Compounds In Zebrafish Research Articles

O36 | Assessment of the antinociceptive efficacy of analgesics, anesthetics and other compounds in zebrafish (Danio rerio)

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 45-45 ORAL PRESENTATIONS O36 | Assessment of the antinociceptive efficacy of analgesics, anesthetics and other compounds in zebrafish (Danio rerio) First published: 11 June 2023 https://doi.org/10.1111/jvp.13202Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 45-45 RelatedInformation

Influence of ultraviolet-aging and adsorbed pollutants on toxicological effects of polyvinyl chloride microplastics to zebrafish

Microplastics (MPs) undergo various aging processes and interact with diverse pollutants in the environment. In the present study, we investigated the influence of ultraviolet (UV) aging on the adsorption of organic pollutants by polyvinyl chloride microplastics (mPVC) and explored toxicity variations among pristine, aged, and pollutant-loaded mPVCs to zebrafish. Irradiation of UV for 30 d significantly changed the physiochemical properties of mPVC, leading to more oxygen-containing groups and free radicals (1O2, ·O2−, and ·OH) on mPVC surfaces. The aging process reduced the adsorption of mPVC against a hydrophobic compound chlorpyrifos (CPF) but enhanced the adsorption against a moderately hydrophilic compound erythromycin (ERY). Ingestion of CPF- and ERY-loaded mPVCs resulted in bioaccumulation of the two compounds in zebrafish, suggesting a carrier effect of mPVCs. In toxicity tests, the aged mPVC caused severer gut damages, stronger oxidative stresses, and greater interference with the gut microbiota in zebrafish than the pristine mPVC. The CPF and ERY-loaded mPVCs produced lower oxidative stresses in zebrafish than mPVCs alone, due to fewer radicals on mPVC surfaces after the adsorption of organic contaminants. Notably, the CPF and ERY-loaded mPVCs presented greater effects on fish swimming behaviors and gut microbial compositions, which was associated with the released CPF and ERY from mPVCs within the zebrafish. Overall, the present study demonstrated significant influences of UV-aging and the adsorbed pollutants on the toxicological effects of MPs and highlighted the necessity to perform toxicity studies of MPs using more environmentally relevant MPs.

Screening of Mineralogenic and Osteogenic Compounds in Zebrafish-Tools to Improve Assay Throughput and Data Accuracy.

Bone disorders affect millions of people worldwide and treatments currently available often produce undesirable secondary effects or have limited efficacy. It is therefore of the utmost interest for patients to develop more efficient drugs with reduced off-target activities. In the long process of drug development, screening and preclinical validation have recently gained momentum with the increased use of zebrafish as a model organism to study pathological processes related to human bone disorders, and the development of zebrafish high-throughput screening assays to identify bone anabolic compounds. In this review, we provided a comprehensive overview of the literature on zebrafish bone-related assays and evaluated their performance towards an integration into screening pipelines for the discovery of mineralogenic/osteogenic compounds. Tools available to standardize fish housing and feeding procedures, synchronize embryo production, and automatize specimen sorting and image acquisition/analysis toward faster and more accurate screening outputs were also presented.

Integrated approach toward absorption, distribution, metabolism, and excretion of Xiaoke pills in zebrafish based on UPLC-HRMS and DESI-MS techniques

