The Toxicologist - Society of Toxicology

247 CO-CARCINOGENESIS OF N, N-
DIETHYLNITROSAMINE AND CARBON
TETRACHLORIDE IN MOUSE LIVER.
G. Ainslie 1 , T. Uehara 1 , O. Kosyk 1 , B. Bradford 1 , G. Boorman 2 and I. Rusyn 1 .
1 Department of Environmental Sciences and Engineering, University of North
Carolina Chapel Hill, Chapel Hill, NC and 2 Covance, Princeton, NJ.
Background: Liver fibrosis and cirrhosis are well known precursor liver disease
states for hepatocellular carcinoma (HCC) in humans. In rodent 2-year bioassays,
however, liver tumors arise on a normal liver which is one of the factors limiting
their utility to predict human HCC. While several animal models have been used
to examine the mechanisms involved in liver fibrosis, the promoting effect by fibrosis
in the process of hepato-carcinogenesis is less understood. This study aimed to
examine the mechanisms by which fibrosis is involved in tumor promotion in the
liver. Methods: Male B6C3F1 mice were treated with a single dose (1 mg/kg) of
N,N-diethylnitrosamine (DEN) or PBS at 15 days of age. Carbon tetrachloride
(CCl4) treatment (0.2 ml/kg; i.p.; twice a week) was started at 8 weeks of age and
continued for 9 or 14 weeks. Results: DEN-treatment alone led to a time-dependent
increase in the incidence of hepatocellular altered foci (HAF, 80 and 95%).
CCl4-treatment alone induced a small number of HAF and HCC only at the later
time point (12.5 and 37.5%, respectively). CCl4-treatment after DEN-initiation
had a significant effect at each time point on both incidence (40 and 100%) and
multiplicity of neoplastic lesions in the liver. Severe liver fibrosis in CCl4-treated
animals was confirmed by Masson’s trichrome stain and an increase in TGFbeta expression.
Cell proliferation, assessed by BrdU immunohistochemistry was dramatically
elevated in all CCl4-treated groups. Liver inflammation was elevated concomitantly
with the fibrotic changes and DNA repair genes were dys-regulated in
both time points. Conclusions: This study provides strong evidence that progressive
liver fibrosis is acting as tumor promoter after DEN-initiation, a mechanism which
is highly relevant to the development of human HCC. Further studies with this
model will enable evaluation of the mechanisms of human liver carcinogenesis.
248 KNOCK DOWN OF AHR IN HUMAN BREAST CANCER
CELL LINE UPREGULATES THE EXPRESSION OF PRO-
APOPTOTIC FACTOR BAK.
G. Goode and S. E. Eltom. Biochemistry & Cancer Biology, Meharry Medical
College, Nashville, TN.
AhR is overexpressed and constitutively activated in several human breast carcinoma
cell lines and the expression levels showed strong correlation with the degree
of the tumor malignancy. The objective of this study is to examine the effect of reducing
the expression levels of AhR in metastatic human breast carcinoma (HBC)
cell lines on their tumorigenic properties and the underlying molecular targets. We
designed a series of short hairpin RNA (shRNA) targeting AhR and cloned them in
pSuper retroviral vectors, and viruses expressing shRNA were used to infect HBC
cell line MDA-MB231 to generate cell lines that permanently express siRNA targeting
AhR. Clonal cell lines with 50-90% AhR knockdown (AhR-KD) were characterized
in vitro for their tumorigenic properties in comparison to the scramble
RNA-expressing control cell lines. RNA and protein extracts from these cell lines
were compared for their gene and protein expression for markers responsible for
modified phenotypes. AhR-KD cells exhibited prolonged doubling times and decreased
cell proliferation. Cell cycle analysis by FACS showed a decreased transition
of these cells from G1 to S and G2/M phases of cell cycle. Most significantly, the
AhR-KD clones of MDA-MB231 cell line which is characterized by fibroblastic
morphology have reverted to their epithelial morphology, indicating a role for AhR
in regulating the epithelial to mesenchymal transition (EMT). Real time RT-PCR
and Western blotting analyses reveals a dramatic upregulation of BAK a pro-apoptotic
factor in AhR-KD cells, at mRNA and protein levels. These results demonstrate
a major role for the AhR in regulating tumorigenic phenotypes of metastatic
breast cancer and promoting cell survival by inhibiting apoptosis. Findings will establish
the potential of AhR as a target for therapeutic intervention of metastatic
breast cancer.
249 A NOVEL SMALL MOLECULE INHIBITOR OF
WNT/β-CATENIN SIGNAL PATHWAY INDUCES CELL
CYCLE ARREST IN HUMAN NON-SMALL LUNG
CANCER CELLS.
J. Choi 1 , S. Lee 1 , Y. Gong 2 , Y. Park 1 and M. Dong 1 . 1 School Lifesciences and
Biotechnology, Korea University, Seoul, Republic of Korea and 2 Department of
Chemistry, Dongguk University, Seoul, Republic of Korea. Sponsor: B. Lee.
