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Intercalating substances do not induce sister-chromatid exchanges (SCEs) in vivo
Mutation Research, 104 (1982)261-266
261
Elsevier BiomedicalPress
Intercalating substances do not induce sister-chromatid exchanges (SCEs) in vivo G t i n t e r Speit Abteilung Klinische Genetik, Oberer Eselsberg, D-7900 Ulm (Federal Republic of Germany)
(Accepted 13 January 1982)
Summary In contrast to findings in vitro, the intercalating substances proflavine, methylene blue and chlorpromazine did not induce sister-chromatid exchanges (SCEs) in the SCE test in vivo in the bone marrow of the Chinese hamster. The distribution of the metaphases of the first, second and third replication cycles in the presence of bromodeoxyuridine (BrdUrd) showed all 3 substances to have no inhibitory effect on the cellular proliferation in the bone marrow.
Intercalating drugs and dyes induce sister-chromatid exchanges (SCEs) in the V79 cell line of the Chinese hamster (Speit and Vogel, 1979). SCEs have often proved themselves to be sensitive indicators of DNA-damaging effects (Perry and Evans, 1975; Popescu et al., 1977). The fact that intercalating agents are able to induce SCEs indicates that they lead to alterations of the DNA. However, the cause of this effect is not known. It has been proposed that the change in the DNA structure caused by the intercalation is the reason for the induction of SCEs (Speit and Vogel, 1979). However, an investigation of acridine derivatives on human lymphocyte cultures has shown that there is no direct relationship between the intercalation and SCE induction (Crossen, 1979). Because these substances are widely used, for instance as fluorescent dyes, antiseptics and psychopharmaceuticals, the demonstration of a possible DNAdamaging effect would be of great significance. To obtain more relevant information with regard to man, 3 of these substances were investigated, in vivo, with the SCE test, a test that constitutes a sensitive measure for DNA damage and that also takes into account mammalian metabolism.
0165-7992/82/0000-0000/$02.75 ©Elsevier Biomedical Press
262 Materials and methods
The experiments were performed on 6-12-month-old Chinese hamsters, as previously described (Speit et al., 1980a, b). A 50-rag BrdUrd tablet was implanted into the posterior abdominal region. The test substances were injected intraperitoneally 1 h later, after having been dissolved in a physiological saline solution under sterile conditions. The following substances were used: proflavine (Fluka), methylene blue (Merck) and chlorpromazine ('Megaphen', Bayer). 24 h later, colchicine (50 mg/kg) was injected, and 2 h thereafter metaphases were prepared from the bone marrow. Differential chromatid staining was done according to a modified fluorescence-plus-Giemsa technique (Epplen et al., 1975). The SCEs in 40 metaphases were counted for each value, and the mean and the standard deviation from the mean were determined. All trials were repeated at least twice. To evaluate the cellular proliferation, the distribution of the metaphases of the first, second and third generations were determined in 200 mitoses each. Cells that had replicated their DNA exclusively before the implantation of BrdUrd could not be distinguished from first metaphases, and cells that had gone through more than 3 cell cycles were counted as third mitoses. Such cells, however, cannot be expected to occur during an experimental period totalling 26 h.
Results and discussion
The acridine derivative proflavine (PF) as well as the phenothiazine derivatives methylene blue (MB) and chlorpromazine (CPZ) did not lead to a significant induction of SCEs in the bone marrow of the Chinese hamster (Table l). CPZ was tested up to a concentration corresponding to 4 times the therapeutic dose in man. At this concentration, the substance had a pronounced pharmacological effect on the hamster (slowing of movement and apathy). PF and MB, which lead to the induction of SCEs at lesser concentrations than CPZ in vitro (Speit and Vogel, 1979), were tested in vivo up to the same concentration level without showing any effect. An increase of the dose was limited by the solubility of the substances. The distribution of the first, second and third mitoses shows that the proliferation of the cells analysed in the bone marrow was not delayed in comparison with the control (Table 2). If replication were inhibited, one would find a decrease of the frequency of second-replication-cycle cells and an increase in the frequency of firstreplication-cycle cells. To exclude artifacts from the experimental procedure, cyclophosphamide (CP) was tested as a positive control. CP led to a pronounced increase in the SCE frequencies (Table l, Fig. 1) without impeding the proliferation of the cells (data not shown). For the intercalating substances examined here, one therefore obtains qualitative differences in the results of investigations in vitro and in vivo. The weak SCE-
263
TABLE I FREQUENCY OF SCEs 1N THE BONE MARROW TREATMENT WITH PF, MB, CPZ AND CP Substance
Dose (mg/kg)
Control
-
3.3 ± 0.3
PF
1 3 6 12
3.3 3.4 4.0 3.8
MB
1 3 6 12
3.2±0.3 3.4±0.3 3.4±0.3 3.1±0.3
CPZ
1 3 10 15
3.5 ± 0.4 3.6 ± 0.4 3.5 ± 0.3
30 60
24.5 ± 2.1 39,8 ± 2.7
CP
OF
CHINESE
HAMSTERS
AFTER
SCEs per metaphase ± S.E.M.
