DEQ-CFW_00004201

Toxicology 276 (2010) 79-84
Effe-ct of prenatal peroxisome pro] iferator-activated receptor CX (PPARU) ago is
on postnatal development
Prajakta S. Palkara, Cherie R. Andersona, Christina H. Ferrya, Frankj. Gonzalezb, Jeffrey M. Petersa,*
I Department of Veterinary and Biomedical Sciences and The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802,
United States
I Laboratory
y of Metabolism, National Cancer Institute, Bethesda, MD 208921, United States
ARTICLE IN FO
Article history:
Received 26 May 2010
Received in revised form 2 July 2010
Accepted 8 July 2010
Available online 15 July 2010
Keywords:
Peroxisome pro] iferator-activated
receptor -a
Postnatal development
Nuclear receptor
Prenatal exposure
1. Introduction
A B S T R A C T
Recent work indicates that PPARu is required for perfluorooctanoic acid (PFOA)-induced postnatal lethal-
ity resulting from prenatal exposure. The present study tested the hypothesis that relatively modest
activation of PPARa during prenatal development will cause postnatal lethality, similar to that observed
with PFOA, a relatively low affinity PPARa agonist. Female wild -type and Pparu-null mice were mated
overnight with males of the same genotype. The presence of a copulatory plug on the morning after
mating was indicative of pregnancy and considered gestation day (GD) 0. Plugged female mice were
fed either a control diet or one containing clofibrate (0.5%) or Wy-14,643 (0.005%) until GD18 or until
parturition. Mice were examined on GD18 or on postnatal day (PND) 20 following the prenatal expo-
sure period. Dietary administration ofclofibrate orWy-14,643 did not affect maternal weight or weight
gain, the average number of implantations, the percentage oflitter loss, the average number oflive /dead
fetuses, average crown -rump length, orthe average fetal weight on GD18 in either genotype. An increase
in relative maternal liver weight and elevated expression of PPARa target genes in maternal and fetal
livers on GD18 were observed, indicative of PPARa-dependent changes in both the maternal and fetal
compartments. However, no defects in postnatal development were observed by either clofibrate or Wy-
14,643 in either genotype by PND20. These results demonstrate that relatively low level activation of
PPARa by clofibrate or Wy- 14,643 during prenatal development does not cause postnatal lethality.
(0 2010 Elsevier Ireland Ltd. All rights reserved.
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated, soluble nuclear receptors that include three isoforms:
PPARu, PPARP (also referred to as PPAR8 or PPARP(8) and PPARy.
PPARot is expressed in most tissues but is noticeably higher in
liver, kidney and heart (Auboeuf et al., 1997; Braissant et al., 1996:
Braissant and Wahli, 4998) where it is known to regulate expression
of proteins required for fatty acid transport, catabolism, and energy
homeostasis (Peters et al., 2005). The fibrate class of hypolipidernic
drugs, phthalate monoesters and perfluorinated compounds are all
known to activate PPARot (Bility et al., 2004; Forman et al., 1997;
Maloney and Waxman, 1999; Wolf et al., 2008a). In addition to
its known essential role in the regulation of lipid homeostasis,
activation of PPAR(x also causes an increase in hepatocyte prolif-
eration leading to hepatocellular carcinoma in rodents (Hays et
* Corresponding author at: Department of Veterinary and Biomedical Sciences,
312 Life Sciences Building, The Pennsylvania State University, University Park, PA
16802, United States. Tel.: [phone]; fax: [phone].
E-mail address: jmp2 [e-mail] (I.M. Peters).
0300-483X/$ - see front matter 0 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox-2010.07.008
al., 2005; Peters et al., 1998, 4997; Reddy et al., 1980); humans
appear to be refractory to these effects (Gonzalez and Shah, 2008;
Klaunig et al., 2003; Peters, 2008; Peters et al., 2005). More recently,
evidence has also surfaced suggesting that PPARu is essential for
modulating postnatal lethality observed in rodents exposed to per-
fluorooctanoic acid (PFOA) during prenatal development (Abbott et
al., 2007).
