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 KuppusamyDagger , Valerie RoubaudDagger , Jay L. ZweierDagger , 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 Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (Obardot 2) production by biological systems. However, its validity as a Obardot 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 Obardot 2. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of Obardot 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 Obardot 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 Obardot 2 in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 µM in all of the Obardot 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 Obardot 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 Obardot 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 Obardot 2. Thus, LDCL still appears to be a valid probe for detecting Obardot 2 production by enzymatic and cellular sources.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The detection and measurement of fluxes of Obardot 2 within cells are of critical importance for investigating the physiological and pathological roles of Obardot 2. Because of its sensitivity lucigenin-derived chemiluminescence (LDCL)1 has frequently been used in the specific detection of Obardot 2 production by both in vitro enzymatic systems and intact cells. For example, LDCL has been used to detect Obardot 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 (1-8). Our recent studies have also demonstrated that LDCL can be used to monitor mitochondrial Obardot 2 production in intact cells (9-11).

As illustrated in Fig. ins;1939f1}1, to detect Obardot 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 Obardot 2. The lucigenin cation radical then reacts with the biologically derived Obardot 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 Obardot 2 can be monitored. However, the validity of lucigenin as a chemilumigenic probe for detecting biological Obardot 2 has recently been questioned based on the observation that in several in vitro enzymatic systems lucigenin may itself act as a source of Obardot 2 via autoxidation of the lucigenin cation radical (13, 14). These include glucose oxidase (GO)/glucose at pH 9.5, XO/NADH, and endothelial nitric oxide synthase/NADPH, systems that either do not produce Obardot 2 or their ability to reduce O2 to Obardot 2 is very limited (13, 14). Because of the opposite charge of the lucigenin cation radical and Obardot 2, the lucigenin cation radical may have a much higher affinity for Obardot 2 than for O2. As such, in cellular systems that produce significant amounts of Obardot 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 Obardot 2 production 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 Obardot 2 in systems that generate Obardot 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 Obardot 2, including GO/glucose and XO/NADH. Our results demonstrate that in the Obardot 2-producing systems examined, significant LDCL was always elicited at lucigenin concentrations far below those that stimulated additional O2 utilization or Obardot 2 formation via the redox cycling of the lucigenin molecule.


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Fig. 1.   Schematic illustration of the reaction pathway leading to LDCL.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- 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 (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% CO2 in RPMI 1640 medium supplemented with penicillin (50 units/ml), streptomycin (50 µg/ml), and 7.5% fetal bovine serum in 150-cm2 tissue culture flasks. The differentiation to monocytes/macrophages was initiated by incubation of cells (3 × 105/ml) with 0.3 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 × 107 cells/sample) were washed once with PBS. The cell pellet was resuspended in 5 ml of sucrose buffer (0.25 M sucrose, 10 mM Hepes, 1 mM EGTA, and 0.5% bovine serum albumin, pH 7.4) 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 XO and 0.5 mM xanthine; 10 µg/ml LADH and 0.5 mM NADH; 8.5 µg/ml GO and 0.5 mM glucose; or 4 µg/ml XO and 0.5 mM NADH in 1 ml of air-saturated PBS containing 0.1 mM DTPA. The concentration of XO used in the XO/xanthine system was 4 µg/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 × 106 cells) were suspended in 2 ml of air-saturated complete PBS (PBS containing 0.5 mM MgCl2, 0.7 mM CaCl2, and 0.1% glucose) followed by the addition of 10 µM rotenone and myxothiazol. The TPA-stimulated LDCL was initiated by adding TPA at 30 ng/ml unless otherwise indicated. For detecting Obardot 2 production from mitochondrial respiration in intact cells, the unstimulated monocytes/macrophages (1 × 106 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 Obardot 2 production in isolated mitochondria, the reaction mixture contained 0.5 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, 2.5 mM KH2PO4, 2.5 mM MgCl2, 0.5 mM EDTA, and 0.1% bovine serum albumin, pH 7.4). Lucigenin was added to initiate the LDCL response. Data from LDCL experiments are expressed as the integrated area under the curve.

