Arbeitsgruppe Mally

 Unsere Arbeitsgruppe untersucht molekulare Wirkmechanismen der Toxizität und Kanzerogenität von Fremdstoffen als Basis für eine wissenschaftlich fundierte Risikobewertung. Im Mittelpunkt dieser Arbeiten, die u.a. von der DFG gefördert werden, stehen dabei kanzerogene Verbindungen, die in Nahrungsmitteln vorkommen (Ochratoxin A, Furan) und daher für den Menschen besondere toxikologische Relevanz besitzen. Darüber hinaus beschäftigen wir uns mit der Identifizierung neuer Biomarker, mit deren Hilfe unerwünschte Wirkungen von Arzneimitteln früher und zuverlässiger erkannt werden sollen, sowie der Entwicklung von alternativen Methoden zur Prüfung auf Toxizität. Ziel dieser Arbeiten, die in enger Zusammenarbeit mit der pharmazeutischen Industrie durchgeführt werden, ist es, die Vorhersagekraft toxikologischer Prüfungen und damit die Sicherheit neuer Arzneimittel zu verbessern.

 Our research activities focus on understanding molecular mechanisms of disease related to exposure to toxic / carcinogenic chemicals and natural food contaminants (e.g. the mycotoxin ochratoxin A, furan) as a basis for risk assessment. A second arm of our work is aimed at improving the predictivity of chemical and preclinical safety testing, which is currently addressed in close collaboration with Europe´s leading pharmaceutical industries as part of a European Union 6th Framework Programme funded project.

Research projects

• Biomonitoring hitzeinduzierter Prozeßkontaminanten: In vivo Metabolismus und Eliminationskinetik von Alkylfuranen zur Identifizierung und Validierung von Biomarkern der Exposition (gefördert durch die DFG)
• Renal safety assessment of allulose in human volunteers via analysis of urinary markers of kidney injury (gefördert durch Tate & Lyle PLC, London, UK und Savanna Ingredients GmbH, Elsdorf, DE)
• The Role of Replication Stress in Ochratoxin A Genotoxicity (Die Bedeutung von replikativem Stress in der Gentoxizät des Mykotoxins Ochratoxin A) (gefördert durch die DFG)
• Biotransformation of HFO-1123  (sponsored AGC Japan Inc.)
• Biomonitoring and exposure assessment of mycotoxins in Nigerian infants and young children (gefördert von Alexander von Humbold Stiftung: Georg Forster-Forschungsstipendium für Postdoktoranden an Frau Dr. Cynthia Adaku Chilaka)
• Furan-Biomonitoring: Evaluation eines Biomarker-basierten Ansatzes zur Abschätuzung der Exposition gegenüber Furan aus Nahrungsmitteln (gefördert durch die DFG)
• Zebrafisch-Embryonen als Teil einer integrativen Teststrategie zur Prüfung auf Nephrotoxizität (mit D. Liedke) gefördert durch Stiftung zur Förderung der Erforschung von Ersatz- und Ergänzungsmethoden zur Einschränkung von Tierversuchen (Stiftung SET)
• Mining the IMI eTOX database: a systematic analysis of preclinical toxicity data, gefördert durch Boehringer Ingelheim Pharma GmbH
• Integrating mechanisms and quantitative in vitro-in vivo extrapolation (QIVIVE)modelling for risk assessment based on in vitro testing (Risk-IT), Verbundprojekt gefördert durch BMBF (Deutschland) und ZonMw (Niederlande)
• Temporal variations in drug metabolism and cellular stress modulated by environmental factors as determinants of idiosyncratic liver toxicity, gefördert durch die DFG
• Innovative Medicines for Europe - InnoMed (mit W. Dekant), gefördert durch die EU - 6th Framework Programme)
• Role of genetic and non-genetic mechanisms in furan risk (mit W. Dekant), gefördert durch die EU - 6th Framework Programme
• Furan in Lebensmitteln: Risikofaktor für die Entstehung hepatobiliärer Tumore beim Menschen? Funktionelle Veränderungen und Protein-Addukte in der Rattenleber, gefördert durch die DFG
• Molekulare Mechanismen der Toxizität und Kanzerogenität des Mykotoxins Ochratoxin A, gefördert durch die DFG
• In vitro toxicity testing: New approaches for chronic toxicity prediction (mit W. Dekant), gefördert von RCC Ltd. Itingen

Staff involved

Completed PhD Thesis

Benedikt Bauer (2024) M.Sc. Toxikologie, Dr. rer. nat.: Zebrafish larvae as an alternative model organism for nephrotoxicity testing

Raphael Dekant (2022) Pharmazeut, Dr. rer. nat.: Species-differences in the in vitro biotransformation of trifluoroethene (HFO-1123)

Sebastian Jarzina (2022) M.Sc. Toxikologie, Dr. rer. nat.: Assessment of systemic toxicity in vitro using the Adverse Outcome Pathway (AOP) concept: nephrotoxicity due to receptor-mediated endocytosis and lysosomal overload and inhibition of mtDNA polymerase-gamma as case studies

Markus Slopianka (2020) M.Sc. Toxikologie, Dr. rer. nat.: Evaluation von Gallensäuren als Biomarker für Lebertoxizität in der präklinischen Arzneimittelentwicklung

Ramm, Susanne (2012) LM-Chem., Dr. rer. nat.: Mechanismen idiosynkratischer Lebertoxizität – Einfluss von Arzneistoff-unabhängigen Stressfaktoren auf die Bildung reaktiver Metaboliten und zellulären Stress

Müller, Stephanie (2012)  LM-Chem., Dr. rer. nat.: Identification of early molecular changes associated with Fumonisin B₁-induced carcinogenesis in vivo and in vitro

Adler, Melanie (2012) Dipl.-Troph., Dr. rer. nat.: New approaches to improve prediction of drug-induced hepatotoxicity

Moro, Sabrina (2011) Pharmazeutin, Dr. rer. nat.: Identification of target proteins of furan reactive metabolites in rat liver

Hoffmann, Dana (2010) Dipl.-Biol., Dr. rer. nat.: Neue Biomarker zum Nachweis von Nierentoxizität

Rached, Eva Katharina (2010) Dipl.-Biol., Dr. rer. nat.: Neue Ansätze zur Entwicklung von Alternativmethoden zur Prüfung auf chronische Nierentoxizität.