± ± ± ±
0.3 0.3 0.4 0.4
3.3 ± 0.3
TABLE 2 THE EFFECT OF I N T E R C A L A T I N G SUBSTANCES ON CELL REPLICATION IN THE BONE MARROW OF CHINESE HAMSTERS Substance
Dose (mg/kg)
Cell cycle (%) 1
Control
2
3
3
91
6
PF
1 12
2 8
93 91
5 1
MB
1 12
9 10
91 87
0 3
CPZ
1 15
12 9
87 90
I
1
inducing action of CPZ in vitro has to date only been observed in V79 cells (Speit and Vogel, 1979), and for MB there is a positive finding (Speit and Vogel, 1979) paralleled by a negative one (Popescu et al., 1977). In contrast to these 2 substances,
264
O
%
4P
b
Fig. 1. Metaphase with sister-chromatid differentiation from the bone marrow of the Chinese hamster showing cyclophosphamide-induced(30 mg/kg) sister chromatid exchanges (SCEs). the SCE induction by PF is unequivocally documented in various studies on cell cultures (Kato, 1974; Popescu et al., 1977; Carrano et al., 1978; Crossen, 1979; Speit and Vogel, 1979). At a lower concentration (6 mg/kg), a negative finding for PF in vivo had already been reported (Nakanishi and Schneider, 1979). T o date only few substances have shown an SCE induction in vitro and a negative result in vivo. Thus the fluorescent dye Hoechst 33258 induced SCEs in C H O cells (Perry and Evans, 1975), but not in the bone marrow of the mouse (Nakanishi and Schneider, 1979). The antitubercular drug isoniazide (INH) induced SCEs in various cell lines of the Chinese hamster (MacRae and Stich, 1979a; Speit et al., 1980a), but not in the bone marrow of Chinese hamsters (Speit et al., 1980a). Positive results were also found for vitamin C in vitro in various systems (Galloway and Painter, 1979; MacRae and Stich, 1979b; Spelt et al., 1980b), but no SCE induction in the bone marrow of the Chinese hamster (Speit et al., 1980b). Such diverse findings could be due to the fact that a substance is 'inactivated' in the mammalian metabolism, as is assumed for INH. On the other hand, there is the
265 possibility that, u n d e r c o n d i t i o n s in vitro, reactions occur that are n o t relevant to the m a m m a l i a n m e t a b o l i s m . T h u s the i n d u c t i o n o f SCEs by v i t a m i n C is p r o b a b l y based o n a reaction with the culture m e d i u m ( M a c R a e a n d Stich, 1979b; Speit et al., 1980b). T h e r e a s o n for the negative f i n d i n g for intercalating substances is u n k n o w n , as is the cause for SCE i n d u c t i o n in vitro. As far as a direct c o m p a r i s o n is possible, the dose is the same for b o t h procedures. However, the d i s t r i b u t i o n o f the test substances in the m a m m a l i a n o r g a n i s m is unclarified a n d it is not k n o w n what c o n c e n t r a t i o n s are present in the b o n e m a r r o w . Because intercalating substances i n h i b i t the p r o l i f e r a t i o n o f cell cultures (Speit a n d Vogel, 1979) w i t h o u t i n f l u e n c i n g the p r o l i f e r a t i o n of b o n e - m a r r o w cells, one can c o n c l u d e that these substances a p p e a r in the b o n e m a r r o w in insufficient c o n c e n t r a t i o n s or in a n ' i n a c t i v e ' form. T o evaluate a possible genetic risk posed by these substances, it would be necessary to recognize the cause o f SCE i n d u c t i o n in vitro as well as the m e c h a n i s m s protecting the m a m m a l i a n o r g a n i s m from such a n effect.
