Yunbo Li, Hong Zhu, Periannan Kuppusamy, Valerie Roubaud, Jay L. Zweier, and Michael A. Trush

THE JURNAL F BILGICAL CHEMISTRY Vol 273, No 4, Issue of January 23, pp 2015 2023, 1998 1998 by The American Society for Biochemistry and Molecular Biology, Inc Printed in USA Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems* (Received for publication, September 29, 1997, and in revised form, November 14, 1997) Yunbo Li, Hong Zhu, Periannan Kuppusamy, Valerie Roubaud, Jay L Zweier, and Michael A Trush From the Division of Toxicological Sciences, Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 and the Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology and the Electron Paramagnetic Resonance Center, The Johns Hopkins School of Medicine, Baltimore, Maryland 21224 Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical ( 2 ) production by biological systems However, its validity as a 2 -detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no 2 Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of 2 has not been carefully investigated We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), 2 consumption by oxygen polarography, and 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 2 in various enzymatic and cellular systems Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 M in all of the 2 -generating systems examined, including xanthine oxidase (X)/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 2 consumption or 2 production in the above systems Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the X/ xanthine and phagocytic NADPH oxidase systems In contrast to the above 2 -generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and X/NADH systems, which do not produce a significant amount of 2 Thus, LDCL still appears to be a valid probe for detecting 2 production by enzymatic and cellular sources The detection and measurement of fluxes of 2 within cells are of critical importance for investigating the physiological * This work was supported by National Institutes of Health Grants ES03760, ES03819, and ES08078 (to M A T) and HL38324 and HL52315 (to J L Z) The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 USC Section 1734 solely to indicate this fact To whom correspondence should be addressed: Rm 7032, Division of Toxicological Sciences, Dept of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, 615 N Wolfe St, Baltimore, MD 21205 Tel: 410-955-4712; Fax: 410-955-0116; E-mail: mtrush@jhsphedu This paper is available on line at http://wwwjbcorg 2015 and pathological roles of 2 Because of its sensitivity lucigenin-derived chemiluminescence (LDCL) 1 has frequently been used in the specific detection of 2 production by both in vitro enzymatic systems and intact cells For example, LDCL has been used to detect 2 production by xanthine oxidase (X) 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 (1 8) ur recent studies have also demonstrated that LDCL can be used to monitor mitochondrial 2 production in intact cells (9 11) As illustrated in Fig 1, to detect 2, lucigenin must first be reduced by one electron to produce the lucigenin cation radical (3, 12) The biological system that reduces lucigenin may also be the same one that produces the 2 The lucigenin cation radical then reacts with the biologically derived 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 (3, 12) Through sensitive measurement of the photon emission, the biological production of 2 can be monitored However, the validity of lucigenin as a chemilumigenic probe for detecting biological 2 has recently been questioned based on the observation that in several in vitro enzymatic systems lucigenin may itself act as a source of 2 via autoxidation of the lucigenin cation radical (13, 14) These include glucose oxidase (G)/glucose at ph 95, X/NADH, and endothelial nitric oxide synthase/nadph, systems that either do not produce 2 or their ability to reduce 2 to 2 is very limited (13, 14) Because of the opposite charge of the lucigenin cation radical and 2, the lucigenin cation radical may have a much higher affinity for 2 than for 2 As such, in cellular systems that produce significant amounts of 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, 2 consumption by oxygen polarography, and 2 production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide (DEPMP) spin trapping Using these end points, we 1 The abbreviations used are: LDCL, lucigenin-derived chemiluminescence; X, xanthine oxidase; G, glucose oxidase; DEPMP, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; LADH, lipoamide dehydrogenase; SD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid; TPA, 12--tetradeconylphorbol-13-acetate; PBS, phosphate-buffered saline; DEPMP-H, DEPMP-superoxide adduct; DEPMP-H, DEPMP-hydroxyl; KCN, potassium cyanide; BPQ, benzo(a)pyrene-1,6-quinone Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

