Document O14x2GQVqDBak06M6ZgNE7X11

t L I EXHIBIT I * DOW-344____ I (J (C Rtprimrd from tho Arckivtt of Pathology Hay J96, Pol. tS Copyright 1961, by Amoriem Modietl Auodatie* Pnmtod tad Pobkthei m tho Umtod Stator of Amerua Pulmonary Ferruginous Bodies Peel Cram, JO; Uwta J. CnOoy, PhD, CO --H O o cn ro Formation ef femigM&i boditt dmeU not bo confused wttb patiganicHy. Faflure to anderatand tbit differentiation may result in tbo wtonoooi generefixatien tbot al (Sbrout dials boro tbo tbitty of ebestos to prodoeo lung damage. Such materials ot fibrous aJumuwm dfieate, liRcon corbido wbitkon, counottc talc, and flan fiber* produce form91nous bodiot osporimontaly which aro indistinguishable from thoae produced by atbottot fiber*. A method of isolation and concentration of fer ruginous bodiot from lungs of animab and carried out in thoae cities. Those bodies are to asbestos bodies, though the natures of tho central fibers have not been identified. Asbestos bodies are golden-brown, ferro-coatcd 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 5p in diameter and 20p to 90p long. Tha core is composed humem it described. Ferruginous bodiot from atbottot fiber* are much more pleomorphic than hat goneraly boon described, catting further doubt on morphological distinction! utad in tho pait in separating o catted as bestos bodiot from pseudoaibettos bodiot. 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, end in the WORLDWTDE attention was refo latter, of material other than asbestos. cused on ferruginous bodies by tho publi cation cf Thomson,1 who found this phenomenon in tho lungs of more than 30% of unselected autopaiod adult hospital patients in Capetown, South Africa. A similar percentage was notad in Miami,* 43% in Pittsburgh,* and 4S% in Mon treal4 The higher percentages reported in Pittsburgh and Montreal aro possibly inherent in the more intensive searches Acnetad ter publication an Oct IT. usr. 7roa the Industrial Hystae foundation. Pittsburgh (Dr. Gram and Dr. daTreviUa); the National Caatar tor Urban and Industrial Health (Dr. Crellop): and the Departamt ot Pathology, British A^xstoaas Kaaoanh Council. Ururwtoty of Coat bridge, Cambridge, Bigland (Dr. Davit). Reprint regueeto to die Industrial Hyfieoe Foun dation. 44M Ftfdi Ave. Pittoburgh 1S2U (Dr. Groat). Since ssbestoe-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, ineluding asbestos. Davis * and Collet (according to e letter in June 1963) demonstrated with the elec tron microscope that ferruginous bodies are formed within macrophages by gran ules of ferritin or a ferritin-like protein Arch PtA--Vol IS, Msv 1KI DOW 06904 */ ST00II622 540 FZRRUG1SOVS BODIES--GROSS XT do not result in the forma tion of ferruginous bodies when inhaled. Respirable fibers, how ever, are apparently ubiq uitous.* 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 l.--AtbMh bodies from th lung f an MbwtM wwtor to Mwtrato lama of th mart tbnpla farm that mg ba found. In addition to aama apparently nakad fiber*. thara ara pala, nanaagmaniad. radRka bodla* with bipolar clubbing. Tha dual waa probably chryaatlla (eoneantrallan mathadi amaari unalainadi x I.UXA. 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 workerT (Fig 1). Although nonsegmented, 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 Hie problem now confronting investi gators concerns the significance of the widespread finding of ferruginous bodies in the lungs of urban population groups. The solution of the problem is, of course, findings as follows: 1. --Ferruginous bodies are developed in the lungs of hamsters in response to the presence of "biologically inert" filament ous aluminum silicate, glass, and silicon linked to the identity of the central fiber 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 microprobe 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 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, in industrial and community environ self-retaining speculum that made the vo ments, are not deposited in the lungs, or cal chords visible and allowed the inser- Arck Ptth--Vol S3, May lttt DOW 06905 S T00I1623 ntutvGnrovs bodizs--gross it al Ml tion of a loaf 18-gauge needle between tha vocal chords under direct observation. AH of tha following war* injected: 1. Ceramic aluminum silicate fibers. This is an uneoatad caramic fiber with a median diameter of 2p. Fifty ptw-tnt of tha fiban ware undar 75p in length, and many filaments ware shorter than 15|i. No free silica was detactad in tha fibers. 2. Silicon carbide whiskers. These ware 99.5+% SiC. Fibar diameter ranged from 0.5p to 3>i, and fibar length that ranged from lOOp to 750}*. 3. Glass fibers, uneoatad. Tha fiban had a mean diameter of 0.4|i and a mean length of 4.4(1. 4. Cosmetic talc. Fifty percent of tha fibrous material in the talc was under 0.2|i in diameter and l|i in length. 5. Attapulgita (fibrous clay mineral). Fifty percent of tha fiban ware undar 0.1(i in diameter and l(i in length. 6. Chrysotile (95% of the asbestos used on this continent is chrysotile). Most of the fiban ware of ultramicroscopic di mensions. Isolation and Concentration of Ferruginous Bodies 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 tha pneance of anthraeotic pigment, Use film is usually grey in color. The film is dissolved by vigonrasly washing with a mixture of one volume of chloroform and two volumes of approximately 50% ethyl alcohol; the total volume should be the 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 anthraeotic material and the ferruginous bodies. In such cases, rehy dration followed by the addition of chloro form will usually effect a good separation. 