摘要
1、PBDEs(多溴联苯醚)及目前相关研究
多溴联苯醚(PBDEs)是一系列含溴原子的芳香族化合物,最初因作为防火阻燃剂被人类利用,现已被普遍认为全球环境污染物及健康危险因子。其结构稳定,具有高生物活性、持久性、脂溶性等特点,可随着废弃的电子产品进入大气、水源、土壤以及人类的食物链中,构成持续性的有机污染物(POPs),对环境和人体造成巨大的潜伏性危害,有学者称之为“化学定时炸弹”。PBDEs存在于空气、土壤、食物链中,可通过呼吸、皮肤或消化道进入机体,甚至可以通过胎盘或母乳进入胎儿和新生儿体内。进入体内后多数在脂肪组织积累,部分低溴PBDEs还能在机体的一些靶器官中积聚,如:肝脏、肾上腺皮质、卵巢、肺及(最初时)脑中,其浓度通过生物积聚作用而逐渐升高。目前动物实验证明PBDEs对机体健康有不良影响,如:破坏甲状腺荷尔蒙,削弱学习和记忆能力,改变行为举止,令听觉神经受损,积聚于靶器官并对其功能结构产生影响,如睾丸、卵巢等,延迟青春期,减低精子数,造成畸形胎儿和引发癌症。
近年PBDEs在环境及有机体中浓度呈逐年增高趋势,大约每隔2—5年增加一倍,而海洋哺乳动物如海豹的体内浓度每隔1.8年就增加一倍。据报道,1992-2002年温哥华妇女母乳中PBDEs的浓度增加了5倍。2008年比利时学者研究人体脂肪组织和肝脏中PBDEs的浓度分别达5.3ng/g/lw和3.6ng/g/lw,比过去十年增加了5-10倍。美国得克萨斯大学公共卫生中心研究表明,北美妇女母乳中PBDEs的浓度可达6-419 ng/g,数量级别比世界其它国家报道的浓度高3-100倍,居全世界第一位。
2、BDE-209(十溴联苯醚)及目前相关研究
BDE-209又称十溴联苯醚(deca-BDE),是含有十个溴原子的一种多溴联苯醚(PBDEs)类化合物,与其它PBDEs不同之处在于其具有光化降解的特性,经紫外光照射后脱溴而最后分解成为低溴PBDEs(penta-BDE、octa-BDE)。2008年北美已开始禁止使用五溴和八溴联苯类物(penta-BDE、octa-BDE),但BDE-209仍允许使用,其在全世界生产、使用量最大,占据了PBDEs的80%。目前在全球范围内,含有PBDEs的产品中主要包括:75%的BDE-209,10%penta-,15%octa-。对于BDE-209(十溴联苯醚即decaBDE)的潜在毒性损害的有关研究较有限,主要集中在啮齿类动物及低级哺乳动物。有报道称怀孕的动物接触了BDE-209同源物,会出现体重不增、肝脏肿大、高胆固醇血症,并且严重损害胎儿健康,造成子代四肢和尿道畸形、心脏肿大、肋骨弯曲、骨骼迟变硬和体重不增等异常。BDE-209暴露可致动物癌变、自主学习行为能力下降及甲状腺平衡紊乱等,且与BDE-209的剂量呈正相关。本课题组前期研究发现母代本大鼠孕期前后及哺乳期持续暴露一定剂量BDE-209(300mg/Kg/d),可导致仔代大鼠的免疫功能显著下降;且可降低子大鼠学习记忆能力,影响仔鼠神经系统发育,但损伤的具体机制尚不明确。
3、目的
各种关于BDE-209可能是一种持续性有机污染物(POPs,即持久的、生物累积的、有毒的)的研究信息,均提示其存在潜在危害(包括对生态环境及人类健康)。2007年西班牙马德里人群抽样调查显示,母体血清、父系血清、脐血清、母乳样品中PBDEs浓度分别为9.7—12ng/g、12 ng/g、15—17 ng/g、5.5—6.1ng/g。其中BDE-47是母体血清、父系血清、脐血清中最主要的一类PBDEs,而在胎盘和母乳中占最主要的是BDE-209,结果提示PBDEs具有其它持续性有机污染物(POPs)类似的特性,即可通过胎盘屏障,但各种不同的同源物透过胎盘的速度各不相同,不同区域、性别、年龄阶层差异不显著,但母体血清、脐血、母乳样品中的差异则具有统计学意义,这说明出生前及出生后的母乳暴露直接影响PBDEs生物积累作用。加拿大学者对啮齿目动物的研究提示,PBDEs可通过破坏胆碱能神经递质系统而具有神经发育毒性;瑞士学者Virberg等研究发现出生后第三天暴露一定剂量的BDE-209,可导致大鼠成年后(2月龄)自发性行为的改变和对烟碱激动剂的诱导反应的下降,并认为这可能与大鼠神经发育中胆碱能系统受破坏有关,但并未从母源性角度研究大鼠在出生前暴露的影响,其具体的机制尚需进一步证实。
因此,本实验在课题组的前期实验基础上,从母胎医学角度,研究不同剂量BDE-209对仔鼠空间学习记忆能力的影响及剂量效应关系;进一步从乙酰胆碱能神经递质、NO神经递质方面探讨BDE-209对机体神经系统产生危害的作用机制,为下一步找到相应干预措施奠定基础。
第一部分不同剂量母源性BDE-209暴露对仔代大鼠空间学习记忆能力和海马组织结构的影响
【目的】
本研究建立不同剂量母源性BDE-209大鼠胃灌暴露模型,Morris水迷宫检测各组仔鼠的空间行为学,立足于母胎医学领域,研究不同剂量的BDE-209暴露对仔代大鼠的空间学习记忆能力的影响及其剂量效应关系,目前尚无相关报道。并从形态学角度比较光镜下各组仔鼠海马组织CA1结构的变化,旨在进一步研究母源性BDE-209对仔鼠神经毒性的结构基础。
【方法】
1、建立BDE-209母源性暴露模型
3月龄的SPF级Wistar大鼠75只(体重250～320 g,其中雌鼠50只,雄鼠25只,购于南方医科大学实验动物中心)。分笼饲养7天,按雌雄比例为2:1合笼。以雌鼠阴道分泌物涂片发现活动的精子作为确定大鼠交配成功。
