Document a43d1NmzbNoEe7nmoMDzKDGJy

Toxic, in Vilro Vol. 7, No. 1, pp. 7-14, 1993 Primed in Great Britain. All rights reserved 0887-2333/93 S6.00 + 0.00 Copyright 1993 Pergamon Press Ltd IN VITRO RESPONSE OF RAT PLEURAL MESOTHELIAL CELLS TO TALC SAMPLES IN GENOTOXICITY ASSAYS (SISTER CHROMATID EXCHANGES AND DNA REPAIR) S. E n d o - C a p r o n *, A . R e n ie r *, X. J a n s o n , L. K h e u a n g * a n d M . C. J a u r a n d *}: INSERM-U139, Laboratoire de Toxicologie Cellulaire et Molculaire de l'Environnement, CHU Henri Mondor, 94010 Crteil and fLaboratoire d'Etude des Particules Inhales, DASS, 11 rue Georges Eastmann, 75013 Paris, France (Received 16 April 1992; revisions received II August 1992) Abstract--The genotoxicity o f three samples of talc has been determined using in vitro cell systems previously developed for testing asbestos fibres. The talc samples used consisted of particles of respirable size in order to test the effect of particles likely to be deposited in the lung. Genotoxicity was tested in cultures of rat pleural msothlial cells (RPMC) using genotoxicity assays for unscheduled DNA synthesis (UDS) and sister chromatid exchanges (SCEs). The effects were compared with those obtained with negative controls (attapulgite and anatase) and positive controls (chrysotile and crocidolite asbestos). In contrast to asbestos, none of the talc samples, nor the negative controls, induced enhancement of UDS or SCEs in treated cultures in comparison with the untreated cultures. INTRODUCTION Talc is a mineral commonly used in various industries including the ceramics, paper, plastics, paints, pharmaceutical and cosmetics industries. It is a magnesium silicate of similar chemical composition to chrysotile asbestos fibres but with a different structure. 1ARC has evaluated the biological efTects of talc (1ARC Working Group, 1987); according to their findings the results obtained in previous exper iments in vivo and in vilro were inadequate to evaluate the carcinogenicity or genotoxicity of talc because of the limited number of studies. Data from animal studies did not show an excess of pleural sarcomas or mesotheliomas after the intrapleural administration of talc (Endo-Capron el al., 1990; Stanton et al., 1977; Wagner el al., 1977). From data from epidemio logical studies, the IARC Working Group (1987) concluded that it was possible that carcinogenicity could result from exposure to some specific samples found to be associated with fibrous tremolite. How ever, epidemiological studies have been updated recently and no evidence of increased risk of lung cancer has been found (Weill et al., 1990). Some authors have examined the association between genital talcum powder exposure and ovarian cancer (Harlow and Weiss, 1989); no appreciable altered risk was {To whom correspondence should be addressed. Abbreviations: FCS = foetal calf serum; HU = hydroxy urea; RPMC = (rat pleural mesothelial cells); SCE = sister chromatid exchange; TEM = transmission electron microscopy; UDS = unscheduled DNA synthesis. observed following exposure to baby powders, which are reported to contain only talc, but an increased risk was associated with the use of talc-containing powders, that is, also containing deodorizing sub stances or a variety of other free and bound silica (Harlow and Weiss, 1989). The present experiments were designed to deter mine whether talc particles of respirable dimensions exerted a genotoxic effect on cultures of rat pleural mesothelial cells (RPMC). Pleural mesothelial cells are an important target for fibrous particles inhaled from our environment and can be used as test models to determine the in vilro effects of particle matter. In addition, talc has been used to overcome pleural effusion (IARC Working Group, 1987). It is therefore of