The Toxicologist - Society of Toxicology

strand breaks in an in vitro model of mutagenesis. The pKZ1 transgenic mouse
model contains a DNA construct allowing for the detection of intrachromosomal
recombination events. Immunocytochemisty probing for H2A.X phosphorylation
was used to assess the ability of BQ to induce DNA double strand breaks in
hematopoietic cells. Primary cultures of gestational day 14 fetal livers were exposed
to 5, 10, 25, or 50 μM BQ and stained for recombination. A significant increase in
recombination events was observed following treatment with 25 and 50 μM BQ at
all time points with the 10 μM treatment displaying a significant increase over control
at 24 hrs. Hematopoietic cells treated with 25 and 50 μM BQ showed as increased
number of fluorescent γ-H2A.X foci at 8 and 24hrs but this increase was
found to be non-significant likely due to the small sample size. These results indicate
that BQ is able to induce intrachromosomal recombination in fetal
hematopoietic cells possibly through the creation of DNA double stand breaks.
Support: CIHR.
849 OBSERVATIONS OF THE EFFECTS OF
NANOPARTICLES ON REPRODUCTION AND
DEVELOPMENT IN DROSOPHILA MELANOGASTER
AND CD-1 MICE.
N. A. Philbrook 1 , V. K. Walker 3, 1 and W. M. Louise 2, 1 . 1 School of Environmental
Studies, Queen’s University, Kingston, ON, Canada, 2 Pharmacology and Toxicology,
Queen’s University, Kingston, ON, Canada and 3 Biology, Queen’s University,
Kingston, ON, Canada.
The excitement surrounding the multiple uses of nanoparticles continues to increase,
while information about their potential toxicity lags behind. Because of the
small size of nanoparticles (>100nm), their chemical properties can change allowing
them to cross cellular membranes and to potentially interfere with cellular
processes. Silver (Ag) and titanium dioxide (TiO2) nanoparticles are becoming
widely used in popular commercial products such as foods and packaging, cosmetics
and medical devices. To investigate any effect on reproduction and development,
these two nanoparticle types were assessed using both Drosophila
melanogaster and mice as models. Male and female Drosophila were housed together
and exposed to varying concentrations of either type of nanoparticle or a vehicle
control in their food (0.005% w/v to 0.5% w/v). The exposure period was 14
days and during this time, males and females were allowed to reproduce while female
fecundity was recorded daily. Information taken included both oviposition
and overall fertility. Both Ag and TiO2 nanoparticles significantly reduced female
fecundity, particularly at 0.1% and 0.5% concentrations. In mice, pregnant CD-1
dams were orally dosed with either nanoparticle (10, 100 or 1000 mg/kg) or a vehicle
control on gestational day (GD) 9. Fetuses were removed from dams on
GD19, and were examined for both incidence of resportions and the incidence of
morphological defects. Defects were observed in mouse fetuses particularly with
TiO2 nanoparticles, though to a lesser extent than in the invertebrate studies.
Together, these studies shed light on the potential toxicological implications of
nanoparticles and future studies will investigate the mechanisms of this toxicity.
850 DEVELOPMENTAL REGULATION OF RETINOIC ACID
SIGNALING: TERATOGENIC ROLE OF MICRORNAS.
J. A. Franzosa, T. L. Tal and R. L. Tanguay. Department of Environmental and
Molecular Toxicology, Oregon State University, Corvallis, OR.
Retinoic acid (RA) is essential for vertebrate development. Improper spatial and
temporal regulation of RA signaling during development results in teratogenic outcomes.
The role of microRNAs (miRNAs), small RNAs that exert post-transcriptional
control over gene expression during development, in regulating RA signaling
has yet to be evaluated. We demonstrate that developmental RA exposure (6-48
hours post-fertilization (hpf)) in zebrafish results in a distinct posterior curved
body axis (EC100=5 nM) suggesting that RA exposure negatively impacts somitogenesis,
a temporally coordinated process controlling the formation of somites that
ultimately mature to form the dermis, muscle and skeleton. To identify if specific
miRNAs are misexpressed following RA exposure, microarray analyses were conducted
at 12, 24, 36, and 48 hpf and miRNA expression levels in vehicle and RA
exposed zebrafish were compared. Numerous miRNAs were differentially expressed
upon exposure to RA. Strikingly, the expression of three miR-19 family members,
whose putative function during development has yet to be investigated, were significantly
repressed upon RA exposure during somitogenesis (12 hpf).
Bioinformatics analyses predicted that miR-19 miRNAs target RA signaling molecules
important for controlling somitogenesis including cyp26, fgf, hox, and her
family members. Consistent with these findings, an antisense oligonucleotide morpholino
was used to repress miR-19 expression and somitogenesis was severely impacted,
phenocopying embryos that were developmentally exposed to RA.
