The Effect Of Maternal BDE-209 Exposure On Severeal Neurotypic And Gliotypic Proteins,Oxidative And Apoptosis In Hippocampus Of Offsping Rats

Polybrominated diphenylethers (PBDEs) are a widely used class of organic brominated flame retardants. Over the previous 20 years, concentrations of PBDEs have been increasing in sediment, fish, bile, human milk, serum and adipose tissue due to the persistence and bioaccumulation of these materials. Especially in human tissue, the concentrations of PBDEs were found to be 8.61—46.05 ng/g lipid; mean, 19.33 ng/g lipid in blood; 6.2- 419 ng/g lipid in milk; 17-9630 ng/g lipid in adipose tissue. The most widely used congener of the PBDEs is BDE-209, and the world wide use of this congener was 56100 metric tones for the year 2001, and the levels tend to increase over time .It is also interesting to note the fact that levels of BDE-209 markedly exceed the levels of all other congeners ,especially since BDE-209 is the only PBDE still in production.The toxic effects of PBDEs are currently a rising public concern. Their structural similarity to PCBs has caused attention to focus on the potential effects to the developing nervous system. The most important risk of PBDEs exposure is developmental neurotoxicity. Mouse receiving PBDEs during the brain spurt, manifested spontaneous deranged behavior, learning and memory defects, and dysfunction in the cholinergic system in adult, all of which worsen with age, calls for further studies of the neurotoxicity of PBDEs.Hippocampus play a central role in the process of learning and memory. Therefore, the present study explores the effects of different concentrations of the matreal BDE-209 on oxidative stress, signaling proteins, and apoptosis in hippocampus of offsping rats, and examines the correlation between the presence of ROS and the levels of signaling proteins and apoptosis. The results provide useful information for further study of the mechanism of BDE-209 induced neurotoxicity.Part I EFFECT OF MATERNAL BDE-209 EXPOSURE ON SPACIAL LEARNING AND THE MICROSTRUCTURE INHIPPOCAMPUS OF OFFSPRING RATS[Objective]To evaluate the effect of the maternal BDE-209 on the offspring rats's learning andmemory ability and the change of the microstructure in hippocampus.[Material and Methods]1 .Fifty female rats were be divided in 5 groups freedly. The experimental group weregiven BDE-209 in doses of 100(A),300(B),600(C),1200(D) mg/ kg·d by oralgavage when they are in gestational and lactational. The control group wasadministered only with the same capacity of Peanut oil at the same time.2.Twenty male offspring rats of each group will be exzamed by the method of 100 ofthe offspring rats will be examined of their learning and memory ability by the method of Morris water maze when they are 28 days old.3. Hippocampus were obtained from five offspring rats of each group after Morriswater maze. The change of microstructure in hippocampus were observe by lightmicroscope after HE staining.[Results]1.Offspring rats of the experimental group have lower learning and memory abilitycompared with the control group .There is singnifcant difference in learning andmemory ability between the group C,D and the group E (P<0.05, <0.01),but there isnot in the group A and B.2. Obvious histomorphology changs were found in group C and D in hippocampus ofthe offspring rats.[Conclusions]1. High dose maternal BDE-209 exposure will diminish the offsprings' ability oflearning and memory.2.High dose maternal BDE-209 exposure can affect the the development of nerve celland damage the microstructure of hippocampus.Part II EFFECT OF MATERNAL BDE-209 EXPOSURE ON THE CAMKⅡCONTENT AND THE EXPRESSION OF GAP-43,BDNF IN HIPPOCAMPUS OF OFFSPING RATS[Objective]The previous work confirmed that neurotypic and gliotypic proteins can serve as sensitive indicators of time- and region-specific effects of chemicals on the developing nervous system. In the present study ,several biochemical indices were choosed to characterize the effects of BDE-209 on neonatal brain development. We hypothesized that the effects of PBDE on critical developmental processes would be reflected by changes in the biochemical substrates underlying them. The levels of several proteins involved in neuronal survival, growth, and synaptogenesis were examined. There are signaling proteins that are highly enriched in the nervous system and regulate neuronal processes which peak during the brain growth spurt. In this study CaMKⅡ,GAP-43 and BDNF were measured to observe the effect of the maternal BDE-209[Material and Methods]1. Hippocampus were obtained from ten offspring rats of each group after Morris water maze.2. The levels of CaMK II in hippocampus were examined using sandwich ELISA procedures.3. The expression of GAP-43 and BDNF in the hippocampus were examined by means of immunohistochemistry.[Results]1.The CaMKⅡactivity in hippocampus decreased in different level. The group C,D had significant difference from control group(P=0.031, P=0.005); The group A and B had no significant difference from control group(P=0.526, P=0.284); In the experiment groups, the group A had significant difference from the group D (P=0.025) .But there is no significant difference of CaMKⅡactivity between the group B,C and the group D(P=0.068, P=0.474).2.There is singnifcant difference of the expression of GAP-43 in hippocampus between the group C,D and the group E (P=0.013, P=0.000). There were no significant differences between the group A,B and the group E (P=0.177, P=0.093). In the experiment groups, the group A,B had significant difference from the group D (P=0.011, P=0.025) .But there is no significant difference between the group C and the group D (P=0.157).3.There is singnifcant difference of the expression of BDNF in hippocampus between the experiment group (group B,C,D) and the control group (P=0.033, P=0.005, P=0.001);There was no significant differences between the group A and the control group(P=0.066). In the experiment groups, the group A,B,C had no significant difference from the group D (P=0.052, P=0.103, P=0.409) .