Toxic Effect And Mechanism Of Ultrafine Carbon Black On Mouse Splenocytes And Two Kinds Of Enzymes

Nowadays,the air pollution problem has become the primary factor that affects the health of local residents and the air quality in cities,which presents a character of combined pollution:multi-media effects and multi-source superposition.As the core of air pollutants,the health effects of atmospheric particulates,especially ultrafine particles(UFPs)on human body have drawn the attention of governments and organizations in the world.With small particle diameter,large specific surface-area,high number concentrations and adsorption of various pollutants,UFPs present unique biological effects in the body,which is believed to be closely linked to the respiratory,immune and cardiovascular diseases of human.Inflammatory response and oxidative stress is the main toxic effects of UFPs in the body.Humans can be exposed to these particles by routes like inhalation and oral instillation.Once inside,UFPs can act on alveolar epithelial cells and macrophages with changes of cell membrane permeability,active of relevant signaling pathways,transcription and expression of inflammatory cytokines,which eventually induce oxidative stress and inflammation in the organism.In addition,some of UFPs can penetrate cellular barrier of the epithelial and endothelial cells and thereafter act on tissues and organs,such as liver and spleen via blood circulatory system,causing further oxidative damage.At present,most toxicological studies on UFPs focus on the biological effects on lung cells,the toxic effects and mechanism of action caused by the accumulation of UFPs in other organs and tissues have not been elucidated yet,especially the research gap need to be filled about the formation mechanism of complexes in the process of interactions of UFPs with biological macromolecules.Therefore,it is of great importance to explore the biological effects and toxicity mechanisms of UFPs into the blood circulation at the molecular and cellular levels.In view of the complex composition of UFPs and the difficulties to study dose-effect relationships of each composition,ultrafine carbon black(UFCB)with stable properties was selected as a model to study the biological effects of UFPs into blood circulatory system and the mechanism of oxidative stress induced by the accumulation of UFPs in spleen.This study mainly includes the following four parts:The first part briefly introduced the characteristics and current situation of air pollution in our country and the latest research progress of atmospheric particle pollution.Then intake patterns,biological effects and toxicity mechanism of UFPs and UFCB were elaborated from multiple aspects such as molecular toxicity,cytotoxicity,physical and chemical properties.The latest research progress and current issue of pollutants in epidemiology and nanotoxicology on were also reviewed.Finally we summarized the toxicity evaluation methods adopted in the study.In the second part,mouse splenocytes were selected as research target to explore the vitro oxidative damage under short-term exposure to UFCB via testing oxidative stress parameters like reactive oxygen species(ROS),cell vitality,malondialdehyde(MDA)and antioxidase activity.Low dose of UFCB(<5-15 ug/ml)induced a weak oxidative stress in splenocytes.The activity of CAT and SOD slightly increased with ROS and MDA level in a stable range,which means a weak lipid peroxidation and oxidative damage under the regulation of antioxidant system.When reached to a higher concentration(>15 ug/ml),UFCB can induce cells to produce and accumulate excessive ROS,leading to a decrease of antioxidase activity.Lipid peroxidation and oxidation stress thereafter resulted in the decrease of cell vitality and spleen oxidative damage.In the third part,the most ubiquitous digestive enzyme a-amylase was chosen as the target protein in blood.Multiple spectroscopic methods and enzymatic activity detection techniques were applied to investigate the effect on the structure and function of a-amylase under short-term UFCB exposure at the molecular level.The experimental results showed that UFCB could bind to a-amylase in biological fluids to form complexes called the protein-corona after invading the biological environment.Their comlbination altered the conformation of ?-amylase and the microenvironment of amino acid residues,causing the regular increase of fluorescence and skeletal contraction of the protein.In addition,enzyme activity experiments showed that with the addition of UFCB,the enzymatic activity decreased to 84.7%compared to the control group,which means the binding sites of UFCB may be located near the enzyme activity center,which affected the expression of a-amylase activity.In the fourth part,another common digestive enzyme in blood lipase was selected as research target to study the toxic effects of UFCB on lipase.Similar to a-amylase,the nanoparticles UFCB could react with lipase to form the protein-corona in biological fluids and the binding sites were located near the active site,resulting in the activity decline(77.9%compared to the control group)of lipase.Beyond that,the secondary conformation of lipase and the microenvironment of amino acid residues were changed by the combination,leading to the fluorescence sensitization and skeletal contraction of the protein.In this dissertation,we investigated the potential biomacromolecule toxicity and cellular oxidative stress effects of UFCB after entering the blood circulation of the body from the molecular and cellular levels,and established the interaction model between UFCB and biological macromolecules.Such explorations would contribute to the improvement and better comprehension about the toxic mechanism and the biological effects of UFCB in the blood circulation system.This research could also provide the basic data and theoretical basis for the toxicity evaluation of nanomaterials.