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Lung Asbestos Content in Chrysotile Workers with Mesothelioma \NDREW CHURG, BARRY WIGGS, LISA OEPAOLI, BRIGITA KAMPE, and BONNIE STEVENS Introduction \lthough the association of mesothe- ioma with asbestos exposure is well esablished, there is considerable conroversy concerning the relative carcino- SUMMARY Th rol* of chryiotil* asbestos In ths genesis of mesotheliomas in humans is d;J[)u| ed. We analyzed the asbestos content of the lung In 6 long-term chrysotile mintrs and mj||(ri who had pleural mesotheliomas. In five patients, only chrysotile ore components (chrysotile 4n<J tremollte/acttnollte/anthophylllte types of amphibole asbestos) were found, while the sixth piti*,, presented both chrysotile ore components and amosite, a type of asbestos that is not derive^ :enic effects of the different types of from the mining process. The mean number of flberslg dry lung for the 5 patients with mesothsiio. isbestos. It is generally agreed that comnercial forms ot amphibole asbestos, ' imOSlte"and crociaoutf i especialtv_cro- iclotitej, can induce mesothelioma (1-8), >ut it has been suggested tnai. in humans rc syS- rhry<?r.T;ifl ic glthor.vwry wpalf.lv or IQt at all rarrinriffpnin in this regard (AJtV or example, McDonald and associates ma containing only chrysotile ore components was higher(chrysotile $4 x I0` and trsmolitgrou() 540 x 10*) than in a group of long-term chrysotile miner control subjects who had no ntj,,(oj related dfseate (chrysotile 23 x 10*,tremollte group 58 x t0`), but some patients with masothnio. ma had fiber burdens near the mean of the control range. Fiber sizes and aspect ratios in th mesothelioma group were approximately the same as those In the control subjects, and analyst, of fiber distribution failed to show any preferential localization In the periphery of the lung. Ho* ever, the concentration ratio of tremollte In the lungs of the mesothelioma cases competed to the control cases was 9.3, while the ratio of chrysotile was only 2.8. pur findings provirt. i^n evidence that chrysotile mine dust (chrysotile and amphibole components)can produce mssothttiJ. ny in humynij theoreatorrelollve amounts of tremollte group ampniboies present In the mll.T., 6) found only IS cases of mesothelioma with mesothelioma raise the possibility that these fibers may be important in im pathogenesis n a series of 4,547 deaths in Quebec ^JgUbe-tumera- ~ " am rev respir tats UMtlStj-ronto :hrysotile industry workers, compared to > of 56 deaths (1) in a series of workers vho had assembled gas masks incor porating crocidolite asbestos. Berry and elusions. Langer and McCaughey (10) Methods . 'Jewhouse (8) studied a series of 13,000 isbestos factory workers exposed to hrysotiie and crocidolite, and conclud- have suggested, based on the observation of chrysotile fibers in a tissue section of lung, that a case of mesothelioma in a The 6 cases analyzed in this study are derived from a series of approximately 90 sequentuj autopsies of long-term workers in the Que ;d that only those exposed to crocidolite brake repair worker was produced by bec chrysotile industry; the autopsies were per ieveloped mesothelioma. McDonald and -ry (7) found a mesothelioma 'rate of 1.4/1,000 deaths in a factory processing >nly chrysotile, compared to 12.6/ 1,000 deaths in a factory processing chrysotile and amphiboles. Similar conclusions were reached by Acheson and coworkers (4,5) in 2 different exposure groups. How ever, many of these studies are confound chrysotile asbestos; although this case may indeed represent a chrysotileinduced mesothelioma, the volume of tis sue sampled is so small that amosite or crocidolite might have been missed. Chrysotile miners and millers represent a population with, theoretically, exposure