微囊藻毒素导致鲫鱼淋巴细胞凋亡的研究

采用离体细胞培养诱导方法,以鲫鱼(Garassiusauratus)为实验材料,研究了两种微囊藻毒素microcystinLR(MCLR)和microcystinRR(MCRR)在低浓度下(1nmol·L-1,5nmol·L-1和10nmol·L-1)对鲫鱼淋巴细胞的毒性效应.结果表明,鲫鱼淋巴细胞分别经两种微囊藻毒素体外诱导2h后,出现细胞核固缩的典型细胞凋亡形态学特征;琼脂糖凝胶电泳检测到明显的细胞凋亡的生物化学特征梯状DNA(DNAladder);并且两种藻毒素诱导的细胞凋亡呈时间和剂量效应关系.该研究表明微囊藻毒素可导致鱼类淋巴细胞产生凋亡,因而可能影响鱼类免疫功能.     

Caspase-3在微囊藻毒素诱导的细胞凋亡中的作用

在已证实微囊藻毒素LR(MCLR)可以诱导大鼠肾细胞凋亡的基础上,采用荧光染料FAM标记的特异性的caspase-3的抑制剂来检测活细胞中caspase-3的酶活性,探讨caspase-3在MCLR诱导的细胞凋亡中所起的作用.结果表明,MCLR可以诱导caspase-3活性的增强.因此推测caspase-3在MCLR诱导的细胞凋亡中可能起重要作用.     

微囊藻毒素LR对BRL-3A凋亡相关蛋白表达的影响

用蛋白印迹法研究了微囊藻毒素LR(MCLR)暴露时大鼠BRL-3A细胞p53、Bcl-2和Bax的表达情况.结果表明,与对照组相比,各实验组p53和Bax表达均明显升高.除了10nmol/L LR暴露1h实验组外,其它实验组Bcl-2表达均下降.在低浓度组(10nmol/L),随暴露时间的延长,p53和Bax表达逐渐升高,Bcl-2表达逐渐下降,而在中浓度(100nmol/L)和高浓度组(1000nmol/L),蛋白表达与暴露时间的一致性没有低浓度组(10nmol/L)明显.表明p53、Bcl-2和Bax在MCLR诱导的细胞凋亡中可能起重要作用.     

微囊藻毒素毒理学研究综述

微囊藻毒素常见于富营养化的水体中，结构是单环多肽链，其中均含有特殊的必要需活性氨基酸基团。ＭＣＹＳＴ的化学性质稳定，但可被生物降解和ＵＶ光解。利福平等化学保护剂可预防其毒作用。该文就ＭＣＹＳＴ的理化性质，暴露剂量，毒作用及机理，预防等各方面的研究，作一综述。     

微囊藻毒素研究进展

蓝藻水华使水的感官性状恶化，水体自净能力降低，其中的蓝绿藻会产生对健康有潜在威胁的微囊藻毒素，是淡水水体中危害最大的一类。就国内外近年来对微囊藻毒素的毒理、危害和流行病学等方面的研究作一综述。     

程海微囊藻毒素的结构分析

从中国云南程海湖获得的“水华”藻类样品中分离得到具有肝毒性的微囊藻毒素，用ＮＭＲ，ＭＳ等多种结构分析手段鉴定，证明主毒素是一种环状七肽，含有Ａｄｄａ，β－Ｍａｓｐ，Ｍｄｈａ和Ｇｌｕ各一分子以及２个Ａｒｇ残基。相对分子质量１０３８即为Ｍｉｃｒｏｃｙｓｔｉｎ－ＲＲ。进一步利用二维核磁共振技术测定发现此毒素有两部分，它们是同分异构体，区别仅在于Ａｄｄａ上的２个双键的构型差异。     

微囊藻毒素的提取和提纯研究

对采自云南滇池水华蓝藻细胞中的微囊藻毒素 (Microcystins,MCs)的提取与提纯方法进行了研究 .结果表明 ,在藻细胞干重浓度为 2 0g·L-1下 ,与不同甲醇浓度提取液相比 ,4 0 %甲醇溶液可以最有效地从藻细胞中提取出MC RR和MC LR .将MCs提取液过Waters固相萃取小柱后 ,用 70 %甲醇溶液洗脱吸附于柱上的MCs ,可以分别获得 7 3%和 3 5 %纯度的MC RR和MC LR .通过观察洗脱液的颜色变化 ,收集蓝绿色和橘黄色后面流出的基本无色的洗脱液 ,可以获得纯度为 2 8 6 %和12 9%的MC RR和MC LR .     

