Sinomenine Hydrochloride Ameliorates Fish Foodborne Enteritis via α7nAchR-Mediated Anti-Inflammatory Effect Whilst Altering Microbiota Composition.

Foodborne intestinal inflammation is a major health and welfare issue in aquaculture. To prevent enteritis, various additives have been incorporated into the fish diet. Considering anti-inflammatory immune regulation, an effective natural compound could potentially treat or prevent intestinal inflammation. Our previous study has revealed galantamine's effect on soybean induced enteritis (SBMIE) and has highlighted the possible role of the cholinergic anti-inflammatory pathway in the fish gut. To further activate the intestinal cholinergic related anti-inflammatory function, α7nAchR signaling was considered. In this study, sinomenine, a typical agonist of α7nAChR in mammals, was tested to treat fish foodborne enteritis via its potential anti-inflammation effect using the zebrafish foodborne enteritis model. After sinomenine's dietary inclusion, results suggested that there was an alleviation of intestinal inflammation at a pathological level. This outcome was demonstrated through the improved morphology of intestinal villi. At a molecular level, SN suppressed inflammatory cytokines' expression (especially for tnf-α) and upregulated anti-inflammation-related functions (indicated by expression of il-10, il-22, and foxp3a). To systematically understand sinomenine's intestinal effect on SBMIE, transcriptomic analysis was done on the SBMIE adult fish model. DEGs (sinomenine vs soybean meal groups) were enriched in GO terms related to the negative regulation of lymphocyte/leukocyte activation and alpha-beta T cell proliferation, as well as the regulation of lymphocyte migration. The KEGG pathways for glycolysis and insulin signaling indicated metabolic adjustments of α7nAchR mediated anti-inflammatory effect. To demonstrate the immune cells' response, in the SBMIE larva model, inflammatory gatherings of neutrophils, macrophages, and lymphocytes caused by soybean meal could be relieved significantly with the inclusion of sinomenine. This was consistent within the sinomenine group as CD4+ or Foxp3+ lymphocytes were found with a higher proportion at the base of mucosal folds, which may suggest the Treg population. Echoing, the sinomenine group's 16s sequencing result, there were fewer enteritis-related TM7, Sphingomonas and Shigella, but more Cetobacterium, which were related to glucose metabolism. Our findings indicate that sinomenine hydrochloride could be important in the prevention of fish foodborne enteritis at both immune and microbiota levels.

Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae.

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 µM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.

The function of zebrafish gpbar1 in antiviral response and lipid metabolism.

G protein-coupled bile acids receptor 1 (GPBAR1 or TGR5) has been widely studied as a metabolic regulator involved in bile acids synthesis, glucose metabolism and energy homeostasis. Several recent studies have shown that mammalian GPBAR1 is also involved in antiviral innate immune responses. However, the functions of piscine GPBAR1 in antibacterial or antiviral immune responses and lipid metabolism remain unclear. In the present study, we report the functional characterization of zebrafish gpbar1. Similar to mammalian GPBAR1, zebrafish gpbar1 contains similar domain composition, shows a dose-dependent activation by bile acids including INT777, LCA, DCA, CDCA and CA, and can be induced by viral infection. Compared with corresponding control groups, a significant antiviral activity against spring viremia of carp virus (SVCV) infection was observed in ZF4 cells overexpressing zebrafish gpbar1 with INT777 treatment, but not in ZF4 cells overexpressing zebrafish gpbar1 without INT777 treatment. The activation of zebrafish gpbar1 had no significant antibacterial effect against Edwardsiella piscicida infection in ZF4 cells in vitro. Transcriptome analysis revealed that zebrafish gpbar1 activation played a crucial role in activating RLR signaling pathway and inducing the production of ISGs, but not for bile acid biosynthesis and transportation. The co-occurrence analysis for antiviral-related and bile acids metabolism-related DEGs suggested a strong interaction among 2 bile acid receptors (gpbar1 and nr1h4), slco2b1 and the antiviral DEGs. The lipidomic analysis showed that zebrafish gpbar1 activation in ZF4 cells resulted a change of glycerophospholipids, but none of bile acids nor their derivatives, which were different from mammalian GPBAR1. All together, these results firstly demonstrate the conserved antiviral role of gpbar1 and its function in regulating glycerophospholipids metabolism in teleost.

Hypothalamic Pomc Neurons Innervate the Spinal Cord and Modulate the Excitability of Premotor Circuits.

