Gastric aspiration during the perioperative period is a major risk factor in the development of postoperative acute respiratory distress syndrome (1). The acute lung injury caused by acid aspiration is neutrophil-dependent and associated with large increases in serine proteinases. These enzymes seem to be involved in the pathogenesis of the lung injury (2,3). We demonstrated that intratracheal instillation of low pH saline “primes” the lungs to the deleterious effects of increased ambient oxygen concentrations (hyperoxia) (4–6). The additional lung injury caused by concomitant exposure to hyperoxia, as assessed by a number of functional variables, is mediated, in part, by the endogenous generation of reactive species of oxygen (ROS) (7). This hyperoxia-induced component can be effectively decreased by the administration of deferoxamine, an iron chelator, which inhibits the generation of ROS (4,5).
We also demonstrated that lung antioxidant levels, as assessed by the capacity of lung tissue to inhibit standardized oxidant generation, are decreased after acid aspiration (7). This variable reflects the ability of the organ to resist oxidative stress and is further decreased in the presence of hyperoxia (7). The antioxidant reserve capacity primarily represents the sum of the innate antioxidants capable of scavenging ROS and includes the recognized isoforms of superoxide dismutase (SOD), catalase, glutathione peroxidase, ascorbic acid, and multiple amino acids. We hypothesized that inactivation of SOD activity is, in part, responsible for the decrease in lung antioxidant capacity.
We hypothesized that serine proteinases released in response to acid aspiration are responsible for degradation of this antioxidant enzyme. Aprotinin is a nonspecific serine proteinase inhibitor that decreases several cellular and chemical effector responses associated with acute inflammation (8,9). Clinically, in addition to decreasing surgical bleeding, this drug also diminishes diffuse inflammatory elements associated with cardiopulmonary bypass surgery. These findings raise the question as to whether aprotinin may be beneficial in attenuating acute inflammatory organ damage. The results are not conclusive. Thus, we undertook a study of this drug in a model of acute inflammatory lung injury.
In the present study, an extensively characterized rat acid aspiration model was used to study the hypothesis that SOD, a critical proximal innate antioxidant, is reduced after aspiration injury as a result of degradation by proteinases. It was hypothesized that inhibition of proteinases would lead to maintenance of normal antioxidant capacity and a reduction in lung injury. Based on previous observations, we have predicted that proteinases or ROS mediate the decrease in antioxidant capacity associated with aspiration of the low pH component of gastric contents and that this change contributes to the pathogenesis of the lung injury.
The experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of the State University of New York at Buffalo based on the National Institute of Health guidelines. Anesthesia was induced in male Long-Evans rats (Harlan Sprague Dawley, Indianapolis, IN), 250–300 g, with 2% halothane in air in a bell jar and maintained via a nose cone. After the establishment of a surgical depth of anesthesia, the rats were placed in a 60-degree upright position and hung from their upper incisors. The trachea was exposed through a midline skin incision, and the injury vehicle was instilled intratracheally using a 22-gauge needle. Injury was induced by instillation of 1.2 mL/kg of HCl-acidified normal saline, pH value of 1.25 (acid-injured [ACID]) (10). A sham injury (SHAM) was used as a control and included a tracheotomy without instillation of an injury vehicle. Halothane exposure lasted no longer than 10 min. Rats were randomized to receive either 10,000 U/kg of aprotinin (Bayer Corp., West Haven, CT) or an equal volume of isotonic saline injected IV via the dorsal penile vein at the time of injury.
The rats were further randomized to exposure with air or 98% oxygen for 5 h after injury and then killed. At the time of harvest, the lungs were perfused with a right ventricular injection of 25 mL of 2.5% sucrose in Tris-buffered saline (50 mM of Tris, 0.5 M of NaCl, 50 mM of KCl, 10 mM of Na2HPO4, and 0.1% Triton X-100, with a pH value of 7.5) while inflated to the functional residual volume. The lungs were then excised and homogenized in 1:3 vol/vol of either phosphate buffered saline for activity assay or in cell lysis buffer (10 mM of Na2HPO4, 0.5 M of NaCl, 0.25% Tween-20, 10 mM of 2-mercaptoethanol, 10 mM of EDTA, 10 mM of EGTA, 1% Triton X-100, and 0.5% NP-40, with a pH value of 7.0) for enzyme-linked immunosorbent assay (ELISA).
