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Types, numbers, sizes, and distribution of mineral particles in the lungs of urban male cigarette smokers
ENVIRONMENTAL RESEARCH 42, 121-129 (1987)
Types, Numbers, Sizes, and Distribution of Mineral Particles in the Lungs of Urban Male Cigarette Smokers 1 A N D R E W C H U R G 2 AND BARRY WIGGS
Department of Pathology and UBC Health Sciences Centre Hospital, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 Received July 8, 1985 We analyzed the exogenous mineral particle concentration, size, type, and distribution for particles larger than 0.1 Ixm in the left lungs of 10 long-term male cigarette smokers. The mean number of particles found was 465 _+ 295 × 106/g dry lung, of which 80% were kaolinite, micas, feldspars, free silica, and talc. Lead particles were extremely rare, despite their ubiquity in urban air. Overall there were no differences in particle concentration in upper vs lower lobes or central vs peripheral sampling sites. However, a significant correlation was found for upper lobe (r = 0.68), but not lower lobe (r = 0.08), particle concentration and amount of cigarette smoking. Overall, the geometric mean particle size was 0.6 ___ 2.1 Ixm; 56% of the particles in the upper lobes were larger than 0.75 ixm in diameter, compared to 17% in the lower lobes, and the mean upper lobe particle size was greater than the mean lower lobe particle size for all individual mineral types. There was a remarkable homogeneity of mean particle size from patient to patient (mean intercase arithmetic particle size _+ SD of 0.8 _~ 0.1 Ixm). Particle size was not affected by the amount of smoking. We conclude that (1) contrary to some published acute deposition data, there are no longterm differences in upper vs lower lobe particle concentration; (2) total upper lobe particle retention is influenced by the amount of smoking as measured by pack-years, whereas total lower lobe particle retention appears to be independent by the amount of smoking; (3) particles retained in the upper lobe are somewhat larger than those retained in the lower lobe, but the amount of smoking does not appear to influence retained particle size; (4) the size of long-term retained particles most likely reflects largely atmospheric particle burden; and (5) in the absence of overwhelming dust loads, the lung is able to regulate retained particle concentration and size in a fairly narrow range. © 1987AcademicPress, Inc.
INTRODUCTION Mineral particles consititute one fraction of the air pollutants to which everyone in the population is exposed. Little information is available concerning either the burden of such particles in the lungs or the possible pathologic effects (if any) which they might produce. In humans and animals, many types of mineral particles, either in pure form or derived from cigarette smoke, evoke inflammatory reactions in the lung and such reactions may be important in the genesis of both fibrotic pneumoconioses and emphysema (Brody et al., 1984; Kilburn and McKenzie, 1975; Hunnighake and Crystal, 1983). A few mineral particles, for example, asbestos, are known carcinogens. There is also some suggestion in the t Supported by a grant from the National Cancer Institute of Canada and Grant MA7820 from the Medical Research Council of Canada. z To whom correspondence should be addressed at: Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, B.C., Canada V6T 1W5. 121 0013-9351/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CHURG AND WIGGS
literature that many other types of mineral particles might play a role in pulmonary carcinogenesis, since a variety of "inert" particles appear to redirect carcinogen metabolism and increase the yield of lung tumors in experimental animal systems (Stenback et al., 1976; Saffiotti et al., 1972; Lubawy and Isaac, 1980; Warshowsky et al., 1983). More direct evidence for the carcinogenic effects of mineral particles was provided by Schlesinger and Lippmann (1978) who showed that, in a cast of the human bronchial tree, particle deposition varies from lobe to lobe in a fashion which parallels the known lobar distribution of lung cancer. In this paper we examine the types, numbers, sizes, and distribution of exogenous mineral particles in the lungs of current male smokers. MATERIALS AND METHODS
For this study, we used autopsy material from 10 male current cigarette smokers who were greater than 50 years old and who had no pulmonary disease other than chronic airflow obstruction by either history or pathologic examination. Patients with lung cancer were purposely excluded, since, in a previous study, we have shown that such patients tend to have increased particle burdens (Churg and Wiggs, 1985). For each case, occupational dust exposure was ruled out by administering a standardized questionnaire to relatives (spouses or children); the same questionnaire was used to determine smoking history and residence. All patients had been residents of the Vancouver area for at least 10 years. For each case, the available pathologic material consisted of a midsagittal slice of left lung. Routine histologic sections were reviewed to confirm the lack of pulmonary abnormalities (except for emphysema and small airways changes related to cigarette smoke). For mineralogic analysis, essentially the same procedure was employed which we have used previously (Churg and Wiggs, 1985). This consists of dissolving a 3to 5-g piece of lung in laundry bleach, further treating the sediment with hydrogen peroxide to destroy remaining carbonaceous debris, and collecting the mineral particles on membrane filters. The filters are then dissolved in acetone in order to transfer the particles to coated electron microscope grids. Details of the procedure are available in Churg and Wiggs (1985) and are largely the same as for asbestos fibers. However, because the concentration of mineral particles is some 500 times greater than the concentration of asbestos fibers (Churgs and Wiggs, 1985), a correspondingly smaller aliquot of digestate (i.e., a greater dilution of the sample) is used to prepare the membrane filters. For this study, four samples were prepared from each lung: one each from periphreal (defined as a strip of subpleural lung of approximately 0.5-cm thickness) upper and lower lobes; and one each from central (defined as deeper than 3 cm from the pleural surface) upper and lower lobes. Only grossly normal or mildly emphysematous lung was used for each sample. An adjacent piece of lung was selected and dried to constant weight at 60°C in order to express final particle concentrations per gram of dry lung. For each site, 100 to 200 particles were measured, and identified using a combination of energy dispersive X-ray spectroscopy, supplemented by electron optical morphology and electron diffraction. Only particles greater than 0.1 p~m in diam-
