Regulation of cellular gas exchange, oxygen sensing, and metabolic control.

Cells must continuously monitor and couple their metabolic requirements for ATP utilization with their ability to take up O2 for mitochondrial respiration. When O2 uptake and delivery move out of homeostasis, cells have elaborate and diverse sensing and response systems to compensate. In this review, we explore the biophysics of O2 and gas diffusion in the cell, how intracellular O2 is regulated, how intracellular O2 levels are sensed and how sensing systems impact mitochondrial respiration and shifts in metabolic pathways. Particular attention is paid to how O2 affects the redox state of the cell, as well as the NO, H2S, and CO concentrations. We also explore how these agents can affect various aspects of gas exchange and activate acute signaling pathways that promote survival. Two kinds of challenges to gas exchange are also discussed in detail: when insufficient O2 is available for respiration (hypoxia) and when metabolic requirements test the limits of gas exchange (exercising skeletal muscle). This review also focuses on responses to acute hypoxia in the context of the original "unifying theory of hypoxia tolerance" as expressed by Hochachka and colleagues. It includes discourse on the regulation of mitochondrial electron transport, metabolic suppression, shifts in metabolic pathways, and recruitment of cell survival pathways preventing collapse of membrane potential and nuclear apoptosis. Regarding exercise, the issues discussed relate to the O2 sensitivity of metabolic rate, O2 kinetics in exercise, and influences of available O2 on glycolysis and lactate production.

Hyperthermia induces injury to the intestinal mucosa in the mouse: evidence for an oxidative stress mechanism.

Loss of the intestinal barrier is critical to the clinical course of heat illness, but the underlying mechanisms are still poorly understood. We tested the hypothesis that conditions characteristic of mild heatstroke in mice are associated with injury to the epithelial lining of the intestinal tract and comprise a critical component of barrier dysfunction. Anesthetized mice were gavaged with 4 kDa FITC-dextran (FD-4) and exposed to increasing core temperatures, briefly reaching 42.4°C, followed by 30 min recovery. Arterial samples were collected to measure FD-4 concentration in plasma (in vivo gastrointestinal permeability). The small intestines were then removed to measure histological evidence of injury. Hyperthermia resulted in a ≈2.5-fold elevation in plasma FD-4 and was always associated with significant histological evidence of injury to the epithelial lining compared with matched controls, particularly in the duodenum. When isolated intestinal segments from control animals were exposed to ≥41.5°C, marked increases in permeability were observed within 60 min. These changes were associated with release of lactate dehydrogenase, evidence of protein oxidation via carbonyl formation and histological damage. Coincubation with N-acetylcysteine protected in vitro permeability during hyperthermia and reduced histological damage and protein oxidation. Chelation of intracellular Ca(2+) to block tight junction opening during 41.5°C exposure failed to reduce the permeability of in vitro segments. The results demonstrate that hyperthermia exposure in mouse intestine, at temperatures at or below those necessary to induce mild heatstroke, cause rapid and substantial injury to the intestinal lining that may be attributed, in part, to oxidative stress.

Superoxide scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm.

Acute exposure to severe hypoxia depresses contractile function and induces adaptations in skeletal muscle that are only partially understood. Previous studies have demonstrated that antioxidants (AOXs) given during hypoxia partially protect contractile function, but this has not been a universal finding. This study confirms that specific AOXs, known to act primarily as superoxide scavengers, protect contractile function in severe hypoxia. Furthermore, the hypothesis is tested that the mechanism of protection involves preservation of high-energy phosphates (ATP, creatine phosphate) and reductions of P(i). Rat diaphragm muscle strips were treated with AOXs and subjected to 30 min of hypoxia. Contractile function was examined by using twitch and tetanic stimulations and the degree of elevation in passive force occurring during hypoxia (contracture). High-energy phosphates were measured at the end of 30-min hypoxia exposure. Treatment with the superoxide scavengers 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron, 10 mM) or Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (50 microM) suppressed contracture during hypoxia and protected maximum tetanic force. N-acetylcysteine (10 or 18 mM) had no influence on tetanic force production. Contracture during hypoxia without AOXs was also shown to be dependent on the extracellular Ca(2+) concentration. Although hypoxia resulted in only small reductions in ATP concentration, creatine phosphate concentration was decreased to approximately 10% of control. There were no consistent influences of the AOX treatments on high-energy phosphates during hypoxia. The results demonstrate that superoxide scavengers can protect contractile function and reduce contracture in hypoxia through a mechanism that does not involve preservation of high-energy phosphates.

