Cell proliferation in nasal respiratory epithelium of people exposed to urban pollution
L. Calderón-Garcidueñas1,2,5,
A. Rodriguez-Alcaraz3,
R. Garcia2,
G. Barragan2,
A. Villarreal-Calderón2 and
M.C. Madden4
1 Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA,
2 Instituto Nacional de Pediatria, 14410 Mexico City, Mexico,
3 Soc Mex ORL y CCC, Mexico City, Mexico and
4 EPA Human Studies Division, Chapel Hill, NC 27599-7310, USA
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Abstract
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The nasal passages are a common portal of entry and are a prime site for toxicant-induced pathology. Sustained increases in regenerative cell proliferation can be a significant driving force in chemical carcinogenesis. The atmosphere in Mexico City contains a complex mixture of air pollutants and its residents are exposed chronically and sequentially to numerous toxicants and potential carcinogens. We were concerned that exposure to Mexico City's atmosphere might induce cytotoxicity and increase nasal respiratory epithelial cell proliferation. Nasal biopsies were obtained for DNA cell cycle analysis from 195 volunteers. The control population consisted of 16 adults and 27 children that were residents in a Caribbean island with low pollution. The exposed Mexico City population consisted of 109 adults and 43 children. Sixty-one of the adult subjects were newly arrived in Mexico City and were followed for 25 days from their arrival. Control children, control adult and exposed Mexico City children all had similar percentages of cells in the replicative DNA synthesis phase (S phase) of the cell cycle (%S). A significant increase in %S in nasal epithelial cells was seen in exposed adult residents in Mexico City biopsied at three different dates compared with control adults. Newly arrived adults exhibited a control level of cell turnover at day 2 after coming to the city. However, at days 7, 14 and 25 they exhibited significant increases in %S. These data demonstrate an increased and sustained nasal cell turnover rate in the adult population observable in as little as 1 week of residence in Mexico City. This increase in cell proliferation is in agreement with other reports of induced pathological changes in the nasal passages of Mexico City dwellers. These observations suggest an increased potential risk factor of developing nasal neoplasms for residents of large cities with heavy pollution.
Abbreviations: CV, coefficient of variation; LI, labeling index; NTE, nasal transitional epithelium; O3, ozone; PI, propidium iodide; PMN, polymorphonuclear leukocyte; RBC, red blood cell; SSB, single strand break; SWMMC, Southwest Metropolitan Mexico City; US NAAQS, US National Ambient Air Quality Standard.
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Introduction
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Induced cell proliferation is a major mechanistic consideration in chemical carcinogenesis for both genotoxic and nongenotoxic agents (15). DNA replication is required to convert a DNA adduct to a true mutation. Increased cell proliferation decreases the time that is available for DNA repair of adducts from exogenous and endogenous sources. DNA-reactive chemicals are far more effective mutagens and carcinogens when there is also increased cell turnover. DNA synthesis is involved in chromosomal aberrations, insertions, deletions and gene amplification, which in turn are important mechanisms in chemical carcinogenesis. Growth factors associated with tissue regeneration can provide a selective growth advantage to and thereby clonally expand initiated cell populations (15).
Formaldehyde-induced nasal cancer in rats illustrates the importance of cell turnover as a driving force in cancer. Formaldehyde is a weak mutagen. However, it is the patterns of formaldehyde induced necrosis and regenerative cell proliferation that are the determining factors in the non-linearity in concentration response to tumor induction and the site specificity of formaldehyde-induced squamous cell carcinomas in the nasal passages of rats (6,7). The concentration-dependent increases in cell proliferation correlate strongly with the tumor response curve, supporting the proposal that sustained increases in cell proliferation are crucial in formaldehyde carcinogenesis (8).
