RESUMEN
Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Óxidos N-Cíclicos/análisis , Óxidos N-Cíclicos/química , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Radicales Libres/análisis , Peroxidación de Lípido , Lípidos/química , 4-Cloro-7-nitrobenzofurazano/análisis , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/farmacología , 4-Cloro-7-nitrobenzofurazano/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Óxidos N-Cíclicos/farmacología , Óxidos N-Cíclicos/uso terapéutico , Dietilnitrosamina , Modelos Animales de Enfermedad , Colorantes Fluorescentes/farmacología , Colorantes Fluorescentes/uso terapéutico , Depuradores de Radicales Libres/análisis , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Depuradores de Radicales Libres/uso terapéutico , Radicales Libres/química , Radicales Libres/metabolismo , Inflamación/prevención & control , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/química , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Ratas , Espectrometría de FluorescenciaRESUMEN
Haemin (iron (III)-protoporphyrin IX) is a degradation product of haemoglobin in circulating erythrocytes. Haemin may play a key oxidising agent for lipoprotein oxidation in patients with haemolytic anaemia. In this study, kinetic changes in chemical composition and target sites of haemin-induced LDL and HDL oxidation were investigated. Haemin initially induced the loss of α-tocopherol, followed by accumulation of lipid hydroperoxide (LP) and alteration of core lipid fluidity. The absence of LP in HDL was explained by the antioxidant activity of PON in addition to α-tocopherol. The target site of haemin was evaluated by ESR spin labelling with 5- and 16-doxyl steric acids. In the presence of t-BuOOH and haemin, ESR signal decay of the doxyl moiety demonstrated the initiation phase and the propagation phase of lipid peroxidation. The results of the lag time and the rate of signal decay indicated that haemin is located near the 16th carbon atom of the fatty acid chain in the phospholipid layer. The analyses of motion parameters, order parameter (S) of 5-DS and rotational correlation time (τ) of 16-DS, supported the observation that the lipid properties changed near the hydrophobic region rather than at the surface region of lipoproteins. Moreover, ESR spin labelling demonstrated that haemin molecules but not iron ions caused lipoprotein oxidation. In conclusion, haemin is a potent inducer of lipoprotein oxidation, and the target site for this oxidation is near the hydrophobic core of the lipoprotein leading to the loss of antioxidant activities and changes in lipid composition and physical properties.
Asunto(s)
Hemina/metabolismo , Peroxidación de Lípido/inmunología , Lipoproteínas/metabolismo , Adulto , Humanos , Cinética , Masculino , Adulto JovenRESUMEN
Oxidized low density lipoprotein (Ox-LDL) is implicated in a variety of oxidative diseases. To clarify the mechanisms involved and facilitate the investigation of therapeutics, we previously developed a detection method for lipid-derived radicals using the fluorescent probe 2,2,6-trimethyl-6-pentyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidine-1-oxyl (NBD-Pen). In this study, NBD-Pen was used to detect lipid-derived radicals in Ox-LDL from in vitro and in vivo samples using an iron overloaded mouse model. By following the timeline of lipid radical generation using this method, the iron overloaded mice could be successfully treated with the antioxidant Trolox, resulting in successful lowering of the plasma lipid peroxidation, aspartate transaminase and alanine transaminase levels. Furthermore, using a combination therapy of the chelating agent deferoxamine (DFX) and Trolox, liver injury and oxidative stress markers were also reduced in iron overloaded mice. The NBD-Pen method is highly sensitive as well as selective and is suitable for targeting minimally modified LDL compared with other existing methods.