微囊藻毒素-LR致小鼠肝细胞DNA-蛋白质交联作用的研究

为探讨微囊藻毒素-LR致小鼠肝细胞的DNA-蛋白质交联作用,将20只昆明雄性小鼠随机分为4组:1个对照组和3个染毒组,采用腹腔注射进行染毒7d,染毒剂量分别为3.0、6.0和12.0μg·kg-1,检测小鼠肝细胞DNA-蛋白质交联程度.结果显示,3.0、6.0和12.0μg·kg-1微囊藻毒素-LR均可导致小鼠肝细胞显著的DNA-蛋白质交联作用(与对照组相比,p<0.01,p<0.01,p<0.05),当微囊藻毒素为6.0μg·kg-1时,这种作用最明显.     

DNA-Protein Crosslinks Induced by Microcystin-LR in Hepatic Cells of Mice

为探讨微囊藻毒素-LR致小鼠肝细胞的DNA-蛋白质交联作用,将20只昆明雄性小鼠随机分为4组：1个对照组和3个染毒组,采用腹腔注射进行染毒7d,染毒剂量分别为3.0、6.0和12.0μg·kg-1,检测小鼠肝细胞DNA-蛋白质交联程度. 结果显示,3.0、6.0和12.0μg·kg-1微囊藻毒素-LR均可导致小鼠肝细胞显著的DNA-蛋白质交联作用（与对照组相比,p<0.01,p<0.01,p<0.05）,当微囊藻毒素为6.0μg·kg-1时,这种作用最明显.     

己酸二乙氨基乙醇酯的致突变性研究

采用鼠伤寒沙门氏菌回复突变试验(Ames试验),用TA97,TA98,TA100和TA102菌株,加与不加S9,剂量分别设为每皿1000μg,500μg和250μg;小鼠骨髓多染红细胞微核试验,剂量设为雄性、雌性小鼠给药剂量均为250,500和1000mg*kg-1;小鼠睾丸精母细胞染色体畸变试验,剂量设为625mg*kg-1,1250mg*kg-1和2500mg*kg-1.结果表明,Ames试验中,各测试浓度的诱发回变菌落数均未超过自发回变菌落数的2倍;小鼠骨髓多染红细胞微核试验和小鼠睾丸精母细胞染色体畸变试验,各剂量组和溶剂对照组的微核率进行统计学处理,未见有显着性差异(P＞0.05),表明各项试验结果均为阴性.因此,己酸二乙氨基乙醇酯无致突变作用.     

气相色谱质谱法测定纺织品中N,N-二甲基甲酰胺、N,N-二甲基乙酰胺和N-甲基吡咯烷酮溶剂残留

建立了气相色谱质谱法测定纺织品中N,N-二甲基甲酰胺(DMF)、N,N-二甲基乙酰胺(DMAC)和N-甲基吡咯烷酮(NMP)的方法.纺织品中N,N-二甲基甲酰胺、N,N-二甲基乙酰胺和N-甲基吡咯烷酮经丙酮超声萃取,用0.45μm滤膜过滤后直接进样,将提取物进行GC-MS定性定量分析.结果表明,在0.1—10μg·mL-1线性范围内,DMF、DMAC和NMP线性方程分别是y=75041x+8039,y=71622x+12371,y=96278x+8548,相关系数均大于0.9995;方法检出限DMF和DMAC为5 mg·kg-1,NMP为3 mg·kg-1;平均回收率为86.3%—92.0%;相对标准偏差(n=6)为1.4%—5.0%.该方法简便、快速、灵敏度高、重复性好,可用于测定纺织品中N,N-二甲基甲酰胺、N,N-二甲基乙酰胺和N-甲基吡咯烷酮.     