Invited review: Adaptive responses of skeletal muscle to intermittent hypoxia: the known and the unknown.

Intermittent hypoxia (IH) describes conditions of repeated, transient reductions in O2 that may trigger unique adaptations. Rest periods during IH may avoid potentially detrimental effects of long-term O2 deprivation. For skeletal muscle, IH can occur in conditions of obstructive sleep apnea, transient altitude exposures (with or without exercise), intermittent claudication, cardiopulmonary resuscitation, neonatal blood flow obstruction, and diving responses of marine animals. Although it is likely that adaptations in these conditions vary, some patterns emerge. Low levels of hypoxia shift metabolic enzyme activity toward greater aerobic poise; extreme hypoxia shifts metabolism toward greater anaerobic potential. Some conditions of IH may also inhibit lactate release during exercise. Many related cellular phenomena could be involved in the response, including activation of specific O2 sensors, reactive oxygen and nitrogen species, preconditioning, hypoxia-induced transcription factors, regulation of ion channels, and influences of paracrine/hormonal stimuli. The net effect of a variety of adaptive programs to IH may be to preserve contractile function and cell integrity in hypoxia or anoxia, a response that does not always translate into improvements in exercise performance.

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia.

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20-36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress (P < 0.005), twitch half-relaxation time (P < 0.02), and tetanic stress at 20 or 30 Hz (P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals (P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.

NMR spin trapping: detection of free radical reactions with a new fluorinated DMPO analog.

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.

Unique in vivo applications of spin traps.

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.

Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle.

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

Surfactant treatment impairs gas exchange in a canine model of acute lung injury.

UNLABELLED: The effectiveness of surfactant (SURF) treatment in acute lung injury in the adult is controversial. In this study, we tested the effectiveness of early surfactant treatment in a commonly used animal model of acute lung injury, phorbol-myristate acetate (PMA), to see if it would attenuate the progression of lung injury. We measured the effect on lung compliance and whether positive end-expiratory pressure (PEEP) (10 cm H2O) during SURF administration had a synergistic effect. METHODS: Four groups of anesthetized dogs were studied: a) normals; b) PMA injury only; c) PMA injury + SURF; and d) PMA + SURF + PEEP. Lung injury was induced with 25-30 microg/kg of PMA. Responses were measured over 7 hrs. Surfactant was administered in the form of Survanta, 4 x 25 mg/kg doses via tracheal instillation 2.5 hrs after PMA. For the group receiving PEEP, 10 cm H2O PEEP was begun 1.5 hrs after PMA, 1 hr before SURF. Postmortem, the left lung was excised and inflated three times to total lung capacity (volume at 30 cm H2O) and expiratory compliance was measured with 25-100 mL volume increments. The trachea was then clamped and trapped volume was determined by water displacement. RESULTS: PMA-induced lung injury significantly reduced expiratory compliance and total lung capacity (p < .05 from normal). Wet/dry lung weights did not differ between groups. SURF without PEEP further decreased lung compliance as compared with PMA only. CONCLUSIONS: SURF administration after PMA injury causes marked reductions in lung compliance when no PEEP is coadministered. However, the loss of static expiratory lung compliance appears partly ameliorated by application of PEEP + SURF. Given that tracheal instillation of SURF is known to acutely elevate lung impedance in the first few hours after administration, coadministration of PEEP appears to be critically important in counteracting these early effects of surfactant instillation on the lung.

Increased susceptibility to pulmonary emphysema among HIV-seropositive smokers.

