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Mineralogic Parameters Related to Amosite Asbestos-induced Fibrosis in Humans1-3 PLAINTIFF'S EXHIBIT ANDREW CHURG, JOANNE WRIGHT, BARRY WIGGS, and LISA DEPAOLI Introduction Epidemiologic studies as well as analy ses of lung asbestos content have suggest ed that asbestosis (diffuse interstitial fibrosis) is associated witn exposure to or retention of very large amounts of ashestos ri-8). Analytic studies quantifving either asbestos bodies or uncoated asbestos fibers have further indicated that on a case by case basis^greater asbestos burdens are associated with pathologi cally greater degrees ot norosiS"f3^8). These reports have generally consid ered only the issue of fiber number and evaluated only one sample per case, so that the effects of intrapulmonary fiber distribution and its relationship to local degrees of fibrosis are unknown. The ef fects of parameters related to fiber size (length, width, aspect ratio, etc.) have mostly been ignored. Because it is gener ally believed that long fibers are more fibrogenic than short ones (9-12), this question is of interest, but there is dis agreement about which parameter may be most useful to measure. Timbrell and colleagues (13) have proposed that one should examine total fiber size parameters, particularly total surface area and mass (ire., total mass as the sum of the masses of all fibers in a given sample), rather than mean fiber sizes to obtain correla tions with fibrosis (see Discussion). We have previously examined the is sues of local fiber burden and size and their relationship to fibrosis in the lungs of a series of chrysotile asbestos miners and millers, and found that the grade of fibrosis correlated positively with fiber concentration but negatively with fiber length (8). In order to determine wheth er similar effects are seen with amphibole asbestos, in this study we have ex amined the lungs of workers with heavy exposure to the commercial amphibole amosite and measured the same param eters. In addition, we have compared, on a fiber-for-fiber basis, the fibrogenic ef fects of chrysotile asbestos and its natu ral contaminant, tremolite, with amosite. SUMMARY We have pravloualy *frown that In tiro lungs of a group of chryaotlla mlnsrs and mlllsn, grad# of IntanrtHlal flbroala faabaatoafa) la dlraetty proportional to tramollta flbar or chryaotlla flbar cancantratlon but Is Invarsaly proportional to main llbar langth and tangttMalatad paramatara. To compart tiro affacta of tha eommarelal amphlbole aabaatoa amoalta on paranchymal flbroala, wa histologically graded flbroala In fourdifferentaftaa in tha lungsof SO shipyard and Insulation workers with heavy amoalta exposure and measured by analytic electron microscopy fiber concentration andsize In comspariding portions of lung tissue. Fibrosis grade was foundto be strongly positively correlated with amoeba concentration and negatively correlated with mean fiber size paramatara, Including flbar langth, width, surface area, and mass. A comparison of our present teauKs with our data on tha chryaotlla mlnsta and miller* showed that tha regression lines of flbroala grads versus concentration tor amoalta, chryaotlla, and tramollta were statistically different. These find ings Indicate that amoalta cancantratlon, Ilka chryaotlla and tramollta concentration. Is closely end directly related to fibrosis at the local lung level. Furthermore, these observations again rales tha possibility that short fiber* may be more Important than la commonly believed In the genesis of fibrosis in man. Last, tha concentration comparison data Indicate that flbar for flbar, amoalta la more fibrogsnlc than la chryaotlla or tramollta, and Indirectly suggest that tramollta It more fibre- genic than It chryaotlla. am rev RESWt ns two; i42:mi-iue Method* Specimen Selection and Preparation Twenty specimens of autopsy lungs from heavily exposed insulators and/or shipyard workers were selected from a larger group of such specimens. To be selected, we required at least a midsagittal slice of left or right lung so that