The Toxicologist - Society of Toxicology

224 NON-HUMAN RELEVANCE OF A CAR-MEDIATED
TUMOR FORMATION IN RATS INDUCED BY THE
HERBICIDE METAZACHLOR.
C. Wiemann 1 , N. Honarvar 1 , M. Göttel 1 , S. Gröters 2 , E. Fabian 2 , B. van
Ravenzwaay 2 and I. Fegert 1 . 1 Regulatory Toxicology Pesticides, BASF SE,
Ludwigshafen, Germany and 2 Experimental Toxicology and Ecology, BASF SE,
Ludwigshafen, Germany. Sponsor: R. Landsiedel.
Assessment of human relevance of liver tumors seen in rodents can be performed
according to the WHO/IPCS proposal. An analysis was performed on the slightly
increased incidence of benign liver tumors in female Wistar rats induced by
metazachlor, a herbicide that did not give indication of genotoxic effects. In order
to address the suggested Mode of Action (MoA) via the activation of the
Constitutive Androstane Receptor (CAR) a series of mechanistic studies were performed
also excluding other possible mechanisms. A Metazachlor-induced CARmediated
MOA similar to that of phenobarbital was confirmed by showing 1) the
induction of CAR-regulated Cytochrome P450 isoenzymes (CYP) of the CYP 2B
family on mRNA and activity level, 2) nuclear accumulation of CAR on protein
level 3) CAR-dependent activation of a phenobarbital responsive enhancer modulespecific
CYP 2B promotor region in transfected primary hepatocytes. Centrilobular
hypertrophy and liver cell proliferation, considered as further characteristic key
events for this MoA, were also observed in the Metazachlor-induced liver tissue. An
Arylhydrocarbon Receptor-mediated mechanism was excluded by the absence of
the respective mRNA and enzyme activity of the CYP 1A family (7-
Ethoxyresorufin-O-deethylase). A peroxisome proliferator activated receptor α-mediated
mechanism was excluded by lacking mRNA (CYP 4A1) and enzyme activity
(CN- -insensitive palmitoyl CoA oxidation and lauric acid 12-hydroxylation) induction.
The involvement of sustained cytotoxicity as a MoA was excluded since no
early onset of inflammation and no necrosis in the liver were identified after treatment
with metazachlor. The issue of non-relevance to humans was investigated in
vitro in primary hepatocytes as well as in a transgenic mouse model expressing
solely the human PXR/CAR genes. In conclusion, this database supports the exclusive
association of the Metazachlor hepatotumorigenicity in the rat with CAR-mediated
phenobarbital-like mechanisms.
225 LIGAND-DEPENDANT ACTIVATION AND
DEACTIVATION OF NUCLEAR RECEPTOR SUBFAMILY
4 MEMBERS IN PANCREATIC CANCER CELLS.
X. Li 1 and S. Safe 2, 3 . 1 College of Medicine, Texas A&M Health Science Center,
College Station, TX, 2 Veterinary Physiology and Pharmacology, Texas A&M
University, College Station, TX and 3 Institute of Biosciences & Technology, Texas
A&M Health Science Center, Houston, TX.
Nuclear receptor related 1 (Nurr1) and nerve growth factor IB (Nur77 or TR3) are
members of the nuclear receptor subfamily 4. Expression levels and modulation of
activities of these transcription factors induces apoptosis in various cancer cell lines.
Therefore these receptors are potential drug targets for clinical treatment of tumors.
A group of methylene-substituted diindolylmethanes (C-DIMs) were screened for
ligand-dependent activation and deactivation of Nurr1 and TR3 in Panc1 and
Panc28 pancreatic cancer cell lines. Significant induction of Nurr1 activity was observed
for compounds such as 1,1-bis(3’-indolyl)-1-(p-trifluoromethylphenyl)methane
(DIM-C-pPhCF3) and 1,1-bis(3’-indolyl)-1-(p-bromophenyl)methane
(DIM-C-pPhBr). Increased and decreased TR3 dependent luciferase activities were
also observed after treatment with 1,1-bis(3’-indolyl)-1-(p-benzophenyl)-methane
(DIM-C-pPhC6H5) and 1,1-bis(3’-indolyl)-1-(p-hydroxyphenyl)-methane
(DIM-C-pPhOH) respectively. These preliminary results were further examined by
using response element sequences linked to a luciferase reporter gene. The study has
identified, for the first time, several C-DIMs which activate or deactivate Nurr1
and TR3. Their effects on gene expression and inhibition of cancer cell growth are
currently being investigated.
226 CANNABINOIDS INDUCE TYROSINE PHOSPHATASES
AND INHIBIT SP TRANSCRIPTION FACTORS IN
COLON AND PROSTATE CANCER CELLS.
S. Sreevalsan 1 , N. Kaminski 3 and S. Safe 1, 2 . 1 Veterinary Physiology and
Pharmacology, Texas A&M University, College Station, TX, 2 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.
