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Am J Physiol Heart Circ Physiol 276: H984-H992, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 3, H984-H992, March 1999

Role of nitric oxide-derived oxidants in vascular injury from carbon monoxide in the rat

Stephen R. Thom1,2, Donald Fisher1, Y. Anne Xu1, Sarah Garner1, and Harry Ischiropoulos1,3

1 Institute for Environmental Medicine and 2 Departments of Emergency Medicine and 3 Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068


    ABSTRACT
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Studies were conducted with rats to investigate whether exposure to CO at concentrations frequently found in the environment caused nitric oxide (NO)-mediated vessel wall changes. Exposure to CO at concentrations of 50 parts per million or higher for 1 h increased the concentration of nitrotyrosine in the aorta. Immunologically reactive nitrotyrosine was localized in a discrete fashion along the endothelial lining, and this was inhibited by pretreatment with the NO synthase (NOS) inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME). The CO-induced elevations of aortic nitrotyrosine were not altered by neutropenia or thrombocytopenia, and CO caused no change in the concentration of endothelial NOS. Consequences from NO-derived stress on the vasculature included an enhanced transcapillary efflux of albumin within the first 3 h after CO exposure and leukocyte sequestration that became apparent 18 h after CO exposure. Oxidized plasma low-density lipoprotein was found immediately after CO exposure, but this was not inhibited by L-NAME pretreatment. We conclude that exposure to relatively low CO concentrations can alter vascular status by several mechanisms and that many changes are linked to NO-derived oxidants.

myeloperoxidase; nitrotyrosine; lipoprotein oxidation; endothelium; xanthine oxidase


    INTRODUCTION
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

CARBON MONOXIDE (CO) is a well-recognized environmental toxicant, and there is a growing body of evidence which indicates that CO may also serve a physiological role in signal transduction (23). In this regard, similarities between CO and nitric oxide (NO) have been recognized for a number of years (1). Some aspects of toxicity associated with CO and NO also overlap. When vascular endothelial cells in culture are exposed to between 20 and 100 parts per million (ppm) CO they suffer oxidative stress and a form of delayed death due to generation of NO-derived oxidants (53). Both endothelial cells and platelets have been found to liberate NO to the surrounding medium when exposed to CO (52, 53). These effects occur because CO increases the intracellular steady-state concentration of NO by inhibiting NO binding to hemoproteins, without changing the activity of NO synthase (NOS) or mitochondrial function (52, 53). In a model of brain injury mediated by high concentrations of CO, vascular oxidative changes triggered by reactive nitrogen species were found to initiate a biochemical cascade leading to leukocyte adherence and activation, followed by brain lipid peroxidation (12).

We hypothesized that rats exposed to concentrations of CO frequently found in contaminated environments would exhibit evidence of vascular oxidative injury and that this injury would be mediated by NO-derived oxidants. The concentrations of CO present in modern environments pose a health risk, although the pathogenesis remains unclear. Ambient CO levels in air pollution have been correlated with hospital admissions, mortality, and morbidity due to cardiovascular and respiratory diseases (16, 28). Outdoor CO levels in urban environments have been decreasing in recent years due to automobile catalytic converters; atmospheric levels may range from 2 to 40 ppm (61). Cigarette smoke, a frequently cited source of indoor CO, contains levels in mainstream and sidestream smoke ranging from ~35 to 1,000 ppm (56). Smokers and workers in certain occupational settings typically have elevated blood carboxyhemoglobin (COHb) levels in the range of 3-11% (39).

One potential health risk from chronic exposure to CO may be acceleration of atherosclerosis, although this is controversial (42). We considered that recent in vitro observations may contribute new information to this issue because CO exposure causes endothelial cells to generate peroxynitrite (49). Peroxynitrite is a potent oxidizing and nitrating agent produced when NO reacts with superoxide radical (11). A link between peroxynitrite and development of atherosclerosis has been suggested because nitrated proteins have been found in low-density lipoprotein (LDL) of human atherosclerotic intima and atherosclerotic coronary arteries (20, 58). Vascular insults from CO have been reported in studies when both human beings and experimental animals were exposed to CO for ~1 h or more (24, 31, 41). The mechanism for this effect is not clear, and moreover vascular injury has not been reproducible in all studies. Capillary leakage has not been found after brief exposures of 5-8 min to 20,000 ppm CO, sufficient to cause COHb levels of 56-90% (40, 45).

