The Toxicologist - Society of Toxicology

7d. 28d after TMT injury there was a 4-fold increase in spDiI+ cells in the dentate
gyrus granular and subgranular zone. Markers for astrocytes (GFAP) or neurons
(NeuN) rarely co-localized suggesting the migrated spDiI+ cells remain in an undifferentiated
state. TMT injury prevented the physiological loss of newly born
(BrdU+) cells after 28d, either by increasing cell survival or recruitment of BrdU+
cells from the SVZ. Less than 15% of BrdU+ cells co-labeled with spDiI. To assess
the role of cell proliferation in recovery from injury, mitosis was reduced by cranial
exposure to 10gy gamma radiation 48hr prior to TMT injection, decreasing newlyborn
cells by 70%. TMT-induced tremors increased in severity and duration in irradiated
mice, but tremors in all mice ceased by 5d post-TMT. TMT exposure
caused prominent thrombomodulin staining in the parenchyma of the hippocampus
dentate gyrus, indicating extravasation of blood-borne factors specifically at the
site of injury. Data suggest that repair/recovery from TMT injury may include migration
of SVZ cells into the injury site in addition to increased proliferation and
survival of new neurons produced from endogenous hippocampal stem cells.
Supported by ES011256, ES05022, and ES07148.
1376 EFFECTS OF DIFFERENT FORMS OF COPPER ON
HUMAN CNS DERIVED CELL LINES.
S. S. Shaligram and A. Campbell. Pharmaceutical Sciences, Western University of
Health Sciences, Pomona, CA.
Copper is considered an essential metal for living organisms. However, excess copper
is toxic owing to its ability to generate reactive oxygen species (ROS). This
metal is directly linked to Menkes and Wilson’s diseases. It is also implicated in agerelated
neurodegenerative disorders such as Alzheimer’s disease. Copper is known to
cause oxidative stress in the CNS. The purpose of this study was to study cellular response
to soluble (copper sulfate) or insoluble (copper oxide) forms of this redoxactive
metal. We examined the formation of reactive oxygen species in human cell
lines derived from the CNS after dosing them with different concentrations (0.5
μM- 625 μM) of copper sulfate or copper oxide. We assessed cell number and ROS
formation after either 4 hr or 24 hr of copper exposure. In the SK-N-SH cells (neuronal),
the number of cells decreased on exposure to the highest concentration of
copper sulfate after 4 hr. After 24 hr exposure, the cell number increased at lower
doses but decreased nonsignificantly at higher doses. With copper oxide, we saw a
dose-dependent decrease in cell number after 4 hr and 24 hr. In A-172 cells
(glioblastoma), the cell number decreased at higher doses with copper sulfate after
4 hr but showed no change at 24 hr. Exposure to copper oxide caused a dose-dependent
decrease in cell number at both time points but the effect was more pronounced
after 24 hr exposure. The neuronal cells showed a decrease in ROS formation
after 24 hr with copper sulfate but showed no change after 4 hr, while the
glioblastoma cells showed an increase in ROS formation at the higher concentrations
of copper oxide after 24 hr exposure. Based on these results, we conclude that
both forms of copper have some effect on the given cell lines but the response to
copper oxide is more pronounced.
1377 MECHANISM OF BRAIN COPPER (CU) TRANSPORT
AND ACCUMULATION: INFLUENCE OF IRON
DEFICIENCY.
A. D. Monnot 1 , S. Ho 1 , G. Robinson 2 , Y. Pushkar 2 and W. Zheng 1 . 1 Health
Sciences, Purdue University, West Lafayette, IN and 2 Physics, Purdue University, West
Lafayette, IN.
Cu and iron (Fe) are essential for brain development and metabolism. Previous
studies from this lab have shown an inverse physiologic relationship between Fe and
Cu in the central nervous system. Despite its critical role in normal brain function,
knowledge on Cu transport, distribution, homeostatic regulation, and the impact
of Fe status, remains largely unexplored. This study was designed to investigate the
regulation of Cu transport by Fe status at the blood-brain barrier (BBB) and bloodcerebrospinal
fluid barrier (BCB), as well as the distribution of Cu in the brain.
Rats were divided into 3 groups with special diets at libertum for 4 weeks: control
(35 mg Fe/kg), Fe deficient (FeD) (3-5 mg Fe/kg), and Fe overload (FeO) (20 g carbonyl
Fe/kg) groups. Serum Fe status, either FeD or FeO, was confirmed by total
Fe, unsaturated Fe binding capacity, total Fe binding capacity, and transferrin saturation
in serum. In situ brain perfusion of 64Cu demonstrated that the rate of Cu
transport was significantly faster in brain parenchyma (+92%) and capillaries
(+500%) in FeD rats than those in controls. In contrast, FeO treatment significantly
reduced the rate of 64Cu transported into brain parenchyma (~50% of controls).
