Research On The Effect And Mechanism Of Earthworm Oxidative Stress Induced By Toluene And Benzoic Acid

In the production process of oil fields,the produced floor oil and oily sewage pose a serious threat to the soil environment.The pollutants of crude oil and petroleum-based processed products entering the soil are mostly hydrocarbons(alkanes,naphthenes and aromatic hydrocarbons),of which toluene It is a common aromatic hydrocarbon substance.Toluene and its oxidation product benzoic acid will cause a series of effects on soil organisms.Toluene and benzoic acid can enter the living body through channels such as inhalation of the respiratory tract,ingestion of the food chain through the digestive tract,and skin contact,causing damage to tissues and organs of the living body.Before entering a living body and reaching tissues and organs through tissue fluid and blood,toluene and benzoic acid will first contact and interact with proteins in tissue fluid and blood,and then interact with proteins in the target organ after reaching the target organ,and show certain Biological effect.Toluene has carcinogenic,teratogenic,mutagenic,reproductive toxicity,neurotoxicity,liver toxicity,blood toxicity and immunotoxicity;benzoic acid has neurotoxicity,blood toxicity,teratogenicity,and has certain damage to organs and tissues.Since the current research on toluene and benzoic acid tends to study the toxicity and pathogenic mechanism of human body,there is very little research on the toxicity of soil ecosystem organisms,and it is urgent to establish a new method and model of pollutant toxicity evaluation,and To provide technical support and theoretical reference for preventing the impact of pollution on soil biological health,and the previous studies on the toxic effects of pollutants on earthworms are mostly at the animal level,and the research on antioxidant enzymes is only focused on the description of pollutants on enzymes in the body The effect of apparent activity,and the reasons for the change in apparent activity were not further explored.Based on cytotoxicity and molecular toxicology,this paper evaluates the toxic effect and mechanism of oxidative damage induced by toluene and benzoic acid at the primary level of earthworm cells at the cellular level,and studies the key to oxidative stress at the molecular level The effects of the enzyme catalase(CAT)and superoxide dismutase(SOD)on the structure and function of the two proteins.The article is mainly divided into the following three partsIn the first part.selecting somatic cells of earthworms as target cells.selecting four important biomarkers(T-AOC,MDA.CAT,and SOD)to measure oxidative stress,and studying toluene-and benzoic acid-induced earthworms at the cellular level The mechanism of oxidative stress in somatic cells.The results show that.with the addition of toluene and benzoic acid,the total antioxidant capacity of somatic cells increased slightly compared with the control group.When the concentration of benzoic acid reached 6 × 10-5 M and the concentration of toluene reached 15 × 10-5 M.The antioxidant capacity is reduced and is lower than that of the control group without the addition of small molecules.We believe that the antioxidant system of somatic cells is not enough to cope with the oxidative stress caused by small molecules.reduce.With the increase of the concentration of toluene and benzoic acid,the amount of MDA produced in somatic cells showed an upward trend.Statistical significance.MDA results showed that toluene and benzoic acid induced oxidative stress in earthworm cells.CAT enzyme activity test results showed that the relative activity of CAT in the earthworm body cells under different concentrations of toluene exposure showed a tendency to rise slightly and then decrease,inhibiting the enzyme activity.Under different concentrations of benzoic acid exposure,the relative activity of CAT in earthworm body cells showed a slight first increase and then a slight decrease trend.There was no significant difference,indicating that benzoic acid had little effect on CAT enzyme activity.The SOD enzyme activity test results showed that the relative activity of SOD in earthworm cells under different concentrations of toluene exposure showed a slight increase and then a slight decrease trend,there was no significant difference,indicating that benzoic acid had little effect on CAT enzyme activity.The relative activity of SOD in earthworm body cells under different concentrations of benzoic acid exposure showed an upward trend,rising to 109.3%of the blank control group and was statistically significant.The activity of the enzyme is closely related to the structure,and the change of the enzyme structure is the main reason that affects its functional expression in the bodyIn the second part.the key enzyme of oxidative stress,CAT.was selected as the research object.Various methods of spectrometry,molecular docking and enzyme activity determination were used to explore the effects of toluene and benzoic acid on the structure and function of CAT.The results show that the interaction between toluene and benzoic acid and the CAT molecule has caused the microenvironment of the aromatic amino acid residues of the CAT molecule to be affected to a certain extent.The skeleton structure of CAT is loose,unfolding occurs,and the hydrogen bonding network of CAT is destroyed.It affects the secondary structure of the protein,causing misfolding and partial denaturation of the peptide chain.The docking results show that toluene interacts with Gln 167 and Asp 127 on the channel of the substrate into the CAT active center,preventing the substrate from smoothly entering the enzyme active center,resulting in a decrease in CAT enzyme activity;Therefore,it has little effect on enzyme activity,which is consistent with the results of enzyme activity measurement.In the third part,selecting the key enzyme of oxidative stress,SOD,as the research object,using a variety of spectroscopy,molecular docking,and enzyme activity determination methods to explore the effects of toluene and benzoic acid on the structure and function of SOD.The results showed that the interaction between toluene and benzoic acid and the SOD molecule both led to the loose structure of the SOD,and unfolding occurred,affecting the secondary structure of the protein,resulting in the wrong folding and partial denaturation of the peptide chain,but the SOD molecule aromatic amino acid The microenvironment in which the residues are located is not affected.The result of docking showed that toluene did not directly bind to the SOD active center,and it was bound at the interface of the two subunits of SOD.so it had little effect on enzyme activity,which was consistent with the results of enzyme activity measurement;the result of docking showed benzene Formic acid is more likely to collide and bind on the molecular surface of SOD.After the benzoic acid interacts with SOD,it induces the conformational change of SOD.The pocket near the active site of SOD increases,increasing the contact area of the substrate,which in turn leads to enzyme activity rise.