Document kNQVkYn4eZmLaYRL2QDBvzJb

PLAINTIFF'S EXHIBIT ASA-697 Reprinted from the Archives of Pathology May 1968, Vol. 85 Copyright 1968, by American Medical Association Printed and Published in the United States of America . 539 Pulmonary Ferruginous Bodies Development in Response to Filamentous Dusts and a Method of Isolation and Concentration Paul Gross, MU; and Robert T. P. deTreville, MD, DSc, Pittsburgh; Lewis J. Cralley, PhD, Cincinnati; and J. M. G. Davis, PhD, Cambridge, England Formation of ferruginous bodies should not be confused with pathogenicity. Failure to un derstand this differentiation may result in the erroneous generalization that all fibrous dusts share the ability of abestos to produce lung damage. Such materials as fibrous aluminum silicate, silicon carbide whiskers, cosmetic talc, and glass fibers produce ferruginous bodies experimentally which are indistinguishable from those produced by asbestos fibers. A method of isolation and concentration of fer ruginous bodies from lungs of animals and humans is described. Ferruginous bodies from asbestos fibers are much more pleomorphic than has generally been described, casting further doubt on morphological distinctions used in the past in separating so-called as bestos bodies from pseudoasbestos bodies. WcORLDWIDE attention was refo cused on ferruginous bodies by the publi cation of Thomson,1 who found this phenomenon in the lungs of more than 30% of unselected autopsied adult hospital patients in Capetown, South Africa. A similar percentage was noted in Miami,2 43% in Pittsburgh,3 and 48% in Mon treal:4 The higher percentages reported in Pittsburgh and Montreal are possibly inherent in the more intensive searches Accepted for publication on Oct 17, 1967. From the Industrial Hygiene Foundation. Pitts burgh (Dr. Gross and Dr. deTreville); the National Center for Urban and Industrial Health (Dr. Cral ley); and the Department of Pathology, British Asbestosis Research Council, University of Cam bridge, Cambridge, England (Dr. Davis). Reprint requests to the Industrial Hygiene Foun dation, 4100 Fifth Ave, Pittsburgh 1S2I3 (Dr. Gross). carried out in these cities. These bodies are similar to asbestos bodies, though the natures of the central fibers have not been identified. Asbestos bodies are golden-brown, ferro-coated formations found in the lungs of persons who have inhaled asbestos dust. They are generally described as sym metrical, segmented structures, usually with clubbed ends, 3p to 5n in diameter and 20p to 50p long. The core is composed of a transparent colorless asbestos fiber that is not always demonstrable. Apparently the only difference between an asbestos body and a pseudoasbestos body is that in the former, the central fi ber is composed of asbestos, and in the latter, of material other than asbestos. Since asbestos-like bodies can form in response to respirable, transparent, color less fibers deposited in the lungs and com posed of materials other than asbestos; and since basing classification of these structures upon identification of the cen tral fiber presents difficulties, a generic term, "ferruginous," has been proposed for all bodies formed in response to the presence (in body tissues) of a broad spectrum of fibers, including asbestos. Davis 5 and Collet (according to a letter in June 1966) demonstrated with the elec tron microscope that ferruginous bodies are formed within macrophages by gran ules of ferritin or a ferritin-like protein Arch Path--Vol 85, May 1968 ASARCO ELP 0003246 J 1 540 FERRUGINOUS BODIES--GROSS ET AL do not result in the forma tion of ferruginous bodies when inhaled. Respirable fibers, how ever, are apparently ubiq uitous.8 They may be mineral, animal, or vege table in nature and of either natural or synthetic origin. They are dissemi nated by industrial proces sing, community activities, personal habits, and the action of natural forces. Fig 1.