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Am J Physiol Lung Cell Mol Physiol 291: L644-L650, 2006. First published April 28, 2006; doi:10.1152/ajplung.00027.2006
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Cyclic exposure to ozone alters distal airway development in infant rhesus monkeys

Michelle V. Fanucchi, Charles G. Plopper, Michael J. Evans, Dallas M. Hyde, Laura S. Van Winkle, Laurel J. Gershwin, and Edward S. Schelegle

Center for Comparative Respiratory Biology and Medicine, California National Primate Research Center, University of California, Davis, California

Submitted 20 January 2006 ; accepted in final form 19 April 2006


   ABSTRACT
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Inner city children exposed to high levels of ozone suffer from an increased prevalence of respiratory diseases. Lung development in children is a long-term process, and there is a significant period of time during development when children growing up in urban areas are exposed to oxidant air pollution. This study was designed to test whether repeating cycles of injury and repair caused by episodes of ozone exposure lead to chronic airway disease and decreased lung function by altering normal lung maturation. We evaluated postnatal lung morphogenesis and function of infant monkeys after 5 mo of episodic exposure of 0.5 parts per million ozone beginning at 1 mo of age. Nonhuman primates were chosen because their airway structure and postnatal lung development is similar to those of humans. Airway morphology and structure were evaluated at the end of the 5-mo exposure period. Compared with control infants, ozone-exposed animals had four fewer nonalveolarized airway generations, hyperplastic bronchiolar epithelium, and altered smooth muscle bundle orientation in terminal and respiratory bronchioles. These results suggest that episodic exposure to environmental ozone compromises postnatal morphogenesis of tracheobronchial airways.

oxidant air pollution; adverse effects; growth and development; lung

OZONE IS THE PRINCIPAL OXIDANT air pollutant in photochemical smog. Almost half (49%) of the population in the United States lives in areas that have unhealthful levels of ozone (2). In controlled human studies (using healthy young to middle-aged adults), health effects of ozone include reductions in pulmonary function measurements, increased airway reactivity, and pulmonary inflammation (reviewed in Ref. 1). Exposure to occupational ozone by mail carriers in Taiwan has also been associated with reductions in acute lung function (9).

Children may be more at risk to the adverse effects of air pollution than adults are for several reasons. First, children have a higher minute ventilation and a higher level of activity compared with adults (reviewed in Ref. 26). Second, children spend more time outdoors than adults do, increasing exposure to air pollutants (reviewed in Ref. 1). Third, lung development is a long-term process. Although the human lung needs to be sufficiently formed at birth to perform its primary function, gas exchange, lung growth continues for an extensive period (8–12 years) after birth (6). During this time, there are multifold increases in overall lung size, active cellular differentiation, cell division, and alveolar formation. As a result, airways change in size and shape with maturation, altering deposition patterns. In addition, lung function continues to change, increasing until late adolescence in both males and females, when it plateaus (3, 17, 39).

Although it is well recognized and accepted that air pollution affects lung function and growth in children (7, 15, 17, 23, 29, 32, 33, 43), results from epidemiological studies differ in the contribution that the oxidant pollutant ozone plays in these alterations. Epidemiologic studies differ in design, methods of assigning exposure, methods of assessing lung function, and populations studied. To study directly the effect that ozone has on lung growth, controlled experimental studies are needed.

Chronic airway disease and decreased lung function in children exposed to ambient air pollution may be due to repeating cycles of injury and repair, which alter normal lung maturation. To address how repeated exposures to episodes of ozone affects airway development, we exposed infant rhesus monkeys to cyclic episodes of 0.5 parts per million ozone for 5 mo. This concentration of ozone is equivalent to high environmental concentrations found in Mexico City. Nonhuman primates were chosen as a model because their airway structure and postnatal lung development is similar to those of humans. The distal portion of the airway tree in nonhuman primates is similar to that described in humans (20, 50), consisting of a terminal bronchiole (the most distal nonalveolarized conducting airway) followed by several generations of respiratory bronchioles (small airways with alveolar outpocketings in the walls) (36) opening into the alveolar region (46). After evaluating airway architecture, epithelial composition, and airway wall smooth muscle bundle orientation, we conclude that exposure to environmental ozone during the early postnatal period alters the development of the distal pulmonary airways. Some of the results from these studies have been previously reported in abstract form (14).


