RESUMEN
Previous studies demonstrated that low-level postnatal and early life exposure to the environmental contaminant, trichloroethylene (TCE), in the drinking water of MRL+/+ mice altered glutathione redox homeostasis and increased biomarkers of oxidative stress indicating a more oxidized state. Plasma metabolites along the interrelated transmethylation pathway were also altered indicating impaired methylation capacity. Here we extend these findings to further characterize the impact of TCE exposure in mice exposed to water only or two doses of TCE in the drinking water (0, 2, and 28mg/kg/day) postnatally from birth until 6weeks of age on redox homeostasis and biomarkers of oxidative stress in the cerebellum. In addition, pathway intermediates involved in methyl metabolism and global DNA methylation patterns were examined in cerebellar tissue. Because the cerebellum is functionally important for coordinating motor activity, including exploratory and social approach behaviors, these parameters were evaluated in the present study. Mice exposed to 28mg/kg/day TCE exhibited increased locomotor activity over time as compared with control mice. In the novel object exploration test, these mice were more likely to enter the zone with the novel object as compared to control mice. Similar results were obtained in a second test when an unfamiliar mouse was introduced into the testing arena. The results show for the first time that postnatal exposure to TCE causes key metabolic changes in the cerebellum that may contribute to global DNA methylation deficits and behavioral alterations in TCE-exposed mice.
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Conducta Animal/efectos de los fármacos , Cerebelo/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Tricloroetileno/toxicidad , Animales , Animales Recién Nacidos , Cerebelo/química , Cerebelo/metabolismo , Cisteína/análisis , Glutatión/análisis , Glutatión/metabolismo , Masculino , Ratones , Ratones Endogámicos MRL lpr , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
INTRODUCTION: We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. METHODS: Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group). RESULTS: The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002). CONCLUSIONS: Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Sulfatos de Condroitina/metabolismo , Proteínas de la Membrana/metabolismo , Selectina-P/metabolismo , Sulfotransferasas/metabolismo , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Selectina-P/genética , Proteoglicanos/análisis , Interferencia de ARN , ARN Interferente Pequeño , Sulfotransferasas/genéticaRESUMEN
DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.
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Algoritmos , Islas de CpG , Metilación de ADN , Genómica/métodos , Sulfitos/química , Animales , Electroforesis en Gel de Agar , Colorantes Fluorescentes , Ratones , Reacción en Cadena de la Polimerasa , Timina/análisisRESUMEN
The onset and progression of cancer is associated with the methylation-dependent silencing of specific genes, however, the mechanism and its regulation have not been established. We previously demonstrated that reduction of mitochondrial DNA content induces cancer progression. Here we found that mitochondrial DNA-deficient LNrho0-8 activates the hypermethylation of the nuclear DNA promoters including the promoter CpG islands of the endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin. These are unmethylated and the corresponding gene products are expressed in the parental LNCaP containing mitochondrial DNA. The absence of mitochondrial DNA induced DNA methyltransferase 1 expression which was responsible for the methylation patterns observed. Inhibition of DNA methyltransferase eliminated hypermethylation and expressed gene products in LNrho0-8. These studies demonstrate loss or reduction of mitochondrial DNA resulted in the induction of DNA methyltransferase 1, hypermethylation of the promoters of endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin, and reduction of the corresponding gene products.
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Núcleo Celular/genética , Islas de CpG/genética , Metilación de ADN , ADN Mitocondrial/genética , ADN de Neoplasias/genética , Mitocondrias/genética , Neoplasias de la Próstata/genética , Línea Celular Tumoral , Humanos , MasculinoRESUMEN
Epigenetics affects health, appearance and behavior and propagates mammalian phenotypes across generations. Nutrients, drugs and behavior can all direct changes in epigenetics. In at least some cases, these directed changes are propagated across generations. This range of influences on epigenetics suggests that epigenetics is highly interactive with the environment. Changes in the environment may regularly change epigenetics and influence our future responses to the environment. The current research challenge is to understand these influences and use them to direct epigenetics toward improved health and longevity.