Measurement of O2 Consumption-- O2 consumption was measured polarographically with a Clark-type oxygen electrode (YSI-53, Yellow Springs, OH) at 37 °C in 2.5 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 Obardot 2 by Ferricytochrome c Reduction-- The generation of Obardot 2 was measured indirectly by the reduction of ferricytochrome at 550 nm as described previously (21). Non-Obardot 2-dependent reduction of cytochrome c was corrected for by deducting all activity not inhibited by SOD. 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 Obardot 2-- For DEPMPO spin trapping measurement of Obardot 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, 0.5 G; scan time, 30 s; microwave power, 20 mW; and microwave frequency, 9.78 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 < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

LDCL, O2 Consumption, and DEPMPO-OOH Spin Adduct Formation by the XO/Xanthine and LADH/NADH Systems-- Both the XO/xanthine and the LADH/NADH systems consume O2 and produce Obardot 2 as detected by SOD-inhibitable cytochrome c reduction and DEPMPO spin trapping (Table I, Figs. ins;1939f2}2 and 3). DEPMPO reacts with Obardot 2 to form a relatively stable DEPMPO-OOH adduct (half-life ~15 min) with characteristic hyperfine splittings that give rise to 12 resolved peaks (15, 23). The hyperfine splitting constants of the DEPMPO spin adduct formed in the XO/xanthine and LADH/NADH systems (Figs. 2 and 3) are similar to the reported values for DEPMPO-OOH (15, 23). As shown in Figs. 2 and 3, marked LDCL was also elicited in both the XO/xanthine and LADH/NADH systems. With the XO/xanthine system, the LDCL response reached a plateau at concentrations of lucigenin above 20 µM. No stimulation of either additional O2 consumption or DEPMPO-OOH formation was detected at up to 100 µM lucigenin with the XO/xanthine system (Fig. 2). Moreover, when the concentration of XO was varied, a significant linear correlation (r = 0.98) between the LDCL and SOD-inhibitable cytochrome c reduction by the XO/xanthine system was observed (Fig. ins;1939f4}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 µM lucigenin (Fig. 3). No stimulation of additional O2 consumption was observed in the presence of a lucigenin concentration up to 20 µM. However, both O2 consumption (Fig. 3B) and DEPMPO-OOH formation (Fig. 3C) were elevated by ~30% in the presence of 50 µM lucigenin. 100 µM lucigenin stimulated further O2 consumption (Fig. 3B). Based on the above results lucigenin does not appear to redox cycle with the XO/xanthine system, although it does with the LADH/NADH system at concentrations of 50 and 100 µM.

                              
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Table I
Oxygen consumption and Obardot 2 production by various systems used in this study
The O2 consumption and Obardot 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.


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Fig. 2.   LDCL (panel A) and the effects of lucigenin on O2 consumption (panel B) and Obardot 2 production (panel C) in the XO plus xanthine system. Measurement of LDCL and O2 consumption and the DEPMPO spin trapping detection of Obardot 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 XO/xanthine plus 10 mM DEPMPO; b is as in a but with 5 µM lucigenin; c is as in a but with 50 µM lucigenin. The spectrum of DEPMPO-OOH corresponds to an exchange between two conformers X and Y of the DEPMPO-OOH adduct with the following parameters: X (43%): aN = 13.13 G; aP = 55.61 G; aHbeta  = 13.11 G; aHgamma  = 0.71, 0.42, 0.7, 0.25, and 0.6 G. Y (57%): aN = 13.08 G; aP = 45.85 G; aHbeta  = 9.53 G; aHgamma  = 1.05, 0.42, 0.7, 0.25, and 0.6 G. Values in panels A and B represent the mean ± S.E. from at least three experiments. ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 × 105.


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Fig. 3.   LDCL (panel A) and the effects of lucigenin on O2 consumption (panel B) and Obardot 2 production (panel C) in the LADH plus NADH system. Measurement of LDCL and O2 consumption, and the DEPMPO spin trapping detection of Obardot 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 DEPMPO; b is as in a but with 5 µM lucigenin; c is as in a but with 50 µM lucigenin. Values in A and B represent the mean ± S.E. from at least three experiments. ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 × 105. *, significantly different from 0 µM lucigenin.


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Fig. 4.   Correlation between LDCL and SOD-inhibitable cytochrome c reduction in the XO plus xanthine system. LDCL at 5 µM lucigenin and cytochrome c reduction were measured for 15 min after incubation of 0.5 mM xanthine with various concentrations of XO (0.5, 1, 2, and 4 µg/ml), as described under "Experimental procedures." Values represent the mean from three experiments with the S.E. less than 10% of the mean.

