Evidences showing 2,4-D ethyl ester and pencycuron-induced DNA damage in cyanobacteria and detection by PCR

The impact of 2,4-D ethyl ester and pencycuron in inducing DNA damage in three species of cyanobacteria-Anabaena fertilissima, Aulosira fertilissima, and Westiellopsis prolifica as evidenced by PCR-based assays: RAPD and 16S rRNA amplification was examined. Exposure of genomic DNA (in vitro) to pencycuron for 4 days did not produce severe damage in DNA fragments of all three cyanobacterial species whereas exposure to 2,4-D ethyl ester markedly inhibited the template activity of genomic DNA compared to untreated cultures of A. fertilissima. In A. fertilissima a single band of approximately 1000?bp was observed even after 16 days of exposure to 60?ppm pencycuron which suggests that certain segments of DNA are resistant to pencycuron DNA damaging effects. However, a significant effect was observed in the case of W. prolifica for 2,4-D ethyl ester and pencycuron where complete disappearance of fragments was not recorded even after 16 days of incubation and interestingly some new DNA bands were induced. Similar to the effects with RAPD profile, amplification of rRNA was significantly inhibited following exposure of genomic DNA to 2,4-D ethyl ester and pencycuron. Our findings clearly demonstrate that pesticide concentrations affected cyanobacterial DNA and lethality of these microbes might be due to irreversible DNA damage. Thus, it is postulated that PCR assays may be conveniently used for screening DNA damage produced by 2,4-D ethyl ester and pencycuron in all three cyanobacteria examined in this study.     

间接竞争ELISA方法测定水中2，4-D的研究

以2,4-D-BSA为包被抗原,采用自行制备的2,4-D单克隆特异性抗体6D11建立了水中2,4-D的间接竞争 ELISA检测方法．本研究比较了包被抗原2,4-D-BSA浓度分别为240ng·mL-1、120ng·mL-1和60ng·mL-1的反应体系和竞争反应时间为60min和15min的间接竞争ELISA剂量-反应曲线,确定了当包被抗原浓度为60 ng·mL-1、竞争反应时间为15min时,剂量-反应曲线的IC50值较低．采用上述实验条件分别测定了由PBS缓冲溶液、饮用水、清华大学地下水和圆明园福海地表水配制的2,4-D标准溶液的剂量．反应曲线,发现实际水样的基质效应对检测结果的影响较大;采用实际水样和PBS缓冲溶液配水在含有5％乙醇的PBS缓冲体系中反应的方法,基本上消除了基质效应对检测结果的干扰．采用上述优化试验条件,测定2,4-D浓度分别为0．5mg·L-1、0．125mg·L-1和0．03mg·L-1的加标样品,测定数据的准确度符合痕量有机污染物定量检测对准确度的要求,但是平行样品测定数据之间的变异系数较大,需要进一步改进检测方法,用于实际水样的检测．     

Effect of 2,4-dichlorophenoxyacetic acid on growth, protein and chlorophyll-a content of Chlorella vulgaris and Spirulina platensis cells

In this study, effect of different 2,4 -dichlorophenoxyacetic acid (2,4-D) concentrations (0.0, 9.10(-5), 9.10(-4), 9.10(-3) and 9.10(-2) mM) on growth rate, content of protein and chlorophyll-a in Chlorella vulgaris and Spirulina platensis cells was investigated. The most stimulatory effect on growth rate, protein and pigment ratio of C. vulgaris and S. platensis was observed at 9.10(-4) mM concentrations of 2,4-D. The results show that low concentrations of 2,4-D have hormonal effect due to being a synthetic auxin. Cell number protein and pigment rates were inhibited at 9.10(-2) mM concentration in C. vulgaris. Such parameters were inhibited in S. platensis, both at 9.10(-3) and 9.10(-2) mM 2,4-D concentrations. This is due to herbicidal effect of high concentrations of 2,4-D. S. platensis was found to be more sensitive than S. vulgaris to 2,4-D applications. The use of algae as bio-indicators in herbicide contaminated fresh water habitats, was discussed.     

