(Received for publication, April 7, 1995; and in revised form, June 2, 1995)
From the
3-Morpholinosydnonimine (SIN-1) is widely used to generate
nitric oxide (NO) and superoxide radical (O). The effect of
SOD on the toxicity of SIN-1 is complex, depending on what is the
ultimate species responsible for toxicity. SIN-1 (<1 mM)
was only slightly toxic to HepG2 cells. Copper,zinc superoxide
dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD)
increased the toxicity of SIN-1. Catalase abolished, while sodium azide
potentiated, this toxicity, suggesting a key role for
H
O
in the overall mechanism. Depletion of GSH
from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD.
Although Me
SO, sodium formate, and mannitol had no
protective effect, iron chelators, thiourea and urate protected the
cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The
cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose
response being most pronounced at lower concentrations (10-100
units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of
H
O
in a concentration-dependent manner. In
contrast, Cu,Zn-SOD increased H
O
accumulation
from SIN-1 at low but not high concentrations of the enzyme, suggesting
that high concentrations of the Cu,Zn-SOD interacted with the
H
O
. EPR spin trapping studies demonstrated the
formation of hydroxyl radical from the decomposition of
H
O
by high concentrations of the Cu,Zn-SOD. The
cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly
enhanced by SOD; catalase had no effect. Thus, the oxidants responsible
for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these
conditions are different, with H
O
derived from
O dismutation playing a major role with SIN-1. These results suggest
that the potentiation of SIN-1 toxicity by SOD is due to enhanced
production of H
O
, followed by site-specific
damage of critical cellular sites by a transition metal-catalyzed
reaction. These results also emphasize that the role of SOD as a
protectant against oxidant damage is complex and dependent, in part, on
the subsequent fate and reactivity of the generated
H
O
.
Nitric oxide (NO) ()and other reactive nitrogen
oxidative metabolites (NO
) are produced by a variety of
mammalian cells. These agents may be important for host defense, but
under certain conditions, NO and NO
may cause tissue damage
by still to be clarified mechanisms. Both NO and superoxide anion
radical (O) are known to be generated by macrophages, neutrophils, and
endothelial cells(1, 2, 3) . The production
of these two radicals under physiological conditions can lead to the
formation of peroxynitrite (ONOO
) and other reactive
species that are
cytotoxic(4, 5, 6, 7, 8) .
The cytotoxicity of ONOO
may be mediated by the
ability of this compound to initiate lipid peroxidation (9, 10) , cause oxidation of protein and non-protein
sulfhydryls(11) , produce nitration of tyrosine residues in
proteins(5) , or react with sugars (4) and
DNA(12) . Superoxide dismutase (SOD) present in the cytoplasm
and mitochondria within cells and in the extracellular space dismutates
two O into H
O
and O
(13) .
The protective effect of SOD against oxygen-derived free radicals in vivo, in intact cells and in vitro studies is well
documented(14, 15) . However, in some studies a
bell-shaped dose-response curve for the protective effect of SOD was
observed. At low concentrations, SOD was usually protective, but at
very high concentrations, its protective effect decreased or was
reversed such that SOD potentiated
toxicity(16, 17, 18) .
SIN-1
(3-morpholinosydnonimine), the active metabolite of the vasodilatatory
drug molsidomine, is frequently used as a model compound for a
continuous release of O, NO and/or NO, and other potent
oxidants such as ONOO
and hydroxyl radical
(OH)(16, 19, 20) . These SIN-1-derived strong
oxidants degrade deoxoyribose(6) , oxidize low density
lipoproteins(21) , mediate loss of microsomal
-tocopherol(22) , damage surfactant protein
A(23) , and inhibit glyceraldehyde-3-phosphate dehydrogenase (24) and hepatic gluoneogenesis(25) . Cytotoxic effects
of SIN-1 have been demonstrated in a variety of cell
lines(26, 27, 28) . For example, the
LD
for SIN-1 cytotoxicity against Escherichia coli was 0.5 mM(29) , and 1 mM SIN-1 was very
toxic to neurons(30) . SOD had a protective effect against
SIN-1 toxicity to cultured neurons (30) and to E.
coli(29) , but had no protective effect with rat hepatoma
cells(28) , and SOD actually increased SIN-1 toxicity to Leishmania major(31) . The effect of SOD on the
toxicity of SIN-1 is complex, e.g. if ONOO
is the cause of toxicity, SOD will be protective since
dismutation of O will prevent formation of ONOO
.
