Research On Cadmium Induced Oxidative Stress-related Toxic Effects And Its Underlying Mechanisms

Cadmium is a typical heavy metal pollutant. Cadmium enters into the environment mainly through fossil fuel combustion, phosphatic fertilizer, natural sources, steel foundry, municipal solid waste incineration, cement and nonferrous metals productions and other industrial productions. With the rapid developments of modern industry, the problem of environmental cadmium pollution currently exists in most areas of the whole world and continues to increase in the future. Due to the common presence of cadmium in the environment and its potential toxic effects, the adverse effects of cadmium on the environment and human health have raised wide attention and obtained numerous on-going researches.As a non-essential element to the organism, cadmium can not be biologically degraded in the organism. For non-occupationally exposed people, cadmium enters into their body mainly through contaminated food and tobacco smoking. After entering into the body, cadmium distributes to other organs through blood circulation system and induces the damage of multisystems and multiorgans including bone, liver, kidney and reproductive system. Metal-induced oxidative stress is closely associated with a variety of diseases. In vitro and in vivo studies have demonstrated that cadmium could disrupt the balance of the celluar redox state and lead to oxidative stress. The overproduction of reactive oxygen species (ROS) induced by cadmium could lead to varieties of nonspecific damages, leading to cell death and tissue damage. These are considered as the general mechanism of common damages and diseases.Currently, cadmium induced oxidative stress-related toxic effects and its underlying mechanisms have not been completely clarified. The methodology of related research still needs to be further established and completed. Depending on the difference of experimental conditions and study objects, it remains controversial for cadmium induced oxidative stress-related toxic effects and its underlying mechanisms. To cure the above problems, this paper investigated cadmium induced oxidative stress-related toxic effects and its underlying mechanisms from the level of animal, cell and molecule. This paper is mainly consisted with the following six parts:In chapter one, the characteristics and current situation of environmental cadmium pollution as well as the research development of cadmium toxicity were simply introduced. The induction mechanism of oxidative stress, oxidative damage and the related regulated mechanism were overviewed. The effects and mechanism of cadmium-induced oxidative stress were also summarized. Through reviewing this knowledge, the research developments and the current scientific problems in the toxicology filed were concluded and experimental protocols were set for these problems.In chapter two, in vivo animal experiments were conducted and zebrafish was chosen as the study object. Cadmium induced oxidative stress-related toxic effects were investigated in the liver of zebrafish through measuring the biomarker changes during this process, including the activity change of antioxidant enzyme, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) as well as the level change of reduced glutathione (GSH), oxidized glutathione (GSSG) and the product of lipid peroxidation malondialdehyde (MDA). Results showed that cadmium exposure led to the increase of GPx activity, the decrease of GR and GST activity. The activity of CAT decreased and the activity of SOD increased. The level of GSH decreased and the level of GSSG increased, resulting in the decrease of GSH/GSSG ratio. The level of MDA increased with the concentration of cadmium. These results suggested that cadmium exposure induced amount of ROS production in the liver of zebrafish, disrupted the balance of redox state, induced oxidative stress in zebrafish livers and resulted oxidative damage.In chapter three, the mouse primary hepatocytes were chosen as the study object and exposed to cadmium in vitro. Cadmium induced oxidative stress-related toxic effects and its underlying mechanisms in mouse primary hepatocytes were investigated at the cellular level. The cellular Cd2+ level were measured in situ. The cell viability, apoptosis percentage, the activation of ERK signaling pathway and caspase-3 were measured after 6 or 24 h cadmium exposure. The response of oxidative stress biomarker ROS, GSH, CAT and SOD, DNA damage and histone phosphorylation were also investigated. After 6 h cadmium exposure, no free Cd2+ exists in the hepatocytes and cadmium exhibits no overt toxicity to the hepatocytes. After 24 h cadmium exposure, ROS level elevated and other oxidative stress-related effects occurred in a part of the hepatocytes. The increase of apoptosis percentage, DNA oxidative damage and other toxic effects were also observed in a part of the hepatocytes. The antioxidant N-acetyl-L-cysteine (NAC) prevented the decrease of cell viability, the increase of apoptosis percentage and DNA oxidative damage, suggesting that cadmium-induced cellular oxidative stress had adverse effects on the hepatocytes. Oxidative stress-mediated apoptosis reduced the cell viability of the hepatocytes. The ERK signaling pathway was activated after exposure to cadmium. NAC prevented the phosphorylation of key protein in ERK pathway. NAC and ERK pathway inhibitor PD98059 prevented apoptosis and the activation of caspase-3. These results suggested that oxidative stress-mediated the activation of downstream ERK signaling pathway participated cadmium-induced mouse primary hepatocytes. This study also demonstrated