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We evaluated the accumulation of okadaic acid (OA), a diarrhetic toxin, and the antioxidant responses in the marine annelid Laeonereis acuta exposed to the benthic toxigenic dinoflagellate Prorocentrum cf. lima. Nontoxic Tetraselmis sp. was used as a control diet. Living cells of the two algae were supplied as food to animals kept in agar medium for 72 h. To assess the significance of the observed effects, our experimental design treated the algal species (diet), algal cell densities, and exposure time as fixed factors. Responses of the organisms were assessed through oxidative stress biomarkers (glutathione-S-transferase [GST], catalase [CAT], reduced glutathione [GSH] and lipid peroxidation [LPO]). Toxin accumulation was measured by LC-MS/MS in whole-body homogenates after 12, 24 and 72 h of exposure. Worms exposed to the toxigenic dinoflagellate gradually accumulated OA, with toxin levels directly related to the cell density of Prorocentrum cf. lima. Worms fed with Prorocentrum cf. lima exhibited decreased CAT activity, increased LPO levels – both interactively affected by algal species and time – and decreased GSH levels, which were interactively affected by algal species and cell density. Higher LPO levels, along with the inhibition of CAT and GSH, clearly indicated an oxidative stress situation in worms exposed to the toxigenic dinoflagellate. Laeonereis acuta accumulated moderate OA levels and may act as a vector of OA to food webs in estuarine areas under high Prorocentrum cf. lima abundance.

Anti-inflammatory agents may be alternatives for the treatment of Alzheimer's disease. Then, we propose the nanotechnology as a strategy to curcumin and meloxicam co-nanoencapsulation for neurodegenerative diseases treatment. In this study, we developed and validated an analytical method by HPLC-DAD for simultaneous quantification of curcumin and meloxicam in the same formulation. We applied this method to characterize curcumin, meloxican and curcumin plus meloxican loaded lipid-core nanocapsules (LNC): drug content, encapsulation efficiency, photostability and drug's distribution. Additionally, toxicity levels of the nanoparticles were evaluated in vivo (mice). Therefore a C18-RP column was used with a guard column packed with the same material as the stationary phase. Acetonitrile: methanol: water: triethylamine (52:5:43:0.3 v/v/v/v), was used as the mobile phase in the 1 mL min−1 flow. Detection was 424 nm (curcumin) and 365 nm (meloxicam). The method was established in accordance with the current national and international guidelines, showing good linearity (r2 > 0.999). The formulations showed exclusively nanosized particles, negative zeta potential and slightly acidic pH, with content and encapsulation rate close to 100%. The distributions of meloxicam and curcumin in the LNC (alone or co-encapsulated) were type III and type VI, respectively. LNC were able to protect both drugs against UVA degradation. In vivo experiments showed no toxicity in relation to the parameters determined of all LNC evaluated in mice. In this work, we showed the possibility to carry meloxicam and curcumin in LNC with good technological characteristics and without toxicity in vivo with prospects for the treatment of Alzheimer's disease in sequential pharmacological studies. Keywords: Lipid-core nanocapsules, HPLC-PDA, Alzheimer’s disease, Curcumin, Meloxicam

The objective of this study was to demonstrate the pH effects on growth, survival, water quality, proximal composition of bioflocs and oxidative stress of Nile tilapia in biofloc technology (BFT) system. Twenty‐five fish (3.68 ± 0.93 g) were distributed in each tank (useful vol. 37.5 L), utilizing treatments with pH 8.3, 7.5 and 6.5 at 60 days. During the experiment, the oxidation of total ammonia was similar among the treatments. However, the NO₂⁻‐N oxidation was slower at pH 6.5 (10.1 ± 1.0 mg/L) compared to pH 7.5 (7.0 ± 0.6 mg/L) and 8.3 (7.1 ± 1.5 mg/L). The final weight was higher for pH 7.5 treatment (44.1 ± 0.9 g) compared to pH 8.3 (37.1 ± 3.9 g), while the pH 6.5 (40.4 ± 4.1 g) was like to the other treatments. Moreover, the survival, daily growth rate and the food conversion rate were not affected by treatments. When evaluating physiological and biochemical parameters, no alterations were detected, therefore, indicating that fish have a good health status. Thus, the present study demonstrates that BFT for a Nile tilapia nursery, utilizing pH 6.5–7.5, promotes the best results in terms of growth, net yield and efficiency.

The aim of this work was to test the insecticidal effect of the essential oil of Illicium verum (Hook) by observing the survival, biochemical parameters (acetylcholinesterase (AChE) activity, glutathione s-transferase (GST) activity and the concentration of reactive oxygen species (ROS)) and locomotor capacity of the Coleoptera Alphitobius diaperinus (Panzer), a pest of beef poultry. The sublethal concentrations (100% survival of A. diaperinus during 96 h of exposure) of I. verum essential oil selected for analysis were 0.5% and 1%. The selected sublethal concentrations did not show significant increases in ROS levels after 24 h of exposure to the essential oil. However, increases in GST activity were seen following exposure to 0.5% I. verum essential oil, while decreases in AChE activity were observed following exposure to concentrations of 0.5% and 1%. These results correlate with the observed behavior of A. diaperinus; when placed into an arena, these insects typically demonstrate aversion to stimuli and refuge-seeking behavior. Following exposure to 0.5% I. verum essential oil, the insects showed loss of refuge-seeking capacity and, following exposure to a concentration of 1%, loss of locomotor capacity. Overall, these results indicate that I. verum essential oil can be used as an alternative to conventional insecticides.

