Flow cytometry in the analysis of hematological parameters of tilapias: applications in environmental aquatic toxicology.

Blood tissue has been used to assess animal health and the environment in which they live. This tissue is easily acquired and has the ability to respond to various adverse conditions. Several techniques have been employed in the detection of xenobiotic-induced cell damage in blood cells. In general, traditionally used technologies, such as cellular analysis in blood smears, are time-consuming and require great analytical capacity. The present study proposes flow cytometry as a method to detect changes in blood cell populations. Tilapia (Oreochromis niloticus) was selected as a model for plotting the profile of fish blood cell populations after exposure to xenobiotics without euthanizing animals or using cell markers. Populations of erythrocytes and lymphocytes were detected only by combining the techniques of FACSAria cell sorting and light microscopy. Systemic deleterious effects were found through blood analysis, such as an increased lymphocyte-rich population at 48 h of exposure followed by a subsequent decrease. Moreover, the time-dependent expression of Nrf2 suggests its participation in increased membrane disruption, indicating it has a central role in erythrocyte lifespan. The present results shed light on the viability of using flow cytometry for blood analysis of living fish.

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.

An imaging flow cytometry-based technique to quantify erythrocyte nuclear alterations.

Morphological nuclear alterations are indicative of DNA damage and have been considered excellent markers of exposure to several pollutants in aquatic environments. Flow cytometry is a powerful technique for measuring cell phenotypes in large numbers of cells in a short period of time. This technique is suited to the study of cell populations and subset identification as a function of its high-throughput and multi-parameter characteristics. We used the quantification of erythrocyte nuclear alterations to compare the techniques of imaging flow cytometry and light microscopy. The comparison used blood samples of the fish Oreochromis niloticus assayed using cadmium as a nuclear alteration-inducing agent. The results showed that imaging flow cytometry has higher sensitivity than light microscopy for detecting and quantifying erythrocytic nuclear alterations. We conclude that imaging flow cytometry can produce fast and reliable results and could potentially be useful in studies involving fish erythrocytes under normal and impacted environmental conditions.

Potential of mucoadhesive nanocapsules in drug release and toxicology in zebrafish.

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.

Reactive oxygen species generating photosynthesized ferromagnetic iron oxide nanorods as promising antileishmanial agent.

Aim: To investigate the photodynamic therapeutic potential of ferromagnetic iron oxide nanorods (FIONs), using Trigonella foenum-graecum as a reducing agent, against Leishmania tropica. Materials & methods: FIONs were characterized using ultraviolet visible spectroscopy, x-ray diffraction and scanning electron microscopy. Results: FIONs showed excellent activity against L. tropica promastigotes and amastigotes (IC50 0.036 ± 0.003 and 0.072 ± 0.001 µg/ml, respectively) upon 15 min pre-incubation light-emitting diode light (84 lm/W) exposure, resulting in reactive oxygen species generation and induction of cell death via apoptosis. FIONs were found to be highly biocompatible with human erythrocytes (LD50 779 ± 21 µg/ml) and significantly selective (selectivity index >1000) against murine peritoneal macrophages (CC50 102.7 ± 2.9 µg/ml). Conclusion: Due to their noteworthy in vitro antileishmanial properties, FIONs should be further investigated in an in vivo model of the disease.

Nanoparticle phosphate-based composites as vehicles for antimony delivery to macrophages: possible use in leishmaniasis.

Pentavalent antimonial drugs such as N-methylglucamine antimonate (Glucantime®) are used for treating leishmaniasis but produce severe side effects, including cardiotoxicity and hepatotoxicity. We characterized the physicochemical properties of 3 nanoparticle phosphate-based composites (NPCs; NPC0, NPC3, and NPC5) as Sb(v) carriers for specifically targeting macrophages and reducing systemic side effects. NPCs were synthesized in liquid media and sterilized at 25 kGy before use. Macrophage viability and NPC toxicity, independent of Sb uptake, were evaluated to assess NPC safety in visceral leishmaniasis treatment. NPC zeta potential, conductivity, diameter, Sb content, and crystallinity were determined using electrophoretic light scattering, scanning electron microscopy (SEM), conductance, graphite furnace atomic absorption spectrometry (GFAAS), and X-ray diffraction, respectively. In vitro NPC cytotoxicity against murine peritoneal macrophages was evaluated using MTT assays, and Sb amounts internalized by macrophages were determined using GFAAS. The rate of macrophage infection caused by Leishmania infantum was assayed in vitro, by using Glucantime® as a reference drug. NPCs featured negative zeta potentials (-15.5 to -19.5 mV), mean diameters around 180 nm, and a low dissolution constant in Milli-Q water (<0.0197 mS cm-1), and were prepared using 0.0 (NPC0) to 36.2 µg mL-1 Sb (NPC5). NPC5 exhibited characteristic crystalline peaks resembling mopungite, but other NPCs exhibited predominantly amorphous structures. Cell viability was not markedly affected at any NPC concentration tested. Light microscopy, SEM, and GFAAS data revealed NPC internalization and intracellular Sb retention. Amastigote infection was reduced by both Sb-containing NPC3 and Sb-lacking NPC0, but NPC3 was more effective. These data indicate the potential of NPCs as Sb nanocarriers for specifically targeting macrophages and lowering Sb dosage without reducing leishmanicidal activity.

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.

Enzymatic, analytical and structural aspects of electron-dense granules in cells of Ucides cordatus (Crustacea, Decapoda) hepatopancreas.

Electron-dense granules (EDGs) are singular structures found in the tissues of several vertebrate and invertebrate organisms. Two types of EDGs were observed in hepatopancreatic cells of the crab Ucides cordatus: (1) a non-mineralized EDG, found mainly inside vacuoles, which reacted positively to acid phosphatase and D-amino acid oxidase, possibly formed by degradation of lipid membranes, and (2) a mineralized EDG surrounded by endoplasmic reticulum membranes that gave a positive reaction for glucose-6-phosphatase. In this study we show the fine structure and composition of the mineralized EDGs using cytochemistry, analytical transmission electron microscopy and field-emission scanning electron microscopy. They are formed of microvesicle-like structures that are arranged in concentric spherical layers in the most mineralized portions of the granule. Analytical microscopy of mineralized EDGs indicated that they are composed of amorphous calcium-magnesium phosphate. Isolated EDGs treated with NaOCl lose several elements, including P, when compared with EDGs treated with deionized water. Removal of the organic matrix by NaOCl induced marked changes in the mineralized EDGs, showing that the organic matrix plays an important role in its elemental composition and structure.