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We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on oxidative stress, lung damage, and
mortality induced by an endotoxin (lipopolysaccharide, or LPS) in the rat. Continuous intravenous infusion of 275 mg NAC/kg in 48 h, starting 24 h before LPS challenge, decreased hydrogen peroxide
(H2O2) concentrations in whole blood (p < 0.01). This decrease was accompanied by fewer histologic
abnormalities of the lung and decreased mortality (p < 0.025), compared with rats receiving LPS
alone. N-Acetylserine, which has no sulfhydryl group, did not protect rats against LPS toxicity. Improved survival was not associated with an increase in pulmonary reduced glutathione, nor with inhibition of serum tumor necrosis factor (TNF) activity. In vitro, TNF production and DNA binding of nuclear factor kappa B (NF-
B) in human Mono Mac 6 cells was only inhibited at concentrations of NAC
above 20 mM. High-dose NAC treatment (550 and 950 mg/kg in 48 h) decreased lung GSH (p < 0.05) and resulted in a significantly smaller number of surviving animals when compared with the
low-dose NAC group (p < 0.025). In vitro, NAC increased hydroxyl radical generation in a system with
Fe(III)-citrate and H2O2 by reducing ferric iron to its catalytic, active Fe2+ form. We conclude that low-dose NAC protects against LPS toxicity by scavenging H2O2, while higher doses may have the opposite effect. Sprong RC, Winkelhuyzen-Janssen AML, Aarsman CJM, van Oirschot JFLM, van der
Bruggen T, van Asbeck BS. Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality.
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INTRODUCTION |
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N-Acetylcysteine (NAC), which is a scavenger of hydrogen peroxide (H2O2), hypochloric acid (HOCl), and hydroxyl radical (·OH) in vitro (1), has been successfully used in various models (2, 3) of adult respiratory distress syndrome (ARDS). Evaluation of the protective effects of NAC against lung damage induced by the endotoxin lipopolysaccharide (LPS) has been mainly directed to the hemodynamic effects of LPS, such as increased pulmonary arterial pressure (3, 4). Although sufficient studies support the involvement of reactive oxygen species (ROS) in ARDS (5), direct evidence for an antioxidant effect of NAC in LPS toxicity is still lacking. Recently, diminished peroxide levels in the expired breath of NAC-treated rats were reported in an interleukin (IL)-1 model of lung injury (8).
There is increasing evidence that H2O2, directly or indirectly
via its reduction product ·OH, acts as a messenger molecule in
the synthesis and activation of inflammatory mediators. Oxidant scavengers, such as dimethyl sulfoxide and dimethylthiourea, inhibited LPS-stimulated IL-8 release in human whole
blood (9). N-Acetylcysteine has been reported to diminish the
expression of the vascular cell adhesion molecule VCAM-1 on
endothelial cells in vitro (10) and to decrease TNF levels in endotoxic mice (2) and dogs (3). Several lines of evidence suggest
that TNF gene expression is controlled by the transcription factor nuclear factor B (NF-B), which activity can be induced
by H2O2 (11). Antioxidants have been shown to inhibit the activity of NF-B in several cell lines, including the human monocytic cell line Mono MAC 6 (11, 12) and murine peritoneal macrophages (13).
Here, we report the effect of NAC in a rat model of LPS
toxicity by monitoring oxidative stress, lung damage, and endogenous antioxidant protection in lungs. Oxidative stress was
assessed from peroxide concentrations in deproteinized whole
blood. The effect of NAC on the inflammatory mediator TNF
was evaluated in vivo by measuring serum TNF activity, and in
vitro using LPS-induced TNF production and NF-B activity
in Mono Mac 6 cells.
Toxicity of NAC is low, although adverse effects, including
anaphylactic responses, have been observed in accidental high-dose NAC infusion in humans. No adverse effects of NAC in
LPS toxicity have been reported so far. Nevertheless, we
found that high doses of NAC aggravate LPS toxicity. To understand this finding, its mechanism was further investigated.
Autoxidation of thiols is known to occur in the presence of
transition metals, such as iron(III), which then reduces to its
catalytically active form Fe2+ (14). In addition, autoxidation of
sulfhydryl groups may yield superoxide (O
2 ·) and H2O2 (15,
16). Thus, factors necessary for ·OH generation according to
the Fenton reaction (17)
i.e., H2O2 and Fe2+ ionscan be
generated during thiol oxidation. N-Acetylcysteine, therefore,
may also display a similar pro-oxidant activity. Therefore, the
iron-reducing capacity of NAC and its potentiating effect on
·OH generation were also studied.
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Animals. Pathogen-free male Wistar rats weighing approximately 250 g (Iffa-Credo, Brussels, Belgium) were used for this study. They were given water and standard food ad libitum. All animal studies performed followed the institutional guidelines concerning the care and handling of animals.
