Document 3J36BBwK3vDBem2KwqGMQXqNn

- J*K" IUil4kh{n-*. ll'r,K,!.v >i^ii1w I'liI'Iishctf by (Klnnl ^imwi silv IV,-v. IV11 Iff A.1A!|rJa'](^|^ Tremolite and Mesothelioma VICTOR L. ROGGLI*, ROBIN T. VOLLMER, KELLY J. BUTNOR and THOMAS A. SPORN Departments ofPathology, Duke University and Durham VA Medical Centers, Durham, NC 27710, USA Received 16 October 2001; in final form 17 January 2002 Background: Exposure to chrysotile dust has been associated with the development of meso thelioma and recent studies have implicated contaminating tremolite fibers as the likely etio logical factor. Tremolite also contaminates talc, the most common non-asbestos mineral fiber in our control cases. Methods: We examined 312 cases or mesothelioma for which fiber burden analyses of lung parenchyma had been performed by means of scanning electron microscopy to determine the content of tremolite, non-commercial amphiboles, talc and chrysotile. The vast majority of these patients were exposed to dust from products containing asbestos. Results: Tremolite was identified in 166 of 312 cases (53%) and was increased above back ground levels in 81 cases (26%). Fibrous talc was identified in 193 cases (62%) and correlated strongly with the tremolite content (P < 0.0001). Chrysotile was identified in only 32 cases (10%), but still correlated strongly with the tremolite content (P < 0.0001). Talc levels explained less of the tremolite deviance for cases with an increased tremolite level than for cases with a normal range tremolite level (22 versus 42%). In 14 cases (4.5%) non-commercial amphibole fibers (tremolite, actinolite and/or anthophyllite) were the only fiber types found above back ground. Conclusions: We conclude that tremolite in lung tissue samples from mesothelioma victims derives from both talc and chrysotile and thal tremolite accounts for a considerable fraction of the excess fiber burden In end-users of asbestos products. Keywords? asbestos; mesothelioma; tremolite; talc; chrysotile; amphiboles INTRODUCTION Tremolite is a hydrated calcium magnesium silicate [CanMgjSijOj^OHy belonging to the amphibole group of minerals. It is a known contaminant of chry sotile asbestos and also contaminates fibrous talc and vermiculite (McDonald et at, 1989; Langer et al., 1991). In addition, tremolite is the most common amphibole fiber found in lung samples from our control populations (Srebroand Roggli, 1994). Due to the fragility of chrysotile asbestos and its tendency to be removed from the lungs at much greater rates than amphiboles,. it has been suspected that contaminating tremolite might be responsible for mesotheliomas occurring in chrysotile miners and millers (McDonald et al., 1989). Recent studies have leant further support to this hypothesis in that *Author to whom correspondence should be addressed. Tel'. + 1-919-286-0411 ext. 6615; fax; +I-9I9-2B6-68I8; e-mail; roxgli.v@durhain.va.gov mesotheliomas in Thetford chrysotile miners and millers were much more likely to occur in the five central mines, where there is greater contamination by tremolite as compared with (lie more peripheral mines (McDonald el al., 1997)- Furthermore, it has been suggested thal processed (i.e. milled) chrysotile contains Hide or no tremolite contaminant (Churg, 1988; Craighead, 1995). Our laboratory has performed fiber analyses on a large number of mesothelioma patients, most of whom were exposed to asbestos-containing products as end-users. We have previously shown that amosite asbestos is the most common fiber type found in the lungs of these workers (Roggli et al., 1993), It is the purpose of this study to evaluate the relationship between levels of tremolite, non-commercial amphi boles, tale and chrysotile and to compare these find ings with the occupational exposure information in a series of 312 mesothelioma cases for which a lung fiber burden analysis had been performed. 