Study Of Toxic Effects Of Particulate Matters From Different Areas On Alveolar Macrophages

Objective:In recent years, the degree of air pollution has been significant increased in the Pearl River Delta region. For the large amount of population coming from other provinces increased the requirement of energy support and the number of personal vehicles in this area. According to the statistical results provided by the weather department, the haze weather was recorded among these cities frequently in the decade. And both of the haze weather duration dates and the total number of haze weather influenced days across the whole year were increased year by year. Although many epidemiological investigation and laboratory research about the particle-induced health effects could be found in domestic and foreign academic magazines. But the systematic research for the health threaten to people exposed to haze in the Pearl River Delta region have not been found exactly, especially the studies about cell toxicity in vitro. The exposure limits to the particulate matter in the environment and occupational places have already been instituted. But the research for the comparative differences and correlations in the biological effects which caused by atmospheric particulate matter in the environment and occupational inhaled quartz was merely to be found. In order to know the cytotoxicity of haze particulate matter and the different characters of atmospheric particulate matter in the environment and occupational respiratory quartz, we cultured primary rat alveolar macrophages in our study, took alveolar macrophages exposure to the collected atmospheric particulate matter in different cities and the Chinese standard quartz. Methods:Guangzhou city, Shenzhen city and Dongguan City were chosen as the representative sampling sites which were influenced by the haze weather in the Pearl River Delta. Zhaoqing city was chosen to be the control city which had not been reported influenced by the haze weather in Guangdong province. The environmental high-volume samplers were used to collect the PM2.5 fraction, and the elutriator samplers were used to collect the respiratory fraction. The sampling time was from the end of August to the end of December. This lasted for half a year. Continuous sampling was executed during this time except the rainy and windy days. According to the weather reports, these sample filter membranes which were collected in the right haze days were chose as the haze samples of particulate matter. These chosen membranes were deal by ultrasonic processing and the wet scratch method. Then the suspension of particulate matter was gotten. After the vacuum freeze-drying and weight process, the particle was re-suspended by NS to be the prepared sample. China CDC standard quartz (SiO2 was more than 95%) was prepared quartz suspension by grinding and ultrasonic treatment. The rats was Lavaged to obtain rat primary alveolar macrophages. The cell concentration was 1×106/ml or 5×105/ml. Six doses (1, 10, 50, 100, 200, 300μg / ml) of atmospheric particulate matter and quartz were exposed to the AM. And the AM without exposure to particulate matters was as the blank control. The cell was cultured in the condition of 37℃, 5%CO₂ for 24 hours. By detecting tumor necrosis factor (TNF-α) and nitric oxide (NO) release, intracellular superoxide dismutase (SOD) activity, malondialdehyde (MDA) formation, lactate dehydrogenase (LDH) leakage rate and cell survival (MTT method) to evaluate the extent of the affected AMs after 24 hours.Results:1. The TNF-αrelease by AM which was induced by the ambient particle matter collected in Guangzhou city was the highest one. PM collected in Dongguan city was after it. Both of the above were significant higher than the TNF-αrelease by PM collected in Zhaoqing city(P<0.001). The LDH leakage rate which caused by PM collected in Dongguan city was the highest. PM collected in Guangzhou and Shenzhen cities were after it. The LDH leakage rate which was promoted by all of these three ones were significant higher than the one caused by PM collected in Zhaoqing city(P<0.05). The activity of intracellular SOD which affected by PM collected in Shenzhen and Dongguan cities were significant higher than the compared city Zhaoqing(P<0.05). And the productions of intracellular MDA which were induced by PM collected in Guangzhou, Shenzhen and Dongguan cities were significant higher than PM collected in Zhaoqing city(P<0.05). The variation in the rate of AM survival and NO release were not observable.2. The TNF-αrelease by AM which was induced by the PM2.5 fraction was higher than the respiratory fraction. The same was observed in the inhibition ratio of intracellular SOD. All of the LDH leakage rate, NO release and the rate of AM death were higher in respiratory fraction -induced than PM2.5-induced. The variation between the PM2.5 fraction and respiratory fraction in the production of intracellular MDA was not significant.3. With the increase in the concentration of PM, the TNF-αrelease by AM was increased. When the concentration of PM was more than 200μg/ml, the TNF-αrelease reached the max value. The TNF-αrelease induced by quartz reached the highest value when the concentration of PM was 50μg/ml. The TNF-αrelease induced by quartz was reduced when the concentration of PM was over 100μg/ml. The leakage rate of intracellular LDH induced by PM was significant increased when the concentration of PM was between 10 to 50μg/ml. While the concentration of PM was kept on added, the increase of LDH leakage rate could not be observed. The LDH leakage rate induced by quartz was increased in two ranges, and could be the max value when the concentration of quartz was 300μg/ml. One of the two ranges was among 10 to 50μg/ml, and the other was in 200 to 300μg/ml. The NO release induced by PM was firmly increased with the adding of PM concentration. When the concentration of PM was higher than 200μg/ml, the NO release detected would reach the max value. The NO release induced by quartz was the max when the concentration of quartz was 50μg/ml. With the adding of quartz, the detection of NO release was decreased. The inhibition of intracellular SOD had an increased trend with the adding of concentration of PM. In the other side, the inhibition of intracellular SOD which was caused by quartz would get the max value when the concentration of quartz was10μg/ml. But the inhibition of SOD affected by quartz would be decreased when the concentration was higher than 10μg/ml. The production of intracellular MDA which was induced by PM collected in the haze affecting cities was significant higher than the one induced by quart(zP<0.05). The death ratio of AM caused by quartz was increased with the adding of quartz's concentration. When the concentration was between 10 to 50μg/ml, the death rate would be significant decreased. And the ability of causing cell death by PM was weaker than the quartz's.4. The NO release which was induced by the PM_2.5 fraction from Guangzhou and Dongguan had direct correlation with the release of TNF-α. The correlation indexes were 0.949 and 0.906 to each area. Conclusion:There is a significant regional variation and size variation in the early AM-induced biological effects caused by atmospheric particulate matter. Compared with the control urban PM_2.5, the haze affected urban air PM_2.5 had the greater effects on AM in the inflammatory response, oxidation strain and cellular toxicity. While atmospheric particulate matter in small size showed the stronger performance of membrane injury, lipid peroxidation and induction of cell death capacity than PM in larger size collected in the same cities, except the abilities in promoting the release of inflammatory factors and oxidative strain. Atmospheric particulate matter occurred in the biological effects induced by AM had a significant dose-dependent increasing. With the only difference was that the quartz in the lower dose levels could be observed a certain degree of dose - effect relationship. The inflammatory response induced by PM could regulate the production and release of other cytokines in AM. However, further epidemiological investigation about the finding for relationship between haze affected atmospheric particulate matter and the human health expose in this area should be perform in order to verify the cell toxicity results.