环境接触镉人群外周血淋巴细胞中金属硫蛋白基因表达的改变

为探讨外周血淋巴细胞中金属中金属硫蛋白基因表达作为镉接触的生物标志物的可行性,选择环境接触镉的人群,进行接触评定和效应评定,分离淋巴细胞进行外培养,测定ＭＴ基因表达水平。结果表明环境接触镉人群的基础和诱导ＭＴ有达水平 尿镉、血镉和镉摄入量水平上升。     

Examining the suitability of mussel digestive gland to serve as a biomonitoring target organ

In coastal marine areas, the level of metallothioneins (MTs) is monitored as the specific response to trace heavy metals and is considered as a biomarker of metal exposure. Laboratory experiments with bivalves indicate that MT synthesis is induced by Cd, Cu, Hg, Ag and Zn ions. The results of our study, conducted with the mussel Mytilus galloprovincialis, deployed 12 months in the "hot spot" area of the Kastela Bay, Dalmatia, Croatia, are evaluated considering the recommendation of MED POL Programme for estimating exposure of mussels to trace heavy metals by means of MTs. Our data clearly indicate that the MT level in the digestive gland of the Mediterranean mussel depends on the biotic and abiotic parameters. Beside MT level, a number of parameters, like digestive gland mass, shell mass, cytosolic metal concentration is needed in order to define if MT level in the digestive gland of mussels is indicative as a biomarker of cadmium exposure.     

Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. I. Molecular, biochemical, and methodical background

The biomarker concept is an important approach of modern ecotoxicology based on the detection and quantification of molecular, biochemical, cellular, or physiological alterations in organisms as the result of exposure to toxic compounds. In the present study, the biomarker potential of two metallothionein (MT) isoforms from hepatopancreas and mantle tissue of the Roman snail, Helix pomatia L., is evaluated. This species possesses two MT isoforms with specific functions: The Cd-specific isoform (Cd-MT) is predominantly induced and expressed in the hepatopancreas upon Cd exposure. It binds and inactivates this potentially toxic trace element. The second isoform is Cu-specific (Cu-MT), primarily expressed in the mantle tissue and responsible for the homeostatic regulation of Cu. The differential expression and metal-specificity of the two isoforms make them potential candidates for a dual MT biomarker system based on an isoform-specific approach. This study shows that Cd-MT and Cu-MT of H. pomatia can reliably be quantified by a differential metal saturation approach that could be used for environmental biomarker studies. The approach works by virtue of the following preconditions: The two isoforms differ from each other with respect to their molecular and biochemical features, they show metal-specific binding preferences, they are organ-specific, they exhibit distinct induction patterns in response to the metals to which they bind, and their biological and functional background is thoroughly known.     

Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure

Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.     

Assimilation and bioconcentration of Ag and Cd by the marine black bream after waterborne and dietary metal exposure

We determined the aqueous uptake and dietary assimilation of Cd and Ag by the marine black bream Acanthopagrus schlegeli following one to four weeks' exposure (or conditioning) to waterborne or dietary Cd or Ag at different concentrations. The concentrations of metals and metallothioneins (MT) in different tissues also were determined. The viscera contained the highest Ag, Cd, and MT concentrations after metal exposure. After exposure to waterborne metals, the metal and MT concentrations in the gills were higher than those in the remaining tissues (mainly muscles and bones), but this pattern was reversed following exposure to dietary metals. The assimilation efficiencies (AEs) of Cd and Ag ranged from 6 to 24% and 15 to 30%, respectively. The rate constant of uptake from the dissolved phase (ku) of Cd and Ag ranged from 2.2 to 7.5 and 8.0 to 31.7 L kg(-1) d(-l), respectively. In all the exposure experiments, the ku and AE increased with induced MT concentration and tissue metal concentration. Increasing metal accumulation may have been due to the increased available binding sites following the induction of MT in the fish. Furthermore, the MT induced by either Cd or Ag was not specific, but was able to bind with both metals and enhance bioaccumulation. Exposure to dissolved and dietary metals may increase metal accumulation, which potentially may lead to metal toxicity, although the fish may develop a tolerance to metals due to the apparent induction of MT.     

