Zebrafish Research Articles

Transcriptomic and functional effects from a chemical mixture based on the exposure profile in Baltic Sea salmon, on metabolic and immune functions in zebrafish embryo

The Baltic Sea is one of the world’s most contaminated seas with long-standing adverse health status of its wildlife such as the Baltic Sea salmon, resulting in reduced fecundity and increased mortality. While adverse health effects have been reported among wild fish from the Baltic Sea, the toxicity mechanisms underlying these adversities, and the chemical effect drivers mediating them are poorly understood. To address this knowledge gap, we utilized the zebrafish (Danio rerio) embryo model to determine molecular and functional effects brought on by exposure to a technical mixture including 9 organohalogen compounds detected in serum from wild-caught Baltic Sea salmon. To align with the salmon exposure scenario, an internal dose regimen was opted to establish same relative proportions of the compounds in the zebrafish (whole body) as observed in the salmon serum. Through transcriptomic profiling, we identified dose-dependent effects on immune system and metabolism as two critical functions overlapping with adverse effects observed in wild fish from the Baltic Sea. We then determined likely effect drivers by comparing gene responses of the mixture with those of individual mixture components. Aligned with our transcriptome results, the number of total macrophages was reduced and the zebrafish’s ability to respond to a tissue damage suppressed in a dose-dependent manner. This study brings forth a key advancement in delineating the impact of chemical pollutants on the health of wild fish in the Baltic Sea.

Assessment of the effect of tricaine (MS-222)-induced anesthesia on brain-wide neuronal activity of zebrafish (Danio rerio) larvae.

Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg(elavl3:H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine's anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch.

Multiparameter analysis of Diuron and its metabolites (DCA and DCPMU) in different stages of zebrafish (Danio rerio) development

IntroductionDiuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a prominent herbicide in Brazilian agriculture, particularly in sugarcane farming. This herbicide persists in the environment and is transformed into potentially toxic metabolites, such as 3,4-dichloroaniline (DCA) and 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), raising concerns that encompass environmental contamination and One Health, which unites environmental and human health. Zebrafish are an appropriate experimental model to evaluate toxicity: they metabolically and genetically resemble humans and can be applied in ecotoxicology.MethodsIn this study, we have employed multiparameter evaluation to characterize the toxicity of environmentally relevant concentrations (0.5–100 μM) of Diuron, DCA, or DCPMU in Brazil, a developing country. In the early stages of zebrafish development, we performed the fish embryo toxicity test according to the guidelines established in OECD Protocol No. 236, extended to 144 h post fertilization (hpf). We also investigated the oxygen consumption rate in zebrafish embryos at 24 hpf, evaluated the acetylcholinesterase (AChE) enzymatic activity in zebrafish larvae at 96 hpf, and used the Comet Assay to assess DNA fragmentation in zebrafish larvae at 96 hpf. We subjected adult zebrafish to acute exposure for 96 h in accordance with OECD protocol no. 203 for the Acute Toxicity Test in Fish. Following this exposure, we analyzed mutagenicity by using the micronucleus test, which was complemented by quantification of nuclear anomalies (NA) in erythrocytes and further confirmed by histopathological investigations.Results and discussionExposure to any of the tested Diuron concentrations damaged DNA, and NA became significant beyond 10 μM Diuron. At 10 μM, DCA altered AChE activity, suggesting neuromuscular toxicity. Between 0.5 and 5 μM, DCA was potentially genotoxic, and 5–10 μM DCA induced nuclear pleomorphism. At all the tested concentrations, DCPMU damaged DNA, and NA emerged between 1 and 10 μM DCPMU. Adult zebrafish exposed to any of the tested Diuron, DCA, or DCPMU concentrations for 96 h did not exhibit significant histopathological alterations. Erythrocyte morphology analysis did not reveal mutagenicity. Although we observed non-mutagenic genotoxicity, chromosomal instability is of concern. The findings of this study contribute to our understanding of how Diuron and its metabolites affect zebrafish and highlights the need for multiparametric assessment to elucidate how herbicides and their metabolites impact the environment and human health. This endeavor informs regulatory measures for safely using Diuron and similar herbicides and reveals substantial implications for developing nations grappling with escalating water contamination due to pesticide use.

