两类含氟污染物转运过程中毒性作用机理的研究
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摘要
近年来,随着新型环境污染物的提出,含氟污染物作为其中重要的一大类正逐步引起国内外的重视。在众多的含氟污染物中,有两类因其自身的特点,受到较高的关注。一类是全氟化合物(PFAAs),被广泛地应用于工业生产和生活消费领域。它们的难降解性,存在持久性和强生物蓄积性,已对全球范围的环境和生态系统造成了污染,并对包括人类在内的生态系统中的各种生物构成了潜在的威胁。另一类是氟喹诺酮类药物(FQs),作为一种人用和兽用药物,由于该类药物在体内的不完全吸收和传统水处理方法的弊端,使得该类药物的原形化合物及其代谢产物进入各种水体、土壤等环境介质中。长期富集后的药物残留物通过食物链影响动植物和微生物的正常生命活动,对生物体造成严重的毒性胁迫,进而对人类健康造成不良影响。
这两类外源化合物经呼吸道、消化道和皮肤接触等途径进入生物体后,可能引发机体各组织和器官的损伤,导致机体代谢紊乱。这些污染物进入机体后首先与体液接触,通过循环系统进行运输,在这些过程中污染物会与组织液、血液中的蛋白和酶分子发生相互作用,诱发蛋白的结构和功能变化,进而引起细胞损伤。蛋白质是生物体各种功能的直接执行者,而细胞是生物体结构和功能的基本单位,因此,从蛋白质分子和细胞两个层面建立这两类含氟污染物转运过程毒性微观机制的研究是十分必要的。
本论文在已有的研究结果的基础上,以分析化学和环境毒理学为背景,选取了具有代表性的全氟化合物(全氟戊酸PFPA、全氟辛酸PFOA和全氟癸酸PFDA)和氟喹诺酮类药物(环丙沙星CPFX和恩诺沙星ENFX),1、从分子和细胞两个层面考察了这两类含氟污染物转运过程中的毒性作用机理及同类含氟污染物同系物之间的毒性作用规律。论文主要包括以下五个部分：
论文第一章简要介绍了新型环境污染物的概念,阐明了全氟化合物和氟喹诺酮类药物这两类含氟污染物的使用现状,概述了这两类污染物的具体环境分布特点及污染现状,对目前两类含氟污染物展开的毒性研究进展和毒性评价方法进行了具体介绍。在文献综述的基础上,分析了目前这两类含氟污染物毒性评价中存在的问题,提出了在体外条件下从分子和细胞两个层面建立两类含氟污染物的转运过程毒性的评价方法。
论文第二章以转运过程中的胞外蛋白为靶分子,从分子水平上评价了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)分别对牛血清白蛋白(BSA)的毒性作用机理及同系物之间的作用规律,并基于FQs的药理学特性,采用光谱学方法考察了CPFX和ENFX对转运过程的非特异性免疫蛋白溶菌酶的毒性作用。
1)利用荧光光谱、同步荧光光谱、圆二色谱等光谱学手段和热力学计算评价了不同碳链长度的三种全氟化合物(PFPA、PFOA和PFDA)对BSA的毒性作用规律。从荧光光谱和圆二色谱的结果可以发现,PFPA对BSA的结构影响不大,而PFOA和PFDA均通过氢键和范德华力与BSA结合,使BSA的内源荧光发生了明显的猝灭作用,并使蛋白的骨架变得松散。其中,碳链较长的PFDA对BSA的毒性作用更强。
2)以BSA为靶分子,利用多种光谱学手段及分子模拟手段评价了ENFX及其主要代谢产物CPFX的毒性作用特点。研究发现,二者均能使BSA的内源荧光发生明显的猝灭作用,并生成了FQ-BSA复合物。热力学公式计算表明,CPFX和ENFX均通过静电作用与BSA发生结合,并且CPFX与BSA之间的作用力要大于ENFX。利用分子对接模拟技术进一步确定CPFX (ENFX)与BSA的结合位置位于BSA位点Ⅱ区的正电荷氨基酸残基区域。而紫外可见吸收光谱和圆二色谱的研究结果表明,CPFX和ENFX使BSA的二级结构和氨基酸残基周围的微环境发生了改变。
3)溶菌酶作为体液内重要的非特异性免疫蛋白,参与机体内的多项免疫反应。摄入后的污染物会对其结构和功能的表达造成影响。因此,本部分从分子水平探讨了两种具有药理特性的CPFX和ENFX对溶菌酶结构的影响。光谱学手段结合分子模拟技术的研究结果表明,两种FQs均与溶菌酶活性中心附近的Trp62和Trp63氨基酸残基发生了作用,诱发溶菌酶的荧光发生了明显的猝灭。通过热力学常数计算分析得出,它们之间的结合力主要为氢键和范德华力,并且ENFX的结合要大于CPFX,此结论在分子模拟结果中得到了验证。另外,结合的FQs均使酶分子的二级结构和色氨基酸残基的微环境发生了改变。
论文第三章采用光谱学结合分子模拟手段,以转运过程中几种胞内蛋白为研究对象,分别探讨了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)对牛血红蛋白(BHb)的毒性影响机制及规律。另外,从分子层面考察了CPFX和ENFX对细胞内的抗氧化酶(过氧化氢酶CAT和铜锌超氧化物歧化酶Cu/ZnSOD)的结构和功能影响。
1)通过多种光谱学手段,研究了PFPA、PFOA和PFDA与BHb的毒性作用机理。结果表明,PFPA和PFOA对BHb荧光光谱的影响很小,而PFDA能够使BHb的荧光发生增敏作用。通过紫外可见吸收光谱和圆二色谱可以发现,PFDA使BHb的结构发生了改变,并伴随着血红素的暴露和高铁血红素的形成,从而使血红蛋白的传输氧能力下降。由此可见,随着碳链增长,PFAAs对BHb的毒性作用增强。
2)光谱学手段与分子模拟技术的研究结果表明,CPFX和ENFX都通过静电力与BHb发生相互作用并形成复合物,而FQs与BHb的结合位置位于BHb四个亚基之间形成的空穴中。其中,CPFX与BHb的结合作用要大于ENFX。这种结合作用诱导了BHb的结构和氨基酸残基微环境发生改变,骨架变得松散。
3)通过多种光谱学手段、酶活性评价和分子模拟技术,研究了CPFX和ENFX对CAT结构和功能的影响。结果发现,两种FQs均可以通过静电作用与CAT发生结合作用形成复合物,并且二者分别与CAT结合时只有一个结合位点,均位于CAT分子中心的疏水腔内。两种FQs改变了CAT的二级结构,从而导致CAT活性中心附近的微环境发生了改变,进而对CAT的活性产生了抑制作用,且CPFX对CAT的结构和功能影响要明显大于ENFX。
4)以Cu/ZnSOD作为靶分子,采用荧光光谱、时间-分辨荧光光谱、紫外可见吸收光谱、同步荧光光谱、圆二色谱、酶活性分析和分子模拟技术,考察了CPFX和ENFX对Cu/ZnSOD毒性作用机理。FQs与Cu/ZnSOD之间有较强的相互作用,通过氢键与范德华力与酶分子1：1结合。FQs的结合作用诱发了酶分子的构象改变,空间结构变得松散,但由于结合部位对活性中心的影响相对较小,所以Cu/ZnSOD的酶分子活性变化不大。
论文第四章以人血红细胞为研究对象,通过体外染毒,从细胞水平分别考察了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)对红细胞抗氧化能力的影响,探讨了两类含氟污染物诱发红细胞氧化应激效应的毒性作用规律。另外,结合第三章中抗氧化酶分子的实验结果,通过比较分子和细胞水平的酶活性指标,探讨了CPFX和ENFX诱发红细胞的氧化应激机制与抗氧化酶分子结构和功能改变之间的相关性。
