Maternal Exposure To Decabrominated Diphenyl Ether(bde-209) During Pregnancy Disrupts Proliferation,differentiation And Apoptosis Of Hippocampal Neural Stem Cells In Fetal Rats,and Impairs Learning And Memory Capability Of Offsping Rats

【Background】1.Polybrominated Diphenly Ethers (PBDEs)PBDEs are a series of aromatic compounds with bromine atoms.PBDEs have a total of 209 kinds of isomers according to the different number and location of bromine atoms on the benzene ring.PBDEs are often used as flame retardant additives to be added into electron equipment or electrical appliance,automatic controller,building materials,textile product and other fields. Products containing PBDEs are likely to release PBDEs into the environment in the production,use and disposal process.PBDEs are difficult to be dissolved,long residence time,having strong lipotropic and hydrophobic property,bioaccumulation and biological amplification. PBDEs in air, water and soil can be concentrated in human and other higher organisms through food chains to impair their health,and lead to global pollution through the long-distance migration of "grasshopper jump effect".In recent years,levels of PBDEs in environment and human tissues,or blood and breastmilk are increased year by year.The potential adverse impact of PBDEs on the environment and human health has been the concern of researchers.PBDEs can be decomposed into the low bromine diphenly ethers with strong toxicity,cause thyroid and sex hormone secretion disorder,neurobehavior changes, capability of learning and memory decrease,liver damage and induce cancer,etc.PBDEs can permeate through placental barrier and be transmitted from mothers to fetuses by milk,affect fetal growth and development. Has been confirmed by Animal studies that pregnant rats exposed to PBDEs may cause activity and spontaneous behavior change, capability of learning and memory decrease of offsping rats. There is indication that young animals may have a reduced ability to excrete PBDEs, and the young have higher tissue (including brain) concentrations than the adult. PBDEs can damage neuronal cell structure and function,its mechanism of toxicity relates to cellular oxidative stress and DNA damage,etc. Thus,the European Union have banned the application of all PBDEs in electrical and electronic products as flame retardants.All states of USA made a bill restricting the application of brominated flame retardants.However,other countries including China continued to large-scale use of PBDEs.2.Brominated diphenly ethers-209(BDE-209,decaBDE)BDE-209 is a kind of high bromine PBDES containing 10 bromine atoms, which has good thermal stability,less addition amount,cheap and other advantages.BDE-209 is the most widely used PBDEs.it is also the major flame retardants used in China,and meanwhile it is one of the major pollutants in developed regions in China.More and more studies show that BDE-209 has similar biotoxicity with other PBDEs, including neurodevelopmental,immunotoxicity,endocrine toxicity and carcinogenicity,and BDE-209 can permeate through placental barrier and be transmitted from mothers to fetuses by milk.However,some researchers also believe that the metabolism of BDE-209 in body is very fast,having low accumulation,and will not bring impact on the body under normal exposure level.Due to inconsistency of the results, researches on the biological toxicity of BDE-209 are becoming necessary.3.neural stem cells(NSCs)Stemple isolated stem cells from neural crest in 1992 is the earliest and most direct evidence,identified that produce neurons and Schwann cells.These cells can self-renewal and produce the same cells with multiple differentiation potential. Now the concept of NCSs is widely accepted made by Mckay in 1997: NSCs,a kind of multi-directional neural precursor cells,have self-renewal and proliferation capability,and can differentiate into neurons,astrocytes and oligodendrocytes,and shows the corresponding specialized morphological structure,phenotype and biological characteristics.NSCs have self-renewal,proliferation,differentiation capability, and multi-differentiation potential.It can differentiate into different nerve cells under the different environments and factors.There are two growth modes:one is symmetric division into two stem cells,the other is assymmetric division into a stem cell and a progenitor cell. progenitor cell can be divided into multiple neural cells under certain conditions.During embryonic period,NSCs in the mammals are mainly distributed in cerebral cortex, striate body, hippocampus, subependymal layer,mesencephalon and so on.While,the NSCs in adults are mainly confined to hippocampal dentate gyrus, striate body and subependymal layer around lateral cerebral ventricle,but these NSCs are normally in resting state.The regulation of NSC proliferation and differentiation is the core of recent researches on NSCs. Recent studies reveal that proliferation, differentiation,apoptosis and migration of NSCs are co-regulated by a variety of factors,and gene expression of NSCs is regulated not only by inherent molecular processes,but also by exogenous signals including cytokines,micro-environment and so on.Based on previous studies and the potential hazard of BDE-209 on maternal and fetal health,this study aimed to investigate the impact of maternal exposure to different concentrations of BDE-209 on the proliferation,differentiation and apoptosis of NSCs in fetal rats, learning and memory in offspring rats.Part IMATERNAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER(BDE-209) DURING PREGNANCY IMPAIRS LEARNING AND MEMORY CAPABILITY OF OFFSPING RATS[Objective]:The SD rats on pregnancy day 1 were intragastrically administrated with different concentrations of BDE-209 every day.After 14 days,there were conventionally bred up to weaning at 25 days after parturition.The learning and memory capabilities of offspring rats were detected by Morris water maze assay.