2016 Validation of Lucigenin as a Superoxide-detecting Probe FIG 1Schematic illustration of the reaction pathway leading to LDCL TABLE I xygen consumption and 2 production by various systems used in this study The 2 consumption and 2 production were measured as described under Experimental Procedures Phagocytic NADPH oxidase was activated with 30 ng/ml TPA Values represent the mean from at least three experiments with a standard error less than 10% of the individual mean ND, not detectable System Enzyme/substrate 2 consumption 2 production g/ml/mm nmol 2 nmol cytochrome c reduced X/xanthine 40/05 807/15 min 200/15 min LADH/NADH 100/05 850/15 min 136/15 min G/glucose 85/05 1300/15 min ND X/NADH 40/05 39/15 min 138/15 min NADH 0/05 ND 137/15 min Phagocytic NADPH oxidase 961/30 min/10 6 cells 817/30 min/10 6 cells FIG 2 LDCL (panel A) and the effects of lucigenin on 2 consumption (panel B) and 2 production (panel C) in the X plus xanthine system Measurement of LDCL and 2 consumption and the DEPMP spin trapping detection of 2 were as described under Experimental Procedures In panel A, LDCL data represent the integrated area under the curve over a period of 15 min In panel C, a is X/xanthine plus 10 mm DEPMP; b is as in a but with 5 M lucigenin; c is as in a but with 50 M lucigenin The spectrum of DEPMP- H corresponds to an exchange between two conformers X and Y of the DEPMP- H adduct with the following parameters: X (43%): a N 1313 G; a P 5561 G; a H 1311 G; a H 071, 042, 07, 025, and 06 G Y (57%): a N 1308 G; a P 4585 G; a H 953 G; a H 105, 042, 07, 025, and 06 G Values in panels A and B represent the mean SE from at least three experiments ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 10 5 Downloaded from http://wwwjbcorg/ by guest on January 14, 2019 have characterized the potential of lucigenin to undergo redox cycling and as such to act as an additional source of 2 in systems that generate 2, including X/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 2, including G/ glucose and X/NADH ur results demonstrate that in the 2 -producing systems examined, significant LDCL was always elicited at lucigenin concentrations far below those that stimulated additional 2 utilization or 2 formation via the redox cycling of the lucigenin molecule EXPERIMENTAL PRCEDURES Materials Lucigenin, X from buttermilk, xanthine, LADH (type III) from porcine heart, NADH, glucose, superoxide dismutase (SD), 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--Tetradeconylphorbol-13-acetate (TPA)

Validation of Lucigenin as a Superoxide-detecting Probe 2017 FIG 3 LDCL (panel A) and the effects of lucigenin on 2 consumption (panel B) and 2 production (panel C) in the LADH plus NADH system Measurement of LDCL and 2 consumption, and the DEPMP spin trapping detection of 2, were as described under Experimental Procedures In panel A, LDCL data represent the integrated area under the curve over a period of 15 min In panel C, a is LADH/NADH plus 10 mm DEPMP; b is as in a but with 5 M lucigenin; c is as in a but with 50 M lucigenin Values in A and B represent the mean SE from at least three experiments ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 10 5 *, significantly different from 0 M lucigenin was from LC laboratories (Woburn, MA) Fetal bovine serum was from Biowhittaker (Walkersville, MD) Dulbecco s phosphate-buffered saline (PBS, ph 74) was from Life Technologies, Inc Tissue culture flasks were from Corning Costar Co (Cambridge, MA) DEPMP was synthesized and prepared as reported (15) Culture and Differentiation of ML-1 Cells to Monocytes/Macrophages 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% C 2 in RPMI 1640 medium supplemented with penicillin (50 units/ml), streptomycin (50 g/ml), and 75% fetal bovine serum in 150-cm 2 tissue culture flasks The differentiation to monocytes/macrophages was initiated by incubation of cells (3 10 5 /ml) with 03 ng/ml TPA for 3 days, and then the medium was removed The cells were fed with fresh media without further addition of TPA The cells were cultured for another 3 days Cells at this time were characteristic of monocytes/macrophages (16, 17) and were harvested for further experiments Isolation of Mitochondria from Monocytes/Macrophages Mitochondria were isolated from the freshly harvested monocytes/macrophages according to the method of Rickwood et al (18) with minor modifications Briefly, cells (4 6 10 7 cells/sample) were washed once with PBS The cell pellet was resuspended in 5 ml of sucrose buffer (025 M sucrose, 10 mm Hepes, 1 mm EGTA, and 05% bovine serum albumin, ph 74) and homogenized in a Dounce tissue grinder on ice The homogenate was centrifuged at 1,500 g for 10 min at 4 C The supernatant was collected and centrifuged at 10,000 g for 10 min at 4 C The resulting mitochondrial pellet was washed once with 5 ml of sucrose buffer and then resuspended in 1 ml of