Antonio De la Vieja

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.

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.

The Na⁺/I⁻ symporter (NIS) is the plasma membrane (PM) protein that actively mediates iodide (I⁻) transport into the thyroid gland. Pathogenic variants in the SLC5A5 gene cause iodide transport defects (ITDs). A heterozygous G288S NIS variant was identified in a Spanish family in which female carriers developed thyroid dysfunction during pregnancy. Here, we characterized the functional significance of the G288S variant and other substitutions at residue 288 of human NIS. Human NIS (hNIS) expression and maturation were analyzed by immunoblotting, its subcellular localization was analyzed by immunofluorescence and flow cytometry, and its activity was analyzed by radioiodide uptake assays. The G288S variant does not affect hNIS maturation, membrane trafficking, or I⁻ uptake capacity, but significantly reduces I⁻ affinity while preserving substantial transport activity. In contrast, substitutions introducing charged residues (arginine, aspartic acid, or glutamic acid) or proline severely disrupted NIS maturation, plasma membrane targeting, and iodide transport. Because the variant was identified in heterozygosity, we evaluated residue 288 substitutions under heterozygous-like conditions. Co-expression of the patient-derived G288S variant with WT NIS produced an intermediate apparent K_m without reducing V_max compared with WT, consistent with a modest co-expression-dependent kinetic effect rather than a strong dominant-negative mechanism. In contrast, the severely disruptive G288E substitution reduced cell-surface NIS expression under co-expression conditions, providing proof-of-principle evidence that severe alteration of residue 288 can impair NIS plasma membrane delivery. These findings highlight residue 288 as a key determinant of hNIS functionality and underscore the need to carefully evaluate heterozygous SLC5A5/NIS variants, as they may become clinically relevant under conditions of increased physiological iodine demand and contribute to partial iodide transport impairment.

Despite decades of intense research, cancer remains a major global health challenge, with a clear need for new and more efficient therapies. Among emerging approaches, high atomic number (Z) nanomaterials capable of modulating the interaction between radiation and matter have attracted growing interest as radiotherapy enhancers. In this work, ultra‐small Platinum‐based nanoparticles with a metallic core diameter of 2–3 nm were synthesized from H ₂ PtCl ₆ using polyvinylpyrrolidone as stabilizer. This nanomaterial exhibits low acute toxicity, catalase‐like activity and enhances DNA damage under pre‐clinical X‐ray irradiation, increasing the antitumoral effect of X‐ray alone. In vitro and in vivo studies, including intratumoral and intravenous administration, demonstrate improved tumor control and prolonged survival without detectable systemic toxicity under the tested conditions, establishing ultra‐small Pt‐nanoparticles as efficient materials for radiotherapy enhancement.

Nanoplastics are increasingly detected in the environments, raising concerns about their capacity to disrupt endocrine regulation during vertebrate development. In this study, we investigated developmental and endocrine toxicity of 30 nm polystyrene nanoplastics (PSNP) in zebrafish embryos and larvae (0-120 hpf), with emphasis on hypothalamus-pituitary-thyroid (HPT) axis disruption and downstream neurodevelopmental outcomes. Embryos were exposed throughout development to environmentally relevant concentrations of PSNP (0-3 mg/L). Confocal time-lapse imaging revealed rapid accumulation in the chorion followed by progressive internalization and widespread distribution, with only partial elimination after prolonged depuration, indicating sustained internal exposure. Whole-body endocrine profiling revealed a dose-dependent decrease in TSH, T4 and T3 levels at 120 hpf, consistent with suppression of hypothalamic-pituitary drive within the HPT-axis and systemic thyroid hormone depletion. This hormonal decrease was accompanied by transcriptional rewiring across thyroid signaling pathways, including marked downregulation of Tshb and apparent compensatory regulation of thyroid-related synthesis/transport genes. Neurodevelopmental programs were concurrently dysregulated, with significant induction of genes linked to oligodendrocyte lineage specification, myelination, synaptogenesis and neuronal differentiation, and embryos displayed altered early motor output (increased tail coiling frequency with shortened bursts at 24 hpf). At the organism level, PSNP exposure induced multisystem developmental phenotypes, including reduced pigmentation, impaired eye and otolith development, swim bladder deflation, and cardiovascular alterations (tachycardia and pericardial edema), alongside stimulus-dependent anxiety-like and locomotor changes, some of which persisted after PSNP depuration. Collectively, these data strongly suggest sustained PSNP internal exposure disrupts thyroid hormone homeostasis through multi-level HPT-axis alterations, propagating across molecular, morphological and behavioral domains during early vertebrate development, with implications for aquatic health and endocrine-relevant hazard assessment.

The nervous system is highly vulnerable to chemical disruption, yet current regulatory guidelines do not include behavioral endpoints that capture changes in stress-related responses. Zebrafish larvae, up to 5 days old, have emerged as a promising model to bridge this gap, offering genetic and neurochemical similarity to humans together with high throughput potential. In this work, we have developed and evaluated a larval thigmotaxis assay as a new approach methodology (NAM) to detect behavioral alterations caused by neuroactive substances. Thigmotaxis, or edge-preference behavior, was studied in zebrafish larvae exposed to a range of model compounds and challenged with both visual (light/dark) and acoustic (sound/silence) stimuli. We compared 24 round well plates, commonly used in behavioral assays, with 96 square well plates to increase throughput. The two formats showed equivalent results, supporting the use of the higher-capacity system. Classical controls confirmed assay performance with caffeine increasing thigmotaxis, while diazepam decreased it. Additional neuroactive substances with diverse modes of action (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) produced stimulus-dependent responses, whereas negative controls (saccharin, amoxicillin) had little or no effect. Benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Overall, this multiplexed visual–acoustic thigmotaxis assay proved reproducible, scalable, and sensitive. In neurotoxicity testing this method could be used both as a stand-alone assay or as part of a broader behavioral NAM battery to assess potential effects on the vertebrate nervous system. This method provides a practical and ethical tool to improve chemical safety assessment both in ecotoxicology and human toxicology. GRAPHICAL

Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large scale is still challenging. One of the fluids from which they could be isolated in large quantities is milk. Moreover, regeneration is a well-known property of milk. The present work seeks to optimize a method for isolating exosomes from bovine and human milk, comparing different storage conditions and different extraction protocols. We found differences in the yield extraction associated with pre-storage milk conditions and observed some differences according to the processing agent. When we removed milk fat globules and added rennet before freezing, we obtained a cleaner final fraction. In summary, we attempted to optimize a rennet-based new milk–exosome isolation method and concluded that pre-treatment, followed by freezing of samples, yielded the best exosome population.