The Toxicologist - Society of Toxicology

down the doses may produce more toxic metabolites. This can cause neuron damage
and therefore lead to behavioral changes. The zebrafish (Danio rerio) can be
used to test toxicological properties of compounds with more translational relevance
than supplementary non-mammalian models given that it is a vertebrate.
Because zebrafish mature rapidly, they are also beneficial for studying long-term effects
of exposure to compounds in the juvenile stage. This pilot study was conducted
to examine the effects of acetaminophen in comparison to the CB1 receptor
agonist WIN 55,212 or 5-HT1A partial agonist buspirone on social behavior, also
known as shoaling tendency, in zebrafish. Fish were exposed by bath to either 1 g/L
acetaminophen, 1 mg/L buspirone or 0.1 mg/L WIN 55,212 that were dissolved in
200μl DMSO or home tank water (+/- DMSO) in a volume of 250 ml contained
in a 600 ml beaker for 15 minutes. Fish were then placed in a social interaction and
novelty test for 10 minutes each following 20 minutes of acclimation to test the effect
of these drugs on shoaling tendency. We found that WIN 55,212, acetaminophen
and buspirone all increased shoaling tendency, which is consistent with their
targeting of neural circuits that might be involved in autism and low social interaction.
Future studies will involve exposure of young-juvenile zebrafish to these same
compounds and testing as adults.
1882 TOXICITY EVALUATION OF INHALED LOXAPINE IN
THE RAT.
B. E. Stewart 1 , M. Garley 1 and A. Koester 2 . 1 Alexza Pharmaceuticals, Inc.,
Mountain View, CA and 2 Battelle, Columbus, OH.
The potential toxicity of inhaled loxapine was evaluated in male and female
Sprague Dawley rats. Loxapine was delivered as repeated nose-only inhalation doses
for 14 days followed by a 14-day, posttreatment recovery period (n=10/sex/group
main study, n=5/sex/group recovery). Toxicokinetics was evaluated in additional
animals (n=3/sex/timepoint). Mean daily dose levels were 1.7, 6.4 and 13 mg/kg
for males and females combined (10 minute daily exposure). A separate placebo
group of animals had the same dosing schedule. All animals survived until scheduled
necropsy. Dose related CNS clinical signs were consistent with the extended
pharmacology of loxapine, an antipsychotic with D 2 dopamine receptor antagonist
activity. Treated animals in the mid and high dose groups showed significant gender-specific
decreases in mean body weights and weight gain patterns compared to
animals in the control group; weight decreases were considered secondary to
lethargy and decreased activity. Histological changes related to inhalation exposure
included squamous metaplasia of the larynx, which was considered a reversible, non
specific effect due to particle impaction. Changes related to the extended pharmacology
of loxapine at these doses included mammary hyperplasia in both sexes, and
ovarian follicular cysts and mucification of vaginal epithelium in females. These
histological reproductive tissue effects were anticipated based on the pharmacology
of loxapine and the reported results of oral exposure studies in rats. Mammary hyperplasia
in males and ovarian follicular cysts in females were no longer seen by the
end of the 14-day posttreatment period, and the incidences of the remaining reproductive
tissue changes were markedly reduced, indicating ongoing recovery by Day
29 of the study. Based on the persistence of the clinical signs until the morning following
dosing, the slow recovery of body weight decreases and the partial recovery
of the histological findings at the high dose, the no-observed-adverse-effect level
(NOAEL) was considered to be 1.7 mg/kg/d, the low dose in the study.
1883 TOXICITY EVALUATION OF INHALED LOXAPINE IN
THE DOG.
M. K. Garley 1 , B. E. Stewart 1 and R. Matheson 2 . 1 Alexza Pharmaceuticals, Inc.,
Mountain View, CA and 2 Charles River Laboratories Preclinical Services Montreal,
Senneville, QC, Canada.
The potential toxicity of inhaled loxapine was evaluated in male and female beagle
dogs. Loxapine was delivered as repeated oral inhalation doses for 28 days followed
by a 14-day, posttreatment recovery period (n=3/sex/group main study;
n=2/sex/group recovery phase for placebo control and high dose groups only).
Toxicokinetics of loxapine and active metabolite 7-OH-loxapine was evaluated in
all groups. Mean daily dose levels were 0.12, 0.95 and 1.8 mg/kg for males and females
combined (11-18 minute exposure daily). All doses are expressed as loxapine
free base and dose calculations assume a deposition fraction of 1. A separate group
of animals received the placebo control with the same dosing schedule. All animals
survived until scheduled necropsy. Treatment-related CNS clinical signs were observed
in all loxapine treatment groups and included decreased activity, lying on
side, weakness, tremors and/or uncoordination. Incidence and severity of these observations
was dose-related and consistent with the pharmacology of loxapine, an
antipsychotic with D 2 dopamine receptor antagonist activity. Incidence, severity
and duration of clinical signs decreased with repeated exposure. There were no
treatment-related changes in body weight. Slight decreases in food consumption
noted throughout the treatment period were considered related to decreased activ-
ity following treatment and not considered of toxicological significance. There were
no treatment-related changes in clinical pathology or immunoglobulins and no
treatment-related ocular or cardiac changes. No treatment-related organ weight,
gross or histological changes to the respiratory system or other organ systems were
observed during the main study or recovery phase. There were no treatment-related
changes during the recovery phase. The no-observed-adverse-effect level (NOAEL)
was considered to be 1.8 mg/kg/d, the high dose in the study. This dose corresponds
to Day 28 loxapine plasma AUC last values of 467 and 577 ng h/mL in males
and females, respectively.
