Study On The Molecular Mechanism And Regulation Of Polybrominated Diphenyl Ethers (BDE-47 And BDE-209)-Induced Cytotoxicity And Apoptosis In Neuro-2a Cells

Polybrominated diphenyl ethers (PBDEs), extensively used in the past few decades as brominated flame retardants in a variety of consumer products, have become world-wide persistent environmental pollutants. PBDEs can release from products and enter the environment, causing environment pollution and harm to human health, which has caused worldwide concern. PBDEs exhibit many negative biological effects, inhibit the cells growth and induce cell apoptosis, and the mechanisms involved in PBDEs-induced apoptosis are most likely related to oxidative stress and relative gene regulation at molecular level. The present study examined the neurotoxicity and the potential neurotoxic mechanisms of BDE-47 and BDE-209 in human neuroblastoma (Neuro-2a) cells in vitro. Relative inhibitors of intracellular reactive oxygen species (ROS) and Ca2+ were used to study the relationship between intracellular ROS and Ca2+ level and PBDEs-induced Neuro-2a cells apoptosis. To insight the ROS-mediated and Ca2+ -mediated signal pathway, we examined the expression of relative genes and proteins levels of p38 MAPK signaling pathway and Nrf2-ARE pathway. Compared the toxic ity and mechanisms of BDE-47 and BDE-209, try to provide foundational data and theory for preventing and controlling the damage caused by PBDEs.1、BDE-47 and BDE-209 induced Cytotoxicity in Neuro-2a Cells (1) MTT assays and Trypan blue dye exclusion test results showed BDE-47 and BDE-209 inhibited the proliferation of Neuro-2a cells, and caused cells death. LDH release assay indicated BDE-47 and BDE-209 could induce the leakage of lactate dehydrogenase from Neuro-2a cells. Flow cytometry found BDE-47 and BDE-209 significantly arrested cell cycle and high concentrations of BDE-47 arrested cells in G0/G1 phase. BDE-47 and BDE-209-induced apoptosis in Neuro-2a cells were confirmed using optical microscope observation, Hoechst33342 staining and Annexin V-FITC/PI double staining methods. The obtained results inferred the occurrence of PBDE-mediated cytotoxicity in Neuro-2a cells, and the cytotoxicity was greater with BDE-47 than with BDE-209.(2) BDE-47 significantly induced cell cycle arrest in the G1 phase after 48 h of treatment. The results of qRT-PCR and Western Blot showed that BDE-47 increased the expressions p53 and p21mRNA and caused a decrease in cyclin D1, cyclin E, CDK2, CDK4 mRNA expressions accompanied with a decrease in pRb protein levels, promotes de-phosphorylation of Rb protein. Rb de-phosphorylation inhibited the entry of cell cycle from G1 phase to S phase, and further inhibited Neuro-2a cell growth. BDE-209 had no significant effect on Neuro-2a cell cycle.2、The mechanisms of apoptosis in BDE-47 and BDE-209-treated Neuro-2a cells were involved the extrinsic and intrinsic apoptosis pathways.(1) The expresion of Fas and FADD mRNA were detedted by qRT-PCR. Activaties of Caspase-8 and -3 in BDE-47 and BDE-209-induced apoptosis were detected by Caspase activity assay. The result showed BDE-47 and BDE-209 induced apoptosis in Neuro-2a cells may through death receptors Fas and adaptor protein FADD activation of Caspase-8 and the downstream effector cleaved caspase-3 activation the extrinsic apoptotic pathway.(2) The results of qRT-PCR, Caspase activity assay and Western Blot showed that BDE-47 and BDE-209 induced apoptosis in Neuro-2a cells may through the mitochondrial apoptotic pathway, which involved increased Bax/Bcl-2 ratio, disrupted mitochondrial membrane potential, promoted the release of cytochrome C (Cyt C), and the activation of Caspase-9.