专论 正式出版 版本 1 Vol 11 (2) : 125-142 2020
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PM2.5的健康危害、毒理效应与作用机制的研究
Update on the toxicological effects and mechanism of PM2.5
: 2019 - 04 - 25
: 2019 - 09 - 12
: 2019 - 09 - 19
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摘要&关键词
摘要:大气颗粒物对人体健康的影响在世界范围内引起关注,尤其是细粒子因粒径小、复杂,对人体健康影响尤为突出。PM2.5的毒理效应与作用机制是当前研究的热点,分析其研究现状与存在问题能更好地把握其核心领域并开展更进一步的研究。基于文献检索与分析,系统梳理了PM2.5危害导致的疾病类型;介绍了PM2.5毒理研究的主要实验手段;讨论当前认知的PM2.5毒理效应;汇总PM2.5毒理作用机制研究方法的原理、特点、检测指标与测定方法,解析目前研究认为的五种主要的PM2.5毒理作用机制。在分析已有研究成果的基础之上,就PM2.5毒理实验、方法和研究内容方面存在的不足进行讨论,并提出今后需要重点开展的研究方向,以期为PM2.5健康危害的准确评估和有效防治提供科学依据。
关键词:PM2.5;健康危害;毒理实验;健康效应;作用机制
Abstract & Keywords
Abstract: Background, aim, and scope The review deals with publications concerning the toxicological effects and mechanism of PM2.5. The impact of atmospheric particulate matter on human health has attracted worldwide attention. In particular, fine particles (PM2.5) have a greater impact on human health due to their small particle size and complex composition. As a consequence, identifying the mechanism of action of PM2.5 has always been a major objective of atmospheric environmental science research. Materials and methods The review describes the types of diseases caused by PM2.5, the experimental methods used in PM2.5 toxicology research, and the toxicological effects of PM2.5 (including the results of in vitro and in vivo experiments on PM2.5 characterised by different components, sources, and seasons), as well as the consensus mechanism of action of PM2.5. This review emphasizes that the mechanisms of action of PM2.5 vary according to component, source, and season. Results Several publications have been identified that PM2.5 can induce a variety of diseases. The mechanisms of action involve oxidative damage, inflammatory damage, DNA damage and mutations, apoptosis, and autophagy. Discussion Research on the underlying mechanism of PM2.5 might help to provide a scientific basis for accurately assessing the health hazards and effective prevention of PM2.5. The components of PM2.5 and the mechanisms of action of PM2.5 can vary, as can the target cells. Conclusions Air pollution is one of the main causes of global morbidity and mortality. The discussion of air pollution control and the pathogenesis of PM2.5 is an extremely important issue. Oxidative stress, inflammatory damage, DNA damage and mutations, apoptosis, and autophagy are the main mechanisms of PM2.5-induced disease. The results of PM2.5 with different components, sources, and seasons from in vitro and animal experiments provide a scientific basis for the accurate assessment and effective prevention of PM2.5 health hazards. Recommendations and perspectives In order to better explore the mechanism of action of PM2.5, toxicity research methods employing in vitro and in vivo experiments should be developed and improved, and the interactions between different PM2.5 components should be addressed. Intervention studies on PM2.5 health hazards and finding effective ways of reducing or preventing the health risks of PM2.5 are critical. 李莎:注意in vitro和in vivo要斜体。通篇注意。
