Document NGQEdG2OE90w3dvgnmdY6RpLV

-it 1 'll 'nil /- VcaaaJL*- J. Cancer Envinmmmtat Health Perspectives. Vol. 34. pp. 37-46, mo activity of (P71). peritoneal iKorrCX: THIS UktSSXXL MAY BE PBOTECTED^ j_ ^ T, , BY COPYRIGHT LA (:mi17, tf.S. CODS) UcH 1 '-V 1 0 is-J. NL V. T. E-, 17 lumora r NOT Cl. 1 '' j L- 1-- i 0779). *4 ju> and ol. AnaL Comparative Studies on tSie Cytotoxicity iali*n;uit jral (IumJ s. 58: 97 of Amphibole and Serpentine Asbestos ullowirtj 1969). asbestos resethe- {I976J. by John E. Craighead,* Brooke T. &3ossman,* and Bruce J- Bradley* Thechemical and physiol properties of serpentine and amphibole asbestos arc considered in the context of ihrir interaction with tissue of the tracheobronchial tree end tongs. /1 rhnt studies in cultures of several types areevaluated and work with the erythrocyte hemolysis system is rrvirwed. Although fibers of the two nutjor mineral types dilTer substantially, it is likely they are modified by secretions and membranes of cells after inhalation to the respiratory tract. Investigations using virgin asbestos might not provide aa accurate picture of events in wow. Asbestos is not one, but a family of, hydrated mental composition, but have a basic Si,Oj. com silicate minerals having a fibrous crystalline struc position. Within the individual classes of asbestos, i ture. The types of asbestos differ mineralogically structural variability is common. Moreover, inor and, as might be expected, these chemical and ganic and organic chemicals of a variety of types i physical differences are reflected in the mechanism contaminate the mineral. Some of these foreign sub l i of interaction of fibers with cells. In this paper, we stances are found in association with the naturally * consider comparatively the characteristics of ser- occurring fiber, whereas others are added during I `pentine and amphibole asbestos and attempt to relate milling and industrial processing. i these features to the pathogenetic effects on tissues The biological effects of the various asbestos min 1 of the respiratory tract. erals appear to relate to their chemical and crystal /i Structural and Chemical Properties lographic properties. The fundamental Si:Oi sub units of chrysotile are tetrahedra linked to form extensive thin sheets of fixed composition. On the apical oxygens are located hydrated magnesium i1 1 [1 I Serpentine asbestos derives its name from the pliable, curled, "serpentlike" property of the fiber (Fig. J). Chrysotile, the only commercially impor tant mineral of this type, comprises over 90% of the asbestos mined in the world today. The members of the amphibole asbestos group are more numerous, but have fewer commercial uses. The amphiboles (crocidolite, amosite, and anlhophyllite) exhibit a coarse, fibrous structure (Fig. 2). The mineralogy of asbestos is complex and in completely defined. The types vary in major cle- molecules which account for the highly polar, basic property of the mineral. The layers of this silicate characteristically form concentric cylinders that parallel the fiber axis. In nature, these structures are arranged as loose bundles. This property accounts for the tendency of chrysotile to fragment into sub fibrils and segments along both the long and short axes (Fig. 3). Amphibole asbestos is formed of chains of tetra hedra linked into SuOtt units. The chains arc four tetrahedra wide and are situated parallel to the fiber axis. Cations of various types bind the units, al though these minerals cleave <nlong parallel planes *Dcpartmcnt oT Pathology. University of Verrucat. College of Medicine, llurlington, Vermont 05405. and are believed to break down .into subunits in tis sue (Fig. 4). j v urt I February 1980 n THIS DOCUMENT WAS NOT A RECORD OF 7"b1T0(>2063*1 I --------------- 2124 37 PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES AND CANNOf BE AUTHENTICATED BY PPG INDUSTRIES. INC. NOTE- ^']T P! r) -J -.