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Asbestos Fibers Contributing to the Induction of Human Malignant Mesothelioma YASUNOSUKE SUZUKI AND STEVEN R. YUEN Department of Community and Preventive Medicine, Mount Sinai School ofMedicine, New York, New York 10029, USA Abstract: To elucidate the features of the asbestos fibers contributing to the induction of human malignant mesothelioma, we used high-resolution analyti cal electron microscopy to determine the type, number, and dimensions of as bestos fibers in lung and mesothelial tissues in 168 cases of mesothelioma. Results: 1. Asbestos fibers were present in almost all of the lung and mesothelial tissues from the mesothelioma cases. 2. The most common types of asbestos fibers in lung were either an admixture of chrysotile with amphiboles, amphibole alone, and occasionally chrysotile alone. In mesothelial tissues, most asbes tos fibers were chrysotile. 3. In lung, amosite fibers were greatest in number followed by chrysotile, crocidolite, tremolite/actinolite, and anthophyllite. In mesothelial tissues, chrysotile fibers were 30.3 times more common than am phiboles. 4. In some mesothelioma cases, the only asbestos fibers detected in ei ther lung or mesothelial tissue were chrysotile fibers. 5. The average number of asbestos fibers in both lung and mesothelial tissues was two orders of mag nitude greater than the number found in the general population. 6. The major ity of asbestos fibers in lung and mesothelial tissues were shorter than 5 pm in length. Conclusions: 1) Fiber analysis ofboth lung and mesothelial tissues must be done to determine the types of asbestos fibers associated with the induction of human malignant mesothelioma; 2) short, thin asbestos fibers should be in cluded in the list of fiber types contributing to the induction of human malig nant mesothelioma; 3) Results support the induction of human malignant mesothelioma by chrysotile. Keywords: asbestos, fibers, mesothelioma, chrysotile, amphiboles INTRODUCTION It is well accepted that asbestos fibers are the cause of virtually all cases of human malignant mesothelioma.1-4 It is also known that all asbestos types, including chryso tile and amphiboles, have been shown in epidemiological and toxicological studies to be fully capable of inducing the tumor.5-9 In addition to heavy (occupational) asbes tos exposure, milder asbestos exposure (bystanders and family contact) can also in duce the tumor.10-12 Presently, no data are available to support a threshold limit for exposure to asbestos below which there is no risk of malignant mesothelioma.4 Address for correspondence: Yasunosuke Suzuki, M.D., Department of Community and Pre ventive Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029. Voice: 212-241-4777; fax: 212-996-0407. yasunosuke.suzuki@mssm.edu Ann. N.Y. Acad. Sci. 982: 160-176 (2002). 2002 New York Academy of Sciences. 160 SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 161 Human malignant mesothelioma develops after a long latency period: it takes fif teen years or more from the first asbestos exposure to death from malignant me sothelioma. Latency periods greater than 40 years have been reported.2-5 There is no cure for human malignant mesothelioma. Asbestos fibers are durable in nature and are not easily digested or dissolved by either phagocytic cells or the tissue fluid. Asbestos fibers are identified as asbestos bodies by light microscope or as naked asbestos fibers by an electron microscope. Some inhaled asbestos fibers translocate from lung into regional lymph nodes,1315 pleural and peritoneal mesothelial tissues,16-24 and other organs.13,25 Fibers may pass from lung to other organs by direct migration26,27 via lymphatic capillary sys tem,13-15,19 and by hematogenous spread.28,29 Up to now, most investigators have focused exclusively on asbestos fibers in the lung for identification of asbestos fibers that contribute to induction of human ma lignant mesothelioma.30-34 We questioned the adequacy of such an approach be cause the primary site of malignant mesothelioma is not the lung but the mesothelial tissue and because the type and number of asbestos fibers in lung may not be iden tical to those in mesothelial tissue owing to possible translocation of lung fibers to other organs including mesothelial tissues. Short, thin asbestos fibers, i.e., 0.06 pm long and 0.02-0.03 pm wide, can easily be identified by the high resolution analytical electron microscope, a transmission