The Toxicologist - Society of Toxicology

scribed in the literature. We have optimized the way in which users can view and
evaluate the automated results. Commercially available software is cumbersome for
quickly evaluating both the overall health of a particular culture and cell level details
of the entire population. This step is crucial for evaluating pharmaceutical
compounds in development as each class of compound causes various morphologies
which can tax the ability of the automated algorithm. Our interface generates
well level images and individual thumbprints of cells in question. We can review the
calls made by the system and make changes which are then immediately reflected in
the output data. These changes can be tracked for audit. Now that the operational
performance for DNA damage detection is optimized, HCI is providing us with a
multitude of ways to follow up positive results (i.e. cytoskeletal rearrangement, centrosome
enumeration). It also affords us the ability to evaluate the individual mechanism(s)
by which test chemicals cause damage. This thorough understanding provides
for high confidence in our risk management strategies.
33 HIGH CONTENT SCREENING (HCS) IN EARLY SAFETY
ASSESSMENT: FROM DATA TO PREDICTIVE MODELS.
M. Kansy 1 and A. Hoffman 2 . 1 Non-Clinical Safety, F. Hoffmann-La Roche Ltd., Basel,
Switzerland and 2 Discovery Technologies, F. Hoffmann-La Roche Ltd., Nutely, NJ.
High content screening (HCS) enables the simultaneous determination of multiple
features of molecular phenotypes which are assumed to be relevant to therapeutic
and toxic activities of compounds. High content screening has been used to a larger
extend in ligand-target prediction to identify or confirm mechanism of actions. In
addition HCS approaches have been used in identifying potential toxic liabilities of
drugs and drug candidates. Validation studies evaluating HCS as predictive tool in
toxicity prediction were usually performed with relatively small data sets of toxic
and assumed “non-toxic” compounds. Published validation studies on larger compound
sets including hundreds of compounds are rarely described in literature.
However, the application of HCS in the lead optimization phase for toxicity assessments
and compound selection/optimization is assumed to positively impact the
identification of high quality candidate molecules in early drug discovery. We will
describe the result of our efforts to evaluate High Content Imaging (HCI) approaches
in their capability to classify toxic effects of development and marketed
compounds. Based on safety profiles determined in rats, more than 235 compounds
were grouped into toxicity severity classes (high, medium, low). The effect
of different compounds at concentrations from 0.3 to 100 μmolar on HEPG2 cells
was determined in vitro by applying HCI. More than 60000 data points were generated.
In order to separate toxic from non toxic compounds, obtained data were
analyzed with computational tools and methods like e.g. Naive Bayesian, support
vector machine (SVM) classification, kernel ridge regression (Kridge) and specific
multivariate data analysis tools. The analysis indicated a weak but significant relationship
between HCI observations and rat overall toxicity, allowing a separation of
toxic from non toxic compounds. Derived models can be obtained with a much
smaller set of HCS observations, usually cell viability parameter, without significant
loss of accuracy.
34 RIBOTOXIC STRESS: MECHANISMS AND MODELS
FOR HUMAN DISEASE.
J. J. Pestka 1 , V. L. Tesh 2 , B. E. Magun 3 , Y. Moon 4 and N. Tumer 5 . 1 Michigan
State University, East Lansing, MI, 2 Texas A&M University Health Science Center,
College Station, TX, 3 Oregon Health and Science University, Portland, OR, 4 Pusan
National University School of Medicine, Yangsan, Republic of Korea and 5 Rutgers
University, New Brunswick, NJ.
Many xenobiotics evoke toxicity and cause disease by modifying critical mitogenactivated
protein kinase (MAPK) signaling pathways that regulate growth, differentiation,
and cell survival. A number of plant, fungal, bacterial, and algal toxins can
aberrantly activate the P38, ERK, and JNK MAPKs by targeting the ribosome via a
process termed the ribotoxic stress response. Examples of ribotoxic agents include
natural toxins produced by plants (ricin), fungi (trichothecenes), bacteria (Shiga
toxins), and algae (palytoxin). These agents can be encountered in food and water
and are of further concern because of their potential use in chemical terrorism.
Cells involved in the innate immunity appear to be particularly sensitive to ribotoxic
stress. Ribotoxic stress is not completely understood but possible mechanisms
activation of intracellular signaling pathways by sensors of damage-associated
molecular patterns (DAMPs) or endoplasmic reticulum stress. From a translational
perspective, exposure of experimental animals to ribotoxic stressors can result in
downstream pathologic sequelae that remarkably mimic clinical signs associated
with inflammatory human diseases such as acute respiratory distress, ulcerative colitis,
IgA nephropathy, and hemolytic uremic syndrome. To address these issues, our
panel of experts will explore commonalities and differences in upstream mechanisms
of ribotoxic stress by different biotoxins and relate these to downstream sequelae
associated with human inflammatory diseases.
