| ObjectiveInhalable particles, a collection of different types of chemicals as principal atmospheric pollutants, cause many healthy issues, among which toxic effects on cardiovascular system has become one hot topic. According to WHO report, the pollution of fine particles (PM2.5) results in at least 0.8 million deaths and 6.4 million disability adjusted life year (DALY) losses every year around the world,65% of which mainly occur in Asia Countries (China and India) where PM2.5 pollution is much more serious than that in other districts. Owing to urban economic development model and fuel structure depending on coal, pollution in China is characterized by the combination of coal and petrol pollution, yet coal-fired pollution is the dominant type. In addition, PM2.5 in inhalable particles generated by coal is mainly responsible for coal-fired pollution. Although a large variety of researches have focus on the toxicity of fine particles on respiratory system, few studies have been carried out on the effect of PM2.5 on cardiovascular system.To explore the effects of different compositions of fine particles on the cardiovascular system, elements and ion analysis and their toxicity on cardiovascular system were performed in this study. In addition, oxidative damage of different compositions in PM2.5 on human umbilical vein endothelial cells EA.hy926 was also carried out to provide in vitro experimental data for subsequent studies that investigate the mechanism on how PM2.5 induces cardiovascular toxicity.Methods1. Cell CultivationHuman umbilical vein endothelial cells are widely used in studies of endothelial functions. Because HUVEC cells in primary culture is time-consuming and easily stained with miscellaneous cell, it begins to shrank after 2or 3 passages cells transformation and it is difficult to be maintained for long-term purpose. As a human umbilical vein endothelial cells fusion with A549 cell, EA.hy926 cell is the most recognized cell line on endothelial function research.The frozen EA.hy926 cells were shifted to DMEM culture medium containing 20% fetal bovine serum and 1% double-antibody, and then were incubated at 37℃, 5% CO2 conditions. Fresh medium was changed every third day. When cells reach 80% confluence, they were passaged with 0.25% trypsin with DMEM culture medium containing 10% fetal bovine serum and 1% double-antibody, incubating in 37℃,5% CO2 conditions to Exponential phase of growth. Cells were grown to confluence and used for experiment in the second to fifth cell passages. Cells were grown to 80% confluency in 96-well or 24-well plates, then cells were treated with the total coal particles, inorganic compositions and organic extracts of fine particles(final concentrations were 10,25,50,100 and 200 mg/ml)for 24 h, respectively.2. Coal-fired PM2.5 collection and composition extractionThe samples were derived from Datong coal, diluted in channel with fixed source to collect PM2.5. The total coal particles and inorganic compositions were extracted from PM2.5 in water-bath ultrasonic, and organic compositions were extracted from in the Soxhlet Apparatus, which were diluted to 100,250,500,1000,2000μg/mL as exposed liquor at -20℃store for use.3. Cytotoxicity testTo evaluate the cytotoxicity of the different compositions of coal-fired PM2.5 on EA.hy926, MTS Assay (Cell Proliferation Assay) was adopted. According to pre-test results, EA.hy926 cells were exposed to samples at the dose of 10,25,50,100 and 200μg/mL for 24 hours. PBS was taken as solvent control in the total particle of PM2.5 and inorganic compositions group, while DMSO was used as solvent control in organic compositions group. One hour before the end of the test, MTS (the new tetrazolium compound better than MTT, to detect principles the same to MTT) was added to the culture. Optical density (OD) was quantified at 490 nm using Model 680 microplate reader (Bio-Rad) after incubation for one hour at 37℃.4. Detection of oxidative damageTo evaluate oxidative damages of different compositions of coal-fired PM2.5 exposed to the EA.hy926 cells for 24h, the assays of superoxide dismutase (SOD), malondialdehyde (MDA), Glutathione peroxidase (GSH-Px), catalase (CAT), and NO were carried out using assay kits according to the manufacturer's instructions.Results1 Determination of emission factors for coal-fired PM2.51.1 The soluble ion content in coal-fired emissions of PM2.5Bulk determination of soluble ions of PM2.5 from Datong combustion Coal