【作者】 孙宁；

【导师】 高灿柱； 刘汝涛；

【作者基本信息】 山东大学 ， 环境工程（专业学位）， 2022， 硕士

【摘要】 芘(Pyr)是存在于石油烃中的一种多环芳香烃,在生产染料、杀虫剂、合成树脂以及增塑剂领域应用广泛。近年来,随着石油需求增长,其开采、运输以及使用过程导致大量的芘进入土壤并造成环境污染。检测数据表明在石油烃污染过的土壤中芘的浓度不容小觑。芘也先后被美国、欧洲、日本等国家环境保护局列为“十六种优先控制多环芳烃”。土壤作为大多数污染物最终的“汇”,对其中的生物也存在着不可估量的危害和潜在隐患。并且这些持久性有机物经过食物链传递最终富集至人体内,对人类健康造成威胁。为深入探究芘的毒性和致毒机理,本研究以土壤指示生物蚯蚓为研究对象,系统地研究芘对蚯蚓个体、组织器官、细胞、大分子蛋白的一系列氧化应激相关的影响并探索生物毒理学效应机制。本研究在探索芘诱发蚯蚓氧化损伤的同时,尝试搭建一条跨生物层级的氧化应激毒性通路。实验从个体、细胞、分子三个层面展开,探究芘暴露诱导蚯蚓氧化损伤的精准剂量-效应关系、疏导各层级间致毒机制的逻辑通路。以表观毒性为切入口,逐渐深入至微观层面,结合分子模拟技术探索致毒机理。此外,为验证氧化应激毒性损伤的猜测,在细胞和分子层面探索了非酶抗氧化剂维生素C的保护作用和机理。第一章介绍了多环芳烃芘的性质、来源以及研究进展,从相关基础研究和氧化应激的末端毒性效应分析芘对蚯蚓的毒性作用以及生物体内的抗氧化防御体系。第二章主要研究了土壤中的芘对蚯蚓个体、组织器官以及细胞的影响,从芘对蚯蚓生长繁殖、肌肉形态、细胞内氧化应激水平等方面展开探索。个体、组织器官水平的研究结果表明,芘的暴露提高了蚯蚓的死亡率,抑制了体重增长和产卵率。当芘浓度为600 mg/kg时,蚯蚓的精囊和纵肌出现明显的萎缩。细胞水平的研究结果表明,芘能够诱导细胞膜损伤并增加Ca2+内流,这些引发了线粒体膜去极化和活性氧水平激增。当染毒28 d时,900 mg/kg芘暴露下的蚯蚓死亡率为91.67%,其DNA的橄榄尾距(OTM)达到15,这些数据表明芘在蚯蚓体内诱发的氧化应激对细胞内的蛋白质、脂质和DNA都造成了严重的损伤。第三章主要研究了芘诱发蚯蚓体腔细胞内的氧化应激效应以及维生素C对蚯蚓的保护作用。检测发现芘引起了蚯蚓体腔细胞活力的显著降低和严重的氧化损伤。细胞内持续高水平的活性氧抑制了抗氧化系统中酶的活性,在活性氧超出空白组3倍多时,过氧化氢酶活性仅为正常值的61.11%。氧化损伤在损害细胞内蛋白质的同时对脂质也造成了不可逆的伤害。然而,维生素C的加入在很大程度上维持了细胞内活性氧水平并抑制脂质过氧化的发生,这对调节细胞内的氧化还原平衡和缓解毒性效应起到了很好的保护作用。相比较而言,维生素C对脂质的保护作用强于对蛋白质的保护。第四章主要在分子层面上探索了芘与直接抗氧化酶过氧化氢酶和超氧化物歧化酶的相互作用以及维生素C的保护机理。结合光谱技术和计算机模拟技术建立了作用模型,探究了芘、酶、维生素C三者间的作用机理。荧光和圆二色光谱测试表明芘会改变过氧化氢酶中氨基酸残基的微环境并增加二级结构中的α-螺旋的占比。分子模拟结果表明,芘在过氧化氢酶活性中心附近的主要结合力是氢键力。此外,芘对过氧化氢酶活性的抑制率可以达到空白组的69.9%,但维生素C的加入可以更好的维持过氧化氢酶的活性。同时,芘主要通过改变酪氨酸Tyr 108的微环境和二级结构来抑制超氧化物歧化酶的活性。因此,直接抗氧化酶的功能减弱可能是细胞内活性氧过度增加的根本原因。第五章节是本研究的主要结论、创新点以及展望,对日后更全面的研究提出设想。本研究有助于我们深入理解芘对生态环境潜在的毒性威胁以及在生物体内的作用机理,研究中探索的毒性作用途径将为后续的毒理学研究提供科学思路与基础信息支撑。更多还原

