PERSPECTIVE
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Historical Note |
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Free molecular oxygen probably appeared on the earth's
surface some 2 billion years ago as a result of photosynthetic microorganisms acquiring the ability to split water
(6). It is the most abundant element in the earth's crust,
and the second most abundant element in the biosphere.
Oxygen is an unusual molecule in that it has two unpaired
electrons with parallel spins. It is therefore a biradical. To
overcome spin restriction, oxygen prefers to accept electrons one at a time, and the sequential addition of electrons leads to the formation of ROS. As a consequence of
aerobic metabolism, all aerobic organisms are subject to a
certain level of physiologic oxidative stress as ROS are
produced continuously in numerous biologic processes. In
rats, an average of about 10 trillion oxygen molecules are
processed by each cell daily under basal conditions, and
the leakage of partially reduced oxygen molecules is about 2% (7). Under basal conditions, human cells produce about
one tenth the ROS of those of rats or 2 billion O2
and
H2O2 molecules per cell per day.
Once regarded as merely waste byproducts of aerobic metabolism or molecules of defense produced by host inflammatory cells against invading organisms, ROS are now understood to be major mediators of human disease. A considerable body of recent literature describes roles for ROS in the pathogenesis of pulmonary diseases such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, asthma and interstitial pulmonary fibrosis. Initially, the involvement of ROS in illness was conceptualized as the chemistry of "scorched earth," in which critical cell proteins and lipids were indiscriminately oxidized and rendered metabolically inactive for their roles in normal cell function (8). The realization that ROS operate as signaling molecules, controlling even gene expression, has been more recent. As one example of the signaling properties of ROS in the airway, treatment of tracheal myocytes with exogenous H2O2 has been shown to activate extracellular signal-regulated kinases (9) via successive activation of protein kinase C, Raf-1, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (10). This provides a mechanism by which ROS directly effect transduction of mitogenic signals to the nucleus.
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ROS and Apoptosis |
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The studies by Lavrentiadou and colleagues provide a direct link between two important aspects of mammalian stress responses: the generation of ROS and activation of the sphingomyelin/ceramide cycle leading to apoptosis. Apoptosis is currently a hot area in biology. It represents the orchestrated collapse of a cell and includes membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation, and DNA degradation. It is an essential part of life. It is estimated that about 10 billion of our cells will die on a normal day just to counter the new cells that arise through mitosis. In the lung, epithelial cells of the airway and the alveolar compartments are constantly exposed to airborne environmental stresses. Their ability to adapt to injury from these insults is essential in maintaining lung function. One response to cellular injury is to give up the fight and die to permit neighboring cells to replicate and replace the injured cell. Apoptosis is also a mechanism by which the lung purges itself of pathogen-invaded cells (11).
One of the most reproducible inducers of apoptosis is
mild oxidative stress. Lavrentiadou and coworkers demonstrate that either administration of exogenous H2O2 or enhancement of endogenously generated H2O2 (by administration of aminotriazole) are effective in depleting cellular
GSH and initiating ceramide-induced apoptosis. Both scenarios are relevant to lung epithelium. H2O2 is a ubiquitous molecule, freely miscible and able to cross cell membranes readily. It is present in several air pollutants,
including the vapor phase of cigarette smoke. It is detected
in exhaled air of humans (12), although it is uncertain
where this H2O2 originates. Amounts of exhaled H2O2 appear greater in subjects with lung inflammation (13)
and in cigarette smokers (16). Importantly, several agonists increase generation of H2O2 by epithelial cells. These
include cytokines (TNF-
, IL-1, and Fas ligand), cytotoxic
agents and ionizing radiation, and infections (e.g., HIV or bacteria).
With regard to endogenous sources of ROS, a key question that remains unanswered is: what are the intracellular sites of their generation that contribute to apoptosis in lung epithelium? These sites are poorly characterized. ROS generation in nonphagocytic cells is frequently considered to be an "accidental" byproduct of mitochondrial respiration. Clearly, the mitochondrial electron transport chain is an important source of ROS in apoptosis. Results of a recent study in an apoptosis model show that ceramide exerts a direct effect on mitochondria leading to production of H2O2 by inhibition of electron flow at the ubiquinone pool of complex III (17). However, apoptosis is a highly complex biochemical process and the increase in mitochondrial ROS generation occurs relatively late in the course. The emerging picture is that an early burst of ROS production is also involved in triggering the apoptosis pathway in a variety of models. Lavrentiadou and colleagues demonstrate this by the administration of aminotriazole, a specific inhibitor of catalase, that led to rapid accumulation of ceramide, a proximal event in their apoptosis model.