Locomotion requires energy, yet animals need to increase locomotion in order to find and consume food in energy-deprived states. While such energy homeostatic coordination suggests brain origin, whether the central melanocortin 4 receptor (Mc4r) system directly modulates locomotion through motor circuits is unknown. Here, we report that hypothalamic Pomc neurons in zebrafish and mice have long-range projections into spinal cord regions harboring Mc4r-expressing V2a interneurons, crucial components of the premotor networks. Furthermore, in zebrafish, Mc4r activation decreases the excitability of spinal V2a neurons as well as swimming and foraging, while systemic or V2a neuron-specific blockage of Mc4r promotes locomotion. In contrast, in mice, electrophysiological recordings revealed that two-thirds of V2a neurons in lamina X are excited by the Mc4r agonist α-MSH, and acute inhibition of Mc4r signaling reduces locomotor activity. In addition, we found other Mc4r neurons in spinal lamina X that are inhibited by α-MSH, which is in line with previous studies in rodents where Mc4r agonists reduced locomotor activity. Collectively, our studies identify spinal V2a interneurons as evolutionary conserved second-order neurons of the central Mc4r system, providing a direct anatomical and functional link between energy homeostasis and locomotor control systems. The net effects of this modulatory system on locomotor activity can vary between different vertebrate species and, possibly, even within one species. We discuss the biological sense of this phenomenon in light of the ambiguity of locomotion on energy balance and the different living conditions of the different species.

An agonist for membrane progestin receptor (mPR) induces oocyte maturation and ovulation in zebrafish in vivo.

The maturation and ovulation of fish oocytes are well-characterized biological processes induced by progestins via coordination of nongenomic actions and genomic actions. Previously, we established a procedure that enables the induction of oocyte maturation and ovulation in live zebrafish by simple administration of the natural teleost maturation-inducing hormone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20ß-DHP) into the surrounding water. By this in vivo assay, the potencies of chemicals in inducing or preventing oocyte maturation and ovulation can be evaluated. The potencies of compounds in inducing ovulation of zebrafish oocytes also can be evaluated in vivo with improved in vitro assays. Here, we attempted to evaluate the effect of Org OD 02-0 (Org OD 02), a selective agonist for membrane progestin receptor (mPR), on fish oocyte maturation and ovulation with in vitro and in vivo assays. As reported previously, Org OD 02 triggered oocyte maturation in vitro. The same Org OD 02 triggered oocyte maturation within several hours in vivo. Surprisingly, Org OD 02 even induced ovulation both in in vivo and in vitro. Eggs from Org OD 02-induced ovulation could be fertilized by artificial insemination. The juveniles developed normally. These results indicated that Org OD 02 triggered physiological ovulation in live zebrafish. In summary, we have demonstrated the effect of Org OD 02 on fish oocyte maturation and ovulation in vitro and in vivo. The results suggested that Org OD 02 acted as an agonist not only of mPR but also of nuclear progesterone receptor (nPR).

The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Rapid effects of estradiol and its receptor agonists on object recognition and object placement in adult male zebrafish.

In recent years, there has been a growing appreciation that 17ß-estradiol (E2) can rapidly modulate learning and memory processes by binding to membrane estrogen receptors and cause the activation of a number of signaling cascades within the central nervous system. In this study, we sought to investigate the effects of post-training administration of E2 (100 ng/g, 1 µg/g, 10 µg/g) and involvement of the estrogen receptors (ERs) using selective ER agonists on the consolidation of object recognition (OR) and object placement memory (OP) in adult male zebrafish. The general activation of ERs with the highest E2 dose improved consolidation of memory in both learning tasks within 1.45 h of administration. Activation of classical ERs (ERα and ERß) improved consolidation of OR memory, but had no effect on fish performance in OP task. On the other hand, activation of G protein-coupled ER1 impaired and enhanced consolidation of OR and OP memories, respectively. Memory improvement in both tasks was accompanied by a marked up-regulation in the expression of genes encoding ionotropic and metabotropic glutamate receptors in a task-dependent manner. In contrast, the down-regulation in the expression of certain ionotropic glutamate receptors was observed in fish with impaired OR memory. Moreover, our study also revealed an increase in the transcript abundance of genes associated with synaptic protein synthesis (brain-derived neurotrophic factor, synaptophysin, and the mechanistic target of rapamycin). These results suggest that E2 may affect consolidation of memory in zebrafish likely through rapid changes in synaptic morphology and function.

Synaptic mitochondria regulate hair-cell synapse size and function.

Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca2+ through CaV1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca2+ influx through CaV1.3 channels initiates mitochondrial-Ca2+ (mito-Ca2+) uptake adjacent to ribbons. Block of mito-Ca2+ uptake in mature cells depresses presynaptic-Ca2+ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca2+ uptake coincides with spontaneous rises in presynaptic-Ca2+ influx. Spontaneous mito-Ca2+ loading lowers cellular NAD+/NADH redox and downregulates ribbon size. Direct application of NAD+ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca2+ couple to confer proper presynaptic function and formation.

Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish.

The Keap1-Nrf2-ARE system serves as a premier defence mechanism to curb oxidative stress, which remains as one of the major causes of ageing and pathogenesis in various diseases. Nrf2 is the principal master regulator of the cellular defence system, and its activation remains the prospective therapeutic approach against chronic diseases. One of the recent strategies is to disrupt Keap1-Nrf2 protein-protein interaction (PPI) that alters the docking of Keap1 with Nrf2 by compounds occupying a position in the Keap1 blocking the interface with Nrf2. In this study, we made an attempt to identify the compounds with anticancer, antioxidant and anti-inflammatory properties to disrupt Keap1a/b-Nrf2 PPI through in silico molecular docking in zebrafish. The phylogenetic analysis of Keap1 proteins revealed the existence of orthologous Keap1-Nrf2-ARE system in lower vertebrates that includes zebrafish. The DGR domains of zebrafish Keap1a and Keap1b were modelled with Modeller 9.19 using Keap1 of Mus musculus (PDB ID:5CGJ) as template. Based on the docking calculations, top hit compounds were identified to disrupt both Keap1a and Keap1b interaction with Nrf2 which include quercetin 3,4'-diglucoside, flavin adenine dinucleotide disodium salt hydrate, salvianolic acid A, tunicamycin and esculin. The LC50 of esculin in 3 dpf zebrafish larvae is 5 mmol/L, and the qRT-PCR results showed that esculin significantly increased the transcription of Nrf2 target genes-Gstpi, Nqo1, Hmox1a and Prdx1 in 3 dpf zebrafish larvae. These potential hits could serve as safer Nrf2 activators due to their non-covalent disruption of Keap1-Nrf2 PPI and be developed into efficacious preventive/therapeutic agents for various diseases.

Functional characterization of the cannabinoid receptors 1 and 2 in zebrafish larvae using behavioral analysis.

RATIONALE: The endocannabinoid system (ECS) comprises the cannabinoids anandamide and 2-arachidonoylglycerol and the cannabinoid receptors 1 and 2 (Cnr1 and Cnr2). The function of these receptors in relation to zebrafish larval behavior is poorly understood, even though the zebrafish larva has become a versatile animal model in biomedical research. OBJECTIVES: The objective of the present study is to characterize the function of Cnr1 and Cnr2 in relation to behavior in zebrafish. METHODS: Behavioral analysis of zebrafish larvae was performed using a visual motor response (VMR) test, which allows locomotor activity to be determined under basal conditions and upon a dark challenge. RESULTS: Treatment with the non-specific Cnr agonists WIN55,212-2 and CP55,940 resulted in a decrease in locomotion. This was observed for both basal and challenge-induced locomotion, although the potency for these two effects was different, which suggests different mechanisms of action. In addition, WIN55,212-2 increased the reaction time of the startle response after the dark challenge. Using the Cnr1 antagonist AM251 and a cnr1-/- mutant line, it was shown that the effects were mediated by Cnr1 and not Cnr2. Interestingly, administration of the antagonist AM251 alone does not have an effect on locomotion, which indicates that endogenous cannabinoid activity does not affect locomotor activity of zebrafish larvae. Upon repeated dark challenges, the WIN55,212-2 effect on the locomotor activity decreased, probably due to desensitization of Cnr1. CONCLUSIONS: Taken together, these results show that Cnr1 activation by exogenous endocannabinoids modulates both basal and challenge-induced locomotor activity in zebrafish larvae and that these behavioral effects can be used as a readout to monitor the Cnr1 responsiveness in the zebrafish larva model system.

Glucococorticoid receptor activation exacerbates aminoglycoside-induced damage to the zebrafish lateral line.