Keywords:  PM2.5; health hazards; toxicological experiments; health effects; mechanisms of action
环境空气污染被确定为全球疾病负担的主要风险因素Shang et al,2017;Wang et al,2018。颗粒物,特别是空气动力学直径小于2.5μm的颗粒PM2.5,粒径小,比表面积大,易吸附重金属、酸性氧化物、有机物、细菌和病毒等,对人体健康带来严重危害。PM2.5的来源多种多样,对人体的影响也不尽相同PM2.5在多种疾病中的流行病学研究由来已久,但其发病机制尚不清楚。本文综述PM2.5的健康危害、毒理效应及其毒理研究方法与作用机制,以期为PM2.5健康危害的准确评估和有效防治提供科学依据。
1   PM2.5的健康危害
1.1   呼吸系统疾病
PM2.5能较长时间停留在空气中,主要经呼吸进入人体,对呼吸系统影响最直接。PM2.5会造成呼吸道粘膜上皮细胞的损伤,还有部分沉积在肺部,激活免疫细胞,引发炎症反应,引起呼吸系统疾病。流行病学研究表明,上呼吸道刺激、儿童急性呼吸道感染、成人慢性支气管炎、慢性肺纤维化、、肺癌等疾病的发生率都与PM2.5的浓度有密切联系吕广娜和李荣山,2013。
1.2   胃肠道影响
PM2.5还能经口与食物、水一同进入消化道,对胃肠道产生影响Beamish et al,2011。流行病学研究报道颗粒物暴露对胃肠道疾病的不良影响,包括克罗恩病、炎症性肠病和消化道癌症Ananthakrishnan et al,2011;Beamish et al,2011。
1.3   心血管疾病
流行病学调查显示,暴露于高浓度PM2.5环境,会导致血压升高,诱发心律失常、缺血性心肌炎、心梗、心衰等心血管疾病Szyszkowicz et al,2012。研究显示,PM2.5的浓度与居民的总死亡率及由呼吸系统疾病、心血管疾病等疾病引发的死亡率之间呈现长期的正相关,且均有显著意义等。
1.4   免疫系统异常
PM2.5还会引发人体免疫系统的异常。研究发现,PM2.5的吸入水平与和细胞管型相关,且能影响系统性红斑狼疮等自身免疫风湿病的疾病活动。
1.5   代谢综合征
代谢综合征由一,包括腹部肥胖、血脂异常、血压升高和高血糖。一项大型横断面研究发现,人体长期暴露于PM1、PM2.5、PM10的环境中,代谢综合征风险增加,且显著相关Yang et al,2018。
1.6   癌症
PM2.5还可能导致癌症的发生。除了目前众所周知的肺癌,等研究癌症总体及某些肺外器官癌症发病率、死亡率与PM2.5暴露浓度的关系。胰腺癌PC是一种复杂的恶性肿瘤,生活方式的改变和工业发展造成的环境污染与PC有关。
2   PM2.5的毒理研究实验
是现有的PM2.5毒理研究的重要实验手段。
2.1   体外实验
体外毒理学实验是对体外细胞培养、染毒,再观察和检测细胞对毒物的反应。往往根据靶器官或机制探讨的需要,选定体外细胞的种类。经文献检索,对当前PM2.5毒理实验的体外细胞进行汇总,见表1。
表1   PM2.5毒理研究体外实验常用细胞
细胞名称
Cell name
靶器官
Target organ
参考文献
References
单培养
Cell onoculture systems
人肺腺癌细胞A549
Human lung adenocarcinoma cells

Lung
Chen and Lippmann, 2009; Gualtieri et al, 2009; Wang et al, 2011; Lippmann et al, 2013; Pavagadhi et al, 2013; Alessandria et al, 2014
人胚胎肺上皮细胞L132
Human embryonic pulmonary epithelial cells
Saint-Georges et al,2008
人类胚胎肺成纤维细胞HEL12469
Human embryonic lung fibroblasts
Líbalová et al,2012
人支气管上皮细胞
Human bronchial epithelial cells
HBE
Human bronchial epithelial
支气管
Bronchus
丁晓洁,2014;华秋翰,2016;张世鑫等,2016;张鑫,2016
16-HBE
Human bronchial epithelial
Baulig et al,2007;Rumelhard et al,2007;覃辉艳等,2012;平飞飞等,2015
BEAS-2B
Immortalized human bronchial epithelial cell line
Oh et al2011 et al2012Longhin et al2013
正常人鼻上皮细胞NHNE
Normal human nasal epithelial cells
鼻腔
Nasal cavity
Rumelhard et al2007
小鼠单核巨噬细胞白血病细胞RAW264.7
Mouse onocyte acrophage eukemia ells
血液
Blood
Cavanagh et al2009Jalava et al2009Steenhof et al2011
人脐静脉内皮细胞HUVEC
Human umbilical vein endothelial cells
血管
Vascellum
范兰兰,2013;秦梦楠,2016
肝癌细胞HepG2
Hepatoma cell

Liver
et al2010
大鼠肝脏上皮细胞WB-F344
Rat hepatic epithelial cells
Topinka et al2008Andrysík et al2011
