Mutation Research, 282 (1992) 61-67 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00



Cytotoxicity of tin on human peripheral lymphocytes in vitro Bani Bandana Ganguly (Ghosh), Geeta Talukdar and Archana Sharma Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, Calcutta - 700 019 (India) (Received 18 October 1991) (Revision received 19 December 1991) (Accepted 2 January 1992)

Keywords: Tin; Human chromosomes

Summary The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20-40 years of age. The endpoints observed were chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics. The maximum concentrations which reduced the replicative index by about 50%, of stannic chloride and trimethyltin chloride were 40/zg and 2/~g per culture respectively. The tested doses were 20/zg and 10/xg of stannic chloride and 1 ~g and 0.5 tzg of trimethyltin chloride. Both doses of stannic chloride induced a much higher frequency of chromosomal abnormalities (P < 0.05-P < 0.001) and a greater reduction of cell cycle kinetics than the corresponding relative doses of trimethyltin chloride. The frequencies of SCEs/cell induced by the latter were, however, slightly higher than those induced by the former.

Chromosomal aberrations caused by occupational exposure have formed the basis of a large number of studies. The importance of these studies cannot be overemphasized since industrial workers are exposed to a large number of agents which are known to be mutagenic and also carcinogenic (Barrett and Elmore, 1985). As a result of its wide use in industries, agricultural and general biocides, tin in both inorganic and organic forms is widely dispersed throughout the ecosphere where it can accumulate in the food chain (Hodge et al., 1979) via the tin geocycle

Correspondence: Dr. Bani Bandana Ganguly, Biological Sciences Division, Biochemistry Unit, Indian Statistical Institute, 203 Barrackpore Trunk Road, Calcutta - 700 035 (India).

(Wood, 1974; Ridley et al., 1977; Craig, 1988). Organotins have been shown to exhibit antitumor effects (Cardin and Roy, 1985, 1986). Toxic effects of tin halides in experimental animals are well documented (WHO, 1980). Organotin compounds produce acute immunosuppression and depletion of thymocytes and thymus-dependent cortical lymphocytes in experimental animals (Penninks, 1985; Snoeij, 1987) and also damage the cerebral nervous system resulting in cerebral edema and pycnosis in the amygdaloid tissues (Watanabe, 1980; Chang et al., 1983; Besser et al., 1987). Effects of tin compounds on lymphoid cells have been assessed with respect to different endpoints (Snoeij et al., 1986). However, the comparative clastogenicity of tin compounds in vitro and

62 in vivo has not yet been studied (Sharma and Talukder, 1987). This aspect requires emphasis in monitoring the toxicity of this metal. In this study organic and inorganic tin chloride were compared with respect to the induction of chromosomal aberrations, sister-chromatid exchanges and their effects on cell cycle kinetics in human peripheral blood lymphocytes in vitro. Materials and methods

Chemicals Inorganic stannic chloride (SnC14 • 5H20; Loba Chemie) and organic trimethyltin chloride (CH3)3-SnC1; Fluka AG (Buchs, Switzerland) were used. The doses were selected according to Preston et al. (1987) following administration of different concentrations to determine the maximum one that reduced replicative index by about 50%. The final concentrations used were 20 and 10 /xg, and 1 and 0.5 ~g per 5 ml of culture for stannic chloride and trimethyltin chloride respectively. In vitro collection of cells 5 ml venous blood was collected from 27 healthy normal male voluntary donors 30-40 years of age. These individuals did not smoke and had not been exposed to radiation or drugs. 0.3 ml of heparinized whole blood was inoculated into the culture medium (RPMI-1640, Gibco) containing 20% heat-inactivated human AB serum, phytohemagglutinin (Gibco, 0.2 ml/5 ml culture) and 5-bromodeoxyuridine (Sigma, 8 ~g/ml). The chemical to be tested was added just after inoculation. Control and treated cultures were maintained in replicate sets. Two different concentrations of the same chemical were tested on the same sample. Treatment period was 72 h at 37°C in the dark. After 70 h of inoculation, cells were pretreated with colchicine for 2 h followed by hypotonic treatment in 0.09% NaC1 in deionized water preheated at 37°C and fixation in 3:1 methanol:acetic acid (see Sharma and Talukder, 1974; Sharma and Sharma, 1980). Cells were collected on clean slides, air-dried and stained in Hoechst 33258 (5 /zg/ml) followed by photo-illumination in sunlight, mounted in 2 × SSC buffer (Goto et al., 1975; Ghosh, 1988), washed thoroughly and

stained in 2% Giemsa in phosphate buffer for 8-10 min.