multiple samples could be obtained. Initial screening of histologic sections was car ried out to ensure that the 20 cases represent ed a range of interstitial fibrosis from none to severe. An initial sample was analyzed to ensure that the patient had been exposed to amosite asbestos. To examine the relationship between degree of fibrosis and asbestos content, we followed a protocol similar to that used in our previ ous study of chrysotile miners (8). Four blocks of fixed tissue, approximately 1 x 1 x 0.5 cm, were selected (two upper lobe; two lower lobe; selection random within the lobes). A piece measuring 1 x l x 0.2 cm was trimmed off each block, dehydrated in alcohol, and embedded in paraffin. A 5-pm section was cut and stained with hematoxylin and eosin and graded for interstitial fibrosis as described below. Mineralogic Analysis The remaining portion of each tissue block was dissolved in bleach and the asbestos fibers collected and transferred to coated electron microscope grids as per our published pro tocol (14). For each sample, approximately 75 sequentially encountered amosite fibers were identified and measured using a Philips 400T electron microscope equipped with a Kevex 7000 energy dispersive X-ray spectrome ter. At the time of tissue sampling, an addi tional piece of tissue was taken and dried to constant weight to allow final expression of results in terms of fibers/g dry lung. Using an algorithm relating weight of lung dissolved and area of grid examined, the concentration of fibers/g dry lung was determined. Further details of the analytic procedure can be found in reference 14. Amosite surface area and mass were calculated using a height-to-width ratio of 2.5 (15) and a density of 3.4. All size, sur face, and mass data were expressed as geo metric means because fiber sizes are usually log normally distributed. Additionally, theto- {Received in originalform December 14,1989 and in revised form April 19, 1990) ' From the Departmentof Pathology and Univer sity Hospital, University ofBritish Columbia. Van couver, British Columbia, Canada. * Supported by Grant No. MA6907 and MA7820 from the Medical Research Council of Canada. 1 Correspondence and requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC V6T 2BS. Canada. 1331 1332 CHURG. WRIGHT. WIGGS. AND OEPAOU Rg. 2 (right). Grada vstaus moan fiber length In mlcrom: r -026, p < 001. tal length, width, and so forth of all fibers in each site were calculated (see below for the significance of these values). Grading of Interstitial Fibrosis Fibrosis was graded in exactly the same fash ion (16) as used in our previous study of chrysotile miners (8). In brief, each hematox ylin and eosin-stained section was examined in its entirety using a lOx objective; and each microscopic field was assigned a grade of zero (no fibrosis) to 4 (most severe fibrosis). The value for each field was recorded and an av erage taken over all the fields to provide a value for each site. Some of the cases graded for the study on chrysotile miners (8) were regraded to ensure consistency between that study and the present one. Statistical Analysis Using simple regression, the relationship of the various measured parameters to grade of interstitial fibrosis was examined. The SYSTAT system (17) was used for this purpose. Initial inspection showed that the concentration data could be normalized by a log transformation, and the analyses using concentration were per formed on the transformed data. Bonferroni corrections for multiple comparisons were used as appropriate. To evaluate the question of whether amosite; chrysotile, and tremolite produce different amounts of fibrosis on a fiber-for-fiber basis, comparisons were made using the method of Feldman (18) for examin ing families of lines. - Fteaulta - Demographic Data The mean age of the patients SD was 68 9yr; mean pack-years of smoking, 29 26; mean years of asbestos ex posure, 24 13; and mean years since first exposure, 44 4. Correlations with Concentration Data and Size Data A strong, positive correlation (r = 0.62) was seen between fibrosis