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Cytotoxic and genotoxic effects of calcium silicates on human lymphocytes in vitro
Mutation Research, 300 (1993) 45-48 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
45
MUTGEN 01877
Cytotoxic and genotoxic effects of calcium silicates on human lymphocytes in vitro M. A s l a m a n d Q. R a h m a n Industrial Toxicology Research Centre, Lucknow-226001, India (Received 29 April 1992) (Revision received 10 December 1992) (Accepted 10 December 1992)
Keywords: Calcium silicate; Human lymphocytes; Sister-chromatid exchanges; Chromosomal aberrations; Proliferation rate index
Summary Calcium silicate has been considered as a possible replacement for asbestos due to its heat and fire resistance. We describe the cytotoxic and genotoxic potential of calcium silicate using peripheral human blood lymphocytes. Calcium silicates at concentrations of 10 and 100/zg/ml significantly increased the frequencies of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs). The increases in CAs and SCEs were dose-dependent, though not linearly. A significant decrease in the proliferation rate index was observed with increased dose of calcium silicates. The induction of chromatid-type aberrations indicates that the clastogenic activity of calcium silicate is S-phase-dependent.
Several epidemiological and experimental studies have shown the carcinogenic and genotoxic properties of asbestos (Edelman, 1988; Mossman, 1988; Daniel, 1983). These properties have restricted its use and efforts have been made to find suitable substitutes, usually other fibrous materials. Among the substitutes, calcium silicate has been considered because of its high thermal resistance. Several epidemiological and experimental studies are available on calcium silicate (Mossman, 1988; Aslam, 1990; Aslam et al., 1992; Skaug et al., 1984; Skaug and Gylseth, 1983; Skaug and Haugen, 1989), but apparently no report exists on its possible carcinogenicity and
Correspondence to: Dr. M. Aslam, Fibre Toxicology Section, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P.O. Box 80, Lucknow-226001, U.P., India.
genotoxicity. In view of this, a series of in vitro and in vivo experiments have been carried out in our laboratory. The present paper deals with experiments to evaluate chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs) and proliferation rate index (PRI) in human peripheral blood lymphocytes (PBLs) cultured with calcium silicates in vitro. Materials and methods Calcium silicate samples, namely Kemolit ASB-3, Kemolit-N and Kemolit A-60, were obtained from the Director, Wolkem Pvt. Ltd., Udaipur, Rajasthan, India. The purity of calcium silicate samples is approximately 97%. The differences between the three samples are the percentage of calcium oxide and silica oxide. Na, Mg, AI, K and Fe were also found in trace amounts.
46 serum, 0.03% L-glutamine, 100 IU penicillin, 100 /~g/ml streptomycin, 3% P H A - M and 5 txg/ml BrdUrd according to the method of Moorhead et al. (1960). Before incubation, 0.35 ml of calcium silicate suspension was added to cultures in a final volume of 7.0 ml. Equal volumes of medium only were added to the control cultures. All cultures were incubated in the dark at 37°C in a 5% C O 2 / 9 5 % O 2 atmosphere for 48 or 72 h to study CAs and sister-chromatid differentiation (SCD) respectively.
Sources of other chemicals were: 5-bromo-2-deoxyuridine (BrdUrd), bisbenzamide (Hoechst 33258) and colchicine were purchased from Sigma Chemical Company (St. Louis, MO, USA), fetal calf serum (FCS) from Sera (UK), medium RPMI-1640, L-glutamine, penicillin, streptomycin and phytohaemoagglutinin-M (PHA-M) from Gibco Laboratories (Grand Island, NY, USA). Other chemicals used were of analytical grade with high purity. Calcium silicate with a particle size of less than 30/zm was prepared according to the method of Zaidi (1969).
