| Aflatoxin and fumonisin are the most common mycotoxins in cereal, among them,aflatoxin B1(AFB1), as a class of secondary metabolites produced by Aspergillus flavus andparasitic fungus, is considered as the most toxic mycotoxins known currently. AFB1can bemutagenic by causing DNA damage, and can also cause induced liver cancer. Fumonisin B1(FB1) is a major component of fumonisin which can lead to hepatotoxic and kidney damage,and studies have shown that the incidence of human esophageal cancer is also associated withFB1. Given the fact that two toxins often coexist in a variety of cereals and their products, andtheir common target cells are liver cells, the human hepatoma cell line HepG2was chosen asthe object to study their joint toxicity and apoptosis pathway induced by AFB1and FB1, andtheir mixture. The study would provided a foundation for the further study on the overalltoxicity when a variety of mycotoxins are co-existed as well as their mechanism of jointaction. Meanwhile, the study can also provided a reference for human risk assessment forcoexistence of different mycotoxins.HepG2cell lines which was in the logarithmic growth phase was treated with differentconcentration of AFB1(0.1、10、50、100μmol/L) and FB1(0.1、7、14、70μmol/L) for24hor48h to analyze their joint effect on HepG2cells through measuring the change of cellactivity, ATP content, degree of DNA damage, change of mitochondrial membranepermeability and active oxygen content. The results showed that, in the SRB assay, the ratioof the theoretical value and the actual measured value of the inhibition rate caused by mixedtoxins on HepG2are close to1, and there was no significant difference(p>0.05) between thetheoretical value compared with the actual value of the other four indexes, and the overalltoxicity was greater than a single toxin of either one. Therefore, the joint toxic effect of AFB1and FB1on HepG2cell lines was additive to each other.Selected the corresponding concentration of the three toxin group AFB1、FB1andAFB1+FB1, which caused inhibition rate for10%,30%,50%towards HepG2. After reactingwith cells for48h, the changes of HepG2cells’ nuclear morphology, apoptosis rate, cycledistribution and mitochondrial membrane potential was futher investigated. The resultsshowed that, after reacting with various of concentration of AFB1and FB1for48h, thestained nuclei of HepG2appeared some phenomenon of apoptosis such as fragmental thickdense or half moon agglomeration by laser scanning confocal microscope. In the assay ofapoptosis rate, various degrees of apoptosis for HepG2could be caused by AFB1and FB1mixed and separate groups, and there were significant differences(p <0.01) compared with thecontrol group. Meanwhile, the apoptosis rate in each group was tconcentration-dependent.Cellcycle assay showed, FB1mainly impacted on the G0/G1phase in the cell cycle, AFB1andAFB1+FB1groups mainly impacted the S phase. Detection of mitochondrial membranepotential by JC-1showed that, after the two toxins acting alone or mixed on HepG2cells for48h, the mitochondrial membrane potential had a different degree of reduction compared tothe control group, and the degree of reduction for membrane potential increases as the toxins’concentration increasd. The T-test showed no significant difference for the extent of decrease in mitochondrial membrane potential and apoptosis rate caused by the three toxingroups(p>0.05), further evidencing the additive joint effect of the two toxins.The expression of protein related to apoptosis including caspase-3, caspase-8, caspase-9,bcl-2, bcl-x and p53were measured by immunohistochemistry to analyze the molecularmechanism of apoptosis in HepG2caused by the three groups of toxin. The results showed,all of AFB1、FB1and AFB1+FB1induced apoptosis under the control of p53protein. On onehand, they used the death receptor to activate apoptosis promoter caspase-8and executorcaspases to enter in the apoptosis program, on the other hand, the stimulation of toxins wouldlead to an increased expression of pro-apoptosis protein Bax, a decreased expression ofanti-apoptosis protein Bcl-2, and it would also cause the decline of mitochondrial membranepotential, release of cytochrome C, activation of prompter caspase-9and executive caspases,and eventually led to cell crash by proteolytic enzyme. All these results showed that thesetoxin groups can cause the expression of apoptosis pathway to cause cell death. |
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