Reactive Oxygen Species, Inflammation, and Lung Disease

Current Pharmaceutical Design, 2012, 18, 3889-3900
3889
Reactive Oxygen Species, Inflammation, and Lung Diseases

Di Paola Rosanna1 and Cuzzocrea Salvatore1,2,*
1
Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Via C. Valeria
Gazzi 98100 Messina, Italy; 2University of Manchester
Abstract: Reactive oxygen species (ROS) are well recognized for playing a dual role as both deleterious and beneficial species. ROS are
products of normal cellular metabolism and under physiological conditions, participate in maintenance of cellular redox homeostasis.
Overproduction of ROS, results in oxidative stress. Oxidative stress is a deleterious process that leads to lung damage and consequently
to various disease states. The lung is a highly specialized organ that facilitates uptake of oxygen and release of carbon dioxide.
Persistent inhalation of the invading pathogens or toxic agents may result in overwhelming production of ROS. Oxidants initiate a number of pathologic processes, including inammation of the airways, which may contribute to the pathogenesis and/or exacerbation of airways disease.
During inflammation, enhanced ROS production may induce recurring DNA damage, inhibition of apoptosis, and activation of protooncogenes by initiating signal transduction pathways. Therefore, it is conceivable that chronic inflammation-induced production of ROS
in the lung may predispose individuals to lung diseases. In this review, we discuss mechanisms of oxidant stress in the lung, the role of
oxidants in lung disease pathogenesis and exacerbation.
Keywords: Oxidative stress, inflammation, DNA damage, lung diseases.

1. INTRODUCTION
Oxidative stress plays an important role in the pathogenesis of
various lung disorders such as asthma, chronic obstructive lung
disease (COPD), acute lung injury (ALI), pulmonary fibrosis and
lung cancer [1, 2]. Oxidative stress results from an oxidant/antioxidant imbalance in favor of oxidants and causes oxidation of
proteins, DNA and lipids, as well as inducing a variety of cellular
responses through the generation of secondary metabolic ROS [3].
Biological systems are continuously exposed to oxidants, either
generated endogenously by metabolic reactions (e.g. from mitochondrial electron transport during respiration or during activation
of phagocytes), or derived from exogenous sources (e.g. air pollutants and cigarette smoke) [4, 5]. The lung is exposed to high levels
of oxygen. The adult human lung provides an enormous surface
area of approximately 140 m2 to air. This makes the lung vulnerable
to a wide range of toxicants and infectious agents with the potential
to induce oxidative damage [6]. Inhalation of such toxic air pollutants and microorganisms results in lung injury and generation of
reactive oxygen (ROS), leading to cascades of signaling events that
trigger production of pro-inflammatory mediators [7]. Inflammation
is the primary reaction of a tissue to eliminate pathogenic insult and
injured tissue components in order to restore normal physiological
functions or replace the irreparable tissue with scar tissue. In this
review, the key events triggered by lung inflammation-induced by
ROS generation and their purported role in the genesis of lung diseases are discussed.
2. REACTIVE OXYGEN SPECIES: PRINCIPAL SOURCES
ROS are oxygen-containing molecules that are capable of either
accepting or donating a free electron, thus they are, to some extent,
unstable and react with other molecules. This reaction may lead to
the generation of other, sometimes even more reactive molecules.
The first step in the complex chain of ROS is a one electron reduction of molecular oxygen, leading to production of superoxide O2.
O2 is unstable and quickly undergoes another reduction to
*Address correspondence to this author at the Department of Clinical and
Experimental Medicine and Pharmacology, School of Medicine, University
of Messina, Via C. Valeria Gazzi 98100 Messina, Italy; Tel: (39) 090
2213644; Fax: 0902213300; E-mail: salvator@unime.it
1873-4286/12 $58.00+.00
hydrogen peroxide H2O2, either spontaneously or in a much faster

reaction catalyzed by superoxide dismutase (SOD). H2O2 is relatively stable and can migrate from its site of origin; therefore it is
capable of affecting a large scale of important cellular molecules. It
can also turn into highly reactive hydroxyl radical OH (Fentons
reaction, catalyzed by free iron). Superoxide (O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), lipid peroxides (ROOH),
ozone (O3) and hydroxyl radical (OH) are some of the major ROS
in living systems. ROS can be produced from both endogenous and
exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation [8]. Mitochondria have long been known to
generate significant quantities of hydrogen peroxide. Generation of
the superoxide radical by mitochondria was first reported more than
three decades ago by Loschen et al. [9]. Mitochondria generate
approximately 23 nmol of superoxide/min per mg of protein, the
ubiquitous presence of which indicates it to be the most important
physiological source of this radical in living organisms [8]. In the
mitochondria O2- can be generated enzymatically during oxidative
phosphorylation in the electron transfer chain, as a byproduct of
normal cellular aerobic metabolism, with the rate of respiration
responsible for the rate of generation of ROS [10]. The mitochondrial enzyme superoxide dismutase (mSOD) rapidly scavenges
O2- accelerating the removal of this radical by dismutation to generate H2O2, a less reactive molecule. Mitochondria are involved
also in the generation of nitric oxide (NO) via the nitric oxide synthase (NOS) reaction. Superoxide anion and NO react to form
another harmful oxidant, peroxinitrite (ONOO_) which is also a
potential source for more powerful and aggressive hydroxyl radical
(OH). Several non-mitochondrial sources of ROS exist in the cell.
The endoplasmic reticulum is a source of ROS, where resident cytocrome P-450 oxidizes unsaturated fatty acids and xenobiotics
such as pollutants, drugs and toxins to generate O2- and H2O2.
Xanthine oxidase (XO), a highly versatile enzyme that is widely
distributed among species (from bacteria to man) and within the
various tissues of mammals [11]. Some cells contain xanthine oxidoreductase (XOR), which catalyses the transformation of hypoxanthine to xanthine and xanthine to uric acid. Under physiological conditions, the XOR is present in the form of xanthine dehydrogenase (XD). XD can be modified, either reversibly (by oxidation
of sulfhydryls) or irreversibly (by mild proteolysis) to xanthine
2012 Bentham Science Publishers
3890 Current Pharmaceutical Design, 2012, Vol. 18, No. 26
oxidase (XO),generating large amounts of H2O2 as well as O2. It is

