Cytological value of sputum in workers daily exposed to air pollution.

Abstract.

In this study, quantitative modifications of dust

cells, siderocytes, Curschmann’s spirals and asbestos bodies

and qualitative modifications (cellular changes and

inflammatory infiltrate) in the sputum of 164 traffic police

officers and 218 railway workers, occupationally exposed to

environmental pollution, and the sputum of 119 inhabitants of

a rural area, were evaluated. The results were correlated with

time of exposure and smoking habits. Seventy-three (45%)

traffic police officers (TPO), 76 (35%) railway workers (RW)

and 29 (24%) of the rural population (RP) were smokers. The

sputum, collected over a 3-day period, was smeared on glass

slides and stained according to the Papanicolaou, Perl and

yellow eosin methods. The results of the qualitative cytological

diagnosis revealed a statistically significant difference between

the TPO, RW and the RP (p<0.001). The results of the

qualitative and quantitative cytological examinations were not

significantly correlated to time of occupational exposure, which

was considered to be a continuous variable. The qualitative

cytological examination of sputa was not statistically significant

for the smoking habits of the TPO and the RP, but was

significant for the RW (p<0.0067). In the TPO, the number of

dust cells was higher in smokers, and the relative risk (RR) was

3.95. In the RW, the RR was 2.84. The results of our study

revealed that for the RW, the qualitative-quantitative

cytological alterations in sputum were due much more to

smoking habits than to occupational exposure, while the

presence of asbestos bodies correlated with work activity. The

qualitative-quantitative cytological examinations of the TPO

differed significantly from that of the other two populations.

This could be explained by exposure to urban air pollution, or

by a remarkable synergistic effect between smoking and

environmental urban pollution. It can be concluded that the

qualitative and quantitative cytological analysis of sputum

appears to be a reproducible and sensitive method for the

verification of pulmonary changes that seem to reflect different

environmental conditions and lifestyle habits.

The damaging effects of occupational or environmental

exposure to air pollutants may be difficult to evaluate since

workers may be exposed to more than one toxic substance

in their place of work. Moreover, exposure may depend not

only on professional habits, but also on personal ones.

The physiopathological effects of air pollution have been

extensively studied. Major air pollutants include noxious

gases, such as ozone (O

), sulphur dioxide (SO

) and

nitrogen dioxide (NO

), or particulates such as sulphates,

nitrates, diesel exhaust, anthracotic dust and asbestos fibres.

All these substances may be found alone or in combinations

(1-3). This list is not all-inclusive, but is representative of the

major air pollutants present in the industrial world and those

most frequently studied. The effects of these agents on the

airways include bronchoconstriction, increased non-specific

airway responsiveness and mucociliary dysfunction (1).

Many investigators have demonstrated that the effects of

smoking on the bronchial epithelium are progressive and

predictable over a period of decades (4-7). The biological

effects of cigarette smoke begin early in the patient's

smoking history and include an increase in the production

of bronchial mucous, paralysis of ciliary motion and the

stimulation of an immunological host response by the

presence of increased production of alveolar macrophages

and neutrophils (8, 9). Overproduction of mucous

subsequently leads to the formation of Curschmann's spirals

and other non-cellular bodies (10, 11). The increase in

alveolar macrophages and neutrophils has been shown to

release a high level of elastase, an enzyme that may

contribute to the pathogenesis of emphysema (12). Chronic

Microbiologia e Virologia, Presidio Addolorata, Azienda Ospedaliera S.Giovanni Addolorata, Via S. Stefano Rotondo n.5, 00184 Roma, Italy. Tel/Fax: ++39 06 77054256, e-mail: maria.cenci@libero.it

Key Words: Sputum, air pollution, lung cancer.

