The Toxicologist - Society of Toxicology

SIN-1 resulted in an 8.6 fold increase in SN compared to CI-1044 alone, and a 4.6
fold increase from SIN-1 alone. Mesenteric and periarterial inflammation in the
CI-1044 group was increased compared to vehicle controls, L-NAME or SIN-1
groups. Mild arterial fibrinoid necrosis was also present in a subset of animals
treated with CI-1044. Co-administration of CI-1044 and L-NAME resulted in reduced
mesenteric and periarterial inflammation compared to CI-1044 alone.
Furthermore, arterial medial injury was not noted. Co-administration of CI-1044
and SIN-1 resulted in a marked increase in mesenteric arterial injury and inflammation
compared to SIN-1 or CI-1044 alone. The present study suggests a link between
NO modulation and DIVI. Future studies will be aimed at further defining
the pathogenesis of this unique type of injury.
1745 INVOLVEMENT OF SHEAR STRESS IN FENOLDOPAM
AND DOPAMINE INDUCED MESENTERIC MEDIAL
ARTERIAL NECROSIS.
D. Dalmas, M. Scicchitano, K. Roland, D. Mullins, T. Chordia, K. Frazier and
H. Thomas. Safety Assessment, GlaxoSmithKline, King of Prussia, PA.
Extrapolation and relevance of drug-induced rodent vascular injury to humans has
hampered drug development due to lack of understanding of the pathologic mechanisms
involved. Although vasodilatation & increased shear stress (SS) have been
hypothesized to be involved in the pathogenesis of these lesions, the exact role of SS
on primary target cells, smooth muscle (SMC) and endothelial cells (EC) in vivo
remain unclear. Dopaminergic DA1 agonists such as Fenoldopam and Dopamine
reproducibly induce mesenteric medial arterial necrosis (MAN) in rats following
single vasotoxic doses. To investigate the involvement of SS in the development of
MAN, rats were given vehicle, Dopamine or Fenoldopam for 4 days followed by
necropsy 24 hours later. Yohimbine, an α2 adrenoreceptor antagonist, also vasoactive
but lacking MAN histologic lesions, was given to rats for 4 days for comparison.
To evaluate the timecourse of lesion development, rats were also given vehicle
or a single vasotoxic dose of Fenoldopam and necropsied 1-, 4-, 6-, 12- or 24 hours
postdose. Mesentery from each rat was collected and frozen in OCT, then EC and
SMC were microdissected from sections of mesenteric arteries of each rat, and
RNA was amplified and analyzed. Regulation of 37 shear stress responsive genes
were evaluated using TaqMan gene expression profiling. Many of the genes evaluated
were confirmed to be differentially expressed by Dopamine and Fenoldopam
in EC- and/or SMC-enriched samples as compared to controls or Yohimbine following
4 daily doses. A number of SS responsive genes were also shown to be differentially
regulated beginning 1- and/or 4-hours post-Fenoldopam treatment and
prior to histological evidence of MAN (which was initially observed beginning at
12-hours). Evaluation of this panel of genes has provided evidence of the involvement
and regulation of SS responsive genes in both EC and SMC during the development
of Dopamine and Fenoldopam-induced vascular injury.
every timepoint analyzed except recovery animals. EMPs with phenotypes of
CD45-CD42d-CD31+CD54+, CD45-CD42d-CD31+CD146+, CD45-CD42d-
CD54+CD146+ and CD45-CD42d-CD106+Flk-1+ were increased significantly
24 hours after drug treatment and correlated with histopathology data. EMP levels
dropped to baseline levels after 10 days of recovery. The present study suggests that
EMPs potentially represent a novel biomarker for DIVI observed in preclinical
species.
1747 A NOVEL MECHANISM FOR ALCOHOLIC
CARDIOMYOPATHY: SUPEROXIDE GENERATION IS A
PIVOTAL MEDIATOR FOR SUPPRESSION OF GAPDH
THAT TRIGGERS CARDIAC IMBALANCE OF ENERGY
UTILIZATION, OXIDATIVE STRESS, AND
REMODELING.
Y. Tan 1 , Z. Zhou 2 and L. Cai 1 . 1 Pediatrics, University of Louisville, Louisville, KY
and 2 Medicine, University of Louisville, Louisville, KY.
We tried to define whether ethanol may induce NADPH oxidase-dependent superoxide
that inhibits GAPDH, thereby diverting upstream metabolites from glycolysis
into pathways of glucose overutilization, resulting in an imbalance of glucose
and fatty acid utilization, cardiac oxidative stress and structural remodeling. Male
C57/BL6 mice were pair-fed an alcohol or isocaloric maltose dextrin liquid diet for
two months. Chronic alcohol feeding caused a significant increase in NADPH oxidase
expression and a decrease in GAPDH expression, and also caused a cardiac
glucose overutilization, shown by increased GSK-3β phosphorylation and hexokinase
II expression, and fatty acid metabolism disorder, evidenced by increased
PPARα (a positive mediator for fatty acid uptake and utilization) and decreased
PGC-1α (an essential regulator for the optimal cardiac mitochondrial fatty acid oxidation).
