Enhanced applications of the zebrafish (Danio rerio) embryo toxicity test as a model to mechanistically differentiate metal toxicity effects in fish

The preservation of the environment and the containment of environmental pollution is one of the greatest challenges faced by our society. Metals as environmental pollutants, are a serious global problem. They are released in various forms during their extraction (mining), processing (chemical and metal industry) and, finally, due to improper disposal of metal-containing goods. Million tons of electronic waste materials containing metals such as cadmium, copper, chromium, silver, nickel and cobalt end up in the environment every year. Once metals have entered the environment, they persist because they are neither chemically nor biologically degradable. Surface waters and their sediments are major sinks of metal pollution. They are directly contaminated by the discharge of industrial effluents, leaching and surface runoffs from storage or landfill sites or agricultural fields (sprayed with metal-containing pesticides). Therefore, aquatic organisms as fish are particularly affected by metal exposure. This exposure can lead to severe impairment of fish development, although the mechanistic action of which is not well understood. This PhD-thesis investigates the effects of the environmentally relevant metals cadmium, cobalt and copper on the embryonic development of the zebrafish (Danio rerio) at different biological levels. The zebrafish is an established model organism representative of vertebrates, and specifically aquatic vertebrates. Zebrafish early larval stages are increasingly endorsed as alternatives to animal testing using fish due to their many advantages. The uptake of heavy metals and metal ions in fish mainly takes place via the gills and the intestines and leads primarily to an imbalance of ion homeostasis, especially of Na+ and Ca2+. However, since the uptake of food and gills are absent in fish embryos, heavy metal uptake and effects in the early life stages are still largely unknown. In order to address some of the gaps in this knowledge, effects of metal exposure were studied morphologically (by fish embryo toxicity tests and microscopic imaging) molecularly (by transcriptome analysis) and functionally (by behavioural observations) on zebrafish embryos and eleutheroembryos. Specifically, the morphological development, the death of the hair cells of the lateral line organ's neuromasts, the impairment of motor neuron development and the touch-evoked escape response were studied in wild-type zebrafish embryos, following exposure to different concentrations of either cadmium (CdCl2), cobalt (CoSO4) or copper (CuSO4). With this approach, it was intended to link cellular, morphological and functional aspects of adverse effects of a non-essential metal (cadmium) in comparison to essential metals (cobalt and copper). Motor neuron damage was investigated by immunofluorescence staining of primary motor neurons (PMNs) and secondary motor neurons (SMNs). In vivo staining using the vital dye DASPEI were used to quantify neuromast damage. The consequences of metal exposure were also assessed functionally by testing the escape response behaviour following tactile stimulation. The median effective concentration (EC50) values for morphological effects at 72 hours post fertilization (hpf) were 14.6 mg/L for cadmium and 0.018 mg/L for copper, whereas no morphological effects were found in the cobalt exposed embryos up to 45.8 mg/L. All three metals caused a concentration-dependent reduction in the number of normal PMNs and SMNs, and in the fluorescence intensity of neuromasts (i.e. increase in hair cell death). Even the lowest metal concentrations (cadmium 2 mg/L, copper 0.01 mg/L and cobalt 0.8 mg/L) resulted in neuromast damage. The results demonstrate that the neuromast cells were more sensitive to metal exposure than morphological traits, or the response to tactile stimulation and motor neuron damage.The molecular basis of metal toxicity in zebrafish embryo was also investigated on a broader scale by applying genome-wide transcriptomics analysis. This was aimed to improve the mechanistic understanding of adverse effects caused by different metals. This study was designed to compare exposure effects of three different exposure concentrations of the three metals at two different developmental stage (pre-hatch and post-hatch). Thus, embryos were exposed to low, sub-toxic concentrations of copper, cadmium and cobalt until 48 and 96 hpf and subsequently subjected to microarray analysis to determine the changes in the transcriptome profiles. With regard to the effects seen at transcriptome level, the embryos reacted differently to the non-essential metal cadmium compared to the essential metals cobalt and copper. For example, gene specific transcription as well as GO terms hinted at detoxification processes in cadmium treated embryos only. Possible indicators of a detoxification response like glutathione-s-transferases and metallothionein2, were identified and heme oxygenase, another possible indicator of oxidative stress and detoxification, was significantly regulated after cadmium exposure. In contrast, no detoxification related gene transcripts or ontology terms were found regulated for essential metals cobalt and copper. Also in terms of the overall transcriptome response pattern and strength and pathway modulation. This thesis clearly showed a different reaction of the embryos to the essential compared to the non-essential metals. Furthermore, the results of this thesis point out the neuro- and ototoxic potential of cadmium, cobalt and copper. The additional study of the embryos' escape response after tactile stimulation allowed a correlation with the effects on the motor neurons for all three metals. The findings suggested that the motor neuron damage may have caused to interrupt the innervation of the embryos' tail muscles, which subsequently inhibited the essential muscle contraction required for the escape response. The transcriptome analysis at low concentration levels enabled us to relate GO terms of the nervours system development to the defect on the motor neurons for all three metals. Therefore, the transcriptional changes related to the nervous system development and the motor neuron damage, which resulted in the altered escape response, seemed to be connected by similar processes for all three metals. However, the no-hatch effect, which may have partly been caused by impaired tail muscle innervation due to an inhibited signal transduction in the nervous system (e.g. between the sensory neurons and motor neurons), was observed only in embryos exposed to copper. Based on the transcriptome level results, this assumption of signal transduction inhibition in the nervous system was strengthened since copper regulated Wnt and Notch signal transduction pathways. Further, additional GO terms of neuron development and differentiation associated with the deformation of the neurons for copper but not for cadmium and cobalt. Both molecular mechanisms seem to be related to both, the effect on the escape response and the no-hatch effect. Interestingly, Wnt signaling pathways were found regulated also in cobalt treated embryos, where a no-hatch effect was observed at higher carbonate hardness. In contrast, cadmium did not affect the hatching of the embryos, and also the Wnt signaling pathways were unaltered. However, the significant upregulation of cldnb mRNA may suggest an ototoxic potential of cadmium, which the embryos try to compensate by degeneration. The tight junction protein Claudin b is highly expressed in the neuromast. Overall, this thesis demonstrates that a combination of fluorescence staining endpoints, gene expression analysis, behaviour assessment and zebrafish embryo toxicity tests can help to specify, quantify and elucidate mechanisms and connections defining the exposure effects of metals like copper, cadmium and cobalt. Moreover, it was shown that additional endpoint assessment procedures are available, which display higher sensitivity to detected metal toxicity in zebrafish embryos than current test procedures applied in the ecological risk assessment.