Document 5n5yE19jGQEBk0YLrv9vQ5LR

rr i ENVIRONMENTAL HEALTH PERSPECTIVES, 1983{September); 51; 35 -_39; w- ft kthke N L00e ll HF.C 17 19H y. M. CLVRDON Environmental Health Perspectives VoL 51. pp. 35-39.1933 -j n % QC Some Observations on the in Vitro Cytotoxicity of Chrysotile Prepared by the Wet Dispersion Process O by I. P. Gormley,* R. E. Bolton,* G. M. Brown,* % On- J. M. G. Davis* and A. Wright* -*04 "`""I Saapfe* of the chryootik taken daring *ad after treat-cat by the wet Atapen--* peace-- have bee* tested far their cytotoxic effect in nitre and the reaalU ee--pared with hath a U1CC ehrysatiie A --i*t aad a daat prepared fraa a standard ehryeatiie textile yarm. Resvlta were abtaiaed fraa three dilfereat to oitre aaaay ayeteaa atibziag P388D,, V7P-4 aad AMS ceiia. A saaple which (till eaataiaed the wettiag agent need in the wet dispema* praceaa fatted U shew artnrity ta any af these assays. The other saaples, however, were all settee with these dasts abtaiaed by stilling the final prodset aad by saapling the sir af the factory ceaaieleatly peweiag signiftcaatiy -are eytotpeje than the standard ehryeatiie eaatraia. Preluaiaary resalts freffl r*r*|lel ia ataa stadv sag" gest that these a--pk are alas --r aetiee in proaaciBg cssthrUsssas in rata. Introduction In industrialized countries, man is continuously introducing alternative processes far the production and exploitation of new mineral substances. In many eases, the hazards to health resulting from the use of minerals are well established, but, when new minerals or processes are involved, their poten tially pathogenic effects are unknown. As epidemio logical studies often take many years to complete, during which time a considerable number of people are exposed to a possibly hazardous situation, much effort has been expended in investigating short term laboratory techniques that are capable of de tecting pathogenic minerals. These techniques have involved both m vivo and m vitro approaches. The effects of mineral dusts in animal*, mainly rats, have been extensively studied, with the ani mals being exposed to a given dust by inhalation or intratracheal instillation and then investigated for , disease over their lifespan. As macrophages are con . sidered to be the ceils most likely to first come into - contact with mineral particles in the lung, most of the w vitro assays have been based on the cytotox icity of a given mineral for these cells. Both animal `Institute of Occupations] Medicine. 6 Roxburgh Place. Edinburgh EH8 9SU. Scotland. macrophages (1-3) and mare recently permanent lines of maerophagelike cells U-6) have been used to investigate the cytotoxicity of mineral dusts and this effect seems to give an indication of the fibrotk potential of a given mineral dust In addition, it has been suggested that assays using phagocytic cells can also be used to give an indication of the malig nant potential of such dusts (7, 81 New farms of cy totoxicity assay have also been developed by Cham berlain and Brown (9) using other types of perma nent cell lines, and it is suggested that these may in dicate the carcinogenic potential of such dusts rather than their fibrogenicity. The wet dispersion process is now commonly used by industry to produce an asbestos fabric from chrysotile, and an experimental sample of this mate rial was found to be particularly cytotoxic in an tn vitro assay with P333D, cells, a mouse macrophage like cell line (J0L This material was subsequently found to induce mesotheliomas in rats at an earlier time than UICC chrysotile A dll It seemed possi ble that these phenomena might be related to wet dispersion processes in general and so the present study was undertaken using several different sam ples, some of which had been treated in order to ob serve the effects of extraneous chemicals used dur ing the chrysotile treatment. UCC 009029 A 0 4 3 74 1 f 36 GORMLEYETAL. Materials and Methods Dust Samples The wet dispersion (WD) process is one used by a number of manufacturers in order to prepare a par ticularly durable cfarysotile yarn that can be spun or woven into doth. Although the technique varies slightly horn manufacturer to manufacturer, the ba sic process consists of the disaggregation, using a wetting agent, of bundles of chrysotile fibers into a slurry consisting of predominantly single fibrils. This slurry is then passed through fine nozzles, and the addition of electrolytes at this point causes the fibrils to reaggregate with a very firm bonding be tween them. The resulting strands of yarn can then be woven or spun to produce asbestos textile prod ucts (12). The samples used in the present study consisted ofc (1) the original experimental sample tested previously (10) [original WD chrysotile (WDC)]; (2) a finished textile yarn from a long-run ning factory WD process