The Toxicologist - Society of Toxicology

and a bronchoalveolar lavage (BAL) investigation performed. A BAL total and differential
cell count was used to evaluate the efficacy of FP. Delivered doses of
0.0103, 0.117 and 0.863 mg/kg were achieved, which were within 14% of target.
This resulted in a dose dependent inhibition of BAL neutrophils of 40%, 79% and
98% respectively compared with the lactose/LPS control group. In conclusion, the
results give confidence that the CBAG is a viable alternative to IT methodology for
studies in early drug development and has the added advantage of producing results
representative of inhaled exposures.
(1) Paul G, Somers G, Moore S and Goodway R, Respiratory Drug Delivery 2012,
Vol. 2, 525-530.
220 Distinct Inflammatory Macrophage Subpopulations and
Myeloid-Derived Suppressor Cells Accumulate in the Lung
and Spleen following Exposure of Mice to Inhaled Ozone.
D. L. Laskin 1 , H. M. Choi 1 , J. D. Laskin 2 and M. Mandal 1 . Pharmacology and
Toxicology, University of Arizona, Tucson, AZ.
Ozone is an ubiquitous urban air pollutant known to damage the lung. Activated
macrophages (MP) and inflammatory mediators they produce have been implicated
in ozone toxicity. However, the phenotype and origin of these cells have not
been established. In these studies, techniques in flow cytometry were used to assess
macrophage subpopulations in the lung, spleen and bone marrow following ozone
inhalation. Exposure of C57Bl/6 male mice to ozone (0.8 ppm, 3 h) resulted in increased
bronchoalveolar lavage (BAL) protein levels after 24-72 h, indicative of
alveolar epithelial injury. This was correlated with a rapid and persistent increase in
the percentage of CD11b+F4/80+ inflammatory macrophages in BAL. An increase
in F4/80 negative CD11b+Ly6C+Ly6G+ myeloid-derived suppressor cells
(MDSCs) was also observed in BAL, a response most prominent 24 h post ozone
exposure. Conversely, F4/80 positive CD11b+Ly6C+Ly6G+ MDSCs decreased in
BAL after ozone exposure. We also found that ozone exposure resulted in a persistent
decrease in CD11b+F4/80+ inflammatory macrophages, and a transient increase
in CD11b-F4/80+Ly6C+Ly6G+ MDSCs in the spleen. In contrast, there
were no changes in bone marrow cell subpopulations after ozone inhalation. Taken
together, these results suggest that the spleen is a source of inflammatory MP in the
lung following ozone exposure; moreover, subpopulations of MDSCs originating in
the lung and the spleen may contribute to early inflammatory responses in the lung
and to processes of injury and repair. Supported by NIH grants GM034310,
ES004738, CA132624, AR055073, ES007148, ES005022.
221 Compare In Vitro Endothelial Cell Release of Endothelium
Derived Vasodilators in Response to Diesel, Biodiesel Blend
and Biodiesel Neat Combustion Extract.
L. Bhavaraju 1 , A. Williams 2 , T. Kormos 3 and M. Madden 4 . 1 Toxicology,
University of North Carolina at Chapel Hill, Chapel Hill, NC; 2 National Renewable
Energy Laboratory, Golden, CO; 3 NERL, ORD, US EPA, Research Triangle Park,
NC; 4 EPHD, NHEERL, US EPA, Chapel Hill, NC.
Diesel exhaust exposure in controlled human chamber studies found exposure induced
inhibition of vasodilation. Particles emitted in exhaust can translocate into
the vascular system however when particles are disolved in solvents of various polarity
the insoluble fraction separates from the soluble fraction. We collected the
soluble fraction of combusted particles dissolved in DMSO for evaluation of the
extract to interfere with the release of endothelium derived vasodilators.
Endothelium dependent vasodilation is dependent on 6-keto PGF1alpha (6keto) a
vaso-active metabolite of arachidonic acid. We have investigated the effects of
diesel, biodiesel blend and biodiesel neat for a change in 6 keto release from three
cell lines: endothelial hybrid cell line (EA hy 926 cells), primary human umbilical
vein endothelial cells (HUVEC) and primary human coronary artery endothelial
cells (HCAEC). ELISA results of EA hy 926 cells with extract exposure for 24hrs
indicate a statistically significant (p≤0.009) increase in 6keto from control and
100μg/mL of biodiesel neat (B100). However ELISA results from HUVEC and
HCAEC exposed to extract for 6, 8 and 24hrs indicate no statistically significant
change in 6keto release. QPCR data from extract exposure indicates there is no increase
in markers of inflammation. However there is a measureable increase in
heme oxygenase-1(HO-1) gene expression. HUVEC and HCAEC with 8hr extract
exposure to B100 at 100ug/mL have over two fold increase in HO-1. The B100
particle composition analysis indicates high levels of Zn and Fe compared to
biodiesel blend and diesel. In our work we address a possible mechanism for attenuation
of vaso-active arachidonic acid metabolites in endothelial cells exposed to
diesel, biodiesel blend and biodiesel neat particle extracts. [This is an abstract of a
proposed presentation and may not necessarily reflect official US EPA Policy.]
