Document g9EXGnL891qpv1vJqz1K72L3

The Interactions Between Asbestos Fibers and J\4etaphase Chromosomes ofRat Pleural Mesothelial Cells in Culture A Scanning and Transmission Electron Microscopic Study N. S. WANG, MD, PhD, M. C. JAURAND, ScD, L. MAGNE, BS, L. KHEUANG, BS, M. C. PINCHON, andj. BIGNON, MD From INSERM U 139, Creteil, Frame, and the Department of Pathology, McGill University, Montreal, Quebec, Canada Rat pleural mesothelial cells (PMCs) in culture at the exponential growing phase were exposed to 5 jig/ml of chrysotile (CH) or crocidolite (CR).asbcstos fibers: the cells and their chromosomes were studied 48 hours thereafter by light, scanning, and transmission elec tron microscopy (LM, SEM, TEM). PMCs phagocytized both CH and CR. Mild vacuolar cytoplasmic changes by LM and a few small surface blebbings by SEM were present, mainly in cells treated with CH. Metaphase chromosomes were well separated and re tained surface details by SEM in the control group. Chromosomes were frequently entangled with, adher ent to, and severed or pierced by long and thin curvilin ear CH with occasional chromatin fibers threading over the partly severed asbestos. Similar chromosomal changes were much less frequently found in CRtreated cells; TEM confirmed the same findings. CH and CR have different physicochemical properties and also appear to have direct, intricate, but different inter actions with chromosomes, as well as the cytoplasm, of PMCs. (Am J Pathol 1987, 126:343-349) EPIDEMIOLOGIC and experimental evidence indi cate that asbestos fibers induce mesotheliomas.1,2 The detailed mechanisms of their carcinogenesis are not completely clear: both physical and chemical proper ties of the fibers have been implicated.3,4 The two major classes of asbestos, the serpentines and the amphiboles, have different physicochemical properties and also appear to cause different incidences of meso theliomas in humans1,5,6; their tumorigenic effects in experimental animals are, however, similar.2,3 Rat pleural mesothelial cells (PMCs) in culture phagocytize both chrysotile (serpentine, CH) and cro cidolite (amphibole, CR) asbestos fibers.7 Vacuolar changes of the cytoplasm, prolonged population dou bling times, and polyploidy, however, have been found mainly in CH and less commonly in CR- lfcated cells, especially at a low dosage (5 /ig/ml cul ture medium).7 By light microscopy (LM), CH also induces chromosomal aberrations, including frag mentation and breaks in PMCs.8 CR also induces chromosomal changes in PMCs and other types of cells in culture but with a lower incidence than CH.9,10 These findings indicate that these two types of as bestos cause different degrees or patterns of cytoplas mic and nuclear changes in cultured cells and also suggest the possibility of a direct interaction between asbestos fibers and chromosomes.8 The present study investigated the metaphase chromosomes of PMCs in culture by light, scanning, and transmission electron microscopy (LM, SEM, TEM) for their possible inter actions with CH and CR. Materials and Methods Rat pleural mesothelial cells (PMCs) were cultured in Falcon flasks with or without glass coverslips as detailed previously.7 Briefly, PMCs were obtained and cultured in Ham's F-10 medium (Biopro, France) supplemented with 10% fetal bovine serum, 10 mM Supported by INSERM funds. Accepted for publication September 19, 1986. Address reprint requests to Dr. M. C. Jaurand, INSERM U 139, CHU Henri Mondor, 94010 Creteil Cedex, France. 343 344 WANG ET AL AJP February |ft, Hepes, 100 U/ml penicillin, and 50 //g/ml streptomy cin. The culture medium of PMCs, between 12 and 20 passages and 24 hours after plating, was replaced by a complete culture medium containing 5 /zg/ml of UICC chrysotile A or crocidolite. The asbestos fibers were dispersed by sonication in the complete culture medium for 5 minutes (50 kHz, 20 W) immediately before the medium change. In the control group the complete culture medium, without fibers, was re placed at the same time. After 48 hours of incubation, some of PMCs grown on coverslips were fixed with 3%glutaraldehyde in 0.1 M Na-cacodylate buffered solution for LM, SEM, and TEM. The coverslips were examined directly by an inverted phase-contrast microscope or stained rou tinely with the Giemsa stain for LM. For SEM, the cells were postfixed in 1% osmium