Introduction

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Mucociliary clearance is a crucial component of nonspecific defense mechanisms in human airways (1). Normal function of the mucociliary system depends on the efficacy of ciliary beating, the numbers and integrity of ciliated and goblet cells, and the properties of mucus (1). Ciliary alterations that contribute to functional deficits in mucociliary clearance have been described in a number of diseases, including primary ciliary dyskinesia (2, 3), and in subjects with chronic respiratory diseases (4).

The contribution of air pollutant exposure to airway epithelial injury is well documented. Long-term exposure of rodents to ozone causes marked changes in nasal surface epithelial components (5). Bonnet monkeys exposed to 0.15 or 0.30 ppm ozone for 6 or 90 d exhibited nasal ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia (6). Interactive effects between ozone and formaldehyde have been described on the nasal respiratory epithelium of Wistar rats, including disarrangement and loss of cilia (7). Light and electron microscopic alterations in human nasal epithelium have also been described in individuals exposed to ozone and nitrogen dioxide in controlled chamber studies (8, 9), as well as in field studies (10, 11).

Southwest Metropolitan Mexico City (SWMMC) children are repeatedly exposed to high levels of a complex mixture of air pollutants, including ozone, particulate matter, and aldehydes. Their nasal biopsies exhibit a wide range of histopathology, including marked changes in ciliated and goblet cell populations, basal cell hyperplasia, squamous metaplasia, and mild dysplasias (12). The objectives of the present study were to determine the nature, magnitude, and extent of ultrastructural alterations in the nasal epithelium of children in SWMMC, and to address the potential impact between an altered nasal mucociliary apparatus and the likelihood for lower respiratory tract damage in the context of chronic exposure to air pollutants.

	Materials and Methods

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Study Areas

Mexico City is located in a high mountain basin, 2,250 m above sea level. Sunshine, light winds, temperature inversions, 20 million inhabitants, heavy traffic, urban leakage of liquefied petroleum gas, and intense industrial activity contribute to make Mexico City an ideal environment in which complex photochemical reactions produce oxidant chemicals and particulate matter. Air quality data is provided by an automated surface network of 33 monitoring stations in and around Mexico City; hourly near-surface measurement of monitored pollutants include ozone (O₃), particulate matter (PM₁₀), SO₂, NO₂, NOx, and CO. Southwest Mexico City's atmosphere is characterized by high ozone concentrations of up to 0.48 ppm (13), with average maximal concentrations of 0.250 ppm and excesses > 0.08 ppm O₃, an average of 4 ± 1 h/d. The number of hours children enrolled in the study have been exposed to O₃ at concentrations above the standards are 40, 30, 740, 959, 1,224, 1,403, 1,561, 1,395, 1,146, 1,061, 1,249, 1,080, 1,123, 1,203, 1,342, and 1,284 h/yr for the years 1984-1999. Both PM₁₀ and PM_2.5 in SWMMC routinely exceed their respective annual arithmetic means above the current annual U.S. standards (PM₁₀, 50 µg/m³ and PM_2.5, 15 µg/m³) with average values of 78 and 21.6 µg/m³ for PM₁₀ and PM_2.5, respectively (14). Other pollutants detected in SWMMC include formaldehyde and acetaldehyde, with outdoor values in the range of 5.9 to 110 and 2 to 66.7 parts per billion per volume (ppbv), respectively (15). Mutagenic particulate matter (16), alkane hydrocarbons (13), volatile organic compounds, and peroxyacetyl nitrate (17) are also found in the SWMMC atmosphere. Veracruz, the control city, is located on the Gulf of Mexico, 14 m above sea level; it has a tropical climate with average temperatures in the range of 24 to 36°C and is in compliance for all air criteria pollutants.

