Document baMp3YpO5Bk33oBdqnw05mGqg

PLAINTIFF'S EXHIBIT Industrial' Health 2001, 39, 150-160 SA-479 Original Article Asbestos Tissue Burden Study on Human Malignant Mesothelioma Yasunosuke SUZUKI* and Steven R. YUEN Department of Community and Preventive Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place 10029, New York, New York, U.S.A. Received December 15, 2000 and accepted February 13, 2001 * ~i Abstract: Asbestos fibers in the lung and mesothelial tissues (mesotheliomatous tissue and hyaline plaque) taken from 151 human malignant mesothelioma cases were identified and characterized by high resolution analytical electron microscopy. Asbestos fibers were present in almost all of the lung tissue as well as in the mesothelial tissue. The most common asbestos types seen in the lung were an admixture of chrysotile with ampbiboles followed by amphlboles alone and chrysotile alone. The majority of asbestos types seen in the mesothelial tissues were chrysotile alone, followed by chrysotile plus amphibole and amphibole alone. A disproportion of asbestos types between the lung and mesothelial tissues was frequently observed. The most common pattern of the disproportion was chrysotile plus amphibole(s) in the lung and chrysotile only in the mesothelial tissues, followed by amphibole(s) in the lung and chrysotile only in the mesothelial tissues. Such a disproportion was considered to have been caused by chrysotile fiber's strong capacity to translocate from the lung to mesothelial tissues. The number of asbestos fibers in the lung was dS6.4 x.104 fibers/dry gram in maximum, 0.08 x JO4 fibers/dry gram in minimum and 105 x 104 fibers/dry gram on average; in the mesothelial tissues it was 240.0 x 10` fibers/dry gram in maximum, 0.03 x 104 fibers/dry gram in minimum and 49.84 x 10` fibers/dry gram on average. These numbers were greater than those seen in the general population. The majority of asbestos fibers detected in the lung and mesothelial tissues were shorter than 5 pm in length. Asbestos fibers fit to Stanton's hypothetical dimensions (efi.O /un-in length and <0.25 pm in diameter) were only 4.0%, since the majority of these fibers were shorter (<8 pm) and thinner (<0.25 pm) fibers. We concluded that such short, thin asbestos fibers should not be excluded from those contributing to the induction of human malignant mesothelioma. The present study supports that chrysotile asbestos can induce human malignant mesothelioma, since, in some of the mesothelioma cases, asbestos fibers detected in both the lung and mesothelial tissues, or lung tissue alone or mesothelial tissues alone were exclusively chrysotile fibers. Key words: Mesothelioma, Type and Dimensions of Asbestos Fibers. Chrysotile, Translocation Introduction I( is well known that human malignant mesothelioma is caused almost exclusively by exposure to asbestos. It is also known that inhaled asbestos fibers are durable in the lung and persist in the lung and that a part of the fibers'are transformed into asbestos bodies after hemosiderin deposits To whom correspondence should be addressed. on the surface of the fibers1'. Interestingly, however, some of intrapulmonary asbestos fibers, particularly chrysotile fibers are cleared from the lung2'. It is also known that asbestos fibers are capable of translocating from the lung into other tissues including lymph nodes and mesothelial tissue3-1'. There were reports that asbestos bodies were found in various organs other than the lung, supporting that asbestos fibers were disseminated from the lung to other organs'-", Asbestos fibers in human tissues can be identified and RECEIVED TiVIE SEP. 25. -2:06PM ASBESTOS TISSUE BURDEN STUDY ON HUMAN MALIGNANT MESOTHELIOMA 151 characterized by a high resolution analytical electron Indeed, such cases have been reported elsewhere6-201. To microscope, even if they are short and thin in dimension obtain clear answers to the above problems and to identify (>0.1 pm in length and 0.03 pm in width). ; type, number and dimensions of asbestos fibers contributing Up to the present, to clarify asbestos fibers associated to the induction of human malignant mesothelioma, we have with the induction of human malignant mesothelioma, done asbestos fiber analyses on the lung and mesothelial researchers have been focusing almost exclusively on tissues (mesotheliomatous tissue in the primary site or pleural asbestos fibers in the lung tissue taken from mesothelioma hyaline plaque), which were taken from 151 cases of human patientsia-u). malignant mesothelioma. We