The imbalanced energy utilization was associated with significant increases
in cardiac oxidative damage, shown by 3-nitrotyrosine accumulation, cell
death and neutrophil infiltration, and cardiac remodeling, mirrored by cardiac hypertrophy
and fibrosis (TGF-β1 and CTGF expression). To define the critical role
of superoxide, cardiac H9c2 cells were treated with 2.5% (v/v) ethanol for 120
hours, which induced significant oxidative damage, cell death, and fibrosis, as observed
in the hearts of alcoholic mice. However, pre-incubation of cells with superoxide
dismutase mimetic significantly attenuated ethanol-decreased GAPDH expression,
along with improvement of the above described pathogenic changes.
Therefore, alcohol activation of NADPH oxidase-related superoxide generation
plays a critical role in depressing GADPH expression. The decreased expression and
function of GAPDH may be the key mediator leading to cardiac imbalance of glucose
and fatty acid utilization, oxidative damage, inflammation and remodeling
that eventually results in alcoholic cardiomyopathy.
1746 IDENTIFICATION AND ANALYSIS OF CIRCULATING
ENDOTHELIAL MICROPARTICLES AS A POTENTIAL
BIOMARKER OF DRUG-INDUCED VASCULAR INJURY.
B. Enerson, C. Conley, C. Huang, M. Kate and S. Sokolowski. Drug Safety
Research and Development, Pfizer Global Research and Development, Groton, CT.
Sponsor: M. Lawton.
The finding of acute drug-induced vascular injury (DIVI) in preclinical toxicity
studies often leads to delays in, or termination of, drug development projects because
the relevance of this finding to humans is unclear. Furthermore, there are no
sensitive and specific DIVI biomarkers that readily translate into a clinical setting.
Endothelial microparticles (EMPs) are small vesicles that are released from endothelial
cells into circulating blood and are found at elevated levels in a number of
human diseases associated with vascular or endothelial dysfunction. The purpose of
this study was to identify and quantify by flow cytometry circulating EMPs in
plasma of rats treated with phosphodiesterase 4 (PDE4) inhibitor CI-1044. In this
time-course study, platelet-poor plasma (PPP) was collected from male Sprague
Dawley rats (5/time point) 4, 8, 16, and 24 hours following a single dose, 24 hours
following either 2 or 3 daily doses, and 10-days following 3 daily doses of 80
mg/kg/day CI-1044. Detection of EMPs in PPP was accomplished using markers
specific for endothelial cells (CD31, CD106, CD146, CD54, Flk-1, vWF).
Additional exclusion markers (CD45 and CD42d) and the apoptotic marker
Annexin V were included to further characterize the origin of microparticles. All
stained PPP samples were processed concurrently with microbeads to set a baseline
for size and EMPs were identified based on size and labeling with specific markers.
Administration of CI-1044 to rats resulted in increased plasma levels of EMPs at
1748 POLYCHLORINATED BIPHENYL INDUCED VCAM-1
EXPRESSION IS ABOLISHED IN AORTIC
ENDOTHELIAL CELLS ISOLATED FROM CAVEOLIN-1
DEFICIENT MICE.
S. Han 1 , M. Toborek 2 and B. Hennig 1 . 1 Animal and Food Sciences, University of
Kentucky, Lexington, KY and 2 Department of Neurosurgery, University of Kentucky,
Lexington, KY.
Environmental chemical contaminants, such as polychlorinated biphenyls (PCBs)
are known to be atherogenic in human and the underlying mechanism is becoming
elucidated. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for
adhesion and uptake of monocytes in the initial stage of atherosclerosis development
in endothelium. PCBs may be proatherogenic by causing upregulation of
VCAM-1. Caveolae are particularly abundant in endothelial cell membrane and are
involved in trafficking and signal transduction. The objective of this study was to
investigate the role of caveolae in PCB-induced endothelial cell dysfunction.
Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1 deficient
mice and background C57BL/6 mice were treated with coplanar PCBs, such as
PCB77 and PCB126. In addition, siRNA gene silencing technique was used to
knockdown caveolin-1 in porcine vascular endothelial cells. Expression of VCAM-
1 was observed as a proatherogenic marker in cells. Both coplanar PCBs increased
mRNA and protein levels of VCAM-1 and number of adhered monocytes in
porcine endothelial cells. In MAECs, VCAM-1 mRNA and protein levels were increased
after exposure to both coplanar PCBs in cells with caveolin-1, whereas not
significantly altered in cells absent caveolin-1. Furthermore, number of adhered
monocyte was significantly increased by PCB exposure only in caveolin-1 containing
MAECs. Similarly, caveolin-1 silencing using siRNA technique in porcine en-
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