The Toxicologist - Society of Toxicology

876 DEVELOPMENT AND EVALUATION OF A GENOMIC
SIGNATURE FOR THE PREDICTION OF NON-
GENOTOXIC HEPATOCARCINOGENS IN THE RAT.
R. T. Dunn 1 , A. Adai 2 , A. Olaharski 3 , G. H. Searfoss 4 , J. Sina 5 , J. Aubrecht 6 ,
E. Boitier 7 , P. Nioi 1 , D. Jacobson Kram 8 , N. Raghavan 9 , B. Car 10 , S. Chen 10 ,
Y. Yang 11 , A. Kinkaid 12 , J. Sherlock 12 , S. Auerbach 13 and M. Fielden 1 . 1 Amgen,
Thousand Oaks, CA, 2 Asuragen, Inc., Austin, TX, 3 F. Hoffman-LaRoche, Nutley,
NJ, 4 Lilly Research Laboratories, Indianapolis, IN, 5 Merck, West Point, PA, 6 Pfizer,
Groton, CT, 7 sanofi aventis, Vitry-Sur-Seine Cedex, France, 8 U.S. FDA, Silver
Spring, MD, 9 J&J PRD, Raritan, NJ, 10 BMS, Princeton, NJ, 11 Abbott, Abbott
Park, IL, 12 Life Technologies, Foster City, CA and 13 NIEHS-NTP, Research Triangle
Park, NC.
Evaluating risk of chemical carcinogenesis is challenging due to the protracted nature
of the pathology and the translatability of animal models. The Predictive Safety
Testing Consortium of the Critical Path Institute facilitates early risk assessment of
new chemical entities by advancing prediction and mechanistic insight into rodent
non-genotoxic hepatocarcinogenity. Extending upon prior microarray studies, we
investigated a higher-throughput q-PCR signature to predict the potential for nongenotoxic
compounds to induce liver tumors in rat. Using rat liver RNA from a
training set of 72 compounds, a predictive model was developed on the TaqMan®
Array using only 23 genes, thus providing an economical and standardized assay
protocol. Testing on over 900 diverse rat liver RNA samples confirmed the interlaboratory
reproducibility of the assay and its ability to classify known non-genotoxic
hepatocarcinogens. Based on variables inherent to the model and empirical
data, we found that repeat dosing and the use of male S-D rats to be important for
optimal classification. When tested under these conditions, signature sensitivity
and specificity was 71% and 65%, respectively. Exploratory evaluations showed
that prediction of carcinogenicity in other tissues based on liver gene expression is
unlikely with the current model; however different modes of action for non-genotoxic
hepatocarcinogens can be discriminated based on the expression of specific
genes. These results define an early preclinical testing paradigm that enables a
proactive risk assessment for non-genotoxic hepatocarcinogens.
877 REDOX STATES OF THIOREDOXIN-1 DIFFERENTIATE
HUMAN CANCER FROM ITS ADJACENT BENIGN
TISSUES.
W. Shan 1, 2 , W. Zhong 2, 3 and T. D. Oberley 2, 3 . 1 Molecular and Environmental
Toxicology Center, University of Wisconsin, Madison, WI, 2 Department of Pathology
and Laboratory Medicine, University of Wisconsin, Madison, WI and 3 Pathology and
Laboratory Medicine Service, William S. Middleton Veterans Memorial Hospital,
Madison, WI. Sponsor: C. Bradfield.
Our previous study using prostate cancer cell lines showed that thioredoxin 1
(Trx1) redox states could function as a biomarker of redox imbalance and was important
in determining the response of prostate cancer cells to ROS-generating
chemotherapeutic agents. In this study, we examined Trx1 redox states in several
human cancer tissues including human kidney, liver and prostate cancer tissues. We
found that Trx1 oxidation was consistently higher in cancer tissues tested compared
to their adjacent benign tissues despite differential protein expression levels. The reduced
form of Trx1 in benign tissues adjacent to tumors was lower in high-grade
prostate tumors than low-grade prostate tumors. Our results suggested an oxidative
environment in cancer tissues, and in high-grade prostate tumors, the adjacent benign
tissues may be in precancerous oxidative stress conditions. The increased oxidation
of Trx1 in cancer tissues was accompanied by a decrease in thioredoxin reductase
1 (TrxR1) protein levels, suggesting loss of TrxR1 may be a direct cause of
increased Trx1 oxidation. This is the first study to detect Trx1 redox states in human
tissues. Our results suggest that Trx1 redox states may be an important biomarker
in determining the redox state of tissues and differentiates tumor tissues from their
benign counterparts, and thus could be a useful adjunct for clinical diagnosis and
treatment of cancer by analyzing and manipulating tumor redox states.
878 TROVAFLOXACIN PROMOTES DNA DOUBLE-STRAND
BREAKS IN A MACROPHAGE CELL LINE.