electron microscope with an energy dispersive X-ray spectrometer, but not by scan ning electron microscope with resolution of 0.3 to 0.4 pm. It is noteworthy that the number and dimensions of asbestos fibers obtained from a high resolution analytical electron microscope will be different from those obtained by a scanning electron microscope. It has been proposed on the basis of animal studies that long (greater than 8 pm in length) and thin (less than 0.25 pm in width) mineral fibers were strongly carci nogenic for the induction of malignant mesothelioma in rats (Stanton's hypothesis)35 and that shorter fibers pose less risk. Stanton's hypothetical dimensions were derived from his experimental studies using direct administration of heavy doses of various mineral fibers of different dimensions into rats pleural cavities. Stanton stated that direct application of his results to the problem in man would be unwise.36 However his hypothetical model of asbestos fibers' relative carcinogenicity has been directly applied to the counting of the asbestos fibers in man. To evaluate airborne fibrous dusts in industrial atmospheres, the current Occupa tional Safety and Health Administration (OSHA) method by light microscopy (phase microscopy) counts only those asbestos fibers that are longer than 5 pm with an aspect ratio larger than 3 to 1, assuming that fibers shorter than 5 pm are not car cinogenic. Even using the electron microscopic level, using the same assumption, some investigators have neglected to count short asbestos fibers (< 5 pm).37-40 Our previous tissue burden studies22 using high resolution analytical electron mi croscopy revealed that the majority of asbestos fibers from human lung and me sothelial tissues of human mesothelioma patients were less than 5 pm long (81.4%) and less than 0.25 pm in diameter. The narrow width of asbestos fibers (< 0.25 pm in diameter, another parameter in Stanton's hypothesis) has been emphasized as an important parameter not only for the fibers' carcinogenicity, but also for the fibers' ability to penetrate into the peripheral part of the lung by an aerodynamic mechanism.35,36,41-46 Both OSHA's method using light microscopy and some asbestos tis 162 ANNALS NEW YORK ACADEMY OF SCIENCES sue burden studies using electron microscopy did not pay serious attention to fiber diameter when counting of asbestos fibers. It was noteworthy that our study22 re vealed that only 4% of all asbestos fibers detected in the lung and mesothelial tissues from mesothelioma patients fit Stanton's criteria. An asbestos tissue burden study is an effective approach to clarify whether chrysotile fibers are capable of inducing human malignant mesothelioma. If the as bestos fiber type seen in both the lung and mesothelial tissues is solely chrysotile, such mesothelioma cases can be considered to have been caused by chrysotile expo sure. Indeed, such cases have been reported elsewhere.21,22,47 Our objective in this study was to characterize the features of the asbestos fibers contributing to the induction of human malignant mesothelioma. To achieve this goal, the type, number, and dimensions of the asbestos fibers in both lung and mesothelial tissues taken from human malignant mesothelioma cases were investigated. MATERIALS AND METHODS Both lung and mesothelial tissues (the mesotheliomatous and/or fibroplastic se rosal tissues) from 168 cases of human malignant mesothelioma (164 males and 4 females; 156 pleural and 12 peritoneal; definite or probable diagnostic certainty) were used. The mesotheliomatous tissue was selected from the primary serosal tu mor where the tumor was intimately associated with fibrosis and/or hyaline plaque. Asbestos fibers were studied in both the lung and mesothelial tissues in 74 of the 168 cases, exclusively in lung in 45 of the 168 cases, and exclusively in mesothelial tis sue in the remaining 49 cases. Patients' occupational history was diverse and included asbestos insulators, pipe fitters, electricians, shipyard workers, U.S. Navy servicemen, sheet metal workers, power plant workers, boiler men, brake lining mechanics, fire fighters, family mem bers of asbestos workers, etc. To prepare electron microscopic specimens, bulk tissues were digested using bleach or KOH solution, or a low temperature ashing technique of 25 pm thick sec tion, or both were used. Details of these techniques have been reported else where.18,47-49 A high-resolution analytical electron microscope (JEOL 100CX equipped