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Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

Establishing new animal models for the study of inflammation is very important in the process of discovering new drugs, since the inflammatory event is the basis of many pathological processes. Whereas rodent models have been the primary focus of inflammation research, we defend the zebrafish (Danio rerio) test as a feasible alternative for preclinical studies. Moreover, despite all the technological development already achieved by humanity, nature can still be considered a relevant source of new medicines. In this context, the aim of this work was to evaluate the anti-inflammatory effect of a substance isolated from the medicinal plant Annona crassilfora Mart, the peltatoside, in an inflammatory model of zebrafish. It was determined: (i) total leukocyte count in the coelomate exudate; (ii) N-acetyl-β-d-glucuronidase (NAG); (iii) myeloperoxidase (MPO); (iv) and the histology of liver, intestine and mesentery. Peltotoside (25, 50 and 100 μg) and dexamethasone (25 μg) were administered intracelomatically (i.c.) 30 min before carrageenan (i.c.). Pretreatment with peltatoside at three doses significantly inhibited leukocyte recruitment in the coelomic cavity, and inhibited NAG and MPO activity against the action of Cg, in a similar manner as dexamethasone. However, some microlesions in the evaluated organs were detected. The dose of 25 μg showed an anti-inflammatory effect with lower undesirable effects in the tissues. Our results suggest that the zebrafish test was satisfactory in performing our analyzes and that the peltotoside has a modulatory action in reducing leukocyte migration.

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research., Graphical abstract Unlabelled Image

Advances in vaccine development depend on animal models to test innovative therapies. Recent studies have reported the successful introduction of teleost fish as a new vertebrate model in scientific research, with emphasis on the species Danio rerio (zebrafish). This chapter aims to give an overview of important aspects related to the immune system of fish, as well as the current progress of the successful use of these animals in studies for the development of vaccines, assisting in the determination of efficacy and clinical safety. Among the advantages of using fish for the development of vaccines and immunomodulatory drugs, it is worth highlighting the reproductive capacity of these animals resulting in a high number of individuals belonging to the same spawning, transparent embryos, low cost of breeding and high genetic similarity that favor translational responses to vertebrate organisms like humans.

Made available in DSpace on 2022-04-28T19:45:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) This study aims to demonstrate the applicability and importance of zebrafish (Danio rerio) model to study acute and chronic inflammatory responses induced by different stimuli: carrageenan phlogogen (nonimmune); acute infection by bacteria (immune); foreign body reaction (chronic inflammation by round glass coverslip implantation); reaction induced by xenotransplantation. In addition to the advantages of presenting low breeding cost, high prolificity, transparent embryos, high number of individuals belonging to the same spawning and high genetic similarity that favor translational responses to vertebrate organisms like humans, zebrafish proved to be an excellent tool, allowing the evaluation of edema formation, accumulation of inflammatory cells in the exudate, mediators, signaling pathways, gene expression and production of specific proteins. Our studies demonstrated the versatility of fish models to investigate the inflammatory response and its pathophysiology, essential for the successful development of studies to discover innovative pharmacological strategies. Laboratory of Animal Pharmacology and Toxicology Brazil University (UB) Department of Preventive Veterinary Medicine São Paulo State University (UNESP) Department of Morphology Federal University of Minas Gerais (UFMG) Department of Physiology and Pharmacology Federal University of Minas Gerais (UFMG) Department of Biochemistry and Immunology Federal University of Minas Gerais (UFMG) Department of zootechnics at the Veterinary School Federal University of Minas Gerais (UFMG) Postgraduate Program in Health Sciences - PROCISA Federal University of Roraima Immunochemistry Laboratory Butantan Institute Department of Clinical and Toxicological Analyses Federal University of Minas Gerais (UFMG) Department of Pharmacology University of São Paulo (ICB-USP) Department of Preventive Veterinary Medicine São Paulo State University (UNESP) FAPESP: 2013/25971-9 FAPESP: 2018/07098-0 FAPESP: 2019/19939-1 CNPq: 301473 / 2016-1 CNPq: 307526/2018-6 CNPq: 477816/2013-4

