF JARID1A DEMETHYLASE.
X. Zhou 1 , H. Sun 1 , H. Chen 1 , J. Zavadil 2 , T. Kluz 1 and M. Costa 1 .
1
Department of Environmental Medicine, Nelson Institute of Environmental
Medicine, New York University School of Medicine, Tuxedo, NY and 2 Department of
Pathology, New York University Cancer Institute and Center for Health Informatics
and Bioinformatics, New York University Langone Medical Center, New York, NY.
Cancer cells experience severe hypoxia, resulting from reduced oxygen supply from
blood vessels because of the rapid cell proliferation. Histone H3 Lysine 4 (H3K4)
tri-methylation at the promoter region of genes has been linked to transcriptional
activation. In the present study, we found that hypoxia (1 % oxygen) increased
H3K4 tri-methylation in both normal human bronchial epithelial Beas-2B cells
and human lung carcinoma A549 cells. Hypoxia increased H3K4 tri-methylation
by inhibiting H3K4 demethylating activity. In support of this, knocking down
JARID1A, which is the major demethylase in Beas-2B cells, attenuated the increase
of H3K4 tri-methylation induced by hypoxia. However, the mRNA and protein
levels of JARID1A were not affected by hypoxia. GeneChip and pathway analysis
in JARID1A knockdown Beas-2B cells revealed that JARID1A regulates the expression
of hundreds of genes involved in different cellular functions, including tu-
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