Experimental procedures. After anesthetizing the rats with 0.5 ml/ kg Hypnorm intramuscularly (10 mg/ml fluanison and 0.315 mg/ml fentanyl citrate) and 2.5 mg/kg Diazepam intravenously, animals were cannulated. A siliconized silastic cannula was inserted in the jugular vein, the end of the cannula reaching into the right atrium. The other end was passed subcutaneously to the skull, where it was fixed to a metal cannula (0.8 mm). During recovery, the system was filled with 50% polyvinylpyrrolidone and 500 U/ml heparin in saline to prevent occlusion by blood. After 7 d, the system was connected to a miniature single-channel swivel (Alice King Chatham Medical Arts, Los Angeles, CA). Continuous infusion of 2.0 ml NAC in 24 h (N-acetyl-L-cysteine, 5 g/25 ml, pH 6.95; Fluimucil infusion solution, Zambon, Amersfoort, The Netherlands) was maintained with a perfusion pump, modified for the use of 8 syringes. Different doses of NAC were given for 48 h: Group 1 received a loading dose of 75 mg/kg in 1 h followed by 100 mg/ kg in 24 h (total dose 275 mg/kg in 48 h); Group 2 received a loading dose of 150 mg/kg in 1 h followed by a maintenance dose of 200 mg/kg in 24 h (total dose 550 mg/kg in 48 h); Group 3 received a loading dose of 150 mg/kg in 1 h followed by a maintenance dose of 400 mg/kg in 24 h (total dose 950 mg/kg in 48 h). Control rats received either saline for 48 h or N-acetyl-D,L-serine (NAS; Sigma, St. Louis, MO), with a structure similar to that of NAC but lacking the sulfhydryl group, in amounts equimolar to 275 mg NAC/kg in 48 h. The total volume infused in both NAC and control animals was 4 ml and the pH ranged from 6.95 to 6.52 for the NAC-containing solutions, compared to a pH of 5.97 for saline. All solutions were endotoxin-free as measured by the Limulus amoebocyte lysate assay. A lethal dose of 5 mg/kg LPS (phenol extract from Salmonella ryphimurium, L6511; Sigma) was given intraperitoneally 24 h after NAC infusion was started. This time point is referred to as t = 0 h. Rats were either sacrificed at times stated below or used for survival experiments. In order to investigate whether the endogenous antioxidant glutathione is important in the defense mechanism against LPS, it was depleted in rats. Therefore, rats were intraperitoneally injected with L-buthionine-[S,R]-sulfoximine (BSO; Sigma) at a dose of 6 mmol/kg each day for 2 d. On the third day, LPS was intraperitoneally injected together with a third dose of BSO. Survival was measured 24 h after LPS injection. Blood samples taken from the intravenous system were collected in heparin tubes for H2O2 determination. Serum was stored at
70° C
until assayed for TNF.
Histology. For histological examination, the lungs were removed
and fixed ex vivo by intratracheal instillation of phosphate-buffered 10% formalin at 20 cm H2O. Sections were stained with hematoxylin and eosin. Histologic assessment was performed in a blind fashion and
judged by three independent observers to assure that the presented data are representative.
Preparation of tissue homogenates. For the determination of pulmonary acid-soluble sulfhydryl and glutathione concentrations, rats
were killed with 45 mg/kg of Nembutal (Ceva, Paris, France) mixed
with 250 U heparin/kg to prevent blood clotting. Lungs were perfused
blood-free in situ with saline via the pulmonary artery, and bronchoalveolar lavage was performed eight times with 10 ml saline. Lungs were
frozen in liquid nitrogen until homogenization with an Ultra-turrax
T25 (IKA Labortechnik, Staufen, West Germany) in two volumes
(wt/vol) ice-cold 50 mM phosphate buffer, pH 7.4. After centrifugation (48,000 × g, 20 min, 4° C), tissue supernatant was used immediately for glutathione assay or stored at 20° C.
Hydrogen peroxide concentrations in whole blood. Blood samples
drawn at t = 0 h and t = 3 h after LPS injection were immediately deproteinized using 2.5 ml ice-cold 5% trichloric acid per 1 ml whole
blood. Samples were quickly centrifuged (2,100 × g, 10 min) and supernatants were adjusted to pH 7.0 by adding 3 M K-phosphate, pH
13.0. Hydrogen peroxide was measured in deproteinized samples using the oxidation of phenol red by horseradish peroxidase (18). Addition of exogenous catalase (100 µg/ml, thymol-free, from bovine liver;
Sigma) to the pH-adjusted deproteinized sample totally abolished
peroxidase-mediated oxidation of phenol red, indicating that other
blood components do not interact with the H2O2 assay.
Total glutathione, reduced glutathione, and protein measurement.
Total glutathione, defined as the sum of reduced glutathione (GSH)
and oxidized glutathione (GSSG), was determined enzymatically in
deproteinized lung supernatants, as described by Tietze (19), and expressed as µmol per g protein according to Bradford (20). Reduced
glutathione was measured following the method of Prins and Loos (21).
