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Asbestos bodies in lung parenchyma in relation to ingestion and inhalation of mineral fibers
ENVIRONMENTAL
RESEARCH
14, 286-304 (1977)
Asbestos Bodies in Lung Parenchyma in Relation Ingestion and Inhalation of Mineral Fibers’ OSCAR AUERBACH,” E. CUYLER HAMMOND,? IRVING J. SELIKOFF,$ VERTA R. PARKS,~ HAVEN D. KASLOW,” AND LAWRENCE GARFINKEL * VA Hospital, East Orange. New Jersey and Professor of Pathology, Medicine and Dentistry of New, Jersey. Ne+cs Jersey Medical School, Jersey. t Departmetrt of Epidemiology and Statistics. American Cancer New, York. $ Environmental Sciences L&oratory, Mt. Sinai Hospital, York, 0 Senior Medical Investigator Laboratory. VA Hospital. East Jersey,” Department of Epidemiology and Statistics, American Cancer New York, ’ Department of Epidemiology and Statistics. American Cancer Society. Inc., Nelt, York
to
#
College of Neu,ark, New Society, Inc., New York, Neti. Orange, Neuj Society, Inc.,
Received April 21. 1977
This is one of several studies initiated by various investigators under unusual circumstances which, in the eyes of a large number of people, constituted an emergency. In 1973, the people of Duluth, Minnesota were informed by the news media that their drinking water was, and for several years had been. heavily contaminated with asbestos fibers. It was well known that workers with occupational exposure to asbestos dust are subject to a greatly increased risk of lung cancer, pleural mesothelioma, peritoneal mesothelioma, and to a lesser degree. cancer of the gastrointestinal tract (l-3). Thus, there was reason to fear that ingestion of asbestos fibers might lead to an increased risk of cancer of the sites of direct contact (i.e., tissues of the gastrointestinal tract) and perhaps to cancer of other sites as well. A perusal of official mortality statistics indicated that, up to that date, death rates from cancer of the sites and types named above had not been unusually high in Duluth. However, this was not reassuring since, even under conditions of heavy occupational exposure to asbestos dust, the carcinogenic effects do not usually become discernible until about 20 years or longer after onset of exposure. Since direct epidemiological evidence, one way or the other, would not become available until many years in the future, there was an urgent need to obtain as much indirect evidence as possible even though such evidence probably would not be conclusive. Among the issues were [l] whether asbestos fibers ingested with Duluth drinking water penetrated and became lodged in tissues lining the gastrointestinal tract and [2] whether the ingested fibers entered the bloodstream and were thereby carried to lung, pleural, and peritoneal tissues. Because of technical problems, to be described later, we were unable at the time to investigate either one of these Supported in part by a Center Grant. “ES00928” of the National Institute of Environmental Health Sciences of the U.S. Department of Health, Education and Welfare to the Mt. Sinai School of Medicine of the City University of New York. 286 CopyrIght 0 1977 hy Academx Press. Inc All rughts of reproduction in any form reserved.
ASBESTOS
BODIES
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PARENCHYMA
287
two questions. Instead, by force of circumstances, we took an indirect approach to the latter question. In so doing, we realized that this approach would provide only limited information. When asbestos fibers are inhaled, many of them are, by biological processes, coated with a material containing iron. These coated fibers have a unique appearance under the microscope and are usually called “asbestos bodies” (see Fig. 1). Since some other types of mineral fibers can be similarly coated, some authors prefer the more general term “ferruginous bodies” (4). Whether or not very small asbestos fibers become coated, only those which become 5 pm or larger could be recognized as asbestos bodies at the light microscopic level. In the coating process fibers less than 5 pm in length could become bodies large enough to become identifiable by light microscopy. We reasoned [I] if large numbers of asbestos fibers were taken into the bloodstream (as a result of drinking the contaminated Duluth water) some of them would probably lodge in the lungs and become coated (56): as a result, [2] more asbestos bodies would be found in lung tissue of Duluth residents who died after the water had become contaminated: and [3] more asbestos bodies would be found in lung tissue of Duluth residents who died in recent years than in lung tissue of residents of a city in which the drinking water was not heavily contaminated with asbestos fibers. The present study was based upon the above hypotheses. Strongly positive findings would constitute suggestive evidence that the contamination of Duluth drinking water might lead to increased death rates from cancer of the sites named above in future years. Negative findings would not rule out the possibility that asbestos fibers of submicroscopic size entered the bloodstream (from the contaminated water), and so would not rule out the possibility of increased risk of cancer. (The asbestos fibers contained in Duluth drinking water were typically very small as compared with many of the fibers inhaled by occupationally exposed workers, although they, too, also inhale a great number of very small fibers.) BACKGROUND
In 1955 a taconite ore processing plant was established at Silver Bay, Minnesota on the shore of Lake Superior about 55 miles from Duluth, Minn. The process consists essentially of crushing the ore to a very fine powder. suspending it in water, and magnetically extracting the iron particles. The remaining materials are discharged into the lake. Since September of 1960 the volume of lake water permitted for this use has been sufficient to carry 67,000 or more tons of tailings into Lake Superior per day (7). With the passage of time, such of the pulverized material as remained suspended in water spread through the lake (8). We do not know how long it took for an appreciable concentration of particles to reach Duluth. Since the waters of Lake Superior had long been noted for their purity, the water system of Duluth drew its supply directly from the lake and distributed it without filtration throughout the city. In August of 1973, samples of water drawn from city taps, contained about 14 x 10fi amphibole fibers per liter of water (9). In the spring of 1975. following unusual weather conditions, some of the samples of
288
AUERBACH
FIG.
1. Montage
of asbestos
bodies
of various
ET AL.
shapes.
Arrow:
Clump
of asbestos
bodies.
ASBESTOS
BODIES
IN
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PARENCHYMA
289
tap water contained as many as 300 to 600 x IO6 fibers per liter (9). Most of the fibers were of such small size that they could not be seen by light microscopy, that is, 5 pm or less. However, a small percentage were 5 pm long or longer and when the enormous number present in the water is considered, even this small percentage would result in the ingestion of a significant number of fibers that would be visible by light microscopy. In 1972 the Environmental Protection Agency (EPA) brought suit in Federal District Court to prohibit further contamination of the lake: but initially the possibility of a health hazard was not an issue. The situation changed when, in 1973, it became known that many of the contaminating particles were asbestos fibers. Mineralogically they are of the cummingtonite-grunerite series ( 10). The trial in Federal Court was scheduled to commence August 1, 1973. HOWever, shortly before that time, the EPA called upon us (I.J.S., E.C.H., and associates) to investigate the possible health effects especially with respect to cancer of the types associated with occupational exposure to asbestos dust and to serve as expert witnesses in the legal case. Shortly thereafter the Court ordered us to initiate as quickly as possible any such studies as we thought might help throw light on the matter. The scope of studies feasible at that date was limited by time constraints and various other practical considerations. Duluth was included in a long-term prospective epidemiological study in progress since 1959 (11); and we felt that this would eventually provide a definitive answer. Members of our group (Drs. Arthur M. Langer, William J. Nicholson, Arthur N. Rohl, and colleagues) immediately began a series of mineralogical studies. The problem was to design and carry out a pertinent short-term biological investigation. St. Mary’s Hospital (Sister Marybelle Leick, administrator and Drs. A. C. Aufderheide and J. P. Knoedler, pathologists) offered us splendid cooperation as did St. Luke’s Hospital (Dr. Volker Goldschmidt, pathologist) at a somewhat later date. Our first thought was to search for asbestos fibers (by electron microscope and ancillary means) in specimens of colon-rectum and lung tissue taken routinely at autopsy from residents of Duluth during the preceding year and similar specimens taken at autopsy prior to the opening of the taconite processing plant and at several intermediate periods. This plan had to be abandoned when we found that the formalin used to fix the specimens had been diluted with city tap water. Electron microscopic examination of the diluted formalin then in use in the hospital showed that it contained very large numbers of asbestos fibers. Thus, if asbestos fibers had been found in the preserved tissues of patients who had died during the past few years, there would have been no way of knowing whether they had been present before the death of the patient or whether they came from the asbestos-contaminated formalin. For the above reason, we decided instead to search, by light microscopy, for asbestos bodies in specimens of lung tissue. If found, they could not have come from the contaminated formalin. (There was none in the contaminated formalin; and they cannot be formed in lung tissue after the death of the patient.) Although this plan had the limitation that the procedure could not reveal the presence of sublight microscopic bodies, the presence of bodies 5 pm and longer would be established.
290
AUEKBACH
ETAL
MATERIAL
The plan was to examine specimens of lung tissue taken routinely at autopsy (and preserved in paraffin blocks) from female residents of Duluth who died during four periods of time (1972-1973, 1966-1967, 1960-1961, and 1953-1955), who had resided in Duluth for at least 15 years prior to death, who had used city water during that period, who had never been occupationally exposed to asbestos dust, and who had not lived within 1 mile of a shipyard, ore-loading dock, or steel mill. (Women were selected because they are less likely than men to be either occupationally exposed to asbestos dust or to be exposed while making home repairs.) St. Mary’s Hospital provided us with a list of all women who had come to autopsy in that hospital during the specified years, from whom lung tissue specimens were available, and who were residents of Duluth at the time of death. By telephone interviews with relatives and friends of the deceased, through the cooperation of the Duluth Public Library, and by a search of city directories and newspapers from prior years, we then ascertained which of them met the other specifications outlined above. In case of doubt, usually resulting from inability to find a relative or friend able to supply the pertinent information, the woman was excluded. The Duluth City Planning Department located and pinpointed the residences of the prospective subjects with relation to shipyards, docks, etc. The Duluth Water and Gas Department provided exact information on the water supply to residences of the prospective subjects. It turned out that, after this screening, the number of suitable subjects available from St. Mary’s Hospital was insufficiently large for a statistically valid study. For this reason, we obtained additional material in the same way from St. Luke’s Hospital. Staff members of both hospitals provided us with invaluable assistance. As shown in Table 1, the total sample contained 282 Duluth women in four groups as follows: Group A, who died 1972-1973, 96 women of whom 48 were from St. Mary’s and 48 from St. Luke’s; Group B, 19661967.66 women, 48 from St. Mary’s and 18 from St. Luke’s; Group C, 1960-1961, 54 women, 36 from St. Mary’s and 18 from St. Luke’s: Group D, 1953-1955. 66 women, 36 from St. Mary’s and 30 from St. Luke’s. We had hoped to have the same number of subjects (66) in Groups B, C, and D; but were able to find only 54 for Group C, years 1960-1961. A fifth group of subjects (Group E, New York City women) was included for comparison with Group A. Group E. We had available specimens of lung tissue from all adults who came to autopsy in two New York City Hospitals (Mt. Sinai and Elmhurst) during the years 1966-1968. For most of these we also had available information provided by relatives and friends on residence history, occupation, occupational exposures, and possible household exposures. From this material, we selected a sample of 96 women matched on age with the 96 Duluth women of Group A. Thus Groups A and E together consisted of 96 matched pairs of women. The New York sample was confined to women recorded as being housewives or white collar workers with no known occupational exposure to asbestos ‘dust. Other groups. Four groups of men were included for the following reasons, partly, but not entirely, related to the main purpose of the study: Groups F and G
ASBESTOS
BODIES
IN
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TABLE SUBJECTS
291
PARENCHYMA
I
INCLUDED
IN STUDY
Age at death SO+
Mean we
14
21
35
26
72.5
14 20 8 18
21 13 16 21
35 20 19 17
26 13 11 10
72.6 66.8 68.9 64.8
8 14
5 7
1 5
2 2
-
56.8 60.4
19 19 438
2 2 90
6 6 110
IO 10 150
1 1 88
69.8 68.8 69.1
matched
on age.
