Assessment Of Tris(1,3-dichloro-2-propyl) Phosphate Toxicology In PC12 Cells By Using Digital Gene Expression Profiling

As a new type of environmental organic pollutants,tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)has been paid more and more attention by researchers.The production volume and consumption of TDCIPP has been at a high level and increases year by year.It is used as an additive to various products,such as textiles,furniture,electronics equipment and many baby products.Therefore,TDCIPP is easier to release to the environment.Biomonitoring studies detected TDCIPP in water,air,soil,and dust,even in the tissues of fishes,and birds samples.The level of TDCIPP in some samples exceeded the level in polybrominated diphenyl ethers.As a result,TDCIPP can be taken up or absorbed by humans through multiple routes,including ingestion,inhalation,and skin absorption.Furthermore,TDCIPP and its metabolites have been detected in human urine,breast milk and human placenta.This has led to great concern over its potential toxicity and environmental health effects.Previous studies demonstrated that TDCIPP caused adverse effects on thyroid hormone imbalance and produced the neurotoxicity.In the study,we used the PC12 cell line,a widely used in vitro model for neurotoxicity,to investigate the toxicity and the possible molecular mechanism of the effects of TDCIPP.ObjectiveTDCIPP-induced injury in PC12 cells was established to investigate its toxicity.The mechanism of the effects of TDCIPP was investigated by screening and verifying differentially expressed genes based on digital gene expression profiling and other molecular biology methods.In order to provide information,this study focused on the toxicology mechanism of TDCIPP.Methods 1.TDCIPP induced the toxicology in PC12 cells.1.1 Experimental classification and the treatment:Differentiated PC12 cells were divided into 5 groups: normal control group and TDCIPP exposure groups with TDCIPP concentration of 7.5,15,30,60 ?M,respectively.There are 3-6 biological repeats in each group.1.2 Effects of TDCIPP on cell viability in PC12 cells: After differention into neural cells,the cells were exposed to TDCIPP for 24 h or 72 h according to the experimental group,then cell counting kit-8 was used to measure PC12 cell viability.1.3 Effects of TDCIPP on oxidative stress in PC12 cells: Differentiated PC12 cells were exposed to different concentration for 72 h.A ROS assay kit was used to measure the levels of intracellular reactive oxygen specie(ROS);SOD,GSH,and MDA concentrations were measured using commercial ELISA kits.1.4 Effects of TDCIPP on cell apoptosis in PC12 cells: Differentiated PC12 cells were exposed to different concentration for 72 h.The fluorescein isothiocyanate(FITC)Annexin V Apoptosis Detection Kit ?was used to detect the levels of apoptosis.Then,the expression of apoptosis related protein Bcl-2 and Bax were detected by western blot.2.The molecular mechanism of TDCIPP exposure on PC12 cells.2.1 Experimental classification and the treatment: The experimental groups used for DGE sequencing analysis were normal control group(C)and 60 ?M TDCIPP exposure group(H);experimental groups for qRT-PCR and Western blot were compared with section 1.1.for 72 h.There are 3-6 biological repeats in each group.2.2 Differentiated PC12 cells were exposed to different concentration for 72 h.Digital gene expression profiling(DGE)was employed to investigate the changes of transcriptomic and screen differentially expressed genes.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway(KEGG)analysis were performed to obtain function and pathway information.2.3 Differentiated PC12 cells were exposed to different concentration for 72 h.Six differentially expressed genes(Myc,p21,Lamb3,col1a1,THBS,and CREB)were selected to validate the results of the DGE assay by quantitative real-time polymerase chain reaction(qRT-PCR).2.4 Differentiated PC12 cells were exposed to different concentration for 72 h.phosphatidylinositol 3-kinase(PI3K),phosphorylated(phospho)-PI3 K,protein kinase B(Akt),phospho-Akt,Myc,and p21 protein expression were measured by western blot.Results 1.TDCIPP induced the toxicology in PC12 cells.1.1 Effects of TDCIPP on cell viability in PC12 cells: After exposure to different concentration of TDCIPP for 24 h and 72 h,the cell viability decreased with increasing concentrations of TDCIPP.At the same exposure concentration,the cell viability at 72 h was less than at 24 h.Exposure of PC12 cells to 60 ?M TDCIPP for 72 h,the cell viability decreased to(58.15 ? 0.78)%.1.2 Effects of TDCIPP on oxidative stress in PC12 cells: Compared with the control group,ROS levels were increased by TDCIPP treatment in a concentration-dependent manner.Cells exposed to 30 and 60 ?M TDCIPP showed significant 1.8-and 2.7-fold increases over the ROS levels observed in untreated control cells(P < 0.01),respectively.The content of SOD and GSH decreased with the increase of TDCIPP exposure concentration,while the content of MDA increased.1.3 Effects of TDCIPP on cell apoptosis in PC12 cells: Compared with the control group,exposure to TDCIPP induced apoptosis in PC12 cells in a concentration-dependent manner.Treatment with 15,30,and 60 ?M TDCIPP significantly increased(P < 0.01).Moreover,the protein relative expression of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax were decreased and increased,respectively,leading to a significant concentration-dependent decrease in the Bcl-2/Bax ratio in the presence of 7.5,15,30,or 60 ?M TDCIPP(P < 0.05).2.The molecular mechanism of TDCIPP exposure on PC12 cells.2.1 The depth and breadth of DGE sequencing data were better.We identified 161 genes with significantly different expression levels in PC12 cells exposed to 60 ?M TDCIPP,as compared with control cells;49 of these were upregulated and 112 were downregulated by TDCIPP.Functional and pathway analysis of the transcriptional profile demonstrated that genes showing significant TDCIPP-associated changes in expression were involved in carboxylic acid metabolism,amino acid metabolism,phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,and extracellular matrix–receptor interactions,2.2 The qRT-PCR results of 6 selected genes and DGE sequencing results were tested by Pearson correlation and simple linear regression analysis.The results showed a significantly positive correlation,indicating that the DGE results were authentic.2.3 TDCIPP exposure decreased phospho-PI3 K and phospho-Akt expression in a concentration-dependent manner.In contrast,the levels of non-phospho-PI3 K and non-phospho-Akt showed no concentration-dependent changes.Myc mRNA and protein expression levels decreased,while p21 were significantly up-regulated in a TDCIPP concentration-dependent manner.Conclusion 1.TDCIPP induced the toxicology in PC12 cells.Exposure to 7.5,15,30,and 60 ?M TDCIPP for 72 h inhibited cell viability,and destroyed the stability of oxidation and antioxidant systems.ROS levels were increased by TDCIPP treatment in a concentration-dependent manner.The content of SOD and GSH decreased with the increase of TDCIPP exposure concentration,while the content of MDA increased.It can promote the apoptosis of PC12 cells and cause cell damage.2.The molecular mechanism of TDCIPP exposure on PC12 cells.TDCIPP exposure can reduce cell viability and induce apoptosis in PC12 cells by inhibiting activation of the PI3K/Akt signaling pathway using DGE technology.Phospho-PI3 K and phospho-Akt expression decreased.Myc mRNA and protein expression levels decreased,while p21 were significantly up-regulated in a TDCIPP concentration-dependent manner.