The Toxicologist - Society of Toxicology

1306 DEVELOPMENT OF AN IN VITRO ASSAY FOR
RESPIRATORY SENSITIZATION CONSIDERING THE
VISION ON TOXICITY TESTING IN THE 21ST
CENTURY.
S. Verstraelen 1 , J. Hooyberghs 1 , I. Nelissen 1 , H. Witters 1 , G. Schoeters 1, 2 , P.
Van Cauwenberge 3 and R. Van Den Heuvel 1 . 1 Environmental Risk and Health,
Flemish Institute for Technological Research - Centre for Advanced R&D on
Alternative Methods (VITO - CARDAM), Mol, Belgium, 2 University of Antwerp,
Antwerp, Belgium and 3 Department of Otorhinolaryngology, University of Ghent,
Ghent, Belgium. Sponsor: B. De Wever.
Despite regulatory requirements there is no established protocol for the identification
of chemical respiratory sensitizers. New tests should be based on mechanistic
understanding and should be preferentially restricted to in vitro assays.
To contribute to the development of a respiratory sensitization assay considering
the vision on toxicity testing in the 21st century, we performed transcriptomics experiments
and studied alterations in gene expression in a BEAS-2B lung model
after exposure to the respiratory sensitizers ammonium hexachloroplatinate IV,
hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and
methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Agilent
Whole Human Genome arrays used in this study revealed markers that are able to
discriminate respiratory sensitizers from respiratory non-sensitizers. One of the interesting
genes is CASP9, which is known to be associated with asthma and/or respiratory
sensitization. When categorizing the 1000 most discriminative genes into
biological Gene Ontology terms, 20 genes were associated with immune function.
The majority of these genes have already been studied in the context of airway inflammation,
asthma, and respiratory sensitization (e.g. CCL24, CEBPB, MIF, and
TLR4). We also hypothesized on possible toxicity pathways that could be associated
with respiratory sensitization based on pathway analysis. Within this cellular
model the phosphatase and tensin homolog (PTEN) pathway was identified as possibly
specific for respiratory sensitization.
These preliminary transcriptomics data add a new aspect to the action in the field
to develop an in vitro respiratory sensitization assay, taking into account the new vision
on mechanism-based toxicity testing.
1307 SELECTIVE MEASUREMENT OF ALT ISOFORMS IN
RAT SERUM BY LC/MS.
C. Drupa and J. Colangelo. DSRD, Pfizer, Groton, CT.
Alanine aminotransferase (ALT) activity in serum has long been used as a biomarker
of hepatotoxicity in clinical and preclinical studies and increases often correlate
with observed histopathological findings in the liver. Occasionally, increases
in serum ALT have been measured with no concurrent liver histopathological findings
in preclinical species and can complicate the safety profile of a drug candidate.
To date, there are two known forms of ALT which are differentially expressed in tissues
and subcellular compartments which the chemistry analyzer used to measure
serum ALT activity can not distinguish. We have developed a liquid chromatography/mass
spectrometry (LC/MS) based assay to selectively and independently
measure total ALT, ALT 1 and ALT 2 levels in rat serum using peptide quantitation
with stable isotope dilution. Immunoprecipitation is employed to enrich the ALT
and remove interfering proteins from the serum sample after which the samples are
digested and signature peptides are quantified. Standard curves are constructed by
fortifying ALT depleted control rat serum with known amounts of recombinant rat
ALT 1 and ALT 2. The limits of quantitation achieved with this assay are 20, 10
and 10 ng/mL for total ALT, ALT 1 and ALT 2, respectively. In addition to serum,
this assay has been used to measure ALT isoforms in tissue homogenates. The ALT
protein levels determined by LC/MS have been compared with ALT activity levels
measured by other assays. This data is being used to understand the role of each
ALT isoform in disease progression in the rat.
1308 SERUM CARDIAC TROPONIN I CONCENTRATIONS
ARE TRANSIENTLY INCREASED IN RATS DOSED
WITH ROSIGLITAZONE, A PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR γ AGONIST.
G. M. Hirkaler 1 , R. Fernandes 1 , D. Coluccio 1 , C. Kanwal 2 , E. Rasmussen 3 , T.
Visalli 1 , M. O. Bachynsky 4 , H. Hilton 2 , R. Nicklaus 1 , J. Hoflack 5 , A. Buness 5 ,
F. Herting 6 , L. Suter-Dick 5 and I. Mikaelian 1 . 1 Toxicology, Hoffmann-La Roche,
Nutley, NJ, 2 RNA, Hoffmann-La Roche, Nutley, NJ, 3 InS, Hoffmann-La Roche,
Nutley, NJ, 4 Phar, Hoffmann-La Roche, Nutley, NJ, 5 PRNB, Hoffmann-La Roche,
Basel, Switzerland and 6 TR-D, Hoffmann-La Roche, Penzberg, Germany.
