【作者】 黄莉； 戴丽军； 邱剑峰； 刘寒英； 冯媛瑜；

【Author】 HUANG Li DAI Lijun QIU Jianfeng LIU Hanying FENG Yuanyu (Experimental Animal Research Center, Guangzhou Medical College, Guangzhou 510180 ,China)

【机构】 广州医学院实验动物研究中心；

【摘要】 目的建立一套动物模型实验性方案,用于筛选和研究对人类和动物生殖和早期胚胎发育有影响的环境芳香烃类污染物。方法利用TCDD作为初始研究标准化合物,用NIH小鼠建立动物早期生殖和发育动物模型和毒性评估方案进行: 1．剂量-反应评估(DRE):以0,2,50和100ng·(kg·d)-1等4个剂量的2,3,7,8-四氯苯对二噁英(TCDD),在小鼠妊娠早期的1-8d经口腔灌服,于小鼠妊娠的第9天观察胚胎着床数量和生长发育情况,研究TCDD对NIH小鼠妊娠早期生殖和胚胎发育的毒性影响;2．着床前后毒性比较(PPP):以同样剂量分别给正常怀孕小鼠在妊娠早期的1-3d,和胚泡着床后期的4-8d经口腔染毒,于小鼠妊娠的第9天观察胚胎附植数量和生长发育情况;3．子宫蜕膜细胞反应(DCR):假孕小鼠以与DRE同样剂量和时间染毒,第9天观察子宫增重情况;4．胚胎转移速率(ETR)分析:于小鼠妊娠第1天,第2天和第3 天上午灌喂同样剂量的TCDD,于第1天,第2天和第3天下午冲洗输卵管和子宫观察胚胎位置和转移速度。结果剂量- 反应评估结果发现50和100ng·(kg·d)-1剂量的TCDD引起了第9天胚胎数量的显著减少,并且具有明显剂量依赖性。着床前后毒性比较发现剂量100 ng·(kg·d)-1的TCDD使胚胎着床前期染毒影响显著大于着床后期染毒,对第9天的胚胎重量的检测发现,50ng·(kg·d)-1和100 ng·(kg·d)-1剂量的TCDD在妊娠早期的不同时期给药均影响了着床后胚胎发育,使得着床后的胚胎发育迟缓,重量减轻。子宫蜕膜细胞反应检测结果发现同等剂量TCDD处理的假孕小鼠,子宫蜕膜生长受到了明显抑制(P<0．01):其中以连续染毒对子宫的蜕膜化抑制最为明显。胚胎转移速率分析结果发现,50和100 ng·(kg·d)-1 TCDD处理的妊娠小鼠,胚胎着床数量明显减少(P<0．05),两组剂量未影响胚胎在输卵管中的转移速度, 但造成胚胎在着床前丢失,分裂卵发育迟缓。另外,对妊娠第18天胚胎成活率观察发现,TCDD可造成出生前第18天胎儿死亡,出生前胚胎成活数量低于妊娠中期第9天的胚胎成活数量。结论 1．TCDD对胚胎的毒性剂量远小于对成年个体的剂量;2．实验证明,随剂量的增加,TCDD的胚胎着床前毒性明显大于着床后,妊娠的第1-6天是最敏感时期。3．TCDD对胚胎的毒性影响可能是多方面的:强烈抑制子宫蜕膜细胞反应,表现出典型的母体毒性;虽然没有影响胚胎转运速度,但造成着床前分裂卵的丢失或发育的不同步;早期染毒降低了第9天的胚胎着床数,同时使出生前第18天胚胎成活数继续减少。说明TCDD具有持续毒性的特点。因此,该方案可作为胚胎毒理学的分析项目,用于筛选对动物妊娠早期胚胎发育具有毒性的芳香烃类污染的化合物。更多还原

【Abstract】 Objective To establish an experimental animal model and protocol for screening and study the toxic of halogenated aromatic hydrocarbon on the early pregnant embryos. Methods A series of experiments are Included: 1. Dose-response evaluation.Groups of pregnant NIH mice were dosed orally with 0, 2.0, 50 and 100 ng ·kg-1·d-1 of TCDD on days 1-8 of early pregnancy. The animals were sacrificed on day 9, the numbers of embryo implantation sites, the weightof embryo were recorded. 2. Pre- versus Post- implantation Analysis. The pregnant animals were dosed with same amount of TCDD but on days 1-3 and 4-8 of early pregnancy respectively, the numbers of embryo implantation sites, development retardation and death of post-implantation embryos. Conclusion 1 .The TCDD dosage need for impair the embryos of early pregnant mouse is much lower than that of used in development toxic experiment reported, and toxicity of TCDD on the embryos is much sensitive than the adults. This demand that risk assessment of toxic chemical shall include early pregnant toxic screening assays for guaranteeing the reproductive health of exposed populations. 2. The experiment proved that pre-implantation embryos are more sensitive than post-implantation ones, suggested TCDD may exert its effect before implantation, or at this process. 3. The toxicity of TCDD on the early embryo can be of omnifarious. It can strongly inhibit DCR, which represent mother toxicity; It the weight of embryo were recorded. 3. Decidua cell response. Groups of pseudo-pregnant NIH mice were dosed orally with same amount of TCDD on Days 1-8, 1-3 and 4-8 of early pregnancy. The animals were sacrificed on day 9, and the weights of the uterus of pseudo-pregnant mice were recorded. 4. Embryo Transport Rate Analysis. The pregnant animals were dosed with same amount of TCDD but on days 1 -3 at 8:00am, animals were sacrificed on day 1,day 2,and day 3 afternoon to check the position of embryo. Results The results showed that the number of implantation sites decreased significantly(P<0.05=with the occurrence of post-implant embryo death and retardation of embryo development when the mice were dosed with 50 and 100 ng · kg-1 · d-1 TCDD; And this treatment inhibited uterus deciduas growth of pseudo-pregnant mice significantly (P<0.01=. Pre- versus Post-implantation analysis revealed that pre-implantation stage is more sensitive than post-implantation stage. The embryo transport rate was not changed in TCDD treated animals. But the embryo in the oviduct was observed to be un-synchronization in development with a proportion of embryo loss. A group of TCDD treated animals was allowed to had the fetus until d18. The survival rate was compared with d9 data. The result showed that the d18 survival rate is significantly lower than d9, TCDD caused cause un-synchronizated cleavage of pre-implanted zygote and pre-implanted embryo loss; It also decreases implantation sites in the uterus, and continued to effecting the ut更多还原