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

Comparative analysis of the transcriptome responses of zebrafish embryos after exposure to low concentrations of cadmium, cobalt and copper

Metal toxicity is a global environmental challenge. Fish are particularly prone to metal exposure, which can be lethal or cause sublethal physiological impairments. The objective of this study was to investigate how adverse effects of chronic exposure to non-toxic levels of essential and non-essential metals in early life stage zebrafish may be explained by changes in the transcriptome. We therefore studied the effects of three different metals at low concentrations in zebrafish embryos by transcriptomics analysis. The study design compared exposure effects caused by different metals at different developmental stages (pre-hatch and post-hatch). Wild-type embryos were exposed to solutions of low concentrations of copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) until 96 h post-fertilization (hpf) and microarray experiments were carried out to determine transcriptome profiles at 48 and 96 hpf. We found that the toxic metal cadmium affected the expression of more genes at 96 hpf than 48 hpf. The opposite effect was observed for the essential metals cobalt and copper, which also showed enrichment of different GO terms. Genes involved in neuromast and motor neuron development were significantly enriched, agreeing with our previous results showing motor neuron and neuromast damage in the embryos. Our data provide evidence that the response of the transcriptome of fish embryos to metal exposure differs for essential and non-essential metals

Concentration dependent transcriptome responses of zebrafish embryos after exposure to cadmium, cobalt and copper

Environmental metals are known to cause harmful effects to fish of which many molecular mechanisms still require elucidation. Particularly concentration dependence of gene expression effects is unclear. Focusing on this matter, zebrafish embryo toxicity tests were used in combination with transcriptomics. Embryos were exposed to three concentrations of copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) from just after fertilization until the end of the 48hpf pre- and 96hpf post-hatch stage. The RNA was then analyzed on Agilent's Zebrafish (V3, 4×44K) arrays. Enrichment for GO terms of biological processes illustrated for cadmium that most affected GO terms were represented in all three concentrations, while for cobalt and copper most GO terms were represented in the lowest test concentration only. This suggested a different response to the non-essential cadmium than cobalt and copper. In cobalt and copper treated embryos, many developmental and cellular processes as well as the Wnt and Notch signaling pathways, were found significantly enriched. Also, different exposure concentrations affected varied functional networks. In contrast, the largest clusters of enriched GO terms for all three concentrations of cadmium included responses to cadmium ion, metal ion, xenobiotic stimulus, stress and chemicals. However, concentration dependence of mRNA levels was evident for several genes in all metal exposures. Some of these genes may be indicative of the mechanisms of action of the individual metals in zebrafish embryos. Real-time quantitative RT-PCR (qRT-PCR) verified the microarray data for mmp9, mt2, cldnb and nkx2.2a

The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes

Silver nanoparticles (AgNPs) are widely believed to be retained in the sewage sludge during sewage treatment. The AgNPs and their derivatives, however, re-enter the environment with the sludge and via the effluent. AgNP were shown to occur in surface water, while evidence of a potential toxicity of AgNPs in aquatic organisms is growing. This study aims to examine the toxicity of AgNPs to the embryos of the aquatic vertebrate model zebrafish (Danio rerio) before and after sewage treatment plants (STPs) processes. Embryos were treated with AgNP (particle size:[90 %\20 nm) andAgNO3 in ISO water for 48 h and consequently displayed effects such as delayed development, tail malformations and edema. For AgNP, the embryoswere smaller than the controls with conspicuously smaller yolk sacs. The corresponding EC50 values of 48 hours post fertilization (hpf) were determined as 73 mikrog/l for AgNO3 and 1.1 mg/l for AgNP. Wholemount immunostainings of primary and secondary motor neurons also revealed secondary neurotoxic effects. A TEM analysis confirmed uptake of the AgNPs, and the distribution within the embryo suggested absorption across the skin. Embryos were also exposed (for 48 h) to effluents of AgNPspikedmodel STPwith AgNPinfluent concentrations of 4 and 16 mg/l. These embryos exhibited the same malformations than for AgNO3 and AgNPs, but the embryo toxicity of thesewage treatment effluent was higher (EC50 = 142 mikrog/l; 48 hpf). On the other hand, control STP effluent spiked with AgNPs afterwards was less toxic (EC50 = 2.9 mg/l; 48 hpf) than AgNPs in ISO water. This observation of an increased fish embryo toxicity of STP effluents with increasing AgNP influent concentrations identifies the accumulation of AgNP in the STP as a potential source of effluent toxicity