Efficacy of bioactive compounds from Curcuma longa L. against mosquito larvae

To identify larvicidal compounds from the ethanolic extracts of Curcuma longa root, the active compounds were isolated using activity‐guided fractionation with column chromatography and identified based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. The dipping method was used to determine the larvicidal activities of each compound against 4th‐instar larvae of Culex pipiens pallens. Two compounds were isolated and identified, ar‐turmerone and 8‐hydroxyl‐ar‐turmerone. The two compounds exhibited larvicidal activities against the 4th‐instar larvae of C. pipiens pallens after 24 hr of treatment with LC₅₀ values of 138.86 and 257.68 ppm, respectively. The larvicidal activities of ar‐turmerone and 8‐hydroxyl‐ar‐turmerone against C. pipiens pallens are reported herein for the first time. The elucidation of the structure of these phytochemicals and their insecticidal activities are important for assessing the potential of this plant as a botanical insecticide.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

BACKGROUND: The phenotype of embryos exposed to ethanol is complex and likely due to multiple alterations in developmental pathways. We have previously demonstrated that Sonic hedgehog signaling (Shh‐s) was reduced in both chicken and zebrafish embryos when exposed to ethanol. METHODS: There are many tissues affected by embryonic ethanol exposure, and in this article we explore the development of axial tissues, using zebrafish embryos. We then compare these effects to the phenotypes produced by exposure to two drugs that also inhibit Shh‐s: cyclopamine and forskolin. RESULTS: We found alterations in the development of the notochord and somites produced by all three compounds, although only ethanol produced developmental delay of epiboly. Upon observation of early developing embryos, muscle pioneer cells were completely lost in cyclopamine‐treated embryos, and reduced, but less so, in embryos treated with forskolin and ethanol. Ethanol treatment produced a dose‐dependent reduction in total body length that may be linked to epiboly delay seen earlier during development. Despite the differences between cyclopamine and forskolin, we found that shh mRNA injection rescued the short body length, the alteration in somite shape, and the cyclopia produced by ethanol exposure. CONCLUSIONS: Taken together, each teratogen produced a unique set of phenotypic changes in the body axis, suggesting that each compound affects Shh‐s and also produces a distinctive set of molecular alterations. However, addition of exogenous Shh to ethanol treated zebrafish prevented many of the gross physical phenotypes, suggesting that the suppression of Shh‐s is one of the major effects of ethanol exposure. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

双酚A对斑马鱼胚胎的毒性作用

Bisphenol A(BPA)is a chemical estrogen-like sub-stance with properties that are of environmental concern.It is widely used in the chemical industry to manufactureepoxy-and polyester-styrene resins.It has been reportedthat BPA ranges between0and33μg in each plasticcup[1].After atwo-weekexposureto0.5%bisphenol Aithas beenreportedthat disattachments betweensertoli cellsand spermatogonia were observed while spermatogoniawere arrangedin disorder and displacement of spermatogo-nia away fromthe basement membrance ...     

Noninvasive Imaging of Ethanol‐Induced Developmental Defects in Zebrafish Embryos Using Optical Coherence Tomography

In this article, we report the use of optical coherence tomography for noninvasive cross‐sectional real‐time imaging of ethanol‐induced developmental defects in zebrafish embryos larvae. For ethanol concentration of over 300 mM, developmental defects of eye (shrinkage and retinal abnormalities), malformation of the notochord and ataxia arising due to the toxic effects of ethanol were observed in OCT images from 3 days post fertilization onwards. The results suggest that OCT could be a valuable tool for noninvasive assessment of birth defects in small animal systems.     

Developmental effects of trichloroacetate in Zebrafish embryos: Association with the production of superoxide anion

To assess the developmental toxicity of trichloroacetate (TCA), zebrafish embryos were exposed to 8 to 48 mM of TCA and evaluated for developmental milestones from 8‐ to 144‐hour postfertilization (hpf). All developmental toxicities are reported in this paper. Embryos were found to have developed edema in response to 16 to 48 mM of TCA exposure at 32‐ to 80‐hpf, experienced delay in hatching success in response to 24 to 48 mM at 80‐hpf. Lordosis was observed in developing embryos exposed to 40 to 48 mM at 55‐ to 144‐hpf. The observed toxic effects of TCA exposure were found to be concentration and exposure period independent. Effects were found to be associated with increases in superoxide anion production, but these increases were also found to be concentration and time independent. TCA resulted in concentration‐dependent increases in embryonic lethality at 144‐hpf, with an LC₅₀ determined to be 29.7 mM.     

