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

Both lucigenin and luminol have widely been used as chemilumigenic probes f or detecting reactive oxygen species (ROS) production by various cellular s ystems. 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 H2O2: formation, respectively, by isolated intact mitochondria. Mitoc hondria were isolated from monocytes/macrophages differentiated from monobl astic ML-1 cells. Incubation of the substrate-supported mitochondria with l ucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondr ia in the presence of horseradish peroxidase (HRP). The lucigenin-derived C L 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, lumi nol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect m itochondrial 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 l uminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS produc tion by mitochondria. (C) 1999 Elsevier Science B.V. All rights reserved.