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1 Institute for Environmental Medicine, and Departments of 2 Emergency Medicine and 3 Biochemistry and Biophysics, The University of Pennsylvania, Philadelphia, Pennsylvania 19104-6068
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Thom, Stephen R., Melissa Kang, Donald Fisher, and Harry
Ischiropoulos. Release of glutathione from erythrocytes and other
markers of oxidative stress in carbon monoxide poisoning. J. Appl. Physiol. 82(5):
1424-1432, 1997.
Rats exposed to CO in a manner known to cause
oxidative stress in brain exhibited a twofold increase in plasma levels
of oxidized proteins, thiobarbituric acid-reactive substances (TBARS),
oxidized glutathione (GSSG), and reduced glutathione
(GSH). Changes were neither directly related to hypoxic
stress from carboxyhemoglobin nor significantly influenced by
circulating platelets or neutrophils. Treatment with the nitric oxide
synthase inhibitor
N
-nitro-L-arginine methyl
ester inhibited elevations in GSH and GSSG but not changes
in oxidized proteins or TBARS, suggesting that two oxidative mechanisms
may be operating in this model and that GSH and GSSG elevations
involved nitric oxide-derived oxidants. Elevations of blood GSH and
GSSG occurred at different anatomic sites, indicating that no single
organ was the source of the increased peptides. Animals that underwent
exchange transfusion with a hemoglobin-containing saline solution did
not exhibit elevations in GSH and GSSG, suggesting that blood-borne
cells released these peptides in response to oxidative stress. In in
vitro studies, erythrocytes, but not platelets and leukocytes,
responded to oxidative stress from peroxynitrite by releasing GSH,
whereas no release was observed in response to nitric oxide or
superoxide. Glucose, maltose, and cytochalasin B, agents that protect
extracellular components of the hexose transport protein complex from
oxidative stress, prevented GSH release. The data indicate that nitric
oxide-derived oxidants are involved in CO-mediated oxidative stress
within the vascular compartment and that elevations of several
compounds may be useful for identifying exposures to CO likely to
precipitate brain injury.
peroxynitrite; nitric oxide; oxidized proteins; thiobarbituric acid
reactive substances; hexose transport
INTRODUCTION OXIDATIVE STRESS in the body occurs when the production
of free radicals overwhelms the antioxidant defense systems, and
oxidative damage of cells is the result. In recent years, reductions in the level of plasma antioxidants such as vitamin E, as well as increases in oxidized glutathione (GSSG) and products of lipid peroxidation, have been used as "biomarkers" of oxidative injury (15, 21, 22, 24, 37). These changes can provide insights into
pathophysiology, and, in a clinical situation, they may be useful to
assess the severity of an injury.
Clinical observations suggest that the pathophysiology of CO poisoning
is not based solely on the hypoxic stress mediated by an elevation in
the blood carboxyhemoglobin (HbCO) level (11). In
particular, the risk of permanent brain injury appears to be higher
when CO poisoning involves a pattern in which the exposure occurs over
a span of time, referred to as a "soak", and then an
individual suffers an interval of unconsciousness, presumably due to a
further elevation in the environmental CO level (6, 20). Unfortunately,
the pattern of CO poisoning that a patient has suffered is not always
clear in an emergency setting, and there are currently no reliable
laboratory tests that predict which exposures are likely to
precipitate neurological injuries.
We have found that several different oxidative processes occur in
association with CO poisoning in a rat model. Perivascular oxidative
changes mediated by nitric oxide (· NO) are
indicated by deposits of nitrotyrosine, and these changes occur even
when exposures last for only relatively brief time periods of <1 h (13). A more extensive process of brain lipid peroxidation occurs when
exposure to CO follows a pattern similar to that seen among patients at
high risk for developing brain injuries (29). This pattern involves
exposing rats to 1,000 parts/million (ppm) of CO for 40 min as the soak
and then exposure to CO at 3,000 ppm, which eventually causes
hypotension and cerebral hypoperfusion due to cardiac dysfunction. We
have found that cerebral hypoperfusion is manifested grossly as
unconsciousness (17, 29). After rats are exposed to CO, a cascade of
cellular and biochemical events occurs. Vascular oxidative changes
attributable to · NO contribute to leukocyte attachment to
the brain microvasculature; these leukocytes become activated and cause
conversion of endothelial xanthine dehydrogenase to oxidase; and the
oxygen radicals that are generated cause brain lipid peroxidation (13,
29-31). Others have poisoned rats with CO according to a similar
pattern and found functional neurological impairments and
histopathology in the hippocampus (35).
