Chronotropic and vasoactive properties of the gut bacterial short-chain fatty acids in larval zebrafish.

Short-chain fatty acids (SCFAs) produced by the gut bacteria have been associated with cardiovascular dysfunction in humans and rodents. However, studies exploring effects of SCFAs on cardiovascular parameters in the zebrafish, an increasingly popular model in cardiovascular research, remain limited. Here, we performed fecal bacterial 16S sequencing and gas chromatography/mass spectrometry (GC-MS) to determine the composition and abundance of gut microbiota and SCFAs in adult zebrafish. Following this, the acute effects of major SCFAs on heart rate and vascular tone were measured in anesthetized zebrafish larvae using fecal concentrations of butyrate, acetate, and propionate. Finally, we investigated if coincubation with butyrate may lessen the effects of angiotensin II (ANG II) and phenylephrine (PE) on vascular tone in anesthetized zebrafish larvae. We found that the abundance in Proteobacteria, Firmicutes, and Fusobacteria phyla in the adult zebrafish resembled those reported in rodents and humans. SCFA levels with highest concentration of acetate (27.43 µM), followed by butyrate (2.19 µM) and propionate (1.65 µM) were observed in the fecal samples of adult zebrafish. Immersion in butyrate and acetate produced a ∼20% decrease in heart rate (HR), respectively, with no observed effects of propionate. Butyrate alone also produced an ∼25% decrease in the cross-sectional width of the dorsal aorta (DA) at 60 min (*P < 0.05), suggesting compensatory vasoconstriction, with no effects of either acetate or propionate. In addition, butyrate significantly alleviated the decrease in DA cross-sectional width produced by both ANG II and PE. We demonstrate the potential for zebrafish in investigation of host-microbiota interactions in cardiovascular health.NEW & NOTEWORTHY We highlight the presence of a core gut microbiota and demonstrate in vivo short-chain fatty acid production in adult zebrafish. In addition, we show cardio-beneficial vasoactive and chronotropic properties of butyrate, and chronotropic properties of acetate in anesthetized zebrafish larvae.

Development of Liquid Chromatography-Tandem Mass Spectrometry Analytical Methods for the Study of Whole-Body, Head, and Trunk Uptake and Elimination of Methamphetamine in 5-Day Postfertilization Zebrafish Larvae Using the Quick, Easy, Cheap, Effective, Rugged, and Safe Technique.

Synthetic cathinones are drugs of abuse substituted for amphetamine-like stimulant drugs such as methamphetamine. In this study, methamphetamine was studied as a prototypical amphetamine-like drug as a first step toward establishing methods to study this entire drug class. The internal concentration of methamphetamine in zebrafish larvae was determined using matrix-matched calibration along with extraction and purification of samples using the quick, easy, cheap, effective, rugged, and safe technique in liquid chromatography-tandem mass spectrometry. Whole-body and head/trunk uptake and elimination in 5-day postfertilization zebrafish larvae were determined. A gradient method was developed using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases, 10 min of total run time, and a 0.3 mL/min flow rate. The limit of quantification was 60 ng/mL, linearity with r2 = 0.9991, and recovery values from 92% to 120%. The internal concentration of methamphetamine was quantifiable in whole-body homogenates within 15 min of uptake analysis. The internal concentration increased with time, whereas a biphasic elimination pattern was shown. With increasing length of exposure, a higher accumulation of drugs was found in the head than in the trunk.

Small fish, big discoveries: zebrafish shed light on microbial biomarkers for neuro-immune-cardiovascular health.

Zebrafish (Danio rerio) have emerged as a powerful model to study the gut microbiome in the context of human conditions, including hypertension, cardiovascular disease, neurological disorders, and immune dysfunction. Here, we highlight zebrafish as a tool to bridge the gap in knowledge in linking the gut microbiome and physiological homeostasis of cardiovascular, neural, and immune systems, both independently and as an integrated axis. Drawing on zebrafish studies to date, we discuss challenges in microbiota transplant techniques and gnotobiotic husbandry practices. We present advantages and current limitations in zebrafish microbiome research and discuss the use of zebrafish in identification of microbial enterotypes in health and disease. We also highlight the versatility of zebrafish studies to further explore the function of human conditions relevant to gut dysbiosis and reveal novel therapeutic targets.

Methamphetamine-induced lethal toxicity in zebrafish larvae.

