The Toxicologist - Society of Toxicology

survival using both smoking regimes, there was no clear difference in toxicity between
cells exposed to smoke under either ISO or HCI regimes, at exposures of up
to 30 minute duration.
Further work is required to define the precise dose of both vapour phase and particulate
phase constituents to which the cells are exposed under the stated conditions
and to further define time and concentration dependencies.
970 APPLICATION OF SILAC QUANTITATIVE
PROTEOMICS FOR PATHWAY DISCOVERY OF
IDIOSYNCRATIC DRUG-INDUCED LIVER INJURY
COMPOUNDS.
K. L. Leach 1 , S. Nadanaciva 1 , C. Vazquez-Martin 2 , A. Lamond 2 and P. Ajuh 2 .
1 Compound Safety Prediction, Pfizer, Groton, CT and 2 Dundee Cell Products,
Dundee, United Kingdom.
SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) quantitative
proteomics (QP) is a mass spectrometry-based proteomics method that provides accurate
and reliable quantitation of changes in protein levels in cells. Drug-induced
liver injury (DILI) accounts for 50% of all cases of liver failures in the US, and a
significant fraction of those are caused by idiosyncratic DILI (I-DILI) drugs. The
cellular mechanisms which contribute to I-DILI are not known. We have applied
SILAC QP technology to identify novel cellular proteins and/or pathways that are
affected by I-DILI drugs. HepG2 cells were treated with the I-DILI drugs nefazodone
(40 uM), glafenine (100 uM) or nimesulide (100 uM) for 72 hours and the
cellular proteome was analyzed by SILAC QP. Buspirone (100 uM) and rosiglitazone
(100 uM) are not known to cause DILI and were used as control compounds.
Over 1800 proteins were identified by at least two peptides per protein in each of
the cell lysates. The antidepressant nefazodone caused an increase (2-fold or greater)
in the level of 34 proteins and a decrease in the level of 47 proteins whereas buspirone
caused much fewer overall changes in protein levels (increases in 14 proteins
and decreases in 15 proteins). Nefazodone caused an increase in a number of mitochondrial
proteins, including mitochondrial fission protein 1, NADH dehydrogenase,
and voltage-dependent anion-selective channel protein, suggesting that
changes in mitochondrial function might contribute to the mechanism of toxicity
for nefazodone. The cyclooxygenase inhibitors, glafenine and nimesulide, both
caused 3-11 fold increases in 4 proteins in the aldo-keto reducatase family. These
enzymes are activated by stress caused by electrophiles and reactive oxygen species,
and are involved in the bioactivation and detoxification of a number of drugs.
Taken together, these results suggest that I-DILI drugs may quantitatively change
several proteins and activate specific cellular signaling pathways.
971 EXPERIENCES WITH CYTOTOXICITY ASSAYS TO
SELECT STARTING DOSES FOR ACUTE ORAL
TOXICITY TESTING.
K. Hempel, A. Schrage, S. Kolle, M. Schulz, B. van Ravenzwaay and R.
Landsiedel. BASF SE, Ludwigshafen, Germany. Sponsor: A. Doi.
Acute oral toxicity testing is essential for the toxicological assessment of new chemicals.
With the objective of reducing and refining animal experiments necessary for
determination of acute oral toxicity, we have implemented cytotoxicity data to estimate
acute oral starting doses in rats. According to the ICCVAM Validation Study
in 2006, we used the neutral red uptake (NRU) method in Balb/c 3T3 fibroblasts
after 48 hours exposure to determine the cytotoxicity of about 120 test substances
including a broad variety of chemicals and formulations. The predicted starting
doses, estimated with the ICCVAM prediction model, were then used in rat acute
oral toxicity studies. Comparing the predicted LD50 and the in vivo determined
GHS classification, the cytotoxicity assay showed a good prediction only for the
low toxic substances (83%, GHS Cat. 4, > 300 – 2000 mg/kg body weight). The
overall concordance was rather low (36%), mainly because 76% of the tested substances
were classified as low toxic in vitro, but only 34% in vivo. Expanding the
prediction +/- one category greatly enhanced the overall concordance to 82% with
only 8% overpredicted (in vitro Cat. 3, > 50 – 300 mg/kg, in vivo Cat 5, > 2000
mg/kg) and 10% underpredicted test substances (in vitro Cat. 4, in vivo Cat 1-2, ≤
50 mg/kg). So far, the use of cytotoxicity data to predict starting doses did not sufficiently
contribute to the refinement and reduction in acute oral toxicity testing.
As the predictivity of the in vitro test highly depends on specific properties of the
tested substances, further analysis considering molecular weight, pH, log POW, solubility,
metabolic breakdown and mode of action are necessary and will be presented.
Using this data the applicability domain of the cytotoxicity assay for the
prediction of acute oral toxicity in rats might be refined.
972 FURAN AND 1, 3-PROPANEDIOL PRODUCE DNA
CROSS-LINKS IN TURKEY FOETAL LIVER.