1966-1968
96
1972- 1973 1%6- 1%7 1960-1961 1953-1955
96 66 54 66
E
N.Y.C.
women
A B C D
Duluth Duluth Duluth Duluth
women women women women
F G
Paterson Insulation
H I
Paterson “neighborhood” N.Y.C. men Total Groups
70-79
160
Group
Note:
60-69
Total number
asbestos factory workers men
A and E women
men men
were
matched
on age and Groups
H and I men were
consisted of men who had been occupationally exposed to asbestos dust. The main purpose was to obtain an estimate of the number of asbestos bodies associated with occupational exposure in comparison with the number to be found in Duluth and New York City women without such exposure. Group F was of special interest because these men died years after having been heavily exposed for relatively short periods of time. Group H was included to see whether residence in a neighborhood with elevated levels of asbestos dust in the air was associated with an increased number of asbestos bodies in lung tissue. Group I was included for comparison with Group H. Group F. An amosite (grunerite) asbestos insulation factory was in operation in Paterson, New Jersey during years 1941-1954. According to accounts of men who had worked there, they were exposed to “clouds” of dust. Unfortunately, no counts were made of the number of asbestos fibers in the air. For this study, lung tissue (taken at autopsy) was obtained from 8 men who had worked in the factory for from 2 months to 2 years. Group G. This group consisted of 14 members of the insulation workers union. They had been occupationally exposed to asbestos dust (chrysotile and amosite) for 25 to 52 years prior to death. Group H. Lung tissue, taken at autopsy, was obtained from 19 men who had lived within a radius of ‘/2 mile from the amosite asbestos factory mentioned above during the time that the factory was in operation. None of these men had worked in the factory and none was a member of the insulation workers union. According to newspaper accounts written while the plant was in operation, people living in this neighborhood had complained of dust from the factory; asbestos dust can still be found in attics and other undusted places in houses still standing there. Group 1. Nineteen men who died and came to autopsy at Mount Sinai and Elmhurst Hospitals (New York City) during 1966-1968 were matched on age with
292
AUERBACH
ET AL.
the 19 men of Group H. None was recorded as having been occupationally exposed to asbestos dust. However, according to members of their families, some of these men had probably been exposed to asbestos dust while making home repairs and improvements. STATISTICAL DESIGN As described above, there were 96 Group A subjects, 66 Group B subjects, 54 Group C subjects, and 66 Group D subjects. By random numbers, these 282 subjects were assigned to 6 sets (sets # 1 through #6) such that there were 16 A, 11 B, 9 C. and 11 D subjects in each set. By random numbers, 1 of the 8 Group F subjects, 2 of the 14 Group G subjects, and 3 of the 19 Group H subjects were assigned to each of the 6 sets. Each of the 96 Group E subjects was then assigned to the set containing the Group A woman with whom she had been matched on age; and each of 18 of the Group I men was assigned to the set containing a Group H man with whom he had been matched on age. There then remained 6 male subjects (2 of Group F, 2 of Group G, 1 of Group H. and 1 of Group I). By random numbers, 1 of these 6 men was assigned to each of the 6 sets. Thus there was a total of 73 subjects in each set. By random numbers, the subjects within each set were put in random order. While in this order, a serial number was placed on each record such that serial number 1 was given to the first subject of set # 1 and serial number 438 was given to the last subject of set #6. As will be described, one slide containing ashed lung tissue was prepared for each subject. This was done in order according to serial number; and each slide was labeled only with the serial number. The slides were later examined microscopically in the same order. The serial number gave the examiner no clue as to the identity of the subject. DESIGN
OF GRID SLIDES
Since asbestos bodies are difficult to see when embedded in lung tissue, our procedure is to mount a section of tissue on a glass slide, ash it in activated oxygen, put on a drop or two of mounting medium, and then set a cover slip in place. In past studies we had used ordinary microscope slides and cover slips. The following difficulties were encountered. (1) Often, after ashing, so little material (visible to the naked eye) remains that it is difficult to place the cover slip in the correct position. (2) If the specimen (or a portion of it) contains very little noncombustible material, it is difficult to focus the microscope on the proper plane; and if out of focus, such particles as are present may not be seen. (3) Without guidelines, it is difficult to scan microscopically the entire area under the cover slip without skipping some areas. (4) While the location of objects may be recorded for later review by reading the coordinates from the mechanical stage, this is time consuming. It also has the disadvantage that in order to find the object again by the recorded coordinates, the same microscope must be used. To overcome these difficulties one of us (E.C.H.) designed a new type of slide as described below (see Figs. 3 and 4). A square grid as shown in Fig. 3, is “printed” on a 25 x 75 mm glass slide by
FIG.
2. Cluster
of asbestos
bodies
photograpl
293
hed at three
different
focal
planes
294
AKJERBACH
ET AL
c
3
II I c D c ,
I) . e D c C L
3
FIG. 3. 18 x 18 mm grid printed on a 25 x 75 mm microscope The interior lines are 100 pm in width.
slide by the chrome
-sputter
process.
the chrome-sputter process. The extremely thin layer of chromium withstands the ashing procedure. The grids are divided into 18 rectangular areas by lines. The heavy outer boundary lines are 400 pm in width and the inner lines are 100 pm in width. The entire square measures 18 x 18 mm between the centers of the outer boundary lines. (The overall dimensions are 18.4 x 18.4 mm.) To help in locating an area, the numbers 1, 2, and 3 are printed in visible size at the top and bottom of the square and the letters A, B, C, D, E and F are printed at each side. Coordinates, invisible to the naked eye but visible under the microscope are printed within the grid lines as illustrated in Fig. 4. The horizontal coordinates are identified as Al, A2, A3, A4, A5: Bl, B2 . . . through F3, F4, F5. Under the microscope, it is easy to record the location of an object to within about oneor two-tenths of the distance between the printed coordinates. For example, the dot shown in Fig. 4 would be recorded as at location Dl .O, 28.7. This is sufficiently accurate for finding an object a second time. The grid lines and coordinates afford
FIG. 4. Magnification of a portion object located at coordinates D1.O.
of the interior 28.7.
grid lines showing
coordinates.
Arrow
points
to an
ASBESTOS
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PARENCHYMA
295
a means of accurately focusing the microscope at a plane just above the surface of the slide. Dr. Thomas S. Ely of Eastman Kodak suggested the use of the chromesputter process after we had tried ordinary photographic methods and found them to be unsatisfactory. The slides were manufactured for us by Qualitron. Inc. of Danbury, Connecticut to whom we are indebted for assistance in details of the design. PREPARATION
OF SPECIMENS
As previously described, the specimens of lung tissue had been taken routinely at autopsy and embedded in paraffin blocks at various points in time between 1953 and 1973. They varied considerably in shape and size, some being very small and others being fairly large. Furthermore, some of the blocks contained tissue specimens from more than one organ. In such an instance, it was necessary to cut and stain a thin section to determine which specimen was lung tissue. The lung tissue specimen having been identified, it was removed from the original block and reembedded in paraffin. For quantitative estimates, it is essential that each tissue specimen examined be of the same volume as nearly as possible. We decided to use 20 mm3 of paraffinembedded tissue as the standard. This was accomplished as follows. Either a microscopic section taken from the block or the surface of the block itself was projected on paper at a fixed setting of the magnifier. The outline of the tissue was traced and the area (making adjustment for magnification) was determined by means of a planimeter. From this was calculated the depth to which the embedded tissue had to be cut in order to obtain a volume of 20 mm3. In practice, tissue areas varied from 56 to 444 mm’. Therefore the corresponding required depth of cut ranged from 357 to 45 pm. A sliding (rather than rotary) microtome was found to be most accurate for cutting sections of specified thickness. The microtome feed mechanism was calibrated gravimetrically using tissuemat paraffin and was found to be accurate within 1% at settings from 20 to 160 pm. It was found to be unwise to make cuts thicker than 40 pm each because of apparently hindered paraffin extraction and retarded low-temperature oxidation. Therefore, when a total depth of over 40 pm was required, multiple sections were cut such that together they totaled the required depth. Excess paraffin around the edges of the tissue specimen was removed. The sections were then placed within the grid square of a slide as evenly as possible but no closer than 2 or 3 mm from the outer boundary lines. Then the slide was placed on a hot plate to melt down and fix the sections. After cooling, the paraffin was extracted by several drops of xylene, the process being repeated three times. The slide was then heated briefly to drive off the xylene. Slides, prepared as above, were placed, one each, in the five chambers of a low-temperature asher. Within 2 to 3 minutes after starting the pump the vacuum is sufficient (below 0.1 mm Hg) to strike the glow discharge and admit the oxidant. The burn is timed from that point. As oxidation proceeds, the color of the glow discharge changes; and apparently at or near the end of active oxidation it becomes very dull and dark red. Most of the samples reached this point in about 15 to 20 minutes but sometimes longer. After 45 minutes, instrument shut down is
296
AUERBACH
ET AL.
