Document 5Vm7MyevMgzq7y6rd1azrgO5

Mutation Research. 320(1994)253-259 0 1994 Elsevier Science B.V. All rights reserved 0165-1218/94/S07.00 MUTGEN 1972 In vitro genotoxicity studies of chrysotile asbestos fibers dispersed in simulated pulmonary surfactant J. Lu a, M.J. Keane b, T. Ong a-b and W.E. Wallace a'b-* J West Virginia University and h Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health. 944 Chestnut Ridge Road, Morgantown. WV 26505-2808, USA (Received 15 March 1993) (Revision received 4 August 1993) (Accepted IS August 1993) Keywords: Chrysotile asbestos; Dipalmitoyl lecithin; Micronucleated cells; Multinucleated cells; Sister-chromatid exchange Summary Micronucleus (MN) formation and sister-chromatid exchange (SCE) assays were performed for asbestos in cultured Chinese hamster lung (V79) cells to determine the effect of surfactant treatment on the genotoxicity of two chrysotile asbestos samples of different fiber lengths. The cells were challenged in vitro with NIEHS intermediate- and short-length chrysotile fibers in both their native state and with surfactant pretreatment. For the surfactant pretreatment, the fibers were incubated in a simulated pulmonary surfactant which was prepared by ultrasonically dispersing dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, in minimal essential medium (MEM). Chrysotile asbestos was ultrasonically mixed into the prepared surfactant dispersion or into MEM. V79 cells were exposed to DPL-treated intermediate-length chrysotile (TICA), intermediate-length chrysotile (ICA), DPL-treated short-length chrysotile (TSCA) or short-length chrysotile (SCA) fibers for 48 h. For each treatment, 2000 mononucleated cells were scored for MN formation, and 30 M2 metaphase cells were scored for SCE induction. The results showed that all samples, TICA, ICA, TSCA and SCA, caused significant elevation in the frequency of cells with micronuclei and of cells with two or more nuclei. The increase in micronucleus frequency was greatest in cells challenged with untreated intermediate-length fibers, and was greater for untreated than for DPL-treated short-length fibers. For the short-length fiber samples, DPL surfactant treatment decreased activity for multiple nucleus formation, while DPL treatment did not result in consistent changes in that activity for intermediate-length fibers. Results of SCE assays were either negative or inconclusive. Cells were more viable following TICA and TSCA than following ICA and SCA challenge as measured by cell counts after 48 h of incubation. Corresponding author. SSDl 0165 -1 218< 93 )Ed 1 03 - L :?4 Asbestos is known to induce plural mesothe lioma and carcinoma in the lungs of humans and rats (Davis. 1970: Stanton and Wrench. 1972: Wagner et al., 1973. 1982; Harington et ah. 1975; Selikoff. 1979). In vitro, asbestos induces chromo somal aberrations and micronuclei in cultured mammalian cells (Sincock and Seabright, 1975: Brown et al.. 1979; Babu et al., 1980, 1981: Os- himura et al.. 1984; Hesterberg et al.. 1985. I986a.b). The biological effects of asbestos are influenced by several factors, which include size, durability, chemistry, and physiochemistry of fibers. Jaurand et al. (1980, 1988) and Vallyathan et al. (1987) indicated that the surface of asbestos fibers is one of the critical factors in determining their in vitro toxicity. Surface modifications, therefore, may affect the genotoxicity or cytotoxi city of native asbestos fibers. When inhaled into the deep lung airspaces, fibers first contact the pulmonary surfactant on the inner surfaces of the lung alveoli or bronchi oles. Studies have shown that the initial response of the rat lung to chrysotile asbestos is a progres sive increase in the amount of pulmonary surfac tant generated (Tetley et al., 1977; Oblin et al., 1978). It was reported that chrysotile asbestos can absorb dipalmitovl lec^hm (DPL) which is a