The Toxicologist - Society of Toxicology

aries targeting all transcription factors, protein kinases and phosophatases and
(de)-ubiquitinases. The siRNA screens resulted in novel players that control the
DDR response. We have integrated these functional critical components of the
DDR with the phospho-proteomics and transcriptomics data to generate a comprehensive
understanding of the DDR signaling network. This information not only
increases the level of understanding of the DDR program but also may identify improved
mechanism-based biomarkers for genotoxicity. In addition, these novel
DDR regulators may be future candidate drug targets for either increasing sensitivity
of otherwise drug resistant tumor cells or protect normal cells from DNA-damaging
anticancer drugs. Sponsored by the Netherlands Toxicogenomics Center
2653 GLUCOSE ENHANCES MENADIONE MEDIATED
HYDROGEN PEROXIDE FORMATION IN BETA CELLS.
M. A. Palo 1 , T. L. Womack 1 , E. Heart 2 and J. P. Gray 1, 2 . 1 Science - Chemistry,
United States Coast Guard Academy, New London, CT and 2 Biocurrents Research
Center, Marine Biological Laboratory, Woods Hole, MA.
During tumor development, cancer cells undergo dramatic changes in cellular metabolism,
including decreased mitochondrial respiration and enhanced glycolysis.
They also express elevated levels of glucose transporters to support changes in glycolysis.
Redox cycling chemicals such as menadione are enzymatically reduced by
cellular reductases and chemically reduce oxygen, resulting in the formation of reactive
oxygen species including superoxide anion and hydrogen peroxide. In the
current work, we hypothesized that redox cycling in cancer cells is glucose-dependent.
However, because cancer cells constitutively express multiple glucose transporters,
we utilized the beta cell model INS-1 832/13, which expresses a unique
glucose transporter (GLUT2) that rapidly equilibrates glucose. In these cells, menadione
was found to stimulate formation of ROS; this was concentration-dependent
in the range of 0.1-10 μM. To test the importance of glucose for chemical redox cycling,
cells were treated with 10 μM menadione and increasing concentrations of
glucose (2-16 mM). We found that the rate of hydrogen peroxide formation increased
five-fold over this concentration range. Concentrations of glucose inducing
maximal release of hydrogen peroxide directly correlated with maximal insulin release
from these cells. The non-metabolizable sugar derivative 2-deoxy-D-glucose
did not support increases in hydrogen peroxide formation suggesting that glucose
metabolism is required for the generation of ROS. Taken together, these data suggest
that INS-1 832/13 cells are a useful model for investigating the interaction of
glucose metabolism and redox cycling. The glucose-dependent formation of reducing
agents supports chemical redox cycling, and the overexpression of glucose transporters
in cancer cells might be exploited for chemotherapeutic design.
2654 THE DURATION OF THE EVALUATION PERIOD OF
THE POSITIVE CONTROL GROUP FOR RASH2
SHORT-TERM CARCINOGENICITY STUDIES.
S. A. Shah, M. A. Paranjpe and E. A. Zahalka. Mammalian Toxicology, BioReliance
Corporation, Rockville, MD.
Short-term (26-week) transgenic mouse studies have been accepted by the regulatory
agencies as an alternative model in place of the conventional 2-year mouse carcinogenicity
models, for the last 14 years. In these studies a positive control group
is used to demonstrate the validity of the test system’s susceptibility to carcinogenicity
and to ensure the use of the proper transgenic mouse strain. The duration
period of the observations following treatment is currently set at 17 weeks in our
study protocols. At the end of this period mice are evaluated macro- and microscopically.
Shortening this evaluation period without impacting the objectives of
the positive control group in these studies is of considerable interest, especially from
an animal welfare perspective. Accordingly, we have designed a study to address this
issue using the RasH2 model since it is currently the most widely used model in the
industry. The study included five groups treated with Urethane (1000 mg/kg), and
one control group (saline). Each group consisted of 10mice/sex. Mice were administered
a total of 3 intraperitoneal injections of Urethane (1000 mg/kg) on study
days 1, 3, and 5. Animals were evaluated macroscopically on weeks 8, 10, 12, 14 or
16 following treatment. All animals treated with Urethane responded positively to
the treatment, as was evident by post-dose clinical observations (i.e. ataxia, decreased
motor activity, prostrate positioning, labored/rapid/shallow breathing).
Macroscopic observations at necropsy revealed that control animals were tumor free
at 16 weeks, while urethane-treated mice showed tumors in the lungs and spleen as
early as 10 weeks post treatment. At 8 weeks post treatment only the lung responded
positively. In conclusion, we have demonstrated that it may not be necessary
to retain the positive control animals on study beyond week 10 for scoring tumors.
Ongoing microscopic evaluations will enable confirmation of these findings.
2655 CYTOTOXICITY OF ARSENICALS ON HUMAN
BRONCHIAL EPITHELIAL CELLS IN VITRO.
L. Arnold, S. Kakiuchi-Kiyota, K. L. Pennington and S. M. Cohen. University of
Nebraska Medical Center, Omaha, NE.
