Document RJqNdMVnE4bxNL0w3xwZ8Rb0V

Environmental Health Perspectives VoL 51, pp. 153-158, 1983 Comparative Toxicities of Different Forms of Asbestos on Rat Pleural Mesothelial Cells by M. C. Jaurand,* I. Bastie-Sigeac,* A Renter* and J. Bignon* The effects of UICC erocidoiite and chrysotile A, either oxalic acid-leached or unleached, on the via bility, morphology and growth characteristics of rat pleural mesothelial cells (PMC) were examined; .'. ; DQ12 quartz particles were also used. ., When asbestos fibers were added for 48 hr at the beginning of exponential growth, 20 or 50 pg/ mL of chrysotile fibers were cytotoxic and no growth occurred; with 5 or 10 pg/mL a latent period was observed, and the mean populaton doubling time was increased. Chrysotile ingestion was associ- ated with morphological changes (spreading, intense vacuolation); moreover, a large proportion of the cells was binucleated (more than 30% with 10 pg/mL). The oxalic acid-leached chrysotile inhibited growth at a concentration of 50 pg/mL; with 5 or 10 pg/mL, no spreading occurred, but a shrinkage of.. . - some cells was observed. A few large vacuoles were seen in the cytoplasm of the cells; there were fewer binucleated cells. Addition of 5 or 10 pg/mL of erocidoiite, either leached or unleached, did not significantly change the growth rate, in spite of the presence of a large number of fibers inside the ' , cells which persisted when the cells reached confluency. With 20 or 50 pg/mL, the mean population doubling time was increased in a dose-dependent manner. A slight vacuolation of the cells occurred. , The sample of quartz did not modify the parameters studied in this report. . .. The results confirm the different in cilro reactivities of the two kinds of unleached asbestos fibers. Leaching of chrysotile fibers decreased their reactivity; alternatively, leaching of erocidoiite in- ' creased the effects on PMC. .. . : . . : ., : introduction _ When erocidoiite or chrysotile fibers are injected into the pleural cavity of rats, they both induce meso thelioma U). However, when tested with in vitro systems, these fibers give different responses (2). In these in vitro experiments, the cells used did not come from the pleura. The aiiia of the work reported here was to study the in vitro effects of asbestos fibers on the morphology and growth characteristics of rat pleural mesothelial cells in short-term experi- Oients. :. Since acid-leached chrysotile fibers. have been found to be nontumorigenic (3,4), leached chrysotile ' .mHrd: leached erocidoiite were also tested. In order to >, . .. '' . . tv, GrouPe de Recherches et d'Etudes sur les Affections Res et l'Environnement (INSERM U 139 et ERA CNRS 4jr- Centre Hospitalier Intercommunal, 40, Ave de Ver- 94010 Cre'teil Cedex, France. ` : compare the results with the activity of noncareinogenic particles, a sample of quartz was also used. . Material and Methods Asbestos Fibers and Control Particles The asbestos fibers were obtained from the UICC. Chrysotile A and erocidoiite were used. Chrysotile and erocidoiite were leached with 0.1 N oxalic acid (5). DQ12 quartz from Dorentrup was chosen as a control. The particles were dispersed by sonication in the culture medium (50 kHz, 20 W, 5 min.) ' ".v;: ' y.y.: The size parameters of the samples were deter mined by electron microscopy as described else where 4). The leached chrysotile had a slightly higher mean diameter than'the; unleached samples (0.091 and 0.082 pm) and a lower mean length (1.8 'and 3.1 pm). ' ' ' " " ' 154 JAURANDETAL. Table 1. Percentage of binucleated PMC 72 hr after contact with the particles. Concentration 6-8 -12 passages, 11-14-15 passages, 8-9-10-13 passages, chrysotile (ChP leached Cha crocidolite (CrP Control 5 pm/mLb 10 pg/mL 20 (ig/mL 50 pg/mL 1.7 0.8 29.3 0.2' 37.3 2.5' ndc ndc 1.1 1.6 8.2 0.8 12.7 4.5' 19.8 5.3' 19.3 4.0' 1.7 0.8 2.2 0.9 4.3 1.2 7.5 0.4 16.5 10.0t a For each treatment different cell strains were used b When expressed as pg/cm! the concentrations were from 1 to 10 pg/cm1. ' nd = not determined. * p < 0.1. tp < 0.5. 