However, if NO itself is the toxic agent, SOD can potentiate NO
toxicity by preventing its reaction with O, thereby elevating steady
state concentrations of NO. The role of H
O
, the
product of O dismutation by SOD, has generally not been considered as
important as nitrogenous metabolites in SIN-1 toxicity. It was of
interest that the Cu,Zn-SOD did not provide more than 50% protection
against SIN-1 toxicity to E. coli even when added in
excess(29) , perhaps due to the toxicity of
H
O
produced from O dismutation. In the current
report, the effect of SOD on SIN-1 cytotoxicity in a human hepatoma
liver cell line (HepG2) was determined. It was observed that Cu,Zn-SOD
and Mn-SOD enhanced SIN-1-mediated cytotoxicity to HepG2 cells, and
that this potentiation was due to an increase in H
O
formation caused by SOD-catalyzed dismutation of the O released
from SIN-1. The toxicity of SIN-1 under these reaction conditions and
concentrations appeared to be independent of NO or NO
.
In separate experiments evaluating
iron-catalyzed decomposition of HO
, 1 mM SIN-1 was co-incubated with either Cu,Zn-SOD (100 or 1000
units/ml) or Mn-SOD (100 or 1000 units/ml) for 24 h at 37 °C in the
absence of cells. At the end of the incubation period, 100 mM DMPO and 600 mM Me
SO were added and
decomposition of H
O
was initiated by the
addition of 40 µM FeSO
. In the presence of
Me
SO, OH radicals are converted to CH
radicals,
which were spin-trapped with DMPO (DMPO/CH
adducts), thus
providing a characteristic OH radical fingerprint(37) . The
samples were transferred to a EPR flat cell and measured immediately
after the addition of the last compound.
Figure 1:
The effect of SIN-1, in the absence and
presence of Cu,Zn-SOD, on the viability of HepG2 cells. HepG2 cells
were incubated with the following concentrations of SIN-1 for the
indicated time periods: A, 0.1 mM SIN-1; B,
0.316 mM SIN-1; C, 1 mM SIN-1; D, 3
mM SIN-1. The concentration of Cu,Zn-SOD added to the medium
was as follows: no addition (), 10 units/ml (
), 100
units/ml (
), or 1000 units/ml (
). Results are mean
± S.E. from three experiments.
A more detailed study of the concentration dependence of the cytotoxic effect of Cu,Zn-SOD in the presence of 0.316 mM SIN-1 is shown in Fig. 2. The potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD was biphasic; toxicity increased in a concentration-dependent manner up to 100 units of Cu,Zn-SOD/ml but decreased at higher concentrations (Fig. 2A). Boiled Cu,Zn-SOD did not potentiate the cytotoxicity of SIN-1, and Cu,Zn-SOD in the absence of SIN-1 had no cytotoxic effect (Fig. 2A). Another type of superoxide dismutase, Mn-SOD, also potentiated the cytotoxicity of SIN-1, but in contrast to the Cu,Zn-SOD, the potentiation of SIN-1 cytotoxicity by the Mn-SOD did not decrease at higher concentrations of this isoform (Fig. 2B).
Figure 2:
Effect of SOD on the cytotoxicity of SIN-1
to HepG2 cells. Panel A, effect of catalase and azide on the
potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Viability of the HepG2
cells was determined after a 24-h incubation with 0.316 mM SIN-1 in the presence of the indicated concentrations of
Cu,Zn-SOD. Additions were as follows: SIN-1 (), SIN-1 plus 0.316
mM azide (
), SIN-1 plus catalase (200 units/ml)
(
), SIN-1 plus boiled Cu,Zn-SOD (
), Cu,Zn-SOD in the
absence of SIN-1 (
). Results are mean ± S.E. from three
experiments. Panel B, comparison of the effects of Cu,Zn-SOD
(
) and Mn-SOD (
) on the cytotoxicity of SIN-1 to HepG2
cells. The cytotoxic effect of 0.316 mM SIN-1 was determined
in the presence of the indicated concentrations of Cu,Zn-SOD or Mn-SOD.
