Search Results (631)

Parkinson’s disease (PD), impacting millions worldwide, leads to motor deficits and various non-motor symptoms. Although there is no cure, treatment primarily involves dopamine replacement therapy, especially L-dopa for motor symptoms, and additional drugs are required to address non-motor effects. This underscores the increasing demand for dual-acting drugs that can effectively target both symptom types in PD. This study explored the potential effects of a standardised Mesembryanthemum tortuosum extract, Zembrin^®, in treating PD, utilising in vivo and in silico models. Zebrafish larvae were subjected to pre-treatment with reserpine, followed by exposure to Zembrin^®, with selegiline and L-dopa as positive controls. The in vivo component of this study monitored locomotion and oxidative stress, while the in silico component identified potential drug targets for the treatment of PD. Reserpine induced hypolocomotion and oxidative stress in zebrafish larvae, and Zembrin^® (12.5 µg/mL) effectively enhanced locomotion and reduced oxidative stress. The molecular docking, molecular dynamics simulations, and binding free energy calculations revealed that four mesembrine alkaloids (mesembranol, mesembrenol, mesembrenone, and mesembrine) form stable and energetically favourable complexes with monoamine oxidase B (MAO-B) and dopamine transporter (DAT), which are significant targets for addressing both the motor and non-motor effects of PD. Full article

Chemical euthanasia protocols are routinely applied across zebrafish developmental stages, but the effects of formulation, time exposure, and developmental stage remain unclear and could introduce variability in transcriptional results and interfere with interlaboratory reproducibility of molecular endpoints. Wild-type AB zebrafish were exposed at three developmental windows (6 hpf, gastrulation; 30 hpf, pharyngula; 100 hpf, pre-feeding larva) to three operational euthanasia formulations (clove oil 0.1% v/v, tricaine/MS-222 1 g/L, or lidocaine 1 g/L), for 3, 10, or 25 min. Expression of ten genes covering immediate-early activation, cellular stress, metabolism, neuroendocrine signalling, apoptosis, and inflammation was quantified by qPCR. Expression responses were analysed using a three-way factorial framework including the developmental phase, formulation, and exposure time. The developmental phase explained substantial developmental context in several genes. The Phase × Treatment interaction was significant after global FDR correction for nine of ten genes, whereas treatment main effects did not provide a stable cross-stage ranking of formulations. Descriptively, gene expression varied across formulations and stages, with several Phase III markers showing positive shifts, but no operational formulation showed universally consistent effects after multiple-testing correction, while effect-size magnitudes should be interpreted cautiously. Acute transcriptional responses to operational chemical euthanasia formulations in zebrafish embryos and larvae were context-dependent. Developmental stage, rather than a universal formulation effect, emerged as the most consistent modulator. Because euthanasia efficacy and non-recovery were not experimental endpoints, these transcriptional data do not identify a best formulation for each developmental phase. These results support stage-aware reporting of euthanasia conditions. Full article

Introduction: Aquatic contaminants can alter fish behavior before overt toxicity becomes evident, making neurobehavioral endpoints relevant for ecosystem protection and for hazard prioritization within a One Health framework. We recently developed a high-throughput visual-acoustic zebrafish larval thigmotaxis assay in which edge preference is interpreted as an anxiety-like behavioral endpoint, thereby adding spatial phenotyping beyond conventional locomotion metrics. Objective: To evaluate assay performance in a multisubstance screening challenge and determine whether it can discriminate distinct behavioral fingerprints without prior knowledge of chemical identity. Methodology: Zebrafish larvae were exposed for 1 h at 120 hpf. For each substance, 24 larvae were tested per condition, with six concentrations per substance, plus positive and negative controls. Larvae were challenged using alternating light/dark and tapping/quiet paradigms. The primary endpoint was the percentage of time spent at the edge as a proxy for anxiety-like behavior, while total distance and mean total velocity when moving were used as contextual locomotor metrics; edge distance and edge velocity were used as supportive spatial metrics. Data from 37 substances were analyzed through a standardized automated workflow. Results: Controls performed as expected and supported assay stability across runs. The chemical screening revealed heterogeneous but reproducible behavioral fingerprints. Seven substances produced weak/minimal acute responses, ten showed predominantly suppressive profiles, three predominantly activating profiles, nine showed prominent thigmotaxis-specific anxiety-like signals not explained by locomotion alone, and eight displayed mixed or stimulus-dependent patterns, including non-monotonic responses. Several substances altered edge preference while distance and velocity changed less, differently, or in the opposite direction, indicating behavioral reorganization rather than simple hypo- or hyperactivity. The multi-stimulus design was critical because some effects were evident only under specific sensory contexts. Conclusions: The multisubstance challenge supports the discriminatory capacity, robustness, and added value of the assay for high-throughput neurobehavioral screening. By capturing anxiety-like behavior through thigmotaxis and complementing it with locomotor context, the method improves phenotypic resolution for aquatic pollution assessment and offers a sensitive fish-based NAM to prioritize chemicals of concern for both environmental and human health-oriented testing strategies. Full article