only to chrysotile mine dust; in this pa per we analyze the mineral content in the formed from late 1980 through the end ofl98J at the Hopital General de la Region de l'Amiante at Thetford Mines, Quebec. Cases and exposure histories were obtained through the courtesy of Dr. M. Poulin and Mr. C. Pratte of that institution. All tissues were fixed m formalin. The 6 patients with mesothelioma repre sent ail the mesotheliomas present in the se ries of 90 cases. The diagnosis in all cases was ed by uncertainties regarding the exact lungs of 6 such workers and demonstrate confirmed by finding the following; a typical ype of Tiber to which individual work- that chrysotile mine dust can produce gross appearance of tumor encasing the lung, ;rs may have been exposed. Because more than 90fo of the as- Jf"; Ticpft.ii/nrlH.wj^/-;T/--t1rycoljU tfyfi mesotheliomas. We also examine the rela tionship of fiber number, fiber type, and fiber size and shape to the development no evidence of another primary at autopsy, and the typical histologic and histochemicai features (II) of mesothelioma. All of the question is of considerable importance. of mesothelioma. Mineralogic evaluation of lung tissue is Throughout this oaoer. we shall use jne method of producing exact data re- the term "chrysotile ore components*1, and' iarding lung mineral burden, but to our ' ""chrysotile mine dust", to signify both (Received in originalform October 20, 1983 m3 cnowledge, no series of occupationally , jSHrygnrilc -)ch^rrnr^H-th.--sm^hlholfQ in revised form May 21, 1984) exposed workers with mesothelioma have tremolit^. actinolite. and anfhnpfiyllitR. seen shown to have only chrysotile ore tomponents (see definition subsequent) .n their lungs. Wagner and associates (9) attempted such an analysis on lungs from Which are integral oarts.of the chrysotile pre. These latter forms of asbestos are essentially contaminants of the ore, and are not purposely mined. By compari ' From the Department of Pathology, Univer sity of British Columbia, Vancouver, B.C.. Canada 1 Supported by Grants No. MT6907 and MAJEO from the Medical Research Council of Canada*!*! a grant from the National Cancer Institute oi workers in an asbestos factory, and found that, in addition to chrysotile, amosite, md especially crocidolite, were also presI ent, thus ruling out any definitive con- son, the amphibole minerals amosite and crocidolite are forms of asbestos that are completely unrelated to chrysotile and which are mined and used commercially. Canada. ' Requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology University of British Columbia, 2211 Wesbtoot Mall, Vancouver, B.C. V6T IWJ, Canada. 1042 TABLE 1 there is considerable overlap in the 2 DEMOGRAPHIC DATA groups. ;,S' ' Age/Sex 58/M 52/M 60/M 67/M 65/M 68/M Employment m Chrysolite Industry id years (1952 to 1980) 10 years(1946 to 1956) 36 years (1947 to 1983) 42 years (1937 to 1979) 40 years (1935 to 1975) 47 years (1928 to 1975) Date of Death 1983 1982 1983 1983 1983 1981 The distribution of fiber sizes in the Details of Occupation Mechanic in mine, no mill work Fiber bagger in mill/mechanic in mine Fiber bagger and sorter in mill/ patients with mesothelioma compared to the control group is shown in table 3. The aspect (length to width) ratios for vari ous fibers is shown in table 4, the ratios of numbers of fibers in central and pe miner ripheral sites are listed in table 5, and the Underground miner only Fiber separator, mill No details available fiber size distribution and aspect ratios for fibers in central versus peripheral sites - are shown in tables 6 and 7. In all these tables, the mesothelioma group is com posed of patients I to 5 (those contain -ors in this report were of pleural cavity (mean SD)/g dry lung, and of the ing