Tumor necrosis factor levels in sera. Blood samples were drawn exactly 90 min after LPS injection. Preliminary studies with blood samples drawn every 15 min for the first 180 min after LPS injection
showed that peak values of TNF occur at 90 min after LPS injection.
Tumor necrosis factor was determined in heated (30 min at 56° C) serum using the murine L929-fibroblast cytotoxicity assay (22). L cells
were cultured (37° C, 5% CO2) in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and
gentamycin (10 µg/ml). Confluent cells were spliced with a trypsin
ethylene diaminetetracetic acid (EDTA) solution (Gibco, Paisley,
UK). Cells were seeded in a 96-well plate (Nunc, Roskilde, Denmark)
at a density of 2 × 104 cells/well. After overnight incubation, various
concentrations of RhuTNF (12.5-1600 pg/ml, activity approximately
1 µg TNF
/ml = 40,000 IU/ml; NIBSC, Hertfordshire, UK) and rat
sera at different dilutions were added in duplicate. Actinomycin D
(1 µg/ml final concentration in a total volume of 200 µl) was added
and cells were incubated for 18 h. After fixation with 18.3% glutaraldehyde, cells were stained with 0.05% methylene blue for 30 min. The
amount of TNF U/ml represents the reciprocal of the TNF dilution
causing 50% cytotoxicity under the conditions described. To address
the possibility that NAC in serum inhibits Rhu TNF-mediated L929
cell lysis in vitro, causing an artifact in the bioassay, cells were similarly incubated in triplicate with Rhu TNF (0-625 pg/ml) in the presence of NAC at concentrations ranging from 0-150 mM. The NAC-mediated protection was measured as an increase in the concentration of Rhu TNF that was needed to induced 50% lysis (LC50). The lowest concentration of NAC at which an inhibition of the LC50 of L929 cells
by TNF was observed was 10 mM. In the presence of 10 mM NAC,
the LC50 of L929 cells was reached at 273.6 ± 175.9 pg TNF/ml, compared with 70.9 ± 22.7 pg TNF/ml when NAC was not added to the incubation medium (p = 0.026), and at 82.1 ± 34.5 pg TNF/ml in the
presence of 1 mM NAC (mean ± SD, n = 7).
Tumor necrosis factor production by Mono Mac 6 cells in vitro. In
order to investigate whether NAC reduces TNF production in vitro,
Mono Mac 6 cells (1 × 106 cells/ml) were preincubated with NAC at
concentrations of 0-30 mM in IMDM supplemented with 10% FBS
for 30 min (37° C, 5% CO2). After 2 h of incubation with 30 ng LPS/
ml, the amount of TNF released in the supernatant was measured using ELISA.
Nuclear extracts and electrophoretic mobility shift assay (EMSA).
Mono Mac 6 cells (106 cells/ml) were preincubated with 0-30 mM
NAC in IMDM supplemented with 10% FBS for 30 min at 37° C, then
subsequently incubated with 30 ng LPS/ml for 2 h. Nuclear extracts
were made according to Schreiber and colleagues (23). Electrophoretic mobility shift assays were performed as described by Dignam
and colleagues (24). Binding of NF-B to the 32P-labeled double-stranded oligonucleotide representing the 605 NF-B site of the human TNF gene (23) was carried out with 10 µg nuclear protein. Samples were separated on nondenaturing polyacrylamide gels in 1× TBE
buffer (89 mM Tris-HCl, 89 mM borate, 2 mM EDTA, pH 8.3). Supershift assays with a polyclonal antibody directed against the p50
subunit of NF-B (Santa Cruz Biotechical Inc., Santa Cruz, CA) were
performed to investigate whether the DNA binding factor was indeed
NF-B.
Ferricytochrome c reduction assay. The reduction of ferricytochrome c (75 µM, horse heart type II; Sigma) to ferrocytochrome c in
the presence of various concentrations of NAC was performed in
Hanks balanced salt solution (HBSS, pH 7.4). The change in absorbance at 550 nm was continuously monitored for 30 min using a diode
array spectrophotometer (Hewlett Packard 8452A; Hewlett Packard,
Palo Alto, CA).
Hydroxyl radical generation during autoxidation of N-acetylcysteine. The generation of ·OH was determined by the oxidation of
2-keto-4-methylthiobutyric acid (KMB) to ethylene (25). Briefly, ethylene was assayed by gas chromatography (Model GC-9a; Shimadzu, Kyoto, Japan) using a stainless steel column packed with Carbosieve G (Supelco, S.A., Gland, Switzerland) and a flame ionization detector. The oven was heated from 145° C to 195° C (programmed to rise at 6° C/min). Nitrogen (N2) was used as a carrier (flow rate, 50 cm3/
min). Glucose oxidase (100 mU/ml; Sigma), 5 mM glucose, 50 µM ferric citrate, 10 mM KMB, and various concentrations of NAC were incubated at 37° C in a shaking water bath. The assay was performed in
HBSS. The glass tubes were sealed with rubber stoppers. After 30 min, a sample of 0.5 ml of headspace gas was taken using an airtight
syringe and immediately analyzed. Ethylene production was determined by comparing it with a calibration chromatogram of a known
amount of ethylene standard (Supelco S.A.) injected into the column.
