天津野生鲫鱼卵黄蛋白原的研究

用HPLC方法提纯经雌二醇诱导的鲫鱼卵黄蛋白原,用SDS-PAGE凝胶电泳测定其分子量为160Kda左右.在建立卵黄蛋白原(VTG)的HPLC分析方法(检测下限为0.025mg/mL)的基础上,对北京排污河天津段的野生和养殖鲫鱼血浆中的VTG进行检测.在所捕获的12尾野生鲫鱼中(其中2尾为雄性鲫鱼),都检测出了VTG,浓度范围为0.284～5.971mg/mL;对同期捕获的35尾天津养殖鲫鱼包括1尾雄性鲫鱼中的VTG也进行了检测,浓度范围为0.119～0.250mg/mL.而同期在实验室饲养的雌雄鲫鱼血浆中,均未检测出VTG.结果表明,北京排污河(天津段)的野生鲫鱼受到类雌激素物质的污染.     

壬基酚对鲫鱼(Carassius auratus)的雌激素效应研究

实验研究了环境雌激素4-壬基酚(NP)对鲫鱼(Carassiusauratus)的剂量-效应关系。雄性鲫鱼腹腔注射不同剂量的17β-雌二醇(E2)(质量分数分别为0 01,0 10,1 00mg kg(注射剂量 鱼体重,下同))和4-壬基酚(NP)(质量分数分别为1,10,50,100,200mg kg),用ELISA方法测定鲫鱼血浆中卵黄蛋白原含量。结果显示,与对照相比,在注射后14d内,0 01mg kgE2处理组和1 00mg kgNP处理组鲫鱼血浆卵黄蛋白原含量没有显著差异;其他各处理组在注射后8d,血浆卵黄蛋白原含量显著升高(P<0 05),且与暴露浓度呈依赖关系;注射后14d,10,50,100和200mg kgNP处理组的含量依次是对照组的10,59,340和435倍。研究表明,NP对鲫鱼具有明显的雌激素效应。      

氟对斑马鱼生殖毒性研究

为了探讨氟对鱼类的生殖毒性效应,以成年斑马鱼为试验对象,氟化钠暴露45 d和90 d后,运用组织切片观察斑马鱼性腺组织的发育情况,采用酶联免疫分析法测定性腺中雌二醇（Estradiol,E2）、卵黄蛋白原（Vitellogenin,VTG）和睾酮（Testosterone,T）的含量,并使用实时荧光定量PCR方法检测斑马鱼生殖相关基因的表达水平.结果显示：氟暴露会阻滞成年斑马鱼性腺发育,造成卵泡发育不良,精巢生精能力下降;同时降低雌性斑马鱼性腺中E2和VTG的含量,增加T的含量;雌性斑马鱼孕酮受体（Prg）和糖皮质激素受体（GR）基因相对表达量随氟浓度的增加呈先降后升的趋势,随着染毒剂量与时间的增加,雄激素受体（AR）基因相对表达量逐渐增加,而CYP19a基因相对表达量逐渐减少.雄性斑马鱼性腺中E2、VTG、T的含量及生殖相关基因表达的变化趋势则与雌性斑马鱼相反.综上所述,氟暴露可以通过影响斑马鱼体内性腺发育、E2、VTG、T含量及生殖相关基因表达来影响其生殖功能.实验结果可为进一步研究氟化物对水生动物的生殖毒性效应提供理论依据.     

酚类化合物对金鱼幼鱼的雌激素效应研究

采用金鱼（Carassius auratus）幼鱼的卵黄蛋白原（VTG）作为雌激素污染的生物标志物,评价了几种酚类化合物的雌激素效应.实验表明鲤鱼（Cyprinus carpio）VTG多克隆抗体对金鱼VTG有可识别性以及肝脏匀浆法检测金鱼体内VTG的可行性,并用酶联免疫吸附法半定量地测定了几种重要酚类物质的雌激素效应.试验结果为：半静态暴露15 d后,己烯雌酚、辛基酚、壬基酚、叔丁基试验组金鱼体内VTG均有较为明显增加;五氯酚组稍有增长,四氯酚和三氯酚与空白组无明显区别;受试化学物质雌激素效应的相对强弱为：己烯雌酚＞辛基酚＞壬基酚＞叔丁基酚＞五氯酚.     

