卡培他滨对非靶标生物斑马鱼胚胎的发育毒性研究

为评价抗肿瘤药物卡培他滨(CAP)对非靶标生物的毒性, 以斑马鱼胚胎为受试生物, 研究了CAP对斑马鱼胚胎的发育毒性及对其抗氧化酶系的影响。结果表明, 直接暴露于卡培他滨中, 造成斑马鱼胚胎死亡率和畸形率增加, 且其机能有所下降。当暴露浓度高于20 μg·L-1时, 处理后的斑马鱼胚胎死亡率和畸形率显著升高, 与对照组相比有极显著差异。CAP浓度为0.2 μg·L-1时, 超氧化物歧化酶 (SOD) 活性和过氧化氢酶 (CAT) 活性均显著升高, 表明机体遭受一定程度的氧化损伤; 当浓度高于20 μg·L-1时, SOD和CAT活性显著降低, 表明斑马鱼仔鱼所受氧化损伤超出其自我修复能力, 引发致死性伤害。本文从发育毒性及氧化应激着手, 探究了CAP对非靶标生物的潜在危害, 为其生态效应提供一定的科学依据。     

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。     

迷迭香酸对氯化镉致斑马鱼胚胎发育毒性的保护作用北大核心CSCD

通过考察氯化镉(CdCl2)对斑马鱼胚胎发育的毒性效应及迷迭香酸(rosmarinic acid,RA)的干预作用,探讨镉的发育毒性机理。将受精1 h后的斑马鱼胚胎暴露于不同浓度的CdCl2或含不同浓度RA的CdCl2溶液中,观察胚胎死亡、孵化及幼鱼畸形的情况。采用吖啶橙染色,定性观察胚胎细胞凋亡情况;以单细胞凝胶电泳法检测胚胎细胞的DNA损伤;以活性氧(reactive oxygen species,ROS)荧光探针DCFH-DA染色法检测胚胎的ROS水平,硫代巴比土酸比色法测定胚胎脂质过氧化水平,5,5-二硫二硝基苯甲酸比色法测定胚胎的还原谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平;光泽精化学发光法检测胚胎还原型辅酶(nicotinamide adenine dinucleotide phosphate,NADPH)Ⅱ氧化酶(NADPH oxidase,Nox)活性。结果显示,RA浓度依赖性地抑制了CdCl2诱导的胚胎死亡和幼鱼畸型,同时提高了胚胎孵化率。RA可浓度依赖性地改善CdCl2诱导的胚胎ROS和MDA(malonic dialdehyde)水平升高、GSH/GSSG比值降低和Nox活性升高,抑制CdCl2诱导的胚胎细胞凋亡。RT-PCR结果显示,RA对CdCl2诱导的胚胎Cu/Zn-Sod基因表达上调和Bcl-2/Bax水平下调也有明显改善作用。结果说明,CdCl2对斑马鱼胚胎发育具有毒性,可活化Nox,促进ROS生成,造成胚胎细胞氧化胁迫损伤以至胚胎细胞凋亡,而RA可抑制Nox活化,改善氧化胁迫状态,从而对CdCl2的发育毒性起保护作用。     

~（14）C－涕灭威在旱田土壤中的降解

研究了１４Ｃ－涕灭威在５种土壤中（４ｐｐｍ,互．２２μＣｉ·５０ｇ－１土壤干重）的生物降解．模拟试验为密闭系统,土壤中水分含量为２２％,气温２０—３０Ｃ℃在供试的５种土壤中,北京肖家河的土壤降解最快,为施人放射剂量的５１．３％,以“ＣＯ：形式从土壤进出;２６．０％与土壤结合,只有２１．６％可以被抽出．取自浙江义乌的土壤降解较慢,收集到的１４ＣＯ２为施入量的２３．３％．土壤中加入杀菌剂红霉素或敌茵丹降解作用明显减慢．土壤提取物中涕灭威亚砜、涕灭威亚砜肟被确认是主要的代谢产物,还发现了少量的涕灭威砜,涕灭威亚砜腈涕灭威砜腈和涕灭威砜肟等降解物．     