prin- cipal constituer surfactant, and that the absorpt. pholipid changes the surface propc .e asbestos, result- ing in the suppre. hemolytic activity (Light and Wei. 1977a.b; Jaurand et al., 1980). It has also been reported that DPL surfactant-disper sion reduces the in vitro cytotoxicity of diesel exhaust particles (Wallace et al.. 1987: Keane et al.. 1991). Therefore, the question arises as to whether pulmonary surfactant can affect the genotoxicity of chrysotile asbestos. In this study. DPL was ultrasonically dispersed in minimal essential medium (MEM) to model phospholipid surfactant dispersion in the pul monary alveolar hypophasc. Cultured Chinese hamster lung cells. V79. were challenged with untreated chrysotile asbestos and with chrysotile asbestos treated by incubation in DPL surfactant dispersion. Micronucleus (MN) formation and sister-chromatid exchange (SCE) were used as in vitro sjenotoxicity assay systems. r Material and methods DPL (1 mg /ml) dispersion Dipalmitoyl lecithin was obtained from Cal- biochem (La Jolla, CA). Minimal essential medium (Gibco. Grand Island, NY) without fetal bovine serum (FBS; Gibco) was added to DPL that had been weighed into a sterile centrifuge tube, and the contents were ultrasonicated for 10 min at 30 W (Heat Systems Inc., Plainvicw, NY). Preparation of chrysotile asbestos NIEHS intermediate length and short length chrysotile asbestos samples (15 mg), autoclaved 15 min at 121C and separately mixed in either a DPL dispersion or in their native state in MEM without FBS, were sonicated for 10 min at 30 W. The samples were incubated 60 min at 37C in a rotary drum incubator, then centrifuged for 10 min at 1200. g. After centrifugation, the super natant was discarded, and the pellet resuspended in MEM without FBS. The mixing ratio for treat ment in DPL dispersion was 500 mg DPL/g asbestos, which has been shown in this laboratory previously to supress membranolytic activity in the hemolysis assay (unpublished data). The mean fiber length for the short length sample was 11.6 /am, with 98% of fibers less than 10 pm. The intermediate length sample mean length was 101 Aim, with 65% of the fibers greater than 10 pm. Additional physical and chemical characteriza tions of the fiber samples have been given by Campbell ,et al. (1980). Both asbestos samples were analyzed for specific surface area by Nadsorption using the BET rpethod with a multi point analyzer (Micromeritics, Norcross, GA). The short chrysotile measured 41.4 nr/g and the intermediate 22.6 nr/g. Examination of dis persed fibers by scanning electron microscopy indicated no aggregation of fibers. Treatments 4.0 x 10' V79 cells were seeded into each 100 mm dish in 10 ml MEM supplemented with 10% FBS. 1% penicillin-streptomycin, and 1% iglutamine. After overnight incubation at 37'C and 5% C02. DPL-treated intermediate- (TICA). DPL-treated short- (TSCA). untreated intermedi- n Cal-ential it fetal > DPL trifuge for 10 . NY). length e laved liter a MEM 30 W. C in a lor 10 -uperended treat)PL/g ratory -ty in mean -11.6 . The - 101 < ^int. eriza.n by nples w N\ miltiGA). J the diswopy 100- ;oa iC A). vdi- I ; 1 ate- (ICA) and untreated short-length (SCA) chrysotile asbestos fibers were added into the culture cell dishes to give concentrations of 3.3 100 Aig/ml. respectively. The concentrations se lected were based on other studies reported in the literature (Lavappa et al., 1975; Sincock and Sebright, 1975; Livingston et al.. 1980; Babu et al., 1981; Casey, 1983). Blank and DPL controls were included in each experiment. Micronucleus assay After 48-h treatment, cells were rinsed 5 times with phosphate-buffered saline, left to incubate an additional 24 h, then harvested by trypsinization. The contents of each dish were transferred into a 15-ml centrifuge tube and centrifuged at 285 g for 6 min. The supernatant was removed and the pellet resuspended in MEM to the de sired dilution. Slides were prepared on a Cytospin and stained with Diff-Quik solution (American Scientific Products, McGaw Park. IL). The slides were coded before scoring. 