RESUMEN
Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 microM). Changes on a post-translational level were studied using a 1 h exposure to 30 microM PBDE-99. The effects of 24 h exposure to 3 and 30 microM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 microM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 microM, but not 10 or 30 microM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Retardadores de Llama/toxicidad , Éteres Difenilos Halogenados/toxicidad , Proteínas del Tejido Nervioso/biosíntesis , Animales , Células Cultivadas , Corteza Cerebral/citología , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Femenino , Feto/citología , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
BACKGROUND: Endogenous peptides such as neuropeptides are involved in numerous biological processes in the fully developed brain but very little is known about their role in brain development. Japanese quail is a commonly used bird model for studying sexual dimorphic brain development, especially adult male copulatory behavior in relation to manipulations of the embryonic endocrine system. This study uses a label-free liquid chromatography mass spectrometry approach to analyze the influence of age (embryonic days 12 vs 17), sex and embryonic day 3 ethinylestradiol exposure on the expression of multiple endogenous peptides in the developing diencephalon. RESULTS: We identified a total of 65 peptides whereof 38 were sufficiently present in all groups for statistical analysis. Age was the most defining variable in the data and sex had the least impact. Most identified peptides were more highly expressed in embryonic day 17. The top candidates for EE2 exposure and sex effects were neuropeptide K (downregulated by EE2 in males and females), gastrin-releasing peptide (more highly expressed in control and EE2 exposed males) and gonadotropin-inhibiting hormone related protein 2 (more highly expressed in control males and displaying interaction effects between age and sex). We also report a new potential secretogranin-2 derived neuropeptide and previously unknown phosphorylations in the C-terminal flanking protachykinin 1 neuropeptide. CONCLUSIONS: This study is the first larger study on endogenous peptides in the developing brain and implies a previously unknown role for a number of neuropeptides in middle to late avian embryogenesis. It demonstrates the power of label-free liquid chromatography mass spectrometry to analyze the expression of multiple endogenous peptides and the potential to detect new putative peptide candidates in a developmental model.
Asunto(s)
Coturnix/embriología , Diencéfalo/química , Diencéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Neuropéptidos/análisis , Animales , Cromatografía Liquida , Coturnix/genética , Coturnix/metabolismo , Diencéfalo/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Masculino , Espectrometría de Masas , Neuropéptidos/genética , Caracteres SexualesRESUMEN
Polybrominated diphenyl ethers (PBDEs) are environmental contaminants found in human and animal tissues worldwide. Neonatal exposure to the flame retardant 2,2', 4,4',5-pentabromodiphenyl ether (PBDE-99) disrupts normal brain development in mice, and results in disturbed spontaneous behavior in the adult. The mechanisms underlying the late effects of early exposure are not clear. To gain insight into the initial neurodevelopmental damage inflicted by PBDE-99, we investigated the short-term effects of PBDE-99 on protein expression in the developing cerebral cortex of neonatal mice, and the cytotoxic and apoptotic effects of PBDE-99 in primary cultures of fetal rat cortical cells. We used two-dimensional difference gel electrophoresis (2D-DIGE) to analyze protein samples isolated from the cortex of NMRI mice 24h after exposure to a single oral dose of 12 mg/kg PBDE-99 on post-natal day 10. Protein resolution was enhanced by sample pre-fractionation. In the cell model, we determined cell viability using the trypan blue exclusion assay, and apoptosis using immunocytochemical detection of cleaved caspase-3. We determined the identity of 111 differentially expressed proteins, 32 (29%) of which are known to be cytoskeleton-related. Similar to previous findings in the striatum, we found elevated levels of the neuron growth-associated protein Gap43 in the cortex. In cultured cortical cells, a high concentration of PBDE-99 (30 microM) induced cell death without any apparent increase in caspase-3 activity. These results indicate that the permanent neurological damage induced by PBDE-99 during the brain growth spurt involve detrimental effects on cytoskeletal regulation and neuronal maturation in the developing cerebral cortex.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Retardadores de Llama/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Éteres Fenílicos/toxicidad , Bifenilos Polibrominados/toxicidad , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional/métodos , Embrión de Mamíferos , Femenino , Éteres Difenilos Halogenados , Ratones , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma. RESULTS: Here, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4-1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds. CONCLUSION: The pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.
