Lush Prize for Science - 2015
Research Paper

Prepared by TJM Consultancy July 2015
www.tjmconsultancy.co.uk

1. Executive Summary
1.1 What is the Lush Science Prize?
Now in its fourth year, the Lush Prize supports animal-free testing by awarding
money prizes totalling £250,000 to the most effective projects and individuals who
have been working towards the goal of replacing animals in product or ingredientsafety testing. Prizes are awarded for developments in five strategic areas: science;
lobbying; training; public awareness; and Young Researchers. Should there be amajor breakthrough in 21st Century Toxicology - the area which holds out most hopefor a ‘Eureka’ moment leading to the replacement of animal tests – a Black Box Prizeequivalent to the entire annual fund of £250,000 will be awarded to the individual(s)or team(s) responsible.
1.2 Methodology
In order to obtain an overview of developments in the field of animal replacement in
toxicity pathway research, we firstly reviewed the recent work of the relevantscientific institutions and projects in this area, including AXLR8; OECD; HamnerInstitutes; Human Toxome Project; ECVAM; US Tox21 Programme, the ToxCast
programme; and the Human Toxicology Project Consortium (see section 4). We
also assessed recent developments in toxicity testing research by reviewing therelevant literature (see section 5).
In our search for candidate prize winners, we identified conferences focusing on
animal replacement in toxicity testing that have been held in the preceding 12months. These included WC9, the Society of Toxicology annual conference, and the
SEURAT-1 annual conference. There were a total of 3370 abstracts from oral andposter presentations from these conferences, but many were not relevant to theLush Science prize. Relevant abstracts were scored using the system derived inprevious years (see section 3) in which 3 points are awarded for projects identifying
new toxicity pathways, assays, or biomarkers; 2 points for reporting new knowledge
or tools; and 1 point for abstracts which stand out in some other way. We thenperformed literature searches using PubMed, Google Scholar and Terkko Feed
Navigator to identify projects describing recent advances in toxicity testing research.
One additional relevant project was identified directly from the Hamner Institutes
website. In all, searches yielded in excess of 2200 projects which we assessed as
described (see Section 3).
Overall, from all sources combined, from 50 abstracts which scored 1 or more, 2
scored 1; 32 scored 2; 11 scored 2 +1 (i.e. total 3); and 4 scored 3 because they
appeared to be reporting a new toxicity pathway, assay, or biomarker. A single
project scored the maximum possible marks of 3 for reporting a new toxicity pathway
feature plus an additional mark for standing out as well (use of high-throughput
techniques). The titles and authors of those abstracts scoring 1 or more are shownin section 6.3, whilst full abstracts of those projects scoring at least 3 (either as 2+1,
3 or 3+1) are given in Section 6.4.

Lush Prize for Science, 2015 – Research Paper2

1.3 Projects recommended for the shortlist
Research papers for each Prize category identify projects that merit particularconsideration because of their outstanding work in the field. There were 16 projects
which received scores of at least 3 for reporting new pathways, assays, orbiomarkers of toxicity. The full abstracts are given in Section 6.4.

Lush Prize for Science, 2015 – Research Paper

32. Background
The objectives of 21st Century Toxicity Testing (tt21c) research can be summarised
as: improving safety testing of chemicals and contaminants by using more relevant
and predictive human models; and simplifying and automating tests so that many
more chemicals can be tested for safety. The Science background papers for the
2012, 2013, and 2014 Lush Prizes provide an overview, and links to further
resources, describing the concept of 21st Century Toxicology.
The Lush Science Prize seeks to reward those researchers making 'outstanding
contributions' to tt21c research. In previous years, the focus of the award has beenon research aimed at elucidating key pathways in which perturbation results intoxicity. For 2015, we have continued this approach, and further extended it to coveradditional aspects of tt21c, beyond toxicity pathways. In particular, we have alsosought to identify significant contributions to the development of new in vitro assaysthat advance tt21c, and the discovery of biomarkers that signal early activation of
toxicity pathways.
The brief for prize applicants, taken from the Lush prize website
http://www.lushprize.org/ was as follows:
Science Prize
For individuals, research teams or institutions for work conducted on relevant toxicity
pathways. Outstanding research producing an effective non-animal safety test basedon an approach other than toxicity pathways, where none existed before, may also
be considered.
There is a £50,000 prize fund shared between all the winners of the Science Prize.
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 science androbotics.
It offers better relevance to humans (rather than using mice, rats and rabbits), andwill explain the underlying causes of toxicity. Unlike animal methods, the new tests
will help predict human variability and differential effects on embryos, children andadults. And as the superior scientific basis of the new approach is recognised,outdated animal tests will be replaced.

Lush Prize for Science, 2015 – Research Paper

43. Methodology
The primary purpose of this report is to assist the Lush Prize judging panel bydescribing significant advances in animal-free toxicity testing that have taken placeover the preceding year, and by identifying key projects that are making major
contributions to the field of animal-free toxicology research. From these, along withadditional nominations received directly via the Lush Prize website, the panel may
select the winners of this year’s Lush Prize for Science. In this section we describe
how we identified projects that might be worthy of consideration as potential prize
winners, and then how we scored each project to create a shortlist for the panel’s
consideration.
Of the “3 Rs”, Lush Prize’s interest focuses primarily on Replacement, so our searchfor potential prize winners targeted projects working towards the replacement ofanimals in product testing, and we excluded research aimed at either Refining or
Reducing the use of animals in experimentation. We also excluded research thatwas linked to animal testing in other ways. We considered projects based anywherein the world, but restricted the search as far as possible to work reported in the year
preceding the award (i.e. June 2014 – July 2015).
In the identification of key developments in the area of toxicology research, and in
the search for candidate prize winners, we followed three separate strands ofinvestigation. We started firstly by reviewing the recent research of some key
institutions and collaborative projects working in the area of animal replacement in
toxicity pathway research. These included OECD; Hamner Institutes; Human
Toxome Project; ECVAM; US Tox21 Programme, the ToxCast programme; and EU
Horizon 2020 (see section 4).
Secondly, we identified relevant conferences held in the preceding 12 months andassessed abstracts, where available, for oral and poster conference presentations.
Scientific conferences provide the forum in which the most up-to-date science isshared, reporting on recent developments and work-in-progress, without the lag timerequired for formal presentation as a journal publication. The relevant conferencesfor 2014 - 2015 included the 9th World Congress on Alternatives & Animal Use in theLife Sciences (WC9), held in Prague in August 2014, the EU SEURAT-1 Project’s 5th
Annual Meeting, held in Barcelona in January 2015, and finally the Society of
Toxicology’s (SoT’s) 54th Annual Meeting, held in San Diego in March 2015. The
European Society for Alternatives to Animal Testing (EUSAAT) did not hold ameeting in 2014 – the next EUSAAT meeting is in September 2015. Proceedings
from the SEURAT-1 2015 meeting were kindly provided by Sara Vinklatova,
Administrator, COACH SEURAT-1. Due to confidentiality issues, several SEURAT
abstracts were not available for our review.
Thirdly, we conducted a review of the recent literature. For this we used three
separate sources. Firstly, we searched PubMed for research published from01/06/2014 to date, combining search terms “toxicity pathway,” “toxicity assay” and“toxicity biomarker”, excluding any review articles and clinical trials, and restricting
the subject matter to “humans”. As a second literature source, we searched Google
Scholar for relevant papers published in the period 2014 to 2015, combining searchterms “toxicity pathway,” “toxicity assay” and “toxicity biomarker”, and restricting theLush Prize for Science, 2015 – Research Paper

5subject matter to “humans”. As a final literature source, we searched Terkko Feed
Navigator combining search terms “toxicity pathway,” “toxicity assay” and “toxicity
biomarker” and considered only those papers that were published in 2014 or 2015.
As per last year, our selection procedure was a three stage process. In the firststage, we reviewed the project title and rejected any articles which were clearly
reviews or which were obviously unsuitable through using animal models or being
overly focussed on disease. In the second stage, we assessed the abstracts which
passed the initial filter and further eliminated those which reported findings fromclinical trials, those focusing on cancer research, all research that included animal
subjects, and any work conducted on non-human cell lines. In the third stage,
projects were scored using a system devised and successfully applied in previous
years. In this system, points are awarded according to the following criteria:
Does the work appear to be reporting: discovery of a new pathway; asignificant advance in assay technology or approach; or a new biomarker
for early activation of toxicity?

