© 2002 American Thoracic Society
Environmental Oxidant Pollutant Effects on Biologic SystemsA Focus on Micronutrient Antioxidant–Oxidant InteractionsDivision of Pulmonary and Critical Care Medicine, Division of Nephrology, and Center for Comparative Lung Biology and Medicine, University of California School of Medicine, Davis, California; Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany; and Department of Pathology, School of Medicine, University of Vermont, Burlington, Vermont Correspondence and requests for reprints should be addressed to Carroll E. Cross, M.D., Pulmonary and Critical Care Medicine, University of California, Davis, 4150 V Street, Suite 3400, Sacramento, CA 95817. E-mail: cecross{at}ucdavis.edu ABSTRACT Oxidative atmospheric pollutants represent a significant source of stress to both terrestrial plants and animals. The biosurfaces of plants and surface-living organisms are directly exposed to these pollutant stresses. These surfaces, including respiratory tract surfaces, contain integrated antioxidant systems that would be expected to provide a primary defense against environmental threats caused by atmospheric reactive oxygen species. When the biosurface antioxidant defenses are overwhelmed, oxidative stress to the cellular components of the exposed biosurfaces can be expected, inducing inflammatory, adaptive, injurious, and reparative processes. Studies of mutants and/or transformed plants and insects, with specific alterations in key components of antioxidant defense systems, offer opportunities to dissect the complex systems that maintain surface defenses against environmental oxidants. In this article, we use a comparative approach to consider interactions of atmospheric oxidant pollutants with selected biosystems, with focus on O3 as the pollutant; plants, flies, skin, and lungs as the exposed biosystems; and nonenzymatic micronutrient antioxidants as significant contributors to overall antioxidant defense strategies of these varied biosystems. Parallelisms among several living organisms, with regard to their protective strategies against environmental atmospheric oxidants, are presented.
Key Words: ascorbic acid • flies • ozone • plants • respiratory tract lining fluids Most biologic systems are equipped with elaborate mechanisms for protection against the toxicity of reactive oxygen species (ROS). Toxicity arising from imbalances of biologic pro-oxidant and antioxidant processes is usually designated as oxidative stress. Prevalent among the oxidative stresses are contributions of cells generating ROS as part of normal aerobic metabolism, the ROS generated secondary to plant and animal responses to injury and invading organisms, and the ROS generated in polluted atmospheres. The environmental oxidant pollutants, including ozone (O3), oxides of nitrogen, and particulates with chemically active surfaces (e.g., containing redox-cycling substances), represent an important source of oxidative stress to terrestrial plants and invertebrates and to vertebrate organisms including humans. Among the many elaborate, redundant and overlapping mechanisms for combating these oxidant hazards are the plant-based antioxidants such as ascorbic acid, tocopherols, carotenoids, and polyphenols (1). These defense mechanisms are of considerable clinical interest because of the potential that they can be augmented by increased dietary consumption or supplementations, and because of the general, although uncritical, widespread belief that these micronutrients confer health benefits (2). It is the outermost extracellular surface and respiratory apparatus constituents of plants and animals that are initially exposed to atmospheric pollutant oxidants, and in situations in which environmental oxidative stress fails to reach levels that penetrate these extracellular defenses to reach underlying cell membrane surfaces, these extracellular defenses are solely responsible for protecting the biologic systems from the oxidative pollutant. Failure of this outermost defense allows oxidants to penetrate to the surface of plasma membranes, where their biologic effects include damage to proteins and lipids, with subsequent elicitation of adaptive, inflammatory, injurious, and reparative responses. Thus, surface antioxidants serve to regulate or minimize changes in organismal function occurring as consequence of atmospheric environmental oxidants. It can be expected that, to some degree, the surface low molecular weight antioxidants, such as antioxidant enzyme systems, might constitute an evolutionally conserved ROS defense system. Augmentation of extracellular surface antioxidants might minimize the adverse biologic effects of exposure to elevated levels of pollutant oxidants. Although whole genome sequencing studies and research in developmental genetics indicate that plants and animals have in large measure evolved independently, a similar logic underlies many of their metabolic processes. Comparative study of the biosystem responses to oxidative air pollutants in both plants and animals is thus informative.