The nasal cavity is a common portal of entry to the human body for air pollutants and it is a well-known target site for air pollutant-induced toxicity and carcinogenicity (9). The human mucosal epithelium of the upper respiratory airway, including the nose, is normally in a steady state of cell renewal (10). Nasal cell turnover is dependent on the concentration, dose and duration of the exposure to the irritant and on its physicochemical properties (11). Ozone, the most important irritant oxidant gas in photochemical smog, produces upper respiratory lesions in rats, monkeys and humans (1218). In rats a single 6 h inhalation exposure to 0.8 p.p.m. ozone induces cellular injury followed by a 10-fold increase in DNA synthesis, regenerative cell proliferation and secretory cell metaplasia in nasal transitional epithelium (NTE) (19). Overt cell death and necrosis in ozone-exposed animals are not prerequisites for toxicant-induced increases in cell proliferation (19). Nasal cancers in experimental animals are the result of chronic exposure to a wide range of inhaled chemicals and although the evidence for nasal carcinogenicity of inhaled chemical mixtures in experimental animals is limited, there is ample evidence in humans that occupational exposure to certain chemical mixtures is associated with increased risk of nasal cancer (20).
The atmosphere in Southwest Metropolitan Mexico City (SWMMC) is a complex mixture of air pollutants, including ozone, particulate matter, formaldehyde, acetaldehyde, nitrogen oxides, sulfur dioxide, volatile organic compounds, nonmethane organic compounds, alkaline hydrocarbons and other pollutants which have not been totally characterized (2128). We have reported that SWMMC is a location where residents have significant nasal pathological changes (29). In the adult population nasal biopsies often exhibit a wide range of histopathological changes including marked decreases in the numbers of ciliated and goblet cells, basal cell hyperplasia, squamous metaplasia and dysplasias, submucosal vascular proliferation and mild to moderate chronic inflammatory infiltrates. Dysplastic lesions in antral squamous epithelium and in squamous metaplastic epithelium located predominantly on the posterior inferior turbinates display p53 nuclear accumulation (30). In addition, the nasal epithelium of both SWMMC-resident children and adults exhibits DNA damage as evidenced by the detection of increased single strand breaks (SSBs) (31,32). Levels of 8-hydroxydeoxyguanosine are increased 3-fold in nasal cells of SWMMC children compared with control children living in a low-polluted environment (33).
In the course of studying the nasal epithelial changes in Mexico City residents it became a matter of concern that increased and sustained nasal cell turnover could contribute to the potential of induced nasal cancer, in particular when these residents have increased DNA oxidative damage and are exposed to inhaled mutagens and to agents which inhibit DNA repair. Cancers of the nasal passages are relatively rare in humans, with estimations of <1 case/100 000 (34). Increased incidences of nasal cancer have been reported in nickel refining, leather and wood furniture manufacturing workers, so there is a potential risk of developing nasal neoplasms on exposure to inhaled carcinogens (34). To date no epidemiological reports addressing the incidence of nasal neoplasms for Mexico City residents or residents of highly polluted cities are available. The purpose of this research was to examine whether residents in SWMCC exhibited a pattern of increased nasal cell turnover.
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Materials and methods
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Study area
Mexico City has 20 million residents with associated production of air pollutants from automobiles, leakage of petroleum gas and industrial activity. The climate is mild and tropical with year-round sunshine, light winds and temperature inversions. Each of these factors contribute to create an environment in which complex photochemical reactions produce oxidant chemicals and other toxic compounds. Monitored pollutants in Mexico City include ozone (O3), PM10, SO2, NO2, NOx , CO and lead (Pb). The SWMMC atmosphere is characterized by high ozone concentrations of up to 0.48 p.p.m. (28). This is well above the US National Ambient Air Quality Standard (US NAAQS) for ozone: 0.08 p.p.m. as a 1 h maximum concentration, not to be exceeded more than three times in a year. An average of 3 ± 1 h/day above the US NAAQS for ozone is recorded in SWMMC year-round (21). NO2 concentrations do not usually exceed the annual arithmetic mean of 0.053 p.p.m., while SO2 levels exceed the 24 h primary standard of 0.14 p.p.m. in the winter months. PM10 usually averages <150 µg/m3 in 24 h, except when meteorological conditions are unfavorable. Other pollutants detected in SWMMC include formaldehyde and acetaldehyde, with outdoor values in the range of 5.9110 p.p.b.v. and 266.7 p.p.b.v., respectively (26), benzo[a]pyrene and benzo[k]fluoranthene (22), mutagenic particulate matter (27) and alkane hydrocarbons (28).
