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OCT 11 2005 15:40 FR C.STI ICIST 348 613 952 9303 TO 12145201181 ' P. 03/16 SCF-FA-6506 i Chrysotile Asbestos in the Lungs of Persons in New York City Arthur M. Longer, PhD; Irving J, Setikoff, MD; and Antonio Sastre, New York , , i j When inhaled, chrysolite tends to split into unit fibrils 200 to 400 Angstroms in diameter, invisible with optical microscopy. Further, it Is altered chemically and physically in vivo. Therefore, at* tempts to identity unaltered chrysotile in the core of "asbestos bodies" have many pitfalls, especially when such attempts are limited by optical mi croscopy. High-magnification electron-microscopic examination of representative small samples of lung unequivocally showed chrysotile asbestos to be present in 24 of 28 consecutive New York city autopsy cases. Our data demonstrate that chrysotile fibers and fibrils are present in the lungs of New York city residents. Similar obser vations have been made in London. We antici pate that what is now known for New York and London wilt be found In other cities as well. Asbestos Bodies: Their Nature and Occur rence.--Soon after the recognition of ashestosis, examination of lung tissue obtained from individuals with this disease demon strated the presence of coated fibers which were first termed "curious bodies"1-2 and, subsequently, "asbestos bodies."3 Since these first individuals had been heavily exposed to asbestos at their work, it was assumed, un doubtedly with good reason, that the central fibrous core of these unusual structures was asbestos. Submitted for publication May 22, 1970; accepted June 26. From the Environmental Sciences laboratory. Mount Sinai School of Medicine of the City Univer sity of New York. Reprint requests to Environmental Sciences Labor atory, Mount Sinai School of Medicine, Fifth Ave and 100th St, New York 10P29 (Dr. Selikofl). When what appeared to be identical structures were next found in lungs of people not known to be occupationally exposed, the "asbestos-core" assumption was applied to these as well. It is of interest that the first descrip tion of such nonoccupational bodies in 1929 was made after examination of the lung tis sue of a person who had lived across die road from an asbestos plant.4 In recent years a number of investigations, starting with the Capetown study of Thom son et al,5 have demonstrated the presence of what appear to be "asbestos bodies" in the lungs (both in tissue sections and in ex pressed tissue fluid) of a large proportion of urban dwellers who had no recorded occupa tional exposure to asbestos. The percentage of instances in which they are found varies with the technique used in the investigation,6 the amount of lung tissue examined, and the diligence and persistence with which they are sought. The percentage of positive findings has been reported to be as high as 97%T or 100%.8 The question whether the cores of the "asbestos bodies" observed in these nonoccupationally exposed individuals are neces sarily asbestos has been revived. Although "asbestos bodies" occurring in the general population appear morphologically identical to asbestos bodies observed in asbestos workers, this does not guarantee that they necessarily have the same core. For epidemiological studies it would be useful to have unequivo cal identification of the fibrous core.9 This has not been easily accomplished because of technical factors.19 t I j j j "as ast lor esf dotifi rgk dobo scr "ft th< fib ha: the als the asl fui in' fir rei mi tic un lo' m; lir sir U) ha SO' ini ex q* W( ul th Wt ar to to as ta at ai si* lu e Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM P-EXHIBIT-249 i OCT 11 2005 15:41 FR CISTI 1CIST 613 952 9303 TO 12145201181 CHRYSOTILE ASBESTOS--LARGER ET AL P. 04/16 349 ' ! I ! Lungs k City e, New York jntical struc>f people not ised, the "as>lied to these first descrip>dies in 1929 the lung tisd across the ' j j 1 1 ; : nvestigations, dy of Thom e presence of dies" in the and in ex proportion of >rded occupale percentage found varies investigation,6 ined, and the i which they of positive x as high as cores of the lese nonoccus are necesred. Although t the general lly identical to estos workers, ey necessarily ndemiological ave unequivois core.9 This ied because of The possibility that what appear to be .usbestos bodies" may have other than an .sbestos core has been accepted almost as long as asbestos bodies have been known,11 especially when exposure to asbestos is not documented. When other exposures are iden- tified, appropriate designations are useful ("talc bodies," "graphite bodies," "fibrous glass bodies," etc). When exposure is not documented, one can consider implicit the bodies' nonspecifirity12-13 or even use a de scriptive term which is noncommittal, as "ferruginous body."11 In such circumstances, the uncertainty concerning the nature of the fibrous core of random "asbestos bodies" has hampered evaluation of the significance of their demonstration in the lungs of individu als in the general population. Do they or do they not reflect nonoccupational community asbestos exposure? Investigations have been pursued to obtain further information on the problem. These investigations have taken two directions: first, the study of asbestos bodies by more refined analytical techniques, as electron microprobe