分组:给交配成功后的雌鼠进行动物编号,采用随机数字表法将妊娠雌鼠分为五组,每组10只。胃灌时间自确定大鼠交配成功开始,直至其自然分娩且哺乳仔代大鼠的第21天。实验组(A):胃灌一定剂量BDE-209(100mg/kg/d)+精练花生油;实验组(B):胃灌一定剂量BDE-209(300mg/kg/d)+精练花生油;实验组(C):胃灌一定剂量BDE-209(600mg/kg/d)+精练花生油;实验组(D):胃灌一定剂量BDE-209(1200mg/kg/d)+精练花生油;对照组(E):胃灌等量的精练花生油。
每日傍晚避光胃灌一次,喂养方法按SPF级饲养,自由采食,饮水,采光,自然日夜周期。自然妊娠,分娩第一代仔代大鼠为相应的A、B、C、D、E组,即本实验的研究对象。
2、Morris水迷宫实验对仔鼠进行空间学习行为的检测:给动物编号,按随机数表法随机取各组第一代断乳后的雄性仔鼠(体重120～150 g)各20只。每天固定时间每只仔鼠训练2次,上、下午各1次,每次分别从四个不同的入水点入水,共8天,其中前3天训练让仔鼠熟悉水迷宫及实验室环境,不记录成绩,后5天为正式测试。训练时将其面向侧壁放入水中,待找到平台后,让其在平台上停留30 s,记录大鼠寻台时间即逃避潜伏期(Escape latency)。如果在120 s内未找到平台,需用手将其引至平台,这时潜伏期记为120 s,每次训练间隔最少60 s。通过记录各组仔鼠的逃避潜伏期,比较仔鼠空间学习记忆能力。
3、仔鼠海马组织切片制作:Morris水迷宫试验结束后,从各组分别随机选取5只仔鼠观察其海马组织结构。固定动物,10%水合氯醛4ml/kg腹腔麻醉后,打开胸腔经左心室插穿刺针至升主动脉,依次应用生理盐水200ml、4%多聚甲醛磷酸盐缓冲液200ml灌注。灌注结束后,取海马组织,放于4%多聚甲醛磷酸盐缓冲液4℃冰箱中固定24小时,常规石蜡包埋,存于4℃冰箱。多聚赖胺酸预处理载玻片备用,用轮转式切片机对蜡块进行连续切片,片厚4μm,捞组织片于载玻片上,置于60℃烤干备用。再经脱蜡、HE染色、封片等步骤后制成病理切片,在光镜下观察海马组织CA1区的结构。
【结果】
1、母源性BDE-209对仔代大鼠空间学习记忆能力的影响
1.1主效应时间因素(day)差异有统计学意义(F=37.351,P=0.000),各组潜伏期有随时间变化的趋势;时间和分组的交互效应无显著差异(F=0.163,P=1.000),说明时间因素的作用不随着分组的不同而不同。
1.2分组因素差异有统计学意义(F=10.971,P=0.000),说明分组因素起作用。
1.3每个时间点上5个分组之间的两两比较:
第1-2天,总P均大于0.05,说明各组潜伏期差异无统计学意义,提示各组仔代大鼠空间学习记忆能力无显著差异。
第3-5天时,与对照组E组相比,①A组与E组潜伏期无显著差异(P均大于0.05),提示一定剂量母源性BDE-209(100mg/kg/d)暴露对仔鼠空间学习记忆能力无显著影响;②而B、C、D组潜伏期均延长,差异有统计学意义(P均小于0.05),说明暴露一定剂量的母源性BDE-209(≥300mg/kg/d)可导致仔鼠空间学习记忆能力下降。
2、HE染色光镜下观察各组仔鼠海马组织CA1区结构的变化:E组仔鼠海马各层组织结构层次清晰,神经细胞排列整齐,细胞轮廓清楚,细胞间隙正常,核居中,A、B组与对照组E无显著差异。但C、D组神经细胞数量显著减少,排列紊乱,细胞核固缩过半,细胞水肿。
【结论】
提示低剂量母源性BDE-209暴露对仔鼠空间学习记忆能力无显著影响;达到一定剂量浓度的BDE-209(≥300mg/kg/d)方可导致仔鼠的空间学习记忆能力障碍且随着暴露剂量的增加,影响越显著。可见BDE-209对大鼠空间学习记忆能力影响与暴露剂量有着密切联系,即存在剂量效应关系;光镜下发现一定剂量的BDE-209的母源性暴露损伤仔鼠海马组织结构(神经细胞数量显著减少,排列紊乱),这可能是其导致仔鼠神经系统损伤的结构基础。
第二部分母源性BDE-209对仔代大鼠海马胆碱能神经递质的影响
【目的】
本研究通过比较各组仔代大鼠海马组织胆碱能神经递质的变化,如乙酰胆碱纤维量(ACH)、乙酰胆碱脂酶(ACHE)活性、毒蕈碱受体(M-ACHR)含量、烟碱受体(N-ACHR)含量,从胆碱能神经递质方面探讨BDE-209对仔鼠空间学习记忆损伤的可能机制,为下一步的干预奠定基础。
【方法】
1、动物的处理与标本的收集:
动物模型同第一章。Morris水迷宫试验结束后,按随机数字表从每组分别随机选取10只仔鼠进行取材。固定动物,10%水合氯醛4ml/kg腹腔麻醉满意后,断头处死,剥出全脑,掀开大脑皮层,用眼科镊小心分离海马组织,4℃生理盐水漂洗去血液,放入冻存管,立即置于-170℃液氮内保存。将液氮灌中组织块取出,解冻,4℃预冷的生理盐水漂洗,滤纸拭干,称重,放入5 ml的小烧杯内,移液管量加入冷生理盐水(按9:1的比例加入),眼科剪尽快剪碎组织块(将盛有组织的小烧杯放入冰水中)。组织捣碎机10000～15000r/min上下研磨制成10%组织匀浆,将制备好的10%匀浆用低速离心机3000r/min离心10～15分钟,将离心好的匀浆留取上清液。取匀浆液备检,严格按各检测指标的相关试剂盒的要求步骤操作。
