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1.
Ecotoxicol Environ Saf ; 203: 110994, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888603

RESUMO

The effects of cyanobacteria (Aphanizomenon flos-aquae (90%), Microcystis aeruginosa) and dense Elodea canadensis beds on the health endpoints of the amphipod Gmelinoides fasciatus and bivalve mollusc Unio pictorum were examined in mesocosms with simulated summer conditions (July-August 2018) in the environment of the Rybinsk Reservoir (Volga River Basin, Russia). Four treatments were conducted, including one control and three treatments with influencing factors, cyanobacteria and dense elodea beds (separately and combined). After 20 days of exposure, we evaluated the frequency of malformed and dead embryos in amphipods, heart rate (HR) and its recovery (HRR) after stress tests in molluscs as well as heat tolerance (critical thermal maximum or CTMax) in both amphipods and molluscs. The significant effect, such as elevated number of malformed embryos, was recorded after exposure with cyanobacteria (separately and combined with elodea) and presence of microcystins (MC) in water (0.17 µg/l, 40% of the most toxic MC-LR contribution). This study provided evidence that an elevated number (>5% of the total number per female) of malformed embryos in amphipods showed noticeable toxicity effects in the presence of cyanobacteria. The decreased oxygen under the influence of dense elodea beds led to a decrease in HR (and an increase in HRR) in molluscs. The notable effects on all studied biomarkers, embryo malformation frequency and heat tolerance in the amphipod G. fasciatus, as well as the heat tolerance and heart rate in the mollusc U. pictorum, were found when both factors (elodea and cyanobacteria) were combined. The applied endpoints could be further developed for environmental monitoring, but the obtained results support the importance of the combined use of several biomarkers and species, especially in the case of multi-factor environmental stress.


Assuntos
Anfípodes/efeitos dos fármacos , Bivalves/efeitos dos fármacos , Cianobactérias/metabolismo , Monitoramento Ambiental/métodos , Hydrocharitaceae/metabolismo , Poluentes Químicos da Água/toxicidade , Anfípodes/metabolismo , Animais , Aphanizomenon/metabolismo , Biomarcadores/análise , Bivalves/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Microcistinas/metabolismo , Microcistinas/toxicidade , Microcystis/metabolismo , Federação Russa , Poluentes Químicos da Água/metabolismo
2.
Ecotoxicol Environ Saf ; 203: 111043, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888597

RESUMO

Intraspecific difference in toxicity brings uncertainty to ecological risk assessment (ERA) and water quality criteria (WQC) of chemicals. Here, we compared intraspecies sensitivity to toxicants for Mesocyclops leuckarti of which toxicity data was obtained from published literatures, and zebrafish Danio rerio of which toxicity data was done in this study). Due to the internal concentration of chemicals not measured, simplified toxicokinetic-toxicodynamic (TK-TD) models were used, and we investigated whether TK-TD parameters estimated by Bayesian method might represent the differences in sensitivity between life-stages of 2 species. The results demonstrated that the difference in TK-TD parameters (background mortality m0, no effect concentration NEC, the killing rate ks, and the dominant rate kd) could represent the toxicity difference between life-stages of individual species. The TK-TD model could predict toxicity in individual species (Cyprinus carpio L., Enchytraeus crypticus, Folsomia candida, Hyalella Azteca) exposed to different chemical concentrations and successfully extrapolate toxicity between different life stages of Mesocyclops leuckarti and Danio rerio by scaling several TK-TD parameters. The modified TK-TD model on the extrapolation toxicity of chemicals between life stages for species could be useful for the ERA and for deriving and revising WQC for chemicals.


