大气细颗粒物致肺损伤的易感性研究
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摘要
颗粒物、臭氧、一氧化碳、二氧化硫、氮氧化物和铅这6种污染物被美国环境保护署(Environmental Protection Agency，EPA)定义为常见污染物，也被称为标准污染物。其中颗粒物和臭氧是对人类健康威胁最普遍的两种，而颗粒物又因其复杂的组成成分以及多样的毒作用机制，受到越来越多学者的关注。一般来说，颗粒物可以依据其粒径的大小，分为总悬浮颗粒物、可吸入颗粒物、细颗粒物和超细颗粒物四种。细颗粒物，即PM_(2.5)，是指空气动力学等效直径≤2.5μm的颗粒物，主要来自于燃煤燃油等高温燃烧过程以及机动车尾气的排放。大气细颗粒物表面吸附了大量有毒有害物质，可通过呼吸而沉积在肺泡，甚至可以通过气血屏障进入血液循环，引起机体呼吸、心血管、免疫等系统的损害。流行病学研究表明，不同地区来源的大气细颗粒物毒性大小存在一定的差异，以及不同的人群对于大气细颗粒物的致肺损伤作用的敏感程度不同，但是此方面的毒理学研究非常有限。本次研究通过综合分析大气细颗粒物成分的特异性毒性、遗传易感性和疾病易感性三个方面，探讨大气细颗粒物成分毒性的差异以及其致肺损伤的易感性因素和机制。
本研究对上海市某城区大气细颗粒物及其有机物成分进行连续24h的监测，共1天，结果发现细颗粒物及其有机物成分在一天中出现两个高峰，分别是6：00～10：00时间段和15：00～20：00时间段。相关分析表明，大气中的细颗粒物和多环芳烃存在明显的相关性，相关系数为0.231(P＜0.01)。运用高效液相色谱法对2006年3、6、9和12月份4个细颗粒物样品中的有机成分种类分析结果发现，细颗粒物中有机物的种类繁多，含量不均一，其中Phenanthrene(菲)、Anthracene(蒽)、Fluoranthene(荧蒽)、Pyrene(芘)、Benzo[a]anthracene(苯并[a]蒽)、Benzo[b]nuoranthene(苯并[b]荧蒽)、Benzo[a]pyrene(苯并[a]芘)和Benzo[g，h，i]perylene(苯并[ghi]花)8个有机物在4个样品中均能检测到。同时，对2006年2-12月份中细颗粒物的无机离子成分的研究表明，阴离子成分主要以SO_4~(2-)、NO_3~-和Cl~-为主，其年平均值所占比例超过了99％；检测了13种金属成分，其中Al＞Na＞K＞Mg，这四种金属成分占到了总成分的90％以上。对其余的9种金属成分进一步分析结果表明，各种金属所占比例依次为：Zn＞Pb＞Fe＞Mn＞Cr＞Cu＞Ni＞V＞Cd。为了比较细颗粒物不同成分的毒性大小，利用人类肺癌上皮细胞株A549细胞，分别对SO_4~(2-)、SO_3~(2-)、NO_3~-和Cl~-4种阴离子和7种金属离子的毒性进行了检测，结果发现在染毒剂量为0.01mmol／L、0.05mmol／L、0.1mmol／L、0.5mmol／L、1.0mmol／L、5.0mmol／L和10.0mmol／L的范围内，阴离子中仅有SO_3~(2-)和NO_3~-离子对细胞活性有抑制作用：而7种金属离子对细胞均有毒性，其大小依次为：Cu~+＞Cu~(2+)＞Ni~(2+)＞pb~(2+)＞Zn~(2+)＞Fe~(3+)＞Fe~(2+)。同时，还对细颗粒物的水溶成分和非水溶成分的毒性进行了检测，结果表明在染毒剂量为100μg／mL、200μg／mL和300μg／mL的范围内，水溶成分的细胞毒性明显高于非水溶成分，表现为抑制细胞活性，导致细胞膜损伤增加，并且诱导细胞凋亡和细胞活性氧(ROS)的产生。使用N-乙酰半胱氨酸(NAC)和抗坏血酸(Vit C)阻断活性氧(ROS)产生的研究结果表明，与未加上述抗氧化剂的染毒组相比，加抗氧化剂的染毒组细胞的活性明显增强，提示氧化应激损伤可能是细颗粒物毒作用的重要机制之一。综上所述，细颗粒物组成成分的不同可以导致其对细胞毒性的差异。