tetroxide, dehy drated in graded acetone, critical-point-dried with C02, and coated with a layer of gold." For TEM, the cells were processed as above and then detached from thecoverslipin Epon.7 Sixty to 100-nm-thick sections were stained with lead citrate and uranium acetate. To study chromosomes in metaphase, mitosis was arrested by adding 0.4//g/ml of Colcemid to the cul ture medium for 2-3 hours. PMCs in mitosis on the Falcon flask were detached with 0.25% trypsin for 1 minute, incubated in 75 mMKCl solution at 37 C for 15 minutes, fixed with two changes of 3:1 methanol/ acetic acid, spread on glass slides or coverslips, and air-dried (detached preparation.8 PMCs in mitosis grown on coverslips were prepared similarly, except that the cells remained in situ throughout the process ing (in situ preparation). A portion of PMCs in meta phase was also detached and fixed in 3% glutaraldehyde as above for routine TEM. The coverslips with air-dried metaphase chromo somes were processed further, 2-7 days later, by washing with 1% Triton X-100 for 1 minute, incu bated with 0.025% trypsin for 30 seconds at room temperature, washed with three changes of phos phate-buffered normal saline solution, refixed with the glutaraldehyde solution, and processed for LM, SEM, and TEM as above. Results By LM all preparations showed near confluent cell growth with many cells in mitotic process. As de tailed in the previous study, there was no evidence of cytolysis, and asbestos fibers were readily found in the cytoplasm of most cells in both experimental groups.7 Mild to moderate vacuolar changes of the cytoplasm were found in some CH-treated but seldom in CRtreated or control cells. By SEM surface microvilli and pinocytic vesicles were moderate in number and unevenly distributed but appeared similar in all three groups. A few small surface blebbings of 0.5-1 // in size were, however, noted in some CH-treated but rarely in CR-treatcd or control cells. The CH fibers were mostly in groups, curvilinear, with fragmented or frayed ends, and partly enveloped by or inserted into the PMCs (Fig ures 1 and 2). The CR fibers, on the other hand, were mostly isolated and straight, showed less fragmenta tion, and were more likely to lie adjacent to or glued on, instead of inserted into, the PMCs (Figures 3 and 4). Both types of fibers were frequently long and thin and quite varied in size, ranging from more than I Op to less than 1 p in length and more than 1 // to less than 0.1 // in diameter. The variations in the size were more striking in CH than in CR. By TEM most intracellular fibers were membrane-bound, and more in number than suspected by SEM. Focal vacuolar changes and surface blebbings of the cytoplasm were mainly seen in the CH-treated cells. Intranuclear fibers were short and thin and were found only very rarely in CHtreated cells. Metaphase chromosomes, prepared by the de tached and in situ methods for SEM, appeared similar within the same groups and will be described together. The chromosomes in the control groups were mostly well separated, rod or oval in shape, with the detailed surface appearance of chromatin (Figures 5 and 6). Fibers connecting chromatids and adjacent chromo somes were frequently seen. Variations in the separa tion and surface details of chromosomes were, how ever, present between slides and within the same slide. Polyploidy was noted in all, but more in the CHtreated groups. CH fibers were found in 21 of 23 groups of metaphase chromosomes: CH fibers ap peared to sever, puncture, or adhere to and mingle with chromosomes, causing deformities (Figures 7 and 8). Occasionally chromatin fibers appeared to thread over the asbestos fiber in the partly severed chromosome (Figure 7). Usually chromosomal alter ations were multiple within the same cell: chromo somes that appeared to be uninvolved at low magnifi cation might have small, thin, and long fibers inserted in them (Figures 9 and 10). Contrary to CH, most CR fibers were located adjacent to or between metaphase chromosomes; obvious fiber-chromosome interac tions, similar to those observed in the CH group, were found only in 2 of 21 groups of metaphase chromo somes (Figure 11). By TEM asbestos fibers were present mostly adjacent to the chromosomes but oc casionally were also found within a chromosome^g' Jj***1 nd 2--Curvilinear chrysolite