The aim of this study was to evaluate whether there may be plasma
markers of oxidative stress in rats that have been exposed to CO.
Changes in the concentration of reduced glutathione (GSH) and GSSG in
plasma led to further investigations, including a study of the effect
of peroxynitrite on isolated red blood cells (RBC). Peroxynitrite is a
strong oxidant that is generated by the near-diffusion limited reaction
between superoxide and · NO (12). Production of both
superoxide and · NO is increased during CO poisoning (13, 33,
38). RBC have been shown to liberate GSSG in response to oxidative
stress, and reducing equivalents from extracellular GSH can be
transduced across the cell membrane via the hexose transport protein
complex (7, 16). However, GSH itself has not been found to be
transported across the RBC plasma membrane.
MATERIALS AND METHODS
-nitro-L-arginine methyl
ester (L-NAME; 1 mg/kg ip) was injected 2 h before CO exposure. At the end of CO
exposures, rats were anesthetized by an intraperitoneal injection with
ketamine (73.5 mg/kg) and xylazine (1.5 mg/kg). Heparinized (10 U/ml)
blood was obtained from rats by aortic puncture. Blood HbCO and total
hemoglobin (Hb) levels were measured by using a CO-oximeter (model 282;
Instrumentation Laboratories, Lexington, MA).
The number of circulating cells was depleted in rats by performing an
exchange transfusion with the use of stroma-free Hb solution. A
cell-free Hb solution (1.2 g/dl) was prepared by hypotonic lysis of rat
RBC. RBC were separated from platelets and leukocytes by placing
heparinized rat blood in a tube, underlayering the blood
with 3 ml Histopaque 1077 (Sigma Chemical) and then Histopaque 1119, and then centrifuging at 700 g for 30 min. The RBC pellet was resuspended in 8 ml deionized water, sonicated
(Heat Systems-Ultrasonics sonicator model W220-F at a setting of 6) for
40 s on ice, and then dialyzed overnight against hypertonic saline (2.7 g NaCl/100 ml) at 4°C. The dialysate was sonicated a second time
and then centrifuged at 1,000 g for 10 min. The supernatant solution was analyzed for Hb content, oxygenated,
and transfused into rats.
Rats were prepared by placing catheters into a femoral artery and vein
while under ketamine-xylazine anesthesia. After the rats'
recovery from surgery, a total of 12 ml of blood was removed and
replaced with 12 ml of oxyhemoglobin solution in the following manner.
Aliquots of 2 ml blood were removed by using the arterial catheter, and
2 ml of Hb solution were then infused through the venous catheter. This
process was repeated a total of six times, and then an additional 3 ml
sterile saline solution were infused. The blood pressure of the rats
was directly measured before and after this procedure. Before
transfusion, the mean blood pressure was 108 ± 5 mmHg
(n = 12), and after the transfusion
the blood pressure was 106 ± 5 mmHg
(n = 12; no significant difference). After the transfusion procedure, the rats all walked around their cages, ate food, and drank water in a seemingly normal fashion. They
were left unmanipulated for ~30 min and then randomly selected to be
exposed to CO poisoning or else remained in the air environment as
control animals.
Immediately after CO poisoning, rats were anesthetized, and blood was
removed for analysis. Blood was also removed from control animals at
this same time interval. All animals were killed by an overdose of
anesthetic within 2 h after exchange transfusion.
Analytical procedures.
Heparinized blood was drawn into a closed system and centrifuged for 10 min at 300 g at 4°C; the plasma
was collected under nitrogen and stored at 
20°C until
assayed. Plasma was deproteinized and assayed for thiobarbituric
acid-reactive substances (TBARS) as described in a previous publication
(15). The protein fraction was solubilized by 6 M guanidine
hydrochloride and reacted with 0.05% (wt/vol) 2-4
dinitrophenylhydrazine (DNPH) to assay for oxidized proteins by
following our published procedures (15). Plasma potassium was directly
measured by using a potassium-selective electrode (Fisher Scientific),
and free Hb was measured using the benzidine assay (34). Immediately
after blood was removed from rats, total glutathione and GSSG were
assayed by a spectrophotometric assay, utilizing GSSG reductase and
following the methods described by Adams et al. (2).