Cannabinoids are being developed for cancer chemotherapy however the mechanisms
associated with their antineoplastic activity are unclear. In this study we examined
the effects of two agents that activate both CB1 and CB2 receptors, namely
WIN 55,212-2 (WIN), a synthetic aminoalkylindole derivative and cannabidiol
48 SOT 2011 ANNUAL MEETING
(CBD), a major constituent in the plant Cannabis sativa. Both CBD and WIN inhibited
growth of SW480 colon cancer cells with IC50 values of 5.54 and 9.01 uM
respectively and co-treatment of 15 uM CBD with CB1 and CB2 receptor antagonists
AM251 and AM630 respectively blocked the growth inhibitory effects of
CBD. In contrast, neither CB receptor antagonists inhibited the effects of WIN
demonstrating that the anti-proliferative activity of CBD and WIN were receptordependent
and independent respectively. The effects of WIN and CBD on expression
of Sp transcription factors, Sp regulated genes and kinases were also investigated.
WIN and CBD downregulated p-Akt expression and CBD also inhibited
p-MAPK expression suggesting that WIN/CBD, like other phyto-derived-anticancer
drugs may induce phosphatase activity. Treatment of SW480 cells with 15
uM CBD or 7.5 uM WIN induce expression of MKP-1, MKP-5, s-PAcP, c-PAcP
and SHP-1 but not PTEN, PP2A, PTP1B, MKP-2 and DEP-1. Moreover phosphatase
inhibition with sodium orthovanadate indicates that CBs primarily induce
tyrosine phosphatases that have previously been characterized for anticancer activities
and the role of CB-induced phosphatases on pro-carcinogenic pathways/genes
is being investigated.
227 HOPS (HUMULUS LUPULUS) INHIBITS ESTROGEN
METABOLISM IN NON-TUMORIGENIC HUMAN
BREAST EPITHELIAL CELLS (MCF-10A CELLS).
L. P. Hemachandra, R. P. Chandrasena, B. M. Dietz, S. Chen, G. F. Pauli, N.
R. Farnsworth, G. R. Thatcher and J. L. Bolton. Medicinal Chemistry &
Pharmacognosy, University of Illinois at Chicago, Chicago, IL.
Long-term exposure to estrogen increases the risk of hormone dependent cancers in
women. Estrogen induced cell proliferation in estrogen receptor positive cells (hormonal
pathway), and the formation of reactive estrogen o-quinones mediated by
cytochrome P450s (chemical pathway) are believed to contribute to the estrogen
carcinogenesis mechanism. Estrogens are oxidized by P450 1A1/1A2 to the catechol
metabolites, 2-hydroxyestradiol, and by P450 1B1 to 4- hydroxyestradiol.
Both catechols are further oxidized to form electrophilic o-quinones that can react
with DNA and proteins. For years, plant extracts have been used worldwide, as dietary
supplements for women’s health particularly for menopausal symptom relief.
Mounting evidence suggests that botanical dietary supplements have chemopreventive
properties which could specifically protect women from the carcinogenic effects
of endogenous estrogens through modulation of both hormonal and chemical
mechanisms. The current study focuses on elucidating the influence of these extensively
utilized botanicals on estrogen metabolism in normal breast epithelial cells
(MCF-10A). MCF-10A cells are estrogen receptor negative non-tumorigenic
human breast epithelial cells, which can be transformed into a malignant phenotype
with estradiol. After treatment of MCF-10A cells with estradiol and botanical
extracts, estrogen metabolites were analyzed using a sensitive LC-MS/MS method.
Among the botanical extracts tested, hops (Humulus lupulus) showed a significant
reduction in catechol estrogen formation whereas black cohosh (Cimicifuga racemosa)
had little effect. These data suggest that the hop extract might possess
chemopreventive activity through inhibition of genotoxic estrogen quinone formation
in addition to its known antioxidant and induction of detoxification enzyme
capabilities.
228 ENDOCRINE TOXICANTS PROMOTE RESISTANCE TO
ANOIKIS AND INVASIVENESS OF BREAST CANCER
CELLS IN VITRO .
W. G. Foster and H. L. Cameron. Obstetrics and Gynecology, McMaster University,
Hamilton, ON, Canada.
Epidemiological studies suggest that environmental toxicants (ETs) can enhance
breast cancer tumour aggressiveness and decrease survival in women with the highest
exposures. We hypothesize that ETs can promote breast cancer progression by
increasing breast cancer cell survival and invasiveness. The effect of increasing concentrations
of two high priority and unregulated ETs, bisphenol A (BPA) and
dibutyl phthalate (DBP), a phthalate used in cosmetics and enteric-coated slow-release
capsules, on characteristics of proliferation, the invasive and metastatic potential
of breast cancer cells in vitro. The human breast cancer cell line MDA-MB-231
was cultured and treated with BPA, DBP or its major metabolite monobutyl phthalate
(MBP) for 1–9 days. Proliferation rate was measured by BrdU incorporation
and subsequent immunochemical detection by spectrophotometer. Viability was
measured as reductive capacity of the tetrazolium salt MTS to its formazan product
and quantified by spectrophotometry. Invasiveness was measured by seeding cells in
wells coated with extracellular matrix. Cells that degraded and penetrated into the
lower compartment were then quantified by fluorescent labelling and fluorescent
spectrophotometry. Resistance to anoikis was measured by seeding cells in ultra low
binding plates and quantifying the number of viable cells at day 9. None of the ETs
affected proliferation or viability at any tested concentration. Bisphenol A increased