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M I N E R A L P A R T I C L E S IN L U N G
eter were counted. Particles which analyzed as pure iron, iron plus phosphorus, or calcium plus phosphorus were ignored, since we were not able to determine i f these particles were endogenous or exogenous in origin. Results were converted to particles/gram dry lung using an algorithm relating amount of lung tissue used, filter area, and numbers of electron microscope squares counted. A total of approximately 5600 particles was analyzed in this study. Because particle size distributions are usually log normal, statistical comparisons between sizes were performed using t tests on the geometric size data. R ES U LTS
The 10 patients in this study had smoked from 15 to 100 pack years with a mean of 45 _+ 24 pack-years; Table 1 lists the smoking amounts, and particle load for each case. There was a correlation of r = 0.68 (P < 0.03) between number of pack-years smoked and upper lobe particle concentration, but no correlation for lower lobe particle concentration (r = 0.08). Table 2 shows the mean number of particles over all of the cases, divided by site. Considering all particles, there was an approximately even distribution between all four sample sites, and between upper and lower lobes as well as central and peripheral sites. Table 3 shows the different types of minerals encountered in the 10 cases. Kaolinite, silica (quartz and polymorphs), micas, feldspars, and talc constituted approximately 80% of the total. Their relative amounts varied from site to site; kaolinite was the most frequently found mineral in all but the peripheral lower lobe. Overall, the mean arithmetic particle size was 0.8 _+ 0.8 Ixm, and the geometric mean size 0.6 - 2.1 ~m (Table 4). There were no differences in particle size between central and peripheral sites. The size distributions of particles in TABLE 1 SMOKING AND TOTAL PARTICLE LOAD Particles ( x 106/gm dry lung) Case
Smoking (pack-years)
Total
U p p e r lobe
L o w e r lobe
1 2 3 4 5 6 7 8 9 10
15 22 27 35 40 45 50 60 60 100
326 347 348 183 264 296 1086 305 837 663
244 126 401 262 444 195 794 144 1148 1050
408 569 294 103 84 386 1377 467 527 276
M e a n ± SD
45 ± 24
465 ± 295
481 ± 380
450 ± 364
Note. Correlation coefficients for smoking and: upper lobe particles = 0.68, P < 0.03; lower lobe particles = 0.08, P = NS.
124
C H U R G A N D WIGGS TABLE 2 MEAN NUMBER OF PARTICLES BY SITE
All sites
465 _+ 295
Peripheral upper Peripheral lower Central upper Central lower
496 407 465 491
Upper lobes Lower lobes
481 _+ 380 450 _+ 364
Central Peripheral
478 +_ 456 453 +- 260
_+ +_ +_ -+
252 209 271 287
Note. Values x 106/g dry lung _+ SD.
upper and lower lobes is shown in Fig. l; these distributions are statistically different (Chi square = 677, P = 0.0001) with distinctly smaller particles in the lower lobe. In the upper lobes 56% of the particles were larger than 0.75 Ixm, compared to 17% in the lower lobes. The mean arithmetic upper lobe particle diameter was 0.9 _+ 0.9 txm and the geometric mean diameter 0.6 - 2.3 p~m. For the lower lobes corresponding values were 0.7 _+ 0.6 and 0.5 _+ 1.9 txm. There were no significant differences in particle size between central and peripheral sites. Table 4 also shows a detailed breakdown of the values for arithmetic and geometric mean sizes for each case for all sites and divided by upper and lower lobes. For 7 of 10 cases the mean particle size was slightly larger in the upper than the lower lobe; in the other 3 cases, sizes were equal in upper and lower lobes. Overall, there was remarkably little variation in mean particle size from case to case (see Discussion). Particle size by mineral type is shown in Table 5, and by mineral type and upper or lower lobe in Table 6. There was little variation in mean particle size from TABLE 3 PERCENTAGES OF DIFFERENT MINERALS COMPRISING SITE TOTALS
Percentage of Mineral, Kaolinite Silica
Total number x 106/g
Total
PU
PL
CU
CL
Misc
129 91 75 44 31 29 20 46
28 20 16 9 7 6 4 10
31 14 17 8 8 10 3 9
19 26 16 12 9 3 4 11
32 13 15 7 7 7 2 17
27 22 17 11 4 4 7 8
Tot al
465
100
100
100
100
100
Mica
Feldspars Talc Titanium Aluminum
Note. PU, peripheral upper tobe; CU, central upper lobe; PL, peripheral lower lobe; CL, central
lower lobe.
MINERAL PARTICLES IN LUNG
125
TABLE 4 MEAN PARTICLESIZES BY CASE AND SITE (ARITHMETICAND GEOMETRICMEAN SIZES ± SD IN ~m) All sites (N = 5616)
Upper lobes (N = 3092)
Lower lobes (N = 2524)
Arithmetic
Geometric
Arithmetic
Geometric
Arithmetic
Geometric
1 0.8 2 0.7 3 0.9 4 0.7 5 1.0 6 1.0 7 0.8 8 0.7 9 0.6 10 0.7 Mean over all particles 0.8 ~ 0.8 Mean over all cases 0.8 ± 0.l
0.6 0.6 0.7 0.6 0.7 0.7 0.6 0.5 0.4 0.4
1.2 0.7 0.7 0:9 1.2 1.3 1.0 0.7 0.6 0.7
0.8 0.6 0.6 0.7 0.8 1.0 0.7 0.5 0.4 0.5
0.5 0.7 0.7 0.7 0.7 0.8 0.7 0.7 0.5 0.6
0.5 0.6 0.5 0.5 0.6 0.6 0.5 0.5 0.4 0.4
0.6 ± 2.1
0.9 ± 0.9
0.6 ± 2.3
0.7 ± 0.6
0.5 ± 1.9
Case
0.9 ± 0.3
0.7 ± 0.1
m i n e r a l t o m i n e r a l , b u t f o r all m i n e r a l s u p p e r l o b e p a r t i c l e s w e r e s l i g h t l y l a r g e r o n a v e r a g e t h a n l o w e r l o b e p a r t i c l e s . T a b l e s 4, 5, a n d 6 a l s o s h o w t h e i n t e r c a s e s t a n d a r d d e v i a t i o n s a n d r a n g e o f m e a n p a r t i c l e s i z e s , in o r d e r to g i v e a n i n d i c a t i o n o f t h e v a r i a t i o n in p a r t i c l e s i z e f r o m p a t i e n t to p a t i e n t ( s e e D i s c u s s i o n ) . O v e r a l l t h e m e a n i n t e r c a s e p a r t i c l e s i z e _+ S D w a s 0.8 _+ 0.1 Ixm.