0#柴油对斑马鱼基因组DNA和环境DNA遗传毒性的RAPD比较

研究利用随机扩增多态性DNA (Random Amplified Polymorphic DNA, RAPD)技术, 以斑马鱼基因组DNA和其养殖水体中的环境DNA (environmental DNA, eDNA)为模板, 检测0#柴油可溶性组分对斑马鱼(Danio rerio)遗传毒性的影响。结果显示, 通过基因组DNA和eDNA扩增的RAPD图谱均可检测到0#柴油对斑马鱼的遗传毒性。在未受到柴油暴露时, 斑马鱼基因组DNA和水环境中eDNA在96h内的RAPD图谱均无明显变化; 在不同浓度的柴油暴露下, 随着暴露时间(0、24h、48h、72h、96h)延长, 基因组DNA和eDNA的多态性位点减少, 模板稳定性降低; 随着柴油浓度(15%、50%、100%)的增加, 基因组DNA和eDNA的多态性位点也减少, 模板稳定性降低。这表明0#柴油对斑马鱼基因组DNA和eDNA的遗传毒性均呈现时间-效应和浓度-效应关系, 并且无论以斑马鱼基因组DNA还是eDNA为模板, 柴油暴露组和未进行暴露的对照组的RAPD扩增图谱条带变化趋势一致。研究结果为通过RAPD技术检测柴油对水生生物的遗传毒性提供了新的研究思路和技术手段。     

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.     

1-苯基-2-硫脲对斑马鱼胚胎发育与黑色素生成的影响

目的1-苯基-2-硫脲（PTU）可抑制斑马鱼胚胎黑色素的产生,保持斑马鱼透明,便于形态观察和信号检测。本文研究了PTU对斑马鱼胚胎发育的影响和抑制斑马鱼胚胎黑色素生成,保持斑马鱼透明性的最佳浓度。方法用不同浓度PTU处理23hpf（受精后,hourspostfertilization,hpf）斑马鱼胚胎,作用57h后观察80hpf斑马鱼的形态学、生理学改变,计算死亡率和孵化率,测量心率和静脉窦-动脉球之间的距离。结果浓度为0．197mmo]／L、0．296mmol／LPTU可以有效抑制黑色素生成,保持斑马鱼整体透明,对斑马鱼心血管系统结构和生理功能无影响,且不影响斑马鱼正常孵化过程。随着PTU浓度的增加,斑马鱼死亡率增加,孵化率下降,出现心包水肿,心脏畸形等改变,心率下降,静脉窦-动脉球之间的距离增大。结论浓度不高于0．296mmol／L的PTU溶液能有效抑制斑马鱼黑色素生成,对斑马鱼心血管毒性研究无影响。     

14C-涕灭威在旱田土壤中的降解

研究了¹⁴C-涕灭威在5种土壤中(4ppm,1.22μCi·50g^-1 土壤干重)的生物降解。模拟试验为密闭系统,土壤中水分含量为22%,气温20-30℃在供试的5种土壤中,北京肖家河的土壤降解最快,为施人放射剂量的51.3%,以“¹⁴CO₂ 形式从土壤进出;26.0%与土壤结合,只有21.6%可以被抽出。取自浙江义乌的土壤降解较慢,收集到的¹⁴CO₂ 为施入量的23.3%.土壤中加入杀菌剂红霉素或敌茵丹降解作用明显减慢。土壤提取物中涕灭威亚砜、涕灭威亚砜肟被确认是主要的代谢产物,还发现了少量的涕灭威砜,涕灭威亚砜腈涕灭威砜腈和涕灭威砜肟等降解物。     