Impact of Biotransformation on Internal Concentrations and Specificity Classification of Organic Chemicals in the Zebrafish Embryo (Danio rerio).

Internal concentrations (ICs) are crucial for linking exposure to effects in the development of New Approach Methodologies. ICs of chemicals in aquatic organisms are primarily driven by hydrophobicity and modulated by biotransformation and efflux. Comparing the predicted baseline to observed toxicity enables the estimation of effect specificity, but biological processes can lead to overestimating ICs and bias the specificity assessment. To evaluate the prediction of a mass balance model (MBM) and the impact of biotransformation on ICs, experimental ICs of 63 chemicals in zebrafish embryos were compared to predictions with physicochemical properties as input parameters. Experimental ICs of 79% (50 of 63) of the chemicals deviated less than 10-fold from predictions, and the remaining 13 deviated up to a factor of 90. Using experimental ICs changed the classification for 19 chemicals, with ICs 5 to 90 times lower than predicted, showing the bias of specificity classification. Uptake kinetics of pirinixic acid, genistein, dexamethasone, ethoprophos, atorvastatin, and niflumic acid were studied over a 96 h exposure period, and transformation products (TPs) were elucidated using suspect- and nontarget screening with UPLC-HRMS. 35 TPs (5 to 8 TPs per compound) were tentatively identified and semiquantified based on peak areas, suggesting that biotransformation may partly account for the overpredictions of ICs.

Low-Basicity 5-HT6 Receptor Ligands from the Group of Cyclic Arylguanidine Derivatives and Their Antiproliferative Activity Evaluation

The serotonin 5-HT6 receptor (5-HT6R), expressed almost exclusively in the brain, affects the Cdk5 signaling as well as the mTOR pathway. Due to the association of 5-HT6R signaling with pathways involved in cancer progression, we decided to check the usefulness of 5-HT6R ligands in the treatment of CNS tumors. For this purpose, a new group of low-base 5-HT6R ligands was developed, belonging to arylsulfonamide derivatives of cyclic arylguanidines. The selected group of molecules was also tested for their antiproliferative activity on astrocytoma (1321N1) and glioblastoma (U87MG, LN-229, U-251) cell lines. Some of the molecules were subjected to ADMET tests in vitro, including lipophilicity, drug binding to plasma proteins, affinity for phospholipids, drug–drug interaction (DDI), the penetration of the membrane (PAMPA), metabolic stability, and hepatotoxicity as well as in vivo cardiotoxicity in the Danio Rerio model. Two antagonists with an affinity constant Ki < 50 nM (PR 68 Ki = 37 nM) were selected. These compounds were characterized by very high selectivity. An analysis of pharmacokinetic parameters for the lead compound PR 68 confirmed favorable properties for administration, including passive diffusion and acceptable metabolic stability (metabolized in 49%, MLMs). The compound did not exhibit the potential for drug–drug interactions.

Thyroid, neurodevelopmental, and kidney toxicities of common organic UV filters in embryo-larval zebrafish (Danio rerio), and their potential links

Organic UV filters (OUVFs) have been commonly used in sunscreen and many consumer products. Following dermal application, these compounds can enter circulation and may cause systemic effects in humans. In the present study, we chose four OUVFs frequently detected in the environment, i.e., avobenzone (AVB), benzophenone-3 (BP-3), octocrylene (OC), and octyl methoxycinnamate (OMC), and evaluated their thyroid, neurodevelopmental, and kidney toxicities. For this purpose, zebrafish embryos (<4 h post fertilization, hpf) were exposed to sublethal concentrations of AVB, BP-3, OC, or OMC until 120 hpf. Exposure to all OUVFs decreased thyroid hormone (TH) levels, probably by enhanced metabolism and excretion of THs (ugt1ab and/or sult1 st5) in the larval fish. Exposure to the OUVFs also induced hypoactivities and/or anxiety-like behaviors: Regulatory changes of mbp, gfap, c-fos, syn2a, sty1a, and stxbp1b genes, support the changes in normal neurobehavior of the larval fish. Moreover, the OUVFs exposure caused increased proteinuria in the fish, along with transcriptional changes of wt1, nephrin, podocin, and cdh17 genes, which could explain the observed reduction in kidney functions. Principal component analysis (PCA) implied the potential interplay of THs with neurogenesis, or podocyte differentiation of the larval fish. Toxicological consequences of altered TH homeostasis, neurobehavior, and kidney function at the early life stage warrant further investigations not only in humans but also in aquatic ecosystems.