analysis15-16 or electron diffrac tion17; second, the appreciation that the unique morphology of chrysotile asbestos al lows its specific identification by highmagnification electron microscopy. Both lines of study have centered on chrysotile, since more than 90% of asbestos used in the United States is of this variety, and both have sought to answer the question: is chry sotile asbestos to be found in the lungs of individuals not known to be occupationally exposed to asbestos, and, if it is, how fre quently? It was recently reported that analyses were made of 28 asbestos bodies microman ulated from the lungs of 28 individuals in the general population of Pittsburgh.17 It was stated that chrysotile was not found in any instance, and it was inferred that asbes tos contamination of urban air was unlikely to be responsible for the formation of the asbestos bodies. Unfortunately, since few de tails were given, it is difficult to fully evalu ate the report. We have investigated the problem in another way and come to another conclu sion; chrysotile was specifically identified in lung tissue in 24 of 28 consecutive cases examined at autopsy in New York city. This suggests that it is commonly present in the lungs of urban dwellers at this time. Mineral Fibers: Their Occurrence in Lung, Recovery, and Species.---It has been frequently noted that there is lack of corre spondence between the ease of recovery of chrysotile asbestos from the lungs of experi mental animals and the difficulty experi enced in its recovery from the lungs of hu mans occupationally exposed to this mineral. This observation may be explained by a number of interrelated factors. The first factor is the amounts of asbestos involved. In a series of experiments by W. E. Smith, MD, and associates (unpublished data), 25 mg of chrysotile asbestos were injected intratracheally into golden Syrian hamsters. Six hundred and fifty-one days later, some of the animals were killed and lung tissue forwarded to our laboratory for mineralogical analysis. The tissue was ashed in a low-temperature ashing device, and the unashed residue was water dispersed and examined by means of polarized light mi croscopy and electron microscopy. Every where within the partially digested tissue debris, chrysotile fibers were observed. The optical properties remained remarkably simi lar to the natural material before injection; asbestos bodies tended to form about the thinner fiber bundles. By electron microscopy (Fig 1, A), a large number of thick fibers were found that were not broken open into individual fibrils. Several of the larger fibers demonstrated incipient coatings. Electron diffraction patterns obtained on the thicker fibers (Fig 1, B) indicated that the chryso tile had apparently not altered structurally. High-magnification examination of the fibers with the electron microscope demonstrated the ultimate structure in the form of the internal capillary (Fig 1, C). The recovery of chrysotile from experi mental animals is technically simple. The 25-mg do6e injected into the lungs of a ham ster of a young or a modest age (50 to 100-gm range of animal weight) is massive. Considering the weight of a hamster and its ratio of lung weight, the amount of chryso tile injected would equal several percent of its lung weight (This would correspond to huge amounts of chrysotile asbestos in the lungs of humans, in the order of 4 to 5 gm per human lung.) This massive dose, cou- Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM OCT 1 1 2005 15:41 FB C1ST1 ICIST 613 952 9303 TO 12145201181 j 350 CHRYSOTILE ASBESTOS--LANGER ET AL P. 05/16 pled with relatively short-term biological res been made with amphibole asbestos types, idence, insured recovery of characteristic eg, amosite.10 fibers. This chemical and physical instability cf In contrast to experimental animal chrysotile has made the quantitative extrac- studies, the amount of asbestos recovered tion of asbestos fibers from the lungs from lungs of persons occupationally ex of exposed individuals a problem that posed to the fiber has ranged from reported has not yet been completely solved. (See values of 0.6% to 0.001% of lung weight18-19 Nagelschmidt20 for discussion.) As an alter Nagelschmidt noted20 that the recovery of native, it has been considered useful to at chrysotile asbestos from lung tissue is always least obtain those fibers which had become low and recovery of amphibole asbestos gen coated (the "asbestos bodies"). While there erally high for human cases. are no data to quantitatively relate the pres The second reason for the difficulty in ence of such asbestos bodies to the remain extracting unaltered chrysotile fibers from der of the asbestos present, it has been human lungs lies in the fact that the nature thought that it may at least reflect, to an of chrysotile asbestos is such that it tends to undetermined degree, the presence of asbes break down chemically and physically after tos in general. Moreover, individual asbestos prolonged biological residence. This is espe bodies can be removed for analysis of their cially true of chrysotile fibers that have sepa coating and, more germane to the current rated into their fine unit fibrils. problems, of their central, fibrous cores. Chrysotile asbestos is chemically unstable A useful method to obtain individual as- even in distilled water.2122 