RATIONALE: The use of novel psychoactive substances has been steadily increasing in recent years. Given the rapid emergence of new substances and their constantly changing chemical structure, it is necessary to develop an efficient and expeditious approach to examine the mechanisms underlying their pharmacological and toxicological effects. Zebrafish (Danio rerio) have become a popular experimental subject for drug screening due to their amenability to high-throughput approaches. OBJECTIVES: In this study, we used methamphetamine (METH) as an exemplary psychoactive substance to investigate its acute toxicity and possible underlying mechanisms in 5-day post-fertilization (5 dpf) zebrafish larvae. METHODS: Lethality and toxicity of different concentrations of METH were examined in 5-dpf zebrafish larvae using a 96-well plate format. RESULTS: METH induced lethality in zebrafish larvae in a dose-dependent manner, which was associated with initial sympathomimetic activation, followed by cardiotoxicity. This was evidenced by significant heart rate increases at low doses, followed by decreased cardiac function at high doses and later time points. Levels of ammonia in the excreted water were increased but decreased internally. There was also evidence of seizures. Co-administration of the glutamate AMPA receptor antagonist GYKI-52466 and the dopamine D2 receptor antagonist raclopride significantly attenuated METH-induced lethality, suggesting that this lethality may be mediated synergistically or independently by glutamatergic and dopaminergic systems. CONCLUSIONS: These experiments provide a baseline for the study of the toxicity of related amphetamine compounds in 5-dpf zebrafish as well as a new high-throughput approach for investigating the toxicities of rapidly emerging new psychoactive substances.

Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.

Differential Toxicity of mDia Formin-Directed Functional Agonists and Antagonists in Developing Zebrafish.

The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5-10 µM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10-20 and 50 µM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

Alcohol consumption impairs the ependymal cilia motility in the brain ventricles.

Ependymal cilia protrude into the central canal of the brain ventricles and spinal cord to circulate the cerebral spinal fluid (CSF). Ependymal cilia dysfunction can hinder the movement of CSF leading to an abnormal accumulation of CSF within the brain known as hydrocephalus. Although the etiology of hydrocephalus was studied before, the effects of ethanol ingestion on ependymal cilia function have not been investigated in vivo. Here, we report three distinct types of ependymal cilia, type-I, type-II and type-III classified based upon their beating frequency, their beating angle, and their distinct localization within the mouse brain-lateral ventricle. Our studies show for the first time that oral gavage of ethanol decreased the beating frequency of all three types of ependymal cilia in both the third and the lateral rat brain ventricles in vivo. Furthermore, we show for the first time that hydin, a hydrocephalus-inducing gene product whose mutation impairs ciliary motility, and polycystin-2, whose ablation is associated with hydrocephalus are colocalized to the ependymal cilia. Thus, our studies reinforce the presence of three types of ependymal cilia in the brain ventricles and demonstrate the involvement of ethanol as a risk factor for the impairment of ependymal cilia motility in the brain.

HM015k, a Novel Silybin Derivative, Multi-Targets Metastatic Ovarian Cancer Cells and Is Safe in Zebrafish Toxicity Studies.

This study was designed to determine the in vitro mechanisms by which the novel silybin derivative, (E)-3-(3-(benzyloxy) phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (HM015k or 15k), produces its anticancer efficacy in ovarian cancer cells. Compound 15k induced apoptosis in ovarian cancer cells in a time-dependent manner by significantly upregulating the expression of Bax and Bak and downregulating the expression of Bcl-2. Interestingly, 15k induced the cleavage of Bax p21 into its more efficacious cleaved form, Bax p18. In addition, caspase 3 and caspase 9 were cleaved to their active forms, inducing the cleavage of poly ADP ribose polymerase (PARP) and ß-catenin. Furthermore, in OV2008 cells, 15k induced significant cleavage in nuclear ß-catenin to primarily inactive fragments of lower molecular weight. Furthermore, 15k reversed the metastatic potential of OV2008 cells by inhibiting their migration and invasiveness. The mesenchymal phenotype in OV2008 was reversed by 15k, causing cells to be rounder with epithelial-like phenotypes. The 15k-induced reversal was further confirmed by significant upregulation of the E-cadherin expression, an epithelial marker, while N-cadherin, a mesenchymal marker, was downregulated in OV2008 cells. Compound 15k inhibited the expression of the oncogenic c-Myc protein, downregulated proteins DVL3 and DVL2 and significantly upregulated cyclin B1. Also, 15k significantly downregulated the expression levels of ABCG2 and ABCB1 transporters in resistant ABCG2 overexpressing H460/MX20 and resistant ABCB1 overexpressing MDCK/MDR1 cells, respectively. Finally, 15k was safe in zebrafish in vivo model at concentrations up to 10 µM and induced no major toxicities in cardiac, morphology and swimming position parameters. Overall, 15k is a multi-targeted inhibitor with efficacy against metastatic and resistant ovarian cancer. Future in vivo studies will be conducted to determine the efficacy of 15k in tumor-bearing animals.