The Toxicologist - Society of Toxicology

eleased from necrotic cells where it binds to receptor for advanced glycation end
products (RAGE) on macrophages and stimulates inflammatory mediator production.
In the present studies, we analyzed the potential role of HMGB1 in silica induced
inflammation and fibrogenesis. C57BL/6J mice were treated with silica (1
mg/kg Min-U-Sil 5) by aspiration. After 7 d, we found that silica administration resulted
in increased expression of cycloxygenase-2 and inducible nitric oxide synthase
in the lung, enzymes mediating the production of proinflammatory/cytotoxic
prostaglandins and reactive nitrogen species. This was associated with increased expression
of HMGB1 in macrophages and the appearance of soluble RAGE in bronchoalveolar
lavage. We also found that Wnt1 and nuclear β-catenin protein expression
were upregulated in the lung of silica-treated animals, while glycogen synthase
kinase-β3 (GSK-β3), which functions to negatively regulate β-catenin signaling,
was down regulated. This was correlated with increased proliferating cell nuclear
antigen staining in the lung and the development of fibrotic lesions. These data
suggest that HMGB1 and downstream signaling pathways may be important in
both silica-induced inflammation and fibrosis. Supported by NIH grants
CA132624, ES004738, ES005022, AR055073, GM034310 and HL074115.
1666 USING MULTIPARAMETRIC HIGH CONTENT
IMAGING TO ASSESS MECHANISMS OF CELLULAR
STRESS AND TOXICITY.
D. M. Miller 1 , B. A. Samson 1 , S. J. Hong 2 and A. M. Peters 1 . 1 Cellular Imaging
and Analysis, Thermo Fisher Scientific, Pittsburgh, PA and 2 High Content Reagent
Development, Thermo Fisher Scientific, Rockford, IL. Sponsor: J. Haskins.
Understanding cellular toxicity, including effects on a cell’s biochemistry, is key to
understanding the undesired side effects that a compound may have. High-content
image analysis, coupled with antibody-based staining or engineered cell lines expressing
a GFP-tagged protein of interest, serve as a useful tool to help understand
the underlying mechanisms of toxicity. In this study, several markers of cellular
stress, genotoxicity, apoptosis and general toxicity were evaluated. As models of systemic
toxicity, HepG2 (liver) and HK-2 (kidney) cell lines were treated with compound
and stained with antibodies against various cellular targets such as p-H2AX,
p-ATM, p53, cJun, activated caspase 3 and a marker of cell proliferation. In addition,
cell density, morphology and DNA content were also analyzed, providing a
multiparametric approach to understanding the underlying mechanisms of toxicity.
Compounds screened in 96-well microplates demonstrated a range of responses in
these targets for both cell lines. Additional markers were studied in engineered cell
lines that stably express GFP-chimeric protein, that when activated, translocate
from the cytoplasm to the nucleus of the cell. These stable cell lines, expressing
GFP chimeras of ATF6, p53-Hdm2, Rad51, and HIF-1a, were plated into 96-well
microplates, treated with compounds for a set period of time, fixed, stained with
Hoechst (to identify nuclei) and analyzed with a high content imaging platform.
The compounds tested showed varying response for the targets of interest. Overall,
the multiparametric approach proved to be a useful tool for studying mechanisms
of stress and toxicity. The compounds tested showed cell line- and target-specific
profiles for markers of cellular toxicity.
1667 MALDI-MS-BASED DRUG AND PROTEIN IMAGING TO
SIMULTANEOUSLY DETERMINE DRUG DISPOSITION
AND PROTEIN MODIFICATION IN CELLS.
N. Mastrandrea, J. D. Cohen, M. J. Kimzey, T. J. Monks and S. S. Lau.
Pharm/Toxicology University of Arizona, Tucson, AZ.
Understanding the efficiency of drug delivery to target tissues and mechanism of
drug action is pertinent in developing an efficacious therapeutic regimen. Our rodent
models of renal cell carcinoma (RCC) exhibit increased levels of 4EBP1 phosphorylation
at sites critical for the release of eIF4E, facilitating eIF4E-mediated initiation
of cap-dependent translation of proteins that likely contribute to tumor
progression. We used MALDI-MS coupled to Western blot and MTS assays to determine
cellular drug disposition, drug effects, and drug toxicity profile, respectively,
for two potential drugs for the treatment of RCC. Combination treatments
were performed with erlotinib, an EGFR tyrosine kinase inhibitor, and MP470, a
novel receptor tyrosine kinase inhibitor, using QTRRE cells, a chemical transformed
primary renal epithelial cell line derived from the Eker rat. The 20μM combination
treatment resulted in a maximal 30% decrease in cell viability at 2 h.
Western data revealed that combination therapy resulted in a more profound decrease
in p4EBP1-Ser65, -Thr70, and -Thr37/46 than with either drug as a single
treatment. MALDI-MS data also effectively confirmed drug uptake into cells, revealing
394.35 m/z (erlotinib) and 448.2 m/z (MP470). Moreover, we detected induction
of several proteins (8559.4 m/z, 7883.6 m/z and 9153.6 m/z) 1.5 h after
drug treatment, the identity of which is currently under investigation. The rapid
analysis of intact protein levels can be important in determining drug mechanism
of action. Taken together we have demonstrated the value of applying MALDI-
TOF imaging techniques to simultaneously visualize differential protein expression
and localization of drugs in the same sample. We are extending the utility of these
methodologies to analyze nude mice xenograft tumor tissue and human tissues following
drug treatment, to unravel signaling pathways and posttranslational modifications
to key proteins associated with the progression of RCC (GM070890,
ES006694, ES007091, ES016652).
