The Toxicologist - Society of Toxicology

2631 LIVER TOXICITY FOLLOWING KAVA KAVA
ADMINISTRATION IN F344 RATS AND B6C3F1 MICE.
M. Behl 1 , R. S. Chhabra 1 , A. Nyska 2 , L. Fomby 3 , B. R. Sparrow 3 , M. R.
Hejmancik 3 and P. C. Chan 1 . 1 NTP, NIH, Research Triangle Park, NC, 2 Timrat
and Sackler School of Medicine, Tel Aviv University, Timrat, Israel and 3 Battelle,
Columbus, OH.
Kava Kava is an herbal supplement extensively being used as an alternative to antianxiety
drugs such as Xanax® and Valium®. It has also been proposed for use to
help children with hyperactivity. The reports on hepatotoxicity associated with
kava-containing dietary supplements in humans are of widespread concern, resulting
in its ban in several European countries. Although the US FDA has issued
warnings about the potential hepatotoxic effects of Kava, it continues to be widely
used in the United States due to lack of sufficient toxicity data. The purpose of the
NTP studies was to characterize the toxicity and carcinogenicity of Kava in male
and female F344 rats and B6C3F1 mice. The animals were administered kava root
extract orally by gavage in corn oil for two weeks, thirteen weeks or two years.
Results from the 2-week and 13-week studies in rats and mice administered kava at
doses of 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg body weight revealed dose-related increases
in the incidences of heptocellular hypertrophy. This was accompanied by
significant induction of hepatic drug metabolizing enzymes. In the chronic studies,
rats were gavaged 0.1, 0.3, 1.0 g/kg Kava and mice with 0.25, 0.5, 1.0 g/kg. Doserelated
increases in hepatocellular hypertrophy were seen in rats and mice. Also,
there were significant increases in incidences of centrilobular fatty changes. No carcinogenic
activity was observed in rats. In male mice, there was a significant doserelated
increase in the incidence of hepatoblastomas. However, the combined incidences
of hepatocellular adenoma, carcinoma, and hepatoblastoma were not
statistically different from controls. In female mice, there was a significant increase
in the combined incidence of hepatocellular adenoma and carcinoma in the 0.25
and 0.5 g/kg groups, but not in the 1.0 g/kg group. In conclusion, the data indicate
that chronic administration of kava causes liver toxicity in rats and mice and hepatocellular
neoplasms in mice.
2632 USING DNA-BASED MEASUREMENT OF
ONCOMUTATION TO UNDERSTAND CARCINOGEN
MODE OF ACTION.
Y. Wang, F. Meng, P. B. McKinzie, M. B. Myers and B. L. Parsons. Division of
Genetic and Molecular Toxicology, National Center for Toxicological Research/U.S.
FDA, Jefferson, AR.
Oncogene mutations are considered key events in carcinogenesis and are being developed
as quantitative biomarkers of carcinogenic effect. The allele-specific competitive
blocker PCR (ACB-PCR) assay is being used to measure levels of oncogenic
point mutations in animal and human tissues. This assay measures the ratio of mutant
to wild-type allele (mutant fraction) using standards that range from 10 -1 to 10 -
5 . We will present data from studies employing model carcinogens, including
benzo[a]pyrene, N-hydroxy-2-acetylaminofluorene, aristolochic acid, and
azoxymethane which establish four different approaches whereby DNA-based
measurement of oncomutation can inform chemical mode of action (MOA). First,
induction of two different oncogene mutations, one corresponding to the mutational
specificity of the chemical, and the other a frequent spontaneous mutation,
has been used to evaluate the extent to which a chemical induces de novo oncogene
mutation versus amplification of preexisting oncomutation. For example, using
ACB-PCR it was shown that both K-Ras codon 12 GGT to GAT and GGT to TGT
mutations increase as a function of benzo[a]pyrene dose in A/J mouse lung. Second,
as part of dose-response assessment, oncomutation induction can be integrated with
the dose-dependence of other chemical effects. ACB-PCR was used to describe the
dose-response relationship between aristolochic acid and induction of H-Ras codon
61 CTA mutation in Big Blue rat liver and kidney. Third, oncomutation induction
can be integrated with additional endpoints like DNA-adduct formation and/or
neutral reporter gene mutation, to establish whether a chemical is carcinogenic
through a mutagenic MOA. Fourth, the timing of induction of tumor-associated
mutation can be compared to that of other chemical-specific effects, to establish
whether induction of mutation is an early biomarker of carcinogenesis.
564 SOT 2011 ANNUAL MEETING
2633 ANALYSIS OF PEROXISOME PROLIFERATOR-
ACTIVATED RECEPTOR GAMMA (PPARγ) AGONIST
TROGLITAZONE-INDUCED TRANSCRIPTIONAL
CHANGES IN MOUSE ENDOTHELIAL CELLS.
