Bde-209, N-acetyl Cysteine ​​on The Bde-209 Induced Oxidative Damage, Neonatal Rat Hippocampal Neurons Of The Hippocampal Neurons Of The Dna Methylation Level

Polybrominated diphenly ethers (PBDEs) are a class of aromatic series which contain bromine atoms.From 20th century,we found that polycholorinated biphenyls(PCBs) had harmful effects on human health.So PCBs were banned to used as additive flame retardamts.PBDEs are chemically similar to PCBs,and instead of PCBs, they are usd as additive flame retardants in a variety of consumer products..PBDEs are persistent organic pollutants,bioaccumulate in the environment and biomagnify up the food chain.In the past three decades, we found that the levels of PCBs in biota have been decreasing,levels of PBDEs have significantly increased. They have been detected in human adipose tissue, serum and breast milk.PBDEs have low acute toxicity,with oral LD50s of >5g/kg.Upon chronic exposure,target organs are the liver,the kidney and the thyroid gland. Lower brominated congeners have been banned in United States and Europe.And in our country,we not only produce PBDEs in quantity, BDE-209 especially,but also we received a large of electronic wastes.BDE-209 has became an important environmental contaminants in our country.Therefor, it is necessary for us to have a research on BDE-209.Decabrominated diphenyl ether(BDE-209)is the most widely used congener of PBDEs,which are less potent than other lower brominated congeners. But BDE-209 can be broken down to the lower brominated congeners and induct more toxicity.So there are many disputes on the toxicity of BDE-209. In many animals experiments,we found that an increased incidence of hepatocellular carcinomas and of thyroid adenomas have been observed in rodents upon exposure to BED-209. Pregnant mice were exposed to BDE-209,which can impair male rat reproductive functions.The current greatest concern for potential adverse health effects of BDE-209 relates to their developmental neurotoxicity.Because animal studies have indicated that pre-and poste-natal exposure to PBDEs may cause long-lasting behavioral alterations,particularly in the domains of motor activity and cognitive behavior.And PBDEs are excreted in milk, and relatively high concentrations are found.Infants and toddlers have the highest body burden of PBDEs, due to exposure via maternal milk and house dust.Intensive study of BDE-209 is very signigicance for environmental health and improving birth outcome and child development.Our research group found that PBDE-209 could induce oxidative stress in hippocampal neurons of fetal rats and may be one of neurotoxic mechanisms of BDE-209.But we are not clear what mechanisms BDE-209 decreased the ability of learning and memory by and whether BDE-209 has genotoxicity.In this study, we observe protective effects of N-acetyl-cysteineon on oxidative stress which induced by BDE-209 on hippocampal neurons of neonatal rats in primary culture.And we found out the effect of BDE-209 on the global DNA methylation of hippocampal neurons initially.DNA methylation is an important mechanisms of genes modification.It is very important for central nervous systerm to keep DNA methylation in tune.To assay the state of DNA methylation correctly,it will help us to understand the mechanisma of toxicity inducted by BDE-209.The study is divided into three parts for the specific experiments.Part I Effects of N-acetyl-cysteineon on the changes of morphology induced by BDE-209 in hippocampal neurons of neonatal rats[Objective]:We use hippocampus of neonatal rats for primary neurons culture.Identification of neurons in the purity.To observe effects of N-acetyl-cysteineon on the changes of morphology and cell survival induced by BDE-209.[Material and Methods]:1. The SD rats of post-natal hour 24,From fetal rat hippocampus neurons in a primary cell culture.2. Nerve cells purity of identification that the hippocampus Neurons of training 7 d .3. After 7 days culture,cell were divided into 11 groups,That were, two control groups and experimental I, II, III Groups. In I group,there were three groups which exposed to BDE-209 solely,whose concentrations were. In II groups,there were three groups which exposed to BDE-209 and NAC at the same time,the BDE-209 concentrations were also 10, 30 and 50ug / ml .In III groups, cells were exposed to BDE-209 after exposing to NAC for 24 hours the BDE-209 concentrations were 10, 30 and 50ug / ml. NAC concentrations was 0.1mmol/L. Each section will parallel three.The control group joined with the 1‰DMSO medium and 0.1mmol/L NAC.The inverted microscope under the observation of cells afer 24h.4. MTT colorimetric analysis detecting neuron survival.