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N-acetylcysteine ??for BDE-209 in neonatal rat hippocampal neurons oxidative damage and preliminary study BDE-209 on hippocampal neurons influence the level of DNA methylation

Author: ChenJingSi
Tutor: ChenDunJin
School: Guangzhou Medical College
Course: Obstetrics and Gynaecology
Keywords: BDE-209 NAC Hippocampal neurons Morphology Cell viability Superoxide dismutase (SOD) Malondialdehyde (MDA) Nitric oxide (NO) The overall level of DNA methylation
CLC: R114
Type: Master's thesis
Year: 2010
Downloads: 60
Quote: 0
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Abstract

Polybrominated diphenyl ethers (Plybrominated diphenyl ethers, PBDEs) are some of the class of aromatic chemicals bromine atoms, from the 1980s onwards, it was found that flame retardants PCBs (Polychlorinated biphenyl, PCBs) harmful to human health, and PBDEs The chemical structure similar to the PCB, have similar flame-retardant effect, it is as polychlorinated biphenyls (PCBs) alternatives are widely used in a variety of consumer species. But as PBDEs and PCBs is an ongoing environmental organic pollutants. With industrial development, PBDEs worldwide increasing demand, the concentration of PBDEs in the environment are increasing year by year, is higher than the concentration of PCBs in the environment. PBDEs can be deposited in the water and soil, and through the food chain into animals and humans. In human milk, fat, blood, liver and other organs can be detected PBDEs. Acute toxicity of PBDEs is low, oral median lethal dose of gt; 5g/kg. PBDEs and chronic exposure, the effect of the target organ is the liver, kidney, thyroid, nervous system. At home and abroad for low PBDEs related toxicity study more, and more positive. From the large number of animal studies have found that PBDEs might liver toxicity, reproductive toxicity, thyroid toxicity, neurodevelopmental toxicity and potential carcinogenicity and so on. Therefore, in the EU and the U.S. have banned, decabromodiphenyl ether (decabrominated BDE, BDE-209 or DeBDE) less toxic polybrominated diphenyl ethers low in the United States and Europe are still allowed production use. In China, not only PBDEs in the production, use and export of power, which decabromodiphenyl ether (BDE-209) is manufactured using the largest, but also receive electronic waste power. BDE-209 has become an important environmental contaminants. Therefore, our in-depth on decabromodiphenyl ether (BDE-209) of a certain urgency and necessity. BDE-209 is the most widely used PBDEs, brominated its lower toxicity is low, but it can enter the environment, after photolysis, pyrolysis, biological and microbiological degradation processes into bromo-p-dioxin, polybrominated dibenzofurans and lower brominated diphenyl ethers, causing greater toxicity. Thus, for BDE-209 whether there is damage to the body there are still a lot of controversy, however, related experiments carried out at home and abroad. In animal studies, BDE-209 can increase the incidence of hepatocellular carcinoma, the thyroid hormone secretion. BDE-209 with fetal toxicity, exposure during pregnancy, BDE-209, can be male offspring reproductive dysfunction. The developmental neurotoxicity in recent years people are most concerned about parts. This is because in animal studies found that exposure during pregnancy and postpartum in a variety of PBDEs (including BDE-47, 153,209, etc.), the persistence will produce behavior change, particularly in the muscle activity and cognition, learning, memory capacity; And in recent years found levels of PBDEs in breast milk increasing breastfed infants body contains levels of PBDEs than non-breastfed infants is high, but the concentration of PBDEs in vivo infants than adults to be high, which in addition to breastfeeding related with the outside and the contact housing in the garbage and infants less able to excrete PBDEs have a relationship. Seen on the BDE-209 in-depth study of the environment on human health and prenatal and postnatal care has an important significance. Our research team in the early experiments on primary fetal rat hippocampal neuron cells after exposure to BDE-209 was found in a certain dose of BDE-209 under the effect of fetal rat primary hippocampal neurons apoptosis and can occur oxidative damage, and there was a certain dose - response relationship, suggesting that oxidative stress may be caused by BDE-209 one of the important mechanisms of neurotoxicity. But BDE-209 is how to cause decline in learning and memory mechanism is still unclear whether there are any genotoxic remains unclear. The main subject of research at the cellular level antioxidant N-acetylcysteine ??(NAC) for BDE-209 induced primary cultured hippocampal neuronal oxidative damage and BDE-209 Preliminary study on primary cultured hippocampal neurons overall methylation of the DNA level. DNA methylation is an important mechanism for regulating gene expression, and in many poisons and diseases play an important role; normal DNA methylation is important for the central nervous system function, abnormal DNA methylation and cancer, mental retardation , immune deficiencies and other diseases have some relevance; on DNA methylation status of correct assessment, help us to BDE-209 in-depth understanding of toxicological mechanisms. This topic is divided into the following three parts: The first part of N-acetylcysteine ??on BDE-209 in neonatal rat hippocampal neurons morphologic changes 【Purpose】 take newborn within 24 hours of the original SD rat hippocampal tissue neuronal cell cultures, identifying the purity of neurons was observed N-acetylcysteine ??on BDE-209 induced primary cultured neonatal rat hippocampal neuronal morphology changes and the impact of lower cell viability. [Materials and Methods 1. Purchase newborn within 24 hours of SD rats hippocampal tissue taken for primary neuronal cell cultures. 2 hippocampal neurons cultured for 7 days neuronal cell purity identification. 3 7-day primary cultured neonatal rat hippocampal neurons were divided into 11 groups, the control group and the experimental group and the control group was divided into blank control group (1 ‰ DMSO-containing culture medium) and NAC group (containing 0.1mmol / L NAC in the culture medium). Experimental group is divided into group I (BDE-209 alone treated group), II group (BDE-209 and NAC simultaneously) and group III (NAC for 24 hours after the first, BDE-209 re-exposure), the major groups are containing three concentrations of BDE-209 group, BDE-209 concentrations were 10,30,50 ug / ml. NAC concentration was 0.1mmol / L. Each set three parallel samples, experiments repeated three times. After 24h exposure, under an inverted microscope for cell morphology. 