Score 3

If it is working with an apparently previously understood pathway, assay
technology, or biomarker, does it bring new knowledge or tools?

Score 2

Does it stand out in any other way?

Score 1

Projects awarded a score of 2 or 3 could also receive an extra 1 point if they also
stood out in some other way, so the maximum possible score is 4.
At each stage of our search, research projects were carefully excluded based on our
selection criteria, in order to achieve a manageable shortlist of excellent work which
fully met the prize brief.

Lush Prize for Science, 2015 – Research Paper

64. Significant Institutional and Project Developments
This section summarises significant events or news focussing on 21st century
toxicology from selected Institutions and major collaborative projects, reported within
the last year.
4.1 Tox21
Tox21 is a collaborative effort among the National Institutes of Health (NIH), theEnvironmental Protection Agency (EPA), and the Food and Drug Administration(FDA). NIH partners include NCATS (National Center for Advancing Translational
Sciences) and the National Toxicology Program, administered by the National
Institute of Environmental Health Sciences. The Tox21 initiative is designed to
improve current toxicity assessment methods, which are slow and costly, by utilisingrobotic systems and high-throughput technologies to screen thousands ofcompounds.
In addition to testing thousands of chemicals for their effects on a few human celllines, the Tox21 programme has recently used its technology to look at differences inhuman sensitivity to compounds1. NCATS' screening capabilities have been used toscreen the effect of 179 chemicals on more than 1000 individuals, using
lymphoblastoid cells from 1,086 people from the 1,000 Genomes Project2. The
study found that the toxicodynamic factor for risk assessment of chemicals variedmuch more than previously thought, with half of the chemicals having a much larger
factor than usually applied.
4.2 ToxCast
The US EPA's ToxCast (Toxicity Forecaster) programme aims to use highthroughput screening tools to test many thousands of chemicals in in vitro assays.
ToxCast has published an expanded model of endocrine profiling using its data on
1858 chemicals. It should be noted, however, that the EPA ToxCast programme
continues to use animal models for some of its work (seehttp://pubs.acs.org/doi/abs/10.1021/tx500501h for an example).
4.3 AXLR8/Horizon 2020
The EU AXLR8 Project’s mission was to “accelerate the transition to a toxicity
pathway-based paradigm for chemical safety assessment through internationally coordinated research and technology development”. As reported in the 2014 Sciencebackground paper, AXLR8 published its final report in December 2013 andrecommended that the EU invest €250 million over the seven years of the Horizon
2020 funding round, focussing on three pillars of work:

1 http://ehp.niehs.nih.gov/1408775/
2 http://www.1000genomes.org/

Lush Prize for Science, 2015 – Research Paper

7

•
•
•

Adverse Outcome Pathway (AOP) discovery and informatics
Enabling technologies and infrastructures
Implementation

The EU's call for proposals under the Horizon 2020 'Personalising health and care
(PHC)' programme opened on July 30th 2014. One of the topics for proposals is'New approaches to improve predictive human safety testing' (PHC-33-2015). The
call closed on February 24th 2015, and 28 proposals were submitted. These
proposals are competing with 54 others for a share of €88 million of PHC-allocatedfunds. Clearly this is considerably short of AXLR8's recommendations so far.
4.4 The Organisation for Economic Co-operation and Development (OECD)The OECD launched its AOP development programme in 2012. Within thisprogramme is a 'Knowledge Base project (AOP-KB)' – co-led by the European
Commission's Joint Research Centre and the US Environmental Protection Agency.
In September 2014, the first element of the AOP-KB was released – the AOP Wiki
(https://aopkb.org). The AOP Wiki is an interactive and virtual encyclopaedia for
AOP development, structured in accordance with the OECD's original guidance
document. Other modules of the AOP-KB are under development, and it is expectedthat the next module released will be Effectopedia – a knowledge aggregation andmodelling platform designed for collaborative development and utilisation of AOPs.
The other significant development this year is the publication of two new Test
Guidelines (442c and 442d) for non-animal tests for skin sensitivity, which occurredin February 2015. Guideline 442c is an in chemico test (Direct Peptide Reactivity
Assay, DRPA) for discriminating between skin sensitizing chemicals and nonsensitizers. Guideline 442d is an in vitro assay using human keratinocytes(KeratinoSens™) for the same purpose.
4.5 Hamner Institutes
Based in North Carolina, USA, The Hamner Institutes for Health Sciences bring
together safety assessments for chemicals and drugs, world-class education andtraining, and accelerated technology development.
The Hamner is focussing on case studies that show a pathway for the
implementation of new methods for safety assessment. The oestrogen pathway
case study emphasises the need to develop more comprehensive assessments ofthe various oestrogen receptors and to focus on the 'adverse' responses, rather thanrelying on more simple pathway perturbations such as binding, transactivation etc.
The p53-DNA damage pathway case study has indicated that DNA-reactive
compounds have threshold responses at low doses: this work is due to be publishedthis year.
The Hamner has announced the formation of the Comprehensive Center for Liverand Metabolic Disease Research (CCLMDR), a pre-competitive, collaborative
partnership between The Hamner and a diverse set of stakeholders across industry
and academia. The Center aims to pool resources and co-develop platforms

Lush Prize for Science, 2015 – Research Paper

8focussed on liver and metabolic disease research. The CCLMDR’s vision is to be therecognized global leader and ultimate collaborative research resource for thedevelopment of strategies and next-generation technologies aimed at conquering
liver and liver-related metabolic diseases. The Steering Committee for the CCLMDR
is headed by Dr. Paul Watkins, director of the Hamner-University of North Carolina
Institute for Drug Safety Sciences, and Dr. Edward LeCluyse, associate investigatorat The Hamner.
4.6 European Union Reference Laboratory for alternatives to animal testing(EURL EVCAM)
The role of EURL EVCAM is the validation of methods which reduce, refine or
replace the use of animals in safety testing and in efficacy/potency testing ofchemicals, biologicals and vaccines.
In March 2015, EURL EVCAM announced the validation of the human Cell Line
Activation Test (h-CLAT) for skin sensitization. The h-CLAT test identifies chemicals
that can trigger skin allergies – estimated to affect 20% of the population in Europe.
Having validated h-CLAT, ECVAM recommends its use as part of an integrated
approach to chemical testing and assessment, along with other non-animal methods
and in silico predictions. The ECVAM validation and recommendation of h-CLAT
should help in the development of an OECD Test Guideline for h-CLAT, applicableglobally.
In April 2015, ECVAM scientists published work (in collaboration with CAAT) whichshows gender differences in drug sensitivity and hepatotoxicity between male- andfemale- derived primary human hepatocytes. This is the first time that genderdifferences in drug-induced liver injury have been demonstrated at the cellular level –see Section 5 for more details.
The Joint Research Centre (JRC), which hosts EURL ECVAM, has stated its
intention to collaborate with those consortia that are awarded funding under the EU
Horizon 2020 programme call for 'New approaches to improve predictive humansafety testing'.
Although not a complete move to eradicate animal use, EURL ECVAM has releaseda strategy document for the replacement, reduction, and refinement of the use ofanimals in the assessment of acute mammalian systemic toxicity3. EURL ECVAM
has also published a state-of-the-art review on 'Alternative methods for regulatory
toxicology'4.
4.7 HSI-Human Toxicology Project Consortium (HTPC)
3 https://eurl-ecvam.jrc.ec.europa.eu/news/acute-mammalian-systemic-toxicity-testing-eurl-ecvamreleases-itsstrategy?utm_medium=twitter&utm_term=%23animalfreetests&utm_source=twitterfeed
4
http://publications.jrc.ec.europa.eu/repository/bitstream/111111111/32662/1/echa_jrc_sla_rep
ort_public_05-09-14_withcover%20ipo.pdf

Lush Prize for Science, 2015 – Research Paper

9The HSI-Human Toxicology Project Consortium is a group of stakeholders currently
drawn from the corporate and public interest communities that share the objective of
accelerating the implementation of a biological pathway-based approach to
toxicology, as described in the National Research Council’s 2007 report on “Toxicity
Testing in the 21st Century.” Its work in the 2014/2015 period has focussed mainly
on dissemination of advances in assays and technologies for tt21c, such as organson-a-chip.
4.8 Human Toxome Project
The Human Toxome Project is a five-year collaborative project, funded by the NIH
NIEHS, that aims to map comprehensively the pathways of endocrine disruption inhumans, as a first step to mapping all of the toxicity pathways that make up thehuman 'toxome'. The institutional collaborators are: CAAT; Bloomberg School of
Public Health; EPA; Agilent Technologies; Hamner Institute for Chemical Safety
Sciences; Brown University; and Georgetown University Medical Center. Progress
to date is summarised in a recent ALTEX article5. The focus of the project is todevelop methods for deducing and validating molecular pathways of toxicity, using
oestrogen endocrine disruption as a model.