speculated that this process gains importance especially after
ischemia-reoxygenation, when the XD-XO transformation is rapid
and hypoxic tissue contains vast amounts of substrate for XO.
However, latest research suggests that the XD-XO conversion may
not be of clinical significance and that total XOR activity is important, even XD being able to generate ROS [12]. Recently it has
been discovered that XOR can transform nitrates and nitrites to
nitrites and NO, respectively (even in anoxia, in contrast to NOS).
The enzyme itself is able to catalyse the reaction of NO with O2,
thus generating highly reactive peroxynitrite as well [13]. On the
other hand, it has to be noted that XOR is a source of uric acid,
which is known to be potent oxidant scavenger. ROS can be generated as by-products during metabolism of arachidonic acid, which
to some degree takes place in practically every cell. Enzymes participating in the process are cyclooxygenase, lipooxygenase and
cytochrome P-450 [14]. Arachidonic acid may be a source of ROS
even by a non-enzymatic process. Practically all cells possess P450
cytochrome oxidase, a heme-containing enzyme system localized in
mitochondria or microsomes. It is a superfamily of isoenzymes that
mostly function as monooxygenase, but it can catalyse an intramolecular transfer of oxygen as well. P450 isoenzymes participate in the metabolism of steroid hormones, cholesterol and its
catabolism to bile acids, arachidonic acid and eicosanoids [15]. It
catalyses the hydroxylation of vitamin D3 and retinoic acid, and
plays an important role in the metabolism of many xenobiotics. The
underlying concept of its activity is a multi step transfer of 2 electrons to a substrate while binding one oxygen atom to it, the second
being reduced to water. Part of the oxygen involved is inevitably
reduced to superoxide. Lysosomal membrane contains an electron
transport system, which helps ensure optimal intralysosomal pH by
pumping protons. This system promotes a three-electron reduction
of oxygen, thus leading to generation of highly reactive OH.
Membrane associated oxidases such as NADPH oxidases generate
ROS by reacting with the intracellular NADPH, to reduce molecular oxygen superoxide anion (O2-) [16]. This reaction is typical of
activated phagosomes in neutrophils and macrophages during the
respiratory burst, in response to xenobiotics such as particles of
pollution, inflammatory agents or infectious conditions. Upon activation, neutrophils initiate an oxidative burst by consuming molecular oxygen resulting in the formation ROS as a defense mechanism to achieve localized microbicidal function. As a consequence
of the activation of neutrophils and oxidative burst, myeloperoxidase (MPO), a hemeprotein present in the azurophil granules in
neutrophils, is released either into the lysosomes of the phagocytic
cell or into the extracellular space. MPO catalyzes the formation
HOCl from H2O2 and chloride ions, a further strong oxidizing and
chlorinating species. MPO activation may lead to irreversible protein and lipid modification, increasing levels of oxidized low density lipoprotein, through free radical formation. Additional endogenous sources of cellular reactive oxygen species are neutrophils,
eosinophils and macrophages. Activated macrophages initiate an
increase in oxygen uptake that gives rise to a variety of reactive
oxygen species, including superoxide anion, nitric oxide and hydrogen peroxide [17].
Neutrophils have been known to produce ROS as a part of their
bactericidal activity. In the cytoplasmic membrane they contain
NADPH oxidase, which generates large amounts of O2. Its activation is responsible for the respiratory burst. Another enzyme contributing to the complex defence mechanism is myeloperoxidase,
which catalyzes the reaction of H2O2 with halide anions upon neutrophil stimulation [18]. A chloride anion is by far the most frequent one, therefore MPO produces mostly hypochlorous acid
HOCl, which is highly reactive [19]. Xanthine oxido reductase has
been detected in neutrophils as well. However, neutrophils present
only minor cellular type in lungs under physiologic conditions and
it is only after stimulation during inflammation that they migrate
into pulmonary circulation in vast amounts.
Rosanna and Salvatore
Eosinophils are also a potent cellular source of ROS, especially

