(Received for publication, July 27, 1995; and in revised form, November 20, 1995)
From the
The mechanism of cytotoxicity of the NO donor
3-morpholino-sydnonimine toward a human ovarian cancer cell line
(OVCAR) was examined. It was found that the NO-mediated loss of cell
viability was dependent on both NO and hydrogen peroxide
(HO
). Somewhat surprisingly, superoxide
(O
) and its reaction product with NO,
peroxynitrite (
OONO), did not appear to be directly
involved in the observed NO-mediated cytotoxicity against this cancer
cell line. The toxicity of NO/H
O
may be due to
the production of a potent oxidant formed via a trace metal-,
H
O
-, and NO-dependent process. Because the
combination of NO and H
O
was found to be
particularly cytotoxic, the effect of NO on cellular defense mechanisms
involving H
O
degradation was investigated. It
was found that NO was able to inhibit catalase activity but had no
effect on the activity of the glutathione peroxidase
(GSHPx)-glutathione reductase system. It might therefore be expected
that cells that utilize primarily the GSHPx-glutathione reductase
system for degrading H
O
would be somewhat
resistant to the cytotoxic effects of NO. Consistent with this idea, it
was found that ebselen, a compound with GSHPx-like activity, was able
to protect cells against NO toxicity. Also, lowering endogenous GSHPx
activity via selenium depletion resulted in an increased susceptibility
of the target cells to NO-mediated toxicity. Thus, a possible
NO/H
O
/metal-mediated mechanism for cellular
toxicity is presented as well as a possible explanation for cell
resistance/susceptibility to this NO-initiated process.
The cytotoxic actions of activated macrophages against human
cancer cell lines both in vitro and in vivo have
been, at least partially, attributed to their ability to generate
nitric oxide (NO) ()(for example see Hibbs et
al.(1988), Stuehr and Nathan(1989), and Farias-Eisner et
al.(1994)). Although the cytotoxic/cytostatic activity of NO is
well established, the chemical mechanism by which NO elicits its
cytotoxic action is less well understood. NO is capable of degrading
certain iron-containing prosthetic groups, which results in an
inhibition of the mitochondrial respiratory chain, DNA synthesis, and
aconitase activity (Hibbs et al., 1988). Along with NO
generation, activated macrophages also produce superoxide
(O
). The reaction of NO with
O
is extremely rapid (Huie and Padmaja,
1993) and results in the generation of peroxynitrite (
OONO), which is a potent chemical oxidant when in
the protonated form (Koppenol et al., 1992). It has been
demonstrated that
OONO can be formed from
macrophage-derived NO (Ischiropoulus et al., 1992) and is
capable of, for example, lipid peroxidation (Radi et al.,
1991a), oxidation of sulfhydryl functions (Radi et al., 1991b)
and aconitase inhibition (Hausladen and Fridovich, 1994; Castro et
al., 1994). It has therefore been proposed that
OONO is responsible for a significant portion of
macrophage derived cytotoxicity through a direct reaction of
OONO with critical cellular components (Koppenol et al., 1992). However, a recent report utilizing NO donor
compounds indicated that NO was particularly tumoricidal in the
presence of hydrogen peroxide (H
O
) and not
O
. Thus, it has been suggested that
OONO may not be the only mechanism responsible for
the cytotoxic actions of NO (at least in the hepatoma cell line
utilized in that study) (Ioannidis and de Groot, 1993).
HO
is formed as an indirect product of
macrophage activation (via the dismutation of
O
). Therefore, we have performed a
detailed examination of the chemistry and enzymology of possible
NO/H
O
interactions with tumor cell components
in order to evaluate the possible role of NO/H
O
in macrophage-mediated tumoricidal activity. Herein, we present
evidence confirming the original observations of Ioannidis and de
Groot(1993) indicating that the combination of NO and
H
O
was particularly cytotoxic to a human
ovarian cancer cell line, and we propose a mechanism, based on a novel
chemical process involving both NO and H
O
, for
macrophage and/or NO-mediated tumoricidal activity. Furthermore, we
present evidence supporting a hypothesis that may explain cell
susceptibility/resistance toward NO-mediated cytotoxicity.
