Letters, 51 (1990) 12>145
Cellular, biochemical and functional effects of ozone: new research and perspectives on ozone health effects
Elaine S. Wright, Daniel Dziedzic and Candace S. Wheeler Biomedical Science Department. General Motors Research Laboratories, Warren, MI (U.S.A.) (Received
3 May 1989)
Key words: Ozone; Effects; Lung
a toxic component
research efforts for several decades. risks to human provided
to ozone exposure. emerged
on the range of biochemical,
While the response
which may aid attempts
has been the focus of considerable
In spite of this large body of work, questions
to ozone exposure
remain as to the potential
to ozone. Newer studies in animals functional
to apply the results of these studies to decisions
some generalities regarding
troposphere by the reaction of hydrocarbons and oxides of nitrogen in the presence of sunlight. While ground-level ozone declined during the early years of this decade in most areas of the United States [l], more than 60 cities are currently classified as
Address for correspondence: Elaine S. Wright, Laboratories,
MI 48090, U.S.A.
@ 1990 Elsevier Science Publishers
out of compliance with the National Ambient Air Quality Standard (NAAQS) for ozone (daily I h average of 0.12 ppm not to be exceeded more than once per year). Thus, in spite of recent progress segments of the U.S. population exceed the current
in controlling oLone precursors in the atmosphere, continue to breathe ozone at concentrations which
This raises two distinct
but related questions
public health which must be addressed: (I) does the present standard protect the public health with an adequate margin of safety, and (2) is there excess risk of adverse health effects for populations living in areas where the current standard is not met? Recent epidemiologic studies have suggested a relationship between air pollution levels and deficits in pulmonary function [2.3]. But these studies are limited by confounding variables and difficulty in establishing exposure history. In controlled laboratory studies, decrements in pulmonary function parameters in human subjects have been measured under a variety of exposure conditions and concentrations of ozone as low as 0.12 ppm [&6]. These changes are reversible. and have been measured at low concentrations only when subjects are exercised strenuously to increase total inhaled dose. There is a high variability in responsiveness to oLone among individuals for reasons which are still unclear . The mechanisms by which irritants such as oLone induce decrements in pulmonary function parameters are not understood. Finally, it is not known whether these small, transient functional changes are in any way related to the potential development of chronic disease [Xl. Ozone is a reactive molecule and the products and stoichiometry of its reactions with cellular and extracellular components of the respiratory tract have not been fully elucidated. A promising new approach involves the USCof oxygen- IX as a tracer for both the chemical reactions and the binding of ozone-derived oxygen to tissue components as an estimate of total tissue dose . Although some studies suggest that the mechanism by which ozone causes damage involves the initiation of peroxidation of membrane lipids [IO]. other evidence does not support this view. The primary oronolysis products of unsaturated fatty acids are aldehydes and hydrogen peroxide [I 11. In erythrocyte ghosts. ozone appeared to react predominantly with membrane proteins extending into the extracellular fluid, with sullhydryl enzymes most sensitive to inactivation by ozone [ 121. A full understanding of the mechanisms by which ozone initiates damage at the cellular and subcellular level awaits further invcstigation. Animal studies of chemical, biochemical and morphologic responses of the respiratory system to ozone exposure suggest that the response of the lung to ozone is a dynamic one, involving a complex cascade of events. The morphologic and biochemical changes measured after ozone exposure are the results of both the direct effects of the initial interactions of ozone or the products of ozone-tissue reactions on cells, and the secondary responses of lung tissue to that initial damage. These secondary responses include influx and proliferation of inflammatory cells, proliferation and morphologic alteration of epithelial cells, and changes in synthesis and/or content of cellular and extracellular proteins. These effects, with varying degrees of severity,
have been observed
after acute and chronic
to a range of ozone
concentrations. Ozone can induce an apparent
state of adaptation
in the lung, wherein certain phy-
siological and/or cellular responses are attenuated even though exposure is continued. Furthermore, for a limited period of time after the cessation of exposure, a tolerant state exists so that re-exposure to a lethal concentration is survived and damage from sublethal doses may be less severe. Whether adaptation is beneficial and protects the lung from adverse effects of further exposure, or whether the induction of such a state represents an adverse effect per se, is subject to discussion. The complexity of the lung’s response to ozone, typified by the phenomena of adaptation and tolerance, has made it extremely difficult to use simple algorithms to predict the effects of chronic or lifetime exposure to low levels of ozone from the results of high-level acute studies. A number of recent studies have examined changes in the structure and function of the respiratory system after prolonged exposures to ozone in animals. This review will focus on recent contributions to the scientific literature which have significantly expanded our understanding of the various effects of ozone and their relationship to overall lung function, with particular emphasis on the effects of low-level and long-term exposures. MORPHOLOGICAL
Terminal airways andgas
The ozone lesion is focal in nature, and is most pronounced in the region of the central acinus where the terminal airways join the proximal alveoli in the gas exchange region of the lung [ 133151. Distal alveoli are less affected, although the lesions become more widespread as exposure concentration increases. Because of the focal nature of the ozone lesion, sophisticated sampling and measurement techniques have been applied to quantitate the small changes that occur in response to low-level exposure to ozone [ 16201. By sampling selectively from the affected regions in the central acinus, the ‘dilution’ effect of the remaining mass of unaffected alveolar tissue is minimized and sensitivity of quantitative morphometric measurements is increased. The most sensitive cell in the alveolus appears to be the epithelial type I cell, the squamous cell which lines the alveolar septum . Exposure to high ozone concentrations produced necrosis and sloughing of type I cells from the basement membrane . After exposure to concentrations of 0.50 ppm or less, electron-microscopic morphometric studies have shown increased volume and thickness and decreased surface area of type I cells in affected regions [17, 201. Type I cell morphology returned to normal after approximately a week in clean air  and tolerance to reexposure to a lethal concentration of ozone persisted only while type I cell morphology was altered . ‘Thicker’ type I cells appear to persist as long as exposure continues, at least for periods of up to 6 weeks [ 171. The alveolar type II cell, the progenitor of the type I cell and the cellular site of
after the initiation sponse is transient
a wave ofcell division
1 3 days
of exposure to ozone concentrations as low as 0.25 ppm. This reand subsides after 1 week even if exposure continues [22,24-261.