Pollutant measurements
Atmospheric pollutants and meteorological conditions were monitored at the Pedregal station located in SWMMC, downwind of the major diurnal emissions in Metropolitan Mexico City. Data for O3, PM10, NO2, NOx , SO2, temperature and relative humidity were reviewed for the project year. The maximal concentrations, numbers of hours equal or above the US NAAQS and the time of ocurrence of pollutant peaks were recorded. Data from Isla Mujeres, the control site, were obtained from the Capitania del Puerto.
Study population
This project was approved by the Instituto Nacional de Pediatria Review Boards for Human Studies and informed written consent was obtained from subjects or their parents. Subjects were recruited from a children's hospital personnel and their relatives, a security corporation and a children's Sunday school by posted advertisements and word of mouth. Two hundred and ten non-smoking subjects, or their parents in the case of children, were asked to complete a questionnaire form, with the assistance of a physician. The questionnaire included a personal medical history (including recent respiratory illness), history of exposure to potential toxics (solvents, paints, heavy metals, photocopying machines), occupational history, outdoor exposure, physical activities, length of residency in Mexico City or in the control city, place of residency and place of work within the city and traveling outside their residency area or outside the city. We selected 198 subjects based on a negative history of allergic and respiratory diseases, exposure to potential toxic substances at home or in the work environment, negative history of active or passive repeated tobacco exposure, no recent respiratory illnesses (last 3 months) and permanent residency in SWMMC or in the control city with no traveling outside the city for periods of >2 days every month. The original study group of 198 subjects underwent a complete medical history with a physical examination and three subjects, including one child were found to have upper viral respiratory infections, and were excluded from the study. A group of 195 Mexican volunteers was recruited and included a control population of 16 male adults and 27 children and an exposed SWMMC population of 43 children and 109 male adults. Sixty-one of the adult volunteers were newly arrived in Mexico City. They were sampled four times in a 25 day period from their arrival.
Control subjects were permanent residents on Isla Mujeres, a Caribbean Island. Both children and adults seldom left their small island and had never been to a large city. They had no known exposures to local sources of anthropomorphic air pollutants or toxic substances. The group of control children included 17 boys and 10 girls. Their ages ranged from 6 to 15 years with an average age of 9.9 ± 0.5 years and an average daily outdoor exposure of 6.52 ± 0.3 h. The control adult group had an average age of 26.6 ± 8 years with a daily outdoor exposure of 11.2 ± 0.5 h. Exposed SWMMC subjects were divided into three groups. Group 1: children (n = 43) were aged 611 with an average age 9.1 ± 0.3 years and an outdoor daily exposure of 7.7 ± 1.3 h; these children were permanent SWMMC residents; they attended school and lived in the same neighborhood and seldom travelled outside the SW area. Group 2: adults (n = 48) were life-long residents in SWMMC who lived and worked in the area; their average age was 30.4 ± 9.5 years with a daily outdoor exposure of either <6 or >10 h/day. Group 3: adults (n = 61) had an average age of 23.7 ± 4.2; they were newly arrived volunteers to Mexico City from low-polluted small coastal towns. They all arrived in SWMMC on the same day and were followed for a 25 day period. Their criteria for inclusion in the study were the same as for the rest of the volunteers. These subjects were immediately housed in well-ventilated buildings that used open windows for cooling and without local sources of toxic substances. The living-working housing premises were enclosed, so vehicular traffic was minimal. The adult control group and the exposed group 3 had similar well-balanced dietary intake, level of physical activity and exercise routine outdoors. These subjects stayed outdoors from 6:00 to 18:00 h, with only brief intermittent periods indoors (<1 h). Six days per week from 8:00 to 14:00 h they exercised moderately outdoors and had intermittent light exercise the remainder of the outdoor time.
