Organic Phosphorus Flame Retardants TCEP Induced Mitochondrial Peroxidation Damage In N2a Cells

Tris (2-chloroethyl) phosphate (TCEP) , an excellent flame retardant plasticizer, is widely used in the manufacturing process of building materials, furniture, children's toys,electronic products, food packaging bags and other products. TCEP is mainly added to the material with non-chemical bonds it is easy to evaporate into the environment after long-term use, and extremely difficult to degrade under natural conditions. TCEP can enter the body through air inhalation, dietary intake and skin contact.A large body of evidencesshowed that TCEP had reproductive and developmental toxicity, which can affect the heart, liver, kidney, testicular and other organs.In recent years, there have been few reports about TCEP caused neurotoxicity, and the involved mechanismsare not clear.Brain is the most active organ of metabolic activity,which demands enough ATP to provide energy. Mitochondria can generate ATP via respiratory chain,then plays important role in the energy supply of brain. Reactive oxygen free radical (ROS) was released by mitochondia during ATP synthesis, which can cause cell peroxidation damage.It has been reported that TCEP can induce abnormal expression of oxidative stress-related proteins in cells.The ROS in cells is mainly produced by mitochondria.But till now, there has been no report about the effect of TCEP exposure on neuronal mitochondrial function.Therefore, we used mouse neuroblastoma N2a cellsas the experimental model, to investigate TCEP induced mitochondria peroxidation damage and possible mechanism, and the effect of TCEP on the expression and activity of autophagy regulating protein HDAC6 were also measured.We treated N2a cells for 24 hours with different concentrations of TCEP (25, 50, 75,100 ?M). The effect of TCEP on cell viability was determined by CCK-8;The changes of ROS level, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured by DCFH-DA method, TBARS colorimetry and Hydroxylamine method respectively. Meanwhile, we also pretreated cells with 25or 50?M antioxidant Vitamin E(VitE) for lhour then added 50 ?M TCEP in the medium for 24 hours to explore the protect effect of VitE on TCEP induced peroxidative damage .In order to clarify the possible mechanisminvolving in this process, we used JC-1 technique to detect mitochondrial membrane potential changes, we also detected the activity of Ca2+- ATPase,cytochromec(Cytc) oxidase which acted as themarker of mitochondrial function;The apoptosis-related proteins Bcl-2,Caspase-9 and Caspase-3 were detected by immunoblotting to investigate the changes of mitochondrial-dependent apoptosis. The content of HDAC6 and Ac-tubulin were detected by immunoblotting and the changes of HDAC6 activity were measured by Colorimetricanalysis, the changes of cytoskeleton were observed by immuno- fluorescence labeling technique. The results are showed tas following: (1) With the increasing of TCEP concentration, the cell activity decreased gradually.(2) Compared with the control group, the levels of ROS, MDA and SOD activity increased in a TCEP concentration dependent manner, but the relative ratio of SOD to MDA decreasedprogressively.(3) Compared with the control group, the level of ROS in mitochondria increased gradually with the TCEP concentration increase.(4) After 50 ?M TCEP treatment, the number of cells emitting red fluorescence signal reduced significantly, suggesting that the mitochondrial membrane potential decreased;In addition,the activity of Ca2+-ATPase(P<0.01) and Cyt c oxidase(P<0.05)were significantly lower than those in the control group.(5) After 50 ?M TCEP treatment, the content of anti-apoptotic factor Bcl-2 decreased significantly (P<0.01), the active fragment of Caspase-9 and the content of activated Caspase-3 protein increased significantly(P<0.01). (6) Compared with the control group, the expression of HDAC6 did not change significantly after 50 ?M TCEP treatment.However, the activity of HDAC6 decreased significantly (P<0.01), and the acetylation modification of tubulin increased dramatically(P<0.01). (7) Following 50 ?M TCEP treatment, the microtubule density decreased obviously, and microtubule network structure disintegrated significantly. (8)VitE pretreatment can prevent N2a cellsfrom TCEP induced peroxidation damage, apoptotic activation and HDAC6 depression, it also effectivelyrepressed the TCEP induced microtubule network collapse.The above results indicate that TCEP exposure can lead to peroxidation to N2a cells by attacking mitochondria, then triggered endogenous apoptosis pathway.Inaddition, TCEP can downregulate HDAC6 activity, injury the microtubule network, these abnormal changes may impair the degradation of damaged mitochondria by autophagic pathway. This study provides some new data for the neurotoxicity research of TCEP.