The Toxicologist - Society of Toxicology

Of the >40,000 genes, 682 and 1,514 were significantly altered on day 2 and day 4,
respectively. Many are known to affect uterine morphology and function, including
transcription factors, secreted signaling molecules, hormonally responsive genes,
and immune response genes. To validate and extend the microarray findings, real
time RT-PCR was performed on altered genes at multiple time points from neonatal
genistein treatment to early pregnancy. To test alterations in hormone responsiveness,
control and genistein-treated mice were ovariectomized and then treated
with estradiol or progesterone. These studies suggested that abnormalities in expression
of transcription factors (FoxA2, Hox) and secreted signaling factors
(Wnt7a) during neonatal treatment led to permanent alterations in uterine morphology.
Furthermore, uterine hormone responses were dampened and/or delayed.
Taken together, these data suggest that exposure to genistein during female reproductive
tract development contributes to infertility by permanently altering gene
expression that controls normal uterine morphology and critical cellular responses
to hormonal cues of pregnancy. These findings may have implications for the use of
soy-based infant formula and subsequent female reproductive health.
2198 NEONATAL GENISTEIN EXPOSURE PERMANENTLY
DISRUPTS OVIDUCT GENE EXPRESSION AND TISSUE
ARCHITECTURE DURING PREGNANCY.
E. Padilla-Banks, W. Jefferson, S. M. Wagner and C. J. Williams. NIEHS,
Research Triangle Park, NC .
Approximately 20% of US infants consume soy-based infant formula and have
high circulating levels of genistein (GEN). Mice exposed neonatally to similar
serum GEN levels are infertile due in part to abnormal embryo development in the
oviduct. Histological analysis of the oviduct on day 2 of pregnancy revealed abnormal
vascular development, muscle disorganization and excessive epithelial proliferation.
Many epithelial cells were abnormally distended with intracellular mucopolysaccharide
material. Microarray analysis identified 1126 oviductal genes
significantly altered by neonatal GEN treatment, including transcription factors,
signaling molecules, and vascular development factors. To validate and extend the
microarray findings, real time RT-PCR was performed on oviducts collected on
postnatal day 5 (during treatment) and several additional time points through day
4 of pregnancy. The transcription factors Pitx1, Nkx3.1, and Six1 were aberrantly
expressed at all of these time points. Pitx1 and Six1 protein overexpression was confirmed
by immunohistochemistry and immunoblotting. In contrast, Wnt7a was
significantly downregulated during neonatal GEN treatment. The documented role
of Wnt7a in female reproductive tract development suggests that Wnt7a downregulation
in the neonatal period may explain some of the morphological abnormalities
observed. To determine how the altered oviductal environment impacted
preimplantation embryo development, embryos flushed from oviducts/uteri on
days 1-4 of pregnancy were analyzed. Fertilization was significantly delayed in
GEN treated mice, but developmental timing was recovered by the blastocyst stage.
Significantly, there was a lower trophectoderm/inner cell mass ratio in blastocysts
from GEN treated mice, suggesting that the abnormal oviductal environment affected
blastocyst differentiation. This alteration did not affect the number of live
pups delivered following blastocyst transfer experiments. Taken together, these
findings have implications for the use of soy-based infant formula and subsequent
female reproductive health.
2199 DELAYED TEMPORAL INCREASE OF HEPATIC HSP70
IN APOE-/- MICE WITH ACCELERATED
ATHEROSCLEROSIS INDUCED BY IN UTERO ARSENIC
EXPOSURE.
N. N. Ngalame, M. E. Feil, A. F. Micciche and J. States. Pharmacology/Toxicology,
University of Louisville, Louisville, KY.
Early life toxic exposures can predispose to adult disease. Chronic arsenic (As) exposure
is associated with increased cardiovascular disease (CVD). We showed that
in utero As exposure accelerates atherosclerosis underlying CVD in ApoE-/- mice,
but the mechanism is unknown. As is a hepatotoxicant, and liver disease increases
atherosclerosis risk. We hypothesize that in utero As exposure primes the liver for
susceptibility to other environmental insults, predisposing to liver disease and accelerated
atherosclerosis. Microarray analyses showed As increased Hsc70 and
Hsp70 mRNA expression in livers of 10 wk old mice. We examined developmental
expression of Hsc70 and Hsp70 and effects of As on DNA methylation in liver.
Pregnant mice were given drinking water with 85 mg/L NaAsO2 (49 ppm As) from
gestation day (GD) 8 - 18. Livers were obtained from GD18, and 3, 10 and 24 wk
old mice. As content in GD18 liver analyzed by ICP-MS was 350 ng/g indicating
As crossed placenta and reached fetal liver. Age-dependent Hsc70 and Hsp70 expression
and global DNA methylation were determined by western blot analyses
and methyl acceptance assay respectively. Gene-specific methylation was determined
in 10 wk old livers by bisulfite sequencing. As did not alter expression of
472 SOT 2011 ANNUAL MEETING
Hsc70. However, As induced Hsp70 at age 3 wk with increase at 10 wk, but return
to unexposed levels by 24 wk. Global DNA methylation was age-dependent, with
As having no effects. To determine if increased Hsp70 expression was due to epigenetic
effects of As, we analyzed Hsp70 promoter region and part of its CpG island.
The promoter region was 100% unmethylated in DNA of exposed and unexposed
mice. However, CpG island showed differential methylation with region-specific
hyper- and hypomethylation relative to unexposed mice. These results show in
utero As exposure induces temporal Hsp70 expression increase, suggesting a temporal
state of stress in the livers which can predispose the mice to developing atherosclerosis.
Supported in part by USPHS grants ES015812 and ES014443.
2200 CORRELATION BETWEEN POLYBROMINATED
DIPHENYL ETHERS (PBDES) AND PERSISTENT
ORGANIC POLLUTANTS (POPS) OR HEAVY METALS
DETECTED IN UMBILICAL CORDS.
Y. Igoshi 1 , S. Ochiai 1 , E. Todaka 1, 2 , Y. Matsuno 1 , S. Suzuki 4 , N. Shimojo 3 , Y.
Kohno 3 and C. Mori 1, 2 . 1 Bioenvironmental Medicine, Chiba University, Chiba City,
Chiba, Japan, 2 Preventive Medical Science, Chiba University, Chiba, Japan,
3 Pediatrics, Chiba University, Chiba, Japan and 4 Pediatrics, Shimoshizu National
Hospital, Yotsukaido, Japan.
The purpose of this study was to clarify the relationship between the levels of polybrominated
diphenyl ethers (PBDEs) and persistent organic pollutants (POPs) including
polychlorinated biphenyls (PCBs), pesticides, and heavy metals in human
umbilical cord. Eighty-six umbilical cords were collected at a maternity clinic in
Chiba City, Japan, and PBDEs, POPs and heavy metals in the umbilical cords were
analyzed by GC/MS. As the results, PBDEs, PCBs, lead, mercury, p,p’-DDT, p,p’-
DDE, trans-nonachlor, oxychlordane, HCB, mirex, and β-HCH were detected
from all the samples. Significant correlation (P