Sources of ROS that may provide the early oxidant burst
needed to initiate proximal events of apoptosis in nonphagocytic cells include: the molybdenum hydroxylases (xanthine
oxidoreductase and aldehyde oxidase); NADPH or NADH
oxidoreductases (including the phagocytic cell oxidase; cytochrome P-450 and nitric oxide synthase); and arachidonic acid metabolizing enzymes (such as cyclooxygenase). Involvement of many of these enzyme systems in the
generation of ROS in nonphagocytic cells has been well documented. Of particular interest, however, is the recent
identification of several novel NADPH oxidoreductases in
nonphagocytic cells, including epithelium. NADPH oxidase
is responsible for the respiratory burst of professional
phagocytes (polymorphonuclear leukocytes and monocytes/
macrophages). In phagocytes, the oxidase consists of at least
six proteins, including two membrane proteins, gp9l and
p22, that bind a flavin adenine nucleotide (FAD) and form a unique cytochrome. The gp91 subunit appears to contain
all factors necessary for transporting electrons from NADPH
via FAD and then heme to molecular O2. Recently, evidence has been accumulating that a low-activity NAD(P)H
oxidase(s) is present in nonphagocytic cells and that this
oxidase generates ROS as signaling intermediates. Initially, the molecular composition of this oxidase proceeded on
the assumption that the enzyme was structurally similar to
the neutrophil oxidase. However, within the past eighteen
months, six new homologs of gp91 were identified (18). Unlike gp91, these new proteins (now officially classified as
the Nox family) are not expressed in phagocytes. Their
biochemical properties and physiologic functions still require characterization. However, H2O production by Nox homologs has been demonstrated to participate in regulation of cell proliferation and induction of the transformed
phenotype (19), raising the possibility that these homologs
may also be involved in generating ROS that function as
signaling molecules in apoptosis.
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Dual Role for ROS in Apoptosis |
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While one of the most reproducible inducers of apoptosis is mild oxidative stress, to consider ROS only as proapoptotic is too narrow a view. However, the potential for inhibition of this process by ROS, and the consequences thereof, have been largely ignored. The key enzymes in apoptosis, caspases, are themselves cysteine-dependent enzymes and thus potentially redox sensitive. Indeed, Hampton and Orrenius have demonstrated that prolonged or excessive oxidative stress can actually prevent caspase activation and resultant apoptosis (20). Thus, while an early burst of ROS production has been hypothesized to be involved in the triggering of apotosis in a variety of models, excessive ROS production at the early stage may actually block apoptosis.
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GSH and Apoptosis |
|---|
An important finding in the report by Lavrentiadou and coworkers is the critical role of GSH in preventing lung epithelial cell apoptosis. In their model, inhibitory effects were observed by both extracellular and intracellular GSH. The effects of extracellular GSH are particularly pertinent to lung epithelium. As pointed out in the article, several research groups have determined that alveolar epithelial lining fluid contains GSH at concentrations more than 100-fold greater than those in plasma (from 90 to 500 µM versus 1-2 µM) (21). Importantly, alterations in the levels of GSH in the lung lining fluid have been shown in various inflammatory conditions. For example, GSH levels are decreased in the epithelial lining fluid in idiopathic pulmonary fibrosis ( IPF ) (22), acute respiratory distress syndrome (23), cystic fibrosis (24), lung allograft (25), and human immunodeficiency virus (HIV)-positive patients (26). The reductions in GSH, which may be profound, may contribute to the epithelial cell apoptosis observed in many of these disorders.
In the Lavrentiadou article, the effect of GSH on the apoptotic process was noted at multiple levels. GSH inhibited both early (before ceramide production) and late (after ceramide production) events leading to apoptosis. The mechanisms by which GSH depletion induced apoptosis were not determined. GSH serves several vital functions, including: (1) detoxifying electrophiles; (2) maintaining the essential thiol status of proteins by preventing oxidation of -SH groups or by reducing disulfide bonds induced by oxidant stress; (3) scavenging free radicals; (4) providing a reservoir for cysteine; and (5) modulating critical cellular processes such as DNA synthesis, microtubular- related processes, and immune function (27). A recent observation has shown that GSH or similar molecules inhibit the activity of magnesium-dependent neutral sphingomyelinase (31). This is a plausible and likely explanation for the early effects of GSH shown in the Lavrentiadou study. Somewhat surprising, the SH group of GSH was not required to inhibit neutral sphingomyelinase as S-methyl GSH and GSSG inhibited the enzyme at lower concentrations than GSH (31). Thus, the ability of GSH to prevent activation of sphingomyelinase was not due to the intrinsic antioxidant properties of the molecule, but, rather, seemed attributable to GSH functioning as a specific allosteric regulator of the enzyme. Regarding the ability of GSH to inhibit apoptosis after ceramide generation, recent studies have shown that mitochondrial GSH is critical in defending against both physiologically- and pathologically-generated oxidative stress (32). This critical role of GSH is probably because mitochondria have no catalase. The effects of GSH in the downstream events (after ceramide generation) observed by Lavrentiadou and coworkers in lung epithelial cells is likely due, in part, to its ability to scavenge mitochondrial-generated ROS.
In summary, ROS have pleiotropic activities. They can function as cell toxins or signaling molecules. In the latter role, they may influence diverse cell processes ranging from cellular proliferation to apoptosis. What determines which of the myriad of activities of ROS predominate? The answer to this question is not known. The magnitude of ROS generation, the site and source of their generation, the phase of the cell cycle, the state of activation of transmodulating signals, and the antioxidant status of the cell all likely influence the final outcome.
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Footnotes |
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Address correspondence to: John R. Hoidal, Department of Medicine, University of Utah Medical Center, 30 N. 1900 East Rm 4C1 04 5011, Salt Lake City, UT 84132-2406.
(Received in original form October 12, 2001).
Abbreviations: flavin adenine nucleotide, FAD; glutathione, GSH; reactive oxygen species, ROS.| |
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