447 -t4.S' V Rvggii MATERIALS AM) METHODS Case selection The study group consisted of ati mesothelioma cases in the database of one of the authors (V.L.R.) for which a Tiber burden study with identification of fiber types had been performed. The diagnosis of mesothelioma was based on previously published criteria (Roggli el ai, 1992a). These included the gross distribution of tumor, histological appearance and immunohislochemical findings. In some cases histochemistry and electron microscopy were also performed. In addition, occupational information was reviewed for cases with a tremolite content that exceeded our background range (see below). In all cases the diagnosis of mesothelioma was made inde pendently of asbestos exposure history or determin ation of tissue mineral fiber content. Mineralfiber analysis Fiber analysis was performed on lung tissue samples obtained either at time of surgery (lobectomy or pneumonectomy) or at autopsy. Most lung samples were formalin fixed, and a few were paraffin embedded. For the latter cases a correction factor (0.7) was applied to the final calculation so that the results were comparable to the formalin fixed tissue results (Roggli el ah, 1986). The tissue was digested in sodium hypochlorite solution and the residue collected on a 0.4 pm pore size polycarbonate filter as previously described (Roggli, 1992). The filter was mounted on a carbon disc with colloidal graphite and sputter coated with gold or platinum for examination by scanning electron microscopy. Scanning electron microscopy was performed with a JEOL ISM 6400 scanning electron microscope operated at an accelerating voltage of 20 kV. screen magnification of lOOOx and a scan rate of 10 s/frame. Both coated (asbestos body) and uncoatcd fibers >5 pm in length were counted, with a fiber defined as a mineral particle with an aspect (length to width) ratio of at least 3:1 and roughly parallel sides. A total of 100 fields (filter area of -2.37 mm5) or 200 fibers, whichever occurred first, were counted for each sample. Blank filters were also examined and all reagents were pre-filtered to avoid contamination with fibers (Roggli, 1992). ' The elemental composition of individual mineral fibers was determined by means of energy dispersive X-ray analysis (EDXA). Asbestos fibers were classi fied as amosite, crocidolile, tremolite, anthophyllite, actinolite, chrysolite or talc based on their morphology and X-ray spectra as previously described (Fig. I; Roggli, 1989; Roggli el ai, 1992b). For each case 5 50 fibers (average 20 fibers) were classified by EDXA. The detection limit for each fiber type varied somewhat from case to ease, depending on the concentration of fibers in the lung tissue and the actual number of fibers analyzed by EDXA for that case. Statistical analysis For the purposes of examining the relationship between levels of tremolite or non-commercial amphibole fibers (combined tremolite, actinolite and antho phyllite) and talc or chrysotile only those cases in which at least one of these four fiber types was iden tified were included. These criteria were met by 248 cases, three of which were individuals exposed to tremolite/actinolite environmentally in Turkey. Since this environmental exposure is not related to cither chrysotile or talc, these three cases were excluded Fig. 1. Energy dispersive X-ray spectra for tremolite (a), actinolite (b), anthophyllite (c) and chrysotile (d). Characteristic elemental composition for each Tiber type is shown. The gold peak is due to sputter coating of the sample to reduce charging artifacts. 1 ik:mtiliiv' jiiiJ mi.,'.i>i!iL.'ho4ii,i > 2 4 6 B 10 12 U>9(Trem) 4 6 fi 10 12 LogfTAA) Fig. 2. Frequency distribution for four of the fiber types analyzed in this study: ircinolile, TAA (tremolite, actinolite and anthophyllite), talc and chrysotile. The values are tog normally distributed. from the analysis, leaving 245 cases. As fiber burden levels have been shown to be log normally distrib uted (Fig. 2), a log transformation of the fiber counts was performed. Zero values were recorded as half the detection limit for the purposes of this analysis. A general linear model was used to examine the relation between chrysotile and talc as independent variables and tremolite or non-commercial amphiboles as the dependent variables (McCullagh and Nelder, 1989). Residual deviance (residual sum of squares) was deter mined using the S-Plus statistical package (MathSoft Inc., Seattle, WA), Univariate analysis was also used to examine the relationship between total non-commer cial amphiboles, tremolite, talc and chrysotile. Statis tical significance was considered for P values <0.05. RESULTS Tremolite was detected in' 166 of 312 cases (53%), with a median value of 2840 fibers/g wet lung tissue for fibers >5 pm in length. The range of values was 120-292000 fibers/g. Non-commercial amphibole fibers of any type (tremolite, aclinolite or anthophyllite) were delected i n 210 cases (67%), with a median value of 3160 and a range of 26-454000 fibers/g. Talc was identified in 193 cases (62%), with a median value of 3910 and a range of280-138000 fibers/g. In contrast, chrysotile was identified in only 32 cases (10%), with a median value of 1800 and a range of 540-124000 fibere/g. These values are compared with our background range in Table 1. Tremolite exceeded our background range in 81 cases (26%). Exposure information was available for 70 of these cases, and the exposure categories for these cases are summarized in Table 2. The occupa tional categories in this table are generally considered to be end-users of asbestos-containing products or bystanders ofend-users. Of interest is the observation that six of these cases occurred as household contacts of asbestos workers, whereas three of the cases were building occupants. The one brake mechanic with excess tremoiite also had excess levels of amosite, suggesting an alternative means of exposure. There were 14 cases (4.5%) in which non-commercial_amphibole fibers were the only fiber type found in excess in the lung tissue samples. These cases are of particular interest and are summarized in Table 3. Three of these cases occurred in individuals from Turkey with environmental exposures to tremolite/actinoiite (Zeren el ctl.t 2000). Three additional cases were house hold contacts of asbestos workers and three were building occupants. One case worked in a pattern shop in a silica plant, where the primary exposure was to talc. The correlation among tremolite, non-commercial amphibole fibers, talc and chrysotile is summarized in Table 4. Multivariate analysis of the 245 cases for which at least one of these mineral species was 450 ^ l.. Kv4;:l! ri til liihk 1. Noii-aimmcivul .impliiMi:. Lili: mid Juj.miIiIi: i<>inviai,Uii'ii> ill 2H\ uniMHlsvIioina Mesothelioma No. of Median Range Controls*1 Median Range Tremolite 166 2840 120-292000 <600 <170-2540 TA A' 210 3160 26-454000 <600 <170-2540 Tafc 193 3910 280-138000 <600 210-10200 Slid 19 ci'iltrnl.i1' Chrysolite 32 1800 540-124000 <600 <80-1000 TAA, tremolite + aetinolite + anthophyl Lite. Values represent the median and range for fibers 5 pm in length per gram of wet lung tissue us determined by analytical scanning electron microscopy. ''Controls include 19 cases with no evidence of asbestos-related disease, no history ofasbestos exposure and normal lungs at autopsy. Table 2. Exposure information for 81 mesothelioma cases with elevated tremolite content Exposure category Shipyard worker Household contact*1 Pipefmer/welder Electrician1" Plumber/constnjction Sleamfitter/boiler worker Power plant worker US Navyj'merchant marine Railroad worker Building occupant Insulator . Oil refinery worker Other4* No information No.1 12 to 9 5 5 4 4 4 3 3 2 2 9 11 When the multivariate analysis was restricted to the SI cases with an elevated tremolite burden, talc explained only 22% of the deviance of tremolite concentrations (P < 0.0001), while an additional 6% was accounted for by chrysoti le (P < 0.03). When the analysis was restricted to only the 32 cases in which chrysotile was actually detected, talc explained 54% of the deviance of tremolite concentrations (P < 0.0001), while an additional 4% was accounted for by chrysotilc (P = 0.12) (see Table 4). Figure 3 shows the comparison between the predicted log concentration of tremolite based on the log concentrations of talc and chrysotile and the observed log concentration of tremolite. The association is highly significant, although there is still considerable scatter of the data around the line of perfect agree ment. `Total exceeds 81 because a few esses had more than one exposure. 'Contacts include shipyard worker (four), insulator (two), chemicul plant worker, pipefiitcr/wcldcr, tool grinder and glass company worker and power plant worker (one each). `Shipyard, paper ntilj and chemicai plant. ^Includes silica plant/pattern shop, machinist in a glass manufacturing plant, brake mechanic, steel worker, asbestos textile plant worker, dry cleaning plant assembler, painter/plasterer, sheet metal worker and truck driver (insulation). detected showed that talc accounted for 42% of the residual deviance of tremolite concentrations, while an additional 16% was accounted for by Chrysotilc (P < 0.0001). Similarly, talc accounted for 36% of the deviance of non-commercial amphibolc concentra tions, while an additional 12% was accounted for by chrysotile (P < 0.0001). In addition, a strong correla tion was observed using pair-wise regressions between tremolite and non-commercial