Type IT cell proliferation is a general response to oxidants and other lung toxicants , and is often described as a repair mechanism. However, it is apparently not necessary for the type I cell to be lost from the epithelium, since type II cell proliferation has been observed in the absence of type I cell necrosis. The alveolar macrophage, a cellular component of the inflammatory response induced by toxic insult to the lung. may have a role in the induction of epithelial cell proliferation [27,28]. Six weeks of exposure to 0.25 ppm also affects the two predominant cell types in the terminal bronchiole, the Clara cell and the ciliated cell . The surface area of both cells was decreased. There were no changes in absolute or relative cell numbers of either cell type. although Clara cell proliferation has been observed after exposures to higher ozone concentrations [30,3 11. Earlier findings  suggesting that chronic high-level ozone exposure leads to an apparent local restructuring in the regions of the central acinus, have recently been confirmed in both rats and monkeys. Extended exposure to a high concentration of ozone (0.95 ppm ozone for 90 days, 8 h/d) produced a 3.4-fold increase in respiratory bronchiolar cell volume in rat lungs . As new respiratory bronchiolar segments were formed, the lesion areas appeared to shift distally. Analogous findings have been reported in primates after 1 year of daily (8 h/d) exposures to 0.64 ppm . The functional significance of this anatomical restructuring is not clear, although the practical result appears to be an increase in the volume of the more resistant epithelial structures in the terminal airways; the site of continuing cellular injury remains the epithelium of the alveoli proximal to the terminal airways. Trachea and large airways After 3 days of exposure of rats to a high concentration of ozone (0.96 ppm, 8 h/night) light and electron microscopy showed increases in numerical density of abnormal and necrotic cells . Abnormal ciliated cells had shortened or damaged cilia. Labeling index (fraction of cells incorporating 3H-thymidine) was also increased, consistent with the typical proliferative repair response which occurs in other regions of the respiratory tree during the first week after exposure is initiated. After 60 days of exposure, the number of cells with shortened cilia increased, but no excess of necrotic cells relative to controls was observed. Labeling index had returned to control values as had the numerical density of intermediate cells. Seven days after the conclusion of the 60-day exposure, the normal morphology of cells in the tracheal epithelium had been partially restored, and by 42 days post-exposure, all parameters had returned to control values. Thus, prolonged exposure to a high concentration of ozone resulted in an apparent state of morphologic adaptation in that the severity of damage declined with continued exposure. Furthermore, all changes induced by chronic exposure to ozone in the trachea appeared to be fully restored to normal when animals were removed from the ozone and allowed to recover in clean air.
