Key points are not available for this paper at this time.
Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O·̄2) production by biological systems. However, its validity as a O·̄2-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitroenzymatic systems, which produce little or no O·̄2. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O·̄2 has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O·̄2 production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O·̄2 in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 μm in all of the O·̄2-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O·̄2 production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/xanthine and phagocytic NADPH oxidase systems. In contrast to the above O·̄2-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O·̄2. Thus, LDCL still appears to be a valid probe for detecting O·̄2 production by enzymatic and cellular sources. Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O·̄2) production by biological systems. However, its validity as a O·̄2-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitroenzymatic systems, which produce little or no O·̄2. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O·̄2 has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O·̄2 production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O·̄2 in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 μm in all of the O·̄2-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O·̄2 production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/xanthine and phagocytic NADPH oxidase systems. In contrast to the above O·̄2-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O·̄2. Thus, LDCL still appears to be a valid probe for detecting O·̄2 production by enzymatic and cellular sources. The detection and measurement of fluxes of O·̄2 within cells are of critical importance for investigating the physiological and pathological roles of O·̄2. Because of its sensitivity lucigenin-derived chemiluminescence (LDCL) 1The abbreviations used are: LDCL, lucigenin-derived chemiluminescence; XO, xanthine oxidase; GO, glucose oxidase; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; LADH, lipoamide dehydrogenase; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; TPA, 12-O-tetradeconylphorbol-13-acetate; PBS, phosphate-buffered saline; DEPMPO-OOH, DEPMPO-superoxide adduct; DEPMPO-OH, DEPMPO-hydroxyl; KCN, potassium cyanide; BPQ, benzo(a)pyrene-1,6-quinone.1The abbreviations used are: LDCL, lucigenin-derived chemiluminescence; XO, xanthine oxidase; GO, glucose oxidase; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; LADH, lipoamide dehydrogenase; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; TPA, 12-O-tetradeconylphorbol-13-acetate; PBS, phosphate-buffered saline; DEPMPO-OOH, DEPMPO-superoxide adduct; DEPMPO-OH, DEPMPO-hydroxyl; KCN, potassium cyanide; BPQ, benzo(a)pyrene-1,6-quinone. has frequently been used in the specific detection of O·̄2 production by bothin vitro enzymatic systems and intact cells. For example, LDCL has been used to detect O·̄2 production by xanthine oxidase (XO) plus xanthine or hypoxanthine, NADPH-cytochrome P450 reductase in microsomes, NADPH oxidase in phagocytic cells, and a possible diphenyleneiodinium-sensitive NAD(P)H oxidase in endothelial, fibroblastic, and vascular smooth muscle cells (1Greenlee L. Fridovich I. Handler P. Biochemistry. 1962; 1: 779-783Crossref PubMed Scopus (60) Google Scholar, 2Trush M.A. Wilson M.E. Van Dyke K. Methods Enzymol. 1978; 57: 462-494Crossref Scopus (221) Google Scholar, 3Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (441) Google Scholar, 4Twerdok L.E. Mosebrook D.R. Trush M.A. Toxicol. Appl. Pharmacol. 1992; 112: 266-272Crossref PubMed Scopus (17) Google Scholar, 5Storch J. Ferber E. Anal. Biochem. 1988; 169: 262-267Crossref PubMed Scopus (59) Google Scholar, 6Mohazzab-H K.M. Kaminski P.M. Wolin M.S. Am. J. Physiol. 