钙和铅相互作用对鲫鱼毒性效应的影响

以鲫鱼为材料,研究了钙和铅相互作用对鲫鱼毒性效应的影响.试验分为5组,分别为空白对照Ⅰ组、单独染铅Ⅱ组(0.1 mg·L-1)及铅钙联合组(Ⅲ组:0.1 mg·L-1铅+2 mmol·L-1钙,Ⅳ组:0.1 mg·L-1铅+4 mmol·L-1钙,Ⅴ组:0.1 mg·L-1铅+6 mmol·L-1钙),处理35 d后,检测鲫鱼肌肉、鳃中铅含量,以及肝胰脏中总抗氧化能力(T-AOC)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量.结果表明,与Ⅰ组比较,Ⅱ、Ⅲ组鲫鱼肌肉、鳃中铅含量增高(p0.05),Ⅴ组鲫鱼肌肉中铅含量降低(p0.05),与Ⅱ组比较,Ⅳ、Ⅴ组鲫鱼肌肉、鳃中铅含量降低(p0.05);与Ⅰ组比较,Ⅱ组CAT活性降低(p0.05),Ⅲ、Ⅳ和Ⅴ组CAT活性升高(p0.05),与Ⅱ组比较,Ⅳ、Ⅴ组CAT活性升高(p0.05);与Ⅰ组比较,Ⅱ、Ⅲ和Ⅴ组T-AOC活性降低(p0.05),与Ⅱ组比较,Ⅲ、Ⅳ和Ⅴ组T-AOC活性增高(p0.05);与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ和Ⅴ组MDA含量升高(p0.05),与Ⅱ组比较,Ⅲ、Ⅳ和Ⅴ组MDA含量降低(p0.05).由此可见,钙可竞争鲫鱼对铅的吸收,降低铅在其肌肉、鳃中的蓄积,提高鲫鱼肝胰脏的抗氧化能力,表明钙能降低铅的毒性效应.     

火焰原子吸收法直接测定降水中钙

本文研究了用火焰原子吸收法直接测定降水中钙,进行了仪器工作条件选择实验,确定其最佳的工作条件,同时还做了不同浓度的钾、钠、铁、铝和无机酸对测定钙的影响实验,以及铝对钙测定的消除干扰实验,建立了火焰原子吸收法直接测定降水中钙的方法。方法的相对误差在0.3%以下,最低检出浓度为0.028mg/L,加标回收实验在96～103%之间,能够满足于降水中钙测定的要求.     

苯并芘对稀有鮈鲫的内分泌干扰效应研究

为评价典型有机污染物对长江三峡库区上游特有珍稀鱼类的环境风险,本实验以稀有鮈鲫为对象,研究了低剂量多环芳烃(PAHs)暴露而引起的内分泌干扰效应.将成年稀有鮈鲫暴露于不同浓度(0、0.1、0.3、1.0和3.0μg·L-1)的苯并芘(Ba P)中28 d,测定了体质系数、性激素含量、下丘脑-垂体-性腺(HPG)轴和肝脏中与繁殖相关基因的表达.结果显示:在雌鱼中,Ba P暴露后,睾酮(T)的含量显著升高,雌二醇(E2)的水平显著下降并改变了T/E2的比值;在雄鱼中,T和E2的含量都显著下降.雌雄鱼性激素的改变可能与类固醇合成相关基因(Cyp17、Cyp19a)表达量的改变有关.Ba P暴露显著抑制了雌雄鱼肝脏中卵黄蛋白原(VTG)基因的表达.以上检测到的激素或者基因表达的改变主要发生在高剂量(3.0μg·L-1)Ba P暴露下,而长江上游库区水体中多环芳烃含量较低(100 ng·L-1),因此推测,库区存在的多环芳烃不会对三峡库区上游水体中的鱼类产生内分泌干扰效应.     

