The Toxicologist - Society of Toxicology

2539 PREDICTION OF HEPATOTOXICITY IN ZEBRAFISH.
C. Callol, C. Quevedo and A. Letamendia. Biobide S.L., San Sebastian, Spain.
Sponsor: S. Tripathi.
Drug-induced hepatotoxicity is one of the most important causes of drug withdrawal
in the process of Drug Discovery. The low correlation of the in vitro cytotoxicity
studies, with human hepatotoxicity, especially in the early stages of Drug
Discovery, make human hepatotoxicity difficult to predict. Therefore, the finding
of new methods and models to predict hepatotoxicity is a growing area of research.
The zebrafish model has been extensively studied in the last decades for its ability to
regenerate. Specifically, the liver is one of the organs with the highest regenerative
capacity and research over the past 10 years have characterized many of the mechanisms
that occur during this process leading to an important knowledge of the signaling
pathways that govern zebrafish liver function and development.
Therefore, because of the functional and development similarities between the liver
of zebrafish and mammals, we propose to analyze the potential of zebrafish for prediction
of hepatotoxic agents. For this purpose, the following aspects have been
studied: i) The stage at which adult liver markers are expressed has been identified.
ii) Several techniques have been developed for assessing the hepatotoxicity of a
compound in zebrafish. iii) These techniques will be validated by testing different
drugs. iv) The results will be compared with those obtained in mammals.
Our results indicate that zebrafish embryos could be used as an alternative model in
the Drug Discovery process complementing the information of in vitro models and
mammals and providing pharmaceutical industry of new assays which might decrease
the current huge gap between in vitro and in vivo assays.
2540 PREDICTION OF DRUG-INDUCED LIVER INJURY
WITH HIGH SENSITIVITY AND SPECIFICITY WITH
HIGH-CONTENT CELL-BASED IMAGING.
R. N. Ghosh, S. J. Hong, B. A. Samson, K. Corkan, M. Pietila, C. Vasudevan
and J. R. Haskins. Thermo Fisher Scientific, Pittsburgh, PA.
Drug-induced liver injury is a major reason for drug failure and withdrawal from
the market. An assay system that enabled early and accurate prediction of drug-induced
hepatotoxicity with high sensitivity and specificity would benefit drug development
by improving the safety profile of newly developed drugs. Developing an in
vitro assay that accurately predicts the toxicology of drug candidates remains a challenge
in drug development. A critical factor contributing to the predictive nature of
such assays is the ability to measure early indicators of cell stress and toxicity in liver
cells. We report here a predictive, automated cell-based high-content imaging assay
that simultaneously quantifies the multiplexed response from five different cellular
properties in a single assay platform: cell loss, DNA content, intracellular reduced
glutathione level, intracellular reactive oxygen species level and mitochondrial
membrane potential changes. In this assay, liver cells (HepG2 cells, primary hepatocytes)
were treated with a panel of compounds, including those known to be idiosyncratic
human hepatotoxicants such as troglitazone, dantrolene, and tetracycline,
as well as non-hepatotoxic compounds including rosiglitazone, aspirin, fluoxetine
and melatonin. The cells were then labeled with specific fluorescent probes for the
distinct cellular properties and automatically imaged and quantitatively analyzed
using the Thermo Scientific ToxInsight IVT platform. By combining the individual
responses from the five assay targets, we defined several multiplexed toxicity indices
that accurately predicted drug-induced hepatotoxicity with greater than 80% specificity
and sensitivity on the hepatic models tested. Additionally, the different multiplexed
toxicity indices had better specificity and sensitivity in determining compound
hepatotoxicity than measuring the individual assay target responses. We
believe our approach shows an effective method of predicting hepatoxicity of compounds
and thus reduce drug attrition.
2541 GENE EXPRESSION PROFILING OF AN IN VITRO
HUMAN SKIN MODEL AFTER PSORALEN PLUS
ULTRAVIOLET LIGHT-INDUCED PHOTOTOXICITY.
L. F. Pratt 1 , G. L. DeGeorge 1 and M. Cunningham 2 . 1 MB Research Laboratories,
Spinnerstown, PA and 2 Nanomics Biosciences, Inc., Cary, NC.
To reduce the number of animals for safety screening of potential irritating chemicals
and phototoxins, efforts have been made to develop more predictive in vitro assays.
