Toxic Effects And Mechanisms Of Microplastics On Human Cells

Currently,microplastics have become a class of pollutants that are ubiquitous and continuously produced in soils,atmospheric,freshwater and oceans.Due to the long-term exposure to the environment,microplastics can be further aged under the influence of external factors.And aging will significantly change the physical and chemical properties of microplastics,which ultimately influence the environmental behavior and health risks of microplastics.Microplastics are exposed basically via inhalation,ingestion,and dermal exposure into human body,due to their small size and wide distribution,microplastics can directly penetrate cell membrane and accumulate in tissues and organs,posing a potential threat to human beings.Microplastics entering any biological fluid will immediately be covered by a corona of biomolecules.The corona confers biological identity to the microplastics,as it is this that interacts with the cellular machinery,thereby determining the microplastics destiny in and impacts on organisms.Therefore,the study on the environmental behavior and health risks of microplastics have become the research focus in the environmental field.The current health risks of microplastics are mainly focused on their toxicological research which based on primary or commercially available model with short-term exposure.However,there is a lack of in-depth understanding of the toxic effects caused by the long-term exposure and the aged microplastics.Therefore,in the present study,human cells and polystyrene microplastics(PS-MPs)(a commonly used plastic polymer)were selected to investigate the biological effects of microplastics(at low concentration)at both short-term exposure and long-term exposure.Transcriptomics were used to analysis the possible cytotoxic mechanisms of short-term and long-term PS-MPs exposure.The changes in physical and chemical characteristics and aging mechanism of PS-MPs during chlorine disinfection process were investigated.Moreover,the cytotoxicity results were used to clarify the potential mechanisms of the toxicity induced by pristine PS-MPs and chlorinated PS-MPs.The adsorption mechanisms of HSA and anti-cancer lead compounds by different microplastics were analyzed,and biological effects of microplastics induced by the formation of molecular corona were also evaluated.This provides effective information for the accurate assessing the health risks of microplastics.The main research contents and results of this paper are as follows:(1)The effects of short-term and long-term exposure of microplastics on human gastric mucosal epithelial cells(GES-1)were investigated,and the underlying molecular mechanisms were also clarified.The effects of microplastics on proliferation,EdU,clone formation and migration were evaluated.It was found that both short-term and long-term exposure did not have major adverse effects on cell proliferation,DNA duplication and migration.Transcriptome analysis revealed that a total of 488 differentially expressed genes(200 up-regulated and 288 down-regulated)were identified at short-term exposure.The gene functions and pathways for cytocyine-cytocyine receptor were identified.A total of 1504 differentially expressed genes(733 up-regulated and 771 down-regulated)were identified in the transcriptome at long-term exposure.The gene functions and pathways for inflammatory pathways including MAPK and TNF were identified.Moreover,RT-qPCR was also used to verify the differentially expressed genes found in transcriptomics.It was found that PS-MPs could induce stress response in long-term treated GES-1 cells such as triggering the inflammatory response through noclassic NF-κB.(2)The effects and mechanisms of chlorine disinfection on the age of microplastics were evaluated.In this study,the chlorination of microplastics,utilizing PS-MPs as a model,was investigated under chlorine with simulated chlorine disinfection for different time and dosage.Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS)and energy dispersive X-ray spectroscopy(EDS)were used to analysis the changes in chemical properties of PS-MPs during the process of chlorine disinfection.It was found that abundant carbon-chlorine bonds and persistent free radicals were generated on the surface of chlorinated PS-MPs.In addition,the aging of PS-MPs by chlorine disinfection involved the process of degradation,oxidation and chlorination.Furthermore,the degree of degradation,oxidation and chlorination for PS-MPs increases with the increasing free chlorine concentration and exposure time.Scanning electron microscope(SEM)combined with curvature differences analysis revealed that chlorine disinfection did not change the size of pristine PS-MPs.But could induce the formation of some cracks and pits on the surface of PS-MPs and increase the roughness of PS-MPs,suggesting that chlorine disinfection could induce the structural damage of PS-MPs.Chlorine disinfection could also decrease the Zeta potential and hydrodynamic diameter,which may contribute to the change of stability in the environment.Moreover toxic effects of PS-MPs after chlorine disinfection were evaluated,GES-1 cells were selected as the model,and cell viability was applied to evaluate the cytotoxicity of pristine and chlorinated PS-MPs.It was found that both the pristine PS-MPs and chlorinated PS-MPs had almost no impact on cell viability upon low concentration(1 mg/L)exposure.However,GES-1 cell viability were reduced obviously after exposure to high concentration(especially at 100 mg/L).Additionally,there are significant differences between pristine PS-MPs and chlorinated PS-MPs groups at high concentration exposure,chlorinated PS-MPs posed a higher cytotoxicity to GES-1 cells than pristine PS-MPs.(3)The underlying mechanisms responsible for the cytotoxicity of chlorinated PS-MPs were disclosed.Compared with pristine PS-MPs,chlorinated PS-MPs markedly changed morphology and submicroscopic of GES-1 cells.MD results showed that the increased cell membrane permeability caused by chlorinated PS-MPs might due to the presence of C-Cl bonds.Moreover,chlorinated PS-MPs could also induce cell oxidative stress,inflammatory response and apoptosis.A centrifugation-elution strategy that similar with immunoprecipitation was used to validate the potential affinitive proteins.Proteomics analysis indicated that chlorinated PS-MPs and pristine PS-MPs had obvious interactions with intracellular proteins,however,they were different in terms of category and abundances of bonded proteins,likely due to the difference in surface properties between these two particles.Compared to pristine PS-MPs,chlorinated PS-MPs could react with more distinct proteins,particularly mitochondrial,plasma membrane and cytoplasmic proteins,which might be the key issue to initiate the cell damage.Furthermore,we found that the regulation of PI3K/AKT and Bcl-2/Bax pathways,oxidative stress-triggered mitochondrial depolarization,and the activation of caspase cascade were identified as the underlying mechanisms for the enhanced apoptosis ratio in GES-1 cells when exposed to chlorinated PS-MPs.(4)The adsorption mechanisms of HSA and anti-cancer lead compounds by different microplastics were analyzed,and biological effects of microplastics induced by the formation of molecular corona were also evaluated.Protein adsorption experiment revealed that there exist different adsorption mechanism between HSA and different microplastics(pristine PS-MPs,PS-Cl-MPs and PS-NH2-MPs),likely due to the difference in surface properties of microplastics such as surface charge and roughness.Moreover,results of molecular dynamics(MD)and FTIR further demonstrated that the secondary structure of HSA were changed a lot induced by pristine PS-MPs,PS-Cl-MPs and PS-NH2-MPs,leading to the decrease in α-helix and increase in β-turn and random coil.These changes may have negative effects on protein activity.Zeta potential and hydrodynamic diameter results demonstrated that protein corona(especially on the surface of PS-Cl-MPs and PS-NH2-MPs)could accelerated the agglomeration of microplastics,which may have negative effects on their stability in human body.CCK-8 results revealed that the formation of protein corona could alleviate the cytotoxicity induced by microplastics.Moreover,the inhibition rate of cancer cells was used to evaluated the effects of PS-MPs,PS-NH2-MPs and PS-Cl-MPs on the anti-cancer viability of anti-cancer lead compounds.Molecular docking were also applied to investigate the interaction mechanism between microplastics and anti-cancer lead compounds.Results suggested that PS-MPs have negative effects on the anti-cancer activity of lead compounds through the formation of hydrophobic effect and pi-pi stacking between them.