started, another 50 to 60 minutes then being required to repressurize the oxidation chambers. After noting the location of the greatest concentration of ash on the grid, a small drop of Namount from a capillary tube is applied on this spot. An 18 x 18 mm cover slip is then placed over the grid. All of the slides were prepared in this way by one of us (H.D.K.), this being done in order by serial number as previously described, Technical difficulties were encountered from time to time, sometimes resulting in slides of very poor quality. The two principal difficulties were as follows. Some tissue sections contained a relatively large amount of material which withstood ashing; and this material may have been very unevenly distributed on the grid. After ashing, some of the grids were coated with a more or less evenly distributed layer of a yellow or yellow-brown material. In extreme cases, the coating was so dense as to make microscopic examination difficult if not impossible. Each slide, immediately after preparation, was examined by H.D.K. for defects as described above. If the quality was very poor, he prepared a new slide by repeating the entire process. In some instances, the first reader, after a brief preliminary look under the microscope, decided that the quality was too poor for valid readings. In such cases, another slide was made. EXAMINATION
OF SLIDES
The entire area of each grid was examined microscopically by one of us (V.R.P.) under phase contrast using a 25x Neofluor objective (N.A. 0.60) and 12.5x wide-angle eyepieces. Every object found at this magnification which gave the slightest suggestion of possibly being an asbestos body was studied closely at higher magnification (500x) under both phase contrast and ordinary light. Figure 1 is a montage showing asbestos bodies of various different shapes. The same general shapes shown here can be found in objects varying considerably in overall size. In slides in which less than about a dozen asbestos bodies were found, they were almost always scattered, usually just one, and seldom more than two being observed within the same microscopic field of view. In slides containing a larger number, clumps of asbestos bodies were often found within the same high-power microscopic field. (Such a clump is shown by the arrow in Fig. 1). At any one focal plane, it was often difficult to count the number of coated fibers present; but by focusing up and down the actual number could usually be ascertained. However, in some instances, what appeared to be fragments were observed; and it was virtually impossible to ascertain whether two or more fragments had originally come from one or more than one coated fiber. Some slides contained a very large number of asbestos bodies. In such slides there were many separate microscopic fields each of which contained one or more clumps containing many asbestos bodies, Figure 2 shows one such clump photographed at three different focal planes. No better than a crude estimate could be made of the number of coated fibers within such a clump. In many instances it was difficult to decide whether an object should be classified as an asbestos body. For example, the object might be in a field containing so much material which could not be ashed as to make visualization difficult; or
ASBESTOS
BODIES
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PARENCHYMA
297
the apparent shape of the object might be such as to leave at least some doubt as to whether or not it was truly a coated fiber. For this reason, the following types of objects were recorded: (I) “definite” asbestos body, (2) “probable“ asbestos body, (3) “possible” asbestos body, and (4) “unusual object” which might be a coated fiber of some sort but which did not have the appearance of an asbestos body. Notes were also made as to the quality of the slide (from the standpoint of visualization) and whether or not a large number of uncoated fibers or nonfibrous particles were present. “Definite” meant an object which, in the opinion of V.R.P., exhibited all of the characteristics of one or another of the various types of asbestos bodies. “Probable“ indicated some uncertainty. “Possible” was used simply to call an object (which could possibly be an asbestos body) to the attention of the later reviewer. A pencil sketch of each object was drawn and its coordinates were recorded. The time required to examine a single grid in this way averaged about 4 hours. However, up to 8 hours was required in some instances, as for example, grids with a large amount of unashed material but without a very large number of asbestos bodies. In some instances it was quickly apparent that the specimen contained hundreds of asbestos bodies. Typically in such instances, such a large quantity of other unashed material was present (including fibers, granules, and what appeared to be broken fragments of asbestos bodies) that an accurate count could not be made. To expedite the process, such slides were initially recorded as “T.N.C.” (“too numerous to count”). At a later date these slides were examined again to obtain a rough estimate of the number. In Tables 2-4, the estimated number of asbestos bodies in such cases are indicated as >200 or >999. After a slide has been examined in this way by V.R.P., every object which she had recorded as a “definite,” “probable,” or “possible” asbestos body or an “unusual object” was reexamined by O.A. Disagreement in classification was extremely rare. RESULTS-DULUTH
AND NEW YORK WOMEN
Table 2 shows the percentage distribution of Groups E, A, B, C, and D subjects by the number of “definite” asbestos bodies found in the ashed specimens of lung tissue. No definite asbestos bodies were found in ashed sections from 33.3% of the Group A women (Duluth 1972-1973). This percentage with no definite asbestos bodies was greater for Group A than for any of the other four groups: (New York women, 24.0%; Duluth women 1966-1967, 30.3%: Duluth women 1960-1961, 24.1%; and Duluth women 1953-1955, 25.8%). Likewise, the percent of women with two or more definite asbestos bodies found was less for Group A women (Duluth 1972-1973) than for women in any of the other four groups, Less than 9% of the ashed sections from any of the five groups (E, A, B, C, and D) had over 24 definite asbestos bodies; but in all groups except Group B, some sections contained 25 to 99. one in Group C contained 186 definite asbestos bodies and one in Group D contained more than 200 asbestos bodies. With such a wide range of values and with such skewed distribution of values, the mean numbers of asbestos bodies are not very illuminating. Although the mean number found in the
298
AUERBACH
ET AL
TABLE PEKCEI\‘T
DIS~RIBUWON
ASBESTOS
BODIES
Group City Year of death Number of asbestos bodies 0 I 2-4 5-9 10-24 25-99 IOO- 186 >200 Total Number of women Mean no. asbestos bodies
OF FE~MALE
FO~IND IN E N.Y.C. 1966-68
-
2
SUBJECTS
ASHED
BY NUMBER
SPECIMENS
A Duluth 1972-73
OF
B Duluth 1966-67
cl0
5%
9%
24.0 9.4 18.8 20.8 18.8 8.3 -
33.3 8.3 16.7 22.9 12.5 6.3
30.3 10.6 28.8 18.2 12.1 -
100.0 96 8.21
100.0 96 6.54
OF DEFINXE
LUKC
TISSUE
C Duluth 1960-61
%i 24.1
D Duluth 1953-55
9z 25.8
100.0 66 3.53
7.4 22.2 25.9 14.8 3.7 1.9 100.0 54 8.94
24.2 21.2 12.1 3.0 1.5 100.0 66 9.33*
27. I 72.9 3.83
22.5 71.5 8.02
20.0 80.0 12.33*
-
12.1
Age standardized figures*” % with 0 asbestos bodies % with I+ asbestos bodies Mean no. asbestos bodies
24.0 76.0 8.21
33.3 66.7 6.54
* Mean based upon the arbitrary assumption that the one section designated by “>200” contains 225 asbestos bodies. ** Standardized for age on age distribution of Groups A and E (~60. 60-69, 70-79. 80+).
actually
New York City women was slightly greater than the mean number found in the Duluth 1972-1973 women, the difference is not statistically significant. The mean values for groups A, B, C, and D do rzof suggest that the mean number of asbestos bodies in lung tissue of Duluth women increased consistently with time from 1953-1955to 1972-1973. Somewhat fewer asbestos bodies were found in women under the age of 60 than in older women. As previously described Groups E and A women had been matched on age; and, as shown in Table 1, their mean ages were slightly older than the mean ages of women of Groups B, C, and D. For this reason, at the bottom of Table 2, we have shown percentages standardized on age to the age distribution of women of Groups A and E. Age standardization did not greatly change the picture. As previously described, there were 96 pairs of Group A and Group E women matched on age; and the two women in each pair were placed in the same set. In 48 of these pairs, more definite asbestos bodies were found in the ashed section from the Group E woman (New York City) than in the ashed section from the Group A woman (Duluth 1972-1973); in 9 pairs, no definite asbestos bodies were found in the ashed section from either of the two women; in one pair. just one definite asbestos body was found in each of the two ashed sections: and in the
ASBESTOS
BODIES
IN
LUNG
TABLE PLRC:EST DISTRIBUIION ASBESTOS
Group City Year
of death
Number
of asbestos bodies 0 I Z-4 5-9 10-24 25-99 100-210 >210 Total Number of women Mean no. asbestos bodies
or
BODIES
FEMALE
Focxo E N.Y.C. 1966-68
3
SUB,JEC-I-S
BY NUMBER
OF DEHNI
L‘E AM)
SPECIMENS
OF LUYG
TISSUE
IN ASHED
A Duluth 19x-13
B Duluth 1966-67
C Duluth 1960-61
PROHARLF.
D Duluth 1953-5.5
7i
%
%
%
9.4 4.2 19.8 10.8 30.2 14.6 I.0 -
21.9 10.4 16.7 15.6 94.0 11.5 -
19.7 7.6 15.2 17.3 27.3 3.0 -
IOO.0 96 15.55
100.0 96 10.05
100.0 66 7.42
13.0 5.5 18.5 27.8 27.8 5.5 I.9 100.0 54 11.89
18.2 4.5 16.7 ‘8.8 75.8 1.5 3.0 1.5 100.0 66 14.20*
17.5 82.5 7.99
12.3 87.7 11.85
14.1 85.9 17.01
Age standardized ‘Z with 0 asbestos bodies r/c with I+ asbestos bodies Mean no. asbestos bodies
299
PARENCHYMA
9.4 90.6 15.55
5%
figures** 11.9 78. I 10.05
* Mean based upon the arbitrary assumption that the one section designated by “110” actually contains 225 asbestos bodies. ** Standardized for age on age distribution of Groups A and E (~60. 60-69, 70-79, SO+).
remaining 38 pairs, more were found in Group A woman than in the Group E women. This difference between Groups E and A is not statistically significant. “Probable” asbestos bodies (as previously defined) were found in some of the ashed sections in which no definite asbestos bodies were found. Table 3 shows the findings for the “definite” and “probable” categories combined. No asbestos bodies (either “definite” or “probable”) were found in ashed sections from 9.4% of the Group E women, 21.9% of the Group A women (Duluth 1972-1973) 19.7% of Group B; 13.0% of Group C; and 18.2% of Group D. In 58 of 96 matched pairs, more asbestos bodies (“definite” plus “probable”) were found in the Group E woman (New York) than in the Group A woman (Duluth); in 4 pairs, no asbestos bodies were found in either of the two women: and in the remaining 34 pairs, more were found in the Group A woman than in the Group E woman. This difference between Group E and Group A is statistically significant fP = cO.02). RESULTS-PATERSON
As previously matched on age asbestos factory 1954. The Group 1971.
“NEIGHBORHOOD” MEN AND N.Y.C. MEN 19 men who died in New York City (Group I) were
described. with the 19 men (Group H) who lived within half a mile of an in Paterson, N. J., which was in operation between 1941 and H men included here died at various times between 1958 and
300
AUERBACH
ETAL
Table 4 shows the number of definite asbestos bodies found in each pair of men (one man being of Group H and the other being of Group I). It also shows the difference between the two men of a pair in respect to the number of asbestos bodies found. One or more definite asbestos bodies were found in ashed sections from all (100%) of the Group H men (Paterson) and from 17 (89%) of the Group I men. The mean number of definite asbestos bodies was 20.3 in Group H and 8.9 in Group I, a difference of 11.4 (over two to one). This difference (based upon only 19 pairs of men) is not statistically significant (P = >0.05). In 13 of the 19 pairs of men, more definite asbestos bodies were found in the Group H man than in the Group I man; in 2 pairs, the two men had the same number of asbestos bodies; and in 4 pairs the Group I man had more asbestos bodies than the Group H man. This difference is of borderline statistical significance. (The calculated value of P is slightly less than 0.05 or greater than 0.05 depending on which test is used.) Table 4 also shows the results for these two groups of subjects for definite plus probable asbestos bodies. One or more asbestos bodies (definite or probable) were TABLE NUMBER OF DEFINITE PROBABLE ASBESTOS (PATERSON
Definite Pair number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Mean no. of asbestos bodies H>I H=I H
4
ASBESTOS BODIES AND NUMBER OF DEFINITE AND BODIES IN MATCHED PAIRS OF MEN IN GROUP H NEIGHBORHOOD)
asbestos
AND
bodies
GROUP
I (N.Y.C.)