In vitro assays.
RBC were separated by centrifuging heparinized blood underlayered with
Histopaque, as described above. The RBC layer was removed and combined
in a ratio of ~1:2 with buffer (100 mM potassium phosphate, 0.9%
NaCl, and 0.1 mM diethylenetriaminepentaacetic acid, pH 7.4).
Leukocytes and platelets used for GSH- and GSSG-leakage studies were
initially prepared simply by centrifuging heparinized blood and
removing the supernatant and buffy coat. Cells were combined with a
volume of buffer equal to the initial blood volume, and assays were
performed with free radical generators. When no GSH or GSSG leakage was
measured, we repeated our studies with more concentrated suspensions of
cells prepared from the upper layers of isolated RBC preparations. The
platelet- and leukocyte-containing bands were obtained after 12 ml of
blood were centrifuged with the use of Histopaque. The layers were
combined with 8 ml buffer and centrifuged at 700 g for 10 min, and then the pellet was
resuspended in 5 ml of buffer. A total of five studies were performed
with suspensions of leukocytes and platelets prepared with an average of 18,240 ± 2,920 leukocytes and 0.7 ± 0.3 × 106 platelets/µl.
Both the RBC and leukocyte-platelet preparations were incubated at room
temperature (25°C) with free radical generators. The concentration
of GSH and GSSG in the leukocyte-platelet suspensions was assayed every
5 min. As outlined in RESULTS, a
rather complex pattern was observed with RBC preparations, and the
initial rates of release of GSH and GSSG were taken as the change in
concentration over the first 3 min of incubation. Stock solutions of
SIN-1 were prepared in 50 mM phosphate buffer, pH 5.0, and solutions of
DENO were prepared in 50 mM phosphate buffer, pH 8.5. Superoxide
radical was generated in solutions containing 4 mM hypoxanthine and
xanthine oxidase (0.017 U/ml). The rate of peroxynitrite formation from SIN-1 was assessed by measuring the oxidation of dihydrorhodamine 123 to rhodamine, following published methods (14). The half-life of DENO
was determined based on its characteristic ultraviolet absorption
spectrum
(E250 = 6,500), and liberation of · NO was measured directly with a
· NO-selective polarographic probe (Iso-NO, World Precision
Instruments, Sarasota, FL). The rate of superoxide generation was
assessed as the superoxide dismutase-inhibitable rate of cytochrome
c reduction (18).
Statistics.
Statistical significance was determined by analysis of variance
followed by Scheffé's test (26). The level of significance was
taken as P < 0.05. Results are
expressed as means ± SE.
RESULTS
Influence of platelets and neutrophils on plasma indicators of CO-induced oxidative stress. Platelets liberate relatively large amounts of · NO during CO poisoning (33). Because platelet-derived · NO does contribute to perivascular oxidative stress (13), we evaluated whether oxidized products in plasma were elevated in thrombocytopenic rats (Fig. 1). Elevated levels of GSSG, TBARS, and oxidized proteins were found in rats with circulating platelet counts that were only 16 ± 8% (n = 6) of the control level (Fig. 1). We conclude that platelets do not appear to contribute significantly to the changes observed in plasma. Because neutrophils are involved in brain oxidative changes that are detected ~1.5 h after CO poisoning (31), we also examined plasma indicators in rats made neutropenic by injections of antineutrophil antiserum. Neutropenia (neutrophil count <200 cells/µl blood) did not alter elevations in plasma indicators in response to CO poisoning (Fig. 1). Plasma GSH is increased in CO poisoning. The ratio of GSSG to GSH in plasma is often a sensitive indicator of systemic oxidative stress (9, 21). The GSSG-GSH ratio in control rats was 0.026 ± 0.005 (n = 14), and the level was insignificantly different in rats exposed to the standard CO model (0.030 ± 0.005; n = 15). We found that, in rats after CO poisoning, this ratio remained unchanged because the concentration of GSH in plasma also doubled (Table 1). Our initial hypothesis for the rise in both GSH and GSSG was that hemolysis may have occurred. To evaluate this possibility, we measured the free Hb and potassium concentrations in plasma. No change in either parameter was found after CO poisoning. The free Hb concentration was 3.6 ± 1.0 mg/100 ml plasma (n = 8) in control rats and 3.0 ± 0.5 mg/100 ml (n = 8) in rats poisoned with CO. The plasma potassium concentration was 4.4 ± 0.3 meq/l (n = 8) in control rats and 4.4 ± 0.3 meq/l (n = 11) after CO poisoning. We conclude from these data that the elevations in GSH and GSSG were not due to overt RBC damage that caused hemolysis.