Statistical analysis. Statistical significance was determined using
Student's t test, with significance defined as p < 0.05. Unless otherwise stated, the standard deviation was taken as an estimate of variance. Statistical differences in the survival of rats were determined using Chi-Square distribution.
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Effects of Various N-Acetylcysteine Concentrations on Lipopolysaccharide-induced Mortality
In the first series of experiments, the protective effect of NAC against LPS toxicity was evaluated 24 h after endotoxin challenge. No deaths were recorded after this time point. Intraperitoneal injection of rats with 5 mg LPS/kg induced extensive hemorrhages in lung, liver, gut, and kidney. Survival of control rats (n = 39) that received saline and were challenged with LPS was 53% (Table 1). In contrast, when rats (n = 29) were treated with NAC by continuous intravenous administration at a concentration of 275 mg NAC/kg in 48 h, starting 24 h before LPS injection, there was a significant decrease in mortality (83% survival; p < 0.025). Treatment with an equimolar concentration of NAS, which has a structure similar to NAC but lacks the sulfhydryl group, was used to evaluate whether that group is crucial for protection. Survival after LPS challenge (40%, n = 25) was not influenced by NAS treatment. The protective effect of NAC was only noticed in the group of animals treated with 275 mg NAC/kg in 48 h. Survival of rats receiving 550 mg/kg in 48 h (63%, n = 46) was not significantly improved, whereas a marked increase in mortality (21%, n = 25) was observed at a dose of 950 mg NAC/kg in 48 h. At these doses, a significantly (p < 0.025) smaller number of rats survived the LPS challenge than survived after receiving 275 mg NAC/kg in 48 h. This difference could not be attributed to a direct toxic effect of NAC itself, since there were no deaths among animals (n = 10) receiving 950 mg of NAC/kg in 48 h alone.
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To validate the protective role of antioxidants against LPS toxicity, the effect of glutathione depletion on LPS-induced mortality was investigated. Treatment with BSO for 2 d reduced total glutathione in the lungs (6.0 ± 1.5 µmol/g protein, n = 8, versus 13.3 ± 3.2, n = 14 in saline-treated rats, p < 0.01) and significantly (p < 0.005) enhanced LPS-induced mortality (6% survival, n = 16).
Lung Histology of N-Acetylcysteine; and N-Acetylserine Treated Rats after Lipopolysaccharide Challenge
Lungs from LPS-injected rats treated with either NAC, NAS, or saline were investigated for histologic abnormalities. Control animals, which were treated with saline but not with LPS, had no histologic abnormalities in their lungs (Figure 1A). Lungs of control LPS-treated rats receiving a continuous infusion of saline showed thickening of the alveolar capillary membrane, shedding of bronchiolar epithelium, and diffuse alveolar damage characterized by alveolar and interstitial hemorrhage and edema as well as extensive interstitial and alveolar infiltration with leukocytes (Figure 1B). A similar array of histological changes was observed in NAS-treated rats, in rats treated with 550 and 950 mg NAC/kg in 48 h, and in rats treated with 275 mg NAC/kg in 48 h that died from LPS (not shown). Rats treated with NAS and saline that survived the LPS challenge also showed intra-alveolar edema and hemorrhage, infiltrated leukocytes, and thickening of the alveolar capillary membranes (not shown). In marked contrast, animals treated with 275 mg NAC/kg in 48 h that survived showed no lung lesions (Figure 1C).
