In Vivo Toxicity Evaluation of Sugar Adulterated Heterotrigona itama Honey Using Zebrafish Model.

Honey is prone to be adulterated through mixing with sugars, cheap and low-quality honey, and other adulterants. Consumption of adulterated honey may cause several health issues such as weight gain, diabetes, and liver and kidney dysfunction. Therefore, studying the impact of consumption of adulterated honey on consumers is critical since there is a lack of study in this field. Hence, the aims of this paper were: (1) to determine the lethal concentration (LC50) of adulterated honey using zebrafish embryo, (2) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish, (3) to determine the effects of adulterated honey on histological changes of zebrafish, and (4) to screen the metabolites profile of adulterated honey by using zebrafish blood serum. The LC50 of Heterotrigona itama honey (acacia honey) and its sugar adulterants (light corn sugar, cane sugar, inverted sugar, and palm sugar in the proportion of 1-3% (w/w) from the total volume) was determined by the toxicological assessment of honey samples on zebrafish embryos (different exposure concentrations in 24, 48, 72, and 96 h postfertilization (hpf)). Pure H. itama honey represents the LC50 of 34.40 ± 1.84 (mg/mL) at 96 hpf, while the inverted sugar represents the lowest LC50 (5.03 ± 0.92 mg/mL) among sugar adulterants. The highest concentration (3%) of sugar adulterants were used to study the toxicology of adulterated honey using adult zebrafish in terms of acute, prolong-acute, and sub-acute tests. The results of the LD50 from the sub-acute toxicity test of pure H. itama honey was 2.33 ± 0.24 (mg/mL). The histological studies of internal organs showed a lesion in the liver, kidney, and spleen of adulterated treated-honey groups compared to the control group. Furthermore, the LC-MS/MS results revealed three endogenous metabolites in both the pure and adulterated honey treated groups, as follows: (1) S-Cysteinosuccinic acid, (2) 2,3-Diphosphoglyceric acid, and (3) Cysteinyl-Tyrosine. The results of this study demonstrated that adulterated honey caused mortality, which contributes to higher toxicity, and also suggested that the zebrafish toxicity test could be a standard method for assessing the potential toxicity of other hazardous food additives. The information gained from this research will permit an evaluation of the potential risk associated with the consumption of adulterated compared to pure honey.

Meis1, Hi1α, and GATA1 are integrated into a hierarchical regulatory network to mediate primitive erythropoiesis.

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.

Transcriptomic analysis reveals potential mechanisms of toxicity in a combined exposure to dibutyl phthalate and diisobutyl phthalate in zebrafish (Danio rerio) ovary.

Phthalate esters (PAEs), which are notable plasticizers, can be prolific contaminants in aquatic environments, and have been shown to induce reproductive toxicity. However, the studies concerning their toxicity towards aquatic species are based on individual chemicals, and the combined toxicity of PAEs to aquatic organisms remains unclear. The aim of this study was to explore the potential toxicity mechanisms associated with combined exposure to dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) in adult female zebrafish ovaries. Zebrafish were exposed to DBP, DiBP and their mixtures for 30 days, and their effects on ovarian histology, plasma sex hormones and ovarian transcriptomics were investigated. Plasma estradiol (E2) levels were significantly decreased by 38.9% in the DBP-1133 exposure group and 41.0% in the DiBP-1038 exposure group. The percentage of late/mature oocytes was also significantly decreased by 17.3% under DBP-1133 exposure and 16.2% under DiBP-1038 exposure, while that under combined exposure was not significantly affected. Nevertheless, transcriptome sequencing revealed 2564 differentially expressed genes (DEGs) in zebrafish ovaries after exposure to the mixtures. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were involved in the neuroactive ligand-receptor interaction, GnRH, progesterone-mediated oocyte maturation, oocyte meiosis and steroid hormone biosynthesis signaling pathways. These results revealed that combined exposure exerts potential reproductive toxicity at the molecular level.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure.

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.

Chronic exposure to environmental concentrations of phenanthrene impairs zebrafish reproduction.

Phenanthrene (PHE) is a tricyclic polycyclic aromatic hydrocarbon which distributed extensively in the aquatic environment. However, the knowledge about its impact on fish reproduction is still limited, particularly under a chronic exposure regime. In this study, we exposed zebrafish (Danio rerio) embryos to environmentally relevant concentrations (0.2, 1.0, and 5.0 µg/L) of PHE for 4 months and assessed the impact on reproduction. The results demonstrated that egg production was decreased in fish exposed to PHE, with a significant reduction at 5.0 µg/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or E2 in male fish. In addition, plasma vitellogenin levels were significantly inhibited after PHE exposure in female fish. The transcription of hypothalamic-pituitary-gonadal (HPG) axis related genes (GnRH2, FSHß, LHß, 17ß-HSD, CYP11A1, and CYP19a) were significantly altered in a sex-specific manner. In addition, embryos derived from exposed parents exhibited increased malformation and decreased hatching success in the F1 generation. Taken together, these results demonstrate that chronic exposure to environmentally relevant concentration of PHE could cause adverse effects on reproduction and impair the development of offspring, ultimately leading to fish population decline in aquatic environment.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Analysis of O-Acetylated Sialic Acids in Dried Blood Spots.