5 http://altweb.jhsph.edu/altex/32_2/Bouhifd.pdf

Lush Prize for Science, 2015 – Research Paper

105. Literature Highlights
We have undertaken an extensive literature search to identify potential Lush Prize
for Science nominees. Details of the search and our results are given elsewhere inthis document (see Sections 3 and 6). In the course of the research for this Science
Prize paper, three particular reports that did not specifically meet the criteria of the
prize stood out. One report that does meet the criteria for consideration for thescience prize is also discussed here, because although it does not describe a newpathway or assay per se, its impact is highly significant.
The first was a roundtable discussion between leading figures in the field on thesubject “A Vision of Toxicity Testing in the 21st Century”, reported in the first editionof the new journal Applied In Vitro Toxicology6. Two particular points stand out, bothmade by Mel Andersen, one of the authors of the seminal 2007 EPA policy
document 'Toxicity Testing in the 21st Century'. The first is that disappointingly little
progress has been made on establishing the global consensus that in vitro testing,rather than high-dose animal studies, are the preferred way to approach tt21c.
Andersen observes that, in the EU, animal testing is preferred, but this has had tochange because of pressure to implement the 3Rs, while in the US he suggests thatanimal testing is perceived to be better, but that it is too slow and costly. Andersen
believes that more effort needs to be made to build the consensus that testing must
focus on human-relevant biology. Secondly, Andersen highlights the perception that
AOPs may not be as helpful as hoped in persuading regulators to adopt tt21c: there
is a risk that they may reinforce a focus on high-dose animal effects as the adverse
outcome of interest.
The second was a workshop report from the transatlantic think tank for toxicology (t4)on the state-of-the-art of 3D cell culture and 'organs-on-a-chip' in safety testing7.
The review summarises the current technology for creating organotypic models, andoffers consensus recommendations for the successful implementation of 3D models
in routine screening.
Thirdly, Mennecozzi et al8 report for the first time gender differences, in terms ofsusceptibility to drug-induced liver effects, at the cellular level. While no new
pathways were described, the study highlighted the value of using human-derived
cells (in this case cryo-preserved human hepatocytes from pre-menopausal andpost-menopausal women, and from men) to show subtle but significant differences
between populations.
Finally, Yang et al9 have impressively demonstrated the power of in silico modellingfor drug safety analysis. The drug troglitazone was introduced in 1997 as atreatment for type 2 diabetes. It was withdrawn from the market in 2000 after 63
people died from liver failure after taking it. Preclinical studies had shown no danger
to the liver in rats, and trials had not highlighted a significant risk to humans. It was
only when the drug was used in a large enough population that individuals appeared
who were unable to process the drug safely. The Yang paper, published last year,
6789

http://online.liebertpub.com/doi/pdf/10.1089/aivt.2014.1501
http://www.altex.ch/All-issues/Issue.50.html?iid=151&aid=6
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122786
http://onlinelibrary.wiley.com/doi/10.1038/clpt.2014.158/abstract

Lush Prize for Science, 2015 – Research Paper

11has used the Hamner Institutes DILIsym software model of drug-induced liver injury
to model the effects of troglitazone in rat and human populations. The findings werethat the DILIsym model successfully predicted that troglitazone would, very rarely,
cause life-threatening liver injury in humans, and that the accumulation of bile acids
would be the likely cause of such liver injury. The model also predicted that rats
would respond differently to the drug than humans. This study powerfully illustrates
that animal-based toxicity testing cannot be relied upon to predict effectively the risks
to humans, and also demonstrates that modern tt21c methods, such as in silico
modelling, can successfully predict potentially life-threatening toxicities.

Lush Prize for Science, 2015 – Research Paper

126. Toxicity Pathway Abstracts
6.1 Conference Abstract Selection
As described in the Methodology, we reviewed abstracts from the WC9 2014
meeting, from the SoT’s 2015 meeting, and also from the EU SEURAT-1 2015
meeting.
From the 891 abstracts which comprised the WC9 2014 conference presentationand poster proceedings, we identified 37 abstracts which passed through ourselection process to the final (3rd) scoring stage. Of these, 11 scored 1 or more.
From the 2468 SoT abstracts, and based on the Abstract book keyword index, 53
were passed to the scoring stage, of which 3 scored 1 or more. From 11 abstracts
made available to us from the SEURAT-1 meeting all were reviewed, but only 3scored 1 or more.
6.2 Published Abstract Selection
From the PubMed search, we identified 364 relevant titles from the “Toxicity
pathway” search, 1283 from the “Toxicity assay” search, and 345 from the “Toxicity
biomarker” search: a combined total of 1992 articles.
Stages 1 and 2 of the selection process (review of titles and, if necessary abstracts,
to reject review articles, results of clinical trials, articles reporting use of animal
subjects or non-human cell lines, or those on cancer research or overly focussed onother disease), reduced these 1992 titles to just 99 abstracts (38 + 32 + 29 for the 3
separate searches, respectively). These final 99 abstracts were passed to the thirdassessment stage, to be scored as potential prize winners. Of these, 27 scored 1 or
more.
The Google Scholar search identified 355 possibly relevant abstracts, of which only
8 survived selection stages 1 and 2 and were passed on to the scoring stage. Of
these 8, a final 4 scored 1 or more.
The Terkko FeedNavigator search yielded 32 papers, of which we identified 2 novel
papers to pass through to the scoring stage. Of these, 1 scored 1 or more.
We also identified one further relevant project, directly from the Hamner Institutes
website, which scored 1 or more.
6.3 Scores
From the three separate sources of potential shortlisted projects, we thus identified a
total of 50 abstracts describing work which scored at least one point according to ourgiven criteria. Of these, 2 scored 1 for standing out in some way (for example forproviding opportunities for data sharing, or for combining methodologies to give“added value”); 32 scored 2 for bringing new knowledge or tools to a previously
identified pathway, assay, or biomarker of toxicity; 11 scored 2 for bringing new

Lush Prize for Science, 2015 – Research Paper

13knowledge or tools to a previously identified pathway, assay, or biomarker of toxicity
but with an additional 1 (i.e. total 3) because they stood out in some way; and 4scored 3 because they appeared to be reporting a new toxicity pathway, assay, or
biomarker. Only one scored the maximum possible 4 marks with 3 awarded forreporting a new toxicity pathway plus an additional mark for standing out - in thiscase by using a high throughput approach.
The Table lists details (Title, Authors, contribution (pathway, assay, or biomarker),source, and score) of all the abstracts scoring 1 or more. All of the abstracts for
those projects scoring a total of 3 or more are shown in full in Section 6.4.
Title
Paired image- & FACS-based
toxicity assays for HCS of
spheroid-type tumor cell
cultures.
Certain Phase I and II enzymes
as toxicity biomarker: An
overview.
Toxicity mechanisms
identification via gene set
enrichment analysis of timeseries toxicogenomics data:
Impact of time andconcentration.
Systematic proteomic approach
to characterise the impacts ofchemical interactions on protein
and cytotoxicity responses tometal mixture exposures.
Profiling dose-dependent
activation of p53-mediatedsignalling pathways bychemicals with distinct
mechanisms of DNA damage.
Transciptional profiling andbiological pathway analysis ofhuman equivalence PCB
exposure in vitro: Indicator of
disease and disorderdevelopment in humans.
Potential of extracellular
microRNAs as biomarkers ofacetaminophen toxicity inchildren.

