New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: a comparison between hypoxic and normoxic cells

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O₂) and normoxic (21% O₂) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.     

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: A comparison between hypoxic and normoxic cells

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O₂) and normoxic (21% O₂) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.     

Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.     

Exercise delays the hypoxic thermal response in rats.

Exercise exacerbates acute mountain sickness. In infants and small mammals, hypoxia elicits a decrease in body temperature (Tb) [hypoxic thermal response (HTR)], which may protect against hypoxic tissue damage. We postulated that exercise would counteract the HTR and promote hypoxic tissue damage. Tb was measured by telemetry in rats (n = 28) exercising or sedentary in either normoxia or hypoxia (10% O2, 24 h) at 25 degrees C ambient temperature (Ta). After 24 h of normoxia, rats walked at 10 m/min on a treadmill (30 min exercise, 30 min rest) for 6 h followed by 18 h of rest in either hypoxia or normoxia. Exercising normoxic rats increased Tb ( degrees C) vs. baseline (39.68 +/- 0.99 vs. 38.90 +/- 0.95, mean +/- SD, P < 0.05) and vs. sedentary normoxic rats (38.0 +/- 0.09, P < 0.05). Sedentary hypoxic rats decreased Tb (36.15 +/- 0.97 vs. 38.0 +/- 0.36, P < 0.05) whereas Tb was maintained in the exercising hypoxic rats during the initial 6 h of exercise (37.61 +/- 0.55 vs. 37.72 +/- 1.25, not significant). After exercise, Tb in hypoxic rats reached a nadir similar to that in sedentary hypoxic rats (35.05 +/- 1.69 vs. 35.03 +/- 1.32, respectively). Tb reached its nadir significantly later in exercising hypoxic vs. sedentary hypoxic rats (10.51 +/- 1.61 vs. 5.36 +/- 1.83 h, respectively; P = 0.002). Significantly greater histopathological damage and water contents were observed in brain and lungs in the exercising hypoxic vs. sedentary hypoxic and normoxic rats. Thus exercise early in hypoxia delays but does not prevent the HTR. Counteracting the HTR early in hypoxia by exercise exacerbates brain and lung damage and edema in the absence of ischemia.     

Impact of iron and vitamin C-containing supplements on preterm human milk: in vitro

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.     

Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas

We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice. The aim of this study was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa. Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1-0.3% O2) for 1 h, with or without a recovery period of normoxia (21% O2) for 2 h, and then inoculated with P. aeruginosa containing the PA-I reporter construct. Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P < 0.001). Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers. PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. We conclude that the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.     

低氧中强度训练可通过上调HIF-1α来提高机体抗氧化能力

低氧环境和运动训练均可导致人体体重降低,然而,低氧结合中强度训练对肥胖人群能量代谢及氧化应激的影响尚不清楚。本研究招募了60名无系统运动训练史的健康男性大学生,将受试者分为低氧组和常氧组,每组30名。在一个110 m^2的训练室内通过低氧训练系统模拟人工低氧环境(海拔高度:2 500 m,氧浓度:15%)。两组受试者进行1个月的低氧/常氧中强度骑行训练。此外,对低氧和常氧中强度训练的大鼠进行力竭跑台运动测试,苏木精和伊红(HE)染色评价大鼠骨骼肌形态学变化,RT-PCR检测低氧诱导因子1α(HIF-1α) mRNA的表达。研究显示,运动后低氧组的体重、脂肪重量和BMI均显著低于常氧组(p<0.05)。运动后低氧组的血清TC、HDL-C和LDL-C含量均显著低于常氧组(p<0.05),而总TG含量与常氧组无显著差异(p>0.05)。运动后,低氧组的游离脂肪酸含量显著高于常氧组(p<0.05),两组血糖无显著差异(p>0.05)。运动后,低氧组的SOD和GSH-PX水平显著高于常氧组(p<0.05),而MDA水平显著低于常氧组(p<0.05)。运动后,低氧组的IL-1β、IL-6和TNF-α水平显著低于常氧组(p<0.05)。力竭运动后,低氧组大鼠的骨骼肌形态学改变异常情况明显低于常氧组。低氧组的HIF-1αm RNA水平显著高于常氧组。本研究表明,与常氧相比,低氧中强度训练可有效降低肥胖人群的血脂水平,促进脂肪动员,减弱氧化应激损伤,抑制促炎细胞因子表达,从而促进体重减轻,并防止糖尿病、高血脂等肥胖相关疾病的发生。此外,低氧中强度可通过上调HIF-1α来提高机体抗氧化能力并减弱运动损伤。     

