Cisplatin Toxicity Causes Neutrophil-Mediated Inflammation in Zebrafish Larvae.

Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (Danio rerio), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin (il)-12, il6, and il8 increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils.

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.

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.

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.