The Toxicologist - Society of Toxicology

for six weeks, with 10 mg/kg azoxymethane (AOM), an alkylating colonotropic
carcinogen. All mice were terminated eight months following the last AOM treatment.
We show that the mutation had no effect on AOM induced tumor initiation
based on tumor multiplicity (1.2 vs 1.1) and tumor incidence between the two
genotypes, 50% for the wild type and 47% for mutants respectively. Surprisingly,
the tumor burden was 14.2 mm3 in mutants compared to 22.5mm3 in wild type
(p value 0.05). In contrast, there was 39% gross liver injury incidence in wild type
and 85% gross liver lesions in mutants. We conclude that the XRCC1 L360R point
mutation increases sensitivity to AOM-induced liver toxicity but suppresses AOMinduced
tumor progression in the colon. Further investigation will be of interest to
determine the clinical relevance of this point mutation in colon cancer.
136 DIFFERENTIAL DNA REPAIR IN HEMATOPOIETIC
STEM AND PROGENITOR CELLS FOLLOWING
GENOTOXIC DAMAGE BY BENZENE METABOLITES.
D. Alexander 1 , M. Zimmer 3 , M. T. Smith 2 , E. C. Forsberg 3 and M. Camps 1 .
1
Microbiology and Environmental Toxicology, University of California Santa Cruz,
Santa Cruz, CA, 2 Public Health, University of California Berkeley, Berkeley, CA and
3
School of Engineering, University of California Santa Cruz, Santa Cruz, CA.
To gain insight into the mechanisms of benzene-induced leukemia, we quantified
DNA damage and repair in highly purified populations of murine hematopoietic
stem cells (HSC) and myeloid progenitors (MP) in response to alkylating agents
and quinone metabolites of benzene. Acute myeloid leukemia is a paradigm for
cancer stem cells with a hierarchy analogous to that seen in normal hematopoiesis.
Thus, either HSC or MP are the ultimate targets for the genotoxic insults that give
rise to environmentally induced leukemias. Our goal is to determine whether a cellintrinsic
difference in metabolic activation of DNA damaging metabolites by HSC
or MP contributes to hematotoxicity and benzene-induced leukemia. Applying a
COMET assay, we observed a similar pattern of dose dependent DNA damage for
both HSC and MP in response to treatment with direct alkylating agents and ionizing
irradiation (IR). Although HSC and MP exhibited equal damage immediately
after treatment, HSC showed evidence of DNA repair with lower levels of cell
elimination after 24hrs in culture as compared to MP. Patterns of DNA damage
and repair following hydroquinone (HQ) treatment of HSC and MP were analogous
to that found with IR and alkylation. Our observations of repair in HSC are
consistent with findings that long-lived HSC have robust intrinsic cell-type specific
survival mechanisms, however, their largely quiescent status may force them to initiate
DNA repair using error-prone repair mechanisms, which then render them
susceptible to transformation. In contrast, short-lived MP appear molecularly
poised to undergo apoptosis and are therefore eliminated in response to DNA damage.
DNA damage by benzene metabolites in combination with HSC quiescence
may be the key to understanding why HSC are a target for leukemic transformation
by benzene.
137 POTENTIATING EFFECT OF DIETARY FAT ON
BENZO(A)PYRENE (BAP) BIOTRANSFORMATION AND
COLON TUMORS IN APC MIN MICE.
D. L. Harris 1 , D. B. Hood 2 , L. J. Roberts 3 and A. Ramesh 1 . 1 Biochemistry &
Cancer Biology, Meharry Medical College, Nashville, TN, 2 Neuroscience &
Pharmacology, Meharry Medical College, Nashville, TN and 3 Pharmacology,
Vanderbilt University, Nashville, TN.
Our studies thus far have shown formation of colon tumors in Apc Min mice subsequent
to ingestion of fat containing BaP, an environmental toxicant. These findings
have human health relevance in that in US alone, around 60,000 lives/year are lost
to colon cancer. Diet and environment have been implicated in the development of
sporadic colon tumors. Since biotransformation of toxicants is the prime driving
force for carcinogenesis, the objective of this study was to determine how dietary fat
potentiates the development of colon tumors through altered BaP biotransformation,
using a mouse model. Benzo(a)pyrene was administered to Apc Min mice in unsaturated-
(peanut oil) and saturated- (coconut oil) fats at doses of 50 and 100
μg/kg via oral gavage over a 60-day period. Blood, colon and liver were collected at
the end of exposure period. The expression of BaP biotransformation enzymes
(CYP1A1, CYP1B1 and GST) in liver and colon were assayed at the level of
mRNA and activities. Tissue samples were analyzed by reverse phase-HPLC for BaP
metabolites, and 32 P-postlabeling method for BaP- DNA adducts. BaP exposure
through dietary fat altered its metabolic fate in a dose-dependent manner, with 100
μg/kg dose group registering an elevated expression of BaP biotransformation enzymes,
greater concentration of BaP metabolites, BaP-DNA adducts and more adenomas
compared to the 50 μg/kg dose group (p < 0.05). This effect was more pronounced
for saturated fat group compared to unsaturated fat group (p < 0.05).
These findings establish that saturated fat causes sustained induction of BaP biotransformation
enzymes and extensive metabolism of this toxicant. As a consequence,
the reactive metabolites such as epoxides and quinones generated in colon
and liver bind with DNA, form adducts resulting in colon tumors in a subchronic
exposure regimen (supported by NIH grants S11ES014156, 1F31ES017391-01,
5T32 HL007735-14 and RO3CA130112-01).
