Insights on the toxicity of selected rare earth elements in rainbow trout hepatocytes.

The increasing extraction of rare earth elements (REEs) for technology applications raised concerns for contamination and toxicity in the environment. The purpose of this study was to examine the toxicity of the following REEs in primary cultures of rainbow trout hepatocytes: yttrium (Y), samarium (Sm), gadolinium (Gd), terbium (Tb) and lutetium (Lu). Hepatocytes were exposed to increasing concentrations of the above elements for 24 h at 15 °C and they were analyzed for viability, metallothioneins (MT), glutathione-S-transferase (GST) and arachidonate cyclooxygenase (COX) as markers of oxidative stress and inflammation. The results revealed that the cytoxicity of REEs were as follows in decreasing order: Y > Sm > Lu > Tb > Gd in concordance with published rainbow trout mortality data. While effects on GST and COX activities were marginal, MT levels were more strongly increased with the 2 most toxic REEs (Y and Sm) and Gd, while MT levels were decreased in the least toxic ones (Tb, Lu). While cell viability followed published trout mortality data, it also followed the redox potential and the glutathione affinity constant (log k). The capacity to induce/decrease MT levels was associated with ionic radius, log k (glutathione) and electronegativity. A proposed mechanism of toxicity for REEs is presented based on the chemical properties of REEs, namely the glutathione binding constant and ionic radius, in light of the observed effects in trout hepatocytes.

Gene expression changes and toxicity of selected rare earth elements in rainbow trout juveniles.

Rare earth elements (REEs) are increasingly used in electronics industry and other areas of our economy and questions were raised about their impacts to the environment. The purpose of this study was to examine the lethal and sublethal toxicity of REEs in juvenile rainbow (Oncorhynchus mykiss) trout. The fish were exposed to increasing concentrations (0.064, 0.32, 1.6, 8 and 40 mg/L) of the following 7 REEs for 96 h at 15 °C: cerium (CeCl3), erbium (ErCl3), gadolinium (GdCl3), lanthanum (LaCl3), neodymium (NdCl3), samarium (SmCl3) and yttrium (YCl3). The mortality were determined and in the surviving fish, 10 target gene transcripts were measured in the liver to track changes in oxidative stress, DNA repair, tissue growth/proliferation, protein chaperoning, xenobiotic biotransformation and ammonia metabolism. The data revealed that Y, Sm, Er and Gd formed a distinct group based on toxicity (mortality) and gene expression changes. Electronegativity was significantly correlated (r = -0.8, p < 0.01) with the lethal concentration (LC50). Gene expression changes occurred at concentration circa 120 times lower than the LC50 and the following transcripts in protein chaperoning (heat shock proteins), DNA repair (growth arrest DNA Damage) and CYP1A1 gene expression involved in the metabolism of coplanar aromatic hydrocarbons were involved. In conclusion, the study revealed that the more electronegative REEs were the most toxic to trout juveniles and produced sublethal effects at concentrations 2 orders of magnitude lower than the lethal concentrations. The toxicity of REEs depends on the elements were toxicity involves specific pathways at the gene expression level.

Influence of cadmium on oxidative stress and NADH oscillations in mussel mitochondria.

Biological organisms evolved to take advantage of recurring environmental factors which enabled them to assimilate and process metabolic energy for survival. Mitochondria display non-linear oscillations in NADH levels (i.e. wave behavior) that result from the balance between NADH production (aerobic glycolysis) and oxidation for ATP synthesis. The purpose of this study was to examine the effects of cadmium (Cd) on mitochondrial NADH oscillations in quagga mussels Dreissena bugensis exposed to 50 and 100 µg/L CdCl2 for 7 days at 15 °C. Metallothionein (MT) levels, thioredoxin reductase (TrxR) activity and NADH oxidation rate were also determined, as were oscillations in NADH and the formation of dissipative structures (turbidity), in isolated mitochondria suspensions. The results show that exposure to Cd readily induced MT levels at both concentrations tested and that TrxR and NADH oxidase activity was induced at 100 µg/L Cd only. In control mussels, NADH levels oscillated in mitochondria suspensions with a natural period of 2 to 2.5 min for up to 40 min. Exposure to Cd increased the complexity of the frequency profile of NADH oscillations and reduced the amplitudes of the natural signal with a period of 2 to 2.5 min. The formation of dissipative structures decreased in response to a Cd concentration of 100 µg/L but increased at a level of 50 µg/L. The amplitudes at the natural frequency were significantly correlated with NADH oxidase activity (r = -0.91) and with the formation of dissipative structures (r = -0.59). We conclude that Cd could alter the natural frequency in oscillations of NADH in mitochondria, thereby contributing to an increase in NADH oxidation rate and disruption of the spatial organization of mitochondria in suspension. In conclusion, changes in the wave behavior of NADH in mitochondria are proposed as a novel biomarker of toxicity in aquatic organisms.

