镉诱导金属硫蛋白在华溪蟹组织中的表达

采用体外暴露染毒法,研究了不同浓度与时间条件下,镉诱导河南华溪蟹(Sinopotamon henanense)金属硫蛋白(metallothionein,MT)在肝胰腺、肌肉、鳃和卵巢中的表达差异。镉浓度分别为0、14·5mg/L、29mg/L和58mg/L;处理时间依次为1d、3d和5d。利用镉血红蛋白饱和法和火焰原子吸收分光光度法(AAS)测定MT的蛋白含量。结果显示,用不同的染毒浓度和处理时间,镉在组织中诱导产生MT的含量有较大差异,其中肝胰腺MT的诱导量最大,变化规律也最明显;肌肉中也有较大量MT的表达;而鳃和卵巢MT的诱导量均较低。此外,本文分析了镉的浓度与时间梯度对诱导MT表达的影响与毒性效应机制。结论:组织不同,染毒浓度及时间不同,镉诱导MT的表达也不同,具有一定的组织差异性和规律性。     

镉离子对文蛤肝胰腺超微结构的影响

本文以文蛤(Meretrix meretrix)为实验对象,将其在不同浓度镉溶液(0、1.5 mg/L、3 mg/L、6 mg/L)中染毒5 d,并利用透射电镜技术分析文蛤肝胰腺细胞的超微结构变化。结果表明：镉对文蛤肝胰腺细胞超微结构的影响较大,主要表现在对内膜系统和线粒体的破坏上,其中线粒体嵴减少,肿胀变形,逐渐空泡化;粗面内质网由原先的板层状结构解体,形成许多大小不同的小泡。微绒毛脱落并逐渐减少,部分空泡化,高尔基体膜也有扩张的现象。膜性结构形成的空泡中出现沉积物,核膜出现内陷。镉离子破坏了文蛤肝胰腺细胞膜和主要细胞器的结构,影响了肝胰腺细胞的正常生理机能,随着镉溶液浓度的升高,对肝胰腺细胞结构的破坏性也增强。     

斧文蛤金属硫蛋白基因的克隆与表达分析

金属硫蛋白（Metallothionein,MT）是一类富含半胱氨酸的小分子蛋白质,参与机体重金属解毒、维持金属元素代谢平衡以及清除自由基等生理功能。为了解斧文蛤金属硫蛋白（Ml-MT）的分子生物学特征及其在重金属Cd2+胁迫下的响应机制,本文采用RACE技术从斧文蛤（Meretrix lamarckii）总RNA反转录产物中获得了636 bp的Ml-MT cDNA基因序列。该序列包含65 bp的5非编码区（UTR）和340 bp的3非编码区（UTR）以及231 bp的开放阅读框（ORF）,可编码76个氨基酸;其中半胱氨酸占27%,不含芳香族氨基酸,含16个MT所特有的Cys-Xn-Cys结构,预测的分子量约为7.704 kD,理论等电点7.138。MT氨基酸序列比对分析表明:斧文蛤金属硫蛋白（Ml-MT）与丽文蛤（Meretrix lusoria）的相似性高达88%,与文蛤（Meretrix meretrix）的同源性为87%。实时荧光定量（qRT-PCR）检测MT在斧文蛤5种组织中均有表达,但存在组织特异性,其中内脏团表达量最高,其次依次为鳃丝、闭壳肌、外套膜、斧足。在Cd2+（0.13 mg/L）胁迫0、6h、12h、24h、48h、72h和96h下,斧文蛤内脏团MT呈现出不同程度的上调表达,具体表现为高-低-高-低的波浪式变化,除6h以外,其他时间点均与对照组存在极显著差异（P0.01）。本研究表明:MT基因在维护机体正常生理功能及斧文蛤抵御重金属Cd2+胁迫过程中发挥着重要的分子调控作用。     

