Studies Of Neurotoxic Effect And Mechanism Of PBDE-47 And/or PCB153

Polybrominated diphenyl ethers (PBDEs), as additive flame retardants, are widespreadused in electrical equipment, wire, furniture, and textile. PBDEs and polychlorinatedbiphenyls (PCBs) belong to the halogenating hydrocarbon compound. According to thedifferent atomic number and the position of bromine and chlorine atom in the benzene ring,both the PBDEs and PCBs theoretically include 10 main categories and 209 kinds ofhomologs. At present the production and use of the two compounds have been restricted,PCBs is forbidden in many countries to produce and to use in particular, but PBDEs andPCBs exist in all the places in the earth biosphere such as air, water, soil, each kind ofsediments, as well as wild animal, fish, domesticated fowl and in humanity's organism.Furthermore, the concentration of PBDEs being detected has the trend of escalation year byyear.Because the molecular conformation has the similarity, PBDEs is similar to PCBs intoxicity. To date, some in vitro and in vivo studies have reported that the functionary targetorgans of PBDEs and PCBs are mainly the fatty tissue, the thyroid gland, the reproductiongrowth and the central nervous system. PBDEs and PCBs have neurotoxicity,hepatotoxicity, reproduction toxicity, immunity toxicity, and carcinogenicity. And as a kindof endocrine interferent, which possess analogical estrogen activeness, and can cause thehyperplasia of the thyroid gland, leading to the change of thyroid metabolism.The massive animal experimentation studies indicated that animals exposed to lowdose PBDEs in the critical period of cerebrum growth, such as grown-up mouse brainfunction to be unusual, behavior disorder, studying and memory decrease will happen, andit will become more serious every day with the increasing of age. Therefore, the recent years the developmental neurotoxic effects of PBDEs get more and more attention frompeople. However, the neurotoxic effects of PBDEs are still at the starting stage. At presentthe studies are also mainly concentrated in nerve behavior, cognition function, learning andmemory and so on. The further research on the neurotoxic effective mechanism of PBDEsis to be advantageous in seeking the target spot of the neurotoxic effect and also providesthe theory basis for the prevention and control of neurotoxic effect of PBDEs.Regarding to the in vitro experimental studies, Madia et al reported that both PBDE-99and Aroclor1254 may induce the astrocytoma cells to apoptosis and possess obvious celltoxicity. Our early in vitro experimental result indicated that PBDE-47, a typicalpredominant congner of PBDEs, may induce the SH-SY5Y cell to apoptosis. Manyresearches confirmed that apoptosis plays an extremely vital roles in multicellularorganism's basic vital activity. At present apoptosis include three classic pathways:mitochondria pathway, death receptor pathway, and endoplasmic reticulum pathway. Allthese signal transduction pathways can activate apoptosis performer Caspase3, which canhydrolyze various cell component to lead to cell apoptosis. So far, the action of apoptosis inthe neurotoxicity induced by PBDEs is not yet reported.After synthetically analyze all the announced monitoring results from the global crowd,Hites et al reported that the closest 5 kinds of PBDEs homolog to the crowd are PBDE-47(2, 2', 4, 4'-tetrabromodiphenyl ether, PBDE-47), 99, 100, 153, 154. But Bi et al foundthat PBDE-47 and PBDE-153 are the most main homologs after analyzed the 21 pairs ofbaby umbilical cord and the mother vein blood samples from an austral city in China. Asthe PCBs homolog, 2, 2', 4, 4', 5, 5'-six chlorine biphenyl (2, 2', 4, 4', 5, 5'hexachlorobiphenyl, PCB153) is one of most content homolog in the body tissue and thebreast milk. Therefore, PCB153 is one of PCBs homolog which has the closest relationswith human health. The epidemiology investigation discovered that PCB153 and PBDE-47may simultaneously exist in the environment and human body organize, but not yet see thecombined toxic effects and the mechanism reported at present.Basis above reasons, whether PBDE-47 and/or PCB153 induce nerve cell apoptosisthrough three classics signal pathways, then initiates a series of neurotoxic effect issupposed, which is the main content of this study. The research experiment will go on both in vitro and in vivo, 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT),spectrophotometric method, Morris maze experiment, ultramicro pathology detect, flowcytometry (FCM), real time fluorescent quantitation reverse transcriptase polymerase chainreaction (real time RT-PCR), and Western blot and so on will be used to study theneurotoxic effect and the mechanism of PBDE-47 and/or PCB153 through detecting theneurocyte survival rate, lactate dehydrogenase (LDH) leakage, intracellular free Ca²⁺concentration ([Ca²⁺]_i, the SD rat's learning and memory capability, ultrastructure ofhippocampal neuron, and the mRNA and protein expression levels ofX-chromosome-linked inhibitor of apoptosis protein (XIAP), cysteine containing