The Toxicologist - Society of Toxicology

into preclinical studies. We evaluated a new method for diagnosing phospholipidosis
that combines LAMP-2 (lysosomal associated membrane protein-2) immunohistochemical
staining with digital image analysis. In cell culture assays, LAMP-2
staining is specific for drug-induced phospholipidosis as detected by quantitation of
fluorescent phospholipid accumulation. In Fisher rats treated with coralgil, a drug
known to induce generalized phospholipidosis, a significant increase in LAMP-2
staining was measured in tissues with a reported finding of vacuolated macrophages
(spleen, mesenteric lymph node, and lung) and in tissues with a reported finding of
microvesicular vacuolation (liver), both of which were confirmed as phospholipidosis
by TEM. No increase in LAMP-2 staining was seen in skeletal muscle, where
stress-induced vacuolation was observed. Because an influx of macrophages - a
common pathology of phospholipidosis - can increase LAMP-2 staining, as a control
for potential false positive results, immunohistochemistry with CD68 (ED1), a
macrophage specific lysosomal protein, was performed to calculate macrophage cell
counts. The quantitation of LAMP-2 immunohistochemical staining, in addition
to other immunohistochemical approaches, may be an effective tool for diagnosing
phospholipidosis in preclinical studies.
1453 BIOCHEMICAL MODULATION OF COPPER
NANOPARTICLE UPTAKE DIMINISHES
CYTOTOXICITY.
J. B. Lin, A. Schrand and S. M. Hussain. Applied Biotechnology, AFRL/RHPB,
Wright-Patterson AFB, OH.
Copper particles have been heavily researched for their applications as antimicrobial
and catalytic agents. Nonetheless, their toxicity has limited the feasibility of
their wide-spread application in living systems. This study demonstrates that dosedependent
cytotoxicity of micro- (~480nm) and nano-sized metallic Cu particles
(~80-90nm) in the murine neuroblastoma (N2A) cell line is linked to their endocytic
uptake. The addition of a chemical to inhibit endocytosis (Dynasore, 25 μM)
resulted in the rapid remediation of cytotoxicity as measured with multiple toxicity
endpoints (mitochondrial function, lysosome number). However, it was critical
that the cells were pre-treated with the inhibitor for 1 h prior to exposure to a combination
of both the inhibitor and copper particles for 3 h. Evaluation of multiple
physicochemical parameters and conditions (ie. size in solution, chemical interactions)
were used to develop a treatment paradigm leading to significant decreases in
toxicity at particle concentrations > 50 μg/mL. Application of this method to other
cell types may prove to be a viable method for modulating the uptake of micro- and
nano-based particles and their subsequent cytotoxicity.
1454 EFFECT OF ENGINEERED TITANIUM DIOXIDE
NANOPARTICLE SHAPE ON TOXICITY IN VIVO.
D. W. Porter 1, 2 , M. G. Wolfarth 1 , N. Wu 3 , A. Holian 4 , A. Hubbs 1 , K. A.
Funk 5 and V. Castranova 1, 2 . 1 HELD/PPRB, NIOSH, Morgantown, WV,
2 Physiology and Pharmacology, West Virgina University, Morgantown, WV,
3 Mechanical and Aerospace Engineering, West Virgina University, Morgantown, WV,
4 Center for Environmental Health Sciences, University of Montana, Missoula, MT
and 5 Experimental Pathology Laboratories, Inc., Sterling, VA.
The hypothesis of this study is that nanoparticle (NP) shape will affect pulmonary
inflammation and fibrosis. To test this hypothesis, three types of anatase TiO 2 NPs
were used: nanospheres (NS), short nanobelts (NB-1) and long nanobelts (NB-2).
For in vivo studies, we exposed C57Bl/6 mice by pharyngeal aspiration to TiO 2
nanoparticles (0-30 μg/mouse). Whole lung lavage (WLL) and histopathology
studies were conducted. WLL demonstrated that TiO 2 NPs induced dose- and
time-dependent pulmonary inflammation (WLL PMNs) and damage (WLL fluid
albumin concentration and lactate dehydrogenase activity). Cytokine and
chemokine inflammatory mediators also showed dose- and time-dependent
changes in WLL fluid. The pattern of responses was NS∠NB-1∠NB-2. Pulmonary
clearance of NS and NB-2 indicated that relative to initial deposition, at 28 days
post-exposure 69% of NS were cleared verusus 54% for NB-2, and at 112 days
post-exposure 96% of NS were cleared versus 80% for NB-2. Histopathology studies
showed that at 28 days post-exposure, TiO 2 NPs were observed in alveolar
macrophages within the lung, and to a lesser degree within the tracheal-bronchiolar
lymph nodes (TBLN). There was an increase in the incidence of the latter finding
at 112 days post-exposure, suggesting NP clearance from the lung to the lymph
nodes. Persistent alveolitis was observed in mice exposed to NB-1 and NB-2.
Interstitial fibrosis was only observed in the NB-1 and NB-2 groups at 28 days
post-exposure, and this change persisted at an increased incidence at 112 days postexposure.
Taken together, the data suggest that NP shape does affect lung toxicity
and exposure to TiO 2 nanobelts may result in adverse health outcomes. Supported
in part by grant RC2 ES-018742 (A. Holian, PI) and NSF grant CBET-0834233
(N. Wu, PI).
312 SOT 2011 ANNUAL MEETING
1455 INFLUENCE OF THE AGGLOMERATION STATE ON IN
VIVO PULMONARY INFLAMMATION AND
CYTOTOXICITY OF INHALED NANO-AEROSOLS OF
TIO2.
A. Noel 1 , K. Maghni 2 , Y. Cloutier 3 , C. Dion 3 , R. Tardif 1 and G. Truchon 3 .
1 Environmental and Occupational Health, University of Montreal, Montreal, QC,
Canada, 2 University of Montreal, Montreal, QC, Canada and 3 Institut de Recherche
Robert-Sauvé en Santé et en Sécurité du Travail, Montreal, QC, Canada.
Nanoparticle (NP) size may be a critical parameter related to toxicity. Few studies
have investigated the influence of the agglomeration state on the correlation between
the characteristics of the NP exposure dose (including agglomerates size in
aerosols) and the toxic effects. The objective of this study was to evaluate in vivo the
pulmonary toxicity of nano-TiO2 aerosols of different agglomeration states,
strongly (>100 nm) and lightly agglomerated (