Study On The Mechanism Of Deoxynivalenol-induced Toxicity Of Human Kidney Epithelial Cells

Vomitoxin,also known as Deoxynivalenol（DON）,with a molecular weight of 296.32and a molecular formula of C₁₅H₂₀O₆,is a secondary metabolite produced by some bacteria such as Aspergillus and Penicillium,which is widely distributed in contaminated wheat,corn and other common grains and food feed.Although the national standard（2761-2011）stipulates that the limit of DON in cereals and cereal products is 1000μg/kg,the European Union has more stringent requirements,stipulating that the limit of DON residues is 750μg/kg.Although countries have set strict standards for the limit of DON in food,incidents of food contamination caused by DON are still frequently reported worldwide.The current research on DON is mostly focused on the rapid detection and degradation techniques of DON,but the risk assessment of DON on human health and its specific toxic effects and molecular mechanisms have not yet been reported in detail in the literature.Many animal models have shown that DON is enterotoxic,hepatotoxic,embryotoxic and immunotoxic.However,there are still few reports on the nephrotoxicity of DON.It is well known that the kidney plays an important role in human metabolism as a target organ for water-soluble substances,and DON,as a water-soluble toxin,has a practical reference value for the study of its nephrotoxic effects on humans.Therefore,in this study,human renal tubular epithelial cells（Human renal tubular epithelial cells,HK2）were used as the test cell line to investigate the cytotoxic mechanism of DON from the aspects of morphological appearance,physiology and biochemistry,oxidative stress,expression of apoptotic enzymes and apoptotic proteins and differential gene analysis after DON treatment,in order to provide a theoretical basis for better food safety standards and methods to reduce the toxicity of fungal toxins.The main findings are as follows:1.The cytotoxic effects of DON on HK2 were investigated in a comprehensive manner.MTT experiments results showed that the survival rate of HK2 cells decreased as the DON concentration increased.The correlation curve between the DON concentration and the survival rate of HK2 cells showed that the IC₅₀was 38.15μM,so the IC₅₀for this experiment was determined to be about 40μM.The subsequent experiments also used this concentration as the dosing concentration,and the optimal dosing time was determined to be 24 h.The Taipan blue staining further results showed that DON had a toxic effect on HK2 cells.Hoechst 33258 and DAPI fluorescence staining results showed that DON had an apoptogenic effect on HK2 cells;the results of AV/PI double staining method showed that as the concentration of DON increased,the number of viable cells decreased and apoptotic cells dominated.This means that DON has a significant toxic effect on HK2 cells and can trigger apoptosis.2.The effects of DON on intracellular antioxidant and inflammatory indices in HK2cells were assessed comprehensively.With increasing DON concentration,intracellular malondialdehyde（MDA）content,reactive oxygen radical（ROS）level,interleukin-6（IL-6）,interleukin-1β（IL-1β）and tumour necrosis factor（TNF-α）levels gradually increased;while intracellular superoxide dismutase（SOD）activity,catalase（CAT）activity,glutathione peroxidase（GSSG）content and adenosine triphosphate（ATP）levels decreased.This means that DON can cause significant changes in both antioxidant and inflammatory indicators in HK2 cells.3.The effects of DON on apoptosis-related enzymes and proteins in HK2 cells were comprehensively analyzed.The results showed that DON induced cells to promote the expression of caspase-2,caspase-3,caspase-6,caspase-8 and caspase-9 apoptotic enzymes,as well as the Bax,caspase-2,caspase-3,caspase-6,caspase-8 and caspase-9 apoptotic proteins and suppressed the expression of Bcl-2 apoptotic protein.This means that DON upregulated the expression of pro-apoptotic proteins and enzymes and downregulated the expression of anti-apoptotic proteins in HK2 cells.4.A comparative analysis of the genomic expression of DON-induced HK2 cells at 24h was studied using transcriptome sequencing technology（RNA-seq）.Using untreated HK2cells as control,a total of 5962 differentially expressed genes were induced by 40μM DON at 24 h.Among them,2813 genes were up-regulated and 3149 genes were down-regulated.The differentially expressed genes were also classified at three levels:biological process,cellular component and molecular function.The results showed that DON induced up-or down-regulation of differentially expressed genes in several pathways,including MAPK signalling pathway,apoptosis pathway,PI3K-Akt signalling pathway,calcium pathway and oxidative phosphorylation pathway in HK2 cells,causing changes in various metabolic pathways.This means that DON has toxic effects on HK2 cells mainly through MAPK signalling pathway,apoptosis pathway,PI3K-Akt signalling pathway,calcium pathway and oxidative phosphorylation pathway.