which was milled in order to generate dust (milled WDCh (3) a sample from the milled process in (2) above from which the wet ting agent had not been removed (Unextracted WDCh (4) a sample of material from the process in (2) above from which all traces of the wetting agent had been removed by heat cleaning (heat cleaned WDCh (5) a dust sample collected from the factory air at the plant producing sample (21 This sample was collected from a section of the factory where only WDC products were handled (factory WDCX The WDC samples were compared to both a U2CC chrysotile A sample and to dust prepared from a standard chrysotile textile yarn made from exactly the same type of chrysotile used in the WD process. All of the samples tested were collected from an airborne dust cloud before use in either m vitro or m vivo studies. A number of these samples were extremely difficult to suspend in tissue culture medium due to their tendency to form tangled masses of fibers during the suspension process. The samples used in vitro were therefore ultrasonicsted and, in some cases, farced through a hypodermic sy ringe needle (21 gauge) in an attempt to disperse them. . Cytotoxicity Assays P388Dj Assay. The techniques used have been fully described previously (5, 101 In brief, 5 x 10* viable cells of the macrophagelike cell line P388D, were exposed to 10 and 50 pg/mL of the dust in 5 mL of culture medium. After 24 and 48 hr the cultures were assayed for cell viability by using the trypan blue exclusion technique, lactate dehydroge nase release Iff), Af-acety1-0-D-giucosaminidase (14), lactic add (25) and cellular protein content (16). The controls consisted of undusted cells and cells ex posed to 80 pig titanium dioxide/mL (nontoxic control) or 20 ^g quartz DQ^mL (17) (toxic control). Vt&4 Ceil Assay. The reduction in colony for mation by V79-4 cells was measured using the techniques described by Chamberlain and Brown (31 Giant Cell Formation in A549 Cultures. The for mation of giant cells in A549 cells was also mea sured by using the techniques described by Cham berlain and Brown (9). The measurements of ceil size were made by using a Graphic Instruments GDS1 image analyzer. However, the WD chrysotile samples were very cytotoxic when added to these cells, and so a higher cell inoculum (2j0 x 10* cells/25 cm1 tissue culture flask) was used with lower dust concentrations (25 and 50 fig/mLI In both the V79-4 and A549 assays, a sample of UICC croddolite was added as an additional control so that the results could be compared with those re ported by Chamberlain and Brown (9). Animal Experiments The techniques used in this part of the study have been fully described by Bolton et &L (11). The five WDC samples and the two chrysotile reference samples were each injected into groups of thirtytwo 10-week-oid male AF/HAN random-bred SPF Wistar laboratory rats. The elutriated dust samples were dry heat-sterilized at 60C for 30 min, mixed with sterile Dulbecco's phosphate buffered saline, and 2 mL of PBS containing 25 mg of the dust were then injected intraperitoneally. The animals were observed daily and killed when distressed or mori bund. Results and Discussion The mean results from a series of three indepen dent experiments using the P388D, assay are shown in Table 1. The results for the biochemical estima tions were in good agreement with those for the cellular viability as reported previously (10, 18), and so only the viability results are reported. These are presented as the percentage of those found for the titanium dioxide controls. It can be seen from Table 1 that, although there were slight differences (prob ably due to experimental variation) in the ranking of the dusts according to their cytotoxicity, four out of the five samples of WDC were mare cytotoxic than the UICC chrysotile control The only excep tion to this was the sample of unextracted WDC. It was noteworthy that three samples of the finished product--heat cleaned WDC, milled WDC and the environmentally collected factory sample--were al ii n 40-7 ? UCC 009030 Time, hr 24 48 IN VtTRO CYTOTOXICITY OF CHRYSOTILE Tabic 1. Order af cytetoxieity of WOC sanpiot baaed an viability carnation*. Sample (10 fig/mL) Unextraeted WDC UICC ehryaotile A Milled chrvsotile Original WDC Heat-cleaned WDC Factory WDC Milled WDC Unextraeted WDC UICC ehryaotile A Milled ehryaotile Original WDC Factory WDC Heat-cleaned WDC Milled WDC Viability 96 14 84 6 79 a 10 68 a 11 62 a 10 50 a 8 44 a 6 75 a 4 52 a 6 43 a 12 30 11 25 a 3 25 a 5 22 a 4 Sample <50 pgimL) Unextraeted WDC UICC ehryaotile A Original WDC Milled ehryaotile Milled WDC Heat-cleaned WDC Factory WDC Unextraeted WDC UICC chrysotile A Milled chrysotile Original WDC Factory WDC Milled WDC Heat-cleaned WDC Viability* 87 a 9 47 a 8 34 a 3 34 a 14 25 a 4 22 a 7 20 a 8 59 a 21 a 18 a 17 