Cytological Value of Sputum in Workers Daily

Exposed to Air Pollution

MAURO ALDERISIO

, MARIA CENCI

, PIERPAOLO MUDU

,

ALDO VECCHIONE

and MARIA ROSARIA GIOVAGNOLI

1

_{National Institute of Health,}

_{S.Giovanni Addolorata Hospital and}

_{S. Andrea Hospital, "La Sapienza" University, Rome, Italy}

irritation induces epithelial cells to lose their adhesive

ability, with the likelihood of eventual metaplastic and

dysplastic cellular transformation. These changes may

progress, finally, to chronic obstructive pulmonary disease,

to malignancy, or both.

In this study, the effects of air pollution and smoking

habits on cellular changes of the pulmonary epithelium were

examined by cytological monitoring of inflammatory or

metaplastic changes of columnar cells, and by evaluation of

the presence and/or the number of dust cells, siderocytes,

neutrophils, mucous spirals and asbestos bodies.

Materials and Methods

Study population. The study was performed on 3 different populations (traffic police officers [TPO], railway works [RW] and a rural population [RP]) exposed to various degrees of air pollution, both at work and at home. A comparison of the 3 populations is provided in Table I.

One hundred and sixty-four TPO (32% of the total population studied) were exposed to high levels of urban pollution (gaseous and dust), and possibly to asbestos fibres. The TPO were 31 to 65 years old (mean age 46 years±6.921); 150 were men and 14 were women. The TPO were divided into 2 groups according to their place of work (central city or suburban). The time of exposure to traffic pollution (expressed in years) and smoking habits were also considered.

Two hundred and eighteen RW (43.5%) had a high risk of exposure to asbestos fibres and iron powders. The RW were 27 to 82 years old (mean age: 44±9.045 years); 215 were men and 3 were women. All were residents of Rome, Italy. The RW worked in machine shops, dismantling asbestos panels used for insulation. The workers were divided into 3 groups according to their risk of exposure to asbestos: low, medium and high. The time of exposure (expressed in years) and smoking habits were recorded.

There were 119 people (23.85) in the RP studied, who were from 32 to 74 years old (mean age: 50±9.897). Sixty-six were men and 53 were women. They lived in the country, far from any industrial activity, 7 to 10 km from the small Umbrian city, Perugia. This population was considered to have low exposure to atmospheric pollutants. Their "exposure" was measured as years of life in the rural environment.

Data collection. All participants in the investigation were asked to fill out a questionnaire. Information was given regarding their work, any previous exposure to atmospheric pollution (due to previous jobs, place of residence etc.), hobbies, smoking habits and state of health (heart or pulmonary pathologies). The last part of the questionnaire was completed with the help of the family physician for the RP, and with the help of the medical staff for the TPO and RW. Individuals were considered to be "smokers" if they smoked more than 5 cigarettes per day. People who had not smoked during the previous 10 years were considered "ex-smokers". Sample collection. Each person was instructed to cough vigorously on 3 consecutive mornings. The sputum produced was collected in a plastic cup with a fixative consisting of 70% ethanol. The jar was sent to the Cytological Laboratory of Rome University, "La Sapienza", Italy.

Sample processing. The collected material was examined and the most representative parts were smeared on the slides. Four slides were made from each sputum sample. All the specimens were immediately post-fixed with Surgipath sprayfix (60071, Richmond, IL, USA). Two samples were stained for cytological analysis (qualitative and quantitative evaluation) using Papanicolaou’s procedure. The other 2 slides were stained according to Perl’s method and utilized for the count of pigmented alveolar macrophages and siderocytes.

The remaining sputum was placed in a 2% sodium hypochloride solution for 24 h at 37Æ C, and smears were stained with eosin for the evaluation of asbestos bodies. The eosin stain was performed by treatment with 95% ethanol. Then the specimens were immersed for 20 sec in yellow eosin 1% water solution (Carlo Erba, code 446632, Milan, Italy) before the slides were dehydrated and mounted.

Sample analysis.

a) Qualitative cytology: The smears, stained according to Papanicolaou's procedure, were evaluated using an optical microscope. Sputa without macrophages were considered not representative of pulmonary origin (inadequate). Inadequate and within normal limits (WNL) results were considered as normal samples, since a healthy individual may not be able to produce adequate sputum.