Def.
and prob.
asb. bodies
Group I
Diff. (H-1)
Group H
Group I
Diff. (H-1)
2 8 3 4 13 4 8 74 8 8 19 4 118 7 32 37 10 23 3
2 5 2 1 13 2 5 37 0 0 32 8 4 9 2 38 7 1 I
0 +3 +1 +3 0 +2 +3 i37 +8 +8 -13 -4 1-114 7 +30 -1 +3 +22 +2
7 20 4 5 26 4 34 >999 II 8 36 5 148 7 43 65 14 35 5
2 17 12 2 13 3 13 50 3 0 62 10 10 44 3 63 9 2 7
+5 +3 -8 +3 +13 +1 +21 +950 +8 +8 -26 -5 +38 -37 +40 +2 +5 +33 -2
20.3
8.9 13 2 4
Group H
+11.4
77.7
17.1 14 0 5
+60.6
ASBESTOS
BODIES
IN
LUNG
301
PARENCHYMA
found in ashed sections from all of the Group H men and all but one of the Group I men. In the ashed section from one of the Group H men, 74 definite asbestos bodies were found plus more probable asbestos bodies than could be counted with accuracy. The total number probably exceeded 999 (although this is only a rough guess). If this case is included (and the estimated number taken as lOOO), then the mean number was 77.7 for the 19 Group H men, 17.1 for the 19 Group I men. If the one extreme case is excluded, then the mean number was 26.5 for the remaining 18 Group H men and 17.1 for the 19 Group H men. These differences are not statistically significant. In 14 of the 19 pairs, more asbestos bodies (definite plus probable) were found in the Group H man than in the Group I man; and in the remaining 5 pairs more were found in the Group I man than in the Group H man. This difference is not statistically significant. RESULTS-OCCUPATIONALLY
EXPOSED
MEN
Most of the ashed sections from the occupationally exposed men (Groups F and G) were extremely difficult to evaluate. Typically, the slides from these subjects contained many clusters of fibers such as illustrated in Fig. 2. In addition, they typically contained large quantities of material which could not be ashed including uncoated fibers and what appeared to be fragments of asbestos bodies and nonfibrous mineral particles. Group G consisted of 14 members of the insulation workers union who had been occupationally exposed to asbestos from 25 to 52 years. These ages at time of death ranged from 43 up to 76. Because of the difficulties described above, accurate counts of asbestos bodies could not be made. Three of the sections perhaps contained less than 1000 asbestos bodies (but this is not certain). The others contained a large number of asbestos bodies. Group F consisted of eight men who had worked in the Paterson amosite asbestos factory for periods ranging from 2 months up to 2 years. The slides were extremely difficult to evaluate. One may have contained no more than perhaps 50 asbestos bodies. All of the others certainly contained well over 100 asbestos bodies. Two probably contained at least 1000 and perhaps several thousand asbestos bodies. DISCUSSION
AND CONCLUSION
In previous studies (12,13,14,15,16) asbestos fibers have been found in the air of a number of cities; and, considering the widespread use of asbestos during the last half century, it seems likely that at least some asbestos fibers are present in the air of a11 modern cities. Several investigators ( 17,18) have reported finding asbestos bodies (ferruginous bodies) in lung tissue of men and women with no known occupational exposure to asbestos dust. In some such instances (18,19) it was found that the subject had probably been exposed to asbestos fibers in any one of several ways, as for example, the use of asbestos products in making home repairs or the wife of an asbestos worker handling her husband’s dusty work clothes. Another possible source of asbestos fibers giving rise to asbestos bodies is in the
302
AL’ERBACH
DAL
use of some grades of talcum powder which sometimes contain a considerable amount of asbestos (20). For these reasons, when asbestos bodies are found in lung tissue of persons with no occupational exposure to asbestos dust, it is difficult if not impossible to prove with certainty whether the fibers giving rise to the asbestos bodies came from the general air or whether they came from some other source such as mentioned above. It would be surprising indeed if the amount of asbestos dust in the general air does not vary somewhat from one city to another, from one neighborhood to another, and from one decade to another. It would also be surprising if, among people with no ascertainable exposure to asbestos dust (other than from the general air), there is absolutely no variation from community to community or from decade to decade in the degree of exposure to asbestos dust. (For example, both the use of talcum powder and the quality of the talcum powder probably varies to some degree with location and with time.) In this study we determined the number of asbestos bodies in lung tissue specimens taken at autopsy from 96 women who had lived in Duluth, Minnesota for at least 15 years and who died in the period 1972-1973 and 96 women who died in New York City in 1966-1968. The two groups were matched on age and none of them was known to have been occupationally exposed to asbestos dust. The proportion of these subjects with one or more asbestos bodies (in the lung tissue sample) was somewhat greater in the New York group than in the Duluth group: and the mean number of asbestos bodies was somewhat greater in the New York group than in the Duluth group. The New York group may have had somewhat greater exposure to asbestos dust than the Duluth group: and it is conceivable that this was approximately compensated by (in the Duluth group) some asbestos bodies being formed about asbestos fibers coming from ingestion of water contaminated with asbestos fibers. All of the Duluth women in this study died in that city and had lived there for at least 15 years prior to death. The taconite ore processing plant opened in October 1955; so contamination of Duluth drinking water with asbestos fibers from that source could not have started before that date. Thus, the women who died in 1953-1954 had no exposure to asbestos from that source and those who died in 1955 either had no exposure or, at most, only a very slight exposure. We do not know the exact date at which the Duluth water first became appreciably contaminated. However, the women who died in 1972-1973 must have been exposed to quantities of asbestos in their drinking water for at least several years. Those who died in 19661967 could have been exposed for no more than about 11 or 12 years. and those who died in 1960-1961 could have been exposed for no more than about 5 or 6 years. Therefore, if drinking water contaminated with asbestos fibers (in the numbers and fiber sizes present in Duluth water in the early 1970s) greatly increases the occurrence of asbestos bodies in lung tissue, then one or more asbestos bodies should have been found in a larger percentage of ashed specimens from the women who died in 1972-1973 than in ashedspecimens from the women who died in earlier periods (1966-1967, 1960-1961, and 1953-1955). This was not the case.
ASBESTOS
BODIES
IN
LUNG
PARENCHYMA
303
The evidence summarized above indicates that the ingestion of Duluth water contaminated with asbestos fibers did rrol result in a great increase in the number of asbestos bodies present in lung tissue and of sufficient size to be clearly seen under a light microscope. No further inferences are justified. Specifically, this study provides no evidence as to whether asbestos fibers of smaller sizes are taken into the bloodstream as a result of drinking Duluth water. “Neighborhood”
Exposure
Two groups of men were matched on age (a total of 19 matched pairs). One group consisted of 19 men who had lived within half a mile of an amosite asbestos factory which was in operation in Paterson, New Jersey from 1942 to 1954. None of these men had worked in the factory and none were members of the insulation workers union. The other group consisted of 19 men who died in New York City. According to members of their families none of the New York men had been occupationally exposed to asbestos dust. No information is available on the number of asbestos fibers present in the air within half a mile of the asbestos factory; but accounts derived from the time the factory was in operation indicate that there was enough to be of annoyance to residents in the area. Presumably, it exceeded, to a considerable degree, the amount of asbestos dust present in the general air of New York City. The number of asbestos bodies found in ashed sections of lung tissue from the “Paterson neighborhood” men was considerably greater than the number found in sections from the New York City men. However, based on a sample of only 19 pairs, the difference was not statistically significant. Therefore, it can only be said the findings were not inconsistent with the hypothesis that the increased number of asbestos fibers in the “Paterson neighborhood“ was accompanied by an increase in the number of asbestos bodies in lung tissue. REFERENCES 1. Selikoff, I. J., Churg, J.. and Hammond. E. C. (1964). Asbestos exposure and neop1asia.J. Amer. Med.
Assoc.
188, 23.
1,. Selikoff, 1. J., Hammond. E. C.. and Churg. J. (1968). Asbestos exposure. smoking and neoplasia. J. Amer. Med. Assoc. 204, 106. 3. Bogovski. P. rf NI. (Eds.) (1973). The Biological Effects of Asbestos.” Lyon, France. International Agency for Research on Cancer 1973. 4. Gross, P., Cralley, L. J., and de Treville. R. T. P. (1967). Asbestos bodies: Their nonspecificity. Amer.
Ind.
H.vg.
Assoc.
J. 28, 541-542.
5. Cunningham, H. M. and Pontefract, R. D. (1973). Asbestos fibers in beverages, drinking water and tissues: Their passage through the intestinal wall and movement through the body. J. A.ssoc. Ofj’. Ad. Chrm. 56, 976-981. 6. Auerbach, 0. ( 1977). “Extra-Pulmonary Asbestosis.” Presented at the Symposium on Oecupational Pulmonary Diseases at Baylor College of Medicine. Houston, Texas on February 26. 7. Ulrich. S.. Berg. T. J., and Hedlund, D. (1972). Superior Polluter. Save Lake Superior Association and Northern Environmental Council, Duluth, Minn.. Appendix, pg. f.. October. 8. “Asbestos in the Great Lakes Basin.” ( 1975). A Report to the International Joint Commission from the Great Lakes Advisory Board, February. 9. Cook, P. N.. Rubin, I. B., Maggiore. C. J., and Nicholson. W. J. (1976). X-ray diffraction and electron beam analysis of asbestiform minerals in Lake Superior Waters. In “Proceedings, International Conference on Environmental Sensing and Assessment,” Paper 34-l. Vol. 2. Institute of Electrical and Electronics Engineers, New York.
