Expression of the PEPT1, CAT,SOD2 and GPX1 genes in the zebrafish intestine supplemented with methionine dipeptide under predation risk

This study evaluated the effect of methionine supplementation, predation risk and their interaction on gut histology, whole-body cortisol levels, and intestinal gene expression in zebrafish. A total of 360 one-year-old animals were maintained under two environmental conditions and fed diets containing different methionine sources. Fish were fed either a control diet (CTL, without methionine supplementation), a diet supplemented with dl -methionine (DLM), or a diet supplemented with methionine dipeptide (MM) in the absence (AP) of a predator or in the presence of the predator (PP) for 48 h or 20 days. Predator-induced stress for 20 days resulted in lower body weight. Zebrafish fed methionine-supplemented diets had higher weight gain than control fish. We found no effect of predation stress or methionine supplementation on cortisol level. Predation risk and methionine supplementation showed no interaction effect on dipeptide transporter gene expression. After 48 h of predation pressure, zebrafish had higher mRNA expression of SOD2, CAT and GPX1 in the gut. After 20 days of exposure to the predator, zebrafish fed methionine-supplemented diets had lower expression of GPX1, SOD2 and CAT than those diet CTL. Methionine dipeptide and free methionine supplementation improved growth, intestinal health and survivability of zebrafish both conditions.     

Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

纳米ZnO和常规ZnO、ZnSO4对斑马鱼毒性效应的比较

为比较纳米ZnO、常规ZnO和ZnSO_4对斑马鱼氧化应激毒性的强弱,探究纳米ZnO的毒性作用与其释放的Zn2+和本身特性的关系,研究了纳米ZnO、常规ZnO、ZnSO_4对斑马鱼肝脏、肠、鳃组织中抗氧化酶活性及炎症凋亡基因表达的影响。实验将斑马鱼分别暴露于纳米ZnO、常规ZnO、ZnSO_4水体中,在4、24和96 h后,用分光光度法检测斑马鱼肝脏、肠、鳃中过氧化氢酶(CAT)、谷胱甘肽(GSH)、谷胱甘肽硫转移酶(GST)、活性氧自由基(ROS)的变化;采用荧光定量PCR技术,测定实验组中肝脏、肠和鳃中Bax、Bcl-2、TNF-α及IL-6的mRNA相对表达量。结果显示,纳米ZnO、常规ZnO、ZnSO_4均引起斑马鱼各组织的氧化应激反应,且使组织中凋亡基因和炎症基因表达水平发生变化,激活生物体内的细胞坏死和细胞凋亡途径,引起细胞死亡或机体炎症,其中纳米ZnO的致氧化损伤作用最强。研究表明,纳米ZnO对斑马鱼的氧化应激毒性强于常规ZnO和ZnSO_4,而纳米颗粒本身特性是导致纳米ZnO毒性作用的主要原因。     

Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance

Owing to the increasing importance of zebrafish as a vertebrate model in many fields of research, efforts must be made to breed and maintain this species in laboratory. Zebrafish larvae are traditionally reared on cultured live paramecia during the first 9 days of exogenous feeding, followed by a combination of paramecia and artemia nauplii until day 21, making larval rearing expensive, labour intensive and unpredictable. Thus, a trial was conducted with zebrafish larvae in order to evaluate the suitability of artificial diets as an alternative to live food during the first 21 days of exogenous feeding. Five dietary treatments were tested: (1) artemia nauplii; (2) a commercial; (3) a purified; (4) a practical diet, all delivered continuously; (5) the same practical diet delivered manually. The best overall larval performance was achieved in the group fed artemia nauplii (86% survival, 14.3 mm standard length, 46.1 mg wet weight). Compared with existing results obtained with the traditional live food schedule, our results suggest that paramecia might not be the most suitable first food for zebrafish, and that artemia nauplii could be used as the only live food. The present work demonstrates that zebrafish larvae can be reared without live food with a significant growth and a high survival, provided that an appropriate artificial diet is presented in a continuous way. Among the diets tested, the practical diet, if continuously delivered, led to the best performance assuring a mean standard length of 72% of that obtained with artemia and a similar survival rate. Moreover, the purified diet, supporting over 50% survival and an appreciable growth, could be useful in some toxicological studies in which a well‐defined diet is needed.     

双酚A和壬基酚长期暴露对斑马鱼繁殖的影响

内分泌干扰物的研究目前正逐渐成为国际研究新的热点，但长期暴露条件下对生殖情况的研究目前较少。本文研究了环境类雌激素双酚A（BPA）以及和壬基酚（NP）联合的作用，对暴露一个世代的斑马鱼（F1）（Danio rerio）的生殖情况、子代质量的影响。将健康的受精卵48h后暴露于200，500，1000μg·L^-1 BPA以及三个浓度与30μg·L^-1 NP的联合，同时设置空白对照和30μg·L^-1 NP对照。130dph将成鱼置于清水喂养20d，统计每天的产卵量、产卵次数、畸形率、孵化率、卵径、破膜时间以及耐饥时间。以此为终点评估暴露后斑马鱼的生殖能力和子代质量。与对照相比NP，BPA可以抑制斑马鱼的生殖，两种类雌激素联合作用可以极显著地抑制斑马鱼的生殖（P〈0．01）；BPA各浓度组导致F1代畸形率上升（P〈0．05）；高浓度BPA抑制产卵量（P〈0．05）；BPA200，500μg·L^-1会延长F1代的破膜时间和耐饥时间（P〈0．01），而BPA1000μg·L^-1则产生相反的效果（P〈0．05）。双酚A对斑马鱼的生殖有明显的抑制作用，双酚A和壬基酚有协同效应。长时间暴露于双酚A会影响斑马鱼的生殖能力和子代质量。     

