Mutation Research, 260 (1991) 181-185
181
© 1991 ElsevierSciencePublishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100094K MUTGEN 01657
Studies on modulation of the effects of colchicine by L-cysteine using bone marrow of Swiss mice Surendra Ghaskadbi and V.G. Vaidya Department of Zoology, M.A.C.S. Research Institute, Pune-411 004 (lndia)
(Received 22 March 1990) (Revision received8 November 1990) (Accepted 16 November 1990) Keywords: Colchicine;L-Cysteine;Modulatoryeffects; Micronuclei,induced; (Swiss mice)
Summary Colchicine (COL) elevates the frequency of micronucleated polychromatic erythrocytes (PE), the ratio of normochromatic to polychromatic erythrocytes ( N / P E ) and the frequency of large PE due to spindle disruption. Simultaneous i.p. injection of L-cysteine (CYS) does not influence the effects of COL while if administered 1 h prior to COL, CYS suppresses the N / P E ratio and frequency of large PE but not the frequency of micronucleated PE elevated by COL. Preincubation of CYS with COL at 37 ° C for 1 h results in a significant decrease in all the COL effects. The modulatory effect of exogenous CYS appears to be due to its competition with the endogenous tubulin cysteine residues for interacting with COL.
Colchicine (COL), a mitotic inhibitor, brings about induction of micronuclei in the polychromatic erythrocytes (PE) of the bone marrow of Swiss mice (Tsuchimoto and Matter, 1979; Asano et al., 1989). This effect of COL is due to spindle disruption (Malling and Wassom, 1977) which occurs after binding of COL to tubulin dimers in spindle microtubules (Mareel and De Mets, 1984). Both polymerization of tubulin and binding of spindle poisons to tubulin have been shown to involve sulfhydryl groups of cysteine residues in the tubulin molecule (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976; Luduena and Roach, 1981; Postingl et al., 1983; Bai et al., 1989). The Correspondence: Dr. Surendra Ghaskadbi, Department of Zoology, M.A.C.S.Research Institute, Pune-411 004 (India). Abbreviations: COL, colchicine; CYS, L-cysteinehydrochlo-
ride; PE, polychromatic erythrocytes; N/PE ratio, normochromatic to polychromaticerythrocyteratio.
present work was undertaken to find out if exogenous L-cysteine (CYS) is capable of modulating the spindle-disrupting effects of COL. Further, it was also intended to examine the hypothesized role of tubulin cysteine residues in COL tubulin binding (Luduena and Roach, 1981). The in vivo micronucleus test in Swiss mice is a suitable modal system to study modulation of clastogenicity of chemicals (Ghaskadbi et al., 1987; Ghaskadbi and Vaidya, 1989) and the same was employed in the present study. Materials and methods Animals
A Swiss albino mouse stock, originally obtained from the National Institute of Virology, Pune, was inbred in this laboratory for several generations. Chemicals
Colchicine (batch 130) and L-cysteine hydrochloride (batch T 811980) were obtained from
182
Romali, Bombay and Sisco Laboratories, Bombay, respectively.
Treatments and the micronucleus assay Male mice, 6-8 weeks old and weighing about 20 g, were intraperitoneally administered an aqueous solution of COL at 2 different dosages, viz., 1.25 and 2.5 m g / k g body weight at 0 and 24 h. The animals were killed by cervical dislocation at 30 h and bone marrow smears were prepared according to the method of Schmid (1975). For each dose and for control at least 3 mice were used. Coded slides were used for scanning 20003000 PE from each mouse to assess the frequency of micronucleated cells. Normochromatic erythrocytes in the corresponding microscopic fields were counted to determine the ratio of normochromatic to polychromatic erythrocytes ( N / P E ) . The frequency of larger-than-normal PE was determined by scanning 200-300 PE from each mouse. (These are presumed to result from PE where spindle formation was totally blocked as a consequence of which the chromosomes reassembled into a tetraploid nucleus which was later expelled in toto from the erythroblasts; these cells will henceforth be referred to as 'large PE'; Maier and Schmid, 1976.) At 1.25 mg/kg, COL was found to exert maximum effect on the frequency of micronucleated PE and this dose was used in all subsequent experiments. Three sets of experiments were carried out to evaluate the ability of CYS to modulate the effects of COL. In the first set of experiments, mice were administered, at 0 and 24 h, COL (1.25 m g / k g ) and were simultaneously injected with 40, 80 and 160 m g / k g CYS. In the second set, 80 and 160
m g / k g CYS was injected 1 h prior to the injection of COL. In the third set, COL (final dose 1.25 m g / k g ) and CYS (final doses 80 and 160 m g / k g ) solutions were mixed and incubated at 37 ° C for 1 h and the mixture was then injected. COL incubated alone in a similar manner served as control for this set of experiments. The bone marrow smears from all experiments were scanned as before to evaluate the frequencies of micronucleated and large PE as well as the N / P E ratio.
