Lush Science Prize 2013

Background Paper

Lush Prize Science Prize – background paper – August 20131

1 Executive Summary
1.1 Building on the work of the 2012 Background Paper
The Lush Science Prize is designed to reward 'outstanding contributions' to 21 st
Century Toxicology Research. In the 2012 Background Paper for the Lush
Science Prize it was proposed that, in order to have a rational set of criteria to
identify possible winners from what was potentially a very wide area, there was aneed to focus in on a specific type of research to have an impact. Because of theLush Prize's wider goal to encourage a breakthrough event (with a Black Box
Prize) around a 'proof of concept toxicity pathway study', the focus chosen wason researchers working to understand 'toxicity pathways'.
This approach won approval from the Judges and the wider Prize community andtherefore it has formed the basis for research for this 2013 Background Paper.
It should be noted that the wording on the nominations forms was changedslightly this year and this has been reproduced in Section 2 below.

1.2 Methodology
We looked at some key institutional developments in the area which included theOECD's Adverse Outcome Pathway Project, the Hamner Institute's work and the
Human Toxome Project (Section 4 below). We also looked at the phenomenon ofcollaborative computer projects in this area (Section 5) and performed a brief
literature review (Section 6).
However, in order to identify specifically 'toxicity pathway' breakthroughs, we
focused, as last year, mainly on work published in peer-reviewed journals.
The journal search methodology we used is explained in Section 3 below and the51 abstracts we selected for 'scoring' appear in Section 7. By this method two
projects received the highest score and 21 projects received the next highest.
Short descriptions of each of those receiving the lower score appear in an
appendix at the end of this document.

1.3 1.3 Projects recommended for the shortlist
These are the two projects which received the highest scores.
In the event that insufficient nominations are received in the Science category thisyear, these projects could go forward to be considered by the judges, as
happened last year. More details appear in the Conclusions.

Lush Prize Science Prize – background paper – August 20132

1.3.1

Human embryonic stem cell-derived test systems for developmental neurotoxicity:a transcriptomics approach

Krug AK, Kolde R, Gaspar JA, Rempel E, Balmer NV, Meganathan K, Vojnits K,
Baquié M, Waldmann T, Ensenat-Waser R, Jagtap S, Evans RM, Julien S,
Peterson H, Zagoura D, Kadereit S, Gerhard D, Sotiriadou I, Heke M, Natarajan
K, Henry M, Winkler J, Marchan R, Stoppini L, Bosgra S, Westerhout J, Verwei M,
Vilo J, Kortenkamp A, Hescheler J, Hothorn L, Bremer S, van Thriel C, Krause
KH, Hengstler JG, Rahnenführer J, Leist M, Sachinidis A.
Department of Biology, University of Konstanz, 78457 Constance, Germany.
“Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT)
often manifest themselves in functional deficits that are not necessarily based oncell death, but rather on minor changes relating to cell differentiation orcommunication. The fields of DNT/RT would greatly benefit from in vitro tests thatallow the identification of toxicant-induced changes of the cellular proteostasis, orof its underlying transcriptome network. Therefore, the ‘human embryonic stem
cell (hESC)-derived novel alternative test systems (ESNATS)’ European
commission research project established RT tests based on defineddifferentiation protocols of hESC and their progeny. In conclusion, the ESNATS
assay battery allows classification of human DNT/RT toxicants on the basis of
their transcriptome profiles.”

1.3.2

Profiling Environmental Chemicals for Activity in the Antioxidant Response
Element Signaling Pathway Using a High Throughput Screening Approach

Sunita J. Shukla, Ruili Huang, Steven O. Simmons, Raymond R. Tice, Kristine L.
Witt, Danielle VanLeer, Ram Ramabhadran, Christopher P. Austin, Menghang Xia
NIH Chemical Genomics Center USA, National Institutes of Health, Department
of Health and Human Services, Rockville, Maryland, USA, U.S. Environmental
Protection Agency, National Institute of Environmental Health Sciences, National
Institutes of Health, Department of Health and Human Services, USA“Oxidative stress has been implicated in the pathogenesis of a variety of diseases
ranging from cancer to neurodegeneration, highlighting the need to identify
chemicals that can induce this effect. The antioxidant response element (ARE)signaling pathway plays an important role in the amelioration of oxidative stress.
Thus, assays that detect the up-regulation of this pathway could be useful for
identifying chemicals that induce oxidative stress.
We used cell-based reporter methods and informatics tools to efficiently screen alarge collection of environmental chemicals and identify compounds that induce
oxidative stress. We utilized two cell-based ARE assay reporters, β-lactamaseand luciferase, to screen a U.S. National Toxicology Program 1,408-compound
library (NTP 1408, which contains 1,340 unique compounds) for their ability toLush Prize Science Prize – background paper – August 20133

induce oxidative stress in HepG2 cells using quantitative highthroughput screening (qHTS).
Our results support the robustness of using two different cell-based approaches
for identifying compounds that induce ARE signaling. Together, these methods
are useful for prioritizing chemicals for further in-depth mechanism-based toxicity
testing.”

1.4 Other Conclusions
At Section 5 below we note how the way that the Science Prize research, with its
current focus on 'toxicity pathway breakthroughs', will always overlook someequally, if not more, important collaborative meta-initiatives (such as the Human
Toxome Project) and some purely computational contributions. Some of these, it
should be noted, are already very substantially funded.
It may be that one year, the Prize could choose to focus on and invite applications
from, for example, purely computational projects in order to compensate for this.

2 Background
The Lush Prize website science prize nominations form text has been updated
slightly from last year and currently reads as follows
Science
21st Century Toxicology Research
21st Century Toxicology is a new approach to safety testing which is exciting
regulators, toxicologists, campaigners and companies around the world. It has
become possible because of advances in biology, genetics, computer scienceand robotics.
21st-Century Toxicology focuses on human ‘toxicity pathways’, the sequences ofmolecular changes within the body’s cells that follow exposure to a toxic
chemical. As these molecular pathways are elucidated for different groups ofchemicals and different toxic effects, computer technology will help identify the
key steps that can then be used to design non-animal safety tests.
Many of these new tests will be done robotically, providing more cost-effective
chemical assessment and helping to clear the backlog of untested substances.
They offer better relevance to humans (rather than using mice, rats and rabbits),
and will explain the underlying causes of toxicity. Unlike animal methods, the newtests will help predict human variability and differential effects on embryos,children and adults. And as the superior scientific basis of the new approach isrecognised, outdated animal tests will be replaced.
For more information on this subject see:
Humane Society International (video)
Lush Prize Science Prize – background paper – August 20134

National Academy of Sciences Introduction (3pp pdf)
Human Toxicology Project Document (2pp pdf)
US National Academy of Sciences (Book 196pp)
Anyone can nominate individuals, research teams or institutions for work
conducted on relevant toxicity pathways. Applicants can also nominate
themselves. Outstanding research producing an effective non-animal safety test
based on an approach other than toxicity pathways, where none existed before,
may also be considered. You can nominate an individual, team or institution
below.

3 Methodology
In 2012, two recent major conferences on alternatives to animal testing had
published a list of submitted papers:•

the 8th World Congress on Alternatives and Animal Use in the Life
Sciences (WC8 at Montreal); and•the European Society for Alternatives to Animal Testing (EUSAAT 14 atLinz).

The 2012 Background Paper was therefore able to explore the 381 research
papers submitted in order to identify potential prize winning projects. For this2013 Paper, a method using conference papers was not possible as these orsimilar events were not taking place this time round.
After a review of key institutions and publications (Section 4 and 6 below) we
therefore decided to look at 'toxicity pathway' research published in peerreviewed journals during 2012/13. In the 2012 Background Paper we identified
how “an exploration of the journal aggregating website sciencedirect.com
revealed that although there were only 21 journals with 'toxicology' in the title,
there were 1078 with 'biochemistry, genetics and molecular biology' in the title. In
order to reduce the research area to a manageable size, we reviewed only five
key sources for new research published in the 2012/13 period:
Two directly relevant journals•

ALTEX Journals - looking at abstracts or citations (4 found)•Toxicology In Vitro Abstracts – looking at abstracts (15 found)

Lush Prize Science Prize – background paper – August 2013

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One key annual review of progress in the area of 21st century
toxicology research• AXLR8 Progress Report 2012 (7 found)Two broader electronic searches for 'toxicity pathway' (2012/13) among the wide
range of journals searchable at:• Google Scholar (6 selected)• Terkko – Helsinki University Medical Library Search Engine (19 selected)
We then scored each abstract according to the method used in the 2012 Science
Prize Background Paper as follows:
Does it appear to be reporting a new pathway discovery?