BSA体系中2,4-D和阿特拉津对性激素的干扰机理

在2,4-D和阿特拉津以及天然性激素(雌二醇、睾酮)中加入牛血清白蛋白(BSA),通过紫外吸收峰的变化、温度与体系荧光强度的关系、以及计算扩散碰撞猝灭常数Kq等方法确定2,4-D和阿特拉津均可以发生类似天然性激素和BSA间的基态络合.并通过双倒数公式计算出各自二元体系的结合常数.2,4-D、阿特拉津、雌二醇、睾酮与BSA的结合常数(K)均在104数量级,可以认为2,4-D和阿特拉津具有与天然性激素相近的对BSA结合能力,并可通过竞争占据结合位点的方式干扰正常性激素与BSA的结合.     

除草剂2，4-D在土壤中吸附常数的测定

介绍除草剂２,４－Ｄ在４种不同有机碳含量、不同ｐＨ值的土壤中吸附常数的测定．结果表明,４种土壤对２,４－Ｄ的吸附等温线均符合非线形的Ｆｒｅｕｎｄｌｉｃｈ吸附等温式,其有机碳吸附常数的平均值为２３９．６．２,４－Ｄ的吸附常数与土壤有机碳含量呈正相关,土壤的ｐＨ值对２,４－Ｄ的吸附也有一定的影响．     

Soil mycofloral responses following the exposure to 2, 4-D

2, 4-dichlorophenoxyacetic acid (2,4-D) is a post emergence herbicide. The tests were conducted to study the toxicity of 2,4-D using EC50 value in four dilutions. 2,4-D was applied at concentration of 25, 50, 75, 100, 200 and 300 mg l(-1) in the potato Dextrose agar medium. The effect of this herbicide was evaluated as the colony forming unit (CFU). EC50 value for 10(-3) dilution of soil was 138 +/- 5.944 mg l(-1). Soil physico-chemical parameters and mycofloral properties were also evaluated. Qualitatively 10 genera of fungi were observed in untreated soil, whereas 4 genera were found in 2,4-D administered dose.     

Umweltmonitoring von Herbiziden in aquatischen Systemen

Herbicides of the chlorinated phenoxyalkanoic acid group like 2,4-D, 2,4-DP and MCPP, belong to the most commonly used pesticides. This is also true for the Land Brandenburg. Therefore, this substance group was integrated in the current state monitoring programme for ground and surface water quality. Starting in 2001, the analysis was carried out complying with DIN 38407-20 by gas chromatography and mass spectrometric detection after enrichment with solid phase extraction and following derivatization with diazomethane-forming methyl esters. Quantifying 2,4-D considering selected ions in a surface water sample from the river Oder, a remarkable peak pattern was observed. A second peak with identical spectrum was found about 0.7 minutes earlier than the 2,4-D calibrated peak. Its intensity suggested a concentration 10–20 fold higher than the 2,4-D level. This unknown peak was identified as 2,6-D which is a structure isomer of the herbicide 2,4-D. This substance has not been described in literature or chemical substance databases known to us yet. Structure isomers have been found in other samples too. We suppose that the chlorinated phenoxyalkanoic acids chlorinated in the 6-position of the phenyl ring are formed as a technical by-product and show a higher persistance than the commonly known agents.     

除草剂对优质小麦品质和旗叶保护酶的调控效应

通过田间试验研究了4种除草剂在正常剂量下对2个优质小麦品种品质性状、蛋白质组分和旗叶保护酶系的影响.结果表明,整个籽粒灌浆过程中,2,4-D丁酯处理使籽粒蛋白质含量一直较高,世玛处理在灌浆前中期籽粒蛋白质含量较高,但灌浆末期籽粒蛋白质含量最低,巨星和骠马处理影响较小.2,4-D丁酯处理使收获时籽粒蛋白质含量及组分最高,湿面筋含量、沉降值、吸水率、稳定时间和评价值等加工品质最好,世玛处理则使营养品质和加工品质最差,巨星和骠马处理影响相对较小.籽粒灌浆期间,2,4-D丁酯处理使旗叶SOD、CAT和POD活性一直较低,尤其是籽粒灌浆末期活性最低;其它除草剂使旗叶SOD、CAT和POD活性在不同灌浆时期存在差异:灌浆前中期,骠马处理使3种保护酶活性最高;灌浆后期,世玛处理使旗叶中保护酶活性下降缓慢,且活性最高.籽粒灌浆期前期,4种除草剂对旗叶中MDA含量的影响是:世玛＞骠马＞CK＞巨星＞2,4-D丁酯,灌浆中后期,2,4-D丁酯和巨星处理使MDA含量快速提高,世玛和骠马处理使MDA含量升高相对较慢,到灌浆末期,2,4-D丁酯使MDA含量最高,世玛处理最低.图5表2参22     

