Evaluation of new alternative strategies to predict neurotoxicity with human based test systems

Krug, Anne K.

Evaluation of new alternative strategies to predict neurotoxicity with human based test systems

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Dissertation_Anne-Krug_flat.pdf(8.58 MB)

Date

2013

Publication type

Dissertation

Abstract

Animal experiments are still the ‘gold standard’ in safety evaluation defined by the OECD (Organisation for Economic Co-operation and Development) or the US EPA (Environmental Protection Agency). Millions of animals are used each year to assess the risk of chemical toxicities for human health. But animal experiments are expensive, time-consuming and have a restricted prediction capacity regarding human toxicity. Hence the demand for validated alternative strategies is high. Validated differentiation protocols of embryonic stem cells or immortalized human organ specific cell lines provide the possibility to recapitulate human development and to study organ specific toxicity of different developmental stages (immature to mature) in vitro. In the framework of this doctoral thesis, we provide insights into the development and evaluation of test systems established specifically to assess neurodevelopmental toxicity as well as neurotoxicity in vitro.

In a first step we evaluated an assay based on neurite outgrowth assessment to detect putative developmentally neurotoxic chemicals. This assay was based on a human mesencephalic neuronal precursor cell line, called LUHMES. In the study, the model has been challenged for its reliability and consistency using more than 50 compounds and combinations of them. We proved the applicability of the assay for screening, and suggest that the test has the potential to be used for identification and potency-ranking of putative developmental toxicants with regard to effects on neurite growth.
In a second step we used different human stem cell-based test systems to mimic several stages of the early human neurodevelopment in vitro. We analysed the transcriptome changes of these test systems after exposure to two developmental toxicants, valproic acid and methylmercury. Both toxicants induced test system and compound specific transcriptome changes. A common toxicant specific signature of transcription factor binding sites was identified for the different test systems, which we suggest as classifier for compound grouping in future experiments.

In a last step we used a well described model compound 1-methyl-4-phenylpyridinium (MPP+) to analyse the suitability of Omics combinations to monitor the MPP+ induced changes on LUHMES. We found early large adaptive metabolome and transcriptome changes which taken together lead to the identification of novel pathways involved in early MPP+ toxicity. The findings of this thesis contribute to alternative test-strategy development in neurotoxicity and disclose important considerations when developing in vitro test systems.

Summary in another language

Tierversuche sind nach wie vor der Goldstandard für die Sicherheitsbewertung, die von der OECD bzw. der US EPA vorgeschrieben wird. Millionen Tiere werden jedes Jahr benötigt, um die Gefahr von chemischen Substanzen für die menschliche Gesundheit abzuschätzen. Aber Tierversuche sind teuer, zeitintensiv und haben eine beschränkte Voraussagekraft bezüglich menschlicher Toxizität. Daher ist die Nachfrage an validierten Alternativstrategien hoch. Differenzierungsprotokolle embryonaler Stammzellen oder humaner Organ spezifischer Zelllinien, ermöglichen die Rekapitulation humaner Entwicklung und die Untersuchung Organ spezifischer Toxizität während unterschiedlicher Entwicklungsstadien in vitro. Im Rahmen dieser Doktorarbeit bieten wir Einblick in die Entwicklung und Evaluierung von Testsystemen, die spezifisch für die Untersuchung neuronaler Entwicklungstoxizität und Neurotoxizität in vitro hergestellt wurden.

Im ersten Teil dieser Arbeit galt es einen Assay zu bewerten, der auf die Untersuchung von Neuritenwachstum ausgelegt ist, um mögliche neuronale Entwicklungstoxikantien zu identifizieren. Der Assay basiert auf humanen neuronalen Vorläuferzellen, den LUHMES. Das vorgelegte Modell wurde auf seine Verlässlichkeit und Konsistenz getestet, indem mehr als 50 verschiedene Substanzen und Kombinationen dieser eingesetzt wurden. Wir zeigen die Anwendbarkeit dieses Assays für Screenings und untermauern sein Potenzial für die Identifikation möglicher Entwicklungstoxikantien in Hinsicht auf gestörtes Neuritenwachstum.
Im zweiten Teil verwendeten wir Testsysteme, basierend auf humanen Stammzellen, um verschiedene Stadien der frühen humanen neuronalen Entwicklung in vitro darzustellen. Wir analysierten die Veränderungen auf Transkriptionsebene nachdem die Test-Systeme zwei Entwicklungstoxikantien, Methylquecksilber und Valproinsäure, ausgesetzt waren. Beide Substanzen induzierten testsystem- und substanzspezifische Veränderungen in der Transkription. Eine substanzspezifische und Testsystem übergreifende Signatur in Transkriptionsfaktor-Bindestellen wurden identifiziert, die wir als Klassifikator für zukünftige Experimente vorschlagen um ähnliche Substanzen zu gruppieren.

Im letzten Schritt benutzten wir die viel beschriebene Substanz 1-Methyl-4-Phenylpridinium (MPP+) um die Anwendbarkeit von Omics-Kombinationen zu analysieren und um die MPP+-induzierten Veränderungen in LUHMES zu überprüfen. Wir beobachteten erhebliche Anpassungen auf metabolischer und transkriptioneller Ebene, die zusammengenommen zur Identifikation neuer Reaktionswege führten, die in die MPP+-Toxizität involviert sind. Die Erkenntnisse, die aus dieser Arbeit gewonnen wurden, tragen zur Entwicklung von alternativen Teststrategien bei und weisen auf wichtige Ansichten hin, wenn solche in vitro Systeme entwickelt werden.

Subject (DDC)

570 Biosciences, Biology

Cite This

ISO 690

KRUG, Anne K., 2013. Evaluation of new alternative strategies to predict neurotoxicity with human based test systems [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Krug2013Evalu-25743,
  year={2013},
  title={Evaluation of new alternative strategies to predict neurotoxicity with human based test systems},
  author={Krug, Anne K.},
  address={Konstanz},
  school={Universität Konstanz}
}

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Examination date of dissertation

December 17, 2013