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638 Reprinted from the Archives of Environmental Health April 1968, Volume 16 Copyright 1968. American Medical Association PLAINTIFF'S EXHIBIT Experimental Studies on the Progressiveness of the Pulmonary Fibrosis Caused by Chry6otile Dust Paul Gran. MD, and Robert T. P. dr Trrrillc. Pitt,burgh unreliability of symptoms as a crite termined. This is particularly true of asbes- rion for the progression of pulmonary fibro tosis. sis was underlined in a medical and psychi It is the purpose of this paper to define atric 6tudy of coal miners with respiratory some of the anatomic stigmata of progres complaints. Here, a very significant associa sion of asbeslosis and to determine whether tion was observed between a recognizable or not asbestosis caused by chrysotile dust is psychoneurotic factor and an apparent progressive. worsening of the clinical condition: of 40 patients studied, only 12.5% had disability Methods and Materials on "physical" grounds alone; 35% had dis ability on psychiatric grounds alone; and the remainder on both.1 Furthermore, discerning clinicians recog nize that opinions concerning the progres sion of a pulmonary fibrosis based upon the Lung burdens of chrysotile asbestos dust were imposed upon rats, hamsters, and guinea pigs by exposure in inhalation chambers as well as by intratracheal injection. Most of the ani mals exposed to dust in an inhalation chamber and reported in this study were part of a larger symptomatology (eg, shortness of breath) investigation that will be reported separately. and a roentgenologic evaluation, even when reenforced by lung function studies may be quite erroneous. In the presence of a superadded coincidental pulmonary illness, such signs and symptoms may be reversible in whole or in part between such episodes, which include covert and overt acute pneumonitides, pulmonary edema, and allergic The inhalation chamber in which all animals (except four guinea pigs) were exposed to dust, measured 8x8x8 feel. The animals were housed in wire cages that were suspended in racks. Periodically, these cages were rotated so that inequalities in dust exposure caused by po sition were largely obviated. The exposures were for Bix hours per day, five days per week. The total exposure varied, as listed in the table. or chronic bronchitis--with or without em The chrysotile asbestos was ballmilled and physema, or emphysema without bronchitis. then Ted into a hammennill (modified from a Such diagnostic difficulties were the subject of a recent symposium on emphysema in >ndustry. As uncertain as are the clinical criteria of progression of pneumoconiosis, very little help has come from postmortem studies of human lungs or those of experimental ani mals in defining the criteria by which design by Holt and Young:). This was provided with inlet and outlet tubing so arranged that the comminuted asbestos was fed back continu ally into the hammennill. At the same time, the ultrafines were allowed to waft upward into the inhalation chamber. Two of these hammermills provided the chamber with sufficient dust to av erage 86 mg/cu m with a range of 42 mg/cu m to 146 mg/cu m. The asbestos dust cloud was progression of pneumoconiosis may be de- evaluated by sampling with a two-stage sire se Submitled (or publication Aug 9. 1966; accepted Aug 15. From the Industrial Hygiene Foundation. Mellon Institute. Pittsburgh. Read before the Hatch Symposium. Graduate School of Public Health, University of Pittsburgh, Julv IB-19. 1966. Reprint requests to the Industrial Health Foundstion. Mellon Institute, 4400 Fifth Ave. Pittsburgh 15213 (Dr. Grose). lective device, similar in design to that pro posed by Wright' and operated at 20 li ters/minute. The first stage of the instrument was a horizontal elutriator with selector char acteristics recommended by the Johannesburg Pneumoconiosis Conference of 1959. namely acceptance of all particles having terminal set tling velocities greater than that of a 7.1s sphere of density 1 gm/cu cm. 50% acceptance Arch Environ Health--Vol lh. Nor 1967 SCF-FA-5980 8005 1458 PRODUCED BY FORD of particles, the terminal settling velocity of Anlmtlt Exposed to, or Injected Intratrachaally which it equivalent to 6s spherical particles of With, Chrysolite Asbestox Oust 1 gm/cu cm density, and dropping to xero ac ceptance of particles having terminal settling velocities equal to a Is sphere of 1 gm/cu cm density. All particles penetrating the first stage elutriator were considered "respirable" and were collected on a permeable-membrane filter which acted as the instrument's second stage. The filter was dried and weighed prior to and after collection of the sample. In this man ner, it was found that 63% to 79% of the sus pended asbestos dust in the exposure chamber was respirable, ie, its terminal settling velocity was less than that of a 7.1a sphere of 1 gm/cu cm density. The exhaust air from the inhalation chamber was drawn by suction through a pair of elec trostatic precipitators. The chamber air was consequently always under negative pressure as long as the hammermills were operating. Inhalation Exposure (Months) at Dust Intre- Time. Concentration trachaal tod of Special (average) injection Exposure No. (86 mg/ (20 mg/ No. 3.b-mg to Death Animals cu m) cu m) Injections (Months) Rati Total 2 6 6 6 4 4 5 b 4 )0 52 0.25 O.b 1 2 4 