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1.
Toxics ; 8(4)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233497

RESUMO

(1) Background: Amikacin is an aminoglycoside antibiotic used for treating gram-negative bacterial infections in cancer patients. In this study, our aims are to investigate the migratory inhibition effects of amikacin in human MDA-MB-231 cells. (2) Methods: We used a wound-healing assay, trans-well analysis, Western blotting, immunostaining and siRNA knockdown approaches to investigate how amikacin influenced MDA-MB-231 cell migration and invasion. (3) Results: Wound healing showed that the MDA-MB-231 cell migration rates decreased to 44.4% in the presence of amikacin. Trans-well analysis showed that amikacin treatment led to invasion inhibition. Western blotting demonstrated that amikacin induced thioredoxin-interacting protein (TXNIP) up-regulation. TXNIP was knocked down using siRNA in MDA-MB-231 cell. Using immunostaining analysis, we found that inhibition of TXNIP expression led to MDA-MB-231 pseudopodia extension; however, amikacin treatment attenuated the cell extension formation. (4) Conclusions: We observed inhibition of migration and invasion in MDA-MB-231 cells treated with amikacin. This suggests inhibition might be mediated by up-regulation of TXNIP.

2.
Environ Toxicol ; 33(12): 1321-1328, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30259639

RESUMO

4-methylimidazole (4-MI) is an imidazole-derived organic chemical compound that can be used as a raw material in the manufacture of diverse chemicals and has been identified as an ingredient of caramel color in soybean sauce, beers, and other soft drinks. The aim of the present study was to investigate the teratogenic effects of 4-MI during zebrafish embryogenesis. Zebrafish embryos were treated with different dosages of 4-MI (0-120 mM) for different exposure durations (12-60 hours). The percentages of embryos with malformed phenotypes increased as the exposure dosages and duration time of 4-MI increased. We also used immunofluorescence and transmission microscopy to evaluate the subtle changes in the myofibril alignment and ultrastructure of muscle organization. Our data showed that 4-MI treatment disturbs muscle fiber alignment. Electron microscopy data indicated that Z-lines were undetectable in the 4-MI-treated embryos. Although the thick and thin filaments were visible, they were all disorganized. In addition, zebrafish embryos treated by 4-MI exhibited aberrant expression of 2 muscle-specific genes, myod and myogenin. Taken together, we concluded that early exposure to 4-MI affects zebrafish myogenesis, especially in myofibril alignment.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Imidazóis/toxicidade , Desenvolvimento Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Miofibrilas/fisiologia , Proteínas de Peixe-Zebra/metabolismo
3.
J Appl Toxicol ; 35(3): 287-94, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25186829

RESUMO

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.


Assuntos
Compostos de Boro/toxicidade , Cafeína/toxicidade , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Citosol/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica de Transmissão , Miofibrilas/ultraestrutura
4.
BMC Cancer ; 14: 815, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25376302

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignancy with poor survival outcome. New treatment options for the disease are needed. In this study, we identified and evaluated tumor vascular PLVAP as a therapeutic target for treatment of HCC. METHODS: Genes showing extreme differential expression between paired human HCC and adjacent non-tumorous liver tissue were investigated. PLVAP was identified as one of such genes with potential to serve as a therapeutic target for treatment of HCC. A recombinant monoclonal anti-PLVAP Fab fragment co-expressing extracellular domain of human tissue factor (TF) was developed. The potential therapeutic effect and toxicity to treat HCC were studied using a Hep3B HCC xenograft model in SCID mice. RESULTS: PLVAP was identified as a gene specifically expressed in vascular endothelial cells of HCC but not in non-tumorous liver tissues. This finding was confirmed by RT-PCR analysis of micro-dissected cells and immunohistochemical staining of tissue sections. Infusion of recombinant monoclonal anti-PLVAP Fab-TF into the main tumor feeding artery induced tumor vascular thrombosis and extensive tumor necrosis at doses between 2.5 µg and 12 µg. Tumor growth was suppressed for 40 days after a single treatment. Systemic administration did not induce tumor necrosis. Little systemic toxicity was noted for this therapeutic agent. CONCLUSIONS: The results of this study suggest that anti-PLVAP Fab-TF may be used to treat HCC cases for which transcatheter arterial chemoembolization (TACE) is currently used and potentially avoid the drawback of high viscosity of chemoembolic emulsion for TACE to improve therapeutic outcome. Anti-PLVAP Fab-TF may become a viable therapeutic agent in patients with advanced disease and compromised liver function.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Transporte/análise , Células Endoteliais/química , Neoplasias Hepáticas/química , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Membrana/análise , Animais , Anticorpos Monoclonais/efeitos adversos , Antígenos de Superfície/imunologia , Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Células Endoteliais/metabolismo , Feminino , Xenoenxertos , Humanos , Fígado/química , Neoplasias Hepáticas/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , RNA Mensageiro/metabolismo , Proteínas Recombinantes/imunologia
5.
J Toxicol Pathol ; 27(1): 19-24, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24791063