Effects of humic acid fractions with different polarities on photodegradation of 2,4-D in aqueous environments

Four fractions (A, B, C, and D) of humic acids (HAs) were separated based on the polarity from weak to strong. UV-vis absorption and Fourier transform infrared spectroscopy (FTIR) analysis show that the fractions C and Dpossessedmore aromatic C=C content. The influences of HAs and their fractions on the photolysis were investigated by the photodegradation of 2,4-D solutions under simulated solar light irradiation. The degradation rate of 2,4-D was found to decrease in the presence of bulk HAs or their fractions especially at high HAs concentration. The fractions of strong polarity C and D retarded the degradation rate more than the fractions of weak polarity A and B. This could be attributed to the different absorption intensity of the four HAs fractions in the order of D ? C > A > B, and the stronger π-π electron donor-acceptor interactions between the strong polar fractions and 2,4-D.     

2,4-D二甲胺盐对莲草直胸跳甲生存和繁殖的影响

为探究除草剂在防治喜旱莲子草(Alternanthera philoxeroides)的过程中对其天敌昆虫的影响,采用不同浓度的2,4-D二甲胺盐浸叶处理的喜旱莲子草饲喂莲草直胸跳甲,连续饲喂7d后,考察了莲草直胸跳甲的繁殖、存活和发育情况。结果表明,2,4-D二甲胺盐可显著降低莲草直胸跳甲成虫的取食量,且抑制作用随药剂浓度升高和取食时间延长而显著增强;较高浓度(1.72～3.44g·L~(-1))的2,4-D二甲胺盐还会导致莲草直胸跳甲雌成虫存活率和产卵量的明显降低,卵孵化率、幼虫存活率和蛹羽化率也显著降低。可见,高浓度(1.72～3.44g·L~(-1))的2,4-D二甲胺盐对莲草直胸跳甲的生长发育和繁殖有显著不利影响,在防治喜旱莲子草时,应将2,4-D二甲胺盐的施用浓度控制在0.22～0.86g·L~(-1)内。     

Additive und synergistische Kombinationswirkungen von Xenobiotika in subtoxischen Konzentrationen auf menschliche Fibroblasten

The toxic combination effects of xenobiotics of different substance classes were investigated in human fibroblasts. Subtoxic concentrations of 4-chloroaniline enhanced the toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D, dimethylammonium salt) in an additive manner. A mixture of 4-chloroaniline, dicofol and 4-chlorophenol increased the toxicity of 2,4-D synergistically. The concentrations of the components used in the mixture were one third of their ?no observed effect concentrations” (NOEC). The combination of 4-chloroaniline, dicofol and 4-chlorophenol (without 2,4-D) at subtoxic concentrations also showed synergistic effects. The great differences in the lipophilicity of the combined substances might be the cause of the synergistic action.     

Effects of chemical contaminants on the health of Mytilus edulis from Puget Sound,Washington. II. Cytochemical detection of subcellular changes in digestive cells

Activities of the enzymes NADPH-dependent ferrihemoprotein reductase (NFR), NADH-dependent DT-diaphorase (DTD), gamma-glutamyl transpeptidase (GGT), and catalase (CAT) and peroxisome proliferation (PP) in the digestive cells of Mytilus edulis from nine sites in Puget Sound, Washington (USA) sampled in September 1992 were measured cytochemically using image analysis. Mussels from these areas are known to be exposed to a wide range of chemical contaminants. At urban-associated sites, mussels generally showed increased activities of NFR, DTD, and CAT, suppressed GGT activity, and peroxisome proliferation, relative to mussels from the non-urban reference sites. Significant positive relationships were observed between tissue concentrations of polyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and DDTs and activities of NFR, DTD, CAT, and peroxisome proliferation. Structural changes in the digestive gland of mussels also appear to be more responsive to chemical contaminant exposure than changes in enzyme activity. These relationships suggest that NFR, CAT, and the induction of peroxisome proliferation represent complimentary indicators of biological effects from chemical-contaminant exposure in the marine bivalve M. edulis. The current findings support the use of selected cytochemically measured subcellular responses as biomarkers of contaminant exposure in environmental monitoring programs.     