Polystyrene nanoplastics mediate skeletal toxicity through oxidative stress and the BMP pathway in zebrafish (Danio rerio)

The widespread presence of micro(nano)plastics (MNPs) has generated public concern. Studies have indicated that MNPs can accumulate in mammalian bones; however, research on the skeletal toxicity and underlying molecular mechanisms of MNPs in aquatic organisms remains limited. We subjected zebrafish embryos to three varying levels (1, 10, 100 μg/mL) of polystyrene nanoplastics (PSNPs) exposure over a period of 7 days in our research. The results revealed that PSNPs significantly reduced the body length and hatching rate of zebrafish, leading to skeletal deformities. mRNA level analysis showed significant upregulation of sp7, sparc, and smad1 genes transcription by PSNPs. Moreover, PSNPs markedly downregulated the mRNA levels associated with runx2a, bmp2a, and bmp4. Further investigations demonstrated that PSNPs dramatically increased ROS levels in zebrafish larvae, with significant downregulation of transcription levels of sod1 and cat genes, resulting in a sharp increase in transcription levels of apoptosis-related regulatory genes bcl-2 and bax. Furthermore, PSNPs led to a marked rise in Caspase 3 activity in zebrafish larvae, suggesting the initiation of apoptosis. PSNPs also notably inhibited alkaline phosphatase (AKP) activity. Compared to a 4-day exposure, a 7-day exposure to PSNPs intensified abnormalities across multiple indicators. In summary, our research indicates that PSNPs cause significant oxidative stress in zebrafish larvae, resulting in apoptosis. Moreover, PSNPs disrupt the transcription of genes related to skeletal development through the bone morphogenetic protein (BMP) pathway, further disrupting skeletal development processes and ultimately resulting in skeletal deformities in zebrafish larvae. This study provides new insights into the skeletal toxicity of MNPs.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated adult zebrafish as a model for Parkinson’s Disease

Dopamine (DA) is a catecholamine neurotransmitter that works to regulate cognitive functions. Patients affected by Parkinson’s Disease (PD) experience a loss of dopaminergic neurons and downregulated neural DA production. This leads to cognitive and physical decline that is the hallmark of PD for which no cure currently exists. Danio rerio, or zebrafish, have become an increasingly popular disease model used in PD pharmaceutical development. This model still requires extensive development to better characterize which PD features are adequately represented. Furthermore, the great majority of PD zebrafish models have been performed in embryos, which may not be relevant towards age-related human PD. As an improvement, mature D. rerio were treated with neurotoxic prodrug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through intraperitoneal injection to induce parkinsonism. Behavioral analysis confirmed disparities in movement between saline-injected control and the MPTP-injected experimental group, with swim distance and speed significantly lowered seven days after MPTP injection. Simultaneously, cognitive decline was apparent in MPTP-injected zebrafish, demonstrated by decreased alternation in a y-maze. RT-qPCR confirmed trends consistent with downregulation in Parkinsonian genetic markers, specifically DA transporter (DAT), MAO-B, PINK1. In summary, mature zebrafish injected with MPTP present with similar movement and cognitive decline as compared to human disease. Despite its benefits, this model does not appear to recapitulate pathophysiology of the disease with the full profile of expected gene downregulation. Because of this, it is important that researchers looking for pharmacological interventions for PD only use this zebrafish model when targeting the human-relevant PD symptoms and causes that are represented.