Acknowledgements I t h a n k Mrs. T h e a T r a u t m a n n for excellent technical assistance a n d Dr. W. Vogel for useful discussions.
References Carrano, A.V., L.H. Thompson, P.A. Lindl and J.L. Minkler (1978) Sister-chromatid exchange as an indicator of mutagenesis, Nature (London), 271, 551-553. Crossen, P.E. (1979) The effect of acridine compounds on sister-chromatid exchange formation in cultured human lymphocytes, Mutation Res., 68, 295-299. Epplen, J.T., J.W. Siebers and W. Vogel (1975) DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique, Cytogenet. Cell Genet., 15, 177-185. Galloway, S.M., and R.B. Painter (1979) Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests, Mutation Res., 60, 321-327. Kato, H. (1974) Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair, Exp. Cell Res., 85,239-247. MacRae, W.D., and H.F. Stich (1979a) Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compounds, Mutation Res., 68, 351-365. MacRae, W.D., and H.F. Stich (1979b) Induction of sister-chromatid exchanges in Chinese hamster cells by the reducing agents bisulfite and ascorbic acid, Toxicology, 13, 167-174. Nakanishi, Y., and E.L. Schneider (1979) In vivo sister-chromatid exchange: a sensitive measure of DNA damage, Mutation Res., 60, 329-337. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sisterchromatid exchange, Nature (London), 258, 121-125. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister-chromatid exchange and chromosome aberration analysis with the use of several carcinogens and noncarcinogens: brief communication, J. Natl. Cancer Inst., 59, 289-292.
266 Speit, G., and W. Vogel (1979) The effect on sister-chromatid exchanges of drugs and dyes by intercalation and photoactivation, Mutation Res., 59, 223-229. Speit, G., C. Wick and M. Wolf (1980a) Induction of sister-chromatid exchanges by hydroxylamine, hydrazine and isoniazid and their inhibition by cysteine, Hum. Genet., 54, 155-158. Speit, G., M. Wolf and W. Vogel (1980b) The SCE-inducing capacity of vitamin C: Investigations in vitro and in vivo, Mutation Res., 78, 273-278.
261
Elsevier BiomedicalPress
Intercalating substances do not induce sister-chromatid exchanges (SCEs) in vivo G t i n t e r Speit Abteilung Klinische Genetik, Oberer Eselsberg, D-7900 Ulm (Federal Republic of Germany)
(Accepted 13 January 1982)
Summary In contrast to findings in vitro, the intercalating substances proflavine, methylene blue and chlorpromazine did not induce sister-chromatid exchanges (SCEs) in the SCE test in vivo in the bone marrow of the Chinese hamster. The distribution of the metaphases of the first, second and third replication cycles in the presence of bromodeoxyuridine (BrdUrd) showed all 3 substances to have no inhibitory effect on the cellular proliferation in the bone marrow.
Intercalating drugs and dyes induce sister-chromatid exchanges (SCEs) in the V79 cell line of the Chinese hamster (Speit and Vogel, 1979). SCEs have often proved themselves to be sensitive indicators of DNA-damaging effects (Perry and Evans, 1975; Popescu et al., 1977). The fact that intercalating agents are able to induce SCEs indicates that they lead to alterations of the DNA. However, the cause of this effect is not known. It has been proposed that the change in the DNA structure caused by the intercalation is the reason for the induction of SCEs (Speit and Vogel, 1979). However, an investigation of acridine derivatives on human lymphocyte cultures has shown that there is no direct relationship between the intercalation and SCE induction (Crossen, 1979). Because these substances are widely used, for instance as fluorescent dyes, antiseptics and psychopharmaceuticals, the demonstration of a possible DNAdamaging effect would be of great significance. To obtain more relevant information with regard to man, 3 of these substances were investigated, in vivo, with the SCE test, a test that constitutes a sensitive measure for DNA damage and that also takes into account mammalian metabolism.