chrysotile ore components only). Be iriein- For comparison purposes, we used 9 piously published long-term chrysotile in- justrv workers from the same autopsy series tJ2); these 9 patients had neither mesothelio ma nor asbestos-induced parenchymal or air- *av fibrosis. For mineralogic analysis, we followed the same procedure as previously reported (12). Fhis consists, in brief, of dissolving 3- to 5-g tremolite group of amphiboles, 540 840 fibers/g dry lung. These values are compared in table 2 to those found in the reference chrysotile miners (12), and a group of 25 men without occupational asbestos exposure (13). Although the pa tients with mesothelioma have higher mean level, that le control subjects, cause distributions of aspect ratios are often skewed, geometric means are in cluded in tables 4 and 7 as well. There were no differences found for fiber size distribution or fiber aspect ratios either between mesothelioma and control groups, or between central and periph eral sample sites. There was no tendency asnples of formalin fixed lung in bleach, col lecting the mineral particulate sediment on a Millipore filter, and transferring the parti cles to a coated electron microscope grid. The TABLE 2 arid is then scanned in the electron micro scope, and every fiber encountered is identi NUMBERS OF FIBERS FOR ALL SITES (ALL COUNTS AS FIBERS x 10*/G DRY LUNG) fied by a combination of morphology, elec ;ton diffraction, and energy dispersive X-ray ,pectroscopy. For the present investigation, Case Chrysotile Tremolite/Actinolite/ Anthophyllite Amosite or Crocidolite asubpleural 0.5-cm deep sample and anoth- 1 63 244 0 er piece from 3 cm deep within the lung were obtained and processed in order to compare central and peripheral distribution of fibers. Approximately 100 fibers were examined for each case. Results were calculated using an algorithm relating weight of tissue used and 2 3' 4 5 Mean - SD' 6 8 69 10 169 64 65 44 44 320 62 2023 540 840 54 0 0 0 0 0 70 (amosite) 1 grid area, and expressed as fibers/g dry lung. Reference population of 9 chrysotile miners without mesothelioma (12). A blank control was created for each test Mean: 23 58 0.02 ample. This was prepared by running the en:>ne preparatory procedure without tissue, and canning approximately 50 grid squares in the electron microscope to detect contaminants Reference population of 25 men without occupational asbestos exposure (13): Mean: 0.6 0.3 0.01 ' Mean values for 5 cases containing only chrysotile ore components. from solutions and air. In this study, only rare particles of very short fiber chrysotile were TABLE 3 found in the blanks, and these values were FIBER SIZE DISTRIBUTION FOR ALL SAMPLE SITES subtracted from the test values. Percent of fibers in size range - Results Demographic data and occupational de tails are shown in table 1, and results of mineralogic analysis are shown in table 2. Tremolite, actinolite, and anthophyllite are grouped into 1 category, as we have done previously (12). Five of the 6 Patients with mperithelioma had only cfirygnrilf* rsrr* cnmpnnentS in theitlungS. The sixth patient also had substantial lev els of amosite, and is excluded from the calculations of mean fiber number, size, etc., in order to deal with a mineralogically homogeneous population. For the 5 patients with mesothelioma, the mean number of chrysotile fibers was 64 65 Chrysotile Mesothelioma Patients 1 to 5 Mining control group (12) Tremolitefactinolite/anthophyllite Mesothelioma Patients 1 to 5 Mining control group (12) 0.5 to 5 i 89 85 87 94 5 to 10 u 11 6 10 + TABLE 4 MEAN FIBER ASPECT RATIOS FOR ALL SAMPLE SITES' Mesothelioma Patients 1 to 5 Control Group (12) Chrysotile Tremolitefactinolite anthophyllite 92 103 (60) 14 12(11) 96 * 120(53) 13 12 (9) ` values are mean + SO: geometric mean in parentheses. I 1044 CHURa WIGGS. OEPAOU. XAUPE. TABLE S MEAN VALUES OF RATIO OF NUMBERS OF CENTRAL .... ................ TO PERIPHERAL FIBERS , ent in the ore in much larger n,., ttefcamphiboicTTEr accumula>r tissue. Whether this phenomenoiITnS Mesothelioma patients Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mean SD Control group: Mean SD _ . Chrysotile 7.5 1,0 0.4 0.6 3.6 2.6 3.0 __ 2.3, - 3.1 Tremolite/Actmolite/ Anlhophylltte 5.9 0.5 0.1 0.5 u 1.6 * 2.4 . 2-0 a 1.1 chemical leaching of the fibers hanced clearanceof chrysotile com 'Cnto amphibole, or low pulmonary (L3"* tion of chrysotile compared to am bole, is not clear. Our subsequent *' ments must, therefore, be interpret!?" light of the fact that we cannot deter!?:r what chrysotile load may have been ^ ent in these lungs, although the stud!5'" Rowlands and associates (15) su., TABLE 6 DISTRIBUTION OF FIBER SIZES IN PERIPHERAL VERSUS CENTRAL SAMPLES OF MESOTHELIOMA PATIENTSJ__TO___ that estimates of fiber burden dcrj ' from mineralogic analysis correlate with estimates of dust exposure in n, ^ ' Workers. ^ Percent in Size Range 0.5 to 5 u _ .5 tq 10 a ,1Q+. The types, sizes, and distribution 0i fibers that induce, mesothelioma ha, been the subject of considerable di'scn? Chrysotile Central Peripheral Tremolite/Actmolite/Anthophylfite Central Peripheral 87 95 88 88 . . 12 1 4i 11 12-- i r. 2 sion based on animal experiments (I7jg> measurements of mine dust size, and tli oretical calculations of fiber deposit;,), in the lung (see Harington (19) for ^ .view). Animal studies (17,181 haw^ ` that mesothelioma canbe induced hy ^ TABLE 7 ASPECT RATIOS FOR FIBERS IN CENTRAL VERSUS PERIPHERAL SITES IN MESOTHELIOMA PATIENTS 1 TO 5' --- - --__ _ ' - * -TT-H.at1-Central. . .. * Pe-*r-i-p-her-al - Chrysotile 92 101 (57) 92 106(82) T. remoU_te/Actinolite--/A__n__t_h__o__p: hyiW_--te 13 12111) .......1.4.....1..2...(1.0)' * Values are mean * SO: geometric mean in parentheses. tually any insolubleminerai fibenfitT~ sufficiently small (particularly, diamei . je&ihan 0.25 u and letmluutater ih^ 8. u) and.has.a. high aspect (length to widthlratio. In contrast to the sitnari^ jnjmmans, cTTrysrujlernTnearstoheft? ~as_effective as crocidolite in indm-j^ mesothelioma in animals (18). It has alstTBeen postulated that, "in Humans who for fibers to concentrate in the periph ery of the lung (table 5). The ratios of numbers of fibers of each type in mesothelioma patients 1 to 5 ver sus the reference cases are shown in table 8. The ratio of tremolite (9.3) between the groups is considerably higher than that of chrysotile (2.8). likely that the worker was employed in one of the asbestos processing factories in Quebec that used amosite at various times (14), but we were unable to obtain enough occupational details to verify this possibility. Amosite in chrysotile miners has been demonstrated previously: Rowlands and associates (15) found it in Discussion the lungs of 2 of 47 Quebec chrysotile workers, an observation that emphasizes As noted in Introduction, epidemiolog again the usefulness of confirming ic studies suggest that chrysotile is a rel epidemiologic studies with mineralogic atively weak mesothelial carcinogen in analysis. Nonetheless, our data tom the man; however, it is clear that, even in analysis of Patients 1 to 5 provide strong many factories that processed largely ~gvfdence that chrysotile mine dust com- chrysotile, amphibole was also used at 'ponents (as defined) can induce one point (5,8,9), and hence, actual tis mesotheliomas sue analysis is required to demonstrate Which Of the minerals in fhp chrvso- that only chrysotile or chrysotile ore com tile ore is producing the tumors is a mat ponents are present before a given case ter of considerable importance. As in our can be definitely ascribed to chrysotile previous study of chrysotile miners (12) dust inhalation. The requirement for such ahd the study ot Rowlands arid associ analysis is well illustrated by our Patient ates (to), we found more ampbibofe 5, a long-term worker in the chrysotile (tremolite/actinolite/anthophyllite) 'de .ndustry, whose lungs contained large rived from-rhe-ftfB4aodyUran chrysotile unounts of amosite asbestos as well as in these lungs, an observation that reflects .