Document 8JXz5Kr6b607nVvqVz9gJBvd

V.VVJWn itl "n*w. - r~ Reprinted from' Volume 31, Mmrch-April, 1970 i4rM EfC\ The Pulmonary Response to Fibrous Dusts of Diverse Compositions PAUL GROSS, MJ>,, ROBERT T. P. deTREVBLLRMJ)^ LEWIS J. CRALLEY, PHJD-t WUXIAM T. GRANQUIST, PHJ>4 od FRED L. PUND5ACK, PHJ>. Industrial Hygiene Research Unit, Department of Occupational Health, Graduate School of Public Health, Vnioenity of Pittsburgh, Pittsburgh, PentuyUienia 15213 Fibrous quartz, chrygotile asbestos, od trtme&tc talc duct, all of rtcpir&ble particle : fixe, injected iBCratncbeally, produced polypoid proliferative inflammations with- ! in smaller air-conducting tabes as wdl as mote peripherally. With time, the biflam- ( matory tissue became converted into collagenous scan which often canted permanent deformities of bronchi and bronchioles. After intratracheal injection of a fibrous * 3 dost such as synthetic chrysotilc, ceramic aluminum sBfoatc, silicon carbide, glass, or brucite, the main pulmonary response acts a macrophage reaction with minimal stxotml ' participation. In addition, within 4 days after the injection, there were foci of poly* I paid proliferative inflammation but limited to the mote peripheral respiratory bron- j chiole and alveolar ducts. Because these polypoid lesions did not collageaae and did : j not destroy the anatomic integrity of the dr spaces, and because the lessons were { : reversible, the dusts railing forth tills type of response must be dined as biologically i "inert." Furthermore, the polypoid lesions axe believed to be attifactual in the sense ; tint their production is determined by the method of introducing the dust into the ( lungs, since aaeh lesions sure not seen in tahalfag high concentrations of the same dusts. f introduction TyriTH THE INCREASING production " and use of fibrous materials, both the naturally occurring and those industrially pro duced, the dust created by their fragmenta tion is becoming more prevalent. We know that one type of naturally occurring fibrous dust, namely asbestos, is biologically active and is capable of causing extensive and fatal scarring of the lungs, and some kinds of this mineral have been associated with the pro duction of cancer. Inasmuch as it is not known exactly what it is about the asbestos dust particle that is responsible for its pathogenicity, the simplest explanation, which seemed to be attractive *IoduxUul Hygiene Foundation, 5231 Ceane Ave., Pitts* burgh, Pecmnfoni* 15232. _. _ tBuitttt of Occupational Safety and Health. Department of Health, Education, and Welfare, 10(4 Broadway, Gn- cumati. Ohio 45202. IMtOon Institute, 4400 Fifth Ave.. Pittsburgh, Peaasvferaata 15215. , iReeearch and Development, lohos-MaavflU Research and Engineering Center, Manviile, Hew Jersey 088)5, to many in the past, was that the patho genicity of asbestos, and, therefore, of all fibrous dusts, was related to the fibrous shape of the particles. According to this theory, when the fibers are inhaled, their sharp ends traumatize the cells they contact, and fibrosis results from the multiple traumata. Although some years ago we had investi gated the pulmonary response to one indus trially produced fibrous dust, namely ceramic aluminum silicate fibers, and found it to be biologically "inert"1 in recentyears the needle like character of fibrous dust has again been implicated as the pathogenic factor. This has occurred in connection with fibrous glass dust, the pathogenic potential of which has been questioned in spite of the fact that nonfibrous glass dust has been found to be biologically "inert-"1 This paper is concerned with a study of the pathogenic potential of fibrous dusts not here tofore documented and with the pathologic C35794 0210 SA-589 126 March-Aprit, 1970 Table. I Tabular Protocol of Rats Injected Intratraeheally with Dusts Dun Mtcroquam* Fiber Diameter U) 1.2 No. e! Rau 62' Natural chrysotile* Talc (tfemolilc)4 0.05-0.2 0.2 >1 0.14.2 55 50 Synthetic chrysolite* 55 Ceramic aluminum silicate' Clast* 20 1 B0 75 BcucStc 24 15 Silicon carbide* 034.0 22 Amorphous magnesium silicate* 10 Dk 75 tat 103 me + 14 siy 25 tnjc 14 k + 45 <uk 103 tujc 103 mt 103 me 33 me 75 mjr Mortality* 26% u 6 tmthi 45% ia 15 Bwatkt 90% ia IB moaths 100% In 24 months 40% ia 6 months 44% ia 6 months 22% ia 12 months 55% ia 24 months 72% m 18 months 85% ia 18 month* 67% in 12 months 100% in 18 moaths 47% in 12 CBonths 73% ia 24 months 18 rats sacrificed at In tervals (oo deaths in C months) 10% in 6 moaths Range of mortality m month* of different croups. High nickel, 35 rau; medium nickel, 31 rats; and low nickel, 27 raU. Bail-milled dust given to 40 nu and hammer-milled dust to 15 rats. *Tale with high and low natural nickel content (given to 25 rats each). One batch prepared at Melton Institute, the other at JohrwManviUe Research aud Engi neering Center. 