Sieber, Max (2009) Dipl.-Chem., Dr. rer. nat.: Evaluation of 1H NMR- and GC/MS-based metabonomics for the asssessment of liver and kidney toxicity

The objective of this project is to assess the biotransformation of trifluoroethylene (HFO-1123) in vitro to understand species differences in the toxicity of HFO-1123.

Mycotoxins are toxic secondary metabolites produced by various toxigenic fungi especially Aspergillus, Fusarium, Penicillium, and Alternaria in diverse agricultural commodities such as cereals, nuts, cocoa and coffee beans and others. While mycotoxin contamination of food is a global challenge to food security and food safety, it pose a greater problem to economically developing regions such as sub-Saharan Africa. This is due to several favourable conditions (environmental condition, poor agricultural practices and socio-economic factors) in the region that favour the growth of fungi and subsequently production of mycotoxins.  Human exposure to mycotoxins have been linked to several health effects ranging from acute toxicity to chronic health disorders. Infants and young children are more at risk and susceptible to mycotoxin contamination because of their higher intake/body weight ratio, higher metabolic rate and lower detoxification capacity. In order to protect consumers, especially infant and young children from the toxic effects of mycotoxins, there is an urgent need to assess the risk associated with ingestion of mycotoxins. This procedure involves a systematic evaluation through hazard identification, dose-response assessment, exposure assessment, and risk characterization toward providing baseline data for effective regulation. So far, the developed countries have achieved success in minimizing the risk associated with dietary exposure to mycotoxins through establishment of regulatory limits including the European Union (EU) legislations. However, this is not the case in the developing countries like Nigeria, reason which is attributed to lack of sufficient scientific data (occurrence, exposure, and toxicological). Generating data toward solving this problem becomes imperative. Our research seeks to assess mycotoxin exposure in Nigerian infants and young children using a multi-mycotoxin based LC-MS/MS for biomarkers. Within the global international context, this study is in line with the sustainable development goal of zero hunger, which incorporates the need to achieve food security and food safety, and improve nutrition, while promoting good health and well-being of the populace and ensuring healthy lifestyle by 2030.

Furan kommt in verschiedenen hitzebehandelten Lebensmitteln vor. Bedingt durch die hohe Flüchtigkeit von Furan kann eine verlässliche Ermittlung der Exposition über Verzehrsdaten und den Gehalt an Furan in Lebensmitteln nicht sichergestellt werden. Vor dem Hintergrund des geringen Abstandes zwischen der bisher geschätzten humanen Furan-Exposition und Dosen, die im Tierversuch adverse Effekte auslösen, ist eine verbesserte Expositionsabschätzung als Basis für eine wissenschaftlich fundierte Risikobewertung essentiell. Ziel des beantragten Forschungsvorhaben ist es, unter Berücksichtigung des Beitrags einer möglichen endogenen Bildung von Furan die Validität eines Biomarker-basierten Ansatzes zur Abschätzung der Exposition gegenüber Furan aus Nahrungsmitteln zu prüfen. Nach Gabe von Isotopen-markiertem [3,4-¹³C]-Furan an Ratten soll erstmals in einem für den Menschen relevanten Dosisbereich die Korrelation zwischen aufgenommener Dosis und Konzentration verschiedener Furan-Metabolite im Urin über die Zeit exakt ermittelt und die externe Exposition gegenüber einer möglichen endogenen Bildung bzw. Hintergrundbelastung durch Furan im Futter abgegrenzt werden. Anhand der tierexperimentell gewonnene Daten sollen Aussagen getroffen werden, welche Metabolite isoliert bzw. in Kombination geeignet sind, unter realistischen Expositionsszenarien die externe Furan-Exposition verlässlich zu ermitteln. Um die Validität der untersuchten Metabolite zur Verwendung als Biomarker der Exposition im Humanbiomonitoring zu überprüfen, sollen diese abschließend im Rahmen einer Proof-of-Concept Studie im Urin von Probanden nach Aufnahme von Furan über die Nahrung (nach Verzehr von furanarmen Lebensmitteln bzw. Lebensmittel mit bekannt hohen Gehalt an Furan) ermittelt werden und daraus die Furan-Exposition der Probanden abgeschätzt werden.

Während der 2007 veröffentlichte Bericht des US National Research Council (NRC) “Toxicity Testing in the 21st Century: A Vision and A Strategy“ (Tox21) einen vollständigen Paradigmenwechsel in der toxikologischen Prüfung und Risikobewertung weg von apikalen Endpunkten für Toxizität im Tier hin zu in vitro Hochdurchsatzverfahren in Zell-basierten Testsystemen vorsieht, zeigen erste Erfahrungen, dass Zell-basierte Systeme, die komplexe Vorgänge im Organismus in der Regel nicht oder nur unzureichend widerspiegeln, alleine derzeit keine hinreichend verlässlichen Aussagen über mögliche Gesundheitsrisiken von Arzneimitteln und Chemikalien erlauben.  Es besteht jedoch Konsens, dass ein erster Schritt hin zu tierversuchsfreien Methoden in der Entwicklung von mehrstufigen Teststrategien liegt, die moderne in silico Methoden, in vitro Bioaktivitätsassays und quantitative in vitro-in vivo Extrapolation (Stufe 1) mit Untersuchungen an alternativen Modellorganismen (Stufe 2) und – wenn nötig – konventionellen Guideline-Studien (Stufe 3) integrieren. Untersuchungen an Embryonen des Zebrabärblings (Danio rerio), die nach den Regelungen des Tierschutzgesetzes bis zum 5. Tag nach der Befruchtung nicht als Tierversuch sondern als in vitro Tests eingestuft werden, eignen sich in besonderer Weise als Alternativmodell für Toxizitätsscreening, das einerseits Kosten-effizient und Mittel- bis Hochdurchsatz-fähig ist, auf der anderen Seite - anders als Zell-basierte Assays - die physiologischen Gegebenheiten im Gesamttier, insbesondere Kinetik und Biotransformation, besser widerspiegelt.