LDCL, O2 Consumption, and DEPMPO-OOH Spin Adduct Formation in the GO/Glucose and XO/NADH Systems-- Neither GO/glucose nor the XO/NADH system produces a significant amount of Obardot 2 as detected by SOD-inhibitable cytochrome c reduction and DEPMPO spin trapping (Table I, Figs. ins;1939f5}5 and 6). Neither a significant LDCL nor stimulation of additional O2 consumption was detected in the GO/glucose system at a concentration of lucigenin up to 100 µM (Fig. 5). A weak DEPMPO-hydroxyl (DEPMPO-OH) signal was observed at 50 µM lucigenin (Fig. 5C). However, the formation of this spin adduct was not inhibited by SOD (data not shown), suggesting that Obardot 2 was not produced. The XO/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 µM but not 5 µM lucigenin. Lucigenin at 20 and 50 µM but not 5 µM also stimulated additional O2 consumption (Fig. 6B). A DEPMPO-OOH adduct was also detected at 50 µM lucigenin (Fig. 6C).


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Fig. 5.   LDCL (panel A) and the effects of lucigenin on O2 consumption (panel B) and Obardot 2 production (panel C) in the GO plus glucose system. Measurement of LDCL and O2 consumption and the DEPMPO spin trapping detection of Obardot 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 GO/glucose plus 10 mM DEPMPO; b is as in a but with 5 µM lucigenin; c is as in a but with 50 µM lucigenin. The DEPMPO-hydroxyl adduct (DEPMPO-OH) has the following parameters: aN = 14.05 G; aP = 47.29 G; aHbeta  = 13.40 G; aHgamma  = 0.6 (×3) G. This spin adduct was not SOD-inhibitable. Values in panels A and B represent the mean ± S.E. from at least three experiments. ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 × 105.


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Fig. 6.   LDCL (panel A) and the effects of lucigenin on O2 consumption (panel B) and Obardot 2 production (panel C) in the XO plus NADH system. Measurement of LDCL and O2 consumption, and the DEPMPO spin trapping detection of Obardot 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 XO/NADH plus 10 mM DEPMPO; b is as in a but with 5 µM lucigenin; c is as in a but with 50 µM lucigenin. Spectrum c corresponds to the addition of two spin adducts, DEPMPO-OOH and DEPMPO-OH. Values in panels A and B represent the mean ± S.E. from at least three experiments. ESR spectra represent the averaged signal of 10 scans of 30 s with receiver gain being 1 × 105. *, significantly different from 0 µM lucigenin.

Detection of Mitochondrial Obardot 2 Production by LDCL with Isolated Mitochondria and Intact Monocytes/Macrophages-- The mitochondrial electron transport system is known to be able to reduce O2 to Obardot 2 univalently (24-26). As shown in Fig. ins;1939f7}7, with succinate-driven isolated mitochondria a linear relationship existed between LDCL and the concentration of lucigenin up to 20 µM. 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 µM rotenone/myxothiazol (data not shown). KCN is a mitochondrial cytochrome oxidase inhibitor that causes electrons to build up leading to enhanced production of Obardot 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 Obardot 2, KCN-resistant O2 consumption was determined in the presence of various concentrations of lucigenin. KCN was used to inhibit the O2 utilization by mitochondrial respiration so that the O2 consumption caused by the redox cycling of lucigenin could be detected. No stimulation of KCN-resistant O2 consumption was observed at a concentration of lucigenin up to 20 µM. 50 µM and 100 µM lucigenin slightly stimulated the KCN-resistant O2 consumption (Fig. 7). In contrast, the presence of 5 µM benzo(a)pyrene-1,6-quinone (BPQ) resulted in a marked KCN-resistant O2 consumption (Fig. 7). BPQ has been shown to redox cycle in mitochondria (29). Detection of mitochondrial Obardot 2 production by LDCL in unstimulated intact monocytic cells has been demonstrated previously (9-11, 16, 17). Shown in Fig. ins;1939f8}8 are representative LDCL responses observed with 5 µM 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 O2 consumption was detected in the presence of up to 50 µM lucigenin (Table II). In contrast, incubation of cells with 5 µM BPQ resulted in a marked stimulation of KCN-resistant O2 consumption (Table II).


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Fig. 7.   LDCL (panel A) and KCN-resistant O2 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 0.2 mM KCN. KCN-resistant O2 consumption was measured after incubation of the mitochondria with either the indicated concentrations of lucigenin or 5 µM 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 ± S.E. from at least three experiments. *, significantly different from 20 µM lucigenin. #, significantly different from 50 and 100 µM lucigenin. ND, not detectable.


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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 µM lucigenin in the presence or absence of 0.2 mM KCN or 10 µM rotenone/myxothiazol, as described under "Experimental Procedures."