Stability and Efficacy of Live-Attenuated Vibrio harveyi Vaccines Under Different Storage Conditions in Zebrafish (Danio rerio) Models

Graphical Abstract Highlight Research The LD50 (median lethal dose) of Vibrio parahaemolyticus and harveyi in zebrafish was determined to be 1 x 106 CFU/mL, while for V. alginolyticus it was found to be 1 x 105 CFU/mL. The LAVh vaccine demonstrated cross-protection against various pathogenic strains of Vibrio, leading to an average of 80% survival rate in vaccinated individuals. The analysis of the LAVh vaccine emphasized its versatility, as it can be quickly deployed and stored as a freeze-dried powder. The LAVh vaccine can be easily accessible and user-friendly in various aquaculture environments, such as offshore and remote farms. Abstract Vibriosis poses a significant threat to marine teleosts, causing substantial losses in the global aquaculture industry. Previous work in our lab led to the development of a live-attenuated V. harveyi vaccine (LAVh) candidate that targets the serine endoprotease gene with a three-point knockout and has shown promise in protecting against vibriosis. However, further investigation is necessary to evaluate the stability and efficacy of its various storage conditions for broader applications. This study aims to determine how well the three different LAVh vaccine storage (fresh, stale, and freeze-dried LAVh) worked against vibriosis. A total of 1000 adult zebrafish (Danio rerio) (mean weight: 0.20±0.5 g) were divided into four groups. Groups 1, 2, and 3 were intraperitoneally injected with different LAVh vaccine storage (fresh, stale, and freeze-dried, respectively), while Group 4 received 0.01 M phosphate-buffered saline (PBS) and served as the unvaccinated control. Fish were monitored for 21 days post-vaccination for safety, stability, efficacy, and antibody analysis. The results showed that a modest dosage of 1 x 104 CFU/mL of LAVh vaccine from all storage conditions provided 80% survival upon intraperitoneal challenge with pathogenic strains of pathogenic V. harveyi, V. alginolyticus, and V. parahaemolyticus. This dosage induced significant antibody production and conferred cross-protection against different Vibrio spp., indicating the LAVh vaccine’s potential for commercial application. The LAVh vaccine demonstrated high effectiveness and suitability for storage as a freeze-dried powder. This study might offer significant insights into practical strategies for reducing vibriosis, especially in aquaculture settings with limited infrastructure.

Protective role of melatonin against radiation-induced disruptions in behavior rhythm of zebrafish (danio rerio)

Ionizing radiation, as an increasingly serious environmental pollutant, has aroused widespread public concern. Melatonin, as an indole heterocyclic compound, is known to have anti-inflammatory and antioxidant effects. However, few studies have considered the comprehensive impact of melatonin on radiation damage. In this study, we used zebrafish as experimental materials and employed methods such as acridine orange staining, enzyme-linked immunosorbent assay (ELISA), video tracking for automated behavior analysis, microscope imaging, and real-time fluorescence quantitative analysis. Zebrafish embryos at 2 h post-fertilization (hpf) were treated under four different experimental conditions to assess their growth, development, and metabolic consequences. Our findings indicate that 0.10 Gy gamma radiation significantly augments body length, eye area, spine width, and tail fin length in zebrafish, along with a marked increase in oxidative stress (P < 0.05). Moreover, it enhances cumulative swimming distance, time, and average speed, suggesting elevated activity levels. We observed circadian rhythm phase shifts, peak increases, and cycle shortening, accompanied by abnormal expression of genes pivotal to biological rhythms, exercise, melatonin synthesis, apoptosis/anti-apoptosis, and oxidation/antioxidant balance. The inclusion of melatonin (1 × 10-5 mol/L MLT) ameliorated these radiation-induced anomalies, while its independent effect on zebrafish was negligible. Melatonin can regulate oxidative stress responses, hinders apoptosis responses, and reprogramming the expression of rhythm-related genes in zebrafish embryos after reprogramming radiation stimulation. Overall, our research highlights melatonin's critical role in countering the biological damage inflicted by gamma radiation, proposing its potential as a therapeutic agent in radiation protection.

