Zinc oxide nanoparticles disrupt development and function of the olfactory sensory system impairing olfaction-mediated behaviour in zebrafish.

Zinc (Zn) is an essential metal present in numerous enzymes throughout the body, playing a vital role in animal and human health. However, the increasing use of zinc oxide nanomaterials (ZnONPs) in a diverse range of products has raised concerns regarding their potential impacts on health and the environment. Despite these concerns, the toxicity of ZnONP exposure on animal health remain poorly understood. To help address this knowledge gap, we have developed a highly sensitive oxidative stress (OS) biosensor zebrafish capable of detecting cell/tissue-specific OS responses to low doses of various oxidative stressors, including Zn, in a live fish embryo. Using live-imaging analysis with this biosensor zebrafish embryo, we discovered that the olfactory sensory neurons in the brain are especially sensitive to ZnOP exposure. Furthermore, through studies monitoring neutrophil migration and neuronal activation in the embryonic brain and via behaviour analysis, we have found that sub-lethal doses of ZnONPs (ranging from 0.033 to 1 mg/L nominal concentrations), which had no visible effect on embryo growth or morphology, cause significant localised inflammation, disrupting the neurophysiology of olfactory brain tissues and ultimately impaired olfaction-mediated behaviour. Collectively, these findings establish a potent and important effect mechanism for ZnONP toxicity, indicating the olfactory sensory system as the primary target for ZnONPs as an environmental toxicant in aquatic environments. Our result also highlights that even low doses of ZnONPs can have detrimental effects on the olfactory sensory system, surpassing previous expectations. The importance of olfaction in environment sensing, sex behaviours and overall fitness across species raises concerns about the potential impact of ZnONPs on olfaction-mediated brain function and behaviour in animals and humans. Our study emphasises the need for greater consideration of the potential risks associated with these nanomaterials.

The influence of size and surface chemistry on the bioavailability, tissue distribution and toxicity of gold nanoparticles in zebrafish (Danio rerio).

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

Real-time drug testing of paediatric diffuse midline glioma to support clinical decision making: The Zurich DIPG/DMG centre experience.

BACKGROUND: Children diagnosed with diffuse midline gliomas (DMG) have an extremely poor overall survival: 9-12 months from diagnosis with currently no curative treatment options. Given DMG molecular heterogeneity, surgical biopsies are needed for molecular profiling and as part of enrolment into molecular-based and precision medicine type clinical interventions. In this study, we describe the results of real time profiling and drug testing at the diffuse intrinsic pontine glioma/DMG Research Centre at University Children's Hospital Zurich. METHOD: Biopsies were taken using a frame based stereotactic robot system (NeuroMate®, Renishaw) at University Children's Hospital Zurich. Tissue samples were evaluated to confirm diagnosis by H3K27M and H3K27 trimethylation loss. Genomic analyses were done using a variety of platforms (INFORM, Oncomine, UCSF500 gene panel). Cell lines were developed by mechanical tissue dissociation and verified by either sequencing or immunofluorescence staining confirming H3K27M mutation and used afterwards for drug testing. RESULTS: Twenty-five robot-assisted primary biopsies were successfully performed. Median hospital stay was 2 days (range 1-4 days). Nine low-passage patient-derived cells were developed, whereas 8 cell lines were used to inform response to clinically relevant drugs. Genome and RNA expression were used to further guide treatment strategies with targeted agents such as dual PI3K/mTOR inhibitor paxalisib. CONCLUSION: We established a systematic workflow for safe, robot-assisted brainstem biopsies and in-house tissue processing, followed by real-time drug testing. This provides valuable insights into tumour prognostic and individual treatment strategies targeting relevant vulnerabilities in these tumours in a clinically meaningful time frame.

Imipridones affect tumor bioenergetics and promote cell lineage differentiation in diffuse midline gliomas.

BACKGROUND: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs. METHODS: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq. RESULTS: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state. CONCLUSION: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).

Occurrence of Fungi and Fungal Toxins in Fish Feed During Storage.