铜对梨形环棱螺抗氧化酶活性和金属硫蛋白含量的影响

本实验采用暴露重金属的方法,研究了不同浓度硫酸铜（Cu2+ 分别为0、0.005、0.01、0.02、0.05 mg/L）在不同暴露时间（0—14d）下对梨形环棱螺（Bellamya purificata）过氧化氢酶(CAT)、超氧化物歧化酶（SOD）、谷胱甘肽硫转移酶（GST）的活性、还原性谷胱甘肽（GSH）和金属硫蛋白（MT）含量的影响,以探讨Cu2+ 对梨形环棱螺的氧化损伤及其防御作用的机理,并为水环境Cu2+ 污染的早期诊断及生态风险评价提供科学的依据。结果表明：Cu2+对梨形环棱螺肝脏和鳃中CAT、SOD、GST、GSH 和MT 均有明显影响,表现出时间剂量效应。SOD在前4天、CAT在前3天酶活性总体上表现出诱导趋势, GST在前4天酶活性处于诱导状态,随着暴露时间的延长,酶活性下降,到第5天时表现出抑制趋势;随着时间的进一步增长,至14d时, 0.005 mg/L剂量组酶活性维持在正常值附近波动, 0.01 mg/L剂量组酶活性被诱导, 0.02 mg/L剂量组酶活性在肝脏中表现为诱导而在鳃中则被抑制,0.05 mg/L剂量组酶活性被抑制。肝脏和鳃GSH含量的变化与GST相似,在短时间内表现出诱导效应,肝脏GSH在暴露的前5天、鳃GSH在暴露的前4天均处于诱导状态,随着暴露时间的延长,0.005 mg/L剂量组表现出诱导,0.05 mg/L剂量组则受到抑制。MT在整个实验期间均处于诱导状态,各剂量组在0.5d被极显著诱导,随后MT含量出现起伏波动,有上升和下降,至第14天时达到一稳定水平。其中,0.01 mg/L剂量组肝脏的MT在整个实验期间均被极显著地诱导（P <0.01）,0.01 mg/L 剂量组的鳃组织MT除第10天外也被极显著诱导（P <0.01）。在暴露14d时,除0.05 mg/L剂量组的肝脏MT外,其余处于极显著诱导状态（P <0.01）。     

苯并[a]芘暴露对三疣梭子蟹鳃、肝胰腺组织毒理学指标的影响

本文研究了三疣梭子蟹(Portunus trituberculatus)在对苯并[a]芘(BaP)富集(15 d)、释放(15d)过程中其鳃和肝胰腺组织的4种毒理学指标的响应.4种毒理学指标分别为7-乙氧基异吩噁唑酮-脱乙基酶(EROD)、谷胱甘肽硫转移酶(GST)、超氧化物歧化酶(SOD)和脂质过氧化(LPO).设置了0.05 μg/L和0.45 μg/L两个实验组以及海水和丙酮对照组.结果显示,在富集阶段,与海水对照组相比,第1天时0.05 μg/L和0.45μg/L实验组鳃、肝胰腺组织的各毒理指标均显著受到诱导(P＜0.05),诱导程度随BaP暴露浓度的增加而增大.而后鳃、肝胰腺组织的EROD、GST活性以及鳃组织的SOD活性达到峰值后下降,肝胰腺组织的SOD活性以及鳃、肝胰腺组织的丙二醛(MDA)含量则持续增加.鳃组织的EROD、GST、SOD活性到达峰值时间早于肝胰腺组织,其活性以及MDA含量也低于肝胰腺组织.在释放阶段,0.45pg/L实验组鳃组织的SOD活性,0.05μ-g/L和0.45 μg/L两个实验组肝胰腺组织的SOD活性均依然显著高于同期海水对照组水平(P＜0.05),其余各浓度实验组鳃、肝胰腺组织均能恢复到同期海水对照组水平(P＞ 0.05).实验结果表明,三疣梭子蟹的鳃组织对于BaP暴露响应时间比肝胰腺组织更早,但均具有一定的恢复能力.     

