Assessment of the Novel Anti-Seizure Potential of Validamycin A Using Zebrafish Epilepsy Model.

Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

Toxicity assessment of di(2-ethylhexyl) phthalate using zebrafish embryos: Cardiotoxic potential.

Plasticizers are considered as newly emerged contaminants. They are added to plastics to increase their flexibility and softness. Phthalate plasticizers including the Di-2-ethylhexyl phthalates (DEHP) are toxic and induce adverse effects on the different organization levels of the environment. In the current study, we investigated the potential toxicity of DEHP using Zebrafish as a biological model. Five ascending concentrations of DEHP were tested in embryos throughout 96 hpf: 0.0086, 0.086, 0.86, 8.6, and 86 mg/L. Embryotoxicity assessments revealed limited lethal effects on DEHP-exposed embryos, yet notable anticipation of the hatching process was observed at 48 hpf. Although DEHP showed negligible influence on the length and pericardial area of exposed embryos, it led to multiple bodily deformities. Gene expression analyses of key cardiogenic and inflammatory genes evidenced alterations in tbx20, bcl2, and il1b expression in Zebrafish embryos at 96 h post-fertilization. Results from the cardiac function analysis displayed that DEHP significantly affected the arterial pulse and linear velocity within the Posterior Cardinal Vein (PCV) of exposed fish. These findings strongly advance that even at low concentrations, DEHP can be considered as potential toxic agent, capable of inducing cardiotoxic effects.

Comparative toxicological assessment of 2 bisphenols using a systems approach: evaluation of the behavioral and transcriptomic responses of Danio rerio to bisphenol A and tetrabromobisphenol A.

The zebrafish (Danio rerio) is becoming a critical component of new approach methods (NAMs) in chemical risk assessment. As a whole organism in vitro NAM, the zebrafish model offers significant advantages over individual cell-line testing, including toxicokinetic and toxicodynamic competencies. A transcriptomic approach not only allows for insight into mechanism of action for both apical endpoints and unobservable adverse outcomes, but also changes in gene expression induced by lower, environmentally relevant concentrations. In this study, we used a larval zebrafish model to assess the behavioral and transcriptomic alterations caused by subphenotypic concentrations of 2 chemicals with the same structural backbone, the endocrine-disrupting chemicals bisphenol A and tetrabromobisphenol A. Following assessment of behavioral toxicity, we used a transcriptomic approach to identify molecular pathways associated with previously described phenotypes. We also determined the transcriptomic point of departure for each chemical by modeling gene expression changes as continuous systems which allows for the identification of a single concentration at which toxic effects can be predicted. This can then be investigated with confirmatory cell-based testing in an integrated approach to testing and assessment to determine risk to human health and the environment with greater confidence. This paper demonstrates the impact of using a multi-faceted approach for evaluating the physiological and neurotoxic effects of exposure to structurally related chemicals. By comparing phenotypic effects with transcriptomic outcomes, we were able to differentiate, characterize, and rank the toxicities of related bisphenols, which demonstrates methodological advantages unique to the larval zebrafish NAM.

Diving into drug-screening: zebrafish embryos as an in vivo platform for antimicrobial drug discovery and assessment.

The rise of multidrug-resistant bacteria underlines the need for innovative treatments, yet the introduction of new drugs has stagnated despite numerous antimicrobial discoveries. A major hurdle is a poor correlation between promising in vitro data and in vivo efficacy in animal models, which is essential for clinical development. Early in vivo testing is hindered by the expense and complexity of existing animal models. Therefore, there is a pressing need for cost-effective, rapid preclinical models with high translational value. To overcome these challenges, zebrafish embryos have emerged as an attractive model for infectious disease studies, offering advantages such as ethical alignment, rapid development, ease of maintenance, and genetic manipulability. The zebrafish embryo infection model, involving microinjection or immersion of pathogens and potential antibiotic hit compounds, provides a promising solution for early-stage drug screening. It offers a cost-effective and rapid means of assessing the efficacy, toxicity and mechanism of action of compounds in a whole-organism context. This review discusses the experimental design of this model, but also its benefits and challenges. Additionally, it highlights recently identified compounds in the zebrafish embryo infection model and discusses the relevance of the model in predicting the compound's clinical potential.

Quantitative assessment and monitoring of microplastics and nanoplastics distributions and lipid metabolism in live zebrafish using hyperspectral stimulated Raman scattering microscopy.

Microplastics (MP) and nanoplastics (NP) pollutions pose a rising environmental threat to humans and other living species, given their escalating presence in essential resources that living subjects ingest and/or inhale. Herein, to elucidate the potential health implications of MP/NP, we report for the first time by using label-free hyperspectral stimulated Raman scattering (SRS) imaging technique developed to quantitatively monitor the bioaccumulation and metabolic toxicity of MP/NP within live zebrafish larvae during their early developmental stages. Zebrafish embryos are exposed to environmentally related concentrations (3-60 µg/ml) of polystyrene (PS) beads with two typical sizes (2 µm and 50 nm). Zebrafish are administered isotope-tagged fatty acids through microinjection and dietary intake for in vivo tracking of lipid metabolism dynamics. In vivo 3D quantitative vibrational imaging of PS beads and intrinsic biomolecules across key zebrafish organs reveals that gut and liver are the primary target organs of MP/NP, while only 50 nm PS beads readily aggregate and adhere to the brain and blood vessels. The 50 nm PS beads are also found to induce more pronounced hepatic inflammatory response compared to 2 µm counterparts, characterized by increased biogenesis of lipid droplets and upregulation of arachidonic acid detected in zebrafish liver. Furthermore, Raman-tagged SRS imaging of fatty acids uncovers that MP/NP exposure significantly reduces yolk lipid utilization and promotes dietary lipid storage in zebrafish, possibly associated with developmental delays and more pronounced food dilution effects in zebrafish larvae exposed to 2 µm PS beads. The hyperspectral SRS imaging in this work shows that MP/NP exposure perturbs the development and lipid metabolism in zebrafish larvae, furthering the understanding of MP/NP ingestions and consequent toxicity in different organs in living species.

Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.

BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.

Preliminary insight on diarylpentanoids as potential antimalarials: In silico, in vitro pLDH and in vivo zebrafish toxicity assessment.

Malaria remains a major public health problem worldwide, including in Southeast Asia. Chemotherapeutic agents such as chloroquine (CQ) are effective, but problems with drug resistance and toxicity have necessitated a continuous search for new effective antimalarial agents. Here we report on a virtual screening of ∼300 diarylpentanoids and derivatives, in search of potential Plasmodium falciparum lactate dehydrogenase (PfLDH) inhibitors with acceptable drug-like properties. Several molecules with binding affinities comparable to CQ were chosen for in vitro validation of antimalarial efficacy. Among them, MS33A, MS33C and MS34C are the most promising against CQ-sensitive (3D7) with EC50 values of 1.6, 2.5 and 3.1 µM, respectively. Meanwhile, MS87 (EC50 of 1.85 µM) shown the most active against the CQ-resistant Gombak A strain, and MS33A and MS33C the most effective P. knowlesi inhibitors (EC50 of 3.6 and 5.1 µM, respectively). The in vitro cytotoxicity of selected diarylpentanoids (MS33A, MS33C, MS34C and MS87) was tested on Vero mammalian cells to evaluate parasite selectivity (SI), showing moderate to low cytotoxicity (CC50 > 82 µM). In addition, MS87 exhibited a high SI and the lowest resistance index (RI), suggesting that MS87 may exert effective parasite inhibition with low resistance potential in the CQ-resistant P. falciparum strain. Furthermore, the in vivo toxicity of the molecules on early embryonic development, the cardiovascular system, heart rate, motor activity and apoptosis were assessed in a zebrafish animal model. The overall results indicate the preliminary potential of diarylpentanoids, which need further investigation for their development as new antimalarial agents.

Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

BACKGROUND: Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. METHODS: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. RESULTS: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. CONCLUSIONS: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

A comprehensive assessment of palmatine as anticonvulsant agent - In vivo and in silico studies.

Previously, we demonstrated that palmatine (PALM) - an isoquinoline alkaloid from Berberis sibrica radix, exerted antiseizure activity in the pentylenetetrazole (PTZ)-induced seizure assay in larval zebrafish. The aim of the present study was to more precisely characterize PALM as a potential anticonvulsant drug candidate. A range of zebrafish and mouse seizure/epilepsy models were applied in the investigation. Immunostaining analysis was conducted to assess the changes in mouse brains, while in silico molecular modelling was performed to determine potential targets for PALM. Accordingly, PALM had anticonvulsant effect in ethyl 2-ketopent-4-enoate (EKP)-induced seizure assay in zebrafish larvae as well as in the 6 Hz-induced psychomotor seizure threshold and timed infusion PTZ tests in mice. The protective effect in the EKP-induced seizure assay was confirmed in the local field potential recordings. PALM did not affect seizures in the gabra1a knockout line of zebrafish larvae. In the scn1Lab-/- zebrafish line, pretreatment with PALM potentiated seizure-like behaviour of larvae. Repetitive treatment with PALM, however, did not reduce development of PTZ-induced seizure activity nor prevent the loss of parvalbumin-interneurons in the hippocampus of the PTZ kindled mice. In silico molecular modelling revealed that the noted anticonvulsant effect of PALM in EKP-induced seizure assay might result from its interactions with glutamic acid decarboxylase and/or via AMPA receptor non-competitive antagonism. Our study has demonstrated the anticonvulsant activity of PALM in some experimental models of seizures, including a model of pharmacoresistant seizures induced by EKP. These results indicate that PALM might be a suitable new drug candidate but the precise mechanism of its anticonvulsant activity has to be determined.

DbGTi: Thermostable trypsin inhibitor from Dioscorea bulbifera L. ground tubers: assessment of antioxidant and antibacterial properties and cytotoxicity evaluation using zebrafish model.

Oxidative stress disorders and diseases caused by drug-resistant bacteria have emerged as significant public health concerns. Plant-based medications like protease inhibitors are growing despite adverse effects therapies. Consecutively, in this study, trypsin inhibitors from Dioscorea bulbifera L. (DbGTi trypsin inhibitor) ground tubers were isolated, purified, characterized, and evaluated for their potential cytotoxicity, antibacterial, and antioxidant activities. DbGTi protein was purified by Q-Sepharose matrix, followed by trypsin inhibitory activity. The molecular weight of the DbGTi protein was found to be approximately 31 kDa by SDS-PAGE electrophoresis. The secondary structure analysis by circular dichroism (CD) spectroscopy revealed that the DbGTi protein predominantly comprises ß sheets followed by α helix. DbGTi protein showed competitive type of inhibition with Vmax = 2.1372 × 10-1 µM/min, Km = 1.1805 × 102 µM, & Ki = 8.4 × 10-9 M and was stable up to 70 °C. DbGTi protein exhibited 58 % similarity with Dioscorin protein isolated from Dioscorea alata L. as revealed by LC-MS/MS analysis. DbGTi protein showed a non-toxic effect, analyzed by MTT, Haemolytic assay and in vivo studies on zebrafish model. DbGTi protein significantly inhibited K. pneumoniae and has excellent antioxidant properties, confirmed by various antioxidant assays. The results of anti-microbial, cytotoxicity and antioxidant assays demonstrate its bioactive potential and non-toxic nature.