A rapid protocol for inducing acute pancreatitis in zebrafish models.

Acute pancreatitis (AP) is an inflammatory disorder that occurs in the exocrine pancreas associated with tissue injury and necrosis. Experimental models of AP typically involve rodents, such as rats or mice. However, rodents exhibit divergent pathophysiological responses after the establishment of AP between themselves and in comparison, with human. The experiments conducted for this manuscript aimed to standardize a new AP model in zebrafish and validate it. Here, we provide a protocol for inducing AP in zebrafish through intraperitoneal injections of synthetic caerulein. Details are provided for solution preparation, pre-injection procedures, injection technique, and monitoring animal survival. Subsequently, validation was performed through biochemical and histological analyses of pancreatic tissue. The administered dose of caerulein for AP induction was 10 µg/kg applied four times in the intraperitoneal region. The histological validation study demonstrated the presence of necrosis within the first 12 h post-injection, accompanied by an excess of zymogen granules in the extracellular milieu. These observations align with those reported in conventional rodent models. We have standardized and validated the AP model in zebrafish. This model can contribute to preclinical and clinical studies of new drugs for AP treatment. Therefore, this novel model expands the toolkit for exploring faster and more effective preventive and therapeutic strategies for AP.

Using structural equation modeling to assess the genotoxic and mutagenic effects of heavy metal contamination in the freshwater ecosystems: A study involving Oreochromis niloticus in an urban river.

Chemical pollutants represent a leading problem for aquatic ecosystems, as they can induce genetic, biochemical, and physiological changes in the species of these ecosystems, thus compromising their adaptability and survival. The Capibaribe River runs through the state of Pernambuco, located in Northeastern Brazil, and passes through areas of agricultural cultivation, densely populated cities, and industrial centers, primarily textiles. Despite its importance, few ecotoxicological studies have been conducted on its environment, and knowledge about pollution patterns and their effects on its biota is still being determined. The objective of this study was to evaluate the water quality and the damage supposed to be caused by pollutants on the DNA specimens of Nile tilapia (Oreochromis niloticus) obtained from seven strategic points of Capibaribe. Tilapia specimens and water were collected during the rainy and dry seasons from 2015 to 2017. The following characteristics were analyzed: physicochemical (six), metal concentration (seven), local pluviosity, micronuclei, and comet assay. The physicochemical and heavy metal analyses were exploratory, whereas the ecotoxicological analyses were hypothetical. To verify this hypothesis, we compared the groups of fish collected to the results of the micronuclei test and comet assay. We created a Structural Equation Model (SEM) to determine how each metal's micronuclei variables, damage index, pluviosity, and concentration were related. Our results demonstrated that the highest values for markers of genetic damage were detected at points with the highest heavy metal concentrations, especially iron, zinc, manganese, chromium, and cadmium. The SEM demonstrated that metals could explain the findings of the genotoxicity markers. Moreover, other pollutants, such as pesticides, should be considered, mainly where the river passes through rural areas. The results presented here demonstrate that the Capibaribe River has different degrees of contamination and confirm our hypothesis.

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing.

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

Biocompatibility and immunostimulatory properties of fish collagen and shrimp chitosan towards peripheral blood mononuclear cells (PBMCs).

Several naturally occurring biopolymers are commercially produced from livestock and farmed animals processing wastes, including aquatic organisms. These wastes are considered valuable coproducts of fishery processing industry, from which biopolymers may be recovered and exploited for their bioactive potential. The aim of this work was to prepare polymeric films from collagen and chitosan solutions, extracted from fishery discards, and investigate the cytotoxicity and immunomodulatory activity towards human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy donors and treated with Chitosan, Collagen, Chitosan+Collagen solutions and Chitosan+Collagen film in order to measure the changes in cell viability, cytosolic calcium concentration ([Ca2+]cyt), mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) levels, differentiation and activation of T CD8+ and CD4+ lymphocytes, and cytokine production. Results showed that collagen and chitosan preparations did not show cytotoxic effect, while cellular IL-6, IL-10, and TNF-α release was observed. Chitosan and collagen were able to promote non-cytotoxic PBMCs activation through cytosolic and mitochondrial ROS production. There was a noteworthy phenotyping of lymphocytes T CD8+ and CD4+ counting and an increase of [Ca2+] cyt and ΔΨm levels. These results suggest that chitosan/collagen-based biomaterials produce immunostimulatory effects on PBMC with potential to biomedical approaches.

Brain acetylcholinesterase of three perciformes: From the characterization to the in vitro effect of metal ions and pesticides.

