The Toxicologist - Society of Toxicology

measurement of individual bile acids has biological significance with regard to determining
their role in hepatotoxicity and liver disease. Previous studies, in which 21
individual bile acids (IBA) were evaluated, demonstrated that 3 IBA were potential
biomarkers of DILI in the rat model. To investigate these further, an LC/MS/MS
method to quantitate the 3 IBA of taurocholic acid, glycocholic acid and cholic acid
was optimized and validated. Three deuterated internal standards used for the
method and very good accuracy, precision, linearity and reproducibility were
achieved. Serum samples from rats dosed with known toxicants, including hepatotoxicants,
cardiac toxicants, and skeletal muscle toxicants, were analyzed and quantitated
for the three bile acids. Results were evaluated against histopathology findings,
clinical chemistry parameters and other proposed biomarkers. The
concentration of the bile acids were observed to increase significantly in the animals
dosed with all hepatotoxins on fold change and p-value, but not statistically increased
in other toxins dosed animals relative to control animals, even alanine amimotransferase
(ALT) elevated. Also, cholic acid was observed to response prior to the
onset of ALT elevations in rats dosed with hepatotoxins. The data indicated that the
bile acids can potentially be an earlier, more specific and more sensitive biomarker of
hepatotoxicity in preclinical species. The relationships of the individual bile acids to
the specific disease states and their predictivity will be further investigated.
438 UNTARGETED METABOLOMICS IDENTIFIED A
PANEL OF MARKERS FOR DRUG-INDUCED LIVER
INJURY.
L. Guo 1 , N. Masutomi 2 , M. Miyake 2 , M. Yamazaki 2 , H. Sato 2 , K. A. Lawton 1 ,
M. V. Milburn 1 and J. A. Ryals 1 . 1 Metabolon, Durham, NC and 2 Mitsubishi Tanabe
Pharmacology Corporation, Chiba, Japan.
Drug-induced liver injury (DILI) is a major cause for compound attrition during
drug development and drug withdraws from market. The current practices using
histopathological analyses in preclinical animal models may have limitations in sensitivity
and discordance with effects in humans. In order to identify biomarkers for
improved DILI detection, we performed an untargeted metabolomic profiling of
plasma, urine, and liver samples from rats treated with 13 known liver toxicants at
two doses (a low dose and a high dose) and two time points. Nine of the toxicants
(acetaminophen, methapyrilene, ticlopidine, bendazac, ethionine, cyclosporine A,
naphthylisothiocyanate, tetracycline, and carbon tetrachloride) caused various
types and degrees of heptocellular damages and altered clinical chemistry, especially
at the high dose and at the later time point. The other 4 toxicants (carbamazepine,
flutamide, chlorzoxasone, and nimesulide) did not cause any apparent liver injury
to the rats, thus they were grouped as “human specific toxin” as they were known to
induce liver injury in human. Statistical analysis and pathway mapping of the
nearly 1,900 metabolites detected across the three matrices revealed various diverse
metabolic perturbations from each toxicant. A small panel of plasma and urine biomarkers
was found to show common responses to most or all the toxicants, including
the human specific toxins. The changes of these biomarkers were apparently
specific to DILI but not to non-specific metabolic interferences such as cellular energetic
state and altered gut flora, thus they could be potentially used as DILI biomarkers
during preclinical stage.
439 EVALUATION OF ALT ISOZYMES AS REFINED
BIOMARKERS OF DRUG-INDUCED LIVER DAMAGE:
CHARACTERIZATION AND DEVELOPMENT OF
METHODS FOR ANALYZING ALT1 AND ALT2
ISOZYMES.
M. S. Mondal 1 , J. Gabriels 1 , K. Zhu 2 and F. Pognan 3 . 1 Investigative Toxicology,
Preclinical Safety, Novartis Institute of Biomedical Research, Inc., Cambridge, MA,
2 Analytical Sciences, Novartis Institute of Biomedical Research, Inc., Cambridge, MA
and 3 Investigative Toxicology, Preclinical Safety, Novartis Institute of Biomedical
Research, Inc., Basel, Switzerland.
In clinical and preclinical practices, the total serum alanine aminotransferase (ALT)
activity serves as a routine surrogate marker for liver injury. However, serum ALT
activity can sometimes be elevated upon drug treatment without detectable liver
histopathology findings. Conversely, it is also possible to observe histopathologic
liver damage in vivo, with no sign for elevated total ALT activity in serum. In drug
development, these uncertainties undermine the diagnostic value of total serum
ALT activity measurement, creating risks for false positive or false negative evaluations.
To improve upon these uncertainties, we have conducted an investigation
over a wide range of organs of the two isozymes - ALT1and ALT2 – that are expressed
differentially in various tissues and serum. Using large-scale two dimensional
gel electrophoresis (2DGE), Westerns/Mass spectrometry methodologies and
functional activity measurements in various tissues, we have established specific
methods to detect and identify ALT1 and ALT2 isozymes. Based on our analysis,
94 SOT 2011 ANNUAL MEETING
we propose to correlate the total ALT activity with the levels of ALT1 and ALT2
isozymes in various tissues and serum. Furthermore, in this presentation, we discuss
how ALT1 and ALT2 levels and their activities could be used as a possible specific
signature for liver damage and how these analyses can help diagnose the mechanisms
of ambiguous liver injury.