Made available in DSpace on 2021-06-25T15:04:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Goiano Federal Institute Although the toxicity of conventional microplastic types (i.e., petroleum derivatives) in different organisms is already known, knowledge about the effects of alternative biopolymers on aquatic vertebrates remains incipient. Thus, the aim of the present study is to test the hypothesis that the exposure of adult Danio rerio individuals to this pollutant for 30 days is enough to cause polylactic acid biomicroplastics (BioMPs of PLA) accumulation in their bodies, which leads to behavioral/neurotoxic, biochemical, and morphological changes. Based on our results, PLA BioMPs at concentrations of 2.5 and 5 mg/L accumulated in the liver, brain, gills and carcass of the assessed animals. However, such an accumulation was not able to cause locomotor damages or to trigger anxiety-like behavior in them. On the other hand, it was enough to cause behavioral changes (in shoal) predictive of co-specific social interaction and antipredatory defensive response deficit likely related to cholinergic changes inferred by increased acetylcholinesterase activity and REDOX imbalance. This imbalance was featured by increased production of reactive species. We observed that the treatments have affected animals' pigmentation pattern. Therefore, our study highlights the toxicological potential of the herein assessed biopolymer, and this finding puts in check the innocuousness of this material, as well as expands our knowledge about how PLA BioMPs can affect the ichthyofauna in freshwater environments. (C) 2021 Elsevier Ltd. All rights reserved. Goiano Fed Univ, Postgrad Program Cerrado Nat Resources Conservat, Urutai Campus, Urutai, Brazil Goiano Fed Inst, Biol Res Lab, Urutai Campus, Urutai, Brazil Univ Fed Uberlandia, Postgrad Program Ecol & Nat Resources Conservat, Uberlandia, MG, Brazil Sao Paulo State Univ, Morphol Dept, Reprod & Mol Biol Grp, Botucatu, SP, Brazil Fed Univ Jatai, Dept Biol Sci, Zebrafish Res & Reprod Lab LABFISH, Jatai, Brazil Univ Sao Paulo, Inst Biomed Sci, Dept Pharmacol, Sao Paulo, Brazil Univ Fed Goias, Postgrad Program Biotechnol & Biodivers, Goiania, Go, Brazil Sao Paulo State Univ, Morphol Dept, Reprod & Mol Biol Grp, Botucatu, SP, Brazil CNPq: 426531/2018-3 Goiano Federal Institute: 23219.001551.2020-92

We aim at evaluating the toxicity of naturally-aged polystyrene microplastics (MPs) in Danio rerio at intermediate development stage. Animal models were stactically exposed to 4 × 104 and 4 × 106 microparticles/m3 for five days – this concentration is environmentally relevant. We evaluated MP’s impact on animals’ nutritional status and REDOX balance, as well as its potential neuro- and cytotoxic action on them. Initially, MPs did not induce any change in total carbohydrates, triglycerides and total cholesterol levels. MP accumulation was associated with oxidative stress induction, which was inferred by the nitrite and thiobarbituric acid reactive substances levels. Furthermore, we observed that such stress was not counterbalanced by increase in the assessed enzymatic (total glutathione, catalase and superoxide dismutase) and non-enzymatic (total thiols, reduced glutathione and DPPH radical scavenging activity) antioxidants. The association between high acetylcholinesterase activity and numerical changes in neuroblasts distributed on animals' body surface confirmed MP’s neurotoxic potential. MP’s ability to induce apoptosis and necrosis processes in animals' erythrocytes suggested its cytotoxic action; therefore, the present study is pioneer in providing insight on how MPs can affect young freshwater fish at environmental concentrations. It is essential knowing the magnitude of these pollutants’ impact on the ichthyofauna.

SummaryEstablishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.

