Combined Toxicity Evaluation And Intervention Of Aflatoxin B₁ And Cyclopiazonic Acid Toxin Based On A Novel Spheroid Model

In recent years,the increasing risk of synergistic toxicity from coexisting pollution with multiple mycotoxins in food has triggered widespread global concern.Cyclopiazonic acid（CPA）,an emerging toxin,is widely polluting peanuts,figs,corn and other cereals and oils,and has a co-exposure pathway with aflatoxin B₁（AFB₁）,the most widespread and toxic toxin,and some reports showed that the two might have synergistic toxicity.Therefore,it is important to investigate the synergistic toxicity evaluation and intervention control of AFB₁ and CPA.At present,the in vitro toxicity evaluation is still based on the adherent cell model,but the adherent-grown cells differ greatly from the in vivo microenvironment and have many shortcomings.The evaluation model constructed by multicell co-culture could better simulate the actual physiological environment in vivo compared with single cells,and could improve the accuracy of toxin toxicity evaluation.Therefore,we constructed and optimized a co-cultured three-dimensional（3D）hepatocyte spheroid model,compared the differences between the adherent model and the hepatocyte spheroid model,and selected the evaluation model that is closer to the in vivo microenvironment;for the toxicity evaluation of AFB₁ and CPA,we discovered the synergistic toxicity effect of CPA and AFB₁ and applied astilbin intervention to alleviate the synergistic toxicity of the toxin,which laid the foundation for toxicity evaluation and control.A summary of our work is as follows:1.Construction of a novel scaffold-free hepatocyte spheroid model and analysis of the advantages of its application to AFB₁ toxicity evaluation.Three different hepatocyte spheroid models were constructed by optimizing the cell initial number,cell ratio,culture time and other conditions with hepatic parenchymal cell Hep G2,endothelial cell EA.hy 926 and hepatic stellate cell LX-2.AFB₁ was used as the research object to compare the advantages of the hepatocyte spheroid evaluation model by testing several indicators.The results showed that AFB₁ caused significant cell membrane damage to adherent cells（0.01μg/m L,24 h;p<0.01）,which could also significantly affect the activity,proliferation activity and oxidative stress level of adherent cells（1.0μg/m L,24 h;p<0.05）.In contrast,hepatocyte spheroids were less susceptible to AFB₁.Combined with the analysis of gene expression,it was found that the cytochrome P450 enzyme and drug metabolizing enzyme activities of hepatocyte spheroids were higher than those of adherent cells,and that hepatocyte spheroids formed by triple co-cultured were better than those formed by monoculture.Therefore,it was hypothesized that triple co-cultured hepatocyte spheroids with strong liver function and high metabolic activity could be better self-regulated to cope with damage induced by external stimuli,more realistically mimicking the in vivo microenvironment,and could be used as an evaluation model for subsequent toxicity studies.2.The combined toxicity and mechanism of AFB₁ and CPA were investigated based on the above-mentioned co-cultured hepatocyte spheroid.Based on the toxic effects of AFB₁ and CPA exposure separately,two modes of equal toxicity exposure and different ratio exposure were selected to stimulate for 48 h to investigate the combined toxic effects of AFB₁ and CPA.CI model analysis showed that the combined toxicity of AFB₁+CPA was more influenced by the dose of toxin and less by the concentration ratio.At the of IC₅₀,AFB₁ and CPA exerted synergistic effects at low doses(<1/8 IC₅₀)and shifted to antagonistic effects at high doses(>1/8 IC₅₀).A similar trend of combined effect was obtained for mitochondrial and reactive oxygen species（ROS）levels tests.Gene expression analysis showed that the shift in the combined effect might be related to AKT gene and the biological activity of CPA.Compared to normal cells,the activity of cells overexpressing AKT gene（13.28-fold）was significantly increased by toxin treatment for 48 h（AFB₁:3.06%,p<0.001;AFB₁+CPA:3.36%,p<0.05）.3.Metabolomics was used to analyze the metabolic synergistic mechanism of AFB₁ and CPA on the hepatocyte spheroid.Based on the previous experiments,two dose combinations of0.625μg/m L AFB₁+3.125μg/m L CPA(1/16 IC₅₀,synergistic)and 5μg/m L AFB₁+25μg/m L CPA(1/2 IC₅₀,antagonistic)were selected to study the metabolic combined mechanism of AFB₁and CPA.The results showed that the metabolic profile of the toxin group was significantly different from that of the control group and multiple metabolites and metabolic pathways were affected by the toxin.At the synergistic concentration point of low doses,the metabolic levels of energy metabolism-related substances such as ethanolamine and L-aspartic acid were significantly down-regulated（p<0.001）,while high doses of toxin also significantly affected the metabolic levels of antioxidant substances（p<0.01）.The results of metabolic pathway enrichment also indicated that AFB₁ and CPA perturb amino acid metabolism（alanine,aspartic acid,glycine,serine,etc.）to a greater extent and might further disrupt the intracellular redox homeostasis.In addition,the CPA-containing toxin group disturbed further metabolic pathways and might also affect lipid and sugar metabolism via the tricarboxylic acid（TCA）cycle.4.To investigate the effect of astilbin intervention on AFB₁ and its combined toxicity with CPA.It was found in the previous study that AFB₁ and CPA co-exposure affects intracellular redox levels and have synergistic toxic effects.Therefore,astilbin,a plant extract with antioxidant activity,was chosen to intervene at the synergistic concentration point(1/16 IC₅₀)of AFB₁ and CPA.The results showed that astilbin helped to maintain cellular activity after toxin exposure,and the highest reduction of cellular inhibition in hepatocyte spheroid was 9.36%（p<0.001）.The early intervention was less effective than simultaneous co-exposure with toxin.In addition,astilbin increased the intracellular mitochondrial content and promoted energy metabolism in the combined toxin-treated group.The antioxidant capacity was more remarkable,with an exposure concentration of 10μg/m L significantly reducing the toxin-induced increase in intracellular ROS levels（p<0.001）.Metabolomic analysis revealed that astilbin intervention affected intracellular amino acid metabolism,glucose metabolism and lipid metabolism,resulting in a metabolic profile more similar to that of the control group.In conclusion,we constructed a 3D co-cultured hepatocyte spheroid model and used it for toxicity evaluation of mycotoxins,providing an novel model for toxicity evaluation with higher biomimetic.The article also explored the combined toxic effects of the emerging mycotoxin CPA when co-exposed with AFB₁,providing some theoretical basis for the scientific formulation of CPA limit standards.In addition,the AKT gene overexpression and astilbin treatment provide ideas for the inhibition or mitigation of AFB₁ and CPA toxicity.