2、检测各组仔鼠海马组织中乙酰胆碱纤维量(ACH)、乙酰胆碱脂酶(ACHE)活性、毒蕈碱受体(M-ACHR)含量、烟碱受体(N-ACHR)含量。
【结果】
1、仔鼠海马组织乙酰胆碱纤维量(ACH):各组组间ACH含量差异有统计学意义(F=7.220,P=7.220);与对照组E组相比,B、C、D组ACH含量下降,差异有统计学意义(P值均小于0.05),A组与E组比较无显著差异(P=0.637)。实验组间比较,D组ACH含量较A、B、C组均下降,差异有统计学意义(P值均小于0.05)。
2、仔鼠海马组织乙酰胆碱脂酶(ACHE)活性:各组组间ACHE含量差异有统计学意义(F=9.524,P=0.000);与对照组E组相比,B、C、D组ACHE活性下降,差异有统计学意义(P值均小于0.01),A组与E组比较无显著差异(P=0.173)。实验组间比较,D组ACHE活性较A、B、C组均下降,差异有统计学意义(P值均小于0.05)。
3、仔鼠海马组织毒蕈碱受体(M-ACHR)含量:各组组间M-ACHR含量差异有统计学意义(F=3.838,P=0.009);与对照组E组相比,仅C、D组M受体表达含量下降,差异有统计学意义(P值均小于0.05),A、B组均与E组无显著差异(P=0.161,P=0.116)。实验组间比较,D组M受体含量较A、B组均下降,差异有统计学意义(P值均小于0.05)。
4、仔鼠海马组织烟碱受体(N-ACHR)含量:各组组间N-ACHR含量差异有统计学意义(F=4.055,P=0.007);与对照组E组相比,仅D组的N受体的表达含量下降,差异有统计学意义(P=0.001),A、B、C组均与E组无显著差异(P值均大于0.05)。实验组间比较,D组N受体的含量较A、B组均下降,差异有统计学意义(P值均小于0.01)。
【结论】
实验结果中不同剂量母源性BDE-209对仔鼠海马组织ACH含量、ACHE活性、M-ACHR含量、N-ACHR含量的影响并不完全相同,一定剂量母源性BDE-209可不同程度降低上述各相应指标,提示胆碱能神经递质是母源性BDE-209损伤仔鼠空间学习记忆能力的可能机制。
第三部分母源性BDE-209对仔代大鼠海马一氧化氮神经递质的影响
【目的】
母源性BDE-209暴露对仔代大鼠的学习记忆的损伤的具体机制,目前尚无相关报道。本研究通过比较各组仔代大鼠海马组织一氧化氮(NO)含量、神经元型一氧化氮合酶(nNOS)活性、环鸟苷酸(cGMP)含量,从NO神经递质方面探讨BDE-209对仔鼠学习记忆影响的可能机制,为下一步的干预奠定基础。
【方法】
动物模型同第一章。Morris水迷宫试验结束后,采用随机数字表从各组分别随机选取10只进行取材。固定动物,10%水合氯醛4ml/kg腹腔麻醉满意后,断头处死,剥出全脑,掀开大脑皮层,用眼科镊小心分离海马组织,4℃生理盐水漂洗去血液,放入冻存管,立即置于-170℃液氮内保存。将液氮灌中组织块取出,解冻,4℃预冷的生理盐水漂洗,滤纸拭干,称重,放入5 ml的小烧杯内,移液管量加入冷生理盐水(按9:1的比例加入),眼科剪尽快剪碎组织块(将盛有组织的小烧杯放入冰水中)。组织捣碎机10000～15000r/min上下研磨制成10%组织匀浆,将制备好的10%匀浆用低速离心机3000r/min离心10～15分钟,将离心好的匀浆留取上清液。取匀浆液备检,严格按一氧化氮(NO)、神经元型一氧化氮合酶(nNOS)、环鸟苷酸(cGMP)测定试剂盒的要求步骤操作。
【结果】
1、仔鼠海马组织NO含量:各组组间NO含量差异有统计学意义(F=0.6323,P=0.000);与对照组E相比,C、D组NO含量明显增加,差异有统计学意义(P＜0.05),A、B组与E组差异无统计学意义(P=0.515,P=0.135)。实验组间比较,D组NO含量较A、B组增加,差异有统计学意义(P＜0.05)。
2、仔鼠海马组织nNOS活性:各组组间nNOS活性有统计学意义(F=11.928,P=0.000);与对照组E相比,C、D组nNOS活性明显升高,差异有统计学意义(P＜0.05),A、B组与E组差异无统计学意义(P=0.965,P=0.112)。实验组间比较,D组nNOS活性较A、B组升高,差异有统计学意义(P＜0.05)。
3、仔鼠海马组织cGMP含量:各组组间cGMP含量差异有统计学意义(F=4.119,P=0.00);与对照组E相比,B、C、D组cGMP含量明显升高,差异有统计学意义(P＜0.05),A组与E组差异无统计学意义(P=0.133)。实验组间比较,D组cGMP含量较A组增加,差异有统计学意义(P=0.045)。
【结论】
实验结果显示不同剂量母源性BDE-209暴露能不同程度影响仔代大鼠海马组织一氧化氮(NO)、神经元型一氧化氮合酶(nNOS)、环鸟苷酸(cGMP),提示NO神经信号递质,尤其NO/CGMP通路途径可能是一定剂量母源性BDE-209损伤仔鼠空间学习记忆能力的机制之一。
全文小结
1、在大鼠神经系统发育的关键期,通过经口胃灌方法成功建立不同剂量母源性BDE-209大鼠慢性染毒模型,为仔代大鼠空间学习行为学研究及相关机制的探讨奠定了基础。此模型具有便于操作、成功率高和仿真程度好等特点。
2、一定剂量母源性BDE-209(≥300mg/kg/d)暴露可导致仔鼠的空间学习记忆能力障碍,且存在剂量效应关系。