共培养
Toxicity of Co-Culture Cell Model
肺泡巨噬细胞AM和L132细胞
Alveolar macrophage and uman embryonic pulmonary epithelial cells

Lung
Abbas et al2009
A549细胞和AM细胞
Human lung adenocarcinoma cells and lveolar macrophage
André et al201
BEAS-2B细胞和A549细胞
Immortalized human bronchial epithelial cell line and Human lung adenocarcinoma cells
支气管和肺
Bronchus and ung
Gualtieri 2010
人T细胞和人单核源性巨噬细胞MDMs Human T lymphocyte and uman monocyte-derived macrophages血液
Blood
Long et al2005
PM2.5毒理研究的体外细胞培养、染毒以单种细胞实验为主,通过细胞培养、染毒,指标检测,表征细胞的反应,从而探讨PM2.5的毒理机制。文献报道采用的体外细胞种类有:肺腺癌细胞A549、胚胎肺上皮细胞L132、人支气管上皮细胞HBE、16-HBE、BEAS-2B、正常人鼻上皮细胞NHNE、小鼠单核巨噬细胞白血病细胞RAW264.7、人脐静脉内皮细胞HUVEC、肝癌细胞HepG2、人类胚胎肺成纤维细胞HEL12469、肝脏上皮细胞WB-F344。华秋翰2016采用人支气管上皮细胞进行PM2.5染毒探讨其对细胞的氧化损伤效应,结果显示不同浓度的PM2.5均能使支气管上皮细胞存活率降低,并且促进以及细胞中的升高,细胞中的活性降低刘婷2015研究了冬季灰霾对肺泡巨噬细胞的毒理机制,将肺泡巨噬细胞分别以0、33、100、300μg/mL染毒,实验结果表明随着PM2.5浓度的升高肺泡巨噬细胞的存活率、SOD活性、均下降。
除了单细胞培养染毒,已有少量多细胞共培养染毒的报道,如AM与L132细胞共培养、A549与AM共培养、BEAS-2B与A549共培养、人T细胞与MDMs共培养Abbas et al2009通过人AM与L132细胞的共培养模型研究大气颗粒物中、组分诱导的不良健康影响机制,结果表明在单培养L132细胞时,经PM染毒后,基因表达增加,而在与AM共培养时几乎不受影响。
et al201研究了单培养和共培养A549与人肺泡巨噬细胞中PAHs的代谢活化和DNA加合物生成,发现在肺泡巨噬细胞中PAH诱导CYP1A1基因表达,而在L132细胞中则不然。然而,暴露于PM2.5的人AM、L132共细胞模型中未观察到明显的DNA加合物的生成。
Gualtieri 2010选用A549与BEAS-2B共培养,进行了PM毒性试验。暴露于冬季PM2.5的BEAS-2B细胞中,由于有丝分裂延迟/停滞而减少增殖,而在A549中没有观察到这种影响,以上结果表明体外细胞对PM的反应可能与细胞系有关,并提示不同的PM特性可能触发不同的机制,如炎症、细胞周期的扰动和细胞死亡。
Long et al2005研究了人T细胞和MDMs对合成C和C/Fe颗粒1 m的反应,以研究PM2.5碳颗粒和铁对细胞的损伤。结果表明T细胞并未表现出超微结构变化和对两种颗粒的吞噬能力,MDMs对两种类型的颗粒均有较强的吞噬能力。
2.2   动物试验
体内毒理学实验是以动物多为小鼠为染毒对象,观察动物染毒后的反应,检测动物器官、血液的指标。目前PM2.5的体内实验有以下几种染毒方法:气管滴注染毒、空气浓缩富集系统吸入染毒、重污染环境下直接吸入染毒和尾静脉注射等。
2.2.1   气管滴注
气管滴注是PM2.5毒理研究体内试验应用较多的一种染毒方法。采集PM2.5颗粒,配制含PM2.5的液体,以气管滴注方式进入动物体内华秋翰,2016。优点方法经济、使用毒物少、剂量准确便于控制,操作方便;缺点是与人实际接触毒物的条件和方式有差异,对动物的机械损伤较大。
2.2.2   空气富集浓缩系统吸入
Ying et al2014应用空气富集浓缩系统对实验小鼠染毒,研究浓缩环境PM2.5颗粒的心血管毒性。采用这种方法染毒,精准地控制染毒剂量,但应用成本较高且系统结构复杂。
2.2.3   重污染环境下直接吸入
Chen et al2013开展高污染PM2.5环境中小鼠自主呼吸染毒实验,研究环境颗粒物对ApoE敲除小鼠动脉硬化的影响,采用自然条件染毒。该法需要PM2.5高浓度的自然条件,限于PM2.5污染严重地区的污染期。
2.2.4   尾静脉注射
此外,还有研究采用尾静脉注射的方法进行PM2.5体内染毒。孙萌等2011在研究PM2.5不同成分对大鼠肾血管环收缩舒张反应影响的实验中,使用此法染毒。
3   PM2.5的毒理效应
3.1   不同组分的毒理效应
颗粒物是一种复杂的、非均匀的、不同维数的颗粒混合物,其化学组分随时间和空间的变化而变化,PM2.5的化学组分不同,其对人体健康的影响和作用机制也随之不同。
3.1.1   水溶性离子毒理效应
水溶性无机离子是PM2.5的主要成分et al,200,它们还控制颗粒酸度;颗粒酸度是影响气溶胶形成和健康效应的重要理化性质Grassian2001;,2010。Zou et al2016利用人肺上皮A549体外检测水溶性离子的毒性作用结果表明水溶性离子可显著降低A549的存活率,但对LDH释放的影响较小Okeson 2004;et al,2006;Deng et al,2007。经水溶性离子染毒后的A549形态发生改变,通过透射电镜观察A549在水溶性离子处理后的细胞发生细胞核核肿胀和线粒体增殖,说明PM2.5中的水溶性离子可以诱导A549内ROS生成、线粒体倍增,可能通过氧化应激引起细胞损伤。
3.1.2   无机元素毒理效应
颗粒无机元素主要来自天然矿物尘和人为来源的含金属元素的烟尘,金属组分会引起炎症和癌症,主要是由于Fenton反应产生ROS导致DNA损伤且过渡金属通过ROS的生成、线粒体功能障碍、丝裂原活化蛋白激酶MAPK、人体抑癌基因p53和含半胱氨酸的天冬氨酸蛋白水解酶caspase的激活或下调抗凋亡蛋白Bcl-2Pulido,2003的表达促进细胞凋亡。
Chen et al2018采集南京某地PM2.5进行体外毒性实验,发现铜等过渡金属可能是PM2.5细胞毒性的重要因素。Sørensen et al2005关注水溶性过渡金属与氧化DNA损伤之间的关系,选用8-oxodG来表征DNA损伤的程度,结果表明淋巴细胞中8-oxodG浓度与钒和铬浓度显著相关,PM2.5中的钒和铬可能在诱导氧化DNA损伤中起主要作用。
