RESUMO
Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and ß-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Nitrocompostos/farmacologia , Ácidos Oleicos/farmacologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organogênese/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Oestradiol (E2) is a critical factor for multiple systems' development during the embryonic period. Here, we aimed to investigate the effects of oestradiol on intrahepatic bile duct development, which may allow a better understanding of congenital bile duct dysplasia. DLK+ hepatoblasts were extracted from the C57BL/6CrSlc foetal mice and randomly divided into control group, oestradiol groups (1, 10, 100 nM) and oestradiol (10 nM) + DAPT (inhibitor of Notch signalling; 40 µM) group for in vitro experiments. For in vivo analysis, pregnant mice were divided into control group, oestradiol (intraperitoneal injection of 0.6 mg/kg/day) ± DAPT (subcutaneous injection of 10 mg/kg/day) groups and tamoxifen (gavage administration of 0.4 mg/kg/day) group. The results showed that oestradiol promoted hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development during the embryonic period. Tamoxifen, an antioestrogenic drug, inhibited the above processes. Moreover, oestradiol promoted the expression of Notch signalling pathway-associated proteins and genes both in vitro and in vivo. Notably, DAPT addition inhibited the oestradiol-mediated effects. In conclusion, oestradiol can promote hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development of C57BL/6CrSlc mice during embryonic period via the Notch signalling pathway.
Assuntos
Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/metabolismo , Estradiol/metabolismo , Organogênese , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Diferenciação Celular , Células Cultivadas , Estradiol/farmacologia , Expressão Gênica , Hepatócitos/metabolismo , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
The brominated flame retardants (BFRs), 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane (TBECH) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) bind to the androgen receptor (AR). in vitro bioassays have shown that TBECH is a potent androgen agonist while DPTE is a potent AR antagonist. Both TBECH and DPTE alter gene expression associated with AR regulation. However, it remains to be determined if TBECH and DPTE can affect the prostate. For this reason, we exposed CD1 mice to a 1:1 mixture of TBECH diastereomers α and ß, a 1:1 mixture of γ and δ, and to DPTE, and tested their effects on prostate growth, histology and gene expression profiles. Castrated mice were used to study the androgenic effects of TBECHαß and TBECHγδ while the antagonistic effects of DPTE were studied in non-castrated mice. We observed that testosterone and TBECHγδ increased body and prostate weights while TBECHαß affected neither of them; and that DPTE had no effect on body weight but reduced prostate weight drastically. Histomorphometric analysis of the prostate revealed epithelial and glandular alterations in the TBECHγδ group comparable to those in testosterone group while alterations in the TBECHαß group were less pronounced. DPTE displayed androgen antagonist activity reminiscent of castration. The transcription profile of the prostate was altered by castration and exposure to testosterone and to TBECHγδ reversed several of these changes. Testosterone and TBECHγδ also regulated the expression of several androgen responsive genes implicated in prostate growth and cancer. While DPTE resulted in a drastic reduction in prostate weight, it only affected a small number of genes. The results indicate that TBECHγδ and DPTE are of high human health concern as they may contribute to changes in prostate growth, histology and function.
Assuntos
Cicloexanos/toxicidade , Disruptores Endócrinos/toxicidade , Retardadores de Chama/toxicidade , Hidrocarbonetos Bromados/toxicidade , Próstata/efeitos dos fármacos , Antagonistas de Androgênios , Antagonistas de Receptores de Andrógenos , Androgênios , Animais , Linhagem Celular Tumoral , Disruptores Endócrinos/metabolismo , Expressão Gênica/efeitos dos fármacos , Halogenação , Humanos , Masculino , Camundongos , Organogênese/efeitos dos fármacos , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Receptores Androgênicos/metabolismoRESUMO
Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.