An aqueous solu bestos bodies for study is to isolate them by tion with a pH below 10.8 will cause the micromanipulation.6 It should be noted, fiber to be leached of some of its magnesium. however, that micromanipulation must be Chemical instability in a biological environ done under the optical microscope, generally ment has been demonstrated several times. using low magnification. This inevitably pro Morgan and Holmes,38 following the duces a biased selection of large asbestos "daughter" decay products of radioactive bodies from lung tissue,1018-28 since by de chrysotile asbestos, showed that magnesium finition the only bodies that can be manipu was rapidly leached from the chrysotile lated are those that can be seen at such low fibers while in biological residence. This was magnifications! Micromanipulation of as- also indicated by the work of Langer et al.10 bestos bodies precludes small asbestos bodies Electron microprobe studies of chrysotile as from analysis. Micromanipulation of asbes- bestos fibers removed from the lungs of ham tos bodies from human lung tissue, therefore, sters indicated a wide range of magnesium leads to particle selection. On the basis of values in contrast to the narrow range in the the physical and chemical differences ob fibers before injection. Similar results have served experimentally and determined theo- been obtained in analyses of fibers and retically for chrysotile and amphibole types, bodies removed from lung tissue of a Cana it may be concluded that the large recover dian chrysotile miner. able fibers and bodies, selected by using the These findings are consistent with optical optical microscope, are more likely to be studies previously reported,20-34 in which amphibole asbestos than unaltered chryso chrysotile asbestos removed from the lungs tile. of workmen exposed to this fiber was found Timbrell et al27 observed that what is seen not to possess the same optical characteris by light microscopic methods often differs tics as did the materials to. which they were from what may be observed by electron mi- exposed. Rather, the findings reflected chem croscope methods and that respirable-size ically degraded fibers. fractions need not have the same size distri Marked physical changes in chrysotile bution or properties as those observed in the may also take place in vivo. The chrysotile gross overall sample. Similarly, Lynch and fiber composed of bundles of fibrils--breaks Ayer28 in describing the inadequacies of the open, and the individual unit fibrils are sepa imjpinger method of particle counting, con rated. This has been demonstrated by Suzu cluded that only about one in 100 fibers ki and Churg.25 No such observations have present in the air could be seen by light ; ' ' ( . | ' ' j | j I ! 1 \ : i j ! j Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM OCT 11 2005 15:42 FR CISTI ICIST 613 952 9303 TO 12145201181 P. 06/16 estos types, , istability of itive extrac- , the lungs oblem that solved. (See As an alter- useful to at had become While there ' ate the presthe remain- it has been eflect, to an 1 ice of asbesdual asbestos lysis of their the current ts cores. ndividual aslate them by 1 be noted, , j 1 ; | ion must be >pe, generally revitably proarge asbestos since by de n be manipu- j' n at such low ation of assbestos bodies don of asbes- sue, therefore, i the basis of ifferences obermined theophibole types, large recoverI by using the likely to be ' j i Itered chryso- it what is seen ; often differs y electron mirespirable-size me size distribserved in the y, Lynch and quacies of the counting, conin 100 fibers seen by light i ! Fig 1.--Chrysotlle extracted from hamster tung. Size range is smaller than light microscopy shows; fiber bundles are broken open (A, C). Chrysotlle electron diffraction patterns (B) are obtainable. Arrow (A) indicates where on asbestos body area was selected for diffraction; arrows (C) show characteristic capillaries. Arch Environ Health--Vof 22, March 1971 .^RECEIVED TIME OCT. 11. 2:43PM OCT 1 1 2005 15:42 FR C1ST1 ICIST 613 952 9303 TO 12145201181 352 CHRYSOTILE ASBESTOS--LANGER ET AL P. 07/16 Table 1.--Epidemiological Data on 28 Studied Cases Case Age 1 66 2 70 3 45 Sex F M M Occupation Domestic Truck driver Home repairs Cause of Death Polycystic kidneys Lung cancer Glomerulonephritis 4 82 M Tailor 5 80 M Linen supplier 6 53 M Bartender 7 75 M Porter, office 8 27 M Computer operator 9 46 M Appliance factory 10 48 M Plumber 11 61 M Attorney 12 77 M Lens polisher 13 81 M Tailor Arteriosclerotic heart disease Colon cancer Hypernephroma Hypertension Melanoma, eye Sepsis Chronic glomerulo nephritis Reticulum cell sarcoma Pancreatic cancer Pancreatic cancer 14 50 F 8roker IS 48 F Housewife 16 61 F Housewife 17 68 M Chauffeur 18 80 F Office secretary 19 42 F Grocery clerk 20 59 F Bookkeeper 21 79 F Housewife 22 58 F Stenographer 23 76 F Housewife 24 75 F Seamstress Myocardial infarction Myocardial infarction Lung cancer Heart block Myocardial infarction Cirrhosis of the liver Myeloid metaplasia Bronchopneumonia Stroke Myocardial infarction Glomerulonephritis 25 45 M Musician 26 72 M Salesman 27 64 F Housewife 28 76 M Dress manufacturer * NA, not available. Aortic Insufficiency Metastatic carcinoma, primary site not determined Cryptococcal meningo encephalitis Myocardial infarction Asbestos Exposure Comments 0 Used asbestos ironing pads 0 Some used Lived near shipyard in repairs 0 0 0 0 0 0 Material in trade 0 0 0 NA* 0 0 0 0 0 0 NA* 0 0 0 0 0 Asbestos ironing board covers Construction laborer, 1940"s . .. ... Oil burner repairs Home repairs, asbestos cement Asbestos ironing board covers; lived near shipyard ... -. ... ... ... Asbestos Ironing board covers Riveter, WWII: asbestos gloves .. Asbestos ironing board covers ... Once lived near yard with building materials ... ... 