Study On Susceptibility Of Lung Injury Caused By Atmospheric Fine Particles

Six air pollutants, i.e., Particulate matter, ozone, carbon monoxide, sulfur oxides, nitrogen oxides and lead, are common or criteria pollutants set by US Environmental Protection Agency (EPA). Among the six pollutants, particulate matter and ozone pose the most widespread health threats. Because of its complicated components and various toxicity mechanisms, Particulate matter has caused more and more attention. Generally, based on the size of particle matter, it can be classified into four categories, including total suspended particles (TSP), inhalable particles (PM₁₀), fine particles (PM_2.5) and ultrafine particles (UFP). Fine particles are particles with aerodynamic diameters less than 2.5 micrometer (PM_2.5). Fine particles originate mainly from man-made pollution, especially emissions from industrial, residential coal and oil combustion as well as vehicle exhaust. Fine particles are so small that they can penetrate into the deepest parts of the respiratory tract. Furthermore, a lot of toxic compounds adsorbed on fine particles can deposit in the alveoli through inhalation, even penetrate the air-blood barrier into blood circulation. Therefore, fine particles may result in a wide range of damages to respiratory, cardiovascular, immune and other systems. Recent epidemiologic investigations suggested that the toxicity of Particulate matter from different areas is diverse and different sub-populations may have different susceptibility to lung injury caused by fine particles, while little toxicological research has been done in these aspects. In this thesis, toxicity of different components of fine particles is presented, and the host factors about genetics and the presence of pre-existent cardiopulmonary disease are researched on the susceptibility of lung injury caused by fine particles.In this study, fine particles and polycyclic aromatic hydrocarbons (PAHs) were monitored in certain urban districts of Shanghai. Two peak periods with highest concentration of fine particles and PAHs were identified through 24-hour monitoring: approximately from 6: 00 to 10: 00 and from 15: 00 to 20: 00 respectively. We found significant correlation between fine particles and PAHs with a correlation coefficient of 0.231 (P < 0.01). We used high performance liquid chromatography (HPLC) in analyzing fine particles samples of collected in Shanghai urban area in March, June, September and December of 2006 for PAHs. Many kinds of PAHs in fine particles were identified with different proportions. Phenanthrene, anthracene, fluoranthene, pyrene, benzo [a] anthracene, benzo [b] fluoranthene, benzo [a] pyrene and benzo [g, h, i] perylene were found in all samples. Meanwhile, the results of analysis for inorganic ions in fine particles sampled from February to December in 2006 showed that the anions were mainly SO₄^2-, NO₃^- and Cl^-, accounting for over 99% of anions. Thirteen kinds of metal ions were detected in the fine particles samples, in which the annual percentage of Al, Na, K and Mg accounted for over 90% of metal irons and the percentages were in ascending order: Al > Na > K > Mg. Further analysis of the other 9 metal ions showed that the percentages were in the order of Zn > Pb > Fe > Mn > Cr > Cu > Ni > V > Cd. Because diverse components of fine particles could cause different lung injury, components' toxicity of the fine particles was tested systematically. According to the results of treatment of human lung adenocarcinoma cell line (A549 cells) with anions of SO₄^2-, SO₃^2-, NO₃^-, Cl^- and seven metal ions (exposed doses: 0.01 mmol/L, 0.05 mmol/L, 0.1 mmol/L, 0.5 mmol/L, 1.0 mmol/L, 5.0 mmol/L and 10.0 mmol/L respectively), we found that only SO₃^2- and NO₃^- anions could inhibit cell activity in the anions determined, and all metal ions could inhibit cell activity in the following order of inhibition: Cu⁺ > Cu²⁺ > Ni²⁺ > Pb²⁺ > Zn²⁺ > Fe³⁺ > Fe²⁺. Furthermore, by experimenting with A549 cells exposed to soluble and insoluble components of fine particles (exposed doses: 100μg/mL, 200μg/mL and 300μg/mL respectively), we found that soluble components showed more toxicity to cells than that of insoluble components. Specifically, as compared with cells exposed to insoluble components, soluble components showed more inhibition of cell activity, greater injury of cell membranes, and higher rates of apoptosis and levels of reactive oxygen species (ROS). Meanwhile, according to the results of treatment with N-Acetyl-L-cysteine (NAC) and Vitamin C, levels of ROS in cells decreased greatly, while cell activity increased significantly. Therefore, oxidative stress was suggested as one of the mechanisms of lung injury caused by fine particles. In summary, different components of fine particles could lead to different