The presence of normal or abnormal epithelial cells, inflammatory leucocytic infiltrate, metaplastic changes, reactive changes, dysplastic/neoplastic cells and Curschmann's spirals were analysed. Curschmann's spirals are casts of inspissated mucous in the shape of small bronchioles. Their characteristic coiled appearance, with a dark central axis and a translucent periphery, are easily recognized in sputum cytology (13). They are present in the event of bronchiolar obstruction.

b) Quantitative parameters: Pulmonary macrophages usually contain a variable number of particles of grey, brown or black granular material or dust, hence the name "dust cells" is occasionally used (13). In haemorrhages in the pulmonary parenchyma, haemosiderin may be ingested by the macrophages (siderocytes or "heart failure cells"). In the absence of heart or pulmonary pathologies, siderocytes may be considered as a sign of iron particles present in the atmosphere. Both the dark pigment and the iron particles within siderocytes are easily identified with an iron-specific stain as Perl’s method. The count of the alveolar macrophages and siderocytes in the smears was carried out using a light microscope with 10X ocular and 20X objective. A total of 20 fields per slide were analysed, chosen randomly at 3-mm intervals. The total number of dust cells and siderocytes were evaluated for each subject. Moreover, the range and the average number of these cells were calculated in the 3 populations examined.

The asbestos bodies were counted in all smears obtained. Asbestos bodies appear pink-orange on a pink-light background in smears stained by the eosin method.

Statistical methods. Statistical analysis was performed in accordance with the procedure of the Statistical Analysis System (SPSS, Vers. 7.5, Chicago, IL, USA). The total number of dust cells and siderocytes was evaluated for each subject. Furthermore, the range and the average number of dust cells and siderocytes were calculated for each of the 3 populations. The results were correlated relative to time of exposure, smoking habits and heart/pulmonary diseases.

Initially, a descriptive analysis of the variables was performed, and it was found that non-normality of the data existed with respect to the comparison of the means. The Mann Whitney test was, therefore, used. The dependence among various risk factors and the presence of pathological elements (e.g. dust cells, siderocytes, metaplasia) was evaluated by the Chi-square (¯2_{) test. Logistic} regression was performed on metaplasia, siderocytes and dust cells as outcome and 3 predictors: groups, age and smoke.

Results with a probability of less than to 0.05 were considered significant. A logistic regression with a stepwise procedure was then applied.

Results

Traffic police officers.

a) Qualitative cytology: The cytological results of analyses

done on the sputa of the TPO are reported in Table IIA.

Forty-nine TPO (29.88%) had inadequate samples and 14

(8.54%) were WNL. Seventy-two subjects (43.90%)

showed a heavy inflammatory infiltrate and 29 (17.68%)

metaplastic changes in their sputum. One hundred and one

(61.58%) TPO showed pathological smears and 63

(38.42%) normal ones, considering WNL and inadequate

samples together. There was a significant difference

between the cytological examinations of the TPO in

comparison to the other populations considered

(p<0.001).

b) Quantitative parameters: The mean number of dust cells,

the number of subjects with more than 50 dust cells and the

number of subjects with siderocytes differed significantly

from those observed in the RW and the RP. On the

contrary, the mean number of siderocytes was not

significantly different from that of the RP.

Table I. Characteristics of the three groups examined.

Traffic Police Railway Rural Total

Officers Workers Population

no. % no. % no. % no. %

Cases 164 32.7 218 43.5 119 23.8 501 100 Mean age SD Range (years) 46 6.921 44±9.045 50±9.897 31-65 27-82 32-74 Sex M 150 91.5 215 98.6 66 55.5 431 86.0 F 14 8.5 3 1.4 53 44.5 70 14.0 central city 75 45.7 0 0 0 0 75 15.0 Place of work outskirts 80 48.8 0 0 0 0 80 16.0 office 9 5.5 59 27.1 0 0 58 13.6 rural area 0 0 0 0 119 100 119 23.7 Professional asbestos exposure low risk 0 0 95 43.6 0 0 95 19.0 high risk 0 0 64 29.3 0 0 64 12.8 Exposure to air pollution due to present job 115 70.1 159 72.9 0 0 274 54.7 previous job 4 2.4 109 50.0 41 34.4 154 30.7