Data analysis Slides were coded for data entry and observed according to standard procedure (Forni, 1984; WHO, 1985; Preston et al., 1987). For each experimental set 50, 100 and 150 metaphases were observed for sister-chromatid exchanges (M2), chromosomal abnormalities (M~) and cell cycle kinetics respectively. For cell cycle kinetics, the replicative index (RI) was calculated as RI = 1 M1% + 2 M2% + 3 M3%/100 by scoring first(M 0, second- (M2) and third- (M 3) division metaphases from differentially stained plates (Schneider and Lewis, 1981). Data were analyzed statistically following Student's t test. Results

In comparison to control sets, the two concentrations of both compounds significantly elevated the numbers of chromosomal aberrations and sister-chromatid exchanges in relation to the doses given. The rate of cell generation cycle was lowered to levels directly proportional to the concentrations used. Stannic chloride significantly increased the frequency of chromosomal abnormalities (P < 0.05-P < 0.001) with the higher dose, as compared to that of trimethyltin chloride (Table 1); however, with the lower dose the effect was similar. Trimethyltin chloride induced a slightly higher frequency of sister-chromatid exchanges. The replicative index was more depressed by stannic chloride with both doses (Table 2). The first-cycle metaphases (M~) remained at a much higher frequency even after 72 h (two cell cycles). Chromosomal abnormalities induced were chromatid and chromosome breaks and gaps, dicentrics, ring and quadriradial and triradial exchange configurations (Fig. 1). Discussion

Significant elevations of chromosome aberrations, sister-chromatid exchanges and micronucleus counts in peripheral blood lymphocytes of tin miners and in patients with lung cancer who

63 TABLE 1 D A T A O N C H R O M O S O M E A B E R R A T I O N S I N D U C E D BY I N O R G A N I C A N D O R G A N I C T I N ON H U M A N L Y M P H O CYTES IN V I T R O Chemical

Stannic chloride

Trimethyl tin chloride

Dose (/~g/culture)

N u m b e r of samples

Types of aberrations (%) G' G" B' B"



















































CAs/cell (mean±SD) Incl. gaps

Excl. gaps

0.051 + 0.012 0.154 a" +0.011 0.127 a"b" _+0.016

0.044 ± 0.007 0.140 a" _+0.013 0.112 a"b" _+0.018

0.046 ± 0.010 0.138 a" + 0.027 0.117 a"b' ± 0.031

0.040 _+0.013 0.109 a"c" ± 0.018 0.094 ~"c' _+0.028

Damaged cells (%) (mean ± SD) 4.55±0.93 13.18+ 1.78 a" 11.18± 1.79 a"b'

3.69±0.95 9.94_+1.84 a"c" 8.81±2.88 a"c'

G ' , chromatid gap; G " , chromosome gap; B', chromatid break, B", chromosome break; DIC, dicentric, RR, rearrangements. a Individual dose was compared with control., a" p < 0.001. b Two doses of the same chemical were compared, b' p < 0.05; b" P < 0.001. c Relative doses of the two chemicals were compared, c' p < 0.05; c" p < 0.001.

had worked in the Yunnan tin mines (China) showed the carcinogenic effect of tin (Hu et al., 1987). Inorganic stannous and stannic chlorides induced prolonged suppression of DNA synthesis in human white blood cells (McLean et al., 1983). The relatively mild action of stannic chloride has been attributed to difficulty in entering the nucleus.