The recognition images of cattle brand in an automatic way is a necessity to governmental organs responsible for this activity. To help this process, this work presents a method that consists in using Bag of Visual Words for extracting of characteristics from images of cattle brand and Support Vector Machines Multi-Class for classification. This method consists of six stages: a) select database of images; b) extract points of interest (SURF); c) create vocabulary (K-means); d) create vector of image characteristics (visual words); e) train and sort images (SVM); f) evaluate the classification results. The accuracy of the method was tested on database of municipal city hall, where it achieved satisfactory results, reporting 86.02% of accuracy and 56.705 seconds of processing time, respectively.

Chlorothalonil is an active biocide applied in antifouling paints, and also used as fungicide in agricultural activities with the purpose to protect plants from foliar and seed diseases. Thus, the aim of this study was to evaluate the effects of chlorothalonil exposure on biochemical biomarkers of oxidative metabolism as well as on cholinesterases in the estuarine polychaete Laeonereis acuta. Animals were exposed for 24 and 96 h to the following nominal concentrations of chlorothalonil: 0.1, 10.0 and 100.0 μg/L. The antioxidant capacity against peroxyl radicals (ACAP) and the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), glutamate cysteine ligase (GCL), acetylcholinesterase (AChE) and propionylcholinesterase (PChE) were evaluated in whole-body tissue. In addition, the levels of reduced glutathione (GSH), lipid peroxidation (LPO), glycogen and lactate levels were also analyzed. A reduction in ACAP levels was observed in animals exposed to the higher chlorothalonil concentration, concomitantly with an induction of GST activity as well as diminution in GSH content in these animals. This disturbance in the redox state of animal tissues leads to an oxidative stress situation, resulting in an induction in LPO levels. It was also demonstrated that chlorothalonil exposure causes alteration in AChE activity, possibly related to damage to membrane lipids. These results demonstrated that chlorothalonil possesses harmful effects to estuarine animals and its use as antifouling biocide has to be carefully reconsidered in risk analysis studies.

Reproductive effects caused by the exposure to environmentally relevant dosages of atrazine on wild animals are poorly understood. This study evaluated the effects of three dosages of atrazine on sperm parameters of adult Calomys laucha males. Adult mice were orally exposed to dosages of 0 (water and vehicle control), 0.1, 1, and 10 mg/kg of animal weight for a 21-day period. Following exposure, analyses were performed to determine sperm motility parameters, plasma membrane integrity and fluidity, mitochondrial functionality, acrosome integrity, DNA damage, lipid peroxidation, and production of reactive oxygen species (ROS) in the sperm samples. Total and progressive motility were reduced in all dosages in comparison to control groups. Membrane integrity and mitochondrial functionality of sperm were reduced in all dosages, and the sperm membrane fluidity increased in the higher dosages of atrazine (1 and 10 mg/kg), in comparison with the vehicle control. A decrease in the acrosome integrity was noted at 10 mg/kg of atrazine, compared to the control groups. The integrity of DNA, ROS generation, and lipid peroxidation of sperm showed no significant differences when compared with the control groups. These results suggest that exposure to low dosages of atrazine can affect sperm parameters of Calomys laucha and therefore reduce the reproductive capacity of wild rodent species.

It has been demonstrated that glyphosate-based herbicides are toxic to animals. In the present study, reactive oxygen species (ROS) generation, antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO), as well as the activity and expression of the acetylcholinesterase (AChE) enzyme, were evaluated in Danio rerio males exposed to 5 or 10 mg/L of glyphosate for 24 and 96 h. An increase in ACAP in gills after 24 h was observed in the animals exposed to 5 mg/L of glyphosate. A decrease in LPO was observed in brain tissue of animals exposed to 10 mg/L after 24 h, while an increase was observed in muscle after 96 h. No significant alterations were observed in ROS generation. AChE activity was not altered in muscles or brains of animals exposed to either glyphosate concentration for 24 or 96 h. However, gene expression of this enzyme in the brain was reduced after 24 h and was enhanced in both brain and muscle tissues after 96 h. Thus, contrary to previous findings that had attributed the imbalance in the oxidative state of animals exposed to glyphosate-based herbicides to surfactants and other inert compounds, the present study demonstrated that glyphosate per se promotes this same effect in zebrafish males. Although glyphosate concentrations did not alter AChE activity, this study demonstrated for the first time that this molecule affects ache expression in male zebrafish D. rerio.