The aim of this presentation is to highlight parallels between plants and animals in the antioxidant micronutrient mechanisms for protecting their biosurfaces from oxidizing atmosphere pollutants, a topic that we have covered previously (3). Our presentation highlights O3 as the representative environmental oxidant and on ascorbic acid (vitamin C) and THE PLANT WORLD O3 is one of the most ubiquitous and damaging air pollutants to which plants are exposed (4). There is arguably more evidence that present-day O3 levels cause greater damage to the vitality of plants than of animals (5). It is less arguable that understanding of the mechanisms underlying O3-related phytotoxicity of plants will necessarily be helpful to those concerned with the health of humans. In both plants and animals the effects of O3 depend on the concentration and the duration of exposure, the genetic background, and the phase of growth. As illustrated in Figure 1A , environmental gases, including O3, are exposed to plants via their outer cell wall and gain entrance to the plant respiratory system, designed to optimize leaf gas exchange (e.g., the uptake of CO2 for photosynthesis and elimination of water vapor and O2 for transpiration), via stomata (pores on the leaf surface), the outer cell wall (cuticle) being practically impermeable to O3. The stomata are composed of guard cells, which in pairs surround the stomatal pores, which provide an entrance to the inner air pockets and the extracellular apoplastic fluids (6, 7). By regulating the size of the stomatal opening, the guard cells surrounding the stomatal aperture control both the influx of CO2 and atmospheric pollutants, as well as the outflux of water. Guard cells are important, much-studied cells in overall plant biology and physiology, which have evolved over millions of years (8). Of intriguing interest to the respiratory disease community is that a plant anion channel cystic fibrosis transmembrane conductance regulator homology appears likely to participate in guard cell regulation of the stomatal opening (9).
Rooted in one place, plants have learned to cope with environmental stresses by methods other than flight. It can be expected that guard cell signaling pathways would integrate with environmental conditions to regulate stomatal apertures. If O3, or other reactive pollutants, gain entrance to the air–aqueous phase of the apoplast, damage to the underlying plasma membrane and cytosolic constituents would be expected to occur unless components of the extraprotoplastic matrix apoplastic fluids detoxify O3 and its highly reactive products. A decade of work has indeed indicated that antioxidant micronutrients, especially ascorbic acid, are present in millimolar concentrations in apoplastic fluid compartments compared with the micromolar concentrations on the surfaces of the human respiratory tract (10–13). In combination with the ascorbate peroxidase system, these micronutrients constitute an important antioxidant defense system at the surface of plant cells. Efforts have led to major advances in understandings of the plant guard cell signal transduction pathways regulating the stomatal aperture (6, 14) and of how guard cells respond to stress, stress hormones, and even O3 (14–16). As an example of one mechanism, it is now known that O3, which causes oxidative stress in guard cells, downregulates potassium ion inward channels. This appears to relate to signal transduction systems that eventually cause osmotically driven changes in guard cell volumes, which in turn regulate the apertures of the associated stomatal openings, as illustrated in Figure 1B. Plant stress hormones, signal transduction systems involving kinases and phosphatases with positive and negative regulatory effects, ion channel modifications, transcellular calcium fluxes, nicotinamide adenine dinucleotide phosphate oxidase-like pathways, and hydrogen peroxide and actin filament organizations all are likely to participate in the complex network of signaling cassettes regulating the stomatal aperture. That these processes can be modulated by catalase and nicotinamide adenine dinucleotide phosphate oxidase inhibitors (17) is of interest because nicotinamide adenine dinucleotide phosphate oxidase activation also appears to be involved in O3-induced hyperresponsiveness in human airways (18). As in animals, the quantitative fate of the O3 reaching the gas-exchanging compartments in plants is unknown (19). The mechanisms of injury, however, may in several ways be similar. In humans, for example, inflammatory cell responses appear to contribute to