3、一定剂量的BDE-209(≥600mg/kg/d)的母源性暴露可损伤仔鼠海马组织结构,这可能是其导致仔鼠神经系统损伤的基础,进而降低仔鼠空间学习记忆能力。
4、一定剂量母源性BDE-209暴露不同程度影响仔代大鼠海马组织ACH含量、ACHE活性、M-ACHR含量、N-ACHR含量,提示乙酰胆碱能神经递质是一定剂量母源性BDE-209损伤仔鼠学习记忆能力的可能机制。
5、一定剂量母源性BDE-209暴露不同程度影响仔代大鼠海马组织一氧化氮(NO)含量、神经元型一氧化氮合酶(nNOS)活性、环鸟苷酸(cGMP)含量,提示NO神经信号递质,尤其NO/CGMP途径是可能是一定剂量母源性BDE-209损伤仔鼠空间学习记忆能力的机制之一。
综上所述,一定剂量浓度母源性BDE-209暴露可能通过破坏胆碱能神经递质及一氧化氮神经递质机制而导致仔鼠学习记忆能力的下降。而具体的引起生物毒性的临界剂量浓度的确定、以及是否还有其它机制及各个机制间是否相互关联等问题仍待进一步研究。因BDE-209的生物累积作用,随着对其更加深入的研究,对于目前欧美等发达国家唯一可生产的BDE-209是否能继续商业上使用将会引起更加广泛的关注及限制。
1.About Polybrominated diphenly ethers(PBDEs) and the current study
Polybromine diphenyl ethers(PBDEs) is a series of aromatic compounds including bromine.At first extensively used as fire retardant chemicals,they have been considered as the the global environmental pollutant and danger to health recently.For the stable structure and the character of high biological activity, liposolubility,they can ingress in the atmosphere、water resource、soil and the food chain of human with the dumped electricity,so people call them as Persistent Organic Pollutants(POPs) and some experts claimed it as the "chemical time bomb". The can do potentially harm to the environment and human being enomously.As PBDEs exist in the air,soil and chain food,they can ingress into organism by respiration,skin and digestive tract,even into the fetals and newborns by the placenta and maternal milk.They accumulate in the fat tissue,and part of PBDEs gather in the target organs,such as liver,the adrenal cortex,ovary,lungs and the brain(initial),which the concentration increase gradually by the biology accumulation.At present animal experiments have demonstrated that PBDEs have harmful effects to the organic health.For example,they can disrupt the hormones in thyroid,decrease the ability of learning and study,alter the behaviour,destroy the sence of heating,postpone the adolescence,reduce the sperma amount,result in the fetus anomalies and cancer,influence the function of target organs,including testicle,ovary, and so on.
