The Toxicologist - Society of Toxicology

1461 PHOTO-ACTIVATION OF MULTISIZED TITANIUM
DIOXIDE NANOPARTICLES (TIO 2 NPS) RESULTS IN
DIFFERENT ROS PRODUCTION AND OXIDATIVE
TOXICITY PROFILES IN ZEBRAFISH EMBRYOS.
O. Bar-Ilan1, 2 , K. Louis2 , S. Yang2 , J. Pedersen2 , R. Hamers2 , R. E. Peterson1, 2
and W. Heideman1, 2 . 1Division of Pharmaceutical Sciences, University of Wisconsin,
Madison, WI and 2NSEC, University of Wisconsin, Madison, WI.
When TiO 2 NPs absorb photons of sufficient energy, electrons are excited from valence
to conduction band, generating electron-hole pairs. These photo-activated (p-
) TiO 2 NPs can interact with H 2 O or O 2 , creating reactive oxygen species (ROS).
This can overwhelm neutralizing antioxidant cellular defenses, resulting in ROS
damage to important cellular macromolecules, leading to death. To investigate the
environmental implications of TiO 2 NPs photochemistry, we used zebrafish as a
model aquatic organism for exposure to two commercial and four lab-synthesized
preparations of TiO 2 NPs ranging in modal size from approximately 6-30 nm. We
hypothesized that photo-activation of TiO 2 NPs would induce oxidative toxicity in
zebrafish embryos, and that the potency in producing this toxicity would be affected
by size and phase type of the particles. Continuous exposure to TiO 2 NPs
during development resulted in significant mortality and incidence of malformations
(stunted growth and craniofacial, yolk, tail, and circulatory malformations),
which were dependent on illumination. Differences in toxicity and ROS production
were found between commercial and lab-made TiO 2 NPs, as well as between
otherwise identical preparations of different sizes. A transgenic zebrafish reporter
line Tg(are:eGFP) was created, coupling a GFP reporter with antioxidant response
elements (AREs) to help assess the production of ROS in vivo. ARE activation was
detected only in embryos exposed to TiO 2 NPs and illumination. Assessment of
DNA damage by ELISA for 8-OHdG also showed increased levels of oxidized bases
in embryos exposed to p-TiO 2 NPs. Pulse exposures, TEM, and ICP-OES revealed
uptake/association of the TiO 2 NPs by embryos, with the chorion as a protective
barrier. Rescue of the toxic effects by pre-exposure to N-acetylcysteine for oxidative
stress protection demonstrated that p-TiO 2 NPs lead to oxidative toxicity in zebrafish
embryos. NSF award DMR-0425880.
1462 PULMONARY TOXICITY OF BIODIESEL PARTICULATE
MATTER.
A. A. Shvedova 1 , E. Kisin 1 , A. R. Murray 1 , A. Tkach 1 , D. Schwegler-Berry 1 , S.
H. Young 1 , V. E. Kagan 2 and A. D. Bugarski 3 . 1 PPRB, NIOSH, Morgantown, WV,
2 Department of Environmental and Occupational Health, University of Pittsburgh,
Pittsburgh, PA and 3 OMSHR, NIOSH, Pittsburgh, PA.
Biodiesel is the only alternative fuel that currently meets requirements of the Clean
Air Act. The use of biodiesel results in a substantial reduction of unburned hydrocarbons,
carbon monoxide, and particulate matter (PM) as compared to diesel
emissions. The broad use of biodiesel in mining and other industries urge assessments
of their health effects. We hypothesized that biodiesel PM is toxic to the lung
and the toxicity is dependent on at least three major mechanisms: direct reactivity
of biodiesel PM electrophiles towards critical biomolecules, induction of robust inflammatory
response associated with the nano-sized components of biodiesel PM,
and triggering of oxidative stress pathways via depletion of essential antioxidant
molecules and activation of oxidative burst in inflammatory cells. In the current
study, we compare toxic effects of neat or blended biodiesel PM in vitro and in vivo.
Here, we report that biodiesel PM is cytotoxic to RAW 264.7 macrophages and
triggering oxidative stress pathways via depletion of essential antioxidant molecules
such as GSH. Our preliminary in vivo data indicating that pharyngeal aspiration
exposures to biodiesel PM resulted in an augmentation of biomarkers of inflammation
and oxidative stress as evidenced by significant depletion of antioxidants in
mouse lungs, recruitment of inflammatory cells, changes in permeability of the
lung epithelium, pulmonary cell damage and increase in pro-inflammatory cytokines
as assessed in the bronchoalveolar lavage (BAL) fluid. Further investigations
are warranted to elucidate the toxic properties of biodiesel PM in vitro and in vivo
and the possible mechanisms involved in such toxicity.
1463 ENGINEERED SILICA NANOPARTICLES ACT AS
ADJUVANTS TO ENHANCE ALLERGIC AIRWAY
DISEASE IN MICE.
J. R. Harkema 1 , N. Rowley 1 , L. Bramble 1 , Q. Zhang 2 , D. Jackson-Humbles 1 ,
G. Baker 2 , J. Wagner 1 and M. Worden 3 . 1 Pathobiology, Michigan State University,
East Lansing, MI, 2 Chemistry, Michigan State University, East Lansing, MI and
3 Chemical Engineering, Michigan State University, East Lansing, MI.
With the increasing production and use of engineered nanoparticles (ENP; < 100
nm mean diameter), there is growing public concern about potential health effects
due to occupational/environmental exposure. Nanosized particles found in envi-
314 SOT 2011 ANNUAL MEETING
ronmental air pollution have been associated with increased morbidity of asthma.