Nasal biopsies
Samples of nasal respiratory epithelium were obtained between 7:00 and 9:00 h, from the ventral surface of the inferior nasal turbinate, under direct visual inspection, with a disposable plastic curette (Rhino-Probe; ASI, Arlington, TX). The nasal sample obtained by this procedure using 23 scrapes/site, was in the form of oval clusters of well-oriented epithelial cells, yielding up to 250 000 cells as determined by an hemocytometer count. The sample was immediately immersed in 1 ml of cold RPMI 1640 medium (Gibco, Grand Island, NY). Cell viability was assessed by the propidium iodide (PI) exclusion assay and the trypan blue exclusion method. DNA cell cycle analysis was carried out with a flow cytometer. SWMMC samples were easily disaggregated by gently shaking the glass tube. Samples from individuals in the control group and the first sample of group 3 required mincing with a scalpel blade and vortexing for 10 s. Fifty microliters of the single cell suspension was taken to perform a cell count with differential, trypan blue dye exclusion and cytological observations; 100 µl of the suspension was used for the viability test with the PI assay and 200 µl was used for the DNA cell cycle analysis in an EPICS Profile II Coulter flow cytometer (Coulter, Hialeah, FL). The PI method was used for DNA cell cycle analysis (35). Briefly, unfixed single cell suspensions of fresh nasal cells were stained with a solution containing 50 µg/ml PI, 50 µg/ml RNase and 0.1% Tween 20 (Sigma-Aldrich, St Louis, MO) and were analyzed after 30 min of staining. PI fluorescence was detected through a 675 nm LP filter when excited with a 488 nm laser light. Ten thousand events were collected for each sample and the PI signals were recorded as linear simplified data. Processed samples were analyzed using the Multicycle analysis software, a cell cycle analysis program that is based on a procedure of curve fitting that separates the G0/G1, S and G2/M phases of a cell cycle in one DNA histogram. The DNA synthesis phase (%S or the portion of the cell cycle in which DNA is replicated) was determined for each biopsy sample. The cell cycle analysis model used was a zero-order S phase with sliced nuclei debris correction, which results in increased reproducibility (36). The coefficient of variation (CV) was calculated by the Multicycle using the full width at half maximum method.
Statistical analysis
Statistics were performed using the program Instat (GraphPad, San Diego, CA). The following statistical procedures were used. (i) One way analysis of variance (ANOVA) and the Tukey-Kramer multiple comparisons test were used to establish the differences in values of synthesis phase (S phase) of the cell cycle (%S) between the control and exposed children's group and also the control adults versus group 2 adults and group 3 adults on the different sampling days. (ii) ANOVA with Dunnett multiple comparisons test for the cell viabilities between the adult control sample and the different sampling dates in the adult groups. (iii) The unpaired t-test for the cell viabilities betweeen children controls and the exposed group. Results are given as means ± SD. A P value of < 0.05 was used to indicate statistical significance.
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Results
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Air quality data
Permanent residents in SWMMC are chronically exposed to a complex mixture of pollutants including high O3 concentrations which are recorded all year long. The numbers of hours SWMMC residents who have been exposed to ozone above the NAAQS for the years 19871996 are: 740, 959, 1224, 1403, 1561, 1395, 1146, 1061, 1249 and 1080 h, respectively. SWMMC volunteers were studied at different times in 1995 and 1996: group 1 exposed children were sampled the first week in October 1995, a month with 101 h with O3 > 0.08 p.p.m., an average of 3.25 h/day with O3 > 0.08 p.p.m., a maximal O3 peak of 0.260 p.p.m. (October 17, 1995) and no other monitored pollutants above their respective NAAQS. Group 2 exposed adults were biopsied in October and November 1996, with 103 and 135 h with O3 > 0.08 p.p.m., an average of 3.32 and 4.5 h/day with O3 > 0.08 p.p.m. and maximal O3 peaks of 0.323 and 0.253 p.p.m., respectively. Volunteers had their nasal biopsies on October 15, 1996 (0.301 p.p.m. O3 peak, 7 h with O3 > 0.08 p.p.m. and a cumulative O3 value of 1.515 p.p.m. from 8001800 h), November 5, 1996 (O3 peak of 0.199 p.p.m., 7 h with O3 > 0.08 p.p.m. and a cumulative O3 value of 1.058 p.p.m. from 8:00 to 18:00 h) and November 13, 1996 (O3 peak of 0.059 p.p.m., 0 h with O3 > 0.08 p.p.m. and a cumulative O3 value of 0.501 p.p.m. from 8:00 to 18:00 h) (Figure 1