Risk exposure SD (years) 13.390±8.908 15.583±6.788 0

Range (years) 0-43 0-42 Heart/pulmonary 37 22.6 10 4.6 25 21.0 72 14.4 pathologies Smoking habit smoker 73 44.5 76 34.9 29 24.4 178 35.5 ex-smokers 50 30.5 48 22.0 32 26.9 130 25.9 non-smokers 41 25.0 94 43.1 58 48.7 193 38.6 ex+non-smokers 91 55.5 142 65.1 90 75.7 323 64.5

Table IIA. Cytological examination of the traffic police officers.

Traffic Police Officers

no. % ¯2 _P Cases 164 32.7 Cytological examination inadequate 49 29.9 WNL 14 8.5 27.39^ 0.001^ HII 72 43.9 125.8* 0.001* metaplasia 29 17.7

Mean number of dust cells±SD 103.811±147.393 0.0001^≈

Range 0-999 0.001* ≈

Subjects with >50 dust cells 83 50.6 60.88^ 0.0001^

57.42* 0.001*

Subjects with siderocytes 28 17.0 11.09^ 0.004^

25.90* 0.001*

Mean number of siderocytes±SD 0.988±4.074 NS^≈

Range 0-39 0.001*

Mean number of spirals±SD 0.244±0.544 NS^≈

Range 0-2 NS* ≈

Subjects with asbestos bodies 1 0.6 NS^≈

NS* ≈ WNL: within normal limits; HII: heavy inflammatory infiltrate; NS: not significant; ^compared to rural population;

*compared to railway workers; ≈ Mann-Whitney test. Table IIB. Cytological examination of the railway workers.

Railway Workers no. % ¯2 _P Cases 218 43.5 Cytological exam inadequate 134 61.5 WNL 65 29.8 30.2^ 0.001^ HII 18 8.3 125.8* 0.001* metaplasia 1 0.5

Mean number of dust cells±SD 24.200±53.192 NS^≈

Range 0-510 0.001* ≈

Subjects with >50 dust cells 32 14.7 4.66^ 0.03^

57.42* 0.001*

Mean number of siderocytes±SD 0.490±3.943 0.001^≈

Range 0-50 0.001* ≈

Subjects with siderocytes 5 2.3 14.66^ 0.001^

25.90* 0.001*

Mean number of spirals±SD 0.014±0.151 NS^≈

Range 0-2 NS* ≈

Subjects with asbestos bodies 4 1.8 NS^≈

NS* ≈ WNL: within normal limits; HII: heavy inflammatory infiltrate; NS: not significant; ^compared to the rural population;

Asbestos bodies were observed in one case: an officer

who checked illegal house construction. For the TPO, the

number of people with more than 50 dust cells was higher

for smokers. The relative risk was 3.95. The difference

between ex-smokers, non-smokers and smokers was

statistically significant (p<0.005), Table III.

Railway workers.

a) Qualitative cytology: The cytological results of the analyses

of the RW sputa are provided in Table IIB. One hundred

and ninety-nine of the 218 RW (91.3%) showed no

pathological smears (inadequate and WNL), while there

were 19 (8.7%) pathological smears (heavy inflammatory

infiltrate and metaplasia). There was a significant difference

between the cytological results of RW sputa and those of

the TPO and of the RP (p<0.001).

b) Quantitative parameters: The difference in the mean

number of dust cells and number of subjects with more

than 50 dust cells was statistically significant between the

RW and TPO, independent of personal habits. No

difference was seen when compared to the RP. The number

of subjects with siderocytes and the mean number of

siderocytes also differed significantly when compared to the

TPO and to the RP.

Four cases showed asbestos bodies in the sputum. Two

people were in the high risk group and 2 belonged to the

mild risk group.

The cytological analyses revealed a higher percentage of

cases with heavy inflammatory infiltrates in sputa of

smokers (13.2% vs. 4.2% of ex-smokers and 6.4% of

non-smokers) (p<0.0067), Table III.