6-8-16 passages, leached Cra 1.8 1.4 3.4 3.1 8.2 1.2* 13.8 1.3 25.9 2.6' 17 -10 passages" quartz ' 0.2 0.3 1.1 1.5 0.6 0.8 0.6 0.8 2.2 3.1 -* Rat Pleural Mesothelial Cells (PMC) PMC were cultured as described elsewhere (6). The cells were used between the 6th and 17th passage. The PMC came from six different rats. Morphological Studies PMC morphology was observed by use of phasecontrast microscopy and electron microscopy as de scribed by Jaurand et al. (7). Growth Analysis The growth curve analysis was performed as de scribed elsewhere (7). Briefly, PMC were plated at 5 x 105 cells per flask, and 24 hr after plating the particles were added to the culture medium for 48 hr. The difference between the number of treated and untreated cells was assessed by the F test. The experiments were carried out in triplicate or quadruplicate for each particle type. The percentage of mitosis or of binucleated cells, following either 48 hr or 72 hr of incubation with the particles, was determined. The difference between the percentage observed in treated and untreated cells was assessed by the z test. Results and Discussion Morphological Studies The results describing the effects of chrysotile and crocidolite fibers have been reported elsewhere (7). Chrysolite-treated PMC had numerous vacuoles; there was an increase in the cell volume, and a large proportion of binucleated cells appeared (Table 1). Crocidolite fibers did not induce such morphological changes except at the highest concentration (50 pg/mL), but these effects were less than with 5 pg/ mL) of chrysotile fibers. PMC treated with acid-leached chrysotile also showed a vacuolation of the cytoplasm but the vac uoles were less numerous and generally larger than with unleached chrysotile (Fig. 1). Shrinkage of the cells was sometimes observed. After 72-hr contact with acid-leached fibers, some binucleated cells were observed, but their percentage was less than with the untreated chrysotile (Table 1). The difference between the number of binucleated cells in treated and untreated cultures was significant with 10 pg/mL of leached chrysotile fibers. With the unleached fibers, it was significant with 5 pg/mL; moreover, it was twice the percentage observed in the leached chrysotile-treated culture. Thus, the effect of leached fibers was less than the effect of the unleached. Very few vacuoles were seen in the cultures treated with leached crocidolite (Fig.. 2). Electron microscopy revealed the presence of intracellular fibers (Fig. 3). The number of binucleated cells was less than in the cultures treated with either leached or unleached chrysotile (Table 1). However, the leached sample of crocidolite had more effect on PMC than the unleached sample, since 10 pg/mL induced a significant increase in the proportion of binucleated cells as compared to the control cultures. Treatment with 50 pg/mL of crocidolite fibers was necessary to obtain a significant in crease of this parameter. The particles of quartz did not modify the mor phology of the culture (Fig. 4), in spite of the pres ence of intraphagosomal particles (Fig. 5). When the proportion of binucleated PMC was ex amined, a differential effect of leaching was ob served as the proportion of binucleated cells de creased with leaching of chrysotile and increased with leaching of crocidolite. Table 2 shows that there was no effect, due to the treatment with the particles, on the percentage of mitosis determined 48 hr after their additionHowever, during this time, the cells were in contact with the particles. This result means that in the fiy groups of treated PMC there was no inhibition of passage to the M phase and that the toxicity may TOXICITIES IN PLEURAL MESOTHELIAL CELLS 155 ? Figure 1. Phase contrast microscopy of PMC in culture u. (x 500); 24 hr following trypsination, PMC were treated with (a) chrysotile, 10 pg/mL, or (b) leached chrysotile, 50 pg/ mL for 48 hr. Figure 2. Phase contrast microscopy of PMC in culture (x 310): (a) PMC at the 8th passage treated with 20 pg/ mL of leached crocidolite for 48 hr; (b) control culture. 156 JA URAND ETAL. Figure 3. Electron microscopy of PMC at the 17th passage treated with 10 pg/mL, of leached crocidolite fibers for 48 hr : x 9000). : Figure 5. Electron microscopy of PMC at the 4th passage treated at confluency with 5 pg/mL of DQ12 for 48 hr (x 55000). Figure 4. ^ Phase contrast microscopy of PMC at the 20th pas sage treated with 10 pg/mL of DQ12 for 48 hr (x 310). not be dependent on the phase of the cell cycle. Our previous results (7) have shown that the phases of cell mitosis can be observed in chrysotile-treated PMC. The presence of binucleated PMC may result from an inhibition of cell division or from a fusion of the PMC; however, no indication of fusion between PMC was observed. When mitosis occurred in a chrysotile-treated culture, the cells adhered and spread on the plastic at the end of telophase, rather than before the beginning of mitosis. An incomplete synthesis of the plasma membrane may, therefore, result in a binucleated cell. Growth Analysis The results concerning the mean population dou bling time 0 are expressed in Table 3. DQ12 quartz had no significant effect on the value of 0. Leached crocidolite and chrysotile, either leached or un leached, were cytotoxic, but the concentration needed to prevent growth depended on the nature of the fibers. Unleached crocidolite had some effect if added at the highest concentration (50 pg/mL). TOXICITIES IN PEEURAt, MES'OTHELIAL CELLS 157 Concentration ''Table'2. "Pejcentagc'of cells in Mitosis after 18 hrof contact with the particles:! r." non-:; oirisT 6 - 8 -12 passages, [-yllol4,-;15'passages,0 8 - 9 5.10.-13 passages,__ .6..-j&.-.16.passages,____ 10 -.11 - 17_passages^ ,. chrysotile (Ch)3 .... leached,Ch"f.;. crocidolite (Cr)3 - " leached Cr3 Jr-or-Jam":quartz3 Control v-;., 7,,;;1.4Q-3-v 5pg/mL ., ' - " 1.6i0.6lOpg/mL' ' ' 20pgimL ' 1 ^ -2:(VT^7o 0+1.3 " 1.3 0.0 7 qxy y.2flt 0.8'c:;9rf* bin; o.9h?o:2':do isdi 50pg/mL ;r.; to:.'. nd :7o;j'i7J na-mO/f 0.8 evrlosi . For each treatment, diffefenit'cellstfaihs were used'.ai'bin ;: :v ' hso'bMi ri G-.v 1.4 0.3 0.7 0.2 1.0 0.1 2.1 0.2 i:o"d:3 y t .. - - 7 :7 . L ' .: `.'l 1: 0.6 0.6 1.7 0.3 1.7 0.3 1.5 0.7 1.4 1.2 y'iJor.v'l;8) 0.7 - r.3 bo;ioj0.8I 0.3 o !)i:,oi:2.3'i2.4 t0.21+0.3 170 E3 .? iydi; h+r!7.9si Table 3.' ` Mean pbpuiafiondoUblinVtimerin hours of PMC incubated for 48 hr with1 the particies; y-'i-'c-3 < l*J - _ ?. firyinu-.o n o) . y i'jywy .c v > r. .,: 6 - 8 -12yl4passages 11 -14 -15 passages 9 -10 = 8-13 passages - 69116 passages 3iLltt.-,:11 k 17.passages1 Concentration - chrysotile (Ch):1. i- leached Ch3 , crocidolite tCr)3 ; leached.Gr3 yroril,'JS 9.