Diphenyliodonium, an NAD(P)H oxidase inhibitor, also induces apoptosis
Nianyu Li, Kathy Ragheb, Gretchen Lawler, J. Paul
Robinson, Purdue University Cytometry Laboratories, ,
West Lafayette, IN 47907
Abstract
The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are
well-known phagocyte NAD(P)H oxidase inhibitors. However, at high concentrations
they could also suppress ATP production by inhibiting mitochondria complex I NADH
reductase. Since mitochondria have been shown to play a crucial role in producing
superoxide and inducing apoptosis, we therefore investigated the effect of iodonium
compounds on mitochondria-derived superoxide and on apoptosis. Undifferentiated HL60 cells, in which the functional NADPH oxidase is not present, were incubated with
various concentrations of iodonium compounds before assay. Superoxide production was
studied by flow cytometry using hydroethidine as the fluorescent probe. Inhibition of
oxygen consumption and DNA fragmentation was also investigated. Our results showed
that superoxide production of HL-60 cells was increasd in one hour with both rotenone
and iodonium compounds. The degree of respiration inhibition was not proportional to
the amount of superoxide produced. Polyacrylamide gel assay also showed that the
production of superoxide induced by both rotenone and iodonium compounds was able to
trigger DNA fragmentation at 24 hours. These observations suggest that IDP can induce
apoptosis through its effect on mitochondria, possibly by inducing superoxide production.
Key words: Apoptosis; Iodonium compounds; HL-60 cells; Flow cytometry
Introduction
Mitochondria are usually known as the site where oxidative phosphorylation occurs
in eukaryotes. Recently it was recognized that this organelle played an important role in
signal transduction pathways including oxidative stress and apoptosis. Mitochondria are
involved in apoptosis on at lease three aspects. First, upon stimulation, cytochrome c is
released from mitochondria to the cytosol. Once in the cytosol, cytochrome c can lead to
the activation of caspase 3. Cytochrome c release was found during chemotherapeutic
reagent or staurosporine treatment, UV irradiation, growth factor withdrawal, Fas ligation
(Apo-1, CD95/CD95L), and tumor necrosis factor receptor activation (Adachi et al.,
1997; Kharbanda et al., 1997; Kluck et al., 1997; Yang et al., 1997; Bossy-Wetzel et al.,
1998). However, in some other cases, mitochondria permeability transition pores can
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open and release another apoptogenic protein, apoptosis inducing factor (AIF), to the
cytosol. The second way in whick the mitochondria to regulate apoptosis is by disrupting
the respiratory chain. On the one hand, this respiratory chain is so vital to the mammalian
cell that breaking the chain will certainly lead to cell death, apoptosis or necrosis. On the
other hand, during apoptosis when cytochrome c is released from mitochondria to the
cytosol, the respiratory chain will be broken by the loss of the electron carrier cytochrome c. Previous studies showed that inhibitors of the respiratory chain were able
to induce apoptosis. These inhibitors include the complex I inhibitor rotenone (Marchetti
et al., 1996), the complex III inhibitor antimycin A (Marchetti et al., 1996), the complex
VI inhibitor cyanide (Pastorino et al., 1993, Shimizu et al., 1996), the complex V
inhibitor oligomycin (Castedo et al., 1996), and the protonophore inhibitor mClCCP
(Zamzami et al., 1996). Finally, mitochondria are one of the major sites for production of
reactive oxygen species are produced.
Reactive oxygen species (ROS) usually include superoxide anions, hydrogen
peroxide, organic peroxides and radicals. Most of them are small and highly reactive
molecules. ROS can be produced in vivo by many enzyme systems including NADPH
oxidase, NADH oxidase, xanthine oxidase, 5-lipoxygenase and the mitochondria
respiratory chain complexes. Among them, mitochondria respiratory chain is the most
important ROS producing enzyme system. The mitochondria-derived superoxide is
important not only because mitochondria respiratory chain components are present on all
eukaryotic cells, but also because the superoxide produced in mitochondrial can readily
influence the mitochondria function without having to diffuse a long way from the
cytosol. The production of ROS by mitochondria is usually enhanced when the
mitochondria respiratory chain is interrupted. Early reports showed that complex I
inhibitor retenone and 1-methyl-4-phenylpyridinium could enhance ROS production in
mitochondria (Turrens and Boversis, 1980). However, some recent data showed that this
kind of upstream respiratory chain inhibitors could prevent ROS accumulation and thus
protect cells from apoptosis (Schulze-Osthoff et al., 1992; Quillet-Mary et al., 1997).