Viability was determined after a 24-h incubation period. Results are
mean ± S.E. from three experiments.
Since SOD increases the rate of
production of HO
by catalyzing O dismutation,
the effect of catalase on the SIN-1 plus SOD toxicity was determined.
Catalase (200 units/ml) abolished the SOD-mediated cytotoxic effect of
SIN-1 at all Cu,Zn-SOD concentrations tested (Fig. 2A).
The possible involvement of intracellular H
O
and catalase in the cytotoxic effect of SOD plus SIN-1 was
studied using sodium azide, an inhibitor of the intracellular catalase.
Sodium azide slightly increased the toxicity of SIN-1 in the absence of
SOD and further potentiated the cytotoxic effect produced by the
combination of SIN-1 plus Cu,Zn-SOD (Fig. 2A).
Since
HO
can be a precursor for formation of OH, the
possible involvement of OH-like species in the SOD-mediated cytotoxic
effect of SIN-1 was evaluated using OH scavengers. Viability of the
HepG2 cells was lowered by 10% by 0.632 mM SIN-1 and by 90% in
the presence of SIN-1 plus 100 units/ml Cu,Zn-SOD (Fig. 3).
Thiourea and uric acid were very effective in protecting the HepG2
cells against this SOD-mediated cytotoxicity. However, other OH
scavengers tested, including Me
SO, sodium formate, and
mannitol, had no protective effect (Fig. 3). Similarly, ethanol
(10-100 mM) and benzoic acid (0.5-10 mM)
also had no protective effect (data not shown).
Figure 3:
Effect of hydroxyl radical scavengers on
the potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Viability of the
HepG2 cells was determined after a 24-h incubation without SIN-1 (C), with 0.632 mM SIN-1 (S), or with 0.632
mM SIN-1 plus 100 units/ml Cu,Zn-SOD (SS). All the
other incubations contained SIN-1 plus Cu,Zn-SOD plus the indicated
antioxidants at the following concentrations: thiourea, 0.1, 0.5, 1,
and 5 mM, respectively; urate, 0.1, 0.5, and 1 mM,
respectively; MeSO, 0.1, 0.316, 1, and 3.16 mM,
respectively; formate, 0.1, 0.316, 1, and 3.16 mM,
respectively; mannitol, 1, 3.16, and 10 mM, respectively.
Results are mean ± S.E. from four
experiments.
Formation of OH and
other potent oxidizing species from HO
requires
catalysis by transition metals such as iron. The role of transition
metals in the SOD plus SIN-1 cytotoxicity was studied using the iron
chelators deferoxamine and 2,2`-dipyridyl. As shown in Fig. 4,
the cytotoxicity of SOD plus SIN-1 was reduced in a
concentration-dependent manner by co-incubation of the samples with
these two iron chelators. In contrast, EDTA and DTPA had only a small
protective effect; the viability of the cells exposed to the SIN-1 plus
SOD combination increased from 20% in the absence of chelator to 34 and
39% in the presence of 1 mM EDTA or 1 mM DTPA,
respectively (data not shown).
Figure 4: Effect of iron chelators on the potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Experiments were carried out as described in the legend to Fig. 3: control (no SIN-1 added) (C), 0.632 mM SIN-1 (S), and 0.632 mM SIN-1 plus 100 units/ml Cu,Zn-SOD, (SS). All the other incubations contained SIN-1 plus Cu,Zn-SOD plus the indicated chelators at the following concentrations: deferoxamine, 0.01, 0.0316, 0.1, and 0.316 mM, respectively; 2,2`-dipyridyl, 0.01, 0.0316, 0.1, and 0.316 mM, respectively. Results are mean ± S.E. from three experiments.
The role of intracellular glutathione
in the protection of HepG2 cells against the toxicity of SIN-1 itself
or the potentiation of SIN-1 toxicity by SOD was studied using BSO,
which inhibits GSH synthesis (38) . GSH functions to help
remove HO
via glutathione peroxidase activity;
GSH can also scavenge peroxynitrite (29) . BSO at a
concentration (0.1 mM) that depleted cellular GSH by 85 to 90%
increased the cytotoxic effect of SIN-1 itself from about 5% loss of
viability in the absence of BSO treatment to about 50% loss of
viability after BSO treatment (Fig. 5). In addition, the BSO
treatment also increased the potentiation of SIN-1 cytotoxicity
produced by Cu,Zn-SOD (Fig. 5), analogous to the potentiation
produced by azide (Fig. 2A).