1994; 266: H2568-H2572PubMed Google Scholar, 7Bhunia A.K. Han H. Snowden A. Chatterjee S. J. Biol. Chem. 1997; 272: 15642-15649Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 8Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearson E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1418) Google Scholar). Our recent studies have also demonstrated that LDCL can be used to monitor mitochondrial O·̄2 production in intact cells (9Rembish S.J. Yang Y. Esterline R.L. Seacat A. Trush M.A. Goldberg A.M. In Vitro Toxicology: Mechanisms and New Technology. Alternative Methods in Toxicology. Mary Ann Liebert, Inc., New York1991: 463-469Google Scholar, 10Rembish S.J. Trush M.A. Free Radical Biol. Med. 1994; 17: 117-126Crossref PubMed Scopus (88) Google Scholar, 11Rembish S.J. Yang Y. Trush M.A. Res. Commun. Mol. Pathol. Pharmacol. 1994; 85: 115-129PubMed Google Scholar). As illustrated in Fig. 1, to detect O·̄2, lucigenin must first be reduced by one electron to produce the lucigenin cation radical (3Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (441) Google Scholar, 12Faulkner K. Fridovich I. Free Radical Biol. Med. 1993; 15: 447-451Crossref PubMed Scopus (321) Google Scholar). The biological system that reduces lucigenin may also be the same one that produces the O·̄2. The lucigenin cation radical then reacts with the biologically derived O·̄2 to yield an unstable dioxetane intermediate. The lucigenin dioxetane decomposes to produce two molecules of N-methylacridone, one of which is in an electronically excited state, which upon relaxation to the ground state emits a photon (3Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (441) Google Scholar, 12Faulkner K. Fridovich I. Free Radical Biol. Med. 1993; 15: 447-451Crossref PubMed Scopus (321) Google Scholar). Through sensitive measurement of the photon emission, the biological production of O·̄2 can be monitored. However, the validity of lucigenin as a chemilumigenic probe for detecting biological O·̄2 has recently been questioned based on the observation that in several in vitro enzymatic systems lucigenin may itself act as a source of O·̄2 via autoxidation of the lucigenin cation radical (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar, 14Vasquez-Vivar J. Hogg N. Pritchard Jr., K.A. Martasek P. Kalyanaraman B. FEBS Lett. 1997; 403: 127-130Crossref PubMed Scopus (187) Google Scholar). These include glucose oxidase (GO)/glucose at pH 9.5, XO/NADH, and endothelial nitric oxide synthase/NADPH, systems that either do not produce O·̄2 or their ability to reduce O2 to O·̄2 is very limited (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar, 14Vasquez-Vivar J. Hogg N. Pritchard Jr., K.A. Martasek P. Kalyanaraman B. FEBS Lett. 1997; 403: 127-130Crossref PubMed Scopus (187) Google Scholar). Because of the opposite charge of the lucigenin cation radical and O·̄2, the lucigenin cation radical may have a much higher affinity for O·̄2 than for O2. As such, in cellular systems that produce significant amounts of O·̄2 under physiological conditions, the propensity of lucigenin to undergo redox cycling may be very limited. In this study, we examined and correlated three end points, including sensitive measurement of LDCL, O2 consumption by oxygen polarography, and O·̄2production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) spin trapping. Using these end points, we have characterized the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O·̄2 in systems that generate O·̄2, including XO/xanthine, lipoamide dehydrogenase (LADH)/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase, as well as in systems that produce little or no O·̄2, including GO/glucose and XO/NADH. Our results demonstrate that in the O·̄2-producing systems examined, significant LDCL was always elicited at lucigenin concentrations far below those that stimulated additional O2 utilization or O·̄2 formation via the redox cycling of the lucigenin molecule. Lucigenin, XO from buttermilk, xanthine, LADH (type III) from porcine heart, NADH, glucose, superoxide dismutase (SOD), diethylenetriaminepentaacetic acid (DTPA), succinate, rotenone, myxothiazol, cytochrome c, RPMI 1640, penicillin/streptomycin, and bovine serum albumin were from Sigma. Glucose oxidase (grade I) was from Boehringer Mannheim. 