低质量浓度除草剂2，4滴丁酯处理对土壤动物群落结构的影响

土壤动物是土壤污染的敏感指示生物,对土壤环境变化表现出比较灵敏的反映,为了解低质量浓度除草剂对土壤生态系统的影响,进一步推动除草剂使用的安全性评价研究,采用除草剂2,4滴丁酯对土壤动物群落进行室内染毒模拟试验,参照农田施用常规参考质量浓度,设置4个质量浓度梯度和一个不喷施除草剂的对照质量浓度,质量浓度最高限于参考喷施质量浓度的3~4倍,一次染毒后分别于12、24、48和72 h后对土壤动物进行分离并对群落结构特征进行分析。试验共获得土壤动物18655个,隶属于2门,4纲,12目,群落结构分析结果表明低质量浓度2,4滴丁酯导致土壤动物个体数和类群数有所减少,但减少的程度并不大。个体数量变化与质量浓度增高之间呈现较明显负相关（r=-0.856,P〈0.05）,种类数变化与质量浓度之间不具有明显的相关性,土壤动物种类数的减少主要取决于稀有类群和极稀有类群,数量的变化则主要是优势类群和常见类群的增减。2,4滴丁酯的施加使得土壤动物群落多样性指数和优势度指数降低,均匀度指数增高。土壤动物个体数量和类群数量变化与染毒后经历的时间之间并未表现出明显的规律性,染毒后不同时间土壤动物优势度指数相差较大,而多样性指数和均匀度指数在不同历时时间段内相差不大。综合各分析结果能够看出,低质量浓度2,4滴丁酯处理会对土壤动物群落造成一定影响,但影响程度并不大。     

麻疯树胚乳愈伤组织诱导及其污染消除

经过比较分析各种消毒方法,获得了较为理想的麻疯树(Jatrophacurcas)胚乳愈伤组织,并动态观察其类型、形态特征、增殖速率和形态发生能力,为麻疯树胚乳植株再生及愈伤组织和细胞培养生产药用成分奠定了基础.以麻疯树成熟胚乳为外植体进行愈伤组织的诱导,测试0.4mg/L的分裂素(BA、KT与TDZ)与2.0mg/L的生长素(IAA、IBA、NAA与2,4D)搭配组合的诱导效果.结果表明,对胚乳愈伤诱导效果而言,2.0mg/L的2,4D效果最好,NAA的效果次之,IBA又次之,IAA的效果最差.BA和KT诱导效果差异不显著,TDZ的添加可促进胚乳愈伤组织的诱导.选择适宜的消毒剂(种类、浓度)和消毒(处理)时间对胚乳外植体的接种成功具有至关重要的作用.在本试验所采取的灭菌方式中,用70%酒精处理30s,然后用10%NaClO处理25min,灭菌效果最佳,同时对胚乳外植体的生长影响最小.图3表1参23     

A pulsed switching peroxi-coagulation process to control hydroxyl radical production and to enhance 2,4-Dichlorophenoxyacetic acid degradation

• A new pulsed switching peroxi-coagulation (PSPC) system was developed. • The EC_T for 2,4-D removal in the PSPC was lower than that in the EF. • The iron consumption for 2,4-D removal in the PSPC was lower than that in the PC. The aim of this study was to develop a new pulsed switching peroxi-coagulation system to control hydroxyl radical (?OH) production and to enhance 2,4-Dichlorophenoxyacetic acid (2,4-D) degradation. The system was constructed with a sacrifice iron anode, a Pt anode, and a gas diffusion cathode. Production of H₂O₂ and Fe²⁺ was controlled separately by time delayers with different pulsed switching frequencies. Under current densities of 5.0 mA/cm² (H₂O₂) and 0.5 mA/cm² (Fe²⁺), the ?OH production was optimized with the pulsed switching frequency of 1.0 s (H₂O₂):0.3 s (Fe²⁺) and the ratio of H₂O₂ to Fe²⁺ molar concentrations of 6.6. Under the optimal condition, 2,4-D with an initial concentration of 500 mg/L was completely removed in the system within 240 min. The energy consumption for the 2,4-D removal in the system was much lower than that in the electro-Fenton process (68±6 vs. 136±10 kWh/kg TOC). The iron consumption in the system was ~20 times as low as that in the peroxi-coagulation process (196±20 vs. 3940±400 mg/L) within 240 min. The system should be a promising peroxi-coagulation method for organic pollutants removal in wastewater.     