a 10 a 8a 6a 7 5 7 5 3 2 1 37 Viability - too. live cells for test duaUno. live cells for TiO, control) x 100% * SD. The dusts are ranked in order of increasing cytotoxicity. Table 2. Cytotoxicity af WDC samples by the V79-4 aasay. Sample LD_ pg ol dust* Unextraeted WDC Titanium dioxide Original WDC Quartz DQU Heat-cleaned WDC UICC chrysotile A UICC croddolite Milled ehryaotile Factory WDC Milled WDC 87j0 a 19.1 84.8 a 14J 46.2 a 39.2 28J a 12.4 20.4 a 15^ 13J5 a 5.6 11.6 a 2JS 9.7 * 4.6 8.4 a 53 5.0 a 3.4 The concentration of dust causing a 50% reduction in the cloning efficiency of V79-4 cells. ways the most cytotoxic, regardless of time or dose, and these three samples reduced the viability to ap proximately half of that seen with the UICC chryso- tfle sample. . The concentration of dust causing a 50% reduc tion in the cloning efficiency of the V79-4 cells (9) was calculated for each dust sample from five inde pendent experiments, and the results are shown in Table 2. The results are broadly in agreement with those obtained using the P388D, assay in that the unextracted WDC sample was the least cytotoxic, whereas the factory and milled WDC were the most cytotoxic. The result for UICC croddolite was in good agreement with that originally described by Chamberlain and Brown (9), who reported an LD of 9 ptgfmL for this substance. However, quartz DQK was always found to be active in this system with a mean LD. of 2L8 pg/mL, in contrast to the results reported for Min-U-SO and South African silica re ported by Chamberlain and Brown (9). The results obtained when the samples of WDC were tested for their ability to cause giant cell for mation in A549 cells are shown in Tables 3 and 4. Although these large cells have been described as "giant cells" (9), they are not considered to be of shnibu- origin to the classical foreign body giant cell commonly found associated with dust m vivo. These results, which are taken from a single representa tive experiment in which 100 cells were measured for each sample, are broadly in agreement with those reported above with the unextracted sample having the least effect while the other four samples caused more giant cell formation than UICC chrysotile A or milled chrysotile. The quartz sample was not active in this assay system and so was in agree ment with the results reported by Chamberlain and Brown (9). In contrast to their results, however, it should be noted that UICC croddolite had only a minimal effect in our experiments. This result was to be expected in view of the increased cell number and decreased dust dose which was used in this study to overcome the marked toxidty of the WDC samples. When the results of all three assay systems were considered, the unextracted WDC sample was al ways found to be the least aetive of the WDC sam ples. The remaining four samples were more active than either UICC chrysotile A or nulled chrysotile in the P388D, and A549 assay, and two samples -- factory and milled WDC--were more cytotoxic than the standard chrysotile samples in all three assay systems. Only preliminary data from the animal experi mentation are currently available far comparison with the cytotoxidty results. The mean induction period for the first 15 tumors after intraperitoneal injections of 25 mg of the samples is shown in Table 5. These data suggest that three of the WDC sam ples induced tumors at a faster rate than either UCC 009031 A 0 4 3 7 fj 38 Sample (25 uftmL) Titanium dioxide Unextrmcted WDC Quarts DQ Undustad control UICC croeidoiite UICC chrysotile A Milled chrysotile Heat-cleaned WDC Factory WDC Milled WDC Original WDC Sample (50 pg/mL) Quartz DQa Titanium dioxide Undnstod control UICC croddoUte Uaoxtraetod WDC Milled chrysotile UICC ebrysotile A Factory WDC Hoat-eieanod WDC Original WDC Milled WDC GORMLEYETAL. Table 1 Giant cell formation in A549 cells. Sise. pin x SD 17.78 2.40 1843 341 18.18 x 348 1842 249 1942 346 2048 449 2148 x 549 22.71 x 5.09 2448 x 5.71 2441 x 542 25.08 x 645 Colls in size range. % 0-25 pm 25-40 pm > 40 pm 100 0 98 2 98 1 99 1 95 5 83 17 78 22 74 26 56 44 60 39 56 41 0 0 1 0 0 0 0 0 0 1 3 Table 4. Giant cell formation in A549 cella. Size, pm x SD 1744 x 548 1740 L94 1842 x 249 1949 x 343 19.75 x 4.41 22.46 x 449 2341 5.43 23.49 x 646 2449 x 549 24.72 x 642 2846 x 742 Cells in size range. % --3^3. ---------- siflrsm---------- > 40 pm 99 1 99 1 99 1 93 7 92 7 72 28 69 31 63 36 57 41 62 35 44 50 0 0 0 0 1 0 0 1 2 3 6 Cells >25 pm. % 0 2 2 1 5 17 22 26 44 40 44 Cells > 25 pm. % 1 1 1 7 8 28 31 37 43 38 56 Table 5. Preliminary data on mesothelioma prodaction by WDC samples in rata. Sample Heat-cleaned WDC Milled chrysotile Unextrmcted WDC UICC chrysotile A Milled WDC Factory WDC Original WDC Mean tumor induction time far 15 tumors in each group, days 367 357 355 354 300 282 276 milled