【作者】 李春辉；

【导师】 王志萍；

【作者基本信息】 山东大学 ， 公共卫生（专业学位）， 2013， 硕士

【摘要】 研究背景铬及其化合物应用广泛。铬是提高钢耐腐蚀性最常用的添加元素之一,铬盐可用于镀铬、冶金工业生产合金,制作颜料、染料、油漆、橡胶和陶瓷等。工业废水中的六价铬严重污染周围环境的河流、土壤和地下水源,成为一种主要的水环境污染物质之一,如不进行有效处理,将会对环境造成极大的危害。近年来,随着纳米技术的迅猛发展,纳米材料的应用前景越来越广阔。实验研究表明,纳米氢氧化镁(nano-magnesium hydroxide, NMH)用于含铬废水的处理,得到较好的效果,吸附速度快,吸附比高,还可回收利用,可多次用于含铬废水的处理。纳米尺度的化学品具有独特的特征,这使得纳米材料的毒性也表现出其独特性。近年来,生殖毒性和发育毒性已日益成为整体毒理学的重要组成部分。虽然纳米材料的生殖毒性和胚胎毒性已有报道,但是NMH对雌性生殖毒性和胚胎毒性的研究较少。本研究分别于小鼠胚胎围植入期和器官形成期暴露于NMH,观察其对生殖系统和胎儿生长发育的毒性作用,研究结果为NMH吸附剂对孕妇和胎儿安全性评价提供可靠依据。研究目的建立胚胎围植入期和器官形成期NMH吸附剂暴露小鼠模型,通过测量孕鼠孕期体重变化、各脏器重量、脏器系数、以及小鼠身长、尾长及骨化点评价NMH的生殖毒性和胚胎毒性；并通过检测孕鼠血清降钙素(calcitonin, CT)、甲状旁腺素(parathyroid hormone, PTH)和生长激素(growth hormone, GH)的水平,观察孕鼠胎盘病理结构变化及孕鼠血清和胎盘组织血管内皮生长因子(vascular endothelial growthfactor, VEGF)、胎盘生长因子(placenta growthfactor, PLGF)、可溶性血管内皮生长因子受体1(sFlt-1)变化,进一步探索其毒作用机制,为NMH吸附剂对孕妇和胎儿安全性评价提供可靠依据。研究方法1.建立胚胎围植入期和器官形成期暴露小鼠模型ICR小鼠按雌雄1：1合笼,次日检查阴栓,查见阴栓日记为孕第1天(GD1)。实验分为胚胎围植入期和胚胎器官形成期两个暴露模型,每个模型中孕鼠按体重随机分为NMH、NMH-铬复合物(nanocomposite)、重铬酸钾(阳性对照)和蒸馏水(阴性对照)4组。胚胎围植入期暴露模型于GD1-GD10每天灌胃一次；器官形成期暴露模型于GD7-GD16每天灌胃一次。根据灌胃当天孕鼠体重确定灌胃容量,每次灌胃容积为O.1m1/10g体重。暴露剂量为NMH577.4336mg/Kg.bw,NMH-铬复合物为583.7636mg/Kg.bw,重铬酸钾为6.33mg/Kg.bw。GD19终止试验,称取宫胚窝重、胎鼠窝重及子宫、卵巢、胎盘、肝脏、脾脏、肾脏和肺脏的重量,并计数胚胎总数、活胎数和死胎数,低温保存各脏器标本和血清。2.测量胎鼠发育指标测量胎鼠身长和尾长；观察外观畸形；将1/2胎鼠放入Bouins液固定用作胎鼠内脏畸形检查,另外1/2胎鼠经茜素红染色用于胎鼠骨骼畸形观察。3.测定母鼠血清激素水平采用酶联免疫吸附剂测定法(enzyme linked immunosorbent assay, ELISA)测定两模型各组母鼠血清CT、PTH和GH水平。4.观察母鼠胎盘病理改变分别采用肉眼、光镜和电镜观察母鼠胎盘病理结构改变。5.测定母鼠血清和胎盘组织中胎盘功能因子水平采用ELISA方法测定两模型各组母鼠血清和胎盘组织中VEGF、PLGF和sFlt-1水平。6.统计分析方法采用Excel建立数据库,用SPSS19.0软件进行统计分析。对数据进行方差齐性检验：若方差齐,采用单因素方差分析(ONE-WAY ANOVA)进行F检验,同时选择Dunnett-t test进行处理组与对照组各指标均数之间的比较；若方差不齐,采用非参数统计方法(Kruskal-Wallis H法)。采用两个独立样本t检验(Independent-Samples T Test)进行处理组和对照组指标均数比较,双侧检验,检验水准a=0.05。研究结果1.母体毒性孕鼠暴露于NMH、NMH-铬复合物和重铬酸钾与阴性对照组比较,孕鼠GD1体重组间差异没有统计学意义；两个模型各组间孕鼠孕期体重变化趋势、GD19体重和体重净增重、母鼠各脏器重量及脏器系数组间差异均无统计学意义。2.胚胎毒性NMH、NMH-铬复合物和重铬酸钾于胚胎围植入期和器官形成期暴露对小鼠胚胎植入数未见明显影响,未见明显胎鼠外观畸形、内脏畸形及骨骼畸形,未观察到明显的致畸效应。两阶段暴露均可导致明显的胚胎发育毒性。(1)胚胎围植入期暴露模型中,NMH-铬复合物组和重铬酸钾组胎鼠均重、胎鼠身长和胎鼠尾长较阴性对照显著降低(P<0.01),NMH组胎鼠身长显著降低(P<0.05)。器官形成期暴露模型中,NMH-铬复合物组和重铬酸钾组胎鼠均重和胎鼠身长均显著低于阴性对照组(P<0.05),重铬酸钾组胎鼠尾长显著低于阴性对照组(P<0.01),NMH组胎鼠发育指标未见明显改变。(2)胚胎围植入期和器官形成期暴露于NMH、NMH-铬复合物和重铬酸钾,与阴性对照组比较,均可导致胎鼠骨化不全率显著升高(P<0.01),尾椎骨骨化点数显著降低(P<0.01)和第二趾骨骨化点数明显减少(P<0.01),少数胎鼠出现第二指骨骨化不全,肋骨发育不全,胸骨粘连。3.对母鼠血清激素含量的影响胚胎围植入期暴露NMH、NMH-铬复合物和重铬酸钾3种物质,母鼠血清激素CT、PTH和GH水平与对照组比较,差异均无统计学意义；器官形成期暴露后各组母鼠血清PTH水平未见明显变化,但NMH-铬复合物组和重铬酸钾组母鼠血清CT水平和GH水平较对照组均明显降低(P<0.01)。4.对母鼠胎盘结构和功能的影响肉眼和光镜观察母鼠胎盘结构,两模型各处理组与阴性对照组对比,未见明显病理改变。电镜下观察两模型NMH组、NMH-铬复合物组和重铬酸钾组孕鼠胎盘组织均发现细胞死亡现象。胚胎围植入期暴露于3种物质,母鼠血清和胎盘组织中VEGF. PLGF和sFlt-1水平与对照组比较,差异均无统计学意义。器官形成期暴露,NMH-铬复合物组和重铬酸钾组母鼠血清VEGF水平和PLGF水平与对照组相比显著降低(P<0.05), NMH组没有明显改变；另外,母鼠胎盘组织中VEGF水平在NMH组、NMH-铬复合物组和重铬酸钾组较阴性对照组明显降低(P<0.05), PLGF水平在重铬酸钾组显著降低(P<0.01)；母鼠血清和胎盘组织中sFlt-1水平均未见明显变化。结论1.围植入期和器官形成期暴露于NMH、NMH-铬复合物和重铬酸钾能够导致胎鼠生长迟缓、骨化异常。三种物质在该暴露剂量下母体毒性作用相对较小,无明显致畸效应。2.两模型各暴露组孕鼠胎盘组织结构均发生病理损伤,这可能是导致胎鼠发育迟缓和骨化不全的重要机制。3.器官形成期暴露于NMH、NMH-铬复合物和重铬酸钾导致孕鼠胎盘功能损伤,表现为胎盘功能因子VEGF和PLGF水平明显降低。4.器官形成期暴露于NMH-铬复合物和重铬酸钾可降低母鼠血清CT和GH水平,可能是导致胎鼠生长迟缓和骨化不全的原因之一。NMH对血清激素水平影响较小。更多还原