微囊藻毒素-RR的臭氧降解研究

采用臭氧氧化的方法对微囊藻毒素-RR(MC-RR)进行降解.结果表明,在O3∶MC-RR(物质量比)为6的条件下,MC-RR的去除率最高可达到83.0%;pH上升、水中NOM含量增加都能显著降低MC-RR的臭氧降解效果.使用HPLC-MS考察了MC-RR的臭氧降解产物,并在此基础上探讨了MC-RR的臭氧氧化降解途径:主要通过Adda途径和Mdha途径来完成对MC-RR的降解和脱毒作用.臭氧氧化的Adda途径是通过对MC-RR上Adda侧链的进攻,断开具有活性的Adda支链,而达到脱毒的目的,其中Adda途径过程中的苯环羟基化作用对整个过程有促进作用;臭氧氧化的Mdha途径是通过对MC-RR肽环上面Mdha和Ala的断键,打开环状肽链,使藻毒素失去活性.在整个过程中Adda途径占主导地位.     

低剂量微囊藻毒素亚急性肝毒性及其机理

为了研究低剂量微囊藻毒素 (MC)的亚急性肝毒性及其作用机理 ,将SD大鼠随机分为 4组 ,每组雌雄各 1 0只 ,分别经腹腔注射MC 0 ,4,8,1 2 μg·(kg·d) - 1,3 5天后观察各组大鼠血清酶学和肝脏病理学变化 .用原位末端标记法 (TUNEL)观察肝细胞凋亡 ,并用ABC免疫组化技术检测肝脏增殖细胞核抗原 (PCNA) .结果表明染毒组大鼠血清γ 谷氨酰转移酶 (GGT)活力和全血谷胱甘肽 (GSH)浓度下降 ,血清乳酸脱氢酶 (LDH)和谷草转氨酶(AST)升高 ,谷丙转氨酶 (ALT)未见显著变化 .染毒组大鼠肝脏出现特征性的形态学变化 ,并有活跃的肝细胞凋亡和增生并存的现象 .说明微囊藻毒素引起的氧化损伤和肝细胞凋亡可能是其致肝脏毒性的原因     

微囊藻毒素-LR致肝细胞连接通讯下调的研究

不同剂量(0, 10, 50,100, 200, 500, 1000nmol/L)的微囊藻毒素-LR作用于肝细胞株BRL-3A 24 h.采用激光扫描共焦显微镜,按荧光光漂白后再恢复技术测定BRL-3A的间隙连接细胞间通讯功能,四唑盐法测定BRL-3A细胞增殖,同时分析胆汁酸运输系统抑制剂利福平对两者的阻滞作用.结果表明,微囊藻毒素-LR可以显著地抑制BRL-3A细胞GJIC功能,并促进BRL-3A细胞增殖,两者均呈剂量效应关系.利福平(30靘ol/L)对微囊藻毒素-LR诱导的BRL-3A细胞GJIC功能下调和增殖有明显的抑制作用.微囊藻毒素-LR可诱导BRL-3A细胞膜泡形成.说明微囊藻毒素-LR肝脏毒性及致癌性可能与抑制肝细胞GJIC功能有关.     

天津野生鲫鱼卵黄蛋白原的研究

用HPLC方法提纯经雌二醇诱导的鲫鱼卵黄蛋白原,用SDS-PAGE凝胶电泳测定其分子量为160Kda左右.在建立卵黄蛋白原(VTG)的HPLC分析方法(检测下限为0.025mg/mL)的基础上,对北京排污河天津段的野生和养殖鲫鱼血浆中的VTG进行检测.在所捕获的12尾野生鲫鱼中(其中2尾为雄性鲫鱼),都检测出了VTG,浓度范围为0.284～5.971mg/mL;对同期捕获的35尾天津养殖鲫鱼包括1尾雄性鲫鱼中的VTG也进行了检测,浓度范围为0.119～0.250mg/mL.而同期在实验室饲养的雌雄鲫鱼血浆中,均未检测出VTG.结果表明,北京排污河(天津段)的野生鲫鱼受到类雌激素物质的污染.     

Oxidative stress and apoptosis of Carassius auratus lymphocytes induced by nonplanar （PCB153） and coplanar （PCB169） polychlorinated biphenyl congeners in vitro

