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 (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 vitro
enzymatic 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.
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INTRODUCTION |
The detection and measurement of fluxes of O
2 within
cells are of critical importance for investigating the physiological and pathological roles of O
2. Because of its sensitivity
lucigenin-derived chemiluminescence
(LDCL)1 has frequently been
used in the specific detection of O
2 production by both
in vitro enzymatic systems and intact cells. For example, LDCL has been used to detect O
2 production by xanthine oxidase (XO) plus xanthine or hypoxanthine, NADPH-cytochrome P450 reductase in
microsomes, NADPH oxidase in phagocytic cells, and a possible diphenyleneiodinium-sensitive NAD(P)H oxidase in endothelial, fibroblastic, and vascular smooth muscle cells (1-8). 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. ins;1939f1}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 O2 to O
2 is very limited
(13, 14). Because of the opposite charge of the lucigenin cation
radical and O
2, the lucigenin cation radical may have a much
higher affinity for O
2 than for O2. As such, in
cellular systems that produce significant amounts of O
2 under
physiological conditions, the propensity of lucigenin to undergo redox
cycling may be very limited. In this study, we examined and correlated three end points, including sensitive measurement of LDCL,
O2 consumption by oxygen polarography, and O
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 O
2 in systems that generate O
2, including XO/xanthine, lipoamide dehydrogenase
(LADH)/NADH, isolated mitochondria, mitochondria in intact cells, and
phagocytic NADPH oxidase, as well as in systems that produce little or
no O
2, including GO/glucose and XO/NADH. Our results
demonstrate that in the O
2-producing systems examined,
significant LDCL was always elicited at lucigenin concentrations far
below those that stimulated additional O2 utilization or
O
2 formation via the redox cycling of the lucigenin
molecule.
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EXPERIMENTAL PROCEDURES |
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 O
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
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 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 O
2 by Ferricytochrome c Reduction--
The
generation of O
2 was measured indirectly by the reduction
of ferricytochrome at 550 nm as described previously (21). Non-O
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 O
2--
For DEPMPO spin trapping
measurement of O
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.
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RESULTS |
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 O
2 as detected by SOD-inhibitable cytochrome
c reduction and DEPMPO spin trapping (Table
I, Figs. ins;1939f2}2
and 3). DEPMPO reacts with O
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 O 2 production by various systems used
in this study
The O2 consumption and O 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
O 2 production (panel C) in the XO plus xanthine system. Measurement of LDCL and O2 consumption and the
DEPMPO spin trapping detection of O 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; aH = 13.11 G;
aH = 0.71, 0.42, 0.7, 0.25, and
0.6 G. Y (57%): aN = 13.08 G;
aP = 45.85 G;
aH = 9.53 G;
aH = 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
O 2 production (panel C) in the LADH plus NADH system. Measurement of LDCL and O2 consumption, and
the DEPMPO spin trapping detection of O 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.
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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
O
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 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 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
O 2 production (panel C) in the GO plus glucose system. Measurement of LDCL and O2 consumption and the
DEPMPO spin trapping detection of O 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;
aH = 13.40 G;
aH = 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
O 2 production (panel C) in the XO plus NADH system. Measurement of LDCL and O2 consumption, and
the DEPMPO spin trapping detection of O 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.
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Detection of Mitochondrial O
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 O
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 O
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 O
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 O
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.
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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 O
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 O
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 O
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 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 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 O
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
O 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
O 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 O 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.
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DISCUSSION |
Recently, the use of LDCL for detecting O
2 in biological
systems has been questioned (13, 14, 31). To validate lucigenin as a
O
2-detecting probe, in this study we have characterized the
potential of lucigenin to undergo redox cycling in systems that produce
significant amounts of O
2 as well as in systems that produce
little or no O
2. LDCL was observed in the
O
2-producing XO/xanthine system 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 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 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 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 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
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 O
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 O
2 autoxidizes and in so doing
consumes O2, producing O
2.
It has been long known that the mitochondrial electron transport chain
is able to univalently reduce O2 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 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 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. 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 O
2 production by phagocytic cells after activation of
their membrane NADPH oxidase by soluble and particulate stimuli (2-4).
When mitochondrial respiration and O
2 formation were inhibited
in the monocytes/macrophages by rotenone/myxothiazol, O
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 O
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 nonO
2-generating cellular
system.
In summary, this study demonstrates that in the
O
2-producing systems examined, marked LDCL was always observed
at lucigenin concentrations far below those that stimulated additional
O2 consumption and O
2 formation. Because of the
opposite charge of the lucigenin cation radical and O
2 and the
unstable dioxetane intermediate produced from the reaction of lucigenin
cation radical with O
2 (3, 7, Fig. 1), the molecular binding
affinity and the rate constant of reaction between lucigenin cation
radical and O
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 O
2-generating systems. As depicted in Fig.
ins;1939f12}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 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 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
O2 consumption by oxygen polarography or alternatively via
detection of the stimulation of O
2 formation by DEPMPO spin
trapping techniques.

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