金属硫蛋白基因亚型表达在镉接触中作为生物标志物的研究

目的探讨人外周血淋巴细胞（HPBIs）中金属硫蛋白（MT）基因业型的表达在镉接触中作为生物标志物的可行性。方法选择职业接触镉的人群，进行接触评定和效应评定，分离淋巴细胞，用RT-PCR测定MT基因亚型的表达水平。结果接触组工人的MT-1A、1E、1F、1X和MT-2A mRNA表达水平明显高于对照组，差异有统计学意义（P〈0．05）。随血镉水平增高，MT-1A1E、1F、1X和MT-2A mRNA表达水平都明显增高（P〈0．05）。随尿镉水平增高，MT-1A、1E、1F、1X和MT-2A mRNA表达水平有升高趋势，且MT-1A和MT-2A mRNA表达水平明显升高（P〈0．05）。MT-1A、1E、1F、1X和MT-2A mRNA表达水平与血镉水平明显相关（P〈0．05），同时MT-1A、1F、MT-2A mRNA表达水平与尿镉水平明显相关（P〈0．05）。血镉与MT-1E、MT-1F、MT-1X、MT-2A mRNA基础表达水平存在剂量-效应关系；血镉、β2-MG和尿金属硫蛋白（UMT）与MT-1A基础表达水平存在剂量-效应关系。结论HPBLs中MT基因亚型的表达可作为镉接触性生物标志物，其中MT-1A还可作为镉致肾毒性的效应性生物标志物。     

Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. II. A differential biomarker approach under laboratory and field conditions

The two function-specific metallothionein (MT) isoforms characterized from the midgut gland and mantle tissue of Helix pomatia differ substantially in their metal-binding preferences, as well as molecular and biochemical features. These differences make them potential candidates for biomarker studies based on a differential, isoform-specific approach. To prove this hypothesis, induction experiments with two metals (Cd and Cu) that are normally bound by the two isoforms were compared with a range of organic chemicals and physical stressors under laboratory conditions to test the responsiveness of the two isoforms to the stressors applied. In addition, field studies were conducted with Roman snails and substrate samples collected from different metal-contaminated sites in Austria to test the suitability of the two isoforms as biomarkers under field conditions. The results of these combined laboratory and field studies confirmed the validity of the biomarker approach with the two metal- and tissue-specific isoforms. It is demonstrated that the Cd-binding MT specifically and exclusively responds to Cd exposure by increasing concentrations, whereas the Cu-binding MT isoform decreases in its concentration upon exposure to physical stress (X-ray irradiation and cold). This suggests researchers should adopt, under certain preconditions, a dual biomarker approach by combining the simultaneous quantification of Cd-MT concentrations in the midgut gland as a biomarker for Cd pollution and of Cu-MT concentration in the mantle as a biomarker for the impairment of snails by additional physical stressors.     

In situ potential use of metallothionein as a biomarker of cadmium contamination in Ruditapes decussatus

Cadmium (Cd) and metallothionein (MT) concentrations were analysed in the gills and digestive gland of clams Ruditapes decussatus collected from two sites of the Gulf of Gabès (Tunisia) which differ by their degree of pollution. The effect of biotic (maturity stages, protein, sex and body mass) and abiotic factors (temperature, salinity, site and season) on Cd and MT concentrations showed that in the reference site “Bordj d’Ungha” physiological changes caused by gamete development and maturity contributed more to changes in MT concentrations than the bioavailable Cd concentrations; whereas, in the contaminated site “El Hofra”, MT induction responded to raised Cd bioavailabilities rather than to physiological changes. With a view to using MTs as metal exposure biomarker in monitoring program, our results show that the analysis in the digestive gland seem to be more relevant than in gills, and that males appear as the most promising candidates compared to females in the determination of this biomarker.     

Differential metallothionein induction patterns in fed and starved carp (Cyprinus carpio) during waterborne copper exposure

Starved and fed carp (Cyprinus carpio) were exposed to sublethal waterborne copper exposure (1 microM) for 28 d in softened Antwerp, Belgium, city tap water. Copper accumulation in liver and gill tissues was determined, and changes in branchial Na+/K+-adenosine 5'-triphosphatase (ATPase) activity and metallothionein (MT) induction in gill and liver tissues were investigated following 28-d copper exposure. Gill Na+/K+-ATPase activity in exposed fish, both starved and fed, was at its lowest values after 3 d of exposure, after which it slowly recovered to preexposure values. No significant differences in branchial Na+/K+-ATPase activity were found between starved and fed fish. Copper accumulation in the liver and gills of the exposed starved carp was significantly higher than that in the exposed fed carp. The highest MT induction was found in liver tissues. Different patterns of MT induction were observed in the starved and fed carp during copper exposure. Before exposure, MT concentrations in the livers of the starved fish were significantly higher than those in the fed ones. Copper exposure significantly increased MT concentration in the liver of the fed fish, but no changes occurred in the starved fish. In contrast, copper exposure increased MT concentrations in the gills of the starved fish during the first week of exposure, whereas only a slight increase in MT concentration in the gills of the fed fish was observed. When taking into account the role of feeding status in MT induction, hepatic MT was a more relevant indicator for long-term monitoring of copper pollution in carp, but gill MT provided useful information regarding short-term copper toxicity.     