Made available in DSpace on 2021-06-25T11:46:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-20 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Instituto Federal Goiano Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundacao de Amaparo a Pesquisa do Estado de Goias (FAPEG, Brazil) Artemisinin extracted from Artemisia annua L. plants has a range of properties that qualifies it to treat several diseases, such as malaria and cancer. However, it has short half-life, which requires making continuous use of it, which has motivated the association of artemisinin (ART) with polymeric nanoparticles to increase its therapeutic efficiency. However, the ecotoxicological safety of this association has been questioned, given the scarcity of studies in this area. Thus, in this work the toxicity of Poly (epsilon-Caprolactone) nanocapsules added with ART (ART-NANO) in zebrafish (Danio rerio), embryos and adults was studied. Different endpoints were analyzed in organisms exposed to ART-NANO, including those predictive of embryotoxicity and histopatoxicity. Embryotoxicity was analyzed based on Organization for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity applied to zebrafish (Danio rerio) at 96 hpf under five nominal logarithmic concentrations (0.125 to 2.0 mg/L). Our results demonstrate, mainly, that fertilized eggs presented increased coagulation, lack of heart rate, vitelline sac displacement and lack of somite formation. On the other hand, adult individuals (exposed to the same concentrations and evaluated after 24 and 96 h of exposure) have shown increased pericarditis. Therefore, the treatment based on ART, poly (epsilon-caprolactone) nanocapsules and on their combination at different concentrations have shown toxic effects on zebrafish embryos and adult individuals. (C) 2020 Elsevier B.V. All rights reserved. Univ Sao Paulo, Biomed Sci Inst, Pharmacol Dept, Sao Paulo, SP, Brazil Fed Univ Rio Janeiro UFRJ, Biodivers & Sustainabil Inst NUPEM, Integrated Translat Biosci Lab LIBT, Macae, RJ, Brazil Univ Fed Sao Carlos, Dept Genet & Evolut, Sao Carlos, SP, Brazil Univ Estadual Campinas, Pharmaceut Sci Sch, Campinas, SP, Brazil Univ Fed Minas Gerais, Belo Horizonte, MG, Brazil Sao Paulo State Univ, Jaboticabal, SP, Brazil Univ Fed Alfenas, Motric Sci Inst, Alfenas, MG, Brazil Livestock & Supply, Minist Agr, Sao Carlos, SP, Brazil Sao Paulo State Univ, Botucatu, SP, Brazil Goiano Fed Inst, Biol Sci Dept, Urutai, Go, Brazil Sao Paulo State Univ, Jaboticabal, SP, Brazil Sao Paulo State Univ, Botucatu, SP, Brazil CNPq: 426531/2018-3 Instituto Federal Goiano: 23219.000865.2020-78 CNPq: 307743/2018-7

Made available in DSpace on 2021-06-25T11:04:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Mucoadhesive polymeric nanocapsules have attracted interest of researchers from different fields from natural sciences because of their ability to interact with the mucosa and increase drug permeation. Anesthesia by immersion causes absorption through the skin and gills of fish, so it is important to evaluate the exposure of these organs to drug nanosystems. Benzocaine (BENZ) is one of the most popular anesthetic agents used in fish anesthesia, but it has drawbacks because of its low bioavailability, resulting in weak absorption after immersion. Here we describe method developed for preparing and characterizing chitosan-coated PLGA mucoadhesive nanoparticles containing BENZ (NPMAs) for zebrafish immersion anesthesia. We determined the lowest effective concentration, characterized the interaction of the mucoadhesive system with fish, measured the anesthetic efficacy, and evaluated possible toxic effects in embryos and adults exposed to the nanoformulations. This study opens perspectives for using nanoformulations prepared with BENZ in aquaculture, allowing reduction of dosage as well as promoting more effective anesthesia and improved interaction with the mucoadhesive system of fish. Department of Pharmacology University of São Paulo-ICB/USP Laboratório Integrado de Biociências Translacionais (LIBT) Instituto de Biodiversidade e Sustentabilidade (NUPEM) Universidade Federal do Rio de Janeiro (UFRJ) Department of Morphology ICB-UFMG Aquiculture Laboratory (Laqua) UFMG Department of Preventive Veterinary Medicine São Paulo State University Department of Postgraduate in Health Sciences PROCISA Federal University of Roraima (UFRR) Institute of Chemistry São Paulo State University (Unesp) Laboratory of Animal Pharmacology and Toxicology Brasil University Department of Clinical and Toxicological Analyses Faculty of Pharmacy-UFMG Laboratory of Applied Immunology Federal University of São Carlos School of Veterinary Medicine Department of Clinic and Veterinary Surgery UFMG Faculty of Medicine São Leopoldo Mandic Institute of Science and Technology of Sorocaba São Paulo State University Department of Preventive Veterinary Medicine São Paulo State University Institute of Chemistry São Paulo State University (Unesp) Institute of Science and Technology of Sorocaba São Paulo State University

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 μg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems., Graphical abstract Unlabelled Image

In order to search for new alternatives for rapid tests, our research group prospected a strategy to develop a rapid COVID-19 patient detection and tracking kit, using zebrafish model