interest to determine the effects of pure talc on RPMC. In previous experiments, we have used RPMC to study the genotoxicity of asbestos fibres. Enhance ment of unscheduled DNA synthesis (UDS; Renier et al., 1990) and sister chromatid exchanges (SCE; Achard el al., 1987) have been observed in cultured RPMC after exposure to chrysotile or crocidolite fibres, but not after exposure to a non-carcinogenic sample of attapulgite. Identical tests were applied in this study in order to determine the efTects of pure talc. m a ter ia ls and m e t h o d s Particles and test compounds. Three samples of European talc provided by Eurotalc (Brussels, Belgium) were studied. One sample each of French talc (no. 7841). Italian talc (no. 5726) and Spanish 7 8 S. Endo-Capron et al. talc (no. 5725) have been tested. Samples contained 90-95% of talc, the other compounds being chlorite and dolomite. Anatase (a gift from P. Sebastien, Cerchar, France) and attapulgite (from Mormoiron, France) were tested as negative reference particles; Rhodesian chrysotile and crocidolite from the Union Internationale Contre le Cancer (UICC) as positive reference particles. The particles were dispersed in culture medium at a concentration of 560/ig/ml by sonication for 5 min (20 KHz, 3 W). Chemicals used as controls, mitomycin C (Choay, Paris, France) and K2C r0 4 (Aldrich Chemical Co., Milwaukee, MO, USA), were solubilized in water and in culture medium, respectively. Transmission electron microscopy (TEM). Particles at a concentration of I00^g/m l were dispersed in culture medium. An aliquot of the suspension was filtered through a 0.40-pM pore size Nuclepore filter. The filters were transferred to electron microscopic grids and dissolved according to the method routinely used in the laboratory (Sbastien et al., 1978). The size of the particles was determined following a systematic scanning of the grid at two magnifications ( x 33,000 and x 26,000). Cell culture. Rat pleural msothlial cells (RPMC) were obtained as described elsewhere (Jaurand et al., 1981). Briefly, primary RPMC cultures were obtained by scraping the parietal pleura and allowing the cells to grow in multiwell tissue culture plates. The cultures were maintained in complete medium (i.e. Ham's F10 medium; Flow Laboratories, Irvine, Ayrshire, Scot land) supplemented with 2 mM-L-glutamine (Flow Laboratories), 1mM-vitamin C (Sigma Chemical Co., St Louis, MO, USA), IOitim-HEPES (Seromed, Berlin, Germany), 10% foetal calf serum (FCS; from Boehringer, Mielan, France), 100 U penicillin ml and 50 pg streptomycin ml (both antibiotica from Flow Laboratories). When the cells reached confluence they were subcultured. From passage 5, RPMC were subcultured approximately every week by standard trypsinization and used between passages 5 and 15. Ultrastructural analysis. 24 hr after plating, talc was added to the RPMC in the tissue culture dishes at a concentration of \0 pg/cm2. Electron micro scopic studies were carried out according to standard methods previously described (Jaurand et al., 1979). The solid compound concentration was expressed as pg/cm2to take into consideration the particle settling; in these culture conditions, 1pg/cm2 is equivalent to 5 pg/m\. Unscheduled DNA synthesis (UDS). RPMC were cultured in 24-well cluster dishes (Falcon, France); 8 x I04cells were plated per well in complete medium. Ceils reached confluence after 4 days of incubation. The medium of the confluent culture was replaced with RPMI (Flow Laboratories) containing 1% FCS (Boehringer), 5 mM-hydroxyurca (HU; Sigma) to arrest cells in G l, 100 U penicillin/ml and 50 /zg streptomycin/ml (both from Flow Laboratories). The cells were incubated for 24 hr in a humidified atmos phere of 5% C 0 2 in air at 37C. The cells were then treated for 24 hr with the