Impacts of graphene oxide contamination on a food web: Threats to somatic and reproductive health of organisms

Contamination of aquatic food webs with nanomaterials poses a significant ecological and human health challenge. Ingestion of nanomaterials alongside food disrupts digestion and impairs physiological processes, with potential consequences for organism fitness and survival. Complex interactions between nanomaterials and biota further exacerbate the issue, influencing life-history strategies and ecosystem dynamics. Accumulation of nanomaterials within autotrophic and detritus-based food webs raises concerns about biomagnification, especially for top-level consumers and seafood-dependent human populations. Understanding the extent and impact of nanomaterial contamination on aquatic biota is crucial for effective mitigation strategies. To address this challenge, we conducted a comprehensive study evaluating the bioaccumulation effects of graphene oxide (GO), a commonly used nanomaterial, within an aquatic food chain. Using a gnotobiotic freshwater microcosm, we investigated the effects of micro- and nano-scale GO sheets on key organisms: green algae (Chlorella vulgaris), brine shrimp (Artemia salina), and zebrafish (Danio rerio). Two feeding regimes, direct ingestion and trophic transfer, were employed to assess GO uptake and transfer within the food web. Direct exposure involved individual organisms being exposed to either nano- or micro-scale GO sheets, while trophic transfer involved a sequential exposure pathway: algae exposed to GO sheets, artemias feeding on the algae, and zebrafish consuming the artemias. Our study provides critical insights into nanomaterial contamination in aquatic ecosystems. Physicochemical properties of GO sheets, including ζ-potential and dispersion, were influenced by salt culture media, resulting in aggregation under salt conditions. Microscopic imaging confirmed the bioaccumulation of GO sheets within organisms, indicating prolonged exposure and potential long-term effects. Notably, biodistribution analysis in zebrafish demonstrated the penetration of nano-sized GO into the intestinal wall, signifying direct interaction with vital organs. Exposure to GO resulted in increased zebrafish mortality and impaired reproductive performance, particularly through trophic transfer. These findings emphasize the urgent need to address nanomaterial contamination in aquatic food webs to protect ecosystem components and human consumers. Our study highlights the importance of developing effective mitigation strategies to preserve the integrity of aquatic ecosystems, ensure resource sustainability, and safeguard human well-being. In conclusion, our study provides crucial insights into the impact of nanomaterial pollution on aquatic biota. By recognizing the challenges posed by nanomaterial contamination and implementing targeted interventions, we can mitigate the adverse effects, preserving the integrity of aquatic ecosystems and safeguarding human health.

Alleviation of bovine serum albumin on the neurotoxicity of silver nanoparticles in zebrafish (Danio rerio) larvae

Silver nanoparticles (AgNPs) are ubiquitous in the aquatic environment and have attracted extensive attention to their toxic effects on aquatic species. However, responses of the nervous system to AgNPs are little known, especially co-existing with the ubiquitous natural organic matter (NOM), which is critical for the ability to act in aquatic species. Here, this study investigated the neurotoxicology of environmentally relevant AgNPs with or without bovine serum albumin (BSA; a classical NOM) to zebrafish (Danio rerio) using visualized transgenic zebrafish. Exposure to AgNPs reduced the locomotor behavior of zebrafish by 28%–45%, including swimming distance and velocity, exhibiting obvious behavioral inhibition. The visualized transgenic zebrafish treated with AgNPs showed developmental retardation in the early development of the heart, central nervous, and motor nerve, as well as the related neurodevelopment genes, which may be responsible for the lowered locomotor behavior. In addition, AgNPs can specifically interfere with the cholinergic system and affect neuronal signaling, ultimately leading to behavioral abnormalities. However, the co-existing BSA alleviated the neurotoxicity of AgNPs in zebrafish, which may partially be attributed to the increased size and electronegativity of AgNPs caused by BSA, thus reducing the direct interaction of AgNPs with cells. The interaction between BSA and the released Ag+ from AgNPs may also be responsible for the alleviation of the neurodevelopment dysfunction in zebrafish. These findings provide valuable insights into the toxicity and risks of AgNPs in natural aquatic environments.

Comparative study of the growth, stress status and reproductive capabilities of four wild-type zebrafish (Danio rerio) lines

