TCBQ Induces Caspase 8/T-Bid-dependent Apoptotic Signaling Pathway In PC12 Cells

Tetrachlorobenzoquinone (TCBQ) was a common preservative and pesticide products in the past. It can harm to human health through the food chain. Previous reports reveal that:TCBQ can induce reactive oxygen stress and genotoxicity in HepG2 cells, but it is not clear of its neurotoxicity. So it is important for clinic supervision to study its toxicity mechanism and new measures to attenuate its toxicity.The quinoid structure can produce large amounts of reactive oxygen species (ROS) in vivo oxidation. The oxidative stress can damage oxidative enzymes, amino acids and protein, and can cause disease such as senility, cancer, mutation and inflammation. ROS, which affect the biochemical processes in the cells, is an important factor inducing apoptosis. Cell apoptosis is an active process, which is an order response to physiological and pathological stimuli, environmental condition changes or palliative injury. It’s closely related with the development of multicellular organisms, differentiation, homeostasis and diseases. Apoptosis mainly includes three signal transduction pathways:death receptor pathway, mitochondrial dependent pathway and endoplasmic reticulum pathway.Rat adrenal chromaffin myeloma cells (PC 12 cells) are usually used in the study of neural biology and neural toxicology, so we selected PC 12 cells as the vireo neurotoxicity model. AO/EB staining assay was used to identify apoptosis and necrosis, DAPI and Hoechst staining assay was used to detect the morphology change at different concentrations of TCBQ. JC-1 fluorescence probe was used to test the mitochondrion cell membrane potential, TCBQ can decrease the cell membrane potential, and cause the release of cytochrome c. After TCBQ treated, the death receptor can be activated and the pro-Caspase 8 can be activated to the active Caspase-8, which cleaved Bid to active t-Bid(truncated Bid), and activated the mitochondrial apoptotic pathway. Immunofluorescent double staining experiment observed that Bid could translocate from the nucleus or cytoplasm to mitochondria after treatment with TCBQ.Cell apoptosis may be related to oxidative stress, so we observed the levels of oxidative stress in PC12 cells exposed to TCBQ. The level of ROS was detected by DCHF-DA fluorescent probe, the results showed that TCBQ can up-regulate ROS level, while the antioxidant NAC significantly down-regulated the level of ROS. We used the kits to detect the GSH, MDA and protein carbonyl content, the results revealed that MDA and protein carbonyl content was concentration dependent-increased, GSH content was concentration dependent-decrease. The WB experiment further revealed that NAC can significantly inhibit Caspase 8/t-Bid pathway expression induced by TCBQ.Overall, our study suggested TCBQ induced PC 12 cells apoptosis through the Caspase 8/t-Bid signaling pathway. Through this research, we can further understand the cytotoxicity mechanism of TCBQ and provide important theoretical basis for the neurotoxicity of TCBQ, so as to provide new ideas for prevention and treatment of neurodegenerative diseases induced by TCBQ.