The Study On Inhalation Bioaccessibility Of Atmospheric Particulate Matters And Proteomics Of Cells Exposed To Atmospheric Particulate Matters

Atmospheric particulate matters(PM)are main components of air pollution,which have been reported to be associated with human adverse health effects.Inhalation of toxic elements in PM is an important exposure pathway to humans.Although risk assessment related to this pathway is usually based on total concentration of element,bioaccessible concentration based method can more accurately assess risk.Environmental proteomics takes advantage of environmental proteomics technologies to investigate reponses of cells and organisms to environmental stimuli/factors including environmental pollutants,etc.Environmental proteomics can identify signal pathways,molecular mechanisms and biomarkers of exposure.The thesis is composed of the introduction(Chapter 1)and research reports(Chapter 2-5).The research reports mainly focused on two parts including in vitro inhalation bioaccessibility of potentially toxic elements(PTEs)in PM(Chapter 2 and3),toxicoproteomic analyses of respiratory system cells following exposure to PM(Chapter 4 and 5).The contents of the thesis are as follows:(1)The origin and composition of PM were described briefly.This chapter summarized sampling and exposure technologies of PM.This chapter also introduced epidemiological studies,toxicology investigations of in vivo animals and in vitro cells of PM.In vitro inhalation and oral bioaccessibility of PTEs in PM were elucidated.Toxicoproteomic analyses of cells or organisms following exposure to environmental pollutants were reviewed as key points.Finally,the main research contents of this thesis were briefly introduced.(2)The total and bioaccessible concentrations of elements in PM were determined by inductively coupled plasma optical emission spectrometer(ICP-OES)and inductively coupled plasma mass spectrometry(ICP-MS).The crustal elements,such as K,Ca,Mg and Fe,were mainly distributed in the coarse fractions of atmospheric particulate matters while toxic elements such as As,Cd,Pb and Sb were enriched more in the fine fractions in both winter and spring.As,Pb,V and Mn showed higher inhalation bioaccessibility extracted by the artificial lysosomal fluid(ALF),while V,As,Sr and Cd showed higher inhalation bioaccessibility using the simulated lung fluid(SLF),suggesting differences in elemental inhalation bioaccessibility between ALF and SLF extraction.There were similar potential carcinogenic and accumulative non-carcinogenic risks via inhalation exposure to indoor and outdoor particle-bound toxic elements based on their bioaccessible concentrations.Therefore,the potential health risks to humans posed by toxic elements in office rooms cannot be neglected via inhalation exposure of the airborne fine particles.(3)Total suspended particulate(TSP)and fine particulate(PM_2.5)were sampled using quartz fiber filters and polytetrafluoroethylene(PTFE)filters.Particles collected using the filters were characterized by a scanning electron microscopy coupled with energy dispersive X-ray spectroscopy(SEM-EDX)and an attenuated total reflection FT-IR spectroscopy(ATR-FT-IR).The in vitro inhalation bioaccessibility of TSP-and PM_2.5-bound potentially toxic elements(PTEs)on the quartz fiber and PTFE filters with different physico-chemical properties were extracted by four different kinds of simulated lung fluids.SEM-EDX spectra under different magnifications show the heterogeneous distribution and agglomeration of PM_2.5 collected on the quartz fiber filters and PTFE filters.Results of ATR-FT-IR show different surface functional groups of PM_2.5 collected using quartz fiber and PTFE filters.24 h was the optimal interval time for the analyses of in vitro inhalation bioaccessibility of TSP-bound PTEs.The in vitro inhalation bioaccessibility of TSP-and PM_2.5-bound PTEs varied greatly among four in vitro inhalation bioaccessibility procedures.The in vitro inhalation bioaccessibility of TSP-and PM_2.5-bound PTEs didn't show strong dependence on the quartz fiber filters and PTFE filters with different properties.The in vitro inhalation bioaccessibility procedures have greater influences on the in vitro inhalation bioaccessibility of particle-bound PTEs than the materials of the sampling filters.(4)In vitro cytotoxicity assays and toxicoproteomic analyses were carried out to investigate toxic effects of PM collected using polytetrafluoroethylene(PTFE)filters extracted with acetone for PM_2.1 and water for PM_2.1 and PM₁₀ on A549 human lung epithelial cells.The cytotoxicity assays based on cell viability,cell apoptosis and reactive oxygen species generation indicated that the toxicity of PM_2.1 extracted with acetone was higher than that of PM_2.1 and PM₁₀ extracted with water.i TRAQ labeling and LC-MS/MS analyses indicated that the number of differentially expressed proteins in A549 cells affected by PM_2.1 extracted with acetone was noticeably higher than that of differentially expressed proteins in the other two groups.Hierarchical cluster analyses showed that the influences of the extracts of PM_2.1 and PM₁₀ using water on the proteome of A549 cells were similar,whereas significantly different from the effect of PM_2.1 extracted with acetone.Pathways analyses indicated that PM_2.1 extracted with acetone influenced the expression of proteins involved in 14pathways including glycolysis/gluconeogenesis,pentose phosphate pathway,proteasome,etc.PM_2.1 extracted with water affected the expression of proteins involved in 3 pathways including non-homologous end-joining,ribosome and endocytosis.However,PM₁₀ extracted with water affected the expression of proteins involved in only spliceosome pathway.The extracts of PM using different extractants to detach PM from PTFE filters influenced the cytotoxic effects of PM and the proteome of A549 cells.Therefore,extractants should be assessed carefully before the investigations on cytotoxicity to improve the compatibility or comparability of experimental results among research teams.(5)On the basis of development of novel agar membranes,low disturbance and accurate in situ exposure,in vitro cytotoxicity assays and toxicoproteomic analyses were carried out to investigate toxic effects of PM collected using agar membranes on A549 human lung epithelial cells and BEAS-2B human bronchial epithelial cells.PM collected using polytetrafluoroethylene(PTFE)filter extracted with water was set as control group.Cytotoxicity assays and toxicoproteomic analyses indicated that the toxicity of PM_0.4-2.1 collected using PTFE filters to A549 and BEAS-2B cells was higher than that of PM_0.4-2.1 collected using agar membranes.i TRAQ labeling and LC-MS/MS analyses indicated that the number of differentially expressed proteins in A549 and BEAS-2B cells affected by PM_0.4-2.1 collected using PTFE filters was higher than that of differentially expressed proteins affected by PM_0.4-2.1 collected using agar membranes.Pathway analyses indicated that PM_0.4-2.1 collected using PTFE filters influenced the expression of proteins involved in 4 pathways in A549cells.PM_0.4-2.1 collected using agar membranes affected the expression of proteins involved in 3 pathways.Pathway analyses indicated that PM_0.4-2.1 collected using PTFE filters influenced the expression of proteins involved in 13 pathways in BEAS-2B cells.PM_0.4-2.1 collected using agar membranes affected the expression of proteins involved in 8 pathways.The PM collected using different filters and different exposure methods influence the cytotoxic effects of PM and the proteome of cells due to different efficiencies of sampling and extraction.Interestingly,agar membranes and direct exposure approach can provide a simple and comparable method.