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Effect of N-Acetyclysteine on Lipopolysaccharide-induced Oxidative Stress
Because the oxidant scavenger NAC protects against LPS toxicity in vivo, reactive oxygen species (ROS) involvement is most likely. Further investigations were therefore conducted to test the theory that NAC protects against LPS toxicity by scavenging oxygen metabolites. First, oxidative stress was monitored in LPS-challenged rats by measuring H2O2 levels in deproteinized whole blood using the oxidation of phenol red by horseradish peroxidase at t = 0 h at t = 3 h after LPS challenge. This method specifically measures H2O2, since addition of catalase to the deproteinized pH-adjusted sample totally abolished the oxidation of phenol red (not shown). As shown in Figure 2, a baseline peroxide concentration (at t = 0 h) of 226 ± 93 µM (n = 28) is present in deproteinized blood of control saline-treated rats. Three hours after LPS injection, H2O2 levels were significantly elevated, up to 388 ± 125 µM (p < 0.01, n = 18), and remained increased for at least 6 h, suggesting that LPS induces oxidative stress in rats. As shown previously by carbon monoxide treatment of whole blood to prevent ROS generation during trichloroacetate treatment (due to autoxidation of heme-ferrous-oxygen complexes), the H2O2 increase after LPS challenge can be ascribed to endogenous H2O2 generation (26). When rats were treated with 275 mg NAC/kg in 48 h, baseline H2O2 levels (144 ± 49 µM, n = 16) measured 24 h after NAC infusion (at t = 0 h) were significantly (p < 0.01) lower than in control saline-treated rats. Although NAC-treated rats showed a slight increase in the peroxide concentrations at t = 3 h after LPS injection (257 ± 56 µM, n = 19), circulating H2O2 concentrations in saline-treated LPS-challenged rats were significantly (p < 0.01) higher. In contrast, an equimolar concentration of NAS could not reduce H2O2 levels in either control (239 ± 83 µM, n = 6) or LPS-treated rats (349 ± 71 µM, n = 9), implying an important role for the sulfhydryl group of NAC in its antioxidant activity. When higher doses of NAC were administered, no decrease in whole-blood H2O2 was observed (not shown). At 550 mg NAC/kg in 48 h, the H2O2 concentrations before and after LPS injection were 278 ± 93 µM and 389 ± 192 µM (n = 4), respectively. In addition, treatment with 950 mg NAC/kg in 48 h did also not result in diminished baseline H2O2 concentrations (241 ± 125 µM, n = 5) but, on the contrary, increased LPS-induced oxidative stress (528 ± 80 µM, n = 3).
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p < 0.01 when compared with
values obtained from saline-treated rats.
Effect of N-Acetylcysteine on Total and Reduced Glutathione Concentrations
N-Acetylcysteine may act as a precursor of glutathione, which plays an important role in the defense of the lung against oxidants. Therefore, the effect of NAC on pulmonary total glutathione and GSH concentrations was measured several times after LPS injection. Because total glutathione is the sum of reduced (GSH) and oxidized (GSSG) glutathione, and the measurement is specific whereas the method we used for measuring GSH also measures non-GSH sulfhydryls, the combination of both determinations allows proper analysis of the glutathione status, including GSSG formation, which is always a result of oxidation of GSH. Treatment with 275 mg NAC/kg in 48 h did not result in augmentation of the total glutathione (Figure 3A) and GSH (Figure 3B) in the lungs during the first 24 h of infusion (measured at t = 0 h) when compared with saline-treated rats. When LPS was injected, pulmonary total glutathione levels increased significantly (p < 0.05) after 6 h in both saline-treated rats and animals receiving 275 mg NAC/kg in 48 h. Similarly, GSH concentrations were augmented significantly (p < 0.05) in both groups of rats 6 h after LPS injection. Reduced glutathione equaled total glutathione levels in saline-treated rats, whereas reduced glutathione levels in rats receiving 275 mg NAC/kg in 48 h were markedly higher than total glutathione levels, suggesting the presence of non-GSH sulfhydryls. After 12 h of LPS injection, pulmonary GSH significantly (p < 0.02) dropped in animals treated with 275 mg NAC/kg in 48 h to levels comparable to those at t = 0 h, suggesting oxidation of both the initial LPS-enhanced GSH and NAC-related non-GSH sulfhydryls.
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Because 950 mg NAC/kg in 48 h aggravated LPS toxicity,
we also investigated the influence of this dose on pulmonary
glutathione levels. In contrast to treatment with the lower
dose of NAC, animals treated with 950 mg NAC/kg in 48 h
showed a significant (p < 0.02) increase in pulmonary total
glutathione after 24 h of infusion, whereas GSH was not significantly increased. This finding indicates the presence of
augmented GSSG by oxidation primarily of enhanced GSH as
a result of high-dose NAC-induced oxidative stress, since the
animals had not yet received LPS. When LPS was injected
(t = 0 h), total glutathione content of the lung was not altered
at either 6 h or 12 h after LPS challenge, butobviously as a
result of LPS-induced oxidative stress and GSSG formation
pulmonary GSH was significantly (p < 0.05) decreased.
Effect of N-Acetylcysteine on Tumor Necrosis Factor Activity
Because many symptoms of LPS toxicity can be ascribed to the cytokine TNF and a decrease in TNF activity has been described in endotoxin mice (2) and dogs (3), we assessed serum levels of bioactive TNF to investigate interference of NAC with TNF production. Tumor necrosis factor activity was not detectable in serum of control rats (< 8 U TNF/ml serum, detection limit). After injection with LPS, TNF activity was quickly elevated, with peak values at 90 min (16.7 × 103 ± 9.7 × 103 U/ml, n = 12). Peak values of TNF were not altered in rats infused with either 275 mg NAC/kg in 48 h (17.1 × 103 ± 8.1 × 103 U/ml, n = 11) or 950 mg NAC/kg in 48 h (14.2 × 103 ± 7.9 × 103 U/ml, n = 8). As shown in the METHODS sections, the bioassay was not influenced by serum NAC concentrations below 10 mM. At higher serum concentrations of NAC, we observed inhibition of RhaTNF-mediated L929 cell lysis.
