Study On The Mechanism Of Intestinal Toxicity And Method Of Toxicity Screening Of Combined Exposure To Vomitoxin And Heavy Metal Cadmium In Food

Vomitoxin(DON)is the most polluted trichothecene mycotoxins in grain food such as wheat.It has intestinal toxicity such as vomiting and anorexia.Cadmium(Cd)is one of the most polluted toxic heavy metals in food and soil.It migrates from soil to crops rapidly,and can contaminate most food.DON and Cd are exposed together in the food chain to produce accumulation.Surveys showed that main grain-producing areas such as the middle reaches of the Yellow River and southeast coast were simultaneously the areas with medium to high-levels of pollution.Cereal food,like wheat and bread,were heavily contaminated by DON and Cd.Therefore,the population faces the risk of co-exposure to DON+Cd.Dietary intake will cause the intestine to be an organ where high levels of DON and Cd act simultaneously,which both toxic to the intestine.Therefore,it is necessary to study the joint intestinal toxicity induced by DON+Cd.In this paper,the intestinal epithelial cell HT-29 model was combined with the chronic exposure research model C.elegans,the joint intestinal toxicity of DON+Cd was evaluated by in vivo and in vitro models,and the mechanism of action was explored.A visual joint toxicity screening model based on the mechanism of action was established.The main research contents are as follows:1.Establishment and evaluation of in vitro model of intestinal toxicity of DON and Cd.The proliferation activity measurement of eight cell lines found that intestinal cells such as HT-29 and Caco2 cells showed a certain resistance to DON.The IC₅₀ values of DON against HT-29 cells fitted by Compu Syn for 12,24,and 48 h were 167.08,143.74,and 15.95?M,respectively,and corresponding IC₅₀ values of Cd were 157.47,44.42,and 6.60?M,respectively.Based on the IC₅₀ value analysis,DON was less toxic to HT-29 cells than Cd.However,based on the analysis of Fa-dose curve,DON at low dose level was more toxic than Cd.Considering the toxicity law of this time dimension,the in vitro multi-dimensional co-exposure model simulated the actual average dietary exposure ratio(1:1,?M:?M)and the reasonable intestinal concentration of DON(0.84-33.78?M),and based on the toxicological end effect(IC₅₀:IC₅₀,1/16 IC₅₀-IC₅₀),to analyze the overall toxicity trend of DON+Cd from the aspects of ratio,time and dose.The results showed that the overall toxicity of co-exposure to DON+Cd at the equimolar ratio for 24 and 48 h became stronger(?>1),and the overall toxicity at 12 h became weaker;the overall toxicity of co-exposure at the IC₅₀ ratio for 12 and 24 h became stronger(?>1),and the overall toxicity became weaker at 48 h(?<1),compared with the single toxin.This co-exposure model provided a basis for the subsequent comprehensive analysis of the joint effects of DON+Cd in the time dimension and the ratio dimension.2.Research on joint effects of DON+Cd based on the toxicity law of HT-29 model.CI model analysis showed that the joint effects of DON+Cd presented a time-oriented and dose-oriented change law.The two exposure ratios showed similar interaction trends in the long-term exposure(24,48 h),but there was difference in the short-term exposure(12 h).At the equimolar ratio,the low-dose of DON+Cd(DON?6.75?M)changed from synergism to antagonism with the increase of exposure time(12-48 h),while the high-dose showed the opposite trend of joint effects.At the ratio of IC₅₀,low or medium high level of DON+Cd(DON?1/2 IC₅₀)showed synergism to antagonism with the time of exposure(12-48 h),while the highest dose(IC₅₀)always showed synergism.Such change law of the joint effects provided the basis of dose and exposure time for the follow-up exploration of the joint toxicity mechanism of DON+Cd.3.Analysis of mRNA regulation and joint toxicity mechanism based on the joint effects of DON+Cd on HT-29 model.According to the above-mentioned changes of antagonism and synergism of DON+Cd in the time dimension,RNA-seq,flow cytometry,and RT-qPCR analysis were combined to reveal toxicity mechanism at the molecular level and the level of cellular functional response.At the molecular level,the overlap results analysis of DESeq showed that the regulation effect of DON on mRNA was affected by Cd.This regulatory effect was not an additive outcome,and the response in the time dimension confirmed to the aforementioned toxic change trend(DON vs DON+Cd,5133 