Traditional Chinese medicine (TCM) has been used in clinical settings for over 2000 years in China. The study of the absorption, distribution, metabolism, and excretion (ADME) of TCM in vivo could be beneficial for the discovery of the active components in TCM. However, the conventional strategies used for ADME research are based on rodent models and have the characteristics of lengthy experimental periods, complex processes, and extensive data processing, which make it difficult to perform rapid analyses and high-throughput ADME screening of the medicinal components of TCM. In this study, an integrated high-throughput research strategy for the in vivo ADME analysis of TCM was established based on a zebrafish model. Accordingly, a combination of ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS), desorption electrospray ionization-mass spectrometry (DESI-MS) imaging, and in-house non-targeted precise-and-thorough background-subtraction (PATBS) data post-processing techniques were successfully applied for the analysis of the metabolism of zebrafish exposed to Xiaoke pills. A total of 49 compounds related to Xiaoke pills (including 13 prototypical components and 36 metabolites) were detected in zebrafish. In total, 32 of them, including puerarin, daidzein, deoxyschizandrin, formononetin, and glibenclamide, which have been identified to have hypoglycemic activity in our previous studies and are phase I and phase II metabolites resulting from the hydroxylation, demethylation, glucuronidation, sulfation, and glycosylation of the prototypical components in vivo, were found in rats treated with Xiaoke pills. Furthermore, the overall distribution of the known compounds in zebrafish exposed to Xiaoke pills was explored using DESI-MS. In summary, this study provides a practical approach for the high-throughput screening of the active components of TCM using a zebrafish model.

ZEBRAFISH AS MODELS OF TYROSINE KINASE‐DRIVEN CANCER

The protein kinase gene family encodes proteins involved in several essential functions, with cell growth and migration being essential outcomes. Mutations in kinases underlie a broad range of diseases, primarily inflammation and cancer. Tyrosine kinase inhibitors (TKIs) in particular, are successfully used as a targeted therapy for cancer and inflammation‐driven disorders. Current unmet needs include addressing the adverse effects of TKIs, the emergence of resistance, and the necessity of newer TKIs. Experiments using accessible animal models such as zebrafish can help address these needs. Zebrafish studies can help in efficient characterization of toxicological and pharmacological effects of inhibition using known or potential TKIs, via the combination of phenotypic and molecular analysis. The recent application of CRISPR‐Cas9 further enables the development of genetically engineered cancer models in zebrafish. Following our previous work showing conservation of zebrafish tyrosine kinases compared to humans, we are now studying the effects of chemical or genetic tyrosine kinase modulation in zebrafish.To identify the phenotypic endpoints of tyrosine kinase inhibition in zebrafish, we selected a few drugs approved by the USFDA for treating cancer and inflammation‐related disorders in humans. We evaluated phenotypic effects including adverse effects to identify NOELs and detectable and consistent phenotypic endpoints of such compounds in zebrafish. These effects provide a basis to evaluate novel TKI compounds. Further, tyrosine kinase genes involved in inflammation and cancer were selected for targeted genetic modification in zebrafish using CRISPR Cas9. Our initial data demonstrate the development of MET (HGFR) homozygous mutant zebrafish and the phenotypes associated with this mutant. Detailed characterization of this mutant including molecular analysis is under way. The characteristics of this mutant provide the basis to identify MET‐specific inhibitors. Development of zebrafish models carrying oncogenic TK mutants, and detailed characterization of the effects of TKIs in zebrafish are ongoing.In summary, our work is focussed on identifying TKI treatment‐related phenotypes in zebrafish, and generation of TK‐crispant zebrafish as cancer models.Support or Funding InformationDepartment of Biotechnology and the Indian Council of Medical Research, Govt. of India

Investigating the utility of adult zebrafish ex vivo whole hearts to pharmacologically screen hERG channel activator compounds.

There is significant interest in the potential utility of small-molecule activator compounds to mitigate cardiac arrhythmia caused by loss of function of hERG1a voltage-gated potassium channels. Zebrafish (Danio rerio) have been proposed as a cost-effective, high-throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker and types 1 and 2 activator compounds on isolated zkcnh6a (zERG3) channels in the Xenopus oocyte expression system as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that under the conditions tested, these channels are blocked by hERG1a channel blockers (dofetilide and terfenadine), and activated by type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activators with higher affinity than hKCNH2a channels (except NS1643), with differences accounted for by different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the action potential duration (APD), whereas hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings lead us to suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds.