indicated dose of particles (1 pg/cm2 is equivalent to 5^g/m l) in 1% FCS medium containing 5mM-HU and [methyl-2U] thymidine (Amersham, les Ulis, France) at 4 ^Ci/ml. The amount of radioactivity incorporated into DNA was determined as described elsewhere (Renier et al., 1990). Six wells were used per treatment. After treat ment, cells were washed three times with phosphate buffered saline. Acid-soluble material was removed by rinsing with 10% cold trichloracetic acid for 10 min and incubated in a mixture of 0.2 M-NaOH and 1% sodium dodecyl sulphate. Aliquots of 200 p I were mixed with scintillation fluid (Pico-fluor, Pack ard) and radioactivity was measured with a Beckman LS 6000SC scintillation counter. Cell DNA content was determined according to West et al. (1985) in separate wells treated with the minerals in the same conditions as described above. Results are expressed as dpm/^g DNA. All studies were carried out with coded samples. Sister chromatid exchanges (SCEs). RPMC were plated at a density of 2 x 106 cells per 75-cm2 flask in RPMI medium supplemented with 10% FCS. Cells were treated cither with test chemicals or with several concentrations of particles plus 5 pg bromodeoxyuridine/ml 24 hr after the plating of the culture. In these culture conditions, 1pg/cm2 is equivalent to 7.5 pg/m\. The cultures were incubated with the test compound at 37C for 48 hr in the dark. 2 hr before harvesting cells, colchicine (Sigma) at a final con centration of 0.2/ig/ml was added to each culture. Mctaphase cells were then detached with 0.25% trypsin (Eurobio, Paris, France), collected in 15-ml corex tubes and centrifuged at I500rpm for 7 min. The supernatant was removed. Cells were treated with 0.075 m-KCI at 37C for 30 min before fixation in methanol-glacial acetic acid (3:1, v/v). The fixative was changed three times and the last fixation step lasted for one night. The cell suspension was dropped onto an ice-cold slide. Cells were stained by the fluorescence plus Giemsa technique (Perry and Wolff, 1974). 30 metaphases exhibiting 37-42 chromosomes were counted per assay. All studies were carried out with coded samples. Statistical analysis. The significance of UDS data was evaluated using Student's r-test. The number of SCEs observed in treated cell cultures was com pared with that in the untreated cultures using the Mann-Whitney test. RESULTS TEM study o f particles The size distribution of the talc samples is reported in Fig. 1. The characteristics of the talc, anatase, crocidolite and chrysotile particles are reported in Table 1. The mean size of the three talc samples was in the ranking order 5725 = 7841 < 5726. The number Percentage of particles * P W tJ * >m.->^rv:. '<yvft * ? ' - Genotoxicily of a talc in msothlial cells It Table I. Characteristics of the particle samples Sample Mean length No. of panicles///g No. of particles of length > 4 n m//t g Talc ^725 Talc 5726 Talc 7841 Anatase Crocjdolite UICC Chrysotile UICC 2.6 4.0 2.6 0.7 3.1 3.2 13.0 x 10* 9.8 x 10* 3.3 x 10* 2.2 x 10' 3.0 x 10* 1.1 x 10' 2.1 x 10* 2.8 x 10* 0.4 x 10* 0 5.1 x 105* 2.8 x 10** Fibres having a diameter < 1.5 /Jill of particles per unit weight was in the ranking order 5725 > 5726 > 7841. Therefore, the number of par ticles having a size greater than 4 /tm is approximately the same in two samples and smallest in sample no. 7841. TEM study showed that none of the three samples of talc contained asbestos fibres (Plate 1). The rtiean length of crocidolite and chrysotile fibres is between those of talc samples 5725 and 7841 and that of sample 5726. The number of crocidolite or chrysotile particles having a length greater than 4 /rm is 2 0 f100 times more than that of talc. Anatase is a very imall particle having an average size of less than 1jim with no particle larger than 