Theme II – Predictive Toxicology

100 Pages·2014·1.55 MB·English

Theme II, SeSSIon II-1a
Theme II – Predictive Toxicology
Coordinators
Nathalie Alépée, L’Oréal, Aulnay-sous-Bois, France
Kevin Crofton, US EPA, Research Triangle Park, NC, USA
Rodger Curren, IIVS, Gaithersburg, MD, USA
Carl Westmoreland, Unilever, Sharnbrook, UK
Session II-1a: Pathways approaches in toxicology
Co-chairs
Kim Boekelheide, Brown University, USA
Maurice Whelan, EURL ECVAM, JRC, Italy
Session II-1a: Oral presentations
II-1a-123 working to provide practical examples of how the tt21C vision can
Pathway activation from be implemented to facilitate chemical safety assessments for several
toxicity pathways, including the DNA damage response pathway. the
tissue samples as a predictor
project studies p53-mediated DNA damage stress response in human
of in vivo toxicity profile cells to determine the response circuitry and the dose-response behav-
iour for pathway activation following chemical induced DNA damage.
W. Buck and M. Liguori this research has three overarching goals: (1) map the key determi-
nants of cellular fate following DNA damage, (2) identify dose-depen-
Research and Development, AbbVie, North Chicago, USA
dent thresholds associated with adaptation and toxicity (clastogenic-
[email protected] ity) and (3) perform a safety assessment based on predicted regions
of safety. Initial work involved validation of the in vitro model and
Pharmaceutical safety assessment has historically had good success collection of dose-response data at the gene (transcriptomics), protein
in avoiding serious injury to first in human volunteers and patients. (p53, p-p53, p-H2Ax, MDM2, etc.), and cellular (cell cycle arrest,
Present guidance from regulatory authorities specify reporting animal apoptosis, micronucleus) level using prototype chemicals. Biokinetic
studies dosed high enough to induce toxicity up to predefined limit models have been used to facilitate in vitro to in vivo extrapolation and
dose levels. Forward looking toxicologists anticipate that a finite num- the determination of safe levels of human exposure.
ber of toxicity pathways may be defined in in vitro biological culture
systems that would encompass all risk of human toxicity, and that Reference
these in vitro tests could replace the need for animal studies. As tech- Adeleye, Y., Andersen, M., Clewell, R. et al. (2014). Toxicology. epub
nologies advance, it may prove useful to demonstrate the translatabil- ahead of print. doi: 10.1016/j.tox.2014.02.007
ity of in vitro and in vivo toxicity pathways by showing predictivity of
in vitro systems for same-species in vivo equivalents. As a first step,
we retrospectively analyzed in vivo gene expression patterns from ani- II-1a-613
mals dosed for 5-7 days with a compound for activation of pathways
Cell-based risk assessment
of toxicity. this pathway activation in vivo was evaluated for its ability
to predict toxicity in the same tissue and same species in repeat dose relies on a quantitative
toxicity studies. All authors are employees of AbbVie. the design, understanding of toxicity pathways
study conduct, and financial support for this research was provided by
AbbVie. AbbVie participated in the interpretation of data, review, and K. Boekelheide
approval of the publication.
Department of Pathology & lab Medicine, Brown University,
Providence, RI, USA
II-1a-251 [email protected]
Can cancer safety assessment
At its core, the paradigm shift in toxicity testing now taking place relies
be conducted solely on the basis on the power of modern molecular biology to identify perturbations in
of in vitro studies? cellular function, and to integrate these perturbations into a quantita-
tive systematic framework (Bhattacharya et al., 2011). In this explana-
P. Carmichael1, Y. Adeleye1, R. Clewell2, B. Sun2, A. White1 tory framework, cells and computational systems biological models are
used to understand how biological pathways act and interact to main-
and M. Andersen2
tain cellular homeostasis. toxicant-induced alterations in biological
1SeAC, Unilever, Sharnbrook, UK; 2Chemical Safety Sciences, pathways are the signals used to develop models of the dynamics of
the Hamner Institutes, Research triangle Park, USA disruption of cellular functions, and are key to distinguishing adaptive
[email protected] versus adverse responses, and points of departure for risk assessment
(Boekelheide and Andersen, 2010). the critical biological pathways
“toxicity testing in the 21st Century (tt21C): A Vision and A Strate- that are most responsive to toxicant exposure have been called toxicity
gy” called for a change in toxicology; focusing on evaluating changes pathways, and the ongoing description of their dynamics is a key step
in signaling pathways using human relevant cells rather than relying forward (Andersen et al., 2011). Requirements for success of this cel-
on high dose animal studies. to support this transition, we have been lular framework for risk assessment include a focus on human biology,
Altex Proceedings 3, 1/14, Prague 2014 53
Theme II, SeSSIon II-1b
broad coverage of cell types, and an understanding of how endocrine, thyroperoxidase inhibition, as well as the inclusion of other available
extracellular matrix, and intercellular interactions modify cellular re- HtS assays for markers of hepatic catabolism, will also be discussed.
sponses. taking this systematic cell-based approach to perturbed hu- Assay development for thyroid-disruptor screening based on AOPs
man biology, an approach that relies on mechanisms and pathways, has would provide predictive tools for new chemical entity development,
inherent value, contributing both to improved toxicity testing and to the drastically reduce animal tissue use, and inform prioritization testing
fundamental molecular elucidation of human disease. schemes.
References
Andersen, M. e., Clewell, H. J., Carmichael, P. l. and Boekelheide, K. II-1a-712
(2011). ALTEX 28, 175-182. Advancing integrated approaches
Bhattacharya, S., Zhang, Q., Carmichael, P. l. et al. (2011). PLoS One
to animal-free chemical safety
6, e20887.
Boekelheide, K. and Andersen, M. e. (2010). ALTEX 27, 243-252. assessment using an Adverse Outcome
Pathway framework
II-1a-696
M. Whelan
Using adverse outcome pathway
eU Reference laboratory for Alternatives to Animal
analysis to identify gaps in testing (eURl eCVAM), european Commission Joint Research
high-throughput screening for thyroid Centre, Ispra, Italy
[email protected]
disruption
Reducing and ultimately replacing animal testing for determining
K. Paul
complex human health and environmental effects of chemicals de-
Human Safety Regulatory toxicology, Bayer CropScience, pends on our ability to effectively combine information derived from
Research triangle Park, NC, USA multiple complementary alternative methods. Although considerable
[email protected] scientific challenges have yet to be overcome, the rapid emergence
of increasingly sophisticated experimental and computational tools
Common findings in safety studies include rodent thyroid histological is paving the way towards credible solutions to predictive toxicity.