304
.4SBESTOS
BODIES
IN
LUNG
PARENCHYMA
10. Nicholson. W. J. (1974). Analysis of amphibole asbestiform fibers in municipal water supplies. Environ. Health Perspect. 9, 165-172. 11. Hammond, E. C. (1966). Smoking in relation to the death rates of one million men and women. Epidemiological study of cancer and other chronic diseases. Nat. Cancer Inst. Manog. 19, 269-285, January. 12. Thomson, J. G.. Path, F. C., and Graves, W. M. Jr. (1966). Asbestos as an urban air contaminant. Arch. Pathol. 81, 458-W. 13. Cauna, D., Totten, R. S., and Gross. P. (196.5). Asbestos bodies in human lungs at autopsy. J. Amer. Med. Assoc. 192, 371-373. 14. Anjilvel. L. and Thurlbeck, W. M. (1966). The incidence of asbestos bodies in the lungs of random necropsies in Montreal. Canado Med. Assoc. J. 9.5, 1179-I 182. 15. Selikoff. 1. J., Nicholson, W. J.. and Langer. A. M. (1972). Asbestos air pollution. Arch. Environ. Health 25, I-13. 16. Nicholson. W. J. and Pundsack, F. L. (1973). Asbestos in the environment. In “Biological Effects of Asbestos,” International Agency for Research on Cancer, Publication No. 8, Lyon, pp. 126130. 17. Yazicioglu, S. ( 1976). Pleural calcification associated with exposure to chrysotile asbestos. Chest 70, 43-47. 18. Langer, A. M., Selikoff, I. J.. and Sastre. A. (1971). Chrysotile asbestos in the lungs of persons in New York City. Arch. Environ. Health 22, 348-361. 19. Anderson. H. A., Lilis, R., Daum, S. M., Fischbein, A. S.. and Selikoff, I. J. (1976). Householdcontact asbestos neoplastic risk. Ann. N. Y. Acad. Sci. 271, 31 l-323. 20. Rohl, A. N., Langer. A. M., Selikoff, 1. J.. Tordini, A., Klimentidis. R., Bowes. D. R.. and Skinner, D. L. (1976). Consumer talcums and powders: mineral and chemical characterization. J. Toxic&. En,?ron. Health 2. 255-284.
RESEARCH
14, 286-304 (1977)
Asbestos Bodies in Lung Parenchyma in Relation Ingestion and Inhalation of Mineral Fibers’ OSCAR AUERBACH,” E. CUYLER HAMMOND,? IRVING J. SELIKOFF,$ VERTA R. PARKS,~ HAVEN D. KASLOW,” AND LAWRENCE GARFINKEL * VA Hospital, East Orange. New Jersey and Professor of Pathology, Medicine and Dentistry of New, Jersey. Ne+cs Jersey Medical School, Jersey. t Departmetrt of Epidemiology and Statistics. American Cancer New, York. $ Environmental Sciences L&oratory, Mt. Sinai Hospital, York, 0 Senior Medical Investigator Laboratory. VA Hospital. East Jersey,” Department of Epidemiology and Statistics, American Cancer New York, ’ Department of Epidemiology and Statistics. American Cancer Society. Inc., Nelt, York
to
#
College of Neu,ark, New Society, Inc., New York, Neti. Orange, Neuj Society, Inc.,
Received April 21. 1977
This is one of several studies initiated by various investigators under unusual circumstances which, in the eyes of a large number of people, constituted an emergency. In 1973, the people of Duluth, Minnesota were informed by the news media that their drinking water was, and for several years had been. heavily contaminated with asbestos fibers. It was well known that workers with occupational exposure to asbestos dust are subject to a greatly increased risk of lung cancer, pleural mesothelioma, peritoneal mesothelioma, and to a lesser degree. cancer of the gastrointestinal tract (l-3). Thus, there was reason to fear that ingestion of asbestos fibers might lead to an increased risk of cancer of the sites of direct contact (i.e., tissues of the gastrointestinal tract) and perhaps to cancer of other sites as well. A perusal of official mortality statistics indicated that, up to that date, death rates from cancer of the sites and types named above had not been unusually high in Duluth. However, this was not reassuring since, even under conditions of heavy occupational exposure to asbestos dust, the carcinogenic effects do not usually become discernible until about 20 years or longer after onset of exposure. Since direct epidemiological evidence, one way or the other, would not become available until many years in the future, there was an urgent need to obtain as much indirect evidence as possible even though such evidence probably would not be conclusive. Among the issues were [l] whether asbestos fibers ingested with Duluth drinking water penetrated and became lodged in tissues lining the gastrointestinal tract and [2] whether the ingested fibers entered the bloodstream and were thereby carried to lung, pleural, and peritoneal tissues. Because of technical problems, to be described later, we were unable at the time to investigate either one of these Supported in part by a Center Grant. “ES00928” of the National Institute of Environmental Health Sciences of the U.S. Department of Health, Education and Welfare to the Mt. Sinai School of Medicine of the City University of New York. 286 CopyrIght 0 1977 hy Academx Press. Inc All rughts of reproduction in any form reserved.
ASBESTOS
BODIES
IN
LUNG
PARENCHYMA
287
two questions. Instead, by force of circumstances, we took an indirect approach to the latter question. In so doing, we realized that this approach would provide only limited information. When asbestos fibers are inhaled, many of them are, by biological processes, coated with a material containing iron. These coated fibers have a unique appearance under the microscope and are usually called “asbestos bodies” (see Fig. 1). Since some other types of mineral fibers can be similarly coated, some authors prefer the more general term “ferruginous bodies” (4). Whether or not very small asbestos fibers become coated, only those which become 5 pm or larger could be recognized as asbestos bodies at the light microscopic level. In the coating process fibers less than 5 pm in length could become bodies large enough to become identifiable by light microscopy. We reasoned [I] if large numbers of asbestos fibers were taken into the bloodstream (as a result of drinking the contaminated Duluth water) some of them would probably lodge in the lungs and become coated (56): as a result, [2] more asbestos bodies would be found in lung tissue of Duluth residents who died after the water had become contaminated: and [3] more asbestos bodies would be found in lung tissue of Duluth residents who died in recent years than in lung tissue of residents of a city in which the drinking water was not heavily contaminated with asbestos fibers. The present study was based upon the above hypotheses. Strongly positive findings would constitute suggestive evidence that the contamination of Duluth drinking water might lead to increased death rates from cancer of the sites named above in future years. Negative findings would not rule out the possibility that asbestos fibers of submicroscopic size entered the bloodstream (from the contaminated water), and so would not rule out the possibility of increased risk of cancer. (The asbestos fibers contained in Duluth drinking water were typically very small as compared with many of the fibers inhaled by occupationally exposed workers, although they, too, also inhale a great number of very small fibers.) BACKGROUND
In 1955 a taconite ore processing plant was established at Silver Bay, Minnesota on the shore of Lake Superior about 55 miles from Duluth, Minn. The process consists essentially of crushing the ore to a very fine powder. suspending it in water, and magnetically extracting the iron particles. The remaining materials are discharged into the lake. Since September of 1960 the volume of lake water permitted for this use has been sufficient to carry 67,000 or more tons of tailings into Lake Superior per day (7). With the passage of time, such of the pulverized material as remained suspended in water spread through the lake (8). We do not know how long it took for an appreciable concentration of particles to reach Duluth. Since the waters of Lake Superior had long been noted for their purity, the water system of Duluth drew its supply directly from the lake and distributed it without filtration throughout the city. In August of 1973, samples of water drawn from city taps, contained about 14 x 10fi amphibole fibers per liter of water (9). In the spring of 1975. following unusual weather conditions, some of the samples of
288
AUERBACH
FIG.
1. Montage
of asbestos
bodies
of various
ET AL.
shapes.
Arrow:
Clump
of asbestos
bodies.
ASBESTOS
BODIES
IN
LUNG
PARENCHYMA
289
tap water contained as many as 300 to 600 x IO6 fibers per liter (9). Most of the fibers were of such small size that they could not be seen by light microscopy, that is, 5 pm or less. However, a small percentage were 5 pm long or longer and when the enormous number present in the water is considered, even this small percentage would result in the ingestion of a significant number of fibers that would be visible by light microscopy. In 1972 the Environmental Protection Agency (EPA) brought suit in Federal District Court to prohibit further contamination of the lake: but initially the possibility of a health hazard was not an issue. The situation changed when, in 1973, it became known that many of the contaminating particles were asbestos fibers. Mineralogically they are of the cummingtonite-grunerite series ( 10). The trial in Federal Court was scheduled to commence August 1, 1973. HOWever, shortly before that time, the EPA called upon us (I.J.S., E.C.H., and associates) to investigate the possible health effects especially with respect to cancer of the types associated with occupational exposure to asbestos dust and to serve as expert witnesses in the legal case. Shortly thereafter the Court ordered us to initiate as quickly as possible any such studies as we thought might help throw light on the matter. The scope of studies feasible at that date was limited by time constraints and various other practical considerations. Duluth was included in a long-term prospective epidemiological study in progress since 1959 (11); and we felt that this would eventually provide a definitive answer. Members of our group (Drs. Arthur M. Langer, William J. Nicholson, Arthur N. Rohl, and colleagues) immediately began a series of mineralogical studies. The problem was to design and carry out a pertinent short-term biological investigation. St. Mary’s Hospital (Sister Marybelle Leick, administrator and Drs. A. C. Aufderheide and J. P. Knoedler, pathologists) offered us splendid cooperation as did St. Luke’s Hospital (Dr. Volker Goldschmidt, pathologist) at a somewhat later date. Our first thought was to search for asbestos fibers (by electron microscope and ancillary means) in specimens of colon-rectum and lung tissue taken routinely at autopsy from residents of Duluth during the preceding year and similar specimens taken at autopsy prior to the opening of the taconite processing plant and at several intermediate periods. This plan had to be abandoned when we found that the formalin used to fix the specimens had been diluted with city tap water. Electron microscopic examination of the diluted formalin then in use in the hospital showed that it contained very large numbers of asbestos fibers. Thus, if asbestos fibers had been found in the preserved tissues of patients who had died during the past few years, there would have been no way of knowing whether they had been present before the death of the patient or whether they came from the asbestos-contaminated formalin. For the above reason, we decided instead to search, by light microscopy, for asbestos bodies in specimens of lung tissue. If found, they could not have come from the contaminated formalin. (There was none in the contaminated formalin; and they cannot be formed in lung tissue after the death of the patient.) Although this plan had the limitation that the procedure could not reveal the presence of sublight microscopic bodies, the presence of bodies 5 pm and longer would be established.