Increased cyp26a1 expression was observed in RA exposed embryos and miR-19
morphants at 12 hpf. Collectively, these results indicate that some of the teratogenic
effects of RA exposure result from inappropriate miR-19 expression and subsequent
misregulation of RA signaling during somitogenesis. This research was supported
by NIH P30 ES00210, NIEHS T32 ES07060, and an OSU Linus Pauling
Institute Pilot Project Grant.
851 EVALUATION OF THE EFFECTS OF SURGERY AND
CONTINUOUS INTRAVENOUS INFUSION ON
EMBRYOFETAL DEVELOPMENT IN PREGNANT
SPRAGUE-DAWLEY RATS.
S. K. Sahambi 1 , A. LeBlanc 2 , C. Gordon 1 , E. Lebel 1 and G. Washer 1 . 1 Study
Management, LAB Research Inc., Laval, QC, Canada and 2 Toxicology, LAB Research
Inc., Laval, QC, Canada.
Early embryonic development is susceptible to interference by a multitude of
chemical, biochemical or physiological factors. Maternal stress arising from laboratory
animal manipulation during early gestation may impact the well being of the
developing embryo. As a part of the developing reproductive-embryofetal toxicology
facility in our laboratory, the objective of this study was to assess the impact of
surgical cannulation of timed-pregnant Sprague-Dawley (SD) rats on Gestation
Day (GD) 0, followed by continuous intravenous infusion through GD 6 to 15, on
pregnancy outcome and embryofetal development. The females were surgically
cannulated with an intravenous catheter inserted into the femoral vein and advanced
into the vena cava, and administered saline at a dose rate of 2.5 mL/kg/hour
from GD 6 to 15. Control animals were time-mated non- catheterized, and untreated.
Maternal body weights (BW) and food consumption (FC) were monitored
throughout the gestation period. Pups were delivered by cesarean on GD21 and an
external as well as internal exam was performed, followed by processing for skeletal
exam and head exam (using Wilson’s technique). Maternal BW and FC were comparable
in cannulated and non-cannulated animals, though the former showing
slightly lower values. Litter size and mean fetal body weights were comparable between
the cannulated and non-cannulated animals and there were no external or
visceral anomalies or malformations to indicate an effect of the surgical or infusion
procedures. Similarly, skeletal and Wilson’s examinations did not reveal differences
from controls. The above results indicate that surgical manipulation of Sprague-
Dawley rats on GD0 for cannulation followed by intravenous infusion from GD6-
15 does not impact pregnancy outcome and embryofetal development.
852 EMBRYOFETAL TOXICITY OF BORIC ACID:
VALIDATION OF PROCEDURES AND EVALUATION
METHODS, INCLUDING CONTINUOUS
INTRAVENOUS INFUSION IN NEW ZEALAND WHITE
RABBITS.
C. Gordon 1 , S. K. Sahambi 1 , A. LeBlanc 2 , E. Lebel 1 and G. Washer 1 . 1 Study
Management, LAB Research Inc., Laval, QC, Canada and 2 Toxicology, LAB Research
Inc., Laval, QC, Canada.
As a part of the developing reproductive-embryofetal toxicology facility in our laboratory,
this study was conducted to (i) confirm the effects of a known embryotoxicant
(ii) assess the effects of surgical cannulation of does on gestation day (GD) 0
and (iii) assess the effects of continuous intravenous infusion from GD 6 to 19, on
pregnancy outcome and embryofetal development in New Zealand White (NZW)
rabbits. Boric Acid (BA, identified as a Class 2 embryotoxicant by European Centre
for the Validation of Alternative Methods, was selected for part (i). Timed-pregnant
NZW rabbits were orally administered (5 mL/kg) 0, 125, 200, and 250 mg/kg BA
in water (doses selected based on literature as well as a pilot study in non-pregnant
adult female NZW rabbits) from GD 7 to 19. For part (ii and iii) a separate group
of animals was intravenously infused with saline via a surgically implanted catheter
from GD 7 to 19 (3 mg/mL/hr; animals surgically cannulated on GD0). Maternal
body weights and food consumption were monitored throughout the gestation period.
Pups were delivered by cesarean on GD29 and an external as well as internal
exam was performed. The fetuses were processed for skeletal (Alizarin Red S staining)
and head examination (Wilson’s technique). BA treatment resulted in vaginal
bleeding at 200 and 250 mg/kg and severe developmental toxicity with complete
loss of surviving fetuses at 250 mg/kg. Fetal survival was not affected at 125 mg/kg
BA. Despite decreased appetence in the infusion animals throughout gestation,
mean litter size and fetal weights were comparable to the control animals. These results
(for BA) were consistent with the published literature and thus demonstrated
that the laboratory’s procedures and evaluation methods were suitable for identification
of BA-induced effects (part i) as well as early gestational maternal manipulation
(part ii) and gestational infusion (part iii) on embryofetal development and
pregnancy outcome in NZW rabbits.
SOT 2010 ANNUAL MEETING 181