Study On Cytotoxicity Induced By Polystyrene Plastic Exposure And The Mechanism Of Bile Acid Metabolism Disorders In Mice

Plastics are widely used in various fields such as agriculture,medicine and transportation.However,with the rapid increase in plastic production and consumption,plastic pollution is also growing.The aging of discarded plastic products in the environment through weathering,mechanical stress,ultraviolet irradiation,microorganisms and other external factors has resulted in smaller particle size microplastics（MPs）and nanoplastics（NPs）,as well as changes in the physicochemical properties of the material such as surface groups.In addition,it has been shown that the small size effect and high surface activity of nanoparticles make it easy for them to enter the human circulatory system through inhalation,ingestion and skin contact,and to rapidly adsorb proteins in the bloodstream,forming nanoparticle-protein complexes known as“protein corona”.The formation of protein corona can alter the properties of the nanomaterial and affect the subsequent biological effects.At the same time,the composition of the protein corona on the surface of the nanoparticles is influenced by their particle size,surface charge and other physicochemical properties.Therefore,further studies are needed to determine whether aging-induced changes in surface properties affect the composition of the protein corona on nanoplastics surfaces and subsequent biological effects such as cytotoxicity and cellular uptake,which will help to provide a proper understanding of the toxicity of micro-and nanoplastics in the real environment.In addition to studies at the cellular level,it is also important to explore the toxic effects of micro-and nanoplastics at the animal level.Exposure to micro-and nanoplastics induces abnormalities in bile acid metabolism in fish and elevated bile acid levels in mouse liver,but the regulatory mechanisms that affect bile acid metabolism in mammals are unclear.Therefore,more research is needed to demonstrate the mechanisms by which micro-and nanoplastics induce disturbances in bile acid metabolism in mice,which will facilitate further understanding of how micro-and nanoplastics interfere with organismal metabolism and value the toxic effects they cause.Part I:Polystyrene nanoplastics without surface modification（PS NPs）and aminodized polystyrene nanoplastics（PS-NH₂ NPs）were used as nanoplastic models in this section to investigate the effects of aging induced by ultraviolet irradiation（UV）and ozone（O₃）treatments on the physicochemical properties of the material and the adsorption of plasma proteins.First,the aging process of PS NPs and PS-NH₂ NPs was accelerated using UV and O₃ treatments,two aged polystyrene nanoplastics（PS-UV/PS-NH₂-UV and PS-O₃/PS-NH₂-O₃）were obtained and subsequently characterised for their physicochemical properties.The morphology of the pristine and aged nanoplastic particles was observed by SEM,which revealed that the pristine PS NPs and PS-NH₂NPs were spherical with smooth surfaces,whereas wrinkles and tiny fragments appeared on the surface of aged materials.DLS analysis showed that the hydrodynamic size of PS NPs and PS-NH₂NPs became smaller,the absolute value of zeta potential decreased and the PDI value increased after aging,indicating that the aging treatment reduced the stability and homogeneity of the material.The FTIR spectroscopy and XPS analysis revealed a significant increase in the total O/C content on the surfaces of PS NPs and PS-NH₂ NPs after UV or O₃ treatment,as well as an increase in the content of oxygen-containing groups in the aged materials to varying degrees.In addition,the water contact angle of both materials was significantly reduced after aging compared to the pristine PS NPs and PS-NH₂ NPs,indicates an increase in hydrophilicity on the surface of the material after aging,which is consistent with an increase in surface oxygen-containing groups.Subsequently,we chose to incubate mouse plasma（MP）in vitro to simulate the circulatory environment of nanoplastics after they enter the body,and to analyse the interaction of PS NPs,PS-NH₂NPs and plasma proteins before and after aging.The results of BCA quantification showed that incubation time had little effect on the total amount of proteins adsorbed on the surface of the material,but aging caused a reduction in the total amount of proteins adsorbed on the surface of PS-NH₂ NPs.SDS-PAGE of the proteins adsorbed on the surface of the material before and after aging revealed a strong interaction between NPs and MP,which correlated with plasma protein concentration,and aging altered the protein adsorption properties of the materials.In addition,it was found that the adsorption of proteins on the surface of the material before and after aging would further alter the physicochemical properties of the material in terms of particle size and potential.The above results showed that both UV and O₃ aging methods can change the physicochemical