erade and amo site fiber concentration (figure 1). Sig nificant negative correlations were seen between fibrosis grade and all measured mean size parameters except aspect ratio (figures 2-6). Bv contrast, a significant positive correlation was seen between fibrosis grade and total fiber length, width, and aspect ratio, but no correla tion was found between fibrosis grade and total fiber surtacc'or massTfigtircs 7-rll). Comparison of Asbestos Fiber Types The regression data for fibrosis grade and log fiber concentration for amosite, chrysotile, and tremolite are shown in ta ble 1. The latter data were derived from our previous report (8). Reinspection of the chrysotile data from that report showed that four sites had extremely small fiber concentrations (< 10,000 Fig. 4 (right). Grada varau* maan flbar aipact ratio: r -O01, p valut is not significant. AMOUTE-MOUCCD nWCMIS Fig. 5 (fell). Grade versus mean fiber surface tret in microns squared: r - -032, p < 0003 Fig, 6 (right). Grade versus mean fiber mtss x 10*'1 g: r . -033, p < 0003. 133 Total Fiber Length Fig. 7 (left). Grade versus total fiber length in microns: r 037, p < 0001. Fig. 8 (right). Grade versus total fiber width in microns: r - 033, p < 0,003. Total Fiber Aspect Fig. 9 (htt). Grade versus fiber aspect ratio: r 040, p < 0001. Fig. 10 (right). Grade versus total liber surface area in microns squared: r 0/ p value is not significant. 1334 CHURG, WRH3HT. WtOGS. AHO OCMOU 4r mean fiber length was negative, i.e.. the fenftcr. the tiber. the tower the tibrosfs grade, A negative correlation of roughly 3 stmilafmignitudc was found for1 most I Fig. 11. Grads versus total fiber mass x 10-'1 g: r 0.07, p value is not significant. of the other parameters relatca to mean fiber size. Length thus appears to be a suitable representative parameter to mea sure the effects of mean fiber st2c on fibrostsT.- "SevCraTaiffcrent groups have found a positive correlation between fibrosis grade and fiber concentration at the whole case 0 1---------------------------------- 1----------------------------------1 0 50 100 level by counting asbestos bodies, uncdated fibers by light microscopy, and un Total Fiber Mass ' coated fibers by electron microscopy (3-8). In most of these studies, the sam ples for mineral analysis were not taken fibers/g dry lung) but considerably great er than zero fibrosis grades. It was felt in retrospect that these data, which as a group formed outliers from the main mass of points, were unlikely to be cor rect, and these points were dropped for this analysis. This procedure had the ef fect of bringing the intercept of the chrysotile regression line through the origin at about the same point as the tremolite and amosite regression lines, a finding which makes biologic sense. Analysis of these data by the method of Feldman (18) showed that all three regression lines were significantly differ ent from each other (each p < 0.01), i.e., for a given fiber concentration, amosite produced more fibrosis than did chrysotile, which produced more fibrosis than tremolite. A similar analysis of the ef fect of mean fiber length and fibrosis grade, also shown in table 1, indicated no difference between amosite and chrys otile, but a significant difference existed between tremolite and the other fibers (p < 0.05 comparing tremolite to chrysotile or amosite). Diacussion In this study, we have used the lungs of workers exposed to amosite asbestos to examine the correlation of fibrosis grade and a variety of mineralogic parameters. We have selected this group of workers because their amosite exposure was, historically and analytically, quite high and, in a very broad sense, reasonably homogeneous, so that the effects of this specific fiber type on fibrosis can be evaluated. However, this study popula tion is not ideal: all of the workers histor ically had chrysotile exposure; generally at a very high level, as well. As is usually true of remote chrysotile exposure (6-12, 14,15), most ofthe chrysotfie had disap peared from these lungs by the time of analysis, and there were too few chryso tile