Combined cytotoxicity of aflatoxin B1 and deoxynivalenol to hepatoma HepG2/C3A cells

Co-occurrence of aflatoxin B1 (AFB1) and deoxynivalenol (DON) is one of the most common mycotoxin contaminations in cereal crops and food ingredients. However, the mechanism of their combined toxicity is poorly understood. In the current investigation, the hepatoma HepG2/C3A cell line was used to explore the combined cytotoxicity of AFB1 and DON. The values of IC50, based on a sulforhodamine B (SRB) assay, were 4.5 mM and 18.7 mM for DON and AFB1, respectively. The analysis of cytotoxicity endpoints using the combination index (CI) theory revealed that the changes of mitochondrial membrane permeability and ATP resulted from an additive cytotoxic effect (CI≈1) of AFB1 and DON. However, the endpoints of double strand DNA (ds-DNA), reactive oxygen species (ROS) and cell viability (SRB) were synergistically (CI<1) affected in a dose-dependent manner. RNA-seq analysis demonstrated a number of uniquely expressed genes in their combination (AFB1: 2.5 mM, DON: 0.56 mM), indicating a synergistic interaction between AFB1 and DON at a molecular level. Additional transcriptomics analysis showed that the endoplasmic reticulum stress-associated JNK/p38/MAPK pathway was induced by DON, whereas the p53 signalling pathway was activated by AFB1. The expression profiles of apoptosis-related genes caspase-3, Bax and Bcl-2 suggested a mitochondria-mediated apoptosis pathway that was shared between AFB1 and DON. Thus, different cytotoxicity pathways and their converging at the apoptotic process might be the mechanism of the additive/synergistic cytotoxicity of AFB1 and DON to HepG2/C3A cells.