BackgroundZebrafish are widely used in various research fields and to fulfil the diverse research needs, numerous zebrafish lines are available, each with a unique domestication background, potentially resulting in intraspecies differences in specific biological functions. Few studies have compared multiple zebrafish lines under identical conditions to investigate both inter- and intra-line variability related to different functions. However, such variability could pose a challenge for the reproducibility of results in studies utilising zebrafish, particularly when the line used is not clearly specified. This study assessed growth, stress status (cortisol, serotonin) and reproductive capabilities (maturity, fecundity, fertilisation rate, sperm quality) of four commonly used wild-type zebrafish lines (AB, SJD, TU, WIK) using standardized protocols.ResultsThe stress markers levels were found to be similar across the lines, indicating that the endocrine stress status is robust to diverse domestication histories. Variations were observed in the growth and reproductive parameters. The lines exhibited differences in the timing of puberty (86 dpf for AB and SJD lines vs. 107 dpf for the WIK line) despite achieving similar sizes, suggesting that there are line-specific variations in the induction of maturation. Additionally, the AB line demonstrated higher sperm quality than did the other lines and higher fecundity and fertilization rates than did the SJD line. The AB line also exhibiting a smaller adult size but a heavier brain relative to its body weight.ConclusionThese findings emphasize the importance of line selection for zebrafish research, indicating that researchers should consider line-specific traits to ensure the biological relevance and reproducibility of the results.

Characterization of NK-lysin A, a potent antimicrobial peptide from the zebrafish Danio rerio

Antimicrobial peptides (AMPs) are important players of the innate immune system with a major role in the defense against invading pathogens. AMPs belonging to the family of saposin-like proteins (SAPLIPs) include the porcine NK-lysin and the human granulysin. In the zebrafish Danio rerio, transcript analyses of NK-lysin encoding genes have been reported, but biochemical characterizations at the protein level are missing so far. Here, we present the recombinant expression, purification, and characterization of one of these homologs, namely of NK-lysin A (DaNKlA). To remove the affinity tag from DaNKlA, we made use of a self-splicing intein. Recombinant DaNKlA depolarized liposomes over a broad pH range and showed a preference for negatively charged lipids. DaNKlA inhibited the growth of and killed different Gram-positive and Gram-negative bacteria, including the fish pathogenic bacterium Vibrio anguillarum, by membrane permeabilization but displayed substantially lower activity against yeast cells. Structural modelling and bioinformatic comparison of DaNKlA with characterized SAPLIPs suggest membrane destabilization accompanied by strong electrostatic interactions as the mode of action.

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

AbstractInternal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p &lt; 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p &lt; 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p &lt; 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

Immunotoxic response of bio-based plastic on early life stage zebrafish (Danio rerio): A safe alternative to petroleum-based plastics?

Bio-based plastics are marketed as environmentally friendly alternatives to petroleum-based plastics, although they require specific composting conditions for degradation, which leads to their accumulation in the environment and potential risks to aquatic organisms. We hypothesized that the accumulation of bio-based plastics may induce immunotoxic responses in fish. Our research focused on the accumulation and immunotoxicity of 80 nm polylactic acid (PLA) and polystyrene (PS) (0.1–10 mg/L) on early life stage zebrafish (Danio rerio) exposed for 7 days. Compared to PS, there was a higher accumulation of PLA in larvae. Exposure to PLA resulted in a significant increase in neutrophils and macrophages, while immune protein levels such as Complement 3 (C3), Immunoglobulin M (IgM), and C-reactive protein (CRP) were significantly reduced. Furthermore, the mRNA expression of pro-inflammatory cytokines, including tnf-α and il-6, were significantly elevated in PLA treatments. Additionally, PLA-exposed zebrafish were more susceptible to infection by Vibrio parahaemolyticus. Interestingly, at the same concentration, exposures to PS did not induce significant changes in macrophages or immune protein levels, C3 and IgM. This suggests that PLA has a greater immunotoxic response relative to PS. Our research findings contradict the popular belief that bio-based plastics are non-toxic and harmless, which may have potential risk to aquatic organisms.

THE EFFECT OF LEAD ON THE DEVELOPMENT OF EMBRYOS AND LARVAE OF DANIO RERIO

The early stages of development of aquatic organisms, including fish, are inherently vulnerable to the effects of lead (Pb) and other water-polluting metals. However, reports of harmful effects of environmentally significant Pb levels are limited. To this end, we exposed the embryos of old Danio rerio fish 2.5 hours after fertilization (hours) to a range of Pb concentrations covering environmentally significant levels (10 μm; 20 μm; 40 μm; 60 μm), up to 120h. Exposure negatively affected the development and survival of the embryos of danio fish, causing mortality associated with embryo coagulation, depending on concentration. At 24 hpf, the highest level of exposure (60 μm Pb) led to impaired embryo activity, characterized by a decrease in burst activity and the number of movements performed by embryos per minute. At 72 hpf, all the embryos hatched in all exposure concentrations. Morphological abnormalities and an increase in mRNA expression were observed. These results indicate that the Pb content within ecologically significant levels may be harmful to developing danio fish.