sucrose buffer The mitochondrial protein was measured with Bio-Rad protein assay dye based on the method of Bradford (19) with bovine serum albumin as the standard Measurement of LDCL LDCL was monitored with a Berthold LB9505 luminomitor at 37 C For enzymatic systems, the reaction mixtures contained X and 05 mm xanthine; 10 g/ml LADH and 05 mm NADH; 85 g/ml G and 05 mm glucose; or 4 g/ml X and 05 mm NADH in 1 ml of air-saturated PBS containing 01 mm DTPA The concentration of X used in the X/xanthine system was 4 g/ml unless otherwise indicated The LDCL was initiated by adding various concentrations of lucigenin For phagocytic NADPH oxidase system, either undifferentiated ML-1 cells or the differentiated monocytes/macrophages (1 10 6 cells) were suspended in 2 ml of air-saturated complete PBS (PBS containing 05 mm MgCl 2, 07 mm CaCl 2, and 01% glucose) followed by the addition of 10 M rotenone and myxothiazol The TPAstimulated LDCL was initiated by adding TPA at 30 ng/ml unless otherwise indicated For detecting 2 production from mitochondrial respiration in intact cells, the unstimulated monocytes/macrophages (1 10 6 cells) were suspended in 2 ml of air-saturated complete PBS The LDCL response was initiated by adding various concentrations of lucigenin For detection of 2 production in isolated mitochondria, the FIG 4Correlation between LDCL and SD-inhibitable cytochrome c reduction in the X plus xanthine system LDCL at 5 M lucigenin and cytochrome c reduction were measured for 15 min after incubation of 05 mm xanthine with various concentrations of X (05, 1, 2, and 4 g/ml), as described under Experimental procedures Values represent the mean from three experiments with the SE less than 10% of the mean reaction mixture contained 05 mg/ml mitochondria in the presence of 6 mm succinate in 1 ml of air-saturated respiration buffer (70 mm sucrose, 220 mm mannitol, 2 mm Hepes, 25 mm KH 2 P 4, 25 mm MgCl 2, 05 mm EDTA, and 01% bovine serum albumin, ph 74) Lucigenin was added to initiate the LDCL response Data from LDCL experiments are expressed as the integrated area under the curve Measurement of 2 Consumption 2 consumption was measured polarographically with a Clark-type oxygen electrode (YSI-53, Yellow Springs, H) at 37 C in 25 ml of reaction mixture as described previously (20) The buffers and the concentrations of the enzymes/substrates, cells, and mitochondria were identical to these used for measurement of LDCL as described above Detection of 2 by Ferricytochrome c Reduction The generation of 2 was measured indirectly by the reduction of ferricytochrome at 550 nm as described previously (21) Non- 2 -dependent reduction of cyto- Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

2018 Validation of Lucigenin as a Superoxide-detecting Probe FIG 5 LDCL (panel A) and the effects of lucigenin on 2 consumption (panel B) and 2 production (panel C) in the G plus glucose system Measurement of LDCL and 2 consumption and the DEPMP spin trapping detection of 2 were as described under Experimental Procedures In panel A, LDCL data represent the integrated area under the curve over a period of 15 min In panel C, a is G/glucose plus 10 mm DEPMP; b is as in a but with 5 M lucigenin; c is as in a but with 50 M lucigenin The DEPMP-hydroxyl adduct (DEPMP- H) has the following parameters: a N 1405 G; a P 4729 G; a H 1340 G; a H 06 ( 3) G This spin adduct was not SD-inhibitable Values in panels A and B represent the mean SE from at least three experiments ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 10 5 FIG 6 LDCL (panel A) and the effects of lucigenin on 2 consumption (panel B) and 2 production (panel C) in the X plus NADH system Measurement of LDCL and 2 consumption, and the DEPMP spin trapping detection of 2, were as described under Experimental Procedures In panel A, LDCL data represent the integrated area under the curve over a period of 15 min In panel C, a is X/NADH plus 10 mm DEPMP; b is as in a but with 5 M lucigenin; c is as in a but with 50 M lucigenin Spectrum c corresponds to the addition of two spin adducts, DEPMP-H and DEPMP- H Values in panels A and B represent the mean SE from at least three experiments ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 10 5 *, significantly different from 0 M lucigenin Downloaded from http://wwwjbcorg/ by guest on January 14, 2019 chrome c was corrected for by deducting all activity not inhibited by SD The buffers and the concentrations of the enzymes/substrates and cells were identical to these used for measurement of LDCL as described above ESR Measurement of 2 For DEPMP spin trapping measurement of 2, ESR spectra were recorded at room temperature with a spectrometer (model ER 300, IBM-Bruker) operating at X-band with a TM 110 cavity and TM flat cell as described previously (22, 23) Briefly, the spectrometer settings were: modulation frequency, 100 khz; modulation