'Named Ftberinx, obtained (raa Carborundum Company. (Groups of 15 rats given different kinds of fibrous glass: 1, etched glass; 2. utteooted; I, coated with starch binder; I, coated with resin. kSlicon carbide whiskers from Carbonsodum Company. ^Prepared by meting sodium silicate with MfCh and washing precipitate. effects of these, as well as of previously in vestigated dusts that have not been reported. This study is part of a more basic investiga tion being conducted in cooperation with the U.S. Public Health Service (Grant No. 1 R01 UI-00849-01) and industry, the purpose of which is to determine the locus of pathogen icity of asbestos dust. Method end Materials ' A tabular summary of the types of dust studied, the number of rats employed, and the dose of dust administered is given in Table I. Included under any one type of dust may be two or more materials from different sources of slightly different compositions but grouped together because, for the purpose of this study, no significant difference was noted. For instance, microquartz prepared at the Johns-Manville Research and Engineering Center consisted of resintered, acid-leached glass fibers with an originally high alkali con tent. The average diameter of the fiber was 1.2 #t. Two batches had been prepared: one with a metallic nickel content of 0.1 1% and tile other, of 3.1%. The natural chrysotile was also of two kinds. One had been ball-milled and then hammermilled. In the latter process, besides being reduced to submicronic dimensions, it also acquired an increased nickel content from the nickel-steel alloy of the hammers. The other was comminuted by ball milling only. The talc dust was of the tremolite variety, and there were two kinds. One had a high natural nickel content and contained fibers with an average diameter of 0.2 n; the other had a low nickel content and contained fibers with an average diameter of 0.1 ft. Two batches of synthetic chrysotile were employed. One, prepared at the Mellon In stitute, Pittsburgh, Pennsylvania, had a purity of about 90%. The impurities consisted large ly of brudte (Mg(OH)j). The diameter of the tubular ciystals averaged 0.02 /i and their length varied from 0.08 to 0.17 fi. The other batch was synthesized at the Johns-Manville Research and Engineering Center, Manville, New Jersey. Its purity was 99.4%. The aver age diameter of the crystals was 0.03 to 0.04 C35794 0211 American Industrial Hygiene Association Journal 127 with a solution of sodium silicate. The result ing precipitate was washed with abundant water, and a standard suspension was pre pared. All dusts were suspended in water, the con centrations depending on the amount sus pended in 1 ml of water which could be in jected without killing the rats. Most sus pensions contained 3.5 mg of dust per milli liter. Several suspensions contained 25 mg of dust per milliliter. A total of 42+ rats was injected intra tracheally with these dusts. In some groups the total dose was administered by as many as four injections. The injections were made under light ether anesthesia with the aid of an illuminated laryngeal speculum which al lowed the introduction of a spinal-tvpe needle between the vocal chords under direct ob servation. Figure 1. Tubular crystal* of synthetic chrysotile prepared at Mellon Institute, Pittsburgh, Pennsyl vania. fi with a length of I /r or less, although a few fibers were up to 5 p in length. Both lots gave x-ray diffraction patterns typical of chrysotile (Figures 1 and 2). Five different varieties of fibrous glass were injected into rats. These averaged about 1 p in diameter. One was etched, two were un coated, one was coated with a textile-type of binder (mostly starch), and the last was coated with a phenol-formaldehyde resin type of binder ( used largely for insulation). The ceramic aluminum silicate fibers (Fiberfrax from Carborundum Company) had an average diameter of 2.0 /i. Two batches were used: one had been hammer-milled to in crease its nickel content, and the other was briefly comminuted in a glass tissue grinder. The silicon carbide whiskers, also obtained from the Carborundum Company, had a fiber diameter ranging between 0.5 and 3 /i and a length ranging between 100 and 750 is. Amorphous magnesium silicate was used as a nonfibrous control dust. It was prepared by reacting a solution of magnesium chloride Ficure 2. Crystals of synthetic cbsysotile pre pared at the Johns-Manville Research and Engi neering Center, Manviilc, New Jersey. Note that these crystals arc longer and necdle-like; also the magnification is approximately one-tenth of that in Figure 1. 