abnormalities, increased gland weight, and/or tumors. the presence of However, relying on conventional trial-and-error approaches to iden-
these rodent thyroid abnormalities may suggest a chemical-induced tify the optimum method-combinations for different protection goals
perturbation of thyroid hormones (tHs) that is relevant across spe- seems a futile pursuit since we simply have too many tools in our
cies. A primary toxicological concern is that maternal tHs modulate toolbox. Progress towards reduction and replacement of animal test-
neurodevelopment. Adverse outcome pathway (AOP) analysis dem- ing is far more likely if the design of integrated approaches is based on
onstrates that tH disruption occurs via several molecular-initiating understanding of the underlying biological system and the toxicologi-
events (MIes), including: inhibition of glandular iodine uptake, cal pathways that lead to its failure. the Adverse Outcome Pathway
tH synthesis, and/or peripheral deiodination of tHs; increased he- (AOP) framework provides a systematic, practical, and scientifically
patic catabolism of tHs; and, interaction with tH receptors. How- sound approach to describe the sequential chain of causally linked key
ever, high-throughput screening (HtS) assay data are unavailable for events, occurring at different levels of biological organisation, that lead
many MIes. the need for HtS assay development is highlighted by to an adverse health or ecotoxicological effect. An AOP represents a
comparison of thyroid-related HtS data from the US ePA toxCast conveniently packaged distillation of curated mechanistic knowledge
program and the in vivo thyroid endpoints reported in toxRefDB. that can be readily used to design integrated testing strategies and to
Analysis of the added predictivity of a new high-throughput assay for guide method development and validation.
Session II-1b: Pathways approaches in toxicology
Co-chairs
Katie Paul, Bayer CropScience, USA
Mathieu Vinken, Vrije Universiteit Brussel, Belgium
Session II-1b: Oral presentations
II-1b-010 2Systems toxicology Unit, Institute for Health and Consumer
Protection, european Commission, Joint Research Center, european
Development of an adverse outcome
Commission, Ispra, Italy; 3endocrinology, AlgemeenStedelijk
pathway from drug-mediated Ziekenhuis, Aalst, Belgium; 4National Center for Computational
bile salt export pump inhibition to Toxicology, Office of Research and Development, US
environmental Protection Agency, Research triangle Park, North
cholestatic liver injury
Carolina, USA; 5Pharmacology, toxicology and therapeutics,
University of Kansas Medical Center, Kansas City, USA;
M. Vinken1, B. Landesmann2, M. Goumenou2, S. Vinken3, 6the Humane Society of the United States, the Humane Society
I. Shah4, H. Jaeschke5, C. Willett6, M. Whelan2 and V. Rogiers1 of the United States, Washington DC, USA
1toxicology, Vrije Universiteit Brussel, Brussels, Belgium; [email protected]
54 Altex Proceedings 3, 1/14, Prague 2014
Theme II, SeSSIon II-1b
Adverse outcome pathways (AOPs) have been recently introduced in MIes and key cellular events (Kleinstreuer et al., 2013), and com-
human risk assessment as pragmatic tools with multiple applications. pound hazard predictions will be discussed.
AOPs intend to provide a mechanistic representation of pertinent
toxicological effects. AOPs are typically composed of a molecular This abstract does not necessarily reflect EPA policy. This project
initiating event, a series of intermediate steps and key events, and an was funded in whole or in part with Federal funds from the EPA,
adverse outcome (Vinken, 2013; Vinken et al., 2014). In this study, CSS Research Program, Lockheed-Martin contracting, and NIEHS,
an AOP framework is proposed for cholestasis triggered by drug-me- NIH under Contract No.HHSN27320140003C.
diated inhibition of the bile salt export pump transporter protein. For
this purpose, an in-depth survey of relevant scientific literature was References
carried out in order to identify intermediate steps and key events. the Kavlock, R. et al. (2012). Chem Res Toxicol 25, 1287-1302.
latter include bile accumulation, the induction of oxidative stress and Kleinstreuer, N. et al. (2013). PLoS Comput Biol 9, e1002996.
inflammation, and the activation of specific nuclear receptors. Collec- Knudsen, t. B. and Kleinstreuer, N. C. (2011). Birth Defects Res C
tively, these mechanisms drive both a deteriorative cellular response, Embryo Today 93, 312-323.
which underlies directly caused cholestatic injury, and an adaptive
cellular response, which is aimed at counteracting cholestatic insults.
AOP development was performed according to OeCD guidance, in-
II-1b-468
cluding consideration of the Bradford Hill criteria for weight of evi-
Building on shared experience
dence assessment and the OeCD key questions for evaluating AOP
confidence (Vinken et al., 2013). The postulated AOP is expected to to advance practical application of
serve as the basis for the development of new in vitro tests and the
pathway-based toxicology:
characterization of novel biomarkers of drug-induced cholestasis
(Vinken, 2013). repeat-dose liver toxicity
References C. Willett1, J. Caverly Rae2, K. O. Goyak3, B. Landesmann4,
Vinken, M. (2013). Toxicology 312, 158-165. G. Minsavage3 and C. Westmoreland5
Vinken, M., landesmann, B., Goumenou, M. et al. (2013). Toxicol Sci
1Animal Research Issues, Humane Society of the United States,
136, 97-106.
Gaithersburg, MD, USA; 2Haskell Global Centers for Health and
Vinken, M., Whelan, M. and Rogiers, V. (2014). Arch Toxicol 88,
environmental Sciences, DuPont, Newark, De, USA; 3Biomedical
1-2.
Sciences, exxonMobil, Annandale, NJ, USA; 4Institute for Health
and Consumer Protection Joint Research Centre, european
Commission, Ispra, Italy; 5Safety & environmental Assurance
II-1b-376 Centre, Unilever, Colworth, UK
Constructing, quantifying, and [email protected]
validating an adverse
In January 2013, the Human toxicology Project Consortium con-
outcome pathway for vascular
vened a workshop to exchange experience regarding the usefulness
development toxicity of pathways as an organizing principle for applying different types of
information at all levels of biological organization, using two liver-
N. Kleinstreuer1, T. B. Knudsen2, T. Tal3, T. Heinonen4, J. specific pathways (fibrosis and steatosis) as examples. Conclusions
Franzosa2, N. Baker5, S. Padilla3, E. Carney6 and W. Casey7 included that applicability of the pathway depends on the confidence
in, and completeness of, the pathway, where qualitative pathways are
1NICeAtM, Integrated laboratory Systems, Inc., Research
useful for prioritization and read across but more developed pathways
triangle Park, NC, USA; 2ORD/NCCt, U.S. environmental
may be used quantitatively. Additionally, the quality assurance meth-
Protection Agency, Research triangle Park, NC, USA;
odologies described in OeCD guidance to assess data quality causal
3ORD/IStD, U.S. environmental Protection Agency, Research
relationships, and completeness are appropriate and essential. Also,
triangle Park, NC, USA; 4FICAM, University of tampere,
pathway development should be an iterative, interactive, multidisci-
tampere, Finland; 5ePA Contractor, lockheed-Martin, Research
plinary and public process. Confidence in the prototype liver pathways
triangle Park, NC, USA; 6Predictive toxicology Division,
can be increased by including information from traditional toxicologi-
the Dow Chemical Company, Midland, MI, USA; 7NICeAtM,
cal sources such as histology and knowledge from pathologists and
NIH/NIeHS/DNtP, Research triangle Park, NC, USA
clinicians familiar with the biological outcomes at the tissue and indi-
[email protected] vidual levels. Bioinformatics and modeling approaches can be used to
both inform the understanding of the pathway and to identify potential
embryonic vascular disruption (Knudsen and Kleinstreuer, 2011) predictive markers or signatures for liver toxicity. this presentation
leads to a range of adverse prenatal outcomes. the adverse outcome will discuss workshop findings and recommendations as well as recent
pathway (AOP) for embryonic vascular disruption was recently en- developments since the workshop.