amplitude, 05 G; scan time, 30 s; microwave power, 20 mw; and microwave frequency, 978 GHz The microwave frequency and magnetic field were measured precisely with a source-locking microwave counter (model 575, EIP Instruments, San Jose, CA) and an NMR gaussmeter (model ER 035 M, Bruker Instruments, Billerica, MA), respectively ESR data collections were performed, and the digital spectral data were transferred to a personal computer for analysis using software developed in the Electron Paramagnetic Resonance Center Spectral simulations were performed on the personal computer and matched directly with experimental data to extract the spectral parameters Statistical Analysis Student s t test was used Statistical significance was considered at p 005 RESULTS LDCL, 2 Consumption, and DEPMP-H Spin Adduct Formation by the X/Xanthine and LADH/NADH Systems Both the X/xanthine and the LADH/NADH systems consume 2 and produce 2 as detected by SD-inhibitable cytochrome c reduction and DEPMP spin trapping (Table I, Figs 2 and 3) DEPMP reacts with 2 to form a relatively stable DEPMP- H adduct (half-life 15 min) with characteristic hyperfine splittings that give rise to 12 resolved peaks (15, 23) The hyperfine splitting constants of the DEPMP spin adduct formed in the X/xanthine and LADH/NADH systems (Figs 2

Validation of Lucigenin as a Superoxide-detecting Probe 2019 FIG 7 LDCL (panel A) and KCN-resistant 2 consumption (panel B) in isolated mitochondria driven by succinate LDCL was monitored continuously for 60 min after incubation of mitochondria with the indicated concentrations of lucigenin in the presence or absence of 02 mm KCN KCN-resistant 2 consumption was measured after incubation of the mitochondria with either the indicated concentrations of lucigenin or 5 M BPQ Values in panel A represent the average from two experiments with range less than 10% of the average Values in panel B represent the mean SE from at least three experiments *, significantly different from 20 M lucigenin #, significantly different from 50 and 100 M lucigenin ND, not detectable FIG 8 Representative profiles of LDCL response elicited by unstimulated monocytes/macrophages LDCL was monitored continuously for 30 min after incubation of the cells with 5 M lucigenin in the presence or absence of 02 mm KCN or 10 M rotenone/myxothiazol, as described under Experimental Procedures and 3) are similar to the reported values for DEPMP-H (15, 23) As shown in Figs 2 and 3, marked LDCL was also elicited in both the X/xanthine and LADH/NADH systems With the X/xanthine system, the LDCL response reached a plateau at concentrations of lucigenin above 20 M No stimulation of either additional 2 consumption or DEPMP-H formation was detected at up to 100 M lucigenin with the X/xanthine system (Fig 2) Moreover, when the concentration of X was varied, a significant linear correlation (r 098) between the LDCL and SD-inhibitable cytochrome c reduction by the X/xanthine system was observed (Fig 4) With the LADH/NADH system, varying the lucigenin concentration resulted in a biphasic LDCL response with the second phase occurring between 20 and 50 M lucigenin (Fig 3) No stimulation of additional 2 consumption was observed in the presence of a lucigenin concentration up to 20 M However, both 2 consumption (Fig 3B) and DEPMP-H formation (Fig 3C) were elevated by 30% in the presence of 50 M lucigenin 100 M lucigenin stimulated further 2 consumption (Fig 3B) Based on the above results lucigenin does not appear to redox cycle with the X/xanthine system, although it does with the LADH/NADH system at concentrations of 50 and 100 M LDCL, 2 Consumption, and DEPMP-H Spin Adduct Formation in the G/Glucose and X/NADH Systems Neither G/glucose nor the X/NADH system produces a significant amount of 2 as detected by SD-inhibitable cytochrome c reduction and DEPMP spin trapping (Table I, Figs 5 and 6) Neither a significant LDCL nor stimulation of additional 2 consumption was detected in the G/glucose system at a concentration of lucigenin up to 100 M (Fig 5) A weak DEPMPhydroxyl (DEPMP-H) signal was observed at 50 M lucigenin (Fig 5C) However, the formation of this spin adduct was not inhibited by SD (data not shown), suggesting that 2 was not produced The X/NADH system was previously shown to catalyze the one electron reduction of lucigenin (13) We examined whether redox cycling of lucigenin by this system could lead to LDCL As shown in Fig 6, significant LDCL was detected in the presence of 20 and 50 M but not 5 M lucigenin Lucigenin at 20 and 50 M but not 5 M also stimulated additional 2 consumption (Fig 6B) A DEPMP-H adduct was also detected at 50 M lucigenin (Fig 6C) Detection of Mitochondrial 2 Production by LDCL with Isolated Mitochondria and Intact Monocytes/Macrophages The mitochondrial electron transport system is known to be able to reduce 2 to 2 univalently (24 26) As shown in