The Toxicologist - Society of Toxicology

effect on uterine weight or vaginal patency in any treatment group. Results from
this three-generation study thus far do not provide evidence for a transgenerational
epigenetic inheritance phenomenon.
2193 MATERNAL DHA SUPPLEMENTATION ATTENUATES
HYPEROXIA-INDUCED LUNG DEVELOPMENTAL
DEFICITS IN ADULT MICE.
L. K. Rogers 1, 2 , K. Dingess 1, 2 , M. Velten 1 and T. E. Tipple 1, 2 . 1 Center for
Perinatal Research, The Research Institute at Nationwide Children’s Hospital,
Columbus, OH and 2 Department of Pediatrics, The Ohio State University,
Columbus, OH.
Preterm infants often require life-saving supportive therapies such as supplemental
O2. Newborn mice exposed to hyperoxia have demonstrated decreased alveolarization
similar to newborn infants. Docosahexanoic acid (DHA) is a long chain fatty
acid that attenuates inflammatory responses. We tested the hypothesis that DHA
administration to pregnant and nursing dams would decrease inflammatory responses
and preserve lung growth which would result in improved lung function at
28 days of age. Dams were fed a purified control or DHA-supplemented diet beginning
at E16. After birth, the pups were exposed to >85% O2 or RA for 14 days.
After 14 days, both groups were returned to standard diets and RA until 28 days.
Lung structure was assessed by morphometric analyses and pulmonary function
was assessed by FlexiVent (SCIREQ). Protein levels of COX-2 and solulable receptor
for advanced glycation end products (sRAGE) were analyzed by western blot.
Mice that were DHA supplemented and exposed to hyperoxia during the newborn
period demonstrated 1) increased alveolarization (alveolar number, 103.9±1.9 vs
65.1±3.5; alveolar area, 1516±51 vs 2058±69 microns2) and 2) improved lung
function (tissue elastance, 24.9±1.6 vs 32.0±2.1 ml/cmH2O; tissue compliance,
0.042 vs 0.035 ml/cmH2O, DHA vs control diet, respectively) compared to the
control diet hyperoxia exposed mice at 28 days of age. Neonatal hyperoxia exposure
caused a sustained increase in COX-2 and sRAGE protein expression at 28 days
that was attenuated in the DHA supplemented mice. Hyperoxia exposure during
the neonatal period causes sustained lung deficits in adulthood. DHA administration
to the dam dampened inflammation in the pups and improved lung growth
and function in adulthood potentially though through RAGE or COX-2 mediated
signaling pathways. We speculate that maternal DHA supplementation will be a
beneficial intervention in preventing lung disease in premature infants.
2194 PRENATAL AND POSTNATAL CIGARETTE SMOKE
EXPOSURE INCREASES SUSCEPTIBILITY TO ADULT
ONSET DISEASE.
J. L. Lyon and T. Gordon. Environmental Medicine, New York University School of
Medicine, Tuxedo, NY.
Rationale: The CDC reports that an estimated 10-12% of the women who delivered
a child, smoked during pregnancy, and of the 45% of female smokers that quit
during pregnancy, more than half relapsed after delivery, potentially exposing more
than 2 million babies to cigarette smoke (CS) during early development. CS is a
complex chemical mixture with carcinogenic properties, and many of the chemical
constituents can pass the placental barrier. CS condensate has been shown to alter
the methylation patterns of DNA, potentially altering gene expression patterns.
Given the delicate nature of fetal and neonatal development, CS exposure during
these periods may change the expression patterns of critical genes, predisposing the
offspring to diseases later in life. This study investigates the relationship between
prenatal and early postnatal CS exposure and adult onset disease. Methods:
Pregnant C57BL/6J mice were exposed via whole body exposure to 5mg/m3 mainstream
CS for 3 hrs/day, 5 days per week during gestation. For postnatal exposures,
the offspring were exposed to 5 mg/m3 environmental tobacco smoke for 3 hrs/day,
5 days/ week, from day 4 post partum until day 23 post partum. Total RNA was extracted
from the liver, and hybridized to Affymetrix Mouse Gene 1.0 ST arrays to
analyze differential gene expression. Results were confirmed using real time RT-
PCR. Results: Prenatal and postnatal CS exposures resulted in gene expression
changes. A predominance of upregulation was noted in both groups. The apoptosis
related genes growth arrest and DNA damage inducible gamma 45 and polo-like
kinase 3 were found significantly upregulated in both exposures. Two genes involved
in the bioavailability of insulin like growth factor, insulin-like growth factor
binding protein (IGFBP) 1 and 2, were upregulated in the postnatal (IGFBP-1)
and prenatal groups (IGFBP-1 and IGFBP-2). Conclusion: Prenatal and postnatal
CS exposure resulted in changes in gene expression in pathways that could result in
adult onset diseases such as cancer and diabetes.