Asunto(s)
Pollos/genética , Compensación de Dosificación (Genética) , Expresión Génica , Genes Ligados a X , Animales , Embrión de Pollo , Femenino , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Caracteres SexualesRESUMEN
There are indications that Cd-induced malformations in rodents are related to a disrupted flux of Zn to the developing embryo. The aim of the present study was to detect ZnT-1 (Slc30a1) and MT (Mt1) protein in structures within the decidua, yolk sac and embryo of mice and to determine whether Cd affects ZnT-1 or MT-1 gene expression in these tissues. ZnT-1 was detected in the placental labyrinth, in the ventral part around the floor plate, in the inner cell layers of the rhombencephalon and in the ventral area of the otic vesicle. MT protein was detected in the yolk sac and in the surface ectoderm of some embryonic areas, such as the pharyngeal arches. ZnT-1 and MT-1 transcripts were most abundant in the decidua and yolk sac, whereas the abundance of these genes was relatively low in the embryo. Cd exposure down-regulated ZnT-1 and up-regulated MT-1 gene expression in all structures investigated, indicating that maternal Cd exposure may alter Zn homeostasis in the conceptus.
Asunto(s)
Cloruro de Cadmio/toxicidad , Proteínas de Transporte de Catión/metabolismo , Decidua/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Metalotioneína/metabolismo , Teratógenos/toxicidad , Animales , Proteínas de Transporte de Catión/genética , Decidua/metabolismo , Decidua/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Exposición Materna , Metalotioneína/genética , Ratones , Ratones Endogámicos C57BL , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saco Vitelino/efectos de los fármacos , Saco Vitelino/metabolismo , Saco Vitelino/patologíaRESUMEN
There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Biomarcadores , Evaluación Preclínica de Medicamentos/métodos , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Xenobióticos/toxicidad , Animales , Línea Celular Tumoral , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Técnicas In Vitro , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Toxicidad/métodos , Toxicogenética/métodos , Ácido Valproico/toxicidad , Xenobióticos/clasificaciónRESUMEN
BACKGROUND: Differentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. Genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms. RESULTS: Using cDNA microarrays and real-time PCR we found gene expression differences between the male and female embryonic brain (or whole head) that may be independent of morphological differentiation of the gonads. Genes located on the sex chromosomes (ZZ in males and ZW in females) were common among the differentially expressed genes, several of which (WPKCI-8, HINT, MHM non-coding RNA) have previously been implicated in avian sex determination. A majority of the identified genes were more highly expressed in males. Three of these genes (CDK7, CCNH and BTF2-P44) encode subunits of the transcription factor IIH complex, indicating a role for this complex in neuronal differentiation. CONCLUSION: In conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Expresión Génica/fisiología , Caracteres Sexuales , Factores de Edad , Animales , Embrión de Pollo , Hibridación in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Cromosomas Sexuales/genética , Procesos de Determinación del Sexo , Diferenciación Sexual/fisiologíaRESUMEN
Exposure to the brominated flame retardant 2,2 ,4,4 ,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., alpha-enolase; gamma-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit (Atp5b); and alpha-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.