苯并[a]芘暴露对三疣梭子蟹鳃、肝胰腺组织毒理学指标的影响1

本文研究了三疣梭子蟹(Portunus trituberculatus)在对苯并[a]芘(Ba P)富集(15 d)、释放(15 d)过程中其鳃和肝胰腺组织的4种毒理学指标的响应。4种毒理学指标分别为7-乙氧基异吩噁唑酮-脱乙基酶(EROD)、谷胱甘肽硫转移酶(GST)、超氧化物歧化酶(SOD)和脂质过氧化(LPO)。设置了0.05μg/L和0.45μg/L两个实验组以及海水和丙酮对照组。结果显示,在富集阶段,与海水对照组相比,第1天时0.05μg/L和0.45μg/L实验组鳃、肝胰腺组织的各毒理指标均显著受到诱导(P0.05),诱导程度随Ba P暴露浓度的增加而增大。而后鳃、肝胰腺组织的EROD、GST活性以及鳃组织的SOD活性达到峰值后下降,肝胰腺组织的SOD活性以及鳃、肝胰腺组织的丙二醛(MDA)含量则持续增加。鳃组织的EROD、GST、SOD活性到达峰值时间早于肝胰腺组织,其活性以及MDA含量也低于肝胰腺组织。在释放阶段,0.45μg/L实验组鳃组织的SOD活性,0.05μg/L和0.45μg/L两个实验组肝胰腺组织的SOD活性均依然显著高于同期海水对照组水平(P0.05),其余各浓度实验组鳃、肝胰腺组织均能恢复到同期海水对照组水平(P0.05)。实验结果表明,三疣梭子蟹的鳃组织对于Ba P暴露响应时间比肝胰腺组织更早,但均具有一定的恢复能力。     

重组金属硫蛋白HcMT的原核表达及其与金属离子竞争反应

[目的]在不同p H范围内和不同时间下检测重组HcMT结合的锌、铜离子的含量,对金属离子竞争结合金属硫蛋白的特征进行分析。[方法]构建重组表达载体,将重组质粒pET-32a-Hc MT进行原核表达,诱导时加入Zn~(2+),获得菌体后超声破碎,上清中添加Cu~(2+),分离纯化融合蛋白Trx A-Hc MT,检测金属硫蛋白溶液紫外吸收情况。样品硝化后用ICP-OES检测金属硫蛋白结合的金属离子含量。[结果]在pH 3～9的范围内,重组Hc MT上结合的Zn~(2+)含量随着p H值升高而升高。加入Cu~(2+)后,重组Hc MT上结合的Cu~(2+)浓度上升,Zn~(2+)浓度下降。在p H 3时,重组Hc MT结合金属离子的比值Cu~(2+)/Zn~(2+)为最小值0. 68,在pH 8时Cu~(2+)/Zn~(2+)为最大值1. 48,有利于Cu~(2+)竞争。当pH 8时,不同时间下Zn-Hc MT与Cu~(2+)的竞争结果表明1 h时锌铜有竞争高峰。[结论]证明了铜离子可以取代结合在重组HcMT上的锌离子,在p H 7～9范围中结合1 h竞争效率最高,该研究为重组Hc MT在重金属解毒方面的应用奠定基础。     