--Asbestos bodies from the lung of an asbestos worker to illustrate some of the more simple forms that may be found. In addition to some apparently naked fibers, there are pale, nonsegmented, rodlike bodies with bipolar clubbing. The dust was probably chrysotile (concentration method; smear; unstained; X 1.100). Also, we have recently re ported experimental pro duction of ferruginous bodies with ceramic fi bers of aluminum silicate. that are precipitated upon and around some foreign materials. An asbestos body, therefore, is only one kind of ferruginous body; one in which the central filament is an asbestos fiber. As will be seen later, the appearance and dimensions of these bodies are so varied as to defy the reasonably short description These bodies were indistinguishable by light microscope from many of those iso lated from an asbestotic lung of a known asbestos worker T (Fig 1). Although non segmented, they were golden-yellow, sym metrical, clubbed bodies, staining deep blue with Peris' test and exhibiting a central transparent filament. usually employed. The present paper discusses our further The problem now confronting investi findings as follows: gators concerns the significance of the 1. --Ferruginous bodies are developed in widespread finding of ferruginous bodies in the lungs of urban population groups. The solution of the problem is, of course, linked to the identity of the central fiber the lungs of hamsters in response to the presence of "biologically inert" filament ous aluminum silicate, glass, and silicon carbide particles. about which the ferruginous body forms and which is at present unknown. It is hoped that recent analytical advances, such as electron diffraction and micro probe will provide techniques for defini tive identification of the central fiber. 2. --A simple method is given for isolat ing ferruginous bodies and bare fibers (including asbestos bodies) from lungs. The method is given in detail and the re sults obtained are described briefly. Up to this point, the identification of ferruginous bodies in the lungs of un selected autopsied hospital patients as as bestos bodies1 has been based on the hypothesis that transparent fibers of re spirable size composed of materials other than asbestos either are not encountered in industrial and community environ The Production of Ferruginous Bodies Groups of 12 hamsters each were in jected intratracheally with 3.5 mg of fibers contained in 0.5 ml of aqueous suspen sions. This was done under light ether anesthesia with the aid of an illuminated, self-retaining speculum that made the vo- ' ments, are not deposited in the lungs, or cal chords visible and allowed the inser- Arch Patk--Vol 85, May 1968 ASARCO ELP 000324-7 finM- in FERRUGINOUS BODIES--GROSS ET AD 541 tion of a long 18-gauge needle between, the vocal chords under direct observation. All of the following were injected: 1. Ceramic aluminum silicate fibers. This is an uncoated ceramic fiber with a median diameter of 2p. Fifty percent of the fibers were under 75u in length, and many filaments were shorter than 15+ No free silica was detected in the fibers. 2. Silicon carbide whiskers. These were 99.5+% SiC. Fiber diameter ranged from 0.5tt to 3(i, and fiber length that ranged from lOOp. to 750+ 3. Glass fibers, uncoated. The fibers had a mean diameter of 0.4u and a mean length of 4.4+ 4. Cosmetic talc. Fifty percent of the fibrous material in the talc was under 0.2u in diameter and l[i in length. 5. Attapulgite (fibrous clay mineral). Fifty percent of the fibers were under O.lu in diameter and l|i in length. 6. Chrysotile (95% of the asbestos used on this continent is chrysotile). Most of the fibers were of ultramicroscopic di mensions. Isolation and Concentration of Ferruginous Bodies Samples of lung tissue cut into thin strips 3 to 4 mm thick, or fragments about 0.5 cc in volume, are placed in clean glass or plastic containers. To the tissue is added about 20 times the tissue volume of commercial 5%'sodium hypochlorite solution. This is allowed to stand undis turbed at room temperature for several hours until all chemical action has ceased. More hypochlorite solution is then added at frequent intervals until the tissue has been digested. For small lungs, such as those of rats and guinea pigs, the frequency and amount of addition of fresh hypochlorite solution should be such that all lung tissue is digested in approximately 24 hours. For human lungs, unless quantitative recovery of ferruginous bodies is desired, complete digestion is not necessary. In the case of human lungs, the fer ruginous bodies and bare fibers are often associated with a sticky lipidic film ad herent to the bottom of the container. The stickiness allows one to pour off all the fluid and undigested lung tissue without loss of the bodies and fibers. Because of the presence of anthracotic pigment, the film is usually gray in color. The film is dissolved by vigorously washing with a mixture of one volume of chloroform and two volumes of approximately 50% ethyl alcohol; the total volume should