Metabolism In Zebrafish Research Articles

Carbon Nanotubes Induce Metabolomic Profile Disturbances in Zebrafish: NMR-Based Metabolomics Platform.

The present study aims to investigate the metabolic effects of single-walled carbon nanotubes (SWCNT) on zebrafish (Danio rerio) using 1H nuclear magnetic resonance (1H-NMR) spectroscopy. However, there is no significant information available regarding the characterization of organic molecules, and metabolites with SWCNT exposure. Noninvasive biofluid methods have improved our understanding of SWCNT metabolism in zebrafish in recent years. Here, we used targeted metabolomics to quantify a set of metabolites within biological systems. SWCNT at various concentrations was given to zebrafish, and the metabolites were extracted using two immiscible solvent systems, methanol and chloroform. Metabolomics profiling was used in association with univariate and multivariate data analysis to determine metabolomic phenotyping. The metabolites, malate, oxalacetate, phenylaniline, taurine, sn-glycero-3-phosphate, glycine, N-acetyl mate, lactate, ATP, AMP, valine, pyruvate, ADP, serine, niacinamide are significantly impacted. The metabolism of amino acids, energy and nucleotides are influenced by SWCNT which might indicate a disturbance in metabolic reaction networks. In conclusion, using high-throughput analytical methods, we provide a perspective of metabolic impacts and the underlying associated metabolic pathways.

Effect of dark environment on intestinal flora and expression of genes related to liver metabolism in zebrafish (Danio rerio)

To explore the effects of dark environment on intestinal flora and expression of genes related to liver metabolism in zebrafish, a total of 60 zebrafish were fed for 21 days (24 h dark treatments or 14/10 h light/dark cycle), and the influence of dark environment on gut microbes and liver gene expression was studied using sequencing analysis of intestinal flora and liver. The results showed that the body weight of fish was significantly increased in the dark group than that in the control group (P < 0.05). Compared with the control group, dark environment treatment changed the composition of dominant flora, increased the abundance of unconventional bacteria and reduced probiotics in the intestine of zebrafish. Of these, the ratio of Bacteroidetes to Firmicutes in the intestine was reduced. The genome expression of the liver showed significant changes, and liver metabolites were also affected. Meanwhile, dark environment decreased gene expression associated with changes in blood glucose, lipid metabolism and immunization. Dark environment also caused liver steatosis as observed by histological study. This study shows that dark environment treatment has an important impact on liver metabolism and intestinal microbes in zebrafish.

Dietary Nitrate Supplementation Alters Protein and Lipid Metabolism in Zebrafish (Danio rerio) Livers

ObjectivesDietary nitrate supplementation shows protective effects against cardio-metabolic disease, decreases pulmonary oxygen uptake, and improves exercise performance in animal models and humans. However, the biological effect of nitrate on energy metabolism in the liver is not well understood. The objective of this study was to elucidate changes in liver metabolism associated with nitrate treatment and exercise. MethodsFish were exposed to sodium nitrate (606.9 mg/L), or control water, for 21 days and analyzed at intervals during a strenuous exercise test. We utilized untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine the effect of nitrate treatment and exercise on the liver metabolome. We measured gene expression of 31 genes linked to energy metabolism and redox signaling. ResultsIn the absence of exercise, nitrate treatment upregulated expression of genes central to nutrient sensing (pgc1a and sirt3), protein synthesis (mtor) and purine metabolism (pnp5a and ampd1) and downregulated expression of genes involved in mitochondrial fat oxidation (acaca, cpt2 and hadh). Upregulation of these genes was associated with an increased abundance of metabolites involved in endogenous nitric oxide metabolism, dopamine biosynthesis, branched chain amino acid metabolism, and lipid metabolism in nitrate-treated livers at rest, compared to rested controls. As expected, the availability of these metabolites was diminished in nitrate-treated livers relative to rested controls. We found no significant change in gene of metabolites directly linked to glycolysis. ConclusionsThe main novel finding of this study was that sub-chronic nitrate treatment altered dopamine biosynthesis, protein synthesis and lipid metabolism in zebrafish liver without exercise. This is significant because dietary nitrate is emerging as an interesting therapeutic modality for metabolic syndrome and non-alcoholic fatty liver disease by preventing lipid accumulation in the liver. Funding SourcesCelia Strickland and G. Kenneth Austin III Endowment and National Institutes of Health.

