A multibiomarker approach to assess toxic effects of wastewater treatment plant effluents and activated defence mechanisms in marine (Ruditapes philippinarum) and fresh water (Corbicula fluminea) bivalve species.

Since it has been demonstrated that urban effluents can have adverse effects on aquatic organisms, a multibiomarker study was used to evaluate the effects of wastewater treatment plant (WWTP) effluents discharged into the marine and freshwater environments on clams in Cádiz, Spain. One bioassay was performed in the Bay of Cádiz, exposing Ruditapes philippinarum (marine) to a reference site as well as two sites close to WWTP discharges for 14 days. A second bioassay was performed in the Guadalete River, exposing Corbicula fluminea (fresh water) to three sites for 21 days. The biomarkers analysed included defence mechanisms and various toxic effects. Results indicated that WWTP effluents activated defence mechanisms and induced toxic effects in clams exposed to both environments, thus indicating bioavailability of contaminants present in water. Elevated enzymatic activity was found in clams deployed in La Puntilla and El Trocadero compared to control clams and those exposed to the reference site, and 96% of clams deployed at G2 in the Guadalete River died before day 7. Clams exposed to G1 and G3 indicated significant differences in all biomarkers analysed with respect to control clams (p < 0.05). Both species were sensitive to contaminants present in studied sites. This is the first time that these species were used in cages to assess the environmental risk of wastewater effluent discharges in freshwater and marine column environments. The multibiomarker approach provided important ecotoxicological information and is useful for the assessment of the bioavailability and effect of contaminants from WWTP effluents on marine and fresh water invertebrates.

Animal model studies yield translational solutions for cochlear drug delivery.

The field of hearing and deafness research is about to enter an era where new cochlear drug delivery methodologies will become more innovative and plentiful. The present report provides a representative review of previous studies where efficacious results have been obtained with animal models, primarily rodents, for protection against acute hearing loss such as acoustic trauma due to noise overexposure, antibiotic use and cancer chemotherapies. These approaches were initiated using systemic injections or oral administrations of otoprotectants. Now, exciting new options for local drug delivery, which opens up the possibilities for utilization of novel otoprotective drugs or compounds that might not be suitable for systemic use, or might interfere with the efficacious actions of chemotherapeutic agents or antibiotics, are being developed. These include interesting use of nanoparticles (with or without magnetic field supplementation), hydrogels, cochlear micropumps, and new transtympanic injectable compounds, sometimes in combination with cochlear implants.

The effects of human drugs in Corbicula fluminea. Assessment of neurotoxicity, inflammation, gametogenic activity, and energy status.

The constant release of pharmaceuticals products to aquatic environment even at low concentrations (ng L-1 to µg L-1) could lead to unknown chronic effects to non-target organisms. The aim of this study was to evaluate neurotoxic responses, inflammation, gametogenic activity and energy status on the fresh water clam C. fluminea after exposure to different concentrations of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ), novobiocin (NOV) and tamoxifen (TMX) for 21 days under laboratory conditions. During the assay, water was spiked every two days with CAF (0; 0.1; 5; 15; 50µgL-1), IBU (0; 0.1; 5; 10; 50µgL-1), CBZ, NOV, and TMX (0.1, 1, 10, 50µgL-1). After the exposure period, dopamine levels (DOP), monoamine oxidase activity (MAO), arachidonic acid cyclooxygenase activity (COX), vitellogenin-like proteins (VTG), mitochondrial electron transport (MET), total lipids (TLP), and energy expenditure (MET/TLP) were determined in gonad tissues, and acetyl cholinesterase activity (AChE) was determined in digestive gland tissues. Results showed a concentration-dependence response on biomarkers tested, except for MAO. Environmental concentrations of pharmaceuticals induced significant changes (p < 0.05) in the neurotoxic responses analyzed (CAF, CBZ and NOV increased DOP levels and CBZ inhibited AChE activity), inflammation (CAF induced COX), and energy status (MET and TLP increased after exposure to CBZ, NOV and TMX). Responses of clams were related to the mechanism of action (MoA) of pharmaceuticals. Biomarkers applied and the model organism C. fluminea constituted a suitable tool for environmental risk assessment of pharmaceutical in aquatic environment.

Are the TiO2 NPs a "Trojan horse" for personal care products (PCPs) in the clam Ruditapes philippinarum?

In recent years, increasing quantities of personal care products (PCPs) are being released into the environment. However, data about bioaccumulation and toxicity are scarce; and extraction and analytical approaches are not well developed. In this work, the marine clam Ruditapes philippinarum, selected as model organism, has been employed to investigate bioaccumulation, antioxidant enzyme activities and DNA damage due to exposure to TiO2 nanoparticles and bulk TiO2 (inorganic compounds that are frequent components of PCPs, plastics, paints and coatings, foods and disinfectant water treatments). We have also studied the joint effect of both forms of inorganic TiO2 combined with four organic compounds (mixture exposures) commonly used in PCPs: an antimicrobial (triclosan), a fragrance (OTNE) and two UV filters (benzophenone-3 and octocrylene). Bioaccumulation of the inorganic compound, TiO2, was almost immediate and constant over exposure time. With respect to the organic compounds in mixtures, they were mediated by TiO2 and bioaccumulation is driven by reduced size of the particles. In fact, nanoparticles can be considered as a vector to organic compounds, such as triclosan and benzophenone-3. After a week of depuration, TiO2 NPs and TiO2 bulk in clams showed similar levels of concentration. Some organic compounds with bioactivity (Log Kow >3), like OTNE, showed low depuration after one week. The joint action of the organic compound mixture and either of the two forms of TiO2 provoked changes in enzyme activity responses. However, for the mixtures, DNA damage was found only after the depuration period.

