大气中典型硝基多环芳烃的细胞毒性及作用机制研究

发布时间：2018-09-08 20:57

【摘要】：大量的流行病学和毒理学研究表明,大气颗粒物浓度的上升与心肺系统疾病发病率和死亡率增加显著相关,大气颗粒物污染已成为影响人类健康的主要环境因素之一。柴油尾气颗粒物、城市空气颗粒物及交通源颗粒物的毒性较大,主要与其表面含有大量的有毒有害物质有关,如过渡金属、有机物等。在健康影响中最为关注的有机物是多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)及其衍生物。研究发现硝基多环芳烃(nitro-PAHs)的毒性效应可能强于其母体多环芳烃。Nitro-PAHs不仅在大气颗粒物中被检出,汽车尾气、木材燃烧烟气颗粒物及大气气体样品中同样有检出。Nitro-PAHs具有急性毒性、免疫毒性和致癌毒性,在大气环境中广泛存在,具有潜在的健康风险。本研究以体外培养人肺上皮细胞A549为研究对象,探讨nitro-PAHs及其对应氨基代谢产物(amino-PAHs)的细胞毒理作用及其致毒机制,进一步明确nitro-PAHs对人体的有害影响,明确nitro-PAHs在大气颗粒物生物毒性方面的重要作用。同时探讨1-硝基芘(1-NP)与其它多环芳烃及多环芳烃衍生物的联合细胞毒性。本研究首先以两种大气中典型的硝基多环芳烃及其相应的氨基取代多环芳烃,包括1-硝基芘(1-NP)、3-硝基荧蒽(3-NF)、1-氨基芘(1-AP)、3-氨基荧蒽(3-AF)的氧化还原性质作为切入点,研究它们在细胞体系和非细胞体系的氧化还原能力。对参与氧化应激的超氧化物歧化酶(Superoxide Dismutase,SOD)的活性、谷胱甘肽(glutathione,GSH)的含量及活性氧自由基(Reactive Oxygen Species,ROS)的产生规律进行了探讨。结果表明1-NP、3-NF、1-AP、3-AF均可与二硫苏糖醇(DTT)在非细胞体系发生反应。均可促进胞内的ROS的生成,降低GSH活性,影响SOD的活性,导致细胞内氧化应激的产生,破坏胞内的氧化还原平衡。然后分别通过细胞增殖实验、凋亡率的检测、胞外乳酸脱氢酶(LDH)检测、线粒体膜电位检测、胞内钙离子(Ca2+)浓度变化检测、彗星实验、细胞周期/凋亡实验,系统地检测四种PAHs衍生物在一定剂量范围内对细胞生长、细胞凋亡和细胞周期、细胞膜通透性、DNA损伤、线粒体膜电位、胞内钙离子(Ca2+)浓度变化等的影响,确定时间-效应及剂量-效应关系。最后通过检测炎症因子(IL-6、MCP-1)、细胞色素P450酶(Cyp1a1、Cyp1b1、Cyp1a12、Cyp1b12)和血红素加氧酶(HO-1)的m RNA表达情况,来进一步讨论nitro-及amino-PAHs的促炎作用。结果表明1-NP、3-NF、1-AP、3-AF可促进炎症因子、细胞色素P450酶和血红素加氧酶m RNA的表达。除1-NP外,均可促进炎症因子IL-6和血红素加氧酶(HO-1)的表达,1-AP和3-AF可诱导至少一种细胞色素P450酶的表达。最后,评价1-NP分别与1,2-萘醌(1,2-NQ)和苯并[a]芘B[a]p对A549细胞的联合毒性作用。联合染毒的结果表明,1,2-NQ预染A549细胞24 h,能明显减弱1-NP造成的DNA损伤和ROS升高。表明,用1,2-NQ预处理A549细胞可能通过抑制产生ROS,而降低DNA损伤。B[a]p预染A549细胞24 h,能明显减弱1-NP造成的ROS升高,但DNA损伤加剧。B[a]p和1-NP同时作用于A549细胞,能明显减弱1-NP造成的ROS升高和细胞增殖抑制作用,DNA损伤与1-NP单独染毒组相近。表明,B[a]与1-NP联合作用可能通过抑制ROS生成降低对细胞的增殖抑制,对DNA的损伤加剧可能是通过其它的作用途径。本论文的研究结果表明nitro-PAHs可对细胞功能产生有害影响。不仅可以导致细胞凋亡、细胞膜通透性改变,还能对线粒体、DNA和Ca2+浓度等细胞内重要的细胞器和信号转导系统产生影响,可产生ROS和炎症因子。表明nitro-PAHs是大气颗粒物中对人体健康影响的重要组分。因此,在评估大气颗粒物对人体健康影响的过程中,除了大气颗粒物的粒径和浓度,还应注意化学物质组成的重要影响。
[Abstract]:A large number of epidemiological and toxicological studies have shown that the increase of atmospheric particulate matter concentration is significantly related to the increase of morbidity and mortality of cardiopulmonary diseases. Atmospheric particulate matter pollution has become one of the main environmental factors affecting human health. Diesel exhaust particulate matter, urban air particulate matter and traffic source particulate matter are more toxic, mainly. Polycyclic Aromatic Hydrocarbons (PAHs) and their derivatives are the most concerned organic compounds in health effects. Nitro-PAHs are found to be more toxic than their parent polycyclic aromatic hydrocarbons. Nitro-PAHs are not Nitro-PAHs have acute toxicity, immunotoxicity and carcinogenicity. They are widely present in the atmosphere and have potential health risks. In this study, human lung epithelial cells A549 were cultured in vitro to explore the effects of n-PAHs on human lung epithelial cells. The cytotoxicity and toxicity mechanism of itro-PAHs and their corresponding amino-PAHs were further clarified. The