Hyperoxic Mice Research Articles

DNA Damage Induced by Hyperoxia

Inspired oxygen, an essential therapy for cardiorespiratory disorders, has the potential to generate reactive oxygen species that damage cellular DNA. Although DNA damage is implicated in diverse pulmonary disorders, including neoplasia and acute lung injury, the type and magnitude of DNA lesion caused by oxygen in vivo is unclear. We used single-cell gel electrophoresis (SCGE) to quantitate two distinct forms of DNA damage, base adduction and disruption of the phosphodiester backbone, in the lungs of mice. Both lesions were induced by oxygen, but a marked difference between the two was found. With 40 h of oxygen exposure, oxidized base adducts increased 3- to 4-fold in the entire population of lung cells. This lesion displayed temporal characteristics (a progressive increase over the first 24 h) consistent with a direct effect of reactive oxygen species attack upon DNA. DNA strand breaks, on the other hand, occurred in < 10% of pulmonary cells, which acquired severe levels of the lesion; dividing cells were preferentially affected. Characteristics of these cells suggested that DNA strand breakage was secondary to cell death, rather than a primary effect of reactive oxygen species attack on DNA. By analysis of IL-6- and IL-11-overexpressing transgenic animals, which are resistant to hyperoxia, we found that DNA strand breaks, but not base damage, correlated with acute lung injury. Analysis of purified alveolar type 2 preparations from hyperoxic mice indicated that strand breaks preferentially affected this cell type.

The role of endothelin-1 in hyperoxia-induced lung injury in mice

BackgroundAs prolonged hyperoxia induces extensive lung tissue damage, we set out to investigate the involvement of endothelin-1 (ET-1) receptors in these adverse changes.MethodsExperiments were performed on four groups of mice: control animals kept in room air and a group of mice exposed to hyperoxia for 60 h were not subjected to ET-1 receptor blockade, whereas the dual ETA/ETB-receptor blocker tezosantan (TEZ) was administered via an intraperitoneal pump (10 mg/kg/day for 6 days) to other groups of normal and hyperoxic mice. The respiratory system impedance (Zrs) was measured by means of forced oscillations in the anesthetized, paralyzed and mechanically ventilated mice before and after the iv injection of ET-1 (2 μg). Changes in the airway resistance (Raw) and in the tissue damping (G) and elastance (H) of a constant-phase tissue compartment were identified from Zrs by model fitting.ResultsThe plasma ET-1 level increased in the mice exposed to hyperoxia (3.3 ± 1.6 pg/ml) relative to those exposed to room air (1.6 ± 0.3 pg/ml, p < 0.05). TEZ administration prevented the hyperoxia-induced increases in G (13.1 ± 1.7 vs. 9.6 ± 0.3 cmH2O/l, p < 0.05) and H (59 ± 9 vs. 41 ± 5 cmH2O/l, p < 0.05) and inhibited the lung responses to ET-1. Hyperoxia decreased the reactivity of the airways to ET-1, whereas it elevated the reactivity of the tissues.ConclusionThese findings substantiate the involvement of the ET-1 receptors in the physiopathogenesis of hyperoxia-induced lung damage. Dual ET-1 receptor antagonism may well be of value in the prevention of hyperoxia-induced parenchymal damage.