RESUMO

D-serine is a well-known activator of N-methyl-D-aspartate receptors; however, little is known about the teratogenic effects of D-serine overdose during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity, since they have transparent eggs, making the organogenesis of zebrafish embryos easier to be observed. After D-serine injection (100-1000 ppm), the most evident defective phenotypes were bent trunk phenotypes, including malformed somite boundary, twisted body axis and shorter body length. As the injection dosages increased, the rates of embryos with bent trunk phenotypes decreased (0% for 0 ppm, n=573; 59.9~84.3% for 100-1000 ppm of D-serine, n=383-451). In addition, D-serine-injected embryos exhibited significantly reduced the frequencies of spontaneous in-chorion contraction (21.7 for 0 ppm vs. 18.3-0.9 for 100-1000 ppm D-serine, n=30) in comparison with mock-treated controls (0 ppm). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1, Zn5 and α-bungarotoxin to detect morphological changes in muscle fibers, primary motor axons, secondary motor axon projections and neuromuscular junctions, respectively. Our data show that overdose of D-serine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.

6.
Molecules ; 19(1): 641-50, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24402197

RESUMO

The aim of this study was to investigate the in vivo toxicities of some novel synthetic chalcones. Chalcone and four chalcone analogues 1a-d were evaluated using zebrafish embryos following antibody staining to visualize their morphological changes and muscle fiber alignment. Results showed that embryos treated with 3'-hydroxychalcone (compound 1b) displayed a high percentage of muscle defects (96.6%), especially myofibril misalignment. Ultrastructural analysis revealed that compound 1b-treated embryos displayed many muscle defect phenotypes, including breakage and collapse of myofibrils, reduced cell numbers, and disorganized thick (myosin) and thin (actin) filaments. Taken together, our results provide in vivo evidence of the myotoxic effects of the synthesized chalcone analogues on developing zebrafish embryos.


Assuntos
Anormalidades Induzidas por Medicamentos/patologia , Chalcona/análogos & derivados , Chalcona/toxicidade , Fibras Musculares Esqueléticas/patologia , Teratogênios/toxicidade , Animais , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/anormalidades , Peixe-Zebra
7.
J Toxicol Pathol ; 26(1): 79-82, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23723573

RESUMO

Despite its medical use, little is known about the mechanisms underlying amikacin-induced embryotoxicity, including fin reduction, in zebrafish. In this study, we examined the expression of well-known autophagy markers mTOR (target of rapamycin), atg10 (autophagy-related gene), atg12 and LC3 (mammalian homolog of Atg8) in amikacin-treated zebrafish embryos. Our results indicated that the mRNA expression level of atg12 in the amikacin-treated group was significantly increased by 1.5-fold (p<0.05) compared with the corresponding mock control group, while the expression levels of atg10 and mTOR were significantly decreased by 0.74-fold (p<0.05) and 0.58-fold (p<0.05), respectively. Western blot analysis revealed that LC3 protein expression was induced by amikacin. Taken together, these data suggest that amikacin-induced fin reduction is mediated by fin cell autophagy.