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Metilación de ADN , ADN/fisiología , Ambiente , Epigénesis Genética/fisiología , Animales , HumanosRESUMEN
Trichloroethylene (TCE) is a widespread environmental toxicant with immunotoxic and neurotoxic potential. Previous studies have shown that continuous developmental exposure to TCE encompassing gestation and early life as well as postnatal only exposure in the drinking water of MRL+/+ mice promoted CD4+ T cell immunotoxicity, glutathione depletion and oxidative stress in the cerebellum, as well increased locomotor activity in male offspring. The purpose of this study was to characterize the effects of exclusively prenatal exposure on these parameters. Another goal was to investigate potential plasma oxidative stress/inflammatory biomarkers to possibly be used as predictors of TCE-mediated neurotoxicity. In the current study, 6 week old male offspring of dams exposed gestationally to 0, 0.01, and 0.1mg/ml TCE in the drinking water were evaluated. Our results confirmed that the oxidized phenotype in plasma and cerebellum was maintained after exclusively prenatal exposure. A Phenotypic analysis by flow cytometry revealed that TCE exposure expanded the effector/memory subset of peripheral CD4+ T cells in association with increased production of pro-inflammatory cytokines IFN-γ and IL-17. Serum biomarkers of oxidative stress and inflammation were also elevated in plasma suggesting that systemic effects are important and may be used to predict neurotoxicity in our model. These results suggested that the prenatal period is a critical stage of life by which the developing CNS and immune system are susceptible to long-lasting changes mediated by TCE.
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Anestésicos por Inhalación/toxicidad , Encefalitis/etiología , Síndromes de Neurotoxicidad/etiología , Estrés Oxidativo/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Tricloroetileno/toxicidad , Animales , Linfocitos T CD4-Positivos/patología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Femenino , Isoflurano/toxicidad , Masculino , Ratones , Ratones Transgénicos , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Tirosina/análogos & derivados , Tirosina/efectos de los fármacosRESUMEN
Exposure to industrial solvent and water pollutant trichloroethylene (TCE) can promote autoimmunity, and expand effector/memory (CD62L) CD4+ T cells. In order to better understand etiology reduced representation bisulfite sequencing was used to study how a 40-week exposure to TCE in drinking water altered methylation of â¼337 770 CpG sites across the entire genome of effector/memory CD4+ T cells from MRL+/+ mice. Regardless of TCE exposure, 62% of CpG sites in autosomal chromosomes were hypomethylated (0-15% methylation), and 25% were hypermethylated (85-100% methylation). In contrast, only 6% of the CpGs on the X chromosome were hypomethylated, and 51% had mid-range methylation levels. In terms of TCE impact, TCE altered (≥ 10%) the methylation of 233 CpG sites in effector/memory CD4+ T cells. Approximately 31.7% of these differentially methylated sites occurred in regions known to bind one or more Polycomb group (PcG) proteins, namely Ezh2, Suz12, Mtf2 or Jarid2. In comparison, only 23.3% of CpG sites not differentially methylated by TCE were found in PcG protein binding regions. Transcriptomics revealed that TCE altered the expression of â¼560 genes in the same effector/memory CD4+ T cells. At least 80% of the immune genes altered by TCE had binding sites for PcG proteins flanking their transcription start site, or were regulated by other transcription factors that were in turn ordered by PcG proteins at their own transcription start site. Thus, PcG proteins, and the differential methylation of their binding sites, may represent a new mechanism by which TCE could alter the function of effector/memory CD4+ T cells.