Is the step-wise tiered approach for ERA of pharmaceuticals useful for the assessment of cancer therapeutic drugs present in marine environment?

Methotrexate (MTX) and tamoxifen (TMX) cancer therapeutic drugs have been detected within the aquatic environment. Nevertheless, MTX and TMX research is essentially bio-medically orientated, with few studies addressing the question of its toxicity in fresh water organisms, and none to its' effect in the marine environment. To the authors' knowledge, Environmental Risk Assessments (ERA) for pharmaceuticals has mainly been designed for freshwater and terrestrial environments (European Medicines Agency-EMEA guideline, 2006). Therefore, the purpose of this research was (1) to assess effect of MTX and TMX in marine organism using the EMEA guideline, (2) to develop an ERA methodology for marine environment, and (3) to evaluate the suitability of including a biomarker approach in Phase III. To reach these aims, a risk assessment of MTX and TMX was performed following EMEA guideline, including a 2-tier approach during Phase III, applying lysosomal membrane stability (LMS) as a screening biomarker in tier-1 and a battery of biochemical biomarkers in tier-2. Results from Phase II indicated that MTX was not toxic for bacteria, microalgae and sea urchin at the concentrations tested, thus no further assessment was required, while TMX indicated a possible risk. Therefore, Phase III was performed for only TMX. Ruditapes philippinarum were exposed during 14 days to TMX (0.1, 1, 10, 50 µg L(-1)). At the end of the experiment, clams exposed to environmental concentration indicated significant changes in LMS compared to the control (p<0.01); thus a second tier was applied. A significant induction of biomarkers (activity of Ethoxyresorufin O-deethylase [EROD], glutathione S-transferase [GST], glutathione peroxidase [GPX], and lipid peroxidation [LPO] levels) was observed in digestive gland tissues of clams compared with control (p<0.01). Finally, this study indicated that MTX was not toxic at an environmental concentration, whilst TMX was potentially toxic for marine biota. This study has shown the necessity to create specific guidelines in order to evaluate effects of pharmaceuticals in marine environment which includes sensitive endpoints. The inadequacy of current EMEA guideline to predict chemotherapy agents toxicity in Phase II was displayed whilst the usefulness of other tests were demonstrated. The 2-tier approach, applied in Phase III, appears to be suitable for an ERA of cancer therapeutic drugs in the marine environment.

Are standard tests sensitive enough to evaluate effects of human pharmaceuticals in aquatic biota? Facing changes in research approaches when performing risk assessment of drugs.

Nowadays, the presence of pharmaceutical products in aquatic environments is not only common, but is also of significant concern regarding the adverse effect they may produce to aquatic biota. In order to determine the adverse effects of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ) and novobiocin (NOV), at environmental occurring concentrations, standardized endpoints applied in current guidelines were evaluated in four organisms including bioluminescence response in Vibrio fischeri, growth inhibition in Isochrysis galbana (marine water) and Pseudokirchneriella subcapitata (fresh water) and fertilization and embryo-larval development in Paracentrotus lividus. To reach this aim bioassays were implemented by exposing organisms to water spiked with drugs dissolved in DMSO (0.001% v/v). Risk characterization was performed, calculating the environmental impact of drugs by calculating environmental concentration and predicted no effect concentration ratio (MEC/PNEC). Results indicate that acute toxicity was found above environmental concentrations in the order of mg L(-1) for bacteria bioluminescence, microalgae growth inhibition and sea urchin fertilization. However, teratogenicity was observed on sea urchin after exposure to environmental concentrations of drugs at 0.00001 mg L(-1); at this concentration CBZ and IBU were found to reduce significantly the embryo-larval development compared to controls (p<0.01). The risk calculated for selected drugs suggested they are harmless for aquatic environment except when applying the embryo-larval development endpoint. Endpoints applied in this study showed the necessity of using more sensitive responses, when assessing risk of pharmaceuticals in aquatic environments, since endpoints applied in current guidelines may not be suitable.

Early responses measured in the brachyuran crab Carcinus maenas exposed to carbamazepine and novobiocin: application of a 2-tier approach.

One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50µgL(-1). Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50µgL(-1)). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50µgL(-1)). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50µgL(-1) (p<0.05). Crabs exposed to NOV (50µgL(-1)) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment.

Identification of biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of Carcinus maenas.

