The Toxicologist - Society of Toxicology

cell lines (ATCC) were cultured in MEM media with 10% FBS and antibiotics in
24-well tissue culture plates. At near confluence, the cells were treated with NM at
0, 50, 100, 250, 500 and 1000 μg/ml in triplicate at each concentration. U-PA activity
was carried out by fibrin zymography, MMPs by gelatinase zymography and
TIMPs by reverse zymography. Results: U-PA activity was detected in both breast
cancer cell lines, showing two bands corresponding to 55 and 33 kD. NM significantly
reduced u-PA activity at 250 μg/ml. However, no bands corresponding to u-
PA were detected for Hela and SKOV3 cell lines. MDA-MB-231 and MCF-7
showed one band corresponding to MMP-9, Hela showed two bands, an intense
band corresponding to MMP-2 and a faint band corresponding to MMP-9, and
SKOV3 showed only a band corresponding to MMP-2. NM inhibited their expression
in all cell lines at 100 μg/ml and blocked expression at 250 μg/ml. Activity
of TIMPs was up regulated in all cancer cell lines in a dose–dependent manner.
Minimum activity was expressed at 50 and maximum at 1000 μg/ml. Analysis of
correlation revealed a positive correlation between u-PA and MMPs and a negative
correlation between u-PA /MMPs and TIMPs. Conclusions: These findings suggest
that NM could potentially be developed as a new anticancer agent that inhibits u-
PA and MMPs and increases TIMPs.
2004 THE MAMMARY SECRETORY EPITHELIAL CELL
SPECIFIC ROLE OF PEROXISOME PROLIFERATOR-
ACTIVATED RECEPTOR (PPAR)γ IN DMBA-MEDIATED
BREAST TUMOURIGENESIS.
A. J. Apostoli 1 , N. T. Peterson 1 , S. K. SenGupta 1 and C. J. Nicol 1, 2 . 1 Pathology &
Molecular Medicine, Queen’s University, Kingston, ON, Canada and 2 Division of
Cancer Biology & Genetics, CRI, Queen’s University, Kingston, ON, Canada.
Peroxisome proliferator-activated receptor (PPAR)γ plays a role in tumourigenesis.
Previous studies with PPARγ(+/-) mice suggest PPARγ normally suppresses dimethylbenz[a]anthracene
(DMBA)-induced breast, and other, tumour progression;
however, the PPARγ-dependent mechanisms and cell types critical to this process
remain unknown. We evaluated whether mammary secretory epithelial (MSE) cellspecific
PPARγ signaling normally acts to prevent DMBA-mediated breast tumour
progression. To do this, we first crossed our previously described PPARγ floxed
mice to transgenic mice expressing Cre recombinase under the control of the whey
acidic protein (WAP) promoter and generated a colony of conditional MSE cellspecific
PPARγ knockout mice (PPARγ-MSE KO). Eight week old female PPARγ-
MSE KO mice (n=17) and their congenic wildtype (PPARγ-WT) controls (n=7)
were mated to induce pregnancy and allowed to lactate for 3 days to ensure WAP
promoter activation. Subsequently, all mice were treated by gavage once/week for 6
weeks with 1 mg DMBA and monitored weekly for 25 weeks. Tumour and tissue
samples were collected at necropsy, and portions of each were fixed and frozen for
future analyses. Preliminary data suggests that total and mammary tumour incidences
were modestly (~10%) higher among PPARγ-MSE KOs compared to
PPARγ-WT controls, although mammary tumour multiplicity (2.1 vs 2.0 tumours/mouse
respectively) were not different. More interestingly, PPARγ-MSE KO
mice had a decreased median survival (week 17 vs week 23 respectively), a 2-fold
decrease in mammary tumour latency, and a 1.6-fold increase in mammary tumour
volumes compared to PPARγ-WT controls. These results suggest that normal
PPARγ signaling in MSE cells contributes to the suppression of DMBA-mediated
breast tumour growth. They also add evidence supporting a protective role for
PPARγ activation in reducing environmental chemical-mediated breast tumour
progression.
2005 EVALUATION OF INTRAUTERINE ADMINISTRATION
OF QUINACRINE IN A LIFETIME CANCER BIOASSAY
IN RATS.
D. Creasy 1 , A. Cancel 2 , J. Dillberger 3 , C. Kelly 1 , H. Bolte 1 and D. Sokol 2 .
1
Huntingdon Life Sciences, East Millstone, NJ, 2 Family Health International,
Research Triangle Park, NC and 3 J. Dillberger LLC, Nashville, IN.
