酵母双杂交法筛检酚类物质的内分泌干扰作用

 利用酵母双杂交系统对4种天然和合成的雌激素,4种植物性激素以及14种常用于自来水供水系统中的酚类物质进行了内分泌干扰作用的筛检测定.结果表明,这些物质的作用强度与其结构有很大的关系.14种人工合成的化学物质中,有8种显示出b-半乳糖甘酶诱导活性.从与雌激素受体结合活性值的比较结果可以推测,没有显示出b-半乳糖甘酶诱导活性的物质可能具有抗雌激素(antagonist)作用.     

典型酚类污染物内分泌干扰效应研究——对斑马鱼发育及核受体介导基因调控的分子影响

以斑马鱼为研究对象,观察2,4,6-三氯酚（2,4,6-trichlorophenol）、4-氯-3-甲酚（4-chloro-3-methylphenol）和4-硝基苯酚（4-nitrophenol）3种酚类污染物对斑马鱼胚胎发育早期阶段形态学指标的影响,借助q-RT-PCR方法对斑马鱼胚胎分别暴露于0.1,0.5和2.5mg/L至120hpf幼鱼8个重要受体（雄激素受体（AR）、甲状腺激素受体（TR）、芳香烃受体（AhR）、雌激素受体（ER）、糖皮质激素受体（GR）、孕烷X受体（PXR）、盐皮质激素受体（MR）、过氧化物酶体增殖物激活受体（PPAR））相关基因的转录水平进行了研究;进一步采用分子对接和分子动力学模拟的方法研究污染物与筛选出的影响受体（ER、AhR、PXR、GR以及MR）之间的相互作用过程.结果表明,在5和10mg/L的浓度下3种酚类污染物都显著影响斑马鱼的发育,造成脊柱弯曲、心包水肿等不良影响;基因结果显示2,4,6-三氯酚暴露导致ER的基因下调,雌激素受体α（er1）基因在2.5mg/L浓度下下调2.1倍.4-氯-3-甲酚显著影响ER、AhR以及PXR的相关基因,表现为在0.5和2.5mg/L暴露er1基因显著下调1.8和2.2倍,ahr2和pxr基因在0.1和2.5mg/L的浓度下分别下调1.7和1.8倍和上调3.5和3.2倍.4-硝基苯酚暴露使GR、MR核心受体基因发生调控变化,糖皮质激素受体基因（gr）在低中浓度下分别显著上调2.0和2.1倍,最高浓度下下调1.9倍.盐皮质激素受体基因（mr）在中高浓度下分别下调1.9和2.1倍,在最低浓度下显著上调1.7倍;分子计算结果显示它们与相关受体通过疏水和氢键等相互作用而稳定结合,且均方根偏差（RMSD）在5ns后稳定.这3个典型酚类污染物主要通过斑马鱼ER、AhR、PXR、GR、MR受体介导产生内分泌干扰效应并影响其早期生长发育.     

酚类化合物雌激素效应的比较研究

采用重组人雌激素受体基因片段(hER)及共激活因子(GRIP1)双杂交酵母检测体系,检测9种酚类物质的类雌激素和抗雌激素效应.结果表明,所检测的化合物中有3种同时表现出雌激素诱导与抑制活性;2种只表现出雌激素诱导活性;1种只表现出雌激素抑制活性;3种均没有雌激素诱导与抑制活性.结构分析表明:酚类中产生雌激素活性的物质具有取代基位于对位且邻位没有大的取代基团的结构;烷基在对位的酚类物质也能显示出雌激素拮抗活性.所采用的双杂交酵母检测体系适用于外源物质的雌激素活性检测,具有较高的灵敏度;结合外源物的雌激素诱导与抑制活性,用于分析污染物的体内作用方式.     