Also, the result of chemiluminscence showed that both retenone and
diphenyleneiodonium were able to inhibit the ROS production in mitochondria (Li and
Trush, 1998).
Diphenyleneiodonium (DPI) and diphenyiodonium (IDP) are well known as
flavoprotein inhibitors. They have been reported to inhibit phagocyte NADPH oxidase
(Cross and Jones, 1986), nitric oxide synthase (Stuehr et al., 1991), xanthine oxidase
(Sanders et al.), and P-450 NADPH reductase. They could also inhibit mitochondria
respiratory chain complex I NADH reductase. These compounds are widely used to study
the protein-bound flavin, to inhibit superoxide production, and to inhibit apoptosis in
many instances. However, due to their function on mitochondria respiratory chain
complex I, the iodonium compounds also have the potential to increase the production of
superoxide and influence mitochondria function. Therefore we investigated the effect of
iodonium compounds on superoxide production and furthermore the effect of these
chemicals on apoptosis.
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Materials and Methods
Collection of neutrophils
Peripheral blood was collected from healthy adult volunteers by standard
venipuncture methods in a sterile tube containing preservative-free heparin. Preparation
of human neutrophils from normal venous blood was performed as previously described
(Robinson, 1993). Briefly, fresh venous blood was placed in a sterile 50ml centrifuge
tube and diluted with NH4Cl lysing solution (dilution 16.7:1). Tubes were centrifuged at
400  g for 10 minutes at 4ºC. The supernatant was decanted and cells were washed twice
with HBSS two times. Hanks’s balanced salt solution (HBSS) was composed of 137mM
NaCl, 5.4mM KCl, 0.7mM, 1.2 mM NaHCO3, 12.2 mM glucose (0.22%), 27.5 mM Tris,
1.87 mM Ca2+ and 0.8 mM Mg2+, adjusted to pH 7.4. The cell concentration was
determined by a Coulter counter (Beckman-Coulter Corp., Hialeah, FL).and was adjusted
to 2.0 × 106 cells/mL in HBSS
Growth and Differentiation of HL-60 cells
HL60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf
serum (FCS) and 10% new-born bovine serum (NBS). Cells at a density of 1  106
cells/ml were pushed into differentiation by the addition of 1.3% DMSO. After 3-4 days
of culture (72-96 hours), cells were harvested by centrifugation at 250g for 10 minutes.
Measurement of Superoxide production
Measurement of O2- in neutrophils and HL-60 cells was performed as described
before with some modification (Robinson, 1993). Cells were treated with various
reagents and collected at different time points. After washing twice with PBS, the cells
were resuspended in PBS containing either 10 M or 1 M of HE. In the case of
ethidium bromide, 10 M of dye was present. After 10 minutes’ incubation at 37ºC, the
cells were washed and resuspended in PBS. The cell concentration was adjusted to 2 
106 per ml. The cell suspension was placed into 12 × 75 mm tubes for assay. (Coulter
cytometry, Hialeah, FL). All studies were carried out using an air-cooled 15 mW argon
laser (Cyonics Model 2201, San Jose, CA) operating at a wavelength of 488 nm.
Fluorescent signals were collected by using a 610 nm long pass absorbance filter.
Respiration measurement
Oxygen consumption was measured with a Clark oxygen electrode (model 5300: Yellow
spring Instrument Co., Yellow spring, OH) as described before (Rustin et al., 1994).