[Material and Methods]:1. The SD rats on pregnancy day 1 were randomly divided into control group (group A),arachis oil group(vehicle,group B),experimental groups (group C, D, E),and with 5 rats in each group. The rats in the group A were conventionally bred,10,30,50 mg/kg*d BDE-209 in arachis oil was intragastrically administrated in group C,D and E,respectively,while equal amount of purified arachis oil was given in group B.The experimental rats were administrated with corresponding drugs every morning from the purchase day to 14 days after pregnancy.there were conventionally bred up to weaning at 25 days after parturition, and 20 randomly selected male offspring rats of group A, B, C, D and E were selected.2. The learning and memory capabilities of offspring rats were detected by Morris water maze assay.[Results]:The learning and memory capabilities of offspring rats were detected by Morris water maze:Statistical analyses showed that each group escape latency changed by time in each group(F=71.686,P=0.000),and time factor played different roles in different groups (F=6.863,P=0.000) while grouping factor played roles (F=32.352, P=0.000).The fourth day of tests, there was no significant difference in escape latency between group A and B (P>0.05) while the escape latency in the group D and E was significantly longer than the group A (P<0.05).[Conclusions]:maternal exposure to some low concentrations of BDE-209 during pregnancy might impair the learning and memory capability of offspring rats. It indicated that the low concentrations of BDE-209 can produce neurodevelopmental toxicity. Part IIMATERNAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER(BDE-209) DURING PREGNANCY DISRUPTS PROLIFERATION HIPPOCAMPAL NEURAL STEM CELLS IN FETAL RATS[Objective]:The SD rats on pregnancy day 1 were intragastrically administrated with different concentrations of BDE-209 every day.On the 14th day,the hippocampus of fetus rats in each group was sterilely harvested,and then NSCs were isolated and cultured in vitro. this study aimed to investigate the impact of maternal exposure to some concentrations of BDE-209 during pregnancy on the proliferation of NSCs in fetal rats.[Material and Methods]:1. The SD rats on pregnancy day 1 were randomly divided into control group (group A),arachis oil group(vehicle, group B),experimental groups (group C, D, E),and with 5 rats in each group. The rats in the group A were conventionally bred,10,30,50 mg/kg*d BDE-209 in arachis oil was intragastrically administrated in group C, D and E, respectively, while equal amount of purified arachis oil was given in group B.The experimental rats were administrated with corresponding drugs every morning from the purchase day to 14 days after pregnancy.2. The embryos of 14-pregnancy-day SD rats were sterilely harvested,and then NSCs were isolated and cultured in vitro. The cell growth was observed every day under an inverted microscope,and the culture medium was changed every 3-4 days,and passage was performed every 7-10 days.3. On the 7th day,the identification of NSCs was detected by immunofluorescence in vitro, the purity of NSCs were detected using flow cytometry.4. The proliferation of NSCs was detected using CCK-8 assay. Each section will parallel six.[Results]:1. Neurospheres were observed after hippocampal NSCs were cultured with serum-free medium for 24h.the neurospheres were gradually from small to large,irregular to regular after 3-5 days,single cell less.the neurospheres can increase to 200μm on the 7th day,the surface shows single cell with highlight. 2. immunofluorescence staining revealed that NSCs-specific Nestin presented positive,while DAPI staining showed nuclei presented blue fluorescence.3. Flow cytometry revealed that the purity of NSCs was 98.02% in neurospheres.4. The NSCs was observed under inverted microscope,the form of neurospheres was significantly slow in each experimental group compared with the control group,the cell bodies is samll and irregular with the increase of BDE-209 concentration during pregnancy.5. Growth curve revealed that cell proliferation was significantly decreased in each experimental group compared with the control group. Statistical analyses showed there was no significant difference in cell proliferation between group A and B (P>0.01), however, there was a significant difference between any two of remaining groups (P<0.01).