富马酸二甲酯对斑马鱼胚胎早期发育的影响

为研究富马酸二甲酯对斑马鱼(Danio rerio)胚胎早期发育的影响,选取不同发育阶段的斑马鱼胚胎,用富马酸二甲酯进行染毒处理,观察胚胎形态发育的异常,计算其对不同发育时期胚胎的24 h、48 h半数致死浓度(LC50)和胚胎72 h孵化率,并考察富马酸二甲酯对胚胎血管发育的影响。结果表明,富马酸二甲酯影响斑马鱼胚胎的早期发育,呈剂量依赖性特点,并与开始处理的时间点有关。富马酸二甲酯引起2 hpf(受精后2 h,2 hours post-fertilization)、10 hpf、24 hpf斑马鱼胚胎死亡的24 h LC50值分别为:13.33μmol/L、17.98μmol/L、32.50μmol/L,48 h LC50值分别为:13.31μmol/L、16.35μmol/L、22.50μmol/L;长期低浓度富马酸二甲酯(≥6μmol/L)作用引起胚胎72 h孵化率下降。27.5μmol/L富马酸二甲酯作用后会显著降低胚胎血管内皮生长因子受体2(VEGFR2)的表达水平。     

迷迭香酸对硫酸铜致斑马鱼胚胎毒性的抑制作用

本文探讨硫酸铜(CuSO_4)对斑马鱼(Danio rerio)胚胎发育的毒性效应,使用迷迭香酸(RA)抑制CuSO_4对斑马鱼胚胎发育的毒性并探讨其作用机制。收集受精后1 h(1 hpf)的斑马鱼胚胎暴露于不同浓度的CuSO_4溶液,或含有不同浓度迷迭香酸的CuSO_4溶液,对照组培养在E3培养液中,观察胚胎死亡、孵化及畸形情况,计算胚胎死亡率、孵化率和畸形率;以活性氧(ROS)荧光探针DCFH-DA染色法检测迷迭香酸保护下胚胎的活性氧水平。对实验数据进行方差分析。结果显示:(1)CuSO_4浓度超过一定量时能诱导斑马鱼胚胎死亡和畸形,胚胎孵化率也降低。CuSO_4对96 hpf斑马鱼胚胎的半致死浓度(LC50)为7.7μmol/L,半致畸浓度(EC50)为1.9μmol/L。(2)在96 hpf,迷迭香酸与8μmol/L CuSO_4共同处理组斑马鱼胚胎的死亡率明显降低,孵化率升高。迷迭香酸与1.6μmol/LCuSO_4共同处理组斑马鱼胚胎的畸形率降低。(3)CuSO_4单独处理组的活性氧含量明显高于迷迭香酸与CuSO_4共同处理组和对照组。结果表明,CuSO_4暴露对斑马鱼胚胎发育的毒性效应可能与活性氧升高导致的氧化应激相关;迷迭香酸抑制CuSO_4对斑马鱼胚胎发育的毒性作用,可能与减少活性氧生成有关。     

壬基酚对金鱼（Carassiusauratus ）胚胎发育毒性的初步研究

目的:观察环境激素壬基酚(Nonylphenol,NP)对金鱼胚胎发育的毒性效应。方法:在繁殖季节选取雌雄健康成年金鱼,采用人工受精的方法获取金鱼受精卵于23℃培养箱孵育。将正常发育的囊胚早期金鱼胚胎(50枚胚胎/培养皿)分别暴露在1μmol·L~(-1)、4μmol·L~(-1)、8μmol·L~(-1)、12μmol·L~(-1)和16μmol·L~(-1)的NP实验液中,设空白对照。通过显微成像技术观察NP暴露对金鱼胚胎发育形态、不同发育阶段胚胎死亡率、受精后72小时(72 hpf)孵化率及畸形率的影响。从原肠期开始每隔12 h观察一次。结果:低浓度NP主要影响金鱼胚胎晚期发育,使其尾部脊索弯曲上翘;高浓度NP可导致金鱼胚胎卵凝结、发育延迟、组织坏死、尾部卷曲、体轴缩短并出现血栓。各发育阶段胚胎死亡率和72 hpf畸形率随NP浓度升高而增加,72 hpf孵化率则随NP浓度升高而降低;NP对金鱼胚胎24 h、48 h、72 h的半数致死浓度(LC50)分别为:16.28μmol·L~(-1)、8.27μmol·L~(-1)和6.52μmol·L~(-1)。结论:NP对金鱼胚胎具有明显发育毒性,金鱼胚胎对低浓度NP也具有较高敏感性,在评价环境激素胚胎发育毒性方面具有潜在应用价值。     

