Cadmium-resistant Chinese hamster V79 cells with decreased accumulation of cadmium

Cells resistant to 3 x 10(-5) M CdCl2 (Cdr cells) were isolated from cultures of Chinese hamster V79 cells by a procedure that involved stepwise increase in the concentration of Cd2+ and subsequent mass selection. Cdr cells grew as fast as wild-type cells (Cds) in medium without cadmium. Cdr cells were not cross-resistant to other divalent metal ions, such as Hg2+, Ni2+, Pb2+, and Zn2+. Both Cds and Cdr cells induced similar levels of metallothioneins (MT) in response to zinc. Depletion of glutathione (GSH) did not significantly influence the sensitivity of Cdr cells to Cd2+ but markedly enhanced the sensitivity to Cd2+ of Cds cells. Furthermore, the rate of synthesis of GSH after depletion did not differ greatly between sensitive and resistant cells. The rate of uptake of 109Cd2+ by Cdr cells was only 10-15% that by Cds cells. The difference in rates of uptake between Cds and Cdr cells was observed irrespective of the presence or absence of serum in the culture medium. These results indicate that, in this system, resistance to Cd2+ is attributable neither to increased inducibility of MT nor to increases in intracellular levels of GSH, and that only a decrease in the rate of uptake of Cd2+ contributes to the acquisition of resistance to Cd2+. Uptake of Cd2+ by cells was dependent on temperature and the rate of uptake of Cd2+ by Cdr cells was lower at all temperatures examined than the rate of uptake by Cds cells. Cycloheximide did not suppress the uptake of Cd2+, suggesting that uptake does not require synthesis of cell proteins de novo. Preincubation of cells with N-ethylmaleimide suppressed the uptake of Cd2+ to some extent, a result that suggests the involvement of surface SH groups in the uptake of Cd2+ by these cells.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

Identification of metaflothionein in Pleurodeles waltl

The characterization of metallothionein in the Urodele amphibian species Pleurodeles waltl was achieved. A simple and rapid method for identification of metallothionein, based on its strong affinity for cadmium (109Cd), was used. We were able to show that metallothionein is constitutively synthesized in liver, ovary and brain. The property of metallothionein to strongly bind essential (Zn, Cu) as well as toxic (Cd, Hg) metals is consistent with a dual role in cellular metabolism, ie. homeostatis and detoxification of heavy metal ions.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals

We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals. Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution. Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals. Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.     

Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes.

Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C-reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct metal-binding configurations in metallothionein

In a study of the binding stoichiometry of various metals to rat liver metallothionein, the protein appears to coordinate metals in 2 distinct configurations. Ions of at least 18 different metals were shown to associate with the protein suggesting that there is little specificity in binding. Most metals exhibited saturation binding at 7 mol eq forming M7-metallothionein. These included Bi(III), Cd(II), Co(II), Hg(II), In(III), Ni(II), Pb(II), Sb(III), and Zn(II). Others metals including Os(III), Pd(II), Pt(IV), Re(V), Rh(III), and Tl(III) give a positive indication of binding, but stoichiometries were unclear. Ag(I) and Cu(I) bound in clusters as M12-metallothionein. This binding stoichiometry was determined in 3 ways: (a) by determining the equivalence point in Cu- and Ag-titrated samples where resistance to proteolysis is maximal; (b) by determining the point where Zn ions are completely displaced from Zn7-metallothionein; and (c) by direct binding studies. Ag-reconstituted protein, recovered from gel filtration, had an average Ag content of 11.5 g atoms/mol of protein. A similar stoichiometry for the Cu-protein resulted from displacement of Zn from Zn7-metallothionein by Cu(I). The M12-protein was converted to the M7-protein by displacement of Ag(I) or Cu(I) with 7 mol eq of Hg(II). Whereas the distribution of metals in the 2 domains of M7-metallothionein is M4 alpha and M3 beta, the arrangement in the M12-molecule is probably M6 alpha and M6 beta. We propose that metallothionein ligates Ag(I) and Cu(I) in a trigonal geometry by bridging thiolates. This is in contradistinction to a tetrahedral binding geometry in the M7-protein. Distinct binding configurations may result in different tertiary structures for M7- and M12-proteins which may relate to metabolic specificity of Zn-metallothionein and Cu-metallothionein, respectively.     

