Impacts of waterborne copper and silver on the early life stage (ELS) of zebrafish (Danio rerio): physiological, biochemical and molecular responses
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Toxic metals are major pollutants of the aquatic environment and are able to cause survival impairment of the early life stage of the aquatic organisms. They can affect the osmoregulatory system and electrolyte balance in fishes as well as the expression of genes which are essential in the formation and development of the organs at the early embryonic stages of development. There are a lack of studies concerning the toxic effects of waterborne copper and silver on the osmoregulation, electrolytes balance and expression the genes which are responsible for the formation and development of heart and metal binding proteins in the early life stage of zebrafish. The current study aimed to assess the toxic effects of waterborne concentrations of copper as an essential trace element, and silver as a non-essential trace element, on biochemical processes and the molecular biology of the early life stages (ELS) of zebrafish. The first experiment of the current study (Chapter 3) aimed: 1. to determine the time of nkx2.5 gene expression, a gene involved in cardiac development, relative to the time of embryonic development. 2. To assess the toxic concentration of the copper and most vulnerable and sensitive stage of the embryos < 1 hour post fertilization (hpf) exposed to the copper via water route. The result of the experiment showed that the expression of the nkx2.5 gene reached a maximum at 16 hpf. The first 10 hpf of the embryonic development was the most vulnerable and critical stage of the developing embryos, and characterized by increased mortality as copper concentration increased, and delayed and decreased hatching success. Exposure of embryos for 72 hpf to a concentration of 500 µg L-1 Cu increased heart rate, whereas the exposure of the embryos at the blastula stage only, showed decreases in heart rate. The third part of the experiment evaluated the protective effect of calcium as a major cation of water hardness on Cu toxicity. Embryos age < 2 hpf were exposed to copper (0, 100, 250, and 500 µg L-1), with or without added calcium (40 mg L-1). An increase in embryonic Cu accumulation was observed in live and dead embryos exposed to Cu, with and without added calcium. Calcium concentration increased with embryonic copper tissue concentration in dead embryos. Na+ and K+ concentrations were higher in live embryos compared to dead embryos, and a 4 fold decrease in Na+K+-ATPase activity was seen in live embryos exposed to copper compared to controls. There was no effect of copper on total glutathione. Expression of nkx2.5 as one of the essential genes for the formation and development of the heart increased significantly; approximately 10 fold in the presence of Cu+Ca in comparison to the unexposed control or Cu exposure alone. Whereas expression of mt2 increased significantly 6 fold compared to the control during Cu exposure without added Ca2+. The second experiment (Chapter 4) aimed to investigate the effect of the dissolved Ag+ as AgNO3 on the survival of the early life stage of zebrafish. Embryos < 2 hpf were exposed to silver 0 (no added Ag), 2.5, 5, 7.5, 10 and 15 µg L-1 Ag as AgNO3 for up to 72 h. Although, the survival was not affected by increasing concentrations of total silver, a decrease in hatching and increase in heart beat was observed. A significant increase in embryonic silver accumulation in both live embryos (at 24 and 72 hpf) and dead embryos (at 24 hpf) was observed. The accumulation of silver in 24 hpf live embryos was more significant than in dead embryos. Dead and live embryos at 72 hpf exposed to Ag had lower Na+ and K+ concentrations. Live embryos also showed a transient increase in Ca2+ concentration at 24 h. Four fold increases in Na+K+-ATPase activity, Mt2, and total glutathione concentration were seen in embryos after 72 h of exposure to AgNO3 compared to controls. In contrast, nkx2.5 gene expression was significantly decreased by 3 fold in 24 h aged embryos exposed to silver compared to controls. Due to the lack of studies that investigate the effect of silver on protein expression profiles during the early stages of development of zebrafish, the third experiment (Chapter 5) aimed to investigate the effect of silver on the changes of the expressed proteins of zebrafish embryos at the segmentation stage (24 hpf). The proteomics analysis successfully identified total of 810 proteins in the embryonic homogenate and quantified changes in their abundance in response to silver exposure. MS analysis showed the induction of new proteins which were absent in control embryonic homogenates. Also the analysis revealed there were increased expression of proteins such as zona pellucida glycoprotein, ATP synthase subunit α and β, stressed proteins such as metal chaperones and heat shock proteins, antioxidant proteins such as catalase (CAT), superoxide dismutase (Cu-Zn), glutathione S-transferase M, and glutathione S-transferase and proteins related to muscular development such as myosin heavy polypeptide 2, actin alpha 1 skeletal muscle, slow myosin heavy chain 1, actin cytoplasmic 1, and tropomyosin proteins. Overall, the thesis confirmed that the early life stages of zebrafish are sensitive to metals and that there are critical windows of toxicity during development. Metal exposure at early stages of the development initiate several disturbances in biochemical processes as well as changes in molecular biology that affect fish survival.
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