3.1.3   有机提取物毒理效应
颗粒中的有机物与肺毒性密切相关Lidén et al,2003;Billet et al,2007。颗粒中的有机提取物包含已知的致癌化合物,如PAH,可形成诱变性DNA加合物,通过自由基的生成,导致DNA损伤的形成Feilberg et al,2001;SchnelleKreis et al,2001;Danielsen et al,2009。PAHs是大气中主要的有毒有机污染物,其吸入被证实会导致肺癌、男性精液质量下降和DNA损伤Yang et al,2017;Zhang et al,2009;Wang et al,2011;Pieterse et al,2013;Yue et al,2015。Ferecatu et al2010发现低浓度的PM对人体支气管上皮细胞有抗凋亡作用,与吸附在颗粒上的水溶性组分和成分有关,诱导的细胞凋亡主要是通过线粒体途径介导的。PAHs可能激活细胞内信号转导通路,AhR通路部分参与了微粒的抗凋亡作用,导致机体炎症状态持续时间延长以及延迟损伤组织修复,2008,例如支气管管壁增厚et al,2003和气道重塑。
张玲2014用A549模拟醌类化合物进入呼吸系统的早期接触,探索五种醌类化合物1,2-萘醌、9,10-菲醌、2-甲基蒽醌MAQ、苊醌ACQ和2-甲基-1,4-萘醌MNQ对A549的细胞毒理作用及其致毒机制,进一步明确醌类化合物对人体的有害影响。结果表明五种醌类化合物均不同程度地造成细胞死亡、细胞膜和线粒体损伤,除PQ外的四种醌类化合物均可诱导的表达,1,2-NQ和MNQ可导致DNA损伤,1,2-NQ还会造成Ca2+浓度升高。
3.1.4   内毒素毒理效应
内毒素是革兰氏阴性细菌细胞壁中的一种成分,又叫脂多糖LPS唐虹等,2005,当细菌死亡溶解或用人工方法破坏细菌细胞后会被释放出来。其毒性成分主要为类脂质A,内毒素进入机体后可以引起发热、微循环障碍、内毒素休克及播散性血管内凝血等。PM2.5能较长时间悬浮于空气中,各种生物组分在自然力和人类社会活动中都有可能被大量地播散到空气中,进而被吸附于PM2.5上,被吸附的微生物受各种物理化学因素影响就有可能破裂死亡并释放内毒素。研究表明内毒素是室内环境中的一种健康隐患Teeuw et al,1994;Michel et al,1996;Cândida Rizzo et al,;空气颗粒悬浮液中存在的可溶性内毒素可能促进肺泡巨噬细胞释放,引起进一步的炎症Imrich et al,1999。内毒素与颗粒物组分在对机体造成损伤时可能存在协同作用,但目前国内外对内毒素的健康影响研究尚少。
3.1.5   碳组分毒理效应
根据Gualtieri et al2009研究,在城市地区,颗粒有机碳OC的水溶性和不溶性比例相等,而元素碳EC是完全不溶于水。Steenhof et al2011发现荷兰某地收集的颗粒物元素碳、有机碳等组分,对RAW264.7细胞的毒性实验表明氧化潜能大,单质碳和有机碳的促炎活性最高。
3.1.6   微生物毒理效应
PM2.5吸附的微生物可能与过敏性疾病和呼吸系统疾病有关系et al,2014。巨天珍等2003每立方米空气可培养出的活性微生物可达1.14×105/m3,可培养出的细菌浓度可达4.63×104/m3;杜睿和周宇光2010研究夏季真菌气溶胶浓度可达4.66×105CFU/m3。细菌是PM2.5和PM10污染物中最丰富的原核微生物,其中放线菌门、变形菌门、绿腐菌门、厚壁菌门、拟杆菌门和广古菌门相对丰度1%。在已鉴定的微生物种类中,有几种已知可引起人类过敏和呼吸道疾病,包括肺炎链球菌、烟曲霉和人体腺病毒平均基因组覆盖率分别为2.0%、14.5%和6.5%。其中,肺炎链球菌是社会获得性肺炎CAP最常见的病因,已从近50%的CAP病例中分离出来Cao et al,2014。
3.2   不同季节PM2.5的毒理效应
颗粒物暴露和毒性与季节分布有关,本质上与颗粒物的化学组分有关。Perrone et al2013研究了意大利北部伦巴第地区的米兰市区、乡村和偏远山区的四个不同季节的PM2.5和PM1的化学成分以及对A549的生物效应。结果表明春夏季的PM浓度高于秋冬季,且夏秋季的PM在诱导ROS生成方面比其他季节更显著,而春季米兰市区的PM更能导致DNA损伤。这可能归因于米兰春夏季季节特点是光化学产生的二次气溶胶较多,且富含硫酸盐和二次有机化合物,因此对细胞毒性较为显著。
Chen et al2018检测了南京某地PM2.5对A549的细胞毒性,结果表明冬季PM2.5毒性高于夏季,主要表现在冬季PM2.5较夏季在低浓度时明显抑制细胞活力且冬季LDH水平明显高于夏季,有趣的是冬季低浓度PM2.5降低SOD水平高于夏季,而夏季高浓度PM2.5降低SOD水平高于冬季,可能的原因是夏季PM2.5样品中大多数测量金属的颗粒积累量高于冬季,但冬季PM2.5中典型过渡金属如Cu、Mn和Co的累积量较高,它们可能与PM2.5细胞毒性差异有关。
3.3   不同来源PM2.5的毒理效应
颗粒物包含多种化合物,除受季节影响外,颗粒物化学性质还取决于排放源Dingenen et al,2004;Stone et al,2009,不同来源的颗粒物具有独特的性状。
3.3.1综合源
Seagrave et al2006采集了美国东南部四个具有代表性地点的环境颗粒物,分别是阿拉巴马州伯明翰BHM的基地一个未开发的建筑用地,靠近重型交通和工业乔治亚州亚特兰大市杰佛逊街JST一块城市用地,位于停车场、城市街道、仓库和仓库之间佛罗里达州彭萨科拉混合了城市和住宅,靠近一所小学阿拉巴马州森特维尔CTR位于农村和森林中,靠近塔拉迪加国家森林,研究发现在冬季采集的颗粒物中,JST位点的颗粒物毒性明显高于其他样本,引发炎症最明显,而BHM位点次之,柴油和汽油的燃烧排放在BHM和JST两位点中贡献较大,可见汽车排放尾气和工业排放废气毒性较大。
3.3.2地铁源
有研究表明地下铁路空气中粗颗粒物和细颗粒物含有高浓度的过渡金属,其毒性高于其他来源的颗粒et al,2006,研究还发现地下铁路工人的血液中可能会出现系统性炎症和促凝血因子的标记Bigert et al,2008,这可能颗粒金属的影响。Loxham et al2015研究了地下火车站粗、细、超细颗粒对人源代支气管上皮细胞的毒性,发现地铁颗粒物由于铁能够诱导细胞发生急性促炎反应和抗氧化反应。