Chromosome preparation and staining Colchicine (0.1 tzg/ml) was added to the cultures 3 h prior to harvesting. The cells were then treated with hypotonic solution (75 mM KC1) at 37°C for 20 min and centrifuged at 1200 rpm for 10 min. Freshly prepared fixative (methanol:glacial acetic acid, 3 : 1 ) was added to the pellets. After three changes of fixative, the cell suspension was dropped onto precleaned chilled glass slides. Slides were stained with 4% Giemsa in phosphate buffer (pH 6.8) for 10-15 min and 100 metaphases were scored for aberration studies. Differential chromatid staining was performed according to Perry and Wolff (1974) with a slight modification. Slides were treated with 5 0 / z g / m l bisbenzamide (Hoeehst 33258) in the dark for 15
Lymphocyte culture Peripheral venous blood was collected prior to breakfast from a 30-year-old male non-smoking volunteer donor, who was not on any medication, in heparinised vials under sterile conditions. PBLs were obtained by Ficoll-Hypaque gradient centrifugation (Boyum, 1968). The sterilised calcium silicate samples were suspended at a concentration of 2 m g / m l in RPMI-1640 medium and maintained under constant stirring. Dust suspensions of different concentrations ( 0 . 1 - 1 0 0 / ~ g / m l ) were prepared by serial dilution. The washed PBLs were suspended and cultured in RPMI-1640 medium supplemented with 10% foetal calf
TABLE 1 PERCENTAGE OF ABERRANT METAPHASESIN HUMAN LYMPHOCYTESCULTURED WITH CALCIUMSILICATES Agent
Dose (/xg/ml)
Control Kemolit ASB-3
0.0
Excluding gaps
Gaps Chromatid
Breaks Isochromatid Chromatid
4
-
4
-
-
5 6 9*
-
5 5 7
1 2
-
11 *
2
7
2
2
-
4 6
1 1
-
1
7
2
1
100.0
5 7 10 * 13 *
3
8
2
2
0.1 1.0 10.0 100.0
6 8 12 * 15 *
2 2 3
5 4 8 9
1 2 2 3
2 1 2
0.1 1.0 10.0 100.0
Kemolit-N
0.1 1.0 10.0
Kemolit A-60
Damaged Including gaps
* p < 0.005 (x 2 test).
Isochromatid
-
1
1 1
47 min, e x p o s e d to b r i g h t sunlight in t h e p r e s e n c e o f 2 x S S C for 2 h a n d t h e n s t a i n e d with 4 % G i e m s a in p h o s p h a t e b u f f e r ( p H 6.8). A n a l y s e s o f S C E s w e r e c a r r i e d o u t f r o m 50 cells at s e c o n d ( M 2) mitosis.
Evaluation of cell cycle kinetics Cell cycle kinetics w e r e d e t e r m i n e d from 200 m e t a p h a s e s by m e a n s o f t h e relative f r e q u e n c i e s o f first (M1) , s e c o n d (M2), t h i r d a n d s u b s e q u e n t ( M 3) division m e t a p h a s e cells. P R I was c a l c u l a t e d by t h e m e t h o d o f K r i s h n a et al. (1986) using t h e f o r m u l a PRI =
1M1+2M2+3M
3
100
where MI, M z and M 3 represent proportions of first, s e c o n d a n d t h i r d plus s u b s e q u e n t division m e t a p h a s e s respectively.
a b e r r a n t m e t a p h a s e s at h i g h e r doses, i.e., 10 a n d 1 0 0 / z g / m l , w h e n c o m p a r e d with t h e i r r e s p e c t i v e controls ( T a b l e 1). T h e p e r c e n t a b e r r a n t m e t a phases showed a dose-dependent response, t h o u g h n o t linearly. G a p - t y p e a b e r r a t i o n s w e r e m o r e c o m m o n t h a n o t h e r types. C h r o m a t i d - t y p e aberrations rather than chromosome-type were g e n e r a l l y observed. A n i n c r e a s e in M 1 cells a n d a d e c r e a s e in M 3 a n d s u b s e q u e n t m e t a p h a s e s w e r e o b s e r v e d at t h e h i g h e r c o n c e n t r a t i o n s with all types o f calcium silicate used. This r e s p o n s e was not d i r e c t l y p r o p o r t i o n a l to t h e c o n c e n t r a t i o n . N o m a r k e d effect on p r o l i f e r a t i o n c o u l d b e o b s e r v e d at lower c o n c e n t r a t i o n s . T a b l e 2 clearly i n d i c a t e s t h a t as t h e silicate c o n c e n t r a t i o n i n c r e a s e s t h e r e is a d e c l i n e in P R I values. T a b l e 2 also shows t h a t t h e f r e q u e n c y o f S C E s i n c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s b u t n o t directly p r o p o r t i o n a l to the concentration.