tered into the AOP wiki and accepted as part of the OeCD workplan.
the AOP was built based on molecular initiating events (MIes) af-
fecting genes from critical pathways (hypoxia/growth factor signal- II-1b-499
ing, chemokine networks, extracellular matrix interactions and ves- Promises and challenges in
sel remodeling/stabilization) with evidence of abnormal embryonic
constructing an adverse outcome
vascular development in the mammalian phenotype browser of the
Mouse Genome Informatics database (http://www.informatics.jax. pathway for chemical
org). ePA toxCast high throughput screening data (Kavlock et al.,
sensitization of the respiratory tract
2012) for assays mapping to targets in the AOP were used to prioritize
>1000 chemicals for their potential to disrupt vascular development.
K. Sullivan1, S. Cochrane2, S. Enoch3, I. Kimber4, G.
A subset of these chemicals are being tested for developmental effects
Patlewicz5, E. Roggen6, K. Sewald7 and J. Ezendam8
across a wide range of vascular-specific model systems. Preliminary
results from functional validation of AOP targets, quantification of 1toxicology and Regulatory testing, Physicians Committee
Altex Proceedings 3, 1/14, Prague 2014 55
Theme II, SeSSIon II-1c
for Responsible Medicine, Washington, USA; 2Safety and II-1b-843
environmental Assurance Centre, Unilever, Sharnbrook, UK; An adverse outcome pathway (AOP)
3School of Pharmacy and Biomolecular Sciences, liverpool John
Moores University, liverpool, UK; 4Faculty of life Sciences, framework for screening
University of Manchester, Manchester, UK; 5Quantitative for potential endocrine disruption
Assessment & Consulting, DuPont Haskell Global Centers for
Health and environmental Sciences, Newark, USA; 6Department D. Dix
of toxicology, Novozymes A/S, Bagsvaerd, Denmark;
Office of Science Coordination and Policy, USEPA, Washington
7Immunotoxicology, Fraunhofer Institut für toxikologie und
DC, USA
experimentelle Medizin IteM, Hannover, Germany; 8Innovative
testing Strategies, National Institute for Public Health and the [email protected]
environment, Bilthoven, the Netherlands
the US ePA is applying an adverse outcome pathway (AOP) frame-
[email protected]
work for screening potential endocrine disrupting chemicals. Rather
Increased interest in toxicological modes of action and a desire to de- than considering a single molecular initiating event (MIe) and subse-
velop mechanistically-based assays for improved characterization of quent events leading to a specific adverse outcome, a more holistic ap-
toxic hazards has fueled interest in the OeCD Adverse Outcome Path- proach is being used to evaluate chemical interactions along the estro-
way program. this program outlines and models AOPs to support the gen (e), androgen (A), and thyroid (t) hormone pathways. endpoints
development of new regulatory tools for toxicology. of ePA’s endocrine Disruptor Screening Program (eDSP) assessment
One endpoint of particular regulatory interest is chemical sensitiza- tools (e.g., in silico models, in vitro and in vivo assays), including
tion of the respiratory tract, or chemical respiratory allergy. An expert MIes, as well as intermediate and terminal events, are mapped to key
group was formed to define an AOP for this endpoint. The group first events along the AOP. linking assessment tools to steps along an AOP
aimed to identify commonalities and differences between chemical will assist in evaluating interactions and adverse consequences of ex-
sensitization of the respiratory tract and skin sensitization resulting in posure to endocrine disrupting chemicals. examples of the pathway
allergic contact dermatitis. Important differences may include differ- concept and maps of endpoints to steps along the AOP will be pro-
ential selectivity of protein binding, dendritic cell interactions, and the vided, with recognition that chemicals may activate multiple MIes
quality of immune responses associated with the acquisition of sensi- and/or hormone pathways with varied outcomes across different life
tization. However, experimental evidence confirming some of these stages and taxa. this effort is intended to illustrate the utility of this
differences may not always be available. Furthermore, determining conceptual framework for linking assay endpoints with outcomes of
the kinds of evidence to include in an AOP can be challenging. For regulatory interest. this, coupled with estimates of exposure can be
example, should this AOP include relevant evidence from work with used for risk-based prioritization of chemicals for eDSP screening.
biological allergens, such as proteins? this presentation will give an
overview of the AOP and discuss some challenges faced in its con- This abstract does not necessarily reflect US EPA policy.
struction.
Session II-1c: Pathways approaches in toxicology
Co-chairs
Ralf Herwig, MPI Molecular Genetics, Germany
Lena Smirnova, CAAT, USA
Session II-1c: Oral presentations
II-1c-212 aim at refining and quantifying the proposed toxicity pathways by
evaluating the causal relationships within the MoA (OeCD, 2013) and
PPARγ-related hepatotoxic mode-
at in silico modeling of the molecular initiating event (MIe) associ-
of-action: quantitative characterization ated with the PPARγ activation.
and in silico study of the molecular Methods: the MoA is assessed according to the AOP/OeCD prin-
ciples. Identification and quantification of structure-activity relation-
initiating event involving receptor
ships are performed to model interactions of PPARγ with its agonists.
activation Results: In this study (i) key events were evaluated by weights-of-
evidence and their contribution to the MoA was assessed, (ii) PPARγ
M. Al Sharif1, I. Tsakovska1, P. Alov1, V. Vitcheva2 ligand database was created with structural and biological data that
and I. Pajeva1 could be used to characterize the chemical space of PPARγ agonists,
and (iii) MIe-based in silico models were developed.