Chromatographic analysis Preliminary chemical analysis of the incubated C O L - C Y S mixture was carried out by thin-layer and two-dimensional paper chromatography to check the interaction between COL and CYS, if any. The solvent system for thin-layer chromatography (TLC) contained butanol : acetic acid : water ( 4 : 1 : 1 ) . The solvent system for the first dimension paper chromatography was the same as that for TLC while for the second dimension it consisted of butanol : acetic acid : water (20 : 5 : 1). The resulting separation was visualized by spraying first DragendorfFs reagent followed by ninhydrin reagent (Stahl, 1969). Results and discussion
IntraperitoneaUy administered COL was found to affect all 3 parameters studied, viz., frequencies of micronucleated and large PE and the N / P E ratio (Table 1). The effect on the frequency of micronucleated PE was more pronounced at 1.25 m g / k g than at 2.5 mg/kg. The observed effect and its biphasic nature are in good agreement with earlier reports (Tsuchimoto and Matter, 1979;
TABLE 1 EFFECTS OF C O L C H I C I N E ON T H E F R E Q U E N C Y OF M I C R O N U C L E A T E D F R E Q U E N C Y OF L A R G E PE IN T H E BONE M A R R O W O F SWISS M I C E
PE, T H E N / P E
RATIO AND THE
Dose (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%o) (mean 5: SD)
N/PE (mean ___SD)
Large PE (%) (mean 5: SD)
0 1.25 2.50
15,000 12,000 12,000
61 309 154
4.07 5:0.70 25.75 5:3.65 a 12.83 + 3.00 a
1.03 4- 0.07 2.25 +0.12 3.24 + 0.27
1.11 + 0.30 58.67 + 3.84 a 72.00 5:4.00 a
Significant compared to control ( P = 0.01).
183 TABLE 2 I N F L U E N C E O F S I M U L T A N E O U S A D M I N I S T R A T I O N O F L-CYSTEINE O N T H E E F F E C T S O F C O L C H I C I N E IN T H E B O N E M A R R O W O F SWISS MICE Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) a (mean ± SD)
N/PE (mean ± SD)
Large PE (%) a (mean ± SD)
1.25 1.25 1.25 1.25
0 40 80 160
12,000 9,000 9,000 9,000
309 239 246 228
25.75 ± 3.65 26.56 5- 3.69 27.33 5- 2.54 25.33 + 2.83
2.25 ± 0.12 2.14±0.11 2.17 d- 0.12 2A6 + 0.14
58.67 ± 58.11 + 60.67 + 56.89 ±
3.84 5.69 4.50 3.96
a N o n e of the values are significant compared to control ( P = 0.05).
Asano et al., 1989). For subsequent experiments to study the modulation of COL effects by CYS, the most effective dose of COL (1.25 mg/kg) was maintained.
Simultaneous i.p. injection of CYS at varying doses along with COL did not significantly influence the effects of COL (Table 2). All 3 parameters studied remained unaffected by CYS at all 3
TABLE 3 I N F L U E N C E OF P R I O R A D M I N I S T R A T I O N O F L-CYSTEINE O N T H E E F F E C T S O F C O L C H I C I N E IN T H E BONE M A R R O W O F SWISS M I C E Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
1.25 1.25 1.25
0 80 160
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) a (mean ± SD)
N/PE (mean ± SD)
Large PE (%) (mean ± SD)
12,000 9,000 8,000
309 212 160
25.75 ± 3.65 23.56 ± 2.45 20.00 + 2.87
2.25 ± 0.12 1.96 ± 0.12 1.71 ± 0.10
58.67 ± 3.84 49.78 _ 4.73 b 42.00 ± 7.87 b
a None of the values are significant compared to control ( P = 0.05). b Significant compared to control ( P = 0.01).
