Study On The Multi-Generational And Combined Toxic Effects Of Deoxynivalenol(DON)

Deoxynivalenol?DON,vomitoxin?is one of the most widely distributed trichothecene mycotoxins commonly found in cereal food and feeds.Significant acute and potential chronic toxic effects of DON have been observed in animals and human populations.A large amount of studies have been done since it's discovered.Recently,few studies have found that DON could be transferred to fetus via placenta from pregnant parent exposed to DON.In real-world scenario,DON is very stable and regional to some specific areas.Thus,humans and animals may probably be unavoidably exposed across multiple generations continually in nature.However,almost none research was done on this aspect.Moreover,an increasing number of studies,to date,have been reported on the combined toxic effects of DON together with other commonly found mycotoxins,but the quantity is still limited.And also,due to the different tested models,treated concentrations,exposed ways,and the varying combinations as well as the ratio of each mycotoxin in the mixtures,outcomes are usually inconsistent.The specific studies and their corresponding outcomes in this whole dissertation are as follows:1.Cell viability was determined via rasazurin assay in HepG2,RAW 264.7 and BF-2 cell lines as well C.elegans and zebrafish,respectively.Results showed that the three cell lines were sensitive to DON and the corresponding ranking in a decreasing order was RAW 264.7(IC₅₀=0.11?g/m L)>HepG2(IC₅₀=0.67?g/mL)>BF-2(IC₅₀=4.73?g/mL).Thereafter,the cellular mechanism were detected by high content screening?HCS?,showing that DON could induce the generation of reactive oxygen species?ROS?,the increase of[Ca²⁺]_i influx,the decrease of mitochondrial membrane potentials?MMP?,and the damages of cell membrane.All the tested concentrations of DON didn't cause the death of both C.elegans and zebrafish.The EC₅₀?95%CI?of DON for the growth,brood size and feeding of C.elegans were 372.4?333.34-571.77?,406.75?342.39-511.46?and 360.30?295.42-473.89??g/m L,respectively.In addition,DON can cause damages on the liver of zebrafish at the concentrations of 20 and 40?g/mL.2.In the assay of investigating the trans-generational toxicity of DON,body length,brood size and food intake,as the endpoints,were detected to assess the toxic effects of DON on the development and reproduction of C.elegans.The results demonstrated that no inhibition was observed in the growth of offspring,but the endpoints of brood size and food intake were affected,showing a gradual recovery trend in the filial generations.3.In order to evaluate the multi-generational toxic effects of DON in C.elegans,body length,brood size and food intake were chosen to be the endpoints in this study.Besides,a mixed-effect model with regression analysis was developed to assess whether the concentration of DON,generation of C.elegans?time period of exposure?and their interactions have significantly different influence on the tested endpoints.The one-to-four generations of C.elegans were named as P₀,F₁,F₂ and F₃.Results displayed that parental exposure to DON could increase the sensitivity of offspring.Specifically,F₂ was the most sensitive generation followed by F₃ and F_1.Furthermore,based on the mixed-effect model,concentration,generation and concentration*generation can be tread as the significantly influential factors for growth and food intake while only concentration and generation alone could significantly affect the brood size of DON-exposed C.elegans.4.The mechanisms of multi-generational effects of DON were studies by miRNA-Seq.Results revealed that DON induced 162,177,217 and 225 significantly differentially expressed genes in P₀,F₁,F₂ and F₃.Gene Ontology?GO?analysis demonstrated that DON mainly affected the synthesis of protein,transcription,sex determination,balance of redox state,activity of kinase and biotransformation,indicating the developmental and reproductive toxicities of DON.Moreover,according to the KEGG pathway analysis,DON showed it multi-generational toxic effect via pantothenate and CoA biosynthesis to a large extent.Additionally,glycerophospholipid metabolism,synthesis and degradation of ketone bodies,butanoate metabolism,arginine biosynthesis,2-oxocarboxylic acid metabolism as well as alanine,asparate and glutamate metabolism also could be interfered by successive DON-exposures.Although the repeated exposure of DON showed the severer toxic effects on the offspring,the degree of toxicities in different generation of C.elegans fluctuated,which hinted that self-repair mechanisms in C.elegans could be correspondingly triggered to some degree with the varying exposed dosages and time period.5.The types of combined toxicity of DON with AFB₁ and ZEN were determined in HepG2,RAW 264.7 and BF-2 cell lines as well C.elegans and zebrafish,respectively,based Chou-Talaly mathematical model.Results demonstrated:1)the mixture of DON+AFB₁,DON+ZEN&DON+AFB₁+ZEN,AFB₁+ZEN showed addition,synergism and antagonism,respectively,in HepG2 cells;2)DON+AFB₁ showed synergism-to-addition as the concentration increased,DON+ZEN and DON+AFB₁+ZEN showed addition-to-synergism with the increasing concentration,and AFB₁+ZEN showed antagonism in RAW 264.7 cells;3)DON+AFB₁ and AFB₁+ZEN showed synergistic effects whereas DON+ZEN and DON+AFB₁+ZEN showed antagonistic effects in BF-2 cells.As for C.elegans,DON+AFB₁showed addition-to-synergism,DON+ZEN showed antagonism,DON+AFB₁+ZEN showed antagonism-to-synergism for growth of 24 hrs and only antagonism for growth of 24 hrs and brood size,and AFB₁+ZEN showed synergism for growth of 48 hrs while antagonism-to-synergism for growth of 24 hrs and brood size as the concentration increased.For zebrafish,synergistic effects were detected in the mixtures of DON+AFB₁ and AFB₁+ZEN while antagonistic effects in the mixtures of DON+ZEN and DON+AFB₁+ZEN.All the above suggested that the type of interaction among multiple mycotoxins were co-determined by concentration,time,tested model and the composition of mixtures.Altogether,DON showed strong cytotoxicity for RAW 264.6 cell line,followed by HepG2 and BF-2 cell lines,and injured C.elegans to some degree,but zebrafish was not sensitive to DON exposure.Thus,in this currnet study,RAW 264.7 cell line could be regarded as the most sensitive experimental model for assessing the toxic effects of DON.Besides,DON has both trans-generational and multi-generational toxic effects on C.elegans.Results of miRNA-Seq showed that successive DON exposure could mainly affect several bio metabolic pathways in which the pantothenate and CoA biosynthesis pathway was the primary one.Finally,the toxic potentials of DON alone could be changed when co-contaminated with other mycotoxins.Therefore,this study could provide theoretical basis for more objective and comprehensive risk assessment as well as standards-making,and also raise new ideas and methods for studying the toxic effects of other foodborne toxic substances.