). Group 3 newly arrived adults were sampled in February and March 1996, with 162 and 120 h with O3 > 0.08 p.p.m., an average of 5.58 and 3.87 h/day with O3 > 0.08 p.p.m. and maximal O3 peaks of 0.288 and 0.225 p.p.m., respectively. The sampling dates for this group were: February 20, 1996 (O3 0.263 p.p.m., 8 h with O3 > 0.08 p.p.m. and a cumulative value of 1.588 p.p.m. from 6:00 to 18:00 h), February 26, 1996 (O3 peak 0.139 p.p.m., 7 h >0.08 p.p.m. and a cumulative O3 value of 1.06 p.p.m.), March 6, 1996 (O3 peak 0.147 p.p.m., 5 h with O3 > 0.08 p.p.m. and a cumulative O3 value of 0.923 p.p.m.), March 15, 1996 (O3 peak 0.140 p.p.m., 3 h O3 >0.08 p.p.m. and a cumulative value of 0.912 p.p.m.) (Figure 2
). The ozone values in parentheses correspond to the data recorded the day previous to the nasal biopsy sampling. PM10 concentrations were on average 5580 µg/m3 in each of the studied months; SO2 and NO2 values were below their respective NAAQS. Metereological conditions in SWMMC for the Fall of 1995 and 1996 as well as February and March of 1996 were similar; there were cool/neutral ranges defining the neutral comfort zone, average temperature 21.5°C, relative humidity 52%. The control population was sampled on October 18, 1996, with atmospheric and metereological conditions average for the season: 24°C, relative humidity 82%, O3 concentrations <0.026 p.p.m. and PM10 averaged 7 µg/m3.

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Fig. 1. Southwest Metropolitan Mexico City (SWMMC) ozone concentrations for October 15, November 5 and November 13, 1996 applicable to group 2 exposed adults. Note that for November 13 the ozone concentrations did not go above the NAAQS (0.08 p.p.m.), while for October 15 the maximal ozone peak reached 0.3 p.p.m..The data were obtained from the Pedregal monitoring station, the station closest to the volunteers' work and residency quarters.
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Fig. 2. SWMMC number of hours with ozone >0.08 p.p.m. (A) and maximal ozone peaks (B) for February and March 1996; data applicable to group 3 subjects' exposures. *Sampling dates.
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DNA cell cycle analysis
We analyzed all biopsy samples available and the data included samples with a CV G0/G1 peak <5. The average of %S phase cells was defined as the average S phase value of a DNA-diploid cell cycle. Control children, control adults and group 1 SWMMC-exposed children had similar %S values: 13.2 ± 2.9, 14.5 ± 4.2 and 15.2 ± 6.8, respectively (P = not significant) (Figure 3
). There was a significant difference in %S between control adults and exposed adults in group 2 with %S values of 29.6 ± 12, 24.4 ± 8.8 and 26 ± 10.3 for October 15, November 5 and November 13, 1996, respectively (Figure 3
). Among group 2 subjects there were no differences in %S between volunteers exposed <6 h or >10 h/day outdoors. For group 3, the subjects newly arrived in Mexico City sampled on days 2, 7, 14 and 25, there was a significant difference in %S in each of the later days compared with day 2: 7.4 ± 3, 24 ± 17, 23 ± 13 and 21.3 ± 10 for days 2, 7, 15 and 25, respectively (Figure 4
).

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Fig. 3. Percentage of nasal respiratory epithelial cells in the replicative DNA synthesis S phase of the cell cycle (%S phase), as determined by DNA cell cycle analysis in a flow cytometer. Data are presented for control and SWMMC exposed children (group 1) and control and SWMMC exposed adults (group 2). *Significant difference between control and exposed groups, P < 0.05.
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Fig. 4. Percentage of nasal respiratory epithelial cells in the replicative DNA synthesis phase of the cell cycle (%S phase) for SWMMC exposed group 3 newly arrived adults. *Significant difference between day 2 and days 7, 15 and 25 from arrival, P < 0.05.