overall oxidant-induced injury (20). In plants, O3 can induce the "hypersensitivity reaction," a phagocyte-free version of nicotinamide adenine dinucleotide phosphate-related sudden and dramatic increase production ROS that is mechanistically similar to the inflammatory "oxidative burst" of animal phagocytes, and that participates in the generation of pathogen stress responses, including adaptation and apoptosis (21, 22). The magnitude of this "burst, which amplifies O3 effects, has been postulated to relate to the O3 sensitivity of both plants and animals. An increased tolerance to atmospheric O3 exposure has been observed in plants in which ascorbic acid synthesis pathways have been augmented (23) and in transgenic plants overexpressing glutathione synthetic pathways (24). The pool of ascorbic acid is not the sole antioxidant defense in the apoplast compartments, and additional apoplastic antioxidant constituents and pathways are likely to complement ascorbic acid in the detoxification of O3 (11). As is the case for the human respiratory tract, ascorbic acid kinetics and metabolism at respiratory plasma membrane cell surfaces are not fully understood; they may involve both transport and redox enzyme processes (25) and may interrelate with peroxidases (19). Plant intracellular oxygen concentrations are high because of the activity of chloroplasts, and to meet this challenge they can be expected to have as sophisticated antioxidant defense systems as animals. Because oxidation products are relatively easy to measure in plants (e.g., isoprostane-like compounds) (26), plants offer a helpful experimental model to investigate the role of supplemental antioxidants (e.g., via nutritional supplementation or genetic engineering). The role of nitric oxide in plants has received much attention (27), and it is becoming apparent that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in animals (27, 28). As in animals, nitric oxide participates in redox signaling during activation of the "hypersensitivity" response in plants (29). Increasing applications of plant genomic and proteomic technologies are impacting all fields of plant biology (30). When combined with interdisciplinary approaches, these new methods will yield revelations pertinent to both the plant and animal worlds and to the disciplines of environmental agriculture and air pollution toxicology. THE INSECT WORLD The Drosophila (fruit fly) genome is well characterized, is highly homologous to the genome of vertebrates (yet much less redundant), and has yielded valuable insights into many complex biologic problems. Like plants, fruit flies and other insects have adapted to a wide range of ecologic niches and microhabits and offer an excellent model system for studying the molecular mechanisms of response to atmospheric oxidizing pollutants. Insects might be expected to be particularly vulnerable to atmospheric oxidative pollutant exposure by virtue of their greatly increased metabolism and related gas exchange requirements. As illustrated in Figure 2A , insects use an extensive tracheal tubular respiratory system (31, 32), which distributes atmospheric gases, including their contained pollutants, to nearly all tissues within the organism. Analogous to plants, and unlike vertebrates, it is likely that gas transfer into the tubular breathing system is largely diffusive, although a portion of thoracoabdominal respiratory gas exchange could be conductive, secondary to skeletal muscle contractions including wing kinematics (33). As is the case for the plant stomatal opening system, the area of the Drosophila spiracle opening, shown in Figure 2B, is regulated by environmental and metabolic factors such as humidity and respiratory gas fluxes (34). Analogous to the plant control of stomatal openings, changes in the pattern of spiracle openings potentially modulate respiratory water loss from the tracheal system. The area of spiracle opening is known to vary to about the same degree as the area of the plant stomatal opening, for example, their average opening areas can be reduced to about 80% of their maximum opening areas (34). Although the terminal tubules are known to contain water (34), few data relate to spiracle responses to O3 or to the low molecular weight antioxidants that might be protective within the insect respiratory tract-lining fluids. In fact, insects have received surprisingly little attention in terms of systematic work concerning the role of antioxidant micronutrients in antioxidant protection, despite the known importance of these micronutrients in plants and vertebrates.