Nasopharyngeal regions Rats exposed to 0.25 or 0.50 ppm ozone for up to 24 months
the respiratory epithelium, primarily goblet cell hyperplasia, in the anterior portion of the nasal cavity. No effects were observed at the light-microscopic level with exposure
to 0.12 ppm . In a quantitative
were exposed to 0.15 ppm ozone (8 h/d) for 6 days or to 0.15 or 0.30 ppm for 90 days . Changes in both acute and chronic studies were limited to the nasal mucosa anterior to the nasal turbinates. After 6 days of exposure, neutrophil infiltration and proliferation of transitional and respiratory epithelial cells were evident. After 90 days, the number and proportion of epithelial cells had returned to control levels and the inflammatory response had disappeared. However, ultrastructural changes in goblet cells were evident that had not been present in the acute study. After both 6 and 90 days, there was secretory cell hyperplasia in transitional epithelium, as well as loss of cilia and the necroses of ciliated cells. In companion experiments , histochemical methods were employed to demonstrate that a 6-day exposure resulted in significant increases in both acidic and neutral glycoconjugates stored in transitional and respiratory epithelium. After 90 days of exposure, the amount of stored mucosubstance had decreased. Only in the transitional epithelium did total mucosubstance remain greater than controls, and there was a shift in acidic mucosubstances from sialomucins to sulfomucins. The relative decrease in stored mucosubstances after 90 days was not due to a decrease in cell number, but rather to a decrease in production or storage. Thus, the effects of a 6-day exposure to ozone on the morphology and secretory function of the nasal region are qualitatively and quantitatively different from the effects observed after exposure for 3 months. FIBROSIS
Pulmonary fibrosis, defined generally as the abnormal deposition of collagen in the lung, or irreversible ‘scarring’, is a possible severe outcome from chronic exposure to ozone. Numerous studies have suggested that short-term exposure to high concentrations of ozone increase collagen synthesis and produce morphologic changes consistent with developing fibrosis . However, significant effects have been detected only at exposure concentrations 30.50 ppm, regardless of exposure duration. Furthermore, chronic exposures to ozone concentrations near ambient levels have not produced changes as severe as would be predicted by simple linear extrapolation from the results of these acute studies . When deposition of excess collagen has been shown, related functional consequences of these changes have been small or unmeasurable. True irreversible fibrosis with associated decrements in lung function would clearly represent an adverse health effect. As yet there is little experimental evidence to support the hypothesis that exposure to environmentally relevant concentrations of ozone, even for prolonged periods, results in an irreversible fibrotic change.
The fibrogenic potential of ozone and other agents has been evaluated quantitatively by measuring collagen synthesis as incorporation of radiolabeled proline into hydroxyproline, or by measuring total lung collagen as hydroxyproline . Increased
of rats to 0.12, 0.25 or 0.50
ppm ozone continuously for up to 1 year , 0.50 ppm for from 3 days to 6 months , 0.80 ppm for l-7 days , and of monkeys to 1.5 ppm for I week . In none of these studies was there an increase in the percent of total protein synthesis devoted to collagen. This suggests that the observed increases in apparent collagen synthesis were part of an overall increase in protein synthesis in the lung, rather than a specific induction of connective tissue protein synthesis. Total lung collagen content in mice increased after exposure to I .O ppm for up to 40 days, but the effect disappeared with IO days recovery in clean air . In the same study, 5 days of exposure to a range of doses up to I .7 ppm produced no increase in total lung collagen. Lung collagen content increased after [email protected]
days of exposure of rats to 0.50 ppm but not after 6 months . No effect of exposure to 0.80 ppm ozone for 37 days on lung hydroxyproline content was detected, nor was there a positive interaction between ozone and silica, a known fibrogenic agent [5 I]. Intermittent exposure over a period of 3 months (6 h/d, 5 d/wk) caused a significant (28%) increase in lung collagen content in rats exposed to 2.0 ppm; however, in this study lung collagen content decreased significantly in rats exposed to 0.20 or 0.80 ppm . Longer exposures have also produced mixed results. After 18 months exposure of rats to 0.12, 0.25 or 0.50 ppm ozone (20 h/d) lung collagen was 28% greater at the highest dose of ozone relative to age-matched controls. However, this increase was not statistically significant. and was superimposed on a highly significant age-related increase in total lung collagen in controls and all ozone-exposed groups . Monkeys exposed for 1 year to 0.64 ppm (8 h/d) showed an increase of approximately 26% in lung collagen when data were normalized to body weight . One year of exposure (8 h/d) of monkeys to 0.25 ppm had no effect on lung collagen content. but when animals were exposed only during alternate months, a 17% increase in total lung collagen was reported . It should be noted that while dramatic increases in total collagen with prolonged exposures have not been demonstated, nature of the collagen which is laid down in ozone-exposed lungs respect to the types of molecular cross-links present .
the qualitative may differ with
Measures of metabolic function and biochemical constituents of the lung are of necessity performed on whole lung tissue, with results thus being an ‘average’ over the entire lung. Because subtle or focal changes may not be detected with biochemical analysis of the whole lung, and because the biological significance of such changes is not clear, it is important to consider morphologic and functional findings in concert with the results of biochemical studies . Thickening of the alveolar interstitium is a common finding in studies of prolonged ozone exposure [32- 34,361. This thickening has been attributed to interstitial edema. infiltration of fibroblasts and inflammatory cells and/or deposition of collagen fibrils.