PFOA is one of a number of perfluorinated compounds that are
capable of causing activation of PPAR(x (Wolf et al., 2008a). Per -
fluorinated compounds are not extensively metabolized in vivo
because of the strong covalent bond between carbon and fluorine
atoms (Ullrich and Diehl, 1971) and are hence environmentally per-
sistent (Lion et al., 2010). Recent studies show that exposure to
PFOA during prenatal development results in dose -dependent full -
litter resorptions, as well as delayed development and postnatal
lethality in CD-1 mice (Lau et al., 2006) and 129/Sv mice (Abbott et
al., 2007). These effects are mediated by PPARu, as they are found in
wild -type mice but not in Pparci-null mice (Abbott et al., 2007). Evi-
dence also exists suggesting that these effects are due to gestational
exposure to PFOA that may cause alterations in mammary gland
function but are not due to lactational exposure of PFOA (Lau et al.,
2006; White et al., 2007; Wolf et al., 2007). The present study was
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P.S. Palkar et al. / Toxicology 276 (2010) 79-84
designed to test the hypothesis that relatively modest activation of
PPARa during prenatal development will cause postnatal lethality,
similar to that observed with PFOA, a relatively low affinity PPAR(x
agonist.
2. Materials and methods
2.1. Animalstudies
Animal experiments were approved by the Institutional Animal Care and Use
Committee at The Pennsylvania State University, which conforms to the Guide for
the Care and Use of Laboratory Animals published by the National Institutes of
Health. Male and female wild -type and Pparce-null mice (Lee et al., 1995) on a 129iSv
genetic background (Akiyama et al., 2001) were used for this study.
2.1,1. Study design
Female wild -type or Ppara-null mice were mated overnight with male mice of
the same genotype, and examined for the presence of a copulatoryplug after mating.
The presence of a copulatory plug was considered indicative of successful mating
and designated gestation day (GD) 0. Pregnant female mice were weighed and ran-
domly assigned to one of three groups and fed either a control diet, a diet containing
0.5% clofibrate (Dyets, Inc., Bethlehem, PA) or a diet containing 0.005% Wy-14,643
(Dyets, Inc., Bethlehem, PA). Mice were fed these diets until GD18 or until partu-
rition. After parturition, all groups of mice were fed the control diet. Mice were
examined on either GD18 or on postnatal day (PND) 20. The dietary concentrations
of PPARa agonists were chosen in an attempt to model the relative ability of PFOA
to activate PPARu in the liver and cause approximately a doubling of relative liver
weight as shown by previous studies in rodent models (Lee et al., 1995; Marsman et
al., 1992; Wolfet al., 2008b), an effectwhich is known to be associated with increased
developmental delays and neonatal lethality (Abbott et al., 2007). The rationale that
this approach would achieve low level activation of PPARa is based in part on sev-
eral relationships. Dietary clofibrate at a dose of 0.5% causes an increase in rat liver
weight of —1.5-fold after 3 weeks of treatment, while 0.005% Wy-14,643 causes an
increase in rat liver weight of —2-fold after 3 weeks of treatment (Marsman et al.,
1992). This is consistent with the fact that clofibrate is less effective for increas-
ing PPARa-dependent reporter activity as compared to Wy-14,643 (Shearer and
Hoekstra, 2003). It is also known that PFOA is less effective at activating PPARa
as compared to Wy-14,643 (Maloney and Waxman, 1999) and that doses of PFOA,
capable of causing a modest (- 1.5-2-fold) increase in maternal liver weight, cause
marked developmental delay and neonatal lethality (Abbott et al., 2007). Clofibrate
was chosen as one model PPARa agonist because it is a relatively less effective ago-
nist (e.g. one that would cause low level activation) as compared to Wy-14,643 based
on cell based reporter assays, and is more comparable with the PFOA in terms of acti-
vating PPARa based on similar cell based reporter assays (Maloney and Waxman,
1999; Shearer and Hoekstra, 2003). The very low dietary level of the PPARa agonist
Wy-14,643 was selected in part because it is more effective at activating PPARa, and
should thus more closely model PPARu activation observed in response to PFOA.
These relationships were collectively used to establish a dosing paradigm that was
predicted to cause low level activation of PPARu.