Assuntos
Córtex Cerebral/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Organogênese , Transdução de Sinais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organogênese/efeitos dos fármacos , Fator de Transcrição PAX6/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Redox regulation during metazoan development ensures that coordinated metabolic reprogramming and developmental signaling are orchestrated with high fidelity in the hypoxic embryonic environment. Valproic acid (VPA), an anti-seizure medication, is known to increase markers of oxidation and also increase the risk of neural tube defects (NTDs) when taken during pregnancy. It is unknown, however, whether oxidation plays a direct role in failed neural tube closure (NTC). Spatial and temporal fluctuations in total glutathione (GSH) and total cysteine (Cys) redox steady states were seen during a 24 h period of CD-1 mouse organogenesis in untreated conceptuses and following exposure to VPA and the Nrf2 antioxidant pathway inducer, 1,2-dithiole-3-thione (D3T). Glutathione, glutathione disulfide (GSSG), and Cys, cystine (CySS) concentrations, measured in conceptal tissues (embryo/visceral yolk sac) and fluids (yolk sac fluid/amniotic fluid) showed that VPA did not cause extensive and prolonged oxidation during the period of NTC, but instead produced transient periods of oxidation, as assessed by GSH:GSSG redox potentials, which revealed oxidation in all four conceptal compartments at 4, 10, and 14 h, corresponding to the period of heartbeat activation and NTC. Other changes were tissue and time specific. VPA treatment also reduced total FITC-Ab clearance from the medium over 3 h, indicating potential disruption of nutritive amino acid supply. Overall, these results indicated that VPA's ability to affect cellular redox status may be limited to tissue-specific windows of sensitivity during the period of NTC. The safety evaluation of drugs used during pregnancy should consider time and tissue specific redox factors.
Assuntos
Anticonvulsivantes/toxicidade , Antineoplásicos/toxicidade , Embrião de Mamíferos/efeitos dos fármacos , Tionas/toxicidade , Tiofenos/toxicidade , Ácido Valproico/toxicidade , Aminoácidos/metabolismo , Animais , Cisteína/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Troca Materno-Fetal , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Organogênese/efeitos dos fármacos , Oxirredução , GravidezRESUMO
The development of the female reproductive tract can be divided into three parts consisting of Müllerian duct organogenesis, pre-sexual maturation organ development, and post-sexual maturation hormonal regulation. In primates, Müllerian duct organogenesis proceeds in an estrogen independent fashion based on transcriptional pathways that are suppressed in males by the presence of AMH and SRY. However, clinical experience indicates that exposure to xenoestrogens such as diethylstilbestrol (DES) during critical periods including late organogenesis and pre-sexual maturational development can have substantial effects on uterine morphology, and confer increased risk of disease states later in life. Recent evidence has demonstrated that these effects are in part due to epigenetic regulation of gene expression, both in the form of aberrant CpG methylation, and accompanying histone modifications. While xenoestrogens and selective estrogen receptor modulators (SERMS) both can induce non-canonical binding confirmations in estrogen receptors, the primate specific fetal estrogens Estriol and Estetrol may act in a similar fashion to alter gene expression through tissue specific epigenetic modulation.
Assuntos
Metilação de DNA/genética , Estrogênios/genética , Genitália Feminina/crescimento & desenvolvimento , Organogênese/genética , Animais , Metilação de DNA/efeitos dos fármacos , Dietilestilbestrol/farmacologia , Epigênese Genética/genética , Estradiol/metabolismo , Estrogênios/metabolismo , Feminino , Genitália Feminina/metabolismo , Humanos , Ductos Paramesonéfricos/efeitos dos fármacos , Ductos Paramesonéfricos/crescimento & desenvolvimento , Ductos Paramesonéfricos/metabolismo , Organogênese/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimentoRESUMO
The developing uterus is highly sensitive to a brief exposure to different substances, in particular those with endocrine-disrupting activity. Thus, exposure to environmental, nutritional, chemical, and other xenobiotic factors affecting signaling events during critical organizational periods can alter the normal course of uterine development with lasting consequences. In this chapter, we provide an experimental protocol to evaluate the development of the rat uterus as a toxicity biomarker at two different developmental time points: (1) the neonatal period, on postnatal day (PND) 8, and (2) the prepubertal period, on PND21. In this experimental approach, we propose to assess: (1) uterine morphology and cytodifferentiation, (2) uterine cell proliferation, and (3) the expression of proteins involved in uterine organogenetic differentiation. All these morphological and molecular markers are useful tools to determine the consequences of exposure to toxicants with the potential to disrupt the uterine development.