Several studies suggest that Antarctic endemic stenothermic fish species, such as Notothenia coriiceps, have a limited ability to acclimate to warmer conditions. In the present study, we evaluated the effects of exposure to short- and long-term temperature increases on the occurrence of erythrocytic nuclear abnormalities (ENAs) and changes in the leukocyte profile of N. coriiceps. For the experiments, 30 specimens of N. coriiceps were captured in Admiralty Bay and transferred to the Commandant Ferraz Brazilian Antarctic Station where they were acclimated to water at 0 °C for 6 days and subsequently subjected to treatments of temperature increases (2–4 °C) on a short- (1 day) or long- (6 days) term basis. At the end of exposure, peripheral blood samples were collected and blood smears were prepared for qualitative and quantitative analysis of the ENAs and the leukocyte profile. Significant responses were observed for both ENAs and leukocyte profile. The responses of ENAs were characterized by an increased incidence of bilobed nuclei in erythroblasts following 1-day exposure to water at 2–4 °C, as well as increased incidence of bilobed nuclei in erythrocytes following 1-day and 6-day exposure to water at 4 °C. In the leukocyte profile, 1-day exposure to water at 2 °C promoted a stress response characterized by diminished levels of circulating lymphocytes (lymphopenia) and elevated levels of circulating monocytes (monocytosis). In conclusion, our results indicate that elevation of water temperature during short periods provoked cellular and physiological stress in N. coriiceps. Furthermore, we conclude that in terms of nuclear alterations, erythrocytes and erythroblasts respond differently to thermal stress. Finally, we conclude that the methodology proposed was efficient in quantifying thermal stress in N. coriiceps and we recommend the utilization of this technique in the biomonitoring of thermal stress in this species and other notothenioids.

In biofloc technology (BFT) systems, nutrient cycling for microorganisms takes place. This results in minimal or null water exchange. The sum of fish biomass and microorganisms favors alkalinity consumption and, consequently, pH reduction. It is in this context that the present study evaluates alkalinity and pH maintenance using sodium bicarbonate (NaHCO3), calcium carbonate (CaCO3) or calcium hydroxide (Ca(OH)2) on BFT during a Nile tilapia (Oreochromis niloticus) nursery. In this study, 25 fishes/tank was distributed in nine experimental units (useful vol. 37.5 L) and the performance was evaluated at 60 days. During the assay, the temperature, oxygen and pH were maintained within the ideal range levels for Nile tilapia growth. All alkalizing compounds were able to pH and alkalinity correction, but when using NaHCO3, the alkalinity and pH were more elevated than the other treatments. Furthermore, at the beginning these assay, the total ammonia (TAN; NH3 + NH4+) and NO2−-N accumulate and it caused a peak, but mostly experiment remained to very low levels because of the total nitrification activity, resulting in NO3−-N accumulation. Because the non water exchange, at the final experiment the ion Na+ accumulate when utilized NaHCO3, resulting in level similar to brackish water. While using CaCO3 or Ca(OH)2, the Ca2 + ion accumulate, resulting in extremely hard water. Despite this, the fish survival was similar between treatments (about 80%). Moreover, the final weight, daily growth rate and net yield for NaHCO3 and Ca(OH)2 they were higher than CaCO3 treatment. This may have been because of the higher total suspended solids (TSS) and lower protein content of the bioflocs in this treatment. In order to assess the possible physiologic alterations of the fish associated with the production system, the hematocrit, glycemia and plasmatic osmolality were evaluated. Furthermore, the antioxidant capacity against peroxy radicals (ACAP), lipid peroxidation (LPO) and catalase (CAT) and superoxide dismutase (SOD) activities on the gills and liver were also evaluated. There were no differences in biochemical/physiological parameters when the different alkalizing compounds were utilized. The results demonstrate that the use of sodium bicarbonate, hydroxide or calcium carbonate is effective on the alkalinity and pH adjustments of the final proportion of 14.64 ± 0.49, 7.18 ± 0.32 e 24.09 ± 2.32% in relation to the feed consumption, respectively. Thus, the study demonstrates that the use of NaHCO3 and Ca(OH)2 are recommended for alkalinity and pH correction during Nile tilapia nursery on BFT systems, because of the higher growth and net yield, and this sum to less amount of these compounds may represent important economic gain. Statement of relevance The results of this manuscript demonstrate: − Amount of alkalizing compound utilized for maintenance of alkalinity and pH − Best growth to utilize NaHCO3 and Ca(OH)2 − The importance of evaluate the ionic concentration, specially Na+ and Ca+ 2, because the imbalance ionic − The difficulty to solids control in BFT system.

The objective of the present study was to determine the effects of Roundup on oxidative status in adult Danio rerio liver and gills. Reactive oxygen species (ROS) and antioxidant capacity (ACAP) were measured in fish after exposure to Roundup (5 and 10 mg/L) for 24, 48, 72, and 96 h. Furthermore, gene expression related to antioxidant response was evaluated after 24 and 96 h. In gills, an increase in ACAP was observed after 96 h in the highest concentration. In the liver, a reduction in ROS and ACAP was observed after 24 h, whereas an increase in ACAP was observed after 48 h in the highest concentration. Exposure to the lowest concentration caused a reduction in ROS after 72 and 96 h. Regarding gene expression, a reduction in superoxide dismutase 2 (sod2) and glutathione S-transferase (gstπ) was observed. An increase in uncoupling protein 1 (ucp1) expression was observed in gills of animals exposed to the highest concentration after 24 h. Glutathione peroxidase (gpx) gene expression was reduced in the gills of animals exposed to the lowest concentration; however, it was induced in liver tissue after 96 h of exposure to the highest concentration. These results indicate that zebrafish exposure to Roundup alters oxidative status and causes a response in terms of antioxidant defense system gene expression.