The time course of toxicity was determined for 5 mM SIN-1. Thus, cell viability was monitored by the method described above over a time period of 48 h with time points at 6, 12, 18, 24, and 48 h.
The concentration of NO in the
catalase solution was determined by analyzing a 1-ml aliquot from the
cuvette using a previously described procedure (Fukuto et al.,
1992). Briefly, the 1-ml sample was introduced into a 10-ml flask, the
gaseous contents of the solution were sparged with N, and
the gas stream was passed into a chemiluminescence detector (Antek 720,
Houston, TX). Quantitation of the NO evolved was accomplished by
comparison of the detector response with that of known standards of
authentic NO. Also, the expected concentration of NO in the catalase
solution was calculated using Henry's law and Henry's
coefficient for NO previously reported (Shaw and Vosper, 1977). The two
values were found to be in close agreement, 15.5 µM (experimentally determined) and 20 µM (calculated).
Thus, in a typical experiment, the appropriate metal salt was placed
into a 10-ml flask equipped with a serum-capped stopcock. The salt was
then taken up in 2 ml of water and degassed. Then NO gas was injected
into the reaction headspace. When required, the addition of
HO
to these solutions was also made via
injection of a degassed stock solution in water. Reaction times were
chosen on the basis of the spectroscopic studies described above. That
is, because the reduction of Fe(III) by NO was found to be slow, such
reactions were allowed to run for 15 min, whereas the reaction between
H
O
and Fe(II), which was found to be extremely
fast, was run for only 1 min. Reactions were quenched by the addition
of strong base (NO
is unstable under
acidic conditions, especially in the presence of
H
O
). The reaction flask was then degassed again
to remove any unreacted NO. Aliquots of the solution were then analyzed
immediately. Control experiments were performed to assure the stability
of measured species, NO
and
NO
, under the assay and reaction
conditions.
Figure 1:
The effect of
superoxide dismutase (200 units/ml), HO
, and
catalase (400 units/ml) on SIN-1-mediated loss of OVCAR cell viability.
Each shaded column represents the mean (± S.E.) value
(percentage) of the total lactate dehydrogenase (LDH) activity
in the supernatant of 0.5
10
OVCAR cells/well. *, p < 0.05 compared with control (minimum essential medium (MEM) alone), SIN-1 (2.5 mM), and SIN-1 (5.0
mM) + superoxide dismutase + catalase. All values
represent the mean of at least two experiments performed in
triplicate.
The above data
implicate both NO and HO
as being involved in
the observed cytotoxicity. Because cells are normally able to keep
H
O
levels at a minimum by utilizing enzymes
that specifically degrade H
O
to innocuous
species, the effect of NO on these enzymes, catalase and the
GSHPx-glutathione reductase system, was examined.
Figure 2:
The effect of ebselen on the
SIN-1/superoxide dismutase-mediated loss of OVCAR cell viability. Each
column represents the mean (± S.E.) value (percentage) of total
lactate dehydrogenase (LDH) activity in the supernatant of 0.5
10
cells/well. *, p < 0.05 compared
with SIN-1 (2.5 mM) with superoxide dismutase. The final
concentrations of the enzymes and reagents were: SIN-1, 2.5
mM; superoxide dismutase, 200 units/ml; catalase, 400
units/ml; and ebselen, 10 µM. All values represent the
mean of at least two experiments performed in triplicate. MEM,
minimum essential medium; SOD, superoxide
dismutase.
Figure 3: The effect of selenium (Se) depletion on the susceptibility of OVCAR cells to SIN-1 mediated cytotoxicity. The cells without selenium were determined to have approximately 28% less GSHPx activity compared with the cells with selenium. *, p < 0.0005 when compared with cells cultured in the presence of 25 nM selenious acid. All values represent the mean of at least two experiments performed in triplicate. LDH, lactate dehydrogenase.
Figure 4:
Effect of added ferric ion on the
decomposition of NO in the presence of hydrogen peroxide. , NO
+ H
O
, trace Fe
added at
35 min;
, NO without H
O
, trace
Fe
added at 35 min;
, NO without
H
O
or added
Fe
.