We have investigated whether exposure to CO caused physiological and biochemical evidence of vascular injury and whether changes were mediated by NO-derived oxidants. The role of circulating polymorphonuclear leukocytes (PMN) and platelets and the importance of the duration of exposure and concentration of CO were examined.


    METHODS AND MATERIALS
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Animals and reagents. Wistar male rats (Charles River Laboratories, Wilmington, MA) weighing 200-290 g were fed a standard diet and water ad libitum. For selected experiments rats were fed a standard laboratory rat chow supplemented with 4% cholesterol and 2% sodium deoxycholate for 6 wk (North Penn Feeds, Lansdale, PA). 125I-labeled BSA was purchased from ICN Pharmaceuticals (Irvine, CA). Antibodies against myeloperoxidase were purchased from Dako (Carpinteria, CA), and anti-endothelial NOS and anti-inducible NOS were from Affinity BioReagents (Golden, CO). Unless otherwise specified, other reagents were purchased from Sigma Chemical (St. Louis, MO).

Animal manipulations. Rats were exposed to CO in a 7-liter Plexiglas chamber. A mixture of CO and air was flushed through the chamber at a rate of 8-12 l/min. Premixed gas supplies containing 100, 1,000 or 3,000 ppm CO in air were purchased from Air Products (Camden, NJ). When a concentration of 50 ppm CO was desired, a gas supply was prepared using a gas-mixing chamber (Calibrated Instruments, Ardsley, NY). A supply of compressed air containing 100 ppm CO was attached to one port of the device and purified; compressed air was attached to the second port. The concentration of CO was selected by setting the mixing ratio between the CO supply and the compressed air, and the concentration delivered was verified using a gas chromatography CO detector (Trace Analytical Reduction Gas Analyzer, Menlo Park, CA). Some rats were subjected to CO according to a standard model that leads to brain injury and has been described in previous publications (12). In brief, rats were placed in the Plexiglas chamber and a small volume of pure CO was injected into a gas stream of 1,000 ppm CO and air to compensate for the volume of air already in the chamber. This procedure allowed the rats to be exposed to 1,000 ppm CO from the very beginning of the study. Rats were exposed to 1,000 ppm CO for 40 min, the gas was switched to 3,000 ppm CO in air, and another CO bolus was added to rapidly achieve the 3,000 ppm concentration. Rats remained in the chamber for up to 20 min, until they lost consciousness, and then they were removed from the chamber to breathe room air.

Rats were made thrombocytopenic by an intraperitoneal injection of 1.5 ml/kg of rat plasma-adsorbed rabbit anti-rat platelet antiserum (Inter-cell Technologies, Hopewell, NJ) given 2 h before exposure to CO, as described in previous studies (12, 52). Circulating PMN were reduced to counts of <100 cells/µl blood by an intraperitoneal injection of 4 ml/kg anti-neutrophil antiserum (Inter-cell Technologies) administered 24 h before study (12). Nomega -nitro-L-arginine methyl ester (L-NAME, 1 mg/kg ip) was injected 2 h before CO exposure.

Immunohistochemistry. Staining was carried out with an affinity purified monoclonal anti-nitrotyrosine antibody obtained from Dr. J. S. Beckman. This antibody has been characterized and described in a previous publication (12). Rats were anesthetized by an intraperitoneal injection with ketamine (73.5 mg/kg) and xylazine (1.5 mg/kg). The abdominal aorta was exposed and fixed in situ by infusing 4% paraformaldehyde in a 0.1 M sodium cacodylate buffer. The aorta was removed, left in fixative for 3 h, and then cryoprotected by 1-h incubations in 10, 20, and 30% sucrose in 0.1 M sodium cacodylate buffer. Aortic samples were placed on tissue holders covered with OTC Tissue Tek Compound (OTC; Sakura Finetek, Torrance, CA), covered with a thin layer of OTC, and frozen with distilled Freon 22 in liquid nitrogen. Eight micronmeter-thin sections were prepared and placed onto poly-L-lysine coated slides. Before staining, slides were soaked in Antigen Retrieval Citra Solution (BioGenex, San Ramon, CA), heated in a microwave oven and processed according to the protocol recommended by BioGenex. Slides were stained with a 1:100 dilution of anti-nitrotyrosine antibody and counterstained following procedures exactly as described in a previous publication (12).