RESUMEN
PURPOSE: Enhanced prognostication power is becoming more desirable in clinical oncology. In this study, we explored the prognostic potential of multigene hypermethylation profiling in non-small-cell lung cancer. EXPERIMENTAL DESIGN: We evaluated a panel of eight genes (p16, APC, ATM, hMLH1, MGMT, DAPK, ECAD, and RASSF1A) using methylation-specific PCR in 105 archived specimens of non-small-cell lung cancer representing all stages of the illness. We analyzed the effect of gene methylation status on outcome individually in a cumulative manner and in a combinatorial approach using recursive partitioning to identify methylation profiles, which affect overall survival. RESULTS: In this data set, tumors harboring promoter hypermethylation at two or more genes exhibit similar survival trends to others in the cohort. Using recursive partitioning, three genes (APC, ATM, and RASSF1A) emerged as determinants of prognostic groups. This designation retained its statistical significance even when disease stage and age were entered into a multivariate analysis. Using this approach, patients whose tumors were hypermethylated at APC and those hypermethylated at only ATM (not also at APC or RASSF1A) enjoyed substantially longer 1- and 2-year survival than patients in the remaining groups. In 32 adjacent histologically normal lung tissue specimens, we detected similar methylation abnormalities. CONCLUSION: Assessment of promoter hypermethylation aberrations may facilitate prognostic profiling of lung tumors, but validation in independent data sets is needed to verify these profiles. This system uses material that is abundantly available with linked outcome data and can be used to generate reliable epigenetic determinants.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Metilación de ADN , Neoplasias Pulmonares/patología , Proteínas Adaptadoras Transductoras de Señales , Proteína de la Poliposis Adenomatosa del Colon/genética , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Cadherinas/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas Portadoras , Proteínas de Ciclo Celular/genética , Estudios de Cohortes , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , O(6)-Metilguanina-ADN Metiltransferasa/genética , Reacción en Cadena de la Polimerasa , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Análisis de Supervivencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
AIM: Autoimmune disease and CD4(+) T-cell alterations are induced in mice exposed to the water pollutant trichloroethylene (TCE). We examined here whether TCE altered gene-specific DNA methylation in CD4(+) T cells as a possible mechanism of immunotoxicity. MATERIALS & METHODS: Naive and effector/memory CD4(+) T cells from mice exposed to TCE (0.5 mg/ml in drinking water) for 40 weeks were examined by bisulfite next-generation DNA sequencing. RESULTS: A probabilistic model calculated from multiple genes showed that TCE decreased methylation control in CD4(+) T cells. Data from individual genes fitted to a quadratic regression model showed that TCE increased gene-specific methylation variance in both CD4 subsets. CONCLUSION: TCE increased epigenetic drift of specific CpG sites in CD4(+) T cells.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Tricloroetileno/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Islas de CpG , Metilación de ADN , Exposición a Riesgos Ambientales , Femenino , RatonesRESUMEN
CD4+ T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4+ T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4+ T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4+ T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4+ T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4+ T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4+ T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4+ T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Interferón gamma/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Solventes/toxicidad , Tricloroetileno/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Islas de CpG/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Exones/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hormesis , Memoria Inmunológica/efectos de los fármacos , Interferón gamma/agonistas , Interferón gamma/antagonistas & inhibidores , Interferón gamma/genética , Intrones/efectos de los fármacos , Ratones Endogámicos MRL lpr , Reproducibilidad de los Resultados , Solventes/administración & dosificación , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Factores de Tiempo , Tricloroetileno/administración & dosificación , Contaminantes Químicos del Agua/administración & dosificaciónRESUMEN
Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2'-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the methylation status of this gene also has potential as a prognostic biomarker.
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Neoplasias de la Mama/genética , Metilación de ADN , Sulfotransferasas/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Islas de CpG , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Metástasis de la Neoplasia , Pronóstico , Células Tumorales CultivadasRESUMEN
The incidence and severity of obesity and type 2 diabetes are increasing in Western societies. The progression of obesity to type 2 diabetes is gradual with overlapping symptoms of insulin resistance, hyperinsulinemia, hyperglycemia, dyslipidemias, ion imbalance, and inflammation; this complex syndrome has been called diabesity. We describe here comparisons of gene expression in livers of A/a (agouti) vs. A(vy)/A (obese yellow) segregants (i.e., littermates) from BALB/cStCrlfC3H/Nctr x VYWffC3Hf/Nctr-A(vy)/a matings in response to 70% and 100% of ad libitum caloric intakes of a reproducible diet. Twenty-eight (28) genes regulated by diet, genotype, or diet x genotype interactions mapped to diabesity quantitative trait loci. A subset of the identified genes is linked to abnormal physiological signs observed in obesity and diabetes.
