| Background and ObjectiveAir pollution has been widely acknowledged as one of the major risk factors for respiratory diseases. Traffic-related air pollution is the major source of air pollution in urban areas. The components of traffic-related air pollution are very complex. Short-term exposure to traffic-related air pollution could reduce the pulmonary functions, while long-term exposure is closely associated with the development and progression of pulmonary diseases. Althought many studies have investigated the effects of air pollution on pulmonary diseases, the exact mechanisms are still unclear.Traffic-related air pollution is a complex mixture containing various hazardous materials such as particulate matters and gases, which could induce toxic effects. The epigenetic effects induced by traffic-related air pollution has attracted more and more attention in recent years. Abnormal global and gene-specific gene methylation changes, as well as histone deaetylase expression changes have been discorvered in patients with pulmonary diseases. While the specific components of traffic-related air pollution, such as particulate matter, carbon black, and benzene could cause global hypomethylation, tumor suppressor gene hypermethylation, and increased activity of histone acetylatrasfase. These findings demonstrate that both the pulmonary diseases and air pollution suggest epigenetic changes play important roles in the effects of traffic-related air pollution on pulmonary diseases.Only few studies investigated the epigenetic effects of traffic-related air pollution, and most of the previous studies only investigated the effects of single component, while the interactions among the components in the mixture were not investigated. In addition, the studies mainly used ambient air pollution level to reflect the exposure level, and the epigenetic changes were investigated only in the blood. In the present study, Wistar rats were exposed to traffic-related air pollution for different time at the traffic sites, and then the DNA methylation and histone H3K9 acetylation levels were measured in both the blood and lung tissues to explore the early effects, accumulative effects, interactions, and dose-effect responses of the traffic-related air pollution on epigenetic changes.Materials and MethodsIn the present study, a busy traffic tunnel and crossroad between two busy main roads were selected for the exposure, while the campus of the University was selected as the control. The levels of traffic-related air pollution at different sites were measured during the exposure. Wistar rats were randomly divided into 8 groups in both spring and autumn, and then exposed to the traffic-related air pollution at the three sites for 4 h,7 d, 14 d, and 28 d. The rats were sacraficed after the exposure completed, the blood and lung were collected to extract the genomic DNA and total histone. The global methylation (LINE-1 and Alu) and methylation of the promoters of specific genes (p16, iNOS, and APC) were quantified with pyrosequencing; the histone H3K9 acetylation level was quantified with quantitative ELISA.SPSS 19.0 software was used for the statistical analyses. Independent Mest was used to compare the data between spring and autumn, as well as between male and female rats. One-way analysis of variances (ANOVA) folowed by post-hoc Bonferroni test was used to compare the levels of air pollution, DNA methylation, and histone H3K9 acetylation among different groups. Multiple linear regression was used to evaluate the dose-effect responses and accumulative effects, and generalized linear model was used to investigate the interactions of season and sex with air pollutants.Results(1) Traffic-related air pollution levels:The levels of PM3.5, PM10, and NO2 in the tunnel group were significantly higher than in the crossroad group and control group (P<0.01), and the levels in the crossroad group was significantly higher than in the control group (P<0.01).(2) DNA methylation:No significant difference in the DNA methylation level was found between males and females. After exposed for 4 h, the methylation of LINE-1 and iNOS in the blood and lung tissue DNA of the rats in the tunnel and crossroad groups were significantly lower than in the control group; the methylation of APC promoter in the lung tissue DNA of the rats in the tunnel group was significantly higher than in the control group in autumn. After exposed for 7 d, the methylation of LINE-1 and iNOS in the blood and lung tissue DNA of the rats in the tunnel and crossroad groups were significantly lower than in the control group; the methylation of p16 promoter, both in blood and lung tissue DNA, and APC promoter, in only lung tissue DNA, were significantly higher in the tunnel group.(3) Histone H3K9 acetylation:No significant difference in the H3K9 acetylation level was found between males and females. After exposed for 4 h, no significant difference was found in either the tunnel or crossroad group as comparing with the control group. However, after exposed for 7 d, the H3K9 acetylation levels in the tunnel and crossroad groups were significantly higher than in the control group, and the level in the tunnel group was also significantly higher than in the crossroad group.