Analytical procedures. Heparinized blood (10 U/ml) was drawn into a closed system, and blood COHb levels were measured using a CO-oximeter model 282 (Instrumentation Laboratories, Lexington, MA).

The influx of 125I-BSA into skeletal muscle was measured using essentially the same techniques as those described in a previous publication (51). Briefly, rats were injected intravenously with 125I-BSA having a specific activity of ~800,000 cpm. Three hours later rats were anesthetized, and samples of anterior thigh skeletal muscle were removed, rinsed with PBS to clear away all blood, weighed, homogenized in a Polytron blender, and counted in a gamma counter (Wallac 1,470). The amount of 125I-BSA in muscle was expressed as 100 × (radioactive counts present in 1 g of homogenized tissue/radioactive count in 1 ml of blood). Counts were corrected for residual intramuscular blood, which was assessed as the change in absorbance at 415 nm before and after muscle homogenates were incubated with 13 mM sodium dithionite (47). Nitrotyrosine concentration in aorta was measured in tissue obtained from anesthetized rats, after it was rinsed and homogenized in PBS, using a solid-phase radiochemical assay (12).

Myeloperoxidase concentrations in aorta were measured using a new solid-phase radiochemical assay. Tissue was perfused to remove blood, removed from the rats, and immediately frozen in liquid nitrogen. The frozen tissue was weighed, placed in 2 ml PBS, homogenized with a Polytron, subjected to two freeze-thaw cycles with liquid nitrogen, and then made to 1% (wt/vol) cetyltrimethylammonium bromide (CTAB). Control experiments were also done using isolated rat PMN that had been obtained from heparinized blood by density-gradient centrifugation using materials and procedures from Cardinal Associates (Santa Fe, NM). This process resulted in suspensions of rat PMN that exceeded 95% purity. Suspensions of PMN in 1 ml of PBS were subjected to the same freeze-thaw procedures as the tissue samples and suspended in 2 ml of CTAB solution. Homogenized samples of tissue or PMN were centrifuged at 1,000 g for 10 min, and supernatants were made up to a volume of 10 ml with 1% CTAB containing 1 mM CaCl2, MnCl2, and MgCl2 and passed through 0.45-µm polycarbonate filters. Filtered samples were placed in test tubes with 0.5 ml concanavalin A gel and incubated overnight at 4°C with constant shaking. The samples were then centrifuged at 1,000 g for 5 min, and the pellet was washed twice with wash buffer [0.1 M sodium acetate, pH 6.0, containing 0.1 M NaCl and 0.05% (wt/vol) CTAB]. The washed pellets were resuspended in 300 µl elution buffer (wash buffer that contained 0.5 M methyl-D-mannopyranoside), incubated for 10 min, and centrifuged at 1,000 g for 5 min. The supernatant was saved, and the pellet was subjected to two additional washes with elution buffer. All supernatants were combined (900 µl) and frozen at -20°C until analysis.

Proteins in the concanavalin A eluates were immobilized by spotting from 4 to 8 concentration-dependent dilutions of the tissue extracts onto nitrocellulose paper using the 96-well Bio-Dot microfiltration unit (Bio-Rad, Hercules, CA). Human myeloperoxidase (Sigma) was prepared in PBS, and 8-12 different concentrations were also placed onto the same paper. After being blocked with 2% gelatin, the nitrocellulose was incubated for 15 h with a solution containing 1:100 dilution rabbit anti-human myeloperoxidase (Dako) followed by a 3-h incubation in a solution containing donkey anti-rabbit 125I-labeled IgG (0.1-0.2 mCi/ml). The blot was extensively washed in Tween-TBS and dried. The radioactivity of each sample was measured directly by beta scanning using an Ambis 400 imaging detector. The net counts of radioactivity (corrected for background counts from a sample blank) were obtained using the AMBIS image analysis software (v4.1) and then plotted on a semilogarithmic plot. The counts obtained with human myeloperoxidase were used to assure that the techniques for individual experiments were comparable. The data, recorded as counts per minute, were converted to number of rat PMN using a relationship generated by correlating 125I cpm obtained with extracts prepared using a range of numbers of PMN. The semilogarithmic plot was subjected to analysis by least-squares linear regression (n = 20, r2 = 0.88).