mRNA/24 h,2643 mRNA/12 h).At the level of shared pathways,the signal cascade of TNF-MAPK-AP-1-IL-1B participated in the synergistic and antagonistic effects of DON+Cd.At the level of cellular functional response,DON+Cd up-regulated ROS,Ca²⁺,and MMP,which aggravated the oxidative stress imbalance,leading to compensatory up-regulation of tight junction Claudin-2 and Claudin-5.At the level of non-shared pathways,fat synthesis and metabolism(glycerophospholipids,arachidonic acid,steroids,glycosphingolipids),amino acids(phenylalanine,tyrosine),protein digestion and absorption,glycans,and immunity were related to synergism of DON+Cd;Glycosphingolipids,glycan,glycine,serine,and threonine,and immunity were related to antagonism of DON+Cd;Eight of the above ten kinds of toxicity pathways were related to fat metabolism,indicating that fat metabolism was an important toxic mechanism of the joint effects of DON+Cd.The above-mentioned mechanism analysis of TNF,MAPK,oxidative stress,and fat metabolism was verified by RT-qPCR,and will be further verified in the follow-up in vivo model and in vitro screening model.4.Construction of DON+Cd chronic co-exposure C.elegans model and analysis of its intestinal toxicity mechanism.On the basis of the aforementioned toxicity mechanism analysis of in vitro model,the intestinal toxicity of DON+Cd under stimulated actual average pollution levels on C.elegans was further analyzed to verify the in vitro toxicity mechanism of oxidative stress and fat metabolism.The results showed that the oxidative stress and fat metabolism interference of DON on C.elegans were important mechanisms of its intestinal toxicity,and Cd significantly enhanced the growth inhibition and feeding inhibition induced by DON,and the inhibition rate increased from 22.38%and 16.80%to 33.92%and 40.45%,respectively.Cd aggravated the intestinal ROS stress and lipofuscin accumulation,and weakened the intestinal hsp16.2 stress caused by DON.DON+Cd co-exposure significantly regulated genes of lipolysis and synthesis,oxidative stress,and metallothionein,such as POD-2(1.97×10³ times),CTL-2(1.62 times),MTL-1(11.81 times),and MTL-2(15.68 times).Through the correlation analysis of molecular stress indicators,it was found that ROS was highly negatively correlated with food intake level and body length development,and it was clear that there were 23 pairs of highly correlated genes in 12 stress molecules.It was confirmed that the intestinal toxicity effect of DON+Cd co-exposure was the joint effects of oxidative stress and fat metabolism,which was consistent with the toxicity mechanism results of HT-29model.5.Establishment of sensor screening method of joint toxicity of DON+Cd based on MAPK/AP-1 signal transduction.Based on the response of the aforementioned TNF-MAPK-AP-1-IL-1B pathway to DON+Cd,the AP-1 family stress situation was further analyzed.The specific binding site AP-1 site was used as a toxic pathway marker to establish joint toxicity screening model of DON+Cd.The results showed that DON significantly up-regulated six AP-1 transcription factor subunits,such as FOSL2,FOSB,FOS,and FOSL1.DON+Cd significantly affected the regulation of DON on FOSB and FOS.The subculture of the AP-1site-mCherry-HEK293 model based on the AP-1 site tandem sequence showed good viability,and the mCherry signal responded stably to positive substrates.Compared with DON or Cd,0.5-10.0?M of DON+Cd induced a significantly enhanced mCherry signal.This result further validated the MAPK/AP-1 toxicity pathway in the aforementioned HT-29 model,and this fluorescent cell sensor can be further used for simple visual screening of DON,Cd and their joint toxicity.In summary,this paper designed in vivo and in vitro models for DON+Cd co-exposure which simulated actual dietary exposure and toxicological end-point effects,and investigated the law of joint intestinal toxicity under short-term and long-term co-exposure.The work found that chronic co-exposure to DON+Cd at an average pollution level led to a synergistic enhancement of intestinal toxicity.The toxic mechanisms of synergism and antagonism of DON+Cd were clarified.Based on the transcription factor of the obtained toxicity signal pathway,a fluorescent cell sensor for simple,rapid,and visual screening of joint toxicity of DON+Cd close to the actual pollution level was successfully developed.This research lays the foundation for the joint toxicity research and joint toxicity screening of DON+Cd.