Molecular cloning and expression of novel metallothionein (MT) gene in the polychaete Perinereis nuntia exposed to metals

To report a novel metallothionein (MT) gene and evaluate its potency as a biomarker, we clone this MT gene and measured the expression levels in the metal-exposed polychaete Perinereis nuntia. Accumulated metal contents and metallothionein-like proteins (MTLPs), which have been recognized as potential biomarkers, were compared with the relative mRNA expressions of the MT gene of P. nuntia (Pn-MT). In addition, the metal-binding affinity was estimated by recombinant Pn-MT protein. Pn-MT having high cysteine residues with three metal response elements in the promoter region closely clusters with those of other invertebrates. The accumulation patterns of metals were dependent on the exposure times in lead (Pb), cadmium (Cd), and copper (Cu) exposure. Particularly, both MTLP levels and relative mRNA expressions of MT were increased with accumulated metal contents and exposure time in P. nuntia exposed to Pb and Cd. There was no significant modulation of the Pn-MT gene in polychaetes exposed to Zn and As. However, the metal-binding ability of the recombinant Pn-MT protein provides a clear evidence for a high affinity of MT to several metal elements. These results suggest that Pn-MT would play an important role in the detoxification and/or sequestration of specific metals (e.g., Pb and Cd) in P. nuntia and have potential as a molecular biomarker in the monitoring of the marine environment using a polychaete.     

Antioxidant defense gene analysis in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Trifolium repens</Emphasis> exposed to Cd and/or Pb

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.     

Cloning of a river pufferfish (Takifugu obscurus) metallothionein cDNA and study of its induction profile in cadmium-exposed fish

We report here the full-length cDNA sequence of metallothionein (MT) gene from an anadromous river pufferfish, Takifugu obscurus (order: Tetradotiformes; family: Tetradontidae). Phylogenetic relationship analysis revealed that the identified MT has high sequence similarity with many Perciformes fish species. The tissue distribution and concentration- and time-dependent expression of MT mRNA were studied in fish exposed to cadmium. Liver showed the highest level of MT gene expression followed by other tissues (brain, gill and kidney) in response to cadmium exposure. Muscle showed a weak expression response of MT gene. Time-course study revealed highest early phase (at 6h) expression in the brain and late persistence of induction in the intestine. MT mRNA expression showed a concentration-dependent expression in all the tissues. However, induction in brain and liver occurred at much lower concentrations as compared to other tissues. Our results demonstrate that MT in T. obscurus is induced by cadmium exposure which indicates that it plays a functionally conserved function of metal detoxification.     

Metallothionein-like proteins in the freshwater oligochaete Limnodrilus udekemianus and their role as a homeostatic mechanism against cadmium toxicity

The purpose of this study was to determine if metallothioneins are present in the aquatic oligochaete Limnodrilus udekemianus and to determine the interplay between the presence of these proteins, cadmium (Cd) exposure, and Cd toxicity. The latter was geared specifically towards evaluating the role of metallothionein as a homeostatic mechanism against Cd toxicity. These issues are important for evaluating the usefulness of the quantification of metallothioneins as a biomonitoring tool. Worms in sediment were exposed to Cd under static conditions, with Cd initially added to the aqueous phase. Survival was monitored while respiration (as a measure of sublethal Cd effects) was determined immediately following exposure. Metallothioneins were separated from the cytosol by gel permeation high performance liquid chromatography (HPLC) while Cd levels were quantified in whole worms, cytosol and cytosolic fractions. Also, a Cd-saturation assay was used to determine the amounts of Cd bound to metallothionein and the total Cd-binding capacity of the metallothionein. Limnodrilus udekemianus has a metallothionein-like protein (an inducible cytosolic protein with an apparent molecular weight of approximately 15 kD that binds high levels of Cd and shows a red shift upon Cd binding). Sediment Cd levels above 60 microg/g were lethal to the worms (in 8-day exposures). Respiration rates at 13 and 41 microg/g Cd were not significantly different from controls, though cytosolic Cd levels were substantially increased in the 41 microg/g exposure. In this latter cytosol, Cd levels were significantly elevated in the low molecular weight pool (which includes metallothioneins) but not in the other pools, while the Cd-saturation assay also showed that worms in this group had significantly elevated levels of metallothionein-bound Cd. However, in all treatments the metallothionein was far from saturated by Cd. These observations indicate that no 'spill-over' of Cd was evident as lethal levels of Cd were approached. The overall cytosolic Cd distribution, and the degree of metallothionein saturation in Limnodrilus udekemianus thus do not appear to be good predictors of Cd toxicity in this species.     