Introduction: Aquatic chemical pollution is among the most worrying threats to ecosystem health. There is an ever-increasing variety of pollutant substances detected across the source-to-sea continuum, causing loss of biodiversity and ecological disequilibrium. Achieving cleaner and healthier systems relies on carrying out sustained, cost-effective, diagnosis and aquatic effects monitoring, within the adaptive management cycle. The available methods are, however, cumbersome, which creates a clear need for innovative expeditious approaches for low-cost surveillance monitoring. In the last decade, Raman Spectroscopy (RS) has gained wide recognition for application to biological questions, for its ability to uncover the complexity of molecules and their interactions. Various fields, from pharmacology to disease diagnosis and prognosis, have suffered an innovation revolution through the application of RS. In this technique inelastic light scattering of a small part of photons of an incident electromagnetic monochromatic light beam (ranging from near-infrared to visible or ultraviolet) is caused by the molecular vibration of chemical bonds. This results in shifts in energy, which indicate discrete vibrational modes of polarisable molecules, providing qualitative and quantitative assessments of the chemical composition and molecular structure of the sample. The technique shows high sensitivity, no need for sample preparation and the possibility of use in non-invasive and label-free analysis. Objective: The aim of this work is to present and discuss evidence about the application of Raman Spectroscopy (RS) to environmental diagnosis and aquatic effect monitoring of pollution. Methodology: The technique was applied to different biological models, i.e., diatoms, zebrafish embryos and larvae and freshwater snails. Quality assessments with diatoms were tested in environmental monitoring, while assessments with other models were done upon exposure to metals and organic contaminants. Results and conclusions: The Raman spectra obtained from the samples analysed comprised bands detected within the 800 to 2000 cm⁻¹ wavenumber range. These were related to bond vibrations of carbohydrates, DNA phosphate groups, proteins or CH, NH and OH stretching in lipids and proteins. Data analysis using chemometric methods clearly distinguished pollutant exposure from control sites or treatments, pointing out the potential for surveyance monitoring. The next steps include the comparison with other sensitive methods (e.g., locomotion and avoidance behaviours, omics methods) to assess efficiency and bring further mechanistic understanding. Full article