诺氟沙星对斑马鱼胚胎发育的毒性作用及对TGF-β1的影响

旨在通过观察不同浓度诺氟沙星对斑马鱼胚胎不同发育时期的毒性作用,以及对TGF-β1基因表达的影响。配制诺氟沙星浓度为0、10、20、40μmol/L,将斑马鱼胚胎暴露在上述浓度的诺氟沙星中。观察在胚胎不同发育时期,诺氟沙星对斑马鱼脊柱弯曲、心包囊肿、卵黄囊肿和死亡率的影响,以及使用实时定量聚合酶链式反应(q PCR)检测其对TGF-β1基因表达的影响。结果显示,诺氟沙星对斑马鱼的胚胎发育有明显影响,主要表现在脊柱弯曲、心包囊肿和卵黄囊肿,随着诺氟沙星暴露浓度的增大,胚胎的发育延迟,孵化时间延长,胚胎死亡率增加;当诺氟沙星暴露浓度为40μmol/L时,96 hpf的胚胎死亡率达到76.45%;与正常状态相比,暴露于不同浓度诺氟沙星的斑马鱼胚胎中TGF-β1基因的m RNA表达随发育时间延长而增加趋势减缓。说明诺氟沙星对斑马鱼胚胎发育的致畸作用与致死作用有显著的影响。提示水体中残留的诺氟沙星对鱼类的生殖与发育具有潜在的危害。     

硫代硫酸钠干扰斑马鱼胚胎发育并致畸

硫的衍生物潜在的威胁着胚胎的发育过程.斑马鱼被用于研究不同浓度（1×10-6～1 mol/L）的硫代硫酸钠（sodium thiosulfate, STS）对胚胎发育的影响,在解剖显微镜下实时观察斑马鱼胚胎发育的全过程.采用Western 印迹法检测乙酰化的微管蛋白——α-微管蛋白（acetylated tubulin, α-tubulin）和神经元增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达,分别检测STS暴露后胚胎的运动神经元功能,神经元的增殖状态.发育中的斑马鱼胚胎暴露于0.1～1 mol/L STS,呈现出严重的发育迟缓,并且伴随多脏器畸形;暴露于10 μmol/L～10 mmol/L STS,胚胎呈现循环系统,神经系统以及颌面部畸形.胚胎在48 hpf (hours post fertilization)时,对STS的暴露敏感高于24 hpf和96 hpf.STS可能干扰细胞的增殖及运动神经元的正常分化.STS可能干扰正常的细胞骨架结构,并在胚胎发育晚期影响细胞增殖,对胚胎神经系统、循环系统及颌面部有致畸作用.     

叶酸缺乏对斑马鱼背主动脉发育的影响

目的观察叶酸缺乏斑马鱼胚胎的背主动脉（DA）发育情况,初步探讨叶酸缺乏后胚胎DA发育异常的机理。方法采用将二氢叶酸还原酶（DHFR）功能阻断的方法构建叶酸缺乏斑马鱼模型,分别应用DHFR抑制剂甲氨蝶呤（MTX）以及DHFR基因knock-down技术处理斑马鱼胚胎。在胚胎发育至48 hpf时在显微镜下观察胚胎的整体发育情况,在60 hpf时应用荧光显微造影的方法观察胚胎的背主动脉发育状况。利用胚胎整体原位杂交和real-time PCR的方法检测影响DA发育的关键因子ephrinB2、Ang-1和Radar的表达情况,利用TUNEL法检测胚胎底索的凋亡情况。结果MTX处理组胚胎以及DHFR knock-down组胚胎有相似的胚胎发育异常表型。荧光显微造影显示叶酸缺乏组胚胎的DA发育异常。叶酸缺乏组胚胎的ephrinB2、Ang-1和Radar表达减弱,底索凋亡增加。结论叶酸缺乏可导致斑马鱼胚胎背主动脉发育异常,其机理与ephrinB2、Ang-1和Radar的表达减弱以及底索凋亡增加有关。     

