Acta Pathol. Jpn. 36(12) : 1781-1791, 1986

A QUANTITATIVE AND HISTOLOGICAL STUDY ON PULMONARY EFFECTS O F ASBESTOS EXPOSURE

IN GENERAL AUTOPSIED LUNGS

Hiroshi KOBAYASHI, Hisashi WATANABE, Wei Ming ZHANG, and Yoshihisa OHNISHI

Department of Pathology, school of Medicine, Niigata University, Niigatu

We demonstrated the distribution of asbestos bodies (ABs) in the lungs of 656 consecutive autopsy cases by KOH digestion method. Nine patients includ- ing one case with asbestosis had more than a thousand ABs. These patients were all blue-collar workers except the two cases without occupational history. The tissue sections for these patients revealed the constant presence of more than one or two ABs. In addition, five of these subjects had parenchymal and/ or peribronchiolar fibrosis. On the basis of the result of AB counts, we selected 17 subjects for enumeration and typing of asbestos fibers (AFs). The number of AFs roughly correlated with that of ABs. However, the ratio of AFs to ABs ranged from 13 to 3,700. The fiber types in our selected subjects were almost always amphibole (crocidolite, amosite). These findings indicate that modest or secondary exposure to asbestos causes virtual asbestosis to several subjects among the general population. ACTA PATHOL. JPN. 36 : 1781 - 1791, 1986.

Introduction It has been well defined that asbestosis, pulmonary carcinoma, and mesothelioma

often occur among the people with high asbestos exposure who engage in asbestos mining, milling, and m a n u f a c t ~ r i n g . ~ J ~ J ~ J ~ ~ ~ ~ * ~ ~ Asbestos has a wide variety of uses and is applied to thousands of industrial products. Its consumption has been vastly increasing since the last three decades.1° Thus, disease related to asbestos is corisid- ered to be a major public health problem.

The diagnosis of the diseases caused by asbestos exposure is more easier in persons whose occupational history is definitely obtained. However, there are frequently much difficulties to determine whether a disease is related to asbestos exposure in the cases without known employmental data.4 Indeed, even in autopsy materials, pathologists may sometimes overlook a “virtual” asbestosis if only a few ABs and slight fibrosis are noticed.

Accepted for publication March 3, 1986.

Mailing address : Hiroshi KOBAYASHI, MD, Department of Pathology, School of Medicine, Niigata University, Asahimachidori 1-757, Niigata 951, JAPAN.

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1782 PATHOLOGICAL EFFECTS OF ASBESTOS EXPOSURE Acta Pathol. Jpn.

From these viewpoints, we believe that it is of great practical importance to obtain a quantitative information on ABs and AFs from the general population and to examine the relation between their burdens and pulmonary changes. In this paper, we report the AB and AF content of lungs from 656 consecutive autopsy cases from 1977 to 1981, and demonstrate certain pulmonary alterations probably due to asbestos exposure in several of these cases.

Matem'al and Methods Six hundred and fifty six cases were selected only because of the age over twenty years among all

autopsied materials from 1977 to 1981 in the department of pathology, School of Medicine, Niigata University. In addition, one surgically resected lung diagnosed as asbestosis with pulmonary car- cinoma was available for this study. Occupational data were obtained by review of the subject's chart.

(1) A B counts in general autopsy cases. KOH digestion method' modified by us was applied. We took certain pieces of wet lung tissue

from the right lower lobe of 656 patients' lungs, avoiding as much as possible major bronchi or large vessels and the areas of hemorrhage, pneumonia, and consolidation. It was divided into two parts. One half was weighed and dried overnight a t 60'C to be reweighed. The reduction in weight due to drying allowed the equivalent dry weight of the second- haf to be gauged without drying, second half was cut into small pieces. 0.5 g of them was then melted by immersion in about 10 ml of 40% KOH in a clean 40ml conical centrifuge tube, which was kept in a bath of boiling water for 20 to 30 minutes. Following centrifugation at 1,700 rpm for 20 minutes, the supernatant was discarded. The sediment was resuspended in 99% butyl alcohol and filtered through a 0.8 p pore sized membrane filter (Toyo Roshi Co. Ltd. Tokyo). The filter was dried, and mounted on a slide. We counted all femiginous bodies with transparent, colorless cores as asbestos bodies. The observed number of AB was converted into counts per gram of dried lung.