Among all the 209 kinds of polychlorinated biphenyls (PCBs) congeners, nonplanar and coplanar PCB congeners have di erent levels of toxicity on mammal cells such as neuronal cells, but little is known about their toxicity on fish cells although PCB congeners usually have high bioaccumulation abilities in the detected fish bodies. This study showed that 2,2’,4,4’,5,5’-hexacholorbiphenyl (PCB153, nonplanar congener) and 3,3’,4,4’,5,5’-hexacholorbiphenyl (PCB169, coplanar congener) caused apoptosis on the isolated crucian carp (Carassius auratus) lymphocytes and the induced cytotoxicity was structure-dependent. According to the laser confocal microscope observations, apoptosis was clearly distinguished by condensation of nucleus, shrinkage and formation of apoptotic bodies. DNA fragmentation was detected by agarose gel electrophoresis. These typical morphological and biochemical characteristics indicate the occurrence of apoptosis on fish lymphocytes. According to the flow cytometry analysis, after the cells were exposed to 10 mol/L PCBs for 3 h, the apoptotic percentage induced by PCB153 was 23.41%, while that induced by PCB169 was even higher (31.03%). Furthermore, incubating PCBs with fish lymphocytes enhanced levels of reactive oxygen species (ROS) and malondialdehyde (MDA), clearly indicating the presence of oxidative stress and lipid peroxidation. Our data also demonstrate that the di erent cytotoxic e ects induced by coplanar and nonplanar PCBs were correlated with their structural characteristics and the coplanar congener was more cytotoxic than nonplanar congener. This study suggests that cytotoxicity mechanisms of the PCB congeners on fish lymphocytes depend on their planarity and chemical structures.     

滴水湖及其鲫鱼体内PAHs分布特征与影响因素分析

通过测定滴水湖水体、颗粒物和沉积物体系PAHs含量,探讨其分布与组成特征、影响因素及污染来源.结果表明,滴水湖水体中溶解态、颗粒态和沉积物中PAHs平均浓度分别为16.78ng/L、33.02ng/g和40.98ng/g.统计分析表明,水体酸碱度以及电导率与溶解态低环PAHs之间存在显著相关性,总有机碳(TOC)与沉积物中高环PAHs浓度之间存在显著相关性.溶解态的PAHs来源主要表现为草、木和煤的高温燃烧,部分样点表现为石油源;颗粒态PAHs则主要表现为高温燃烧以及石油泄漏源;而沉积物PAHs的来源则较复杂,除了草、木及煤的高温燃烧源和石油泄漏源,还有部分样点表现为石油的高温燃烧源.鲫鱼肌肉、卵部以及鳃部PAHs含量的测定结果表明,鲫鱼不同部位对PAHs的富集能力具有较大差异.鳃部总PAHs含量最高,其次为鲫肉部分,鲫卵所含PAHs浓度最少.与国内外其他研究相比较,滴水湖鲫鱼体内PAHs含量处于较低水平,但鲫鱼部分样品的BaP等当量浓度略高于EPA规定的可食性生物器官中PAHs含量的上限值.     

碘代乙酸体外暴露诱导斑马鱼(Danio rerio)淋巴细胞内源性凋亡可能机理研究

以斑马鱼(Danio rerio)为试验生物,经淋巴细胞体外暴露试验,从细胞内源性凋亡角度,研究了饮用水消毒副产物—碘代乙酸诱导斑马鱼淋巴细胞损伤的致毒机理.结果表明,在碘代乙酸的环境浓度1μg·L-1下暴露24、36、48和96 h后,细胞凋亡率从对照组的3.52%分别增加到15.89%、22.47%、40.76%和52.13%,与对照组差异均为极显著(p0.01);线粒体膜电位分别较对照组下降了32.9%、50.1%、68.6%和81.5%,与对照组差异均为极显著(p0.01);细胞色素C的相对释放量分别增加了0.85、1.37、1.86和2.66倍,与对照组差异均为极显著(p0.01).暴露36、48和96 h后,Caspase-3酶活性分别增加了0.49、0.86和1.43倍,与对照组差异均为极显著(p0.01);Caspase-9酶活性分别增加了0.73、1.41和1.88倍,与对照组差异均为极显著(p0.01);抑制凋亡Bcl-2基因的相对表达量分别下降27.0%、35.3%和52.3%,与对照组差异均为极显著(p0.01);而促进凋亡的Bax基因相对表达量分别增加1.1、2.3和3.2倍,与对照组差异均为极显著(p0.01).碘代乙酸体外诱导斑马鱼淋巴细胞凋亡的可能机制是,线粒体膜电位的崩溃导致细胞色素C持续从线粒体中释放,进而引起细胞凋亡下游通路的变化;Bcl-2基因和Bax基因明显参与了对凋亡的调控.此外,Caspase-9酶的活化导致Caspase-3酶活化,最终引起细胞凋亡.     

鞘氨醇单胞菌USTB-05对微囊藻毒素的生物降解

研究了云南滇池水华蓝藻细胞中微囊藻毒素(MCs)的分析和鞘氨醇单胞菌USTB-05在细胞水平和酶水平下对MC-YR、RR和LR的生物降解.结果表明,云南滇池水华蓝藻细胞中MC-YR、RR和LR的含量分别为0.16, 0.96, 0.47mg/g.在初始浓度分别为19.5mg/L MC-YR、79.5mg/L MC-RR和43.6mg/L MC-LR下,鞘氨醇单胞菌USTB-05在2d内可将上述3种MCs全部降解,鞘氨醇单胞菌USTB-05粗酶液可以以更快的速率对MC-YR、MC-RR和MC-LR进行高效酶催化降解,在10h内可以将初始浓度分别为14.8mg/L MC-YR、28.4mg/L MC-RR和19.5mg/L MC-LR全部降解.同时发现了MC-YR降解过程中的2个中间和1个最终代谢产物.     