Study Population

The study was approved by the Review Board for Human Studies at the Instituto Nacional de Pediatria in Mexico City, and informed written consent was obtained from the parents of all children enrolled in the study. Children in Mexico City were recruited from the Instituto Nacional de Pediatria personnel, and at the control city from a Sunday School by word of mouth. Participation was limited to children who fulfilled the following requirements: (1) nonsmoking households and negative personal smoking history and environmental tobacco exposure; (2) lifelong residency in SWMMC or the control city; (3) no known exposures to local sources of air pollutants; (4) no indoor pets; (5) negative family history of atopic diseases; (6) unremarkable clinical histories; (7) no respiratory illnesses or febrile episodes in the previous three months; and (8) physically active children, regular participants in a variety of outdoor physical activities. The study group consisted of 11 control children from Veracruz and 15 children from SWMMC. A detailed clinical history was obtained from the parents of each participant and each child was given a complete physical examination by a senior pediatrician (G.V.S.) and an ear, nose, and throat (ENT) exam by a senior ENT physician (A.R.A.). The control children included six boys and five girls (6 to 15 yr of age) with an average daily outdoor time of 7.6 ± 1.5 h, and the exposed children included nine boys and six girls (5 to 15 yr of age) with an average outdoor time of 5.4 ± 2 h/d. These children have two to three hours of physical education per week and a 30-min recess period every school day. After school hours, the maximal physical activity period takes place between 1:00 and 6:00 P.M., coinciding with the pollutant peaks. Children were of the same socioeconomic level and within their city attended the same school district and lived in the same neighborhood.

Nasal Biopsies

Samples of nasal epithelium were obtained with a disposable plastic curette (Rhino-Probe; ASI, Arlington, TX) under direct visual inspection. The nasal sample obtained by this procedure using two to three scrapes was in the form of oval clusters of epithelial cells. Samples were obtained from the inferior medial surface of the posterior portion of the inferior nasal turbinate. SWMMC samples disaggregated easily, while Veracruz samples remained intact. Occasional tearing of the ipsilateral eye and sneezing were recorded after the procedure. The tip of the plastic curette was immediately immersed into a vial of cold 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The specimens were fixed overnight at 4°C, rinsed in the same buffer, and postfixed in 1% phosphate-buffered osmium tetroxide. Samples were dehydrated through a graded ethanol series, infiltrated, and embedded in an epoxy resin. The structure of the nasal mucosa was first evaluated by light microscopy in 1-µm-thick epoxy resin-embedded sections, followed by identification of representative areas for each specimen. Histopathologic analysis of 1-µm-thick toluidine blue- stained sections included an evaluation of epithelial shedding, necrotic cells, basal cell and goblet cell hyperplasia, shortened or absent cilia, squamous metaplasia, mononuclear and polymorphonuclear (PMN) infiltration, intracytoplasmic PM, PM in intercellular spaces, and wide intercellular spaces. The histopathology severity of the selected parameters was assessed semiquantitatively from 0 to 3 (0, no pathologic change and 3, the most severe pathologic change). A minimum of two 1-µm-thick toluidine blue samples was available per child and the samples were evaluated blindly and independently by two experienced observers. The observers had no access to the data related to the geographic source of the biopsies at any point in the evaluation. Ultrathin sections were cut with an LKB-Huxley ultramicrotome and poststained with uranyl acetate and lead citrate. The grids containing sections were examined and photographed in a Zeiss EM-900 (Oberkochen, Germany) transmission electron microscope at an accelerating voltage of 50 kV.