Authors
Trumpi et al

Category
Assay

SourceGoogleScholarScore
2

Hassan et al

Biomarker

GoogleScholar2

Gao et al

Pathway/
assay

GoogleScholar2+1

Ge et alPathway

GoogleScholar

2+1

Clewell et al

Pathway

TerkkoFeed
Navigator

3

Ghosh et al

Pathway/
biomarker

PubMed2

Yang et al

Biomarker

PubMed2

Lush Prize for Science, 2015 – Research Paper

14TitleIdentification of novel
biomarkers for doxorubicininduced toxicity in humancardiomycytes derived frompluripotent stem cells.
Identification of lipidomic
biomarkers for coexposure to
subtoxic doses ofbenzo[a]pyrene and cadmium:The toxicological cascade
approach.
OxLDL induces injury anddefenestration of human liver
sinusoidal endothelial cells via
LOX1.
How useful are clinical liverfunction tests in in vitro humanhepatotoxicity assays?
Molecular mechanisms of 3'3dichlorobenzidine-mediatedtoxicity in HepG2 cells.
Development of a pluripotent
stem cell-derived neuronalmodel to identify chemicallyinduced pathway perturbations
in relation to neurotoxicity:
Effects of CREB pathway
inhibition.
Toxicity of silver nanoparticles
to human dermal fibroblasts on
microRNA level.
More than just hormones:
H295R cells as predictors of
reproductive toxicity.
Performance of the N/TERT
epidermal model for skinsensitizer identification via Nrf2Keap1_ARE pathway activation.
Systems biology modelling ofomics data: effect of
cyclosporinA on the Nrf2
pathway in human renal cells.
Silica nanoparticles induce
cytokine activation responses inlung epithelial cells throughactivation of a
p38/TACE/TGFa/EGFRpathway and NfkB signalling.

Authors
Holmgren etalCategory
Biomarker

Source
PubMedScore
2+1

Jungnickel etalBiomarker/
assay

PubMed3

Zhang et al

Pathway

PubMed2

Borlak et al

Assay

PubMed2

Chen et al

Pathway

PubMed2

Pistollato et al Pathway/
model

PubMed1

Huang et al

Pathway

PubMed2Maglich et al

Assay

PubMed3

AlloulRamdhani etal

Assay/
model

PubMed2

Hamon et al

Modelling/
predictive

PubMed2+1

Skuland et al

Pathway

PubMed2

Lush Prize for Science, 2015 – Research Paper

15TitleEvaluation of cytotoxicity and
DNA damage response withanalysis of intracellular ATM
signalling pathways.
The acquisition of cancer stem
cell-like properties andneoplastic transformation ofhuman keratinocytes induced byarsenite involves epigenetic
silencing of let-7c via
Ras/NFkB.
Replication stress by Py-lm
polyamides induces a noncanonical ATR-dependent
checkpoint response.
Modulation of the DNA repair
system and ATR-p53-mediatedapoptosis is relevant fortributyltin-induced genotoxic
effects in human hepatoma G2
cells.
Induction of glutathione
synthesis in human hepatocytes
by acute and chronic arsenic
exposure: Differential roles of
mitogen-activated protein
kinases.
AP-1 activation attenuates thearsenite-induced apoptotic
response in human bronchial
epithelial cells by up-regulating
HO-1 expression.
MicroRNA regulation of DNA
repair gene expression in 4aminobiphenyl-treated HepG2
cells.
Topological, functional, anddynamic properties of theprotein interaction networks
rewired by benzo(a)pyrene.
Heavy metal-induced
metallothionein expression isregulated by specific protein
phosphatase 2A complexes.
Evaluation of the MultiImmunoToxAssay composed of
3 human cytokine reporter cellsby examining immunologicaleffects of drugs.

Authors
Bandi et al

Category
Assay

Source
PubMedScore
2

Jiang et al

Pathway

PubMed2

Martinez et al

Pathway

PubMed2

Li et alPathway

PubMed2

Hou et al

Pathway

PubMed2

Aodengqimuge et al

Pathway

PubMed2

Huan et al

Pathway

PubMed2Ba et alPathway

PubMed2+1

Chen et al

Pathway

PubMed2

Kimura et al

Assay

PubMed3

Lush Prize for Science, 2015 – Research Paper

16TitleThe low molecular weight DNA
diffusion assay as an indicatorof cytotoxicity for the in vitro
comet assay.
A cell-based biosensor system
HepG2CDKN1A-DsRed for
rapid and simple detection of
genotoxic agents.
Identification of microRNAs
involved in growth arrest andcell death in hydrogen peroxidetreated human dermal papillacells.
Body-on-a-chip simulation withgastrointestinal tract and livertissues suggests that ingested
nanoparticles have the potential
to cause liver injury.
Systems pharmacology
modelling predicts delayed
presentation and species
differences in bile acid-mediatedtroglitazone hepatotoxicity.
Novel computational
approaches for high content
image analyses (HCA) oforganoid 3D neurospherecultures in vitro.
Human beating heart-on-a-chip
for cardiotoxicity testing as an
example case of the Dutchorgan-on-a-chip initiative.
Neurotoxicity in vitro:
Assessment of the predictivity ofneuronal networks coped [sic] tomicroelectrode arrays for
identification of neurotoxicants.
LUHMES 3D neuronal model for(developmental) neurotoxicity
testing.
Using high-content imaging toanalyze cell-state trajectories
and biological tipping points forchemical exposures.

Authors
Speit et al

Category
Assay

Source
PubMedScore
2

Blagus et al

Assay

PubMed2

Kim et al

Pathway

PubMed2

Esch et al

Assay

PubMed2+1

Yang et al

Pathway

HamnerInstitutes
website2+1

Fritsche et al

Assay

WC9

2

Van de Stolpe Assay& van denEijndenWC92

Ramirez et al

Assay

WC9

2+1

Smirnova et
al

Assay

WC9

2+1

Shah

Assay/
pathwayWC93+1

Lush Prize for Science, 2015 – Research Paper

17TitleIdentification of cornifelin andearly gene response-1 as new
biomarkers for in vitro eye
irritation using a 3D
reconstructed human cornea
model, MCTT HCE™.
C24:1-ceramide may be a novel
lipid biomarker for eye irritationin 3D human corneal epithelial
model, MCTT HCE™.
SDF-1 induced migration of
THP-1 cells through amonolayer of human coronary
artery endothelial cells
promotes differentiation of
monocytes to an adhesive
phenotype.
Development of EYEIRR-IS®,an toxicogenomic assay using
the Skinethic HCE model forevaluating chemical ocular
irritation potency.
New biomarkers for endocrine
disruption evaluation on
microplate using a humanplacental cell line.
Nano aerosol chamber for
realistic in vitro toxicity studies –NACIVT.
Prediction of Chemical
Respiratory Sensitizers Using
GARD, a Novel In Vitro Assay
Based on a Genomic Biomarker
Signature.
Organelle Imaging Toxicology:
Novel Analysis of the Sandwich
High-Content Screening Project.
Activation of the NLRP3Inflammasome As a Potential
Biomarker of Idiosyncratic Drug
Reactions.
A HepaRG 3D system forstudying cholestatic drug
hepatotoxicity.
Repeated dose toxicity ofvalproic acid in a HepaRG 3D
system.

Authors
Choi et al

Category
Biomarker

Source
WC9

Score
2

Lee et al

Biomarker

WC92

Van derToorn et al

Assay

WC9

2

Cottrez et al

Assay/
biomarker

WC92

Wakx et al

Assay/
biomarker

WC92

Jeannet et al

Assay

WC9

2

Forreryd et al

Assay

SoT 2015

2

Irwin &
LiccioneAssay

SoT 2015

2

Weston &Uetrecht

Biomarker

SoT 2015

2

Fredriksson
et al

Pathway

SEURAT-1
2015

2+1

Fredriksson
et alModel/
assay

SEURAT-1
20151

Lush Prize for Science, 2015 – Research Paper

18

TitleHepatic toxicity evaluation using
oxygen-based phosphorescentparticles and microfluidicsbased glucose and lactate
sensors.

