Hydrogen peroxide production by alveolar macrophages is increased and its concentration is elevated in the breath of rats exposed to hypoxia: relationship to lung lipid peroxidation

Hypoxic exposure triggers a generation of reactive oxygen species that initiate free radical damage to the lung. Hydrogen peroxide is the product of alveolar macrophages detectable in the expired breath. We evaluated the significance of breath H(2)O(2) concentration for the assessment of lung damage after hypoxic exposure and during posthypoxic period. Adult male rats were exposed to normobaric hypoxia (10 % O(2)) for 3 hours or 5 days. Immediately after the hypoxic exposure and then after 7 days or 14 days of air breathing, H(2)O(2) was determined in the breath condensate and in isolated lung macrophages. Lipid peroxidation was measured in lung homogenates. Three-hour hypoxia did not cause immediate increase in the breath H(2)O(2); 5-day hypoxia increased breath H(2)O(2) level to 458 %. After 7 days of subsequent air breathing H2O2 was elevated in both groups exposed to hypoxia. Increased production of H(2)O(2) by macrophages was observed after 5 days of hypoxia and during the 7 days of subsequent air breathing. Lipid peroxidation increased in the periods of enhanced H(2)O(2) generation by macrophages. As the major increase (1040 %) in the breath H(2)O(2) concentration found 7 days after 3 hours of hypoxia was not accompanied by lipid peroxidation, it can be concluded that the breath H(2)O(2) is not a reliable indicator of lung oxidative damage.     

Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate-ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts

The effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate-ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50-500 microM H2O2 for 20 min or 100 microM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250-500 microM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50-500 microM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant-related DNA damage is a function of the type of oxidative stress used and may be cell-specific.     

Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.     

Superoxide radical production in response to environmental hypoxia in cultured shrimp

Markers of oxidative stress in response to hypoxia and reoxygenation were assessed in Pacific white shrimp (Litopenaeus vannamei). Adult shrimp were either exposed to hypoxia (1 mg O(2)/L) for 6, 12, or 24 h followed by 1-h reoxygenation, or exposed to hypoxia for 24 h followed by 1- to 6-h reoxygenation. In all cases, shrimp maintained at constant normoxia were used as controls. Spectrophotometric techniques were applied to analyze lactate concentration, superoxide radical (O(2)(*-)) production, lipid peroxidation (TBARS), and antioxidant capacity status in muscle, hepatopancreas, and gill samples. Results indicate differences among tissues, even under control conditions. O(2)(*-) production and TBARS levels were higher in hepatopancreas than in gill or muscle. No effect of exposure to hypoxia was found. However, reoxygenation following exposure to hypoxia was found to affect the oxidative metabolism of muscle and hepatopancreas from cultured shrimp. Lactate concentration and O(2)(*-) production increased while antioxidant capacity decreased in hepatopancreas and muscle in the first hours of reoxygenation. This could translate into tissue damage, which may significantly jeopardize the commercial aquaculture product.     

Maturation of baseline breathing and of hypercapnic and hypoxic ventilatory responses in newborn mice

Breathing during the first postnatal hours has not been examined in mice, the preferred mammalian species for genetic studies. We used whole body plethysmography to measure ventilation (VE), breath duration (T(TOT)), and tidal volume (VT) in mice delivered vaginally (VD) or by cesarean section (CS). In experiment 1, 101 VD and 100 CS pups aged 1, 6, 12, 24, or 48 h were exposed to 8% CO2 or 10% O2 for 90 s. In experiment 2, 31 VD pups aged 1, 12, or 24 h were exposed to 10% O2 for 5 min. Baseline breathing maturation was delayed in CS pups, but VE responses to hypercapnia and hypoxia were not significantly different between VD and CS pups [at postnatal age of 1 h (H1): 48 +/- 44 and 18 +/- 32%, respectively, in VD and CS pups combined]. The VE increase induced by hypoxia was greater at H12 (46 +/- 27%) because of T(TOT) response maturation. At all ages, hypoxic decline was ascribable mainly to a VT decrease, and posthypoxic decline was ascribable to a T(TOT) increase with apneas, suggesting different underlying neuronal mechanisms.     

