The Toxicologist - Society of Toxicology

167 STUDIES OF LONG-TERM PROTEIN CHANGES IN
RATS NEONATALLY TREATED WITH THE
CYANOBACTERIAL TOXIN β-N-METHYLAMINO-L-
ALANINE (BMAA) USING MALDI IMAGING MASS
SPECTROMETRY.
O. Karlsson, E. Roman, E. Brittebo and M. Andersson. Pharmaceutical
Biosciences, Uppsala University, Uppsala, Sweden. Sponsor: M. Stigson.
Most cyanobacteria (blue-green algae) found in aquatic and terrestrial environments
can produce the toxin BMAA. Dietary exposure to BMAA has been suggested
to be involved in the etiology of amyotrophic lateral sclerosis/Parkinsonismdementia
complex. Our previous studies have revealed an uptake of BMAA in the
hippocampus and striatum of neonatal mice. Furthermore, neonatal rats treated
with BMAA displayed acute but transient motoric disturbances and failed to show
habituation at juvenile age. In addition, neonatal BMAA administration induced
long-term learning impairments in adult rats.
The aim of the present study was to search for molecular correlates of changed
learning behavior in the brain of the adult rats used in the behavior studies. Wistar
rats were given BMAA on postnatal day 9–10 (200 or 600 mg/kg; sc injection) and
Matrix Assisted Laser Desorption Ionization (MALDI) Imaging Mass
Spectrometry (IMS) was used to assess regional distribution and protein levels at
adult age. Primarily MALDI IMS is focused on brain structures that are important
for spatial learning and memory formation, including hippocampus and striatum
that were previously demonstrated to have a selective uptake after neonatal exposure.
Brain sections were thaw-mounted onto MALDI compatible glass slides and
picoliter-sized droplets of sinapinic acid and 2,4,-dihydroxyacetophenone matrix
were deposited in arrays across the sections using a robotic microdispenser. Mass
spectra were acquired from each matrix deposition on a Bruker Ultraflex II
MALDI-TOF operated in linear mode. There were more than 300 small proteins
in the mass range between 2000-20 000 Da in every mass spectrum. The preliminary
data evaluation revealed that the BMAA-treated groups expressed a lower level
of 18 kDa Myelin basic protein (MBP) but not of the 14 kDa MBP isoform in the
striatum. Other proteins of unknown identity also demonstrated treatment-related
changes and could be involved in the BMAA-induced learning impairments.
168 EFFECT OF DEVELOPMENTAL CHLORPYRIFOS
EXPOSURE ON ENDOCANNABINOID
METABOLIZING ENZYMES IN THE BRAIN OF
JUVENILE RATS.
R. L. Carr and M. K. Ross. Center for Environmental Health Sciences, Mississippi
State University, Mississippi State, MS.
The endogenous cannabinoids 2-arachidonylglycerol (2-AG) and anandamide
(AEA) play vital roles during nervous system development including regulating axonal
guidance and synaptogenesis. The degradation of 2-AG and AEA is mediated
by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively.
Both enzymes are highly susceptible to inhibition by organophosphate
compounds in vitro, and acute in vivo exposure of adult animals to the agricultural
insecticide chlorpyrifos (CPS) results in moderate inhibition of both MAGL and
FAAH at 30 mg/kg and elevated the levels of the endocannabinoids at 100 mg/kg.
However, the effects of repeated exposure to lower levels of CPS, especially during
development, on endocannabinoid metabolism in the brain is not known. To determine
this, rat pups were exposed daily from days 10-16 to CPS (5 mg/kg) either
orally dissolved in corn oil (PO-Oil) or subcutaneously dissolved in DMSO (SC-
DMSO). A representative vehicle/route control group was present for each treatment.
At 6 hrs post-exposure, the activities of acetylcholinesterase (AChE), MAGL,
and FAAH were determined in the forebrain. AChE inhibition in the SC-DMSO
and PO-Oil groups was 75% and 50%, respectively. No significant effect on
MAGL activity was observed in either treatment group. However, FAAH activity
was virtually eliminated with inhibition ranging from 93% in the PO-Oil group to
100% in the SC-DMSO group. This extensive inhibition of FAAH suggests the potential
for accumulation of AEA resulting in activation of endocannabinoid transmission
during brain maturation. This could have implications for the appropriate
development of the endocannabinoid system leading to permanent alterations in
neuronal brain circuits and behavioral responses.
169 PHENCYCLIDINE (PCP) INDUCED CELL DEATH AND
CHANGES IN GENE EXPRESSION.
F. Liu 1 , N. Sadovova 3 , X. Zou 1 , X. Zhang 1 , L. Shi 2 , L. Guo 2 , F. Qian 4 , Z.
Wen 4 , T. Patterson 1 , J. Hanig 5 , M. Paule 1 , W. Slikker 1 and C. Wang 1 .