For GD18 analyses, pregnant mice were euthanized by overexposure to carbon
dioxide, and livers were carefully dissected and snap frozen until later use. Gravid
uterine weights were recorded. For each litter, the number of live fetuses, dead
fetuses and resorption sites were counted. The sex of each fetus was determined,
crown to rump length was measured, and fetal and fetal liver weights were recorded.
Fetal livers were snap frozen after weighing for RNA analysis.
For PND20 analysis, pregnant mice were allowed to deliver their litters and day
of parturition was recorded. Pups were weighed on the clay of delivery and on PND7,
PND14 and PND20. The pups were observed daily to determine postnatal lethality,
and the onset ofeye openingwas examined as a measure of postnatal development.
Dams and pups were euthanized by overexposure to carbon dioxide on PND20 and
livers were obtained by dissection and snap frozen after weighing for RNA analysis.
2.2. Quantitative real-time PCR (gPCR) analysis
Total RNA was isolated from liver samples rising Ribozol (Amresco, Solon, OH).
For maternal fiver, four independent samples from four mice from each group were
used. For fetal liver, samples from one fetus randomly chosen from each of four
individual litters were used. For neonatal liver, samples from one pup represent-
ing each of four litters were used. The cDNA was generated using 2.5 µg total RNA
with Multi scribe Reverse Transcriptase kit (Applied Biosystems, Foster City, CA). The
mRNAs encoding the known PPARa target genes, cytochrome P450 4a10 (Cyp4a10)
and acyl-CoA oxidase 1 (Aco), were measured using gPCR analysis. The sequence
for the forward and reverse primers used to quantify mRNAs for Cyp4a10, Aco and
internal control, giyceraidehyde 3-phosphate dehydrogenase (Gapdh) are described
previously (Foreman et al., 2009). PCR reactions were carried out using SYBRI Green
Supermix for IQ(Quanta Biosciences, Gaithersburg, MD) in the iCycler and detected
using the MyiQ Real -Time PCR Detection System (Rio-Rad Laboratories, Hercules,
CA). The conditions used for PCR were 95 °C for 15 s, 94 °C for 10 s, 60 °C for 30 s,
and 72 °C for 30 s, repeated for 45 cycles. The PCR included a no template reaction
control for detecting contamination and genomic amplification. All reactions had
>85% efficiency. Relative expression levels of mRNA were analyzed for statistical
significance using ANOVA and post hoc tests.
2.3. Statistical analysis
Data were analyzed for statistical significance using analysis ofvariance and the
Tukey's post hoc test (Prism 5.0a, GraphPad Software Inc., San Diego, CA). The cri-
terion used to determine statistical significance was P <0.05. For fetal and neonatal
endpoints, statistical analysis revealed essentially identical results when the indi-
vidual or litter was used as the statistical unit (data not shown). Figure legends
indicate whether the individual or litter was used as the statistical unit.
3. Results
3.1. Effect of prenatal PPAR(x agonisrn on maternal and fetal
endpoints on GD18
Prenatal exposure to PFOA in pregnant female mice causes an
increase in resorptions and postnatal lethality in surviving offspring
(Abbott et al., 2007; Lau et al., 2006). The increase in postnatal
lethality in mice was associated with doses of PFOA where relative
liver weight is twice that of control as observed in non -pregnant
mice (Wolf et al., 2008b). Thus, the effect of prenatal exposure to
the PPARa agonists clofibrate and Wy-14,64.3 at doses that are also
associated with causing approximately a doubling of liver weight
in non -pregnant mice and rats (Lee et al., 1995; Marsman et al.,
1992), was determined in wild -type and Ppara-null mice. Average
maternal weight and average maternal weight gain during preg-
nancy were not influenced by exposure to 0.5% clofibrate or 0.005%
Wy-14,64.3 as compared to controls in both genotypes (Table 1). No
differences in the average number of implants per dam, the aver-
age number of live or dead fetuses per litter, the average number
of resorptions per litter, the percentage of litter loss, the average
fetal weight or the average crown to rump length were observed in
litters examined from mice of both genotypes treated with either
clofibrate or Wy-14,643 as compared to control (Table 2). Addition-
ally, no difference in the distribution of male and female fetuses
was observed by either treatment in either genotype compared to
control (Table 2).
