Response of antioxidant systems to copper in the gills of the clam Ruditapes decussatus

Copper (Cu) is an essential element for biological systems, however, when present in excess, is toxic. Metallothioneins can play an important role in Cu homeostasis and detoxification. Moreover, Cu can catalyse the production of toxic hydroxyl radicals that cause lipid peroxidation but defence systems in the cells can limit the oxidative damage. The present study was performed to investigate the effect of three Cu concentrations (0.5, 2.5 and 25 μg l⁻¹) on the response of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathion peroxidase and total glutathion peroxidase), total proteins, metallothioneins (MT), malondialdehyde (MDA) concentrations in the gills of the clam, Ruditapes decussatus. The activity of antioxidant enzymes and total proteins, MT and MDA concentrations were measured in the gills of the clams after 1, 3, 7, 14, 21 and 28 days of Cu exposure. Results indicate that Cu only induces an imbalance in the oxygen metabolism during the first week of Cu exposure due to a decrease in mitochondrial SOD and CAT, selenium-dependent and total glutathion peroxidase activities. Cu also causes lipid peroxidation, measured by the MDA formation, that was Cu dependent. In the gills of clams exposed to 25 μg Cu/l, the excess of Cu triggers the induction of MT synthesis after 3 days of exposure.     

Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure

Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.     

Subcellular distribution of trace elements in the liver of sea turtles

Subcellular distribution of Cu, Zn, Se, Rb, Mo, Ag, Cd and Pb was determined in the liver of green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) from Yaeyama Islands, Japan. Also, hepatic cytosol from sea turtles was applied on a Sephadex G-75 column and elution profiles of trace elements were examined. Copper, Zn, Se, Rb, Ag and Cd were largely present in cytosol in the liver of both species, indicating that cytosol was the significant site for the accumulation of these elements in sea turtles. In contrast, Mo and Pb were accumulated specifically in nuclear and mitochondrial fraction and microsomal fraction, respectively. Gel filtration analysis showed that Cu, Zn, Ag and Cd were bound to metallothionein (MT) in the cytosol of sea turtles. To our knowledge, this is the first report on the association of trace elements with MT in sea turtles.     

Transformation of Tobacco with ScMTII Gene‐Enhanced Cadmium and Zinc Accumulation

Genetic transformation is gaining importance for developing plant types suitable to metal accumulate and/or hyperaccumulate. In this study, the transgenic tobacco plant which transferred the ScMTII gene from Saccharomyces cerevisiae to wild type tobacco cultivar Petite Havana (SR1) was grown on soils with low and high cadmium (Cd) and zinc (Zn) concentrations in a growth chamber for 6 weeks and compared to wild type tobacco for Cd and Zn accumulation. Cadmium and Zn accumulations in the transgenic and wild type tobacco plants were increased with the increasing Cd and Zn concentrations. Unlike Zn, the transgenic plant accumulated significantly higher amount of Cd compared to the wild type control plants. Shoot Cd concentrations of transgenic tobacco in higher Cd dosages reached the above the hyperaccumulation threshold value of 100 mg Cd kg^?1 in the dry weight (DW). Transgenic tobacco accumulated 354, 400, 372, and 457 mg Cd kg^?1 DW, for 10, 20, 40, and 80 mg Cd kg^?1 soil treatments, respectively. These values are 3.5–4.5‐fold higher than that of Cd hyperaccumulation threshold value. With 10 mg kg^?1 Cd treatment, the bioconcentration factor (BCF) of transgenic tobacco plants for Cd reached up to 35 in which the threshold value for BCF should be at least 10. Our results showed that the transgenic tobacco may be used as a good Cd hyperaccumulator plant and for phytoextraction of Cd contaminated soils, but not for Zn.     

镉对褐菖鲉金属结合蛋白的诱导

金属结合蛋白是一类富含半胱氨酸、不含芳香族氨基酸和组氨酸的低分子量蛋白质。在紫外254nm处具强的吸收。用不同剂量的氯化镉诱导褐菖，通过凝胶过滤从褐菖肝脏提取金属结合蛋白，并测定其结合的稿以及硫醇含量与紫外254nm处的吸收值。结果表明，镉能诱导褐菖金属结合蛋白的合成。     

Mechanism of cadmium resistance and adsorption of a yeast strain Rhodotorula sp. Y11

The mechanism of cadmium resistance of a yeast strain Rhodotorula sp. Y11 isolated from mine soil was investigated. We found that the yeast cells treated with different methods showed different cadmium-adsorption models. Grown in medium supplied with 100 mg/L of cadmium, 3.29% of the cell-absorbed cadmium was accounted in the cytoplasm. However, only 1% was taken into the cytoplasm and 99% was bound to the cell wall using the lyophilized biomass to adsorb cadmium in double distilled water. Treatments with alkali, ethanol-chloroform and proteinase showed different influences on the biosorption of whole cells and isolated cell walls. FT-IR analysis showed that acetyl of chitin was the active compound in the cells to absorb cadmium. The production of Metallothioneins, proteins related to the resistance to heavy metal in yeast, was evidently induced by cadmium, achieving 638.8 μg/g wet weight, which was about 85 folds higher than that in the uninduced biomass and was also much higher than that reported previously. The molecular weight of Metallothioneins was 6500 Da estimated by SDS-PAGE.     