【Abstract】 BackgroundChromium compounds are widely used in industry:Chromium is the most widely used adding element in steel to improve corrosion resistance. Chromium can be used for chrome plating, alloy production in metallurgy industry, as well as pigment, dyestuff, paint, rubber and ceramic manufacture. The industrial production and use of chromium compounds lead to the contamination of surface water and groundwater, so the people exposure to chromium through the water. Hexavalent chromium in the wastewater is highly toxic, which will seriously pollute the surroundings of rivers, soil and groundwater sources. Cr (VI) is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects. Studies have shown that the embryo and fetus in mice and rats after exposure to hexavalent chromium can produce toxic effects, including pregnancy complications.Chromium has become one of the major environmental pollutants of the water, which will cause great harm to the environment if no effective measures to take. In recent years, with the rapid development of nanotechnology, nanomaterials have increasingly broad application prospects. Experimental studies have shown that NMH is used for the treatment of wastewater containing chromium, which gets good results, fast absorption and high adsorption. NMH recovered after roasting, after many times for the treatment of waste-water containing chromium.Nanomaterials have unique characteristics, which makes the toxicity of nanomaterials also demonstrate its uniqueness. In recent years, reproductive and developmental toxicity has increasingly become recognized as an important part of overall toxicology. Although the reproductive toxicology of nanomaterials has been reported, there has been no study on female reproductive toxicity and fetal toxicity resulting from NMH. Therefore, the present study investigated the effect of NMH on reproductive system and fetal development in ICR mice exposed by gavage for GD1-GD10and GD7-GD16respectively. It is significant whether NMH used to process wastewater containing chromium safely for pregnant women or not.ObjectiveThe model of phase of peri-implantation and organogenesis period were established to observe the weight change of pregnant mice, each organ weight, organ coefficient, fetal mice in length, tail length, and ossification points to evaluate the reproductive toxicity and embryotoxicity of NMH adsorbent. By detecting calcitonin (CT), parathyroid hormone (PTH) and growth hormone (GH) levels of pregnant mice serum, placental pathology structural changes of pregnant mice and vascular endothelial growth factor(VEGF),soluble vascular endothelial growth factor receptor(sFlt-1), placental growth factor (PLGF) of serum and placenta slurries of pregnant mice to further explore the mechanism of its toxicity. This study will provide a reliable basis for whether it is safe to pregnant women and the fetus for the use of NMH to process wastewater containing chromium.Methods1. Establishment of animal modelsDesign for the exposure:Pregnant mice were gavage