The Toxicologist - Society of Toxicology

arbecued pork and beef meat, contributing to a substantial dietary intake of this
toxicant. Studies conducted in our lab have shown that BaP contributes to colon
cancer development in animal models. Through metabolic activation, BaP is biotransformed
into reactive metabolites that bind with DNA, cause DNA damage
and associated changes in cell cycle. The purpose of this study was to determine the
concentrations of BaP that are most toxic to HT-29 human colon cells. Cells were
cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12
(DMEM/F12) supplemented with 10% FBS and antibiotics (penicillin/streptomycin).
After revival of cells and growing them to confluence, cells were synchronized
overnight by serum starvation method. The HT-29 cells were exposed to 1, 5,
10, 25, 50, and 100μM BaP over the course of 10 days and then counted using a
Coulter Counter. Based on a concentration – response study 5, 10, and 25μM BaP
in DMSO (vehicle for BaP; 0.01%); 4 days after exposure were selected as optimal
concentrations to analyze the effect of BaP on cell growth, viability, biochemical,
and molecular endpoints. The expression of both Phase I and II drug metabolizing
enzymes were analyzed by western blot and RT-PCR. Analysis of cell cycle via
FACS showed a distinct inhibition of the S and G2-phase in cells treated with increasing
concentrations of BaP. An increase in CYP 1A1 and 1B1 expression also
occurred in a BaP concentration-dependent manner. On the other hand, not much
difference was seen in Phase II metabolizing enzyme expression levels regardless of
BaP concentrations. Our results suggest that BaP cytotoxicity is controlled by metabolic
activation. This study was funded by the NIH grants 5T32HL007735-12,
1RO1CA142845-01A1, 1RO3CA130112-01, 1S11ES014156-01A1 and
Southern Regional Education Board.
2649 EFFECTS OF SUBCHRONIC SODIUM ARSENITE ON
MALIGNANT TRANSFORMATION MARKERS IN MCF-
7 CELLS.
A. D. Rios-Perez 1 , R. Ruiz-Ramos 1 , L. T. Lopez-Carrillo 2 and M. E. Cebrian 1 .
1 Toxicology, CINVESTAV, Mexico City, DF, Mexico and 2 CISP, INSP, Cuernavaca,
Morelos, Mexico.
Inorganic arsenic (iAs) is a well-known human carcinogen, however its mechanisms
still require further investigation. We have reported that arsenite induced cell proliferation,
increased S-phase cell recruitment, and ROS generation in MCF-7 cells,
promoting genomic instability by damaging DNA and inducing oncogene expression
and cell cycle regulatory proteins. We also showed induced protein levels of
metallothioneins 1 and 2, PCNA and p53 in the presence of decreased p21 levels,
all of which contributed to explain cell cycle disruption. Regarding other potential
mechanisms of As-induced cancer, S100 proteins are small molecules of the EF
hand type, which have been functionally linked with cell growth, motility, invasiveness,
matrix metalloprotease (MMP) overexpression and alterations in p53 activity.
Furthermore, alterations in S100 expression have been associated with gastric, colorectal,
lung and breast cancer, and have been proposed as staging and prognostic
tools. The aim of this study was to evaluate the effect of sodium arsenite over
S100A4/7 expression and its relationship with MMP9/13. Cells exposed to arsenic
(0-10 uM during 0-8 h) showed increased S phase recruitment, as evaluated by
FACS. In addition, treated cells also showed S100A4, S100A7 gene overexpression,
and increased protein levels, as analyzed by PCR-RT and Western Blot. In addition,
cells subchronically exposed to iAs (0.25 uM; 8 weeks) showed S100A7, MMP9
and MMP13, gene overexpression. Only S100A7 was down regulated at 1 uM,
while S100A4 was downregulated at 0.25 and 1 uM. These findings suggest that arsenic
is able to deregulate S100A7 and MMP9/13 gene expression and their potential
relationship. Further studies are required on the mechanisms underlying a potential
malignant transformation induced by As in MCF-7.
2650 DOES HELICOBACTER PYLORI PARTICIPATE IN T
HELPER-1 MEDIATED IMMUNE ACTIVATION IN
COLORECTAL CANCER PATIENTS?
B. A. Engin 1 , A. Karakaya 1 and A. Engin 2 . 1 Department of Toxicology, Gazi
University, Faculty of Pharmacy, Ankara, Turkey and 2 Department of General Surgery,
Gazi University, Faculty of Medicine, Ankara, Turkey.
Controversial findings are present on the association between increased risk of colorectal
carcinoma and chronic infection. Colonization of Helicobacter pylori
(H.pylori) increases risk of the development of colorectal cancer by 2.2 fold.
However, it is not clear whether the H.pylori stimulate the cell-mediated immunity
or tryptophan degredation pathway of cancer patients. The aim of this study was to
evaluate the cellular immune activation by measuring serum neopterin and estimate
T-cell responsiveness by calculation of kynurenine to tryptophan ratio. Thus, in this
study, ninety seven patients (19 were H.pylori positive and 78 were H.pylori negative)
with colorectal cancer diagnosis conducted as cancer-group. One hundred
eight cancer-free individuals (37 were H.pylori positive and 71 were H.pylori negative)
were constituted the control group. H.pylori IgG seropositivity was detected
568 SOT 2011 ANNUAL MEETING
by enzyme linked immunosorbent assay (ELISA). Serum tryptophan and
kynurenin were measured by high performance liquid chromatography.