1)通过测定红细胞中氧化应激的五种生物学指标(GSH、MDA、CAT、SOD和GSH-Px)发现,PFPA、PFOA和PFDA诱发红细胞发生了氧化应激作用,表现为GSH含量的降低,MDA含量随着染毒浓度的增加而增大,而CAT、SOD、GSH-Px的酶活性在低染毒浓度下变化不大,随着作用浓度的增加,酶活性相应受到抑制,同时随着碳链长度的增加,这五种指标的变化程度逐渐增大,这说明PFDA的毒性作用要明显大于PFOA和PFPA,与分子水平的考察结果相一致。
2)从CPFX和ENFX对人血红细胞的抗氧化能力的考察中发现,CPFX和ENFX均能影响红细胞的抗氧化能力,表现为GSH含量随着染毒浓度的增加而下降,MDA水平呈现上升的趋势,其中,CPFX的毒性作用要大于ENFX。而CAT、SOD和GSH-Px的酶活性在低浓度染毒剂量下表现为诱导促进效应,而高浓度下表现为抑制效应,并且具有统计学显著性特点。
3)通过比较分子和细胞水平上关于两种FQs对抗氧化酶(CAT和Cu/ZnSOD)活性的影响规律发现,细胞实验中的酶活性抑制现象与分子水平的考察结果一致,说明了分子水平的酶结构和功能改变也可以从一定程度反映出细胞中的抗氧化酶的变化程度,验证了分子毒性实验的可信度。另外,基于抗氧化酶在细胞防御系统的重要作用,并且酶结构决定其功能的表达,结合光谱表征的蛋白构象分析,可以判定,FQs对两种抗氧化酶结构和功能的影响与其诱发细胞的氧化应激效应机制之间存在密切的联系。这为氧化应激机制的研究提供了新的思路,有利于疾病的早期诊断、预防和治疗。
论文第五章对各部分的研究结果进行归纳总结,并分析了两类含氟污染物的转运过程毒性评价方法的优势和不足,对未来的研究方向进行了展望。本论文从分子和细胞两个层面对两大类含氟污染物转运过程中的毒性作用机理进行了系统的评价,丰富了环境污染物体内转运过程中毒性通路的评价方法,具有很强的参考借鉴价值。通过探讨和比较全氟化合物以及氟喹诺酮类药物同系物间的毒性作用规律,为认识和评估这两大类含氟污染物的环境风险提供了新的方法和基础数据,为相关疾病的早期诊断、预防和治疗提供了科学的判定依据。
In recent years, the fluorine containing pollutants, as one representative class of the new emerging contaminants have attracted much concern for their global distribution and potential threats to the environment and humans. Among these many fluorinecontaining compounds, there are two types, which are broadly exposed in environment, leading to efforts to better understand the hazards that may be inherent in these compounds. One is known as the perfluoroalkyl acids (PFAAs), which are widely used in consuming and industrial applications, ranging from surfactants and emulsifiers to textiles, carpets, and paper products. But these fully fluorinated hydrocarbons are exceedingly stable and resistant to degradation, extensive amounts of data have become available describing the concentrations of PFAAs in the environment, wildlife and human tissues in many different geographic locations throughout the world, posing potential threats to both ecosystems and humans. Another type of fluorine containing pollutants belongs to fluoroquinolones (FQs), which are frequently used in many human and veterinary applications. However, due to the incomplete metabolism and relative ineffectiveness of conventional water treatment technologies in removing them, they have recently been detected in wastewaters, surface and ground water and in drinking water as well. These residues in the environment could enter the body by food chain or diet, affecting the normal life of plants, animals and microorganisms in the ecosystem and even human health.
These two types of exogenous fluorine containing pollutants normally enter into the organism mainly by the respiratory, digestive and skin contact, leading to damage of the tissues and organs of the body, causing the metabolic disorders in body. They firstly contact with the body fluids, and then being transported through the circulatory system in body. In this process, exogenous contaminants have great potential to interact with the proteins and enzymes in the tissue fluids and blood, which could result in the alteration of protein's structure and function, and thus induce the damages to cells. Proteins are the essential components in the life with various physiological functions in vivo. Cells are the basic unit of biological structure and function. Therefore, it is of great importance to study the toxic micro-mechanism of these two fluorine containing pollutants during the transport process.
