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1.
Ecotoxicol Environ Saf ; 183: 109566, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31437728

RESUMO

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants. Biomonitoring studies have shown widespread presence of PBDEs in humans and their accumulation in food chain cause concern to human health, especially for foetus and infant development. The early-life stages are generally considered more sensitive to exposure to toxic compounds than juvenile or adults. For this reason the aim of this study was to evaluate the effects of the three most environmentally relevant BDE (BDE- 47, 99 and 209) on zebrafish embryos. The fish embryo toxicity (FET) OECD tests on zebrafish were performed followed by histopathogical examination to assess morphological changes. The gene expression of the thyroid stimulating hormone ß (Tshß), the transport proteins transthyretin (Ttr) and thyroxine-binding globulin (Tbg) as well as the enzyme iodothyronine deiodinase 1 (Dio1) was also assessed by Real-time PCR. BDE-47 and BDE-99 showed an increase of the severity of the effects at the lower concentrations while for the BDE-209 the effects were higher to the high concentrations. Although all compounds did not show any acute toxicity for none of the concentrations tested, they reported interesting sub-acute lesions, including yolk and pericardial edema, tail and head malformation, reduced and extremely reduced heart beat rate, blood stasis and spinal curvature, with the highest percentage recorded for BDE-209. Cardiac edema, damage of eye structure and hydrocephaly were confirmed also by histophatological examination. Furthermore, a toxic and dose-dependent liver vacuolization in BDE-209 was observed in all experimental groups. Although no statistically significant difference in gene expression was observed, BDE-209 up-regulated only Dio1 while the other congeners induced Tshß, Ttr, Tbg and Dio1. Overall, this research highlighted that exposure to BDE-47, BDE-99 and BDE-209 at realistic concentrations caused lethal and sub-lethal alterations and impaired genes involved in thyroid hormones homeostasis leading to abnormal development of zebrafish embryos.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Monitoramento Ambiental/métodos , Retardadores de Chama/toxicidade , Expressão Gênica/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Peixe-Zebra , Adulto , Animais , Relação Dose-Resposta a Droga , Embrião não Mamífero/patologia , Retardadores de Chama/análise , Éteres Difenil Halogenados/análise , Humanos , Hormônios Tireóideos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
2.
Life Sci ; 235: 116791, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31465732

RESUMO

AIMS: Prostate cancer (PCa) incidence rates are rising in China currently. Cancer-associated fibroblasts (CAFs), as a major component of tumor microenvironment, are crucial for tumor progression. This study was aimed to explore the promotion effect of patient-derived CAFs on the proliferation and migration of prostate cancer cells. MAIN METHODS: CAFs were isolated from tumor tissues of PCa patients. The promotion effect of CAFs on the proliferation and migration of PC-3 and LNCaP cells were evaluated in vitro and in vivo. The concentration of TGF-ß1 was measured by Luminex assay. The blocking activity of LY2109761 on the promotion effect of CAFs was also evaluated. KEY FINDINGS: CAFs could significantly promote the proliferation and migration of PC-3 and LNCaP cells both in vitro and in vivo. TGF-ß1 was identified as a highly increased factor in CAFs-CM compared with the normal culture medium of these two cancer cell lines. TGF-ß receptor inhibitor LY2109761 could suppress the CAFs-induced cellular proliferation and migration of PC-3 cells but not LNCaP cells. SIGNIFICANCE: Our study suggested a crucial role for CAFs and TGF-ß signaling in the progression of PCa. Zebrafish xenograft model was an ideal animal model for the study of CAFs and cancer cell interaction.


Assuntos
Fibroblastos Associados a Câncer , Movimento Celular , Proliferação de Células , Embrião não Mamífero/patologia , Neoplasias da Próstata/patologia , Pirazóis/farmacologia , Pirróis/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
3.
Toxicol Lett ; 314: 43-52, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31310794