[Conclusions]:It was found that hippocampal NSCs cultured in vitro with the serum-free medium had the capabilities of self-renewal, proliferation and differentiation. Maternal exposure to some concentrations of BDE-209 during pregnancy might impair the proliferation of hippocampal NSCs.With the increase of the BDE-209 concentration,the proliferation of NSCs were significantly decreased.Part IIIMATERNAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER(BDE-209) DURING PREGNANCY DISRUPTS APOPTOSIS OF HIPPOCAMPAL NEURAL STEM CELLS IN FETAL RATS[Objective]:The SD rats on pregnancy day 1 were intragastrically administrated with different concentrations of BDE-209 every day.On the 14th day,the hippocampus of fetus rats in each group was sterilely harvested,and then NSCs were isolated and cultured in vitro.this study aimed to investigate the impact of maternal exposure to some concentrations of BDE-209 during pregnancy on the apoptosis of NSCs in fetal rats. [Material and Methods]:1. The SD rats on pregnancy day 1 were randomly divided into control group (group A),arachis oil group(vehicle, group B),experimental groups (group C,D,E),and with 5 rats in each group. The rats in the group A were conventionally bred,10,30,50 mg/kg*d BDE-209 in arachis oil was intragastrically administrated in group C,D and E, respectively, while equal amount of purified arachis oil was given in group B.The experimental rats were administrated with corresponding drugs every morning from the purchase day to 14 days after pregnancy.2. The embryos of 14-pregnancy-day SD rats were sterilely harvested,and then NSCs were isolated and cultured in vitro. The cell growth was observed every day under an inverted microscope,and the culture medium was changed every 3-4 days,and passage was performed every 7-10 days.3. Hippocampus apoptosis of NSCs in secondary passage was detected by flow cytometry. Each section will parallel six.[Results]:Apoptosis rate of NSCs in secondary passage was detected by flow cytometry in each group.It was found that there was no significant difference in apoptosis rate between group A and B (P>0.05) while there was significant difference between any two of remaining groups (P<0.05).[Conclusions]:Maternal exposure to some concentrations of BDE-209 during pregnancy might impair the apoptosis of hippocampal NSCs.With the increase of the BDE-209 concentration,the apoptosis of NSCs were significantly decreased. [Key word]:PBDE-209,hippocampal neural stem cells,apoptosisPartⅣMATERNAL EXPOSURE TO DECABROMINATED DIPHENYL ETHER(BDE-209) DURING PREGNANCY DISRUPTS DIFFERENTIATION OF HIPPOCAMPAL NEURAL STEM CELLS IN FETAL RATS[Objective]:The SD rats on pregnancy day 1 were intragastrically administrated with different concentrations of BDE-209 every day.On the 14th day,the hippocampus of fetus rats in each group was sterilely harvested,and then NSCs were isolated and cultured in vitro.this study aimed to investigate the impact of maternal exposure to some concentrations of BDE-209 during pregnancy on the differentiation of NSCs in fetal rats.[Material and Methods]:1. The SD rats on pregnancy day 1 were randomly divided into control group (group A),arachis oil group(vehicle, group B),experimental groups (group C,D,E),and with 5 rats in each group.The rats in the group A were conventionally bred,10,30,50 mg/kg*d BDE-209 in arachis oil was intragastrically administrated in group C,D and E,respectively, while equal amount of purified arachis oil was given in group B.The experimental rats were administrated with corresponding drugs every morning from the purchase day to 14 days after pregnancy.2. The embryos of 14-pregnancy-day SD rats were sterilely harvested,and then NSCs were isolated and cultured in vitro. The cell growth was observed every day under an inverted microscope,the culture medium was changed every 3-4 days,and passage was performed every 7-10 days.3. Hippocampal NSCs cultured with the serum-free medium in vitro were induced to differentiate with 10 % fetal bovine serum in secondary passage.The differentiation of neurospheres was observed everyday under an inverted microscope.4. Hippocampus differentiation of NSCs in secondary passage was detected by immunofluorescence on the 7th day.Each section will parallel six.[Results]:1. The majority of well-differentiated cells grew in adherence with walls, and many processes around the neurospheres were observed 24 h later, and the processes gradually extended outwards.3 days later,the processes around the neuospheres further increased and presented radial extension.7 days later,the extended processes was cross-linked and presented a network.2. Immunofluorescence staining showed that the cytoplasm of NeuN- positive stained cells presented green fluorescence,and cell body was triangle,round or conical,and nuclei were large and rounded;the cytoplasm of GFAP-positive stained cells presented red fluorescence, and cell body was long spindle-shaped or polygonal,with long processes, and nuclei were smaller oval-shaped; DAPI- positive stained nuclei presented blue fluorescence.3. The proportion of hippocampus NSCs differentiated into neurons in offspring rats after maternal exposure to different concentrations of BDE-209 during pregnancy:Statistical analyses showed no significant difference between group A and B (P>0.05),however,there was a significant difference between any two of remaining groups (P<0.05).[Conclusions]:Maternal exposure to some concentrations of BDE-209 during pregnancy might impair the differentiation of hippocampal NSCs.With the increase of the BDE-209 concentration,the differentiation of NSCs was significantly decreased.