3.3.3烹饪油烟
烹饪油烟COF是亚洲室内空气污染的主要来源,有研究调查COF对A549的影响,并探讨COF衍生的PM2.5暴露的反应的凋亡机制。201采集了厨房油烟并制备了其衍生的PM2.5,对A549进行体外实验,结果表明COF衍生的PM2.5降低了肺泡细胞的活力和功能,并显著诱导ROS的产生;此外,E试验表明分泌IL-6和TNF-α增加,/Bcl-2 mRNA比率增加。值得注意的是,COF衍生的PM2.5诱导了STAT1的磷酸化以及MAPK途径的NF-和ERK1/2p38JNK活化。COF衍生的PM2.5导致A549细胞发炎、凋亡和细胞损伤,MAPK/NF-/STAT1途径可能是造成氧化还原失衡导致肺实质损伤的原因。
3.3.4沙尘暴
沙尘暴是全世界干旱地区最常见的自然灾害,沙尘暴是PM2.5的重要来源。发生沙尘暴时,PM2.5浓度较非沙尘暴时高出35倍,沙尘暴PM2.5对人体健康产生危害。Lee et al2014沙尘暴天气较非沙尘暴天气造成65岁以上老人的死亡率增加4.39%5.00%。Li2014发现沙尘暴可明显增加大气中PM2.5的浓度,引发以儿童哮喘为代表的呼吸系统疾病入院率明显增加。Crooks et al2016研究美国县级人群19932005年因呼吸系统疾病、心血管疾病和非意外事件导致的死亡率时发现,沙尘暴造成人群总死亡率增加7.4%,沙尘暴与中老年人心血管疾病的死亡率密切相关。国内学者对20072011年兰州春季沙尘暴与呼吸系统疾病急诊入院率关系的研究表明,沙尘暴可明显增加老年人慢性阻塞性肺疾病等呼吸系统疾病的急诊入院率Ma et al,2016。Teng et al2016在143063名急性患者的入院研究中提出,沙尘暴能对中老年人急性发病产生延迟效应,增加了急性的发病率。针对沙尘暴PM2.5的生物学效应,研究提出了许多作用机制假设,其中主要包括炎症反应、氧化应激、免疫毒性、DNA损伤等。在Watanabe et al2015的研究中发现,沙尘暴PM2.5会造成细胞内、、和TNF-水平升高。Kouassi et al2010研究发现,PM2.5中携带了大量的过渡金属元素如钙、钠、镁、钛、铝、铁、锰、铬、铅、锌、铜等,会诱导细胞发生脂质过氧化,并造成胞内抗氧化系统紊乱,造成机体发生氧化应激损伤。另外沙尘暴PM2.5过度暴露引发的胞内氧化应激反应和细胞因子释放紊乱,使免疫球蛋白表达失调,进而导致免疫毒性损伤。
4   PM2.5的毒理作用机制
4.1   PM2.5的毒理作用机制研究方法
氧化损伤、炎性损伤和DNA损伤及突变是目前研究认为的PM2.5主要的毒理作用机制,围绕PM2.5如何引起氧化损伤、炎症损伤和DNA损伤及突变,研究者开展了实验研究,表2汇总了PM2.5毒理作用机制研究方法的原理、特点、检测指标和测定方法。
表2   PM2.5毒理作用机制研究方法
实验分类
Experimental lassification
指标Index实验原理
Experimental rinciple
测定方法
Test ethods
毒理机制
Toxic echanism
特点
Characteristics
参考文献
References
细胞
功能学
Cellular functionology
细胞
存活率
Cell survival rate
MTT试剂盒
MTT assay
微板比色法
Colorimetric method
氧化损伤
Oxidative damage
优点:简单、快捷
Advantages: imple and fast
Perrone et al,2013;Chen et al,2018
缺点:

Disadvantage:

omplexoperation steps

t is not suitable for suspension cell activity detection
CCK-8试剂盒
Cell counting kit-8
优点:灵敏度高;重复性优于MTT;对细胞毒性小

华秋翰,2016;Shang et al,2017

Disadvantage: mpurities are easy to dye, affecting OD value
细胞
凋亡
Apoptosis
AnnexinV-FITC-PI试剂盒
Annexin V-FITC/PI poptosis etection it
流式细胞仪
Flow cytometer
优点:图片美观;结果更为直观
Advantages: beautiful pictures;more intuitive results
徐大琴,2008;刘婷,2015;华秋翰,2016
荧光显微镜
Fluorescence microscope
氧化应
激实验
Oxidative stress experiment
SOD
Superoxide dismutase
超氧化物歧化酶
测定试剂盒
Superoxide dismutase assay kit
羟胺法Hydroxylamine method优点:既可以定性又可以定量
Advantages: ualitative and quantitative
Oh et al,2011;华秋翰,2016;Chen et al,2018
分光光度法
Spectrophotometry
比色法
Colorimetric method
ACP
Acid phosphatase
酸性磷酸酶
测定试剂盒
Acid phosphatase assay kit
分光光度法
Spectrophotometry
刘婷,2015;华秋翰,2016
比色法
Spectrophotometric Colorimetry
微板法
Colorimetric method
ALP
Alkaline phosphatase
碱性磷酸酶
测定试剂盒
Alkaline phosphatase assay kit
可见光比色法
Visible light colorimetry
华秋翰,2016
微板法
Colorimetric method
钙钴法
Calcium cobalt method
偶氮偶联法
Azo coupling method
MDA
Malondialdehyde
丙二醛
测定试剂盒
Plant malondialdehyde (MDA) assay kit
微板法
Colorimetric method
缺点:操作步骤复杂
Disadvantages:omplexoperation steps
李娟等,2014;刘婷,2015;华秋翰,2016;Chen et al,2018