be the minimal amount needed to remove all the film. The wash fluid is centrifuged at 2,000 rpm for about five minutes. Because .of their high specific gravity, the fer ruginous bodies and the insoluble mineral particles settle to the bottom of the tube. On the other hand, most of the carbonace ous material collects at the interphase be tween the chloroform and the aqueous alcohol. If too much alcohol is used, the carbonaceous material will lose some of the water that lowers its specific gravity. As a result, there will be no separation between the anthracotic material and the ferruginous bodies. In such cases, rehy dration followed by the addition of chloro form will usually effect a good separation. All of the fluid and the carbonaceous, more viscid material above the sediment at the bottom of the tube are discarded, and the walls of the tube are cleaned of the adherent anthracotic material. The sediment is washed several times with water to remove all hypochlorite and other water-soluble materials. It is then stored in an aqueous or alcoholic medium. When smears are made of the suspen sions, it may be advisable to dehydrate the smear and use a mounting medium to render much of the mineral dust associ ated with the ferruginous bodies less con spicuous. The naked fibers remain visible. In the case of small animal lungs, chloroform and the supernatant fluid were poured into centrifuge tubes in a propor tion of 1:2. After centrifuging, the super natant fluid was carefully removed and discarded except for about 1 ml left un- Arch Path--Vol 85, May 1968 J ASARCO ELP 0003248 J l 542 FERRUGINOUS BODIES--GROSS ET AL disturbed on the bottom. The film on the bottom of the original container in which the lung tissue was digested was then re moved, as in the case of human lungs. This fluid was added to a pool of whatever sediment was obtained from the super natant fluid. The subsequent procedure was the same as with human lungs. We have worked with formalin-fixed tissue only, but there appears to be no reason why this method should not work equally well with fresh lung tissue. Results Paraffin Sections.--Initially the fer ruginous bodies were sought only in paraffin sections that had been stained with hematoxylin and eosin or Peris' test for iron. Because of the paucity and small size of these bodies in the sections, the hematoxylin made the search more diffi cult. Subsequently, replicate sections that were given the Peris' test only, or also lightly counterstained with eosin and cleared, proved to be satisfactory. The injected fibers generally were con fined to the air spaces, where they were associated with free macrophages. Some of the filaments passed through the bodies of one to three, and even four, macro phages so that these cells appeared to be impaled as though upon a spit (Fig 3 bot tom left). When the sections had been stained for iron, the dust-containing areas, under low magnification, were usually marked by a granular deep blue colora tion. In sections of lungs of hamsters killed one month after an intratracheal injection of aluminum silicate and glass fibers, oc casional ferruginous bodies were found. These were non-segmented, light-yellow structures with bipolar clubbing and a transparent central filament (Fig 2 top left, and 3 top left). These, subsequent to the Peris' test, took on a deep blue color that often obscured the central filament. Very similar non-segmented bodies were seen in suspensions of isolated and con centrated human asbestos bodies derived from an asbestotic lung of a worker known to have been exposed to chrysotile asbestos dust for 30 years (Fig 1). The ferruginous bodies that formed in response to chrysotile asbestos were smaller than those that formed in response to aluminum silicate and glass, and also different from the latter two in being seg mented (Fig 4 bottom left). The ferrugin ous bodies were more readily found in the lung sections of hamsters injected with aluminum silicate filaments than in lung sections of animals injected with filament ous glass or chrysotile dust. No ferrugin ous bodies were found in the lung sections of hamsters injected with silicon carbide filaments at this time (one month after the injection). Examination of lung sections of ham sters killed six months after the intra tracheal injection of the filamentous dusts revealed no more ferruginous bodies than were encountered five months earlier. No segmented forms were found except in association with chrysotile dust. No fer ruginous bodies were seen in lung sec tions of hamsters injected with silicon carbide. Dust-containing alveoli found in sections of lung from a hamster injected with talc were easily identified because of the blue coloration caused by the pres ence of iron; however, ferruginous bodies could not be identified. Similarly, no ferruginous bodies were found in lung sections from hamsters injected intratracheally with attapulgite. Lung Digests.