Introduction: Chemical pollution of water bodies constitutes a global problem with huge impacts on fish populations. Consequently, the assessment of the effects of contaminants, especially on the nervous system, has become essential. The zebrafish (Danio rerio) has emerged as a prominent vertebrate model in ecotoxicology and neuroscience, in large part owing to the availability of genetic resources, including a high level of genome sequencing and annotation, plus the similarity of its neuron types and neurotransmitters to other vertebrates, including humans, and its stereotyped behaviour. Objective: The main objective of this mini-review is to present a synthesis of the key behavioural assays used in zebrafish larvae to assess neurotoxicity, focusing on developmental neurotoxicity. Methodology: A literature review was conducted based on the ScienceDirect and PubMed databases, covering publications between 2000 and 2025, selecting relevant studies on larval (up to 120 hpf) behaviour and contaminant exposure. The methodology was based on the analysis of behavioural tests applied to larvae, which evaluate responses to various stimuli, including visual, acoustic, tactile, and social stimuli. Results: Established, commonly used key assays include the light/dark test and locomotor, touch, photomotor, acoustic, and social response tests. The literature results confirm that zebrafish larvae exhibit complex behavioural patterns comparable to those of higher vertebrates, making them suitable for neurobehavioural studies. Changes in locomotor behaviour, responses to stimuli, or social patterns are extremely sensitive indicators of early neurotoxic effects, often before morphological changes are observed. Furthermore, the developing nervous system is particularly sensitive to chemicals, with high potential for irreversible effects, even with short-term exposures. Conclusions: Overall, our findings demonstrate that behavioural assays in zebrafish larvae constitute an effective, sensitive, and economically viable tool for assessing the neurotoxicity of compounds, contributing to a better understanding of the mechanisms of action and advancing environmental protection and public health strategies, considering also the “one health” approach. Full article

Cisplatin is a widely used platinum-based chemotherapeutic agent that often causes irreversible hair cell loss, leading to hearing impairment. To date, effective strategies for preventing cisplatin-induced ototoxicity remain limited. Corchorus olitorius L. (COL) is rich in bioactive phytochemicals with antioxidant and anti-inflammatory properties; however, the protective role of COL stem against cisplatin-induced hearing loss has not been explored. This study aimed to determine whether COL stem extract treatment could mitigate cisplatin-induced hair cell damage in the lateral line system of zebrafish. Herein, we use 7-day post-fertilization (dpf) transgenic zebrafish larvae as a high-throughput screening platform to assessed COL stem extract against cisplatin-induced hair cell injury. Endpoints included mechanotransduction (MET) function, reactive oxygen species (ROS) production, apoptotic and inflammatory responses, and locomotor behavior. Antioxidant capacity and acute toxicity were also evaluated. Pretreatment with COL stem extract preserved hair cell viability, restored MET function, reduced ROS accumulation, upregulated Nrf-2-dependent cytoprotective genes, suppressed apoptosis, and attenuated macrophage infiltration. The recovery of swimming behavior correlated with hair cell protection, confirming the phenotypic relevance. This study demonstrates, for the first time, that COL stem exerts potent otoprotective effects through antioxidative, anti-apoptotic, and anti-inflammatory mechanisms, contributes to maintain mechanosensory function and swimming behavior. The findings support COL stem as a promising candidate for otoprotection and validate zebrafish-based high-throughput screening for novel therapeutic discovery. Full article

Spermatogonial stem cells (SSCs) are pivotal in surrogate broodstock technology. However, species-specific protocols for the efficient enrichment and long-term preservation of SSCs in olive flounder (Paralichthys olivaceus) are not yet fully established. In this study, we evaluated and optimized methods for the isolation and cryopreservation of P. olivaceus SSCs. First, we compared two enrichment methods, including Percoll density gradient centrifugation (PDGC) and differential plating (DP). Although SSCs enriched by both methods showed increased expression of SSC-specific marker genes, PDGC resulted in significantly greater enrichment than DP. A combination of PDGC and DP did not further improve enrichment efficiency, suggesting that PDGC alone is sufficient in P. olivaceus. Second, we optimized cryopreservation conditions according to various cryoprotectants. Among the conditions, SSCs cryopreservation using 1.3 M propylene glycol (PG) as a permeating agent and 0.2 M raffinose (Raf) as a non-permeating cryoprotectant provided the highest cell viability (56.1%), demonstrating a synergistic protective effect. Finally, preliminary in vivo migration and localization ability of the cryopreserved SSCs was confirmed through xenotransplantation into zebrafish (Danio rerio) larvae. PKH26-labeled donor cells exhibited successful initial localization and short-term persistence within the presumptive gonadal ridge of the recipients at 5 days post-transplantation. These findings provide an optimized protocol for the handling and preservation of P. olivaceus germline resources, contributing to the technical advancement of surrogate reproduction strategies in this species. Full article

