Synergistic cytotoxicity from nitric oxide and hyperoxia in
cultured lung cells
Pramod
Narula,
Jing
Xu,
Jeffrey A.
Kazzaz,
Carolyn G.
Robbins,
Jonathan M.
Davis, and
Stuart
Horowitz.
CardioPulmonary Research Institute, Departments of Pediatrics,
Pulmonary Medicine, and Thoracic and Cardiovascular Surgery,
Winthrop-University Hospital, State University of New York at Stony
Brook School of Medicine, Mineola, New York 11501
 |
ABSTRACT |
Exogenous nitric oxide (NO) is being tested
clinically for the treatment of pulmonary hypertension in infants and
children. In most cases, these patients receive simultaneous oxygen
(O2) therapy. However, little is
known about the combined toxicity of NO+hyperoxia. To test this
potential toxicity, human alveolar epithelial cells (A549 cells) and
human lung microvascular endothelial lung cells were cultured in room
air (control), hyperoxia (95% O2), NO (derived from chemical
donors), or combined hyperoxia+ NO. Control cells grew normally over a
6-day study period. In contrast, cell death from hyperoxia was evident
after 4-5 days, whereas cells neither died nor divided in NO
alone. However, cells exposed to both NO and hyperoxia began to die on
day 2 and died rapidly thereafter.
This cytotoxic effect was clearly synergistic, and cell death did not
occur via apoptosis. As an indicator of peroxynitrite formation,
nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine
antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to
NO or NO+hyperoxia. These results indicate that combined NO+hyperoxia
has a synergistic cytotoxic effect on alveolar epithelial and lung
vascular endothelial cells in culture.
apoptosis; oxygen toxicity; persistent pulmonary hypertension of
the newborn; lung injury
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INTRODUCTION |
NITRIC OXIDE (NO) is an inorganic, highly reactive, and
rapidly diffusible gas that was discovered to be the
endothelium-derived relaxation factor (13, 18). Endogenous
NO is produced by many cell types and is important in regulating normal
basal vasomotor tone. In the lung, NO produced by the pulmonary
endothelium is responsible for the fall of pulmonary vascular
resistance soon after the birth of the newborn infant (1). High
vascular pressure persists when this response fails to occur, resulting
in persistent pulmonary hypertension of the newborn. Exogenously
administered NO is also a potent vasodilator (5, 7, 12, 16, 20, 22).
Its mechanism of action is primarily through the activation of
guanylate cyclase, which increases the production of guanosine 3',5'-cyclic monophosphate and results in smooth muscle
relaxation (24). A dynamic cycle exists in which hemoglobin is
S-nitrosylated in the lung when red
blood cells are oxygenated, and the NO group is released during
arterial-venous transit. Changes in oxygen (O2) tension might also regulate
NO delivery because NO release is facilitated by deoxygenation (14).
Various early reports (16, 20) showed that inhalation of NO together
with high inspired concentrations of
O2 significantly improved
oxygenation in infants with persistent pulmonary hypertension of the
newborn and in some cases reduced the need for extracorporeal membrane
oxygenation. NO has also been reported to improve oxygenation in
premature infants with respiratory distress syndrome and in full-term
infants with congenital heart disease and diaphragmatic hernia (5, 12).
Clinical use of NO in the United States is currently limited to
experimental protocols for treating extremely sick patients with
primary diseases of the lungs or heart. These patients are invariably
receiving high concentrations of
O2 (hyperoxia) to maintain
adequate oxygenation. Hyperoxia alone is cytotoxic (9). In most
instances, NO is added to the existing flow of O2. Recent evidence suggests that
the combined use of NO+hyperoxia has adverse effects on lung surfactant
(10, 11, 19). However, there is a paucity of studies addressing whether
the combination of inhaled NO+hyperoxia can injure cells or organs.
Here we report that this combination produces synergistic cytotoxicity
in cultured alveolar epithelial and pulmonary microvascular endothelial
cells.
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METHODS |
Cell culture and cytotoxicity assays.