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Restricción Calórica/métodos , Ingestión de Energía/genética , Genes/genética , Obesidad/genética , Animales , Cruzamiento , Mapeo Cromosómico/métodos , Dieta , Femenino , Regulación de la Expresión Génica/genética , Ligamiento Genético/genética , Marcadores Genéticos/genética , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos , Ratones Obesos , Sitios de Carácter Cuantitativo/genética , Aumento de Peso/genéticaRESUMEN
Previous studies have shown that short-term (4 weeks) or chronic (32 weeks) exposure to trichloroethylene (TCE) in drinking water of female MRL+/+ mice generated CD4(+) T cells that secreted increased levels of interferon (IFN)-γ and expressed an activated (CD44(hi)CD62L(lo)) phenotype. In contrast, the current study of subchronic TCE exposure showed that midway in the disease process both of these parameters of CD4(+) T cell activation were reversed. This phase of the disease process may represent an attempt by the body to counteract the inflammatory effects of TCE. The decrease in CD4(+) T cell production of IFN-γ following subchronic TCE exposure could not be attributed to skewing toward a Th2 or Th17 phenotype or to an increase in Treg cells. Instead, the suppression corresponded to alterations in markers used to assess DNA methylation, namely increased expression of retrotransposons Iap (intracisternal A particle) and Muerv (murine endogenous retrovirus). Also observed was an increase in the expression of Dnmt1 (DNA methyltransferase-1) and decreased expression of several genes known to be downregulated by DNA methylation, namely Ifng, Il2, and Cdkn1a. CD4(+) T cells from a second study in which MRL+/+ mice were treated for 17 weeks with TCE showed a similar increase in Iap and decrease in Cdkn1a. In addition, DNA collected from the CD4(+) T cells in the second study showed TCE-decreased global DNA methylation. Thus, these results described the biphasic nature of TCE-induced alterations in CD4(+) T cell function and suggested that these changes represented potentially reversible alterations in epigenetic processes.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Solventes/toxicidad , Tricloroetileno/toxicidad , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Epigénesis Genética/genética , Epigénesis Genética/inmunología , Femenino , Genes de Partícula A Intracisternal/genética , Genes de Partícula A Intracisternal/inmunología , Estudio de Asociación del Genoma Completo , Inmunofenotipificación , Interferón gamma/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos MRL lpr , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/patología , Factores de TiempoRESUMEN
Previous studies have shown that continuous exposure throughout gestation until the juvenile period to environmentally relevant doses of trichloroethylene (TCE) in the drinking water of MRL+/+ mice promoted adverse behavior associated with glutathione depletion in the cerebellum indicating increased sensitivity to oxidative stress. The purpose of this study was to extend our findings and further characterize the impact of TCE exposure on redox homeostasis and biomarkers of oxidative stress in the hippocampus, a brain region prone to oxidative stress. Instead of a continuous exposure, the mice were exposed to water only or two environmentally relevant doses of TCE in the drinking water postnatally from birth until 6 weeks of age. Biomarkers of plasma metabolites in the transsulfuration pathway and the transmethylation pathway of the methionine cycle were also examined. Gene expression of neurotrophins was examined to investigate a possible relationship between oxidative stress, redox imbalance and neurotrophic factor expression with TCE exposure. Our results show that hippocampi isolated from male mice exposed to TCE showed altered glutathione redox homeostasis indicating a more oxidized state. Also observed was a significant, dose dependent increase in glutathione precursors. Plasma from the TCE treated mice showed alterations in metabolites in the transsulfuration and transmethylation pathways indicating redox imbalance and altered methylation capacity. 3-Nitrotyrosine, a biomarker of protein oxidative stress, was also significantly higher in plasma and hippocampus of TCE-exposed mice compared to controls. In contrast, expression of key neurotrophic factors in the hippocampus (BDNF, NGF, and NT-3) was significantly reduced compared to controls. Our results demonstrate that low-level postnatal and early life TCE exposure modulates neurotrophin gene expression in the mouse hippocampus and may provide a mechanism for TCE-mediated neurotoxicity.