Smoking habits correlated significantly to the presence of

more than 50 dust cells in the sputum and to the mean

number of dust cells. The latter was higher in smokers and

the RR, evaluated by means of logistic regression, was 2.84.

The difference between smokers, ex-smokers and

non-smokers was statistically significant (p<0.022 and p<0.005,

respectively), Table III.

Rural population. The results of the analyses of the RP sputa

in relation to the other 2 groups examined are presented in

Table IIC. In Table III, the considered variables are related

Rural Population no. % ¯2 _P Cases 119 23.8 Cytological examination inadequate 59 49.6 WNL 23 19.3 30.2^ 0.001^ HII 30 25.2 27.39* 0.001* metaplasia 7 5.9

Mean number of dust cells±SD 12.479±23.380 NS^≈

Range 0-132 0.0001* ≈

Subjects with >50 dust cells 8 6.7 4.66^ 0.03^

60.88* 0.0001*

Mean number of siderocytes±SD 5.311±21.162 0.001^≈

Range 0-120 NS* ≈

Subjects with siderocytes 15 12.6 14.66^ 0.001^

11.09* 0.004*

Mean number of spirals±SD 0.076±0.653 NS^≈

Range 0-7 NS* ≈

Subjects with asbestos bodies 0 0

WNL: within normal limits; HII: heavy inflammatory infiltrate; NS: not significant; ^compared to railway workers; *compared to traffic police officers; ≈ Mann-Whitney test.

to smoking habits. In smokers, the mean number of dust

cells was 7.27. There were 2 (6.9%) people with more than

50 dust cells. One person had siderocytes (3.45%).

General results. No statistically significant correlation was

found between the state of health, district of work,

previous job and qualitative and quantitative cytology in

the 3 populations analysed. The number of spirals in the

sputum was almost the same in the 3 populations

examined.

The results of logistic regression with stepwise analysis

performed on metaplasia, siderocytes and dust cells as

outcome and 3 predictors, groups, age and smoke, are

provided in Table IV.

Compared with the RP, the TPO showed a worse

qualitative cytological examination (RR 4.48). The RR for

the presence of metaplastic changes in the sputum was 4.42.

Compared to the RW, the RR was 16.79 for the cytological

examination, 46.61 for the presence of metaplastic changes

and 8.88 for the presence of the siderocytes in the sputum.

The RW in comparison to the RP showed a slightly

lower risk for both the cytological examination and the

metaplastic changes, as well as for the presence of

siderocytes in the sputum.

Table III. Cytological data in the traffic police officers, railway workers and the rural population in relation to smoking habits. Traffic Police Officers Railway Workers Rural Population

Smokers Ex-smokers Non-smokers P Smokers Ex-smokers Non-smokers P Smokers Ex-smokers Non-smokers P n. 73 n. 50 n. 41 n. 76 n. 48 n. 94 n. 29 n. 32 n. 58 44.5% 30.5% 25.0% 34.9% 22.0% 43.1% 24.37% 26.89% 48.74% Cytological examination Inadequate no. 20 13 16 36 36 62 19 15 25 % 27.4 26.0 39.0 47.4% 75.0 66.0 65.5 46.8 43.1 WNL no. 10 1 3 *N.S. 30 9 26 ¯2 _{=17.79 3 5 15 *N.S.} % 13.7 2.0 7.3 39.5 18.8 27.7 *p<0.0067 10.3 15.6 25.9 HII no. 27 28 17 10 2 6 7 10 13 % 37.0 56.0 41.5 13.2 4.2 6.4 24.1 31.3 22.4 Metaplasia nÔ. 16 8 5 0 1 0 0 2 5 % 21.9 16.0 12.2 2.1 6.3 8.6 Subjects with >50 dust cells nÔ. 46 24 13 ¯2_{=10.49 18 5 9 ¯}2_{=7.57 2 2 4 N.S.} % 63.0 48.0 32.0 p<0.005 23.7 10.4 9.6 p<0.022 6.9 6.3 6.9 Mean 133.7 80.8 78.6 N.S. 41.4 13.7 15.6 p<0.005 7.27 10.9 15.9 N.S. number of dust cells Subjects with siderocytes no. 6 7 5 N.S. 2 2 1 N.S. 1 5 9 N.S. % 21.9 14.0 12.2 2.6 4.2 1.1 3.45 15.6 15.5