Aberrant activation of the Wnt/β-catenin signal pathway is linked to a high frequency
of numerous tumors. In previous study, we screened the chemical library to
develop the new drug for lung cancer therapy as a target of Wnt2/β-catenin path-
way using a cell-based assay in A549/Wnt2 cells stably expressed wnt2. We chose
one compound, KROR-759, which was reduced to below 50% of the luciferase activity
at the cell proliferation IC50 concentration was taken. 759 reduced βcatenin-TCF/LEF
dependent transcriptional activity in a dose-dependent manner
with an IC50 value of about 2μM. To further address the action mechanism of
KROR-759, we performed DNA microarray analysis by using Illumina BeadArray
Technology, after treatment of A549 cells with 1uM KROR-759 for 24h. The data
demonstrated that KROR-759 had a very selective effect on global gene transcription.
KROR-759 was down-regulated the expressions of Wnt/β-catenin signaling
pathway target genes, such as E2F, cyclin D1 and mini-chromosome maintenance
(MCM), which play critical roles in cell growth, proliferation, and differentiation.
KROR-759 induced accumulation of sub-G1 phase and arrests in the G1 phase of
the cell cycle. Induction of G1 arrest by KROR-759 was correlated with inhibition
of E2F, cyclin D1 and MCM expression. Moreover, mini-chromosome maintenance
(MCM) proteins were markedly down-regulated in A549 cells treated with
KROR-759. Collectively, these results suggested that KROR-759 possessed a potential
anti-cancer activity against lung cancer cells by inhibiting their proliferation
and inducing cell cycle G1 arrest.
250 ARISTOLOCHIC ACID NEPHROPATHY: AN
ENVIRONMENTAL AND IATROGENIC DISEASE.
A. Grollman 1 , B. Jelakovic 2 , Y. Pu 3 and C. Chen 3 . 1 State University of New York
at Stony Brook, Stony Brook, NY, 2 University of Zagreb, Zagreb, Croatia and
3 National Taiwan University, Taiwan, China. Sponsor: J. Swenberg.
Aristolochic acid (AA), a principal component of Aristolochia species, was shown
recently to be the toxin responsible for the clinical syndromes known as Chinese
herb nephropathy and endemic (Balkan) nephropathy (EN). Both disorders are associated
with a high incidence of upper urinary tract urothelial carcinomas (UUC)
and appear to constitute a single disease entity, designated aristolochic acid
nephropathy (AAN). Based on the traditional use of Aristolochia in herbal remedies,
we posited that AAN represents a long-overlooked iatrogenic disease and an
international public health problem of considerable magnitude (Adv Mol Tox 3,
211, 2010). Pursuing this hypothesis, we conducted studies of 100 patients with
UUC residing in Taiwan, or in regions of Croatia, Bosnia and Serbia where EN is
prevalent. DNA was obtained, with informed consent, from renal cortical and
tumor tissues following nephroureterectomy. Aristolactam (Al)-DNA adducts in
the renal cortex were quantified by 32P- post-labeling techniques. Chip-sequencing
technology was utilized to establish the pattern of mutations in the TP-53 gene. Al-
DNA adducts were detected in the majority of patients with UUC. The TP53 mutation
spectrum was dominated by A:TÇT:A transversions located almost exclusively
on the non-transcribed DNA strand, reflecting a failure to excise AL-DNA
adducts by global genomic nucleotide excision repair. This factor may account for
the remarkable persistence of these adducts in human tissues. Thus, aristolochic
acid joins aflatoxin and vinyl chloride as one the few human carcinogens with a definitive
TP53 mutational signature. Coupled with the use of AL-DNA adducts as
biomarkers, the presence of a mutational signature establishes an etiological role for
AA in UUC. Public health authorities in countries where Aristolochia has been
used are encouraged to initiate screening programs to detect UUC and to implement
measures that will reduce human exposure to this nephrotoxic and carcinogenic
herb. (Supported by NIEHS).
251 VITAMIN E DEFICIENCY IN MICE TREATED WITH
DICHLOROACETATE OR TRICHLOROACETATE
MODULATES THE BIOMARKERS OF PHAGOCYTIC
ACTIVATION AND ANTIOXIDANT ENZYME
ACTIVITIES IN THE PERITONEAL LAVAGE CELLS.
E. Hassoun and A. Al-Dieri. Pharmacology, Pharmacy, University of Toledo,
Toledo, OH.
Dichloroacetate (DCA) and trichloroacetate (TCA) are water chlorination by products.
Previous studies in our lab have indicated induction of phagocytic activation
in the peritoneal lavage cells of mice after subchronic exposure to the compounds.
To study the effects of marginal vitamin E deficiency on DCA- and TCA-induced
phagocytic activation, groups of mice were kept on either regular diet supplemented
with vitamin E (E-normal group) or low vitamin E diet (E-deficient
group). The two groups of mice were divided into subgroups and were treated daily,
by gavage, with water (control groups), 77 mg DCA/kg/day and 77mg TCA/day
for 13 weeks. Mice were killed at the end of the treatment period and peritoneal
lavage cells (PLCs) were collected and assayed for biomarkers of phagocytic activation,
including superoxide anion (SA) production and myeloperoxidase activity,
and for activities of the antioxidant enzymes superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GSH-PX). DCA and TCA, in general, induced
SOT 2011 ANNUAL MEETING 53