The Preliminary Study Of The Toxic Effect Of Airborne PM_2.5on Human Bronchial Epithelial Cells

Air pollution has been recognized as a human carcinogen. With the development of China’s urbanization and industrialization, particulate matter (PM) air pollution has been one of the the major environmental factors which are harmful to public health. PM, a mixture of solid particles and liquid droplets existing in the air, is important air pollution. Inhalable PM can be divided into two classes based on aerodynamic diameter:coarse particles PM10with a diameter≤10μm and fine particles PM2.5with a diameter≤2.5μm. Numerous epidemiological and toxicological studies have shown that, PM2.5which closely associated with many different cardiopulmonary diseases and even the occurrence of lung cancer is more harmful than PM10. PM2.5air pollution is a continuing challenge to public health in China. It was recently demonstrated that PM2.5was the fourth leading risk factor of disease burden in China.In2011, the top four causes of death in China were stroke, lung disease, coronary heart disease and lung cancer, all were linked with PM2.5.In the past decades, numerous investigations on PM2.5and human health have reported. However, the complex mechanisms underlying PM2.5-induced adverse health effects remain incompletely understood. To reveal the potential health effects of PM2.5, we collected the airborne PM2.5from a community located in the center of the downtown area of Wuhan, and combined in vitro, flow cytometry, genome wide microarray and bioinformatics analysis, we carried out the following research:(1) investigate the toxic effect of PM2.5on HBE cells;(2) explore the changes in gene expression and DNA methylation profiling of human bronchial epithelial cells exposed to PM2.5.Part I The toxic effect of PM2.5on HBE cellsObjective:PM2.5is a major component of air pollution, mainly from vehicle emission, fossil fuels (oil, coal, etc.) combustion and burning of biomass and other fuels, there are a large number of toxic and hazardous substances (such as PAHs and transition metals) absorbed on the surface. PM2.5can be deposited in the respiratory airway and alveolar, and even into the bloodstream through the blood barrier, causing more extensive damage to the body’s respiratory and cardiovascular systems. In this study, we use in vitro experiments to explore the impact of PM2.5on the cell viability, cell cycle and apoptosis of HBE cells.Methods:Lung is the main target organ of PM2.5. Therefore, we used HBE cells as the object of this study. Using Cell Counting Kit8(CCK-8) cell proliferation assay to evaluate the cell viability of HBE cells exposed to airbone PM2.5. There are9goups in this experiment, namely:control (0μg/ml),100μg/ml,200μg/ml,300μg/ml,400μg/ml,500μg/ml,600μg/ml,700μg/ml and800μg/ml, the exposure time is set to24h and48h. According to the results of CCK-8assay, we chose the exposure time of24h to apply in the following experiments. To make the exposure concentration of PM2.5consistent between cells and human beings as much as possible, we predicted our daily exposure concentration of PM2.5. The mean concentration of PM2.5during our sampling time in Wuhan was115μg/m3, a person breathed20m3of air containing a PM2.5concentration of115μg/m3for24h, in a volume of12.4ml of airway and lung lining fluids would have a PM2.5concentration in the lining fluid of185μg/ml. Of course, the actual concentration may be a bit lower than the dose269ug/ml, because not all the inhaled PM2.5can get into the lining fluids. However, it may also be higher, because PM2.5was inhaled everyday and could not be all excreted, so there would be a basal concentration in the lining fluid. The maximum concentration of PM2.5we ever measured in Wuhan was358μg/m3, correspondingly the PM2.5concentration in the lining fluid would be577μg/ml. Given the amount of PM2.5one person inhaled daily, the relatively low-dose (200μg/ml) and high-dose (500μg/ml) of PM2.5were chosen to treat HBE cells for the following experiments. We performed flow cytometric analysis to examine the influence of PM2.5on the cell cycle and apoptotic behavior of HBE cells.Results:The results of CCK-8assay showed that after24h of incubation, the cell viability of cells exposed to different concentrations of PM2.5all decreased compared to the control. All the concentrations of PM2.5except100μg/ml significantly decreased HBE cell numbers. After48h of incubation, the cell viability of cells exposed to different concentrations of PM2.5all significantly decreased compared to the control. In addition, PM2.5decreased the cell viability in a dose-dependent manner (r=0.99, P<0.01). There was no significant difference (P>0.05) between the cell viability of24h treated and48h treated cells exposed to the same concentration of PM2.5. The result of cell cycle assay showed that, the proportion of cells in the G1phase of the low-dose group was71.54%, the high-dose group was67.58%, which were significantly decreased (P<0.05) compared with the control goup (76.42%). As the concentrations gone up, there was a tendency to increase in the S phase cells (control group:14.13%, low-dose group:19.18%, high-dose group:23.45%). The proportion of cells in the S phase of the high-dose group was significantly higher than the control group. In the G2phase, there was no significant difference between treated cells and control cells but a tendency to decrease with the exposure concentration raised (control group:9.44%, low-dose group:9.28%, high-dose group:8.96%). The result of apoptosis assay showed that, total cellular apoptosis rate in the high-dose group (3.70%) was significantly higher than the control group (3.17%), however there was no significant difference between treated and control groups.Conclusion:Airborne PM2.5can decrease the cell viability of HBE cells in a dose-dependent manner. In addition, PM2.5can induce cell cycle arrest in HBE cells.Part Ⅱ Changes in gene expression profiling of HBE cells exposed to