Electron Micrograph Analysis

A series of electron micrographs was obtained from each sample in the two cohorts. The majority of micrographs were obtained at plate magnifications of ×1,100 to ×12,000. All sets of electron micrographs from both groups were evaluated blindly and independently by two experienced observers, and each set of micrographs was assessed based on the following criteria: (1) presence of neutrophilic infiltrates in the nasal epithelium; (2) alterations in the distribution of ciliated and goblet cells; (3) presence of squamous metaplastic cells; and (4) changes in ciliary ultrastructure. Next, a semiquantitative morphometric assessment of abnormal cilia was undertaken. The areas with cross-sections of cilia were chosen for each biopsy, and electron micrographs were exposed at the primary magnification of ×15,000. The final magnification of the cilia prints taken was ×45,000. Ciliary findings were divided into five different groups: (1) supernumerary central and peripheral tubules; (2) ciliary microtubular discontinuities; (3) ciliary disorientation; (4) compound cilia; and (5) number of microvilli and short cilia (< 5 µm). Ciliary orientation was estimated by measuring the angles between the plane defined by the central tubules and a reference line (18).

Statistical Analysis

Statistics were performed using the statistics program Instat (Graph Pad, San Diego, CA). Power calculations indicated that a sample size of 10 children per group (controls and exposed) was sufficient to demonstrate an effect with an  of 0.05 and a power of 0.8. Differences between control and exposed children in terms of outdoor exposure hours were tested with the unpaired t test, whereas the histopathology and electron micrograph parameter differences were tested with the Fisher exact test. A P value of < 0.05 was used to indicate statistical significance.

	Results

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There was a significant difference in the outdoor exposure time between children in the low polluted city versus those in SWMMC; even though children in the control city spend considerably more time outdoors (P = 0.005), their nasal biopsies were unremarkable (Figure 1). The major histopathology parameters evaluated on the 1-µm-thick toluidine blue sections are illustrated in Table 1. Every child in the SWMMC group displayed an abnormal nasal biopsy. Major findings included lack of cohesion between cells (Figure 2), epithelial shedding, necrotic cells, PMN epithelial infiltration, and short or absent cilia (Figure 3). A marked reduction in the numbers of ciliated cells and increased numbers of microvilli studded cells were seen in all samples (Figure 3).

View larger version (144K): [in this window] [in a new window]   Figure 1.   Control biopsy. Unremarkable respiratory nasal epithelium is seen; a pseudostratified columnar epithelium is comprised of basal, ciliated, and mucous cells. The integrity of the tissue is maintained.

View larger version (196K): [in this window] [in a new window]   Figure 2.   SWMMC biopsies are characterized by the extreme friability of the tissue. The epithelial cells are separated by wide intercellular spaces (asterisks), and cell debris is present on the luminal surface (L).

View larger version (216K): [in this window] [in a new window]   Figure 3.   Epithelial cells with numerous microvilli and large lysosomal bodies are separated by wide intercellular spaces (asterisks) occupied by low electron-dense granular material. A necrotic epithelial cell is seen on the luminal surface (arrow).

The cytoplasm of ciliated cells was less electron dense than nonciliated cells; there was little granular endoplasmic reticulum, variable amounts of glycogen, and a Golgi apparatus that varied from a few cisternae to multiple complexes. PMN epithelial infiltration was seen in every SWMMC biopsy; the neutrophils were scattered and no microabscess formation was observed (Figure 4). Wide intercellular spaces, occupied by low electron-dense granular material, reflected potential deficiencies in epithelial junction integrity (Figure 4). Cells undergoing differentiation toward a ciliated cell phenotype, as evidenced by the presence of migratory kinetosomes, were numerous in every biopsy (Figure 5), as were ciliated cells with abnormal cilia, including cilia with absent central microtubules, supernumerary central and peripheral tubules, ciliary microtubular discontinuities, and compound cilia (Table 2 and Figure 6). Dyskinesia associated with these abnormal cilia was suggested by the altered orientation of the central microtubules in closely adjacent cilia (Figure 7). Ciliary alterations were observed in every SWMMC biopsy and involved > 40% of cilia.

View larger version (194K): [in this window] [in a new window]   Figure 4.   A multinucleated PMN (arrow) is seen infiltrating the nasal epithelium. Notice the wide intercellular spaces (asterisks) occupied by electron-dense granular material reflecting potential deficiencies in epithelial junction integrity.