1
Division of Neurotoxicology, National Center for Toxicological Research, Jefferson,
AR, 2
Division of Systems Toxicology, National Center for Toxicological Research/U.S.
Food & Drug Administration, Jefferson, AR, 3
Toxicologic Pathology Associates,
Jefferson, AR, 4
Z-Tech, an ICF International Company at NCTR, Jefferson, AR and
5
5Center for Drug Evaluation and Research/U.S. Food & Drug Administration,
Silver Spring, MD.
Repetitive administration (perinatal) of PCP, an N-methyl-D-aspartate (NMDA)
receptor antagonist, may result in cortical apoptosis and long-lasting behavioral
deficits. The present study examined the potential neurotoxic effects of PCP on
postnatal day (PND) 7 rat pups and the possible underlying mechanism. PND 7
rats were subcutaneously administered 10 mg/kg PCP or saline (control) on PNDs
7, 9, and 11, and 24 hours later the rats were sacrificed and brain tissues were harvested
for RNA extraction and morphological assessments. RNA samples were collected
from frontal cortical levels and hybridized to Illumina Rat Ref-12 Expression
BeadChips containing 22,226 probes. The criteria used for selecting the differentially
expressed genes (DEGs) were a fold-change greater than 1.4 (up or down) and
a P-value less than 0.05 when comparing the PCP treatment group to the control
group. Based on these criteria, 19 genes were up-regulated and 24 genes were down
regulated. Among them, 6 up-regulated genes are pro-apoptotic, and 8 down-regulated
genes are anti-apoptotic factors, which may have the potential to induce apoptosis.
Using histochemical approaches, elevated neuronal cell death was evident in
PCP-treated brains. Consistent with previous observations, DNA microarray data
indicated a significant up-regulation of NMDA receptor NR1 subunit mRNA in
the PCP group. These findings support the working hypothesis that the apoptosis
produced by PCP is, at least in part, through the up-regulation of NMDA receptors,
which makes neurons possessing these receptors more vulnerable, after PCP
washout, to the excitotoxic effects of endogenous glutamate. Supported by
NCTR/NTP IAG # 244-07-0007 (E-2155).
170 RECOMMENDATIONS FOR DEVELOPING
ALTERNATIVE TEST METHODS FOR
DEVELOPMENTAL NEUROTOXICITY.
K. Crofton 1 , L. Buzanska 2 , S. Coecke 2 , H. Knaut 3 , P. Lein 4 , W. Mundy 1 , A.
Price 2 , A. Seiler 5 and A. Goldberg 6 . 1 Integrated Systems Toxicology, U.S. EPA,
Research Triangle Park, NC, 2 Joint Research Council, Ispra, Italy, 3 Skirball Institute,
New York University School of Medicine, NYC, NY, 4 Molecular Biosciences, U.S.
Davis, Davis, CA, 5 ZEBET, Berlin, Germany and 6 CAAT, Johns Hopkins University,
Baltimore, MD.
There is great interest in developing alternative methods for developmental neurotoxicity
testing (DNT) that are cost-efficient, use fewer animals and are based on
current scientific knowledge of the developing nervous system. Alternative methods
will require demonstration of their predictive ability as well as the development and
acceptance of a strategy for data interpretation. Therefore, an international group of
experts developed a set of recommendations to facilitate development of alternative
DNT test methods. The intent of the document is to engage the research community
in the development of in vitro and alternative DNT methods, to stimulate discussion
and to facilitate comparative evaluations of candidate models. Definitions
for endpoint, test system and test method were adopted from previously published
OECD guidance documents. Fourteen criteria deemed important for DNT test
method development are proposed to address issues of relevance to human neurodevelopment,
specificity, sensitivity and comparison of data within and between
laboratories. These criteria are applicable to new approaches and are meant to stimulate
input of new scientific knowledge into the development of alternative DNT
methods. This abstract does not necessarily reflect the policy of the U.S. EPA.
171 ASSESSING LATER-LIFE BEHAVIORAL PHENOTYPES
IN RESPONSE TO PRENATAL EXPOSURE TO
BENZOPYRENE.
M. McCallister 1 , M. Maguire 1 , A. Ramesh 3 and D. B. Hood 1, 2 . 1 Center for
Molecular and Behavioral Neuroscience, Meharry Medical College, Nashville, TN,
2
Department of Pharmacology and The Brain Institute, Vanderbilt University School
of Medicine, Nashville, TN and 3 Department of Biochemistry and Cancer Biology,
Meharry Medical College, Nashville, TN.
Benzo(a)pyrene is an environmental contaminant that is a member of the polycyclic
aromatic hydrocarbon (PAH) family. It was reported that environmental levels
encountered in the air of New York City can affect child IQ scores adversely.
36 SOT 2010 ANNUAL MEETING