fi I 2 A 6 Hamster* ) J-13 13-24.5 11.25 24 11.25-23 0.5-21 3.5-17 21 21 21 21 When the hammermills were shut down, fresh air was pulled through the chamber before the 9 b 1 13.5-21 2 12.5-16.5 door was opened. Total 14 Periodically, dust from the electrostatic pre Guinea Pigs cipitators was collected and weighed. Since a record of the air flow through the chamber was 6 O.bb.b 43 0 0-4 kept, it was possible to calculate the average Total 10 dust concentration in the chamber air by divid ing the weight of the dust collected within a lion of the lung dust burden as shown in the time period by the volume of air flowing table. through the chamber within that time. All animals were autopsied. the lungs were As seen in the table, four guinea pigs were exposed to a concentration of chrysotile dust averaging about 20 mg/cu m. For this exposure, a smaller chamber was used and the dust was prepared by atomizing a suspension of ballmilled chrysotile asbestos at a pressure of 100 lb/sq in and impinging the emergent jet a nearsonic velocity against a tool-steel baffle. The av erage fiber length of this dust was 0.92s as de termined by measurements from electron pho tomicrographs. Inasmuch as other data on the size distribution of the dust are not available, it is not possible to state what percentage of the dust suspended in the chamber air was respira ble. These animals were exposed for three months to this dust. For intratracheal injections, the dust collect ed from the electrostatic precipitators was sus pended in water so that 1 ml contained 3.5 mg removed and expanded with 4% formaldehyde solution under a pressure of 12 cm water. Paraffin sections were cut at 6a. Cleared un stained sections were examined under darkfield conditions4 and appropriate fields were photographed on 35 mm film. These sections were then stained with hematoxylin and eosin and the same fields rephotographed. After de colorize tion and silver impregnation (Gordon and Sweets), the fields were photographed for the third time. A fourth photograph of the same fields was made after the sections had been subjected to microincineration at 600 C for an hour and treated with concentrated hy drochloric acid after cooling. Representative lesions in the lungs of control animals were also photographed for purposes of comparison with the experimental lessons. There were 16 rats, 29 hamsters, and 13 guinea pigs that had been part of the shipments of the dust. The rats and hamsters were injected in animals later put on test, but had been set tratracheal)}', some repeatedly, as listed in the aside away from the dust exposure as laborato table. The injections were made under light ether anesthesia with the aid of an illuminated speculum that allowed visualization of the vo cal chords. ry- controls. The results of this investigation are based largely upon a comparative study of about 700 fields selected from the sections of 76 animals. Some animals died at various times following the imposition of the dust burden. Others were killed approximately two years after the imposi- Results Rais (Intratracheal Injection).--Immedi- Arch Environ Health--Vol Jo, .Voo J9S? 8005 1459 PRODUCED BY FORD 640 EXPERIMENTAL ASBESTOSIS--GROSS A DE TREVILLE Fig 1.--Ash pattern of odd-insoluble material in lung of rat immediately following Intretrecheal injec tion of chiysotlle dust. Alveolar ducts , are outlined by fairly thick, continuous coating of dust. Some dust, mostly in the form of discontinuous deposits. Is also found In some distal alveoli. Section was subjected to microincineration followed by treatment with concen trated hydrochloric acid (X 150). ately after the intratracheal injection of chryaotile, the dust was seen applied as a uniform and continuous, dense coating upon the respiratory surfaces of the alveolar ducts and their evaginating alveoli (Fig 1). The lumens were everywhere widely patent. Sur face coating by the dust of some of the distal alveoli was also observed, but not regularly. After 72 hours, the proximal portion of the raoemus6 was a nearly solid, cellular structure in which the lumens of the respira tory bronchiole and alveolar ducts were oblit erated by a polypoid mass of ovoid and plump spindle-shaped cells which also oblit erated the lumens of the evaginating al veoli. A highly significant change in the distribution of the asbestos dust was now Fig Ash pattern of acid-insoluble materiel, three days after intratracheal Injection of chryaotile dust In rat. Dust, now in the form of flocculent masses of splculated aggregates, seems to form casts of alveolar ducts and fills their lumens. Very little dust is noted in the peripheral alveoli (X 150). observed. The dust permeated the occlusive inflammatory tissue and thereby formed a cast of what was once the lumen (Fig 2). It was, however, no longer oompactly disposed; rather, it was distributed in a flocculent manner. Nevertheless, the total amount of dust in the lumens of the alveolar ducts ap peared to be considerably greater three days after the intratracheal injection than that present immediately after the injection. At the same time, less asbestos dust was ob served in the more peripherally situated al veoli. It is probable that the increase in the amount of dust in the alveolar ducts was de rived from the more peripheral alveoli. Al though some of the finer dust may have been intracellular, the greater bulk, by far, was extracellular, being imprisoned in the inter- Arch Erwiron Health--VoI 15. Nov 1967 8005 1460 PRODUCED BY FORD EXPERIMENTAL ASBESTOSIS--GROSS A DE TREV1LLE 641 Fig 3.--Minimal, moderately healed asbestotie lesion in rat exposed to chrysotile dust for one month, then postured and killed 24 months later. Lesion consists of a partially thickened alveolar duct. Mural thickening is caused by hypocelUiler connective tissue that has ob literated alveoli near bottom of field (hematoxylin and eosin, (X 250). slices of the precollagenous stroma of the inflammatory tissue. . When examined one year or longer after the intratracheal dust injection, asbestotie fibrosis was found in all rats so injected. The characteristic lesion was sharply limited to the alveolar duct as well as to the short re spiratory bronchiole and consisted of moder ate to severe collagenous thickening of the wall, often also associated with striking hy perplasia of smooth muscle (in more than one third of the injected animals). The col lagenous mural thickening involved much of the wall and resulted in the obliteration of many of the evaginating alveoli. Generally speaking, the involvement of the alveolar ducts, even in animals injected with but a Fig 4--Field tame at in Fig 3 after tilver Impregnelion chows that (car is composed on nonbranching, largely parallel, thick collagen fibers that ere fairly densely arranged (Gordon and Sweets. (X 2S0). single dose of 3.5 mg of chrysotile dust, was greater than the minimal lesion observed in rats that had inhaled high concentrations of the dust for one month. As judged from the examination of a single section of both lungs of the rats, greater dust dosage was often, but not always, associated with an increased incidence of thickened alveolar ducts. The occurrence of larger collagenous scars con taining small remnants of air spaces seemed also to be related to the multiplicity of the asbestos dust injections, as was a metaplasia to the columnar variety of the epithelium lining the surviving evaginating alveoli. The sharply delimited mural thickening of the alveolar ducts (Fig 3) and respiratory bronchioles, with the collagenized, nonarboresoent, thick stromal fibers (Fig 4), and the associated reduced oellularity (Fig 3), Arch Environ Health--Vol IS, Nov 1957 8005 1461 PRODUCED BY FORD 642 EXPERIMENTAL ASBESTOS1S--GROSS & DE TREVJLLE Tig 5.--Ash pattern of aeld-lntotuble material of tame field at In Fig 3 after microincineration and treatment with concentrated hydrochloric add. allowing the pretence of tangled mattes of atbettot fibert In the regiont of the tear tittue (X ISO). represented a healed or healing inflamma tion. Within this scar tissue, apparently sequestered compact masses of asbestos fibers were often strikingly prominent after micro incineration (Fig 5.) On the other hand, many scars were seen that contained little or no demonstrable dust The sharp delimita tion of the scars was accentuated by the ad jacent normal delicate alveolar walls. These were characterized by single, nonbranching argyrophilic fibers. Nevertheless, in some of the rats, there were also fod of a more active inflammation. Such foci were marked by cellular thickening of alveolar walls and by considerable desquamation. Here, the stroma was arborescent, the side brandies tending to give structural support to the increased num ber of alveolar cells. Since similar inflamma tory foci of cellular alveolar mural thicken ing 6.--Hyperplasia of smooth musela In rat Infected twice with 3.3 mg chiysotile dust end killed 21 months later. Well-defined bundles of smooth muscle era found In relation to alveolar ducts where the only normelly present muscle Is sltueted around mouths of evagineting alveoli (hematoxylin and eosin, X ISO). ing were also found in the lungs of control rats not exposed to, or injected with, dust, it is believed that they were caused by un related spontaneous disease. No asbestos bodies were found in rats ex posed to or injected with chrysotile asbestos dust. Rats (Inhalation).--The characteristic minimal lesion, as found a year or more aft er the pulmonary deposition of chrysotile dust, consisted of focal collagenous thicken ing affecting patches of alveolar duct wall between some of the evagimting alveoli (Fig 3 and 4). With more extensive deposi tion of the chrysotile fibers, the mural fibro sis tended first to narrow the mouths of the evaginating alveoli and then to dose them Arch Environ Health--Vof IB, Nov 1967 8005 1462 PRODUCED BY FORD HFM -013205 EXPERIMENTAL ASBBSTOSIS--GROSS A DE TREV1LLE 643 1 i Fig 7.--One of smaller cellular foci in lungs of hamster injected twice Intratraeheally wtth 3.5 mg chrysotite dust. Animal died one year later. The alveo* lor structure of cellular tissue is obliterated by cellular proliferations. There is a suggestion of a polypoid mass slightly to the right end above the center of the field (hematoxylin and eosin. X 350). Fig 6.--Silver impregnation of same field as In Fig 7 shows a plethora of branching and interconnecting argyrophllic fibers forming thick alveolar walls and projecting Into air spaces as polypoid masses of vari ous sues. Largest polypoid mass ts one referred to In Fig 7. It fills the lumen of alveolar duct (Gordon and Sweets, x 150). off entirely. The fibrosis also dipped into some of the evaginating alveoli, partially or completely obliterating them. In this man ner, the respiratory bronchiole and adjoin ing alveolar ducts may become converted into relatively smooth-walled tubes with few or no evaginations. Most of the wall thick ness of these tubes was attributable to the fibrotically obliterated alveoli that original ly evaginated from the former. The oellularity of the thickened structures varied considerably depending upon the time interval between the deposition of the dust upon the parenchymal surfaces and the death of the animal. In the florid stage of the inflammation, as seen in a rat exposed to the chrysotile dust for four months and killed two weeks later, the oells in the affect ed regions were large and numerous while the stroma consisted of an arborescent net work of argyrophilic fibers in which the oells were enmeshed. In time, the argyrophilic fibers became thickened, condensed, and lost their arborescence as well as their argyrophilia; the latter coincident with their col lagenous conversion (Fig 4), During this prooess of stromal maturation, the oellulari ty of the tissue diminished and the oells elongated to resemble fibrocytes. In some of the rats examined a year or longer after the dust exposure, there were, in addition to the collagenous fod, cellular foci of more active inflammation that had no ap parent relation to the proximal portion of the Arch Environ Health--Vol 15, Nov 1967 h 8005 U63 PRODUCED BY FORD 644 EXPERIMENTAL ASBESTOSIS--GROSS A DE TREVILLE Fig 9.--Mb pattern of acld-lnsoluble material show* that region of polypoid mast In alveolar duct is densely permeated by tangled masses of asbestos fibers. Small er masses and aggregates of dust are found In periph eral alveoli and Other air spaces (X 150). Fig 10.--Numerous luminetcent asbestos bodies in region of asbestotic Inflammation, from the lung of e hamster that received intratracheal Injection of 3.5 mg chrysotile dust and died 13 VS months later (unstained section; 12.5/0.3 Lain objective and 0.9 Lettz dart- held condenser X 150). raoemus. Some of these foci may also con tain variable numbers of polymorphonuclear leukocytes. Similar cellular fod of active inflammation were also found in some of the unexposed laboratory control rats of compar able age. Lung sections of all 18 tats exposed one month or longer to heavy concentrations of chrysotile dust exhibited minimal fibrotic pulmonary lesions one year or longer after the exposure. In contrast, in but one of six rats exposed to the same dust for only two weeks were similar changes found. The oth er five exposed animals had lungs that did not differ from those of unexposed rats. In neither of two rats exposed to the same dust for only one week, was there any evidence of asbestotic fibrosis 11 and 13 months, respec tively, after exposure. Compared with the amount of acid-insolu ble ash seen in the lung sections shortly aft er the dust exposure, it appeared that only a small fraction of this amount of dust could be seen in the lungs of rats one or more years after the exposure. Nevertheless, in view of the ultramicroscopic size of much of the chrysotile dust, the failure of this method to demonstrate mineral dust in the tissue must not be construed to indicate necessari ly that no dust was present. It is also of in terest that the darkfield examination of the unstained sections with dry objectives was negative for luminescent material, even in regions later shown to contain abundant add-insoluble mineral ash. In the lungs of a number of rats exposed to chrysotile dust two months or more, there Arch Environ Health--Vol 1$, Nov 1967 8005 U64 PRODUCED BY FORD EXPERIMENTAL A8BE8T0SJB--OROSB A DE TREVJLLE 646 were bundles of smooth muscle that often encroaching upon the lumens. Occasionally, constituted masses 50p to 100/i in thick the argyrophilic stroma extended into the ness (Fig 6). These muscle bundles were lumen of an alveolar duct or a bronchiole in sharply delimited and related to the wall of a polypoid manner, occluding it The con thickened alveolar ducts. They had no con densation of thin argyrophilic fibers to form nection with blood or lymph vessels nor parallel thick fibers devoid of arboreeoenoe could a relationship to the muscularis of a and the transformation into collagen, char terminal bronchiole be demonstrated. In the acteristic of the lesions in rats, was not seen newly formed connective tissue about some at all or to a very minor extent in these of the alveolar ducts and respiratory bron hamsters. For the most part, the occlusion of chioles, there were scattered gland-like the air spaces was earned mainly by massed structures. These pseudo-glands represent alveolar cells, but the contribution of the ar surviving units of the evaginating alveoli gyrophilic stroma should not be dismissed. that have become lined by tall columnar epi It should also be emphasized that in con thelium, occasionally ciliated. No acid-insol trast to the situation in rat lungs where the uble mineral ash was demonstrable in the involvement was sharply limited to the al area- occupied by the proliferated muscle veolar duct and its evaginating alveoli, there fibers. The regions, pervaded by the mare was no such delimitation in the hamster cellular and active inflammation, some con lung. Here, the cellular as well as the stro taining leukocytes, were also devoid of de mal proliferation extended without abate monstrable mineral ash. - ment to the peripheral alveoli, thus account Hamsters (Intratracheal Injection).--Of ing for the diffuseness of the consolidation. 19 hamsters, 16 died as a result of the aabes- Following incineration, abundant arid- totic inflammation that followed the intra insoluble mineral dust was observed in the tracheal injection of chrysotile dust. The involved raoemi. Many individual fibers three survivors had received only one injec could be identified; some straight or wavy, tion and were killed 21 months after the im others coiled and forming tangled masses. position of the lung dust burden. The aver Compared with the amount of dust found in age survival of the hamsters that had one rat lungs, the chrysotile dust in the hamster intratracheal injection was 16% months, lungs was much more abundant and more with a range of 13% to 20Vi months. The diffusely distributed (Fig 9). Asbestos bod animals that had received two intratracheal ies were found in abundance throughout the injections survived, on the average, 14% affected regions. They were considerably months from the time of the first injection, smaller than those seen in human lung6 but with a range of 12% to 16% months. The were readily recognized. They were best three survivors that were killed after 21 seen under dark-field illumination with dry months were mariatad and also had exten objectives in unstained sections (Fig 10). sive asbestotic disease. Many of the lung sections showed exten sive consolidation with relatively few patent air spaces. In other lung sections, the consol idation was patchy, Beemingly concentrated about larger air spaces with relatively little aerated tissue between the consolidated por However, there was no parallelism between the number of luminescent asbestos bodies seen in the unstained sections and the amount of dust found in the same field after microincineration. The amount of dust pres ent was much greater than might be sus pected from the number of asbestos bodies tions. The consolidated portions were com observed. Most of the dust and the asbestos posed of masses of cells with vesicular round or oval nuclei--apparently alveolar cells (Fig 7). Leukocytes were not seen. Al veoli or larger air spaces usually could not be identified in these regions except after silver impregnation (Fig 8). The argyrophil- bodies were situated in the air spaces. When observed within tissue, the asbestos bodies and the dust were found trapped within the network oi inflammatory argyrophilic fibers and associated oells that tended to occlude the air spaces. ic mural stroma of all air 6paoes in the solid Guinea Pigs (Inhalation) ^Barely recog ified regions was greatly thickened by ar nizable, minimal mural thickening of an oc borescent fibers that formed a loose network casional alveolar duct and respiratory bron- Arch Environ Health--Vol IS, Nov 1967 8005 1465 PRODUCED BY FORD 640 EXPERIMENTAL ASBESTOSJB--GROSS A DE TREV1LLE chiole was found in the lungs of guinea pigs disappearance of chryaotile dust from the that had been exposed for three months to lesion, either by dissolution or by transport, an atmosphere containing 20 mg/cu m of is not a aine qua non of healing; but that chryeolile dust. Similar to the appearanoe of healing, at least in the rat, does take place-- such lesions in hamsters, the mural thicken even in the presence of this dust One would ing consisted of proliferated alveolar cells like to think of the formation of the asbestos and their supporting stroma of interconnect body as a protective mechanism by which ing argyrophilic fibers. The lung sections of the asbestos fiber becomes sequestrated and guinea pigs that had inhaled high concentra the tissues safeguarded from further irritant tions (66 mg/cu m) of chiysotile dust for action by the fiber. To what extent this two weeks or longer, manifested similar mechanism may apply is not known; but it minimal mural asbestotic thickening in the does seem that in rats, in which asbestos proximal portion of the raoemus. However, bodies are not demonstrable (with the opti the number of units (raoemi) involved was cal microscope),11 healing occurs in the pres much greater than that in the animals ex ence of apparently naked fibers. posed to the lower dust concentration for The diminution, in time, of the amount three months. No evidenoe of healing (sig of dust demonstrable in the tissue and its nificant reduction in oellularity of the lesion apparent disappearance in some of the scars, and collagenization of the stroma) was seen poses an interesting question in regard to in animals examined up to seven months aft the mechanism by which the asbestos fibers er the beginning of the exposure. Following disappear. It is commonly believed that microincineration and treatment with acid, chryaotile fibers have a relatively high solu the amount of ash seen in the guinea pig bility in tissue fluid. This would seem to ac lung sections was comparable to that noted count for the inability to demonstrate asbes in rats that had inhaled chrysotile dust. As tos fibers in some of the asbestos bodies bestos bodies found in the lung sections found in human asbestotic lungs. However, were few and very small. dissolution of the fibers, particularly of the coarser ones, would require a long time--eo Comment that it would be difficult to explain the fail ure of peripheral