The Toxicologist - Society of Toxicology

2161 MEMBRANE-MEDIATED MODES OF ENTRY OF C 60
INTO CULTURED IMMORTALIZED MACROPHAGES.
K. A. Russ 1 , I. C. Speirs 1 , P. Elvati 2 , J. A. Fernandez 1 , A. Violi 2 and M. A.
Philbert 1 . 1 Department of EHS, Toxicology, University of Michigan, Ann Arbor, MI
and 2 Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI.
Hydrophobic fullerenes such as C 60 have been studied in several cell types and are
cytotoxic. This study examines the interactions of C 60 with cultured RAW 264.7
immortalized macrophages and its effects on inflammatory potential and cell death
pathways. Cells were exposed to five concentrations of fullerenes (0.1, 0.2, 0.76, 1,
4 μg/mL) for 24 hours and were monitored for viability, alterations in cell proliferation,
and activation of caspases. An inverted “U-shaped” dose response for viability
and activation of caspases was observed, as was a “U-shaped” curve for cell proliferation.
Cells exposed to 0.1 μg/mL C 60 for 24 hours showed decreased global
expression of cytokines/chemokines. 24 hour exposure to 4 μg/mL C 60 caused a
global increase in cytokine/chemokine gene expression. Examination of cells by
transmission electron microscopy (0.1 and 0.36 μg/mL Tb-endohedral C 60 ) revealed
the presence of at least three different pathways by which these nanoscale
structures gain access to the cell. There was clear morphological evidence of endocytosis
(a mechanism well described by others), diffusion into the membrane, and
an unknown pathway unbound by lipid membranes leading to deposition in the
nucleus. Based on the data, we hypothesize that the increased metabolic activity is
due to the increase in energy required to endocytose or exocytose particles, and the
increase in cell growth could be attributed to particles interacting with growth receptors
in the cell membrane. Predictive computational models of the interaction(s)
between C 60 and lipid bilayers confirm the most favored disposition of C 60 .
Additional investigation is required to determine the biological and toxicological
significance of each pathway and their role in the ultimate fate of the cell. This
work was supported by in part by 2R01 ES008846 (MAP), NCI R33 CA125297
(MAP), NIEHS Training Grant T32-ES007062-26 and NSF-CAREER Award
CBET 0644639 (AV).
2162 IN VITRO TESTING STRATEGIES FOR ASSESSING
PARTICLE-INDUCED GENETIC DAMAGE USING
ALVEOLAR CELL LINES.
K. P. Glover, A. Myhre, M. Laskowski, K. L. Reed, E. Donner and D. B.
Warheit. DuPont Haskell Global Centers for Health and Environmental Sciences,
Newark, DE.
New in vitro testing strategies are necessary to better simulate in vivo exposure conditions,
and there might be a need to refine and develop endpoints specific to particle
toxicity. Results are shown from studies with alveolar cell lines, such as
macrophages, that can retain characteristics of cells that are exposed through in vivo
inhalation exposure and therefore more appropriate for in vitro testing. The purpose
of this experiment was to analyze 5 different particle types with different levels
of toxicity (carbonyl iron (CI), crystalline silica (CS), amorphous silica (AS) and
fine and nanosized zinc oxide (ZnOf and ZnOn)) for their genotoxic potential in
two cell lines; the rat alveolar epithelial cell line, L2, and the rat alveolar
macrophage cell line, NR8383. The cells were exposed to CI, AS and CS at 10 –
100 μg/cm 2 . However, ZnO was 10 fold more toxic than the other particles and
was used at 1 – 10 μg/cm 2 . Particle induced cytotoxicity was measured through the
trypan blue exclusion method and the MTT assay. Genetic damage was assessed
using the In vitro MicroFlow® Micronucleus assay kit (Litron Laboratories,
Rochester, NY) and the alkaline comet assay. In the in vitro micronucleus assay, a
positive response was observed for CI and both ZnO particles (≥ 2 fold increase relative
to the negative control) at particle exposure conditions that induce very high
toxicity (< 50% survival). According to the comet assay both L2 and NR8383 cells
treated with CI had significantly increased mean tail moments compared to the
control. However, this effect was not dose dependent and the highest response was
not observed at the highest dose level tested, 100 μg/cm 2 . More research efforts
need to focus on how to accurately determine potential particle induced genetic
damage using modified assay conditions that account for the unique properties of
particles in the traditionally validated assays.
2163 DEVELOPING AN IN VITRO MODEL OF THE
ALVEOLO-CAPILLARY BARRIER TO STUDY TOXICITY
AND TRANSLOCATION OF NANOPARTICLES.
K. Luyts, B. Nemery and P. H. Hoet. Research Unit for Lung Toxicology,
K.U.Leuven, Leuven, Belgium.
Due to their small size, nanoparticles (NP) can penetrate deep into the lungs and
reach the alveoli where they can cross the alveolar barrier and reach the pulmonary
vessels. Therefore, an in vitro model of the alveolo-capillary barrier might be a use-
464 SOT 2011 ANNUAL MEETING
ful tool to study the effects and translocation of NP. The human pulmonary epithelial
16HBE14o- cell line (epi) and human endothelial EAHY926 cell line (endo)
were seeded on opposite sides of a permeable membrane support creating a bilayer.