电子垃圾处理地土壤中芳烃受体效应物质的分布规律

利用大鼠肝癌细胞H4IIE离体生物测试(EROD测试)评估了位于我国南方的典型电子垃圾处理地(某镇)三个自然村共23个土壤样品有机提取物的芳烃受体效应,并研究了这些地区土壤中芳烃受体效应物质的污染水平和空间分布规律.结果表明,在所研究地区中.塑料回收村芳烃受体效应最高,其次是电路板回收村和电器拆解村.与文献数据相比,该镇土壤有机组分的芳烃受体效应较传统工业城市天津的污染土壤高,且空间分布规律与该镇电子垃圾处理业的区域分工在某种程度上有密切关系.研究结果表明,粗放型的电子垃圾处理手段导致了芳烃受体效应物质在该镇土壤环境中的积累,且离体生物效应标记方法可用来快速筛选和甄别芳烃受体效应物质污染的高风险区.     

应用重组孕激素基因酵母测定饮用水中内分泌干扰物的方法

研究了用重组孕激素受体基因酵母测定饮用水中内分泌干扰物的方法,并利用该种方法检测了南方某水厂不同处理工艺过程水样对孕激素受体活性的抑制水平.结果表明,重组孕激素受体基因酵母能够与孕激素专一性的结合,诱导产生明显的剂量-效应关系,EC₅₀值为0.5 nmol/L,具有较高灵敏度;环境内分泌干扰物五氯酚和壬基酚具有孕激素受体抑制活性,其IC₅₀值分别为2.4μmol/L和3.7μmol/L;重组孕激素受体基因/报道基因的酵母技术是一种筛选和定量分析具有孕激素受体抑制活性的内分泌干扰物的快速、有效方法.结合固相萃取的前处理技术,重组孕激素受体基因酵母对水厂不同处理工艺水样检测出明显的孕激素受体抑制活性,抑制率均大于58%,表明重组孕激素受体基因酵母检测技术能够快速监测和鉴别水样中具有抑制孕激素受体活性的物质.     

苯醚甲环唑对斑马鱼抗氧化酶的影响

以斑马鱼为模式生物研究了三唑类杀菌剂苯醚甲环唑对斑马鱼脑和肝脏中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)四种抗氧化酶活性的影响.结果发现:苯醚甲环唑暴露后,斑马鱼脑中CAT酶活性随暴露时间延长呈现出先升高后降低的趋势.50μg/L及更高浓度的苯醚甲环唑能够显著抑制斑马鱼脑和肝脏中GPx的活性,且对脑中GPx活性的抑制程度更强.另外,500μg/L苯醚甲环唑处理后,斑马鱼肝脏和脑中GR活性表现出不同的变化趋势,肝脏中GR活性下降,而脑中GR活性升高.上述结果表明50μg/L的苯醚甲环唑即可影响斑马鱼的抗氧化系统,其对农业水域中的鱼类影响值得重视.     

a-萘酚对小球藻谷胱甘肽及其还原酶的影响

研究了a-萘酚对2种小球藻谷胱甘肽(包括还型谷胱甘肽GSH与氧化型谷胱甘肽GSSG)含量及其还原酶(GR)活性的影响.结果表明,低浓度a-萘酚对藻细胞的谷胱甘肽水平和GR活性有显著的激发作用,随着a-萘酚浓度的升高,普通小球藻和蛋白核小球藻的GSH含量、谷胱甘肽总量及GR活性均有所提高,并分别在5mg/L和10mg/L时达到最大值,而GSSG均不断下降且在相同浓度下(2mg/L)至最小后开始上升.GSSG/GSH先降后升的变化说明藻细胞在a-萘酚胁迫下膜脂过氧化加剧,而GSH、GR在清除活性氧、消除过氧化方面起了重要作用     

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.     

α-萘酚对小球藻谷胱甘肽及其还原酶的影响

研究了--萘酚对2种小球藻谷胱甘肽-包括还型谷胱甘肽GSH与氧化型谷胱甘肽GSSG-含量及其还原酶-GR-活性的影响.结果表明,低浓度--萘酚对藻细胞的谷胱甘肽水平和GR活性有显著的激发作用,随着--萘酚浓度的升高,普通小球藻和蛋白核小球藻的GSH含量、谷胱甘肽总量及GR活性均有所提高,并分别在5mg/L和10mg/L时达到最大值,而GSSG均不断下降且在相同浓度下-2 mg/L-至最小后开始上升.GSSG/GSH先降后升的变化说明藻细胞在--萘酚胁迫下膜脂过氧化加剧,而GSH、GR在清除活性氧、消除过氧化方面起了重要作用.     

