Research on the Mechanism of UFCB Inducing Oxidative Stress and Genotoxicity on Molecular,cellular and Animal Level
Supervisor:liu ru tao
As a typical nanoparticle,ultrafine particle is mainly from the industry waste gas,vehicle emission,artificial synthesis and even the natural activities such as volcanos and tides.Anthropogenic activity has been the main ultrafine particle source which lead to the fact that ultrafine particle exposures to human beings mostly happens in the polluted area.Exposure to ultrafine particles could cause severe inflammation in the respiratory tract and even induce damages to organs all over the body by crossing the lung-blood barrier.As a kind of widely used ultrafine particle,ultrafine carbon black(UFCB)has been used in rubber,painting,dyes and printing.Therefore,it is important to figure out the potential toxicity of UFCB to the environment and organisms.UFCB is a carbon-based particle with an average diameter less than 100 nm and the diameter of the UFCB employed in our research are 13 nm(FW200)and 50 nm(SB100),and this two UFCB were used to cellular toxicology and molecular/organism toxicology based on their dispersity in different solutions.The current studies on the toxicity of UFCB focuses on its ability to induce inflammation in the respiratory system of the mouse and most of the target animals are mammal,the possibility of UFCB leaking into the water has increased stagerringly since the use of UFCB in agriculture,land and water management.In addition,as the UFCB might penetrate into the circle system,it may further lead toxicity to the micromolecules and cells of the organism.However,little attention was paid to the toxicity of UFCB to biomacromolecule,cell or aquatic organism.Herein,we investigated the toxicity of UFCB on molecular,cellular and organism level by using antioxidant enzymes,mouse hepatocytes and zebrafish as the target biological model.Our work has six chaptersChapter 1 is the introduction of our work.It discusses the widespread of ultrafine particles in the environment and their potential negative effects to human health.This chapter also talk about the formation of protein corona and its effect on the fate and toxicity of ultrafine particles in the organism.The definition of UFCB and its connection with ultrafine particles were proposed.The fact that UFCB has been used as a model in the study of ultrafine particle toxicity and the extensive use of UFCB in our daily life has motivated tons of effort on the research of UFCB toxicity.The current study on the toxicity of UFCB was summarized.The problems in the investigation of UFCB toxicity waiting to be solved and hypothesis need to test and verify were proposed in the end of the chapter.Chapter 2 talked about the dispersion effect of FW200 and SB 100 in different dispersants and selected the proper dispersants for corresponding toxicological studies.Five different dispersants(ultrapure water,tween 80,tween 20,ethanol,complete medium)were employed to disperse FW200 and SB100,DLS(dynamic light scattering),TEM(transmission electron microscope),SEM(scanning electron microscope)were used to characterized the UFCB in the five different dispersants.The results of DLS showed that ethanol,tween 20 and tween 80 could disperse both FW200 and SB100 very well and ethanol exhibited the highest dispersing ability among the three dispersants.On the other hand,FW200 and SB 100 could be dispersed in complete medium and ultrapure water well,respectively.The complete medium and ultrapure water have a similar ability to disperse FW200 and SB 100 with tween 80 or tween 20.Thereafter,FW200 and SB100 were dispersed in complete medium and ultrapure water for the cellular and molecular/organism toxicity study.Chapter 3 revealed the mechanism of UFCB inducing oxidative stress and genotoxicity on molecular level by studying the interaction between UFCB with antioxidant proteins and DNA molecule.Antioxidant proteins play a key role in scavenging reactive oxygen species(ROS)in organism and exotic chemicals could cause oxidative stress by impacting antioxidant proteins in organism.To figure out the mechanism of inducing oxidative stress and genotoxicity of UFCB,we investigated the interaction between SB 100 and four different biomacromolecules including SOD(superoxide dismutase),LYZ(lysozyme),TF(transferrin)and DNA(herring sperm DNA).UV-vis absorption,stead-state fluorescence spectra,synchronous fluorescence spectra,3D fluorescence spectra,resonance light scattering and circular dichroism were used to investigate the interaction mechanism between SB 100 and the four biomacromolecules and the conformational changes of the biomacromolecules caused by SB100.The enzyme activity of SOD and LYZ was researched to figure out the effect of SB 100 on the enzyme function,which could help build the correlation between the enzyme function and its structure change.The chapter includes four sections:(1)toxicity of SB100 to SOD;(2)toxicity of SB100 to LYZ;(3)toxicity of SB100 to TF;(4)toxicity of SB100 to DNA.