In order to elucidate further the effect of NAC on TNF, in
vitro studies with Mono Mac 6 cells were performed. Although this seems to be an indirect approach to examining the
in vivo events in rats, human Mono MAC 6 cells are an appropriate model for investigating the effect of antioxidants on
TNF production, since it has been shown that antioxidants act
in a similar way on NF-B activation and TNF production in
murine (13) and rat (26) peritoneal macrophages. N-Acetylcysteine at concentrations of 0-10 mM did not alter TNF production in LPS-stimulated Mono Mac 6 cells (Figure 4). At
concentrations of 20 mM and 30 mM, however, NAC markedly reduced TNF production (Figure 4).
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Effect of N-Acetylcysteine on Nuclear
Factor-B Activation
In order to study whether the effects of NAC on TNF production of LPS-stimulated Mono Mac 6 cells were due to a reduction in NF-B activity, binding of NF-B to the NF-B consensus locus of the human TNF promoter was determined. As
shown in Figure 5, NAC at concentrations of 10 mM and
lower did not attenuate NF-B activation, while concentrations of 20 and 30 mM markedly reduced NF-B DNA-binding. Incubation of the nuclear extracts with an antibody
against the p50 part of NF-B resulted in a supershift of the
upper band (not shown), indicating that the transcription factor located in the upper band is indeed NF-B.
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Pro-oxidant Activity of N-Acetylcysteine in a Cell-free System
Thiols are very reactive and highly susceptible to oxidation
to their disulfide form in the presence of a transition metal (16), and NAC at high concentrations worsened LPS toxicity; therefore, we considered the possibility that NAC might act as a pro-oxidant. In order to explore this idea, the conversion of ferricytochrome c to ferrocytochrome c by NAC was monitored. N-Acetylcysteine dose-dependently reduced ferricytochrome c (Figure 6). Cytochrome c reduction was not affected
by superoxide dismutase, or SOD (not shown), indicating that
O2 · is not involved in the NAC-mediated reduction of ferricytochrome c. Thus, in the presence of NAC, catalytically active
iron can be formed which, in the presence of H2O2 may favor
the generation of ·OH. To further investigate this possibility,
we measured the formation of ethylene from KMB in a cell-free system consisting of the H2O2-generating system glucose
plus glucose oxidase and 50 µM Fe(III)-citrate, in the absence
or presence of NAC. In the absence of NAC, 65 ± 14 pmol
ethylene (mean ± SEM; n = 5) was detectable. N-Acetylcysteine (10 mM) without H2O2 and iron did not generate ethylene from KMB (less than 75 pmol ethylele), whereas the
amount of ethylene produced by NAC plus H2O2 was 274 ± 127 pmol ethylene (n = 8). However, when 10 mM NAC was
added to the complete system, we measured a significant (p < 0.01) increase in the ethylene concentration (3,639 ± 790 pmol
ethylene, n = 9; Figure 7A), suggesting production of ·OH. In
the absence of H2O2, that is, the reaction between 50 µM
Fe(III)-citrate and 10 mM NAC, ·OH (6,957 ± 1,409 pmol
ethylene, n = 8) was also generated (Figure 7B). This reaction
could be prevented by the addition of catalase (100 µg/ml;
1,437 ± 494 pmol ethylene, n = 8, p < 0.005), indicating H2O2
formation, presumably by the reaction of O2 · with NAC (see
Reference 16). Indeed, when SOD (100 µg/ml) was added, the
formation of ethylene markedly increased (9,481 ± 1,786 pmol ethylene, n = 8). These data suggest the reduction of
Fe(III)-citrate by NAC to catalytically active Fe2+ ions, which
catalyze ·OH generation from H2O2 in the Fenton reaction.
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In the present study, we show that continuous infusion with the oxidant scavenger NAC improved survival of endotoxemic rats and resulted in fewer histologic abnormalities in the lung. Other investigators have consistently shown that NAC, when administered before LPS challenge, protects animals against the hemodynamic (3, 4) and lethal (2) effects of LPS. We have now demonstrated that the NAC-mediated protection against LPS toxicity is associated with decreased H2O2 concentrations in whole blood. This is in line with another study in which NAC decreased expired H2O2 in rats treated with IL-1 (8).
