Mário S. Diniz

Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: A weighted indices approach

Young juvenile Solea senegalensis were exposed to three sediments with distinct contamination profiles collected from a Portuguese estuary subjected to anthropogenic sources of contamination (the Sado estuary, western Portugal). Sediments were surveyed for metals (cadmium, chromium, copper, nickel, lead and zinc), a metalloid (arsenic) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide, dichloro-diphenyl-trichloroethane plus its metabolites), as well as total organic matter, redox potential and particle fine fraction. The fish were exposed to freshly collected sediments in a 28-day laboratorial assay and collected for histological analyses at days 0 (T(0)), 14 (T(14)) and 28 (T(28)). Individual weighted histopathological indices were obtained, based on presence/absence data of eight and nine liver and gill pathologies, respectively, and on their biological significance. Although livers sustained more severe lesions, the sediments essentially contaminated by organic substances caused more damage to both organs than the sediments contaminated by both metallic and organic contaminants, suggesting a possible synergistic effect. Correlation analyses showed that some alterations are linked, forming distinctive histopathological patterns that are in accordance with the severity of lesions and sediment characteristics. The presence of large eosinophilic bodies in liver and degeneration of mucous cells in gills (a first-time described alteration) were some of the most noticeable alterations observed and were related to sediment organic contaminants. Body size has been found to be negatively correlated with histopathological damage in livers following longer term exposures. It is concluded that histopathological indices provide reliable and discriminatory data even when biomonitoring as complex media as natural sediments. It is also concluded that the effects of contamination may result not only from toxicant concentrations but also from their interactions, relative potency and sediment characteristics that ultimately determine bioavailability.

Influence of temperature in thermal and oxidative stress responses in estuarine fish

The influence of increasing temperatures in thermal and oxidative stress responses were studied in the muscle of several estuarine fish species (Diplodus vulgaris, Diplodus sargus, Dicentrarchus labrax, Gobius niger and Liza ramada). Selected fish were collected in July at the Tagus estuary (24±0.9°C; salinity of 30±4‰; pH=8). Fish were subjected to a temperature increase of 1°C.h(-1) until they reached their Critical Thermal Maximum (CTMax), starting at 24°C (control temperature). Muscle samples were collected during the trial and results showed that oxidative stress biomarkers are highly sensitive to temperature. Results from stress oxidative enzymes show alterations with increasing temperature in all tested species. Catalase (CAT; EC 1.11.1.6) activity significantly increased in L. ramada, D. labrax and decreased in D. vulgaris. Glutathione S-transferase (GST; EC 2.5.1.18) activity increased in L. ramada, D. sargus, D. vulgaris, and D. labrax. In G. niger it showed a cycle of increase-decrease. Lipid peroxidation (LPO) increased in L. ramada, D. sargus and D. labrax. With respect to correlation analysis (Pearson; Spearman r), the results showed that oxidation products and antioxidant defenses were correlated in L. ramada (LPO-CAT and LPO-GST, D. sargus (LPO-CAT), and D. labrax (LPO-CAT). Oxidative biomarkers were correlated with thermal stress biomarker (Hsp70) in L. ramada (CAT-Hsp70), D. vulgaris (LPO-Hsp70), D. labrax (GST-Hsp70) and G. niger (LPO-Hsp70). In conclusion, oxidative stress does occur with increasing temperatures and there seems to be a relation between thermal stress response and oxidative stress response. The results suggest that oxidative stress biomarkers should be applied with caution, particularly in field multi-species/multi-environment studies.

Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques.

Recent tools addressed to accelerate the different steps of the sample treatment for protein identification in modern workflows are reviewed and critically commented in this manuscript. Heating, microspin columns, ultrasonic energy, high pressure, infrared energy, microwave energy, alternating electric fields and microreactors are outlined as useful tools that can be used to accelerate all or some of the following steps for in-gel or in-liquid based approaches for protein identification: (i) protein dissolution/denaturation, (ii) protein reduction, (iii) protein alkylation and (iv) protein digestion. The advantages and drawbacks, along with the main differences among the different tools are also commented. Future prospects for hyphenation of methods are also discussed. Researchers are informed also in this work regarding the main problems to be found when implementing any of the above mentioned methods.

Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis.

Abstract Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3′-Cy3 and 5′-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.

Ocean Warming Enhances Malformations, Premature Hatching, Metabolic Suppression and Oxidative Stress in the Early Life Stages of a Keystone Squid

Background The knowledge about the capacity of organisms’ early life stages to adapt to elevated temperatures is very limited but crucial to understand how marine biota will respond to global warming. Here we provide a comprehensive and integrated view of biological responses to future warming during the early ontogeny of a keystone invertebrate, the squid Loligo vulgaris. Methodology/Principal Findings Recently-spawned egg masses were collected and reared until hatching at present day and projected near future (+2°C) temperatures, to investigate the ability of early stages to undergo thermal acclimation, namely phenotypic altering of morphological, behavioural, biochemical and physiological features. Our findings showed that under the projected near-future warming, the abiotic conditions inside the eggs promoted metabolic suppression, which was followed by premature hatching. Concomitantly, the less developed newborns showed greater incidence of malformations. After hatching, the metabolic burst associated with the transition from an encapsulated embryo to a planktonic stage increased linearly with temperature. However, the greater exposure to environmental stress by the hatchlings seemed to be compensated by physiological mechanisms that reduce the negative effects on fitness. Heat shock proteins (HSP70/HSC70) and antioxidant enzymes activities constituted an integrated stress response to ocean warming in hatchlings (but not in embryos). Conclusions/Significance The stressful abiotic conditions inside eggs are expected to be aggravated under the projected near-future ocean warming, with deleterious effects on embryo survival and growth. Greater feeding challenges and the lower thermal tolerance limits of the hatchlings are strictly connected to high metabolic demands associated with the planktonic life strategy. Yet, we found some evidence that, in the future, the early stages might support higher energy demands by adjusting some cellular functional properties to increase their thermal tolerance windows.

Biological treatment of the effluent from a bleached kraft pulp mill using basidiomycete and zygomycete fungi

Three white-rot fungi (Pleurotus sajor caju, Trametes versicolor and Phanerochaete chrysosporium) and one soft-rot fungi (Rhizopus oryzae) species confirmed their potential for future applications in the biological treatment of effluents derived from the secondary treatment of a bleached kraft pulp mill processing Eucalyptus globulus. Among the four species P. sajor caju and R. oryzae were the most effective in the biodegradation of organic compounds present in the effluent, being responsible for the reduction of relative absorbance (25-46% at 250 nm and 72-74% at 465 nm) and of chemical oxygen demand levels (74 to 81%) after 10 days of incubation. Laccase (Lac), lignin (Lip) and manganese peroxidases (MnP) expression varied among fungal species, where Lac and LiP activities were correlated with the degradation of organic compounds in the effluent treated with P. sajor caju. The first two axes of a principal component analysis explained 88.9% of the total variation among sub-samples treated with the four fungus species, after different incubation periods. All the variables measured contributed positively to the first component except for the MnP enzyme activity which was the only variable contributing negatively to the first component. Absorbances at 465 nm, LiP and Lac enzyme activities were the variables with more weight on the second component. P. sajor caju revealed to be the only species able to perform the biological treatment without promoting an increment in the toxicity of the effluent to the Vibrio fischeri, as it was assessed by the Microtox assay. The opposite was recorded for the treatments with the other three species of fungus. EC(50-5 min) values ranging between 28 and 57% (effluent concentrations) were recorded even after 10 to 13 days of treatment with P. chrysosporium, R. oryzae or with T. versicolor.

Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction.

