Cytotoxicity And Degradation Of Mycotoxins In Milk

Aflatoxins(AFs)is a secondary metabolite of Aspergillus species sush as Aspergillus flavus and Aspergillus parasiticus,including aflatoxin B1(AFB1),aflatoxin B2(AFB2),aflatoxin G1(AFG1)and aflatoxin G2(AFG2).Natural environment such as drought,high temperature,storage time and conditions affect the growth and emergence of AFs,among them AFB1 was the most toxic,which has carcinogenic,teratogenic,mutagenic effects.Feed and its raw materials often contain a variety of mycotoxins,and one kind of mycotoxins also can produce a variety of toxins.The toxicity of Ochratoxin A(OTA)is same as AFM1,also exist in milk.Lactoferrin(LF)is an active ingredient in milk,which also can be found in human saliva,tears,bile,plasma,mucous membrane and genital secretions,and have multiple physiological functions such as broad-spectrum antibacterial infection,regulate bone marrow cell growth,promote normal cell growth and regulate the balance of iron and so on,its antioxidant activity on the body has a positive effect.Therefore,this study focuses on researching the combination toxic of OTA and AFM1,discussing the damage of AFM1 and AFB1 on Caco-2,HEK293 T,Hep-G2 and SK-N-SH cells,and discussing the inhibitory effect and its mechanism of LF on cytotoxicity for AFs.The main research contents and results are as follows:1.Differentiated Caco-2(human colorectal adenocarcinoma)cells were cultured and treated in the following groups: AFM1(12 ?M),OTA(6 ?M),AFM1(12 ?M)+OTA(6 ?M).Cell viability and proliferation were detected by Alamar-Blue method,the three group data was 95.59%,93.14% and 81.57%,respectively.Cell apoptotic rate was quantified by Annexin V-FITC/PI staining through flow cytometry,apoptotic rate of the three group was 25.68%,33.7% and 37.4%,respectively.The results showed that AFM1 and OTA have a synergistic effect in inhibiting the proliferation of differentiated Caco-2 cells,and inducing cell apoptosis.What's more,Intracellular ROS(reactive oxygen species,ROS)release was detected by active oxygen fluorescent probe,AFM1 group was 1.37-fold of control group level,in OTA group was 1.79-fold of control group level,and the one in combined group was 6.21-fold of control group level,and the mitochondrial membrane potential was detected by a JC-1 probe.Additionally,the protein of Caspase-3 and Caspase-9 were detected by Western blotting.Expression levels of mitochondrial membrane potential and apoptotic proteins suggested apoptosis of differentiated Caco-2 cells was induced through mitochondrial pathways,moreover,this induction seemed to be more obvious in the combination group.2.The Caco-2,HEK293 T,Hep-G2 and SK-N-SH cells were exposed to 0.05-4 ?g/m L AFB1 or AFM1 for 24 h,respectively.Cell viability was in dose-dependent mannersin four cells(p< 0.05),which treated with AFB1 was lower than AFM1 in the same condition on Caco-2,HEK293 T and SK-N-SH cells,suggested that cytotoxicity of AFB1 was higher than AFM1,however the results were opposite to HEK293 T cells.3.The Caco-2,HEK293 T,Hep-G2 and SK-N-SH cells were also exposed to 10-100 ?g/m L LF,but the survival rate was not in dose-dependent(p< 0.05),the optimal concentration was 100 ?g/m L.Considering about Lactate dehydrogenase(LDH)and glutathione(GSH)release which promoted to play an antioxidant effect,the optimum concentration was 1000 ?g/m L.4.The purpose of this study was to evaluate the AFB1 and AFM1 induced cell toxicity by determining cell viability,membrane permeability and genotoxicity,and then investigate the capacity of LF to protect cells against AFB1 and AFM1.Data showed that 4 ?g/m LAFB1 or AFM1 could significantly inhibit Caco-2 cells,HEK293 T cells,Hep-G2 cells,and SK-N-SH cell growth,increase LDH and cause genetic damage(p<0.05).In comparison,AFB1 was found to be more toxic than AFM1 on all four cells especially on Hep-G2 cells.Significant reductions in cytotoxicity and oxidative DNA damage were observed when cells were pretreated with 10,100 or 1000 ?g/m LLF then exposed to 4 ?g/m LAFB1 or AFM1.Our data suggested that AFB1 or AFM1 induces DNA damage in Caco-2 cells,HEK293 T cells,Hep-G2 cells,and SK-N-SH cells,whereas that the antioxidant activity of LF may contribute to the inhibition of AFB1 or AFM1-induced cytotoxicity and DNA damage by reducing oxidative stress.5.Protein expression was detected by Western blot,and the results were shown that LF inhibited AFB1 or AFM1-induced cytotoxicity and DNA damage by reducing oxidative stress,and the mechanism was related to the expression of ERK1/2 and JNK proteins in MAPK signaling pathway.