Briefly, 5  107 cells were collected and resuspended in a medium containing 0.3 M
mannitol, 10 mM KCl, 5 mM MgCl2, 1 mg/ml BSA, 10 mM KH2PO4 and 0.01%
digitonin. 1.6 ml of cell suspension was injected into the respiration chamber which was
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then sealed. Respiration was calculated as the rate of changes in the oxygen concentration
after the addition of various mitochondria complex I inhibitors, assuming the initial
oxygen concentration as 6.8 mg O2 / liter.
DNA fragmentation
The DNA fragmentation assay was performed according to the method described
previously with some modification (Herrmann et al., 1994) Untreated or treated HL-60
cells were washed twice with PBS (4C, pH 7.4) and collected by centrifugation at 500g
for 5 minutes. Cell concentration was adjusted to 2  107 / ml. The cell pellets were
treated with 0.5 ml lysis buffer (10 mM Tris-HCL, ph7.4, 10 mM EDTA, 0.5% sodium
dodecyl sulfate) for 10 minutes on ice. After treated with Rnase A (final concentration
100 g/ml) for 1hour at 37C, the cells were incubated at 50C for 4 hours in the present
of 100 g/ml proteinase K. DNA was then precipitated with 50 l of 3M sodium acetate
(pH 5.2) and 1ml of cold (4C) 100% ethanol. Finally, DNA was dissolved in TrisEDTA. For analysis, 10 to 20 l of DNA was loaded on a 1.2% agarose gel containing 10
g/ml ethidium bromide. Electrophoresis was performed in 0.5  Tris-boric-EDTA
buffer at 70 V for 2 hours. DNA was visualized under ultraviolet light and photographed.
Results and Discussion
The inhibition of superoxide production of NADPH oxidase by iodonium
compounds
Hydroethidine has been shown to be very useful in the study of cellular oxidative
metabolism. A major advantage of hydroethidine is its ability to distinguish between
superoxide and hydrogen peroxide (Robinson, 1998). The dye enters the cell freely and
reacts specifically with superoxide to produce ethidium bromide. Consistent with
previous reports, our result showed that IDP could inhibit superoxide production by
NADPH oxidase both in neutrophils and in dimethylsulfoxide (DMSO) differentiated
HL-60 cells (Figure 1, 2). Similarly, DPI could also inhibit NADPH oxidase both in
neutrophils and in differentiated HL-60 cells (data not shown).
Stimulation of superoxide production by iodonium compounds and mitochondria
complex I inhibitor rotenone
Iodonium compounds are not only NADPH oxidase inhibitors, but also
mitochondria complex I NADH reductase inhibitors. It is suspected that they acted on a
site between NADH and the Fe-S clusters (Majander et. al, 1994), in contrast to rotenone,
whose inhibition site is between the Fe-S clusters and ubiquinone (Palmer et. al, 1968).
Since rotenone was reported to be a good candidate to mimic the TNF function on
mitochondria complex I, including superoxide production, mitochondria membrane
permeability transition, cytochrome c release and apoptosis (Higuchi et. al, 1998), we
thus investigated the possibility that iodonium compounds have similar function.
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Again hydroethidine is used to study superoxide production in the presence
mitochondria complex I inhibitors. Previous results on mitochondria derived superoxide
production were contradictory. Early reports showed that rotenone and 1-methyl-4phenylpyridinium could enhance ROS production both in intact mitochondria and in
submitochondrial particles (Turrens and Boversis, 1980). Dichlorodihydrofluorescein
derivatives or dihydrorhodamine-123 based assay also showed that rotenone could
enhance mitochondrial ROS production. However, recent chemiluminscence data showed
that both rotenone and diphenyleneiodonium were able to inhibit ROS production in
mitochondria (Li and Trush, 1998). Our result clearly showed similarly to rotenone, both
DPI and IDP could induce superoxide on undifferentiated HL-60 cells. It was found that
10 nM to 1 M rotenone could induce superoxide production through a dose-dependent
manner (Figure 3). Higher concentration of rotenone could not further increase
superoxide production. Similarly, DPI or IDP could induce superoxide production in a
concentration range of 1 to 100 M and 10 to 200 M, respectively (Figure 4, 5).
Superoxide levels did not increase after one hour (Figure 6, only IDP data are shown).