Neuroinflammation, mediated by microglia and astrocytes, is an abnormal immune reaction in central nervous system (CNS) disorders. Stimulation of TRPV1 has been found to enhance microglial activation, resulting in a pro-inflammatory response. Natural anthraquinones such as physcion and rhein are commonly found in rhubarb, a medicinal plant recognized for its dual role in culinary and therapeutic applications. The therapeutic potential and mechanisms of these anthraquinones remain largely unexplored. This research aims to examine how anthraquinones protect against neuroinflammation and delineate the underlying mechanisms in lipopolysaccharide (LPS)-mediated cellular and zebrafish models. Among the representative anthraquinone analogs, physcion and rhein showed potent functional inhibitory activity against the TRPV1 channel. The production of nitric oxide (NO) and secretion of pro-inflammatory factors triggered by LPS were significantly reduced in BV-2 cells through regulation of iNOS, IL-6, IL-1β, and TNF-α mRNA expression. Moreover, physcion and rhein inhibited calcium influx and exerted anti-neuroinflammatory effects, which were closely associated with the suppression of Ca²⁺/CAMKK2/AKT and the PI3K/AKT-mediated NF-κB activation pathways. Furthermore, physcion and rhein reduced LPS-driven neutrophil recruitment to the brain and ameliorated locomotor deficits in zebrafish larvae, with the restoration of IL-1β, IL-6, and TNF-α transcript levels to baseline. In conclusion, natural-derived anthraquinones from rhubarb, physcion and rhein, acted as functional inhibitors of TRPV1-mediated calcium dynamics and significantly reduced LPS-mediated neuroinflammation in microglial cells and zebrafish larvae, suggesting promise as therapeutics for neurological disorders. Full article

Emerging evidence links environmental exposures and circadian dysregulation to autism spectrum disorder (ASD), yet whether circadian phase modulates vulnerability to developmental toxicants remains unclear. Here, we investigated whether embryonic bisphenol A (BPA) exposure induces circadian phase-dependent ASD-related behavioral alterations via disruption of nr1d1 rhythmicity in zebrafish. In control larvae, nr1d1 exhibited significant circadian oscillation, whereas BPA exposure reduced expression levels and dampened oscillation amplitude. Two-way ANOVA revealed significant treatment × phase interactions in nr1d1 expression. Pharmacological activation of Nr1d1 partially restored rhythmic expression. Behavioral assessments conducted at defined circadian phases demonstrated a significant treatment × phase interaction in social preference. BPA-exposed larvae exhibited reduced social preference selectively at circadian time 15 (CT15), corresponding to the trough phase of nr1d1 expression, whereas no differences were observed at circadian time 3 (CT3). In contrast, tactile hyper-responsiveness showed a significant treatment effect but no phase interaction. BPA exposure also induced phase-dependent alterations in ASD-related genes, including α-nrxn2a and β-nrxn3a, with significant treatment × phase interactions. At the molecular level, BPA increased reactive oxygen species, impaired antioxidant defense, enhanced neuroinflammatory responses, and disrupted excitatory–inhibitory balance. Several of these endpoints exhibited phase-dependent modulation and were partially attenuated by Nr1d1 activation. These findings indicate that circadian phase modulates embryonic susceptibility to BPA-induced ASD-related behavioral and molecular alterations. Disruption of nr1d1 rhythmicity may contribute to time-of-day-specific neurodevelopmental vulnerability following environmental exposure. Full article

