Research On The Toxicity Mechanism Of Two Types Of Fluorine Containing Pollutants During Their Transport Proces

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.