Human alveolar epithelial A549 cells (American Type Culture Collection
CCL-185) were grown in Ham's F-12-K medium
(GIBCO BRL, Life Technologies,
Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, 100 U/ml of penicillin, and 100 µg/ml of
streptomycin. Human microvascular endothelial lung cells (HMVEC-L)
(Clonetics, San Diego, CA) were grown in endothelial growth medium
(Microvascular, BulletKit, Clonetics). Cells were maintained at
37°C in 95% room air-5% CO2
in a humidified chamber. A549 cells were seeded at 6 × 104 cells/ml, and HMVEC-L were
seeded at 4.8 × 104
cells/ml. Cells adhered overnight in 35-mm culture dishes. The next
day, the dishes were divided into four groups, and cells were grown in
95% room air-5% CO2 (control),
room air+NO, 95% O2-5%
CO2 (hyperoxia), and hyperoxia+NO.
NO was generated in culture medium using one of two NO donors:
S-nitroso-N-acetylpenicillamine (SNAP; Biomolecular Research Laboratories, Plymouth Meeting, PA) or
2,2'-(hydroxynitrosohydrazono)-bis-ethanamine (DETA
NONOate; Cayman Chemical, Ann Arbor, MI). NO levels generated in
culture medium were measured using the Iso-NO meter (World Precision
Instruments, Sarasota, FL). Media, NO donors, and gasses were refreshed
once each day. The total cell count and number of live cells were
determined daily with a hemacytometer; live cells were distinguished by
the exclusion of trypan blue dye.
Assays of apoptosis.
Cells were grown on glass coverslips and treated as described above for
3 days. The protocol utilized for the terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling
(TUNEL) assay was as described previously (15).
Fluorescent TUNEL reagents were obtained from Boehringer Mannheim.
Cells were dual labeled with 4 µg/ml of the DNA-binding dye
4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Boehringer Mannheim, Indianapolis, IN) for 5 min at room temperature.
With the use of a Nikon Optiphot microscope with epifluorescence and an
ultraviolet-2A filter (Nikon Instruments, Melville, NY), apoptotic nuclei were identified as smaller and brighter objects than their normal counterparts (15).
Western blot analysis.
Cells were washed three times with phosphate-buffered saline (PBS) and
lysed in 300 µl of lysis buffer [10 mM
tris(hydroxymethyl)aminomethane (Tris) hydrochloride, pH 6.8, 10 µg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluoride] per
dish. Cellular proteins were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis using 4-20% acrylamide
gradient gels (BioRad Laboratories, Hercules, CA) under reducing
conditions (17). Proteins were transferred to Immobilon polyvinylidene
fluoride membranes (Millipore, Bedford, MA) at 24 V for 30 min using an electrophoretic blotting apparatus (Idea Scientific,
Minneapolis, MN). Nonspecific binding to the membrane was blocked by
incubation with 5% nonfat dry milk overnight at 4°C.
Nitrotyrosine-containing proteins were immunodetected using 1 µg/ml
of rabbit polyclonal anti-nitrotyrosine antibody (Upstate
Biotechnology, Lake Placid, NY) in PBS and 0.1% Tween 20. A goat
anti-rabbit secondary antibody [immunoglobulin G (IgG)] conjugated to alkaline phosphatase (Boehringer Mannheim) was used to
detect the primary antibody and was visualized by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphate buffer (100 mM Tris · HCl, pH 9.5, and 50 mM MgCl2).
Immunocytochemistry.
Cells cultured on coverslips were rinsed with ice-cold PBS and fixed
and permeabilized for 30 min in 2% paraformaldehyde and 0.1% Triton
X-100. Cells were incubated for 1 h at 4°C with 3 µg/ml of the
same rabbit polyclonal anti-nitrotyrosine antibody. Primary antibody
was detected with 240 µg/ml of goat anti-rabbit IgG conjugated with
rhodamine (Organon Teknika, Durham, NC). The cellular localization of
the antigen was visualized using a G-2A filter (Nikon) attached to a
fluorescent microscope.
 |
RESULTS |
To compare the cytotoxicity of combined NO+hyperoxia with either agent
alone, cells were divided into four groups: room air (control), room
air+NO, hyperoxia (95% O2-5%
CO2), and a combination of
hyperoxia+NO. A series of preliminary experiments was performed to
identify conditions under which the NO donors would not be overtly
cytotoxic to A549 cells or HMVEC-L. On the basis of these experiments,
we determined that the highest concentration that caused virtually no
cell death over the time course of 1 wk was 2 mM SNAP or 0.5 mM DETA
NONOate (data not shown). In the culture medium used, 2 mM SNAP yielded
peak levels of NO (4.4 µM) after 2 h, and NO levels gradually
declined to baseline over the next 10 h (Fig.
