Differential Neuroprotective Effects of N-Acetylcysteine against Dithianon Toxicity in Glutamatergic, Dopaminergic, and GABAergic Neurons: Assessment Using Zebrafish.

Despite the widespread agricultural use of dithianon as an antifungal agent, its neurotoxic implications for humans and wildlife have not been comprehensively explored. Using zebrafish embryonic development as our model, we found that dithianon treatment induced behavioral alterations in zebrafish larvae that appeared normal. Detailed quantitative analyses showed that dithianon at ≥0.0001 µgmL-1 induced cytoplasmic and mitochondrial antioxidant responses sequentially, followed by the disruption of mitochondrial and cellular homeostasis. Additionally, dithianon at 0.01 and 0.1 µgmL-1 downregulated the expressions of glutamatergic (slc17a6b), GABAergic (gad1b), and dopaminergic (th) neuronal markers. Contrarily, dithianon upregulated the expression of the oligodendrocyte marker (olig2) at concentrations of 0.001 and 0.01 µgmL-1, concurrently suppressing the gene expression of the glucose transporter slc2a1a/glut1. Particularly, dithianon-induced increase in reactive oxygen species (ROS) production was reduced by both N-acetylcysteine (NAC) and betaine; however, only NAC prevented dithianon-induced mortality of zebrafish embryos. Moreover, NAC specifically prevented dithianon-induced alterations in glutamatergic and dopaminergic neurons while leaving GABAergic neurons unaffected, demonstrating that the major neurotransmission systems in the central nervous system differentially respond to the protective effects. Our findings contribute to a better understanding of the neurotoxic potential of dithianon and to developing preventive strategies.

TNNT1 myopathy with novel compound heterozygous mutations.

Nemaline myopathies are clinically and genetically heterogeneous disorders caused by several different genes. One of them is TNNT1, which was initially described in Amish families and has not been reported in Asian populations. Although most TNNT1 myopathies are caused by loss-of-function mutations, several recent studies have shown that missense mutations can also be pathogenic. A 16-year-old Korean boy with progressive muscle weakness visited the Seoul National University Hospital. He showed generalized myopathy, which was predominant in the paraspinal and neck muscles. Moreover, nemaline rods were observed in a muscle biopsy. Whole-exome sequencing of DNA samples of the patient and his younger brother, who had a similar phenotype, revealed novel compound heterozygous mutations in TNNT1 (c.724G>C (p.Ala242Pro) and c.611+1G>A). Sanger sequencing of cDNA extracted from muscle samples of the patient confirmed partial or total skipping of exon 11 in the splicing variant. The impact of the missense variant on muscle integrity and locomotor activity was verified using a zebrafish loss-of-function model. Here, we reported novel familial cases of TNNT1 myopathy with intermediate clinical presentations caused by compound heterozygous mutations and demonstrated their functional defects using an animal model.

Ipconazole Disrupts Mitochondrial Homeostasis and Alters GABAergic Neuronal Development in Zebrafish.

Ipconazole, a demethylation inhibitor of fungal ergosterol biosynthesis, is widely used in modern agriculture for foliar and seed treatment, and is authorized for use in livestock feed. Waste from ipconazole treatment enters rivers and groundwater through disposal and rain, posing potential toxicity to humans and other organisms. Its metabolites remain stable under standard hydrolysis conditions; however, their neurodevelopmental toxicity is unknown. We investigated the potential neurodevelopmental toxicity of ipconazole pesticides in zebrafish (Danio rerio). Our behavioral monitoring demonstrated that the locomotive activity of ipconazole-exposed zebrafish larvae was reduced during early development, even when morphological abnormalities were undetected. Molecular profiling demonstrated that the mitochondrial-specific antioxidants, superoxide dismutases 1 and 2, and the genes essential for mitochondrial genome maintenance and functions were specifically reduced in ipconazole-treated (0.02 µg/mL) embryos, suggesting underlying ipconazole-driven oxidative stress. Consistently, ipconazole treatment substantially reduced hsp70 expression and increased ERK1/2 phosphorylation in a dose-dependent manner. Interrupted gad1b expression confirmed that GABAergic inhibitory neurons were dysregulated at 0.02 µg/mL ipconazole, whereas glutamatergic excitatory and dopaminergic neurons remained unaffected, resulting in an uncoordinated neural network. Additionally, ipconazole-treated (2 µg/mL) embryos exhibited caspase-independent cell death. This suggests that ipconazole has the potential to alter neurodevelopment by dysregulating mitochondrial homeostasis.