Authors
Bavli et al

Category
Pathway/model

SourceSEURAT-1
2015

Score
2+1

6.4 Nominated Abstracts
This year there were only 5 projects which received the highest scores of either 3 or
3+1 for reporting new pathways, assays, or biomarkers of toxicity. A further 11
abstracts received a score of 2+1. Since the scope of the selection process has
been expanded this year, it seems appropriate to include those abstracts that scored
2+1 as they do stand out as making significant contributions and advances to theadoption of non-animal testing protocols. The 16 abstracts are given below. Due tospace constraints, we have given the affiliations of the first author only, althoughmany of these projects were collaborative efforts between several partners. For
abstracts from published work, we provide the digital object identifier (DOI) whichallows the original document to be located online. For conference abstracts, we givethe abstract or poster number for identification.
We consider all worthy of being considered by the judges as potential prize winners.
6.4.1 Using high-content imaging to analyze cell-state trajectories andbiological tipping points for chemical exposures
I. ShahNational Center for Computational Toxicology, US EPA, Research Triangle Park,
USA. shah.imran@epa.gov
WC9 Abstract I-6-875
Score 3+1 (3 for discovery of low concentration 'tipping points', +1 for using highthroughput approach)
Abstract
Translating results obtained from high-throughput screening to risk assessment isvital for reducing dependence on animal testing. We studied the effects of 976
chemicals (ToxCast Phase I and II) in HepG2 cells using high-content imaging (HCI)
to measure dose- and time-dependent perturbations in p53, JNK, oxidative stress,
cytoskeleton, mitochondria, and cell cycle. A novel computational model wasdeveloped to describe the dynamic response of the system as cell-state trajectories
based on multidimensional HCI datastreams. Cell-state trajectories produced by 10
concentrations (0.4 to 200 µM) of 976 chemicals showed resilience of the HepG2
system in many cases, however, we also found “tipping points” in system recovery.
Further analysis of trajectories identified dose-dependent transitions, or criticalpoints, in system recovery for 340/976 chemicals. The critical concentration was
generally 5-times lower than the concentration that produced 50% cell loss. We
believe that HCI can be used to reconstruct cell state trajectories, and provide insight
into adaptation and resilience for in vitro systems. With additional research, cellular

Lush Prize for Science, 2015 – Research Paper

19tipping points could be used to define an in vitro point of departure (PoD) for riskbased prioritization of environmental chemicals.
6.4.2 Profiling Dose-Dependent Activation of p53-Mediated Signaling
Pathways by Chemicals with Distinct Mechanisms of DNA Damage
R A. Clewell, Bin Sun, Y Adeleye, P Carmichael, A Efremenko, P D. McMullen, S
Pendse, O. J. Trask, A White, M E. Andersen.
The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina
27709. rclewell@thehamner.org
Toxicol. Sci. (November 2014) 142 (1): 56-73. doi: 10.1093/toxsci/kfu153
Score 3 (Pathway)
Abstract
As part of a larger effort to provide proof-of-concept in vitro-only risk assessments,
we have developed a suite of high-throughput assays for key readouts in the p53
DNA damage response toxicity pathway: double-strand break DNA damage (pH2AX), permanent chromosomal damage (micronuclei), p53 activation, p53transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Doseresponse studies were performed with these protein and cell fate assays, together
with whole genome transcriptomics, for three prototype chemicals: etoposide,
quercetin, and methyl methanesulfonate. Data were collected in a human cell line
expressing wild-type p53 (HT1080) and results were confirmed in a second p53
competent cell line (HCT 116). At chemical concentrations causing similar increases
in p53 protein expression, p53-mediated protein expression and cellular processes
showed substantial chemical-specific differences. These chemical-specificdifferences in the p53 transcriptional response appear to be determined byaugmentation of the p53 response by co-regulators. More importantly, doseresponse data for each of the chemicals indicate that the p53 transcriptional
response does not prevent micronuclei induction at low concentrations. In fact, theno observed effect levels and benchmark doses for micronuclei induction were lessthan or equal to those for p53-mediated gene transcription regardless of the test
chemical, indicating that p53's post-translational responses may be more important
than transcriptional activation in the response to low dose DNA damage. This effort
demonstrates the process of defining key assays required for a pathway-based, invitro-only risk assessment, using the p53-mediated DNA damage response pathway
as a prototype.
6.4.3 Identification of lipidomic biomarkers for coexposure to subtoxic dosesof benzo[a]pyrene and cadmium: the toxicological cascade biomarker
approach
H Jungnickel, S Potratz, S Baumann, P Tarnow, M von Bergen, A Luch.
Department of Product Safety, German Federal Institute for Risk Assessment (BfR) ,Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany
Environ Sci Technol. 2014 Sep 2;48(17):10423-31. doi: 10.1021/es502419w
Score 3 (Biomarker)
Abstract

Lush Prize for Science, 2015 – Research Paper

20The search for model bioassay systems indicating activation of different toxicologicalsignaling pathways is one of the paramount goals of modern toxicology. Especially
coexposure scenarios need to be investigated with respect to synergistic andinterdependent effects for the activation of toxicological signaling pathways. The
present study introduces an experimental in vitro model system for nontoxic and lowdose coexposures of human mammary carcinoma MCF-7 cells against polycyclicaromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP) and heavy metals suchas cadmium. For the first time, a multivariate model that identifies 18 metabolic
biomarkers has been shown to be sufficient to separate BP-treated cells from
coexposed or control cells. A "toxicological pathway color code model" is introduced
to visualize the results. Different biomarker subsets can be associated with specific
HER2 signaling steps. A tiered cascade biomarker approach is proposed that could
be used to identify profiles associated with tumorigenic potency of environmental
toxicants in coexposure scenarios, including possible synergistic or additive effects.
6.4.4 More than just hormones: H295R cells as predictors of reproductive
toxicity
JM Maglich, M Kuhn, RE Chapin, MT Pletcher.
Pfizer Global Research Development, Pfizer Inc., Cambridge, MA 02420, UnitedStates. Mathew.Pletcher@pfizer.com.
Reprod Toxicol. 2014 Jun;45:77-86. doi: 10.1016/j.reprotox.2013.12.009
Score 3 (Assay)
Abstract
Many of the commonly observed reproductive toxicities associated with therapeutic
compounds can be traced to a disruption of the steroidogenic pathway. We sought todevelop an in vitro assay that would predict reproductive toxicity and be highthroughput in nature. H295R cells, previously validated as having an intact andfunctional steroidogenic pathway, were treated with 83 known-positive and 79
known-negative proprietary and public-domain compounds. The assay measured theexpression of the key enzymes STAR, 3βHSD2, CYP17A1, CYP11B2, CYP19A1,
CYP21A2, and CYP11A1 and the hormones DHEA, progesterone, testosterone, andcortisol. We found that a Random Forest model yielded a receiver operating
characteristic area under the curve (ROC AUC) of 0.845, with sensitivity of 0.724
and specificity of 0.758 for predicting in vivo reproductive toxicity with this in vitro
assay system.
6.4.5 Evaluation of the Multi-ImmunoTox Assay composed of 3 humancytokine reporter cells by examining immunological effects of drugs
Y Kimura, C Fujimura, Y Ito, T Takahashi, S Aiba.
Department of Dermatology, Tohoku University Graduate School of Medicine,
Sendai 980-8574, Japan. saiba@med.tohoku.ac.jp
Toxicol In Vitro. 2014 Aug;28(5):759-68. doi: 10.1016/j.tiv.2014.02.013
Score 3 (Assay)
Abstract
We established a luciferase reporter assay system, the Multi-ImmunoTox Assay(MITA), to evaluate the effects on key predictivein vitro components of the human