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Contaminantes Ambientales/toxicidad , Glutatión/metabolismo , Hipocampo/efectos de los fármacos , Factores de Crecimiento Nervioso/metabolismo , Síndromes de Neurotoxicidad/etiología , Estrés Oxidativo/efectos de los fármacos , Tricloroetileno/toxicidad , Factores de Edad , Animales , Biomarcadores/sangre , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Homeostasis , Masculino , Metilación , Ratones , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/genética , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/metabolismo , Oxidación-Reducción , Tirosina/análogos & derivados , Tirosina/sangreRESUMEN
C57BL/6J (B6) mice are susceptible to in utero growth retardation and a number of morphological malformations following prenatal alcohol exposure, while DBA/2J (D2) mice are relatively resistant. We have previously shown that genomic imprinting may play a role in differential sensitivity between B6 and D2. The best-characterized mechanism mediating genomic imprinting is differential DNA methylation. In the present study we examined DNA methylation and gene expression, in both embryonic and placental tissue, at the mouse Igf2 locus following in utero ethanol exposure. We also examined the effects of a methyl-supplemented diet on methylation and ethanol teratogenesis. In embryos from susceptible B6 mice, we found small decreases in DNA methylation at four CpG sites in one of the differentially methylated regions of the Igf2 locus; only one of the four sites showed a statistically significant decrease. We observed no significant decreases in methylation in placentae. All Igf2 transcripts showed approximately 1.5-fold decreases following intrauterine alcohol exposure. Placing dams on a methyl-supplemented diet before pregnancy and throughout gestation brought methylation back up to control levels. Methyl supplementation also resulted in lower prenatal mortality, greater prenatal growth, and decreased digit malformations; it dramatically reduced vertebral malformations. Thus, although prenatal alcohol had only small effects on DNA methylation at the Igf2 locus, placing dams on a methyl-supplemented diet partially ameliorated ethanol teratogenesis.
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Metilación de ADN/efectos de los fármacos , Dieta , Etanol/toxicidad , Expresión Génica/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/genética , Intercambio Materno-Fetal , Anomalías Inducidas por Medicamentos/etiología , Anomalías Inducidas por Medicamentos/prevención & control , Animales , Betaína/administración & dosificación , Colina/administración & dosificación , Resistencia a Medicamentos , Etanol/administración & dosificación , Femenino , Muerte Fetal/prevención & control , Desarrollo Fetal/efectos de los fármacos , Ácido Fólico/administración & dosificación , Impresión Genómica , Masculino , Metanol/administración & dosificación , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Embarazo , Especificidad de la Especie , Vitamina B 12/administración & dosificaciónRESUMEN
Coat-color proportions and patterns in mice are used as assays for many processes such as transgene expression, chimerism, and epigenetics. In many studies, coat-color readouts are estimated from subjective scoring of individual mice. Here we show a method by which mouse coat color is quantified as the proportion of coat shown in one or more digital images. We use the yellow-agouti mouse model of epigenetic variegation to demonstrate this method. We apply this method to live mice using a conventional digital camera for data collection. We use a raster graphics editing program to convert agouti regions of the coat to a standard, uniform, brown color and the yellow regions of the coat to a standard, uniform, yellow color. We use a second program to quantify the proportions of these standard colors. This method provides quantification that relates directly to the visual appearance of the live animal. It also provides an objective analysis with a traceable record, and it should allow for precise comparisons of mouse coats and mouse cohorts within and between studies.