(2) AF counts in the selected cases. On the basis of the result of AB counts, we selected 17 subjects for enumeration of fibers : two

patients had more AB counts than lo4 AB/g ; eight patients, ranging from lo3 to less than lo4 AB/ g ; seven patients, 500 to less than lo3 AB/g. The 0.5 g of lung tissue was melted aa described above and centrifuged a t 1,700 rpm for 20 minutes, collecting the sediment on a 0.8 p pore sized membrane. In certain cases i t was diluted with distilled water ta be suitable for electron microscopy if the sediment was too much. As previously described: pieces of the filter were cut out and placed on carbon coated, 300 mesh, copper electron microscope grids. The filter waa dissolved by placing the grids on acetone-impregnated polyurethane foam sponges. Uncoated asbestos fibers were identified by electron microscopy using, a combination of morphology, electron diffraction, and electron microprobe analysis.

(3) A B count in tissue sections and pathological data. Four to twelve paraffin-embedded blocks of lung tissue were available in the 17 cases. Sections

were cut a t a thickness of 5 p m and stained with hematoxylin-eosin. All fields of each section was scanned a t a magnification of ~ 2 0 0 and the number of AB was counted.

We, also, examined the pulmonary changes including parenchymal and peribronchiolar fibrosis in the specimens of 17 cases.

Only fibers longer than 1 p were counted.

Pathological information was obtained by review of the patients' protocals.

36/12) : 1986 H. KOBAYASHI st ol.

The distribution of AB counts for the 656 autopsy cases and one case from surgical resection is shown in Fig. 1. Two patients had lung parenchymal asbestos counts of more than 104/g ; they were employed in an occupation with definite exposure to asbestos. Eight patients had asbestos counts ranging from lo3 to lo4 AB/g ; they were all male and all blue-collar workers except two patients with unknown employmental history as shown in Tablesl, 2. Nine patients had asbestos counts ranging from 500 to lo3 AB/ g ; only one of them was female. Thirty five patients had asbestos counts ranging from 100 to 500AB/g. The remaining 92% of au-

400

P 300- d O

d 8 200-

*

1783

- Asbestos Body in Autopsy Lung -

r,

n n <10 10-49 50-99 100-499 500-999 10%

Table 1. Asbestos Bodies in Males and Females

Asbestos <10 49-10 99-50 499-100 999-500 103s bodies

M : F 283: 160 81:31 42:6 29:6 8 : 1 9 : o M/F 1.77 2.6 7.0 4.8 8.0 -

Table2. Asbestos Bodies and h p a t b n s

<10 49-10 99-50 499-100 999-500 103s Asbestos bodies

Blue-collar 24 11 4 6 0 7 White-collar 77 23 12 4 1 0 Farmer 38 9 1 3 2 0 Other 46 11 5 4 5 0 Unknown 98 27 20 12 3 2

Blue-collar workers included manual laborers, workers in construction and automo- bile factories, steel mills, dockyards, and similar jobs. White-collar included public servants, professional, and clerical workers. Others means butchers, fishmongers, greengrocers, fishers, cooks, salesmen, and businessmen. Unknown includes the peoples without known occupational history and housewives.

