Exploring The Toxicity Of Copper Ions By Mouse Primary Hepatocytes And Oxidative Stress Proteins

Copper （Cu） is an essential micronutrient for all organisms,which plays a key role in biological functions. However, the massive utilization of copper in energy industry, machinery manufacturing, construction, transportation military weapons, etc., has led to concerns regarding its potential health impact. Copper intakes is mainly through the gastrointestinal, respiratory and skin tracts after widely contacted. Then, it is transported via the blood circulation to the various organs and accumulated in the liver, kidney, brain and other organs, causing oxidative damage in cells, organs and body.Many varied diseases is closely associated with oxidative stress. Multiple studies have shown that copper could disturb the intracellular redox balance, generate reactive oxygen species, induce oxidative stress, and subsequently cause irreversible damage, leading to a variety of diseases. Toxic effects of heavy metal pollutants are often reflected in the molecular, cellular and animal levels, single-level evaluation is not sufficient to fully clarify the toxic effect of copper. Therefore, it is necessary to explore mechanisms of oxidative damage caused by copper from multiple levels and reveal the microscopic mechanism of health damage after copper exposed.This paper aims to explore the mechanisms of oxidative damage caused by copper and combined at cellular and molecular levels with the knowledge of molecular and cellular toxicology. This paper includes the following five parts:The first part briefly introduced the toxic effects of copper and the relationship between copper toxicity and oxidative stress. Then, we briefly described the methods and techniques at cellular and molecular level. Finally, we summarized research purposes, contents and significance of this paper.In the second part, mouse primary hepatocytes were chosen to elucidate the vitro oxidative damage of short-term copper exposure （10-200μM） by single-cell analysis. We evaluated the toxicity of copper by reactive oxygen species （ROS）, glutathione （GSH） and oxidative DNA damage at the single-cell level. Oxidative damage induced by copper was verified by the morphological changes, persistent elevations of excessive ROS and malondialdehyde （MDA）, a decrease in GSH level and the oxidative DNA damage. Furthermore, the average ROS generation, GSH consumption and the indicators in DNA damage did not significantly change at relative low concentrations （10 or 50 μM） but we can find the alteration of parameters in some single cells clearly.In the third part, we selected oxidative stress directly related enzyme catalase（CAT） as research target to elucidate the toxic effects of copper on antioxidative enzyme from cellular and molecular level. Experimental results showed that copper exposure inhibited the CAT activity in primary hepatocytes as a result of oxidative stress. And Cu²⁺also can induce the decrease of CAT activity in vitro by directly interacting with CAT. During the molecular level study, the fluorescence of CAT was quenched and the secondary structure content of CAT was changed by the addition of Cu²⁺. Molecular simulation study indicated that Cu²⁺was located between two P-sheets and two random coils of CAT near to the heme group, and interacts with His74 and Ser 113 residues near ahydrophilic area. The decrease of a-helix and the binding of His74 are considered to be the major reason for the inhibition of CAT activity caused by Cu²⁺.In the fourth part, we selected oxidative stress related enzyme lysozyme as research target to investigate the toxic effects of copper on lysozyme, which was based on variety spectroscopy methods, isothermal titration calorimetry （FTC） and molecular docking methods. Results indicated that the binding interaction was a spontaneous process with approximately three thermodynamical binding sites at 298 K and the hydrophobic force is the predominant driven force. Multi-spectroscopic measurements proved that copper quenched the intrinsic fluorescence of lysozyme in a static process accompanied by complex formation and conformational changes.The enzyme activity was obviously inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites.In the fifth part, the effect of Cu²⁺on the structure and function of Human Serum albumin（HSA） in vitro were investigated by biophysical methods at molecular level. Experimental results showed that:Multi-spectroscopic measurements proved that Cu²⁺ quenched the intrinsic fluorescence of HSA in a dynamic process accompanied by the formation of complex and alteration of secondary structure. Cu²⁺interacted with HSA spontaneously through hydrophobic forces with approximately 1 thermodynamic identical binding sites at 298 K. The esterase activity of HSA was inhibited obviously at the concentration of 8×10^-5 M.