Recent years it is reported that the concentration of PBDEs has been increased largely in the environment and organic body with the years,and double every two to five years,meanwhile they double in the ocean mammals such as seals every one point eight years.It is claimed that the concentration of PBDEs in the meternal milk had have five times between the year of 1992 and 2002.In the year of 2008,the scholars in Belgium indicated that the density of PBDEs in the human livers and fat tissues have arrived to 5.3ng/g/lw and 3.6ng/g/lw,which has been five to ten times over past.The study of public health center in Texas US,have manifestated that the concentration of PBDEs in the meternal milk of North America has reached to6—419 ng/g,which has the three to ten times in the order of magnitude and dominate the first in the wideworld.
2.About BDE-209 and the current study
BDE-209 belongs to ten bromines associations phenylate(deca-BDE),includes ten bromines atoms.As one congener of the deca-bromine biphenyl ethers,BDE-209 is the most widely used,in commercial field are five bromines associations phenylate,eight bromines associations phenylate and ten bromines associations phenylate.BDE-209 has the photochemical degradation the characteristic,after can pass through the ultraviolet ray illumination to decompose the debrominate but finally to decompose into low bromine PBDBs(penta-BDE,octa-BDE).For ten bromines associations phenylate(deca-BDE is BDE-209) potential toxic damage of the study are unclear and limited information.The overseas scholar discovered ten bromines PBDEs(deca-BDE is BDE-209) has the carcinogenicity,and presents the correlation with the deca-BDE dosage.Thought BDE-209 mainly causes the liver malignant tumor,the non- white courage and uprightness leukemia,breast cancer and the thyroid cancer and so on.At present some country environmental protection organizations thought deca-BDE has the characteristic of the potential hazard to the human health and the environment and it is chemical substance which one kind receives pays attention highly.The health and the environment risk scientific committee(SCHER) proposed ten bromines diphenyl oxide(deca-BDE) to environment discharging,will be possible in the future to constitute serious issue. European Union later has come to reach the agreement about the comprehensive prohibition of using PBDE and gathering the bromine biphenyl(PBB) last year on July 1,2006.The PBDE-209 commercialization produced and applies still has the dispute until now.