Asunto(s)
Cuerpo Estriado/química , Hipocampo/química , Éteres Fenílicos/toxicidad , Bifenilos Polibrominados/toxicidad , Administración Oral , Animales , Animales Recién Nacidos , Retardadores de Llama , Éteres Difenilos Halogenados , Masculino , Ratones , Proteína Quinasa C/metabolismo , Proteómica , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Cadmium (Cd) administered to female C57BL/6 mice on gestation day 8 induces a high incidence of anterior neural tube defects (exencephaly). This adverse effect can be attenuated by maternal pretreatment with zinc (Zn). In this study we used replicated microarray analysis and real-time PCR to investigate gene expression changes induced in the embryo 5 and 10h after maternal Cd exposure in the absence or presence of Zn pretreatment. We report nine genes with a transcriptional response induced by Cd, none of which was influenced by Zn pretreatment, and two genes induced only by combined maternal Cd exposure and Zn pretreatment. We discuss the results in relation to the possibility that Cd is largely excluded from the embryo, that the teratogenic effects of Cd may be secondary to toxicity in extraembryonic tissues, and that the primary protective role of Zn may not be to reverse Cd-induced transcription in the embryo.
Asunto(s)
Cloruro de Cadmio/toxicidad , Cloruros/farmacología , Embrión de Mamíferos/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Compuestos de Zinc/farmacología , Animales , Teorema de Bayes , Cloruro de Cadmio/administración & dosificación , Cloruros/administración & dosificación , Interacciones Farmacológicas , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Edad Gestacional , Inyecciones Intraperitoneales , Masculino , Exposición Materna/efectos adversos , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Regulación hacia Arriba/genética , Compuestos de Zinc/administración & dosificaciónRESUMEN
Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.
Asunto(s)
Anticonvulsivantes/toxicidad , Desarrollo Embrionario y Fetal/efectos de los fármacos , Perfilación de la Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/farmacología , Defectos del Tubo Neural/diagnóstico , Defectos del Tubo Neural/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Toxicogenética/métodos , Ácido Valproico/toxicidad , Animales , Apoptosis , Bioensayo/métodos , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Femenino , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cadmium is a potent teratogen in laboratory animals, causing exencephaly when administered at early stages of development. Due to its heterogenicity with respect to molecular targets, the mechanisms behind cadmium toxicity are not well understood. In the present study, C57BL/6 pregnant mice were treated with saline, cadmium, or zinc plus cadmium at 8 days post-coitus and studied 24 h after exposure. Cadmium induced significant DNA damage in the embryonic cells. Cadmium also induced embryonic growth retardation, as well as a significant upregulation of p53, p21, and Bax transcription levels. At the same time, there was a downregulation of Bcl-2, shifting the equilibrium Bcl-2/Bax toward the apoptotic pathway. There was an increase in apoptotically stained cells in the cadmium-treated embryos, and pro-caspase-3 was significantly activated. Zinc pretreatment maintained DNA damage at the control levels. It also prevented cadmium-induced effects on the expression levels of p53 and p21. The cadmium-induced decrease in Bcl-2 was inhibited, whereas the Bax levels were maintained closer to the control values. The Bad transcripts did not change at any experimental condition. Morphologically, zinc could maintain the embryological development, where apoptotic areas were as in the controls, and decrease por-caspase-3 activation. In summary, cadmium administered to pregnant mice increased primary DNA damage and activated the apoptotic pathway. These effects could be ameliorated by zinc pretreatment, and, because of that, it is possible that the mechanisms of cadmium-induced teratogenicity are related to zinc metabolism.
Asunto(s)
Apoptosis/efectos de los fármacos , Cloruro de Cadmio/toxicidad , Cloruros/farmacología , Daño del ADN , Embrión de Mamíferos/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Compuestos de Zinc/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Embarazo , Factores de Transcripción/biosíntesisRESUMEN
Cadmium accumulation in the mouse gut endoderm occurs until the closure of the vitelline duct (day 9 post-coitus; p.c.), producing anterior neural tube defects (NTD). The anterior part of the primitive endoderm, designated as the primary signaling center for anterior patterning, expresses several transcription factors of importance for head formation. Here, we studied the expression levels of some of these transcription factors (Hesx1, HNF3beta, Cerl, Otx2 and Sox2), and cell death induced after single cadmium administration to dams on days 7, 8 and 9 p.c. Stage specific down-regulation of Hesx1, Cerl, and Sox2, and an up-regulation of HNF3beta were observed. No effect was seen in Otx2 expression levels. Cell death was increased in the neuroepithelium of the cranial neural folds, and in areas where neural crest cells migrate, but not in the gut endoderm. It is proposed that cadmium-induced NTD is due to interference with head-inductive signals from the endoderm to the adjacent layers.
