A Study On The Cytotoxic Effects Of Microplastics/nanoplastics Toward Human Alveolar Epithelial Cells A549 And Their Removal

With the large-scale production and usage of plastic products around the world,the pollution problem of microplastics/nanoplastics（MPs/NPs）in recent years has become more serious,and the potential harm and impact on human health caused by this has attracted much attention.NPs,in particular,may pose greater health threats due to their smaller particle size and larger specific surface area.The biotoxicity of MPs/NPs is affected by their physicochemical properties and environmental behaviors.Particle size and surface property as important physicochemical characteristics of MPs/NPs have been reported to affect their toxicity.MPs/NPs have specific environmental behaviors in the actual environment,including the adsorption behavior of MPs/NPs with environmental pollutants and the aging effect of MPs/NPs.At the same time,due to the stability and refractory degradation properties of MPs/NPs,they can cause long-term exposure to organisms as a persistent organic pollutant in the environment.MPs/NPs are widely distributed in the air,and respiratory exposure is one of the main ways of MPs/NPs exposure on human body,which will bring health risks to humans.However,studies on the toxicity of MPs/NPs to human respiratory exposure are lacking,especially based on the characteristics and environmental behaviors of MPs/NPs.The systematic toxicity studies conducted on their particle size and surface properties,combined pollution,aging effects,and long-term exposure are of great necessity.In addition,in view of the serious pollution and adverse effects of MPs/NPs,the development of a simple,green,and effective removal technology to eliminate the harm of MPs/NPs is an urgent problem to be solved.In this study,human alveolar epithelial cells A549 were used as the in vitro model of human respiratory exposure,and polystyrene plastic particles were used as typical MPs/NPs to systematically study the toxic effects and mechanisms of MPs/NPs under different characteristics and environmental behaviors.The removal of NPs with different forms was preliminarily achieved by the preparation of modified biochar.The main contents and results of this study are as follows:（1）The toxic effects and mechanisms of MPs/NPs with different particle sizes and surface properties on A549 cells were studied.The toxicity of MPs/NPs on A549 cells were researched by using MPs/NPs with different particle sizes（20 nm,100 nm and 1μm）and different surface properties（pristine（PS）,aminated（PS-NH₂）and carboxylated（PS-COOH））.On the one hand,our results showed that particle size severely affected the cytotoxicity of MPs/NPs,and their cytotoxicity increased with decreasing particle size.The uptake of MPs/NPs in A549 cells showed that MPs/NPs with three particle sizes could be taken by A549 cells.The uptake efficiency of MPs/NPs was inversely proportional to the particle size,and the uptake efficiency of NPs with the minimum particle size of 20 nm was as high as 83.86%.Cell viability assay showed that the cell viability decreased with the increase of MPs/NPs concentration and decreased with the decrease of MPs/NPs particle size.At the same time,the reduction of particle size increased the level of ROS in A549 cells and reduced the antioxidant capacity of cells.NPs with smaller particle size promoted the up-regulation of IL-1β,IL-6,IL-8 and TNF-αgene expression up to 3.50-fold that of control.On the other hand,our results suggested that the surface properties altered the cytotoxicity of NPs,and the magnitude of the toxicity followed the order of PS-NH₂>PS-COOH>PS.The cellular uptake efficiency of PS-COOH and PS-NH₂ was significantly enhanced due to the interaction between the surface groups of NPs and the cell membrane.Compared with the same concentration of PS,PS-COOH and PS-NH₂ decreased the cell viability of A549 cells,and the cell viability of PS-NH₂ group was the lowest.In addition,PS-COOH and PS-NH₂ significantly inhibited the activities of antioxidant enzymes SOD,CAT and GSH-Px,and promoted the production of MDA,indicating that oxidative damage has occurred in cells,and antioxidant enzymes have been unable to maintain the balance between oxidants and antioxidants.PS-COOH and PS-NH₂ significantly promoted the gene expression of IL-1β,IL-6,IL-8 and TNF-α,and stimulated the inflammatory response.In all toxic effects,PS-NH₂ exhibited greater toxicity than PS-COOH.（2）The combined toxicity of NPs and phthalates（PAEs）on A549 cells and its mechanism were investigated.MPs,especially NPs,have high specific surface area and hydrophobicity,which are easy to adsorb a large number of toxic pollutants in the surrounding environment.As typical plasticizers,PAEs are commonly used in plastic production,and they are easy to coexist with NPs in the actual environment,causing combined pollution.Dibutyl phthalate（DBP）and dioctyl phthalate（DEHP）were selected as two typical PAEs,and they were combined with lesser-concentration（20μg/m L）and greater-concentration（200μg/m L）NPs（100 nm）respectively,were to explore the combined toxicity of NPs and PAEs.When the two PAEs were co-exposed with lesser concentration（20μg/m L）of NPs,the cell viability of A549 cells increased significantly,and the increase was more significant in the co-exposed group of NPs and DEHP.However,when both PAEs were co-exposed with greater concentrations（200μg/m L）of NPs,cell viability was decreased.Studies have shown that when PAEs were co-exposed to lesser concentrations of NPs,the free phase concentration(C_free)of PAEs decreased due to the adsorption of NPs,which could in turn lead to a decrease in their bioavailability,and further cause the weakening of the combined toxicity of NPs and PAEs.However,when greater concentration of NPs were co-exposed with PAEs,the toxic effects may be dominated by NPs,thus resulting in a decrease in the viability of A549 cells.Further studies found that oxidative stress and inflammatory responses were the main mechanisms for the combined toxicity.