Role Of Brominated Diphenly Ethers-209 In Proliferation, Differentiation And Apoptosis Of Neural Stem Cells In Vitro

【Background】1.About Polybrominated diphenly ethers (PBDEs)PBDEs are a group of brominated flame retardant(BFR) chemicals that are added to manufactured products including paints, plastics, textiles, and electronic appliances and other fields,end products widely used for PBDEs include electronics, computers, and foam padding in furniture . Since 1970, production and use of PBDEs has resulted in environmental pollution globally, Some data showed that 80% of e-waste in developed countries were brougt to China, India and Pakistan and other countries, which also accounted for 90% of e-waste in China. For exterior air, urban PBDE concentrations were 10 times greater than rural concentrations, indicating an urban rural gradient and greater PBDE sources in urban areas, There formed collection and distribution center for dismantling e-waste in some areas in China, PBDEs and other persistent toxic pollutants continuously release to the local environment so that they result in serious pollution and endangering human health because of the original disassembling means (open burning, baking, and pickling, etc.).PBDEs have the property of low degradation, environmental stability, high fat-solubility and biological amplification functions, which can be transferred through food chain to allow the organisms of higher-level grade to be poisoned and finally bring dangers to human health. Temporal studies have shown that PBDEs are increasing in the environment and human breast milk at an exponential rate, which is consistent with the increase in global demand since the early 1980s.PBDEs are present in the environment and concentrate along the food chain and eventually accumulate in the body through food, breast milk, atmosphere and room dust, etc. The current study suggests that, PBDEs (mainly low-bromo-class) mainly accumulate in adipose tissue in the body which cause adverse effects on the health. The half-life of PBDEs in the adipose tissue of rodents is 19-119 d, the more brominated congeners of PBDEs are, the longer half-life is. Polybrominated diphenyl ethers are also carcinogenicity, reproductive toxicity, neurotoxicity and endocrine toxicity in animals test, therefore, in the United States and europe and other places, low-brominated PBDEs such as BDE-47, 99,153 and so on have been produced and used restrictly. However, the high bromide PBDEs, such as BDE-209, is still widely used because of its good flame-retardant, bio-toxicity of uncertainty. Understanding of the toxicity of PBDEs is far less PCBs. At present, the laboratory evidence of the toxicity of PBDEs is relatively more, while the research is mainly based on professional population, so it is lack of data. the distribution of PBDEs are not identical in different occupational populations ,BDE-153 is often now the major PBDE congener in nonoccupationally exposed populations, such as in the Netherlands, the United Kingdom, Sweden and China. BDE-209 and BDE-183 have been specifically related to occupational exposure。2. About BDE-209BDE-209 is a kind of high bromine PBDEs containing 10 bromine atoms, which has good thermal stability, less addition amount, cheap and other advantages. The deca-BDE formulation made up 83.3% of the PBDE global demand in 2001,followed by BDE-206, BDE-208, BDE-207, BDE-203, BDE-47, and BDE-153 ,In China, the domestic production of deca-BDE was approximately 25 000 t in 2004,The estimated domestic production of predominant commercial deca-BDE mixture in China increased by 200% between 2000 and 2005, from 10 000 to about 30 000 metric tons. Due to its biological accumulation effect, the concentration of BDE-209 in the blood will continue to rise. BDE-209 were detected in various environmental media ( air, sediment, soil, indoor air, as well as a variety of organisms, etc.) and in human with the increase of the concentration every year. After release to the environment, BDE-209 is transformed into polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans and low-brominated biphenyl ether by photolysis, pyrolysis, biological and microbiological degradation and so on, which are more easily enter the organism and more toxic. Some research also proved that BDE-209 has a neural developmental toxicity, immunotoxicity, endocrine toxicity and carcinogenecity .Some Researchers think PBDEs exposure in the perinatal period can affect the capacity spontaneous behavior and learning and memory of next generation . In our preliminary experiments, we have confirmed that maternal BDE-209 exposure during the period of pregnancy can decrease learning and memory in mice. Also in vitro, BDE-209 induced apoptosis and oxidative stress and led to changes in nerve cell structure and function in vitro in primary cultured hippocampal neurons of rat in a concentration-dependent