0 Asbestos ironing board covers 0 microscopic methods. This was determined by comparison of light microscopic samples with the same material studied by electron microscopy. Asbestos and Asbestos Bodies.--Thus, sev eral considerations explain the difficulty of demonstrating chrysotile asbestos in human lung tissue if one is limited to those particles visible by optical microscopy: (1) Chrysotile tends to break into fine fibrils, often smaller in diameter than 0.5/t, the limit of resolution of the light microscope, and so not visible by this instrument. (2) It may be inhaled as Abrils or fibers; the latter, once inhaled, may separate into the unit fibrils and thus be come "invisible" (3) Chrysotile's chemical and physical nature makes it subject to attack in tissue fluids, in contrast to amphibole asbestos. These factors make for "survivor populations" of the latter when fibers are extracted from human lung. All in all, with out the electron,; microscope, one must feel insecure when searching for chrysotile asbes tos in lung tissue, and techniques based upon light optical microscopy are studded with pitfalls. - In summary, chrysotile asbestos is unlike ly to remain as large fiber bundles in man. "Asbestos bodies" in man, that are easily visible in the light microscopic size range, are not likely to be chrysotile nucleated or, if so nucleated, not to be unaltered chrysotile. If I ! ' ! Arch Environ Health~Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM OCT 11 ^005 15:43 FR CISTI IC1ST ! . 613 952 9303 TO 12145201181 ' CHRYSOTILE ASBESTOS--LANGER ET AL P. 08/16 353 ents -onifig pads ard { board covers >orer, 1940`s rs sbestos cement g board covers; pyard ig board covers asbestos gloves ig board covers yard with rials >g board covers Table 2.--Comparison of Particle Counts Obtained by Light and Electron Microscopy Optical Microscopy Electron Microscopy Asbestos Bodies Case 0} Ashing KOH Digestion Asbestos Bodies Chrysotile Fibers Fibrils Other Fibers Thick* Thin* Platy Particles (Clays. Talc) 13 0 1 14 40 7l + 21 0 0 0 9 15 0 0 30 0 0 0 4 4 1 0 4 3 0 0 1 2 31 0 0 5 3 4 0 l 15 1 2 0 60 0 0 9 203 0 4 + 70 0 0 1 132 0 1 + 83 2 0 36 308 13 10 + 9 0 0 0 0 37 3 0 0 10 1 32 0 1 24 11 0 0 11 9 3 1 8 99 43 7 4- 12 1 0 0 5 114 14 11 0 13 0 2 O 3 17 O a 4- 14 0 0 0 5 74 4 i . + 15 1 2 0 2 . 55 9 4 4- 16 0 1 O 6 64 6 2 0 17 0 2 0 2 SS 2 0 4- 18 0 0 0 1 15 4 5 . + 19 1 0 0 0 7 22 0 20 1 5 1 5 23 0 3 0 21 0 0 0 9 14 3 1 + 22 2 1 0 3 18 8 2 + 23 O 0 0 6 10 25 3 + 24 0 4 O 5 45 2 0 25 1 4 0 16 254 76 + 26 0 1 0 3 23 9 0 + 27 1 0 0 4 77 1 0 + 28 0 0 0 18 168 13 3 + Thick fibers, those with length:diameter ratio < 10:1; thin fibers, length:diameter ratio > 10:1. ubject to atto amphibole for "survivor :n fibers are ! in all, withne must feel Table 3.--Distribution of Chrysotile in ____ 28 Cases Studied Group 1 2 3 4 5 "Blank grids" No. of Chrysotile Fibers and Fibrils 9 10-50 51-99 100-200 201 Cases 4 11 6 4 3 9 Men 3 5 1 4 3 Women 1 6 5 0 0 M| mately 45% have been found to have what appear to be asbestos bodies in their lungs. A still larger proportion had "uncoated" fibers present (I. J. Sehkoff, MD, unpub lished data). This is in keeping with our observation that asbestos bodies represent a small proportion of fibrous particles present in the lung tissue of asbestos workers. For the present investigation, 28 consecu tive cases were chosen (Table 1). All had ysotile asbes- been longtime residents of New York city; 3 based upon | we are to seek chrysotile, it would appear none had been asbestos workers. In each, the tudded with , necessary to include a search at a submicro lungs had been frozen at autopsy. From scopic level. each, inorganic materials were extracted for ios is unlike- study. dles in man. Present Study This investigation was undertaken to de it are easily termine the presence or absence of submi- ize range, are ated or, if so dirysotile. If Our laboratory has been concerned with an investigation of 3,000 consecutive deaths in New York city. Of this number, approxi croscopic uncoated asbestos fibers in the lungs of urban dwellers who died in New York city. Comparison of light microscopic Arch Environ Health--Vol 22, March 1971 ^RECEIVED TIME OCT. 11. 2:43PM OCT 1 1 2005 15:43 FR CISTI ICIST 613 952 9303 TO 12145201181 354 CHRYSOTILE ASBESTOS--LANGER ET AL P. 09/16 Fig 2.