TABLE 4 I N F L U E N C E O F L-CYSTEINE P R E I N C U B A T E D W I T H C O L C H I C I N E (37 ° C, 1 h) O N T H E E F F E C T S O F C O L C H I C I N E IN T H E BONE M A R R O W O F SWISS MICE Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) (mean ± SD)
N/PE (mean ± SD)
Large PE (%) (mean ± SD)
1.25 1.25 a 1.25 1.25
0 0 80 160
12,000 12,000 9,000 8,000
309 335 217 120
25.75 + 3.65 27.92 __2.72 24.11 + 4.82 15.00 ± 1.66 b
2.25 ± 2.15 ± 1.99 ± 1.44 ±
58.67 + 3.84 57.42 _+5.30 52.55 _+8.60 c 31.50 ± 6.24 b
a Colchicine was incubated at 37 ° C for 1 h in the absence of L-cysteine. b Significant compared to control ( P = 0.01). ¢ Significant compared to control ( P = 0.05).
0.12 0.07 0.13 0.07
184 dose levels. When CYS was administered 1 h prior to COL, significant suppression was evident in the N/PE ratio and the frequency of large PE which were found to be elevated by COL. The lowering of the frequency of micronucleated PE, however, was not significant at either dose of CYS (Table 3). The third set of experiments in which mixtures of COL and CYS were incubated at 37 o C for 1 h prior to i.p. administration yielded the following interesting results. At 160 m g / k g , CYS significantly suppressed the frequency of large PE, the N/PE ratio as well as the frequency of micronucleated PE (Table 4). Preliminary chemical analysis of the preincubated C O L - C Y S mixture by chromatographic techniques revealed formation of a new complex (data not shown), Its further characterization has yet to be undertaken. The present results show that CYS can modulate the effects of COL in the bone marrow of Swiss mice. CYS is not effective when administered simultaneously with COL into the intraperitoneal cavity, probably due to the quick action of the latter. When injected 1 h prior to COL or when allowed to react with COL at 37 ° C for 1 h in vitro, CYS is capable of suppressing the effects of COL. These results can be interpreted on the basis of the following information. As already mentioned, polymerization of tubulin into microtubules involves the sulfhydryl groups of the cysteine residues (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976). Furthermore, the binding of many mitotic inhibitors, including COL, to tubulin also involves the cysteine residues of tubulin (Luduena and Roach, 1981; Postingl et al., 1983; Bai et al., 1989). Thus the suppression of COL effects by C ¥ S observed in the present study could be due to the competition between exogenous and endogenous cysteine residues for interaction with COL. This argument is further strengthened by the observation that incubation of the mixture of COL and CYS at 37 ° C results in the formation of a new chemical complex. In conclusion, the present results demonstrate that the mutagenic effects by COL through spindle dysfunction can be modified by exogenous CYS in a dose-dependent manner. The interaction between the 2 compounds can occur in a test tube as well as in the body of a living animal. Such
chemical modulation of the effects of COL by exogenous CYS can be monitored by using quantitative parameters in the bone marrow cells. Further, the results of the present study are also consistent with the hypothesis (Luduena and Roach, 1981) that binding between COL and its target molecule tubulin involves cysteine residues present in the tubulin molecule.
Acknowledgement We thank Dr. S.B. Bhosale of the Department of Chemistry for useful discussions and for his help in the chromatographic analysis.