fibers left to even attempt to count and correlate with fibrosis grade. To the extent that past chrysotile exposure played a role in producing fibrosis, our amosite results may therefore be in error; how ever, the comparative data on amosite, tremolite, and chrysotile discussed below suggest that this is not a serious problem. Using this group of lungs, we have shown that interstitial fibrosis (asbestosis) can be related to the concentration and size ofamosite fibers at the local lev el. The correlation between fibrosis grade and concentration was positive, i.e., the greater the fihfr mncentrafinn, the great er the fibrosis grade. In contrast, the. correlation between fibrosis grade and from the same areas as were used to grade interstitial fibrosis, and generally only one sample was obtained per case. The present sampling scheme has the advan tage of examining multiple areas within the same lung and allows examination oLhistologic and mineralogic samples- that are virtually identical. Because mul tiple points are sampled in each case, this protocol also permits us to use a newly developed statistical method (18) for ex amining series of lines and thus to com pare fibrosis as a function of concentra tion and size parameters among the different asbestos minerals. Using this sampling protocol (8), we previously showed that for chrysotile as bestos and its natural contaminant trem olite, positive correlations were present between fibrosis grade and fiber concen tration. The finding of a similar effect in pur present study of amosite-exnosed workers suggests that for all types of as bestos, the degree ot local fibrosis is dusely related to tne local tioer cOncHP- tration. What our present and previous studies do not explain, however, is the distinct lower zonal distribution ot as- bestosis in. man: thus far, studieslrom our laboratory and from other laborato ries have failed to find a consistently" gtguei fibercoiujentratiou in the lower- lung zones (19-22). ` Much less information is available con TABLE 1 REGRESSION EQUATIONS FOR FIBROSIS GRADE AS A FUNCTION OF FIBER CONCENTRATION OR LENGTH AS DETERMINED BY THE METHOD OF FELDMAN* Fibrosis Grade as a Function of Log Fiber Concentration Fibrosis Grade as a Function of Geometric Mean Fiber Length cerning the relationship of fibrosis grade and parameters related to fiber size in man. In our study of chrysotile workers (8), we found a negative correlation be tween mean fiber length add leriglhrelatcd parameters (aspect ratio, surface intercept as Slope as AFiorotis Grads/ Intercept as Slope u AFibroais Grade! ares, mass) and fibrosis grade: aritTtKc~ Fiber Type Fibrosis Grade ALog Concentration Fibrosis Grade AMsan Length same effect _ic.pMgnt--in-fhi.-nat? Amosite Chrysotile Tremolile 1.20 O.SS 0.59 0.21 0.17 0.1S 2.04 -0.17 airin'iirr-rYnOTrd-wonkert^-Tiiis.is.a very 1.72 -0.22 surprising finding because animal studies 2.53 -1.05 'gonsistently indicate that long fibers are' * Sec reference 16 ' more fibrogcnic (and more carcinogen- ic) than are short fibers J9-12). Timbrell though surface area does not appear to References (277has suggested that fibrotic lungs re tain more fibers than do nonfibrotic lungs, and it is conceivable that what we are observing is a very local increase in be one of the parameters that is useful, but we were unable to find any correla tions of total size parameters and fibro sis when we examined chrysotile and 1. Doll R, Peto J. Asbestos: effecis on healri exposure to asbestos. London: Her Majesty's S tionery Office. 1985. 2. Report of the Royal Commission on Matt fiber retention in any area in which fibro sis develops. However, to produce a nega tive correlation with fibrosis grade, pref erential retention of short fibers would tremolite (8). The reasons for these dis crepancies are unclear. It should be ap preciated that total fiber parameters in the present study correlate strongly with of Health and Safety Arising from the Use of / bestos in Ontario. Toronto: Queen's Printer for C tario. 1984. 