【Abstract】 Pyrene(Pyr)is a kind of polycyclic aromatic hydrocarbon in petroleum hydrocarbons.In addition,due to the leakage of petroleum in the production and exploitation process,a large number of Pyr into the soil and cause environmental pollution.According to the investigation,the concentration of Pyr in petroleum hydrocarbon contaminated soil can not be ignored.Pyr is also on the list of 16 priority PAHs issued by the U.S.Environmental Protection Agency.As the final "sink" of most of the pollutants,soil also has immeasurable harm hidden danger to the living things in it,and affect human health and safety ultimately.Therefore,starting from earthworms,the soil organism most easily exposed to petroleum hydrocarbon pollution,this study systematically studied a series of effects of Pyr on earthworms,animals and organs,cells and macromolecular proteins,and explored the biotoxicological effectmechanism.In this study,oxidative damage of Pyr on earthworms was explored,and the oxidative stress toxicity pathway was established.Toxicity experiments were carried out from animal,cellular and molecular levels to analyze the relationship between toxicity dose-response relationship,and to explore the logical pathway of toxicity mechanism.At the same time,the protective effect and mechanism of non-enzymatic antioxidant vitamin C were investigated at molecular and cellular levels.In the first chapter,the properties,sources and research progress of Pyr were introduced.The toxicity of Pyr to earthworms and the antioxidant defense ability of earthworms were analyzed from various basic studies.The main technical methods involved in the experiment and the research significance are introduced.In the second chapter,the effects of Pyr on animal,tissues,organs and cells of earthworms were studied systematically from the aspects of oxidative stress effect,gene damage and oxidative stress toxicity pathway.Experimental results show that Pyr can increase the mortality of earthworms and inhibit weight gain and oviposition.At the same time,when the Pyr concentration reached 600 mg/kg,the seminal vesicle and longitudinal muscle of earthworm showed obvious atrophy.At the cellular level,Pyr induces cell membrane damage and increases Ca2+ influx,which triggers mitochondrial membrane depolarization and reactive oxygen species levels surge.When the mortality rate of earthworm was 91.67%,the olive tail motion(OTM)of earthworm DNA reached 15.It means the oxidative stress caused by Pyr in earthworms caused serious damage to proteins,lipids and DNA.In chapter 3,the oxidative stress effect of Pyr on coelomic cells of earthworm and the protective effect of vitamin C on oxidative damage induced by Pyr were studied.It was found that Pyr caused significant decrease in cell viability and severe oxidative damage in the coelom of earthworms,and decrease in the activities of direct antioxidant enzymes catalase and superoxide dismutase.The catalase activity was only 61.11%of the normal value when reactive oxygen species were more than 3 times of the blank group.Oxidative damage not only damages proteins in cells but also causes irreversible damage to lipids.However,the addition of vitamin C significantly improved the level of intracellular ROS and lipid peroxidation,which played a good protective role in regulating the intracellular redox balance and alleviating the toxic effects.In contrast,vitamin C has a stronger protective effect on lipid than on protein activity.In the fourth chapter,the interaction between Pyr and macromolecular proteins was studied.Catalase and superoxide dismutase can protect organisms.In order to reveal the potential effect of Pyr on CAT enzyme and the protective effect of vitamin C,multi-spectral technology and molecular docking simulation technology were used to establish the interaction model and explore the interaction mechanism.Fluorescence and circular dichroism analysis showed that Pyr induced microenvironmental changes in CAT amino acid residues and increased α-helix in secondary structure.Molecular simulation results show that the main binding force of Pyr around the active center of CAT is hydrogen bonding force.Compared with the blank group,the inhibition effect of Pyr on catalase activity could reach 69.9%,but the addition of vitamin C could better maintain the original catalase activity.Meanwhile,Pyr inhibits Cu/Zn-superoxide dismutase activity mainly by changing the microenvironment and secondary structure of Tyr 108.Therefore,the reduced function of direct antioxidant enzymes may be the root cause of the excessive increase of intracellular ROS.The fifth chapter is the main conclusions,innovations and prospects of this study.This study will help us to have an in-depth understanding of the potential toxic threat of Pyr to the ecology and the mechanism of its application in organisms.The toxicity pathway explored in the experiment will provide scientific ideas and basic information support for subsequent toxicological research.更多还原