RESUMEN
Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of human disease states and drug toxicities, but development of imaging tools to study ROS biology in vivo remains a challenge. Here we synthesized and validated a novel PET tracer (12) and its (18)F radiolabeled version [(18)F]12 to allow PET ( positron emission tomography) imaging of superoxide in vivo. Initial analysis of ROS reaction kinetics found that compound 12 was rapidly and selectively oxidized by superoxide, but not other ROS. Cell culture studies in EMT6 cells exposed to the cancer chemotherapeutic agent Doxorubicin (DOX), which activates the superoxide-generating enzyme, NADPH oxidase, showed that compound 12 was a sensitive and specific probe for superoxide in cells. The microPET imaging of heart in mice with DOX-induced cardiac inflammation observed 2-fold greater oxidation of [(18)F]12 in the DOX-treated mice compared to controls (p = 0.02), the results were confirmed by distribution studies on organs subsequently removed from the mice and HPLC analysis of [(18)F] radioactivity compounds. These data indicate that compound 12 is a useful PET tracer to imaging ROS in vivo.
Asunto(s)
Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Superóxidos/análisis , Animales , Línea Celular Tumoral , Doxorrubicina/farmacología , Etidio/análogos & derivados , Etidio/síntesis química , Etidio/química , Fluorescencia , Radioisótopos de Flúor , Corazón/efectos de los fármacos , Humanos , Ratones , Radiofármacos/química , Distribución Tisular/efectos de los fármacosRESUMEN
Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2(+/-)) mice had no evidence of increased renal disease, and Ampka2(-/-) mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocondrias/metabolismo , Superóxidos/metabolismo , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Activación Enzimática/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Ribonucleótidos/farmacología , Rotenona/farmacología , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.
Asunto(s)
Mapeo Encefálico , Encéfalo/metabolismo , Etidio/análogos & derivados , Colorantes Fluorescentes/administración & dosificación , NADPH Oxidasas/metabolismo , Superóxidos/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Encéfalo/patología , Encéfalo/fisiología , Mapeo Encefálico/instrumentación , Mapeo Encefálico/métodos , Etidio/administración & dosificación , Etidio/farmacocinética , Colorantes Fluorescentes/farmacocinética , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos , Microscopía Confocal , Mitocondrias/metabolismo , Dispositivos Ópticos , Oxidación-Reducción , Distribución TisularRESUMEN
RNA and DNA oligonucleotides radiolabeled with (32)P or (33)P often require gel electrophoresis to remove undesired side and/or degradation products. Common ways to visualize these molecules after electrophoresis are by ultraviolet (UV) shadowing, which necessarily reduces the specific activity of the oligonucleotide, and by autoradiography using film, which is cumbersome and increases the cost of generating the radiolabeled molecule. A more cost-effective method is to physically inject the gel with a "Dip-N-Dot" solution of dye and radionuclide after electrophoresis but prior to phosphorimaging. The gel can be overlaid on its computer-generated image, allowing the labeled molecules to be visualized quickly.