Fig 7, with succinate-driven isolated mitochondria a linear relationship existed between LDCL and the concentration of lucigenin up to 20 M To test whether LDCL was derived from the mitochondrial electron transport chain, the effects of several inhibitors known to affect mitochondrial respiration were determined The LDCL was elevated markedly in the presence of KCN (Fig 7) and was abolished completely by 10 M rotenone/myxothiazol (data not shown) KCN is a mitochondrial cytochrome oxidase inhibitor that causes electrons to build up leading to enhanced production of 2 (10) Rotenone and myxothiazol are specific inhibitors of mitochondrial NADH-coenzyme Q reductase and coenzyme Q-cytochrome c reductase, respectively (27, 28) To examine whether lucigenin undergoes redox cycling while being used to detect mitochondrial 2, KCN-resistant 2 consumption was determined in the presence of various concentrations of lucigenin KCN was used to inhibit the 2 utilization by mitochondrial respiration so that the 2 consumption caused by the redox cycling of lucigenin could be detected No Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

2020 Validation of Lucigenin as a Superoxide-detecting Probe stimulation of KCN-resistant 2 consumption was observed at a concentration of lucigenin up to 20 M 50 M and 100 M lucigenin slightly stimulated the KCN-resistant 2 consumption (Fig 7) In contrast, the presence of 5 M benzo(a)pyrene- 1,6-quinone (BPQ) resulted in a marked KCN-resistant 2 consumption (Fig 7) BPQ has been shown to redox cycle in mitochondria (29) Detection of mitochondrial 2 production by LDCL in unstimulated intact monocytic cells has been demonstrated previously (9 11, 16, 17) Shown in Fig 8 are representative LDCL responses observed with 5 M lucigenin in unstimulated monocytes/macrophages in the presence or absence of KCN or rotenone/myxothiazol As shown, LDCL was stimulated markedly by KCN and was abolished completely by rotenone/myxothiazol (Fig 8 and Table II), indicating that LDCL in the unstimulated monocytes/macrophages was derived totally from mitochondrial respiration With the intact cells no stimulation of KCN-resistant 2 consumption was detected in the presence of up to 50 M lucigenin (Table II) In contrast, incubation of cells with 5 M BPQ resulted in a marked stimulation of KCN-resistant 2 consumption (Table II) LDCL, 2 Consumption, and DEPMP-H Spin Adduct Formation by the TPA-stimulated Phagocytic NADPH xidase System LDCL has frequently been used to detect the 2 production by phagocytic NADPH oxidase (2 4) Undifferentiated monoblastic ML-1 cells lack a functional NADPH oxidase activity, whereas differentiation of ML-1 cells to monocytes/ macrophages results in the expression of membrane NADPH oxidase and the maturation of mitochondrial respiration (16, 17) Because monocytes/macrophages exhibit such a strong mitochondrial respiration and LDCL due to the mitochondrial electron transport chain (16, 17, Figs 7 and 8), we have observed that it is difficult to assess the contribution of NADPH oxidase-derived 2 to LDCL (11, 30) As such, 10 M rotenone and myxothiazol were added to the monocytes/macrophages to block mitochondrial respiration and its accompanying 2 production As shown in Fig 9, under these experimental conditions, LDCL as well as SD-inhibitable cytochrome c reduction, 2 consumption, and DEPMP spin trapping all equally reported a TPA-stimulated 2 -producing activity by NADPH oxidase in the monocytes/macrophages but not in the undifferentiated ML-1 cells In data not shown, no LDCL was detected in TPA-stimulated undifferentiated ML-1 cells even at 100 M lucigenin Fig 10A shows the relationship between the lucigenin concentration and the LDCL elicited after TPA activation of NADPH oxidase in the monocytes/macrophages Lucigenin at up to 50 M did not stimulate any additional 2 utilization or DEPMP-H formation (Fig 10, B and C) In fact, the DEPMP-H formation was slightly reduced in the presence of 50 M lucigenin, which may result from the competition by FIG 9 Detection of TPA-stimulated NADPH oxidase activity in the undifferentiated ML-1 cells and the monocytes/macrophages differentiated from ML-1 cells The 2 -producing activity of the TPA (30 ng/ml)-stimulated NADPH oxidase was assessed by LDCL at 5 M lucigenin (panel A), SD-inhibitable cytochrome c reduction (panel B), 2 consumption (panel C), and DEPMP spin trapping (panel D), as described under Experimental Procedures In panels A, B, and C measurements were for 30 min Values represent the mean SE from at least three experiments In panel D the ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 10 5 ND, not detectable Downloaded from http://wwwjbcorg/ by guest on January 14, 2019 TABLE II LDCL- and KCN-resistant 2 consumption in unstimulated monocytes/macrophages LDCL was monitored continuously for 30 min after incubation of the cells with the indicated concentrations of lucigenin in the presence or absence of 02 mm KCN or 10 M rotenone (RT)/myxothiazol (MYX) KCN-resistant 2 consumption was measured after incubation of the cells with either the indicated concentrations of lucigenin or 5 M BPQ Values represent the mean SE from at least three experiments *, significantly different from control ND, not detectable Control KCN Integrated CL ( 10 6 ) % Stimulation by KCN RT/MYX % Inhibition by RT/MYX KCN-resistant 2 consumption nmol 2 /min/10 6 cells Lucigenin ( M) 1 121 18 222 29* 835 02 01* 983 ND 5 436 57 1101 38* 1525 16 12* 963 ND 50 4700 231 11690 467* 1487 55 06* 988 ND BPQ ( M) 5 62 06