--Chrysotile morphology range in recovered mineral dusts. Rare well-formed fibrils with undeformed capillaries and thick, electron-dense walls without amorphous coatings, (A); more often, electron-dense wall encapsulated in amorphous coating (B); very often, fibrils with deformed internal capillaries and thin crystalline walls encapsulated in thick amorphous covering (C). findings with those by electron microscopy was made. Study was further undertaken to determine whether the fibers found were or were not chrysotile asbestos. Separation Techniques and Preparation. --From each of the 28 frozen lungs, 1 cc of tissue was cut and placed in a thick-walled centrifuge tube. A 40% KOH solution was added to the tube until the solution entirely covered the lung specimen (3 ml required). The centrifuge tubes were then placed in a hot water bath, and the water heated to boiling. Digestion was carried out for one horn: starting from the time the water began to boil. After one hour very little lung resi due remained in most instances. However, in some it was necessary to continue the diges tion for an additional hour. It was found that this was sufficient to complete the diges tion. The tubes were centrifuged at 15,000 to 17,000 rpm for approximately 30 minutes (head design indicates F > 30,000 g). The supernatant was next decanted, and the resi due washed with distilled water. Washing, centrifugation, and decantation were repeat ed 3 times until the residue was free of KOH. Examination of the supernatant with a polarized light microscope indicated that no optically visible asbestos bodies or fibers were present in the supernatant after any of the spinning periods. The residues were examined by polarized light microscopy. The mounting medium used in all cases was a highly viscous liquid which minimized particle migration20; scan ning was commonly done under 250X, 400X, and 500X magnification. High-magnification examination was occasionally undertaken at 1.000X. Each of the 28 cases examined mi croscopically showed some residual undigest ed organic materials present, despite the ap parently "complete" digestion. Tissue extracts were also prepared for study by electron microscopy. Small splits (approximately 1 mg) of the washed resi dues from the 28 specimens were pipetted into 28 smaller test tubes. Each of the latter 28 test tubes was filled with 2 ml of distilled water. The residue and medium were agi tated (for dispersal) for 30 seconds. Small proportions of the dispersant residues were removed, and one drop pipetted onto a poly vinyl methylal (Formvar) coated 200-mesh copper electron microscope (EM) locator grid. The drop of water was allowed to remain quietly on top of the grid for 15 minutes to allow the settling of solid materi als. At the end of that time, the liquid drop was drawn off with wetted filter paper. The amount of material that actually set tled out in this time onto the polyvinyl methylal grid was invisible to the unaided Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. II. 2:43PM e e n a ti e fi Si t! a t i OCT 11 2005 15:44 FR CISTI ICIST 613 952 9303 TO 12145201181 I CHRYSOTILE ASBESTOS--LANCER ET AL P. 10/16 355 ibrils with lore often, 3d internal by polarized ing medium 'iscous liquid ition20; scan250X, 400X, magnification ndertaken at ocamined miual undigestspite the ap- prepared for Small splits washed resi- * Fig 3.--Chrysotile fiber size range (thickness, length); fibers several fibrils thick (A to F) to fibril bundles (6, H), Fibrils range from well-formed (A) to highly deformed (C). Fiber length ranges from several thousand Angstroms to several microns. /ere pipetted of the latter ll of distilled im were agiconds. Small esidues were L onto a polyjed 200-mesh EM) locator ' allowed to grid for 15 soil'd materi- 3 liquid drop paper, eye. Only 9 of 248 squares of each grid were examined under the EM beam. The tech nique used in this investigation provides an answer to the qualitative question: is chryso tile a ubiquitous contaminant of the tissue examined? It is evident that only an in finitesimal fraction of lung tissue was scanned (conservatively estimated at 10- of the total lung burden). Quantitative studies are in progress; these will be useful for epi demiological investigations concerned with the biological significance of the current qualitative findings. the electron microscope (RCA EMU 3G). A fibril is defined as the smallest characteristic fiber unit of chrysotile asbestos, with a thick ness of 200 to 400 Angstroms. Scanning was done at magnifications of 31,000X with an acceleration voltage of 100 kv. Identification of chrysotile was confirmed using the 7X binocular attachment, or at 217,000X. Identification Criteria.--In order to justify identification of chrysotile asbestos as it ap pears after extraction from lungs, it was necessary to review and extend information concerning the morphological nature of chry actually set- Instrumentation.--Identification and loca sotile. The range of morphological character ie polyvinyl tion of asbestos fibers and fibrils required the istics of chrysotile was studied in consider the unaided use of the highest magnification available on able detail, and the findings are described Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM OCT 11 2005 15:44 FR Cl ST I ICI ST i 613 952 9303 TO 12145201181 P. f i -4 Fig 4.--Chrysotlle field et magnification as on EM viewing screen (insert); and photo graphic enlargement. Even with photographic plate's high contrast, lower-magnification count only 45 fibers and fibrils were counted; enlargement shows 59 actually present. elsewhere (A. M. Langer, PhD, unpublished data).80 Background Problems.