View larger version (191K): [in this window] [in a new window]   Figure 5.   Cells undergoing differentiation toward a ciliated cell phenotype, as evidenced by the presence of migratory kinetosomes (arrows), are numerous.

View larger version (177K): [in this window] [in a new window]   Figure 6.   Abnormal cilia with absent central microtubules and supernumerary peripheral tubules. A compound cilium is present (arrow).

View larger version (196K): [in this window] [in a new window]   Figure 7.   Abnormal cilia with supernumerary peripheral tubules and ciliary microtubular discontinuities. There is a marked altered orientation of the central microtubules in closely adjacent cilia. Acquired ciliary dyskinesia is suggested based on these abnormally oriented cilia. Black bars indicate the direction of the ciliary beat.

The presence of cells consistent in morphology with lymphocytes was not associated with an increase in the intercellular spaces (Figure 8). The majority of goblet cells were covered by microvilli and exhibited multiple small secretory granules in their apical aspect. Mucous cells containing coarse electron-dense, flocculent material and large secretory granules having an electron-dense core and a lucent halo (biphasic granules), as well as small nucleated granules, were commonly seen (Figure 9). Abnormal mitochondria exhibiting a condensed matrix and compressed cristae were conspicuous in squamous metaplastic cells filled with keratin bundles (Figure 10).

View larger version (188K): [in this window] [in a new window]   Figure 8.   A cell consistent in its morphology with a lymphocyte (arrow) is seen infiltrating the epithelium.

View larger version (181K): [in this window] [in a new window]   Figure 9.   Goblet cells exhibiting small and large secretory granules. Large secretory granules contain an electron-dense center (arrows); fusion of numerous coarse, electron-lucent granules is evident. Goblet cells are covered by numerous microvilli.

View larger version (212K): [in this window] [in a new window]   Figure 10.   Abnormal mitochondria (arrows) exhibiting a condensed matrix and compressed cristae are conspicuous in this squamous metaplastic cell filled with keratin bundles (asterisks).

An unexpected finding was the presence of PM in five of 15 SWMMC children. The fine granular, densely osmiophilic PM could be identified in the 1-µm-thick toluidine blue sections. PM was localized in the widely abnormal intercellular spaces, in the cytoplasm of adjacent epithelial cells, and surrounding the abnormal cilia. Deposition of dark osmiophilic granular material could be seen at the cellular borders and inside large lysosomal bodies in neighboring cells (Figure 11). The contents of the secondary lysosomes were similar to the PM seen in the intercellular spaces (Figure 11).

View larger version (219K): [in this window] [in a new window]   Figure 11.   Deposition of dark osmiophilic granular material could be seen at the epithelial borders (white arrows) and inside large lysosomal bodies in neighboring epithelial cells (black arrows).

	Discussion

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The electron microscopic findings observed in the nasal epithelium of children growing up in a polluted environment are likely a reflection of sustained airway epithelial injury. These children have lifetime exposures to significant concentrations of pollutants such as O₃, PM, formaldehyde, and acetaldehyde. Because the children had no exposure to tobacco and had negative histories of upper and/or lower respiratory diseases (either acute or chronic), the findings described here may be attributed in large part to the chronic and sequential exposure to air pollutants in a pattern that does not allow recovery periods.

Most striking in SWMMC children are the severe ultrastructural alterations in the morphology of ciliated and mucous cells, with patchy replacement of the diseased mucociliary epithelium by squamous metaplastic cells. Cells undergoing differentiation toward a ciliated cell phenotype together with the different stages of mucous cell secretion are indirect evidence of attempts to restore and protect this epithelium against the effects of continuous exposure to inhaled pollutants. The presence of an intraepithelial inflammatory infiltrate supports the view that the nasal epithelium is likely responding to the injury with the production of inflammatory cytokines such as interleukin (IL)-6 and IL-8, which are enhancing the migration of both PMN and mononuclear cells (19, 20).

