Cytotoxicity Effects And Molecular Mechanism Of Organophosphate Flame Retardant (TDCIPP) On Human Neuroblastoma (SH-SY5Y) Cell

Organophosphorus flame retardants(OPFRs)are mass produced chemicals mainly used as replacements for brominated flame retardants.OPFRs are ubiquitous in the environment and now they are considered as emerging contaminants.Here,a typical OPFR,tris(1,3-dichloro-2-propyl)phosphate(TDCIPP),was selected to investigate neurotoxicity effects and the underlying mechanism on human neuroblastoma cell(SH-SY5Y cell).As followes:1.Investigeted the cytotoxicity of TDCIPP on SH-SY5Y cell.MTT assay showed that no obersved effect concentration(NOEC)of TDCIPP to SH-SY5Y cells was 2.5 ?M,while the lowest observed effect concentration(LOEC)of TDCIPP to SH-SY5Y cells was 10 uM.The cell vibility of TDCIPP(10-200 ?M)was significant decreased in a concentration-dependent manner;the median lethal dose was 100 ?M.LDH assay showed the cell viability was markedly decreased in a time dependent manner under the treatment of TDCIPP(100 M),when the cells were treated for 24 h,the cell viability was about 50%.2.Explored the role of TDCIPP on SH-SY5Y cell apoptosis.In Hochect 33342 assay,the results showed that TDCIPP induced the change of nuclear morphology,mainly showed as nuclear condensation and disruption.Then,the apoptotic ratio was assayed by AnneixV FITC/PI;the results showed that the apoptotic ratio markedly increased in a concentration-dependent manner.Furthermore,the protein marker of apoptosis Caspase3/cleaved Caspase3 were assayed by western blot and the results showed that the expression of Caspase3/cleaved Caspase3 significantly increased in a concentration-dependent manner.3.Investigated the role of autophagy in TDCIPP induced SH-SY5Y cell apoptosis and the signaling pathway.The results showed that TDCIPP can induce autophagy in SH-SY5Y cells,mainly performance was the increase of autophagy marker:the ratio of microtubule associated protein light chain 3(LC3)II/LC3I and the degration of Sequestosome 1(p62/SQSTMl),while the increase of autophagosome was also assayed by laser scanning confocal microscope and flow cytometry.Furthermore,autophagy inhibitor 3-Methyladenine(3MA)and Chloroquine(CQ)markedly increased the apoptotic ratio,while autophagy inducer Rapamycin(Rapa)significantly reduced the apoptotic ratio compared to TDCIPP treated alone.These results showed that autophagy played a protective role in TDCIPP induced SH-SY5Y cell apoptosis.Subsequently,we found that Reactive oxygen species(ROS)and Adenosine Monophosphate Activated Protein Kinase(AMPK)/Manmal target of rapamacy(mTOR)/Unc-51 kinase(ulkl)signaling pathway were involved in TDCIPP induced autophagy in SH-SY5Y cells.An antioxidant NAC can alleviate TDCIPP induced activation of AMPK and autophagy.4.Uncovered the mechanism of TDCIPP induced apoptosis.The results showed that TDCIPP led to an increase of intracellular ROS generation,[Ca2]i and decrease of mitochondrial membrane potential(MMP).Meanwhile,the results also showed that the expression of endoplasmic reticulum stress(ER stress)marker was significantly increased,such as:eukaryotic translation initiation factor 2a subunit(EIF2a),activation transcription factor(ATF4),glucose-regulated protein(GRP78),and the proapoptotic factor C/EBP homologous protein(CHOP).Phenyl butyric acid(PBA),an ER stress inhibitor can significantly inhibit TDCIPP induced ER stress.At the same time,the expression of apoptotic related gene bal-2 was significantly up-regulated and bax was significantly down regulated.These results showed that the inhibition of ER stress led to a protect role to TDCIPP induced SH-SY5Y cell apoptosis.Anymore,antioxidant NAC can effectively eliminate the generation of intracellular ROS and inhibit ER stress,led to an increase in cell viability.5.Explored the effect of low concentration TDCIPP on SH-SY5Y cells differentiation and the related mechanism.The results showed that TDCIPP(0-2.5 ?M)have no significant cytotoxicity on SH-SY5Y for 5 d.TDCIPP(2.5 ?M)can induce SH-SY5Y cells differentiated into a neural-like cell after treated for 3 d and in a time dependent manner.The results also showed that autophagy was involved in TDCIPP induced SH-SY5Y cell differentiation.Autophagy inhibitor 3MA markedly decreased the ratio of neurite bearing cell and shortened the lengtion of neurite.Autophagy inhibitor 3MA also inhibited the increase expression of cytoskeletal components:neurofilaments-H(NF-H),neurofilaments-L(NF-L)and ?-tubullin in TDCIPP treated cells.Taken together,these results showed that the relative low concentration TDCIPP can induce the differentiation of SH-SY5Y cells and the mechanism is related to the autophagy regulated cytoskeletal components(NF-H,NF-L and(3-tubullin).