libers (CH) are frequently inserted into the mesothetial cells. Chrysolite fibers appear to fray easily (arrow). Microvilli pinocytic vesicles (small arrow) are more commonly found in the peripheral portion of the cytoplasm. (Figure 1. SEM. X6340: Figure 2, SEM ***00) Figures 3 and 4--Crocidolite fibers are mostly straight and isolated individually (arrows). They usually do not fray and more frequently appear *'**nt to. rather than inserted into or phagocytized by. the mesothefial cell in culture. (Figure 3, SEM, X5500; Figure 4. SEM, XI 9,000) 346 WANG ET AL Figures 5 and 6 -- By SEM chromosomes appear ovoid or rod-shaped, usually well separated from each other, in the control rat mesothelial celts. Surface details of chromosomes are discernible, as shown in Figure 6 from a chromosome (A) in Figure 5. Fate chromosome fibers (arrows) connecting chromatids or adjacent chromosomes are frequently present. [In situ preparation, SEM, Figure 5, X4880; Figure 6, X39.000) Figure 7--Long and thin chrysotile fibers intermingle with and also appear to sever, puncture, or adhere to chromosomes in multiple places [arrows). [In silu preparation, SEM, X6100) Figur* 8--Deformities of chromosomes, in addition to other alterations, shown in Figure 7, are prominent in areas with many chrysotile fibers. A repairlike change [arrow) can be seen over a chrysotile fiber that partly severs a chromosome. (SEM, detached preparation, X48.800) . ure 12), especially in the CH-treated group, both in the detached and in situ preparations. Discussion Chromosomal aberrations can be induced by a va riety of agents, and apparently also by a variety of mechanisms.12 Our SEM findings suggest that as bestos fibers can induced chromosomal changes by direct physical or mechanical damage. The severing or puncturing of chromosomes could occur between prometaphase and metaphase when the nuclear membrane breaks down and the condensed chromo somes go through a stage of violent rotating and oscil- I VoL 126 No. 2 SEM OF ASBESTOS INDUCED CHROMOSOMAL CHANGES 347 Figures 9 and 10 -- Fine chrysotile fiber (arrows) appear mainly scattered between chromosomes at a low magnification. A higher magnification of a ^omosome in Figure 9 (large arrow), however, shows a thin fiber that is inserted into the chromosome (Figure 10, arrows). The surface details o( "komosomes are discernible. (SEM. in situ preparation. Figure 9.X5850; Figure 10. X73.200) Figure 11 --Although the shapes ol chromosomes are ^Xhewhat altered, crocidolite fibers are mostly found adjacent to or between chromosomes. Direct interactions between the two. as seen in chrysotile (Figures * 6nd 8). are rare. (SEM, in situ preparation, X7800) Figure 12 -- Many small bundles of chrysotile fibers are seen around, in direct contact with (arrows), or '"thin, the chromosomes. The chromosomes (c) are loosened up into networks after the trypsin treatment. (TEM. X33.000) 348 WANG ET AL AJP February lating movements, to and fro between the two poles, before aligning themselves at the equator.13-14 The fre quently long and thin curvilinear CH fibers with frag mented, serrated, or frayed surfaces and ends would appear to become entangled easily with and cause more damage to chromosomes than the usually single, unfragmented. and straight CR fibers. The small inserted CH fiber in apparently normal chro mosomes (Figure 10) supported this assumption. The violent movements of chromosomes between promclaphase and metaphase and physical properties of the asbestos fibers, however, were probably not the only factors in their interactions. Despite the fact that CR does not fray or disintegrate as readily as CH does, as many long and thin CR fibers were found among or abutted to. but not interacting with, chromosomes as CH fibers. The surface properties of CH and CR are quite different. The surface of CH is rich in magnesium ions, positively charged, and high in electron transfer potential and catalytic activities to generate hy droxyl and superoxide radicals from H202. and shows high affinity to polar proteins, including chromosomes.415'17 The negatively charged surface of CR has much less of these activities and also causes less cytotoxicity and hemolysis than CH. The differ ent surface properties of CH and CR. therefore, may be responsible for the difference in their interactions with chromosomes. Although