In Ergänzung zu bestehenden Forschungsvorhaben, die in silico und in vitro Prädiktionsmodelle (BMBF Projekt Risk-IT, Fkz031L0019A) für schädigende Wirkung auf die Niere als ein wichtiges, exemplarisches Zielorgan für systemische Toxizität von Fremdstoffen adressieren, wird im Projekt die grundsätzliche Eignung von Embryonen des Zebrabärblings als Teil einer mehrstufigen, für regulatorische Entscheidungen geeigneten Teststrategie zur Prüfung auf Nephrotoxizität untersucht, um Einschränkungen von in vitro Tests zu überwinden und die Lücke zwischen Zell-basierten Hochdurchsatz Screening Assays und Toxizitätsprüfungen am Tier zu schließen. Damit soll die zur toxikologischen Prüfung zur Verfügung stehende in silico/in vitro Toolbox sinnvoll ergänzt werden, so dass insgesamt eine verlässliche Bewertung des Risikos basierend auf tierversuchsfreien Methoden erreicht werden kann.

While toxicity observed during preclinical stages of drug development continues to be a leading cause of drug attrition, a systematic evaluation of the frequency of a particular treatment related toxicity finding across studies, the concordance between histopathological and clinical chemistry findings, the impact of study duration, species-specificity of a particular toxicity finding, and finally incidences in control animals, has been hampered by the lack of access to sufficiently large and representative data sets of high-quality in vivo preclinical safety data generated by pharmaceutical companies during drug development.

The Innovative Medicines Initiative (IMI) JU eTOX project was initiated to develop innovative strategies and novel software tools to better predict the safety and the side-effects of new candidate medicines. Core to this project, the eTOXsys database was established by extracting in vivo preclinical safety data from systemic toxicity reports of the participating pharmaceutical companies. The eTOXsys database, currently the largest and most detailed preclinical database, provides a unique opportunity to systematically analyse preclinical toxicity data across multiple companies and extract information which may help improve testing and decision making in drug development.

The overall objective of this project is to mine the eTOXsys database in order to obtain reliable data on the frequency of different types of toxicity findings and consistency across study duration and species.

Risk-IT integrates expert knowledge gained from previous systems toxicology projects with innovative in vitro data generation technologies (High Content Screening, Metabolomics) and reverse physiologically-based pharmacokinetic modelling to address the unmet need for non-animal based approaches to predict repeat dose systemic toxicity. Focusing on kidney toxicity as an exemplary area and key target organ of chemical/drug induced toxicity, Risk-IT aims to provide a proof-of-concept for the successful integration of new mechanism-based in vitro methods and toxicokinetic modelling into a tiered testing strategy that is fit-for purposes of regulatory decision making. It is anticipated that the project will directly impact the 3Rs by developing a ready-to-use battery of in vitro assays predictive of nephrotoxicity for use in drug and chemical safety assessment. Moreover, by assessing the confidence in risk assessment based on in vitro data, the outcomes of Risk-IT are expected to boost similar activities in other areas of systemic toxicity.

Project Participants

•             Department of Toxicology, University of Würzburg, Coordinator (A. Mally)

•        Institute for Risk Assessment Sciences, Toxicology Division, Utrecht University, Netherlands (N. Kramer) 

•        Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen (S. di Fiore)

•        BASF, Experimental Toxicology & Ecology, Ludwigshafen, Germany (B. van Ravenzwaay)  

Main objectives

The overall objective of Risk-IT is to accelerate the transition of chemical safety testing from animal-based approaches with limited predictivity for human risk to more predictive, animal-sparing solutions by developing new mechanism-based in vitro methods and providing a proof-of-concept for the successful integration of mechanistic endpoints and QIVIVE modelling for regulatory purposes.

The main scientific aims of Risk-IT are:

• to integrate expert knowledge and existing and newly developed ‘omics data to develop a systems-toxicology-based mechanistic framework for drug/chemical-induced nephrotoxicity in line with the AOP concept
• to use the developed mechanistic framework to establish a fit-for-purpose, battery of mechanism-based in vitro assays for prediction of human  drug/chemical safety for nephrotoxicity
• to provide a proof-of-concept for the successful integration of new mechanism-based in vitro methods and QIVIVE into a tiered testing strategy that is fit-for-purposes of regulatory decision making
• to assess the confidence of risk assessment based on in vitro data

Disseminations

Talks/presentations at national/international events

A. Mally (UW). Poster presentation at EUROTOX 2020, Copenhagen, DNK, 6 – 9.09.2020. Title: “Application of the adverse outcome pathway conceptual framework for translation of mechanistic data into regulatory decisions: adverse outcome pathways for kidney injury as case study”. For more information see: http://www.eurotox-congress.com/2020/

Barbara Birk, Saskia Sperber, Svenja Wallisch, Michael Herold, Volker Haake, Meike Amma, Tilmann Walk, Hennicke Kamp, Bennard van Ravenzwaay (BASF). Short talk at the 86th Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT), 2.03. – 5.03.2020 in Leipzig. Talk title (B. Birk): In vitro Metabolome and biochemical pathway analysis of nephrotoxicants in NRK-52e cells. For more information see: https://www.gpts-kongress.de/

A. Mally, B. Birk, S. Di Fiore, B. Ellinger, S. Jarzina, P. Reiser, F. Taverne, N. Kramer (UW, BASF, FH-IME, UU). Poster presentation at EUROTOX 2019, Helsinki, FI, 8 – 11.09.2019. Poster title: “Application of adverse outcome pathways and quantitative in vitro-in vivo extrapolation (QIVIVE) modelling for risk assessment based on in vitro data”. For more information see: http://www.eurotox-congress.com/2019/?s=abstracts

S. Sperber, V. Starck, B. Birk, V. Haake, H. Kamp, T. Walk, B. van Ravenzwaay (BASF). Poster presentation at the 58^th Annual Meeting of the Society of Toxicology, Baltimore, March 10-14, 2019. Poster title " Mode-of-action prediction of liver toxicants using metabolomics in vitro". For more information related to the Congress see: https://www.toxicology.org/events/am/AM2019/General-Information.asp