have questioned the adequacy ofsuch an approach since; I * a) the primary site of malignant mesothelioma is not the lung Materials and Methods but the mesothelial tissue (pleural or peritoneal). Accoidingly, asbestos fibers translocated into the mesothelial tissue should A total of 151 malignant mesothelioma cases of which be considered as a more important contributory factor for tbe diagnostic certainty was confirmed as definite or probable induction of malignant mesothelioma, and b) there is evidence by the author were used as materials. The diagnostic certainty that type and number of asbestos fibers are frequently different was decided by a systematic analysis consisting of gross between the lung and the mesothelial tissue in mesothelioma appearances, histology, histochemistry, immunocytochemistry cases*-*5. Therefore, it may not be logical to say that and electron microscopy (in some cases only). Occupational intrapulmonary asbestos fibers can be blindly used as a definite history of these mesothelioma patients, who were living in marker for the induction of the tumor. the United States, were diverse and included insulation It has been emphasized from animal studies that long workers, pipe fitters, electricians, shipyard workers, U.S. (greater than 8 pm in length) and thin (less than 0.25 pm in Navy servicemen, sheet metal workers, power plant workers, width) mineral fibers were strongly carcinogenic for the boiler men, brake lining mechanics, a fire fighter, a housewife, induction of pleural mesothelioma in rats. (Stanton's etc. Asbestos fibers in both the lung and mesothelial tissues hypothesis)15'. His hypothetical dimensions have been (primary mesotheliomatous tissue, or fibrotic serosa including directly applied to the counting of the asbestos fibers in human pleural hyaline plaque, or both) were investigated in 64 of case. The current Occupational and Health Administration the 151 cases. In 43 ofthe 151 cases, the fibers were exclusively (OSHA) method by light microscopy counts asbestos fibers investigated in the lung. In the rest 44 cases, the fibers were that are longer than 5 pm in length with on aspect ratio of also exclusively investigated in mesothelial tissue, larger than 3 to 1, assuming that all fibers shorter than 5 pm The mesotheliomatous tissue was selected from the are not carcinogenic. Further, even on the electron primary serosal (pleural or peritoneal) tumor where the rumor microscopic level, using the same assumption, some was intimately associated with fibrosis and or hyaline plaque. investigators have neglected to count short asbestos fibers To prepare electron microscopic specimens, either a digestion (< 5 pm) in their tissue burden studies1'- However, technique of the bulk tissues using bleach or KOH solution, our previous studies revealed that the majority of asbestos or a low temperature ashing technique of 25 pm thick section, fibers in human lung and mesothelial tissues taken from or both were used. Details of these techniques have been mesothelioma patients did not fit Stanton's hypothetical reported elsewhere5-6-20-3!|. dimensions; less than 2% of chrysotile fibers and less than A high resolution analytical electron microscope was used 10% of arnosite fibers in these tissues fit with Stanton's for the identification and characterization of asbestos fibers, criteria. Short, thin asbestos fibers were the majority among in these tissues; ultrastructure, energy dispersive X ray asbestos fibers detected in these tissues5-6'. It was strongly spectrometry and selected area electron diffraction (in a suggested that short, thin asbestos fibers are contriburive to limited numbers of these cases) were utilized for these the induction of malignant mesothelioma and that they should purposes. Asbestos fibers measured and those with an aspect not be categorically excluded from carcinogenic fibers5-6'. ratio of 3:1 and greater were counted in this study, even if Asbestos tissue burden study is an effective approach to they were shorter than 1 pm in length. clarify whether chrysotile fibers are capable of inducing human malignant mesothelioma. If the asbestos type seen in the lung and mesothelial tissues of mesothelioma cases Observations is solely chrysotile, such mesothelioma cases can be A. In 64 of the 151 cases, asbestos fiber analysis was considered to have been caused by chrysotile exposure. performed in both the lung and mesothelial tissues, using : 2:06PM 152 Y SUZUKI et al. digested bulk samples, ashed sections or both. Results were Results were as follows: as follows. 