-4 Asbestos in the Respiratory Tract The fate of inhaled asbestos in the human respira tory tract is incompletely defined. It is recognized that relatively long fibers either are removed from the inspired air in the upper respiratory tract or eliminated by the mucociliary escalator system of the tracheobronchial tree. The aerodynamic proper ties offibers are complex, but studies by Harris et al. (7) indicate that the depth of penetration of a fiber into the lung parenchyma relates to its crosssectional diameter. Of importance is the fragmenta tion offibers that is thought to occur after inhalation. Because fibers of small size cannot be recognized in tissue by traditional morphological techniques, the distribution and density of these deposits in the lungs is undefined, and their biologic significance unclear. Fibers or both serpentine and amphibolc asbestos exhibit complex surface deposits of iron-containing proteins (ferruginous bodies) when observed in lung tissue by light microscopy. These fibers are believed to be chemically inert. It seems unlikely that similar "coats" form on small fiber fragments in tissue, but this remains to be determined. Studies' in several laboratories have demonstrated the uptake of both albumin and sialic acid by chrysotile from biologic 38 THIS DOCUMEffT WAS NO.T A RECORD OF PPG INDUSTRIES, INC. DID NOT'COME FROM IT'S FILES AND CANNOT BE-AUTi-iS , : BY PPG' INDUSTRIES, INC. Environmental Health Perspectives 7 BB 00 2Q632_! 0^25  J --*'- iA'^ * "3j OTE: T: IT CL. kthUiitii * 'J* 'JJ* '5! 33 <4 -- F "T \ . ' >': -sih* y\ .i * -. r v , > v*/-:1-'': " -34*.*4* *L*t \r * '.%'1j i.;- *.; ^ r -..v:.: .-x'<U l :,^^6.vV' i.3 ' # . /^*V ^ ^'i - v*F y "'.ijS#orr*f <">>> f s; // A> * i t-.... .:>^V. >'-{.* .. T * vf ,- ' A.-L'/*.' ; \v 1 T: 'Mg?1*.-. .,, .._' '" W_TOiwV i.W.T--V,~ Fictne 1. Scanning electron micrograph of the serpentine asbestos, chrysotile. Note the variability in length and diameter of individual fibers as well as their twisted and curled "serpentlike" properties. Asbestos in the Respiratory Tract The fate of inhaled asbestos in the human respira tory tract is incompletely defined. It is recognized that relatively long fibers either are removed from the inspired air in the upper respiratory tract or eliminated by the mucociliary escalator system of the tracheobronchial tree. The aerodynamic proper ties of fibers are complex, but studies by Harris et al. (/) indicate that the depth of penetration of a fiber into the lung parenchyma relates to its erosssectionul diameter. Of importance is the fragmenta tion of fibers that is thought to occur after inhalation. Because fibers of small size cannot be recognized in tissue by traditional morphological techniques, the distribution and density of these deposits in the lungs is undefined, and their biologic significance unclear: Fibers of both serpentine and amphibole asbestos exhibit complex surface deposits of iron-containing proteins (ferruginous bodies) when observed in lung tissue by light microscopy. These fibers are believed to be chemically inert. It seems unlikely that similar "coats" form on small fiher fragments in tissue, but this remains to be determined. Studies in several laboratories have demonstrated the uptake of both albumin and sialic acid by chrysotile from biologic 38 THIS DOCUMENT^ WAS NOT A RECORD OF PPG INDOSTRIES, INC. DID NOT COME FROM IT'S FILES AND* CANNOT BE AUTHENTlCAtCD BY PPG INDUSTRIES, INC: . J Il W"J> -r-rng* V" V* 1/" Environmental Health Perspectives TbiT0020 633 V ------------ 2126  --- VOTE: I! o 7s *! r-1 i i n ~ TM <- 1 !OT CO. :*- ;-- ^\ 4 i Figure 3. Hypothetical representation of the freementation of a chrysolite fiber into fibrils along the long and short axes. Chrysolite fibers are comprised of numerous subunits that fragment when exposed to physical forces and biological fluids in vivo. Fkrjke 4. Diagrammatic representation of an amphiholc asbestos fiber ax represented by a stack of 2 x 4 in. pine boards. Fibers of crocidolite also fragment into subunit fibrils when exposed u> physical forces and biologic fluids in viva. 40 THIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES AND CANNOT BE AUTHENTICA. ll. BY PPG INDUSTRIES, INC. Environmental Health Perspectives' 7" Bb"* 00 20634 | 3127 tract secretions and fluids on the intrinsic structural and chemical integrity of the fiber is lacking. A de crease in the Mg:Si ratio of chrysotile fibers in pul monary tissues has been demonstrated (S). This sug gests that Mg** is removed in tissues, as is the case when the fiber is treated with acid in vitro. NOTE: n" n} IS T C *' T p ' NOT C: changes in the cells fail to develop and the interact! n of fiber with erythrocytes is less intimate. (Fig. 6). Since the fibers lack the strong positive charge of chrysotile, these observations suggest that ionic surface properties or the asbestos fibers are intrinsic to the cellular interactions of asbestos. Interactions cf Asbestos With Cells ]t generally is assumed that alveolar macrophages phagocytize the bulk of the small asbestos particles entering the acini of the lungs. Because chrysotile asbestos