Former uranium mine-induced effects in caged roach: a multiparametric approach for the evaluation of in situ metal toxicity

Ecotoxicology - To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private...     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography-electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC-MS-MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C18 column (125 x 3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate-acetonitrile as the mobile phase at a flow-rate of 0.7 ml min(-1). Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC-MS-MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL-2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

Sensitive and fast identification of urinary human,synthetic and animal insulin by means of nano‐UPLC coupled with high‐resolution/high‐accuracy mass spectrometry

The double‐chain polypeptide insulin and its synthetic (Insulin Glulisine, Insulin Aspart, Insulin Glargine, or Insulin Lispro) or animal analogues (porcine insulin or bovine insulin) are potential performance‐enhancing agents in elite sports or potentially effective toxins in forensic science. The present study demonstrates an analytical method to purify the insulins simultaneously from urine specimens with an approach based on immunoaffinity isolation, using coated magnetic beads (anti‐mouse) and a primary anti‐insulin antibody (IgG, monoclonal). The extracts were purified sufficiently for separation by means of nano‐flow liquid chromatography coupled with nano‐scale high‐resolution, high‐accuracy ESI‐MS/MS. Elucidation of collision‐induced dissociations with product ion experiments using the fivefold protonated precursor ion of each target analyte enabled all synthetic and animal insulins to be differentiated from their human counterpart, which was particularly important for Lispro, possessing the same molecular mass as human insulin. The method was fully validated for specificity, limit of detection (LOD, 0.5 fmol/mL), precision (<20%), recovery (approximately 30%) and linearity (2–40 fmol/mL) for all target analytes. Copyright © 2009 John Wiley & Sons, Ltd.     

Multi-Biomarkers Approach in Different Organs of <Emphasis Type="Italic">Anodonta cygnea</Emphasis> from the Dnister Basin (Ukraine)

The aim of this study was to compare environmental quality in two sites in western Ukraine—rural (R) and urbanized (U)—with the usage of the resident bivalve mollusk Anodonta cygnea. The study was realized during three seasons. The metal uptake and a set of biochemical markers were determined. For each season, Cd and metallothioneins (MTs) contents in the digestive gland and gills of the mollusc were higher at the U site, reflecting its chronic pollution. The oxidative stress in the mollusk was observed at the U site during spring and at the R site during summer and autumn according to the differences in Mn–superoxide dismutase and catalase activities, O₂^•− production, lipid peroxidation, and glutathione levels. The elevated vitellogenin-like protein levels in the hemolymph and the ethoxyresorufin-O-deethylase activity in the digestive gland in summer–autumn suggested pollutions by organic substances at the R site. The acetylcholinesterase activity was similar in both groups. The centroid grouping analysis of biomarkers and morphological and water indexes demonstrated the clear differentiation of general response in each group in spring and, at the R site, in summer and autumn but its similarity at the U site in summer and autumn.     

Juvenile roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis>) increase their anaerobic metabolism in response to copper exposure in laboratory conditions

This study aims to determine the potential impairment of cell energy synthesis processes (glycolysis and respiratory chain pathways) by copper in juvenile roach at different regulation levels by using a multi-marker approach. Juvenile roach were exposed to 0, 10, 50, and 100 µg/L of copper for 7 days in laboratory conditions. The glycolysis pathway was assessed by measuring the relative expression levels of 4 genes encoding glycolysis enzymes. The respiratory chain was studied by assessing the electron transport system and cytochrome c oxidase gene expression. Muscle mitochondria ultrastructure was studied, and antioxidant responses were measured. Furthermore, the main energy reserves—carbohydrates, lipids, and proteins—were measured, and cellular energy was evaluated by measuring ATP, ADP, AMP and IMP concentrations. This study revealed a disturbance of the cell energy metabolism due to copper exposure, with a significant decrease in adenylate energy charge in roach exposed to 10 μg/L of copper after 1 day. Moreover, ATP concentrations significantly decreased in roach exposed to 10 μg/L of copper after 1 day. This significant decrease persisted in roach exposed to 50 µg/L of copper after 7 days. AMP concentrations increased in all contaminated fish after 1 day of exposure. In parallel, the relative expression of 3 genes encoding for glycolysis enzymes increased in all contaminated fish after 1 day of copper exposure. Focusing on the respiratory chain, cytochrome c oxidase gene expression also increased in all contaminated fish at the two time-points. The activity of the electron transport system was not disturbed by copper, except in roach exposed to 100 µg/L of copper after 1 day. Copper induced a metabolic stress. Juvenile roach seemed to respond to the ensuing high energy demand by increasing their anaerobic metabolism, but the energy produced by the anaerobic metabolism is unable to compensate for the stress induced by copper after 7 days. This multi-marker approach allows us to reach a greater understanding of the effects of copper on the physiological responses of juvenile roach.     

Antipeptide antibody against bovine IGF‐BP‐2: Application to the detection of bovine somatotropin‐treated cows

The aim of this study was to develop a method to identify cows illegally treated with bovine somatotropin (BST). Using the Western ligand blotting technique, BST treatment was shown to cause a dramatic decrease of IGF‐BP‐2 in the serum of treated cows. Measurement of the insulin‐like growth factor‐binding protein‐2 (IGF‐BP‐2) concentration in cow plasma could thus be a good way to identify BST‐treated animals. In order to develop an immunoassay specific for bovine IGF‐BP‐2, polyclonal antibodies against synthetic peptides of bovine IGF‐BP‐2 were produced. Three peptides were chosen in the [150–190] region of the molecule, which display no identity with other IGF‐BPs in serum: [154–165], [171–184] and [163–176]. Two antisera were obtained which reacted specifically with the entire bovine IGF‐BP‐2 molecule, both of which came from rabbits immunized with the [163–176] peptide.     

The production and characterisation of dinitrocarbanilide antibodies raised using antigen mimics

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised.The antibodies exhibited an IC(50) range of 2.3-7.6 ng/ml using a competitive ELISA procedure. Serum from one rabbit, R555, exhibited an IC(50) of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole.     

Preliminary study on the presence of prednisolone in porcine urine and liver – How to distinguish endogenous from therapeutically administered prednisolone

In animal breeding in Europe, synthetic corticosteroids are not allowed as growth‐promoting agents. However, prednisolone residues have recently been found in porcine urine samples collected at slaughterhouses. The aim of this work was therefore to look for prednisolone in porcine urine and liver, to determine if detected residues might be of endogenous origin, and to check the possible relation with stress. An analytical method developed in‐house was validated, combining immunoaffinity‐based purification and ultra‐high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS). This method was applied to urine and liver samples collected from sows experimentally treated either with prednisolone or tetracosactide hexaacetate (synthetic analogue of ACTH). Thanks to the performance of the analytical method, both cortisol and prednisolone were detected in all pig urine samples collected before or after administration of prednisolone or tetracosactide hexaacetate. High levels of prednisolone were found in porcine urine just after prednisolone administration, decreasing quickly to within the range detected in non‐treated animals. In urine, the cortisol level varied depending on the time lapse between administration and sampling. On the other hand, prednisolone was detected also in liver samples of treated pigs. In this matrix, the cortisol level remained constant and prednisolone/cortisol level could be used to detect prednisolone administration at least 4 days after injection. In conclusion, the best indicator for detecting illicit prednisolone administration to pigs seems to be the prednisolone/cortisol ratio in liver samples. This preliminary work must be confirmed by a larger‐scale study and metabolites should also be included. Copyright © 2013 John Wiley & Sons, Ltd.     

In situ effects of metal contamination from former uranium mining sites on the health of the three-spined stickleback (<Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>, L.)

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.