Research On The Mouse Hepatomegaly,Oxidative Stress And Related Effects Induced By Perfluorooctanoic Acid And Perfluorooctane Sulfonate

Due to the thermal stability,chemical stability and surfactant activity,perfluorinated compounds(PFCs)are widely used in various industrial processes and products,such as water and oil repellents,fire fighting foams,lubricants,surfactants and coatings.Among all PFCs,the most commonly used and most typical are perfluorooctanoic acid(PFOA)and perfluorooctane sulfonate(PFOS).Because carbon-fluorine bonds(C-F)are very stable,PFOA and PFOS are difficult to be naturally converted and degraded,which leads to their persistence in the environment and human body.The widespread use of PFOA and PFOS has raised concerns about their toxic effects and human health risks,and along the accumulation of the food chain,the toxicity of PFOA and PFOS can be amplified.After PFOA and PFOS enter the human body,they are distributed to various organs in the body through the blood circulation system,and cause multiple body damage.The liver is the largest detoxifying organ in the human body and is closely related to the occurrence and development of various diseases.Meanwhile,liver is also the main target organ for PFOA and PFOS,and PFOA and PFOS can cause hepatomegaly in mice.In addition,the oxidative stress effect can occur in many tissues,and the occurrence of most body damage is closely related to it.PFOA and PFOS can induce oxidative stress in the body,and even further lead to cell death,and tissue damage in severe cases.At present,the correlation between PFOA and PFOS-induced hepatomegaly effects and oxidative stress effects in mice is unknown;In addition,there are few studies on PFOA and PFOS at the subcellular level caused by oxidative stress;The molecular mechanism of PFOA and PFOS binding to antioxidant enzymes is not yet clear;The toxic effects of pollutants can be evaluated at the animal,cellular,subcellular,and molecular levels.Previous studies have often focused on only one of these levels and ignored the combinations of multiple levels.Aiming at the above problems,this study,based on the background of environmental toxicology and modern instrumental analysis science,investigated the toxic effects and mechanisms of PFOA and PFOS induced hepatomegaly and oxidative stress in mice at the animal,cellular,subcellular and molecular levels.First,the characteristics of PFCs,the pollution situation,and the toxic effects of PFOA and PFOS on various systems were summarized.The oxidative stress effects induced by PFOA and PFOS in various organisms and the mechanism of regulation of cell apoptosis were introduced.The interactions of PFOA and PFOS with biological macromolecules were summrized.Through the review of the existing research,the latest research progress in the field of PFOA and PFOS toxicity was summarized,the problems that have not been solved by the current research were put forward,and the main content and significance of this study were introduced.At the animal level,C57BL/6 mice were taken as the research objects to study the toxic effects and mechanisms of hepatomegaly and oxidative stress induced by PFOA and PFOS in mice.Mice were exposed to PFOA or PFOS with or without N-acetyl-L-cysteine(NAC)for 21 days.Then the hepatomegaly,the liver pathology and and pathological damage of the mice were detected and analyzed.The ultrastructural damage of mouse liver was observed by transmission electron microscope,and the changes of catalase(CAT),superoxide dismutase(SOD)activity and lipid peroxidation level(malonaldehyde MDA)in mouse liver were measured.The results showed that after exposure to PFOA and PFOS,livers of mice were significantly enlarged and the liver body index was significantly increased,but the symptoms of hepatomegaly of the mice were milder in the NAC groups,that is,the stronger the oxidative stress effect,the more severe the symptoms of hepatomegaly.Pathological section results of liver tissue showed that PFOA and PFOS could cause pathological phenomena such as hepatocyte edema inflammatory cell infiltration,vacuole degeneration,and enlarged nuclei in mice.Transmission electron microscope observation of the ultrastructure of mouse liver cells revealed that PFOA and PFOS caused mitochondria swell and vacuole and ribosomes fall off the endoplasmic reticulum.NAC can alleviate the damage to mitochondria of hepacytes in mice,thereby making hepatomegaly milder.In addition,both PFOA and PFOS exposure could destroy the redox balance in mouse liver,change the activities of antioxidant enzymes CAT and SOD,and produce lipid peroxidation,thereby inducing oxidative stress effects and causing oxidative damage to tissues.NAC can remove excess ROS in the body,thereby alleviating the toxic effects of PFOA and PFOS on hepatomegaly and oxidative damage in mice..At the cellular and subcellular levels,the study subjects were primary hepatocytes of normal mice.After in vitro exposure,the toxic effects and mechanisms of oxidative stress and cell apoptosis caused by PFOA and PFOS in mouse primary hepatocytes were evaluated and analyzed.After exposure to PFOA or PFOS for 24 h,the cell viability,the intracellular ROS content,the activity of antioxidant enzymes CAT,SOD,glutathione(GSH)content,cell membrane damage,mitochondrial membrane potential,intracellular Ca2+concentration,the activation of caspase-3,and the proportion of cell apoptosis were measured.The results of the study proved that after 24 hours of PFOA and PFOS exposure,excessive ROS accumulated in the primary liver cells of mice,and the activities of CAT and SOD were changed,indicating that PFOA and PFOS weakened the antioxidant capacity of the cells and destroyed the redox balance,thereby inducing oxidative stress,which has a toxic effect on mouse liver cells.PFOA and PFOS also damaged the cell membrane,changed the permeability of the cell membrane,reduced the mitochondrial membrane potential in hepatocytes,and further activated the intracellular Ca2+and caspase-3 activity,and eventually induced cell apoptosis.The results indicated that oxidative stress regulated the apoptosis in mouse primary hepatocytes induced by PFOA and PFOS,and also demonstrated that the pathway of PFOA and PFOS-induced apoptosis was mediated by