<juaftz*.3Qfiij Control 5 pg/m L 10pg;'mL \ 20 pgmL, 50pg/mL . ; 29.7 -4.1 ,v32.3. 5.5 rr-, - .33.2 11.3 - , 38.6 t 4.4 o . 42.7;;20.9'.;`; - '42.6 6.0 1: ' ' i - bo ' /! .M ' ! ; ' 87.0.-> 1516 ; . ' : -<0. : ;':-i->bi273.249:,-iV7 '' / :; r: n.>.7t-v . .or.<0 ;i.,v, 29.6 3.8 31.8 4.3 32.8 3.4 35.5 5.8 90.7-5.1 > . : . .'46.3 :9.3;- ]o r.rM.O 5.0*.'ndo . . .49.3 8.1 : 71'.,- 42.7,- 8.9,-,;ib,., . , 50.7 11.9 % ` * 46.3 + 7.2 11 M "' -744.33 ! '-'ri',-3'--49'.o+`l6,.5 50.5 1417 " : >r:t .in "in 3 For each treatment, different cellstrains \v,ere used. y:.y a: stnocmtqni.- io 29iq;ri2 b -ar.-rc;-.'!) oikocy'nb 4i x106cells '' `or; ;I .ooor! bvi'toqs'i ?i b?>:j i>ii;osv~ris hgrbbs; L-br.i ion i'xOJOJV? i jjnizur.0 oo' on* ri' ii'nos. 7ac r'fio OJfc , .. (6) 96 144 hr fna iu: 6. Growth curves of one strain of PMC treated with (a) leached-chrysotile or (6) leached-crocidolite: () control culture; 5 (jg/mL; (O) 10 pg/mL; (A) 20,pg/mL; (A) 50.pg/mli. = ;; j The growth curves showed that a lag time oc curred with the chrysotile-treated cultures but this was not observed with;leached-chrysotile or with crocidolite, either leached or unleached (Fig. 6). It is known that the number of cells N during the exponential growth is ` " : ....... . . N =. n0 29$ ... d) where N0 is the number of PMCat the beginning of growth, t the time and 0 the population doubling tlrne. Then the derivative of Eq. (1) may be written dN/dt = 1/0 (/V log 2) (2) hen the particles are introduced into the medium, an 'nhibition can occur; it may be proportional to a power (x) of the particle concentration c, and it causes a decrease in the number of PMC: dNidt = kNck .'V- (3) where k is a constant. Then, the .variation in the num ber of PMC will be ;: i dNidt = Ml/0 log 2 U kc*) I (4) If 0' is the mean population doubling time in'the presence of the particles, the variation of 0' with c can be written - 0' = a0/(a- cx) !' (5) In this relationship, when c -+ 0, then 0 '-* 0; and whenc*-*- a, 9' . From the value a it is possible to deduce a1/1, which, is the value of the concentration at which no growth occurs (cj;m). _ From the values reported in Table 3, the .calcula tion gives the values ofegm expressed in Table 4. When the particles are compared by weight, the toxicity will increase in the order Cr < LCr < LCh < Ch, where Cr, Ch are. crocidolite and chrysolite, 158 JAURANDETAL. Table 4. Calculated values of the concentration where no growth occurred. Treatment Chrysotile Leached Ch Crocidolite Leached Cr ' clim- Pg/mL 22.6 28.8 237 51 respectively, and LCr and LCh are the respective leached fibers. Our results are in good agreement with previous data reported by Neugut et al. (3) and Reiss et al. (9) using chrysotile and amphiboles on various cell lines. These authors found a higher toxicity of chrysotile than of crocidolite. It seems that proliferative epithelioid cell lines react to asbestos treatment by somewhat similar responses, possibly related to the amount of particles ingested. Reiss et al. (9) found that the toxicity of the leached samples of amphiboles increased, whereas that of chrysotile decreased with leaching. A similar result is reported here. It is not known if the batch of ' leached chrysotile used for the PMC treatment was carcinogenic. However, other leached samples did not induce mesothelioma (3, -4), suggesting that this cytotoxicity was not related to the probability of causing mesothelioma. There were some differences in the size of the fi ber samples, as determined by electron microscopic and optical measurements, and