1668 ANNEXINS: AN EARLY RESPONSE TO
ENVIRONMENTAL TOXICANTS AND A POTENTIAL
NEW BIOMARKER OF TUMORIGENESIS.
B. L. Upham 1 , P. Babica 1 , J. Park 1 , I. Sovadinova 1 , D. A. Whitten 2 , C. G.
Wilson 2 , J. E. Trosko 1 and L. Blaha 3 . 1 Pediatrics/Human Development, Michigan
State University, East Lansing, MI, 2 RTSF Proteomic Core, Michigan State
University, East Lansing, MI and 3 Masaryk University, Brno, Czech Republic.
Lower molecular weight polycyclic aromatic hydrocarbons (PAHs) with specific
structural features are potent in vitro inhibitors of gap junctional intercellular communication
(GJIC), and activators of arachidonic acid (AA) release and mitogenactivated
protein kinases (MAPKs), which are cellular events linked with tumor
promotion and other pathologies. We previously found that inhibition of GJIC in
a rat liver oval-like epithelial cell line (WB-F344) is preceded with the activation of
phosphatidylcholine-specific phospholipase C (PC-PLC). We used advanced proteomics
techniques (SILAC – Stable Isotopes Labeling with Amino Acids in Cell
Culture, ZOOM® isoelectric fractionation, 1-DE, 2-DE and mass spectroscopic
identification) to further identify early upstream biochemical signaling events.
Among the proteins specifically affected within a 5 min exposure time to 1-MeA,
annexins A1, A3 and A5 showed significant responses characterized by their disappearance
from plasma membrane, while A2, A4, A6, A7 and A11 were not affected.
Immunostaining experiments on annexin A3 (AnxA3) indicated a translocation
from the plasma membrane within 30 s of exposure followed by reintegration back
into the plasma membrane after 60 min. Translocation of AnxA3 from the plasma
membrane was effectively prevented by pre-treatment of the cells with PC-PLC inhibitor,
D609. Knock-down of AnxA3 by siRNA did not prevent 1-MeA induced
inhibition of GJIC but did stimulate the 1-MeA induced release of AA. We hypothesize
that annexins closely interact with phospholipases in the plasma membrane
until removed from the membrane in response to 1-MeA, and the subsequent
phospholipase-induced events then regulate the release of lipid derived second messengers.
Support: NIEHS grant #R01 ES013268-01A2 to upham.
1669 CYTOTOXICITY OF CYCLODEXTRINS:
IMPLICATIONS IN CELLULAR CHOLESTEROL LIPID
RAFT STUDIES.
S. R. Kotha 1 , A. H. Hinzey 1 , M. A. Kline 1 , E. S. O’Connor Butler 1 , R. M.
Uppu 2 and N. L. Parinandi 1 . 1 Pulmonary, Allergy, Critical Care, and Sleep
Medicine, The Ohio State University College of Medicine, Columbus, OH and
2
Department of Environmental Toxicology, Southern University and A&M College,
Baton Rouge, LA.
Membrane cholesterol (lipid raft-associated) has been emerging as a pivotal player
in cellular signaling cascades. Cyclodextrins, such as methyl-β-cyclodextrin
(MβCD) and hydroxypropyl cyclodextrin (HPCD), have been widely used as tools
to deplete membrane raft-associated cholesterol in cell culture models to study the
role of cholesterol lipid rafts in cell signaling cascades. However, the adverse effects
of cyclodextrins are not thoroughly established in cell culture models. In order to
establish the adverse effects of two well-known cyclodextrins, MβCD and HPCD,
bovine pulmonary artery endothelial cells (BPAECs) were treated with different
concentrations of MβCD and HPCD (2% and 5%, wt/vol) for 15-120 min and
the loss of membrane cholesterol, cell viability (lactate dehydrogenase release and
MTT reduction), cell morphology, protein alterations, changes in phospholipid
fatty acid composition, cell replication, and cytoskeletal alterations were determined.
The results revealed that both MβCD and HPCD caused significant loss of
membrane cholesterol, loss of cell viability, altered cell morphology, loss of membrane
fatty acids, altered proteins, and induction of actin cytoskeletal rearrangement
in BPAECs. However MβCD caused a greater extent of cytotoxicity as compared
to that caused by HPCD under identical conditions. Removal of cholesterol
by cyclodextrin (especially MβCD) treatment, caused loss of fluidity of the cell
membrane and leakage of vital cellular components, and thus led to cytotoxicity
and biochemical alteration in ECs. Also, the study offered a safer method of cholesterol
removal by HPCD treatment, without extensive loss of cell viability, for studies
on the role of lipid rafts in endothelial cell signaling.
SOT 2010 ANNUAL MEETING 355