The embryonic stage of lung development (26 days ≈ 6 weeks) begins when the respiratory diverticulum, or lung bud, appears as an outgrowth from the ventral wall of the foregut. This stage is followed by the separation of the lung bud from the foregut, thus forming the trachea (windpipe) and bronchial buds, which successively enlarge at the beginning of the fifth week to form the main bronchi. The embryonic stage is marked by the formation of the lobular and segmental sections of the respiratory tree as columnar-epithelium-lined tubes evident by the end of the fifth or sixth week 5.

The pseudoglandular stage roughly begins at the fifth/sixth week of gestation and lasts up until 16th/17th week. What marks this period are the histological appearance of the fetal lungs as an exocrine gland and the completion of the proliferation of the primitive airways. At this stage, cartilage is formed around the larger airways and smooth muscles begin to envelop airways and blood vessels. Upon completion of this stage, acinar outlines first begin to appear as epithelial tubes continue to grow and branch. The undifferentiated columnar epithelial cells lining the tubular glandular structures are destined to evolve into the many cell types that populate the airways, including serous, goblet, ciliated, Clara and alveolar cells 568.

The canalicular development stage comprises the period commencing on the 16th/17th week and continuing to the 25th-27th weeks of gestation. The enlargement of the lumina of bronchi and terminal bronchioles characterizes the canalicular stage, in addition to the formation of capillaries at the site of the future air space and the appearance of surfactant, representing the major developments that are absolutely crucial to extra-uterine life. During this stage, in addition, the acini subdivisions are formed, and the epithelial lining begins to differentiate into alveolar type I (ATI) and II (ATII) cells 56.

The saccular stage, or terminal sac stage (28th-35th week of gestation), represents the development of terminal air sacs from alveolar ducts, refinement of gas exchange sites, a decrease in the thickness of the interstitium, thinning of the epithelium and separation of the terminal air units. This stage also marks the terminal differentiation stages of alveolar ATI and ATII epithelial cells.

The final 5 weeks of fetal lung development, termed the alveolar period, encompass the alveolar stage in which millions of alveoli are formed, with the surface area increased by thinning of the septal walls and attenuation of the cuboidal epithelium. The terminal subsaccules are now separated by loose connective tissue and cellular maturation continues specifically with ATII cells developing a greater density of lamellar bodies 56.

Concomitant with the development of various lung structures is the cellular differentiation of ATI and ATII cells occurring as the alveolar epithelium matures. During the first four months of gestation the epithelial lining is more or less columnar to cuboidal 567. By six months, ATI and ATII cells can be relatively distinguished in the more localized differentiated zones of pseudo-cuboidal cells.

Accumulating evidence in recent years has linked the pathogenesis of some human diseases to increased oxidative stress 5815222733343536. In particular, ROS, which are partially reduced metabolites of oxygen consumption, may contribute to alveolar-capillary membrane disturbances and the development of lung injury 58102235. A wealth of data has drawn attention to both the significance of maintaining reducing conditions in cells and the fight against the damaging effect of ROS intermediates 172235373839. Oxidants, for instance, can cause carcinogenesis, sclerosis, Alzheimer's disease and other neurological disorders, acute lung injury and chronic lung diseases 58172227333738. Oxidative cell injury involves the modification of cellular macromolecules by ROS, often leading to cell death and the lysis of sensitive cells, resulting in microvascular and alveolar perturbations 5817223839. Oxidative stress appears to increase in the lung, the level of antioxidants in some experimental models, and hypoxia and hyperoxia modulate fetal lung growth 141617212327. Furthermore, there is growing evidence supporting the concept of cross-talk between oxidative stress and upregulation of a proinflammatory signal through the participation of cytokines 343536394041424344.

Cytokines are peptide hormones that participate in autocrine and paracrine signaling 42434546. They are major participants in the pathophysiology of respiratory distress and have been recognized as signaling molecules responsive to dynamic variation in pO₂34353940414244. Examples of such cytokines are interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, transforming growth factor, and granulocyte-macrophage colony-stimulating factor. Cytokines and other inflammatory mediators play important (not necessarily inflammation-related) roles not only during fetal life, but also in the initiation of labor and in neonatal immunity and diseases 36373840424546. Hematopoietic growth factors, for example, regulate the maturation of progenitors in fetal and neonatal hematopoietic organs 364245. Cytokines act as extrahematopoietic growth factors and as modulators of fetomaternal tolerance and are involved in selective apoptosis during tissue remodeling 34363738. Inter-regulation of cytokine networks is therefore critical for normal function and maturation of neonatal host defenses. Neonates initially depend on natural (innate) immunity and antigen-specific immunity develops later in life 36424546. Cytokines regulate innate immunity and connect it with antigen-specific adaptive immunity 34364546. This integral association between oxidative stress and a proinflammatory state may affect cellular redox equilibrium, thereby imposing a direct role in modulating the pattern of gene expression in lung tissues; accordingly, this could be pivotal in determining cellular fate under these conditions 231314161718192627343940414243444546.