DIETARY FLAXSEED SUPPLEMENTATION PROTECTS AGAINST ACUTE LUNG INJURY AND INFLAMMATION IN MICE

Background Flaxseed, a nutritional supplement, has high contents of omega-3 fatty acids and lignans with known anti-inflammatory and anti-oxidant properties. Its use in acute lung disease has never been evaluated. In this study we tested flaxseed (FS) diets in three experimental murine models of acute lung injury (ALI). Methods Mice were fed 10% FS for several weeks to determine kinetics of lignan accumulation in blood using liquid chromatography tandem mass spectrometry. Mice fed control and 10% FS diets were challenged by Hyperoxia (80% O2), intratracheal (i.t.) LPS or acid aspiration (AA). Bronchoalveolar lavage (BAL) was evaluated for white blood cells (WBC), neutrophils (PMN), and proteins 24 hours post i.t. challenge of acid or LPS or 6 days after hyperoxia. Lung lipid peroxidation was determined by malondialdehyde (MDA) tissue levels. Results Plasma FS lignans, enterodiol and enterolactone, peaked in the first week and decreased to steady state by 2–3 weeks. Following hyperoxia, FS mice showed a significant decrease in BAL proteins, a measure of lung edema, and a trend with AA while lung inflammation and MDA levels were significantly decreased in both models. In strong contrast, LPS injury and inflammation was not ameliorated by FS. Remarkably, serum lignan levels decreased dramatically in both AA and hyperoxic mice but not in LPS mice. Conclusion Dietary FS is protective against experimental ALI, inflammation and lung lipid peroxidation in vivo. Funded by: AICR (MCS), NIH-HL64063 (YA), U. Penn Research Foundation (MCS, YA).

Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis.

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.

Neonatal Exposure to 65% Oxygen Durably Impairs Lung Architecture and Breathing Pattern in Adult Mice

To test the hypothesis that exposure to hyperoxia during the postnatal period of rapid alveolar multiplication by septation would cause permanent impairments, even with moderate levels of hyperoxia. We exposed mouse pups to 65% O(2) (hyperoxic mice) or normoxia (normoxic mice) during their first postnatal month, and we analyzed lung histology, pulmonary mechanics, blood gas, and breathing pattern during normoxia or in response to chemical stimuli in adulthood, when they reached 7 to 8 months of postnatal age. Hyperoxic mice had fewer and larger alveoli than normoxic mice (number of alveoli per unit surface area of parenchyma, 266 +/- 62/mm(2) vs 578 +/- 77/mm(2), p < 0.0001) [mean +/- SD], the cause being impaired alveolarization (radial alveolar count, 5.8 +/- 0.2 in hyperoxic mice vs 10.5 +/- 0.5 in normoxic mice, p < 0.0001). Respiratory system compliance was higher in hyperoxic mice (0.098 +/- 0.006 mL/cm H(2)O) than in normoxic mice (0.064 +/- 0.006 mL/cm H(2)O, p < 0.016). Baseline tidal volume (VT) and breath duration (TTOT]) measured noninvasively by whole-body plethysmography were larger in hyperoxic mice than in normoxic mice (VT, + 15%, p < 0.01; TTOT, + 12%, p < 0.01). Despite these impairments, blood gas, baseline minute ventilation E, and E responses to hypoxia and hypercapnia were normal in hyperoxic mice, compared with normoxic mice. Hyperoxic exposure during lung septation in mice may cause irreversible lung injury and breathing pattern abnormalities in adulthood at O(2) concentrations lower than previously thought. However, ventilatory function and body growth were preserved, and ventilatory function showed no major abnormalities, at least at rest, despite early oxygen-induced injuries.

Mitochondrial thiol status in the liver is altered by exposure to hyperoxia

Patients with poorly functioning lungs often require treatment with high concentrations of supplemental oxygen, which, although often necessary to sustain life, can cause lung injury. The mechanisms responsible for hyperoxic lung injury have been investigated intensely and most probably involve oxidant stress responses, but the details are not well understood. In the present studies, we exposed adult male C57/Bl6 mice to >95% O 2 for up to 72 h and obtained lung and liver samples for assessment of lung injury, measurements of tissue concentrations of coenzyme A (CoASH) and the corresponding mixed disulfide with glutathione (CoASSG), as possible biomarkers of intramitochondrial thiol redox status. Subcellular fractions were prepared from both tissues for determination of glutathione reductase (GR) activities. Lung injury in the hyperoxic mice was demonstrated by increases in lung weight to body weight ratios at 48 h and by increases in bronchoalveolar lavage protein concentrations at 72 h. Lung CoASH concentrations declined in the hyperoxic mice, but CoASSG concentrations were not increased nor were CoASH/CoASSG ratios decreased, as would be expected for an oxidant shift in mitochondrial thiol–disulfide status. Interestingly, CoASSG concentrations increased (from 6.72±0.54 to 14.10±1.10 nmol/g of liver in air-breathing controls and 72 h of hyperoxia, respectively, P<0.05), and CoASH/CoASSG ratios decreased in the livers of mice exposed to hyperoxia. Some apparent effects of duration of hyperoxia on GR activities in lung or liver cytosolic, mitochondrial, or nuclear fractions were observed, but the changes were not consistent or progressive. Yields of isolated hepatic nuclear protein were decreased in the hyperoxic mice within 24 h of exposure, and by 72 h of hyperoxia, protein recoveries in purified nuclear fractions had declined from 41.8 to 14.8 mg of protein/g animal body weight. Concentrations of 10-formyltetrahydrafolate dehydrogenase were diminished in hepatic mitochondria of hyperoxic mice. A second protein in hepatic mitochondria of ≈25 kDa showed apparent decreases in thiol content, as determined by fluorescence intensities of monobromobimane derivatives separated by SDS-PAGE. The mechanisms responsible for the observed effects and the possible implications for the adverse effects of hyperoxic therapies are not known and need to be investigated.