2000 mononucleated cells from each group were scored at random for MN, and another 2000 cells were scored from each group for determining the per centage of cells with one nucleus and with two or more nuclei. Sister-chromatid exchange assay After 17 h of asbestos challenge, 15 /a M bro- modeoxyuridine (BrdU; Gibco) was added to each dish. Colcemid (Gibco) was added (0.15 p.g/ml) 3 h before the end of culturing. Cells were har vested and slides prepared 48 h after the initial asbestos challenge. The slides were allowed to air dry and were stained by the modified fluores cence plus Giemsa method of Perry and Wolff (1974) and Goto et al. (1978). Slides were stained for 15. min with 1(F? Hoechst 33258, rinsed with distilled water, then placed in a pool of Sorenson's buffer (pH 6.8) at 56-60C on a slide warmer and exposed to "black" light of approximate 355 nm at a distance of less than 1.0 cm for 15 min. Slides were rinsed in distilled water and stained with 5c'c Giemsa for 15-20 min. After a rinse, they were allowed to air dry and then were coded. 30 well-differentiated second division (M2) cells, with 20-22 chromosomes from each dose and control, were scored for SCE. Cell replicative index (RI) --- was obtained by determining the numbers of Ml. M2 and M3 cells in the first 100 mitotic cells observed. Results Cells with MS' and multinucleated cells Table 1 and Fig. 1 show that all doses of TICA. ICA. TSCA and SCA induced a statisti cally significant elevated percentage of micronucleated cell (MNC) over the DPL or blank con trol after a 48-h treatment ip < 0.01). Untreated short-length fibers induced greater frequencies of TABLE 1 FREQUENCIES OF MICRONUCLEATED AND MULTINUCLEATED V79 CELLS AFTER TREATMENT WITH CHRYSOTILE ASBESTOS Treatment TICA ICA TSCA SCA DPL Blank Concentration 1 nmr/ enr enr Micronucleated cells Number rr 1.27 4.2 12.7 28.7 95 287 78 "* 158 >' 178 c 5.9 7.9 8.9 1.27 4.2 12." 28.7 95 287 157 " 178 " 2111' 7.9 8.9 1(1.6 H.42 1.27 4.2 12.7 17.5 53 175 527 58 1 65 75 7t) * 1.9 3.3 5.7 4.0 0.42 L27 4.2 12.~ 17.5 55 175 527 56 d 92 ' 145 c 155 - 1.8 4.6 *7 Q 7.8 1.27 16 0.8 - IS 0.9 Q >2 Nuclei 5.5 ! 26.4 56.5 : 19.1 26.5 20.4 2.6 : 4.6 ' 8.5 ' l.s 12.9 15.9 19.5 n.4 1.5 Based on 2000 mononucleated cells scored. ' TICA. DPL-lreated intermediate-length chrysotile a-he-'.ov ICA. intermediate-length chrysotile asbestos. TSCA. DPL-ireated short-length chrysotile ashe-to-. SCA. short-length chrysotile ashestos. DPL. dipalmitoyl lecithin. To convert dose- to tig, nil or mm- ml. multiply do-e tigures by '.s?. ' i> - 0.05. compared to proper control. p - tUU. compared to proper control. ' /) II.U5. compared to name chrysotile at the same do-e. - p n til. compared to native chrysotile at the -ante do-e 256 ' MNC than short-length chrysotile treated with TABLE 2 DPL. For example, at 4.2 /zg/cm2 and 12.7 jxg/ SISTER-CHROMATID EXCHANGE FREQUENCIES AF cm2 the number of MNC in SCA-challenged cul TER ASBESTOS TREATMENTS " | ture was about 2 times greater than that of % TSCA-challenged culture. The frequencies of Treatmen b Concentration SCEs/cell RI i MNC induced by DPL-treated and untreated in M-g/cm mm-Am- wean su, termediate-length fibers, however, were not sig TICA 1.3 29 7.33 2.48 1.99 nificantly different. Intermediate-length chrysotile asbestos induced a greater frequency of MNC 4.2 95 12.7 287 7.43 2.43 7.77 2.65 1.97 1.92 than did short-length fibers on a mass or surface area basis. The frequency of MNC in SCA-chailenged cultures at 12.7 /zg/cm2 was of the same ICA 1.3 29 4.2 95 12.7 287 7.13 2.32 8.17 2.96 c 6.93 1.98 1.93 1.97 1.93 order as that in ICA-challenged cultures at 1.3 TSCA 0.4 18 7.30 2.15 1.97 . !g/cm2 (7.8% and 7.9% respectively). Results are