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Environmental and Molecular Mutagenesis 30:11-20 (1997) Research Articles DNA Damage in Nasal Respiratory Epithelium From Children Exposed to Urban Pollution Uiian Caider6n-Garciduefias,I* Norma Osnaya,1 Antonio Rodriguez-Alcaraz,2 and Anna Viilarreai-Calder6n3 ~ Experimentai Pathology Section, Instituto Nacional de Pediatrla, Mexico City, Mexico 2Soc Mex ORL y CCC, Mexico City, Mexico SUniversidad Aut6noma Metropolitana, Xochimilco, Mexico City, Mexico The nasal cavity is the most common portal of entry to the human body and a well-known target site for a wide range of air pollutants and chemically induced toxicity a~d carcinogenicity. DNA single- sirand breaks (SSB) can be used as a biomarker of oxidant exposure and as an indicator of the carcino- genicity and mutagenicity of a substance. We ex- amined the utility of using the alkaline single cell gel eleclmphoresls assay (SCGE) for measuring DNA damage in children's nasal epithelium exposed to air pollutants. We studied 148 children, ages 6- 12, including 19 control children from a low pol- luted Paciffc port and 129 children from $ou~mest Metropolitan Mexico City, an urban polluted area with high ozone concentrations year-round. Three sets of two nasal biopsies were taken in a 3-month period. All exposed children had upper respiraton/ symptoms and DNA damage in their nasal cells. Key words: DNA strand breaks; ozone; urban nasal epithelium Eleven- and twelve.year-aids had the most DNA damage, and more than 30% of children aged 9- 12 exhibited patchy areas of squamous metaplasia over high-flow nasal regions. These areas had the greatest numbers of damaged DNA cells (P 0.001) and a large number of DNA tails > 80 (P < 0.001) when compared to the contralateral macroscopically normal site in the same child. The youngest children with significantly less outdoor ex. posure displayed patchy areas of goblet cell hyper- plasia and had the least DNA damage. These find- ings suggest that SCGE can be used to monitor DNA damage in children's nasal epithelium and, fisrther, the identification of DNA damage in nasal proliferative epithelium could be regarded as a sen- tinel lesion, most likely due to severe and" sustained cell injun/. Environ. Mol. Mutagen. 30:11-20, 1997 © 1997Wiley.Llss, Inc. pollution; comet assay; children's DNA damage; INTRODUCTION Exposure to 0-, concentrations that exceed the current US Environmental Protection Agency National Ambient Mr Quality Standard (120 ppb) is a daily occurrence for ,~-AIHons of p~ople throughout the world. Southwest Met- ropolitan Mexico City (SWMMC) is a highly polluted urban environment with daily ozone concentrations > 0.12 ppm all year long. School children am an espe- cially highly exposed group because they engage in play and coml~itive outdoor physical activities in the after- noon, when O3 levels am at their peak. Ozone., the product of volatile hydzocaflxm degradation to nitrogen oxides, is a potent nonradical oxidant and a ajor component of photochemical smog; concerns con- unue to increase about its potential health hazard for hu- rnans [Lippman, 1993; Bascom et al., 1996]. The potential health effe~-ts on children receiving a lifetime exposure to a complex mixture of pollutants, including high ozone @ ] 997 Wiley-Hss, Inc. concentrations are of concern, given the capability of ozone alone to cause respiratory epithelial damage in experimen- tal animals and humans [Tepper et al., 1989; Chang et al., 1991; Calderdn-Garciduefias et al., 1992, 1994; Pino et al., 1992; Harkema et al., 1993]. Although exposure to 03 primarily affects the terminal bronchioles and alveolL the resp/ratory eFitheiinm in the nose, the first part of the respiratory airway, which conditions between I0,000 and 20,000 liters of air, receives the highest Os concentration during nasal breathing [Henderson et al., 1993]. Significant nasal epithelial lesions have been described both in animals and in humans exposed to Os [Harkema et al., 1987, 1989; Calder6n-Crarciduefias et al., 1992]. *Correspondence to: Dr. LiHan Calderon-Garciduefias, Cerm del Vigio lante 96, Romero de Terreros, Coyoacan 04310, Mexico DF, Mexico; F_,-mail: lcaldera@mail~.rnain.cona~.mx. Received 28 August 1996; revised and a~cepted 26 December 1996.