Rosiglitazone, a PPARγ agonist of the thiazolidinedione class, is a major insulinsensitizing
drug widely used to treat type-2 diabetes. Rosiglitazone causes myocardial
hypertrophy in rodents and increased risk of myocardial infarction in man. To
better characterize its cardiac effects and determine if cardiac troponin I (cTnI) can
serve as an early biomarker for cardiac liability, male Wistar rats were orally administered
0, 10 or 80 mg/kg/day rosiglitazone. Myocardial gene expression profiling,
hematology, histopathology and clinical chemistry, including measurement of cTnI
concentrations with the Singulex Erenna® Ultrasensitive Immunoassay, were evaluated
after 6, 24, 168 and 336 hours of dosing. Heart weight mildly increased after
168 (~10%) and 336 (~15%) hours of dosing at 80 mg/kg/day in the absence of
microscopic changes. At the transcriptomics level, gene categories typically associated
with myocardial damage were not over-represented. Most importantly, cTnI
transiently increased from 2±2 pg/mL in vehicle-treated rats to 19±4 pg/mL in 5/9
rats after 168 hours of dosing at 80 mg/kg/day, returning to 3±2 pg/mL after 336
hours of dosing underscoring the temporal nature of cTnI increases. This is the first
study detecting serum cTnI increases in rats administered rosiglitazone. This effect
may be linked to functional changes because rosiglitazone-induced myocardial hypertrophy
is postulated to be the result of positive inotropic and lusitropic effects
without changes in heart rate, ventricular pressures and hematocrit. In light of reported
cardiac events in patients chronically dosed with PPARγ agonists, our results
support cTnI as the earliest biomarker heretofore of cardiac liability associated with
these compounds.
1309 DISTRIBUTION ANALYSIS OF THE ALT ISOZYMES IN
CANINE TISSUES.
Y. Sudo 1 , Y. Takai 1 , M. Aoki 1 , K. Hirai 1 , Y. Matsumoto 1 , N. Inui 1 , Y.
Miyamoto 1 , H. Hamajou 1 , E. Maeda 1 , T. Ito 2 , S. Ohkubo 2 , R. Fukuda 1 and K.
Takami 1 . 1 Development Research Center, Takeda Pharmaceutical Company Limited,
Osaka, Japan and 2 Discovery Research Center, Takeda Pharmaceutical Company
Limited, Osaka, Japan.
Serum alanine aminotransferase (ALT, also known as glutamate pyruvate transaminase,
GPT) is widely used as a marker for hepatic damage in clinical and non-clinical
practice. However, it is thought that ALT elevation in the serum could be
caused by effects on other organs other than only due to hepatic damage. In recent
years, it has been shown in humans, mice and rats that ALT has two isozymes
(ALT1 and ALT2) which are expressed differently in the various organs. But the
isozymes in dogs, which are commonly employed in drug development, are not
well studied. To comprehend the non-clinical data more precisely, in the present
study we investigated the sequences of the canine ALT isozymes and their expression
in various organs. Sequence analysis provided the sequences of canine ALT1
and ALT2, which are highly preserved in the other mammals. Based on the sequences,
gene expression levels in the organs were analyzed by quantitative realtime
PCR and specific polyclonal antibodies induced in rabbits, and then the expression
of the isozymes was analyzed by western blotting and
immunohistochemistry. These analyses showed that the isozymes are expressed differently
in an organ-dependent manner; ALT2 is plentifully expressed in muscle,
adipose tissue and kidney cortex while ALT1 is dominant in liver, heart and gastric
mucosa. In addition, immunohistochemistry demonstrated characteristic distribution
in the canine organs or cells. Strong ALT1 immunoreactivity was noted in the
hepatocytes, myocardiocytes, parietal cells, etc, while ALT2 reactivity was seen in
the proximal renal tubules, striated muscle, neuronal cell bodies, etc. The present
findings could be of great value for better understanding of serum ALT elevation in
canines, especially in the case of absence of histopathological changes in the liver.
1310 CYTOKINES OF INFLAMMATION AND OXIDATIVE
STRESS IN THE URINE OF COCAINE USERS.
M. M. Bourgeois and I. S. Richards. EOH, University of South Florida COPH,
Tampa, FL.
Cocaine is a powerful sympathomimetic associated with systemic inflammation,
oxidative stress and vascular dysfunction. Its use has been linked to renal disease,
rhabdomyolysis, vasoconstriction, acute myocardial ischemia and infarction.
Systemic vascular damage has been linked to the induction of inflammatory and
oxidative stress pathways. This study examines the expression of inflammatory,
stress and vascular biomarkers in the urine of cocaine users. Commercially available
ELISA test kits were used to assay urine specimens for markers representative of
these pathways, including several interleukins, myeloperoxidase, high sensitivity c
reactive protein, myoglobin, proatrial natriuretic peptide, creatine kinase MB
isoenzyme, aldosterone, microalbumin, neutrophil gelatinase associated lipocalin,
vascular endothelial growth factor and heat shock protein 90. Several of these have
not yet been validated in urine; however, urine specimens are non-invasive and
could prove useful for qualitative diagnostic testing once appropriate reference
ranges have been identified. Increased expression of these markers is associated with
SOT 2010 ANNUAL MEETING 279