in some allergic or infectious diseases [20]. Similar to neutrophils,
their membrane contains NADPH oxidase which generates superoxide. Cytoplasmic peroxidase (eosinophilic peroxidase, analogue
of myeloperoxidase in neutrophils) contributes significantly to bactericidal activity or damaging effects of eosinophils, producing
mostly hypochlorous acid (HOCl).
Alveolar macrophages are phagocyting cells present in the
lungs in large numbers and therefore form the first line of lung
defence against infection. Similar to neutrophils, their main source
of ROS is the membrane NADPH oxidase, generating O2[21]. It
was suggested that alveolar macrophages are the major source of
ROS under physiologic conditions [22].
Peripheral monocytes-macrophages attracted by inflammatory
cytokines contribute substantially to the damage in pulmonary diseases recently it has been shown that after stimulation and differentiation into macrophages these cells are capable of producing
superoxide by XOR [23] which plays a significant role in acute
lung injury (as opposed to invading neutrophils, where XOR is
silent). Mast cells are also present in lungs, but so far the reports
about their ROS production are rather confusing. However, there is
certain data proving that they might be able to generate ROS [24].
Type II pneumocytes form a part of alveolar epithelium. These
cuboidal cells with very active metabolism produce surfactant and
are thought to function as precursor cells for type I pneumocytes in
the case of increased destruction o falveolar epithelium. Recent
studies show that even type II epithelial cells possess enzymatic
properties for production of some ROS [25].
Endothelial cells, thanks to rich lung vascularization, present
another substantial cellular mass. In recent years there has been
growing evidence that even these cells can present a source of ROS
and participate in oxidative stress and lung injury under pathological conditions. They contain xanthine oxido reductase complex
which seems to become stimulated mostly after hypoxia [26].
Moreover, it has been shown that membrane-bound NADPH oxidase is also present, with some differences from the phagocytic
type[27]. Endothelial cells are capable of releasing substantial
amounts of O2into the extracellular space, possibly via membrane
anion channels [28]. Formation of another ROS, highly reactive
OH, can be catalyzed by iron ions present in proximity of the endothelial surface [28].
Smooth muscle cells (either in airway or in vessel walls) may
act as another ROS source as it has been shown that their membrane contains NADPH-like oxidase generating O2[29]. ROS
produced by airway smooth muscle cells play a significant role in
airway hyper reactivity.
Lung fibroblasts have also been proved to produce ROS, especially after stimulation by inflammatory cytokines. Thannickal [30]
reports presence of two different systems, both membrane-bound.
The first is NADPH oxidase (phagocyte-like) which generates O 2
intracellularly. The second enzyme is NADH oxidase, which
produces H2 O 2 directly into the extracellular space [31]. It is evident that cells of practically every type present in the lungs are
capable of producing some ROS. The role of different cell types in
the pathogenesis of specific diseases is far from being clear. While
it is believed that during inflammation phagocytes are the main
source of the oxidative stress, the role of different cell types in a
variety of other conditions is still to be discovered.
NADPH oxidase Major sources of O2- in the body are NADPH
oxidases. NADPH oxidases are a family of enzyme complexes
whose primary function is to catalyze the transfer of electrons from
NADPH to molecular oxygen via their Nox catalytic subunit,
generating O2 and H2O2. Nox enzymes contribute to numerous
biological and pathological processes, including hearing and balance (Nox3), blood pressure regulation, inflammation, cell growth
(Nox1/Nox2), and differentiation (Nox4). The Nox proteins vary in
ROS and Lung Injury
terms of their mode of activation and localization. Nox1 is expressed in smooth muscle cells, but is also present in other vascular
cells. Nox2, previously known as gp91phox, is present in endothelial and phagocytic cells. Nox3 is expressed in the brain and inner
ear. Nox4 is constitutively expressed and active in vascular smooth
muscle and endothelial cells. Nox5has been identified in human
immature lymphatic tissues and in human endothelial cells. NOX
enzymes have six transmembrane regions at the N-terminal half. In
the third and fifth transmembrane domains, four heme-binding histidines are conserved. In the C-terminal cytosolic region, they have
an FAD binding domain and an NADPH-binding domain. In addition to these domains, NOX5 has EF-hands, the Ca2+-binding domains, at the N-terminus. In addition to EF-hands, DUOX enzymes
have a membrane-spanning region and a peroxidase-like domain at
the N-terminus. The Duox1/Duox2 proteins are described as having
a dual nature due to an extracellular peroxidase domain in addition
to the EF-hand Ca2+ binding and gp91phox homology domains.
Originally isolated from the thyroid, they produce the H2O2 that is
used to oxidize iodide during thyroid hormone synthesis. It is important that Nox isoforms not only have different regulation and specific subcellular localization but also generate distinct ROS. For
example, Nox4 is responsible for the basal production of H2O2,
Nox1 and Nox2 generate O2 , and Nox5 produces H2O2 in a Ca2+dependent fashion. An increase number of studies demonstrated
that activation of NADPH oxidases may increase production of
mitochondrial ROS and vice versa: increase in mitochondrial ROS
may activate NADPH oxidases, and this represent an ongoing feedforward cycle.The cross talk between mitochondrial ROS and
NADPH oxidases probably plays an important role in normal
physiological redox cell signaling. Under normal physiological
conditions, production of ROS is highly restricted to specific subcellular sites and is down-regulated by a number of negative feedback mechanisms. Production of ROS in excessive amounts because of overstimulation by Angiotensin II (AngII), high glucose,
fat, or hypoxia results in oxidative stress and transforms this feed
forward redox signaling into a vicious cycle (Fig. 4), which contributes to the development of many chronic inflammatory disease
such as COPD, lung fibrosis, and lung carcer.
Reactive oxygen species can be produced by a host of exogenous processes. Environmental agents including non-genotoxic
carcinogens can directly generate or indirectly induce reactive oxygen species in cells. The induction of oxidative stress and damage
has been observed following exposure to various xenobiotics. These
involve chlorinated compounds, metal (redox and non-redox) ions,
radiation and barbiturates. For example 2-butoxyethanol is known
to produce ROS indirectly, which causes cancer in mice [32]. The
human lung with a surface area of 40-120 m2 is exposed to between
10,000 and 20,000 liters of ambient air each day [33] and ambient
air contains a wide range of pollutants. At least six main air pollutants have been as noxious for human health, these are: lead, carbon
monoxide (CO), sulfur dioxide (SO2), nitrogen dioxide (NO2),
ozone (O3) and particulate matter (PM). Many types of inhaled
particles have the ability to generate free radicals in biological systems and to activate oxidative stress-response signalling pathways
in cells [34]. Ambient particulate matter may also induce oxidative
DNA damage in lung epithelial cells [35]. In addition, a recent
study revealed that inhalation of exogenous H2O2 increased lung
vascular permeability in an animal model [36]. Cigarettes smoke
has been linked to a variety of chronic lung disorders and is a major
cause of morbidity and mortality. Overall estimates suggested that
active smoking is responsible about 90% of lung cancer cases [37].
3. MECHANISMS OF OXIDANT-INDUCED LUNG INJURY
The mechanisms whereby oxidants exert their pathological
effects on the lungs have been the focus of numerous studies and
are still the subject of debate. Despite the diversity of these agents
and the multitude of complex mechanisms that exist, several common themes have been identified. Many ambient air pollutants may
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induce oxidative stress in the lung that arises when ROS overwhelm
antioxidant defenses. After this imbalance is reached, ROS readily
reacts with proteins, lipids, and DNA, resulting in a number of
pathological consequences.
Lipid peroxidation: A primary consequence of oxidative stress
is lipid peroxidation, or the oxidative degeneration of lipids. Lipid
peroxidation is caused by a free radical chain reaction mainly involving membrane polyunsaturated fatty acids. If not quenched, this
reaction can permanently damage cell membranes, ultimately leading to cell death. Exposures to oxidant air pollutants cause lipid
peroxidation in human beings and rodents [38-40]. Lipid peroxidation process consists of three stages: initiation, propagation and
termination [41]. The end products of lipid peroxidation can lead to
subsequent pathological consequences. Once formed, peroxyl radicals can be rearranged via a cyclisation reaction to endoperoxides
(precursors of malondialdehyde) with the final product of the peroxidation process being malondialdehyde (MDA) [42]. MDA is
mutagenic in bacterial and mammalian cells and carcinogenic in
rats. The major aldehyde product of lipid peroxidation other than
malondialdehyde is 4-hydroxy-2-nonenal (HNE). HNE-protein
adducts have been found in the lungs of mice and human beings
after O3 exposure [43] and can induce cell death of alveolar macrophages in mice [44]. Another secondary mediator can be generated
by a reaction of O3 with unsaturated fatty lipids. Ozone can react
directly with unsaturated fatty lipids in the epithelial lining fluid
and cell membranes to produce lipid ozonation products (LOPs),
which also have pathological downstream effects [45]. These products are small, diffusible, and relatively stable, studies have shown
that exposure of bronchial epithelial cells to LOPs caused activation
of phospholipases A2, C, and D as well as the induction of inflammatory mediators such as platelet-activating factor, prostaglandin
E2, IL-6, and IL-8[45]. The deleterious process of the peroxidation
of lipids is also very important in arteriosclerosis, cancer and inflammation.
Proteins : The side chains of all amino acid residues of proteins
are susceptible to oxidation by ionizing radiation and by the action
of ROS [46]. The amino acid residue side chains that are most vulnerable to attack by various ROS lead to the formation of the following products: glutamic semialdehyde from arginine; 4-hydroxyglutamate from glutamate; 2-oxo-histidine from histidine; 3,4dihydroxy phenylalanine from tyrosine, Tyrtyr cross-linked proteins, 3-nitro-tyrosine; [46]. Protein oxidation by ROS is associated
with the formation of many different kinds of inter- and intraprotein cross-linkages, including those formed, (i) by addition of
lysine amino groups to the carbonyl group of an oxidized protein;
(ii) by interaction of two carbon-centred radicals obtained by the
hydroxyl radical-driven abstraction of hydrogens from the polypeptide backbone; (iii) by the oxidation of sulphydryl groups of cysteine residues to form SS crosslinks; (iv) the oxidation of tyrosine residues to form tyrtyr cross-links.
Amino acid composition, particularly cysteine and methionine
residues, can render proteins more susceptible to oxidation [47]. For
example, oxidation of methionine residues in a-1-antitrypsin by
ROSs in vitro results in loss of anti neutrophil elastase activity [48].
Without protection from a-1-antitrypsin, the alveolar matrix is susceptible to destruction by neutrophil elastase, which can eventually
contribute to emphysema. Oxidation of multiple methionine residues by ROSs impairs rapid sodium channel inactivation [49].
ROSs also oxidizes methionine residues in surfactant protein (SP)
B, leading to inactivation [50]. Inactivation of SP-B reduced the
ability of the surfactant film to reduce lung surface tension during
breathing, which can contribute to respiratory distress syndrome.
Similarly, acute exposure of guinea pigs to O3 altered SP-A function, contributing to the inflammatory response. Another study
found that in vitro and in vivo O3 exposure caused oxidative modifications in SP-A that reduced the ability to enhance phagocytosis
of bacteria [51]. Oxidative modification of surfactant proteins may
also render the lung more susceptible to lipid peroxidation, inflammation, and oxidative damage because these proteins have been
reported to inhibit these processes [52].
Cytokine and growth factor signaling: A variety of cytokines
and growth factors that bind to receptors of different classes have
been reported to generate ROS in non-phagocytic cells. Growth
factor tyrosine kinases receptors play a key role in the transmission
of information from outside the cell into the cytoplasm and nucleus
[53]. ROS production results from the activation of signalling
through the epidermal growth factor (EGF), PDGF, and vascular
endothelial growth factor (VEGF) receptors. Moreover, ROS induce VEGF expression in vitro and in vivo [54].
DNA Damage
Several studies showed that ROS directly interact with DNA,
producing structural alterations including small-scale insertions,
DNA base pair deletions, base modifications, chromosomal
changes/loss, microsatellite instability, and translocation of segments [55]. As a result of the normal aerobic metabolism, even
without oxidative stress, the oxidants generated are known to produce hundreds of hits per cell per day, inducing more than 100 different oxidative modifications in DNA [56]. In general, OH radical
is the major source of DNA damage that attacks phosphate, deoxyribose, and base sites, resulting in strand breakage. These oxidatively damaged DNA lesions are efficiently excised by a DNA glycosylase (AP lyase). The hydroxylation of guanine in the 8-position
is the frequent mutagenic lesion induced by oxidative species. The
presence of 8-OH-G in human urine was first reported by Ames and
co-workers [57]. This oxidized DNA product is important because
it is both relatively easily formed and is mutagenic and carcinogenic. It is a good biomarker of oxidative stress of an organism and
a potential biomarker of carcinogenesis. Furthermore, it was established that DNA damage produced by activated phagocytes is a
result of attack by OH radical [58]. RNS generated by inflammatory cytokines may induce point mutations and may also damage
some DNA repair proteins [59]. Epidemiologic and experimental
studies have shown that exposure to air pollutants increases the risk
of DNA damage resulting from free radical formation [60]. Prahalad et al demonstrated that PM can cause DNA damage and that
this effect was inhibited by an OH scavenger and metal ion chelators, suggesting a role for PM-generated free radicals and metals
adsorbed onto the particles [61]. Further evidence also showed that
PM caused increased DNA oxidative damage to human airway
epithelial cells and was associated with the amount of water-soluble
metals contained on these particles [62]. DNA damage has also
been shown in lung epithelial cells exposed to O3, and this effect
was reduced by pretreatment with vitamins C and E [63]. It had also
been reported that DNA backbone cleavages caused by O3 were
dependent on hydroxyl radicals, whereas DNA base modifications
were mainly caused by a direct effect of O3 [64]. Furthermore,
DNA-protein cross-link has been shown in the lungs of mice exposed to SO2 [65]. DNA damage may alter gene and protein expression as well as cell death.
Cell Signaling
Cells communicate with each other and respond to extracellular
stimuli through biological mechanisms called cell signalling or
signal transduction [66]. Signal transduction is a process enabling
information to be transmitted from the outside of a cell to various
functional elements inside the cell. Signal transduction is triggered
by extracellular signals such as hormones, growth factors, cytokines
and neurotransmitters [67]. Signals sent to the transcription machinery responsible for expression of certain genes are normally
transmitted to the cell nucleus by a class of proteins called transcription factors. These signal transduction processes can induce
various biological functions, such as muscle contraction, gene expression, cell growth and nerve transmission [66]. The initiation
and/or proper functioning of several signal transduction pathways

