The Toxicologist - Society of Toxicology

Fluvastatin) and 3 FB (Ciprofibrate, Fenofibrate and Gemfibrozil) were studied,
using a high-content toxicity screening (HCS) by two different systems for the
analysis of assorted drugs in HepG2 cells. Both systems were used as implemented
via: Laser Scanning Cytometry (LSC) and with the HyperCyt instrument matched
with a CyAn cytometer. HCS data comprised of measurement of mitochondrial
membrane potential (MMP, by TMRM), plasma membrane permeability (ToPro-
3), and DNA content, nuclear circularity and area (Hoechst 33342). Disruption of
MMP is the one of the earliest indicators for the mitochondrial toxicity. An altered
mitochondrial response in HepG2 was observed after 24h. A decline of MMP was
accompanied by decreasing cell viability. ST and TZD presented a variable MMP
response within the class of agents with simvastatin and rosiglitazone having the
most influence on MMP. FB and BG responses were consistent. Similar IC50 values
were obtained for the eleven compounds by both systems. Statistical analysis
tools were developed for ungating and these were compared with traditional gating.
Funding: Postdoc Research Mobility Grant (EX2008-0452) USA.
1609 EFFECTS OF GESTATIONAL EXPOSURE TO PFOA ON
PPAR PROTEIN AND MRNA EXPRESSION IN VITAL
ORGANS OF FETAL AND POSTNATAL MICE.
A. M. Watkins, C. R. Wood, K. P. Das, C. Lau and B. D. Abbott. U.S. EPA,
Research Triangle Park, NC.
Perfluorooctanoic acid (PFOA) is developmentally toxic, causing in utero and
neonatal mortality, and altering development and growth in mice. PFOA activates
peroxisome proliferator-activated receptor (PPAR)α and PPARα signaling is required
for toxicity. This study examines the expression of PPARα, β, and γ in fetal
and postnatal mice. Timed pregnant CD-1 mice were dosed orally from gestational
day (GD) 1-17 with vehicle or 5 mg PFOA/kg. Tissues were collected on GD14,
GD17, postnatal day (PND) 1, 7, 14, 21, and 28, and divided for preparation of
RNA and protein. qPCR and Western blot data were normalized to internal controls
(GAPDH, β-actin). PFOA exposure altered PPAR expression in liver, decreasing
expression of mRNA and protein for PPARα and β on PND14. Other changes
in liver included decreased mRNA for PPARα, β, and γ on PND1, decreased
PPARβ mRNA on PND1-21, and increased PPARα mRNA on PND21 and
PPARγ mRNA on PND21-28. In the heart, PFOA exposure decreased PPARα
mRNA on PND14 and protein on PND21. Heart PPARβ mRNA decreased on
PND14 and 28, and PPARγ mRNA increased on PND1. In PFOA-exposed kidney,
PPARγ mRNA was increased on PND14 and PPARβ protein decreased on
PND28. In the stomach, PFOA exposure decreased PPARγ mRNA and protein on
PND14 and mRNA remained depressed on PND21. PFOA increased PPARβ protein
levels in stomach on PND21. In the intestine, PFOA increased PPARγ mRNA
on PND1 and 14, while PPARβ mRNA was reduced on PND21. In spleen, thymus,
and lung, PFOA increased PPARα mRNA on PND1 and PPARβ mRNA was
also increased on PND1 in spleen and thymus. PFOA decreased adrenal expression
of PPARγ mRNA on PND14. In addition, PPAR-, CAR-, and PXR-regulated
genes were examined and effects of PFOA on expression differed between tissues.
The profiles of PPAR expression and the responses to PFOA are complex. These results
will be valuable for the further elucidation of PFOA’s mode of action for developmental
toxicity.
[This abstract does not necessarily reflect US EPA policy.]
1610 DEVELOPMENTAL TOXICITY OF LEFLUNOMIDE AND
ITS METABOLITE TERIFLUNOMIDE IN ZEBRAFISH
EMBRYOS.
A. Mattsson, K. Vikholm, E. Ullerås and A. Oskarsson. Biomedical Sciences and
Veterinary Public Health, The Swedish University of Agricultural Sciences, Uppsala,
Sweden.
Leflunomide is a prodrug used in treatment of rheumatoid arthritis. Leflunomide is
rapidly metabolized to the active metabolite teriflunomide, which is long-lived in
plasma and induces embryolethality and teratogenicity in rats and rabbits at plasma
levels that are within the therapeutic range. We have studied developmental toxicity
of leflunomide and teriflunomide in zebrafish embryos. Embryos were exposed to
leflunomide (0.1 - 5 μg/mL) and teriflunomide (0.1 - 10 μg/mL) from 2 hours post
fertilization (hpf) and observed at 8, 24, 78 and 72 hpf for mortality and developmental
parameters. The two compounds induced similar effects, but leflunomide
was more potent. A dark-appearing yolk and heart malformations were the most
sensitive endpoints for both leflunomide and teriflunomide (LOAEL: 0.1μg/mL
and 0.5μg/mL, respectively). Effects at higher concentrations included oedema,
low blood circulation, developmental delay, lack of pigmentation, various malformations
and lethality. The impact of chorion as a protective barrier was investigated
by exposing dechorinated and intact embryos to leflunomide and teriflunomide at
0.1 and 0.5 μg/mL, respectively. No differences in effects were found between intact
and dechorinated embryos. Sensitivity at various developmental stages was
346 SOT 2011 ANNUAL MEETING
studied by exposing embryos to leflunomide (2 and 5 μg/mL) for 2 hrs starting at
2, 12, or 24 hpf. Most abnormalities induced were the same as after continuous exposure,
which stated at 2 hpf. However, starting exposure at later developmental
stages resulted in less pronounced effects. The results show that both leflunomide
and its metabolite teriflunomide are potent teratogens in zebrafish, leflunomide
being the more potent of the two. The LOAEL of teriflunomide in the present
study (0.5 μg/mL) is approximately 100 times lower than steady-state plasma levels
in patients (Brent, 2001). Two hours exposure was sufficient to cause teratogenic effects
and the chorion did not act as a protective barrier against the compounds.
