Deleterious Effects of Polypropylene Microplastic Ingestion in Nile Tilapia (Oreochromis niloticus).

The effect of daily ingestion of polypropylene microplastic on the health of tilapia, Oreochromis niloticus, was evaluated. 60 fish (± 200 g) were placed in 6 aquariums (n = 10, 100 L each), constituting the following treatments: Control (without the addition of polymer), fed with 100 and 500 µg of polypropylene/kg of body weight (b.w.), respectively. After 30 days of feeding, the animals were submitted to blood collection for hemogram and biochemical study and later euthanized for gut microbiological analysis, somatic index of liver, spleen, heart, kidney, stomach, and intestine. In the serum biochemical study, an increase in cholesterol and serum Aspartate Aminotransferase (AST) activity levels was observed in animals treated with 500 µg of polypropylene. Tilapia-fed polypropylene in the diet showed an increase in thrombocyte and total leukocyte counts, marked by a significant increase in the number of circulating lymphocytes. The results of the somatic study revealed a significant increase in the stomach, liver, and heart of tilapia fed with the polymer. Increase in the number of Gram-negative microorganisms and decrease in mesophilic aerobic microorganisms were observed in the gut of fish exposed to the polymer, including a dose-response effect was observed for these analyses. Therefore, tilapias fed daily with diets containing polypropylene for 30 consecutive days showed deleterious effects, resulting in systemic inflammatory disturbs by altering liver functions, leukocyte profile, and organ morphometry, as well as changes in the intestinal microbiota. Such results demonstrate the impairment of fish health, highlighting the need for further studies that evaluate the impact of microplastics on aquatic organisms.

A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

Device implant based on poly (lactic acid) with vitamin E for vaccine delivery system in Tilapia: Study for biocompatibility and biodegradation.

The use of Poly (lactic acid) (PLA) as a slow-release vehicle for vaccines has attracted the attention of researchers, since its insertion improves the uptake of them, and reduces side effects or by stimulating recruited defense cells, assisting immunity without the need for booster vaccine doses. Seeking to develop new strategies for the administration of drugs and vaccines in aquaculture, we evaluated the biocompatibility and biodegradation of polymeric PLA devices and PLA plus vitamin E devices, implanted through subcutaneous (SC) and intraperitoneal (IP) routes in Nile tilapia. To carry out this study, 84 male tilapia (initial 243.82 ± 56.74 g; final 400.71 ± 100.54 g) were randomly distributed in 3 tanks (n = 28 fish per treatment/tank). The devices were prepared in two formulations: neat PLA (containing 100% PLA) and PLAVE (PLA plus vitamin E) implanted using a commercial AnimalTag® applicator, and non-implanted fish (control). Fish were sampled 15, 30, 60, and 120 days post-implantation (DPI). Blood analysis was used to access blood cells and blood smear for differential leucocytes count. Serum biochemistry to evaluated changes in serum proteins and glycemia. Histopathological investigation using hematoxylin-eosin (H&E) was used to assess polymer-tissue interaction. Histochemistry and immunohistochemistry was used to detection immune cells and phagocytes in capsule, and analyses of melanomacrophage centers (MMCs) to morphometric evaluation and percentage amount of melanin, hemosiderin and lipofucsin pigments. Histopathological study revealed an increase of capsular formation and inflammatory cell infiltration in PLAVE-implanted tilapia through SC route (15 DPI). Tilapia implanted with PLAVE and PLA (SC) presented mast cells and eosinophilic granular cells during 15, 30, and 60 DPI, with a decrease in these cells in the fibrous capsule around the polymer at 120 DPI. PLAVE implanted tilapia SC at 60 DPI showed significantly phagocytosis points than other groups. Phagocytic cells (F4/80+) were observed near to biopolymers in phagocytosis sites. Lipofuscin at 120 DPI in spleen melanomacrophage centers were significantly high in PLAVE implanted tilapias when compared to fish with PLA implants and control. The serum biochemical study of tilapia did not reveal changes in cytotoxicity and liver function in implanted fish. The absence of side effects in hematological and biochemical findings, including the absence of mortality after device implantation, proves its clinical safety. PLA implants in tilapia have demonstrated biocompatibility, biodegradation, clinical safety, and excellent evolution of foreign body inflammatory responses.

Antagonism of cysteinyl leukotriene receptors by zafirlukast modulated acute inflammatory reaction in tilapia, Oreochromis niloticus.

