Joint Cytotoxicity And Mechanism Of Mycotoxins（AFB₁、DON） In HepG2/C3A Cells

It is of high possibility for human and animals to be exposed to multiple mycotoxins since food and feeds can be contaminated with various fungal species and there are so many different mycotoxins all over the world. Deoxynivalenol（DON） and Aflatoxin B1（AFB₁）, which are commonly co-existed in cereal-based ingredients or foods, are chosen to study their combinative cytotoxicity and toxic mechanisms on HepG2/C3 A cells.According to the IC50 values of DON and AFB₁ on HepG2/C3 A cells after treatment for 24 h and 48 h measured by a SRB method, different concentrations of DON, or/and AFB₁ were chosen to analyze the type of combined effects between DON and AFB₁ through five cytotoxicity endpoints including cell proliferation inhibition rate（IR）, double-stranded DNA content, reactive oxygen species（ROS） level, ATP content and mitochondrial membrane permeability（MMP）. The results showed that the values of IC50, based on the sulforhodamine B（SRB） assay, were 4.90±1.20 μmol/L and 4.50±0.90 μmol/L for DON（24 h and 48 h）, 58.30±6.70 μmol/L and 18.70±3.80 μmol/L for AFB₁（24 h and 48 h）, respectively. DNA content and ATP content were decreased in a dose-and time-dependent manner, while the cell proliferation inhibition, the level of intracellular ROS and mitochondrial membrane permeability were increased. Statistically analysis of five cytotoxicity endpoints revealed an additive cytotoxicitic relationship between DON and AFB₁ on the HepG2/C3 A cells.Cell apoptosis and possible toxicity mechanism were studied by measurement of cell cycle distribution, cell apoptosis, and the mRNA level of cell apoptosis related genes after 24 h treatment of DON and/or AFB₁ at relatively lower dosages. The results showed that 0.56 μmol/L DON induced HepG2/C3 A cell arrest at G₂/M phase while 2.5 μmol/L AFB₁ and their combined group induced cell arrest at S and G₂/M phases. The increase of apoptosis rate was dose-related and the combined effect was significantly higher than individual exposure. The apoptosis rate of HepG2/C3 A cells arrested in S（by AFB₁） or G₂/M（by DON） phase after exposure to mycotoxins also showed an additive apoptotic effect. In addition, the increased expression of caspase-3, Bax, GADD153 and c-JUN as well as the decrease expression of Bcl-2 further supported their additive toxic effect. The mRNA level of Bax was significantly induced by AFB₁ while DON caused the increase of c-JUN mRNA level indicating DON might induce apoptosis of HepG2/C3 A cells through MAPK pathway in contrast to p53-mediated apoptosis by AFB₁ as reported in literature.RNA-Seq, currently the advanced method for transcrptomics study, after exposed to 0.56 μmol/L DON, 2.5 μmol/L AFB₁ and their combination for 24 h was carried out for a deep understanding the molecular mechanism of their combinative cytotoxicity through analyzing the gene expression of the whole genome of liver cells The results showed DON mainly induced cell apoptosis through JNK and p38 MAPK and endoplasmic reticulum stress pathway which caused the DNA damage, AFB₁ mainly induced the apoptosis by mitochondrial damage through p53 pathway, while their combined effect was mainly mediated by JNK and p38 MAPK pathway, endoplasmic reticulum stress pathway and p53 pathway. In their combination effect, AFB₁ played a leading role that cell apoptosis was mainly induced depending on the vital role of p53 pathway.In this study, the main novelty was that transcriptomics was used the first time for studying the combined cytotoxicity mechanism of DON and AFB₁ in HepG2/C3 A cells, and the whole genome sequencing was carried out using the modern RNA-Seq technology, which provided the basis for further molecular understanding of the cytotoxicity mechanisms of DON, AFB₁ and their combinations.