aspartatespecific protease 3 (Caspase3), cysteine containing aspartate specific protease 12(Caspase12), death associated protein kinase (DAPK), and Cytochrome C in SD rat and inSH-SY5Y cells.Part 1 Studies of toxic effect and mechanism of PBDE-47 and/or PCB153in SH-SY5Y cellsObjective: PBDE-47 and PCB153 can coexist in the environment and human tissuesas dominant congeners of PBDEs and PCBs, respectively. The aims of presental experimentare to explore the neurotoxic effect and the mechanism of PBDE-47 and the mode of actionin combination with PCB153 in SH-SY5Y cells.Methods: MTT, spectrophotometric method, flow cytometry, real time fluorescentquantitation RT-PCR, and Western blot were used to detect the cell viability, LDH leakage,[Ca²⁺]_I, apoptosis, and mRNA and protein expression levels of DAPK, XIAP, Caspase3,Caspase12, and Cytochrome C in SH-SY5Y cells treated with PBDE-47 (0, 1, 5 and 10μmol/L) and/or PCB 153 (0 and 5μmol/L) for 24h.Results: Compared to controls, the cell viabilities were clearly decreased (P＜0.05),and LDH leakage, [Ca²⁺]_i and apoptosis were significantly increased (P＜0.05).Furthermore, expression levels of DAPK and Caspase3 mRNA, Caspase12, as well asCytochrome C mRNA and proteins were markedly increased (P＜0.05), whilePro-caspase3 proteins were significantly decreased (P＜0.05). A positive correlationbetween [Ca²⁺])i and percentage of apoptotic cells (r=0.86, P＜0.05). An interaction between PBDE-47 and PCB153 were existed in decreasing SH-SY5Y cell survival,increasing the [Ca²⁺]_i, inducing the apoptosis and LDH leakage, as well as inducing theXIAP, Caspase3 and Cytochrome C mRNA expression, and XIAP, Pro-caspase3,Caspase12 and Cytochrome C proteins expression.Conclusions: PBDE-47 has the obvious toxic effect on SH-SY5Y cell. All threeclassic apoptosis pathway possibly participate in the process of SH-SY5Y cell apoptosisinduced by PBDE-47. Moreover, the calcium disequilibrium in the cell may be one of themain induction factors. The toxic effect is supposed to increase when SH-SY5Y cells areexposed to PBDE-47 and PCB 153 together in certain dosage.Part 2 Studies of developmental neurotoxic effect and mechanism ofPBDE-47 and/or PCB153 in ratsObjective: Because PBDE-47 and PCB153 may coexist in the environment media andhuman tissues, this experiment is to explore the developmental neurotoxic effect and themechanism of PBDE-47 alone and/or combined with PCB153 in the SD rats.Methods: Morris water maze, ultramicropathology technique detect,chemoluminescence, real time RT-PCR and Western blot were used to detect the learningand memory capabilities of rats, ultrastructure of neurons in the hippocampal CA1 region,the level of thyroid hormone and the expression levels of mRNA and proteins of XIAP,DAPK, Caspase3, Caspase12 and Cytochrome C in hippocampus in 2-month-old ratsexposed to a single oral dose of PBDE-47 (0, 1, 5 and 10 mg/kg) and/or PCB153 (0 and 5mg/kg) on post natal day (PND) 10.Results: The results suggested that the total distance swam (distance travelled by ratsto reach the escape platform) and the latency period (time travelled by rats to reach anescape platform) were significantly increased in place navigation test and the ratios(distance (time) in platform quadrant/total distance (time) swam) in spatial probe test werenotably decreased in all treated groups in water maze experiment (P＜0.05) whencompared to the control group. Additionally, compared with the control group,ultrastructure of neurons was prominently changed in the treated groups. Theconcentrations of T₄ in serum were obviously depressed in 5 mg/kg PBDE-47 group than that of the control (P＜0.05). Compares with the control group, the mRNA and the proteinexpression levels of XIAP in PBDE-47 treated groups were remarkably decreased (P＜0.05), the mRNA and protein expression levels of Caspase12 and Cytochrome C (femalebig mouse Cytochrome C mRNA expression exception) in PBDE-47 treated groups wereremarkably increased (P＜0.05). PBDE-47 caused Caspase3 mRNA expression levelsobviously increased and caused Pro-caspase3 protein expression levels remarkablydecreased (P＜0.05). PBDE-47 caused DAPK mRNA expression level of the female ratsremarkably decreased, but caused DAPK mRNA expression level of the male ratsobviously increased (P＜0.05). Furthermore, an interaction was found between PBDE-47and PCB153 in decreased the capability of learning and memory, as well as inducingCaspase3, XIAP, Caspase12 and Cytochrome C (only in male rats) mRNA expression, andPro-caspase3, XIAP, Caspase12 and Cytochrome C proteins expression in hippocampus.Conclusions: PBDE-47 has the obvious developmental neurotoxic effect. All threeclassic apoptosis pathway possibly participate in the process of the developmentalneurotoxic effect induced by PBDE-47. Both affecting the development of thyroid glandand reducing the thyroxine secretion are possibly one of the reasons of developmental toxiceffects induced by PBDE-47. In the critical period of development, the toxic effect issupposed to increase when SD rat is exposed to PBDE-47 and PCB153 together in certaindosage.