RESUMO

Thioredoxin is an evolutionarily conserved antioxidant protein that plays a crucial role for fundamental cellular processes and embryonic development. Growing evidence support that Thioredoxin influences cellular response to chemicals insults, particularly those accompanying oxidative stress. The mechanisms underlying the functions of Thioredoxin1 in the embryonic development under the environmental toxicant exposure remain, however, largely unexplored. We report here that thioredoxin1 becomes differentially expressed in zebrafish embryos after exposure to 9 out of 11 environmental chemicals. In situ gene expression analysis show that thioredoxin1 is expressed in neurons, olfactory epithelia, liver and swim bladder under normal conditions. After MeHg exposure, however, thioredoxin1 is ectopically induced in the hair cells of the lateral line and in epithelia cells of the pharynx. Knockdown of Thioredoxin1 induces hydrocephalus and increases cell apoptosis in the brain ventricular epithelia cells. In comparison with 5% malformation in embryos injected with control morpholino, MeHg induces more than 77% defects in Thioredoxin1 knockdown embryos. Our data suggest that there is an association between hydrocephalus and Thioredoxin1 malfunction in embryonic development, and provide valuable information to elucidate the protective role of Thioredoxin1 against chemicals disruption.


Assuntos
Encéfalo/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Hidrocefalia/induzido quimicamente , Tiorredoxinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Hidrocefalia/embriologia , Hidrocefalia/genética , Hidrocefalia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxinas/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Chemosphere ; 233: 336-346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31176896

RESUMO

6-benzylaminopurine (6-BA) is widely used in agriculture and horticulture as plant growth regulator. Its excessive use may pose a potential risk to both environment and human health, which is causing great concern. This study was undertaken to assess the acute developmental toxicity of 6-BA to zebrafish embryos based on OECD protocols and mortality, hatching rate and malformation were investigated. Results showed that the 96 h-LC50 and 96 h- EC50 values were 63.29 mg/L and 41.86 mg/L, respectively. No mortality or teratogenic effects were found at concentrations lower than 10 mg/L 6-BA at concentrations higher than 50 mg/L significantly inhibited hatchability and embryo development, induced serious toxicity characterized by morphologic abnormalities (elongated pericardium, heart and yolk sac edema, spine curvature) and functional failure (slow spontaneous movement and heart rate, growth retardation, yolk sac absorption retention). Moreover, 6-BA-induced apoptosis was observed in embryos by the acridine orange staining and confirmed by the apoptotic-related genes, all of which p53 was significantly up-regulated at concentrations higher than 10 mg/L, bax at concentrations higher than 12.5 mg/L, while bcl2 was down-regulated at concentrations higher than 25 mg/L. As for genes of cardiac development, qPCR results demonstrated that nkx2.5, gata5, and amhc were significantly down-regulated at concentrations higher than 25 mg/L, vmhc and atp2a2a at concentration of 50 mg/L, in contrast, hand2 was up-regulated at concentration of 50 mg/L. Our data indicate that 6-BA induces a dose-dependent toxicity resulting in apoptosis through the involvement of p53-dependent pathways and hindering normal heart development in zebrafish embryos.


Assuntos
Compostos de Benzil/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Purinas/toxicidade , Peixe-Zebra/embriologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Benzil/administração & dosagem , Relação Dose-Resposta a Droga , Embrião não Mamífero/patologia , Feminino , Humanos , Masculino , Purinas/administração & dosagem , Testes de Toxicidade Aguda
5.
Mol Biol Cell ; 30(12): 1353-1358, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31145669

RESUMO

Embryonic tissues heal wounds rapidly and without scarring, in a process conserved across species and driven by collective cell movements. The mechanisms of coordinated cell movement during embryonic wound closure also drive tissue development and cancer metastasis; therefore, embryonic wound repair has received considerable attention as a model of collective cell migration. During wound closure, a supracellular actomyosin cable at the wound edge coordinates cells, while actin-based protrusions contribute to cell crawling and seamless wound healing. Other cytoskeletal networks are reorganized during wound repair: microtubules extend into protrusions and along cell-cell boundaries as cells stretch into damaged regions, septins accumulate at the wound margin, and intermediate filaments become polarized in the cells adjacent to the wound. Thus, diverse cytoskeletal networks work in concert to maintain tissue structure, while also driving and organizing cell movements to promote rapid repair. Understanding the signals that coordinate the dynamics of different cytoskeletal networks, and how adhesions between cells or with the extracellular matrix integrate forces across cells, will be important to elucidate the mechanisms of efficient embryonic wound healing and may have far-reaching implications for developmental and cancer cell biology.


