The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.     

Antioxidant responses to oxidant-mediated lung diseases

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated throughout the human body. Enzymatic and nonenzymatic antioxidants detoxify ROS and RNS and minimize damage to biomolecules. An imbalance between the production of ROS and RNS and antioxidant capacity leads to a state of "oxidative stress" that contributes to the pathogenesis of a number of human diseases by damaging lipids, protein, and DNA. In general, lung diseases are related to inflammatory processes that generate increased ROS and RNS. The susceptibility of the lung to oxidative injury depends largely on its ability to upregulate protective ROS and RNS scavenging systems. Unfortunately, the primary intracellular antioxidants are expressed at low levels in the human lung and are not acutely induced when exposed to oxidative stresses such as cigarette smoke and hyperoxia. However, the response of extracellular antioxidant enzymes, the critical primary defense against exogenous oxidative stress, increases rapidly and in proportion to oxidative stress. In this paper, we review how antioxidants in the lung respond to oxidative stress in several lung diseases and focus on the mechanisms that upregulate extracellular glutathione peroxidase.     

Pharmacological antioxidant strategies as therapeutic interventions for COPD

Cigarette/tobacco smoke/biomass fuel-induced oxidative and aldehyde/carbonyl stress are intimately associated with the progression and exacerbation of chronic obstructive pulmonary disease (COPD). Therefore, targeting systemic and local oxidative stress with antioxidants/redox modulating agents, or boosting the endogenous levels of antioxidants are likely to have beneficial effects in the treatment/management of COPD. Various antioxidant agents, such as thiol molecules (glutathione and mucolytic drugs, such as N-acetyl-L-cysteine and N-acystelyn, erdosteine, fudosteine, ergothioneine, and carbocysteine), have been reported to modulate various cellular and biochemical aspects of COPD. These antioxidants have been found to scavenge and detoxify free radicals and oxidants, regulate of glutathione biosynthesis, control nuclear factor-kappaB (NF-kappaB) activation, and hence inhibiting inflammatory gene expression. Synthetic molecules, such as specific spin traps like α-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a superoxide dismutase mimetic M40419, iNOS and myeloperoxidase inhibitors, lipid peroxidation inhibitors/blockers edaravone, and lazaroids/tirilazad have also been shown to have beneficial effects by inhibiting cigarette smoke-induced inflammatory responses and other carbonyl/oxidative stress-induced cellular alterations. A variety of oxidants, free radicals, and carbonyls/aldehydes are implicated in the pathogenesis of COPD, it is therefore, possible that therapeutic administration or supplementation of multiple antioxidants and/or boosting the endogenous levels of antioxidants will be beneficial in the treatment of COPD. This review discusses various novel pharmacological approaches adopted to enhance lung antioxidant levels, and various emerging beneficial and/or prophylactic effects of antioxidant therapeutics in halting or intervening the progression of COPD. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

同型半胱氨酸相关性慢性阻塞性肺疾病临床研究进展

在世界慢性疾病中,慢性阻塞性肺疾病(COPD)发病率和死亡率均较高,鉴于对患者生活造成严重伤害,越来越受到人们关注。同型半胱氨酸(Homocysteinemia,Hcy)对全身器官损害多有报道,随着研究的深入,Hcy目前对于肺部疾病的影响也受到重视。部分学者提出同型半胱氨酸可能是COPD发病机制中又一重要因素。Hcy在体内可以刺激产生大量的ROS和自由离子,并引起内皮细胞应激,还可降低肺脏内还原型谷胱甘肽含量。研究表明同型半胱氨酸水平在慢性阻塞性肺疾病中处于高表达状态,且其高表达水平与患者疾病的程度成相关性。本文将通过总结Hcy在肺脏及体内的代谢、各种应激反应等方面阑述同型半胱氨酸水平与COPD的相关性,并总结高水平的同型半胱氨酸相关的COPD治疗方法。     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Pycnogenol enhances endothelial cell antioxidant defenses

Summary

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂^?), and hydroxyl radical (OH^?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H₂O₂ and O₂^? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H₂O₂. Pycnogenol caused a concentration-dependent enhancement of H₂O₂ and O₂^? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.     