WNL= within normal limits; HII= heavy inflammatory infiltrate

The presence or absence of dust cells was considered, as

well as the number of subjects with more or less than 50

dust cells in the sputum. For the latter, the relative risk for

TPO as compared to the RP was 14.73 and was 5.68 in

comparison to the RW. For all 3 groups, smoking gave a

2-fold increase in the number of dust cells found in the

sputum.

Discussion

Atmospheric pollution and occupational exposure are some

of the main environmental problems seen by researchers

who analysed air quality (1-3). The results of their analyses,

however, only partially reflect the effects that dust particles

may have on the lungs of the exposed subjects. Sputum

represents a secretion produced from the lower respiratory

tract to trap harmful inhaled pollutants, and it contains both

cellular and non-cellular elements, thus reflecting

environmental injury to the lungs.

The results of our study showed that, in several cases the

TPO had an abundance of inflammatory infiltrate, almost

double that of the RP. These results confirm the findings of

Nobutumo (14), who compared the sputa of 2 different

populations (i.e., 1 rural and 1 urban). We observed,

moreover, metaplastic changes in the pulmonary epithelium

in many individuals exposed to urban pollution. (15).

Metaplasia is the result of a chronic and persistent irritating

stimulus, which, in its early stages, may be reversible (16).

Lung cancer frequently arises on the metaplastic epithelium

(13). According to the Wald’s test, only the "groups" reliably

predicted metaplasia. The TPO had a diagnosis of

"metaplasia" in 17.7% of the cases, while the RW showed a

percentage close to zero. Groups and age reliably predicted

siderocytes.

Being a TPO, once again, was the factor that considerably

increased the log of the odds for macrophages. The log of

the odds of showing sputum with more than 50 dust cells

was positively related to groups, smoking and age, the most

Outcome Variables in model Coefficient b Standard Wald test P Improvement Exponent b

error ¯2_{p values} Cytolology^ age 0.0434 0.0127 11.6781 0.0006 0.0007 1.0443 groups~ (1) 1.5018 0.2713 30.6520 0.0001 0.0001 4.4899 groups~ (2) –1.3176 0.3207 16.8822 0.0001 0.2678 groups~ (3) 2.8208 0.2890 95.3740 0.0001 0.0001 16.791 Metaplasia age 0.0444 0.0219 4.1150 0.0425 0.0456 1.0454 groups~ (1) 1.4872 0.4690 10.0555 0.0015 0.0001 4.4246 groups~ (2) –2.3398 1.0831 4.6669 0.0307 0.0001 0.0963 groups~ (3) 3.8419 1.0230 14.1049 0.0002 46.612 Siderocytes age 0.0631 0.0176 12.8804 0.0003 0.0005 1.0651 groups~ (1) 0.8874 0.3890 5.2054 0.0225 0.0001 2.4288 groups~ (2) –1.3051 0.5514 5.6029 0.0179 0.2711 groups~ (3) 2.1840 0.5080 18.518 0.0001 0.0001 8.8810

Dust cells¨ age 0.0452 0.0113 15.8505 0.0001 0.0001 1.0462

smoke* 0.5285 0.2024 6.8155 0.0090 0.0087 1.6964

groups~ (1) 0.8483 0.2601 10.6397 0.0011 0.0001 2.3357 groups~ (3) 1.3070 0.2220 34.5890 0.0001 0.0001 2.1660

