The Toxicologist - Society of Toxicology

positive cells, caspase-3 activation and protein nitration) and cardiac remodeling
(shown by hypertrophy and fibrosis). Mechanistic study in which cardiac H9c2
cells were treated with 100-400 mM alcohol for 24h revealed that alcohol exposure
induced cell death, nitrosative damage and fibrosis, along with NADPH oxidase
p47phox (NOX) activation in a dose-dependent manner. Pre-treatment of alcoholtreated
cells with NOX inhibitor (apycinin), peroxynitrite scavenger (uriate), NOS
inhibitor (L-NAME) and superoxide dismutase mimic (MnTMPyP) significantly
abolished alcohol induced cell death. Furthermore, exposure to alcohol significantly
up-regulated the expression of angiotensin II (AngII) and its type 1 receptor
(AT1) in vitro and in vivo, either PKCα/β1 inhibitor or AT1 blocker completely
prevented alcoholic NOX activation, and AT1 blocker inhibited the expression of
PKCβ1, implying that alcoholic NOX activation is dependent of PKCα/β1 activation
via AT1.To validate the in vitro findings, alcohol-fed and pair-fed mice were
treated with MnTMPyP (5 mg/Kg) daily for 2 months, which did not change
blood pressure, alcohol-induced cardiac PKC and NOX activation, but significantly
prevented alcohol-induced cardiac nitrosative damage and cell death, along
with a prevention of cardiac fibrosis and dysfunction. These results suggest that alcohol-induced
cardiac cell death is mediated by up-regulated Ang II interaction
with AT1 to induce PKCα/β1-dependent NOX activation, and the cardiac cell
death plays a critical role in the development of alcoholic cardiomyopathy.
211 ASSESSMENT OF THE VARIATION IN TROPONIN I
LEVELS IN THE DOG DURING THE PERIOD PRIOR
TO TOXICOLOGICAL STUDY DOSING.
C. J. Betts, T. Child, S. J. Bickerton, M. Moores, N. Derbyshire, H. T.
Thomas, M. C. Jacobsen and S. Robinson. Safety Assessment, AstraZeneca,
Macclesfield, Cheshire, United Kingdom.
Cardiotoxicity is major cause of attrition of novel compounds during preclinical
and clinical development. Thus cardiovascular toxicity is a primary safety concern
for the pharma industry and there is a drive to progress the assessment and use of
predictive biomarkers to reduce attrition in late stage development. Cardiac troponin
is a direct marker for cardiac myocyte damage and is used in the clinic for
monitoring cardiac injury. This marker can be readily assessed in preclinical toxicity
study species using species-specific ELISA methodologies or automated analysers
developed for the assessment of troponin in the clinic. The objective of this study
was to assess the levels of troponin I (TNI) in the period prior to the start of compound
administration. Animals were acclimatised for at least 3 weeks prior to dosing
and were sham dosed over the period of sample collection. Twelve female dogs
were sampled daily for 7 days prior to the start of dosing by oral gavage. Blood was
taken at the same time each day and analysed using an Ultrasensitive human TNI
assay validated for use with dog plasma on the Siemens Centaur CP analyser. In the
majority of animals TNI levels remained close to the assay limits of detection
(0.006 ng/mL) for the period prior to study start. However, 5 out of 12 animals
demonstrated sporadic, marked increases in TNI to levels considered indicative of
cardiac toxicity, but which returned to baseline values at the next sample point.
This analysis highlights that plasma levels of TNI can vary markedly in individual
animals during the acclimatisation period prior to toxicological study start. The
cause of this release is not clear, although it coincides with the onset of handling
and dosing procedures. Thus to prevent sporadic, non-dose related increases in TNI
during the early dosing period animals should be acclimatised over an extended period
of time with sham dosing prior to study start if possible.
212 EGCG DECREASES POLYCHLORINATED BIPHENYL-
INDUCED EXPRESSION OF CYP1A1 AND
ENDOTHELIAL INFLAMMATORY PARAMETERS VIA
INDUCTION OF ANTIOXIDANT ELEMENTS.
B. Hennig 1 , S. Han 1 and M. Toborek 2 . 1 Animal and Food Sciences, University of
Kentucky, Lexington, KY and 2 Department of Neurosurgery, University of Kentucky,
Lexington, KY.
Epigallocatechin gallate (EGCG) protects against vascular diseases such as atherosclerosis
via its antioxidant and anti-inflammatory functions. Polychlorinated
biphenyls (PCB) are persistent and widespread environmental contaminants that
induce oxidative stress and inflammation in vascular endothelial cells. Although
PCBs are no longer produced, they are still detected in human tissues and thus a
risk for vascular dysfunction. We hypothesized that EGCG may protect endothelial
cells against PCB-induced cell damage via its antioxidant and anti-inflammatory
properties. To test this hypothesis, primary vascular endothelial cells were pretreated
with EGCG (25 and 50 μM), followed by exposure to coplanar PCB126
(0.25 μM). Exposure to PCB126 significantly increased cytochrome P450 1A1
(Cyp1A1) mRNA and protein expression and superoxide production, events which
were significantly attenuated following pretreatment with EGCG. Similarly,
EGCG also attenuated PCB126-mediated induction of adhesion molecules such as
monocyte chemotactic protein-1 (MCP-1). Furthermore, EGCG decreased en-
dogenous or base-line levels of Cyp1A1 and MCP-1 in endothelial cells. Most of
all, treatment of EGCG upregulated expression of phase II antioxidant enzymes,
glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1
(NQO1), in a dose-dependent manner via Nrf2 signaling. Pretreatment of all-trans
retinoic acid, an inhibitor for antioxidant response element (ARE)-driven gene expression,
resulted in an increase of Cyp1A1 and MCP-1 expression, suggesting
ARE-driven antioxidant enzymes play an important role against PCB-induced inflammatory
responses in endothelial cells. These data show that EGCG may reduce
cardiovascular disease risks caused by PCB exposure via elevated expression of antioxidant
genes. (This work was supported in part by NIH/NIEHS grant
P42ES007380 and the UK AES)
213 QUANTITATIVE PROTEOMICS REVEALS RAPID
CHANGES IN NA+/K+ ATPASE AND NCX1 LEVELS IN
ADULT ZEBRAFISH HEART FOLLOWING TCDD
EXPOSURE.
K. Lanham1 , J. Zhang2 , R. Peterson2 , W. Heideman2 and L. Li2 . 1Biomolecular Chemistry, University of Wisconsin Madison, Madison, WI and 2School of Pharmacy,
University of Wisconsin Madison, Madison, WI.
The hearts of zebrafish embryos and larva are acutely sensitive to TCDD. Within
hours of exposure to TCDD cardiac output is reduced, which is followed by severe
heart malformations and pericardial edema. Interestingly, juvenile and adult zebrafish
hearts are refractory to the acute effects of TCDD exposure, but it is unknown
how this occurs. One possibility is that the mechanism responsible for toxicity
in the embryo is still active, but the adult has found a way to compensate for
the disruption. To investigate this possibility we used a proteomic approach to determine
whether there are any early alterations in the adult heart consistent with a
toxic response to TCDD. Adult zebrafish were exposed to TCDD (1 ng/mL), or vehicle,
for 1 hour by static water born exposure, and whole hearts were collected at
24 hours post-exposure. Quantitative mass spectrometry based on the spectral
counting approach was employed to evaluate the protein expression changes induced
by TCDD. Using the NSAF (normalized spectral abundance factor) approach
we found 105 proteins with altered abundance following TCDD exposure,
60 of these were over expressed (>1.5 fold) and 45 were under expressed (