46 SOT 2013 ANNUAL MEETING
222 Role of CD36 in Ozone (O 3 )-Induced Lung Injury,
Inflammation, and Vascular Dysfunction.
S. Robertson, S. N. Lucas, P. Hall, M. Paffett and M. J. Campen. University of
New Mexico, Albuquerque, NM.
Ground level O 3 can damage the cardiovascular system. A lack of a clear mechanism
explaining O 3 -induced vascular health effects hinders the effectiveness of policies
for achieving better health. Evidence suggests that inhaled pollutants evoke a
systemic inflammatory response that causes endothelial injury and dysfunction.
Using serum from O 3 -exposed mice, we found that circulating components impaired
acetylcholine (ACh) vasorelaxation in aortas from naïve wild type (WT)
mice. However, the mechanistic interaction(s) between circulating factors and endothelial
cells is unknown. To address this issue we turned our attention to pattern
recognition receptors (PRRs), such as CD36 (cluster of differentiation 36), as mediators
of vascular abnormalities following O 3 exposure. PRRs are capable of detecting
danger signals released by stressed or injured cells. We hypothesized that activation
of endothelial CD36 following acute O 3 exposure mediates cross-talk
between lung-derived circulating factors and vascular endothelium, culminating in
endothelial dysfunction.
Female C57 wild type (WT) and CD36 knockout (KO) mice were exposed to filtered
air (FA) or 1 ppm O 3 for 4 h. Indices of pulmonary (quantified by lavage inflammatory
cells) inflammation was assessed 24 h later. The effects of exposure on
ACh-induced vasorelaxation were studied using the aortic ring preparation. Parallel
experiments were performed in aortas from naïve WT mice incubated with serum
from exposed mice.
O 3 -induced infiltration of macrophages and neutrophils into the airspace in WT
mice were absent in CD36 KO mice. ACh-evoked vasorelaxation of thoracic aorta
of WT mice, but not CD36 KO mice, was significantly reduced after inhalation of
O 3 . Ex vivo assays utilizing homologous serum demonstrated that the vascular damage
caused by O 3 -induced circulating factors was dependent on vascular CD36 receptor
expression.
Collectively, our data demonstrate that an as yet unidentified circulating factor, or
factors, induced by O 3 exposure leads to vascular dysfunction mediated, in part, by
CD36 binding in the vascular tissue.
223 In Vitro Endothelial Cell Model to Assess the Impact of
Systemic Inflammation on Vascular Health.
M. Aragon, E. S. Colombo, M. J. Campen and S. Lucas. Department of
Pharmaceutical Sciences, University of New Mexico, Albuquerque, NM.
Assessing the adverse vascular health effects of systemic inflammation caused by inhaled
toxins has presented a substantial research challenge. Current models rely on
anatomically disputable direct application of xenobiotics, especially airborne particulate
matter, on cultured endothelial cells. Such assay systems fail to account for the
complex interactions and toxicokinetics that occur in vivo. We have developed a
model that takes these factors into account to better elucidate the mechanisms involved
in a living system. This approach utilizes plasma or serum from exposed animals
as the endothelial stimulus, as this is the component in direct contact with endothelial
cells. Briefly, the serum/plasma isolated from exposed animals is incubated
on endothelial cells and canonical activation pathways are assessed. In this way the
endothelial cells act as a “biosensor”, expressing markers of inflammation, especially
cell surface adhesion molecules. In addition, nitric oxide (NO) bioavailability via
electron paramagnetic resonance can be directly measured in the supernatant. We
have characterized this model paradigm using primary endothelial cells from rats
and mice, using known mediators (IL-6, TNF-α) to assess the range of response in
order to compare endothelial cell responses to serum obtained from ozone-exposed
rodents. Serum obtained 24h following exposure to 1ppm ozone for 4h caused a 2fold
increase in vascular cell adhesion molecule-1 cell surface expression on rat aortic
endothelial cells, as compared to serum from filtered air-exposed rats. Similarly,
we observe reductions in NO generation and elevated mRNA expression of specific
markers of endothelial cell activation. The potential for this assay extends beyond
the toxic effects of air pollution, with potential applications to drug safety and efficacy,
as well as having prognostic value for vascular disease.