New insights into bisphenols induced obesity in zebrafish (Danio rerio): Activation of cannabinoid receptor CB1

Bisphenols (BPs), as widely used plastic additives, penetrate into our daily lives. BPs are considered endocrine disruptors and could potentially induce obesity. In this study, the effects of bisphenol A (BPA) and tetrabromobisphenol A (TBBPA) on food intake and lipid metabolism in zebrafish were determined. Moreover, the impact of BPA and TBBPA on the endocannabinoid system (ECS) of zebrafish was further explored by metabolomics, transcriptomics, and molecular docking analysis. Here we show that exposure to BPA and TBBPA at concentrations commonly found in the environment (20, 100, and 500 μg/L) led to hyperphagia and obesity in adult male zebrafish. Metabolomics and histopathological analysis revealed significant lipid accumulation in the liver of zebrafish exposed to BPA and TBBPA. The expression of ECS-related genes, in conjunction with RNA-Seq results, further indicated that BPA and TBBPA increased appetite and induced obesity by activating cannabinoid receptor type 1(CB1). Furthermore, molecular docking revealed that six representative BPs including BPA and TBBPA could bind to the CB1 receptor. Collectively, these findings indicate that CB1 may be a potential target for BPs to induce obesity.

Unraveling the mechanism of long-term bisphenol S exposure disrupted ovarian lipids metabolism, oocytes maturation, and offspring development of zebrafish

Bisphenol S (BPS) acts as a xenoestrogen and disturbs the female reproductive system; however, the underlying mechanism has not been elucidated. In this study, the effect of chronic BPS exposure (1 μg/L and 100 μg/L) on ovarian lipid metabolism in zebrafish was investigated to determine its influence on adult reproductive capacity and offspring development. The results showed that long-term (240 days) exposure to BPS induced lipid accumulation in the ovaries by promoting the transport of more lipids from the circulation to the ovaries and by upregulating triacylglycerol synthesis-related genes. Significantly increased expression of cpt2, acadm, acadl, and pparα, which are involved in β-oxidation in the ovarian mitochondria, indicated that more energy was provided for oocyte maturation in exposed zebrafish ovaries. Thus, the proportion of full-grown stage oocytes in ovaries and egg reproduction were elevated at an accelerated rate, which earlier than normal reproductive cycle (8–10 days posts pawning). Moreover, the maternally BPS-exposed F1 embryos (2 h post-spawning, hpf) showed higher neutral lipid levels, impaired hatching capacity, and increased occurrence of larval deformities. All these findings demonstrated that stimulated lipid synthesis and β-oxidation in zebrafish ovaries significantly contribute to BPS-induced oocyte precociousness with subsequent effects on the development of unexposed offspring. This study provides new insight into the impact of xenoestrogens on oviparous reproduction in females and offspring development from the perspective of ovarian lipid metabolism.

