Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems*

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.

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)(2)(3)(4)(5)(6)(7)(8). Our recent studies have also demonstrated that LDCL can be used to monitor mitochondrial O 2 . production in intact cells (9 -11). As illustrated in Fig. 1, to detect O 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 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 (3,12). 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 (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 O 2 . or their ability to reduce O 2 to O 2 . is very limited (13,14). Because (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% CO 2 in RPMI 1640 medium supplemented with penicillin (50 units/ml), streptomycin (50 g/ml), and 7.5% fetal bovine serum in 150-cm 2 tissue culture flasks. The differentiation to monocytes/macrophages was initiated by incubation of cells (3 ϫ 10 5 /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 ϫ 10 7 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 ϫ g for 10 min at 4°C. The supernatant was collected and centrifuged at 10,000 ϫ 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 g/ml LADH and 0.5 mM NADH; 8.5 g/ml GO and 0.5 mM glucose; or 4 g/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 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 ϫ 10 6 cells) were suspended in 2 ml of air-saturated complete PBS (PBS containing 0.5 mM MgCl 2 , 0.7 mM CaCl 2 , and 0.1% 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 O 2 . production from mitochondrial respiration in intact cells, the unstimulated monocytes/macrophages (1 ϫ 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 O 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 KH 2 PO 4 , 2.5 mM MgCl 2 , 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 O 2 Consumption-O 2 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 chrome 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  (Fig. 2). Moreover, when the concentration of XO was varied, a significant linear correlation (r ϭ 0.98) between the LDCL and SOD-inhibitable cytochrome c reduction by the XO/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 O 2 consumption was observed in the presence of a lucigenin concentration up to 20 M. However, both O 2 consumption (Fig. 3B) and DEPMPO-OOH formation (Fig. 3C) were elevated by ϳ30% in the presence of 50 M lucigenin. 100 M lucigenin stimulated further O 2 consumption (Fig. 3B) Neither a significant LDCL nor stimulation of additional O 2 consumption was detected in the GO/glucose system at a concentration of lucigenin up to 100 M (Fig. 5). A weak DEPMPOhydroxyl (DEPMPO-OH) signal was observed at 50 M lucigenin (Fig. 5C). However, the formation of this spin adduct was not inhibited by SOD (data not shown), suggesting that O 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 M but not 5 M lucigenin. Lucigenin at 20 and 50 M but not 5 M also stimulated additional O 2 consumption (Fig. 6B). A DEPMPO-OOH adduct was also detected at 50 M lucigenin (Fig. 6C).   (Fig. 7). In contrast, the presence of 5 M benzo(a)pyrene-1,6-quinone (BPQ) resulted in a marked KCN-resistant O 2 consumption (Fig. 7). BPQ has been shown to redox cycle in mitochondria (29). Detection of mitochondrial O 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 O 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 O 2 consumption (Table II).

LDCL, O 2 Consumption, and DEPMPO-OOH Spin Adduct Formation by the TPA-stimulated Phagocytic NADPH Oxidase
System-LDCL has frequently been used to detect the O 2 . production by phagocytic NADPH oxidase (2)(3)(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 O 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 O 2 . production. As shown in Fig. 9, under these experimental conditions, LDCL as well as SOD-inhibitable cytochrome c reduction, O 2 consumption, and DEPMPO spin trapping all equally reported a TPA-stimulated O 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 O 2 utilization or DEPMPO-OOH formation (Fig. 10, B and C). In fact, the DEPMPO-OOH formation was slightly reduced in the presence of 50 M lucigenin, which may result from the competition by  -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 O 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 O 2 . in the reaction pathway leading to LDCL (Fig. 1). The failure of lucigenin at up to 100 M to stimulate additional O 2 consumption and DEPMPO-OOH adduct formation in the XO/xanthine system indicates that lucigenin at these concentrations does not undergo redox cycling in this O 2 .
-generating system. The validity of using LDCL for detecting O 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 O 2 . production (32). Stimulation of additional O 2 consumption and DEPMPO-OOH 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 XO/xanthine system. Based on cytochrome c reduction and O 2 consumption, the LADH/NADH system was less efficient than the XO/xanthine system with regard to O 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 O 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 H 2 O 2 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 O 2 to H 2 O 2 , 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 O 2 . as determined by SOD-inhibitable cytochrome c reduction and DEPMPO spin trapping (Table I and Fig. 6). In the presence of 5 M 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 O 2 utilization and DEPMPO-OOH adduct formation observed at 20 and 50 M 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 O 2 . autoxidizes and in so doing consumes O 2 , producing O 2 . .
It has been long known that the mitochondrial electron transport chain is able to univalently reduce O 2 to O 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 O 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 O 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 O 2 consumption indicates that lucigenin does not undergo redox cycling at these concentrations in this cellular system. The ability of lucigenin to detect mitochondrial O 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. . production by the respiratory burst was supported further by the significant linear relationship between LDCL and SOD-inhibitable cytochrome c reduction or O 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. depicted in Fig. 12, the relative rate of production of the lucigenin cation radical and O 2 . by biological one-electron reduction systems both appear to determine whether LDCL will reflect only biological O 2 . or O 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 O 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 O 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 O 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 O 2 consumption by oxygen polarography or alternatively via detection of the stimulation of O 2 .
formation by DEPMPO spin trapping techniques.