1). As expected, control untreated cells
increased in number during the entire experiment when cultured in room
air alone. Figure 2 shows that there was no
appreciable cell death in SNAP alone during the experiment, although
the cell population did not increase. Similarly, cell death was minimal
in the hyperoxia group until day 4.
Unlike these relatively modest cytotoxic effects, cells exposed to the
combination of NO+hyperoxia died rapidly (Fig. 2). Cell death in large
numbers was evident as early as day 2,
and the remaining cells died soon thereafter. This cytotoxicity was
clearly synergistic, with the death rate exceeding any additive effect.
To determine whether the synergistic cytotoxicity was due specifically
to NO+hyperoxia and not to a nonspecific artifact of the NO donor SNAP,
the experiment was repeated using a different NO donor, DETA NONOate. A
concentration of 0.5 mM was chosen on the basis of several preliminary
experiments that showed no cytotoxicity at this concentration over a
period of 6 days. NO release from 0.5 mM DETA NONOate was slower but
more sustained than with 2 mM SNAP (Fig. 1). Figure
3 shows that the NO donor alone had few cytotoxic effects but was synergistic when used in combination with
hyperoxia. This observation confirms the earlier results with SNAP and
supports the conclusion that synergistic cytotoxicity was due to
hyperoxia+NO and not a by-product of either NO donor. To determine
whether cell death synergy is limited to these transformed cells or
occurs in any primary lung cells, the experiments were repeated using
primary HMVEC-L. Figure 4 shows that
synergistic cytotoxicity was evident in these primary cells, suggesting
that the effects of O2+NO might be
widespread. HMVEC-L cultured under these conditions were relatively
resistant to both NO and hyperoxia, and the synergistic death did
not occur until day 6.

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Fig. 1.
Production of nitric oxide (NO) during cell culture in 2 mM
S-nitroso-N-acetylpenicillamine
(SNAP) or 0.5 mM 2,2'-(hydroxynitrosohydrazono)-bis-ethanamine
(DETA NONOate). SNAP or DETA NONOate were added to culture media as
described in METHODS, and NO levels
were monitored over a 24-h time period. Results are of a typical
experiment reproduced at least 3 times.
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Fig. 2.
Synergistic cytotoxicity of NO from 2 mM SNAP+hyperoxia in cultured
human alveolar epithelial A549 cells. Cells were cultured in 95% room
air-5% CO2 (control) or 95%
O2-5%
CO2 (hyperoxia) in presence or
absence of 2 mM SNAP as described in
METHODS. Cells (total and live) were
counted each day. Values are means ± SD.
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Fig. 3.
Synergistic cytotoxicity of NO from 0.5 mM DETA NONOate+hyperoxia in
cultured A549 cells. Cells were cultured in 95% room air-5%
CO2 (control) or 95%
O2-5%
CO2 (hyperoxia) in presence or
absence of 0.5 mM DETA NONOate as described in
METHODS. Cells (total and live) were
counted each day. Values are means ± SD.
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Fig. 4.
Synergistic cytotoxicity of NO from 2 mM SNAP+hyperoxia in cultured
human microvascular endothelial lung cells (HMVEC-L). Cells were
cultured in 95% room air-5% CO2
(control) or 95% O2-5%
CO2 (hyperoxia) in presence or
absence of 2 mM SNAP as described in
METHODS. Cells (total and live) were
counted each day. Values are means ± SD.
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To determine whether combined NO+hyperoxia causes cell death via
apoptosis, two different assays of apoptosis were utilized. DAPI is a
DNA-binding dye that is fluorescent when bound to DNA and excited by
ultraviolet light. Apoptotic nuclei are clearly distinguished from
normal nuclei on the basis of their intense fluorescence and much
smaller size (15). Figure 5 shows that apoptosis was not evident after culture in NO alone, hyperoxia alone,
or combined NO+hyperoxia. As a positive control, cells were exposed to
a level of
H2O2
that is known to induce widespread apoptosis in these cells (15).