rely on ROS as signalling molecules, which may act at different
levels of the signal transduction cascade. ROS thus play a very
important physiological role as second messengers [68]. ROS were
shown to function as important modulators of signaling mechanisms in several pulmonary diseases including cancer. ROSmediated signaling activates pathophysiological events such as cell
proliferation, apoptosis, cytokines, and transcription of several
genes that are often kept at baseline activity or silent. The signaling
cascades triggered by ROS lead to the activation and phosphorylation of MAPKs, including ERK. This consequently results in activation of transcription factors including NF-B and AP-1 that may
lead to the induction of early response genes such as c-jun and cfos, which are involved in inflammatory influx, inhibition of apoptosis, cell proliferation, transformation, differentiation, and other
changes [69].
Serine/Threonine Kinases
Air pollutants and ROS can activate mitogen-activated protein
kinase (MAPK) signaling, which may ultimately promote inflammation. There are four known MAPK families (MAPKs): extracellular-regulated (ERKs), c-jun-NH2- terminal kinase (JNKs), p38
MAPK and the big MAPK- 1 (BMAPK-1). MAPK relay signals
generated by exogenous and endogenous stimuli into the cell by
phosphorylation of proteins. MAPK-catalyzed phosphorylation of
substrate proteins functions as a switch to turn on or off the activity
of the substrate protein. The substrates include other protein
kinases, phospholipases, transcription factors and cytoskeletal proteins. During this process of intracellular communication, MAPK
interact with upstream mediators, including growth factor receptors,
G-proteins, tyrosine kinases and ROS [70]. Disregulation of MAPK
function has been reported for skin, breast and cervical cancers in
humans. Products of NOX1 activity, superoxide, hydrogen peroxide
can activate the MAPK cascade at the level of MEK and ERK1/2.
The experimental studies on the up regulation of MAPKs by H2O2
treatment have shown that the activation of each signalling pathway
is type- and stimulus-specific. For example, it has been reported
that endogenous H2O2 production by the respiratory burst induces
ERK but not p38 kinase activity [71]. Conversely, exogenous H2O2
activates p38 kinase, but not ERK in rat macrophages [72]. The
ERK pathway has most commonly been associated with the regulation of cell proliferation. The balance between ERK and JNK activation is a key factor for cell survival since both a decrease in ERK
and an increase in JNK is required for the induction of apoptosis.
Moreover, inhibition of c-Jun N terminal kinase in mice attenuated
O3-induced inflammation and hyper-responsiveness [73]. In addition, end products of lipid peroxidation activate extracellular signalregulated kinase p44/42 (Erk1/2), c-Jun N terminal kinase, and
p38MAPK, and activation can be blocked by N-acetyl cysteine
[74]. Activation of these kinases was also accompanied by increased DNA binding activity of the transcription factor activator
protein 1, which can lead to the transcription of stress response
genes including phase II enzymes.
Activator Protein 1 and Nuclear Factor Kappa B
AP-1 and NF-B are important transcription factors that are
sensitive to ROS. Oxidants and some inflammatory cytokines such
as TNF- were reported to (1) activate NF-B and AP-1 and (2)
modulate the expression of both pro-inflammatory and antioxidant
genes [75]. This oxidant-mediated gene expression was proposed to
be regulated by the degree of acetylation of histones to facilitate
DNA binding. In contrast, NO was implicated in the suppression
of NF-B activation by limiting degradation of IB [76]. AP-1 is
composed of oncogene proteins such as c-jun and c-fos and is activated by oxidants such as H2O2. Activation plays an important role
in the induction of neoplastic transformation and initiation of several genes involved in cell proliferation, differentiation, apoptosis,
inflammation, and carcinogenesis [77]. It was reported by Wilhelm
ROS and Lung Injury
et al. (1997) that perturbation of cellular thiol redox status induces