--We have ob served that polyvinyl methylal grids which were scanned at the low magnification of 20.000X were void of "background" fibrils. However, other "blank" grids, scanned at higher magnifications of 31,OOOX were found to Sometimes contain a background of single, isolated chrysotile fibrils. The range of val ues observed in these control grids was zero to two fibrils per field, and no more than nine background fibrils for nine fields per grid scanned. Observations Lung Residues.--Light Microscopy.--The results of optical microscopic scanning for the 28 cases are given in Table 2. Two preparation techniques are compared, KOH Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM OCT 1 1 2005 15:45 FR CISTI ICIST > 613 952 9303 TO 12145201181 P. 12/16 ' *!' >hoto- atlon jsent. rids was zero o more than ie fields per jscopy.--The scanning for ible 2. Two ipared, KOH Fig 5.--Chrysotlle field In partially digested tissue. Although tissue was electron transmissive, count obtained on screen was 20 compared with count of 31 on photograph. AH short and thin fibrils were "missed" on viewing screen. and ashing (Table 2). A comparison of as bestos body count obtained with the low temperature ashing technique and the KOH digestion method shows 14 of 28 positive by the ashing technique and 13 of 28 positive by the KOH technique. However, agreement was present in only eight cases of both group totals. Combining both techniques, 19 of 28 cases, or 68% of the cases, were positive for defiinite asbestos bodies. Of the eight in agreement, the number of bodies obtained with the KOH technique was higher in five of tire eight cases. These observations were expected because the amount of tissue sam pled in each instance differed (1,000 cu mm, KOH; 17.5 cu mm, ashing); different por tions of the same lung were examined for each technique. . RECEIVED TIME OCT. Arch Environ Health--Vol 22, March 1971 2:43PM OCT 1 1 2005 15:47 FR CIST1 ICIST 613 952 9303 TO 12145201181 358 CHRYSOTILE ASBESTOS--LANGER ET AL P. 13/16 Lung Residues.--Electron Microscopy.-- A number of substances were observed to be present on the prepared grids: chrysotile as bestos (fibrils and fibers); polygonal platy particles (clay and talc minerals); electrondense fibrous particles (resembling amiphibole asbestos minerals); fibrous glass; as bestos bodies; and diatom fragments. The occurrence of chrysotile in our cases is shown in Table . 3. There are characteristic morphological ap pearances to the chrysotile particles found in human lung tissue. The fibrils range from those with relatively undeformed internal capillaries, thick crystalline electron-dense walls, and thin amorphous edges (Fig 2, A) to others with, thinner crystalline walls sur rounding slightly deformed capillaries which are in turn surrounded by thicker edges of amorphous material Fig 2, B. Some fibrils are highly deformed and are encapsulated by thick-walled amorphous material (Fig 2, C). Many of the fibrils observed appear de formed. The size of the fibrils found in human lung range from several hundred Angstroms to several microns in length. The average size of fibrils observed from human lung tissue appears to be 0.2/x to 0.3/x in length. We have also found chrysotile fibers to be present. A fiber is defined as any chrysotile aggregate greater than one fibril in thickness. The range in morphological characteristics of the fibers may be seen in Fig 3. It is of interest to note that some of the morphologi cal characteristics of the fibers are similar to those described by Suzuki and Churg in their study of the origin of asbestos bodies in hamsters.25 Tables 2 and 3 demonstrate that 24 of the 28 cases had asbestos fibers in their lungs in numbers higher than background counts could explain. Those with high fiber counts had high fibril counts as well. Three of the four "negative" cases were observed by light microscopy to possess asbestos bodies (Table 2). Chrysotile fibers and fibrils tend to occur in fields. Fibrils and small thin fibers, which apparently consist of only two or three unit fibrils still bonded, predominate the fields. We found only three cases to contain more than 200 chrysotile fibers and fibrils , in the grids studied by us (Tables 2 and 3). It may be of interest that we have observed these Fig 6.--Example of association of platy particles with chrysotile asbestos. Arrows Indicate location Of chrysotile fibrils. Note also presence of partially digested tissue. Fig 7.--Asbestos body observed by means of EM. Photomicrograph shows parallel bundle of chrysotile fibrils coated with electron-dense material which resembles incipient bodies described in Suzuki and Churg.B highly positive cases only among men. Scanning and Counting.--Photographic recording of EM fields consistently gave more accurate counts than simple visual scanning of the EM screen, a function of increased magnification, contrast, particle size, and search in. partially digested debris, Figure 4 demonstrates the difference in . , . i : O! :! an vvf gr sit or sc i.i 'I :h T sc pl cc v; F b rr a a c r i t. Y t I RECEIVED TIME OCT. Arch Environ Health--Vo( 22t March 1971 2:43PM OCT 11 2005 15:47 RR CISTI ICIST 613 952 9303 TO 12145201181 ! CHRYSOTILE ASBESTOS--LANGER ET AL P. 14/16 359 platy particles idlcate location nee of partially ' means of EM. lie of chrysotlle material which in Suzuki and A? 1 men. Photographic stently gave imple visual . function of "ast, particle jested debris, difference in \ (Hunting obtained at different magni- electron beam (compare with naturally oc r. .rations. At the low magnification