Role of metallothioneins in superoxide radical generation during copper redox cycling: defining the fundamental function of metallothioneins.

In order to demonstrate the in vivo antioxidant properties of metallothioneins (MTs), the bacteria Escherichia coli was used as a cell reactor in which we compared the metal binding and antioxidative functions of MTs from different species, with different structures and polypeptide lengths. No protective effects of cytoplasmic MTs from cadmium (Cd) or zinc (Zn) contamination were observed in a wild-type E. coli strain, although these MTs can efficiently bind both Cd and Zn. To test their antioxidant properties, MTs were expressed within the cytoplasm of a sodA sodB deficient mutated strain (QC1726). However, a paradoxical MT toxicity was found when this strain was contaminated with Cd and Zn, suggesting that in a wild-type strain, superoxide dismutase counteracts MT toxicity. The most toxic MT was the one with the strongest Cd and Zn binding capacities. This toxic effect was linked to the generation of superoxide radicals, since a Cd-contaminated QC1726 strain expressing oyster MT isoforms produced 75-85% more O(2)*(-) than the control QC1726 strain. Conversely, under anaerobiosis or in the presence of a copper chelator, MTs protected QC1726 strain from Cd and Zn contamination. A model is proposed to explain the observed MT toxicity.

Cytochrome c oxydase subunit I gene is up-regulated by cadmium in freshwater and marine bivalves.

Inhibition of the mitochondrial electron transfer chain and induction of reactive oxygen species (ROS) production are one of the roots of cadmium (Cd) toxicity. To appreciate the impact of Cd on mitochondria, we focused on the expression of CoxI gene which encodes the subunit I of the Cytochrome c oxidase (complex IV of the respiratory chain). CoxI gene expression was studied by real-time quantitative PCR in three species: two freshwater bivalves (Corbicula fluminea and Dreissena polymorpha) and one marine bivalve (diploid or triploid Crassostrea gigas). Bivalves were exposed for 10 or 14 days to 0.13 microM Cd(2+) and 15.3 microM Zn(2+) in controlled laboratory conditions. We demonstrate that in the three mollusk species CoxI gene was up-regulated by Cd. Zinc (Zn), which is known to have antioxidant properties, had no effect on CoxI gene expression. In the presence of Cd and Zn, CoxI gene inducibility was lower than after a single Cd exposure, in each species; result that could not be fully explained by a decreased Cd accumulation. CoxI gene induction by Cd was 4.8-fold higher in triploid oysters than in diploid ones, indicating a possible influence of triploidy on animal responses to Cd contamination.

Heterologous expression of bacterial and human multidrug resistance proteins protect Escherichia coli against mercury and zinc contamination.

In order to determine the role of multidrug resistance proteins in mercury and zinc resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA genes were expressed in an Escherichia coli tolC mutant which is hypersensitive to metals. The three transporters conferred an increased mercury and zinc resistance to E. coli as compared to the control bacteria. This improved resistance correlated with a decreased zinc and mercury bioaccumulation. Indeed, quantification of intracellular metal concentrations by atomic absorption spectrometry (AAS) showed a 2.1-, 3-, and 5.1-fold decrease in zinc in cells expressing hMDR1, omrA, and lmrA, respectively, and a 2.7-, 7.5-, and 7.7-fold decrease in mercury in cells expressing omrA, lmrA, and hMDR1, respectively, as compared to the control bacteria. This means that hMDR1, LmrA, and OmrA proteins which are specialised in xenobiotic scavenging, their main known function, are nevertheless able to confer some resistance against metals. Our results show that the tolC mutated strain is well adapted to the study of MDR transporter activity and could be used to screen substrates and competitive hMDR1 inhibitors.

cDNA cloning and gene expression of ribosomal S9 protein gene in the mollusk Corbicula fluminea: a new potential biomarker of metal contamination up-regulated by cadmium and repressed by zinc.

Biological indicators can be used to assess polluted sites, but their success depends on their specificity. The aim of the present study was to identify a specific cadmium biomarker of the freshwater bivalve Corbicula fluminea. Differential display technique was used to identify new molecular indicators for cadmium exposure. Gene expression of C. fluminea transplanted into a cadmium- and zinc-contaminated river was compared to gene expression of bivalves from a reference site. One differentially expressed band was identified, and the entire cDNA sequence coding for the ribosomal protein S9 (rpS9) was cloned. The rpS9 gene expression was studied by real-time reverse transcription-polymerase chain reaction. After controlled cadmium (15 microg/L) and/or zinc (1 mg/L) laboratory contaminations of the bivalves, this gene was shown to be up-regulated by cadmium and down-regulated by zinc. A 48-d transplantation of animals into a polymetallic field contamination in the Lot river basin (Midi-Pyrénées region, France) showed that rpS9 gene regulation was correlated with the fluctuating cadmium and zinc water contents. To our knowledge, the existence of a gene that is up-regulated by cadmium and repressed by zinc is demonstrated here for the first time.

Impact of cadmium contamination and oxygenation levels on biochemical responses in the Asiatic clam Corbicula fluminea.

The aim of this work was to evaluate the potential utility of several biochemical parameters as indicators of the toxic effects of cadmium in the freshwater clam Corbicula fluminea under two levels of oxygenation (normoxia 21 kPa and hypoxia 4 kPa). These variations in oxygenation are representative of the natural environments of bivalves living at the bottom of the water column, where hypoxic episodes may occur regularly. Cadmium accumulation, metallothionein synthesis, MXR protein induction, lipoperoxidation and antioxidant enzyme activities (catalase, glutathione reductase and total and selenium-dependent glutathione peroxidases) were assessed in the gills of C. fluminea in four experimental conditions: normoxia, hypoxia, normoxia with cadmium and hypoxia with cadmium ([Cd]=30 microg l(-1)) over a 14-day period. Behavioural reactions were also followed for the duration of the experiment by monitoring clam activity and valve movements. This study is a first report on biochemical responses under cadmium contamination and hypoxia and will enable us to determine better biomarkers for C. fluminea as they were measured simultaneously. In metal-exposed animals, we found an increasing accumulation of cadmium in the gills with time, and this was more severe in hypoxic conditions. Metallothionein synthesis occurred in contaminated clams and was precocious in hypoxic conditions. MXR protein induction appeared promising due to its quick and significant response to metal with a strong impact from hypoxic contamination. On the other hand, in our experimental conditions, antioxidant parameters did not show decisive responses to contamination and hypoxia, except glutathione peroxidases which decreased systematically with time in a cadmium-independent manner. Lipid peroxidation, expressed as malondialdehyde content, was not stimulated by normoxic contamination, as has been shown in other studies, but was stimulated under hypoxic cadmium contamination. Our study confirms the importance of a multi-biomarker approach in environmental studies as some are not appropriate to all organisms.

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli.

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 microM verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent (109)Cd(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities.