Effects ofan Ambient Level of Ozone on PrimateNasal Epithelial Mucosubstances

Quantitative Histochemistry

JACK R. HARKEMA, DVM, PhD,CHARLES G. PLOPPER, PhD,

DALLAS M. HYDE, PhD,JUDITH A. ST. GEORGE, PhD, and

DONALD L. DUNGWORTH, BVSc, PhD

Despite the absorption ofinhaled oxidant gases by thenasal cavity, little effort has been made to characterizethe effects ofthese oxidants on the nasal mucosa. Thisstudy defines the effects of ambient concentrations ofozone on the character and amount ofmucosubstancesin epithelium of nasal mucosa. Bonnet monkeys wereexposed to 0.00 or 0.15 ppm 03 (8 hr/day) for 6 or 90days, anesthetized, and exsanguinated. Nasal cavitieswere fixed with Karnovsky's fixative, decalcified, andprocessed for light microscopy, and sections werestained with alcian blue (pH 2.5)/periodic acid-Schiffor high iron diamine. Volume densities of secretorymaterial in nasal epithelium were determined with the

SECRETORY CELL hyperplasia and mucus hyper-secretion are important characteristics ofhuman res-piratory disorders, especially chronic bronchitis andcystic fibrosis.'3 These changes in secretory patternsalso occur in animals experimentally exposed tochemical irritants such as sulfur dioxide and tobaccosmoke.4" Histochemically, the degree of sulfation ofglycoconjugates in airway secretory cells is increasedboth in chronic bronchitis and in cystic fibrosis,3'7 andthere is a shift in composition from neutral to acidicmucosubstances in laboratory animals exposed tochemical irritants."Although there is a considerable body of informa-

tion concerning the effects of inhaled irritants on thesecretory apparatus of the trachea and more distalairways, less is known about their effects on the nasalcavity. Recently we demonstrated that exposures toambient levels ofozone, the major respiratory irritantin photochemical smog, could induce morphologicalterations to the nonolfactory nasal epithelium ofnonhuman primates.8 Quantitative changes were evi-dent only in the transitional and respiratory epithelia

From the California Primate Research Center and DepartmentsofAnatomy and Pathology, School of Veterinary Medicine,University of California, Davis, California

use of a Quantimet 900 image analyzer. After 6 days'exposure there were significant increases in both acidicand neutral glycoconjugates stored in transitional andrespiratory epithelium. After 90 days there was signifi-cantly less mucosubstance than at 6 days. Only in thetransitional epithelium did the total and sulfated mu-cosubstance remain greater than that ofcontrols. Naso-pharyngeal epithelium was minimally affected after 6days of03 and unchanged after 90 days. It is concludedthat exposures to ambient levels of 03 induce signifi-cant changes in the stored secretory product of nasalepithelium. (Am J Pathol 1987, 127:90-96)

in the anterior nonturbinate-containing aspect ofthenasal cavity. Transitional epithelium is a nonciliated,cuboidal/columnar, stratified epithelium which linesthe luminal surface of the anterior nasal cavity be-tween the nasal vestibule and the nasal turbinate re-gion (Figure 1). The respiratory epithelium, which is aciliated, pseudostratified epithelium with goblet cells,lines the remainder ofthe main chamber ofthe nasalcavity, except for a relatively small area in the dorso-posterior aspect of the airway covered by olfactoryepithelium (Figure 1). One of the most significantchanges observed in this study was secretory cell hy-perplasia in these two surface epithelia ofthe anteriornasal cavity, after both 6-day and 90-day ozone expo-sures. Ciliated cell necrosis and shortened cilia were

Supported by Grant POI-ES00628 and in part by GrantsDRROO169 and HL28978 from the National Institutes ofHealth.Accepted for publication November 5, 1986.Address reprint requests to Jack R. Harkema, DVM,

PhD, Inhalation Toxicology Research Institute, P.O. Box5890, Albuquerque, NM 87185.