8.
Molecules ; 18(2): 2052-60, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23385341

RESUMO

The aim of this study was to investigate novel chalcones with potent anti-inflammatory activities in vivo. Chalcone and two chalcone analogues (compound 5 and 9) were evaluated using a caudal fin-wounded transgenic zebrafish line "Tg(mpx:gfp)" to visualize the effect of neutrophil recruitment dynamically. Results showed that treatment with compound 9 not only affected wound-induced neutrophil recruitment, but also affected Mpx enzymatic activity. Moreover, protein expression levels of pro-inflammatory factors (Mpx, NFκB, and TNFα) were also regulated by compound 9. Taken together, our results provide in vivo evidence of the anti-inflammatory effects of synthesized chalcone analogues on wound-induced inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Chalcona/análogos & derivados , Chalcona/farmacologia , Peixe-Zebra/metabolismo , Nadadeiras de Animais/cirurgia , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Chalcona/síntese química , Chalcona/química , Larva/efeitos dos fármacos , Larva/enzimologia , Modelos Animais , Infiltração de Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Cicatrização/efeitos dos fármacos
9.
Drug Chem Toxicol ; 35(4): 361-5, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22313413

RESUMO

Caffeine is a widely consumed substance that occurs in numerous dietary sources, but teratogenic effects of caffeine intake during embryonic development are still not clear. In the present study, we used the zebrafish as a model to assess caffeine-induced toxicity on embryonic vascular development. A green fluorescent vascular endothelium transgenic line, Tg(fli1:egfp), was utilized for the sensitive detection of vascular development, including vasculo- and angiogenesis. Caffeine-treated embryos showed no defects in vasculogenesis, but revealed dose-dependent (250-350 ppm) developmental defects in intersegmental vessels, dorsal longitudinal anastomotic vessels, and subintestinal vein sprouting. Further, real-time polymerase chain reaction analysis of caffeine-treated embryos showed an upregulation of nrp1a along with a downregulation of sema3aa and sema3c. In conclusion, caffeine treatment induces defects of angiogenesis in zebrafish embryos.


Assuntos
Cafeína/toxicidade , Endotélio Vascular/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Peixe-Zebra/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Cafeína/administração & dosagem , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fatores de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Reação em Cadeia da Polimerase em Tempo Real , Teratogênios/toxicidade , Regulação para Cima/efeitos dos fármacos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
10.
Toxicol Mech Methods ; 22(2): 151-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22242631

RESUMO

We used zebrafish as a model to assess amikacin-induced embryotoxicity. We exposed zebrafish embryos to amikacin, using different amikacin doses (0-10 ppm), durations (12-48 h), and onsets (0, 24, 48 hpf). Amikacin-induced embryonic toxicity and reduced survival rate were found dependent on the exposure dose, duration and onset. Based on immunostaining with neuron-specific antibodies, amikacin reduced the number and size of zebrafish neuromasts. In addition, Amikacin caused pelvic, dorsal and anal fin defects in dose-dependent and duration-dependent manners. Proliferating cell nuclear antigen immunostaining revealed that amikacin-induced fin defects were not due to reduction of proliferating mesenchymal cells. TUNEL assay demonstrated that amikacin-induced fin defects might not associate with apoptosis. Therefore, further investigations are required to elucidate if other cell death pathways are involved in amikacin-induced fin defects.


Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Amicacina/toxicidade , Nadadeiras de Animais/anormalidades , Antibacterianos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Peixe-Zebra
11.
Birth Defects Res B Dev Reprod Toxicol ; 92(2): 139-47, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21416579

RESUMO

The objective of this study was to investigate the embryotoxicity of diclofenac. Zebrafish (Danio rerio) embryos at 12 hpf were treated with different dosages of diclofenac (0-2,000 ppm) for different time courses (12-72 hr). Results showed no evident differences in survival rates or morphological changes between the mock-treated control (0 ppm) zebrafish embryos and those with 1-ppm diclofenac-exposure (12-24, 12-36 hpf). In contrast, after higher doses (5 and 10 ppm) of exposure, embryos displayed some defective phenotypes, including malformed somite boundary, a twisted body axis, and shorter body length. In addition, diclofenac-treated embryos exhibited significantly reduced frequencies of spontaneous in-chorion contractions in comparison with mock-control littermates (mock-control: 13.20 ± 2.24 vs. 5-10 ppm diclofenac: 6.66 ± 1.35-3.03 ± 1.84). Subtle changes were easily observed by staining with specific monoclonal antibodies F59 and phalloidin to detect morphological changes in muscle fibers and formation of F-actin, respectively. Our data show that diclofenac treatment disturbs actin organization and muscle fiber alignment, thus causing malformed somite phenotypes.


Assuntos
Actinas/efeitos dos fármacos , Diclofenaco/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Actinas/metabolismo , Animais , Embrião não Mamífero/anormalidades , Modelos Lineares , Modelos Logísticos , Miofibrilas/metabolismo , Miofibrilas/patologia , Fenótipo , Somitos/efeitos dos fármacos , Somitos/patologia , Peixe-Zebra/anormalidades
12.
Protein Expr Purif ; 75(1): 21-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20627128

RESUMO

Capsulin is one of the transcription factors involved in regulating cell differentiation but its biochemical properties and structural characteristics are still unclear. In the present study, we cloned capsulin from zebrafish, which produces large numbers of transparent embryos and has well-characterized developmental stages. By alignment, the deduced amino acid sequence of zebrafish Capsulin, which contains a putative bHLH motif, shares very high homology to that of other species with an 72-82% identity. Zebrafish Capsulin was also targeted to the nucleus of mammalian cells when overexpressed by transient transfection. In order to characterize the structural and biochemical properties of zebrafish Capsulin, a recombinant zebrafish Capsulin protein was expressed and purified in Escherichia coli. By circular dichroism spectroscopy, Capsulin was shown to be 55% α-helical. The size distribution assay by analytical ultracentrifugation indicated that it existed as a monomer-dimer mixture. The results suggested that the recombinant Capsulin has a well-organized and functional structure. Finally, endogenous Capsulin was distributed mainly in the epicardial cells of zebrafish by immunohistochemistry analysis using antibodies raised against zebrafish Capsulin. The present study not only helps us to comparatively analyze capsulin genes across species, but it also provides valuable structural information for further studies of Capsulin biological function in the future.


Assuntos
Escherichia coli/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Linhagem Celular , Núcleo Celular/ultraestrutura , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Pericárdio/citologia , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Transfecção , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/isolamento & purificação
13.
Toxicol Mech Methods ; 21(1): 63-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21067310

RESUMO

This study applied broccoli and cauliflower extracts (whole, floret, and stem) to zebrafish larvae in parallel to receive 100 mJ/cm(2) of UVB six times, and recorded their fin malformation phenotypes. Chemopreventive effects of each group, including UVB, whole-, floret-, and stem-extracts of broccoli and cauliflower on fin development were evaluated using Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression. Results showed that (1) zebrafish fins in the UVB + whole broccoli extract group are 6.20~9.32-times more likely to return to normal fins than ones in the UVB only group, but fins in the UVB + whole cauliflower extract group are only 5.13~11.10-times more likely to recover, indicated that whole broccoli and cauliflower extract had similar chemopreventive ability on fin development; and (2) the broccoli stem has the highest antioxidant capacity among other groups. In conclusion, zebrafish can be used as a system for evaluating the efficacy of other UVB protective compounds.