Differentiating copper and arsenic toxicity using biochemical biomarkers in Asellus aquaticus and Dreissena polymorpha

Biomarkers of metal exposure are well known, but how a suite of such biomarkers will respond if the metal is also an oxidizing agent or causes oxidative stress is unclear. This study compares the effects of copper and arsenic, two metals with different oxidizing potential, on freshwater invertebrates. Dreissena polymorpha and Asellus aquaticus were exposed to nominal concentrations of copper (100 microg/L) or arsenic (80 microg/L) over 7 days, and physiological stress was examined by measuring metallothionein (MT) induction, thiobarbituric acid reactive substances (TBARS), and Na(+)/K(+)-ATPase activity. Both species showed increased levels of MT during 7-day Cu exposure tests and transient changes in lipid peroxidation (TBARS) which decreased to control levels by day 7. Arsenic had no effect on TBARS and only a transitory effect on MT in D. polymorpha over 7 days, although it initially induced lipid peroxidation in A. aquaticus on day 3. No inhibition of the Na(+)/K(+)-ATPase enzyme was observed for exposed organisms, and baseline values reported here, for A. aquaticus, 1.1 micromol Pi/mg/h, and for D. polymorpha, 0.38 micromol Pi/mg/h, are probably the first reported for these species.     

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata)

The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.     

Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.     

Expression of Hepatic Metallothionein Messenger RNA in Feral and Caged Fish Species Correlates with Muscle Mercury Levels

Metallothioneins (MTs) are low-molecular-weight cytosolic proteins that are induced by cellular stress as well as exposure to various heavy metals including mercury. Excessive residues of mercury have recently been identified in various fish species of the lower Quachita River system in Arkansas. Fillets of mature largemouth bass (Micropterus salmoides) collected from Woodard Lake, an ox-bow lake of the Ouachita River, possessed muscle residues of mercury ranging from 0.3 to 1.0 ppm (μg/g). To assess the usefulness of using MT expression as a biomarker of mercury exposure, livers and filets were obtained from feral bass of Woodard Lake. Ouachita served as a control site having mercury residues below detection. Analyses using a ribonuclease protection assay with winter flounder MT cDNA revealed that bass had significantly elevated levels of MT mRNA which correlated (r² = 0.756) with the levels of mercury in muscle fillets. To further explore the water quality of Woodard Lake, 10 juvenile channel catfish were housed in cages and placed where feral collections were made in both sites for 2 weeks. Mercury was not detected in muscle or liver and no significant difference in hepatic MT mRNA was observed. These data demonstrate that MT mRNA expression can be used as a tool to assess exposure to heavy metals and suggest that the elevated levels of mercury in large predatory fish may be due to trophic magnification rather than a single point-source exposure.     

Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha

The effects of metal mixture (Cd+Cu) versus single-metal exposure on total MT response and bioaccumulation were investigated in the freshwater bivalve Dreissena polymorpha. A two-month exposure period, including two levels of contamination, was chosen for each of the two metals: 5, 10 microg/L for Cu, and 2, 20 microg/L for Cd, with mixtures of, respectively, 5 microg/L Cu+2 microg/L Cd, 5 microg/L Cu+20 microg/L Cd, 10 microg/L Cu+2 microg/L Cd, and 10 microg/L Cu+20 microg/L Cd. Total MT contents were assessed by an Ag-saturation method, and metals contents were determined by atomic absorption spectrometry.Results at the whole-organism level showed a significant and early increase of total MT biosynthesis after exposure to Cd. This increase was significantly correlated with Cd bioaccumulation. By contrast, Cu did not modify total MT response, and mussels limited Cu bioaccumulation. The mixture either did not influence or only weakly influenced metal accumulation and MT response to Cu and Cd after long-term exposure. Our results suggest that the form of MT existing in D. polymorpha was not Cu-inducible. This could limit the use of MT in D. polymorpha as a biomarker of heavy metal pollution in freshwater ecosystems.     

Screening and testing for endocrine disruption in fish-biomarkers as "signposts," not "traffic lights," in risk assessment

Biomarkers are currently best used as mechanistic "signposts" rather than as "traffic lights" in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect concentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38-55% (comparable to published CVs of 19-58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the "biomarkers as signposts" approach is a pragmatic way forward in the current risk assessment of EDCs.     