Susceptibility to oxidative stress of mussels (Mytilus galloprovincialis) in the Venice Lagoon (Italy)

The aim of this study was to evaluate the susceptibility to pollutant mediated oxidative stress of the Mediterranean mussel Mytilus galloprovincialis in the Venice lagoon (Italy).

In June 2003, mussels from a farm were transplanted to eight sites in the lagoon for five weeks. Oxidative stress responses were measured by: (i) total oxyradical scavenging capacity (TOSC) assay, for an overall evaluation of the oxidative stress response capability; (ii) catalase (CAT), as a key enzyme involved in the antioxidant defence system; (iii) malondialdehyde (MDA), as an indicator of lipid peroxidation, to evaluate an oxidative damage; (iv) metallothioneins (MTs), as they play a role in the antioxidant defence.

The TOSC analysis revealed a reduced capability to eliminate: (i) peroxyl radical in mussels transplanted at Palude della Rosa, Valle Millecampi and Chioggia; (ii) hydroxyl radical at Campalto and Valle Millecampi; (iii) peroxynitrite at Valle Millecampi.

Inhibition in CAT activity, observed in all the monitored sites, confirms the presence of an oxidative pressure in transplanted mussels.

In addition, Pearson correlation analysis was performed in order to observe possible links between the various parameters. The PCA was a powerful tool to discriminate impacted sites, suggesting that the mussels transplanted throughout the Venice lagoon were subjected to different levels of oxidative pressure. Furthermore, it provided an easy and useful tool to summarize the obtained results.     

青蛤(Cyclina sinensis)金属硫蛋白及硫氧还蛋白基因的克隆与表达分析

利用构建的cDNA文库及高通量测序方法,获得金属硫蛋白及硫氧还蛋白基因全长。结果表明,青蛤金属硫蛋白和硫氧还蛋白的cDNA全长分别为776bp和804bp,金属硫蛋白基因编码74个氨基酸,包含7个CXC特征结构;硫氧还蛋白基因编码165氨基酸,包含5个氨基酸构成的活性中心。采用实时定量PCR方法分析了两种基因在Cd2+胁迫下的表达变化,结果显示,金属硫蛋白基因在Cd2+胁迫下6—12hmRNA表达量急剧上升,与对照组有显著性差异(P<0.01)。硫氧还蛋白基因在Cd2+胁迫下mRNA在6h明显升高,在6—24h与对照组出现显著性差异(P<0.05)。说明Cd2+能够诱导青蛤金属硫蛋白和硫氧还蛋白基因表达的时序性升高,二者的转录过程反映了贝类抵御重金属胁迫的分子调控过程。     

Bivalve population health: Multistress to identify hot spots

This study investigated some stress (metals, parasites) and response (immunity, metallothionein) factors in two cockle and two Manila clam populations. Data from eight seasons were averaged to obtain global baseline values. Stress/response characteristics of each population were compared to population health status that was determined through population dynamics parameters. Four different scenarios were discussed: (1) a lightly stressed cockle population with correct population health but with a risk of deterioration (hot spot); (2) a lightly stressed introduced cockle population threatened of extinction. In this case ecological factors were suspected; (3) a moderately stressed clam population with moderate adaptative response. The population was sustainable but the level of stress should not increase (hotspot); and (4) a stressed clam population and unfavourable ecological conditions preventing clam settlement. This monitoring highlighted that the discrepancy between population health and stress levels could be due to insufficient response by bivalves and/or by unfavourable ecological factors.     

Metallothioneins and cytosolic metals in Neomysis integer exposed to cadmium at different salinities

In the present study the induction of metallothioneins (MTs) and its relation to cytosolic metal concentrations (Zn, Cu and Cd) in the euryhaline crustacean Neomysis integer exposed to Cd at different salinities was studied. N. integer was exposed to the same free cadmium ion activity of 5.74 x 10(-9) mol l(-1) (i.e. 1/5 of the 96 h LC(50) value expressed as cadmium activity) in hypo-osmotic (5 psu), isosmotic (16 psu) and hyper-osmotic media (25 psu) for 7 days. In this way, the effect of salinity on cadmium speciation was eliminated and therefore the physiological effect of salinity on Cd accumulation and MT induction could be studied. The accumulation of cytosolic Cd in N. integer changed with salinity from 1.11+/-0.05 micromol l(-1) at 5 psu up to 1.43+/-0.17 micromol l(-1) at 25 psu. This could indicate that the physiological response of euryhaline estuarine invertebrates like N. integer to salinity changes can influence the rate of trace metal uptake from solution. While the salinity changes did not cause significant differences in cytosolic Zn concentrations (mean value of all tested salinities: 34.4+/-2.8 micromol l(-1)), an inverse relationship between salinity and cytosolic Cu concentration was observed. The highest concentration of 15.7+/-2.3 micromol Cul(-1) was determined at 5 psu and the lowest 10.9+/-1.4 micromol Cul(-1) at 25 psu. This could point to a possible relationship between the copper concentration and the hemocyanin metabolism in N. integer. This is the first time that differential pulse voltammetry method was applied to MT assays with N. integer. Although the exposure to Cd resulted in a higher Cd cytosolic concentration, no subsequent MT increase was detected. The significant positive correlation between MT levels and cytosolic Cu concentrations (Spearman correlation coefficient r(s)=0.356, p=0.009) implies a strong relationship between MT and Cu in N. integer.