12 Calder6n-Garc|duefias et al. Ozone produces damage by both radical and nonradical mediamd processes. Ozone-induced free radical formation involves the reaction of O~ with olefinic compounds or electron donors [Pryor, 1994]." Recent studies have sug- gested that damage to DNA could be produced aRer expo- sure to ambient levels of O3; ozone exposure in vivo of Hsher 344 rats induces DNA damage in alveolar macro- phages [Hanley et al., 1993]. A similar induction is seen in bronchioalveolar macrophages and tracheal epithelial cells of guinea pigs exposed to 1.0 ppm O3 for 2 hours and in healthy human volunteers exposed m 0.4 ppm O3 for 2 hours [Lee et al., 1995]. Hydrogen peroxide has been shown to induce DNA single-su'and breaks (SSB) in the human lung cell line A549 [Baker and He, 1991] and in the adeno SV40 wansformed human bronchial epio thelial cells (BEAS) in a dose-dependent manner [Mc- Donald and Ducore, 1993]. Further, O3 degrades unsatu- rated fatty acids, like arachidonic acid (AA), m hydrogen peroxide and carbonyl compounds [Madden et al., 1993]. Ozonized AA potentiates the formation of DNA SSB in cultured human lung cells [Kozumbo et al., 1996]. Ozone exposure also causes airway inflammation; activated neu- trophils exer~ deleterious effects, including generation of oxygen radicals that mediate cellular injury, that is, nu- cieic acid base damage., DNA smmd crosslinkage, and DNA SSB that may result in celluiar repair, proliferation, differentiation, or mansformation [Cochmne, 1991]. In a recent study, we reported the induction of DNA damage in nasal respiratory cells of newly arrived young males to Mexico City and the presence of significant num- bers of SSB in nasal cells of 12-year-olds and adults with a lifetime exposure to the city's polluted environment [Calderdn-Garcidueflas et al., 1996]. The exposed chil- dren studied showed two interesting findings. First, they all had statistically significant numbers of DNA-damaged nasal cells compared with children of the same age and socioeconomic status living in a low polluted environ- merit. Second, the Mexico City children displayed patches of macroscopicaUy abnormal mucosa over high-flow na- sal areas [Hahn et al., 1993] located on the inferior and middle mrbinates. In the present study we extended the documentation of DNA-damaged nasal cells (detection of DNA damage by the single cell gel electrophoresis assay [SCGE], pH > 13) to include elementary school children ages 6 through 12; to determine if the areas ofmacrosoop- ically abnormal mucosa were exclusive of older children or could be detected in younger ones, to define these abnormal mucosal areas histologically and to look for any differences in DNA damage between the macroscopically abnormal and normal nasal epithelium. MATERIAI.S AND METHODS Pollutont Methodolow/ A~nosphtric pollutants and memomlogical conditions wer, moni- tor~ at the P~dr,gal station, located in SWMMC downwind of the major diurnal emissions in Meu'opolimn Mexico City and 3 miles or less from the children's neighborhood. Ozone w~s monitored by using a Beckman 950 chemilumine~ance analyzer with a calibration routine in accordance with USEPA procedures. We also monitored NO~. $O~, mmpemmm, mladve humidity, wind speed, and rain evenm. The maximal concenmnions, number of hours equal to or above the N AAQS. and the time of occurrence of pollutant pesks, were recorded. Dam from Manzauilio, the Pacific port connol area. were obtained from the Sludy Population The projec~ was approved by the Institute Nacional de Pediatda Re- view Boards for Human Studies. and informed written consent was obtained from the children's parenm. The study group consismd of 148 chikL, en. including a comzol population of 19 children and an exposed Southwest Meu-opoiitsn Mexico City gmop of 129 cldldren. All partici- pants in the study had a personal negative history of active smoking and environmental tobacco smoke exposure. The information obtained from each child and pax~nt (usually the mother) included age, sex, place and length of residency, socioeconomic status, daily outdoor time, level of physical activity and time of day when it took place, dietary intake, honsehuld cooking methods, patents' ocm~padonal history, history of toxic exposures and toba~,o e~xposum, family history of respiratory disease, personal history of allergies, and rt;spiratory and otolaryn- gology symptoms, including epistaxis, rhinorre~ quality and quantity of mumm, nasal dryness, nasal obsmmtion, cough, thoracic pain. and recent respiratory illness (in tim previous 3 months). Children with a history of earonos~.throat surgical pro~edmes, in nend of treatment for atopiv or inf~tions rhinitis, bronchitis, asthma, allvrgi~ diseases, or known expostmm to harmful substan~ (solvents. paints, metals, photo- copying machines) wet, ~luded from the study. The contsol children included 9 girls and I0 boys. aged 11.21 + 0..5 years, with a daylight exposure of 6.26 + 0.98 hours. These children lived in a low-polluted Pacific port; they seidom lefft their small town, and they had never been to a larg~ city. The exposed group incluck;d 65 girls and 64 boys. ages 6-12. an averag~ of 21 children per age group. These children were lifetime residents in SWMMC. all attended the same school, had the same socioeconomic level and lived in the same neighborhood. 2063633612 : W~ ~btalned ~amples ~f n~al r~pi~ory epitheiinm with ~ dispos~bl~ pl~ti¢ cur~tm ~hino-P~b~o ASL ,4~llngmn, Text) under dir~:t visual in~.-'tion. Th~ first se~ ~f binpdes w~ obtained from the inferio~ surf~:~ of the po~t-'~ior portion ~f the inferior n~al turbinm~ ~2 biopsie~ per child, 14g children). The s~coad ~t (32 childnm) w~ I~t~" ~ ~bnm'm~l mucosal ~ p~t on the anm~ior h~ad of the inferio~ and middl~ turbin~ and ~h~:m'iz~ by irregular p~:hes whid~ho~y d~'~scd muco~. The third se~ w~ ~ 4 ~ ~ ~bnm-mal muco~l ~ ~ d~eribed ~bove and the ¢ontmlm- ~ idend~d m~ro~:opically ne~'mal ~tomi~l ~ 04 child,s). Occ~don~l m~-ing of th~ ipdlm~ral ~y~ and sn~..zin~ wer~ ~ th~ p~u~- One biopsy sampl~ per child w~ immediamly h~o m~r~:d in I ml of cold RPIVl116~0 medinm (GIbo~, Grand Island, and ~-ved for the viability pmpidium iodid~ ~PI) ~clu~ion ~ el~¢u'opbo~is a~.~y. The s~c~nd biop~ was fixed in nentra110% f~rmaldehyd~. gently sh~k~u~ the glass mb~; ¢onm~l ~nples required mincing with scalp~l blad~ and vo~g f~ I0 s~ond~. F~ the vi~bllity pl of th~ ~ su~:~mdon w~ t~ with th~ PI as.~y in an EPIC~ P~file II C~ult~ Fl~w C~om~er ~Conlt~, I-liale~h, FLL Obs~-vadon~

were made as to the overall adequacy of the single-call separation, distribution of cell types, and ciliary motility. Single Cell Gel Electmphoresis Assay We asse~ed DNA damage by the S~GE assay [Singh e¢ al.. 1988]. Briefly. the single na~l cell suspension volume was adjus~d to 50,000 callgS0 ~L Thes~ cells wer~ mixed with 50 ~i of low malting agsrose at 37"~ and then placed on precienned microscope slides (Fisher fully f~osted slides, Haber Scientific, Pittsburgh, PA), which were already covered with a thin layer of 0-5% normal.malting agarose. The slides were kept at 4"C for 5 minutes to allow solidification of the agarose and then immersed in a ft~hly made cold 4C lysin$ solution (1% .-:dium um:osinate, 2.5 M NaC1, I00 ~ NarEDTA, 10 raM, Tris pH 10, and 1% Triton X-100) for 1 hour to lyse the cells, The slides were then removed f~om the lysing solution and placed on a horizontal gel electrophomsis unit (Easy Cast, Model B2 Owl Scientific Inc. Wood- burn, MA). The unit was filled with fresh alectrophoredc alkaline buffer (I mM EDTA and 300 ram NaOH), and the slides were allowed to set for 20 minutes to pennk unwinding of DNA before alectmphoresis. Electmphonmis wm conducted for the next 20 minutes at 25 V and 300 mA by u~ing an alecm3phoresls-compact power supply (Buchler Insummeat~ Kansas CIW, MO). Aft~ alecu~phonmls, the slides were washed gently with 0.4 M Tri~ pH 7.5 and then stained with 25 ttl of 20 ttg/ml ethidium bromide in distilled water. Observations were made ~ing an Olympus AH-2 microscope equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. We analyzed a minimum of 50 randomly selected cells per sample, and the score was based on the observations of one slide reader, thus minimizing variability due to subjective scoring. To quantimm DNA migration, we measm~i with an ocular micmmemr the DNA pauem length of individual cells in two replicate slides from the same nasal sample. For each cell the length of the image (nucleus plus migrated DNA) was measured in micrometers at a 250-fold magnification. To elaborate the histograms of the distribu- tion of DNA mlgnuion we arbiwarily divided the scale into four groups: <10 ~m, 10-40 lun, 41-80 ttm, 81-120 ttm and >121 p.m. DNA from =vntrol cells (human fresh lymphocytes) appeared as a round pattern that did not migrate in the gel The SCGE assay utilizes the principle that cells with damaged DNA will migrate a greater distance in an alectro- phomfic fiald under the alkaline conditions used. Comet tails am caused by the increased migration of the smaller DNA fragments toward the L~ght Microscopy Formaldehyde.fixed samples were processed in paraffin, cut at 5 ttm ~-~d stained with hematoxylin-cosin and alcian blue periodic acid Schiff .. ,B/PAS pH 2.5). Cell suspensions used for the SCGE assay were stained with Papanicolau stain. Statistical Analysis Data capture was 100% for the 148 participant chikkren, Statistical evaluation of dam consis~i of nonparametri¢ KruskalLWallh to test for differences in the numbers of calls with DNA tails < 10 pm between control and exposed g~oups; differences in the numbers of cells with t~ils <10 tun among the exposed groups themselves and differences in :" n~ DNA taft lcogth among exposed ~oups. The Z2 test was used for differences in the frequency and pwcenmg¢ of undamaged DNA cells in mam'oscopicaily normal vs. abnormal biopsies and ANOVA analysis for multiple comparisons for the outdoor ¢xposom analysis of SWMMC children. All values are reposed as ±SD. P < 0.05 was considered significant. Children's Nasal Mucosa and DNA Damage 13 RESULTS Air Quality Data SWMMC residents are recurrently exposed to a com- plex mixture of air pollutants, ozone being the main pol- lutant. Since the fall of 1986, O3 concentrations on and above the current NAAQS (0.12 ppm as I hour maximum concentration, not to be exceeded more than once per year) have been recorded in SWMMC throughout all sea- sons, an average of 3 - 1 hour/day [Garc~a-Guti&'rez et al., 1991]. Exposed children were studied in a 3-month period (September-November 1995) characterized by an average of 82 hours/month with O3 > 0.12 ppm, the highest O3 value was recorded on September 22 at 1400 hours (0.286 ppm). NO~ concentrations were on average 0-~49, 0.908, and 1.283 pproJday, while SO~ values were on average 0.257, 0.318, and 0.380 ppra/day, for Septem- ber, October, and November, respectively. The conu'ol population on the Pacific pore was sampled on January 1995 with aunospheric and meteorological conditions av- erage for the season: 26°C; relative humidity 87%; wind speed 9-18 ~ and no detectable air pollutants. Study Population Clinical Data Nasal and respiratory symptoms were absent in the control children. The youngest SWIVIMC children had the least epistaxis (7/22) and chest discomfort (3/22) and denied nasal dryness; while 11-year-olds had the highest incidence of epistaxis (21/26), nasal obstruction (19/26), nasal dryness (20/26), cough (25/26), and chest discom- fort (24/26). The finding by direct ENT endoscopic visual inspection of a macroscopically abnormal nasal mucosa, predominantly over the anterior head of the inferior and middle turbinates (irregular, sharply demarcated, geo- graphical patches of thinner, depressed, opaque, whitish- gray mucosa), was uncommon in first graders (1/22, 4_~%). The highest frequency was observed in 9- to 12- year-olds (30-35%) These patches of abnormal mucosa decreased in size and number toward the middle and pos- terior head of the affected mrbinates. The outdoor expo- sure time was significantly higher in grades 2-6 than in first graders (P < 0.001)~ older children spend more dine outdoors and engage in physical activities. Single-Cell Gel Electrophomsis Assay A signi~cant difference (P < 0.0001) in the numbers of DNA tails < 10 ~ (interpreted as undamaged cells), was observed between control and exposed children. The numbers of nasal cells with DNA damage in control chil- dren was low (17 __ 6.07%) and all of them migrated in the range of 10-40 ~n. By contrast, SWMMC children showed 82.16 __. 6.4% of nasal cells with DNA damage. The largest numbers of damaged cells among the Mexico

14 C.alder6n-Garddueflas et al. 0 DNA Tall Length ~m) Hg. 1. Di~ribution of comet ~ in nasal cells of the 14 SWMMC childt~ that had an area of whitish. ~ nasal mucosa and a contralateral grossly unremarkable epithelium, both are~ were biopsied and subjected to the SCGE assay. Macroscopically abnormal mtw, osal ,ageas con~spond histologically to squamous metaplastic epithelium and show a marked decnutse in the numbe~ of undamaged DNA ceils (< I0 gin) as well as a significant diffeze~ce in the numbe~ of DNA tails 81-120 ltm when compared to the contralate~al macroscopically normal ~ite in the same child (data from 14 children. 28 biopsies). City children were observed in 11- and 12-year-olds, and, furthermore, these two groups had significantly more ceils with DNA tails in-the range of 81-120 pan than younger children (P < 0.001). A similar trend was observed for the three older children groups; they had a statistically significant number of ceils zrfigrating in the range of 40- 80 tun (P = 0.03, 0.02, and 0.003 for ages 10, 11, and 12, respectively) when compared with children age 6 through 9. We also compared the SCGE findings in the 32 biopsy samples taken from abnormal nasal areas vs. the results of the 129 exposed original samples. The dif- ferences between these two sets of samples were statisti- cally significant in the numbers of ceils with DNA tails < 10 I.tm, markedly decreased in the 32 biopsies (P = 0.001) and in the numbers of cells with DNA migration lengths in the range of 80-120 p.m, significantly elevated in the macroscopically abnormal nasal biopsies (P ffi 0.009). We then proceeded to analyze the 28 samples taken from the 14 children that had an area of whitish- gray, depressed mucosa and a conwalateral grossly unre- markable epithelium. Again, the macroscopically ~bnor- real mucosa had the least numbers of ceils with tails < 10 p.m (P < 0.001) and a large number of DNA tails >81 ttm (P < 0.001) (Fig. 1). Cell Viabilities and Histopaibology Viable nasal cells were 72.5 __. 6.3% for control chil- dren and 63.4 _ 7.2% for exposed ones. There were no differences in cell viability between school grades or in r~lafion to gender or anatomical location of the biopsies. An average of 2 mm of nasal epithelium was present in the 194 biopsies examined. Control children had normal mucociliary epithelium, which in the fresh s~at~ showed strong, regular beating movements, while exposed chil- ciren, for the most part, had weak, irregular ciliary beating movements with multifocal loss of cilia or attenuated cilia (Hg. 2A). Patchy areas of an apparent goblet cell hyper- plasi~. ~ (Fig. 2B) were present in the first set of biopsies in all exposed children. These areas had a marked ten- dency to decrease in the older children (18/22, 15/23, 10/23, 9/22, 2/26, and 3/19, respectively, for ages 6-12). Goblet cells in the exposed children exhibited increased alcianophilic mucous (Fig 2C). No attempt was made to quantify the numbers of goblet ceils in the biopsy sam- pies, since we did not have a basal lamina included in them. A mild neutrophilic intraepitheJial infiltrate was present in all of the exposed biopsies (Fig 2A). The mac- roscopicaily abnormal mucosal samples were character- ized by no identifiable mucociliary epithelium; instead, there was a metapiasti¢ squamous epithelium with mild to moderate variation in nuclear size and prominent nucleoli; disc~te areas with microvessel proliferation were also seen impinging upon the basal layer (Fig. 2D). DISCUSSION 20636336 14 The present study documents the results of the single cell gel electrophoresis assay in detecting DNA damage in nasal respiratory epithelium of two groups of elemenr~y school children, a group from a low-polluted Pacific coastal area and a highty exposed group from Southwest Mem3poiltan Mexico City. The single ceil gel electropho- resis assay under alkaline conditions (pH > 13) is capable of detecting single- and double-swand breaks, alkali-labile sites, and DNA repair sites LTice, 1995]. In our study, children living in a low-polluted environment had sig- rdflcantly less DNA nasal damage than SWMMC ct~l- dren. Further, exposed SWMMC older elementary schoot children had greater DNA migration when compared to younger ones. Since migration length correlates cLirectty to DNA fragment size, this parameter is expected to be proportional to the extent of DNA damage. Moreover, more than 30% of SWMMC children ages 9-12 exhibited patchy areas of sqnamous metapiasia over high-flow nasal regions, with statistically significant DNA damage com- pared to biopsies of SWMMC children with still identifi- able respiratory epithelium or with goblet cell hyper- piasia. The atmosphere of SWMMC is a complex mixture of air pollutants, many of which have not been identified. However, ozone is the key index pollutant for the area and the only monitored pollutant that exceeds the NAAQS every day, all year round. Ozone, therefore has been the

focus of our attention because it 'is highly reactive and interacts with a wide variety of organic molecules, includ- ing unsaturated fatty acids, proteins, and nucleic acids to produce toxic free radical intermediates, initiates cascades of free radical reactions that damage genetic integrity, and causes, at relatively high concentrations, extensive DNA damage as reflected by strand breaks, DNA in- terstrand crosslinks, and DNA protein crosslinks [Ha- melin, 1985; Victorin, 1992; Feig et al., 1994]. Nelson and Kastan [1994] have reported that, since DNA damage may predispose cells to genomie alterations associated with neoplastic transformation, and broken DNA ends :nay be potentially recombinogenic, resulting in chromo- some segment deletions, translocations and or duplication in cells that traverse the cell cycle, the detection of DNA damage in nasal proliferating epithelium can then be re- garded as a sentinel lesion, the result of severe and sus- tained cell injury. These authors have also emphasized that DNA strand breaks are a considerable threat to cell viability and cell genome integrity and that different types of cells appear to be endowed with different DNA repair capacities and vary in their propensity to generate DNA strand breaks after exposure to genotoxic agents. These observations are extremely relevant to our findings, since in the exposed children, squamous metaplastic epithelium located over high flow nasal areas displayed the most DNA damage. This metaplastie epithelium lacks goblet cells, and mucus with its known antioxidant effect is markedly decreased [Cross et al., 1984]; this increases the exposure of the underlying cells to ozone and other pollutants. By sharp contrast, the nasal respiratory epithe- .ium with identifiable ciliated and goblet cells and the patchy areas with goblet cell hyperplasia, had signifi- candy less DNA damage. Thus, mucus cells appear to reduce the direct or indirect toxic effect of 03 and other pollutants on nasal tissues. Two points of interest are worth discussing here: first, the finding of goblet cell hyperplasia in the SWMMC-exposed younger children and, second, the progressive diminution of this important protective mechanism as the children grow older in their ?olluted environment. Harkema and colleagues [1987] described a mucus cell hyperplasia in the nasal transi- tional epithelium (TE) of monkeys exposed to 0.15 ppm 03, 8 hours/day for 6 days, while in Fisher rats (F344/ N) exposed to 0.8 ppm 03, 6 hours/day for 7 days, they described a mucus cell metaplasia in the TE lining the lateral aspects of the aasoturbinates, the lateral wall and the maxilloturbinates [Harkema et al.. 1989]. Therefore, the nasal goblet cell hyperplasia and metaplasia seen in O3-exposed monkeys and rats, as well as the extensive ::.'onchiolar metaplasia observed in the terminal bronchi- ¢~les of rats exposed chronically to 0.5 and 1.0 ppm O3 [Stockstill et al., 1995], may represent an adaptive mecha- nism to the continued ozone insult. Ozone could affect epithelial secretory cells directly with a resulting in- Children's Nasal Mucosa and DNA Damage 15 creased proliferation or could cause proliferation of undif- ferentiated cells selectively into goblet cells [Harkema and Hotehkiss, 1995]. Regardless of the mechanisms, it seems that goblet cell hyperplasia most likely represents a protective, adaptive mechanism that apparently de- creases in humans with prolonged pollution exposure. The second point of interest is the fact that in experi- mental animals, nasal epithelial lesions develop in specific sites of the nasal passages for many inhaled toxicants [Morgan and.Monticello, 1990]. According to these re- searchers, th~distfibution of chemically induced lesions in the respiratory tract is influenced by one or more of the following factors: (1) regional uptake patterns, (2) cellular susceptibility to a given dose of a chemical, and (3) local clearance processes by the mucosa and the vas- culature. While the squamous epithelium in the rat nasal vestibule is fairy resistant to inhaled gaseous irritants [Jiang et al., 1986], in nonhuman primates, the transitional epithelium, located between the anterior squamous epithe- lium and the most posterior respiratory epithelium and the ethmoid turbinate, is the major target site for O3 toxicity [Harkema et al., 1987; Hatch et al., 1995]. Therefore, to find areas of squamous metaplasia with severe DNA damage over the anterior heads of the anterior and middle turbinates coincides with a lesion pattern that reflects the major inspiratory airflow streams [Kimbell and Morgan, 1991; Hahn et al., 1993; Kimbell et al., 1993]. This pattern might be expected for materials delivered in high local concentrations as a result of inspiratory airflow with a diminishing anterior-to-posterior severity gradient due to the scrubbing action of airway secretions [Kimbell and Morgan, 1991]. Kimbell and coworkers [1993] concluded that, while airflow patterns and aiiavay diffusion play a role in determining ozone lesion location, airway lining and wall factors are also important determinants. Regional uptake of inhaled gases is influenced by the nature of the nasal lining, with complex interactions be- tween inspired gas and lining layers [Morris et al., 1986; Bogdanffy et al., 1987]. Although the greatest concern during assessment of nasal toxicity is the cancerous re- sponse, the relationship between adaptive responses (i.e., squamous metaplasia) and human nasal carcinogenesis remains problematic. Monticello and Morgan [1995] em- phasize the difference in rodents between adaptive squa- mous metaplasia of the nasal transitional or respiratory epithelia and the squamous metaplasia with prominent keratinization, an ominous finding with respect to cancer. Chlorine gas is a good example of an upper respiratory toxicant, but not a carcinogen in rats and mice. It induces lesions confined to the nose in F344 rats and B6C3F1 mice, the lesions are more severe in the anterior nasal cavity and include mucosal inflammation, respiratory epi- thelial hyperplasia, squamous metaplasia, and goblet cell hypertrophy and hyperplasia [Wolf et al., 1995]. On the other hand, formaldehyde, a major air pollutant and a Ix.', o 0~

16 Calder6n-Garciduefias et al. F~g. 2, Nasal biopsies from exposed SWMMC children. A: Biopsy from a 7-year-old girl shows identifiable respiratory epi~elium with short cilia, mild variation in nuclear size, prominent nucleoli and a fex¢ scattered neutrophils. H&E x l14. B: Biopsy from a 6-year-old boy showing an apparent increase in goblet cells, short cilia, and deciliated areas, along with scattered PMNs. H&E x90. C: Same biopsy as Figure B. showing numerous goblet cells identified by the abundant alcianophi- lie material stained blue with the PAS-AB pH 2.5 stain ×90. D: Squa- indUS metaplastie epithelium with moderate variation in nuclear size has replaced the normal respiratory, epithelium over the anterior head of the middle turbinate in this 12-year-old boy. Discrete areas with microvesset proliferation are seen impinging upon the basal layer, H&E ×90.