138 THE ROLES OF CR(VI) AND ITS REDUCTIVE
INTERMEDIATES IN APOPTOSIS THROUGH THE
MITOCHONDRIAL INTRINSIC PATHWAY.
A. O. Chiu 1 , R. Hill 2 , P. W. Lee 2 , N. H. Chiu 3 and J. D. Robertson 4 .
1
NCEADC, U.S. EPA, Washington, DC, 2 Microbiology/Immunology, Dalhousie
University, Halifax, NS, Canada, 3 Office of Science & Technology, Office of Water,
U.S. EPA, Washington, DC and 4 Pharmacology, Toxicology, Therapeutics, University
of Kansas Medical Center, Kansas City, KS. Sponsor: J. Landolph.
Hexavalent Cr(VI) chromium enters the cell as the divalent anion CrO4(2-)
through the chloride-phosphate-anionic-intracellular-channel (ClIC1) and interacts
with glutathione (GSH). It is established that reduction process of Cr(VI) produces
φx174 DNA-double-strand-breaks in vitro, via Comet assays as well as the H2AX
posphoryla-tion. While ROS are formed intracellularly from Cr(VI) exposure in the
cytoplasm causing p53-independent apoptosis initially, it was followed with a subsequent
p53-dependent increase of apoptosis. The persistence of unrepaired DNA
double-strand-breaks from the exposure to Cr(VI) brings about an increase of p53-
phosphorylation and its localization in the cytoplasm leading to apoptosis via the
mitochondrial pathway. We also report here that caspase2 is activated from the exposure
of cells to Cr(VI) and Cr(V) model compound, but not to exposure to
Cr(IV) model compound. Caspase-2 is the initiator-caspase in the nucleus, which
activates the intrinsic-mitochondrial-apoptotic pathway with Bax/p53/PUMA from
exposure to genotoxic agents. The implication the differential responses of caspase-2
activation to Cr(VI) and its reductive intermediates Cr(V) and Cr(IV) on the mode
of action of Cr(VI) induced apoptosis, cell arrest and carcinogenesis is discussed.
Disclaimer: The opinions and conclusions in this paper are those of the authors and
do not necessarily reflect their affiliated institutions.
139 PROFILING THE MODE OF ACTION FOR LIVER
TUMORS IN B6C3F1 MICE EXPOSED TO METHYL
ISOBUTYL KETONE (MIBK).
D. Geter 1 , N. A. Berdasco 1 , W. Gulledge 2 , R. Gingell 3 and S. Green 4 . 1 The Dow
Chemical Company, Midland, MI, 2 American Chemistry Council, Arlington, VA,
3
Shell Oil Company, Houston, TX and 4 Eastman Chemical Company, Kingsport, TN.
MIBK is used primarily as a solvent in the production of paints, pesticide formulations,
adhesives, wax/oil separation, leather finishing, textile coating, and specialty
surfactants for inks. Chronic exposure to high levels of methyl isobutyl ketone
(MIBK) to B6C3F1 mice in a recent NTP study resulted in a significant induction
of hepatocellular adenomas, but not carcinomas. In order to examine a possible
mode of action of MIBK, liver-specific clinical chemistry, histopathology, gene expression,
cytochrome P450 enzymatic activity, and hepatocellular proliferation
were measured in male B6C3F1 mice exposed to 1800 ppm (NTP high dose) of
MIBK via inhalation for 7 days. No treatment-related effects were observed for
clinical signs, body weights, liver weights, or clinical chemistry measurements in
MIBK exposed animals. Treatment-related histopathologic changes in the liver consisted
of very slight hepatocyte hypertrophy (enlargement) with increased cytoplasmic
eosinophilia in the centrilobular/midzonal regions of the hepatic lobule. These
changes were consistent with possible increased smooth endoplasmic reticulum and
induction of cytochrome P450 enzymes. Significant transcript induction was observed
for Cyp2b10 (constitutive androstane receptor [CAR] associated gene), however
no changes were observed in Cyp1a1 or Cyp3a11, and a slight decrease in expression
was seen for Cyp4a10 (peroxisome proliferator-activated receptor alpha
[PPARalpha] associated gene). A significant induction of Cyp2b enzyme activity
(PROD) and hepatocellular proliferation were also observed. Aryl hydrocarbon receptor
(AhR) related enzyme activity (EROD) was not altered in this study. The responses
observed in this study support a CAR-mediated mode of action for the observed
liver tumors, which is of little to no relevance to humans.
140 CANNABINOIDS DECREASE CANCER CELL GROWTH
AND INHIBIT SP TRANSCRIPTION FACTORS.
S. Sreevalsan 1 , S. Safe 1, 2 and N. E. Kaminski 3 . 1 Veterinary Physiology and
Pharmacology, Texas A & M University, College Station, TX, 2 Center for
Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A
& M Health Science Center, Houston, TX and 3 Center for Integrative Toxicology,
Michigan State University, East Lansing, MI.
In addition to their psychotropic effects cannabinoids (CB) and cannabimimetics
are effective anti-cancer agents and inhibit growth of multiple cancer cells and tumors.
However, the mechanisms of their antineoplastic activity remain unclear. In
SOT 2010 ANNUAL MEETING 29