日本沼虾MT基因克隆、组织差异性表达及与饲料铜、锌含量的相关性

为了更全面理解日本沼虾(Macrobrachium nipponense)的铜/锌营养生理作用,研究利用RACE技术从日本沼虾肝胰腺中克隆获得一金属硫蛋白基因c DNA全长(mn-MT),并对该基因分子特征、组织表达谱和饲料铜/锌水平对其表达的影响进行分析。结果显示:(1)mn-MT c DNA全长665 bp,含编码59个氨基酸的180 bp的开放阅读框,预测该多肽的理论分子量为6.085 k D,等电点为7.73。该蛋白中半胱氨酸含量最高(30.5%),其次是赖氨酸(16.95%)和丝氨酸(10.17%)。相似性分析显示mn-MT氨基酸序列与美洲海螯虾、斑节对虾和中华绒螯蟹MT的相似性分别达到78%、75%和75%。(2)q RT-PCR分析显示,mn-MT m RNA在肝胰腺、血细胞、鳃、胃、卵巢、肠和肌肉中都有表达,其中肝胰腺中表达量最高。(3)用4组铜添加量分别为0、20、40及160 mg/kg的饲料和3组锌添加水平分别为0、35和210 mg/kg的饲料饲喂初重为(0.101±0.002)g日本沼虾56d后,分析各组虾肝胰腺的mn-MT m RNA表达。mn-MT m RNA表达随饲料铜水平的提高而升高,到40 mg/kg组达到最高(P0.05),而后开始下降;饲料中高锌(210 mg/kg)显著提高mn-MT表达(P0.05),0和35 mg/kg组间差异不显著(P0.05)。结果表明饲料中铜/锌均可影响mn-MT表达,且呈现不同的剂量依赖效应。     

金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

文蛤SRBI基因克隆及其在不同壳色群体中的差异表达

为了研究SRBI基因的结构、功能以及在贝类壳色形成中的作用, 利用SMART RACE技术克隆得到文蛤(Meretrix meretrix) SRBI (Mm-SRBI)基因的全长序列, 并对其内含子特征及不同组织、不同壳色群体外套膜中的表达差异进行了分析。结果表明: Mm-SRBI基因cDNA全长1676 bp, 开放阅读框1515 bp, 编码504个氨基酸, 结构域预测发现有一个CD36结构域; 氨基酸序列比对发现, 与华贵栉孔扇贝的同源性最高(55%), 与其他物种的相似性在34%—40%, 表明该基因变异较大; 在Mm-SRBI基因中扩增出12个内含子, 均存在于开放阅读框中, 且都遵循GT-AG原则; 荧光定量PCR (qRT-PCR)结果表明, Mm-SRBI在闭壳肌、外套膜、斧足、鳃、内脏团和水管6个组织均有表达, 其中在外套膜中表达量显著高于其他组织(P<0.01), 这可能与外套膜中类胡萝卜素含量较高有关; 不同壳色群体外套膜中基因表达分析表明,Mm-SRBI在黑斑和红壳文蛤中的表达量显著高于白壳文蛤(P<0.05)。实验结果为文蛤壳色形成研究奠定了基础。     

Induction of metallothionein by exposure to normobaric 100% oxygen atmosphere in rats with different zinc supply

The objective of this study was to determine the effects of an oxygen enriched environment on the induction of the metalloprotein metallothionein (MT) and its relation to zinc metabolism in rats supplied with different levels of dietary zinc. Male albino rats were fed purified diets based on maize starch, egg white, saccharose and soybean oil differing in the concentration of zinc (1; 20; 100; 500 mg Zn/kg diet). At a dietary zinc supply of 1 mg/kg, the rats developed a zinc deficiency indicated by visual and biochemical parameters. At the end of the 37-day feeding period, half of the rats were exposed to 100% oxygen for 12 h.

The oxygen treatment significantly reduced plasma zinc in the zinc supplemented rats and reduced it in tendency in the zinc deficient rats. The MT concentration was increased in the zinc supplemented groups in the liver, kidney and lung. The oxygen treatment elevated the metallothionein concentration in the two high zinc supplemented groups (100 and 500 mg Zn/kg diet) in the liver. The response of the zinc concentration in plasma and of hepatic metallothionein levels to oxygen exposure indicates a role of metallothionein in zinc distribution or interactions with other trace elements to support antioxidant capacity, rather than an impact on direct scavenging activity of free radicals.     

