Exploring Mechanisms Of Oxidative Damage And Cell Apoptosis Induced By Lead Combined At The Molecular, Cellular And Organismal Levls

The type and usage of lead-containing products are growing rapidly with the fast development of modern industry, which results in serious environmental pollution. Lead still possesses great threats to human health owing to its widespread distribution in the environment caused by human activities, although various actions have been taken to cut down the use and distribution of lead by the governments. Lead can enter the human body mainly through the respiratory, gastrointestinal and skin tracts after widely contacted with water, air and food chains containing lead, causing cognitive dysfunction, hematological disorders, neurological damage, renal impairment and immunological pathologies.Oxidative stress is defined as an imbalance of oxidant and antioxidant status of cells and tissues altered by oxidants, and abundant evidence has indicated oxidative stress is a trigger of many varied diseases. Multiple studies have shown that lead toxicity is related to oxidative stress because it generates reactive oxygen species (ROS), interferes with antioxidant enzyme activities, and breaks the balance of the pro-oxidant/antioxidant defense system, resulting in oxidative damage of proteins, nucleic acids and lipid compounds, cell apoptosis and necrosis, and metabolic disorders of tissues and organs of humans, and causes harmful diseases or cancers. However, mechanisms of oxidative damage caused by lead remain unknown. Therefore, it is of great importance to study mechanisms of oxidative damage caused by lead combined at the molecular, cellular and organismal levels to understand harmful effects of lead exposure to human health.This thesis aims to study mechanisms of oxidative damage caused by lead combined at the organismal, cellular and molecular levels with the knowledge of molecular and cellular toxicology and instrument analysis, which includes the following five chapters:The first chapter briefly introduced toxic effects of lead after entered into human bodies, and the relationship between lead toxicity and oxidative stress. Then we reviewed response of several biomarkers of oxidative stress, including anti-oxidant enzymes, GSH/GSSG ratios and lipid peroxidation, after lead exposure at the experimental animal level, relationship between cell apoptosis and lead-induced oxidative damage at the cellular level, and oxidative damage of proteins and DNA induced by lead at the functional molecular level. At last research problems in the toxic evaluation of lead at these three levels were analyzed according to literature reviews above, and new methods to investigate mechanisms of oxidative damage and cell apoptosis caused by lead combined at molecular, cellular and organismal levels were also established.Chapter two studied molecular mechanisms of oxidative stress in zebrafish livers induced by lead, and part of this work has been published in the Journal of Physical Chemistry B. Results are listed below:(1) Zebrafish were exposed to lead (0,10 and 50 mg/L) for 24 h in 1.5-L containers at 28 ℃. Liver homogenates of zebrafish were used to measure biomarkers of oxidative stress, including contents of reduced glutathione (GSH) and oxidized glutathione (GSSG); the related enzyme activities of glutathione reductase and glutathione peroxidase and changes in lipid peroxide and antioxidant enzyme activities of catalase and superoxide dismutase. Lead exposure resulted in a decrease of GSH level and an increase of GSSG content, down-regulations of glutathione reductase and glutathione peroxidase, an increase of malondialdehyde (MDA) content, and up-regulation of CAT activity and down-regulation of superoxide dismutase (SOD) activity, but little change in the enzyme contents. Results above provide sufficient evidence of oxidative stress in zebrafish livers after lead exposure.(2) Then we investigated and compared activities two important anti-oxidant enzymes (SOD, CAT) affected in lead-treated zebrafish livers and explored the mechanism of changes of CAT and SOD activities by lead at the molecular level using multiple spectroscopic techniques, isothermal titration calorimetric (ITC) measurement, molecular docking study and ICP-AES detection. Results on enzyme activities affected by lead showed the same trends in animal and molecular experiments, while the amount of catalase and SOD showed few changes. Results from Pb-SOD interactions showed lead exposure decreased SOD activities in zebrafish livers due to direct interactions between lead and SOD, resulting in conformational and functional changes of the enzyme. To be specific, Studies at the molecular level indicated that lead bound into the active site channel of SOD, hindered the path of the catalytic substrate (O2-·), damaged its skeleton conformation and secondary structure, and interacted with the enzymatically related residue (Arg 141) through electrostatic forces (△H<0, AS>0), and caused the release of Cu2+ and Zn2+ from the catalytic pocket of SOD. Moreover, results from Pb-CAT interactions showed that lead bound in the largest cavity of catalase (with a volume of 9650 A3), which is far away from catalytic areas of CAT, and should be a reasonable explanation of the up-regulation of catalase activity.Chapter three evaluated mechanisms of DNA damage induced by lead at the cellular and molecular levels, and this work has been published in Journal of Photochemistry and Photobiology B-Biology, which is shown below in detail:(1) The primary liver cells obtained from health male mice (C57BL6-J) were incubated in lead solutions under standard cell culture condition (37 ℃,5%CO2) for 24 h. Results from comet assay showed lead at 1-10 μM induced significant increase in the values of OTM,%tail DNA and tail length, which indicated that lead caused a dose-dependent increase in the frequency of DNA strand breaks in mice liver cells. However, Signals of DNA-protein crosslinks (DPC) were not significantly detected until exposed at 100 μM Pb2+, and the DPC coefficient reached μp to 12.6% in Pb2+ exposure at 100μM. Under low Pb2+(<10μM) exposure, we found significant increase in Olive tail moment of DNA compared with the control, but few changes in amount of DPCs, which can be inferred that generation of DPCs need aggravated DNA damage and considerable fragments of DNA, so DPC can be regarded as an indicator of DNA damage in severe levels.