Asunto(s)
Cadmio/toxicidad , Sistema Nervioso Central/embriología , Endodermo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Teratógenos/toxicidad , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo , Muerte Celular/efectos de los fármacos , Sistema Nervioso Central/citología , Regulación hacia Abajo , Ratones , Ratones Endogámicos C57BL , Defectos del Tubo Neural/embriología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Regulación hacia ArribaAsunto(s)
Vacunas contra la Influenza/efectos adversos , Conservadores Farmacéuticos/efectos adversos , Timerosal/efectos adversos , Encéfalo/efectos de los fármacos , Humanos , Vacunas contra la Influenza/química , Conservadores Farmacéuticos/análisis , Conservadores Farmacéuticos/toxicidad , Factores de Riesgo , Timerosal/análisis , Timerosal/toxicidadAsunto(s)
Alternativas a las Pruebas en Animales/tendencias , Implantación del Embrión/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Endometrio/citología , Endometrio/efectos de los fármacos , Femenino , Humanos , Técnicas de Cultivo de Órganos , Placenta/fisiología , Embarazo , Reproducibilidad de los Resultados , Seguridad , Toxinas Biológicas/toxicidad , Trofoblastos/efectos de los fármacos , Trofoblastos/fisiologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) affects anterior horn cells of the spinal cord causing an indolent slow and steady deterioration of muscle strength leading inevitably to death in respiratory failure. ALS is a model condition for neurodegenerative disorders. Exposure to different agents dispersed in the environment has been suggested to cause neurodegeneration but no convincing evidence for such a link has yet been presented. Respiratory exposure to metallic mercury (Hg(0)) from different sources may be suspected. Body distribution of metallic mercury is fast and depends on solubility properties. Routes of transport, metabolism, excretion and biological half-life determine the overall toxic effects. Inhalation experiments were performed in 1984 where small marmoset monkeys (Callithrix jacchus) were exposed to (203) Hg(0 vapour) mixed into the breathing air (4-5 µg/l). After 1 hr of exposure, they were killed and whole body autoradiograms prepared to study the distribution of mercury within organs. Autoradiograms showed that Hg was deposited inside the spinal cord. Areas of enhanced accumulation anatomically corresponding to motor nuclei could be observed. This study describes a reinvestigation, with new emphasis on the spinal cord, of these classical metal exposure data in a primate, focusing on their relevance for the causation of neurodegenerative disorders. A comparison with more recent rodent experiments with similar findings is included. The hypothesis that long-time low-dose respiratory exposure to metals, for example, Hg, contributes to neurodegenerative disorders is forwarded and discussed.