Western analysis was performed after the aorta was homogenized in 0.1 M phosphate buffer containing 2 mM MgCl2. The preparation was diluted to a concentration of 100 µg protein/10 µl in 0.1 M phosphate buffer containing 2% SDS, 10% glycerol, 5% beta -mercaptoethanol, and 0.00125% bromphenol blue and boiled for 5 min. Equal amounts of preparation from different homogenates (100 µg) were subjected to 12% SDS-PAGE and proteins transferred to a 0.45-µm polyvinylidene difluoride membrane (Bio-Rad). Membranes were incubated for 2 h with PBS containing 0.1% Tween 20 and 5% nonfat dry milk and then for 2 h with either rabbit anti-endothelial or anti-inducible NOS (1:1,000 dilution). Membranes were washed with Tween-TBS, incubated for 3 h with horseradish peroxidase-conjugated anti-rabbit IgG (1:3,000 dilution; Boehringer Mannheim, Ridgefield, CT), and washed again for 25 min with Tween-TBS. Imaging of the membranes was carried out by enhanced chemiluminescence (ECL) using ECL reagents and procedures obtained from Amersham Life Sciences on Kodak Reflection Film (Eastman Kodak, Rochester, NY). The film was developed and scanned on a Microtek Scanmaker E6 (Redondo Beach, CA) with transparency adaptor and analyzed using the SigmaScan program (Jandel, Chicago, IL).

LDL was isolated from ~10 ml of rat blood using a potassium bromide density gradient and ultracentrifugation as described by van't Hooft and van Tol (55). The LDL fraction was withdrawn, 0.025% (wt/vol) butylated hydroxytoluene was added, and the samples were frozen at -20°C overnight. Fractions were assayed for thiobarbituric acid-reactive substances (TBARS) according to published procedures using 200-µl LDL samples (~60 µg protein) (48). Xanthine dehydrogenase and oxidase were assayed in aortic homogenates following the fluorometric assay described in a previous publication (48). Plasma cholesterol and triglycerides were assayed using commercial kits (Sigma).

Statistics. Statistical analysis was determined by ANOVA followed by Scheffé's test (43). The level of significance was taken as P < 0.05. Results are expressed as means ± SE.


    RESULTS
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Immunohistochemical evidence of aortic oxidative stress. We first sought evidence for production of NO-derived oxidants in the aorta by staining frozen aortic sections with an antibody that specifically recognizes nitrotyrosine. The predominant site of nitrotyrosine staining was the endothelial lining. Representative sections from the aorta of a rat killed immediately after exposure to 100 ppm CO for 1 h are shown in Fig. 1. We typically found irregularly shaped patches of nitrotyrosine in the aortic intima of CO-exposed rats but not control animals.


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  		Fig. 1.   Immunofluorescence photomicrographs from aortic frozen sections. Panels show serial thin sections from a rat killed immediately after exposure to 100 parts per million (ppm) CO for 1 h and sections from a control rat.

Quantitative evaluation of aortic nitrotyrosine. The nitrotyrosine concentration in homogenates of aortas from rats exposed to 50, 100, 1,000, or 1,000 followed by 3,000 ppm CO for 1 h were increased over control (Fig. 2). When rats were pretreated with L-NAME there was no significant increase in the nitrotyrosine concentration, indicating that NOS activity was required for nitrotyrosine formation.