alveoli to beoome involved A chronic pulmonary inflammation may by inflammation. Furthermore, high solubil be termed progressive if it extends from its ity would not be consistent with the sharp original 6ite into adjoining, previously nor inflammatory localization of the asbestotic mal air spaces, and if it remains active, re lesion in the proximal portion of the racemtaining its argyrophilic precollagenous stro us in rats. It is much more reasonable to ma and high oellularity. Such a pulmonary explain this sharp localization of the in inflammation may be considered healed if flammation on a fairly prompt removal of its argyrophilic stroma has been completely the inhaled or injected irritant dust from the collagenized while its oellularity has beoome peripheral alveoli and the subsequent stag considerably reduced. nation of the dust in the proximal portions In the rat that has inhaled high concen of the raoemus.7 The transport of the chrys trations of chrysotile asbestos fibers for only otile dust from the peripheral alveoli is ef a few months or has been injected intra fected by the alveolar clearance mechanism tracheally with this dust, the asbestotic in consisting of a proximally moving film of flammation remains sharply limited to the alveolar fluid.' This transport is dramatical proximal portion of the raoemus and heals 1 ly illustrated by the increase in, and concen by becoming transformed into a hypooellu- tration of dust in the lumen of the alveolar lar collagenous scar. Thus, it would appear duct within 72 hours after the intratracheal proper to classify asbestosis caused by chrys injection of chrysotile dust (cf Fig I and otile dust as nonprogressive in the rat. 2). It may be of interest at this point to It is of basic interest that appreciable note that the localization of the early asbes amounts of ashesto6 fibers are demonstrable totic lesion to the proximal portion of the within the scars of healed or healing raoemus was first described by Vorwald et inflammation. This would suggest that the al, and reoently confirmed by Holt et al. Arch Environ Health--Vol IS. Nov 1967 8005 1466 PRODUCED BY FORD EXPERIMENTAL ASBBSTOSIS--GROSS A BE TREV1LLE 647 The reaction of the hamster lung to chrys the considerably larger amount of acid-insol olite dust is the antithesis of that observed uble mineral ash demonstrable in the ham in rats. The lesion extends from the proxi ster lung sections than in those of rata Fur mal portion of the racemus to the peripheral ther evidence is seen in the diffuseness of the alveoli, thereby producing extensive consoli dust distribution in the hamster (Fig 9) in dations in the hamster lungs. The consolida contrast to its restriction to the proximal tions are composed of obliterated air spaces, portion of the racemus as in the rat (Fig 5). the lumens of which are filled with masses of The reactivity of hamster tissue to chryso- alveolar cells, mostly desquamated (Fig*6). tile fibers is significantly different from that Although the inflammation involves the en of rat tissue, the former being much more tire rooemus in this animal, the proximal florid. This is seen not only in the plentiful portion shows more severe involvement production of asbestos bodies in hamster than the peripheral portion. It is this ex lungs but also in the failure of argyrophilic tensiveness of asbestotic involvement with precollagenous fibers to mature into colla consequent pulmonary inadequacy that ac gen as well as in the undiminishing oellulari- counts for the high mortality of the ham ty of the inflammatory tissue even after a sters. lapse of nearly two years. This difference in With the previously given definition of the reactivity of lung tissue in the rats on the progression in mind, it is seen that the one hand and in hamsters on the other is not diffuse involvement of the racemus in the unique to chryBotile dust inasmuch as simi hamster would fit the first requirement for lar and quite comparable differences in progressiveness of the disease, if it can be these two species have been noted in their shown that the inflammation originally was reactivity to quartz dust.10 confined to the proximal portion of the ra It is highly probable that interspecies cemus and then extended into the peripheral differences in the efficiency of the pulmo air spaces. This information, unfortunately, nary clearance mechanism play an impor is not available from the present investiga tant role in the susceptibility of the different tion. However, the finding of a more severe species to certain pulmonary diseases. It is involvement of the proximal portion of the recognized, for example, that horses and racemus favors the probability that such an mules working during their lifetime in mines extension took place. The second require exposed to quartz dust will have accumulat ment of progression, ie, that the inflamma ed a negligible amount of silica in the lungs tion remain active and nonhealing, is a con and will demonstrate no silicosis;1' whereas spicuous feature of the asbestotic hamster miners in the same workings will have vary lungs. The failure of the argyrophilic fibers ing degrees of silicosis as well as a sig to lose their arborescenoe and to oollagenize nificantly elevated pulmonary silica content. and the persistence of the extreme cellulari- We have recently exposed rats and mioe si ty of the involved