PPAR(x agonists are known to increase replicative DNA syn-
thesis and hyperplasia in the liver through a PPARa-dependent
mechanism (Peters et al., 1998). Compared to controls, relative
maternal liver weight on GD18 was increased by clofibrate and
Wy-14,643 in wild -type mice but not in similarly treated Ppara-
null mice (Fig. IA). In contrast, relative fetal liver weight on GD18
was increased only modestly in wild -type mice by clofibrate but
not by Wy-14,643 as compared to control, while relative fetal liver
weight was unchanged by clofibrate and Wy-14,643 in Pparu-null
mice (Fig. 113). To determine the relative efficacy of clofibrate and
Wy-14,643 to activate PPAR(x in maternal and fetal liver, expres-
sion of the well characterized PPARa target genes Aco and Cyp4a10
was quantified. Expression ofAco and Cyp4a 10 mRNA was increased
by clofibrate and Wy-14,643 in both maternal liver and fetal liver
as compared to control, and these effects were not found in simi-
larly treated Pparcx-null mice (Fig.1 C-F). Interestingly, the relative
increase in expression of Aco and Cyp4a10 mRNA was higher in
Wy- 14,643 -treated fetuses as compared to the increase observed
in maternal liver (Fig. 1C-F). These data clearly demonstrate that
the doses of clofibrate and Wy-14,643 effectively activated PPARa
causing modest maternal liver hepatomegaly and increased expres-
sion of target genes known to modulate lipid catabolism.
3.2. Effect of prenatal PPARcx agonisrn on postnatal development
Since prenatal exposure to PFOA led to reduced survival of pups
and delayed development (as assessed by the onset of eye opening)
in wild -type mice but not in Pparu-null mice (Abbott et al., 2007),
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81
Table 7
Effect of prenatal PPARa agonism on pregnancy outcome on GD18.
Genotype
Wild -type
Ppara-n ul f
Diet
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Control
Clofibrate
Vly-14.643
Control
Clofibrate
Wy-14,643
Number of dams
9
8
7
8
10
13
Maternal weight (g) on GD18
34.3 + 2.6a
32.5 f 4.01
35.5- 6.71
30.8 3.51
30.4 - 4.9,
30.7 f 2.81
Maternal weight gain (g) on GD18
12.4 - 23a
10.63 L 1.81
12.9- 731
10.3 2.21
9.1 - 3.7,
11.5 f 2.51
Gravid uterus weight (g) on GD18
9.7 - 1.5'
7.4 f 1.1'
8.7 _y 3.91
7.0 1.91
6.3 - 3.4'
8.1 f 2.71
Implants per uterus (1)
8.2-2.1a
5.4 L2.1a
7.0-2.21
6.6 1.51
7.1-2.6a
6.4f2.41
Number of live fetuses per litter
7.0 -1.2a
4.612.0'
5.7 2.81
5.0 -1.71
4.7 - 2.81
5.812.2'
Number of dead fetuses per litter (D)
0.0 _ 0.01
0.010.0,
0.1= 0.46
0.1- 0.41
0.3 - 0.71
0.1 10.3a
Number of resorptions per litter(R)
1.2±1.61
0.811.01
1.1 1.26
1.5 1.51
2.1±2.01
0.711.01
%Litter loss =I(0-tR)/1'1001
12,4-15,26
123f17.4a
25.1-34.61
20.8,
36,1-33,81
9.4f13.01
Values represent the mean S.E.10. Values within a row with different letters are significantly different, P <- 0.05.
Table 2
Effect of prenatal PPARa agomsno, on fetal endpoints
on GD18,
Genotype Diet
Number of
Crown to rump
Body weight
Ratio of female
Ratio of finale
fetuses/litter'
length (mm)b
(g)b
fetuses to total
fetuses to total
number of
number of
fetusesa
fetusesa
Wild -type Control
7.0 f 1.2a
20.1 f 1.4a
1.0 f 0.2a
47 51
53 f 5a
Clofibrate
4.6 f 2.Oa
19.6 4- 1.2a
1.0 4- 0.2a
38 + 71
62 4 7a
Wy-14,643
5.7 f 2.8a
20.1 4- 1.1a
1.0 4- 0.1a
40 + 71
40 4 7a
PPAR a -null Control
5.0 4- 1.7a
20.2 f 09a
1.0 4 0.2a
61 _ 81
39 4- 8a
Clofibrate
4.7 -L 2.8a
18.9 _L 1.5a
0.9 _L O.la
49 91
51 _L 9a
Wy-14,643
5.8 -L 2.2a
20.2 -L 1.0a
1.0 _L O.la
51 5a
49 _L 5a
Values represent the mean+ S.E.M. Values within a column with different letters are significantly different, P<_ 0.05
a The statistical unit was the litter.