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Cell viabilities and cytological observations
Control adults and children had similar nasal cell viabilities as evaluated by the PI exclusion assay: 72.5 ± 6.3% for children and 76.2 ± 8.2% for adults. SWMMC children residents in group 1 had 62.8 ± 4.3 (P < 0.05 compared with control children). Group 2: 43.0 ± 2.3, 39.4 ± 3.7 and 59.2 ± 3.4 cell viability, respectively, for the three sampling dates (P < 0.01 compared with control adults and P = not significant when comparing the three different dates) (Figure 5A
). Group 3 had the following viabilities for days 2, 7, 14 and 25, respectively, of residency in Mexico City: 42.0 ± 9.8, 58.0 ± 14.0, 57.5 ± 19.0 and 57.0 ± 14.0 (P < 0.01 compared with the adult control group) (Figure 5B
). Fresh cell observations made while performing the trypan blue exclusion viability test showed control children and adults with unremarkable ciliated cells, with abundant cilia and strong, regular beats along with normal goblet cells and very few scattered polymorphonuclear leukocytes (PMNs). Samples from group 1 exposed children had ciliated cells with very few short cilia or deciliated areas along with clusters of goblet cells or isolated goblet cells with vacuolated cytoplasm and also scattered PMNs. Samples from group 2 subjects had ciliated cells with very short cilia in irregular patches and deciliated cells and few goblet cells were seen. PMNs were present in small numbers (<5%) at all sampling dates. Group 3 samples varied considerably according to the sampling date. On day 2, there were red blood cells (RBCs) in moderate numbers, many of the ciliated cells showed blebs on their cytoplasm; clusters of dead and alive ciliated cells, with remarkably normal cilia, were seen in a checkerboard pattern and naked nuclei were common. Very few PMNs were identified. On day 7, RBCs were present in moderate numbers, PMNs were few (<5%) and ciliated cells exhibited short cilia; blebs were still numerous. Epithelial binucleated cells were identified as deciliated cells by their shape and the presence of the blepharoplast line opposite to the basal nuclei. On days 15 and 25, ciliated cells with deciliated patches or cells with no cilia at all exhibited abundant yellow pigment in their cytoplasm; a few naked nuclei and isolated squamous cells as well as clusters of squamous cells were present, RBCs were crenated and a few blebs were seen in both epithelial cells and PMNs. In summary, cell viabilities were lower in exposed subjects and the cytological abnormal changes were present in various degrees in all exposed subjects.

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Fig. 5. Nasal cell viabilities (expressed as % of total) as determined by the PI exclusion method. (A) Control children and adults and exposed SWMMC group 1 children and group 2 adults. (B) Exposed group 3 newly arrived adults. *Significant difference from control group, P < 0.05.
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Discussion
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This study demostrates that increased and sustained cell proliferation is present in the nasal respiratory epithelium of lifelong adult residents of Mexico City and in newly arrived adult individuals in a period as short as 1 week. The increase in nasal cell proliferation as evaluated by the percentage of cells in the replicative DNA synthesis phase of the cell cycle (S phase) was persistent through days with different levels of pollutants. Specifically, concentrations of ozonea major air pollutant for the SWMMC areavaried from maximal peaks of 0.323 p.p.m. to days where ozone concentrations were well below the US NAAQS. Although there are a few interspaced days with ozone concentrations below the standard by far, the average day in SWMMC has 34 h with ozone concentrations >0.08 p.p.m. all year round, including weekends. More importantly, major species of organic compounds involved in photochemical air pollution, such as aldehydes and alkanes, are concomitantly elevated with ozone, but are not monitored on a regular basis (26,28). The nasal epithelium is therefore exposed chronically and sequentially to different toxicants and potential carcinogens.