Assuntos
Carpas/metabolismo , Copépodes/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Modelos Biológicos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Teorema de Bayes , Bioacumulação , Carpas/crescimento & desenvolvimento , Copépodes/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Larva/metabolismo , Medição de Risco , Especificidade da Espécie , Toxicocinética , Peixe-Zebra/crescimento & desenvolvimento
3.
Ecotoxicol Environ Saf ; 205: 111339, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32961491

RESUMO

Famoxadone-cymoxanil is a new protective and therapeutic fungicide, but little research has been done on it or its toxicity in aquatic organisms. In this study, we used zebrafish to investigate the cardiotoxicity of famoxadone-cymoxanil and the potential mechanisms involved. Zebrafish embryos were exposed to different concentrations of famoxadone-cymoxanil until 72 h post-fertilization (hpf), then changes of heart morphology in zebrafish embryos were observed. We also detected the levels of oxidative stress, myocardial-cell proliferation and apoptosis, ATPase activity, and the expression of genes related to the cardiac development and calcium-signaling pathway. After famoxadone-cymoxanil exposure, pericardial edema, cardiac linearization, and reductions in the heart rate and cardiac output positively correlated with concentration. Although myocardial-cell apoptosis was not detected, proliferation of the cells was severely reduced and ATPase activity significantly decreased, resulting in a severe deficiency in heart function. In addition, indicators of oxidative stress changed significantly after exposure of the embryos to the fungicide. To better understand the possible molecular mechanisms of cardiovascular toxicity in zebrafish, we studied the transcriptional levels of cardiac development, calcium-signaling pathways, and genes associated with myocardial contractility. The mRNA expression levels of key genes in heart development were significantly down-regulated, while the expression of genes related to the calcium-signaling pathway (ATPase [atp2a1], cardiac troponin C [tnnc1a], and calcium channel [cacna1a]) was significantly inhibited. Expression of klf2a, a major endocardial flow-responsive gene, was also significantly inhibited. Mechanistically, famoxadone-cymoxanil toxicity might be due to the downregulation of genes associated with the calcium-signaling pathway and cardiac muscle contraction. Our results found that famoxadone-cymoxanil exposure causes cardiac developmental toxicity and severe energy deficiency in zebrafish.


Assuntos
Acetamidas/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Fungicidas Industriais/toxicidade , Coração/efeitos dos fármacos , Estrobilurinas/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Cardiotoxicidade , Regulação para Baixo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/embriologia , Frequência Cardíaca/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Ecotoxicol Environ Saf ; 205: 111165, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32836160

RESUMO

BACKGROUND: Bisphenol A (BPA) is a well-known xenobiotic endocrine disrupting chemical, with estrogenic activity and many other potential biological effects. Although multiple toxicities have been reported for BPA, molecular mechanisms underlying the transgenerational toxic effects of BPA are still underestimated. METHODS: Parental F0 fish were exposed to 1.0 µM BPA or control (0.1% DMSO, v/v) for 7 days. Eggs (F1) were collected and kept in control medium until 4.5 or 120 h post fertilization (hpf). RNA sequencing (RNA-seq) was conducted on embryos and larvae, to discover differentially expressed genes (DEGs), and then KEGG pathway, GO enrichment and GSEA were performed to interpret functional ontology. Histopathology was performed to explore the morphological and structural alterations in liver tissues of zebrafish larvae (120 hpf) after parental BPA exposure. RESULTS: Parental BPA exposure induced global transcriptomic changes in zebrafish embryos and larvae. For embryos, epigenetic regulation genes were decidedly affected, highlighted epigenotoxicity might involve in the transgenerational effects during embryogenesis and early development. By further investigation on its delayed effects, our RNA-Seq data of larvae suggested ROS metabolic process, apoptosis, p53 and MAPK signaling pathway were concentrated, indicating defensive cellular processes still involved in protecting against BPA toxicity. Furthermore, parental BPA-treated larvae manifested hepatic injury by histopathological analysis. CONCLUSIONS: Parental BPA exposure led to global transcriptomic changes involved in epigenetic regulation, oxidative stress, apoptosis and DNA damage of offspring. These findings advanced the field of the parental-mediated subsequent generational toxic effects of BPA.