A 28-day bioassay was performed with Carcinus maenas to evaluate chronic effects caused by exposure to caffeine and ibuprofen (0.1-50 µg L(-1)) in sea water. Lysosomal membrane stability (LMS) was evaluated in hemolymph applying the neutral red retention assay (NRRA); several biomarkers including ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPX), lipid peroxidation (LPO) and DNA damage were studied in gill, hepatopancreas, muscle and gonad tissues. In crabs exposed to environmental concentrations of the drugs, retention time was reduced by 50%. EROD and DBFOD activities were induced by caffeine in muscle and hepatopancreas tissues (p < 0.05); GST activity was activated by ibuprofen in gill, hepatopancreas and muscle at the highest concentrations tested (p < 0.05). All tissues showed GPX activity and LPO induction (p < 0.05). Crabs exposed to caffeine and ibuprofen showed evidence of DNA damage mainly in hepatopancreas tissues (p < 0.05). Environmental concentrations of pharmaceuticals induce LMS and the biochemical responses studied in this crab. This methodology is a suitable technique for assessing pharmaceutical toxicity in the marine environment.

Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals.

The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked every 2 days with various concentrations (from 0.1 to 50 µg · L(-1)) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p < 0.05) after exposure to environmental concentrations of each pharmaceutical (caffeine = 15 µg · L(-1); carbamazepine = 1 µg · L(-1); ibuprofen = 5 µg · L(-1); and novobiocin = 0.1 µg · L(-1)) when compared with control organisms. The predicted "no environmental effect" concentration/measured environmental concentration results showed that the selected pharmaceuticals are toxic at environmental concentrations and need further assessment. LMS monitoring in crabs is a sensitive tool for evaluating exposure to concentrations of selected drugs under laboratory conditions and provides a robust tier 1 testing approach (screening biomarker) for rapid assessment of marine pollution and environmental impact assessments for analyzing pharmaceutical contamination in aquatic environments.

Global structure and dynamics of human apolipoprotein CII in complex with micelles: evidence for increased mobility of the helix involved in the activation of lipoprotein lipase.

Apolipoprotein CII (apoCII), a surface constituent of plasma lipoproteins, is the activator for lipoprotein lipase (LPL) and is therefore central for lipid transport in blood. The three-dimensional structure of (13)C-, (15)N-enriched human full-length apoCII in complex with sodium dodecyl sulfate (SDS) micelles is reported. In addition to the structure determination, (15)N-relaxation measurements have been performed at two magnetic fields to characterize the dynamics of the backbone of apoCII in the complex. The relaxation data also provided global structural constraints, viz. the orientation of helices in the complex. In addition, global constraints were derived from the fact that apoCII helices are attached to the surface of the SDS micelle and that the hydrophobic moments of each helix faces the interior of the micelle. These three categories of global constraints, together with the local classical NMR constraints, were sufficient to define the 3D structure of the apoCII-SDS micelle complex. To our knowledge, this presents the first example in which the global structure of a protein-SDS micelle complex has been determined. The C-terminal helix of apoCII is known to be responsible for the activation of LPL. This helix is distinguished from the other helices by a higher degree of internal motion on the nanosecond time scale as shown by the relaxation data. The overall structure and the internal dynamics, combined with previous mutation data, give important clues toward a possible mechanism for the activation of LPL by apoCII.

Nitroxide side-chain dynamics in a spin-labeled helix-forming peptide revealed by high-frequency (139.5-GHz) EPR spectroscopy.

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

A neural network approach to the rapid computation of rotational correlation times from slow motional ESR spectra.

We explore the use of feed forward artificial neural networks for determining rotational correlation times from slow motional nitroxide electron spin resonance spectra. This approach is rapid and potentially eliminates the need for traditional iterative fitting procedures. Two networks are examined: the radial basis network and the multilayer perceptron. Although the radial basis network trains rapidly and performs well on simulated spectra, it is less satisfactory when applied to experimental spectra. In contrast, the multilayer perceptron trains slowly but is excellent at extracting correlation times from experimental spectra. In addition, the multilayer perceptron operates well in the presence of noise as long as the signal-to-noise ratio is greater than approximately 200/1. These findings suggest neural networks offer a promising approach for rapidly extracting correlation times without the need for iterative simulations.

Synthesis and conformational studies of peptides containing TOAC, a spin-labelled C alpha, alpha-disubstituted glycine.

A variety of host L-alanine homo-peptides (to the pentamer) containing one or two spin-labelled TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) residues were synthesized by solution methods and fully characterized. The conformational features of the terminally blocked, doubly spin-labelled TOAC-(Ala)2-TOAC-Ala-pentapeptide were examined in the crystal state by X-ray diffraction and in solution using a combination of techniques (Fourier transform infrared, circular dichroism, cyclic voltammetry and electron spin resonance) in comparison with singly labelled shorter peptides. The 3(10)-helical structure of the pentapeptide, promoted by the two C alpha, alpha-disubstituted glycines under favourable experimental conditions, allows an interaction to take place between the two nitroxide TOAC side chains spaced by one turn of the helix. Taken together, these results suggest that TOAC is an excellent probe for exploring bends and helices in doubly labelled peptides.

Short alanine-based peptides may form 3(10)-helices and not alpha-helices in aqueous solution.

Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.