   METHODS
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Animals and ozone exposure. Twelve male infant rhesus monkeys (Macaca mulatta) were removed from their mothers at birth and raised as social groups in housing supplied with chemically, biologically, and radiologically filtered air as previously described (38). All monkeys were from the breeding colony at the California National Primate Research Center at the University of California, Davis. Care and housing of animals before, during, and after treatment complied with the provisions of the Institute of Laboratory Animal Resources and conformed to practices established by the American Association for Accreditation of Laboratory Animal Care. Animal protocols were reviewed and approved by the University of California, Davis, Institutional Animal Use and Care Committee. Monkeys were housed in stainless steel open-mesh cages. Before exposure, animals were given an appropriate period to become acclimated to the chamber environment and housed in small socialized groups within the chambers. Six monkeys were exposed to 5 days of 0.5 parts per million ozone for 8 h/day and then 9 days of filtered air for a total of 11 cycles beginning at 30 days of age until 6 mo of age. Six control monkeys were exposed to filtered air only. Ozone was generated as previously described (51). The monkeys were exposed to ozone in large stainless steel and glass units (3.5 m3 in volume). Each chamber had a flow rate of 2.1 m3 per minute for a complete air change every 2 min. Ozone was generated from vaporized liquid medical-grade oxygen by electric discharge ozonizers. The ozone concentration was monitored with an ultraviolet ozone analyzer (model 1003; Ahm Dasibi, Glendale, CA).

Gross dissection and tissue fixation. All animals were killed after the 11th cycle at 6 mo of age. The monkeys were weighed and then sedated with Telazol (8 mg/kg im) and anesthetized with Diprivan (0.1–0.2 mg/kg/min iv), with the dose adjusted as necessary by the attending veterinarian. The monkeys were euthanized with an overdose of pentobarbital sodium followed by exsanguination through the abdominal aorta. Three lung lobes (described below) were immediately inflation fixed via bronchial cannula for 4 h at 30-cm fluid pressure. The left cranial lobe was fixed with 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The right middle lobe was fixed with 1% glutaraldehyde-1% paraformaldehyde in cacodylate buffer (adjusted to pH 7.4, 330 mosM), and lung lobe volume was measured by fluid displacement after fixation. The right cranial lobe was fixed with 4% paraformaldehyde. After fixation, the left cranial lobe was separated into the cranial and caudal segments.

Evaluation of distal airways. The caudal segment of the left cranial lobe was microdissected by placing one blade of a pair of microdissecting spring scissors into the airway lumen and cutting along the axial path of the airway tree. This exposes the luminal surface of the airway and leaves the parenchyma attached to the airway wall as described in detail previously (35). Airway branches were numbered and recorded in a three-dimensional manner (Fig. 1, A and B). To determine whether postnatal ozone exposure alters alveolarization of the distal conducting airway, the number of airway generations in the main axial pathway from the lobar bronchus to the junction with the first alveolar outpocketing was counted in each of the four lobes.


Figure 1
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  		Fig. 1. Position of first airway outpocketing in filtered-air control and ozone-exposed infant monkeys. Airway microdissections of control (A) and ozone-exposed (B) monkey lungs show the main axial pathway through the left cranial lobe. Intrapulmonary airway branches are indicated by number. *Location of the most proximal alveolar outpocketing. Low-magnification Alcian blue/periodic acid-Schiff stained paraffin sections of conducting airway generation 10 in filtered-air (C) and ozone-exposed monkeys (D). Alveolar outpocketings are indicated by large arrowheads. Magnification bars = 6 mm (A and B) and 0.25 mm (C and D).

 
To determine whether postnatal ozone exposure alters the overall growth of the distal airways, the length and diameter were measured for the most precisely defined distal bronchioles: the last strictly conducting airway (the terminal bronchiole) and the first airway generation with alveolar outpocketings (respiratory bronchiole). The length was determined (using an eye-piece micrometer mounted in a Wild M-8 stereoscope) as the distance between the proximal bifurcation ridge and the distal bifurcation ridge, parallel to the long axis, using images of both halves of the airway to define the exact branch point. Internal airway size (diameter) was determined by direct measurement of the maximum distance of the lumen perpendicular to the long axis with the use of images of both halves of the airway.

