Toxic Effects Of Aflatoxin M1 And Its Coexistence With Aflatoxin B1 In Differentiated Caco-2 Cells

Mycotoxins contamination in crops and food occurs worldwide posing a serious threat to human health.The gastrointestinal tissue is the first target to be exposed after the mycotoxins they were taken in to the body.As the largest digestive organ,the intestine is of great significance for the absorption,transport,and metabolism of nutrients.It also resists harmful substances from entering and causing damage or dysfunction,which depends on the intact and perfect structure,and effective functions of the epithelial cells.After ingestion of contaminants,such as toxins,intestinal epithelial cells are exposed to toxins at relatively higher concentrations than other tissues and cells;they are also more susceptible to contaminants due to the high protein turnover rate and metabolic activities.Among these mycotoxins,aflatoxins(AFs)are the most common and toxic,of which aflatoxin B1(AFB1)is the most threatening component and the strongest carcinogen in mammals.Aflatoxin M1(AFM1)is the hydroxylated metabolite of AFB1 and is the only mycotoxin with specific established maximum residue limit in milk and various dairy products worldwide.With the increasement of daily consuming proportion of cow milk and various dairy products,and the growing diversity and complexity of human diet,human could be exposed to these two mycotoxins.Consequently,the potential risk of exposure to both of the toxins could not to be underestimated,which may also become a new content of food safety risk assessment.However,compared with the individual effects of AFB1 or AFM1,the potential risks and toxicity mechanisms of their combination need to be explored further.Thus,in order to study the toxic effects of AFM1 and its cytotoxicity in the presence of AFB1 on intestinal epithelial cells,the effects of exposure to 0.5-8 μg/mL AFM1 for 48 h on Caco-2 cell viability was first investigated.The changes of transcriptome levels after differentiated Caco-2 cells were exposed to different concentrations of AFM1 were also analyzed.Based on the results of this experiment and literature data,the toxicity mechanism of AFM1 on Caco-2 cells at a concentration that did not cause significant reduction to cell viability was studied.Subsequently,the transepithelial electrical resistance(TEER)and FITC-dextran(4 and 40 kDa)paracellular flux were measured;then the changes in transcription and protein levels of tight junction were examined,and immunofluorescence staining was performed to observe the localization of tight junction proteins.Combined with the results of our experiment,the combined effects of AFM1 and AFB1 in differentiated Caco-2 cells were discussed.The possible mechanisms of toxins changing the subcellular localization of tight junction proteins were explored.The main results are as follows:1.AFM1 posed cytotoxicity in differentiated Caco-2 cells with a dose-dependent effect: AFM1 at concentrations higher than 4 μg/mL significantly decreased cell viability;the disturbance of 0.0005 and 0.005 μg/mL AFM1 to the whole transcriptome on Caco-2 cells were negligible.By trend analysis,differentially expressed genes(DEGs)were mainly clustered into profile 0 and profile 3,two profiles presenting downward trend.GO term annotation indicated that in the biological process,DEGs were mainly enriched into cellular processes and metabolic processes;in the molecular function class,DEGs were clustered for binding and catalytic activity.STRING analysis revealed that these candidate DEGs were involved in the regulation of cell cycle process.q-PCR was conducted to verify the candidate DEGs,the expression trend was consistent with that in RNA-seq.AFM1 could cause cell cycle arrest in Caco-2 cells,which was related to the dosage applied in the study.It was demonstrated that disturbed cell cycle phase distribution was not associated with cell apoptosis(or DNA damage),decreased cell viability,or cell membrane breakage.AFM1-induced cell cycle block at S-G2 phase in differentiated Caco-2 cells is linked to the perturbation of CDK1,p-Akt,and p-AMPK signaling molecules.2.Caco-2 cell viability in AFB1 and AFM1 collectively treated group were significantly lower than that in the control group and in the single toxin treated group(p<0.05).AFM1 significantly reduced the TEER values and increased the permeability of differentiated Caco-2 cell monolayers in a dose-dependent manner.Toxins at relatively lower concentrations tend to promote mRNA levels of tight junction,while at higher doses,toxins could inhibit CLDN3,CLDN4,OCLN and TJP1 expression levels and tight junction protein abundance in differentiated Caco-2 cells.TEER,FITC-dextran(4 and 40 kDa)and TJ proteins(C-3,C-4,Occ and ZO-1)were analyzed to prove significant correlation.AFM1 and AFB1 influenced the distribution of TJ protein individually and collectively,and TJ proteins were found a certain degree of internalization-from membrane localization to cytoplasmic localization.Toxin-induced cell membrane damage could cause changes in localization of tight junction proteins,and clathrin-mediated endocytosis may have a role in this biological process.Through the two parts of experiments,we came to conclude that AFM1 could cause cell cycle arrest;its cytotoxicity was enhanced in the presence of AFB1,impairing the tight junction between differentiated Caco-2 cells.These demonstrated the toxic effects of AFM1 in differentiated Caco-2 cells.