The zebrafish Danio rerio is a popular model organism for

The zebrafish (Danio rerio) is a popular model organism for virtually any biological function of vertebrates, and has, therefore, received a wide distribution in particular in developmental biology and toxicology (Strähle et al., 2012). With respect to AChE, the zebrafish has, e.g., been identified as a suitable model to mirror human exposure to AChE inhibitors (Koenig et al., 2016). Likewise, the zebrafish model has gained increasing attention for AChE inhibition testing in environmental and toxicological studies (Chen et al., 2017). Given the increasing use of zebrafish for AChE inhibition studies, the absence of non-animal-derived extracts for zebrafish AChE (zfAChE) experimentation has become a challenge. Therefore, the purpose of the present study was to develop an alternative to the standard approach of extracting the enzyme from either adult or embryonic zebrafish. For this end, a genetically engineered stable cell line of zebrafish origin expressing zfAChE at high amounts along with tdTomato fluorescent protein for easy identification and tracking of transgenic EG00229 synthesis was generated. As a basis, the permanent adult zebrafish liver-derived cell line ZF-L (Ghosh et al., 1994) was used. The present communication describes the protocol used for the generation of stably transfected ZF-L cells and the homologous expression of zfAChE. For extracts from transformed cells, a routine protocol for an in vitro inhibition assay is presented together with technical details on the short- and mid-term stability of enzyme preparations stored under varying conditions. In order to analyze the suitability of the generated extracts for the recording of zfAChE inhibition potential, a selection of model AChE inhibitors with different modes of action was tested.
Material & methods
Results
Discussion
Conclusions In the present study, ZF-L cells were successfully and stably transfected. zfAChE was expressed at high levels in stably transfected lines, the extracts of whose can be used for in vitro assessment of zfAChE-inhibition. The in vitro protocol based on extracts from stably transfected ZF-L cells seems amenable to medium- and high-throughput screening. Since enzyme preparations did not prove stable for extended periods of storage, the use of fresh preparations for in vitro zfAChE inhibition assays is recommended. The cell-based in vitro test system presented here appears as a valuable complementation to earlier approaches based on zebrafish embryos, which are also regarded experiments with non-protected models according to current animal welfare legislation at least in the EU. In contrast to zebrafish embryos, however, the ZF-L-based approach presented is a truly animal-free source for zfAChE enzyme preparations. Especially if the plain interaction of a compound with zfAChE is of interest, the zebrafish acetylcholinesterase from genetically engineered zebrafish liver (ZF-L) cells represent an interesting approach that avoids problems associated with the not yet completely characterized aspects of embryonal bioavailability, biotransformation and compensation mechanisms. More elaborate comparisons between the two test systems with focus on the impact of bioavailability and biotransformation as well as with respect to the relative sensitivity of the two alternative systems are subject of further ongoing research.
Acknowledgements The present study was funded by the German Federal Ministry for Science and Research (BMBF), contract no. 03F0735A within the Joint Programming Initiative Healthy and Productive Seas and Oceans (JPI Oceans) project EPHEMARE (“Ecotoxicological effects of microplastics in marine ecosystems”). The authors thank Prof. Dr. Karl Fent and Dr. Susanne Faltermann for enabling this study by providing ZF-L cells and details on their cultivation method.
Introduction Pharmaceuticals are widely used as therapeutic agents in human and veterinary medicines, and are increasingly found in aquatic ecosystems (Ternes et al., 2001). Therefore, these compounds are a growing concern as an emerging contaminant in aquatic environments. Pharmaceuticals have often been detected in sewage treatment plant (STP) effluents, drinking water, groundwater, and seawater (Fent et al., 2006). Among these aquatic contaminants, the most common pharmaceutical groups include analgesics, antibiotics, anti-epileptics, β-blockers, and lipid regulators (Fent et al., 2006; Kümmerer, 2009). In Korea, several pharmaceuticals including acetaminophen, carbamazepine, diclofenac, ibuprofen, and salicylic acid have been detected from treated wastewater (Park, 2005; Han et al., 2006). Of these, pharmaceuticals such as acetaminophen and carbamazepine have been the most frequently reported worldwide (Ternes et al., 2001; Kolpin et al., 2002; Fent et al., 2006). Sulfonamides including sulfamethoxazole are known as the second largest group in Korea and other countries (Thiele-Bruhn, 2003; National Veterinary Research and Quarantine Service, 2005; Park, 2005). Antibiotics such as oxytetracycline, sulfamethoxazole, and trimethoprim have been used extensively as therapeutic drugs in aquaculture (Kümmerer, 2009).