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  		Fig. 2.   Aortic nitrotyrosine and arterial carboxyhemoglobin (COHb) levels in rats exposed to CO for 1 h. Values are means ± SE. Solid bars, nitrotyrosine concentrations; hatched bars, COHb values. Rats were exposed to indicated concentrations of CO for 1 h and then anesthetized. Blood was obtained anaerobically from abdominal aorta for COHb determination, and aortas were taken and processed as described in METHODS AND MATERIALS. Columns labeled STD CO and STD CO + Nomega -nitro-L-arginine methyl ester (L-NAME) are data from rats exposed to standard CO protocol, 1,000 ppm CO for 40 min and then 3,000 ppm for up to 20 min until the rats fell unconscious. Rats were injected with L-NAME (1 mg/kg ip) 2 h before CO exposure. * Significantly greater than control concentration of nitrotyrosine. + Significantly less than concentration of nitrotyrosine after standard model. COHb level for group exposed to 50 ppm CO was not significantly greater than control; values for all other CO-exposed groups were significantly greater than control. There was no significant difference in COHb values in rats exposed to 1,000 ppm CO, STD CO, or STD CO + L-NAME.

COHb in CO-mediated oxidative stress. An hypoxic stress from elevations of COHb is an obvious possible effect of CO exposure. Therefore, we evaluated the COHb levels that resulted from the 1-h exposures to CO (Fig. 2). The COHb concentration after exposure to 50 ppm CO was not significantly greater than control, whereas values were significantly increased by exposure to 100 and 1,000 ppm CO. When rats were exposed to a pattern of CO that causes brain oxidative injury, 1,000 ppm CO for 40 min and then 3,000 ppm for up to 20 min until rats fell unconscious (Fig. 2), the concentrations of COHb in blood and aortic nitrotyrosine were significantly increased. However, the magnitude of the increases was not significantly different from exposure to 1,000 ppm CO for 1 h (Fig. 2).

When rats were exposed to 1,000 ppm CO for only 40 min, the aortic nitrotyrosine level was 5.9 ± 0.9 (n = 7) ng/µg protein [not significantly greater than control (NS)]. However, the COHb concentration that resulted from exposure for 40 min to 1,000 ppm CO was 49 ± 2% (n = 6). This value was significantly greater than control and insignificantly different from exposure to 1,000 ppm for 60 min (Fig. 2). If rats were exposed to pure CO for ~2 min, the COHb level was 79 ± 1% (n = 7), a value significantly greater than after 1,000 ppm CO for 1 h (Fig. 2). However, the aortic nitrotyrosine concentration in rats exposed to pure CO for 2 min was 1.4 ± 0.5 ng/µg protein (NS vs. control). We conclude that the CO-mediated increase in aortic nitrotyrosine concentration depended on the duration of CO exposure and was not correlated with the COHb concentration.

Role of circulating platelets and PMN in CO-mediated aortic nitrotyrosine. Platelet-derived NO plays a role in CO-mediated oxidative stress (12, 52), and thrombocytopenic rats have lower concentrations of nitrotyrosine in brain after some patterns of CO exposure (12). Nitrotyrosine can also be produced by peroxynitrite generated by circulating PMN or by myeloperoxidase-H2O2 and nitrite (7, 54). We were interested in assessing the involvement of platelets and PMN in aortic oxidative stress. After injection of anti-platelet antiserum rats had circulating platelet counts <20% of normal. However, they exhibited a mean aortic nitrotyrosine concentration of 13.4 ± 4.6 ng/µg (n = 4) protein after exposure for 1 h to 1,000 ppm CO. This value was significantly greater than control but not significantly different from rats with normal numbers of platelets (Fig. 2). Neutropenic rats (see METHODS AND MATERIALS) exposed to CO according to the standard model had a mean aortic nitrotyrosine concentration of 13.3 ± 3.9 ng/µg (n = 4) protein, a value significantly greater than control but not significantly different from CO-exposed rats with normal numbers of PMN (Fig. 2). We conclude that neither platelets nor PMN were responsible for CO-mediated protein nitration.