DISCUSSION In this study we have examined the types, numbers,
sizes, and distribution of
50-
40-
o 30-
mmmUpper Lobe ~ Lower Lobe
OI~0 20-
10-
~-
llt II
II
<.~5 as'..so .5;-;.zs .7o:;.o lo;'-tzs ~g-~ 1.51:l.zs;vg-zo zoo'-z25>2'.2s Size Range (/~)
FIG. 1. Particle sizes in upper and lower lobes.
126
CHURG AND WIGGS
TABLE 5 MEAN PARTICLE SIZES FOR SELECTED MINERAI~S AND ALL SITES (ARITHMETIC AND GEOMETRIC MEANS ± STANDARD DEVIATIONS IN l&m) Over all particles Mineral All Kaolinite Silica Mica Feldspars Talc
Over all cases
Arithmetic
Geometric
Arithmetic
0.8 0.7 0.8 0.8 1.0 1.1
0.6 0.5 0.6 0.6 0.7 0.8
0.8 0.7 0.8 0.9 1.0 1.1
± ± ± ± ± ±
0.8 0.9 0.8 0.7 0.9 0.9
± ± ± ± ±. ±
2.1 2.1 2.1 2.0 2.2 2.9
± ± ± ± ± ±
0.1 0.2 0.4 0.2 0.2 0.3
Range of case means 0.6-1.0 0.5-1.1 0.7-1.8 0.6-1.2 0.7-1.4 0.8-1.8
m i n e r a l p a r t i c l e s i n t h e l u n g s o f 10 m a l e c i g a r e t t e s m o k e r s . A n u m b e r o f i n t e r r e lated points emerge. F i r s t is t h e c o r r e l a t i o n o f t h e a m o u n t o f s m o k i n g a s m e a s u r e d b y p a c k - y e a r s o f smoking with numbers of retained particles. A number of studies have shown that s m o k i n g r e d u c e s a c u t e p a r t i c l e c l e a r a n c e ( r e v i e w e d i n L i p p m a n et al., 1980), a n d B o h n i n g e t al. ( 1 9 8 2 ) f o u n d a c l e a r r e l a t i o n s h i p b e t w e e n t h e s l o w i n g o f l o n g - t e r m particle clearance and the number of cigarettes smoked. The present findings thus r e p r e s e n t t h e l o g i c a l o u t c o m e o f t h e o b s e r v a t i o n s o f B o h n i n g et al. ( 1 9 8 2 ) i n t h a t persistent, smoke-mediated reduction in long-term clearance should produce a smoke-related
increase
in long-term
particle burden.
What
is s u r p r i s i n g is t h a t
TABLE 6 MEAN PARTICLE SIZES FOR SELECTED MINERALS IN UPPER AND LOWER LOBES (ARITHMETIC AND GEOMETRIC MEANS -- STANDARD DEVIATIONS IN p.m) Over all particles Mineral All particles Upper lobe Lower lobe Kaolinite Upper lobe Lower lobe Silica Upper lobe Lower lobe Mica Upper lobe Lower lobe Feldspars Upper lobe Lower lobe Talc Upper lobe Lower lobe
Over all cases
Arithmetic
Geometric
Arithmetic
Range of case means
0.9 ± 0.9 0.7 ± 0.6
0.6 -+ 2.3 0.5 _+ 1.9
0.9 -+ 0.3 0.7 -+ 0.1
0.6-1.3 0.6-0.8
0.8 ± 1.0 0.6 ± 0.6
0.5 -- 2.2 0.5 ± 1.9
0.8 ± 0.3 0.6 - 0.2
0.5-1.3 0.5-1.2
1.0 ± 0.9 0.7 ± 0.7
0.7 _+ 2.3 0.5 ± 1.8
1.0 ± 0.4 0.7 ± 0.3
0.5-1.9 0.5-1.7
0.9 _-2-0.7 0.7 ± 0.6
0.7 ± 2.1 0.5 _+ 1.9
1.0 ± 0.3 0.7 ± 0.2
0.6-1.4 0.5-1.1
1.2 _+ 1.1 0.8 -+ 0.8
0.8 + 2.4 0.7 ± 1.9
1.1 _+ 0.4 0,9 -+ 1.2
0.5-1.8 0.5-1.3
1.2 ± 0.9 0.9 + 1.0
0.9 + 2.0 0.7 _+ 1.9
1.2 -- 0.4 1.0 ± 0.5
0.7-2.2 0.6-2.3
M I N E R A L P A R T I C L E S IN L U N G
127
this correlation was present only for the upper lobes and not for the lower lobes. This observation may imply that there is a baseline particle retention which is independent of cigarette smoking, and that cigarette smoke adds an effect which is most marked in the upper lobes. It is interesting in this context that both the inflammatory effects of cigarette smoke, as measured by centrilobular emphysema, and the carcinogenic effects, as measured by numbers of lung cancers, are also greatest in the upper lobes. A second observation is that the overall concentration of particles within the lung is relatively even; there are no significant or even suggestive differences among the various sampling sites, between upper and lower lobes, or between central and peripheral sites for these particles. Thirdly, there are differences in size between lobes, with the upper lobes having slightly larger mean sizes, and most of the particles greater than 1 ~m in diameter. This finding implies either preferential deposition or preferential retention of large particles in the upper lobe. It is possible that this size difference is a smoking effect, but, if so, it does not correlate with the amount of smoking. It is interesting in this context to compare the data of Schlesinger and Lippmann (1978) examining particle deposition in a cast of the human bronchial tree. They found that the upper lobe deposition for both left and right lungs was considerably greater than the lower lobe deposition (about twofold); this distribution applied to all particle sizes used (median aerodynamic diameters of 0.26 to 7.0 ixm) and not just to larger particles. One possible explanation for these discrepancies is that the lungs which we examined show the results of long,tel~m deposition and clearance, and that over the long term larger particles are preferentially cleared from the lower lobes, thus changing both the ultimate sizes and concentrations of particles in the lower lobes. Another is that the Schlesinger and Lippmann data apply only to the airways themselves and not to the parenchyma as a whole. A fourth conclusion is that, as in our previous study of patients with and without lung cancer (Churg and Wiggs, 1985), we found that 80% of the particles were represented by five minerals: talc, kaolinite, micas, feldspars, and crystalline silica (Table 3). Smaller amounts of aluminum (presumably aluminum oxide), titanium (rutile), chlorites, tin, spinels, calcium oxide, and heavy metals, as well as unidentified particles made up the other 20%. In a qualitative sense, the minerals found in these lungs are a good match to those reported in air samples from many areas (Berry et al., 1980; Windom et al., 1967; Corn, 1976) and urban air is presumably their source. All minerals were found in all the sampling sites, and there was no statistically significant localization of any mineral in any site. It is surprising that we failed to find other than rare lead particles in our lungs, despite their high frequency in the fine particle fraction of the air (Corn, 1976). This observation suggests that the lead must completely and rapidly dissolve in the lung, whereas other metals such as iron, aluminum, and titanium do not. Our fifth conclusion is that there is a remarkable homogeneity of mean particle size from mineral to mineral, and within upper or lower lobes (Tables 5 and 6). Most likely the sizes of particles present in these lungs reflect the sizes of particles which are present in the air, although exact information on particles measured by projected area diameter and identified by mineral type is hard to obtain.