Validation of Lucigenin as a Superoxide-detecting Probe 2021 FIG 10 LDCL (panel A) and the effects of lucigenin on 2 consumption (panel B) and 2 production (panel C) in the TPA-stimulated monocytes/ macrophages NADPH oxidase system Measurement of LDCL and 2 consumption and the DEPMP spin trapping detection of 2 were as described under Experimental Procedures In panel A, LDCL data represent the integrated area under the curve over a period of 30 min In panel C, a is 1 10 6 cells, 30 ng/ml TPA plus 10 mm DEPMP; b is as in a but with 5 M lucigenin; c is as in a but with 50 M lucigenin Values in panels A and B represent the mean SE from at least three experiments In panel C, ESR measurement was as described in the legend of Fig 9 the lucigenin cation radical for 2 When the monocytes/macrophages were stimulated with various concentrations of TPA (19 15 ng/ml), a significant linear relationship was observed between LDCL and SD-inhibitable cytochrome c reduction (r 099) or 2 consumption (r 099) by the respiratory burst (Fig 11) DISCUSSIN Recently, the use of LDCL for detecting 2 in biological systems has been questioned (13, 14, 31) To validate lucigenin asa 2 -detecting probe, in this study we have characterized the potential of lucigenin to undergo redox cycling in systems that produce significant amounts of 2 as well as in systems that produce little or no 2 LDCL was observed in the 2 -producing X/xanthine system more than 3 decades ago (1) The univalent reduction of lucigenin by X has also been shown to precede its reaction with 2 (1) The complete inhibition of the LDCL by SD but not by catalase in the X/xanthine system at physiological ph indicates the specific involvement of 2 in the reaction pathway leading to LDCL (Fig 1) The failure of lucigenin at up to 100 M to stimulate additional 2 consumption and DEPMP-H adduct formation in the X/xanthine system indicates that lucigenin at these concentrations does not undergo redox cycling in this 2 -generating system The validity of using LDCL for detecting 2 production by the X/xanthine system was strengthened further by the significant linear correlation between the LDCL and the SD-inhibitable cytochrome c reduction (Fig 4), a standard assay for measuring 2 production (32) Stimulation of additional 2 consumption and DEPMP-H adduct formation by lucigenin at 50 M and above in the LADH/NADH system suggests that lucigenin is more likely to undergo redox cycling in this system than in the X/xanthine system Based on cytochrome c reduction and 2 consumption, the LADH/NADH system was less efficient than the X/xanthine system with regard to 2 production (Table I) This may account, at least in part, for the redox cycling of lucigenin at high concentrations in the LADH/ NADH system LDCL and SD-inhibitable cytochrome c reduction were also observed previously in the LADH plus NADH system (33) There is no 2 production by the G/glucose system However, a significant LDCL response has recently been FIG 11 Correlation between LDCL and SD-inhibitable cytochrome c reduction (panel A) or 2 consumption (panel B) by the respiratory burst resulting from TPA stimulation of ML-1 cell-derived monocytes/macrophages LDCL at 5 M lucigenin, cytochrome c reduction, and 2 consumption were measured over a period of 30 min in monocytes/macrophages stimulated with various concentrations of TPA, as described under Experimental Procedures TPA concentrations used for LDCL/cytochrome c reduction were 19, 38, and 75 ng/ml; TPA concentrations used for LDCL/ 2 consumption were 19, 38, 75, and 15 ng/ml Values represent the mean from at least three experiments with a SE less than 10% of the mean Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