A. M. Jeffrey 1 , J. Duan 1 , K. D. Brunnemann 1 , J. R. Schlatter 2 and G. M.
Wiliams 1 . 1 Department of Pathology, New York Medical College, Valhalla, NY and
2 Swiss Federal Office of Public Health, Stauffacherstrasse 101, Zürich, CH-8004,
Switzerland.
Furan is a contaminant formed in food as a result of heating. It is hepatocarcinogenic
in rats and mice [1]. The potential genotoxicity of furan in the liver is unclear.
Cordelli, et al [2] found evidence of liver DNA single strand breaks (SSBs) when
B6C3F1 mice were administered a single dose of furan. Genotoxicity can be assessed
in an in ovo turkey liver assay [3]. In this system, we tested furan and 1,3propane
diol (PD), as a positive control, for their ability to cause DNA-protein
cross-links (DPXLs) and DNA fragmentation detectable in the alkaline Single Cell
Gel Electrophoresis (COMET) assay. A single injection of furan into turkey eggs at
23 days of development (about 5 days before hatching) produced a reduction in
COMET tail length at doses of 2 to 15 μmoles/egg (approximately 75g) in hepatocytes
isolated from the foetus 24 hr later. When the same cells were treated with
proteinase K to digest the DPXLs, an increase in tail length was observed compared
to the corresponding negative controls, indicating the presence of DNA SSBs. PD
causes DNA cross-linking in rat liver [4], which the authors considered consistent
with conversion of PD to malondialdehyde (MDA). In the eggs, PD showed little
toxicity and was used at high doses (up to 300 μmoles/egg), at which it induced
both DPXLs and DNA SSBs. This diol, with probable metabolic formation of
MDA, showed much less toxicity compared to glutaraldehyde, a known direct acting
cross linking agent, which caused significant egg mortality at doses lower than
those needed to yield clear evidence of DPXLs and SSBs. In conclusion, furan produced
DNA cross-linking and SSBs in foetal turkey liver indicating that these lesions
may be involved in its rodent hepatocarcinogenicity. 1. Natl. Toxicol.
Program.Tech. Rep. Ser. 402, 1-286 (1993). 2. Cordelli, E., et al, Mutagenesis 25,
305-314 (2010). 3. Perrone, C. E., et al, Arch. Toxicol. 78, 589-598 (2004). 4.
Summerfield, F. W. and Tappel, A. L. Chem. Biol. Interact. 50, 87-96 (1984).
973 RETINOIC ACID DIFFERENTIATED H9C2 RAT
CARDIAC CELLS AS A MODEL FOR TOXICITY
SCREENING.
H. P. Behrsing, J. Hamre III, M. J. Furniss, D. Mesa and R. E. Parchment.
SAIC-Frederick/NCI-Frederick, Frederick, MD.
The H9C2 rat embryonic myoblastic cell line has been used to study the effects of
toxicants on cardiac muscle, but often in an undifferentiated state. Published articles
have indicated that differentiation and method thereof (e.g. inclusion of
retinoic acid) has an impact on the expression of cardiac markers (e.g. L-type calcium
channel). As part of an effort to establish robust in vitro models for screening
of chemotherapeutic compounds, we have characterized the H9C2 cells when undifferentiated
and differentiated, in glucose- or galactose-based media.
Characterization included staining the cells for muscle-specific markers (cardiac
tropinin, myosin light chain 2, and myogenin), and exposure to known cardiac toxicants
for IC50 determination using the live cell esterase calcein AM assay, and/or
ATP assay. Cells were also treated using etoposide and topotecan and assayed for
gamma-H2AX expression, using DAPI and ToPro3 for data normalization.
Staining results indicate that differentiation of H9C2 results in the increased expression
of cardiac -specific markers, with retinoic acid inclusive conditions yielding
a greater increase of expression. Toxicity testing using known mitochondrial
toxicants produced expected results, but the differentiated H9C2 acquired a greater
sensitivity (e.g. lower IC50 values) than undifferentiated cells for some compounds
tested. Gamma-H2AX staining of cells exposed for 6 hr to etoposide (100 μM max,
2-fold dilutions) showed a concentration-dependent increase of gamma-H2AX
staining but 6 hr exposure to topotecan (25 μM max, 2-fold dilutions) did not. In
conclusion, the H9C2 rat cardiac cell line exhibits favorable characteristics as a cardiac
model for toxicity testing when differentiated using retinoic acid. The state of
differentiation can impact the cell’s expression of organ specific biomarkers and
sensitivity to toxicants. Funded by NCI Contract No. HHSN261200800001E.
974 IDENTIFICATION AND CLASSIFICATION OF SKIN
SENSITIZER USING PEPTIDE BINDING ASSAY BY LC-MS.
T. Morimoto, T. Higaki, M. Ota, K. Inawaka and S. Kawamura. Sumitomo
Chemical Co., Ltd., Osaka, Japan. Sponsor: T. Yamada.
We are developing Peptide Binding Assay using LC-MS as an alternative method
for the skin sensitization test. In this study, we evaluated the predictive capacities of
identification and classification of skin sensitizers in the Peptide Binding Assay. Test
chemical and glutathione(GSH:tripeptide) were dissolved in acetonitrile and PBS,
SOT 2011 ANNUAL MEETING 207