Score 3

If it is working with apparently previously understood pathway
research, does it bring new knowledge or tools?

Score 2

Does it stand out in any other way?

Score 1Each of the 51 abstracts that were initially identified appears in Section 7 below,
along with their scoring. There were 21 projects scoring three points and two
scoring four. Short descriptive extracts from the abstracts scoring 3 or more
appear in Appendix 1 below with links to the journal publisher in each case.
It should be noted that the researcher working on this paper is a journalist and nota toxicologist and that the high volume of publications in this area means that only
cursory analyses of each project was possible during this process.

4 Key Institutional Developments
4.1 OECD Adverse Outcome Pathways
One of the key elements of the Lush Prize is to focus attention on 21st Century
Toxicology and its promise to learn about human toxicity pathways. It is therefore
very significant to see that, since the last Background Paper, the OECD – not an
organisation short of access to financial resources – has announced a project
specifically in this area. Its website describes the project as follows:“In 2012, the OECD launched a new programme on the development of Adverse
Outcome Pathways. An Adverse Outcome Pathway (AOP) is an analytical constructthat describes a sequential chain of causally linked events at different levels of
biological organization that lead to an adverse health or ecotoxicological effect.
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6

AOPs are the central element of a toxicological knowledge framework
being built to support chemical risk assessment based on mechanistic reasoning.
Most of the AOP development and review is intended to take place via a web-based
IT management tool (“AOP Knowledge Base”) which is in development and is being
tested until the end of 2013. More information will be made publicly available after
the testing phase.”1

There is also an open call for projects, and 26 were already listed on the website
in August 2013. Most of these has 2013 start dates and will not have outputs yet.
However they may be promising routes to identifying possible pathway
breakthroughs in future years. We look at the web-based management tool in
section 5.1 below.

4.2 The Hamner Institute and proof-of-concept toxicity pathway work
The US-based Hamner Institute is openly committed to working on the very proofof-concept toxicity pathways that the Lush Black Box Prize is seeking toencourage.
“While all these research programmes are contributing to advancing risk
assessment sciences with in vitro methods, proof-of-concept studies appear
particularly important for assessing whether the specific recommendations of the
NRC report are feasible based on current biological knowledge....At the Hamner,
we have initiated programmes to develop case study approaches with six toxicity
pathways: three receptor mediated pathways—aryl hydrocarbon (AhR),
peroxisome proliferator-activated receptor-alpha (PPARα), and estrogen receptor(ER)—and three reactivity based pathways—DNA-damage, oxidative stress andmitochondrial toxicity. Our approaches are furthest along with PPARa and p53.
The most extensive programme focuses on estrogen pathway signalling.” 2
Disappointingly, it appears that its use of 'limited' in vivo studies may make itineligible for the Prize. “We are conducting similar in vitro studies with rat
hepatocytes. Limited
in vivo studies in rats will focus on dose response of gene expression in rat livers
after treatment with GW7647 and allow determination of the relevance of thedose-response seen in vitro in primary hepatocytes with that occurring in vivo.”3

4.3 The Human Toxome Project
Also mentioned in the 2012 Background Paper, the Center for Alternatives toAnimal Testing at Johns Hopkins University's programme to assess the human
toxome and annotate the pathways associated with toxic responses continues. It
has been funded with US $6 million over five years from the US National

1
2
3

www.oecd.org/env/ehs/testing/molecularscreeningandtoxicogenomics.htm
AXLR8 2012 Progress Report pp142-7
AXLR8 2012 Progress Report pp145

Lush Prize Science Prize – background paper – August 20137

Institutes of Health, plus extra support from the Environmental
Protection Agency and the Food and Drug Administration.4
In this project, “studies in validated cell systems define pathways of toxicity (PoTs)
in human cells in vitro in order to develop mode-of-action based mechanistic
information for assessing risks posed by exposure to various substances. The
eventual goal is to map all pathways of what is called ‘the human toxome’. The
ongoing project at Johns Hopkins focuses on estrogen pathway signalling in
MCF-7 cells. Validated methods already exist for using these cells to assess
estrogenicity. Using the reverse dosimetry approach developed jointly between
the Hamner Institutes and staff at the US EPA National Center for Computational
Toxicology (NCCT), concentrations that elicit cellular responses become the basis
for estimating safe human exposure conditions (or exposures that pose a deminimis risk level).”5
They appear to visualise a similar collaborative web tool to those worked on byothers (see section 5 below). “Beyond that, this project will develop a common,
community-accessible framework and databases that will enable the toxicology
community at large to comprehensively and cooperatively map the human
toxome using integrated testing strategies that combine “omics” data withcomputational models.”65 Collaborative computational systems
5.1 OECD Adverse Outcome Pathway Wiki
The announcement, in 2012, of the Adverse Outcome Pathway Wiki project by
the OECD appears to be an important step towards mapping toxicity pathways in
a common format. This was described on the JRC's website as follows:“An IT system to capture, manage and share Adverse Outcome Pathways - AOP
Wiki - has been made available for testing to an initial user group comprising current
contributors to the OECD's AOP Development Programme, including members of
the OECD Extended Advisory Group on Molecular Screening and Toxicogenomics(EAG MST) and the WHO/IPCS Mode of Action project group.
The Wiki leads AOP developers through the steps necessary to capture the scientificinformation needed to document and evaluate an AOP following recently publishedOECD guidance and provides an ideal collaboration space for international AOPdevelopment project teams. A Wikipedia-like system of user-friendly tables, dropdown-boxes and built-in functionality for automatic cross-referencing between
related pages makes the tool extremely easy to use. A series of webinars areplanned to familiarise the first user group with the process of entering information
into the AOP Wiki and keeping their AOPs updated.

4
5
6

http://www.nature.com/news/big-biology-the-omes-puzzle-1.12484
AXLR8 2012 Progress Report pp147
http://humantoxome.com homepage viewed August 2013

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The beta-testing phase will last until the end of 2013, when the next
development cycle will start. The system is expected to become freely available(read access) to the international scientific community by January 2014."7

5.2 Effectopedia: The Online Encyclopaedia and Graphical Editor for AOPs
To some extent the OECD project appears to be working in the same area as aproject of the International QSAR Foundation (which has received OECDfunding). The effectopedia project is, however, already active and is described in
the following poster, presented at the EPAA annual conference by Gilly Stoddartof PETA.8“The 21st Century shift to more prospective hazard identification and hypothesis
generation requires greater strategic application of systems biology, QSAR andarchived toxicological data in the form of adverse outcome pathways (AOPs). AOPs
describe the causal linkages among biological responses to chemicals over time.
The complexity of integrating science can be a barrier to progress in terms of the
toxicity pathways and networks involved as well as the need to organize knowledge
from many disciplines.
Effectopedia is an open-knowledge aggregation and collaboration tool fordelineating AOPs in an encyclopedic and predictive manner. It includes discrete
cause-effect studies and critical reviews that are relevant to toxicology. To achieve
human and machine interpretability, Effectopedia uses an ontology-enhanced,
natural language interface that offers clarifying questions and special tags to define
the semantic knowledge while preserving the natural language description of the
AOP’s elements. Effectopedia serves as a graphical editor to delineate causal
linkages at any level of biological organization and species. It creates a common
organizational space that (1) helps experts identify gaps in knowledge of causal
linkages of biological responses and (2) acts as a web-based
conference room for dialogue and synthesis by experts with interest in specific
AOPs. Effectopedia’s live documents are instantly open for discussions andfeedback, whilst giving credit to original authors and reviewers. New contributions
are immediately distributed to interested parties. Uncoupling the contribution andreview processes permits organizations to define their own seals of approval andassociate them with special interest pathways without slowing down the Wikiinspired contributions.”