On the basis of the existing research, three representative PFAAs, perfluoropentanoic acid (PFPA), perfluoroctanoic acid (PFOA), and perfluorodecanoic acid (PFDA), and two FQs, ciprofloxacin (CPFX) and enrofloxacin (ENFX), are selected as the objects. We evaluated the toxic effects of there fluorine-containing pollutants during the transport process from the molecular and cellular perspectives. This paper was divided into five parts:
The first chapter briefly introduced the concept of the new emerging contaminants and described the environmental distribution and pollution characteristics. Besides, the advances and methods in the toxicity studies of PFAAs and FQs were also reviewed. Based on the literature review, the problems in the toxicity evaluation of these two fluorine containing pollutants were also analyzed and the new evaluation methods of exploring their toxic mechanism of these two types of contaminants during the transport process were established from the molecular and cellular level.
In the second chapter, the extracellular proteins were selected as the target molecules. The toxic effects of three PFAAs (PFPA, PFOA and PFDA) and two FQs (CPFX and ENFX) on bovine serum albumin (BSA) were evaluated by means of multiple spectroscopic and computational methods. Besides, for the pharmacological properties of FQs, the interactions between CPFX (ENFX) and the immune lysozyme were also investigated from the molecular level. The results were as follows:
1) The effects of three PFAAs on BSA were characterized by fluorescence spectroscopy, synchronous fluorescence spectroscopy, and circular dichroism (CD). On the basis of the fluorescence spectra and CD data, we concluded that PFPA had little effect on BSA. However, PFOA and PFDA exhibited remarkable fluorescence quenching, which was attributed to the formation of a moderately strong complex. Furthermore, the BSA conformation was slightly altered in the presence of PFOA and PFDA. These results indicated that PFAAs indeed impact the conformation of BSA, and PFAAs with longer carbon chains were more toxic, especially at lower concentrations.
2) We studied the effects of CPFX and ENFX exposure to BSA by several spectroscopic techniques and molecular docking. It could be concluded from the fluorescence spectra that the quenching effect of BSA by two FQs was mainly due to complex formation. The number of binding sites, the binding constants, the thermodynamic parameters and binding subdomain were measured, indicating that two FQs could spontaneously bind with BSA on subdomain ⅢA through electrostatic forces. Furthermore, the conformation of BSA was demonstrably changed in the presence of CPFX and ENFX.