Indolamine-2,3-dioxygenase (IDO) activity was calculated by kynurenine/tryptophan.
Serum neopterin was detected by ELISA. The measured serum parameters of
H.pylori negative control group compared to the positive control group were not
significantly different (p>0.05). There was an increased cellular immune activation
in H.pylori negative colorectal cancer patients compared to the H.pylori negative
controls, as well as in H.pylori positive cancer patients compared to H.pylori positive
controls (both, p0,05). However, colorectal
cancer causes the stimulation of macrophages and increases the kynurenine synthesis
independent of the H.pylori existence.
2651 TOXICOGENOMIC PROFILING OF FORMALDEHYDE-
EXPOSED NORMAL AND TRANSFORMED HUMAN
ORAL KERATINOCYTES.
R. Ceder 1 , M. Merne 1 , J. Nilsson 1 , C. Staab 2 , J. Höög 2 , C. M. Thompson 3 and
R. C. Grafström 1, 4 . 1 Institute of Environmental Medicine, Karolinska Institutet,
Stockholm, Sweden, 2 Department of Medical Biochemistry and Biophysics, Karolinska
Institutet, Stockholm, Sweden, 3 ToxStrategies, Inc., Katy, TX and 4 Medical
Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland.
Toxicogenomic assessments in vitro might serve to generate novel toxicity biomarkers
and support the replacement of animals in toxicity testing. Toxicity of formaldehyde
(FA), a classified human carcinogen, was studied in a cell culture model for
cancer development that included normal (NOK), immortalized (SVpgC2a) and
malignant (SqCC/Y1) cells. Repeated 1h FA exposures of SVpgC2a induced transformation
as indicated from cell crisis and the generation of a novel line (SVpgC3a)
with altered morphology (e.g., multifocal, soft agar growth, but non-tumorigenic
in athymic nude mice). Assessment of cytotoxicity (MMT method) and genotoxicity
(DNA protein crosslinks) indicated similar initial damage levels in all lines
whereas the longer-term consequences differed (cell number and cloning assessments
indicated a sensitivity order of NOK>SVpgC2a>SVpgC3a≈SqCC/Y1). FA
induced cell death primarily by terminal differentiation in NOK whereas differently
SVpgC2a, SVpgC3a and SqCC/Y1 died by other means e.g., apoptosis.
Interestingly, over a dose range, the highest levels of micronuclei in SVpgC2a were
observed at the FA concentration that induced cell transformation. Proteomics and
transcriptomics profiling of SVpgC3a versus SVpgC2a showed that cell transformation
coupled with multiple changes in single genes, molecular networks and
gene ontologies. Likewise, transcript profiling of the respective cell lines for up to
48h following 1h FA exposure indicated multiple genomic changes, some of which
overlapped to results from cancer-inducing protocols in vivo. In conclusion, the
sensitivity to formaldehyde toxicity might differ between stages in cancer development,
and specific gene expression changes couple to the respective phenotypes.
Generation of the SVpgC3a line extended the current cancer model of normal, immortal
and malignant cells.
2652 A GENOME-WIDE CELL SIGNALING RNA-
INTERFERENCE SCREEN TO IDENTIFY NOVEL
REGULATORS OF THE DNA DAMAGE RESPONSE.
B. van de Water 1 , J. Carreras 1 , L. von Stechow 1 , R. Sidappa 1 , A. Pines 2 , J.
Olsen 3 , H. Vrieling 2 , L. Mullenders 2 and E. Danen 1 . 1 Division of Toxicology,
Leiden University, Leiden, Netherlands, 2 Department of Toxicogenetics, Leiden
University Medical Center, Leiden, Netherlands and 3 Panum Institute, University of
Copenhagen, Copenhagen, Denmark.
The DNA damage response (DDR) involves a complex of signaling events that participate
in lesion recognition, cell cycle arrest, damage repair, and ultimately cell
cycle re-entry or, if repair fails, initiation of cell death. The DDR is critical for
maintenance of genomic integrity and its failure is associated cancer. In a highly
simplified model DNA-damage causes activation of the sensor kinases (e.g. ATM,
ATR, DNA-PK) that activate p53 and the checkpoint kinases (i.e. Chk1, Chk2),
which in turn regulate effector pathways that the above programs. In reality, the
process is much more complex with many additional regulators, extensive crosstalk,
and multiple positive and negative feedback loops. We have taken a “systems
approach” to map the DDR signal transduction network in more detail. For this we
used embryonic stem cells treated with the DNA cross-linking agent cisplatin and
systematically determined the differential changes at the transcriptome and (phospho)-proteome
level. These findings were combined with high-throughput RNAinterference-based
functional genomics approaching using genome wide siRNA li-