References Asano, N., T. Morita and Y. Watanabe (1989) Micronucleus test with colchicine given by intraperitoneal injection and oral gavage, Mutation Res., 223, 391-394. Bai, R.-I., C. Duannu and E. Hamel (1989) Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of fl-tubulin, Biochim. Biophys. Acta, 994, 12-20. Ghaskadbi, S., and V.G. Vaidya (1989) In vivo antimutagenic effect of ascorbic acid against mutagenicityof the common antiamebic drug diiodohydroxyquinoline, Mutation Res., 222, 219-222. Ghaskadbi, S., S.V. Pavaskar and V.G. Vaidya (1987) Bioantimutagenic effect of L-cysteine on diiodohydroxyquinolineinduced micronuclei in Swiss mice, Mutation Res., 187, 219-222. Kuriyama, R., and H. Sakai (1974) Role of tubulin -SH groups in polymerization to microtubules. Functional -SH groups in tubulin for polymerization, J. Biochem. (Tokyo), 76, 651-654. Luduena, R.F., and M.C. Roach (1981) Interaction of tubulin with drugs and alkylating agents. 2. Effects of colchicine, podophyllotoxin, and vinblastine on the alkylation of tubulin, Biochemistry, 20, AAA.A.-4450. Maier, P., and W. Schmid(1976) Ten model mutagensevaluated in the micronucleus test, Mutation Res., 40, 325-338. Mailing, H.V., and J.S. Wassom (1977) Action of mutagenic agents, in: J.G. Wilson and F.C. Fraser (Eds.), Handbook of Teratology, Vol. 1, Plenum, New York, pp. 99-152. Mareel, M.M., and M. De Mets (1984) Effect of microtubule inhibitors on invasion and on related activities of tumor cells, Int. Rev. Cytol., 90, 125-168. Mellon, M.G., and L.I. Rebhun (1976) Sulfhydryls and the in vitro polymerization of tubulin, J. Cell Biol., 70, 226-238. Postingl, H., E. Krauhs and M. Little (1983) Tubulin amino acid sequence and consequences, J. Submicrosc. Cytol., 15, 359-362.
185 Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Stahl, E. (1969) Thin-Layer Chromatography: A Laboratory Handbook, 2nd edn., Allen and Unwin, London, Springer Verlag, Berlin.
Tsuchimoto, T., and B.E. Matter (1979) In vivo cytogenetic screening methods for mutagens, with special reference to the micronucleus test, Arch. Toxicol., 42, 239-248.
181
© 1991 ElsevierSciencePublishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100094K MUTGEN 01657
Studies on modulation of the effects of colchicine by L-cysteine using bone marrow of Swiss mice Surendra Ghaskadbi and V.G. Vaidya Department of Zoology, M.A.C.S. Research Institute, Pune-411 004 (lndia)
(Received 22 March 1990) (Revision received8 November 1990) (Accepted 16 November 1990) Keywords: Colchicine;L-Cysteine;Modulatoryeffects; Micronuclei,induced; (Swiss mice)
Summary Colchicine (COL) elevates the frequency of micronucleated polychromatic erythrocytes (PE), the ratio of normochromatic to polychromatic erythrocytes ( N / P E ) and the frequency of large PE due to spindle disruption. Simultaneous i.p. injection of L-cysteine (CYS) does not influence the effects of COL while if administered 1 h prior to COL, CYS suppresses the N / P E ratio and frequency of large PE but not the frequency of micronucleated PE elevated by COL. Preincubation of CYS with COL at 37 ° C for 1 h results in a significant decrease in all the COL effects. The modulatory effect of exogenous CYS appears to be due to its competition with the endogenous tubulin cysteine residues for interacting with COL.
Colchicine (COL), a mitotic inhibitor, brings about induction of micronuclei in the polychromatic erythrocytes (PE) of the bone marrow of Swiss mice (Tsuchimoto and Matter, 1979; Asano et al., 1989). This effect of COL is due to spindle disruption (Malling and Wassom, 1977) which occurs after binding of COL to tubulin dimers in spindle microtubules (Mareel and De Mets, 1984). Both polymerization of tubulin and binding of spindle poisons to tubulin have been shown to involve sulfhydryl groups of cysteine residues in the tubulin molecule (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976; Luduena and Roach, 1981; Postingl et al., 1983; Bai et al., 1989). The Correspondence: Dr. Surendra Ghaskadbi, Department of Zoology, M.A.C.S.Research Institute, Pune-411 004 (India). Abbreviations: COL, colchicine; CYS, L-cysteinehydrochlo-
ride; PE, polychromatic erythrocytes; N/PE ratio, normochromatic to polychromaticerythrocyteratio.