It should be noted that similar – though not necessarily collaborative – databases
covering similar areas also exist. The Max Planck Institute for Molecular
Genetics, for example, manages the ConsensusPathDB resource(cpdb.molgen.mpg.de), a meta-database that integrates the content of 22
different interaction databases and that comprises 2,144 predefined humanpathways of which 1,695 had more than five members and were used forpathway response analysis.9

http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing-safety-assessmentchemicals/improved_safety_assessment_chemicals/first-release-of-aop-wiki/8
http://ec.europa.eu/enterprise/epaa/3_events/ann-conf-2012/poster-book.pdf
9
AXLR8 2012 Progress Report p177
7

Lush Prize Science Prize – background paper – August 20139

As we mentioned in the 2012 Background Paper, the way the
Science Prize currently focuses on 'toxicity pathway' breakthroughs means thatsome of these equally valuable collaborative meta-initiatives and computational
contributions will be overlooked. It may be that one year, the Prize could choose
to focus on and invite applications from purely computational projects in order tocompensate for this.

6 Brief Literature Review
A wider literature review of key 21st Century Toxicology texts took place in the2012 Science Background Paper and has not been repeated here. This year webegan by absorbing the AXLR8 2012 Progress Report which is designed to reportannually on alternative testing strategies. It also describes in detail some of thevery substantially funded EU projects such as SEURAT 1 (systematic dose
replacements) which are still under way. It has been used as a key resource
throughout this Background Paper and is not otherwise discussed separately
here.
We have also systematically reviewed the ALTEX journal which has reproduced anumber of discursive analyses of current developments in 21 st Century
Toxicology, but the following 'roadmap' review from Basketter et al (ALTEX 29,
1/12 3) stands out as a useful summary of progress in five key areas.
The main conclusions and recommendations of the roadmap were summarised
as follows:“1. Toxicokinetics– Represents a necessary complement to all in vitro approaches to allow QIVIVE.
– Need for “in vitro kinetics” of chemicals in the experimental systems with thegoal of producing proper kinetic parameters for QIVIVE.
– In silico approaches need to be further optimised.
– Need for more comprehensive data collections, especially in vitro data frombarrier models.
– Problems mainly in the fields of bioavailability and urinary excretion.
– Achievable with reasonable investment.

2. Sensitisation– Reasonably good animal model (LLNA) capable of generating potency and dose
response information.

Lush Prize Science Prize – background paper – August 201310

– Multiple in vitro assays available but unclear which test methods
provide potency information.
– The need to build mechanistic understanding to enable data integration forpotency determination for hazard characterisation & risk assessment remains animportant in vitro challenge.

3. Repeated dose testing– Tox-21c approaches based on PoT represent the key perspective; need tofocus on defining levels that cause. -Adverse effects rather than just hazard
identification.
– Need for data sharing from industry.
– Need for models for PoT identification (e.g., stem cells).
– Need for co-cultures, 3D models, and long-term models.
– Human disease knowledge and known toxicants must be exploited.
– Increased focus on modelling of inflammatory/immunological damage.

4. Carcinogenicity– Possible abolition of current test via an objective assessment with tools ofevidence-based toxicology (EBT).
– Important ongoing work to optimise genetic toxicity battery.
– Further evaluation of cell transformation assay required.
– ITS including non-genotoxic modes of action should be developed.
– Tox-21c approaches based on PoT (including metabolomics) represent a key
opportunity.

5. Reproductive Toxicity– Analysis of current animal tests by EBT approaches.
– Validation of (human) embryonic stem cell test variants.
– Validation of zebrafish egg test for teratogenicity.
– Extension of ITS approaches, extending the approach of ReProTect– Extension of the ToxCast program currently pioneering PoT-based assessments

Lush Prize Science Prize – background paper – August 201311

– Tox-21c approaches based on PoT, especially mapping the PoT for
reproductive toxicity for a Human Toxome database.”10

7 Searching for Toxicity Pathway Journal Abstracts
This section contains a number of tables which display in the first column anabstract title and in the second column a 'score' using the 2012 Background
Paper methodology.
Does it appear to be reporting a new pathway discovery?

Score 3

If it is working with apparently previously understood pathway
research, does it bring new knowledge or tools?

Score 2

Does it stand out in any other way?

Score 1

7.1 ALTEXALTEX 2012 and 2013 was searched both for its own published research and fornews of other potentially useful publications or breakthroughs. The following fourpieces of research stood out with their scores appearing in the final column.
Human embryonic stem cell-derived test systems for developmental
neurotoxicity: a transcriptomics approach3+1

A 3-dimensional human embryonic stem cell (hESC)-derived model todetect developmental neurotoxicity of nanoparticles

2

Perspectives on validation of high-throughput assays supporting 21 st
century toxicity testing1

A 155-plex high-throughput in vitro coregulator binding assay for(anti-)estrogenicity testing evaluated with 23 reference compounds

3

7.2 Toxicology In Vitro Abstracts
We searched for 'pathway' in Toxicology In Vitro from August 2012 to present.
Quite a few are chemicals being tested against known pathways.
Cytotoxic responses in BC3H1 myoblast cell lines exposed to 1desulfoyessotoxin
102

t4 Report A Roadmap for the Development of Alternative (Non-Animal) Methods for Systemic Toxicity
Testing http://www.altex.ch/resources/altex_2012_1_003_092_Basketter_full_final.pdf

Lush Prize Science Prize – background paper – August 201312

Cooperation of bisphenol A and leptin in inhibition of caspase-3
expression and activity in OVCAR-3 ovarian cancer cells

2

Assessing dose-dependent differences in DNA-damage, p53 response
and genotoxicity for quercetin and curcumin

2Time course study of Aβ formation and neurite outgrowth disruption indifferentiated human neuroblastoma cells exposed to H 2O2: Protectiverole of autophagy

2Ethanol-induced apoptosis in human liver adenocarcinoma cells (SKHep1): Fas- and mitochondria-mediated pathways and interaction with
MAPK signaling system

3?

Induction of HepG2 cell apoptosis by Irgarol 1051 throughmitochondrial dysfunction and oxidative stresses

2Melamine activates NFκB/COX-2/PGE2 pathway and increases
NADPH oxidase–dependent ROS production in macrophages andhuman embryonic kidney cells

2Applicability of a keratinocyte gene signature to predict skin sensitizing
potential

3

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent
and ROS-independent pathways

3

Sodium arsenite induces cyclooxygenase-2 expression in humanuroepithelial cells through MAPK pathway activation and reactive
oxygen species induction

3

Cerium oxide nanoparticles induce cytotoxicity in human hepatoma
SMMC-7721 cells via oxidative stress and the activation of MAPK
signaling pathways

2

Methylglyoxal induces DNA crosslinks in ECV304 cells via a reactive
oxygen species-independent protein carbonylation pathway

2

T-2 toxin enhances catabolic activity of hypertrophic chondrocytes
through ROS-NF-κB-HIF-2α pathway

2

Critical roles of Rho-associated kinase in membrane blebbing andmitochondrial pathway of apoptosis caused by 1-butanol2

Impacts of low doses of pesticide mixtures on liver cell defence
systems2

Lush Prize Science Prize – background paper – August 201313

7.3 AXLR8 Citations from 2012
A Computational Model Predicting Disruption of Blood Vessel
Development

2+1

Putting the parts together: combining in vitro methods to test for skinsensitizing potentials

2+1

Allergic contact dermatitis: a commentary on the relationship between T
lymphocytes and skin sensitising potency.

2

An in vitro method for detecting chemical sensitization using humanreconstructed skin models and its applicability to cosmetic,
pharmaceutical, and medical device safety testing.

