Study On The Combined Toxicity And Toxic Mechanism Of Deoxynivalenol And Its Acetylated Derivatives

Vomitoxin,also known as deoxynivanenol(DON),is a kind of trichothecenes produced by Fusarium.Trichothecenes are widely detected in grains around the world,so the toxicity research of them have always been valued by experts and scholars.Two-dimensional planar cell culture as a toxicological method is often used for in vitro toxicity evaluation of mycotoxins.Compared with planar cell culture,three-dimensional cell culture can better simulate the in vivo environment,and can assess the cytotoxicity more accurately.In addition,the current studies on trichothecenes are mostly focused on individual toxin,and there is still a lack of comprehensive studies on the types and mechanisms of the combined toxicity of the toxins.Here,the toxic effects of DON,3-acetyldeoxynivalenol(3-ADON)and 15-acetyldeoxynivalenol(15-ADON)on target cells A549 and Caco-2 were studied.A three-dimensional cytotoxicity evaluation model based on electrochemical methods was constructed through 3D printing technology to realize the individual and combined toxicity evaluation of DON and its derivatives.Then,metabolomics methods are used to further explain the toxic effects and damage mechanisms of trichothecenes on target cells.The main contents of this study are as follows.1.To determine the effects of different cell lines,toxin dose,stimulation time and toxin combinations on the combined cytoxicity of DON,3-ADON and 15-ADON.It was found that the activities of A549 cells and Caco-2 cells were inhibited by all three toxins in a time-dependent and dose-dependent manner.Compared with Caco-2 cells,A549 cells were more sensitive to the three toxins.Under the stimulation over 48 hours,both the two kind of cells were more insensitive to the toxin.The mixture of DON+3-ADON and 3-ADON+15-ADON produced almost complete antagonistic cytotoxicity(IC₁₀?IC₉₀)on A549 cells and Caco-2 cells,while the combination of DON+15-ADON and DON+3-ADON+15-ADON had synergistic effect on low cell inhibition level,and antagonistic effect on medium and high cell inhibition level.2.An electrochemical cell biosensor based on Carbon Nanofibers/gelatin methacryl(Gel MA)composite conductive hydrogel was developed for the combined toxicity assessment of DON,3-ADON and 15-ADON.In order to realize the unified and mass production of toxicity evaluation tools,3D bioprinting technology was introduced into the construction of cell-based biosensor,and an 8-channel three-dimensional"honeycomb"electrochemical biosensor was developed for the cytotoxicity evaluation of DON,3-ADON and 15-ADON.Carbon nanofibers with excellent biocompatibility were incorporated into Gel MA hydrogels to enhance the conductivity of the sensor.Under this evaluation method,the LOD of DON,3-ADON and 15-ADON were 0.07,0.10 and 0.06?g/ml,respectively.The results of the constructed electrochemical method are in good agreement with the results of the CCK8 method.3.Through unsupervised PCA,clustering heat map analysis and pathway analysis,the intracellular metabolic profile changes of the two target cells after treated with DON,3-ADON and 15-ADON were analyzed.Based on the preliminary experiments,it can be found that different cell lines,toxin dose,stimulation time and toxin combinations have a greater impact on the toxic effect,so this chapter selects these three dimensions to research into the cellular metabolomics.The results showed that as the concentration of toxins increased,the number of significantly different metabolites in cells increased,indicating that high-dose toxins aggravated the disturbance of toxins to the normal metabolic dynamics of cells.In addition,the mixture of the toxins did not increase the number of significantly different metabolites compared to individual toxin,which may be related to the antagonistic effect of toxins coexisting.The results of enrichment analysis of metabolic pathways showed that DON and its derivatives mainly disturb the target cells'phenylalanine,tyrosine,tryptophan,alanine,aspartic acid,glutamic acid,taurine and low Taurine metabolism pathway,thereby affecting the synthesis of amino acids and proteins,resulting in cytotoxicity.In summary,different cell lines,stimulation time,stimulation dose and toxin combination will affect the combined cytotoxicity of DON,3-ADON and 15-ADON.In the three-dimensional cell culture system,there was a dose-effect relationship between toxins and cell activity measured by electrochemical methods.The bioprinting-based toxicity evaluation model construction method provides a new thought for uniform and batch production of toxin toxicity evaluation tools.The metabolic disorder of target cells caused by toxins indicates that the toxins mainly affect the normal growth of cells by disturbing the energy metabolism pathway of cells.This thesis objectively and comprehensively evaluates the combined exposure risk of DON,3-ADON and 15-ADON,which can provide a theoretical basis for scientifically formulating the limits of trichothecenes.