Although few studies characterizing insect responses to oxidative air pollutants have been reported, mutations and/or overexpression of antioxidant enzyme systems and tolerance to oxidant stress in insects have been the subject of extensive research (35, 36), as have their genomic responses to oxidative stress (37) and their formation of oxidative protein and lipid products after oxidant exposure (38, 39). Their metabolism of ascorbate, vitamin E, and urate has been only partially characterized (40), including how they relate to oxidative stress (41–43). Interestingly, augmentations of antioxidant enzyme systems in whole insects, or even in motor cortex alone, increase the insect life span (36). However, paradoxically, exogenous augmentations of micronutrient antioxidants failed to affect life span and high dietary levels (2%) of ascorbic acid and/or vitamin E actually decreased life span (40). Augmentations of exogenous micronutrient antioxidants actually caused an adaptive decrease in selective endogenous antioxidant enzyme systems, a worrisome theoretic construct suggesting that intracellular antioxidants are actually decreased by exogenously administered antioxidants reaching intracellular compartments (40). Thus, the case of saturation of some transport systems in vertebrates (e.g., ascorbate cellular transport systems) might be protective against the accumulation of excessive intracellular concentrations of micronutrient antioxidants that could potentially adaptively decrease cellular antioxidant enzyme levels. It is apparent that additional scrutiny of the genomic, molecular, cellular, and anatomic responses of insects to oxidative pollutant exposures could enhance the understanding of inhalation toxicology in humans. THE SKIN WORLD Cutaneous tissues of animals, like respiratory tracts, are directly exposed to environmental toxicants such as O3. In these tissues it is the outermost barrier, the stratum corneum, that is the site of the atmospheric/cutaneous tissue boundary. The stratum corneum is composed of a unique system of structural, anucleated cells (corneocytes) embedded in a lipid-enriched intercellular matrix forming stacks of bilayers that are rich in ceramides, cholesterol, and fatty acids and consisting of only 15% water (44). Cutaneous cells at stratum corneum–epidermis interfaces even contain lamellar bodies responsible for lipid secretion into the outer protective layers of the skin (analogous to the protective surfactant mechanisms of the lower respiratory tract). This lipid–protein biphasic structure, which has a thickness of 3–5 µm in mice and is considerably thicker in humans, has a fairly rapid turnover and is believed to be the crucial determinant of the skin permeability barrier, which protects the body from harmful infectious and environmental agents while preventing excessive loss of water and aqueous solute movements through the skin. These extracellular lipid-enriched lamellar membranes, augmented by the oily complex lipids comprising sebum, which is secreted from sebaceous glands, serve as sacrificial reactants with environmental oxidants, desquamating and protecting underlying cellular layers in an analogous (albeit a more efficient) manner to the respiratory tract mucociliary apparatus, which continually removes oxidized surface proteins and lipids from airways and is replenished by secretions of the respiratory tract epithelial cells and glands. Like respiratory tract tissues, the skin contains an impressive network of extracellular and intracellular antioxidants (45). Figure 3 schematically depicts the various layers of the skin, illustrating reactions expected to occur on exposure to atmospheric O3. When hairless mice are skin exposed to high concentrations of O3 (e.g., 10 ppm for 2 hours), no significant depletions of vitamin E could be observed when full-thickness skin was analyzed, whereas vitamins E and C were found to be significantly depleted when only the epidermis was analyzed (46). However, when only the outermost stratum corneum was sampled, exposure to O3 at 1 ppm for 2 hours decreased vitamin E and vitamin C to about 80% and glutathione (GSH) and uric acid to 40–45% (47), unsurprisingly demonstrating that the stratum corneum represents the primary cutaneous target for environmental exposure and conclusively demonstrating that O3, either because of its direct reaction or via the generation of other reactive species at or near the biomolecular surface, is capable of locally depleting the micronutrient antioxidants as well as GSH and uric acid. It is not yet possible to estimate the degree of protection or "scavenging" of O3 by antioxidant micronutrients, although we have been able to demonstrate, using techniques whereby portions stratum corneum are removed by a tape-stripping technique, that exposure to concentrates of O3 as low as 1 ppm leads to generation of malondialdehyde (as determined by high-pressure liquid chromatography-based methods) and protein carbonyls (46).