Lush Prize for Science, 2015 – Research Paper

21

immune system. The system is composed of 3 stable reporter cell lines transfectedwith 3 luciferase genes, SLG, SLO, and SLR, under the control of 4 cytokine
promoters, IL-2, IFN-γ, IL-1β, and IL-8, and the G3PDH promoter. We first compared
the effects of dexamethasone, cyclosporine, and tacrolimus on these cell lines
stimulated with phorbol 12-myristate 13-acetate and ionomycin, or
lipopolysaccharides, with those on mRNA expression by the mother cell lines andhuman whole blood cells after stimulation. The results demonstrated that MITA
correctly reflected the change of mRNA of the mother cell lines and whole blood
cells. Next, we evaluated other immunosuppressive drugs, off-label
immunosuppressive drugs, and non-immunomodulatory drugs. Although MITA did
not detect immunosuppressive effects of either alkylating agents or antimetabolites,
it could demonstrate those of the off-label immunosuppressive drugs, sulfasalazine,
chloroquine, minocycline, and nicotinamide. Compared with the publishedimmunological effects of the drugs, these data suggest that MITA can present a
novel high-throughput approach to detect immunological effects of chemicals otherthan those that induce immunosuppressive effects through their inhibitory action oncell division.
6.4.6 Identification of novel biomarkers for doxorubicin-induced toxicity inhuman cardiomyocytes derived from pluripotent stem cells
G Holmgren, J Synnergren, Y Bogestål, C Améen, K Åkesson, S Holmgren, A
Lindahl, P Sartipy.
Systems Biology Research Center, School of Bioscience, University of Skövde, Box408, Kanikegränd 3A, SE-541 28 Skövde, Sweden. gustav.holmgren@his.se
Toxicology. 2015 Feb 3;328:102-11. doi: 10.1016/j.tox.2014.12.018
Score 2+1 (Biomarker + use of stem cells)
Abstract
Doxorubicin is a chemotherapeutic agent indicated for the treatment of a variety ofcancer types, including leukaemia, lymphomas, and many solid tumours. The use of
doxorubicin is, however, associated with severe cardiotoxicity, often resulting in early
discontinuation of the treatment. Importantly, the toxic symptoms can occur several
years after the termination of the doxorubicin administration. In this study, the toxic
effects of doxorubicin exposure have been investigated in cardiomyocytes derived
from human embryonic stem cells (hESC). The cells were exposed to different
concentrations of doxorubicin for up to 2 days, followed by a 12 day recovery period.
Notably, the cell morphology was altered during drug treatment and the cells showeda reduced contractile ability, most prominent at the highest concentration of
doxorubicin at the later time points. A general cytotoxic response measured as
Lactate dehydrogenase leakage was observed after 2 days' exposure compared to
the vehicle control, but this response was absent during the recovery period. A
similar dose-dependant pattern was observed for the release of cardiac specifictroponin T (cTnT) after 1 day and 2 days of treatment with doxorubicin. Global
transcriptional profiles in the cells revealed clusters of genes that were differentiallyexpressed during doxorubicin exposure, a pattern that in some cases was sustainedeven throughout the recovery period, suggesting that these genes could be used assensitive biomarkers for doxorubicin-induced toxicity in human cardiomyocytes. The
results from this study show that cTnT release can be used as a measurement of
acute cardiotoxicity due to doxorubicin. However, for the late onset of doxorubicin-

Lush Prize for Science, 2015 – Research Paper

22induced cardiomyopathy, cTnT release might not be the most optimal biomarker. As
an alternative, some of the genes that we identified as differentially expressed after
doxorubicin exposure could serve as more relevant biomarkers, and may also help
to explain the cellular mechanisms behind the late onset apoptosis associated withdoxorubicin-induced cardiomyopathy.
6.4.7 Systems biology modeling of omics data: effect of cyclosporine a on the
Nrf2 pathway in human renal cells
J Hamon, P Jennings, FY Bois
Mathematical Modeling for Systems Toxicology, Université de Technologie deCompiègne, BP 20529, 60205 Compiègne Cedex, France. frederic.bois@utc.fr.
BMC Syst Biol. 2014 Jun 25;8:76. doi: 10.1186/1752-0509-8-76
Score 2+1 (Pathway model + predictive modelling)
Abstract
BACKGROUND:
Incorporation of omic data streams for building improved systems biology models
has great potential for improving their predictions of biological outcomes. We haverecently shown that cyclosporine A (CsA) strongly activates the nuclear factor(erythroid-derived 2)-like 2 pathway (Nrf2) in renal proximal tubular epithelial cells(RPTECs) exposed in vitro. We present here a quantitative calibration of adifferential equation model of the Nrf2 pathway with a subset of the omics data wecollected.
RESULTS:In vitro pharmacokinetic data on CsA exchange between cells, culture medium andvial walls, and data on the time course of omics markers in response to CsA
exposure were reasonably well fitted with a coupled PK-systems biology model.
Posterior statistical distributions of the model parameter values were obtained byMarkov chain Monte Carlo sampling in a Bayesian framework. A complex cyclic
pattern of ROS production and control emerged at 5 µM CsA repeated exposure.
Plateau responses were found at 15 µM exposures. Shortly above those exposurelevels, the model predicts a disproportionate increase in cellular ROS quantity whichis consistent with an in vitro EC50 of about 40 µM for CsA in RPTECs.
CONCLUSIONS:
The model proposed can be used to analyze and predict cellular response tooxidative stress, provided sufficient data to set its parameters to cell-specific values.
Omics data can be used to that effect in a Bayesian statistical framework which
retains prior information about the likely parameter values.
6.4.8 Topological, functional, and dynamic properties of the protein
interaction networks rewired by benzo(a)pyrene
Q Ba, J Li, C Huang, J Li, R Chu, Y Wu, H Wang
Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
huiwang@sibs.ac.cn.
Toxicol Appl Pharmacol. 2015 Mar 1;283(2):83-91. doi: 10.1016/j.taap.2015.01.006
Score 2+1 (Pathway + novel approach)Lush Prize for Science, 2015 – Research Paper

23

Abstract
Benzo(a)pyrene is a common environmental and foodborne pollutant that has been
identified as a human carcinogen. Although the carcinogenicity of benzo(a)pyrene
has been extensively reported, its precise molecular mechanisms and the influenceon system-level protein networks are not well understood. To investigate the systemlevel influence of benzo(a)pyrene on protein interactions and regulatory networks, abenzo(a)pyrene-rewired protein interaction network was constructed based on 769
key proteins derived from more than 500 literature reports. The protein interaction
network rewired by benzo(a)pyrene was a scale-free, highly-connected biologicalsystem. Ten modules were identified, and 25 signaling pathways were enriched,most of which belong to the human diseases category, especially cancer andinfectious disease. In addition, two lung-specific and two liver-specific pathways
were identified. Three pathways were specific in short and medium-term networks(<48h), and five pathways were enriched only in the medium-term network (6h-48h).
Finally, the expression of linker genes in the network was validated by Western
blotting. These findings establish the overall, tissue- and time-specificbenzo(a)pyrene-rewired protein interaction networks and provide insights into the
biological effects and molecular mechanisms of action of benzo(a)pyrene.
6.4.9 Body-on-a-chip simulation with gastrointestinal tract and liver tissues
suggests that ingested nanoparticles have the potential to cause liver injury
MB Esch, GJ Mahler, T Stokol, ML Shuler.
Department of Biomedical Engineering, 305 Weill Hall, Cornell University, Ithaca, NY
14853, USA. mls50@cornell.edu.
Lab Chip. 2014 Aug 21;14(16):3081-92. doi: 10.1039/c4lc00371c
Score 2+1 (Assay + microfluidics)
Abstract
The use of nanoparticles in medical applications is highly anticipated, and at thesame time little is known about how these nanoparticles affect human tissues. Here
we have simulated the oral uptake of 50 nm carboxylated polystyrene nanoparticles
with a microscale body-on-a-chip system (also referred to as multi-tissue
microphysiological system or micro Cell Culture Analog). Using the 'GI tract-liverother tissues' system allowed us to observe compounding effects and detect livertissue injury at lower nanoparticle concentrations than was expected fromexperiments with single tissues. To construct this system, we combined in vitro
models of the human intestinal epithelium, represented by a co-culture ofenterocytes (Caco-2) and mucin-producing cells (TH29-MTX), and the liver,
represented by HepG2/C3A cells, within one microfluidic device. The device also
contained chambers that together represented the liquid portions of all other organsof the human body. Measuring the transport of 50 nm carboxylated polystyrene
nanoparticles across the Caco-2/HT29-MTX co-culture, we found that this multi-celllayer presents an effective barrier to 90.5 ± 2.9% of the nanoparticles. Further, oursimulation suggests that a larger fraction of the 9.5 ± 2.9% nanoparticles thattravelled across the Caco-2/HT29-MTX cell layer were not large nanoparticle
aggregates, but primarily single nanoparticles and small aggregates. After crossing
the GI tract epithelium, nanoparticles that were administered in high doses estimatedin terms of possible daily human consumption (240 and 480 × 10(11) nanoparticles
mL(-1)) induced the release of aspartate aminotransferase (AST), an intracellular