Additionally, Cu clearance from the CSF was greater in FeD than in control
animals as demonstrated by ventriculo-cisternal brain perfusion, suggesting an increased
Cu uptake from the CSF by the BCB. Results from mRNA and protein expression
studies indicated that FeD was responsible for the up-regulation of
DMT1, but not CTR1, at both the BBB and BCB, which may contribute to the
enhanced Cu uptake found at the barriers. X-ray fluorescence imaging indicated
Cu accumulation in the subventricular zone (SVZ) of the lateral ventricles. An elevated
Cu level (+25%), although not statistically significant, was found in the SVZ
of FeD. These results suggest that DMT1 regulation at the brain barriers, but not at
SVZ, contribute to Cu accumulation and regulation during FeD in the brain.
(Supported by NIH/RO1-ES008146)
1378 PERSISTING NEUROCHEMICAL EFFECTS OF
DEVELOPMENTAL COPPER EXPOSURE IN WILDTYPE
AND METALLOTHIONEIN 1 AND 2 KNOCKOUT MICE.
A. Petro 1 , H. Sexton 1 , C. Miranda 1 , A. Rastogi 1 , J. H. Freedman 3 and E. D.
Levin 1, 2 . 1 Psychiatry, Duke University Medical Center, Durham, NC, 2 Integrated
Toxicology and Environmental Health Program, Duke University, Durham, NC and
3 NIEHS, Research Triangle Park, NC.
Metallothioneins are small proteins, which are crucial for the distribution of heavy
and transition metals. This series of studies was conducted to determine the role of
metallothioneins on metal-induced neurotoxicity during development. Previously,
we found that knockout of MT 1 and 2 potentiated the cognitive impairment
caused by developmental mercury exposure. The current study examined the neurocognitive
effects of developmental copper supplementation. Wildtype and MT 1
and 2 knockout mice (MTKO) were given 10 or 50 mg/l of supplemental copper
during gestation and until weaning. Mice of both genotypes with no added copper
served as controls. When the mice were young adults they were trained for 18 sessions
on the win-shift 8-arm radial maze test of spatial learning and memory. Male
MTKO mice with no added copper were significantly impaired on radial-arm maze
choice accuracy relative to wildtype controls. This MTKO-induced impairment
was significantly attenuated by addition of 10 mg/l but not 50 mg/l of copper supplementation
during development. Neurochemical analyses showed that MTKO
caused a significant overall increase in serotonin in all of the regions studied: the
frontal cortex, posterior cortex, hippocampus, striatum, midbrain, and brainstem.
MTKO also caused a significant increase in norepinepherine in the brainstem and
hippocampus. In wildtype mice, copper supplementation during development
caused a significant decline in dopamine and norepinepherine levels in the midbrain
and dopamine in the frontal cortex. These effects were blocked by knockout
of MT 1 and 2.
(Supported by NIH ES10356)
1379 PERSISTENT OLFACTORY DEFICITS FOLLOWING
INTRANASAL CADMIUM EXPOSURE CAN BE
REHABILITATED BY TRAINING ON AN OLFACTORY
DETECTION TASK.
D. J. Turkel, A. H. Moberly, L. Czarnecki and J. P. McGann. Psychology
Department, Rutgers University, Piscataway, NJ.
We have previously shown that acute intranasal exposure to cadmium disrupts olfactory
nerve function, including a large decrease in odorant-evoked neurotransmitter
release, reduced innervation of the olfactory bulb, and profound impairment
on an olfactory detection task in the mouse. These deficits persist for at least four
weeks. However, some olfactory nerve function remains intact following cadmium
exposure, which could in principle be sufficient to detect an odorant. We hypothesized
that post-exposure olfactory training could potentially rehabilitate detection
performance. C57Bl/6 mice were trained to perform an odorant-guided go/no-go
odor detection task in which they sampled at an odor-port and then received a sucrose
reward for a nose poke in a dipper port if and only if the odorant was presented.
Incorrect responses were punished by a lengthened intertrial interval. After
mice achieved criterion performance, they were anesthetized and received bilateral
intranasal instillations of either 20 μg cadmium/side or vehicle control. They were
randomly assigned to either a 2-day delay or 10-day delay group. Cadmium-exposed
mice tested at either time point were profoundly impaired on the detection
task, with an average d’ of about 0.15 in both groups. After testing, the worst performing
mice from the 2-day group were trained daily on the discrimination task
until they again achieved criterion performance. Remarkably, by Day 10 this
group’s performance was not only significantly better than that of the untrained
group at Day 10, but comparable to baseline performance. Rehabilitated mice
tested without odorants exhibited no discrimination, confirming that they were indeed
using their damaged olfactory systems to perform the task. This recovery of
detection ability suggests that downstream brain regions can successfully learn to
interpret olfactory degraded sensory input. In addition to informing potential therapies,
these data suggest that rapid sensory learning could potentially mask the effects
of serious pathology.
SOT 2011 ANNUAL MEETING 295