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Color del Cabello , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Color , Recolección de Datos/métodos , Bases de Datos Factuales , Epigénesis Genética , Métodos , RatonesRESUMEN
CONTEXT: Even among cases of non-small cell lung cancer (NSCLC) in the most favorable stage (IA), the disease-specific mortality is 25% or greater. One plausible explanation implicates the simplistic standard pathologic procedures used to designate lymph node involvement. A more sensitive assessment of the nodal status may improve staging. OBJECTIVE: To determine the prognostic impact of detecting an abnormal molecular event (promoter hypermethylation in a set of relevant genes) in histologically uninvolved lymph nodes in resected NSCLC. DESIGN: In this retrospective analysis of archived material, we examined DNA extracted from lymph nodes of stage I NSCLC (n = 180). Patients underwent surgery between 1991 and 1995 in a single institution. Methylation-specific polymerase chain reaction was used to detect promoter hypermethylation in a panel of 8 genes. Survival data were extracted from the computerized database at the Tumor Registry. RESULTS: Evidence of promoter hypermethylation in at least 1 gene was detected in 67% of these N0 nodes. The most commonly hypermethylated gene was E-cadherin (53%). The hypermethylation frequency for the remaining genes were as follows: APC, 5%; p16, 9%; MGMT, 11%; hMLH1, 15%; RASSF1A, 4%; DAP kinase, 9%; and ATM, 19%. The presence of promoter hypermethylation in 2 or more genes did not influence the overall, median, or 5-year survival rates. CONCLUSIONS: Identifying promoter hypermethylation (in our panel) in N0 lymph nodes in stage I NSCLC cannot be recommended for clinical decision making. Molecular abnormalities, including those found in cancer by qualitative methylation-specific polymerase chain reaction, are not synonymous with established, histologically detectable metastasis.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Neoplasias Pulmonares/genética , Ganglios Linfáticos/patología , Regiones Promotoras Genéticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/secundario , ADN de Neoplasias/análisis , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Adult tumors can be characterized by hypermethylation of CpG islands associated with 5'-upstream and coding regions of specific genes. This hypermethylation can also be part of the aging process. In contrast, much less is known about gene hypermethylation in childhood cancers, where methylation changes are not part of the aging process but likely represent developmental dysregulation. PAX3 is an important gene in muscle development and muscle-producing neoplasms such as rhabdomyosarcomas. PROCEDURES: We examined the methylation status of a PAX3 5'-CpG island in rhabdomyosarcoma subtypes and in normal fetal skeletal muscle. PAX3 methylation was analyzed in 15 embryonal rhabdomyosarcomas, 12 alveolar rhabdomyosarcomas, and in six normal skeletal muscle samples, using semi-quantitative PCR analysis of DNA digested with methyl-sensitive restriction enzymes. RESULTS: The CpG island in the upstream region of the human PAX3 gene was hypermethylated in the majority of ERMS examined (13 of 15 tumors, mean of 52% methylation), whereas most ARMS (9 of 12 tumors) and all normal muscle samples showed relative hypomethylation (both 18% mean methylation). Various CpG sites differ in contribution to overall PAX3 CpG island methylation, with methylation at a HaeII site being inversely correlated with PAX3 expression. CONCLUSIONS: PAX3 CpG island methylation appears to distinguish embryonal subtype of rhabdomyosarcoma from alveolar, and methylation at certain sites within this CpG island is inversely correlated with PAX3 expression. In addition to exemplifying developmental dysregulation, methylation of PAX3 has potential in the development of an epigenetic profile for the diagnosis of rhabdomyosarcoma.
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Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Rabdomiosarcoma/genética , Factores de Transcripción/genética , Niño , Humanos , Desarrollo de Músculos/genética , Músculo Esquelético/química , Músculo Esquelético/embriología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Rabdomiosarcoma/patología , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/genética , Rabdomiosarcoma Embrionario/patologíaRESUMEN
Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countries.
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Genómica , Fenómenos Fisiológicos de la Nutrición/fisiología , Animales , Modelos Animales de Enfermedad , Ingestión de Alimentos , Ambiente , Variación Genética/genética , Genoma Humano , Humanos , Cooperación Internacional , Fenotipo , InvestigaciónRESUMEN
This study was designed to determine if maternal dietary methyl supplements increase DNA methylation and methylation-dependent epigenetic phenotypes in mammalian offspring. Female mice of two strains were fed two levels of dietary methyl supplement or control diet prior to and during pregnancy. Offspring of these mice vary in phenotype, which is epigenetically determined and affects health and 2-y survival. Phenotype and DNA methylation of a long terminal repeat (LTR) controlling expression of the agouti gene were assayed in the resulting offspring. Methyl supplements increase the level of DNA methylation in the agouti LTR and change the phenotype of offspring in the healthy, longer-lived direction. This shows that methyl supplements have strong effects on DNA methylation and phenotype and are likely to affect long-term health. Optimum dietary supplements for the health and longevity of offspring should be intensively investigated. This should lead to public policy guidance that teaches optimal, rather than minimal, dose levels of maternal supplements.