2022 Validation of Lucigenin as a Superoxide-detecting Probe FIG 12 Hypothetical model depicting how biological 2 affects the ability of lucigenin to undergo redox cycling shown to be elicited by the G/glucose system at ph 95 (13) The H 2 2 produced by the G/glucose at ph 95 was thought to reduce lucigenin to its cation radical, followed by autoxidation of the lucigenin cation radical, leading to an LDCL response (13) Data presented in Fig 5 however, clearly demonstrated that this does not happen at a physiological ph Because G/ glucose ordinarily catalyzes the two electron reduction of 2 to H 2 2, this enzymatic system is unlikely to be able to reduce lucigenin univalently to its cation radical at physiological ph In contrast to X/xanthine, X plus NADH did not produce a significant amount of 2 as determined by SD-inhibitable cytochrome c reduction and DEPMP spin trapping (Table I and Fig 6) In the presence of 5 M lucigenin, a strong LDCL response was elicited from the X/xanthine system, whereas no significant LDCL was observed with the X/NADH system (Fig 6) n the other hand, the significant LDCL response and the stimulation of additional 2 utilization and DEPMP-H adduct formation observed at 20 and 50 M lucigenin in the X/NADH (Fig 6) suggest that lucigenin undergoes redox cycling in this enzymatic system The univalent reduction of lucigenin by the X/NADH system has been demonstrated previously (13) It is likely that the lucigenin cation radical formed in the X/NADH system in the absence of enzymatic 2 autoxidizes and in so doing consumes 2, producing 2 It has been long known that the mitochondrial electron transport chain is able to univalently reduce 2 to 2 (24 26) and is a major source of cellular reactive oxygen species (34, 35) The selective accumulation of the positively charged lucigenin molecule by mitochondria in cells makes lucigenin an ideal probe for detecting 2 derived from mitochondrial respiration (10) The stimulation by KCN and complete inhibition by rotenone/myxothiazol of LDCL (Fig 8 and Table II) suggest that LDCL in the unstimulated monocytes/macrophages is totally derived from mitochondrial respiration KCN-resistant 2 consumption is used frequently to assess the ability of a chemical to undergo redox cycling in cells The failure of lucigenin at up to 50 M to stimulate any detectable KCN-resistant 2 consumption indicates that lucigenin does not undergo redox cycling at these concentrations in this cellular system The ability of lucigenin to detect mitochondrial 2 in intact cells was strengthened further by the observation that a strong LDCL response could also be elicited by succinate-driven isolated mitochondria (Fig 7) In data not shown, uptake and accumulation of the positively charged lucigenin molecule by isolated mitochondria occur through a process dependent on the mitochondrial membrane potential Stimulation of KCNresistant 2 consumption by lucigenin at 50 M and above (Fig 7) suggests that redox cycling of lucigenin occurs at high concentrations in the isolated mitochondria However, comparison of the KCN-resistant 2 consumption induced by lucigenin and BPQ in both intact cells and isolated mitochondria indicates that lucigenin is not as good a redox cycling chemical as BPQ Another major application of LDCL with cellular systems has been to measure 2 production by phagocytic cells after activation of their membrane NADPH oxidase by soluble and particulate stimuli (2 4) When mitochondrial respiration and 2 formation were inhibited in the monocytes/macrophages by rotenone/myxothiazol, 2 produced by the TPA-stimulated NADPH oxidase was detected by LDCL as well as SD-inhibitable cytochrome c reduction and DEPMP spin trapping (Table I, Figs 9 and 10) Failure of lucigenin at up to 50 M to stimulate either additional 2 utilization or DEPMP-H adduct formation in the TPA-stimulated monocytes/macrophages suggests that lucigenin at these concentrations does not undergo redox cycling in this cellular system The validity of using LDCL to detect 2 production by the respiratory burst was supported further by the significant linear relationship between LDCL and SD-inhibitable cytochrome c reduction or 2 utilization by the membrane NADPH oxidase in the TPAstimulated monocytes/macrophages (Fig 11) Moreover, no LDCL was elicited in the TPA-stimulated undifferentiated ML-1 cells (Fig 9), which lack a functional membrane NADPH oxidase The failure of elicitation of LDCL at up to 100 M lucigenin in the undifferentiated ML-1 cells suggests that lucigenin does not undergo redox cycling in this non- 2 -generating cellular system In summary, this study demonstrates that in the 2 -producing systems examined, marked LDCL was always observed at lucigenin concentrations far below those that stimulated additional 2 consumption and 2 formation Because of the opposite charge of the lucigenin cation radical and 2 and the unstable dioxetane intermediate produced from the reaction of lucigenin cation radical with 2 (3, 7, Fig 1), the molecular binding affinity and the rate constant of reaction between lucigenin cation radical and 2 may be much higher than those between the lucigenin cation radical and 2 This may explain the inability of lucigenin below certain concentrations to undergo redox cycling in the 2 -generating systems As Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