Environmental monitoring by gene expression biomarkers in Barbus graellsii: laboratory and field studies

Barbel (Barbus graellsii) is a freshwater fish used as a sentinel species in environmental monitoring programmes and ecotoxicological studies in northern Spain, particularly in the Ebro River basin, the largest freshwater resource in Spain. We developed specific primers for the quantification of CYP1A and metallothionein (MT)-1 and -2 gene expression by QRT-PCR in barbel in order to assess their suitability in biological effect monitoring of dioxin-like compounds and metals. All three genes responded as expected in laboratory tests, using model inducers. Hepatic CYP1A mRNA levels showed a twofold induction in fish injected intraperitoneally with beta-naphthoflavone, whereas MT-1 and MT-2 gene expression was strongly induced by cadmium (15- and 13-fold, respectively) and mercury (five- and eightfold). Barbel populations from different sites on the Ebro River basin showed a good correlation between the historical records of organochlorine pollution, CYP1A expression levels and EROD activity. Nevertheless, although metallothionein protein levels in the liver of wild fish correlated with hepatic levels of mercury, MT-1 and MT-2 gene expression did not correlate with the mercury content or with the concentration of metals in sediments from the sites inhabited by the fish. These results demonstrate the utility of barbel CYP1A-mRNA expression, but not that of MT-1 or MT-2, as a biomarker in field studies. The tools and protocols developed here are likely to apply to other species of the genus Barbus, with some 700 species distributed throughout most of the Old World.     

Single and double metallothionein knockout in the nematode C. elegans reveals cadmium dependent and independent toxic effects on life history traits

The genome of the nematode Caenorhabditis elegans contains two metallothionein genes, both involved in metal homeostasis and/or detoxification. Single metallothionein knockout mutants have been created and now, for the first time, a double mutant has been isolated. Life history studies in the presence or absence of cadmium showed that all metallothionein mutants are viable. Although cadmium did not influence longevity, a dose dependent reduction in total brood size and volumetric growth was observed in wild type animals, which was magnified in single knockouts and further exacerbated in the double knockout. However, the metallothionein deletion caused two effects that are independent of cadmium exposure, namely all knockout strains displayed a reduced total brood size and the deletion of both metallothionein loci caused a significant reduction in volumetric growth. In summary, metallothionein is undoubtedly an important player in cadmium detoxification, but evidently also an important factor in cadmium independent pathways.     

Variation of zinc and copper in metallothionein-like protein in killifish (Oryzias latipes) exposed to cadmium

Killifish (Oryzias latipes) were exposed for 0, 24, 48 and 168 hours to cadmium solution. The hepatic cytoplasm was fractionated by using Sephadex G-75 and the content of zinc, copper and cadmium in the fractions was measured. Cadmium content in the metallothionein fraction increased with increase of exposure time. Zinc content in the metallothionein fraction was reduced at early exposure time and increased after prolonged exposure. Copper content in the metallothionein fraction was unchanged. It is shown that zinc bound to thionein in the liver is partially replaced by cadmium and that at least two isomers of metallothionein occur in this fish.     

Cadmium as a possible cause of bladder cancer: a review of accumulated evidence

Bladder cancer is a significant disease, the rates of which have increased over the few last years. However, its etiology remains as yet undefined. Cadmium, a widespread environmental carcinogen that has received considerable interest, presents evidence as a possible cause of bladder cancer. A literature review was conducted from the years 1984–2013 to study the accumulated evidence for cadmium as a possible cause of bladder cancer, including routes of cadmium exposure, accumulation, toxicity, carcinogenicity, and evidence from epidemiological and experimental studies. Special reference is devoted to cadmium nephrotoxicity, which illustrates how cadmium exerts its effects on the transitional epithelium of the urinary tract. Mechanisms of carcinogenesis are discussed. The effects of cadmium on gene expression in urothelial cells exposed to cadmium are also addressed. Despite different methodologies, several epidemiologic and nephrotoxicity studies of cadmium indicate that occupational exposure to cadmium is associated with increased risk of bladder cancer and provide additional evidence that cadmium is a potential toxic element in urothelial cells. In vitro studies provide further evidence that cadmium is involved in urothelial carcinogenesis. Animal studies encounter several problems such as morphology differences between species. Among the complex mechanisms of cadmium carcinogenesis, gene expression deregulation is the subject of recent studies on bladder cadmium-induced carcinogenesis. Further research, however, will be required to promise a better understanding of the mechanisms underlying cadmium carcinogenesis and to establish the precise role of cadmium in this important malignancy.     

Sperm count in earthworms (Lumbricus terrestris) as a biomarker for environmental toxicology: effects of cadmium and chlordane

Earthworms, Lumbricus terrestris, exposed in artificial soil to sublethal concentrations of technical chlordane (6.25, 12.5, and 25 ppm) and cadmium nitrate (100, 200, and 300 ppm) exhibited significant reduction in spermatozoa from testes and seminal vesicles. The onset time of reduction varied with exposure concentration, but absolute depression in sperm count was independent of exposure concentration or exposure duration after reduction was first manifested, demonstrating a threshold effect. Earthworm sperm counts show potential as a rapid-measurement endpoint biomarker for measuring sublethal effects of chemical pollutants on reproduction.     