tissues nearly two years multaneously to quartz dust in the same in after the intrapulmonary dust deposition is halation chamber for the same length of incontrovertible evidence of nonhealing. time. All animals were killed immediately Two possible explanations come to mind after the end of the dust exposure. Well- for the difference in the disease produced in defined silicotic nodules were present in the the rat and the hamster by the same chryso rat lungs; whereas, the mouse lungs were ab lite dust. One explanation is that the reactiv solutely normal, and no add-insoluble dust ity of hamster tissue to the dust is greater could be demonstrated in the mouse-lung than that of rat tissue. The other is that the sections after miaoindneration (unpub hamster's alveolar clearance mechanism is lished data). not as capable as that of rats in transporting The above observations are relevant to the dust from the peripheral air spaces prox the present investigation insofar as such in- imally. As will be seen below, both explana terspedes differences tend to make extrapo tions apply. lation from animal data to human disease There is some evidence that the hamster's unreliable. The questions to be resolved are pulmonary clearance mechanism is not as whether the efficiency of the clearance effective as that of the rat. This is seen in mechanism of man approximates that of the Arch Environ Health--VoJ IS, Nov 1967 t i 8005 1467 PRODUCED BY FORD 648 EXPERIMENTAL ASBESTOS18--GROSS A 1>E TREV1LLE rat or of the hamatpr unit whether tissue exposed to the different dust concentrations reactivity of the rat or of the hamster more is too small, and the time allowed for matu closely resemble that of mnn. ration of the lesion too short to permit A study of the ash-pattern (microindn- definitive conclusions. eration) of a number of lungG from asbestos The results of this investigation also have workers (exclusive of cases of ailiooasbcsto- given some insight into the relative patho sis) has disclosed no instance in which the genicity of chrysotile asbestos dust far rats amount of mineral dust in the lung sections insofar as a single intratracheal injection of has been more than scanty.12-1* Other in 8.5 mg of this dust has produced minimal vestigators have found upon chemical analy asbestotic lesions in rats. This result may be sis that the silicate content of manifestly as- compared with that of King, et al1* who bestotic lungs was surprisingly low.14-1* found that a quantity of more than 2 mg and These observations approximate the findings lees than 5 mg of quartz dust injected intra in the rat. Another similarity of human tracheally into rats was the smallest amount chrysotile asbestosis to findings in asbestotic of ailica capable of producing demonstrable rat lungs is the complete oollagenization silicosis in this animal. Thus, it seems that with associated aoellidarity that may be for the rat, chrysotile has about the same or found in "burned-out" cases of human as der of pathogenicity as quartz du6t. bestosis.18 Nevertheless, in 6pite of the simi For the hamster, the smallest amount of larity of the oollagenization in man and intratracheal!)- injected chrysotile dust ca rat, there is an important and significant pable of producing minimal lesions has not difference. The early disease in rats is multi been determined. This dose will apparently focal, affecting the proximal portion of the be smaller than that found for rats. raoemus; whereas in man such multifocal By the inhalation technique, using a very distribution has not been described--only high concentration of respirable cluysotile diffuse involvement. A possible explanation dust (86 mg/cu m), the exposure time re of the diffuseness of asbestotic involvement quired to produoe minimal asbestosis in rats in human lungs could be that the inflamma and guinea pigs seems to be somewhere be tion, initially confined to the proximal por tween 60 and 120 hours. tion of the racemus because of continued The hyperplasia of smooth muscle ob exposure, spreads peripherally, thereby be served in the asbestotic rats is very similar coming confluent with that of neighboring raoemi. The fact that an asbestotic inflam mation may "burn out" in man, does not rule out the possibility that this inflamma tion remained active and progressive for same years alter exposure had ceased before finally attaining the terminal healed stage. There is, unfortunately, no information on the activity of the asbestotic inflammatory to such hyperplasia observed in human as bestosis cases studied in this laboratory and is also similar to that found in bronchiolar emphysema of the lung.11 Although the ex act origin of the muscle was not determined, its location made it appear most likely that it was derived from the circular muscle in the alveolar ducts that regulate the openings of the evaginating alveoli. process in human lungs at various intervals following removal from further exposure to Summary and Conclusions the specific dust. Rats, hamsters, and guinea pigs were giv In guinea pigs, the results of the inhala en various lung dust burdens of chrysotile tion of chrysotile dust are very similar to asbestos; some, by inhalation and others, by those observed in rets with respect to the intratracheal injection. An attempt was site and extent of the lesion, but conversion made to determine the smallest amount of