stress-activated signal transduction pathways by JNK and p38
kinase that in turn provide a signal for AP-1 activation. This activation led to the induction of genes for cytokines, chemokines, and
various pro-inflammatory mediators that play an important role in
the inflammatory response [78, 79]. NF-B is another vitally important transcription factor that governs the expression of several
genes involved in cell development, intercellular communications,
apoptosis, and carcinogenesis [80]. In cells, NF-B is normally
bound to the inhibitory protein IB in the cytoplasm. ROS activate
NF-B by rapid phosphorylation, ubiquitination, and subsequent
proteasomal degradation of the inhibitory protein (IB). This is
followed by the translocation of NF-B to the nucleus, where it
activates gene transcription. NF-B regulates the expression of
many genes involved in lung inflammation including iNOS, IL-1,
TNF- and IL-6, IL-8, E-selectin, vascular cell adhesion molecule1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) [80,
81]. Persistent production and elevated levels of ROS result in the
activation of NF-B and AP-1, which through the activation of
various proinflammatory cytokines produces chronic inflammation
that subsequently culminates in tumor development.
iNOS Over-expression
Lung cells, such as macrophages play an important role not
only in killing foreign organisms but also in initiating the immune
response. These cells have the capacity of secreting a large number
of multifunctional compounds which include enzymes, prostaglandins, cytokines and reactive oxygen and nitrogen metabolites. Nitric
oxide (NO) is generated during immune and in inflammatory responses in different organs where its function is extremely complex
and, especially in lung tissue still not well understood. NO appears
to be involved in a wide spectrum of physiological processes within
the human airways. The small messenger molecule acts as a vasodilator through the activation of soluble guanylate cyclases in vascular smooth muscle cells, and the same mechanism may also account
for the bronchodilator effect of the free radical. In certain types of
inflammation, for example in asthma, high levels of NO are produced. NO has also been implicated as a pro-inflammatory or immunosuppressive agent via its inhibitory or apoptotic effects on
cells, and in particular NO generation may participate in the progression of acute lung injury. There are three types of nitric oxide
synthases (NOS): two of them are constitutively expressed while
the inducible form (iNOS) is expressed only in activated cells. At
high concentrations, like those produced under aerobic conditions,
NO is rapidly oxidized to reactive nitrogen oxide species (RNOS).
These species are unstable and rapidly react with thiol or amine
groups, to form nitrosothiols or nitrosoamines, or are hydrolyzed
and excreted as nitrites. It has been shown that high RNOS concentrations induce cell toxicity by nitrosating DNA and tyrosine residues, and inducing lipid peroxidation. Under conditions of combined nitrosative and oxidative stress, superoxide anions induce the
oxidative inactivation of NO promoting peroxynitrite formation.
This highly reactive oxidant molecule is thought to mediate many
of the most severe toxic effects of NO.
iNOS is an inflammatory and immunomodulatory mediators
that cause many biological effects in mammals. Several agents that
produce oxidative stress induce iNOS expression. A number of
studies have indicated that iNOS pathways are also co-induced in
vivo in different models that involve both inflammation and oxidative stress conditions. The transcriptional activator nuclear factorB (NF-B) controls the expression of several pro-inflammatory
cytokines, as well as the expression of inducible enzymes, such as
iNOS.
Nuclear Factor Erythroid-2 Related Factor (NRF)2,
Another important transcription factor involved in the response
to oxidative stress is NRF2. NRF2 contributes to the oxidative
3893
stress response through its binding of antioxidant response elements, leading to the induction of various genes involved in mitigating oxidative damage[82]. Oxidant-induced activation of NRF2
leads to the transcription of genes for antioxidants, DNA damage
recognition, glutathione homeostasis, free radical metabolism, and a
number of other elements involved in the oxidative stress response
[83]. Mutations in NRF2 or a disruption of the signaling pathway
would likely render individuals more susceptible to the adverse
effects of pollutant exposure. Further investigation of pollutantinduced activation of NRF2 and the consequences of NRF2 mutation in the response to pollutant exposure is needed to elucidate
fully the role of NRF2 in mitigating the effects of oxidant pollutant
exposure.
p53
The nuclear factor plays a key role in protecting a cell from
tumorigenesis [84]. Due to its ability to halt the cell cycle or initiate
apoptosis if cell is damaged, it is often called a tumor suppressor.
Mutations in p53 leading to its inactivation has been found in more
than half of human cancers[85] . Many studies have been devoted
to mutations in p53 caused by direct action of ROS [187]. ROS
have been correlated with p53-mediated apoptosis [86]. Upon over
expression of p53, levels of ROS rise, and inhibition of ROS by
antioxidants inhibits apoptosis in smooth muscle cells.
4. INFLAMMATION AND LUNG DISEASES: ROLE OF
OXIDATIVE STRESS
ROS and Inflammation
ROS are involved in a wide spectrum of diseases, including
chronic inflammation, and in a wide variety of cancers. Chronic
inflammation is induced by biological, chemical, and physical factors and is in turn associated with an increased risk of several human cancers [87]. The link between inflammation and cancer has
been suggested by epidemiological and experimental data [88, 89]
and confirmed by anti-inflammatory therapies that show efficacy in
cancer prevention and treatment [90]. Virchow first noted that inflammatory cells are present within tumors and that tumors arise at
sites of chronic inflammation [91]. This inflammation is now regarded as a secret killer for diseases such as cancer. The exact
mechanisms by which a wound-healing process turns into cancer
are topics of intense research [90, 92], and possible mechanisms
include induction of genomic instability, alterations in epigenetic
events and subsequent inappropriate gene expression, enhanced
proliferation of initiated cells, resistance to apoptosis, aggressive
tumor basement membrane, and metastasis [93]. The sources of
inflammation are widespread and include microbial and viral infections; exposure to allergens, radiation, and toxic chemicals; autoimmune and chronic diseases; obesity; consumption of alcohol;
tobacco use; and a high-calorie diet [94]. In general, the longer the
inflammation persists, the higher the risk of cancer. Two stages of
inflammation exist, acute and chronic inflammation. Acute inflammation is an initial stage of inflammation (innate immunity), which
is mediated through the activation of the immune system. This type
of inflammation persists only for a short time and is usually beneficial for the host. If the inflammation lasts for a longer period of
time, the second stage of inflammation, or chronic inflammation,
sets in and may predispose the host to various chronic illnesses,
including cancer [95]. During inflammation, mast cells and leukocytes are recruited to the site of damage, which leads to a respiratory burst due to an increased uptake of oxygen and, thus, an increased release and accumulation of ROS at the site of damage
[92]. On the other hand, inflammatory cells also produce soluble
mediators, such as metabolites of arachidonic acid, cytokines, and
chemokines, which act by further recruiting inflammatory cells to
the site of damage and producing more reactive species. These key
mediators can activate signal transduction cascades as well as inducing changes in transcription factors, such as nuclear factor B
(NF-B), signal transducer and activator of transcription 3