administration to NMH (577.4336mg/Kg.bw), nanocomposite (583.7636mg/Kg.bw) and K2Cr2O7(6.33mg/Kg.bw) for one time everyday at GD1-GD10during phase of peri-implantation and GD7-GD16during organogenesis period. The day that the female mice mated successfully was identified as the first day of gestation(GD1). The gavage volume was0.1ml/10g body weight. Mice in the control group were were given a gavage of distilled water. Collection of the observation indexes:Mice were sacrificed on GD19. The body weights during the pregnancy were recorded. The embryo and uterus from each litter were harvested and weighted and the number of embryos on GD19in each litter was counted. The weights of uterus, ovary, liver, spleen, kidney and lung were taken down and then these tissues were frozen and preserved in-80℃refrigerator for further analysis.2. Measurement of the development indicators of fetal miceThe body length and tail length of the fetal mice were measured. After detecting of appearance of deformity, half of the fetal mice were added to Bouin’s fluid for the use of fetal mice visceral malformations examination, and the other half of the fetal mice for bone examination after alizarin red staining.3. Detection of CT, PTH and GH concentration in maternal serumConcentration levels of CT, PTH and GH in maternal serum were determined by enzyme-linked immunosorbent assay (ELISA). The detection of hormone levels was operated according to the instructions of the ELISA kits.4. Detection of maternal placental pathologyRespectively, we detected maternal placental pathology changes with the naked eye, light microscopy and electron microscopy.5. Detection of VEGF, PLGF and sFlt-1in maternal serum and placentaThe detection of VEGF, PLGF and sFlt-1in maternal serum and placenta slurries was the same as the methods used in part3.6. Statistical analysis methodA computerized statistical program (SPSS19.0) was used for statistical treatment. The datas were first analyzed by homogeneity test for variance, followed by one-way analysis of variance (ANOVA) if the variances were homogenous, and then the different toxic effects between groups were determined by Dunnett’s t test. If the variance were not equal, the data would be analyzed by a non-parametric analysis of variance (Kruskal-Wallis H test). Statistical significance was determined at level of α=0.05.Results 1. Maternal toxicity induced by NMH exposure during phase of peri-implantation and organogenesis periodWe observed the maternal body weight during pregnancy, and organic weights and indices of the two experimental models stated above. None of these indicators show any significant variety among four groups at the administered doses. Neither did weights and coefficients of reproductive organs show much distinction among four groups. These results suggest that the dose we used may be not enough to exert evident effects on the general condition of pregnant mice and their basic organs.2. Embryotoxicity induced by NMH exposure during phase of peri-implantation and organogenesis