| Micro/nanoplastics(MPs/NPs)have become a worldwide concern as newly emerging pollutants in recent several years.They have also been found in many real environments and generated enormous publicity in the related research areas.They may induce severe biological risks because of their larger specific surface area and smaller size.At present,the toxicity assessment of MPs/NPs is not limited to the basic research on the physicochemical properties of MPs/NPs.More studies have focused on the environmental relevance of MPs/NPs(realistic MPs/NPs and their complex environmental transformation).In this thesis,different MPs/NPs model particles are prepared to assess the toxicity effects of MPs/NPs from the perspectives of the protein corona,combined contamination and source authenticity.The main contents are outlined below.1.Influence of the size benzo[a]pyrene nanoclusters and absorption on microplastics on their cellular toxicityBenzo[a]pyrene(Bap)is one of organic pollutants in atmospheric haze,which is usually enriched in fine water drops and particulate matters.The understanding of Bap’s form in water is of great importance to unveil its real biological effects toward the respiratory system.It is found that Bap existed as self-aggregated nanoclusters(NCs)with tunable sizes rather than free molecules in water.Bap NCs of smaller size enter A549 lung epithelial cells at a faster internalization speed.The size of Bap NCs a dominant parameter for determining Bap’s cytotoxicity.Polystyrene(PS)microparticles(with a diameter of 190 nm),as a model for MPs,could adsorb Bap nanoclusters and serve as carriers that enter the cells.The stably adsorbed Bap on PS MPs to produce PS@Bap composite,which reduces the size effect of Bap NCs cytotoxicity.Bap of PS@Bap composites was stably adsorbed on PS at the early stages of endocytosis until it was detached during the lysosomal transport and maturation process.The dissociated Bap might bypass the lysosome pathway and be released to the cytosol with nanocluster structure,or relocation into the endoplasmic reticulum.While the detached PS was inclined to bound to mitochondria or be excreted out of the cell upon the lysosomal pathway.Moreover,PS@Bap composites resulted in a significant loss of mitochondrial membrane potential and induced apoptosis through a mitochondria-involved apoptosis pathway.2.Influence of Mucin corona on the intracellular behavior of microplasticsResearches on the effects of microplastics on human health have focused on dietary exposure pathways.However,only a few reports focus on human exposure by inhalation.After being inhaled into the respiratory system,microplastics can interact with Mucin-rich mucus to form “protein corona”.The coverage of protein corona may alter the physicochemical properties of MPs,endowing it with new biological characteristics,and thus alter its interaction with lung cells.Herein,190 nm PS microparticles were prepared as microplastics model and their interaction with Mucin and the resultant biological responses were studied.The Mucin corona would stably adsorb on PS MPs at the early stages of endocytosis until degraded during the lysosomal transport and maturation process.Mucin corona can increase the uptake of PS MPs and delay intracellular trafficking of PS,thereby increasing intracellular bioaccumulation.After coverage onto the PS surface,Mucin corona exhibits no obvious cytotoxicity as PS MPs.3.Influence of Mucin corona on the intracellular behavior of PS@Bap compositesThe impact of protein corona on biological response of combined contaminant nanoparticles(CCMPs)was still poorly understood.In order to clarify how protein corona affects the cellular level events of CCMPs,polystyrene-benzopyrene(PS@Bap)MPs were prepared as CCMPs model and their interaction with Mucin and the resultant biological responses were studied.Herein,Mucin and PS were labeled with FITC and Nile red,combined with Bap’s spontaneous blue fluorescence,and their intracellular fate was detected in situ and dynamically by fluorescence imaging.Combined with cell viability detection,reactive oxygen species(ROS)generation,impaired mitochondrial function and cell apoptosis,we further explain the underlying mechanism of influence of Mucin corona on CCMPs.It was observed that Mucin corona stably attached to the CCMPs surface,which significantly altered the fate of the CCMPs in A549 lung epithelial cells.The Mucin corona is stably adsorbed on PS@Bap at the early stages of endocytosis until degraded during the lysosomal transport and maturation process.Mucin corona can delay intracellular trafficking of PS@Bap and the progress of Bap detached from PS.Mucin corona enhances the uptake of PS@Bap but reduces the cytotoxicity elicited by PS@Bap,as indicated by cell viability,generation of ROS,impairment on mitochondrial function,and further cell apoptosis.In addition,in vivo study also verified the enhanced effect of PS on the development of an acute lung inflammatory response induced by Bap.4.Realistic PVC micro/nanoplastics and the size-and concentration-dependent toxicological impacts on Zebrafish EmbryosCurrent toxicity research mainly employed chemically synthesized polystyrene MPs/NPs as model particles.The cellar toxicity of other types of MPs/NPs,such as polyvinyl chloride(PVC)MPs/NPs are rarely investigated,which limits our understanding of their real environmental and biological effects.Herein,we have proposed a simple and universal method for preparing PVC micro/nanoplastics.This method is based on the mechanical breakdown with an immersion blender,using PVC wire bushings as raw materials,combined with differential centrifugation,and can easily obtain PVC MPs/NPs of different diameters ranging from several nanometers to hundred nanometers.We investigate the bioaccumulation and in vivo toxicity of the produced PVC MPs/NPs using developing zebrafish embryos as animal models.This study demonstrates size-dependent distribution and the size-and concentration-dependent toxicity of PVC MPs/NPs in terms of hatching rate,heart rate,mortality and malformation and ROS generation.Further mechanism studies have shown that PVC MPs/NPs induce malformations and growth inhibition by interfering with the hypothalamic-pituitary-thyroid axis(HPT axis).5.Physical and chemical damage induced by environmental related PVC MPs/NPsThe mechanical breakdown method is adopted to break the PVC wire bushings to obtain PVC MPs/NPs,which preserves PVC’s chemical properties,and mimic the mechanical breakdown process of plastics in nature.Zebrafish embryos were used as animal models to investigate the bioaccumulation and in vivo toxicity of PVC MPs/NPs.We speculated the source of toxicity of PVC MPs/NPs from the physical effects,chemical factors and interactive effects of physical and chemical factors.The toxicity mechanism of environment-related PVC MPs/NPs was studied by using MPs/NPs preparing from commercial additive-free PVC powder as a reference for the physical factors of MPs/NPs and using PVC MPs/NPs leachate as a reference for the chemical factors of MPs/NPs.The size-dependent uptake and accumulation of PVC MPs/NPs were observed.From the hatching rate,heart rate,mortality,deformity and ROS generation,it also reveals that physical factors of nanoparticles can inhibit incubation,increase mortality and reduce heart rate,and chemical factors can induce growth inhibition and induction of deformity by interfering with the HPT axis,and the combined effect of physical and chemical factors can cause more serious embryo development toxicity.This work investigated the toxicity sources of environmental MPs/NPs in aquatic organisms and provided a new perspective for toxicity assessment of MPs/NPs. |
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