Studies On The Toxicity And Mechanism Of Iodoacetic Acid To Antioxidant Enzymes

In the process of drinking water disinfection,disinfectants will react with natural organic matter in the water to form disinfection by-products(DBPs),which poses a potential hazard to human health.With the deepening of the epidemiological and toxicological research of DBPs,people pay more attention to the health risks of DBPs.Iodoacetic acid(IAA)is a typical DBPs,which has the characteristics of low dose and high toxicity,especially higher than similar DBPs in terms of cytotoxicity,genetic toxicity and teratogenicity.Therefore,IAA has attracted increasing attention from researchers in the field of DBPs toxicity research.The mechanism of toxic action of IAA is very complicated.In the current toxicological research on IAA,existing research data have proved that the oxidative damage caused by oxidative stress induced by IAA is one of the important mechanisms of IAA's toxic effect.Oxidative stress refers to the state of imbalance between the oxidative and antioxidant systems in the body.As an important component of the antioxidant system,antioxidant enzymes'main function is to remove excess reactive oxygen species(ROS)in the body and maintain the balance of redox levels.Toxicology research at the molecular level found that after entering the body,small molecule pollutants may change the molecular structure of the enzyme through interaction with the enzyme,affecting the function of the enzyme.Therefore,IAA may change the structure and function of the enzyme by interacting with antioxidant enzymes,leading to the effect of ROS clearance,thereby inducing oxidative stress.Therefore,this study used mouse primary hepatocytes as a cell model to investigate the effects of IAA on the activity of two typical antioxidant enzymes in the cell-catalase(CAT)and superoxide dismutase(SOD).Using CAT and SOD as biomacromolecule models of enzymes,the interaction mechanism between IAA and two enzymes was explored,and the cell-level and molecular-level studies were used to elaborate the toxic effects and mechanism of IAA's anti-oxidase activity.The main contents of the study include the following three parts:Part ?:A review on the status of toxicity study of disinfection by-products IAA,the oxidative stress,and the relationship between IAA toxicity and oxidative stress.The shortcomings of the current research on the toxicity mechanism of IAA were clarified,and the main research contents of this paper were determined.Part ?:First,the mouse primary hepatocytes were used as the cellular research model.The intracellular CAT activity was measured under experimental conditions that simulated physiological environment.The effect of IAA exposure on the CAT activity of the cells was studied at the cellular level.The experimental results showed that intracellular CAT activity increases with the increasing of IAA concentration.Then use CAT molecules as molecular research models,and use various methods such as molecular enzyme activity measurement,isothermal titration calorimetry,ultraviolet-visible absorption spectroscopy,and molecular simulation techniques to explore the changes of structure and function of CAT after the interactions between CAT and IAA.The results showed that the molecular CAT activity is inhibited under IAA exposure.IAA is combined with CAT under the driving of van der Waals force and hydrogen bonding force.The binding constant is(1.57±0.481)×10~3,and the number of binding sites is about 4.The interaction with IAA changed the secondary structure of CAT,resulting in a loose protein backbone and changing the volume of protein aggregates formed by CAT aggregation.When IAA binds to CAT,it preferentially binding to the site on the surface of the CAT molecule,and the binding at this site will not directly affect the activity of CAT.When the binding of CAT surface sites reaches saturation,IAA will bind to sites near the active center of CAT and interact with key amino acid residues HIS 74 and TYR 357,which affect the enzyme activity,resulting in changes of the structure around the Heme group.After that,the microenvironment polarity of amino acid residues was enhanced,and the substrate's entry into the active center was effected.The results of comprehensive cellular and molecular experiments showed that the reasons for the change of cellular CAT activity are as follows:on the one hand,the response of the cell's self-protection mechanism to IAA stimulation,which increases the content of CAT,and promotes CAT activity in the cell.On the other hand,IAA inhibited the molecular CAT activity by changing the structure of CAT and interactions with HIS 74 and TYR 357.Part ?:First,the mouse primary hepatocytes were used as the cellular research model.Under the experimental conditions that simulated physiological environment,the intracellular SOD activity was measured.The effect of IAA exposure on the SOD activity of the cells was studied at the cellular level.The experimental results showed that intracellular SOD activity increases with increasing IAA concentration.Then,using SOD molecules as molecular research models,various methods such as molecular enzyme activity measurement,isothermal titration calorimetry,ultraviolet-visible absorption spectroscopy,UV-visible absorption spectroscopy,Zeta potential measurement,and molecular simulation techniques were used to explore the changes of structure and function of SOD after the interactions between CAT and IAA.The results showed that the molecular SOD activity was promoted under IAA exposure.IAA is combined with SOD under electrostatic force.The number of binding sites is about 7.The binding constant is(7.09±2.88)×10~2.After interacting with IAA,the conformation of SOD changed,mainly reflected on the changes of secondary structure,including the increasing of?-helix and?-turn content,as while as the decrease of?-sheet and random coil content.These changes resulting in misfolding of the peptide chain and making the protein backbone loose.In addition,the endogenous fluorescence of SOD is quenched,leading to changes in the microenvironment of tyrosine residues.When IAA binds to SOD,it preferentially binds to the binding site near the active center of SOD,changing the molecular structure around the site.The results of comprehensive cell and molecular experiments show that the reasons for the changes in cell SOD activity are as follows:on the one hand,the amount of SOD generated by the cell to eliminate the adverse effects of oxidative stress induced by IAA increases.On the other hand,molecular spatial structure was changed,which improved the molecular enzyme activity.