Barbara Birk, Saskia Sperber, Svenja Wallisch, Hans-Albrecht Huener, Andreas Verlohner, Tilmann Walk, Volker Haake, Michael Spitzer, Hennicke Kamp, Bennard van Ravenzwaay (BASF). Poster presentation at the 85^th Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT), 25. - 28.02.2019, Stuttgart. Poster title "In vitro metabolome analysis predicts nephrotoxicity and related modes of action". For more information related to the Congress see: https://www.gpts-kongress.de/

A. Mally  (UW), Invited speaker at the 20th International Congress on In Vitro Toxicology (ESTIV2018), Berlin, DE, 15 – 18.10.2018 . Talk title "Integration of mechanism-based in vitro methods and quantitative in vitro-in vivo extrapolation (QIVIVE) modelling for prediction of nephrotoxicity". For mor information related to the Congress see: https://www.estiv2018.com/

S. Jarzina, P. Reiser, J. Scherer, B. Ellinger, S. DiFiore, A. Mally  (UW, FH-IME), Poster presentation at the 20th International Congress on In Vitro Toxicology (ESTIV2018), Berlin, DE, 15 – 18.10.2018 . Poster title "Application of the Adverse outcome pathways (AOP) concept to systemic toxicity assessment in vitro: Kidney injury due to lysosomal overload and inhibition of mtDNA polymerase-γ as case studies". For mor information related to the Congress see: https://www.estiv2018.com/

A. Mally (UW) Session chair of the joint workshop  of Innosystox projects "Systems-biology-based 3R methods in toxicology" at EUROTOX 2018, Brussels, 03.09.208. Talk title (A. Mally): "Integration of mechanism-based in vitro methods and quantitative in vitro-in vivo extrapolation (QIVIVE) modelling for prediction of nephrotoxicity" (UW, BASF, UU, FH-IME). For abstract see: http://eventclass.org/contxt_eurotox2018/

S. Jarzina, P. Reiser, J. Scherer, S. DiFiore, B. Ellinger, A. Mally  (UW, FH-IME), Poster presentation at EUROTOX 2018, Brussels, BE, 02 – 05.09.2018. Poster title "The Adverse Outcome Pathway (AOP) concept as a basis for the development of mechanism-based animal sparing approaches: Kidney toxicity due to lysosomal overload and inhibition of mtDNA polymerase-γ as case studies" For mor information related to the Congress see: http://www.eurotox-congress.com/2018/

N. Kramer, F. Taverne, A. Mally, S. Jarzina, B. Birk, S. DiFiore, B. Ellinger, R. Gehring (UU, UW, BASF, FH-IME), Poster presentation at EUROTOX 2018, Brussels, BE, 02 – 05.09.2018. Poster title "In Vitro Kinetics and Quantitative In Vitro-In Vivo Extrapolation of Nephrotoxicants" For mor information related to the Congress see: http://www.eurotox-congress.com/2018/

B. Birk, S. Sperber, H.-A. Huener, A. Verlohner, T. Walk, V. Haake, M. Spitzer, H. Kamp, B. van Ravenzwaay  (BASF), Poster presentation at EUROTOX 2018, Brussels, BE, 02 – 05.09.2018. Poster title "Is the prediction of nephrotoxicity and its mode of action by metabolome analysis in vitro in NRK-52e cells feasible?" For mor information related to the Congress see: http://www.eurotox-congress.com/2018/

S. Di Fiore, F. Schmelter, B. Ellinger, S. Jarzina, A. Mally (FH-IME, UU), Poster presentation at EUROTOX 2018, Brussels, BE, 02 – 05.09.2018. Poster title "A High-Content Imaging approach for AOP development of kidney proximal tubule toxicity". For mor information related to the Congress see: http://www.eurotox-congress.com/2018/

Angela Mally, Invited article "The future of chemical safety assessment – how systems toxicology may help to avoid animal testing". Online-journal systembiologie.de 2018, 12:43-45 (available online at systembiologie.de)

Angela Mally, Sebastian Jarzina, Pia Reiser, Femke Taverne, Barbara Birk, Stefano DiFiore, Bernhard Ellinger, Nynke Kramer (UW, UU, FH-IME), Poster presentation at the 57^th Annual Meeting of the Society of Toxicology, San Antonio, TX, March 11-15, 2018. Poster title "Mapping Adverse Outcome Pathways for kidney injury: Opportunities for the development of mechanism-based animal-sparing approaches to assessment of nephrotoxicity".
For mor information related to the Congress see: http://www.toxicology.org/events/am/AM2018/General-Information.asp

Barbara Birk, Saskia Sperber, Hans-Albrecht Huener, Andreas Verlohner, Tilmann Walk, Volker Haake, Michael Spitzer , Tzutzuy Ramirez-Hernandez, Hennicke Kamp and Bennard van Ravenzwaay (BASF), Poster presentation at the 57^th Annual Meeting of the Society of Toxicology, San Antonio, TX, March 11-15, 2018. Poster title "Metabolome analysis in vitro in NRK-52e cells –a potential tool for investigation of mode of action of nephrotoxicants".
For mor information related to the Congress see: http://www.toxicology.org/events/am/AM2018/General-Information.asp

Sebastian Jarzina (UW), Short presentation at the 84^th Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT), 26.02 - 01.03.2018, Göttingen. Presentation title "Application of the adverse outcome pathway concept for prediction of systemic toxicity: Kidney injury due to receptor mediated endocytosis and lysosomal overload as a case study". For mor information related to the Congress see: http://www.gpts-kongress.de/

Barbara Birk, Saskia Sperber, Hans-Albrecht Huener, Andreas Verlohner, Tilmann Walk, Volker Haake, Michael Spitzer , Tzutzuy Ramirez-Hernandez, Hennicke Kamp and Bennard van Ravenzwaay (BASF), Poster presentation at the 84^th Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT), 26.02 - 01.03.2018, Göttingen. Poster title "A promising tool for investigation of the mode of action of nephrotoxicants: metabolome analysis in vitro in NRK-52e cells". For more information related to the Congress see: http://www.gpts-kongress.de/