3. Ashcroft T, Heppieston AO. The optical ai electron microscope determination of pulmona have to occur. Such an effect might be seen if fibrosis simply impaired clearance of all types of fibers because short fibers generally outnumber long fibers in dust clouds; however, one does have to ques fiber concentration, so in practice the to tal fiber size parameters act as a substi tute for fiber number. A question ofconsiderable interest for the understanding ofasbestos-related dis asbestos fibre concentration and its relation to a human pathological reaction. J Clin Pathol 197 26:224-34. 4. Whitwell F, Scott J, Grimshaw M. Relatior ship between occupations and asbestos fibre con lent of the lungs in patients with pleural mesothe tion if given the variability of the dust clouds inhaled by workers under differ ent conditions, the general person-toperson variability of clearance of any ease is whether all asbestos types are equally fibrogenic. Epidemiologic studies have shed little light on this issue in hu mans. It is interesting that in two animal lioma, lung cancer, and other disease. Thorax 1977 32:377-86. 5- RoggliVL, Pratt PC, Brody AR.Asbestoseontent of lung tissue in asbestos-associated diseases: a study of 110 cases. Br J Ind Med 1986; 43:18-29. dust and the necessity that fiber clear inhalation experiments examining the fi- 6. Wagner JC, Newhouse ML, Corrin B, Rossiter ance impairment be exactly proportion al to local degree of fibrosis, whether this effect would be consistent enough to pro duce the type of negative correlation brogenicity of long fiber chrysotile or amosite, Davis and coworkers (9, 10) found a roughly equal mean fibrosis grade after approximately 30 months of CER, Griffiths DM. Correlation between fibrecon tent of the lung and disease in East London facto ry workers. Br J Ind Med 1988; 45:305-8. 7. Green FHY, Harley R, Vallyathan V, Dement J, Pooley F, Althouse R. Pulmonary fibrosis and found here. The other possibility is that, at least in hum&riS. Short fibers aic of themselves fllOre important generators of exposure to both fiber types even though the measured amosite exposure in terms of fibers/ml was only about half the mea sured chrysotile exposure This finding asbestosexposure in chrysotile asbestos textile work ers. In: Wagner JC, ed. Biological effects of chryso tile. Philadelphia: Lippineott Publishers, 1986: 59-68. 8. Churg A, Wright JL, DePaoli L, Wiggs B. fibroflntt&n. has generally been believed implies that amosite is more t'ibrogcmc and/or that, once impaired clearance, is tfian is chrysotile. established for any reason, short fibers ` I he present analyses were designed to become more dangerous than thev-would allow a direct examination of this ques otherwise be. The situation in man may tion in humans. Using the data on Mineralogic correlates of fibrosis in chrysotile miners and millers. Am Rev Respir Dis 1989; 139:891-6. 9. Davis JMG, Addison J, Bolton RE, Donald son K, Jones AD, Smith T. The pathogenicity of long versus short fibre samples of amosite asbestos also be complicated by other types ofcxnnsiires: tor examnli* we have-saoMOun guinea pigs that cigarette smoke increases retention of short fibers to a greater degregrftan retention ofIN ft*I-JKf:M *Kl*. changes the way in which specific cells ih tnc lung handle tong or shqrt fibers J24, 25), and it is possible that cigarette ^Mimkgatggchangcs the retention pattern chrysotile and tremolite from our previ ous study, and employing the newly de veloped statistical method of evaluating families of lines proposed by Feldman (18), we found that on a fiber-for-fiber basis, amosite was ronn* was either chrysotile or tremolite (table 1). Although the regression data in table ' 1 suggest at first glance that chrysotile administered to rats by inhalation and imraperi- toneal injection. Br J Exp Pathol 1986; 67:415-30. 10. Davis JMG, Jones AJF. Comparison of the pathogenicity of long and short fibres of chryso tileasbestos in rats. Br J Exp Pathoi 1988; 69:717-37. 11. Adamson 1YR, Bowden DH. Response of mouse lung to crocidoliteasbestos. 1. Mineral fibro sis reaction to short fibres. J Pathol 1987; 152: 99-107. 