The Toxicologist - Society of Toxicology

419 SOCIAL MEDIA AND INFORMATICS ESSENTIALS FOR
TOXICOLOGISTS.
L. Burgoon 2 , S. Bhatia 1 , P. Wexler 3 and M. Price 4 . 1 Human Health Sciences,
RIFM, Woodcliff Lake, NJ, 2 Biostatistics and Bioinformatics Research Core (BBRC),
U.S. EPA, Durham, NC, 3 Toxicology and Environmental Health Information
Program, National Library of Medicine, Bethesda, MD and 4 Membership and Social
Media Marketing, Society of Toxicology, Reston, VA.
How many times a day do we use Google or a search engine? Its use has become so
intuitive that we have coined this term as a verb and simply cannot imagine functioning
without it. Information that was once confined to libraries and various periodicals
is now free and easily accessible since the advent of Internet technology.
Furthermore, it has broken cultural and language barriers and enabled one to communicate
and collaborate with people all over the world. The combination of social
media, open source programs, and bioinformatics has transformed the role of the
computer in the modern scientist’s life. Video journals, blogging, open access journals,
social network websites, and podcasts have become the new channels of communication
- enabling first hand transfer of free knowledge and an ease by which to
carry out many collaborative efforts. A familiarity with simple Internet searching,
word processing, and expansive spreadsheets is simply not an adequate preparation.
Furthermore, the software tools used to deal with data arising from in silico models,
toxicogenomics, or high-throughput screens require an understanding of basic concepts
in computer science, database design, bioinformatics, and statistics. To bridge
the gap between these two worlds our panel of experts will provide toxicologists
with the basic knowledge of the informatics and various open source tools available.
In closing, we’ll discuss innovative strategies including the use of social media as a
communication, collaboration, networking, and a career advancement tool.
420 DEVELOPING A SCREENING SYSTEM FOR
HEPATOTOXICITY BY COMBINING THE HEPARG
CELL MODEL WITH HIGH CONTENT IMAGING.
M. Mennecozzi, M. Tsaneva, L. Saavedra and M. Whelan. Sistems Toxicology Unit,
European Commission-Joint Research Centre, Ispra, Varese, Italy. Sponsor: D. Dix.
Although it is evident that liver toxicity is still a major issue in the pharmaceutical
sector, there is still no established in vitro screening system to reliably identify potentially
hepatotoxic drug candidates early in the development phase. Moreover, in
the safety assessment of industrial chemicals and cosmetic ingredients, tighter restrictions
on the use of animals has generated considerable demand for alternative
methods for assessing systemic toxicity, with hepatotoxicity being a central element
of any resulting testing strategy. It is clear that hepatotoxicity encompasses a range
of complex processes and toxicological pathways, and thus it is a major challenge to
capture the key biological events in a screening system. We have addressed the
problem by combining a highly relevant cellular model, HepaRG, with high content
imaging. The HepaRG cell line is derived from a human hepatocellular carcinoma
and when seeded at low density, differentiates into bipotent hepatic progenitors
and divides before acquiring morphological and functional characteristics of
human hepatocytes. Differentiated HepaRG cells express the major liver functions,
including P450s, phase II enzymes, transporters and nuclear receptors at levels
comparable to those found in primary hepatocytes. The high content imaging approach
we adopted is based on automatic analysis of image-sets acquired with an
epifluorescent microscope, for the quantification of biomarkers expressed by
treated HepaRG cells. A quantitative high throughput screening format was employed
using a 96-well plate format, which facilitates the testing of 80+ chemicals in
one experimental run. We exposed HepaRG cells to serial concentrations of chemicals/drugs
and analyzed multiple cellular phenotypic changes including cell loss,
nuclear size, nuclear morphology, DNA content, mitochondrial membrane potential,
and intracellular accumulation of lipids. Dose-response data were acquired and
analyzed to characterize both the technical performance of the system and its ability
to predict hepatotoxicity.
421 USING PRECISION-CUT LIVER SLICES TO PROFILE
BIOCHEMICAL CHANGES OF DRUG INDUCED
HEPATOTOXICITY.
R. Hernandez, J. W. Lawrence and M. A. Lafleur. Amgen, Thousand Oaks, CA.
Sponsor: S. Sawant.
Precision cut liver slices (PCLS) in dynamic organ culture provide a model that has
the potential to improve in vitro hepatotoxicity testing since it contains all hepatic
cell types and native matrix. Moreover, we have demonstrated that the use of certain
culture medium constituents, such as glucocorticoids, are not required.
Glucocorticoids are potent anti-inflammatory agents and prevent a vital portion of
liver reactions to hepatotoxicants. We have developed the PCLS model into a useful
90 SOT 2011 ANNUAL MEETING
tool to rapidly profile various pathways involved in toxicity. Several endpoints
(LDH release, alkaline phosphatase, glycogen, triglycerides, cholesterol, TBARS,
glutathione, ATP content, and caspase induction) were measured for a series of
agents with known hepatic toxicity liabilities and several non-hepatotoxic agents.