Lush Prize for Science, 2015 – Research Paper

24

enzyme of the liver that indicates liver cell injury. Our results indicate that body-on-achip devices are highly relevant in vitro models for evaluating nanoparticle
interactions with human tissues.
6.4.10 Toxicity Mechanisms Identification via Gene Set Enrichment Analysis ofTime-Series Toxicogenomics Data: Impact of Time and Concentration
C Gao, D Weisman, J Lan, N Gou, AZ Gu.
Department of Civil and Environmental Engineering, Northeastern University,
Boston, Massachusetts 02115, United States. april@coe.neu.edu
Environ. Sci. Technol., 2015, 49 (7), pp 4618–4626. DOI: 10.1021/es505199f
Score 2+1 (Pathway + approach)
Abstract
The advance in high-throughput “toxicogenomics” technologies, which allows forconcurrent monitoring of cellular responses globally upon exposure to chemical
toxicants, presents promises for next-generation toxicity assessment. It is
recognized that cellular responses to toxicants have a highly dynamic nature, andexhibit both temporal complexity and dose-response shifts. Most current geneenrichment or pathway analysis lack the recognition of the inherent correlation withintime series data, and may potentially miss important pathways or yield biased andinconsistent results that ignore dynamic patterns and time-sensitivity. In this study,
we investigated the application of two score metrics for GSEA (gene set enrichment
analysis) to rank the genes that consider the temporal gene expression profile. One
applies a novel time series CPCA (common principal components analysis) togenerate scores for genes based on their contributions to the common temporalvariation among treatments for a given chemical at different concentrations. Anotherone employs an integrated altered gene expression quantifier-TELI (transcriptional
effect level index) that integrates altered gene expression magnitude over the
exposure time. By comparing the GSEA results using two different ranking metrics
for examining the dynamic responses of reporter cells treated with various doselevels of three model toxicants, mitomycin C, hydrogen peroxide, and lead nitrate,
the analysis identified and revealed different toxicity mechanisms of these chemicals
that exhibit chemical-specific, as well as time-aware and dose-sensitive nature. The
ability, advantages, and disadvantages of varying ranking metrics were discussed.
These findings support the notion that toxicity bioassays should account for the cells’
complex dynamic responses, thereby implying that both data acquisition and data
analysis should look beyond simple traditional end point responses.
6.4.11 Systematic Proteomic Approach to Characterize the Impacts ofChemical Interactions on Protein and Cytotoxicity Responses to Metal Mixture
Exposures
Y Ge, M Bruno, K Wallace, S Leavitt, D Andrews, M A Spassova, M Xi, A Roy, N
Haykal-Coates, W Lefew, A Swank, W M Winnik, C Chen, J Woodard, A Farraj, K Y
Teichman, J A Ross.
National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, 109 T. W. Alexander Drive, Research Triangle Park, North
Carolina 27709, United States. ge.yue@epa.gov
J. Proteome Res., 2015, 14 (1), pp 183–192. DOI: 10.1021/pr500795d

Lush Prize for Science, 2015 – Research Paper

25

Score 2+1 (Pathway + proteomics)
Abstract
Chemical interactions have posed a big challenge in toxicity characterization andhuman health risk assessment of environmental mixtures. To characterize theimpacts of chemical interactions on protein and cytotoxicity responses toenvironmental mixtures, we established a systems biology approach integrating
proteomics, bioinformatics, statistics, and computational toxicology to measure
expression or phosphorylation levels of 21 critical toxicity pathway regulators and445 downstream proteins in human BEAS-2B cells treated with 4 concentrations ofnickel, 2 concentrations each of cadmium and chromium, as well as 12 definedbinary and 8 defined ternary mixtures of these metals in vitro. Multivariate statisticalanalysis and mathematical modeling of the metal-mediated proteomic response
patterns showed a high correlation between changes in protein expression or
phosphorylation and cellular toxic responses to both individual metals and metal
mixtures. Of the identified correlated proteins, only a small set of proteins including
HIF-1α is likely to be responsible for selective cytotoxic responses to different metals
and metals mixtures. Furthermore, support vector machine learning was utilized tocomputationally predict protein responses to uncharacterized metal mixtures using
experimentally generated protein response profiles corresponding to known metal
mixtures. This study provides a novel proteomic approach for characterization andprediction of toxicities of metal and other chemical mixtures.
6.4.12 Systems Pharmacology Modeling Predicts Delayed Presentation andSpecies Differences in Bile Acid–Mediated Troglitazone Hepatotoxicity
K Yang, J L Woodhead, P B Watkins, B A Howell, K L R Brouwer
Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman
School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina, USA. kbrouwer@unc.edu
Clinical Pharmacology & Therapeutics (2014); 96 5, 589–598.
doi:10.1038/clpt.2014.158
Score 2+1 (Pathway + importance)
Abstract
Troglitazone (TGZ) causes delayed, life-threatening drug-induced liver injury in somepatients but was not hepatotoxic in rats. This study investigated altered bile acidhomeostasis as a mechanism of TGZ hepatotoxicity using a systems pharmacology
model incorporating drug/metabolite disposition, bile acid
physiology/pathophysiology, hepatocyte life cycle, and liver injury biomarkers. In thesimulated human population, TGZ (200–600  mg/day × 6 months) resulted in delayed
increases in serum alanine transaminase >3× the upper limit of normal in 0.3–5.1%,
with concomitant bilirubin elevations >2× the upper limit of normal in 0.3–3.6%, of the
population. By contrast, pioglitazone (15–45  mg/day × 6 months) did not elicit
hepatotoxicity, consistent with clinical data. TGZ was not hepatotoxic in thesimulated rat population. In summary, mechanistic modeling based only on bile acideffects accurately predicted the incidence, delayed presentation, and species
differences in TGZ hepatotoxicity, in addition to predicting the relative liver safety of
pioglitazone. Systems pharmacology models integrating physiology andLush Prize for Science, 2015 – Research Paper