3

Profiling Environmental Chemicals for Activity in the Antioxidant
Response Element Signaling Pathway Using a High Throughput
Screening Approach

3+1

Focus on Stem Cells as Sources of Human Target Cells for In Vitro
Research and Testing1

Development of new structural alerts suitable for chemical category
formation for assigning covalent and non-covalent mechanisms
relevant to DNA binding

7.4 Google Scholar
Google scholar produced 251 results for a search of 'toxicity pathway' between
2012-2013 (200 were scanned). Ignoring animal tests and disease-related
research (e.g. cancer, Alzheimer’s) the following abstracts were scored.
Quantitative High-Throughput Screening for Chemical Toxicity in a
Population-Based In Vitro Model

1

Predictive toxicology using systemic biology and liver microfluidic “onchip” approaches: Application to acetaminophen injury

2

Mechanisms of toxicity of triphenyltin chloride (TPTC) determined by alive cell reporter array

3

Effects of usnic acid exposure on human hepatoblastoma HepG2 cells
in culture2

A systematic study of mitochondrial toxicity of environmental chemicals
using quantitative high throughput screening

3

Lush Prize Science Prize – background paper – August 201314

Reactivity of Chemical Sensitizers Toward Amino Acids In Cellulo Playsa Role in the Activation of the Nrf2-ARE Pathway in Human Monocyte
Dendritic Cells and the THP-1 Cell Line3

7.5 Terkko – Helsinki University Medical Library
Terkko has a particularly useful 'feed navigator' which indexes journals from Pub
med, Elsevier, Singer, Science Direct, Oxford Journals, ALTEX etc.
In August 2013 we searched LifeSciencesJournals – Pharmacology, toxicology
and pharmaceutics for “toxicity pathway”
http://www.terkko.helsinki.fi/feednavigator
With around 450 results, we eliminated by reading the titles only: clearly cancer or
disease related studies, some nanotech research, some heavy metals toxicity
research and some pesticides research.
It is clear that much toxicity pathway research is happening in cancer researchcommunities which are looking to ameliorate drug side effects as well as looking
to create toxic damage to cancer cells. Reading the remaining abstracts, we theneliminated those using animal testing in the broader research which left thefollowing 19 abstracts.
The proteasome cap RPT5/Rpt5p subunit prevents aggregation of
unfolded ricin A chain2

Use and validation of HT/HC assays to support 21 st century toxicity
evaluations

1

Neuregulin-1β regulates tyrosine kinase receptor expression in cultured
dorsal root ganglion neurons with excitotoxicity induced by glutamate

3

Modeling drug- and chemical-induced hepatotoxicity with systems
biology approaches.

3

Gene and protein responses of human lung tissue explants exposed toambient particulate matter of different sizes.

3

Unraveling amyloid toxicity pathway in NIH3T3 cells by a combined
proteomic and 1H-NMR metabonomic approach3

Differential Mitochondrial Toxicity Screening and Multi-Parametric Data
Analysis

2

Lush Prize Science Prize – background paper – August 201315

Discovery of Inhibitors of Microglial Neurotoxicity Acting ThroughMultiple Mechanisms Using a Stem-Cell-Based Phenotypic Assay

3

Metabolomic Response of Human Embryonic Stem Cell-Derived Germlike Cells After Exposure to Steroid Hormones

2

Concentration-dependent induction of reactive oxygen species, cell
cycle arrest and apoptosis in human liver cells after nickel
nanoparticles exposure

2

Protein kinase CK2-dependent phosphorylation of the human
Regulators of Calcineurin reveals a novel mechanism regulating thecalcineurin-NFATc signaling pathway.

3

A signaling cascade mediated by ceramide, src and PDGFRβcoordinates the activation of the redox-sensitive neutral
sphingomyelinase-2 and sphingosine kinase-1

3

Toxicometabolomics

1

Evaluation of a human neurite growth assay as specific screen fordevelopmental neurotoxicants.

2

Perspectives for integrating human and environmental risk assessment
and synergies with socio-economic analysis.

1

Global Gene Expression Profiling Reveals Functional Importance of
Sirt2 in Endothelial Cells under Oxidative Stress.

3

Compound selection for in vitro modeling of developmental
neurotoxicity.

1

A 3-dimensional human embryonic stem cell (hESC)-derived model todetect developmental neurotoxicity of nanoparticles.

2

Mechanism of the toxicity induced by natural humic acid on humanvascular endothelial cells

2

8 Conclusions
Using the methodology described in Section 7 above, there were 21 projects
scoring three and two scoring four. Short descriptive extracts from the abstracts
for each of these scoring 3 or more appear in Appendix 1 below with links to thejournal publisher in each case.

Lush Prize Science Prize – background paper – August 201316

The two abstracts which scored highest (4) are reproduced in full
here as follows. It will be worth considering whether they should be added to the2013 Prize Shortlist.

8.1 Human embryonic stem cell-derived test systems for developmental
neurotoxicity: a transcriptomics approach

Archives of Toxicology January 2013, Volume 87, Issue 1, pp 123-143
Krug AK, Kolde R, Gaspar JA, Rempel E, Balmer NV, Meganathan K, Vojnits K,
Baquié M, Waldmann T, Ensenat-Waser R, Jagtap S, Evans RM, Julien S,
Peterson H, Zagoura D, Kadereit S, Gerhard D, Sotiriadou I, Heke M, Natarajan
K, Henry M, Winkler J, Marchan R, Stoppini L, Bosgra S, Westerhout J, Verwei M,
Vilo J, Kortenkamp A, Hescheler J, Hothorn L, Bremer S, van Thriel C, Krause
KH, Hengstler JG, Rahnenführer J, Leist M, Sachinidis A.
Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT)
often manifest themselves in functional deficits that are not necessarily based oncell death, but rather on minor changes relating to cell differentiation orcommunication. The fields of DNT/RT would greatly benefit from in vitro tests thatallow the identification of toxicant-induced changes of the cellular proteostasis, orof its underlying transcriptome network. Therefore, the ‘human embryonic stem
cell (hESC)-derived novel alternative test systems (ESNATS)’ European
commission research project established RT tests based on defineddifferentiation protocols of hESC and their progeny. Valproic acid (VPA) andmethylmercury (MeHg) were used as positive control compounds to address thefollowing fundamental questions: (1) Does transcriptome analysis allow
discrimination of the two compounds? (2) How does analysis of enriched
transcription factor binding sites (TFBS) and of individual probe sets (PS)
distinguish between test systems? (3) Can batch effects be controlled? (4) How
many DNA microarrays are needed? (5) Is the highest non-cytotoxic
concentration optimal and relevant for the study of transcriptome changes? VPA
triggered vast transcriptional changes, whereas MeHg altered fewer transcripts.
To attenuate batch effects, analysis has been focused on the 500 PS with highest
variability. The test systems differed significantly in their responses (<20 %overlap). Moreover, within one test system, little overlap between the PS changedby the two compounds has been observed. However, using TFBS enrichment, a
relatively large ‘common response’ to VPA and MeHg could be distinguished from‘compound-specific’ responses. In conclusion, the ESNATS assay battery allows
classification of human DNT/RT toxicants on the basis of their transcriptome
profiles.
The full article is freely available on the web at:
http://link.springer.com/article/10.1007%2Fs00204-012-0967-3#page-1

Lush Prize Science Prize – background paper – August 201317

8.2 Profiling Environmental Chemicals for Activity in the

Antioxidant Response Element Signaling Pathway Using a HighThroughput Screening Approach

Environ. Health Perspect. 120:1150-1156 (2012).
http://dx.DOI.org/10.1289/ehp.1104709
Sunita J. Shukla1, Ruili Huang1, Steven O. Simmons2, Raymond R. Tice3, Kristine
L. Witt3, Danielle VanLeer1, Ram Ramabhadran2, Christopher P. Austin1,
Menghang Xia1
NIH Chemical Genomics Center, National Institutes of Health, Department of
Health and Human Services, Rockville, Maryland, USA; 2U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina, USA; 3Division of the
National Toxicology Program, National Institute of Environmental Health
Sciences, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, North Carolina, USA
1

BACKGROUND:
Oxidative stress has been implicated in the pathogenesis of a variety of diseases
ranging from cancer to neurodegeneration, highlighting the need to identify
chemicals that can induce this effect. The antioxidant response element (ARE)signaling pathway plays an important role in the amelioration of oxidative stress.
Thus, assays that detect the up-regulation of this pathway could be useful for
identifying chemicals that induce oxidative stress.
OBJECTIVES:
We used cell-based reporter methods and informatics tools to efficiently screen alarge collection of environmental chemicals and identify compounds that induce
oxidative stress.
METHODS:We utilized two cell-based ARE assay reporters, β-lactamase and luciferase, toscreen a U.S. National Toxicology Program 1,408-compound library (NTP 1408,
which contains 1,340 unique compounds) for their ability to induce oxidative
stress in HepG2 cells using quantitative high throughput screening (qHTS).
RESULTS:
Roughly 3% (34 of 1,340) of the unique compounds demonstrated activity acrossboth cell-based assays. Based on biological activity and structure-activity
relationship profiles, we selected 50 compounds for retesting in the two ARE
assays and in an additional follow-up assay that employed a mutated ARE linked
to β-lactamase. Using this strategy, we identified 30 compounds that
demonstrated activity in the ARE-bla and ARE-luc assays and were able todetermine structural features conferring compound activity across assays.