' 128
CHURG AND WIGGS
Using electron microscopic measurements of projected area diameter, Jacobs et al. (1962) found that 75 to 80% of the atmospheric particles were smaller than 1 Ixm; Waller et al. (1963) suggested that the mean diameter was about 0.1 ~xm. These data include a large variety of particles which are smaller than our counting limit of 0.1 txm, and also include particles of iron, which make up a substantial fraction of the identifiable burden of atmopsheric particles (Corn, 1976), but which are excluded from our analysis because we cannot differentiate the exogenous inhaled iron from endogenous iron resulting from hemorrhage into the lung and ferrugination of other exogenous minerals. A related conclusion is the remarkable homogeneity of retained particle size from patient to patient, as represented by the intercase mean and standard deviation (Tables 4 to 6). Overall, the arithmetic mean particle size in all 10 patients was 0.8 ~xm with a range of 0.6 to 1.0 ~zm and an intercase standard deviation of 0.1 ~xm. Similar values are seen for the upper and lower lobes and for each major mineral type. Again, this observation probably reflects the atmospheric origin of most of the particles, but it also implies a homogeneity of long-term particle retention which is considerably greater than that seen in acute deposition and clearance studies (Lippmann et at., 1980). A similar conclusion may apply to the total particle concentration: despite the differences which appear to relate to smoking, there was only about a fivefold difference in total particle concentration from greatest to least in our 10 patients. These data may imply that, in the absence of massive (occupational) dust exposure, the lung is able to regulate particle burden to a fairly narrow equilibrium range of size and number.
REFERENCES Berry, J. P., and Galle, urban atmosphere. Brody, A. R., Warheit, deposition pattern
P. (1980). Chemical and crystallographic microanalysis of fine particles in the Environ. Res. 12, 150-164. D. B., Chang, L. Y., Roe, M. W., George, G., and Hill, L. H. (1984). Initial of inhaled minerals and consequent pathogenic events at the alveolar level. Ann. N.Y. Acad. Sci. 420, 108-120. Bohning, D. E., Atkins, H. L., and Cohn, S. H. (1982). Long-term particle clearance in man: Normal and impaired. Ann. Occup. Hyg. 26, 259-271. Churg, A., and Wiggs, B. (1985). Mineral particles, mineral fibers, and lung cancer. Environ. Res. 37, 364-372. Corn, M. (1976). Aerosols and the primary air pollutants--nonviable particles. In "Air Pollution" (A. C. Stern, Ed.), 3rd. ed., Vol. 1, pp. 77-168. Academic Press, New York. Flint, L., Maxwell, K. W., and Renzetti, A. D. (1971). Influence of cigarette smoke on guinea pigs. Arch. Environ. Health 22, 366-369. Hunnighake, G. W., and Crystal, R. G. (1983). Cigarette smoking and lung destruction: Accumulation of neutrophils in the lungs of cigarette smokers. Amer. Rev. Respir. Dis. 128, 833-838. Jacobs, M. B., Manoharan, A., and Goldwater, L. J. (1962). Comparison of dust counts of indoor and outdoor air. Int. J. Air Water Pollut. 6, 205-213. Kilburn, K. H., and McKenzie, W. (1975). Leukocyte recruitment to airways by cigarette smoke and particle phase in contrast to cytotoxicity of vapor. Science 189, 634-637. Lippmann, M., Yeates, D. B., and Albert, R. E. (1980). Deposition, retention, and clearance of inhaled particles. Brit. J. Indust. Med. 37, 337-362. Lubawy, W. C., Isaac, R. S. (1980). Acute tobacco smoke exposure alters the profile of metabolites produced by benzo(a)pyrene by the isolated perfused rabbit lung. Toxicology 18, 37-47. Safiotti, U., Montesano, R., Sellakumar, A. R., Cetis, E, and Kaufman, D. G. (1972). Respiratory
MINERAL PARTICLES IN LUNG
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tract carcinogenesis in hamsters induced by benzo(a)pyrene and ferric oxide. Cancer Res. 32, 1073-1081. Schlesinger, R. B., and Lippmann, M. (1978). Selective particle deposition and bronchogenic carcinoma. Environ. Res. 15,424-431. Stenback, E, Rowland, J., and Sellakumar, A. (1976). Carcinogenicity of benzo(a)pyrene and suts in the hamster lung. Oncology 33, 29-34. Waller, R. E., Brooks, A. G. E, and Cartwright, J. (1963). An electron microscope study of particles in town air. Int. J. Air. Water Pollut. 7, 779-786. Warshowsky, D., Bingham, E., and Niemeier, R. W. (1983). Influence of airborne particulate on the metabolism of benzo(a)pyrene in the isolated perfused lung. J. Toxicol. Environ. Health 11, 503 -517. Windom, H., Griffin, J., and Goldberg, E. D. (1967). Talc in atmospheric dusts. Environ. Sci. Technol. 1, 923-926.