90

EFFECTS OF OZONE ON NASAL MUCOSUBSTANCES

2

B L

S H-. 4. go TO OO

Figure 1-Diagrammatic representation of tissue sections taken from thenasal cavity and nasopharynx of the bonnet monkey. A-Diagram oflateral wall indicating levels from which transverse sections (shown below)were taken. Distribution of the four epithelial populations is shown. S, squa-mous epithelium; T, transitional epithelium; R, respiratory epithelium; 0, ol-factory epithelium; LW, lateral wall; NL, nasal lumen.

also observed at 6 or 90 days of exposure to 0.15 or

0.30 ppm ozone. An intraepithelial influx of neutro-phils was present only at 6 days ofexposure, and ultra-structural changes in goblet cells were evident only at90 days.The purposes ofthe present study were 1) to charac-

terize possible histochemical changes in mucosub-stances in the surface epithelium ofthe monkey nasalcavity after short and long-term exposure to ozone;2) to determine whether there were changes in theamount of surface epithelial mucosubstance in theanterior nasal cavity and nasopharynx after these ex-

posures, and 3) to compare our findings with thosefrom previous studies in which the effects of differentirritants on the respiratory tracts of laboratory ani-mals or man were examined.

Materials and MethodsAnimals and Exposures

Fourteen male and 6 female colony-born bonnetmonkeys (Macaca radiata) between 2 years, 1 monthand 6 years, 5 months of age and weighing between2.4 and 11.0 kg were used in this study. The animalswere shown to be free ofclinical respiratory disease onthe basis of physical examination, thoracic radio-

graphs, and complete blood counts. These animalshad been raised indoors and were placed in 4.2-cumchambers for 1 week prior to exposure. Monkeysweredivided into three experimental groups. Group 1 con-sisted of 8 animals that were exposed to chemicallyand biologically filtered air for 6 or 90 days, 8 hr/day.Group 2 contained 4 monkeys that were exposed to0.15 ppm ozone, 8 hr/day, for 6 days. Eight animalsconstituted the third experimental group, which wasexposed to 0.15 ppm ozone, 8 hr/day, for 90 days.Ozone was generated by silent arc discharge andmonitored every half hour by a DASIBI UV ozonemonitor, which was calibrated by a DASIBI absoluteozone photometer. Recorded concentrations ofozone were based on the ultraviolet photometricscale.

After exposure, monkeys were deeply anesthetizedwith ketamine and sodium pentabarbitol and euth-anized by exsanguination via the abdominal aorta.

Immediately after death, the head from each mon-key was removed from the carcass and the nasal cavi-ties were flushed retrograde through the nasopharyn-geal orifice with 100-200 ml of a glutaraldehyde andparaformaldehyde fixative in cacodylate buffer at 440mOsm, pH 7.4.9 After this intranasal flush, the eyes,integument, and musculature were removed from thehead and then immersed in a large volume ofthe samefixative.After fixation, the right halves of the nasal cavities

in seven ofthe animals were split down their longitu-dinal midlines from the anterior nares to the posteriorends ofthe nasopharynx, which exposed the mucosalsurfaces ofseptal and lateral walls. The location ofthenasal turbinates and their orientation with respect tothe teeth and palatine structures could then be deter-mined. The lateral wall ofthe nasal cavity was decal-cified with a 13% formic acid solution for 7-10 daysand rinsed in tap water for at least 4 hours. Afterlandmarks had been determined from the first 7monkeys, the right halves of the nasal cavities of theremaining monkeys were left intact prior to decalcifi-cation.

After decalcification, we took two tissue blocks ap-proximately 5 mm in thickness from each monkey bytransverse section of the right lateral walls, using spe-cific teeth as landmarks (Figure 1). The anterior sur-face of the first tissue specimen was made by cuttingperpendicular to the longitudinal axis ofthe hard pal-ate, between the first and second premolars. The ante-rior surface ofthe second tissue specimen was taken atthe level ofthe nasopharynx between the anterior endand the posterior end of the soft palate.The anterior surface of each specimen was photo-

graphed before the tissue was further dissected into

91Vol. 127 * No. I

92 HARKEMA ET AL

smaller pieces, which were embedded in glycol meth-acrylate. These smaller pieces were coded and photo-graphed for later identification and orientation.Three 2-,u serial sections for the anterior surface ofeach of these tissues were cut with a Sorval J-B4 mi-crotome and glass knives. Sections were stained withalcian blue (pH 2.5)/periodic acid-Schiffs sequence(AB/PAS) for demonstration of acid (blue) and neu-tral (red) mucosubstances.'0 The acidic mucins werecharacterized as being sulfated with the use of highiron diamine (HID) on an adjacent section.

Morphometry

Because of the variation in tissue selection, not allepithelial regions could be morphometrically evalu-ated in all animals. As a result, the total number ofanimals evaluated in Figures 3, 4, and 5 will not addup to 19. The methods used for morphometric esti-mation of epithelial mucosubstances are identical tothose previously published by this group.'2"13Volume densities of secretory material in the sur-

face epithelium were determined with the use of aCambridge Instruments Quantimet 900 Image Ana-lyzer at a final magnification of 360X. Defined re-gional areas of surface epithelium (ie, transitionaland/or respiratory) in each tissue block were outlinedwith a light pen. Differentiation of stain reactivitywithin these areas was based on stain intensity. Threelevels ofdetection were used: one for AB-positive, onefor AB-positive/PAS-positive, and one for HID-posi-tive material. Stored mucosubstance was quantitatedas the volume of stored mucosubstance per surfacearea of epithelial basal lamina. Eight to 12 fields perslide were evaluated, and the data were processed forstatistical analysis (analysis of variance and a two-tailed Student t-test'4) with a DEC LST 11/73 mini-computer.

ResultsQualitative Changes

Histochemically, there were apparent qualitativedifferences in the stored secretory product (stained byconventional carbohydrate histochemical methods)in the transitional and respiratory nasal epitheliumfrom ozone-exposed and nonexposed bonnet mon-keys (Figure 2). More AB and PAS stainable muco-substance was evident in the transitional and respira-tory epithelium of the anterior nasal cavity fromanimals exposed to 0.15 ppm ozone for 6 days (Figure2B and H), compared with that present in these sameepithelial locations from animals exposed to 0.00ppm ozone for 6 or 90 days or those exposed to 0.15

ppm ozone for 90 days. There also appeared to bemore mucosubstances that stained HID-positive inthe transitional epithelium of some animals exposedto ozone for 6 days (Figure 2E). Differences betweencontrol and longer term ozone-exposed animals inthese epithelial regions were less apparent except forrelatively more HID-stained material in transitionalepithelium from animals exposed to ozone for 90days (Figure 2F). No differences in histochemicalstaining were noticeable by light-microscopic obser-vation between the nasopharyngeal epithelium ofmonkeys in the different experimental groups (Figure2J, K, and L).

Quantitative Changes

Figures 3, 4, and 5 compare the quantitativechanges in the volume of mucosubstances persquared surface area of basal lamina in the three epi-thelial regions. Compared with unexposed controls,there was a significant increase in total stained muco-substance in the transitional epithelium after both6-day and 90-day exposure to 0.15 ppm ozone (Figure3). Increases in both acidic and neutral glycoconju-gates within this epithelial population accounted forthe increase in total stained mucosubstance. Whenmonkeys exposed for 6 or 90 days were compared, asignificant decrease in both acidic and neutral muco-substances in the exposed transitional epithelium wasobserved after 90 days. The amount ofstored sulfated(HID-positive) glycoconjugates within the transi-tional epithelium was also elevated after ozone expo-sure, but only after 90 days were these increases statis-tically significant.