微塑料对红鲫鱼幼鱼抗氧化酶系统的影响

为了研究不同微塑料对鱼类的毒性效应,探讨其可能的作用机理,文章以红鲫鱼为目标生物,采用半静态毒性实验方法,对不同暴露时间下不同浓度聚乙烯微塑料对红鲫鱼幼鱼肝脏组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽硫转移酶(GST)活性以及丙二醛(MDA)含量进行了测定。结果表明:经聚乙烯微塑料暴露后,红鲫鱼幼鱼肝脏组织SOD活性、GST活性和MDA含量均随暴露时间延长"先升高后降低"的趋势,而CAT活性则表现为"先降低再升高然后再降低"的波浪型变化趋势。在相同处理时间下,不同浓度微塑料对抗氧化酶的作用效果表现为在处理前期诱导作用比较高且3个处理组之间差异明显,处理后期3组浓度的抗氧化酶活性之间差异逐渐减小。     

垃圾焚烧飞灰浸出液中铅的检测方法对比及干扰排除

采用HJ/T 300-2007醋酸缓冲溶液法对飞灰样品进行毒性浸出,对浸出液中的铅含量分别采用火焰原子吸收分光光度法(AAS)、电感耦合等离子体发射光谱法(ICP-AES)、电感耦合等离子体质谱法(ICP-MS)3种方法进行检测对比。结果表明:AAS和ICP-AES测试时浸出液中含量1%的钙对铅产生严重干扰,使测定结果偏高5倍以上。干扰程度与钙浓度呈正比;ICP-MS测试结果准确度高,基本无干扰、并对干扰排除方法进行了介绍。     

A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen

A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol(E_2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry(LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41(MAb-sea bass VTG 41)was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay(ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E_2-treated sea bass and others EDCs(Nonylphenol, Benzo[a]pyrene and CdCl_2). Moreover, cross-reactivity was also found in E_2-treated coral grouper(Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.     

Assessing the anti-estrogenic activity of sodium pentachlorophenol in primary cultures of juvenile goldfish (Carassius auratus) hepatocytes using vitellogenin as a biomarker

Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.     

四溴双酚A的鲫鱼血清蛋白生物标志物研究

以鲫鱼为供试生物,对四溴双酚A(TBBPA)的急性毒性、血清蛋白潜在生物标志物以及相应的毒性靶器官组织病理进行了研究,结果表明TBBPA对鲫鱼96 h的半致死浓度LC50是3.13 mg/L,TBBPA诱导下鲫鱼血清蛋白可区分为11个组分,各组分随时间和浓度的变化规律存在差异。组分11条带清晰,表达量变化幅度最大,在低浓度TBBPA暴露下表达量先降低后升高,第16天达到最高;高浓度时表达量在第2天达到最高。组分11的分子量为24 kDa,可能为鲫鱼斯坦尼小体(CS)中降血钙因子的解聚物。组织病理切片结果显示,鲫鱼肝脏和肾脏均表现出时间-剂量依赖性,且与组分11的诱导差异性表达呈现相应的病理损伤。因此,鲫鱼血清蛋白组分11对TBBPA的诱导具有浓度和时间响应敏感性,且易于识别和鉴定,可作为TBBPA暴露的鲫鱼血清蛋白潜在生物标志物。     

The acute lethality and endocrine effect of 1,2,3,7,8-PeCDD in juvenile goldfish （ Carassius auratus ） in vivo

1,2,3,7,8-PeCDD was administrated to juvenile goldfish （Carassius auratus） by peritoneal injections to explore the acute lethality and endocrine effects of 1,2,3,7,8-PeCDD in vivo. The value of acute median lethal dosage （LD50） of 1,2,3,7,8-PeCDD was determined in acute lethality tests. The endocrine effect of 1,2,3,7,8-PeCDD, whose exposed concentrations were determined based on the LD50 （1.84 mg/kg）, was studied by measuring the plasma vitellogenin （Vtg） content in juvenile male goldfish with enzyme-linked immunosorbent assays （ELISA）. Due to its significant induction of the plasma Vtg after one week＇s exposure in vivo in the 1/2 LD50 and LD30 groups, 1,2,3,7,8-PeCDD might be one of the important contributors to the estrogenic effect of PCDDs in the environment. The values of 1/2 LD50 and LD30 were within the range of the effective dosages of 1,2,3,7,8-PeCDD, indicating that there was a certain relationship between the estrogenic effective dosages and the LD50.     