0165-7992/82/0000-0000/$02.75 ©Elsevier Biomedical Press
262 Materials and methods
The experiments were performed on 6-12-month-old Chinese hamsters, as previously described (Speit et al., 1980a, b). A 50-rag BrdUrd tablet was implanted into the posterior abdominal region. The test substances were injected intraperitoneally 1 h later, after having been dissolved in a physiological saline solution under sterile conditions. The following substances were used: proflavine (Fluka), methylene blue (Merck) and chlorpromazine ('Megaphen', Bayer). 24 h later, colchicine (50 mg/kg) was injected, and 2 h thereafter metaphases were prepared from the bone marrow. Differential chromatid staining was done according to a modified fluorescence-plus-Giemsa technique (Epplen et al., 1975). The SCEs in 40 metaphases were counted for each value, and the mean and the standard deviation from the mean were determined. All trials were repeated at least twice. To evaluate the cellular proliferation, the distribution of the metaphases of the first, second and third generations were determined in 200 mitoses each. Cells that had replicated their DNA exclusively before the implantation of BrdUrd could not be distinguished from first metaphases, and cells that had gone through more than 3 cell cycles were counted as third mitoses. Such cells, however, cannot be expected to occur during an experimental period totalling 26 h.
Results and discussion
The acridine derivative proflavine (PF) as well as the phenothiazine derivatives methylene blue (MB) and chlorpromazine (CPZ) did not lead to a significant induction of SCEs in the bone marrow of the Chinese hamster (Table l). CPZ was tested up to a concentration corresponding to 4 times the therapeutic dose in man. At this concentration, the substance had a pronounced pharmacological effect on the hamster (slowing of movement and apathy). PF and MB, which lead to the induction of SCEs at lesser concentrations than CPZ in vitro (Speit and Vogel, 1979), were tested in vivo up to the same concentration level without showing any effect. An increase of the dose was limited by the solubility of the substances. The distribution of the first, second and third mitoses shows that the proliferation of the cells analysed in the bone marrow was not delayed in comparison with the control (Table 2). If replication were inhibited, one would find a decrease of the frequency of second-replication-cycle cells and an increase in the frequency of firstreplication-cycle cells. To exclude artifacts from the experimental procedure, cyclophosphamide (CP) was tested as a positive control. CP led to a pronounced increase in the SCE frequencies (Table l, Fig. 1) without impeding the proliferation of the cells (data not shown). For the intercalating substances examined here, one therefore obtains qualitative differences in the results of investigations in vitro and in vivo. The weak SCE-
263
TABLE I FREQUENCY OF SCEs 1N THE BONE MARROW TREATMENT WITH PF, MB, CPZ AND CP Substance
Dose (mg/kg)
Control
-
3.3 ± 0.3
PF
1 3 6 12
3.3 3.4 4.0 3.8
MB
1 3 6 12
3.2±0.3 3.4±0.3 3.4±0.3 3.1±0.3
CPZ
1 3 10 15
3.5 ± 0.4 3.6 ± 0.4 3.5 ± 0.3
30 60
24.5 ± 2.1 39,8 ± 2.7
CP
OF
CHINESE
HAMSTERS
AFTER
SCEs per metaphase ± S.E.M.
± ± ± ±
0.3 0.3 0.4 0.4
3.3 ± 0.3
TABLE 2 THE EFFECT OF I N T E R C A L A T I N G SUBSTANCES ON CELL REPLICATION IN THE BONE MARROW OF CHINESE HAMSTERS Substance
Dose (mg/kg)
Cell cycle (%) 1
Control
2
3
3
91
6
PF
1 12
2 8
93 91
5 1
MB
1 12
9 10
91 87
0 3
CPZ
1 15
12 9
87 90
I
1
inducing action of CPZ in vitro has to date only been observed in V79 cells (Speit and Vogel, 1979), and for MB there is a positive finding (Speit and Vogel, 1979) paralleled by a negative one (Popescu et al., 1977). In contrast to these 2 substances,
264
O
%
4P
b