with an EDX spectrometer) was used for the identification and characterization of asbes tos fibers in these tissues; ultrastructure, energy dispersive X-ray spectrophotometry and, in a limited number of cases, selected area electron diffraction were utilized. Asbestos fibers were measured in printed electron micrographs, and those with an aspect ratio of 3:1 and greater were counted even if they were shorter than 1 pm in length. RESULTS 1. Types of asbestos fibers in lung and mesothelial tissues in the 168 malignant me sothelioma cases studied are outlined below. SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 163 TABLE 1. Type of asbestos fibers in lung and mesothelial tissues in 168 malignant mesothelioma cases A. Asbestos tissue burden study was performed in both the lung and mesothelial tissues: 74 of 168 cases Lung tissue Mesothelial tissue No. of cases C+A C A C+A A A C A C C C C+A C+A C C+A A 19 18 16 9 4 3 2 2 1 Total 74 B. Asbestos tissue burden study was performed in the lung tissues alone: 45 of 168 cases Lung tissue No. of cases A C+A C - 19 15 11 0 Total 45 C. Asbestos tissue burden study was performed in mesothelial tissues alone: 49 of 168 cases Mesothelial tissue No. of cases C C+A A 35 7 6 1 Total 49 C = chrysotile; A = amphibole(s); C + A = chrysotile and amphibole(s); - = not detected. A. In 74 of the 168 cases, asbestos fiber analysis was performed in both the lung and mesothelial tissues, using digested bulk samples, ashed sections or both. Results are summarized in Table 1A. 1) Types of asbestos fibers detected in the lung were quite often different from those in the mesothelial tissue. The combination of asbestos type between the lung and mesothelial tissues was as follow: (i) chrysotile plus amphibole(s) in lung, and chrysotile alone in mesothelial tissues: 19/74 (25.7%); (ii) chrysotile in lung, and chrysotile in mesothelial tissues: 18/74 (24.3%); (iii) amphibole(s) in lung, and chrysotile in mesothelial tissues: 16/74 (21.6%); 164 ANNALS NEW YORK ACADEMY OF SCIENCES (iv) chrysotile plus amphibole(s) in lung, and chrysotile plus amphibole(s) in mesothelial tissues: 9/74 (12.2%); (v) amphibole(s) in lung, and chrysotile plus amphibole(s) in mesothelial tissues: 4/74 (5.4%); (vi) amphibole in lung, and no asbestos fibers in mesothelial tissues: 3/74 (4.0%); (vii) no asbestos fibers in lung, and chrysotile in mesothelial tissues: 2/74 (2.7%); (viii) chrysotile in lung, and chrysotile plus amphibole(s) in mesothelial tis sues: 2/74 (2.7%); and (ix) amphibole in lung, and amphibole in mesothelial tissues: 1/74 (1.4%). In summary, a disproportion of the type of asbestos fibers between the two tissues was seen in the majority (46 of 74; 62.2%) of cases. 2) Asbestos types identified in lung were chrysotile (49/74; 66.2%) and amosite (49/74; 66.2%), followed by tremolite (15/74; 20.3%), crocidolite (13/74; 17.6%) and anthophyllite (12/74; 16.4%). 3) Chrysotile was the most common asbestos type detected in mesothelial tis sues. It was present in 70 of the 74 cases (94.6%); chrysotile was exclusively detect ed in 55 of the 74 cases (74.3%). 4) When chrysotile was exclusively seen in the lung, the asbestos type detected in mesothelial tissues was also exclusively chrysotile in 18 of 20 cases (90%). 5) When amphibole(s) was exclusively found in lung, the asbestos type(s) in mesothelial tissues was exclusively amphibole(s), although this occurred rarely (1/24; 4.2%). Other asbestos types found in mesothelial tissues were chrysotile alone (16/ 24; 66.7%), chrysotile plus amphibole(s) (4/24; 16.7%), and none (3/24; 12.5%). B. In 45 of the 168 cases, an asbestos tissue burden study was carried out exclu sively in the lung using digested bulk samples, ashed tissue section, or both. Results are summarized in Table 1B. 1) Asbestos types detected in lung of the 45 cases varied. They were amphibole(s) alone (19/45; 42.2%) followed by chrysotile plus amphibole(s) (15/45; 33.3%), and chrysotile only (11/45;24.5%). 2) The subtype of amphiboles in the lung of 34 of the 45 cases was amosite alone (18/34; 52.9%), followed by amosite plus tremolite/actinolite (5/34; 14.7%), croci dolite alone (4/34; 11.8%), tremolite alone (2/34; 5.9%), amosite plus crocidolite (2/ 34; 5.9%), amosite plus crocidolite plus anthophyllite (2/34; 5.9%), and amosite plus anthophyllite (1/34; 2.9%). C. In 49 of the 168 cases, an asbestos tissue burden study was done on mesothelial tissues only, using digested bulk samples, ashed sections, or both. Results are summarized in Table 1C. 1) Again, chrysotile fibers were the major asbestos type detected in mesothelial tissues. 