manner. However, some researchers also believe that the metabolism of BDE-209 in the body is very fast, having low accumulation, and will not bring impact on the body under normal exposure level and that BDE-209 is a hero to prevent a large number of fire. Due to the inconsistency of the results, researches on the biological toxicity of BDE-209 are becoming necessary.3. About neural stem cells (NSCs)The formation of new memories,recalling old memory and learning capacity-building are related to hippocampus of the brain. In recent years, the discovery of neural stem cells provides a new perspective for the study of learning and memory mechanism. It has been reported that the more proliferation of neural stem cell was and the stronger of learning and memory was in animal experiment. The proliferation of neural stem cell may play regulatory role in learning and memory. In the period of mammalian embryo, neural stem cells (neural stem cell, NSCs) are mainly distributed in the hippocampus, large annoying cortex, striatum, ependyma, and in the lower brain areas and there are NSCs in SVZ (subventficalar zone. SVZ), hippocampus dentate gyrus (dentategyrus, DG), striatum, spinal cord, etc in the adult.Neural stem cells are self-renewal and able to differentiate, it can differentiate into various types of nerve cells by asymmetric division. It is found that the biological characteristics of NSC are: (1) possessing self-renewal capacity (2) able to generate functional differentiated potential and differentiate into neurons, astrocytes and oligodendrocytes; (3) in a highly undifferentiated state; (4) possessing proliferation and differentiation capacity and can proliferate and differentiate in some environment with a signal change. (1) and (2) are the two basic characteristics of NSC. The regulation of proliferation and differentiation is the core issue of NSCs research, Recent research data shows that the proliferation and differentiation, migration are regulated by a variety of related factors such as①endogenous factors: a series of factors that can signal to the outside world and regulate self-NSCs through signal transduction pathways .Including some proteins,various factors (such as EGF, FGF, IGF, etc.),ions,hormones and neurotransmitters in NSCs,They are the essential element of existence and differentiation of NSCs . A number of transcription factors and gene activation or silencing may also affect the direction of differentiation of NSCs.②exogenous factors: glucose, amino acids, vitamins and others maintain the basic elements of cell survival. Co-ordination between various factors, osmotic pressure, pH, oxygen concentration and temperature have an important impact on the NSCs proliferation, migration and differentiation. Another,Certain pathological conditions such as cerebral ischemia is also an activate factor of NSCs.we selected different concentrations of BDE-209 as the target compounds, cultured neonatal rat hippocampal neural stem cells in vitro, exposed to different concentrations of BDE-209, observed the morphology of NSCs, detected proliferation, differentiation and apoptosis of NSCs ,so that to explore the role of BDE-209 on neural stem cells and to explore the effect of environmental factors on neuro-developmental toxicity and to explore the mechanism of neurotoxicity of BDE-209, this issue is divided into the following four parts for the specific experiments. Role of Brominated diphenly ethers-209 in morphology of neuralstem cells in Vitro【Objective】The hippocampal NSCs of neonatal SD rat postnatal day 1 were subcultured in serum-free DMEM , identificated the purity of NSCs, measured the number of neurosphere and diameter of neurospheres, tested the effect of BDE-209 on morphology of NSCs in Vitro.【Materials and Methods】1. The hippocampal NSCs of neonatal SD rat postnatal day 1 were subcultured in vitro.2. Identification the purity and identification of NSCs.3. Subcultured 3-4 generations of NSCs,dissipated into a single cell suspension and exposed to BDE-209, grouped as follows :blank control group, DMSO control group, the experimental A Group: the concentration of BDE-209 is 10ug/ml, experimental B Group: the concentration of BDE-209 is 30ug/ml, experimental C Group: the concentration of BDE-209 is 50ug/ml. Each section will parallel three. The DMSO control group joined with the 1‰DMSO medium.4.When exposed to BDE-209 after 72h, we observed the morphology of NSCs under microscope and measured the diameter and the number of neurospheres in each group by U.S. Image-Pro-Plus6.0 image analysis software.