shown in curring clay minerals). ' the upper right of Fig 4, the number of fibers In the extracts examined, only three of 28 i and fibrils counted was only 45. However, cases showed what appeared to be asbestos ' when the magnification was increased photo- bodies on the EM level. In each of these graphically, the count increased to 59. Occa- three cases, asbestos bodies were observed . sionally, even at the highest magnification, with the light microscope. Figure 7 shows organic debris (partially digested tissue) ob- one of these bodies. The asbestos body is . scures chrysotile from view. morphologically like asbestos bodies ob- Some fiber fields were counted by scan- served by Suzuki and Churg25 and appears ning and were then photographed; recount- to be nucleated on chrysotile. It is a paradox ing of the fields photographed indicates that that, although asbestos bodies were readily . they were initially "undercounted" (Fig 5). observed with the light microscope, very few The findings reported here are based upon were observed with the EM. The reasons scanning values, and are not corrected for may lie in the preparation technique: the photographic results; the fiber and fibril water drop is drawn off the EM substrate by I counts given, therefore, reflect minimum means of a wet filter paper; die moisture is I values. : quickly and strongly "sorbed" into the blot- There exists a close association of undi- ting material; nearly all of the larger parti- I gested tissue and chrysotile fibers and fibrils, cles are "pulled" along with the water, as I Chrysotile fibrils occur not only next to, but evidenced by brown discoloration of the | in the midst of, partially digested tissue, wetted filter paper. It is likely that only the j Fibrils inside the undigested tissue tend to smallest and most highly surface-charged | be thin-walled and relatively small.-Clay particles remain attached to the substrate. ' minerals are present in the undigested tissue Diatom fragments, other fibrous materials as well. Asbestos "counts" from photographs including fibrous glass and amphibole asbes- again demonstrated the presence of more tos types and sheet silicates were observed to chrysotile than could be counted directly on be present in the lung dust residues. The the screen. occurrence of fibrous particles (other than Figure 6 demonstrates the close associa- chrysotile and currently unidentified) is rela tion of chrysotile with well-formed polygonal tively frequent. Diatom fragments, although plates, morphologically similar to clay and occasionally observed, were rare, talc minerals. This association is striking because chrysotile fibrils tend to be both Comment ` "surface-" and edge-sorbed." Sheet silicates i tend to have a negative surface charge and a Unaltered chrysotile is uncommonly found positive edge charge. This suggests that one as the core fiber in asbestos bodies removed of the following mechanisms is at work: (1) from lungs of people in the general popula- The chrysotile fibrils possess a range of sur- tion. In an electron microprobe study of 16 face properties. (2) Different sheet silicates cases from which such asbestos bodies were are involved in the association. (3) Both 1 recovered and analyzed,18 none were chemi- and 2. (4) The association is artifact, cally equivalent to unchanged chrysotile. FifThe morphology of the sheet silicates indi- teen of 16 analyses, however, were consistent cates that they may be kaolinite (Compare with magnesium-leached chrysotile, and one | with kaolinite plates shown in Beutelspacher was consistent with amphibole (amosite). ; and van der Marei,81 Fig 19 to 32, pages 53 We have similarly examined asbestos bodies i to 59; Fig 259 to 260, Page 271; Fig 238, A from the lungs of a Canadian chrysotile and B, page 252. Several of the sheet materi- miner and from hamsters injected with chryals resemble bentonites. Fig 130, page 137, as sotile, all of which gave results consistent well as labile chlorite. Fig 152 and 153, page with magnesium-leached fibers. 161). This association may be important in This could well have been predicted, in determining the origin of the chrysotile that biological environments would seem ide- source. The nature of the pitted surface tex- al for this. Indeed, with the rapid splitting of ture is likely artifact produced under the the fibers into their unit fibrils, the surface Arch Environ Health--Vol 22, March 1971 (RECEIVED TIME OCT. 11. 2:43PM OCT 1 1 2005 15:48 FR CISTI ICIST 613 952 9303 TO 12145201181 360 CHRYSOTJLE ASBESTOS--LANGER ET AL P. 1 area is greatly increased, augmenting the leaching process (air-milling of the fibers increases the surface area from 4 sq m/gm to almost 60 sq m/gm) ,32 There are other good reasons why the large fibers which we inevitably choose by optical microscopy will tend not to be unal tered (or even, in many instances, altered) chrysotile. The optical microscope delivers a select, biased population. First, we can only study what the microscope sees--and it only sees large fibers, those thicker than 0.5yx in diameter. Chrysotile, it has been noted, tends to split into finer fibrils in biological environments, and degradation to smaller particles occurs. Amphlboles (anthophyllite, tremolite, amosite, croddolite) resist such physical and chemical attack. Therefore, selection of large asbestos