Sialic acid is a family of N- and O-substitutions of neuraminic acid. Plasma or serum sialic acid has been established as a potential disease marker. For example, the presence of 9- O-acetyl on the sialic acid of some glycans and glycoconjugates (e.g., 9- O-acetyl GD3 ganglioside) could be related to cancer occurrence. A variety of assays are available to measure serum or plasma sialic acid; however, sample preparation and storage can alter the O-acetylation profile due to the loss of O-acetyl groups and/or the migration of O-acetyl groups. Herein, we report dried blood spot (DBS) sampling, in combination with diamino-4,5-methylenedioxybenzene derivatization, for profiling sialic acids in blood samples with minimal alteration in O-acetylation patterns. The feasibility of the method was first evaluated by analyzing sialic acids in crucian carp blood and comparing with traditional blood/plasma sample preparation procedures. A total of 19 different sialic acids were identified by using liquid chromatography-Orbitrap mass spectrometry, including four mono-O-acetylated N-acetylneuraminic acids, four mono-O-acetylated N-glycolylneuraminic acids, six di-O-acetylated N-acetylneuraminic acids, and three tri-O-acetylated N-acetylneuraminic acids. The long-term storage study indicated that DBS sampling could effectively preserve the O-acetylation information for at least 6 weeks. Thus, it is demonstrated that this method is a valuable tool for the study of sialic acid diversity, especially for the characterization of isomeric structures.

Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis.

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ±â€¯1.3 ng/L and 470.3 ±â€¯2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17ß-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish.

Higher Anti-angiogenesis Activity, Better Cellular Uptake and Longer Half-life of a Novel Glyco-modified Endostatin by Polysulfated Heparin.

BACKGROUND: Endostatin (ES) is a promising anti-angiogenesis protein and has been approved for the treatment of non-small cell lung cancer, but short half-life, poor stability and nonspecific delivery caused great pain to patients and produced unsatisfactory treatment effectiveness. OBJECTIVE: In this work, in order to overcome these disadvantages, ES was covalently modified by polysulfated heparin (PSH) with the expectancy of longer half-life, higher anti-angiogenesis activity and better cellular uptake. METHODS: To characterize the cellular uptake, flow cytometry and confocal laser scanning microscopy were used to study the intracellular localization of fluorescein isothiocyanate-labeled ES and PSH-ES in EAhy926 endothelial cells. Zebrafish model was used to study the anti-angiogenesis activities of ES and its derivatives in vivo. The 125I-radiolabeled ES and PSH-ES were administered to healthy BALC/c mice for the pharmacokinetics study. RESULTS: Compared with ES, better cellular uptake effects were detected in PSH-ES group. Both ES and PSH-ES showed inhibition on the intersegmental vessels formation, while PSH-ES displayed a higher one. The half-life of PSH-ES was lengthened and area under the curve (AUC) was increased. At the same time, ES and PSH-ES were both widely and rapidly distributed in the lungs, livers, kidneys and hearts with little difference. CONCLUSION: The results indicated that PSH displayed good properties as a novel glyco-modifier for protein and peptide. The results also showed that PSH-ES displayed better cellular uptake, higher antiangiogenesis activity and prolonged half-life, which would lead to better anti-tumour effects.

Estrogenic effects associated with bisphenol a exposure in male zebrafish (Danio rerio) is associated with changes of endogenous 17ß-estradiol and gene specific DNA methylation levels.

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17ß-estradiol (E2), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500µg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1a's promoters were determined. Our results indicated that for the 500 and 1500µg/L treatment groups, VTG might be induced mainly by the elevated E2 levels; increases of E2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish.

Quizalofop-P-ethyl exposure increases estrogen axis activity in male and slightly decreases estrogen axis activity in female zebrafish (Danio rerio).