of argyrophilic stroma to collagen is not en chryBotile dust that would produce a recog countered in the sections of guinea pigs that nizable minimal asbestotic lesion. The time had been killed up to seven months after the lapse between the imposition of the lung initiation of the dust exposure. The amount dust burden and the death of the animal of dust found in the lung sections is scanty varied from zero to 24 months. and is limited to the proximal portion of the The following conclusions have been racemus. However, the number of animals reached: Arch Environ Health--Vol IS. Nov 1967 8005 1468 PRODUCED BY FORD EXPERIMENTAL ASBESTOSIS--GROSS A DE TREV1LLE 649 1. The characteristic early asbestotic le 9. The early chrysotile asbestotic lesion sion is situated in, and sharply localized to, in guinea pigs resembles that of rats, but it the proximal portion of the pulmonary ra- has not been adequately studied to permit cemus which, in the rat, consists of a very further characterization. short respiratory bronchiole and adjoining alveolar ducts. 2. Asbestosis in rats, caused by chrysotile This study was supported in part by Public Health service grant OH 00132. Johns-Manville Company, through the courtesy of Kenneth W. Smith. MD. supplied the chrysotile asbestosis. dust, is nonprogreesive. 8. The evidence for the nonprogieeaive- References ness of chiysotQe asbestosis in rats consists of the observations that the minima] asbestotic lesion is limited to the wall of the respiratory bronchiole and of the adjoining alveolar ducts and does not extend into ad joining normal alveoli and that the minimal asbestotic lesions heal by becoming collagenized and hypooellular. 4. The localization of the minimal asbes totic lesions is attributed to the proximal L Roes, W>., et al: Emotional Aspects of Raspiratory Disorders Among Coal Miners, JAMA. 156464 (Oct 2) 2954. Z Holt, P.F., and Young, D.K.: A Dust-Feed Mechanism Suitable for Fibrous Dust, Ann Oeeup Hyg 2:249, I960. 9. Wright, B.M.: A Size-Selecting Sampler, for Air-Borne Dust, Brit J Industr Med 11:284,1954. 4. Gross, P,, and Tolker, EB.: Dust Particles in Lung Sections: Some Notes on Methods of Their Visualization, Arch Environ Health 12:213. 1966. transport of dust from peripheral air spaces by the alveolar clearance mechanism and subsequent stagnation of the transported dust in the proximal portion of the recemus. 5. In rats chrysotile asbestotic lesions heal in the absence of demonstrable (optical microscope) asbestos bodies and in the pres ence of chrysotile fibers, the latter becoming trapped in the scar tissue. 6. voc Hayek, H.: The Human Lung, VJ2. Krahl (trans). New York: Hefner Publishing Company, Inc, p 172. 6. Vorwald, A.J.; Durban, T.M.; and Pratt, P.C.: Experimental Studies of Asbestosis, Arch Industr Hyg 9:1, 2951. 7. Gross, P.; P6txer. E.A.; and Hatch, T.F.: Al veolar Clearance; Its Relation to Lesions of the Re spiratory Bronchiole, Amer Rev Resp DU, 94:10, 1966. . 6. Hatch. T.F.. and Gross, P.: Pulmonary Deposi 6. There is a considerable reduction in the amount of chrysotile dust found in the lung sections of rats one year or more after the imposition of the lung dust burden as tion and Retention of Inhaled Aerosols, New York: Academic Press, Inc., 2964, p 69. 8. Holt. PS,; Mills. J.; and Young, D-K-: Early Effects of Chrysotile Asbestos Dust on Rat Lung. J Path Bact 97:15. 3964. compared with the amount of dust present shortly after the dust burden was imposed. 7. The reaction of hamster lungs to chrys otile dust is the antithesis of that observed in rats. The lesion is progressive, extending 10. Groes, P.; deViliiera, AJ.i and deTreville, R.T.P.: Experimental Silicosis, Arch Path 64:87-94. 1967. 11. Einbrodt, HJ.; Klosterkdtter, W.; and Metze, H-: Vergleicbende Uotersuchungen fiber die Korn* grtrasen retiaierter Staube in den Lungen von Mensch into peripheral air spaces bo as to involve the entire raoemus and thereby producing extensive consolidations leading to the death of the animals. The lesions do not heal, since the precollagenous stroma and und Tier. Beitr Silikoseforsch 6:491, 1963. 12. Gross, P.. and Smith, K.W.: The Topographic Distribution of Mineral Dusts in Some Pneumo- oooiotic Lungs, DU Chest 95:140, 1959. 13. Groes, P.: 'Pathology I the Pneumoco nioses.*' in Lanza, AJ.L (ed.): The Pneumoconioses, the high cellularity persists through the twoyear period of observation. At the same time, the amount of asbestos dust demon strable in the sections remains large and New York: Grunt and Stratton, Inc., 1963, p 51. 14. Knox, J.F., and Beattie, J.: Mineral Content of the Lungs After Exposure to Asbestos Dust. Arch Industr Hyg 10:23. 1950. 15. Nagelschmidt. G.: Some Observations of the diffusely distributed. 8. The progressiveness of the chrysotile asbestotic lesion in hamsters is ascribed to a less effective pulmonary clearance mecha nism and to a greater reactivity of the pul Dust Cootent and Composition in Lungs With As- bestoeU, Ann HY A cad Sci 132:64-76, 1965. 16. Ray, S.C.; King, E.J.; and Harrison. C.V.: The Action of Variable Amounts of Quartz on the Lungs of Rats, Brit J Industr Med 6:62, 1951. 17. Sieberl. F.T.. and Fisher, E.R.: Bronchiolar monary tissue than exist in rats. Emphysema. Amer J Path 93:1137, 1957. Arch Em*iron Hrofth--VoI 35, Not' 195? Printed end Puthshed w me united States of America 8005 1469 PRODUCED BY FORD