Assuntos
Citoesqueleto/metabolismo , Embrião não Mamífero/patologia , Cicatrização , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Epitélio/patologia , Humanos
6.
J Assist Reprod Genet ; 36(8): 1549-1554, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31129863

RESUMO

While mitotic errors commonly cause aneuploid clones soon after conception, the embryos often normalize as clones are rapidly eliminated. Although generally considered benign, evidence suggests clone elimination as the primary cause of the vertebral, ano-rectal, cardiac, tracheo-esophageal, renal, and limb (VACTERL) association of anomalies, and possibly other adverse outcomes as well. Here, clone elimination-related development disruption at specific locations is used as the basis of a comprehensive theoretical VACTERL association model that also elucidates mitotic mosaic aneuploidy effects. For the association, the model explains random temporal and spatial origins during a limited time frame and overlapping clusters of component anomalies. It supports early developmental effects involving the stage of determination, where the position in a specific morphogen field controls what a cell will become and where it will be located. Developmental properties related to determination also create specific vulnerabilities to the midline and distal defects, the latter explaining exclusively radial and tibial defects with duplications and deficiencies. The model also supports isolated anomalies as part of the association and, for mosaic mitotic aneuploidy, indicates that clone elimination nears completion at the time of lower limb determination. Although mosaic clone elimination may cause other defects, occurrences in different developmental fields separate them from VACTERL anomalies. Clone elimination may also be related to risks for a single umbilical artery and for non-structural adverse pregnancy outcomes such as losses, prematurity, and growth delays, while a paucity of clone lethality in non-humans explains the rarity of the association and of single umbilical arteries in animals.


Assuntos
Anormalidades Múltiplas/etiologia , Canal Anal/anormalidades , Aneuploidia , Embrião de Mamíferos/patologia , Embrião não Mamífero/patologia , Esôfago/anormalidades , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Rim/anormalidades , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Coluna Vertebral/anormalidades , Traqueia/anormalidades , Canal Anal/patologia , Animais , Esôfago/patologia , Feminino , Rim/patologia , Gravidez , Coluna Vertebral/patologia , Traqueia/patologia
7.
Dis Model Mech ; 12(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31043432

RESUMO

DYRK1A is a major causative gene in Down syndrome (DS). Reduced incidence of solid tumors such as neuroblastoma in DS patients and increased vascular anomalies in DS fetuses suggest a potential role of DYRK1A in angiogenic processes, but in vivo evidence is still scarce. Here, we used zebrafish dyrk1aa mutant embryos to understand DYRK1A function in cerebral vasculature formation. Zebrafish dyrk1aa mutants exhibited cerebral hemorrhage and defects in angiogenesis of central arteries in the developing hindbrain. Such phenotypes were rescued by wild-type dyrk1aa mRNA, but not by a kinase-dead form, indicating the importance of DYRK1A kinase activity. Chemical screening using a bioactive small molecule library identified a calcium chelator, EGTA, as one of the hits that most robustly rescued the hemorrhage. Vascular defects of mutants were also rescued by independent modulation of calcium signaling by FK506. Furthermore, the transcriptomic analyses supported the alterations of calcium signaling networks in dyrk1aa mutants. Together, our results suggest that DYRK1A plays an essential role in angiogenesis and in maintenance of the developing cerebral vasculature via regulation of calcium signaling, which may have therapeutic potential for DYRK1A-related vascular diseases.


Assuntos
Vasos Sanguíneos/patologia , Sinalização do Cálcio , Técnicas de Inativação de Genes , Proteínas Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Hemorragia Cerebral/patologia , Ácido Egtázico/farmacologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Harmina/farmacologia , Espaço Intracelular/metabolismo , Mutação/genética , Fenótipo , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidores
9.
Cell Mol Life Sci ; 76(13): 2593-2614, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30830239

RESUMO

Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.