mitochondria.At the molecular level,the indirect antioxidant enzyme lysozyme(LYZ)was used as the research object,and a variety of spectroscopy(fluorescence emission,synchronous fluorescence,ultraviolet-visible,circular dichroism,resonance light scattering,and three-dimensional fluorescence spectra)methods were employed to detect the effects of PFOA and PFOS on the structure and conformation of LYZ;the effects of PFOA and PFOS on the function of LYZ were studied;the molecular docking technique was used to simulate the binding sites of PFOA and PFOS on LYZ,and the action models of PFOA-LYZ and PFOS-LYZ were established.The results showed that LYZ combined and interacted with PFOA and PFOS,resulting in a decrease in the enzyme activity of the LYZ molecule.The microenvironment of aromatic amino acids and secondary structure of LYZ were altered by PFOA and PFOS,resulting in unfolding of the LYZ molecule.After the combination of PFOA and PFOS,new complexes were formed,which increased the particle size of the system.The binding sites of PFOA and PFOS and LYZ were located near the active center of LYZ,and the binding sites included tryptophan(Trp 63 and Trp 108).At the same time,due to the high polarity of the sulfonic acid group of PFOS,PFOS can be directly bonded with Asp 52 by hydrogen bonding,which led to a greater influence of PFOS on the activity of LYZ.At the molecular level,in addition to indirect antioxidant enzymes,the direct antioxidant enzyme CAT and SOD were chosen as the research objects,and a variety of spectroscopy methods(fluorescence emission,synchronous fluorescence,ultraviolet-visible,circular dichroism,resonance light scattering spectra),enzyme activity,isothermal titration calorimetry and molecular docking were employed to detect the effects of PFOA and PFOS on the structure and conformation of CAT and SOD.The effects of PFOA and PFOS on the structure and conformation of the two enzymes were investigated by using various spectroscopic methods;the enzyme activities of CAT and SOD were detected after combining with PFOA and PFOS;and molecular simulation technology was used to simulate the binding sites of PFOA and PFOS on enzymes;the thermodynamic parameters and binding constants of PFOA,PFOS and SOD were investigated by using isothermal titration calorimetry technology.(1)The structure of CAT was changed after interacting with PFOA or PFOS.The results showed that both PFOA and PFOS affected the secondary structure of CAT by reducing the a-helix and increasing the β-sheet contents.PFOA could loosen and expand the CAT skeleton.PFOS had little effect on the CAT skeleton,but it could affect the microenvironment of aromatic amino acids and heme groups of CAT.PFOA and PFOS exposure resulted in a decrease of the CAT particle size,and PFOS had a greater effect.Because PFOA cannot preferentially bind to the active center of CAT,PFOA had little effects on the activity of CAT.But when PFOS interacted with CAT,the enzyme activity of CAT eventually decreased to 71.81%.Molecular simulation results showed that the binding site of PFOA on CAT was on the surface of the enzyme,while the binding site of PFOS was close to the active center of CAT,and two important amino acid residues related to enzyme activity were at the binding site of PFOS,so PFOS had a greater influence on the enzyme activity of CAT than PFOA.(2)The combinations of PFOA and PFOS with SOD increased the enzyme activity of SOD.The binding constants of PFOA-SOD and PFOS-SOD were 1.68 ± 0.13 and 1.13 ± 0.32(105 M-1),and the interaction processes were spontaneous.PFOS could bind to Tyr residues and affect the microenvironment of Tyr,but the effect of PFOA on Tyr was not obvious.PFOA could cause the skeleton of SOD loose and unfolding,but PFOS could tighten the skeleton of SOD.PFOA and PFOS also changed the secondary structure of SOD,causing changes in the percentages of α-helix,β-sheet,turn,and random coil contents.In addition,both PFOA and PFOS could form complexes with SOD to increase the particle size,but as the concentration of PFOS further increased,the particle size of the PFOS-SOD complexes became smaller.The results of molecular docking showed that PFOA and PFOS increased the pocket near the active center of the enzyme molecule by changing the structure of SOD,so that the SOD could more fully contact the reaction substrate,resulting in an increase in enzyme activity.Finally,the main conclusions and innovations of this paper were summarized and summarized,and the shortcomings of this study were analyzed,and the research direction of this research field was prospected.This study established a multi-level comprehensive toxicity evaluation method,including four levels of animal,cell,subcellular and molecular.The toxic effects and mechanisms of hepatomegaly and oxidative stress induced by PFOA and PFOS in mice were evaluated.PFOA and PFOS could induce oxidative stress by disrupting the antioxidant defense system in the mouse liver and cause hepatomegaly,tissue and ultrastructural damage.But antioxidants can significantly alleviate toxic effects of PFOA and PFOS on hepatomegaly and oxidative damage in mice.The stronger the oxidative stress effect,the more severe the symptoms of hepatomegaly and liver injury caused by PFOA and PFOS can be treated by suppressing oxidative stress effects.It is proved that PFOA and PFOS destroyed the antioxidant defense system and caused excessive accumulation of ROS,thereby inducing oxidative stress effects and further mitochondrial-mediated apoptosis in mouse primary hepatocytes.Binding models of the interactions between PFOA or PFOS and antioxidant enzymes were established.The specific mechanism of PFOA and PFOS affecting protein structure and function was analyzed.It is proved that PFOA and PFOS could combine with LYZ,CAT,SOD and affect the structure of the enzymes,change the enzyme activity,and make the enzyme unable to perform there functions normally.Studies at the animal,cellular,subcellular,and molecular levels have comprehensively and comprehensively evaluated the toxic effects and mechanism of PFOA and PFOS-induced hepatomegaly and oxidative stress in mice.This study provided a scientific reference for evaluating the toxicity of PFCs and treating diseases caused by related toxic substances.