TBA法
TBA method
LDH
Lactate dehydrogenase
乳酸脱氢酶
测定试剂盒
Lactate dehydrogenase assay kit
比色法
Colorimetric method
范兰兰2013;焦周光等,2016;Chen et al,2018
微板法
Colorimetric method
GSH-Px
Glutathione peroxidase
谷胱甘肽过氧化物酶
测定试剂盒Glutathione peroxidase (GSH-PX) assay kit
比色法
Colorimetric method
刘婷,2015;张世鑫等,2016
NO
Nitric xide
一氧化氮
测定试剂盒
Nitric xide (NO) assay kit
微板法
Colorimetric method
华秋翰,2016
硝酸还原酶法Nitrate reductase method
蛋白
组学
Proteomics
IL-6
Interleukin- 6
待测物与酶连接,观测酶
与底物的颜色反应,定量抗体或抗原
To observe the color reaction between enzyme and substrate, to quantify antibodies or antigens.

ELISA

Enzyme-linked immunosorbent assay
炎性损伤
Inflammatory injury
优点:既可以定性也能非常精确的定量;
操作简单;
Advantages:t can be qualitative as well as very precise quantitative
imple operation
Soukup and Becker,2001;Becker et al,2005;焦周光等,2016;Chen et al,2018
IL-8
Interleukin- 8
Perrone et al,2013;Chen et al,2018
IL-1β
Interleukin- 1
缺点:成本高
Disadvantage: xpensive
Shang et al,2017
TNF-α
Tumor ecrosis actor
焦周光等,2016
Cleaved caspase-3聚丙烯酰胺凝胶电泳分离的目的基因表达蛋白的检测
Detection of target gene expression proteins separated by polyacrylamide gel electrophoresis
Western
blotting
优点:成本低
Advantages: ow cost
刘婷,2015
Bcl-2
B-cell lymphoma-2
et al 2010;刘婷,2015
Bax
B-cell lymphoma-2-ssociated X
刘婷,2015
p53
Tumor suppressor gene of 53kDa
缺点:western blot更偏向于定性的检测,定量也只能是通过比较的半定量,误差大;操作复杂;
Disadvantages: estern blot is more inclined to qualitative detection, quantitative can only be semi-quantitative by comparison, with large error and complex operation;
刘婷,2015
转录
组学
Transcriptomics
TNF-α
Tumor ecrosis actor
荧光定量技术反转录PCR,Real-time PCR定量分析
Quantitative nalysis of eal-time PCR and everse ranscription PCR with luorescence uantitative echnology
q-PCR
Quantitative real time polymerase chain reaction
炎性损伤
Inflammatory injury
优点:灵敏度高
Advantages: igh sensitivity
焦周光等,2016
IL-6
Interleukin- 6
焦周光等,016
IL-8
Interleukin- 8
Perrone et al,2013;Chen et al,2018
Bax / Bcl-2
B-cell lymphoma-2/ B-cell lymphoma-2-ssociated X
刘婷,2015
氧化应激
及DNA
氧化损伤
Oxidative stress and DNA oxidative damage
羟基脱氧鸟苷
8-
8-Hydroxy-2 eoxyguanosine
经高效液相色谱分离后电化学检测器收集电信号定量检测
Quantitative detection of electrical signals collected by electrochemical detector after separation by high performance liquid chromatography
HPLC-ECD
High erformance iquid hromatography-lectrochemistry
DNA氧化损伤及其突变
DNA Oxidative damage and mutation
优点:快速、需样量少、检测范围宽和高灵敏度
Advantages: fast, less sample requirement, wide detection range and high sensitivity
袭著革,2003;王旗等,2005
缺点:存在酶解不完全、洗涤不彻底等问题干扰实验结果
Disadvantage: he problems of incomplete enzymatic hydrolysis and incomplete washing interfere with the experimental results
8-OHdG与酶连接,观测酶与底物的颜色反应,定量抗体或抗原
8-OHdG binds to enzymes, observes the color reaction between enzymes and substrates, and quantifies antibodies or antigens.