Teneurin C-Terminal Associated Peptide (TCAP)-3 Increases Metabolic Activity in Zebrafish

Teneurin C-terminal associated peptides (TCAP), bioactive peptides located on the C-terminal end of teneurin proteins, have been shown to regulate stress axis functions due to the high conservation between TCAP and corticotropin releasing factor (CRF). Additionally, recent work demonstrated that TCAP can increase metabolism in rats via glucose metabolism. These metabolic actions are not well described in other organisms, including teleosts. Here we investigated the expression of a tcap isoform, tcap-3, and the potential role of TCAP-3 as a regulator of metabolism across zebrafish life-stages. Using pcr-based analyses, tcap-3 appears to be independently transcribed, in relation to teneurin-3, in muscle tissue of adult zebrafish. Resazurin, respirometry chambers, and mitochondrial metabolism analyses were used to study the metabolic effects of synthetic rainbow trout TCAP-3 (rtTCAP-3) in larval and adult zebrafish. Overall, metabolic activity was enhanced by 48 h of rtTCAP-3 treatment in larvae (bath immersion) and adults (intraperitoneal injections). This metabolic activity increase was due to mitochondrial uncoupling, as mitochondrial respiration increase by rtTCAP-3 was due to proton leak. Additionally, rtTCAP-3 protected larval fish from reduced metabolic activity induced by low temperatures. Subsequently, rtTCAP-3 increased metabolic output in adult zebrafish subjected to accelerated swimming speeds, demonstrating the potent role of rtTCAP-3 in zebrafish metabolism regulation during metabolic challenges. Collectively, these results demonstrate the conserved roles for rtTCAP-3 as a metabolic activator in zebrafish.

Zebrafish as an Emerging Model for Dyslipidemia and Associated Diseases.

Dyslipidemia related diseases such as hyperlipidemia and atherosclerosis are the leading cause of death in humans. While cellular and molecular basis on the pathophysiology of dyslipidemia has been extensively investigated over decades, we still lack comprehensive understanding on the etiology of dyslipidemia due to the complexity and the innate multimodality of the diseases. While mouse has been the model organism of choice to investigate the pathophysiology of human dyslipidemia, zebrafish, a small freshwater fish which has traditionally used to study vertebrate development, has recently emerged as an alternative model organism. In this review, we will provide comprehensive perspective on zebrafish as a model organism for human dyslipidemia; we will discuss the attributes of zebrafish as a model, and compare the lipid metabolism in zebrafish and humans. In addition, we will summarize current landscape of zebrafish-based dyslipidemia research.

Comparative Redox-active Properties of Asparagine Metabolism in Zebrafish and Mouse Fibroblasts

Comparative Redox-active Properties of Asparagine Metabolism in Zebrafish and Mouse Fibroblasts

Early leucine programming on protein utilization and mTOR signaling by DNA methylation in zebrafish (Danio rerio)

BackgroundEarly nutritional programming affects a series of metabolism, growth and development in mammals. Fish also exhibit the developmental plasticity by early nutritional programming. However, little is known about the effect of early amino acid programming on growth and metabolism.MethodsIn the present study, zebrafish (Danio rerio) was used as the experimental animal to study whether early leucine stimulation can programmatically affect the mechanistic target of rapamycin (mTOR) signaling pathway, growth and metabolism in the later life, and to undercover the mechanism of epigenetic regulation. Zebrafish larvas at 3 days post hatching (dph) were raised with 1.0% leucine from 3 to 13 dph during the critical developmental stage, then back to normal water for 70 days (83 dph).ResultsThe growth performance and crude protein content of zebrafish in the early leucine programming group were increased, and consistent with the activation of the mTOR signaling pathway and the high expression of genes involved in the metabolism of amino acid and glycolipid. Furthermore, we compared the DNA methylation profiles between the control and leucine-stimulated zebrafish, and found that the methylation levels of CG-differentially methylated regions (DMGs) and CHH-DMGs of genes involved in mTOR signaling pathway were different between the two groups. With quantitative PCR analysis, the decreased methylation levels of CG type of Growth factor receptor-bound protein 10 (Grb10), eukaryotic translation initiation factor 4E (eIF4E) and mTOR genes of mTOR signaling pathway in the leucine programming group, might contribute to the enhanced gene expression.ConclusionsThe early leucine programming could improve the protein synthesis and growth, which might be attributed to the methylation of genes in mTOR pathway and the expression of genes involved in protein synthesis and glycolipid metabolism in zebrafish. These results could be beneficial for better understanding of the epigenetic regulatory mechanism of early nutritional programming.

