The Toxicologist - Society of Toxicology

ment. Paradoxically, buthionine sulfoximine, which inhibits glutathione synthesis,
prevented the AQ-induced ALT increase. Cyclosporine also decreased AQ-induced
ALT elevation. Conclusion: An AQ-induced liver injury model was developed in
rats that is characterized by a delayed onset of mild liver injury, activated Kupffer
cells/macrophages, apoptotic cells and lymphocyte infiltration in the liver. The effect
of cyclosporin is consistent with an immune mechanism. This work was supported
by grants from CIHR.
424 DEVELOPMENT OF AN ANIMAL MODEL OF
ISONIAZID-INDUCED LIVER INJURY.
I. G. Metushi 1 and J. P. Uetrecht 2, 1 . 1 Pharmacology, University of Toronto, Toronto,
ON, Canada and 2 Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
Background: Idiosyncratic drug reactions are a major medical problem. A classic example
is isoniazid (INH)-induced liver toxicity. Previous studies suggested that this
toxicity is due to bioactivation of acetylhydrazine based on the observation that no
covalent binding of the parent drug was observed and acute toxicity in an animal
model was prevented by inhibition of the hydrolysis of N-acetylisoniazid. However,
it seemed unlikely that INH would not also be oxidized to a reactive metabolite.
We set out to determine if INH is oxidized to a reactive metabolite and to develop
an animal model with characteristics more like the liver injury that occurs in humans.
Methods and Results: An anti-INH antibody was generated by reacting
Protein Blue with an active ester of isonicotinic acid followed by immunization of
rabbits with this antigen. This antibody has a high affinity for isonicotinic acidmodified
protein and it detected many modified proteins from the livers of rats
treated with INH on a western blot. Attempts were made to develop a mouse
model by treating C57BL and Cbl-b (-/-) mice with INH (200 mg/kg/day in food)
and co-treated with either INH-modified hepatic protein, 6-formylindolo[3,2b]carbazole
or anti-CD25 antibody to try to prevent immune tolerance. Although
these treatments did not lead to an increase in ALT, we found that INH interferes
with the ALT assay by reacting with pyridoxal, the cofactor in the assay, so we
changed to a sorbitol dehydrogenase (SDH) assay to test for liver injury. Treatment
of Brown Norway rats with INH (200 or 400 mg/kg/day by oral gavage) for 1 week
led to an increase in serum SDH levels. Conclusions: These studies demonstrate
that bioactivation of INH itself leads to covalent binding and further work is being
carried out to determine the mechanism of injury in this animal model and to see if
it is consistent with INH-induced liver injury in humans. This research was supported
by grants from CIHR.
425 APPROACH FOR DETECTING FLUTAMIDE-INDUCED
HEPATOTOXICITY USING GLUTATHIONE
DEPLETED RATS.
H. Horie, I. Miyawaki, I. Matsumoto, T. Kai, M. Aoki, T. Yamada, J. Kimura,
H. Funabashi and T. Seki. Dainippon Sumitomo Pharmacology Co., Ltd., Osaka,
Osaka, Japan.
AIM: Flutamide, a nonsteroidal antiandrogen drug primarily used for treatment of
prostate cancer, is associated with idiosyncratic drug-induced liver injury (DILI) in
human patients. In pre-clinical phase, however, it is known that obvious hepatotoxicity
is not found in flutamide-treated rodents. Glutathione (GSH) plays an important
role in protection against oxygen stress and reactive hepatotoxic metabolite.
Generally the glutathione s-transferase activity of rodents is higher than that of
human, which may be contributing the species difference in incidence of DILI. In
this study, we aimed to detect flutamide-induced hepatotoxicity using hepatic GSH
depleted rats. METHODS: For depletion of hepatic GSH, we used 2 GSH-depleting
agents, D,L-buthionine sulfoximine (BSO) and diethyl maleate (DEM).
Sprague-Dawley male rats were intraperitoneally injected with BSO-DEM mixture
(500 mg/kg each in corn oil) or vehicle 1 h before flutamide treatment. Flutamide
(300 mg/kg in 0.5% methylcellulose) or vehicle was orally treated by gavage. Rats
were sacrifice 6 and 24 h after treatment of flutamide to collect serum and liver
samples. Measurement of serum hepatic enzyme activities, hepatic GSH level and
gene expression, and histopathological evaluation of the liver were carried out. RE-
SULTS: Slight centrilobular vacuolation in the hepatocytes was noted by flutamide
treatment, and the hepatic alteration was markedly accelerated by depletion of hepatic
GSH (ex. centrilobular vacuolation and necrosis in the hepatocytes, increases
in AST, ALT and LDH). Expression of GSH synthesis-related genes were increased
by GSH depletion, and the changes were accelerated by flutamide treatment.