1784 PATHOLOGICAL EFFECTS OF ASBESTOS EXPOSURE Acta Pathol. Jpn.

Table 3. The Number and Types of Asbestos Fiber in the Selected Cases

Case AB ( x 103/g) AF ( x 103/g) AF/AB AF type

Resected 1,700 172,000 100 A 71 (79) 30 93,700 3,100 A

3 (79) 10 (80) 76 (80) 10 (78) 56 (78)

141 (78) 58 (79)

154 (77)

4.1 4.9 5.0 1.5 3.6 8.1 2.6 3.4

1,560 620

64 940 282

1,870 N.D 189

380 130 13

630 78

230 -

56 ~~~

148 (78) 0.7 52 70 A, c 47 (81) 0.8 58 73 A 35 (78) 0.8 91 110 A, c

1 (78) 0.8 34 42 A 112 (77) 0.7 2,590 3,700 A 128 (78) 0.5 178 360 A 233 (81) 0.6 43 71 A, c

AB : asbestos body, AF : asbestos fiber, A : amphibole, C : chrysotile.

2. A F counts in selected cases.

The number of AB/g, AF/g, AF/AB and fiber variety in each case is listed in Table 3. The number of AB/g roughly correlated with that of AF/g. However, the ratio of AFs to ABs varied widely, that is, from 3,700 to 13. The amphiboles were identified in all examined cases (Fig. 2). These amphiboles were almost always crocidolite (Fig. 3) and/or amosite (Fig. 4) except Case 76(80) in which calcium amphiboles were the major fiber types. On the other hand, chrysotile was only occasionally identified. In Case 58( 79) who had silicosis, the exact AF counting could not be done because of the contamination of numerous silicotic particles.

3. AB counts in tissue sections and pathological data.

AB counts in tissue sections, occupational and pathological data are given in Table 4. In each tissue section more than one or two ABs were usually found in the patients whose number of AB exceeded a thousandlg dry weight. Meanwhile AB was some- times present in one or two of several sections whose number of AB in extracted lung was less than a thousand. Pulmonary carcinomas had developed in three of eight patients whose AB counts ranged from lo3 to lo4 AB per gram dry weight. One of these eight patients had diffuse parenchymal fibrosis. And three patients showed

36/12) : 1986 H. KOBAYASHI rt crl. 1785

Fig. 2. Electron micrograph illustrating amphibole fibers. They are straight and have light

Fig. 3. X-ray energy spectra of crocidolite had distinct peaks for silicon and iron, and low peaks

Fig. 4. X-ray energy spectra of amosite had no peak for sodium.

and dark bendings that appear to cross the fiber axis at right angles.

for magnesium and sodium.

x 11,500.

1786 PATHOLOGICAL EFFECTS OF ASBESTOS EXPOSURE Acta Pa th1 . Jpn . Table4. Occupational and Pathlogzcal Data and Asbestos

Body Counts in Tissue Sections

Pathological diagnosis Case Occupation Pulmonary

findings AB count in tissue section

Resected

71 (79)

3 (79)

10 (80)

76 (80) 10 (78) 56 (78)

141 (78) 58 (79) 154 (77)

148 (78)

47 (81) 35 (78)

1 (78)

112 (77)

128 (78) 233 (81)

AF spraying

AF spraying

construction worker

worker unknown welder steel mill worker plumber miner unknown

farmer

unknown .house wife

farmer

acupuncturist

unknown office-man

factory

Asbestosis, pulmonary ca. Asbestosis, pulmonary ca. Pulmonary ca.

Pulmonary ca.

Agranulocytosis Gastric ca. Pulmonary fibrosis and ca. Gastric ca. Silicosis Renal ca., Reticulosis Verrucous endocarditis Uhl’s anomaly Ovarian ca.

Myocardial infarction Esophageal ca.

Esophageal ca. Hepatoma, liver cirrhosis

Diffuse fibrosis

Diffuse fibrosis

Mild fibrosis

Mild to moderate fibrosis Pulmonary abscess Mild fibrosis Diffuse fibrosis

( -1 Diffuse (silicosis)

( - 1

Bronchopneumonia

Mild perichronchiolar fibrosis Pulmonary infarctions Radiation pneumonitis Bronchopneumonia

( -1

too much to count 7891 8

91 4

441 7

16/12

23/ 8

la/ 5

11/ 5

6/ 4 31 5

2/ 7

Q/ 5 6/ 4

Q/ 4

1/ 4

o/ 5 11 7

slight to moderate fibrosis in the areas including terminal, respiratory bronchioles, and alveolar ducts (Figs. 5, 6).