Juvenile Solea senegalensis were exposed to fresh sediments from three stations of the Sado estuary (Portugal) in 28-day laboratory assays. Sediments revealed distinct levels of total organic matter, fine fraction, redox potential, trace elements (arsenic, cadmium, chromium, copper, nickel, lead and zinc) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide: dichloro diphenyl trichloroethane). Organisms were surveyed for contaminant bioaccumulation and induction of two hepatic biochemical biomarkers: metallothionein (MT) and cytochrome P450 (CYP1A), as potential indicators of exposure to metallic and organic contaminants, respectively. Using an integrative approach it was established that, although bioaccumulation is in general accordance with sediment contamination, lethality and biomarker responses are not linearly dependent of the cumulative concentrations of sediment contaminants but rather of their bioavailability and synergistic effects in organisms. It is concluded that metals and organic contaminants modulate both MT and CYP1A induction and it is suggested that reactive oxygen species may be the link between responses and effects of toxicity.

Oxidative stress and histological changes following exposure to diamond nanoparticles in the freshwater Asian clam Corbicula fluminea (Müller, 1774).

Recently, the scientific community became aware of the potential ability of nanoparticles to cause toxicity in living organisms. Therefore, many of the implications for aquatic ecosystems and its effects on living organisms are still to be evaluated and fully understood. In this study, the toxicity of nanodiamonds (NDs) was assessed in the freshwater bivalve (Corbicula fluminea) following exposure to different nominal concentrations of NDs (0.01, 0.1, 1, and 10 mg l(-1)) throughout 14 days. The NDs were characterized (gravimetry, pH, zeta potential, electron microscopy, and atomic force microscopy) confirming manufacturer information and showing NDs with a size of 4-6 nm. Oxidative stress enzymes activities (glutathione-S-transferase, catalase) and lipid peroxidation were determined. The results show a trend to increase in GST activities after seven days of exposure in bivalves exposed to NDs concentrations (>0.1 mg l(-1)), while for catalase a significant increase was found in bivalves exposed from 0.01 to 1.0 mg l(-1) following an exposure of 14 days. The histological analysis revealed alterations in digestive gland cells, such as vacuolization and thickening. The lipid peroxidation showed a trend to increase for the different tested NDs concentrations which is compatible with the observed cellular damage.

Ecotoxicity of ketoprofen, diclofenac, atenolol and their photolysis byproducts in zebrafish (Danio rerio).

The occurrence of pharmaceutical compounds in wastewater treatment plants and surface waters has been detected worldwide, constituting a potential risk for aquatic ecosystems. Adult zebrafish, of both sexes, were exposed to three common pharmaceutical compounds (atenolol, ketoprofen and diclofenac) and their UV photolysis by-products over seven days. The results show that diclofenac was removed to concentrations<LOD after 5 min of UV irradiation. The oxidative stress response of zebrafish to pharmaceuticals and their photolysis by-products was evaluated through oxidative stress enzymes (glutathione-S-transferase, catalase, superoxide dismutase) and lipid peroxidation. Results suggest that the photolysis by-products of diclofenac were more toxic than those from the other compounds tested, showing an increase in GST and CAT levels, which are also supported by higher MDA levels. Overall, the toxicity of waters containing atenolol and ketoprofen was reduced after the parent compounds were transformed by photolysis, whereas the toxicity increased significantly from the by-products generated through diclofenac photolysis. Therefore, diclofenac photolysis would possibly necessitate higher irradiation time to ensure that the associated by-products are completely degraded to harmless form(s).

Effects of diamond nanoparticle exposure on the internal structure and reproduction of Daphnia magna

Nanomaterials have significant technological advantages but their release into the environment also carry potential ecotoxicological risks. Carbon-based nanoparticles and particularly diamond nanoparticles have numerous industrial and medical applications. The aim of the present study was to evaluate the toxic effects of diamond nanoparticles with an average particle size of 20 nm on the survival, reproduction and tissue structure of the freshwater crustacean Daphnia magna. The chronic toxicity test results showed 100% mortality at concentrations higher than 12.5 mg l(-1) and that reproduction inhibition occurred in concentrations higher than 1.3 mg l(-1). Light microscopy showed that diamond nanoparticles adhere to the exoskeleton surface and accumulate within the gastrointestinal tract, suggesting that food absorption by the gut cells may be blocked. The results support the use of chronic approaches in environmental protection as part of an integrated environmental monitoring and assessment strategy.