Assuntos
Movimento Celular , Embrião não Mamífero/patologia , Neoplasias/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina , Animais , Embrião não Mamífero/metabolismo , Humanos , Microtúbulos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Transporte Proteico , Transdução de Sinais , Células Tumorais Cultivadas , Peixe-Zebra , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/genética
10.
Am J Hum Genet ; 104(3): 503-519, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827500

RESUMO

Although the use of model systems for studying the mechanism of mutations that have a large effect is common, we highlight here the ways that zebrafish-model-system studies of a gene, GRIK5, that contributes to the polygenic liability to develop eye diseases have helped to illuminate a mechanism that implicates vascular biology in eye disease. A gene-expression prediction derived from a reference transcriptome panel applied to BioVU, a large electronic health record (EHR)-linked biobank at Vanderbilt University Medical Center, implicated reduced GRIK5 expression in diverse eye diseases. We tested the function of GRIK5 by depletion of its ortholog in zebrafish, and we observed reduced blood vessel numbers and integrity in the eye and increased vascular permeability. Analyses of EHRs in >2.6 million Vanderbilt subjects revealed significant comorbidity of eye and vascular diseases (relative risks 2-15); this comorbidity was confirmed in 150 million individuals from a large insurance claims dataset. Subsequent studies in >60,000 genotyped BioVU participants confirmed the association of reduced genetically predicted expression of GRIK5 with comorbid vascular and eye diseases. Our studies pioneer an approach that allows a rapid iteration of the discovery of gene-phenotype relationships to the primary genetic mechanism contributing to the pathophysiology of human disease. Our findings also add dimension to the understanding of the biology driven by glutamate receptors such as GRIK5 (also referred to as GLUK5 in protein form) and to mechanisms contributing to human eye diseases.


Assuntos
Bancos de Espécimes Biológicos , Registros Eletrônicos de Saúde , Embrião não Mamífero/patologia , Oftalmopatias/patologia , Regulação da Expressão Gênica , Receptores de Ácido Caínico/genética , Doenças Vasculares/patologia , Animais , Embrião não Mamífero/metabolismo , Oftalmopatias/genética , Oftalmopatias/metabolismo , Genótipo , Humanos , Fenótipo , Receptores de Ácido Caínico/metabolismo , Doenças Vasculares/genética , Doenças Vasculares/metabolismo , Peixe-Zebra
11.
Aquat Toxicol ; 209: 168-177, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30784778

RESUMO

This study aimed to assess the sublethal effects of a platinum-based compound, cisplatin, using a zebrafish model. Zebrafish embryos were incubated in different concentrations of cisplatin at 0-96 h post-fertilization. Using a non-invasive, scanning ion-selective electrode technique (SIET), we measured the functions of hair cells (Ca2+ influx) and ionocytes ([H+] gradients). The survival rate, hatching rate, phenotype, body length, whole-body ion (Na+, Cl-, and Ca2+) and Pt contents were also determined. The effects of cisplatin on zebrafish embryos were demonstrated as first impairing hair cell function (at 1 µM of cisplatin), the hair cell number, and body ion content of Cl- (at 10 µM of cisplatin), then decreasing ionocyte acid secretion and overall body ion contents of Na+ and Ca2+ (at 50 µM of cisplatin). The body length and ionocyte density decreased at 100 µM of cisplatin, and survival decreased at 500 µM of cisplatin. As the cisplatin concentration increased, the accumulation of Pt in fish embryos also increased. These results revealed that hair cells are significantly more susceptible to cisplatin toxicity than ionocytes. By determining the lowest observed effective concentration of cisplatin that caused in vivo functional alterations of zebrafish hair cells and skin ionocytes, this model demonstrated 500-fold greater sensitivity than by detecting changes in survival, for early assessment of the effects of platinum-based chemotherapeutic drugs on fish.


Assuntos
Cisplatino/toxicidade , Embrião não Mamífero/patologia , Exposição Ambiental , Cabelo/patologia , Pele/patologia , Peixe-Zebra/embriologia , Animais , Cálcio/metabolismo , Cloretos/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Eletrodos Íon-Seletivos , Íons , Sódio/metabolismo , Análise de Sobrevida , Peixe-Zebra/anatomia & histologia , Proteínas de Peixe-Zebra/metabolismo
12.
J Biochem Mol Toxicol ; 33(5): e22289, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30657620

RESUMO

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 µg/L TCC from 4-hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development-related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.