ELISA实验
Enzyme-linked mmunosorbent assay
优点:操作方便、灵敏度高、重复性好
Advantages: easy operation, high sensitivity and good repeatability
郑全美等,2002
缺点:存在交叉反应,导致测定值偏高,且不能精确定量
Disadvantage: here is a cross-reaction, which leads to a high value and can not be accurately quantified
32 P标记,层析分离,经放射自显影测定
32 P labeling, chromatographic separation and autoradiographic determination
32 P后标记法
32 P ost-labeling
优点:灵敏度高
Advantages: igh sensitivity
潘洪志等,2004
缺点:特异性低,且对操作人员有一定的放射性损害
Disadvantage: ow specificity and certain radioactive damage to operators
经色相色谱分离,质谱定性定量检测
Separation by hromatography and ualitative and uantitative etection by ass pectrometry
GC-MS
Gas Chromatography-ass pectrometer
优点:灵敏度较高且能精确的分析出8-OHdG的浓度
Advantages: igh sensitivity and accurate analysis of 8-OHdG concentration
梅素容等,2006;梅素容等,2007
缺点:成本较高
Disadvantage: igh cost
彗星实验
Comet assay
单细胞凝胶电泳实验,观测DNA迁移形貌
The migration morphology of DNA was observed by single cell gel electrophoresis
单细胞凝胶
电泳仪
inglecellgeleletrophoresis
优点:图片美观;结果更为直观
Advantages:eautiful picturesmore intuitive results
范兰兰,2013
缺点:成本较高
Disadvantage: igh cost
由表2可知,PM2.5主要的毒理机制为氧化损伤、炎性损伤及DNA氧化损伤及其突变,氧化损伤可进行体外细胞功能学实验和氧化应激实验,体外细胞功能学实验主要分为细胞存活率实验和细胞凋亡实验,细胞存活率实验分为MTT实验和CCK-8实验,MTT实验简单快捷,但是也存在着操作步骤复杂、不适合悬浮细胞活性检测等缺点。目前,CCK-8实验因其灵敏度高、对细胞毒性小等优点逐步替代MTT实验,但缺点是杂质较易染色,影响OD值。细胞凋亡实验的主要实验方法为流式细胞仪和荧光显微镜观察,优点是图片美观,缺点是操作步骤复杂。氧化应激实验主要以检测各类酶活性以及酶含量为目的,主要方法为分光光度法、比色法、微板法等,该类实验既可定量亦可定性,但是操作步骤复杂。炎性损伤主要从两个水平检测观察,一是从蛋白组学水平,二是从转录组学水平。ELISA和Western blotting是两个广泛应用的从蛋白组学水平上观察炎性损伤的实验方法,两个实验原理类似,都是利用抗原抗体结合的原理,但是ELISA既可定性又可精确定量,而Western blotting偏向于定性。转录组学观察炎性损伤主要利用q-PCR法,优点是灵敏度高。DNA氧化损伤及其突变主要以检测8-OHdG和彗星实验为主,8-OHdG是DNA氧化损伤中最常用的生物标志物,主要的检测方法为高效液相色谱分离后电化学检测器收集电信号定量检测、ELISA法、32P后标记法、GC-MS等方法,其中:高效液相色谱分离法快速、需样量少、检测范围宽和灵敏度高,但也存在酶解不完全、洗涤不彻底、干扰实验结果等缺点;ELISA法操作方便、灵敏度高,但也存在交叉反应,导致测定值偏高等问题;32P后标记法特异性低,且对操作人员有一定的放射性损害;GC-MS灵敏度较高且能精确分析出8-OHdG的浓度,但是成本较高。彗星实验可直接观测DNA迁移形貌,结果更直观,但成本较高。
4.2   氧化损伤
氧化损伤,是指PM2.5进入人体后,颗粒物本身或表面吸附物质通过反应如过渡金属Fenton的反应或干扰某些相关酶的功能,生成大量的活性氧ROS,包括·OH、H2O2、HO2-等,使机体处于氧化应激状态,可以引起细胞膜的脂质过氧化、蛋白质氧化或水解以及DNA的损伤,造成细胞损伤甚至凋亡et al,2011。
PM2.5中的可溶性组分和不可溶性组分不仅对细胞生长有极强的抑制作用,而且能诱导细胞产生大量活性氧,且可溶成分诱导的过氧化氢多于不可溶成分et al,2011。
氧化损伤在细胞自噬过程中的作用不容小觑。环境中除草剂和多环芳烃等污染物使得ROS水平增高,此时引起细胞自噬Moore,2008,导致溶酶体膜损伤,溶酶体区室结构的变化抑制其与包裹了损伤物质的自噬小囊泡的融合,同时使细胞中的水解酶类释放,又加剧细胞损伤Kiffin et al,2006。
4.3   炎性损伤
炎性损伤,是指PM2.5进入人体后,机体炎症相关因子基因转录水平增高,诱导炎症细胞产生大量的细胞因子或黏附因子,如、IL-6和和TNF-α等,使机体产生炎症反应,造成炎性损伤et al,2011。