2) Asbestos types seen in mesothelial tissues were chrysotile alone (35/49; 71.4%) followed by chrysotile with amphibole (7/49; 14.3%), no asbestos fibers de tected (6/49; 12.3%), and amphibole alone (1/49; 2.0%). Findings presented in 1, A, B and C are summarized as follows. (1) Asbestos fibers were present in almost all of the lung tissue (117/119; 98.3%) as well as in the mesothelial tissue (114/123; 92.7%). SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 165 TABLE 2. Type and number of asbestos fibers in lung parenchyma, pleural plaque, and mesotheliomatous tissues among 22 cases of mesothelioma Case # Occupation Site Disease Chry Amos Croc Anth Tr/Ac DL Total # i insulation L pl meso 28.3 P 12.1 2 insulation L pl meso 28.6 P 39.2 T 62.1 3 insulation L pe meso 24 P 36.3 T 14.8 4 insulation L pe meso 111 P 31.8 T 16.5 5 insulation L pe meso 25.5 P 29.4 T 12.6 6 insulation L pe meso 91.9 T 50.1 T 43.7 7 insulation L pe meso 18.8 T 90 8 insulation L pe meso 1.5 T/P 17 9 engineer L pl meso <DL T 22.5 10 aircraft inspector L pl meso 61 T 120 ii power plant L pl meso <DL T 240 12 shipyard/ power plant L pl meso <DL T 51.3 13 power plant L pl meso <DL 125 1.29 194 0.6 <DL 139 6.34 <DL 282 6.81 0.52 120 1.8 1.76 213 1.79 <DL 415 14 7.1 <DL 2.5 <DL <DL <DL 47 <DL 2.6 <DL 1.3 <DL <DL <DL <DL <DL 7.37 <DL <DL 25.6 <DL <DL <DL <DL <DL 86.4 <DL <DL 11.3 <DL <DL <DL 0.53 0.22 <DL <DL <DL <DL <DL <DL <DL 2.83 <DL 3 <DL <DL <DL <DL <DL 4.3 <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL 0.22 <DL <DL <DL <DL <DL <DL 0.15 <DL <DL 3 <DL <DL 11.4 <DL <DL <DL <DL <DL <DL <DL <DL 3.68 <DL <DL 11.3 <DL <DL <DL <DL <DL 0.7 <DL <DL <DL <DL <DL 0.15 2.83 0.16 1.5 0.6 1.27 1.26 0.58 0.76 2.13 0.76 0.17 0.77 0.6 0.44 1.84 0.6 0.48 3.75 1.42 0.29 0.26 0.18 0.22 0.35 156.1 13.4 228.6 39.8 62.1 181.8 42.6 14.8 422.9 38.6 17 145.5 31.2 14.4 395 51.9 43.7 456.44 104 8.5 17 3 22.94 61.7 0.35 120 2.9 47 2.9 240 0.22 2.6 0.27 51.3 0.15 1.6 14 welder T 2.6 L pl meso 0.62 0.3 <DL <DL <DL <DL 0.26 <DL <DL 0.15 0.26 2.9 0.88 166 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 2. Type and number of asbestos fibers in lung parenchyma, pleural plaque, and mesotheliomatous tissues among 22 cases of mesothelioma (Continued) Case # Occupation Site Disease Chry Amos Croc Anth Tr/Ac DL Total # T 0.7 0.4 <DL 0.3 <DL 0.09 1.4 15 US Navy L pl meso 27 <DL <DL <DL <DL 4.4 27 T 22 <DL <DL <DL <DL 0.88 22 16 electrician L pl meso <DL 19.2 2.9 <DL <DL 1.45 22.1 T/P 228.2 1.8 <DL <DL <DL 2.9 230 17 firefighter L pl meso 32.5 1.4 <DL <DL <DL 1.77 33.9 T/P 16.6 <DL <DL <DL <DL 0.22 16.6 18 US Navy/ railroad L pl meso <DL 0.08 <DL <DL <DL 0.02 0.08 T 0.06 <DL <DL <DL <DL 0.03 0.06 19 US Navy L pl meso <DL 0.52 <DL <DL <DL 0.03 0.53 T 2.6 <DL <DL <DL <DL 0.11 2.6 20 sheetmetal L pl meso 0.49 <DL <DL 0.04 <DL 0.04 0.53 T 0.19 <DL <DL 0.04 <DL 0.04 0.23 21 roofer L pl meso 1.5 0.03 <DL <DL <DL 0.03 1.53 T 0.3 <DL <DL <DL <DL 0.03 0.3 22 boiler L pl meso <DL mechanic 0.54 0.07 <DL <DL 0.02 0.6 T <DL 0.15 <DL <DL <DL 0.03 0.15 Note: Figures represent asbestos fibers X 106/gram (dry tissue). ABBREVIATIONS: L, lung; P, plaque; T/P, tumor/plaque; DL, detection limit; <DL, under detec tion limit (no detection); Chry, chrysotile; Amos, amosite; Croc, crocidolite; Anth, anthophyllite; Tr/Ac, tremolite/actinolite; pl, pleura; pe, peritoneum; meso, mesothelioma. (2) A disproportion in the types of asbestos fibers between lung and mesothelial tissues was common and was present in 49 of 74 cases (66.2%). (3) The most common asbestos types in lung were an admixture of chrysotile with amphiboles (43/119 36.1%) or amphiboles alone (43/119; 36.1%). Chrysotile alone was seen occasionally (31/119; 26.1%). Rarely, no asbes tos fibers were seen (2/119; 1.7%). (4) In mesothelial tissues, the major asbestos type was chrysotile alone (90/ 123; 73.2%) followed by chrysotile plus amphibole (22/123; 17.9%), no asbestos fibers detected (9/123; 7.3%), and amphibole alone (2/123; 1.6%). The amphiboles included anthophyllite mixed with chrysotile in 15 cases and amphiboles alone in 1 case, followed by tremolite mixed with chrysotile in 4 cases, amosite mixed with chrysotile in 3, and amosite alone in 1 case. 2. Quantitative analysis of asbestos fibers in the tissues (number of fibers/dry gram) was performed in both digested lung and digested mesothelial