Assuntos
Anti-Infecciosos/efeitos adversos , Carbanilidas/efeitos adversos , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Peixe-Zebra/embriologia , Animais , Anti-Infecciosos/farmacologia , Carbanilidas/farmacologia , Embrião não Mamífero/patologia , Disruptores Endócrinos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Poluentes Químicos da Água/farmacologia
13.
Toxicol Lett ; 302: 83-91, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30282005

RESUMO

INTRODUCTION: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC). METHOD: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (1) microRNA-122 (miR-122) expression (a hepatic marker), (2) free GSH concentration (a marker of oxidative stress) and (3) NAC antioxidant intervention. RESULTS: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had a significantly higher amount of GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively). CONCLUSION: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Entorpecentes/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Embrião não Mamífero/efeitos dos fármacos , Fígado/efeitos dos fármacos , Xenopus laevis/embriologia , Animais , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/embriologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Glutationa/metabolismo , Fígado/embriologia , Fígado/metabolismo , Fígado/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Oxidativo/efeitos dos fármacos
14.
FASEB J ; 33(1): 696-710, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30044923

RESUMO

The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I ( ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b ( tnni1b) ( Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.


Assuntos
Nó Atrioventricular/patologia , Embrião não Mamífero/patologia , Valvas Cardíacas/patologia , Miocárdio/patologia , Troponina I/antagonistas & inibidores , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Nó Atrioventricular/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Miocárdio/metabolismo , Organogênese , Ratos , Troponina I/genética , Troponina I/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
16.
Toxicol Sci ; 167(1): 15-25, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30011007

RESUMO

There is a clear need to establish and validate new methodologies to more quickly and efficiently screen chemicals for potential toxic effects, particularly on development. The emergence of alternative animal systems for rapid toxicology screens presents valuable opportunities to evaluate how systems complement each other. In this article, we compare a chemical library of 87-compounds in 2 such systems, developing zebrafish and freshwater planarians, by screening for developmental neurotoxic effects. We show that the systems' toxicological profiles are complementary to each other, with zebrafish yielding more detailed morphological endpoints and planarians more behavioral endpoints. Overall, zebrafish was more sensitive to this chemical library, yielding 86/87 hits, compared with 50/87 hits in planarians. The difference in sensitivity could not be attributed to molecular weight, log Kow, or the bioconcentration factor. Of the 87 chemicals, 28 had previously been evaluated in mammalian developmental neuro- (DNT), neuro-, or developmental toxicity studies. Of the 28, 20 were hits in the planarian, and 27 were hits in zebrafish. Eighteen of the 28 had previously been identified as DNT hits in mammals and were highly associated with activity in zebrafish and planarian behavioral assays in this study. Only 1 chemical (of 28) was a false negative in both zebrafish and planarian systems. The differences in endpoint coverage and system sensitivity illustrate the value of a dual systems approach to rapidly query a large chemical-bioactivity space and provide weight-of-evidence for prioritization of chemicals for further testing.


Assuntos
Alternativas aos Testes com Animais , Síndromes Neurotóxicas/etiologia , Planárias , Bibliotecas de Moléculas Pequenas/toxicidade , Testes de Toxicidade/métodos , Peixe-Zebra , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/patologia , Determinação de Ponto Final , Modelos Biológicos , Sensibilidade e Especificidade , Bibliotecas de Moléculas Pequenas/química , Especificidade da Espécie
17.
Nanoscale ; 11(7): 3173-3185, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30534785

RESUMO

As nano- and micro-sized plastics accumulate in the environment and the food chain of animals, including humans, it is imperative to assess the effects of nanoplastics in living organisms in a systematic manner, especially because of their ability to adsorb potential toxicants such as pollutants, heavy metals, and organic macromolecules that coexist in the environment. Using the zebrafish embryo as an animal model, we investigated the bioaccumulation and in vivo toxicity of polystyrene (PS) nanoplastics individually or in combination with the Au ion. We showed that smaller PS nanoplastics readily penetrated the chorion and developing embryos and accumulated throughout the whole body, mostly in lipid-rich regions such as in yolk lipids. We also showed that PS nanoplastics induced only marginal effects on the survival, hatching rate, developmental abnormalities, and cell death of zebrafish embryos but that these effects were synergistically exacerbated by the Au ion in a dose- and size-dependent manner. Such exacerbation of toxicity was well correlated with the production of reactive oxygen species and the pro-inflammatory responses synergized by the presence of PS, supporting the combined toxicity of PS and Au ions. The synergistic effect of PS on toxicity appeared to relate to mitochondrial damage as determined by ultrastructural analysis. Taken together, the effects of PS nanoplastics were marginal but could be a trigger for exacerbating the toxicity induced by other toxicants such as metal ions.