present work was undertaken to find out if exogenous L-cysteine (CYS) is capable of modulating the spindle-disrupting effects of COL. Further, it was also intended to examine the hypothesized role of tubulin cysteine residues in COL tubulin binding (Luduena and Roach, 1981). The in vivo micronucleus test in Swiss mice is a suitable modal system to study modulation of clastogenicity of chemicals (Ghaskadbi et al., 1987; Ghaskadbi and Vaidya, 1989) and the same was employed in the present study. Materials and methods Animals
A Swiss albino mouse stock, originally obtained from the National Institute of Virology, Pune, was inbred in this laboratory for several generations. Chemicals
Colchicine (batch 130) and L-cysteine hydrochloride (batch T 811980) were obtained from
182
Romali, Bombay and Sisco Laboratories, Bombay, respectively.
Treatments and the micronucleus assay Male mice, 6-8 weeks old and weighing about 20 g, were intraperitoneally administered an aqueous solution of COL at 2 different dosages, viz., 1.25 and 2.5 m g / k g body weight at 0 and 24 h. The animals were killed by cervical dislocation at 30 h and bone marrow smears were prepared according to the method of Schmid (1975). For each dose and for control at least 3 mice were used. Coded slides were used for scanning 20003000 PE from each mouse to assess the frequency of micronucleated cells. Normochromatic erythrocytes in the corresponding microscopic fields were counted to determine the ratio of normochromatic to polychromatic erythrocytes ( N / P E ) . The frequency of larger-than-normal PE was determined by scanning 200-300 PE from each mouse. (These are presumed to result from PE where spindle formation was totally blocked as a consequence of which the chromosomes reassembled into a tetraploid nucleus which was later expelled in toto from the erythroblasts; these cells will henceforth be referred to as 'large PE'; Maier and Schmid, 1976.) At 1.25 mg/kg, COL was found to exert maximum effect on the frequency of micronucleated PE and this dose was used in all subsequent experiments. Three sets of experiments were carried out to evaluate the ability of CYS to modulate the effects of COL. In the first set of experiments, mice were administered, at 0 and 24 h, COL (1.25 m g / k g ) and were simultaneously injected with 40, 80 and 160 m g / k g CYS. In the second set, 80 and 160
m g / k g CYS was injected 1 h prior to the injection of COL. In the third set, COL (final dose 1.25 m g / k g ) and CYS (final doses 80 and 160 m g / k g ) solutions were mixed and incubated at 37 ° C for 1 h and the mixture was then injected. COL incubated alone in a similar manner served as control for this set of experiments. The bone marrow smears from all experiments were scanned as before to evaluate the frequencies of micronucleated and large PE as well as the N / P E ratio.
Chromatographic analysis Preliminary chemical analysis of the incubated C O L - C Y S mixture was carried out by thin-layer and two-dimensional paper chromatography to check the interaction between COL and CYS, if any. The solvent system for thin-layer chromatography (TLC) contained butanol : acetic acid : water ( 4 : 1 : 1 ) . The solvent system for the first dimension paper chromatography was the same as that for TLC while for the second dimension it consisted of butanol : acetic acid : water (20 : 5 : 1). The resulting separation was visualized by spraying first DragendorfFs reagent followed by ninhydrin reagent (Stahl, 1969). Results and discussion
IntraperitoneaUy administered COL was found to affect all 3 parameters studied, viz., frequencies of micronucleated and large PE and the N / P E ratio (Table 1). The effect on the frequency of micronucleated PE was more pronounced at 1.25 m g / k g than at 2.5 mg/kg. The observed effect and its biphasic nature are in good agreement with earlier reports (Tsuchimoto and Matter, 1979;
TABLE 1 EFFECTS OF C O L C H I C I N E ON T H E F R E Q U E N C Y OF M I C R O N U C L E A T E D F R E Q U E N C Y OF L A R G E PE IN T H E BONE M A R R O W O F SWISS M I C E
PE, T H E N / P E
RATIO AND THE
Dose (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%o) (mean 5: SD)
N/PE (mean ___SD)
Large PE (%) (mean 5: SD)
0 1.25 2.50
15,000 12,000 12,000
61 309 154
4.07 5:0.70 25.75 5:3.65 a 12.83 + 3.00 a
1.03 4- 0.07 2.25 +0.12 3.24 + 0.27
1.11 + 0.30 58.67 + 3.84 a 72.00 5:4.00 a
Significant compared to control ( P = 0.01).