Angela Mally, Invited article "Die Zukunft der Sicherheitsbewertung von Chemikalien - Wie die Systemtoxikologie zur Vermeidung von Tierversuchen beiträgt". Online-journal systembiologie.de 2017, 12:43-45 (available online at systembiologie.de)

Femke J. Taverne, Nynke I. Kramer, Bas J. Blaauboer (UU), Poster presentation at the 10^th World Congress on Alternatives and Animal Use in the Life Sciences (3Rs In Action), Seattle WA, 20 – 24.08.2017. Poster title "Comparing cytotoxicity and in vitro kinetics of PFOA in different cell lines". For more information related to the Congress see: http://wc10seattle.org/2017/home.aspx

Barbara Birk et al. (BASF), Poster presentation at the 10^th World Congress on Alternatives and Animal Use in the Life Sciences (3Rs In Action), Seattle WA, 20 – 24.08.2017. Poster title “Investigation of nephrotoxicity and its mode of action by metabolomics in vitro”.
For more information related to the Congress see: http://wc10seattle.org/2017/home.aspx

Angela Mally (Uni Würzburg), Invited Talk OPENTOX EURO 2016, Rheinfelden, Germany: Adverse outcome pathways as a mechanistic basis for the development of animal-sparing approaches to asessment of repeat dose systemic toxicity.
For more information related to the conference see: http://www.opentox.net/events/opentox-euro-2016 and http://www.opentox.net/events/opentox-euro-16/assessment-of-repeat-dose-systemic-toxicity

Press release

05.09.2016, einBLICK - Das Online-Magazin der Universität Würzburg

Zellkulturen auf dem Prüfstand

Wie gut sind Zellkulturen, wenn man die von Chemikalien ausgehenden Gesundheitsrisiken überprüfen will? Das erforscht ein Team am Lehrstuhl für Toxikologie in Kooperation mit der BASF SE und anderen Partnern.
Will die Industrie Pflanzenschutzmittel, Klebstoffe oder andere Chemikalien auf den Markt bringen, muss sie prüfen, ob die Substanzen die Gesundheit des Menschen gefährden können....
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Summary

Idiosyncratic drug reactions (IDRs) are a rare but major complication of drug therapy and drug development. Although a basic understanding of the chemical and biological events leading to idiosyncratic drug toxicity is still lacking, it appears that an individual´s susceptibility to rare adverse drug reactions is determined by the interaction of metabolic, genetic and environmental factors. Thus, genetic or environmental factors that increase reactive metabolite formation, alter cellular stress and immune response, or a combination of these may be critical determinants in the response to an otherwise non-toxic drug. The objectives of this project are to explore the relationship between reactive metabolite formation and molecular signaling after continuous exposure to a model drug associated with drug idiosyncrasy and to test the hypothesis that various environmental factors may reduce the threshold for liver toxicity through modulation of drug metabolism and/or sensitization of the liver through up-regulation of co-stimulatory factors. It is expected that results from these studies will not only provide a better understanding of the basic mechanisms related to idiosyncratic drug reactions, but will also help establish animal models more relevant to the human situation with improved sensitivity to predict potential drug hazards. Moreover, results from these studies may contribute to a more fundamental understanding as to how environmental factors or co-exposure to chemicals may introduce temporal variability into the susceptibility of an individual by altering the threshold of toxicity of a compound.

Innovative Medicines for Europe

Objective

The discovery and development of new drugs is very costly and attrition rates are high. Initiatives to reduce the rate of attrition during later phases are clearly desirable and if successfully implemented will reduce development costs. The InnoMed proposal addresses the complex issues associated with the future of biomédical research in the EU, and addresses ways of achieving accelerated development of, safe and more effective medicines, aiming to revitalize the European biopharmaceutical research environment. InnoMed's wide consortium base, being led by the European Federation of Pharmaceutical Industry and Associations (EFPIA), guarantee's a commitment from all the stakeholders needed to change the process of drug development in Europe. The course for addressing the necessary changes is to first develop a Strategic Research Agenda (SRA) that will encompass the whole path from discovery of a new drug target to the validation and approval stages of a new drug compound. This will be agreed by all the relevant stakeholders via meetings and workshops. Four key bottlenecks in the drug development process will be addressed: Safety Efficacy Knowledge Management Training and Education This comprehensive strategy with a detailed roadmap will lead to the deployment of a European Technology Platform (ETP). This European Technology Platform will deliver added value to the drug discovery and development process and to individual stakeholders. To ensure the relevance of the ETP InnoMed will validate it using two research proposals, AddNeuroMed, which will develop and validate novel surrogate markers based upon in vitro and in vivo models in animals and humans, using Alzheimer's disease as a testing platform. PredTox will deliver new biomarkers of toxicity and a greater understanding of mechanisms of toxicity. Both of these projects will demonstrate the viability and necessity of the ETP.

PredTox: Summary Results

EU Framework 6 Project: Predictive Toxicology (PredTox)—overview and outcome. Suter L, Schroeder S, Meyer K, Gautier JC, Amberg A, Wendt M, Gmuender H, Mally A, Boitier E, Ellinger-Ziegelbauer H, Matheis K, Pfannkuch F. Toxicol Appl Pharmacol. 2011 Apr 15;252(2):73-84. doi: 10.1016/j.taap.2010.10.008. Epub 2010 Oct 16. PMID:20955723
 

PredTox: Selected publications

Development of a pharmaceutical hepatotoxicity biomarker panel using a discovery to targeted proteomics approach. Collins BC, Miller CA, Sposny A, Hewitt P, Wells M, Gallagher WM, Pennington SR. Mol Cell Proteomics. 2012 Aug;11(8):394-410. doi:10.1074/mcp.M111.016493. Epub 2012 Apr 23. PMID: 22527513

Integrated transcriptomic and proteomic evaluation of gentamicin nephrotoxicity in rats. Com E, Boitier E, Marchandeau JP, Brandenburg A, Schroeder S, Hoffmann D, Mally A, Gautier JC. Toxicol Appl Pharmacol. 2012 Jan 1;258(1):124-33. doi: 10.1016/j.taap.2011.10.015. Epub 2011 Oct 29. PMID: 22061828