翟文慧等2015测定了2013年北京城区PM2.5对A549的炎症损伤作用,发现随着PM2.5浓度的升高,IL-6、TNF-α表达水平明显增高;随着PM2.5干预时间的延长,IL-6、TNF-α表达水平亦明显增高。H et al2017于2013年收集了日本工业区和市区的PM2.5提取物,研究发现上述PM2.5的有机提取物可以刺激气道上皮细胞产生IL-6,并引起炎症反应。
4.4   DNA损伤及其突变
目前关于PM2.5对DNA的损伤机制主要分为氧化损伤机制和DNA加合物形成机制。
自由基氧化损伤机制: PM2.5诱导机体产生自由基的途径可分为以下几类:①吸附的重金属元素进入体内后释放转运金属离子,导致大量自由基生成;②PM2.5作用于肺泡上皮细胞和巨噬细胞等机体各类吞噬细胞后导致大量活性氧簇生成;③PM2.5→自身表面化学效应→自由基生成;④颗粒物本身含有丰富的自由基,每浓度多为1×1016×1017个自旋;⑤PM2.5导致细胞内Ca2+浓度升高或超载,体内Ca2+稳态失衡导致DNA降解、促进自由基生成;⑥PM2.5所含的PAHs在体内代谢产生羟基自由基、超氧阴离子2013。上述途径生成的自由基可引起DNA氧化应激损伤。
DNA加合物形成机制: PM2.5富集了多种具有“致畸、致癌、致突变”效应的物质,主要是PAHs,例如:苯并[b]荧蒽、苯并[k]荧蒽、苯并[a]芘、二苯并[ah]蒽、苯并[ghi]芘等,PAHs在体内生物转化为二氢二醇环氧苯并芘并与DNA亲和位点鸟嘌呤外环氨基端共价结合成BPDE-DNA加合物。另外PM2.5中也有一些金属成分可引起DNA形成加合物,例如Fe2+。DNA加合物改变DNA的结构,如不能及时修复,则诱导基因突变SchnelleKreis et al,2001;Danielsen et al,2009,活化癌基因抑制正常基因的表达,增加机体患癌的风险。
4.5   细胞凋亡
Liu et al2015研究了烹调油烟源性的PM2.5对原代胎儿肺泡Ⅱ型上皮细胞的影响,发现PM2.5能够引起肺泡Ⅱ型上皮细胞毒性作用,其机制可能与增强氧化应激损伤,通过内质网途径诱导细胞过度凋亡等有关。刘婷等2015发现太原市灰霾PM2.5可引起肺泡巨噬细胞发生氧化应激损伤,并引起细胞凋亡。
4.6   细胞自噬
细胞自噬又称型程序性细胞死亡,是细胞对持续性内外刺激的一种非损伤性应答反应,可将生理或病理引起破损的细胞器及蛋白质等大分子在单位膜包裹的囊泡中大量降解。当细胞出现氧化应激损伤,可出现线粒体DNA损伤及线粒体肿胀等线粒体损伤,从而导致自噬的发生。Deng et al2013用PM2.5干预人体外培养的A549细胞,观察到A549发生了自噬,随着PM2.5干预浓度和干预时间的增加,自噬相关的微管结合蛋白轻链3LC3明显积累,自噬相关蛋白Atg5和BeclinlmRNA的表达明显增多。Liu et al2015用PM2.5干预体外培养的BEAS-2B细胞,利用电子显微镜、免疫荧光染色及免疫印迹等方法确定了PM2.5能够诱导BEAS-2B细胞自噬,其机制可能与抑制PI3K/A/mTOR信号转导通路有关。
5   研究展望
PM2.5的健康效应越来越被重视,PM2.5毒理研究已取得不少成果,但仍存在一些不足:1体外毒理实验单一细胞培养的方式,难以代表组织、器官的真实状况,且很多实验细胞系为癌细胞系,不能代表正常组织的真实情况;2细胞间通过微环境的相互作用状况在单一细胞培养中不能实现,毒物进入机体后要经过肝脏的代谢转化才会体现毒性,现有实验方法不足以模拟体内真实情况;3PM2.5的组成因季节、区域和来源的不同而存在较大差异4PM2.5的成分较为复杂,PM2.5完全颗粒、单纯颗粒毒理差异尚不清楚,PM2.5多组分、单组分毒理差异仍有许多不确定性,PM2.5与污染物的致毒作用是协同还是拮抗尚不明确;5动物体内毒理实验多采用高剂量和急性染毒,很难真实反映实际暴露情况。
基于现有研究结果与归纳的研究不足,可以考虑在以下方面开展后续研究:发展和完善体外细胞毒理研究方法,开展多细胞共培养的体外毒理实验,更好地模拟人体组织、器官的真实暴露情况;关注不同人群,针对不同人群主要的活动场所,加强PM2.5时空分布和理化特征的研究,增加与人体健康息息相关的代表型室内环境(室内源)PM2.5的毒理研究,系统分析PM2.5在不同阶段、不同地点、不同浓度条件下对多细胞共存培养的毒理作用;研究PM2.5不同组分的单独致毒作用和多组分共同致毒作用机制,探讨PM2.5与污染物的联合毒理机制;急性染毒和慢性染毒相结合用于动物体内毒理实验,染毒剂量与人体真实暴露环境中PM2.5的呼吸剂量作参照,真实反映实际暴露情况,系统阐述PM2.5暴露对人群或个体健康的影响与毒理机制;验证PM2.5毒理作用机制的假说,开展PM2.5健康危害的干预研究,寻找能减弱或预防PM2.5健康危害的有效手段。
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稿件与作者信息
黄 虹
HUANG Hong
万雪莹
WAN Xueying
陈廷涛
CHEN Tingtao
邹长伟
ZOU Changwei
irishhjx@163.com
国家自然科学基金(41765009);南昌大学研究生创新专项资金(CX2018239)
National Natural Science Foundation of China (41765009); Postgraduate Innovation Special Fund project of Nanchang University (CX2018239)
出版历史
出版时间: 2019年9月19日 (版本1
参考文献列表中查看
地球环境学报
Journal of Earth Environment