3) We investigated the interaction mechanism between the two FQs and lysozyme by the spectroscopic and molecular docking methods. As shown in by the fluorescence spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of lysozyme, which was attributed to the formation of a moderately strong complex. Thermodynamic analysis indicated that van der Waals forces and hydrogen bonds were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore, data obtained by UV-vis absorption, synchronous fluorescence and CD suggested that both CPFX and ENFX could lead to the conformational and some microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two FQs had specific interactions with the residues of Trp62and Trp63.
In the third chapter, the toxicity during the transport process was determined from the intracellular proteins. Firstly, the mechanisms of three PFAAs and two FQs targeting to the molecule bovine hemoglobin (BHb) were evaluated using spectroscopic and computational methods. Secondly, the toxic effects of two FQs on the structure and function of antioxidant enzymes were also explored from the molecular level. Catalase (CAT) and copper-zinc superoxide dismutase (Cu/ZnSOD) were selected to determine their oxidative stress effects from the macromolecular perspective. It covered four sections:
1) We determined the effects of PFDA binding to BHb. Using fluorescence spectroscopy, we found that PFDA greatly enhanced the fluorescence intensity of BHb, while PFOA and PFPA have minimal effects on the fluorescence. UV-vis absorption spectroscopy showed that PFDA induced the unfolding of the hemoproteins accompanied by exposure of the heme pocket and facilitating the formation of hemichrome. Additionally, as shown by the CD data, PFDA altered the secondary structure of BHb.
2) The present study evaluated the binding mechanism of both CPFX and ENFX to BHb using the spectroscopic and docking methods. From fluorescence spectra, we found that both FQs could bind with BHb to form a complex mainly through electrostatic interactions, which was also verified by the molecular docking study. As shown by the synchronous fluorescence, UV-visible absorption and CD data, both CPFX and ENFX could lead to the conformational and microenvironmental changes of BHb, which may affect its physiological functions. The work is beneficial for understanding the biological toxicity of FQs in vivo.
3) The binding modes of two FQs (CPFX and ENFX) to the important antioxidant enzyme CAT were characterized by means of spectroscopic and molecular docking methods from the molecular level in vitro. Using fluorescence spectroscopy, both CPFX and ENFX could react with CAT to form a complex mainly through electrostatic forces with only one binding site. And the specific binding interactions were observed by molecular docking. On the basis of UV-vis absorption, synchronous fluorescence and CD data, the conformational and micro-environmental alterations were induced with the additions of two FQs. Furthermore, the activity of CAT molecule in vitro was inhibited with increasing concentrations of two FQs.
4) The non-covalent toxic interaction of two FQs with Cu/ZnSOD was investigated by the fluorescence spectroscopy, UV-vis absorption and CD spectroscopy at physiological pH7.4. Both FQs can interact with Cu/ZnSOD to form a complex mainly by van der Waals' interactions and hydrogen bonds with one binding site. The binding of two FQs can result in change of the micro-environment of tryptophan residues and the secondary structure of Cu/ZnSOD. The activity of Cu/ZnSOD was also slightly affected for the bound FQs.
In the fourth chapter, we investigated the three PFAAs and two FQs on the oxidative capacity of human erythrocytes at the cellular level. Five biomarkers were selected to evaluate the involvement of oxidative stress induced by the two fluorine containing pollutants. It was divided into three parts:
1) The oxidative stress effects of erythrocytes in the presence and absence of PFPA, PFOA and PFDA were examined. After incubated with the three PFAAs, the contents of GSH decreased and the contents of MDA increased with the addition of PFAAs. At lower doses of three PFAAs, the activity of CAT, SOD and GSH-Px increased. However, at higher doses, the PFAAs inhibited their activity. All these results confirmed that three PFAAs could induce the oxidative stress of erythrocytes. And PFDA posed more of a threat than the other two PFAAs.
2) The cellular tests were carried out to evaluate the effects of two FQs on the antioxidant capacity of erythrocytes. The results confirmed an enhanced oxidative stress in FQs treated erythrocytes from the depletion of GSH contents and increase of MDA. Besides, CPFX posed more of an oxidative threat than ENFX. The activity of CAT, SOD and GSH-Px were elevated at lower concentrations of FQs. When the erythrocytes were incubated with higher doses, the activity of three antioxidant enzymes showed a decline trend.
3) As mentioned in the third chapter, both FQs could result in the alterations of both structure and function of CAT and SOD. Meanwhile, the cellular data also confirmed that the activity of CAT and SOD were induced by CPFX and ENFX. The activity tests in the molecular study were consistent with the cellular activity measurements. All these results suggested that the structural and functional changes of CAT and SOD were closely associated with increased risk of oxidative stress induced by both FQs. The established methods in this work could help to comprehensively understand the oxidative stress induced cellular damage of other pollutants via antioxidant effects.