Results
Discussion
K e m o l i t A-60, K e m o l i t - N a n d K e m o l i t A S B - 3 i n d u c e d significantly i n c r e a s e d f r e q u e n c i e s o f
T h e results i n d i c a t e t h a t c a l c i u m silicates significantly i n d u c e d cell cycle d e l a y a n d genotoxic-
TABLE 2 SCE FREQUENCIES, DISTRIBUTION OF M 1, M 2 AND TREATED HUMAN LYMPHOCYTES Agent
M3
CELLS AND PRI IN CONTROL AND CALCIUM SILICATE-
Dose (/zg/ml)
SCE/metaphase (mean 5: SE)
M1
Metaphases (%) M2
M3
PRI
Kemolit ASB-3
0.0 0.1 1.0 10.0 100.0
8.2 5:2.6 9.1 ± 1.6 9.8 _+2.1 11.3 5:1.3 * 14.0± 1.8 *
18 18 25 28 39
25 27 28 27 34
57 55 47 45 27
2.39 2.37 2.22 2.17 1.88 t
Kemolit-N
0.0 0.1 1.0 10.0 100.0
7.8 ± 2.5 8.9 _+2.3 9.9 ± 2.3 11.5 ± 1.4 * 14.8± 1.8 *
15 18 21 32 38
29 29 36 30 35
56 53 43 38 27
2.41 2.35 2.22 2.06 t 1.89 t
Kemolit A-60
0.0 0.1 1.0 10.0 100.0
7.9 5:2.3 8.8 ± 2.1 10.2 ± 2.4 12.4±2.4 * 15.7 ± 2.5 *
17 18 22 30 42
27 32 40 38 33
56 50 38 32 25
2.39 2.32 2.16 2.02 t 1.83 *
For SCEs, 50 metaphases of the second cell division were analysed. For cell cycle kinetics, a total of 200 metaphases per treatment were analysed. * p < 0.05 (Student's t-test); t p < 0.05 (X 2 test).
48 ity. A significant i n c r e a s e in t h e f r e q u e n c y of c h r o m a t i d - t y p e a b e r r a t i o n s , (gaps a n d b r e a k s ) was observed rather than chromosome-type aberrations. This i n d i c a t e s t h a t t h e clastogenicity of calcium silicate occurs m o s t likely by an S - p h a s e dependent mechanism. It has o f t e n b e e n o b s e r v e d t h a t S C E s can b e i n d u c e d at d o s e s insufficient to give rise to C A s (Wolff, 1982; D e e n et al., 1989). So, it has b e e n s u g g e s t e d t h a t t h e S C E assay is m o r e sensitive t h a n t h e C A assay ( H a d n a g y et al., 1989). T h e f r e q u e n c y of S C E s was significantly i n c r e a s e d at h i g h e r c o n c e n t r a t i o n s a n d e x h i b i t e d a positive dose response. The method of BrdUrd labelling for S C E analysis was also u s e d s i m u l t a n e o u s l y for investigation of c e l l u l a r kinetics ( A b e l , 1987; H a d n a g y et al., 1989). C a l c i u m silicates a l t e r e d cell kinetics causing cell cycle d e l a y o f v a r i o u s d e g r e e s . T h e d e c r e a s e in P R I v a l u e s i n d i c a t e s that t h e s e silicates i n d u c e d p r o l o n g a t i o n o f cell cycle d u r a t i o n . Cell cycle delay, in a d d i t i o n to SCE, m a y r e p r e s e n t a n o t h e r level o f biological d a m a g e resulting f r o m silicate t r e a t m e n t . H o w ever, t h e d a t a o b t a i n e d d o not allow us to assess t h e specific stage at which the d e l a y o c c u r r e d .