Assuntos
Compostos Benzidrílicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Larva/efeitos dos fármacos , Fenóis/toxicidade , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Larva/genética , Análise de Sequência de RNA , Peixe-Zebra/metabolismo
5.
Ecotoxicol Environ Saf ; 204: 111068, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32745784

RESUMO

Herein, eight common endocrine disrupting chemicals (EDCs) were exposed to zebrafish (Danio rerio) to investigate the relationship between different EDCs and their activated estrogen receptors. Under acute exposure, we identified five major malformation types whose incidence and deformity modes differed among EDCs. Luciferase analysis divided the EDC receptors into four categories: (i) triclosan (TCS), 17ß-estradiol (E2) and estriol (E3) mainly activated GPER expression; (ii) bisphenol A (BPA), p-(tert-octyl) phenol (POP), 17α-ethynylestradiol (EE2), E2 and E3 activated ERß expression; (iii) E2 and E3 acted on both GPER and ERß; and (iv) estrone (E1) and 9,9-bis(4-hydroxyphenyl)fluorene (BHPF) had little effect on the two receptors. In vivo immunofluorescence experiments on 96-hpf larvae provided evidence that TCS and POP acted on GPER and ERß, respectively, while E2 acted on the two receptors simultaneously. Luciferase activities in the promoter regions of gper (-986 to -488) and erß (-1998 to -1496) were higher than those in other regions, identifying these key regions as targets for transcription activity. TCS promoted GPER expression by acting on the JUND transcription factor, while POP promoted ERß expression by activating the Foxl1 transcription factor. In contrast, E2 mainly regulated transcription of GPER and ERß by Arid3a. These findings provide compelling evidence that different EDCs possess varying estrogen receptors, leading to differential regulatory pathways and abnormality symptoms. These results offer an experimental strategy and fundamental information to assess the molecular mechanisms of EDC-induced estrogen effects.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Receptor beta de Estrogênio/metabolismo , Fenóis/toxicidade , Receptores Acoplados a Proteínas-G/metabolismo , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Compostos Benzidrílicos/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Fenóis/metabolismo , Poluentes Químicos da Água/metabolismo
6.
Chemosphere ; 260: 127609, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32693259

RESUMO

The environmental contaminant 3,3'-dichlorobiphenyl (PCB-11) is widely detected in environmental samples, and this parent compound along with its metabolites 4-OH-PCB-11 and 4-PCB-11-Sulfate are detected in human serum. Our previous research in zebrafish (Danio rerio) embryos shows exposure to 20 µM PCB-11 inhibits Cyp1a enzyme activity and perturbs lipid metabolism pathways. In this study, wildtype AB embryos underwent acute exposures from 1 to 4 days post fertilization (dpf) to 0.002-20 µM 4-OH-PCB-11 or 0.2-20 µM 4-PCB-11-Sulfate, with and without co-exposures to 100 µg/L benzo[a]pyrene (B[a]P) or 5 nM 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and were assessed for in vivo EROD activity and morphometrics. Chronic exposures from 1 to 15 dpf to assess lipid accumulation using Oil-Red-O staining were also conducted with 0.2 µM parent or metabolite compounds, alongside a co-exposure experiment of 0.002-0.2 µM 4-PCB-11-Sulfate and 10 µg/L B[a]P. For acute experiments, 2 and 20 µM 4-OH-PCB-11 was lethal but no Cyp1a or morphological effects were observed at lower concentrations; 20 µM 4-PCB-11-Sulfate significantly lowered the Cyp1a activity of B[a]P and PCB-126 but did not alter morphological development. For chronic experiments, 0.2 µM 4-PCB-11-Sulfate significantly increased lipid accumulation 30% in single exposures and 44% in co-exposures with B[a]P. Further long-term studies would better elucidate the effects of this contaminant, particularly in the context of environmentally-relevant mixtures.