In the fifth chapter, the main findings in the above parts were concluded and the advantages as well as the weakness were also reviewed. We also provided some new ideas on exploring the toxicity of fluorine containing pollutants in the future. In this paper, a new method was established to evaluate the toxicity of pollutants from the functional macromolecular and cellular level, which could provide the methodological reference and technical support for the toxicity evaluation of other pollutants. In addition, this work sheds light on the relationship of the chain lengths and functional groups of PFAAs and FQs to their molecular toxicology in vitro. It will also complement studies on the environmental risk assessment of PFAA and FQ pollution.
这两类外源化合物经呼吸道、消化道和皮肤接触等途径进入生物体后,可能引发机体各组织和器官的损伤,导致机体代谢紊乱。这些污染物进入机体后首先与体液接触,通过循环系统进行运输,在这些过程中污染物会与组织液、血液中的蛋白和酶分子发生相互作用,诱发蛋白的结构和功能变化,进而引起细胞损伤。蛋白质是生物体各种功能的直接执行者,而细胞是生物体结构和功能的基本单位,因此,从蛋白质分子和细胞两个层面建立这两类含氟污染物转运过程毒性微观机制的研究是十分必要的。
本论文在已有的研究结果的基础上,以分析化学和环境毒理学为背景,选取了具有代表性的全氟化合物(全氟戊酸PFPA、全氟辛酸PFOA和全氟癸酸PFDA)和氟喹诺酮类药物(环丙沙星CPFX和恩诺沙星ENFX),1、从分子和细胞两个层面考察了这两类含氟污染物转运过程中的毒性作用机理及同类含氟污染物同系物之间的毒性作用规律。论文主要包括以下五个部分：
论文第一章简要介绍了新型环境污染物的概念,阐明了全氟化合物和氟喹诺酮类药物这两类含氟污染物的使用现状,概述了这两类污染物的具体环境分布特点及污染现状,对目前两类含氟污染物展开的毒性研究进展和毒性评价方法进行了具体介绍。在文献综述的基础上,分析了目前这两类含氟污染物毒性评价中存在的问题,提出了在体外条件下从分子和细胞两个层面建立两类含氟污染物的转运过程毒性的评价方法。
论文第二章以转运过程中的胞外蛋白为靶分子,从分子水平上评价了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)分别对牛血清白蛋白(BSA)的毒性作用机理及同系物之间的作用规律,并基于FQs的药理学特性,采用光谱学方法考察了CPFX和ENFX对转运过程的非特异性免疫蛋白溶菌酶的毒性作用。
1)利用荧光光谱、同步荧光光谱、圆二色谱等光谱学手段和热力学计算评价了不同碳链长度的三种全氟化合物(PFPA、PFOA和PFDA)对BSA的毒性作用规律。从荧光光谱和圆二色谱的结果可以发现,PFPA对BSA的结构影响不大,而PFOA和PFDA均通过氢键和范德华力与BSA结合,使BSA的内源荧光发生了明显的猝灭作用,并使蛋白的骨架变得松散。其中,碳链较长的PFDA对BSA的毒性作用更强。
2)以BSA为靶分子,利用多种光谱学手段及分子模拟手段评价了ENFX及其主要代谢产物CPFX的毒性作用特点。研究发现,二者均能使BSA的内源荧光发生明显的猝灭作用,并生成了FQ-BSA复合物。热力学公式计算表明,CPFX和ENFX均通过静电作用与BSA发生结合,并且CPFX与BSA之间的作用力要大于ENFX。利用分子对接模拟技术进一步确定CPFX (ENFX)与BSA的结合位置位于BSA位点Ⅱ区的正电荷氨基酸残基区域。而紫外可见吸收光谱和圆二色谱的研究结果表明,CPFX和ENFX使BSA的二级结构和氨基酸残基周围的微环境发生了改变。
3)溶菌酶作为体液内重要的非特异性免疫蛋白,参与机体内的多项免疫反应。摄入后的污染物会对其结构和功能的表达造成影响。因此,本部分从分子水平探讨了两种具有药理特性的CPFX和ENFX对溶菌酶结构的影响。光谱学手段结合分子模拟技术的研究结果表明,两种FQs均与溶菌酶活性中心附近的Trp62和Trp63氨基酸残基发生了作用,诱发溶菌酶的荧光发生了明显的猝灭。通过热力学常数计算分析得出,它们之间的结合力主要为氢键和范德华力,并且ENFX的结合要大于CPFX,此结论在分子模拟结果中得到了验证。另外,结合的FQs均使酶分子的二级结构和色氨基酸残基的微环境发生了改变。
论文第三章采用光谱学结合分子模拟手段,以转运过程中几种胞内蛋白为研究对象,分别探讨了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)对牛血红蛋白(BHb)的毒性影响机制及规律。另外,从分子层面考察了CPFX和ENFX对细胞内的抗氧化酶(过氧化氢酶CAT和铜锌超氧化物歧化酶Cu/ZnSOD)的结构和功能影响。
1)通过多种光谱学手段,研究了PFPA、PFOA和PFDA与BHb的毒性作用机理。结果表明,PFPA和PFOA对BHb荧光光谱的影响很小,而PFDA能够使BHb的荧光发生增敏作用。通过紫外可见吸收光谱和圆二色谱可以发现,PFDA使BHb的结构发生了改变,并伴随着血红素的暴露和高铁血红素的形成,从而使血红蛋白的传输氧能力下降。由此可见,随着碳链增长,PFAAs对BHb的毒性作用增强。
2)光谱学手段与分子模拟技术的研究结果表明,CPFX和ENFX都通过静电力与BHb发生相互作用并形成复合物,而FQs与BHb的结合位置位于BHb四个亚基之间形成的空穴中。其中,CPFX与BHb的结合作用要大于ENFX。这种结合作用诱导了BHb的结构和氨基酸残基微环境发生改变,骨架变得松散。
3)通过多种光谱学手段、酶活性评价和分子模拟技术,研究了CPFX和ENFX对CAT结构和功能的影响。结果发现,两种FQs均可以通过静电作用与CAT发生结合作用形成复合物,并且二者分别与CAT结合时只有一个结合位点,均位于CAT分子中心的疏水腔内。两种FQs改变了CAT的二级结构,从而导致CAT活性中心附近的微环境发生了改变,进而对CAT的活性产生了抑制作用,且CPFX对CAT的结构和功能影响要明显大于ENFX。
