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Clastogenic effects of ftorafur
301
Mutation Research, 88 (1981) 301--306 © Elsevier/North-Holland Biomedical Press
CLASTOGENIC EFFECTS OF FTORAFUR II. CYTOGENETIC ANALYSIS OF BONE MARROW IN MICE
P. GOETZ and BO~ENA NOVOTNA
Research Institute of Child Development, Genetical Department, Faculty of Pediatrics, Department for Congenital Defects, Institute of Experimental Medicine, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 28 May 1980) (Revision received 23 September 1980) (Accepted 29 September 1980)
Summary Ftorafur was administered i.p. to C57 B16 mice in single doses of 40, 80 and 160 mg/kg b.w. Bone-marrow cells were examined 6, 12, 24 and 48 h after administration. The highest frequency of chromosomal aberrations was present after 24 h, as well as a dose--effect relationship, both in the frequency of abnormal metaphases and the frequency of breaks, mainly chromatid ones. The cytostatic and clastogenic mechanism of action is discussed.
Ftorafur (FT), synthesized by Hiller et al. (1967) as the analogue of 5-fluorouracil (5-FU), showed an anti-tumour activity both in experimental tumours (Pallavicini and Cohen, 1975; Garibjanian et al., 1976) and in tumours in man, particularly in breast and gastro-intestinal turnouts (Blokhina and Voznyj, 1972; Karev et al., 1972; Hattori et al., 1973; Cichy et al., 1974; Okazaki et al., 1974; Valdivieso et al., 1976). A lower toxicity of FT as compared with that of 5-FU found in mice with the same anti-turnout activity (Hrsak and Pavicic, 1974; Johnson et al., 1976), suggested the possibility of different effects on t u m o u r and non-turnout cells. This hypothesis was confirmed by the results of clastogenic activity of FT in human and hamster tumour and non-turnout cells in vitro, even with regard to the difference between the turnout karyotype and the karyotype of normal tissues (Volgareva et al., 1977, 1978, 1979; Sokova, 1978; Sokova and Volgareva, 1979). Nevertheless, the studies in vivo did not indicate different frequencies of chromosomal aberrations in turnout and non-turnout Djungarian hamster cells after the application of FT (Sokova and Volgareva, 1977). In the present study,
302
therefore, we focused our interest on the FT effects on bone-marrow cells in C57 B16 mice with the aim of characterizing its clastogenic effect on nontumour proliferating tissue. Material and methods Ftorafur (Medeexport USSR series 950678, a 10-ml ampoule of 4% solution), was freshly diluted with physiological saline and given intra-peritoneally in single doses of 40, 80 and 160 mg/kg b.w. to C57 Bl6 mice. Bone-marrow cells were examined 6, 12, 24 and 48 h after the administration. Each experimental and control groups consisted o f 5 animals; 50 cells were analysed in all concentrations and from each animal, according to the recommendation of Bochkov et al. (1976). Gaps, breaks and exchanges were evaluated. Cells showing any of these abnormalities were considered abnormal. Statistical assessment was done by an arcsin transformation t test. Results The results of the cytogenetic analysis are shown in Table 1. The highest frequencies of aberrant metaphases were present 24 h after the application of FT, with doses of 40 and 80 mg/kg b.w. With both doses, the difference between intervals of 6 and 12 h after the application was not significant. Both doses resulted in a decrease of the frequency of abnormal metaphases 48 h after the
TABLE 1 FTORAFUR,
Interval after appHcation
BONE MARROW,
Dose in mg/kg
N
Aberrant cells
Gaps %
%
Control
M I C E C 5 7 BI6
A b e r r a n t cells e x c l u d i n g gaps G/C
abs.
Breaks %
%
Exchanges B/C
%
E/C
--
--
abs.
250
1.6
4
1.2
0.012
0.4
1
0.4
0.004
40 80
250 250
7.6 13.6
19 34
6.4 8.8
0.068 0.088
1.2
3 18
1.2
0.012
--
--
7.2
7.2
0.076
--
--
40 80
250 250
8.4 18.0
21 45
5.6 8.0
0.056 0.080
2.8
7
2.8
0.032
--
--
10.8
27
10.8
0.112
--
--
24 h
40 80 160
250 250 250
19.2 44.8 55.2
48 112 138
11.2 22.8 20.0
0.116 0.28 0.26
11.2 32.0 79.2
28 80 198
11.2 28.8 55.2
0.136 0.48 1.056
--2.8
--0.028
48 h
40
250
14.0
35
8.4
0.088
8.0
20
8.0
0.112
--
--
80
250
25.2
63
12.8
0.132
13.6
34
14.0
0.172
--
--
6h
12 h
G / C , gaps per cell.
B / C , b r e a k s per cell. E / C , e x c h a n g e s per cell.