183 TABLE 2 I N F L U E N C E O F S I M U L T A N E O U S A D M I N I S T R A T I O N O F L-CYSTEINE O N T H E E F F E C T S O F C O L C H I C I N E IN T H E B O N E M A R R O W O F SWISS MICE Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) a (mean ± SD)
N/PE (mean ± SD)
Large PE (%) a (mean ± SD)
1.25 1.25 1.25 1.25
0 40 80 160
12,000 9,000 9,000 9,000
309 239 246 228
25.75 ± 3.65 26.56 5- 3.69 27.33 5- 2.54 25.33 + 2.83
2.25 ± 0.12 2.14±0.11 2.17 d- 0.12 2A6 + 0.14
58.67 ± 58.11 + 60.67 + 56.89 ±
3.84 5.69 4.50 3.96
a N o n e of the values are significant compared to control ( P = 0.05).
Asano et al., 1989). For subsequent experiments to study the modulation of COL effects by CYS, the most effective dose of COL (1.25 mg/kg) was maintained.
Simultaneous i.p. injection of CYS at varying doses along with COL did not significantly influence the effects of COL (Table 2). All 3 parameters studied remained unaffected by CYS at all 3
TABLE 3 I N F L U E N C E OF P R I O R A D M I N I S T R A T I O N O F L-CYSTEINE O N T H E E F F E C T S O F C O L C H I C I N E IN T H E BONE M A R R O W O F SWISS M I C E Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
1.25 1.25 1.25
0 80 160
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) a (mean ± SD)
N/PE (mean ± SD)
Large PE (%) (mean ± SD)
12,000 9,000 8,000
309 212 160
25.75 ± 3.65 23.56 ± 2.45 20.00 + 2.87
2.25 ± 0.12 1.96 ± 0.12 1.71 ± 0.10
58.67 ± 3.84 49.78 _ 4.73 b 42.00 ± 7.87 b
a None of the values are significant compared to control ( P = 0.05). b Significant compared to control ( P = 0.01).
TABLE 4 I N F L U E N C E O F L-CYSTEINE P R E I N C U B A T E D W I T H C O L C H I C I N E (37 ° C, 1 h) O N T H E E F F E C T S O F C O L C H I C I N E IN T H E BONE M A R R O W O F SWISS MICE Dose of colchicine (mg/kg)
Dose of L-cysteine (mg/kg)
Total PE scanned
Total micronucleated PE
Micronucleated PE (%0) (mean ± SD)
N/PE (mean ± SD)
Large PE (%) (mean ± SD)
1.25 1.25 a 1.25 1.25
0 0 80 160
12,000 12,000 9,000 8,000
309 335 217 120
25.75 + 3.65 27.92 __2.72 24.11 + 4.82 15.00 ± 1.66 b
2.25 ± 2.15 ± 1.99 ± 1.44 ±
58.67 + 3.84 57.42 _+5.30 52.55 _+8.60 c 31.50 ± 6.24 b
a Colchicine was incubated at 37 ° C for 1 h in the absence of L-cysteine. b Significant compared to control ( P = 0.01). ¢ Significant compared to control ( P = 0.05).