Estuarine environments gather pollution from large regions including urban and industrial zones. The monitoring of environmental quality in these areas constitutes a real requirement for global sustainable development. Therefore, the aim of this study was to characterize the physicochemical and kinetic parameters of brain acetylcholinesterase (AChE) in the species Centropomus undecimalis, Diapterus auratus and Diapterus rhombeus and to assess the effects (in vitro) of pesticides and metal ions on their respective activities in order to investigate them as potential biomarkers. Physicochemical properties such as thermostability, optimal pH and temperature, as well as kinetic parameters were investigated. AChE was pointed as the predominant cholinesterase (ChE) in the brains of the species under study. The highest optimum pH value was observed for C. undecimalis (8.0), and the lowest for D. rhombeus and D. auratus, with 7.2 and 7.0, respectively. The optimal temperature was 35 °C for the three species. The AChEs of the three species presented moderate thermostability, since they retained 61%, 72% and 67% of the activity up to 45 °C (C. undecimalis, D. auratus and D. rhombeus, respectively). The carbamate carbofuran showed to be the strongest inhibitor even at very low concentrations (IC50: 0.182, 0.174 and 0.203 µmol/L - C. undecimalis, D. auratus and D. rhombeus, respectively), followed by dichlorvos and carbaryl. According to the findings, the AChE of these species may be proposed as in vitro biomarker of exposure to carbofuran and dichlorvos (all three species) and carbaryl (D. auratus and D. Rhombeus), as well as for exceeding limit concentrations of Hg2+ (D. rhombeus) and As3+ (D. auratus) in biomonitoring programs located or not at estuarine environments.

Evaluation of antioxidant and antibacterial capacity of green microalgae Scenedesmus subspicatus.

Microalgae are considered one of the most promising raw materials for the development of high value products for pharmaceuticals, nutraceuticals, and cosmetic industries, as well as being potential sources of protein, vitamins, and minerals for human consumption. Hence, the present research focuses extraction of antioxidant and antimicrobial compounds from Scenedesmus subspicatus using solvents of different polarities. Different solvents such as ethanol, methanol, butanol, acetone, dimethyl sulfoxide, and water were used to extract compounds from the green microalgae S. subspicatus and then they were examined for phytochemical screening, antioxidant activity, and antimicrobial properties. In vitro free radical quenching and total antioxidant activity of extracts were investigated with 1,1-diphenyl-2-picryl hydrazyl and compared with catequin and gallic acid as positive controls. The antimicrobial activity was evaluated in gram-negative and gram-positive bacteria. Aqueous extracts and dimethyl sulfoxide presented better performance in phytochemical analysis. This result showed consistency in the sequential tests. The antioxidant activity was also better using the two solvents cited above. The extracts acetone, water, and dimethyl sulfoxide showed ability to inhibit the growth of Bacillus subtilis. However, only dimethyl sulfoxide inhibited the growth of Klebsiella pneumoniae and Escherichia coli. Use of the aqueous extract, proven its effectiveness, is an economic protocol and avoids the use of toxic substances.

Total carotenoids and antioxidant activity of fillets and shells (in natura or cooked) of "Vila Franca" shrimp (Litopenaeus Schmitti) in different intervals of storage under freezing / Carotenóides totais e atividade antioxidante dos filés e cascas (in natura ou cozida) do camarão "Vila Franca" (Litopenaeus schmitti) em diferentes intervalos de armazenamento sob congelamento

ABSTRACT Shrimps are sources of carotenoids, astaxanthin is the predominant, responsible for their special and desirable properties, as well as for their instability under heat treatment during the domestic preparation, industrial processing or storage under freezing. These can cause discoloration and reduce the beneficial health properties. This study aimed to evaluate the effect of heat treatment and storage under freezing (0, 45 and 90 days) on the levels of total carotenoids and stability of the antioxidant activity of ethanolic extracts of fillets and shells, raw and cooked, of the white shrimp ("Vila Franca") Litopenaeus schmitti (Burkenroad, 1938). The antioxidant ability of the extracts was evaluated using the radicals DPPH• (2,2-diphenyl-1-picryl-hydrazyl) and ABTS+• (2,2'-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid), as well as by the iron reducing power (FRAP) test. The extracts of cooked or in natura shrimps (fillets and shells) represent dietary sources of carotenoids, displaying antioxidant activity through all the tested methods, after heat treatment and storage under freezing. The antioxidant activity of the extracts was superior to the one of ascorbic acid, mainly in the cooked fillet and shells. The samples of shrimp shells seemed a valuable source of carotenoids, whose antioxidant activity was verified even 90 days after freezing, and can be used in food products as functional natural supplement, adding value to this waste.