Aminoglycoside antibiotics have potent antibacterial properties but cause hearing loss in up to 25% of patients. These drugs are commonly administered in patients with high glucocorticoid stress hormone levels and can be combined with exogenous glucocorticoid treatment. However, the interaction of stress and aminoglycoside-induced hearing loss has not been fully explored. In this study, we investigated the effect of the glucocorticoid stress hormone cortisol on hair cells in the zebrafish lateral line as an important step toward understanding how physiological stressors modulate hair cell survival. We found that 24-hr cortisol incubation sensitized hair cells to neomycin damage. Pharmacological and genetic manipulation demonstrates that sensitization depended on the action of the glucocorticoid receptor but not the mineralocorticoid receptor. Blocking endogenous cortisol production reduced hair cell susceptibility to neomycin, further evidence that glucocorticoids modulate aminoglycoside ototoxicity. Glucocorticoid transcriptional activity was apparent in lateral line hair cells, suggesting a direct action of cortisol in these aminoglycoside-sensitive cells. Our work shows that the stress hormone cortisol can increase hair cell sensitivity to aminoglycoside damage, which highlights the importance of recognizing stress and the impacts of glucocorticoid signaling in both ototoxicity research and clinical practice.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

Conditional Overexpression of rtn4al in Muscle of Adult Zebrafish Displays Defects Similar to Human Amyotrophic Lateral Sclerosis.

The protein level of muscle-specific human NogoA is abnormally upregulated in amyotrophic lateral sclerosis (ALS) mice and patients. On the other hand, while the presence of miR-206 in muscle cells delays onset and death in ALS, the relationship between these two phenomena remains unclear. Mammalian NogoA protein, also known as Reticulon 4a (Rtn4a), plays an important role in inhibiting the outgrowth of motor neurons. Our group previously identified zebrafish rtn4al as the target gene of miR-206 and found that knockdown of miR-206 increases rtn4al mRNA and Rtn4al protein in zebrafish embryos. It can be concluded from these results that neurite outgrowth of motor neurons is inhibited by Rtn4a1, which is entirely consistent with overexpression of either human NogoA or zebrafish homolog Rtn4al. Since an animal model able to express NogoA/rtn4al at the mature stage is unavailable, we generated a zebrafish transgenic line, Tg(Zα:TetON-Rtn4al), which conditionally and specifically overexpresses Rtn4al in the muscle tissue. After doxycycline induction, adult zebrafish displayed denervation at neuromuscular junction during the first week, then muscle disintegration and split myofibers during the third week, and, finally, significant weight loss in the sixth week. These results suggest that this zebrafish transgenic line, representing the inducible overexpression of Rtn4a1 in muscle, may provide an alternative animal model with which to study ALS because it exhibits ALS-like phenotype.

Effects of calcium and estrogen on the development of the ceratohyal cartilage in zebrafish (Danio rerio) larvae upon embryo and maternal cadmium exposure.

The present study is to investigate the reason why the ceratohyal cartilage (CH) angle of zebrafish larvae were larger compared to the control group after their female parents were treated with cadmium (F-Cd). However, the CH angle was smaller compared to the control group when embryos were directly exposed to Cd2+ for 72 h (D-Cd). Results showed that calcium contents of larvae were lower than the control, but the transporter isoforms trpv4 and trpv6 mRNA expressions were significantly increased upon D-Cd treatment. Furthermore, external Ca2+ added during D-Cd treatment reveals that the CH angles of larvae did not appear significantly different compared to the control. On the other hand, E2 (17ß-estradiol) contents were higher around 1.9 folds in the ovaries of females; CH angle were over 25°, and Cd2+ contents were higher around 6 folds than the control group on larvae treated through F-Cd treatment; CH angles and E2 levels on larvae were higher than the control after the larvae were treated with 1.84 µM E2 (D-E2); Estradiol receptor (ER) isoforms ERß1 and ERα mRNA expressions significantly increased when 0 hpf embryos were either treated with D-E2 or D-Cd. According to the results, we suggested that the CH angle of larvae become larger upon F-Cd treatment due to maternal Cd2+ inducing E2 levels. However, the CH angle of larvae appeared to be smaller compared to the control upon D-Cd treatment. We suggested that the CH angle decreased due to the decrease of Ca2+ contents upon Cd2+ exposure.

Evaluation of the interaction between proliferation, oxidant-antioxidant status, Wnt pathway, and apoptosis in zebrafish embryos exposed to silver nanoparticles used in textile industry.