1QSAR and Molecular Modelling, Institute of Biophysics
Conclusion: This is the first attempt to quantify the developed MoA.
and Biomedical Engineering, BAS, Sofia, Bulgaria; 2Faculty of
Further efforts are necessary to refine and update it considering the
Pharmacy, Medical University, Sofia, Bulgaria
wide range of downstream events, the complex interconnections of
[email protected] PPARγ with other transcriptional regulators and the tissue specificity
of the effects (liver vs. adipose tissue). Structural descriptors of impor-
Aim: In line with the new predictive toxicology concepts a MoA from tance for the PPARγ agonistic activity were identified.
ligand-dependent dysregulation of PPARγ to nonalcoholic fatty liver
disease has been developed (Al Sharif et al., 2014). In this study we Acknowledgments to: (i) the European Community’s 7th Framework
56 Altex Proceedings 3, 1/14, Prague 2014
Theme II, SeSSIon II-1c
Program (FP7/2007-2013) COSMOS Project under grant agree- the level of entire pathways rather than isolated gene markers (Yildi-
ment n°266835 and from Cosmetics Europe; (ii) the project rimman et al., 2011; Doktorova et al., 2013). In our presentation we
BG051PO001-3.3.06-0040, scheme BG051PO001-3.3.06 – “Support demonstrate, based on data from the field of carcinogenicity and liver
for the development of PhD students and PhD graduates, post-doctor- toxicity, that this approach improves hazard prediction to a large ex-
al students, and young scientists” of the operative program “Human tent. Furthermore, we describe a novel resource, toxDB, that holds
Resources Development” of the European Social Fund. functionality for network based interpretation of toxicogenomics data.
toxDB combines statistical network methodology and utilizes the
References ConsensusPathDB, a large resource of human molecular interactions
Al Sharif, M., Alov, P., Vitcheva, V. et al. (2014). PPAR Res 2014, and pathways (Kamburov et al., 2013). toxDB can be used as a tool
432647. for deriving more stable network module signatures for hazard predic-
OeCD (2013). Guidance Document on Developing and Assessing tion and adverse outcome pathway identification.
Adverse Outcome Pathways, Series on Testing and Assessment No.
184, Paris, France OeCD. References
Doktorova, t., Yildirimman, R., Vinken, M. et al. (2013). Carcinogen-
esis 34, 1393-1402.
II-1c-391 Kamburov, A., Stelzl, U., lehrach, H. and Herwig, R. (2013). Nucleic
Acids Res 41(Database issue), D793-800.
Metabolomics in vitro: a new
van Delft, J., Gaj, S., lienhard, M. et al. (2012). Toxicol Sci 130, 427-
approach for systemic toxicity – first 439.
applications for chemical grouping Yildirimman, R., Brolén, G., Vilardell, M. et al. (2011). Toxicol Sci
124, 278-290.
T. Ramirez1, W. Mellert1, A. Strigun2, H. Kamp1,
T. Walk2 and B. van Ravenzwaay1
II-1c-838
1experimental toxicology and ecology, BASF Se, ludwigshafen
am Rhein, Germany; 2Analytics, metanomics GmbH, Berlin, Pathways of toxicity as a predictive
Germany tool for safety assessment
[email protected]
H. Hogberg, R. Bachinski, M. Bouhifd, G. Harris,
the use of alternatives to animal testing has considerably increased A. Kleensang, S. Odwin-DaCosta, D. Pamies, L. Smirnova,
in the last years but is still mostly limited to topical applications. Me- L. Zhao and T. Hartung
tabolomics in vitro is a novel approach to identify systemic toxicity
CAAt, eHS, Johns Hopkins University, Baltimore, USA
by determining modes of action and (dis)similarities in the profiles of
chemicals. In vitro metabolomics allows for the acquisition of quan- [email protected]
titative information about the multi-parametric metabolic response of
the cellular systems in normal and patho-physiological conditions. the National Research Council report, “tox-21c” has created an at-
Our data demonstrate a highly stable and reproducible method. the mosphere of departure in the US. It suggests moving away from tra-
purpose of this study was to investigate if toxicological equivalence ditional (animal) testing to modern technologies based on toxicity
between pure enantiomers and their corresponding racemates can be pathways.
demonstrated using this technology. We therefore exposed HepG2 the current developments on OeCD level are to organize our
cells at two concentrations for 48 h to different, hepatotoxic, active knowledge on hazard manifestations as Adverse Outcome Pathways
ingredients and determined the metabolome of both the pure and race- (AOP). the concept of Pathways of toxicity (Pot) is part of the AOP
mic compounds. the results obtained indicate that there was no bio- and describes the molecular definition of mechanism and the per-
logically relevant difference between the two forms of the respective turbed networks.
compounds. As such equivalence had been demonstrated in the past the NIH is funding a transformative research grant, led by CAAt
using in vivo studies, the present results can be seen as a proof of that involves several members of the tox-21c panel. the goal is to
concept for the use of this in vitro technology. We thus demonstrate develop a public database of pathways, the Human toxome, to enable
straightforward applicability and conclude that in vitro metabolomics scientific collaboration and exchange.
is (more than) a promising tool to address systemic toxicity. An area of toxicology where tox-21c and the Human toxome could
have a significant impact is developmental neurotoxicity (DNT). Cur-
rent animal tests for DNt have several limitations: high costs, time
consuming and uses large numbers of animals. Consequently, only
II-1c-521
very few substances have been tested for DNt. this is a concern as
Pathway level interpretation
evidence shows that exposures to environmental chemicals contribute
of toxicogenomics data to the increasing incidence of neurodevelopmental disorders in chil-
dren. DNtox-21c is a CAAt project funded by the FDA that aims to
C. Hardt, M. Beber, A. Kamburov and R. Herwig find pathways of developmental neurotoxicity using a metabolomics
approach.
Vertebrate Genomics, Max Planck Institute for molecular Genetics,
Berlin, Germany
[email protected]
A major goal of toxicogenomics is the prediction of hazard (and ul-
timately risk) of chemicals based on genome-wide transcriptomics
either derived from microarray or alternatively from high-throughput
sequencing experiments (van Delft et al., 2012). expression signatures
derived from single gene markers are highly variable and thus lack
stability across large numbers of chemicals. to address this point we
have developed an approach that evaluates transcriptomics data at
Altex Proceedings 3, 1/14, Prague 2014 57
Theme II, SeSSIon II-1
Session II-1: Poster presentations
II-1-103 mitochondrial dysfunctions: disruptive AtP production and biogen-
In vitro skin sensitization testing esis, By employing two human cell lines (AC16 cardiomyocytes and
HepG2 hepatocytes) with two chemicals (doxorubicin and paraceta-
strategy – in-house post-validation
mol) at varied concentrations over several time points, substantial
with “real-life” compounds concentration-dependent and time-course response data have been
generated using western blot, flow cytometry, confocal microscopy,
K. Guth1, S. Kolle1, N. Honarvar1, B. Wareing1, A. Mehling2, real-time PCR, ATP colorimetric/fluorometric assays. Our pathway
B. van Ravenzwaay1 and R. Landsiedel1 data outcomes with observed adversity (i.e., reduced AtP production
& mitochondria number) demonstrate that ROS-mediated mitochon-
1experimental toxicology and ecology, BASF Se, ludwigshafen,
drial toxicity follows with the perturbations of the two pathways in a
Germany; 2BASF Personal Care and Nutrition GmbH, Düsseldorf,
concentration-dependent & time-course manor. the study allows us to
Germany
begin to understand how cellular pathway perturbations can be used,
[email protected] as part of evidence, to inform our risk assessment decision for chemi-
cals, especially for identification of biological pathway altering doses
Allergic contact dermatitis (ACD) is a skin disease triggered by re- (BPAD), critical input into novel mechanistically-derived non-animal
peated contact to an allergen. the prerequisite for the development approaches for safety assessments (Adeleye et al., 2014).
