Exploration Of The Mechanisms About The Two Typical OPFRs-induced Neurotoxicity In PC12 Cells

With the halted out of the Polybrominated diphenyl ehers(PBDEs) in world widely, one of the replacements, the organophosphate flame retardants(OPFRs), as a kind of additive began vastly to be added to instead of being chemically bonded to varieties of commercial products, such as foams, paints, plastics, textiles and furniture, even including baby’s toys. Consequently, the increasement of the production and usage of OPFRs lead to the ubiquitous environmental contaminants and the high potential of detectability in biota samples. Information regarding the distribution of the OPFRs is limited now, but there are some studies have indicated the toxicity of TDCPP/TCEP in fish, chicken, human and rat cells.In the present study, we investigated the toxic effects of the two typical OPFRs, TDCPP and TCEP, using the PC12 cell line, a widely used in vitro model for neurotoxicity. This study aims to observe the changes in cell morphology under the stimulation of TDCPP and TCEP to investigate the effects of various concentrations of TDCPP and TCEP on cell viability and apoptosis. To further explore which proteins are possible markers of the toxicity and the possible mechanisms of the effects of TDCPP and TCEP on PC12 cells, we used western blotting to detect changes in protein synthesis of the related proteins, including CAMK2A/CAMK2B(calcium(Ca2+)/calmodulin dependent kinase type 2), GAP43(growth associated protein-43), tubulin-α, tubulin-β, NF-H(neurofilament). Then we chose the Ca MK2 as an indicator to explore the possible signaling pathways that may be involved in the neurotoxicity of TDCPP. The alterations in the Ca2+ levels in PC12 cells were detected by flow cytometry, the changes in the activations of Ca MK2 and the mitogen-activated protein kinases(MAPKs) JNK, ERK1/2, and p38 signaling pathways were semi-quantified by western blotting, and the relationship between Ca MK2 and the MAPK pathways was determined using an inhibitor of Ca MK2. So our study has three parts, the groups and main results of each part are shown as follows:Firstly, the TDCPP/TCEP induced the neurotoxicity in PC12 cells.To test the cell viability, the cells were grouped into the control group, the 1μM, 5μM, 15μM, 20μM, 25μM, 50μM and 75μM TDCPP groups, or the control group, the 15μM, 20μM, 40μM, 80μM, 150μM, 200μM and 400μM TCEP groups, persisted 6 days. To find the time-dependent effect of the cell viability, PC12 cells were grouped into six groups which exposure with 50μM TDCPP persisted 1-6 days each other, and the control groups were set to compare in each experiment. All of the cultures, before and after treatment with NGF, TDCPP or TCEP, were examined under an inverted phase contrast microscope to examine cell morphology. To test the cell apoptosis the cells were grouped into the control group, the 5μM, 15μM, 25μM, 50μM TDCPP groups, persisted 3 or 6 days.The results indicated that the PC12 cells exposed to TDCPP/TCEP were tend to become rounded, have attenuated branches and decreased axon number and failed to form reticular connections. There was a dose and time-dependent effect in the cell viability and apoptosis. The results suggested the neurotoxicity induced by the TDCPP/TCEP in PC12 cells.Secondly, the useful biomarkers of the TDCPP/TCEP-induced neurotoxicity in PC12 cells.PC12 cells were cultured on six-well plates and subjected to the treatment as the groups of the control group, the DMSO group, the 5μM, 15μM, 25μM, 50μM TDCPP or the 40μM, 80μM, 150μM, 200μM TCEP groups.The results showed that the translation level of the regulatory proteins---Ca MK2 and GAP43 in PC12 cells exposed to TDCPP had decreased about 20%-40% compared to the normal cells, and there was a dose-dependent effect relationship with the TDCPP. Then the skeleton protein---tubulin and NF-H had reduced about 30%, and also had a dose-dependent effect relationship with the TDCPP. GAP43 in PC12 cells exposed to TCEP also had decreased about 50% compared to the normal cells, but the CAMK2 had a trend to increased; the skeleton protein---tubulin had reduced about 20%-30%, but NF-H had increased one-fold. So, with the expourse of OPFRs, the expression levels of the proteins associated with the neural cell growth in PC12 were all changed, the growth and the normal form of PC12 cells were damaged, the neural cell functions was blocked, which suggested that these proteins might be useful biomarkers of the neurotoxicity of TDCPP/TCEP in PC12 cells.Thirdly, the might mechanisms of the TDCPP-induced neurotoxicity in PC12 cells.To test the intracellular Ca2+ level, the cells were grouped into the control group, the 5μM, 15μM, 25μM, 50μM TDCPP groups, each group was treated 0-6 days, all of the samples were analyzed using a FACScan flow cytometer. To test the expression levels of the Ca MK2 and MAPK pathways proteins and the phosphorylation level changes, the cells were grouped into:the control group, the 5μM, 15μM, 25μM, 50μM TDCPP groups, each group was treated 4 days, or treated with 50μM TDCPP for 0-6 days. To explore the relationship between the Ca MK2 and the MAPK pathways, PC12 cells were grouped into the control group, the control group+KN93, 25μM TDCPP, 25μM TDCPP+10μM KN93, 50μM TDCPP, 50μM TDCPP+10μM KN93.Results showed that TDCPP induced a dose-dependent elevation of Ca2+ in the PC12 cells during the each period. And the elevation of Ca2+ was peaked at 3rd and 6th d respectively. And exposure of neuronal cells to TDCPP resulted in phospho-Ca MK2 and MAPK pathways proteins increase in a time and concentration-dependent manner. There was a dramatically increased phosphorylation of Ca MK2 at 15 μM TDCPP for 4 d and significantly increased at 4–5 d post-exposure to 50 μM TDCPP. Significantly increase of phospho-JNK was emerged at 25, 50 μM TDCPP for 4 d and at 3–5 d post-exposure to 50 μM TDCPP, respectively. The phospho-ERK1/2 was increased at 25 μM TDCPP for 4 d and at 5–6 d post-exposure to 50 μM TDCPP, respectively. Then the significantly increase of phospho-p38 was also emergy at 25, 50 μM TDCPP for 4 d and at 3–6 d post-exposure to 50 μM TDCPP, respectively.In conclusion, up to now, the information about the mechanisms of neurotoxicity is limited. In the present study, we determined the neurotoxicity of TDCPP/TCEP, found the CAMK2, GAP43, tubulin and NF-H might be the useful biomarkers of the neurotoxicity induced by TDCPP/TCEP, and the neurotoxicity of TDCPP might be elicited by the elevation of the intracellular Ca2+, the phosphorylation of Ca MK2, and the activation of MAPK pathways. The investigation of the relationship between the signaling pathways further elucidated the MAPK pathways were partially activated by the Ca MK2 phosphorylation. These might be the mechanisms of the TDCPP/TCEP-induced neurotoxicity in PC12 cells. From these studies, the basic theory of the neurotoxicity of the OPFRs will be enriched, and the assessment of the damage of nervous system will be supported by more basic data.