Validation of Lucigenin as a Superoxide-detecting Probe 2023 depicted in Fig 12, the relative rate of production of the lucigenin cation radical and 2 by biological one-electron reduction systems both appear to determine whether LDCL will reflect only biological 2 or 2 arising from both biological source and autoxidation of the lucigenin cation radical In addition, the rate of production of the lucigenin cation radical is in turn determined by the lucigenin concentration used As such, when careful measurement of 2 consumption is used as a corollary approach to LDCL (Figs 2, 3, 5 7, 10), a safe non-redox cycling concentration of lucigenin can be determined which sensitively and reliably detects 2 production by enzymatic and cellular systems This safe non-redox cycling concentration of lucigenin may vary with different experimental systems and conditions Accordingly, we recommend that whenever LDCL is used to detect 2 production by an enzymatic or cellular system under a particular experimental condition, a safe non-redox cycling concentration of lucigenin be determined through measurement of the stimulation of 2 consumption by oxygen polarography or alternatively via detection of the stimulation of 2 formation by DEPMP spin trapping techniques REFERENCES 1 Greenlee, L, Fridovich, I, and Handler, P (1962) Biochemistry 1, 779 783 2 Trush, M A, Wilson, M E, and Van Dyke, K (1978) Methods Enzymol 57, 462 494 3 Allen, R C (1986) Methods Enzymol 133, 449 493 4 Twerdok, L E, Mosebrook, D R, and Trush, M A (1992) Toxicol Appl Pharmacol 112, 266 272 5 Storch, J, and Ferber E (1988) Anal Biochem 169, 262 267 6 Mohazzab-H, K M, Kaminski, P M, and Wolin, M S (1994) Am J Physiol 266, H2568 H2572 7 Bhunia, A K, Han, H, Snowden, A, and Chatterjee, S (1997) J Biol Chem 272, 15642 15649 8 Irani, K, Xia, Y, Zweier, J L, Sollott, S J, Der, C J, Fearson, E R, Sundaresan, M, Finkel, T, and Goldschmidt-Clermont, P J (1997) Science 275, 1649 1652 9 Rembish, S J, Yang, Y, Esterline, R L, Seacat, A, and Trush, M A (1991) in In Vitro Toxicology: Mechanisms and New Technology Alternative Methods in Toxicology (Goldberg, A M, ed) pp 463 469, Mary Ann Liebert, Inc, New York 10 Rembish, S J, and Trush, M A (1994) Free Radical Biol Med 17, 117 126 11 Rembish, S J, Yang, Y, and Trush, M A (1994) Res Commun Mol Pathol Pharmacol 85, 115 129 12 Faulkner, K, and Fridovich, I (1993) Free Radical Biol Med 15, 447 451 13 Liochev, S I, and Fridovich, I (1997) Arch Biochem Biophys 337, 115 120 14 Vasquez-Vivar, J, Hogg, N, Pritchard, K A, Jr, Martasek, P, and Kalyanaraman, B (1997) FEBS Lett 403, 127 130 15 Frejaville, C, Karoui, H, Tuccio, B, Le Moigne, F, Culcasi, M, Pietri, S, Lauricella, R, and Tordo, P (1995) J Med Chem 38, 258 265 16 Rembish, S J, Craig, R W, and Trush, M A (1992) Toxicologist 12, 281 (abstr) 17 Rembish, S J (1994) An in Vitro Mononuclear Cell of Differention Model and Its Application to Toxicology PhD thesis, The Johns Hopkins University, Baltimore 18 Rickwood, D, Wilson, M T, and Datley-Usmar, V M (1987) in Mitochondria: A Practical Approach (Darley-Usmar, V M, Rickwood, D, and Wilson, M T, eds) pp 1 16, IRL Press, xford 19 Bradford, M M (1976) Anal Biochem 72, 248 254 20 Li, Y, and Trush, M A (1993) Arch Biochem Biophys 300, 346 355 21 Kensler, T W, and Trush, M A (1981) Cancer Res 44, 216 222 22 Li, Y, Kuppusamy, P, Zweier, J L, and Trush, M A (1996) Mol Pharmacol 49, 412 421 23 Roubaud, V, Sankarapandi, S, Kuppusamy, P, Tordo, P, and Zweier, J L (1997) Anal Biochem 247, 404 411 24 Turrens, J F, and Boveris, A (1980) Biochem J 191, 421 427 25 Turrens, J F, Alexandre, A, and Lehninger, A L (1985) Arch Biochem Biophys 237, 408 414 26 Nohl, H, and Jorden, W (1986) Biochem Biophys Res Commun 138, 533 539 27 Slater, E C (1967) Methods Enzymol 10, 48 57 28 Von Jagow, G, and Link, T A (1986) Methods Enzymol 126, 253 271 29 Trush, M A, Zhu, H, and Li, Y (1997) Acta Haematol 98, (Suppl 1) 90 (abstr) 30 Esterline, R L, and Trush, M A (1989) Biochem Biophys Res Commun 159, 584 591 31 Fridovich, I (1997) J Biol Chem 272, 18515 18517 32 McCord, J M, and Fridovich, I (1969) J Biol Chem 244, 6049 6055 33 Grinblat, L, Sreider, C M, and Stoppani, A (1991) Biochem Int 23, 83 92 34 Sohal, R S, and Brunk, U T (1992) Mut Res 275, 295 304 35 Shigenaga, M K, Hagen, T M, and Ames, B N (1994) Proc Natl Acad Sci U S A 91, 10771 10778 Downloaded from http://wwwjbcorg/ by guest on January 14, 2019

Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems Yunbo Li, Hong Zhu, Periannan Kuppusamy, Valerie Roubaud, Jay L Zweier and Michael A Trush J Biol Chem 1998, 273:2015-2023 doi: 101074/jbc27342015 Access the most updated version of this article at http://wwwjbcorg/content/273/4/2015 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 32 references, 7 of which can be accessed free at http://wwwjbcorg/content/273/4/2015fullhtml#ref-list-1 Downloaded from http://wwwjbcorg/ by guest on January 14, 2019