asbestos fibers damage cells and chromo somes by direct contact, other mechanisms of toxicity also exist. Toxic cytoplasmic changes, including va cuolization and surface blcbhing. as seen in our CHtreated cells, have been seen in the mesolhelial cells of amosite-exposed guinea pigs.18 Direct contact be tween mcsothclial cells and fibers was not seen in that study. Cytoplasmic vacuolization, is. therefore, sus pected to be due to chemically mediated stimuli cither directly from the fibers or secondarily from parenchyma-asbestos interactions.18 Abnormalities of chromosomes, in addition, could also be induced by the interference of microtubules, microfilaments, or other structural components of the spindle appa ratus by CH and CR at cell division.9 This last possi bility cannot be evaluated in our materials because the structural components of the spindles were dis solved away in the procedures of tissue preparation. Both physical and chemical properties of asbestos fibers, therefore, might directly or indirectly affect their affinity and interactions with chromosomes, as they do with the cell membrane. All our specimens went through the step of air-dry ing. which was necessary for the adherence, but more importantly, for the spreading of chromosomes on the coverslip.19 Air-drying is carefully avoided in any fine structural studies of biologic material because it obliterates structural details. This rule apparently does not apply in chromosomal studies,19 probably indicating the resilient properties of DNA after denaturation. The interaction between chromosomes and as bestos fibers might occur during the processing of the tissue, including air-drying. This was. however, un likely because of the same and consistent results ob tained by the detached and in situ processing methods in our and other studies.10 Threading of the chroma tin liber over the CH fiber (Figure 8) also argued fora prefixation event of probable chromosomal repair. Although the obvious interactions between asbestos fibers and chromosomes were readily discernible, precise interpretations of other chromosomal details were difficult, in part because of the inhomogcncity of the preparations. The application ofSEM in diagnos tic cytogenetic studies, however, appears feasible, with some improvements in the methodology. The role asbestos may play in human carcinogene sis is not completely clarified. Neither CH or CR in duces any detectable activities in common gene mu tation assays at doses that arc cytotoxic and cell-transforming.20 Asbestos is. therefore, proposed to act primarily as a promoter or cocarcinogcn.21" Cancer may also occur secondarily to the induction of inflammation or fibrosis by asbestos.23 Asbestos, however, induces mesothelioma in viva2-1 and chro mosomal aberrations and cell transformation, in w vitro systems, with or without other carcinogenic contaminants.8-20-24-25 Reduction of the fiber length of as bestos. however, reduces the incidence of the cell transformation,24 as well as the induction of mesothe lioma.2 These observations suggest that asbestos could still be a complete carcinogen and that the length, or the length and diameter (aspect) ratio, of the fibers could play an important role in carcinogenesis.3-4-20-24 Asbestos-mediated chromosomal changes have been suggested to play a role in asbestos carcino genesis.9-I0-20 Our findings of the direct and intricate interactions between the long and thin fibers and chromosomes could explain and support further this concept. Both CH and CR induced direct chromosomal al terations, but with a striking difference in the inci dence. The significance of this difference in the inci dence is unclear. The cytotoxicity of CH and that of CR are different, and the difference might be reflected in the incidence ofchromosomal alterations. A higher dose of CR, therefore, might be necessary for the same incidence of chromosomal alterations induced by a lower dose of CH. The dosage of CH and CR used in Vol I2 ' No. 2 SEM OF ASBESTOS INDUCED CHROMOSOMAL CHANGES 349 this study was, however, chosen with the considera tion of maximal exposure with minimal cytotoxicity for this cell model7: the dosage was high for both CH and CR, compared with conceivable situations in human exposure. ' The direct interaction of the asbestos fibers with chromosomes is. however, merely one of the many aspects of the inhalational carcinogenesis also gov erned by the physicochemical properties of the fibers. The same properties dictate the inhalability, deposi tion. migration, as well asdurability ofthe fibers in the lung: all of them are sequential in the eventual devel opment of cancer. References 1. Wagner JC. SleggsCA. MarchandP: Diffuse mesotheli oma and asbestos exposure in the North Western Cape Province. Br J Ind Med 1960, 17:260-272 2. Wagner JC. Berry' G: Mesotheliomas in rats following innoculation with asbestos. Br J Cancer 1969. 