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Mercurio/farmacocinética , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Células del Asta Anterior/efectos de los fármacos , Células del Asta Anterior/metabolismo , Células del Asta Anterior/fisiopatología , Autorradiografía/métodos , Callithrix , Relación Dosis-Respuesta a Droga , Semivida , Exposición por Inhalación , Masculino , Mercurio/toxicidad , Metalotioneína/metabolismo , Ratones , Ratones de la Cepa 129RESUMEN
As to the alignment of "Horizon 2020", ir is a more integrated approach to European science policy than expressed in the proposals previously drafted, and specifically considers: (i) promoting excellence in Science, (ii) establishing a sound industrial leadership and (iii) expressing an ambition to address current and future societal challenges. In this respect, the quest for a knowledge-based economy in Europe should result in proposals for industrial and employment policies that will consolidate the major European advantages in the biomedical, healthcare and pharmaceutical sectors. Horizon 2020 also provides the possibility of adopting a more flexible and simplified management route to drive European research through innovation, research and development. What should be additionally considered? Unmet medical needs, under pressure from demographic changes, await the generation of new medicines and health technologies which will evolve into a driver for a unified European policy. We believe that this should be focused on harnessing pharmaceutical knowledge for clinical use, as part of a response to accommodate patient needs and economic growth based on a robust, scientific approach. The bolder ambition for European research is to unlock key bottlenecks currently undermining European competitiveness. The historical lack of an appropriate business/innovation environment with reduced access to adequate risk finance instruments has severed the path for economic growth and industrial development. These issues are of critical importance and a solution is urgently needed to foster translation from the university to the healthcare sector through the generation and support of start-ups, spin-offs, university-industry consortia, and other platforms, which support translational research. The ultimate goal is implementation of holistic programmes: the 'bench to bedside' paradigm of medicines and other healthcare products. The European Research Council supports the basic biomedical research programmes of long term importance for development of medicines; however, fundamental research initiatives on medicines development will not be competitive in such an environment. In order to strengthen the long term outlook, we must foster innovative research within the university sector, EUFEPS proposes that a fund for such research be set up within Horizon 2020, which would be open for individual research groups and which would include Public-Public Partnerships (complementing already existing Public-Private Partnerships). How do we look for implementation? There is an established research agenda for medicines research that is globally focused, and which incorporates a cooperative model between universities and industry, facilitating integration of complex technologies. Regulatory Science will play an important role in this integration. This agenda uses tools arising from systems approaches (including discovery with systems biology and also systems pharmacology) and has the potential for providing better knowledge management, as well as technological innovation (including manufacturing). It also addresses the drive towards personalised medicines and can, with support from both public and private sectors, foster translation of knowledge to new technologies and from that, to new medicinal products and complex integrated systems. This is a part of a strategy capable of solving unmet medical needs, which would increase the quality of life of patients suffering from chronic and debilitating diseases. The instruments to allow the development of a research agenda should strengthen existing partnerships such as the IMI-JU model; allow for the creation of European-network infrastructures that can bring together existing competences with adequate European coordination, thus promoting advanced training and continuous professional development for the pharmaceutical sciences. This will be the cornerstone of a knowledge management strategy, providing education and training for healthcare professionals and scientists. A key role for EUFEPS is to help the research community to embrace these new holistic policies applied to the spectrum of pharmaceutical, medical and cognate sciences.
Asunto(s)
Investigación Biomédica , Descubrimiento de Drogas , Industria Farmacéutica , Europa (Continente) , Asociación entre el Sector Público-Privado , UniversidadesRESUMEN
Microvascular pericytes are of key importance in neoformation of blood vessels, in stabilization of newly formed vessels as well as maintenance of angiostasis in resting tissues. Furthermore, pericytes are capable of differentiating into pro-fibrotic collagen type I producing fibroblasts. The present study investigates the effects of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on pericyte proliferation, cell viability, migration and differentiation. The results show that HDAC inhibition through exposure of pericytes to VPA in vitro causes the inhibition of pericyte proliferation and migration with no effect on cell viability. Pericyte exposure to the potent HDAC inhibitor Trichostatin A caused similar effects on pericyte proliferation, migration and cell viability. HDAC inhibition also inhibited pericyte differentiation into collagen type I producing fibroblasts. Given the importance of pericytes in blood vessel biology a qPCR array focusing on the expression of mRNAs coding for proteins that regulate angiogenesis was performed. The results showed that HDAC inhibition promoted transcription of genes involved in vessel stabilization/maturation in human microvascular pericytes. The present in vitro study demonstrates that VPA influences several aspects of microvascular pericyte biology and suggests an alternative mechanism by which HDAC inhibition affects blood vessels. The results raise the possibility that HDAC inhibition inhibits angiogenesis partly through promoting a pericyte phenotype associated with stabilization/maturation of blood vessels.