麻黄愈伤组织细胞的悬浮培养

用中麻黄幼苗的３种外植体进行了离体培养。不同外植体诱导愈伤组织的结果表明：最佳培养基为ＭＳ＋２ｍｇ／Ｌ２，４－Ｄ＋１ｍｇ／Ｌ６－ＢＡ；最佳外植体为下胚轴；用源于下胚轴的愈伤组织进行悬浮培养，从第３代开始建立起稳定的悬浮系。愈伤组织、悬浮培养细胞及培养液中都含有麻黄碱，悬浮培养液中的麻黄碱含量高于愈伤组织和悬浮培养细胞。表２参１２     

Gene expression profiling of human liver carcinoma (HepG2) cells exposed to the marine toxin okadaic acid

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning. In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration-dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.     

没食子酸丙酯对K562细胞的增殖抑制和诱导凋亡作用

观察没食子酸丙酯(Propyl gallate,PG)对人慢性髓系白血病K562细胞株的增殖抑制及诱导凋亡作用.采用倒置显微镜观察细胞形态及数目的变化,乳酸脱氢酶(Lactate dehydrogenase,LDH)释放检测细胞活性,流式细胞术PI染色法及DNA倍体分析检测细胞凋亡作用.结果表明,没食子酸丙酯能有效地抑制K562细胞增殖,药物处理24 h时细胞形态发生变化,细胞数目减少;细胞培养液中LDH活性分析显示,20~300μg/mL没食子酸丙酯处理24 h对细胞毒性作用不明显;但处理时间达48 h时,没食子酸丙酯对细胞毒性明显增加;亚二倍体峰(凋亡峰)的出现及DNA片段化分析表明,200μg/mL PG处理细胞24 h时,能引起K562细胞凋亡.结果表明,PG具有明显的体外抗肿瘤活性,其抗癌活性与其抑制细胞增殖、诱导细胞凋亡有关.图5参18     

硝基甲苯对小鼠睾丸生殖细胞DNA的损伤作用

为提供硝基甲苯的环境遗传毒理学依据和建立鱼类生殖细胞的培养方法,采用昆明小鼠睾丸支持细胞/生殖细胞共培养法以及彗星实验,研究了2,4-二硝基甲苯(2,4-DNT)、2,6-二硝基甲苯(2,6-DNT)、对硝基甲苯(4-NT)对小鼠睾丸生殖细胞DNA的损伤作用.结果表明,3种受试硝基甲苯化合物均能够诱导小鼠睾丸生殖细胞DNA单链断裂,而且其受损率与剂量对数具有明显的剂量-效应关系.2,4-DNT、2,6-DNT以及4-NT的各个剂量浓度引起细胞DNA损伤的程度,与对照组相比,均具有显着性差异(p<0.01,p<0.05).受试化合物的毒性顺序为2,6-DNT>2,4-DNT>4-NT,DNA的损伤作用二硝基甲苯大于单硝基甲苯.提示在体外条件下,2,4-DNT、2,6-DNT和4-NT具有生殖毒性,可以引起小鼠睾丸生殖细胞DNA损伤.     

Nuclear events during early development in gametophytes of Macrocystis pyrifera,and the temporal effects of a marine contaminant

The time course of DNA synthesis in developing haploid gametophytes of the giant kelp Macrocystis pyrifera was determined, and the effects of arsenic (As) on the temporally distinct nuclear events, DNA synthesis and subsequent nuclear division/translocation, were investigated to establish which of these specific events may be disrupted by this contaminant. Experiments were carried out on material collected from kelp beds near Santa Barbara, California from 1993–1994. Timing of DNA synthesis was determined during development by use of the fluorochrome, DAPI (4,6-diamidino-2-phenylindole), and single-cell microspectrofluorometry. Zoospores, which result from meiosis, had already undergone two rounds of DNA synthesis at the time of release. The developing gametophytes underwent an additional two rounds of replication of DNA by 16 h of development, and following the first nuclear division/translocation, the gametophyte contained eight times the minimum DNA level throughout subsequent development. Both DNA synthesis and nuclear division/translocation, were found to be inhibited by As. Phosphate enrichment reduced the inhibitory effects of As on division/translocation of the nucleus, supporting the hypothesis that As interferes with phosphorylation. Gametophytes were more severely affected by As under light conditions, as opposed to dark, suggesting that photosynthesis may be more sensitive than dark metabolism.