To identify activation pathways and effector mechanisms of innate immunity in fish has become relevant for the sanitary management of intensive fish farming. However, little is known about the blocking of cysteinyl leukotrienes receptors (CysLTRs) and their effects in teleost fish. Our study evaluated the anti-inflammatory effect of 250 and 500 µg zafirlukast (antagonist of CysLTRs)/kg b.w., administered orally in the diet, during acute inflammatory reaction induced by Aeromonas hydrophila bacterins in Oreochromis niloticus. 80 tilapia were distributed in 10 aquariums (100L of water each, n = 8) to constitute three treatments: Control (inoculated with A. hydrophila bacterin and untreated); Treated with 250 µg or 500 µg of zafirlukast/kg b.w. and inoculated. To be evaluated in three periods: 6, 24 and 48 h post-inoculation (HPI), totaling nine aquariums. A tenth group was sampled without any stimulus to constitute reference values (Physiological standards). Tilapia treated with zafirlukast demonstrated dose-response effect in the decrease of accumulated inflammatory cells, strongly influenced by granulocytes and macrophages. Zafirlukast treated-tilapia showed decrease in blood leukocyte counts (mainly neutrophils, and monocytes) and reactive oxygen species production. Treatment with zafirlukast resulted in down-regulation of ceruloplasmin, complement 3, alpha2-macroglobulin, transferrin and apolipoprotein A1, as well as up-regulation of haptoglobin. Our study provided convincing results in the pathophysiology of tilapia inflammatory reaction, considering that treatment with zafirlukast, antagonist of cysteinyl leukotriene receptors, resulted in a dose-response effect by suppressing the dynamics between leukocytes in the bloodstream and cell accumulation in the inflamed focus, as well as modulated the leukocyte oxidative burst and the acute phase protein response.

Modular Label-Free Electrochemical Biosensor Loading Nature-Inspired Peptide toward the Widespread Use of COVID-19 Antibody Tests.

Limitations of the recognition elements in terms of synthesis, cost, availability, and stability have impaired the translation of biosensors into practical use. Inspired by nature to mimic the molecular recognition of the anti-SARS-CoV-2 S protein antibody (AbS) by the S protein binding site, we synthesized the peptide sequence of Asn-Asn-Ala-Thr-Asn-COOH (abbreviated as PEP2003) to create COVID-19 screening label-free (LF) biosensors based on a carbon electrode, gold nanoparticles (AuNPs), and electrochemical impedance spectroscopy. The PEP2003 is easily obtained by chemical synthesis, and it can be adsorbed on electrodes while maintaining its ability for AbS recognition, further leading to a sensitivity 3.4-fold higher than the full-length S protein, which is in agreement with the increase in the target-to-receptor size ratio. Peptide-loaded LF devices based on noncovalent immobilization were developed by affording fast and simple analyses, along with a modular functionalization. From studies by molecular docking, the peptide-AbS binding was found to be driven by hydrogen bonds and hydrophobic interactions. Moreover, the peptide is not amenable to denaturation, thus addressing the trade-off between scalability, cost, and robustness. The biosensor preserves 95.1% of the initial signal for 20 days when stored dry at 4 °C. With the aid of two simple equations fitted by machine learning (ML), the method was able to make the COVID-19 screening of 39 biological samples into healthy and infected groups with 100.0% accuracy. By taking advantage of peptide-related merits combined with advances in surface chemistry and ML-aided accuracy, this platform is promising to bring COVID-19 biosensors into mainstream use toward straightforward, fast, and accurate analyses at the point of care, with social and economic impacts being achieved.

Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms.

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Shedding light on the toxicity of SARS-CoV-2-derived peptide in non-target COVID-19 organisms: A study involving inbred and outbred mice.