for 1 year to 0.64 ppm (8 h/d) produced
of this effect in monkeys in stainable
exposed fibrils and
fibroblasts that were not statistically significant . After 24 months of exposure of rats to 0.25 or 0.50 ppm (but not 0.12 ppm), a qualitative increase in stainable collagen was observed in the peribronchiolar/alveolar duct region. Increased collagen deposition in alveolar septae observed after 78 weeks was attributed to age rather than ozone exposure since the changes occurred in controls and all exposure groups and in alveoli distal to the regions affected by ozone . Diffuse accumulation of interstitial collagen in aged rats has been described previously . Attempts to relate ozone-induced changes in ventilatory function with either biochemical or histologic evidence of fibrosis have met with limited success. In monkeys exposed daily during alternate months for 1 year to 0.64 ppm (8 h/d), chest wall compliance and inspiratory capacity increased, but no change in lung compliance was detected; continuous daily exposures produced no functional changes . These results are consistent with an effect of ozone on the timing of lung development in the monkey but are not consistent with functionally significant fibrosis. Resistive changes were measured in rats after exposure to 0.20 or 0.80 ppm over a period of 3 months (6 h/d, 5 d/wk) in the presence of decreases (rather than increases) in lung collagen and elastin content . Chronic exposure of rats to ozone administered in a complex diurnal pattern simulating urban conditions (13 h of ozone at 0.06 ppm with a 9-h spike peaking at 0.25 ppm, 5 d/wk for up to 18 months) produced changes in lung function, including a significant decrease in total lung capacity, which were reversible after 6 months recovery in clean air . Morphometric evaluation of the lungs of animals from this study showed increased volume density of the interstitium in centriacinar regions that was not fully reversed after 4 months of recovery in clean air . However, the increase in collagen fibril content in the interstitium observed after 18 months of exposure was no longer present after the recovery period, suggesting that the increased interstitial matrix volume deposition of connective tissue protein. Exposure of rats to 0.50 ppm ozone (20 matory and fibrotic changes in the central in ventilatory function parameters. After the functional and inflammatory changes logic evidence of minimal fibrosis remained
may not have been due solely to excess
h/d) for 52 weeks caused mild focal inflamacinus regions as well as restrictive changes 3-6 months of recovery in clean air, both had completely resolved, but some histo. The authors suggested that the func-
tional changes observed were more likely attributable to the smoldering inflammatory response that persisted as long as ozone was present, rather than to connective tissue deposition, a conclusion that is not inconsistent with the findings of others who have attempted to measure functional changes in response to chronic ozone exposure.
of a number
[60,61]. In brief, clearance
from the deep lung and airways of cellular
on the function
on the tree
ciliated cells, and
secretory cells, and on the physical and chemical properties of fluids  lining the alveoli and airways, all of which have been shown to be affected by ozone exposure. It is not unreasonable to suspect that one or more aspects of lung clearance would be compromised by ozone exposure. Recent studies of the effects of ozone exposure on the rate at which materials are removed from the lung have shown that acute exposure can increase or decrease clearance rates, depending on the concentration and duration of the ozone exposure and the time at which clearance measurements are made. Single acute exposures of animals and humans to ozone concentrations less than 0.60 ppm have been shown to accelerate clearance of particles (1.65 pm mass median aerodynamic diameter) from the tracheobronchial tree [63--661. Single acute exposures to ozone concentrations greater than 0.60 ppm caused a delay in particle clearance during the first 50 h after exposure, but enhanced long-term clearance rates (measured between 50 and 300 h post-exposure) [63,65]. Thus, single acute exposure to ozone at concentrations greater than 0.60 ppm appears to retard clearance, while lower concentrations have the opposite effect. On the other hand. repeated exposures (2 h,id for 14 d) to 0.10 and 0.60 ppm ozone produced significant acceleration in alveolar clearance of latex particles , but had no effect on tracheobronchial clearance . These results taken together are consistent with the development of an adapted state, suggested by morphologic studies [32,35,37] and demonstrated by Frager et al. . In the latter study. acute exposure to I .2 ppm ozone caused a significant retardation in tracheobronchial clearance; when animals were pre-exposed to 0.80 ppm, the I .2 ppm ozone challenge 3 days later had no effect. There has only been one study of the effects of low levels of ozone on long-term clearance . After 6 weeks of exposure to a complex exposure regimen designed to mimic ambient pollutant conditions rats inhaled chrysotile asbestos for 4 h. Preexposure to ozone had no effect on deposition of asbestos or on lung burdens after 24 h of recovery in clean air. However, after an additional month in clean air, more of the originally deposited asbestos was retained in the ozone-exposed animals. Because other studies have suggested that low-level exposures increase rather than decrease clearance up to 2 weeks after exposure, it is unclear whether this result is unique to asbestos, or whether other fibers and particles would also show a delay in long-term clearance from this region after long-term ozone exposure. ALVEOLAR
(AM) is the primary
lower lung and plays a critical role in defending inhaled
the alveoli and distal airways against
out this important