expressed in both p.g/cm2 and mm2/cm2 to allow comparison from both a mass and surface 1.3 53 4.2 175 12.7 515 6.90 1.86 6.50 1.89 7.20 1.61 1.97 1.94 1.98 area basis. The TICA, 1CA. TSCA and SCA treatments all caused a significant increase in the number of cells with 2 or more nuclei over the DPL or blank control (p<0.01. Table 1). The intermediatelength chrysotile samples induced higher frequen SCA Blank DPL 0.4 18 1.3 53 4.2 175 12.7 527 10.0 - 6.27 2.42 7.03 1.65 6.50 2.52 7.43 2.50 6.47 + 2.35 7.13+1.50 1.99 2.00 1.93 1.93 1.99 2.00 cies of multinucleated cells than the respective J 30 M2 metaphases containing 21 1 chromosomes were short samples, and treated fibers were generally less active than untreated fibers,of respective size, with the exception of the highest-concentration scored for each group. h Abbreviations, see Table 1. c p < 0.05, compared to proper control. ICA sample. Increases in TICA, TSCA and SCA concentrations, with the exception of 12.7 /zg to the micronucleus assay were studied by the ICA/ cm2, increased the number of multinucle immunofluorescent kinetochore staining method ated cells, while monocleated cells became less of Channarayappa et al. (1992) to investigate frequent with an increase in concentration (trend whether clastogenic or aneuploidogenic effects analysis, p < 0.01). were associated with the micronuclei in this study. Several fiber samples that responded positively The limited results indicated that both effects were present in similar proportions (data not shown) in the samples studied. SCE results Table 2 shows that there was no dose-related 2Oa. a increase in SCE frequency induced by any of the 4 chrysotile asbestos samples. Nor was the cell replicative index affected by the chrysotile as bestos samples used in the experiment. a TICA * ICA TSCA ~ SCA i 2 4 6 8 10 12 14 CHR1SOTILF. CONCENTRATION (pL'm? ' Fin. 1. Frequencies of niicronucleated cells after asbestos treatments. Relative viability of cells When harvested, viable cells in each of the cultures were counted with a hemacytometer un der the microscope. The relative viability of cells was calculated from the number of cells that excluded trypan blue after 48 h of incubation 5 COE V) U>pi U5l CE Fig. will cell sho' 12.' TS( ICY hig! ITUU the 2 cr Dis chi tio; stc cel al.. al.. hat gr< ka: ak fo: sa- U1- fn st I ru ur in> o\ sarc bc 100 \ \ > 60 TICA * ICA TSCA SCA 0 2 4 6 8 10 12 14 CHRVSOTILE CONCENTRATION (iig/cm ) Fig. 2. Viability of V79 cells exposed to chrysotile fibers suspended in medium and simulated surfactant. with chrysotile fibers divided by the number of cells in the medium control or DPL control. As shown in Fig. 2, the relative viabilities of cells at 12.7 jug/cm2 were 15.1 and 34.4% for TICA and TSCA challenge, and were 3.8 and 12.1% for ICA and SCA challenge, respectively. At the higher doses of untreated chrysotile treatment, many cells were morphologically distorted, and the cell membrane was often broken in cells with 2 or more nuclei. Discussion Chrysotile asbestos has been shown to induce chromosomal aberrations and micronuclei forma tion in the Syrian hamster cell, the Chinese ham ster ovary cell, and the rat pleural mesothelial cell (Sincock and Seabright, 1975; Oshimura et al,, 1984; Hesterberg et ah, 1986a,b; Jaurand et ah, 1988). In this study, we used the Chinese hamster lung cell line. V79, since it has a rapid growth rate, high plating efficiency and stable karyotype (Bradley et ah, 1981). The intermedi ate-length fiber sample appears to be more active for micronucleus induction than the shorter fiber sample. However, only two sets of fibers were used, and thpy were not size separated fractions from a common sample, thus the role of other structural or compositional factors cannot be ruled out. In this study, both DPL-treated and untreated chrysotile asbestos caused a significant increase in the frequency of MNC in V79 cells over the controls in a dose-related manner for samples of both lengths: Treatment with DPL reduced activity of the short-length chrysotile as bestos for induction of MN. 257 It has been suggested that the surface proper ties of asbestos, especially the surface charge, affect the in vitro toxicity (Jaurand et al,, 1980. 