Multidimensional chemobehavior analysis of flavonoids and neuroactive compounds in zebrafish

The comparative analysis of complex behavioral phenotypes is valuable as a reductionist tool for both drug discovery and defining chemical bioactivity. Flavonoids are a diverse class of chemicals that elicit robust neuroactive and hormonal actions, though bioactivity information is limited for many, particularly for neurobehavioral endpoints. Here, we used a zebrafish larval chemomotor response (LCR) bioassay to comparatively evaluate a suite of 24 flavonoids, and in addition a panel of 30 model neuroactive compounds representing diverse modes of action (e.g. caffeine, chlorpyrifos, methamphetamine, nicotine, picrotoxin). Naïve larval zebrafish were exposed to concentration ranges of each compound at 120 hour post-fertilization (hpf) and locomotor activity measured for 5 h. The model neuroactive compounds were largely behaviorally bioactive (20 of 30) with most effects phenotypic of their known modes of action. Flavonoids rapidly and broadly elicited hyperactive locomotor effects (22 of 24). Multidimensional analyses compared responses over time and identified three distinct bioactive groups of flavonoids based on efficacy and potency. Using GABAergics to modulate hyperactive responses, two flavonoids, (S)-equol and kaempferol were tested for GABAA receptor antagonism, as well as a known GABAA receptor antagonist, picrotoxin. Pharmacological intervention with positive allosteric modulators of the GABAA receptor, alfaxalone and chlormethiazole, ameliorated the hyperactive response to picrotoxin, but not for (S)-equol or kaempferol. Taken together, these studies demonstrate that flavonoids are differentially bioactive and that the chemobehavioral effects likely do not involve a GABAA receptor mediated mode of action. Overall, the integrative zebrafish platform provides a useful framework for comparatively evaluating high-content chemobehavioral data for sets of structurally- and mechanistically-related flavonoids and neuroactive compounds.

Geometric Morphometrics as a Tool to Evaluate Teratogenic Effects in Zebrafish (Danio rerio).

Geometric morphometrics allows for the characterization of shape using Cartesian geometric coordinates rather than linear or volumetric measurements, which are dependent upon size and are insufficient to capture geometric shape. By using landmarks on specimens, variations in position, orientation and scale between specimens can be removed to better compare variations in shape. This method has primarily been used in the fields of evolutionary biology and taxonomy. Here we describe how geometric morphometrics can be used to delineate variations in shape caused by teratogenic compounds in zebrafish.

Bioaccumulation Behavior of Pharmaceuticals and Personal Care Products in Adult Zebrafish (Danio rerio): Influence of Physical-Chemical Properties and Biotransformation.

The factors influencing bioaccumulation of pharmaceuticals and personal care products (PPCPs) in aquatic organisms are not well understood. The present study involved a comprehensive laboratory investigation to assess the bioaccumulation behavior of several PPCPs in adult zebrafish (Danio rerio). The studied PPCPs included several ionogenic organic compounds (IOCs) such as weak acids and weak bases. Experiments involved two exposure groups (high and low) and a control group, with a 6 day aqueous exposure, followed by a 7 day depuration phase under flow-through conditions. Uptake rate constants (ku) ranged between 0.19 and 8610 L·kg-1·d-1, while depuration rate constants (kd) ranged between 0.14 and 5.14 d-1 in different fish tissues. Steady-state bioconcentration factor (BCFss) values varied widely among the studied PPCPs, ranging from 0.09 to 6,460. In many cases, BCFss values of individual PPCPs differed substantially among different fish tissues. Positive linear relationships were observed between log BCFss values and physical-chemical properties such as octanol-water distribution coefficients (log Dow), membrane-water distribution coefficients (log Dmw), albumin-water distribution coefficients (log DBSAw), and muscle protein-water distribution coefficients (log Dmpw), indicating the importance of lipid-, phospholipid-, and protein-water partitioning. The results also showed that for many PPCPs, the estimated whole-body metabolism rate constant (km) values were comparable to the observed depuration rate (kd), indicating that metabolism plays a major role in the overall elimination of these compounds in zebrafish. An exception was sertraline, which exhibited a kd value (0.4-0.5 d-1) that was much higher than the estimated whole-body km (0.03 d-1). Overall, the results help to better understand the influence of physical-chemical properties and biotransformation on bioaccumulation behavior of these contaminants of concern in aquatic organisms.