Histopathological assessment of laterality defects in zebrafish development.

Laterality defects during embryonic development underlie the aetiology of various clinical symptoms of neuropathological and cardiovascular disorders; however, experimental approaches to understand the underlying mechanisms are limited due to the complex organ systems of vertebrate models. Zebrafish have the ability to survive even when the heart stops beating for a while during early embryonic development and those adults with cardiac abnormalities. Therefore, we induced laterality defects and investigated the occurrence of situs solitus, situs inversus, and situs ambiguus in zebrafish development. Histopathological analysis revealed heterotaxy in both embryos and juvenile fish. Additionally, randomization of left-right asymmetry of the brain and heart in individual zebrafish embryos under artificial experimental pressure further demonstrated the advantage of transparent zebrafish embryos as an experimental tool to select or reduce the embryos with laterality defects during early embryonic development for long-term studies, including behavioural and cognitive neuroscience investigations.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

Velutin, an Aglycone Extracted from Korean Mistletoe, with Improved Inhibitory Activity against Melanin Biosynthesis.

The abnormal regulation of melanin synthesis leads to a wide range of pigmentary disorders. Although various melanin biosynthesis inhibitors have been developed, their efficacy and long-term safety needs to be further improved, and thus the goal of this study is to develop promising natural compound inhibitors of melanin biosynthesis. Here, we obtained aglycone flavonoid extract through the microwave-assisted hydrolysis of glycoside extract from Korean mistletoe in acidic condition. The aglycone extract inhibited tyrosinase activity more efficiently with better antioxidant activity than glycoside extract in vitro. The microwave-assisted aglycone extract of mistletoe was further analyzed for in vivo activity, and the results showed the aglycone extract inhibited both early melanocyte development and melanin synthesis more efficiently in zebrafish embryo in a dose-dependent manner. Our in vivo toxicity assay quantitatively measured cell death in zebrafish embryos and showed that the microwave-assisted aglycone extract of mistletoe had no significant effect on cell death (p < 0.001), indicating that aglycone extract is more biocompatible than glycoside extract. Furthermore, our in vitro and in vivo analyses successfully identified and characterized velutin, an aglycone of a homoflavoyadorinin B glycoside, as a major inhibitory component in the microwave-assisted mistletoe extract. Ultimately, this study showed that the novel natural compound inhibitor velutin, which was generated through microwave-assisted extraction from mistletoe, improved the efficacy of melanin biosynthesis inhibition with little toxicity.

3D light-sheet assay assessing novel valproate-associated cardiotoxicity and folic acid relief in zebrafish embryogenesis.

Precise in vivo toxicological assays to determine the cardiotoxicity of pharmaceuticals and their waste products are essential in order to evaluate their risks to humans and the environment following industrial release. In the present study, we aimed to develop the sensitive imaging-based cardiotoxicity assay and combined 3D light-sheet microscopy with a zebrafish model to identify hidden cardiovascular anomalies induced by valproic acid (VPA) exposure. The zebrafish model is advantageous for this assessment because its embryos remain transparent. The 3D spatial localization of fluorescence-labeled cardiac cells in and around the heart using light-sheet technology revealed dislocalization of the heart from the outflow tract in two-day-old zebrafish embryos treated with 50 µM and 100 µM VPA (P < 0.01) and those embryos exposed to 20 µM VPA presented hypoplastic distal ventricles (P < 0.01). These two observed phenotypes are second heart field-derived cardiac defects. Quantitative analysis of the light-sheet imaging demonstrated that folic acid (FA) supplementation significantly increased the numbers of endocardial and myocardial cells (P < 0.05) and the accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate increased in response to the cellular changes occurring in embryonic heart development (P < 0.05). The present study disclosed the cellular mechanism underlying the role of FA in spontaneous cellular changes in cardiogenesis and in VPA-associated cardiotoxicity. The 3D light-sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicities in both humans and animals.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.