Evaluation of general stress, detoxification pathways, and genotoxicity in rainbow trout exposed to rare earth elements dysprosium and lutetium.

Rare earth elements (REEs) have been recently identified as emergent contaminants because of their numerous and increasing applications in technology. The impact of REEs on downstream ecosystems, notably aquatic organisms, is of particular concern, but has to date been largely overlooked. The purpose of this study was thus to evaluate the toxicity of lanthanide metals, lutetium (Lu) and dysprosium (Dy) in rainbow trout after 96 h of exposure. The lethal concentration (LC50) was determined and the expression of 14 genes involved in different pathways such as oxidative stress, xenobiotic detoxification, mitochondrial respiration, DNA repair, protein folding and turnover, inflammation, calcium binding and ammonia metabolism were quantified in surviving fish. In parallel, lipid peroxidation (LPO), DNA damage (DSB), metallothionein level (MT) and cyclooxygenase activity (COX) were examined. The acute 96 h-LC50 data revealed that Lu was more toxic than Dy (1.9 and 11.0 mg/L, respectively) and was able to affect all investigated pathways by changing the expression of the studied genes, to the exception of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). It also induced a decrease in DNA repair at concentrations 29 times below the LC50. This suggests that Lu could trigger a general stress to disrupt the cell homeostasis leading to genotoxicity without promoting oxidative stress. However, Dy induced modulation in the expression of genes involved in the protection against oxidative stress, detoxification, mitochondrial respiration, immunomodulation, protein turnover and an increase in the DNA strand breaks at concentrations 170 times lower than LC50. Changes in mRNA level transcripts could represent an early signal to prevent against toxicity of Dy, which exhibited inflammatory and genotoxic effects. This study thus provides useful knowledge enhancing our understanding of survival strategies developed by rainbow trout to cope with the presence of lanthanides in the environment.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Biological fabrication of cellulose fibers with tailored properties.

Cotton is a promising basis for wearable smart textiles. Current approaches that rely on fiber coatings suffer from function loss during wear. We present an approach that allows biological incorporation of exogenous molecules into cotton fibers to tailor the material's functionality. In vitro model cultures of upland cotton (Gossypium hirsutum) are incubated with 6-carboxyfluorescein-glucose and dysprosium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-glucose, where the glucose moiety acts as a carrier capable of traveling from the vascular connection to the outermost cell layer of the ovule epidermis, becoming incorporated into the cellulose fibers. This yields fibers with unnatural properties such as fluorescence or magnetism. Combining biological systems with the appropriate molecular design offers numerous possibilities to grow functional composite materials and implements a material-farming concept.

Evidence for in vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

Dysprosium-sensitized chemiluminescence system for the determination of enoxacin in pharmaceutical preparations and biological fluids with flow-injection sampling.

A novel trivalence dysprosium(Dy(3+))-sensitized chemiluminescence method was developed for the first time for the determination of enoxacin (ENX) using flow-injection sampling based on the chemiluminescence (CL) associated with the reaction of the Dy(3+)-cerium(Ce(IV))-S(2)O(3) (2-)-ENX system and the Dy(3+)-MnO(4) (-) S(2)O(3) (2-)-ENX system. The analytical conditions for CL emission were investigated and optimized. The relationship between the CL intensity of ENX and its concentration has good linearity, with a correlation coefficient of 0.9984-0.9994. The limit of detection (LOD, 3sigma) was 0.20 ng/mL for the Dy(3+)-ENX-S(2)O(3)(2-)-Ce(IV)-H(2)SO(4) system and 0.22 ng/mL for the Dy(3+)-ENX-S(2)O(3)(2-)-MnO(4) (-)-HNO(3) system. The relative standard deviation (RSD, n = 11) was 1.8% for 11 determinations of 60 ng/mL ENX. The proposed method was applied to the analysis of ENX in injections, serum and urine samples with a recovery of 98%-105%. A possible mechanism for this sensitized CL reaction is discussed by comparing the CL spectra with the fluorescence emission spectra. The proposed method represents a wide linear range, high sensitivity and accuracy, and can be used for the routine determination of ENX in pharmaceutical preparations and biological fluids.

The 33 kDa protein of photosystem II is a low-affinity calcium- and lanthanide-binding protein.

We have shown that the isolated 33 kDa protein of photosystem II contains one calcium and one lanthanide low-affinity binding site with binding constants (K(D)) on the order of 10(-5) M. Binding of calcium or lanthanides to this site induces conformational changes in the protein that manifest in fluorescence emission spectra of the protein, circular dichroism spectra, and calorimetric thermograms where the phase transitions are shifted to lower temperatures. The role of calcium binding to the 33 kDa protein in the attainment of its native structure and the significance of this interaction for the oxygen evolution process are discussed.

Lanthanide induced residual dipolar couplings for the conformational investigation of peripheral 15NH2 moieties.