35 RICIN MEDIATES ACUTE RESPIRATORY DISTRESS
THROUGH IL-1BETA.
B. Magun. Oregon Health and Science University, Portland, OR. Sponsor: J. Pestka.
Pulmonary toxicity induced by aerosolized ricin is characterized by accumulation of
inflammatory cells and destruction of alveoli. Ricin-induced morbidity is strongly
reduced in mice that lack IL-1 or IL-1R, or in mice receiving recombinant IL-1 receptor
antagonist (IL-1RA), demonstrating a key role of IL-1 in orchestrating inflammatory
responses. Ricin-mediated activation of JNK and p38 through ZAK, a
MAP3K, is responsible in part for the proinflammatory effects of ricin. We have
identified two inhibitors of ZAK that are currently employed by as cancer therapeutics
and that have very limited toxicity in humans. In addition, ricin activates
the NALP3 inflammasome in macrophages through a signaling pathway that does
not involve the activation of ZAK, JNK, or p38. Our evidence suggests that all inhibitors
of protein translation will activate the NALP3 inflammasome in
macrophages, suggesting a novel role of translation inhibition in mediating inflammatory
responses.
36 MUCOSAL RIBOTOXIC STRESS AND INTESTINAL
INFLAMMATORY DISEASES.
Y. Moon. Department of Microbiology and Immunology, Pusan National University
School of Medicine, Yangsan, Republic of Korea.
Inflammatory bowel diseases (IBD) are aggravated by disrupted epithelial barrier
integrity and the following microbial translocation. Many muco-active ribotoxic
stress agents are known to cause IBD-like symptom by triggering pro-inflammatory
responses and mucosal intolerance to the dietary xenobiotic. Studies are aimed at
addressing the potential mechanistic association of the chemical ribotoxic stress
with human intestinal inflammatory diseases. There will be special focus on epithelial
recognition of the mucosal ribotoxic stress in the complex gut environment and
its subsequent contribution to the disease progress in human intestine (This work
was supported by the Korea Research Foundation (KRF) grant funded by the Korea
government (MEST) (No. 2009-0087028)).
37 SHIGA TOXINS AND THE HEMOLYTIC UREMIC
SYNDROME.
V. L. Tesh. Microbial and Molecular Pathogenesis, Texas A&M University Health
Science Center, College Station, TX. Sponsor: J. Pestka.
Shiga toxins are a family of genetically and structurally conserved proteins expressed
by the enteric pathogens Shigella dysenteriae serotype 1 and certain
serotypes of Escherichia coli. The capacity of Shiga toxin-producing bacteria to
cause widespread disease is highlighted by recent outbreaks of bloody diarrhea associated
with the ingestion of contaminated beef products or spinach. Unfortunately,
10-15% of patients with bloody diarrhea will develop life-threatening complications,
chief among them acute renal failure (hemolytic uremic syndrome). Shiga
toxins possess an AB5 structure with a single A-subunit in non-covalent association
with a pentameric ring of B-subunits. Shiga toxins bind to human cells through Bsubunit
interaction with the membrane glycolipid globotriaosylceramide.
Following toxin binding and internalization, the holotoxin is routed to the endoplasmic
reticulum (ER) where an A-subunit fragment retrotranslocates into the cytoplasm
and cleaves a single adenine residue from the 28S rRNA subunit, thereby
mediating protein synthesis inhibition. More recently, Shiga toxins have been
shown to be signaling molecules par excellence. In addition to inhibiting protein
synthesis, Shiga toxins activate all major mitogen-activated protein kinase (MAPK)
pathways and the Src/PI3K/Akt/mTOR pathway in human macrophages or
macrophage-like cell lines. Activation of these pathways is involved in both the positive
and negative regulation of cytokine expression in response to intoxication. The
toxins also activate the ER stress response, a “quality control” system which normally
monitors levels of mis-folded proteins in the ER lumen. Shiga toxin-mediated
activation of this system triggers intracellular signaling events that result in
apoptotic cell death. Signaling pathways activated in human macrophages by Shiga
toxins will be reviewed along with potential roles of toxin-induced signaling in
pathogenesis. Disruption of these signaling pathways may provide targets for future
interventional strategies to prevent or ameliorate disease.
SOT 2011 ANNUAL MEETING 7