440 SORBITOL DEHYDROGENASE AND GLUTAMATE
DEHYDROGENASE ARE NOT SUPERIOR TO
TRADITIONAL BIOMARKERS OF LIVER INJURY: A
HEALTHY VOLUNTEER STUDY OF HEPARINS.
A. H. Harrill 1 , S. Eaddy 1 , J. Roach 2 , I. D. Fier 2 and P. B. Watkins 1, 3 . 1 University
of North Carolina-Hamner Institute for Drug Safety Sciences, The Hamner Institutes,
Research Triangle Park, NC, 2 Momenta Pharmaceuticals, Cambridge, MA and
3 University of North Carolina Chapel Hill, Chapel Hill, NC.
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are
routinely incorporated into clinical drug development as markers of liver injury.
However, some agents, such as heparins, cause ALT and AST elevations, yet rarely,
if ever, cause serious liver injury. Sorbitol dehydrogenase (SDH) and glutamate dehydrogenase
(GLDH) have been proposed as alternative biomarkers of liver injury
and, if specific for serious liver injury, should not be elevated by heparins. To test
this hypothesis, we measured SDH and GLDH in serial serum samples obtained in
48 healthy adult subjects who received one of four heparin preparations (s.c.) twice
daily for 4.5 days: unfractionated heparin (150 IU/kg, n=12), enoxaparin sodium
(100 IU/kg, n=12), dalteparin sodium (120 IU/kg, n=12), or M118 (a novel
LMWH - 125 IU/kg, n=12). Blood was collected on study Days -1 (baseline), 1, 2,
3, 4, 5, 6 and 11. Mean serum ALT and AST levels increased following treatment
and peaked on Day 6 (mean fold change over subject baseline ± SEM across all
treatments for ALT: 7.8 ± 0.6, and AST: 5.9 ± 0.5). At each time point, there was
no significant difference (P>0.05) in the serum ALT or AST level observed between
each drug tested. Mean serum levels of GLDH and SDH also increased during
treatment with each heparin, and peaked on Day 6 (mean fold change ± SEM for
GLDH and SDH were 14.4 ± 2.5 and 6.2 ± 0.5, respectively). Fold change in
GLDH and SDH were significantly correlated with fold change in ALT levels at all
time points; on Day 6, Spearman’s rank correlation coefficients were 0.76 and 0.86
for GLDH and SDH respectively. We conclude that GLDH and SDH are not superior
to ALT as biomarkers for serious liver injury potential in this model. Efforts
are ongoing to use this sera bank to identify liver specific biomarkers that are not elevated
by heparins, including miRNA and purine nucleoside phosphorylase.
441 IMPORTANCE OF IL-6 SIGNALING FOR
MITOCHONDRIAL STAT3 EXPRESSION AND
MITOCHONDRIAL FUNCTION IN MOUSE LIVER.
F. Boess, A. Roth, K. Schad, C. Zihlmann, M. Bellot, A. Olaharski, L. Suter,
S. Platz, T. Weiser, T. Singer and L. Mueller. F. Hoffmann-La Roche AG, Basel,
Switzerland.
Signal Transducer and Activator of Transcription-3 (STAT3) is an important downstream
effector in the signaling pathway of several cytokines, including interleukin-
6 (IL-6), an important target for the treatment of various inflammatory diseases. In
addition to its function in cell growth or apoptosis, a potential novel function of the
serine-phosphorylated STAT3 as an essential player for the proper function of the
mitochondrial respiratory chain has been proposed by Wegrzyn et al. (2009). As IL-
6 is one of the major factors regulating STAT3 activity, the aim of this study was to
investigate signaling of mitochondrial STAT3 and mitochondrial function in a situation
of absence of IL-6 in order to support safety assessment of therapies targeting
IL-6. Method: Liver tissue was harvested from IL-6 knockout (B6.129S2-
Il6tm1Kopf) and wild type-mice (C57/B6) at 6, 16 and 30 weeks of age. Protein
levels of STAT3, including phosphorylated forms were analyzed in total tissue extracts
and subcellular fractions (nucleus, mitochondria, cytosol). In addition,
freshly isolated liver mitochondria were used for fluorescence-based live monitoring
of mitochondrial oxygen consumption.
Results: Protein expression of STAT3, Ser727-pSTAT3 or Tyr705-pSTAT3 in
whole liver extracts was unchanged in WT compared to KO animals at all ages.
Analysis of STAT3 and its phosphorylated forms in subcellular fractions of liver tissues
did not suggest significant differences in distribution and expression in cytoplasm,
mitochondria or nucleus between genotypes and across different ages.
Measurements of the respiratory function of liver mitochondria under various conditions
revealed no differences between mitochondria form WT and KO animals.
Conclusion: The absence of IL-6 in IL-6 KO animals does not lead to any changes
in STAT3 expression or phosphorylation status and does not alter mitochondrial
function. Hence, absence of IL-6 signaling does not impair mitochondrial function
under normal conditions.