The Toxicologist - Society of Toxicology

manganism. Recent work from our laboratory identified activated microglia expressing
inducible nitric oxide (iNOS) in the basal ganglia of mice exposed to manganese
(Mn) before the appearance of activated astrocytes; however, the role of activated
microglia in manganism is poorly understood. In this study we postulated
that Mn directly activates microglia, resulting in increased expression of tumor
necrosis factor α (TNF- α), interleukin 1-beta (IL-1β) and iNOS that ultimately
lead to increased astrocyte activation. Primary microglia and astrocytes were isolated
from C57Bl/6 mice in mixed cultures then purified for experiments.
Immunofluorescence staining for microglia and astrocytes was performed using
ionized calcium binding adaptor protein-1 (IBA-1) and glial fibrillary acidic protein
(GFAP), respectively. This method yielded culture purities of 97% for microglia
and 99% for astrocytes. Treatment of microglia with Mn induced a dose-dependent
expression of TNFα, iNOS mRNA and protein. Furthermore, a
quantitative PCR array revealed increased expression of proinflammatory mediators
in microglia when treated with Mn. Treatment of astrocytes with conditioned
media from Mn-treated microglia or via co-culture with microglia caused an activated
phenotype characterized by increased iNOS, TNFα, and IL-1β expression.
Collectively, these data indicate that Mn activates microglia in a dose-dependent
manner resulting in increased production of proinflammatory mediators that enhance
activation of astrocytes, suggesting a complex pattern of glial-glial interactions
underlying a neuroinflammatory phenotype in this model.
2130 NOVEL NADPH OXIDASE MODULATOR PROTECTS
AGAINST NEUROINFLAMMATION ASSOCIATED
WITH MANGANESE NEUROTOXICITY.
C. Hogan1 , R. Gordon1 , A. Kanthasamy1 , V. Anantharam1 , B. Kalyanaraman2 and A. G. Kanthasamy1 . 1Biomedical Sciences, Iowa Center for Advanced
Neurotoxicology, Iowa State University, Ames, IA and 2Biophys, Medical College of
Wisconsin, Milwaukee, WI.
Sustained neuroinflammation has become increasingly evident as a causative factor
in the progression of many neurodegenerative diseases including Parkinson’s disease
(PD). Exposure to environmental neurotoxicants such as metals and pesticides are
considered as potential risk factors in the pathogenesis of PD. Microglia are a large
part of the innate immune system of the CNS and are capable of producing rapid
neuroinflammatory responses when confronted with pathogens or neurotoxic insults.
Metal exposure has been shown to trigger activation of both microglia and astroglia
in order to counter neurotoxic insults. Recently, studies have shown that
manganese (Mn) exposure by itself or in combination with other inflammatory
stimuli can produce neuroinflammatory responses in the nigrostriatal dopaminergic
system. We and others have shown that Mn can augment lipopolysaccharide
(LPS)-induced cytokine production, iNOS activation, and ROS generation in microglial
cells. In the present study, we sought to investigate the anti-inflammatory
potential of a novel NADPH oxidase inhibitor MIA-4025 against microglial activation
in response to Mn/LPS-induced inflammatory events. MIA-4025 treatment
significantly blocked Mn/LPS-induced iNOS activation and ROS generation in
both primary microglia and BV2 microglia cell models. Measurement of cytokine
release in the supernatant of MIA-4025 treated BV2 cell lines and primary microglia
cells revealed that the compound effectively suppressed the release of TNFα,
IL-1β, IL-6, IL-10 and IL-12. Expression of phox proteins, components of the
NADPH oxidase complex and a main contributor to ROS production, was also decreased
with MIA-4025 treatment. Collectively, these results suggest that our translational
approach with MIA-4025 can suppress neuroinflammatory events associated
with neurotoxic metal exposure. (Supported by NIH grants ES10586,
NS065167 & NS039958.)
2131 PROTEIN KINASE C IS INVOLVED IN MANGANESE-
MEDIATED DISRUPTION OF GLUTAMINE TURNOVER
IN ASTROCYTES.
M. Sidoryk-Wegrzynowicz1 , E. Lee2 and M. Aschner1 . 1Department of Pediatrics,
Vanderbilt University Medical Center, Nashville, TN and 2Department of Physiology,
Meharry Medical College, Nashville, TN.
Manganese (Mn) is an essential trace element for normal human development and
is required for proper functioning of a variety of physiological processes. Chronic
exposure to Mn can cause manganism, a neurodegenerative disorder similar to
Parkinson’s disease. Mn neurotoxicity is characterized by impairment both in the
expression and activity of glutamine (Gln) transporters in astrocytes. Since protein
kinase C (PKC) activation leads to downregulation in a number of neurotransmitter
transporters and Mn increases PKC activity, we hypothesized that the PKC signaling
pathway may contribute in Mn-mediated disruption of glutamine turnover.
We have found that Mn exposure elevates phosphorylation of PKCα and PKCδ
isoforms. PKCs activity (measured by phosphorylation of substrate peptide using
radiolabeled ATP) was found to be increased after Mn treatment. As was previously
observed, Mn caused a decrease in Gln uptake. This effect was blocked by PKCs inhibitor.