4)以Cu/ZnSOD作为靶分子,采用荧光光谱、时间-分辨荧光光谱、紫外可见吸收光谱、同步荧光光谱、圆二色谱、酶活性分析和分子模拟技术,考察了CPFX和ENFX对Cu/ZnSOD毒性作用机理。FQs与Cu/ZnSOD之间有较强的相互作用,通过氢键与范德华力与酶分子1：1结合。FQs的结合作用诱发了酶分子的构象改变,空间结构变得松散,但由于结合部位对活性中心的影响相对较小,所以Cu/ZnSOD的酶分子活性变化不大。
论文第四章以人血红细胞为研究对象,通过体外染毒,从细胞水平分别考察了三种全氟化合物(PFPA、PFOA和PFDA)和两种氟喹诺酮类药物(CPFX和ENFX)对红细胞抗氧化能力的影响,探讨了两类含氟污染物诱发红细胞氧化应激效应的毒性作用规律。另外,结合第三章中抗氧化酶分子的实验结果,通过比较分子和细胞水平的酶活性指标,探讨了CPFX和ENFX诱发红细胞的氧化应激机制与抗氧化酶分子结构和功能改变之间的相关性。
1)通过测定红细胞中氧化应激的五种生物学指标(GSH、MDA、CAT、SOD和GSH-Px)发现,PFPA、PFOA和PFDA诱发红细胞发生了氧化应激作用,表现为GSH含量的降低,MDA含量随着染毒浓度的增加而增大,而CAT、SOD、GSH-Px的酶活性在低染毒浓度下变化不大,随着作用浓度的增加,酶活性相应受到抑制,同时随着碳链长度的增加,这五种指标的变化程度逐渐增大,这说明PFDA的毒性作用要明显大于PFOA和PFPA,与分子水平的考察结果相一致。
2)从CPFX和ENFX对人血红细胞的抗氧化能力的考察中发现,CPFX和ENFX均能影响红细胞的抗氧化能力,表现为GSH含量随着染毒浓度的增加而下降,MDA水平呈现上升的趋势,其中,CPFX的毒性作用要大于ENFX。而CAT、SOD和GSH-Px的酶活性在低浓度染毒剂量下表现为诱导促进效应,而高浓度下表现为抑制效应,并且具有统计学显著性特点。
3)通过比较分子和细胞水平上关于两种FQs对抗氧化酶(CAT和Cu/ZnSOD)活性的影响规律发现,细胞实验中的酶活性抑制现象与分子水平的考察结果一致,说明了分子水平的酶结构和功能改变也可以从一定程度反映出细胞中的抗氧化酶的变化程度,验证了分子毒性实验的可信度。另外,基于抗氧化酶在细胞防御系统的重要作用,并且酶结构决定其功能的表达,结合光谱表征的蛋白构象分析,可以判定,FQs对两种抗氧化酶结构和功能的影响与其诱发细胞的氧化应激效应机制之间存在密切的联系。这为氧化应激机制的研究提供了新的思路,有利于疾病的早期诊断、预防和治疗。
论文第五章对各部分的研究结果进行归纳总结,并分析了两类含氟污染物的转运过程毒性评价方法的优势和不足,对未来的研究方向进行了展望。本论文从分子和细胞两个层面对两大类含氟污染物转运过程中的毒性作用机理进行了系统的评价,丰富了环境污染物体内转运过程中毒性通路的评价方法,具有很强的参考借鉴价值。通过探讨和比较全氟化合物以及氟喹诺酮类药物同系物间的毒性作用规律,为认识和评估这两大类含氟污染物的环境风险提供了新的方法和基础数据,为相关疾病的早期诊断、预防和治疗提供了科学的判定依据。
In recent years, the fluorine containing pollutants, as one representative class of the new emerging contaminants have attracted much concern for their global distribution and potential threats to the environment and humans. Among these many fluorinecontaining compounds, there are two types, which are broadly exposed in environment, leading to efforts to better understand the hazards that may be inherent in these compounds. One is known as the perfluoroalkyl acids (PFAAs), which are widely used in consuming and industrial applications, ranging from surfactants and emulsifiers to textiles, carpets, and paper products. But these fully fluorinated hydrocarbons are exceedingly stable and resistant to degradation, extensive amounts of data have become available describing the concentrations of PFAAs in the environment, wildlife and human tissues in many different geographic locations throughout the world, posing potential threats to both ecosystems and humans. Another type of fluorine containing pollutants belongs to fluoroquinolones (FQs), which are frequently used in many human and veterinary applications. However, due to the incomplete metabolism and relative ineffectiveness of conventional water treatment technologies in removing them, they have recently been detected in wastewaters, surface and ground water and in drinking water as well. These residues in the environment could enter the body by food chain or diet, affecting the normal life of plants, animals and microorganisms in the ecosystem and even human health.