SOD activity was examined using a commercially available kit (SOD-525; Bioxytech, Portland, OR) that is based on SOD-mediated increases in the rate of auto-oxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo(c) fluorane in an aqueous alkaline solution to yield a chromophore with maximum absorbency at the 525-nm wavelength, as described by the manufacturer. SOD activity was examined in lung homogenates with or without the addition of 50 mM of diethyl dithiocarbamate, a specific inhibitor of Cu/Zn SOD. This allowed us to calculate enzymatic activity related to either isoenzyme.
The concentration of Cu/Zn SOD immune reactive protein was determined in lung homogenate samples using a commercially available ELISA kit (Kamiya BioMedical, Seattle, WA). Standards and samples were prepared as recommended in the packet insert. Plates were read at 450 nm using a microplate reader (BioRad, Hercules, CA).
Lung injury was assessed by determination of alveolar-capillary protein leakage (the protein permeability index [PI]), the Pao2/Fio2 (P-F [fraction of inspired oxygen] ratio), and the alveolar-arterial oxygen gradient. The protein PI was defined as the ratio of radioactivity (cpm) in the harvested lungs to the radioactivity of 1 mL of blood from the same rat. Pao2 was obtained from an arterial blood gas drawn from the descending aorta at the time of death with the rats breathing 2% halothane in oxygen (4).
Myeloperoxidase (MPO) activity was measured in lung homogenates as a surrogate marker of neutrophil infiltration. In brief, lung homogenates were processed in a buffer containing 0.5% hexadecyl-trimethyl-ammonium bromide and 5 mM of EDTA, with a pH value of 6.0, by subjecting it to repeated freeze/thaw and sonication cycles to solubilize the neutrophil granules. MPO activity was quantified by measuring the rate of absorbance change at 460 nm of a reaction containing 50 μL of sample and 1.5 mL of assay buffer (50 mM of KH2PO4, 0.004% H2O2, and 525 μM of o-dianisidine hydrochloride, with a pH value of 6.0 over the linear portion of the curve (11).
The pulmonary inflammatory response was further assessed by measurement of inflammatory cytokines (interleukin [IL]-1β, interferon [IFN]γ, IL-10, and macrophage chemoattractant protein [MCP]-1) in the lung tissue homogenates by a cytometric bead array assay (microsphere-based capture-detecting antibody sandwich ELISA) customized for rat cytokines. Macrophage inflammatory protein [MIP]-2, a major rodent chemokine, was also measured in lung tissue homogenates using a commercially available ELISA kit (Biosource International, San Diego, CA) to determine the chemotactic activity in the lungs. Tumor necrosis factor α (TNFα) was measured using the WEHI bioassay technique. As previously reported by our laboratory, WEHI 164, subclone 13 cells (a TNFα susceptible cell line) were used in a cytocidal bioassay. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Sigma Chemical, St. Louis, MO) was used as the cell viability indicator, assessed at 570 nm (Bio-Rad) (11).
The number of rats in each group was selected based on power analysis, accepting 20% β error and 5% α error. Data were expressed as mean ± sd and analyzed by factorial analysis of variance. Two-factor analysis of variance was used to test for interaction between ACID and hyperoxia with respect to Cu/Zn SOD protein concentration among the groups receiving similar treatments. The Bonferroni/Dunn test was then used as the post hoc test to determine where the differences occurred. The Pearson correlation test was used for simple regression analysis of Cu/Zn SOD and SOD bioactivity. Null hypotheses were rejected at P < 0.05.
Total SOD enzymatic activity was assessed after aspiration of the low pH vehicle with exposure to either room air or increased ambient oxygen concentration, with or without treatment with a serine antiproteinase (Fig. 1). There were no changes in total SOD activity among rats in the control groups receiving the serine antiproteinase. However, after acid aspiration and a 5-h exposure to 98% oxygen (injured), a significant decrease in the enzymatic activity of the antioxidant was observed (from 52.6 ± 7.9 to 33.3 ± 6.4 U/mg of protein; P < 0.05). This decrease was attenuated in injured rats in which serine proteinase activity was inhibited (50.7 ± 6.4; P < 0.05).
