RESUMO
Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.
Assuntos
Desenvolvimento Embrionário/genética , Epigênese Genética , Metionina Adenosiltransferase/genética , Metionina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Sirtuína 1/genética , Acetilação , Animais , Apoptose , Diferenciação Celular , Embrião de Mamíferos , Histonas/genética , Histonas/metabolismo , Metabolômica , Metionina/administração & dosagem , Metionina Adenosiltransferase/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Células-Tronco Embrionárias Murinas/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , S-Adenosilmetionina/metabolismo , Sirtuína 1/deficiênciaRESUMO
BACKGROUND: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3). OBJECTIVES: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses. METHODS: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species. RESULTS: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen. CONCLUSIONS: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.
Assuntos
Disruptores Endócrinos/toxicidade , Útero/efeitos dos fármacos , Animais , Biomarcadores/análise , Diarileptanoides/toxicidade , Estrogênios/toxicidade , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Fitoestrógenos/toxicidade , Útero/metabolismoRESUMO
Because of possible side effects of herbal medicines containing ephedrine and guarana-derived caffeine, including increased risk of stroke, myocardial infarction, and sudden death, the Food and Drug Administration recently banned the sale of ephedra-containing products, specifically over-the-counter dietary supplements. We report cardiac in 7- and 14-week-old male F344 rats exposed by gavage to ephedrine(25 mg/kg) and caffeine (30 mg/kg) administered in combination for one or two days. The ephedrine-caffeine dosage was approximately 12- and 1.4-fold, respectively, above average human exposure, based on a mg/m2 body surface-area comparison. Several (5/7) of the exposed 14-week-old rats died or were sacrificed in extremis 4-5 h after the first dosing. In these hearts, changes were observed chiefly in the interventricular septum but also left and right ventricular walls. Massive interstitial hemorrhage, with degeneration of myofibers, occurred at the subendocardial myocardium of the left ventricle and interventricular septum. Immunostaining for cleaved caspase-3 and hyperphosphorylated H2A.X, a histone variant that becomes hyperphosphorylated during apoptosis, indicated multifocal generalized positive staining of degenerating myofibers and fragmenting nuclei, respectively. The Barbeito-Lopez trichrome stain revealed generalized patchy yellow myofibers consistent with degeneration and/or coagulative necrosis. In ephedrine-caffeine-treated animals terminated after the second dosing, foci of myocardial degeneration and necrosis were already infiltrated by mixed inflammatory cells. The myocardial necrosis may occur secondarily to intense diffuse vasoconstriction of the coronary arterial system with decreased myocardial perfusion. Our work shows the direct relationship between combined ephedrine and caffeine exposure and cardiac pathology.
Assuntos
Adrenérgicos/toxicidade , Cafeína/toxicidade , Estimulantes do Sistema Nervoso Central/toxicidade , Morte Súbita/etiologia , Efedrina/toxicidade , Infarto do Miocárdio/induzido quimicamente , Doença Aguda , Administração Oral , Animais , Cafeína/administração & dosagem , Caspase 3 , Caspases/metabolismo , Estimulantes do Sistema Nervoso Central/administração & dosagem , Combinação de Medicamentos , Interações Medicamentosas , Efedrina/administração & dosagem , Coração/efeitos dos fármacos , Hemorragia/induzido quimicamente , Hemorragia/patologia , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Necrose , Ratos , Ratos Endogâmicos F344 , Coloração e RotulagemRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects the thyroid morphologically and/or functionally in adult animals. Recently, the National Toxicology Program conducted a 2-year gavage study of TCDD in female Harlan Sprague-Dawley rats. The only treatment-related alterations found in thyroid follicles were decreased luminal size and increased height of the follicular epithelial cells, without prominent protrusion into the lumen. The present study elucidated the nature of these follicular lesions. Thyroid glands of 10 rats each from the control, high (100 ng/kg/day)-dose, and stop-study (100 ng/kg/day, 30 weeks; vehicle to study termination) groups in the 2-year study were evaluated microscopically. Twenty randomly selected follicles were measured morphometrically in each animal. TCDD treatment significantly decreased the mean ratio of luminal/epithelial areas and increased the mean sectional epithelial height of the high-dose group compared to controls. Thyroid sections were immunostained with antibody against minichromosome maintenance (MCM) proteins, a novel cell-cycle biomarker. The MCM labeling index of the high-dose group was significantly higher than that of the control; however, the TUNEL labeling index was also higher in the high-dose group than the control. All data from the stop group were comparable to those from controls. These results indicate that the follicular cell response was hypertrophic and reversible. This information should contribute to diagnosis of nonneoplastic thyroid follicular lesions in rats.