Assuntos
Testes de Toxicidade , Útero/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Microscopia , Organogênese/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Útero/metabolismo , Útero/patologiaRESUMO
Primary cilia function as critical signaling hubs whose absence leads to severe disorders collectively known as ciliopathies; our knowledge of ciliogenesis remains limited. We show that Smo induces ciliogenesis through two distinct yet essential noncanonical Hh pathways in several cell types, including neurons. Surprisingly, ligand activation of Smo induces autophagy via an LKB1-AMPK axis to remove the satellite pool of OFD1. This is required, but not sufficient, for ciliogenesis. Additionally, Smo activates the Gαi-LGN-NuMA-dynein axis, causing accumulation of a portion of OFD1 at centrioles in early ciliogenesis. Both pathways are critical for redistribution of BBS4 from satellites to centrioles, which is also mediated by OFD1 centriolar translocation. Notably, different Smo agonists, which activate Smo distinctly, activate one or the other of these pathways; only in combination they recapitulate the activity of Hh ligand. These studies provide new insight into physiological stimuli (Hh) that activate autophagy and promote ciliogenesis and introduce a novel role for the Gαi-LGN-NuMA-dynein complex in this process.
Assuntos
Autofagia , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Organogênese , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Adenilato Quinase/metabolismo , Autofagia/efeitos dos fármacos , Corpos Basais/efeitos dos fármacos , Corpos Basais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Centríolos/efeitos dos fármacos , Centríolos/metabolismo , Cílios/efeitos dos fármacos , Dineínas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organogênese/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Piridinas/farmacologia , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Soro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/agonistas , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismoRESUMO
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/genéticaRESUMO
The aim of this study was to evaluate the impact of prenatal testosterone exposure on prostate development in male and female neonatal gerbils. Pregnant females were exposed to subcutaneous injections of testosterone cypionate (500 µg/animal) at gestational days 20 and 22. Male and female pups were then euthanized at postnatal day 1. Morphological analysis showed that females were severely affected by androgen exposure. We also observed that male and female urogenital sinus (UGS) responded differentially to testosterone treatment, demonstrating heterogeneous immunostaining for the androgen receptor (AR), estrogen receptor alpha (ERα), and proliferating cell nuclear antigen (PCNA). Smooth muscle α-actin (α-SMA) analysis showed that testosterone delays the myodifferentiation, allowing buds to reach the ectopic mesenchymes of the female UGS. Our data showed that abnormal testosterone exposure disrupted prostate organogenesis, altered the expression patterns of important markers, and demonstrated that female UGS was particularly influenced by androgen exposure during a critical window in the developmental period.
Assuntos
Organogênese/efeitos dos fármacos , Próstata/crescimento & desenvolvimento , Testosterona/farmacologia , Animais , Receptor alfa de Estrogênio/metabolismo , Feminino , Gerbillinae , Imageamento Tridimensional , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Próstata/anatomia & histologia , Próstata/diagnóstico por imagem , Próstata/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Testosterona/sangueRESUMO
Nutrition is an important source of exogenous AGEs and thermally processed foods present in western-style diets contain a large amount of these pro-inflammatory AGEs. Additionally, the intake of dietary AGEs could upregulate ovarian gene expression of inflammatory macrophage markers. The objective of this study was to investigate the effect of diet rich in AGEs on estrous cyclicity and ovarian function in a mouse model. Six-week old C57BL/6 J female mice were randomly subjected to either a diet low in AGEs (L-AGE) or a diet high in AGEs (H-AGE) for a total of 13 weeks. Experiments performed included daily vaginal smears to assess estrous cyclicity, ovarian superovulation with gonadotropins to assess the number of oocytes released, whole ovarian tissue mRNA quantification by RT-PCR to quantify genes involved in folliculogenesis, steroidogenesis, and macrophage markers, and ovarian morphology for follicle count. Outcome measures included estrous cyclicity, number of oocytes following superovulation, expression of genes involved in folliculogenesis, steroidogenesis, and macrophage infiltration as well as the number of primordial, primary, secondary, antral follicles and corpora lutea. Compared to mice on L-AGE diet, mice on H-AGE spent significantly longer time in the diestrus phase, had similar number of oocytes released following ovarian superovulation, and showed significant alterations in genes involved in steroidogenesis (increase in Star mRNA expression levels) and folliculogenesis (increase in Gdf-9 and Fshr mRNA expression levels). Mouse macrophage marker F4/80 mRNA expression was upregulated in mice on H-AGE diet compared to mice on L-AGE diet. Finally, mice on H-AGE diet had significantly fewer corpora lutea in their ovaries. These results indicate that the ingestion of high amounts of dietary AGEs could disrupt folliculogenesis and steroidogenesis that might lead to abnormal estrous cyclicity. Intake of dietary AGEs could also upregulate ovarian gene expression of inflammatory macrophage markers.
