Study On The Toxicity Of Atmospheric Ultrafine Particles Surrounding Urban Roads On Cultured Neurocytes

ObjectiveThe problem of urban air pollution is getting more and more serious in China. Particulate matters in the atmosphere contain a large number of ultrafine particles(UFPs). Results of some epidemiological studies showed that there may be a correlation between ultrafine particles and nervous system diseases. Long-term living in the area polluted by particulate matters may lead to a decline in cognitive function. The concentration of UFPs in the air near urban roads is significantly higher than other areas. Compared with other occupational groups, urban road related workers were exposed to higher dose of UFPs, so their central nervous system may have more opportunity to be affected by UFPs. In this study, we collected UFPs near urban road and put them in the culture medium of human brain glioma cells (U251).We observed the toxicity of UFPs on U251 in different concentrations, and investigated their toxical mechanisms in several aspects including oxidative stress, mitochondrial function and apoptosis level. We want to provide experimental data and theoretical references for researches about effects of urban road air pollution on CNS of related exposed population.Method1. UFPs collection This study atmospheric UFPs were collected near a main traffic road in a city by a Dekati DLPI-impact multistage particles sampler.2. UFPs suspension preparationUFPs were placed in a glass bottle, sterilized by high-pressure steam (121℃,30min), dried, and put into certain volume of culture medium to prepare UFPs mother solution of 6400μg/ml. The mother solution was suspended for 30 minutes by sonication before used, then diluted to required concentration with cell culture media.3. CCK-8 assayU251 cells were exposed to UFPs for 48h, thenCCK-8 reagent was added and fully mixed. Incubated for 60 minutes at 37℃.The absorbance at 450 nm was recorded. This test was repeated three times.4. Measurement of oxidative stress indicatorsU251 cells were exposed to UFPs for 48h.Oxidative stress Kits were used to determine the T-SOD activity, GSH content, total antioxidant capacity (T-AOC) and MDA content in U251 cells.5. Measurement of mitochondrial membrane potential (MMP)U251 cells were exposed to UFPs for 48h. Mitochondrial membrane potential (MMP) was tested by fluorescent dyes JC-1.Fluorescence microscope and microplate reader were used to detect the changes of fluorescence intensity before and after contamination. Judging depolarization degree of mitochondrial membrane by ratio of red and green fluorescence intensity.6. Detection of ATP contentAfter being exposed to UFPs for 48h, ATP assay kit was used to determine the ATP level in U251 cells.7. Detection of apoptosis levelU251 cells were exposed to different concentrations of UFPs for 48h. The apoptosis level induced by UFPs was measured by flow cytometry with Annexin-V (FITC labeled) and Propidium Iodide (PI).Result1. Effects of UFPs on livability of U251 cellsThe results showed that cell livability was decreased by UFPs exposure in a dose-dependent manner. The difference was statistically significant for every experimental group, compared with the untreated group (P< 0.05).2. Effects of UFPs on indicators of oxidative stress in U251 cellsThe indicators of oxidative stress in U251 cells were changed after exposed to UFPs for 48h. In the U251 cells treated with 20μg/ml、40μg/ml UFPs, T-AOC was decreased, compared to the untreated cells (P< 0.05). In the U251 cells treated with 10μg/ml、20μg/ml、40μg/ml UFPs, T-SOD was decreased, MDA was increased, compared to the untreated control cells (P< 0.05). GSH in each treated group was decreased, but the differences showed no statistical significance (P>0.05).3. Effects of UFPs on MMP in U251 cellsU251 cells were exposed to different concentrations of UFPs for 48h. Changes of MMP was observed by fluorescence microscope. Accompanied by the increase of UFPs’ dosage, MMP of U251 cells decreased gradually. In the U251 cells treated with 20μg/ml、40μg/ml UFPs, the MMP decreased significantly, compared with 0μg/ml group (P<0.05).4. Effects of UFPs on ATP content in U251 cellsUFPs led to a concentration-dependent decrease in intracellular ATP content. The difference was statistically significant in 40μg/ml group, compared with the untreated group (P<0.05).5. Effect of UFPs on apoptosis level in U251 cellsApoptosis rate in U251 cells was evaluated after exposure to UFPs for 48h. The results show that apoptosis rate was increased by UFPs exposure in a dose-dependent manner. In the U251 cells treated with 20μg/ml and 40μg/ml UFPs, apoptosis rate was increased1.6% and 6.85% respectively, compared with the untreated control cells (P< 0.05). Early apoptosis rate was generally higher than the late apoptosis rate in each treated group, both of them increased in a dose-dependent manner. In the U251 cells treated with 20μg/ml, and 40μg/ml UFPs, early apoptosis rate was increased significantly, compared with the untreated cells (P< 0.05).As for the late apoptosis, the 40μg/ml group was higher than the untreated group (P< 0.05).Conclusion1. Exposure to UFPs may induce decline of cell livability, MMP and ATP content in U251 cells, and the levels of oxidative stress and apoptosis were elevated at the same time.2. The toxicity of UFPs on neurocytes may be related to enhancement of oxidative stress, mitochondrial damage and raise of apoptosis level.