The Toxicologist - Society of Toxicology

28 HIGH CONTENT IMAGING – APPLICATIONS IN
TOXICOLOGY AND TOXICITY TESTING.
W. Mundy. Integrated Systems Toxicology Division, U.S. EPA, Research Triangle
Park, NC.
High Content Imaging (HCI) often referred as high content screening (HCS),
combines state-of-the-art microscopy, robotics, and computer-assisted image analysis
to measure multiple cellular and sub-cellular features. The technology produces
information (digital images and metadata) on cellular response or morphological
changes at a high level of detail. Assays are amenable to high-throughput automation
generating hundreds of measurements per cell and millions of data points from
a single microtiter plate. Introduction of this technology in the pharmaceutical industry
led to analysis of cell signaling and processes such as receptor internalization.
The technology has expanded to include applications in angiogenesis, wound healing,
cell cycle regulation, cell death, neurodegeneration, regeneration, and genotoxicity,
etc. Using these approaches, measurements are made at the single cell or population
level. HCI assays are generally conducted using cell lines, primary cells, and
stem cells. Ex vivo tissue and whole organisms such as Drosophila, C. elegans, and zebrafish
can also be imaged providing knowledge from intact specimens. Novel
strategies for toxicity testing are likely to expand with HCI studies examining biologically
significant cellular perturbations by linking genetic, phenotypic, or functional
cellular changes with adverse outcomes from exposure to environmental
chemicals or pharmaceutical compounds. HCI is likely to become a key technology
for developing toxicity pathway assays as outlined by the 2007 NRC report Toxicity
Testing in the 21st Century: A Vision and A Strategy. Furthermore, it is now evident
toxicology programs in the pharmaceutical industry have adopted this technology
in an effort to expedite the fate of compound candidate selection in preclinic
and clinical trials using key measurements generated with HCI. Therefore it
is important to provide an overview of this technology and its applications in toxicity
testing relative to the HCI field from academia, biopharma, and government
agencies with examples and case studies to gain knowledge and insight into toxicity
pathways.
29 AN INTRODUCTION TO HIGH CONTENT IMAGING
TECHNOLOGIES AND APPLICATIONS IN
TOXICOLOGY.
O. Trask. The Hamner Institutes for Health Sciences, Research Triangle Park, NC.
Sponsor: R. Thomas.
High content imaging (HCI) technology has provided great promise to the scientific
community with a plethora of the different instrumentation platforms covering
a vast array of biological applications to study disease including signaling cascades,
MOA, and phenotypic characterization in immortalized and primary cells,
ex-vivo tissue, and small organisms such as zebrafish, C. elegans and drosophila.
From the onset HCI has bestowed investigators with a number of underlying cellular
measurements which have been exploited to determine cell death/loss and toxicity
assessments in multiple disciplines across the biopharmaceutical industry, academia
and government agencies. An introduction and broad stroke overview of
commonly used HCI assays in toxicology such as micronucleus, comet assay, apoptosis,
cell loss, functional biomarkers, and others will be covered. An often overlooked
aspect of HCI technology occurs during data analysis of primary endpoint
assays, the other “high content” or multiparametric data is not commonly used. I
will provide the audience with key examples how embedded data is extracted from
digital images at a single cell level or as global population statistics to measure features
such as DNA content or cell loss as an indicator of cytotoxicity during compound
treatment respectively. To a degree with the exception of flow cytometry
these key secondary high content measurements are not typically obtained by other
cell based technologies. It is well known with all technologies there are limitations,
knowing the caveats of the technology and approaches are critical for a productive
outcome including the importance of institutional infrastructure to support the implementation
of the technology is a key component for success.
30 HIGH CONTENT ANALYSIS OF CYTOTOXICITY FOR
PREDICTION, MECHANISTIC ELUCIDATION, AND
BLOOD BIOMARKERS OF HUMAN TOXICITY.