Similar patterns of ozone-induced, histochemicalchange were evident in the nasal respiratory epithe-lium of the anterior lateral wall (Figure 4). Totalstored secretory product in this ciliated surface epithe-lium was elevated after 6 days' exposure. This changewas primarily due to a significant elevation in neutralglycoconjugate, compared with that of controls. Theamount of acidic mucosubstances within the respira-tory epithelium of monkeys exposed for 6 days wasalso relatively greater, compared with that ofthe con-trol group, but this was not statistically significant. Asin the transitional epithelium, there was a significantdrop in total and neutral mucosubstances between 6and 90 days of exposure. Although there was a slighttendency for an increase in sulfated mucosubstancesin the respiratory epithelium after ozone exposure,there were no significant differences between any ofthe experimental groups.No significant histochemical changes were evident

in the nasopharyngeal epithelium after ozone expo-

AJP * April 1987

EFFECI7S OF OZONE ON NASAL MUCOSUBSTANCES

Figure 2-Light micrographs of surface epithelium from the lateral walls of the nasal cavity and nasopharynx of bonnet monkeys exposed to 0.00 ppm or 0.15ppm ozone for 6 or 90 days. A-Transitional epithelium, 0.00 ppm. (AB/PAS) B-Transitional epithelium, 0.15 ppm for 6 days. (AB/PAS) C-Tran-sitional epithelium, 0.15 ppm for 90 days. (AB/PAS) D-Transitional epithelium, 0.00 ppm. (HID) E-Transitional epithelium, 0.15 ppm for 6 days.(HID) F-Transitional epithelium, 0.15 ppm for 90 days. (HID) G-Nasal respiratory epithelium, 0.00 ppm. (AB/PAS) H-Nasal respiratory epithe-lium, 0.15 ppm for 6 days. (AB/PAS) I-Nasal respiratory epithelium, 0.15 ppm for 90 days. (AB/PAS) J-Nasopharyngeal respiratory epithelium, 0.00ppm. (AB/PAS) K-Nasopharyngeal respiratory epithelium, 0.15 ppm for 6 days. (AB/PAS) L-Nasopharyngeal respiratory epithelium, 0.15 ppm for90 days. (AB/PAS)

sure, except for an increase in the amount of storedPAS-staining material in the surface epithelium ofanimals exposed for 6 days (Figure 5). After 90 days ofexposure this increase was no longer apparent, theamount ofPAS staining material being similar to thatin control monkeys.

Discussion

The results of this study indicate that exposure tolow concentrations of ozone induces significantquantitative changes in the stored secretory productin the surface epithelium of the anterior nasal cavity.

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94 HARKEMA ET AL

VOLUMESURFACE AREA

mm3 x 10-3mm2

180

160

14.0

120

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80

60

40

20

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10.0

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J 6/90d, OUVO ,-pm

0 6d, 0.15ppmC 90d, 0 /5 ppm

n=3 n=4 n=4TOTAL MUCOSUBSTANCE

n=3 n=4 n=4ACIDIC(AB)

n=3 n=3 n=3SULFATED

(HID)

*t

n=3 n=4 n=4NEUTRAL

(PAS)

EJ 6/90d, 000 ppm

M 6d, 0/15ppm0 90d, 0/15ppm

Figure 3- Ozone-inducedchanges in the volume of tran-sitional epithelial mucosub-stance per surface area of basallamina. *Significantly different(P < 0.05) from control group(0.00 ppm ozone). Significantlydifferent (P < 0.05) from 6-dayor 90-day 0.15 ppm ozone-ex-posed group.

Figure 4- Ozone-inducedchanges in respiratory epithelialmucosubstance per surfacearea of basal lamina in the ante-rior lateral wall of the nasal cav-ity. *Significantly different (P <0.05) from control group (0.00ppm ozone). Significantly differ-ent (P < 0.05) from 6-day or 90-day 0.15 ppm ozone-exposedgroup.