1988; Vallyathan et al,, 1985). Chrysotile asbestos can adsorb phospholipids on its surface, masking or reducing the surface charge of the fibers (Light and Wei, 1970a,b; Jaurand et al., 1980). The decrease in clastogenic activity of DPL-treated fibers seen here may be due to DPL masking fiber surface sites or due to DPL modification of the fiber surface area or composition. To deter mine if the latter occurs over more extended times in vivo, the surface composition of chrysotile asbestos could be analyzed after a long period of retention in the lungs of exposed animals during the course of an animal carcinogen study. Recent data have indicated that long fibers have the higher genotoxic and carcinogenic po tency as compared with shorter fibers (Stanton et al., 1981; Pott et al., 1984; Hesterberg et al., 1986a). The results of this study are not inconsis tent with those findings. The frequencies of MNC in ICA- and TICA-challenged cultures at a dose of 1.3 ixg/cm2 or 29 mm2/cm2 were comparable to those in SCA- and TSCA-challenged cultures at a dose of 12.7 q,g/cm2 or 527 mm2/cm2, respectively (Table 1 and Fig. 1). Hesterberg (1986a) indicated that cells may selectively inter nalize longer fibers, and that the surface area per fiber could be an important factor in phagocyto sis, whereas the mass or the length of fiber could be a critical factor affecting the induction of cytogenetic damage. The presence of binucleated cells in asbestostreated cultures have been reported (Sincock and Seabright, 1975; Casey. 1983; Hesterberg et al.. 1985. 1986a). The initiation of cytokinesis is de pendent on the successful completion of earlier events of mitosis. Thus possible interference of chrysotile asbestos with the structure or function of the mitotic apparatus could affect cytokinesis, resulting in binucleation or multinucleation. In this study, we found a common trend between the percentage of cells with micronuclei and the per centage of cells with > 2 nuclei. Whether there is a relationship between the two effects is not known. It is known that micronuclei are formed either by acentric chromosomal fragments due to chromosomal breakage or by centric chromo- 25S somes lagging behind in mitosis due to spindle damage. However, it is not apparent as to how chrysotile asbestos induces multinucleated cells. Neither is it clear as to how DPL treatment reduces the activity of the short-length chrysotile asbestos sample to induce MNC and multinucle Indian variety of chrysotile asbestos. Environ. Res. M 416-422. "' Babu. K.A., S.K. Nigam. B.C. Lakkad, D.K. Bhatt. A.B Karnik. K.N. Thakore. S.K. Kashyap and S.K. Chatterjee (1981) Effect of chrysotile asbestos (AP-1) on sister-chro matid exchanges in Chinese hamster ovary cells. Environ. Res.. 24. 325 -329. ' ated cells observed in the present study. DPL treated intermediate-length fibers appeared to be as active as untreated fibers, but this could be due to cytotoxic effects of the untreated fibers. Bradley. M.D.. B. Bhuyan. M.C. Francis, R. Langenbach, A. Peterson and E. Huberma (1981) Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis of the literature. Mutation Res., 87. 