To ensure that the histological evaluation of the airways compared exactly the same intrapulmonary airway generations in each monkey, the branching patterns of the axial path in the cranial segment of the left cranial lobe and the right cranial lobe were recorded as described above. Because the airway lumen was exposed by one cut and the rest of the lung remained intact and attached, the lobe could then be cut into 1-cm-thick slices perpendicular to the long axis of the main airway, washed in 0.2 M phosphate buffer for 30 min, and embedded in paraffin. Paraffin sections (5–6 µm thick) were deparaffinized, and mucous cells were identified by positive staining as stained with Alcian blue-periodic acid-Schiff.

Airway-specific epithelial composition was evaluated by high-resolution light microscopy (35). Again, the airway branching pattern of the axial path in the right middle lobe was recorded, and the lobe was sliced 1 mm thick for high-resolution light microscopy. The pieces were embedded in Araldite 502, following a process that allows selection of specific areas from large tissue faces (35). Areas containing airways of interest were isolated from the large blocks and remounted. Sections 0.5 µm thick were cut with glass knives and stained with a solution of 1% toluidine blue. The surface epithelia of microdissected airways of the left cranial lobe, caudal segment (costal half), were also evaluated by scanning electron microscopy as described previously (44). Airways were imaged with a Philips SEM 501 microscope (FEI, Hillsboro, OR).

Distal airway smooth muscle bundles were evaluated as described previously (40, 45). Smooth muscle bundle orientation, bundle abundance, and profile length and diameter were measured. The microdissected airways of the left cranial lobe, caudal segment (mediastinal half), were permeabilized with 0.3% Triton X-100, washed with PBS, incubated in 0.066 µM Alexa Fluor 568 phalloidin (a probe for polymerized actin; Molecular Probes, Eugene, OR) for 20 min, and then washed again with PBS. Distal airway generations were imaged with laser scanning confocal microscopy [Bio-Rad MRC 1024 ES (Hercules, CA) mounted on an Olympus BX50WI microscope (Melville, NY)] as described previously (40). A smooth muscle bundle was identified as a group of transversely oriented smooth muscle cells stained with phalloidin and separated from each other by large spaces. Briefly, a x10 long-working-distance water-immersion objective was used, and a series of images was taken through each terminal and respiratory bronchiole at focal planes that were 20–40 µm apart with a focus depth of 20 µm. With the use of LaserSharp 2000 software (Bio-Rad Cell Sciences Division, Hemp Hempstead, UK), 5–10 images were stacked together to produce three-dimensional composites of specific areas of the airway tree GoGoGo(Fig. 5). The final magnification was x170. Smooth muscle bundle orientation was measured as the angle of deviation ({theta}) from perpendicular to the long axis of the airway segment. The long axis of each airway was defined as the plane parallel to the walls of the airway. Zero degrees was perpendicular to the long axis of the airway, and 90° was parallel to the long axis. {theta} was measured for each smooth muscle bundle. Bundle angle mean and SD were also calculated. Bundle abundance and profile size were determined by defining the boundaries of each airway segment on composite images. Three linear probes were oriented parallel to the long axis of the airway and superimposed over the image, and the number of intercepts of the probes with smooth muscle bundles was counted. Number per unit length of airway was calculated by dividing the total number of intercepts by the total length of the probes. Relative smooth muscle abundance was calculated as the number of smooth muscle bundles per 100 µm of airway length. The average size of each bundle was estimated as the mean of the absolute values for the length of the probe covering each bundle.


Figure 2
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  		Fig. 2. Number of airway branches down the axial path (beginning at the trachea) of 4 different lung lobes to the first proximal alveolar outpocketings in filtered-air and ozone-exposed infant monkeys. *Significant difference (P < 0.05) from the filtered-air control lobe. RB, respiratory bronchiole.

 

Figure 3
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  		Fig. 3. Histological comparison of midlevel airway and alveolar outpocketings in filtered-air control and ozone-exposed monkeys. Airway generation 10 in both filtered-air controls (A) and ozone-exposed (B) monkeys consists of pseudostratified epithelium made up of basal cells (arrowheads), mucous cells (arrows), and ciliated cells. The airway wall is surrounded by smooth muscle (SM). The first alveolar outpocketing in both filtered-air control (C; generation 14) and ozone-exposed monkeys (D; generation 10) is lined by alveolar epithelium. Magnification bars = 15 µm.

 

Figure 4
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  		Fig. 4. Effect of postnatal ozone exposure on the composition of airway epithelial lining in respiratory bronchioles. Low-magnification scanning electron micrographs show the most proximal airway generation with alveolar outpocketings in filtered-air (A) and ozone-exposed (B) monkeys. Airway surfaces are covered by ciliated epithelium (cil), cuboidal and squamous epithelium (cub), and alveolar outpocketings (alv). C and D are higher magnifications of boxed areas indicated in A and B. Magnification bars = 200 µm (A and B) and 80 µm (C and D).