Spectroscopic studies were then
performed to determine the possible interactions of NO and iron in
solution. Thus, the addition of excess NO gas to a 1 mM Fe(III) (FeCl) solution resulted in the gradual
formation of an apparent NO adduct as indicated by the appearance of
absorbances at 436 and 578 nm. Significantly, the addition of NO to a 1
mM Fe(II) (FeSO
) solution resulted in the rapid
formation of the same adduct as evidenced by the identical UV-visible
spectrum. The absorbance spectrum of the apparent NO adduct is
identical to that of the so-called ``brown ring'' complex,
Fe(II)NO(H
O)
with
=
436 and 578 nm (Littlejohn and Chang, 1982). Thus, it is apparent that
NO is able to reduce Fe(III) to Fe(II) in water to generate an
NO (or equivalent) species. Reaction of
NO with water should yield, as the nitrogen oxide
product, NO
( and ). Complexation of Fe(II) by another equivalent of NO
would then give the observed brown ring complex ().
In the absence of any oxidizing agents, the Fe(II)-NO complex
would be the terminal product. However, the formation of an Fe(II)
species in the presence of an oxidizing agent like HO
should lead to the formation of a potent oxidizing species like
OH via a Fenton process (). (Note: the addition of
H
O
to the brown ring complex results in a rapid
disappearance of the complex as evidenced by the immediate loss of the
absorbances at 436 and 578 nm; data not shown.)
Therefore, a solution of Fe(III), NO, and HO
should be capable of reacting by the process defined by the sum
of , , and ().
Thus, represents a process by which NO and
HO
can react, in the presence of a catalytic
amount of a trace metal, to generate a potent oxidizing species like
OH.
This is apparently the case under the conditions of these
experiments because a 0.2 mM solution of Fe(II)SO in water was found to react under anaerobic conditions with NO
(excess) and H
O
(1 equivalent) to give
NO
as the primary nitrogen oxide with
NO
present in only trace amounts. The
results of these studies are summarized in Table 2.
SIN-1 is cytotoxic toward OVCAR cells (Fig. 1). It
might be expected that the NO donor SIN-1 would be an especially
cytotoxic NO donor because it is capable of stoichiometric generation
of O along with NO, and many studies
have implicated the NO-O
reaction
product,
OONO, as being a particularly potent and
destructive oxidant (Koppenol et al., 1992). If this were the
case, superoxide dismutase should attenuate the cytotoxicity of SIN-1.
However, we found that superoxide dismutase did not attenuate
SIN-1-mediated cytotoxicity and, in some cases, actually potentiated
it. This observed potentiation by superoxide dismutase could be
attributed to an increase in the relative rate of H
O
generation from O
dismutation versus trapping by NO, provided that H
O
contributes to the overall cellular toxicity of NO. Furthermore,
our results suggest that NO-mediated cytotoxicity is somewhat dependent
on the presence of H
O
because catalase was able
to protect against the toxicity of SIN-1 (Fig. 1). These results
are consistent with the previous work of Ioannidis and de Groot(1993),
who originally found that H
O
enhances the
toxicity of NO toward a hepatoma cell line. Interestingly, they also
found that
OONO did not appear to play a role in the
observed cytotoxicity in their system. The above mentioned results
indicate that H
O
may be an important mediator
of the tumoricidal activity of NO. Significantly, it has been
previously demonstrated that activated macrophage-mediated cytotoxicity
toward tumor cells could be inhibited by catalase, whereas superoxide
dismutase had no effect (Weiss and Slivka, 1982) (also, preliminary
results in our laboratory with OVCAR cells confirm this observation).
Thus, on the basis of the effects of both catalase and superoxide
dismutase, SIN-1 appears to serve as reasonable model for the activity
exhibited by activated macrophages.
