Cell-based Biosensor Evaluation Method Establishment And The Combined Toxicity Study With Mycotoxins

With the trends of global warming,the contamination of cereals with fungi has become more and more serious.The secondary metabolites produced by mycotoxins not only reduce the nutritional value of foods,but also inhibit the growth and reproductive performance of animals.As a matter of fact,when humans and animals are being exposed to mycotoxins,it is usually a mixture of compounds and not a single agent.Due to the combined effects caused by the coexistence of mycotoxins,it has brought huge safety risks and economic losses to people's daily life.In recent years,the investigation on the combined toxicity of mycotoxins has become a hot topic.In this paper,deoxynivalenol?DON?,zearalenone?ZEN?,and aflatoxin B₁?AFB₁?,which have the most widespread contamination and a tremendous threat to human health were chosen as objects,and a novel cell-based electrochemical biosensor was constructed for evaluating the toxicity of mycotoxins,then the combined toxicity of them was studied by traditional cytotoxic experiments comprehensively.At last,metabolomics was used in a cellular metabolic level to investigate the toxicity and damage mechanism of the co-exist mycotoxins.Firstly,an electrochemical biosensor for evaluating toxicity of mycotoxins was established.In this study,screen-printed carbon electrode was used as the working electrode,combined with the ultra-sensitive electrochemical impedance spectroscopy?EIS?.The developed method was much more sensitive than conventional cytotoxicity evaluation methods such as cell counting kit8?CCK-8?at low dose of toxins.Besides,the mechanism of the toxicity evaluation for the biosensor was studied,which indicated that the impedance value correlated with cell morphology,[Ca²⁺]_i concentrations,apoptosis and necrotic cells ratios.In this chapter,a low cost and protable Hep G2 cell-based electrochemical biosensor was developed,which could be used for evaluating the cytotoxicity of mycotoxins.The combined toxicity of co-exist DON,ZEN,and AFB₁ was determined.Based on three mathematical model including concentration addition?CA?,independent action?IA?,and Chou-Talalay's combination-index?CI?methods,being exposed to DON+ZEN or ZEN+AFB₁ shows antagonism effects.Being exposed to DON+AFB₁ or DON+ZEN+AFB₁ shows synergistic or additive effect.In addition,the results show that the binary and ternary mixtures can inhibit cell activity,lead to increasing the intracellular ROS levels and damaging the cells by oxidative stress,induce Caco-2 cell apoptosis or necrosis through significantly upregulating p53 gene,Bax,caspase-3,caspase-8,caspase-9 and downregulating Bcl-2 gene.The combined effects of the three mycotoxins was explored at the cellular and gene level through the probable similar interaction sites and signaling pathways,thus increasing the cytotoxicity of individual toxin.The exact determination of the combination type of mycotoxins could provide references for establishing and updating limit standards in the future.Finally,based on the synergistic dose of DON+AFB₁ and DON+ZEN+AFB₁,metabolomics was used to further analyze the mechanism of the combined effects of mycotoxin DON,ZEN,and AFB₁ on cellular level.By using gas chromatography-mass spectrometry technology?GC-MS?to detect cellular metabolic product of the Caco-2 cells stimulated by combined toxins,the significant metabolic differences of Caco-2 cell metabolites were screened by the multivariate variables statistical analysis.Pathway analysis showed that coexistence of low-dose mycotoxins could interfer cellular amino acid metabolism and protein synthesis,meanwhile,high doses of mycotoxins increased the effect on lipid and glucose metabolism,and enhanced cytotoxicity.Among them,the coexistence of mycotoxins below the limited dose of national standards has caused serious disorders on cellular metabolism,which deserves attention and needs further study.