Figure 5 shows widespread apoptosis under these conditions. To
independently confirm these observations, apoptosis was also studied
with the TUNEL assay, which labels 3'-COOH ends of DNA in
chromatin, which result from endonucleolytic cleavage occurring during
apoptosis (15). Figure 5 shows that cells cultured in NO+hyperoxia, in
NO alone, and in hyperoxia alone were all TUNEL negative, whereas cells
exposed to
H2O2
(positive control) clearly demonstrate apoptosis. These data confirm
the observation that hyperoxia does not cause epithelial cell apoptosis (15) and indicate that combined NO+hyperoxia also causes cell death via
nonapoptosis.

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Fig. 5.
Nonapoptotic cell death from NO+hyperoxia. A549 cells were cultured for
3 days on coverslips in room air in absence
(A and
F) or presence
(B and
G) of 2 mM SNAP and in hyperoxia in
absence (C and
H) or presence
(D and
I) of 2 mM SNAP for 3 days as
described in METHODS.
E and
J: a typical field of cells exposed to
5 mM
H2O2
for 3 h (positive control for apoptosis). Cells in
A-E were terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeled. Cells in
F-J were stained with DNA-binding
dye 4',6-diamidino-2-phenylindole dihydrochloride. Only
H2O2-treated
cells showed apoptosis.
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Hyperoxia is known to cause intracellular accumulation of reactive
O2-derived intermediates including
the superoxide anion (
) (23).
Superoxide can react rapidly with NO to form peroxynitrite
(ONOO
), an extremely
reactive and toxic molecule (3). Although
ONOO
is too reactive to
measure directly, one of its reaction products, nitrotyrosine, which
forms when ONOO
donates a
nitro group to tyrosine residues in cellular proteins, is stable and
detectable in tissues and cells. To determine whether the synergistic
cytotoxicity of NO+hyperoxia is associated with increased
nitrotyrosine, immunocytochemistry was performed using anti-nitrotyrosine antibodies. Nitrotyrosine was barely detected by
immunofluorescence in cells grown in room air alone or in hyperoxia (Fig. 6). Cells cultured in NO showed
diffuse immunofluorescence, suggesting the accumulation of
nitrotyrosine. However, cells grown in combined NO+hyperoxia showed a
more intense signal, perhaps because the intracellular
immunofluorescence pattern became eccentric (Fig. 6). Cells incubated
with secondary antibodies alone showed no signal (not shown).
To gain a further understanding of the nature of the proteins modified
by ONOO
, cellular proteins
were size fractionated on polyacrylamide gels and assayed on Western
blots using anti-nitrotyrosine antibodies. Figure
7 shows that immunoreactive proteins were
barely detected in either control cells cultured in room air or cells
exposed to hyperoxia alone. In contrast, cells exposed to combined
NO+hyperoxia showed a notable increase in the levels of two protein
bands, migrating at 25 and 35 kDa. Interestingly, cells cultured in NO alone also showed immunoreactive proteins at 25 and 35 kDa. The specificity of this result was confirmed with control assays by incubating Western blots with nonimmune rabbit IgG or with
anti-nitrotyrosine antibodies preabsorbed with excess (10 mM)
nitrotyrosine (data not shown).

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Fig. 7.
Western blot for nitrotyrosine-containing proteins. Cells were cultured
for 3 days, and Western blots were prepared as described in
METHODS. Blots were incubated with
anti-nitrotyrosine antibody. Two proteins, at 25 and 35 kDa, were
specifically enriched after treatment with NO alone or NO+hyperoxia.
These specific bands were not detected when blots were incubated with
nonimmune rabbit IgG or with anti-nitrotyrosine antibodies preabsorbed
with excess (10 mM) nitrotyrosine (data not shown).