(STAT3), hypoxia-inducible factor-1 (HIF-1), activator protein-1
(AP-1), nuclear factor of activated T cells, and NF-E2 related factor-2 (Nrf2), which mediate immediate cellular stress responses.
Induction of cyclo-oxygenase-2 and inducible nitric oxide synthase
(iNOS), aberrant expression of inflammatory cytokines (tumor
necrosis factor (TNF), interleukin-1 (IL-1), IL-6) and chemokines
(IL-8, CXC chemokine receptor 4 (CXCR4)), as well as alterations
in the expression of specific microRNAs have also been reported to
play a role in oxidative stress-induced inflammation [96]. This sustained inflammatory/ oxidative environment leads to a vicious circle, which can damage healthy neighboring epithelial and stromal
cells and over a long period of time may lead to carcinogenesis
[97].
ROS and Lung Diseases
Acute Lung Injury (ALI)
Acute lung injury (ALI) and its most severe form, the acute
respiratory distress syndrome (ARDS) are frequent complications in
critically ill patients and are responsible for significant morbidity
and mortality [98]. In the context of ALI/ARDS, there are many
potential sources of ROS, including itinerant and resident leukocytes (neutrophils, monocytes, and macrophages), parenchymal
cells (endothelial and epithelial cells, fibroblasts, and myocytes),
circulating oxidant-generating enzymes (xanthine oxidase), and
inhaled gases with high concentrations of oxygen that are often
used during mechanical ventilation There is much experimental
evidence supporting the role of oxidants and oxidative injury in the
pathogenesis of ALI/ ARDS. Evidence of oxidative stress and injury is common to most experimental models of lung injury and is
corroborated in studies of patients with ALI/ARDS, underscoring
the physiologic importance of these processes [99].
Markers of oxidative damage have been detected in patients
with ARDS at levels significantly in excess of those found in controls. These include loss of thiol groups [100, 101] with increased
carbonyl formation in plasma proteins [102]; increased levels of
lipid peroxidation products have also been detected in plasma [103]
and exhaled breath [104] from patients with ARDS. Markers of
ROS and RNS formation have also been detected at elevated levels
in lung lining fluid from patients with ARDS [105]. Oxidants in the
form of H2O2 have also been directly detected in the exhaled breath
of patients with ARDS [106]. Hypoxanthine, a product of purine
catabolism, is a substrate for the ROS-producing enzyme xanthine
oxidase, and is a marker of ischemia. This is elevated in the plasma
of patients with established ARDS and correlates with poor survival
rates [107]. Tissue culture experiments and in vivo models of
ARDS also strongly suggest that ROS and RNS contribute to the
pathogenesis of the syndrome [108]. Inflammatory mediators such
as cytokines, chemokines, and adhesion molecules expressed during
ARDS can also indirectly mediate production of ROSs [109] and
thereby lead to further damage. High concentrations of TNF- and
IL-1 have been detected in high concentrations of BALF of patients with ARDS. IL-6 levels in the circulation are known to be a
detector of ARDS of different etiologies such a sepsis and acute
pancreatitis. Recently, studies have suggested that pathogenesis of
ARDS involves enhanced production of ROSs and diminished antioxidant levels [106]. Patients with ARDS have high levels of hydrogen peroxide in exhaled air and urine [110] and high circulating
levels of 4-HNE [111]. Levels of antioxidant defense system including enzymes like superoxide dismutase and catalase as well as
other scavengers like glutathione and vitamins E and C have been
shown to drop with increasing levels of ROSs [112]. Iron is also
known to be a mediator of oxidative stress because it can catalyze
pro-oxidant reactions. Decreased plasma iron binding activity leading to decreased ability to prevent iron dependent ROS formation
has been detected in patients with ARDS. Transferrin receptor protein levels were found to be significantly increased in lung biopsies
of patients with ARDS, implicating iron as a mediator of oxidative

stress [113].
Asthma
Asthma is a chronic inflammatory airway disease, and oxidative
stress may be involved in its pathogenesis [114]. In bronchial
asthma, oxidative stress aggravates airway inflammation by inducing diverse proinflammatory mediators, enhancing bronchial hyperresponsiveness, stimulating bronchospasm, and increasing mucin
secretion [115, 116] .
Alveolar macrophages from asthmatic subjects show increased
release of O2 - and other ROS compared with those of healthy
controls [117, 118]. Eosinophils, alveolar macrophages and neutrophils from asthmatic patients produce more ROS than those from
normal subjects [119]. ROS are involved in airway smooth muscle
contraction, impairment of adrenergic receptor function, decreased numbers and function of epithelial cilia, increased mucus
production, altered release of inflammatory mediators, influx of
inflammatory cells and increased vascular permeability. Reactive
oxygen species cause direct contraction of airway smooth muscle
preparations and this effect is enhanced when the epithelium is
injured or removed [120]. Overproduction of ROS or depression of
the protective system also results in bronchial hyperreactivity which
is characteristic of asthma [120, 121]. Animal models have shown
that ROS contribute to airway hyperresponsiveness by increasing
vagal tone due to damage of oxidant-sensitive b-adrenergic receptors, as well as decreasing mucociliary clearance [122, 123]. H2O2
causes contraction of airway smooth muscle and has been implicated in airway hyperresponsiveness in animal models [36]. Reactive oxygen species appear to directly stimulate histamine release
from mast cells and mucus secretion from airway epithelial cells
[124]. Increased generation of ROS can result in direct oxidant
damage and shedding of epithelial cells [125]. Many studies suggest that levels of oxidative stress are increased in children and in
adults with asthma, not only in their lungs but also in the circulation. Excessive production of oxidants occurs spontaneously or
after stimulation in blood leucocytes of stable asthmatics compared
with normal subjects [1, 126, 127]. Levels of eosinophil peroxidase
(EPO) and/or MPO are increased in the peripheral blood, induced
sputum, and bronchoalveolar lavage (BAL) fluid from patients with
stable asthma [128]. Recently, increased levels of many direct and
indirect markers of oxidative stress (including malondialdehyde,
thiobarbituric acid reactive products (TBARs), and H2O2) have
been found in the urine, plasma, sputum, BAL fluid, and lung tissues of patients with asthma [126, 127]. Their level is often related
to the severity of the disease and is inversely related to the degree
of stability[126, 127]. Furthermore, analysis of exhaled breath and
exhaled condensate has recently allowed direct assessment of H2O2
and nitric oxide (NO) and measurement of several indirect byproducts of oxidation in those samples [129, 130]. The latter are footprints of oxidation on different substrates such as proteins (nitrotyrosine), lipids (isoprostanes and ethane), and DNA (hydroxydeoxyguanosine) [129, 130]. There is a lot of interest in the effect of these
new non-invasive markers on oxidative stress and in their possible
clinical application [129-131] as their concentrations are significantly increased in asthma. The expression of nitrotyrosine is increased also in bronchial and bronchiolar epithelial cells, in smooth
muscle cells, and in eosinophils of bronchial airways and lung parenchyma of patients with stable asthma [132-134]. Excessive production of 3-bromotyrosine, another marker of oxidative stress, has
been reported in the airways of patients with severe asthma [135].
A significant increase in 3-bromotyrosine has also been described
in the proteins of sputum of asthmatic subjects. External oxidant
stimuli also worsen existing allergic disease. For example, O3 may
evoke asthma exacerbations. Ozone inhalation was shown to increase airway hyperresponsiveness (AHR); induce higher IL-5,
GM-CSF, and granulocyte-colony stimulating factor levels; and
ROS and Lung Injury
indirectly enhance the longevity of eosinophils via suppressing