久效磷农药对金鱼肝细胞DNA的损伤及其机制研究

以金鱼(Carassius auratus)为模式生物,研究了久效磷农药暴露导致的DNA损伤类型及其作用机制.结果表明:0.01,0.10,1.00mg/L的久效磷农药暴露24,48,96,168h均导致金鱼肝细胞DNA损伤程度显著升高,48h损伤最严重;暴露48h采用碱性、pH值12.1和中性彗星电泳发现DNA损伤类型主要为碱不稳定位点形成,其次为单/双链断裂;采用Endo Ⅲ酶和FPG酶处理的碱性彗星电泳,发现DNA出现氧化损伤;暴露24h肝脏谷胱甘肽过氧化物酶(GSH-Px)活性显著降低,丙二醛(MDA)含量显著升高达到峰值,96~168h超氧化物歧化酶(SOD)和GSH-Px活性显著升高,MDA含量与24h相比有所降低,表明活性氧自由基(ROS)含量在短期暴露升高(24h)、在96~168h暴露逐渐降低.影响抗氧化酶活性、干扰ROS清除过程是久效磷农药造成DNA损伤的主要机制.     

The dynamic changes of arsenic biotransformation and bioaccumulation in muscle of freshwater food fish crucian carp during chronic dietborne exposure

Dietary uptake is the major way that inorganic arsenic (iAs) enters into benthic fish; however, the metabolic process of dietborne iAs in fish muscle following chronic exposure remains unclear. This was a 40-day study on chronic dietborne iAs [arsenite (AsIII) and arsenate (AsV)] exposure in the benthic freshwater food fish, the crucian carp (Carassius auratus), which determined the temporal profiles of iAs metabolism and toxicokinetics during exposure. We found that an adaptive response occurred in the fish body after iAs dietary exposure, which was associated with decreased As accumulation and increased As transformation into a non-toxic As form (arsenobetaine). The bioavailability of dietary AsIII was lower than that of AsV, probably because AsIII has a lower ability to pass through fish tissues. Dietary AsV exhibited a high potential for transformation into AsIII species, which then accumulated in fish muscle. The largely produced AsIII considered more toxic at the earlier stage of AsV exposure should attract sufficient attention to human exposure assessment. Therefore, the pristine As species and exposure duration had significant effects on As bioaccumulation and biotransformation in fish. The behavior determined for dietborne arsenic in food fish is crucial for not only arsenic ecotoxicology but also food safety.     

Body mass dependence of glycogen stores in the anoxia-tolerant crucian carp (<Emphasis Type="Italic">Carassius carassius</Emphasis> L.)

Glycogen is a vital energy substrate for anaerobic organisms, and the size of glycogen stores can be a limiting factor for anoxia tolerance of animals. To this end, glycogen stores in 12 different tissues of the crucian carp (Carassius carassius L.), an anoxia-tolerant fish species, were examined. Glycogen content of different tissues was 2–10 times higher in winter (0.68–18.20% of tissue wet weight) than in summer (0.12–4.23%). In scale, bone and brain glycogen stores were strongly dependent on body mass (range between 0.6 and 785 g), small fish having significantly more glycogen than large fish (p < 0.05). In fin and skin, size dependence was evident in winter, but not in summer, while in other tissues (ventricle, atrium, intestine, liver, muscle, and spleen), no size dependence was found. The liver was much bigger in small than large fish (p < 0.001), and there was a prominent enlargement of the liver in winter irrespective of fish size. As a consequence, the whole body glycogen reserves, measured as a sum of glycogen from different tissues, varied from 6.1% of the body mass in the 1-g fish to 2.0% in the 800-g fish. Since anaerobic metabolic rate scales down with body size, the whole body glycogen reserves could provide energy for approximately 79 and 88 days of anoxia in small and large fish, respectively. There was, however, a drastic difference in tissue distribution of glycogen between large and small fish: in the small fish, the liver was the major glycogen store (68% of the stores), while in the large fish, the white myotomal muscle was the principal deposit of glycogen (57%). Since muscle glycogen is considered to be unavailable for blood glucose regulation, its usefulness in anoxia tolerance of the large crucian carp might be limited, although not excluded. Therefore, mobilization of muscle glycogen under anoxia needs to be rigorously tested.