BACKGROUND: Previous uncontrolled reports have suggested that HIV-seropositive persons develop an accelerated form of emphysema. OBJECTIVE: To characterize the risk for emphysema in a stable HIV-seropositive outpatient population. DESIGN: Controlled, cross-sectional analysis. SETTING: Midwestern urban community. PARTICIPANTS: HIV-seropositive persons (n = 114) without AIDS-related pulmonary complications and HIV-seronegative controls (n = 44), matched for age and smoking history. MEASUREMENTS: Measurement of pulmonary function, bronchoalveolar lavage, and high-resolution computed tomography of the chest. RESULTS: The incidence of emphysema was 15% (17 of 114) in the HIV-seropositive group compared with 2% (1 of 44) in the HIV-seronegative group (P = 0.025). The incidence of emphysema in participants with a smoking history of 12 pack-years or greater was 37% (14 of 38 persons) in the HIV-seropositive group compared with 0% (0 of 14 persons) in the HIV-seronegative group (P = 0.011). The percentage of cytotoxic lymphocytes in lavage fluid was much higher in HIV-seropositive smokers with emphysema. CONCLUSIONS: Infection with HIV accelerates the onset of smoking-induced emphysema. The results of this study support the emerging concept that cytotoxic lymphocytes may have an important role in emphysema pathogenesis.

Oxidants and skeletal muscle function: physiologic and pathophysiologic implications.

Previous studies have demonstrated that skeletal muscles generate considerable reactive oxygen during intense muscle contraction. However, the significance of this phenomenon and whether it represents normal physiology or pathology are poorly understood. Treatment with exogenous antioxidants suggests that normal redox tone during contraction is influencing ongoing contractile function, both at rest and during intense exercise. This could represent the influence of redox-sensitive proteins responsible for excitation-contraction coupling or redox-sensitive metabolic enzymes. Some conditions associated with intense exercise, such as local tissue hypoxia or elevated tissue temperatures, could also contribute to reactive oxygen production. Evidence that muscle conditioning results in upregulation of antioxidant defenses also suggests a close relationship between reactive oxygen and contractile activity. Therefore, there appears to be a significant role for reactive oxygen in normal muscle physiology. However, a number of conditions may lead to an imbalance of oxidant production and antioxidant defense, and these, presumably, do create conditions of oxidant stress. Ischemia-reperfusion, severe hypoxia, severe heat stress, septic shock, and stretch-induced injury may all lead to oxidant-mediated injury to myocytes, resulting in mechanical dysfunction.

Focal air trapping in patients with HIV infection: CT evaluation and correlation with pulmonary function test results.

OBJECTIVE: HIV-positive individuals commonly have symptoms of airway disease. We evaluated thin-section CT scans of HIV-infected individuals during inspiration and expiration for evidence of focal air trapping. We also correlated imaging findings with pulmonary function test results. SUBJECTS AND METHODS: Fifty-nine subjects, 48 of whom were HIV-positive and 11 of whom were HIV-negative, underwent thin-section CT of the thorax during inspiration and expiration. All subjects also underwent pulmonary function tests. Two radiologists, who were unaware of the subjects' HIV status and smoking history and of the results of pulmonary function tests, evaluated the CT scans for the presence and severity of focal air trapping. RESULTS: Expiratory CT revealed focal air trapping in 33 subjects: 30 were HIV-positive and three were HIV-negative (p = .0338). The mean values of forced expiratory volume in 1 sec (FEV1), forced mid expiratory flow, and diffusion capacity (DL(CO)) were significantly lower for subjects with focal air trapping (mean = 88.85, 84.52, and 80.80, respectively) than for those with normal findings on CT (mean = 100.84, 99.24, and 95.82, respectively; p = .001, p = .021, and p = .003, respectively). We found no significant differences in smoking history between HIV-positive and HIV-negative subjects. Severe air trapping on expiratory CT scans was seen in three subjects: All three had HIV infection, low CD4 counts, and abnormally decreased FEV1 and DL(CO) values. CONCLUSION: Focal air trapping was a common finding on thoracic CT scans obtained during expiration in HIV-positive subjects. In addition, focal air trapping was associated with significantly lower FEV1, forced mid expiratory flow, and DL(CO) values than those found for subjects in whom CT revealed no focal air trapping. These results suggest that small airways disease may accompany a decline in pulmonary function in HIV-positive individuals.

