A comparison of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines

Mutation Research, 101 (1982) 257-268 Elsevier BiomedicalPress

257

A comparison.of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines Demonstration of growth inhibition in primary human fibroblasts A.M. Sincock I, J.D.A. Delhanty and G. Casey 2 The Galton Laboratory, Department of Genetics and Biometry, University College London, London (Great Britain)

(Received 17 September 1981) (Revisionreceived25 November 1981) (Accepted 26 November 198l)

Summary Asbestos and fine glass fibre, which induce high levels of chromosome aberrations and polyploidy in Chinese hamster permanent cell lines, were found to cause no increase in chromosome damage or polyploidy in primary human fibroblasts or in human lymphoblastoid lines. In common with permanent cell lines of hamster or human origin, treatment of primary human fibroblasts with higher doses of asbestos or fine glass resulted in almost total growth inhibition, showing that the primary cells are not unaffected by these agents. The reason for lack of evident cytogenetic damage in primary cells may lie in the greater spontaneous karyotype instability of transformed (permanent) cell lines or may be connected with the less efficient D N A repair capacity of Chinese hamster ovary cells.

Asbestos is known to produce neoplasia of the human lung (Selikoff et al., 1964) and other thoracic disorders, principally asbestosis (Selikoff et al., 1965). In rats, following intrapleural inoculation, both asbestos (Wagner et al., 1973) and more recently glass fibre (Stanton et al., 1977) have been shown to produce lung tumours. N o reports to date have provided any direct evidence that neoplastic changes occur in the human lung following occupational exposure to glass fibres (Shulte, 1976). As the cellular level asbestos has been shown to be taken up by lung macrophages (Davis, 1963) and Chinese hamster lung cells (Huang et al., 1978). Both asbestos Present address: Fund for the Replacementof Animals in Medical Experiments,London SW20. 2 Present address: Institute of Cancer Research, Royal Marsden Hospital. 0165-1218/82/0000-0000/$02.75 © ElsevierBiomedicalPress

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259 (Sincock and Seabright, 1975; Huang et al., 1978) and glass fibre (Sincock, 1977) have been shown to induce chromosome aberrations after short periods of exposure in Chinese hamster cells. In Syrian hamster embryo cells chrysotile was found to induce dose-related chromosome aberrations and inhibition of the mitotic index (Lavappa et al., 1975). However, in vivo treatment of Rhesus monkeys and Swiss albino mice failed to induce chromosome damage in bone-marrow cells (Lavappa et al., 1975). The present study is a comparison of the response to asbestos and glass fibre in established Chinese hamster cells, primary human fibroblasts and human lymphoblastoid cell lines. The aim was to discover whether human cells could be used to monitor the cytogenetic effects of asbestos and glass dusts and possible asbestos substitutes instead of the ubiquitous Chinese hamster cells.

Materials and methods

Cell cultures used for chromosome studies are listed in Table 1, together with tissue of origin, growth media and source of the cells. The human fibroblasts did not exceed the 15th passage. Cells were regularly tested for mycoplasma contamination with no positive results (Chen, 1977). The dusts employed consisted of the U.I.C.C. sample crocidolite (Timbrell et al., 1971) and S.F.A.-chrysotile, a superfine sample from a Canadian mine (Wagner et al., 1973). The mean particle lengths of the asbestos samples fell within the limits of 1.1-3.4 # while the mean particle diameters varied between 0.15-0.47/~. Glass fibres code 100 and 110 (Johns Manville) had been prepared by chopping and grinding. Their mean particle lengths varied between 2.7 and 26 #, and mean particle diameters between 0.12 and 1.9 # (Timbrell, 1976). Asbestos and glass dusts were autoclaved dry before use and suspended in sterile phosphate-buffered saline. Chromosome studies Human fibroblasts and CHO ceils were mixed with dusts while still in suspension and seeded at a density of 1.37 × 105 per 25 cm2 flask. Lymphoblastoid ceils were set up at a density of 7.5 × 105 in 10 ml of medium in flat bottomed 20-ml plastic containers; dusts were then added to give the appropriate concentrations. Treatment was for 48 or 72 h prior to harvest in the case of CHO cells and fibroblasts, 72 h for lymphoblastoid lines, at concentrations ranging between 0.01 and 0.1 mg/ml. From time to time throughout this period flasks were agitated in order to resuspend the fibres. At the end of the exposure period chromosomes were prepared and stained by standard methods. Metaphases were scored for gaps, chromosome and chromatid breaks, marker chromosomes indicating gross rearrangements and consistent addition or loss of chromosomes. A minimum of 50 cells was scored per treatment for each cell line. Polyploidy was estimated by examining 200 dividing cells per treatment. Lymphoblastoid preparations were also assessed for micronuclei formation; 2000 interphase cells were examined for each treatment.