Intrauterine instillation of quinacrine has been studied as an easy and inexpensive
method of female sterilization for over 25 years. Quinacrine is both mutagenic and
clastogenic in vitro, raising concerns about its carcinogenic potential but there is
limited data on its tumorigenic response in the female reproductive tract of laboratory
animals. A study was conducted to investigate if quinacrine induces a tumorigenic
response in Sprague Dawley rats, using a dosing regime similar to that used
clinically. Two intrauterine instillations, at doses of 0/0, 10/10, 70/70 and 70/250-
350 mg/kg (first dose/second dose), were administered early in life and approximately
21 days apart. Rats were then monitored for 23 months after the second
dose and observed for viability, clinical signs, and changes in body weight and food
consumption. At necropsy, selected tissues were processed and examined for
histopathologic abnormalities. Acute quinacrine toxicity occurred during the dosing
period but did not affect long term survival. The incidence of uncommon tumors
of the reproductive tract (tumors with less than 1% historical background incidence)
was dose-related and was significantly increased at doses that were approximate
multiples of 1X, 8X and 40X the locally delivered uterine human dose
(mg/quinacrine/g uterine weight). There was also an increase in the incidence of
uterine degenerative and inflammatory lesions in the dose groups that had increased
reproductive tract tumors. While it is possible that the chronic tissue damage
associated with quinacrine exposure was contributory to the tumorigenic response,
it cannot be used to exclude the relevance of the carcinogenic response in
rats, because tissue damage and inflammation are a prerequisite for successful sterilization
in humans. We conclude that two doses of quinacrine administered approximately
21 days apart into the uterus of young sexually mature rats increased the
lifetime risk of tumor development in the reproductive tract.
2006 DOSE-RELATED EFFECTS OF QUERCETIN IN THE
HUMAN BREAST CARCINOMA MCF-7 CELL LINE.
B. Obinaju and F. L. Martin. Lancaster Environment Centre, Lancaster University,
Lancaster, United Kingdom.
Quercetin has been epidemiologically associated with bioactive protective effects.
This compound has also been shown to be mutagenic and toxic in short-term in
vitro test systems. This study evaluated quercetin for toxicity in the MCF-7 cell line
in the presence or absence of benzo[a]pyrene (B[a]P), a known DNA-reactive carcinogen.
In the clonogenic assay, 2.5 × 10 2 cells were seeded into 25 cm 2 flasks in
the presence or absence of graded concentrations of quercetin ± B[a]P. In the cytokinesis
block micronucleus (CBMN) assay, 3 ml aliquot (≈2 × 10 4 cells) was
seeded into 30 mm petri dishes containing 20 mm coverslips; cells were treated
with graded concentrations of quercetin ± B[a]P for 24 h (Yared et al., 2002). All
test agents were evaluated at micromolar (μM) concentrations. Results were obtained
from at least three independent experiments and analyzed for significant
mean differences using a Student’s t-test. For quantitative real-time transcriptase
polymerase chain reaction (RT-PCR), total RNA extraction was performed using
the Qiagen RNeasy ® kit in combination with Qiagen RNase-free DNase kit.
Quercetin induced dose-related increases in cytotoxicity in the clonogenic assay
and micronucleus formation in CBMN assay. However in the presence of B[a]P,
quercetin exerted significant protective effects demonstrated by enhanced % survival
in the clonogenic assay and reductions in micronucleus formation in the
CBMN assay. RT-PCR revealed down-regulated levels of expression of P21 WAF1/CIP1
and CYP1A1 in cells incubated in the presence of both quercetin and B[a]P compared
to levels of induction noted in cells exposed to B[a]P alone. This study provides
evidence of quercetin-induced genotoxicity/cytotoxicity in vitro. It also points
to quercetin-associated protective effects against a prototypical carcinogen
(i.e.,B[a]P)that might be important in an epidemiological setting.
Reference: Yared, E., McMillan, T.J., and Martin, F.L. (2002) Genotoxic effects of
oestrogens in breast cells as detected by the micronucleus assay and the Comet
assay. Mutagenesis 17, 345-352.
2007 SECOND GENERATION CURCUMIN ANLOGS AS
NOVEL DRUGS FOR THE TREATMENT OF ER-
NEGATIVE BREAST CANCER.
S. Taurin 1 , L. Larsen 2 , B. Yadav 1 , T. J. Somers-Edgar 1 and R. J. Rosengren 1 .
1
Pharmacology & Toxicology, Univeristy of Otago, Dunedin, New Zealand and 2 Plant
and Food Research Limited, Dunedin, New Zealand.
We have synthesized analogs of curcumin in order to develop potential drugs for
the treatment of ER-negative breast cancer. These compounds were examined for
their ability to elicit cytotoxicity in ER-negative breast cancer cell lines (MDA-MB-
231 (ER-/Her-2-) and SkBr3 (ER-/Her-2+). This was followed by apoptosis, cell
cycle arrest and mechanistic studies, which examined the protein expression of key
cell proliferative proteins that are initiated by the epidermal growth factor receptor
(EGFR). Six of the derivatives demonstrated increased cytotoxic potency compared
to curcumin. Specifically, 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone
(BMHPC), and other cyclohexanone derivatives abbreviated RL90,
RL91, RL92, RL53 and RL71 elicited EC50 values between 0.26 and 2.6 μM in
MDA-MB-231 cells and between 0.18 and 1.2 μM in SKBr3 cells. All other compounds
examined were less potent than curcumin (EC50 values of 7.57 and 2.35
μM in MDA-MB-231 and SKBr3 cells, respectively). Mechanistic analyses demonstrated
that all of the derivatives significantly increased the number of G2/M-phase
cells as well as the number of cells undergoing apoptosis compared to control in
both cell lines. Furthermore, RL90, RL91 and RL92 significantly decreased the
protein expression of the phosphorylated forms of Akt, JNK and mTOR in both
SOT 2010 ANNUAL MEETING 427