铅和邻苯二甲酸二丁酯联合暴露对斑马鱼胚胎的影响

为了研究铅(Pb)和邻苯二甲酸二丁酯(DBP)单独及联合暴露对斑马鱼胚胎神经发育和细胞凋亡相关基因表达的影响,考察两者对斑马鱼胚胎神经系统的毒性作用。将720个斑马鱼胚胎按3×3析因设计随机分为9组,以Pb(0、0.01 mg/L和1 mg/L)和DBP(0、0.005 mg/L和0.5 mg/L)单独及联合对斑马鱼胚胎暴露120 h。观察、记录胚胎的孵化、死亡情况,采用荧光定量PCR测定斑马鱼胚胎神经发育相关基因(NR1A、NR2A、NR2D)和神经细胞凋亡相关基因(bcl-2、c-fos)的表达变化情况。与空白对照组相比,各暴露组均可致斑马鱼胚胎孵化率降低、死亡率增高(P0.05);联合暴露组NR1A、NR2A、NR2D、c-fos基因表达水平显著升高,bcl-2基因表达水平显著下降(P0.05)。Pb和DBP均可影响斑马鱼胚胎的发育,且具有一定的神经毒性,而二者联合暴露毒性复杂,对斑马鱼胚胎孵化率和死亡率的影响有交互作用,对神经发育和细胞凋亡相关基因无交互作用,其机制有待进一步研究。     

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。     

Processing of a psoralen DNA interstrand cross-link by XPF-ERCC1 complex in vitro

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5' to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3'-side of an ICL. The ICL-specific 3'-incision, along with the 5'-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.     

Study on the fluorescent enhancement effect in terbium-gadolinium-protein-sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level

The co-luminescence effect in a terbium-gadolinium-protein-sodium dodecyl benzene sulfonate (SDBS) system is reported here. Based on it, the sensitive quantitative analysis of protein at nanogram levels is established. The co-luminescence mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy and NMR measurement. It is considered that protein could be unfolded by SDBS, then a efficacious intramolecular fluorescent energy transfer occurs from unfolded protein to rare earth ions through SDBS acting as a "transfer bridge" to enhance the emission fluorescence of Tb3+ in this ternary complex of Tb-SDBS-BSA, where energy transfer from protein to SDBS by aromatic ring stacking is the most important step. Cooperating with the intramolecular energy transfer above is the intermolecular energy transfer between the simultaneous existing complexes of both Tb3+ and Gd3+. The fluorescence quantum yield is increased by an energy-insulating sheath, which is considered to be another reason for the resulting enhancement of the fluorescence. F?rster theory is used to calculate the distribution of enhancing factors and has led to a greater understanding of the mechanisms of energy transfer.     

十二烷基苯磺酸钠对引发内置物感染表皮葡萄球菌生物被膜的作用

【背景】随着医用内置物的广泛使用,由表皮葡萄球菌生物被膜导致的医院获得性感染不断增多,目前鲜见关于表面活性剂针对表皮葡萄球菌生物被膜作用的报道。【目的】通过研究阴离子型表面活性剂十二烷基苯磺酸钠(sodium dodecyl benzene sulfonate,SDBS)分别对ATCC 35984 (产膜表皮葡萄球菌标准株)生物被膜的清除、生物被膜内细菌代谢和形成生物被膜的关键物质多糖胞间黏附素(polysaccharide intercellular adhesion,PIA)产生的影响,为临床使用SDBS防治由表皮葡萄球菌生物被膜引起的相关感染提供可靠的理论及实践依据。【方法】利用XTT减低法,评价SDBS对ATCC 35984已形成生物被膜的清除效率及对生物被膜内细菌代谢的影响;激光共聚焦显微镜观察SDBS对生物被膜作用的效果;采用刚果红培养基观察SDBS对PIA产生的影响。【结果】浓度为256、128、64、32、16 mg/L的SDBS在作用6、12、24 h时,对ATCC 35984的生物被膜均有显著的清除效率(P<0.01);浓度为32 mg/L时对生物被膜内细菌的...     

Iron(II)-mimosine catalyzed cleavage of DNA

Mimosine, DNA breaKs, Free Radicals, Fenton Reaction Supercoiled plasmid DNA was treated in vitro with H2O2, DTT and either Fe (II), Fe (II)-EDTA or Fe (II)-mimosine. The rate of DNA break formation was followed by the conversion of the supercoiled form into relaxed-circular and linear forms. In the concentration interval of 0-4 microM Fe (II), Fe (II)-EDTA slowed-down the formation of DNA breaks, while Fe (II)-mimosine enhanced the rate of break formation up to several times. A conclusion is drawn that this enhancement is due to the increased affinity of the Fe (II)-mimosine complex to DNA.     