Peroxisomal proliferator-activated receptor α-b deficiency induces the reprogramming of nutrient metabolism in zebrafish.

The pparab subtype in zebrafish is much more highly expressed in tissues with high oxidative activity than pparaa. The pparab deficiency in zebrafish reduces fatty acid β-oxidation both in liver and muscle, illustrating its functional homology as a mammalian peroxisome proliferator-activated receptor α (PPARα). pparab deficiency promotes metabolic reprogramming by increasing glucose utilization and inhibiting amino acid breakdown. The present study brings new insights into the comprehensive regulatory roles of PPARα in the cellular fuel selection and provides a valuable animal model for PPARα studies from a viewpoint of comparative physiology. Dysfunction of lipid metabolism is involved in the pathogenesis of several chronic metabolic diseases. Peroxisome proliferator-activated receptor α (PPARα) is essential for normal metabolic homeostasis and, in particular, for the regulation of fatty acid β-oxidation (FAO). However, little is known about its regulation roles in systemic nutrient metabolism. To explore the underlying modulation role of PPARα in metabolic homeostasis, we generated a pparab-knockout zebrafish (Danio rerio) model. The pparab mutants demonstrated lower expression of key enzymes involved in FAO, as well as lower mitochondrial and peroxisomal FAO in tissues, which was associated with lipid accumulation in liver and visceral mass. Conversely, glucose utilization was higher because they demonstrated lower blood glucose and tissue glycogen concentrations, as well as activation of the phosphoinositide 3-kinase/AKT pathway. In addition, pparab-deficient zebrafish demonstrated activation of AKT/mammalian target of rapamycin signalling and higher protein content, implying greater protein synthesis and/or lower amino acid breakdown. These data clearly revealed that pparab deletion reduces FAO but increases glucose utilization and protein deposition to maintain energy homeostasis. The present study provides new insights into the comprehensive regulatory role of PPARα in systemic energy metabolism in fish, and this pparab-deficient zebrafish also constitutes a valuable model for investigating the functions of PPARα in mammals from comparative physiology aspects.

UPLC-TOF-MS/MS metabolomics analysis of zebrafish metabolism by spirotetramat

Spirotetramat, a member of tetronic and tetramic acid derivatives, is a unique insecticide and acaricide. Although the effect on zebrafish embryos lipid biosynthesis of spirotetramat has been characterized, the energy metabolism and toxic effect mechanism warrant further investigation. To investigate the toxic mechanism of spirotetramat on energy metabolism, zebrafish embryos were exposed to 100, 500 and 1000 µg/L of spirotetramat for 4 days. Untargeted metabolomics showed the synthesis and degradation of ketone pathway metabolites (R)-3-Hydroxybutyric acid and Acetoacetate significantly decreased, as well as increasing the abundance of Anti-Acetyl Coenzyme A Carboxylase protein (ACC1). Down-regulation of the genes related to ß-oxidation and the tricarboxylic acid cycle in the embryos show decreased energy metabolism. Carnitine palmitoyltransferase 1 (CPT- I) significantly decreased while citrate synthase (CS) significantly increased. Additionally, mitochondrial lesions in embryos were found using electron microscopy. Our study provides novel and robust perspectives, which show that spirotetramat treatment in embryos leads to metabolic disturbances that adversely affect cellular energy homeostasis.

Fat-1 transgenic zebrafish are protected from abnormal lipid deposition induced by high-vegetable oil feeding.