A comparative integrated transcript analysis and functional characterization of differential mechanisms for induction of liver hypertrophy in the rat. Boitier E, Amberg A, Barbié V, Blichenberg A, Brandenburg A, Gmuender H, Gruhler A, McCarthy D, Meyer K, Riefke B, Raschke M, Schoonen W, Sieber M, Suter L, Thomas CE, Sajot N. Toxicol Appl Pharmacol. 2011 Apr 15;252(2):85-96. doi: 10.1016/j.taap.2011.01.021. Epub 2011 Feb 26. PMID:21315101

Cross-study and cross-omics comparisons of three nephrotoxic compounds reveal mechanistic insights and new candidate biomarkers. Matheis KA, Com E, Gautier JC, Guerreiro N, Brandenburg A, Gmuender H, Sposny A, Hewitt P, Amberg A, Boernsen O, Riefke B, Hoffmann D, Mally A, Kalkuhl A, Suter L, Dieterle F, Staedtler F. Toxicol Appl Pharmacol. 2011 Apr 15;252(2):112-22. doi: 10.1016/j.taap.2010.11.006. Epub 2010 Nov 21. PMID:21081137

Tissue microarrays and digital image analysis. Ryan D, Mulrane L, Rexhepaj E, Gallagher WM. Methods Mol Biol. 2011;691:97-112. doi: 10.1007/978-1-60761-849-2_6. PMID: 20972749

The enhanced value of combining conventional and "omics" analyses in early assessment of drug-induced hepatobiliary injury. Ellinger-Ziegelbauer H, Adler M, Amberg A, Brandenburg A, Callanan JJ, Connor S, Fountoulakis M, Gmuender H, Gruhler A, Hewitt P, Hodson M, Matheis KA, McCarthy D, Raschke M, Riefke B, Schmitt CS, Sieber M, Sposny A, Suter L, Sweatman B, Mally A. Toxicol Appl Pharmacol. 2011 Apr 15;252(2):97-111. doi: 10.1016/j.taap.2010.09.022. Epub 2010 Oct 1. PMID: 20888850

Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity. Hoffmann D, Fuchs TC, Henzler T, Matheis KA, Herget T, Dekant W, Hewitt P, Mally A. Toxicology. 2010 Nov 9;277(1-3):49-58. doi: 10.1016/j.tox.2010.08.013. Epub 2010 Sep 9. PMID: 20816719

Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies. Adler M, Hoffmann D, Ellinger-Ziegelbauer H, Hewitt P, Matheis K, Mulrane L, Gallagher WM, Callanan JJ, Suter L, Fountoulakis MM, Dekant W, Mally, A. Toxicol Lett. 2010 Jun 16;196(1):1-11. doi: 10.1016/j.toxlet.2010.03.018. Epub 2010 Apr 1. PMID: 20362651

Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies. Adler M, Hoffmann D, Ellinger-Ziegelbauer H, Hewitt P, Matheis K, Mulrane L, Gallagher WM, Callanan JJ, Suter L, Fountoulakis MM, Dekant W, Mally A. Toxicol Lett. 2010 Jun 16;196(1):1-11. doi: 10.1016/j.toxlet.2010.03.018. Epub 2010 Apr 1. PMID:20362651

Use of SELDI MS to discover and identify potential biomarkers of toxicity in InnoMed PredTox: a multi-site, multi-compound study. Collins BC, Sposny A, McCarthy D, Brandenburg A, Woodbury R, Pennington SR, Gautier JC, Hewitt P, Gallagher WM. Proteomics. 2010 Apr;10(8):1592-608. doi: 10.1002/pmic.200900608. PMID: 20162557

Performance of novel kidney biomarkers in preclinical toxicity studies. Hoffmann D, Adler M, Vaidya VS, Rached E, Mulrane L, Gallagher WM, Callanan JJ, Gautier JC, Matheis K, Staedtler F, Dieterle F, Brandenburg A, Sposny A, Hewitt P, Ellinger-Ziegelbauer H, Bonventre JV, Dekant W, Mally A. Toxicol Sci. 2010 Jul;116(1):8-22. doi: 10.1093/toxsci/kfq029. Epub 2010 Jan 29. PMID: 20118187

Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity. Sieber M, Hoffmann D, Adler M, Vaidya VS, Clement M, Bonventre JV, Zidek N, Rached E, Amberg A, Callanan JJ, Dekant W, Mally A. Toxicol Sci. 2009 Jun;109(2):336-49. doi: 10.1093/toxsci/kfp070. Epub 2009 Apr 6. PMID: 19349640

Creation of a digital slide and tissue microarray resource from a multi-institutional predictive toxicology study in the rat: an initial report from the PredTox group. Mulrane L, Rexhepaj E, Smart V, Callanan JJ, Orhan D, Eldem T, Mally A, Schroeder S, Meyer K, Wendt M, O'Shea D, Gallagher WM. Exp Toxicol Pathol. 2008 Aug;60(4-5):235-45. doi: 10.1016/j.etp.2007.12.004. Epub 2008 May 13. PMID: 18479893