23:567 581 3. Stanton MF, Wrench C: Mechanisms of mesothelioma with asbestos and fibrousglass. J Natl Cancer Inst 1972. 28:797-821 4. Langer AM. Nolan RP: Physicochemical properties of minerals relevant to biological activities. In Vitro Ef fects of Mineral Dusls. Edited by, J Bignon. NATAO ASI series. Vol G3. Berlin. Springcr-Vcrlag, 1985. pp 10-24 5. Jones JSP, Pooley RD. Smith PG: Factory population exposed to crocidolite asbestos, a continuing survev. 1NSERM 1976. 52:117-120 ' 6. McDonald JC. Liddell FDK. Gibbs GW, Eyssen GE, McDonald AD: Dust exposure and mortality in chrv- sotile mining 1910-75. Br J Ind Med 1980. 33:11-24 7. Jaurand MC, Kheuang L, Magne L, Bignon J, Stoebner P: Effect of chrysolite and crocidolite on the morphol ogy and growth of rat pleural mesothelial cells. Env Res 1983. 30:255-269 8. Jaurand MC, Kheuang L, Magne L, Bignon J: Chro mosomal changes induced by chrysotile fibers or Benzo (3-4) pvrene in rat pleural mesothelial cells. Mutation Res 1986. 169:141-148 9. Hestcrberg TW. Barret J: Induction by asbestos libers of anaphase abnormalities: Mechanism foraneuploidy induction and possible carcinogenesis. Carcinogenesis 1985.6:473-475 ' 10. Sincock A. Seabright M: Induction of chromosome changes in Chinese hamster cells by exposure to as bestos fibers. Nature 1975, 257:56-59 U. Wang NS: The surface appearance of some lung tumors, mesothelioma, and their precursor lesions. Scanning Electron Microscopy 1980, 111:79-88 12. Savage JRK: Classification and relationships of in duced chromosomal structural changes. J Med Genet 1975, 12:102-122 13. Nicklas BB: Chromosomal movement: Current models and experiments in living cells. Molecules and Cell Movement. Edited by S Inoue, RE Stephens New York, Raven Press, 1975, pp 97 -118 14. Begg DA, Ellis GW: Micromanipulation studies of chromosomal movement. J Cell Biol 1979, 82:538 -- 554 15. Haringlon JS, Miller K, Macnab G: Hemolysis by as bestos. Environ Res 1971, 4:95-1 17 16. Mossman BT. Marsh JP: Mechanisms of toxic injury' bv asbestos fibers: role of oxvgen-frcc radicals.4 pp 66 74 17. Weitzman SA, Graceffa P: Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen per oxide. Arch Biochem Biophvs 1984, 228:373-377 18. Dodson RF, Ford JO: Early response of the visceral pleura following asbestos exposure: An ultrastructural study. J Toxicol Environ Health 1985, 15:673-686 19. Harrison CJ, Allen TD, Britch M. Harris R: High reso lution scanning electron microscopy of human meta phase chromosomes. J Cell Sci 1982. 56:409-422 20. Oshimura M. Hestcrberg TW. Tsutsui T. Barret JC: Correlation ofasbestos induced cytogenetic elfects with cell transformation of Syrian hamster embryo cells in culture. Cancer Res 1984. 44:5017-5022 21. Mossman BT, Light WG. Wei ET: Asbestos: Mecha nisms of toxicity and carcinogenecily in the respiratory tract. Ann Rev Pharmacol Toxicol 1983, 23:595-615 22. Topping CD, Nettcsheim P: Two stage carcinogenesis studies with asbestos in Fisher 344 rats. J Nall Cancer Inst 1980. 65:627-630 23. Haringlon JS. Allison AC. Badami DV: Mineral fibers: Chemical, physicochemical and biological properties. Adv Pharmacol Chcmother 1975. 12:291-402 24. HesterbcrgTW, Barret JC: Dependence ofasbestos and mineral dust-induced transformation of mammalian cells in culture on fiber dimension. Cancer Res 1984, 44:2170-2180 25. Paterour MJ. Bignon J, Jaurand MC: In vitro transfor mation of rat pleural mesothelial cells by chrysotile fibers and/or bcnzo(a)pvrcne. Carcinogenesis 1985. 6:523-529 Acknowledgments The authors are most grateful to Prof. J. Diebold and M. Reyes and Drs. F. Capron and P. Bruneval, Department d'Anatomie Pathologique, Faculte Broussais-Hotcl Dieu. Universite Paris, for permission and assistance in using their scanning electron microscopy facilities.