maintain active mobility, phagocytic activity, membrane integrity and enzymatic capacity. The effects of ozone on AM function have been implicated as a factor in increasing susceptibility to infection in animal models . Ozone exposure in vivo has also been shown to alter the number and morphology of AM, depending on the dose and the duration of the exposure. A transient decrease in the absolute number of AM has been observed following acute ozone exposures to relatively high ozone concentrations [70,7 11. The initial decrease in lavaged cells may result from a destruction or lysis of exposed macrophages. A decline in the number of macrophages recovered by bronchoalveolar lavage may also be due to a change in functional characteristics of the macrophage population such as increased stickiness resulting from spreading and more filopodia, or to an increase in the viscosity of the intraluminal exudate. AM recovered from rats exposed for 16 h to 0.05, 0.10, 0.20 or 0.40 ppm ozone showed no differences in cell yield or cell viability after ozone exposure. However, adherence of macrophages to a nylon filter increased after exposure to the two lower concentrations, an effect which the authors interpreted as reflecting increased cell activation, a defensive reaction of the cell . Increases in AM numbers have also been observed after in vivo ozone exposure . Recruitment and persistence of AM may result from: (1) chemotaxis and migration of cells into the lung alveoli, (2) local proliferation, or (3) decreased cell efflux or turnover. While the ultimate source of AM has long been considered to be the bone marrow, attention has recently been directed towards local cell division within the lung of intra-alveolar macrophages as a source of these phagocytic cells. Evidence is accumulating that proliferation of resident AM, induced by single acute exposures to ozone as well as by continuous low-level exposures, contribute to the increased number of AM observed in the lung [26,69,74]. The degree to which in situ AM proliferation contributes to the increase in numbers of AM with exposure relative to other mechanisms such as influx from the vascular and interstitial compartments is not clear. The dynamic character of the AM population is an important factor in attempting to interpret the results of studies of AM function. Among the functional properties of AM affected by ozone is random migration, or mobility. After exposure of rats to 1 ppm ozone for 4 h a decrease in the surface area cleared by the lavaged AM as they moved across gold-colloid-coated glass coverslips was measured . In another study, cells were obtained by bronchopulmonary lavage from rhesus monkeys exposed to 0.80 ppm continuously for 7 days . Both the number of cells randomly migrating and the distance they migrated was decreased due to ozone exposure. The addition of surfactant isolated from control animals enhanced migration but did not restore it to pre-exposure levels. AM mobility was not altered, however, following an acute (2-h) exposure to 0.10 or 1.2 ppm ozone or to a 13-day (2 h/d) exposure to 0.10 ppm ozone .
One of the primary functions of the AM is the maintenance of sterility in the lung by phagocytosis of foreign particulate material. This function may be measured in vitro by incubating AM with test particles and measuring the number of particles taken up by the cells over time. AM recovered by lavage from rat lungs and exposed in culture by diffusion of ozone through a teflon film showed dose-related decreases in phagocytosis . Cells exposed in culture on a rocker panel to a range of ozone concentrations in air (0.20 6. I ppm) for 2 h showed no changes in viability or adherence, but phagocytosis was significantly depressed following exposure to concentrations of 0.60 ppm or greater . Thus, ozone can apparently directly induce decreases in AM phagocytic activity. Changes in the phagocytic activity of AM have also been observed following in vivo exposure to oLone. A marked depression in the phagocytic activity of AM lavaged from ozone-exposed rabbits was observed immediately and 24 h after acute exposure to 0. IO ppm and at all time-points (immediately, 24 h, and 7 d) after exposure to 1.2 ppm ozone. Repeated exposures (2 h;‘d for 13 d) to 0.10 ppm reduced the number of phagocytically active AM on day 3 and 7 but returned to control levels by day 14 . While impairment of AM function has been reported after only a few hours of ozone exposure . enhanced phagocytic function has been observed after longer exposures . Enhanced phagocytosis of inert microspheres was observed after 3, 7 or 20 days of exposure of rats to 0.80 ppm ozone, with the greatest increase observed on day 3. The difference in response between these studies and others which showed impairment of phagocytic function may be explained by differences in exposure conditions, species or assay conditions and indicates the importance of dose (both concentration and duration of exposure) on the subsequent functional changes observed. While the initial changes were in opposite directions, the effects in both studies were maximal after 3 days of exposure and subsided to approximately control values after 2 or 3 weeks of continued exposure. This may be due to a shift in cell population after several days of exposure, leading the authors to suggest that there had been an adaptive response with subchronic exposure. The influence of ozone on the secretory activity of AM and their ability to release factors which stimulate the migration of neutrophils or other cell types into the lung has been examined. Culture medium recovered from monolayer cultures of rabbit AM immediately, 2 and 6 h after exposure to air or 0. IO, 0.30 or 1.2 ppm ozone for 2 h was used in Boyden chambers to test for chemotactic activity using blood-derived monocytes and neutrophils as the responding cell types. Exposure to the highest ozone concentration increased monocyte chemotaxis. while exposure to both 0.30 and I .2 ppm increased neutrophil chemotaxis [X0]. Thus, ozone can apparently affect AM directly, with resultant increases in the release of chemotactic factors. This result in vitro is consistent with in vivo studies showing that acute exposure to 1.2 ppm ozone or subchronic exposure (7 or I4 d, 2 h/d) to 0.10 ppm results in an increase in polymorphonuclear leukocytes recoverable from the lung by lavage [7l], and sug-
gests that AM recruitment the overall pulmonary
