Joint Toxicity Of Aflatoxin B₁and Sterigmatocystin In HepG2Cells

Different types of mycotoxins often co-exist in nature, it is possible to simultaneouslyexpose two kinds or more types of toxin in our daily lives, but there are few studies on jointtoxicity of different mycotoxins exposed to a living body. Here, aflatoxin B1(AFB1) andsterigmatocystin (ST) were selected to study their joint toxicity. Considering two toxins haveliver toxicity, HepG2cells were chosen as the cell model in this study. After HepG2cellswere treated by ST and AFB1alone and their combination, the damage of internal parts ofHepG2cells, the degree of apoptosis and changes in related protein expression wereexamined, to investigate the mechanism and the type of joint toxicity of ST and AFB1.Different concentrations of AFB1, ST and their mixture dissolved in DMSO were addedto the HepG2cells, and control group was also included by adding DMSO(0.1%). After24h,48h treatment, five indicators were measured:the cell proliferation, the ATP content,DNAcontent, mitochondrial membrance potential and reactive oxygen content. The combinedeffect of the two toxins was analyzed. We found that when HepG2cells were treated by thetwo toxins individually and combined, the cell proliferation, DNA content, ATP content aredecreased in a dose-dependent manner, while the content of intracellular ROS andmitochondrial membrane potential was increased along with the toxin dose. Statistically,analysis of the results showd an additive toxicity between AFB1and ST on the HepG2cells.The concentration of AFB1, ST and their mixture were determined when the cellproliferation inhibition rate was10%,30%and50%. After48h treatment, the morphologicalchanges of cell was observed after Hoechst33258staining, and the changes in mitochondrialmembrane potential, the cell cycle, and apoptosis rate were detected by flow cytometry with afluorescent dye. The Hoechst33258stain indicated a phenomenon of apoptosis.AFB1, ST andmixtures could induce HepG2cell cycle arrest. Different concentrations of the individualtoxin induce different cycle arrest, concentrated in the G0/G1phase and S phase, and theirmixture also induced G0/G1phase and S phase arrest. The increase of apoptosis rate and thedecreased of mitochondrial membrane potential were dose-related. After two toxins combinedexposure, the apoptosis are not significantly increase or decrease than individual effects,supporting an additive toxicity effect by AFB1and ST.The immunohistochemistry staining was used to detect the expression of Bax, Bcl-2,Caspase-3, Caspase-8, Caspase-9and p53after AFB1and ST treatment. The expression ofBcl-2protein shows a dose-dependent decrease. The expression of Bax, Caspase-3, Caspase-8,p53and Caspase-9are increased with the increasing concentrations of the toxins.The results showed that AFB1, ST can cause damage to DNA and mitochondria of thecells, and induce the apoptosis. The overall results indicated an additive toxicity effects onHepG2cells.