Asunto(s)
Regulación de la Expresión Génica , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Ácido Valproico/farmacología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Colágeno Tipo I/metabolismo , Femenino , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Microcirculación , Neovascularización Fisiológica , Pericitos/citología , Fenotipo , Placenta/efectos de los fármacos , Reacción en Cadena de la Polimerasa/métodos , EmbarazoRESUMEN
Prediction of developmental toxicity in vitro could be based on short-time toxicogenomic endpoints in embryo-derived cell lines. Microarray studies in P19 mouse embryocarcinoma cells and mouse embryos have indicated that valproic acid (VPA), an inducer of neural tube defects, deregulates the expression of many genes, including those critically involved in neural tube development. In this study, we exposed undifferentiated R1 mouse embryonic stem cells to VPA and VPA analogs for 6 h and used CodeLink whole-genome expression microarrays to define VPA-responsive genes correlating with teratogenicity. Compared with the nonteratogenic analog 2-ethyl-4-methylpentanoic acid, VPA and the teratogenic VPA analog (S)-2-pentyl-4-pentynoic acid deregulated a much larger number of genes. Five genes (of â¼2500 array probes correlating with the separation) were sufficient to effectively separate teratogens from nonteratogens. A large fraction of the target genes correlating with teratogenicity are functionally related to embryonic development and morphogenesis, including neural tube formation and closure. Similar responses in R1 were found for most genes previously identified as VPA responsive in P19 and embryos. A subset of target genes was evaluated as candidate markers predictive of potential teratogenicity against a range of known teratogens using TaqMan expression arrays. These marker genes showed a positive predictive value for the teratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoic acid are known histone deacetylase (HDAC) inhibitors but not for compounds that are likely to act by other mechanisms. This indicates that HDAC inhibition may be a major mechanism by which VPA induces gene deregulation and possibly teratogenicity.
Asunto(s)
Células Madre Embrionarias/efectos de los fármacos , Ácidos Grasos Insaturados/toxicidad , Regulación del Desarrollo de la Expresión Génica , Ácido Valproico/análogos & derivados , Anomalías Inducidas por Medicamentos/genética , Animales , Butiratos/toxicidad , Línea Celular , Desarrollo Embrionario/genética , Células Madre Embrionarias/metabolismo , Estudios de Asociación Genética , Marcadores Genéticos , Inhibidores de Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/toxicidad , Ratones , Análisis por Micromatrices , Defectos del Tubo Neural/inducido químicamente , Defectos del Tubo Neural/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Teratógenos/toxicidad , Toxicogenética , Ácido Valproico/toxicidadRESUMEN
Cell-based in vitro assays would potentially reduce animal testing in preclinical drug development. Mouse embryos exposed to the teratogenic drug valproic acid (VPA) in utero for 1.5, 3 or 6h on gestational day 8 were analyzed using microarrays. Significant effects on gene expression were observed already at 1.5h, and 85 probes were deregulated across all time points. To find transcriptional markers of VPA-induced developmental toxicity, the in vivo data were compared to previous in vitro data on embryonal carcinoma P19 cells exposed to VPA for 1.5, 6 or 24h. Maximal concordance between embryos and cells was at the 6-h time points, with 163 genes showing similar deregulation. Developmentally important Gene Ontology terms, such as "organ morphogenesis" and "tube development" were overrepresented among putative VPA target genes. The genes Gja1, Hap1, Sall2, H1f0,Cyp26a1, Fgf15, Otx2, and Lin7b emerged as candidate in vitro markers of potential VPA-induced teratogenicity.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Teratógenos/toxicidad , Transcripción Genética/efectos de los fármacos , Ácido Valproico/toxicidad , Alternativas a las Pruebas en Animales , Animales , Biomarcadores/análisis , Desarrollo Embrionario/genética , Femenino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Embarazo , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.