Pharmacological Manipulation of Shoaling Behavior in Zebrafish

Background: The zebrafish has been a favorite of developmental biologists and geneticists for decades, however, recently, it has gained popularity among behavior researchers too. The reason for the popularity of this species is that while it is a simple vertebrate, it possesses numerous features that make it translationally relevant. Objective: Social interaction is an essential component of human as well as zebrafish behavior; yet, the biological mechanisms underlying social behavior and its dysfunction remain poorly understood in both species. This review focuses on pharmacological manipulation of social behavior in zebrafish, studies where the ultimate goal is to understand the psychopharmacology of human behavior. Methods: We focused specifically on quantification of social interaction, specifically shoaling, in zebrafish, and compared it to rodent literature where appropriate. If no social studies exist for specific classes of compounds in zebrafish or if other behaviors may help with interpretation of social responses, we also highlighted non-social behaviors (locomotion, anxiety, etc). Results: We discussed findings on the effect of exposure of zebrafish to alcohol, psychostimulants, depressants and other drugs of abuse. Comparison of studies that have quantified and manipulated zebrafish social behavior of either single zebrafish or groups of zebrafish high-light both growth and gaps in this rapidly evolving field. Conclusion: Although new in psychopharmacology research, the zebrafish will be an important tool with which analysis and modeling of human social behavior and disorders involving social abnormalities including Fetal Alcohol Spectrum Disorders, Autism spectrum disorders, and Schizophrenia may be facilitated. Keywords: Zebrafish, social behavior, alcohol, psychostimulants, antidepressants, anxiolytics, shoaling.

Zebrafish Reel in Phenotypic Suppressors of Autism

Chemical genetics can help decipher novel pathways underlying neurodevelopmental psychiatric impairments. Hoffman et al. (2016) utilized behavioral profiling of psychoactive compounds in zebrafish and identified estrogens as suppressors of a phenotype resulting from loss of an autism risk gene.

Analysis of 17 neurotransmitters, metabolites and precursors in zebrafish through the life cycle using ultrahigh performance liquid chromatography-tandem mass spectrometry.

An ultrahigh performance liquid chromatography-tandem mass spectrometry method for the identification and quantification of neurotransmitters, metabolites and precursors at different stages in zebrafish life was developed. Betaine, glutamine, glutamic acid, γ-aminobutyric acid, phosphocholine, glycerophosphocholine, cytidine 5'-diphosphocholine, choline, acetylcholine, dopamine, norepinephrine, serotonin, tyrosine, epinephrine, tryptophan, 5-hydroxyindolacetic acid and agmatine were selected as analytes. The method consisted of a simple deproteinization of samples using methanol and formic acid, subsequent injection onto the chromatographic equipment and quantification with a triple quadrupole mass spectrometer detector using an electrospray ionization interface in positive mode. Limits of detection ranged from 0.02 to 11ngmL(-1) and limits of quantification from 0.1 to 38ngmL(-1), depending on the analyte. The method was validated according to US Food and Drugs Administration (FDA) guideline for bioanalytical assays. Precision, expressed as relative standard deviation (%RSD), was lower than 15% in all cases, and the determination coefficient (R(2)) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85% and 115%. The method was applied to determine of these compounds in zebrafish from early stages of development to adulthood and showed the time-course of neurotransmitters and others neurocompounds through the life cycle. The possibility of measuring up to 17 compounds related with the main neurotransmitter systems in a simple analytical method will complement and reinforce the use of zebrafish in multiple applications in the field of neurosciences. The proposed method will facilitate future studies related with brain development.