Types, Numbers, Sizes, and Distribution of Mineral Particles in the Lungs of Urban Male Cigarette Smokers 1 A N D R E W C H U R G 2 AND BARRY WIGGS
Department of Pathology and UBC Health Sciences Centre Hospital, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 Received July 8, 1985 We analyzed the exogenous mineral particle concentration, size, type, and distribution for particles larger than 0.1 Ixm in the left lungs of 10 long-term male cigarette smokers. The mean number of particles found was 465 _+ 295 × 106/g dry lung, of which 80% were kaolinite, micas, feldspars, free silica, and talc. Lead particles were extremely rare, despite their ubiquity in urban air. Overall there were no differences in particle concentration in upper vs lower lobes or central vs peripheral sampling sites. However, a significant correlation was found for upper lobe (r = 0.68), but not lower lobe (r = 0.08), particle concentration and amount of cigarette smoking. Overall, the geometric mean particle size was 0.6 ___ 2.1 Ixm; 56% of the particles in the upper lobes were larger than 0.75 ixm in diameter, compared to 17% in the lower lobes, and the mean upper lobe particle size was greater than the mean lower lobe particle size for all individual mineral types. There was a remarkable homogeneity of mean particle size from patient to patient (mean intercase arithmetic particle size _+ SD of 0.8 _~ 0.1 Ixm). Particle size was not affected by the amount of smoking. We conclude that (1) contrary to some published acute deposition data, there are no longterm differences in upper vs lower lobe particle concentration; (2) total upper lobe particle retention is influenced by the amount of smoking as measured by pack-years, whereas total lower lobe particle retention appears to be independent by the amount of smoking; (3) particles retained in the upper lobe are somewhat larger than those retained in the lower lobe, but the amount of smoking does not appear to influence retained particle size; (4) the size of long-term retained particles most likely reflects largely atmospheric particle burden; and (5) in the absence of overwhelming dust loads, the lung is able to regulate retained particle concentration and size in a fairly narrow range. © 1987AcademicPress, Inc.
INTRODUCTION Mineral particles consititute one fraction of the air pollutants to which everyone in the population is exposed. Little information is available concerning either the burden of such particles in the lungs or the possible pathologic effects (if any) which they might produce. In humans and animals, many types of mineral particles, either in pure form or derived from cigarette smoke, evoke inflammatory reactions in the lung and such reactions may be important in the genesis of both fibrotic pneumoconioses and emphysema (Brody et al., 1984; Kilburn and McKenzie, 1975; Hunnighake and Crystal, 1983). A few mineral particles, for example, asbestos, are known carcinogens. There is also some suggestion in the t Supported by a grant from the National Cancer Institute of Canada and Grant MA7820 from the Medical Research Council of Canada. z To whom correspondence should be addressed at: Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, B.C., Canada V6T 1W5. 121 0013-9351/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
122
CHURG AND WIGGS
literature that many other types of mineral particles might play a role in pulmonary carcinogenesis, since a variety of "inert" particles appear to redirect carcinogen metabolism and increase the yield of lung tumors in experimental animal systems (Stenback et al., 1976; Saffiotti et al., 1972; Lubawy and Isaac, 1980; Warshowsky et al., 1983). More direct evidence for the carcinogenic effects of mineral particles was provided by Schlesinger and Lippmann (1978) who showed that, in a cast of the human bronchial tree, particle deposition varies from lobe to lobe in a fashion which parallels the known lobar distribution of lung cancer. In this paper we examine the types, numbers, sizes, and distribution of exogenous mineral particles in the lungs of current male smokers. MATERIALS AND METHODS
For this study, we used autopsy material from 10 male current cigarette smokers who were greater than 50 years old and who had no pulmonary disease other than chronic airflow obstruction by either history or pathologic examination. Patients with lung cancer were purposely excluded, since, in a previous study, we have shown that such patients tend to have increased particle burdens (Churg and Wiggs, 1985). For each case, occupational dust exposure was ruled out by administering a standardized questionnaire to relatives (spouses or children); the same questionnaire was used to determine smoking history and residence. All patients had been residents of the Vancouver area for at least 10 years. For each case, the available pathologic material consisted of a midsagittal slice of left lung. Routine histologic sections were reviewed to confirm the lack of pulmonary abnormalities (except for emphysema and small airways changes related to cigarette smoke). For mineralogic analysis, essentially the same procedure was employed which we have used previously (Churg and Wiggs, 1985). This consists of dissolving a 3to 5-g piece of lung in laundry bleach, further treating the sediment with hydrogen peroxide to destroy remaining carbonaceous debris, and collecting the mineral particles on membrane filters. The filters are then dissolved in acetone in order to transfer the particles to coated electron microscope grids. Details of the procedure are available in Churg and Wiggs (1985) and are largely the same as for asbestos fibers. However, because the concentration of mineral particles is some 500 times greater than the concentration of asbestos fibers (Churgs and Wiggs, 1985), a correspondingly smaller aliquot of digestate (i.e., a greater dilution of the sample) is used to prepare the membrane filters. For this study, four samples were prepared from each lung: one each from periphreal (defined as a strip of subpleural lung of approximately 0.5-cm thickness) upper and lower lobes; and one each from central (defined as deeper than 3 cm from the pleural surface) upper and lower lobes. Only grossly normal or mildly emphysematous lung was used for each sample. An adjacent piece of lung was selected and dried to constant weight at 60°C in order to express final particle concentrations per gram of dry lung. For each site, 100 to 200 particles were measured, and identified using a combination of energy dispersive X-ray spectroscopy, supplemented by electron optical morphology and electron diffraction. Only particles greater than 0.1 p~m in diam-