Aortic leukocyte sequestration due to CO exposure. Leukocyte adherence may be increased after endothelium has been subjected to an oxidative stress (57). We sought evidence of leukocyte sequestration by measuring the myeloperoxidase concentration in aortic homogenates. No increase in myeloperoxidase was found in samples obtained immediately after CO exposure. However, 18 h after a 1-h exposure to CO, myeloperoxidase was significantly increased (Fig. 3). Myeloperoxidase activity was not increased in rats that had been treated with L-NAME, indicating that NO-derived oxidants were required for development of this effect.


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  		Fig. 3.   Polymorphonuclear leukocytes (PMN) in aortic homogenates. Values reflect number of PMN quantified on basis of measurements of myeloperoxidase concentration using solid-phase radioimmunoassay (see METHODS AND MATERIALS). Aortas were obtained from rats after they were exposed to 100 ppm CO for 1 h. Some rats were killed immediately after CO exposure, and some were killed 18 h after exposure. Where indicated, rats were injected with L-NAME at 1 mg/kg (ip) 2 h before CO exposure and then killed 18 h after exposure. * Significantly greater than control.

Duration of aortic nitrotyrosine elevations after CO exposure. Because of the evidence for delayed leukocyte sequestration, we were interested in evaluating the duration of time that aortic nitrotyrosine concentration remained elevated. The nitrotyrosine concentration in aortas obtained 45 min after rats were exposed to 100 ppm CO for 1 h was 4.8 ± 1.7 ng/µg protein (n = 11; NS vs. control). We conclude that production of NO-derived oxidants must cease relatively soon after CO exposure and that the nitrotyrosine epitope is removed either by proteolysis or an alternative intracellular mechanism (9, 14).

Western analysis for NOS. Analysis of aortic homogenates for NOS was performed on Western blots (Fig. 4). The concentration of NOS in aortic tissue was evaluated by comparing the density of the bands obtained using homogenates from CO-exposed rats with the mean density for bands from control rats run on the same gel. Mean density among control bands was 1.0 ± 0.12 (n = 5), the density was 0.92 ± 0.12 (n = 5, NS vs. control) among aortic samples obtained within 2 h after exposure to 100 ppm CO, and 1.08 ± 0.09 (n = 4, NS vs. control) for aortic samples obtained 18 h after 100 ppm CO. No bands were detectable when gels were stained using antibody against the inducible form of NOS.


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  		Fig. 4.   Western blot of aortic homogenates. Tissue homogenates were subjected to polyacrylamide gel electrophoresis and processed as described in METHODS AND MATERIALS. Samples were taken from control rats or from rats exposed to 100 ppm CO for 1 h.

Albumin efflux from microvasculature. The influx of albumin into skeletal muscle was increased during the 3-h interval after exposure to 100 ppm CO (Fig. 5). There was no significant increase in counts after CO exposure when rats were pretreated with L-NAME, indicating that the change depended on NO. The elevation of albumin efflux from the microvasculature into muscle was not detectable when rats were injected with 125I-BSA at 18 h after CO exposure.


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  		Fig. 5.   Vascular efflux of albumin caused by CO. Values reflect 125I-labeled BSA in leg skeletal muscle obtained and processed as described in METHODS AND MATERIALS. Samples were taken from control rats or from rats injected with 125I-BSA either immediately or 18 h after exposure to 100 ppm CO. Tissue 125I-BSA concentration was expressed as 100 × (cpm in 1 g muscle/cpm in 1 ml blood taken from same rat). * Significantly greater than control.

Effect of high-cholesterol diet on aortic nitrotyrosine concentration and endothelial oxidant production. Hypercholesterolemia can increase endothelial cell production of superoxide by enhancing xanthine oxidase activity, and hypercholesterolemia also enhances production of NO (27, 29, 30, 38). We examined the impact of hypercholesterolemia on CO-mediated aortic oxidative stress after rats were fed a cholesterol-supplemented diet for 6 wk. The serum cholesterol level of control rats was 58 ± 3 mg/dl (n = 8), and the cholesterol level of rats fed the supplemented diet was 404 ± 39 mg/dl (n = 10, P < 0.05). The high-cholesterol diet did not cause morphological changes in the aorta and did not change the nitrotyrosine level. The nitrotyrosine concentration in aortas from rats fed the cholesterol-supplemented diet was 3.1 ± 0.1 ng nitrotyrosine/µg protein (n = 3, NS vs. control). Hypercholesterolemic rats exposed to 100 ppm CO for 60 min had nitrotyrosine levels that were significantly greater than air-exposed rats fed the same diet, 9.7 ± 0.9 ng/µg protein (n = 4, P < 0.05 vs. control), but the concentration was not significantly different than in CO-exposed rats fed the standard diet (Fig. 2).