4/tm . Table 3. Unscheduled DNA synthesis in pleural msothlial cells treated with different talc samples at several doses Thymidine incorporation (dpm/pg DNA)*t Talc sample Dose Experiment Experiment Experiment (H ilcm 1) I 2 3 No. 5725 No. 5726 No 7841 0 10 20 50 0 10 20 50 0 10 20 50 1726 189 1174 + 285 1711 72 1833 144 1652 306 1681 364 1419 186 1527 357 1532 23 1321 4 0 1293 12 1271 36 1299 100 1310 120 1289 189 1232 38 1328 249 1190 64 1223 + 54 1323 118 974 6 6 1053 120 928 + 60 923 98 6779 324 5963 740 5628 908 6032 524 6708 357 6049 666 6086 534 5405 420 6401 360 6162 516 6300 241 6450 315 Experiments 1 and 2 were carried out with a specific activity of methyl* H of 20-30 Ci/mmol; experiment 3 was carried out with a specific activity of 40-60 Ci/mmol. fValues are means SD for six replicates. Anatase did not enhance UDS in RPMC. Cells treated with crocidolite at 10/tg/cm2 or chrysotile at 4 or 10 ng/cm2 always showed a significant enhancement of UDS compared with untreated cells. None of the talc samples tested here enhanced UDS. Sister chromatid exchanges Structural and ultrastructural studies The numbers of SCEs for reference particles, Nd structural change has been observed following chemicals and talc samples are shown in Table 4. treatment of RPMC with talc (Plate 2). It appeared The control particles, attapulgite and anatase, did that the number of cells was reduced compared with not induce a significant modification in the number I that of untreated cells but no sign of cytolysis was of SCEs. In contrast, increased numbers of SCEs detected. TEM studies have indicated a capacity of were observed when RPMC were treated with the RPMC to ingest talc and anatase particles. Plate 3 genotoxic chemicals mitomycin C and K.2C r0 4. A showis that talc particles were located in the peri statistically significant enhancement of SCEs was nuclear region and organelles did not seem changed obtained in cells treated with 2ng mitomycin C/ml in comparison with untreated cells. (P < 0.005) or 0.5 /tg K.2C r0 4/ml (P <0.005). The mean number of SCEs was significantly increased Unscheduled DNA synthesis (UDS) by chrysotile at 1/rg/cm2 (P < 0.005) or crocidolite at 2 ig/m2 (P < 0.05), with significant increases Tables 2 and 3 show the effect of treatment of occurring in two out of four and three out of RPMC with reference particles or talc samples. eight experiments with chrysotile and crocidolite, Tabic 2. Unscheduled DNA synthesis in pleural mesothelial cells treated with different reference particles at several doses Particle Thymidine incorporation (dpm//4g DNA)t Dose (H g/cm !) Experiment 1 Experiment 2 Experiment 3 Crocidolite! Chrysotile! Anatase 0 4 10 0 4 10 0 2 4 10 1299 100 1625 191 1668 53*** 1495 106 1744 188** 1646 124** 1316+ 153 1271 61 1380 276 1318 264 1327 57 1425 926 1489 203* 1362 117 1498 + 116* 1598 64** 6169 760 6096 705 6535 565 6405 480 6086 299 9572 463" 8323 308*" 5632 326 7590 + 649" * 8157 341 ** 6579 413 6785 650 7214 301 7764 456" fValues are means SD of six replicates and those marked with asterisks differ significantly (Student's /-lest) from the corresponding value for untreated cells (*P < 0.05; / ><0.01; / <0.00l). ^Experiments 1 and 2 were carried out with a specific activity of methyl of 20-30 Ci/mmol; experiment 3 was carried out with a specific activity of 40-60 Ci/mmol. Experiment I was carried out with a specific activity of methyl-*H of 20-30 Ci/mmol, experiments 2 and 3 were carried out with a specific activity of 40-60 Ci/mmol. ' m m ....................................................................... r i I M I r~ T T 1 j TTiTTI-ili r ^ . ..... IT FrfnMliUMii 12 S. En do-C apron et a i Table 4. SCE induction in RPMC treated with reference particles, chemicals and talc samples Treatmcnl No. of experi- Dose