Coordinator

EUROPEAN FEDERATION OF PHARMACEUTICAL INDUSTRIES AND ASSOCIATIONS, Belgium

Participants

F. HOFFMANN-LA ROCHE LTD, Switzerland

BOEHRINGER INGELHEIM GMBH, Germany

EUROPEAN FEDERATION OF BIOTECHNOLOGY, Belgium

INSTITUT DE RECHERCHES INTERNATIONALES SERVIER, France

ELI LILLY AND COMPANY, United States

MERCK KGAA, Germany

NOVO NORDISK A/S, Denmark

PHARMIDEX PHARMACEUTICAL SERVICES LIMITED, United Kingdom

JULIUS-MAXIMILLIAMS-UNIVERSITAET WUERZBURG, Germany

CAPSANT NEUROTECHNOLOGIES LTD, United Kingdom

UNIVERSITY OF KUOPIO, Finland

UNIVERSITA DEGLI STUDI DI PERUGIA, Italy

ARISTOTLE UNIVERSITY OF THESSALONIKI, Greece

ROSKILDE UNIVERSITETSCENTER, Denmark

ASTRAZENECA AB, Sweden

KUNGLIGA TEKNISKA HOEGSKOLAN, Sweden

KAROLINSKA INSTITUTET, Sweden

NOVARTIS PHARMA AG, Switzerland

N.V. ORGANON, Netherlands

GENEDATA AG, Switzerland

KING'S COLLEGE, LONDON, United Kingdom

ISTITUTO DI RICERCHE BIOMEDICHE "A. MARXER" RBM SPA, Italy

CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE, France

GLAXOSMITHKLINE RESEARCH & DEVELOPMENT LTD, United Kingdom

UNIVERSITY COLLEGE DUBLIN, NATIONAL UNIVERSITY OF IRELAND, DUBLIN, Ireland

PROTEOME SCIENCES PLC, United Kingdom

HACETTEPE UNIVERSITESI, Turkey

UNIVERSITY COLLEGE LONDON, United Kingdom

UNIVERSITY OF SOUTHAMPTON, United Kingdom

HUNTER FLEMING LIMITED, United Kingdom

SANOFI-AVENTIS DEUTSCHLAND GMBH, Germany

ARTTIC IN BRUSSELS SPRL, Belgium

JANSSEN PHARMACEUTICA N.V, Belgium

BIOWISDOM LIMITED, United Kingdom

CEREBRICON LTD., Finland

EUROPEAN FEDERATION FOR PHARMACEUTICAL SCIENCES, Sweden

DEPARTMENT OF OLD AGE PSYCHIATRY & PSYCHOTIC DISORDERS, MEDICAL UNIVERSITY OF LODZ, Poland

CIPHERGEN BIOSYSTEMS, INC., United States

NYCOMED GMBH, Germany

AVENTIS PHARMA RECHERCHE-DÉVELOPPEMENT, France

BAYER PHARMA AG, Germany

Summary

The proposal will address modes of action for tumor induction in liver by the food contaminant furan, which is formed by processing of food resulting in widespread human exposure. There is uncertainty regarding the relevance of tumors induced in rodents to human risk assessment because the mechanisms are unclear. The research will address the role of DNA and protein binding of furan, oxidative DNA damage, non-genotoxic alteration of proliferation and apoptosis, cytogenetics and cytotoxicity in furan-mediated liver toxicity and carcinogenicity. A combination of in vivo and in vitro systems, analytical chemistry, cell biology and “omics” technologies will be applied. In rodents in vivo, extent and dose-dependence of covalent binding to DNA, cytogenetic changes and geno- and cytotoxicity in target cells in the liver will be addressed after oral administration of furan. In addition, the induction of oxidative DNA-modifications and mechanisms of mutations will be investigated in genetically modified rodent models. These in vivo studies will characterize the mode of action of furan and also address irreversible metaplasia, changes in cell signaling and inflammation. The interaction of these effects with possible genetic changes in liver cells including aspects of forced cell proliferation will be included. The in vivo work will be complemented by studying mode of mutation of furan and its metabolite cis-2-butene-1,4-dial in cell culture models resembling the target cells. The content of furan in food will be determined and human exposures will be assessed using probabilistic modeling. Mechanisms of formation of furan in food may open ways to reduce exposures. The results will provide data on the mode-of-action of furan induced liver carcinogenesis as a basis for a conclusive assessment of health risks in humans due to dietary exposure. Combining these findings will provide a risk/benefit analysis and a scientific basis to justify limits for human furan exposures.

Project Participants

Participant 1 (coordinator), University of Würzburg, Germany
Participant 2, Istituto Superiore di Sanita', Italy
Participant 3, University of Kaiserslautern, Germany
Participant 4, University of Birmingham, U. K.
Participant 5, Università degli Studi della Tuscia
Participant 6, Free University of Amsterdam, Netherlands
Participant 7, Euro Environmental Containers, U. K.
Participant 8, Nestlé Research Center Lausanne, Switzerland

Project Objectives

After the discovery of the formation of acrylamide during heat processing of the food, more recent studies have also indicated that the simple organic molecule furan is present in a wide variety of foods and may also be formed during heat processing. Like acrylamide, furan until recently has also been known only as an industrial chemical and most of the toxicity data elaborated up to now are not a suitable basis for a the required detailed risk assessment of human exposures to furan with food.

Since the mechanisms of carcinogenic activity of furan in rodents are not well understood, the objectives of this proposal are to generate relevant mechanistic information as a support for the ongoing risk assessment of human furan exposures with food. The importance of mode-of-action research on furan is underscored by the comparatively small difference in the estimated human exposures and the doses of furan, which cause carcinoma in the liver of experimental animals.

A detailed elucidation of genotoxic and non-genotoxic mechanisms and their possible dose-response relationships and interconnectivity are of fundamental importance for a reliable risk assessment.

The final outcome of the proposed research will be a careful assessment of the genotoxic potential of furan in the target cells in vivo for carcinogenicity, generation of information on the potential relevance of DNA lesions and the tissue environment changes that may exacerbate mutations. The methods applied will include state-of-the-art analytical procedures to quantify furan induced DNA-adducts in very low concentrations and other DNA-damage or cytogenetic changes, biomarkers, gene-arrays and modern cell biology to characterize furan induced changes in cellular function in the target cells of carcinogenesis in relevant species.