 

Figure 5
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  		Fig. 5. Effect of postnatal ozone exposure on smooth muscle bundle orientation ({theta}) in distal bronchioles. A: representative composite map of laser scanning confocal images of smooth muscle in distal bronchioles of the rhesus monkey. Smooth muscle is identified with Alexa Fluor 568 phalloidin, a probe for filamentous actin. B: diagram illustrating basis of measurement of smooth muscle bundle orientation in an airway. Arrow depicts the axis of airflow. Bundle angles ({theta}) were measured from a line perpendicular to the axis of the airway, at which {theta} = 0. Smooth muscle bundle {theta} was measured in the terminal bronchioles (C and D) and the first respiratory bronchioles (E and F) of filtered-air control (C and E) and ozone-exposed (D and F) monkeys. For each airway, the graph summarizes the percentage of bundles whose orientation ({theta}) fell within the following ranges: 0–14.9° (gray), 15–29.9° (white), and >30° (black).

 
Statistics. Data are reported as means (SD). Differences between groups were determined with Student's t-test (SigmaStat 3.0; SPSS, Chicago, IL) (18). P < 0.05 was considered statistically significant.


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Overall lung growth. At the end of the exposure protocol, there was no difference in overall body weight (1.55 [SD 0.171] and 1.71 kg [SD 0.14], respectively) or lung growth [8.20, [SD 0.89] and 9.73 ml [SD 0.78] (right middle lobe volume), respectively] between filtered-air controls and ozone-exposed infant monkeys.

Airway architecture. Respiratory bronchioles are differentiated from terminal bronchioles by the presence of alveolar outpocketings. In filtered-air control infant monkeys, the most proximal alveolar outpocketings occurred after an average of 13 or 14 generations of branching airways down the axial path beginning at the trachea. However, in ozone-exposed infant monkeys, the most proximal alveolar outpocketings occurred after an average of only 10 generations of airway branching starting at the trachea (Fig. 2). Infant monkeys exposed to ozone had significantly fewer generations of purely conducting airway generations (with no alveolar outpocketings) in three of the four lobes evaluated. Ozone-exposed monkeys had an average loss of three conducting airway generations per axial path for each lobe (Fig. 2).

The length and diameter of the last strictly conducting (terminal) bronchiole and the first respiratory bronchiole, at the boundary of the conducting airways and the gas-exchange area, were measured. This area was chosen because it is the most precisely defined area between the strictly conducting airways (terminal bronchiole) and the conducting airways and gas-exchange area (respiratory bronchiole). Terminal bronchioles of ozone-exposed monkeys were an average of 38% narrower and 45% shorter than terminal bronchioles of filtered-air control monkeys. In contrast, although the most proximal respiratory bronchioles of ozone-exposed monkeys were 41% narrower than the bronchioles of filtered-air control monkeys, the length of this airway did not differ significantly (Table 1). Therefore, after ozone exposure, both the terminal bronchiole and most proximal respiratory bronchiole were smaller than their filtered-air counterparts.


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  		Table 1. Effect of postnatal ozone exposure on the growth of distal airways in infant rhesus monkeys

 
Airway epithelial composition. In filtered-air control infant monkeys, nonrespiratory, strictly conducting airways were lined by a layer of continuous pseudostratified epithelium composed of mucous cells, ciliated cells, and basal cells and surrounded by a continuous layer of smooth muscle (Figs. 1C and 3A) as were the nonrespiratory, strictly conducting airways of ozone-exposed monkeys (Fig. 3B). The only difference at this level was that the strictly conducting airway continued to airway generation 13 in filtered-air monkeys and only to generation 10 in ozone-exposed monkeys. This generation in ozone-exposed monkeys was lined by a discontinuous layer of pseudostratified epithelium punctuated by alveolar outpocketings and surrounded by a discontinuous layer of smooth muscle (Fig. 1D). Within the alveolar outpocketings of both filtered-air control (Fig. 3C) and ozone-exposed monkeys (Fig. 3D), cuboidal cells with large nuclei and very little cytoplasm (Type II cell-like) and squamous cells with smaller nuclei and thin cytoplasm (Type I cell-like) were present (Fig. 3, C and D). Alveolar outpocketings in the first respiratory bronchioles of filtered-air monkeys were surrounded by areas of low, nonciliated, cuboidal epithelium (Fig. 4, A and C). In contrast, alveolar outpocketings in the first respiratory bronchioles of ozone-exposed monkeys were surrounded by ciliated epithelium (Fig. 4, B and D).