The herbicide Quizalofop-P-ethyl (QpE) exerts toxic effects in fish, but limited information is currently available on its effects on the endocrine system. In the current study, adult zebrafish (Danio rerio) were exposed to different concentrations (0, 2, 20, 200µg/L) of QpE for 30days. In males, QpE exposure significantly increased plasma estradiol (E2) and vitellogenin (VTG) levels, concomitant with up-regulation of hepatic esr1 and vtg gene expression. In females, plasma sex hormone levels and VTG concentrations were not altered significantly, but an increased expression of hepatic esr1 in addition to decreased expression of hepatic vtg, esr2a and esr2b was observed. Marked histological lesions were also observed in the gonads of both males and females. Moreover, QpE exposure significantly increased transcriptional profiles of some genes in the HPG axis and liver in males, while the majority of these genes were down-regulated in females. Docking studies showed QpE forming stable interactions with the ligand-binding domain (LBD) of zebrafish ESR1 and ESR2a, suggesting QpE may bind to estrogen receptors (ESRs). This study for the first time reveals QpE as an endocrine-disrupting chemical (EDC) disrupting the zebrafish endocrine system in a sex-specific manner, whereby it increases estrogen axis activity in males and slightly decreases estrogen axis activity in females, which may be accounted for by QpE regulating steroidogenesis and/or activating ESR(s).

A novel enzyme-linked immunosorbent assay based on anti-lipovitellin monoclonal antibodies for quantification of zebrafish (Danio rerio) vitellogenin.

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95-250ng/mL, with a detection limit of 0.78ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E2-treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17ß-estradiol.

Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.

This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis.

Since its introduction in early 1980s, the zebrafish (Danio rerio) has become an invaluable vertebrate animal model system to study many human disorders in almost all systems, from hepatic and brain pathology, to autoimmune and psychiatric disorders. Hematopoiesis between zebrafish and mammals is highly conserved, making the zebrafish an attractive model to study hematopoietic development and blood disorders. Unique attributes of the zebrafish include the ability to perform large-scale genetic and chemical screens in vivo, study development at the cellular level, and use transgenic fish to dissect mechanisms of disease or drug effects. This review summarizes major discoveries that helped define molecular control of hematopoiesis in vertebrates and specific contributions from studies in zebrafish.

Inflammatory response and blood hypercoagulable state induced by low level co-exposure with silica nanoparticles and benzo[a]pyrene in zebrafish (Danio rerio) embryos.

Given the severe situation of world-wide particulate matter air pollution, it is urgent to explore the combined effects of particulate matter components on cardiovascular system. Using zebrafish model, this study was aimed to determine whether the low level co-exposure to silica nanoparticles (SiNPs) and benzo[a]pyrene (B[a]P) had a pronounced cardiovascular toxicity than the single exposure to either SiNPs or B[a]P alone. The FTIR and TGA analysis showed that the co-exposure system possessed of high absorption and thermal stability. Embryos exposed to SiNPs or B[a]P alone did not show cardiac toxicity phenotype at the NOAEL level. However, embryos co-exposed to SiNPs and B[a]P exhibited pericardial edema and bradycardia. While ROS generation remained unaffected, the co-exposure induced significant neutrophil-mediated inflammation and caused erythrocyte aggregation in caudal vein of embryos. Microarray analysis and STC analysis were performed to screen the cardiovascular-related differential expression genes and the expression trend of genes in each group. The co-exposure of SiNPs and B[a]P significantly enhanced the expression of proinflammatory and procoagulant genes. Moreover, the co-exposure markedly increased the phosphorylated AP-1/c-Jun and induced TF expression, but not NF-κB p65. This study for the first time demonstrated the inflammatory response and blood hypercoagulable state were triggered by the combination of SiNPs and B[a]P at low level exposure.

Long-term exposure to triphenylphosphate alters hormone balance and HPG, HPI, and HPT gene expression in zebrafish (Danio rerio).

With the global decline in the use of polybrominated diphenyl ethers, the demand for alternative flame retardants, such as triphenylphosphate (TPP), has increased substantially. Triphenylphosphate is now detected in various environments including aquatic ecosystems worldwide. However, studies on the toxicological consequences of chronic TPP exposure on aquatic organisms are scarce. The zebrafish model was used to investigate the effects of long-term TPP exposure on the endocrine system. Zebrafish embryos were exposed to 5 µg/L, 50 µg/L, or 500 µg/L TPP for 120 d, and hormonal and transcriptional responses were measured along the hypothalamic-pituitary-gonad (HPG) axis, the hypothalamic-pituitary-interrenal (HPI) axis, and the hypothalamic-pituitary-thyroid (HPT) axis. Exposure to TPP significantly increased plasma 17ß-estradiol, but decreased 11-ketotestosterone in both sexes. Gene expression data support these changes. In the HPI axis, plasma cortisol and proopiomelanocortin (pomc) and mineralocorticoid receptor transcripts increased in females, but in males cortisol decreased whereas pomc increased (p < 0.05). Thyroxine and triiodothyronine increased, and thyrotrophin-releasing hormone receptor 2 (trhr2) and trh expression were affected only in females (p < 0.05). In summary, long-term exposure to TPP enhanced estrogenicity in both males and females, potentially through influencing the HPG axis, but modulated the HPI, and HPT axes differently by sex, suggesting that both genomic and nongenomic responses might be involved. Environ Toxicol Chem 2016;35:2288-2296. © 2016 SETAC.