Flavonols are dietary polyphenols whose biological activity is influenced by structural modifications such as O-methylation. This study compared two quercetin derivatives, isorhamnetin (3′-O-methylquercetin) and rhamnetin (7-O-methylquercetin). Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays. Cyclooxygenase-2 (COX-2) inhibitory activity was assessed in vitro and supported by molecular docking simulations. In vivo effects included developmental toxicity, behavioral assessment, and locomotor responses in a 6-hydroxydopamine (6-OHDA) model. The results demonstrated that rhamnetin exhibited significantly stronger radical-scavenging and reducing activity in DPPH, ABTS, and FRAP assays, whereas no significant differences were observed in the CUPRAC assay. Isorhamnetin showed stronger COX-2 inhibition, with docking results suggesting a different mode of binding when analyzing possible interactions with enzyme active site. In zebrafish larvae, rhamnetin showed lower observable developmental toxicity within the tested concentration range, whereas isorhamnetin induced developmental abnormalities at higher concentrations. Both flavonols attenuated 6-OHDA-associated locomotor deficits and modulated antioxidant enzyme activity under oxidative stress conditions. In conclusion, our findings indicate that the position of O-methylation influences flavonol antioxidant properties, COX-2 interactions, and organism-level responses. Full article

6:2 Chlorinated polyfluoroether sulfonate (F-53B), an emerging per- and polyfluoroalkyl substance used as a perfluorooctane sulfonate substitute in electroplating, may co-occur with Cr(VI) in industrially affected aquatic environments. However, their combined developmental toxicity in vertebrates remains poorly understood. This study compared the effects of F-53B and Cr(VI) alone and in combination on zebrafish embryos and larvae exposed from 0 to 120 hpf at environmentally relevant concentrations (10 and 100 μg/L). Developmental toxicity, ocular morphology, retinal histology, locomotor behavior, and ocular-development-related gene expression were assessed. Single Cr(VI) exposure produced prominent effects on hatching, locomotor activity, retinal-layer thickness, and selected transcriptional responses related to retinoic acid signaling and ocular development. F-53B alone caused developmental and ocular alterations but generally produced weaker or more selective responses than Cr(VI). Co-exposure did not uniformly intensify the toxicity of either compound. Instead, the mixture enhanced some endpoints, including malformation, heart rate, and specific ocular or retinal alterations while showing weaker or divergent responses for other endpoints, such as locomotor activity and selected transcriptional markers. These findings indicate that F-53B/Cr(VI) co-exposure produces endpoint-dependent toxicity rather than a consistent synergistic pattern. Overall, the study highlights the importance of comparing single and combined exposures across multiple biological endpoints when assessing the developmental risks of co-occurring contaminants. Full article

Gatifloxacin (GTFX), a fourth-generation fluoroquinolone, causes metabolic disturbances in mammals, but its hepatotoxic mechanisms in aquatic vertebrates remain unclear. This study investigated whether GTFX induces liver injury in zebrafish larvae through oxidative stress or alternative pathways. Larvae at 3 days post-fertilization were exposed to 0.2–2.3 mg/mL GTFX for 48 h. Liver morphology, histopathology, intracellular reactive oxygen species (ROS), and expression of lipid metabolism (pparg) and xenobiotic biotransformation genes (cyp1a, cyp1b1) were assessed. GTFX exposure caused concentration-dependent reductions in liver area, increased hepatic opacity, delayed yolk sac absorption, and hepatocyte swelling with cytoplasmic vacuolization. Despite these structural changes, ROS levels did not differ significantly from those of controls. In contrast, transcriptional analysis revealed significant upregulation of pparg, cyp1a, and cyp1b1, indicating disrupted lipid homeostasis and enhanced detoxification responses. Acute high-dose GTFX exposure induced a metabolism-associated hepatotoxic response in zebrafish larvae, which occurred without a statistically significant change in bulk ROS levels. Together, these findings offer mechanistic insight into fluoroquinolone-associated liver injury. Full article