In this study, a sensitive HPLC-UV assay was developed and validated for the determination of LASSBio-1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid-liquid extraction using acetonitrile was employed to extract LASSBio-1736 and IS from 100 μL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb(®) S5 ODS2 C18 column (150 × 4.6 mm, 5 μm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio-1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 μg/mL and linearity between 0.2 and 4 μg/mL was obtained, with an R(2) > 0.99. The accuracy of the method was >90.5%. The relative standard deviations intra and interday were

Dichlofluanid is a fungicide employed as a booster biocide in antifouling paints, but information its toxicity to aquatic organisms is scarce. This study aims to evaluate biomarker responses in the mussel Perna perna exposed to dichlofluanid. Mussels were exposed to 0 (control), 0.1 μg/L (environmental concentration), 10, and 100 μg/L of dichlofluanid for 24 and 96 h. Byssus formation, oxygen consumption, and oxidative stress response were evaluated in gills and digestive glands. The results demonstrated that even the lowest dichlofluanid concentration causes a reduction in byssus biomass and water content. The higher concentrations caused an acute increase in oxygen consumption, which only returned to control levels after 96 h of exposure. ACAP levels and antioxidant enzyme activities were affected in both tissues with a larger effect observed in gill tissues as demonstrated by the IBR index. The overall results demonstrated that environmentally relevant concentrations of dichlofluanid would be deleterious to aquatic organisms.

Graphene has been shown to induce toxicity in mammals and marine crustaceans; however, information regarding oxidative stress in fish is scarce. The aim of this study was to evaluate the mechanism of graphene toxicity in different tissues of Danio rerio, considering different parameters of stress. Animals were injected intraperitoneally (i.p.) with 10 μL of suspensions containing different graphene concentrations (5 and 50 mg/L); the gills, intestine, muscle and brain were analysed 48 h later. There was no significant difference in the expression of the gclc (glutamate cysteine ligase catalytic subunit) and nrf2 (nuclear factor (erythroid-derived 2)-like 2) genes after exposure. In contrast, glutamate cysteine ligase (GCL) and glutathione-S-transferase (GST) activities were modulated and the glutathione (GSH) concentration was reduced in different tissues and at different concentrations. Lipid damage was observed in the gills. Histological analyses were performed to observe if the exposure could induce pathological damage in these tissues. The results showed pathological effects in all tissues, excluding the intestine, after exposure to both concentrations. Overall, these results indicate that graphene induces different grades of toxicological effects that are dependent on the analysed organ, with distinct pathological effects on some and oxidative effects on others. However, the brain and gills seem to be the primary target organs for graphene toxicity.

The automatic recognition of cattle brandings is a need of the government organizations responsible for controlling this activity. To help this process, this work presents a method that consists in using Deep Learning techniques for extracting features from images of cattle branding and Support Vector Machines for their classification. This method consists of six stages: (a) selection of a database of images; (b) selection of a pre-trained CNN; (c) pre-processing of the images, and application of the CNN; (d) extraction of features from the images; (e) training and classification of images (SVM); (f) evaluation of the results obtained in the classification phase. The accuracy of the method was tested on the database of a City Hall, where it achieved satisfactory results, comparable to other methods reported in the literature, with 91.94% of Overall Accuracy, and a processing time of 26.766 s, respectively.

Atrazine is an extensively used herbicide, and has become a common environmental contaminant. Effects on dopaminergic neurotransmission in mammals following exposure to atrazine have been previously demonstrated. Here, the effects of atrazine regarding behavioural and dopaminergic neurotransmission parameters were assessed in the fruit fly D. melanogaster, exposed during embryonic and larval development. Embryos (newly fertilized eggs) were exposed to two atrazine concentrations (10μM and 100μM) in the diet until the adult fly emerged. Negative geotaxis assay, as well as exploratory behaviour, immobility time and number of grooming episodes in an open field system were assessed. Tyrosine hydroxylase (TH) activity and gene expression of the dopaminergic system were also evaluated in newly emerged male and female flies. All analyzed parameters in male flies were not significantly affected by atrazine exposure. However female flies exposed to atrazine at 10μM presented an increase in immobility time and a reduction in exploratory activity in the open field test, which was offset by an increase in the number of grooming episodes. Also, female flies exposed to 100μM of atrazine presented an increase in immobility time. Gene expression of DOPA decarboxylase and dopamine (DA) receptors were also increased only in females. The behavioural effects of atrazine exposure observed in female flies were due to a disturbance in the dopaminergic system.

Lead (Pb) effects on testis histology, as well as sperm quality and oxidative status were evaluated in male Chestnut Capped Blackbird (Chrysomus ruficapillus). Wild blackbirds were captured, immediately sampled (field group) or kept in captivity and treated with a single intraperitoneal injection of saline solution (control) or saline solution with Pb acetate (50 or 100 mg/kg Pb). Seven days after injection, whole blood, ductus deferens and testis samples were collected. Increased Pb concentrations were observed in whole blood and testis of Pb-exposed blackbirds with respect to those from field and control blackbirds. Sperm cells of Pb-exposed blackbirds showed loss of membrane integrity, mitochondrial functionality, and DNA integrity. Also, oxidative damage was observed in testis of blackbirds injected with 100 mg/kg Pb. These findings indicate that Pb is accumulated in testis of C. ruficapillus, inducing severe morphological and biochemical injury that can compromise the reproductive performance of male blackbirds. Although the exposure scenario (Pb acetate, high dosage and intraperitoneal injection) tested in the present study would likely not occur in the wild, it was adequate to show potential and relevant toxic effects of Pb in wild birds.