As the fetus leaves the hypoxic environment and enters the relatively hyperoxic environment during the transition from placental to lung-based respiration, it is imperative that it develops antioxidant mechanisms to guard against the potential harm posed by oxygen-derived species 13141920. Defense mechanisms include, for example, the reduction of ROS by antioxidant enzymes such as catalase, manganese-, copper- and zinc-containing superoxide dismutase (Mn-SOD/Cu-SOD/Zn-SOD), and the redox-sensitive enzyme glutathione peroxidase 2313161718192737. ROS may not, however, pose a real threat to the fetus if these endproducts are detoxified and balanced against the amount of ROS generated 2227373840.

In keeping with this idea, the tripeptide L-γ-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), a ubiquitous thiol, plays an important role in maintaining intracellular redox equilibrium and has evolved as one of the major detoxifying antioxidants and abundant thiols in almost all mammalian cells 1314404147484950. Glutathione determines intracellular redox potential and detoxifies harmful ROS by the glutathione-peroxidase-coupled reaction (Fig. 1). Oxygen signaling across membranes of intercellular compartments may be linked to a certain redox state that might be crucial in regulating the magnitude and pattern of gene expression of oxygen- and redox-responsive transcription factors 23131416171819. Such transcription factors are implicated in determining cellular responses under both physiological and pathophysiological conditions 23161718273751.

Redox-sensitive transcription factors are therefore likely to be differentially regulated by oxygen availability, to bind specific DNA consensus sequences and to activate the expression of several genes, particularly those controlling adaptive homeostasis in a hostile environment 2316171851. Among such factors, HIF-1α and NF-κB, whose activation states are differentially regulated under oxidative stress 52535455 are particularly important. HIF-1α, first identified in vitro through its DNA-binding activity expressed under hypoxic conditions 56 has its concentration and activity increased exponentially when oxygen tensions are decreased over physiologically relevant ranges 515253. The ubiquitous activation of HIF-1α is thus consistent with the significant role that this factor plays in coordinating adaptive responses to hypoxia 5253. NF-κB, on the other hand, was first identified as a transcription factor that regulates antibody release in B cells 57. It is central to the regulation and expression of stress-response genes in the face of inflammatory and oxidative challenge 13141617181927545558. Oxygen and redox regulation of HIF-1α and NF-κB will be comprehensively discussed after a brief discussion of the regulation of oxygen-sensing mechanisms.

Oxygen sensing and its underlying molecular stratagems have been the focus of experimental investigations trying to find an answer to the question: "What is the identity of the oxygen sensor?" 1232930315960616263. The first molecular mechanism to be proposed to underlie oxygen sensing in mammalian cells involves an oxygen sensor that is a heme protein 123596061. Studies on erythropoietin (EPO), a glycoprotein hormone required for the proliferation and differentiation of erythroid cells, demonstrated that EPO production is enhanced under hypoxic conditions 525359616465. EPO expression can be induced by transition metals such as cobalt (Co²⁺) and nickel (Ni²⁺), supporting the hypothesis that these metal atoms can substitute for the iron atom within the heme moiety, and that the oxygen sensor for the induction of EPO is a heme protein 5960616465. Further evidence supporting the notion that the oxygen sensor is a heme protein came with additional studies that utilized carbon monoxide (CO); CO can noncovalently bind to ferrous (Fe²⁺) heme groups in hemoglobin, myoglobin, cytochromes and other heme proteins 5960616263 where its ligation state is structurally identical to that of oxygen. It was subsequently proposed that CO might affect oxygen sensing by locking the sensor in an oxy conformation, which could involve a multisubunit mechanism 59606162636465 (Fig. 2).

In addition to the aforementioned models for oxygen sensing, certain pharmacological studies, led by Fandrey and colleagues 66 suggest that the oxygen sensor might involve a microsomal mixed-function oxidase. Based on these studies, it was proposed that oxygen sensing for EPO involves an interaction between cytochrome P450 and cytochrome P450 reductase, thereby allowing the conversion of molecular oxygen to superoxide anion (O₂^-•) and hydrogen peroxide (H₂O₂) radicals 59616667. Acker 59 has provided support based on spectroscopic evidence for the central role of an oxidase in oxygen sensing. It was reported that b-cytochrome functions as a NAD(P)H oxidase, converting oxygen to O₂^-•. The enzymatic complex in mammalian cells is membrane-bound and transduces the conversion of molecular oxygen to ROS, according to the following equations:

CytFe²⁺ + O₂ → CytFe²⁺O₂

CytFe²⁺O₂ → CytFe³⁺ + O₂^-•

CytFe³⁺ + NAD(P)H → CytFe²⁺ + NAD(P)⁺

A resurgence of interest in mitochondrial physiology has recently developed as a result of new experimental data demonstrating that mitochondria function as important participants in a diverse collection of novel intracellular signaling pathways. Further experiments showed a potential involvement of the mitochondria in oxygen sensing 68. For instance, a spectroscopic photolysis with monochromatic light has identified a CO-binding heme protein falling within the spectrum of the mitochondrial cytochrome a₃69. It was consequently proposed that this heme protein, presumably located on the plasma membrane, has a low affinity for oxygen and a relatively high affinity for CO (Fig. 2). The same model predicted that another heme protein in the mitochondria has a relatively higher affinity for oxygen and a lower affinity for CO 596061626364656667686970. The biochemical reaction, which was proposed as an alternative way of regenerating ferroheme in the oxygen sensor, is given below:

CO + 2Fe³⁺ + H₂O → CO₂ + 2Fe²⁺ + 2H⁺

These aforementioned observations pertaining to the mitochondrion as a possible oxygen sensor were unequivocally supported by novel studies recently reported by Schumacker, Chandel and colleagues 717273747576. Cardiomyocytes are known to suppress contraction and oxygen consumption during hypoxia 71. Cytochrome oxidase undergoes a decrease in V_max during hypoxia, which could alter mitochondrial redox status and increase generation of ROS. Duranteau and colleagues 71 tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of oxygen with the functional response. Contracting cardiomyocytes were superfused under controlled oxygen conditions while fluorescence imaging of 2,7-dichlorofluorescein was used to assess ROS generation. Compared with normoxia, graded increases in 2,7-dichlorofluorescein fluorescence were seen during hypoxia. In addition, the antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H₂O₂ mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible. To test the role of cytochrome oxidase, sodium azide was added during normoxia to reduce the V_max of the enzyme. It was observed that azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible, demonstrating that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggesting that cytochrome oxidase may contribute to this oxygen sensing mechanism 71.

The same group also recently reported that mitochondrial ROS trigger hypoxia-induced transcription. Chandel et al.72 tested whether mitochondria act as oxygen sensors during hypoxia and whether hypoxia and CO activate transcription by increasing generation of ROS. Results showed that wild-type Hep3B cells increased ROS generation during hypoxia or CoCl₂ incubation. Hep3B cells depleted of mitochondrial DNA (ρ⁰ cells) failed to respire, failed to activate mRNA for EPO, glycolytic enzymes or vascular endothelial growth factor (VEGF) during hypoxia and failed to increase ROS generation during hypoxia. The ρ⁰ cells increased ROS generation in response to CoCl₂ and retained the ability to induce expression of these genes. The antioxidants pyrrolidine dithiocarbamate (PDTC) and ebselen, a glutathione peroxidase mimetic, abolished transcriptional activation of these genes during hypoxia or CoCl₂ in wild-type cells and abolished the response to CoCl₂ in ρ⁰ cells 72. It was proposed that hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl₂ activates transcription by stimulating ROS generation via a mitochondria-independent mechanism 727374.

In another interesting observation, Chandel and colleagues reported that mitochondrial ROS play a major role in HIF-1α regulation 75. In this respect, it was observed that hypoxia increased mitochondrial ROS generation at Complex III, which caused the accumulation of HIF-1α protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element 75. Of note, this response was lost in cells depleted of mitochondrial DNA. Furthermore, overexpression of catalase abolished expression of the hypoxic response element-luciferase construct during hypoxia. In addition, exogenous H₂O₂ stabilized HIF-1α protein during normoxia and activated luciferase expression in wild type and ρ0 cells. In fact, isolated mitochondria increased ROS generation during hypoxia, indicating that mitochondria-derived ROS are both required and sufficient to initiate HIF-1α stabilization during hypoxia, thereby implicating this transcription factor as a possible oxygen sensor (see below) 70717273747576.

A nonmitochondrial oxygen sensor has, however, been recently proposed. Ehleben and colleagues applied biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent O₂^-• formation and localization of •OH by three-dimensional (3D) confocal laser scanning microscopy to reveal putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia 477. A cell membrane localized nonmitochondrial cytochrome b558 seemed to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563, probably probing additional metabolic changes. The hydroxyl radical (•OH), a putative second messenger of the oxygen-sensing pathway generated by a Fenton reaction, could be visualized in the perinuclear space of the three human cell lines used.

Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia-induced gene expression, interact on various sides of the oxygen-sensing pathway, confirming the importance of b-type cytochromes and the Fenton reaction. Furthermore, NADPH oxidase isoforms with different gp91 phox subunits, as well as an unusual cytochrome aa3 with a heme:aa3 ratio of 9:91, have been discussed as putative oxygen sensor proteins influencing gene expression and ion channel conductivity 78. ROS are believed to be important second messengers of the oxygen-sensing signal cascade determining the stability of transcription factors or the gating of ion channels. The formation of ROS by a perinuclear Fenton reaction was imaged by one- and two-photon confocal microscopy, revealing both mitochondrial and nonmitochondrial generation.