amphiboles, tremolite and talc, tremolite and chiysotile, non commercial amphiboles and talc and non-commer cial amphiboles and chrysotilc (P < 0.0001 for all comparisons). . DISCUSSION We have found in a large series of patients with malignant mesothelioma in which fiber burden analyses had been performed on lung tissue samples that tremo lite fibers are commonly observed. These tremolite levels exceeded background in >25% of these cases, The levels of tremolite were strongly associated with concentrations of fibrous talc, which accounted for just over 40% of the deviance in tremolite concentra tions. However, when cases with tremolite levels in excess of background were examined, fibrous talc accounted for just over 20% of the deviance. There fore, in these cases some other source must be invoked to explain the tremolite deviance. Although chiysotile concentrations were also strongly correlated with tremolite fiber burden, chryso tile accounted for a smaller percentage of the tremo lite deviance. In this regard it should be noted that chrysotile does not accumulate in lung tissue samples to the extent that the amphiboles do, as it is broken down into smaller fibrils that are more readily cleared from the lungs. These smaller fibrils would have been missed by our technique, since we only counted fibers i'rcmi'hk* and pivM'lli.'lu'm. Jil T.ihio .v Mesothelioma Viises lor iiliidi iiiui-i,im!!in.'mnl ,impl)ihutr> were the mil) tiher type present ill cnCi'm; level* Case Age sC-t Diugjuvtis no. i NDM 5PI 2 67 M EPI 3 NDM EPI 4 54 M EPI 5 56M BPI 6 57 F EPI 7 61 M BPI 8 57 M SFI 9 59 F SPt 10 73 F BPI 11 59 M BPI 12 44 F EPI 13 64 M 14 58 F BPI SPI Exposure Environmental exposure, Turkey Environmental exposure, Turkey Environmental exposure, Turkey Household contact1 Painicr/plitstercr Household contact*1 Shipyard worker Supervised construction Household contact0 Silica plant/pattem shop Radar man, US Navy Dry cleaning plant assembler School administrator Teacher's aide Dumtinn Trcmoliic TAA TjIl- Chrysolite ND Lifetime ND 18 yr 38 yr 1-2 yr 8 days 1 yr 2-3 yr 34 yr 4 Syr ND ND 18 yr 292000 <15900 23500 7460 8110 8100 11700 9550 5030 5540 6060 5440 3280 4330 454000 286000 30400 17700 16200 16200 11700 moo 6330 6070 6060 5440 4580 4330 <24700 <15900 <3010 13100 60800 <B100 <5850 6530 3250 46100 8080 680 3930 6930 <24700 <15900 <3010 <2760 <4060 <8100 <5850 <500 <1130 <3030 <1010 <680 <660 <870 B, biphasic; E, epithelial; ND, no data; PI, pleural mesothelioma; S, sarcomatoid; TAA, tremolite + aclinoliie + anthophyllile. "Father, loolgrinder; mother, glass plant worker. ^Husband, shipyard worker. "Father and mother, shipyard workers. Table 4. Multivariate analysis of tremolite. non-commercial amphibole, talc and chrysotile concentrations* Dependent variable Independent variable Coefficient F statistic P value log(tremolite) log(talc) log(chrysotile) 0.355 0.502 240 89 <0.0001 <0.0001 log(TAA) logCtalc) log(chrysotiJe) 0.347 0.457 170 58 <0.0001 <0.0001 Deviance (%)b 42 16 36 12 TAA, tremolite + acrinolite + anthophyllile, Fiber levels determined by scanning electron microscopy; log transformation of values (see text for details). ''Percent of deviance of dependent variable that is explained by the independent variable. that were >5 Jim in length. Furthermore, chrysotile fibers split longitudinally in vivo to produce long fibers that are <0.1 Jim in diameter (Roggli and B rody, 1984; Coin el a!., 1994), Such long, thin fibers would likewise be missed at the screening magnification that we use. Since chrysotile content is poorly detected by SEM and fiber burden is a poor indicator of total chrysotile exposure, other information must be sought to address this question. The best indicator of chrysotile exposure is an occu pational history (Henderson el at., 1997). In this regard patients in our series with elevated tremolite fiber burdens typically had exposures to both commer cial amphibole fibers (especially amosite) and chryso tile. A substantial proportion of the cases occurred among shipyard workers, pipefitters/welders, elec tricians, plumbers/construction workers, steamfitters/boiler workers and US Navy/merchant marine seamen. Household contacts of asbestos workers and building occupants accounted for six and three cases, respectively. The latter individuals in particular are more likely to be exposed to chrysotile (Gaensler, 1992). Another source of information that sheds some light on this question is published data on