1611 IMPACT OF DIFFERENT OILY VEHICLES ON
TESTICULAR TOXICITY OF DI-BUTYL PHTHALATE
(DBP).
A. J. Martino-Andrade 1, 2 , A. S. Lourenço 2 , B. C. Minatovicz 1 , M. F. Kienast 1 ,
R. N. Morais 1 and P. R. Dalsenter 2 . 1 Physiology, Universidade Federal do Paraná,
Curitiba, Brazil and 2 Pharmacology, Universidade Federal do Paraná, Curitiba,
Brazil. Sponsor: R. Landsiedel.
In rats, reproductive adverse effects resulting from developmental exposures to
DBP and other active phthalates seem to be highly variable depending on rat strain
and other laboratory conditions. One possible component responsible for such
variability could be the type of oily vehicle used in experimental studies. In addition,
there are reports indicating that phthalates could act either as pro-inflammatory
or anti-inflammatory compounds, and that maternal dietary supplementation
with omega-3 fatty acids, such as those present in canola and fish oil, could alter the
toxicity of pthalates. In the present study, we evaluated the impact of different oily
vehicles on testicular effects induced by DBP in male rats fetuses. Pregnant rats
(n=11-13/group) were treated by oral route (gavage) with DBP diluted in different
vehicles from day 13 to 20 of gestation. A total of 6 experimental groups were used
- 3 control groups: 5 mL/Kg of oil (corn, canola or fish); and 3 treated groups: DBP
500 mg/Kg/day diluted in corn, canola or fish oil. Dams were sacrificed on day 20
of gestation and the anogenital distance and intratesticular testosterone levels were
evaluated in male offspring. All DBP treated groups exhibited a significant reduction
in both anogenital distance and intratesticular testosterone levels, when compared
to the groups that received the correspondent vehicles. There were no differences
between the DBP groups, indicating that the three different vehicles used
(corn, canola and fish oil) had no impact on testosterone production and anogenital
distance, an external marker of androgen action. In addition, no significant differences
were observed among vehicle-only groups (controls). It remains to be determined
whether these different vehicles can have any impact on other
phthalate-induced alterations such as gonocyte multinucleation in fetal testis.
Moreover, it is important to establish the impact of longer periods of maternal supplementation
(e.g., prior to pregnancy).
1612 IN UTERO EXPOSURE TO BISPHENOL A INDUCES
CHANGES IN THE DEVELOPING MAMMARY GLAND.
J. LaRocca, J. Pietruska and M. Hixon. Brown University, Providence, RI.
Bisphenol A (BPA), a chemical widely used in the production of plastics, is the subject
of recent controversy as to its effects as a reproductive toxicant. We are investigating
if in utero exposure to low to moderate doses of BPA induces phenotypic
and/or transcriptional changes in the adult mammary gland. Pregnant C57/Bl6
mice were exposed to either sesame oil as a negative control, a low dose (50μg/kg) of
BPA, a moderate dose (1,000μg/kg) of BPA, or diethylstilbestrol (2.0μg/kg) as a
positive control via oral gavage from gestational days 10 to 16. Offspring were sacrificed
at either postnatal day (PND) 25 or PND 56. Whole mount analysis indicated
that both mammary gland ductal outgrowth and the terminal end bud population
were significantly decreased at PND 25 for the 50μg/kg BPA and DES
exposure groups, but were unchanged for the 1,000μg/kg BPA group. However, by
adulthood mammary glands exposed in utero with BPA at both the moderate (50
μg/kg) and high (1,000 μg/kg) dose or DES (2 μg/kg) resulted in increased ductal
outgrowth as compared to sesame oil control glands. Real-time RT-PCR revealed
statistically significant increased expression of progesterone receptor, estrogen receptor
alpha, and g-coupled protein receptor 30 (GPR30) in PND 25 mammary
glands exposed in utero with 1,000μg/kg BPA. Interestingly, by adulthood GPR30
mRNA levels were significantly decreased in all exposure groups. Initial evidence
also suggests that mice exposed to 30 mg/kg Dimethylbenz[α]nthracene at PND 56
that were previously exposed in utero to 1,000 μg/kg BPA have increased mammary
hyperplasia by one year of age. We are currently conducting in vitro experiments
with BPA to differentiate between classical and non-classical estrogenic signaling
pathways. Our results suggest that in utero exposure to BPA alters postnatal mammary
gland development, causing a permanent change in mammary gland structure
and possibly increased susceptibility to cancer development following carcinogen
exposure. Furthermore, the pathways through which BPA elicits its response appear
to be dependent on the dose, resulting in a non-monotomic dose response.