One model in more common use is reconstituted human epidermis (MatTek
Epiderm) resembling in vivo human skin. In this investigation, these human skin
models were exposed to a known skin irritant, 8-methoxypsoralen (8MOP), at a
dose comparable to the EC10. The samples were also either kept in the dark or exposed
to solar simulated light (SSL). Gene activity was analyzed with mRNA mi-
544 SOT 2011 ANNUAL MEETING
croarrays at 1, 6, and 20 hr to determine potential cellular and molecular mechanisms
of action. Two levels of biological control samples were used: a) samples not
treated with 8MOP and kept in the dark or exposed to UV light and b) samples
treated with sodium dodecyl sulfate (SDS) [positive control]. Purified, labeled, and
fractionated cRNA isolated from each of the biological samples were hybridized
onto whole human genome mRNA expression microarrays, each containing
41,000 unique probes. Data analysis was done by a tiered approach. Coefficients of
variation (CV) from all the probes passing quality measures or a total of 11,335
probes for each biological sample within the treatment groups ranged from 18.5-
33.1%. The least variability was observed with the principal components analysis
(PCA) for the negative control samples (those not exposed to 8MOP) and the samples
exposed to 8MOP under dark conditions. The most activity was seen with
8MOP and SSL exposures at 6 and 20 hr as well as exposures to SDS, the positive
control. Several genes in common between treatments with SDS and 8MOP were
CXCL14, fibrillin2, tropomyosin alpha 1, CYP26B1, HSP70B AND VEGF-A.
Funding was provided by NIEHS Grant No. 5-R44-ES-11927-02.
2542 OXIDATIVE STRESS AND HYPOXIA AS FACTORS IN
PHOTOTOXIC DAMAGE TO A RECONSTITUTED
HUMAN SKIN MODEL: GENE EXPRESSION
PROFILING EVIDENCE.
M. Cunningham 2 , L. F. Pratt 1 and G. L. DeGeorge 1 . 1 MB Research Laboratories,
Spinnerstown, PA and 2 Nanomics Biosciences, Inc., Cary, NC.
Currently there is worldwide emphasis on more predictive in vitro assays for phototoxicity,
especially in screening dermatological and pharmaceutical products.
Reconstituted human skin tissues modeling human skin in vivo were exposed to a
known phototoxin, chlorpromazine (CPZ) with and without ultraviolet light, at a
dose correlating with the EC10. The goal was to understand what cellular and genetic
mechanism(s) contribute to the phototoxic response. The skin models were
treated with CPZ in the dark as well as exposed to solar simulated light (SSL) and
gene activity was measured with mRNA expression microarrays at 1, 6, and 20 hr
post-exposure. For comparison, biological control tissues were concurrently treated
with vehicle alone or left untreated CPZ, and kept either in the dark or exposed to
UV light. Purified, labeled, and fractionated cRNA isolated from each of the tissue
samples were hybridized onto whole human genome mRNA expression microarrays.
The microarrays contained 41,000 unique probes corresponding to the full
complement of sequenced human genes. The data was analyzed using a tiered approach.
Coefficients of variation (CV) from all the probes passing quality measures
or a total of 10,299 probes for each tissue sample within the treatment groups
ranged from 5.3 to 19.2%. The least variability was observed with the principal
components analysis (PCA) for the skin model samples not treated to CPZ under
either dark or light conditions. The profiles for each treatment group were more
similar by time point rather than by light/dark exposure. The numbers of downregulated
genes ranged from 10 to 52 and the numbers of up-regulated genes
ranged from 96 to 236. Noteworthy genes which were down-regulated included
genes involved in differentiation and normally expressed in epithelial tissue, such as
CXCL14 and DAPL1. Several genes (e.g. HSPA6, ERO1L and ANGPTL4) related
to oxidative stress and hypoxia were up-regulated. Funding was provided by
NIEHS Grant No. 5-R44-ES-11927-02.
2543 A NEW IN VITRO MODEL FOR IDENTIFYING LIVER
SPECIFIC TOXICITY (LST).
J. M. McKim, B. Wallace, P. C. Wilga, C. Toole, H. Wagner, A. Swanson and
K. Rutherford. CeeTox, Inc., Kalamazoo, MI.
In early drug discovery, the ability to rapidly identify compound liabilities while
optimizing for desired drug qualities is critical for success in preclinical and clinical
safety studies. Cell lines can provide information on general toxicity, but cannot
identify organ specific toxicity. The aim of this study was to develop and optimize
an in vitro system that could identify liver specific toxicity of highly toxic compounds
and of compounds with low toxicity, often categorized as idiosyncratic. To
accomplish this, a two cell model was used in this study, a rat hepatoma (H4IIE)
cell line and rat primary hepatocytes grown in sandwich culture. Test compounds
with known mechanisms of toxicity (rotenone, camptothecin, ANIT) were tested
along with three groups of approved drugs, where one member of each group had
shown after-market liver toxicity or a drug-drug interaction. The three groups evaluated
included the antifungals (ketoconazole, itraconazole, fluconazole), the antidepressants
(nefazodone, trazodone, buspirone), and the antidiabetics (troglitazone,
pioglitazone, and rosiglitazone). The test compounds were applied to the culture