在对遗传因素是否为细颗粒物致肺损伤的易感因素的研究中，运用Affymetrix Mouse430 2.0全基因组表达谱芯片，对两种不同遗传背景的小鼠(对大气细颗粒物敏感的近交系小鼠C57BL／6(B6)和对大气细颗粒物耐受的近交系小鼠C3H／He(C3))进行气管灌注大气细颗粒物染毒，剂量为10mg／kg b.w，每天染毒1次，连续染毒2d，最后一次染毒结束24h后，处死动物，对肺组织基因的表达情况进行筛选。结果表明，相对于C3小鼠，B6小鼠肺部基因Chi313、Chi314、Cxcl2、C4、Hc、Cp和Sirpb1的表达上调，而基因Igh-6、Cap1、Mtap2、Nine7和5830443L24Rik的表达下调。进一步分析发现，上调基因多与免疫炎症反应、化学趋化因子活性、细胞因子活性和补体活化有关；而下调基因多与细胞内吞作用的正向调节、细胞游走、细胞形态形成、核酸合成和新陈代谢、以及微管束聚合以及解聚合作用相关。结合文献报道以及本实验室研究结果，即相同剂量的大气细颗粒物染毒后，B6小鼠肺损伤比C3小鼠严重，从本次研究可以推断，相对于C3品系小鼠，B6品系小鼠对大气细颗粒物染毒产生了更加强烈的肺部炎症反应；同时，B6品系小鼠肺部的防御功能以及体液免疫能力减弱，巨噬细胞的吞噬功能以及游走功能下降，损伤后修复能力也降低。对B6和C3两种品系小鼠的信号通路的研究发现，在B6和C3两种品系小鼠之间存在三条有差异的信号通路：炎症反应通路、基质金属蛋白酶通路和经典的补体激活通路。最后，通过同源性比对，找到了人类的同源性基因，包括：CXCL3、C4、C5、CP、CAP1、MAP2、NME7、MMP2、TIMP1、COL1A1和COL1A2。本部分的研究结果提示，遗传因素是细颗粒物致肺损伤的易感因素之一。
在本次研究中，还运用动物模型研究了心肺疾病患者是否为细颗粒物的易感亚群。在呼吸系统模型的研究中，以慢性支气管炎大鼠作为模型组，与健康大鼠进行了比较，采用气管灌注大气细颗粒物的方法染毒，染毒剂量为1.6mg／kg b.w、8.0 mg／kg b.w和40.0 mg／kg b.w，对照组气管滴注生理盐水，每天染毒1次，连续染毒3d，最后1次染毒后24h后，处死大鼠，取肺泡灌洗液和肺组织进行分析，结果发现在相同的大气细颗粒物染毒剂量下，模型组大鼠肺泡灌洗液中白蛋白(ALB)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)和丙二醛(MDA)的含量比正常组大鼠高，而谷胱苷肽(GSH)的含量相对较低，说明模型组大鼠在肺部血管损伤、肺实质细胞损伤以及氧化应激损伤等方面都比非模型组大鼠严重。在高血压大鼠模型的研究中，发现在高血压模型组和非模型组大鼠中，无论是反映氧化损伤的MDA的含量，炎症相关基因TNF-α、IL-1β、MIP-2、CD44和OPN的表达，还是抗氧化应激相关基因CC16、HO-1和SP-A的表达，均呈现升高的趋势，而与抗氧化损伤相关的蛋白(SOD和GSH)含量却明显降低。同时还发现，在高血压模型组大鼠，SOD和GSH这两个指标的表达明显低于非模型大鼠，而其它指标的表达则明显高于非模型组大鼠，说明高血压模型组大鼠肺部炎症反应更加严重，而抗氧化能力较弱，最终导致肺损伤更加严重。最后，通过对TLR4和NF-κB的检测，发现NF-κB通路是细颗粒物致肺损伤的重要通路之一，而高血压模型组大鼠更加容易激活该通路，这可能也是导致高血压大鼠对大气细颗粒物致肺损伤易感的因素之一。该部分实验结果验证了人群流行病学相应的结论，即患心肺疾病的人群对大气细颗粒物的致肺损伤作用更加易感，因此，心肺疾病的患病状态也是细颗粒物致肺损伤的易感因素之一。
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 (PM_(10)), 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 SO_4~(2-), NO_3~- 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 SO_4~(2-), SO_3~(2-), NO_3~-, 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 SO_3~(2-) and NO_3~- anions could inhibit cell activity in the anions determined, and all metal ions could inhibit cell activity in the following order of inhibition: Cu~+ > Cu~(2+) > Ni~(2+) > Pb~(2+) > Zn~(2+) > Fe~(3+) > Fe~(2+). 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.
