Characteristics Of Microplastics Exposure In Lower Respiratory Tract And Mechanism Of DNA Damage Induced By Microplastics

ObjectiveMicroplastics(MPs)are a type of plastic debris smaller than 5mm,which has been included in one of the new pollutants in China’s New Pollution Control Action Plan.Due to their small size and light weight,MPs are widely distributed in the atmosphere and pose health risks to humans through respiratory exposure.Currently,the distribution and exposure characteristics of MPs in the respiratory system are unclear.Therefore,accurate qualitative and quantitative analysis of the exposure characteristics and distribution of MPs in the respiratory system is particularly vital for assessing the risk of MPs exposure.The only existing research shown that cell viability,cell barrier damage,and cell apoptosis were affected by exposure to MPs,and the explanation for these phenomena were attributed to inflammation and oxidative stress induced by MPs.However,the mechanism of the toxic effects of MPs has not been studied.Animal experiments have suggested that MPs can cause DNA damage,but the mechanism is still unclear.DNA damage is a common indicator for evaluating genetic toxicity.Therefore,it is necessary to investigate the internal exposure of MPs in the lower respiratory tract and their mechanisms of DNA damage.Methods18 bronchoalveolar lavage fluid(BALF)samples were obtained along with 4blank controls.The BALF samples and blank controls were pre-treated using acid digestion,followed by laser infrared imaging to characterize the MPs present in the BALF and blank samples.The representative MPs from BALF samples were marked and further observed under electron microscopy to identify their morphology and features.To explore the potential respiratory toxicity of MPs,polystyrene microplastics(PS-MPs)with size in 20 nm and 1000 nm were selected and exposed to human bronchial epithelial cells(16HBE)at concentrations ranging from 0 to 1000 μg/m L for 24,48,and 72 hours.To confirm the physical properties and stability of PS-MPs,scanning electron microscopy and Zeta potential measurements were performed.The uptake of PS-MPs by 16 HBE and their induction of cytotoxic reactions were further determined using laser confocal scanning microscopy after exposure to PS-MPs at a concentration of 1000 μg/m L for 24,48,and 72 hours.Cell viability was evaluated using the CCK8 assay,and the impact of microplastic exposure on 16 HBE toxicity was determined by evaluating the exposure time of PS-MPs.Comet assay and Western blotting were used to detect DNA damage in16 HBE exposed to gradient concentrations of PS-MPs and to analyze the level of DNA damage induced by PS-MPs with size in 20 nm and 1000 nm.To further investigate the mechanism by which high concentrations of PS-MPs with size in 20 nm induce DNA damage in 16 HBE,proteomics and metabolomics were performed to examine the changes in protein and metabolite levels in cells exposed to 1000 μg/m L concentrations of PS-MPs with size in 20 nm and 1000 nm.Differential expression of proteins and metabolites in the proteomic analysis was analyzed using Kyoto Encyclopedia of Genes and Genomes(KEGG)and Reactome databases,and FEN1,PARP4,and POLE3 proteins and the metabolite hypoxanthine(Hx)with differential expression in the long-chain base repair pathway were selected as target proteins and metabolites.The expression levels of FEN1,PARP4,and POLE3 proteins and Hx were further detected using Western blotting and spectrophotometry in 16 HBE exposed to gradient concentrations of 20 nm and 1000 nm PS-MPs.ResultsMPs were detected in the all 18 BALF samples,and a total of 13 types of MPs were identified.Of the 1634 MPs observed in the samples,86.1% were identified as PE particles,while 7.5% were PET and 1.9% were PP.The size distribution of the MPs ranged mainly from 20-80 μm,with 4.2% of the MPs identified as fibers and95.8% defined as irregular particles.Results from the population study indicated significant variations in both the exposure levels and types of MPs in the lower respiratory tract among individuals.Using a Zeta potential analyzer,the potential of PS-MPs with size in 20 nm and1000nm was found to be-22.0 ± 6.75 and-11.6 ± 4.02,respectively.Results from cell uptake after time gradient and cell activity after time-concentration gradient poisoning experiments suggested that a 48-hour exposure time and a concentration gradient of 0,62.5,125,250,500,and 1000 μg/m L were the optimal conditions for subsequent experiments.Comet and Western blot experiments revealed that 20 nm PS-MPs caused DNA damage in 16 HBE at high concentrations of 500 and 1000μg/m L,while there was no apparent effect at the same concentration of 1000 nm PS-MPs.To investigate the mechanism of DNA damage caused by high concentrations of 20 nm PS-MPs on16 HBE,we compared the expression of differential proteins and metabolites in blank controls and cells exposed to 1000μg/m L of 20 nm and 1000 nm PS-MPs for 48 hours using proteomics.The results showed that there were 288 differential proteins in the20 nm exposure group compared with the control group,140 differential proteins in the 1000 nm exposure group compared with the control group,and 375 differential proteins between the 20 nm and 1000 nm exposure groups.The PCA results of the proteomics showed that there were significant differences between the 20 nm exposure group and the control group,while there were minor differences between the 1000 nm exposure group and the control group.This suggested that the 20 nm PS-MPs had a stronger effect on the protein expression of 16 HBE,which corresponds to the result of20 nm PS-MPs causing DNA damage to 16 HBE.The results of metabolomics showed that there were 35 differential metabolites in the 20 nm exposure group compared with the control group,15 differential metabolites in the 1000 nm exposure group compared with the control group,and 16 differential metabolites between the 20 nm and 1000 nm exposure groups.This indicates that 20 nm exposure group had a stronger effect on the expression of cellular metabolites,which corresponds to the results of proteomics.Using the KEGG database,three proteins in the base excision repair pathway were identified: FEN1,PARP4,and POLE3.The WB experiments with gradient concentrations of PS-MPs with size in 20 nm and 1000 nm indicate that the expression levels of FEN1 protein increase with the concentration of 20 nm PS-MPs after exposure,while the expression levels of PARP4 and POLE3 proteins both decrease at high concentrations of PS-MPs with size in 20 nm.There were no significant changes in the target protein of 16 HBE after exposure to gradient concentrations of 1000 nm PS-MPs.Through the Reactome database,metabolites differentially expressed in the group exposed to 20 nm PS-MPs were enriched with FEN1,PARP4,and POLE3,and the expression of Hx,a metabolite,was measured by spectrophotometry.The results showed that the expression of Hx increased with the concentration of PS-MPs with size in 20 nm,while there was no significant change in the expression level of Hx after exposure to 1000 nm PS-MPs.ConclusionsA variety of microplastics MPs were detected in 18 BALF sample,with the main components being PE,PET,and PP.The particle size of MPs was mainly concentrated in the range of 20-80 μm,indicating the human body may unintentionally inhale microplastics through the respiratory tract.In vitro experiments showed that PS-MPs with size in 20 nm may induce DNA damage in 16 HBE through internalization and translocation,activate the base excision repair pathway,while 1000 nm PS-MPs had no significant DNA damage effect,suggesting that high concentrations of small-sized MPs may cause respiratory DNA damage.