Assuntos
Embrião não Mamífero/metabolismo , Ouro/toxicidade , Nanopartículas/toxicidade , Poliestirenos/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/patologia , Íons/toxicidade
18.
Environ Sci Pollut Res Int ; 26(4): 3869-3881, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30539402

RESUMO

In this paper, the developmental toxicity and apoptosis in zebrafish (Danio rerio) embryos induced by 0.01, 0.05, and 0.10-Gy γ-ray irradiation were investigated and verified by single cell gel electrophoresis, acridine orange staining, flow cytometry, transmission electron microscopy, digital gene expression sequencing, and Western blot analysis. DNA damage, deformity rates, and apoptosis of zebrafish embryos were found to increase significantly with the increase of irradiation dose, and survival and hatching rates significantly decreased when the irradiation dose exceeds 0.10 and 0.05 Gy, respectively. Exposure to 0.10-Gy γ-ray irradiation resulted in the swelling of cell mitochondria of zebrafish embryos and changes in their intracellular vacuoles. mRNA and protein expression levels of Shh (sonic hedgehog 19 KDa) and Smo (smoothened 86 KDa) of Hh signaling pathway associated with the development of early embryos significantly increased with the increase of irradiation dose. Expression of the AKT (56 KDa) and PiK3r3 (55 KDa) genes, which are anti-apoptotic and involved with the PI3K/Akt signaling pathway, significantly decreased, while expression of the bada gene, which is pro-apoptotic, significantly increased. The results show that γ-ray irradiations of 0.01 and 0.05 Gy can induce developmental toxicity and apoptosis in zebrafish embryos via Hh and PI3K/Akt signaling pathways, respectively.


Assuntos
Apoptose/efeitos da radiação , Embrião não Mamífero/efeitos da radiação , Raios gama/efeitos adversos , Peixe-Zebra/crescimento & desenvolvimento , Animais , Dano ao DNA , Relação Dose-Resposta à Radiação , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Peixe-Zebra/genética
19.
Gen Comp Endocrinol ; 279: 53-59, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30395803

RESUMO

Parabens are widely used as antimicrobial preservatives. Recent studies have reported the endocrine disrupting effects of these chemicals, especially methylparaben. Previously, we have reported the alteration in Vtg gene expression upon exposure to environmentally relevant doses of methylparaben in zebrafish (Danio rerio) embryos. However, studies reporting neurobehavioural outcomes on exposure to methylparaben are limited. Therefore, this study was aimed at investigating the methylparaben-induced effects on developmental and neurobehavioural endpoints. Zebrafish embryos were exposed to sub-lethal concentrations of methylparaben: 0.1 ppb, 1 ppb, 10 ppb and 100 ppb. Alterations in developmental landmarks such as heart rate and hatching percentage were observed in embryos exposed to 10 ppb and 100 ppb of methylparaben. Results obtained from the novel tank diving test established that anxiety-like behaviour is induced in larvae exposed to 0.1 ppb and 1 ppb of methylparaben. A significant inhibition in the acetylcholinesterase (AChE) activity was also recorded in methylparaben-exposed groups. An increase in cortisol levels was observed in the exposed groups, which further supports the observations made in the novel tank diving test, establishing methylparaben as an anxiogenic agent even at sub-lethal concentrations. The underlying molecular mechanism needs further elucidation to investigate whether the behavioural effects are proximally or distally induced by early developmental exposure to methylparaben.


Assuntos
Acetilcolinesterase/metabolismo , Ansiedade/patologia , Comportamento Animal , Embrião não Mamífero/enzimologia , Embrião não Mamífero/patologia , Hidrocortisona/metabolismo , Parabenos/toxicidade , Peixe-Zebra/embriologia , Animais , Comportamento Animal/efeitos dos fármacos , Mergulho , Embrião não Mamífero/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/metabolismo , Poluentes Químicos da Água/toxicidade
20.
Ecotoxicol Environ Saf ; 170: 227-237, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529917

RESUMO

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 µM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50-60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 µM maneb. Larvae exposed to 0.5 and 1.0 µM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Maneb/toxicidade , Mitocôndrias/efeitos dos fármacos , Notocorda/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Feminino , Expressão Gênica , Locomoção/efeitos dos fármacos , Masculino , Mitocôndrias/patologia , Notocorda/patologia , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio , Praguicidas/toxicidade , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Peixe-Zebra/metabolismo
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