The high-cholesterol diet increased the activity of xanthine dehydrogenase in aortic homogenates. In rats fed the control diet, xanthine dehydrogenase activity was 88 ± 16 mU/mg protein (n = 5), whereas in the rats fed the high-lipid diet activity was 164 ± 15 mU/mg protein (n = 6, P < 0.05). However, the activity of xanthine oxidase was not significantly different. Rats fed the control diet had an activity of 89 ± 10 mU/mg (n = 5), and in rats fed the high-fat diet activity was 94 ± 15 mU/mg protein (n = 6). This differs somewhat from observations by others where an elevated xanthine oxidase activity was found after a high-cholesterol diet (29). This may be why hypercholesterolemia did not augment aortic nitrotyrosine concentrations in rats after CO exposure.

Oxidation of LDL. Because of the possible association between CO exposure and atherosclerosis, and the fact that peroxynitrite can oxidize LDL (20, 59), we were interested in evaluating whether LDL would be oxidized after CO exposure. As shown in Table 1, there was nearly a sixfold increase in LDL oxidation assessed as the concentration of TBARS in LDL obtained immediately after rats were exposed to CO. However, rats treated with L-NAME had no significant reduction in the TBARS concentration after CO poisoning, suggesting that the oxidative process was different from that which caused aortic nitrotyrosine formation and other changes documented after CO exposure in this study.

                              
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  		Table 1.   LDL oxidation assessed as TBARS concentration


    DISCUSSION
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

The results from this study demonstrate that exposure to CO at concentrations frequently found in modern environments can cause a number of changes in the vasculature. Based on the inhibitory effects of L-NAME, NO-derived oxidants such as peroxynitrite are responsible for development of many of these changes.

Our findings indicate that the duration of exposure to CO has more influence on aortic protein nitration than does the concentration of CO in the breathing mixture. An elevated concentration of nitrotyrosine in aortic homogenates persisted for <45 min after CO poisoning. This suggests that the source of oxidative stress was transient and changes were reversible. There are several mechanisms by which exposure to CO could temporarily increase production of NO-derived oxidants. One mechanism is by increasing the steady-state concentration of NO due to a competition for hemoprotein targets between CO and the NO that is normally produced by cells. This is the mechanism we have identified in studies with endothelial cells and platelets (52, 53). We cannot rule out the possibility that CO may also increase the activity of NOS in vivo. However, we have shown that CO at concentrations used in this study do not alter NOS activity in endothelial cells or platelets (52, 53). Moreover, at higher concentrations, CO will inhibit, not enhance, NOS activity (52, 60).

Vascular oxidative stress could occur if there were an increase in oxygen-based free radicals. Hydroxyl radicals were reported to be generated by brain cell mitochondria when rats were exposed to 2,500 or 10,000 ppm CO (35, 36). CO binding to cytochrome-c oxidase in vivo will occur when COHb is high (~50%), a level that causes both systemic hypotension as well as impaired oxygen delivery (3). CO binding to mitochondrial cytochromes of respiring cells in vitro has only been documented when either the CO concentration was extraordinarily high, or O2 tension extremely low, such that the CO-to-O2 ratio exceeded 12:1 (5). Therefore, oxidative stress due to direct CO binding to mitochondrial constituents is extremely unlikely at the concentrations used in this study. However, there are several mechanisms linked to NO that may contribute to oxidative stress. For example, either NO or peroxynitrite could perturb mitochondrial function and cause enhanced O2-based free radical production. Peroxynitrite appears to inhibit complexes I-III and NO targets cytochrome oxidase (4, 21, 37). Alternatively, exposure to CO may inhibit antioxidant defenses. Mechanisms linked to elevations in NO could be responsible for inhibiting one or more enzymes. NO-derived oxidants can inhibit manganese superoxide dismutase and glyceraldehyde-3-phosphate dehydrogenase and deplete cellular stores of reduced glutathione (13, 22).