of ACD is sensitization. Several in vitro methods including dendritic
cell line activation (e.g., mMUSSt, h-ClAt), keratinocyte activation References
(e.g., luSens, KeratinoSens™) and in chemico (e.g., DPRA) assays Adeleye, Y. et al. (2014). Toxicology. doi:10.1016/j.tox.2014.02.007
have been developed to assess the skin sensitizing potential of com- Guo, Q. et al. (2013). The Chinese of Journal of Pharmacological Bul-
pounds. Although these are not stand-alone methods, they can be used letin 29, 1-5 (in Chinese).
in an integrated testing strategy (ItS) preferably representing different Westmoreland, C. et al. (2010). ALTEX 27, 61-65.
steps of the adverse outcome pathway for skin sensitization to replace Westmoreland, C. et al. (2013). http://bit.ly/1lygfwB (last accessed on
animal tests. One promising strategy that was shown to have a good April 2014)
overall accuracy is to use the results in a weight of evidence (Woe) Yuan, H. et al., (2013). The Proceedings of the 6th Chinese National
approach based on a 2-out-of-3-assessment (Bauch et al., 2012). to Toxicology Congress, Guangzhou, China (the best paper award).
expand the existing data set, 40 additional compounds representing
different chemical classes and formulations (including acrylates, sur-
factants, isocyanates, polyethylene imines, plant extracts and agro- II-1-285
chemical formulations) was tested in mMUSSt, h-ClAt, luSens and
Development of an alternative testing
DPRA followed by an Woe assessment. the results were compared
to in vivo data. When excluding polyethylene imines, the accuracy of strategy for the Fish Early Life-
the WoE approach remains high. The results of this project help define Stage (FELS) test: results of the AOP
the applicability domain of this ItS in which reliable predictions can
‘Acetylcholinesterase inhibition
be expected.
leading to motor activity impairment’
Reference
Bauch, C. et al. (2012). Regul Toxicol Pharmacol 63, 489-504. S. Verstraelen1, P. Berckmans1, L. Vergauwen2, R. Blust3,
F. Dardenne3, D. Villeneuve4, G. Ankley4, D. Knapen2 and
H. Witters1
II-1-183
1environmental Risk and Health Unit – team Applied Bio &
Developing a mitochondrial toxicity
molecular Systems (ABS), Flemish Institute for technological
pathway-based non-animal approach Research (VItO nv), Mol, Belgium; 2Zebrafishlab, Veterinary
Physiology and Biochemistry, Department of Veterinary Sciences,
for chemical safety assessment
University of Antwerp, Antwerp, Belgium; 3Systemic Physiological
and ecotoxicological Research (SPHeRe), Department of Biology,
J. Li1, H. Yuan2, T. Zhang2, J. Guo2, S. Peng2, D. Suarez-
University of Antwerp, Antwerp, Belgium; 4ORD NHeeRl,
Rodriguez1, A. While1, P. Carmichael1 and C. Westmoreland1
Mid-Continent ecology Division, US ePA, Duluth, USA
1Safety and environmental Assurance Centre, Unilever, Bedford, [email protected]
UK; 2evaluation and Research Center for toxicology, Institute of
Disease Control and Prevention, Academy of Military Medical the Fish early life-Stage (FelS) test (OeCD 210) is the primary
Sciences, Beijing, China guideline used to estimate chronic toxicity of chemicals. Industry and
[email protected] regulatory institutions express the need to develop alternative testing
strategies for environmental impact assessment focusing on non-an-
We are at the forefront of research into novel non-animal approaches imal alternatives and mechanistic information (Ankley et al., 2010;
for assessing consumer safety (Westmoreland et al. 2010, 2013). Re- Scholz et al., 2013). the goal of the CeFIC lRI-eCO20 project is to
search collaboration between Unilever and Chinese Academy of Mili- develop an alternative testing strategy to reduce the need for 30 days
tary Medical Sciences is to develop mitochondrial toxicity pathway- FelS tests. the project puts forward 4 adverse outcome pathways
based approaches to risk assessment. Identified as master regulators in (AOPs) linking molecular initiating events to apical endpoints. Pre-
mitochondrial anti-oxidative defence systems (Guo et al., 2013; Yuan liminary results of the AOP “Acetylcholinesterase (AChe) inhibition
et al. 2013), PGC-1α and Nrf2 pathways were studied to understand leading to motor activity impairment” will be presented for chlorpyri-
whether perturbations of both induced by chemicals could result in fos, chlorpyrifos-oxon, and carbaryl using a 5 days ZebraFish embryo
58 Altex Proceedings 3, 1/14, Prague 2014
Theme II, SeSSIon II-1
Korea; 2College of Pharmacy, Chung-Ang University, Seoul,
toxicity (ZFet) test. AChe was inhibited and movement frequency
South Korea
was reduced in zebrafish larvae after 5 days exposure to each of these
3 compounds. These experiments with zebrafish larvae analysing lo- [email protected]
comotor activity and AChe inhibition are further extended to cover
key events at different organisational levels. therefore, neurite out- Since 1960s, the Draize rabbit eye irritation test (Draize et al., 1959)
growth in a human neuronal cell model and motorneuron develop- has been widely used in which chemicals are directly applied to rabbit
ment in zebrafish are under investigation to elaborate on this AOP and eye, and the symptoms and signs of eyes are scored. to resolve this
demonstrate whether any of these approaches, or combinations will ethical issue, it is essential to develop an animal alternative testing
contribute to predict chronic FelS toxicity of selected organophos- methods. Previously, we also reported an eye irritation test method
phates and carbamates. using a new human corneal epithelial model (MCtt HCe™) which is
reconstructed from remaining human tissues after a corneal transplan-
This research is funded by CEFIC Long-Range Research Initiative. tation (Doucet et al., 2006; Cotovio et al., 2010; Jung et al., 2011). to
improve the test accuracy for this model, we explored new biomarkers
References for the eye irritation using toxicogenomics approach. We selected 3
Ankley, G. t., Bennett, R. S., erickson, R. J. et al. (2010). Environ surfactants among the known eye irritants. After the test chemicals
Toxicol Chem 29, 730-741. were treated at various concentrations, we investigated the differen-
Scholz, S., Sela, e., Blaha, l. et al. (2013). Regul Toxicol Pharmacol tially expressed genes through a whole gene microarray. As a result,
67, 506-530. we identified that CNFN (cornifelin) and Egr-1 (early growth response
1) gene are significantly changed in three eye irritants. Significant up-
regulation of CNFN and Egr-1 gene were further confirmed by Q-RT-
PCR. Moreover, expression of CNFN and egr-1 gene was well-corre-
II-1-289
lated with SlS-mediated time-dependent tissue damage, which could
An adverse outcome pathway
be observed by morphology and cell viability. these results indicate
based approach to evaluate novel that CNFN and egr-1 can be novel biomarkers for determining the eye
irritation potential of surfactants.
tobacco products and electronic
nicotine delivery devices in vitro References
Cotovio, J., Grandidier, M. H., lelievre, D. et al. (2010). Toxicol In
F. Lowe, K. McQuillan, A. Bushby, G. Phillips and Vitro 24, 523-537.
D. Breheny Doucet, O., lanvin, M., thillou, C. et al. (2006). Toxicol In Vitro 20,
499-512.
