Catherine Lecomte-Pradines

Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase.

ABSTRACT Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.

Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna.

Acute uranium toxicity (48 h immobilisation test) for Daphnia magna was determined in two different exposure media, differing in pH and alkalinity. LC(50) varied strongly between media, from 390+/-40 microgL(-1)U at pH 7 to 7.8+/-3.2 mgL(-1)U at pH 8. According to the free ion activity model uranium toxicity varies as a function of free uranyl concentration. This assumption was examined by calculating uranium speciation in our water conditions and in those reported in the literature. Predicted changes in free uranyl concentration could not solely explain observed differences in toxicity, which might be due to a competition or a non-competitive inhibition of H(+) for uranium transport and/or the involvement of other bioavailable chemical species of uranium. Chronic effects of uranium at pH 7 on mortality, ingestion and respiration, fecundity and dry mass of females, eggs and neonates were investigated during 21-day exposure experiments. A mortality of 10% was observed at 100 microgL(-1)U and EC(10) for reproduction was 14+/-7 microgL(-1)U. Scope for growth was affected through a reduction in feeding activity and an increase in oxygen consumption at 25 microgL(-1)U after 7 days of exposure. This had strong consequences for somatic growth and reproduction, which decreased, respectively, by 50% and 65% at 50 microgL(-1)U after 7 days and at 25 microgL(-1)U after 21 days. Uranium bioaccumulation was quantified and associated internal alpha dose rates from 2.1 to 13 microGyh(-1) were estimated. Compared to the toxicity of other alpha-emitting radionuclides and stable trace metals, our results confirmed the general assumption that uranium chemical toxicity predominates over its radiotoxicity.

Heavy metal accumulation by recombinant mammalian metallothionein within Escherichia coli protects against elevated metal exposure.

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins known to provide protection against toxic heavy metals such as cadmium. In an attempt to increase the ability of bacterial cells to accumulate heavy metals, sheep MTII was produced in fusion with the maltose binding protein (MBP) and localized to the cytoplasmic or periplasmic compartments of Escherichia coli. For all metals tested, higher levels of bioaccumulation were measured with strains over-expressing MBP-MT in comparison with control strains. A marked bioaccumulation of Cd, As, Hg and Zn was observed in the strain over-expressing MBP-MT in the cytoplasm, whereas Cu was accumulated to higher levels when MBP-MT was over-expressed in the periplasm. Metal export systems may also play a role in this bioaccumulation. To illustrate this, we over-expressed MBP-MT in the cytoplasm of two mutant strains of E. coli affected in metal export. The first, deficient in the transporter ZntA described to export numerous divalent metal ions, showed increasing quantities of Zn, Cd, Hg and Pb being bioaccumulated. The second, strain LF20012, deficient in As export, showed that As was bioaccumulated in the form of arsenite. Furthermore, high quantities of accumulated metals, chelated by MBP-MT in the cytoplasm, conferred greater metal resistance levels to the cells in the presence of added toxic metals, such as Cd or Hg, while other metals showed toxic effects when the export systems were deficient. The strain over-expressing MBP-MT in the cytoplasm, in combination, with disruption of metal export systems, could be used to develop strategies for bioremediation.

Nested interactions in the combined toxicity of uranium and cadmium to the nematode Caenorhabditis elegans

Uranium is a natural, ubiquitous radioactive element for which elevated concentrations can be found in the vicinity of some nuclear fuel cycle facilities or intensive farming areas, and most often in mixtures with other contaminants such as cadmium, due to co-occurrence in geological ores (e.g. U- or P-ore). The study of their combined effects on ecosystems is of interest to better characterize such multi-metallic polluted sites. In the present study, the toxicity of binary mixture of U and Cd on physiological parameters of the soil nematode Caenorhabditis elegans was assessed over time. Descriptive modeling using concentration and response addition reference models was applied to compare observed and expected combined effects and identify possible synergistic or antagonistic interactions. A strong antagonism between U and Cd was identified for length increase and brood size endpoints. The study revealed that the combined effects might be explained by two nested antagonistic interactions. We demonstrate that the first interaction occurred in the exposure medium. We also identified a significant second antagonistic interaction which occurred either during the toxicokinetic or toxicodynamic steps. These findings underline the complexity of interactions that may take place between chemicals and thus, highlight the importance of studying mixtures at various levels to fully understand underlying mechanisms.

Effects of radionuclide contamination on leaf litter decomposition in the Chernobyl exclusion zone.

The effects of radioactive contamination on ecosystem processes such as litter decomposition remain largely unknown. Because radionuclides accumulated in soil and plant biomass can be harmful for organisms, the functioning of ecosystems may be altered by radioactive contamination. Here, we tested the hypothesis that decomposition is impaired by increasing levels of radioactivity in the environment by exposing uncontaminated leaf litter from silver birch and black alder at (i) eleven distant forest sites differing in ambient radiation levels (0.22-15μGyh(-1)) and (ii) along a short distance gradient of radioactive contamination (1.2-29μGyh(-1)) within a single forest in the Chernobyl exclusion zone. In addition to measuring ambient external dose rates, we estimated the average total dose rates (ATDRs) absorbed by decomposers for an accurate estimate of dose-induced ecological consequences of radioactive pollution. Taking into account potential confounding factors (soil pH, moisture, texture, and organic carbon content), the results from the eleven distant forest sites, and from the single forest, showed increased litter mass loss with increasing ATDRs from 0.3 to 150μGyh(-1). This unexpected result may be due to (i) overcompensation of decomposer organisms exposed to radionuclides leading to a higher decomposer abundance (hormetic effect), and/or (ii) from preferred feeding by decomposers on the uncontaminated leaf litter used for our experiment compared to locally produced, contaminated leaf litter. Our data indicate that radio-contamination of forest ecosystems over more than two decades does not necessarily have detrimental effects on organic matter decay. However, further studies are needed to unravel the underlying mechanisms of the results reported here, in order to draw firmer conclusions on how radio-contamination affects decomposition and associated ecosystem processes.

A dynamic energy-based model to analyze sublethal effects of chronic gamma irradiation in the nematode Caenorhabditis elegans

ABSTRACT Understanding how toxic contaminants affect wildlife species at various levels of biological organization (subcellular, histological, physiological, organism, and population levels) is a major research goal in both ecotoxicology and radioecology. A mechanistic understanding of the links between different observed perturbations is necessary to predict the consequences for survival, growth, and reproduction, which are critical for population dynamics. In this context, experimental and modeling studies were conducted using the nematode Caenorhabditis elegans. A chronic exposure to external gamma radiation was conducted under controlled conditions. Results showed that somatic growth and reproduction were reduced with increasing dose rate. Modeling was used to investigate whether radiation effects might be assessed using a mechanistic model based upon the dynamic energy budget (DEB) theory. A DEB theory in toxicology (DEB-tox), specially adapted to the case of gamma radiation, was developed. Modelling results demonstrated the suitability of DEB-tox for the analysis of radiotoxicity and suggested that external gamma radiation predominantly induced a direct reduction in reproductive capacity in C. elegans and produced an increase in costs for growth and maturation, resulting in a delay in growth and spawning observed at the highest tested dose rate.

In situ visualization of carbonylation and its co-localization with proteins, lipids, DNA and RNA in Caenorhabditis elegans

All key biological macromolecules are susceptible to carbonylation - an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.