The protective effect of NAC can be attributed to its sulfhydryl group because NAS, which lacks that group, did not improve survival and had no effect on whole-blood H2O2 levels. The NAC-mediated protection is probably a result of direct scavenging of toxic oxygen species and is not due to increased glutathione synthesis, since NAC (275 mg/kg in 48 h) had no effect on the concentration of pulmonary and hepatic (not shown) total glutathione. In addition, NAC did not influence the level of GSH. However, GSH increased (markedly more than total glutathione) in rats treated with 275 mg NAC/ kg in 48 h at 6 h after LPS injection. This may be due to non-GSH sulfhydryls, either from NAC itself or its deacetylated form cysteine, interfering with the spectrophotometric assessment of reduced DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)], since this phenomenon was not observed in the saline-treated group of animals. These observations agree with another study (27), in which NAC had no effect on the concentration of total glutathione in plasma. N-acetylcysteine only enhanced the concentration of GSH in glutathione-deficient states, such as idiopathic pulmonary fibrosis (28), chronic obstructive pulmonary disease (29), and experimental models of sepsis (2) and ischemia/reperfusion (30). The observation that NAC inhibited the decrease in liver GSH in mice treated with LPS and the glutathione synthesis inhibitor BSO (2) suggests that NAC prevents oxidation of GSH by its own oxidant scavenging capacity, rather than by enhancing the synthesis of GSH.
The augmentation of total glutathione observed in both saline- and NAC-treated animals 6 h after administration of LPS suggests an increased synthesis in response to LPS-mediated oxidant exposure, as has been shown to occur in lungs (31) and in endothelial cells (32) after oxidant stress. It is conceivable that the same condition, oxidative stress, is also responsible for the subsequent fall in GSH in both saline- and low-dose NAC-treated rats as measured 12 h after the LPS challenge. This is consistent with the general opinion that oxidation of GSH is a parameter of oxidative stress.
The observed decrease in H2O2 levels after low-dose NAC
treatment could result in protection against LPS-mediated oxidative stress by interfering with several peroxide-mediated
pathways. First, less substrate is available for the generation
of the highly toxic ·OH and of ROS generated by myeloperoxidase-catalyzed reactions. Second, inflammatory responses
may be blunted, since a second messenger function of H2O2
and H2O2-derived oxidants, such as ·OH, has been suggested
in the synthesis and activation of inflammatory mediators, including IL-8 (9) and TNF (2, 3). The activity of this latter cytokine has been reported to be mediated by the redox-controlled transcription factor NF-B. Our in vitro studies with
Mono Mac 6 cells also support the hypothesis that NF-B and subsequent TNF production can be diminished by NAC. In
our rat model of LPS toxicity, however, serum TNF activity
was not decreased in NAC-treated animals. As shown in our
in vitro studies, inhibition of NF-B activity and TNF production could only be inhibited by high concentrations of NAC
(> 10 mM). These results are consistent with those reported
by others (11) who showed that high concentrations of NAC
(20 and 30 mM) inhibited the activation of NF-B by H2O2 as
well as its activation by TNF and PMA. At the highest dose of
950 mg/kg in 48 h in our study, assuming no disappearance
from the circulation, the plasma NAC concentration would accumulate to 45 mM (calculated from data in reference 33).
However, these concentrations were probably not achieved
because the NAC terminal half-life after intravenous administration is 2 to 2.5 h (34, 35). The fact that NAC had no effect
on TNF in our study, while it decreased TNF activity in mice
(2) and dogs (3), might be explained by the higher doses of
NAC used in those studies (1 g/kg orally administered NAC in
mice [2] and a bolus injection of 150 mg/kg followed by 20 mg/
kg/h in dogs [3]). Lower doses of NAC were not effective in
decreasing TNF activity in mice (2). The highest dose used in
our study (total dose 950 mg/kg in 48 h) was lower than those
used by others. In addition, it has recently been reported that
NAC dose-dependently suppresses LPS-induced NF-B activation in rat lung tissue (36), suggesting TNF- inhibition as well. The authors indeed observed a diminished inflammatory response as measured by cytokine-induced neutrophil
chemoattractant mRNA expression and LPS-induced neutrophilic alveolitis. They administered NAC as a single intraperitoneal injection at concentrations of 200 to 1,000 mg/kg 1 h before the endotoxin challenge. This may have led to peak NAC
plasma concentrations of about 3-15 mM at the time of LPS
injection (calculated from data in references 34, 35), close to
our in vitro NAC concentrations at which inhibition of NF-B
and TNF was observed. However, it is also possible that NAC
was already partly oxidized before entering the blood stream.
In our study, LPS was injected 24 h after the start of intravenous NAC at dosages accumulating to 175-550 mg/kg after a
period of 24 h; therefore, it is possible that higher concentrations would have been necessary to achieve an inhibitory effect on serum TNF levels. As discussed below, when we increased the dose of NAC, protection against LPS toxicity was
apparently overruled by pro-oxidant effects. Comparison of
the two studies suggests that these adverse reactions do not
play a role when high-dose NAC is administered as an intraperitoneal injection. Alternatively, NAC at a concentration of
10 mM and higher may have inhibited recombinant-TNF-induced L929 cell lysis (see METHODS). Thus, depending on local tissue concentrations, NAC may reduce TNF cytotoxicity.