NMR spin trapping: detection of free radical reactions using a phosphorus-containing nitrone spin trap.

This study employs (31)P-nuclear magnetic resonance (NMR) to probe for changes in molecular structure arising from reactions between free radicals and a phosphorus-containing nitrone spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). A number of biologically relevant free radical reactions were detected: a) reactions of DEPMPO with ( small middle dot)OH resulted in a new (31)P-NMR resonance at 27.05 ppm (shifted from the parent compound at 23.67 ppm); evidence suggests that this species is a diamagnetic hydroxy-pyrrolidone reduction product; b) (31)P-NMR spectra of DEPMPO/( small middle dot)CH(3) reactions resulted in peaks at 24.54, 30.83, and 32.31 ppm, while DEPMPO/( small middle dot)CH(2)OH produced peaks at 24.05, 30.80 and 32.52 ppm; in the presence of excess ascorbate, only resonances between 30 and 32 ppm were evident, which we have tentatively assigned to the hydroxylamine isomers of their respective adducts; and c) reaction of DEPMPO with O(2)( small middle dot-), produced by xanthine/xanthine oxidase or stimulated neutrophils, resulted in a single line, indistinguishable from DEPMPO/( small middle dot)OH reaction products. We conclude that NMR spin trapping is a useful approach for detecting free radical reaction pathways. It may have future applications for human free radical biology and imaging. Magn Reson Med 42:228-234, 1999.

The pathophysiology of pulmonary diffusion impairment in human immunodeficiency virus infection.

Numerous reports have demonstrated that prior to the development of acquired immunodeficiency syndrome (AIDS)-related pulmonary complications, human immunodeficiency virus-positive (HIV+) individuals commonly develop unexplained reductions in pulmonary diffusing capacity (DLCO). The potential relevance of this observation is underscored by recent data demonstrating that reductions in DLCO independently predict the subsequent development of opportunistic pneumonia. To delineate the alterations in gas exchange associated with HIV, we investigated a group of HIV+ subjects with unexplained reductions in DLCO, using high-resolution computed tomography (HRCT) of the chest and a separation of diffusing capacity into its membrane (Dm) and capillary blood volume (Vc) components. We compared this abnormal group with HIV+ subjects with more normal gas exchange and also with a group of HIV- volunteers matched for age and smoking history. Compared with other groups, the HIV+ group with diffusion impairment demonstrated prominent reductions in Vc, despite a well-preserved total lung capacity (TLC). HRCT demonstrated virtually no evidence of interstitial fibrosis in any HIV+ subject, but evidence of early emphysema that significantly correlated with DLCO. Our results suggest that the previously reported impairment in pulmonary gas exchange in the HIV+ population involves loss of Vc and likely represents the development of early emphysema.

Cigarette smoking in HIV infection induces a suppressive inflammatory environment in the lung.

Lung lymphocyte numbers are frequently increased in human immunodeficiency virus (HIV)-infected individuals in the absence of lung infection, and may play a critical role in viral surveillance and protection against new infections. In this context, cigarette smoking by HIV-infected individuals has been associated with a relative increase in the peripheral blood CD4(+) T-lymphocyte count as compared with that of nonsmokers. Because lung defense is local, the aim of the present study was to determine whether cigarette smoking had a significant impact on local lung defenses in HIV-infected individuals. The numbers and subtypes of bronchoalveolar lymphocytes and the ability of lung lavage cells to produce proinflammatory cytokines were compared in 58 smokers and 34 nonsmokers. In contrast to a trend toward an increase in peripheral blood CD4(+) cell counts among nonsmokers, smokers had significant depressions in both the percentage and absolute numbers of CD4(+) and CD8(+) cells in their bronchoalveolar lavage fluid (BALF). A decrease in CD4(+)/CD8(+) cell ratios was also seen with smoking. In addition, production of both interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was suppressed with cigarette smoking. These observations show that cigarette smoking is associated with suppression in localized lung defenses, and suggest that smoking cessation may have a positive impact on lung defenses in HIV-infected smokers.

