Investigations On The Toxic Characterization Of Alizarin Red S On Antioxidant Enzymes And Hepatocytes

In recent years,with the development of modern dyeing industry,the chance of dye exposure to human body has gradually increased,and the pollution from textile dyeing industry has attracted more attention.Anthraquinone dyes have the advantages of bright colors and outstanding fastness to sunlight,and are often used for dyeing synthetic fibers.Relevant research indicates that anthraquinone compounds may trigger the generation of reactive oxygen species in the body and induce oxidative stress to cause DNA damage.Alizarin Red S(ARS)is a kind of anthraquinone dyes deriving from alizarin,which is widely used in textile dyeing,staining calcium in biology studies,and in the field of geology.Alizarin Red S,as a calcium ion developer,can quantitatively detect the distribution and accumulation of calcium ions in biological tissues by colorimetry,providing an accurate and quantitative research method for studying bone growth and calcium accumulation.Geologically,alizarin red S can be used to stain thin slices of rock to study the calcium minerals in the thin slicesAt present,studies at molecular level has revealed the effects and the underlying mechanism of interaction between ARS and proteins such as human serum albumin(HSA)and bovine serum albumin(BSA).Other works reported the toxic effect by ARS to enzymes such as lysozyme,acetylcholinesterase,and alpha-amylase of which the activity was inhibited.And at cellular level,studies on the cytotoxicity of alizarin,purpurin,and quinizarin have been described by reckoning the viability,scavenging ability for free radicals and reducing ability.However,toxic effect and mechanism of ARS to cells and antioxidants such as catalase and superoxide dismutase has not been investigated yet.At the molecular level,there are no reports on the changes in the enzyme activities of catalase,superoxide dismutase,lysozyme and other common antioxidant enzymes under the exposure of ARS.At the cellular level,the effect of Alizarin Red S on the oxidative stress effect of hepatocytes is unclear.Therefore,this article evaluates the toxicity of Alizarin Red S on various antioxidant enzymes and hepatocytes from the molecular and cellular levels,and discusses the mechanism behind the experimental phenomenon.This work consists of four main parts:The first part of this work studies the toxic effects and mechanism of ARS on catalase(CAT),a common antioxidant enzyme catalyzing the hydrogen peroxide to oxygen and water,in vitro.CAT distributes ubiquitously in animal tissues,especially in the liver.In this study,the toxicity of ARS was investigated using the spectroscopy methods,enzyme activity assay,isothermal calorimetric titration(ITC)and molecular docking simulation.The results revealed that,ARS bound to CAT spontaneously through electrostatic force and inhibited its enzyme activity in vitro.However,no significant changes to structure and conformation of CAT were observed.Under the exposure of ARS at 5 ×10-6 mol·L-1,the relative enzyme activity of CAT was inhibited to 76.2%.The probable mechanism of activity inhibition by ARS is demonstrated by the molecular docking simulation that ARS contacted with the His 74 residue which located in the active site of CAT.The second part investigated the toxic effects of ARS on superoxide dismutase(SOD)at molecular level,using various spectroscopy methods,enzyme activity assay and molecular docking simulation.SOD is an important antioxidant enzyme that decomposes superoxide anion by-products from the electron transport chain of respiration,protecting cells from oxidative stress damages.Results of this work shows that,ARS could inhibit the activity of SOD slightly without structural and conformational changes to SOD.Under the exposure of ARS whose concentration reached 6×10-6 mol·L-1,the relative enzyme activity of SOD was inhibited to 88.3%.The bind site of ARS to SOD is located between the four monomers of SOD.It is suspected that ARS inhibit the activity of SOD by influencing the overall structure of the four SOD monomers.The third part investigated the toxic effects of ARS on lysozyme(LYZ)at molecular level,using various spectroscopy methods,enzyme activity assay and molecular docking simulation.Lysozyme is related to the body's innate immune system and can hydrolyze the cell wall of bacteria to promote bacterial lysis.In addition,lysozyme can inhibit the expression of oxidative stress-related genes and inhibit oxidative stress substances in the body.LYZ is an indirect antioxidant enzyme widely present in the body.Results of this work shows that,ARS as ligand could interact with the lysozyme receptor,which increased the scattering particle size of the ARS-LYZ system,but did not significantly affect the protein backbone structure of lysozyme molecule and the microenvironment near its aromatic amino acid residues.ARS can also inhibit the activity of lysozyme.When the concentration of Alizarin Red S reached 1×10-5 mol·L-1,the relative activity of lysozyme would decrease to 89.7%comparing to the sample without exposure.The possible reason is that ARS interacted with the key amino acid residue Glu35 of lysozyme by electrostatic forces,which led to the inhibition of lysozyme activity.The last part of this work investigated the toxicity of ARS to primary hepatocytes from mouse.After 24 hours of contacting with ARS,hepatocytes represented significant increment in cell viability assay when ARS concentration is 1×10-4 mol·L-1,while the reactive oxidative species measurements have shown that ROS was slightly increased under ARS exposure,the content of reactive oxygen species in the cells increased from 7.4%to 8.9%.The measurement of intracellular enzyme activity showed that the activities of intracellular CAT and SOD were significantly inhibited under the exposure of ARS.When the concentration of Alizarin Red S reached 2×10-5 mol·L-1,the intracellular activity of CAT and SOD decreased to around 48.3%and 80.3%respectively,comparing to the unexposed conditions.It is suspected that the enzyme activity changes of antioxidant enzymes are one of the reasons for ROS increment observed in liver cells.This study shows that ARS has significant inhibition effect on the enzyme activity of antioxidant enzymes such as CAT,SOD and LYZ.Moreover,it has a slight effect on the oxidative stress of the liver cells,and which therefore causes the increment of ROS in hepatocytes.