Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Bastian Niklas Hölzel - , Institute for Ecology of Waters and Applied Biology (Author)
  • Kurt Pfannkuche - , University of Cologne (Author)
  • Bernhard Allner - , Institute for Ecology of Waters and Applied Biology (Author)
  • Hans Thomas Allner - , Institute for Ecology of Waters and Applied Biology (Author)
  • Jürgen Hescheler - , University of Cologne (Author)
  • Daniel Derichsweiler - , University of Cologne (Author)
  • Henner Hollert - , University Hospital Frankfurt (Author)
  • Andreas Schiwy - , University Hospital Frankfurt (Author)
  • Julia Brendt - , University Hospital Aachen (Author)
  • Michael Schaffeld - , Johannes Gutenberg University Mainz (Author)
  • Alexander Froschauer - , Faculty of Biology, Chair of History of Saxony, Fraunhofer Institute for Molecular Biology and Applied Ecology, Dresden University of Technology (Author)
  • Petra Stahlschmidt-Allner - , Institute for Ecology of Waters and Applied Biology (Author)

Abstract

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

Details

Original languageEnglish
Pages (from-to)3265-3280
Number of pages16
JournalArchives of toxicology
Volume94
Issue number9
Publication statusPublished - Sept 2020
Peer-reviewedYes

External IDs

PubMedCentral PMC7415759
Scopus 85088384587
ORCID /0000-0001-9326-7967/work/142252294

Keywords

Research priority areas of TU Dresden

Sustainable Development Goals

Keywords

  • Adverse Outcome Pathways, Animals, Animals, Genetically Modified, Carcinogens/toxicity, Cell Line, Cell Nucleus, Cell Transformation, Neoplastic, Cells, Cultured, Cyclophosphamide, DNA Damage, Ethyl Methanesulfonate, Green Fluorescent Proteins, Histones, Humans, Mutagenicity Tests, Mutagens/toxicity, Neoplasms, Rats, Stem Cells