apoptosis in a mouse model of allergic asthma [52].
Chronic Obstructive Pulmonary Disease (COPD)
COPD has been described as an organ-specific pathology of the
lung with chronic and acute inflammation of the airways, but recently it has been recognized that a number of patients show features of systemic inflammation [136]. The classical definition of
COPD is a chronic and progressive reduction in airflow, secondary
to an abnormal inflammatory response of the lungs to the inhalation
of noxious particles or toxic gases. This inflammation produces
alterations of varying severity in the bronchi (chronic bronchitis),
bronchioles (obstructive bronchiolitis), lung parenchyma (emphysema), or any combination of the three [137]. COPD can be classified into four classes of severitybased on lung function. Emphysema, chronic bronchitis with airway obstruction, and small airways
disease are the distinct phenotypes of COPD, but most patients
show a combination of different phenotypes [138]. Four principal
mechanisms are responsible for the alterations observed in COPD:
oxidative stress; inflammation; protease-antiprotease imbalance;
and apoptosis [139]. The relative contribution of each of these
mechanisms varies and possibly explains the different forms of
presentation of the disease. Oxidative stress has been attributed a
central role in the pathogenesis of COPD because, in addition to
causing direct injury to the respiratory tract, oxidative stress triggers and exacerbates the three other mechanisms mentioned previously [140, 141]. The importance of oxidative stress has been confirmed by several studies that have identified the presence of oxidative stress/free radical biomarkers in patients with COPD. Oxidative
stress can be measured through direct quantification of the production of oxidants or, indirectly, through quantification of the products resulting from lipid peroxidation, such as 8-isoprostane, 4HNE and MDA, in the alveolar space, in the exhaled air, in the
sputum and in the blood [142]. In addition to their importance as
markers of oxidative stress, isoprostanes, particularly 8-isoprostane,
has been reported as being one of the mediators of the reversible
component of the obstruction observed in most patients with COPD
[143]. Markers of oxidative stress and of DNA damage are significantly higher in patients with COPD, especially in those whose
causative factor is smoking [144]. Patients with COPD present a
high incidence of lung neoplasia, and DNA modifications induced
by the reactive species might be the link between these two conditions. In individuals who develop COPD, there is a marked exacerbation of the inflammatory response, which increases with the progression of the disease [145]. The molecular mechanism of this
exacerbation remains unknown; however, genetic factors, latent
viral infections and prolonged oxidative stress have been reported
as being potentially responsible for the exacerbation [139]. Chronic
inflammation in COPD is associated with an increase in the production of various mediators and proinflammatory proteins, including
cytokines, chemokines, inflammatory enzymes, receptors and adhesion molecules, which are regulated by gene transcription factors
[139]. Among the mediators, those that are chemotactic for inflammatory cells, in particular leukotriene B4 and IL-8, as well as
proinflammatory cytokines, such as TNF-, IL-1 and IL-6, are
noteworthy [146]. Growth factors, including TGF-, which induces
fibrosis in the small airways, are also considered important [146].
The most important cellular elements in the inflammation in COPD
are epithelial cells, neutrophils, alveolar macrophages, CD8 lymphocytes and, during periods of exacerbation, eosinophils [146].
There is strong evidence that an imbalance between proteases and
endogenous antiproteases plays an important role in the pathogenesis of COPD. Three classes of proteases are considered relevant to
the etiopathogenesis of COPD: serine proteases, which can degrade
elastin and certain forms of collagen; cysteine proteases, which
degrade matrix components; and matrix metalloproteinases, which
act on collagen, gelatin and laminin. Oxidants can potentiate the
effects of proteases on COPD through the activation of these en-
3895
zymes. Systemic markers of oxidative stress and elevated plasma

levels of inflammatory mediators have been reported in smokers
and in patients with COPD [147]. The peripheral neutrophils of
patients with COPD release more ROS than do those of normal
nonsmokers [148]. Furthermore, the levels of lipid peroxidation
products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA)
were increased in lungs of patients with COPD, and this increase
was negatively correlated with lung function [149, 150]. These
reactive aldehydes deplete thiol pool, carbonylate proteins or form
aldehyeprotein adducts leading to an alteration of protein function
and causing a variety of cellular and biochemical effects including
immunogenicity and proteolysis thereby inducing lung inflammatatory and autoimmune responses, and injury. Oxidative stress and
chronic inflammation are some of the factors involved in the
mechanism that generates the systemic manifestations (e.g., weight
loss and skeletal muscle dysfunction) observed in some patients
with COPD. Patients with COPD are also at increased risk of cardiovascular disease [151]. One of the probable mechanisms for this
increase is the endothelial damage caused by systemic inflammation and systemic oxidative stress in these patients [151].
Idhiopatic Pulmonary Fibrosis (IPF)
Pulmonary fibrosis is the end result of a diverse group of lung
disorders. Although there are multiple initiating agents for pulmonary fibrosis, including toxins, fibres/particles, autoimmune reactions, drugs and radiation, the aetiology of the majority of cases of
pulmonary fibrosis is unknown. Several studies have suggested that
oxidant antioxidant imbalance in the airways plays a critical role
in the pathogenesis of IPF [152, 153]. For example, pulmonary
inflammatory cells of patients with IPF generate higher levels of
oxidants than those in control patients [152]. Bronchoalveolar lavage fluid of patients with IPF show elevated levels of myeloperoxidase and eosinophil cationic protein, suggesting a pathophysiologic
role for neutrophils and possibly eosinophils in this disease [154].
There is also evidence that increased myeloperoxidase is associated
with epithelial injury in IPF [152]. Mitochondrial generation of
ROS has been suggested to be associated not only with increased
cellular oxidative stress but also with apoptosis of alveolar epithelial cells [155]. Bronchoalveolar lavage fluid of patients with IPF
contains higher levels of 8-isoprostane, a biomarker of oxidative
stress, than that of control subjects [153]. Patients with IPF have
also been shown to have elevated levels of exhaled NO [129]. In
addition, lung specimens of the patients with IPF show elevated
expression of iNOS [156]. These findings suggest that patients with
IPF have increases in both oxidative and nitrosative stress. Oxidants
may contribute to the development of pulmonary fibrosis due to
their effects on the production of cytokines and growth factors such
as TGF-, a key regulator of aberrant repair mechanisms that are
characteristic of many fibrotic diseases including IPF. There are
several potential interactions between TGF- and oxidants/antioxidants in the lung. TGF- not only induces ROS production by activation of NADPH oxidases and/or mitochondrial
dysfunction, but also decreases natural cellular antioxidant production through decreased expression of both catalase and mitochondrial SOD [31, 157]. Increased levels of oxidized proteins have
been reported in human subjects with IPF [158]. In addition, some
studies have reported that various antioxidant enzyme systems protect against lung fibrosis [159]. IPF subjects also have lower antioxidant capacity than healthy subjects [160].
Cystic Fibrosis
Lung dysfunction in cystic fibrosis consists of progressive airflow obstruction caused by the accumulation of thick, viscous secretions, and the gradual destruction of the bronchiolar and larger
airways [161]. Endobronchial infection, particularly in association
with colonization by Pseudomonas aeruginosa, leads to damage of
the airways and the gradual deterioration of pulmonary function.
Oxidative stress, caused by toxic reactive oxygen species (ROS), is
believed to play an important role in the pathophysiology of cystic
sion of DNA methyltransferases, leading to a global hypermethylation of the genome [91]. This leads to promoter silencing of several
genes, such as adenomatous polyposis coli, cyclindependent kinase
inhibitor-2, breast cancer susceptibility gene 1, retinoblastoma protein, murine double minute 2 (MDM2), and the DNA mismatch
repair gene, human mutL homolog 1 [167]. On the other hand, low
or transient levels of ROS can activate cellular proliferation or survival signaling pathways, such as the NF- B, AP-1, extracellular
signal-regulated kinase/mitogen-activated protein kinase, and phosphoinositide 3-kinase/Akt8 virus oncogene cellular homolog pathways. For example, H2O2 is able to degrade IB, the inhibitory
subunit of NF-B [168]. Protein kinase C, which participates in a
variety of pathways regulating transcription and cell cycle control,
is also activated by H2O2 [168]. In addition, ROS induce both the
activation and synthesis of AP-1, a regulator of cell growth, proliferation, and apoptosis [169], and transcription factors such as
STAT3, HIF-1, and p53 [170, 171]. Cancer is a multistage process
defined by at least three stages: initiation, promotion, and progression [172, 173]. Oxidative stress interacts with all three stages of
this process. During the initiation stage, ROS may produce DNA
damage by introducing gene mutations and structural alterations
into the DNA. In the promotion stage, ROS can contribute to abnormal gene expression, blockage of cell-to-cell communication,
and modification of second-messenger systems, thus resulting in an
increase in cell proliferation or a decrease in apoptosis of the initiated cell population. Finally, oxidative stress may also participate in
fibrosis (CF)[162, 163]. It is well-established that when activated,