1. Asbestos types detected in the lung of these 43 cases 1. Types of asbestos fibers detected in the lung were quite varied. They were omphibole(s) alone (18/43; 41.9%), often different from those seen in the mesothelial tissue. followed by chrysotile plus amphibole($) (15/43; 34.9%) The combination of asbestos type between the lung and and chrysotile only (10/43; 23.3%). mesothelial tissues was as follows: 2. Subtype of amphiboles seen in the lung of 33 of the 43 1) Chrysotile plus amphibole(s) in thelung, andchrysotile cases was amosite alone (17/33; 51.5%), followed by alone in the mesothelial tissues. 18/64 cases; 28.1%. amosite plus tremolite (5/33; 15.2%), crocidolite alone 2) Chrysolite in the lung, and chiysotile in the mesothelial (4/33; 12.1%), tremolite alone (2/33; 6.1%), amosite tissues. 15/64; 23.3%. plus crocidolite (2/33; 6.1%), amosite plus crocidolite 3) Amphibole(s) in the lung, and chrysotile in the plus anthophyllite (2/33; 6.1%) and amosite plus mesothelial tissues 13/64; 20.3%. anthophyllite (1/33; 3.0%). 4) Chrysotile plus amphibole(s) in the lung, and chrysotile plus amphibole(s) in the mesothelial tissues. 8/64; C. In 44 of the 151 cases, asbestos tissue burden study was 12.5%. done in the mesothelial tissues only, using digested bulk 5) Amphibole(s) in the lung, and chrysotile plus samples, ashed sections or both. Again, chrysotile fibers amphibole(s) in the mesothelial tissues. 4/64; 6.3%. were the major asbestos type detected in the mesothelial 6) No asbestos fibers in the lung, and chrysotile in the tissues. Asbestos types seen in the mesothelial tissues were mesothelial tissues. 2/64: 3.1%. chrysotile alone (30/44; 68.2%), followed by chrysotile with 7) Chiysotile in the lung, and chysotile plus amphibole(s) amphibole (7/44; 15.9% [4 with tremolite, 2 with amosite In the mesothelial tissues: 2/64; 3.1%. and 1 with anthophyllite]), no asbestos fibers detected (6/ 8) Amphibole in the lung, and no asbestos fibers in the 44; 13.6%) and amosite alone (1/44; 2.3%). mesothelial tissues. 1/64; 1.6%. Findings obtained from A, B and C are summarized as 9) Amphibole in the lurig, and amphibole in the . follows. .' mesothelial tissues. 1/64; 1.6%. 1. Asbestos fibers were present in almost all of the lung In summary, a disproportion of type of asbestos fibers tissue (105/107; 98.1%) as well as in the mesothelial between the two tissues was quite common; it was seen in tissues (101/108; 93.5%). 40 of the 64 cases (62.5%). 2. A disproportion of types of asbestos fibers between the 2. Asbestos types identified in the lung were chrysotile lung and the mesothelial tissues was common; it was (43/64; 67.2%), followed by amosite (43/64; 67.2.%), seen in 41 of the 64 cases (64%). tremolite (13/64t20.3%), anthophyllite (11/64; 17.2%) and crocidolite (10/64; 15.6%). 3. The most common asbestos types seen in the lung were an admixture of chrysotile with amphiboles (41/105; 3. Chrysotile was the most common asbestos type detected 39.1%) followed by amphiboles alone (37/105; 35.2%) in the mesothelial tissues. It was present in 62 of the 64 and chrysotile alone (27/105; 25.7%). cases (96.9%); chrysotile was exclusively detected in 48 of 4. In the mesothelial tissues, the majority of asbestos type the 62 cases (77.4%). ' f _ seen was chrysotile (78/101; 77.2%), followed by 4. When chrysotile was almost exclusively seen in the chrysotile plus amphibole (21/101; 20.8%) and lung, asbestos type detected in the mesothelial tissues was amphibole alone (2/101; 2.0%). also exclusively chrysotile (15/17 cases; 88.2%). The type of asbestos fibers in the lung and mesothelial 5. When amphibole(s) was exclusively observed in the tissues among the 151 mesothelioma cases is shown in Table lung, asbestos type seen in the mesothelial tissues rarely 1. contained amphibole(s) (1/19 cases; 5.3%). Other asbestos type(s) seen in the mesothelial tissues were chrysotile alone (13/19; 68.4%), chrysotile plus amphibole(s) (4/19; 21.0%) D.Quantitative analysis of asbestos fibers in the tissues (number of the fibers/dty gram) was done in both digested and no asbestos fibers (L/19; 5.3%), lung and digested mesothelial tissues taken from 21 mesothelioma cases (Table 2) and from the digested lung B. In 43 of the 151 cases, asbestos tissue burden study was taken from additional 23 mesothelioma cases (Table 3). The carried out in the lung tissue only using digested bulk samples, or ashed tissue sections or both. 21 cases were a part of the 64 cases in A, and the 23 cases were a part of the 43 cases in B. " " A YE) ;v: n. * 2:06FM Industrial Health 2001. 