Despite advances in research on the vaccine and therapeutic strategies of COVID-19, little attention has been paid to the possible (eco)toxicological impacts of the dispersion of SARS-CoV-2 particles in natural environments. Thus, in this study, we aimed to evaluate the behavioral and biochemical consequences of the short exposure of outbred and inbred mice (male Swiss and C57Bl/6 J mice, respectively) to PSPD-2002 (peptide fragments of the Spike protein of SARS-CoV-2) synthesized in the laboratory. Our data demonstrated that after 24 h of intraperitoneal administration of PSPD-2002 (at 580 µg/kg) the animals did not present alterations in their locomotor, anxiolytic-like, or anxiety-like behavior (in the open field test), nor antidepressant-like or depressive behavior in the forced swimming test. However, the C57Bl/6 J mice exposed to PSPD-2002 showed memory deficit in the novel object recognition task, which was associated with higher production of thiobarbituric acid reactive substances, as well as the increased suppression of acetylcholinesterase brain activity, compared to Swiss mice also exposed to peptide fragments. In Swiss mice the reduction in the activity of superoxide dismutase and catalase in the brain was not associated with increased oxidative stress biomarkers (hydrogen peroxide), suggesting that other antioxidant mechanisms may have been activated by exposure to PSPD-2002 to maintain the animals' brain redox homeostasis. Finally, the results of all biomarkers evaluated were applied into the "Integrated Biomarker Response Index" (IBRv2) and the principal component analysis (PCA), and greater sensitivity of C57Bl/6 J mice to PSPD-2002 was revealed. Therefore, our study provides pioneering evidence of mammalian exposure-induced toxicity (non-target SARS-CoV-2 infection) to PSPD-2002, as well as "sheds light" on the influence of genetic profile on susceptibility/resistance to the effects of viral peptide fragments.

Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish.

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.

Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 µg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences.

Can carbon nanofibers affect anurofauna? Study involving neotropical Physalaemus cuvieri (Fitzinger, 1826) tadpoles.

Although carbon nanotubes' (CNTs) toxicity in different experimental systems (in vivo and in vitro) is known, little is known about the toxic effects of carbon nanofibers (CNFs) on aquatic vertebrates. We herein investigated the potential impact of CNFs (1 and 10 mg/L) by using Physalaemus cuvieri tadpoles as experimental model. CNFs were able to induce nutritional deficit in animals after 48-h exposure to them, and this finding was inferred by reductions observed in body concentrations of total soluble carbohydrates, total proteins, and triglycerides. The increased production of hydrogen peroxide, reactive oxygen species and thiobarbituric acid reactive substances in tadpoles exposed to CNFs has suggested REDOX homeostasis change into oxidative stress. This process was correlated to the largest number of apoptotic and necrotic cells in the blood of these animals. On the other hand, the increased superoxide dismutase and catalase activity has suggested that the antioxidant system of animals exposed to CNFs was not enough to maintain REDOX balance. In addition, CNFs induced increase in acetylcholinesterase and butyrylcholinesterase activity, as well as changes in the number of neuromasts evaluated on body surface (which is indicative of the neurotoxic effect of nanomaterials on the assessed model system). To the best of our knowledge, this is the first report on the impact of CNFs on amphibians; therefore, it broadened our understanding about ecotoxicological risks associated with their dispersion in freshwater ecosystems and possible contribution to the decline in the populations of anurofauna species.

Doxorubicin-loaded pH-sensitive micelles: A promising alternative to enhance antitumor activity and reduce toxicity.

Doxorubicin (DOX) is an anthracycline antibiotic widely used in the treatment of cancer, however, it is associated with the occurrence of adverse reactions that limits its clinical use. In this context, the encapsulation of DOX in micelles responsive to pH variations has shown to be a strategy for tumor delivery of the drug, with the potential to increase therapeutic efficacy and to reduce the toxic effects. In addition, radiolabeling nanoparticles with a radioactive isotope is of great use in preclinical studies, since it allows the in vivo monitoring of the nanostructure through the acquisition of quantitative images. Therefore, this study aimed to develop, characterize, and evaluate the antitumor activity of a pH-sensitive micelle composed of DSPE-PEG2000, oleic acid, and DOX. The micelles had a diameter of 13 nm, zeta potential near to neutrality, and high encapsulation percentage. The critical micellar concentration (CMC) was 1.4 × 10-5 mol L-1. The pH-sensitivity was confirmed in vitro through a drug release assay. Cytotoxicity studies confirmed that the encapsulation of DOX into the micelles did not impair the drug cytotoxic activity. Moreover, the incorporation of DSPE-PEG2000-DTPA into the micelles allowed it radiolabeling with the technetium-99 m in high yield and stability, permitting its use to monitor antitumor therapy. In this sense, the pH-sensitive micelles were able to inhibit tumor growth significantly when compared to non-pH-sensitive micelles and the free drug. in vivo toxicity evaluation in the zebrafish model revealed significantly lower toxicity of pH-sensitive micelles compared to the free drug. These results indicate that the developed formulation presents itself as a promising alternative to potentiate the treatment of tumors.