Inorganic arsenic [arsenate (As V ) and arsenite (As III )] is a known human carcinogen,
inducing tumors of the skin, urinary bladder and lung in individuals exposed to
high concentrations, primarily through drinking water. The mechanism by which
inorganic arsenic induces tumors is being studied. In vivo, we have shown that inorganic
arsenic induces cytotoxicity with increased cell proliferation and regenerative
hyperplasia in the urinary bladder of rats and mice. In vitro, the trivalent arsenicals
As III , monomethylarsenous acid (MMA III ) and dimethylarsinous acid
(DMA III ) are highly cytotoxic to human and rat urothelial cells compared to the
corresponding pentavalent forms. In addition, we have shown that the pentavalent
thio-metabolite, dimethylmonothioarsinic acid (DMMTA V ) is cytotoxic to rat and
human urothelial cells at concentrations similar to those of the trivalent arsenicals.
In this study, we examined the cytotoxicities of various arsenicals on human
bronchial epithelial cells (HBEC) isolated from bronchial brush biopsies. The trivalent
arsenicals and DMMTA V were cytotoxic at micromolar concentrations (As III -
5.8 μM, MMA III -1.0 μM, DMA III -1.4 μM, DMMTA V -5.5 μM). As V was also cytotoxic
at micromolar concentrations, but at a much higher level compared to As III
(As V -46.5 μM). The pentavalent organic arsenicals monomethylarsonic acid
(MMA V ) and dimethylarsinic acid (DMA V ) were cytotoxic at millimolar concentrations
(MMA V -6.1 mM, DMA V -1.2 mM). These results show that arsenicals are
cytotoxic to human bronchial epithelial cells at concentrations similar to those that
induce cytotoxicity in human and rat urothelial cells.
2656 FORMATION OF TAMOXIFEN (TAM)-DNA ADDUCTS
IN MONKEYS AND HUMANS.
E. Hernandez Ramon 1 , K. John 1 , R. A. Woodward 2 and M. C. Poirier 1 .
1 NCI/NIH, Bethesda, MD and 2 NICHD/NIH, Poolesville, MD.
Tamoxifen (TAM) is currently given to thousands of women and is a highly-successful
adjuvant therapy for estrogen-receptor-positive breast cancer. However
women receiving TAM, also have an increased risk of endometrial and myometrial
cancer, which may be due to genotoxicity or may result from receptor-related
mechanisms. Controversy has surrounded the issue of whether or not TAM-DNA
adducts form in human tissues. To address this, we have examined TAM-DNA
adduct formation in multiple tissues of Erythrocebus patas (patas) monkeys given
oral TAM dosing for 3 months, and in endometrial and myometrial tissues of
human cancer patients receiving TAM therapy. Adducts were determined by TAM-
DNA chemiluminescence immunoassay (CIA) using an antiserum elicited against
DNA modified with (E)-α-(deoxy-guanosin-N 2 -yl)-tamoxifen (dG-TAM). Oral
dosing with TAM (1.7 mg TAM/kg/day) was given to 3 adult (22 yrs) patas females
for 3 months. Two unexposed patas of similar age were used as controls. DNA, extracted
from several organs, was analyzed by TAM-DNA CIA. The highest TAM-
DNA adduct levels were found in liver (40.3±3.5 adducts/10 8 nucleotides, mean ±
SE). Measurable levels were also found in uterus (35.1±11.7 adducts/10 8 nucleotides),
brain cerebellum (10.1±2.9 adducts/10 8 nucleotides) and esophagus
(13±3 adducts/10 8 nucleotides). However, no adducts were detectable in brain cortex,
ovary and kidney. Four samples of endometrial tumor from patients receiving
TAM therapy had values of 10-16.5 adducts/10 8 nucleotides, while no TAM-DNA
adducts were detected in 8 patients not receiving TAM therapy. These studies are
being expanded to include determination of TAM-DNA adducts by HPLC-
MS/MS and TAM-DNA immunohistochemistry in human and primate. These
data show that TAM-DNA adducts can be formed in primate reproductive organ
tissues as well as human endometrial tumors, suggesting that TAM-DNA adduct
formation may play a role in the development of human endometrial cancer.
2657 INVOLVEMENT OF CONSTITUTIVE ANDROSTANE
RECEPTOR (CAR) IN THE PROCESS OF LIVER
HYPERTROPHY AND HEPATOCARCINOGENESIS-
INDUCED BY CYP2B-INDUCING NON-GENOTOXIC
HEPATOCARCINOGENS IN MICE (2).
K. Inoue 1 , M. Yoshida 1 , Y. Sakamoto 1 , M. Takahashi 1 , Y.Taketa 1 , S.
Hayashi 1 , S. Ozawa 2 and A. Nishikawa 1 . 1 Division of Pathology, National Institute
of Health Sciences, Tokyo, Japan and 2 School of Pharmacy, Iwate Medical University,
Iwate, Japan.
In our short-term study using CAR knockout (KO) mice, it was clarified that
piperonyl butoxide (PBO) induces liver hypertrophy without CAR-dependence,
while decabromodiphenyl ether (DBDE) induces the lesion in a CAR-dependent
manner similarly to phenobarbital (PB). To confirm the involvement of CAR in the
SOT 2011 ANNUAL MEETING 569