The Toxicologist - Society of Toxicology

treated by instillation also showed increases of total cells, viable cells and neutrophils
in both concentration groups, these were however not significant, partly
because of outliers. After the recovery period of one month, animals exposed by inhalation
showed no difference in BAL parameters compared to controls. In the animals
treated by instillation, however, changes in cell numbers in BAL were still
present. Animals showed an increase in oxidative stress (HO-1 expression analysis)
just after treatment, which was concentration dependent for animals exposed by inhalation,
but not in animals treated by instillation. The increased lung weight in animals
exposed by inhalation was not found after a recovery period, whereas an increased
lung weight was still found in animals exposed by instillation after the
recovery period. Inhalation and instillation of silicon dioxide aggregated nanoparticles
showed generally similar effects although the magnitude and its occurrence in
time differed. Therefore, it can be concluded that, although instillation exposure
may be used to assess the effects of nanoparticles, exposure by inhalation is more
representative for the human exposure route.
368 THE ROLE OF HYPOXIA-INDUCIBLE FACTOR IN
MMP-2 AND MMP-9 PRODUCTION BY HUMAN
MONOCYTES EXPOSED TO NICKEL
NANOPARTICLES.
Y. Mo, R. Wan, D. J. Tollerud and Q. Zhang. Environmental and Occupational
Health Sciences, University of Louisville, Louisville, KY.
With development of nanotechnology, nano-size nickel (Nano-Ni) and nano-size
titanium dioxide (Nano-TiO2) particles have been developed and produced for
many years with new formulations and surface properties to meet novel demands.
Several studies have shown that nanoparticles can translocate from the lungs to the
circulatory system. Previous studies in our laboratory have shown that Nano-Ni instilled
into lungs caused a greater inflammatory response as compared with standard-sized
nickel (5 μm) at equivalent mass concentrations. And Nano-Ni caused a
persistent high level of inflammation in lungs even at low doses. To evaluate the potential
systemic effects of metal nanoparticles, we compared the ability of Nano-Ni
and Nano-TiO2 to cause alteration of transcription and activity of MMPs in
human monocytes and explored possible mechanisms. Our results showed that exposure
of U937 cells to Nano-Ni caused dose- and time- response increases in
MMP-2 and MMP-9 mRNA expression and pro-MMP-2 and pro-MMP-9 activity,
but Nano-TiO2 does not. We also demonstrated dose- and time- related increases
in tissue inhibitors of metalloproteinases 1 (TIMP-1), but not to Nano-
TiO2. To determine the potential mechanisms involved, we determined the
expression of hypoxia-inducible factor 1α (HIF-1α) in U937 cells exposed to
Nano-Ni and Nano-TiO2. Our results demonstrated that exposure to Nano-Ni
caused HIF-α accumulation. Further, pre-treatment of U937 cells with HIF-α inhibitor,
17-AAG, prior to exposure to Nano-Ni significantly abolished Nano-Niinduced
pro-MMP-2 and pro-MMP-9 activity. Our results suggest that HIF-α accumulation
may be involved in the MMP-2 and MMP-9 production in U937 cells
exposed to Nano-Ni.
369 C 60
HAS OPPOSING DOSE-DEPENDENT EFFECTS ON
ELECTRON TRANSPORT CHAIN FUNCTION AND
OXIDATIVE STRESS IN ISOLATED BOVINE HEART
MITOCHONDRIA.
S. Rosario and C. Thomas. Chemistry, Central Washington University, Ellensburg, WA.
Buckminster Fullerene, C 60
, is a carbon nanoparticle that has been investigated in
many biomedical applications. After two decades of intensive research, however, its
biological effects remain controversial. This may be due in part to the methods used
to solubilize C 60
in water. In this study we examined the effects of C 60
solubilized in
7.5% bovine serum albumin on isolated bovine heart mitochondrial function. The
aims were to determine the dose-dependent and time-dependent effects of C 60
on
mitochondrial electron transport chain function and oxidative stress. To assess electron
transport through complexes II, III, and IV, succinate oxidase activity was
measured by oxygen consumption after exposure to 2 - 35 ppm C 60
for time points
up to 30 minutes. Hydrogen peroxide production and lipid peroxidation in isolated
mitochondria were measured as indicators of oxidative stress. The ability of C 60
to
produce reactive oxygen species (ROS) in the presence of physiological reductants
(succinate, ascorbate and NADH), but in the absence of mitochondria was also
measured by oxygen consumption. The studies in isolated bovine heart mitochondria
showed that 2 ppm C 60
caused a surprising 50% increase in electron transport
chain function at all time points as compared to controls, and no oxidative stress.
However, higher concentrations of C 60
(10 & 35 ppm) caused approximately 20-
30% electron transport chain inhibition at all times points except for the zero time
point. Oxidative stress measured as H 2
O 2
was observed only at the highest concentration
tested, 35 ppm C 60
. Lipid peroxidation measured as thiobarbituric reactive
substances was observed at all times points. The physiological reductants did not
produce significant amounts of ROS in C 60
solutions in the absence of mitochondria.