26experimental data can evaluate drug-induced liver injury mechanisms and may be
useful to predict the hepatotoxic potential of drug candidates.
6.4.13 Neurotoxicity in vitro: assessment of the predictivity of neuronal
networks coped [sic] to microelectrode arrays for identification ofneurotoxicants
T. Ramirez, T. Weisschu, H. A. Huener, R. Landsiedel, B. van Ravenzwaay
Experimental Toxicology and Ecology, BASF SE, Ludwigshafen am Rhein,
Germany. tzutzuy.ramirez-hernandez@basf.com
WC9 Abstract I-3-389
Score 2+1 (Assay + importance)
Abstract
A challenging aspect to assure the safety of a product is the assessment of its
neurotoxic hazard potential. Currently, only in vivo methods are regulatorily acceptedand so far, no in vitro model has been fully validated. With the advance intechnology and the ability to maintain primary neuronal models for prolonged
periods, a promising test system emerged, combining the use of microelectrodearrays (MEAs) and in vitro culture of 2D neuronal networks (NN). In the presentation,
we report on the in-house validation of the NN MEA assay using a set of 58
compounds of different chemical classes with known neurotoxic and non-neurotoxic
potential with the aim to use it for screening of compounds under development andin the future for its potential application in the regulatory framework. The results
demonstrate that the method presents a good sensitivity of 81% and accuracy above
75%. In order to increase the rate of false predictions we also report in the new
approach integrating the electrophysiological assessment with a panel of cytotoxicity
assays.
6.4.14 LUHMES 3D neuronal model for (developmental) neurotoxicity testing
L. Smirnova, G. Harris, H. Hogberg, D. Pamies, L. Zhao, R. Bachinski, J. Bressler, J.
Delp, M. Leist, T. HartungCenter for Alternative to Animal Experiments (CAAT), the Johns Hopkins University,
Bloomberg School of Public Health, Baltimore, USA. lsmirno1@jhu.edu
WC9 Abstract I-4-803
Score 2+1 (Assay + importance)
Abstract
Broader testing of substances is crucial to respond to surging neurodevelopmental
problems in children, including autism and ADHD. Therefore, our principal goal is todevelop a human relevant, quality-assured, medium- to high-throughput testing
strategy to prioritize chemicals for (developmental) neurotoxicity testing. This work
aims to develop a 3D brain model to identify changes in transcriptome, microRNA
expression, and metabolome after exposure to neurotoxicants. We developed suchmodel based on well characterized LUHMES dopaminergic neuroprogenitor cell line
which can be used to test compounds for neuro(developmental) toxicity effects andmodel chemically induced neuronal disorders (Schildknecht et al., 2013; Scholz etal., 2011). The size of differentiating spheroids, penetration rate and cell death weremonitored up to day 21 of differentiation, to ensure delivery of medium supplements

Lush Prize for Science, 2015 – Research Paper27

to the center of the spheroids. The efficiency of differentiation in 3D wascharacterized by qRT-PCR, flow cytometry and immunocytochemistry. To
demonstrate the modelÅs suitability for neurotoxicity testing, the aggregates weretreated for 24 and 48 hours on day 6 of differentiation with dopaminergic
neurotoxicants, MPP+ and rotenone. Perturbations in energy metabolism and stressresponse were analyzed by gene expression and miRNA profiling. In conclusion, we
have successfully developed a new 3D human model that can be used for(developmental) neurotoxicity testing.
References
Schildknecht, S., Karreman, C., Pöltl, D. et al. (2013). ALTEX 30, 427-444. Scholz,D., Pöltl, D., Genewsky, A. et al. (2011). J Neurochem 119, 957-971.
6.4.15 A HepaRG 3D system for studying cholestatic drug hepatotoxicity
L Fredriksson, D Hendriks, I Johansson, M Ingelman-Sundberg
Section of Pharmacogenetics, Department of Physiology and Pharmacology,
Karolinska Institutet, Stockholm, Sweden. lisa.fredriksson@ki.se
SEURAT-1 2015 Poster 1
Score 2+1 (Pathway + 3D model)
Abstract
Drug-induced cholestasis is the most common cause of fulminant hepatic failure andit would therefore be advantageous to identify this injury at an earlier stage of drug
development. Here we present a 3 dimensional organotypic spheroid model, basedon human HepaRG cells. We show a significant increase in toxicity of cholestatic
compounds chlorpromazine (CPZ), troglitazone and bosentan when co-exposed toan otherwise none-toxic concentrated mix of human bile acids. Importantly, thisenhanced toxicity could not be seen with the non-cholestatic, although hepatotoxic,
compounds paracetamol and tetracycline. Further mechanistic investigation of theenhanced toxicity seen with CPZ and bile acids revealed a role for oxidative stressinduction. This was determined by using a fluorescent probe for oxidative stress in
the intact spheroid as well as by an up-regulation of the oxidative stress-responsive
transcription factor nuclear factor (erythroid-derived 2)-like 2 (NFE2L2; Nrf2) and its
target gene sulfiredoxin-1 (SRXN1) on RNA level. In addition, an observed downregulation of the bile-salt export pump (BSEP) after exposure to this drug with andwithout bile acids can be presented as a possible mechanism to the enhanced
toxicity. This was supported by the observed accumulation of a fluorescent bile acid,
which transport is dependent on BSEP, after exposure to CPZ. In conclusion wehere present an organotypic hepatotoxicity model, based on drug/bile acid coexposures, which is able to distinguish cholestatic from non-cholestatic compounds
and which constitute a promising basis for further mechanistic studies of this effect.
6.4.16 Hepatic Toxicity Evaluation Using Oxygen-based PhosphorescentParticles and Microfluidics-based Glucose and Lactate sensors
D Bavli, S Prill, G Levy, E Ezra, M Jaeger, Y Nahmias
MicroLiver Technologies Laboratory, Alexander Grass Center for Bioengineering,
The Hebrew University of Jerusalem, Israel. danny.bavli@mail.huji.ac.il
SEURAT-1 2015 Poster 8
Score 2+1 (Pathway + fluidics)

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Abstract
The HeMiBio consortium aims to engineer a liver-mimicking microchip, capable offunctioning for over 28 days in vitro, for repeated-dose toxicity study ofpharmaceutical agents and cosmetic compounds. The over goal is to replace the useof animals as an industry standard. Current methods to detect hepatotoxicity utilize
large numbers of cells and measure viability at multiple time points. Our approach is
to confine minute population of cells in microfluidic chips, and continuously monitor
their function over time. Microfluidic liver-on-chip devices offer an alternative foranimal experiments as they can mimic the native microenvironment and support
long-term function under continuous perfusion. Continuous flow enables stable dose
exposure of the cells over time, eliminating the rapid loss of signal due to nonspecific adsorption and metabolism that characterizes both static in vitro assays andin vivo experiments. Here we report the fabrication and validation of a microfluidic
liver bioreactor capable of maintaining metabolically active HepG2 cells for over 28
days in vitro. The cell display epithelial polarization and high CYP450 expression forthe period. Cell viability is continuously monitored automatically in real time using anovel on-chip frequency-based luminescence-quenching oxygen probe seeded
within the self-assembled tissue and integrated off-chip glucose and lactate sensors.
Continuous electrochemical monitoring of glucose and lactate levels provides
additional means of detecting the shift from oxidative phosphorylation to glycolysis inconjunction with oxygen consumption, permitting the unique real-time detection ofminute mitochondrial damage. Using the system developed we were able to detect a
new mechanism of CYP2E1-independent acetaminophen toxicity involving direct
mitochondrial damage. We were also able to show the toxicity modes of action ofother drugs such as the anti-diabetic drug, troglitazone, insecticide rotenone and theantiarrhythmic agent, amiodarone, caused a time-delayed dose-dependent responsewith a TC50 of 285 µM, 12.5 µM and 425 µM respectively. The microfluidic systemprovides major benefits including reduction of required cell number, enhancement ofefficiency by means of multiplexed screening, and providing the ability to maintain
optimum culture conditions as required for long-term studies.

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7. Conclusions
The review of the most recent World Congress, SoT, and SEURAT-1 conference
proceedings, and an extensive literature search, yielded 16 abstracts describing
projects which we consider to be potential candidates for the Lush Prize for Science.
These are given in Section 6.
This year, the scope of the search for candidates for the Lush Prize for Science has
been expanded to new assays and biomarkers of toxicity, in addition to pathway
discovery. Although we reviewed many more papers and abstracts than in 2014
(over 2,200 this year compared with over 1,000 in 2014), the number that received ascore was slightly lower than in 2014. In particular, we were surprised that so few
SoT abstracts met the Lush Prize criteria (53 out of 2468, based on the SoT Abstract
book keyword index). It was also disappointing that a large proportion of these 53
abstracts described whole animal studies. Perhaps the Lush Prize organisation
should consider lobbying the Society of Toxicology, and its conference attendees, atthe next SoT meeting (in New Orleans in March 2016).
The nominated abstracts are very diverse, and cover 3D and microfluidics models for
toxicity assays, high-throughput- and high content- screening, in silico analyses, anddemonstrations of the utility of modern tt21d approaches. We believe that they are
all worthy candidates for the 2015 Science Prize.

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