Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol

Abstract

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide (O −⋅ 2) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O −⋅ 2 and H 2O 2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine- N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O −⋅ 2 production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H 2O 2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.