Ambient nanoparticles have been shown to have adjuvant effects that enhance allergic
airway disease in mice. Similar studies of the adjuvant potential of selected
ENP are lacking. The purpose of the present study was to investigate the adjuvant
potential of silica ENP (SiO2; 90 nm) using a murine model of allergic airway disease.
Female BALB/c mice were intranasally sensitized with 0.02% ovalbumin
(OVA; allergen) or saline (control), and co-sensitized with 0, 10, 100, or 400 μg of
SiO2. OVA-sensitized mice were intranasally challenged with 0.5% OVA two
weeks after sensitization. Mice were sacrificed 24 hours after the last intranasal challenge
and bronchoalveolar lavage fluid (BALF) was collected. Differential cell
counts and cytokines in BALF were determined and left lung lobes were processed
for histopathology and morphometric analyses. OVA-induced pulmonary inflammation
was enhanced by SiO2 co-sensitization. SiO2 caused a dose-dependent increase
of BALF eosinophils in OVA mice (544%, 1,341%, and 2,448% for 10,
100, and 400 μg SiO2 respectively). Similar SiO2 enhancement of OVA-induced
neutrophils in BALF was also induced (72%, 107% and 145%). This pattern of
dose response was also reflected in the amounts of inflammatory cytokines (e.g., IL-
5, IL-6, IL-13, INFγ, KC) in BALF. Intranasal aspiration of SiO2, concurrent with
OVA sensitization, caused an adjuvant-like enhancement of OVA-induced allergic
airway disease in mice. These results suggest that asthmatics may be particularly
susceptible to the adverse health effects caused by airborne exposure to silica ENP.
Funded by NIH 5RC2ES018756-02.
1464 COPPER NANOPARTICLES INDUCE CEREBRAL
MICROVASCULATURE PROLIFERATION OR TOXICITY
THAT AFFECT BLOOD-BRAIN BARRIER
PERMEABILITY.
W. J. Trickler 1 , S. M. Lantz 1 , B. L. Robinson 1 , G. D. Newport 1 , P. C. Howard 2 ,
J. J. Schlager 3 , M. G. Paule 1 , W. Slikker 1 , S. M. Hussain 3 and S. F. Ali 1 .
1 Neurochemistry Laborator, Division of Neurotoxicology, NCTR/U.S. FDA, Jefferson,
AR, 2 Office of Scientific Coordination, NCTR/U.S. FDA, Jefferson, AR and 3 AB
Branch, AFRL, Wright-Patterson Air Force Base, Dayton, OH.
The purpose of the current studies was to determine if systemic exposure to copper
nanoparticles (CuNPs) can induce the release of pro-inflammatory mediators that
influence the restrictive permeability characteristics of the BBB. Our in vitro BBB
model, a primary culture of brain microvessel endothelial cells (BMEC),was isolated
from fresh porcine or rat cerebral cortices.Confluent rat and porcine BMEC
monolayers (10-14 days) were treated with 40 and 60 nm CuNPs. Cytotoxicity was
evaluated using a cell proliferation assay (XTT) in BMEC following CuNPs exposure
(1.5-to-50 μg/ml, 24 hr). The extracellular concentrations of pro-inflammatory
mediators (IL-1β,IL-2, TNFα and PGE2) were evaluated using an ELISA at 0,
2, 4, 6 and 8 hr following CuNPs exposure (25 μg/ml). BMEC cell morphology
was monitored microscopically following CuNPs exposure (5 or 25 μg/ml).
BMECs were cultured in standard 12-well transwell inserts and permeability was
evaluated following 24hr CuNP exposure (25μg/ml) by measuring fluorescein
transport and the apparent permeability. CuNPs induced cytotoxicity at concentrations
≥ 12 μg/ml, while lower concentrations induced cell proliferation in BMECs.
PGE2 release following CuNP exposure was significantly increased (≈7fold and
3fold at 8 hr after 40 and 60 nm, respectively). Qualitatively similar results were observed
for both TNFα (3 fold after 4hrs) and IL-1β (10 fold after 8hrs). The
changes in pro-inflammatory events correlated well with increased fluorescein permeability
where permeability apparent ratios (exposure/control) were 8 fold for 40
and 60 nm CuNPs. These data suggest that interactions of CuNPs with the BBB
may produce pro-inflammatory mediators that could influence BMEC proliferation
at low concentrations or induce BBB toxicity and potential neurotoxicity at
higher concentrations. Supported by WPAFB/NCTR IAG.
1465 IN VITRO TOXICITY OF AMPHIPHILIC POLYMER-
COATED CD/SE/ZNS QUANTUM DOTS IN MULTIPLE
HUMAN AND MOUSE CELL TYPES.
L. McConnachie, C. White, D. Botta, M. Zadworny, D. Cox, R. Beyer, X.
Hu, D. Eaton, X. Gao and T. Kavanagh. University of Washington, Seattle, WA.
Recent developments in nanotechnology have produced an array of engineered
nanoparticles that possess many unique and interesting properties. However, concerns
have been raised regarding their potential to exert deleterious effects on biologic
systems. Therefore, it is critical to understand and characterize their potential
toxicity. Quantum dots (QDs) are nanoscale (2-10 nm) fluorescent crystals with
unique photochemical and photophysical properties. We have synthesized
CdSe/ZnS core/shell QDs with a tri-n-octylphospine oxide, poly(maleic anhydride-alt-1-tetradecene)
(TOPO-PMAT) coating. We investigated their uptake and
toxicity in 5 different cell lines derived from both murine and human origins, in-