RESUMO Os camarões são fontes de carotenóides, sendo a astaxantina o predominante, responsáveis por suas propriedades especiais e desejáveis, e também a causa da sua instabilidade pelo tratamento térmico durante o preparo doméstico, processamento industrial ou armazenamento sob congelamento, que pode causar descoloração e redução de suas propriedades benéficas à saúde. Este trabalho visou avaliar o efeito do tratamento térmico e armazenamento sob congelamento (0, 45 e 90 dias) nos teores de carotenóides totais e na estabilidade da atividade antioxidante dos extratos etanólicos de filé e cascas, cruas ou cozidas, de camarão "Vila Franca" Litopenaeus schmitti (Burkenroad, 1938). A capacidade antioxidante dos extratos foi avaliada utilizando os radicais DPPH• (2,2-difenil-1-picril-hidrazila) e ABTS+• (2,2'-azino-bis-(3-etilbenzotiazolina-6- acido sulfônico), bem como pelo teste do poder redutor do ferro (FRAP). Os extratos do camarão cozido ou in natura (filé e cascas), representam fontes dietéticas de carotenóides, exibindo atividade antioxidante através de todos os métodos testados, após o tratamento térmico e armazenamento sob congelamento. A atividade antioxidante dos extratos foi superior a de ácido ascórbico, principalmente no filé e cascas cozidos. As amostras de cascas de camarão parecem uma valiosa fonte de carotenóides, cuja atividade antioxidante foi verificada até 90 dias após o congelamento, e pode ser utilizado nos produtos alimentares como suplemento natural funcional, agregando valor a esses resíduos.

Cytotoxic Compounds Derived from Marine Sponges. A Review (2010-2012).

Abstract: This extensive review covers research published between 2010 and 2012 regarding new compounds derived from marine sponges, including 62 species from 60 genera belonging to 33 families and 13 orders of the Demospongia class (Porifera). The emphasis is on the cytotoxic activity that bioactive metabolites from sponges may have on cancer cell lines. At least 197 novel chemical structures from 337 compounds isolated have been found to support this work. Details on the source and taxonomy of the sponges, their geographical occurrence, and a range of chemical structures are presented. The compounds discovered from the reviewed marine sponges fall into mainly four chemical classes: terpenoids (41.9%), alkaloids (26.2%), macrolides (8.9%) and peptides (6.3%) which, along with polyketides, sterols, and others show a range of biological activities. The key sponge orders studied in the reviewed research were Dictyoceratida, Haplosclerida, Tetractinellida, Poecilosclerida, and Agelasida. Petrosia, Haliclona (Haplosclerida), Rhabdastrella (Tetractinellida), Coscinoderma and Hyppospongia (Dictyioceratida), were found to be the most promising genera because of their capacity for producing new bioactive compounds. Several of the new compounds and their synthetic analogues have shown in vitro cytotoxic and pro-apoptotic activities against various tumor/cancer cell lines, and some of them will undergo further in vivo evaluation.

Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions.

This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68µM (dichlorvos); 4.35µM (carbaryl) and 0.28µM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7µM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.

Alpha-Tocopherol Counteracts the Effect of Ethanol on Cortical Spreading Depression in Rats of Various Ages, With and Without Ethanol Abstinence.

BACKGROUND: We previously demonstrated that acute and chronic treatment with ethanol (EtOH), respectively, decelerated and accelerated the propagation of cortical spreading depression (CSD) in rats and that the antioxidant carotenoid astaxanthin counteracted these effects. Here, we investigated whether noncarotenoid antioxidants exert the same action by testing α-tocopherol in rats of various ages, with and without 5 to 10 days of EtOH abstinence. METHODS: Male Wistar young adult (60 to 80 days old) and mature adult (150 to 180 days old) rats received per gavage acute (1 day) or chronic (21 days) treatment with 3 g/kg/d EtOH combined with acute (300 mg/kg) or chronic (85 mg/kg/d) treatment with α-tocopherol or vehicle-only treatment (olive oil and water for α-tocopherol and EtOH, respectively). CSD was recorded over 4 hours and the velocity of CSD propagation was calculated. On both ages, animals under chronic EtOH treatment were subjected to CSD recording immediately after EtOH treatment or after a 5- to 10-day period of EtOH abstinence. RESULTS: In both age groups, acute and chronic EtOH exposure decelerated and accelerated CSD, respectively, versus the corresponding control groups. Addition of α-tocopherol counteracted the effects of EtOH on CSD, returning CSD velocities to levels in control groups (p < 0.05). Chronic α-tocopherol (85 mg/kg/d) did not alter CSD. CONCLUSIONS: Our data reinforce the counteracting role of antioxidants on brain processes involved in the action of EtOH on CSD and suggest that this role is not a particular property of carotenoids; furthermore, this general feature of antioxidants is not substantially influenced by age or by 5 to 10 days of EtOH abstinence.