四种拟除虫菊酯类农药的环境雌激素活性研究

将金鱼(Carassius auratus)分别暴露于0.1mg/L甲氰菊酯、氰戊菊酯、溴氰菊酯和0.5mg/L氯氰菊酯21d后,尾静脉取血,离心分离获得血浆,利用Western-blot方法检测金鱼血浆中是否有卵黄原蛋白产生,同时测定金鱼生殖腺指数、γ-谷氨酰转移酶和乳酸脱氢酶活性.结果表明,对照组雄性金鱼血浆中未检测到卵黄原蛋白,4种拟除虫菊酯农药均能诱导雄性金鱼分泌卵黄原蛋白,而且溴氰菊酯和氯氰菊酯明显降低了雄鱼生殖腺指数,4种农药均能够明显抑制精巢γ-谷氨酰转移酶和乳酸脱氢酶活性.由此可见,4种拟除虫菊酯类农药具有潜在的环境雌激素活性和生殖毒性.     

鲫鱼(Carassius auratus)卵黄蛋白原的ELISA检测

采用Sephadex G-200柱层析从鲫鱼(Carassius auratus)成熟鱼卵中提取卵黄脂磷蛋白并制备抗卵黄脂磷蛋白抗血清.用DEAE-cellulose阴离子交换层析和Sephacryl S-300分子筛凝胶层析相结合的两步层析从经17b-雌二醇诱导的雌鱼血浆中提纯卵黄蛋白原.免疫印迹反应显示卵黄脂磷蛋白和卵黄蛋白原都与抗卵黄脂磷蛋白抗血清有特异的反应.以抗卵黄脂磷蛋白抗血清作为抗体,卵黄蛋白原作为竞争抗原建立了间接竞争法酶联免疫吸附反应(ELISA)方法来检测雄鱼血液中卵黄蛋白原含量.该方法可测的最低浓度为7.8ng/mL,工作范围为0.39~25mg/mL,批内和批间误差分别为6.5%和7.5%.     

Estrogenic effects of water from the Yangtze River （Nanjing section） on goldfish （Carassius auratus） after an early life stage exposure

Effects of river water from the Yangtze River (Nanjing section) on fish development, vitellogenin (VTG) induction, gonado-somatic index (GSI) and sex ratio were investigated by exposing goldfish (Carassius auratus) in the early life stage (from fertilization to 28 days post-hatch) to water samples (25%, 50% and 100%) collected from three representative sections. The results showed that there was no significant effect on hatching success for any of the exposure groups, but survival was significantly reduced when compared with the control (P < 0.05). Body lengths, weights of all treated fish did not differ significantly from those of the control. Condition factors (CF) of larval fish exposed to 50% and 100% river water from the Jiangxinzhou section and 100% river water from the Daqiao section were significantly lower than that of the control (P< 0.05). VTG inductions were significant in larval fish exposed to all the dilution series of river water. No significant difference in CF value was observed in any exposure group after 150 days of depuration. VTG was fully eliminated after 75 days of depuration. For both female and male, GSI did not significantly differ between exposure groups and the control after 150 days of convalescence. The highest female:male ratios were observed in response to the treatment with 50% or 100% river water from the Jiangxinzhou section and 100% river water from the Daqiao section (53:47, 56:44 and 54:46, respectively), but no significant difference in sex ratio was observed in any treated group when compared to the control. The results showed that early life stage exposure of river water from the Yangtze River (Nanjing section) had adverse effects on goldfish development and reproductive health, and the effects on CF and VTG were reversible after depuration in clean water.     