4.MTT colorimetric detection of neuronal survival. [Results] 1. Primary hippocampal neurons cultured for 7 days was observed neurons grow well cell in the body, trunk and branches synapses was significantly longer and thicker, and its projection forming dense neural networks. 2 NeuN immunocytochemistry identified neurons classical methods. The results can be seen scattered hippocampal neurons, neurons are subject to 95%. 3 different experimental groups inverted microscope neuronal morphology in the experimental group I 10ug/ml group, 30ug/ml group, 50ug/ml group neuronal cell bodies seen somewhat smaller, deformation, membrane integrity but appear foaming phenomenon, with the exposure dose increased, the foaming phenomenon obviously, their neurites appear shortened 50ug/ml group were seen in the cells appeared shrunken, rounded off, protruding shorten or even disappear. Group II and III in the experimental group were seen neuronal cell body deformation, cell foam has declined, the experimental group II and III between the groups no significant difference in cell morphology. 4. MTT colorimetric detection neuron survival, with the BDE-209 concentrations increased neuronal cell survival was significantly decreased (P lt; 0.05), II and III, the group group of neurons in both cell viability increased, but the group III neuronal cell survival and I group, no statistical significance (P gt; 0.05). Group II and Group I in cell viability compared with significantly higher (P lt; 0.05), was statistically significant (P gt; 0.05). [Conclusion] BDE-209 can cause primary cultured neonatal rat hippocampal neurons cell morphology, cell viability decreased, which BDE-209 have a dose-effect relationship, NAC can improve BDE-209 on hippocampal neuronal cell morphology and cell survival rate effects, NAC antagonism with BDE-209 and NAC concentration and the role of time-related. The second part of N-acetylcysteine ??on BDE-209 neonatal rat hippocampal neurons induced oxidative damage 【Purpose】 To observe the effect of N-acetylcysteine ??against BDE-209 neonatal rat hippocampal neurons induced oxidative damage of . [Materials and Methods 1. Primary cultured 7 days of hippocampal neurons in neonatal rats were divided into eight groups, namely control group and the experimental group, the control group and the experimental group and the control group was divided into blank control group (1 ‰-containing DMSO in the culture medium) and the NAC group (a culture solution containing 0.1mmol/LNAC). Experimental group Group I and II of the group, I group of BDE-209 alone treated group, in which groups of three concentrations were 10 ug / ml group, 30 ug / ml group and 50ug/ml group, II group BDE -209 simultaneously with the NAC group group is also divided into three levels, namely 10ug/ml group, 30ug/ml group and 50ug/ml, NAC concentration was 0.1mmol / L. After 24 hours, cells exposed to culture medium taken for testing. (2) determined using the xanthine oxidase activity of SOD. 3 using the thiobarbituric acid method for the determination of MDA. 4 determined by nitrate reductase NO content. [Results] 1 with the concentration of BDE-209 concentration increases, BDE-209 alone treated group and simultaneously with the NAC group, SOD activity was significantly decreased (P lt; 0.05). Simultaneously with NAC SOD activity in experimental group than BDE-209 exposure group alone SOD activity is high, and there are significant differences (P lt; 0.05) 2. Exposure in BDE-209 alone group, with BDE- 209 concentration, MDA content increased, 30ug/ml 50ug/ml group and compared with the control group, showed significant differences (P lt; 0.05), the BDE-209 alone treated group and the group with the NAC simultaneously compared between the experimental group and the NAC while the role of MDA content than BDE-209 alone treated group of MDA content is low, there are significant differences (P lt; 0.05). 3 In the BDE-209 single exposure group, with BDE-209 concentration increases, NO content were significantly increased (P lt; 0.05), three treated groups showed no significant difference (P gt; 0.05), In BDE-209 alone treated group and between groups simultaneously with NAC compared with the NAC simultaneously NO content in the experimental group than BDE-209 low-exposure group alone, there are significant differences (P lt; 0.05). [Conclusion] BDE-209 can cause hippocampal neurons oxidative stress, which may be of BDE-209 one of the mechanisms of neurotoxicity. And a certain concentration of NAC can improve certain concentration of BDE-209 induced oxidative damage. The third part of the BDE-209 on primary cultured neonatal rat hippocampal neurons overall level of DNA methylation [Objective] To detect BDE-209 on primary cultured neonatal rat hippocampal neurons overall DNA methylation level in order to explore BDE -209 effects on hippocampal neurons in rats. [Materials and Methods 1. Primary cultured seven days of newborn rats hippocampal neurons divided into five groups, namely the control group and the experimental A, B, C group (including BDE-209 concentrations were 10,30,50 ug / ml) in the control group was divided into completely blank control group and the exposure with 1 ‰ DMSO control group. Each set three parallel samples, experiments were repeated three times. 2 cells was extracted DNA3. Adopt an overall ELISA quantitative analysis of DNA methylation [Results] after exposure to BDE-209 were caused by the overall reduction in the level of DNA methylation, which 10ug/ml group and 30ug/ml group compared with the control group, a significant difference (P lt; 0.05), 10ug/ml group compared with DMSO control group there was a marked difference (P lt; 0.05). With increasing concentration of BDE-209 concentration increases, the overall increase in the level of DNA methylation, 10ug/ml group compared with 50ug/ml also significant differences. (P lt; 0.05). [Conclusion] a certain concentration of BDE-209 on hippocampal neurons overall methylation levels have an impact.

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