鸡肾脏对微囊藻毒素-LR氧化胁迫的响应

天然蓝藻中蛋白质含量高达40%,与大豆的蛋白质含量相当,是优质饲料蛋白的潜在来源。然而,天然蓝藻中主要含有一种称为微囊藻毒素(Microcystins,MCs)的生物毒素,对动物和人体的健康具有潜在威胁,且关于MCs对家禽氧化胁迫的研究几乎是一片空白。因此,急需开展蓝藻饲料化利用对家禽氧化胁迫的研究,为蓝藻饲料化利用提供理论依据。该文通过腹腔注射的方式研究了崇仁麻鸡肾脏对微囊藻毒素-LR(MC-LR)氧化胁迫的响应。试验设置了5μg·kg~(-1) MC-LR(低剂量组)、10μg·kg~(-1) MC-LR(中剂量组)和20μg·kg~(-1) MC-LR(高剂量组)3个染毒组,同时腹腔注射生理盐水作为对照组。在腹腔注射后的1、3、12、24、48 h检测了鸡肾脏中谷胱甘肽(GSH)含量以及谷胱甘肽转移酶(GST)、谷胱甘肽过氧化物酶(GPX)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性变化以及肾体比变化。结果表明:在MC-LR的作用下,鸡肾脏抗氧化酶(GST、SOD、CAT和GPX)和GSH含量均存在不同程度的变化,但除了3 h时中和高剂量染毒组肾脏中CAT酶活性(值分别为(96.66±1.13)和(74.01±11.20)U·mg~(-1))显著低于对照组(123.58±11.33)U·mg~(-1)(t=4.253;P=0.024和t=4.432;P=0.022)外,其它染毒组的各类抗氧化酶和GSH指标与对照组之间均无显著性差异(P0.05);鸡肾脏系数从1h就开始呈现毒素处理组高于对照组,并在12、24和48 h时,毒素处理组肾脏系数(0.79%-0.87%)显著高于对照组(0.58%-0.70%)的趋势(t=-6.687--3.495;P=0.018-0.036)。上述结果表明,在MC-LR作用下,鸡肾脏产生了氧化应激现象,但是其抗氧化酶系统可能难以抵御MC-LR危害,MC-LR对鸡肾脏的毒性较大。     

氯化二丁基锡对小鼠外周血液中睾丸酮和雌二醇含量的影响

用放射免疫法分别测定了DBTCl对妊娠d6雌小鼠和成年雄小鼠外周血液中雌二醇和睾丸酮水平的影响.每天按小鼠体重一次腹腔注射0.025～0.400μg/kgd-1DBTCl,共染毒7d.实验条件为(22±2)℃和光暗=12h12h.结果表明,处理组睾丸酮和雌二醇的含量随处理剂量的增加而增加,当w（DBTCl）≥0.05μg/kg时,二者呈明显的剂量-效应关系.雌二醇含量的增加比睾丸酮更为显著CK雄性小鼠睾丸酮浓度为（2.88±0.72）ng/mL,在0.40μg/kg剂量组达到(9.95±2.5)μg/mL时,约为CK的3.5倍;而CK和0.40ng/kg剂量组妊娠小鼠雌二醇浓度分别为(30.32±5.43)ng/mL和(287.57±51.13)ng/mL,相差约9.5倍.文中还就DBTCl对小鼠血液中激素含量的影响机理进行了初步探讨.图1表2参20     

大田软海绵酸对黑鲷胚胎发育及仔鱼的急性毒性

为了研究大田软海绵酸(okadaic acid,OA)对生物胚胎组织发育过程的毒性作用机制,采用静水和半静水实验法,用OA毒素与过滤海水按等对数间距分设5个浓度组,同时以过滤海水为对照组,对人工养殖的黑鲷鱼卵和仔鱼进行了毒性实验。实验结果表明:一定浓度的OA使鱼卵的死亡率和孵出仔鱼的畸形率升高,但对孵化率的影响较弱。OA对黑鲷仔鱼的24、48、72和96h的LC50分别为51.63、45.58、37.97和25.72nmol·L-1,仔鱼体内毒性蓄积程度也随时间逐渐减弱。OA毒素对仔鱼的谷胱甘肽过氧化物酶(GSH-PX)和酸性磷酸酶(ACP)的活性均有诱导作用,与对ACP的影响相比,OA毒素对GSH-PX活性的诱导作用更为突出。此外,OA毒素还会对黑鲷仔鱼的肝脏细胞的核酸代谢及DNA合成有一定的影响,使得RNA和DNA的含量比值发生显著变化。     