Group Research and Development, British American tobacco
Draize, J. H. (1959). Association of Food and Drug Officials of the
(Investments) ltd, Southampton, UK
United States, 46-59.
[email protected] Jung, K. M., lee, S. H., Rye, Y. H. et al. (2011). Toxicol In Vitro 1,
403-410.
Novel “Heat-not-Burn” tobacco products and electronic nicotine
delivery devices are expected to be associated with reduced risk of
smoking-related disease compared to traditional cigarettes. In vitro II-1-501
models have excellent potential to minimise reliance on animal data
Application of alternative
to predict disease risk. Our challenge is to develop an appropriate and
meaningful suite of in vitro models to complement animal and hu- toxicity test methods for safety
man studies, which are able to, collectively, evaluate the potential for assessment of active
reductions in risk.
pharmaceutical ingredient
Adverse outcome pathways are a useful tool for screening single
chemicals against a specific toxicological end point by mapping out intermediates
and testing against the preceding, causative events and interactions.
We have applied these principles to map out a pathway of events which M. Múčková1, M. Bednáriková2, J. Lazová2, V. Šmahovský3
could ultimately result in tobacco-related disease risk. Furthermore, and I. Kakalík3
we propose a suite of in vitro models based upon these pathways,
1Biology, hameln rds a.s., Modra, Slovakia; 2Microbiology, hameln
which collectively, could be used to compare the relative biological
rds a.s., Modra, Slovakia; 3API Development, hameln rds a.s.,
effects of “Heat-not-Burn” tobacco products and electronic nicotine
Modra, Slovakia
delivery devices against traditional cigarettes.
this approach could be useful as part of a weight of evidence pack- [email protected]
age for the comparative risk assessment of future tobacco and nicotine
based products. Chemical synthesis of active pharmaceutical ingredients (APIs) in-
volves handling with many intermediates. In order to establish an ap-
propriate strategy for safety evaluation it is important to identify their
risk and to settle precaution to make the process safer for workers. the
II-1-432
aim of this research was to explore the potential of the alternative test
Identification of cornifelin and early
methods for toxicity testing of four (5, 8, 9 and 17) intermediates of
gene response-1 as new biomarkers synthesis of highly potent APIs with respect to their applicability for
hazard assessment.
for in vitro eye irritation using a 3D
The cytotoxicity was evaluated on three fibroblast cell lines: mouse
reconstructed human cornea model, 3t3 by Neutral Red assay and on mouse l929 and Chinese hamster
MCTT HCE™ V79 using the standard Mtt assay. On the basis of midpoint cyto-
toxicity values, the sequence was intermediate 5 > intermediate 9 >
S. H. Choi1,2, M. R. Lee1, Y. J. Chun2 and K. M. Lim1 intermediate 8 > intermediate 17. Differences in sensitivity between
the NR and Mtt assays were not marked.
1College of Pharmacy, ewha Womans University, Seoul, South
In vitro skin corrosion potential of intermediates was examined us-
Altex Proceedings 3, 1/14, Prague 2014 59
Theme II, SeSSIon II-1
Joint Research Centre, Ispra, Italy; 6Zebrafishlab, University of
ing the in vitro reconstructed human skin model epiDerm™. It was
Antwerp, Wilrijk, Belgium; 7Government Relations, life Sciences
concluded that all 4 intermediates are considered to be non-corrosive
and Chemical Analysis, Agilent technologies, Washington, DC,
to the skin.
USA; 8environmental laboratory, US Army engineer Research and
the predictive potential of in vitro tests was evaluated in compari-
Development Center, Vicksburg, MS, USA
son with the results of a set of alternative in vivo tests.
[email protected]
This work was supported by Ministry of Education SR Agency for
Structural Funds of EU in frame of project code ITMS: Adverse Outcome Pathways (AOPs) offer a pathway-based toxicolog-
26240220061. ical framework to support hazard assessment and reduce uncertainty
in regulatory decision-making. Here we present four case studies dem-
onstrating different levels of knowledge and confidence to examine
II-1-563 how the AOP concept can be used as a qualitative and quantitative tool
in hazard and risk assessment for either human health or ecological
C24:1-ceramide may be a novel lipid
concerns. We also present a conceptual model that enables quantita-
biomarker for eye irritation in 3D tive integration of data into a pathway context, based on the biological
human corneal epithelial model, MCTT relevance of an event to the outcome, the strength of evidence for a
causal relationship between key events, and the ability of a key event
HCE™
to infer that a chemical will cause the adverse outcome. the model
involves weighting values assigned to each event and subsequent anal-
M. Lee1, S. Choi1, K.-M. Joo2, Y.-J. Chun3 and K.-M. Lim1
ysis of the relative contribution of each event to the overall weight-
1College of Pharmacy, ewha Womans University, Seoul, of-evidence assessement. the utility of the model is demonstrated by
South Korea; 2Skin Research Center, AmorePacific R&D Center, examining the pathway for mitochondrial fatty acid beta-oxidation in-
Seoul, South Korea; 3College of Pharmacy, Chung-Ang hibition leading to steatosis. For AOPs to be useful they should result
University, Seoul, South Korea in a risk assessment with higher confidence compared to current ap-
proaches. this necessitates the development of quantitative solutions
[email protected]
to AOP assessment and this study offers one such approach.
eye irritation test is mandatory for cosmetics and pharmaceuticals.
Draize rabbit eye irritation test has been widely used. However, due II-1-830
to an invasive experimental procedure, alternative in vitro tests replac-
The experience of using the alternative
ing Draize test are being actively needed. Recently, 3D reconstituted
human corneal models receive enormous attention since they are mor- in vitro methods for testing and safety
phologically and physiologically similar to human cornea. these 3D assessment of goods for children,
models are employing cell viability as a common endpoint for eye ir-
detergents, cosmetics, including raw
ritant but extra biomarkers are necessary to improve test performance.