Experimental exposure to cadmium affects metallothionein-like protein levels but not survival and growth in wolf spiders from polluted and reference populations

Both local adaptation and acclimation in tolerance mechanisms may allow populations to persist under metal pollution. However, both mechanisms are presumed to incur (energetic) costs and to trade-off with other life-history traits. To test this hypothesis, we exposed Pardosa saltans (Lycosidae) spiderlings originating from metal-polluted and unpolluted sites to a controlled cadmium (Cd) treatment, and compared contents of metal-binding metallothionein-like proteins (MTLPs), internal metal concentrations, and individual survival and growth rates with a reference treatment. While increased MTLP concentrations in offspring originating from both polluted and unpolluted populations upon exposure indicates a plastic tolerance mechanism, survival and growth rates remain largely unaffected, independent of the population of origin. However, MTLP and Cd concentrations were not significantly correlated. We suggest that MTLP production may be an important mechanism enabling P. saltans populations to persist in ecosystems polluted with heavy metals above a certain level.     

Proteomic analysis in the lichen Physcia adscendens exposed to cadmium stress

This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 μM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.     

Cadmium in blood of Tunisian men and risk of bladder cancer: interactions with arsenic exposure and smoking

Prior investigations identified an association between low-level blood arsenic (As) and bladder cancer risk among Tunisian men but questions remain regarding confounding by cadmium (Cd), a well-established bladder carcinogen. A case–control study of Tunisian men was re-examined to assess the levels of cadmium in blood and reparse the association between the simultaneous exposure to these metals and bladder cancer risk. Levels of blood Cd were significantly twice higher among cases than in controls (P?<?0.05) and were positively correlated with smoking and age. Additionally, analysis of metal levels among non-smokers according to the region of residence showed very high blood Cd and As levels for the coastal regions of Sfax and central Tunisia. After controlling for potential confounders, for low blood As levels (<0.67 μg/L), the OR for blood Cd was 4.10 (95 % CI 1.64–10.81), while for higher levels (>0.67 μg/L), it was reduced to 2.10 (CI, 1.06–4.17). Adjustment for Cd exposure did not alter the risk associated to As exposure. This study is the first to report the relationship between Cd exposure and risk of bladder cancer occurrence in interaction with smoking and As exposure. Smoking is shown to be the main exposure source to Cd in the Tunisian population but also environmental pollution seems to be responsible of Cd exposure among non-smokers. Exposure assessment studies encompassing a wider population are needed.     

Correlation of foliar MT2b expression with Cd and Zn concentrations in hybrid aspen (Populus tremula × tremuloides) grown in contaminated soil

Metal uptake and its effect on foliar metallothionein 2b (MT2b) mRNA levels were studied in hybrid aspen (Populus tremula × tremuloides) in field conditions. The trees were planted on a site contaminated with several metals, including cadmium (mean 5.1 mg kg⁻¹), chromium (80 mg kg⁻¹), copper (180 mg kg⁻¹), nickel (81 mg kg⁻¹), vanadium (240 mg kg⁻¹) and zinc (520 mg kg⁻¹). Of the ten trace elements analyzed, only Cd and Zn accumulated in the leaves with maximal foliar concentrations of 35 and 2400 mg kg⁻¹ (dry weight), respectively. There was a strong correlation between Cd and Zn concentrations and bioaccumulation factors (concentration in plant/soil) in the leaves, branches and roots, suggesting similar transport mechanisms for these two metals. The levels of MT2b correlated with Cd and Zn concentrations in the leaves, demonstrating that increased MT2b expression is one of the responses of hybrid aspen to chronic metal exposure.     

Effects of the pharmaceuticals gemfibrozil and diclofenac on biomarker expression in the zebra mussel (Dreissena polymorpha) and their comparison with standardised toxicity tests

Pharmaceuticals, including the lipid regulator gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac have been identified in waste water treatment plant effluents and receiving waters throughout the western world. The acute and chronic toxicity of these compounds was assessed for three freshwater species (Daphnia magna,Pseudokirchneriella subcapitata, Lemna minor) using standardised toxicity tests with toxicity found in the non-environmentally relevant mid mg L⁻¹ concentration range. For the acute endpoints (IC₅₀ and EC₅₀) gemfibrozil showed higher toxicity ranging from 29 to 59 mg L⁻¹ (diclofenac 47-67 mg L⁻¹), while diclofenac was more toxic for the chronic D. magna 21 d endpoints ranging from 10 to 56 mg L⁻¹ (gemfibrozil 32-100 mg L⁻¹). These results were compared with the expression of several biomarkers in the zebra mussel (Dreissena polymorpha) 24 and 96 h after exposure by injection to concentrations of 21 and 21,000 μg L⁻¹ corresponding to nominal concentrations of 1 and 1000 μg L⁻¹. Exposure to gemfibrozil and diclofenac at both concentrations significantly increased the level of lipid peroxidation, a biomarker of damage. At the elevated nominal concentration of 1000 μg L⁻¹ the biomarkers of defence glutathione transferase and metallothionein were significantly elevated for gemfibrozil and diclofenac respectively, as was DNA damage after 96 h exposure to gemfibrozil. No evidence of endocrine disruption was observed using the alkali-labile phosphate technique. Results from this suite of biomarkers indicate these compounds can cause significant stress at environmentally relevant concentrations acting primarily through oxidation pathways with significant destabilization of the lysosomal membrane and that biomarker expression is a more sensitive endpoint than standardised toxicity tests.