mcnis (/jg/cm!)t Attapulgite Anatase 3 9 Chrysotile Crocidolite Mitomycin C KjCrO, Talc no. 5725 4 8 8 8 3 Talc no. 5726 3 Talc no. 7841 3 0 20 0 2 5 0 1 0 2 0 2 0 0.5 0 2 5 10 15 0 2 5 10 15 0 2 5 10 15 No. of SCEs/ metaphascj No. of significant experiments/ no. of experiments 17.6 2.4 19.7+ 1.4 14.6 2.9 12.9 + 3.0 13.9 2.8 15 2 1.6 20.2 3.7** 14.9 + 4.6 16.8 + 5 0* 12.6+ 1.5 47.0+ 12.7* 15.4 3.4 38.6 4.2*` 12.2+ 1.8 12.2+ 1.0 11.8 2.8 11.3 + 0.4 12.6 + 2.4 12.2 + 1.8 12.2 1.8 9.8 1.0 12.2 1.8 12.2 + 2.3 12.1 + 1.1 II 9 l.l 11.0 + 0.8 11.9 + 0.7 11.1 1.3 0/3 0/4 2/4 3/8 4/4 4/4 0/3 0/3 0/3 tExcept mitomycin C (ng/ml) and KjCrO* (/g/ml). {Values are means + SD for the number of experiments shown, and those marked with asterisks diiTer significantly (Mann-Whitney test) from the corresponding values for untreated cells ( P <0.05; **P <0.005). respectively. The number of chromosomes per metaphase and SCE frequencies in RPMC exposed to talc samples 5725, 5726 and 7841 are shown in detail in Tables 5 and 6. No difference in the number of chromosomes per metaphase in treated cells was observed in comparison with untreated cells. More over, treatment with several concentrations, from 2 to 15/ig/cm2, did not increase SCE frequency. Table 5. Number of chromosomes per melaphase in RPMC treated with three talc samples No. of chromosomes/metaphasc* Dose Experiment Experiment Experiment Talc sample (Rg/cm!) 1 2 3 No. 5725 No. 5726 No. 7841 0 40.1 2.6 41.0 1.6 41.2 1.5 2 41.1 + 1.6 41.2 1.1 40.7 1.5 5 40.3 1.7 41.1 1.4 41.0 1.5 10 40.8 1.6 40.8 1.7 40 7 1.6 15 40.2 1.7 40.9 1.6 412 1.8 0 40.4 2.6 41.0 1.6 41.2 1.5 2 40.3 2.0 411 1.3 40.9 1.5 5 40.7 + 1.6 40.9 1.4 40.5 1.9 10 40.7 1.7 40.6+ 1.6 40.6 1.4 15 40.5 1.7 ND 4M 1.7 0 41.1 1.3 41.0 1.6 41.1 + 1.4 2 40 7 2 .1 41.1 1.2 41.2 1.5 5 41.2 1.4 41.0+ 1.7 40.9 1.7 10 40.7 2.0 40 7 1.8 40.7 2.0 15 40 6 2.0 40.7 1.8 41 1 + 1.9 ND = not done Mean SD of 30 metaphases. Table 6. Number of SCEs in RPMC treated with three talc samples No. of SCEs/metaphase* Dose Experiment Experiment Experiment Talc sample (fig/cm1) I 2' 3 No. 5725 No. 5726 No. 7841 0 10.1 3.5 13.3 5.7 13.2 5.8 2 11.4 3.5 11.8 4.1 13.3 4.9 5 9.0 + 3.6 11.6 + 3.9 *14.7 + 6.9 10 11.4 + 3.7 10.9 3.5 11.6 5.6 15 11.6 3.5 10.9 + 3.2 15.3 5.4 0 10.1 3.5 13.3 5.7 13.2 5.8 2 11.0 2.9 11.4 + 4.1 14.3 5.0 5 9.3 3.0 11.0+ 3.6 9.2 6.2 10 10.6 3.4 11.9 + 3.6 14.1 + 5.0 15 10.5 + 2.8 ND 13.8 + 5.4 0 12.0 + 4.3 13.3 + 5.7 1l.l 4,4 2 10.6 3.0 12.2 3.8 12.9 + 4.9 5 10.8 + 4.9 10.3 + 3.6 12.0 4 .0 10 12.1 + 5.4 11.2 + 4.5 12.5 + 6.2 15 11.0 + 3.8 9.9 + 37 12.5 + 3.9 ND = not done Mean SD of 30 melaphases. DISCUSSION In the in vitro studies reported here we investigated the effects of talc in genotoxic assays. We observed that the three talc samples did not increase UDS or SCEs, or produce aneuploidy in RPMC. In contrast, chrysotile and crocidolite fibres consistently enhanced UDS, as well as increasing SCEs in some of the experiments. This is in agreement with previous observations in our laboratory (Achard el at., 1987; Renier et at., 1990). SCE enhancement was also obtained after treatment of RPMC with mitomycin C and K2C r0 4, agents previously known to induce SCE (Darroudi and Natarajan, 1989; Kato and Shimada, 1975; Levis and Bianchi, 1982; Littlefield et al., 1979; Perry, 1980). The negative reference particle, anatase, did not increase cither UDS or the frequency of SCEs in comparison with untreated RPMC. In spite of the fact that talc is a magnesium silicate, as are chrysotile fibres, the in vitro responses of the two particles are different. As far as the mechanisms of genotoxicity of particles are concerned, several factors might account for the different responses, in particular phagocytosis, granulometry and the shape of the particles. Several questions can