tissues taken from 22 SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 167 TABLE 3. Type and number of asbestos fibers in lung parenchyma in 27 additional cases of mesothelioma Case # Occupation Site Chry Amos Croc Anth Tr/Ac DL Total # 1 electrician L (L) 0.04 0.08 0.02 0.02 <DL 0.02 0.16 L (R) 12.1 1.29 <DL <DL <DL 0.03 13.4 2 US Navy L <DL 3.3 <DL <DL <DL 0.17 3.3 3 insulation L <DL 0.6 <DL <DL 0.9 0.26 1.5 4 family contact L-1 <DL 0.11 <DL 0.11 0.33 0.17 0.55 L-2 <DL <DL <DL 0.31 0.31 0.29 0.62 5 jet plane mechanic L 260 <DL <DL <DL <DL 0.22 260 6 mechanic L 76 0.98 <DL 0.16 <DL 0.12 77.1 7 construction L <DL 9.9 <DL <DL <DL 0.13 9.9 8 US Navy L <DL 2.78 <DL 0.22 <DL 0.11 3.0 9 insulation L <DL 7.06 <DL <DL <DL 0.11 7.0 10 insulation L <DL 26 <DL <DL <DL 0.22 26.0 11 construction L 36 7.5 <DL <DL 0.75 0.75 44.3 12 electrician L 1.5 1 0.5 <DL <DL 0.25 3.0 13 pipe fitter L 1.26 0.63 2.8 0.63 <DL 0.33 5.32 14 US Navy L 16 <DL 0.22 <DL <DL 0.22 16.2 15 insulation L 88 <DL <DL <DL <DL 0.44 88.0 16 US Navy L <DL 1.64 0.12 0.5 <DL 0.12 2.26 17 shipyard L 0.66 1.32 <DL <DL <DL n/a 1.98 18 US Navy L 0.94 0.38 <DL <DL <DL n/a 1.32 19 boiler repair L <DL 0.35 <DL 0.07 0.97 0.02 1.39 20 pipe fitter L (R) 3 <DL <DL <DL <DL 0.05 3.0 L (L) 0.03 <DL <DL <DL <DL 0.03 0.03 21 boiler repair L 2.9 <DL <DL <DL 0.07 0.04 3.6 22 shipyard L <DL 0.08 <DL <DL <DL 0.03 0.08 23 shipyard L <DL 0.11 0.1 <DL 0.08 0.02 0.29 24 machinist L <DL 0.06 <DL <DL <DL 0.02 0.06 25 boiler worker L <DL 0.1 <DL 0.04 0.04 0.02 0.18 26 shipfitter L <DL 1.99 <DL <DL 0.07 0.07 2.06 27 pipe coverer L <DL <DL 0.58 <DL <DL 0.05 0.58 Note: Figures represent asbestos fibers X 106/gram (dry tissue). ABBREVIATIONS: L, lung; DL, detection limit; <DL, under detection limit (no detection); Chry, chrysotile; Amos, amosite; Croc, crocidolite; Anth, anthophyllite; Tr/Ac, tremolite/actinolite; (L), left; (R), right; n/a, not available. 168 ANNALS NEW YORK ACADEMY OF SCIENCES mesothelioma cases (Table 2) and from the digested lung alone in an additional 27 mesothelioma cases (Table 3). A. As shown in Table 2, the total number of asbestos fibers detected in lung tis sue was 456.4 x 106 fibers/dry gram maximum, and 0.08 x 106 fibers/dry gram min imum, and 99.9 x 106 fibers/dry gram on average. The average number of intrapulmonary asbestos fibers seen in the 22 cases was greatest for amosite (71.4 x 106 fibers/dry gram) followed by chrysotile (20.6 x 106 fibers/dry gram), and crocidolite (6.1 x 106 fibers/dry gram), tremolite/actinolite (1.37 x 106 fibers/dry gram and 0.48 x 106 fibers/dry gram). B. In the mesothelial tissues, the total number of asbestos fibers was 240 x 106 fibers/dry gram maximum, 0.06 x 106 fibers/dry gram minimum, and 46.5 x 106 fi bers/dry gram on average. The average number of intramesothelial asbestos fibers was greatest for chrysotile (45.2 x 106 fibers/dry gram) followed by amosite (1.3 x 106 fibers/dry gram), anthophyllite (0.03 x 106 fibers/dry gram), crocidolite (0.01 x 106 fibers/dry gram), and tremolite (0.0 x 106 fibers/dry gram). Total number of chrysotile fibers was compared with that of amphiboles in the mesothelial tissues from 22 cases. The total number of chrysotile fibers was 30.3 times greater than the total number of amphiboles fibers in mesothelial tissues C. Asbestos fiber analysis was done exclusively in the lung in an additional 27 mesothelioma cases (Table 3). The total number of asbestos fibers detected in lung was 260 x 106 fibers/dry gram maximum, 0.08 x 106 fibers/dry gram minimum, and 21.0 x 106 fibers/dry gram on average. The average number of intrapulmonary as bestos fibers among asbestos types seen in these 27 cases was greatest for chrysotile (18.2 x 106 fibers/dry gram) followed by tremolite/actinolite (3.18 x 106 fibers/dry gram), amosite (2.46 x 106 fibers/dry gram), crocidolite (0.16 x 106 fibers/dry gram), and anthophyllite (0.06 x 106 fibers/dry gram). Combined data for both the type and number of intrapulmonary asbestos fibers in the 49 mesothelioma cases (22 from Table 2 and 27 from Table 3) was as follows: (i) Total number of asbestos fibers detected in lung of the 49 cases was 456.4 x 106 fibers/dry gram maximum, 0.08 x 106 fibers/dry gram min imum and 56.4 x 106 fibers/dry gram on average. (ii) The most common asbestos type(s) seen in lung in the 49 cases was amphibole alone (23/49; 47.0%), followed by amphibole(s) plus chrysotile (22/49; 44.9%)and chrysotile alone (4/49; 8.1%). (iii) Among the various asbestos