2195 A DIRECT LC/MS/MS METHOD FOR
DETERMINATION OF β-ALANINE IN HUMAN
PLASMA.
C. Chen, C. Cunningham and J. E. Eble. Critical Path Services, Garnet Valley, PA.
Sponsor: A. Chappelle.
Beta-alanine, a non-essential amino acid, is an important factor in building up
muscle mass and strength. Abnormal beta-alanine levels in human diet have been
reported to cause diseases such as cardiovascular disease, or symptoms such as
paraesthesia, resulting from high blood-plasma concentrations of beta-alanine.
Therefore, monitoring beta-alanine levels in human plasma samples may play a role
in disease diagnosis and prevention. An LC-MS/MS method has been developed
for use in this monitoring. The method can detect beta-alanine at level of detection
(LOD) of 2 ppb in water. The limit of detection and quantification (LOQ) in
plasma will be presented. Quantitative analysis of molecules below 100 daltons can
be a challenge. Most of the existing methods for amino acid analysis involve a derivatization
step, which is time-consuming. When electrospray ionization (ESI) is
used as the ionization method, plasma samples present an additional challenge as
they contain phospholipids which can result in matrix effects, specifically ion suppression.
The rapid resolution method presented here uses an internal standard
rather than derivatization for quantitative analysis. The instrument utilized is an
Agilent 1200 HPLC and 6410 Triple Quadrupole MS equipped with a hot box upgrade
operated in positive mode.
2196 DIFFERENTIAL EFFECTS OF ADULT VERSUS
PRENATAL HG EXPOSURE ON THE INNATE IMMUNE
RESPONSE TO CVB3 INFECTION.
K. L. Speiran 1 , D. L. Shirley 1 , E. K. Silbergeld 2 and J. F. Nyland 1, 2 . 1 Pathology,
Microbiology & Immunology, University of South Carolina School of Medicine,
Columbia, SC and 2 Environmental Health Sciences, Johns Hopkins Bloomberg School
of Public Health, Baltimore, MD.
Exposures to mercury (Hg) compounds alter the immune system in adults; prenatal
exposures alter the ontogeny of the immune system. We examined the effects of
exposures to Hg in adults and in fetuses, and interactions of these exposures to later
infection by Coxsackievirus (CVB3), a common infection that can, in susceptible
individuals, lead to autoimmune myocarditis. Because of the role of matrix metalloproteinase
(MMP) activation in the development of the dilated heart phenotype
observed in the end stages of autoimmune myocarditis we examined effects of Hg
on MMP as well as on IL-6 and IL-1β, cytokines that are involved in MMP activation.
In the first study, exposure of adult female mice to Hg changed the innate immune
response to CVB3 and increased subsequent severity of chronic autoimmune
myocarditis. FACS analysis showed that this was accompanied by downregulation
of costimulatory molecule (CD80/86) and upregulation of TLR4 expression on
peritoneal macrophages and mast cells during the innate immune response.
Further, MMP-9 expression was decreased in splenic macrophages. IL-6 levels in
the spleen were elevated. In the second study, we examined the consequences of
prenatal exposure to Hg and later responses to CVB3. Prenatal exposure to Hg
changed the ontogeny of the immune response and subsequent response to adult
infection. Prenatally exposed females downregulated TLR4 and upregulated MMP-
9 expression in splenic macrophages following CVB3 infection. These results
demonstrate the important role of the developmental state of the immune system
on the immunotoxic effects of Hg. They suggest that Hg exposure in utero may
change susceptibility factors relevant to adult immune response to infection.
Funding provided by NIEHS.
2197 NEONATAL GENISTEIN EXPOSURE CAUSES
IMPLANTATION FAILURE BECAUSE OF PERMANENT
ALTERATIONS IN UTERINE MORPHOLOGY AND
STEROID HORMONE RESPONSES.
W. Jefferson, S. M. Wagner, E. Padilla-Banks and C. J. Williams. NIEHS,
Research Triangle Park, NC .
Approximately 20% of US infants consume soy-based infant formulas and have
high serum levels (1-5 uM) of the phytoestrogen genistein. Female mice exposed
neonatally to genistein are infertile in part because the uterus does not support implantation.
We hypothesize that genistein treatment permanently disrupts gene expression
and leads to morphological abnormalities in the uterus that are inconsistent
with successful implantation. Dramatic differences in uterine morphology
were observed in genistein treated mice, including excessive deposition of extracellular
matrix, minimal endometrial stroma, lack of glandular epithelium, and abnormal-appearing
luminal epithelial cells. Microarray analysis was performed on uterine
mRNA from control and genistein-treated mice on days 2 and 4 of pregnancy.
SOT 2011 ANNUAL MEETING 471