The oxido-redox potential of albumin: Methodological approach and relevance to human diseases

In humans, an increased synthesis of reactive oxygen species (ROS) may be a relevant cause of amplification of physiologic processes resulting in inflammatory organ damage or neoplasia. Efficient anti-oxidative systems targeting oxidative stress are thus essential to prevent tissue damage. In plasma, proteins proved to be the first line of defence against ROS and albumin, the highest concentration plasma protein, has a key role in this antioxidant function. Recent studies have clearly documented that albumin oxido-redox potential changes upon oxidation by different oxidants thus becoming a deputy biomarker of this process.ROS react primarily with the free ³⁴Cysteine (³⁴Cys) residue of albumin to form two reversible intermediate derivatives, sulfenic-(SOH-alb) and sulfinic acid (SO₂H-alb), resulting in sulfonic acid (SO₃H-alb), the final stable product of the reaction.Upon stable oxidation (SO₃H-alb), albumin properties are altered: the protein becomes more susceptible to trypsin digestion and is degraded faster compared to the non-oxidized counterpart.The present review focuses on the characterization of albumin chemical changes induced by ROS, their relevance in human pathology and the most recent advances in the approach to oxidation adduct analysis.     

Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr ^−/− lung when compared to wild-type (Cftr ^+/+) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr ^−/− lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr ^−/− mice compared to Cftr ^+/+. Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr ^+/+ lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr ^−/− lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr ^−/− lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.     

Normal alveolar epithelial lining fluid contains high levels of glutathione

The epithelial cells on the alveolar surface of the human lower respiratory tract are vulnerable to toxic oxidants derived from inhaled pollutants or inflammatory cells. Although these lung cells have intracellular antioxidants, these defenses may be insufficient to protect the epithelial surface against oxidants present at the alveolar surface. This study demonstrates that the epithelial lining fluid (ELF) of the lower respiratory tract contains large amounts of the sulfhydryl-containing antioxidant glutathione (GSH). The total glutathione (the reduced form GSH and the disulfide GSSG) concentration of normal ELF was 140-fold higher than that in plasma of the same individuals, and 96% of the glutathione in ELF was in the reduced form. Compared with nonsmokers, cigarette smokers had 80% higher levels of ELF total glutathione, 98% of which was in the reduced form. Studies of cultured lung epithelial cells and fibroblasts demonstrated that these concentrations of reduced glutathione were sufficient to protect these cells against the burden of H2O2 in the range released by alveolar macrophages removed from the lower respiratory tract of nonsmokers and smokers, respectively, suggesting that the glutathione present in the alveolar ELF of normal individuals likely contributes to the protective screen against oxidants in the extracellular milieu of the lower respiratory tract.     

Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury

Summary

Glutathione (GSH) plays several important roles in the protection of cells against oxidative damage, particularly following exposure to xenobiotics. Ferric nitrilotriacetate (Fe-NTA) is a potent depletor of GSH and also enhances tissue lipid peroxidation. In this study, we show the effect of Fe-NTA treatment on hepatic GSH and some of the glutathione metabolizing enzymes, oxidant generation and liver damage. The level of hepatic GSH and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase all decrease following Fe-NTA administration. In these parameters the maximum decrease occurred at 12 h following Fe-NTA treatment. In contrast, γ-glutamyl transpeptidase was increased at this time. Not surprisingly, the increase in the activity of γ-glutamyl transpeptidase and decreases in GSH, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione S-transferase were found to be dependent on the dose of Fe-NTA administered. Fe-NTA administration also enhances the production of H₂O₂ and increases hepatic lipid peroxidation. Parallel to these changes, Fe-NTA enhances liver damage as evidenced by increases in serum transaminases. Once again, the liver damage is dependent on the dose of Fe-NTA and is maximal at 12 h. Pretreatment of animals with antioxidant, butylated hydroxy anisole (BHA), protects against Fe-NTA-mediated hepatotoxicity further supporting the involvement of oxidative stress in Fe-NTA-mediated hepatic damage. In aggregate, our results indicate that Fe-NTA administration eventuates in decreased hepatic GSH, a fall in the activities of glutathione metabolizing enzymes and excessive production of oxidants, all of which are involved in the cascade of events leading to iron-mediated hepatic injury.     