emissions:The highest content is SO42-, and Cl, NO3, Na, NH4, K content is higher that others.1.2 Factor Analysis of PM2.5 emissions from coal-fired elementsThe content change of Datong in PM2.5 emissions from coal-burning:Al, Fe, K, Na, S, Zn and other major elements were higher, and As, B, Co, Cr, Ni, Pb and other trace heavy metal content were distributed at different levels.2 Assessing Cytotoxicity2.1 Dose-effect Cytotoxicity of different compositionsAll particles, inorganic compositions and organic compositions of PM2.5 emissions from Datong coal combustion acted on EA.hy926 cells for 24h, showing that cell survival decreased significantly as the dose increased. Compared to solvent control, the cytotoxic effect of different compositions had statistical significance. 2.2 Cytotoxicity of different compositions at the same doseCell inhibition of all particles, inorganic compositions and organic compositions at the same dose showed a trend of organic compositions> inorganic compositions> all particles with statistical significance. However, at the same dose, all particles and inorganic compositions showed no significant difference in cell inhibition.3 Assessing oxidative damage of different compositions3.1 Superoxide dismutase (SOD)As the dose increased, SOD activity decreased in EA.hy926 exposed to different compositions of coal-fired PM2.5 24h. Each dose group was statistically different (P <0.01) compared with the control group, and there is a dose-response relationship. At the same dose, SOD inhibition of different compositions was:organic compounds> inorganic compositions> all particles, with statistically significance differences among the same dose.3.2 Malondialdehyde (MDA)As the dose increased, MDA levels in supernatants treated with all particles, inorganic compositions and organic compositions increased, respectively. Compared to solvent control, MDA level in each group was statistically different. At the same dose, all compositions of coal-fired PM2.5 showed no difference on MDA level. At the low doses, MDA level of different compositions of coal-fired PM2.5 on EA.hy926 was:organic compositions> all particles> inorganic compositions, while at the high doses:all particles> inorganic compositions> organic compositions with statistically significance. The results can be seen from the above, as the dose increased, MDA level caused by all particles and inorganic compositions increased significantly, while changes of organic compositions is leveling off.3.3 Glutathione peroxidase (GSH-Px)Compared to solvent control, as the dose increased, GSH-Px activity decreased in EA.hy926 exposed to different compositions of coal-fired PM2.5, with statistically significance. At the same dose, GSH-Px inhibition of different compositions was: inorganic compounds> organic compositions> all particles, with no statistically significance differences.3.4 Catalase (CAT)Compared to solvent control, as the dose increased, CAT activity decreased in EA.hy926 exposed to different compositions of coal-fired PM2.5, with statistically significance. At the same dose, CAT inhibition of different compositions was: inorganic compounds> organic compositions> all particles, with no statistically significance differences.3.5 Nitric oxide (NO)Compared to solvent control, as the dose increased, NO level in supernatants increased in EA.hy926 exposed to different compositions of coal-fired PM2.5, with statistically significance. At the same dose, NO level of different compositions was: all particles> inorganic compounds> organic compositions, with statistically significance differences.Conclusion1. Different compositions of coal-fired PM2.5 (all particles, inorganic compositions, organic compositions) showed prohibitive effects on EA.hy926 cells with a dose-response relationship; the cytotoxicity of organic compositions was significantly higher than that of all particles and inorganic compositions, but with no statistically significance difference between all particles and inorganic compositions.2. Different compositions of PM2.5 exerted oxidative damage to EA.hy926, reduced activities of SOD, GSH-Px, CAT, and increased MDA level and NO concentration; there was not consistency between these indexes.3. Discrepancy among the effects of different compositions in PM2.5 may be attributed to metabolic paths of each component in PM2.5. The mechanisms underlying them need further exploration. |
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