ABSTRACT
OBJECTIVE: To explore the molecular mechanism underlying 1,2-dichloroethane(1,2-DCE) induced apoptosis by screening differentially expressed proteins in human astrocytes( HAs). METHODS: HAs were cultured in complete medium with 1,2-DCE at various concentrations of 0-80 or 0-40 mmol/L. After 24 hours,apoptosis of HAs was evaluated using flow cytometry and staining with annexin Ⅴ-fluoresce in isothiocyanate and propidium iodide. An AAH-APO-1-2 protein chip was used to screen differentially expressed proteins and quantitative real-time polymease chain reaction(qRT-PCR) was used to verify related differentially expressed genes(DEGs). RESULTS: At 1,2-DCE concentrations of0-80 mmol/L,the total apoptosis rate of HAs increased with 1,2-DCE concentrations in a dose-dependent manner( P <0. 01). Seven different kinds of proteins were screened out by apoptotic protein chip. Among them,the expression of insulin-like growth factor-binding protein( IGFBP)-1,IGFBP-4 and cytochrome C( Cyto C) were up-regulated,while the expression of P27,cysteine aspartic acid specific protease-3( Caspase-3),B-cell lymphoma-2 interacting mediator of cell death( BIM) and BH3 interacting domain death agonist( BID) were down-regulated compared with the control group. The result of DEGs verified by qRT-PCR showed that the expression of mRNA of IGFBP-1,IGFBP-4 and Cyto C at 1,2-DCE concentrations of 40 mmol/L was up-regulated. This result was in consistent with the trend of target expression in the protein chip. The mRNA expression of Caspase-3,BIM and BID was also up-regulated. CONCLUSION: 1,2-DCE induces apoptosis of HAs through mitochondrial pathway.
ABSTRACT
OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.