Notably, PKC activation by phorbol 12-myristate 13-acetate (PMA) caused
a decrease in Gln uptake mediated by systems ASC and N, and had no effect on
systems A and L. Treatment with PMA significantly decreased SNAT3 (system N)
and ASCT2 (system ASC) protein levels. Additionally, a co-immunoprecipitation
study showed association of SNAT3 and ASCT2 with the PKCδ isoform.
Combined, these findings demonstrate that Mn-induced dysregulation of Gln
homeostasis in primary astrocytes involves PKC signaling.
Acknowledgements: This study was supported by NIH grant ES-10463.
2132 EXPRESSION OF MUTANT HUNTINGTIN INCREASES
ARGINASE ACTIVITY IN MOUSE STRIATUM, BUT
DECREASES ITS SUSCEPTIBILITY TO MANGANESE
EXPOSURE.
M. Wegrzynowicz 1, 2 , H. K. Holt 1, 2 and A. B. Bowman 1, 2, 3 . 1 Department of
Neurology, Vanderbilt University Medical Center, Nashville, TN, 2 Vanderbilt
Kennedy Center, Vanderbilt University Medical Center, Nashville, TN and 3 Center
for Molecular Toxicology, Vanderbilt University Medical Center, Nashville, TN.
Huntington disease (HD), a neurodegenerative disorder principally affecting the
striatum, is caused by mutation in the huntingtin (Htt) gene, but severity of the
disease may be strongly influenced by unidentified environmental factors. We have
reported a gene-environment interaction wherein expression of mutant (mt) HTT
impairs striatal manganese (Mn) accumulation. We hypothesize that mtHTT expression
may alter cellular markers of Mn toxicity.
When testing potential markers we found that basal activity of the Mn-dependent
enzyme, arginase, is elevated in the homogenates of striata from mtHTT-expressing
mice (YAC128Q) comparing to wild-type (wt) littermates. Additionally, we observed
that in vivo exposure to Mn (three 50 mg/kg MnCl 2 subcutaneous injections
over one week) strongly elevates enzyme activity in striatal extracts, however this effect
is less pronounced in mutant mice. Cortical activity was lower than striatal, and
no effect of genotype was found. In vitro supplementation of vehicle-exposed mice
striatum homogenates with 2 μM Mn (a concentration exceeding the values measured
in the homogenates from animals exposed to Mn in vivo) was much less effective
in arginase activation than subcutaneous exposure in vivo. We conclude that increased
activity of arginase, an enzyme known to be neuroprotective, may be a
compensatory response to mtHTT cytotoxicity in the striatum. Impaired activation
of striatal arginase by Mn administered in vivo in YAC128Q mice is probably
due to a mtHTT-induced deficit of Mn accumulation. Interestingly, the effect of
Mn is most likely only partially mediated by increased availability of Mn, a cofactor
of arginase. Inability of in vitro added Mn to replicate the effects of in vivo exposure
suggests that Mn may induce some cellular processes contributing in the activation
of arginase.
Support: NIH ES016931
2133 NEUROTOXIC EFFECTS OF INTRANASAL
MANGANESE EXPOSURE AND INTERACTION WITH
CADMIUM EXPOSURE IN A MOUSE MODEL.
A. H. Moberly, J. Pottackal, L. Czarnecki, T. Rubinstein, D. J. Turkel and J. P.
McGann. Psychology Department, Rutgers University, Piscataway, NJ.
Exposure to aerosolized manganese occurs in a variety of industrial settings, including
mining and welding, but its effects on neurons directly exposed to manganese
has not been explored in vivo. Transgenic mice expressing the fluorescent exocytosis
indicator synaptopHluorin (spH) in olfactory receptor neuron terminals were used
to visualize odorant-evoked neurotransmitter release from the olfactory nerve following
acute toxicant exposure. Mice received a unilateral intranasal instillation of
2, 20, or 200 μg manganese chloride and a vehicle control on the contralateral side.
Two days later they were anesthetized, implanted with a cranial window overlying
the olfactory bulbs, and odorant-evoked neurotransmitter release was quantified bilaterally
via optical imaging of spH signals. At the highest dose, odorant-evoked
sensory input to the olfactory bulb on the Mn-exposed side was reduced to 14% of
control and while epithelial projections to the olfactory bulb were slightly reduced.
The 20 μg dose reduced responses to 58% of control, while the lowest dose had no
significant effect. Because we have previously shown similar effects using intranasal
cadmium exposure, we further hypothesized that mixtures of the two heavy metals
might demonstrate an interaction. To test this, the above experiment was repeated
using instillations of a combination of 20 μg Mn and 2 μg Cd, doses found to produce
intermediate reductions in odorant-evoked responding when presented individually.
Mice exposed to this mixture exhibited odorant-evoked transmitter release
from the olfactory nerve equal to only 11% of control. This decrease suggests that
peripheral exposure to cadmium and manganese can cause pathopysiology comparable
to the sum of their effects individually. We conclude that intranasal Mn exposure
can significantly disrupt olfactory function. Moreover, its neurotoxic effects
can summate with those of Cd, suggesting that total exposure to both should be
considered in assessing risk levels.
SOT 2011 ANNUAL MEETING 457