Assuntos
Dieta , Produtos Finais de Glicação Avançada/efeitos adversos , Ovário/fisiologia , Animais , Biomarcadores/metabolismo , Composição Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Corpo Lúteo/efeitos dos fármacos , Ciclo Estral/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Organogênese/efeitos dos fármacos , Organogênese/genética , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroides/biossíntese , Superovulação/efeitos dos fármacosRESUMO
Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule essential for the development of most organs, including the lung. Despite extensive work detailing effects of RA deficiency in early lung morphogenesis, little is known about how RA regulates late gestational lung maturation. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in the lung. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout development. We find that loss of Cyp26b1 leads to reduction of alveolar type 1 cells, failure of alveolar inflation and early postnatal lethality in mouse. Furthermore, we observe expansion of distal epithelial progenitors, but no appreciable changes in proximal airways, ECs or stromal populations. Exogenous administration of RA during late gestation partially mimics these defects; however, transcriptional analyses comparing Cyp26b1-/- with RA-treated lungs reveal overlapping, but distinct, responses. These data suggest that defects observed in Cyp26b1-/- lungs are caused by both RA-dependent and RA-independent mechanisms. This work reports crucial cellular crosstalk during lung development involving Cyp26b1-expressing endothelium and identifies a novel RA modulator in lung development.
Assuntos
Epitélio/embriologia , Pulmão/embriologia , Alvéolos Pulmonares/embriologia , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/fisiologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Células Endoteliais/citologia , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/efeitos dos fármacos , Gravidez , Prenhez , Transdução de Sinais , Células-Tronco/citologia , Tretinoína/farmacologiaRESUMO
Currently, more than 9 million American adults, including women of childbearing age, use electronic-cigarettes (e-cigs). Further, the prevalence of maternal vaping now approaching 10% is similar to that of maternal smoking. Little, however, is known about the effects of fetal exposures to nicotine-rich e-cig aerosols on lung development. In this study, we assessed whether in utero exposures to e-cig aerosols compromised lung development in mice. A third-generation e-cig device was used to expose pregnant BALB/c mice by inhalation to 36 mg/mL of nicotine cinnamon-flavored e-cig aerosols for 14-31 days. This included exposures for either 12 days before mating plus during gestation (preconception groups) or only during gestation (prenatal groups). Respective control mice were exposed to filtered air. Subgroups of offspring were euthanized at birth or at 4 wk of age. Compared with respective air-exposed controls, both preconception and prenatal exposures to e-cig aerosols significantly decreased the offspring birth weight and body length. In the preconception group, 7 inflammation-related genes were downregulated, including 4 genes common to both dams and fetuses, denoting an e-cig immunosuppressive effect. Lung morphometry assessments of preconception e-cig-exposed offspring showed a significantly increased tissue fraction at birth. This result was supported by the downregulation of 75 lung genes involved in the Wnt signaling, which is essential to lung organogenesis. Thus, our data indicate that maternal vaping impairs pregnancy outcomes, alters fetal lung structure, and dysregulates the Wnt signaling. This study provides experimental evidence for future regulations of e-cig products for pregnant women and developmentally vulnerable populations.