Figure 5: Effect of BSO on SIN-1/SOD cytotoxicity. HepG2 cells were treated without (open bars) or with 0.1 mM BSO (hatched bars) for 2 h, prior to the addition of 0.316 mM SIN-1 plus the indicated concentrations of Cu,Zn-SOD. Viability was determined after a 24-h incubation. Results are mean ± S.E. from three experiments.
Figure 6:
Accumulation of HO
from the decomposition of SIN-1. The formation of
H
O
from 0.316 mM SIN-1 was determined
by assaying for the oxidation of methanol by the
catalase-H
O
compound I complex as described
under ``Experimental Procedures.'' Experiments were
carried out in the absence (blackbar) or presence of
either 100 units/ml Cu,Zn-SOD or Mn-SOD (empty bars) or 1000
units/ml Cu,Zn-SOD or Mn-SOD (hatched bars). Results are mean
± S.E. from three experiments.
Figure 7:
The spectra of DMPO/CH adducts
produced by Fe(II)-catalyzed decomposition of
H
O
. H
O
was formed in
MEM medium during a 24-h incubation of 1 mM SIN-1 with 100
units/ml Cu,Zn-SOD (A), 1000 units/ml Cu,Zn-SOD (B),
100 units/ml Mn-SOD (C), 1000 units/ml Mn-SOD (D), or
in the absence of any SOD (E), After this 24-h incubation, 600
mM Me
SO, 100 mM DMPO, and 40
µM FeSO
were added to samples A-E, and EPR spectra were recorded as described under
``Experimental Procedures.'' PanelF contained 1 mM SIN-1 plus 1000 units/ml Mn-SOD no Fe(II)
was added. This assay measures
OH production from the interaction
of Fe(II) with accumulated H
O
. Each spectrum
represents an average of three scans with the following instrumental
settings: modulation amplitude, 1.25 G; time constant, 0.250 s, scan
rate, 6.25 G/min; gain, 8
10
.
The mechanism of the observed pattern of HO
accumulation in the presence of Cu,Zn-SOD and Mn-SOD was studied.
A strong DMPO/OH signal (a
= a
= 14.9 G) was observed when 50 µM H
O
(comparable concentrations were
produced by the SIN-1 plus SOD system during a 24-h incubation) was
added to the medium containing 1000 units/ml Cu,Zn-SOD and 100 mM DMPO (Fig. 8B). The intensity of the DMPO/OH
signal was considerably smaller when 100 units/ml Cu,Zn-SOD was used (Fig. 8A), and no DMPO/OH signals could be detected
when Mn-SOD (100 or 1000 units/ml) was used (Fig. 8, C and D). The Cu,Zn-SOD concentration-dependent formation
of DMPO/OH adducts is shown in Fig. 9. Formate, but not
Me
SO, inhibited the DMPO/OH signal produced in the
Cu,Zn-SOD/H
O
system (data not shown),
consistent with the decomposition of H
O
within
the channel of Cu,Zn-SOD in the active site of the enzyme(39) .
These data indicate that H
O
produced by
SOD-catalyzed dismutation of O is decomposed to OH radicals in the
presence of 1000 units/ml Cu,Zn-SOD, while much less
H
O
is decomposed by 100 units/ml Cu,Zn-SOD, and
no H
O
decomposition to OH occurs in the
presence of Mn-SOD.
Figure 8:
EPR spectra of DMPO/OH adducts produced by
decomposition of HO
by SOD. Experiments were
carried out as described under ``Experimental
Procedures,'' using DMPO as the
OH spin trapping agent. The
reaction system contained MEM medium, 80 µM H
O
, 100 mM DMPO, and the
indicated additions; 100 units/ml Cu,Zn-SOD (A), 1000 units/ml
Cu,Zn-SOD (B), 100 units/ml Mn-SOD (C), or 1000
units/ml Mn-SOD (D). Each spectrum represents an average of
two scans with the following instrumental settings: modulation
amplitude, 1.25 G; time constant, 0.250 s; scan rate, 6.25 G/min; gain,
4
10
. No DMPO-OH adducts were observed in
the presence of H
O
or Cu,Zn-SOD
alone.