Assuntos
Nadadeiras de Animais/efeitos da radiação , Brassica/química , Extratos Vegetais/farmacologia , Raios Ultravioleta , Animais , Larva/efeitos dos fármacos , Extratos Vegetais/química , Peixe-Zebra/embriologia
14.
Clin Transl Sci ; 3(4): 172-7, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20718818

RESUMO

It is well recognized that an interdisciplinary approach is essential in the development and implementation of solutions to address the current pediatric obesity epidemic. In two half-day meetings that included workshops and focus groups, faculty from diverse fields identified critically important research challenges, and gaps to childhood obesity prevention. The purpose of this white paper is to describe the iterative, interdisciplinary process that unfolded in an academic health center setting with a specific focus on underrepresented minority groups of Black and Hispanic communities, and to summarize the research challenges and gaps related to pediatric obesity that were identified in the process. Although the research challenges and gaps were developed in the context of an urban setting including high-risk populations (the northern Manhattan communities of Washington Heights, Inwood, and Harlem), many of the issues raised are broadly applicable. The processes by which the group identified research gaps and methodological challenges that impede a better understanding of how to prevent and treat obesity in children has resulted in an increase in research and community outreach collaborations and interdisciplinary pursuit of funding opportunities across units within the academic health center and overall university.


Assuntos
Obesidade/prevenção & controle , Obesidade/terapia , Equipe de Assistência ao Paciente , Projetos de Pesquisa , Centros Médicos Acadêmicos , Adolescente , Criança , Relações Comunidade-Instituição , Educação , Feminino , Objetivos , Humanos , Masculino , Cidade de Nova Iorque/epidemiologia , Obesidade/epidemiologia , Prevalência , Pesquisa/tendências
15.
Artigo em Inglês | MEDLINE | ID: mdl-20170747

RESUMO

We used a green fluorescent kidney line, Tg(wt1b:GFP), as a model to access the acetaminophen (AAP)-induced nephrotoxicity dynamically. Zebrafish (Danio rerio) embryos at different developmental stages (12-60hpf) were treated with different dosages of AAP (0-45mM) for different time courses (12-60h). Results showed that zebrafish embryos exhibited no evident differences in survival rates and morphological changes between the mock-treated control (0mM) and 2.25mM AAP-exposure (12-72hpf) groups. In contrast, after higher doses (22.5 and 45mM) of exposure, embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tube, pronephric duct, and a cystic and atrophic glomerulus. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AAP increased. Interestingly, under the same exposure time course (12h) and dose (22.5mM), embryos displayed higher percentages of severe defects at earlier developmental stage of exposure (12-24hpf), whereas embryos displayed higher percentages of mild defects at later exposure (60-72hpf). With an exposure time course less than 24h of 45mM AAP, no embryo survived by the developmental stage of 72hpf. These results indicated that AAP-induced nephrotoxicity depended on the exposure dose, time course and developmental stages. Immunohistochemical experiments showed that the cells' morphologies of the pronephric tube, pronephric duct and glomerulus were disrupted by AAP, and consequently caused cell death. Real-time RT-PCR revealed embryos after AAP treatment decreased the expression of cox2 and bcl2, but increased p53 expression. In conclusion, AAP-induced defects on glomerulus, pronephric tube and pronephric duct could be easily and dynamically observed in vivo during kidney development in this present model.


Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Acetaminofen/toxicidade , Analgésicos não Entorpecentes/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Rim/efeitos dos fármacos , Peixe-Zebra/fisiologia , Anormalidades Induzidas por Medicamentos/patologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Perda do Embrião/induzido quimicamente , Embrião não Mamífero/embriologia , Expressão Gênica/efeitos dos fármacos , Rim/anormalidades , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
16.
Chem Biol Interact ; 182(1): 84-91, 2009 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-19682442