periodWe observed total number of fetal mice, live birth, and absorption fetal, and found little difference among groups. However, the indices of fetus development are significant:the average weight, height, and tail length of fetal mice, and nest of fetal mice weight were diminishing from the group of distilled water, NMH, nanocomposite, to potassium dichromate, indicating the greatest fetal developmental toxicity of potassium dichromate, the weaker one of nanocomposite, and the least one of NMH. The same trend presented in the ossification of fetal mice, which is indicated by the coccygeal vertebra ossification points, the second phalanx of forearms and hindlimbs ossification points, and the abnormality of ossification (rib hypoplasia, sternum adhesion).3. Effects of NMH exposure on concentration of hormonesAlthough PTH level show little distinction among four groups, CT and GH levels are lower in the groups of potassium dichromate and nanocomposite in the model of exposure during organogenesis period.4. Effects of NMH exposure on placenta structure and functionPlacenta weight and coefficient showed little variance among groups. We didn’t see any significant pathological changes in the maternal placenta via visual observation and light microscopy in any group. However, by electron microscopy, we discovered the phenomenon of placenta cell death in the group of NMH, nanocomposite and potassium dichromate in the two models. Here, we observed the decrease of VEGF level in groups of potassium dichromate and nanocomposite and the decrease of PLGF level in group of potassium dichromate in maternal serum, in the model of exposed during organogenetic period, and the level of sFlt-1did not show significant distinction among groups in either models. While in the placenta slurries, VEGF levels in all three groups other than control group are lowered significantly, and PLGF level is dropped only in group of potassium dichromate, in the model of exposed during organogenetic period, and the level of sFlt-1still present little variation.Conclusion1. Exposed to NMH, nanocomposite and potassium dichromate during phase of peri-implantation and organogenesis period, fetal toxicity, especially fetal development toxicity, is the most remarkable, indicated by the shrunken size and reduced ossification.Three substances in the exposure dose have small maternal toxicity effect, no significant teratogenic effects.2. Placenta occurs pathological damage in each exposure groups in two models, which may be an important mechanism of fetal mice developmental retardation and incomplete ossification.3. The period of organogenesis exposed to NMH, nanocomposite and potassium dichromate lead to dysfunction of the placenta, which showed VEGF and PLGF level significantly reduced.4. CT and GH levels are lower in the groups of potassium dichromate and nanocomposite in the model of exposure during organogenesis period, which may cause fetal mice growth retardation and reduced ossification.NMH has less impact on serum hormone levels.更多还原