Dust cellsÆ age 0.0302 0.0140 4.6245 0.0315 0.0339 1.0307

smoke* 0.9335 0.2391 15.2433 0.0001 0.0002 2.5434

groups~ (1) 2.6904 0.4109 42.8700 0.0001 0.0001 14.7370 groups~ (2) 0.9580 0.4266 5.0435 0.0247 0.0001 2.6066

groups~ (3) 1.7370 0.2540 46.7320 0.0001 5.6800

^Inadequate and within normal limits vs. heavy inflammatory infiltrate and metaplasia; ~rural population vs. traffic police officers = groups (1); rural population vs. railway workers = groups (2); railway workers vs. police officers = groups (3); ¨ absence vs. presence; *non-smokers vs. smokers; Æ< 50 and > 50 dust cells.

influential variable being groups. We checked the effects of

all other parameters and observed that the log of the odds

for showing sputum with more than 50 dust cells increased

by a factor of 5.680 for TPO as compared to RW. From the

model we calculated that the odds of showing sputum with

more than 50 dust cells were increased by a factor of 2.729

in smokers. For both the RW and TPO, each unit increase

in age increased the log of the odds for showing sputum

with more than 50 dust cells by 1.030. Age was, thus, a less

influential variable.

For the RW, age and smoking were predictors for

metaplasia, siderocytes and dust cells (as outcomes): smoking

was a more significant predictor than age for dust cells. All

the analyses indicated better conditions for the RW as

compared to the TPO.

Our results confirmed those of previous studies

(15,17-19). Most of the qualitative and quantitative parameters

analysed in the sputa of the TPO showed, moreover, no

statistical difference between smokers and non-smokers,

while air pollution was important. This fact may depend on

the heavy urban pollution of the Roman atmosphere that

could partially mask the effects of smoking on the lung

epithelium. In a previous study (15), we demonstrated that

exposure to a heavy polluted atmosphere and smoking

habits have a synergistic effect on the composition of the

sputum. The synergistic effect between smoking and

atmospheric pollution may have resulted partially from the

fact that both increase the enzymatic activity of the

activated alveolar macrophages, thus reducing the

effectiveness of the ciliated epithelium and the clearance of

both pollutants and mucous, while macrophages, from the

deep lung areas, degenerate (17). Mylius et al. also (17)

observed that there was no statistical difference in the

macrophage numbers of smoking and non-smoking subjects

living in a rural environment. Conversely, Harris et al. (18)

reported that healthy, asymptomatic cigarette smokers, who

lived in a polluted environment, had a higher level of

macrophages than non-smokers did. The present study

confirmed those data, with regard to the RW and the RP.

However, for a population like the TPO, who are exposed

for many hours per day to heavy atmospheric pollution, this

pollution seems to be the prevalent factor.

In the RW smoking had more impact than exposure to

air pollution. This group also had a higher percentage of

subjects with asbestos bodies in the sputum, thus confirming

that, for this group, asbestos exposure was a considerable

risk. This was demonstrated in the Johns Hopkins’ Lung

Projects (20) and in our previous study (21).

In conclusion, the qualitative and quantitative cytological

analysis of sputum appears to be a very sensitive and

reproducible method to identify pulmonary changes that can

be observed even before they can be measured by functional

tests (22-25).

Acknowledgements

This study was supported by the National Institute for Research (CNR Habitat and Environmental Committee Fund CTB 89.05299.13, CTB 90.00621.13 and CTB 91.00080.13). The authors gratefully acknowledge the Municipal District Traffic Commission of Rome for its helpful collaboration, and Dr. M. Bambino of the National Railways Company, Italy, for kindly providing the cytological material.

References

1 Abraham WM, Allegra L and Phipps RJ: Pollutant-induced airway abnormalities. Eur J Respir Dis S146: 117-127, 1986.

2 Ponka A, Savela M and Virtanen M: Mortality and air pollution in Helsinki. Arch Environ Health 53: 281-286, 1998.

3 Nyberg F, Gustavsson P, Jarup L et al: Urban air pollution and lung cancer in Stockholm. Epidemiology 11: 487-495, 2000.