Antiinflammatory properties of inducible nitric oxide synthase in acute hyperoxic lung injury.

The objective of this study was to determine whether endogenous nitric oxide (NO), specifically the inducible NO synthase isoform (iNOS: NOS II), reduces or amplifies lung injury in mice breathing at a high oxygen tension. Previous studies have shown that exogenous (inhaled) NO protects against hyperoxia-induced lung injury, and that endogenous NO derived from iNOS inhibits leukocyte recruitment and protects against lung injury induced by lipopolysaccharide. In the present study, hyperoxia (> 98% O(2) for 72 h) induced acute lung injury in both wild-type and iNOS-deficient mice as determined by elevated albumin and lactate dehydrogenase levels in bronchoalveolar lavage fluid (BALF) and by increased extravascular lung water. Lung injury was greater in iNOS-deficient mice than in wild-type mice and was associated with an increased number of polymorphonuclear leukocytes in BALF. iNOS messenger RNA expression levels increased in the lungs of wild-type hyperoxic mice. Nitrotyrosine, a marker of reactive NO species, was expressed in both wild-type and iNOS-deficient mice in hyperoxia, indicating an iNOS-independent pathway for protein nitration. We conclude that iNOS is capable of reducing pulmonary leukocyte accumulation and lung injury. The data indicate that iNOS induction serves as a protective mechanism to minimize the effects of acute exposure to hyperoxia.

Oxidative stress as a potential pathogenic mechanism in an animal model of Duchenne muscular dystrophy

Dystrophin-deficiency results in degeneration of most, but not all, skeletal muscles. The mechanisms responsible for degeneration of limb muscle and sparing of extraocular muscle are not known. To address the notion that muscle pathology may be free radical-mediated, we evaluated antioxidant enzyme activities and lipid peroxidation products (TBARS) content in mdx and control mice. TBARS content and the activities of total superoxide dismutase, selenium dependent glutathione peroxidase, glucose-6-phosphate dehydrogenase and catalase were consistently higher in both affected and spared muscles of mdx mice. These data suggest that oxidative stress may be constitutively present in mdx muscle, but may not be the principal pathogenic mechanism. To further test the hypothesis of oxidative stress involvement in dystrophinopathies, control strain and mdx mice were subjected to chronic hyperoxia. The pattern of antioxidant enzyme activities and TBARS content from hyperoxic control strain mice was similar to that of normoxic mdx mice, suggesting that a similar level of oxidative stress was induced. In conclusion, this study has provided indirect evidence for oxidative stress in dystrophin-deficient muscle.

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.