This article presents a hybrid method that uses Convolutional Neural Networks (CNN) to segmentation and Support Vector Machines (SVM) to detection the brandings. The experiments were performed using a cattle branding images. Metrics of Overall Accuracy, Recall, Precision, Kappa Coefficient, and Processing Time were used in order to assess the proposed tool. The results obtained here were satisfactory, reaching a Overall Accuracy of 93% in the first experiment with 39 brandings and 1,950 sample images, and 95% of accuracy in the second experiment, with the same 39 brandings, but with 2,730 sample images. The processing time attained in the experiments was 32s and 42s, respectively.

The herbicide atrazine has been used worldwide with subsequent residual contamination of water and food, which may cause adverse effects on non-target organisms. Animal exposure to this herbicide may affect development, reproduction and energy metabolism. Here, the effects of atrazine regarding survival and redox metabolism were assessed in the fruit fly D. melanogaster exposed during embryonic and larval development. The embryos (newly fertilized eggs) were exposed to different atrazine concentrations (10μM and 100μM) in the diet until the adult fly emerged. Pupation and emergence rates, developmental time and sex ratio were determined as well as oxidative stress parameters and gene expression of the antioxidant defence system were evaluated in newly emerged male and female flies. Atrazine exposure reduced pupation and emergence rates in fruit flies without alterations to developmental time and sex ratio. Different redox imbalance patterns were observed between males and females exposed to atrazine. Atrazine caused an increase in oxidative damage, reactive oxygen species generation and antioxidant capacity and decreased thiol-containing molecules. Further, atrazine exposure altered the mRNA expression of antioxidant genes (keap1, sod, sod2, cat, irc, gss, gclm, gclc, trxt, trxr-1 and trxr-2). Reductions in fruit fly larval and pupal viability observed here are likely consequences of the oxidative stress induced by atrazine exposure.

Antioxidant defense system (ADS) and oxidative stress parameters were evaluated in the Antarctic fish Notothenia rossii and N. coriiceps exposed to increasing temperature. Acclimated fish were kept at 0 °C or exposed to 4 °C for 1 day (N. rossii) or to 2 and 4 °C for 1 and 6 days (N. coriiceps). Measurements were assessed in brain, gills, liver, white muscle and erythrocytes. Parameters analyzed included antioxidant capacity against peroxyl radicals (ACAP); reduced glutathione (GSH) and metallothionein-like proteins (MTLP) concentration; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutamate-cysteine ligase (GCL) activity; lipid peroxidation (LPO) level and protein carbonyl (PC) concentration. Increased liver GST activity was observed in N. rossii exposed to 4 °C for 1 day. Increased muscle GPx activity was observed in N. coriiceps after exposure to 2 °C for 1 day. Reduced gill GPx activity and increased liver SOD activity were observed after exposure to 4 °C for 1 day. In N. coriiceps, increased gill GCL activity and reduced gill GPx activity, as well as reduced liver MTLP were observed after exposure to 2 °C for 6 days. Reduced brain SOD activity and increased brain LPO; reduced gill ACAP, GSH concentration and GPx activity, as well as increased gill GCL activity; reduced liver ACAP, MTLP, SOD activity, GST activity and increased liver and erythrocytes LPO were observed after exposure to 4 °C for 6 days. These findings indicate that ADS is more responsive to short-term increasing temperature in the sluggish N. coriiceps than in the active N. rossii. However, responses of N. coriiceps to long-term increasing temperature were transient and did not prevent tissue oxidative damage. Considering the predicted increase in temperature in the Southern Ocean over the next decades, our findings suggest that Antarctic fishes are sensitive to ocean warming, displaying tissue oxidative damage associated with the thermal stress.

Adaptive responses of antioxidant defense systems (ADS) to changes in increased levels of activity are critical, especially in Antarctic fishes. The benthopelagic marbled notothen (Notothenia rossii) shows higher spontaneous activity than the benthonic and sluggish rockcod (N. coriiceps). Therefore, we hypothesize that species-related responses of ADS would occur to counteract different rates of reactive oxygen species formation in these two Antarctic fish. Here we evaluated ADS and oxidative damage in tissues (brain, gills, liver and white muscle) of the two Antarctic fish. Despite no significant differences in lipid and protein oxidative damage were observed, we actually found species- and tissue-specific differences in ADS. Gill metallothionein-like proteins (MTLP) and liver reduced glutathione (GSH) concentrations were higher in N. coriiceps than in N. rossii. Brain and gill antioxidant capacity against peroxyl radicals (ACAP); gill enzyme [glutamate-cysteine ligase (GSL), superoxide dismutase (SOD) and catalase (CAT)] activity; liver GCL and SOD activity; and white muscle CAT activity were higher in N. rossii than in N. coriiceps. Therefore, the more active fish (N. rossii) maintains higher activities of enzymes involved in superoxide ions (O2.-) detoxification and GSH production in peripheral tissues (gills, liver and white muscle). This allows the more active fish (N. rossii) to keep levels of lipid and protein oxidative damage similar to those observed in the sluggish fish (N. coriiceps). It is worth noting that the more active fish also shows a higher brain antioxidant capacity, which could involve other non-enzymatic antioxidants like vitamins C and E. In contrast, N. coriiceps shows lower consumption of non-enzymatic antioxidants in peripheral tissues than N. coriiceps. As hypothesized, our results indicate that differences in ADS profiles between fish species are likely related to their habits and metabolic rates. This would imply in different fish abilities to deal with oxidative stress associated with increasing seawater temperature.