Increased phosphofructokinase content during chronic hypoxia in cultured skeletal muscle (L8) cells

Chronic hypoxia results in increased measured activity of all of the glycolytic enzymes and is associated with an increase in glycolytic capacity. Phosphofructokinase, a rate-limiting glycolytic enzyme, was measured under normoxic and hypoxic conditions to determine the relationship between increased activity and enzyme content. Monoclonal antibodies were used to isolate pure enzyme in rat skeletal muscle cells (L8) cultured hypoxically (PO2 = 14 torr) and normoxically (PO2 = 142 torr). Phosphofructokinase content per cell in cultures maintained under chronic (96 h) hypoxic conditions was twice that of cells cultured under normoxic conditions (0.0675 +/- 0.008 (S.E.) and 0.0345 +/- 0.003 micrograms enzyme protein/microgram DNA, P less than 0.01). Phosphofructokinase activity increased proportionately (hypoxia, 0.020 +/- 0.003; normoxia, 0.010 +/- 0.001 units/microgram DNA). The specific activity (units/mg enzyme protein) of phosphofructokinase in the hypoxic (296 +/- 32) versus the normoxic (290 +/- 15) cultures was not significantly different, indicating that the increased activity was accounted for by an increase in enzyme content. Glycolytic rate appears to be regulated at the level of enzyme content.     

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].     

Hypoxia and high glucose upregulate AT1 receptor expression and potentiate ANG II-induced proliferation in VSM cells

We examined the effect of hypoxia and high glucose (HG) on ANG II type 1 (AT(1)) receptor expression and proliferation in cultured vascular smooth muscle (VSM) cells. Exposure of quiescent cells to hypoxia in a serum-free DME-Ham's F-12 medium for 6-24 h induced a progressive increase in AT(1) mRNA expression. Exposure of cells to 24 h of hypoxia also resulted in a significant increase in ANG II receptor binding as assessed with (125)I-labeled ANG II. Treatment with ANG II (1 microM) for 24 h under normoxic conditions caused an approximately 1.5-fold increase in both DNA synthesis and cell number, which was enhanced to approximately 3.0-fold under hypoxic conditions. An AT(1) receptor antagonist (losartan, 10 microM) blocked the ANG II-induced increase in DNA synthesis under both normoxic and hypoxic conditions. Incubations in HG medium (25 mM) for 12-24 h under normoxic conditions induced an approximately 2.5-fold increase in AT(1) mRNA levels, which was markedly enhanced by hypoxia to approximately 5.5-fold at 12 h and approximately 8.5-fold at 24 h. ANG II under HG-normoxic conditions caused a complete downregulation of AT(1) expression, which was prevented by hypoxia. These results demonstrate an upregulation of AT(1) receptor expression by hypoxia and HG in cultured VSM cells and suggest a mechanism for enhanced ANG II-induced VSM cell proliferation and the development of atherosclerosis in diabetes.     

Poly(ADP-ribose) polymerase activity in the cat carotid body in hypoxia and hyperoxia.

Reactive oxygen species (ROS) induce DNA damage with the ensuing activation of the chromosomal repair enzyme poly(ADP-ribose) polymerase (PARP). ROS also interact with the function of carotid body chemoreceptor cells. The possibility arises that PARP is part of the carotid chemosensing process. This study seeks to determine the presence of PARP and its changes in response to contrasting chemical stimuli, hypoxia and hyperoxia, both capable of generating ROS, in cat carotid bodies. The organs were dissected from anesthetized cats exposed in vivo to acute normoxic (PaO2 approximately 90 mmHg), hypoxic (PaO2 approximately 25 mmHg), and hyperoxic (PaO2 > 400 mmHg) conditions. Carotid body homogenate was the source of PARP and [adenine 14C] NAD was the substrate in the assay. Specimens of the superior cervical ganglion and brainstem were used as reference tissues. We found that PARP activity amounted to 27 pmol/mg protein/min in the normoxic carotid body. The activity level more than doubled in both hypoxic and hyperoxic carotid bodies. Changes of PARP in the reference tissues were qualitatively similar. We conclude that PARP is present in the carotid body but the augmentation of the enzyme activity in both hypoxia and hyperoxia reflects DNA damage, induced likely by ROS and being universal for neural tissues, rather than a specific involvement of PARP in the chemosensing process.     

Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA

Phenolic phytochemicals are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. The aims of this study were to assess the free radical-scavenging activities of several common phenolic phytochemicals, and then, the effects of the most potent phenolic phytochemicals on oxidative damage to DNA in cultured cells. Epigallocatechin gallate (EGCG) scavenged the stable free radical, alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH), most effectively, while quercetin was about half as effective. Genistein, daidzein, hesperetin, and naringenin did not scavenge DPPH appreciably. Jurkat T-lymphocytes that were pre-incubated with relatively low concentrations of either EGCG or quercetin were less susceptible to DNA damage induced by either a reactive oxygen species or a reactive nitrogen species, as evaluated by the comet assay. More specifically, control cells had a comet score of only 17+/-5, indicating minimal DNA damage. Cells challenged with 25 microM hydrogen peroxide (H(2)O(2)) or 100 microM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188+/-6 and 125+/-12, respectively, indicating extensive DNA damage. The H(2)O(2)-induced DNA damage was inhibited with 10 microM of either EGCG (comet score: 113+/-23) or quercetin (comet score: 82+/-7). Similarly, the SIN-1-mediated DNA damage was inhibited with 10 microM of either EGCG (comet score: 79+/-13) or quercetin (comet score: 72+/-17). In contrast, noticeable DNA damage was induced in Jurkat T-lymphocytes by incubating with 10-fold higher concentrations (i.e., 100 microM) of either EGCG (comet score: 56+/-17) or quercetin (comet score: 64+/-13) by themselves. Collectively, these data suggest that low concentrations of EGCG and quercetin scavenged free radicals, thereby inhibiting oxidative damage to cellular DNA. But, high concentrations of either EGCG or quercetin alone induced cellular DNA damage.     

Antioxidant activities of seabuckthorn (Hippophae rhamnoides) during hypoxia induced oxidative stress in glial cells

The present study reports the cytoprotective and antioxidant properties of alcoholic leaf extract of seabuckthorn (SBT) against hypoxia induced oxidative stress in C-6 glioma cells. Exposure of cells to hypoxia for 12 h resulted in a significant increase in cytotoxicity and decrease in mitochondrial transmembrane potential compared to the controls. Further an appreciable increase in nitric oxide and reactive oxygen species (ROS) production was noted which in turn was responsible for fall in intracellular antioxidant levels and GSH/GSSG ratio. There was a significant increase in DNA damage during hypoxia as revealed by comet assay. Pretreatment of cells with alcoholic leaf extract of SBT at 200 μg/ml significantly inhibited cytotoxicity, ROS production and maintained antioxidant levels similar to that of control cells. Further, the leaf extract restored the mitochondrial integrity and prevented the DNA damage induced by hypoxia. These results indicate that the leaf extract of SBT has strong antioxidant and cytoprotective activity against hypoxia induced oxidative injury. (Mol Cell Biochem 278: 9–14, 2005)     

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.     

Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells

Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identify and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25-100 microM) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50-100 and 50-100 microM, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.     

Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells

Endothelial cells are critical targets in both hypoxia-and reoxygenation-mediated lung injury. Reactive O₂ species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H₂O₂ generation and extracellular H₂O₂ and O/₂ release were measured after exposure to normoxia (room air-5% CO₂), hypoxia (0% O₂ -95% N-5% CO₂), or hypoxia followed by normoxia or hyperoxia (95% O₂-5% CO₂). Exposure to hypoxia results in significant reductions in intracellular H₂ O₂ formation and extracellular release of H₂ O₂ and O₂ by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley-Liss, Inc.