The Research Of Oxidative Stress-induced Effects Of4-aminoantipyrine And Its Mechanism

The pollutions caused by residues of drug and drug inermediates pose a threat to the eco-environmental safety and human health. As a raw material or intermediate,4-aminoantipyrine (AAP) has been widely used in the production of analgesic, anti-inflammatory, antibacterial drugs. Moreover, AAP can cause side effects, such as agranulocytosis. Thus, AAP has become an environmental pollutant exposed in the environment.Many studies have shown that oxidative stress is related with all diseases. The hazards of environmental pollutants on organisms are related with oxidative stress. In the process of oxidative stress, reactive oxygen species (ROS) in vivo largely express, participate in and promote the progression of many diseases, cause oxidative damages of biological macromolecules such as DNA/RNA, proteins and lipids, further lead to metabolic disorders of the body and cause diseases. Therefore, the evaluation of toxic effects of environmental pollutants from the point of view of oxidative stress will help people to understand the pathogenesis of environmental pollutants and evaluate on pollutant toxicity comprehensively. That can provide a scientific basis for early diagnosis, prevention and treatment of related diseases.This thesis has a background in environmental toxicology and modern instrumental analytical science. We studied the oxidative stress effects of AAP and its mechanism from three levels of experimental animals, single cell and protein macromolecules. This thesis includes the following five sections:In the first chapter, the characteristics and applications of AAP were described. The research progress of toxic effects of AAP was overviewed. The compositions and characteristics of ROS and antioxidant system were introduced. The inducing factors of environmental oxidative stress were reviewed. The relationships of oxidative stress and diseases were summarized. The analysis and evaluation methods of oxidative stress in environmental toxicology were summarized.In the second chaper, the zebrafish was used as the research target. Acute exposure experiments were carried out from experimental animal level. The AAP-induced half lethal concentration (LC50-24h) was determined. We investigated the effects of AAP on antioxidant system and oxidative damages in zebrafish liver and evaluated on the oxidative stress effects of AAP. The results showed that AAP caused an increase in CAT and GPx activities, a decrease in GR and GST activities and SOD activity did not change significantly. AAP also led to a significant reduction in GSH content, a increase in GSSG content and a significantly decrease in the ratio of GSH/GSSG. The redox equilibrium of liver tissue was broken, indicating that oxidative stress was formed. MDA content gradually increased, indicating that the product of oxidative damage (LOP) increased. The progress resulted in oxidative damages of adipose tissue.In the third chapter, we used human erythrocytes as research targets. The erythrocytes were exposed by AAP in vitro. We investigated the effects of AAP on GSH contents in single human erythrocytes from single cell level and evaluated on the oxidative stress effects of AAP. When cells were exposed in low AAP concentration, although the GSH average content was not significant changed, the single-cell analysis revealed that the cells of low GSH contents began to appear. When the AAP concentration increased, AAP had a significant impact on the GSH content. The number of cells with low GSH content increased. GSH contents reduced up to14.53%. GSH depletion induced the internal environmente of erythrocytes changed into oxidized state.In the fourth chapter, we used transport proteins in the blood (BSA and BHb) as research targets. The interactions of AAP and transport proteins were investigated from protein macromolecule level. The transmission and distribution of pollutants in the body was determined by the binding of pollutants and transport proteins. Based on multi-spectroscopy and molecular simulation techniques, we established the binding model and investigated the binding mechanisms.1) By using spectroscopy and molecular modeling methods, the interaction between AAP and BSA was investigated in simulated physiological conditions. The results showed that BSA was quenched by AAP significantly through a static quenching mechanism. There was a strong interaction between AAP and BSA. The positively charged AAP can spontaneously bind with the negatively charged region of BSA through electrostatic forces. AAP bound to BSA on the subdomain â…¢A. The microenrironment and conformation of BSA were demonstrably changed in the presence of AAP.2) With multiple spectroscopic techniques including fluorescence spectra, synchronous fluorescence spectra, UV-vis absorption spectra and CD spectra, the interaction mechanism of AAP and BHb was investigated. The results showed that there was a strong interaction between AAP and BHb. AAP bound to BHb through van der Waals interactions and hydrogen bonds with approximately one binding site. The microenvironment of BHb was changed by AAP and the skeletal structure of BHb loosened.In the fifth chapter, we selected three kinds of oxidative stress directly related enzymes (SOD, CAT and Hrp) as research targets. Based on spectroscopy and molecular modeling techniques, the mechanism of toxic effects of AAP on antioxidative enzymes was researched from molecular level. Since GPx was expensive and difficult to save, we used Hrp to simulate the GPx.1) The results of spectroscopic, molecular docking and enzyme activity assessment methods showed that AAP can spontaneously bind with Cu/ZnSOD to form AAP-Cu/ZnSOD complex with one binding site mainly through hydrogen bond and van der Waals forces. AAP bound into the Cu/ZnSOD interface of two subdomains. AAP triggered changes in the secondary structure of Cu/ZnSOD. The skeleton structure of Cu/ZnSOD loosened, exposing internal hydrophobic peptide strands to the solution. As the binding of AAP influenced the microenvironment of the activity sites, AAP led to the inhibition of Cu/ZnSOD activity.2) On the basis of spectroscopic and molecular docking results, the positively charged AAP can spontaneously bound with the negatively charged CAT with one binding site mainly through electrostatic forces. The results of molecular docking simulation revealed that AAP bound into CAT central cavity. AAP triggered changes in the microenvironment and conformation of CAT. The skeleton structure of CAT loosened, exposing internal hydrophobic peptide strands to the solution. As the binding of AAP influenced the microenvironment of the activity sites, AAP led to the inhibition of CAT activity.3) The interaction mechanism of AAP and Hrp was investigated by multiple spectroscopic techniques including fluorescence spectra, synchronous fluorescence spectra, UV-vis absorption spectra and CD spectra. We studied the binding parameters (association constants, number of binding sites, thermodynamic parameters and binding forces) of the interaction and the effect of AAP on the conformation of Hrp. There was a strong interaction between AAP and Hrp. AAP bound to Hrp through electrostatic forces with approximately one binding site. The microenvironment, secondary tructure and conformation of Hrp was changed by AAP and the skeletal structure of Hrp loosened.The research results from animal and molecular level showed that antioxidant enzyme activities were affected by two factors. First of all, the direct binding of the AAP and antioxidant enzymes would inhibit the antioxidant enzyme activities. Second, when oxidative stress was generated in tissue, antioxidant enzymes were in the environment of excessive expressed ROS. The substrate content in the catalytic reaction of antioxidant enzymes increased, which would stimulate antioxidant enzyme activity to increase. Third, AAP acute exposure would have an impact on antioxidant enzyme contents. The relative influence degree of these three factors finally determined the apparent changes of the antioxidant enzyme activities in animal experiments.In sixth chapter, finally, each section of the thesis was summarized. We analyzed the advantages and disadvantages of the evaluation method of oxidative stress induced by AAP in this thesis and discussed the future development of the field. The study provided more systematic and comprehensive evaluation results of AAP-induced oxidative stress effects from the perspective of molecular, cellular and experimental animal levels. This research has enriched the research on the evaluation method of oxidative stress effects of environmental pollutants, provided some reference gists for the toxicities and pathogenesis of environmental pollutantst.