290
AUEKBACH
ETAL
MATERIAL
The plan was to examine specimens of lung tissue taken routinely at autopsy (and preserved in paraffin blocks) from female residents of Duluth who died during four periods of time (1972-1973, 1966-1967, 1960-1961, and 1953-1955), who had resided in Duluth for at least 15 years prior to death, who had used city water during that period, who had never been occupationally exposed to asbestos dust, and who had not lived within 1 mile of a shipyard, ore-loading dock, or steel mill. (Women were selected because they are less likely than men to be either occupationally exposed to asbestos dust or to be exposed while making home repairs.) St. Mary’s Hospital provided us with a list of all women who had come to autopsy in that hospital during the specified years, from whom lung tissue specimens were available, and who were residents of Duluth at the time of death. By telephone interviews with relatives and friends of the deceased, through the cooperation of the Duluth Public Library, and by a search of city directories and newspapers from prior years, we then ascertained which of them met the other specifications outlined above. In case of doubt, usually resulting from inability to find a relative or friend able to supply the pertinent information, the woman was excluded. The Duluth City Planning Department located and pinpointed the residences of the prospective subjects with relation to shipyards, docks, etc. The Duluth Water and Gas Department provided exact information on the water supply to residences of the prospective subjects. It turned out that, after this screening, the number of suitable subjects available from St. Mary’s Hospital was insufficiently large for a statistically valid study. For this reason, we obtained additional material in the same way from St. Luke’s Hospital. Staff members of both hospitals provided us with invaluable assistance. As shown in Table 1, the total sample contained 282 Duluth women in four groups as follows: Group A, who died 1972-1973, 96 women of whom 48 were from St. Mary’s and 48 from St. Luke’s; Group B, 19661967.66 women, 48 from St. Mary’s and 18 from St. Luke’s; Group C, 1960-1961, 54 women, 36 from St. Mary’s and 18 from St. Luke’s: Group D, 1953-1955. 66 women, 36 from St. Mary’s and 30 from St. Luke’s. We had hoped to have the same number of subjects (66) in Groups B, C, and D; but were able to find only 54 for Group C, years 1960-1961. A fifth group of subjects (Group E, New York City women) was included for comparison with Group A. Group E. We had available specimens of lung tissue from all adults who came to autopsy in two New York City Hospitals (Mt. Sinai and Elmhurst) during the years 1966-1968. For most of these we also had available information provided by relatives and friends on residence history, occupation, occupational exposures, and possible household exposures. From this material, we selected a sample of 96 women matched on age with the 96 Duluth women of Group A. Thus Groups A and E together consisted of 96 matched pairs of women. The New York sample was confined to women recorded as being housewives or white collar workers with no known occupational exposure to asbestos ‘dust. Other groups. Four groups of men were included for the following reasons, partly, but not entirely, related to the main purpose of the study: Groups F and G
ASBESTOS
BODIES
IN
LUNG
TABLE SUBJECTS
291
PARENCHYMA
I
INCLUDED
IN STUDY
Age at death SO+
Mean we
14
21
35
26
72.5
14 20 8 18
21 13 16 21
35 20 19 17
26 13 11 10
72.6 66.8 68.9 64.8
8 14
5 7
1 5
2 2
-
56.8 60.4
19 19 438
2 2 90
6 6 110
IO 10 150
1 1 88
69.8 68.8 69.1
matched
on age.
1966-1968
96
1972- 1973 1%6- 1%7 1960-1961 1953-1955
96 66 54 66
E
N.Y.C.
women
A B C D
Duluth Duluth Duluth Duluth
women women women women
F G
Paterson Insulation
H I
Paterson “neighborhood” N.Y.C. men Total Groups
70-79
160
Group
Note:
60-69
Total number
asbestos factory workers men
A and E women
men men
were
matched
on age and Groups
H and I men were
consisted of men who had been occupationally exposed to asbestos dust. The main purpose was to obtain an estimate of the number of asbestos bodies associated with occupational exposure in comparison with the number to be found in Duluth and New York City women without such exposure. Group F was of special interest because these men died years after having been heavily exposed for relatively short periods of time. Group H was included to see whether residence in a neighborhood with elevated levels of asbestos dust in the air was associated with an increased number of asbestos bodies in lung tissue. Group I was included for comparison with Group H. Group F. An amosite (grunerite) asbestos insulation factory was in operation in Paterson, New Jersey during years 1941-1954. According to accounts of men who had worked there, they were exposed to “clouds” of dust. Unfortunately, no counts were made of the number of asbestos fibers in the air. For this study, lung tissue (taken at autopsy) was obtained from 8 men who had worked in the factory for from 2 months to 2 years. Group G. This group consisted of 14 members of the insulation workers union. They had been occupationally exposed to asbestos dust (chrysotile and amosite) for 25 to 52 years prior to death. Group H. Lung tissue, taken at autopsy, was obtained from 19 men who had lived within a radius of ‘/2 mile from the amosite asbestos factory mentioned above during the time that the factory was in operation. None of these men had worked in the factory and none was a member of the insulation workers union. According to newspaper accounts written while the plant was in operation, people living in this neighborhood had complained of dust from the factory; asbestos dust can still be found in attics and other undusted places in houses still standing there. Group 1. Nineteen men who died and came to autopsy at Mount Sinai and Elmhurst Hospitals (New York City) during 1966-1968 were matched on age with
292
AUERBACH
ET AL.
the 19 men of Group H. None was recorded as having been occupationally exposed to asbestos dust. However, according to members of their families, some of these men had probably been exposed to asbestos dust while making home repairs and improvements. STATISTICAL DESIGN As described above, there were 96 Group A subjects, 66 Group B subjects, 54 Group C subjects, and 66 Group D subjects. By random numbers, these 282 subjects were assigned to 6 sets (sets # 1 through #6) such that there were 16 A, 11 B, 9 C. and 11 D subjects in each set. By random numbers, 1 of the 8 Group F subjects, 2 of the 14 Group G subjects, and 3 of the 19 Group H subjects were assigned to each of the 6 sets. Each of the 96 Group E subjects was then assigned to the set containing the Group A woman with whom she had been matched on age; and each of 18 of the Group I men was assigned to the set containing a Group H man with whom he had been matched on age. There then remained 6 male subjects (2 of Group F, 2 of Group G, 1 of Group H. and 1 of Group I). By random numbers, 1 of these 6 men was assigned to each of the 6 sets. Thus there was a total of 73 subjects in each set. By random numbers, the subjects within each set were put in random order. While in this order, a serial number was placed on each record such that serial number 1 was given to the first subject of set # 1 and serial number 438 was given to the last subject of set #6. As will be described, one slide containing ashed lung tissue was prepared for each subject. This was done in order according to serial number; and each slide was labeled only with the serial number. The slides were later examined microscopically in the same order. The serial number gave the examiner no clue as to the identity of the subject. DESIGN
OF GRID SLIDES
Since asbestos bodies are difficult to see when embedded in lung tissue, our procedure is to mount a section of tissue on a glass slide, ash it in activated oxygen, put on a drop or two of mounting medium, and then set a cover slip in place. In past studies we had used ordinary microscope slides and cover slips. The following difficulties were encountered. (1) Often, after ashing, so little material (visible to the naked eye) remains that it is difficult to place the cover slip in the correct position. (2) If the specimen (or a portion of it) contains very little noncombustible material, it is difficult to focus the microscope on the proper plane; and if out of focus, such particles as are present may not be seen. (3) Without guidelines, it is difficult to scan microscopically the entire area under the cover slip without skipping some areas. (4) While the location of objects may be recorded for later review by reading the coordinates from the mechanical stage, this is time consuming. It also has the disadvantage that in order to find the object again by the recorded coordinates, the same microscope must be used. To overcome these difficulties one of us (E.C.H.) designed a new type of slide as described below (see Figs. 3 and 4). A square grid as shown in Fig. 3, is “printed” on a 25 x 75 mm glass slide by
FIG.
2. Cluster
of asbestos
bodies
photograpl
293
hed at three
different
focal
planes
294
AKJERBACH
ET AL
c
3
II I c D c ,
I) . e D c C L
3
FIG. 3. 18 x 18 mm grid printed on a 25 x 75 mm microscope The interior lines are 100 pm in width.
slide by the chrome
-sputter
process.
the chrome-sputter process. The extremely thin layer of chromium withstands the ashing procedure. The grids are divided into 18 rectangular areas by lines. The heavy outer boundary lines are 400 pm in width and the inner lines are 100 pm in width. The entire square measures 18 x 18 mm between the centers of the outer boundary lines. (The overall dimensions are 18.4 x 18.4 mm.) To help in locating an area, the numbers 1, 2, and 3 are printed in visible size at the top and bottom of the square and the letters A, B, C, D, E and F are printed at each side. Coordinates, invisible to the naked eye but visible under the microscope are printed within the grid lines as illustrated in Fig. 4. The horizontal coordinates are identified as Al, A2, A3, A4, A5: Bl, B2 . . . through F3, F4, F5. Under the microscope, it is easy to record the location of an object to within about oneor two-tenths of the distance between the printed coordinates. For example, the dot shown in Fig. 4 would be recorded as at location Dl .O, 28.7. This is sufficiently accurate for finding an object a second time. The grid lines and coordinates afford
FIG. 4. Magnification of a portion object located at coordinates D1.O.
of the interior 28.7.
grid lines showing
coordinates.
Arrow
points
to an
ASBESTOS
BODIES
IN
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PARENCHYMA
295
a means of accurately focusing the microscope at a plane just above the surface of the slide. Dr. Thomas S. Ely of Eastman Kodak suggested the use of the chromesputter process after we had tried ordinary photographic methods and found them to be unsatisfactory. The slides were manufactured for us by Qualitron. Inc. of Danbury, Connecticut to whom we are indebted for assistance in details of the design. PREPARATION
OF SPECIMENS
As previously described, the specimens of lung tissue had been taken routinely at autopsy and embedded in paraffin blocks at various points in time between 1953 and 1973. They varied considerably in shape and size, some being very small and others being fairly large. Furthermore, some of the blocks contained tissue specimens from more than one organ. In such an instance, it was necessary to cut and stain a thin section to determine which specimen was lung tissue. The lung tissue specimen having been identified, it was removed from the original block and reembedded in paraffin. For quantitative estimates, it is essential that each tissue specimen examined be of the same volume as nearly as possible. We decided to use 20 mm3 of paraffinembedded tissue as the standard. This was accomplished as follows. Either a microscopic section taken from the block or the surface of the block itself was projected on paper at a fixed setting of the magnifier. The outline of the tissue was traced and the area (making adjustment for magnification) was determined by means of a planimeter. From this was calculated the depth to which the embedded tissue had to be cut in order to obtain a volume of 20 mm3. In practice, tissue areas varied from 56 to 444 mm’. Therefore the corresponding required depth of cut ranged from 357 to 45 pm. A sliding (rather than rotary) microtome was found to be most accurate for cutting sections of specified thickness. The microtome feed mechanism was calibrated gravimetrically using tissuemat paraffin and was found to be accurate within 1% at settings from 20 to 160 pm. It was found to be unwise to make cuts thicker than 40 pm each because of apparently hindered paraffin extraction and retarded low-temperature oxidation. Therefore, when a total depth of over 40 pm was required, multiple sections were cut such that together they totaled the required depth. Excess paraffin around the edges of the tissue specimen was removed. The sections were then placed within the grid square of a slide as evenly as possible but no closer than 2 or 3 mm from the outer boundary lines. Then the slide was placed on a hot plate to melt down and fix the sections. After cooling, the paraffin was extracted by several drops of xylene, the process being repeated three times. The slide was then heated briefly to drive off the xylene. Slides, prepared as above, were placed, one each, in the five chambers of a low-temperature asher. Within 2 to 3 minutes after starting the pump the vacuum is sufficient (below 0.1 mm Hg) to strike the glow discharge and admit the oxidant. The burn is timed from that point. As oxidation proceeds, the color of the glow discharge changes; and apparently at or near the end of active oxidation it becomes very dull and dark red. Most of the samples reached this point in about 15 to 20 minutes but sometimes longer. After 45 minutes, instrument shut down is
296
AUERBACH
ET AL.
