The Toxicologist - Society of Toxicology

process of hepatocarcinogenesis, 6-week-old male CARKO mice (C3H strain origin)
and wild C3H mice were treated with 5,000 ppm PBO, 50,000 ppm DBDE
or 500 ppm PB in diet for 27 weeks after the initiation with diethylnitrosamine
(DEN). Control animals were fed basal diet alone after DEN initiation. As a result,
in wild mice the PBO and PB treatments increased incidences and multiplicities of
altered foci and adenomas in the liver, of which phenotypes were mainly
eosinophilic. In CARKO mice, PBO and PB drastically reduced such eosinophilic
lesions compared with the relevant treated wild mice, and no tumor was induced by
PB. In contrast, DBDE mainly induced basophilic lesions in wild mice.
Interestingly, multiplicity of basophilic lesions was greater in CARKO mice given
DBDE compared with control CARKO mice, and comparable to those in DBDEtreated
wild mice. These results indicate that CAR might play an important role in
the mechanism of hepatocarcinogenesis induced by PBO or PB, while other mechanisms
might exsist as a main pathway of DBDE-induced hepatocarcinogenesis.
Taken together with short-term and two-stage carcinogenicity studies using
CARKO mice, our results suggest a novel possibility that CAR-mediated hepatocarcinogenesis
might follow different process from CAR-mediated liver hypertophy
in mice.
2658 DNA DAMAGE MODULATION IN XPA(-/-)P53(+/-) AND
WILD TYPE C57BL/6 MICE FED BENZO[A]PYRENE
WITHOUT OR WITH CHLOROPHYLLIN (CHL) FOR
28 DAYS.
K. John 1 , M. I. Churchwell 2 , F. A. Beland 2 , M. M. Pratt 3 , G. McMullen 1 and
M. C. Poirier 1 . 1 Center for Cancer Research, National Cancer Institute, NIH,
Bethesda, MD, 2 Division of Biochemical Toxicology, National Center for
Toxicological Research, U.S. FDA, Jefferson, AR and 3 National Center for
Environmental Assessment, Office of Research and Development, U.S. EPA,
Washington, DC.
Chlorophyllin (CHL), a chemopreventive agent, was previously shown to reduce
formation of DNA damage in normal human mammary epithelial cells exposed to
BP (John et al., Cancer Letters, 2010). Here we examined the effects of CHL on
DNA adduct formation in nucleotide excision repair (NER)-deficient [Xpa(-/-)]
and tumor suppressor haploinsufficient [p53(+/-)] mice, and their wild type (WT)
counterparts, fed either control diet, or diet containing 100 ppm BP, or 100 ppm
BP + 0.3% CHL for 28 days. DNA damage (expressed as adducts per 10 8 nucleotides
± SE, n=6-10 mice/group) was measured in liver, lung and esophagus by
chemiluminescence immunoassay (CIA) using antiserum elicited against DNA
modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene
(BPDE). As expected, there was more DNA damage in Xpa(-/-)p53(+/-) mice compared
to WT mice, confirming the absence of normal NER capacity in the transgenics.
In Xpa(-/-)p53(+/-) mice fed BP + CHL there was no change in DNA
adduct levels in esophagi and lungs, compared to mice fed BP alone. However, in
liver the addition of CHL caused a significant reduction [104.9 ± 13.3 for BP vs.
36.7 ± 10.6 for BP + CHL]. In WT mice fed BP + CHL the adduct levels were unchanged
in the lungs and significantly increased in esophagi [15.4 ± 1.5 for BP vs.
65.1 ± 8.7 for BP + CHL] and liver [54.5 ± 10.5 for BP vs. 261 ± 20.3 for BP +
CHL], compared to mice fed BP alone. Analysis of BPdG adducts by HPLC-
MS/MS in Xpa(-/-)p53(+/-)and WT mice is currently in progress. The increase in
DNA damage in esophagi and livers of WT mice suggest that chronic human use of
CHL for chemoprevention of PAH-mediated carcinogenesis should be viewed with
caution as the burden of DNA damage may not be lowered in all organs.
2659 EXPOSURE TO URBAN PARTICULATE MATTER
INDUCES A DECREASE IN E-CADHERIN EXPRESSION
IN MULTICELLULAR TUMOR SPHEROIDS.
F. M. Bernal-Herrera 1 , C. M. García-Cuéllar 2 , R. Morales-Barcenas 2 , Y.
Sánchez-Pérez 2 and Y. I. Chirino 1 . 1 Unidad de Biomedicina, Universidad Nacional
Autonoma de Mexico, Edo. Mex, Mexico and 2 Subdirección de Investigación Básica,
Instituto Nacional de Cancerologia, Mexico, Mexico. Sponsor: H. Glauert.