【Results】1. Identification of NSCs: the NSCs formed neurospheres cultured in vitro after 3-5days , the neurospheres is gradually bigger after 6-7 days , the newly neurospheres showed Nestin-positive by immunocytochemistry that suggesting that the cells cultured in vitro are neural stem cells.2.The 3-4 generations of NSCs were observed under inverted microscope, tens to hundreds of cell aggregated to form neurospheres , refractive strong halo, percussed into a single cell suspension and identificated the purity of NSCs by immunocytochemistry.we observed that the scattered neural stem cells accounted for over 90%.3. We measured the diameter and the number of neurospheres in each group by U.S. Image-Pro-Plus 6.0 image analysis software: the number of neurospheres significantly reduced in experimental group , and the average diameter of neurospheres significantly reduced with a concentration-dependent manner.【Conclusion】The isolated cells formed neurospheres, they are NSCs by immunocytochemical identification. The diameter and the number of neurospheres reduced with the increase of the concentration of BDE-209. Part IIRole of Brominated diphenly ethers-209 in proliferation of neuralstem cells in Vitro【Objective】To explore the effect of BDE-209 on proliferation of NSCs in Vitro.【Materials and Methods】1.Subcultured 3-4 generations of NSCs,dissipated into a single cell suspension and exposed to BDE-209, grouped as follows :blank control group, DMSO control group, the experimental A Group: the concentration of BDE-209 is 10ug/ml, experimental B Group: the concentration of BDE-209 is 30ug/ml, experimental C Group: BDE-209 concentration of 50ug/ml. Each section will parallel three.The DMSO control group joined with the 1‰DMSO medium.2. When exposed to BDE-209 after 24 ,48,72,96,120 hours, record light absorption(A) respectively by MTT assay.3. Map cell growth curve, A value for statistical analysis.【Results】MTT results showed that the general trend of cell growth increased, the results told that BDE-209 showed a dose-dependent proliferative effect on the growth of NSCs , The correlative analysis showed that there was negative correlation between the concentration of BDE-209 and the proliferation capacity of neural stem cells.【Conclusion】Neonatal rat hippocampus NSCs have the capability to self-renewal and proliferate cultured in serum-free medium, BDE-209 suppressed the proliferation capacity of neural stem cell in a concentration-dependent manner, which may be one of the mechanisms of the neurotoxicity induced by BDE-209.Part IIIRole of Brominated diphenly ethers-209 in differentiation of neuralstem cells in Vitro【Objective】To explore the effect of BDE-209 on differentiation of NSCs in Vitro【Materials and Methods】1.Subcultured 3-4 generations of NSCs in serum culture medium and exposed to BDE-209, grouped as follows :blank control group, DMSO control group, the experimental A Group: the concentration of BDE-209 is 10ug/ml, experimental B Group: the concentration of BDE-209 is 30ug/ml, experimental C Group: BDE-209 concentration of 50ug/ml. Each section will parallel three.The DMSO control group joined with the 1‰DMSO medium.2.When exposed to BDE-209 after 7 days, Cells were processed for indirect immunofluorescent staining. The cell number in each experimental condition was estimated, we counted NF-positive, GFAP-positive, DAPI-positive cell respectively to calculate the ratio of NF + / DAPI +, GFAP + / DAPI +, and measured the length of neurite for NF positive cells by American Image Processing Plus System 6.0【Results】Treatment with BDE-209 inhibited the neurite outgrowth and differentiation of NSCs into neurons in a concentration-dependent manner and by contrast enhanced the ratio of differentiation of NSCs into glial cells.【Conclusion】BDE-209 inhibited the neurite outgrowth and differentiation of NSCs into neurons in a concentration-dependent manner and enhanced the ratio of differentiation of NSCs into glial cells. It is demonstrated that BDE-209 may affect the development of the central nervous system.Part IVRole of Brominated diphenly ethers-209 in apoptosis of neural stemcells in Vitro【Objective】To explore the effect of BDE-209 on apoptosis of NSCs in Vitro.【Materials and Methods】1.Subcultured 3-4 generations of NSCs in serum-free medium and exposed to BDE-209, grouped as follows :blank control group, DMSO control group, the experimental A Group: the concentration of BDE-209 is 10ug/ml, experimental B Group: the concentration of BDE-209 is 30ug/ml, experimental C Group: BDE-209 concentration of 50ug/ml. Each section will parallel three.The DMSO control group joined with the 1‰DMSO medium.2.When exposed to BDE-209 after 24 hours, the apoptotic index were processed for indirectimmunofluorescent staining【Results】NSCs were apoptotic induced by different concentrations of BDE-209 after 24 h, Experimental A, B, Cgroup ,the apoptotic index were significantly higher, BDE-209 increased apoptosis occurrence in a dose-dependent manner, there was a P < 0.01 compared with control groups .【Conclusion】our results suggest that BDE-209 may induce apoptosis of hippocampal NSCs in a certain concentration which may be one of the mechanisms of the neurotoxicity induced by BDE-209.