Lush Prize Science Prize – background paper – August 201318

CONCLUSIONS:
Our results support the robustness of using two different cell-based approaches
for identifying compounds that induce ARE signaling. Together, these methods
are useful for prioritizing chemicals for further in-depth mechanism-based toxicity
testing.
www.ncbi.nlm.nih.gov/pubmed/22551509

Lush Prize Science Prize – background paper – August 201319

Appendix I
Extracts from each abstract scoring 3 or more have been reproduced below togive an indication of the nature of the research. A link at the bottom of each piece
connects to a full version of each abstract available on the web. Where multiple
institutions have been involved in the research, sometimes just the first-named
appears in the credits for reasons of space.
•

ALTEX

Archives of Toxicology January 2013, Volume 87, Issue 1, pp 123-143,
Human embryonic stem cell-derived test systems for developmental
neurotoxicity: a transcriptomics approach
Krug AK, Kolde R, Gaspar JA, Rempel E, Balmer NV, Meganathan K, Vojnits K,
Baquié M, Waldmann T, Ensenat-Waser R, Jagtap S, Evans RM, Julien S,
Peterson H, Zagoura D, Kadereit S, Gerhard D, Sotiriadou I, Heke M, Natarajan
K, Henry M, Winkler J, Marchan R, Stoppini L, Bosgra S, Westerhout J, Verwei M,
Vilo J, Kortenkamp A, Hescheler J, Hothorn L, Bremer S, van Thriel C, Krause
KH, Hengstler JG, Rahnenführer J, Leist M, Sachinidis A.
Department of Biology, University of Konstanz, 78457 Constance, Germany.
Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT)
often manifest themselves in functional deficits that are not necessarily based on cell
death, but rather on minor changes relating to cell differentiation or communication.
The fields of DNT/RT would greatly benefit from in vitro tests that allow theidentification of toxicant-induced changes of the cellular proteostasis, or of its
underlying transcriptome network. Therefore, the ‘human embryonic stem cell
(hESC)-derived novel alternative test systems (ESNATS)’ European commission
research project established RT tests based on defined differentiation protocols of
hESC and their progeny. In conclusion, the ESNATS assay battery allows
classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

http://www.ncbi.nlm.nih.gov/pubmed/23179753

ALTEX. 2013;30(2):145-57.
A 155-plex high-throughput in vitro coregulator binding assay for (anti-)
estrogenicity testing evaluated with 23 reference compounds
Si Wang1,2, René Houtman3, Diana Melchers3, Jac Aarts1,2, Ad Peijnenburg2, Rinie
van Beuningen3, Ivonne Rietjens1, and Toine F. Bovee2

Lush Prize Science Prize – background paper – August 201320

1

Division of Toxicology, Wageningen University and ResearchCentre, Wageningen, The Netherlands; 2Toxicology & Bioassays, RIKILT–
Institute of Food Safety, Wageningen University and Research Centre,
Wageningen, The Netherlands; 3PamGene International B.V.’s-Hertogenbosch,
The Netherlands
To further develop an integrated in vitro testing strategy for replacement of in vivo
tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulatorswith estrogen receptor α was assessed using a PamChip® plate. The relative
estrogenic potencies determined, based on ERα binding to coregulator peptides in
the presence of ligands on the PamChip® plate, were compared to the relative
estrogenic potencies as determined in the in vivo uterotrophic assay. Moreover, thiscoregulator binding assay is able to distinguish ER agonists from ER antagonists:
profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct
from those of pure ER agonists, such as dienestrol. Combination of this coregulator
binding assay with other types of in vitro assays, e.g., reporter gene assays and theH295R steroidogenesis assay, will frame an in vitro test panel for screening andprioritization of chemicals, thereby contributing to the reduction and ultimately the
replacement of animal testing for (anti-)estrogenic effects.

http://www.altex.ch/resources/raltex_2013_2_145_157_Wang4f1.pdf•

Toxicology In Vitro

Toxicology In Vitro Volume 27, Issue 6, September 2013, Pages 1820–1829
Ethanol-induced apoptosis in human liver adenocarcinoma cells (SK-Hep1):
Fas- and mitochondria-mediated pathways and interaction with MAPK
signaling system
Yuri Morio, Mayumi Tsuji, Manami Inagaki, Mai Nakagawa, Yuri Asaka, Hideto
Oyamada, Kanji Furuya, Katsuji Oguchi
Department of Pharmacology, School of Medicine, Showa University, Hatanodai
1-5-8, Shinagawa-ku, Tokyo 142-8555, JapanFor studying molecular mechanisms regulating the fate of ethanol-treatedhepatocytes, involvement of Fas in ethanol-induced apoptosis was examined inhuman liver adenocarcinoma (SK-Hep1) cells in which the function of Fasassociated death domain (FADD) protein was knocked down by transfection. In
FADD-knocked down cells, while ethanol-induced increase in generation of reactive
oxygen species (ROS) was unaffected, apoptosis was significantly suppressed,
demonstrating the involvement of Fas in ethanol-induced hepatocyte apoptosis more
directly than in the past reports. We concluded that oxidative stress inflicted byreactive oxygen species (ROS) triggered Fas-mediated and mitochondria-mediatedapoptotic pathways in ethanol-treated SK-Hep1 cells, and that p38 mitogenactivated protein kinase (MAPK) and JNK were promoting mitochondrial pathway,
suggesting interaction between apoptosis and MAPK signaling systems.

www.sciencedirect.com/science/article/pii/S0887233313001355

Lush Prize Science Prize – background paper – August 201321

Toxicology in Vitro Volume 27, Issue 1, February 2013, Pages 314–
322
Applicability of a keratinocyte gene signature to predict skin sensitizing
potential
Jochem W. van der Veen, Tessa E. Pronk, Henk van Loveren, Janine Ezendam
Department of Toxicogenomics, Maastricht University, P.O. Box 616, NL-6200 MD
Maastricht, The Netherlands; Laboratory for Health Protection Research (GBO),
National Institute for Public Health and the Environment (RIVM), P.O. Box 1, NL3720BA Bilthoven, The Netherlands
There is a need to replace animal tests for the identification of skin sensitizers andcurrently many alternative assays are being developed that have very promising
results. In this study a gene signature capable of very accurate identification ofsensitizers was established in the HaCaT human keratinocyte cell line. Thissignature was evaluated in a separate study using six chemicals that are either local
lymph node (LLNA) false-positive or false-negative chemicals in addition to ninesensitizers and four non-sensitizers. Similar studies do not apply these more difficult
to classify chemicals, which show the true potential for human predictions of anassay. Although the gene signature has improved prediction accuracy compared to
the LLNA, the misclassified compounds were comparable between the two assays.
Gene profiling also showed a sensitizer specific response of the Nrf2-keap1 andToll-like receptor signaling pathways. After exposure to non-sensitizing chemicals
that induce either of the pathways the signature misclassified all Nrf2-inducers, while
the Toll-like receptor ligands were correctly classified. In conclusion, we confirm that
keratinocyte based prediction assays may provide essential information on theproperties of compounds. Furthermore, chemical selection is critical for assessmentof the performance of in vitro alternative assays.

www.sciencedirect.com/science/article/pii/S0887233312002305

Toxicology in Vitro Volume 27, Issue 1, February 2013, Pages 330–338
Silver nanoparticles induce toxicity in A549 cells via ROS-dependent andROS-independent pathways
Porntipa Chairuangkitti, Somsong Lawanprasert, Sittiruk Roytrakul, Sasitorn
Aueviriyavit, Duangkamol Phummiratch, Kornphimol Kulthong, Pithi
Chanvorachote, Rawiwan Maniratanachote
Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 PhayathaiRd., Pathumwan, Bangkok 10330, Thailand
Silver nanoparticles (AgNPs) are incorporated into a large number of consumer andmedical products. Several experiments have demonstrated that AgNPs can be toxic
to the vital organs of humans and especially to the lung. The present study
evaluated the in vitro mechanisms of AgNP (<100nm) toxicity in relationship to thegeneration of reactive oxygen species (ROS) in A549 cells. These observations