Identification of phenolic compounds in red wine extract samples and zebrafish embryos by HPLC-ESI-LTQ-Orbitrap-MS

The zebrafish embryo is a highly interesting biological model with applications in different scientific fields, such as biomedicine, pharmacology and toxicology. In this study, we used liquid chromatography/electrospray ionisation-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC/ESI-LTQ-Orbitrap-MS) to identify the polyphenol compounds in a red wine extract and zebrafish embryos. Phenolic compounds and anthocyanin metabolites were determined in zebrafish embryos previously exposed to the red wine extract. Compounds were identified by injection in a high-resolution system (LTQ-Orbitrap) using accurate mass measurements in MS, MS2 and MS3 modes. To our knowledge, this research constitutes the first comprehensive identification of phenolic compounds in zebrafish by HPLC coupled to high-resolution mass spectrometry.

Toxicity Assessment of 7 Anticancer Compounds in Zebrafish

Toxicity is one of the major reasons for failure in drug development. Zebrafish, as an ideal vertebrate model, could also be used to evaluate drug toxicity. In this study, we aimed to show the predictability and highlight novel findings of toxicity in zebrafish model. Seven anticancer compounds, including triptolide (TP), gambogic acid (GA), mycophenolic acid (MPA), curcumin, auranofin, thalidomide, and taxol, were assessed in zebrafish for their toxicity. Three compounds (GA, TP, and taxol) showed highest acute lethality, with 50% lethal concentration ≈ 1 μmol/L. Missing tails, severe pericardial edema, and enlarged yolk sacs were observed in MPA-treated embryos. The development of pectoral fins was severely disturbed in thalidomide-, GA-, and TP-treated embryos. Bradycardia was observed in MPA- and thalidomide-treated groups. Our findings suggested that the zebrafish are a good model for toxicity assessment of anticancer compounds.

Characterization of retinoic acid–induced neurobehavioral effects in developing zebrafish

Retinoic signaling plays an important role in cell proliferation and differentiation. Disruption of retinoic signaling via excessive or deficient retinoic acid can cause teratogenic effects on developing embryos. Similar to retinoic acid, many xenobiotic environmental pollutants have been found to disrupt retinoic signaling through binding and eliciting agonistic activity on retinoic acid receptors. Currently, studies of retinoic acid or retinoic acid-like compounds in aquatic organisms have mainly focused on teratogenicity and few studies have explored their neurobehavioral toxicity. In the present study, the authors used retinoic acid as an example to explore the neurobehavioral toxicity associated with developmental exposure of retinoic acid-like compounds in zebrafish. The findings confirmed retinoic acid's teratogenic effects such as bent spine, malformed tail, and pericardial edema in developing zebrafish with a median effective concentration of 2.47 nM. Retinoic acid-induced cell apoptosis at 24 h postfertilization was consistently found in the eye and tail regions of embryos. Spontaneous movement as characterized by tail bend frequency was significantly increased in zebrafish embryos following exposure to 2 nM and 8 nM retinoic acid. Relatively low-dose retinoic acid exposure of 2 nM led to fast locomotion behavior in the dark period and hyperactivity during light-dark photoperiod stimulation. The 2-nM retinoic acid exposure also led to alterations of neurobehavior- and optic nerve-related genes, with the transforming growth factor-β signal transduction inhibitor noggin (nog) and the spinal cord marker homeobox c3a (hox) being underexpressed and the retinal G protein-coupled receptor a (rgr), the photoreceptor cell marker rhodopsin (rho), and the short wave-sensitive cone pigment opsin 1 (opn1sw1) being overexpressed. Increased expression of opn1sw1 and rho was confirmed by whole-mount in situ hybridization. Whether the misexpression of these genes leads to the neurobehavioral changes merits further study. The findings demonstrated that low-dose retinoic acid exposure perturbed the visual system and optic nerve development and caused hyperactivity in developing zebrafish.

