Variation in metallothionein gene expression is associated with adaptation to copper in the earthworm Dendrobaena octaedra

Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins.     

Yeast metallothionein function in metal ion detoxification

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.     

乐安河—鄱阳湖湿地植物群落特征及其优势植物对重金属Cu、Pb、Cd的富集

选择乐安河—鄱阳湖湿地典型植物群落,采用重要值方法评价各样点植物群落特征并筛选出典型优势植物,通过室内理化测试分析不同生境中优势植物植株及其根区土壤中重金属Cu、Pb、Cd的含量;采用生物富集系数(BCF)方法评价不同优势植物对重金属Cu、Pb、Cd的富集特性。结果表明:研究区湿地植物以草本为主,在各样点共发现124种物种,包括蕨类植物2科2属2种,种子植物40科97属122种,并从中筛选出羊蹄、红蓼、鼠曲草、紫云英、苎麻等5种富集能力较强的优势植物;植物根区土壤中的Cu、Cd含量均超过土壤环境质量三级标准,而且Cu、Cd的最高含量分别为824.03、5.03 mg·kg-1;不同优势植物对Cu、Pb、Cd等3种重金属元素中的1种或2种表现出较强的富集能力,其中优势物种红蓼对Cu具有较强的富集能力,含Cu量最高为148.80 mg·kg-1,另一种优势物种鼠曲草对三种元素的生物富集系数均较高,且对Cd的最高富集含量为15.17 mg·kg-1,对Cd的生物富集系数最高值为19.14,高于其他植物10倍以上,鼠曲草对重金属Cd具有富集植物的基本特征,且对Cu和Cd具有共富集特征并具有较高的耐性,紫云英、羊蹄等对Cd的富集能力也较强。上述5种优势植物种群对鄱阳湖湿地Cu、Pb、Cd等重金属污染物的生态修复具有一定参考价值,可作为鄱阳湖湿地重金属污染修复植物的选择对象。     

Evaluation of cadmium,lead and metallothionein contents in the tissues of mussels (Mytilus galloprovincialis) from the Campania coast (Italy): Levels and seasonal trends

The biological effect of seasonality on cadmium, lead and metallothionein contents was assessed in mussels Mytilus galloprovincialis from natural banks located along the coastline of the Gulf of Naples (Campania, Italy). Heavy metals and metallothionein concentrations were measured in digestive and reproductive glands. The results showed a clear correlation between metallothionein content and the reproductive gland status determined during the seasons; on the contrary, no correlation was found between metallothionein and metal contents. Data allow us to hypothesize that metallothionein functions go beyond metal detoxification, thus opening new scenarios for these proteins in invertebrates. The effect of seasons on metals concentration in mussel tissues showed similar seasonal patterns between the sites, regardless of their anthropogenic impacts. Cadmium content was not strictly related to seasonal periods, whereas lead content was significantly lower in summer. The results also indicate that the metal contents in mussels from the Gulf of Naples do not represent a risk to human health, even in the period of their maximum accumulation, and that the relaying of mussels before marketing could improve the animal stress conditions, but having a slight effect on metal excretion.     

Heavy metal and abiotic stress inducible metallothionein isoforms from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> (SW) D.C. show differences in binding to heavy metals in vitro

Prosopis juliflora is a tree species that grows well in heavy metal laden industrial sites and accumulates heavy metals. To understand the possible contribution of metallothioneins (MTs) in heavy metal accumulation in P. juliflora, we isolated and compared the metal binding ability of three different types of MTs (PjMT1-3). Glutathione S-transferase fusions of PjMTs (GSTMT1-3) were purified from Escherichia coli cells grown in the presence of 0.3 mM cadmium, copper or zinc. Analysis of metal bound fusion proteins using atomic absorption spectrometry showed that PjMT1 bound higher levels of all three heavy metals as compared to PjMT2 and PjMT3. A comparative analysis of the genomic regions (including promoter for all three PjMTs) is also presented. All three PjMTs are induced by H₂O₂ and ABA applications. PjMT1 and PjMT2 are induced by copper and zinc respectively while PjMT3 is induced by copper, zinc and cadmium. Variation in induction of PjMTs in response to metal exposure and their differential binding to metals suggests that each MT has a specific role in P. juliflora. Of the three MTs analyzed, PjMT1 shows maximum heavy metal sequestration and is thus a potential candidate for use in heavy metal phytoremediation.     

Turnover rate of metallothionein and cadmium in Mytilus edulis

The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.     