0 6/90d, 000ppm

0 6d, 0 t5ppm

C3 90d, 0 15 ppm

TOTAL MUCOSUBSTANCE

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n=5 n=4 n=5ACIDIC(AB)

After short-term exposure to 0.15 ppm ozone, therewere significant increases in both acidic and neutralglycoconjugates stored in transitional and respiratoryepithelium of the lateral wall in the anterior cavity ofthe nonhuman primate. After longer term exposure

(90 days), the amount of stored epithelial mucosub-stance was significantly less than at 6 days. Only in thetransitional epithelium did the total and sulfated mu-cosubstance per surface area of basal lamina remaingreater than that of controls after 90 days' exposure.

n=5 n=4 n=5SULFATED

(HID)

n=5 n=4 n=5NEUTRAL(PAS)

Figure 5- Ozone-inducedchanges in respiratory epithelialmucosubstance per surfacearea of basal lamina in the lateralwall of the nasopharynx. *Sig-nificantly different (P < 0.05)from control group (0.00 ppmozone). Significantly different(P < 0.05) from 6-day or 90-day0.15 ppm ozone-exposedgroup.

Compared with nasal epithelium, the nasopharyngealepithelium showed relatively minimal histochemicalchange after short-term ozone exposure (slight rise inPAS-positive material) and no measurable changeafter longer term exposure.We previously described in detail the histopatho-

logic and ultrastructural changes that occurred in thenasal epithelium of these bonnet monkeys.8 Briefly,these epithelial changes include secretory cell hyper-plasia, ciliated cell necrosis, deciliation, and inflam-

VOLUMESURFACE AREAmm3 x 10-mm2

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mm3 109-3mmV1

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Vol. 127 * No. I EFFECTS OF OZONE ON NASAL MUCOSUBSTANCES 95

matory cell infiltration in the anterior nasal cavityafter 6 days' exposure to 0.15 ppm ozone. Secretorycell metaplasia, ongoing loss of ciliated cells, decilia-tion, and ciliogenesis were observed in the nasal epi-thelium of the anterior nasal cavities of monkeys ex-posed for 90 days to 0.15 ppm ozone. In the animalsexposed for a longer term, there were also increasednumbers ofepithelial cells with ciliated intracytoplas-mic lumina and goblet cells with abnormally dilatedgranular endoplasmic reticulum (gER). All of theseozone-induced epithelial changes, after both shortand long-term exposure, were limited to the veryanterior segments ofthe septal and lateral walls ofthenasal cavity, which contain both nonciliated transi-tional and ciliated respiratory epithelium. No histo-logic or ultrastructural changes in surface epitheliumwere evident in the posterior segments of the nasalcavity or nasopharynx in the monkeys exposed toozone, compared with those of controls.This is the first study, to our knowledge, to demon-

strate and quantitate ozone-induced histochemicalchanges in glycoconjugates stored in airway epithe-lium. Like sulfur dioxide, ozone is recognized as arespiratory airway irritant. But unlike sulfur dioxide,which is regarded mainly as an upper respiratory tractirritant, ozone is most often considered a lower respi-ratory tract toxicant. Pulmonary centriacinar lesionsinduced by ozone in nonhuman primates have beenextensively characterized.15-19 Only recently havestudies been reported on upper respiratory tract le-sions ofozone in monkeys.8'20 The present study dem-onstrated that ozone can induce significant changes inthe secretory apparatus ofthe nasal epithelium whichare somewhat analogous to airway changes inducedby sulfur dioxide and other pollutants in the upperrespiratory tract of laboratory animals.The increase in nasal epithelial mucosubstance in

the monkeys exposed to ozone for 6 days is similar totracheal and bronchial changes reported in rats anddogs after exposure to sulfur dioxide or cigarettesmoke.44 In contrast to those studies which reportedincreases only in acidic mucosubstances, the in-creases in total mucosubstance after short-term ozoneexposure in our monkeys were due to increases inboth acidic and neutral mucosubstances. All or partof this increase in stored epithelial mucosubstance isdue to an induced increase in the number ofsecretorycells containing this material, which we have docu-mented in another report.8 Whether there is also anincrease in the amount of secretory product per cellhas yet to be established.Monkeys exposed for a longer term in this study

also had a significant shift in acidic glycoconjugatesfrom sialomucins to sulfomucins, which is a reported