Airway smooth muscle. In the terminal bronchiolar airways (generations 12 and 13) of filtered-air control monkeys, the majority of the smooth muscle bundles (73.3%) around the airway were oriented at an angle <15° from perpendicular to the long axis of the airway and only a very small percentage (2.6%) of bundles were found at an angle >30° (Fig. 5C). In ozone-exposed monkeys, only 43.0% of the terminal bronchiolar (generations 8–11) smooth muscle bundles were oriented at an angle <15° from perpendicular to the long axis of the airway, but 12.2% of bundles were oriented at an angle of >30° (Fig. 5D). There was no significant difference in terminal bronchiolar smooth muscle bundle thickness (27.6 [SD 6.5], 33.2 µm [SD 13.9]) or abundance (1.51 [SD 0.3], 1.49 [SD 0.4] bundles per 100 µm) between filtered-air and ozone-exposed monkeys, respectively. An opposite pattern of smooth muscle bundle orientation was present in the most proximal respiratory bronchiole. Only half (51.6%) of the smooth muscle bundles in the proximal respiratory bronchioles (generations 12 and 13) of filtered-air control monkeys were oriented around the airway at an angle <15° from perpendicular to the long axis of the airway, and 16.4% of bundles were oriented at an angle of >30° (Fig. 5E). In the first respiratory bronchiole (generations 8–11) of ozone-exposed monkeys, 64.1% of the smooth muscle bundles were oriented at an angle <15° from perpendicular to the long axis of the airway, with only 5.5% of the bundles oriented at an angle of >30° (Fig. 5F). As in the terminal bronchioles, however, there were no significant differences in smooth muscle bundle thickness (27.5 [SD 4.6], 22.8 µm [SD 3.5]) or abundance (1.35 [SD 0.3], 1.77 [SD 0.1] bundles per 100 µm) in the first respiratory bronchioles between filtered-air control monkey and ozone-exposed monkeys, respectively.


   DISCUSSION
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Oxidant air pollutants, such as ozone, have been shown to cause persistent structural alterations in the lungs of adults (8, 16, 21, 28, 30, 37), but the effects of air pollutants on children have not been well studied. Biochemical endpoints suggest that neonates are less susceptible to ozone than adults; for example, neonatal rats have fewer alterations in pulmonary enzymes and markedly reduced cellular injury in the central acinus versus results shown in adults (4, 10, 41, 49). However, ozone exposure has been shown to reduce postnatal morphogenesis of the gas-exchange area in rats (42) and to retard the differentiation of the mucociliary apparatus of proximal airways in lambs (31).

The monkeys described in the present study are the same type of monkeys evaluated for pulmonary function by Schelegle et al. (38). In these monkeys, exposure to ozone during postnatal lung development resulted in a marked increase in baseline airway resistance (a twofold increase). Elevated airway resistance in respiratory diseases such as asthma is often associated with remodeling of the airways that conduct air to the gas-exchange area of the lung. To identify the structural alterations that may contribute to the increased airway resistance in ozone-exposed infant monkeys, we analyzed the transition to alveolarized airways in the main axial path distal conducting airways, the composition of the airway cells, and the structure of the smooth muscle surrounding those airways.

Exposure of infant monkeys to cyclic episodes of ozone produced three major alterations: loss of the number of strictly conducting airways, reduction of distal airway size, and alterations in smooth muscle bundle orientation. The number of strictly conducting airways (those without alveoli) along the main axial pathway of four lung lobes was three generations shorter in monkeys exposed to ozone than in monkeys exposed only to filtered air. Our present study corroborates previous experimental studies of growing lungs of juvenile nonhuman primates. Both daily and seasonal (every other month) ozone exposures over 18 mo in young monkeys (7 mo of age at start of exposure) resulted in significant increases in the volume proportion of the respiratory bronchioles vs. that shown in age-matched filtered-air controls (47). This increase in the volume proportion of respiratory bronchioles was also present in young monkeys (6 mo of age at start of exposure) exposed to daily ozone for only 12 mo and persisted after an additional 6-mo recovery period (48). These changes were not associated with changes in the volume proportion of parenchymal or alveolar compartments of the lung (47, 48), suggesting that the increase in respiratory bronchioles may come from alveolarization of conducting airways.