is cytotoxic for these cells in vitro (4). one might conclude that injury to the macrophages also occurs in the respiratory tract. Whether or not this is a correct assumption is a matter for debate. As briefly discussed above, the surface properties of the fiber doubtless arc changed by their interaction with respiratory secretions. Thus, laboratory studies using virgin asbestos may not appropriately simulate events occurring in vivo. Studies by Brody ct al. (P) have demonstrated phagocytosis of chrysotile by macrophages and type l pneumocytes in the lung of rats several days after inhalation exposure. These cells fail to reveal evidence of cytotoxicity when examined ultrastructurally. Erythrocytes The interaction of asbestos with mammalian erythrocytes in vitro has been examined by a number of investigators in an effort to assess the mechanism of cell injury (4-7. 10). We have pursued the model using scanning electron' microscopy to define the structural alterations that occur in cells interfaced with fibers. As can be seen in Figure 5, dramatic configurational changes become evident in the erythrocytes shortly after chrysotile is added to a cell suspension. These studies strongly suggest that hemolysis occurs, at least in .part, consequent to physical distortion of the red cell membrane. Iqjury to the cell also might result from alterations in the intrinsic molecular structure of the bilipid plasma membrane of the cell as has been suggested by Harington (4). Experimental data from our labora tory suggest that both mechanisms play a role in hemolysis (5). When virgin chrysotile asbestos is mixed with, erythocytesm vitro, the cell membrane and the fiber interact intimately and the cell leaks hemoglobin. This fails to occur when fibers are "coated" with either albumin or sialic acid, and after acid digestion. The amphibolc asbestos, crocidolite, hemoly7.es red blood cells after prolonged periods of exposure in vitro. The mechanism is unclear. Configurational Cultured Fibroblasts and TPuImonary Macrophages in Vitro Studies concerned with the toxicity ofasbestosfor cultured cells have been reported from a number of different laboratories. Although general conclusions can be drawn from a review of the literature, indi vidual reports conflict somewhat with another. It seems likely that the contrasting results obtained by various investigators reflect differences in the cell culture systems employed and subtle variables in experimental protocols. The intrinsic properties of the asbestos particle also are important. It is appar ent that results with fibers of different length, cannot be compared. Moreover, weight would seem to be a poor criterion upon which to base an assessment of dosage effects inasmuch as the surface properties of the fibers appear to be critical determinants of cellu lar injury. The work of several investigators clearly demon strates the cytotoxicity of chrysotile both for fibro blasts (11-13) and pulmonary macrophages (14, 15). The effect on fibroblasts doubtless is consequent to the surface charge of the particle and its Mg** con tent. Amphiboles appear to exhibit variable toxicity for reasons that are not evident. The effect could be attributable to either inorganic or organic surface contaminan's. but this is uncertain. The mechanism of macrophage cytotoxicity is more complex, inas much as the fibers interact with membranes of both the cell surface and the phagolysosomes. Release of lysosomal enzymes consequent to ingestion of the panicle has been amply demonstrated. Since many fibers arc too long to be taken up by cells, there might be an accentuated effect on enzyme release due to leakage from the phagosome (exocytosis). Tracheal Epithelial Cell Monolayer Cultures Figure 7 summarizes the results of studies carried out in this laboratory using monolayer cultures of epithelial cells derived from the mucosa of the hamster trachea (16). These cells exhibit the differ entiated properties of respiratory epithelial cells for they produce extracellular mucins (17). and contain both cilia and mucous vacuoles V6). As can he seen, chrysotile inhibits growth of cells over a range of dosages when the mineral is introduced at the time cells are plated US). Equivalent amounts of crocido lite have no effect. Thus, observations using difler- Febmary 198(THIS DOCUMENT WAS NOT A RECORD OF . PPG INDUSTRIES, INC. DID NOT COME FROM ITS FILES AND CANNOT BE AUTHENTICATED BY PPG INDUSTRIES, INC. i_ff_00 2063 5_7 41 3128 |i n-4i . ! i I i i 3 i i i f *3 ; .1 i I ; i j i I is id / i es 1 I *. _ > ,-^T- ; * c \ ^-- LCM. ..,,i_U(-- 5 ._T.