Acknowledgements T h e a u t h o r s a r e t h a n k f u l to Prof. P.K. Ray, D i r e c t o r o f t h e C e n t r e , for his k e e n i n t e r e s t in t h e w o r k a n d to Mr. M. A s h q u i n for his skilled t e c h n i c a l assistance. This w o r k was s u p p o r t e d by t h e D i r e c t o r a t e of E n v i r o n m e n t , G o v e r n m e n t o f U t t a r P r a d e s h , India.
References Abel, G. (1987) Chromosome damage induced in human lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A, Mutation Res., 190, 63-68. Aslam, M. (1990) Ph.D. Thesis, Aligarh Muslim University, Aligarh.
Aslam, M., M. Ashquin and Q. Rahman (1992) In vitro cytotoxic effects of wollastonites on rat hepatocytes. II. Lipid peroxidation and glutathione depletion, Bull. Environ. Contam. Toxicol., 49, 547-554. Boyum, A. (1968) Isolation of leukocytes from human blood, Scand. J. Clin. Invest., 21 (Suppl. 97), 9-29. Daniel, F.B. (1983) In vitro assessment of asbestos geuotoxicity, Environ. Health Perspect., 53, 163-167. Deen, D.F., W.F. Morgan, P.J. Tolfilson and M.F. Barcellos Hoff (1989) Measurement of sister chromatid exchanges and their relationship to DNA damage and cell killing, Pharmacol. Ther., 42, 349-360. Edelman, D.A. (1988) Asbestos exposure, pleural plaques and the risk of lung cancer, Int. Arch. Occup. Environ. Health., 60, 389-393. Hadnagy, W., N.H. Seemayer, R. Tomingas and K. Ivanfy (1989) Comparative study of SCEs and chromosomal aberrations induced by airborne particulates from an urban and a highly industrialized location in human lymphocyte cultures, Mutation Res., 225, 27-32. Krishna, G., J. Nath and T. Ong (1986) Murine bone marrow culture systems for cytogenetic analysis, Mutation Res., 164, 91-99. Moorhead, P.S., P.C. Nowell, W.J. Mellman, D.M. Battips and B.A. Hungerford (1960) Chromosome preparation Leukocytes cultured from human peripheral blood, Exp. Cell Res., 20, 613-616. Mossman, B.T. (1988) Carcinogenic potential of asbestos and non-asbestos fibres, Environ. Carcinogen. Rev. (J. Environ. Sci. Health), C. 6, 151-165. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature, 251, 156-158. Skaug, V., and B. Gylseth (1983) Hemolytic activity of five calcium silicates, Environ. Health Perspect., 51, 195-203. Skaug, V., and A. Haugen (1989) Cytotoxicity of asbestos and calcium silicates to various lung epithelial cell types in vitro, in: B.T. Mossman and R.O. Begin (Eds.), Effect of Mineral Dusts on Cells, Springer, Berlin, pp. 7-13. Skaug, V., R. Davies and B. Gylseth (1984) In vitro macrophage cytotoxicity of five calcium silicates, Br. J. Ind. Med., 41, 116-121. Wolff, S. (1982) Chromosome aberrations, sister chromatid exchanges and the lesions that produce them, in: S. Wolff (Ed.), Sister Chromatid Exchange, Wiley, New York, pp. 41-57. Zaidi, S.H. (1969) In: Experimental Pneumoconiosis, Johns Hopkins University Press, Baltimore, MD, pp. 35-41.