type seen in lung of the 49 cases, amosite fibers were greatest in number (36.9 x 106 fibers/dry gram on average) followed by chrysotile (19.4 x 106 fibers/dry gram on average), crocido lite (3.13 x 106 fibers/dry gram on average), tremolite/actinolite (0.69 x 106fibers/dry gram on average) and anthophyllite (0.27 x 106 fi bers/dry gram on average). Findings obtained from 2A, B and C (based on Tables 2 and 3) are summarized as follows: (1) Except for 3 cases, the number of asbestos fibers in lung tissue of 49 mesothelioma cases (22 from Table 2 group and 27 from Table 3 group) was greater than the average number in lung in the general population (0.44 x 106 fibers/dry gram).22 (2) The number of asbestos fibers in mesothelial tissues taken from 22 mesothelioma cases (Table 2 group) was also greater in the majority of SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 169 TABLE 4. Dimensions of 10,575 asbestos fibers detected in lung and mesothelial tissues: totals for the 168 cases Length Width Tissue N. G.M. G.S.D. Min. Max. N. G.M. G.S.D. Min. Max. Lung Plaque Tumor 1577 48 66 5.08 2.38 4.55 3.12 3.62 3.50 Amosite 0.20 82.4 0.15 28.0 0.30 62.0 1577 48 66 0.19 0.14 0.21 2.47 2.61 2.19 0.02 0.02 0.03 6.50 1.10 1.10 Lung Plaque Tumor 2921 1208 4412 0.42 0.39 0.35 2.26 2.37 1.97 Chrysotile 0.08 18.5 2921 0.08 38.0 1208 0.07 15.0 4412 0.04 0.04 0.04 1.46 1.40 1.36 0.01 0.01 0.01 3.00 0.20 0.70 Crocidolite Lung 230 4.63 2.34 0.40 31.5 230 0.14 1.96 0.03 1.50 Plaque* - - - - - ----- Tumor 2 3.53 1.09 3.33 3.75 2 0.40 1.37 0.32 0.50 Tremolite Lung 54 5.80 2.75 0.60 34.5 54 0.33 2.60 0.05 1.80 Plaque* - - - - - ----- Tumor 11 1.61 2.18 0.40 3.72 11 0.19 2.37 0.03 0.80 Anthophyllite Lung 38 5.57 3.11 0.25 49.6 38 0.54 2.37 0.10 2.90 Plaque* 3 2.30 2.26 1.00 5.10 3 0.23 2.08 0.10 0.40 Tumor 5 4.40 3.01 1.60 26.3 5 0.39 1.78 0.24 1.00 N = number; G.M. = geometric mean; G.S.D. = geometric standard deviation. cases (18/22) than the average number of the general population (0.41 x 106 fibers/dry gram).22 (3) The average number of each type of asbestos fibers in lung (49 cases) was greatest for amosite (36.9 x 106 fibers/dry gram), followed by chrysotile (19.4 x 106 fibers/dry gram), crocidolite (3.13 x 106 fibers/dry gram, tremolite/actinolite (1.69 x 106 fibers/dry gram) and anthophyllite (0.27 x 106 fibers/dry gram). In contrast, in mesothelial tissues (22 cases), the average number of asbestos fibers was greatest for chrysotile (45.2 x 106 fibers/dry gram) followed by amosite (1.3 x 106 fibers/dry gram), anthophyllite (1.03 x 106 fibers/dry gram), crocidolite (0.01 x 106 fibers/dry gram) and tremolite/actinolite (0 x 106 fibers/dry gram). It was obvious that a dispro portion of the average number of asbestos types was present between lung and mesothelial tissues in the 22 cases of malignant mesothelioma. 170 ANNALS NEW YORK ACADEMY OF SCIENCES 3. From the 168 cases of human malignant mesothelioma, dimensions (length and diameter) of a total of 10,575 asbestos fibers detected in lung and mesothelial tissues (mesotheliomatous tissue and fibrotic serosa including hyaline plaque) were mea sured. Findings are summarized in Table 4 and Table 5. A. 1) As shown in Table 4, the 10,575 asbestos fibers consisted of 8,536 chrysotile fibers (2,921 in lung, 1,203 in plaque, 4,412 in tumor), 1,691 amosite fi bers (1,577 in lung, 48 in plaque, 66 in tumor), 232 crocidolite fibers (230 in lung, 0 in plaque, 2 in tumor), 65 tremolite/actinolite fibers (54 in lung, 0 in plaque, 11 in tumor), and 46 anthophyllite fibers (38 in lung, 3 in plaque, 5 in tumor). 2) The chrysotile fibers obtained were generally short in length (geometric mean [G.M.]: 0.42 |im in lung, 0.39 |im in hyaline plaque, 0.35 |im in tumor) and thin in diameter (G.M.: 0.04 |imin lung, 0.04 |imin both plaque and tumor). Amosite fibers were greater in length (G.M.: 5.08 |im in lung, 2.38 |im in plaque, 4.55 |im in tumor) and thicker in diameter (G.M.: 0.19 |im in lung, 0.14 |im in plaque, 0.21 |im in tumor). Although other amphibole fibers, such as crocidolite, tremolite/actinolite and anthophyllite fibers were much less common: crocidolite fiber length was 4.63 |im (G.M.) in lung, not available in plaque, and 3.53 |im (G.M.) in tumor; their di ameter was 0.14 |im (G.M.) in lung, not available in plaque, and 0.40 |im (G.M.) in tumor. Tremolite/actinolite fiber length was 5.80 |im (G.M.) in lung, not available in plaque, and 1.61 |im (G.M.) in tumor; their diameter was 0.33 |im (G.M.) in lung, not available in plaque, and 0.19 |im (G.M.) in tumor. Anthophyllite fiber length was 5.57 |im (G.M.) in lung, 2.30 |im in plaque, and 4.40 |im (G.M.) in tumor; diameter was 0.54 |im (G.M.) in lung, 0.23 |im (G.M.) in plaque, and 0.39 |im (G.M.) in tumor. 