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1 · min1
(n = 5), only 15% of the expected
rate caused by 111 µM SIN-1, as listed in Table 5
(P < 0.05). Incubation of 111 µM
SIN-1 in combination with 1 mg/ml superoxide dismutase, to scavage
superoxide liberated by SIN-1, resulted in a rate of only 1.9 ± 1.8 µmol GSH · g
Hb1 · min1
(n = 4), significantly less than the
rate with SIN-1 by itself (P < 0.05). Incubation with 111 µM SIN-1 C caused no release of GSH from
RBC (0 ± 0 µmol · g
Hb1 · min1,
n = 3). We conclude that
peroxynitrite generated by SIN-1 caused the release of GSH and GSSG
from RBC.
RBC preparations were also incubated with DENO, a compound that
liberates · NO at physiological pH. At pH 7.4 and room
temperature the half-life of DENO was 2.4 min, and 6.5 µM DENO
generated · NO at a rate of 1.8 µmol/min. Under the same
experimental conditions, 111 µM SIN-1 released peroxynitrite at a
rate of ~1 µmol/min (see MATERIALS AND
METHODS). When RBC suspensions were incubated with 6.5 µM DENO, the rate of release of GSH was only 1.4 ± 1.4 µmol · g
Hb1 · min1
(n = 4), which was not significantly
different from control but was significantly different
from samples incubated with 111 µM SIN-1.
The effects of superoxide radical and
H2O2
on GSH release were also assessed. A preparation of 4 mM hypoxanthine
plus xanthine oxidase generated superoxide at 2.3 µmol/min (see
MATERIALS AND METHODS). When RBC
were exposed to this flux, the initial rate of GSH release was 0 ± 0 µmol GSH · g
Hb1 · min1;
n = 4.
We found that the initial rate of GSSG release from RBC was small and
not significantly affected by SIN-1 (Table 5). However, if incubations
were lengthened, a prominent increase was found in the concentration of
GSSG in the suspension. This effect is illustrated in Fig.
2, where temporal changes are shown for GSH and GSSG concentrations in RBC suspensions exposed to 111 µM SIN-1. The concentration of GSSG in control suspensions of RBC incubated in
buffer for 60 min was 12.6 ± 6.9 µmol/g Hb
(n = 5), in contrast to the GSSG
concentration at 60 min in the presence of 111 µM SIN-1 (129 ± 22 µmol/g Hb; n = 7;
P < 0.05). Obviously, a large portion of the GSSG measured at this time may have been derived from
the GSH previously released by the RBC, as well as GSSG directly released by RBC.
) and GSSG (
) in
suspensions of red blood cells exposed to 111 µM
3-morpholinosyndnonimine. Values are means ± SE;
n, 3-8 rats for each point.
Effects of hexoses and cytochalasin B. A thiol-disulfide exchange mechanism exists in RBC that allows transduction of reducing equivalents into cells via sulfur-rich membrane proteins (8). Components of these proteins can be protected from some oxidants by a number of sugars and by cytochalasin B. We found that incubation with glucose, maltose, or cytochalasin B significantly reduced the flux of GSH from RBC exposed to 111 µM SIN-1 (Table 6). The effect of these agents was not caused by scavaging of peroxynitrite, based on assays conducted with 5 µM SIN-1 incubated with dihydrorhodamine 123. The rate of rhodamine 123 formation was monitored as an index for peroxynitrite-mediated reactions. The rate was 4.6 ± 0.3 nmol/min (n = 8) with no additions. In the presence of 1 µM cytochalasin, 5 mM glucose, or 5 mM maltose, the rates were 4.6 ± 0.2 (n = 3), 4.5 ± 0.4 (n = 3), and 4.6 ± 0.8 (n = 3), respectively (no significant differences from control).
