Toxic Effects And Mechanisms Of Ultrafine Carbon Black On Lysozyme And Splenocytes After Lead Loading

There are a large amount of ultra-fine carbon black(UFCB)particles emitted from the chemical production industry and the exhaust of motor vehicles in urban atmospheric particulates,and lead exposure has been an important factor that endangers human health.UFCB has strong adsorption ability,which can adsorb heavy metals such as lead in atmospheric aerosols and organic compounds to cause composite pollution.Studies have shown that UFCB can enter the human body through the respiratory system and skin and deposit in the lungs to cause lung damage.Small-sized UFCB particles can pass through the lung interstitial and reach multiple organs,such as spleen,kidney,liver,and lymph through blood circulation,inducing a variety of diseases.In addition,after UFCB enters the body tissues and cells,it can interact with the biological macromolecules in them,and then induce cell toxicity.Therefore,studying the toxic effects and mechanisms of UFCB before and after loading pollutants is of great significance for strengthening the prevention and control of atmospheric particle pollution and protecting human health.However,current research on the toxic effects of UFCB has mostly focused on its effects on lung,such as alveolar macrophages.There are few studies on the cytotoxicity of UFCB on the body's functional proteins and other organs,and the cytotoxic effects and mechanisms of its adsorption of different pollutants have not yet been elucidated.In addition,UFCB will undergo denaturation in industrial production and after long-term aging in the air.Therefore,UFCB is often modified in laboratory to simulate its state in the atmosphere.The physical and chemical properties of UFCB,such as particle size,surface area,indicated charge,modification method,degree of aggregation,surface free radicals,determine its biological toxicity.However,many experiments on the safety evaluation of UFCB lack comprehensive characterization.In response to the above problems,this study modified UFCB(31 nm),carried lead and characterized it,and studied the toxic effects and mechanisms of UFCB on lysozyme and spleen cells before and after lead loading at the molecular and cellular level.The paper mainly includes the following five parts:The first chapter briefly introduces the source,properties and research progress of biological toxicity of UFCB,and outlines the ways of lead exposure and the harm of lead to human body.What's more,the combined pollution caused by UFCB and other pollutants in the atmosphere is also summarized.Through the literature review,the scientific value and significance of this research were clarified,and the scientific problems that are still to be solved in this field were put forward,and the experimental scheme of this research was designed for these scientific problems.In chapter two,after comparing the modification effect of acid functionalization and ozone oxidation,the ozone oxidation method were used to modify UFCB.Ultrasonic method was used to load lead acetate,and atomic spectrophotometry and xylenol orange spectrophotometry were used to measure the lead ion concentration of the supernatant after loading and after ultrasonication,respectively.In addition,particle size analyzers,scanning electron microscopes,ZETA potentiometers,and Fourier transform infrared spectrometers(FTIR)were used to comprehensively characterize UFCB before and after lead loading.The results show that under the concentration conditions used in this experiment,the ratio of lead load was about 93.2%,and no lead ions were dissociate from UFCB after lead loading(UFCB-Pb)after sonication.The results of characterization experiments show that UFCB and UFCB-Pb are negatively charged on the surface,and the degree of aggregation of UFCB-Pb in aqueous solution is higher than that of UFCB,which is related to the surface free radical COO-.The third chapter took lysozyme(LYZ),which is an important immune protein,as the research object,and used multispectral methods to study the effects of UFCB and UFCB-Pb on the molecular structure of LYZ.What's more,we studied the effects on the function of LYZ through enzyme activity detection,and the mechanism of UFCB and UFCB-Pb interacting with LYZ was explored based on the above experimental results.The results showed that UFCB and UFCB-Pb can interact with LYZ,cause fluorescence sensitization,and affect the hydrophobicity of proteins by changing the microenvironment of tryptophan residues.Compared with UFCB,the decrease of LYZ enzyme activity was more significant with the increase of UFCB-Pb concentration,which may be caused by the change of the secondary structure of LYZ.The interaction force between UFCB and LYZ may be hydrophobic,and UFCB-Pb may enter the active cavity of LYZ and cause toxicity to LYZ.The fourth chapter took splenocytes,which is an important immune cells,as the research object.After UFCB and UFCB-Pb exposure,CCK-8 cell viability test,caspase-3 enzyme viability test,mitochondrial membrane potential test,and reactive oxygen species(ROS)test were used to know the effects and mechanisms of UFCB and UFCB-Pb on cell viability and apoptosis in mouse primary spleen cells.The results showed that UFCB and UFCB-Pb have cytotoxicity to splenocytes,but toxic effects of UFCB-Pb was more significant.The mechanisms of cell injury and apoptosis is that UFCB and UFCB-Pb can induce ROS in spleen cells.The production of ROS causes damage to mitochondrial membrane and then activates caspase,eventually causing cell damage and apoptosis.Chapter 5 summarized the work of the dissertation and the innovations of this research,sorted out the shortcomings of the research and put forward the research prospects.In this study,scientific methods were used to modify UFCB and UFCB-Pb,which were comprehensively characterized.We studied the effects of ultrafine carbon black before and after lead loading on the molecular structure and function of lysozyme at the molecular level.And their effects on the viability and apoptosis of spleen cells and the specific pathway were also studied.The toxicity of ultrafine carbon black before and after lead loading to immune-related lysozyme and spleen cells was studied at the molecular and cellular levels,respectively.It provided a method based on UFCB to study atmospheric fine particles before and after loading pollutants on functional proteins and cells in the body was established.It provided basic data for the toxicity of UFCB to immune-related proteins and cells before and after lead loading.In addition,it provided a scientific basis for the safety application of UFCB and the prevention and control of atmospheric particulate matter.