Effects And Mechanisms Of Oxidative Stress Induced By Naphthalene And Its Metabolites At Animal,Cellular And Molecular Levels

Polycyclic aromatic hydrocarbons(PAHs),as a kind of organic pollutants,exist widely in the environment and could be easy to bioaccumulate in organisms through the biological chain.Most PAHs are carcinogenic and mutagenic.Soil is an important "sink" of PAHs that can accumulate in soil through atmospheric deposition,sewage irrigation,wastewater seepage,and oil spillage in petroleum exploitation,transportation,refining and use process.Soil could also in turn serve as a "source" to transfer PAHs to water and atmosphere through surface runoff and evaporation.Naphthalene(NAP)is a typical environmental pollutant with the simplest structure and the highest solubility among PAHs,and has been classified as a Class 2B carcinogen.NAP has been more frequently detected d in PAHs-contaminated soil.The toxic effects induced by NAP were closely related to the metabolic transformation process in the organism.NAP could be primarily metabolized by cytochrome P450(CYP)family enzymes.The metabolic process could induce the organism to generate a large amount of reactive oxygen species(ROS)and break the balance of the organism’s own redox,resulting in inflammation,cellular oxidative damage,carcinogenic effects,and genotoxicity.At present,the toxicological evaluation related to NAP metabolism mainly use mammals as experimental models.Evaluation of the toxic effects of soil organisms is critical in early-warning and environmental ecological risk assessment for soil pollution,but there is currently a lack of animal and cellular level studies on the toxic effects of NAP and its metabolites on soil organisms,and the dose-response relationship and potential toxic mechanisms of oxidative stress have not been clarified.NAP and its metabolites might bind to important functional proteins in vivo,which further aggravated the toxic effects.However,the mechanisms of interaction between NAP and metabolites with antioxidant enzymes and DNA molecules remain unclear.In view of the above problems,we selected NAP,1naphthol(1-NAO)and 1,4-naphthoquinone(1,4-NQ)as exposure substances to investigate the related toxic effects and potential toxic mechanisms of NAP and metabolites on oxidative stress at animal,cellular and molecular levels.The paper mainly including the following parts:The first chapter briefly described the properties and main sources of NAP,the pollution and distribution of NAP in various environmental media.This chapter summarized the metabolic process in vivo,biological toxicity and potential mechanism of NAP.The research progress of oxidative stress induced by NAP and its metabolites,and the combination of pollutants and biological macromolecules in the evaluation of toxic effects were also summarized.Based on the literature review,the key scientific problems were put forward,and the research purpose and contents were determined according to the scientific problems.In the second chapter,the toxic effects of NAP and its metabolites(1-NAO and 1,4-NQ)on oxidative stress in earthworms were studied at animal level.The contents of lipid peroxidation products(Malondialdehyde,MDA),activities of antioxidant enzymes and DNA damage index(8-Hyoxy-2-deoxyguanosine,8-OHdG)in earthworms at different exposure periods were evaluated.Changes in the tissue structure of earthworms were also observed.By integrating biomarker response information,the oxidative stress status of the tested earthworms at low lethal levels was comprehensively measured,and the differential toxicity and ecological risks of NAP and metabolites were systematically evaluated.The results indicated that NAP and metabolites exposure disturbed the stability of antioxidant enzyme system in earthworms and the activities of SOD,CAT,POD and GST all changed differently,which resulted in the accumulation of lipid peroxides and the damage of earthworm tissue structure.In addition,NAP did not induce significant oxidative damage to DNA,while the 1NAO and 1,4-NQ caused significant damage to DNA through oxidative stress.The comprehensive toxicity of NAP and its metabolites at animal level was 1,4-NQ>1-NAO>NAP.At the initial stage of NAP and metabolites,the antioxidant system of earthworms responded positively to the stimulation,but the ability of earthworms to resist stimulation decreased resulting in obvious oxidative damage with the prolongation of exposure time.In the third chapter,the oxidative stress-related toxic effects of NAP and its metabolites on coelomocytes were studied at the cellular level.Cell viability was measured to compare the cytotoxicity of NAP and its metabolites.Cell membrane damage,oxidative stress response and mitochondrial damage were also evaluated to explore the potential mechanisms.NAP exposure did not significantly affect the cell membrane permeability of coelomocytes,which was