Hypoxia/reoxygenation stimulates Ca2+-dependent ICAM-1 mRNA expression in human aortic endothelial cells

Increased endothelial ICAM-1 expression is found in normal aging and in atherosclerosis and is related to the chronic effects of oxidative stress. We examined the Ca(2+)-dependence of ICAM-1 mRNA expression in human aortic endothelial cells (HAEC) exposed to hypoxia/reoxygenation (H/R) as a model of oxidative stress. HAEC were exposed to glucose-free hypoxia (95% N(2)/5% CO(2)) for 60 min and were then reoxygenated (21% O(2)/5% CO(2)) and observed for up to 6h. Reactive oxygen species (ROS) generation was measured by dichlorofluorescein fluorescence and ICAM-1 mRNA was assessed by Northern blot. Upon reoxygenation after hypoxia, ROS production occurred in HAEC and was inhibited by diphenyleneiodonium and by polyethylene glycol-catalase, suggesting the involvement of NADPH oxidase-derived hydrogen peroxide. Hypoxia alone did not increase either ROS production or ICAM-1 mRNA levels, but a 2.5-fold increase in ICAM-1 mRNA was noted by 30 min of reoxygenation. This was not observed in Ca(2+)-free buffer or in cells treated with diphenyleneiodonium. Thus, H/R upregulates ICAM-1 mRNA in HAEC by a Ca(2+)- and ROS-dependent mechanism. Characterizing the signaling pathways involved in H/R-induced adhesion molecule expression may result in a better understanding of the vascular biology of normal aging and the pathobiology of atherosclerosis.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

Endothelin receptor blockade decreases lung water in young rats exposed to viral infection and hypoxia

Viral respiratory infections may increase the susceptibility of young animals to hypoxia-induced pulmonary edema. Because hypoxia stimulates endothelin production, we hypothesized that an increase in lung endothelin contributes to these alterations in lung water. Weanling rats were infected with Sendai virus, causing a mild respiratory infection. At day 7 after infection, animals were exposed to hypoxia (inspired O(2) fraction = 0.1) for 24 h. Exposure to virus plus hypoxia led to increases in lung water compared with control groups (P < 0.001). Lung endothelin levels were significantly higher in the virus plus hypoxia group than in control groups (P < 0.001). A second group of infected animals received bosentan, a nonselective endothelin receptor antagonist, during exposure to hypoxia. Bosentan-treated animals showed less lung water accumulation, less lung lavage fluid protein, and less perivascular fluid cuffing than untreated animals (P < 0.01). We conclude that the combination of a recent viral respiratory infection and exposure to moderate hypoxia led to increases in endothelin in the lungs of young rats and that endothelin receptor blockade ameliorates the hypoxia-induced increases in lung water found in these animals.     

白藜芦醇减轻神经元氧化应激损伤的作用机制研究

目的:观察白藜芦醇(RSV)对过氧化氢(H2O2)所致的海马神经元HT22细胞损伤的保护作用,并探讨超氧化物歧化酶2(Mn-SOD)在其中的作用。方法:采用体外培养HT22小鼠海马神经元细胞系,H2O2作为损伤因素模拟氧化应激损伤。将细胞分为5组,分别为正常培养组(Control)、150μM H2O2损伤组(H2O2)、25μM白藜芦醇保护组(RSV+H2O2)、SOD2-si RNA干扰组(SOD2-si RNA+RSV+H2O2)和乱序RNA组(SC-si RNA+RSV+H2O2),药物暴露24 h后,应用MTT法检测HT22细胞活力、比色法检测乳酸脱氢酶(Lactate Dehydrogenase,LDH)释放量、相差显微镜观测细胞形态。结果:与对照组相比,H2O2组的活力显著下降(P0.05),LDH释放量明显增加(P0.05),细胞形态明显破坏;25μM的RSV显著恢复了HT22细胞的活力、减少了LDH释放、改善了细胞形态,而SOD2-si RNA显著逆转了RSV引起的上述保护作用,乱序RNA(SC-si RNA)未对上述保护作用产生明显影响。结论:白藜芦醇可能通过上调SOD2减轻H2O2对HT22细胞的氧化应激损伤。     