某污灌区重金属与两种持久性有机污染物(POPs)污染趋势评价

对某污灌区的土壤剖面进行采样分析,利用不同的评价方法进行评价,结果表明,土壤中金属元素Cr、As、HG、Cd、Cu、Pb、Ni、Zn和持久性有机污染物BHC、DDT含量有上升趋势,80%以上的表层土壤样品污染物含量超过土壤底层,85%以上的土壤样品污染物含量明显超过当地土壤的自然背景值.通过污染物在土壤中的残留模型预测,两种有机污染物和元素Cr、As、Hg、Cd、Cu会在土壤中累积,持续的污水灌溉可能使在土壤中累积的这些污染物进入地下水或食物链,对环境健康造成危害.     

混合暴露条件下近江牡蛎对重金属的积累与释放特征

选择近江牡蛎作为试验生物,研究了混合暴露条件下8种重金属在近江牡蛎体内的积累和释放特征.结果表明: 近江牡蛎对重金属Pb、Cu、Ni、Cd、Cr和Hg有很强的累积能力,可较好地指示溶液中的重金属浓度水平,但对重金属Zn和As的积累能力很小,不能真实反映溶液中重金属Zn和As含量的变化水平.在随后35 d的释放阶段,8种重金属在近江牡蛎体内的含量没有明显变化,表明近江牡蛎对重金属的释放能力较差.双箱动力学模型可较好地反映混合暴露条件下近江牡蛎对重金属的积累特征,但不适合对其释放特征进行描述.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

防城港临时性海洋倾倒区沉积物重金属污染现状及其生态风险评价

对防城港临时性海洋倾倒区表层沉积物重金属的含量和分布进行了对比分析,并采用污染指数法和Hakanson潜在生态风险指数法对其重金属的污染特征及潜在生态风险程度进行了评价。结果表明,倾倒区沉积物Hg、Pb、As和Zn的含量显著性升高;总体污染程度为中等污染,各重金属元素的污染程度由高到低排序依次为As(3.59)>Hg(2.19)>Pb(1.42)>Zn(1.20)>Cu(1.05)>Cd(0.91),其中As是主要污染因子;该海域表层沉积物重金属总体潜在生态风险程度为中等,各重金属元素的潜在生态风险程度由高到低排序依次为Hg(87.74)>As(35.89)>Cd(27.27)>Pb(7.08)>Cu(5.23)>Zn(1.20),其中Hg为主要潜在生态风险因子。     

福建省耕地土壤重金属含量和可浸提性研究

测定了福建省6市、县耕地土壤中的Cu、Zn、Cd、Ni、Pb、Hg、As等7种重金属元素的全量和可浸提态含量,并对测定结果进行了数理统计。结果表明,福建省耕地土壤存在着不同程度的重金属污染,但依元素不同而不同,重金属可浸提态含量占全量的10.0?52.2 Cu、Zn、Cd、Pb、Ni的可浸提态含量与全量之间的相关达极显著水平,Zn、Cd、Pb、As等4元素含量之间存在着显著正相关,而Ni则只与Zn元素有极显著正相关,Cu和Hg与其它重金属元素的相关则均不显著。     