manifested in that the activity of cell membrane protein Na+K+-ATPase was inhibited but the release of LDH did not increase significantly.The antioxidant system of coelomocytes for NAP exposure responded,in which SOD activity was activated,CAT activity was inhibited and the GSH content significantly decreased.NAP exposure did not cause the accumulation of ROS and MDA,and obvious damage to mitochondria.WVhile,1NAO and 1,4-NQ exposure inhibited the activity of Na+K+-ATPase,promoted the release of LDH and altered the permeability of cell membrane.The responses of the antioxidant systems SOD,CAT,and GSH were not sufficient to scavenge the excess ROS generated by the exposure of NAP metabolites resulting in the accumulation of MDA and significant mitochondrial damage that was manifested in decreased mitochondrial membrane potential and increased intracellular Ca2+ levels.The cytotoxicity of NAP and its metabolites was NAP<1-NAO<1,4-NQ.In the fourth chapter,we selected CAT and SOD as research object to explore the molecular mechanisms of oxidative stress induced by NAP and metabolites.The thermodynamic and binding parameters of the interaction of NAP and its metabolites with CAT and SOD were determined by isothermal titration calorimetry(ITC).The effects of NAP and its metabolites on the structure of antioxidant enzymes were analyzed by various spectroscopic methods.The interaction model and potential binding sites were identified by molecular simulation technology.The effect of NAP and its metabolites on the function of antioxidant enzymes was evaluated by enzyme activity assays.At the same time,the genotoxicity mechanisms of NAP and its metabolites at the molecular level were discussed by a variety of spectroscopy and molecular simulation techniques with DNA as the research object.Based on a comprehensive study at the molecular level,the interaction mechanisms of NAP and its metabolites with related functional proteins and DNA were also explained.(1)Hydrophobic force was the key binding force of NAP and 1-NAO to CAT,while hydrogen bond and hydrophobic force played a synergistic role in the binding of 1,4-NQ to CAT.NAP had little effect on the skeleton structure of CAT,but still caused changes in the secondary structure.1-NAO and 1,4-NQ changed the skeleton and secondary structure of CAT and quenched the endogenous fluorescence of CAT statically and dynamically,respectively.The interaction model of NAP and its metabolites with CAT showed that NAP preferentially bound to the surface of CAT,while 1-NAO and 1,4-NQ preferentially bound to the active center.In conclusion,the changes in the structure of CAT at the molecular level and the difference in the mode of action between the above toxicants and CAT had different effects on the function of CAT.NAP and 1,4-NQ directly inhibited CAT activity,while 1NAO activated its enzyme activity.Therefore,it was inferred that the alterations in CAT activity at the animal and cellular levels were mediated by free radicals,structural changes of CAT,and differences in intermolecular interaction mechanisms.(2)Hydrophobic force was synergistic with hydrogen bond in the binding of 1-NAO to SOD,while the hydrophobic force was dominant in the binding of 1,4-NQ to SOD.NAP,1NAO,and 1,4-NQ resulted in the loosening of SOD skeleton structure and no obvious change in secondary structure.The binding sites were all far away from the active center.Changes in SOD skeleton structure at the molecular level did not cause significant alterations in SOD enzyme activity.It was inferred that the alterations in SOD activity at animal and cellular levels were mainly mediated by the accumulation of free radicals.(3)Both covalent and non-covalent binding modes played an important role in the binding of NAP and DNA,while the binding of 1-NAO and 1,4-NQ to DNA were dominated by groove binding in the non-covalent mode enhancing by hydrogen bonding.1-NAO and 1,4-NQ were hydrogen-bonded with cytosine(C)and guanine(G),respectively.NAP had little effect on the phosphate skeleton structure of DNA,while 1-NAO and 1,4-NQ caused the axial contraction and conformation change of DNA polydeoxynucleotide chain.While,NAP and its metabolites had little effect on the base accumulation and right-hand helicity of DNA.The fifth chapter summarized the research including the conclusions,the innovations,the shortcomings,and prospects.In this paper,oxidative stress-related toxic effects and mechanisms of earthworms induced by NAP,1-NAO and 1,4-NQ were systematically studied at the animal,cellular and molecular levels.The interaction models of NAP and its metabolites with biological macromolecules were also established.The mechanisms of interaction of NAP and its metabolites with important functional proteins and DNA were elucidated,providing technical support for strengthening the risk control of PAHs and formulating scientific and rational management and quality strategies.