--The smears of the sedi ment derived from the digestion of the lungs from hamsters injected six months previously with aluminum silicate, glass, and chrysotile, respectively, consisted largely of naked filaments, but many fer ruginous bodies were also seen. Although most of the ferruginous bodies that had formed in response to aluminum silicate and glass fibers were non-segmented, a number of segmented forms were also found (Fig 2 top right, bottom left, bottom right; and 3 top right, bottom left, bottom right). A more prolonged search of the Arch Path--Vol 85, Mag 1958 ASARCO ELP 0003249 FERRUGINOUS BODIES-^GROSS ET Ah 543 sediment from lungs of hamsters injected with silicon carbide was necessary before several ferruginous bodies could be found. This search was facilitated by applying Peris' test for iron to the smear. The bod ies were mostly nonsegmented, and except for the clubbed ends, the coating on the filaments was thin. One silicon carbide fiber was found with a club-shaped coat ing at one end, a fusiform coating near the other end, and a very slight thicken ing near the middle (Fig 4 top left). Two segmented ferruginous bodies that had formed around silicon carbide fibers were observed. Unfortunately, the diffusion of the blue pigment of the body rendered its outline indistinct (Fig 4 top right). The sediment from a hamster lung in jected with talc was composed largely of crystalline plates; but upon careful search, a number of non-segmented ferruginous bodies were also found. These too had a transparent central filament (Fig 2 top left, and 3 top left). The Prussian blue re action for iron had also been used in this smear, and the outlines of these bodies were indistinct because of the diffusion of the pigment (Fig 4 bottom left). Unexpectedly, ferruginous bodies were more difficult to find in lung sections after chrysotile injections than after aluminum silicate injections: The same finding was observed with the concentration method. Although few in number, all asbestos bod ies were segmented (Fig 4 bottom right). One of the most interesting observations was that, when stained for iron, filaments that passed through the bodies of one or more, macrophages showed a coating of ferritin or ferritin-like material only on the intracellular portions of the filament (Fig 3 bottom left). The sediments from the lungs of ham sters injected with attapulgite revealed no ferruginous bodies. The failure to find ferruginous bodies in these sediments may be interpreted as a negative control to indicate that our positive findings were not attributable to the accidental inhala tion of fibers in the ambient laboratory air. The sediment from the human asbestotic lung was a rusty, red-brown color. In the smear, a wide variety of asbestos bodies were seen in addition to innumer able naked fibers. As previously indicated, a large number of the asbestos bodies were pale, thin, and nonsegmented. Also of interest was the fact that many of the largest bodies did not have smooth sur faces, but were spiculated. The spicules Fig Z--Ferruginous bodies formed in response to aluminum silicate filaments in a hamster killed five months after an intratracheal injection of 3.5 mg of the dust. Bottom right, naked filament (concentration method; unstained smear; x 1400}. Arch Path--Vol 85, May 1368 ASARCO ELP 0003250 544 FERRUGINOUS BODIES--GROSS ET Ah were often, coarse with squared ends. A few of the bodies lacked symmetry. Some pear-shaped bodies without a central fila ment were also seen. Although most of the bodies were more or less rectilinear, many curvilinear forms were also present. Some of the latter appeared to measure between 180 to 270*. In the larger spicu.lated forms, the central fiber was often obscured by the thick, dense, red-brown coating. Some of the very long thin fibers (50p. to lOOu) often had segments of ferro- Fig 3.