The biliary epithelial cell toxicants, ANIT and 17-AAG, had selective effects by reducing
slice alkaline phosphatase levels without affecting LDH release. Troglitazone
was demonstrated to have oxidative stress-inducing activities on GSH, TBARS, and
NO induction. Agents that affected energy metabolism involving mitochondria reduced
ATP and glycogen and often reduced glutathione content at concentrations
that did not release LDH. Using this system, agents with known oxidative stress
components, energy metabolism changes, and cholangiolytic specific toxicities can
be distinguished. This system allows researchers to profile several pathways in parallel
that are important to hepatic toxicity induction and prioritize more resource demanding
activities in the laboratory.
422 CYTOTOXICITY OF 3-(3, 5-DICHLOROPHENYL)-2, 4-
THIAZOLIDINEDIONE (DCPT) AND ANALOGUES IN
WILD TYPE AND CYP3A4-TRANSFECTED HEPG2 CELLS.
D. M. Frederick, E. Y. Jacinto, T. H. Rushmore, R. Tchao and P. J. Harvison.
Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, PA.
The thiazolidinedione (TZD) ring is found in the glitazones, drugs used in the
treatment of type II diabetes. Liver injury has been reported in the clinic following
use of these drugs; however, they do not produce hepatotoxicity in common laboratory
animal species. In contrast, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione
(DCPT) causes hepatic damage in rats. Previous in vivo work has shown that
DCPT hepatotoxicity is dependent on an intact TZD ring and cytochrome P450
(CYP)-mediated metabolism. To further study the role of biotransformation in
TZD ring-induced toxicity, DCPT and several analogues were tested in vitro using
wild type HepG2 cells and HepG2 cells that were stably transfected with CYP3A4.
Troglitazone (TGZ), rosiglitazone and 5-(3,5-dichlorophenylmethyl)-2,4-thiazolidinedione
(5-DCPMT), a compound with a similar TZD substitution pattern to
the glitazones, were also tested. CYP3A4 transfectants were characterized by an enzyme
marker assay (6β-hydroxylation of testosterone; Km = 81.1 μM, Vmax = 92.7
pmol/min/mg protein). Both cell lines were treated with 0-250 μM of each compound
in Hanks’ balanced salt solution (HBSS; 0.1% DMSO). After 24 hours, cell
viability was measured. Some of the compounds did not exert cytotoxicity in either
cell line. However, DCPT was significantly (p < 0.05) more toxic in CYP3A4 transfectants
than wild type cells at or above 200 μM. In contrast, 5-DCPMT and TGZ
exerted greater cytotoxicity in the wild type cells, but only at intermediate concentrations.
Median lethal concentrations (LC50s) for DCPT (transfectant LC50 =
152.5 μM; wildtype LC50 = 206.8 μM), 5-DCPMT (transfectant LC50 = 19.4
μM; wild type LC50 = 13.8 μM) and TGZ (transfectant LC50 = 24.6 μM; wild
type LC50 = 21.0 μM) were consistent with these findings. Based on these results,
we conclude that all three parent compounds are toxic and that CYP3A4 may contribute
to DPCT-induced cytotoxicity in the transfectant cells. Supported by PHS
grant ES012499.
423 DEVELOPMENT OF AN ANIMAL MODEL OF
AMODIAQUINE-INDUCED LIVER INJURY.
P. Cai, T. Nakgawa and J. Uetrecth. Faculty of Pharmacy, University of Toronto,
Toronto, ON, Canada.
Background: Amodiaquine (AQ) is an antimalarial drug that is no longer used for
prophylaxis because it is associated with life-threatening agranulocytosis and/or hepatotoxicity.
AQ forms a reactive iminoquinone metabolite and these idiosyncratic
reactions appear to be immune-mediated, but how this reactive metabolite might
induce an immune response is unknown. AQ is reported to cause acute liver toxicity
in rats at high dose; the purpose of this study was to develop a model of AQ-induced
idiosyncratic hepatotoxicity at low dose that is delayed similar to the reaction
in humans. Methods and Results: Male Wistar or Brown Norway (BN) rats were
treated with AQ at 62.5 mg/kg/day, 6 days/week for 5 weeks via gavage. Serum
ALT was increased in both Wistar (65.8 U/L ±3.2 in treated vs 26.7 U/L ± 4.2 in
controls) and BN (65.5U/L ± 6.7 in treated vs 36.8 U/L ±3.0 in controls) rats after
4 weeks of treatment, which then returned to almost normal despite continued
treatment. Pathological changes of activated macrophages in the lung, activated
Kupffer cells and lymphocyte infiltration in the liver were found. The number of
ED1 or ED2 stained Kupffer cells was significantly increased in both rat strains,
but the increase was greater in BN rats. Apoptotic cells were found in the liver of
BN rats. Co-treatment with poly I:C (TLR 3), one dose of 10 mg/kg or imiquimod
(TLR 7) one dose 30 mg/kg, appeared to lead to an earlier onset of ALT increase
while co-treatment with LPS (TLR 4) once a week 5 mg/kg appeared to delay the
onset, but in all cases the ALT returned toward normal despite continued treat-