天津典型河流沉积物潜在毒性的离体生物效应评价

采用成组离体生物效应标记方法,包括鼠肝癌细胞H4IIE 测试、双杂交酵母测试和SOS/umu 测试,对天津地区的北塘排污河、永定新河和大沽排污河沉积物样品进行潜在的生物毒性筛选和评价.结果表明,3 条河流沉积物中芳烃受体效应以2,3,7,8-四氯代二毒性当量(TEQ)表示为19~1227ng/kg;类雌激素效应以17β-雌二醇当量表示为40~6203ng/kg;直接、间接遗传毒性以诱导率表示分别为1.1~3.7 和1.0~2.2.与国内外相关研究比较发现,3 条河流均存在芳烃受体活性物质、类雌激素物质和遗传毒性类物质相当水平的累积,重点污染河段为北塘排污河出口闸、永定新河的唐津高速路桥河段、大沽排污河的巨葛庄和唐港桥2 个河段.同时,永定新河的上游和北塘排污河的上游分别存在较高的芳烃受体效应和类雌激素效应.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Degradation of metabolites accumulated of benzo[a]pyrene by coupling Penicillium Chrysogenum with KMnO4

Several main metabolites of benzo[a]pyrene （BaP） formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone （BP 1,6- quinone）, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene （BP 7,8-diol）, 3-hydroxybenzo[a]pyrene （3-OHBP）, were identified by high-performance liquid chromatography （HPLC）. The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that （1） the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; （2） the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; （3） the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% （w/v）, concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.     

Impact of atrazine and nitrogen fertilizers on the sorption of chlorotoluron in soil and model sorbents

Sorption of chlorotoluron in ammonium sulfate, urea and atrazine multi-solutes system was investigated by batch experiments. The results showed application of nitrogen fertilizers to the soil could affect the behavior of chlorotoluron. At the same concentration of N, sorption of chlorotoluron decreased as the concentration of atrazine increased on the day 0 and 6 in soil, respectively. The sorption of chlorotoluron increased from 0 to 6 d when soils were preincubated with deionized water, ammonium sulfate and urea solution for 6 d. That indicated incubation time was one of the most important factors for the sorption of chlorotoluron in nitrogen fertilizers treatments. The individual sorption isotherms of chlorotoluron in rubbery polymer and silica were strictly linear in single solute system, but there were competition sorption between pesticides or between pesticides and nitrogen fertilizers. That indicated the sorption taken place by concurrent solid-phase dissolution mechanism and sorption on the interface of water-organic matter or water-mineral matter.     

Effects of two sludge application on fractionation and phytotoxicity of zinc and copper in the soil

The potential harm of heavy metals is a primary concern in application of sludge to the agricultural land. A pot experiment was conducted to evaluate the effect of two sludges on fractionation of Zn and Cu in soil and their phytotoxicity to pakchoi. The loamy soil was mixed with 0%, 20%, 40%, 60% and 80% (by weight) of digested sewage sludge (SS) and composted sludge (SC). The additions of both sludges caused a significant raise in all fractions, resulting in that exchangeable (EXCH) and organic bound (OM) became predominance of Zn and organic bound Cu occupied the largest portion. There was more available amount of Zn and Cu in SS treatments than SC treatments. During the pot experiment, the concentration of Zn in EXCH, carbonate (CAR) and OM and Cu in EXCH and OM fractions decreased in all treatments, so their bioavailability reduced. Germination rate and plant biomass decreased when the addition rate was high and the best yield appeared in 20% mixtures at the harvest of pakchoi. The two sludges increased tissue contents of Zn and Cu especially in the SS treatments. Zn in pakchoi was not only in relationship to ΔEXCH and ΔCAR forms but also in ΔOM forms in the sludge-soil mixtures. Tissue content of Cu in pakchoi grown on SC-soils could not be predicted by ΔEXCH. These correlation rates between Zn and Cu accumulation in pakchoi and variation of different fractions increased with time, which might indicate that sludges represented stronger impacts on the plant in long-term land application.     