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 tha tissue volume of commercial 5% hypochlorite solution. Ibis 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 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 anthraeotic 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- AreX PtX--Vol tS, May 1WI DOW 06906 S T 0 0 I1624 Ml FERRUGINOUS BODIES--GROSS ET AL disturbed on tbe bottom. The Aim on the from an asbestotic lung of a worker bottom of the original container in which known to have been exposed to chrysotile the lung tissue was digested was then re asbestos dust for 30 years (Fig 1). moved, as in the case of human lungs. This The ferruginous bodies that formed in fluid was added to a pool of whatever response to chrysotile asbestos were sediment was obtained from the super smaller than those that formed in response natant fluid. Hie subsequent procedure to aluminum silicate and glass, and also was the same as with human lungs. different from the latter two in being seg We have worked with formalin-fixed mented (Fig 4 bottom left). The ferrugin tissue only, but there appears to be no ous bodies were more readily found in the reason why this method should not work lung sections of hamsters Injected with equally well with fresh lung tissue. aluminum silicate filaments than In lung sections of animals injected with filament Results ous glass or chrysotile dust No ferrugin Paraffin Sections.--Initially the fer ruginous bodies were sought only in paraffin sections that had been stained with hematoxylin and eoain or Peris' test ous bodies were found in the lung sections of hamsters injected with silicon carbide filaments at this time (one month after the Injection). 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 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. tion. Lung Digests.--Hie smears of the sedi In sections of lungs of hamsters killed ment derived from the digestion of the one month after an intratracheal injection lungs from hamsters injected six months of aluminum silicate and glass fibers, oc previously with aluminum silicate, glass, casional ferruginous bodies were found. and chrysotile, respectively, consisted These were non-segmented, light-yellow largely of naked filaments, but many fer structures with bipolar clubbing and a ruginous bodies were also seen. Although transparent central filament (Fig 2 top most of the ferruginous bodies that had left, and 3 top left). These, subsequent to formed in response to aluminum silicate the Peris' test, took on a deep blue color and glass fibers were non-segmented, a that often obscured the central filament. number of segmented forms were also Very similar non-segmented bodies were found (Fig 2 top right, bottom left, bottom seen in suspensions of isolated and con right; and 3 top right, bottom left, bottom centrated human asbestos bodies derived right). A more prolonged search of the Arch Path-Voi IS, May 1WI DOW 06907 ST0011624 STOOI1625 FtRRUGIXOUS BODIZS--GROSS ST AL MS sediment from lungs of hamsters injected tion of fibers in the ambient laboratory with silicon carbide u Mcaaqr before several ferruginous bodies could be found. This leerch wot facilitated by applying Paris' test for iron to the smear. The bod ies were mostly nonaagmanted, sad except for the dabbed ends, tbs coating oa ths filftncBts was thin. On ctibidt fiber was fooad with a dub-shspad coat- lag at oaa sad, a fusiform eoatiag near the other sod, aad a very slight thicken- ing aaar the middle (Tig 4 top left). Two air. The from the human ashes totic hmg was a rusty, rad-brown color. In the sneer, a wide variety of asbestos bodies ware seen in addition to innumer able naked fibers. As previously indicated, a large ---*-- of the asbestos bodies wars pale, thin, and nonaagmanted. Also of Interest was the fact that many of tea largest bodies did not have smooth su^ faces, but were spiculated. The spicules segmented ferruginous bodies that had formed around silicon carbide fibers were observed. Unfortunately, the dlffndon of the blue pigment of the body rendered its outline indistinct (Fig 4 top right). In Mwmw Mime Me iwne Mr m InlrsBWi Inlsctlw W XS me > ttm ewe. SetM rt(M, mm fiiaiMnt (lenwnween mem* uns4MnaS homo X US* The sediment from a hamster hmg in jected with talc was romposid largely of crystalline plates; but upon careful search, a number of noa-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 weremore 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, ell 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 Arch Puk--Vot SS. Mv IMS DOW 06908 ST0011626 144 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 .^la 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 spiculated forms, the central fiber was often obscured by the thick, dense, red-brown coating. Some of the very long thin fibers (50(i to 100(i) often had segments of ferro- Fl* t--FsmiglnoM bodiM forraad In pmpoam to fllsmantou* (its* duit SodMs (las, Ml aad rl|M, and eattam rliMj are unitalnad. Four macraphasaa era falnlly autllnad. Only lira Iniraoailular partlanf of tha fllamant ara cantad witti Iran (Faria' train, x MSM. 