3.OBJECTIVE:
BDE-209 is possibly one kind of POPs matter(durable,biological accumulation, Toxic),this research information hints of its potential hazards(including the ecological environment and human health).People from all over the world began to pay close attention to PBDEs,which is called the persistent organic pollutant substance(POPs).In recent studies we have shown that PBDE congeners BDE47,99,and 153can induce persistent neurotoxic effects in adult mice.In addition, neonatal PBDE 99 exposure can affect adult spontaneous behaviour and the cholinergic system in rats.H.Viberg et al have shown that neonatal exposure to BDE-209 can induce persistent behavioural neurotoxic effects in adult mice and that there effects get worse woth increasing age.Congener specific knowledge about the toxicity of PBDEs is limited.No complete toxicological evaluation is currently available on any PBDE individual congeners or any of the commercial mixtures.The effects still have been being discussed on the acute/chronic toxicity,irritation, sensitization,genotoxicity,carcinogenicity and teratogenicity.Some observations do indicate that PBDEs might act as potential endocrine disrupters and interfere with brain development.The people mainly through the edible meats,the domesticated fowl,the fish and the dairy products expose in this kind of chemical material.And PCBs were found in organisms exist as a result of greater toxicity,Serious health hazards to the next generation.Internationally banned.At present correlation harm correlation research day by day increases regarding the structure with PCBs the similar PBDEs,But high bromine biphenyl ethers(especially ten Bromine biphenyl ethers or PBDEs-209) toxicity studies are relatively limited,And therefore its potential for toxicity is not yet clear role.Thus,the study on the rodentia animals from the scholar in Canada,has indicated that PBDEs can impact on the cholinergic nerve system to be developmental neurotoxicity.Viberg et al find the changes in spontaneous behaviour and altered response to nicotine in the adult rat,after neonatal exposure to the brominated flame retardant,decabrominated diphenyl ether(BDE 209),but they never study the effect of BDE-209 exposure to the rats prior to births from the meternal point,and did not verify the further mechanism.
Based on the protophase experiment of our topic group,we evaluate the effect of maternal BDE-209 in different dosage on the offsprings' learning and memory ability and its relashionship with the dosage;To discover the mechanisms of the developmental neurotoxicity of BDE-209 from diacetyl-cholinergic system and nitric oxide neurotransmitter conduction and to establish the foundation of corresponding intervention、prevention and therapeutic treasure.This study has been dividied into 3 parts as following.
PartⅠEFFECT Of mAternal BDE-209 EXPOSURE ON LEARNING AND MEMORY ABILITY and the microstructure in hippocampus of offspring rats.
[Objective]
To evaluate the effect of prenatal and lactational BDE-209(brominated Diphenyl Ethers-209) in different dosage on the offspring's learning and memory ability through animal experiments,there is no correlated report so far.The study is to compare the learning and memory ability in each group after make the model of deca-BDE exposure in different dosage by oral gavage,and to observe the changes of the microstructure in hippocampus of offspring rats accounting for the development neurotoxicity induced by BDE-209 and the effect-dosage relationship.
[Material and Methods]
1.Establish the model of maternal BDE-209 exposure:
Three-month-old rats 75 totally were obtained from the southern medical university(the body weight vary from 250 to 320g,including female 50 and male 25), according to two and one,female and male rats were housed individually in plastic cages in a room.The success of mate was determined by the slough plug from the vagina in the female rats.The study time is from the success of mate of the maternal rats to lactation ends of the offspring rats
Group:Peanut oil suspensions of commercial BDE-209 was given in doses of 100(A)、300(B)、600(C)、1200(D)mg/kg·d by oral gavage for the experimental groups.The control group was administered only with the same capacity of Peanut oil at the same time.The animals were supplied with standardized pellet food with an ambient temperature of 22℃,and a 12/12h cycle of light and dark.The time of oral gavage is at dust.The offspring rat were grouped A、B、C、D、E correspongding.
Group A(the group exposed to BDE-209 100mg/ kg·d in gestation and lactation);
Group B(the group exposed to BDE-209 300mg/ kg·d in gestation and lactation);
Group C(the group exposed to BDE-209 600mg/ kg·d in gestation and lactation);
Group D(the group exposed to BDE-209 1200mg/ kg·d in gestation and lactation)
Group E(The control group)
2.100 of the offspring rats will be examined of their learning and memory ability after lactation by the method of Morris water maze:Numbers the animals,picked up randomly the first generation offspring male rats,which contained twenty rats after the period of delactation in each group.Train the rats four times at the fixed time everyday for five days,and put the rats into the water from four different entrance and make the rats face the lateral wall.Let the rats stay on the platforms for thirty seconds after they found the platforms,record the time to search the stage,which is called the escaped latency.If the rats cannot find the stage,then we took them to the stage with hands,and the time is recorded 120 seconds.The interval between the two train is 60 seconds at least.To evaluate the learning and memory ability of the adult rats,by comparing the escaped latency of each group.
3.Make the pathological section of Hippocampus:After the Morris water maze, picked 5 of the first offspring rats,which had participated the behavior test.Throuth the process of drawing material,fixation,washings,dehydration,transparence, immersing in the wax,embedment,slicing and sticking,sheding the wax,dyeing and so on,to observe the microstructure by HE staining through light microscope in the CA1 zone of the Hippocampus.
[Result]
1.The effect of BDE-209 on the learning and memory ability of the offspring rats:
1.1 The main effect:there was significant difference in time factor,which indicated that the latency was time-dependent(F=37.351,P=0.000);the recipro-effect between time and group had no difference,which mean the time don not work as the group factor(F=0.163,P=1.000).
1.2 there was significant difference in group factor,which indicated that the group works(F=10.971,P=0.000).