Immunomodulatory response of mice splenocytes induced by RcaL, a lectin isolated from cobia fish (Rachycentron canadum) serum.

This work reports the isolation of a serum lectin from cobia fish (Rachycentron canadum) named RcaL. Immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production were also performed. RcaL was obtained through precipitation with ammonium sulphate and affinity chromatography on a Concanavalin A-Sepharose 4B column. The ammonium sulphate fraction F3 showed the highest specific hemagglutinating activity and was applied to affinity chromatography. The lectin was eluted with methyl-α-D-mannopyranoside. RcaL showed highest affinity for methyl-α-D-mannopyranoside and D-mannose; eluted fractions of RcaL agglutinated rabbit erythrocytes (titre, 128(-1)) retained 66 % of chromatographed lectin activity, and the obtained purification factor was 1.14. Under reducing conditions, a polypeptide band of 19.2 kDa was revealed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE). PAGE confirmed RcaL as an acidic protein revealed in a single band. Cytotoxic and immunomodulatory assays with RcaL in mice splenocyte cultures showed that the lectin was not cytotoxic and induced higher interferon gamma and nitric oxide production in splenocyte cultures. Purified RcaL induced preferential Th1 response, suggesting that it acts as an immunomodulatory compound.

The use of cortical spreading depression for studying the brain actions of antioxidants.

OBJECTIVES: We review the main adverse effects of reactive oxygen species (ROS) in the mammalian organism, introducing the reader on the worldwide problem of the ROS neurophysiological impact on the developing and the adult brain, and discussing the neuroprotective action of antioxidant molecules. METHODS: We briefly present the electrophysiological phenomenon designated as 'cortical spreading depression' (CSD), as a parameter of normal brain functioning. We highlight recent electrophysiological advances obtained in experimental studies from our laboratory and from others, showing how to investigate the ROS effects on the brain by using the CSD phenomenon. RESULTS: Under conditions such as aging, ROS production by photo-activation of dye molecules and ethanol consumption, we describe the effects, on CSD, of treating animals with (1) antioxidants and (2) with antioxidant-deficient diets. DISCUSSION: The current understanding of how ROS affect brain electrophysiological activity and the possible interaction between these ROS effects and those effects of altered nutritional status of the organism are discussed.

Shrimp carotenoids protect the developing rat cerebral cortex against the effects of ethanol on cortical spreading depression.

Cortical spreading depression is a neural phenomenon present in several animal species. Spreading depression features, like velocity of propagation, depends on several chemical and metabolic factors, as for example, anti-oxidants. Here we studied spreading depression-velocity changes in weaned rat-pups born from dams treated on a daily basis, either during gestation or lactation, with a carotenoid ethanolic extract (30 microg/kg/day) prepared from shrimp waste (heads). These pups were compared with age-mated ones, whose mothers were treated either with the vehicle (ethanol) or with distilled water. Compared to the distilled water-group (mean values, in mm/min, per hour of recording ranging from 3.02+/-0.26 to 3.15+/-0.27 [treatment during gestation; n=7], and from 3.03+/-0.25 to 3.22+/-0.30 [lactation; n=11]), ethanol-treated rats displayed higher spreading depression-velocities (from 3.74+/-0.06 to 3.82+/-0.08 [gestation; n=7], and from 4.26+/-0.32 to 4.33+/-0.34 [lactation; n=11]; p<0.05). Compared to the ethanol-group, carotenoid-treatment lead to lower spreading depression-velocities (p<0.05), ranging from 3.38+/-0.09 to 3.42+/-0.12, n=7 (gestation) and 3.58+/-0.13 to 3.62+/-0.17, n=12 (lactation). Carotenoid-treatment during lactation was shown to be significantly more effective than that during gestation (p<0.05), in lowering spreading depression-velocity. The results suggest a protective action of shrimp carotenoids against the ethanol effects on spreading depression. This protective effect could be related to the carotenoid antioxidant properties, as previously indicated by evidence showing spreading depression-effects of other antioxidants.