Study On The Toxicological Mechanism Of Disinfection By-product Halobenzoquinones Using Phosphoproteomics

Halobenzoquinone is a new type of tap water disinfection by-product that is not strictly controlled.It has the characteristics of many types,high detection rate,high toxicity,and not being eliminated by conventional disinfection methods effectively.Studies have found that the cytotoxicity and genotoxicity of halobenzoquinones are far stronger than the controlled haloacetic acids and trihalomethanes,and they have serious organ toxicity.However,there are not many studies on the site of action and toxic mechanism of halobenzoquinones,resulting in unclear toxicological mechanisms.Based on preliminary work and literature research,this subject has carried out preliminary research on toxicological mechanisms with phosphorylated proteomics methods as the core.Phosphorylation is a kind of protein post-translational modification that is widely distributed in organisms.It participates in almost all life activities in cells,such as signal transmission,cell proliferation and apoptosis,protein activity and function regulation.Phosphoproteomics technology is currently one of the most effective methods for analyzing high-throughput phosphorylation modification,and it has been widely used in biomarker identification of pollutants and environmental toxicology research.Relying on mass spectrometry detection technology,this project uses zebrafish heart as the research object,using phosphoproteomics to study the toxicity and toxicological mechanism of halobenzoquinone disinfection by-products.The main research contents and conclusions are:1.The disinfection by-products of halobenzoquinones and trihalomethanes,which are common in tap water,were selected,and the toxicity differences between the two were compared.The results showed that the 24 h LC₅₀ of 2,6-dichloro-1,4-benzoquinone in adult zebrafish was 1.234?mol/L;the LC₅₀ of chloroform was 773.34?mol/L.The difference between the two is more than 600 times,indicating that the toxicity of 2,6-DCBQ is much higher than that of controlled trihalomethane.2.Optimize the protein pretreatment process and determine the enrichment efficiency of Ti O₂magnetic particles for phosphopeptides.The?-casein hydrolysate samples were analyzed by microfluidic liquid chromatography-quadrupole time-of-flight mass spectrometry(micro LC-QTOF-MS),and the results showed that peptides were detected when using the common digestion system of Lys-C enzyme and Trypsin enzyme The rate is 84.77%,while the peptide detection rate is only34.55%when using Trypsin digestion.The former is significantly better than the latter;the use of Ti O₂ magnetic particles can identify 19 phosphopeptides from standard protein samples with an enrichment efficiency of 50%?88%.3.Zebrafishes are exposed by different concentrations of 2,6-DCBQ,using SWATH quantitative protein technology to analyze the relative abundance of phosphopeptides in heart tissue,and screen the components that are differentially expressed under different exposure concentrations,and then discuss and 2,6-DCBQ exposes related proteins,genes and signal pathways.In the end,we found 14phosphorylated peptides related to 2,6-DCBQ exposure in zebrafish heart tissue,corresponding to 10proteins;further analysis showed that there were 11 related genes and 4 cell signaling pathways.As a result,we have a preliminary understanding of the toxicological mechanism of 2,6-DCBQ's cardiotoxicity,laying the foundation for a comprehensive and in-depth understanding of the health risks of new halogenated benzoquinone disinfection by-products.