In the uninjured control rats, exposure to hyperoxia resulted in increased levels of Cu/Zn SOD protein, whereas it caused a depletion of this protein in acid-injured rats. Pretreatment with aprotinin effectively replenished the protein levels of Cu/Zn SOD in acid-injured hyperoxia-exposed rats (Fig. 2). To further assess the antiproteinase protection of SOD activity, the enzymatic activity of the cytoplasmic isoform, Cu/Zn SOD, and the mitochondrial isoform, Mn SOD, were determined with or without the addition of a specific Mn SOD inhibitor. There were no differences in Cu/Zn activity among control rats treated with the antiproteinase (Fig. 3). However, in the injured groups, there was a significant decrease in Cu/Zn SOD activity in rats exposed to hyperoxia (11.0 ± 2.1 compared to 27.3 ± 4.1 U/mg of protein; P < 0.05). Cu/Zn SOD bioactivity in the control and injured lungs directly correlated to tissue concentrations of immune reactive Cu/Zn SOD protein in the lungs (R2 = 0.8376; P < 0.001). The decrease in Cu/Zn bioactivity after acid aspiration was inhibited in rats treated with the serine antiproteinase (30.0 ± 5.5; P < 0.05).
Changes in Mn SOD bioactivity were much different than Cu/Zn SOD (Fig. 4). Mn SOD bioactivity in SHAM-injured rats increased from 16.2 ± 1.4 U to 25.3 ± 2.6 U with exposure to hyperoxia (P < 0.05). Acid injury caused a modest increase in Mn SOD activity in air-exposed rats that was offset by decreases in Cu/Zn SOD activity. Furthermore, antiproteinase treatment did not change pulmonary Mn SOD enzymatic activity in either SHAM or ACID groups.
Because the administration of the serine antiproteinase preserved Cu/Zn SOD activity after acid aspiration and hyperoxia, we examined the potential protective role of antiproteinase treatment on the additive lung injury associated with hyperoxic exposure after the initial acidic insult. Both leakage of radiolabeled albumin and arterial blood oxygenation did not improve in antiproteinase-treated rats. Paradoxically, there was an increase in leakage of albumin into the lung after acid aspiration and hyperoxia in rats that received antiproteinase therapy compared with untreated injured rats (P < 0.05) (Fig. 5). Similarly, there was no difference in oxygen exchange, as assessed by Po2/Fio2 (265 ± 20 mm Hg in the antiproteinase-treated group compared with 281 ± 25 mm Hg in normal saline-treated controls).
Nonselective inhibition of serine antiproteinases did not alter the levels of inflammatory markers of acute inflammation after acid aspiration. Infiltration of neutrophils into the lung, as assessed by MPO activity, was slightly increased after antiproteinase treatment. There were no differences detected in the lung tissue concentrations of TNFα and IL-1β among the injury/treatment groups (Table 1). MIP-2 levels were increased in the lungs after acid aspiration. Pulmonary concentrations of this neutrophil chemoattractant paralleled MPO activity, although there were no differences among the different injury groups, either treated or untreated. Pulmonary concentrations of MCP-1 were small. However, the highest levels of this monotactic chemokine were present after inhibition of serine antiproteinase activity after acid aspiration and hyperoxia. Finally, not unexpectedly, no changes were detected in IFN-γ nor IL-10 levels at 5 h postaspiration (Table 1).
We investigated the interaction between two major forms of neutrophil-mediated inflammatory injury to the lungs after aspiration of gastric contents. It has previously been shown that both proteinases and ROS are clearly involved in the pathophysiology of acute lung injury (2,3,7). We found that pretreatment with a nonspecific serine proteinase inhibitor effectively restores the cytoplasmic isoform of superoxide dismutase Cu/Zn SOD. Although this enzyme was depleted through the course of gastric aspiration, its restoration did not decrease the extent of acute lung injury and pulmonary cytokine response after aspiration of gastric contents.
The main goal of our work has been to develop potential therapeutic strategies to decrease the severity of acute lung injury and to prevent the evolution of the initial insult to more severe and progressive pulmonary damage. Previously, our laboratory demonstrated a decrease in lung antioxidant levels after acid aspiration and exposure to increased ambient oxygen (7). We hypothesized that a major component of this decrease in antioxidant activity was the inactivation of SOD. This antioxidant is an important proximal enzyme in the oxidant cascade and makes up a significant component of the antioxidant capacity of the lung. Serine proteinases are increased after acid aspiration and play a role in the pathogenesis of this lung injury (12,13). Because interactions between antioxidants and proteinases have been demonstrated (14), we hypothesized that the decrease in antioxidant levels was caused by proteinase degradation. Thus, we predicted that treatment with a nonspecific serine antiproteinase would inhibit the observed decreases in SOD bioactivity in the lungs.