Epidemiological studies have demonstrated that exposure to particulate matter
(PM) with an aerodynamic diameter smaller than 10 μm (PM10) is associated an
augment the risk of lung cancer. To investigate the effects of PM10, several monolayer
cell culture models have been studied, however, the PM10 effects on tumor
cell models has not been completely explored. The loss of E-cadherin is associated
with the invasiveness of tumors, metastatic dissemination and a poor prognosis. We
hypothesized that PM10 exposure could induce a decrease in E-cadherin expression
in an established tumor. To test this hypothesis, multicellular tumor spheroids
(MTS) of lung adenocarcinoma cells were grown in 96-plates (40,000 cell per well
in non-adherent conditions) for 4 days. On day 5, spheroids were exposed to PM10
(5 μg/mL, 10 μg/mL and 20 μg/mL) for 24 h. Then, spheroids were collected to
570 SOT 2011 ANNUAL MEETING
western blot analysisis. The expression of E-cadherin, actin and GAPDH were performed.
We compared the effect of PM10 exposure on the expression of above
mentioned proteins in monolayer cell culture of the same cells. Our results show
that PM10 induces a 20% decrease in E-cadherin expression in the higher concentration
in MTS but monolayer cell culture remains without changes. We also found
that PM10 induces a 10% increase in actin expression in MTS (10 μg/mL and 20
μg/mL) and using phalloidin, a dynamic actin polymerization to filamentous actin
was observed by confocal microscopy in monolayer cell culture. The expression of
GAPDH was used as loading control. Based on these results, we conclude that
PM10 induced a decrease in E-cadherin expression in MTS and PM10 also induced
an increase in actin expression that could be related to changes to filamentous
actin.
2660 STUDIES ON METABOLISM OF 1, 4-DIOXANE.
W. Eck 1 , J. Fishbein 2 , B. Ginevan 2 , N. Koissi 2 and N. Shah 2 . 1 Directorate of
Toxicology, U.S. Army Public Health Command, Aberdeen Proving Ground, MD and
2 Department of Chemistry and Biochemistry, University of Maryland—Baltimore
County, Linthicum, MD.
1,4-Dioxane is a water-miscible, semi-volatile organic compound that is found in
ground water at many military installations as a consequence of being used as a stabilizer
for the degreasing solvent 1,1,1-trichloroethane. Dioxane use continues for
production of small arms ammunition and as a component of various other formulations.
The U.S. Army is facing a significant environmental restoration liability because
of dioxane contamination. The carcinogenic potential of dioxane is dependent
upon its mode of action and the role of dioxane metabolites. Two possible
metabolites have previously been identified—1,4-dioxan-2-one (dioxanone) and 2hydroxyethoxyacetic
acid (HEAA); the former with electrophilic properties capable
of genotoxicity. Questions have remained about the potential role and relationship
between the two metabolites, which can exist in a simple equilibrium with one another.
To better understand the thermodynamic and kinetic parameters of the equilibrium
between dioxanone and HEAA, an analysis of the excreted metabolites
without chemical derivatization was conducted. The equilibrium constant for the
reaction between dioxanone and HEAA was found to greatly favor HEAA. Kinetic
analysis found that the reaction was rapid, with the half-life of the hydrolysis reaction
being approximately 0.4 hours. Further in vivo metabolism was studied in
Sprague-Dawley rats. Analysis of blood and urine samples for up to 8 hours after
oral gavage failed to detect the presence of any dioxanone in either medium, an observation
that is consistent with the physico-chemical parameters of the reaction.
These data suggest that there is a low probability that the dioxanone electrophillic
metabolite is created and responsible for genotoxic induction of cancer.
2661 THE IN VIVO ROLE OF ADIPOCYTE-SPECIFIC PPARγ
IN ENVIRONMENTAL RISK FACTOR-MEDIATED
BREAST TUMOURIGENESIS.
G. Skelhorne-Gross 1 , N. Peterson 1 , R. Rubino 1 , S. SenGupta 1 and C. Nicol 1, 2 .
1 Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada and
2 Division of Cancer Biology and Genetics, Queen’s University, Kingston, ON, Canada.
One in 28 women will die from the growth and spread of breast cancer. The interactions
between genetic and environmental (chemical and western-style high fat
diet (HF)) risk factors in this process remain unclear. HF consumption also correlates
with increased secretion of adipocyte signaling factors linked to breast tumourigenesis.
Peroxisome proliferator-activated receptor (PPAR)γ plays a role in
many cancers and controls the expression of genes essential for normal fat and sugar
metabolism. We have shown using PPARγ heterozygous (+/-) mice that PPARγ
normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene
(DMBA)-mediated breast tumours. Since many breast cell types express PPARγ,
each with unique signal patterns, we aim to define which are required for these
PPARγ-dependent effects. Here we examine the hypothesis that adipocyte-specific
PPARγ signaling stops the progression of multi-risk factor (DMBA+HF)-mediated
breast tumourigenesis. To do this, we generated PPARγ(+/-) and adipocyte-specific
PPARγ knockout (PPARγ-A KO) mice and their wild-type (WT) controls. Eight
week old female mice (n=10, n=12, n=25, respectively) were treated p.o. with 1mg
DMBA once/week and given HF for the duration of the study (25 weeks). Our
data show that DMBA+HF treated PPARγ-WT mice have significantly decreased
overall survival and tumour latency (13 vs 25 weeks), and increased mammary
(43% vs 18%) and skin (86% vs 64%) tumour multiplicity compared to
DMBA+normal diet treated controls (p