128 In order to study the early pulmonary re sponse to the various types of dust, four rats were killed from each group four days after the hist intratracheal dust injection. The rest were allowed to live out their lives. The lungs of all animals were distended with 4<Jo formaldehyde solution under a head of 10 to 12 cm of water. Paraffin sections of the lungs were stained routinely with hema toxylin and eosin. Pertinent fields were pho tographed, and after impregnation with silver the same fields were rephotographed in order to study the relationship between cells and stroma. In order to study the relationship of the dust to the lesions, some sections, cleared unstained, were examined or photographed under dark-field illumination; other sections were subjected to microincineration and were similarly examined or photographed under March-April, 1970 Figure 4. Acid-insoluble ash pattern superim posed on the same field as in Figure 3. (t show* three dense deposits of chrysotile dull in what prob ably was originally the lumen of the bronchiole. The "snow" in the background is artifactual. Mi croincineration, 150X. dark-field illumination. ^ Figure 3. Contracted, densely collagenous scar in the-lung of rat injected intratracheaJIy with 3.S mg of very finely comminuted chrysotile 23 months previously. The scar probably represents an obliter ated bronchiole as judged by the size of associated blood vessels on its right bonier. Hematoxylin and cosin, 150X. Results Four days after the intratracheal injection, the lungs burdened with asbestos, talc, and microquartz showed a proliferative inflamma tion involving widely scattered smaller bronchi and bronchioles. This was characterized hy polypoid processes of avascular fibroblastic, tissue rich in argyrophilic fibers which en meshed large amounts of the injected dust. These polypoid structures originated from one or several widely separated ulcers in the mu cosa and distorted the bronchial lumen, con verting it into disconnected circumferential channels that tended to encircle the central stromal plug. These channels quickly became invested with normal-appearing ciliated col umnar epithelium. Within a few months, the C35794 0213 American Industrial Hygiene Association Journal 129 latter, considerable shrinkage of the lesions occurred months later when the initially argyrophilic stroma became converted into dense collagen. The main pulmonary response to the dusts of synthetic chrysotile, ceramic aluminum sili cate fibers, fibrous glass, brucite, and silicon carbide whiskers was the mobilization of macrophages which, filled with dust, occupied alveoli evaginating off respiratory bronchioles and alveolar ducts along with much extra cellular dust. The walls of these alveoli were thickened by a combination of surface cell enlargement and arborescence of the septal argyrophilic stroma. Perhaps the most interest ing feature of the pulmonary response was the development of fibroblastic tissue pror- Ficure 5. A polypoid mass of inflammatory tissue occupies the lumen of a respiratory bronchiole. Scattered macrophages are seen in many alveoli. Rat injected intratracheally with 3.5 mg of ceramic aluminum silicate and killed four days later. Hema toxylin and eosin, 150X. argvrophilic fibers were replaced by dense collagenous tissue. The lungs injected with asbestos dust had, in addition to the proliferative inflammation in the smaller bronchi and bronchioles, similar lesions in the respiratory bronchioles, and al veolar ducts. These more peripheral polypoid structures originated from one or mote of the evaginating alveoli. Although the former did not become covered with epithelium, as happened in the terminal bronchioles and larger passages, they did become converted into dense collagen and, as a result, under went considerable shrinkage (Figures 3 and 4). The lungs injected with talc showed num erous foci of proliferative inflammation in respiratory bronchioles and alveolar ducts similar to those encountered in lungs injected