Multiple factors can influence the distribution and severity of lung parenchymal lesions induced by air pollutants. One such crucial factor is the integrity of the mucociliary epithelium. Under normal, healthy conditions, a nose with an intact mucociliary apparatus acts as an extremely effective filter of inhaled gases and particles (21). For example, 30 to 40% of inhaled O₃ is scrubbed at this level (22). Based upon formaldehyde permeability data for human skin (23) and the similarity of skin to the squamous epithelium in the nose, the overall mass transfer coefficient of formaldehyde is an order of magnitude less for nonmucus-coated squamous epithelium compared with mucus- coated respiratory epithelium (24). Thus, potentially it could be less uptake of highly reactive gases such as formaldehyde or O₃ in an epithelium devoid of a liquid lining layer. Hence, there are two main concerns in having an altered nasal mucociliary apparatus. The first lies in the possibility for less ozone and formaldehyde to be scrubbed in children's noses, leaving the more distal pulmonary airways vulnerable to increased concentrations of these pollutants. The second concern is the association between poor mucociliary transport rate and prolonged PM retention in the nose, as well as the increased likelihood of recurrent chronic upper and lower respiratory infections, and bronchiectasis (1, 21, 25, 26).

The human nasal mucosa is susceptible to a wide range of air pollutants, including O₃ as evidenced by acute inflammation, exudation markers, and antioxidant consumption elicited upon exposure to ambient concentrations (27, 28). Low concentrations of O₃ and NO₂ result in an increased production of cytokines (IL-1, IL-6, and IL-8) and tumor necrosis factor  in a nasal mucosa organ culture system (29). Formaldehyde significantly reduces the beating frequency of cilia (30) and at relatively low doses (0.5 mg/m³) causes rhinitis and prolonged PMN changes in nasal lavages (31). Occupational formaldehyde exposures have been associated with loss of nasal cilia, goblet cell hyperplasia, squamous metaplasia, and dysplasias (32). Air pollutants are capable of producing free oxygen radicals by several mechanisms and can cause significant alterations in nasal ciliary mobility (33). Thus, it is possible that oxygen-mediated damage to DNA may contribute to the damage observed in the ciliated respiratory nasal epithelium of SWMMC children.

PM clearance from the nose depends largely on intact mucociliary mechanisms, and whereas deposition of particles larger than 3 µm takes place in the anterior part of the nose (devoid of ciliated epithelium), particles between 0.5 and 3 µm are filtered by an intact respiratory nasal mucosa and transported by ciliary propulsion to the nasopharynx (26). Diesel exhaust particles undergo endocytosis in primary cultures of human nasal epithelial cells, resulting in time- and dose-dependent membrane damage. This is followed by an inflammatory response whose extent seems to depend on the content of organic compounds adsorbed to the particles (34). The observation of intercellular deposition of PM and its presence in heterolysosomes in adjacent cells suggest that clearance of particulates may also be compromised in these children. PM collected in Mexico City shows a considerable mutagenic response (16), and we have recently identified an apparent increment in nasal and paranasal malignancies in Mexico City adult residents (35).

It is clear that the nasal epithelium in SWMMC children is fundamentally disordered and that their nasal mucociliary defense mechanisms no longer function optimally. As the first point of contact of the respiratory system with airborne chemicals in the environment, the nose is a readily accessible window that can be easily monitored and that, once altered, may potentially leave the distal acinar airways more vulnerable to reactive gases and PM. Impairment of mucociliary clearance has the potential to increase the contact time between deposited mutagenic PM and an injured epithelium, this increasing the risk for nasal carcinogenesis. Chronic exposures to air pollutants pose significant public health concerns for healthy individuals as well as those at higher risk, such as asthmatics and patients with compromised cardiorespiratory function. A compromised nasal epithelium has a diminished ability to protect itself and the lower respiratory tract.