0.12 0.07 0.13 0.07
184 dose levels. When CYS was administered 1 h prior to COL, significant suppression was evident in the N/PE ratio and the frequency of large PE which were found to be elevated by COL. The lowering of the frequency of micronucleated PE, however, was not significant at either dose of CYS (Table 3). The third set of experiments in which mixtures of COL and CYS were incubated at 37 o C for 1 h prior to i.p. administration yielded the following interesting results. At 160 m g / k g , CYS significantly suppressed the frequency of large PE, the N/PE ratio as well as the frequency of micronucleated PE (Table 4). Preliminary chemical analysis of the preincubated C O L - C Y S mixture by chromatographic techniques revealed formation of a new complex (data not shown), Its further characterization has yet to be undertaken. The present results show that CYS can modulate the effects of COL in the bone marrow of Swiss mice. CYS is not effective when administered simultaneously with COL into the intraperitoneal cavity, probably due to the quick action of the latter. When injected 1 h prior to COL or when allowed to react with COL at 37 ° C for 1 h in vitro, CYS is capable of suppressing the effects of COL. These results can be interpreted on the basis of the following information. As already mentioned, polymerization of tubulin into microtubules involves the sulfhydryl groups of the cysteine residues (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976). Furthermore, the binding of many mitotic inhibitors, including COL, to tubulin also involves the cysteine residues of tubulin (Luduena and Roach, 1981; Postingl et al., 1983; Bai et al., 1989). Thus the suppression of COL effects by C ¥ S observed in the present study could be due to the competition between exogenous and endogenous cysteine residues for interaction with COL. This argument is further strengthened by the observation that incubation of the mixture of COL and CYS at 37 ° C results in the formation of a new chemical complex. In conclusion, the present results demonstrate that the mutagenic effects by COL through spindle dysfunction can be modified by exogenous CYS in a dose-dependent manner. The interaction between the 2 compounds can occur in a test tube as well as in the body of a living animal. Such
chemical modulation of the effects of COL by exogenous CYS can be monitored by using quantitative parameters in the bone marrow cells. Further, the results of the present study are also consistent with the hypothesis (Luduena and Roach, 1981) that binding between COL and its target molecule tubulin involves cysteine residues present in the tubulin molecule.
Acknowledgement We thank Dr. S.B. Bhosale of the Department of Chemistry for useful discussions and for his help in the chromatographic analysis.
References Asano, N., T. Morita and Y. Watanabe (1989) Micronucleus test with colchicine given by intraperitoneal injection and oral gavage, Mutation Res., 223, 391-394. Bai, R.-I., C. Duannu and E. Hamel (1989) Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of fl-tubulin, Biochim. Biophys. Acta, 994, 12-20. Ghaskadbi, S., and V.G. Vaidya (1989) In vivo antimutagenic effect of ascorbic acid against mutagenicityof the common antiamebic drug diiodohydroxyquinoline, Mutation Res., 222, 219-222. Ghaskadbi, S., S.V. Pavaskar and V.G. Vaidya (1987) Bioantimutagenic effect of L-cysteine on diiodohydroxyquinolineinduced micronuclei in Swiss mice, Mutation Res., 187, 219-222. Kuriyama, R., and H. Sakai (1974) Role of tubulin -SH groups in polymerization to microtubules. Functional -SH groups in tubulin for polymerization, J. Biochem. (Tokyo), 76, 651-654. Luduena, R.F., and M.C. Roach (1981) Interaction of tubulin with drugs and alkylating agents. 2. Effects of colchicine, podophyllotoxin, and vinblastine on the alkylation of tubulin, Biochemistry, 20, AAA.A.-4450. Maier, P., and W. Schmid(1976) Ten model mutagensevaluated in the micronucleus test, Mutation Res., 40, 325-338. Mailing, H.V., and J.S. Wassom (1977) Action of mutagenic agents, in: J.G. Wilson and F.C. Fraser (Eds.), Handbook of Teratology, Vol. 1, Plenum, New York, pp. 99-152. Mareel, M.M., and M. De Mets (1984) Effect of microtubule inhibitors on invasion and on related activities of tumor cells, Int. Rev. Cytol., 90, 125-168. Mellon, M.G., and L.I. Rebhun (1976) Sulfhydryls and the in vitro polymerization of tubulin, J. Cell Biol., 70, 226-238. Postingl, H., E. Krauhs and M. Little (1983) Tubulin amino acid sequence and consequences, J. Submicrosc. Cytol., 15, 359-362.
185 Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Stahl, E. (1969) Thin-Layer Chromatography: A Laboratory Handbook, 2nd edn., Allen and Unwin, London, Springer Verlag, Berlin.
Tsuchimoto, T., and B.E. Matter (1979) In vivo cytogenetic screening methods for mutagens, with special reference to the micronucleus test, Arch. Toxicol., 42, 239-248.