39. 150-160" ASBESTOS TISSUE BURDEN STUDY ON HUMAN MALIGNANT MESOTHELIOMA 153 fable 1. The type of asbestos libers in the lung and mesothelial tissues among 151 malignant mesothelioma cases. A. 64 of 151 cases (asbestos tissue burden study was performed in both (he lung and mesothelial tissues). Lung tissue Mesothelial tissue C+A C A C+A A -- C A A . C C C C+A C +A C C+A -- A Total No. of Cases is' 15 .13 8 4 2 2 I 1 64 ' B. 43 of 151 cases (asbestos tissue burden study was performed in the lung tissues alone). Lung (issue A C+A C -- Total No. of Cases 18 15 10 0 43 C. 44 of 151 cases (asbestos tissue burden study was performed in the mesothelial (issues alone). Mesothelial tissue C_ C+A -- A Total No, of Cases 30 7 6 1 44 C: Chrysotile, A: Amphibolefs). C + A: Chrysodle and Amphiboic(s), --: Not detected. 1. Table 2 (lung and mesothelial tissues; 21 mesothelioma cases). Total number of asbestos fibers detected in the lung tissue was 456.4 a 10s fibers/dry gram in maximum, 0.08 x 106 fibers/dry gram in minimum, and 105 x 104 fibers/dry gram on average. In the mesothelial tissues, the number of the fibers was 240.0 x 104 fibers/dry gram in maximum, 0.03 x 10* fibers/dry gram in minimum, and 49.8 x 10s fibers/dry gram on average. 2. Table 3 (lung tissue only: 23 additional mesothelioma cases). Total number of asbestos fibers detected in the lung was 260 x 104 fibers/dry gram in maximum, 0.08 X104 fibers/dry gram in minimum, and 24.2 x 10s fibers/ dry gram on average. 3. Our unpublished data on the number of asbestos fibers in the lung obtained from the general population (18 cases. New Yorkers) showed that 4.9 X 10s fibers/dry gram in maximum, 0 (or <detection limit) fibers/dry gram in minimum, and 0.44 x 10s fibers/dry gram on average. In the mesothelial tissues taken from the general population (7 cases. New Yorkers), the number of asbestos,fibers was 2.24 x 104 fibers /dry gram in maximum. 0 (or <detection limit) fibers/dry gram in ' minimum, and 0.41 x 10s fibers/dry gram on average. The vast majority of these asbestos fibers seen in both the lung and the mesothelial tissue were short, thin chrysotile fibers. ' In summary, except for three cases, the number of asbestos fibers in the lung of 44 mesothelioma cases (21 from Table 2 group and 23 from Table 3 group) was greater than the average number of asbestos fibers in the lung taken from the general population. The number of asbestos fibers in the mesothelial tissues taken from the 21 mesothelioma cases (Table 2 group) was also greater in die majority (17/21) than the average number of the general population. . E. Dimensions (length and diameter) of a total of 2884 asbestos fibers which were present in the lung and the mesothelial tissues (mesotheliomarous tissue and hyaline plaque) taken from the 21 mesothelioma cases (Table 2 group) were measured. These 2884 asbestos fibers consisted of 1725 chrysotile fibers (495 in lung, 450 in plaque and 780 in tumor), 1042 amosite fibers (959 in lung, 45 in plaque and 38 in tumor), 78 crocidolite fibers (77 in lung, 0 in plaque and 1 in tumor), 19 tremolite fibers (19 in lung, 0 in plaque and 0 in tumor) and 20 anthophyllite fibers (17 in lung, 1 in plaque and 2 in tumor). Results were summarized in Table 3. Chrysotile fibers were short in length (G.M.: 0.75 /an in lung. 0.61 /an in hyaline plaque and 0.65 /an in tumor) and thin in diameter (G.M.: 0.040 /an in lung, 0.04 /an in plaque and 0-04 /an in tumor). Amosite fibers were greater in length (G.M.: 3.75 /an in lung, 2.20 /an in plaque and 3.28 /an in tumor) and thicker in diameter (G.M; 0.14 /an in lung, 0.13 /an in plaque and 0.16 /an in tumor). Although other amphibole fibers, such as crocidolite. tremolite and anthophyllite fibers were much smaller in number, results were as follows. Crocidolite fibers Length was 3.71 /an (G.M.) in lung, not available in plaque and 3.33 /an (G.M.) in tumor, and their diameter was 0.10 /an (G.M.) in lung. RECEIVED TIME SEP. 25. '2:06PM 154 Y SUZUKI et aL Table 2. The type and number of asbestos fibers in long parenchyma, pleural plaque and mesotheSomatous tissues among 21 cases of mesothelioma Cases No. Occupation Site Disease Chry Amos Abestos fibers (*) Croc Anth Tt/Ac DX. Total * 1 Insulation L Worker P 2 Insulation L Worker P T 3 Insulation L Worker P T 4 Insulation L Worker P T 5 Insulation L Worker P T 6 ( Insulation Worker L T T 7 Insulation L Worker T 8 Insulation L Worker T/P 9 Engineer L T 10 Aircraft L Inspector T 11 Power L Plant T 12 Shipyard & L powerhouse T 13 -- Powerhouse 14 Welder L T L T IS US Navy L T 16 Electrician L T/P 17 Firefighter L T/P 18 US Navy L and railroa T 19 US Navy L T 20 Shcetmeta! L T 21 Roofer L T PI. Meso Pl.