RESUMEN
BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disorder of unknown etiology characterized by organ fibrosis and microcirculation dysfunction. Emerging evidence suggests that SSc is related to increased oxidative stress, which contributes to further tissue and vascular damage. METHODS: Oxidative stress response in the peripheral blood was assessed in patients with SSc (nâ¯=â¯55) and well-matched controls (nâ¯=â¯44) using real-time monitoring of protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also analyzed the relationship between HP generation and SSc clinics, systemic inflammation, and cellular fibronectin, an emerging biomarker of endothelial damage. RESULTS: SSc was characterized by a significantly faster (2-fold) fluorescent product generation in the CBA assay and higher cumulative HP formation (3-fold) compared to controls (p<0.001, both). The dynamics of HP generation were not associated with the form of the disease (diffuse vs. limited SSc), current immunosuppressive therapy use, presence of abnormal nailfold capillaries, and autoantibody profile. Still, it was enhanced in patients with more severe illness and certain clinical manifestations (i.e., pulmonary hypertension, digital ulcers, and cyclophosphamide treatment) and in smokers (current or past). Higher serum CRP, blood eosinophil count, and cellular fibronectin with lower hemoglobin levels were independent determinants of increased HP formation. CONCLUSIONS: Our data indicate a pro-oxidant imbalance in SSc, likely related to systemic inflammation and endothelial injury. However, extensive prospective studies are needed to verify whether it is also associated with clinical disease progression.
Asunto(s)
Endotelio , Inflamación , Esclerodermia Sistémica , Humanos , Estrés Oxidativo , Esclerodermia Sistémica/sangre , Microcirculación , Biomarcadores , Endotelio/lesiones , Estudios de Casos y Controles , Masculino , Femenino , Adulto , Persona de Mediana Edad , AncianoRESUMEN
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
Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(â¢), CO3(â¢-),(â¢)NO2 and GS(â¢)). The rate constants for the Amplex® Red oxidation by N3(â¢) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(â¢-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.