A zebrafish xenotransplant model of anaplastic thyroid cancer to study tumor microenvironment and innate immune cell interactions in vivo.

Anaplastic thyroid cancer (ATC) is of the most aggressive thyroid cancer. While ATC is rare, it accounts for a disproportionately high number of thyroid cancer-related deaths. Here, we developed an ATC xenotransplant model in zebrafish larvae, where we can study tumorigenesis and therapeutic response in vivo. Using both mouse (T4888M) and human (C643)-derived fluorescently labeled ATC cell lines, we show these cell lines display different engraftment rates, mass volume, proliferation, cell death, angiogenic potential, and neutrophil and macrophage recruitment and infiltration. Next, using a PIP-FUCCI reporter to track proliferation in vivo, we observed cells in each phase of the cell cycle. Additionally, we performed long-term non-invasive intravital microscopy over 48 h to understand cellular dynamics in the tumor microenvironment at the single-cell level. Lastly, we tested two drug treatments, AZD2014 and a combination therapy of dabrafenib and trametinib, to show our model could be used as an effective screening platform for new therapeutic compounds for ATC. Altogether, we show that zebrafish xenotransplants make a great model to study thyroid carcinogenesis and the tumor microenvironment, while also being a suitable model to test new therapeutics in vivo.

Toxicological impact of SARS-CoV-2 on the health of the neotropical fish, Poecilia reticulata.

There have been significant impacts of the current COVID-19 pandemic on society including high health and economic costs. However, little is known about the potential ecological risks of this virus despite its presence in freshwater systems. In this study, we aimed to evaluate the exposure of Poecilia reticulata juveniles to two peptides derived from Spike protein of SARS-CoV-2, which was synthesized in the laboratory (named PSPD-2002 and PSPD-2003). For this, the animals were exposed for 35 days to the peptides at a concentration of 40 µg/L and different toxicity biomarkers were assessed. Our data indicated that the peptides were able to induce anxiety-like behavior in the open field test and increased acetylcholinesterase (AChE) activity. The biometric evaluation also revealed that the animals exposed to the peptides displayed alterations in the pattern of growth/development. Furthermore, the increased activity of superoxide dismutase (SOD) and catalase (CAT) enzymes were accompanied by increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and hydrogen peroxide (H2O2), which suggests a redox imbalance induced by SARS-CoV-2 spike protein peptides. Moreover, molecular docking analysis suggested a strong interaction of the peptides with the enzymes AChE, SOD and CAT, allowing us to infer that the observed effects are related to the direct action of the peptides on the functionality of these enzymes. Consequently, our study provided evidence that the presence of SARS-CoV-2 viral particles in the freshwater ecosystems offer a health risk to fish and other aquatic organisms.

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.

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.

Mitochondrial connections with immune system in Zebrafish.

Mitochondria are organelles commonly associated with adenosine triphosphate (ATP) formation through the oxidative phosphorylation (OXPHOS) process. However, mitochondria are also responsible for functions such as calcium homeostasis, apoptosis, autophagy, and production of reactive oxygen species (ROS) that, in conjunction, can lead to different cell fate decisions. Mitochondrial morphology changes rely on nutrients' availability and the bioenergetics demands of the cells, in a process known as mitochondrial dynamics, which includes both fusion and fission. This organelle senses the microenvironment and can modify the cells to either a pro or anti-inflammatory profile. The zebrafish has been increasingly used to research mitochondrial dynamics and its connection with the immune system since the pathways and molecules involved in these processes are conserved on this fish. Several genetic tools and technologies are currently available to analyze the behavior of mitochondria in zebrafish. However, even though zebrafish presents several similar processes known in mammals, the effect of the mitochondria in the immune system has not been so broadly studied in this model. In this review, we summarize the current knowledge in zebrafish studies regarding mitochondrial function and immuno metabolism.

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.

Cyclophosphamide modulated the foreign body inflammatory reaction in tilapia (Oreochromis niloticus).

In order to understand events and mechanisms present in the pathophysiology of tilapia's chronic inflammation and based on the immunomodulatory activity attributed to cyclophosphamide which is widely used to suppress immune responses in human medicine, the present study investigated the effects of cyclophosphamide (CYP) treatment on the modulation of foreign body inflammatory reaction in Nile tilapia (Oreochromis niloticus) with round glass coverslip implanted in the subcutaneous tissue (9 mm of diameter). Forty tilapia (151 ± 10,2 g) were randomly distributed in 5 aquariums (n = 8) with a capacity of 250 L of water each, to compose two treatments (sampled 3 and 6 days post-implantation): implanted/untreated (control) and implanted/treated with 200 mg of CYP kg-1 of b.w., through i.p. route. A fifth group (n = 8) was sampled without any stimulus (naive) to obtain reference values. CYP-treated tilapia showed decrease in macrophage accumulation, giant cell formation and Langhans cells on the glass coverslip when compared to control fish. The treatment with CYP resulted in decrease of leukocyte and thrombocyte counts. Decrease in alpha-2-macroglobulin, ceruloplasmin, albumin and transferrin levels, as well as increase in haptoglobin, complement C3 and apolipoprotein A1 were observed in tilapias during foreign body inflammation. Blood levels of complement C3, alpha-2-macroglobulin, ceruloplasmin and transferrin were modulated by treatment with CYP. Therefore, the treatment with 200 mg of CYP kg-1 of b.w. in tilapia resulted in an anti-inflammatory effect by suppressing the dynamics between leukocytes in the bloodstream and macrophage accumulation with giant cell formation in the inflamed focus, as well as by modulating APPs during foreign body reaction.