Stannic chloride has, however, been found to be a potential clastogen (Ghosh, 1988; Ghosh et al., 1988a) showing an age-related elevation of chromosomal aberrations in lymphocyte culture. Organotin, more particularly alkyltin compounds, are lymphocytotoxic, depending on the number and nature of organic groups and the chain length of alkyl derivatives (Seinen et al.,


Stannic chloride Trimethyl tin chloride

Dose (~g/culture)

N u m b e r of samples

Range of SCEs

SCEs/cell (mean ± SD)

0 20 10

11 11 11

1-24 1-35 4-39

16 16 16

0-23 3-41 4-45

0 1.0 0.5


Cell cycle kinetics Replicative index (mean ± SD)




8.65 + 1.78 14.87 -+ 3.21 a" 14.01 _+3.53 a"

2.43 ± 0.12 2.22 _-+0.06 a" 2.12 _+0.12 a"b'

6.89 20.75 30.55

43.69 36.07 42.31

49.57 43.17 34.78

7.63 + 1.84 16.13 + 5.40 a" 15.35 -+ 3.38 a"

2.45 _+0.25 2.34 _+0.27 2.22 ± 0.40

7.22 11.73 19.43

40.80 42.53 37.34

51.98 45.69 43.23

a Individual dose was compared with control, a" p < 0.001. b Two doses of the same chemical were compared, b' p < 0.05.

64 1977). These compounds showed in vivo genotoxicity in the mouse dominant lethal assay (Epstein et al., 1972) and chromosomal aberrations in bone marrow (Belyaeva et al., 1976). Tests with multi-

pie endpoints like chromium release, trypan blue exclusion and thymidine incorporation indicate the cytotoxicity of the more hydrophilic alkyltins (Snoeij et al., 1986). In contrast, trimethyltin


Fig. 1. (a,b) Breaks and gaps. (c) Triradial rearrangement. (d) Sister-chromatidexchangesin treated human lymphocytesin vitro.


chloride significantly increased chromosomal aberrations, sister-chromatid exchanges and micronucleus counts and delayed the rate of cell division and cell generation cycle, the elevation being more pronounced in the older age group of 40-60 years (Ghosh et al., 1989a, b, 1990, 1991). Compared to most organotin derivatives, inorganic tin and its salts are not highly toxic mainly because of their poor absorption and rapid tissue turnover (Barnes and Stoner, 1959; Cheftel, 1967; Hiles, 1974; National Academy of Sciences, 1973; WHO, 1980). Factors facilitating the toxicity of organotin compounds are their lipid solubility and stability in biological fluids at tissue pH, allowing extensive penetration into the brain and lodgement in the central nervous system (Venugopal and Luckey, 1978). This agrees with the structure-activity relationship models of Babich and Borenfreund (1987) showing that the cytotoxicity of various groups of substances is related to their lipophilicity. In the present study, the higher dose of trimethyltin chloride (1 ~g/culture) was 20 times lower than that of stannic chloride (20 /zg/culture) with a corresponding proportion in the lower doses of the two chemicals used. The significantly higher clastogenicity of stannic chloride may be related to the much larger amounts of the salt used and to the high frequency of inter-individual variation. Inter- and intra-individual variations are important factors in assessing chromosomal damage (Obe and Beek, 1984) and variations in chromosomal aberrations and sister-chromatid exchanges in control populations are well known (Dewdney et al., 1986; Ghosh et al., 1988b; Obe et al., 1984). Similar to these previous findings, in the present experiment, significant differences were observed in the frequencies of chromosomal abnormalities between individuals. Tin compounds readily combine with the dithiol groups of proteins and can form stable complexes with -SH (Venugopal and Luckey, 1978). Tin(IV) can be biomethylated in the environment (Wood et al., 1978; Mushak, 1983) and possibly in the cell, which may be the cause of their toxicity to mammalian systems (McLean et al., 1983). Trimethyltin can be dealkylated to dialkyltin and the anticancer activity of dimethyltin may be due to the binding of the metal to

RNA or free nucleosides (Cardin and Roy, 1985, 1986).

Acknowledgements The authors are grateful to Professor A.K. Sharma, Programme Co-ordinator, Centre for Advanced Study, Department of Botany, University of Calcutta and to the Council of Scientific and Industrial Research, Government of India, for providing financial assistance.

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Communicated by J.M. Gentile

Cytotoxicity of tin on human peripheral lymphocytes in vitro.

The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20...
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