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DISCUSSION
Plasma concentrations of TBARS, oxidized proteins, GSH, and GSSG were significantly increased in rats exposed to CO in a manner known to cause brain oxidative stress. Among the oxidation products measured, glutathione values were unique in that elevations of GSH and GSSG were inhibited in rats pretreated with the nitric oxide synthase inhibitor L-NAME. These data suggest that two mechanisms of action may be operating to cause the elevations in plasma oxidized products. We were particularly interested in the role · NO may play with glutathione elevations, because early perivascular oxidative changes during CO exposure are mediated by · NO-derived oxidants, and these · NO-mediated changes are required for the subsequent cascade of cellular and biochemical changes that lead to brain lipid peroxidation after CO poisoning (13).
Plasma glutathione concentration has been found to be the highest in the hepatic vein (5). We attempted to measure plasma levels by aspirating blood from this vessel. However, the space between the diaphragm and the dome of the liver, where the hepatic vein enters the vena cava, is quite small in the rat. The blood we obtained from this area may therefore be considered to be a mixture of blood from the hepatic vein and the proximal vena cava. We found that the GSH concentration was slightly higher in this blood vs. blood in the distal vena cava, but the value in aortic blood was still higher. We interpret these findings as indicative of hepatic production of GSH. However, other cells, possibly in the lungs, also release significant amounts of GSH in the rat. The pertinent issue in relation to our study is that the GSH level in blood obtained from all vessels was significantly greater after CO poisoning. Hence, no single organ could be identified as the source for the increase in plasma glutathione found after CO poisoning.
Elevations in plasma oxidized proteins, TBARS, and gluthathione did not correlate with HbCO levels, which were similar in all three patterns of CO exposure shown in Fig. 1. Elevations in plasma markers only occurred when rats were exposed to CO according to the pattern that causes brain lipid peroxidation (13, 29-31). We have found that during CO poisoning there is a precipitous increase in generation of · NO in association with unconsciousness and, therefore, during brain hypoperfusion (13, 17). It is likely that plasma markers of oxidative stress were detected after the standard CO exposure model and not the other patterns of CO poisoning, because the standard model of exposure causes the most intense oxidative stress.
RBC appear to be the source of the elevated plasma GSH and GSSG in CO poisoning. RBC release GSH in response to exposure to peroxynitrite but not in response to · NO or superoxide radical. This discrete response, which may be related to the chemical reactivity of peroxynitrite, may be the reason why the phenomenon has not been described in other studies of RBC oxidative stress. The source(s) for the oxidizing species during CO exposure remains unclear. Neutrophils and platelets do not seem to be involved, and the vascular endothelium is a strong candidate. We recently reported that the concentration of nitrotyrosine, a relatively specific marker of peroxynitrite formation in vivo, was significantly increased in the brains of CO-poisoned rats and that nitrotyrosine deposits were particularly intense surrounding cerebral blood vessels (13). We have also reported that cultured endothelial cells release both NO and peroxynitrite in association with exposure to CO (32). Interestingly, we have been unable to measure significant release of GSH or GSSG from cultured vascular endothelial cells exposed to CO (unpublished observation).
RBC have a high antioxidant capacity, and they are able to scavage both internal oxidants generated by Hb autoxidation and the partially reduced oxygen species that diffuse across the RBC membrane from extracellular sources (36). This is achieved through the combined activities of superoxide dismutase, catalase, and the GSH-dependent redox cycle (28). Transport of GSH to the extracellular space is a property shared by many cell types, and it is thought to be a mechanism for transferring reducing compounds to the immediate environment of the cell to protect essential thiol groups on the membrane surface (19). Others have found GSH to be a facile agent for transducing reducing power both to and from the intracellular space in RBC (8, 25). However, in these studies, GSH per se did not traverse the cell membrane.