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DISCUSSION |
Inhaled NO therapy is currently being studied in patients also
receiving concurrent treatment with
O2 and mechanical ventilation, yet
there is a paucity of data from animal or cell culture studies on the
combined effects of these two agents. We have performed a set of
experiments in cultured alveolar epithelial and lung microvascular
endothelial cells that demonstrates that the combination of
NO+hyperoxia is much more cytotoxic than either agent alone. These
observations are important because they demonstrate that the beneficial
effects of inhaled NO therapy have the potential to be accompanied by
deleterious side effects when combined with hyperoxia. Although there
is some evidence that endogenous NO or lower doses of exogenous (10 parts/million) NO improved alveolar liquid clearance, higher doses of
exogenous NO (100 parts/million) increased vascular permeability to
protein, and survival of rats exposed to hyperoxia was not improved by
inhaled NO (8). Together with reports of lung injury and surfactant
dysfunction in animals receiving concurrent inhaled NO+hyperoxia (10,
11, 19), these observations suggest that there is a potential for
damage to lung epithelium and endothelium from this therapy. It may
therefore be important to minimize exposure to these agents when
administered clinically.
The precise relationship between these cell culture experiments and the
experimental use of NO in patients is not yet clear, and the full range
of NO doses and O2 levels is yet
unexplored. The levels used in these experiments are probably high
relative to those experienced by cells in intact lungs. Moreover, the
effects of NO+hyperoxia on the multiple cell types of intact lungs are likely to differ in many ways from the effects we observed in homogeneous cell culture. Because NO and
ONOO
are highly reactive,
we postulated that the cells would die via apoptosis, as is typically
reported to occur in response to oxidative cellular damage (2, 4, 6,
21, 23). However, the mode of cell death by combined NO+hyperoxia was
not apoptosis, as shown by nuclear size, DNA staining intensity, and
TUNEL assay. The capacity of these cells to undergo typical
oxidant-induced apoptosis is well described (15) and was evident in
positive control experiments when the cells were exposed to
H2O2.
The mode of cell death from combined NO+hyperoxia may be similar to the
mode of death from hyperoxia alone (15), although the time course of
cell death was much more rapid when exogenous NO was present. However,
the observed nonapoptotic cell death was substantially slower in
combined NO+hyperoxia relative to oxidant-induced apoptosis (2 or more days vs. 4 h), consistent with the nuclear morphology and
TUNEL assays showing the absence of apoptosis.
The observation that intense immunofluorescence with anti-nitrotyrosine
antibody was correlated with severe cytotoxicity suggested that
ONOO
may have a role in
mediating the cytotoxic effects shown here. The Western blot analysis
further suggested that the specific nitration of two proteins with
molecular masses of 25 and 35 kDa may be relevant. However, nitration
of bands of the same size also occurred in the cells grown in NO alone,
indicating that nitration of these proteins is not sufficient for
synergistic cell death. Moreover, on the basis of the fact that
ONOO
formation requires
superoxide (10), this observation suggests that sufficient superoxide
is produced in these cells to generate some
ONOO
, even when cultured in
room air. The observation that nitrotyrosine immunofluorescence
appeared notably enhanced is difficult to understand in the absence of
a large increase in the abundance of nitrotyrosine-containing proteins
on Western blots. Perhaps
ONOO
-modified proteins
become translocated in cells in NO+hyperoxia, resulting in eccentric
fluorescence. Alternatively, changes in conformation (perhaps increased
denaturation) may underlie the enhanced cellular immunofluorescence.
Further work is needed to identify these proteins and to determine
whether they have a role in cytotoxicity.
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ACKNOWLEDGEMENTS |
We thank Dr. Warren Rosenfeld for reviewing the manuscript and Lisa
Underwood and Sona Narula for administrative help.
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FOOTNOTES |
This work was funded in part by National Heart, Lung, and Blood
Institute Grant HL-02791, the American Lung Association, March of Dimes
Birth Defects Foundation Basic Grant FY-97-0590, and Winthrop-University Hospital.
Address for reprint requests: S. Horowitz, CardioPulmonary Research
Institute, Suites 503-505, Winthrop-Univ. Hospital, 222 Station
Plaza North, Mineola, NY 11501.
Received 4 April 1997; accepted in final form 12 December 1997.
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