High dietary concentration of vegetable oil, particularly those rich in n-6 polyunsaturated fatty acids (PUFAs), can induce negative physiological effects including excessive lipid deposition in teleost fish. Omega-3 desaturase (Fat-1) of Caenorhabditis elegans is able to convert n-6 PUFAs to n-3 PUFAs and thus induces a low n-6/n-3 PUFAs ratio alleviating lipid deposition. In this study, we investigated the effects of dietary n-6 PUFAs on lipid metabolism of fat-1 transgenic zebrafish (Tg:fat-1), to explore the role of fat-1 in fish lipid metabolism. We first generated Tg:fat-1 zebrafish and assayed the effects of a low-fat diet (LFD) and a high-fat diet (HFD) prepared from soybean oil. Wild type zebrafish (WT) fed with HFD (HFD-WT) exhibited increased obesity and lipid deposition, especially in the abdominal cavity and liver. These defects were absent from HFD-Tg:fat-1. For each diet group, Tg:fat-1 exhibited significantly decreased levels of almost all hepatic lipid classes compared with WT. Expression levels of lipid synthesis-related genes and lipid deposition-related genes were markedly lower in the liver of HFD-Tg:fat-1 compared with HFD-WT. In contrast, the steatolysis-related genes significantly upregulated in HFD-Tg:fat-1. Then expression profiles of mitochondrial energy metabolism-related genes and ATP contents in the livers from LFD-WT, LFD-Tg:fat-1, HFD-WT, and HFD-Tg:fat-1 were determined. Our findings suggest that fat-1 protects fish from abnormal lipid deposition induced by high-vegetable oil feeding, through endogenously converting n-6 PUFAs to n-3 PUFAs. KEY POINTS: • fat-1 transgenic zebrafish (Tg:fat-1) can endogenously convert n-6 PUFAs to n-3 PUFAs. • Tg:fat-1 avoid serious abnormal lipid deposition induced by high-vegetable oil feeding. • fat-1 transgenosis effectively improved lipid metabolism and mitochondrial energy metabolism in zebrafish.

Systemic effect of dietary lipid levels and α-lipoic acid supplementation on nutritional metabolism in zebrafish (Danio rerio): focusing on the transcriptional level.

Considering the excessive lipid accumulation status caused by the increased dietary lipid intake in farmed fish, this study aimed to investigate the systemic effect of dietary lipid levels and α-lipoic acid supplementation on nutritional metabolism in zebrafish. A total of 540 male zebrafish (0.17g) were fed with normal (CT) and high lipid level (HL) diets for 6weeks, then fed on 1000mg/kg α-lipoic acid supplementation diets for the second 6weeks. HL diets did not affect whole fish protein content, but increased ASNS expression (P <0.05). Dietary α-lipoic acid increased whole fish protein content, and decreased the expressions of protein catabolism-related genes in muscle of high lipid level groups (P <0.05). Furthermore, HL diets increased the whole fish lipid content and the expressions of gluconeogenesis and lipogenesis-related genes (P <0.05), and α-lipoic acid counteracted these effects and decreased the whole fish triglyceride and cholesterol contents and expressions of lipogenesis-related genes, with the enhanced expressions of lipolytic genes, especially in high lipid groups (P <0.05). HL diets did not affect hepatocyte mitochondrial quantity or the mRNA expressions of mitochondrial biogenesis and electron transport chain-related genes; they were significantly increased by dietary α-lipoic acid (P <0.05). These results indicated that high dietary lipid promotes lipid accumulation, while α-lipoic acid increases protein content in association of enhanced lipid catabolism. Thus, dietary α-lipoic acid supplementation could reduce lipid accumulation under high lipid, which provides a promising new approach in solving the problem of lipid accumulation in farmed fish.

