The Toxicologist - Society of Toxicology

adigms such as Tox 21 and Toxcast, which will utilize high throughput cellbased
assays, limited animal data, informatics and computational tools for prediction
and use of physiologically based pharmacokinetic models for extrapolation
(NRC 2007). Approaches to relate cellular dose of nanomaterials from in vitro tests
to cellular and target tissue specific doses in humans and rodents exposed to nanomaterials
is required to support both adequate design and robust interpretation in
vitro studies of nanomaterials. We have developed in vitro and in vivo particokinetic
models for nanomaterials which capture their unique kinetics in these systems.
These models are used to establish and articulate the particokinetic and physiological
basis for in vitro-in vivo extrapolation of nanomaterial toxicity studies. We
demonstrate that in vitro, the most commonly used metric of dose, nominal media
concentration, is, under most conditions, not suitable for dose-response or for extrapolation
to in vivo studies because ignoring the effects of gravity and diffusion
on delivery to cells introduces errors in dosimetry on the order of multiple orders of
magnitude. Correcting for these errors and applying models of pulmonary
nanoparticle deposition and systemic disposition, we show how computational particokinetic
tools can be used to extrapolate tissue and cellular dose across the in
vitro/in vivo divide based on particle surface area, or particle number s as the metrics
of dose. Conversely, we show how the same tools can be used to design in vitro
nanomaterial toxicity studies which utilize doses which correspond to potential
human exposures at OSHA permissible exposure levels for common particulates as
a proxy for potential human exposure to nanomaterials.
281 THE ROLE OF BRAIN MICROVESSEL ENDOTHELIAL
CELLS IN THE NEUROTOXICITY OF SILVER OR GOLD
NANOPARTICLES.
W. J. Trickler 1 , S. M. Lantz 1 , B. L. Robinson 1 , G. D. Newport 1 , J. J. Schlager 2 ,
S. J. Oldenburg 3 , M. G. Paule 1 , S. M. Hussain 2 and S. F. Ali 1 . 1 Neurochem. Lab.,
Division of Neurotox, NCTR/FDA, Jefferson, AR, 2 Applied Biotechnology Branch,
Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson
AFB, Dayton, OH and 3 NanoComposix, Inc., San Diego, CA.
The purpose of the current studies was to determine what role microvessel endothelial
cells, which functionally comprise the blood-brain barrier (BBB), have in
causing brain pro-inflammation state and subsequent neurotoxicity associated with
exposures to colloidal metallic nanoparticles (NPs) like silver (Ag) and gold (Au).
Our in vitro model of the BBB, a primary culture of rat brain microvessel endothelial
cells (rMVEC), was isolated by a series of enzymatic digestions and differential
centrifugation steps. Confluent rMVEC monolayers (10-14 days) were treated with
various sized Ag (25, 40 or 80 nm) or Au (1.9, 3, 5, 7, 10, 30 or 60 nm) NPs. The
cellular accumulation of the NPs was determined spectrophotometrically at various
time intervals (30, 60 or 90 mins). The cytotoxicity was evaluated by cell proliferation
assay (XTT) in rMVEC during a 24hr exposure to NPs (0.7 to 50 ug/ml). The
extracellular concentrations of proinflammatory mediators (IL-1B, IL-2, TNFα
and PGE2) were evaluated by ELISA at 0, 2, 4, 6 and 8 hours following exposure to
Ag and Au NPs. The cytotoxicity of rMVEC following 24-hr exposure to Ag-NPs
(LD50; below 700 ng/ml (25 nm) and 10ug/ml (40 and 80 nm)) was significantly
higher, when compared to Au-NPs (LD50 ≈ 10 ug/ml, 1.9 nm). Au-NPs above 3
nm in size showed no significant cytotoxic effects. PGE2 release following Ag-NPS
exposure was significantly increased (≈ 8-fold at 8hr) when compared to the control.
These data suggest that brain microvessel endothelial cells may play a significant
role in the elaboration of neurotoxicity associated with Ag and Au NPs. Also,
Ag-NPs are shown to be significantly more toxic than Au-NPs in rMVEC. The interactions
of NPs at the BBB MVEC may produce an initial cascade of proinflammatory
mediators that can induce further brain inflammation and neurotoxicity.
282 INTERNALIZATION OF SIO2 NANOPARTICLES: THE
INFLUENCE OF SIZE ON METAL OXIDE
NANOPARTICLE ENDOCYTOSIS.
J. M. Berg 1 , R. Payne 2 , R. Taylor 2 and C. M. Sayes 1 . 1 Physiology and
Pharmacology, Texas A&M University, College Station, TX and 2 Veterinary
Integrative Biosciences, Texas A&M University, College Station, TX.
The use of metal oxide nanomaterials in a variety of industrial, household, and medicinal
products (e.g. cosmetics, drug carriers, biosensors, and insulators) is increasing
exponentially. However, while exposure to nanoparticles is often intentional,
due to their ability to deliver desirable features, little is known about how these materials
interact with biological processes. Here, the ability of silicon dioxide (SiO2)
nanoparticles to yield a cellular response in a human A549 epithelial cell is studied
with respect to cellular internalization. The influence of primary particle size (15
nm, 80 nm, 3 μm) as well as agglomerate size on nanoparticle endocytosis is examined.