neutrophils are a major source of free radicals, and though considered a potent mechanism for killing organisms, they may also damage the pulmonary epithelium in these patients [163]. The damaging effects of increased free radical production may be exacerbated
by a systemic deficiency of the major respiratory antioxidant, glutathione in patients with cystic fibrosis. Oxidative Indices suggesting that there is increased oxidative stress in CF include elevated
plasma malondialdehyde (MDA) levels [164], elevated breath pentane and ethane, elevated plasma levels of hydroperoxides [164]
and depletion of the major lipoperoxidation substrates, such as linoleic and arachidonic acid [164]. Recent observations have suggested
that 8-isoprostane (of which 8-iso- PGF2a is the most well-known
isomer) is also a useful marker of oxidative stress in CF [165].
Lung Cancer
After an inflammatory stimulus, initiation of carcinogenesis
mediated by ROS may be direct (oxidation, nitration, halogenation
of nuclear DNA, RNA, and lipids) or mediated by the signaling
pathways activated by ROS. Direct effects of ROS, generally attributed to high concentrations at the site of damage, include DNA
strand breaks, point mutations, aberrant DNA cross-linking, and
mutations in proto-oncogenes and tumor-suppressor genes, thus
promoting neoplastic transformation [166]. For example, ROS can
reduce the expression and enzymatic activity of the DNA mismatch
repair genes mutS homologs 2 and 6 and can increase the expres-
ROS
Protein damage
HsN+ Gly Ile Val Cys Glu Gin
Ala
S
Ser
S
Val Cys
Leu
Pro
Arg Asp
Lye
Phe
Tyr
Thr Leu His
Lys
Arginine
Nitric oxide
synthase
O2
Asn -COO-
ONOO-
NO
e-
._ e-
O2
H2O2
Super oxide
dismutase
e-
H2O
Catalase
2+
Fe
OH-
Mitochondrial
damage
DNA damage
Membrane
damage
Inflammation
CANCER
Lipid
peroxidotion
Fig. (1). Role of reactive oxygen species (ROS) in mediating oxidant-induced lung injury and disease conditions. PF, pulmonary fibrosis, COPD, chronic
obstructive pulmonary disease.
ROS and Lung Injury
the progression stage of the cancer process by adding further DNA

alterations to the initiated cell population [174]. In lung cancers,
p53, which is associated with the production of ROS, is often mutated and defective in inducing apoptosis. When mutated, p53 accumulates in the cytoplasm and functions as an oncogene [175].
Proteins and lipids are also major targets for oxidative attack, and
modification of these molecules may increase the risk of mutagenesis, through formation of genotoxic lipid peroxidative by-products
that react with DNA, oxidative modification of DNA polymerase or
inhibition of DNA repair enzymes [176].

[14]
[15]
[16]
[17]
[18]
[19]
5. CONCLUSION
Oxidative stress can cause cellular damage by oxidizing nucleic
acids, proteins, and membrane lipids. ROSs have been implicated in
the pathogenesis of many diseases and important biological processes including carcinogenesis and inflammatory disorders (Fig. 1).
ROS appear to be key regulatory factors in the molecular pathways
leading to the induction of lung diseases, and offer potential points
for therapeutic intervention. Future work to understand the molecular mechanisms of ROS-mediated pathophysiological pathways and
their control by various antioxidants may aid in the design of novel
therapies that target the respective molecular pathways.
[20]
[21]
[22]
[23]
[24]
COMPETING INTERESTS
The authors declare that they have no competing interests.
[25]
ACKNOWLEDGEMENTS
The authors would like to thank Carmelo La Spada and Giovanni Leotta for their excellent technical assistance during this
study, Mrs Caterina Cutrona for secretarial assistance and Miss
Valentina Malvagni for editorial assistance with the manuscript.
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Current Pharmaceutical Design, 2012, 18, 3889-3900

Reactive Oxygen Species, Inflammation, and Lung Diseases

Keywords: Oxidative stress, inflammation, DNA damage, lung diseases.

hydrogen peroxide H2O2, either spontaneously or in a much faster

3890 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

oxidase (XO),generating large amounts of H2O2 as well as O2. It is

Rosanna and Salvatore

Eosinophils are also a potent cellular source of ROS, especially

ROS and Lung Injury

Current Pharmaceutical Design, 2012, Vol. 18, No. 26

3892 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Rosanna and Salvatore

and/or proper functioning of several signal transduction pathways

ROS and Lung Injury

et al. (1997) that perturbation of cellular thiol redox status induces

Current Pharmaceutical Design, 2012, Vol. 18, No. 26

3894 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

(NF-B), signal transducer and activator of transcription 3

Rosanna and Salvatore

of patients with ARDS, implicating iron as a mediator of oxidative

ROS and Lung Injury

indirectly enhance the longevity of eosinophils via suppressing

Current Pharmaceutical Design, 2012, Vol. 18, No. 26

zymes. Systemic markers of oxidative stress and elevated plasma

3896 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Rosanna and Salvatore

fibrosis (CF)[162, 163]. It is well-established that when activated,

Thr Leu His

ROS and Lung Injury

the progression stage of the cancer process by adding further DNA

Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Henricks PA, Nijkamp FP. Reactive oxygen species as mediators in

Ivanov I, Saam J, Kuhn H, Holzhutter HG. Dual role of oxygen

3898 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Ergonul Z, Erdem A, Balkanci ZD, Kilinc K. Vitamin E protects

Rosanna and Salvatore

ROS and Lung Injury

Gonda TA, Tu S, Wang TC. Chronic inflammation, the tumor

Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Omenaas E, Fluge O, Buist AS, Vollmer WM, Gulsvik A. Dietary

3900 Current Pharmaceutical Design, 2012, Vol. 18, No. 26

Received: March 12, 2012

Accepted: March 21, 2012

Rosanna and Salvatore

Koli K, Myllarniemi M, Keski-Oja J, Kinnula VL. Transforming

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