b The statistical unit was the individual.
postnatal development was assessed in the present study follow-
ing prenatal exposure to either clofibrate or Wy-14,643. The day
of parturition was not affected by prenatal exposure to either clofi-
brate or Wy-14,643 in either genotype (Table 3). Postnatal lethality
of pups up until PND20 was not different between clofibrate or
Wy- 14,643 -treated wild -type or Pparu-null mice as compared to
controls (Fig. 2A). Similarly, the onset of eye opening and postna-
tal weight gain was not influenced by prenatal exposure to either
clofibrate or Wy-14,643 in either genotype as compared to con-
trols (Fig. 213 and C). Additionally, no differences in the distribution
of male and female pups were observed by either treatment (data
not shown) and no changes in postnatal weight gain between male
and female pups in the different treatment groups were observed
(Table 3). Relative maternal liver weight (data not shown) and rela-
tive pup liver weight (Fig. 3) were not changed on PND20 following
prenatal exposure to either clofibrate or Wy-14,643 in either geno-
type as compared to control. Similarly, relative expression of the
PPARu target genes Aco and Cyp4a 10 in maternal liver was not dif-
Table 3
Effect of prenatal PPARa agonism on pregnancy outcome and postnatal weight gain
ferent on PND20 following prenatal exposure to either clofibrate
or Wy-14,643 in either genotype as compared to control (data
not shown). Compared to control, relative expression of Aco and
Cyp4al0 mRNA in pup liver was not different on PND20 following
prenatal exposure to either clofibrate or Wy-14,643 in wild -type or
Pparu-null mice (Fig. 313 and C).
4. Discussion
Previous studies demonstrated that prenatal exposure to PFOA
results in dose -dependent full -litter resorptions, delayed develop-
ment and postnatal lethality in mice (Abbott et al., 2007; Lau et
al., 2006). As these effects are found in wild -type mice but not in
Pparex-null mice, this demonstrates that these effects are mediated
by PPARcx (Abbott et al., 2007). Cross -fostering studies established
that gestational exposure to PFOA, rather than lactational expo-
sure to PFOA, is required to elicit defects in postnatal development
including delays in the onset of eye opening and early lethality
Genotype
Treatment group
Day of
Number of
Average number
Pup weights (g)
parturition,
litters
ofpups/litterb
Female
Male
PNDOb
PND14a
PND211
PND14a
PND21a
Wild -type
Control
19.4 4 0.2a
12
4.3 4 0.6
1.4 f 0.3a
6.6 f 0.2a
7.1 + 0.4a
6.2 _- 0.2a
6.7 4- 0.4a
Clofibrate
19.4 4 0.3a
7
3.3 4 0.3
1.5 4- 0.2a
8.6 f 0.3a
10.0 + 03a
8.3 _ 0.2a
9.5 4- 0.3a
Wy-14,643
19.440.2a
7
4.040.9
1.54-O.la
7.2f0.21
8.4+03a
7.2_ 0.3a
8.34-0.4a
Ppara-null
Control
19.6 f 0.2a
8
5.3 10.7
1.5 f 0.3a
6.1 f 0.2a
6.7 0.2a
6.5 ± 0.2a
7.3 -L 0.3a
Clofibrate
19.3 1 0.2a
8
4.6 10.4
1.4 1 0.2a
7.4 -L 0.3a
8.5 0.2a
7.0 + 0.2a
8.2 -L 0.2a
Wy-14,643
19.6f0.2a
11
5.6f0.4
1.5f0.2a
5.9f0.1a
7.2 0.2a
6.0-O.la
7.4f0.3a
Mice were fed either a control diet or one containing 0.5% clofibrate or 0.005% Wy-14,643 during gestation. Mice were allowed to deliver and the day of parturition was
recorded. After parturition, mice were provided only the control diet. The number of pups born per litter and pup weight was recorded on PNDO (clay of delivery). Body
weight was measured for male and female pups until PND21. Values represent the mean f S.E.M. Values within a column with different letters are significantly different,
P < 0.05.