cells into the lung may be important
Exposure to ozone can also affect biochemical functions of the macrophage which may be important for the functional integrity of the cell. Ozone exposure caused a reduction in intracellular concentrations of acid phosphatase and /I-glucuronidase . Although these lysosomal enzymes are not bactericidal, their absence suggests that the mechanism of ozone-induced impairment may involve injury to lysosomal enzymes. Lysozyme, a lysosomal enzyme of known bactericidal activity, was reduced after ozone exposure [82,83]. Lysozyme content was further reduced in ozone-exposed rats with chronic pulmonary infection suggesting a synergistic effect between the ozone exposure and the chronic infection . LYMPHOCYTE
Lymphocytes generate, regulate and carry out immune and non-immune inflammatory reactions. Non-immune inflammation occurs as a response to tissue damage or stimulation by materials which are not antigenic (certain components of bacterial cell walls, for example). Specific immunity is a vital component of host defense against foreign microbes and tumor cells, and is related to pathologic processes such as asthma and other hypersensitivity responses. Evidence now shows that lymphocytes are affected in experimental animals in complex ways during ozone inhalation. Lymphocytes appear in the lung and are activated in the lymph nodes which drain the lung. This reaction in turn affects how the lung responds to ozone and to antigenic materials. Lymphocyte
stimulation during ozone exposure
Lymphocytes infiltrate both the upper airways and the centriacinus of the lung during experimental ozone exposure. The upper airways of mice exposed to ozone at 0.50-0.80 ppm (233/, h/d) for 1-24 days show a two-fold increase in IgA+ B-lymphocytes . In the deep lung, exposure to 0.70 ppm (20 h/d) for 4-14 days causes an interstitial accumulation of Thy I .2+ (T-lymphocyte) clusters within the centriacinar lesions [85,86]. However, the effect of ozone on lymphocytes is not limited to the lung proper. The lung is directly connected to a complex of lymph nodes via lymphatic vessels. In the mouse, for example, 2-3 large nodes are present in the upper mediastinum. Examination of these mediastinal lymph nodes from mice exposed to ozone at 0.25, 0.50 and 0.70 ppm (20 h/d) for l-28 days revealed a phasic, dose-dependent accumulation of lymphocytes within 2 days of exposure . Increased numbers of blastic forms, mitotic figures and enhanced tritiated thymidine uptake appeared in the paracortex. Treatment with anti-T-cell serum inhibited ozone-induced cell accumulation . The total leukocyte accumulation is actually greater in the lymph node complex
than at the principal
lesion site in the lung, the centriacinus.
to 0.70 ppm (20 h/d) for 4 days, the time of maximal response in the lymph node, increases the number of cells recovered by lung lavage by about 370000 cells (3.8 x lo5 cells in controls
vs. 7.5 x lo5 cells in exposed
mice) [D. Dziedzic,
lished]. In contrast, the same exposure increases the number of cells in a single mediastinal node over controls by nearly 10 million cells (2.8 x lo6 cells in controls vs. 12.5 x IO6 in exposed mice) . Similarly, exposure to 0.50 ppm of ozone for 9 days results in an increase in the number of cells recovered by lavage from the lung by about 200000 cells per mouse , while the same level of exposure increased the number of cells in a single mediastinal lymph node by 3.3 million . The magnitude of the lymphocyte response indicates that lymphocyte activation is as integral to the reaction to ozone in the mouse as the PMN and macrophage responses. Modulation of the lung response to ozone by lymphocytes How the lymphocyte affects the lung reaction to ozone was investigated using animal models of lymphocyte deficiency and lymphocyte stimulation. Exposure of athymic nude mice, which lack mature T-cells, to 0.70 ppm of ozone for 20 h/d for up to 14 days revealed significantly more lung damage than euthymic animals which possess normal T-cell function. Lung wet weights and lung lesion size increased and histopathologic changes were more extensive in the athymic animal than in euthymic animals . In contrast, treatment of normal animals with poly I:C, a polynucleotide which stimulates lymphokine secretion, protected against ozone-mediated damage . Thus, during ozone exposure lymphocyte activation can modulate the lung response to ozone, probably reflecting its role as a regulator of inflammatory cells. How lymphocytes function as non-immune inflammatory cells is not completely understood . However, the effect of a variety of pulmonary toxins, including ozone and materials such as asbestos , oxygen [94,95] and bleomycin , can be modulated in animals whose lymphocytes are suppressed or stimulated. This suggests a key role for lymphocytes in ‘non-specific’ lung responses to inhaled materials in general. EfSect of ozone on immunity/host defense Ozone may also affect the way that immunologic responses are generated against antigenic materials through its effect on lymphocytes. Changes in immune function depend on the species, organ, dose and duration of ozone exposure. In the mouse, functional changes accompany the increase in numbers of lymphocytes in the mediastinal lymph nodes. Concanavalin A (Con A) is a plant lectin which stimulates the cell-mediated arm of the immune system. Con A reactivity in vitro was unaffected at days 4 or 7 of a 0.70 ppm exposure (20 h/d) but increased 1.5 and 3.0-fold after 14 and 28 days, respectively . In contrast, splenic cells of mice exposed to 0.2 mg/ m3 of ozone (0.10 ppm) for 103 days (5 h/d, 5 d/wk), showed decreased reactivity to T-cell mitogens in vitro (phytohemagglutinin (PHA), 38% decrease; Con A, 75% decrease) but not to a B-cell mitogen (lipopolysaccharide (LPS)) or to alloantigens