Increasing the NAC dose to 550 and 950 mg/kg in 48h did
not result in improved survival and reduction of circulating
H2O2. At these doses, H2O2 concentrations just before the LPS
challenge (24 h after NAC treatment) were not significantly
different from H2O2 concentrations in animals treated with saline, which contrast with the inhibition of the baseline H2O2
level in the 275 mg NAC/kg in 48 h group. Although treatment with 950 mg NAC/kg/48 h for 24 h significantly increased
pulmonary total glutathione, GSH was not enhanced. This
suggests the presence of oxidized GSH (i.e., GSSG) that was
formed under the influence of high-dose NAC, since the animals had not yet received LPS. Furthermore, the failure of
protection against LPS paralleled a lack of increase in pulmonary total glutathione levels, as was noticed 6 h after LPS injection in low-dose NAC-treated animals and which was a result of the LPS challenge itself. These observations, together
with the decrease in GSH at 6 h and 12 h after LPS injection,
strongly suggest further oxidation of GSH by oxidative stress
induced by high-dose NAC (950 mg/kg in 48 h) as well as by
LPS. A pro-oxidant effect of NAC might indeed account for
the decrease in survival of LPS-challenged rats receiving the
highest dose, since in those animals a drug-associated increase
in while blood H2O2 was observed. This effect of NAC is
supported by the considerable literature reporting that low-molecular-weight thiols are pro-oxidants as well as antioxidants (14). Pro-oxidant activity is the result of transition
metal-dependent autoxidation yielding O2 ·, H2O2, and the reduced form of the transition metal, which may behave as a catalyst in free radical reactions. In the present study we show the
ability of NAC to reduce iron to the ferrous oxidation state in
a cell-free system, which results in ·OH generation in the presence of H2O2. In the absence of H2O2 (i.e., the reaction of
NAC with ferricitrate), ·OH was also generated. Inhibition of
ethylene formation by catalase provides evidence for the generation of H2O2 in this reaction. Consequently, addition of
SOD, which favors H2O2 formation, resulted in increased ·OH
production. Thus, thiols can serve as electron donors for
metal-catalyzed ·OH generation and oxidative damage of biological structures.
The finding that high-dose NAC did not reduce the level of circulating H2O2 but, on the contrary, enhanced LPS-induced oxidative stress is supported by our in vitro observations. However, the exact cause of high-dose NAC-associated mortality is not completely clear. Histological examinations did not reveal an enhancement of lung injury in relation to the increased mortality. This might indicate that in a specific treatment group some animals were able to resist toxicities induced by LPS and high-dose NAC, whereas other animals died, due to natural differences in resistance against endotoxemia and oxidative stress. Furthermore, it is unlikely that the increased mortality was a result of acidosis-related hemodynamic changes or volume overload-related pulmonary edema, which could synergize with LPS-related events, since there were no relevant differences in the pH of NAC-containing solutions and saline or in the total volume infused in low- and high-dose NAC regions or saline controls. N-acetylcysteine-related toxicity can probably also not be ascribed to peak NAC levels, as LPS was injected 24 h after the start of the infusion and at that time a steady-state concentration of NAC in body fluids might be expected.
In our study and those of others (2), LPS was injected after steady-state concentrations of NAC have been achieved. This may limit the therapeutic value of NAC in sepsis. However, other investigators showed that infusion of NAC after intratracheal administration of IL-1 attenuated lung damage in rats (8). In addition, recent clinical trials of NAC suggest that it may shorten the duration of active lung injury (37, 38).
In summary, our data indicate that NAC's protection against endotoxin-related mortality can be attributed to its capacity to directly reduce oxidative stress, rather than augmenting endogenous glutathione stores or reducing TNF production. Because low-dose NAC powerfully protects against LPS toxicity, this drug and other scavengers and antioxidants may be effective in the treatment of acute lung injury induced by gram-negative sepsis. However, at high concentrations, NAC, possibly by its capacity to reduce iron to the catalytically active form, increases oxidative stress and LPS toxicity. Therefore, antioxidant therapy must be critically controlled in order to minimize its unfavorable effects.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. B. S. van Asbeck, Department of Internal Medicine, Room F02.126, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: B.S.vanAsbeck{at}digd.azu.nl
(Received in original form August 18, 1995 and in revised form December 3, 1997).
This work is part of the PhD thesis of R. C. Sprong; it was supported by grants from the Netherlands Organization for Scientific Research (900-512-131), the Praeventiefonds (002820980), and Utrecht Institute for Infection and Immunity.Acknowledgments: The authors greatly appreciate the biotechnical assistance of Mr. C. J. W. M. Brandt and thank Dr. G. H. Jansen for his help in pathological anatomical studies.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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