260

Chromosome studies were performed on all human fibroblast lines listed in Table 1 with the exception of 77LO.

Growth experiments It had been noted that concentrations of asbestos dust higher than 0.02 m g / m l resulted in few scorable mitoses in the human fibroblasts. For this reason normal adult (77LO) and foetal (59LO) human fibroblasts were used in experiments to assess the effect on cell growth of higher concentrations of dusts. Cells from confluent monolayers were seeded at a concentration of approx. 2 × 105/ml and fibres were added at concentrations of 0.05 and 0.1 m g / m l before the cells had plated. Fibres used in these experiments were S.F.A. chrysotile and glass code 1 l0 and 100. Growth was followed over a 4-day period. Each day, 4 25-cm2 flasks were harvested for each treatment and for the controls. Cells were counted by means of a haemacytometer; an aliquot was treated with 0.04% trypan blue to determine viability. An additional aliquot of cells was fixed and stained in order to estimate the mitotic index.

Results

Chromosome studies The Chinese hamster cells exposed to the 2 asbestos fibres showed the expected high level of induced chromosome damage following 48-h and 72-h exposure (Table 2). A lower level of damage was apparent in cells treated with glass code 100, but this was still well above the control level. However, the coarse glass fibre, code

TABLE 2 C H I N E S E H A M S T E R CELLS: R E S U L T S OF C H R O M O S O M E S T U D I E S Cell line

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Duration (h)

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48 48

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11 13

3 0

30 32

29 35

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0.01 0.01

48 48

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0 8

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2 24

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72

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72 72

2 4

7 17

3 1

24 42

29 26

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0.01 0.01

72 72

1 2

0 7

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2 18

5 14

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263 110, p r o d u c e d n o i n c r e a s e d c h r o m o s o m e d a m a g e . T h e o b s e r v e d d a m a g e d u e to b o t h t y p e s of a s b e s t o s a n d glass 100 consisted m a i n l y of c h r o m o s o m e b r e a k s a n d r e a r r a n g e m e n t s , p o l y p l o i d y was c o n s i d e r a b l y i n c r e a s e d following asbestos t r e a t m e n t , b u t n o t m a r k e d l y so with glass 100. A r e p r e s e n t a t i v e s a m p l e of results for h u m a n f i b r o b l a s t s is given in T a b l e 3. I n all, 16 e x p e r i m e n t s o n the 5 different lines were p e r f o r m e d . N o significant c h r o m o s o m e d a m a g e a b o v e c o n t r o l levels was f o u n d in a n y of these experiments. Similarly, n o i n c r e a s e d d a m a g e was o b s e r v e d following t r e a t m e n t o f the 2 h u m a n l y m p h o b l a s t o i d lines ( T a b l e 4). P o l y p l o i d y a n d m i c r o n u c l e i f o r m a t i o n also r e m a i n e d at c o n t r o l levels a f t e r t r e a t m e n t ; i n c r e a s e d m i c r o n u c l e i f o r m a t i o n in the 1526 line is in k e e p i n g with k n o w n c h r o m o s o m e i n s t a b i l i t y in this s y n d r o m e , a t a x i a telangiectasia. Growth experiments T h e results of 2 of the series of growth e x p e r i m e n t s in terms o f total cell n u m b e r h a r v e s t e d each d a y , p e r c e n t a g e viability a n d m i t o t i c i n d e x are given in T a b l e s 5 a n d 6. R e p r e s e t a t i v e g r o w t h curves are shown in Fig. 1. T h e presence o f chrysotile o r glass 100 at either 0.05 or 0.1 m g / m l in cultures of