Heavy metals in sediments, mussels and oysters from Trinidad and Venezuela

The Gulf of Paria is bordered by both Trinidad and Venezuela, from which various metallic pollutants and other contaminants can originate. The Gulf is still a significant source of fish, crabs and shellfish for human consumption to both countries, where concerns over the quality of this marine environment have been long expressed but never properly addressed. In addition, the circulatory current patterns in the Gulf ensure that contaminants originating from either country are likely to affect both countries eventually. Heavy metals were determined in oysters (Crassostrea rhizophorae and C. virginica), green mussels (Perna viridis) and sediments from the Gulf of Paria. Samples were obtained at four sites in Trinidad and three sites in Venezuela in the Gulf of Paria, in addition to comparative samples collected from three sites on the north coast of Venezuela. Edible tissues of twelve shellfish from each location were blended and aliquots digested with concentrated nitric acid, for extraction of cadmium, chromium, copper, lead, nickel and zinc. The solutions were analysed by flame atomic absorption spectroscopy. Mercury was extracted with a mixture of nitric, hydrochloric and sulphuric acids and determined by cold vapour atomic absorption. Sediments were oven-dried at 60'C, before being similarly extracted. Results showed that mercury in sediments at all sites in Trinidad and Venezuela exceeded NOAA and Canadian sediment quality guidelines, while cadmium, copper, nickel, lead and zinc also exceeded these guidelines at several sites. Heavy metal levels in oysters and green mussels varied widely with location. However, oysters from the Gulf of Paria contained significantly higher mean levels of cadmium, copper, nickel and zinc than those from the north coast of Venezuela, but this difference was not apparent in mussels. Cadmium, mercury and zinc in sediments were significantly correlated with those of mussels, but not of oysters, in which copper and zinc at several sites in the Gulf of Paria exceeded local maximum permissible levels (Cu = 20 microg g(-1) wet wt; Zn = 50 microg g(-1) wet wt) for human consumption. These findings indicate that while mussels may be better biological indicators of heavy metal pollution in sediments than oysters, the latter may provide copper and zinc contamination. Further research is needed to determine the most appropriate biological indicators of heavy metal and other pollutants in the local marine environment and to develop protocols for their use.     

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd,Cu and Pb exposure

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.     

Comparative Effects of Direct Cadmium Contamination on Gene Expression in Gills, Liver, Skeletal Muscles and Brain of the Zebrafish (Danio rerio)

The effects of cadmium (Cd) on gene expression were examined in four organs (gills, liver, skeletal muscles and brain) of the zebrafish. Adult male fish were subjected to three different water contamination pressures over periods of 7 and 21 days: control medium (C₀: no Cd added) and two contaminated media (C₁: 1.9 ± 0.6 μg Cd l⁻¹, and C₂: 9.6 ± 2.9 μg Cd l⁻¹). Fourteen genes involved in antioxidant defences, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair and apoptosis were selected and their expression levels investigated by quantitative real-time PCR. Cadmium concentrations were determined in the four organs and metallothionein (MT) protein levels investigated in brain, liver and gills. Although skeletal muscle was a poor Cd-accumulating tissue, many genes were up-regulated at day 7: mt1, cyt, bax, gadd and rad51 genes. Three additional genes, c-jun, pyc and tap, were up-regulated in muscles at day 21 whereas bax, gadd and rad51 had returned to basal levels. Surprisingly, mt1 and c-jun were the only genes displaying a differential induction after 21 days in liver, although this organ accumulated the highest cadmium concentration. In brain, only mt1, mt2 and c-jun genes were up-regulated after 21 days. In gills, the highest response was observed after 7 days, featuring the differential expression of oxidative stress-response hsp70 and mitochondrial sod genes, along with genes involved in mitochondrial metabolism and metal detoxification. Then, after 21 days, the expression of almost every genes returned to basal levels while both mt1 and mt2 genes were up-regulated.     

Rainbow trout metallothionein

Two forms of hepatic metallothionein were isolated and purified from rainbow trout injected intraperitoneally with cadmium chloride. Both forms showed similarities with mammalian metallothioneins, had a high cystein content (30 mol%), and were void of aromatic amino acids and histidine. The molecular weight was estimated to be about 6000 dalton for the apothioneins, and the thiol groups of the cysteine residues complexed with the heavy metals (Cd, Cu, Zn) in a SH/Me⁺⁺ ratio of about 2.4. The amount of copper in metallothionein from rainbow trout was very high, greater than the amount of cadmium and zinc after injections of 3 mg cadmium/kg body weight. The total metal content of cadmium, copper and zinc in metallothionein 1 and 2 were about 7 and 8 atoms per molecule respectively.     

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 μmol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 μmol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by K _m of 1.65±0.54 μmol/L and V _max of 3.9±0.3 pmol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 μmol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 μmol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content. This revised version was published online in July 2006 with corrections to the Cover Date.