Glyphosate based herbicides, including Roundup, are widely employed in agriculture and urban spaces. The objective of this study was to evaluate the toxicological effects of Roundup on the estuarine polychaeta Laeonereis acuta. Biomarkers of oxidative stress as well as acetylcholinesterase and propionilcholinesterase activities were analyzed. Firstly, the LC50 96h for L. acuta was established (8.19mg/L). After, the animals were exposed to two Roundup concentrations: 3.25mg/L (non-observed effect concentration - NOEC) and 5.35mg/L (LC10) for 24h and 96h. Oxygen consumption was determined and the animals were divided into three body regions (anterior, middle and posterior) for biochemical analysis. An inhibition of both cholinesterase isoforms were observed in animals exposed to both Roundup concentrations after 96h. A significant reactive oxygen species (ROS) reduction was observed in the posterior region of animals in both periods, while antioxidant capacity against peroxyl radicals (ACAP) was reduced in the posterior region of animals exposed for 24h. Considering the antioxidant defense system, both GSH levels and enzyme activities (catalase, superoxide dismutase, glutathione s-transferase, glutathione peroxidase and glutamate cysteine ligase) were not altered after exposure. Lipid peroxidation was reduced in all analyzed body regions in both Roundup concentrations after 24h. Animals exposed to the highest concentration presented a reduction in lipid peroxidation in the anterior region after 96h, while animals exposed to the lowest concentration presented a reduction in the middle region. Overall results indicate that Roundup exposure presents toxicity to L. acuta, causing a disruption in ROS and ACAP levels as well as affects the cholinergic system of this invertebrate species.

Glyphosate is a non-selective and post-emergent herbicide that affects plant growth. Animal exposure to this herbicide can lead to adverse effects, such as endocrine disruption, oxidative stress and behavioural disorders. Drosophilids have been utilized previously as an effective tool in toxicological tests. In the present study, the effects of a glyphosate-based herbicide (Roundup [Original]) were investigated regarding oxidative stress, the antioxidant defence system and acetylcholinesterase (AChE) activity in Drosophila melanogaster. Flies (of both genders) that were 1 to 3days old were exposed to different glyphosate concentrations (0.0g/L=control, 1.0g/L, 2.0g/L, 5.0g/L and 10.0g/L) [corrected] in the diet for 24h and 96h. After the exposure periods, reactive oxygen species (ROS) levels, antioxidant capacity against peroxyl radicals (ACAP) and lipid peroxidation (LPO) levels were quantified. In addition, the mRNA expression of antioxidant genes (i.e., keap1, sod, sod2, cat, irc, gclc, gclm, gss, trxt, trxr-1 and trxr-2) was evaluated via RT-PCR. Additionally, AChE activity was evaluated only after the 96h exposure period. The results indicated that Roundup exposure leads to a reduction in ROS levels in flies exposed for 96h. ACAP levels and gene expression of the antioxidant defence system exhibited an increase from 24h, while LPO did not show any significant alterations in both exposure periods. AChE activity was not affected following Roundup exposure. Our data suggest that Roundup exposure causes an early activation of the antioxidant defence system in D. melanogaster, and this can prevent subsequent damage caused by ROS.

Growth hormone (GH) excess causes an increment in the metabolic rate and in reactive oxygen species generation, which accelerate the ageing process in mammals. Considering that there is no information on this subject in fish, the aim of the present study was to evaluate the excess GH effect on senescence in a zebrafish (Danio rerio) transgenic model. In order to reach this objective, we analyzed the phenotype of spinal curvature and expression of genes related to the anti-oxidant defense system and myogenesis in muscle of 8 and 30 months old GH-transgenic males. Gene expression analyses revealed that both superoxide dismutase isoforms were down-regulated only in 30 months old animals, while glutamate cysteine ligase was down-regulated in GH-transgenic zebrafish. Acceleration of the spinal curvature and a reduction in the expression of miogenin at both ages and MyoD in the old fish were also observed. Although neurolipofuscin accumulation was not significant in GH-transgenic zebrafish, the estimation of maximum longevity based on the von Bertalanffy growth function was significantly lower in this group. The results obtained here indicate that GH overexpression reduces the transcription of anti-oxidant defense system and myogenesis-related genes, which probably accelerates senescence in the zebrafish transgenic model used.

