The Toxicologist - Society of Toxicology

(n=50) and non-Tg mouse (n=20) at 7 weeks of age. Citrate buffer at pH 4.5
(10ml/kg) was also injected intraperitoneally in rasH2 (n=10) and non-Tg (n=10)
mouse as a negative control agent. Serum samples were obtained from each mouse
at 2-week intervals, and metabolites were measured by CE-TOFMS. When all data
were classified according to pathological findings, some peaks that suggested possible
carcinogenic substances were found in the MNU dosed rasH2 mouse. At least
one signal was detected as a possible biomarker. Further investigations will be required,
but the CE-TOFMS could be a powerful tool for screening carcinogenic
biomarkers together with the rasH2 model.
522 DEVELOPMENT OF INDUCIBLE IN VIVO RNAi
MOUSE MODELS FOR TARGET VALIDATION.
J. Hitchcock 1 , V. Beuger 2 , H. Kissel 2 , N. Pullen 1 and A. Rossi 1 . 1 Pfizer Ltd.,
Sandwich, Kent, United Kingdom and 2 TaconicArtemis GmbH, Cologne, Germany.
Sponsor: M. Sharpe.
Inducible RNAi systems allow the rapid expression of functional small hairpin (sh)
RNA molecules with tight on/off regulation for the purpose of silencing target
genes. The key advantages of an inducible in-vivo RNAi system include a) the ability
to study the loss of function of essential genes which would otherwise result in
knockout lethality b) less concerns around developmental affects compared with
traditional knockout models. A strategic alliance was formed with TaconicArtemis
to help evaluate the utility of this technology for target validation. The aim of the
project was to develop inducible RNAi mouse models against a specific target gene,
to aid in evaluating and characterising the technology, and to help understand the
safety of the target. The target selected was Cyr61/CCN1, a member of the CCN
family of secreted matricellular proteins, which has a role in proliferation, angiogenesis,
inflammation and fibrosis. Studies of Cyr61 have been hampered by the
lack of relevant animal models, as the murine knockout has an embryonic lethal
phenotype. The first step in generation of the inducible mouse models was the selection
of potent shRNA’s designed against the Cyr61 gene. This proved to be quite
challenging and a number of strategies had to be employed, including the use of
different shRNA selection algorithms, and designing against the untranslated region
(UTR) of the gene. This has been shown elsewhere as a useful approach for
gene knockdown studies (McManus et al, 2002). Three shRNA’s exhibiting potent
knockdown in mouse embryonic stem cells were selected. shRNA-1, shRNA-20
and shRNA-21 caused 73%, 75% and 79% knockdown at the gene level, measured
using qRT-PCR. Furthermore the shRNA’s caused 56%, 71% and 77% knockdown
of Cyr61 protein, as shown by Western blotting. Animal generation using
these potent shRNA’s has since been undertaken and resulting phenotypes are currently
being analysed. Mcmanus MT. et al., 2002. Gene silencing using micro-
RNA designed hairpins. RNA 8:842-850
523 METABOLOMIC ASSESSMENT OF TEMPORAL
CHANGES INDUCED DURING THE COURSE OF A
TYPICAL OVERNIGHT FAST IN THE SD RAT.
D. G. Robertson, S. Stryker and J. Vassallo. Discovery Toxicology, Bristol-Myers
Squibb Company, Princeton, NJ.
Fasting induces marked changes in the serum metabolomic profile of SD rats. This
study examined the temporal metabolic changes induced by fasting of up to 16
hours duration. Groups of 5 male rats were fasted for 2, 4, 8, 12 or 16 hours duration.
A group allowed ad libitum access to food served as a control. All groups were
sacrificed at the end of the dark cycle (DC-6AM). One additional group, allowed
ad libitum access to food, was sacrificed at the end of the light cycle (LC-6 PM).
Animals were weighed at the onset and termination of their fasting periods with
urine collected during the duration of the fast and serum collected at termination of
the fast for metabolic analysis. DC rats gained approximately 5% BW while 16 hr
fasting animals lost approximately 8%BW. From 8-16 hrs of fasting, rats lost approximately
1 g/hr with urine production of ~ 0.9 ml/hr as compared to urine production
of ~0.3 mL/hr in DC controls. Serum GLU and TRIG dropped sharply
after 2 hours (by 38 and 64%, respectively) with little subsequent change. TRIG
levels in LC controls were decreased relative to DC controls (by 67%) while GLU
levels were similar. Pronounced metabolic effects were induced by fasting though in
many case LC did not vary from DC even though food consumption was markedly
decreased during LC. Transcriptional evaluation of fast-twitch muscles indicated
time-dependent induction of muscle specific ubiquitin ligase atrogin-1 with more
than a 5-fold increases at 16 hours compared to DC. Consistent with these
changes, serum 3-methylhistidine, a marker for myofibrillar protein degradation,
showed a time dependent increase (up to 3X) after 8 hours of fasting. With some
notable exceptions, metabolic profiles of LC were more similar to DC than to fasting
animals (at any time point) suggesting complex metabolic interplay between diurnal
metabolic responses and those metabolic changes induced by fasting.