--Ferruginous bodies formed in response to filamentous glass dust. Bodies (top, left and right, and bottom right) are unstained. Four macrophages are faintly outlined. Only the intracellular portions of the filament are coated with iron (Peris' stain, X 1,100). protein at either end with relatively long stretches of naked fiber in between. In an attempt to free asbestos bodies from adherent fine carbon particles in an otherwise "clean" suspension, it was sub jected to ultrasonic vibrations for a few seconds. Unexpectedly, only naked fibers remained, and no asbestos bodies could be found in the suspending fluid. The distinction heretofore made be tween asbestos bodies and pseudo-asbestos bodies may have been based in part on what the observer believed to be an ap pearance consistent or inconsistent with that of asbestos bodies. More often, how ever, this distinction was based on the observer's knowledge that the host had or had not been exposed to respirable asbestos fibers. Regardless of the nature of the central fiber, the bodies that result in response to the presence of filamentous dust in the lung have as a common feature a coating of iron-containing protein (ferritin or fer ritin-like) . Furthermore, it appears, from a study of the pleomorphism of human asbestos bodies, which was so well illus trated by Gloyne and Merewether,8 that differences in size, segmentation, or other morphologic features of the ferruginous coating probably would not serve to dis tinguish between ferruginous bodies of asbestotic origin and those of nonasbestotic origin. Thomson has proposed to differentiate between asbestotic and nonasbestotic fer ruginous bodies on the basis of the trans parency or opacity of the central fiber.2 This proposal seems inappropriate, inas much as we have demonstrated that a number of transparent fibers of respirable size other than asbestos are capable of producing ferruginous bodies that are in distinguishable from those produced by asbestos. At any rate, it is obvious from the pro duction of ferruginous bodies in hamsters in response to respirable, colorless, trans parent filaments of aluminum silicate, glass, and silicon carbide, that such bodies Arch Path--Vol 85, May 1968 I) ASARCO ELP 0003251 J FERRUGINOUS BODIES--GROSS ET AL 545 represent a general reaction to filamentous years ago because of overwhelming evi particles* and are not a specific reaction to dence against its validity. Just as the asbestos fibers. To what extent our find proven biologic "inertness" of diamond ings may have application to the fer dust was the coup de grace for the mecha ruginous bodies that are being found with nistic pathogenetic concept of silicosis,* so increasing prevalence in human lungs1-4 should the proven biologic "inertness" of remains to be determined. The applicabil filamentous aluminum silicate10 have dis- ity depends, of course, upon the identity of_ _credited the mechanistic pathogenetic con the central fiber in the ferruginous bodies. cept of asbestosis. The method of isolation and concentration of uncoated fibers and ferruginous bodies from human lungs which is described in this paper may help in the study of the Extremely thin flakes of glass may also be considered to have sharp cutting edges; yet, glass has also been found biologically nature, identity, and prevalence of inhaled filamentous particles and associated pul monary ferruginous bodies. It is highly probable that the composi tion of the ferruginous body is altered by Fig 4.--Ferruginous bodies formed in response to other filamentous dusts and stained for iron (Peris' test). The central fiber (top, (eft and right) is silicon carbide; and (bottom left) tremolite (talc) (X 1,110). II the method used for isolating it, since it is not likely that with the destruction of proteins leading to the liquefaction of all 10 ft- tissue elements in the lung, the protein in the ferruginous bodies should be spared. In all likelihood, the protein constituent of the bodies is destroyed, and only the iron and other inorganic components re main to retain and maintain the form of the bodies. The fragility of this chemically altered ferruginous coating is indicated by the ease with which it is removed when subjected to ultrasonic vibrations, thereby rendering the central fiber naked. This may further facilitate the identification of these fibers. There appears an increasing tendency to consider all filamentous dusts in the same category as asbestos with regard to their pathogenic potential. This reasoning is probably based on a mechanistic con cept of the pathogenicity of fibrous dust in general, and asbestos dust in particular. Such