自噬抑制剂3-MA上调H2O2损伤的PC12细胞氧化应激水平

为探究自噬抑制剂6-氨基-3-甲基腺嘌呤（3-methyladenine,3-MA）对损伤细胞氧化应激水平的影响,将3-MA作用于H2O2诱导的PC12细胞损伤模型,以自噬增强剂雷帕霉素（rapamycin,Rap）作为对照,探讨自噬与氧化应激的关系。测定线粒体的膜电位和细胞内的活性氧（reactive oxygen species, ROS）与丙二醛(malondialdehyde, MDA)含量,以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性,评价损伤细胞的氧化应激状态。单丹(磺)酰戊二胺（monodansylcadaverine,MDC）染色,观察损伤细胞的自噬情况。蛋白质印迹分析损伤细胞中的自噬相关蛋白质LC3-II/LC3-I比值变化。实验结果显示：与正常组相比,H2O2损伤细胞的ROS水平上升到正常组的141%,MDA含量增加(P<0.001);CAT与SOD酶活力显著降低(P<0.001),差异均有统计学意义,证明损伤细胞氧化应激水平增加;MDC染色结果表明,H2O2组自噬明显增加。Western印迹结果表明,LC3-II/LC3-I值显著升高（P<0.05）;与损伤组相比,3-MA组MDC染色结果表明,自噬水平降低。Western印迹结果表明,LC3-II/LC3-I值下降;细胞内ROS水平升高,增加到正常组的208%。MDA含量增加(P<0.001),CAT、SOD酶活力降低(P<0.001)。综上结果表明,自噬抑制剂可增加H2O2诱导的PC12细胞损伤模型的氧化应激水平,增加细胞凋亡。     

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: similarities in natural and artificial systems

The basidiomycete Moniliophthora perniciosa causes Witches' Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H(2)O(2), paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H(2)O(2) when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.     

Hypoxia-mediated prior induction of monocyte-expressed HSP72 and HSP32 provides protection to the disturbances to redox balance associated with human sub-maximal aerobic exercise

HSP72 is rapidly expressed in response to a variety of stressors in vitro and in vivo (including hypoxia). This project sought a hypoxic stimulus to elicit increases in HSP72 and HSP32 in attempts to confer protection to the sub-maximal aerobic exercise-induced disturbances to redox balance. Eight healthy recreationally active male subjects were exposed to five consecutive days of once-daily hypoxia (2,980?m, 75?min). Seven days prior to the hypoxic acclimation period, subjects performed 60?min of cycling on a cycle ergometer (exercise bout 1—EXB1), and this exercise bout was repeated 1?day post-cessation of the hypoxic period (exercise bout 2—EXB2). Blood samples were taken immediately pre- and post-exercise and 1, 4 and 8?h post-exercise for HSP72 and immediately pre, post and 1?h post-exercise for HSP32, TBARS and glutathione [reduced (GSH), oxidised (GSSG) and total (TGSH)], with additional blood samples obtained immediately pre-day 1 and post-day 5 of the hypoxic acclimation period for the same indices. Monocyte-expressed HSP32 and HSP72 were analysed by flow cytometry, with measures of oxidative stress accessed by commercially available kits. There were significant increases in HSP72 (P?<?0.001), HSP32 (P?=?0.03), GSSG (t?=?9.5, P?<?0.001) and TBARS (t?=?5.6, P?=?0.001) in response to the 5-day hypoxic intervention, whereas no significant changes were observed for GSH (P?=?0.22) and TGSH (P?=?0.25). Exercise-induced significant increases in HSP72 (P?<?0.001) and HSP32 (P?=?0.003) post-exercise in EXB1; this response was absent for HSP72 (P?≥?0.79) and HSP32 (P?≥?0.99) post-EXB2. The hypoxia-mediated increased bio-available HSP32 and HSP72 and favourable alterations in glutathione redox, prior to exercise commencing in EXB2 compared to EXB1, may acquiesce the disturbances to redox balance encountered during the second physiologically identical exercise bout.     