It is not clear why elementary school children in Mexico City did not show an increase in nasal cell turnover as was seen in the adult population. Children may change from predominantly nasal to oral breathing when exposed outdoors possibly due to exercise. This change reduces the probability of the nasal passages reacting to potential toxicants (37). Their mechanisms of mucociliary defense, e.g. goblet cell hyperplasia (32), might confer them with enough protection so there is no severe damage to the epithelium (38). Age-dependent differences in the metabolism of xenobiotics, plasma-derived molecules playing a role in the mucosal repair process, peptide growth factors, cell migration, DNA repair and epithelial regeneration may explain the SWMMC children's nasal cell turnover (2,3,5,3942).
Increased airway epithelial proliferation is a reliable indicator of many airborne chemical toxicants (2,7,43). For example, nasal cell proliferation has been demonstrated with inhalation exposures to ozone (1114,44), formaldehyde (6,4551), propylene oxide (52), hydrazine (53) and various chemical mixtures (54). Formaldehyde, a weakly genotoxic carcinogen in rats and a probable carcinogen in humans (55) induces an increase in nasal cell proliferation up to 18-fold in Rhesus monkey nasal epithelium and these rates remain significantly elevated after 6 weeks of exposure to 6 p.p.m. formaldehyde (49). Swenberg et al. (43) have shown that the degree of respiratory nasal cell proliferation depends more on the exposure concentration than on total dose of formaldehyde, a finding confirmed by Wilmer et al. (56) when rats were exposed to marginally cytotoxic concentrations during a 13 week period. Tumors in formaldehyde-exposed animals are found to be associated with cellular changes ranging from severe necrosis and ulceration to increased cell proliferation, suggesting that nasal toxicity plays a pivotal role in the etiology of the nasal tumors (7,57). Interestingly, differences in nasal cavity volumesurface area relationships during exposure to formaldehyde are determinants of the species different responses to this toxic chemical (48). Monticello et al. (6) in an F344 rat study correlating regional and non-linear formaldehyde-induced nasal cancer with proliferating populations of cells, concluded that target cell population size, cell proliferation and local dosimetry were major determinants of the cancer outcome of formaldehyde exposure. Of special interest are the results of Reuzel et al. (57); these authors administered mixtures of ozone at 0.4 p.p.m. and formaldehyde at 3 p.p.m. to Wistar rats and observed a very distinct synergistic effect on cell proliferation, resulting in a labeling index 45 times the sum of the indices for 0.4 p.p.m. O3 and 3 p.p.m. formaldehyde alone.
Cell proliferation in the nasal passages has also been found to be a sensitive and quantitative indicator of ozone exposure (58). Hotchkiss et al. (12) examined the kinetics of the early cellular responses of the rat NTE to O3, at a concentration of 0.5 p.p.m. for 8 h with sampling from 236 h post-exposure. These investigators reported a rapid loss of cells from the NTE compartment and a subsequent burst of epithelial cell DNA synthesis which resulted in the rapid replacement of NTE cells previously lost from the surface epithelium. A significant increase in epithelial DNA synthesis greatest at ~2024 h post-exposure and persisting above control values by 36 h was documented. Animal studies in monkeys and rats demonstrate that the morphological response to O3 involves epithelial cell injury along the entire respiratory tract, resulting in cell loss and replacement of the damaged epithelium (11,14). Formaldehyde and ozone are not the only irritants present in urban polluted atmospheres that are capable of eliciting such proliferative responses. Diesel engine emissions (59,60) and metals such as nickel (6163) and chromium (64) andmost relevant to the environment in Mexico Citycomplex aerosol mixtures (20) also increase proliferation indices in target populations.
It is crucial to emphasize that nasal epithelial cell proliferation studies in healthy individuals exposed to different urban and rural atmospheric environments are not available in the literature. Proliferative studies are reported for nasal polyps (65), and nasal inverted papillomas (66) and interestingly Kotelnikov et al. (67) described the labeling index (LI)as evaluated by bromodeoxyuridinein nasal non-neoplasic pseudostratified epithelium in patients with head and neck tumors. The LI in four samples was 11.2%. The authors concluded that disordered proliferation may be an early consequence of field cancerization, a consequence that occurs prior to the appearance of hyperplastic or dysplastic changes.