123
M I N E R A L P A R T I C L E S IN L U N G
eter were counted. Particles which analyzed as pure iron, iron plus phosphorus, or calcium plus phosphorus were ignored, since we were not able to determine i f these particles were endogenous or exogenous in origin. Results were converted to particles/gram dry lung using an algorithm relating amount of lung tissue used, filter area, and numbers of electron microscope squares counted. A total of approximately 5600 particles was analyzed in this study. Because particle size distributions are usually log normal, statistical comparisons between sizes were performed using t tests on the geometric size data. R ES U LTS
The 10 patients in this study had smoked from 15 to 100 pack years with a mean of 45 _+ 24 pack-years; Table 1 lists the smoking amounts, and particle load for each case. There was a correlation of r = 0.68 (P < 0.03) between number of pack-years smoked and upper lobe particle concentration, but no correlation for lower lobe particle concentration (r = 0.08). Table 2 shows the mean number of particles over all of the cases, divided by site. Considering all particles, there was an approximately even distribution between all four sample sites, and between upper and lower lobes as well as central and peripheral sites. Table 3 shows the different types of minerals encountered in the 10 cases. Kaolinite, silica (quartz and polymorphs), micas, feldspars, and talc constituted approximately 80% of the total. Their relative amounts varied from site to site; kaolinite was the most frequently found mineral in all but the peripheral lower lobe. Overall, the mean arithmetic particle size was 0.8 _+ 0.8 Ixm, and the geometric mean size 0.6 - 2.1 ~m (Table 4). There were no differences in particle size between central and peripheral sites. The size distributions of particles in TABLE 1 SMOKING AND TOTAL PARTICLE LOAD Particles ( x 106/gm dry lung) Case
Smoking (pack-years)
Total
U p p e r lobe
L o w e r lobe
1 2 3 4 5 6 7 8 9 10
15 22 27 35 40 45 50 60 60 100
326 347 348 183 264 296 1086 305 837 663
244 126 401 262 444 195 794 144 1148 1050
408 569 294 103 84 386 1377 467 527 276
M e a n ± SD
45 ± 24
465 ± 295
481 ± 380
450 ± 364
Note. Correlation coefficients for smoking and: upper lobe particles = 0.68, P < 0.03; lower lobe particles = 0.08, P = NS.
124
C H U R G A N D WIGGS TABLE 2 MEAN NUMBER OF PARTICLES BY SITE
All sites
465 _+ 295
Peripheral upper Peripheral lower Central upper Central lower
496 407 465 491
Upper lobes Lower lobes
481 _+ 380 450 _+ 364
Central Peripheral
478 +_ 456 453 +- 260
_+ +_ +_ -+
252 209 271 287
Note. Values x 106/g dry lung _+ SD.
upper and lower lobes is shown in Fig. l; these distributions are statistically different (Chi square = 677, P = 0.0001) with distinctly smaller particles in the lower lobe. In the upper lobes 56% of the particles were larger than 0.75 Ixm, compared to 17% in the lower lobes. The mean arithmetic upper lobe particle diameter was 0.9 _+ 0.9 txm and the geometric mean diameter 0.6 - 2.3 p~m. For the lower lobes corresponding values were 0.7 _+ 0.6 and 0.5 _+ 1.9 txm. There were no significant differences in particle size between central and peripheral sites. Table 4 also shows a detailed breakdown of the values for arithmetic and geometric mean sizes for each case for all sites and divided by upper and lower lobes. For 7 of 10 cases the mean particle size was slightly larger in the upper than the lower lobe; in the other 3 cases, sizes were equal in upper and lower lobes. Overall, there was remarkably little variation in mean particle size from case to case (see Discussion). Particle size by mineral type is shown in Table 5, and by mineral type and upper or lower lobe in Table 6. There was little variation in mean particle size from TABLE 3 PERCENTAGES OF DIFFERENT MINERALS COMPRISING SITE TOTALS
Percentage of Mineral, Kaolinite Silica
Total number x 106/g
Total
PU
PL
CU
CL
Misc
129 91 75 44 31 29 20 46
28 20 16 9 7 6 4 10
31 14 17 8 8 10 3 9
19 26 16 12 9 3 4 11
32 13 15 7 7 7 2 17
27 22 17 11 4 4 7 8
Tot al
465
100
100
100
100
100
Mica
Feldspars Talc Titanium Aluminum
Note. PU, peripheral upper tobe; CU, central upper lobe; PL, peripheral lower lobe; CL, central
lower lobe.
MINERAL PARTICLES IN LUNG
125
TABLE 4 MEAN PARTICLESIZES BY CASE AND SITE (ARITHMETICAND GEOMETRICMEAN SIZES ± SD IN ~m) All sites (N = 5616)
Upper lobes (N = 3092)
Lower lobes (N = 2524)
Arithmetic
Geometric
Arithmetic
Geometric
Arithmetic
Geometric
1 0.8 2 0.7 3 0.9 4 0.7 5 1.0 6 1.0 7 0.8 8 0.7 9 0.6 10 0.7 Mean over all particles 0.8 ~ 0.8 Mean over all cases 0.8 ± 0.l
0.6 0.6 0.7 0.6 0.7 0.7 0.6 0.5 0.4 0.4
1.2 0.7 0.7 0:9 1.2 1.3 1.0 0.7 0.6 0.7
0.8 0.6 0.6 0.7 0.8 1.0 0.7 0.5 0.4 0.5
0.5 0.7 0.7 0.7 0.7 0.8 0.7 0.7 0.5 0.6
0.5 0.6 0.5 0.5 0.6 0.6 0.5 0.5 0.4 0.4
0.6 ± 2.1
0.9 ± 0.9
0.6 ± 2.3
0.7 ± 0.6
0.5 ± 1.9
Case
0.9 ± 0.3
0.7 ± 0.1
m i n e r a l t o m i n e r a l , b u t f o r all m i n e r a l s u p p e r l o b e p a r t i c l e s w e r e s l i g h t l y l a r g e r o n a v e r a g e t h a n l o w e r l o b e p a r t i c l e s . T a b l e s 4, 5, a n d 6 a l s o s h o w t h e i n t e r c a s e s t a n d a r d d e v i a t i o n s a n d r a n g e o f m e a n p a r t i c l e s i z e s , in o r d e r to g i v e a n i n d i c a t i o n o f t h e v a r i a t i o n in p a r t i c l e s i z e f r o m p a t i e n t to p a t i e n t ( s e e D i s c u s s i o n ) . O v e r a l l t h e m e a n i n t e r c a s e p a r t i c l e s i z e _+ S D w a s 0.8 _+ 0.1 Ixm.