bodies by means of light microscopy, resulting in a biased population of larger particles which were not altered in biological residence, stacks the analytical deck. These considerations indicate that study of asbestos bodies need tell us little about the chrysotile content of human lungs. Rath er, if we want to know whether chrysotile is present in lungs, we should look for chryso tile. In a sense, the wrong question has perhaps been asked; instead of "What is the nature of the core of the asbestos body?", it seems more profitable and more direct to ask the question, "Is chrysotile asbestos present in the lungs of urban dwellers?" The answer to the latter question is une quivocally "yes." Data presented here dem onstrate that tiiis is so in New York citv. Similar EM observations have been recorded in London, where Pooley et al88 have not only found chrysotile asbestos in almost 80% of their cases, but noted it to be the most common and most abundant of all fibers detected. We anticipate that what is now known for New York and London will be found in other cities as welL Conclusions In 28 consecutive cases of urban dwellers who died in New York city, electron microsoopy showed chrysotile asbestos to be pres ent in all examined (28 of 28). Of these, possibly four of 28 may have been made "positive" by the occurrence of background fibril contamination. It is noteworthy that asbestos bodies were found in three of four of these "negative" cases. Chrysotile asbestos often occurred in asso ciation with other substances, including pla- ty particles (clay or talc), fibrous glass, and, occasionally, diatoms. The question has been put forward: Is chrysotile asbestos present in the lungs of urban dwellers at this time? Our data dem onstrate that the answer is unequivocally "yes" in New York city. Similar observations have been made in London. We anticipate that what is now known in these two cities will be found in other urban areas as well. This study was supported in part by Public Health Service grant EC-00160. One of us (A.M.L.) received support by career award ES448-12 from the National Institute of Environmental Sciences. References 1. Stewart M3, quoted by Cooke WE: Asbestos dust and curious bodies found in pulmonary asbestosis. Brit Med J 2:678-580,1929. 2. Stewart MJ, Haddow AC: Demonstration of the peculiar bodies of pulmonary asbestosis ("asbestosis bodies") in material obtained by lung puncture nd in the sputum. J Path Bact 32:172, 1929. 3. Gloyne SR: The presence of the asbestos fiber in the lesions of the asbestos workers. Tubercle 10:404-407,1929. 4. Haddow, cited in Report of annual meeting of British Medical Association, Manchester. Lancet 2*230 1929. 6. 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Williams E: "Curious bodies" found in the lungs of coal-workers. Lancet 2:541-542,1933. 12. von Rflttner JR: t)ber Asbeatose- und Pseudoasbestosekfirperchen. Schweiz Z Allg Path 15:628-631, 1952. 13. Schiller F: AdsorpUonserscheimmgen an Steu ben in vivo: Pseudoesbeetose- und ScbaumankSr- Arch Environ Health--Vol 22. March 1971 RECEIVED TIME OCT. II. 2:43PM OCT 11 2005 15:48 FR CISTI ICIST 613 952 9303 TO 12145201181 T CHRYSOTILE ASBESTOS--LANGER ET AL P. 1 6/16 361 eworthy that three of four urred in assoncluding pla~ ua glass, and, ; forward: Is die lungs of >ur data demunequivocally r observations iVe anticipate ese two cities as as well. >y Public Health pport by career tal Institute of a WE: Asbestos ulmonary asbes- emonstration of bestosis ("asbes>y lung puncture 2:172, 1929. he asbestos fiber orkers. Tubercle mual meeting of lcheater. Lancet ;-erchen. Arch Gewerbepath Gewerbehyg 14:664-672, 22. Nagy B, Bates TD: Stability of chrysotile , 56. asbestos. 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Edward GH, Lynch JR: The method used by the U- S. Public Health Service for enumeration of asbestos dust on membrane filters. Ann Occup Hyg 11:1-6,1968. 30. Berkley C, Churg J, Selikoff U: Detection and localization of mineral fibers in tissue. Ann NY Acad Sci 132:48-63,1965. haled Particles and Vapours. New York, Pergamon 31. Beutelspacher H. van der Marel HW: Atlas of press Inc, 1961, pp 419-433. Electron Microscopy of Clay Minerals and Their 20. Nagelschmidt G: Some observations of the Admixtures. New York, American Elsevier Publish , dust content and composition in lungs with asbesto- ing Co Inc, 1968. sis, made during work on coal miners pneumoconi 32. Spell S, Leineweber JP: Asbestos minerals in osis. Ann NY Acad Sci 132:64-76, 1965. modern technology. Environ Res 2:166-208, 1969. 21. Hargreaves A, Taylor WH: An x-ray exami 33. Pooley FD, Oldham PD, Chang-Hyun UM, at nation of decomposition products of chrysotile (as al: The detection of asbestos in tissues, in Webster bestos) and serpentine. Mineral Mag 27:204-216, I (ed): International Conference on Pneumoconiosis, 1946. to be published. MacDonald RR: d. S Afr Med J itration of asbese techniques, in he Second InterEffects of Asbes58), to be pufa ille RTP: Fenuevhlence at ran`alth 17:327-383, nary ferruginous published, vironmental epis of nonoccupaxwure. Amer J of! IJ: Electron ies, in Webster I Pneumoconiosis, to be published, i" found in the 42,1933. >ae- und Pseudo Path 15:628-631, I I nungen an Stau1 Sehaumanktir- ( Arch Environ Health--Vol 22, March 1971 RECEIVED TIME OCT. 11. 2:43PM ** TOTAL PAGE. 16 **