In ozone-exposed infant monkeys, axial path terminal bronchioles were 50% shorter than axial path terminal bronchioles of filtered-air control animals. In addition, the first respiratory bronchiole of the main axial path from ozone-exposed infants was 60% narrower than that shown in filtered-air control animals. A change in the geometry of the distal airways may have important implications in altering the distribution of air flow. Computational studies suggest that, as diameter decreases, the average air flow velocity falls (34), indicating that the velocity of air flow in the smaller diameter distal airways of ozone-exposed infant monkeys would be less than the airflow in the larger-diameter distal airways of filtered-air control infant monkeys. Airway resistance may also be altered by changes in the geometry of the distal airways. Airway resistance in the giant pouched rat (a large rodent) is disproportionately larger than the airway resistance in the harvest mouse (a small rodent) (19). The difference in airway resistance is amplified by the effects of the rat's disproportionately smaller airways. These data suggest that the decrease in diameter of the distal airways of ozone-exposed infant monkeys may play a large role in the increased airway resistance reported previously (38).

Because changes in airway smooth muscle have been implicated in excessive bronchoconstriction and affect resistance, we evaluated the structure of the smooth muscle surrounding the terminal bronchioles and first respiratory bronchioles in the infant monkeys. The bronchioles are important contributors to airway resistance during the first 5 years of life in humans (22). There is also physiological and pathological evidence that bronchioles play a role in asthma [see Kraft (27) for review]. Three-dimensional analysis of complete airway segments with dyes that identify actin suggested significant changes in one aspect of the smooth muscle bundles surrounding the terminal and the most proximal respiratory bronchioles: orientation of individual bundles. Theoretical studies suggest that smooth muscle orientation (helical pitch) is an important factor in airway contractility (5). Ozone exposure during postnatal development in infant monkeys increases the percentage of smooth muscle bundles in terminal bronchioles oriented at angles >30° and decreases the percentage of angles <15° from the perpendicular to the long axis of the airway. However, in the next most-distal generation, the proximal respiratory bronchiole, postnatal ozone exposure resulted in a decrease in the percentage of smooth muscle bundles oriented at angles >30° and an increase in the percentage <15° from the perpendicular to the long axis of the airway. These changes would enhance the ability of the smooth muscle components in distal airways to produce increases in airway resistance for either less nervous stimulation for contraction or less force of contraction for each response (5). In humans, smooth muscle appears to be one of the earliest components to be changed in remodeled airways in patients suffering from fatal asthma.

The molecular mechanism behind the abnormal development of distal conducting airways in animals exposed to ozone may be related to the depletion of perlecan in the basement membrane zone (11). Perlecan is a proteoglycan responsible for many functions, in particular, regulation of growth factor trafficking between cells of the epithelial-mesenchymal unit (13, 24). Ozone-induced depletion of perlecan from the basement membrane zone in trachea was associated with altered regulation of FGF-2 signaling (11, 12). Depletion of perlecan would also affect regulation of the other growth factors that bind to perlecan, including FGF-1, FGF-7, PDGF, hepatocyte growth factor, heparin binding EGF, VEGF, and TGF-beta (25). The functional consequences of deregulation of these collective molecules are significant because they are the basis for much of the cell-cell interactions in the epithelial mesenchymal trophic unit responsible for development of the airway.

The present study identifies the early postnatal period of lung development as highly susceptible to alterations by oxidant air pollutants. The remodeling in the distal conducting airways of young rhesus monkeys exposed since infancy to cyclic episodes of ozone reported in this study provides a pathophysiological basis for the decrement in small airway function reported in college freshmen who have grown up in polluted areas of California's South Coast Air Basin (29, 43).


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This work was supported by National Institutes of Health Grants ES-00628, ES-05707, ES-11617, and RR-00169.


   ACKNOWLEDGMENTS
 
The authors thank Viviana Wong, Christopher Wallis, Sarah Davis, and the animal care staff of the California National Primate Research Center for their technical assistance and Dr. Suzette M. Smiley-Jewell for editorial expertise.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. V. Fanucchi, School of Veterinary Medicine, Dept. Anatomy, Cell Biology and Physiology, One Shields Ave., Univ. of California, Davis, CA 95616 (e-mail: mvfanucchi{at}ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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