三峡水库蓄水后铜鱼和圆口铜鱼肌肉和肝脏中重金属水平

为了解三峡水库蓄水后鱼体重金属富集现状及其潜在的生态风险和食品安全, 测定了三峡水库上、中、下游不同年龄组铜鱼(Coreius heterodon)和圆口铜鱼(C. guichenoti)肌肉和肝脏中重金属含量。利用等离子吸收光谱法、石墨炉原子吸收光谱法、原子荧光光谱法检测样品中Cu、Zn、Cr、Pb、Cd、Hg、As 等 7种重金属含量。结果表明: 7 种重金属在铜鱼和圆口铜鱼体内的含量水平基本一致, 重金属在铜鱼和圆口铜鱼肌肉中含量大小均为Zn Cu Cr Hg AsPb Cd, 在铜鱼肝脏中含量大小为Zn Cu Pb Cd Cr As Hg, 而在圆口铜鱼肝脏中含量大小为Zn Cu Pb Cr Cd Hg As。铜鱼和圆口铜鱼肝脏中重金属含量显著高于肌肉(P0.05)。两种鱼类间大部分重金属在库区上、中、下游无显著差异(P 0.05)。铜鱼和圆口铜鱼肌肉(可食用部分)中7 种重金属含量均未超过国家食品安全卫生标准, 属于安全食用范围;肝脏中除Cd 和Pb 外的其他元素含量均未超过国家标准。相关结果反映了三峡水库175 m 蓄水后底栖土著经济鱼类重金属的污染状况, 对了解该地区水产品质量安全状况及水产品安全评价提供了参考依据。       

Heavy metal pollution in Antarctica: a molecular ecotoxicological approach to exposure assessment

Trematomus bernacchii sampled from two sites at Ross Island, Antarctica, were assessed for condition (gonadal and hepatic somatic indices and condition factor), pathological state (liver and gill histology), hepatic metal (Zn, Cu, Cd and Ni) concentrations, and metallothionein induction by quantitative competitive (qc) RT-PCR. Fish from a polluted site (Winter Quarters Bay) had pathological anomalies including necrosis and periductal inflammation in their livers, and X-cell disease, epithelial hyperplasia, lamellar fusion and aneurysms in their gills. Such anomalies were less common in fish from a relatively pristine site (Backdoor Bay, Cape Royds). Fish from both sites had similar liver concentrations of Zn, Cu and Cd and qcRT-PCR revealed similar levels of hepatic metallothionein mRNA. Ni in the livers of fish from Winter Quarters Bay were higher than those in fish from Backdoor Bay, but the differences were not great enough to affect hepatic metallothionein mRNA significantly. Despite the polluted state of Winter Quarters Bay waters, it seems that the heavy metals present may have only limited impact on the health status of fish collected from this locality. This may reflect a low bioavailability of the heavy metals in Ross Island marine sediments and suggests that other factors, such as relatively high levels of organic pollutants (PAHs, PCBs) or pathogens from the nearby sewage outlet, may play a more significant role in the aetiology of pathological conditions in fish from Winter Quarters Bay.     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

某农药工业园区周边土壤重金属含量与风险评价

以苏南某农药工业园区周边30km2区域为研究区,采用同心圆法采集土壤样品183个,分析了农药工业园区周边土壤Cd、Cr、Cu、Ni、Pb、Zn、Hg和As8种重金属含量、空间变异性、来源及潜在风险.结果表明:以自然背景值为评价标准,研究区表层土壤Hg、Cu、Cd和Pb平均含量超过自然背景值,其中Hg和Cu含量最高;以国标二级标准为评价标准,土壤Cd、Cr、Ni、Pb、Zn、As6种重金属的单项污染指数平均值均小于1,Hg和Cu分别为1.59和1.05.在农药工业园区周边土壤重金属污染较重的东南方向和西北方向,随着与园区距离的增加,土壤Cd、Ni、Pb、Cr、As、Hg、Zn和Cu含量先上升、后下降、再趋于平稳.通过分析农药工业园区周边土壤重金属综合污染指数发现,距离园区约200～1000m周边土壤污染的风险较大,而1000m以外逐渐达到安全范围.利用地统计学和GIS相结合进行分析发现,8种重金属污染指数有明显的空间变异.依据相关分析与主成分分析结果推测,Zn、Ni、Cr、Pb和As主要来源于成土母质,而Hg、Cu和Cd主要与人类活动有关.