PM2.5Objective:PM2.5can inhibit HBE cells proliferation and induce cell cycle arrest. However, the mechanism of the effect is not fully understood. As the toxic effet of PM2.5, there may be changes in gene expression profiling of HBE cells exposed to PM2.5. To comprehensively understand the impact of PM2.5on HBE cells, we would preliminary study the impact of PM2.5on gene expression profiling of HBE cells.Methods:Using Agilent mRNA single-channel expression profiling microarray to detect the impact of PM2.5on gene expression profiling of HBE cells in this part. And then we used RT-PCR to verify the results of microarray analysis. For the analysis of the data of expression profiling microarray, first of all, we screened differentially expressed genes (DEGs) after PM2.5treatment. Then, the Gene Ontology and pathway analysis were conducted by Molecule Annotation System3.0(MAS3.0).Results:The results showed that there were970and492genes that were differentially expressed in low-dose (200μg/ml) group and high-dose (500μg/ml) group, respectively. Among all the DEGs, many are related to inflammatory response, immune response, response to oxidative stress, and response to DNA damage stimulus. Pathway analysis showed that there were58and13significant KEGG pathways in low-dose group and high-dose group, respectively. In addition,12of the significant KEGG pathways in high-dose group were the same with that in low-dose group, they were:p53signaling pathway, apoptosis, regulation of actin cytoskeleton, MAPK signaling pathway, cytokine-cytokine receptor interaction, ribosome, RNA polymerase, ubiquitin mediated proteolysis, nucleotide excision repair, biosynthesis of steroids, pyrimidine metabolism and purine metabolism. In low-dose group, two pathways, cell cycle and apoptosis were in consistent with the GO analysis. Very good agreement was observed between the microarray data and RT-PCR data (Pearson correlation coefficient=0.965; P<0.001).Conclusion:PM2.5can induce changes in the gene expression of HBE cells, and the exposure concentration has an important influence on the changes. The DEGs involved in many biological processes which may related to the mechanisms underlying PM2.5-induced adverse health effects. Part Ⅲ Changes in DNA methylation profiling of HBE cells exposed to PM2.5Objective:As an important epigenetic modification, DNA methylation can regulate gene expression through a variety of ways. Studies have shown that the air PM can induce changes in DNA methylation status. An epidemiological study by Kile, etc. showed that PM2.5could increase the methylation rate of the promoter region in iNOS gene, and the longer of the exposure time, the higher of its methylation rate. Animal studies found that in the mice and primary mouse alveolar epithelial cells treated by PM2.5, the methylation level of the promoter region in DNA methyltransferase1(DNMT1) and the expression of the tumor suppressor gene p16all increased, which indicated that PM2.5may cause the hypermethylation of the primary tumor suppressor gene through the increased expression of DNMT1, thereby increasing the risk of lung cancer. To comprehensively understand mechanisms underlying the impact of PM2.5on HBE cells from whole genomes and epigenetic perspective, we preliminary study the impact of PM2.5on DNA methylation profiling of HBE cells in this part.Methods:We used NimbleGen MeDIP DNA methylation microarray to detect the impact of PM2.5on DNA methylation profiling of HBE cells. For the analysis of the data of DNA methylation profiling microarray, first of all, we screened differentially methylated genes after PM2.5treatment. And then we conducted Gene Ontology and pathway analysis using these differentially methylated genes. After that, we combined the data of gene expression profiling microarray to find the DEGs related to DNA methylation changes, namely find the hypomethylation genes which were up-regulated and the hypermethylation genes which were down-regulated among the differentially methylated genes.Results:According to the screening criteria (a, the score of significantly enriched sense of methylated regions≥2.0; b, compared to the control, Log-Ratio>1.0or≤-1.0.), there were146differentially methylated genes (98hypermethylation genes and47hypomethylation genes) in low-dose group, and119differentially methylated genes (67hypermethylation genes and52hypomethylation genes) in high-dose group. Pathway analysis showed that the significant KEGG pathways in low-dose group were tight junction, p53signaling pathway, melanoma and small cell lung cancer. And there were5significant KEGG pathways in high-dose group: aminoacyl-tRNA biosynthesis, selenoamino acid metabolism, histidine metabolism, base excision repair and pyrimidine metabolism. According to GO analysis, there were4common biological processes in low-dose group and high-dose group: regulation of transcription, DNA-dependent, transcription, positive regulation of transcription from RNA polymerase II promoter and signal transduction. When combined with the gene expression profiling, there were14hypermethylation genes which were down-regulated (HSPB3、RXRG, CXXC1、CDH4、ACTRT1、CXXC5、 EDNRA、PPM1D、AFF1、MEPE, DNUFB1、MYF5、SDR42E1、RPL23A),and5hypomethylation genes which were up-regulated (SCARNA15、LOC116437、 MAGT1、Clorf159、CREB1) in low-dose group;8hypermethylation genes which were down-regulated (CENPK, LLC1、OR1F2P、FOLR1, NRIP3、PFN3、SOX6, ZFHX4) and6hypomethylation genes which were up-regulated (GGPS1、CPNE2、 WDR33、A4GNT, LYPD3、HMGA2) in high-dose group. MSP assay showed that RXRG in low-dose group and LLC1in high-dose group were both hypermethylated after PM2.5treatment. Meanwhile, by RT-PCR validation, these two genes were also down-regulated compared with the control, suggesting that the microarray results were reliable.Conclusion:PM2.5can induce changes in the DNA methylation of HBE cells. There were many differentially methylated genes, among which some genes’ methylation status may related to the gene expression.