[Result]:1. After primary culture 7 days ,the growth state of cells were well. Nuclei and the nucleolus clearly visible, longer processes and branches, edge clear, The neural networks could been seen obviously.2. Immuocytochemistry identified neurons and glial cells. Results from that Neurons is the main account for 95%.3. Under microscope,in I section showed that neurous have smaller cell bodies,deformation,and the cell membrane integrity with foaming phenomenon.With the dose of BDE-209 increased,foaming phenomenon is more and more clearer.In group 50ug/ml, we could seen shrunken.detached procesesses in the cells,even disappear. The differences of the cells morphology between I section and II setion were not significant4. MTT colorimetric analysis detecting neuron survival. The neuron survival in hippocampus was decreased with increased dose of BDE-209.The rate of livability in II setion were significantly higher than I setion. (P< 0.0 5).There were not difference between I setion and III setion (P>0.0 5).[Conclusions]:The BDE-209 can chang the cell morphology and decrease the rate of survival in primary culture of rat hippocampal neurons in some range of dose N-acetyl-cysteineon can improve the effects of BDE-209 in cell morphology and the rate of survival. Part II Protective effects of N-acetyl-cysteineon on oxidative stress induced by BDE-209 on hippocampal neurons[Objective]:To investigate the effects of N-acetyl-cysteineon on oxidative stress induced by BDE-209 on hippocampal neurons of the neonatal rat in primary culture.[Material and Methods]:1. The SD rats of post-natal hour 24,From fetal rat hippocampus neurons in a primary cell culture.2. After 7 days culture,cell were divided into 8 groups,That were, two control groups and experimental I, II Groups. In I group,there were three groups which exposed to BDE-209 solely,whose concentrations were. In II groups,there were three groups which exposed to BDE-209 and NAC at the same time,the BDE-209 concentrations were also 10, 30 and 50ug / ml .NAC concentrations was 0.1mmol/L. Each section will parallel three.The control group joined with the 1‰DMSO medium and 0.1mmol/L NAC.The inverted microscope under the observation of cells afer 24h.3. Assay activity of SOD by the measuring xanthine oxidase.4. Assay content of MDA by measuring thiobarbituric acid5. Assay content of NO by the method of Nitrate reductase[Result]:1. With the dose of BDE-209 increased,the activity of SOD were decreasing significantly in both two setions. The activity of SOD in section II were higher in section I (P<0.05)2. With the dose of BDE-209 increased,the content of MDA were increasing significantly in setions I. The content of MDA in section II were higher in section I (P<0.05)3. With the dose of BDE-209 increased,the content of NO were increasing significantly in setions I. The content of MDA in section I were higher in section II (P<0.05)[Conclusions]:The BDE-209 can induce oxidative damage on hippocampal neurons which may be one of the toxicity mechanisms.N-acetyl-cysteineon can improve the oxidative damage induced by BDE-209 in some concentrations.Part III Effect of BDE-209 on DNA methylation of hippocampal neurons[Objective]:To evaluate the effect of BDE-209 on global DNA methylation of hippocampal neurons of neonatal rats in primary culture[Material and Methods]:1. The BDE-209 Exposure on the hippocampal neurons of primary culture neonatal rat after 7d .Experiment is divided into five groups,There were, two control group and three experimental Groups(BDE-209 concentrations were 10,30 and 50 ug / ml)Eachsection will parallel three.One control group joined with the 1‰DMSO medium,the other control group were band control.2. Extraction of DNA.3. Assay the global DNA methylation by enzyme-linked immunosorbent measure[Result]:After exposed to BDE-209,the level of global DNA methylation were decreasing Compared with bank control,10ug/ml group and 30ug/ml group had difference significantly.With the dose of BDE-209 increased,the level of global DNA methylation were increasing.[Conclusions]:The BDE-209 has effects on the level of global DNA methylation in some dose.