the repartition of the number of fibers with size is different from one sample to another one. The results may have to be reconsidered taking this observation into con sideration. Another way to compare the effects of the sam ples used in this study was to determine the time, Figure 7. Level at which the number of treated cells was ' lower than that of the untreated cells in all of the cell strains used to determine the gTowth rate (see Table 3). For example, with chrysotile fibers, the difference was sig nificant following 24 hr of incubation with a concentration equal to or higher than 20 pg/mL, or following 48 hr with a concentration equal to or higher than 5 gg/mL. for a given concentration, where the value of p which measures the difference between the number of PMC, in treated cultures and the number of in the control culture is such that p < 0.5. For each treatment, the F test was applied every day, and the results are expressed in Figure 7. It can be seen that chrysotile and leached crocidolite were both ef fective at a concentration of 5 pg/mL, but the time necessary to observe a significant effect was longer with leached crocidolite than with chrysotile. This may be due to similar "toxic power" of the samples and to a different sensitivity of the mesothelial cells (expressed by the lag time and possibly related to the intensity of phagocytosis). With the leached samples, no significant effect was observed after 24 hr of contact with the fibers, and with 10 pg/mL, the relationship was Cr < LCh < LCr < Ch. When considering the effects on growing cells, the comparison between different varieties of fibers must be made carefully, since an effect may be re versed due to the form of the dose-effect relation ship. Research was sponsored by the Institut National de la Santc et de la Recherche Medicale (INSERM). We thank Mrs. L. Kheuang for electron microscopic technical assistance and Mrs. N. Cathelineau for typing the manuscript. REFERENCES 1. Wagner, J. C., and Berry, G. Mesotheliomas in rats follow ing inoculation with asbestos. Brit. J. Cancer 23: 567-581 (1969). 2. Bignon, J., and Jaurand, M. C. Biological in vitro and in vivo responses of chrysotile versus amphiboles. Environ. Health Perspect. 51:159-165 (1983). 3. Morgan, A., Davies, P., Wagner, J. C., Berry, G., and Holmes, A. The biological effects of magnesium-leached chrysotile asbestos. Brit. J. Exptl. Pathol. 58: 465-473 (1977). 4. Monchaux, G., Bignon, J., Jaurand, M. C., Lafuma, J., Sebastien, P., Masse, R., Hirsch, A., and Goni, J. Mesotheli omas in rats following inoculation with acid-leached chryso tile asbestos and other mineral fibres. Carcinogenesis 2: 229-236(1981). 5. Jaurand, M. C., Magne, L., Boulmier, J. L., and Bignon, J In vitro activity of alveolar macrophages and red blood cells with asbestos fibres treated with oxalic acid, sulfur di oxide and benzo 3-4 pyrene. Toxicology 21:323-342 (1981). 6. Jaurand, M. C., Bernaudin, J. F., Renier, A., Kaplan, H-. and Bignon, J. Rat pleural mesothelial cells in culture. In Vitro 17:98-106 (1981). 7. Jaurand, M. C., Renier, A., Kheuang, L,, and Bignon, J. Ef fect of chrysotile and crocidolite on the morphology and grown of rat pleural mesothelial cells. Environ. Res., in press. 8. Neugut, A. I., Eisenberg, P., Silverstein, M., Pulkrabek, Pand Weinstein, J. B. Effects of asbestos on ephithelioid cell lines. Environ. Res. 17: 256-265 (1978). 9. Reiss, B., Solomon, S., Weisburger, J. H., and Williams, G. M. Comparative toxicities of different forms of asbestos in a cell culture assay. Environ. Res. 22:109-129 (1980).