Exploring The Effects And Mechanisms Of Oxidative Damage And Cell Apoptosis Induced By MPA Capped CdTe QDs In Mice

Quantum Dots are nanometer semiconductors that are usually composed of Group IIB-VI A elements(such as CdTe,CdSe,etc.)or III A-V A elements(such as InP,GaAs,etc.).Quantum dots are generally spherical or hemispherical,and their particle size generally ranges from 2 nm to 100 nm.Compared with traditional fluorescent materials,quantum dots have excellent characteristics,such as long fluorescence lifetime,good light stability,narrow and symmetrical emission spectra,emission spectra are continuous and adjustable according to particle size and composition,and better biocompatibility after surface modification,so quantum dots have excellent performance in high sensitivity detection,long-time dynamic observation,multi-indicator detection and so on.At the same time,quantum dots are widely used in single electronic devices,solar cells,and optoelectronic devices due to their unique physical effects and excellent optical properties.With the continuous development of quantum dot application science,quantum dots have more opportunities to enter people's work and life,so more and more people start to pay attention to the biosecurity and environmental hazards of quantum dots.Studies have shown that the exposure of quantum dots can cause the produce of excessive amounts of reactive oxygen species in the body,which leads to the destruction of the redox balance,resulting in oxidative damage of DNA,protein,and biofilm,and then leads to the cell apoptosis or necrosis.That is why oxidative damage is considered to be a general mechanism for a variety of injuries and diseases.Nowadays,whether the exposure of QDs causes changes of oxidative stress levels in experimental animals is still controversial;there are few reports on the mechanism of interaction between QDs and oxidative stress-related biomacromolecules;there is almost no researches about the correlation of biomarkers' biotoxicity at different levels or exposure pathways(molecules,cells,animals).Therefore,it is necessary to elucidate the toxic effects and mechanisms related to oxidative stress induced by quantum dot at different levels.The main research contents and results are as follows:(1)In order to explore the toxic effects of MPA-CdTe quantum dots at the level of experimental animals,we dissolved quantum dots in water and configure them into different concentrations,and then exposed them to different groups of mice by feeding water.After incubation for a period of time at different concentrations of MPA-CdTe quantum dots,mouse livers were eviscerated for the experiments.Firstly,we used quantum dot to incubate the mice for 30 days,and made paraffin sections to observe the effect of quantum dot exposure on the morphology of mouse liver cells.Then,we took the liver of the mice after 120 days of quantum dot incubation,and prepared the cell suspension to measure the cell activity,ROS level and MDA level of the hepatocytes,and evaluated the toxic effects of the quantum dots on the liver cells after exposure to the mice.The results at the animal level showed that quantum dots did not induce the changes in cell morphology and corresponding toxicological indicators in the liver of mice when exposed to QDs by feeding water.(2)Mouse hepatic stellate cell,liver cells and kidney cells were used as research objects.After incubation of QDs in vitro,the related toxic effects and mechanisms induced by MPA-CdTe QDs in mouse hepatic stellate cell,hepatocytes and nephrocytes were studied at the cellular level.The results showed that the exposure of MPA-CdT QDs activated the hepatic stellate cells,and the cells changed from static type to activated type,and the expression of ?-SMA and GFAP increased.The results indicated that the exposure of MPA-CdTe QDs produced stimulation and caused changes in the phenotype of hepatic stellate cells.In addition,another two target organs,liver and kidney,were selected as the research object.Meanwhile,the activation of ERK signaling pathway and Caspase apoptotic factor was introduced in the experiment to explore the effects and mechanisms of intracellular oxidative stress and apoptosis induced by MPA-CdTe QDs.The results showed that the exposure of MPA-CdT QDs led to cell apoptosis(compared with the blank group,when the concentration of quantum dots in the experimental group reached 10×10-6 M,the apoptosis rate of hepatocytes increased from 9.3%to 41.6%,and the apoptosis rate of nephrocytes cells increased from 16.2%to 69.7%)and significant decrease in the viability(compared with the blank group,when the concentration of quantum dots in the experimental group reached 10×10-6 M,the cell viability of hepatocytes decreased from 94.9%to 83.6%,and the cell activity of nephrocytes decreased from 92.6%to 64.1%)of primary hepatocytes and nephrocytes in mice.Comparing to hepatocytes,changes in cellular activity and apoptosis of nephrocytes were more sensitive to the exposure of quantum dots,and presented a more pronounced dose-effect relationship.According to the statistical analysis of the proportion of apoptotic cells and necrotic cells,the cell death caused by MPA-CdTe QDs was mainly through the way of apoptosis.The exposure of the coating agent,MPA,did not cause a decrease in the activity of hepatocytes and nephrocytes.The exposure of QDs caused more significant decrease in the cells activity comparing to the same concentration of Cd2+,indicating that the cytotoxicity of QDs is not