卤代酚类物质对抗氧化酶活性的影响研究及构效分析

选取 7种卤代酚类化合物 ,在对鲤科金鱼进行 4 8h急性毒性试验的基础上 ,同时主要研究受试化合物对金鱼肝脏细胞中超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)和谷胱甘肽过氧化物酶 (GSH PX)活性的变化 ,并对急性毒性进行定量结构 活性相关 (QSAR)分析 .结果表明 ,半致死浓度的对数值与化合物分子的辛醇 水分配系数 (Kow)、最低空轨道能 (Elumo)有较好的相关性 .在实验浓度下 ,7种化合物对金鱼肝脏 3种抗氧化酶活性有显著影响 ,随着浓度的升高基本呈现先激活后抑制的趋势 ,因此金鱼肝脏中SOD、CAT和GSH PX的活性作为水生生物在水环境中受到污染胁迫的大分子水平的生物标志物具有一定可行性     

Immunotoxicity of bisphenol a to Carassius auratus Lymphocytes and macrophages following in vitro exposure

Bisphenol A （BPA） is the monomer component of polycarbonate plastics and classified as an endocrine disrupting chemical （EDC）. The reproductive toxicity of BPA has been extensively studied in mammals; however, relatively little information is available on the immunotoxic responses of fish to BPA. In this study, we investigated the effects of BPA on the immune functions of lymphocytes and macrophages in Carassius auratus. The effects of BPA were compared with those of two natural steroid hormones, estradiol and hydrocortisone. Proliferation of the two types of cells in response to PHA was measured using colorimetric MTT assay. Macrophage respiratory burst stimulated by Con A was measured using chemiluminescence assay. Results showed that BPA （0.054-5.4 mg/L）, estradiol （0.0002-2.0 mg/L） and hydrocortisone （5-50 mg/L） significantly induced Carassius auratus lymphocyte proliferation while higher doses of hydrocortisone （500-5000 mg/L） appeared to be inhibitory. BPA （0.005-50 mg/L）, estradiol （0.005-800 mg/L） and hydrocortisone （0.005-500 mg/L） markedly enhanced macrophage proliferation, whereas higher doses of BPA （500-1000 mg/L） appeared to inhibit cell proliferation. Furthermore, higher dosage of BPA （50 mg/L） and hydrocortisone （50 and 500 mg/L） suppressed the macrophages respiratory burst while estradiol is stimulative all the doses tested （0.05-500 mg/L）. In conclusion, BPA could have immunotoxicity to Carassius auratus and functional changes of lymphocyte and macrophage in Carassius auratus may be different between low and high dosages.     

五氯酚对稀有鮈鲫卵黄蛋白原及p53的诱导效应

采用中国特有鱼种稀有鮈鲫为实验动物,研究五氯酚（PCP）对其肝脏卵黄蛋白原（VTG）、VTG基因及抑癌基因p53的诱导效应,评价PCP的内分泌干扰作用,并从蛋白和基因表达水平上筛选PCP检测的敏感生物标志物.1.5、15、40、80、120、150、160μg.L-1PCP暴露稀有鮈鲫,同时设置空白、溶剂对照和17α-雌二醇（EE2）阳性对照,运用SDS-PAGE,ELISA法检测经PCP暴露后稀有鮈鲫肝脏VTG蛋白的表达差异;克隆稀有鮈鲫新的VTG和p53基因序列片段,运用实时荧光定量PCR方法检测PCP暴露对基因表达的影响.结果表明,40、80、120、160μg.L-1PCP均能诱导雌雄稀有鮈鲫肝脏VTG蛋白的表达,具有显著的浓度-效应关系;1.5、15、150μg.L-1PCP暴露后,雄性稀有鮈鲫肝脏VTG和p53基因表达均明显升高,具有显著的浓度-效应和时间-效应关系.证明PCP具有雌激素效应,稀有鮈鲫肝脏的VTG蛋白、VTG及p53基因可作为PCP检测的敏感分子生物标志物.