Here, we explored new lipid biomarkers for eye irritation using MCtt nanomaterials
HCe model. Surfactants were selected as eye irritants since they are
used in cosmetics and pharmaceutical products. three irritants; so- N. Zavyalov, Е. Skvortsova and O. Guskova
dium lauryl sulfate, benzalkonium and tritonx-100 were selected as
Preventive toxicology Department, FBUZ “the Hygiene and
representative surfactants. After treating 3 irritants at different concen-
epidemiology Center in the city of Moscow”, Moscow, Russian
trations on the model, we extracted lipid in supernatant using methyl-
Federation
tert-butyl ether. And we quantified the amount of ceramides and fatty
acids, representative lipid components in outer surface of body, using [email protected]
sensitive lC/MSMS method. It was found that among diverse fatty
acids and ceramides, C24:1Cer was significantly increased by three the alternative in vitro methods are widely used at the safety assess-
eye irritants. Moreover, C24:1Cer was increased by 3 irritants in a ment of the products In the Hygiene and epidemiologyCenter in the
dose-dependent manner suggesting that it can be a novel lipid bio- city of Moscow (further the Center).
marker for eye irritants. About 40% of all toxicological studies conducted in the Center fall
to the share of alternative in vitro methods.
Generally these work are conducted for control in the market of
II-1-781
Russia, Belarus, Kazakhstan domestic and foreign products such as
Using adverse outcome pathways for cosmetics, household chemicals and detergents, goods for children,
regulatory applications toys, sanitary pads and tampons, Among alternative methods of 58%
it is the share of cytotoxicity tests with use of cattle sperm; 30% – the
C. E. Willett1, P. Antczak2, L. Burgoon3, F. Falciani2, S. share of cytotoxicity with photobacteria model and 12% – ex vivo irri-
tation modified HET-CAM test method with Doppler ultrasonography
Gutsell4, G. Hodges4, A. Kienzler5, D. Knapen6, M. McBride7
on САМ vessels.
and E. J. Perkins8
Now in collaboration with Research Institute of Medical equip-
1Animal Research Issues, the Humane Society of the United States, ment, lomonosov Moscow State University, the Republic Hygiene
Washington, DC, USA; 2Institute of Integrative Biology, University and epidemiology Center of Belarus with support of manufacturers, in
of liverpool, liverpool, UK; 3National Center for environmental particular Unilever Company, SPlAt and Colgate-Palmolive, we con-
Assessment, US environmental Protection Agency, Research ducted the studies in order to compare the results of acute toxicity and
triangle Park, NC, USA; 4Safety and environmental Assurance mucosa irritation effects received on various test – models from oral
Centre, Unilever, Colworth Science Park, Sharnbrook, UK; 5Institute care products and row materials, including those which compounds
for Health and Consumer Protection, european Commission containing nanoparticles (Ag, Zn, ti, hydroxyapatite).
60 Altex Proceedings 3, 1/14, Prague 2014
Theme II, SeSSIon II-2
Session II-2: Systems biology
Co-chairs
Ralf Herwig, MPI Molecular Genetics, Germany
Richard Judson, U.S. EPA, USA
Session II-2: Oral presentations
II-2-362 II-2-395
In vitro data combined with human Development of a mechanistic based
disease data to improve model for Nrf2 and oxidative stress
toxicological hazard assessment: in the context of the Adverse Outcome
the ASAT Knowledge Base Pathway framework
J. Venhorst1, J. Aarts2, A. Boorsma1, F. Caiment3, A. J. White1, M. E. Andersen2, P. L. Carmichael1,
L. Soeteman-Hernandez4, J. van der Veen4, M. Tsamou3, S. Windebank1, R. A. Clewell2, S. Cooper1, C. Courage1,
F. Russel5, G. Groothuis6 and R. Stierum1 B. Sun2, S. Glavin1 and G. Jain1
1Risk Assessment for Products in Development, the Netherlands 1SeAC, Unilever R&D, Bedford, UK; 2Institute for Chemical Safety
Organization for Applied Scientific Research, Zeist, Sciences, the Hamner Institutes for Health Sciences, Research
the Netherlands; 2BU toxicology & Bioassays, RIKIlt-Institute triangle Park, USA
of Food Safety, Wageningen, the Netherlands; 3Department of [email protected]
toxicogenomics, University of Maastricht, Maastricht,
the Netherlands; 4laboratory for Health Protection Research,
Cellular responses caused by prolonged increases in ROS production
National Institute for Public Health and the environment (RIVM),
and oxidative stress are detrimental and have been associated with nu-
Bilthoven, the Netherlands; 5Department of Pharmacology and
merous adverse toxicological outcomes in relation to chemical induced
toxicology, Radboud University Medical Centre, Nijmegen,
toxicity. Cells have evolved complex adaptive responses designed to
the Netherlands; 6Drug Metabolism and toxicology, University of
regulate and maintain cellular homeostasis and limit the consequential
Groningen, Groningen, the Netherlands
increased risk of toxicity. Determining the point when this homeo-
[email protected] static control becomes dysregulated and shifts from an adaptive to an
adverse state is critical to facilitate decision making within an AOP
In line with the Assuring Safety Without Animal testing (ASAt) framework. to better understand the mechanism of action in relation
principle, risk assessment may ultimately become feasible without to adaptive and adverse effects at low doses for oxidative stress, we
the use of animals (Fentem et al., 2004). ASAt assumes that activa- report on studies to develop an integrated Nrf2 and cellular outcome
tion of human disease mechanisms in in vitro models can be used for systems model. time- and dose-dependent changes in biomarkers for
toxicological assessment. therefore, goal of the present research was ROS production, alterations in GSH, and Nrf2 activation were mea-
to demonstrate the integration of public data from the human disease sured in a human keratinocyte (HaCat) cell line following chemical
domain with in vitro toxicology. two diseases, associated with chemi- exposure. the downstream effects on cytotoxicity, lipid and protein
cal exposure, were concerned: hepatocellular carcinoma and allergic damage were measured to understand the cellular consequences at
contact dermatitis (ACD). Data were retrieved from online sources varying exposure concentrations and duration. these data are being
(e.g., GeO, CtD) and expert knowledge. A Knowledge Base for cu- integrated into a computational pathway model of Nrf2 response to
ration, storage and modelling of the data was developed. Using the predict the low dose response behaviour of the system and assess
Knowledge Base for ACD, it was possible to discern sensitizing from where physiologically adaptive/adverse responses are observed, for
non-sensitizing chemicals, as defined by enrichment of disease gene non-animal based safety assessment of pro-oxidant chemicals.
sets in in vitro toxicogenomics datasets. Interestingly, the strongest
sensitizers most profoundly activated these gene sets. the ongoing
incorporation of (reverse) PBPK models in the Knowledge Base to II-2-602
judge the relevance of in vitro concentrations, in relation to realistic in Using alternative approaches
vivo exposure scenarios, will be presented. Finally, the expansion with
to prioritize testing for the universe
models towards other disease areas (cholestasis) will be discussed.
of chemicals with potential for
Funding by ZonMw (Projects: 165600002; 114022001), Janssen
human exposure
Pharmaceutica NV and Simcyp Limited is acknowledged.