Effects of difenoconazole on hepatotoxicity, lipid metabolism and gut microbiota in zebrafish (Danio rerio)

In current study, larvae and adult zebrafish were exposed to difenoconazole to assess its effect on hepatotoxicity, lipid metabolism and gut microbiota. Results demonstrated that difenoconazole could induce hepatotoxicity in zebrafish larvae and adult, 0.400, 1.00, 2.00 mg/L difenoconazole caused yolk retention, yolk sac edema or liver degeneration after embryos exposure for 120 h, hepatocyte vacuolization and neoplasm necrosis were observed in adult liver after 0.400 mg/L difenoconazole exposure for 21 d. RNA sequencing showed that the 41 and 567 differentially expressed genes in zebrafish larvae and liver induced by 0.400 mg/L difenoconazole, were concentrated in pathways related to protein digestion and absorption, pancreatic secretion, steroid biosynthesis, and different metabolic pathways including galactose or sugar metabolism. Difenoconazole exposure caused lipid accumulation in larval yolk sac, and the elevated triglyceride (TG), malondialdehyde (MDA) and reactive oxygen species (ROS) levels in larvae and liver, which further confirmed the lipid metabolism disorders induced by difenoconazole. The results further showed that difenoconazole increased the abundance of gut microbiota such as Firmicutes, Aeromonas, Enterobacteriaceae and Bacteroides, further suggested that gut microbiota might participate in lipid metabolism and hepatotoxicity during zebrafish development. These findings advanced the field of the difenoconazole-induced developmental toxicity in larvae and adult zebrafish, and the imbalance of gut microbiota provided the plausible mode of action for the liver damage and disordered lipid metabolism in zebrafish.

Quantitative Phosphoproteomic Analysis Reveals the Regulatory Networks of Elovl6 on Lipid and Glucose Metabolism in Zebrafish.

Elongation of very long-chain fatty acids protein 6 (Elovl6) has been reported to be associated with clinical treatments of a variety of metabolic diseases. However, there is no systematic and comprehensive study to reveal the regulatory role of Elovl6 in mRNA, protein and phosphorylation levels. We established the first knock-out (KO), elovl6−/−, in zebrafish. Compared with wild type (WT) zebrafish, KO presented significant higher whole-body lipid content and lower content of fasting blood glucose. We utilized RNA-Seq, tandem mass tag (TMT) labeling-based quantitative technology and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to perform the transcriptomic, proteomic and phosphoproteomic analyses of livers from WT and elovl6−/− zebrafish. There were 734 differentially expressed genes (DEG) and 559 differentially expressed proteins (DEP) between elovl6−/− and WT zebrafish, identified out of quantifiable 47251 transcripts and 5525 proteins. Meanwhile, 680 differentially expressed phosphoproteins (DEPP) with 1054 sites were found out of quantifiable 1230 proteins with 3604 sites. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis of the transcriptomic and proteomic data further suggested that the abnormal lipid metabolism and glucose metabolism in KO were mainly related to fatty acid degradation and biosynthesis, glycolysis/gluconeogenesis and PPAR signaling pathway. Based on phosphoproteomic analyses, some kinases critical for lipid metabolism and glucose metabolism, including ribosomal protein S6 kinase (Rps6kb), mitogen-activated protein kinase14 (Mapk14) and V-akt murine thymoma viral oncogene homolog 2-like (Akt2l), were identified. These results allowed us to catch on the regulatory networks of elovl6 on lipid and glucose metabolism in zebrafish. To our knowledge, this is the first multi-omic study of zebrafish lacking elovl6, which provides strong datasets to better understand many lipid/glucose metabolic risks posed to human health.

Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish.