Contaminant Exposure and Biomarker Responses in Spectacled Eiders (Somateria fischeri) from St. Lawrence Island, Alaska

Effects of chemical contaminant exposure may be contributing to the decline of spectacled eiders (Somateria fischeri) nesting in coastal areas of western Alaska. We evaluated chemical exposure and potential effects in 20 male eiders collected near St. Lawrence Island, Alaska. Analytes included metals, trace elements, chlorinated organics, and ¹³⁷Cesium (¹³⁷Cs). Effects of contaminant exposure were evaluated using histopathology and biochemical measures of porphyrin profiles, cytochrome P450 activities, and metallothionein (MT) concentrations. Copper, cadmium, and selenium concentrations were elevated in spectacled eiders when compared to literature values for other marine birds. Only a few samples had trace concentrations of chlorinated organic compounds. Muscle ¹³⁷Cs levels were all below the average minimum quantifiable concentration of 0.079 Bq/g. No histopathological lesions were associated with elevated contaminant concentrations in liver, kidney, or testes. Protoporphyrin was found in highest concentration in both the liver and kidneys, followed by coproporphyrin and uroporphyrin, respectively. Hepatic uroporphyrin concentrations correlated significantly to hepatic arsenic concentrations. Mean activities of hepatic EROD, MROD, BROD, and PROD were consistent with other avian species. Comparisons of cadmium/MT ratios from this study to published literature ratios in seven marine avian species suggest that, although adult male spectacled eiders have elevated liver concentrations of certain MT-inducing metals, their MT concentrations are not as strongly induced as would be predicted based on literature values. Despite elevated metal concentrations, the apparent good health of the St. Lawrence Island birds suggests that should these contaminants be a factor in population declines, they likely act by decreasing fecundity or survival of young rather than via direct health impacts on adult male spectacled eiders. Received: 17 March 1999/Accepted: 7 June 1999     

金属硫蛋白在动物体内的功能及其基因表达调控

金属硫蛋白(metallothioneins,MTs)是一类富含金属和巯基的低分子量蛋白质,在动物体内金属元素的代谢、清除自由基、应激防护等方面发挥重要作用。MT基因的表达是通过MT基因启动子上的几种反应元件由许多生化介质引发的。综述了MT在动物体内的功能及其基因表达的调控作用及主要机制。     

Application of reliability models to studies of biomarker validation.

We present a model of biomarker validation developed in our laboratory, the results of the validation study, and the impact of the estimation of the variance components on the design of future molecular epidemiologic studies. Four different biomarkers of exposure are illustrated: DNA-protein cross-link (DNA-PC), DNA-amino acid cross link (DNA-AA), metallothionein gene expression (MT), and autoantibodies to oxidized DNA bases (DNAox). The general scheme for the validation experiments involves n subjects measured on k occasions, with j replicate samples analyzed on each occasion. Multiple subjects, occasions, and replicates provide information on intersubject, intrasubject, and analytical measurement variability, respectively. The analysis of variance showed a significant effect of batch variability for DNA-PC and MT gene expression, whereas DNAox showed a significant between-subject variability. Among the amino acids tested, cysteine and methionine showed a significant contribution of both batch and between-subject variability, threonine showed between-subject variability only, and tyrosine showed between-batch and between-subject variability. The total variance estimated through the experiment was used to calculate the minimum sample size required for a future epidemiologic study including the same biomarkers used for the reliability study. Such validation studies can detect the various components of variability of a biomarker and indicate needed improvements of the assay, along with possible use in field studies.     

Scenario-based simulation of runoff-related pesticide entries into small streams on a landscape level

This paper reviews recent studies on the influence of trace metal exposure history on subsequent metal uptake in marine invertebrates, particularly bivalves and barnacles. Trace metal exposure may induce specific metal-binding ligands. Metallothionein-like proteins appear to play an important role in mediating metal uptake and hence accumulation, especially for Cd. Other ligands such as sulfide are important for Ag biokinetic changes in bivalves. The incorporation of trace metals into metal-rich granules appears to have little if any effect in modifying trace metal uptake. Furthermore, metals also interact strongly in their accumulation by marine animals. Generally, dissolved Hg uptake is reduced following exposure to other metals such as Ag, Cd, Cu, and Zn. Preexposure of marine mussels to Zn also causes concomitant changes in the uptake of other metals such as Cd sharing similar transport pathways. In general, barnacles are less responsive than mussels to trace metal exposure, presumably because of the dominance of detoxificatory pathways in which the accumulated metals are bound in granules (relatively biologically inactive pools). The tissue body burden and the detoxificatory fate of metals in animals seem to be more important in affecting metal accumulation than the nature of the exposure routes (aqueous vs. dietary) or of the exposure regimes. Trace metal accumulation may also be variable in different natural populations of bivalves as a result of different physicochemical environments and histories of exposure. We finally suggest a few areas for future research in this field.