-hrysotile ore dust. In this instance, it is the failure of chrysotile. originally pres- are inhaling asbestos dust, mesothelio ma is induced by dust that penetrates far into the periphery of the lung (19). Hence, it is reasonable to suppose that patienu with mesothelioma might have either higher aspect ratio fibers, or greater num bers of fibers, or more fibers in the pe riphery of the lung than patients with roughly comparable exposure but no mesotheliomas. In fact, we were unable to find anveonsistent ditterences m fiber size or fiber aSpecrfatio between our 5 natientswiih' mesothelioma and 9 control subjects (12). There was no evidence of a.trenri fnr pe ripheral deposition nf.fihers.jjiihfrfa- tients withuxtesot-helioma-fiable^I One TABLE 8 RATIOS OF NUMBERS OF FIBERS OF DIFFERENT MINERALS IN MESOTHELIOMA PATIENTS 1 TO 5 AND CONTROL GROUPS Mineral Ratio Mesothelioma Reference (121 Chrysotile Tremolite/actinolite/anthophyllite 2.8 9.3 * : and lOTHEUOMA jjso suspect, a priori, that because Our observations in chrysotile mine in- Ttk'* the ore separates the long fibers, dustryworkefS thns raisETfie possibility TSeHom. should be found in millers that the amphibole component of the than miners. But 2 of the 5 pa chrysotile ore is important in the genesis , >'5 s for whom we have detailed occupa-" ~of mesothelioma in this group. This sit: al histories appear to have worked ~ uation may be similar to that'reported :lw in the mines. in mesothelioma patients exposed to mix 'However, we did find differences in to tures of chrysotile and the commercial nt com. let^in >ne ^nPres. ' u4-of . fiber numbers and in the ratios of a* of fibers between the groups (ta l and 8). The mean values of fiber ^orjJlcSeistieffoma^gfeSp^i^e ' higher than those for the amphiboles amosite and crocidolite (21,22), where the amphibole appears to play a major role in tumor induction. Ad ditionally, because elevated levels of tremolite and actinohte amphiboles have Hu8gesu drived ate wen in the^ 13 ffioupTbut 2 of the patientswith r n)fl] " ~ ,tients.2aqd4^had fi at orbelowthenRan -ygereferencg-yrnim. He.nceonecah- beenTound in the lungs of some s5condarTchrysotile industry workerSf which we (23. and Churg A. unpublished data) ~an3 others (9) have analyzed, and inphr- tion of !a have d'scus- (17,18), tndthe. 'sition for re. : shown 1 by vir- r if it is atneter sr than tgth to nation be just ducing ttSsiIy ascribe the mesotheliomas only ' (,;gh overall pnmhercfnr'fther*-- -^jf^aTficularinterest is the fact that ,hc patients with mesothelioma having jn|y chrysotile ore components had a much higher ratio of tremolite group am , ,,hiboles (9.3) than chrysotile fibers (2.8) Compared to the control group (table 8), Jnd that this was not true of Patient 6, n'hom amosite was found. Stanton and associates (17) and Wagner and coworkirs (20) have shown that long, thin, high aspect ratio tremolite induces mesothelio ma in animals, whereas short thick fiber iremolite is less effective or ineffective, an observation that agrees with the the ticular, because some of that amphibole Tsofrelatively long length (23), thesSme Pnnrfminn mighr.apnlv to textlTTWgrkers and others exposed to processed chmfflflelnrr. -- ------ --------------- References t. McDonald AD, McDonald JC. Mesothelio ma after crocidolite exposure during gas mask manufacture. Environ Res 1978; 17:340-6. 