The Toxicity Evaluation Of Ni²⁺ To Organism At The Level Of Molecule And Single Cell

With the industrial and agricultural development, environmental issues have become increasingly prominent. The human has paid much attention to heavy metal pollution specially. Heavy metal ions can be transport into the biological body via air, water, foods and other ways and produce the toxicity to humans. Reports of the toxicity of heavy metal ions emerge continuously. Heavy metal pollution has become a key environmental problem endangering the development of humankind.Protein and DNA are major component of living organisms. Cells are the structural and functional units of living organisms. Various toxic and harmful substances come into human bodies often interact with these biological macromolecules to show their harmfulness, followly structure and function change of cells will occur. Therefore, it is of great importance to investigate the details about the mechanism of toxic action of pollutants to organisms at the level of functional macrobiomolecular and single cells, which will also help more to assess the toxicity of harmful substances in depth. Also, this will provide some reference and technical support for the relevant standard of protecting human health and safety.In this paper, we studied the toxicity mechanism of Ni2+ targeted to bovine hemoglobin (BHb) and calf thymus DNA (ctDNA) in molecule level by spectroscopy methods. At the level of single cells, the effects of Ni2+ on glutathione in single human erythrocytes using fluorescence images based on microfluidic chip without cytolysis was developed. Also, the effects of Ni+ on the morphology of human erythrocytes were investigated by scanning electron microscope. The dissertation consists of the following three parts.In the first part, the toxic interaction between Ni2+ and bovine hemoglobin (BHb) was investigated using fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy and circular dichroism spectroscopy (CD) under simulated physiological conditions. The experimental results showed that both dynamic and static quenching occurred simultaneously when Ni2+ quenched the fluorescence of BHb. There was formation of Ni-BHb complex stabilized by electrostatic interaction, but the binding between Ni2+ and BHb was not strong. The apparent binding constant Ka and binding site number n were 102.8 L mol-1 and 0.640 at 287 K. In addition, UV-vis and CD results showed that the addition of Ni2+ changed the conformation of BHb.In the second part, the interaction between Ni2+ and calf thymus DNA (ctDNA) was investigated in simulated physiological buffer (pH 7.4) using the Neutral Red (NR) dye as a spectral probe by UV-vis absorption and fluorescence spectroscopy, as well as CD spectra. The experimental results showed that the conformational changes in DNA helix induced by Ni2+ are the reason for the fluorescence quenching of the DNA-NR system. From the experiment results, conclusion can be drawn that Ni2+ can cause structural changes of ctDNA and bind with DNA by electrostatic interaction. At the same time, the paper proved that conformation changes of DNA can also lead to the fluorescence decrease of DNA-probe systems.In the third part, the toxicity of Ni2+ to human erythrocytes was investigated at the level of a single cell. The effects of Ni2+ on glutathione in single human erythrocytes using fluorescence images based on microfluidic chip without cytolysis was developed by tagging with the 2,3-naphthalenedicarboxaldehyde (NDA). The effects of Ni2+ on the morphology of human erythrocytes were investigated by scanning electron microscope (SEM).The experiment results showed the GSH in untreated cells obtained from healthy adult male were calculated as 30.5±5.0 amol per cell. The GSH levels after treatment of Ni2+ decreased as comparing with control GSH amount. The GSH depletion reached to a maximum of 23.6% comparing with control GSH mass, indicating obvious toxicity to human erythrocytes.The effects of Ni2+ on the morphology of human erythrocytes were determined by SEM. The paramorphia and neuritogenesis of human erythrocytes were found after treatment of Ni2+The results of our study indicate that after Ni2+ came into the body, they can not only lead to conformational change of the protein, but also interact with the genetic material DNA, thus showing its genotoxicity. At the level of single cells, the GSH levels after treatment of Ni2+ decreased as comparing with control GSH amount. Also, Ni2+ can lead to morphology change of human erythrocytes. Thus, conclusion can be made that Ni2+ obvious toxicity to organism.