Ecotoxicity responses of the freshwater cnidarian Hydra attenuata to 11 rare earth elements.

Lanthanides are the major family of rare earth elements (REEs) owing to the essential properties these metallic species provide in diverse fields of today's world economy. They are now being mined and produced as never before. This raises new environmental concerns in terms of their expected future discharges notably to aquatic systems. Interspecies studies of their ecotoxicity are sparse and effects on aquatic life are still poorly understood. Absence of such information for cnidarians, an ecologically relevant freshwater community, thus prompted the present research on REEs toxicity using Hydra attenuata as our animal model. Lethal and sublethal ecotoxicity data generated with the 11 REEs displayed LC50 values ranging from 0.21 to 0.77 mg L-1and EC50 values ranging from 0.02 to 0.27 mg L-1, thereby confirming the inherent sensitivity of Hydra to REE exposure at environmentally relevant concentrations. Additionally, two properties of REEs were shown to modulate Hydra (sub)lethal toxicity (LC50 and EC50) which decreases with increasing atomic number and with decreasing ionic radius. Compared to studies carried out with different taxonomic groups, Hydra toxicity responses to REEs proved to be among the most sensitive, along with those of other invertebrate species (i.e., Daphnia magna, Ceriodaphnia dubia, Hyalella azteca), suggesting that members of this community are likely more at risk to eventual REE discharges in aquatic environments. Demonstrated Hydra sensitivity to REE exposure strongly justifies their future use in toxicity testing battery approaches to evaluate liquid samples suspected of harbouring REEs.

Evaluation of toxicological effects induced by tributyltin in clam Ruditapes decussatus using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy: Study of metabolic responses in heart tissue and detection of a novel metabolite.

Tributyltin (TBT) is a highly toxic pollutant present in many aquatic ecosystems. Its toxicity in mollusks strongly affects their performance and survival. The main purpose of this study was to elucidate the mechanisms of TBT toxicity in clam Ruditapes decussatus by evaluating the metabolic responses of heart tissues, using high-resolution magic angle-spinning nuclear magnetic resonance (HRMAS NMR), after exposure to TBT (10-9, 10-6 and 10-4 M) during 24 h and 72 h. Results show that responses of clam heart tissue to TBT exposure are not dose dependent. Metabolic profile analyses indicated that TBT 10-6 M, contrary to the two other doses tested, led to a significant depletion of taurine and betaine. Glycine levels decreased in all clam groups treated with the organotin. It is suggested that TBT abolished the cytoprotective effect of taurine, betaine and glycine thereby inducing cardiomyopathie. Moreover, results also showed that TBT induced increase in the level of alanine and succinate suggesting the occurrence of anaerobiosis particularly in clam group exposed to the highest dose of TBT. Taken together, these results demonstrate that TBT is a potential toxin with a variety of deleterious effects on clam and this organotin may affect different pathways depending to the used dose. The main finding of this study was the appearance of an original metabolite after TBT treatment likely N-glycine-N'-alanine. It is the first time that this molecule has been identified as a natural compound. Its exact role is unknown and remains to be elucidated. We suppose that its formation could play an important role in clam defense response by attenuating Ca2+ dependent cell death induced by TBT. Therefore this compound could be a promising biomarker for TBT exposure.

HRMAS NMR as a tool to study metabolic responses in heart clam Ruditapes decussatus exposed to Roundup®.

The essence of this study was to investigate the metabolic responses of heart tissues of carpet-shell clam Ruditapes decussatus after exposure to two doses (0.2 and 1 g/L) of Roundup(®) during 24 and 72 h. The main metabolic changes after Roundup(®) exposure were related to disturbance in energy metabolism and metabolic biomarkers such as alanine, succinate, acetate and propionate, suggesting the occcurence of anaerobiosis and the impairment of oxydative metabolism. Results showed also that peak intensities of amino acids used as biomarker of anaerobiosis in molluscs are time and dose dependent. In the opposite, phosphoarginine and ATP level are dependent to Roundup(®) concentration rather than to the time of exposure. We suggest that changes in energy demands require adjustements in the forward arginine kinase reaction rate. Therefore, the results demonstrate the high applicability of HRMAS NMR to elucidate the mechanism of toxicity of Roundup(®). In addition, (31)P HRMAS NMR appeared to be an effective and simple method to follow bioaccumulation of Roundup(®) formulation.

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus.

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I (K slow)) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I (K fast)) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I (KCa)) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I (Ca)) inhibited by verapamil at 5 × 10(-4) M, T-type Ca(2+) current, rapidly activated and inactivated, and sodium current (I (Na)) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 µM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2'-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.