(3) The results of qRT-PCR and Caspase activity assay showed that BDE-47 and BDE-209 induced apoptosis in Neuro-2a cells may through the Endoplasmic reticulum apoptotic pathway, which involved increased the expression of GRP78 and CHOP mRNA lave Is, and the activation of Caspase-12.3、BDE-47 and BDE-209 induced cells apoptosis via activation signaling pathway mediated by ROS and Ca2+ in Neuro-2a cells(1) Using specific fluorescent dyes 2’,7’-dichlorofluorescein diacetate (DCFH-DA) to detect intracellular ROS level changes, the results showed BDE-47 and BDE-209 increased the intracellular ROS levels in a dose-dependent manner. N-acetyl-L-cysteine (NAC), known ROS scavengers, obviously reduced the apoptotic rate. The degree of apoptosis blocking was positively correlated with the loss of mitochondrial membrane potential (MMP) and ROS production. The ROS-mediated mitochondrial pathway might play a key role in BDE-47 and BDE-209-induced apoptosis of Neuro-2a cells.(2) BDE-47 and BDE-209 increased intracellular ROS levels of Neuro-2a cells disturb the balance intracellular redox state. The results of contents of malondialdehyde (MDA) and the ratio of reduced/oxidized glutathione (GSH/GSSG) detected showed BDE-47 and BDE-209 resulting in intracellular lipid peroxidation injury and reduced GSH/GSSG ratio, caused a state of oxidative stress in Neuro-2a cells. The results of qRT-PCR assay showed BDE-47 and BDE-209 increase the expressions Nrf2 mRNA, further upregulate Nrf2/ARE downstream target genes (HO-1, NQO1, GPX, GCLM and GCLC) expression levels and downregulate GR gene expression levels. The results suggesting Nrf2-ARE pathway may be involved in the BDE-47 and BDE-209 within endogenous stress defense mechanisms through regulate oxidative stress generated by intracellular antioxidant/detoxification enzymes.(3) Fluorescent dyes FIuo-3 AM used to detect intracellular Ca2+ level, the results showed BDE-47 and BDE-209 instantly elevated the intracellular Ca2+ levels with a dose-dependent manner at the early period. The intracellular Ca2+ levels also increased with a dose-dependent manner at 1-24 h. BAPTA, intracellular calcium chelator, and obviously reduced BDE-47 and BDE-209 caused intracellular Ca2+ elevated. The degree of apoptosis blocking was positively correlated with Ca2+ production. Ca2+ increased may be another reason in BDE-47 and BDE-209-induced apoptosis of Neuro-2a cells. Dantrolene (Dan) and Xestospongin C (Xe C), the inhibitors of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R) ware used to investigate the relationship between ER Ca2+ release and cell apoptosis. Dan and Xe C reduced BDE-47 and BDE-209 caused intracellular Ca2+ elevated and block cell apoptosis, indicated BDE-47 and BDE-209 induce an increase of intracellular Ca2+ levels due to the release of Ca2+ from ER through RyR and IP3R, leading to the consequent perturbation of Ca2+ homeostasis.(4) AlphaScreen technology was used to detected intracellular p38 MAPK phosphorylation level. The results showed BDE-47 and BDE-209 increased the p38 MAPK protein phosphorylation in Neuro-2a cells. Flow cytometry analysis showed that p38 MAPK inhibitor SB203580 significantly inhibition of BDE-47 and BDE-209-induced cells apoptosis, suggesting that BDE-47 and BDE-209-intuced Neuro-2a cells apoptosis may be involved p38 MAPK pathway. Meanwhile, antioxidants NAC and intracellular Ca2+ chelator BAPTA can reduce the degree of p38 MAPK protein phosphorylation in BDE-47 and BDE-209 treated Neuro-2a cells, suggesting that BDE-47 and BDE-209 induced cells apoptosis via activation of p38 MAPK pathway mediated by ROS and Ca2+ in Neuro-2a cells.The results showed that cytotoxicity of BDE-47 and BDE-209 involved a variety of elements, such as cell cycle, apoptosis, ROS caused oxidative stress, Ca2+, p38 MAPK pathway, p53 signaling pathway and Nrf2-ARE signaling pathway. Among them, the apoptosis were madiated by the extrinsic and intrinsic apoptosis pathways.