The proposed research has the following major objectives:

1. Characterization and quantitation of DNA- and protein binding of furan in liver of rats and mice over a wide dose range. Both responses after single and after repeated exposures will be assessed. (WPs 1.1 and 1.2)
2. Analysis of biomarkers of toxicity, genotoxicity and epigenetic changes affecting gene expression, tissue structure changes and cell proliferation in target cell populations of furan in order to elucidate cell-specific mechanisms. Again, special attention will be given to determine the dose dependence of the effects. (WPs 2.1. and 2.2)
3. Assessment of the genotoxic and clastogenic potential of furan in rodent liver by comet assay, cytogenetics and biomarkers of genetic damage. (WPs 3.1 and 3.2)
4. Assessment of the role of the accumulation of oxidative DNA damage in the mechanism of neoplastic transformation by means of a genetically modified mouse models. (WP 4)
5. Detailed analysis of gene expression changes. (WP5)
6. Characterization and quantitative assessment of genetic changes induced by furan and cis-2-butene-1,4-dial in mammalian cells with special consideration on mechanisms for induction of gene mutations and cytogenetic changes. (WPs 6.1, 6.2 and 6.3)
7. Characterization of furan and cis-2-butene-1,4-dial induced effects on toxicity parameters, and DNA-damage in cell culture models for the specific target cells of furan in order to elucidate cell-specific mechanisms. (WP 7)
8. Analysis of furan in food as a better basis for exposure assessment from food and mechanisms of formation during food processing. (WPs 8.1 and 8.2)
9. Provision of a risk assessment of furan in food. (WP 9)

The results will provide data on the mode-of-action of furan induced liver carcinogenesis as a basis for a conclusive assessment of health risks in humans due to dietary exposure. Combining these findings will provide a risk/benefit analysis and a scientific basis to justify limits for human furan exposures. All results of the project will be published in peer-reviewed scientific journals to make the scientific and regulatory community aware of the project results and as a measure of project success. Assessment and evaluation of WP results and progress towards the objectives will be monitored by the participants in each workpackage.

Graphical presentation

Workpackages

Summary of project results

Strategies for furan reduction in food systems

The formation of furan from linoleic and linolenic acid, respective triglycerides, and (E,E)‑2,4-decadienal was studied in the presence of various antioxidants, prooxidants, and inert atmosphere. Study was carried out in model systems simulating food processing such as roasting and pressure cooking (sterilization). The formation of furan considerably varied depending on antioxidant, precursor (free PUFA vs. triglyceride) and conditions of heating including pH. Under roasting conditions all precursors except (E,E)‑2,4-decadienal showed unexpected increase (from +37 to +89 %) of furan formation in the presence of BHT and ‑tocopherol. Under pressure cooking conditions, BHT revealed significant effect on furan reductions (from ‑45 to -100 %) in all systems investigated. Contrariwise, effects of ‑tocopherol and ascorbyl palmitate resulted in both decrease and increase of furan formation depending mainly on pH and precursor. When precursors were heated in a nitrogen atmosphere, the furan yields from all precursors were lowered under both roasting (from -87 % to -92 %) and pressure cooking (from -68 % to -98 %) conditions. Under sterilization conditions (pH 4 and 7), ascorbic acid revealed a strong prooxidative effect on linoleic acid leading to a growth of furan formation. The effect was higher at pH 7 than at pH 4 and increased with concentration of ascorbic acid. The generation of furan from ascorbic acid alone was minor compared to PUFA precursors.

It was observed that effects of antioxidants on furan formation considerably varied depending on antioxidant, precursor (free PUFA vs. triglyceride) and conditions of heating including pH. Fortification of foods by ‑tocopherol prior to heating seems to be a promising mitigation strategy, which should be further investigated. Furthermore, it is well known that tocopherols posses a strong synergistic effect with AA, which could allow to develop a very efficient antioxidant system for reduction of furan formation. Nevertheless the concentration of AA should be properly optimized with regard to simultaneous prooxidant properties. Modification of atmospheres within heating systems seems to be also very promising mitigation concept. However, such a strategy would require a significant modification of technological process. Obtained data confirmed that AA in the mixture with PUFA acts as a prooxidant rather than direct furan precursor. This effect is higher at pH 7 than at pH 4 and increases with concentration of ascorbic acid. Surprisingly, no suppression of furan generation was observed as one would expect due to known antioxidant effect of AA at high levels. The results showed that effect of AA is not linked with decrease of pH after its addition as speculated before.

Risk assessment of furan

Due to genotoxicity and DNA-binding of furan established in the project, the current risk assessment for furan requires application of a linear dose response relationship. Exposures to furan in adults are estimated to be less than 0.5 μg/kg bw/day, data from this project also support a similar intake of furan in children. Due to the high incidence of tumors induced by furan in rats even at the lowest applied dose, a linear extrapolation of the animal data to calculate possible human tumor risks is uncertain. When using a margin-of-exposure assessment, MoEs to a dose inducing a significant tumor incidence are calculated as only 1000 to 2000. The low MoEs warrant further reductions in furan-exposures.

Summary

In a recent investigation, the U.S. Food and Drug Administration (FDA) identified the chemical furan in a variety of food items that undergo heat treatment. Furan is a potent hepatotoxicant and liver carcinogen in rodents. Chronic administration of furan to rats at doses as low as 2 mg/kg b.w. results in very high incidences of cholangiocarcinoma. Although data on human intake of furan are limited, it appears that there is a relatively narrow margin between human exposure and doses which cause liver tumors in rodents, suggesting that the presence of furan in food may present a potential risk to human health. However, the presently available data on furan toxicity is insufficient to perform a risk assessment and more research regarding the mechanism of furan carcinogenicity is needed.  In an in vivo study using 14C-labeled furan, covalent binding of furan or its reactive metabolite cis-2-butene-1,4-dial  to DNA could not be established. In contrast, 80% of the radioactivity present in livers was found to be associated with proteins. It has been suggested that chronic inflammation and enhanced biliary cell proliferation in response to furan cytotoxicity, mediated through binding of cis-2-butene-1,4-dial to critical target proteins, is likely to play a key role in furan carcinogenicity. The aim of our work is to identify target proteins of furan reactive intermediates and to characterize the cellular and functional consequences associated with protein damage in livers of rats treated with furan. With the knowledge to be gained in this project, particularly in relation to the dose-response relationships, decisions can be made as to whether furan levels in food need to be controlled.

The mycotoxin and food contaminant ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date. The molecular events leading to renal tumor formation by OTA are still poorly understood, but increasing evidence suggests that OTA is not a mutagenic, DNA-reactive carcinogen. Recent work in our laboratory provide important new evidence in support of a mechanism of OTA carciogenicity involving disruption of mitosis, presumably through interference with key regulators of chromosome separation and progression through mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition and subsequent tumor formation.

Publikationen

Pubmed