Vapors Produced by Electronic Cigarettes and E-Juices with Flavorings Induce Toxicity,Oxidative Stress,and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung

Oxidative stress and inflammatory response are the key events in the pathogenesis of chronic airway diseases. The consumption of electronic cigarettes (e-cigs) with a variety of e-liquids/e-juices is alarmingly increasing without the unrealized potential harmful health effects. We hypothesized that electronic nicotine delivery systems (ENDS)/e-cigs pose health concerns due to oxidative toxicity and inflammatory response in lung cells exposed to their aerosols. The aerosols produced by vaporizing ENDS e-liquids exhibit oxidant reactivity suggesting oxidants or reactive oxygen species (OX/ROS) may be inhaled directly into the lung during a “vaping” session. These OX/ROS are generated through activation of the heating element which is affected by heating element status (new versus used), and occurs during the process of e-liquid vaporization. Unvaporized e-liquids were oxidative in a manner dependent on flavor additives, while flavors containing sweet or fruit flavors were stronger oxidizers than tobacco flavors. In light of OX/ROS generated in ENDS e-liquids and aerosols, the effects of ENDS aerosols on tissues and cells of the lung were measured. Exposure of human airway epithelial cells (H292) in an air-liquid interface to ENDS aerosols from a popular device resulted in increased secretion of inflammatory cytokines, such as IL-6 and IL-8. Furthermore, human lung fibroblasts exhibited stress and morphological change in response to treatment with ENDS/e-liquids. These cells also secrete increased IL-8 in response to a cinnamon flavored e-liquid and are susceptible to loss of cell viability by ENDS e-liquids. Finally, exposure of wild type C57BL/6J mice to aerosols produced from a popular e-cig increase pro-inflammatory cytokines and diminished lung glutathione levels which are critical in maintaining cellular redox balance. Thus, exposure to e-cig aerosols/juices incurs measurable oxidative and inflammatory responses in lung cells and tissues that could lead to unrealized health consequences.     

Synergic prooxidant,apoptotic and TRPV1 channel activator effects of alpha-lipoic acid and cisplatin in MCF-7 breast cancer cells

Background: Resistance to cisplatin (Cisp) in the treatment of breast cancer is a major obstacle. Alpha-lipoic acid (ALA) has both antioxidant and oxidant properties. ALA has been used on stimulation mechanisms of apoptosis and oxidative stress in the treatment of cancer with a combination of chemotherapeutic agents, although its role on molecular mechanisms in the cancer cells has not been clarified yet. The aim of this study was to evaluate if a combination therapy of ALA with Cisp can alter the effect of this chemotherapy drug in the MCF-7 breast cancer cells.

Materials: The MCF-7 cells were divided into four treatment groups as control, Cisp (0.025?mM), ALA (0.05?mM), and Cisp?+?ALA.

Results: Apoptosis, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, lipid peroxidation, PARP1, caspase 3 and 9 expression levels are increased through activating TRPV1 in the cells by the Cisp and ALA treatments, although cell viability, reduced glutathione and glutathione peroxidase (GPx) values were decreased by the treatments. The Cisp and ALA-induced increase of intracellular free Ca²⁺ concentration was decreased with the TRPV1 blocker, capsazepine.

Conclusions: Apoptosis and oxidant effects of Cisp were increased by activation of TRPV1 channels, but its action on the values was further increased by the ALA treatment. Combination therapy of ALA and Cisp could be used as an effective strategy in the treatment of breast cancer.     

Anti-malarial drug artesunate protects against cigarette smoke-induced lung injury in mice

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2 h before 4% cigarette smoke exposure for 1 h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1β, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3 K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smoke-induced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.