. In the rat, 7 days of exposure to 1 ppm ozone enhanced reactivity of spleen cells to PHA, Con A and LPS . Thus, reactivity of lung-related lymphocytes increases while depression of systemic cells occurs. The rat and mouse models show different effects in the spleen response indicating possible species differences. Furthermore, the most consistent changes in the functional response occur in the T-cell population. In vitro changes in the systemic immune system in the mouse parallel changes in antigen processing in vivo. During exposure to 0.80 ppm ozone the primary, IgMantibody response to a T-cell-dependent antigen, sheep red blood cells (SRBC), was suppressed at days 1, 3, 7 and 14, as measured using a plaque-forming cell assay with spleen cells . Exposure to 0.80 ppm also inhibited a T-cell-mediated delayed hypersensitivity response to SRBC as assessed using a footpad swelling assay, although the difference between control and exposed animals was statistically significant only at day 7 and not at days 1, 3 or 14. In addition, the number of circulating lymphocytes in blood was depressed on days 3 and 7, but the difference was significant only on day 3 [loo]. These studies suggest a suppressive effect on systemic immune cell processing in the mouse. However, the significance of changes in the systemic immune response as measured both in vivo and in vitro is difficult to assess since the mechanism by which ozone would affect peripheral splenic, thymic of blood lymphocytes is unknown. The high reactivity of ozone makes it unlikely that systemic effects are attributable to a primary reaction of the ozone molecule, and no reactive products of ozone have as yet been identified in peripheral systemic organs. Non-specific changes occur during ozone inhalation in experimental animals which affect cells of the immune system. The mouse, for example, is a steroid-sensitive species in which exogenously applied steroids at doses as low as lo-’ M result in a significant decrease in thymus, spleen and lymph-node weights. Light- and electron-microscopic evidence shows that lymphocyte death occurs in these animals when exposed to steroids [loll. Changes that are consistent with stress-related effects have been observed in the mouse during ozone exposure, Exposure to ozone at levels of 0.40 to 0.80 ppm can induce weight loss and/or thymic and splenic atrophy [87,99]. Prior adrenalectomy of mice exposed to 0.70 ppm ozone (20 h/d) for 4 days substantially reversed thymic atrophy seen at this level of exposure , suggesting that ozone can produce steroidmediated effects on lymphocytes through an adrenal-mediated stress response. The effects of adrenal steroids at higher and lower levels of ozone exposure in the animal model have not, as yet, been systematically studied. ADAPTATION
In spite of the complexity of the lung’s response to ozone, there are some generalities emerging which merit attention because of their impact on attempts to predict human responses to long-term low-level ozone exposure and, by extension, on attempts to protect human health. Although there is not yet sufficient data available
to allow a direct extrapolation
it is likely that responses
to ozone at the cellular level are qualitatively similar for all mammals. Recent studies of morphology, biochemistry and clearance suggest that adaptation, a diminished response with increasing exposure duration, occurs in chronically exposed lungs. Studies showing diminished responses with continued exposure illustrate the limitations of attempting to predict the degree to which lung tissue would be affected by prolonged low-level exposure from experiments which utilized short-term exposures to higher concentrations. In the absence of information about the mechanism by which ozone elicits a particular change, attempts to predict chronic effects from the results of acute exposures to high ozone concentrations should be approached with caution. Lung lesions observed after 1 week of exposure to 0.20, 0.50 or 0.80 ppm ozone were more severe than those observed after 3 months of continued exposure . Other investigations of lung morphology after prolonged exposure periods have yielded similar results in all regions of the respiratory tree [35,37,38,102]. It has been suggested that the morphologic alterations of epithelial cells may account for the development of tolerance and adaptation [20,35,103]. This hypothesis holds that both adaptation and tolerance are due to changes in cell morphology which result in decreased surface area of ‘sensitive’ cells available for attack by ozone molecules. An alternative hypothesis which has been raised to explain tolerance and adaptdtion is based on the observed increase in lung content of certain enzymes which are purported to protect cells from oxidant attack . However, tolerance can be present in the absence of measurable increases in these enzymes [35, 1051. While these systems have been the subject of much speculation regarding their role in defending the lung against toxic insult in general, their importance in mediating the lung’s response to ozone is not yet clear. Consideration of possible adaptation mechanisms may help to explain apparent differences in results of acute and chronic experiments with respect to collagen synthesis and deposition. Exposure of rats to high ozone concentrations for short periods cause chages in collagen synthesis and deposition which have not been duplicated with continuous chronic exposure to lower concentrations. In spite of a great deal of study, chronic, irreversible fibrosis in rats and monkeys has not been demonstrated clearly with exposures to less than 0.50 ppm ozone. An initial burst of collagen synthesis may occur as part of the initial inflammatory and reparative response of lung tissue to injury, which, like the proliferative responses of epithelial cells and macrophages, subsides as the lung becomes adapted to the continued oxidant insult. This hypothesis does not preclude the possibility that increased synthesis and deposition of normal or abnormal collagen may continue in the centriacinar region with prolonged low-level ozone exposure, and that measurements have not been sensitive enough to quantitate those changes. Particularly in view of known age-related patterns of collagen deposition in the lung, the functional significance of such small localized changes would have to be documented in order to evaluate their impacts as a potential adverse health effect in the human population.