In reference to the aforementioned observation 78 some recent concepts on oxygen sensing mechanisms at the carotid body chemoreceptors have been highlighted 179. Most available evidence suggested that glomus (type I) cells are the initial sites of transduction and they release transmitters in response to hypoxia, which in turn depolarize the nearby afferent nerve ending, leading to an increase in sensory discharge. Two main hypotheses have been advanced to explain the initiation of the transduction process that triggers transmitter release. One hypothesis assumed that a biochemical event associated with a heme protein triggers the transduction cascade. Supporting this idea, it has been shown that hypoxia might affect mitochondrial cytochromes. In addition, there was a body of evidence implicating nonmitochondrial enzymes such as NADPH oxidases, nitric oxide (NO) synthases and heme oxygenases located in glomus cells 79. These proteins could contribute to transduction via generation of ROS, NO and/or CO. The other hypothesis suggested that a K⁺ channel protein is the oxygen sensor and inhibition of this channel and the ensuing depolarization is the initial event in transduction, as indicated by Peers and Kemp 1.

Several oxygen-sensitive K⁺ channels have been identified. Their roles in the initiation of the transduction cascade and/or in cell excitability remain unclear. In addition, recent studies indicated that molecular oxygen and a variety of neurotransmitters might also modulate Ca²⁺ channels 79. Most importantly, it is possible that the carotid body response to oxygen requires multiple sensors, and they work together to shape the overall sensory response of the carotid body over a wide range of arterial oxygen tensions.

The hypothesis that there exists a specific oxygen sensor(s), which relay(s) chemical signals intracellularly, is consistent with the notion that there is a unifying mechanism involved in transducing dynamic changes in pO₂ to the nucleus 70. In response to ΔpO₂, there is a coordinate expression of genes needed to confer appropriate responses to hypoxia or hyperoxia 23131416171819262752535455. The regulation of physiologically important oxygen-responsive and redox-sensitive genes would, therefore, dictate well controlled responses of the cell within a challenging environment and necessarily would determine the specificity of cellular adaptation 1231617181920282959606170717273747576777879.

In order to maintain oxygen homeostasis, a process that is, of course, essential for survival, pO₂ delivery to the mitochondrial electron transport chain must be tightly maintained within a narrow physiological range 23283470. This system may fail with subsequent induction of hypoxia, resulting either in a failure to generate sufficient ATP to sustain metabolic activities or in a hyperoxic condition that contributes to the generation of ROS, which, in excess, could be cytotoxic and often cytocidal 582234. Adaptive responses to hypoxia involve the regulation of gene expression by HIF-1α, the expression, stability and transcriptional activity of which increase exponentially on lowering pO₂5253568081.

HIF-1α is a mammalian transcription factor expressed uniquely in response to physiologically relevant hypoxic conditions 5253566467707172737475767778798081. Studies of the EPO gene led to the identification of a cis-acting hypoxia-response element (HRE) in its 3'-flanking region 5253678081 and HIF-1 was identified through its hypoxia-inducible HRE-binding activity 56. The HIF-1 binding site was subsequently used for purification of the HIF-1α and HIF-1β subunits by DNA affinity chromatography. Both HIF-1 subunits are basic helix-loop-helix PAS (an acronym for the first three family members, namely Per/ARNT/Sim) proteins: HIF-1α is a novel protein; HIF-1β is identical to the aryl hydrocarbon receptor nuclear translocator protein. HIF-1α DNA-binding activity and HIF-1α protein expression are rapidly induced by hypoxia and the magnitude of the response is inversely related to pO₂52535664677071727374757677787980818283.

In hypoxia, multiple systemic responses are induced, including angiogenesis, erythropoiesis and glycolysis 525356717273. HREs containing functionally essential HIF-1-binding sites are identified in genes encoding VEGF, glucose transporter 1, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A and phosphoglycerate kinase 1 51525364657273. HIF-1α is an important mediator for increasing the efficiency of oxygen delivery through EPO and VEGF 5253. A well-controlled process of adaptation to hypoxia enables oxygen to be delivered more efficiently, through upregulation of EPO and VEGF and the expression and activation of glucose transporters and glycolytic enzymes 52536465. EPO is responsible for increasing blood oxygen-carrying capacity by stimulating erythropoiesis; VEGF is a transcriptional regulator of vascularization; and glucose transporters and glycolytic enzymes increase the efficiency of anaerobic generation of ATP 515253.

HIF-1α has also been shown to activate transcription of genes encoding inducible nitric oxide synthase and heme oxygenase-1 (which are responsible for the synthesis of the vasoactive molecules NO and CO, respectively), as well as the gene encoding transferrin (which, like EPO, is essential for erythropoiesis) 5253. Each of these genes contains an HRE sequence of <100 base pairs that includes one or more HIF-1-binding sites containing the core sequence 5'-RCGTG-3' 515253. It is expected that any reduction of tissue oxygenation in vivo and in vitro would therefore provide a mechanistic stimulus for a graded and adaptive response mediated by HIF-1α. Hypoxia signal transduction is schematized in Fig. 3 and the array of proteins encoded by genes directly controlled by HIF-1α is given in Table 1.