Children's Nasal Mucosa and DNA Damage 17 Fig. 2. (Continued) potent carcinogen in F344 rats exposed long-term to high doses, causes DNA SSB and DNA protein crosslinks [Morgan et al., 1986: Reuzel et al.. 1990: Casanova et al., 1991; Cassee and Feron, 1994]. Rhinitis, squamous metaplasia, and hyperplasia of the nasal respiratory epi- thelium along with increases in cell proliferation precede

18 Calder6n-Garciduefias et al. the appearance of squamous cell carcinomas in the nasal passages. The location of the tumors correlates well with the distribution of histological evidence of acute cytotox- icity and with areas of inhibition of nasal mucociliary function. Further, areas where tumors are notably absent, that is, the medial aspect of the maxilloturbinate, are more resistant to DNA damage induced by dimethylnitrosamine than are those of the nasoturbinates [Bermudez, 1991]. The interpretation of squamous metaplasia with severe DNA damage in children exposed to a highly polluted atmosphere is an important challenge to pathologists, tox- icologists, epidemiologists, ENT physicians, and pediatri- cians, as is the assignment of squamous metaplasia and other potentially adaptive changes to a specific disease process. Recently developed tissue microdissection tech- niques that allow the procurement and genetic analysis of selected cell populations [Zhuang et aL, 1996]; the determination of oxidative DNA damage (8-hydroxy- deoxyguanosine) [Cheng et al., 1992; Yarborough et al., 1996], the detection of defects in loci on chromosome 11 [Ward et al., 1993], and the analysis of expression of retinoid receptors and different molecular weight keratins [L. Hu et al., 1991: Lancillotti et al., 1992; X. Hu et al., 1994] could be useful in the investigation of these adap- tive versus adverse nasal mucosal changes. The harmful health consequences of the polluted air to which the SWMMC residents are exposed, and the long delay that will occur before our air is cleaner [Blake and Rowland. 1995] makes it crucial to develop strategic approaches to determine the nasal pathology underlying mechanisms, the mapping of lesion distribution patterns, the identification of chronic air pollutant exposure bio- markers, and the definition of the potential relevance of noncancer responses in a range of disease states such as rhinitis, epistaxis, impairment of nasal flow, and mucocili- ary activity, dysosmia, and pathological states such as dysplasia and cancer. Based on the striking finding of an apparent goblet cell hyperplasia and less DNA damage in the nasal ceils of the youngest children examined and its correlation with a lesser outdoor exposure, the first step to protect children ought to be a drastic reduction in their outdoor exposure time and an avoidance of physical exertion at the maximal O3 concentrations periods. In summary., a lifetime exposure to a complex mixture of air pollutants with O3 as the main pollutant, produces significant nasal lesions in exposed children. The finding of particularly severe nasal cell DNA damage in areas of squamous metaplasia located over high-flow nasal re- gions, opens a series of questions about the relevance of these lesions. Are these squamous metaplastic cells more prone to mutations and malignant transformation? Do they represent innocuous adaptive responses? Or could we eventually apply the statement by Farber and Rubin [1991]: "'an adaptive response reveals its less beneficent aspect in neoplasia, when the stress becomes too severe or prolonged." ACKNOWLEDGMENTS We thank all the children who took part in this study, Gerardo Barragfin for statistical support, Humberto Gar- nica and Donald Joyner for the art work, Leticia Rarnfrez for ..h..istological support, Raquel Garc~a for flow cytometry support, Jessica Villarreal-Calder6n for the control popu- lation clinical assistance, Dr. Michael C. Madden of the U.S. Environmental Protection Agency for support and instruction in the use of the SCGE assay, Drs. 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