Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Use of metallothionein in gills from oysters (Crassostrea gigas) as a biomarker: seasonal and intersite fluctuations

The use of oyster gills for the analytical determination of metallothionein (MT) concentration as a biomarker of metal exposure was investigated. Temporal variations in MT and metal concentrations (which can interfere with inter-site differences) were examined over a 7 month period (from spring to autumn) in Japanese oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site (Gironde estuary, France) as well as in individuals translocated from the clean to the contaminated area. The ratio between the annual average of MT concentrations in specimens from the clean and the metal-rich sites was 1.3. During the last 3 months of the experiment, significant differences were no longer registered between transplants and residents from the Gironde estuary. Metals concentrations in oyster gills differed consistently between the clean and the metal-rich sites (annual average ratios of 1.5, 2.7 and 9.8, respectively, for zinc, copper and cadmium) and a fast increase in metal concentrations (over a few months) was observed in transplants, mainly for cadmium. MT and soluble metal concentrations were found to be positively and significantly correlated over the period of the study. This relationship is a positive argument for a possible use of gill MT concentration as a biomarker of metal pollution in contrast to previous findings on the digestive gland, there being a smaller amount of seasonal variability in the weight of oyster gills.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Prismatic cristae and paracrystalline inclusions in mitochondria of myocardial cells of the oyster Crassostrea virginica Gmelin

Summary Several types of unusual mitochondrial configurations were found in myocardial cells of the oyster Crassostrea virginica Gmelin. These mitochondria include, in order of frequency, prismatic cristae, filamentous paracrystals in honeycomb and herringbone configurations, and paracrystals composed of rows of electron dense particles. The long, parallel, evenly spaced prismatic cristae are square or rhomboidal in cross section. In the space between the prismatic cristae are rodlike structures (4–6 nm in diameter) that are regularly spaced about 12nm apart and appear to pass between adjacent cristae. Filamentous paracrystals are observed in slender, elongated mitochondria. The filament spacing and form of these paracrystals suggest that they are composed of the intercristal rods. Alternatively, filamentous paracrystals might be tangential sections of prismatic cristae and intercristal rods. Particulate paracrystals which consist of dense lines or rows of particles are the least frequent type of unusual configuration. The particles are triangular, possibly pyramidal, in shape; their bases are 10–12 nm thick and repeat in rows every 17–18 nm. There is a close association between particulate paracrystals and prismatic cristae plus intercristal rods. Although similar mitochondrial configurations have been associated with disease or altered metabolism in a number of species, we have found no such association in the oyster as yet.Supported in part by the Mississippi-Alabama Sea Grant Consortium, through NOAA, Dept. of Commerce under grant no. NA 79AA-D-0049We wish to thank Ms. Barbara M. Hyde, Ms. Patricia A. Vermiere and Mr. Robert Allen for their technical assistance     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Metal ion binding properties of <Emphasis Type="Italic">Tricium aestivum</Emphasis> E<Subscript>c</Subscript>-1 metallothionein: evidence supporting two separate metal thiolate clusters

Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (E_c-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant E_c-1 is able to bind a total of six divalent d ¹⁰ metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to Prof. Bernhard Lippert on the occasion of his 60th birthday.     

Cytometric investigations on hemocytes of the American oyster, Crassostrea virginica

Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G(0)/G(1) population. There was no evidence of S or G(2)+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Amplification of a gene for metallothionein by tandem repeat in a strain of cadmium-resistant yeast cells

Abstract In a cadmium-resistant strain of Saccharomyces cerevisiae , cells are protected against cadmium toxicity by the production of large amounts of cadmium-binding metallothionein, as occurs similarly in a copper-resistant strain. The apoprotein of the metallothionein is encoded by the CUP1 gene on chromosome VIII. The CUP1 gene is present as 8–10 copies in the cadmium-resistant strain as a result of tandem repeat of a 2.0-kb fragment of DNA that includes CUP1 , while the wild-type strain contains only a single copy of CUP1 . In the cadmium-resistant strain, some evidence for elongation of chromosome VIII with variations in length (maximum to 200 kb) was obtained. However, the elongation was not due to the tandem repeats of the CUP1 -containing region.     

Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn⁺² (50, 100, or 150 μM) or Cd⁺² (3, 6, or 12 μM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn⁺² toxicity and a lesser but significant effect on Cd⁺² toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn⁺² or Cd⁺² exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.     

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.     

Cryopreservation of Crassostrea gigas vesicular cells: viability and metabolic activity

Cryopreservation is widely used for long-term conservation of various tissues, embryos or gametes. However, few studies have described cryopreservation of invertebrate primary cell cultures and more particularly of marine invertebrate somatic cells. This technique would however be of great interest to facilitate the study of various metabolic processes which vary seasonally. The aim of the present study was to develop a protocol for cryopreservation of Crassostrea gigas vesicular cells. Different parameters were adjusted to improve recovery of cells after freezing. The most efficient cryoprotectant agent was a mix of Me(2)SO, glycerol, and ethylene glycol (4% each). The optimal cooling rate was -1 degrees Cmin(-1) down to -70 degrees C before transfer into liquid nitrogen. In these conditions the percentage of viable cells reached 70% of the control. The glucose metabolism of thawed cells was evaluated using radioactive glucose as a tracer. Immediately after thawing, glucose uptake involving membrane transporters was greatly reduced (24% of control) whereas glucose incorporation into glycogen was less affected (68% of control).     

Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae

Observations of a spontaneous infection in Crassostrea virginica larvae at Milford Laboratory indicate that the causative microorganism, Vibrio sp., was present in the filtered, uv light-treated sea water in very low concentrations. The disease was usually noted within 6–10 days after fertilization of eggs and mortality was usually complete about 6–8 days later. The disease could be induced by adding either washed cells of the pathogen or the broth filtrate. Observations made when the filtrate was employed indicate that the bacterium produced a toxin capable of breaking down the tissue structure of the oyster larvae. Mortalities were prevented with 100 mg of neomycin/liter of oyster egg culture.     

Prevalence of kidney dysfunction in humans – Relationship to cadmium dose, metallothionein, immunological and metabolic factors

Long term cadmium (Cd) exposure in occupational and general environments may give rise to kidney dysfunction. This effect is usually considered to be the critical effect, i. e. the effect that occurs at relatively low level of exposure. The present review focused on studies of the prevalence of cadmium-related kidney dysfunction among population groups residing in cadmium contaminated areas in China. Dose–response relationships were shown between UCd and the prevalence of increased levels of biomarkers in urine of renal tubular dysfunction such as urinary beta-2-microglobulin or N-acetyl-beta-d-glucosaminidase – NAG or urinary albumin, a biomarker of glomerular kidney dysfunction. Factors that influence these dose–response relationships include: 1) Metallothionein mRNA levels in peripheral blood lymphocytes, used as a biomarker of the ability of each person, to synthesize metallothionein (a protein known to provide intracellular protection against cadmium toxicity). 2) The occurrence of increased levels in blood plasma of autoantibodies against metallothionein. 3) Concomitant changes in glucose metabolism i e Type II diabetes. 4) Concomitant exposure to other nephrotoxic agents such as inorganic arsenic. Increased susceptibility in diabetics has been shown also in population groups in Europe. In persons with type II diabetes and increased levels of autoantibodies against metallothionein in blood plasma or in persons with concomitant exposure to environmental inorganic arsenic, indications of Cd-related kidney dysfunction was observed at UCd levels around 1 μg/g creatinine, levels found among “unexposed” population groups in many countries.