A variety of techniques have been employed to determine the endocytic
events responsible for the internalization of SiO2 nanoparticles. Mechanistic evaluation
of endocytosis was undertaken utilizing immunocytochemistry combined
with transmission electron microscopy. Furthermore, inductively coupled plasma –
mass spectroscopy was employed to provide quantitative analysis of nanoparticle
uptake subsequent to the use of specific endocytic inhibitors. Results suggest that
SiO2 nanoparticles exist in multiple agglomerate sizes when inside the cell.
Nanoparticle composition was confirmed utilizing energy dispersive x-ray spectroscopy.
Furthermore, nanoparticle uptake is an active process dependent upon
time, temperature, and dose. Additionally, it was noted that while a majority of intracellular
nanoparticles were enclosed in a vesicular structure, most notably a lysosome,
free cytosolic nanoparticles were also visualized. It is hypothesized that route
of endocytosis and incorporation into vesicular structures will play a role in the toxicological
parameters of nanoparticles. Subsequent work will focus upon the examination
of endocytosis in a co-culture system including both macrophage and dendritic
cell lines.
283 COMPARATIVE TOXICOLOGICAL ANALYSIS OF
QUANTUM DOTS AND WIRES ON HUMAN SKIN
TISSUE.
R. Iyer, J. Gao and J. Hollingsworth. LANL, Los Alamos, NM.
Semiconductor quantum dots (QD) and wires are of growing technological relevance
with applications in photoelectronics, electronic integrated circuits, radiation
tolerant solar cells, and biological labeling. We used an in vitro reconstructed
human skin tissue equivalent, typically referred to as 3-D tissue culture or organotypic
raft cultures, closely representing the complexity and structural integrity of
human skin tissue system. In this study we performed a comparative analysis of different
sized QDs and quantum wires, these quantum dots contain a cadmium/selenide
core with a cadmium sulfide shell coated with polyethylene glycol (PEG)
and are soluble in water. The sizes of these nanoparticles are 3.4 ± 0.2 nm, 5 nm
small dots, 16 ± 5 nm large dots and wires that are 10 nm in diameter and >1micron
in length. In this study, QDs were topically applied to skin tissue at concentrations
of 500 nM to 10 μM to assess penetration, cellular viability, cytotoxicity
and inflammatory responses. Preliminary fluorescent microscopy results indicate
that negatively charged quantum dots (5nm) can penetrate through the skin stratum
corneum layers without inducing significant tissue damage and may potentially
interact with epidermal cells. In contrast, the quantum wires induced massive
skin damage compared to both, small and large dots. The MTT viability assay suggests
that 6 nm quantum dots reduce cell viability in a time and dose-dependent
manner. Reduced cell viability was observed within 6 hrs at concentrations as low as
2.5 nM. The large QDs (16 nm) appear to perfuse the skin tissue at early time
points and at all concentrations, however they induce significantly less tissue damage
than the small QDs (3.4 nm). The transmission electron microscopy (TEM)
images indicate that the QDs were uniformly monodispersed. We will also present
data showing a possible QD-induced inflammatory response by the exposed tissue.
These results implicate that the size and shape properties of QDs impact the ability
of these nanomaterials to penetrate skin tissue and influence cytotoxic and inflammatory
responses.
284 BIOLOGICAL SURFACE ACTIVITY INDEX: A NOVEL
METRIC TO CHARACTERIZE NANOMATERIAL
INTERACTIONS IN BIOLOGICAL SYSTEMS.
X. Xia, N. A. Monteiro-Riviere and J. E. Riviere. Center for Chemical Toxicology
Research and Pharmacokinetics, North Carolina State University, Raleigh, NC.
The behaviors of nanomaterials in biological systems are dictated by the absorbed
surface species. Quantitative assessment of the adsorption properties of the nanomaterials
is a crucial step for developing predictive structure-activity relationship in
nanomedicine and risk assessment of nanomaterials. We have developed a biological
surface activity index (BSAI) approach to characterize the surface activity of
nanomaterials in biological systems. A set of small molecules having diverse physicochemical
properties was used as probe compounds. The adsorption coefficients (k)
of the probe compounds were obtained by measuring the quantities of the probe
compounds adsorbed on the surfaces of the nanomaterials and the equilibrium concentrations
of the probe compounds in the media. The log (k) values were scaled to
a set of solvation molecular descriptors of the probe compounds via multiple linear
regressions to provide a set of five nano-descriptors representing the contributions
of the five types of molecular interactions (hydrophobicity, hydrogen-bond acidity
and basicity, dipolarity/polarizability, and lone pair electrons). A pilot BSAI database
of the nano-descriptors were obtained for 12 nanomaterials including silver
(powder), silver (colloid), TiO 2
, ZnO, CuO, NiO, Fe 2
O 3
, SiO 2
, C 60
(powder),
nC 60
(colloid), multiwalled carbon nanotubes (MWCNT) and hydroxylated
SOT 2010 ANNUAL MEETING 61