12-O-Tetradeconylphorbol-13-acetate (TPA) was from LC laboratories (Woburn, MA). Fetal bovine serum was from Biowhittaker (Walkersville, MD). Dulbecco's phosphate-buffered saline (PBS, pH 7.4) was from Life Technologies, Inc. Tissue culture flasks were from Corning Costar Co. (Cambridge, MA). DEPMPO was synthesized and prepared as reported (15Frejaville C. Karoui H. Tuccio B. Le Moigne F. Culcasi M. Pietri S. Lauricella R. Tordo P. J. Med. Chem. 1995; 38: 258-265Crossref PubMed Scopus (462) Google Scholar). Human monoblastic ML-1 cells were obtained from Dr. Ruth W. Craig, Dartmouth Medical School, NH. The cells were cultured at 37 °C in an atmosphere of 5% CO2 in RPMI with and bovine serum in culture The to was by of cells with for and then the was The cells were with of The cells were cultured for at this were of S.J. Trush M.A. 1992; Scholar, S. in Vitro of and to The and were for were isolated from the to the of Wilson Wilson with cells were with The was in 5 of 1 and bovine serum pH 7.4) and in a on The was at for at The was and at for at The mitochondrial was with 5 of and then in 1 of The mitochondrial was with based on the of Anal. Biochem. PubMed Scopus Google with bovine serum albumin as the LDCL was with a at 37 For enzymatic systems, the XO and LADH and and or XO and in 1 of The of XO used in the XO/xanthine system was The LDCL was by various concentrations of For phagocytic NADPH oxidase either ML-1 cells or the were in of and by the of μm and The LDCL was by at For detecting O·̄2 production from mitochondrial in intact cells, the were in of The LDCL was by various concentrations of For detection of O·̄2 production in isolated mitochondria, the mitochondria in the of in 1 of and bovine serum pH Lucigenin was to the LDCL from LDCL are as the under the was with a oxygen at 37 °C in of as Y. Trush M.A. Arch. Biochem. Biophys. 1993; PubMed Scopus Google Scholar). The and the concentrations of the cells, and mitochondria were to these used for measurement of LDCL as The of O·̄2 was by the reduction of at as Trush M.A. Res. Scholar). reduction of was for by all not by The and the concentrations of the and cells were to these used for measurement of LDCL as For DEPMPO spin trapping measurement of O·̄2, were at with a at with a and as Y. P. Zweier J.L. Trush M.A. Mol. Pharmacol. Google Scholar, S. P. Tordo P. Zweier J.L. Anal. Biochem. 1997; PubMed Scopus Google Scholar). the and The and were with a and an were and the were to a for in the were on the and with to the was was at the XO/xanthine and the systems O2 and produce O·̄2 as by reduction and DEPMPO spin trapping and DEPMPO reacts with O·̄2 to a with that to (15Frejaville C. Karoui H. Tuccio B. Le Moigne F. Culcasi M. Pietri S. Lauricella R. Tordo P. J. Med. Chem. 1995; 38: 258-265Crossref PubMed Scopus (462) Google Scholar, S. P. Tordo P. Zweier J.L. Anal. Biochem. 1997; PubMed Scopus Google Scholar). The of the DEPMPO spin in the XO/xanthine and systems and are to the reported for (15Frejaville C. Karoui H. Tuccio B. Le Moigne F. Culcasi M. Pietri S. Lauricella R. Tordo P. J. Med. Chem. 1995; 38: 258-265Crossref PubMed Scopus (462) Google Scholar, S. P. Tordo P. Zweier J.L. Anal. Biochem. 1997; PubMed Scopus Google Scholar). As in and LDCL was also elicited in the XO/xanthine and systems. the XO/xanthine the LDCL a at concentrations of lucigenin above of either additional or formation was at to μm lucigenin with the XO/xanthine system Moreover, the of XO was a significant linear correlation between the LDCL and cytochrome c reduction by the XO/xanthine system was observed the the lucigenin in a LDCL with the between and μm lucigenin of additional O2 consumption was observed in the of a lucigenin to However, and formation were by in the of μm μm lucigenin stimulated O2 consumption on the above results lucigenin not to redox with the XO/xanthine with the system at concentrations of and consumption and O·̄2 production by various systems used in this cytochrome c NADPH O2 consumption and O·̄2 production were as under NADPH oxidase was with the from at three with a than of the not in a and the