These two types of exogenous fluorine containing pollutants normally enter into the organism mainly by the respiratory, digestive and skin contact, leading to damage of the tissues and organs of the body, causing the metabolic disorders in body. They firstly contact with the body fluids, and then being transported through the circulatory system in body. In this process, exogenous contaminants have great potential to interact with the proteins and enzymes in the tissue fluids and blood, which could result in the alteration of protein's structure and function, and thus induce the damages to cells. Proteins are the essential components in the life with various physiological functions in vivo. Cells are the basic unit of biological structure and function. Therefore, it is of great importance to study the toxic micro-mechanism of these two fluorine containing pollutants during the transport process.
On the basis of the existing research, three representative PFAAs, perfluoropentanoic acid (PFPA), perfluoroctanoic acid (PFOA), and perfluorodecanoic acid (PFDA), and two FQs, ciprofloxacin (CPFX) and enrofloxacin (ENFX), are selected as the objects. We evaluated the toxic effects of there fluorine-containing pollutants during the transport process from the molecular and cellular perspectives. This paper was divided into five parts:
The first chapter briefly introduced the concept of the new emerging contaminants and described the environmental distribution and pollution characteristics. Besides, the advances and methods in the toxicity studies of PFAAs and FQs were also reviewed. Based on the literature review, the problems in the toxicity evaluation of these two fluorine containing pollutants were also analyzed and the new evaluation methods of exploring their toxic mechanism of these two types of contaminants during the transport process were established from the molecular and cellular level.
In the second chapter, the extracellular proteins were selected as the target molecules. The toxic effects of three PFAAs (PFPA, PFOA and PFDA) and two FQs (CPFX and ENFX) on bovine serum albumin (BSA) were evaluated by means of multiple spectroscopic and computational methods. Besides, for the pharmacological properties of FQs, the interactions between CPFX (ENFX) and the immune lysozyme were also investigated from the molecular level. The results were as follows:
1) The effects of three PFAAs on BSA were characterized by fluorescence spectroscopy, synchronous fluorescence spectroscopy, and circular dichroism (CD). On the basis of the fluorescence spectra and CD data, we concluded that PFPA had little effect on BSA. However, PFOA and PFDA exhibited remarkable fluorescence quenching, which was attributed to the formation of a moderately strong complex. Furthermore, the BSA conformation was slightly altered in the presence of PFOA and PFDA. These results indicated that PFAAs indeed impact the conformation of BSA, and PFAAs with longer carbon chains were more toxic, especially at lower concentrations.
2) We studied the effects of CPFX and ENFX exposure to BSA by several spectroscopic techniques and molecular docking. It could be concluded from the fluorescence spectra that the quenching effect of BSA by two FQs was mainly due to complex formation. The number of binding sites, the binding constants, the thermodynamic parameters and binding subdomain were measured, indicating that two FQs could spontaneously bind with BSA on subdomain ⅢA through electrostatic forces. Furthermore, the conformation of BSA was demonstrably changed in the presence of CPFX and ENFX.
3) We investigated the interaction mechanism between the two FQs and lysozyme by the spectroscopic and molecular docking methods. As shown in by the fluorescence spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of lysozyme, which was attributed to the formation of a moderately strong complex. Thermodynamic analysis indicated that van der Waals forces and hydrogen bonds were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore, data obtained by UV-vis absorption, synchronous fluorescence and CD suggested that both CPFX and ENFX could lead to the conformational and some microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two FQs had specific interactions with the residues of Trp62and Trp63.