O3 is known to generate a number of bioreactive species through its reactions with lipids (48, 49). Although measurements of formation of these species within the skin have yet to be made, our studies have provided some evidence that they are likely to be generated in stratum corneum and penetrate underlying cutaneous tissues, in that a number of markers of inflammatory processes in skin tissue were found to be elicited by O3 exposure (50). Unlike the case for O3 exposure of plants or of the respiratory tract of insects and animals, including humans, it is yet unproven that exposure to atmospheric O3 makes a significant contribution to skin pathobiology. However, it may be speculated that reaction with the well-organized interstitial lipids and protein constituents of the outermost stratum corneum barrier, and diffusion of bioreactive products from this tissue into the viable layers of the epidermis, may represent a contributor to skin disorders (as does facial cigarette smoke exposure). Furthermore, a closer examination of oxidant pollutant interactions with skin might provide further kinetic and mechanistic clues concerning oxidant-related reactions occurring at respiratory tract surfaces and conceivably serve, along with plants and insects, as a "biomonitor" of oxidative environmental pollutant exposure. THE RESPIRATORY TRACT WORLD Perceived associations between antioxidant levels in the diet and blood, measured indices of oxidative damage, and a host of clinical conditions that include local or systemic activations of components of the inflammatory–immune system, have captured the attention of the public, the nutripharmaceutical industry, and the medical sciences (1). Not surprisingly, a wide panoply of human respiratory diseases has been suggested to be linked to oxidative stress, supported by an ever-increasing number of studies that archive correlations between antioxidant levels and lung health (51–54). Several studies have indeed demonstrated the amelioration of some O3-induced lung effects by antioxidant supplementations (54, 55), but the case is far from convincing. Important considerations in interpreting such studies include the degree to which antioxidants contained in the respiratory tract lining fluids (RTLFs) might react with, and hence modulate, the impact of atmospheric oxidant pollutants, including O3 (3, 56), and of inflammatory–immune cell "oxidants" on respiratory tract cells. Understudied factors include the kinetics of the antioxidants contained in this compartment; the degree to which antioxidant supplements reach this area by means of dietary supplementation or by direct airway delivery; and, most importantly, the mechanisms, safety, and efficacies of such augmentation strategies in rigorously controlled studies. RTLF antioxidants are not evenly distributed throughout the respiratory tract. Proximal RTLFs are thicker and contain mucin (itself a sacrificial antioxidant, along with other glycoconjugated proteins present in RTLFs) and large concentrations of uric acid, whereas distal RTLFs are thinner and contain higher concentrations of ascorbic acid and GSH (3, 57–59). The problems of determining the actual concentrations of substances in airway surface liquids are nearly legendary and involve mainly imprecise measurements of plasma and interstitial fluid "leaks" occurring during lavage and/or induction and collection methodologies. There is controversy even over the electrolyte composition of RTLFs (e.g., hypotonic versus isotonic). Considerations of the role of RTLF compositions (and therapeutic strategies to augment such compositions) have been heightened by the fact that GSH levels, especially in distal RTLFs, have been shown to be decreased in cystic fibrosis, acquired immunodeficiency syndrome, and interstitial fibrosis (58). As diagrammatically depicted in Figure 4 , there is much theoretic and some experimental evidence that ambient pollutant reactive gases, such as inhaled O3, react with RTLF components and may never directly reach the underlying respiratory tract epithelial cells, at least in areas where they are covered by RTLFs (48). Therefore, the toxic effects of O3 on the underlying epithelial cells may be mediated, at least in part, by products of the reaction of these inhaled toxins with RTLF constituents. In the case of O3, such toxins may include not only products of its reaction with water, but also products from its reactions with lipids and proteins. These products would include lipid hydroperoxides, cholesterol ozonization products, ozonides, aldehydes, and oxidation products proteins or even antioxidants themselves (e.g., thyol and thyol-derived radicals).