NF-κB is an important and widely investigated dimeric transcription factor that is a major participant in signaling pathways governing cellular responses to environmental stresses 131419545558103104. NF-κB is involved in the regulation of a large number of genes that control various aspects of the immune and inflammatory response. It is activated by a variety of stimuli ranging from cytokines, to various forms of radiation, to oxidative stress (such as exposure to H₂O₂). Recent studies have advanced our understanding of the signal transduction pathway leading to NF-κB activation by cytokines and will provide insights for the mechanism by which NF-κB is regulated by oxidative stress. An important question that is yet to be answered is whether ROS play a physiological role in NF-κB activation.

First identified as a factor which regulates the expression of the immunoglobulin κ light chains in B lymphocytes 57 NF-κB is also recognized as a sequence-specific transcription factor involved in the activation of an exceptionally large number of genes in response to inflammation, viral and bacterial infections, and other stressful situations requiring rapid reprogramming of gene expression, such as oxidative challenge 5455. In unstimulated cells under resting conditions (inactive state), NF-κB exists as homodimers or heterodimers of members of the Rel family 5556103104. The dimers of NF-κB are sequestered in the cytosol through noncovalent interactions with inhibitory proteins termed IκBs 5455103104. The translocation and activation of NF-κB in response to various stimuli, such as cytokines (IL-1 and TNF-α), microbial agents (lipopolysaccharide-endotoxin), oxidative challenge (ROS) and irradiation (UV and γ-rays), are sequentially organized at the molecular level 5455104. NF-κB activation occurs through the signal-induced phosphorylation of a multisubunit upstream kinase, termed IκB kinase, by NF-κB inducing kinase 103104105. Stimulation leads to rapid phosphorylation of IκB, thereby marking it for ubiquitinylation and ultimately proteolytic degradation. IκB degradation exposes the nuclear localization signal on NF-κB, thus allowing the nuclear translocation of the subunit and activation of the transcription of its target genes (Fig. 6) 5455. The array of proteins encoded by genes directly controlled by NF-κB is given in Table 2.

IκB-independent pathways, however, have recently been recognized as alternative factors that regulate the activation of NF-κB. As an example, direct phosphorylation of RelA (p65), the major transactivating member of the κB family 14195455103 has been shown to regulate NF-κB activation in one of two of its transactivation domains 106. A further mechanism was revealed for NF-κB regulation with the discovery of transcription factor-IIB/D (TF-IIB/D) and TATA-binding protein (TBP), recognized as two important regulators of NF-κB transcriptional activity. The dominant-negative form of the mitogen-activated protein kinase (MAPKp38) expression vector abrogated the interaction of TF-IID/TBP with a co-transfected His-p65 fusion protein, and selective inhibition of MAPKp38 by SB-203580 down-regulated TF-IID/TBP in vitro106. Finally, modulation of intracellular redox equilibrium constitutes a potential mechanism that can manipulate and dictate the localization and activation of NF-κB 231314265455103104105106. Hyperoxia and other stress conditions mediating signal-transduction pathways involving NF-κB are depicted in a schematized model shown in Fig. 7.

Antioxidant/pro-oxidant equilibrium is likely to regulate HIF-1α redox sensitivity 13145173109110111. For instance, the cysteine residue in the CAD has been shown to be redox-sensitive, thereby affecting its interaction with CREB-binding protein/p300 co-activators. This interaction is directly regulated by redox factor-1 and thioredoxin 109110. HIF-1α ubiquitination and degradation by the proteasome system under normoxic conditions are also regulated by redox modifications of the protein 525382111. Furthermore, selective inhibition of γ-glutamylcysteine synthetase (which results in glutathione depletion) in the alveolar perinatal epithelium abrogated hypoxia-induced nuclear localization, stabilization and activation of HIF-1α 1314. It appears, therefore, that maintenance of glutathione equilibrium (and by inference, the shuttling between reduction and oxidation states) is a prerequisite for HIF-1α stabilization 82109.

This assumption is reinforced by the observation that N-acetyl-L-cysteine (NAC, an antioxidant thiol and a precursor for L-cysteine, the rate-limiting amino acid in the biosynthesis of glutathione 1417112) imposes a reducing environment, thereby protracting HIF-1α stability in the cytosol and subsequently favoring its translocation and activation 1314535482111. On the other hand, imposing an oxidizing environment through the rapid accumulation of GSSG in the nucleus adversely affects HIF-1α activation 1314. PDTC is a nonthiol antioxidant that affects redox potential by scavenging radical species (a reduction property) and directly oxidizing glutathione and related thiols (an oxidation property) 1479113. PDTC favors a GSSG/glutathione equilibrium and subsequent stabilization of HIF-1α protein, but fails to induce its activation. This effect is ostensibly due to the generation of thiyl radicals and thiuram disulfides resulting from PDTC antioxidant reactions, thus leading to oxidation of glutathione 1314111. It is very likely, therefore, that HIF-1α's oxygen responsiveness resides over a permissive range of antioxidant buffering capacities, with the demonstration that the antioxidant/pro-oxidant equilibrium effectively uncouples HIF-1α activity from the normal pattern observed in response to variations in pO₂ in the alveolar epithelium 131482. Redox-mediated pathways regulating HIF-1α transduction mechanisms are shown in Fig. 8.