-Meso ' Pe. Meso Pe. Meso Pe. Meso Pe. Meso Pe. Meso Pe. Meso Pi. Meso PI. Meso PI. Meso PI. Meso PI. Meso PI. Meso PL Meso PI. Meso PI. Meso Pi. Meso PI. Meso PI. Meso PI. Meso 28.3 125 <DL 12.1 1.29 <DL 28.6 39.2 62.1 194 '0.6 <DL <DL <DL <DL 24 139 7.37 36.3 6.34 <DL 14.8 <DL <DL ill , -31.8 165 282 6.81 0.52 25.6 ,t <DL <DL 2S.5 29.4 12.6 120 <DL 1.8 . <DL 1.76 <DL 91.9 50.1 43.7 213 1.79 <DL 86.4 <DL <DL 18.8 415 11.3 90 14 <DL 1.5 7.1 <DL 17 <DL <DL . <DL 22.5 2.5 , <DL 0.53 0.22 61 <DL <DL 120 <DL <DL <DL 240 47 <DL <DL <cDL <DL 51.3 2.6 <DL <DL <DL <DL 1.3 <DL 2.6 0.3 <DL 0.62 0.7 <DL 0.4 <DL <DL 27 <DL 22 . <DL <DL <DL <DL 19.2 2.9 228.2 1.8 <DL 32.5 1.4 <DL 16.6 <DL <DL <DL 0.06 0.08 <DL <DL <DL <DL 2.6 0.52 <DL <DL <DL 0.49 0.19 <DL <DL <DL <DL 1.5 0.03 <DL 0-3 <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 0.26 0.3 <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL 0.04 0.04 <DL <DL <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 <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL <DL 2.83 0.16 1.5 0.6 1.27 1.26 058 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 0.35 2.9 2.9 0.22 0.27 0.15 0.15 0.26 0.09 4.4 0.88 1.45 2.9 1.77 0.22 0.02 0.03 0.03 0.11 0.04 0.04 0.03 0.03 156.1 13.4 228.6 39.8 62.1 181.8 92.6 14 422.9 38.6 17 1455 31.2 J4.4 395 51.9 43.7 456.4 104 85 17 30 22.94 61.7 120 41 240 2.6 51.3 1.6 2.9 0.88 1.4 27 22 22.1 230 33.9 16.6 0.08 0.6 0.52 2.6 052 0.23 1.53 0.03 ( ). x 10 gram (dry tissue), L: Lung. P: Plaque, T: Tumor, T/P: Tumor/Plaque, D.L.: Detection Limit, <DL: under detection limit (no detection). Chry; chrysolite. Amos: amosite. Croc: croeidolitc, Anth: anthophylliie. Tr/Ac: iremstitc/ccunolite, PI: pleura. Pe: peritoneum, Mojo; mesothelioma. iM M rrtilltH hlinW H M m tfatkx& ttJnuijZ X tf eiute* RECEIVED TIME SEP. 25. 12:06PM Industrial Health 2001.39, 150--160 i ASBESTOS' TISSUE BURDEN STUDY ON HUMaX MALIGNANT MESOTHELIOMA 155 Table 3. The type and number of asbestos fibers in tang parenchyma in 23 additional cases of mesothelioma Cases No. Occupation Site Chrj 1 Electrician L(L) L(R) 0.1K 12.1 2 U.S. Navy L - <DL 3 Insulation worker L " <DL 4 Family contact L-l <DL L-2 <DL 5 Jet plane mechanic L 260 6 Mechanic L 76 7 Consmietion worker L <DL 8 U.S, Navy L cDC 9 ' Insulation worker L <DL 10 Insulation worker L '<DC 11 Construction worker L 12 1 Electrician L i? 13 Pipe fitter L 1.26 14 U.S. Navy L lt> 15 Insulation worker L 8$ 16 U.S. Navy L <DL 17 Shipyard L 0.6c- 18 U.S. Navy L o.su 19 Boiler repairman L <DL 20 Pipefitter L(L) L(R) 3 0.03 21 Boiler repairman L 22 Shipyard L 23 Shipyard L 2.9 cDL <DL Amos Croc Abestos fibers () Anth Tr/Ac 0.08 1.29 33 0.02 <DL <DL. 0.02 <DL <DL 0.6 , <DL <DL 0.11 <DL <DL <DL 0.11 0.31 <DL 0.98 <DL <DL <DL ' 0.16 9.92.78 <DL <DL <DL# 0.22 7.06 <DL <DL 26 <DL <DL 7.5 cDL <DL I 0.5 cDL 0-63 2.8 0.63 <DL 0.22 <DL <DL <DL <DL 1.64 0.12 0.5 1.32 <DL cDL ' 0.38 " <DL ' <DL 0.35 <DL 0.07 <DL <DL <DL <DL <DL <DL <DL <DL <DL 0.08 <DL <DL 0.11 0.1 <DL <DL <DL <DL 0.9 0.33 0.31 cDL <DL <OL <DL CDL <DL =DL cDL <DL cDL <DL <DL <DL cDL 0.97 cDL <DL 0.07 <DL 0.08 D.L. Total # 0.02 XlO4 0.16x10* 0.03 x 10* 0.26 x 104 0.17 X I0` 3.3 x 10* 0.26 X 10s 1.5 x 10* 0.17 x 104 0.56 x 10* 0.29 x 104- 0.6 x 10s 0.22 xlO4 260x10* 0.12 x 104 77.1 x 104 0.13x10* 9.9 x 10* 0.11 xlO4 3.0 x 10* 0.11 x 10s 7.0 x 10s 0.22 x 104 26.0 x 10* 0.75 x 10* 43,0 x 10* 0.25 x 104 3.0 x 10* 0.33 x 104 5.32 x 10* 0.22 x 10* 16.2x10* 0.44 x 10* 88.0 X 104 0.12x10* 2.26x10* n/a 1.96X10* n/a L.32x 10* 0.02 x 10* 0.52 x 10* 0.05 x 10* 3.0 x 10* 0.03 x 10* 0.03 x 10* 0.04 x 10s 3.6 x 104 0.03 x 104 0.08 x 104 0.02 x 10* 3.29 x 10* ("): x 104 gram (dry tissue), L: Lung, P: Plaque, T: Tumor. T/P: Tumor/Piaque, D.L: Detection Limit, <DL: under detection limit (no detection). Chry: chrysotile. Amos: amosite, Croc: cnactdolite, Anth: anthophyllite, Tr/Ac: tremolite/acanoliic, (L): left. (R): right, n/a: not available. not available in plaque and 0.32 /on (G.M.) in tumor. Tremolite fibers length was 2.75 /an (G.M.) in lung, and their diameter was 0.19 /an (G.M.) in lung: data was not available in both plaque and tumor, since tremolite fibers were not detected in these tissues. Anthophyllite fibers length was 6.93 /an (G.M.) in lung, 1.00 /an (G.M.) in plaque and 7.98 /an (G.M.) in tumor, and their diameter was 0.55 /an (G.M.) in lung, 0.03 /an (G.M.) in plaque and 0.65 /an in tumor. The above findings for the fiber dimensions are summarized in Table 4. F. Asbestos fibers greater than 5 /an in length were measured in the 2884 fibers. Results were summarized in Table 5A. Only 18.6% (537/2884) of the fibers were longer than 5 /an in length. 