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

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.

Kinetic determination of vitellogenin induction in the epidermis of cyprinid and perciform fishes: Evaluation of sensitive enzyme-linked immunosorbent assays.

Induction of vitellogenin (VTG) in male and immature fish is a standardized endpoint in endocrine-disruption testing. To establish a nondestructive swab sampling method, VTG induction in the epidermis of Cypriniformes and Perciformes species was investigated. Both VTG and estrogen receptor genes are expressed in epidermal cells. Immunoaffinity and mass fingerprint analyses show induction of identical VTG peptides in liver and epidermis. Induction of VTG by estradiol (E2) and bisphenol A (BPA) in the epidermis was quantified with homolog enzyme-linked immunosorbent assays. Initial values in juveniles and males were below 1 ng VTG/mL extraction buffer. Exposure to E2 led to values between 200 ng/mL and 4600 ng/mL in cyprinids and between 10 ng/mL and 81 ng/mL in perciforms. Exposure to BPA increased VTG amounts to 250 ng/mL in fathead minnows, 1360 ng/mL in goldfish, 100 ng/mL in zebrafish, and 12 ng/mL in bluegills. Serum VTG contents demonstrated a similar dose-response pattern in the epidermis and the blood. These results show that VTG induction may be reliably assessed in the skin mucus of fishes, demonstrating the suitability of this biological sample for investigating estrogenic activity in compliance with Organisation for Economic Co-operation and Development standard protocols. This broadens the perspectives in toxicological screening and environmental monitoring, reducing the number of tested animals and minimizing harmful effects for animals, allowing for follow-up of individual induction profiles. Environ Toxicol Chem 2016;35:2916-2930. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Vitellogenin in carp (Cyprinus carpio) and perch (Perca fluviatilis): purification, characterization and development of an ELISA for the detection of estrogenic effects.

Vitellogenin (VTG), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted in the liver in response to circulating estrogens in female fish. Thus, the presence of VTG in male fish is a useful biomarker to identify estrogenic activity of natural or anthropogenic substances in sewage effluents. We report the purification of carp (Cyprinus carpio) and perch (Perca fluviatilis) VTG with the subsequent development and characterization of a specific ELISA for VTG measurement. VTG was purified by combination of ion exchange chromatography and size exclusion chromatography. The purified proteins were used as antigen for antibody production, as standard and tracer in the assay. Carp VTG was stable and showed a characteristic double band after Western blotting of the purified protein and in serum samples, respectively. In perch samples, several bands with lower molecular weight were present and appeared to be degradation products of VTG. The development of the carp VTG ELISA led to a sensitive and valid test system with inter-assay coefficients of variation between 3.0 and 12.3%. In contrast to carp, the described ELISA for perch VTG showed a much higher inter-assay variation up to 24%, possibly attributable to fast protein degradation. In conclusion, the described two-step chromatography is a simple purification method for VTG. Immunological and electrophoretical test systems are valid methods to determine VTG in some species like carp, but for other species like perch in which VTG is not stable, these methods are not applicable.

Reproductive functions of wild fish as bioindicators of reproductive toxicants in the aquatic environment.

BACKGROUND, AIM, AND SCOPE: Impacts on the reproductive health of wild fish are thought to be suitable early-warning tools indicating contamination of surface waters with endocrine-disrupting compounds. Ecotoxicological assessment of these field observations depends on the availability of reliable biomarkers to enable a discrimination of natural variations of reproductive functions from anthropogenic impacts. MATERIALS AND METHODS: Roach and perch were caught at eight sampling sites by electrofishing twice a year in summer (July-September) and late autumn/winter (November-December) over a 2-year period. The sites are characterized by different degrees of anthropogenic impact and are situated within the greater Upper Rhine catchment. Age growths, parasitization and gonadal histology of more than 3,000 fish were examined. RESULTS: The two dominant fish species in German surface waters perch (Perca fluviatilis L.) and roach (Rutilus rutilus L.) differ considerably regarding their suitability for biomonitoring. Even in pristine habitats, perch show several variants of sex differentiation in terms of (1) the time of first sexual maturation, (2) the course of seasonal gonadal recrudescence, and (3) the occurrence of heterologous germ cells (testes ova). A statistically significant elevated proportion of males were observed in fish obtained from a TBT-contaminated marina and suppression of gonadal ripening was observed in females caught in a sewage-contaminated brook. Both effects appear to be due to chemical contamination. The only "natural" alteration of sex differentiation in roach was related to parasitization with Ligula intestinalis (Eucestoda, Pseudophyllidea). Other deviations from the normal pattern of sex differentiation were (1) suppression of ovarian ripening and (2) asynchronic seasonal gonadal recrudescence. These are strong indicators of an anthropogenically induced impact on reproductive health. Feminization phenomena were not observed at either the individual or the population level. DISCUSSION: Interpretation of field monitoring results concerning reproductive health requires large numbers of samples and detailed knowledge of the natural plasticity of sex differentiation in the species under investigation. A better understanding of the mechanisms underlying the plasticity of sex differentiation in perch is indispensable to enable perch to be used as a bioindicator. CONCLUSIONS: Deviation from the strict and probably endogenous control of sex differentiation in roach is a strong and unequivocal warning signal. RECOMMENDATIONS AND PERSPECTIVES: The subject of fish monitoring should be addressed in the context of a broader spectrum of potential risks. Seasonal and ontogenetic integrity of gonadal development and recrudescence are potent biomarkers, provided the natural process is well documented for the species under investigation.