As O3 is relatively insoluble, interactions of O3 with RTLFs is primarily governed by reactive absorption (i.e., the more oxidizable substrate that is present in RTLFs, the more O3 will be absorbed by the RTLFs). Therefore, inhaled O3 may be effectively removed by antioxidants present in the more abundant, proximal RTLFs, thus delivering less inhaled O3 to more distal and susceptible gas-exchanging regions of the lung (56, 59). However, when these toxic gases are not removed, analogous to the previously considered plant and insect situations, reaction of O3 may injure underlying cells. This results in cell activations designed to augment defense systems (e.g., increase vascular permeability, increase mucin secretion, decrease inspiratory tidal volumes, decrease airway diameter), or initiate inflammatory–immune processes. Many laboratories have shown that O3 reacts readily with ascorbic acid, uric acid, and thiols, and exposure of O3 to these molecular species results in their rapid depletion, especially of uric and ascorbic acid, and, to a lesser extent, of GSH. It follows that uric and ascorbic acid provide a protective screen against inhaled O3 and that GSH may be relatively less effective, at least in select model systems (60). Unfortunately, few in vivo data quantitate the degree to which these antioxidants actually react with, and thereby inhibit, the biologic effects of the inhaled O3. O3 in sufficient amounts is known to activate regulators of the expression of mediators of airway inflammation such as cytokines, chemokines, and adhesion genes. Critical in this regard are investigations of the effects of O3 in so-called susceptible populations already known to have inflammatory airway diseases (e.g., subjects with asthma and cigarette smokers). Many of these subjects may actually have augmented RTLF antioxidant levels due to increased glandular secretions and plasma leakages and cellular adaptations to the oxidant stress provided by their chronic inflammatory states. Thus, it may not be surprising that it has been difficult to demonstrate increased O3 sensitivity in these populations (61). Confounding the assessment of the oxidative effects of inhaled oxidants (including cigarette smoke) are the separation of direct effects of the inhaled oxidant, oxidative stress caused by inflammatory cell activation, and the oxidative effects known to be generated by cell injury of almost any sort. This also complicates interpretations of whether an administered agent is an "antioxidant," "antiinflammatory," or "cytoprotective" substance, none of these, or all of these, as best exemplified by vitamin E (62) The conundrum that demonstrations of oxidative stress known to be present in the milieu of inflammatory processes, as demonstrated by reduced levels of certain antioxidants and elevated levels of markers of oxidation, and that might be ameliorated by augmented antioxidant administrations, thereby resulting in helpful disease modifications, must still be considered unsolved (63). Understanding of the role of redox/thiol and oxidant/antioxidant processes at the critical respiratory tract cell plasma membrane surfaces must still be considered problematic (e.g., Lin and coworkers [64]), even though it seems likely that they affect cell injury and repair mechanisms (65), including those related to such human respiratory diseases as asthma (66).
CONCLUSION Acknowledgments The authors are grateful for discussions and collaborations with Lester Packer, Jeremy Barnes, Tom Polefka, and Ken Burtis. This article is dedicated to the memory of Eugene D. Robin, who always took a broad view of the lessons of comparative biologic systems in his view of human lung diseases. FOOTNOTES Supported by National Institutes of Health grant HL47628, and by a gift from the Colgate-Palmolive Company. Received in original form June 14, 2002; accepted in final form August 13, 2002 REFERENCES
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-tocopherol (vitamin E) as representative of the major hydrophilic and lipophilic antioxidant micronutrients, respectively. These antioxidants would be expected to react at various biosurfaces directly exposed to atmospheric oxidant pollutants. We focus on considerations of comparative biologic perspectives by comparing plants, insects, and mammalian skin and airways. 