Discussion There have been many reports on the pulmonary incidence of ABs in the general

p 0 p ~ 1 a t i o n . ~ J ~ J ~ ~ ~ ~ J ~ ~ ~ ~ * ~ ~ The incidence in our study was lower than those in these reports. The main reason for this may be that we examined a lesser amount of tissues (0.5 g) than that in other studies where most of them actually took 5 g of tissues. In reality, the incidence will probably increase if more amount of tissues is investigated. The difference of the incidence may also reflect difference in method or virtual difference in subject population. However, our primary aim of AB counting in the general population was to select the cases with modest or secondary occupational

36(12) : 1986 H. KOBAYASHI et al. 1787

Fig. 5. Slight fibrosis of the terminal air passages. H.E., x 12.5. Fig. 6. Enlargement of boxed mew in Fig. 5 (6a, 6b) shows asbestos body. x 125.

exposure. Since our digestion method is operated in a single centrifuge tube through extraction processes, there is little chance to miss ABs. And, when we try to compare the number of ABs with that of AFs, the same digestion procedure should be applied to both ABs and AFs counting. WARNOCK et stated that most AFs remained at the alcohol-chloroform interface even after centrifugation, and our experience indicab ed that the number of ABs were reduced through successive centrifugations. From

1788 PATHOLOGICAL EFFECTS OF ASBESTOS EXPOSURE Acta Pathol. Jpn.

these accounts, i t may be better not to add chloroform and ethanol to the centrifuge tube for AF counting and only one centrifugation may be best. Moreover, all processes in KOH digestion can be done within a few hours and is convenient to deal with many cases.

The patients whose number of ABs ranged from lo3 to 10' were all blue-collar workers except the two patients, that is, a steel worker, a construction worker, a plumber, a welder, a factory worker, and a miner. CHURG et aL5 indicated that values greater than 100occur most frequently in occupational groups known to handle asbestos-containing products. This value approximately correlates with 1,000 per gram of dried lung because the percentage of dry weight per wet weight is about 10 percent. Furthermore, Fig. 1 showed that the patients' number rose up in the blue- collar group in the range from lo3 to lo4, although i t decreased in the other groups as the number of ABs increased. In addition the construction work may be the origin of asbestos exposure because of the wide spread of use of asbestos as construction materials. The plumbing may also run a high risk of exposure for cutting asbestos- cement pipe. The exact source of exposure in steel milling and welding was not obvious. However, CHURG et aL5 demonstrated the association of high counts and work in the steel mills and stated that large asbestos coated walls and benches were present in the parts of the mill where hot metals were handled. The miner's subject had worked in coal-mining industry and later engaged in tunnelling. Asbestos minerals, especially the amphiboles are major participants in rock formation. The asbestiform amphiboles are probably present with a variety of amount in the earth's

Thus, i t may be not too difficult to assume that he inhaled asbestos fibers with other silicate minerals. Taken together, the most probable sources of many ABs in these subjects are their occupational environments.

ASCHCROFT and HEPPLESTON' examined fiber of the lungs of 35 patients with varying degrees of asbestosis and found that there was a progressive increase in concentration of asbestos fibers both coated and uncoated fibers, with increasing severity of fibrosis. WHITWELL et al.23 also examined the fiber contents of the lungs from 100 patients of pleural mesothelioma who had asbestosis and 100 control subjects (those without industrial disease or lung cancer). Seventy three of these mesoth- elioma patients contained over 100,000 asbestos fibers per gram of dried lung, but there was less than 20,000 fibers per gram of dried lung in 71% of control specimens. Our results revealed that the number of AFs roughly correlated with that of ABs. However, the ratio of AFs to ABs ranged from 13 to 3,700. DODSON et aL9 also found the ratio in amosite workers ranged from 14 to 31,000. Hence, these results suggested that the asbestos bodies were an indicator of asbestos exposure but did not always show the exact degree of exposures in individual cases.