45
MUTGEN 01877
Cytotoxic and genotoxic effects of calcium silicates on human lymphocytes in vitro M. A s l a m a n d Q. R a h m a n Industrial Toxicology Research Centre, Lucknow-226001, India (Received 29 April 1992) (Revision received 10 December 1992) (Accepted 10 December 1992)
Keywords: Calcium silicate; Human lymphocytes; Sister-chromatid exchanges; Chromosomal aberrations; Proliferation rate index
Summary Calcium silicate has been considered as a possible replacement for asbestos due to its heat and fire resistance. We describe the cytotoxic and genotoxic potential of calcium silicate using peripheral human blood lymphocytes. Calcium silicates at concentrations of 10 and 100/zg/ml significantly increased the frequencies of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs). The increases in CAs and SCEs were dose-dependent, though not linearly. A significant decrease in the proliferation rate index was observed with increased dose of calcium silicates. The induction of chromatid-type aberrations indicates that the clastogenic activity of calcium silicate is S-phase-dependent.
Several epidemiological and experimental studies have shown the carcinogenic and genotoxic properties of asbestos (Edelman, 1988; Mossman, 1988; Daniel, 1983). These properties have restricted its use and efforts have been made to find suitable substitutes, usually other fibrous materials. Among the substitutes, calcium silicate has been considered because of its high thermal resistance. Several epidemiological and experimental studies are available on calcium silicate (Mossman, 1988; Aslam, 1990; Aslam et al., 1992; Skaug et al., 1984; Skaug and Gylseth, 1983; Skaug and Haugen, 1989), but apparently no report exists on its possible carcinogenicity and
Correspondence to: Dr. M. Aslam, Fibre Toxicology Section, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P.O. Box 80, Lucknow-226001, U.P., India.
genotoxicity. In view of this, a series of in vitro and in vivo experiments have been carried out in our laboratory. The present paper deals with experiments to evaluate chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs) and proliferation rate index (PRI) in human peripheral blood lymphocytes (PBLs) cultured with calcium silicates in vitro. Materials and methods Calcium silicate samples, namely Kemolit ASB-3, Kemolit-N and Kemolit A-60, were obtained from the Director, Wolkem Pvt. Ltd., Udaipur, Rajasthan, India. The purity of calcium silicate samples is approximately 97%. The differences between the three samples are the percentage of calcium oxide and silica oxide. Na, Mg, AI, K and Fe were also found in trace amounts.
46 serum, 0.03% L-glutamine, 100 IU penicillin, 100 /~g/ml streptomycin, 3% P H A - M and 5 txg/ml BrdUrd according to the method of Moorhead et al. (1960). Before incubation, 0.35 ml of calcium silicate suspension was added to cultures in a final volume of 7.0 ml. Equal volumes of medium only were added to the control cultures. All cultures were incubated in the dark at 37°C in a 5% C O 2 / 9 5 % O 2 atmosphere for 48 or 72 h to study CAs and sister-chromatid differentiation (SCD) respectively.
Sources of other chemicals were: 5-bromo-2-deoxyuridine (BrdUrd), bisbenzamide (Hoechst 33258) and colchicine were purchased from Sigma Chemical Company (St. Louis, MO, USA), fetal calf serum (FCS) from Sera (UK), medium RPMI-1640, L-glutamine, penicillin, streptomycin and phytohaemoagglutinin-M (PHA-M) from Gibco Laboratories (Grand Island, NY, USA). Other chemicals used were of analytical grade with high purity. Calcium silicate with a particle size of less than 30/zm was prepared according to the method of Zaidi (1969).