Zinc-related metallothionein metabolism in bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells (BPAEC) were cultured in vitro under a variety of conditions to investigate how metallothionein (MT) might participate in zinc homeostasis. Experimental conditions included 10% serum to ensure that the in vitro environment would be a better reflection of the in vivo situation than with protein-free medium. MT was increased by acutely high zinc concentrations (100-200 micromol/L) in the extracellular environment. MT was relatively insensitive to moderate changes in zinc concentration (2-50 micromol/L), even after prolonged exposure for 7 to 12 days. BPAEC had reduced MT content when grown in medium containing serum that had been dialyzed to remove components with a molecular mass of less than 1,000, including zinc. Because the principal source of the major minerals in the experimental medium was not the serum, their concentrations in the final medium were not significantly influenced by serum dialysis. Restoring the zinc concentration in the medium containing the dialyzed serum did not restore MT content in BPAEC, suggesting that some small molecular weight molecule other than zinc established their basal MT content. This study did not identify these putative factors in serum, but hormones are likely candidates. Forty-eight-hour incubations of BPAEC with interleukin (IL-6) or dexamethasone increased cellular MT; however, 17beta-estradiol decreased MT, and IL-1 and adenosine 3',5'-cyclic phosphate (cAMP) had no discernible effect. We conclude that extracellular zinc concentrations have relatively little impact on the cellular concentrations of MT and zinc of BPAEC in vitro. Zinc homeostasis by BPAEC is not maintained by changing the MT concentration in response to changes in the extracellular zinc environment. (J. Nutr. Biochem. 10:00-00, 1999).     

Metallothionein levels in willow ptarmigan (<Emphasis Type="Italic">Lagopus lagopus</Emphasis>) populations with different natural loads of cadmium

The tissues of willow ptarmigan in some Norwegian mountain areas contain elevated concentrations of cadmium (Cd). It is not known whether such high Cd levels would have negative impacts in otherwise healthy populations of this species. The aim of the current study was to clarify relationships between hepatic and renal metallothionein (MT) and Cd concentrations in willow ptarmigan to assess effects from this metal. The study reported here was undertaken on willow ptarmigan from the Kongsvoll area, with a naturally high Cd load, and the Essand area, with a naturally low Cd load. Cd values in liver and kidney in willow ptarmigan from Kongsvoll were significantly higher than in willow ptarmigan from Essand. The MT content in both tissues was also highest in willow ptarmigan from Kongsvoll. The MT concentration in kidney was twice that in liver for ptarmigan from both areas and at all times of the year. The MT level in both liver and kidney varied greatly throughout the season, with the highest content in spring (May). The variation was greatest in liver. The total material showed a significant linear relationship between Cd and MT levels in both liver and kidney, but a breakdown of the material into seasons and areas gave a varying degree of significance. MT in willow ptarmigan may be an important mechanism for detoxifying Cd, and populations exposed to high load may “respond” to the loads by increasing MT synthesis in tissues such as liver and kidney. There is no evidence that willow ptarmigan from areas with high natural Cd loads have reached a limit for MT synthesis in either liver or kidney.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

Intracellular metallothionein concentration and the rate of zinc or cadmium influx and MT mRNA accumulation in a CHO Cdr variant

The regulation of metallothionein (MT) gene expression in a cadmium resistant CHO cell line which overproduces MT was examined in this study. Our results show that MT mRNA levels reach a maximum 24-30 h after a primary zinc exposure and, subsequently, MT mRNA concentrations decline. This decrease in MT mRNA levels can be correlated with the accumulation of metallothionein and decreased rates of zinc and cadmium uptake.     

Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta)

We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg(-1) of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species.     

Induction and characterization of metallothionein in different organs ofOstrea edulis L.

In this study, the induction of metallothionein (MT) in one species of oysterOstrea edulis exposed to copper (Cu) and zinc (Zn) was examined. The quantity of total protein (mg protein/mg dry wt) in each sample and the measurement of MT were determined by the Lowry and silver (Ag)-saturation methods. Our results show that the gonads are the organs producing the greatest quantity of MT in the controls and in the groups induced by Cu and Zn, followed by the mantle, gut, and by muscle and plasma. Competition by Zn with respect to Cu for the production of MT has been established.