DISCUSSION In this study we have examined the types, numbers,
sizes, and distribution of
50-
40-
o 30-
mmmUpper Lobe ~ Lower Lobe
OI~0 20-
10-
~-
llt II
II
<.~5 as'..so .5;-;.zs .7o:;.o lo;'-tzs ~g-~ 1.51:l.zs;vg-zo zoo'-z25>2'.2s Size Range (/~)
FIG. 1. Particle sizes in upper and lower lobes.
126
CHURG AND WIGGS
TABLE 5 MEAN PARTICLE SIZES FOR SELECTED MINERAI~S AND ALL SITES (ARITHMETIC AND GEOMETRIC MEANS ± STANDARD DEVIATIONS IN l&m) Over all particles Mineral All Kaolinite Silica Mica Feldspars Talc
Over all cases
Arithmetic
Geometric
Arithmetic
0.8 0.7 0.8 0.8 1.0 1.1
0.6 0.5 0.6 0.6 0.7 0.8
0.8 0.7 0.8 0.9 1.0 1.1
± ± ± ± ± ±
0.8 0.9 0.8 0.7 0.9 0.9
± ± ± ± ±. ±
2.1 2.1 2.1 2.0 2.2 2.9
± ± ± ± ± ±
0.1 0.2 0.4 0.2 0.2 0.3
Range of case means 0.6-1.0 0.5-1.1 0.7-1.8 0.6-1.2 0.7-1.4 0.8-1.8
m i n e r a l p a r t i c l e s i n t h e l u n g s o f 10 m a l e c i g a r e t t e s m o k e r s . A n u m b e r o f i n t e r r e lated points emerge. F i r s t is t h e c o r r e l a t i o n o f t h e a m o u n t o f s m o k i n g a s m e a s u r e d b y p a c k - y e a r s o f smoking with numbers of retained particles. A number of studies have shown that s m o k i n g r e d u c e s a c u t e p a r t i c l e c l e a r a n c e ( r e v i e w e d i n L i p p m a n et al., 1980), a n d B o h n i n g e t al. ( 1 9 8 2 ) f o u n d a c l e a r r e l a t i o n s h i p b e t w e e n t h e s l o w i n g o f l o n g - t e r m particle clearance and the number of cigarettes smoked. The present findings thus r e p r e s e n t t h e l o g i c a l o u t c o m e o f t h e o b s e r v a t i o n s o f B o h n i n g et al. ( 1 9 8 2 ) i n t h a t persistent, smoke-mediated reduction in long-term clearance should produce a smoke-related
increase
in long-term
particle burden.
What
is s u r p r i s i n g is t h a t
TABLE 6 MEAN PARTICLE SIZES FOR SELECTED MINERALS IN UPPER AND LOWER LOBES (ARITHMETIC AND GEOMETRIC MEANS -- STANDARD DEVIATIONS IN p.m) Over all particles Mineral All particles Upper lobe Lower lobe Kaolinite Upper lobe Lower lobe Silica Upper lobe Lower lobe Mica Upper lobe Lower lobe Feldspars Upper lobe Lower lobe Talc Upper lobe Lower lobe
Over all cases
Arithmetic
Geometric
Arithmetic
Range of case means
0.9 ± 0.9 0.7 ± 0.6
0.6 -+ 2.3 0.5 _+ 1.9
0.9 -+ 0.3 0.7 -+ 0.1
0.6-1.3 0.6-0.8
0.8 ± 1.0 0.6 ± 0.6
0.5 -- 2.2 0.5 ± 1.9
0.8 ± 0.3 0.6 - 0.2
0.5-1.3 0.5-1.2
1.0 ± 0.9 0.7 ± 0.7
0.7 _+ 2.3 0.5 ± 1.8
1.0 ± 0.4 0.7 ± 0.3
0.5-1.9 0.5-1.7
0.9 _-2-0.7 0.7 ± 0.6
0.7 ± 2.1 0.5 _+ 1.9
1.0 ± 0.3 0.7 ± 0.2
0.6-1.4 0.5-1.1
1.2 _+ 1.1 0.8 -+ 0.8
0.8 + 2.4 0.7 ± 1.9
1.1 _+ 0.4 0,9 -+ 1.2
0.5-1.8 0.5-1.3
1.2 ± 0.9 0.9 + 1.0
0.9 + 2.0 0.7 _+ 1.9
1.2 -- 0.4 1.0 ± 0.5
0.7-2.2 0.6-2.3
M I N E R A L P A R T I C L E S IN L U N G
127
this correlation was present only for the upper lobes and not for the lower lobes. This observation may imply that there is a baseline particle retention which is independent of cigarette smoking, and that cigarette smoke adds an effect which is most marked in the upper lobes. It is interesting in this context that both the inflammatory effects of cigarette smoke, as measured by centrilobular emphysema, and the carcinogenic effects, as measured by numbers of lung cancers, are also greatest in the upper lobes. A second observation is that the overall concentration of particles within the lung is relatively even; there are no significant or even suggestive differences among the various sampling sites, between upper and lower lobes, or between central and peripheral sites for these particles. Thirdly, there are differences in size between lobes, with the upper lobes having slightly larger mean sizes, and most of the particles greater than 1 ~m in diameter. This finding implies either preferential deposition or preferential retention of large particles in the upper lobe. It is possible that this size difference is a smoking effect, but, if so, it does not correlate with the amount of smoking. It is interesting in this context to compare the data of Schlesinger and Lippmann (1978) examining particle deposition in a cast of the human bronchial tree. They found that the upper lobe deposition for both left and right lungs was considerably greater than the lower lobe deposition (about twofold); this distribution applied to all particle sizes used (median aerodynamic diameters of 0.26 to 7.0 ixm) and not just to larger particles. One possible explanation for these discrepancies is that the lungs which we examined show the results of long,tel~m deposition and clearance, and that over the long term larger particles are preferentially cleared from the lower lobes, thus changing both the ultimate sizes and concentrations of particles in the lower lobes. Another is that the Schlesinger and Lippmann data apply only to the airways themselves and not to the parenchyma as a whole. A fourth conclusion is that, as in our previous study of patients with and without lung cancer (Churg and Wiggs, 1985), we found that 80% of the particles were represented by five minerals: talc, kaolinite, micas, feldspars, and crystalline silica (Table 3). Smaller amounts of aluminum (presumably aluminum oxide), titanium (rutile), chlorites, tin, spinels, calcium oxide, and heavy metals, as well as unidentified particles made up the other 20%. In a qualitative sense, the minerals found in these lungs are a good match to those reported in air samples from many areas (Berry et al., 1980; Windom et al., 1967; Corn, 1976) and urban air is presumably their source. All minerals were found in all the sampling sites, and there was no statistically significant localization of any mineral in any site. It is surprising that we failed to find other than rare lead particles in our lungs, despite their high frequency in the fine particle fraction of the air (Corn, 1976). This observation suggests that the lead must completely and rapidly dissolve in the lung, whereas other metals such as iron, aluminum, and titanium do not. Our fifth conclusion is that there is a remarkable homogeneity of mean particle size from mineral to mineral, and within upper or lower lobes (Tables 5 and 6). Most likely the sizes of particles present in these lungs reflect the sizes of particles which are present in the air, although exact information on particles measured by projected area diameter and identified by mineral type is hard to obtain.