The statistical unit was the individual.
b The statistical unit was the litter.
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82
P.S. Palkar et al. / Toxicology 276 (2010) 79-84
(A)
(C)
(E)
(+""+)
Maternal (B)
(D)
(F)
Z
W
E
0
0 Control
0 Clofibrate (0,5%)
0 Wy-14,643 (0M5%)
Fetal
Fi&l. Effect of prenatal PPARa agonism on maternal and fetal endpoints on GD18. Pregnant female wild -type (+/+)or Pparry-n ull (—/—)in ice were fed either a control diet or
one containing clofibrate (0.5%) or Wy-14,643 (0.005%) until GD18. Relative maternal (A) and fetal (B) liver weight (liver weight (g)/boclyweiglit (g) x 100) on GD18. Relative
expression of the PPARa target genes Aco (C and D) and Cyp4a10 (E and F) in maternal (C and E) and fetal (D and F) liver on GD 18 was measured by qPCR as described in
Section 2. Values are the average normalized fold change as compared to vehicle control and represent the mean S.E.M. The statistical unit was the individual. Values with
different letters are significantly different, P< 0.05, as determined byANOVA and Tukey's test.
(Wolf et al., 2007). Since PFOA is known to cause activation of
PPARa, the present study tested the hypothesis that relatively low
level activation of PPARa during prenatal development will cause
postnatal lethality, similar to that observed with PFOA, a relatively
low affinity PPARa agonist. Dietary administration of clofibrate
and Wy-14,643 during prenatal development caused a PPARa-
dependent increase in maternal liver, consistent with the known
mitogenic activity associated with PPAR(x activation in liver (Peters
et al., 1998). Similarly, a PPARci-dependent increase in expression
of the PPARu target genes, Aco and Cyp4a 10, was also observed in
both maternal and fetal liver on GD18 providing direct evidence
that PPARa activity was increased in both maternal and fetal com-
partments. Surprisingly, prenatal exposure to the PPARa agonists
clofibrate or Wy-1 4,643 did not cause any developmental anoma-
lies assessed on GD 18, nor did it cause any developmental delays in
eye opening or postnatal lethality of pups. These results are similar
to those previously reported with perfluorobutyrate (PFBA) where
no adverse developmental toxicity was observed following prena-
tal exposure (Das et al., 2008). This is of interest because PFBA is a
short -chain perfluorinated chemical that has shorter half-life than
PFOA and a weaker potency for PPARa activation as compared to
PFOA (Chang et al., 2008; Wolf et al., 2008a).
These studies do not dispute the fact that prenatal PFOA expo-
sure in mice causes neonatal lethality through a PPARot-dependent
mechanism (Abbott et al., 2007). Moreover, the reason why prena-
tal exposure to PFOA causes PPARa-dependent postnatal lethality,
while prenatal exposure to either clofibrate orWy-14,643 does not,
cannot be determined from this study. This disparity could be due
in part to differences in gene expression resulting from prenatal
exposure to the different compounds. It is also possible that this
disparity is due in part to differences in bioaccumulation. PFOA is
known to persist in environment and is not metabolized exten-
sively in vivo because of the strong covalent bond between carbon
and fluorine (Ullrich and Diehl, 197 1). In mice, the half-life of PFOA
has been estimated to be 15.6 days (Lou et al., 2009) whereas
clofibrate and Wy-14,643 have comparatively shorter half-lives.