12. Adamson IYR, Bowden DH. Response of mouse lung to crocidolite asbestos. 11. Pulmonary in humans! ' --IrrContrist to the negative correlation of fibrosis grade and mean fiber size parameters, a significant positive corre lation was seen between fibrosis grade is more fibrogenic than tremolite, this may not actually be true. Extensive ani mal and human data indicate that chryso- liirts-rapidly removed from lung (6-12. 14, IS), even in experimental conditions fibrosis after long fibres. J Pathol 1987; 152:109-17. 13. Timbrell V, Ashcroft T, Goldstein B, ttaL Rela tionships between retained amphibolc fibres and fibrosis in human lung tissue specimens. Ann Occup Hyg 1988; 32(Suppl:323-40). 14. Churg A. Quantitative methods for analysis and total fiber length, width, and aspect ratio, and a positive but nonsignificant correlation was seen for grade and total surface area or mass. Timbrell and col in which it produces marked fibrosis (9. JO), so that the amount of chrysotile that must once have been present in our cRrysotilc miner lungs, and which is at of disease induced by asbestos and other mineral particles using the transmission electron microscope. In: Ingram P, Shelburne JD, Roggli VD, eds. Mi croprobe analysis in medicine. New York: Hemi sphere Publishing Company, 1989; 79-%. leagues (13) originally proposed measur ' least m part responsible for fibrosis, was 15. Pooley FD, Mitha R. Determination and in ing this type of parameter because it could be easily determined with a light scattering system and hence was far more economic and faster than using electron enormously greater than the analytic data impl^Tremolite, by contrast, constitutes onlya few percent of the chrysotile ore but, like other amphiboles, is poorly terpretation of the levels of chrysotile asbestos in lung tissue. In: Wagner JC, ed. Biological effects of chrysotile. Philadelphia: Lippineott Publishers, 1986; 12-8. 16. Ashcroft T, Simpson JM, Timbrell V. Simple microscope methods. Timbrell and col leagues found that surface area measured in this fashion gave consistent predictions of degree of fibrosis with different types of asbestos. We have confirmed the idea ihat total rather than mean size param eters may provide predictive information in regard to amosite-induced fibrosis, al cleared from lung. Thus, our regression data can be interpreted as indicating that ~thc vast amount of'(no'W~hisiUle) 'tdirysotite exposure produced only abdht cETffuch fibrosis as the several orders of magnitude-towertfemoute exposure, Lei, tremolite is actually more fibrogenic than is chrysotile on a fiber-for-fiber basis;. method of estimating severity of pulmonary fibrosis on a numerical scale J Clin Pathol 1988; 41:467-70. 17. Wilkinson L. SYSTAT: the system for statis tics. Evanston, 1L: SYSTAT Inc. 1988. 18. Fbldman HA. Families of lines: random ef fects in linear regression analysis. J Appl Physiol 1988; 64:1721-32. 19. Sebastien P, Fondimare A, Bignon J, Monchaux G, Desbordes J, Bonnaud G. Topographic distribution of asbestos fibers in human lung in 1330 CHURO. WRMHT. WKJOS. ANO DfMOU relation to occupational and non-occupational ex posure. In: Walton WH, McGovern B, eds. Inhaled panicles IV. New York: Perganton Press, 1977; 433-44. 20. Morgan A, Holmes A. Distribution and char acteristics of amphiboie asbestos fibres, measured with the light microscope, in the left lung of an insulation worker. Br J Ind Med 1983; 40:45-30. 21. Morgan A, Holmes A. The distribution and characteristics of asbestos fibres in the lungs of Finnish anthophyllite workers. Br J Ind Med 1984; 33:62-75. 22. Churg A. The distribution of amositeasbestos in the periphery of the normal human lung. Br J Ind Med 1990; (in Press). 23. Timbrel! V. Deposition and retention of fibres in the human lung. Ann Occup Hyg 1982; 26: 347-69. 24. McFadden D, Wright JL, Wiggs B, Churg A. Smoking inhibits asbestos clearance. Am Rev Respir Dis 1986; 133:372-4. 25. Churg A, Tron V, Wright JL. Effects of ciga rette smoke on retention of asbestos fibers in vari ous compartments of the guinea pig lung. Am J Pathol 1987; 129:385-93.