Growth hormone overexpression increases growth and consequently increases the metabolic rate in fishes. Therefore, the objective of this study was to evaluate the effects of growth hormone overexpression in zebrafish Danio rerio in terms of growth, oxygen consumption, reactive oxygen species production, lipid hydroperoxide content, antioxidant enzyme activity and glutamate-cysteine ligase catalytic subunit gene expression. The employed models were wild type and transgenic (hemizygous and homozygous) zebrafish expressing the Odonthestes argentinensis growth hormone gene directed by the Cyprinus carpio beta-actin promoter. Higher growth parameters were observed in the hemizygous group. The homozygous group possessed higher oxygen consumption and reactive oxygen species production. Growth hormone transgenesis causes a decrease in glutamate-cysteine ligase catalytic subunit expression, an enzyme responsible for glutathione synthesis. Although the lipid hydroperoxide content was similar between groups, we demonstrate that growth hormone overexpression has the potential to generate oxidative stress in fishes.

In this study, a sensitive HPLC-UV assay was developed and validated for the determination of LASSBio-1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid-liquid extraction using acetonitrile was employed to extract LASSBio-1736 and IS from 100 μL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb(®) S5 ODS2 C18 column (150 × 4.6 mm, 5 μm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio-1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 μg/mL and linearity between 0.2 and 4 μg/mL was obtained, with an R(2) 0.99. The accuracy of the method was90.5%. The relative standard deviations intra and interday were6.19 and7.83%, respectively. The method showed the sensitivity, linearity, precision, accuracy and selectivity required to quantify LASSBio-1736 in preclinical pharmacokinetic studies according to the criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2016 John WileySons, Ltd.

A dissolution method for benzoyl metronidazole (BMZ) oral suspensions was developed and validated using a high-performance liquid chromatography (HPLC) method. After determination of sink conditions, dissolution profiles were evaluated using different dissolution media and agitation speeds. The sample insertion mode in dissolution media was also evaluated. The best conditions were obtained using a paddle, 50 rpm stirring speed, simulated gastric fluid (without pepsin) as the dissolution medium, and sample insertion by a syringe. These conditions were suitable for providing sink conditions and discriminatory power between different formulations. Through the tested conditions, the results can be considered specific, linear, precise, accurate, and robust. The dissolution profiles of five samples were compared using the similarity factor (f 2) and dissolution efficiency. The dissolution kinetics were evaluated and described by the Weibull model. Whereas there is no monograph for this pharmaceutical formulation, the dissolution method proposed can be considered suitable for quality control and dissolution profile comparison of different commercial formulations.

A novel approach for statistical analysis of comet assay data (i.e.: tail moment) is proposed, employing public-domain statistical software, the R system. The analytical strategy takes into account that the distribution of comet assay data, like the tail moment, is usually skewed and do not follow a normal distribution. Probability distributions used to model comet assay data included: the Weibull, the exponential, the logistic, the normal, the log normal and log-logistic distribution. In this approach it was also considered that heterogeneity observed among experimental units is a random feature of the comet assay data. This statistical model can be characterized with a location parameter m(ij), a scale parameter r and a between experimental units variability parameter theta. In the logarithmic scale, the parameter m(ij) depends additively on treatment and random effects, as follows: log(m(ij)) = a0 + a1x(ij) + b(i), where exp(a0) represents approximately the mean value of the control group, exp(a1) can be interpreted as the relative risk of damage with respect to the control group, x(ij) is an indicator of experimental group and exp(b(i)) is the individual risk effects assume to follows a Gamma distribution with mean 1 and variance theta. Model selection is based on Akaike's information criteria (AIC). Real data coming from comet analysis of blood samples taken from the flounder Paralichtys orbignyanus (Teleostei: Paralichtyidae) and from samples of cells suspension obtained from the estuarine polychaeta Laeonereis acuta (Nereididae) were employed. This statistical approach showed that the comet assay data should be analyzed under a modeling framework that take into account the important features of these measurements. Model selection and heterogeneity between experimental units play central points in the analysis of these data.

Polychaeta species like Laeonereis acuta (Nereididae) usually secrete great amounts of mucus that wrap the animal inside. Taking into account that fungi action in the sediment and UV radiation acting on dissolved organic matter in the water produces reactive oxygen species (ROS) like hydrogen peroxide (H(2)O(2)), it was considered that the mucus secretion could represent an antioxidant defense against environmental ROS. Antioxidant enzymes (catalase-CAT; superoxide dismutase-SOD; glutathione peroxidase-GPx and glutathione-S-transferase-GST) and total antioxidant capacity (TOSC) were determined in worms and mucus secretion. Higher (p0.05) CAT, GPx and TOSC values were registered in mucus samples respect worms, SOD activity was similar (p0.05) in both kind of samples, and absence of GST activity was observed in mucus samples, suggesting absence of catalyzed phase II reactions. In assays conducted with hepatoma cell lines exposed to H(2)O(2), it was verified that: (1) mucus co-exposure significantly (p0.05) lowered DNA damage induced by H(2)O(2); (2) ROS production was significantly (p0.05) reduced when cells were exposed simultaneously with mucus samples and H(2)O(2) respect H(2)O(2) alone. It can be concluded that the mucus production contributes substantially to the antioxidant defense system of the worm against environmental ROS through the interception or degradation of H(2)O(2), peroxyl and hydroxyl radicals.