For example, the half-life of clofibrate in humans is 15 h because
it is readily absorbed from gastrointestinal tract, metabolized by
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P.S. Palkar et al. / Toxicology 276 (2010) 79-84
83
(A)
P
Q
3�
10
Postnatal day
Postnatal day
Postnatal day
* (+I+) Control
-a (—I—) contral
(+/+) cl:oftrate
(4—) clafibrate
WY-1 4643
Wy- 14,643
Fi&2. Effect ofprenatal PPARu agonism on postnatal development. Pregnant female
wild -type (+/+)or Ppany-null (--/--)mice were fed either a control diet or one con-
taining clofibrate (0.5%) or VVy-14,643 (0.005%) until parturition, after which mice
were fed control diet until PND20. Mice were observed daily for (A) postnatal lethal-
ity and (B) the onset of eye opening. The pups were weighed on PNDO, 7,14 and 20
(C). Values are the average normalized fold change as compared to vehicle con-
trol and represent the meant S.E.M. The statistical unit for (A) and (B) was the
litter, the statistical unit for (C) was the individual. Values with different letters are
significantly different, P < 0.05, as determined by ANOVA and Tukey's test.
CYP3A4, and excreted (Miller and Spence, 4998). Thus, prenatal
exposure to PFOA could cause accumulation of PFOA in fetal liver
that subsequently influences postnatal development due to more
sustained PPAR(x activity, while clofibrate and Wy-14,643 are less
likely to result in this effect. This idea is supported by the observed
PPARa-dependent increase in relative liver weight in PND22 pups
from PFOA-exposed dams at doses <1.0 mg/kg (Abbott et al., 2007).
In contrast, results from the present studies show that relative liver
weight in PND20 pups from clofibrate orWy-1 4,643-exposed dams
is not different than controls and no changes in expression of the
PPARa target genes Aco and Cyp4al0 levels are found. Combined,
these findings suggest that prenatal exposure to PFOA could cause
accumulation in fetal liver that influences postnatal development
through PPARa-dependent mechanisms, while clofibrate and Wy-
14,643 do not.
Several studies have examined the effects of either prenatal
or neonatal exposures to lactating rodents treated with various
PPARa ligands, including Wy-14,643, nafenopin, clofibrate, ciprofi-
brate, and diethylhexyl phthalate (DEHP) (Cibellietal., 1988; Cimini
et al., 1994; Fahl et al., 1983; Singh and Lazo, 1992; Stefanini
et al., 1989, 1999, 1995; Wilson et al., 1991). Collectively, these
studies show that exposure to PPARa agonists induces both per-
oxisome proliferation and increased expression of PPARa target
genes (e.g. Aco, Cyp4al0) in fetal and neonatal rodents. Interest-
ingly, 14-day-old rat pups exhibit enhanced sensitivity to PPARa
17*1 Control
Fig 3. Effect of prenatal PPARa agonism on pup liver endpoints on PND20. Pregnant
female wild -type (+/+)or Ppara-null (—/—)mice were fed either a control diet of one
containingc1ofibrate (0.5%) or Wy-14,643 (0.005%) until parturition. (A) Relative pup
liver weight (liver weight (g)/body weight (g) x 100) on PND20. Relative expression
ofthe PPARa target genes Aco (B) and Cyp4alO (C) in pup liver was measured on
PND20 by qPCR as described in Section 2. Values are the average normalized fold
change as compared to vehicle control and represent the mean 4- S.E.M. The statis-
tical unit was the individual. Values with different letters are significantly different,
P < 0.05, as determined by ANOVA and Tukey's test.
activity as compared to older rat pups (Dostal et al., 1987). This is
the first evidence suggesting that neonatal rodents are more sen-
sitive than adults to PPARet activation. Results from the present
studies are consistent with this idea because the relative increase
in expression of Aco mRNA resulting from prenatal exposure to
both clofibrate and Wy-14,643 was higher in fetal liver on GD18
as compared to maternal liver. While this effect was not found
with the increase in expression of Cyp4al0 mRNA following pre-
natal exposure to clofibrate, relatively higher Cyp4a10 mRNA was
found in fetal liver on GD18 as compared to maternal liver as a
result of prenatal exposure to Wy- 14,643. The significance of this
apparent difference in sensitivity to PPARa agonism remains to be
determined.
Conflict of interest statement
JMP has been retained as an expert consultant by the 3M Com-
pany. PSP, CRA, CHF and FJG have no competing interests.
DEQ-CFW-00004205

84
P.S. Palkar et al. / Toxicology 276 (2010) 79-84
Acknowledgements
The studies were supported by unrestricted gifts from the 3M
Company and Dupont Haskell Global Centers of Health and Envi-
ronmental Sciences.
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