广西涠洲岛底栖蛎甲藻(Ostreopsis sp.)的产毒特性研究

蛎甲藻属(Ostreopsis Schmidt)部分种类可以产生剧毒的海葵毒素(Palytoxins,PLTXs),对海洋生态环境和人类健康构成严重威胁,而国内迄今尚无关于蛎甲藻毒性的研究报道,因此对采自广西涠洲岛同一种蛎甲藻的两个株系(WZD110、WZD111)进行了产毒特性研究。在小鼠生物毒性实验中,腹腔注射两株藻的毒素粗提取物后,小鼠均出现后腰凹陷、运动减少和抽搐等症状,并于短时间内死亡,呈现明显的PLTXs中毒特征。兔血细胞溶血活性实验结果表明,毒素粗提取物具有延迟溶血的效果,且在加入PLTXs特异抑制剂乌本苷后,延迟溶血活性被抑制。液相色谱-质谱联用毒素定性定量分析结果显示,WZD110株单位细胞PLTXs质量为0.348 pg·cell~(-1),WZD111株单位细胞PLTXs质量为0.081 pg·cell~(-1),两株系均含有较高浓度的OVTX-c和OVTX-d/e,以及少量的OVTX-g和OVTX-f。文章关于蛎甲藻胞内毒素的报道在中国海域的此类研究尚属首次,可为正确认识和评价蛎甲藻赤潮产生的危害提供参考依据。     

微囊藻毒素-LR通过线粒体途径诱导人支气管上皮细胞凋亡

探讨线粒体Caspase依赖性途径是否参与微囊藻毒素-LR(microcystin-LR,MC-LR)诱导人支气管上皮细胞(human bronchial epithelial cells,16HBE)凋亡过程。将处于对数生长期的16HBE分别暴露于终浓度为0(对照组)、2.5、5、10μg·m L-1的微囊藻毒素-LR和10μg·m L-1MC-LR+50μmol·L-1Caspase广谱抑制剂Z-VAD-FMK,持续24 h和48 h。检测细胞凋亡率,线粒体跨膜电位(ΔΨm),Caspase-3和Caspase-9相对表达量。结果显示,与对照组相比,各浓度染毒组细胞凋亡率和Caspase-3、Caspase-9相对表达量均升高,10μg·m L-1MC-LR染毒组线粒体膜电位降低;与10μg·m L-1MC-LR组相比,10μg·m L-1MCLR+50μmol·L-1Z-VAD-FMK组细胞凋亡率明显降低,Caspase-3和Caspase-9相对表达量降低,差异均有统计学意义(P0.05)。且随着MC-LR染毒浓度的升高或染毒时间的延长,16HBE细胞凋亡率和Caspase-3、Caspase-9相对表达量呈升高趋势。研究表明,MC-LR可以通过线粒体Caspase依赖性途径诱导16HBE细胞凋亡。     

天然石生苔藓汞含量及对大气汞污染的生物指示

采集贵州万山汞矿区6属7种优势种类苔藓84个样品,测定其汞含量,同时现场监测部分苔藓采样点近地表大气汞,定量分析苔藓汞和大气汞质量浓度之间的关系.结果显示,研究区苔藓汞质量浓度范围为0.96—126μg·g~(-1),平均值80±46μg·g~(-1),受触媒生产、废触媒回收等涉汞化工厂生产活动影响显著;苔藓汞与大气汞呈正相关关系,表现为线性拟合相关性系数r=0.93(n=12,P=0.01030),多项式拟合相关性系数r=0.96(n=12,P=0.01169),对数拟合相关性系数r=0.96(n=12,P=0.00469);选择对数拟合方程估算苔藓采样点大气汞浓度,发现拟合得到的大气汞空间分布特征和实测苔藓汞空间分布特征基本一致,从而通过定量/半定量方法证明了可以利用苔藓汞指示大气汞污染,为汞污染场地修复过程中的大气汞污染评价提供了新思路.     

Effects of a phycotoxin,okadaic acid,on oyster heart cell survival

Okadaic acid (OA) is a dinoflagellate toxin which accumulates in shellfish producing diarrhetic shellfish poisoning (DSP) in humans. It was found that OA is a highly selective inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A) which produces a marked increase in phosphorylation of several proteins, including p38 mitogen-activated protein (MAP) kinase. The cytotoxicity attributed to OA and the effects on p38 MAP kinase and calcium current were examined in the oyster Crassostrea gigas in this study. Data showed that p38 MAP kinase is strongly expressed in oyster heart and that OA bioaccumulated in cultured heart cells. Hence the effects of OA was tested in vitro and in vivo on oysters. OA was found to (i) exert a positive chronotropic effect on cultured atrial cardiomyocytes which is related to an increase in calcium current via PKC as shown by patch clamp measurements, (ii) produce an activation/phosphorylation of MAP kinase as shown by Westernblot while the non-phosphorylated p38 remained constant during treatment, (iii) did not induce a pro-apoptotic effect. Data suggest that OA may also stimulate the anti-apoptotic pathway by phosphatase inhibition.     