be addressed. First, is the lack of genotoxic action of talc due to the absence of phagocytosis? Phagocytosis seems to play an important role in the genotoxic effect of particles, because fibres phagocytosed could interact with the mitotic spindle (Hesterberg and Barrett, 1985) or chromosomes (Wang et al., 1987). This may then induce aneuploidy by chromosomal missegregation (Hesterberg and Barrett, 1985; Palekar et a!., 1987). Our TEM study showed that RPMC can ingest talc particles. This cellular process has been also observed with chrysotile and crocidolite asbestos fibres (Jaurand et al., 1979 and 1983). Despite phago cytosis, talc did not induce aneuploidy since the number of chromosomes per metaphase in talctreated cells was not different from that in untreated cells (Table 5). Therefore, the lack of chromosomal . .**--,PV *;V. V Genotoxicity of a talc in msothlial cells 13 damage might be related to different mechanical or physicochemical properties of talc in comparison with mineral fibres. Secondly, is the absence of genotoxic action due to the size of the talc particles? From the data reported in the literature, the carcinogenic potency of par ticulate matter seems to be dependent on both shape and dimension. For example, Stanton et al. (1981) have reported that after intrapleural inoculation into the rat, the frequency of pleural sarcomas was dependent on the number of fibres less than 0.25 ptm in diameter and more than 8 /tm in length. Moreover, an in vitro assay has shown that thick glass fibres were more efficient than thin fibres, on a per number basis, in transforming Syrian hamster embryo cells. In addition, no transformation was obtained when the fibre length was reduced to 0.95 /im (Hesterberg and Barrett, 1984). In contrast to asbestos fibres, talc does not have a fibrous shape, but rather a polygonal form. Fibre samples containing long fibres can be deposited in the airways because of their small diam eter, whereas respirable talc particles with a diameter higher than 5/tm do not reach the deep lung. The absence of an in vivo effect of talc might also be due to the small size of the particles. The size and number of particles per unit weight are different in the three talc samples. Granulometric study of the talc samples showed that the mean size was in the ranking order 5725 = 7841 < 5726 and of the same order as that of asbestos fibres. However, the number of long (> 4 /rm ) particles is much higher in asbestos samples than in the talc samples used here. The three talc samples did not enhance UDS or induce SCEs in comparison with untreated RPMC. This is in contrast to the results with asbestos, es pecially with regard to the UDS assay in which a significant response was observed with both types of asbestos fibres. The SCE results seem less convincing; in effect, no consistent positive enhancement of SCEs was found with crocidolite, thus lessening the signifi cance of the negative response obtained with talc. However, our observations are in agreement with in vivo data reported by Stanton et al. (1981) and with our previous results obtained with sample no. 7841, which showed that talc did not produce tumours following intrapleural inoculation (Endo-Capron et al., 1990), as well as with in vitro results that showed that talc did not induce chromosomal effects in mammalian cells in vivo and in vitro (IARC Working Group, 1987). Acknowledgements--This work has been supported by INSERM funds and Eurotalc subvention. REFERENCES Achard S., Perderiset M. and Jaurand M. C. (1987) Sister chromatid exchanges in rat pleural msothlial cells treated with crocidolite, attapulgite or benzo 3-7 pyrene. British Journal o f Industrial Medicine 44, 281-283. Darroudi F. and Natarajan A. T. 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