(1)The interaction between SB 100 and SOD changed the structure of SOD.The results of 3D fluorescence spectra shows that a mixture named "protein corona" was formed between SB 100 and SOD,which increased the diameter of the system.The binding of SB100 and SOD also lead to the fluorescence quench and changed the conformation of SOD by loosening the skeleton of it.According to the results of the fluorescence spectra,SB 100 also made the microenvironment of SOD more hydrophilic.SB 100 changed the secondary structure of SOD by decreasing the amount of a-helix and increasing the amount of β-sheet.SB 100 also have a depressing effect on the enzyme activity of SOD which could be ascribed to the structural change of SOD.We built the cartoon molecular binding model of SB 100 and SOD based on the above analysis.In this model,SB 100 bound to the sites close to substrate tunnel and a-helix of SOD,which explains the reason for the enzyme activity depression of SOD.(2)SB 100 imposed a similar effect on LYZ with SOD,which changed the conformation and enzyme activity as well.The binding of SB100 and LYZ increased the diameter of the mixture and also unfolded the backbone of LYZ molecule.The results of UV-vis absorption shows that SB 1 00 changed the π→π*electronic transitions of C=O in the polypeptide backbone and the fluorescence spectra of LYZ was quenched by SB 100 without changing its hydrophobicity.The CD spectra show that SB100 changed the secondary structure of LYZ with the a-helix increased by 11%.The conformational changes caused by SB100 finally resulted in the decrease of enzyme activity of LYZ.In order to make it easier to understand the interaction mechanism between SB100 and SOD,we built the binding model of SB100-LYZ showing that SB 100 bound close to Trp62、Trp63、Trp108 residues which composes the active center of LYZ.(3)SB 100 interacted with TF in a way that is somewhat different from SOD and LYZ.SB 100 increased the fluorescence intensity of TF by binding with it indicating that SB 100 might change the structure of TF by making the quenchers such as carbonyl groups around the fluorophores far away from them,which could affect the microenvironment of the fluorophores as well.The UV-vis absorption and CD spectra show that SB 100 loosened the carbon skeleton of TF and changed the secondary structure of TF by decreasing the amount of a-helix while increasing that of β-sheet.However,SB 100 did not change the hydrophobicity of TF,which implied that hydrophobic force may not be the main binding force between SB 100 and TF.A binding model of SB 100 and TF molecule was made based on the above analysis to make it easier for people to figure out the binding mode of SB 100 and TF(binding to the a-helix structure of TF).(4)The interaction between SB 100 and DNA was drived by electrostatic force,which lead to changes of phosphate skeleton,base stacking and right-handed helicity of DNA.Chapter 4 studied the in-vitro toxicity of FW200 to mouse hepatocytes by incubating mouse hepatocytes with FW200 for 24 h.After the exposure,FW200 induced large amount of ROS(reactive oxygen species)and lead to the oxidative stress in the hepatocytes,which further stimulated the increase of catalase activity of mouse hepatocytes and the decrease of the amount of GSH(glutathione).The exposure of FW200 increased the amount of MDA(malondialdehyde)indicating a serious lipid peroxidation in the mouse hepatocytes.The oxidative stress and lipid peroxidation induced by FW200 finally resulted in a significant decrease of cell viability,high apoptosis level and severe DNA damage of the mouse hepatocytes.Chapter 5 evaluated the in-vivo toxicity of SB100 to zebrafish.The zerbrafish larvae were exposed to SB 100 by microinjecting the SB 100 solution to the yolk sac of the zebrafish embryo,which mimicked the maternal transfer of nanoparticles to the offspring.We measure a series of endpoints after the injection.SB 100 caused a significant mortality of zebrafish larvae and decreased the hatching rate of the larvae;In order to figure out the effect of SB 100 on the gene expression level of zerbrafish larve,we measured the expression of genes related to oxidative stress(sod1 and sod2),visual system(ppn1lw2,opnlmwl,opn1sw2)and nervous system(gfap,mbp,syn2a).SB100 upregulated the expression of sod1 and sod2,while downregulated genes of both visual and nervous system.This suggests that SB 100 affected the redox balance and function of visual and nervous system of zebrafish larvae.Hence,we investigated if the changes of the visual and nervous gene expression affected the behavior of zebrafish larvae and it turns out that SB100 do resulted in hypoactivity of the larva.This indicated that the expression change of the visual and nervous genes might lead to the behavioral abnormity of the zebrafish larva.Chapter 6 summarized the work and clarified the innovation and significance of the paper.The questions on the toxicity of UFCB that need to be further explored and the hypothesis that need to be clarified in the future were also elicited in this section.Summarily,this paper studied the toxicity of UFCB by using four different antioxidant proteins,DNA,mouse hepatocytes and zebrafish larva as the target model.Our work clarified the interaction mechanism between UFCB and the biomacromolecules and found that UFCB lead the oxidative stress and genotoxicity of mouse hepatocytes by penetrating into the cell,and that UFCB could impact the development of zebrafish by inducing oxidative stress and genotoxicity.We clarified the toxicity of UFCB on molecular,cellular(in vitro)and organism(in vivo)level,which will help people have a compresensive knowledge on the negative effects of UFCB to environmental health and provided novel toxicity-evaluation data for future research and risk assessment of this ubiquitous nanoparticle.Our work can also be used as a good reference for the government to make policies on the restriction of the usage and discharge of the UFCB.