Combined Toxicity Of Aflatoxin M1 And Ochratoxin A On Intestine And Kidney

Aflatoxin M1(AFM1)is the only mycotoxin in milk with specific established maximum residue limit.However,in recent years,it has been found that AFM1 in milk interacts with other mycotoxins,especially ochratoxin A(OTA),the existence of such interaction may pose a serious threat to human health,but their interaction mechanism is not clear.In this study,intestinal and renal models were used to analyze the differences in gene,protein,and metabolite caused by the two toxins and their combinations,the results may contribute to exploring the mechanism of the effect of AFM1 and OTA.Intestinal mucin is the main component of intestinal mucous layer,which plays an important role in maintaining intestinal mucosal homeostasis.For this reason,the effects of AFM1 and OTA on the cellular activity,cellular layer structure,transmepithelial electrical resistance(TEER)and the changes of MUC2,MUC5 AC and MUC5 B mRNA and protein levels of Caco-2/HT29-MTX(100/0;90/10;75/25;0/100)were analyzed in this study.The main results are as follows:Cytotoxicity of AFM1 and OTA shows dose-dependent: single and combined mycotoxin AFM1 and OTA have little effect on cells at low concentration(0.05 μg/mL),while at high concentration(4 μg /mL),the values of cell viability and TEER were significantly reduced,the intestinal mucin mRNA expression level and mucin abundance were significantly changed.In addition,OTA showed toxicity similar to or even higher than AFM1 at the same concentration.The interaction effects of the two mycotoxins on intestinal cell viability and mucin abundance were evaluated,synergistic effects were shown in the experiment.Moreover,there was a significant positive correlation between cell viability and TEER value in all culture models(p < 0.01).The main target organ of OTA is the kidney.OTA not only causes nephrotoxicity,hepattoxicity,neurotoxicity,and immunotoxicity,but also may teratogenic,carcinogenic,mutagenic,and poses a serious threat to human health.There is few data to assess the renal toxicity of AFM1 or it work with OTA.Therefore,in this study,metabolomics technology were used to investigate the effects of AFM1 and OTA on renal function.The main results are as follows:After 35 days of treatment with 3.5 mg/kg b.w.of toxin,compared with the control group,both AFM1 and OTA,alone or in combination,significantly reduced the final weight of the mice,while the OTA group and the combined treatment group significantly reduced the relative renal coefficient of the mice.The abnormality of serum biochemical values also indicated kidney injury in mice.HE staining and Masson staining of kidney sections showed kidney damage in mice.Metabolomics analysis showed that the metabolites and metabolic pathways with significant changes in the combined treatment group were more than those in the single AFM1 group,but there was no significant difference between the OTA group and the combined treatment group.In the HK-2 cell model,the combination of AFM1 and OTA at low toxic concentration showed antagonistic effect,which caused the up-regulation of LysoPC levels through SAPK/JNK cell signaling pathway,leading to cell apoptosis.The above experimental results provide strong evidence that individual and combined AFM1 and OTA damage the intestine and kidney.In addition,the toxicity of OTA is higher than AFM1 at the same concentration.Although it may be caused by different sensitivities of various measurement indicators,sufficient attention should be paid.When the two mycotoxins act in combination,additive,synergistic and antagonistic effects were observed,with synergistic effects in the majority.This study is helpful to optimize the maximum allowable limits of mycotoxins in milk to some extent,but the combined effect of AFM1 and other mycotoxins in milk and dairy products remains to be further studied.