The Ca2 calbindin protein in which one calcium has been substituted with Ce(III), Yb(III) and Dy(III) displays substantial alignment in high magnetic fields due to the high anisotropy of the metal magnetic susceptibility. This property has allowed the measurement of residual dipolar coupling contributions to 1J(HN) and 2J(HH) couplings of asparagine and glutamine NH2 moieties. Such data have been used to aid structural characterization of these groups. The exploitation of auto-orientation of magnetic anisotropic metalloproteins represents a step ahead in the investigation of the conformational space of peripheral residues that are not fixed by the protein folding.

Rare earth elements as nonabsorbable fecal markers in studies of iron absorption.

The use of rare earth elements as nonabsorbable fecal markers for studies of iron absorption from sources labeled extrinsically with stable isotopes was evaluated. On 3 successive days 13 healthy fasting adults were given different stable isotopes of iron with samarium, ytterbium, or dysprosium. On day 1, three meals were given with 57Fe (1 mg per meal) plus samarium (0.33 mg per meal); on day 2, identical meals (taken with a calcium supplement to reduce iron bioavailability) were given with equivalent amounts of 58Fe-labeled iron and ytterbium; on day 3, a well-absorbed reference dose of 54Fe (3 mg) was given with 1 mg Dy. A complete fecal collection was carried out for 5-9 d and each stool was analyzed for rare earth elements by inductively coupled plasma-mass spectrometry and iron isotopes by thermal ionization quadrupole mass spectrometry. Mean recovery of rare earth elements was 101%, indicating that they are totally unabsorbed. The excretory pattern of the iron isotopes and the rare earth elements was very similar; the correlation coefficients between samarium and 57Fe, ytterbium and 58Fe, and dysprosium and 54Fe were 0.992, 0.989, and 0.988, respectively (P < 0.001). Iron absorption was calculated as the difference between isotope dose and fecal excretion. Mean (+/-SEM) iron absorption was 16.7 +/- 2.4%, 4.3 +/- 1.6%, and 40.3 +/- 3.1% on days 1-3, respectively. Predicted values estimated from the first 4 d of pooled feces, using the rare earth element recovery data to produce corrected figures for unabsorbed isotope, were in close agreement: 19.1 +/- 2.1%, 4.6 +/- 1.7%, and 40.8 +/- 3.1%, respectively (P < 0.001). With the diet of medium iron bioavailability and with the highly bioavailable reference dose it was possible to predict iron absorption accurately from only one or two stools, provided that they were sufficiently enriched with isotope and a rare earth element.

Monitoring of the effects of dysprosium shift reagents on cell suspensions.

The effects of two widely used paramagnetic shift reagents for cationic NMR, dysprosium tripolyphosphate [Dy(PPP)2(7-)] and dysprosium triethylenetetramine hexaacetate [Dy(TTHA)3-], on the cell structure of dog and human erythrocytes, dog kidney cortical tubules and rat hepatocytes were investigated. The effect of shift reagents on cell integrity was monitored by measuring the hematocrit values for erythrocytes, by measuring the lactate dehydrogenase (LDH) release and by electron microscopy for cortical tubules and hepatocytes. The quantitation of the dyprosium penetration inside cells was accomplished by atomic absorption, atomic emission and neutron activation. More severe effects were observed with Dy(PPP)2(7-) than with Dy(TTHA)3-, and were dependent on the divalent cation concentration and on the shift reagent concentration. Very serious cell damage was observed after 60 min incubation in the presence of 10 mumol Dy(PPP)2(7-)/mL suspension at low or high divalent cation concentration. The situation was improved at 5 mumol Dy(PPP)2(7-)/mL suspension especially at high divalent cation concentration (2.5 mM). Incubation with Dy3+, PPP5- or Dy(TTHA)3- caused little or no structural effects but dysprosium was found to penetrate slowly inside tubules with Dy(TTHA)3-. Both Dy3+ and Dy(PPP)2(7-) penetrated rapidly inside cells. Dysprosium was found to bind to the isolated cytosol but not to isolated membranes, eliminating the possibility of extracellular membrane binding.

31P- and 1H-NMR investigations of the effect of n-alcohols on the hydrolysis by phospholipase A2 of phospholipid vesicular membranes.

31P- and 1H-NMR spectroscopy of small, unilamellar egg yolk phosphatidylcholine (PC) vesicles in the presence of the lanthanide ion Dy3+ have been used to study the effect of various n-alcohols on the permeability induced by the action of the enzyme phospholipase A2 (PLA2). The method allows the monitoring of the number of PC and lysoPC molecules in the outer and inner monolayers. The results indicate that the initial rate of hydrolysis of PC by PLA2 is increased by all the n-alcohols but in a chain-length dependent manner and that the maximum rate occurs at n = 8 (octan-1-ol). The subsequent rate is dependent upon the rate of transbilayer lipid exchange (flip-flop) of PC molecules from the inner to the outer monolayer. The vesicles only become permeable to the Dy3+ ions when lysoPC is mobilised in the flip-flop process of exchange of lipid molecules between the two monolayers. The n-alcohols affect both the time taken to initiate flip-flop of inner monolayer PC and the subsequent rate of permeability to Dy3+. The n-alcohols are seen to affect all the above rates in an identical chain-length dependent manner, indicating a common cause for all observations which we identify as the degree of clustering of the n-alcohol molecules in the bilayer. The results are discussed in terms of the chain-length dependent mechanism of n-alcohol interactions with the membrane and the mechanism by which the vesicles become permeable to Dy3+ ions.