TABLE 5 RESULTS OF GROWTH EXPERIMENTS FOLLOWING TREATMENT OF NORMAL ADULT HUMAN FIBROBLASTS WITH CHRYSOTILE ASBESTOS AND GLASS FIBRE Cell number loglo 0.05 mg/ml

Viability (%)

0.1 mg/ml 0.05 mg/ml

Day 1

Day 2

Day 3

Day 4

Cells in mitosis (%)

Control Chrysotile Glass 110 Glass 100

6.29 +-0.01 6.24---0.01 6.17±0.01 6.21 ---0.02 6.16±0.01 6.19+-4-0.01 6.23---0.01

93 95 95

Control Chrysotile Glass 110 Glass 100

6.60 ± 0.02 6.32+-0.02 6.30+---0.02 6.60±0.03 6.58-4-0.02 6.30±0.01 6.31 +---0.02

96 96 97

Control Chrysotile Glass 110 Glass 100

6.85 ± 0.02 6.35---0.02 6.33+-0.02 6.77±0.02 6.76---0.03 6.31-4-0.01 6.30±0.02

96 95 94

Control Chrysotile Glass I10 Glass 100

6.89±0.04 6.37+0.03 6.33±0.02 6.83+-0.03 6.80+-0.04 6.30±0.02 6.32-----0.02

93 94 97

0.1 mg/ml

0.05 mg/ml

96 94 98

0 0.3 0.01

95 97 94

0.3 1.5 0.2

98 97 98

0.6 1.5 0

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96

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Cell numbers represent the mean and standard deviation of those from 4 × 25 cm2 pooled flasks which were coded prior to harvest.

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266 TABLE 6 RESULTS OF GROWTH EXPERIMENTS FOLLOWING TREATMENT OF NORMAL FOETAL HUMAN FIBROBLASTS WITH CHRYSOTILE ASBESTOS AND GLASS FIBRE Cell number logl0 0.05 mg/ml

Viability (~)

0.1 mg/ml 0.05 mg/ml

Day 1

Day 2

Day 3

Day 4

Cells in mitosis (%)

Control Chrysotile Glass 110 Glass 100

6.15-+0.01 6.24±0.02 6.17-+0.01 6.21±0.01 6.16-+0.02 6.19---0.01 6.23-+0.02

96 94 97

Control Chrysolite Glass 110 Glass 100

6.63 ± 0.02 6.53 -+0.02 6.30 ± 0.01 6.56±0.02 6.54±0.02 6.31 ±0.02 6.26±0.01

94 94 96

Control Chrysotile Glass 110 Glass 100

6.84±0.03 6.52±0.02 6.33±0.02 6.77-+0.03 6.74±0.03 6.45±0.02 6.31 ±0.02

94 96 98

Control Chrysotile Glass 110 Glass 100

6.95 ± 0.04 6.65 + 0.03 6.45 ± 0.02 6.93±0.04 6.91 ±0.03 6.55-+0.03 6.32±0.02

95 94 97

0.1 mg/ml

0.05 mg/ml

95 94 98

0 0.3 0.2

95 97 98

I. 1 1.5 0.3

96 97 94

0.2 1.4 0.3

96 98 97

0.4 0.4 0.2

98

0.1 mg/ml 0.3

96

0 0.3 0.1 1.5

95

0.2 1.3 0.2 1.8

96

0.4 1.6 0 0.1 0.2 0.2 0

Cell numbers represent the mean and standard deviation of those from 4×25 cm2 pooled flasks which were coded prior to harvest.