81.4 % were shorter than 5 /an as shown in Table 5A. To identify asbestos fibers which fit Stanton's hypothetical dimensions, (8 /an in length and ^0.25 /an in diameter), dimensions of the above 2884 asbestos fibers were examined. Table 5B summarizes which of the 2884 asbestos fibers measured fit into Stanton's criteria of >8 /an in length and <0.25 /an in diameter. Of the 2884 fibers, only 116 fibers (4.0%) fit Stanton's hypothetical dimensions. Results were summarized in Table 5B. Chrysotile fibers (L725 fibers) fit to the dimensions were 0.6% (3/495) in lung, 1.6% (7/436) RECEIVED TiME SEP. 25. 12:06PM 156 Y SUZUKI et al. Table 4. Dimensions of 2,884 asbestos fibers detected in lung and raesothelial tissues. Tbtais for the 21 cases (from Table 2) Case Tissue Length N G.M. G.S.D. Min. Max. Width N. G.M. G.S.D. Min. Max. Amosite Lung Plaque Tumor 959 3.75 3.02 - 0.20 774 45 2.20 3.6T 0.15 28.0 38 3.28 2.81 0.40 25.0 959 . 45 38 0-14 0.13 0.16 2.54 2.45 1.98 0.02 0,02 0.03 2.30 0.68 0.70 Chrysotile Lung Plaque Tumor Crocidolite Lung Plaque TUmor 495 0.75 2.55 0.12 18.5 450 0.61 2.86 0.10 38.0 780 0.65 2.79 0.07 15.0 , 77 3.71 2.23 0.70 36.0 1 3.33 1.0 3.33 3.33 49S 0.04 1.63 0.02 3.00 450 0.04 1.45 0.02 0.20 780 0.04 1.56 0.01 0.70 i 77 0.10 1.65 0.04 0.60 1 0.32 1.0 0.32 0.32 Tremolite Lung Plaque Tumor 19 2.75 2.74 0.60 26.0 19 0.19 2.65 0.05 1.80 Anthophyilite Lung Plaque TUmor 17 6.93 3.06 1.20 49.6 17 0.55 2.13 0.10 150 1 1.00 ' 1.0 1.00 1.00 1 0.03 1.00 0.30 0.30 2 7.98 S.40 2.42 26.3 . 2 . , 0.65 1.85 0.42 1.00 N: number; G.M.: geometric mean; G-S.D.: geometric standard deviation. Table SA. Total number of fibers in lung, plaque and mesotheliomatous tissues greater than S pm in length Amosite Crocidolite Tremolite Anthophyilite Chrysotile - 418/1042 28/78 4/19 9/20 78 / 1725 (40.1%) (35.9) (21.1%) (45.0%) (4.52%) 537/2884 (18.6%) Table 5B. Number of fibers found whose Length > = 8 )Jm & Diameter < = 0.25 pm (From Table 2,21 Mesothelioma cases). Amosite Crocidolite Tremolite Anthophyilite Chrysotile Lung 84/959 (8.8%) 13/77 (16.9%) 0/19 (0.0%) 0/17 (0.0%) 3/495 (0.6%) 100/1567(6.4%) Plaque 2/45 0/0 o/o 0/1 7/450 (4.4%) (0.0%) (0.0%) (0.0%) (1.6%) 9/496 (1.8%) Tumor 3/38 0/1 0/0 0/2 4/780 (7.9%) (0.0%) (0.0%) (0.0%) (05%) 7/821 (0.8%) in plaque and 0.5% (4/780) in tumor. Amosite fibers (1042 fibers) were 8.8% (84/959) in lung, 4.4% (2/45) in plaque and 7.9% (3/38) in tumor. Crocidolite fibers (78 fibers) were 16.9% in the lung (13/77) and 0% in both plaque (0/0) and tumor (0/1). Both tremolite(20 fibers) and anthophyilite (29 fibers) were 0% in these tissues. It was concluded that asbestos fibers fit to the Stanton's hypothesis were proportionally small in number in all types of asbestos fibers detected in both the lung and the mesothelial (issues. Comments LeBouffant er o(.23), discovered a deposition of a large number of short, thin chrysotile fibers in pleural hyaline plaque (fibroric parietal pleura) taken from asbestos workers under a transmission electron microscope. It was an important finding at that time, since pathologists could not obviously identify coated or uncoated asbestos fibers in the hyaline plaque in routine histopathologic slides under a light microscope, although they knew that this unique pleural RECEIVED TJME SEP. 25. 12:06PM Industrial Health 2001, 39. 150-160 ASBESTOS TISSUE BURDEN STUDY ON HUMAN MALIGNANT MESOTHELIOMA 157 alteration was intimately related to exposure to asbestos. Sdbastien et al?\ found a disproportion of type and number of asbestos types between the lung and the parietal pleura among 29 asbestos workers and that most of asbestos fibers seen in the parietal pleura were short chrysotile fibers. Dodson et al.