Citreoviridin Induces ROS-dependent Autophagic Cell Death In Human Liver HepG2 Cells

Objective: Citreoviridin(CIT) is a toxic secondary metabolite in mouldy cereals, such as rice and corn. In the developing world, where climatic and crop storage conditions are frequently conducive to fungal growth and mycotoxin production, therefore, citreoviridin contamination may represent a major public health problem considering the high consumption levels of rice.Liver is one of the primary organs responsible for detoxification and the target for some mycotoxins. CIT mainly concentrates in central nervous system, liver, kidney, and heart after injection in animals. Compared with its neurotoxicity and cardiotoxicity, hepatotoxicity of CIT has been less investigated and remains largely unknown. Autophagy is a hot topic in recent years, autophagy is a highly conserved cellular process in which cytoplasmic materials, including organelles, are sequestered into double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation or recycling. Although autophagy was initially considered a temporary survival mechanism for normal and cancer cells under conditions of nutrient limitation and metabolic stress, several studies have shown that persistent stress may lead to an intensification of autophagy that results in cell death, which is defined as autophagic cell death.The purpose of this study was to investigate whether CIT altered autophagic process and which role autophagy served, cytoprotective or pro-death, in hepatocyte. This study may provide insights to understand the molecular mechanisms of CIT hepatotoxicity.Methods: In this study, the metabolically competent human hepatoma HepG2 cells were used to investigate the underlying mechanisms of CIT hepatotoxicity. HepG2 cells were pretreated with the autophagosome formation inhibitor 3-MA, the autophagosome formation stimulator rapamycin, knockdown of Atg5 with Atg5 siRNA and the ROS inhibitor NAC to assess the changes in cell viability after treatment with CIT. The phase-contrast images were used to observe HepG2 cells. We utilized transmission electron microscope to observe the ultrastructure of CIT-treated HepG2 cells. The autophagosome was stained green under the fluorescence microscope by the Cyto-ID® Autophagy Detection Kit. The expression of microtubule-associated protein light chain 3(LC3) and P62 in HepG2 was shown by the results of Western blot assay. The turnover assay allows the estimation of autophagy flux, which is important for distinguishing whether the accumulation of autophagosomes is due to increases in the formation or blockage of the degradation. 2 ’, 7 ’ – dihydro- dichloro within fluorescein(DCFH) was used to detect intracellular ROS levels in CIT-treated HepG2 cells.Results: In this study, CIT caused a dose-dependent decrease in cell viability in HepG2 cells. The autophagosome formation inhibitor 3-MA, knockdown of Atg5 with Atg5 siRNA and the ROS inhibitor NAC increased the cell viability of CIT-treated HepG2 cells. The autophagosome formation stimulator rapamycin decreased the cell viability of CIT-treated HepG2 cells. The phase-contrast images verified the results derived from the MTT assays. We utilized transmission electron microscope to observe the presence of autophagic vesicles which are double- or multiple-membrane vacuoles containing cytoplasmic cargo with uneven electron-dense to be degraded. CIT increased AVs number significantly. The number of autophagosome-positive green puncta increased significantly compared with the control. The expression of LC3-II in HepG2 cells increased markedly as shown by the results of Western blot assay.The expression of P62 in HepG2 cells decreased markedly. The chloroquine-induced LC3-II accumulation was further elevated by LC3-turnover. These data indicated that the increased autophagosome level was caused by increased formation rather than impaired autophagosome clearance. Pretreatment with 2 mM NAC for 1 h reversed the increased expression level of LC3-II in 5 ?M CIT-treated cells. The treatment of HepG2 cells with CIT resulted in significant increased intracellular ROS levels in this study. The pretreatment of cells with the ROS inhibitor NAC at a concentration of 2 mM caused the ROS level in the CIT-treated cells to decrease significantly.Conclusion: We demonstrated that CIT increased autophagosome formation in HepG2 cells. Autophagy was determined to be involved in the death of HepG2 cells caused by CIT, and ROS was demonstrated to play a vital role in this process.