P. J. O’Brien. University College Dublin, Belfield, Dublin, Ireland. Sponsor: J. Trask.
High content analysis (HCA) of in vitro biochemical and morphological effects of
drugs and chemicals is concordant with potential for human toxicity. Concordance
was greater for cytotoxic effects assessed by HCA than for conventional cytotoxicity
tests and for regulatory animal toxicity studies. Additionally, HCA identified
chronic toxicity potential, and drugs producing idiosyncratic adverse reactions and
6 SOT 2011 ANNUAL MEETING
/ or toxic metabolites were also identified by HCA. Mechanistic information on the
subcellular basis for the toxicity is frequently identified, including various mitochondrial
effects, oxidative stress, calcium dyshomeostasis, phospholipidosis, apoptosis,
and antiproliferative effects, and a fingerprinting of the sequence and pattern
of subcellular events. As these effects are frequently non-specific and affect many
cell types, some toxicities may be detected and monitored by HCA of peripheral
blood cells, such as for anticancer and anti-infective drugs. Critical methodological
and interpretive features are identified that are critical to the effectiveness of the
HCA cytotoxicity assessment, including, the need for multiple days of exposure of
cells to drug, use of a human hepatocyte cell line with metabolic competence, assessment
of multiple prelethal effects in individual live cells, consideration of
hormesis, the need for interpretation of relevance of cytotoxicity concentration
compared to efficacy concentration, and quality management. Limitations of the
HCA include assessment of drugs that act on receptors, transporters, or processes
not found in hepatocytes. HCA may be used in a) screening lead candidates for potential
human toxicity in drug discovery alongside of in vitro assessment of efficacy
and pharmacokinetics, b) monitoring in vivo toxicity of drugs with known toxicity
of known mechanism.
31 QUANTITATIVE IN VITRO MEASUREMENT OF
CELLULAR PROCESSES CRITICAL TO THE
DEVELOPMENT OF NEURAL CONNECTIVITY
USING HCA.
J. A. Harrill. Integrated Systems Toxicology Division, U.S. EPA, Research Triangle
Park, NC.
New methods are needed to screen thousands of environmental chemicals for toxicity,
including developmental neurotoxicity. In vitro, cell-based assays that model
key cellular events have been proposed for high throughput screening of chemicals
for developmental neurotoxicity. While in vitro systems can not fully replicate the
complex temporospatial development of the brain, neuronal cultures can recapitulate
neurodevelopmental processes such as cell proliferation, differentiation,
growth, and synaptogenesis.We have evaluated primary neural cultures as models
for neurite outgrowth, estabalishment of polarity and synaptogenesis: processes
critical for the development of neuronal connectivity. In primary neural cultures,
these events occur sequentially, and the effects of chemicals on specific neurodevelopmental
processes can be investigated using different developmental / exposure
periods. This talk will focus on the development of high content assays which allowed
concentration-response assessments of multiple chemicals in 96 well microtiter
plates, in an efficient and cost-effective manner. The ability of these assays
to detect effects on in vitro neural development was determined using a “training
set” of chemicals with known effects in vitro. Specific effects on neurodevelopment
processes could be separated from cytotoxic effects using parallel analysis of cell
number and morphological measurements. The results demonstrate that HCA assays
can rapidly quantify chemical effects in vitro, distinguish between specific effects
on neurodevelopmental endpoints and nonspecific changes in cell health and
provide comparative data on which developmental events are more sensitive to a
particular chemical insult. This abstract does not necessarily reflect USEPA policy.
32 BEYOND DECREASING ATTRITION: HIGH CONTENT
IMAGING (HCI) IN GENETIC TOXICOLOGY.
E. Rubitski. Genetic Toxicology, Pfizer, Groton, CT. Sponsor: J. Aubrecht.
HCI is transforming the way in which in vitro clastogenicity screening & mechanistic
follow up studies are performed in Genetic Toxicology. The In Vitro
Micronucleus (IVMN) assay has been used for years as a screening tool to predict
the aneugenic and clastogenic potential of chemical agents. Regulatory agencies
have recently approved the IVMN assay (OECD Guideline 487) as an alternative
for both the GLP (Good Laboratory Practices) Human Lymphocyte Assay and
Mouse Lymphoma assays. Since there are advantages to running the assay both
with and without cytochalasin-B (CYB), e.g. to increase sensitivity and specificity
for certain classes of compounds, and because we have two state of the art automated
IVMN platforms capable of evaluating micronuclei in both mononcleated
and binucleated Chinese Hamster Ovary cells, we have evaluated extensive dose-response
relationships for 11 commercially available chemicals. The chemicals included
in our study were suggested in the guideline for validation. Here we report
and contrast the results of each chemical within the context of each IVMN platform
(+/- CYB) and the OECD Proposed Guideline 487. The results of this study
show that the automated system allows for rapid and consistent quantification of
DNA damage with results that are concordant with previous manual studies de-