toxicity to cells.With regard to study on genetic susceptibility of lung injury caused by fine particles, C57BL/6 strain mice (a sensitive strain to fine particles) and C3H/He strain mice (a resistant strain to fine particles) were exposed to fine particles (exposed dose 10mg/kg b. w) by intratracheal instillation once a day for two consecutive days. Twenty-four hours after the last exposure, the mice were sacrificed, and the lungs were taken out. Affymetrix GeneChip 430 2.0 was to run to find out the different expressions of genes between B6 mice and C3 mice. Over expression of the genes of Chi313, Chi314, Cxcl2, C4, Hc, Cp and Sirpb1 in B6 mice exposed to fine particles were observed (up-regulated) compared with that in C3 mice exposed to fine particles, while Igh-6, Cap1, Mtap2, Nme7 and 5830443L24Rik in B6 mice were obviously less expressed (down-regulated) than those in C3 mice. Further analysis suggested that most up-regulated genes were related to immune inflammatory response, chemokine activity, cytokine activity and complement activation, while most down-regulated genes were related to positive regulation of endocytosis, cell migration, cell morphogenesis, synthesis and metabolism of nuclei acid and microtubule bundle formation and depolymerization. The results of our experiments showed that lung injury caused by fine particles was greater in B6 mice than in C3 mice, which was in accordance with the findings reported in other studies. It could be concluded that at the same dose of exposure, inflammation and oxidative stress were more aggravated in B6 mice than that in C3 mice, although the ability of defense and humoral immunity, function of cell phagocytosis and migration, and self-reparation were weakened more in B6 mice. Furthermore, three different signaling pathways between B6 mice and C3 mice were considered, including inflammatory response pathway, matrix metalloproteinases pathway and classical complement activation. Through analysis of genetic homology, human homologous genes were found to be CXCL3, C4, C5, CP, CAP1, MAP2, NME7, MMP2, TIMP1, COL1A1 andCOL1A2. In conclusion, genetic factor was one of the susceptible mechanisms of lung injury caused by fine particles.In order to investigate whether the host factor of the presence of pre-existent cardiopulmonary disease was an additional susceptible factor of lung injury caused by fine particles, two rat models of chronic bronchitis and spontaneously hypertensive rats (SHR) were used. Two rat models were exposed to fine particles by intratracheal instillation once a day for three consecutive days, with doses of 1.6 mg/kg b. w, 8.0 mg/kg b. w and 40.0 mg/kg b. w respectively. Twenty-four hours after the last exposure, the rats were sacrificed, and the bronchoalveolar lavage fluid (BALF) and lung tissue were collected for analysis. According to the results of rat models of chronic bronchitis, concentrations of albumin (ALB), lactate dehydrogenase (LDH), alkaline phosphatase (AKP) and malonaldehyde (MDA) in BALF of rats with chronic bronchitis were higher than those in rats without chronic bronchitis, whereas the glutathione (GSH) was lower. It was suggested that as compared with healthy rats, vascular injury, damage of lung cells and oxidative stress were more severe in rats with chronic bronchitis even under the same exposure. In the rat model of hypertension, not only the expression of MDA, TNF-α, IL-1β, MIP-2, CD44 and OPN related to inflammation, but also the expression of CC16, SP-A and HO-1 related to anti-inflammation were increased, whereas the expression of SOD and GSH related to anti-oxidative damage were decreased in rats with both hypertension and none-hypertension exposed to fine particles. Meanwhile, it was also found that the expressions of these genes were significantly higher in rats with hypertension than those in rats without hypertension except SOD and GSH. These results indicated that in rats with hypertension the inflammation was more severe, although the ability of anti-oxidative damage was weaker when compared with that in rats without hypertension, which was responsible for the differences of lung injury between two kinds of rats. Finally, the indices of TLR4 and NF-κB revealed that the pathway of NF-κB was also one of the mechanisms of lung injury caused by fine particles, and the ability of activating this pathway easier in rats with hypertension might also be one of the susceptible factors. The results proved the conclusion from epidemiologic investigation that after exposure to fine particles, people with cardiopulmonary diseases was more susceptible to lung injury caused by fine particles than that in healthy people. In summary, the host factor of the presence of pre-existent cardiopulmonary disease was an additional susceptible factor of lung injury caused by fine particles.