Phoenixin is a 20-amino acid peptide (PNX-20) cleaved from the small integral membrane protein 20 (SMIM20), with multiple biological roles in mammals. However, its role in nonmammalian vertebrates is poorly understood. This research aimed to determine whether PNX-20 influences feeding and metabolism in zebrafish. The mRNAs encoding SMIM20 and its putative receptor, super conserved receptor expressed in brain 3 (SREB3), are present in both central and peripheral tissues of zebrafish. Immunohistochemical analysis confirmed the presence of PNX-like immunoreactivity in the gut and in zebrafish liver (ZFL) cell line. We also found that short-term fasting (7 days) significantly decreased smim20 mRNA expression in the brain, gut, liver, gonads, and muscle, which suggests a role for PNX-20 in food intake regulation. Indeed, single intraperitoneal injection of 1,000 ng/g body wt PNX-20 reduced feeding in both male and female zebrafish, likely in part by enhancing hypothalamic cart and reducing hypothalamic/gut preproghrelin mRNAs. Furthermore, the present results demonstrated that PNX-20 modulates the expression of genes involved in glucose transport and metabolism in ZFL cells. In general terms, such PNX-induced modulation of gene expression was characterized by the upregulation of glycolytic genes and the downregulation of gluconeogenic genes. A kinetic study of the ATP production rate from both glycolytic and mitochondrial pathways demonstrated that PNX-20-treated ZFL cells exhibited significantly higher ATP production rate associated with glycolysis than control cells. This confirms a positive role for PNX-20 on glycolysis. Together, these results indicate that PNX-20 is an anorexigen with important metabolic roles in zebrafish.

Modulation of steroid metabolism and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to triadimefon

The widely used fungicide triadimefon (TDF) has been detected in aquatic environments, and appears to disrupt steroid homeostasis; however, the toxic effects on fish reproduction triggered by TDF via the key receptor signaling pathways remain largely unknown. The present study showed that TDF (0.069, 0.138, 0.690 mg/L) exposure not only caused disordered germ cell maturation, but also decreased spawned egg production. In order to better understand this reproductive inhibition, we investigated the effects of TDF based on quantitative PCR, Western blot and mass spectrometry methodology in zebrafish. Due to the preferential accumulation of TDF in the liver, a general pattern of up-regulation of genes involved in biotransformation pathway was observed. A significant increase in abcb4 expression appeared to be responsible for TDF excretion. TDF-induced receptors (AhR2 and PXR) changed many genes involved in steroid metabolism, and subsequent disruptions in steroid homeostasis, which might be the key biological pathway in TDF reproductive toxicity. However, due to the different metabolic demands, the transcript profiles involved in steroid metabolism in zebrafish exhibited a sex-specific expression pattern. For example, the increase in gene expression of ahr2 was accompanied by a reduction in the rate of E2 biosynthesis resulting from the diminished cyp19a1a expression, and in turn led to down-regulation of esr1 and vtg1 in the liver, supporting the anti-estrogenic effect of TDF in male fish. In contrast, the increase in E2 production was accompanied by an increase in Esr1 protein expression caused by TDF and paralleled the increase in ahrr1 expression, suggesting that TDF may induce estrogenic activity through AhR-ER interactions in females. In addition, over-induction of cyp3a65 activity mediated through pxr, which helped to accelerate the transformation from TDF to triadimenol in the liver, appeared to elevate T metabolite rate in females. The down-regulation of fshβ transcript in males further suggested that TDF might adversely affect normal gametogenesis and induce reproductive toxicity.

Multiple Stressors in the Environment: The Effects of Exposure to an Antidepressant (Venlafaxine) and Increased Temperature on Zebrafish Metabolism.

Aquatic organisms are continuously exposed to multiple environmental stressors working cumulatively to alter ecosystems. Wastewater-dominated environments are often riddled by a myriad of stressors, such as chemical and thermal stressors. The objective of this study was to examine the effects of an environmentally relevant concentration of a commonly prescribed antidepressant, venlafaxine (VFX) [1.0 μg/L], in addition to a 5°C increase in water temperature on zebrafish metabolism. Fish were chronically exposed (21 days) to one of four conditions: (i) 0 μg/L VFX at 27°C; (ii) 1.0 μg/L VFX at 27°C; (iii) 0 μg/L VFX at 32°C; (iv) 1.0 μg/L VFX at 32°C. Following exposure, whole-body metabolism was assessed by routine metabolic rate (RMR) measurements, whereas tissue-specific metabolism was assessed by measuring the activities of major metabolic enzymes in addition to glucose levels in muscle. RMR was significantly higher in the multi-stressed group relative to Control. The combination of both stressors resulted in elevated pyruvate kinase activity and glucose levels, while lipid metabolism was depressed, as measured by 3-hydroxyacyl CoA dehydrogenase activity. Citrate synthase activity increased with the onset of temperature, but only in the group treatment without VFX. Catalase activity was also elevated with the onset of the temperature stressor, however, that was not the case for the multi-stressed group, potentially indicating a deleterious effect of VFX on the anti-oxidant defense mechanism. The results of this study highlight the importance of multiple-stressor research, as it able to further bridge the gap between field and laboratory studies, as well as have the potential of yielding surprising results that may have not been predicted using a conventional single-stressor approach.

