Evaluation Of Oxidative Damage Of Saccharomyces Cerevisiae Induced By Low-dose Environmental Pollutants TCBPA And Exploration Of ROS Production Mechanism

Tetrachlorobisphenol A(TCBPA)is a structural analog of chlorobisphenols.Because of its excellent flame retardancy,it is widely used as a flame retardant additive in various materials in various countries.TCBPA is widely distributed in the environment and has a large amount of reserves.It is found in water,soil,and even living organisms.Since TCBPA is an organic substance,it is easily attached to organisms and absorbed into organisms,then accumulated in the higher organisms through cascades of the food chain,causing serious damage to the environment and biological health.However,at present,little is known about the biological toxicity and therapeutic mechanism of TCBPA.Although TCBPA is widely distributed in the environment,its concentration is low and generally remains at the microgram level.Therefore,understanding the toxicity of TCBPA to organisms at low concentrations is more important.In this article,the eukaryotic model of Saccharomyces cerevisiae,which is widely used in research,was used as a material to evaluate the biooxidative toxicity of TCBPA at low concentrations,and the mechanism of the induction of oxidative toxicity was preliminary explored,which will allow more people to understand the toxicity of TCBPA and provide a theoretical basis for TCBPA to study the toxic damage of humans and other higher organisms.First,S.cerevisiae was exposed to low concentrations of TCBPA(0,5,10,and 20 ?M),and the effect of TCBPA on the growth and survival of S.cerevisiae was measured using a spectrophotometer and blue-white staining.The cell swelling ratio of S.cerevisiae at logarithmic phase was measured by using a hemacytometer to evaluate the damage of TCBPA to S.cerevzisiae cell membrane.The results showed that TCBPA had no significant effect on the growth,survival and cell membrane of S.cerevisiae at a dose of 5 ?M,but at the doses of 10 and 20 ?M,the growth,survival and cell membrane of S.cerevisiae were significantly affected,and the impact has intensified with the dose increased.Next,we used the fluorescent probe DCFH-DA to detect the reactive oxygen species(ROS)content of S.cerevisiae in the presence of different low concentrations of TCBPA.Thiobarbituric acid method,JC-1 fluorescent probe and UHPLC-MS/MS cascade enzymatic assay was used to analyze the intracellular MDA content,mitochondrial membrane potential,and 8-oxodG levels of S.cerevisiae in the presence of TCBPA to assess the oxidative damages caused by TCBPA.The results showed that TCBPA could increase the intracellular ROS content of S.cerevisiae,and thus causing lipid peroxidation,decrease of mitochondrial membrane potential and DNA damage.Then,the signal pathways related to ROS production were constructed by consulting related references.The intracellular calcium ion concentration of S.cerevisiae exposed to TCBPA was detected with Fluo-2 AM.S.cerevisiae intracellular ROS contents were detected when ROS-related production pathways are inhibited by using DPI inhibited NADPH oxidase,FK506 inhibited calcineurin phosphatase,EGTA chelated calcium,BCK1 and MCK1 knockout strains.The results showed that TCBPA could increase the intracellular calcium content of S.cerevisiae.S.cerevisiae intracellular ROS all have been reduced,when the S.cerevisiae ROS-related production pathways were inhibited.Finally,the real-time fluorescence quantitative PCR technique was used to detect the expression of key enzymes in ROS-related pathways of S.cerevisiae cells.Through comparison of RNA transcription between experimental and blank groups,it was showed that TCBPA promoted the key enzymes expression of tyrosine kinase pathway,calcium-related pathway,MAPK pathway and in respiratory chain of S.cerevisiae.The expression of superoxide dismutase(SOD)in the ROS scavenging pathway was enhanced,while catalase(CAT)expression in the terminal key enzyme was significantly reduced.In summary,TCBPA could activate NADPH oxidase to produce ROS by promoting the expression of key enzymes in ROS-related pathways and activating NADPH oxidase activation pathways of S.cerevisiae.Excessive ROS leads to a series oxidative damages of S.cerevisiae,and eventually induce to the survival rate decrease and growth inhibition of S.cerevisiae.