| An increasing number of organic compounds widely used in consumer products are being defined as emerging pollutants,which have attracted considerable attention in environmental field.Triclocarban is a highly effective and broad-spectrum antibacterial reagent used for a wide range of raw materials for many necessities of life and medicine,such as shampoos,liquid soap and medical disinfectants.The potential ecological risks of triclocarban are caused by its anti-biodegradability,high persistence and the environmental ubiquity.Under the influence of the COVID-19 epidemic,the accumulation of a large amount of medicine results in the greater risks of triclocarban to the environment,ecosystem and human health.Previous studies have shown that TCC is easy to migrate from water to soil environment,and can bioaccumulate through food chain.At the same time,TCC has a long half-life in soil environment,and can induce various toxic effects in soil animals,such as earthworms.However,oxidative stress is an important index to study toxic effects of pollutants,and the oxidative stress effect of TCC has not received widespread attention.Currently,the toxic effects of TCC are only dose-dependent studies at the individual level,and a small amount of oxidative stress damage has been reported.However,the mechanism of TCC inducing oxidative stress effect and functional inhibition of antioxidant enzyme activity in earthworm coelom cells at the cellular and molecular levels has not been clarified.In order to better understand the oxidative stress effect of TCC and enrich the research level,the comprehensive toxic mechanism of TCC can be better analyzed.Based on the above research progress,this study selected earthworm coelomic cells,two typical antioxidant enzymes superoxide dismutase(SOD)and catalase(CAT),and indirect antioxidant enzyme lysozyme(LZM)as the research objects at the cellular and molecular levels to reveal the mechanism of oxidative damage and toxicity of TCC.The research content mainly includes the following five parts:The first chapter discussed the sources of TCC in the environment and the background of toxicology research,introduced the research reports on oxidative stress toxicity of TCC,and summarized the current wide application fields of TCC,suggesting that its potential hazards to ecosystem and human health still deserve attention.According to the current research progress on the toxic effects of TCC,the key scientific problems to be solved in the field of research were proposed,and the main content and research value of this study were introduced.In second chapter,an in vitro experimental model at the cellular level was constructed to detect the oxidative damage indicators in earthworm coelomic cells after exposure to TCC with different particle sizes.The viability of earthworm coelomocytes in the TCC treatment group decreased to 77.3%of the initial level,and the levels of intracellular ROS,GSH and MDA increased to varying degrees,confirming that TCC induced oxidative damage in earthworm coelomocytes.TCC significantly inhibited the activities of SOD(80.8%)and CAT(81.5%).However,we found that the antioxidant melatonin was able to abrogate the inhibitory effects of TCC on SOD and CAT activities,a phenomenon that suggests a potential mechanism by which triclocarban affects SOD/CAT activity by modulating oxidative stress.The third chapter constructed the molecular model of TCC binding directly to SOD and CAT.At molecular level,SOD activities was inhibited and CAT activity was enhanced after binding to triclocarban in vitro.Results confirmed that another key mechanism to interfere with SOD/CAT activity was the binding of SOD/CAT and triclocarban.In order to investigate the mechanism of the interaction between TCC and SOD and CAT on the structure and function of TCC,multi-spectroscopy,isothermal calorimetry(ITC)and molecular simulation techniques were comprehensively studied.The binding of TCC and SOD/CAT caused the SOD/CAT protein skeleton and secondary structure,aggregation behavior,as well as the micro-environment of aromatic amino acids were affected by triclocarban binding.ITC experiments demonstrated that triclocarban bound to SOD via electrostatic interactions at relatively moderate affinity,and triclocarban bound to CAT through van der Waals forces and hydrogen bonding with a higher-affinity.The substrate-binding sites of SOD could be occupied by triclocarban,leading to the inhibition of SOD activity.His 74,Asn 147 and Tyr 357 were exist in the binding pocket around the active center,indicating that augmentation of CAT activity was strongly related with the structure changes in the heme groups.Findings from our study comprehensively revealed the response mechanism of SOD/CAT under the regulation of triclocarban-induced oxidative stress and elucidated the interaction mechanism between the major antioxidases SOD/CAT and triclocarban.In fourth chapter,the dose-effect of TCC on LZM was investigated at the protein level.TCC has an effect on lysozyme residues near the active center,reorienting its amino acid residues in the less polar microenvironment.Meanwhile,the interaction of TCC with lysozyme induced the α-Helix structure of lysozyme and altered the local amide bond around C=O,indicating the disruption of the secondary structure of lysozyme after exposure to TCC.The increased α-Helix content promoted the binding of lysozyme to TCC and the formation of aggregates with larger particle sizes,indicating that the lysozyme protein scaffold and polypeptide chains were stretched.In addition,TCC can spontaneously bind to lysozyme with relatively high affinity through intermolecular interactions,including van der Waals forces and hydrogen bonds,an exothermic reaction.Further molecular analysis revealed that the TCC binding site is located near the active center of lysozyme and interacts with Ala 107,Arg 112,Asn 59,Gln 57,Ile 98,Trp 108,Val 109,and Asp 52,The interaction between TCC and the binding site of lysozyme and its substrate inhibited the effect of the core residue(Asp 52)on lysozyme activity,showing an antagonistic effect on the natural substrate.These structural changes can affect the conformation and microenvironment around the active site,leading to inhibition of molecular activity.This study reveals the response mechanism by which TCC interacts with lysozyme both structurally and functionally and establishes a model for the binding of lysozyme to TCC.The fifth chapter summarized the main experimental content of this paper,summarized the innovation points,put forward the shortcomings of the paper and looked forward to the future research direction.At the cellular level,this study explored the mechanism of oxidative stress-related toxicity induced by TCC in earthworm coelom cells,and confirmed that TCC can induce oxidative damage and inhibit the activity of antioxidant enzymes through antioxidant experiments.By simulating the direct combination model of TCC with SOD and CAT,the molecular mechanism of the effect of TCC on the activities of SOD and CAT was explored,and the effects of TCC on the structure and function were further discussed.Finally,by studying the interaction mechanism between TCC and LZM,the influence of TCC on the activity and conformation of indirect antioxidant enzymes LZM was explored from the molecular level,and the binding model of the interaction between TCC and SOD,CAT and LZM was established.In this paper,the oxidative stress toxicity of TCC was evaluated comprehensively at both cellular and molecular levels,which provided scientific reference for the study of toxic effects of TCC... |
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