                              
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Table II
LDCL- and KCN-resistant O2 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 0.2 mM KCN or 10 µM rotenone (ROT)/myxothiazol (MYX). KCN-resistant O2 consumption was measured after incubation of the cells with either the indicated concentrations of lucigenin or 5 µM BPQ. Values represent the mean ± S.E. from at least three experiments. *, significantly different from control. ND, not detectable.

LDCL, O2 Consumption, and DEPMPO-OOH Spin Adduct Formation by the TPA-stimulated Phagocytic NADPH Oxidase System-- LDCL has frequently been used to detect the Obardot 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 Obardot 2 to LDCL (11, 30). As such, 10 µM rotenone and myxothiazol were added to the monocytes/macrophages to block mitochondrial respiration and its accompanying Obardot 2 production. As shown in Fig. ins;1939f9}9, under these experimental conditions, LDCL as well as SOD-inhibitable cytochrome c reduction, O2 consumption, and DEPMPO spin trapping all equally reported a TPA-stimulated Obardot 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 µM lucigenin. Fig. ins;1939f10}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 µM did not stimulate any additional O2 utilization or DEPMPO-OOH formation (Fig. 10, B and C). In fact, the DEPMPO-OOH formation was slightly reduced in the presence of 50 µM lucigenin, which may result from the competition by the lucigenin cation radical for Obardot 2. When the monocytes/macrophages were stimulated with various concentrations of TPA (1.9-15 ng/ml), a significant linear relationship was observed between LDCL and SOD-inhibitable cytochrome c reduction (r = 0.99) or O2 consumption (r = 0.99) by the respiratory burst (Fig. ins;1939f11}11).


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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 Obardot 2-producing activity of the TPA (30 ng/ml)-stimulated NADPH oxidase was assessed by LDCL at 5 µM lucigenin (panel A), SOD-inhibitable cytochrome c reduction (panel B), O2 consumption (panel C), and DEPMPO spin trapping (panel D), as described under "Experimental Procedures." In panels A, B, and C measurements were for 30 min. Values represent the mean ± S.E. 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 × 105. ND, not detectable.


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Fig. 10.   LDCL (panel A) and the effects of lucigenin on O2 consumption (panel B) and Obardot 2 production (panel C) in the TPA-stimulated monocytes/macrophages NADPH oxidase system. Measurement of LDCL and O2 consumption and the DEPMPO spin trapping detection of Obardot 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 × 106 cells, 30 ng/ml TPA plus 10 mM DEPMPO; b is as in a but with 5 µM lucigenin; c is as in a but with 50 µM lucigenin. Values in panels A and B represent the mean ± S.E. from at least three experiments. In panel C, ESR measurement was as described in the legend of Fig. 9.


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Fig. 11.   Correlation between LDCL and SOD-inhibitable cytochrome c reduction (panel A) or O2 consumption (panel B) by the respiratory burst resulting from TPA stimulation of ML-1 cell-derived monocytes/macrophages. LDCL at 5 µM lucigenin, cytochrome c reduction, and O2 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 1.9, 3.8, and 7.5 ng/ml; TPA concentrations used for LDCL/O2 consumption were 1.9, 3.8, 7.5, and 15 ng/ml. Values represent the mean from at least three experiments with a S.E. less than 10% of the mean.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Recently, the use of LDCL for detecting Obardot 2 in biological systems has been questioned (13, 14, 31). To validate lucigenin as a Obardot 2-detecting probe, in this study we have characterized the potential of lucigenin to undergo redox cycling in systems that produce significant amounts of Obardot 2 as well as in systems that produce little or no Obardot 2. LDCL was observed in the Obardot 2-producing XO/xanthine system more than 3 decades ago (1). The univalent reduction of lucigenin by XO has also been shown to precede its reaction with Obardot 2 (1). The complete inhibition of the LDCL by SOD but not by catalase in the XO/xanthine system at physiological pH indicates the specific involvement of Obardot 2 in the reaction pathway leading to LDCL (Fig. 1). The failure of lucigenin at up to 100 µM to stimulate additional O2 consumption and DEPMPO-OOH adduct formation in the XO/xanthine system indicates that lucigenin at these concentrations does not undergo redox cycling in this Obardot 2-generating system. The validity of using LDCL for detecting Obardot 2 production by the XO/xanthine system was strengthened further by the significant linear correlation between the LDCL and the SOD-inhibitable cytochrome c reduction (Fig. 4), a standard assay for measuring Obardot 2 production (32). Stimulation of additional O2 consumption and DEPMPO-OOH adduct formation by lucigenin at 50 µM and above in the LADH/NADH system suggests that lucigenin is more likely to undergo redox cycling in this system than in the XO/xanthine system. Based on cytochrome c reduction and O2 consumption, the LADH/NADH system was less efficient than the XO/xanthine system with regard to Obardot 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 SOD-inhibitable cytochrome c reduction were also observed previously in the LADH plus NADH system (33). There is no Obardot 2 production by the GO/glucose system. However, a significant LDCL response has recently been shown to be elicited by the GO/glucose system at pH 9.5 (13). The H2O2 produced by the GO/glucose at pH 9.5 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 GO/glucose ordinarily catalyzes the two electron reduction of O2 to H2O2, this enzymatic system is unlikely to be able to reduce lucigenin univalently to its cation radical at physiological pH. In contrast to XO/xanthine, XO plus NADH did not produce a significant amount of Obardot 2 as determined by SOD-inhibitable cytochrome c reduction and DEPMPO spin trapping (Table I and Fig. 6). In the presence of 5 µM lucigenin, a strong LDCL response was elicited from the XO/xanthine system, whereas no significant LDCL was observed with the XO/NADH system (Fig. 6). On the other hand, the significant LDCL response and the stimulation of additional O2 utilization and DEPMPO-OOH adduct formation observed at 20 and 50 µM lucigenin in the XO/NADH (Fig. 6) suggest that lucigenin undergoes redox cycling in this enzymatic system. The univalent reduction of lucigenin by the XO/NADH system has been demonstrated previously (13). It is likely that the lucigenin cation radical formed in the XO/NADH system in the absence of enzymatic Obardot 2 autoxidizes and in so doing consumes O2, producing Obardot 2.