change in the bronchial airways of rats exposed tosulfur dioxide.5 In addition, there was considerablyless stored mucosubstance in nasal epithelium after90 days ofozone exposure, compared with that after 6days. In another report we documented that after 90days of0. 15 ppm 03 the total epithelial cells per milli-meter ofbasal lamina had decreased to the point thatthere was no significant difference from that of thecontrol groups.8 This decrease, however, was due to aloss of epithelial cells other than secretory cells, andthe absolute numbers of secretory cells were not dif-ferent between 6 and 90 days ofexposure. Therefore,the decrease in stored mucosubstance between 6 and90 days cannot be explained by a decrease in secretorycells. It must be due to a decrease in the production orin the storage of the epithelial mucosubstance.Ozone-induced changes in the amount of stored

secretory product in the anterior nasal cavitymay alsoreflect alterations in the overlying mucous layer andmucociliary clearance. We have previously shownthat ciliated cells in these regions of the nasal cavityare sensitive and undergo deciliation and necrosisafter both short-term and long-term exposure to am-bient ozone.8 Therefore, it appears that these lowlevels of ozone alter both arms of the mucociliaryapparatus in the anterior nasal cavity. Ozone has beenshown to decrease mucociliary clearance in the tra-chea,21'22 but its effect on nasal mucociliary clearancehas not been reported. Ifthere is significant functionalimpairment to the nasal clearance, this may allowmore potentially harmful agents to reach the distalairways. Although there is no scientific evidence, ithas been suggested that chronic alterations in normalupper airway function may have a significant role inthe development of small airway disease in thelungs.23 Ozone-induced changes in mucosubstances,together with loss of cilia, may lead to insufficientmodification of inhaled air, which would then resultin greater burdens of airborne pollutants to the lowerairways and subsequent lung disease.

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2. Lamb D, Reid L: The tracheobronchial submucosalglands in cystic fibrosis. Br J Dis Chest 1972, 66:230-247

3. Lev R, Spicer SS: A histochemical comparison ofhuman epithelial mucins in normal and in hypersecre-tory states including pancreatic cystic fibrosis. Am JPathol 1965, 46:23-47

4. Jones R, Phil M, Reid L: Secretory hyperplasia andmodification of intracellular glycoprotein in rat air-ways induced by short periods of exposure to tobacco

96 HARKEMA ET AL AJP * April 1987

smoke, and the effect ofanti-inflammatory agent phen-ylmethyloxidiazole. Lab Invest 1978,39:41-49

5. Lamb D, Reid L: Mitotic rates, goblet cell increase andhistochemical changes in mucous in rat bronchial epi-thelium during exposure to sulfur dioxide. J PatholBacteriol 1968, 96:97-111

6. Spicer SS, Charkrin LW, Wardell JR Jr: Effect ofchronic sulfur dioxide inhalation on the carbohydratehistochemistry and histology of the canine respiratorytract. Am Rev Respir Dis 1974, 110:13-24

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8. Harkema JR, Plopper CG, Hyde DM, St. George JA,Wilson DW, Dungworth DL: Response ofthe macaquenasal epithelium to ambient levels ofozone: A morpho-logic and morphometric study of the transitional andrespiratory epithelium. Am J Pathol (Manuscript sub-mitted)

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17. Eustis SL, Schwartz LW, Kosch PC, Dungworth DL:Chronic bronchiolitis in nonhuman primates after pro-longed ozone exposure. Am J Pathol 1981, 105:121-137

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19. Mellick PW, Dungworth DL, Schwartz LW, Tyler WS:Short term morphologic effects of high ambient levelsofozone on lungs ofrhesus monkeys. Lab Invest 1977,360:82-90

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21. Frager NB, Phalen RF, Kenoyer JL: Adaptations toozone in reference to mucociliary clearance. Arch En-viron Health 1979, 34:51-57

22. Kenoyer JL, Phalen RF, Davis JR. Particle clearancefrom the respiratory tract as a test of toxicity: Effect ofozone on short and long term clearance. Exp Lung Res1981, 2:111-120

23. Proctor DF, Adams GK, Andersen I, Man SFP: Nasalmucociliary clearance in man. Ciba Found Symp 1978,54:219-234