Redox regulation of NF-κB seems to be compartmentalized 131419545562113114. Whereas an oxidizing signal is required for the stabilization and translocation of NF-κB subunits, intriguingly, a reduced environment is critical for an optimum DNA-binding activity and transactivity 145455113114. Recent evidence suggests that NF-κB plays a critical role in the early events controlling the molecular response to ROS 545558103. It has been shown, for example, that the activation of NF-κB by a variety of agents can be blocked by NAC, suggesting that the production of ROS may act as a common pathway for a diverse range of stimuli 131454555862101103114115116. In addition, the inhibitory effects of PDTC suggest post-translational instability and interference with the capacity of NF-κB to bind DNA 545562103104. This is further supported by the observation that phosphorylation of IκB at specific serine residues can be inhibited by dithiocarbamates, pointing to the possibility that NF-κB translocation and subsequent activation is mediated by ROS, which might induce a cytosolic kinase activity 5455103104.

There is another assumption that GSSG-mediated inhibition of NF-κB implicates the formation of an inactive NF-κB/disulfide complex, thereby inhibiting DNA binding activity 14161718545562114115116. Although oxidizing conditions are necessary in the cytosol, to allow optimum translocation and dissociation of NF-κB from inhibitory IκB (resulting in activation of NF-κB), NF-κB must be maintained in a reduced state in the nucleus for activation to occur 13141617181954555862103104114115116. Redox-mediated pathways regulating NF-κB transduction mechanisms are shown in Fig. 9.

At least two distinct forms of death are known by which cells undergo death: the well-characterized, and usually rapid, necrotic tissue damage induced by trauma and noxious stimuli, and the more protracted and morphologically distinct form of cell death that has been termed apoptosis 117118119120121122123124125126.

Apoptosis is characterized by an ordered series of events that take place over a longer period of time than events during necrotic cell death. In apoptosis, cells often shrink, dissociate from surrounding cells and undergo cytoplasmic membrane blebbing, a process in which their chromosomes rapidly condense and aggregate around the nuclear periphery and small apoptotic bodies are formed. During apoptosis cellular organelles retain their definition for a long time; the nucleus in particular displays a distinctive pattern of heterochromatization and eventual fragmentation. In many (but not all) apoptotic cells, specific nucleases cleave the DNA of the condensed chromosomes, thereby producing a characteristic 'ladder' 123124125126127.

Necrotic cell death, on the other hand, is relatively quick and violent, characterized by the swelling of the cytoplasm, the rupturing of cell membranes, the dilation of the mitochondria and the disintegration of subcellular and nuclear components. Necrosis may be considered to be analogous to random acts of violence that culminate in murder, whereas apoptosis is more appropriately referred to as cellular suicide. The cell initiates apoptotic death when it senses that its environment of physical state has been vigorously compromised; this is, indeed, the ultimate self-sacrifice 117118119120121122123124125126127.

If there is simple dichotomy in the modes of cell death, perhaps there is more than one basic genetic program for death and more than one final common pathway (Fig. 10A). Perhaps some ligand-induced cell death results from confused or inappropriate regulation of gene expression rather than from turning on preset genetic programs 119120121122123124125126. Studies of C. elegans development, for example, have contributed significantly to the biomolecular understanding of cell death 119120. Genetic analysis has led to the identification of the cellular genes required for programmed cell death during their development. The isolation and molecular characterization of C. elegans death (ced) genes demonstrated that ced-3 gene is homologous to the gene encoding mammalian IL-1β-converting enzyme (ICE; caspase-1) 126127128129. ICE was originally isolated from mammalian cells as an enzyme essential for the proper processing and biological activation of pro-IL-1β, a cytokine involved in mediating cellular inflammatory processes 36404243444546. The expression of ced-3/ICE rapidly induces apoptosis, demonstrating that this gene encodes a cysteine protease essential for programmed cell death 127128129.

Many of the genes encoding ICE-like proteases, henceforth referred to as caspases, were isolated by molecular cloning. The term caspase is based on a common nomenclature universally adopted: 'c' reflects a cysteine protease mechanism, and 'aspase' refers to the ability of these proteases to cleave a protein following an aspartic acid residue 127128129. Many of these caspases contain a conserved sequence, QAC(R/Q)G, required for the catalytic activity of these enzymes 127128129130. The activation of the caspase proteases has been linked to the aggregation of cell surface receptors, either when receptor-sensitive target cells are exposed to the appropriate ligand, or when the receptors self-aggregate in response to their high cell-surface density. A functional caspase, therefore, can be generated following receptor oligomerization by autocatalysis or by the action of another alerted caspase. Recent evidence suggested that caspases regulate the process of apoptosis by regulating additional cellular processes, such as the progression through the well-defined cell cycle and its regulators 126127128129130.