harmful effects of nitro-PAHs on human body were further clarified. The important role of nitro-PAHs in the biological toxicity of atmospheric particulates was also clarified. The combined cells of 1-nitropyrene (1-NP) with other polycyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbon derivatives were also discussed. Toxicity. In this study, two typical atmospheric NITRO-POLYCYCLIC aromatic hydrocarbons and their corresponding amino-substituted polycyclic aromatic hydrocarbons, including 1-nitropyrene (1-NP), 3-nitrofluoranthracene (3-NF), 1-aminopyrene (1-AP), 3-aminofluoranthracene (3-AF), were used as entry points to study their redox capacity in both cellular and non-cellular systems. The activity of superoxide dismutase (SOD), the content of glutathione (GSH) and the production of reactive oxygen species (ROS) were investigated. The results showed that 1-NP, 3-NF, 1-AP, 3-AF could react with dithiothreitol (DTT) in non-cellular system. Intracellular ROS production reduces GSH activity and affects SOD activity, leading to oxidative stress and disruption of intracellular redox balance. Cell cycle/apoptosis experiments were carried out to systematically examine the effects of four PAHs derivatives on cell growth, cell apoptosis and cell cycle, cell membrane permeability, DNA damage, mitochondrial membrane potential, and intracellular calcium (Ca2+) concentration in a certain range of doses to determine the time-effect and dose-effect relationship. The expression of cytochrome P450 (Cyp1a1, Cyp1b1, Cyp1a12, Cyp1b12) and heme oxygenase (HO-1) m RNA was studied to further discuss the pro-inflammatory effect of Nitro-and amino-PAHs. The results showed that 1-NP, 3-NF, 1-AP, 3-AF could promote the expression of inflammatory factors, cytochrome P450 and heme oxygenase m RNA. The combined toxicity of 1-NP with 1,2-naphthoquinone (1,2-NQ) and benzo [a] pyrene B [a] p, respectively, on A549 cells was evaluated. ROS was elevated in A549 cells pretreated with 1,2-NQ, indicating that ROS production might be inhibited by 1,2-NQ pretreatment, and DNA damage could be reduced by B[a]p pretreatment for 24 hours. The elevation of ROS induced by 1-NP could be significantly attenuated by B[a]p and 1-NP pretreatment. The results showed that the combination of B [a] and 1-NP may inhibit cell proliferation by inhibiting ROS production, and the DNA damage may be aggravated by other pathways. It can also affect important cellular organelles and signal transduction systems, such as mitochondria, DNA and Ca2+ concentration, and produce ROS and inflammatory factors. It shows that nitro-PAHs are important components of atmospheric particulate matter affecting human health. Concentration should also pay attention to the important influence of chemical composition.
【学位授予单位】：上海大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X513

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