RESUMO

Ruthenium (Ru) derivatives have less toxicity and higher water-solubility than cisplatin, giving them great potential as antitumor metallodrugs. In this study, zebrafish were employed as a whole-organism model to screen new Ru compounds for anti-cell proliferation activity. After soaking fish embryos in cisplatin and five Ru derivatives, [Ru(terpy)(bpy)Cl]Cl, [Ru(terpy)(dppz)OH(2)](ClO(4))(2), [Ru(terpy)(tMen)OH(2)](ClO(4))(2), [Ru(terpy)(Me(4)Phen)OH(2)](ClO(4))(2), and Ru(bpy)(2)Cl(2), only cisplatin and [Ru(terpy)(bpy)Cl]Cl-treated embryos displayed obvious phenotypic effects, such as fin-reduction. After further modification of [Ru(terpy)(bpy)Cl]Cl's main structure and the synthesis of two structurally related compounds, [Ru(terpy)(dcbpyH(2))Cl]Cl and [Ru(terpy)(dmbpy)Cl]Cl, only [Ru(terpy)(dmbpy)Cl]Cl exhibited fin-reduction phenotypes. TUNEL assays combined with immunostaining techniques revealed that treatment with cisplatin, [Ru(terpy)(bpy)Cl]Cl, and [Ru(terpy)(dmbpy)Cl]Cl led proliferating fin mesenchymal cells to undergo apoptosis and consequently caused fin-reduction phenotypes. Furthermore, [Ru(terpy)(bpy)Cl]Cl was able to activate the P53-dependent and independent pathways, and induced human hepatoma cells to undergo apoptosis. In summary, it was concluded that the zebrafish model was effective for the screening of phenotype-based antiproliferation metallodrugs.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Compostos Organometálicos/farmacologia , Rutênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Microscopia de Fluorescência , Microscopia de Interferência , NF-kappa B/metabolismo , Fenótipo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra
17.
Transgenic Res ; 18(6): 855-64, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19412740

RESUMO

We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.


Assuntos
Carcinoma Basocelular/tratamento farmacológico , Embrião não Mamífero/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Pele/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Queratina-18/genética , Proteínas Luminescentes/genética , Pele/citologia , Teratogênios , Alcaloides de Veratrum , Peixe-Zebra/embriologia
18.
Mar Biotechnol (NY) ; 11(3): 419-29, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19005726

RESUMO

We used zebrafish as a whole-organism model to screen new compounds for sun protection activity. First of all, we designed a series of UVB exposure experiments and recorded the phenotypic changes of zebrafish embryos. Results showed that 100 mJ/cm(2) of UVB given six times separated by 30 min intervals is the best condition. Fin malformation (reduced and/or absent fin) phenotypes are the most evident consequences after exposure to UVB. Each fin was affected by UVB, including pelvic, ventral, caudal, and dorsal fin, but pelvic fin seemed to be the most sensitive target after UVB exposure. We furthermore carried out "prevention" and "treatment" experiments using green tea extract and/or (-)-epigallocatechin (EGCG) to test this whole-organism model by observing the morphological changes of all fins (especially pelvic fin) after UVB exposure. Effects of UVB, green tea extract and EGCG on fin development were assessed using the Kaplan-Meier analysis, log-rank test and Cox proportional hazards regression. Results showed that a zebrafish pelvic fin in the UVB + green tea (treatment) group is 5.51 (range from 2.39 to 14.90) times, one in the UVB + green tea (prevention) group is 7.04 (range from 3.11 to 18.92) times, and one in the 25 ppm of EGCG (prevention) group is 22.19 (range from 9.40 to 61.50) times more likely to return to normal fin than one in the UVB only group. On the basis of these observations, we believe this model is effective for screening the higher stability and lower toxicity of new compounds, such as small chemicals which are derivative from EGCG or other dietary agents for sun protection.