Assuntos
Pulmão/efeitos dos fármacos , Nicotina/efeitos adversos , Útero/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Administração por Inalação , Aerossóis/efeitos adversos , Animais , Sistemas Eletrônicos de Liberação de Nicotina , Feminino , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Organogênese/efeitos dos fármacos , Gravidez , Resultado da GravidezRESUMO
Gonadal development in medaka (Oryzias latipes) is dependent on the synergy between estrogens and androgens. Disruption of steroid hormone levels can lead to ovo-testis. To determine the sensitive windows for hormonally induced sex reversal in medaka, we developed a novel 42sp50-GFP_ChgH-GFP transgenic medaka line, allowing the identification of female gonadal tissue by fluorescence present in developing oocytes. Germinal transgenesis resulted in a stable line exhibiting a strong green fluorescent protein signal constitutively in the ovaries and in the liver in response to estrogens. The sensitivity of this line to disruption of sex determination following 16-d chronic exposures was in the nanograms per liter range. To identify the developmental period sensitive to exogenous agents, fry were exposed to 24-h pulses of high concentrations of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT) at various time points between days postfertilization (dpf) 0 and 12. Evaluation of phenotype followed by genotyping at 16 dpf revealed sensitivity to E2 between 1 and 8 dpf as well as 2 periods of susceptibility to DHT between 0 and 1 dpf and 4 and 8 dpf. No phenotypic sex reversal was detected after exposure to DHT or E2 on 11 or 12 dpf. The observed effects persisted to at least 24 dpf. The identified sensitive embryonic time periods for disruption of sex determination will aid future research on sex determination and the development of screening assays using early embryonic life stages. Environ Toxicol Chem 2020;39:842-851. © 2020 SETAC.
Assuntos
Animais Geneticamente Modificados/embriologia , Disruptores Endócrinos/toxicidade , Organogênese/efeitos dos fármacos , Oryzias/embriologia , Ovário/embriologia , Processos de Determinação Sexual/efeitos dos fármacos , Animais , Di-Hidrotestosterona/toxicidade , Estradiol/toxicidade , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Oryzias/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismoAssuntos
Desenvolvimento Embrionário , Neoplasias/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Antineoplásicos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Organogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development. METHODS: Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined. RESULTS: Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells. CONCLUSION: Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy. LAY SUMMARY: The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Leucemia Mieloide Aguda/sangue , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Meclizina/administração & dosagem , Fosfatidiletanolaminas/antagonistas & inibidores , Fosfatidiletanolaminas/biossíntese , Piridinas/administração & dosagem , Quinolinas/administração & dosagem , Adulto , Idoso , Animais , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada/métodos , Feminino , Técnicas de Inativação de Genes , Glicólise/efeitos dos fármacos , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Organogênese/efeitos dos fármacos , Organogênese/genética , Organoides/efeitos dos fármacos , Organoides/metabolismo , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Estudos Retrospectivos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The endocrine disruptor Bisphenol A (BPA), a widely employed molecule in plastics, has been shown to affect several biological processes in vertebrates, mostly via binding to nuclear receptors. Neurodevelopmental effects of BPA have been documented in vertebrates and linked to neurodevelopmental disorders, probably because some nuclear receptors are present in the vertebrate brain. Similarly, endocrine disruptors have been shown to affect neurodevelopment in marine invertebrates such as ascidians, mollusks or echinoderms, but whether invertebrate nuclear receptors are involved in the mode-of-action is largely unknown. In this study, we assessed the effect of BPA on larval brain development of the ascidian Phallusia mammillata. We found that BPA is toxic to P. mammillata embryos in a dose-dependent manner (EC50: 11.8µM; LC50: 21µM). Furthermore, micromolar doses of BPA impaired differentiation of the ascidian pigmented cells, by inhibiting otolith movement within the sensory vesicle. We further show that this phenotype is specific to other two bisphenols (BPE and BPF) over a bisphenyl (2,2 DPP). Because in vertebrates the estrogen-related receptor gamma (ERRγ) can bind bisphenols with high affinity but not bisphenyls, we tested whether the ascidian ERR participates in the neurodevelopmental phenotype induced by BPA. Interestingly, P. mammillata ERR is expressed in the larval brain, adjacent to the differentiating otolith. Furthermore, antagonists of vertebrate ERRs also inhibited the otolith movement but not pigmentation. Together our observations suggest that BPA may affect ascidian otolith differentiation by altering Pm-ERR activity whereas otolith pigmentation defects might be due to the known inhibitory effect of bisphenols on tyrosinase enzymatic activity.