马兜铃水提液对斑马鱼胚胎的致畸作用和心脏毒性的研究

目的：探索马兜铃水提液对斑马鱼胚胎的致畸作用和心脏毒性.方法：分别用不同浓度的马兜铃水提液和马兜铃酸A（AA）处理斑马鱼胚胎,观察致畸作用和对心脏发育影响.结果：给药组的斑马鱼胚胎出现畸形和死亡;当水提液中AA含量为0.5 μg/mL时,胚胎心率明显减慢;AA含量为5μg/mL时,胚胎在24～48 hpf之间全部死亡;水提液的LC50为1.43 μg/mL.结论：与AA相比,马兜铃水提液对斑马鱼胚胎有着更强的致畸和心脏毒性,且毒性作用具有时间和浓度依赖性.     

斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用

目的观察二氢叶酸还原酶基因(DHFR)功能阻抑斑马鱼胚胎的颅脑部发育情况,初步探讨二氢叶酸还原酶基因在斑马鱼神经系统发育过程中的作用。方法采用显微注射吗啡啉修饰的反义核苷酸(MO)的方法进行DHFR表达阻抑。胚胎发育至受精后48hpf观察胚胎的颅部发育情况,在60hpf时经石蜡切片进一步观察胚胎的脑发育状况。利用胚胎整体原位杂交的方法检测影响神经系统发育的关键因子ngn1和huc的表达情况。结果显微注射MO可成功的进行DHFR表达阻抑。DHFR表达阻抑组胚胎存在颅脑部发育明显异常和ngn1、huc的表达强度明显减弱,且与显微注射的MO剂量呈正相关。结论DHFR在斑马鱼颅脑发育中有重要作用;其功能阻抑可导致胚胎颅脑部发育异常,其机理与ngn1和huc的的表达减弱有关。     

异甘草素对斑马鱼胚胎发育、血管生成和心脏的影响

目的研究异甘草素对斑马鱼Danio rerio var.胚胎发育、血管生成以及心脏的影响。方法异甘草素处理受精后10 h、24 h的正常斑马鱼胚胎,显微镜下观察并记录药物作用12 h、24 h、36 h、48 h后斑马鱼胚胎发育、血管生成、心率以及心脏形态。结果异甘草素浓度为4μg·mL~(-1)时可轻度抑制斑马鱼胚胎尾部发育,浓度为12μg·mL~(-1)及以上时会严重抑制胚胎发育。异甘草素具有抑制斑马鱼胚胎血管生成作用,浓度为2μg·mL~(-1)时即可抑制血管的生成,8μg·mL~(-1)时甚至完全抑制尾部静脉血管的生成。异甘草素可显著降低斑马鱼胚胎心率,中低剂量下,心率随着作用时间增加呈先降后升的趋势,对心脏形态无影响;高剂量下,心率随作用时间增加而降低,异甘草素致斑马鱼胚胎心包与卵黄囊肿大。结论中低剂量异甘草素具有良好的抗血管生成和减慢心率的作用,高剂量时随着作用时间延长具有一定的抑制发育作用。     

三种农药对斑马鱼的急性毒性和生物浓缩系数

采用斑马鱼(Brachydanio rerio)为试验生物,研究了吡虫啉、三唑磷和哒螨灵的急性毒性和生物浓缩系数．通过毒性试验,获得了这3种农药对斑马鱼的24、48、72和96h LC50值．这3种农药的96h LC50值分别为281．37mg·L^-1、8．37mg·L^-1和11．66μg·L^-1．根据急性毒性数据和有关技术指南,确定生物浓缩试验浓度和持续暴露时间为14d．经0．5和5mg·L^-1吡虫啉暴露后的生物浓缩系数(BCFs)分别为1．52和0．97．经0．02和0．2mg·L^-1三唑磷暴露后的BCFs分别为9．00和8．45．经0．1和1μg·L^-1哒螨灵暴露后的BCFs分别为5600和4920．     