Periods of unfavorable storing conditions can lead to changes in the quality of fish feeds, as well as the development of relevant mycotoxins. In the present study, a commercial fish feed was stored under defined conditions for four weeks. The main findings indicate that even storing fish feeds under unsuitable conditions for a short duration leads to a deterioration in quality. Mycotoxin and fungal contamination were subsequently analyzed. These investigations confirmed that different storage conditions can influence the presence of fungi and mycotoxins on fish feed. Notably, ochratoxin A (OTA) was found in samples after warm (25 °C) and humid (>60% relative humidity) treatment. This confirms the importance of this compound as a typical contaminant of fish feed and reveals how fast this mycotoxin can be formed in fish feed during storage.

New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish.

BACKGROUND: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. OBJECTIVES: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. METHODS: To achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. RESULTS: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. DISCUSSION: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish.

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

Impacts of ocean acidification on sperm develop with exposure time for a polychaete with long lived sperm.

The majority of marine invertebrate species release eggs and sperm into seawater for external fertilisation. Seawater conditions are currently changing at an unprecedented rate as a consequence of ocean acidification (OA). Sperm are thought to be particularly vulnerable to these changes and may be exposed to external environmental conditions for variable periods of time between spawning and fertilisation. Here, we undertook a mechanistic investigation of sperm swimming performance in the coastal polychaete Arenicola marina during an extended exposure to OA conditions (pHNBS 7.77, 1000 µatm pCO2). We found that key fitness-related aspects of sperm functioning declined faster under OA conditions i.e. impacts became apparent with exposure time. Sperm swimming speed (VCL), the number of motile sperm and sperm path linearity all dropped significantly after 4 h under OA conditions whilst remaining constant under ambient conditions at this time point. Our results highlight the importance of sperm exposure duration in ocean acidification experiments and may help towards explaining species specific differences in response.

Sensory systems and ionocytes are targets for silver nanoparticle effects in fish.

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.

Bmp suppression in mangrove killifish embryos causes a split in the body axis.

Bone morphogenetic proteins (Bmp) are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV) axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL) movements, and anterior-posterior (AP) axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM) produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

Manipulation and imaging of Kryptolebias marmoratus embryos.

The self-fertilizing mangrove killifish, Kryptolebias marmoratus, is an upcoming model species for a range of biological disciplines. To further establish this model in the field of developmental biology, we examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. These methodological approaches can be divided into two categories: handling of embryos with and without their chorionic membrane. Embryos still enclosed in their chorion can be manipulated using an agarose bed or a methyl cellulose system, holding them in place and allowing their rotation to more specific angles and positions. Using these methods, we demonstrate microinjection of embryos and monitoring of fluorescent yolk syncytial nuclei (YSN) using both stereo and compound microscopes. For higher magnification imaging using compound microscopes as well as time-lapse analyses, embryos were dechorionated and embedded in low-melting-point agarose. To demonstrate this embedding technique, we further examined fluorescent YSN and also analyzed the yolk surface of K. marmoratus embryos. The latter was observed to provide an excellent imaging platform for study of the behavior and morphology of cells during embryonic development, for various types of cells. Our data demonstrate that K. marmoratus is an excellent model species for research in developmental biology, as methodological approaches for the manipulation and imaging of embryos are efficient and readily available.

Embryonic development of the self-fertilizing mangrove killifish Kryptolebias marmoratus.

The mangrove killifish, Kryptolebias marmoratus, is a self-fertilizing vertebrate offering vast potential as a model species in many biological disciplines. Previous studies have defined developmental stages but lacked visual representations of the various embryonic structures. We offer detailed photographic images of K. marmoratus development with revised descriptions. An improved dechorionation method was developed to provide high resolution photographs, in addition to a microinjection technique enabling cell marking in the yolk syncytial layer. Embryos were also treated with PTU (1-phenyl 2-thiourea), an inhibitor of melanogenesis, to provide optical transparency revealing internal structures in late stages of development. Chemical exposures (PTU and retinoic acid) demonstrated that K. marmoratus embryos were sensitive to chemicals, illustrating further their usefulness in developmental biology studies. Our data suggest that K. marmoratus embryos are easily used and manipulated, supporting the use of this hermaphroditic vertebrate as a strong comparative model system in embryology, evolution, genetics, environmental and medical biology.