三唑磷对斑马鱼H19和Sox2OT基因表达调控的影响

长链非编码RNA (long non-coding RNA, lncRNA) 是指长度大于200 nt且不具有编码蛋白质功能的一类RNA分子。当生物体暴露于环境污染物时，其lncRNA通常会异常表达，并与癌症等疾病的发生相关。采用亚致死浓度的三唑磷处理斑马鱼后，利用人源基因芯片初步筛选出了异常表达的斑马鱼lncRNA，并通过生物信息学软件预测了与lncRNA相互作用的微RNA (micro RNA, miRNA)及其靶基因，最后通过实时荧光定量PCR验证了该miRNA及其靶基因在斑马鱼中的表达，从而构建起了长链非编码RNA-微RNA-信使RNA (lncRNA-miRNA-mRNA) 的调控网络。荧光定量PCR检测结果表明:与空白对照组相比，经三唑磷处理后，斑马鱼lncRNA Sox2OT基因的表达下调了35%；lncRNA H19基因的表达上调了1.41倍，而与其可能存在相互作用的dre-let-7c基因的表达则下调了78%，同时dre-let-7c的靶基因ddx18的表达上调了2.11倍；差异均达显著水平 (P < 0.05)。据此推测，在三唑磷的作用下，lncRNA H19可能作为靶基因ddx18的竞争性内源RNA而与dre-let-7c发生相互作用，从而调控靶基因ddx18的表达。因此，lncRNA H19、dre-let-7c及ddx18都有可能作为生物标志物用于监测三唑磷对环境和生物体的影响。     

四种药物对斑马鱼急性毒性试验

为探究消毒液和农药对斑马鱼的毒性与安全评价,首次选用高锰酸钾、甲醛、精碘和卡螨死四种试剂进行急性毒性试验,以24～96h半致死浓度（LC50）判断斑马鱼对四种药物的敏感性。结果表明,24、48、72、96h的LC50,高锰酸钾分别：勾1．15、1．12、1．12、1．11mg／L;甲醛为48、45、45、40mg／L;精碘分别为7．8×10^-3、5．8×10^-3、5．6×10^-3、5．3×10^-3mg／L;卡螨死分别为1．95×10^-7、1．23×10^-7、0．65×10^-7、0．62×10^-7mg／L。高锰酸钾、甲醛、精碘和卡螨死的安全质量浓度（SC）分别为1．07、11．8、0．96×10^-3和1．48×10^-6mg/L。参照我国化学物质对鱼类毒性分级标准,判定高锰酸钾对斑马鱼急性毒性为II级,甲醛为Ⅲ级,精碘和卡螨死对斑马鱼急性毒性为I级。     

苄嘧磺隆对斑马鱼的急性毒性和遗传毒性研究

1材料与方法 1．1动物供试斑马鱼体重12～17g、体长4—4．5cm,购于宠物市场。实验室驯养7d,每天更换1次暴晒24h的水,选取行动活泼,身体健康的个体进行试验。     

苄嘧磺隆对斑马鱼的急性毒性和遗传毒性研究

[目的]研究了除草剂苄嘧磺隆对斑马鱼的急性毒性和遗传毒性的影响。[方法]通过急性毒性试验计算出半数致死浓度,进而分析该除草剂是否对水生生物存在潜在的毒害;并在急性毒性的基础上进行遗传毒性研究,通过计算微核率来判断除草剂苄嘧磺隆是否存在遗传毒害。[结果]24h和48h的半数致死浓度分别为0．698ml／L和0．637ml／L,安全浓度为0．159ml／L。不同处理浓度和染毒时间对斑马鱼红细胞微核形成的影响试验表明,对照组红细胞微核细胞率为0．0103％,处理组红细胞微核细胞率最高达0．372％．说明除草剂苄嘧磺隆对斑马鱼具有遗传毒性作用。同一检测时间,不同浓度处理组的红细胞微核细胞率与对照相比,具有剂量效应;在同一处理组,随着染毒时间的延长,红细胞微核率在24h达到峰值,在48h及72h时下降。[结论]该研究为人们科学选择和合理利用除草剂提供依据。     

螺虫乙酯对雌性斑马鱼的毒性及氧化应激效应

为评价新型季酮酸衍生物类杀虫剂螺虫乙酯(spirotetramat)对水生生物的毒性,以雌性斑马鱼为受试对象,研究了螺虫乙酯对其的急性毒性及氧化应激效应。急性毒性试验测得螺虫乙酯对雌性斑马鱼的96 h-LC50值为7.21 mg/L,属中等毒性。经不同浓度螺虫乙酯(0、36、360和1 800μg/L)处理28 d后,通过检测铜/锌超氧化物歧化酶(Cu/Zn-SOD)、锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶的活性,Cat、Gpx、Mn-Sod、Cu/Zn-Sod等基因的相对表达量,以及丙二醛(MDA)含量等指标的变化水平,研究了螺虫乙酯对斑马鱼肌肉组织的氧化应激效应。结果表明,螺虫乙酯可引起斑马鱼肌肉组织中CAT、GSH-Px活性和MDA含量,以及Cat、Mn-Sod、Gpx的相对表达量显著升高,但未发现螺虫乙酯对Cu/Zn-SOD活性及Cu/Zn-Sod的相对表达量有明显影响。研究表明,螺虫乙酯能够诱导雌性斑马鱼肌肉组织的氧化应激效应,并可能引起机体的氧化损伤。