| Aflatoxin contamination in dairy products has become a growing concern worldwide.AFB1 and its metabolite AFM1 are frequently contaminated in dairy products and are extremely toxic.Recent studies have found that aflatoxin residues in dairy products are potentially serious threat to the growth and development of infants and children.The specific mechanisms of AFB1 and AFM1-induced developmental disorders are still unknown,and there are few studies on AFM1.In this study,zebrafish was used as the research object to explore the developmental toxicity of AFB1 and AFM1 in dairy products,and based on this,a risk identification method for aflatoxin in dairy products was established based,which can be applied to the identification and monitoring of actual dairy product safety risks,so as to provide basis on the risk assessment of dairy products.The main research contents and results are as follow:The developmental toxicity to zebrafish embryos was preliminarily analyzed by morphological ind ex determination,and the toxic target organs and sensitive toxic effects were clarified.Both AFB1 and AFB1 were found to cause developmental malformations such as spinal curvature and delayed yolk sac absorption in zebrafish embryos,resulting in embryonic malabsorption syndrome,suggesting developm ental disorders in zebrafish embryos.The main target organs of both are the liver and nerves,and the del ayed yolk sac absorption(hepatotoxicity)is the most sensitive indicator of toxic effects.The combined results showed that AFB1 exhibited a stronger developmental toxicity.Adverse effects of AFB1 and AFM1 on development were comprehensively analyzed from the pers pective of endogenous metabolism by means of metabolomic and transcriptomic methods.It was found that both AFB1 and AFM1 caused upregulation of most amino acids and glycerophospholipids and inter fered with 26 amino acid metabolic pathways including phenylalanine metabolism,glycerophospholipid metabolism and fatty acid-related metabolic pathways,which are linked to energy metabolism,oxidativ e stress,liver damage and neurodevelopment.In addition,AFB1 had a greater interference with amino acid and lipid metabolism,and in particular,pathways such as fatty acid degradation were enriched only in the AFB1-treated group,which was strongly associated with inflammation development and develo pmental toxicity.Effects of AFB1 and AFM1 on liver development were analyzed by histopathological examination and transcriptomic analysis was performed to investigate the mechanisms involved.Results showed that AFB1 and AFM1 interfered with the PPAR signalling pathway,thereby downregulated the expression of genes related to fatty acid transport(cd36,fabp and scp2a)and lipid transport(apoa4a),led to increa se expression of lipid metabolism-related genes(ptgs2a and socs3),and resulted in a disruption in the metabolism of the major lipid of the yolk sac(PC)and its precursor fatty acid(arachidonic acid).The disruption of lipid metabolism mediated by the PPAR signalling pathway further exacerbated the inflam matory response,which was associated with greater toxicity of AFB1 to zebrafish embryos.Toxic effects of AFB1 and AFM1 on neurodevelopment and locomotor behaviour were analyzed by behavioural analysis and biochemical index measurements.Results showed that AFB1 and AFM1 in duced malformations in the swim bladder,spinal curvature of the embryos and were accompanied by a s uppression of locomotor behaviour.This mainly due to alterations in synaptic transmission-related gene s(ddc,fosab,etc.)that interfered with neurotransmitter regulation and synaptic transmission,and alterat ions in tail and body development-related genes(mmp9 and igfbp1a)that caused abnormal development of locomotor organs.The greater alteration of fosab,mmp9 and igfbp1 a genes in the AFB1-treated group was associated with greater neurotoxicity and behavioural inhibition.Above results showed that the use of relevant developmental aberrations indicators can sensitively identify the developmental toxicity of AFB1 and AFM1 in the liver and neurology and are reliable for as sessing the risk of AFB1 and AFM1.Therefore,a method for the identification of aflatoxin risk in dairy products was developed by combining these indicators and assessing the toxicity of actual dairy powder samples.The results showed that the method was reliable,with all six dairy powder samples having an overall deformation rate of 10%,which is a low risk.In addition,the toxicity was promoted by the dairy powder matrix at high doses,suggesting that further attention needs to be paid to the promoting effect of the dairy powder matrix. |
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