Enhancment of zebrafish (Danio rerio) immune and antioxidant systems using medicinal plant extracts encapsulated in alginate-chitosan nanocapsules with slow sustained release.

This study aimed to screen 10 medicinal plant extracts on zebrafish (Danio rerio), evaluating their impact on the complement system, immunoglobulin M (IgM) levels, lysozyme, and peroxidase activity, while also enhancing their efficacy through the gradual release using alginate-chitosan nanocapsules. The prepared methanolic extracts were combined with fish feed. The fish were divided into 12 groups, including 10 treatment groups, a positive and a negative control group. Results showed varying impacts of the extracts on the immune and antioxidant systems, with Cinnamon (Cinnamon cassia) and Hypericum (Hypericum perforatum) extracts demonstrating the most significant effects. Subsequently, Cinnamon and Hypericum extract were encapsulated in alginate-chitosan nanocapsules to assess their impact on zebrafish immune parameters, separately and synergistically. Gradual release of the extracts from the nanocapsules was observed, with slower release at pH 2 compared to pH 7. Overall, Cinnamon and Hypericum extracts exhibited substantial immune system enhancement, and their encapsulation in nanocapsules improved their effects on zebrafish immune parameters. These findings suggest using these encapsulated extracts to enhance immune responses in aquatic organisms.

Foxg1a is required for hair cell development and regeneration in the zebrafish lateral line.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammals, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. Our study demonstrates that mutation of foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. In developing and regenerating neuromasts, we find that loss of Foxg1a function results in reduced hair cell numbers, as well as decreased proliferation of neuromast cells. Foxg1a specifically regulates the development and regeneration of Islet1-labeled hair cells. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

A larval zebrafish model of cardiac physiological recovery following cardiac arrest and myocardial hypoxic damage.

Contemporary cardiac injury models in zebrafish larvae include cryoinjury, laser ablation, pharmacological treatment and cardiac dysfunction mutations. Although effective in damaging cardiomyocytes, these models lack the important element of myocardial hypoxia, which induces critical molecular cascades within cardiac muscle. We have developed a novel, tractable, high throughput in vivo model of hypoxia-induced cardiac damage that can subsequently be used in screening cardioactive drugs and testing recovery therapies. Our potentially more realistic model for studying cardiac arrest and recovery involves larval zebrafish (Danio rerio) acutely exposed to severe hypoxia (PO2=5-7 mmHg). Such exposure induces loss of mobility quickly followed by cardiac arrest occurring within 120 min in 5 days post fertilization (dpf) and within 40 min at 10 dpf. Approximately 90% of 5 dpf larvae survive acute hypoxic exposure, but survival fell to 30% by 10 dpf. Upon return to air-saturated water, only a subset of larvae resumed heartbeat, occurring within 4 min (5 dpf) and 6-8 min (8-10 dpf). Heart rate, stroke volume and cardiac output in control larvae before hypoxic exposure were 188±5 bpm, 0.20±0.001 nL and 35.5±2.2 nL/min (n=35), respectively. After briefly falling to zero upon severe hypoxic exposure, heart rate returned to control values by 24 h of recovery. However, reflecting the severe cardiac damage induced by the hypoxic episode, stroke volume and cardiac output remained depressed by ∼50% from control values at 24 h of recovery, and full restoration of cardiac function ultimately required 72 h post-cardiac arrest. Immunohistological staining showed co-localization of Troponin C (identifying cardiomyocytes) and Capase-3 (identifying cellular apoptosis). As an alternative to models employing mechanical or pharmacological damage to the developing myocardium, the highly reproducible cardiac effects of acute hypoxia-induced cardiac arrest in the larval zebrafish represent an alternative, potentially more realistic model that mimics the cellular and molecular consequences of an infarction for studying cardiac tissue hypoxia injury and recovery of function.