The chemical mechanism(s) by
which NO and HO
are cytotoxic is not
immediately obvious, because unlike the NO and
O
, they would not be expected to
directly react with each other to generate a chemically destructive
species. However, we have shown that NO and H
O
,
in the presence of trace metals, are capable of generating a potent
oxidant (, , and ), possibly
hydroxy radical (
OH), which should be capable of indiscriminate
damage to cellular components. There is chemical precedence for the
individual steps in the reaction sequence leading to
OH
generation. The reduction of Fe(III) to Fe(II) by NO ( and ) has been reported previously (Wayland and Olsen, 1974;
Wade and Castro, 1990; Gwost and Coulton, 1973), and we have obtained
spectroscopic evidence that this can occur with simple iron salts. The
reduction of H
O
by Fe(II) to generate
OH () is a well known process generally referred to as the
Fenton reaction (for an example see Goldstein et al. (1993)). (
)Also, analysis of the nitrogen oxide products from the
reaction of NO with Fe(III), NO with
Fe(III)/H
O
, and NO with
Fe(II)/H
O
indicate that
NO
is the primary species generated (Table 2) and is generally consistent with the proposed chemistry (, , , and ). That
is, the reaction of Fe(III) with NO should give, maximally, 1
equivalent of NO
per Fe(III) via (0.5 equivalents found). The reaction of NO with Fe(III)
and 1 equivalent of H
O
(based on Fe(III))
should give maximally 3 equivalents of NO
via the sequence of , , and , and 2 (2.3 equivalents found). Finally, the reaction of
NO with Fe(II) and 1 equivalent of H
O
(based on
Fe(II)) should give, maximally, 2 equivalents of
NO
via the reaction sequence 4, 6, and 2
(although the stoichiometry for this reaction was unexplainably higher
than expected, 2.3, only NO
was detected
indicating again that predominates under these
conditions).
It is possible that OONO could have
been generated in our chemical system via ,
or possibly by the combination of and .
However, because NO (the
thermodynamically stable decomposition product of
OONO) was not generated to any significant extent
under the conditions of our experiments, we believe that
OONO is not the likely oxidant in our chemical
systems.
Therefore, based purely on chemical studies, it is not
unreasonable that the metal catalyzed reduction of HO
by NO () can occur. In fact, we have demonstrated
that this chemical system is capable of oxidizing organic substrates,
such as benzene, via a process that is consistent with
OH
formation. Therefore, our results establish chemical precedence for a
process by which NO and H
O
, in the presence of
trace metals, can lead to the generation of potent oxidants which, if
formed, would be deleterious to cells.
The above reactions are
reminiscent of the well known Haber-Weiss process that instead of NO
utilizes O as the reducing agent (, , ).
Hydroxy radical generated via the Haber-Weiss reaction has been
proposed to be responsible for some of the cytotoxicity associated with
O. However, detractors from this idea
have noted several points that would suggest that the Haber-Weiss
reaction is an unlikely mechanism of
O
-mediated cytotoxicity (for example,
see Freeman, 1994). For example, because intracellular iron
concentrations are kept low by iron binding proteins and the levels of
both O
and H
O
are kept low by the presence of degradative enzymes such as
superoxide dismutase, catalase and GSHPx, it is thought that conditions
capable of supporting significant Haber-Weiss chemistry are not
physiologically attainable. Some of these same criticisms can be raised
as well against the proposal that the
NO/H
O
/iron system (reaction 5) was responsible
for the observed cytotoxicity. However, NO may influence cellular
conditions and processes to allow such chemistry to occur in the cell.
For example, activated macrophages are capable of liberating
significant portion of the bound iron in target tumor cells (Hibbs et al., 1984; Lancaster and Hibbs, 1990) (although it is known
that O
can also release iron from
proteins as well (for examples see Ryan and Aust(1992) and
Fridovich(1995)). Also, NO is able to increase intracellular
H
O
generation through inhibition of
mitochondrial respiration (Bolanos et al., 1994) and possibly
through the inhibition of catalase (Table 1). Thus, NO is capable
of both releasing the iron required for catalysis of as
well as increase the intracellular H
O
levels
required for the generation of the presumed toxic entity. Although it
is clear that NO is responsible for Fe(III) reduction to Fe(II) in the
purely chemical systems, under physiological conditions other reducing
agents, such as ascorbate, may be serving to reduce the released
metals.