This study identified daily variations in oxygen consumption, antioxidant-defense system, and lipoperoxidation in the gills and hepatopancreas of the crab Chasmagnathus granulata (Dana, 1851) (Decapoda, Brachyura). In gills, oxygen consumption was higher in the early afternoon and in the middle of the night (p < 0.05). Lipoperoxidation level and non-proteic sulfhydryl content were higher in the middle of the night (p < 0.05). The enzyme glutathione-S-transferase showed lower activity at the end of photophase and higher values during the night (p < 0.05). The enzyme catalase did not show daily variation in activity (p > 0.05). Oxygen consumption in the hepato pancreas showed a similar profile to that in gills, i.e., higher values in the early afternoon and the middle of the night. Glutathione-S-transferase activity and lipoperoxidation levels showed significant variation, with lower values during the afternoon and peaks in the middle of the night (p < 0.05). Catalase activity was significantly higher (p < 0.05) in the middle of the night. The non-proteic sulfhydryl content remained fairly constant (p > 0.05). The results showed daily variations in aerobic metabolism of the gills and hepatopancreas of C. granulata, with concomitant oxidative damage (lipoperoxidation), but with differences between tissues. Whereas in the gills the defense system focused on catalase and non-proteic sulfhydryl, in the hepatopancreas other non-enzymatic components and other antioxidant enzymes besides catalase and glutathione-S-transferase might be involved.

Worms like polychaetes, oligochaetes, and nematodes, have been widely used in studies of aquatic and soil pollution. Several ecotoxicological characteristics make these animals suitable for use as a biomonitor, including the fact that these organisms live in close contact with sediments. Also important is that they posses little mobility, which could be useful in biomonitoring programs since it can reflect local environmental problems. Several toxicological aspects are reviewed in these zoological groups, including their antioxidant responses and detoxifying mechanisms of heavy metals. Interestingly, the nematodes and oligochaetes species show induction of proteins different from the well-known metallothionein, including cysteine-rich proteins, and phytochelatins, this last group until recently believed to be present only in plants and fungi.

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm(2)) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.

a b s t r a c t Glyphosate is a systemic, non-selective herbicide widely used in agriculture worldwide. It acts as an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase by interrupting the synthesis of essential aromatic amino acids. This pathway is not present in animals, although some studies have shown that the herbicide glyphosate can affect fish reproduction. In this study, the effect of glyphosate on sperm quality of the fish Danio rerio was investigated after 24 and 96 h of exposure at concentrations of 5 mg/L and 10 mg/L. The spermatic cell concentration, sperm motility and motility period were mea- sured employing conventional microscopy. The mitochondrial functionality, membrane integrity and DNA integrity were measured by fluorescence microscopy using specific probes. No significant differ- ences in sperm concentration were observed; however, sperm motility and the motility period were reduced after exposure to both glyphosate concentrations during both exposure periods. The mitochon- drial functionality and membrane and DNA integrity were also reduced at the highest concentration during both exposure periods. The results showed that glyphosate can induce harmful effects on repro- ductive parameters in D. rerio and that this change would reduce the fertility rate of these animals.

Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata.

Growth hormone (GH) transgenesis has been postulated as a biotechnological tool for improving growth performance in fish aquaculture. However, GH is implied in several other physiological processes, and transgenesis-induced GH excess could lead to unpredictable collateral effects, especially on reproductive traits. Here, we have used two-years-old transgenic zebrafish males to evaluate the effects of GH-transgenesis on spermatic parameters and reproductive success. Transgenic spermatozoa were analyzed in terms of motility, motility period, membrane integrity, mitochondrial functionality, DNA integrity, fertility and hatching rate. We have also performed histological analyses in gonad, in order to verify the presence of characteristic cell types from mature testes. The results obtained have shown that, even in transgenic testes present in all cells in normal mature gonads, a significant general decrease was observed in all spermatic and reproductive parameters analyzed. These outcomes raise concerns about the viability of GH-transgenesis appliance to aquaculture and the environmental risks at the light of Trojan gene hypothesis.

The aim of this study was to evaluate the effects of growth hormone (GH) overexpression on the gene expression profile of multiple components of the antioxidant defense system (ADS) of different genotypes of a GH-transgenic zebrafish (Danio rerio) model. Several ADS-related genes were analyzed by semiquantitative reverse transcription-PCR in the liver of hemizygous (HE) and homozygous (HO) transgenic zebrafish. The results showed a significant reduction in the glutamate cysteine ligase catalytic subunit (GCLC) and the gene expression of two glutathione S-transferase (GST) isoforms and an increase in the glutathione reductase gene in the HO group compared to non-transgenic controls. The expression of the Cu, Zn-superoxide dismutase (SOD1) and catalase (CAT) genes was reduced in HO and HE groups, respectively. Among the ten genes analyzed, two were altered in HE transgenic zebrafish and five were altered in HO transgenic zebrafish. These findings indicate a genotype-dependent gene expression profile of the ADS-related genes in the liver of our GH-transgenic zebrafish model and are in agreement with the general effects of GH hypersecretion in the fish and mouse, which involves a reduction in the capability of the tissues to deal with oxidative stress situations. The GH-transgenic zebrafish model used here seems to be an interesting tool for analyzing the effect of different GH expression levels on physiological processes.

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

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