本研究对上海市某城区大气细颗粒物及其有机物成分进行连续24h的监测，共1天，结果发现细颗粒物及其有机物成分在一天中出现两个高峰，分别是6：00～10：00时间段和15：00～20：00时间段。相关分析表明，大气中的细颗粒物和多环芳烃存在明显的相关性，相关系数为0.231(P＜0.01)。运用高效液相色谱法对2006年3、6、9和12月份4个细颗粒物样品中的有机成分种类分析结果发现，细颗粒物中有机物的种类繁多，含量不均一，其中Phenanthrene(菲)、Anthracene(蒽)、Fluoranthene(荧蒽)、Pyrene(芘)、Benzo[a]anthracene(苯并[a]蒽)、Benzo[b]nuoranthene(苯并[b]荧蒽)、Benzo[a]pyrene(苯并[a]芘)和Benzo[g，h，i]perylene(苯并[ghi]花)8个有机物在4个样品中均能检测到。同时，对2006年2-12月份中细颗粒物的无机离子成分的研究表明，阴离子成分主要以SO_4~(2-)、NO_3~-和Cl~-为主，其年平均值所占比例超过了99％；检测了13种金属成分，其中Al＞Na＞K＞Mg，这四种金属成分占到了总成分的90％以上。对其余的9种金属成分进一步分析结果表明，各种金属所占比例依次为：Zn＞Pb＞Fe＞Mn＞Cr＞Cu＞Ni＞V＞Cd。为了比较细颗粒物不同成分的毒性大小，利用人类肺癌上皮细胞株A549细胞，分别对SO_4~(2-)、SO_3~(2-)、NO_3~-和Cl~-4种阴离子和7种金属离子的毒性进行了检测，结果发现在染毒剂量为0.01mmol／L、0.05mmol／L、0.1mmol／L、0.5mmol／L、1.0mmol／L、5.0mmol／L和10.0mmol／L的范围内，阴离子中仅有SO_3~(2-)和NO_3~-离子对细胞活性有抑制作用：而7种金属离子对细胞均有毒性，其大小依次为：Cu~+＞Cu~(2+)＞Ni~(2+)＞pb~(2+)＞Zn~(2+)＞Fe~(3+)＞Fe~(2+)。同时，还对细颗粒物的水溶成分和非水溶成分的毒性进行了检测，结果表明在染毒剂量为100μg／mL、200μg／mL和300μg／mL的范围内，水溶成分的细胞毒性明显高于非水溶成分，表现为抑制细胞活性，导致细胞膜损伤增加，并且诱导细胞凋亡和细胞活性氧(ROS)的产生。使用N-乙酰半胱氨酸(NAC)和抗坏血酸(Vit C)阻断活性氧(ROS)产生的研究结果表明，与未加上述抗氧化剂的染毒组相比，加抗氧化剂的染毒组细胞的活性明显增强，提示氧化应激损伤可能是细颗粒物毒作用的重要机制之一。综上所述，细颗粒物组成成分的不同可以导致其对细胞毒性的差异。
在对遗传因素是否为细颗粒物致肺损伤的易感因素的研究中，运用Affymetrix Mouse430 2.0全基因组表达谱芯片，对两种不同遗传背景的小鼠(对大气细颗粒物敏感的近交系小鼠C57BL／6(B6)和对大气细颗粒物耐受的近交系小鼠C3H／He(C3))进行气管灌注大气细颗粒物染毒，剂量为10mg／kg b.w，每天染毒1次，连续染毒2d，最后一次染毒结束24h后，处死动物，对肺组织基因的表达情况进行筛选。结果表明，相对于C3小鼠，B6小鼠肺部基因Chi313、Chi314、Cxcl2、C4、Hc、Cp和Sirpb1的表达上调，而基因Igh-6、Cap1、Mtap2、Nine7和5830443L24Rik的表达下调。进一步分析发现，上调基因多与免疫炎症反应、化学趋化因子活性、细胞因子活性和补体活化有关；而下调基因多与细胞内吞作用的正向调节、细胞游走、细胞形态形成、核酸合成和新陈代谢、以及微管束聚合以及解聚合作用相关。结合文献报道以及本实验室研究结果，即相同剂量的大气细颗粒物染毒后，B6小鼠肺损伤比C3小鼠严重，从本次研究可以推断，相对于C3品系小鼠，B6品系小鼠对大气细颗粒物染毒产生了更加强烈的肺部炎症反应；同时，B6品系小鼠肺部的防御功能以及体液免疫能力减弱，巨噬细胞的吞噬功能以及游走功能下降，损伤后修复能力也降低。对B6和C3两种品系小鼠的信号通路的研究发现，在B6和C3两种品系小鼠之间存在三条有差异的信号通路：炎症反应通路、基质金属蛋白酶通路和经典的补体激活通路。最后，通过同源性比对，找到了人类的同源性基因，包括：CXCL3、C4、C5、CP、CAP1、MAP2、NME7、MMP2、TIMP1、COL1A1和COL1A2。本部分的研究结果提示，遗传因素是细颗粒物致肺损伤的易感因素之一。