with asbestos (Figures 3 and 4); and, like the Ficure 6. Polypoid masses of inflammatory tissue occupy the lumen of a respiratory bronchiole (mid dle left) and the lumen of an alveolar duct (lower right). The inflammatory tissue is loose and cel lular. Transparent fibers and a giant cell are seen in the polyp in the lower right portion of the field. Rat injected intratracheally with 3.5 tug of glass fibers and killed four days later. Hematoxylin and eosin, 150X. 035794 0214 130 March-Aprtl, 1970 delicate. Along with the disappearance of the intraluminal polypoid inflammatory tissue and the reduction in macrophages, the amount of dust in the sections appeared to undergo a parallel reduction. The lungs of rats injected with amorphous magnesium silicate also showed occasional proliferative polypoid fibroblastic inflamma tion in respiratory bronchioles and alveolar ducts; like the lesions associated with synthetic chrysotile injections, they were no longer found some months later. In the main, the pulmonary response was a macrophage re action with minimal stromal reaction. Giant cells were also prominent. Figure 7. A terminal bronchiole containing a polypoid mars of inflammatory tissue enclosing numerous opaque fibers. It is of interest that the inflammatory tissue is already (S6 hours) covered by bronchiolar epithelium. Numerous leukocytes are present Rat injected intratracheally with 3.5 mg of silicon carbide whiskers and killed four days later. Hematoxylin and eoiin, 3Q0X. esses from one or several of the evaginating alveoli of respiratory bronchioles and alveolar ducts. This inflammatory tissue, consisting of argyrophilic stroma, extended in a polypoid manner into the lumen of the parent struc ture (Figures 5, 6 and 7). Well-developed by the fourth postinjection day, these lesions were less numerous by the fourteenth day and could not be found six months and longer after the injection. Collagenization of these lesions was not observed at any time. Evidence of the dust injections was still present in the form of dust-laden macrophages scattered throughout the section, but these were less numerous, loose, and usually separated from one another, and the walls of the air spaces in which they were found now were thin and Comments According to the commonly accepted defini tion of a fiber--a particle whose length is three times its diameter or longer--synthetic chrysotile certainly is fibrous. In one batch (Mellon Institute), the individual particles when viewed under an electron microscope are tubular crystals, the diameter of which in relation to their length is such that by no stretch of the imagination can they be con sidered needlelike (Figure 1). Nevertheless, this material, injected intratracheally, has pro duced proliferative inflammatory lesions sim ilar to those produced by injected brucitc. Furthermore, identical lesions have been seen in an occasional animal injected with amor phous magnesium silicate. It appears, there fore, that the proliferative inflammation noted four days after synthetic chrysotile injections may be ascribed to the high local concentra tions of magnesium silicate associated with the intratracheal injections. In view of the proved biologic inertness of ceramic aluminum silicate,1 silicon carbide,'-4 and glass,2 it is difficult to explain the pro duction of the proliferative inflammation ob served following intratracheal injection of the needlelike particles on any other basis than that of mechanical trauma. It would seem that the injection under pressure from the syringe causes the fluid to emerge from the needle with high velocity. Also, the fibrous particles, tending to align themselves parallel to the stream, would thereby tend to impinge point first on the mucosa of branching con- C35794 American Industrial Hygiene Association Journal 131 inhaled, even in high concentrations. Ex amples of this contradiction are chrysolite as bestos and fibrous glass. Animals have been exposed to high concentrations of chrysolite asbestos dust in inhalation chambers for more than a year without such polypoid-prolifera tive lesions having been observed.1'* We have under study at the present time rats and hamsters that have inhaled coated and un coated fibrous glass in concentrations approx imating 100 mg/m* for over one year, without detecting any such proliferative lesions7 (Fig ures 8, 9 and 10). We are. therefore, forced to conclude that fibrous dust, when injected intratracheally un der pressure, may produce