Figure 9:
Concentration dependence of
Cu,Zn-SOD-catalyzed decomposition of HO
. The
data shown are amplitudes of the second peak of DMPO/OH EPR signals
obtained as described in the legend to Fig. 8. Each point
represents mean ± S.D. from three
experiments.
Since SIN-1 generates O and NO/NO simultaneously, it was of interest to evaluate the effect of SOD
on the cytotoxicity of other systems which produce only
NO/NO
. Therefore, SNAP and DEA/NO, which do not generate O,
were used as a source of NO. SNAP or DEA/NO alone (1 and 3 mM)
displayed a moderate cytotoxicity toward the HepG2 cells, and this
toxicity, in contrast to the results with SIN-1, was only slightly
potentiated in the presence of Cu,Zn-SOD (100 units/ml). Moreover,
catalase did not protect cells against the cytotoxic effect of either
SNAP or DEA/NO alone or SNAP or DEA/NO plus Cu,Zn-SOD (Fig. 10).
Figure 10: The effect of SNAP or DEA/NO on the viability of HepG2 cells. The cytotoxic effect of either 1 mM (open bars) or 3 mM (hatchedbars) SNAP (upper panel) or DEA/NO (lower panel) on HepG2 cells was determined after a 24-h incubation in the presence of the indicated additions; 200 units/ml catalase (CAT), 100 units/ml Cu,Zn-SOD (SOD), control (absence of SNAP or DEA/NO) (C). Results are mean ± S.E. from four experiments.
SIN-1 is often used as a model for the continuous release of
O and NO in order to mimic the release of these agents by macrophages,
neutrophils, and endothelial
cells(6, 19, 20, 21, 22) .
The generation of these two radicals under physiological conditions can
lead to the formation of ONOO and other reactive
species (4, 5, 8) . Superoxide dismutase
(SOD), ubiquitously present in living aerobic organisms, generally has
protective effects against oxygen-derived free
radicals(14, 15) . However, in some studies, a
bell-shaped dose-response curve of the protective effect of SOD was
observed(16, 17, 18) . An increase in SOD
activity has been postulated to enhance lipid peroxidation (40, 41) and cellular
injury(42, 43) . Mao et al.(44) attributed the toxicity of SOD to the production of
OH radicals as a result of an increased Fenton reaction. With respect
to NO toxicity, the effects of SOD are dependent on whether NO itself
or ONOO
is the actual species. Scavenging of O by SOD
has been shown to increase the half-life of NO(45) . In the
central nervous system, SOD increased the toxic effects of
NO(46) , perhaps by increasing the half-life of NO.
SIN-1
has been found to be cytotoxic to different mammalian cells (26, 27, 28) . SIN-1 induced neuronal cell
death in a dose-dependent manner; however, the neurons were completely
protected if SOD was present in the reaction system(30) .
Brunelli et al.(29) found that SIN-1 killed E.
coli with an LD of 0.5 mM. Cu,Zn-SOD
(50-400 units/ml) provided substantial, but not complete,
protection against this SIN-1 killing. Cu,Zn-SOD and catalase together
completely protected E. coli against SIN-1 toxicity. Assreuy et al.(31) recently reported that SIN-1 (0.3
mM) was efficient in killing L. major, and this
cytotoxicity was enhanced by the Cu,Zn-SOD (500 units/ml). The authors
suggested that the killing of L. major by SIN-1 was dependent
only on NO, and the enhancement of toxicity by SOD was due to
scavenging of O, thereby increasing the half-life of the NO. Ioannidis
and DeGroot (28) found that the cytotoxicity of SIN-1 to Fu5
hepatoma cells was not affected by SOD, but catalase diminished cell
damage; the authors suggested a cooperative toxic action between
H
O
and the tumoricidal activity of NO in the
Fu5 cells. Siegfried et al.(47) reported that SIN-1
had significant cardioprotective effects in a myocardial
ischemia-reperfusion model, perhaps via quenching of O by NO produced
from the SIN-1.