Expression of oxidative stress-related genes was also increased by flutamide treatment.
CONCLUSIONS: We could successfully detect flutamide-induced hepatotoxicity
using GSH-depleted rats. Thus, it is considered that the GSH-depleted rat
could be useful to detect DILI potential of candidate drugs in the pre-clinical phase.
426 A 3-DIMENSIONAL LIVER MODEL FOR THE SAFETY-
EVALUATION OF DRUG CANDIDATES.
R. M. Kostadinova, F. Boess, L. Suter-Dick, T. Weiser, T. Singer and A. Roth.
F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Drug-induced liver injury is a common adverse effect observed in animal studies
and the main cause for acute liver failure and post-marketing drug withdrawals.
Because of species-specific differences in hepatocellular biology, animal experiments
can predict liver toxicity in man only to a certain extent. We therefore see an urgent
need to apply in vitro assays using human cell models to support and qualify data
generated in animal experiments to assess the potential human response. Variety of
human 2D monolayer cell-culture systems are used in drug-toxicity screens.
However, all of these systems do not accurately recapitulate liver structure, physiology
and function, thus limiting the value of prediction of liver toxicity for humans.
We have functionally characterized rat and human in vitro 3-dimensional liver
models (3D) from RegeneMed and demonstrated that cells grown on a 3D scaffold
maintain liver function over a period of several months, as measured by the secretion
of specific markers such as albumin, fibrinogen, transferrin and urea. We
found that the drug-inducible metabolic activity of key members of Cytochrome
P450s was preserved for several months in 3D liver cells. Using cell-type specific
protein markers, we demonstrated by immunocytochemistry that, like in adult
liver, in vitro 3D liver co-cultures contained hepatocytes, hepatic stellate cells, kupffer
cells and endothelial cells. The presence of different cell types allows measuring
the release of cytokines upon treatment with an inflammatory stimulus. To assess
the utility of the 3D liver system for toxicity screening, we monitored the toxicity of
hepatotoxins over time and found that the human and rat 3D models have the potential
to better detect drug-induced liver toxicity as compared to 2D hepatocytes.
In conclusion, liver cells with preserved heterotypic cell-cell interactions and 3D
liver architecture, appear to resemble in vivo liver physiology and metabolism and
we propose that this new model could be used in the future for more accurate evaluation
of drug-induced liver toxicity.
427 A BENCHMARK DRUG LIST FOR THE STUDY OF
DRUG-INDUCED LIVER INJURY.
V. Vijay, M. Chen, Q. Shi and W. Tong. Systems Biology, NCTR/U.S. FDA,
Jefferson, AR.
The identification of relevant biomarkers to predict Drug-Induced Liver Injury
(DILI) is very challenging. An accurate, reliable, and reproducible approach to classify
known drugs corresponding to their DILI potentials is essential for comparative
analysis to identify key DILI mechanisms and risk factors. Such studies could
result in the discovery of biomarkers useful in drug development and regulatory review.
Currently, drug classification for DILI potential has primarily relied on case
reports and consensus among pathologists. These processes are both time-consuming
and difficult for independent groups to accurately reproduce, and thus have
limited potential to be adopted as common practices for annotating drugs to support
perspective studies. The objective of this study is to develop a benchmark
dataset for DILI study in order to validate existing or novel DILI biomarkers and to
predict DILI potential for new compounds. Using drug labels that are drafted by
the manufacturers and approved by the FDA, we developed a systematic and objective
classification scheme for the DILI potential of drugs. This effort is a central
component of the Liver Toxicity Knowledge Base (LTKB) project initiated at the
FDA’s National Center for Toxicological Research. We developed an LTKB benchmark
dataset (LTKB-BD) that contains 155 DILI associated drugs and 65 non-
DILI associated drugs, which will grow over the years as the LTKB project progresses.
All of these drugs are commercially available, and each has over 10 years of
market use. The label information for all these drugs is stable, evidence of their
DILI potential is well-established, and our annotation procedure can be reproduced
by others to augment the list. In addition, the LTKB-BD has good representation
of known drugs in terms of chemical structure, therapeutic categories, and daily
dosage. In summary, the LTKB-BD has the potential to serve as a benchmark
dataset for the scientific community to evaluate the performance characteristics of
existing DILI biomarkers and to develop novel DILI biomarkers using emerging
molecular technologies.
SOT 2011 ANNUAL MEETING 91