31P and 23Na NMR studies of the structure and lability of the sodium shift reagent, bis(tripolyphosphate)dysprosium(III) ([Dy(P3O10)]7-) ion, and its decomposition in the presence of rat muscle.

The 31P NMR of aqueous [Dy(P3O10)2]7- demonstrates that it is in slow exchange with P3O5-10 on the NMR time scale. In the presence of tissue, [Dy(P3O10)2]7- decomposes to PO4 with an accompanying slow change of the tissue 23Na NMR of extracellular Na+ ion in several NMR distinguishable extracellular sites.

Measurement of a wide range of intracellular sodium concentrations in erythrocytes by 23Na nuclear magnetic resonance.

The accuracy of the 23Na nuclear magnetic resonance (NMR) method for measuring the sodium concentration in erythrocytes was tested by comparing the NMR results to those obtained by emission-flame photometry. Comparisons were made on aqueous solutions, hemolysates, gels, ghosts, and intact erythrocytes. The intra- and extracellular 23Na NMR signals were distinguished by addition of the dysprosium tripolyphosphate [Dy(PPP)7-2] shift reagent to the extracellular fluid. The intra- and extracellular volumes of ghosts and cells were determined by the isotope dilution method. Our results indicate that greater than 20% of the intracellular signal remains undetected by NMR in ghosts and cells. When the cells are hemolyzed, the amount of NMR-detectable sodium varies depending on the importance of gel formation. In hemolysates prepared by water addition, the NMR and flame photometry results are identical. The loss of signal in ghosts, cells, and undiluted hemolysates is attributed to partial binding of the Na+ ion to intracellular components, this binding being operative only when these components exist in a gel state. In a second part, 31P NMR was used to monitor the penetration of the shift reagent into the cells during incubation. Our data demonstrate that free Dy3+ can slowly accumulate inside the red cell.

Kinetics of [103Ru]phenanthroline and dysprosium particulate markers in the rumen of steers.

Two rumen cannulated steers consuming 5.5 kg air-dry lucerne (Medicago sativa) hay/d given at two-hourly intervals were used to study the kinetics in the rumen of the two particulate markers, 103Ru-labelled Tris-(1, 10-phenanthroline) ruthenium II chloride ([103Ru]P) and dysprosium (Dy). Provision of markers was achieved by allowing the steers to eat separated stems of the hay on to which had been sprayed solutions of the markers. The intake of large-particle (retained by a 3.2 mm mesh screen) dry matter (DM) in boluses and the rumen large-particle pool size measured by emptying the rumen were used to calculate the turnover rate-constant of this pool (1.02 and 1.19/d for steers A and B respectively). The decline with time of both [103Ru]P and Dy associated with large-particle DM in raft digesta was best described by two-compartmental kinetics. The first compartment apparently reflected a combination of the processes of mixing of labelled particles throughout the rumen contents, physical migration of marker from the labelled hay, and physical breakdown of large particles to small particles. The disappearance with time of [103Ru]P and Dy associated with small particles from the raft, ventral digesta and faeces from 3 to 8 d was well described by a single kinetic compartment with a rate constant similar to that of the second compartment of the large particles from the raft. [103Ru]P was rapidly distributed through both the raft and ventral digesta of the rumen. This observation, taken together with measurements of migration of [103Ru]P, suggested that on entering the rumen much of the [103Ru]P did not remain associated with the original feed material. The majority (63-64%) of Dy entered the raft digesta and mixed only slowly through the rumen contents. Some Dy (18-27%) apparently migrated rapidly from large particles and to small particles immediately after ingestion and there was also evidence for some slow migration from small to large particles. When used under the conditions described for this experiment neither [103Ru]P nor Dy was satisfactory as a marker to trace the passage through the rumen of a particular meal.

Experimental radiation synovectomy by 165Dy ferric hydroxide macroaggregate.

The short half-life beta emitter 165Dy coprecipitated as a macroaggregate with ferric hydroxide (FHMA) has been shown to destroy knee synovium in the antigen-induced arthritic rabbit. Using 153Gd as a gamma tracer for leakage studies revealed that the leakage of this system from rabbit knee joints never exceeded 1.2% over 24 hours. This is such less than the leakage rates reported from any human studies or our rabbit studies using 198Au.