81-142. Campbell, W., C. Huggins and A. Wylie (1980) Chemical and The SCE results were different from those for physical characterization of amosite, chrysotile, crocido- micronucleus induction. No significant difference in the frequency of SCE was found after V79 cells were challenged with all 4 types of chrysotile asbestos samples. Conflicting data on SCE induc lite, and nonfibrous tremolite for oral ingestion studies by the National Institute of Environmental Health Sciences, U.S. Bureau of Mines Report 8452, U.S.G.P.O. 1980-6(13102/41. Casey, G. (1983) Sister-chromatid exchange and cell kinetics tion by asbestos fiber have been reported. Casey in CHO-K1 cells, human fibroblasts and lymphoblastoid (1983) did not find SCE enhancement in as bestos-treated CHO cells, human fibroblasts or lymphoblastoid cells. In contrast, Babu et al. (1980) and Livingston et al. (1980) indicated that cells exposed in vitro to asbestos and glass fiber, Mutation Res., 116, 369-377. Channarayappa, J. Nath and T. Ong (1992) Immunofluorescent staining of kinetochores in micronucleus for detection of aneuploidy inducing agents, J. Tiss. Cult. Meth.. 14, chrysotile asbestos caused an increase in SCE 125-132. frequencies. Recent data have shown that in vitro SCE induction is either absent or very slight after chrysotile asbestos challenge (Jaurand et al., 1985; Rosin et al., 1985; Kelsey et al.. 1986). Davis. i.M.G. (1970) Long-term fibrogenic effects of chrysotile and crocidolite asbestos dust injected into the pleural cavity of experimental animals, Br. J. Exp. Pathol.. 51. 617-627. Goto, K.S., S. Maeda, Y. Kano and T. Sugiyama (1978) In conclusion, the present findings indicate Factors involved in differential Giemsa-staining of sister % P that both short- and intermediate-length chry chromatids. Chromosoma, 66, 351-359. t sotile asbestos fibers, with or without DPL surfac Harington, J.S.. A.C. Allision and D.V. Badami (1975) Min tant treatment, induce micronuclei and multiple eral fibers: chemical, physicochemical and biological prop erties. Adv. Pharmacol. Chemother., 12, 291-402. nuclei but not SCE in V79 cells. Treating Hesterberg, T.W.. and i.C. Barrett (1985) Induction by as chrysotile fibers with simulated pulmonary surfac bestos fibers of anaphase abnormalities: mechanism for tant reduced their cytotoxicity. Intermediatelength chrysotile asbestos induced micronuclei and multiple nuclei in a greater percentage of cells than did the short-length chrysotile asbestos. aneuploidy induction and possibly carcinogenesis. Carcino genesis. 6. 473-475. Hesterberg, T.W.. C.J. Butterick, M. Oshimura, A.R. Brody and J.C. Barrett (1986a) Role of phagocytosis in Syrian hamster cell transformation and cytogenetic effects in The primary effect of treating chrysotile fibers duced by asbestos and short and long glass fibers. Cancer with simulated pulmonary surfactant was to re duce cytotoxicity. DPL treatment had no consis tent significant effect on micronucleus or multi ple nucleus induction by intermediate length Res.. 46. 5795-5802. Hesterberg, T.W.. M. Oshimura, A.R. Brody and J.C. Barrett (1986b) Asbestos and silica induced morphological trans formation of mammalian cells in culture: a possible mech anism. in: D.F. Goldsmith. D.M. Win and C.M. Shy (Eds.i. fibers, while DPL treatment significantly reduced micronucleus and multiple nucleus induction by short-length chrysotile fibers. Silica. Silicosis and Cancer. Draeger. New York. pp. 177 190. Jaurand. M.C. (1985) Comparative responses of cultured cell' to asbestos fibres in relation to carcinogenicity, in: E.G. References Beck and J. Bignon (Eds.). In Vitro Effects of Mineral Dusts. Third International Workshop. Springer. Berlin, pp. 215-220. ~ Babu. K.A.. B.C. Lakkad. S.K. Nigam. D.K. Bhatt. A.B. Jaurand. M.C.. J.H. Thomassin. P. Baillif. L. Maaen. J.C. Karnik. K.N. Thakore. S.K. KashyapWntl S7K. Chatterjee Touray and J. Bignon (19.80) Chemical and photoelectron (J9S0) In vitro etiological and cytogenetic effects of an spectrometry analysis of the absorption of phospholipid Jam Kea \ Keb i Las Liiii Lig! Lis i I Obi- ( Osh Per Pot: model membranes and red blood cell membranes on to chrysotile fibers, Br. J. Ind. Med.. 37. 169-174. Jaurand, M.C.. A. Renter, A. Gaudichet. L. Kheuang, L. Magne and J. Bignon (1988) Short-term tests for the evaluation of potential cancer risk of modified asbestos fibers. Ann. N.Y. Acad. Sci., 534. 