Study On The Toxicity Effect And Mechanism Of Oxidative Stress Induced By Ultrafine Carbon Black With Different Particle Sizes At Cellular And Molecular Levels

Air pollution has become an environmental problem which has attracted major global concern.Studies have demonstrated that long-term or short-term exposure to ambient particulate matter(PM)can cause negative effects on human health,and the toxicity of PM in the atmosphere will increase as its particle size decreases.Therefore,ultrafine particles(UFPs)with smaller particle size pose a greater threat to human health.Ultrafine carbon black(UFCB)is one of the important components of UFPs in the atmosphere.As a widely used nanomaterial,UFCB may be exposed during production and processing.In addition,the incomplete combustion of carbon-containing fuels is another important exposure route for UFCB.UFCB has been classified by the International Agency for Research on Cancer(IARC)as a class 2B carcinogen.Studies have shown that UFCB can penetrate the body barrier and accumulate in the lung interstitium and tissues,causing oxidative damage,pathological damage and inflammatory effects in lung tissue,even inducing genotoxicity.However,there is currently a lack of research on the size-dependent toxic effect of UFCB,and systematic particle size effect evaluation methods and representative basic data.Especially in terms of study subjects,no study has focused on the detoxification organs where UFCB is easily deposited,and the size effect of UFCB at the molecular level has yet to be elucidated.Based on the progress,this study selected mouse primary hepatocytes and two important antioxidant enzymes SOD and CAT as study subjects to explore the size effect mechanism of UFCB at the cellular and molecular levels.The study mainly includes the following five parts:The first chapter discussed the species,sources and study progress of toxic effects of UFPs in the environment,and introduced that oxidative stress is the main mechanism of UFPsmediated cytotoxicity.In view of the current diversification of nanoparticle application routes and production processes,the size toxic effects of UFPs were summarized,and the significance of UFCB as a research model for UFPs was expounded.In the second chapter,UFCB13 nm,UFCB50 nm and UFCB95 nm were selected as the research models for UFPs.The surface morphology and particle size information of UFCB with different particle sizes were characterized by scanning electron microscopy.The results showed that the shape of UFCB was fine spherical particles and the particles showed a highly aggregated state.The particle size basically conformed to the manufacturer information.The assay of dynamic light scattering showed that the average particle size of UFCB 13 nm in aqueous solution was the smallest,while the particle size distribution of UFCB50 nm was the most uniform.The determination of zeta potential showed that compared with UFCB 13 nm and UFCB95 nm,the dispersibility of UFCB50nm was the highest.The third chapter took primary mouse hepatocytes as the study subject,and clarified the size effect of UFCB at the cellular level by using oxidative stress mechanism,apoptosis program and gene damage assay.Firstly,the hepatocytes viability in the exposure group of UFCB13 nm decreased to 22.5%,which was lower than that of UFCB50 nm(45.3%)and UFCB95 nm(55.1%).The changes in intracellular reactive oxygen species levels,SOD and CAT activities and glutathione content all demonstrated that UFCB13 nm induced higher levels of oxidative stress in hepatocytes.In addition,the malondialdehyde content of hepatocytes in the UFCB13 nm exposure group increased to 137.65%,which was higher than the other two groups,indicating that UFCB with smaller size induced more severe oxidative damage in hepatocytes.The exposure of UFCB 13 nm led to the greatest decrease of mitochondrial membrane potential,which marked the destruction of mitochondrial function and the occurrence of early cell apoptosis.In the study,the proportion of apoptotic cells in each exposure group was further determined,and early apoptosis occurred in 25.2%of cells in the UFCB 13 nm exposure group,which was higher than UFCB50nm(15.7%)and UFCB95nm(16.6%).Cell apoptosis is usually programmed death regulated by genes.In the gene damage assay,the exposure of UFCB with smaller particle size aggravated the oxidative damage of DNA.The fourth chapter took SOD and CAT as the research objects at the protein molecular level.The exposure of UFCB50 nm resulted in the maximum change of the SOD and CAT backbone structures and secondary structure.The sensitization phenomenon of UFCB50 nm exposure group was more significant in fluorescence spectrometry,indicating that the microenvironment of aromatic amino acid residues was affected to a greater extent.To verify the consistency of protein structure and function,this study further measured the changes of SOD and CAT activities under the exposure to UFCB.The results showed that compared to UFCB13 nm and UFCB95nm,UFCB50nm induced more significant SOD and CAT inactivation.By simulating the binding model,the study believes that UFCB can interact with protein molecules in an attachment binding manner to form a "protein crown".So UFCB50 nm with better dispersion can bind more protein molecules,thereby inducing more serious structural changes and functional impairment of SOD and CAT.The fifth chapter summarized the main work and innovation,and analyzed the shortcomings of this study.The study in this field was prospected.At the cellular and molecular levels,this study evaluated the toxic effects associated with oxidative stress induced by UFCB with different particle sizes,and elucidated the size effect of UFCB-induced apoptosis and genotoxicity by measuring hepatocyte apoptosis and intracellular DNA damage.By simulating the binding model of UFCB and antioxidant proteins,the mechanism of protein structure changes and functional expression differences was revealed.A regular model of the function of intracellular and extracellular antioxidant proteins changing with the particle size of nanopollutants was constructed.It provides a scientific basis and methodological reference for the study of size effect of atmospheric UFPs at the cellular and molecular levels.