Effects of five consecutive nocturnal hypoxic exposures on the cerebrovascular responses to acute hypoxia and hypercapnia in humans.

The effects of discontinuous hypoxia on cerebrovascular regulation in humans are unknown. We hypothesized that five nocturnal hypoxic exposures (8 h/day) at a simulated altitude of 4,300 m (inspired O2 fraction = approximately 13.8%) would elicit cerebrovascular responses that are similar to those that have been reported during chronic altitude exposures. Twelve male subjects (26.6 +/- 4.1 yr, mean +/- SD) volunteered for this study. The technique of end-tidal forcing was used to examine cerebral blood flow (CBF) and regional cerebral O2 saturation (Sr(O2)) responses to acute variations in O2 and CO2 twice before, immediately after, and 5 days after the overnight hypoxic exposures. Transcranial Doppler ultrasound was used to assess CBF, and near-infrared spectroscopy was used to assess Sr(O2). Throughout the nocturnal hypoxic exposures, end-tidal Pco2 decreased (P < 0.001) whereas arterial O2 saturation increased (P < 0.001) compared with overnight normoxic control measurements. Symptoms associated with altitude illness were significantly greater than control values on the first night (P < 0.001) and second night (P < 0.01) of nocturnal hypoxia. Immediately after the nocturnal hypoxic intervention, the sensitivity of CBF to acute variations in O2 and CO2 increased 116% (P < 0.01) and 33% (P < 0.05), respectively, compared with control values. Sr(O2) was highly correlated with arterial O2 saturation (R2 = 0.94 +/- 0.04). These results show that discontinuous hypoxia elicits increases in the sensitivity of CBF to acute variations in O2 and CO2, which are similar to those observed during chronic hypoxia.     

Respiratory adaptation to chronic hypoxia in newborn rats

Newborn rats were maintained in an hypoxic chamber (10% O2 in N2) from the day of birth up to 2 wk of postnatal life. Body weight (BW) and nose-tail length were less in the hypoxic exposed (H) rats than in control (C) animals growing in air. Hematocrit rose from about 37% to about 51%. Oxygen consumption (VO2), measured with a manometric method, was lower in H than in C rats; the difference remained at 5-7 days even after normalization by BW. At 5-7 days ventilation, measured with an airflow plethysmograph, was much more elevated in H rats (whether breathing 10% O2 or air) than in C rats, with an increase in both tidal volume and frequency. This indicates that the biphasic ventilatory response, characterized by an initial rise and then a fall of ventilation toward normoxic values, commonly observed in newborns during acute hypoxic challenge is an immediate but only transient response. The dry lung weight-to-BW ratio and alveolar size were larger in H than in C rats. Lung volumes at 20 cmH2O were similar, despite the smaller BW of the H rats. Hence, in the rat, chronic hypoxia in the immediate postnatal period increases O2-carrying capacity, decreases metabolic demands, increases alveolar O2 availability, and promotes structural changes in the lung that protect the gas exchange area and optimize the structure-function relationship of the lung. These results may also suggest that the lung structural alterations with chronic hypoxia should not be attributed to changes in VO2 but, eventually, to the ventilatory action of the organ.     