741-753. Keane. M.J., S.G. Xing, J.C. Harrison. T.-M. Ong and W.E. Wallace (1991) Genotoxicity of diesel exhaust particles dispersed in simulated pulmonary surfactant. Mutation Res.. 260. 233-238. Kelsey. K.T.. E. Yano, H.L. Liber and J.B. Little (1986) The in vitro genetic effects of fibrous erionite and crocidolite asbestos. Br. J. Cancer, 54, 107-114. Lavappa, K.S.. M.M. Fu and S.S. Epstein (1975) Cytogenetic studies on chrysotile asbestos. Environ. Res.. 10, 165-173. Light, W.G.. and E.T. Wei (1977a) Surface charge and as bestos toxicity. Nature (London), 26. 537-539. Light, W.G., and E.T. Wei (1977b) Surface charge and haemolytic activity of asbestos. Environ. Res., 13, 135-145. Livingston, G.K., W.N. Rom and M.V. Morris (1980) As bestos-induced sister-chromatid exchanges in cultured Chinese hamster ovarian fibroblast cells, J. Environ. Pathol. Toxicol., 4-2, 373-382. Oblin. A., J.M. Warnet, M.C. Jaurand, J. Bignon and J.K. Claude (1978) Biological effects of chrysotile after S03 absorption. III. Effects on the biochemical components of alveolar washing, Br. J. Exp. Pathol., 59, 32-37. Oshimura. M,, T.W. Hesterberg, T. Tsutsui and J.C. Barrett (1984) Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture. Cancer Res., 44, 5017-5022. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids. Nature (London). 251. 156-158. Pott, F., H.W. Schlipkoter. V. Ziem. K. Spurny and F. Huth (1984) New results from implantation experiments with mineral fibers, in: Biological Effects of Man-made Mineral Fibers, Vol. 2, World Health Organization. Regional Of fice for Europe. Copenhagen, pp. 286-302. 259 Rosin, M.P. (1985) In vitro simulation of concurrent expo sures to asbestos fibers and nitrosamines, in: E.G. Beck and J. Bignon (Eds.). In Vitro Effects of Mineral Dust, Third International Workshop. Springer, Berlin, pp. 253 259. Selikoff. I.J. (1979) Mortality experience of insulation workers in the United States and Canada. 1943-1976, Ann. N.Y. Acad. Sci.. 330. 91-116. Sincock. A.M., and M. Seabright (1975) Induction of Chromo some changes in Chinese hamster cells by exposure to asbestos fibers. Nature (London). 257, 56-58. Stanton, M.F., and C. Wrench (1972) Mechanisms of mesothelioma induction with asbestos and fibrous glass. J. Natl. Cancer. Inst., 48, 797-821. Stanton, M.F., M. Layard. A. Tegeris, E. Miller. M. May, E. Morgan and A. Smith (1981) Relation of particle dimen sion to carcinogenicity in amphibole asbestos and other fibrous minerals. J. Natl. Cancer Inst., 67. 965-975. Tetley, T.D., R.J. Richards and J.L. Harwood (1977) Change in pulmonary surfactant and phosphatidylcholine metab olism in rats exposed to chrysotile asbestos dust, Biochem. J., 166. 323-329. Vallyathan, V., N. Hahon, J. Booth, D. Schwegler and M. Sepulveda (1985) Cytotoxicity of native and surface-mod ified asbestos, in: E.G. Beck and J. Bignon (Eds.). In Vitro Effects of Mineral Dust. Third International Workshop, Springer, Berlin, pp. 159-165. Wagner. J.C.. G. Berry and V. Timbrell (1973) Mesothelioma in rats'after inoculation with asbestos and other materials. Br. J. Cancer, 28. 173-185. Wagner, J.C., F.D. Pooley, G. Berry, R.M.E. Seal, D.E. Munday. J. Morgan and N.J. Clark (1982) A pathological and mineralogical study of asbestos-related deaths in the United Kingdom in 1977. Ann. Occup. Hyg.. 26, 423-431. Wallace, W.E.. M.J. Keane. C.A. Hill, J. Xu and T.-M. Ong (1987) Mutagenicity of diesel exhaust particles and oil shale particles dispersed in lecithin surfactant, J. Toxicol. Environ. Health, 21. 163-171. er tl > !> I J/ J 1 February 1994 Appearing monthly Vol. 320, No. 3 ISSN 0165-1218 MUREAV 320 (3) 165-252 (1994) Managing Editors: M.D. Shelby P.H.M. Lohman FE8 9 1994 NOT TC8E TAKE* FHOM LIBRARY ELSEVIER A Section of .illation eseardh Founding Editor: F.H. Sobels