Antimicrobial textile products are developing rapidly as an important part of functional textiles. Silver nanoparticles (AgNPs) are nanotechnology products with antimicrobial properties. However, exposure to nanoparticles in daily life is an important issue for public health, still being updated. Aim was to evaluate the effects of AgNPs on the development of zebrafish embryos focusing on Wnt pathway, proliferation, oxidant-antioxidant status, and apoptosis. The expressions of ccnd1 and gsk3ß were determined by RT-PCR, whereas ß-catenin and proliferative cell antigen (PCNA) expressions were determined immunohistochemically. Lipid peroxidation, superoxide dismutase, and glutathione-S-transferase activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Oxidant status, apoptosis, immunohistochemical PCNA, and ß catenin staining increased, whereas ccnd1 and antioxidant enzyme activities decreased in AgNPs-exposed embryos in a dose-dependent manner. Our results indicate the interaction of possible mechanisms that may be responsible for the toxic effects of AgNPs in zebrafish embryos.

Cadmium exposure exacerbates severe hyperlipidemia and fatty liver changes in zebrafish via impairment of high-density lipoproteins functionality.

Cadmium (Cd) is a heavy metal with several toxicities that have destructive effect on most organ systems. However, its toxic effects on human lipoproteins are largely remained unknown especially in hyperlipidemic zebrafish model. Treatment of human high-density lipoprotein (HDL) with cadmium chloride (CdCl2, final 12 and 24µM) caused spontaneous formation of multimeric apoA-I as well as increased production of glycated extent products. Cd-HDL3 accelerated uptake of oxidized LDL (oxLDL) into macrophages and induced severe senescence in human dermal fibroblast (HDF) cells. Microinjection of Cd-HDL3 into zebrafish embryos resulted in acute embryonic toxicity with high mortality. Exposure of zebrafish embryos to water containing CdCl2 (final 12 and 24µM) caused early embryonic death along with increased production of oxidized products and impairment of skeletal development. Consumption of CdCl2 (12 and 24µM) by zebrafish for 4weeks resulted in severe elevation of plasma total cholesterol (TC) and triglyceride (TG) levels as well as cholesteryl ester (CE) transfer activity. Furthermore, consumption of CdCl2 resulted in acceleration of fatty liver changes and increased production of reactive oxygen species (ROS). In conclusion, CdCl2 caused structural modification of HDL3 and impaired the beneficial functions of HDL3, including anti-oxidation, anti-atherosclerosis, and anti-senescence effects. Consumption of CdCl2 also resulted in exacerbated hyperlipidemia and fatty liver changes in zebrafish via enhancement of cholesteryl ester transfer protein (CETP) activity.

Early Life Exposure to Low Levels of AHR Agonist PCB126 (3,3',4,4',5-Pentachlorobiphenyl) Reprograms Gene Expression in Adult Brain.

Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, nonembryotoxic levels of 3,3',4,4',5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior.

Clock1a affects mesoderm development and primitive hematopoiesis by regulating Nodal-Smad3 signaling in the zebrafish embryo.

Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.

Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish.

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10µM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.

Contribution of G protein-coupled estrogen receptor 1 (GPER) to 17ß-estradiol-induced developmental toxicity in zebrafish.

Exposure to 17ß-estradiol (E2) influences the regulation of multiple signaling pathways, and E2-mediated disruption of signaling events during early development can lead to malformations such as cardiac defects. In this study, we investigated the potential role of the G-protein estrogen receptor 1 (GPER) in E2-induced developmental toxicity. Zebrafish embryos were exposed to E2 from 2h post fertilization (hpf) to 76 hpf with subsequent transcriptional measurements of heart and neural crest derivatives expressed 2 (hand2), leucine rich repeat containing 10 (lrrc10), and gper at 12, 28 and 76 hpf. Alteration in the expression of lrrc10, hand2 and gper was observed at 12 hpf and 76 hpf, but not at 28 hpf. Expression of these genes was also altered after exposure to G1 (a GPER agonist) at 76 hpf. Expression of lrrc10, hand2 and gper all coincided with the formation of cardiac edema at 76 hpf as well as other developmental abnormalities. While co-exposure of G1 with G36 (a GPER antagonist) rescued G1-induced abnormalities and altered gene expression, co-exposure of E2 with G36, or ICI 182,780 (an estrogen receptor antagonist) did not rescue E2-induced cardiac deformities or gene expression. In addition, no effects on the concentrations of downstream ER and GPER signaling molecules (cAMP or calcium) were observed in embryo homogenates after E2 treatment. These data suggest that the impacts of E2 on embryonic development at this stage are complex and may involve multiple receptor and/or signaling pathways.