Assuntos
Bifenilos Policlorados/toxicidade , Peixe-Zebra/fisiologia , Animais , Benzo(a)pireno/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Embrião não Mamífero/metabolismo , Larva/metabolismo , Lipídeos , Fígado/metabolismo , Bifenilos Policlorados/metabolismo , Sulfatos/metabolismo , Peixe-Zebra/metabolismo
7.
Nat Commun ; 11(1): 3317, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620775

RESUMO

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.


Assuntos
Embrião não Mamífero/metabolismo , Mitose , Receptores de Peptídeos/metabolismo , Esfingolipídeos/metabolismo , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Divisão Celular Assimétrica/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Lipoilação , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismo , Receptores de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Fuso Acromático/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
Aquat Toxicol ; 225: 105525, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32629302

RESUMO

Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 µM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 µM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.


Assuntos
Dipeptídeos/toxicidade , Desinfetantes/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Hipopigmentação/induzido quimicamente , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Expressão Gênica/efeitos dos fármacos , Halogenação , Hipopigmentação/genética , Melanóforos/efeitos dos fármacos , Melanóforos/metabolismo , Purificação da Água , Proteínas de Peixe-Zebra/genética
9.
Int J Nanomedicine ; 15: 4407-4415, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606685

RESUMO

Objective: Silica nanoparticles (SiO2 NPs) have been extensively employed in biomedical field. SiO2 NPs are primarily designed to enter the circulatory system; however, little information is available on potential adverse effects of SiO2 NPs on the nervous system. Methods: The neurotoxicity of SiO2 NPs at different concentrations (3, 6, 12 ng/nL) on zebrafish embryos was determined using immunofluorescence and microarray techniques, and subsequently confirmed by qRT-PCR. Results: SiO2 NPs disrupt the axonal integrity and decrease the length of axons in Tg (NBT: EGFP) transgenic lines. The number of apoptotic cells in the brain and central nervous system of zebrafish embryos was increased in the presence of 12 ng/nL of SiO2 NPs, but the difference did not reach statistical significance. Screening for changes in the expression of genes involved in the neuroactive ligand-receptor interaction pathway was performed by microarray and confirmed by qRT-PCR. These analyses demonstrated that SiO2 NPs markedly downregulated genes associated with neural function (grm6a, drd1b, chrnb3b, adrb2a, grin2ab, npffr2.1, npy8br, gabrd, chrma3, gabrg3, gria3a, grm1a, adra2b, and glra3). Conclusion: The obtained results documented that SiO2 NPs can induce developmental neurotoxicity by affecting the neuroactive ligand-receptor interaction signaling pathway. This new evidence may help to clarify the mechanism of SiO2 NPs-mediated neurotoxicity.


Assuntos
Embrião não Mamífero/metabolismo , Nanopartículas/toxicidade , Neurotoxinas/toxicidade , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Dióxido de Silício/toxicidade , Peixe-Zebra/embriologia , Animais , Apoptose/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Ligantes , Transdução de Sinais/efeitos dos fármacos
10.
Aquat Toxicol ; 226: 105560, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32659603

RESUMO

Triclosan (TCS) is commonly used in home and personal care products (HPCPs), which causes it to be ubiquitously detected in aquatic environments. The toxicity of triclosan to aquatic organisms can vary at different pH values because the ionization states of TCS affect its bioaccumulation properties. The objective of this study was to examine the pH-dependent toxicity of TCS on embryonic zebrafish (Danio rerio) using a metabolomic profiling method based on gas chromatography-mass spectrometry (GC-MS). Exposure experiments were conducted on zebrafish embryos at three pH conditions (6, 7, and 8) and two TCS concentrations (30 µg/L and 300 µg/L). Metabolic profiles were obtained by extracting intracellular metabolites. Univariate (One-way ANOVA) and multivariate (PLS-DA) analyses were conducted to determine the metabolomic changes in TCS-treated embryos. Changes in the metabolic profile revealed that interference in biological pathways were induced by mostly ionized TCS (low pH) and high TCS concentrations. Also, fold changes in metabolite profiles showed that the TCS toxicity was a function of pH. Metabolites including urea, D-glucose, D-galactose, phenylalanine, L-glutamic acid, citric acid, and phosphoric acid showed significant changes under different pH conditions (p-value < 0.05). Our metabolomics study revealed that the responses of metabolites to TCS toxicity were pH-dependent. The differences of the responses could be attributed to the bioaccumulation capability of TCS, which increased as the ionized TCS proportion increased under low pH conditions.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Triclosan/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Concentração de Íons de Hidrogênio , Metabolômica/métodos , Peixe-Zebra/crescimento & desenvolvimento
11.
Aquat Toxicol ; 226: 105562, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32668346