Lush Prize Science Prize – background paper – August 201322

allow us to propose that the in vitro toxic effects of AgNPs on A549
cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent(cell cycle arrest) pathways.

www.sciencedirect.com/science/article/pii/S0887233312002287

Toxicology in Vitro Volume 27, Issue 3, April 2013, Pages 1043–1048
Sodium arsenite induces cyclooxygenase-2 expression in humanuroepithelial cells through MAPK pathway activation and reactive oxygen
species induction
Huihui Wang, Shuhua Xi, Yuanyuan Xu, Fei Wang, Yi Zheng, Bing Li, Xin Li,
Quanmei Zheng, Guifan Sun
Department of Occupational and Environmental Health, School of Public Health,
China Medical University, Shenyang, Liaoning, PR China
Arsenic can induce reactive oxygen species (ROS) leading to oxidative stress andcarcinogenesis. Bladder is one of the major target organs of arsenic, andcyclooxygenase-2 (COX-2) may play an important role in arsenic-induced bladdercancer. However, the mechanism by which arsenic induces COX-2 in bladder cells
remains unclear. This study aimed at investigating arsenic-mediated intracellular
redox status and signaling cascades leading to COX-2 induction in humanuroepithelial cells (SV-HUC-1). These data indicate that arsenite promotes an
induction of ROS, which results in an induction of COX-2 expression throughactivation of the MAPK pathway.

www.sciencedirect.com/science/article/pii/S0887233313000131
•

AXLR8

PLOS Computational Biology- Research Article Published: April 4, 2013
A Computational Model Predicting Disruption of Blood Vessel Development
Nicole Kleinstreuer, David Dix, Michael Rountree, Nancy Baker, Nisha Sipes,
David Reif, Richard Spencer, Thomas Knudsen
National Center for Computational Toxicology, Office of Research andDevelopment, U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina, USA.
Vascular development is a complex process regulated by dynamic biologicalnetworks that vary in topology and state across different tissues and developmental
stages. Simulating these interactions at a systems level requires sufficient biologicaldetail about the relevant molecular pathways and associated cellular behaviors, andtractable computational models that offset mathematical and biological complexity.
Here, we describe a novel multicellular agent-based model of vasculogenesis using

Lush Prize Science Prize – background paper – August 201323

the CompuCell3D (http://www.compucell3d.org/) modelingenvironment supplemented with semi-automatic knowledgebase creation.
The model was shown to recapitulate stereotypical capillary plexus formation andstructural emergence of non-coded cellular behaviors, such as a heterologous
bridging phenomenon linking endothelial tip cells together during formation ofpolygonal endothelial cords. Molecular targets in the computational model were
mapped to signatures of vascular disruption derived from in vitro chemical profilingusing the EPA's ToxCast high-throughput screening (HTS) dataset. These findings
support the utility of cell agent-based models for simulating a morphogenetic seriesof events and for the first time demonstrate the applicability of these models forpredictive toxicology. This work was funded by the U.S. EPA.

www.ploscompbiol.org/article/info%3ADOI%2F10.1371%2Fjournal.pcbi.1002996

Regul. Toxicol. Pharmacol. 2012 Aug;63(3):489-504 (DOI:
10.1016/j.yrtph.2012.05.013. Epub 2012 Jun 1)
Putting the parts together: combining in vitro methods to test for skinsensitizing potentials.
Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, vanRavenzwaay B, Landsiedel R.
BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
Allergic contact dermatitis is a common skin disease and is elicited by repeated skincontact with an allergen. The mechanisms that trigger skin sensitization are complex
and various steps are involved. Therefore, a single in vitro method may not be able
to accurately assess this endpoint. In this study, the predictivities of four in vitro
assays, one in chemico and one in silico method addressing three different steps inthe development of skin sensitization were assessed using 54 test substances ofknown sensitizing potential. The predictivity of single tests and combinations ofthese assays were compared. These data were used to develop an in vitro testing
scheme and prediction model for the detection of skin sensitizers based on protein
reactivity, activation of the Keap-1/Nrf2 signaling pathway and dendritic cell
activation. Copyright © 2012 Elsevier Inc. All rights reserved.

www.ncbi.nlm.nih.gov/pubmed/22659254

Cutaneous and Ocular Toxicology. 2012 Dec;31(4):292-305. (DOI:
10.3109/15569527.2012.667031. Epub 2012 Apr 12)
An in vitro method for detecting chemical sensitization using humanreconstructed skin models and its applicability to cosmetic,
pharmaceutical, and medical device safety testing.
McKim JM Jr, Keller DJ 3rd, Gorski JR.

Lush Prize Science Prize – background paper – August 201324

CeeTox, Inc, Kalamazoo, MI 49008, USA. jmckim@ceetox.com
Chemical sensitization is a serious condition caused by small reactive molecules
and is characterized by a delayed type hypersensitivity known as allergic contact
dermatitis (ACD). Although most of the [non animal] research has focused on purechemicals that possess reasonable solubility properties, it is important for any
successful in vitro method to have the ability to test compounds with low aqueous
solubility. The aim of this research was to demonstrate the functionality andapplicability of the human reconstituted skin models (MatTek Epiderm(®) andSkinEthic RHE) as a test system for the evaluation of chemical sensitization and its
potential use for medical device testing. In addition, the development of the human
3D skin model should allow the in vitro sensitization assay to be used for finishedproduct testing in the personal care, cosmetics, and pharmaceutical industries. Thisapproach combines solubility, chemical reactivity, cytotoxicity, and activation of theNrf2/ARE expression pathway to identify and categorize chemical sensitizers.
The results demonstrated that both the MatTek and SkinEthic models performed in amanner consistent with data previously reported with the human keratinocyte(HaCaT) cell line. In all cases, the human skin models performed as well or better
than the HaCaT cell model previously evaluated. In addition, this study identifies aclear unifying trigger that controls both the Nrf2/ARE pathway and essential
biochemical events required for the development of ACD.

www.ncbi.nlm.nih.gov/pubmed/22494060

Environ. Health Perspect. 120:1150-1156 (2012) (DOI: 10.1289/ehp.1104709)
Profiling Environmental Chemicals for Activity in the Antioxidant Response
Element Signaling Pathway Using a High Throughput Screening Approach
Sunita J. Shukla1, Ruili Huang1, Steven O. Simmons2, Raymond R. Tice3, Kristine
L. Witt3, Danielle VanLeer1, Ram Ramabhadran2, Christopher P. Austin1,
Menghang Xia1
NIH Chemical Genomics Center, National Institutes of Health, Department of
Health and Human Services, Rockville, Maryland, USA; 2U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina, USA; 3Division of the
National Toxicology Program, National Institute of Environmental Health
Sciences, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, North Carolina, USA
1

Oxidative stress has been implicated in the pathogenesis of a variety of diseases
ranging from cancer to neurodegeneration, highlighting the need to identify
chemicals that can induce this effect. The antioxidant response element (ARE)signaling pathway plays an important role in the amelioration of oxidative stress.
Thus, assays that detect the up-regulation of this pathway could be useful for
identifying chemicals that induce oxidative stress.
We used cell-based reporter methods and informatics tools to efficiently screen alarge collection of environmental chemicals and identify compounds that induce
oxidative stress. We utilized two cell-based ARE assay reporters, β-lactamase andLush Prize Science Prize – background paper – August 201325

luciferase, to screen a U.S. National Toxicology Program 1,408compound library (NTP 1408, which contains 1,340 unique compounds) for theirability to induce oxidative stress in HepG2 cells using quantitative high throughput
screening (qHTS).
Our results support the robustness of using two different cell-based approaches for
identifying compounds that induce ARE signaling. Together, these methods areuseful for prioritizing chemicals for further in-depth mechanism-based toxicity
testing.