' 128
CHURG AND WIGGS
Using electron microscopic measurements of projected area diameter, Jacobs et al. (1962) found that 75 to 80% of the atmospheric particles were smaller than 1 Ixm; Waller et al. (1963) suggested that the mean diameter was about 0.1 ~xm. These data include a large variety of particles which are smaller than our counting limit of 0.1 txm, and also include particles of iron, which make up a substantial fraction of the identifiable burden of atmopsheric particles (Corn, 1976), but which are excluded from our analysis because we cannot differentiate the exogenous inhaled iron from endogenous iron resulting from hemorrhage into the lung and ferrugination of other exogenous minerals. A related conclusion is the remarkable homogeneity of retained particle size from patient to patient, as represented by the intercase mean and standard deviation (Tables 4 to 6). Overall, the arithmetic mean particle size in all 10 patients was 0.8 ~xm with a range of 0.6 to 1.0 ~zm and an intercase standard deviation of 0.1 ~xm. Similar values are seen for the upper and lower lobes and for each major mineral type. Again, this observation probably reflects the atmospheric origin of most of the particles, but it also implies a homogeneity of long-term particle retention which is considerably greater than that seen in acute deposition and clearance studies (Lippmann et at., 1980). A similar conclusion may apply to the total particle concentration: despite the differences which appear to relate to smoking, there was only about a fivefold difference in total particle concentration from greatest to least in our 10 patients. These data may imply that, in the absence of massive (occupational) dust exposure, the lung is able to regulate particle burden to a fairly narrow equilibrium range of size and number.
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P. (1980). Chemical and crystallographic microanalysis of fine particles in the Environ. Res. 12, 150-164. D. B., Chang, L. Y., Roe, M. W., George, G., and Hill, L. H. (1984). Initial of inhaled minerals and consequent pathogenic events at the alveolar level. Ann. N.Y. Acad. Sci. 420, 108-120. Bohning, D. E., Atkins, H. L., and Cohn, S. H. (1982). Long-term particle clearance in man: Normal and impaired. Ann. Occup. Hyg. 26, 259-271. Churg, A., and Wiggs, B. (1985). Mineral particles, mineral fibers, and lung cancer. Environ. Res. 37, 364-372. Corn, M. (1976). Aerosols and the primary air pollutants--nonviable particles. In "Air Pollution" (A. C. Stern, Ed.), 3rd. ed., Vol. 1, pp. 77-168. Academic Press, New York. Flint, L., Maxwell, K. W., and Renzetti, A. D. (1971). Influence of cigarette smoke on guinea pigs. Arch. Environ. Health 22, 366-369. Hunnighake, G. W., and Crystal, R. G. (1983). Cigarette smoking and lung destruction: Accumulation of neutrophils in the lungs of cigarette smokers. Amer. Rev. Respir. Dis. 128, 833-838. Jacobs, M. B., Manoharan, A., and Goldwater, L. J. (1962). Comparison of dust counts of indoor and outdoor air. Int. J. Air Water Pollut. 6, 205-213. Kilburn, K. H., and McKenzie, W. (1975). Leukocyte recruitment to airways by cigarette smoke and particle phase in contrast to cytotoxicity of vapor. Science 189, 634-637. Lippmann, M., Yeates, D. B., and Albert, R. E. (1980). Deposition, retention, and clearance of inhaled particles. Brit. J. Indust. Med. 37, 337-362. Lubawy, W. C., Isaac, R. S. (1980). Acute tobacco smoke exposure alters the profile of metabolites produced by benzo(a)pyrene by the isolated perfused rabbit lung. Toxicology 18, 37-47. Safiotti, U., Montesano, R., Sellakumar, A. R., Cetis, E, and Kaufman, D. G. (1972). Respiratory
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tract carcinogenesis in hamsters induced by benzo(a)pyrene and ferric oxide. Cancer Res. 32, 1073-1081. Schlesinger, R. B., and Lippmann, M. (1978). Selective particle deposition and bronchogenic carcinoma. Environ. Res. 15,424-431. Stenback, E, Rowland, J., and Sellakumar, A. (1976). Carcinogenicity of benzo(a)pyrene and suts in the hamster lung. Oncology 33, 29-34. Waller, R. E., Brooks, A. G. E, and Cartwright, J. (1963). An electron microscope study of particles in town air. Int. J. Air. Water Pollut. 7, 779-786. Warshowsky, D., Bingham, E., and Niemeier, R. W. (1983). Influence of airborne particulate on the metabolism of benzo(a)pyrene in the isolated perfused lung. J. Toxicol. Environ. Health 11, 503 -517. Windom, H., Griffin, J., and Goldberg, E. D. (1967). Talc in atmospheric dusts. Environ. Sci. Technol. 1, 923-926.