During production, fish are exposed to multiple environmental, physiological, and physical stressors, which compromise development, productivity, and welfare and urge the implementation of effective and safe stress-mitigating strategies, particularly during early developmental stages. Larval zebrafish (Danio rerio) constitute a powerful model for studying acute stress responses due to the numerous advantages they offer, such as developmental transparency, a conserved hypothalamic–pituitary–interrenal (HPI) axis, and suitability for high-throughput screening. This review examines the potential of natural monoterpenes as stress-reducing compounds and compares their performance with conventional synthetic anesthetics. Evidence from vortex-flow stress paradigms, behavioral profiling and biochemical assays shows that acute stress in zebrafish larvae triggers metabolic disruption, behavioral hyperactivity and enzyme imbalance, with cortisol responses depending on stimulus intensity. Monoterpenes such as thymol and menthol consistently reduce stress-induced hyperactivity, support redox homeostasis and display favorable safety profiles at low doses and short exposures. Nevertheless, as research into these substances is still recent, evidence of any potential adverse effects is still limited. Although individual monoterpenes may act on different subsets of molecular targets, their multimodal mechanisms, including gamma-aminobutyric acid (GABA)ergic enhancement, voltage-gated ion channel and transient receptor potential (TRP) modulation, suggest broader and potentially safer actions compared to single-target anesthetics as tricaine methane sulfonate (MS-222). Collectively, these findings suggest that monoterpenes offer promising natural alternatives for stress mitigation in aquaculture and the refinement of research procedures involving early life stages. Full article

The increasing prevalence of fungal infections caused by Candida species, together with rising antifungal resistance, highlights the urgent need for novel therapeutic agents with improved efficacy and safety. In this study, a series of 3-substituted rhodanine derivatives (3–6) were synthesized and evaluated as potential multifunctional compounds combining antifungal activity and ferric reducing capacity in the FRAP assay. The compounds were characterized using FT-IR and NMR spectroscopy and assessed for their physicochemical and pharmacokinetic profiles through in silico ADME analysis. Biological evaluation revealed that compounds 3 and 5 exhibited the most promising antifungal activity against a panel of clinically relevant Candida strains, with compound 5 demonstrating broad-spectrum, predominantly fungicidal effects. In contrast, compounds bearing a bulky 4-chlorobenzoyl substituent (4 and 6) showed reduced activity, indicating the importance of structural features for antifungal efficacy. Ferric reducing capacity assessment using the FRAP assay confirmed that all compounds possess reducing activity, with compounds 3 and 6 showing the highest potential. Safety evaluation using zebrafish (Danio rerio) embryos and larvae revealed concentration-dependent toxicity for all compounds. Notably, compounds 5 and 6 exhibited significant embryotoxicity and neurobehavioral effects at low micromolar concentrations, whereas compound 3 demonstrated a more favorable safety profile, with minimal impact on development and locomotor activity. Taken together, these results indicate that compound 3 provides a balanced combination of antifungal activity and reduced toxicity, while compound 5 represents a highly active but more toxic derivative. The observed structure–activity relationships emphasize the importance of carefully tuning substituent-dependent properties to optimize both biological activity and safety, supporting the continued investigation of rhodanine-based multifunctional antifungal agents targeting fungal proliferation and ferric reducing properties. Full article

Background/Objectives: Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent chemicals widely detected in aquatic systems and drinking water. Perfluorononanoic acid (PFNA), a long-chain PFAS, has been reported globally in environmental matrices and fish tissues. Although PFNA has been linked to developmental, metabolic, and neurological toxicity, its effects on lipid-related pathways and neurotoxicity remain poorly characterized. Methods: This study evaluated the developmental and neurotoxic effects of PFNA exposure in zebrafish embryos and larvae following a 7-day exposure to environmentally relevant PFNA concentrations. Results: PFNA exposure did not significantly affect survival or deformity rates at the concentrations tested. Apoptosis was significantly increased in larvae exposed to 1 µg/L PFNA compared to controls, whereas reactive oxygen species levels were unaffected. Two concentrations (0.1 µg/L and 10 µg/L) were further examined for transcriptomic responses, and the transcriptome response was largely different for each concentration. Low-dose PFNA exposure primarily affected lipid transport, cholesterol metabolism, sphingolipid signaling, and neurodegeneration-related pathways, whereas high-dose PFNA altered transcripts related to synaptic signaling, axon guidance, and thyroid hormone synthesis. Hypoactivity was observed in the movement of larval zebrafish based on a Visual Motor Response test. Conclusions: Taken together, PFNA exposure induced molecular changes related to neurotoxicity and lipid metabolism in zebrafish, which may contribute to adverse neurodevelopmental outcomes. Full article