Evaluation in zebrafish model of the toxicity of rhodamine B‐conjugated crotamine,a peptide potentially useful for diagnostics and therapeutics

Crotamine is defensin‐like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom‐derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B‐conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB‐crotamine was as low as 4 μM, which effectively kill zebrafish larvae in less than 10 min. With non‐lethal concentrations (<1 μM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB‐crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.     

Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin,Fgf, and sonic hedgehog function

BACKGROUND: Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS: Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS: Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol‐induced decreases in GAD1 and Atonal1a gene expression. CONCLUSIONS: These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.     

New Isocoumarins from a Cold‐Adapted Fungal Strain Mucor sp. and Their Developmental Toxicity to Zebrafish Embryos

Three new isocoumarin derivatives, mucorisocoumarins A–C ( 1 – 3 , resp.), together with seven known compounds, 4 – 10 , were isolated from the cold‐adapted fungal strain Mucor sp. (No. XJ07027‐5). The structures of the new compounds were identified by detailed IR, MS, and 1D‐ and 2D‐NMR analyses. It was noteworthy that compounds 1, 2, 4 , and 5 were successfully resolved by chiral HPLC, indicating that 1 – 7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos.     

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.     

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos

Background  

Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

Background

The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines.

Methodology/Principal Findings

In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification.

Conclusion

Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.     

Repellency of essential oils against Nephotettix cincticeps: Laboratory and glasshouse assays

Four oils from Piper nigrum, Litsea cubeba, Zanthoxylum bungeanum and Curcuma longa were obtained by ethanol extraction. The repellency of these oils and two major compounds (linalool and piperine) was evaluated against female adult and third‐instar nymphs of the rice pest, Nephotettix cincticeps, under laboratory and glasshouse conditions. Paired‐choice and no‐choice assays were used for each treatment, with essential oils evaluated after 24 and 48 hr of exposure and chemical compounds evaluated after 12 and 24 hr of exposure. The potential effects of essential oils on activities of glutathione S tranferase (GST), carboxyl esterase (CarE) and acetyl cholinesterase (AchE) were also evaluated after 48 hr of exposure to leafhoppers. The constituents of the essential oils were determined using GC‐MS. The results showed that the major components in the oils were piperine (34.75%) for P. nigrum, 9,12‐octadecadienoic acid (Z,Z) (18.74%) for L. cubeba, ethanone, 1‐(2‐hydroxy‐4,6‐dimethoxyphenyl) (18.51%) for Z. bungeanum and turmerone (15.89%) for C. longa. In all cases, the essential oils repelled female adults and third‐instar nymphs of N. cincticeps. The repellency of the tested oils and chemicals compounds in the paired‐choice assay was higher than in the no‐choice assay. In all experimental conditions, P. nigrum and C. longa oils were the most and the least potent, respectively. Linalool was the best repellent among the single‐tested compounds under laboratory conditions. In the glasshouse study, the highest repellency was observed in the mixture of linalool and piperine. GST and CarE activities of leafhoppers were significantly enhanced by exposure to the four essentials oils; AchE activity increased significantly only in the P. nigrum and L. cubeba assays. Our results clearly indicate that the tested oils and chemical compounds are promising agents for developing plant‐based pesticides to control N. cincticeps.     

五氯酚对斑马鱼胚胎的毒性效应研究

采用斑马鱼胚胎发育技术,对环境激素类物质五氯酚的毒性进行测定,结果表明,五氯酚(PCP)对胚胎的特定作用时间段是卵产出至发育6 h之内;PCP对胚胎发育有明显的抑制作用,会造成胚胎发育的畸形或死亡,不同时间染毒产生的可观察毒理学终点各异;随着PCP对发育48 h斑马鱼胚胎作用时间的减短,其致死效应敏感性降低,其中0 hpf组的LC0值最小,为70.8μg·L^-1,24hpf组LC0值最大,为831.8μg·L^-1;斑马鱼胚胎对孵化后0时染毒的PCP最为敏感,PCP对胚胎产生急性毒性效应的敏感指标:心胞囊肿、血液循环障碍、无心律＞孵化率降低＞停滞发育作用;斑马鱼胚胎最敏感的指标为48 h血液循环障碍和48 h半致死效应.     

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 gsk3β were determined by RT‐PCR, whereas β‐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 β 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.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neuronsin developing zebrafish

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B)86: 85-91, 2009. © 2009 Wiley-Liss, Inc.