`\ found asbestos fibers (predominantly chrysotile) in pleural hyaline plaque taken ffom-8 bases of shipyard workers. Boutin et al.24>, also found highly concentrated asbestos fibers in black spots (glomerate lymphatic capillaries stained dark due to anthracotic pigmentation) in the parietal pleura. They said thatamphibole outnumbered chrysotile in the black spots. Our previous studies1,4', revealed that the type of asbestosfibers were quite often different between lung and mesothelial tissues in mesothelioma cases and that the major asbestos type seen in the mesothelial tissues were short, thin chrysotile fibers. Our present study based on larger numbers of tissue samples showed the same trend for the disproportion of asbestos types between the two tissues. We have previously suggested that such a disproportion was caused by the strong ability of chrysotile fibers to translocate from the lung to the pleura and peritoneum1,6>. The present study also supported such an idea. To clarify asbestos fibers contributing to the induction of malignant mesothelioma, asbestos tissue burden study should be done in both the lung and mesothelial tissues, because the disproportion of type and number of asbestos fibers between the two tissues is not rare. If asbestos tissue burden study is limited to lung tissue, then translocated asbestos fibers from the lung to the mesothelial tissues will be overlooked. In theqiresent study, it was observed that when asbestos fibers detected in the lung were exclusively chrysotile, asbestos type seen in the mesothelial tissues was also exclusively chrysotile (15/17 cases; 88.2%). The passage route of the fibers has not been fully understood, although three ways of the passage are considered: 1) a direct migration of the fibers from the lung to the parietal pleura through the pleural cavity, and also to the peritoneum through the lung, pleural cavity and diaphragm; 2) through a lymphatic capillary system; and 3) through a blood capillary system. The number of asbestos fibers observed per gram in the lung and the mesothelial tissues varied. It was larger than the average number in the general population in 43/44 cases [97.7%] in the lung and in 17/21 cases [81%] in the mesothelial tissues. The number of fibers (chrysotile in the vast majority) in mesothelial tissues was larger than that seen in the lung in some cases (8/21; 38.1%). Numerical ratio between chrysotile fibers and amphibole(s) fibers in the mesothelial tissues was examined in 12 of the 21 mesothelial tissues in which both asbestos types were present (Table 2). It was approximately 18.0 (chrysotile): 1 (amphibole[s]) in the mesothelial tissues. Our present study revealed that the majority of asbestos fibers detected in die lung and mesothelial tissues were shorter than 5 pm; only 18.6% (537/2884) of the fibers were longer than 5 pm in length. It was also established that asbestos fibers fitting to Stanton's hypothetical dimensions (8 fan in length and <0.25 ftm in diameter) were only 4% (116/ 2884) among .the fibers detected in these tissues. From these findings, it is obvious that if we exclusively count asbestps fibers longer than 5 fan or if we select only asbestos fibers fitting to Stanton's hypothetical dimensions, a large proportion of asbestos fibers in these tissues will be omitted, since the majority are shorter than 5 pm in length, although the diameter of these short fibers does generally fit well to Stanton's width parameters. It shouldbe taken into account that such short, thin asbestos fibers are carcinogenic, since they are the majority in the lung and the mesothelial tissues taken from mesothelioma cases. It has been generally accepted that like other asbestos types, chrysotile fibers are capable of inducing human .malignant mesothelioma11-28'. This conclusion has been. obtained from various sources including molecular biological studies2*-"', animal experiments2, epidemiological studies3^4', case reports45^', and asbestos tissue burden studies4,20'. The present study on asbestos tissue burden further supports the evidence that chrysotile fibers were capable of inducing human malignant mesothelioma, since a) chrysotile was the most common asbestos type seen in the mesothelial tissues which is the original site of the induction of mesothelioma and b) chrysotile was exclusively seen in both the lung and the mesothelial tissues in 15/64 (23.3%) cases, in the lung tissue alone in 10/43 (23.3%) and in the mesothelial tissues alone in 30/44 (68.2%) cases. Summary To identify and characterize asbestos fibers contributing to the induction of human malignant mesothelioma, asbestos fibers in the lung and mesothelial tissues (mesotheliomatous tissue and hyaline plaque) taken from 151 human malignant mesothelioma cases were investigated by a high resolution analytical electron microscope. Results were as follows: 1) Asbestos fibers were present in almost all of the lung tissue as well as in the mesothelial tissue. 2) The most common asbestos types seen in the lung were RECEIVED TIME SEP. 25. 