As noted, multicellular organisms eliminate redundant, damaged, or infected cells by a stereotypic program of cell suicide. The first mammalian regulator of apoptosis emerged when bcl-2, the gene activated by chromosome translocation in human follicular lymphoma 131 was unexpectedly found to permit the survival of cytokine-dependent hematopoietic cells, in a quiescent state in the absence of exogenous cytokine 132. Ced-9 of C. elegans and the mammalian Bcl-2 proved to be functional and structural homologues and their survival function is opposed either by close relatives, like Bax, or by distant cousins such as the mammalian proteins Bik, or Nbk, and nematode Egl-1 127128129130131132. All Bcl-2 family members possess at least one of four conserved motifs known as Bcl-2 homology domains (BH-1,-2,-3, and -4). Most prosurvival family members, which can inhibit apoptosis in the face of a wide variety of cytotoxic insults, contain at least BH-1 and BH-2, and those most similar in structure to Bcl-2 have all four BH domains 127128129130131132.

Pro-apoptotic and anti-apoptotic family members can heterodimerize and seemingly titrate one another's function, suggesting that their relative concentrations may act as a rheostat for the suicide programs 133. Bcl-2 resides on the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum and nuclear envelope, and may register damage to these compartments and affect their behavior, possibly by modifying the flux of small molecules and proteins 133. On the other hand, Bax is cytosolic before an apoptotic stimulus, even though it bears a hydrophobic domain (like most other family members) 133. Biochemical evidence suggested that the prosurvival proteins might function by directly inhibiting the activity of caspases, directly or indirectly preventing the release of cytochrome c from the mitochondria. Along with ATP, this may facilitate structural changes in the procaspase domain, allowing its cleavage and activation 127128129130131132133. Bax, Bax-like proteins and the antisurvival proteins may promote apoptosis by cleaving and activating caspases, but also can initiate caspase-independent death via channel-forming activity, which could promote the mitochondrial permeability transition or puncture the mitochondrial outer membrane 134135136137138139140141.

ROS are generated in all aerobic cells during normal mitochondrial respiration, are used by specialized phagocytic cells to destroy invading pathogens and are byproducts of the intracellular metabolism of toxic drugs and their environmental metabolites 2316171826. Incubating cells, for instance, with exogenous oxidants, free radicals, or added redox-active compounds has been shown to trigger the apoptotic process 142143144145146147148149150151152153154155. It has been hypothesized that the oxidation of cellular molecules could trigger a general protection alert system, and these sensors, in turn, detect and assess the damage, subsequently activating the apoptotic machinery. A classic example of this model is p53-mediated detection of DNA damage 156157158159. Alternatively, low-level oxidative stress is known to activate different protein cascades and transcription factors, such as AP-1, HIF-1α, and NF-κB 23161718265152535455. In addition, the ability of NO to rapidly react with the heme group of guanylate cyclase has been used as a message in activating downstream apoptotic cellular pathways 150155.

Recent studies have focused on the role of H₂O₂ in regulating apoptosis in several cell models. H₂O₂ is an oxidant that has been shown to trigger caspase activation and subsequent apoptosis. H₂O₂-mediated caspase activation is dependent on the release of cytochrome c from the mitochondria, suggesting a key role for H₂O₂ in mitochondrial permeability and leakage 142144147149150153.

The importance of dissecting the pathway from oxidative stress/redox signaling to apoptosis is not restricted to models in which cells are exposed to exogenous oxidants. Schematized pathways of apoptosis in the alveolar model and in the developing lung are given in Fig. 11. Intracellular oxidant production has been detected in cells incubated with a wide range of seemingly independent apoptotic agents, and some of these changes have been suggested to occur sufficiently early to be intricately involved in the activation of apoptosis 160. A recent example is p53-mediated apoptosis, which has been proposed to occur through the increased transcription of pro-oxidant factors, thereby leading to caspase activation and apoptosis 156157158159. Interestingly, antioxidants and glutathione precursors conferred a protective effect against ROS-mediated injury and subsequent apoptosis, implicating a critical role for ROS in initiating the death machinery 142143144145146147148149150151152153154.

It is becoming apparent, therefore, that the redox status of a cell can have complex and multilayered effects on apoptosis (Fig. 10B) 161. It has been postulated that the mitochondria could be the principle sensors and that the release of mitochondrial factors such as cytochrome c is the critical event leading to caspase activation, and hence propagation of apoptosis 142143144145146147148149150151152153154155. The effectors of apoptosis, in particular the caspases, are redox-sensitive, and the cell must maintain a strict reducing environment for these effectors to function. By reasoning, therefore, it has been hypothesized that apoptosis cannot occur in cells subjected to excessive oxidative stress 144145146147148149150151152153154155156157158159160161. The ability of oxidants to inhibit caspase function need not be incompatible with oxidant-dependent caspase activation. First, low levels of oxidants appear sufficient for optimal caspase activation, suggesting specific signaling pathways rather than widespread oxidation. Second, the observed ability of cells to repair or replace oxidized caspases indicates that the full complement of apoptotic effector molecules can be regained at some time after the initial oxidative stress 145146147148149150151152153154155156157158159160161. Elucidation of the pathways of oxidant-induced apoptosis, therefore, may provide alternative therapies to scavenging the initial oxidant in those systems where excessive oxidative stress and redox disequilibrium lead to inevitable cell death.