（3）Toxic effects and mechanisms of aging effect on NPs in A549 cells were studied.Aging is an inevitable process of NPs in the environment.The ultraviolet radiation to NPs for 1 month and 2 months were conducted to simulate the photodegradation effect,and the differences in the toxicity of NPs without and with aging treatment to A549 cells were compared.The results showed that,in terms of physicochemical properties,aging reduced the particle size of NPs and formed abundant oxygen-containing functional groups on the NPs surface.Studies from cell levels on cell viability,oxidative stress,membrane damage,and mitochondrial function showed that the aged NPs exhibited greater cytotoxicity compared with pritine NPs,and this toxicity was positively correlated with aging time.Further transcriptome analysis showed that aged NPs had more severe interference on gene expression,which reconfirmed that aging significantly enhanced the toxic effect of NPs on A549 cells at the molecular level.This part of the study emphasized the effect of aging on the physicochemical properties of NPs,and revealed the enhanced effect of aging on the toxicity of NPs at the cellular and molecular levels.（4）The transcriptomic response and molecular mechanism of long-term exposure of NPs to A549 cells were studied.The chronic toxicity of 2-months aged NPs to A549cells was explored by the long-term exposure（30 d）of low dose（4μg/m L）.Meanwhile,A549 cells were treated to short-term（24 h）exposure at a high dose of NPs（80μg/m L,the same cumulative dose as long-term exposure）for comparative analysis.The highly sensitive transcriptomic technique was used to analyze the effects of short-and long-term exposure of aged NPs on A549 cells.The results showed that 4097 differential expressed genes（DEGs）were found in A549 cells exposed to NPs for a short term,of which 2591 DEGs were up-regulated and 1506 DEGs were down-regulated.1869DEGs were found in A549 cells exposed to NPs for a long term,of which 1309 DEGs were up-regulated and 560 DEGs were down-regulated.The Gene Ontology（GO）database and the Kyoto Encyclopedia of Genes and Genomes（KEGG）database were used for enrichment analysis of the identified DEGs.It was found that the short-term exposure group of aged NPs was significantly enriched to 378 GO terms,while the long-term exposure group was significantly enriched to 818 GO terms.KEGG enrichment analysis showed that 19 KEGG pathways were enriched in the short-term exposure group of aged NPs,while 23 KEGG pathways were enriched in the long-term exposure group.The short-term and long-term exposure groups of aged NPs shared 8KEGG pathways,including DNA replication,cell cycle,IL-17 signaling pathway,MAPK signaling pathway,and TNF signaling pathway.However,15 specific KEGG pathways were enriched in the long-term exposure group of aged NPs and were mainly related to amino acid metabolism,cell growth and death,and signal transduction,including alanine,aspartate and glutamate metabolism,glycine,serine and threonine metabolism,cysteine and methionine metabolism,apoptosis,PI3K/Akt signaling pathway,etc.This part of the study highlights the interference of long-term exposure to aged NPs on gene expression,providing detailed transcriptome sequencing data.（5）The ball-milled magnetic biochar was prepared to remove the aged and surface-modified NPs.A facile,low-cost,and environmentally friendly magnetic biochar was successfully prepared by ball milling and the application of Fe₃O₄nanoparticles,which was easily separated under the action of a magnetic field.The ball-milled magnetic biochars prepared at different pyrolysis temperatures of 300℃（MBC300）,500℃（MBC500）and 700℃（MBC700）were studied for the removal of pristine（PS）,UV-aged（UVPS）,aminated（PS-NH₂）and carboxylated（PS-COOH）NPs.The results showed that the removal rates of PS by MBC300,MBC500 and MBC700 were 3.01,5.76 and 3.10 times higher than the corresponding pristine biochars,respectively,at the same pyrolysis temperature.Among them,MBC500 had the highest removal rate of NPs,indicating that the increase in specific surface area and the increase in oxygen-containing functional groups after ball-milling both played important roles in the adsorption process.By exploring the effect of the surface properties of NPs on its removal rate,it was found that the removal rate of PS-NH₂ was the highest with ball-milled magnetic biochar.The adsorption kinetic experiments showed that the ball-milled magnetic biochar had a faster adsorption rate than the pristine biochar.The Langmuir maximum adsorption capacity of NPs by ball-milled magnetic biochar was 107.71-229.59 mg/g,which was much higher than that of pristine biochar（55.45-80.30 mg/g）.This part of the study verified the simplicity and effectiveness of ball milling technology in the preparation of magnetic biochar.Ball-milled magnetic biochar has great potential for efficient removal of NPs,especially NPs with different forms,from water bodies.In this study,based on the own characteristics and environmental behaviors of MPs/NPs,we systematically studied the toxic effects and mechanisms of MPs/NPs,especially NPs,in A549 cells,which enriched people’s comprehensive understanding of MPs/NPs toxicity,and is of great significance for the risk assessment of respiratory exposure of NPs.This study provides a theoretical basis for the research on human health threats of MPs/NPs,and the pollution prevention and control of MPs/NPs.The preparation of modified biochar efficiently removed NPs of different forms,which provides a solution and technical support for the pollution prevention and control of MPs/NPs.