www.ncbi.nlm.nih.gov/pubmed/22551509•

Google Scholar

Environmental Science and Pollution Research February 2013, Volume 20, Issue2, pp 803-811
Mechanisms of toxicity of triphenyltin chloride (TPTC) determined by a live
cell reporter array
Guanyong Su, Xiaowei Zhang, Jason C. Raine, Liqun Xing, Eric Higley, Markus
Hecker, John P. Giesy, Hongxia Yu
State Key Laboratory of Pollution Control and Resource Reuse & School of theEnvironment, Nanjing University, Nanjing, China.
Triphenyltin chloride (TPTC), which has been extensively used in industry andagriculture, can occur at concentrations in the environment sufficient to be toxic.
Here, potency of TPTC to modulate genes in a library containing 1,820 modified
green fluorescent protein (GFP)-expressing promoter reporter vectors constructedfrom Escherichia coli K12 strains was determined. Responses were 1,230 and 97
times more sensitive than the acute median effect concentration (EC50) required toinhibit growth of cells, which demonstrated that this live cell array represents asensitive method to assess toxic potency of chemicals. The 71 differentiallyexpressed genes could be classified into seven functional groups. Of all the alteredgenes, three groups which encoded for catalytic enzymes, regulatory proteins, andstructural proteins accounted for 28%, 18%, and 14% of all altered genes,
respectively. The pattern of differential expression observed during this study wasused to elucidate the mechanism of toxicity of TPTC. The genes rnC, cld, and glgS
were selected as potential biomarkers for TPTC, since their expression was morethan 2.0-fold greater after exposure to TPTC.

www.ncbi.nlm.nih.gov/pubmed/23128992

Chemical Research in Toxicology, Just Accepted Manuscript (DOI:
10.1021/tx4001754 Publication Date (Web): July 29, 2013)
A systematic study of mitochondrial toxicity of environmental chemicals
using quantitative high throughput screening

Lush Prize Science Prize – background paper – August 201326

Matias S Attene-Ramos1 , Ruili Huang1 , Srilatha Sakamuru1 ,Kristine L Witt2, Gyda C Beeson3, Louie Shou1, Rick G Schnellmann3, Craig Cano
Beeson3,4, Raymond Tice2, Christopher P. Austin1, and Menghang Xia1
1

National Center for Advancing Translational Sciences, National Institutes of
Health, Bethesda, Maryland 20892, United States; 2Division of the National
Toxicology Program, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709, United States; 3Department ofDrug Discovery and Biomedical Sciences, Medical University of South Carolina,Charleston, South Carolina 29425, United States; 4MitoHealth, Charleston, South
Carolina 29403, United States
A goal of the Tox21 program is to transit toxicity testing from traditional in vivo
models to in vitro assays that assess how chemicals affect cellular responses andtoxicity pathways. Here, we evaluated the effect of chemical compounds on
mitochondrial membrane potential in HepG2 cells by screening a library of 1,408
compounds provided by the National Toxicology Program (NTP) in a qHTS platform.
Compounds were screened over 14 concentrations, and results showed that 91 and88 compounds disrupted mitochondrial membrane potential after treatment for one
or five h, respectively. Compounds were further assessed for mechanism of action(MOA) by measuring changes in oxygen consumption rate, which enabled
identification of 20 compounds as uncouplers. This comprehensive approach allows
for evaluation of thousands of environmental chemicals for mitochondrial toxicity andidentification of possible MOAs. Copyright © 2013 American Chemical Society.

http://pubs.acs.org/DOI/abs/10.1021/tx4001754

Toxicological Sciences Volume 133, Issue 2 2013 Jun;:259-74. DOI:
10.1093/toxsci/kft075. Epub 2013 Mar 27.
Reactivity of Chemical Sensitizers Toward Amino Acids In Cellulo Plays aRole in the Activation of the Nrf2-ARE Pathway in Human Monocyte
Dendritic Cells and the THP-1 Cell Line
Migdal C, Botton J, El Ali Z, Azoury ME, Guldemann J, Giménez-Arnau E,
Lepoittevin JP, Kerdine-Römer S, Pallardy M.
INSERM UMR-S 996, Faculté de Pharmacie, UniverSud, 92290 ChâtenayMalabry, France.
Allergic contact dermatitis resulting from skin sensitization is an inflammatory skindisease linked to the use of chemicals termed haptens. Chemical reactivity isnecessary for a chemical to be a sensitizer, allowing both covalent binding toproteins and maturation of dendritic cells (DCs) by mimicking “danger signals.” Theaim of this study was to evaluate how the reactivity of chemical sensitizers towardamino acids translates into a biological response using the activation of the nuclear
factor-erythroid 2-related factor 2 (Nrf2) pathway, which was assessed by the
induction of three Nrf2 target genes (ho-1, nqo1, and il-8) and Nrf2 protein
accumulation.

Lush Prize Science Prize – background paper – August 201327

Regression analysis revealed that ho-1 and nqo1 expressions were
found to be associated with chemical sensitizer reactivity to cysteine, providing
evidence of the importance of chemical reactivity, as a part of danger signals, in DC
biology.

www.ncbi.nlm.nih.gov/pubmed/23535360•

Terkko – Helsinki University Medical Library

Regulatory Peptides Volume 180, 10 January 2013, Pages 33–42
Neuregulin-1β regulates tyrosine kinase receptor expression in cultured
dorsal root ganglion neurons with excitotoxicity induced by glutamate
Weiming Yuea, Lijun Songb, Guangchun Fuc, Yizhao Lid, Huaxiang Liub,a

Department of Thoracic Surgery, Shandong University Qilu Hospital, Jinan
250012, China; bDepartment of Rheumatology, Shandong University Qilu
Hospital, Jinan 250012, China; cDepartment of Internal Medicine, Qingzhou
Municipal Hospital, Qingzhou, 262500, China; dJinan e-Join Science andTechnology, Co., Ltd, Jinan 250100, China.
Neuregulin-1 (NRG-1) signaling regulates neuronal development, migration,
myelination, and synaptic maintenance. Three members of tyrosine kinase receptor(Trk) family, TrkA, TrkB, and TrkC, have been identified in DRG neurons. Whether
NRG-1β and its signaling pathways influence the expression of these Trk receptors
in DRG neurons is still unclear. In the present study, primary cultured DRG neurons
were used to determine the effects of NRG-1β on TrkA, TrkB, and TrkC expressionin DRG neurons with excitotoxicity induced by glutamate (Glu). The results indicatedthat in primary cultured DRG neurons with excitotoxicity induced by Glu, NRG-1β
increased the expression of TrkA and TrkB their mRNAs, but not TrkC and its
mRNA. Inhibitors (LY294002, PD98059) either alone or in combination blocked theeffects of NRG-1β. NRG-1β may play an important role in regulating the expressionof different Trk receptors in DRG neurons through the PI3K/Akt and ERK1/2
signaling pathways.

www.sciencedirect.com/science/article/pii/S0167011512002467

Frontiers in Physiology. 2012;3:462. DOI: 10.3389/fphys.2012.00462. Epub 2012
Dec 14.
Modeling drug- and chemical-induced hepatotoxicity with systems biology
approaches.
Bhattacharya S, Shoda LK, Zhang Q, Woods CG, Howell BA, Siler SQ,
Woodhead JL, Yang Y, McMullen P, Watkins PB, Andersen ME.
Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences
Research Triangle Park, NC, USA.