a d u l t h u m a n fibroblasts results in very little increase in cell n u m b e r o v e r 4 days. T h e foetal fibroblasts a p p e a r m o r e resilient a n d s o m e increase in cell n u m b e r occurs in the p r e s e n c e of these agents, a l t h o u g h c o n s i d e r a b l e i n h i b i t i o n is still evident. C o a r s e glass c o d e 110 has n o effect on g r o w t h in foetal or ad u l t fibroblasts. It is e v i d e n t f r o m viabilities at harvest of t r e a te d cells that n o a p p r e c i a b l e a m o u n t o f cell d e a t h resulted f r o m the presence o f asbestos or glass dusts. L o w e r e d cell n u m b e r s at h ar v es t in cells treated with chrysotile asbestos or glass c o d e 100 ap p ear s to result f r o m g r o w t h inhibition, as e v i d e n c e d by the l o w e r e d m i t o t i c indices in these cultures.

Discussion T h e r e is n o o b v i o u s e x p l a n a t i o n f o r the lack of cells f o l l o w i n g t r e a t m e n t with asbestos a n d fine h a m s t e r cells. H o w e v e r , all the h a m s t e r lines w h i c h cell lines a n d t h e r e f o r e the result o f t r a n s f o r m a t i o n ,

c y t o g e n e t i c d a m a g e to h u m a n glass c o m p a r e d with Ch i n ese h a v e b e e n used are p e r m a n e n t albeit s p o n t a n e o u s .

267

The CHO cells which we used exhibit a background level of karyotype instability, a property of most transformed cells. This may influence the expression of induced chromosome damage as a result of asbestos or glass fibre treatment. We included cells from people with a genetic predisposition to cancer (FPC1 and EN1) and spontaneous chromosome instability (ataxia telangiectasia) in our experiments since they seemed the most likely to exhibit induced damage from a known carcinogenic agent. The human lymphoblastoid lines are also transformed cells, produced as a result of treatment with Epstein-Barr virus and thus might have been expected to behave more like the CHO cells. However, although the chromosomes of lymphoblastoid lines do undergo spontaneous change after some time in culture they are much less labile than the chromosomes of the CHO cells (Steel et al., 1980). If the transformed state has an effect on chromosome stability, with respect to asbestos and fine glass, it is perhaps significant that in vivo treatment of Rhesus monkeys and Swiss albino mice failed to induce chromosome damage in bone-marrow cells (Lavappa et al., 1975). Chinese hamster cell lines and a permanent human-derived cell line have previously been shown to be growth-inhibited by the presence of asbestos and fine glass (Chamberlain and Brown, 1978; Huang et al., 1978). CHO-K1 cells exposed to the same dusts and at the same concentration as we used for chromosome studies exhibited significant mitotic inhibition as did human fibroblasts (G. Casey, manuscript submitted). It is possible that the lack of chromosome aberrations in the human cells is due to their greater proficiency at excision repair following DNA damage. CHO cells are known to have moderate excision repair capability (Wolf, 1979). Without electron microscopy it is impossible to compare relative uptake of fibre by human fibroblasts and CHO cells, which may be of importance. It is of interest that coarse glass fibre, code 110, has no effect on growth of human cells whereas fine glass, code 100, is growth-inhibitory, since fibre diameter has been shown to be related to carcinogenicity (Stanton et al., 1977). During the preparation of this paper our attention was drawn to a report on the cytogenetic effects of Rhodesian chrysotile on human lymphocytes (Valerio et al., 1980). An approximate doubling of chromosome and chromatid breaks was found with treatment. However, previous experiments employing the dusts we have used here on human lymphocytes produced no damage (Sincock and Seabright, unpublished observations).

Acknowledgements We thank John Attwood for efficient laboratory organization and for drawing the figures, Jean Barrie for patient typing and help of all kinds, and the Health and Safety Executive of the U.K. for financial support.

268

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