Leukocyte sequestration in the aorta is another effect of CO exposure. Oxidative stress can cause acute and delayed expression of leukocyte adhesion molecules by endothelial cells (2, 57). The mechanism for CO-induced leukocyte sequestration in the aorta was mediated by NO, based on the inhibitory effect of L-NAME, but it appears to be different from the mechanism shown to occur in brain. Leukocyte sequestration in brain microvasculature has only been found immediately after poisoning by high concentrations of CO, which cause both oxidative stress to the vasculature and an acute cardiac compromise that transiently reduces cerebral blood flow (12, 25, 47, 49, 53). Further studies are necessary to identify the specific mechanism responsible for the delayed leukocyte sequestration in aorta after CO exposure.

Vascular leakage due to CO has been documented in a number of studies (17, 24, 31, 41). These investigations involved durations of exposure to CO greater than 45 min, whereas studies that have failed to find evidence of vascular injury used exposures of 5-8 min (40, 45). Capillary permeability to albumin depends on fenestrations in the capillary wall, the total area of capillary wall that is perfused, and the velocity of blood flow. We found that CO exposure increases the efflux of albumin from capillaries by an NO-dependent process. This change may be caused by alterations in endothelial integrity or by hemodynamic changes. Peripheral vascular resistance is diminished by CO, and CO-associated increases in blood flow can be inhibited by L-NAME (15, 26, 34, 46).

The physiological importance of CO-mediated vascular changes may be greater if exposures were repetitive, as would occur with occupational situations and smoking. Because oxidative stress has been linked to the atherosclerotic process, our results offer the first biochemical mechanism that may explain an association between atherosclerosis and chronic CO exposure. The association between atherosclerosis and CO exposure is a controversial issue that has been supported by some, but not all, animal and epidemiological studies (6, 10, 18, 19, 28, 32, 33, 44).

We investigated the impact of a high-cholesterol diet on CO-mediated aortic oxidative stress. Hypercholesterolemia does not alter antioxidant defenses in the rat (8). Elevations in serum cholesterol have been reported to increase vascular xanthine oxidase activity in rabbits, and also production of NO, which may lead to enhanced production of peroxynitrite (29). Others have suggested that peroxynitrite may be associated with development of atherosclerosis because peroxynitrite can oxidize plasma lipoproteins (20, 59). We failed to find evidence for enhanced CO-mediated vascular stress in hypercholesterolemic rats, based on the aortic nitrotyrosine concentration. Xanthine oxidase activity was not increased, however, which could explain why no further elevation in nitrotyrosine was found.

An additional observation in our study was an enhancement of LDL oxidation caused by CO exposure. Although this may have some relationship to atherosclerosis, the CO-mediated mechanism for LDL oxidation appears to be different from nitrotyrosine formation, as LDL oxidation was not inhibited by L-NAME treatment. We recently reported that CO exposure can cause several oxidative changes in plasma components, some that are dependent on NO-derived oxidants and others that are independent of NO (50). We conclude that exposure to environmentally relevant concentrations of CO can cause vascular insults by more than one mechanism.


    ACKNOWLEDGEMENTS

We are grateful to Dr. J. S. Beckman, University of Alabama at Birmingham, for providing the anti-nitrotyrosine antibody and to Dr. Henry Shuman and Kathleen Notarfrancesco for advice and assistance with the immunohistology.


    FOOTNOTES

This work was supported by National Institutes of Health Grants ES-05211 to S. R. Thom and HL-54926 to H. Ischiropoulos and a grant from the Council for Tobacco Research to S. R. Thom. H. Ischiropoulos is an Established Investigator of the American Heart Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. R. Thom, Inst. for Environmental Medicine, Univ. of Pennsylvania, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068.

Received 18 May 1998; accepted in final form 16 November 1998.


    REFERENCES
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 276(3):H984-H992
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