a mechanistic concept holds that the pulmonary fibrosis in asbestosis is the tis sue response to mechanical trauma pro duced when pulmonary cells are perfor ated or impaled by the fine points of the asbestos fibers. It need only be recalled that a similar mechanistic concept of the pathogenicity of crystalline silica was abandoned many Arch Path--Vol 85, May 1968 ASARCO ELP 0003252 546 FERRUGINOUS BODIES--GROSS ET Ah "inert" when inhaled or injected into the lungs of animals. Silicon carbide is noted for its hardness, sharp edges, and points, which make it an ideal abrasive. When inhaled as a fine dust or injected intratracheally, it has caused a pulmonary re sponse likewise classified as biologically "inert." Gaining increased attention is a newer concept that the potential of extraneous trace metals and other materials associ ated with fibrous minerals can cause the injury previously attributed to fibers. The respirable fibers may provide a transport mechanism for dosing tissues with injuri ous materials associated with the fibers. Cralley et al13 have shown that asbestos textile workers in the past have been ex posed to appreciable concentrations of nickel, chromium, and manganese associ ated with the fibrous mineral, and abraded from the alloy metal in asbestos process ing equipment. They state further that there is evidence that this phenomenon exists in relation to a number of other fi brous minerals. There is also some indi cation that the biological response in the formation of ferruginous bodies may be related to the nature and extent of the layer of metal solute surrounding the fi ber. Additional research needed in these areas is currently underway. Unless the above facts are kept in mind, the finding that ferruginous bodies are formed in response to aluminum silicate, silicon carbide, and glass filaments in the lungs, may be used as still another reason for erroneously classifying these dusts with asbestos in their ability to produce lung damage. This investigation was sponsored by JohnsManville Corporation, New York, and supported in part by Public Health Service research grant UI 86-66-156 from the National Center for Urban and'Industrial Health. All experimental materials used, except chrysotile (supplied by Johns-Manville Corporation, New York) were obtained from and analyzed by the US Department of Health, Education and Welfare, Public Health Service Center for Urban and In dustrial Affairs, Occupational Health Program. References 1. Thomson, J.G.; Kaschula, R.O.C.; and Mac Donald, RJb: Asbestos as a Modem Urban Hazard, S Air Med J 37:77-81 (Jan) 1963. 2. Thomson, J.G.; Path, F.C.; and Graves, WM.: Asbestos as an Urban Air Contaminant, Arch Path 81:458-464 (May) 1966. 3. Cauna, D.; Totten, RB.; and Gross, P.: As bestos Bodies in Human Bungs at Autopsy, JAMA .192:371-373 (May) 1965. 4 Anjilvel. iZ, and Thurlheck, Wit: The Inci dence of Asbestos Bodies in the Lungs at Random Necropsies in Montreal, Canad Med Assoc J 95: 1179-1182, 1966. 5. Davis, JM-Gd Electron-Microscope Studies of Asbestosis in Man and Animals, Ann NY Acad Sci 13238-111, 1965. 6. Cralley, LX, et al: Source and Identification of Respirable Fibers, read before the annual meet ing of American Industrial Hygiene Conference, Chicago, May 1967, JAIHA, to be published. 7. Gross, P.; Cralley, LX; and deTreville, R.TP4 "Asbestos" Bodies: Their Nonspecificity, JAIHA 28:541-542 (Nov-Dec) 1967. A Gloyne, SR., and Merewether, EitA.: "As bestos," International Labour Office Supplement, p 7 (Jan) 1938. 9. King, EX; Yoganathan, M.; and Nagelschmidt, G.: The Effect of Diamond Dust Alone and Mixed With Quartz on the Lungs of Rats, Brit J Industr Med 1532-95, 1958. 10. Gross, P, et ah The Effect of a Synthetic Ceramic Fiber Dust upon the Lungs of Rats, Arch Induat Health 13:161-166 (Feb) 1956. 11. Gardner, L.U.: Studies on the Relation of Mineral Dusts to Tuberculosis, Fart II: The Rela tively Early Lesions in Experimental Pneumoconio sis Produced by Carborundum Inhalation and Their Influence on Tuberculosis, Am Rev Tuberc 7344, 1923. 12. Gross, P.; Westrick, MX.; and McNeroey, J. M.: Experimental Tuberculopneumoconiosis, Arch Indust Health 19320-334 (March) 1959. 13. Cralley, LX; Keenan, R.G.; and Lynch, J.R.: Exposure to Metals in the Manufacture of Asbestos Textile Products, read before the annual meeting of American Industrial Hygiene Conference, Chicago, May 1967, JAXHA 28:452-461 (Sept-Oct) 1967. Arch Path--Vol 85, May 1988 ASARCO ELP 0003253