started, another 50 to 60 minutes then being required to repressurize the oxidation chambers. After noting the location of the greatest concentration of ash on the grid, a small drop of Namount from a capillary tube is applied on this spot. An 18 x 18 mm cover slip is then placed over the grid. All of the slides were prepared in this way by one of us (H.D.K.), this being done in order by serial number as previously described, Technical difficulties were encountered from time to time, sometimes resulting in slides of very poor quality. The two principal difficulties were as follows. Some tissue sections contained a relatively large amount of material which withstood ashing; and this material may have been very unevenly distributed on the grid. After ashing, some of the grids were coated with a more or less evenly distributed layer of a yellow or yellow-brown material. In extreme cases, the coating was so dense as to make microscopic examination difficult if not impossible. Each slide, immediately after preparation, was examined by H.D.K. for defects as described above. If the quality was very poor, he prepared a new slide by repeating the entire process. In some instances, the first reader, after a brief preliminary look under the microscope, decided that the quality was too poor for valid readings. In such cases, another slide was made. EXAMINATION
OF SLIDES
The entire area of each grid was examined microscopically by one of us (V.R.P.) under phase contrast using a 25x Neofluor objective (N.A. 0.60) and 12.5x wide-angle eyepieces. Every object found at this magnification which gave the slightest suggestion of possibly being an asbestos body was studied closely at higher magnification (500x) under both phase contrast and ordinary light. Figure 1 is a montage showing asbestos bodies of various different shapes. The same general shapes shown here can be found in objects varying considerably in overall size. In slides in which less than about a dozen asbestos bodies were found, they were almost always scattered, usually just one, and seldom more than two being observed within the same microscopic field of view. In slides containing a larger number, clumps of asbestos bodies were often found within the same high-power microscopic field. (Such a clump is shown by the arrow in Fig. 1). At any one focal plane, it was often difficult to count the number of coated fibers present; but by focusing up and down the actual number could usually be ascertained. However, in some instances, what appeared to be fragments were observed; and it was virtually impossible to ascertain whether two or more fragments had originally come from one or more than one coated fiber. Some slides contained a very large number of asbestos bodies. In such slides there were many separate microscopic fields each of which contained one or more clumps containing many asbestos bodies, Figure 2 shows one such clump photographed at three different focal planes. No better than a crude estimate could be made of the number of coated fibers within such a clump. In many instances it was difficult to decide whether an object should be classified as an asbestos body. For example, the object might be in a field containing so much material which could not be ashed as to make visualization difficult; or
ASBESTOS
BODIES
IN
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297
the apparent shape of the object might be such as to leave at least some doubt as to whether or not it was truly a coated fiber. For this reason, the following types of objects were recorded: (I) “definite” asbestos body, (2) “probable“ asbestos body, (3) “possible” asbestos body, and (4) “unusual object” which might be a coated fiber of some sort but which did not have the appearance of an asbestos body. Notes were also made as to the quality of the slide (from the standpoint of visualization) and whether or not a large number of uncoated fibers or nonfibrous particles were present. “Definite” meant an object which, in the opinion of V.R.P., exhibited all of the characteristics of one or another of the various types of asbestos bodies. “Probable“ indicated some uncertainty. “Possible” was used simply to call an object (which could possibly be an asbestos body) to the attention of the later reviewer. A pencil sketch of each object was drawn and its coordinates were recorded. The time required to examine a single grid in this way averaged about 4 hours. However, up to 8 hours was required in some instances, as for example, grids with a large amount of unashed material but without a very large number of asbestos bodies. In some instances it was quickly apparent that the specimen contained hundreds of asbestos bodies. Typically in such instances, such a large quantity of other unashed material was present (including fibers, granules, and what appeared to be broken fragments of asbestos bodies) that an accurate count could not be made. To expedite the process, such slides were initially recorded as “T.N.C.” (“too numerous to count”). At a later date these slides were examined again to obtain a rough estimate of the number. In Tables 2-4, the estimated number of asbestos bodies in such cases are indicated as >200 or >999. After a slide has been examined in this way by V.R.P., every object which she had recorded as a “definite,” “probable,” or “possible” asbestos body or an “unusual object” was reexamined by O.A. Disagreement in classification was extremely rare. RESULTS-DULUTH
AND NEW YORK WOMEN
Table 2 shows the percentage distribution of Groups E, A, B, C, and D subjects by the number of “definite” asbestos bodies found in the ashed specimens of lung tissue. No definite asbestos bodies were found in ashed sections from 33.3% of the Group A women (Duluth 1972-1973). This percentage with no definite asbestos bodies was greater for Group A than for any of the other four groups: (New York women, 24.0%; Duluth women 1966-1967, 30.3%: Duluth women 1960-1961, 24.1%; and Duluth women 1953-1955, 25.8%). Likewise, the percent of women with two or more definite asbestos bodies found was less for Group A women (Duluth 1972-1973) than for women in any of the other four groups, Less than 9% of the ashed sections from any of the five groups (E, A, B, C, and D) had over 24 definite asbestos bodies; but in all groups except Group B, some sections contained 25 to 99. one in Group C contained 186 definite asbestos bodies and one in Group D contained more than 200 asbestos bodies. With such a wide range of values and with such skewed distribution of values, the mean numbers of asbestos bodies are not very illuminating. Although the mean number found in the
298
AUERBACH
ET AL
TABLE PEKCEI\‘T
DIS~RIBUWON
ASBESTOS
BODIES
Group City Year of death Number of asbestos bodies 0 I 2-4 5-9 10-24 25-99 IOO- 186 >200 Total Number of women Mean no. asbestos bodies
OF FE~MALE
FO~IND IN E N.Y.C. 1966-68
-
2
SUBJECTS
ASHED
BY NUMBER
SPECIMENS
A Duluth 1972-73
OF
B Duluth 1966-67
cl0
5%
9%
24.0 9.4 18.8 20.8 18.8 8.3 -
33.3 8.3 16.7 22.9 12.5 6.3
30.3 10.6 28.8 18.2 12.1 -
100.0 96 8.21
100.0 96 6.54
OF DEFINXE
LUKC
TISSUE
C Duluth 1960-61
%i 24.1
D Duluth 1953-55
9z 25.8
100.0 66 3.53
7.4 22.2 25.9 14.8 3.7 1.9 100.0 54 8.94
24.2 21.2 12.1 3.0 1.5 100.0 66 9.33*
27. I 72.9 3.83
22.5 71.5 8.02
20.0 80.0 12.33*
-
12.1
Age standardized figures*” % with 0 asbestos bodies % with I+ asbestos bodies Mean no. asbestos bodies
24.0 76.0 8.21
33.3 66.7 6.54
* Mean based upon the arbitrary assumption that the one section designated by “>200” contains 225 asbestos bodies. ** Standardized for age on age distribution of Groups A and E (~60. 60-69, 70-79. 80+).
actually
New York City women was slightly greater than the mean number found in the Duluth 1972-1973 women, the difference is not statistically significant. The mean values for groups A, B, C, and D do rzof suggest that the mean number of asbestos bodies in lung tissue of Duluth women increased consistently with time from 1953-1955to 1972-1973. Somewhat fewer asbestos bodies were found in women under the age of 60 than in older women. As previously described Groups E and A women had been matched on age; and, as shown in Table 1, their mean ages were slightly older than the mean ages of women of Groups B, C, and D. For this reason, at the bottom of Table 2, we have shown percentages standardized on age to the age distribution of women of Groups A and E. Age standardization did not greatly change the picture. As previously described, there were 96 pairs of Group A and Group E women matched on age; and the two women in each pair were placed in the same set. In 48 of these pairs, more definite asbestos bodies were found in the ashed section from the Group E woman (New York City) than in the ashed section from the Group A woman (Duluth 1972-1973); in 9 pairs, no definite asbestos bodies were found in the ashed section from either of the two women; in one pair. just one definite asbestos body was found in each of the two ashed sections: and in the
ASBESTOS
BODIES
IN
LUNG
TABLE PLRC:EST DISTRIBUIION ASBESTOS
Group City Year
of death
Number
of asbestos bodies 0 I Z-4 5-9 10-24 25-99 100-210 >210 Total Number of women Mean no. asbestos bodies
or
BODIES
FEMALE
Focxo E N.Y.C. 1966-68
3
SUB,JEC-I-S
BY NUMBER
OF DEHNI
L‘E AM)
SPECIMENS
OF LUYG
TISSUE
IN ASHED
A Duluth 19x-13
B Duluth 1966-67
C Duluth 1960-61
PROHARLF.
D Duluth 1953-5.5
7i
%
%
%
9.4 4.2 19.8 10.8 30.2 14.6 I.0 -
21.9 10.4 16.7 15.6 94.0 11.5 -
19.7 7.6 15.2 17.3 27.3 3.0 -
IOO.0 96 15.55
100.0 96 10.05
100.0 66 7.42
13.0 5.5 18.5 27.8 27.8 5.5 I.9 100.0 54 11.89
18.2 4.5 16.7 ‘8.8 75.8 1.5 3.0 1.5 100.0 66 14.20*
17.5 82.5 7.99
12.3 87.7 11.85
14.1 85.9 17.01
Age standardized ‘Z with 0 asbestos bodies r/c with I+ asbestos bodies Mean no. asbestos bodies
299
PARENCHYMA
9.4 90.6 15.55
5%
figures** 11.9 78. I 10.05
* Mean based upon the arbitrary assumption that the one section designated by “110” actually contains 225 asbestos bodies. ** Standardized for age on age distribution of Groups A and E (~60. 60-69, 70-79, SO+).
remaining 38 pairs, more were found in Group A woman than in the Group E women. This difference between Groups E and A is not statistically significant. “Probable” asbestos bodies (as previously defined) were found in some of the ashed sections in which no definite asbestos bodies were found. Table 3 shows the findings for the “definite” and “probable” categories combined. No asbestos bodies (either “definite” or “probable”) were found in ashed sections from 9.4% of the Group E women, 21.9% of the Group A women (Duluth 1972-1973) 19.7% of Group B; 13.0% of Group C; and 18.2% of Group D. In 58 of 96 matched pairs, more asbestos bodies (“definite” plus “probable”) were found in the Group E woman (New York) than in the Group A woman (Duluth); in 4 pairs, no asbestos bodies were found in either of the two women: and in the remaining 34 pairs, more were found in the Group A woman than in the Group E woman. This difference between Group E and Group A is statistically significant fP = cO.02). RESULTS-PATERSON
As previously matched on age asbestos factory 1954. The Group 1971.