Chromosome preparation and staining Colchicine (0.1 tzg/ml) was added to the cultures 3 h prior to harvesting. The cells were then treated with hypotonic solution (75 mM KC1) at 37°C for 20 min and centrifuged at 1200 rpm for 10 min. Freshly prepared fixative (methanol:glacial acetic acid, 3 : 1 ) was added to the pellets. After three changes of fixative, the cell suspension was dropped onto precleaned chilled glass slides. Slides were stained with 4% Giemsa in phosphate buffer (pH 6.8) for 10-15 min and 100 metaphases were scored for aberration studies. Differential chromatid staining was performed according to Perry and Wolff (1974) with a slight modification. Slides were treated with 5 0 / z g / m l bisbenzamide (Hoeehst 33258) in the dark for 15
Lymphocyte culture Peripheral venous blood was collected prior to breakfast from a 30-year-old male non-smoking volunteer donor, who was not on any medication, in heparinised vials under sterile conditions. PBLs were obtained by Ficoll-Hypaque gradient centrifugation (Boyum, 1968). The sterilised calcium silicate samples were suspended at a concentration of 2 m g / m l in RPMI-1640 medium and maintained under constant stirring. Dust suspensions of different concentrations ( 0 . 1 - 1 0 0 / ~ g / m l ) were prepared by serial dilution. The washed PBLs were suspended and cultured in RPMI-1640 medium supplemented with 10% foetal calf
TABLE 1 PERCENTAGE OF ABERRANT METAPHASESIN HUMAN LYMPHOCYTESCULTURED WITH CALCIUMSILICATES Agent
Dose (/xg/ml)
Control Kemolit ASB-3
0.0
Excluding gaps
Gaps Chromatid
Breaks Isochromatid Chromatid
4
-
4
-
-
5 6 9*
-
5 5 7
1 2
-
11 *
2
7
2
2
-
4 6
1 1
-
1
7
2
1
100.0
5 7 10 * 13 *
3
8
2
2
0.1 1.0 10.0 100.0
6 8 12 * 15 *
2 2 3
5 4 8 9
1 2 2 3
2 1 2
0.1 1.0 10.0 100.0
Kemolit-N
0.1 1.0 10.0
Kemolit A-60
Damaged Including gaps
* p < 0.005 (x 2 test).
Isochromatid
-
1
1 1
47 min, e x p o s e d to b r i g h t sunlight in t h e p r e s e n c e o f 2 x S S C for 2 h a n d t h e n s t a i n e d with 4 % G i e m s a in p h o s p h a t e b u f f e r ( p H 6.8). A n a l y s e s o f S C E s w e r e c a r r i e d o u t f r o m 50 cells at s e c o n d ( M 2) mitosis.
Evaluation of cell cycle kinetics Cell cycle kinetics w e r e d e t e r m i n e d from 200 m e t a p h a s e s by m e a n s o f t h e relative f r e q u e n c i e s o f first (M1) , s e c o n d (M2), t h i r d a n d s u b s e q u e n t ( M 3) division m e t a p h a s e cells. P R I was c a l c u l a t e d by t h e m e t h o d o f K r i s h n a et al. (1986) using t h e f o r m u l a PRI =
1M1+2M2+3M
3
100
where MI, M z and M 3 represent proportions of first, s e c o n d a n d t h i r d plus s u b s e q u e n t division m e t a p h a s e s respectively.
a b e r r a n t m e t a p h a s e s at h i g h e r doses, i.e., 10 a n d 1 0 0 / z g / m l , w h e n c o m p a r e d with t h e i r r e s p e c t i v e controls ( T a b l e 1). T h e p e r c e n t a b e r r a n t m e t a phases showed a dose-dependent response, t h o u g h n o t linearly. G a p - t y p e a b e r r a t i o n s w e r e m o r e c o m m o n t h a n o t h e r types. C h r o m a t i d - t y p e aberrations rather than chromosome-type were g e n e r a l l y observed. A n i n c r e a s e in M 1 cells a n d a d e c r e a s e in M 3 a n d s u b s e q u e n t m e t a p h a s e s w e r e o b s e r v e d at t h e h i g h e r c o n c e n t r a t i o n s with all types o f calcium silicate used. This r e s p o n s e was not d i r e c t l y p r o p o r t i o n a l to t h e c o n c e n t r a t i o n . N o m a r k e d effect on p r o l i f e r a t i o n c o u l d b e o b s e r v e d at lower c o n c e n t r a t i o n s . T a b l e 2 clearly i n d i c a t e s t h a t as t h e silicate c o n c e n t r a t i o n i n c r e a s e s t h e r e is a d e c l i n e in P R I values. T a b l e 2 also shows t h a t t h e f r e q u e n c y o f S C E s i n c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s b u t n o t directly p r o p o r t i o n a l to the concentration.