RESUMO

Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRß) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17ß-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Esteroides/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Receptores Androgênicos/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
12.
Aquat Toxicol ; 226: 105558, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32673888

RESUMO

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins and dioxin-like compounds (DLCs) in vertebrates. Two clades of the Ahr family exist in teleosts (Ahr1 and Ahr2), and it has been demonstrated that Ahr2 is the main protein involved in mediating the toxicity of dioxins and DLCs in most teleost species. Recently, we characterized the Atlantic cod (Gadus morhua) Ahr1a and Ahr2a receptors. To further explore a possible subfunction partitioning of Ahr1a and Ahr2a in Atlantic cod we have mapped the expression and localization of ahr1a and ahr2a in early developmental stages. Atlantic cod embryos were continuously exposed in a passive-dosing exposure system to the Ahr agonist, benzo[a]pyrene (B[a]P), from five days post fertilization (dpf) until three days post hatching (dph). Expression of ahr1a, ahr2a, and the Ahr-target genes, cyp1a and ahrrb, was assessed in embryos (8 dpf and 10 dpf) and larvae (3 dph) with quantitative real-time PCR analyses (qPCR), while in situ hybridization was used to assess the localization of expression of ahr1a, ahr2a and cyp1a. Quantitative measurements showed an increased cyp1a expression in B[a]P-exposed samples at all sampling points, and for ahr2a at 10 dpf, confirming the activation of the Ahr-signalling pathway. Furthermore, B[a]P strongly induced ahr2a and cyp1a expression in the cardiovascular system and skin, respectively, of embryos and larvae. Induced expression of both ahr2a and cyp1a was also revealed in the liver of B[a]P-exposed larvae. Our results suggest that Ahr2a is the major subtype involved in mediating responses to B[a]P in early developmental stages of Atlantic cod, which involves transcriptional regulation of biotransformation genes, such as cyp1a. The focused expression of ahr1a in the eye of embryos and larvae, and the presence of ahr2a transcripts in the jaws and fin nodes, further indicate evolved specialized roles of the two Ahrs in ontogenesis.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Gadus morhua/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Olho/efeitos dos fármacos , Olho/embriologia , Olho/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Poluentes Químicos da Água/toxicidade
13.
Nat Cell Biol ; 22(7): 803-814, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572169

RESUMO

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.


Assuntos
Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Forminas/metabolismo , Melanoma/patologia , Proteínas Musculares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blástula/citologia , Blástula/metabolismo , Forma Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Forminas/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas Musculares/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
14.
J Vis Exp ; (160)2020 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-32568253

RESUMO

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.


Assuntos
Caenorhabditis elegans/metabolismo , Programas de Rastreamento , Chaperonas Moleculares/metabolismo , Especificidade de Órgãos , Animais , Bioensaio , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Epistasia Genética , Mitocôndrias/metabolismo , Óvulo/metabolismo , Fenótipo , Ligação Proteica , Dobramento de Proteína , Proteoma/metabolismo , Interferência de RNA , Reprodutibilidade dos Testes
15.
Ecotoxicol Environ Saf ; 201: 110820, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32531574