only caused by the release of Cd2+.We suspected that QDs and Cd2+ showed a joint toxicity to the cells.Exposure of quantum dots led to the production of reactive oxygen species(compared with the blank group,when the concentration of quantum dots in the experimental group increased to 10×10-6 M,the ROS content in the hepatocytes increased from 3.5%to 52.0%,and the ROS content in the nephrocytes increased from 6.5%to 32.7%),ROS(such as O2-·,H2O2,etc.),and some of them were decomposed by intracellular antioxidant enzymes(CAT,SOD,etc.),while the undecomposed ROS activated the ERK signaling pathway of the cell(compared with the blank group,when the concentration of quantum dots in the experimental group reached 10×10-6 M,the activation of ERK signaling pathway in hepatocytes increased from 1.5%to 40.4%,and the activation of ERK signaling pathway in nephrocytes increased from 1.4%to 11.2%),which caused regulation of the physiological processes in cells,such as proliferation and apoptosis.At the same time,the accumulation of ROS also caused the activation of Caspases(compared with the blank group,when the concentration of quantum dots in the experimental group reached 10×10-6 M,the Caspase activation in hepatocytes increased from 0.92%to 11.0%,and the Caspase activation in nephrocytes increased from 4.0%to 18.8%),which was a key executive factor that initiated apoptosis and induced apoptosis of cells.The regulation of protease cascade led to hydrolysis of the target protein of Caspases,lipid peroxidation of cells,oxidative damage of membrane proteins and DNA,leading to the changes in the structure and function of cells,and finally induced the apoptosis of cells.(3)Two important antioxidant enzymes,catalase(CAT)and superoxide dismutase(SOD),and two indirect antioxidant proteins,human chorionic gonadotropin(hCG)and transferrin(TRF)were studied in this part.To study the mechanism of toxic effects of quantum dots at the molecular level,the effects of MPA-CdTe QDs on the structure and function of the proteins were studied at the molecular level.The results showed that the interaction between QDs and CAT slightly changed the microenvironment of the enzyme and slightly enhanced its hydrophobicity;at the same time,the quantum dots changed the conformation near the Heme group,the QDs changed the secondary structure of CAT,causing the loose of protein skeleton and the change of spatial structure,which caused the enzyme active center pocket to become larger or become more close to the outer space of the protein,therefore,the contact area or contact chance of CAT with its substrate(H2O2)increased,resulting in the increase of the CAT activity.On the other hand,the interaction between QDs and SOD changed the conformation,secondary structure of SOD,leading to the inhibition of molecular SOD activity Combined with cell experiments,we can find that the effects of quantum dots on the activity of the two enzymes at the molecular level were not the same as those at the cellular level,which indicatd that the QDs can affect the enzyme activity by reacting with the enzyme molecule directly.However,in the cells,it was a more complex physiological process,and there were other factors that affected the enzyme activity.On the other hand,for the two indirect antioxidant proteins,the results showed that although quantum dots did not affect the polarity of hCG,but the interaction between quantum dots and hCG changed the skeleton and secondary structure of hCG,inducing the loose of the protein skeleton and the change of the spatial structure,and then affected its active center.The interaction between QDs and TRF induced the loosing of skeleton structure of TRF and the unfolding of secondary structure,meanwhile,the amino acid residues in the internal hydrophobic region turned outward to the external hydrophilic region.QDs affacted the microenvironment of TRF aromatic amino acid residues,making them hydrophilic.(4)We elucidated the mechanism of DNA damage induced by exposure to MPA-CdTe QDs at cellular and molecular levels.The results showed that DNA damage began to appear(compared with the blank group,when the concentration of quantum dots in the experimental group reached 10×10-6 M,the DNA damage in hepatocytes increased from 4.7%to 46.3%,and the DNA damage in nephrocytes increased from 27.5%to 91.6%)in cells at low concentration of QDs(1×10-6 M);when exposed to the same concentration of QDs,the degree of DNA damage in nephrocytes was higher than that in hepatocytes.The result of DNA damage is consistent with the results of cellular lipid peroxidation experiment,which also indicated that the exposure of QDs led to an increase in intracellular ROS content,thereby causing lipid peroxidation of cells and oxidative damage to the DNA.In addition,the addition of antioxidant,NAC,significantly reduced the degree of DNA damage in hepatocytes and nephrocytes(for the experimental group with a QDs concentration of 10×10-6 M,the degree of DNA damage in hepatocytes decreased from 46.3%to 26.7 after NAC addition,and the degree of DNA damage in nephrocytes decreased from 91.6%to 40.3%),which proved that the intracellular oxidative stress induced by QDs plays an important role in DNA damage.In addition,this chapter studied the molecular mechanism of DNA damage induced by MPA-CdTe QDs using various spectroscopy methods and ITC methods.The result showed that MPA-CdTe QDs spontaneously binded to the phosphate backbone of DNA by hydrophobic force,and the reaction changed the double helix structure of DNA.