The effects of the diarrhetic shellfish poisoning toxins,okadaic acid and dinophysistoxin-1, on the growth of microalgae

The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are potent phosphatase inhibitors produced by certain species of marine dinoflagellates. OA can cause hyperphosphorylation of a broad range of animal and higherpalnt proteins, but little is known regarding the effects of the DSP toxins on marine organisms or their biological function. A variety of microalgae, including a clone ofProrocentrum lima known to produce both OA and DTX-1, were incubated with solutions of OA and in one case DTX-1 or a combination of OA and DTX-1. OA inhibited the growth of all non-DSP-producing test species at micromolar concentrations, butP. lima was not affected even at much higher levels. This differential activity of OA suggests that the DSP toxins may play an allelopathic role and raises questions regarding the strategies adopted by DSP-producing dinoflagellates such asP. lima to avoid autotoxicity. The effects of DTX-1 on microalgal growth were found to be equivalentt to those of OA, and the effects of both toxins in combination were simply additive.     

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.     

海洋底栖甲藻——利玛原甲藻（Prorocentrum lima）产毒特征的研究（Studies on Toxin Production of Prorocentrum lima）

为了解海洋底栖甲藻——利玛原甲藻(Prorocentrum lima)的产毒特征,阐明腹泻性贝毒(Diarrhetic shellfishpoisoning,DSP)复杂的生物合成机制,采用酶联免疫试剂盒,对利玛原甲藻不同生长期、不同营养盐条件下毒素生成情况进行了研究.结果显示,不同生长期单个利玛原甲藻细胞中大田软海绵酸(Okadaic acid,OA)的含量明显不同,平台期时OA含量最高,与对数中期存在显著差异(p<0.05).低N、P条件下藻的生长状况较差,所能到达的藻密度明显低于对照组(f/2培养基);藻细胞中OA的总含量也低于对照组,但单个藻细胞中OA的含量明显高于对照组(p<0.05).以上结果提示,利玛原甲藻DSP毒素的产生可能与藻所处的环境和藻的生理状态等有关.     

采用联用技术测定中国海产品中砷的形态

1　IntroductionAtthebeginningof2 0 thcentury ,highlevelsofarsenic～mg·kg-1 werefoundinmarineorga nisms,whichattractedalotofattention ,becausearsenicisoneofthemostnotoriouselementsonaccountofitshumantoxicity.Forthisreason ,thedeterminationofthetotalamount…     

双酚A对中国林蛙蝌蚪生长发育的毒性效应

为评价双酚A(BPA)对中国林蛙(Rana chensinensis)蝌蚪的急性毒性,将26期的蝌蚪暴露于浓度为2.4×10-5mol·L-1～4.2×10-5mol·L-1BPA的水体中进行急性毒性实验.结果表明,24、48、72、96h蝌蚪的死亡几率(y)与浓度对数(x)的回归方程分别为y=16.915x-4.1157、y=22.11x-6.1905、y=20.766x-5.3871、y=20.715x-5.351;半数致死浓度(LC50)分别为3.47×10-5、3.28×10-5、3.20×10-5、3.16×10-5mol·L-1.安全浓度(SC)为0.88×10-5mol·L-1.为探讨在安全浓度以下BPA对中国林蛙蝌蚪生长发育的影响,将林蛙蝌蚪分别于10-5、10-6、10-7mol·L-1BPA的水体中连续暴露直至完全变态,并设10-8、10-9mol·L-1雌二醇(E2)的阳性对照和空白对照,分别测定并统计蝌蚪发育所需的发育时间、体长和体质量.结果表明,蝌蚪对10-5、10-6、10-7mol·L-1BPA与10-8、10-9mol·L-1E2的效应相似,可延缓林蛙幼体的发育时间,导致体长和体质量降低.推测低浓度BPA抑制蝌蚪生长发育的机制之一是干扰了正常的内分泌活动.     