We found that Cu,Zn-SOD did not protect against the
cytotoxicity to human hepatoma HepG2 cells caused by a high
concentration of (3 mM) SIN-1. Actually, both Cu,Zn-SOD and
Mn-SOD potentiated SIN-1-mediated cytotoxicity against the HepG2 cells,
resulting in toxicity at SIN-1 concentrations that alone had no, or
only a minor, cytotoxic effect. Lack of potentiation by boiled
Cu,Zn-SOD indicated that catalytically active enzyme was required for
potentiating this cytotoxic effect of SIN-1. Catalase abolished the
SIN-1 plus SOD-mediated cytotoxic effect at all Cu,Zn-SOD
concentrations evaluated (Fig. 2), whereas boiled catalase had
no effect. This suggests that HO
, produced by a
SOD-catalyzed dismutation of O, is involved in the overall cytotoxic
effect of SIN-1 plus SOD. The further potentiation of the SIN-1 plus
Cu,Zn-SOD cytotoxicity by sodium azide, an inhibitor of intracellular
catalase, is in accord with this suggestion, as are assays of
H
O
accumulation produced by SIN-1 in the
absence and presence of SOD. Alternatively, catalase may be protective
by reaction with NO thereby decreasing levels of NO and
ONOO
. In preliminary experiments, we found that
catalase had no effect on assays of NO
formation from SIN-1
as detected with the Griess reagent. It is likely that Cu,Zn-SOD and
Mn-SOD are increasing dismutation of O produced from SIN-1 to
H
O
in the culture medium rather than in the
HepG2 cells themselves. For example, the conditioned medium containing
SIN-1 plus Cu,Zn-SOD was cytotoxic when added later to HepG2 cells
indicating formation of a stable toxic product, probably
H
O
. This raises the question as to whether the
SIN-1 plus SOD, i.e. H
O
-mediated,
toxicity is occurring extra- or intracellularly.
The toxicity of
HO
produced by the SIN-1/SOD system appears to
be intracellular and mediated by transition metal-catalyzed
decomposition of H
O
at or near critical
cellular sites, based upon the following results. 1) The presence of
sodium azide, an inhibitor of intracellular catalase, increases cell
killing by the SIN-1/SOD combination (Fig. 2); 2) depletion of
intracellular glutathione by BSO also enhanced the observed toxicity of
SIN-1/SOD (Fig. 5); 3) penetrable metal chelators such as
deferoxamine and 2,2`-dipyridyl were very effective in reducing the
SIN-1/SOD-mediated cytotoxicity, while EDTA and DTPA, which do not
readily cross cellular membranes and hence act to chelate iron
extracellularly(48) , provided only a small protection. The
inhibition of SIN-1/SOD toxicity by metal chelators would argue against
a significant role for ONOO
or singlet oxygen, which
may be produced from the interaction of H
O
with
NO (49) or H
O
with ONOO
(50) in the mechanism of SIN-1 toxicity. The possibility
that the cytotoxicity was mediated by the reaction of NO with
H
O
is not likely since no reaction between
H
O
and NO was found at physiological
pH(51) . The requirement for H
O
and
iron in the overall mechanism by which SIN-1/SOD causes toxicity to the
HepG2 cells would suggest that OH-like species formed via a Fenton-type
reaction may be the oxidants responsible for the cytotoxicity. Indeed,
thiourea and urate, potent OH scavengers, were effective in protecting
the HepG2 cells against SIN-1/SOD toxicity. However, other OH
scavengers such as Me
SO, formate, mannitol, benzoate, and
ethanol did not afford any protection; Me
SO, ethanol, and
formate are permeable agents and should be available to scavenge OH
that is accessible. The inability of OH scavengers to provide
protection against oxidant injury has sometimes been explained by
site-specific damage produced when H
O
is
decomposed by transition metals bound to critical cellular targets (52, 53) . The ability of thiourea and urate to
protect against the SIN-1/SOD cytotoxicity may reflect the ability of
these agents to chelate metals (54) or react with
H
O
(55, 56) .
The
effectiveness of Cu,Zn-SOD to potentiate SIN-1 toxicity displayed an
unusual SOD concentration dependence in that lower concentrations of
SOD enhanced the SIN-1 toxicity to a greater extent than higher
concentrations. The Mn-SOD did not display this biphasic response.