3) The numerical distribution of each type of asbestos fiber was compared between the lung and mesothelial tissues (hyaline plaque plus mesotheliomatous tis sue). Distributions were quite different for chrysotile fibers and amphibole fibers. 34.2% (2,921/8,536) of chrysotile fibers were detected in lung, and 65.8% (5,616/ 8,536) were present in mesothelial tissues. The majority of amphibole fibers were seen in lung (amosite: 93.3%; 1,577/1,691; crocidolite: 99.1%; 230/232; tremolite/ actinolite: 83.0%; 54/65; and anthophyllite: 82.6%; 38/46). Only a small proportion of these amphibole fibers were present in mesothelial tissues. This finding supported that compared with amphibole(s) fibers, chrysotile fibers had a much stronger capac ity to translocate from the lung to the mesothelial tissue. TABLE 5A. Total number of asbestos fibers in lung, plaque, and mesotheliomatous tissues greater than or equal to 5 mm in length from the 168 cases Amosite 873/1691 (51.6%) Crocidolite 112/232 (48.3%) Tremolite 30/65 (45.5%) Anthophyllite 23/46 (50.0%) Chrysotile 83/8541 (1.0%) Fibers > 5 |im 1121/10,575 (10.6%) Fibers < 5 |im 9454/10,575 (89.4%) SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 171 TABLE 5B. Number of fibers found with length greater than or equal to 8 mm and diameter less than or equal to 0.25 mm from the 168 cases Lung Plaque Tumor Amosite 171/1577 (10.8%) 2/48 (4.2%) 7/66 (10.6%) Crocidolite 48/230 (20.9%) 0/0 (0.0%) 0/2 (0.0%) Tremolite 4/54 (7.4%) 0/0 (0.0%) 0/11 (0.0%) Anthophyllite 0/38 (0.0%) 0/3 (0.0%) 0/5 (0.0%) Chrysotile 4/2921 (0.1%) 7/1208 (0.6%) 4/4412 (0.1%) Totals: 247/10,575 (2.3%) B. (1) Asbestos fibers greater than 5 pm in length were counted in the 10,575 fibers. As shown in Table 5A, only 10.6% (1,121/10,575) of fibers were longer than 5 pm, and 89.4% were shorter than 5 pm. The proportion of long fibers, i.e., those > 5 pm, was greatest for amosite (873/1,691; 51.6%) followed by anthophyllite (23/ 46; 50.0%), crocidolite (112/232; 48.3%), tremolite/actinolite (30/65; 45.5%), and chrysotile (83/8,541; 1.0%). (2) Table 5B shows that of the 10,575 fibers, only 247 fibers (2.3%) fit Stanton's hypothetical dimensions (> 8 pm in length and < 0.25 pm in diameter). The propor tion of asbestos fibers that fit the hypothetical dimensions among asbestos types in these tissues was greatest for crocidolite (48/232; 20.7%), followed by amosite (180/ 1,691; 10.6%), tremolite /actinolite (4/65; 6.2%), chrysotile (15/8,541; 0.2%) and anthophyllite (0/46; 0%). None of crocidolite (2), tremolite/actinolite (11) and an thophyllite (5) fibers seen in mesothelial tissues (hyaline plaque and mesotheliomatous tissue) fit Stanton's hypothetical dimensions. On the basis of these findings, it was concluded that asbestos fibers detected in lung and mesothelial tissues from mesothelioma patients were predominantly short and thin; 89.4% of asbestos fibers in these tissues were shorter than 5 pm, and the percentage of asbestos fibers that confirms Stanton's hypothetical dimensions (long er than 8 pm in length and smaller than 0.25 pm in diameter) was only 2.3%. DISCUSSION Translocation of asbestos fibers from the lung to other organs, including the pleu ra and peritoneum, has been well documented.16-24 On the light microscopic level, asbestos bodies translocated from lung of deceased asbestos factory workers have been found in various organs such as kidney, heart, liver, spleen, adrenal, pancreas, brain, prostate, and thyroid.25 Asbestos bodies have also been documented in hilar, mediastinal and abdominal lymph nodes, peritoneal mesotheliomatous tissue, and intestinal wall taken from mesothelioma cases.13 On the level of electron microscopy, in 1973, LeBouffant et al.16 revealed the presence of numerous uncoated short, thin chrysotile fibers in pleural hyaline plaques taken from asbestos workers using a transmission electron microscope. This was an important finding at that time because pathologists could not obviously iden 172 ANNALS NEW YORK ACADEMY OF SCIENCES tify coated or uncoated asbestos fibers in the hyaline plaque in routine histopathological slides under light microscopy although they knew that this unique pleural change was intimately related