Lush Prize Science Prize – background paper – August 201328

We provide an overview of computational systems biology
approaches as applied to the study of chemical- and drug-induced toxicity. We focus
on toxicity of the liver (hepatotoxicity) - a complex phenotypic response with
contributions from a number of different cell types and biological processes. We
describe three case studies of complementary multi-scale computational modelingapproaches to understand perturbation of toxicity pathways in the human liver as aresult of exposure to environmental contaminants and specific drugs. One approach
involves development of a spatial, multicellular "virtual tissue" model of the liver
lobule that combines molecular circuits in individual hepatocytes with cell-cellinteractions and blood-mediated transport of toxicants through hepatic sinusoids, toenable quantitative, mechanistic prediction of hepatic dose-response for activation of
the aryl hydrocarbon receptor toxicity pathway. Simultaneously, methods are being
developing to extract quantitative maps of intracellular signaling and transcriptional
regulatory networks perturbed by environmental contaminants, using a combinationof gene expression and genome-wide protein-DNA interaction data. A predictive
physiological model (DILIsym™) to understand drug-induced liver injury (DILI), themost common adverse event leading to termination of clinical development
programs and regulatory actions on drugs, is also described.

www.ncbi.nlm.nih.gov/pubmed/23248599

Inhalation Toxicology 2012 Dec;24(14):966-75. DOI:
10.3109/08958378.2012.742600.
Gene and protein responses of human lung tissue explants exposed toambient particulate matter of different sizes.
Hoogendoorn B, Berube K, Gregory C, Jones T, Sexton K, Brennan P, Brewis IA,
Murison A, Arthur R, Price H, Morgan H, Matthews IP.
Department of Primary Care and Public Health, Neuadd Meirionnydd, School of
Medicine, Heath Park, Cardiff, UK. hoogendoornb@cardiff.ac.uk
Exposure to ambient particulate air pollution is associated with increased
cardiovascular and respiratory morbidity and mortality. The research was designed
to investigate the effect of different size ranges of ambient particulate matter (PM) on
gene and protein expression in an in vitro model. Eighteen different genes of the 84
on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted
in the greatest number of genes with altered expression, followed by size 1 andlastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentiallyexpressed. Enrichment for gene ontology classification revealed potential changes
to various pathways. This approach not only provides an investigative tool toidentify possible causal pathways but also permits the relationship between particle
size and responses to be explored.

www.ncbi.nlm.nih.gov/pubmed/23216157?dopt=Abstract#

Journal of Cellular Physiology Volume 228, Issue 6, pages 1359–1367, June
2013
Lush Prize Science Prize – background paper – August 201329

Unraveling amyloid toxicity pathway in NIH3T3 cells by a
combined proteomic and 1H-NMR metabonomic approach
Vilasi A, Vilasi S, Romano R, Acernese F, Barone F, Balestrieri ML, Maritato R,
Irace G, Sirangelo I.
Laboratory of Mass Spectrometry and Proteomics, Institute of Protein
Biochemistry-CNR, Naples, Italy.
A range of debilitating human diseases is known to be associated with the formationof stable highly organized protein aggregates known as amyloid fibrils. The earlyprefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result
from an intrinsic ability to impair fundamental cellular processes by interacting withcellular membranes, causing oxidative stress and increase in free Ca2+ that lead to
apoptotic or necrotic cell death. However, specific signaling pathways that underlie
amyloid pathogenicity remain still unclear. The results of our work indicated that cell
exposure to prefibrillar aggregates induces changes of the expression level ofproteins and metabolites involved in stress response. The majority of the proteinsand metabolites detected are reported to be related to oxidative stress, perturbationof calcium homeostasis, apoptotic and survival pathways, and membrane damage.
In conclusion, the combined proteomic and 1H-NMR metabonomic approach,described in this study, contributes to unveil novel proteins and metabolites that
could take part to the general framework of the toxicity induced by amyloid
aggregates.

www.ncbi.nlm.nih.gov/pubmed/23192898

Cell Stem Cell Volume 11, Issue 5, 2 November 2012, Pages 620–632
Discovery of Inhibitors of Microglial Neurotoxicity Acting Through Multiple
Mechanisms Using a Stem-Cell-Based Phenotypic Assay
Höing S, Rudhard Y, Reinhardt P, Glatza M, Stehling M, Wu G, Peiker C, Böcker
A, Parga JA, Bunk E, Schwamborn JC, Slack M, Sterneckert J, Schöler HR.
Department of Cell and Developmental Biology, Max Planck Institute for
Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany.
Stem cells, through their ability to both self-renew and differentiate, can produce a
virtually limitless supply of specialized cells that behave comparably to primary cells.
We took advantage of this property to develop an assay for small-molecule-basedneuroprotection using stem-cell-derived motor neurons and astrocytes, together withactivated microglia as a stress paradigm. Here, we report on the discovery of hit
compounds from a screen of more than 10,000 small molecules. These compounds
act through diverse pathways, including the inhibition of nitric oxide production by
microglia, activation of the Nrf2 pathway in microglia and astrocytes, and direct
protection of neurons from nitric-oxide-induced degeneration. We confirm the activityof these compounds using human neurons.

www.ncbi.nlm.nih.gov/pubmed/23064101

Lush Prize Science Prize – background paper – August 201330

Biochimica et Biophysica Acta 2013 Oct;1833(10):2311-21. DOI:
10.1016/j.bbamcr.2013.05.021. Epub 2013 Jun 1.
Protein kinase CK2-dependent phosphorylation of the human Regulators ofCalcineurin reveals a novel mechanism regulating the calcineurin-NFATc
signaling pathway.
Martínez-Høyer S, Aranguren-Ibáñez A, García-García J, Serrano-Candelas E,
Vilardell J, Nunes V, Aguado F, Oliva B, Itarte E, Pérez-Riba M.
Cellular Signaling group, Cancer and Molecular Genetics Program, Bellvitge
Biomedical Research Institute - IDIBELL, L'Hospitalet de Llobregat, 08908
Barcelona, Catalonia, Spain.
Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin
phosphatase activity and consequently activation of cytosolic Nuclear Factor of
Activated T-cell (NFATc) transcription factor. Due to the chronic toxicity associatedwith their administration, the development of more specific immunosuppressants iscurrently an important unmet medical need. Molecular modeling studies have led us
to identify a positively charged interaction site on the surface of calcineurin where
the phosphorylated serine residue of the CIC motif would normally locate. Finally, we
have also identified RCAN3 as a new phosphoprotein with multiple phosphorylationsites. Therefore, our findings reveal for the first time a novel molecular mechanism
underlying the regulation of calcineurin-NFATc signaling by means ofphosphorylation of the CIC motif of RCAN proteins. Copyright © 2013. Published by
Elsevier B.V.

www.ncbi.nlm.nih.gov/pubmed/23732701

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Volume 1831, Issue 8, August 2013, Pages 1344–1356
A signaling cascade mediated by ceramide, src and PDGFRβ coordinates
the activation of the redox-sensitive neutral sphingomyelinase-2 andsphingosine kinase-1
Cinq-Frais C, Coatrieux C, Grazide MH, Hannun YA, Nègre-Salvayre A, Salvayre
R, Augé N.
Inserm UMR 1048, I2MC, Toulouse, France.
Stress-inducing agents, including oxidative stress, generate the sphingolipid
mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved instress-induced cellular responses. The two redox-sensitive neutral
sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate intransducing stress signaling to ceramide and S1P, respectively; however, whether
these key enzymes are coordinately regulated is not known. We investigatedwhether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In

Lush Prize Science Prize – background paper – August 201331

mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect,
mitogenic at low concentration (5 μM), and anti-proliferative and toxic at highconcentration (100 μM). The results also showed that the toxicity of high H2O2
concentration was comparable in control and nSMase2-deficient cells. Taken
together the results identify a tightly coordinated nSMase2/SK1 pathway thatmediates the mitogenic effects of H2O2 and may sense the degree of oxidative
stress.

www.ncbi.nlm.nih.gov/pubmed/23651497

International Journal of Molecular Sciences 2013 Mar 11;14(3):5633-49. DOI:
10.3390/ijms14035633.
Global Gene Expression Profiling Reveals Functional Importance of Sirt2 inEndothelial Cells under Oxidative Stress.
Liu J, Wu X, Wang X, Zhang Y, Bu P, Zhang Q, Jiang F.
Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese
Ministry of Education and Chinese Ministry of Public Health, Shandong University,
Jinan 250012, Shandong, China. bupeili@medmail.com.cn.
The NAD+-dependent deacetylases Sirt1 and Sirt2 mediate cellular stressresponses and are highly expressed in vascular endothelial cells. In contrast to thewell-documented protective actions of Sirt1, the role of endothelial Sirt2 remains
unknown. Using cDNA microarray and PCR validation, we examined global gene
expression changes in response to Sirt2 knock down in primary human umbilicalvein endothelial cells under oxidative stress. We found that Sirt2 knock downchanged expression of 340 genes, which are mainly involved in cellular processes
including actin binding, cellular amino acid metabolic process, transmembrane
receptor protein serine/threonine kinase signaling, ferrous iron transport, proteintransport and localization, cell morphogenesis, and functions associated withendosome membrane and the trans-Golgi network. Our results may provide a basis
for future studies aiming to dissect the specific signaling pathway(s) that mediates
specific Sirt2 functions in endothelial cells.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3634502/

Lush Prize Science Prize – background paper – August 201332