Intraspecific differences in effects of co-contamination of cadmium and

A hydroponic experiment was carried out to study intraspecific differences in the effects of different concentrations of cadmium (Cd)(0-10 mg/L) and arsenate (As(V)) (0-8 mg/L) on the growth parameters and accumulation of Cd and As in six wheat varieties Jing-9428, Duokang-1, Jingdong-11, Jing-411, Jingdong-8 and Zhongmai-8. The endpoints of wheat seedlings, including seed germination,biomass, root length and shoot height, decreased with increasing the Cd and As concentrations. Significant differences in seed germination, biomass, root length, shoot height and the accumulation of Cd and As were observed between the treatments and among the varieties (p ＜ 0.05). The lethal dosage 50% were about 20, 80, 60, 60, 80 and 20 mg As/L for Jing-9428, Duokang-1, Jingdong-11,Jing-411, Jingdong-8 and Zhongmai-8, respectively, and the corresponding values for Cd were about 30, 80, 20, 40, 60 and 10 mg Cd/L, respectively. Among the six varieties, Duokang-1 was found to be the most resistant to Cd and As toxicity, and Zhongmai-8 was the most sensitive to Cd and As co-contamination. The resistance of the six varieties was found dependant on the seedling uptake of Cd and As. Duokang-1 was the most suitable for cultivation in Cd and As co-contaminated soils.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.     

Removal of heavy metals from sewage sludge by low costing chemical method and recycling in agriculture

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Single and joint effects of pesticides and mercury on soil urease

The influence of two pesticides including chlorimuron-ethyl and furadan and mercury （Hg） on urease activity in 4 soils （meadow burozem and phaeozem） was investigated. The soils were exposed to various concentrations of the two pesticides and Hg individually and simultaneously. Results showed that there was a close relationship between urease activity and organic matter content in soil. Chlorimuron-ethyl and furadan could both activate urease in the 4 soils. The maximum increment of urease activity by chlorimuronethyl was up to 14%-18%. There was almost an equal increase （up to 13%-21%） in the urease activity by furadan. On the contrary, Hg markedly inhibited soil urease activity. A logarithmic equation was used to describe the relationship （P〈0.05） between the concentration of Hg and the activity of soil urease in the 4 tested soils. Semi-effect dose （ED50） values by the stress of Hg based on the inhibition of soil urease in the 4 soils were 88, 5.5, 24 and 20 mg/kg, respectively, according to the calculation of the corresponding equations. The interactive effect of chlorimuron-ethyl or furadan with metal Hg on soil urease was mainly synergic at the highest tested concentrations.     

Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria

A study was conducted to compare the diversity of 2-, 3-, and 4-chlorobenzoate degraders in two pristine soils and one contaminated sewage sludge. These samples contained strikingly different populations of mono-chlorobenzoate degraders. Although fewer cultures were isolated in the uncontaminated soils than contaminated one, the ability of microbial populations to mineralize chlorobenzoate was widespread. The 3- and 4-chlorobenzoate degraders were more diverse than the 2-chlorobenzoate degraders. One of the strains isolated from the sewage sludge was obtained. Based on its phenotype, chemotaxonomic properties and 16S rRNA gene, the organism S-7 was classified as Rhodococcus erythropolis. The strain can grow at temperature from 4 to 37℃. It can utilize several （halo）aromatic compounds. Moreover, strain S-7 can grow and use 3-chlorobenzoate as sole carbon source in a temperatures range of 10-30℃ with stoichiometric release of chloride ions. The psychrotolerant ability was significant for bioremediation in low temperature regions. Catechol and chlorocatechol 1,2-dioxygenase activities were present in cell free extracts of the strain, but no （chloro）catechol 2,3- dioxygenase activities was detected. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-chlorobenzoate. On the basis of these results, we proposed that S-7 degraded 3-chlorobenzoate through the modified ortho-cleave pathway.