R. Judson
Reference National Center for Computational toxicology, U.S. ePA, Research
Fentem, J., Chamberlain, M., Sangster, B. (2004). Altern Lab Anim triangle Park, NC, USA
32, 617-623. [email protected]
One use of alternative methods is to target animal use at only those
chemicals and tests that are absolutely necessary. We discuss priori-
tization of testing based on high-throughput screening assays (HtS),
QSAR modeling, high-throughput toxicokinetics (HttK), and expo-
Altex Proceedings 3, 1/14, Prague 2014 61
Theme II, SeSSIon II-2
sure modeling. Concentration-response HtS provides lowest effective II-2-828
concentrations (leC), the lowest concentrations at which relevant Pathway analysis and mathematical
biological perturbations occur. From the HtS data, QSAR models can
modeling of cellular
be developed to provide predicted leC (pleC) values for the remain-
der of a chemical library. HttK allow us to convert from an leC to a systems for personalized medicine
lowest effective level/dose (lel) that can be compared directly with
exposure estimates. Results of this approach will be illustrated using C. Wierling
work on the Human exposure Universe (HeU), a set of 32K distinct
Vertebrate Genomics, Systems Biology group, Max Planck Institute
chemicals with evidence for potential human exposure. We focus on
for Molecular Genetics, Berlin, Germany
the estrogen receptor, for which 1800 chemicals have been assayed
[email protected]
using HtS, while the remainder of the 32K have been modeled using
a crowd-sourced battery of QSAR models. exposure estimates have
been calculated using far-field and near-field models. Finally, HTTK Complex diseases, such as cancer, non-alcoholic fatty liver disease or
data for selected chemicals are used to quantitatively compare expo- the metabolic syndrome, are hard to understand and their therapy has
sure with activity to set priorities for further testing for estrogenicity. turned out to be difficult and different given individual genetic vari-
ants. First insights to the underlying molecular processes of a disease
This abstract does not necessarily reflect Agency policy. can be obtained by pathway analysis of omics data using tools like
ConsensusPathDB (Kamburov et al., 2011). Mathematical modeling
of related cellular processes allows a deeper understanding of the un-
II-2-674 derlying processes. By screening literature information and pathway
databases we have developed a large mathematical model of differ-
Percellome toxicogenomics project as
ent cellular signaling pathways using the modeling software PyBioS
the 3R-toxicology and the foundation (Wierling et al., 2007; Klipp et al., 2009). Development of detailed
of in vitro- and in silico-toxicology mathematical models is hampered by highly restricted availability of
reaction kinetics along with their respective kinetic parameters. to
J. Kanno, K. Aisaki and S. Kitajima overcome this bottleneck we have introduce an approach based on a
Monte Carlo strategy. Simulating different forms of the model (e.g., a
Division of Cellular & Molecular toxicology, Biological Safety
model that resembles a certain mutation or drug treatment) allows pre-
Research Center, National Institute of Health Sciences, tokyo,
diction of individual perturbation effects (Wierling et al., 2012). the
Japan
established resources, tools, algorithms and models build a foundation
[email protected] for the application of systems biology strategies in medical and phar-
maceutical research and, based on data from high-throughput genome,
Percellome toxicogenomics Project, using fewer animals exposing to transcriptome and proteome analysis, it enables the development of a
lower doses for one time or short period of time, was initiated in 2001, personalized medicine (Röhr et al., 2013).
aiming at mechanistically reinforcing the “safety (uncertainty) factor”
used for the extrapolation of animal data to humans, and eventually References
replacing and making the process in silico. the project was designed Kamburov, A., Pentchev, K., Galicka, H. et al. (2011). Nucleic Acids
not to miss any unpredicted toxicity. For this need, a normalization Res 39, Database issue D712-717.
method designated as “Percellome” (Kanno et al., 2006; Aisaki and Klipp, e., liebermeister, W., Wierling, C. et al. (2009). Systems Biol-
Kanno, 2011), was developed for microarrays and Q-PCR to generate ogy – A Textbook. Weinheim: Wiley-VCH.
absolute copy numbers of mRNAs per one cell (in average). Quanti- Röhr, C., Kerick, M., Fischer, A. et al. (2013). PLoS One 8, e67461.
fied mRNA data of mouse liver (4 time points x 4 dose levels, n=3, Wierling, C., Herwig, R. and lehrach, H. (2007). Brief Funct Geno-
48 per chemical) are obtained on more than 100 chemicals and data- mic Proteomic 6, 240-251.
based with the data from repeated dosage, various organs, inhalation, Wierling, C., Kühn, A., Hache, H. et al. (2012). Mutat Res 746, 163-
etc. Data was visualized in 3D surface graphs (time x dose x mRNA 170.
copy number per cell) corresponding to the probesets of Affymetrix
MOe430 2.0 GeneChip and subjected to comprehensive analysis
by a series of in-house software. We report case studies on estragole II-2-863
(Kanno et al., 2012) and pentachlorophenol (Kanno et al., 2013), dis-
In vitro to in vivo translational
covering unreported networks of PPAR-alpha and interferon signaling
networks, respectively. Further strategy, including systems biology biomarkers
(Garuda Project, http://www.garuda-alliance.org/), for the foundation
of the alternative methods will be discussed. P. Jennings
Physiology and medical physics, Innsbruck Medical University,
References
Innsbruck, Austria
Aisaki, K. and Kanno, J. (2011). In D. R. Boverhaf and B. Bhaskar
[email protected]
Gollapudi (eds.), Application of Toxicogenomics in Safety Evalu-
ation and Risk Assessment (323-329). Hoboken, New Jeresy: John
Wiley & Son. there is need to develop better biomarkers with improved target organ
Kanno, J., Aisaki, K., Igarashi, K. et al. (2006). BMC Genomics 29, specificity, improved sensitivity and which provide deeper mechanis-
64. tic insight, for both preclinical and clinical testing. the use of hu-
Kanno, J., Aisaki, K., Igarashi, K. et al. (2012). In I. Pongratz and V. man in vitro cell cultures is being realised as a rich resource for the
Bergander (eds.), Issues in Toxicology No.11. Hormone-Disruptive discovery of novel mechanistically based biomarkers with potential
Chemical Contaminants in Food (184-198). london: Royal Society transferability to in vivo. Here I will provide examples of liver, renal
of Chemistry. and cardiac biomarkers which have been either discovered in vitro
Kanno, J., Aisaki, K., Igarashi, K. et al. (2013). J Toxicol Sci 38, 643- or where such systems have been used to elucidate key mechanistic
654. information. Many of these biomarkers are more than innocent by-
62 Altex Proceedings 3, 1/14, Prague 2014