S. Kakiuchi-Kiyota1 , L. L. Arnold1 , P. Koza-Taylor2 , S. Suzuki3 and S. M.
Cohen1 . 1Pathology and Microbiology, University of Nebraska Medical Center,
Omaha, NE, 2Pfizer Drug Safety and Development, Groton, CT and 3Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate
School of Medical Sciences, Nagoya, Aichi, Japan.
A common tumor finding in bioassays of nongenotoxic PPARγ agonists aimed at
treating type II diabetes is hemangiosarcoma (HS) in mice, but little is known
about their mode of action. We previously showed that troglitazone (TG) increased
endothelial cell (EC) proliferation in mice at sarcomagenic doses, and TG directly
increased DNA synthesis and inhibited apoptosis in mouse (MFP MVEC) but not
in human microvascular ECs. We postulated that TG altered the expression of
genes involved in proliferation and apoptosis in mouse ECs, contributing to increased
EC proliferation observed in vivo and in HS formation. To identify genes
for which expression changed by TG treatment in mouse ECs, MFP MVEC were
treated with a reduced concentration (1%) of FBS and 5 μM TG, the concentration
of TG that showed the greatest mitogenic effect, for 3 days, and transcriptional
profiles were analyzed by microarray analysis. TG significantly altered the expression
of 694 genes. The top 10 genes up- or down-regulated in TG-treated MFP
MVEC were not detected or predicted as downstream target genes of murine
PPARγ signaling. Ingenuity pathway analysis indicated that TG activated the
‘Cancer, Cellular Growth and Proliferation, Reproductive Disease’ network involving
genes implicated in EC proliferation and survival. Among them, insulin-like
growth factor 1 (IGF1), which is known to function in tumorigenesis, was most
highly up-regulated (log2 transformed ratio: 2.2-fold) in TG-treated MFP MVEC.
These results indicate that TG exerts a direct mitogenic and anti-apoptotic effect on
mouse ECs by modulating the gene network implicated in EC proliferation and
survival, and IGF1 may have an important role in TG-induced HS formation.
2634 RALOXIFENE IS CYTOTOXIC TOWARD VARIOUS
ESTROGEN RECEPTOR NEGATIVE CELL LINES.
S. Taurin, B. D. Yadav, K. B. Tran and R. J. Rosengren. Pharmacology &
Toxicology, Univeristy of Otago, Dunedin, New Zealand.
In recent years, numerous studies have shown that the selective estrogen receptor
modulators (SERMs) tamoxifen and raloxifene reduce the risk of invasive breast
carcinoma. Although long term treatment with tamoxifen increased the risk of endometrial
cancer, raloxifene, a second generation SERM, has been shown to be as
effective as tamoxifen, while reducing incidence of endometrial carcinoma.
However, while SERMs do prevent the development of many estrogen-receptor
(ER)-positive breast cancers, few studies suggest a potential effect of these drugs in
the prevention of the development of ER-negative breast cancers. Although, the
therapeutic efficacy of raloxifene in cancer therapy is thought to arise primarily
from its ability to compete with estrogens; raloxifene has been shown to have effects
through estrogen receptor (ER)-independent mechanisms. In the present study, we
show that raloxifene decreased cell viability of four human ER-negative breast cancer
cell lines MDA-MB-231 (EC50 9.5 μM), MDA-MB-468 (EC50 6.8 μM),
Hs578t (EC50 6.8 μM) and SkBr3 (EC50 10.6 μM). Cell cycle analysis demonstrated
an increased proportion of cells in the G1 phase in all four cell lines.
Raloxifene increased apoptosis in MDA-MB-231, MDA-MB-468 and Hs578t by
8 fold and SkBr3 by 6 fold after 48 h. Raloxifene treatment also effected the expression
and phosphorylation pattern of proteins involved either in cell proliferation
such as NFκB, β-catenin, epidermal growth factor (EGFR), AKT as well as
implicated in protein synthesis such as 4EBP-1 or mTOR. Apoptosis was also induced
as shown by changes in caspase 3. Furthermore, results from a mouse
xenograft tumor model showed that a daily dose of raloxifene (0.85 mg/kg) was sufficient
to reduce the tumor size after 10 weeks of treatment. In conclusion, these results
provide us with the basis for future studies on the mechanism involved in
raloxifene’s inhibition of ER-negative tumor growth.
2635 CYTOTOXIC POTENTIAL OF A NOVEL CURCUMIN
ANALOG RL-71 IN IN VITRO AND IN VIVO MODELS
OF ESTROGEN RECEPTOR NEGATIVE BREAST
CANCER.
B. D. Yadav 1 , S. Taurin 1 , L. Larsen 2 and R. J. Rosengren 1 . 1 Pharmacology &
Toxicology, Univeristy of Otago, Dunedin, New Zealand and 2 Plant and Food
Research Limited, Dunedin, Otago, New Zealand.
Estrogen receptor negative breast cancer accounts for approximately 30% of all
breast cancers. Furthermore, there are limited drug treatments available and patients
often suffer from poor prognosis and decreased survival rates. Therefore in