There is escalating evidence in the nasal respiratory epithelium of Mexico City residents to suggest that considerable nasal damage is inflicted on exposure to its polluted atmosphere. (i) DNA damage in the form of SSBs, is rapidly induced upon arrival of previously unexposed adults to the city (31). Nasal SSBs are present in significant numbers in Mexico City permanent residents, both children and adults, when compared with matched populations living in low-polluted environments (31,32). (ii) In SWMMC children, percentages of nasal DNA damaged (SSBs) cells positively correlate with their age and daily outdoor exposure time (32) and children's nasal epithelial cells show a 3-fold increase in 8-hydroxydeoxyguanosine when compared with matched control children from low-polluted places (33). (iii) The nasal histopathological changes range from adaptative responses to putative preneoplastic lesions. Nuclear accumulation of p53 protein is seen in dysplastic lesions, raising the possibility that p53 mutations are already present or, alternatively, there is binding of viral proteins to normal p53 protein, resulting in increased p53 half-life and interference with p53 function, resulting in a selective advantage for clonal expansion in squamous cells (30). (iv) There is severe dysfunction of the mucociliary nasal apparatusas evidenced clinically by the histories of nasal crusting and dry rhinitismaking the mucociliary defense mechanisms ineffectual (29,32). (v) Fry and Black (68) demonstrated that in healthy adults at least 45% of retained particulate material (210 µm) is deposited in the anterior region of the human nasal passages2 cm behind the tip of the nosethe area with the highest number of dysplastic lesions in the multibiopsy site study performed by this laboratory (30). Particulate matter might also play a role in the nasal pathology specially in view that PM10 collected in Mexico City has shown a considerable mutagenic response in the Salmonella/microsome test (27). (vi) Nasal inflammation is an important component of the nasal pathology in Mexico City residents, both in children and adults (69,70). A wide range of inflammatory responses, from acute infiltration of the mucosa with PMNs to a submucosal mixed chronic inflammatory infiltrate are recorded. Inflammatory cells and their reactive oxygen metabolites can cause mutagenic effects in lung parenchyma (71) and sustained chronic oxidative injury may lead to a non-lethal modification of normal cellular growth control mechanisms, a mechanism by which non-genotoxic carcinogens may function (72). Moreover, the cytolethal-proliferative response described for non-genotoxic carcinogens (5) could be relevant to the nasal mucosa in Mexico City residents, since in the exposed adult population the data suggest that nasal cell low viabilities go along with the increased cell proliferation.
It is in this scenario that increased nasal cell proliferation could be a major determinant of neoplasic outcome in exposed susceptible populations. Mexico City residents are being exposed sequentially to air pollutants on a daily basis, so an increase in nasal cell proliferation, no matter what the stimulus is, will ultimately increase their chance for developing nasal neoplasms. Carcinogenesis in humans is a complex and long process. Cell proliferation is a critical piece of information that ought to be evaluated in nasal epithelia from people exposed to different types of atmospheric pollutants in urban settings. The incidence of nasal neoplasms in Mexico City should be monitored and future studies should address the identification of risk groups among the exposed population and formulate an effective policy to help protect these individuals. Additionally, the reversibility of the myriad of observed nasal lesions (from anatomical to biochemical to molecular) should be examined.
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Acknowledgments
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. We are grateful to all the volunteers that took part in the study. Special thanks go to Dr Alessandra Carnevale, Director of the Instituto Nacional de Pediatria in Mexico City, for her constant support and encouragement. The authors are indebted to Drs Byron E.Butterworth, Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, RTP, NC, Susanne Becker, Human Health Effects Division, US EPA, Chapel Hill, NC, and James A.Swenberg, Environmental Sciences, University of North Carolina at Chapel Hill, NC, for helpful discussions and critical reading of the manuscript. We thank Dr Cindy Lawler of the University of North Carolina for her statistical advice and Jessica Villarreal-Calderón for the control population clinical assistance. This work was supported in part by NIEHS training grant no. T32 ES07126.
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Notes
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5 To whom correspondence should be addressed at: MD#58D, EPA, Research Triangle Park, NC 27711, USA Email: calderon.lilian{at}epamail.epa.gov 
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References
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Received June 1, 1998;
revised September 11, 1998;
accepted October 29, 1998.