Assuntos
Compostos Benzidrílicos/toxicidade , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular/efeitos dos fármacos , Organogênese , Fenóis/toxicidade , Pigmentação , Urocordados/citologia , Animais , Compostos Benzidrílicos/química , Movimento Celular/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/metabolismo , Organogênese/efeitos dos fármacos , Membrana dos Otólitos/citologia , Membrana dos Otólitos/efeitos dos fármacos , Fenóis/química , Pigmentação/efeitos dos fármacos , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Testes de Toxicidade , Urocordados/embriologia , Poluentes Químicos da Água/toxicidade , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
(S)-2,4-Diaminobutanoic acid (DABA) is a noncanonical amino acid often co-produced by cyanobacteria along with ß-N-methylamino-l-alanine (BMAA) in algal blooms. Although BMAA is a well-established neurotoxin, the toxicity of DABA remains unclear. As part of our development of biocompatible materials, we wish to make use of DABA as both a building block and as the end-product of enzymatically induced depolymerization; however, if it is toxic at very low concentrations, this would not be possible. We examined the toxicity of DABA using both in vivo embryonic and adult zebrafish models. At higher sublethal concentrations (700 µm), the fish demonstrated early signs of cardiotoxicity. Adolescent zebrafish were able to tolerate a higher concentration. Post-mortem histological analysis of juvenile zebrafish showed no liver or brain abnormalities associated with hepato- or neurotoxicity. Combined, these results show that DABA exhibits no overt toxicity at concentrations (100-300 µm) within an order of magnitude of those envisioned for its application. This study further highlights the low cost and ease of using zebrafish as an early-stage toxicological screening tool.
Assuntos
Aminobutiratos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião não Mamífero/anormalidades , Coração/efeitos dos fármacos , Coração/embriologia , Frequência Cardíaca/efeitos dos fármacos , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Organogênese/efeitos dos fármacos , Natação , Peixe-Zebra/embriologiaRESUMO
2,4-dinitrotoluene (2,4-DNT) is a common environmental pollutant, and was classified as a group 2B human carcinogenic compound by the International Agency for Research on Cancer. This study determined the toxic effects of 2,4-DNT exposure on zebrafish at the embryo-larvae stage, in terms of organ morphogenesis and the expression pattern of selected target genes related to lipid metabolism and oxygen transportation. The results showed that the 120-h post-fertilization LC50 of 2,4-DNT was 9.59 mg/L with a 95% confidence interval of 8.89-10.44 mg/L. The larvae treated with 2,4-DNT showed toxic symptoms including smaller body, less skin pigment production, yolk malabsorption, and disordered liver development. Further studies on the expression of genes related to lipid transport and metabolism, and respiration indicated that they were significantly affected by 2,4-DNT. It is concluded that 2,4-DNT exposure perturbed liver development and yolk absorption in early-life zebrafish, and disturbed the lipid metabolism /oxygen transport gene expression.
Assuntos
Dinitrobenzenos/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Animais , Transporte Biológico , Dinitrobenzenos/toxicidade , Poluentes Ambientais/farmacologia , Poluentes Ambientais/toxicidade , Larva , Lipólise , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/metabolismo , Organogênese/efeitos dos fármacos , Oxigênio/metabolismo , Peixe-ZebraRESUMO
Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20⯵M MCB for 1â¯h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750⯵M tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24â¯h treatment from 24-48 hpf with 100⯵M of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5⯵M BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60â¯min 100⯵M treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.