Teratological studies with prostaglandin E2 in chick embryos

Prostaglandin E₂ (20–100 μg) was lethal to a relatively high percentage of chick embryos when administered at 48 hours incubation; no such effect was observed after 72 hours incubation. A relatively high incidence of abnormal embryos, which increased with the dose-levels of prostaglandin, was induced at both 48 and 72 hours incubation compared to the controls. However, this difference was statistically significant only in embryos treated with 100 μg prostaglandin at 48 hours incubation. The embryos showed no signs of growth retardation.     

Potential use of Lemna minor for the phytoremediation of isoproturon and glyphosate

Pesticides are being detected in water bodies on an increasingly frequent basis. The present study focused on toxicity and phytoremediation potential of aquatic plants to remove phytosanitary products from contaminated water. We investigated the capacity of Lemna minor (L. minor) to eliminate two herbicides isoproturon and glyphosate from their medium. Since phytoremediation relies on healthy plants, pesticide toxicity was evaluated by exposing plants to 5 concentrations (0-20 microg L(-1) for isoproturon and 0-120 microg L(-1) for glyphosate) in culture media for 4 d using growth rate and chlorophyll a fluorescence as endpoints. At exposure concentrations of 10 microg x L(-1) for isoproturon and 80 microg x L(-1) for glyphosate, effects on growth rate and chlorophyll fluorescence were minor (< 25%), so that this initial concentration was selected to study herbicide removal After a 4-d incubation, removal yields were 25% and 8% for isoproturon and glyphosate, respectively.     

氯丙嗪在斑马鱼胚胎和早期幼鱼发育过程中的神经毒性作用

目的 采用模式动物斑马鱼作为研究对象,观察氯丙嗪（chlorpromazine,CPZ）暴露对胚胎和幼鱼早期神经发育的影响.方法 在一般毒性评价的基础上,通过整体胚胎细胞凋亡检测和脑组织病理学检查,了解CPZ对神经发育的器质性改变;采用神经行为学方法,包括幼鱼触动逃避反应、自发运动以及惊恐逃避反射等,研究氯丙嗪暴露所致的神经发育功能性障碍.结果斑马鱼胚胎受精后6 h（6 hpf）～72 hpf暴露于CPZ（≥5 mg/L）可引起胚胎和幼鱼死亡、致畸和幼鱼孵化延迟,并呈浓度和时间依赖性;采用吖啶橙染色检测36 hpf整体胚胎凋亡细胞,发现凋亡细胞主要集中在胚胎中脑、后脑、丘脑以及中后脑连接区、脊索和尾部等处;脑组织病理学检测发现,7dpf幼鱼颅腔增大、脑体积减小、脑细胞缩小且细胞间隙增宽.6～72 hpf CPZ（≥0.0625 mg/L）暴露后,幼鱼神经行为学研究发现,CPZ（≥0.125 mg/L）可引起3dpf幼鱼触觉运动能力下降;CPZ（≥0 5 mg/L）可浓度依赖性地抑制幼鱼自发运动,并出现僵直不动、震颤或快速刻板式转圈运动等行为改变;光惊恐实验中,暗环境下各暴露组幼鱼对突发强光刺激均表现为惊跳逃避,并且暗-光交替期运动加速度变化与对照组无显著差异;在撤除光源后,1mg/L和2 mg/L暴露组幼鱼暗适应时程缩短,而0.125 mg/L和0.25 mg/L暴露组暗适应时程延长,提示CPZ对外界刺激引发的幼鱼活跃游动有抑制和促进双重毒性作用.结论 CPZ暴露对斑马鱼胚胎和幼鱼具有明显的神经发育毒性作用.模式动物斑马鱼作为一种高通量筛选模型在外源性化合物神经发育毒性评价中具有较好的应用前景.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Expression of sclerostin in the developing zebrafish (Danio rerio) brain and skeleton

Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (D. rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost mRNA was noted during 12h post-fertilization (hpf). At 15hpf, sost mRNA was detected in the developing nervous system and in Kupffer's vesicle. At 18, 20 and 22hpf, expression in rhombic lip precursors was seen. By 24hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28hpf and then diminished in intensity through 44hpf. At 28hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48hpf, sost mRNA was clearly detected in the developing pharyngeal arch cartilage. Sost mRNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.     