Fig. 1. Metaphase with sister-chromatid differentiation from the bone marrow of the Chinese hamster showing cyclophosphamide-induced(30 mg/kg) sister chromatid exchanges (SCEs). the SCE induction by PF is unequivocally documented in various studies on cell cultures (Kato, 1974; Popescu et al., 1977; Carrano et al., 1978; Crossen, 1979; Speit and Vogel, 1979). At a lower concentration (6 mg/kg), a negative finding for PF in vivo had already been reported (Nakanishi and Schneider, 1979). T o date only few substances have shown an SCE induction in vitro and a negative result in vivo. Thus the fluorescent dye Hoechst 33258 induced SCEs in C H O cells (Perry and Evans, 1975), but not in the bone marrow of the mouse (Nakanishi and Schneider, 1979). The antitubercular drug isoniazide (INH) induced SCEs in various cell lines of the Chinese hamster (MacRae and Stich, 1979a; Speit et al., 1980a), but not in the bone marrow of Chinese hamsters (Speit et al., 1980a). Positive results were also found for vitamin C in vitro in various systems (Galloway and Painter, 1979; MacRae and Stich, 1979b; Spelt et al., 1980b), but no SCE induction in the bone marrow of the Chinese hamster (Speit et al., 1980b). Such diverse findings could be due to the fact that a substance is 'inactivated' in the mammalian metabolism, as is assumed for INH. On the other hand, there is the
265 possibility that, u n d e r c o n d i t i o n s in vitro, reactions occur that are n o t relevant to the m a m m a l i a n m e t a b o l i s m . T h u s the i n d u c t i o n o f SCEs by v i t a m i n C is p r o b a b l y based o n a reaction with the culture m e d i u m ( M a c R a e a n d Stich, 1979b; Speit et al., 1980b). T h e r e a s o n for the negative f i n d i n g for intercalating substances is u n k n o w n , as is the cause for SCE i n d u c t i o n in vitro. As far as a direct c o m p a r i s o n is possible, the dose is the same for b o t h procedures. However, the d i s t r i b u t i o n o f the test substances in the m a m m a l i a n o r g a n i s m is unclarified a n d it is not k n o w n what c o n c e n t r a t i o n s are present in the b o n e m a r r o w . Because intercalating substances i n h i b i t the p r o l i f e r a t i o n o f cell cultures (Speit a n d Vogel, 1979) w i t h o u t i n f l u e n c i n g the p r o l i f e r a t i o n of b o n e - m a r r o w cells, one can c o n c l u d e that these substances a p p e a r in the b o n e m a r r o w in insufficient c o n c e n t r a t i o n s or in a n ' i n a c t i v e ' form. T o evaluate a possible genetic risk posed by these substances, it would be necessary to recognize the cause o f SCE i n d u c t i o n in vitro as well as the m e c h a n i s m s protecting the m a m m a l i a n o r g a n i s m from such a n effect.
Acknowledgements I t h a n k Mrs. T h e a T r a u t m a n n for excellent technical assistance a n d Dr. W. Vogel for useful discussions.
References Carrano, A.V., L.H. Thompson, P.A. Lindl and J.L. Minkler (1978) Sister-chromatid exchange as an indicator of mutagenesis, Nature (London), 271, 551-553. Crossen, P.E. (1979) The effect of acridine compounds on sister-chromatid exchange formation in cultured human lymphocytes, Mutation Res., 68, 295-299. Epplen, J.T., J.W. Siebers and W. Vogel (1975) DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique, Cytogenet. Cell Genet., 15, 177-185. Galloway, S.M., and R.B. Painter (1979) Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests, Mutation Res., 60, 321-327. Kato, H. (1974) Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair, Exp. Cell Res., 85,239-247. MacRae, W.D., and H.F. Stich (1979a) Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compounds, Mutation Res., 68, 351-365. MacRae, W.D., and H.F. Stich (1979b) Induction of sister-chromatid exchanges in Chinese hamster cells by the reducing agents bisulfite and ascorbic acid, Toxicology, 13, 167-174. Nakanishi, Y., and E.L. Schneider (1979) In vivo sister-chromatid exchange: a sensitive measure of DNA damage, Mutation Res., 60, 329-337. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sisterchromatid exchange, Nature (London), 258, 121-125. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister-chromatid exchange and chromosome aberration analysis with the use of several carcinogens and noncarcinogens: brief communication, J. Natl. Cancer Inst., 59, 289-292.
266 Speit, G., and W. Vogel (1979) The effect on sister-chromatid exchanges of drugs and dyes by intercalation and photoactivation, Mutation Res., 59, 223-229. Speit, G., C. Wick and M. Wolf (1980a) Induction of sister-chromatid exchanges by hydroxylamine, hydrazine and isoniazid and their inhibition by cysteine, Hum. Genet., 54, 155-158. Speit, G., M. Wolf and W. Vogel (1980b) The SCE-inducing capacity of vitamin C: Investigations in vitro and in vivo, Mutation Res., 78, 273-278.