MICE DEFICIENT IN ICAM-1 EXPRESSION AND WITH REDUCED CD18 EXPRESSION HAVE INCREASES IN HYPEROXIC LUNG INFLAMMATION AND INJURY. • 2337

Treatment with supplemental oxygen is frequently used in premature infants because of abnormal lung function. While frequently essential to sustain life, the treatment may cause lung injury. Recently, there has been renewed interest in lung inflammation in hyperoxic lung injury. Investigators have determined that inhibition of the adhesion molecules, intercellular adhesion molecule-1(ICAM-1), and neutrophil (PMN) β2 integrins with monoclonal antibodies (mab) inhibits lung inflammation and injury in hyperoxic mice. However, mab administration may not be entirely specific so that mabs may have effects other than inhibiting adhesion molecules. We therefore placed mice with absent ICAM-1 expression, and mice with reduced β2-integrin expression in >95% oxygen and measured lung inflammation and injury as a function of hyperoxia-exposure time. In all studies wild-type mice on the same genetic background were used as controls. In the two mutant strains of mice there were increases in lung PMN counts when compared to the wild-type controls. In addition, lung weights were significantly greater in ICAM-1 deficient mice than in C57/BL6 at 72 h of hyperoxia-exposure. When mice were exposed to hyperoxia for 84 hours there were 12/12, 3/6 and 0/6 survivors in C57/BL6, ICAM-1 and β2 integrin mice respectively (P<0.05). In conclusion, mice with defects in ICAM-1 and β2-integrin expression are more susceptible to hyperoxic lung injury and have increased lung inflammation than their wild type counterparts. The apparent contradiction in our results with the mab studies suggests that the role of lung neutrophil accumulation in hyperoxic lung injury is more complex than previously appreciated, and that PMN accumulation occurring late in the course of hyperoxia-exposure is ICAM-1/β2 integrin independent.Table

Lipid peroxidation in brain and lungs from mice exposed to hyperoxia

Lipid peroxidation was measured in brains and lungs from mice which had been exposed to 100% O 2 at 445 kPa, 515 kPa and 585 kPa for 30 min. These treatments produced varying degrees of convulsive activity and negligible to moderate lung damage. Lipid peroxidation was estimated by the thiobarbituric acid method and by analysis of Schiff bases. Lipid peroxidation did not increase in either lungs or brains after hyperbaric oxygen exposure, nor was there any difference between tissues from control or hyperoxic mice in the degree of lipid peroxidation which occurred during in vitro incubation of homogenates at 37° in air for 150 min. Similarly, no increased lipid peroxidation was seen in lung tissue from mice exposed to normobaric 100% O 2 for either 60 or 72 hr.

Impaired surfactant phospholipid metabolism in hyperoxic mouse lungs.

We studied the effect of 98% O2 on the amounts of and in vivo and in vitro synthesis rates of lung phospholipids in the mouse. The alveolar fluid obtained by multiple endobronchial lavages showed a decline in the amounts of all phospholipids between 48 and 72 h. The proportion of phosphatidylcholine that was disaturated did not change up to 96 h. Smaller decreases in phosphatidylcholine of the lung tissue were found at the same times. Between 48 and 72 h the turnover of total and disaturated phosphatidylcholine was greatly decreased, and transit time for both from lung to alveolar fluid was prolonged. At 60 h in vitro incorporation by lung slices of both glycerol and palmitate into disaturated phospholipids was reduced by approximately half, the distribution of label into various phospholipid classes being unchanged. We interpret these data as supporting the hypothesis that hyperoxia impairs the synthesis of surfactant phospholipids in the lung and their transport to the alveolar surface, and that this results in a decrease in the phospholipid content of the alveolar lining layer. These effects could account for altered pulmonary mechanical properties and contribute to the morbidity and mortality of O2 toxicity.

19-Nortestosterone decanoate induced erythropoiesis in the actinomycin-D-treated hyperoxic mouse

The erythropoietic effect of an androgen, 19-nortestosterone decanoate (19-ND), on the actinomycin-D- (ACT) treated mouse has been investigated. The toxic effect of a daily administration of 60 μg/kg body weight of ACT on hematopoietic tissue was partially overcome by a daily 1.25 mg injection of the androgen. When an equivalent amount of ACT was administered in fractionalized doses (20 μg/kg body weight at 8-hour intervals), significant levels of erythropoiesis were maintained by identical androgen therapy. These data suggest that 19-ND increases the absolute number of erythroid precursor cells, thus increasing the absolute number of these elements which remain unaffected by the amount of ACT injected. Such unaffected cells will mature to normal erythrocytes, thereby maintaining significant erythropoiesis.