properties of PS NPs and PS-NH₂ NPs such as morphology,hydrodynamic diameter,potential,water contact angle and increased the content of oxygen-containing groups on the surface,and this change would further alter the adsorption characteristics of the materials on plasma proteins and the physicochemical properties of the materials after adsorption of plasma proteins.Part II:This section explored the effects of aging-induced changes in the physicochemical properties and adsorbed protein corona of polystyrene nanoparticles on the toxic effects and uptake of mouse mononuclear macrophage leukaemia cells（RAW264.7）.The effect of aging and protein corona encapsulation on the cytotoxicity of RAW264.7 was first investigated.Cell viability was measured using CCK-8 and a dose-and time-dependent effect of PS NPs and PS-NH₂ NPs on RAW264.7 cell viability was found.Pristine PS NPs and PS-NH₂ NPs were more toxic to cells,while aging effects and protein corona encapsulation attenuated the cytotoxicity of PS NPs and PS-NH₂NPs.Further studies of PS-NH₂ NPs before and after aging were discarded in subsequent experiments due to the excessive effect of PS-NH₂NPs on cell viability.The relative fluorescence intensity of PI as well as LDH release from cells after exposure to PS NPs before and after aging showed changes corresponding to cytotoxicity,suggesting that aging reduces cell membrane damage caused by the material.Flow cytometry analysis showed that both pristine and aged PS NPs were taken up by RAW264.7 cells after 24 h of exposure and that the cells showed a time-and concentration-dependent uptake of the material.Overall,aged PS NPs were less taken up by cells than pristine PS NPs,the 10%MP pre-incubated material was less taken up than its bare counterpart,and RAW264.7cells took up less PS-UV@MP and PS-O₃@MP than PS@MP.To further investigate the reason for this reduced uptake,the protein corona adsorbed on the surfaces of PS,PS-UV and PS-O₃ were examined using LC-MS/MS,and it was found that the relative ratio of surface-adsorbed dysopsonins versus opsonins proteins increased after incubation of PS-UV and PS-O₃ with 10%MP,which may account for the reduced cellular uptake of the material after aging.In addition,flow cytometry analysis showed that the aging process with encapsulation of plasma protein corona inhibited apoptosis induced by PS NPs.The above experimental results showed that aging significantly reduced the cytotoxicity induced by PS NPs and PS-NH₂ NPs.After aging,changes in the physicochemical properties of the material surface directly affected the cell membrane damage and cellular uptake of PS NPs,thus reducing the cytotoxicity of the material.In addition,the altered composition of the plasma protein corona on the surface of the material also had an impact on the cellular uptake and cytotoxicity of the material.The increased ratio of dysopsonin to opsonin proteins resulted in a much lower uptake of PS-UV@MP and PS-O₃@MP by RAW264.7 cells than PS@MP and therefore caused little cellular damage.At the same time,the aging effect and the covering of the protein corona would also alleviate the apoptosis caused by the material.Part III:This part explored the effects and mechanisms of oral exposure to PS MPs on bile acid metabolism in mice.We used C57BL/6 mice treated with a gradient of PS MPs concentrations（0.05,0.5,5 mg/kg/day for 30 days）and showed that oral exposure to PS MPs caused a dose-dependent increase in liver and serum bile acid levels and related indicators of bile acid sludge such as AKP and AST in mice,suggesting that exposure to PS MPs caused disturbances in bile acid metabolism in mice.The results of H&E staining and Masson staining indicated that cholestasis caused by exposure to PS MPs resulted in liver tissue damage and the development of mild fibrosis in mice.At the same time,the metabolism of lipids in the serum and liver was affected.Further analysis of genes related to bile acid metabolism in mouse liver and intestine revealed that PS MPs caused bile acid sludge in the liver mainly by inhibiting bile acid efflux-related genes BSEP,MRP2and NTCP,while at the same time PS MPs could activate the expression of FXR/FGF15pathway in intestinal tissues,thereby inhibiting the expression of hepatic CYP7A1 and suppressing synthesis of bile acid,which may be a negative feedback regulation caused by bile acid sludge.In addition,exposure to PS MPs also damaged the intestinal barrier,allowing them to accumulate in liver tissue.In vitro experiments further demonstrated that exposure to PS MPs caused damage to Hep G2 and significantly reduced the expression of genes related to bile acid efflux.The results above suggested that oral administration of PS MPs can accumulate in the liver of mice by damaging intestinal tissues,which in turn down-regulates the expression of genes related to bile acid efflux in the liver,causing abnormal bile acid metabolism in mice,as well as causing liver damage and abnormal lipid metabolism in mice.