It has been long known that the mitochondrial electron transport chain is able to univalently reduce O2 to Obardot 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 Obardot 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 O2 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 µM to stimulate any detectable KCN-resistant O2 consumption indicates that lucigenin does not undergo redox cycling at these concentrations in this cellular system. The ability of lucigenin to detect mitochondrial Obardot 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 KCN-resistant O2 consumption by lucigenin at 50 µM and above (Fig. 7) suggests that redox cycling of lucigenin occurs at high concentrations in the isolated mitochondria. However, comparison of the KCN-resistant O2 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 Obardot 2 production by phagocytic cells after activation of their membrane NADPH oxidase by soluble and particulate stimuli (2-4). When mitochondrial respiration and Obardot 2 formation were inhibited in the monocytes/macrophages by rotenone/myxothiazol, Obardot 2 produced by the TPA-stimulated NADPH oxidase was detected by LDCL as well as SOD-inhibitable cytochrome c reduction and DEPMPO spin trapping (Table I, Figs. 9 and 10). Failure of lucigenin at up to 50 µM to stimulate either additional O2 utilization or DEPMPO-OOH 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 Obardot 2 production by the respiratory burst was supported further by the significant linear relationship between LDCL and SOD-inhibitable cytochrome c reduction or O2 utilization by the membrane NADPH oxidase in the TPA-stimulated 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 µM lucigenin in the undifferentiated ML-1 cells suggests that lucigenin does not undergo redox cycling in this nonObardot 2-generating cellular system.

In summary, this study demonstrates that in the Obardot 2-producing systems examined, marked LDCL was always observed at lucigenin concentrations far below those that stimulated additional O2 consumption and Obardot 2 formation. Because of the opposite charge of the lucigenin cation radical and Obardot 2 and the unstable dioxetane intermediate produced from the reaction of lucigenin cation radical with Obardot 2 (3, 7, Fig. 1), the molecular binding affinity and the rate constant of reaction between lucigenin cation radical and Obardot 2 may be much higher than those between the lucigenin cation radical and O2. This may explain the inability of lucigenin below certain concentrations to undergo redox cycling in the Obardot 2-generating systems. As depicted in Fig. ins;1939f12}12, the relative rate of production of the lucigenin cation radical and Obardot 2 by biological one-electron reduction systems both appear to determine whether LDCL will reflect only biological Obardot 2 or Obardot 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 O2 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 Obardot 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 Obardot 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 O2 consumption by oxygen polarography or alternatively via detection of the stimulation of Obardot 2 formation by DEPMPO spin trapping techniques.


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Fig. 12.   Hypothetical model depicting how biological Obardot 2 affects the ability of lucigenin to undergo redox cycling.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 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{at}jhsph.edu.

1 The 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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