1.3 Contrast of groups at every time:There was no significant difference among five groups in the first one-two days(P＞0.05),which indicated that the rats each group have the similar learning and memory ability.In the next three to five days, there was no significant difference between group A and the control group(group E) (P＞0.05),which indicated low dosage of BDE-209 have not influenced the learning and memory ability of the offspring rats compared with control group;The latency of group B、C、D have prolonged compared with control group(P＜0.05),which indicated that BDE-209 in high dosage have diminish the learning and memory ability of the offspring rats.
2.The comparison of pathology in the CA1 zone of hippocampus each group: There are obvious histomorphology changs between the C、D groups and control group E by HE staining using light microscope.
[Conclusions]
1.maternal BDE-209 in low dosage did not affect the special learning and memory ability of the offspring rats,when it got to a certain concentration,maternal BDE-209 exposure diminished the offspring rats' learning and memory ability.It is shown that the effect of maternal BDE-209 on the learning and memory ability of the offspring rats has the close relation with the exposure dosage.
2.A certain dose of maternal BDE-209 exposure can affect the the microstructure of nerve cell(number and structure) in hippocampus,which is possible material ground of the impair to the nervous system.
PartⅡEFFECT OF MATERNAL BDE-209 EXPOSURE ON THE CHOLINERGIC SYSTEM IN HIPPOCAMPUS OF OFFSPING RATS
[Objective]
Previous work has shown that the maternal BDE-209 had affect the learning and memory ability in the offspring rats.In the present study we contrasted the levels of cholinergic system parameter involved in of hippocampus tissue,such as acetylcholine amount、acetylcholinesterase activity、the expression of M-receptor and N-receptor,and approached the potential mechanism of the impair to the offspring rats′learning and memory ability.So as to settle the base for the next intervention and treatment.
[Material and Methods]
1.Handling the animals and collecting the specimens:
The animal model came from part one.After the Morris water maze test,picked up randomly ten offspring rats each group for the study objection(All of the specimens come from the experimental animals based on the groupA;B;C;D;E of the part 1).
Fix the animals,anesthetize them through intraperitoneal injection with chloral hydrate at the 4ml/kg dosage,then open the chest to reveal the heart,intubate through cor sinistrum,cut auricle of right atrium and pour quickly into normal saline with 4% Polyoxymethylene(PFA)stationary liquid about 200ml,get the brain on the ice cap after the decapitation,then dissociate the bilateral hippocampus.Wash the blood in the precooling 4℃normal saline,add nine times as much as the volume,then make the 10%tissue homogenate of brain tissue with the glass machine of homogenate at high speed.Take the homogenate to measure,according to the requirement and step of the correlated kit strictly.
2.The levels of cholinergic system parameter in hippocampus tissue of offspring rats were detected using sandwich ELISA procedure,such as acetylcholine amount、acetylcholinesterase activity、the expression of M-receptor and N-receptor.
[Result]
1.The ACH quantity in hippocampus was decreased with increased dosage of the exposure to BDE-209.The B、C、D groups had significant difference from control group(P＜0.05);There was no significant difference between group A and the control group E(P=0.637);Among the experiment group,the groups A、B、C had significant difference from group D(P＜0.05).
2.There were obviously significant decrease of acetylcholinesterase activity in hippocampus of the offspring rats in the group B、C、D,comparing with the control group E(P＜0.05).but there was no significant difference between the group A and the control group E(P=0.173).Among the experiment group,the groups A、B、C had significant difference from group D(P＜0.05).
3.The expression content of M-Receptor in hippocampus were decreased in the groups of C、D,comparing with the control group E(P＜0.05),but there was no significant difference between the group A、B and the control group(P=0.161, P=0.116);Among the groups,there were significant difference of expression of M-Receptor in hippocampus between the group D and the other experimental groups (P＜0.05)except group C.
4.The expression of content N-Receptor in hippocampus were decreased in the groups of D,comparing with the control group(P=0.001),but there was no significant difference between other experimental groups and the control group (P＞0.05);Among the groups,there were significant difference of expression of M-Receptor in hippocampus between the group D and the other experimental groups (P＜0.05).
[Conclusions]
The present results showed that the exposure of BDE-209 may destroy cholinergic system index involved in the CA1 zone of hippocampus tissue,such as acetylcholine amount,acetylcholinesterase activity,the expression of M-receptor and N-receptor,and the cholinergic neurotransmitter was the possible mechanism of the impair to the offspring rats′learning and memory ability.
PartⅢEEFFECT OF MATERNAL BDE-209 EXPOSURE ON THE NITRIC OXIDE NEUROTRANSMITTER IN HIPPOCAMPUS OF OFFSPRING RATS
[Objective]
Our initial studies have proved that the maternal BDE-209 exposure can damage the learning and memory of the offspring rats.The mechanism by which BDE-209 induce these alterations remain unclear.In This study,by contrasting the levels of Nitric Oxide(NO)、neuro-nitricoxide synthase(nNOS) and the cyclic guanylic acid (cGMP)in the hippocampus of the first offspring rats in each group,we approached the possible mechanism of the impair to the offspring rats′learning and memory ability.So as to settle the base for the next intervention and treatment.