Results
Discussion
K e m o l i t A-60, K e m o l i t - N a n d K e m o l i t A S B - 3 i n d u c e d significantly i n c r e a s e d f r e q u e n c i e s o f
T h e results i n d i c a t e t h a t c a l c i u m silicates significantly i n d u c e d cell cycle d e l a y a n d genotoxic-
TABLE 2 SCE FREQUENCIES, DISTRIBUTION OF M 1, M 2 AND TREATED HUMAN LYMPHOCYTES Agent
M3
CELLS AND PRI IN CONTROL AND CALCIUM SILICATE-
Dose (/zg/ml)
SCE/metaphase (mean 5: SE)
M1
Metaphases (%) M2
M3
PRI
Kemolit ASB-3
0.0 0.1 1.0 10.0 100.0
8.2 5:2.6 9.1 ± 1.6 9.8 _+2.1 11.3 5:1.3 * 14.0± 1.8 *
18 18 25 28 39
25 27 28 27 34
57 55 47 45 27
2.39 2.37 2.22 2.17 1.88 t
Kemolit-N
0.0 0.1 1.0 10.0 100.0
7.8 ± 2.5 8.9 _+2.3 9.9 ± 2.3 11.5 ± 1.4 * 14.8± 1.8 *
15 18 21 32 38
29 29 36 30 35
56 53 43 38 27
2.41 2.35 2.22 2.06 t 1.89 t
Kemolit A-60
0.0 0.1 1.0 10.0 100.0
7.9 5:2.3 8.8 ± 2.1 10.2 ± 2.4 12.4±2.4 * 15.7 ± 2.5 *
17 18 22 30 42
27 32 40 38 33
56 50 38 32 25
2.39 2.32 2.16 2.02 t 1.83 *
For SCEs, 50 metaphases of the second cell division were analysed. For cell cycle kinetics, a total of 200 metaphases per treatment were analysed. * p < 0.05 (Student's t-test); t p < 0.05 (X 2 test).
48 ity. A significant i n c r e a s e in t h e f r e q u e n c y of c h r o m a t i d - t y p e a b e r r a t i o n s , (gaps a n d b r e a k s ) was observed rather than chromosome-type aberrations. This i n d i c a t e s t h a t t h e clastogenicity of calcium silicate occurs m o s t likely by an S - p h a s e dependent mechanism. It has o f t e n b e e n o b s e r v e d t h a t S C E s can b e i n d u c e d at d o s e s insufficient to give rise to C A s (Wolff, 1982; D e e n et al., 1989). So, it has b e e n s u g g e s t e d t h a t t h e S C E assay is m o r e sensitive t h a n t h e C A assay ( H a d n a g y et al., 1989). T h e f r e q u e n c y of S C E s was significantly i n c r e a s e d at h i g h e r c o n c e n t r a t i o n s a n d e x h i b i t e d a positive dose response. The method of BrdUrd labelling for S C E analysis was also u s e d s i m u l t a n e o u s l y for investigation of c e l l u l a r kinetics ( A b e l , 1987; H a d n a g y et al., 1989). C a l c i u m silicates a l t e r e d cell kinetics causing cell cycle d e l a y o f v a r i o u s d e g r e e s . T h e d e c r e a s e in P R I v a l u e s i n d i c a t e s that t h e s e silicates i n d u c e d p r o l o n g a t i o n o f cell cycle d u r a t i o n . Cell cycle delay, in a d d i t i o n to SCE, m a y r e p r e s e n t a n o t h e r level o f biological d a m a g e resulting f r o m silicate t r e a t m e n t . H o w ever, t h e d a t a o b t a i n e d d o not allow us to assess t h e specific stage at which the d e l a y o c c u r r e d .
Acknowledgements T h e a u t h o r s a r e t h a n k f u l to Prof. P.K. Ray, D i r e c t o r o f t h e C e n t r e , for his k e e n i n t e r e s t in t h e w o r k a n d to Mr. M. A s h q u i n for his skilled t e c h n i c a l assistance. This w o r k was s u p p o r t e d by t h e D i r e c t o r a t e of E n v i r o n m e n t , G o v e r n m e n t o f U t t a r P r a d e s h , India.
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