Oxidative phosphorylation of liver mitochondria from mice acclimatized to hypobaric hypoxia

Mice exposed to intermittent hypobaric hypoxia for 20 hours a day, 6 days a week, develop extracellular adaptive responses similar to those found in humans exposed to oxygen tension equivalent to that found at an altitude of 4500 m. Isolated liver mitochondria from these animals show no significant differences in rates of substrate-stimulated respiration, ADP-stimulated respiration and the respiratory control ratio (RCR), when compared with sea level controls. Undetectable or negligible differences in these parameters are also noted when sea level animals are exposed for one hour to severe hypoxia (7% O₂). We therefore conclude that the oxidative phosphorylation capacity of the isolated mouse liver mitochondria remains unaltered in both acute and chronic hypoxia. However thein vivo oxygen consumption by mice at this degree of hypoxia was markedly reduced. Lack of observable changes in oxidative phosphorylation could be accounted for by extracellular adaptations in mitochondria isolated from acclimatized animals. This explanation, however, is not consistent with the lack of changes on oxidative phosphorylation in mitochondria isolated from mice undergoing acute hypoxia at sea level. It is then suggested that isolated mitochondrial preparations are of limited value for investigating biochemical mechanisms underlying the variation of cellular respiration occurringin vivo.     

Effect of two paradigms of chronic intermittent hypoxia on carotid body sensory activity.

Reflexes arising from the carotid bodies may play an important role in cardiorespiratory changes evoked by chronic intermittent hypoxia (CIH). In the present study, we examined whether CIH affects the hypoxic sensing ability of the carotid bodies and, if so, by what mechanisms. Experiments were performed on adult male rats (Sprague-Dawley, 250-300 g) exposed to two paradigms of CIH for 10 days: 1) multiple exposures to short durations of intermittent hypoxia per day (SDIH; 15 s of 5% O(2) + 5 min of 21% O(2), 9 episodes/h, 8 h/day) and 2) single exposure to longer durations of intermittent hypoxia per day [LDIH; 4 h of hypobaric hypoxia (0.4 atm/day) + 20 h of normoxia]. Carotid body sensory response to graded isocapnic hypoxia was examined in both groups of animals under anesthetized conditions. Hypoxic sensory response was significantly enhanced in SDIH but not in LDIH animals. Similar enhancement in hypoxic sensory response was also elicited in ex vivo carotid bodies from SDIH animals, suggesting that the effects were not secondary to cardiovascular changes. SDIH, however, had no significant effect on the hypercapnic sensory response. The effects of SDIH on the hypoxic sensory response completely reversed after SDIH animals were placed in a normoxic environment for an additional 10 days. Previous treatment with systemic administration of O(2)(-)* radical scavenger prevented SDIH-induced augmentation of the hypoxic sensory response. These results demonstrate that SDIH but not LDIH results in selective augmentation of the hypoxic response of the carotid body and O(2)(-)* radicals play an important role in SDIH-induced sensitization of the carotid body.     

Oxidative stress response of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> to hydrogen peroxide,paraquat and pressure

The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor. The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated. The exposure for 1 h of K. marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content. SOD induction by the chemical oxidants was independent of the air pressure values used. A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied. In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa. As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels. This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure. The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure. This behaviour was even more patent with 400 kPa. However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.     

Respiratory mechanics in adult rats hypoxic in the neonatal period

Newborn rats were exposed to 10% O2 from 24 h to 6 days after birth, then returned to normoxia and examined at 50 days of age, i.e., after reaching sexual maturity. Despite the important impairment in somatic growth during hypoxia, at 50 days body weight and nose-tail length were as in control rats never exposed to hypoxia. Hypoxic rats had a bigger chest, with larger anteroposterior diameter, larger surface area of the muscle component of the diaphragm, and heavier and more expanded lungs. None of these structural changes were observed in a third group of rats, which were exposed for 6 days to hypoxia between 35 and 42 days of age, i.e., at a much more advanced stage of postnatal development. In addition, hypoxic rats had higher compliance of the respiratory system and of the lung and lower total pulmonary resistance than control rats. Frequency dependence of compliance was not different. We conclude that in the rat the structural changes induced by neonatal chronic hypoxia are not resolved by the return to normoxia but persist at least until postpuberty with modifications of the mechanical properties of the respiratory system.