RESUMO

Growth hormone (GH)/insulin-like growth factor (IGF) axis plays a critical role in fetal development. However, the effect of arsenite exposure on the GH/IGF axis and its toxic mechanism are still unclear. Zebrafish embryos were exposed to a range of NaAsO2 concentrations (0.0-10.0 mM) between 4 and 120 h post-fertilization (hpf). Development indexes of survival, malformation, hatching rate, heart rate, body length and locomotor behavior were measured. Hormone levels, GH/IGF axis-related genes, and nerve-related genes were also tested. The results showed that survival rate, hatching rate, heart rate, body length and locomotor behavior all decreased, while deformity increased. At 120 hpf, the survival rate of zebrafish in 1.5 mM NaAsO2 group was about 70%, the deformity rate exceeded 20%, and the body length shortened to 3.35 mm, the movement distance of zebrafish decreased approximately 63.6% under light condition and about 52.4% under dark condition. The level of GH increased and those of IGF did not change significantly, while the expression of GH/IGF axis related genes (ghra, ghrb, igf2r, igfbp3, igfbp2a, igfbp5b) and nerve related genes (dlx2, shha, ngn1, elavl3, gfap) decreased. In 1.5 mM NaAsO2 group, the decrease of igfbp3 and igfbp5b was almost obvious, about 78.2% and 72.2%. The expression of nerve genes in 1.5 mM NaAsO2 group all have declined by more than 50%. These findings suggested that arsenite exerted disruptive effects on the endocrine system by interfering with the GH/IGF axis, leading to zebrafish embryonic developmental toxicity.


Assuntos
Arsenitos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Somatomedinas/metabolismo , Peixe-Zebra , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/embriologia , Sistema Endócrino/metabolismo , Hormônio do Crescimento/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Transdução de Sinais , Somatomedinas/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
16.
Nat Commun ; 11(1): 2796, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493965

RESUMO

Cell fate decisions involved in vascular and hematopoietic embryonic development are still poorly understood. An ETS transcription factor Etv2 functions as an evolutionarily conserved master regulator of vasculogenesis. Here we report a single-cell transcriptomic analysis of hematovascular development in wild-type and etv2 mutant zebrafish embryos. Distinct transcriptional signatures of different types of hematopoietic and vascular progenitors are identified using an etv2ci32Gt gene trap line, in which the Gal4 transcriptional activator is integrated into the etv2 gene locus. We observe a cell population with a skeletal muscle signature in etv2-deficient embryos. We demonstrate that multiple etv2ci32Gt; UAS:GFP cells differentiate as skeletal muscle cells instead of contributing to vasculature in etv2-deficient embryos. Wnt and FGF signaling promote the differentiation of these putative multipotent etv2 progenitor cells into skeletal muscle cells. We conclude that etv2 actively represses muscle differentiation in vascular progenitors, thus restricting these cells to a vascular endothelial fate.


Assuntos
Vasos Sanguíneos/citologia , Perfilação da Expressão Gênica , Músculo Esquelético/citologia , Análise de Célula Única , Células-Tronco/metabolismo , Proteínas de Peixe-Zebra/deficiência , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Diferenciação Celular/genética , Movimento Celular , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico , Modelos Biológicos , Mutação/genética , Somitos/metabolismo , Transcrição Genética , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Ecotoxicol Environ Saf ; 201: 110808, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32516676

RESUMO

Cyprodinil is a broad-spectrum pyrimidine amine fungicide that has been reportedly used worldwide. However, toxicity studies of cyprodinil on aquatic organisms, specifically zebrafish (Danio rerio), are lacking. In our present study, we predicted cyprodinil binding to the aryl hydrocarbon receptor (AhR) by using molecular docking simulation. Then, we used recombinant HepG2 cells and Tg(cyp1a1-12DRE:egfp) transgenic zebrafish to further assess the AhR agonistic activity of cyprodinil. Besides, the significant upregulation of cyp1a1 further verified that statement. Moreover, we found that zebrafish exposure to cyprodinil induced developmental toxicity in the larvae, particularly during cardiac development. The expression levels of cardiac development-related genes, namely tbx5, nkx2.5, gata4, and tnnt2, were markedly altered, which might cause the adverse effects of cyprodinil on cardiac function and development. In summary, we found that cyprodinil, as an AhR agonist, induced development toxicity in zebrafish larvae, especially on cardiac. Data here can assess the potential effects on organisms in the aquatic environment and promote the regulation and safe use of cyprodinil.