*" - JJ notfrniki6n.Cilyl.w..i.tht.cih,.ryl.sTMolite.. .Atmapwhiboi*lc fiber* exhibit .a relatively negative surface chan. b*1 therefore wooki not be.expecte^ the erythrocyte plasma membrane after proiongcU exposure. February 1980 jju.j THIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES. AND CANNOT BE AUTHENTICAtED BY PPG INDUSTRIES, INC. | BB 0020637^1 43 3130 - NOTE: TH,1 jo r* I Figure 7. Effect of (- -) chrysolite and (------ ) crocidoiite on Growth of monolayer cell cultures derived from the hamster tracheal epithelium. The effect of chrysolite is attributed to cytoiysis. entiated epithelial cells in monolayers correspond with the results of studies by other investigators using both fibroblasts and macrophages. CYTOTOXICITY m HAMSTIK TKACHES 24 In Ann ASBESTOS EXPOSURE IS * nut (<us) Tracheal Epithelial Organ Cultures In a recent report, Mossman and Craighead de scribed the events that occur when explants of hamster trachea are exposed to crocidoiite (79). These studies document the cytotoxicity of the min eral for the mucociliary mucosa. This effect appears to be consequent to a direct interaction of the fibers with these highly differentiated cells. Interestingly enough, basal cells were not affected, and an intact layer was observed adjacent to the basal lamina after the superficial cells had sloughed. These cells phagocytized the asbestos panicles and responded by undergoing hyperplasia and metaplasia. When chrysotile was introduced into cultures of trachea. W3 : main us-sk) I 1 mum oust * mx/mi irtrUi ia tm Mm JTT Figure 8. Effect of chrysolite and crocidoiite on the intact differ* entiaied respiratory mucosa of hamster tracheal organ cul tures. Concentrations indicate the amount of asBestos (mjf/ral) in the medium. 44 Environmental Health Perspectives S N'W a *etD 0. by PprEf AND C'an^0T BE AUTW?ME FR0M ^31 Y PPG industries, INC UTHNTtCATED f'TS " **> it m( .M'l'j ,ri. "j-t m'~ " *.bjm * 'I I 'I'n-ni-h i r. Ij ITM 7* cytotoxicity was more pronounced. (Fig. S). As with crocidoiite, the basal ceils remained intact. Thus, the differentiated epithelium responded to both types of asbestos in a comparable fashion. Discussion and Summary Asbestos is a generic term referring to a family of hydrated silicate minerals having a basic fibrous composition. Serpentine and amphibole asbestos differ structurally and in chemical composition. These features are reflected in the biologic effects summarized here. The cytotoxicity of the serpentine, chrysotile, appears to relate to its magnesium content. Most, if not all, ofthe effects are believed to be consequent to the strong positive charge this cation introduces on the fiber surface. Chrysotile avidly interacts with the membranes of the cell surface and lysosomes. The mechanism whereby it injures these structures re* mains to be defined. In biological systems, chrysotile would appear to interrelate dynamically with its environment. The cytotoxic properties of the mineral are attenuated by a wide variety of naturally occurring anionic sub stances, and the highly reactive Mg** ions are leached during residence in vivo. Thus, inhaled chrysotile in respiratory tract tissue is not compara ble to the virgin mineral used in most in vitro model systems. The amphiboles, amosite and crocidoiite, ` are less cytotoxic than chrysotile. These minerals also absorb biologic substances from fluids and tis sue//! vivo. Thus, the surface properties of the am phiboles also change in the respiratory tract. Thccytotoxic e/Tects ofthe various asbestos types may, in part, be consequent to the inorganic and organic materials that contaminate the basic miner als. This is a hypothetical consideration, however, for conclusive evidence to indicate that these sub stances iryure cells has not been published as of yet. Asbestos triggers the alternate pathway of the com plement system. It remains to be determined whether or not complement-mediated injury is im portant- Moreover, it is not known if the interaction with complement enhances uptake of particles by alveolar macrophages,' cells having plasma mem brane Ca receptors. In our experimental organ culture system, both crocidoiite and chrysotile were cytotoxic for the differentiated mucociliary cell layer of the tracheal mucosa. At present it is not clear why the basal cell layer is resistant to the injurious effects of asbestos. However, as discussed above, the fiber surface is modified by its interaction with