“NEIGHBORHOOD” MEN AND N.Y.C. MEN 19 men who died in New York City (Group I) were
described. with the 19 men (Group H) who lived within half a mile of an in Paterson, N. J., which was in operation between 1941 and H men included here died at various times between 1958 and
300
AUERBACH
ETAL
Table 4 shows the number of definite asbestos bodies found in each pair of men (one man being of Group H and the other being of Group I). It also shows the difference between the two men of a pair in respect to the number of asbestos bodies found. One or more definite asbestos bodies were found in ashed sections from all (100%) of the Group H men (Paterson) and from 17 (89%) of the Group I men. The mean number of definite asbestos bodies was 20.3 in Group H and 8.9 in Group I, a difference of 11.4 (over two to one). This difference (based upon only 19 pairs of men) is not statistically significant (P = >0.05). In 13 of the 19 pairs of men, more definite asbestos bodies were found in the Group H man than in the Group I man; in 2 pairs, the two men had the same number of asbestos bodies; and in 4 pairs the Group I man had more asbestos bodies than the Group H man. This difference is of borderline statistical significance. (The calculated value of P is slightly less than 0.05 or greater than 0.05 depending on which test is used.) Table 4 also shows the results for these two groups of subjects for definite plus probable asbestos bodies. One or more asbestos bodies (definite or probable) were TABLE NUMBER OF DEFINITE PROBABLE ASBESTOS (PATERSON
Definite Pair number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Mean no. of asbestos bodies H>I H=I H
4
ASBESTOS BODIES AND NUMBER OF DEFINITE AND BODIES IN MATCHED PAIRS OF MEN IN GROUP H NEIGHBORHOOD)
asbestos
AND
bodies
GROUP
I (N.Y.C.)
Def.
and prob.
asb. bodies
Group I
Diff. (H-1)
Group H
Group I
Diff. (H-1)
2 8 3 4 13 4 8 74 8 8 19 4 118 7 32 37 10 23 3
2 5 2 1 13 2 5 37 0 0 32 8 4 9 2 38 7 1 I
0 +3 +1 +3 0 +2 +3 i37 +8 +8 -13 -4 1-114 7 +30 -1 +3 +22 +2
7 20 4 5 26 4 34 >999 II 8 36 5 148 7 43 65 14 35 5
2 17 12 2 13 3 13 50 3 0 62 10 10 44 3 63 9 2 7
+5 +3 -8 +3 +13 +1 +21 +950 +8 +8 -26 -5 +38 -37 +40 +2 +5 +33 -2
20.3
8.9 13 2 4
Group H
+11.4
77.7
17.1 14 0 5
+60.6
ASBESTOS
BODIES
IN
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301
PARENCHYMA
found in ashed sections from all of the Group H men and all but one of the Group I men. In the ashed section from one of the Group H men, 74 definite asbestos bodies were found plus more probable asbestos bodies than could be counted with accuracy. The total number probably exceeded 999 (although this is only a rough guess). If this case is included (and the estimated number taken as lOOO), then the mean number was 77.7 for the 19 Group H men, 17.1 for the 19 Group I men. If the one extreme case is excluded, then the mean number was 26.5 for the remaining 18 Group H men and 17.1 for the 19 Group H men. These differences are not statistically significant. In 14 of the 19 pairs, more asbestos bodies (definite plus probable) were found in the Group H man than in the Group I man; and in the remaining 5 pairs more were found in the Group I man than in the Group H man. This difference is not statistically significant. RESULTS-OCCUPATIONALLY
EXPOSED
MEN
Most of the ashed sections from the occupationally exposed men (Groups F and G) were extremely difficult to evaluate. Typically, the slides from these subjects contained many clusters of fibers such as illustrated in Fig. 2. In addition, they typically contained large quantities of material which could not be ashed including uncoated fibers and what appeared to be fragments of asbestos bodies and nonfibrous mineral particles. Group G consisted of 14 members of the insulation workers union who had been occupationally exposed to asbestos from 25 to 52 years. These ages at time of death ranged from 43 up to 76. Because of the difficulties described above, accurate counts of asbestos bodies could not be made. Three of the sections perhaps contained less than 1000 asbestos bodies (but this is not certain). The others contained a large number of asbestos bodies. Group F consisted of eight men who had worked in the Paterson amosite asbestos factory for periods ranging from 2 months up to 2 years. The slides were extremely difficult to evaluate. One may have contained no more than perhaps 50 asbestos bodies. All of the others certainly contained well over 100 asbestos bodies. Two probably contained at least 1000 and perhaps several thousand asbestos bodies. DISCUSSION
AND CONCLUSION
In previous studies (12,13,14,15,16) asbestos fibers have been found in the air of a number of cities; and, considering the widespread use of asbestos during the last half century, it seems likely that at least some asbestos fibers are present in the air of a11 modern cities. Several investigators ( 17,18) have reported finding asbestos bodies (ferruginous bodies) in lung tissue of men and women with no known occupational exposure to asbestos dust. In some such instances (18,19) it was found that the subject had probably been exposed to asbestos fibers in any one of several ways, as for example, the use of asbestos products in making home repairs or the wife of an asbestos worker handling her husband’s dusty work clothes. Another possible source of asbestos fibers giving rise to asbestos bodies is in the
302
AL’ERBACH
DAL
use of some grades of talcum powder which sometimes contain a considerable amount of asbestos (20). For these reasons, when asbestos bodies are found in lung tissue of persons with no occupational exposure to asbestos dust, it is difficult if not impossible to prove with certainty whether the fibers giving rise to the asbestos bodies came from the general air or whether they came from some other source such as mentioned above. It would be surprising indeed if the amount of asbestos dust in the general air does not vary somewhat from one city to another, from one neighborhood to another, and from one decade to another. It would also be surprising if, among people with no ascertainable exposure to asbestos dust (other than from the general air), there is absolutely no variation from community to community or from decade to decade in the degree of exposure to asbestos dust. (For example, both the use of talcum powder and the quality of the talcum powder probably varies to some degree with location and with time.) In this study we determined the number of asbestos bodies in lung tissue specimens taken at autopsy from 96 women who had lived in Duluth, Minnesota for at least 15 years and who died in the period 1972-1973 and 96 women who died in New York City in 1966-1968. The two groups were matched on age and none of them was known to have been occupationally exposed to asbestos dust. The proportion of these subjects with one or more asbestos bodies (in the lung tissue sample) was somewhat greater in the New York group than in the Duluth group: and the mean number of asbestos bodies was somewhat greater in the New York group than in the Duluth group. The New York group may have had somewhat greater exposure to asbestos dust than the Duluth group: and it is conceivable that this was approximately compensated by (in the Duluth group) some asbestos bodies being formed about asbestos fibers coming from ingestion of water contaminated with asbestos fibers. All of the Duluth women in this study died in that city and had lived there for at least 15 years prior to death. The taconite ore processing plant opened in October 1955; so contamination of Duluth drinking water with asbestos fibers from that source could not have started before that date. Thus, the women who died in 1953-1954 had no exposure to asbestos from that source and those who died in 1955 either had no exposure or, at most, only a very slight exposure. We do not know the exact date at which the Duluth water first became appreciably contaminated. However, the women who died in 1972-1973 must have been exposed to quantities of asbestos in their drinking water for at least several years. Those who died in 19661967 could have been exposed for no more than about 11 or 12 years. and those who died in 1960-1961 could have been exposed for no more than about 5 or 6 years. Therefore, if drinking water contaminated with asbestos fibers (in the numbers and fiber sizes present in Duluth water in the early 1970s) greatly increases the occurrence of asbestos bodies in lung tissue, then one or more asbestos bodies should have been found in a larger percentage of ashed specimens from the women who died in 1972-1973 than in ashedspecimens from the women who died in earlier periods (1966-1967, 1960-1961, and 1953-1955). This was not the case.
ASBESTOS
BODIES
IN
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PARENCHYMA
303
The evidence summarized above indicates that the ingestion of Duluth water contaminated with asbestos fibers did rrol result in a great increase in the number of asbestos bodies present in lung tissue and of sufficient size to be clearly seen under a light microscope. No further inferences are justified. Specifically, this study provides no evidence as to whether asbestos fibers of smaller sizes are taken into the bloodstream as a result of drinking Duluth water. “Neighborhood”
Exposure
Two groups of men were matched on age (a total of 19 matched pairs). One group consisted of 19 men who had lived within half a mile of an amosite asbestos factory which was in operation in Paterson, New Jersey from 1942 to 1954. None of these men had worked in the factory and none were members of the insulation workers union. The other group consisted of 19 men who died in New York City. According to members of their families none of the New York men had been occupationally exposed to asbestos dust. No information is available on the number of asbestos fibers present in the air within half a mile of the asbestos factory; but accounts derived from the time the factory was in operation indicate that there was enough to be of annoyance to residents in the area. Presumably, it exceeded, to a considerable degree, the amount of asbestos dust present in the general air of New York City. The number of asbestos bodies found in ashed sections of lung tissue from the “Paterson neighborhood” men was considerably greater than the number found in sections from the New York City men. However, based on a sample of only 19 pairs, the difference was not statistically significant. Therefore, it can only be said the findings were not inconsistent with the hypothesis that the increased number of asbestos fibers in the “Paterson neighborhood“ was accompanied by an increase in the number of asbestos bodies in lung tissue. REFERENCES 1. Selikoff, I. J., Churg, J.. and Hammond. E. C. (1964). Asbestos exposure and neop1asia.J. Amer. Med.
Assoc.
188, 23.
1,. Selikoff, 1. J., Hammond. E. C.. and Churg. J. (1968). Asbestos exposure. smoking and neoplasia. J. Amer. Med. Assoc. 204, 106. 3. Bogovski. P. rf NI. (Eds.) (1973). The Biological Effects of Asbestos.” Lyon, France. International Agency for Research on Cancer 1973. 4. Gross, P., Cralley, L. J., and de Treville. R. T. P. (1967). Asbestos bodies: Their nonspecificity. Amer.
Ind.
H.vg.
Assoc.
J. 28, 541-542.
5. Cunningham, H. M. and Pontefract, R. D. (1973). Asbestos fibers in beverages, drinking water and tissues: Their passage through the intestinal wall and movement through the body. J. A.ssoc. Ofj’. Ad. Chrm. 56, 976-981. 6. Auerbach, 0. ( 1977). “Extra-Pulmonary Asbestosis.” Presented at the Symposium on Oecupational Pulmonary Diseases at Baylor College of Medicine. Houston, Texas on February 26. 7. Ulrich. S.. Berg. T. J., and Hedlund, D. (1972). Superior Polluter. Save Lake Superior Association and Northern Environmental Council, Duluth, Minn.. Appendix, pg. f.. October. 8. “Asbestos in the Great Lakes Basin.” ( 1975). A Report to the International Joint Commission from the Great Lakes Advisory Board, February. 9. Cook, P. N.. Rubin, I. B., Maggiore. C. J., and Nicholson. W. J. (1976). X-ray diffraction and electron beam analysis of asbestiform minerals in Lake Superior Waters. In “Proceedings, International Conference on Environmental Sensing and Assessment,” Paper 34-l. Vol. 2. Institute of Electrical and Electronics Engineers, New York.
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.4SBESTOS
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IN
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