Exploring The Toxicity And Mechanism Of Perfluorodecanoic Acid On Mouse Primary Nephrocyte And Related Proteins

Due to good physicochemical properties.perfluorinated compounds(PFCs)are widely used in textile,packaging,carpet,aguriculture and other fields.However,as persistent organic pollutants,PFCs have strong bioaccumlation.high stability and can withstand strong light,chemical and microbial decomposition,which is very harmful to the environment,organisms and human body.PFCs can cause a series of toxic effects after entering the human body,which has been reported by many studies on animal,cell and molecular levels.Studies have found that the PFCs with longer carbon chain have higher toxicity.Perfluorodecanoic acid(PFDA)is a kind of PFCs with a long carbon chain and its toxicity can not be ignored.This paper evaluated the toxicity and mechanism of oxidative damage and apoptosis induced by PFDA in mouse primary renal cells at the cellular level.The binding mode of PFDA with key oxidative stress enzyme SOD,anti-apoptotic enzyme LYZ,and transport protein HSA.as well as the influence of PFDA on the structures and functions of the three proteins.were also discussed.This article is mainly divided into the following six parts.In the first part,the pollution,toxicity of PFCs and the main techniques used in this paper are briefly introduced.The content and significance of the research are also introduced.In the second part,the toxicity and mechanism of PIFDA on mouse primary renal cells were evaluated by cell viability test,intracellular ROS production test and cell apoptosis test.The results show that with the increase of PFDA concentration,the toxicity of PFDA increased gradually,the generation of ROS increased obviously.When PFDA concentration was 400 ?M,the content of ROS increased to 3.2 times.Excessive ROS accumulated in the cells could not be cleared,which had toxic effects on the renal cells,ultimately reducing cell viability and leading to apoptosis.In addition,there was a significant dose-effect relationship between total apoptotic cells and early apoptotic cells and PFDA concentration.In the third part,the key enzyme of oxidative stress,superoxide dismutase(SOD),was selected as the research object.Based on spectroscopy and thermodynamics,the influence of PFDA on the structure and function of lysozyme was discussed through the determination of enzyme activity and molecular simulation under simulated physiological conditions.The results show that PFDA can spontaneously bind to SOD driven by Van der Waals' forces and intermolecular hydrogen bond forces.The binding site is located on the molecular surface of SOD,and the carboxyl group of PFDA bonds with Val-A98 by forming a hydrogen bond with 3.11 A bond lengths.As the concentration of PFDA increased,the structure of SOD skeleton began to shrink and the secondary structure changed greatly.When the molar ratio of PFDA to SOD was 8:1.the content of ?-Sheet decreased to 34.6%,which led to the unfolding of SOD molecules.The surfactant properties of PFDA induced fluorescence photosensitization effect of SOD.but it does not change the microenvironment around tyrosine in SOD.In the fourth part,the binding mechanism of PFDA to lysozyme was explored on the basis of spectroscopy and thermodynamics,and by enzyme activity assay and molecular simulation under simulated physiological conditions.The results suggest that the binding of PFDA and lysozyme is a spontaneous interaction driven by Van der Waals' forces and intermolecular hydrogen bond forces,with a binding constant(K)of 1.14 × 104 M-1.This interaction results in the tightening of polypeptide chains,decreases the particle size and reduces the hydrophobicity of the microenvironment around aromatic amino acids in lysozyme.Meanwhile,the addition of PFDA greatly reduces the content of a-helix and changes the structure of lysozyme.Molecular simulation suggests that PFDA prefers to bind to Ser-50 and Trp-62 in the enzyme activity center by forming hydrogen bonds with 2.76 A and 2.68 A bond lengths,respectively.The competitive effect between PFDA and substrate at the active site and the change of lysozyme structure lead to a significant reduction of lysozyme activity.Moreover,the fluorescence sensitization of lysozyme arises from the surfactant property of PFDA and its binding to Trp-62In the forth part,the toxic mechanism of perfluorodecanoic acid(PFDA)on human serum albumin(HSA)was explored,interaction mode of PFDA with HSA was established and a new strategy for the toxicity evaluation of PFDA on the functional protein was provided.The binding of PFDA and HSA forms a complex and PFDA induces the fluorescence sensitization of HSA.Meanwhile.PFDA gives rise to a slight impact on the polypeptide chain and the microenvironment around Trp-214 from the spectroscopic angle.Moreover.the relative esterase activity oF HSA depressed by more than 60%upon 4 × 10-4 M PFDA.The thermodynamic parameters ?G.?H and AS were-7.2683 kcal·mol-1,-2.718 × 104 ± 0.5308 × 104 cal·mol-1 and-66.8 cal·mol-1·K-1.which evidenced a spontaneous interaction in which Van der Waals' force and intermolecular hydrogen bond are the predominant driven force.In addition,molecular simulation was applied to determine the specific binding site,which revealed PFDA most preferably binds with Tyr-411 by hydrogen bond and the distance between hydroxyl of PFDA and Tyr-411 is 2.64 A.in accordance with the conclusion of thermodynamic analysis and HSA functional analysisIn the sixth part,the toxic effect and mechanism of PFDA on the three proteins were compared by combining the experimental results of each part.Due to the surfactant properties of PFDA,the fluorescence of the three proteins showed enhancement trends;PFDA reduced the mobility of the microenvironment around Trp in HSA and increased its hydrophobicity.but had little effect on the fluorophore microenvironment in SOD and LYZ;In terms of molecular skeleton structure,PFDA has a greater influence on LYZ than SOD and HSA;PFDA has a great influence on the secondary structure of SOD and LYZ,but has no obvious influence on the secondary structure of HSA;Under experimental conditions.PFDA had little effect on SOD enzyme activity,but significantly inhibited the activity of LYZ and HSA,especially at high concentrations.The reason for this phenomenon is that PFDA binds to the active site of LYZ and HSA,but the binding site of PFDA and LYZ is located on the surface of LYZ,rather than the active center.