Predicting Metabolism from Gene Expression in an Improved Whole-Genome Metabolic Network Model of Danio rerio.

Zebrafish is a useful modeling organism for the study of vertebrate development, immune response, and metabolism. Metabolic studies can be aided by mathematical reconstructions of the metabolic network of zebrafish. These list the substrates and products of all biochemical reactions that occur in the zebrafish. Mathematical techniques such as flux-balance analysis then make it possible to predict the possible metabolic flux distributions that optimize, for example, the turnover of food into biomass. The only available genome-scale reconstruction of zebrafish metabolism is ZebraGEM. In this study, we present ZebraGEM 2.0, an updated and validated version of ZebraGEM. ZebraGEM 2.0 is extended with gene-protein-reaction associations (GPRs) that are required to integrate genetic data with the metabolic model. To demonstrate the use of these GPRs, we performed an in silico genetic screening for knockouts of metabolic genes and validated the results against published in vivo genetic knockout and knockdown screenings. Among the single knockout simulations, we identified 74 essential genes, whose knockout stopped growth completely. Among these, 11 genes are known have an abnormal knockout or knockdown phenotype in vivo (partial), and 41 have human homologs associated with metabolic diseases. We also added the oxidative phosphorylation pathway, which was unavailable in the published version of ZebraGEM. The updated model performs better than the original model on a predetermined list of metabolic functions. We also determined a minimal feed composition. The oxidative phosphorylation pathways were validated by comparing with published experiments in which key components of the oxidative phosphorylation pathway were pharmacologically inhibited. To test the utility of ZebraGEM2.0 for obtaining new results, we integrated gene expression data from control and Mycobacterium marinum-infected zebrafish larvae. The resulting model predicts impeded growth and altered histidine metabolism in the infected larvae.

Pharmacokinetic study of xylazine in a zebrafish water tank, a human-like surrogate, by liquid chromatography Q-Orbitrap mass spectrometry

This study aims to investigate a zebrafish (Danio Rerio) water tank (ZWT) as an alternative model for the study of the metabolism of xylazine. The ZWT approach for the study of metabolism consisted of two aquariums, where 18 fish and xylazine were added into the first tank. The second one, with 18 fish without drug, served as a negative control. The samples were submitted to a comprehensive analytical method developed for doping control purposes by liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) operating in five different acquisition modes. Glycoconjugate metabolites were evaluated indirectly by extracting the samples with and without the enzymatic hydrolysis step using β-glucuronidase. In total, 11 phase I and II metabolites were detected and characterized, of which four were previously described for humans and two for horses, and five metabolites were described for the first time. The main metabolites were 4-hydroxylated (M2) and oxygenated (M1) derivatives. Both metabolites were suggested as analytical targets for xylazine misuse. Around 79% of para- and meta-hydroxylated derivatives were in glycoconjugate form, whereas for oxo-hydroxylated and sulfone-hydroxylated derivatives of xylazine, around 83% and 70% were metabolized to the glycoconjugate form, respectively. Xylazine was the subject of extensive metabolism in zebrafish. 4-Hydroxylated (M2) and oxygenated (M1) derivatives were the most abundant phase I metabolites as the main targets for doping control.