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 "dean" 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 hung have as a common feature a coating of iron-containing protein (ferritin or ferrltin-like). Furthermore, it appears, from a study of the pleomorphism of human asbestos bodies, which was so well illus trated by Gloyne and Merewether,* 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.* 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 Pnh--Vol is, Jfev IMS DOW 06*30^ S tool 1627 rtutvamous bodixs--cross tr kl US represent a general reaction to filamentous years ago because of overwhelming evi particles and ax* not a specific reaction to dence against its validity. Just as the aabaatoa fibers. To what extant our find* proven biologic "Inertness" of diamond inga may have application to toe 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 hinge14 should the proven biologic "inertnam" of remains to be determined. The applicabil filamentous alumtoun silicate ** have<*1*- ity dependa, of course, upon the identity of credltod the mechanistic pathogenetic con the central fiber in the ferruginous bodies. The method of isolation and concentration of uncoated fibers and ferruginous bodies from human lungs which is described fat this paper may help in the study of the cept of ashsetnsls Extremely thin flakes of glass may also be considered to have sharp cutting edges; yet, glass has also bean found biologically nature, identity, and prevalence of inhaled filamentous pertides and associated pul monary ferruginous bodies. It is highly probable that the mmpoai- Pig . rwn&rmm Beales formas to) rmesnee W odiar fUamanawa Susa ana atslnoa far Iran (Carta1 taaQ. The oanval near (Me, MR ana rtfBO M aiiMon rn'iii ana (kaaan MR) tramoun (taM) <x LUO. tion of the ferruginous body is shared by the method used for isolating it, since it is not likely that with the destruction of proteins leading to the liquefaction of ell 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 end 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. Ibis may further facilitate the identification of these fibers. There appears an increasing tendency, to consider ell filamentous dusts in the same category as asbestos with regard to their pathogenic potential This reasoning is probably based on e mechanistic con cept of the pathogenicity of fibrous dust in general, end asbestoe 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 e similar mechanistic concept of the pathogenicity of crystalline silica was abandoned many Arth Pttk-Vol IS. Ifsv IMS DOW 06910 STOOI 1628 Stf ferruginous bodies-gross et al "Inert" when inhaled or injected into the lung* 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"11J* 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 al11 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 w sponsored by JohnsMtavllle Corporation, Now York, and supported In part by Public Health Service research (rent UI M-U-1K from the National Center to Urban end Industrial Health. All experimental materials used, except ehryaotHe (supplied by Johnt-Manville Corporation, New York) wera obtained from and analysed by the US Department of Health, Education end Welfare, Public Health Service Center for Urban and In dustrial Affair*, Occupational Health Program. References 1. Thomsen, J.O.; Kaachula, R.O.C.; and Mac Donald, RJR.: Asbestos aa a Modern Urban Hazard, 8 Air Med J (Jen) IMS. 1 Thomson, J.G.; Path, Y.C.; and Graves, WMj Asbestos aa an Urban Air Contaminant, Arch Path U:4St-IM (May) IMS. *. Cauna, D.; Totten, RB.; and Gross. P.: As bestos Bodies In Human Lungs et Autopsy, JAMA 1*1371-373 (May) IMS. 1 Anjllvel, L., and Tburlbadt, Yit: The Inci dence of Asbeatoo Bodies In the Lwp at Random Necropsies In Montreal, Canod Med Assoc J IS: uTS-iiSh lies. S. Davte, Hadron-Microscope Studies of Aabeitosla in Man and Animals, Am NT Acad Set 1U:M-U1. IMS. 1 Cralley. L4-. d el: Source and Identification of Respirable Fiber*, reed befor* the annual meet ing of American Industrial Hygiene Conference. Chicago, May 1M7, JAIHA, to he published. I. Grass, p.; Crellty, LJ4 and deTrwilie, R-TPj "Asbestos" Bodies: Their Nonspedftclty, JAIHA KS41-S42 (Nov-Dee) 1MT. I. Gloyne, SJL, and Merewether, EJLA.: "As bestos," International Labour Ogles Supplement, p T (Jen) IMS. t. King, EJ.; Yogensthan, M.; and Nagdachmldt, O.: Tbs Effect of Diamond Dust Alone and Mixed With Quarts on the Lungs of Rats. Brit J fndustr Med 1S4MS, IMS. IS. Gross, P,, et al: The Effect of e Synthetic Ceramic Fiber Dust upon the Lungs of Rata, Areh Induet Health ]3:M1-1M (Feb) MSS. 11. Gardner, L.U.: Studies on the Relation of Mineral Dusts to Tuberculosis, Part II: The Rela tively Early Lesions to Experimental Pneumoconio sis Produced by Carborundum Inhalation and Their Influence on Tuberculosis, Am Ree Tubere m IMS. 12. Grata, P.; Wastrick, ML.; and McNemey, J. M.: Experimental Tubarculopnaumoconioais, Areh fnduet Heahh 11320-334 (March) 1SS. IS. Cralley, LJj Keenan, R.O.; end Lynch, J.R.: Exposure to Metals In the Manufacture of Asbestos Textile Products, read before (he annual meeting of American Industrial Hygiene Conference, Chicago, May 1MT, JAIHA 2MU-U1 (Sept-Oet) 1M7. Arch Path--Vo! IS, May 1M DOW 06911 STOO11628