The behavioral effects of acute Δ9-tetrahydrocannabinol and heroin (diacetylmorphine) exposure in adult zebrafish

The use of psychotropic drugs in clinical and translational brain research continues to grow, and the need for novel experimental models and screens is becoming widely recognized. Mounting evidence supports the utility of zebrafish (Danio rerio) for studying various pharmacological manipulations, as an alternative model complementing the existing rodent paradigms in this field. Here, we explore the effects of acute 20-min exposure to two commonly abused psychotropic compounds, Δ9-tetrahydrocannabinol (THC) and heroin, on adult zebrafish behavior in the novel tank test. Overall, THC administration (30 and 50mg/L) produces an anxiogenic-like reduction of top swimming, paralleled with a slower, continuous bottom swimming. In contrast, heroin exposure (15 and 25mg/L) evoked a hyperlocomotor response (with rapid bouts of bottom swimming and frequent ‘bouncing’ motions) without altering anxiety-sensitive top/bottom endpoints. The behavioral effects of these two compounds in zebrafish seem to parallel the respective rodent and human findings. Collectively, this emphasizes the growing significance of novel emerging aquatic models in translational drug abuse research and small molecule screening.

Predicted drug-induced bradycardia related cardio toxicity using a zebrafish in vivo model is highly correlated with results from in vitro tests

Several in vivo and in vitro studies have assessed methods of evaluating the cardio toxicity of compounds during drug development due to its importance for predicting human toxicity. However, in vivo/in vitro relationships have not yet been reported using a zebrafish model. This study determined the bradycardia of 15 compounds by evaluating the change in heart beat rate (HBR) in zebrafish, hERG fluorescence polarization (hERG-FP), and ionic current change using a patch clamp (hERG-PC). In addition, a model for prediction of drug-induced bradycardia was established using in vivo and in vitro assays designed for high-throughput toxicological screening. The IC50 values correlated well in two in vitro studies (R2=0.9). The change in HBR in zebrafish caused by the compounds could be estimated using the IC50 from the hERG-FP assay [(i.e., % of HBR=19.5×log(IC50, hERG-FP)] or hERG-PC assay [(i.e., % of HBR=19.6×log(IC50, hERG-FP)]. To validate the predictive model, 10 unknown compounds were used and the percentages of the HBR were estimated using the model. The observed and predicted HBR% for the compounds in zebrafish were well-correlated (R2=0.948). Therefore, the proposed models were useful for prediction of drug-induced bradycardia related cardio toxicity.

A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity

IntroductionNumerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish. MethodsHepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology. ResultsAll the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish. DiscussionLarval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.

Zebrafish Chemical Screening Reveals the Impairment of Dopaminergic Neuronal Survival by Cardiac Glycosides

Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA) neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na+/K+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na+/K+ ATPase α3 subunit (ATP1A3). Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

Metabolite Profiling of Four Major Flavonoids of Herba Epimdii in Zebrafish

The zebrafish model organism was applied first in a metabolic study of icariin, baohuoside I, epimedin A and epimedin C, which are flavonoids in Herba Epimedii. Metabolites of these compounds in zebrafish after exposure for 24 h were identified by HPLC-ESI-MS, whereby the separation was performed with a Zorbax C-18 column using a gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds were detected in simultaneous negative and positive ionization modes. Metabolic products of icariin and epimedin C via cleavage of glucose residue instead of rhamnose residues were found, which coincided with the results using regular metabolic analysis methods. In addition, the zebrafish model was used to predict the metabolism of the trace component epimedin A, whose metabolic mechanisms haven’t been clearly elucidated with the current metabolism model. The metabolic pathway of epimedin A in zebrafish was similar to those of its homologue icariin and epimedin C. Our study demonstrated that the zebrafish model can successfully imitate the current models in elucidating metabolic pathways of model flavonoids, which has advantages of lower cost, far less amount of compound needed, easy set up and high performance. This novel model can also be applied in quickly predicting the metabolism of Chinese herb components, especially trace compounds.