The fiber types in our selected subjects were almost always amphiboles (crocidolite, amosite) although some chrysotiles were identified in a few patients with AB counts ranging from 500 to 999. CHURG' revealed that the total number of fibers in the lungs with pleural plaques was about the same as that in their control popula-

36/12) : 1986 H. KOBAYASHI et al. 1789

tion but the commercial amphiboles (crocidolite, amosite) were increased almost 50-fold in the former group4 These data probably suggest the moderate amount of crocidolite and amosite indicated the occupational exposure of asbestos. We could not identify chrysotile fibers in the majority of subjects, whereas CHURG et a1.6 found chrysotile fibers as the vast bulk of background asbestos burden. This difference may reflect the difference in methods, especially the pore sizes of filters. Chrysotile is thinner than amphiboles and likely to be fragmented in the lung. So, more chrysotiles may be collected on the filter with smaller pore size.

Although we know little about the initial stage of asbestosis in man, experimental models of the disease have demonstrated the fibrosis in the terminal, respiratory bronchioles, and alveolar ducts by asbestos e x p o ~ u r e . ~ . ~ ~ Of human materials, WRIGHT and CHURG found a distinctive lesion of marked fibrosis in the walls of respiratory bronchioles and alveolar ducts in some asbestos Our eight subjects whose asbestos counts ranged from lo3 to 9,999 per dry weight had occupational exposure as discussed earlier. Tissue sections of these three patients definitely revealed slight to moderate fibrosis in the terminal and respiratory bronchioles and that of one patient showed severe fibrosis. In addition, our data showed the constant presence of at least one or more asbestos bodies in a tissue section. ROGGLI et al. suggested an average of two asbestos bodies in tissue sections w&s equivalent to approximately 200 bodies per gram of wet fixed lung tissue.15 Our present data was quite similar to their result because the lo3 ABs per gram of dry weight were approximately lo2 per gram of wet fixed lung. From these accounts, we believed that the fibroses in our patients were probably due to asbestos exposure. And it seems reasonable that the minimum criteria for the diagnosis of asbestosis are the demonstration of discrete foci of fibrosis in the walls of respiratory bronchioles with at least two asbestos bodies in tissue sections.8

Lung carcinomas have been linked to exposure to absestos. Our two patients with more than lo4 ABs had pulmonary carcinoma and diffuse parenchymal fibrosis and four of eight patients with ranging from lo3 to lo4 had peribronchiolar and parenchymal fibroses with accumulation of ABs in tissue sections and three of these subjects had developed pulmonary carcinomas. These results may indicate that modest exposure of asbestos with slight interstitial fibrosis also cause pulmonary carcinomas as well as heavy exposure. However, these subjects were few and the difference of the incidence of carcinoma was not statistically significant between these subjects and age-sex matched control.

Finally, asbestos bodies may be difficult to find or may be absent if there are fewer than 100 per gram of wet tissue as quantiated by digestion and centrifugation. And we may sometimes overlook ABs in usual lung specimens if they are few. Thus, we believe that we should intensively try to seek ABs by the simple, convenient, and rapid technique for concentrating ABs as well as in tissue sections. These efforts will elucidate the disease processes of asbestosis and carcinogenesis relating to asbestos exposure.

1790 PATHOLOGICAL EFFECTS OF ASBESTOS EXPOSURE Acta Pathol. Jpn.

Acknowledgment : We thank Dr. Junji AKAI (Faculty of Science, Niigata University) and Miss Eiko NAKAZAWA (Naka Works, Hitachi, Ltd.) for their valuable contribution in determining asbestos fibers. In addition, we thank Mr. Tomiyoshi HASECAWA, Mr. Kazumasa SATO, Mr. Susumu MOMOZAKI, and Mrs. Michiko TANABE for their skillful technical essistances.

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