Effects of nanocapsules of poly-ε-caprolactone containing artemisinin on zebrafish early-life stages and adults.

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 (ε-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 (ε-caprolactone) nanocapsules and on their combination at different concentrations have shown toxic effects on zebrafish embryos and adult individuals.

Long-term dexamethasone treatment increases the engraftment efficiency of human breast cancer cells in adult zebrafish.

The host immune system tends to reject xenogenic-implanted cells making tumor development in adult host animal models difficult. Immune system suppression is used for successful xenotransplantation of human cancer cells in many animal models. The studies of cancer development processes in vivo offer opportunities to understand cancer biology and discover new therapeutic strategies. In this context, zebrafish is a model that has been widely applied in the study of human diseases, such as cancer. However, the long-term immunosuppression of these adult zebrafish is still under study as a xenograft animal model for human cancer. This work aimed to evaluate the effects of 21 days of (long-term) exposure of dexamethasone in zebrafish-transplanted with MGSO-3 cells, human breast tumor cell line. Our results show that the animals, while kept on dexamethasone treatment, remained with a 50% reduction in the number of peripheral lymphocytes. In vitro data demonstrated that up to 7 days of dexamethasone treatment did not alter the morphology, proliferation, or viability of MGSO-3 cells. The animals that received a prolonged dexamethasone treatment allowed the engraftment of tumor cells in 100% of the zebrafish tested. These animals also showed tumor progression over 21 days. The experimental group that received only previous exposure to dexamethasone had their tumors regressed after 14 days. In conclusion, the prolonged use of dexamethasone in zebrafish showed a potential strategy for in vivo monitoring of xenograft tumor growth for development studies, as well as in anticancer drug discovery.

Spleen melanomacrophage centers response of Nile tilapia during Aeromanas hydrophila and Mycobacterium marinum infections.

In order to understand the pathophysiology of melanomcrophage centers (MMCs) formation during the tilapia defense response to bacterial infections, the present study evaluated the response, in terms of area, number and pigment constitution, of splenic MMCs of Oreochromis niloticus subjected to intraperitoneal (i.p.) infection with Aeromonas hydrophila and Mycobacterium marinum. Eighty-four fish (396.9 ±â€¯21.0 g) were randomly distributed into twelve plastic tanks (300 L), to constitute three treatments with 28 animals each: control group (inoculated with PBS); Infected with A. hydrophila (1 × 107 UFC mL-1); Infected with M. marinum (1 × 106 UFC mL-1). The spleen was collected in seven fish per treatment on the 3rd, 7th, 14th and 21st day post-infection (DPI). The results revealed the participation of MMCs in the defense response of tilapia during bacterial infection by A. hydrophila and M. marinum, since there was an increase in the number and size of these cell aggregates. Variation of pigment accumulation with significant increase of hemosiderin, in infected tilapias by A. hydrophila, bacteria responsible for causing hemolytic anemia in fish was also found. On the other hand, M. marinum-infected tilapia had high amount of melanin in MMCs. In general, mycobacterial infections are notoriously difficult to treat, being characterized as a chronic disease. These findings demonstrate different strategies of fish response during the evolution of these bacterial diseases.

Progress in nano-drug delivery of artemisinin and its derivatives: towards to use in immunomodulatory approaches.

In recent years, artemisinin (ART) and its derivatives have highlighted according to their effects on highly aggressive cancers, as well as treatment of malaria and leishmaniasis, besides presenting anti-inflammatory and antibacterial activity. It has also been shown that ART compounds have the ability to modulate the immune response by regulating cell proliferation and cytokine release. These effects may be beneficial and improve the treatment of cancer and parasitic diseases by increasing therapeutic success, but it has some pharmacological limitations such as low bioavailability, short half-life and limited tissue access. Nanotechnology has been explored during the last decades, notably in the design of drug carrier systems which includes polymeric, lipid and inorganic nanoparticles, cyclodextrins inclusion complexes, liposomes, carbon nanotubes, among others. These nanostructured drug delivery systems bring benefits both increased therapeutic efficacy and reduced toxicity. This review article aims to give an overview of the current progress in nanostructured drug carriers used for encapsulation of ART and its derivatives yielding examples of successful outcomes. The data collection suggests future applications of ART and derivatives encapsulated in nano delivery systems in clinical trials and prospects for use of ART loaded nanosystems in immunomodulatory responses.