It has long been known ozone, nitrogen
and oxygen will survive
to lethal con-
centrations. Morphologic and morphometric studies have documented the transient nature of proliferative reactions of macrophages and epithelial cells as well as the fact that overall evidence of damage subsides with prolongation of exposure. The adaptation of biochemical and functional end-points such as synthesis and deposition of collagen and bronchopulmonary clearance have not been well characterized and need more direct investigation if they are to be verified. Complicating the situation is the observation that human subjects exposed repeatedly to ozone in controlled chamber studies show decreasing responsiveness in spirometry tests over time. Since the mechanisms by which ozone induces changes in ventilatory function are not presently known, it would be premature to conclude that the observations of adaptation in animals bears a relationship to the phenomenon in humans. However, it is likely that cellular and biochemical changes observed in chronic studies of low-level exposures of animals to ozone bear at least a qualitative relationship to what might occur in humans. Comparison of the results of morphometric analysis of alveolar cells of human, baboon and rat lungs showed interspecies differences in macrophage and epithelial cell populations [ 1061. The authors suggested that humans normally encounter many pollutants (cigarette smoke, ozone, N02, particulates etc.) in daily life and that this could account for the observed differences relative to rats living in carefully controlled laboratory atmospheres. The same investigators have suggested that subtle changes in epithelial cell characteristics occur with chronic exposure to very low levels of ozone, possibly even as low as 0.06 ppm, a concentration which can be attained in pristine areas in the absence of anthropogenic pollution sources [ 107,108]. If these morphologic changes are related to tolerance and adaptation, it may be that the lungs of most if not all humans are in an ‘adapted’ state. DISCUSSION
While it is not yet possible
to define the conditions
on humans can be expected to occur, there are some general principles which should be considered by policy makers in setting and enforcing clean air standards. First, it is important to appreciate all the complexities involved in extrapolating results of animal experiments to human health effects. Aside from the obvious problems of extrapolating to equivalent inhaled doses between animals and humans, it is likely that changes in similar end-points may be mediated by different mechanisms. For example, acute ventilatory function changes in humans may be mediated neurogenically via irritant receptors, while similar effects after acute high-level exposures or chronic low-level exposures of rats have been attributed to sequelae of chronic inflammation or fibrosis. On the other hand, it is not unlikely that the subtle morphologic changes that have been observed in animals may occur in humans as well. It is not yet known,
models which have been studied
are more or less sensi-
tive to ozone injury than humans. Thus, it is important to exercise care in applying the results of animal studies to possible human health effects where there has not yet been developed a sufficient understanding explain the response under study.
of the underlying
Second, as scientists continue to use more sensitive and elegant measurement techniques, small changes are being documented in response to low ozone concentrations. There is at present no consensus among scientists or clinicians regarding the actual significance for human health of small reversible changes in ventilatory function or subtle reversible morphologic alterations. Clearly, irreversible pulmonary fibrosis with functional consequences, increased morbidity, and cancer  are outcomes from which the public must be protected. However, subtle reversible effects which are not clearly related to pathologic changes are not necessarily adverse. The issue of reversibility itself needs further examination. While irreversible changes are clearly adverse, reversible changes may or may not be, depending on other factors such as the severity of the effect, and whether or not function is compromised even temporarily. Furthermore, the complex issues of synergy with other pollutants, and of the impact of small changes on function already compromised by age or pre-existing disease, must be examined. Finally, a better understanding of tolerance and adaptation is needed. Under some experimental conditions, it appears that ozone exposure results in a lung which is more resistant to environmental challenge. Humans encounter ozone and other pollutants under a wide variety of conditions, however. On the basis of our present limited knowledge, the possible importance of these phenomena in modulating thresholds for adverse health effects of exposure to ozone and other inhaled pollutants is not clear.
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