ROS play a crucial role in the initiation and progression of pathophysiological conditions. The signaling mediators involved in stress-induced lung injury are regulated, at least in part, by ROS that upregulate their secretion as part of antioxidant and immune defense mechanisms 154155156157158159160161162163164. ROS-mediated pathways regulating cytokines are schematized in 12A For example, bronchial epithelial cells produce soluble mediators on exposure to ROS, which stimulate the release of glycoconjugates in vitro165166167. In addition, ROS can induce the production of IL-6 and IL-8 in bronchial and alveolar epithelial cells 3637383940414243444546168. Furthermore, ROS can be released in response to a variety of stimuli, such as TNF-α and lipopolysaccharide 3637383940414243444546169 and they serve as intracellular signals for the activation of redox-sensitive transcription factors 2316171826273637383940414243444546164165166167168169170.

Among the mediators of oxidative stress, cytokines are of particular interest as they serve as signaling cofactors 373839404142434445. IL-1β, for instance, is a pleiotropic cytokine produced in response to various stimuli. It acts as a modulator of redox equilibrium 3637383940414243444546; moreover, there is substantial evidence suggesting that, among other regulatory cytokines, IL-1β participates in transduction signals in oxidative injury via ROS and has stimulatory effects on regulatory transcription factors mediating apoptosis 163164165166167168169170. IL-6 is responsive to inflammatory stimuli and oxidative stress 3637383940414243444546. TNF-α, a stress-induced cytokine and a mediator of oxidative injury, has been implicated in the pathogenesis of respiratory distress 3637383940414243444546164165166167168169170. Cytokines have also been shown to induce oxidative stress in several cell models 3637383940414243444546.

The 'biomarkers' of oxidative stress, such as antioxidant inefficiency, redox disequilibrium and derivation of oxidant radicals, may arise from conditions other than hyperoxia (oxidizing signals) per se, such as hypoxia/re-oxygenation and cytokine-dependent processes 23141617182627. In physiological conditions, the intracellular redox status of thiols is highly reductive. Glutathione, for example, is present in high concentrations in lung epithelial lining fluid and has been reported to maintain the integrity of the airspace epithelium 171. Glutathione depletion has been linked to the pathophysiology of idiopathic pulmonary fibrosis 171 adult respiratory distress syndrome 162172 bronchopulmonary dysplasia 173 and cystic fibrosis 174.

Agents that induce the formation of ROS can affect redox homeostasis by upregulating antioxidant enzymes, particularly glutathione peroxidase and enzymes involved in glutathione recycling and biosynthesis 121621223738394041115116164169170175176177. Furthermore, ROS signaling could be mediated by cytokines, whose participation in cellular pathways is modulated by redox status 353637383940414243444546169170171172173174175176177. Redox-mediated pathways regulating cytokines are schematized in Fig. 12B. Conversely, cytokines, which themselves are mediators of oxidative stress, have the potential to alter redox equilibrium, thereby affecting glutathione/GSSG shuttling and recycling 353637383940414243444546169170171172173174175176177.

The immunopharmacological potential assigned to glutathione stems from established observations. IL-1-induced responses, for instance, occur through modulating the redox dynamic equilibrium 178. In addition, ROS signaling regulating the transcription of IL-4 179 IL-6, IL-8 180 and TNF-α 181182 is regulated by a thiol-dependent mechanism. Interestingly, antioxidants and glutathione precursors have been shown to downregulate cytokine synthesis, activation and downstream processes 353637383940414243444546169170171172173174175176177178. Among the many agents that are used for repletion and depletion of glutathione, NAC and L-buthionine-(S,R)-sulfoximine are, respectively, of particular importance as they exhibit antagonistic effects on a pro-inflammatory signal. NAC, an antioxidant and a glutathione precursor 1314194041111 has the ability to abrogate cytokine biosynthesis and ROS-mediated lung injury 353637383940414243444546. In contrast, L-buthionine-(S,R)-sulfoximine, which depletes glutathione by irreversibly inhibiting γ-glutamylcysteine synthetase (the rate-limiting enzyme in the biosynthesis of glutathione 394148103104), has the potential to enhance cytokine secretion by upregulating ROS 353637383940414243444546181183. It has been reasoned that a differential manipulation of glutathione homeostasis and shuttling antagonistically affects a proinflammatory signal. This has potential consequences for the treatment of respiratory distresses, where cytokines are recognized as major participants in pathophysiology.