New ecological and ultrastructural data on the dinoflagellate Dinophysis sp. from the French coast

In situ phytoplankton sampling over 24h was carried out in July 1985 during part of a monitoring program established by the Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER) to survey the blooming of toxic dinoflagellates off the French coast. Analysis of the data suggested a vertical migration of Dinophysis sp. (acuminata?) populations through the sea-water column. These diurnal displacements should be taken into account in the course of monitoring surveys of diarrhetic shellfish poisoning (DSP). Culture assays which were performed in parallel remained unsuccessful. However, Dinophysis sp. survived in an enriched sea-water medium for 4 to 5 wk under low light intensity (8 to 10 W m^-2) and constant temperature (16° to 18 °C). Cell ultrastructure, in pariticular the organization of chloroplasts, is described. All the combined results tend to confirm the photosynthetic capacity of this species.     

Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima

Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 ± 1 °C, under a photon flux density (PFD) of 90 ± 5 μmol m⁻² s⁻¹ on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by “midnight”. Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell⁻¹, respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH₄ was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO₃, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h). Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase (“morning” of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases (“afternoon”). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events. Received: 3 September 1998 / Accepted: 30 March 1999     

Phytoplankton selection by mussels, and diarrhetic shellfish poisoning

To better understand the dynamics of diarrhetic shellfish poisoning (DSP) contamination a field study was carried out on the feeding behavior of Mytilus galloprovincialis (Lmk) during an important DSP outbreak. The study was focused on the relationships between phytoplankton in seawater and algal cells, or their remnants, in mussel stomachs. During the period studied, M. galloprovincialis seemed to feed selectively on dinoflagellates rather than diatoms. Further selection was observed among different dinoflagellate genera, a preference for the genus Dinophysis being particularly evident. In addition, mussels seemed to open the thecae of Dinophysis cells and digest them more easily than other dinoflagellates. Due to the high variability of the results of phytoplankton analysis in the mussel stomachs, no correlation was found between the abundance of Dinophysis species in the mussels' stomachs and the content of okadaic acid plus dinophysistoxin-1 in their digestive glands, evaluated with an ELISA assay. Conversely, the presence of Dinophysis fortii (the main DSP-causative agent in the area studied) in integrated net samples of the whole water column and the toxin content of the digestive glands presented similar temporal trends. Received: 24 March 1997 / Accepted: 10 December 1997     

Untersuchungen über die heterotrophe Aktivität in der zentralen Ostsee

The distribution of some microbial parameters was studied at 3 stations in the Central Baltic Sea (Bornholm Basin, BB; Danzig Deep, DD; and Gotland Deep, GD) during May 1976. The following analyses were performed: total bacterial numbers and biomass, viable counts and maximum uptake velocity (V _max) of glucose. The values found for thesurface, samples were, total bacterial counts: 0.6x10⁶ (BB); 1.7x10⁶ (DD); 0.4x10⁶ (GD) cells/ml; bacterial biomass: 1.9 g C/l (BB); 6.9 g C/l (DD); 1.6 g C/l (GD); viable counts: 0.37x10³ (BB); 17x10³ (DD); 0.4x10³ (GD) counts/ml; V _max: 0.01 g glucose-C/l (BB), 0.06 g glucose-C/l (DD); 0.01 g glucose-C/l (GD). The relatively high microbial numbers and activities of the Danzig Deep may be associated with the fertilization of the area by the River Vistula. The vertical distribution of the microbial parameters in the Bornholm Basin and Danzig Deep showed high values both in the top layer (0 to 20 m) and in the deep layer (>40 m). In the intermediate layer, however, the values decreased significantly. It is suggested that the high values of the microbial parameters at depth are caused by at least two major processes during the inflow of North Sea water into the deep layers of the Baltic Sea: (1) the North Sea water may already have contained high numbers of bacteria; (2) during the inflow, the high concentrations of bacteria normally located at the sediment-water interface are distributed throughout the whole deep layer by mixing.

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Beitrag Nr. 143 aus dem Sonderforschungsbereich 95 Wechselwirkung Meer-Meeresboden, Universität Kiel.