These cells are assumed to contribute to lifelong seasonal gonadal recrudescence and tissue regeneration being the driving factor for carp to have a more than 20-fold higher life expectancy than mammalian models like mouse and hamster (Levine 1997; Hurd and Ralph 1998; Tarn et al. 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 exhibited 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 Dexamethasone palmitate and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is usually suggested. A vertebrate cell model showing “healthy” stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology. Electronic supplementary material The online version of this article (10.1007/s00204-020-02821-3) contains supplementary material, which is available to authorized users. brain has been established. This approach was triggered by the observation of persistent pluripotent cells in seasonal spawning fish. These cells are assumed to contribute to lifelong seasonal gonadal recrudescence and tissue regeneration being the driving factor for carp to have a more than 20-fold higher life expectancy than mammalian models like mouse and hamster (Levine 1997; Hurd and Ralph 1998; Tarn et al. 2005; Allner et al. 2010). Based on this observation, it was possible to isolate constitutive self-renewing cells from healthy individuals in a reproducible manner. The usage of a H2B-eGFP transgenic variant of this cell type to detect genotoxic effects will be reported in this paper. The dynamic H2B-eGFP signal architecture will be compared with the fixation and staining equivalents of MNi, nuclear buds and nucleoplasmic bridges which are used to assess genotoxicity in test procedures standardised thus far (Fenech 2007; Russo et al. 2019). To improve the impact of in vitro test in the context of replacement of animal experiments a biotechnological metabolisation system ewoS9R is implemented. Future perspectives in coupling MNi based nondestructive genotoxicity assessment with downstream monitoring of carcinogenic transformation of healthy stem cells in a single in vitro live imaging test procedure are discussed. Materials and methods Cell line and culture conditions The KCB cell line has been derived Dexamethasone palmitate from Carp (testing was done in June 2019. The expression cassette of a CMV promoter-driven H2B-eGFP was derived of a H2B-eGFP plasmid Rabbit Polyclonal to SRY (Kanda?et al. 1998). H2B-eGFP was kindle Dexamethasone palmitate provided by Geoff Wahl (Addgene plasmid # 11,680). The sequence is usually flanked Dexamethasone palmitate by two repeats of the sea urchin arylsulfatase insulator (Ars insulator). The Ars insulator was placed in duplicate upstream and downstream of the coding sequence. The Ars insulator sequence was kindly provided by Masao Matsuoka (Hino et al. 2004; Tajima et al. 2006). The transgene sequence harbouring the expression cassette and the four copies of the Ars insulator are further flanked by piggybac terminal repeats. The sequences of piggybac terminal repeats were retrieved from pXL-BacII plasmid. pXL-BacII was kindly provided from Malcom Fraser (Cary?et al. 1989). Dexamethasone palmitate The sequence.