Antioxidants protect rat diaphragmatic muscle function under hypoxic conditions.

In hypoxia, mitochondrial respiration is decreased, thereby leading to a buildup of reducing equivalents that cannot be transferred to O2 at the cytochrome oxidase. This condition, called reductive stress, can paradoxically lead to enhanced formation of reactive O2 species, or a decrease in the ability of the cell to defend against an oxidative stress. We hypothesized that antioxidants would protect tissues under conditions of hypoxia. Rat diaphragm strips were incubated in tissue baths containing one of four antioxidants: N-acetyl-L-cysteine, dimethyl sulfoxide, superoxide dismutase, or Tiron. The strips were directly stimulated in an electrical field. Force-frequency relationships were studied under baseline oxygenation (95% O2-5% CO2), after 30 min of hypoxia (95% N2-5% CO2), and 30 min after reoxygenation. In all tissues, antioxidants markedly attenuated the loss of contractile function during hypoxia (P < 0.01) and also significantly improved recovery on reoxygenation (P < 0.05). We conclude that both intracellular and extracellular antioxidants improve skeletal muscle contractile function in hypoxia and facilitate recovery during reoxygenation in an in vitro system. The strong influence of antioxidants during hypoxic exposure suggests that they can be as effective in protecting cell function in a reducing environment as they have been in oxidizing environments.

Dithiothreitol improves recovery from in vitro diaphragm fatigue.

There is increasing evidence that reactive oxygen species are produced during strenuous skeletal muscle work and that they contribute to the development of muscle fatigue. Although the precise cellular mechanisms underlying such a phenomenon remain obscure, it has been hypothesized that endogenously produced reactive oxygen species may down-regulate force production during fatigue by oxidizing critical sulfhydryl groups on important contractile proteins. To test this hypothesis, we fatigued rat diaphragm strips in vitro for 4 min at 20 Hz stimulation and a duty cycle of 0.33. Following fatigue, the tissue baths were drained and randomly replaced with either physiologic saline or physiologic saline containing the disulfide reducing agent, dithiothreitol (DTT) at varying doses (0.1-5.0 mM). Force-frequency characteristics were then measured over a 90-min recovery period. At the 0.5 and 1.0 mM doses, DTT treatment was associated with significantly greater force production in the recovery period. DTT's effects were observed at most frequencies tested, but appeared more prominent at the higher frequencies. The beneficial effects of DTT were not evident at the 0.1 or 5.0 mM doses and appeared to be specific for fatigued muscle. These recovery-enhancing effects of a potent disulfide reducing agent suggest that important contractile proteins may be oxidized during fatigue; such changes may be readily reversible.

Biological reactions of peroxynitrite: evidence for an alternative pathway of salicylate hydroxylation.

Salicylate hydroxylation has often been used as an assay of hydroxyl radical production in vivo. We have examined here if hydroxylation of salicylate might also occur by its reaction with peroxynitrite. To test this hypothesis, we exposed salicylate to various concentrations of peroxynitrite, in vitro. We observed the hydroxylation of salicylate at 37 degrees C by peroxynitrite at pH 6, 7 and 7.5, where the primary products had similar retention times on HPLC to 2,3- and 2,5-dihydroxybenzoic acid. The product yields were pH dependent with maximal amounts formed at pH 6. Furthermore, the relative concentration of 2,3- to 2,5-dihydroxybenzoic acid increased with decreasing pH. Nitration of salicylate was also observed and both nitration and hydroxylation reaction products were confirmed independently by mass spectrometry. The spin trap N-t-butyl-alpha-phenylnitrone (PBN), with or without dimethyl sulfoxide (DMSO), was incapable of trapping the peroxynitrite decomposition intermediates. Moreover, free radical adducts of the type PBN/.CH3 and PBN/.OH were susceptible to destruction by peroxynitrite (pH 7, 0.1 M phosphate buffer). These results suggest direct peroxynitrite hydroxylation of salicylate and that the presence of hydroxyl radicals is not a prerequisite for hydroxylation reactions.