tremolite and chrysotile lung fiber burdens as determined by transmission electron microscopy. This method is more sensitive in detecting very fine chrysotile fibers and chrysotile fibrils. In a study of five chrysotile miners and millers with malignant pleural meso thelioma, Churg ef at. (1984) noted a ratio of tremo lite to chrysotile ranging from 3.9 to 12 (median 5.5). In a_study of 83 shipyard workers and insulators with mesothelioma, Churg and Vedal (1994) found a geometric mean tremolite to chrysotile ratio of 12.8. There was a strong correlation between tremolite and chrysotile fiber concentrations (r = 0.37, P < 0.001 for the entire series of 144 patients). These findings suggest little, if any, removal of tremolite from endproducts containing chrysotile. Furthermore, a recent study by Tossavainen et at. (2001) using SEM detected tremolite contamination of all six samples of processed Chinese chrysotile that were analyzed (range 0-002-0.310% tremolite by weight). Of particular interest are cases of mesothelioma in which non-commercial amphiboles are the only fiber i V I.. R.^ghi'J Predicted Log(Tremolile) Fig. 3. Regression analysis for the relationship between the observed concentration oftremolite (y-axis. log transformation) and the predicted concentration of tremolite based on talc and chrysolite concentrations (x-axis). The straight line indicates the line of perfect agreement. type found in excess in lung tissue samples. Three such cases in our series occurred among individuals with environmental exposure to tremolite/actinolite in Turkey. When these cases are excluded, meso theliomas associated with non-commercial amphibole Fibers alone accounted for -3.5% of our cases (II of 312), These included six men and five women ranging in age from 44 to 73 yr (median age 58 yr). The exposure duration ranged from 8 days to 34 yr. Five had occupational exposures to asbestos, three were household contacts of asbestos workers and three were occupants of buildings with asbestoscontaining materials. We made no attempt in the present study to discrim inate between true fibers and cleavage fragments. All Fibers counted were at least 5 pm in length and almost all had aspect ratios of 5 or greater. These are the fibers believed to be most active in the induction of mesothelioma. Similarly, we did not identify nonfibrous particles in this study, so we cannot comment upon the lung content of vermiculite. However, it seems unlikely that exposure to vermiculite would explain a substantial portion of the tremolite burden in our patient population, which had such widespread exposure to chrysotile asbestos. Chrysolite was not delectable in nearly 90% of our cases and yet there was a- highly significant correl ation between chrysotile and tremolite content by multivariate analysis. This is partly due to an artifact of the analysis, since undetected chrysotile was recorded as half the detection limit for the purposes of the analysis and tremolite when present was often at the detection limit of our methodology. Also, the association between TAA and talc or chrysotile may be trivial, since most of the TAA fibers detected in our study were ircmnliic. In many of our cases the lungs had a high burden of commercial amphibolc fibers so that the detection limits were unusually high. When one cxcl udes cases In which (he detection limit for tremolite exceeded our upper limit of back ground range for tremolite, then tremolite i$ present in 74% of cases (166 of 225) and exceeds our back ground in 36% (81 of 225). In conclusion, evidence front our series indicates that tremolite and other non-commercial amphibolc fibers are present in the lungs of a substantial propor tion of patients with mesothelioma. In some patients these fibers appear to be the likely cause of the disease. The data are consistent with a derivation of the pulmonary tremolite burden from both talc and chrysotile asbestos. The findings do not support the concept that most or all of the tremolite is removed from chrysotile during the milling process. Acknowledgements--The authors gratefully acknowledge the technical assistance of Eve Whalen and Walter Fennell in preparing the samples for fiber analysis and James Burchetts for performance of the immunohistochemical stains on turner tissue samples. REFERENCES Churg A. (1938) Chrysotile. tremolite, and malignant meso thelioma in man. Chest; 93: 621- 8, Churg A, Vedal S. 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