Resonance light scattering study on the interaction of benproperine phosphate with eriochrome blue black R in the presence of sodium dodecylbenzene sulphonate and its analytical application

The interaction of benproperine phosphate (BPP) with eriochrome blue black R (EBBR) in the presence of sodium dodecylbenzene sulphonate (SDBS) was studied using resonance light scattering (RLS) technology and ultraviolet‐visual (UV‐vis) spectrophotometry. Under optimum conditions, BPP reacts with EBBP and SDBS to form a three‐component complex, which results in strong RLS signal and a new RLS peak. The enhanced RLS intensities are proportional to the concentration of BPP over the range 0.6–28.0 µg/mL, with a detection limit of 0.053 µg/mL. The affecting factors as well as the influence of coexisting substances were investigated. The results indicate that this assay method could be applied to the determination of BPP in pharmaceuticals, serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

重离子射线照射后抗辐射菌基因组DNA的损伤修复

研究了不同种类的重离子射线及同一射线的不同剂量照射后引起的抗辐射菌(Deinococcus radiodu-rans)R1的DNA二条链的切断损伤修复时间。结果表明,抗辐射菌经重离子射线照射后所引起的DNA二条链的切断损伤经过培养能被修复;切断的DNA二条链的修复时间随着照射剂量的增加而延长;高LET的重离子射线照射所引起的损伤修复比低LET的重离子射线需要更长的时间,损伤修复的时间与射线的LET之间存在一定的依存性。由此认为:抗辐射菌经照射后引起的DNA二条链切断损伤与射线的种类及照射的剂量有关,照射的剂量越大,射线的LET越高,则DNA二条链的切断损伤越多,损伤修复所需要的时间越长。     

Nickel(II) and cobalt(II) complexes of hydroxyl-substituted triazamacrocyclic ligand as potential antitumor agents

The stability constants for the formation of nickel(II) and cobalt(II) complexes of the ligand [1,4,7]triazecan-9-ol (L) were presented. Antitumor activity of two complexes was reported. Nuclei of [NiL]-stimulated BEL-7402 cells clearly exhibited condensation and break down into chromatin clumps typical of apoptosis. Also it exhibited perturbation effects to cell cycle, and optimal induction of apoptosis was found by Flow-Cytometric analysis. But CoL complex did not exhibit introduction effects to BEL-7402 cells apoptosis; and could not perturb cell cycle. NiL and CuL complexes could cleave supercoiled DNA (pBR 322 DNA) to nicked and linear DNA, and DNA of cells treated with NiL or CuL complex was obviously damaged; while CoL complex only could cleave supercoiled DNA (pBR 322 DNA) to nicked DNA, and DNA of cells treated with CoL complex had no significant difference with control.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.

No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.

Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.

Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

In Vitro Repair of Gaps in Bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

In-Vitro and In-Vivo Effects of Aldicarb on Survival and Development of Heterodera schachtii

Aqueous solutions of 5-500 μg/ml aldicarb inhibited hatching of Heterodera schachtii. Addition of hatching agents, zinc chloride, or sugarbeet root diffusate, to the aldicarb solutions did not decrease the inhibition of hatching. When cysts were removed from the aldicarb solufions and then treated for 4 wk in sugarbeet root diffusate, larvae hatched and emerged. Treatments of newly hatched larvae of H. schachtii with 5-100 μg/ml aldicarb depressed later development of larvae on sugarbeet (Beta vulgaris). Similar treatments with aldicarb sulfoxide had less effect on larval development, and aldicarb sulfone had no effect. Numbers of treated larvae that survived and developed were inversely proportional to concentration (0.1-5.0 μg/ml) and duration (0-14 days) of aldicarb treatments. Development of H. schachtii on sugarbeet grown in aldicarb-treated soil was inversely proportional to the concentration of aldicarb in the tested range of 0.75 - 3.0 μg aldicarb/g of soil. Transfer of nematode-infected plants to soil with aldicarb retarded nematode development, whereas transfer of plants first grownin treated soil to nematode-infested soil only slightly suppressed nematode development. Development of H. schachtii was inhibited in slices of storage roots of table beet (B. vulgaris), sugarbeet and turnip, (Brassica rapa), that had grown in soil treated with aldicarb.