TBX1, a DiGeorge syndrome candidate gene, is inhibited by retinoic acid

Both retinoic acid (RA) and Tbx1 are definitively indispensable for the development of the pharyngeal arches. The defects produced by a loss of Tbx1 highly resemble those induced by hyper- and hypo-RA. Based on these similarities, the effects of RA on Tbx1 expression pattern were explored during pharyngeal arch development in zebrafish. Whole-mount in situ hybridization and real-time quantitative PCR were used. Zebrafish embryos were treated with 5 x 10(-8)mol/L and 10(-7)mol/L RA at 12.5 hours post fertilization for 1.5 hours, respectively. Whole-mount in situ hybridization showed that Tbx1 was expressed in the cardiac region, pharyngeal arch and otic vesicle between 24 hpf and 72 hpf in zebrafish. Tbx1 expression was obviously reduced, even lost, in the pharyngeal arch and outflow tract in RA treated groups. Real-time quantitative PCR analysis showed that Tbx1 expression rose to a peak level at 36 hpf in wild type group. Repression of Tbx1 expression was most evident at 36 hpf, 24 hours after RA treatment. 10(-7 )mol/L RA caused a more severe effect on the Tbx1 expression level than 5 x 10(-8)mol/L RA.The results suggested that RA could produce an altered Tbx1 expression pattern in zebrafish. In addition, RA could repress Tbx1 expression in a dose-dependent manner.     

DDT对多刺裸腹溞的急性毒性和生命表参数的影响

采用48 h急性毒性试验研究了DDT对多刺裸腹溞的48小时LC₅₀值,采用生命表试验方法研究了暴露于不同浓度（1、8、16、24、32和40 μg·L^-1）DDT溶液中的多刺裸腹溞的生命表统计学参数.结果表明：DDT对多刺裸腹溞48小时LC₅₀值为324 μg·L^-1;浓度在1～40 μg·L^-1的 DDT对多刺裸腹溞的生命期望、净生殖率、世代时间和总繁殖率均没有显著影响(P>0.05),但对种群内禀增长率(r_m)影响显著 (P<0.05).与空白对照组相比,浓度为8、16和40 μg·L^-1的DDT显著地提高了多刺裸腹溞种群r_m.在使用多刺裸腹溞的生命表统计学参数监测亚致死浓度的DDT的生态影响时,r_m是较敏感的指标.     

Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene

Pentachlorophenol (PCP), a probable human carcinogen, has been heavily used as an aseptic and a biocide throughout the world, and is widely present in the environment. Recent survey in Germany revealed that the average PCP amount in the urine of general German populations was 1.04 microg/L while the peak concentration could reach up to 19.1 microg/L. PCP was reported to cause DNA damage, but whether it can be involved in inducing point mutations in genome is unknown. To determine the genotoxicity of PCP on vertebrate, we first performed acute toxicity test on zebrafish for the effect of PCP exposure. The LC50 values of zebrafish exposed to PCP at 24, 48, 72 and 96 h were determined to be 0.196, 0.130, 0.130 and 0.130 mg/L, respectively. We then treated zebrafish with PCP for 10 days at 0 (control), 0.5, 5 and 50 microg/L, respectively, to determine whether PCP could be involved in inducing point mutations. Employing denaturing high-performance liquid chromatography analysis and DNA sequencing, we demonstrated that exposure of PCP to zebrafish at a concentration as low as 5 microg/L for 10 days elevates point mutation rate in p53 gene in liver cells. This is the first direct evidence revealing that PCP can elevate point mutation rate in the vertebrate genomes. The result implies PCP might be involved in carcinogenesis by elevating point mutation rate in the somatic genomes.