[Conclusions]The present result show that biochemical assessment of proteins involved in normal brain development may be useful biomarkers for developmental neurotoxicity. All three of these proteins are known as biochemical substrates for cellular processes (including neurite outgrowth and synaptogenesis) which support the formation of proper connectivity in the nervous system. The relationship between chemical-induced changes in these biochemical substrates of growth and plasticity during the brain growth spurt and possible morphological and functional consequences remains to be determined.Part III EEFFECT OF MATERNAL BDE-209 EXPOSURE ON THE SOD ACTIVITY AND THE MDA CONTENT IN HIPPOCAMPUS OF OFFSPRING RATS[Objective]Our initial studies quantified the maternal BDE-209 exposure can damage the ability of learning and memory of the offsprings rats. The toxic effect is not very clear, many studies suggest that xenobiotics can facilitate the active oxygen derived free radicals formation, which make many tissue systems in oxidative stress situation. The developmental nervous system is especially susceptible to it. In This study, lipid peroxidation of BDE-209 was observed in the hippocampus of offspring rats after the maternal BDE-209 exposure.[Material and Methods]1. Hippocampus were obtained from ten offspring rats of each group after Morris water maze.2. The SOD activity is determined by a hydroxylamine assay which was developed from a xanthine oxidase assay using chemical colorimetry. After reaction, the absorbance at 560 nm was monitored using a spectrophotometer.3. The colorimetric determination of MDA is based on the reaction of one molecule of the reactive aldehyde with two molecules of thiobarbituric acid at low pH (2-3) and 95℃for 45 min. The resultant pink color was extracted by n-butanol, and the absorbance at 532 nm was determined with a spectrophotometer.[Results]1. The SOD activity in hippocampus of the experiment groups of the offsprins rats decreased in different level. The group C,D had significant difference from control group(P=0.016, P= 0.000); There were no significant differences between the group A,B and the group E(P=0.180, P=0.068). In the experiment groups, the group A,B had significant difference from the group D (P=0.003, P=0.013) .But there is no significant difference between the group C and the group D (P=0.059).2. The MDA content in hippocampus of the experiment groups of the offsprins rats increased in different level, the groups C,D had significant difference from the control group(P=0.000, P=0.000); There were no significant difference between the group A,B and the group E (P=0.407, P=0.081). In the experiment groups, the group A,B had significant difference from the group D (P=0.001, P=0.014) .But there is no significant difference between the group C and the group D (P=0.451).[Conclusions]The studies suggest that BDE-209 can facilitate the ROS formation, which makemany tissue systems in oxidative stress situation. Lipid peroxidatic produce MDAincreased obviously, and activity of antioxidant enzyme SOD decreased; whichindicated that maternal BDE-209 exposure can make lipid peroxidation in centralnervous system.Part IV EFFECT OF MATERNAL BDE-209 EXPOSURE ON THE EXPRESSION OF BCL-2 AND BAX GENE IN HIPPOCAMPUS OF OFFSPRING RATS[Objective]Apoptosis is a cellular autonomy death model controlled by genes, which is completely different from cellular necrosis in morphology. Apoptosis contributes to the selective elimination of cells in physiologic and pathologic situations. Some studies have suggested that apoptosis involves the participation, at different levels, of ROS like hydrogen peroxide, superoxide anion, and singlet oxygen. The aim of this study is to evaluate the effect of maternal BDE-209 on expression of Bcl-2 and Bax gene in hippocampus of offspring rats .[Material and Methods]1. Hippocampus were obtained from five offspring rats of each group after Morris water maze.2. The expression of Bcl -2, Bax mRNA in hippocampus of the offspring rats were examined by RT-PCR. [Results]1.The results of PCR indicated that the positive expression of Bcl-2 in hippocampusof the experiment groups of the offsprins rats decreased in different level. Thegroups C,D had significant difference from the group E (P=0.011, P=0.005); Therewere no significant differences between the group A, B and the group E (P=0.459,P=0.174). There is significant difference between the group A and the group D(P=0.025).2. The positive expression of Bax in hippocampus of the experiment groups of theoffsprins rats decreased in different level. The groups C,D had significant differencefrom control group(P=0.032, P=0.002); There were no significant differencesbetween the group A,B and the group E(P=0.525, P=0.167). There is significantdifference between the group A and the group D (P=0.010).[Conclusions]Maternal BDE-209 exposure can decrease the positive expression of Bcl-2, increasethe positive expression of Bax in the offspring rat's hippocampus, indicating thatapoptosis was involved in the neurotoxic mechanism of BDE-209 .As a regulatinggene, Bcl-2 and Bax may participate in hippocampus neuron apoptosis induced byBDE-209.SUMMARY1.High dose maternal BDE-209 exposure will diminish the offsprings' learning andmemory abilityand can affect the development of nerve cell and chang themicrostructure.2.Biochemical assessment of proteins involved in normal brain development may beuseful biomarkers for developmental neurotoxicity. CaMKⅡ,GAP-43 and BDNF areknown biochemical substrates for cellular processes (including neurite outgrowth and synaptogenesis) which support the formation of proper connectivity in the nervous system. The relationship between hemical-induced changes in these biochemical substrates of growth and plasticity during the brain growth spurt and possible morphological and functional consequences remains to be determined.3. Maternal BDE-209 can facilitate the ROS formation, which make many tissue systems in oxidative stress situation. Lipid peroxidatic produce MDA increased obviously, and activity of antioxidant enzyme SOD decreased; which indicated that the maternal BDE-209 can make lipid peroxidation in central nervous system. 4.Matemal BDE-209 exposure can decrease the positive expression of Bcl-2, increase the positive expression of Bax in the offspring rat's hippocampus, indicating that apoptosis was involved in the neurotoxic mechanism of BDE-209 .As a regulating gene, Bcl-2 and Bax may participate in hippocampus neuron apoptosis induced by BDE-209.