4 Saccomanno G, Archer WE, Auerbach O, Saunders RP and Brennan LM: Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 33: 256-270, 1974. 5 Frost JK, Gupta PK, Erozan YS et al: Pulmonary cytologic

alterations in toxic environmental inhalation. Human Pathol 4: 521-536, 1973.

6 Auer G, Ono J, Nasiell M et al: Reversibility of bronchial cell atypia. Cancer Res 42: 4241-4247, 1982.

7 Swank PR, Greenberg SD, Montalvo J et al: The application of visual cell profiles in the study of premalignant atypia in sputum. Acta Cytol 29: 373-378, 1985.

8 Hocking WG and Golge DW: The pulmonary-alveolar macrofage. N Engl J Med 301: 580-587, 1979.

9 Ayres SM: Cigarette smoking and lung disease: an update. Resp Care 21: 632-640, 1976.

10 Fullmer CD, Short JG, Allen A and Walker K: Sputum of chronic cigarette smokers: microscopic observations and incidence of bronchial spirals, fibrils and casts. Rocky Mt Med J 66: 42-46, 1969.

11 Schmitz B and Pfitzer P: Acellular bodies in sputum. Acta Cytol 28: 118-125, 1984.

12 Madison R, Afifi AA and Mittman C: Respiratory impairment in coke oven workers: relationship to work exposure and bronchial inflammation detected by sputum cytology. J Chronic Dis 37: 167-176, 1984.

13 Koss LG: Lung cytology. In: Diagnostic Cytology and Its Histopathologic Bases. (J B Lippincott Company). Philadelphia, pp. 807-811, 1992.

14 Nobutomo K: Air pollution and cytological changes in sputum. Lancet 1: 523-526, 1978.

15 Giovagnoli MR, Alderisio M, Cenci M, Nofroni I and Vecchione A: Carbon and hemosiderin-laden macrophages in sputum of traffic policemen exposed to air pollution. Arch Environ Health 54: 284-290, 1999.

16 Gouveia J, Mathe G, Hercend T et al: Degree of bronchial metaplasia in heavy smokers and its regression after treatment with retinoid. Lancet 1: 710-712, 1982.

17 Mylius EA and Gullvag BM: Alveolar macrophage count as an indicator of lung reaction to industrial air pollution. Acta Cytol 30: 157-162, 1986.

18 Harris JO, Swenson EW and Johnson JE: Human alveolar macrophages. Comparison of phagocytic ability glucose utilization and ultrastructure in smokers and non-smokers. J Clin Invest 69: 2086-2096, 1970.

19 Cenci M, Giovagnoli MR, Alderisio M and Vecchione A: Curschmann's spirals in the sputum of subjects exposed daily to urban environmental pollution. Diagn Cytopathol 19: 349-351, 1998.

20 Churg A and Warnoch ML: Analysis of the cores of ferruginous (asbestos) bodies from the general population I. Patients with and without lung cancer. Lab Invest 37: 280-286, 1977. 21 Alderisio M, Giovagnoli MR, Cenci M and Vecchione A:

Asbestos bodies in the sputum of workers exposed to environmental pollution. Anticancer Res 16: 2965-2968, 1996. 22 Roby TJ, Swan GE, Sorensen KW, Hubbard GA and

Schumann GB: Discriminant analysis of lower respiratory tract components associated with cigarette smoking, based on quantitative sputum cytology. Acta Cytol 34: 147-154, 1990.

23 Roy S, Ray MR, Basu C, Lahiri P and Lahiri T: Abundance of siderophages in sputum: indicator of an adverse lung reaction to air pollution. Acta Cytol 45: 958-964, 2001.

24 Sivertsen I, Sjaastad AK, Svendsen K and Krokje A: Alveolar macrophages as biomarkers of pulmonary irritation in kitchen workers. Ann Occup Hyg 46: 713-717, 2002.

25 Kulkarni NS, Prudon B, Panditi SL, Abebe Y and Grigg J: Carbon loading of alveolar macrophages in adults and children exposed to biomass smoke particles. Sci Total Environ 345: 23-30, 2005.

Received June 30, 2005

Revised October 11, 2005

Accepted October 18, 2005