12:06PM 158 Y SUZUKI et al. an admixture of chrysotile with amphiboles (41/105; the lung and mesothelial tissues (15/64; 23.3%), or in 39.1%), followed by amphiboles alone (37/105; 35.2%) lung tissue alone (10/43; 23.3%) or in mesothelial tissues and chrysotile alone (27/105; 25.7%). The majority (30/44; 68.2%) were exclusively chrysotile fibers. of asbestos types seen in the mesothelial tissues were chrysotile alone (78/101; 77.2%), followed by chrysotile plus amphibole (21/101; 20.8%) and amphibole alone References (2/101; 2.0%). 1) Suzuki Y, Churg J (1969) Structure and development 3) A disproportion of asbestos types between the lung and * of the asbestos body. Am J Path SS, 79-107. mesothelial tissues was frequently observed. The most 2) Wagner JC, Beny G, Skidmore JW, Timbreil V (1974) common pattern of the disproportion was chrysotile The effects of the inhalation of asbestos in rats. Br J plus amphibole(s) in the lung and chrysotile only in Cancer 29, 252-69. mesothelial tissues (18/64), followed by amphibole(s) ^ 3) Sdbastien P, Janson X, Gaudichet A, Hirsch A, Bignon in the lung and chrysotile only in mesothelial tissues J (y>80) Asbestos retention in human respiratoiy tissues: (13/64). It was considered that such a disproportion comparative measurements in lung parenchyma and was caused by chrysotile fibers' strong capacity to in parietal pleura. In: Biological effects of mineral fibres, translocate from the lung to the mesothelial tissues. ed. by Wagner JC, 30, 237--46, IARC Scientific 4) The number of asbestos fibers in both the lung and the Publications, WHO/Intemational Agency for Research mesothelial tissues was various among the on Cancer, Lyon. mesothelioma cases. In the lung (44 cases), it was 456.4 4) Dodson RF, Williams MG, Com CJ, Brollo A, Bianchi x 10s fibers /dry gram in maximum, 0.08 x 10s fibers/ C (1991) A comparison of asbestos burden in lung dry gram in minimum and 77.7 x 10s fibers /dry gram parenchyma, lymph nodes and plaques. Ann NY Acad on average. In the mesothelial tissue (21 cases), it was Sci 643,53-60. 240.0 x 106 fibers/dry gram in maximum. 0.03 x 106 5) KohyamaN, Suzuki Y (1991) Analysis ofasbestos fibers fibers/dry gram in minimum and 49.84 x 10s fibers/ in lung parenchyma, pleural plaque and mesothelioma dry gram on average. These numbers were greater than tissues of North American insulation workers. Ann NY those seen in the general population. Occasionally the Acad Sci 643, 27-52. number of asbestos fibers in the mesothelial tissues 6) Suzuki Y, Yuen S, Ashley R, Calderaro A (1998) was larger than that of those seen in the lung. The Asbestos fibers and human malignant mesothelioma. number of chrysotile fibers was 18.1 times greater than In: Proceeding of the 9th International Conference on that of amphibole fibers in the mesothelial tissues taken Occupational Respiratory Diseases, Kyoto, Japan 13-. from 12 cases-in which both asbestos types were 16 October, 1997, eds. by Chiyotani K, Hosoda Y. detected. Aizawa Y, 709-13, Elsevier. 5) ' The majority (81.4%; 2347/2884) of asbestos fibers 7) Dodson RF, O' Sullivan MF, Huang J, Holiday DB, detected in the lung and mesothelial tissues were shorter Hammar SP (2000) Asbestos in extrapulmonary sites- than 5 /an in length. Asbestos fibers fit to Stanton's omentum and mesentery. Chest 117,486-93. hypothetical dimensions (>8.0 j-im jn length and 20.25 8) Goodwin MC, Jagatic C (1971) Asbestos and [m in diameter) were only 4.0% (116/2884), since the mesothelioma. Environ Res 3, 391--416. majority of these fibers were shorter (<8 /an) and thinner 9) Auerbach O, Conston AS, Garfinkel L, Parks VR, (<0.25 fm) fibers. Such short, thin asbestos fibers Kaslow HD, Hammond EC (1980) Presence of asbestos should not be excluded from those contributing to the bodies in organs other than the lung. Chest 77,133-7. induction ofhuman malignant mesothelioma, since they 10) Churg A, Wiggs B, Depaoli L. Kampe B, Stevens B are the major asbestos fibers detected in the lung and (1984) Lung asbestos content in chrysotile workers the mesothelial tissues in the mesothelioma cases. To assess asbestos fibers associated with the induction of with mesothelioma. Am Rev Resp Dis 130, 1042-5. 11) McDonald JC, Armstrong B, Case BW, Doell D, malignant mesothelioma, asbestos fibers in both the lung and the mesothelial tissues should be investigated. 6) The present study supports that chrysotile asbestos can induce human malignant mesothelioma. 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