在本次研究中，还运用动物模型研究了心肺疾病患者是否为细颗粒物的易感亚群。在呼吸系统模型的研究中，以慢性支气管炎大鼠作为模型组，与健康大鼠进行了比较，采用气管灌注大气细颗粒物的方法染毒，染毒剂量为1.6mg／kg b.w、8.0 mg／kg b.w和40.0 mg／kg b.w，对照组气管滴注生理盐水，每天染毒1次，连续染毒3d，最后1次染毒后24h后，处死大鼠，取肺泡灌洗液和肺组织进行分析，结果发现在相同的大气细颗粒物染毒剂量下，模型组大鼠肺泡灌洗液中白蛋白(ALB)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)和丙二醛(MDA)的含量比正常组大鼠高，而谷胱苷肽(GSH)的含量相对较低，说明模型组大鼠在肺部血管损伤、肺实质细胞损伤以及氧化应激损伤等方面都比非模型组大鼠严重。在高血压大鼠模型的研究中，发现在高血压模型组和非模型组大鼠中，无论是反映氧化损伤的MDA的含量，炎症相关基因TNF-α、IL-1β、MIP-2、CD44和OPN的表达，还是抗氧化应激相关基因CC16、HO-1和SP-A的表达，均呈现升高的趋势，而与抗氧化损伤相关的蛋白(SOD和GSH)含量却明显降低。同时还发现，在高血压模型组大鼠，SOD和GSH这两个指标的表达明显低于非模型大鼠，而其它指标的表达则明显高于非模型组大鼠，说明高血压模型组大鼠肺部炎症反应更加严重，而抗氧化能力较弱，最终导致肺损伤更加严重。最后，通过对TLR4和NF-κB的检测，发现NF-κB通路是细颗粒物致肺损伤的重要通路之一，而高血压模型组大鼠更加容易激活该通路，这可能也是导致高血压大鼠对大气细颗粒物致肺损伤易感的因素之一。该部分实验结果验证了人群流行病学相应的结论，即患心肺疾病的人群对大气细颗粒物的致肺损伤作用更加易感，因此，心肺疾病的患病状态也是细颗粒物致肺损伤的易感因素之一。
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 (PM_(10)), 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 SO_4~(2-), NO_3~- 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 SO_4~(2-), SO_3~(2-), NO_3~-, 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 SO_3~(2-) and NO_3~- anions could inhibit cell activity in the anions determined, and all metal ions could inhibit cell activity in the following order of inhibition: Cu~+ > Cu~(2+) > Ni~(2+) > Pb~(2+) > Zn~(2+) > Fe~(3+) > Fe~(2+). 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.
引文
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