The Toxicologist - Society of Toxicology

gene expression post-transcriptionally and are expected to play an important role in
xenobiotic responses. We used the genotoxic anticancer drug cisplatin (CDDP) as a
test compound in several in vitro models to characterize and compare their genotoxic
response patterns in relation to induced miRNA profiles. Primary mouse hepatocytes,
mouse embryonic stem cells (mESC) and the hepatoma cell line HepG2
are frequently used in vitro models for genotoxicity studies. Cell viability, DNA
damage, apoptosis, cell cycle, gene expression and miRNA profiles were investigated
in these models following exposure to different concentrations of CDDP
measured at several time points. DNA damage could be detected reliably with the
yH2AX staining assay in both the primary mouse hepatocytes and in HepG2 cells.
CDDP caused DNA damage in both cell lines. Flow cytometric analysis in HepG2
cells and mESC revealed that damaged cells were blocked in the S-phase and underwent
apoptosis. Analysis of the gene expression data in MetaCore confirmed
that pathways involved in “DNA damage”, “apoptosis” and “cell cycle” were mainly
affected in HepG2 and mESC. MiRNAs revealed novel mechanisms that could
contribute in the understanding of the toxicity caused by CDDP exposure. These
results are part of an ongoing research project and with the results obtained so far
we have partially characterized toxicogenomic responses in different in vitro systems,
which contribute in understanding the mechanistic properties of CDDP.
1587 USING A TRANSCRIPTIONAL GENE SIGNATURE TO
PREDICT NEPHROTOXICITY IN VITRO .
D. DeSilver 1 , V. Bonato 2 , M. Kuhn 2 and M. T. Pletcher 1 . 1 Compound Safety
Prediction, Pfizer, Inc., Groton, CT and 2 Biostatistics, Pfizer, Inc., Groton, CT.
The kidney is a frequent site of drug toxicity and its injury can be induced by a
wide spectrum of agents. Several urine biomarkers exist for detecting drug-induced
kidney injury including KIM-1 and osteopontin, but there is a lack of in vitro approaches
for early identification of compounds that may act as nephrotoxicants.
Our strategy consisted of evaluating a published computationally-derived, in vivo
gene signature (Fielden et al, 2005) designed to predict late onset drug-induced
renal toxicity using early time point transcriptional data. Despite many of the component
transcripts of the signature being poorly characterized genes and representing
diverse pathways and functionalities, the drug-induced expression changes of
the genes directly correlated with the pathological outcome of a structurally diverse
set of nephrotoxicants. The predictivity of the signature model was ~76%. Our intent
was to translate this approach in vitro, using the same 35 gene signature, with
rat kidney tubule cells dosed at a concentration inducing 20% cytolethality where
possible. Cells were dosed for 24 hours with a training set of 56 compounds. Fold
change data was log transformed, subjected to standard quality control metrics, and
run through several predictive models using 200 different training/test splits. Using
a Support Vector Machines model with Recursive Feature Elimination, we calculate
the sensitivity of the signature to be 70.0% and the specificity to be 71.3%. Despite
the in vivo derivation of the signature and the relatively small data set, our in vitro
application is strikingly similar to the in vivo performance. In addition, we show
that the signature is tissue specific in its prediction with a set of non-nephrotoxicant
compounds that induce liver damage in vivo. This work establishes a tool for the
early detection of a class of high-risk compounds so that they may be removed from
development prior to ever reaching a clinical population.
1588 TOXICOGENOMIC COMPARISON OF THE TOXIC
EFFECTS OF DIETARY TCDD IN RAINBOW TROUT
AND ZEBRAFISH.
Q. Liu 1, 2 , M. L. Rise 3 , C. A. Struble 4 , J. M. Spitsbergen 5 , G. Goetz 1 , R. J.
Hutz 2 and M. J. Carvan 6, 1 . 1 Great Lakes WATER Institute, Milwaukee, WI,
2 Biological Sciences, University of Wisconsin Milwaukee, Milwaukee, WI, 3 Ocean
Sciences Centre, Memorial University of Newfoundland, St. John’s, NF, Canada,
4 Department of Math, Statistics, and Computer Science, Marquette University,
Milwaukee, WI, 5 Microbiology, Oregon State University, Corvallis, OR and 6 School
of Freshwater Sciences, University of Wisconsin Milwaukee, Milwaukee, WI.
The purpose of this project is to use functional genomic and bioinformatic methods
to identify molecular biomarkers conserved across divergent teleost species as
indicators of the impact of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.
We are using zebrafish and rainbow trout as models to investigate the effects of
chronic dietary TCDD exposure on global gene expression anchored to histopathological
analysis. Juvenile rainbow trout and young zebrafish were fed dietary TCDD
at 0, 0.1, 1, 10 and 100ppb for six weeks. No significant mortality was observed in
zebrafish during the six weeks of exposure. However, trout is much more sensitive
to TCDD exposure than zebrafish with an LD50 of 10ppb at the end of six weeks
and 100% lethality for the 100 ppb diet by 40 days. TCDD caused multiple
pathologies in both zebrafish and trout and the severity was both time- and dose-
dependent. Histological analysis revealed that by week six significant lesions were
found in the liver, kidney, gills, skin, and heart, and caused a delay in the development
of the skull and brain. Gene expression analysis was performed using the
cGRASP 16K cDNA microarray for trout and NimbleGen 12X135K arrays for zebrafish.
A number of genes were dysregulated in trout and zebrafish by dietary
TCDD that involve a number of different molecular pathways and some of these
may be useful as biomarkers for TCDD effects in the aquatic environment to assess
the potential impacts of low-level environmental TCDD in the food chain and on
the health of humans and animals. Gene expression network analysis of microarray
data from the two species will provide conserved molecular pathways impacted by
chronic dietary TCDD exposure for improving our understanding of the mechanism
of TCDD toxicity.
1589 IDENTIFICATION OF MECHANISTIC BIOMARKERS
OF CARDIAC AND SKELETAL MUSCLE TOXICITY TO
AID THE DEVELOPMENT OF TISSUE-SPECIFIC IN
VITRO MODELS.
W. Dott 1 , P. Mistry 2 , J. Wright 2 and K. Herbert 1 . 1 Cardiovascular Sciences,
University of Leicester, Leicester, United Kingdom and 2 Syngenta, Bracknell, United
Kingdom. Sponsor: R. Peffer.
The primary objective of this work was to discover early markers of striated muscle
toxicity, and to aid the development and validation of in vitro model systems to understand
and identify muscle toxicants early in the R&D process. In this study, toxicogenomics
was used to identify mechanistically-relevant genomic biomarkers of
xenobiotic-induced striated muscle toxicity in tissues taken from in vivo studies.
Rats were dosed daily via the diet with sulfonyl isoxazoline chemistries for 4 or 28
days. Histological assessments revealed a dose-dependent relationship with myositis
and myodegenerative pathology in striated muscle. Transcription profiling of cardiac
and skeletal muscle tissue samples taken from sub-toxic dose levels was carried
out using Illumina microarray and bioinformatics tools (ArrayTrackTM) to filter
and infer biological meaning from the data. After 4 days, subtle but significant
(p