to exposure to asbestos. Several years later, Sebastian etal.17 found an obvious disproportion in the type and number of asbestos fibers be tween lung and parietal pleura of 29 asbestos workers: most of asbestos fibers seen in the parietal pleura were short chrysotile fibers. Dodson etal.19 also found asbestos fibers (predominantly chrysotile) in pleural hyaline plaque taken from tissues of eight shipyard workers. Boutin etal.20 also found highly concentrated asbestos fi bers in black spots (glomerate lymphatic capillaries stained dark from anthracitic pigmentation) in the parietal pleura and stated that amphiboles outnumbered chrysotile in the black spots. Dodson et al.24 detected asbestos fibers (predominantly amosite fibers) in omentum and mesentery taken from 20 (17 pleural and 3 perito neal) mesothelioma cases; they concluded that asbestos fibers could translocate from the lung to the peritoneal cavity. Our previous studies18,21,22 revealed that the types of asbestos fibers were quite often different between the lung and mesothelial tissues in mesothelioma cases and that the major asbestos type seen in the pleural and peritoneal mesothelial tissues was short, thin chrysotile fibers. The capacity of such short, thin chrysotile fibers to translocate from alveoli to the pulmonary interstitium and finally to the pleura has been documented in experimental animal studies.26,27 Our present study based on a larger number of mesothelioma cases confirmed the same disproportion in fiber types (as shown in Table 1A) and number (as shown in Table 2) of asbestos fibers between lung and mesothelial tissues. We have already suggested that such a disproportion was caused by the strong ability of chrysotile fi bers to translocate from lung to the pleura and peritoneum.18,21,22 We proposed that, to clarify the features of the asbestos fibers contributing to the induction of malignant mesothelioma, asbestos fiber analysis should be done of both lung and mesothelial tissues obtained from deceased mesothelioma patients. This approach is essential to grasp the total picture of asbestos exposure in malignant me sothelioma cases. The number of asbestos fibers (per dry gram) counted in both the digested lung (49 cases) and mesothelial tissues (22 cases) varied among the mesothelioma cases. However, it was greater than the general population average number in lung (0.44 x 106 fibers/dry gram) in 45/49 (91.8%). It was also greater than the general popula tion average number in the mesothelial tissues (0.41 x 106 fibers/dry gram) in 18/22 (81.8%). The numerical ratio between chrysotile fibers and amphibole(s) fibers in the mesothelial tissues was examined in 13 of the 22 mesothelioma cases and was found to be 30.3 (chrysotile):1 (amphibole[s]) in the mesothelial tissues. Our present study revealed that the majority of asbestos fibers detected in the lung and mesothelial tissues were shorter than 5 pm; only 10.5% (1,115/10,575) of the fibers exceeded 5 pm in length. Thinness of asbestos fibers has been emphasized as an important factor in their penetration from the proximal area to the peripheral part in the lung and for their translocation from the lung to the pleura.44-46 It was also suggested that the thinness was related to the carcinogenicity of asbestos fibers.35,36,42,43 The present study sup ports this concept in that the vast majority of asbestos fibers that translocated into the mesothelial tissues, the original site from which malignant mesothelioma devel ops, were very thin (0.04 pm in G.M.). SUZUKI & YUEN: ASBESTOS IN HUMAN MALIGNANT MESOTHELIOMA 173 Our present study also revealed that asbestos fibers fitting Stanton's hypothetical dimensions (> 8 pm in length and < 0.25 pm in width) comprised only 2.3% (247/ 10,575) of the fibers detected in both the lung and mesothelial tissues. From these findings, it is obvious that if we exclusively count only asbestos fibers longer than 5 pm or if we select only asbestos fibers fitting Stanton's hypothetical dimensions, a large proportion of asbestos fibers in these tissues will be omitted. We conclude that short, thin asbestos fibers should be considered carcinogenic because they were the principal type of asbestos fiber encountered in the lung and mesothelial tissues taken from human mesothelioma cases. It has been generally accepted, that like other asbestos types, chrysotile fibers are capable of inducing human malignant mesothelioma. 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