chrysotile or UICC chrysotile A. and two of these, milled and factory WDC, caused a marked re sponse in all three cytotoxicity assays. The third sample, original WDC, was more active than the chrysotQes in two out of the three assay systems. The n two results for the remaining two WDC samples were not in agreement with the m vitro data, as heatrdeaned WDC which was active m vitro had the longest tumor induction time, and the unextracted sample, which was the least active of the WDC samples in vitro, produced mesotheliomas in rats after the same approximate period as the standard chrysotile samples. Obviously a full com parison of the in vivo and in vitro data cannot be made until the in vivo experiments have been com pleted. There are, however, possible reasons for the discrepancies. The unextracted WDC was difficult to suspend for the m vitro work, and separation of the fibers may have been incomplete, whereas ade quate separation for tumor induction may well have occurred during many months of residence in the animal tissues. Alternatively, the wetting agent ma terials present in this sample could have a protec tive effect in the short-term tn vitro tests but be re moved during the longer-term in vivo experiments. The discrepancy between the m vitro and in vivo data for beat-cleaned WDC may be related to the heat treatment, as there is evidence to suggest that this was earned out at a temperature high enough to break down some of the chrysotile to forsterite. Although chrysotile samples heated to 850C can exhibit a lower cytotoxicity (10) and a reduced car cinogenic potential ill), these two effects may not be directly related. The carcinogenic potential may be reduced after a level of heating which still leaves the dust with the ability to damage cells m vitro. An alternative explanation may be that the heat cleaning process alters the solubility of the chryso tile. The apparent enhancement of both cytotoxicity and mesothelioma production resulting from the UCC 009032 AU4377 IS VITRO CYTOTOXICITY OF CHRYSOTILE 39 treatment of chrysotile by the wet dispersion pro cess is difficult to explain. It does, however, seem possible that the bonding between the chrysotile fi brils, while strong under normal conditions, be comes weak in the presence of biological fluids. Thus, within the body or in tissue culture medium, the bundles of fibrils split up into their individual components more rapidly than bundles of conven tionally treated chrysotile. The number of individu al fibrils available for reaction with cells either m vivo or m vitro might, therefore, be far greater than with the same mass of conventional chrysotile. There is evidence to suggest that the manufac ture and use of wet dispersed chrysotile products may be associated with a relatively low level of res pirable dust. They are, therefore, considered to pose a reduced health hazard when compared to stan dard chrysotile materials. Evidence from the pres ent studies suggests, however, that if dust is pro duced from wet dispersed chrysotile, then it may be more dangerous than similar amounts of standard chrysotile dust. We are grateful to Mrs. J. Slight for her skilled technical assistance and to the Asbestos Research Council for financial support. REFERENCES 1. Marks, J. The neutralization of silica toxicity in vitro. Brit. J. Ind. Med. 14:81-84 (1957). 2. Marks, J. and Nagelschmidt, G. Toxicity of dust with use of the dehydrogenase technique. Arch. Ind. Health 2th 383-389(1959). 3. Vigliani, EL C- Pernis. B~ and Monaco. L. A study of the action of quartz particles on macrophages in vitro. In: Inhaled Particles and Vapours (C. N. Davis, Ed.), Pergamon Press, London, 1961. pp. 348-353. 4. Wade, M. J. Lipkin. L. E,, Tucker, R. W,, and Frank, A. L. Asbestos cytotoxicity in a long term macrophage like cell culture. Nature 264: 444-446 (19761. 5. Gormiey. L P,,Collings, P,,Davis. J. M. G-, and Ottery. J. Investigation into the cytotoxicity of respirable dusts from British Collieries. Brit. J. Exp. Pathol. 60: 526-536 11979). . 6. Daniel. H. and Le Bouffant. L. 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W. l-( + )'Lactate- Determination with lactate dehydrogenase and NAD. In: Methods of Enzymatic Analysis, 2nd ed. (H. U. Bergmeyer. Ed.). Academic Pres*. New York. 1974. pp. 1464-1468. 16. Chaykin, S. Biochemistry Laboratory Techniques. Wiley, New York. 1966, p. 17. 17. Robock, K. Standard Quartz DQ12 < 5f<m for ex perimental pneumoconiosis research projects in the Federal Republic of Germany. Ann. Occup. Hyg. 16: 63-66 (1973). 18. Gormiey. L P- Wright. A. CoUings. P,, and Davis. J. M. G. The cytotoxicity of UICC and modified asbestos fibers tn vitro. In: Biological Effects of Mineral Fibers, Vol. 1 (J. C. Wagner, Ed.). IARC Scientific Publication No. 30. Lyon, 1980, p. 427-434. , UCC 009033 ii O