Waterborne fluoride exposure changed the structure and the expressions of steroidogenic-related genes in gonads of adult zebrafish (Danio rerio).

Excessive fluoride in natural water ecosystem has been demonstrated to have adverse effects on reproductive system in humans and mammals, while the most vulnerable aquatic organisms were ignored. In this study, the effects of waterborne fluoride on growth performance, sex steroid hormone, histological structure, and the transcriptional profiles of sex steroid related genes were examined in both female and male zebrafish exposed to different concentrations of 0.79, 18.60, 36.83 mg L(-1) of fluoride for 30 and 60 d to investigate the effects of fluoride on reproductive system and the underlying toxic mechanisms caused by fluoride. The results showed that the body weight was remarkably decreased, the structure of ovary and testis were serious injured, and the T and E2 levels were significantly reduced in male zebrafish. The transcriptional profiles of steroidogenic related genes displayed phenomenal alterations, the expressions of pgr and cyp19a1a were significantly up-regulated, while the transcriptional levels of er, ar and hsd3ß were decreased both in the ovary and testis, and hsd17ß8 were down-regulated just in males. Taken together, these results demonstrated that fluoride could significantly inhibit the growth of zebrafish, and notably affect the reproductive system in both sex zebrafish by impairing the structure of ovary and testis, altering steroid hormone levels and steroidogenic genes expression related to the synthesis of sex hormones in zebrafish.

Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish.

Anti-estrogenic effect of semicarbazide in female zebrafish (Danio rerio) and its potential mechanisms.

Semicarbazide (SMC), a member of the hydrazine family, has various toxic effects and has been detected in organisms, aquatic environments, and food. SMC exposure inhibited the transcription of hepatic vitellogenin and estrogen receptors in female zebrafish (Danio rerio), suggesting that it had anti-estrogenic properties. In order to elucidate the mechanisms underlying these, we exposed female zebrafish to SMC and used enzyme-linked immunosorbent assays to examine plasma 17ß-estradiol (E2) and testosterone (T) levels. Gonad histology was analyzed and the mRNA expression of genes involved in the reproductive axis, the gamma-aminobutyric acid (GABA) shunt, and leptin was quantified by real-time PCR. Zebrafish were exposed to 1, 10, 100, or 1000µg/L SMC in a semi-static system for 96hours or 28 days. Plasma E2 levels were significantly decreased and ovarian maturation was inhibited by SMC, suggesting that its anti-estrogenic effect was exerted by reducing endogenous E2 levels. This was likely due to the SMC-mediated inhibition of cytochrome P450 (CYP) 19A mRNA levels, because this enzyme catalyzes the conversion of T to E2 in the gonads. In addition, down-regulation of the mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, steroidogenic acute regulatory protein, CYP17, and 17beta-hydroxysteroid dehydrogenase was observed; this was predicted to reduce T concentrations and further contribute to the reduced E2 levels. SMC-induced changes in the expression of these steroidogenic genes correlated with decreased transcription of gonadotropic hormones (follicle-stimulating hormone and luteinizing hormone) and significantly elevated leptin expression. Furthermore, SMC also altered expression of the key enzyme in gamma-aminobutyric acid (GABA) synthesis, GABA receptors, and salmon gonadotropin-releasing hormone, thus affecting gonadotropin expression. Overall, SMC acted at multiple sites related to reproduction to reduce plasma E2 levels, consequently exerting an anti-estrogenic effect in female zebrafish. These effects were observed at environmentally relevant concentrations and highlight the importance of controlling SMC contamination.

Effects of Clove Oil as a Euthanasia Agent on Blood Collection Efficiency and Serum Cortisol Levels in Danio rerio.

Zebrafish are an important laboratory animal model for biomedical research and are increasingly being used for behavioral neuroscience. Tricaine methanesulfonate (MS222) is the standard agent used for euthanasia of zebrafish. However, recent studies of zebrafish behavior suggest that MS222 may be aversive, and clove oil might be a possible alternative. In this study, we compared the effects of MS222 or clove oil as a euthanasia agent in zebrafish on the volume of blood collected and on serum levels of cortisol. Greater amounts of serum could be collected and lower serum levels of cortisol were present in fish euthanized with clove oil compared with equipotent dose of MS222. Euthanasia with clove oil did not blunt the expected elevation of serum cortisol levels elicited by an acute premortem stress. According to our findings, clove oil is a fast-acting agent that minimizes the cortisol response to euthanasia in zebrafish and allows the collection of large volumes of blood postmortem. These results represent a significant refinement in euthanasia methods for zebrafish.