Our findings support these hypotheses: (a) administration of a serine antiproteinase restores total lung SOD activity after acid aspiration and hyperoxia because of the inhibition of proteinase degradation of the cytoplasmic isoform Cu/Zn SOD; (b) both the decreases in bioactivity and immune recognizable protein levels correlated; (c) the mitochondrial isoenzyme Mn SOD increased in response to both low pH lung inflammation and increased ambient oxygen exposure; (d) the enzymatic activity of Mn SOD isoenzyme was not affected by serine antiproteinase treatment; and (e) despite the apparent salutary improvement in SOD bioactivity after antiproteinase administration, lung injury was not decreased. In fact, paradoxically, there was a nonsignificant increase in neutrophilic lung injury (MPO activity and protein leakage) consistent with a corresponding chemokine response. Whereas Pao2/Fio2 does not change with antiproteinase treatment, this ratio also trends toward increasing the extent of the lung injury.
Previously, our laboratory, as well as other investigators, has demonstrated that after acid aspiration, an acute inflammatory injury develops (15,16). The lung is sensitized to additional insults, including gastric particulate material and exposure to increased ambient oxygen levels (4,12,17). The inflammatory component of lung injury (as opposed to the direct chemically-induced tissue damage) associated with acid aspiration is neutrophil-dependent (2). Both serine and acid proteinases are increased in the lung tissue after aspiration of low pH solutions, in part, as a result of neutrophil activation (2,3). These chemical inflammatory mediators also seem to be responsible for acid pulmonary injury.
Serine proteinases can interact with innate antioxidant proteins in several ways. Reactive species of oxygen can activate transcription factors, such as nuclear factor-κB and AP-1, as a result of alterations in the cell redox potential (18,19). Activation of these transcription factors is associated with increased expression of proteinases (20). Oxidants can also inactivate innate antiproteinases, thereby resulting in an additional gain of total proteolytic activity in injured tissues (14). Finally, an increase in proteolytic activity can also be expected to inactivate antioxidants. Compartmentalization of proteinases during inflammation may explain the lack of effect on Mn SOD bioactivity because this isoform of SOD is located in the mitochondria (21,22).
Innate antioxidants play a critical role in the pathogenesis of lung injury. Excess superoxide, generated through the oxidative metabolism in the mitochondria or as a byproduct of various cytoplasmic oxidases (e.g., xanthine oxidase or MPO), undergoes dismutation to H2O2 by innate SOD isoforms or is scavenged in conjugation with other reactive species, such as nitric oxide. Generated molecules of H2O2 either decompose or are actively catalyzed to molecular oxygen and water. If H2O2 is not converted to inactive compounds rapidly, hydroxyl radicals (OH−), the most chemically reactive of the oxidants, may form through the Haber-Weiss reaction. Thus, despite its pivotal role in oxidative metabolism, SOD does not determine the ultimate fate of the generated superoxide species. As it can be inferred from the oxidative cascade, increasing SOD in the presence of increased superoxide may also result in the generation of more H2O2. Paradoxically, this could lead to an increased lung injury if H2O2 is not rapidly degraded and OH− is generated (23). Alternatively, the increase in lung injury may not be related to the antiproteinase-induced increase in Cu/Zn SOD. Nonselective inhibition of serine proteinases may affect any number of proteinase cascades, some of which could be protective during an inflammatory response (i.e., degradation of inflammatory peptides). During acute inflammation, specific proteinase species or proteolytic activity may have beneficial effects.
Successful restoration of molecular integrity and enzymatic activity of SOD did not improve the severity of lung injury. Conversely, acute inflammatory lung injury can be decreased by inhibiting the generation of ROS and nitrogen further downstream of superoxide and H2O2 production. The protective effects of deferoxamine on the lung injury, caused by acid aspiration and exposure to increased ambient oxygen, have been shown by our laboratory (4). A decrease in SOD bioactivity is probably more sensitive than alterations in immune reactive protein levels in assessing the effects of inhibiting both the generation of OH− and ROS.
In summary, our findings demonstrate that the cytoplasmic isoenzyme Cu/Zn SOD is decreased after acid aspiration and exposure to hyperoxia. Conversely, the mitochondrial isoform, Mn SOD, is increased. A major cause of the decrease in Cu/Zn SOD seems to be proteinase degradation in the cytoplasmic compartment because a nonspecific serine antiproteinase inhibited the loss of bioactivity and antigenic protein of this antioxidant but had no effect on Mn SOD. Lung injury did not improve. Paradoxically, some variables suggested that the pulmonary damage was worse. We speculate that this was because of increased OH− generation because deferoxamine has been shown to completely inhibit the hyperoxia-induced component of the lung injury.
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