RESUMEN
Superoxide formation is a hallmark of cardiovascular disease with the involvement of different tissues and cell types. Identification of the cellular sources and subcellular localization of superoxide formation is important to understand the underlying disease pathomechanisms. In the present study, we used HPLC quantification of the superoxide-specific oxidation products of hydroethidine (HE or DHE) and its derivative hydropropidine (HPr+) for measurement of intra- and extracellular superoxide formation in isolated leukocytes and tissues of hypertensive rats. Superoxide generation by isolated leukocytes from human subjects as well as tissue samples of hypertensive rats (infusion of angiotensin-II for 7 days) was investigated using HPr+ and HE fluorescent probes with HPLC or plate reader detection. Both fluorescent dyes were used to test for intra- and extracellular superoxide formation using the supernatant or cell/tissue pellet for analysis. We demonstrate the correlation of impaired functional parameters (blood pressure, vascular function, and oxidative burst) and increased superoxide formation in different organ systems of hypertensive rats using the HPr+/HPLC method. In the cell model, the differences between HE and HPr+ and especially the advantage of the extracellular specificity of HPr+, due to its cell impermeability, became evident. Plate reader-based assays showed much higher background signal and were inferior to HPLC based methods. In conclusion, the HPr+/HPLC assay for superoxide determination is highly reliable in isolated immune cells and an animal model of arterial hypertension. In particular, the cell impermeability of HPr+ made it possible to differentiate between intra- and extracellular superoxide formation.
Asunto(s)
Hipertensión , Superóxidos , Animales , Cromatografía Líquida de Alta Presión , Humanos , Hipertensión/inducido químicamente , Fenantridinas , Compuestos de Amonio Cuaternario , RatasRESUMEN
Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.
Asunto(s)
Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , NADPH Oxidasas/química , Adenosina Trifosfato/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula , Línea Celular , Cromatografía Líquida de Alta Presión , Interpretación Estadística de Datos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas , Estructura Molecular , NADPH Oxidasas/antagonistas & inhibidores , Oxidación-Reducción , Fenantridinas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-Actividad , Superóxidos/metabolismoRESUMEN
Detection and quantification of the highly reactive and short-lived superoxide (Oâ¢2-) can be challenging. Here, we present a new mass spectrometry (MS)-based method to detect and quantify Oâ¢2- using three fluorogenic hydroethidine probes: hydroethidine (HE), mito-hydroethidine (mito-HE), and hydropropidine (HPr+), which measure cytosolic, mitochondrial, and extracellular Oâ¢2-, respectively. The probes and their oxidation products were simultaneously quantified by applying multiple reaction monitoring (MRM) with MS that allowed the specific measurement of reactive oxygen species (ROS) distribution within the cell. The advantage of this liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is that coeluting compounds can be precisely distinguished using specific precursor and fragment masses. This method overcomes limitations from spectral overlap of Oâ¢2--specific and nonspecific products in fluorescence spectra or the low specificity associated with chromatography-based approaches. However, our experiments showed that these HE probes can be prone to autoxidation during incubation at 37°C in Hank's solution. Cell treatments with strong oxidants did not significantly increase levels of the Oâ¢2- radical. Thus, subtle changes in ROS levels in cell culture experiments might not be quantifiable. Our findings raise the question of whether HE-based probes can be used for the reliable detection of Oâ¢2- radicals in cell culture. Antioxid. Redox Signal. 00, 000-000.
Asunto(s)
Colorantes Fluorescentes/química , Fenantridinas/química , Especies Reactivas de Oxígeno/análisis , Células Cultivadas , Cromatografía Liquida , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Células Hep G2 , Humanos , Estructura Molecular , Fenantridinas/síntesis química , Fenantridinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Nearly ten years ago, we demonstrated that superoxide radical anion (O2â ¯) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E(+)). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that O2â ¯ reacts with HE to form the other oxidation product, ethidium (E(+)). It became clear that due to the overlapping fluorescence spectra of E(+) and 2-OH-E(+), fluorescence-based techniques using the "red fluorescence" are not suitable for detecting and measuring O2â ¯ in cells using HE or other structurally analogous fluorogenic probes (MitoSOX(TM) Red or hydropropidine). However, using HPLC-based assays, 2-OH-E(+) and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. SCOPE OF REVIEW: The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. MAJOR CONCLUSIONS: HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2â ¯ in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2â ¯-derived marker product and other, non-specific oxidized fluorescent products formed from these probes. GENERAL SIGNIFICANCE: Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.