Assuntos
Extremidades/efeitos da radiação , Modelos Animais , Protetores Solares/normas , Peixe-Zebra , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Camellia sinensis , Catequina/análogos & derivados , Catequina/farmacologia , Primers do DNA/genética , Marcação In Situ das Extremidades Cortadas , Extratos Vegetais/farmacologia , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Raios Ultravioleta/efeitos adversos
19.
Neurotoxicol Teratol ; 30(5): 440-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18508234

RESUMO

Though caffeine is broadly distributed in many plants and foods, little is known about the teratogenic effects of caffeine during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity since they have transparent eggs, making the organogenesis of zebrafish embryos easier to observe. When the exposure doses of caffeine were less than 150 ppm (17.5, 35, 50, 100 and 150 ppm), the zebrafish embryos exhibited no significant differences in survival rates after comparison with vehicle-control (0 ppm) group. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 300 ppm caffeine or higher dosages. The most evident change in embryos treated with caffeine was a shorter body length (vehicle-control: 3.26+/-0.01 mm, n=49; vs 150 ppm of caffeine: 2.67+/-0.03 mm, n=50). In addition, caffeine-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-control: 9.93+/-0.77 vs 17.5-150 ppm caffeine: 5.37+/-0.52-0.10+/-0.06). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1 and Zn5 to detect morphological changes in muscle fibers, primary motor axons and secondary motor axon projections, respectively. Our data show that the treatment of caffeine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.


Assuntos
Cafeína/toxicidade , Discinesia Induzida por Medicamentos/fisiopatologia , Embrião não Mamífero/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Discinesia Induzida por Medicamentos/patologia , Embrião não Mamífero/anormalidades , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Neurônios Motores/patologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/anormalidades , Músculo Esquelético/inervação , Junção Neuromuscular/anormalidades , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiopatologia , Inibidores de Fosfodiesterase/toxicidade , Transtornos das Sensações/induzido quimicamente , Transtornos das Sensações/patologia , Transtornos das Sensações/fisiopatologia , Medula Espinal/anormalidades , Taxa de Sobrevida , Fatores de Tempo , Testes de Toxicidade , Peixe-Zebra
20.
Dev Dyn ; 237(4): 1043-50, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18297736

RESUMO

Mrf4 is a basic helix-loop-helix (bHLH) transcription factor associated with myogenesis. Two mrf4 transcripts, mrf4_tv1 and mrf4_tv2, were identified in zebrafish generated by alternative splicing. To study their biological functions, we separately injected the Mrf4-morpholinos, including MO1 (mrf4_tv1:mrf4_tv2 knockdown), MO2+MO3 (mrf4_tv1:mrf4_tv2 knockdown), MO3 (mrf4_tv1 knockdown), and MO4 (mrf4_tv2 knockdown), into zebrafish embryos to observe mrf4 gene knockdown phenotypes. No phenotypic abnormalities were observed following injection with 0.5 ng of MO1 but those injected with 4.5, 9, or 13.5 ng displayed curved-body phenotypes, such as indistinct somite boundaries, and a lack of uniformly sized cell blocks. Similar results were also observed in the (MO2+MO3)-, MO3-, and MO4-injected groups. To further investigate the molecular mechanisms that lead to curved-body phenotypes, we stained embryos with alpha-bungrotoxin and specific monoclonal antibodies F59, Znp1, and Zn5 to detect morphological changes in acetyl-choline receptor (AChR) clusters, muscle fibers, common path of the primary neurons, and secondary neurons axonal projections, respectively. Our results show that the muscle fibers of mrf4_(tv1:tv2)-morphant aligned disorderly and lost their integrity and attachment, while the defects became milder in either mrf4_tv1-morphant or mrf4_tv2-morphant. On the other hand, reduced axonal projections and AChR clusters were found in both mrf4_tv2-morphant and mrf4_(tv1:tv2)-morphant but distributed normally in the mrf4_tv1-morphant. We conclude that Mrf4_tv2 is involved in alignment of muscle fibers, and Mrf4_tv1 might have cooperative function with Mrf4_tv2 in muscle fiber alignment, without affecting the muscle-nerve connection.


Assuntos
Neurônios Motores/patologia , Miofibrilas/patologia , Fatores de Regulação Miogênica/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Miofibrilas/metabolismo , Fatores de Regulação Miogênica/genética , Neurônios/citologia , Neurônios/fisiologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Isoformas de Proteínas/genética , Receptores Colinérgicos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
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