2. SelikofftJ, Seidman H, Hammond ED. Mor tality effects of cigarette smoking among amosite asbestos factory workers. J Natl Cancer Inst 1980; 65:307-13. 3. Jones JSP, Smith PG, Pooley FD. The conse quences of exposure to asbestos dust in a wartime has al i is who ' thelio- ites far Hence, atients ory of Stanton and associates (17) that tumor frequency is proportional to fiber length and aspect ratio (aspect ratio greater than 32 in the study of Stanton and associates). But as Harington (19) j points out, the animal studies also show gas-mask factory. In: Biological effects of mineral fibers. Wagner JC, ed, Lyon: International Agen cy for Research on Cancer, 1980, pp. 637-S3. 4. Acheson ED, Gardner MJ, Pippard EC, Grime LP. Mortality of two groups of women who manufactured gas masks from chrysotile and crocidolite asbestos: a 40 year follow-up. Br J In either that the requirement for high aspect ra dust Med 1982; 39:344-8. rnumn the peents with but no tio or long length is not absolute, and that large numbers of short lower aspect fibers can induce mesothelioma. Our chrysotile miners would presumably fall into tne latter category because the mean 5. Acheson ED, Gardner MJ, Bennett C, Winter PD. Mesothelioma in a factory using amosite and chrysotile asbestos. Lancet 1981; 2:1403-6. 6. McDonald JC, Liddell FDK, Gibbs GW, Eyssen GE, McDonald AD. Dust exposure and mor tality in chrysotile mining, 1910-1975. Br J Indust I any con- aspect ratio for tremolite amphiboles in Med 1980; 37:11-24. 'r fiber their lungs is about 13 to 14 (table 4). 7. McDonald AD, Fry JS. Mesothelioma and fi s with ts (12). or pe te pa ). One ber type in three American asbestos facto Preliminary report. Seand J Work Enviror. 1982; 8 (Suppl 11:53-8. 8. Berry G, Newhouse ML. Mortality of wi manufacturing friction materials using asb Br J Indust Med 1983; 40:1-7, 9. Wagner JC. Berry G, Pooley FD. Mesot mas and asbestos type in asbestos textile wc a study of lung contents. Br Med J 1982: 2:( 10. Langer A. McCaughey WTE. Mesothe in a brake repair worker. Lancet 1982; 2:1 11. Kannerstein M, McCaughey WTE. Ch Selikoff 1J. A critique of the criteria for the nosis of diffuse malignant mesothelioma. MMed J 1977: 44:485-94. 12. Churg A. Asbestos fibre content of the in patients with and without asbestos airwa ease. Am Rev Respir Dis 1983; 127:470-3. 13. Churg A. Asbestos fibers and pleural p in an autopsy population. Am J Pathol 109:79-96. 14. McDonald AD. Malignant mesothelic Quebec. In: Wagner JC, ed. Biological effi mineral fibres. Lyon: International Agency f search on Cancer. 1980, pp. 673-81. 15. Rowlands N, Gibbs GW, McDonald A bestos fibers in the lungs of chrysotile mine millers. Ann Occup Hyg 1982; 26:411-6. 16. Jaurand MC, Bignon J. Sebastien P, C Leaching of chrysotile asbestos in human Environ Res 1977; 14:245-54. 17. Stanton MF, Layard M, Tegeris A, et at tion of particle dimension to carcinogenicity phibole asbestoses and other fibrous mint Natl Cancer Inst USA 1981; 67: 965-75. 18. Wagner JC. Berry G, Timbrel! V. Meso mata in rats after inoculation with asbest, other minerals. Br J Cancer 1973; 28:173 19. Harington JS. Fiber carcinogenesis: ep ologic observations and the Stanton Hype J Natl Cancer Inst USA 1981: 67:977-89. 20. Wagner JC, Chamberlain M, Brown al. Biological effects of tremolite. Br J Cance 45:352-60. 21. McDonald AD, McDonald JC, Pool Mineral fiber content of lung in mcsotheliai n in North America. Ann Occ Hyg 1982; 26:< 22. Wagner JC, Pooley FD, Berry G, e pathological and mtneralogical study of as related deaths in the United Kingdom in 19' Occ Hyg 1982; 26:423-31, 23. Churg A, Harley RA. Long fiber asbi a chrysotile textile worker. Lancet 1984; OF 'OMA 3UPS io lioma ce (121 I