These effects can probably be related to the levels of
HO
that accumulate in the reaction system,
since H
O
plays a central role in the
cytotoxicity of SIN-1/SOD. Highest levels of H
O
are found when SIN-1 is incubated with the lower concentration of
the Cu,Zn-SOD or with low and high concentrations of the Mn-SOD. The
lowest levels of H
O
are found when SIN-1 is
incubated with the high concentration of Cu,Zn-SOD; these levels are
even lower than those produced by SIN-1 alone, in the absence of SOD.
These results suggest that high concentrations of the Cu,Zn-SOD, but
not the Mn-SOD, can decompose H
O
. The ability
of Cu,Zn-SOD but not Mn-SOD to decompose H
O
in
a Fenton-type reaction was originally reported by Yim et
al.(39) . It should be kept in mind that because of the
high rate constant for the SOD-catalyzed dismutation of O (2
10
M
s
),
only small concentrations of SOD are usually necessary to scavenge the
O formed. Considerably higher concentrations of Cu,Zn-SOD are required
to decompose substantial amounts of the formed
H
O
, probably because of the expected low rate
of this reaction (the reported rate constant of different Fenton
catalysts are in the range 10
to 10
M
s
; Refs. 57 and
58). The ability of high concentrations of the Cu,Zn-SOD, but not the
Mn-SOD, to decompose H
O
, with the subsequent
production of OH (39) was confirmed by the experiments shown in Fig. 7-9. This raises the question as to why OH formed by
the decomposition of H
O
by high concentrations
of Cu,Zn-SOD, did not contribute to the cytotoxicity of SIN-1/SOD, i.e. less toxicity is observed under conditions in which OH is
being produced, and Mn-SOD which does not decompose
H
O
to OH was equally cytotoxic at
concentrations of 100 units/ml and more cytotoxic at 1000 units/ml than
Cu,Zn-SOD at the same concentrations. Most likely the OH produced by
this reaction is formed deep within the channel of the Cu,Zn-SOD (39) and reacts with the enzyme before escaping into the bulk
solution to damage the cells. It must also be considered that although
levels of H
O
found in the presence of SIN-1
plus the high concentration of Cu,Zn-SOD are lower than that produced
by SIN-1 alone, toxicity is observed in the former but not the latter
case (Fig. 1, B and C), which may actually be
a reflection of sufficient concentrations of H
O
crossing the cellular membrane before decomposition by the
extracellular Cu,Zn-SOD occurs or there is a contribution toward
overall toxicity by OH produced by the SIN-1 plus Cu,Zn-SOD
combination.
SOD is generally thought to play a protective role in
biological systems against oxidant action. It is now becoming apparent
that the net effect of SOD is complex and often depends on the fate of
the generated HO
and the availability of
H
O
degradation enzymes such as catalase and
glutathione peroxidase. SIN-1 is the active metabolite of molsidomine,
an antianginal drug(59) , which is used for the treatment of
ischemia reperfusion injury (60) and human erectile
dysfunction(61) . It is also used for the production of O and
NO in a variety of experimental systems. The potentiation of SIN-1
cytotoxicity by SOD therefore requires caution in the use of this agent
and in considering the therapeutic efficacy of SOD in systems that
generate O and NO. Our results indicate that the observed potentiation
of the cytotoxicity of SIN-1 by SOD is linked to the formation of
H
O
by SOD-catalyzed dismutation of O. The
largely extracellularly produced H
O
then
diffuses inside the cells, where it exerts its toxic effect via
reaction with transition metals bound to critical cellular sites. The
Cu,Zn-SOD did not significantly increase or decrease the cytotoxicity
of SNAP or DEA/NO (sources of NO but not O), nor did catalase protect
HepG2 cells against the cytotoxicity of SNAP or DEA/NO (Fig. 10), which suggests no significant role for NO in the
observed cytotoxic effects of SIN-1 at the concentrations utilized (e.g. 0.316 or 1 mM) and under these reaction
conditions. A role for NO in SIN-1 toxicity may occur under different
conditions or concentrations, e.g. in the potent toxic effects
of higher concentrations of SIN-1 (e.g. 3 mM), found
in the absence of SOD (Fig. 1D).