Because NO is known to have a high affinity for the iron in
hemeproteins (for example, see Hoshino et al., 1993), it was
not surprising that catalase, a hemeprotein, was inhibited by NO. ()Based on the equilibrium constant between NO and catalase
of 1.8
10
M
(Hoshino et al., 1993) and assuming that NO inhibition was primarily
competitive, the concentration of NO in our experiment (15.5-20
µM) would predict approximately 73-80% inhibition of
catalase activity. This calculated value is consistent with the
experimentally obtained value of 77% enzyme inhibition. Therefore, in
cells that utilize catalase as its primary method for
H
O
degradation, NO may be expected to raise
intracellular H
O
levels. Under
pathophysiological conditions, it may not be unreasonable to reach
micromolar concentrations of NO near activated macrophages, which
should result in substantial catalase inhibition. It should be noted
that the observed inhibition of catalase by NO is not incongruous with
our finding that catalase protects cultured cells from
NO/H
O
-mediated cytotoxicity. In these in
vitro experiments, catalase was used at a relatively high
concentration (400 units/ml), (
)and some residual activity
should have remained in spite of the presence of NO.
Significantly,
we found that NO does not inhibit GSHPx (or the GSHPx-glutathione
reductase system). ()Thus, cells that primarily utilize
GSHPx to keep H
O
levels low may be expected to
be somewhat resistant to NO-mediated cytotoxicity. Interestingly, it
has been demonstrated that GSHPx levels in macrophages increase when
the cells are cytokine-activated (conditions that also result in the
induction of NO biosynthesis) (Jun et al., 1993). (
)Moreover, it has also been shown that GSHPx activity plays
an important role in macrophage functions under oxidative stress
(Rokutan et al., 1988) and endothelial cells, which synthesize
NO, rely heavily on GSHPx to degrade H
O
(Harlan et al., 1984).
If indeed GSHPx activity were important in
protecting cells from the ravages of NO/HO
/iron
chemistry, it would be expected that ebselen, a compound with
GSHPx-like activity (Muller et al., 1984), would offer some
protection. This was found to be the case (Fig. 2). It should be
noted that ebselen also has antioxidant properties, which are unrelated
to its ability to mimic GSHPx, and this may also play a role in the
observed protective effect (Muller et al., 1984). In view of
this, we also examined the effect of selenium depletion on the
susceptibility of the OVCAR cells to NO-mediated cytotoxicity. As
expected, selenium depletion decreases the activity of the
selenium-dependent GSHPx in OVCAR cells and consequently renders them
more susceptible to SIN-1 mediated toxicity (Fig. 3). Thus,
based on the results of this study, it appears that GSHPx may be vital
to the viability of cells when exposed to significant levels of NO. The
protective effect of GSHPx may be due, in part, to reduction of
intracellular NO/H
O
/iron-oxidizing chemistry
through the elimination of one of the critical reactants,
H
O
. These studies are consistent with previous
observations by others who found that tumor cells high in GSHPx
activity were more resistant to activated macrophage-mediated oxidant
injury (no correlation with catalase levels was observed, however)
(Nathan, 1982). Also, endothelial cells rich in GSHPx were found to be
resistant to activated neutrophil damage, whereas cells that were
catalase-rich and GSHPx-poor were highly susceptible to activated
neutrophil-mediated cytotoxicity (Vercellotti et al., 1988).
Like macrophages, neutrophils are known to generate NO (Wright et
al., 1989).
Thus, it is proposed that NO or activated
macrophage-mediated cytotoxicity can be attributed to the generation of
reactive radical species, such as OH, through a chemical process
involving a trace redox active metal, H
O
, and
NO. Of particular importance is the possibility that this hypothesis
may also be the basis for explaining the differential susceptibility of
cells to NO cytotoxicity. Because NO does not affect the
GSHPx-glutathione reductase system, cells that rely heavily on these
enzymes for handling intracellular H
O
would
have increased resistance to NO cytotoxicity.
This work is dedicated to the memory of Prof. T. Roy Fukuto formally of the Departments of Entomology and Chemistry at the University of California at Riverside. His financial support and guidance made much of this work possible.