It is hypothesized that C 60
’s effects are due to its dissolution in the membrane
which causes physical disruption of electron transport chain components.
370 SUB-ACUTE INHALATION EXPOSURE STUDY OF
COPPER NANOPARTICLES IN MICE.
J. Kim 1 , A. Adamcakova-Dodd 2 , P. T. O’Shaughnessy 2 , V. H. Grassian 3 and P. S.
Thorne 1, 2 . 1 Interdisciplinary Graduate Program in Human Toxicology, The
University of Iowa, Iowa City, IA, 2 Department of Occupational and Environmental
Health, The University of Iowa, Iowa City, IA and 3 Department of Chemistry, The
University of Iowa, Iowa City, IA.
Due to the expanding use of nanoparticles, the potential for human exposure has
increased rapidly. The most critical concern over health and environmental effects
occurs when nanoparticles are aerosolized. Airborne nanoparticles are highly mobile
and can enter the human body via inhalation. The purpose of this study was to
assess the potential effects associated with the inhalation of copper (Cu) nanoparticles
using a murine model of lung inflammation, oxidative stress and host defense
mechanism. Our hypothesis is that inhalation sub-acute exposure of Cu nanoparticles
in mice produces inflammatory responses, oxidative stress and alters host defense
in murine pulmonary infection model of Klebsiella pneumoniae. We examined
pulmonary inflammatory responses, oxidative stress, and lung bacterial clearance of
infected Klebsiella pneumoniae in mice following inhalation sub-acute exposure (4
hr/day, 10 days) to commercially manufactured Cu nanoparticles. Concentration
and aerosol size distribution for sub-acute Cu exposures were 3.5 mg/m 3 and near
200 nm of geometric mean diameter, respectively. The amount of Cu measured in
lungs of Cu-exposed animals was 40 mg/kg lung (dry weight). Immediately following
sub-acute exposure, Cu-exposed mice showed increased inflammation compared
to sham exposed animal as indicated by the number of total cells and neutrophils,
levels of total protein, and LDH activity in lung lavage fluid. Bacterial
clearance from the lung was found to be significantly higher in Cu-exposed mice
than sham-exposed animals. We conclude that inhalation sub-acute exposure of Cu
nanoparticles in mice produces inflammatory responses. Increased lung bacterial
clearance is proposed to be associated with neutrophil recruitment mediated by exposure
of Cu nanoparticles in a murine model and antimicrobial activity of Cu.
371 DEVELOPMENT OF A HIGH-THROUGHPUT ASSAY
TO ASSESS THE IMMUNOMODULATORY POTENTIAL
OF ENGINEERED NANOPARTICLES.
L. Brusch-Richardson 1 , M. Ariza 1 , N. B. Saleh 2 and T. Sabo-Attwood 1, 3 .
1
Environmental Health Sciences, University of South Carolina, Columbia, SC, 2 Civil
and Environmental Engineering, University of South Carolina, Columbia, SC and
3
NanoCenter, University of South Carolina, Columbia, SC. Sponsor: D. Volz.
The increased use of engineered nanoparticles (ENPs) without the similar aggressive
development of tools for determining their biological reactivity is a cause for
concern. While it will be impossible to exhaustively study all properties of ENPs,
some key characteristics of their biological behavior can be chosen as indicators of
potential cytotoxicity for predicting in vivo health outcomes. ENP surface chemistry,
aggregation state, and solubility may influence their ability to modulate immune
defense and inflammatory responses in organisms. Specifically, ENPs may be
recognized by Toll-Like Receptors (TLRs), triggering an immune response through
the Nuclear Factor-kappa B (NF-κB) transcription factor. To test the ability of
ENPs in activating this pathway, Human Embryonic Kidney cells (HEK293) stably
expressing TLRs 2, 4, and 5 were transfected with the NF-κB reporter gene. The
ability of select ENPs to transcriptionally activate NF-κB were determined using a
Luciferase assay. Exposure of HEK293-TLR2 expressing cells to non-toxic doses
(10 ng/ul – 100 ng/ul) of silver ENP (75 nm) resulted in significant activation of
NF-κB in a dose dependent manner compared to unexposed cells. The observed
immune regulating behavior of silver and other ENPs (gold and carbon nanotubes)
is currently being correlated with surface charge, degree of solubility, and aggregate
formation. These results suggest ENPs can impact signaling networks associated
with immune function. Ultimately, this work will lead to a better understanding of
the potential immunomodulatory properties of nanoparticles and allow for the development
of a high throughput screening method applicable to in vivo health effects.
This research may also provide insight into the role of ENPs in inflammatory
diseases and open up opportunities for the development of novel therapeutics
which balance drug efficacy with immune responses.
80 SOT 2010 ANNUAL MEETING