that cadmium-induced the phosphorylation of histone H3 was closely correlated with oxidative stress and the activation of ERK signaling pathway.In chapter four, the antioxidant enzymes CAT and SOD were selected as study objects. Enzyme activity change after cadmium exposure was determined using enzyme activity determination method. The effects of cadmium on protein structure were investigated through measuring fluorescence spectra, UV-vis absorption and circular dichroism. The binding mode of the direct interaction between cadmium and enzymes was explored by isothermal titration calorimetry (ITC), including the calculation of the binding parameters and thermodynamic constants. The possible sites on protein after cadmium binding were simulated by the molecular modelling methods. Combined with these conclusions, the mechanism of the structure and function change of the enzyme was clarified.(1) The direct interaction between cadmium and CAT molecule resulted in the decreased activity of CAT. Cadmium binds to CAT mainly through electrostatic forces with one type of binding sites and binding constant K= (2.69±0.243)×103 M-1. Cadmium statically quenched the intrinsic fluorescence of CAT and formed complexes with CAT, changing the hydrophobicity around the aromatic amino acids. The interaction changed the secondary structure of CAT, leading to the misfolding of the protein. Molecular docking results suggested that cadmium interacts with Gln 167 and Trp 185 located at the entrance to the active site of CAT, hinders the substrate entering into the active site and subsequently results in the decrease of the activity.(2) The direct interaction between cadmium and SOD molecule resulted in the increased activity of SOD. Cadmium binds to SOD mainly through electrostatic forces with two types of binding sites and binding constants K1=(6.07±1.47)×105 M-1 and K2=(1.02±0.132)× 104 M-1. The interaction changed the secondary structure of SOD, leading to the unfolding of the protein. And, cadmium did not interact with the aromatic amino acids of SOD until the aromatic amino acids became exposing to cadmium due to structure and conformation changes induced by cadmium. Molecular docking results suggested that cadmium binds to the interface of the subunit of SOD, which might result in the enlargement of the pocket around the enzyme active sites and then lead to the increase of enzyme activity.In chapter five, the indirect antioxidant proteins lysozyme (LYZ) and transferrin (TF) were selected as study objects. Enzyme activity change after cadmium exposure was determined using enzyme activity determination method. The effects of cadmium on protein structure were investigated through measuring fluorescence spectra, UV-vis absorption and circular dichroism. The binding mode of the direct interaction between cadmium and enzymes was explored by ITC, including the calculation of the binding parameters and thermodynamic constants. The possible sites on protein after cadmium binding were simulated by the molecular modelling methods. Combined with these conclusions, the mechanism of the structure and function change of the enzyme was clarified.(1) Cadmium binds to LYZ mainly through hydrophobic forces, changing the secondary structure of LYZ and the microenvironment of Trp residues. Although complexes were formed, no effects were observed on the activity of LYZ at low cadmium concentrations because cadmium does not preferentially bind to the active site of LYZ. However, at relatively high cadmium concentrations, a moderate loss of activity was observed due to the interaction with Glu 35 and Asp 52 located at the active site of LYZ.(2) The ability of TF to bind cadmium and the potential role of TF in neutralizing cadmium toxicity on the hepatocytes were demonstrated. Cadmium statically quenched the intrinsic fluorescence of TF and formed complexes with TF, leading to a decrease of the a-helix content, a shrinkage of the protein and other structural and conformational changes and TF. Cadmium preferentially binds to the higher binding affinity sites of TF via hydrophobic forces with no release of Fe and no interface to the Fe binding site. Subsequently, cadmium binds to lower binding affinity sites of TF via electrostatic forces and interacts with Tyr 517 and Tyr 95 around the Fe binding site, resulting in the release of the Fe content.In chapter six, every part of this thesis was concluded. The innovation points were also summarized. Subsequently, the development direction and future research in this filed were outlooked according to the shortage of the research protocols. This paper combined the exposure routes of in vivo animal experiments, in vitro cell and molecule experiments and evaluated the oxidative stress induced by cadmium in the liver and the hepatocytes. The mechanism of the apoptosis, genotoxicity and epigenotoxicty induced by cadmium were also explored. We also established a research strategy to explore the binding mode of cadmium interacting with proteins and the mechanism of protein structure and function change. Cadmium induced oxidative stress-related toxic effects and its underlying mechanisms were comprehensively investigated through combining the three exposure routes. This research could provide basic data for understanding cadmium toxicity. This research could help to provide references to the nosogenesis of cadmium-induced liver damage as well as the therapeutic schedule of acute and chronic cadmium exposure. This research could also provide scientific basis and technical supports for the hazardous evaluation of heavy metal toxicity, the establishment of intervene policy and measurements by the government.