The Toxicologist - Society of Toxicology

109 CD4+ T CELL INTRINSIC TNF RECEPTOR 2
SIGNALING: AN EPIGENETIC TARGET FOR
TRANSCRIPTIONAL REGULATION OF IL-2?
J. K. Dey 4 , R. S. Schondelmeyer 3 , E. N. Sellers 3 , P. G. Miller 3 , G. Lin 5 , M. B.
Bonn 4 and S. C. McKarns 1, 2 . 1 Surgery, University of Missouri School of Medicine,
Columbia, MO, 2 Molecular Microbiology & Immunology, University of Missouri
School of Medicine, Columbia, MO, 3 Biochemistry, University of Missouri,
Columbia, MO, 4 Biological Sciences, University of Missouri, Columbia, MO and
5 Biological Engineering, University of Missouri, Columbia, MO.
Interleukin (IL)-2 is a key cytokine with pivotal pleotropic regulatory roles in maintaining
immune homeostasis and peripheral tolerance. Thus, a mechanistic understanding
of IL-2 production is central to our understanding autoimmunity, chronic
inflammatory disorders, and cancer. Histone modifications, DNA cytosine methylation,
and transcription factor occupancy regulate chromatin remodeling and promoter
activity of the IL-2 gene in CD4+ T cells. We have previously demonstrated
that tumor necrosis factor-alpha (TNF)receptor 1 (TNFR1) and TNF receptor 2
(TNFR2) double knockout mice produce less IL-2, and this associates with reduced
c-Rel binding to the proximal IL-2 enhancer/promoter. TNF has dual proinflammatory
and immunoregulatory properties with key physiological and pathological
regulatory roles, and the differential usage of its receptors has been proposed
to contribute to its opposing biological activities. The objective of this study was to
define the TNF receptor(s) responsible for modulating IL-2 production. CD4+ T
cells from TNFR1, TNFR2, and TNFR1/2 double knockout IL-2 GFP knock-in
mice were stimulated and compared with control CD4+ T cells from wild-type
B10.A mice. The frequency of IL-2 producers and a delay in the onset of IL-2 transcription
was observed in the TNFR2 and TNFR1/2-deficient T cells only.
Moreover, stimulation of purified CD4+ T cells with plate-bound anti-CD3 and
anti-CD28 demonstrated a T cell intrinsic regulatory role for TNFR2 on IL-2 promoter
activity. Given that c-Rel is essential for chromatin remodeling of the IL-2
enhancer/promoter, our results suggest that TNFR2 signaling may facilitate epigenetic
regulation of cytokine production.
110 EPIGENETIC MODULATION OF XENOESTROGEN-
INDUCED BREAST CANCER.
L. Wang, W. Jiang, B. Moorthy and S. R. Kondraganti. Department of Pediatrics,
Baylor College of Medicine, Houston, TX.
Dietary phytochemicals inhibit cancer formation by multiple mechanisms. These
mechanisms include epigenetic changes resulting from the inhibition of histone
deacetylase (HDAC) activity. HDACs affect histone acetylation status and results
in cell cycle arrest and/or apoptosis. Targeting the epigenome, including the use of
histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention.
Sulforaphane is an isothiocyanate found in cruciferous vegetables, such as
broccoli and broccoli sprouts. Exposure to xenoestrogens leads to adverse health effects
and have a potential effect on oncogenes, specifically in relation to breast cancer.
3-Methyl-cholanthrene (3-MC) represents a novel subclass of xenoestrogenic
compounds that activates both ER-α and AhR signaling pathways. In the present
study, we investigated the role of sulforaphane in inhibition of xenoestrogen induced
cancer formation. Growing evidence show that sulforaphane acts through
different chemoprotective mechanisms. To understand the anticarcinogenic potency
of sulforaphane, we studied induction of phase 2 detoxification enzymes
along with its ability in inhibition of HDAC activity. The wild type (C57BL/6J) female
mice were exposed to 3-MC and sulforaphane, alone and in combination.
Significant reduction of phase I enzymes along with elevation of several phase II enzymes
were observed in hepatic and mammary tissues of female mice treated with
sulforaphane. Thus, sulforaphane could modulate the Phase I/Phase II enzymatic
systems, a critical mechanism involved in the formation of cancers. Our results also
show the differential expression of receptors on exposure to environmental estrogens.
Our studies show that sulforaphane acted as an HDAC inhibitor in the xenoestrogen
exposed rodent hepatic and mammary tissues. This results in enhanced
histone acetylation, derepression of P21 and Bax, leading to cancer prevention. Our
results show the ability of dietary phytochemicals to reactivate epigenetically-silenced
genes in cancer cells and thus have important implications as epigenetic
modulators in cancer prevention and therapy.
111 THE ROLE OF NICOTINE IN THE INDUCTION OF
GENOMIC INSTABILITY IN BREAST CELLS.
C. L. Clemens and J. W. DuMond. Environmental Science and Technology, Texas
Southern University, Houston, TX.