1884 DOXORUBICIN-INDUCED PLATELET
PROCOAGULANT ACTIVITIES: AN IMPORTANT CLUE
FOR CHEMOTHERAPY-ASSOCIATED THROMBOSIS?
K. Kim, S. Kim, K. Lim, J. Noh, S. Kang, O. Bae and J. Chung. College of
Pharmacy, Seoul National University, Seoul, Republic of Korea.
The prothrombotic risk of doxorubicin (DOX) has been frequently reported, yet
the exact mechanism underlying it has not been fully elucidated. Here, we report
that DOX can induce the procoagulant activity in platelets, an important contributor
to the thrombosis. In freshly isolated human platelets, DOX treatment induced
the procoagulant activity as determined by phosphatidylserine (PS) exposure and
microparticle generation while platelet aggregation was not affected. Consistently
with this, DOX-treated platelets and generated microparticles increased the generation
of thrombin significantly in concentration- and time-dependent manners.
DOX-mediated procoagulant activation was determined to be from intracellular
calcium increase and ATP depletion which resulted in the activation of scramblase
and the inhibition of flippase, enhancing PS externalization ultimately. DOX-induced
reactive oxygen species (ROS) generation was determined to underlie the
procoagulant activation of platelets. Along with the increased procoagulant activity,
apoptotic events were also induced by DOX treatment, such as the dissipation of
mitochondrial membrane potential(ΔΨ), cytochrome c release, mitochondrial
translocation of Bax and caspase-3 activation. A caspase inhibitor, QVD can attenuate
DOX-induced procoagulant activity significantly, suggesting the involvement
of apoptotic pathways in the DOX-mediated procoagulant activation of platelets.
Rat in vivo thrombosis models confirmed the prothrombotic effects of DOX
through procoagulant activity, where DOX administration resulted in increased venous
thrombus formation along with increased PS exposure and ΔΨ dissipation,
reflecting that DOX-enhanced prcoagulant activity of platelets may lead to increased
thrombus formation, indeed.
1885 TOXICOLOGY ASSESSMENT OF SHETA2, A NOVEL
CHEMOPREVENTIVE AGENT, IN RATS AFTER 28-DAY
ORAL ADMINISTRATION.
K. K. Kabirov 1 , I. M. Kapetanovic 2 , A. Banerjee 1 , A. D. Zakharov 1 , D. M.
Benbrook 3 and A. V. Lyubimov 1 . 1 University of Illinois at Chicago, Chicago, IL,
2 National Cancer Institute, Bethesda, MD and 3 University of Oklahoma Health
Science Center, Oklahoma City, OK.
The toxicity of SHetA2 and the functional status of the CNS were studied in
Crl:CD (SD) rats after 28 days of gavage administration (0, 100, 500 and 2000
mg/kg/day). No mortalities were seen. Decreased activity in one male in the 2000
mg/kg/day dose group was observed on days 21 and 24. Body weight gains which
were significantly decreased on day 8 in the 2000 mg/kg/day dose group resulted in
a decrease in total body weight gains (statistically significant in females only). Both
males and females in the 2000 mg/kg/day dose groups had decreased food consumption
on days 1–8 and on days 15–22 (males only). These effects were not seen
at the end of the study. FOB measurements (in week 4) revealed no signs of neurotoxicity.
There were no statistically significant differences in clinical pathology parameters
between the groups administered SHetA2 and the control group. Relative
prostate weights were significantly decreased in the 2000 mg/kg/day dose group.
Relative adrenal gland weights were significantly increased in the 2000 mg/kg/day
females. No pathology or histopathology lesions were seen in the control and 2000
mg/kg/day dose groups. Following gavage administration, peak plasma levels were
seen at the 2 hr time-point in the 100 mg/kg/day dose group (52.9 ng/mL) and at
the 6 hr time-point in the 500 and 2000 mg/kg/day dose groups (195.8 and 246.3
ng/mL, respectively). Following single IV administration at 5 mg/kg, the peak
plasma SHetA2 level was seen at the 5 min time-point (1827.7 ng/mL). PK analysis
showed extremely low (< 1%) systemic bioavailability of SHetA2 after oral administration
for all doses tested. The half-life of SHetA2 was short due to high systemic
clearance. The dose at the level of 2000 mg/kg/day was considered as the
LOAEL. The NOAEL for daily oral administration of SHetA2 by gavage to
Crl:CD (SD) rats was 500 mg/kg/day. The study was performed under Contract
No. N01-CN-43306 from the NCI.
SOT 2011 ANNUAL MEETING 403