In the third chapter, the toxicity during the transport process was determined from the intracellular proteins. Firstly, the mechanisms of three PFAAs and two FQs targeting to the molecule bovine hemoglobin (BHb) were evaluated using spectroscopic and computational methods. Secondly, the toxic effects of two FQs on the structure and function of antioxidant enzymes were also explored from the molecular level. Catalase (CAT) and copper-zinc superoxide dismutase (Cu/ZnSOD) were selected to determine their oxidative stress effects from the macromolecular perspective. It covered four sections:
1) We determined the effects of PFDA binding to BHb. Using fluorescence spectroscopy, we found that PFDA greatly enhanced the fluorescence intensity of BHb, while PFOA and PFPA have minimal effects on the fluorescence. UV-vis absorption spectroscopy showed that PFDA induced the unfolding of the hemoproteins accompanied by exposure of the heme pocket and facilitating the formation of hemichrome. Additionally, as shown by the CD data, PFDA altered the secondary structure of BHb.
2) The present study evaluated the binding mechanism of both CPFX and ENFX to BHb using the spectroscopic and docking methods. From fluorescence spectra, we found that both FQs could bind with BHb to form a complex mainly through electrostatic interactions, which was also verified by the molecular docking study. As shown by the synchronous fluorescence, UV-visible absorption and CD data, both CPFX and ENFX could lead to the conformational and microenvironmental changes of BHb, which may affect its physiological functions. The work is beneficial for understanding the biological toxicity of FQs in vivo.
3) The binding modes of two FQs (CPFX and ENFX) to the important antioxidant enzyme CAT were characterized by means of spectroscopic and molecular docking methods from the molecular level in vitro. Using fluorescence spectroscopy, both CPFX and ENFX could react with CAT to form a complex mainly through electrostatic forces with only one binding site. And the specific binding interactions were observed by molecular docking. On the basis of UV-vis absorption, synchronous fluorescence and CD data, the conformational and micro-environmental alterations were induced with the additions of two FQs. Furthermore, the activity of CAT molecule in vitro was inhibited with increasing concentrations of two FQs.
4) The non-covalent toxic interaction of two FQs with Cu/ZnSOD was investigated by the fluorescence spectroscopy, UV-vis absorption and CD spectroscopy at physiological pH7.4. Both FQs can interact with Cu/ZnSOD to form a complex mainly by van der Waals' interactions and hydrogen bonds with one binding site. The binding of two FQs can result in change of the micro-environment of tryptophan residues and the secondary structure of Cu/ZnSOD. The activity of Cu/ZnSOD was also slightly affected for the bound FQs.
In the fourth chapter, we investigated the three PFAAs and two FQs on the oxidative capacity of human erythrocytes at the cellular level. Five biomarkers were selected to evaluate the involvement of oxidative stress induced by the two fluorine containing pollutants. It was divided into three parts:
1) The oxidative stress effects of erythrocytes in the presence and absence of PFPA, PFOA and PFDA were examined. After incubated with the three PFAAs, the contents of GSH decreased and the contents of MDA increased with the addition of PFAAs. At lower doses of three PFAAs, the activity of CAT, SOD and GSH-Px increased. However, at higher doses, the PFAAs inhibited their activity. All these results confirmed that three PFAAs could induce the oxidative stress of erythrocytes. And PFDA posed more of a threat than the other two PFAAs.
2) The cellular tests were carried out to evaluate the effects of two FQs on the antioxidant capacity of erythrocytes. The results confirmed an enhanced oxidative stress in FQs treated erythrocytes from the depletion of GSH contents and increase of MDA. Besides, CPFX posed more of an oxidative threat than ENFX. The activity of CAT, SOD and GSH-Px were elevated at lower concentrations of FQs. When the erythrocytes were incubated with higher doses, the activity of three antioxidant enzymes showed a decline trend.
3) As mentioned in the third chapter, both FQs could result in the alterations of both structure and function of CAT and SOD. Meanwhile, the cellular data also confirmed that the activity of CAT and SOD were induced by CPFX and ENFX. The activity tests in the molecular study were consistent with the cellular activity measurements. All these results suggested that the structural and functional changes of CAT and SOD were closely associated with increased risk of oxidative stress induced by both FQs. The established methods in this work could help to comprehensively understand the oxidative stress induced cellular damage of other pollutants via antioxidant effects.
In the fifth chapter, the main findings in the above parts were concluded and the advantages as well as the weakness were also reviewed. We also provided some new ideas on exploring the toxicity of fluorine containing pollutants in the future. In this paper, a new method was established to evaluate the toxicity of pollutants from the functional macromolecular and cellular level, which could provide the methodological reference and technical support for the toxicity evaluation of other pollutants. In addition, this work sheds light on the relationship of the chain lengths and functional groups of PFAAs and FQs to their molecular toxicology in vitro. It will also complement studies on the environmental risk assessment of PFAA and FQ pollution.
引文
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