Hydroethidine is widely used to monitor superoxide production in intact cells and
isolated mitochondria. However, it has also been reported that mitochondrial membrane
potential could influence the distribution of ethidium bromide across mitochondria
(Rottenberg et. al, 1984). The enhancement of ethidium fluorescence could indicate loss
of mitochondria membrane potential instead of production of superoxide (Budd et al.,
1997). It was suggested that a low concentration (1 M) of hydroethidine should be used
in place of a relative high concentration (10 M). To evaluate our results on superoxide
production by rotenone and iodonium compounds, a parallel experiment was performed
in which HL-60 cells were loaded with 10 to 25 M ethidium bromide. Flow cytometry
showed when 25 M ethidium bromide was used, basal ethidium fluorescence of HL-60
cells are very close to the basal fluorescence in HL-60 cells which were incubated with
10 M hydroethidine (Figure 7), indicating that intracellular ethidium bromide
concentrations in both populations were very close. However, it was found that 50 to 200
M IDP could not induce a fluorescence enhancement in both 10 and 25 M ethidium
bromide loaded cells, in contrast to 10 M hydroethidine loaded cells (Figure 8A, 8B).
This result indicated that the increasing of ethidium bromide fluorescence induced by
IDP was due to superoxide production. IDP treated HL-60 cells were also studied by
incubating with 1 M of hydroethidine. The result showed that as with 10 M
hydroethidine loaded cells, IDP was still able to enhance ethidium fluorescence in a dosedependent manner (figure 9). Combined all these observation, our conclusion is that
iodonium compounds could induce superoxide production on HL-60 cells, possibly
through its inhibition effect on mitochondria respiratory chain complex I.
Oxygen consumption
The mitochondria complex I impairment by rotenone and iodonium compounds was
estimated by measuring cell respiration. Although all of these three compounds were able
to inhibit the mitochondrial respiratory chain at complex I, they seemed to have different
potency. Cell respiration was inhibited to 65% of control by 10 nM rotenone and to less
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than 50% (44%) of control by 20 nM Rotenone (Figure 10-A). Higher concentration of
rotenone (500 nM to 1 M) could entirely block cell respiration (inhibition >95%). In
contrast to rotenone, iodonium compounds inhibit cell respiration less effectively. DPI
could inhibit cell respiration to 60% at 100 M and 43% at 200 M (Figure 10-B). Large
concentration of DPI (1 mM) could inhibit cell respiration to less than 20%. Like to DPI,
IDP only inhibited cell respiration at high concentrations, to 34% by 1 mM and 28% by 2
mM (Figure 10-C). Rotenone could produce more superoxide at 1M than at 100nM
while the degree of cell respiration inhibition at these concentrations was close. However,
100 M DPI and 1 mM DPI produced same amount of superoxide while their cell
respiration inhibitory effect was different. Similarly to DPI, 200 M DPI and 2 mM DPI
produced same amount of superoxide while inhibiting respiration at different degrees. It
was concluded that the degree of inhibition of cell respiration had no direct relationship
with the amount of superoxide produced.
DNA fragmentation
Rotenone was reported to induce apoptosis on various cell lines. Using a DNA
fragmentation technique we also found that 1 M rotenone could trigger DNA
fragmentation at 24 hours while 100 nM rotenone could not (Figure11). It was also found
that both 100 M DPI and 200 M IDP could trigger DNA fragmentation at 24 hours
(Figure 11). It was found that rotenone and iodonium compounds could induce apoptosis
only at concentrations that sufficient superoxide was produced. The degree of cell
respiratory inhibition did not reflect the ability to induce DNA fragmentation. It was also
found that the DNA fragmentation induced by these mitochondria complex I inhibitors
could be inhibited by the non-specific caspase inhibitor Z-VAD, indicating that caspase is
involved in these process.
Conclusion
Diphenyleneiodonium and diphenyiodonium, despite their inhibitory effect on many
superoxide-producing enzymes, could also enhance superoxide production, possibly by
inhibiting mitochondria complex I. And this enhanced superoxide production could
induce apoptosis on HL-60 cells.
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