(4) Dose-effect responses in affecting DNA methylation:1) In the 4 h exposure window, every 1μg/m3 increase of the PM2.5 resulted in 0.027%(P=0.003) and 0.037% (P=0.001) decrease of the LINE-1 and iNOS methylation in the blood DNA, and 0.041% (P<0.001) decrease of the LINE-1 methylation in the lung tissue DNA. Every 1μg/m3 increase of the PM10 resulted in 0.018%(P<0.001) and 0.024%(P=0.001) decrease of the LINE-1 and iNOS methylation in the blood DNA, and 0.027%(P<0.001) decrease of the LINE-1 methylation in the lung tissue DNA. Every 1μg/m3 increase of the NO2 resulted in 0.076%(P<0.05) and 0.102%(P<0.001) increase of the iNOS methylation in the blood and lung tissue DNA, and 0.088%(P<0.001) decrease of the p16 methylation in the blood DNA, respectively.2) In the 7 d exposure window, every 1 μg/m3 increase of the PM2.5 resulted in 0.064%(P=0.003) and 0.047%(P=0.047) decrease of the LINE-1 and iNOS methylation in the blood DNA,0.033%(P=0.012) and 0.053% (P=0.006) decrease of the LINE-1 and iNOS methylation in the lung tissue DNA, and 0.009%(P=0.046) increase of the APC methylation in the lung tissue DNA. Every 1μg/m3 increase of the PM10 resulted in 0.037%(P=0.003) decrease of the LINE-1 methylation in the blood DNA, and 0.019%(P<0.05) and 0.031%(P<0.01) decrease of the LINE-1 and iNOS methylation in the lung tissue DNA. Every 1μg/m3 increase of the NO2 resulted in 0.093%(P<0.05) increase of the LINE-1 methylation in the blood DNA, and 0.099%(P<0.001) increase of the iNOS methylation in the lung tissue DNA, respectivley.3) The results of the rats exposed in the tunnel for different time showed that every 1μg/m3 increase of the PM2.5 resulted in 0.037%(P=0.001) increase of the p16 methylation in the blood DNA,0.015%(P=0.024) and 0.058%(P<0.001) decrease of the LINE-1 and iNOS methylation in the lung tissue DNA, and 0.008%(P=0.046) and 0.011%(P=0.011) increase of the APC and p16 methylation in the lung tissue DNA. Every 1μg/m3 increase of the PM10 resulted in 0.011%(P=0.020) and 0.020%(P=0.001) decrease of the LINE-1 and iNOS methylation, and 0.019%(P=0.017) increase of the p16 methylation in the blood DNA, and 0.017%(P=0.004) decrease of the iNOS methylation in the lung tissue DNA. Every 1 ug/m3 increase of the NO2 resulted in 0.06’ (P<0.001) and 0.074%(P=0.006) increase of the LINE-1 and iNOS methylation in the lung tissue DNA, respectively.(5) Dose-effect responses in affecting histone H3K9 acetylation:In the 4 h exposure window, 1μg/m3 increase of the major air pollutants did not significantly affect the H3K9 acetylation level. However, in the 7 d exposure window, every 1μg/m3 increase of the PM2.5 resulted in 0.475 (P= 0.014) and 0.468 (P= 0.017) ng/mg pro increase of the H3K9 acetylation in the PBMC and lung tissue histone; every lug/m3 increase of the PM10 resulted in 0.280 (P= 0.014) and 0.276 (P= 0.018) ng/mg pro increase of the H3K9 acetylation in the PBMC and lung tissue histone, respectively. The analyses of the H3K9 acetylation levels in the rats exposed in the tunnel for different time showed that every 1μg/m3 increase of the PM2.5 resulted in 0.638 (P <0.001) and 0.659 (P= 0.001) ng/mg pro increase of the H3K9 acetylation in the PBMC and lung tissue histone; every 1μg/m3 increase of the PM10 resulted in 0.180 (P = 0.016) and 0.189 (P= 0.023) ng/mg pro increase of the H3K9 acetylation in the PBMC and lung tissue histone, respectively.(6) Accumulative effects of in affecting DNA methylation and H3k9 acetylation: The DNA methylation and H3K9 acetylation changed significantly with the exposure time, maintained at a level, and then tended to recover after exposed for 28 d. Every 1 day exposure in the tunnel resulted in 0.607%(P=0.033) and 0.317%(P=0.047) decrease of the LINE-1 methylation in the blood and lung tissue DNA,0.842% (P=0.026) and 0.688%(P=0.017) decrease of the iNOS methylation in the blood and lung tissue DNA,0.248%(P=0.003) increase of the p16 methylation in the lung tissue DNA, and 16.033 (P<0.001) and 15.718 (P<0.001) ng/mg pro increase of the H3K9 acetylation in the PBMC and lung tissue histone.(7) Interactions of season and sex with air pollutants:The results of the generalized linear model showed that season interacted with PM2.5, PM10, and NO2 in affecting the blood LINE-1 methylation, with PM10 and NO2 in affecting the blood p16 methylation, interacted with PM2.5 in affecting lung tissue LINE-1 methylation, and interacted with PM10 in affecting the lung tissue p16 methylation. Sex interacted with PM2.5 in affecting blood APC and lung tissue p16 methylation, and interacted with PM10 in affecting lung tissue LINE-1 methylation. In adition, season also interacted with PM2.5, NO2, and SO2 in affecting PBMC and lung tissue H3K9ac, while not interaction between sex and air pollutants were found in affecting H3K9 acetylation.ConclusionThe present study firstly exposed the rats at the sites of traffic-relation air pollution to investigate the early effects, cumulative effects, dose-effect responses, and interactions of the air pollution in affecting DNA methylation and H3K9 acetylation. The major conclusions are as follows:1) The early effect of traffic-related air pollution exposure mainly included global DNA hypomethylation and changes in the inflammation-related gene, while prolonged exposure could effectively affect the methylation of specific genes.2) Expose to high level of traffic-related air pollution had significant cumulative effects on DNA methylation and H3K9 acetylation in a certain range of exposure time, and then tended to recover.3) Season and sex could interact with air pollutants in affecting DNA methylation and H3K9 acetylation.4) APC methylation could has relatively high sensitivity and specificity in reflecting the early changes in the lung tissues induced by traffic-related air pollution.These findings showed that traffic-related air pollution exposure could effectively induce epigenetic changes. Epidemiological studies with large sample sizes are needed to verify these findings, and further investigate the interactions among the air pollutants. |
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