[Material and Methods]
1.Handling the animals and collecting the specimens:
The animal model came from part one.After the Morris water maze test,picked up randomly ten offspring rats each group for the study objection(All of the specimens come from the experimental animals based on the groupA;B;C;D;E of the part 1).Fix the animals,anesthetize them through intraperitoneal injection with chloral hydrate at the 4ml/kg dosage,then open the chest to reveal the heart,intubate through cor sinistrum,cut auricle of right atrium and pour quickly into normal saline with 4%Polyoxymethylene(PFA)stationary liquid about 200ml,get the brain on the ice cap after the decapitation,then dissociate the bilateral hippocampus.Wash the blood in the precooling 4℃normal saline,add nine times as much as the volume, then make the 10%tissue homogenate of brain tissue with the glass machine of homogenate at high speed.Take the homogenate to measure,according to the requirement and step of the correlated kit strictly.
2.Accoriding to the request and procedure of the respective assay knit,we measure the levels of Nitric Oxide(NO)、neuro-nitricoxide synthase(nNOS) and the cyclic guanylic acid(cGMP) in hippocampus tissue of offspring rats
[Result]
1.The NO content in the BDE-209 treated groups increased by the dosage of BDE-209 exposure concentrations(F=0.6323,P=0.000);the content of C、D groups were higher than the control group(P＜0.05),while there were no significant difference in the group A and group B,respectively comparing with the control group E (P=0.515,P=0.135).Among the experimental groups,there was the highest content of NO in group D,though there was no significant difference between group A and group D.
2.The nNOS activity in the BDE-209 treated groups also was increased by the dosage of exposure to BDE-209(F=11.928,P=0.000);the activity of nNOS in the group C and group D were higher than the control group(P＜0.05),while there were no significant difference in the group A and group B,respectively comparing with the control group E(P=0.965,P=0.112).Among the experimental groups,there was the highest activity of nNOS in group D,though there was no significant difference in group A and group B,respectively comparing with the data of the group D.
3.The cGMP content in the BDE-209 treated groups increased by the dosage of BDE-209 exposure concentrations(F=4.119,P=0.00);the content in group B、group C and group E were significantly higher than the control group E(P＜0.05),while there were no significant difference between group A and the control group E (P=0.133).Among the experimental groups,there was significant content between group D and group A inclusively.
[Conclusions]
The study suggested that BDE-209 can influence the levels of nitric Oxide(NO)、the activity of neuro-nitric oxide synthase(nNOS) and the levels of cyclic guanosine monophosphate(cGMP) in hippocampus of offspring rats.It indicated that the Nitric Oxide as a neurotransmitter,may be the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability,especially the NMDAR-NO-CGMP pathway in the nerve system.
SUMMARY
1.We successfully established the model of BDE-209 exposure in different dosage by oral gavage in the spur period of the brain development of rats,settle the base for the study on the special behavior test of the offspring rats and on the possible mechanism of the impair to the learning and memory ability in offspring rats.The model can be manipulated easily and emulated successfully..
2.It is reasonable to conclude that maternal BDE-209 in certain dosage cant affect the spatial learning and memory ability of the offspring rats,and change the microstructure of the hippocampus cells,meanwhile the effect of maternal BDE-209 on the learning and memory ability has close relation with the exposure dosage.
3.A certain dose maternal BDE-209 exposure can affect the the microstructure of nerve cell(number and structure),which is possible material ground of the impair to the nervous system.
4.The exposure of BDE-209 may effect acetylcholine content、acetylcholinesterase activity、the expression content of M-receptor and N-receptor involved in the hippocampus tissue of offspring rats,and the cholinergic neurotransmitter was the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability.
5、The exposure of BDE-209 may influence the levels of nitric oxide(NO)、the activity of neuro-nitric oxide synthase(nNOS) and the levels of cyclic guanosine monophosphate(cGMP) in hippocampus of offspring rats.It indicated that the Nitric Oxide as a neurotransmitter,may be the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability,especially the NMDAR-NO-CGMP pathway in the nerve system.
In summary,we elucidate that the exposure of BDE-209 may effect the learning and memory ability of offspring rats by the possible mechanism cholinergic neurotransmitter and nitric oxide neurotransmitter pathway.But so far the threshold dose of the BDE-209 to induce the biotoxicity remain unclear.It is still need to be study whether there is other mechanism and the association of all the mechanisms. Because the biological accumulation of BDE-209,with the further study of the toxicity and danger,people will pay more attention to the use of BDE-209,including the developed countries.
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