of lucigenin on O2 consumption and O·̄2 production in the LADH plus of LDCL and O2 and the DEPMPO spin trapping detection of O·̄2, were as under In LDCL the under the a of In a is plus is as in a with 5 c is as in a with μm in and the from at three the of of with 1 from μm The O2 consumption and O·̄2 production were as under NADPH oxidase was with the from at three with a than of the not GO/glucose the XO/NADH system produces a significant amount of O·̄2 as by cytochrome and DEPMPO spin trapping 5 and a significant LDCL of additional was in the GO/glucose system at a of lucigenin to μm was observed at μm lucigenin However, the formation of this spin was not by not that O·̄2 was not The XO/NADH system was to the one electron reduction of lucigenin (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar). We examined redox cycling of lucigenin by this system to As in Fig. significant LDCL was in the of and μm not 5 μm Lucigenin at and μm not 5 μm also stimulated additional O2 consumption was also at μm lucigenin and the of lucigenin on O2 consumption and O·̄2 production in the XO plus of LDCL and O2 and the DEPMPO spin trapping detection of O·̄2, were as under In LDCL the under the a of In a is XO/NADH plus is as in a with 5 c is as in a with μm c to the of two spin and and the from at three the of of with 1 from The mitochondrial electron system is to be to reduce O2 to O·̄2 A. Biochem. J. PubMed Scopus Google Scholar, A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, H. W. Biochem. Biophys. Res. Commun. 1986; PubMed Scopus Google Scholar). As in Fig. with isolated mitochondria a linear between LDCL and the of lucigenin to LDCL was derived from the mitochondrial electron the of several to mitochondrial were The LDCL was in the of and was by not is a mitochondrial cytochrome oxidase that to to production of O·̄2 S.J. Trush M.A. Free Radical Biol. Med. 1994; 17: 117-126Crossref PubMed Scopus (88) Google Scholar). and are specific of mitochondrial reductase and c Methods Enzymol. Scopus Google Scholar, Methods Enzymol. 1986; PubMed Scopus Google Scholar). lucigenin redox cycling used to detect mitochondrial O·̄2, was in the of various concentrations of was used to the O2 utilization by mitochondrial that the O2 consumption by the redox cycling of lucigenin be of O2 consumption was observed at a of lucigenin to μm and μm lucigenin stimulated the O2 consumption In the of 5 μm in a O2 consumption has been to redox in mitochondria M.A. H. Y. 1997; Scholar). of mitochondrial O·̄2 production by LDCL in intact cells has been demonstrated (9Rembish S.J. Yang Y. Esterline R.L. Seacat A. Trush M.A. Goldberg A.M. In Vitro Toxicology: Mechanisms and New Technology. Alternative Methods in Toxicology. Mary Ann Liebert, Inc., New York1991: 463-469Google Scholar, 10Rembish S.J. Trush M.A. Free Radical Biol. Med. 1994; 17: 117-126Crossref PubMed Scopus (88) Google Scholar, 11Rembish S.J. Yang Y. Trush M.A. Res. Commun. Mol. Pathol. Pharmacol. 1994; 85: 115-129PubMed Google Scholar, S.J. Trush M.A. 1992; S. in Vitro of and to The Scholar). in Fig. are LDCL observed with 5 μm lucigenin in in the or of or As LDCL was stimulated by and was by and that LDCL in the was derived from mitochondrial the intact cells no of O2 consumption was in the of to μm lucigenin In of cells with 5 μm in a of O2 consumption of LDCL elicited by LDCL was for of the cells with 5 μm lucigenin in the or of or μm as under and consumption in O2 by by in a LDCL has frequently been used to detect the O·̄2production by phagocytic NADPH oxidase M.A. Wilson M.E. Van Dyke K. Methods Enzymol. 1978; 57: 462-494Crossref Scopus (221) Google Scholar, 3Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (441) Google Scholar, 4Twerdok L.E. Mosebrook D.R. Trush M.A. Toxicol. Appl. Pharmacol. 1992; 112: 266-272Crossref PubMed Scopus (17) Google Scholar). monoblastic ML-1 cells a NADPH oxidase of ML-1 cells to results in the of NADPH oxidase and the of mitochondrial S.J. Trush M.A. 1992; Scholar, S. in Vitro of and to The Scholar). Because such a mitochondrial and LDCL to the mitochondrial electron and we have observed that is to the of NADPH O·̄2 to LDCL S.J. Yang Y. Trush M.A. Res. Commun. Mol. Pathol. Pharmacol. 