mechanical trauma resulting in inflammatory foci which, how ever, resolve and disappear with time. These lesions must be considered artifactual. Figure 8. This field is typical of findings in the lungs of mts that had inhaled fibrous glass dust (100 mg/m1) for 232 days, 6 hours per day. It is noted that there is no fibrosis. The alveolar walls are thin and delicate but small dusters of darkly staining alveolar macrophages are present in alveoli clustered about some alveolar ducts. Hematoxylin and cosin, 15OX. ducting tubes, and the possibility of multiple small traumata, amounting to abrasions, be comes a probability. Nevertheless, we are faced with apparent contradictions. When we first investigated the biologic potential of ceramic aluminum silicate fibers,1 we did not observe the prolifer ative inflammatory lesions described above. The reason for this failure lies in the fact that these lesions disappear with time, and we had not examined the lungs during the first two weeks after the intratracheal injec tion. Another highly significant contradiction lies in the fact that such polypoid intraluminal proliferative lesions as are found following the intratracheal injecuon of certain fibrous dusts are not encountered when the same dusts are Ficure 9. The same field as in Figure^ 8 after decolorization and silver impregnation showing min imal stromal reaction which is limited to the re gions where macrophages are clustered and con sists of arborescent reticulin fibers. Gordon and Sweet, 150X. C35794 0216 132 Mareh-Aptil, 1970 and bronchioles caused by permanent scars' and short-lived reversible lesions. Viewed from another angle, the prolifera tive lesions produced by alt the fibrous dusts investigated except those of quartz, asbestos, and talc have the following characteristics: 1. Significant collagenization in the react ing lung tissue is absent 2. The anatomic integrity of the air spaces is maintained in spite of the presence of dust therein. 3. The lesions am reversible. These features are those of biologicalllv "in ert" dusts' and justify classifying synthetic chrysotile, fibrous glass, brucite, silicon carbide whiskers, and ceramic aluminum silicate in this category in spite of the polypoid prolifer ative inflammation produced when these dusts are injected intratracheally. The proliferative inflammation is considered to be artifactual, dependent on the injection technique. FnukE 10. This is the acid-insoluble ash pattern superimposed on the same photograph ns in Figure 8. The Urge amount of fibrous glass dust demon strable and the insignificant tissue reaction to its presence point to the biologic "inertness11 of this fibrous dust. The "snow" in the ^background is artifactual. Microincineration, 150X. The. difference between the proliferative lesions produced by the intratracheal injection of fibrous quartz, asbestos, and talc on the one hand, and those produced by similar in jections of synthetic chrysotile, silicon carbide whiskers, fibrous ceramic aluminum silicate, fibrous glass, and brucite on the other hand, is the difference between the deformed bronchi References 1. Gross, h. M. L. Wuntch, H. H. Schrckk and J. M. KcXerncv: The Effect* of a Synthetic Ceramic Fiber ).Dun upon the Lunp of Riu, A.M.A. Arrk. turn. HeoUk 13: 161 0956 2, Gross. I*., M. L. Vrstrsck, and J. M. McXustv: Gbu Duct. A Studv of Its Biologic Effects. A-Jl.A. Arrk. M. Hrsltk 21: ID (I960). X Gardner, L C.s Studies on the Relation of Miner*! Dusts to Tuberculosis. III. The Relatively Eariy Lesions in Experimental Pneumoconioeti Produced by Carborun dum Inhalation and Their InBoence on Pulmonary Tu- besoukua. dM. Rei-. Tubrrc. 7i 54+ (1923). 4. Gross. P,, M. L. Wcstsuck and J. XI. McMuurcv: Ex* perimenul Tuberculopoeumoconious. A~M.A. Arth. /ni. Httdh 19: $20 (I959|. ^ 5. Wsonat. J. C.: Asbcitotts in Experiments) Animals. Brit. J. tmJ. Mti. 20: 1 |I9C3). 6. Gross, P.r R. T. P. eeTixvuix, E. B. Toucta, M. KrschaK and M. A. BscvaKs Experimental Asbestos*, The Development erf Lung Cancer in Rats with Pulmo* nre Deposit* o( Chrysotile Atbcaio* Dust. ArcK. Espire*. He*lik 15: 543 (1967). 7. Gross, P., R. T. P. oeTREvitin. E. B. Toiu*. M. Kascmak. and XI. A. Bwvaks Ftbtwa Glass Dost, The Pulmonary Response to Looj-Tere* Inhaleiron of H*yn Concentrations. To be published. 8. Gross. P,, and C, A. Nau: Lignite and the Derived Steam-Activated Carbon, The Pulmonary Response to Their Dusts. Arch. nron. H**ltk 14: 450 (1967). Received June 12. 1969 C35^94 0217