Assuntos
Animais Geneticamente Modificados/metabolismo , Larva/efeitos dos fármacos , Pirimidinas/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fungicidas Industriais/metabolismo , Coração/efeitos dos fármacos , Coração/embriologia , Células Hep G2 , Humanos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Organogênese/efeitos dos fármacos , Organogênese/genética , Ligação Proteica , Peixe-Zebra/genética
18.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510506

RESUMO

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.


Assuntos
Culex/embriologia , Culex/genética , Edição de Genes , Microinjeções/métodos , Mosquitos Vetores/genética , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/patogenicidade , Animais , Culex/virologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/virologia , Feminino , Mutagênese Sítio-Dirigida , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-32427057

RESUMO

Embryos from the marine medaka fish Oryzias javanicus were treated with eight concentrations of benzo[a]pyrene (BaP) (0.001, 0.01, 0.1, 1, 2, 5, 10, or 20 µg L--1) after they had been fertilized. Significant mortality and hatching delays were detected in embryos that had been exposed to 10 and 20 µg L-1 BaP for 4 weeks. The mortality rate after hatching was higher in the medaka that had been previously exposed to > 2 µg L-1 BaP. Significant elevations in intracellular reactive oxygen species and malondialdehyde contents were measured and the mRNA expressions of the antioxidant defense system genes (gst, sod, cat, and gpx) increased in the embryos exposed to 10 and 20 µg L-1 BaP for 1 week. The hsp70, ahr, and cyp1a transcriptional responses were also significantly upregulated in the exposed groups after 1 week. The alterations to the in vivo parameters and molecular components suggested that waterborne BaP had a toxic effect on marine medaka embryos. Finally, fin defects, spinal curvature, and cardiac edema were highly induced when the embryos were exposed to > 5 µg L-1 BaP.


Assuntos
Benzo(a)pireno/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Oryzias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Benzo(a)pireno/análise , Biomarcadores/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Oryzias/genética , Estresse Oxidativo/genética , Poluentes Químicos da Água/análise
20.
Toxicol Appl Pharmacol ; 398: 115029, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376357

RESUMO

Resveratrol (RSV), a natural polyphenolic compound commonly found in food, has antioxidant and aryl hydrocarbon receptor (AHR) antagonist effects. We have recently demonstrated that AHR mediated reactive oxygen species (ROS) generation contributes to the cardiac developmental toxicity of ambient fine particle matter (PM2.5). Thus, we hypothesized that RSV protects against the cardiac developmental toxicity of PM2.5 by inhibiting ROS generation and AHR activity. To test this concept, we exposed zebrafish embryos to extractable organic matter (EOM) from PM2.5 in the presence or absence of RSV. We found that RSV significantly counteracted EOM-induced cardiac malformations in zebrafish embryos. The EOM-induced ROS production, DNA damage and apoptosis in the heart of zebrafish embryos were also counteracted by RSV supplementation. Furthermore, RSV attenuated EOM-induced changes in the expression of genes involved in cardiac development (nkx2.5, sox9b, axin2), oxidative stress (nrf2a, nrf2b, gstp1, gstp2, sod1, sod2, cat) and apoptosis (p53, bax). However, RSV did not suppress EOM-induced AHR activity. In conclusion, our data indicates that RSV protects against the PM2.5-induced heart malformations by inhibiting oxidative stress rather than through AHR antagonism.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/tratamento farmacológico , Material Particulado/efeitos adversos , Substâncias Protetoras/farmacologia , Resveratrol/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Cardiopatias Congênitas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
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