112 SOT 2011 ANNUAL MEETING
524 BACKGROUND OCULAR CHANGES FOLLOWING
TRANSVITREAL SUBRETINAL DOSING IN THE
RHESUS AND CYNOMOLGUS MONKEY.
A. Préfontaine, M. Bussières and M. Vézina. Toxicology, Charles River Preclinical
Services, Montreal, QC, Canada.
Subretinal dosing allows for localized, controlled exposure within the eye and is
typically used for gene or cell-based therapies. The transvitreal approach allows precise
placement of the injection. The background changes for rhesus and cynomolgus
monkeys following a 100 μL subretinal injection of saline are described.
Animals were anesthetized and under an operating microscope a self-sealing 25G
port system allowed both a light source and 41G injection needle into the eye to
create a bleb (injection) adjacent to the macula. Ophthalmology was evaluated up
to 8 times and H&E stained sections were evaluated at 13 wks. There was occasional
slight reflux from the bleb into the vitreous and retinal/choroidal hemorrhage
subsequent to the procedure. Areas of hemorrhage progressively resolved by
Day 5 and the blebs receded by Day 8. Traumatic, procedure-related signs of inflammation;
anterior chamber cell/flare and anterior vitreous chamber cell-like
opacities resolved within 13 wks. There was slight retina/choroid pigment variation
associated with the bleb. Microscopic evaluation of the bleb area indicated very
slight retinal atrophy of the photoreceptors/outer nuclear layer, fibrosis of the
choroid, and retinal folds/rosettes. Needle tract lesion and lens degeneration were
occasionally noted. These changes were considered related to the experimental procedure
(including transient retinal elevation caused by the bleb). There were no microscopic
findings adjacent to the bleb. In conclusion, transvitreal subretinal injection
of 100 μL produced similar and consistent mild findings in both rhesus and
cynomolgus monkeys that were transient or stable over the course of 13 wks.
Characterization of the procedure-related findings facilitates the recognition of test
article-related changes.
525 DOSE RESPONSE OF DIURON-INDUCED URINARY
BLADDER HYPERPLASIA IN MALE WISTAR RATS.
A. F. Cardoso, S. M. Ihlaseh, M. S. da Rocha, M. G. Nascimento, J. V. de
Camargo and M. C. de Oliveira. Pathology, Sao Paulo State University - UNESP -
Medical School, Botucatu, Brazil.
Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a urea-derived herbicide, increased
the incidences of urothelial tumors in both genders of Wistar rats fed 2500
ppm in a two-year bioassay. Accordingly, USEPA has categorized diuron as a
“known/likely” human carcinogen. The accepted non-genotoxic mode of action
(MOA) of diuron encompasses urothelial necrosis induced by direct cell exfoliation
or cytotoxicity caused by diuron and/or its metabolites, followed by regenerative
cell proliferation and sustained urothelial hyperplasia that may favor neoplasia development.
This study, approved by the local Committee for Ethics in Animal
Experimentation, was designed to evaluate the dose-response of diuron regarding
urothelial lesions as detected by histology, immunohistochemistry and scanning
electron microscopy (SEM). Six groups of male Wistar rats were fed diuron for 20
weeks mixed in the diet at 0, 60, 125, 500, 1250, or 2500 ppm. One hour before
euthanized, rats were injected with BrdU (100 mg/kg body weight, i.p.).
Histological analysis of the urinary bladders revealed that the two higher doses induced
a significantly increased incidence of simple hyperplasia, a preneoplastic
urothelial lesion. Urothelial cell proliferation was documented also by BrdU labeling.
By SEM, the incidences and severity of lesions were significantly greater in the
500 and 1250 ppm groups (5/10 and 7/10, respectively) than in the lower-dose and
control groups (respectively, 1/10, 2/10 and 0/10). Although numerically increased,
the incidence of lesions in the 2500 ppm group (4/10) did not differ significantly
from the control. The present study documented a dose-response influence
of diuron on the rat urothelium, with a no observed effect level (NOEL) of
125 ppm. Support: FAPESP (Proc. 06/60506-1 and 07/55901-1).
526 BIOAVAILABILITY AND DISTRIBUTION OF SOIL
BOUND POLYCYCLIC AROMATIC HYDROCARBONS
IN SWINE.
R. E. Peters 1 , S. D. Siciliano 1, 2 and M. Wickstrom 2 . 1 Soil Science, University of
Saskatchewan, Saskatoon, SK, Canada and 2 Toxicology Centre, University of
Saskatchewan, Saskatoon, SK, Canada. Sponsor: L. Weber.
Polycyclic aromatic hydrocarbons (PAHs) are common soil contaminants introduced
to the environment through fuel spills and incomplete combustion. These
contaminants are of concern for human exposure, especially in toddlers who are at
high risk due to the quantity of soil they ingest as well as their sensitivity to carcinogens.
Traditionally, the oral bioavailability of PAHs in soil (defined as PAH ab-