1994; 85: 115-129PubMed Google Scholar, R.L. Trush M.A. Biochem. Biophys. Res. Commun. PubMed Scopus (17) Google Scholar). As such, μm and were to the to mitochondrial and its O·̄2 As in Fig. under these conditions, LDCL as well as cytochrome c O2 and DEPMPO spin trapping all reported a O·̄2-producing by NADPH oxidase in the not in the ML-1 cells. In not no LDCL was in ML-1 cells at μm Fig. the between the lucigenin and the LDCL elicited of NADPH oxidase in the Lucigenin at to μm not additional O2 utilization or formation and In the formation was reduced in the of μm which may from the by the lucigenin cation radical for O·̄2. the were stimulated with various concentrations of a significant linear was observed between LDCL and reduction or by the the use of LDCL for detecting O·̄2 in biological systems has been questioned (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar, 14Vasquez-Vivar J. Hogg N. Pritchard Jr., K.A. Martasek P. Kalyanaraman B. FEBS Lett. 1997; 403: 127-130Crossref PubMed Scopus (187) Google Scholar, I. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). lucigenin as a O·̄2-detecting in this we have characterized the potential of lucigenin to undergo redox cycling in systems that produce significant amounts of O·̄2 as well as in systems that produce little or no O·̄2. LDCL was observed in the O·̄2-producing XO/xanthine system than (1Greenlee L. Fridovich I. Handler P. Biochemistry. 1962; 1: 779-783Crossref PubMed Scopus (60) Google Scholar). The reduction of lucigenin by XO has also been to its with O·̄2 (1Greenlee L. Fridovich I. Handler P. Biochemistry. 1962; 1: 779-783Crossref PubMed Scopus (60) Google Scholar). The of the LDCL by not by in the XO/xanthine system at physiological pH the specific of O·̄2 in the to LDCL The of lucigenin at to μm to additional and formation in the XO/xanthine system that lucigenin at these concentrations not undergo redox cycling in this O·̄2-generating The validity of LDCL for detecting O·̄2 production by the XO/xanthine system was by the significant linear correlation between the LDCL and the cytochrome c reduction a for O·̄2 production Fridovich I. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of additional O2 consumption and formation by lucigenin at μm and above in the system that lucigenin is to undergo redox cycling in this system than in the XO/xanthine on cytochrome c reduction and O2 the system was than the XO/xanthine system with to O·̄2 production may at in for the redox cycling of lucigenin at concentrations in the LDCL and cytochrome were also observed in the LADH plus system L. Biochem. Google Scholar). is no O·̄2 production by the GO/glucose However, a significant LDCL has recently been to be elicited by the GO/glucose system at pH (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar). The by the GO/glucose at pH was to reduce lucigenin to its cation by autoxidation of the lucigenin cation to an LDCL (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar). in Fig. 5 demonstrated that this not at a physiological Because GO/glucose the two electron reduction of O2 to this enzymatic system is to be to reduce lucigenin to its cation radical at physiological In contrast to XO/xanthine, XO plus not produce a significant amount of O·̄2 as by cytochrome c reduction and DEPMPO spin trapping and Fig. In the of 5 a LDCL was elicited from the XO/xanthine no significant LDCL was observed with the XO/NADH system the the significant LDCL and the of additional O2 utilization and formation observed at and μm lucigenin in the XO/NADH that lucigenin redox cycling in this enzymatic The reduction of lucigenin by the XO/NADH system has been demonstrated (13Liochev S.I. Fridovich I. Arch. Biochem. Biophys. 1997; 337: 115-120Crossref PubMed Scopus (199) Google Scholar). is that the lucigenin cation radical in the XO/NADH system in the of enzymatic O·̄2 and in between LDCL and cytochrome c reduction in the XO plus xanthine at 5 μm lucigenin and cytochrome were for of xanthine with various concentrations of XO 1, and as under the from three with the than of the has been that the mitochondrial electron is to reduce O2 to O·̄2 A. Biochem. J. PubMed Scopus Google Scholar, A. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, H. W. Biochem. Biophys. Res. Commun. 1986; PubMed Scopus Google and is a source of cellular oxygen Res. 1992; 275: PubMed Scopus Google Scholar, S. A. 1994; PubMed Scopus Google Scholar). The of the lucigenin by mitochondria in cells lucigenin an probe for detecting O·̄2 derived from mitochondrial S.J. Trush M.A. Free Radical Biol. Med. 1994; 17: 117-126Crossref PubMed Scopus (88) Google Scholar). The by and by of LDCL and that LDCL in the is derived from mitochondrial O2 consumption is used frequently to the ability of a to undergo redox cycling in cells. The of lucigenin at to μm to O2 consumption that lucigenin not undergo redox cycling at these concentrations in this cellular The ability of lucigenin to detect mitochondrial O·̄2 in intact cells was by the observation that a LDCL also be elicited by isolated mitochondria In not and of the lucigenin by isolated mitochondria a on the mitochondrial of O2 consumption by lucigenin at μm and above that redox cycling of lucigenin occurs at concentrations in the isolated However, of the O2 consumption by lucigenin and in intact cells and isolated mitochondria that lucigenin is not as a redox cycling as of LDCL with cellular systems has been to O·̄2 production by phagocytic cells of their NADPH oxidase by and M.A. Wilson M.E. Van Dyke K. Methods Enzymol. 1978; 57: 462-494Crossref Scopus (221) Google Scholar, 3Allen R.C. Methods Enzymol. 1986; 133: 449-493Crossref PubMed Scopus (441) Google Scholar, 4Twerdok L.E. Mosebrook D.R. Trush M.A. Toxicol. Appl. Pharmacol. 1992; 112: 266-272Crossref PubMed Scopus (17) Google Scholar). mitochondrial and O·̄2 formation were in the by by the NADPH oxidase was by LDCL as well as cytochrome c reduction and DEPMPO spin trapping and of lucigenin at to μm to either additional or formation in the that lucigenin at these concentrations not undergo redox cycling in this cellular The validity of LDCL to detect O·̄2 production by the was by the significant linear between LDCL and cytochrome c reduction or O2 utilization by the NADPH oxidase in the Moreover, no LDCL was elicited in the ML-1 cells which a NADPH oxidase. The of of LDCL at to μm lucigenin in the ML-1 cells that lucigenin not undergo redox cycling in this cellular between LDCL and cytochrome c reduction or O2 consumption by the from of ML-1 LDCL at 5 μm cytochrome c and O2 consumption were a of in stimulated with various concentrations of TPA, as under concentrations used for were and concentrations used for consumption were and the from at three with a than of the In this that in the O·̄2-producing systems examined, LDCL was always observed at lucigenin concentrations far below those that stimulated additional O2 consumption and O·̄2 Because of the opposite charge of the lucigenin cation radical and O·̄2 and the unstable dioxetane from the of lucigenin cation radical with O·̄2 Fig. the affinity and the of between lucigenin cation radical and O·̄2 may be much higher than those between the lucigenin cation radical and O2. may the of lucigenin below concentrations to undergo redox cycling in the O·̄2-generating systems. As in Fig. the of production of the lucigenin cation radical and O·̄2 by biological reduction systems to LDCL biological O·̄2 or O·̄2 from biological source and autoxidation of the lucigenin cation In the of production of the lucigenin cation radical is in by the lucigenin As such, measurement of O2 consumption is used as a to LDCL a non-redox cycling of lucigenin can be which and O·̄2 production by enzymatic and cellular systems. non-redox cycling of lucigenin may with systems and we that LDCL is used to detect O·̄2production by an enzymatic or cellular system under a a non-redox cycling of lucigenin be measurement of the of O2 consumption by oxygen or via detection of the of O·̄2 formation by DEPMPO spin trapping
Li et al. (Thu,) studied this question.