biologic substances. This may render the particle nontoxic. Whatever the mechanism, the lack of an apparent effect of usbes- NOTE: NOT CO * `iv'M i i I -I1 tos on basal cells is important for the injured epithelium regenerates from the basal layer. It is of interest in this regard that hyperplasia and squamous metaplasia develop in tracheal organ cultures as a consequence of their interaction with the amphibole asbestos, crocidoiite and amosite- Tire experimental work briefly summarized above indicates that the surface properties and configura tional forms of both amphibole and serpentine as bestos are altered after inhalation into the respira tory tract. This conclusion should be considered in the design of in vivo laboratory experiments to examine the pathogenesis of asbestos-associated diseases. This research was supported by KIH Contract PHS CP 33360, and NIOSH grants PHS R01 00633 end PHS 00888. REFERENCES 1. Harris. R. l_ Timbrel!, V., and Berry. G. The influence of fiber shape in brag deposition -- mathematical estimates. Inhaled Particles. V<rf. IV (Proceedings of an International Symposium, British Occupational Hygiene Society, Edin burgh. Sept. 22-26, 1975), W. H. Walttm. Ed.. Pergamon Press, New York, I9T7, p. 73. 2. Morgan. A. Adsorption of human serum albumin by itsboliform minerals and its application to the measurement of surface areas of dispersed samples of chrysotile. Environ. Res. 7: 330 (1974). 3. Haringion, J. S., Miller, K-. and MacNab. G. Hemolysis by asbestos. Environ. Res. 4: 93 (1971). 4. Harington, J. S-Thc biological effects of mineral fibers, espe cially asbestos, as seen from in vitro and in vivo studies. Amt. Anat. Pathol. 21: 155 (1976). 5. Bradley, B. J- and Craighead. J. E. Unpublished observa tions. 6. Light, W. G. and Wei, E. T. Surface charge and hemolytic activity of asbestos. Environ. Res. 13: 133 (1977). 7. Morgan, A , Davies, P.. Wagner, J. CL, Berry, G,, and Holmes, A. The biological effects of magnesium-leached chrysotile asbestos. Brit. J. ExptL Pathol. 38: 463 (1977). 8. Jaurand, M. C.. Bipon. 3.. Sebastien. P- and Goai. J. Leaching of chrysotile asbestos in human lungs. Environ. Res. 14: 243 (1977). 9. Brody, A. R. (NIEHS. Research Triangle Park, N.C.L per sonal communication. 10. Scimitar. R. J- and Pundsack. F. L. Asbestos hemolysis. Environ. Res. 3: 1 (1970). 11. Richards. R. J_, and Jacoby, F. Light microscope studies on the effects of chrysotile asbestos and fiber glass on the mor phology and reticulin formation of cultured lung fibroblasts. Environ. Res. 11: 112 (1976). 12. Hcxt. P. M,, nod Richards. R. J. Biochemical effects of as- bestifonn minerals on lung fibroblast cultures. Brit. J. Eaptl. Pathol.-57: 231 (1976). 13. Chamberiam. and Brown. B. C The cytotoxic effects of asbestos and other mineral dust in tissue culture ceO lines. Brit. J. ExptL Pathol. 59: 183 (19781, 14. Miller, K.. and Harington. J. S. Some biological effects of asbestos on macrophages. Brit. J. ExptL PnthoL 33: 397 (1972). 15. Allison. A C. Experimental methods -- cell and tissue cul ture effects ofasbestos panicles on macrophages, mesothelial cells and fibroblasts. In: Biological Effects of Asbestos, P. Dogovski. V. Timbrcll. J. C. Gilson, and J. C. Wagner. Eds.. Int. Agency Research Cancer, Lyon. 1973. FebnisYjjj1|9^l0CUMENT WAS N0T A Record of PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES. AND CANNOT RE AUTHENTICATED BY PPG INDUSTRIES, INC. T j* * y ------ .------------------- ~-.^20639*7 no *-0.0^ j--wjim i' ^ ~~' --. 4 i-T.-l > ...............-- f " - f'V-J--~= ~-----------^------ --- "" inifflhJaLi "Mia.* 16. Mossman. B. T.. Ezcrmnn. E. B., Adler. K. B., and Craighead, J. E. Isolation and spontaneous transformation of hamster tracheal epithelium. Submitted. 17. Last.J. A., Kaizu. T.. and Mossman. B. T. Mucus glycopro tein synthesis by an established cell line from hamster tracheal epithelium. Exp. Lung Res., in press. 18. Mossman, B. T.. Craiphend, J. E., and Bradley. B. J. Com parative cytotoxicity of chrysotile and crocidolitc asbestos in hamster tracheal epithelial cells. Fed. Ptoc. 38: 1352 (1979). 19. Mossman. B. T. and Craiphead. J. E. Interaction of crocido lite asbestos with hamster respiratory mucosa m organ cul ture. Lab. Invest. 36: 131 (1977).. NOTE: THIS Or*-:; v Tio NOT C0i\iE Ft'iOiVi rr TILES f / t THIS-DOCUMENT WAS NOT A RECORD OF 46 . PPG INDUSTRIES, INC. OID NOT COME FROM Environmental Health Perspectives IT'S FILES AND CANNOT BE AUTHE: BY PPG INCUST-R'.E$. INC. . ________ __ 04 03 BB 0020640 J Jl JU I