Studies in the United States have indicated that active and passive smoking are associated
with increased breast cancer risk. However, there is little direct evidence of
a direct effect of environmental tobacco smoke in breast carcinogenesis. This study
examines the effects of nicotine on the proliferation, gene regulation and ability to
induce oxidative damage in MCF-7 breast cancer cells, as we postulate here that
nicotine can produce genomic instability in breast cancer cells. Cell proliferation
studies were preformed on MCF-7, a human breast cancer cell line. Cell proliferation
studies were performed at concentrations of 1ng/ml, 10ng/ml, and 100pg/ml
of nicotine hydrogen tartrate salt (purity, ≥98%). Cells were counted after 72 hours
of exposure to either treatment or control. The expression of critical DNA repair
and DNA damage genes was analyzed by Real-Time Reverse Transcriptase
Polymerase Chain Reaction after acute exposure to nicotine. Increased oxidative
stress was measured by Trevigen’s Fragment Length Analysis using Repair Enzymes
(FLARE®) assay. Cell proliferation studies showed a significant increase in cell
growth at 10ng/ml (25%) and at 100 pg/ml (20%) when compared to control cells.
RT-PCR showed a down regulation in gene expression of important regulatory
genes, such BRCA1, EXO1, PRKDC, and other DNA damage repair genes such
as; MRE11A, N4BP2, NBN, PCNA, PMS2, RAD17, and XPA. Exposure also resulted
in an up regulation of gene expression in important cell regulating genes;
DDB1, MAPK12, GADD45G and PPP1R15A, IGHMBP2, TP73 and PCBP4,
and LIG1 up regulation were also detected. Other DNA damage repair genes such
as; BTG2, CIDEA, ERCC2, FANCG, FEN1, MPG, PPP1R15A, SEMA4A,
TREX1, XRCC1, and XRCC3 also had a significant up regulation. Oxidative damage
repair genes such as MUTYH, OGG1 and NTHL1 were significantly up regulated
leading to the use of the FLARE assay using the Fpg enzyme to further confirm
oxidative damaged caused by nicotine. The results of the FLARE assay
confirmed that Nicotine (10 ng/ml) exposure increases oxidative damage in MCF-
7 cells.
112 INDUCTION OF BASE EXCISION REPAIR ENZYMES
APE1 AND NTH1 IN RAT SPLEEN FOLLOWING
ANILINE EXPOSURE.
H. Ma, J. Wang and M. Khan. Pathology, University of Texas Medical Branch,
Galveston, TX.
Mechanisms by which aniline exposure elicits splenotoxic response, especially the
tumorigenic response, are not well-understood. Earlier, we have shown that aniline
exposure leads to oxidative DNA damage and up-regulation of OGG1 and
NEIL1/2 DNA glycosylases in rat spleen. However, contribution of
apurinic/apyrimidinic endonuclease 1 (APE1) which plays a central role in the repair
of oxidized and alkylated bases and endonuclease III homolog 1 (NTH1)
which is capable of removing oxidized pyrimidines, such as thymine glycol (Tg)
from DNA via the base excision repair (BER) pathway, in the repair of aniline-induced
oxidative DNA damage in the spleen is not known. This study was, therefore,
focused on examining if APE1 and NTH1 also contribute to the repair of oxidative
DNA lesions in the spleen, in an experimental condition preceding a
tumorigenic response. To achieve this, male SD rats were subchronically exposed to
aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received
drinking water only. By quantitating the cleavage products, the BER activity of
APE1 and NTH1 were assayed using substrates containing tetrahydrofuran and Tg,
respectively. Aniline treatment led to significant increases in APE1- and NTH1-associated
BER activity in the nuclear extracts of spleen compared to the controls.
Western blot analysis showed that protein expression of APE1 and NTH1 in the
nuclear extracts of spleen from aniline-treated rats was 2.7- and 1.9-fold higher
than controls, respectively. Likewise, real-time PCR analysis for APE1 and NTH1
mRNA expression in the spleen showed 3.2- and 2.9-fold increases, respectively, in
aniline-treated rats compared to controls. Aniline treatment also led to stronger immunoreactivity
for both APE1 and NTH1 in the spleens, confined to the red pulp
areas. These results, thus, show that aniline exposure is associated with an induction
of APE1 and NTH1 in the spleen. The increased repair activity of APE1 and
NTH1 could be an important mechanism for the removal of oxidative DNA lesions.
Supported by NIH ES06476.
113 ENHANCED NUCLEOTIDE EXCISION REPAIR OF
COMBINED 17ALPHA-ETHINYLESTRADIOL AND UV
IN ZEBRAFISH LIVER CELLS.
S. Tang 1 , E. G. Notch 2 , V. Allagadda 1 and G. D. Mayer 1 . 1 The Institute of
Environmental and Human Health, Texas Tech University, Lubbock, TX and
2 Department of Physiology, Dartmouth Medical School, Hanover, NH.
Ultraviolet (UV) radiation damages cellular molecules, and has been indicated to
induce nucleotide excision repair (NER) to remove DNA lesions including cyclobutane
pyrimidine dimmers (CPDs). Recent studies suggest that exposure to
17alpha-Ethinylestradiol (EE2) decreases mRNA abundance of key NER genes
(XPC and XPA) and inhibits cellular NER repair capacity in zebrafish (Danio rerio)
liver (ZFL) cells. However, the impact of co-exposure of UV light and EE2 on NER
is still unknown. The aim of this study was to evaluate the combined effects of EE2
SOT 2011 ANNUAL MEETING 23