Reducing equivalents from GSH outside of RBC are transferred across the membrane to the intracellular space by a process involving thiol groups on the exterior membrane surface (7). Sulfur-containing constituents of the hexose-transport mechanism of RBC are located both inside and outside of the membrane as well as within the nonpolar interior of the membrane (1). We hypothesized that the hexose-transport complex may be involved with the transfer of GSH out of RBC in response to peroxynitrite-mediated oxidative stress. To test this hypothesis, RBC were incubated with either a transportable sugar (glucose), an impermeant sugar (maltose), or cytochalasin B. All three of these agents have been shown to protect extracellular sulfhydryls of the hexose-transport mechanism from oxidative stress (1). Because these agents inhibited peroxynitrite-mediated GSH release from RBC, we hypothesize that the hexose-transport complex is involved with GSH transfer.
The pattern for GSH and GSSG release from RBC, as depicted in Fig. 2, is likely to have resulted from the cumulative stress of peroxynitrite. Thus the initial release of GSH may be a defensive mechanism that occurred in response to oxidative attack on the sulfur-containing moieties of the membrane. However, as the flux of peroxynitrite was maintained over time, more intracellular GSSG accumulated while intracellular sulfhydryl targets were attacked. With this progressive rise in intracellular GSSG, the cells would be expected to begin pumping GSSG out to the surrounding medium. Several types of cells, including RBC, hepatocyte, and lens cells of the eye, transport GSSG out of the cell when the intracellular concentration is high, as for example during oxidative stress (4, 23, 27). This action has been theorized to be an "emergency" mechanism to rapidly reestablish thiol/disulfide homeostasis. Therefore, with regard to the pattern of GSH release from RBC, the GSSG measured in the extracellular space would be the combination of GSSG directly released by the cells as well as GSSG resulting from oxidation of the GSH initially pumped out. This process offers an explanation for why the extracellular concentration of GSSG after 60-min incubation with SIN-1 (see Fig. 2) was found to exceed the highest concentration of GSH that was measured.
It was surprising that we did not observe a change in the plasma
GSSG-GSH ratio after CO poisoning, as the GSH released by RBC would
eventually become oxidized. The kidney plays a major role in clearance
of plasma glutathione (5, 10), and renal clearance is likely to have
prevented the GSSG-GSH ratio from being altered. When the GSH and GSSG
concentrations in blood from the renal vein and aorta were compared
(Table 2), renal vein GSH concentration was found to be 17% of the
concentration found in blood from the aorta, whereas GSSG concentration
in renal vein blood was only 0.5% of the level in aortic blood. This
suggests that the relative uptake of GSSG by the kidney is greater than uptake of GSH, which would diminish the rise in plasma
GSSG concentration relative to the amount of GSH released by RBC.
However, the 
-glutamyl cycle is still poorly understood, and this
issue requires additional investigations, including measurements of
urinary glutathione and an analysis of renal -glutamyl
transpeptidase activity.
The physiological significance of an elevation in plasma glutathione will also require additional study. It seems reasonable that this elevation may serve as a defensive response to oxidative stress. Further work is necessary to characterize the mechanism of GSH release by RBC. With regard to the CO model, an elevation of the plasma GSH level in response to oxidative stress is unusual and offers suggestive evidence for involvement of peroxynitrite. From a clinical perspective, if plasma levels of oxidized products could be shown to be correlated with development of neurological morbidity in patients, this laboratory measurement would fill an important void in the emergency evaluation of CO-poisoning victims. Objective parameters could then be used to identify patients who should receive more advanced, aggressive treatment.
ACKNOWLEDGEMENTS
This work was supported by National Institute of Environmental Health Sciences Grant ES-05211 (to S. R. Thom), by a grant from the Council for Tobacco Research (to S. R. Thom), and by a grant from the Pennsylvania Lung and Southeastern Pennsylvania Heart Associations (to H. Ischiropoulos). H. Ischiropoulos is a Parker B. Francis Fellow in Pulmonary Research.
FOOTNOTES
Address for reprint requests: S. R. Thom, Univ. of Pennsylvania, Institute for Environmental Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: SThom{at}mail.med.upenn.edu).
Received 8 December 1995; accepted in final form 18 December 1996.
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