Two conformations were tested: 16HBE14o- on apical side/EAHY926 on basolateral
side (epi/endo) and the other way around (endo/epi). Permeable supports with
different pore size were used: 0.4 and 3 μm. Transepithelial electrical resistance
(TEER) measurements were performed daily to check cell layer integrity and to
compare with monocultures (epi or endo on apical side). Using 0.4 μm pore supports,
the TEER values of the epi/endo bilayer were significantly higher compared
to the monolayers starting from three days after seeding. From five days after seeding
the TEER values of the endo/epi model were significantly higher compared to
both monolayers. Both conformations reached their maximum TEER five days
after seeding: 1596 ± 106.8 Ω.cm2 (epi/endo) and 1559 ± 311.1 Ω.cm2
(endo/epi) compared to 814.8 ± 285.8 Ω.cm2 (epi) and 5.198 ± 0.3160 Ω.cm2
(endo). Using 3 μm pore supports, maximum TEER values were lower (375.7 ±
140.7 Ω.cm2 epi/endo; 571.9 ± 109.7 Ω.cm2 endo/epi) compared to those obtained
with 0.4 μm pore supports and obtained six days after seeding. The results
indicate a tight epithelial barrier and the presence of endothelial cells in the model
accounts for significantly higher TEER values compared to the epi monolayer. This
makes the model suitable to study the effects of NP (and other substances) on the
alveolo-capillary barrier (inflammation, oxidative stress) in two different conformations.
Moreover, 3 μm pore supports allow the study of interactions between epithelial
and endothelial cells as well as particle translocation. EU-FP7: ENPRA;
FWO:F.OS47.08
2164 CYTOTOXICITY AND GENOTOXICITY OF ACUTE AND
CHRONIC EXPOSURE TO SILVER NANOPARTICLES IN
HUMAN LUNG CELLS.
H. Xie 1, 2, 3 , D. McGovern 1, 2 , A. Sample 1, 2 , S. Abramson 1, 2 , A. Perez 1, 2 , M. D.
Mason 4 , G. Craig 4 and J. P. Wise 1, 2, 3 . 1 Wise Laboratory of Environmental and
Genetic Toxicology, University of Southern Maine, Portland, ME, 2 Maine Center for
Toxicology and Environmental Health, University of Southern Maine, Portland, ME,
3 Department of Applied Medical Science, University of Southern Maine, Portland,
ME and 4 Department of Chemical and Biological Engineering, University of Maine,
Orono, ME.
Nanoparticles, such as those made of silver, have shown great promise in a broad
range of applications in industry and biomedicine due to their unique physical and
chemical properties. Human exposure occurs in manufacturing settings as well as
through consumer products and nanoparticles are being released into our environment.
But whether these materials are safe for human exposure is not yet clear. It is
essential to understand if and how nanoparticles induce damage in normal human
cells. To address this issue, we investigated the cytotoxicity and genotoxicity of silver
nanoparticles in human lung cells. We found that bare silver nanoparticles are
toxic to human lung cells after 120 h exposure. There is no induction of chromosome
damage or DNA double strand breaks in cells exposed to silver nanoparticles
for 24 h, however, after 120 h exposure, we noted a weak induction of chromosome
damage and DNA double strand breaks. Further work will include assessing cytotoxicity
and genotoxicity of pegylated and fluoroscein isothiocyanate functionalized
(FITC) silver nanoparticles to see if alterations to the base nanoparticles render
them more or less toxic and determine if cellular transformation occurs after silver
nanoparticle exposure.
2165 INFLUENCE OF SURFACE FUNCTIONALIZATION ON
PROTEIN ADSORPTION AND NANOPARTICLE
UPTAKE BY INTESTINAL EPITHELIAL CELLS.
P. N. Wiecinski 1 , K. M. Louis 2 , R. M. Albrecht 3 and J. A. Pedersen 1, 4 .
1 Molecular and Environmental Toxicology, University of Wisconsin-Madison,
Madison, WI, 2 Chemistry, University of Wisconsin-Madison, Madison, WI, 3 Animal
Science, University of Wisconsin-Madison, Madison, WI and 4 Soil Science, University
of Wisconsin-Madison, Madison, WI. Sponsor: R. Peterson.
The surface chemistry of nanoparticles (NPs) is expected to influence their uptake
and bioavailability. Many engineered NPs are functionalized to improve dispersibility
in aqueous media, enhance biocompatibility or facilitate incorporation into
nano-composite materials. Additional coatings may be acquired during exposure to
environmental and biological systems. Ingestion of NPs is expected to be an important
exposure route to a variety of organisms, including humans. We have synthesized
fluorescent europium-doped yttrium vanadate (YVO 4 :Eu) NPs to examine
the influence of surface chemistry on dietary protein adsorption and uptake using
intestinal cell culture models. The benefits of YVO 4 :Eu NPs include chemical stability
under experimental conditions, low toxicity, the fluorescence of Eu facilitating
tracking, and ease of functionalization to produce water-stable NPs. Here, we
present results for citrate-stabilized and poly(acrylic acid) (PAA)-coated YVO 4 :Eu