(2) Recent studies showed that lead could change the secondary structure of DNA by covalently binding with the oxygen atom of nucleic acid or nitrogen atom of base pairs of DNA. However, the direct binding mode between lead and DNA remains unclear. So direct interactions between Pb2+ and DNA were explored to determine the binding mode between them using spectra analysis, isothermal titration calorimetry studies and molecular docking investigations, which indicated that Pb2+ could bind to DNA with four binding sites to form Pb2+ -DNA complex by minor groove binding effects and electrostatic forces, resulting in damage to the structure of DNA double helix.In Chapter four, the primary hepatocytes and nephrocytes of mice were used to investigate effects of oxidative damage by lead on cell apoptosis and related signal pathways. Meanwhile, lysozyme and human chorionic gonadotropin (HCG) were selected as the anti-oxidant and anti-apoptotic proteins to study toxic effects of lead on the anti-oxidant system. Part of work in this chapter has been published in Journal of Luminescence and the Journal of Physical Chemistry B. Results are illustrated as follows:(1) Firstly, we detected and compared cell viabilities of primary hepatocytes and nephrocytes of mice after lead exposure in 6 h and 12 h using the Muse Cell Analyzer probed by cell viability & death reagent. Results showed that lead significantly decreased cell viabilities of hepatocytes and nephrocytes in the dose-dependent manner, but kidney cells were more sensitive that liver cells and presented higher death rate after lead incubation. In order to investigate mechamnism of cell damage caused by lead, the intracellular contents of ROS and GSH were measured after 12 h lead incubation using CM-H2DCFDA and NDA as fluorescence probes through cytometer and fluorescence microscope, respectively. Moreover, apoptotic status of hepatocytes and nephrocytes were detected using the Annexin V & 7-AAD cell agent by Muse Cell Analyzer. Results illustrated that lead caused decreased GSH/GSSG ratios both in the liver and kidney cells, and ROS contents remained stable in kidney cells, and presented greatly increase in liver cells, which indicated that liver cells suffered more serious oxidative damage than that in kidney cells. However, lead caused more percentages of apoptotic nephrocyte numbers than liver cells. So oxidative damage does not always accompanied by occurrence of cell apoptosis, the mechanism of which was further studied through activations of related apoptotic signal pathways. Activations of ERK and Caspase-3 were measured through Muse Cell Analyzer and microplate reader detector.(2) Lysozyme and HCG can ameliorate toxins-enhanced cell apoptosis and protect cell from oxidative damage, and were selected as the target proteins to study cytotoxic mechanism of lead. The activity of lysozyme was inhibited by the addition of lead acetate. The SERS results revealed that the environment of the tryptophan (Trp) residues became more hydrophobic. Synchronous fluorescence spectra confirmed that the conformation of lysozyme had been changed and both tyrosine and tryptophan residues were located in a more hydrophobic environment. The decreased lysozyme activity and fluorescence quenching results showed that lead acetate entered the largest pocket of lyoszyme, and affected the residues in this pocket, including the catalytic residues (Glu 35, Asp 52) and the most dominant fluorophores (Trp 62, Trp 108). The fluorescence measurements suggested different lead acetate species had varying influences on the micro-environment of lysozyme. In addition, the UV-vis spectra indicated the backbone of lysozyme was changed and circular dichroism showed the secondary structure of lysozyme displayed a decrease in a-helix and an increase in P-sheet with the increasing amount of lead acetate. Results of Pb-HCG system:fluorescence measurements showed that lead acetate dynamically quenched HCG fluorescence through collisional mechanism with the association constant (Ksv) in the magnitude of 103 L/mol at the detected temperatures (298 K,303 K and 310 K). ITC and molecular docking results revealed lead acetate could bind into 5 binding sites of HCG through electrostatic effects (△H<0, △S>0) and hydrophobic forces (△H>0, △S>0). The conformational investigation of HCG by UV-vis absorption spectroscopy, circular dichroism spectroscopy and ELISA indicated lead acetate changed the secondary structure of HCG by loosening and destruction of HCG skeleton and increasing the hydrophobicity around Tyr residues, and resulted in the decreased bioactivities of HCG.Chapter five summarized main studies and reviewed the advantages and highlights of mechanisms of lead toxicity on oxidative damage, and prospected for exploring lead toxicity in the future. In this thesis, mechanisms of oxidative damage and cell apoptosis after lead exposure have been systematacially studied combined at the macromolecular, cellular and organismal levels, which can diversify the toxic research on the evaluation of oxidative stress affected by environmental pollutants, and provide scientific references for early diagnosis and treatment of harmful deseases caused by environmental pollutants.