The Cytotoxic Of Chloropropanol And Apoptosis Pathway On HEK293 Cells

3-MCPD(3-Monochloropropanel-1,2-diol) is one of the hazardous contaminants in the food processing industry. Recently, there are amounts of investigations indicating that 3-MCPD has liver and kidney toxicity, carcinogenicity and reproductive toxicity, however, there is few papers about the investigation of molecular mechanism about 3-MCPD in vitro. For this reason, this paper use genomics and in vitro alternative technology, combined with time-dose effects, to explore cellular oxidative damage induced by 3-MCPD and molecular mechanism of apoptosis at three levels of cell,protein and gene, revealed the mechanism of toxicity of 3-MCPD initially.In this study, we used a series of concentrations (1.25-80 mM) of 3-MCPD to stimulate PC12, Mast cell, HepG2 and HEK293 cells for 12 h,24 h,36 h.CCK-8 colorimetric method was used to test 3-MCPD on cell proliferation inhibition. We screened out the most sensitive cells was the human embryonic kidney cell HEK293 and we screened the best stimulation time, and calculate the value of IC50. We observed the morphological changes of apoptotic cells and detect changes in cell cycle; We detect the cell apopotosis rate and cell DNA damage; Detect intracellular reactive oxygen species (ROS) and changes in calcium levels; Caspase kit to detect intracellular Caspase enzyme activity; JC-1 fluorescent staining to detect the changes of mitochondrial membrane potential; Western blot to detect the cell apoptosis and Bcl-2, Bax, p53 and cytochrome C protein expression level; Through the PCR array technology we detected the changes of multiple genes of apoptosis, combining with gene Q-RT-PCR we analysed the significant changing gene and validated the reliability of gene chip.The results showes that, in the high concentration of 3-MCPD (concentration greater than 10 mM) has obvious inhibitory effect on PC 12, mast cell, HepG2, HEK293 cell growth (P<0.05), and its effect is concentration dependent, among them HEK293 cells was the most sensitive to 3-MCPD. HEK293 cells treated by 3-MCPD showed a slow growth, shrinkage, the typical characteristics of apoptosis such as cell membrane fusion. Cell cycle assay showed 3-MCPD mainly impacted on the d/M phase in the cell cycle.3-MCPD of 20 mM can reduce apoptosis rate up to 25%. Through single cell gel electrophoresis assay we observed that the low concentration (5 mM) of 3-MCPD can cause DNA damage in cells (P<0.05) and high concentration (20 mM) 3-MCPD caused serious cell DNA tail(P<0.01). Increased ROS and Ca2+concentration indicates that the cell oxidative damaged and cell homeostasis;Caspase enzyme activity test results showed that Caspase-3 activity increased significantly (P<0.05), Caspase-8, Caspase-9 did not change significantly. JC-1 staining showed mitochondrial membrane potential decreased.Westem blot results showed that pro-apoptotic proteins Bax, P53 and cytochrome C expression increased, decreased the expression of anti apoptotic protein Bcl-2.Expression of P53-DNA damage related protein increased; 3-MCPD of 20 mM stimulated HEK293 for 12 h, apoptosis gene chip identified 28 apoptosis related gene changes. Q-RT-PCR validated the levels of mRNA of death receptor pathway and mitochondrial pathway in a dose-dependent.Through the study, we can confirm 3-MCPD has significant cell toxicity, and has time and concentration dependent and cell selectivity. Low concentration of 3-MCPD(5 mM) can induce cell cycle arrest, arrest cell cycle at G2/M phase and effect the cell proliferation. The high concentration of 3-MCPD(10 mM,20 mM) induced DNA damage through the production of peroxides, and active p53 and Caspase-3 gene to induce cell apoptosis. The 3-MCPD active the mitochondrial signaling pathway by releaseing cytochrome C, increasing the ratio of Bax/Bcl-2 in mitochondria, and activated caspase-9 which represent the activation of the mitochondrial apoptotic pathway; By upregulating the expression of FasL,,CD27, TR1,TNFR1 DR5 and other death receptor gene family to induce death receptor pathway, The activation of Caspase-8 and upregulate the expression of Bid gene was also confirmed that the two pathway does connected. This study lays a foundation role for toxicology study of 3-MCPD, and provides the basis for make the limits of 3-MCPD and its homologues.