K. L. Poulsen 1, 2 , K. Beggs 1, 2 , R. Singhal 1 , P. E. Ganey 1, 2 and R. A. Roth 1, 2 .
1 Pharmacology and Toxicology, Michigan State University, East Lansing, MI and
2 Center for Integrative Toxicology, Michigan State University, East Lansing, MI.
Trovafloxacin (TVX) is a drug with idiosyncratic drug-induced liver injury (IDILI)
liability in human patients. A hepatotoxic interaction between bacterial
lipopolysaccharide (LPS) and an otherwise nontoxic dose of TVX was established
in a mouse model of IDILI. Characteristic of this model was a significant prolongation
of the LPS-induced increase in tumor necrosis factor-alpha (TNF) concen-
188 SOT 2011 ANNUAL MEETING
tration in the plasma of animals cotreated with TVX, and neutralization of TNF
protected from TVX/LPS hepatotoxicity. In vitro, pretreatment of RAW 264.7
murine macrophage-like cells with TVX enhanced LPS-induced TNF release into
cell culture supernatants. It has been previously suggested that TVX, a fluoroquinolone
antibiotic that inhibits bacterial topoisomerase activity, inhibits eukaryotic
topoisomerase. Since topoisomerase inhibitors and other agents that cause
DNA damage can enhance inflammatory cytokine expression, we hypothesized
that DNA strand breaks caused by inhibition of eukaryotic topoisomerase by TVX
led to this enhancement of TNF production. In a cell-free system using recombinant
human topoisomerase IIalpha, TVX (1 – 300 μM) increased retention of concatenated
DNA and reduced the appearance of decatenated DNA, indicating the
effectiveness of TVX as a topoisomerase II poison. The phosphorylation of histone
H2AX (gammaH2AX) on Ser139 was evaluated by western blotting and used as a
marker of DNA double-strand breaks (DSB). TVX (1 - 300 μM) caused a dose-dependent
increase in gammaH2AX signal in RAW 264.7 cells within 2 hours.
Increases in ubiquitinated H2AX were also observed, further implicating DSB as
well as activation of DSB repair pathways. These findings suggest that TVX treatment
results in DSB, which might activate macrophages to synthesize and release
greater amounts of TNF in response to an inflammatory stimulus such as LPS.
(Supported by NIH grant R01 DK061315 and T32 ES007255)
879 EFFECT OF IONIZING RADIATION ON K- ras CODON
12 POINT MUTATIONS IN LUNG AND LIVER OF THE
F344 RAT.
P. B. McKinzie, J. G. Shaddock, M. G. Pearce and V. N. Dobrovolsky. Division
of Genetic and Molecular Toxicology, National Center for Toxicological Research,
Jefferson, AR. Sponsor: B. Parsons.
Epidemiological studies indicate that ionizing radiation increases the incidence of
human cancer. Previous studies have shown an increase in hprt point mutations
with irradiation doses between 1-3 Gy. We examined whether 1-3 Gy whole-body
irradiation of rats similarly induces K-ras codon 12 GGT to GAT mutations in
lung and liver in an adjunct study of Pig-A mutation analysis. All animals were
housed and treated according to the National Center for Toxicological Research
Institutional Animal Care and Use Committee guidelines. Two-month old rats
were exposed to 0, 1, 2, and 3 Gy delivered in 3 equal daily doses using an RS-2000
Biological Irradiator (Rad Source), and the organs harvested 16 weeks later.
Genomic DNA from 0.5-1.5 g of tissue was isolated from each sample, and prepared
for use in an Allele-Specific Competitive Blocker PCR (ACB-PCR) assay for
K-ras codon 12 GGT to GAT mutation. The ACB-PCR assay can quantify basepair
mutations to a sensitivity of 1 mutant allele out of 100,000 wild-type alleles
(10 -5 ) and was used to quantify the K-ras codon 12 GGT to GAT mutant fraction
(MF) in the lung and liver of F344 rats exposed to X-irradiation. The geometric
mean K-ras codon 12 GGT to GAT MF in lung tissue exposed to 0, 1, 2, and 3 Gy
was 2.3 x 10 -5 , 3.6 x 10 -5 , 3.8 x 10 -5 , and 4.1 x 10 -5 , respectively, and in liver tissue
the geometric mean MF was 7.9 x 10 -5 ,14x10 -5 , 4.6 x 10 -5 , and 6.5 x 10 -5 respectively.
There was no statistical difference between any of the exposure groups, including
the controls, for either tissue studied. The data suggests that X-irradiation
does not induce K-ras codon 12 GGT to GAT mutation, a common cancer-associated
oncogene mutation. We conclude that high levels of X-irradiation does not
exert its carcinogenic effect through K-ras codon 12 GGT to GAT point mutations,
which is consistent with ionizing radiation being classified as a clastogenic
mutagen.
880 ASSOCIATION OF ESTROGEN METABOLISM AND RISK
OF NON-HODGKIN LYMPHOMA: DETECTION OF
NOVEL BIOMARKERS FROM CASE-CONTROL STUDY.
L. Yang 1, 2 , N. W. Gaikwad 3 , D. D. Weisenburger 3 , J. Vose 3 , E. Rogan 3 and E.
Cavalieri 3 . 1 Department of Pharmacology and Chemical Biology, University of
Pittsburtgh, Pittsburgh, PA, 2 Department of Environmental, Agricultural and
Occupational Health, College of Public Health, University of Nebraska Medical
Center, Omaha, NE and 3 Eppley Institute for Research in Cancer and Allied Diseases,
University of Nebraska Medical Center, Omaha, NE.
The precise molecular mechanism by which estrogens play a role in the initiation of
non-Hodgkin lymphoma (NHL) has been in question. We hypothesize that specific
estrogen metabolites, catechol estrogen-3,4-quinones [E1(E2)-3,4-Q], can
bind with DNA to form 4-OHE1(E2)-N7Gua and 4-OHE1(E2)-N3Ade adducts;
apurinic sites that are formed by depurination of these adducts can induce mutations
by error-prone repair. When homeostasis of estrogen metabolism is disrupted
and oxidative estrogen metabolism prevails in the body, then the risk of NHL will
be increased. To test this hypothesis, NHL case-control study has been conducted
to investigate how imbalance of estrogen metabolism affects the etiology of NHL
and try to find potential early biomarkers in urine samples. This was accomplished