303 application, probably due to a rapid elimination of the substance from the organism and the elimination or repair of damaged cells. The same frequency changes as in abnormal metaphases could be seen in the number of breaks related to the cell. With both doses the maximal frequency of chromosomal breaks occurred 24 h after the application; after 48 h the decrease appeared. The value of chromosomal breaks 6 h after application of the dose of 40 mg/kg b.w. did n o t differ significantly from the control level; after 12 h the difference was at the 5% significance level. After the dose of 40 mg/kg b.w. the numbers of breaks after 6 and 12 h did n o t differ significantly from each other. When the dose of 80 mg/kg b.w. was applied, 6 h after application the number of breaks was significantly higher when compared with the controls, b u t no significant difference between the intervals of 6 and 12 h was proved. The highest frequency of chromosomal aberrations was found 24 h after the application. This interval was also found to be most suitable for follow-up of the dose--effect relationship. Both in the frequency of abnormal metaphases and the frequency of chromosomal breaks a significant dose--effect relationship was found. Discussion On the basis of our results, it is obvious that FT is a substance that induces chromosomal aberrations in C57 B16 mice with a maximal frequency 24 h after the application as happens with the majority of chemical clastogens. A significant increase of the frequency of breaks, mostly chromatid ones, occurred in (11% of cells) 24 h after the application even when the dose of 40 mg/kg b.w. was applied. This dosage is the therapeutic dose currently used in man, and a single application represents a b o u t 1/20 of the LD 50 for mice. The dosage of 80 mg/kg b.w. induced breaks in 32% of cells. For man this dose is semi-lethal or even lethal, as was confirmed by the death from t h r o m b o c y t o penia and leukopenia of 2 patients, after the application of a dose of 80 mg FT/kg b.w. in the infusion for a period of 8 days (Smart et al., 1975). Chromosomal rearrangements in our material were found only after the application o f 160 mg FT/kg b.w. and in 3% of cells only. Sokova et al. (1976) did not find increased frequencies of aberrant metaphases in bone-marrow cells of Djungarian hamsters after a single i.p. administration of FT at doses of 200 and 300 mg/kg b.w. when compared with controls. The difference between this finding and our results could be explained by a different species sensitivity to the mutagen. Different frequencies of chromosomal aberrations after identical doses of radiation and/or chemical mutagens were reported both for various species (Goetz et al., 1975, 1976; Ldonard et al., 1976) and in different strains of mice (Surkova and Malashenko, 1975a, b). Different sensitivity of Djungarian hamsters and mice C57 to FT is also indicated by different values of the LD 50. In hamsters, the value stated by Sokova et al. (1976) is 240 mg/kg b.w.; in C57 mice, Johnson et al. (1976) gave the value of 880 mg/kg b.w. The doses applied to hamsters seem to have such cytotoxic effects that they would make the detection of chromosomal aberrations almost impossible.
304 The clastogenic effect of FT on the cells of proliferating tissues in vivo can be presumed from its metabolic conversion and cytostatic effects. In the organism, FT acts as the depot form of 5-FU. The cytological effect is ensured by its metabolite 5-fluoro-2'-deoxyuridine-5-monophosphate which inhibits the activity of thymidylate synthetase; that is, it inhibits the incorporation of thymine into DNA and its synthesis (Pallavicini et al., 1979). The importance of the metabolic activation in vivo was proved by a low inhibition of deoxyuridine incorporation into DNA in vitro, and by the acceleration of the FT-5-FU conversio.n after the addition of the homogenates of various tissues, particularly of liver (Fujita et al., 1972; Meirena and Belousova, 1974). The conversion of FT to 5-FU is species-specific, and it occurs most rapidly in mice (Fujita et al., 1972; Lu et al., 1975). Pallavicini et al. (1979) proved that the inhibition of t u m o u r growth in mice influenced by FT and 5-FU depends on the extent of suppression of the deoxyuridine incorporation into DNA. Changes of the incorporation of deoxyuridine into t u m o u r cells and proliferating cells of the small intestine were similar after the application of FT. If one supposes that chromosomal damage is a consequence of blockage of DNA synthesis by FT metabolites (Volgareva et al., 1977) an increase in the frequency of abnormal metaphases, particularly with trials in vivo, may be expected, and that both in the proliferating cells of bone marrow as shown by our own results, as well as in t u m o u r cells (Volgareva et al., 1977, 1978). Very low frequencies of chromosomal aberrations in non-tumour cells cultivated in vitro in culture media with different FT concentrations (Sokova et al., 1976; Sokova and Volgareva, 1979; Volgareva et al., 1978, 1979) might well add support to the hypothesis that an extension of mutagenic activity of FT is caused by metabolic activation in vivo. However, the arrangement of such experiments is itself of paramount importance. For example, when media (FT) were removed at different intervals before the fixation of embryonal fibroblasts from Djungarian hamsters, an increased frequency of chromosomal aberrations was also found (Volgareva et al., 1979). This finding, together with the positive clastogenic effects of FT manifested in different t u m o u r lines, in vitro (Sokova et al., 1976; Sokova and Volgareva, 1979; Volgareva et al., 1978, 1979) shows that the mutagenic mechanism does n o t have to be fully dependent upon the metabolic activation in vivo. Moreover, on the basis of cytological and metabolic variations, the t u m o u r cells in particular may have different sensitivities altogether. According to Volgareva et al. (1978), a more intensive antimetabolic effect of FT on non-tumour cells causes a blockage of the mitotic cycle, so that cells bearing chromosomal aberration do n o t reach the stage of metaphase. A different reactivity of t u m o u r cells was also reported by Pallavicini et al. (1979), who proved the presence'of moderate rates of DNA synthesis within S10 2F m a m m a r y t u m o u r cells and an almost complete inhibition of thymidylate synthetase activity caused by 5-FU. Incomplete inhibition of the mitotic cycle of t u m o u r cells could explain the increased frequencies of metaphases with chromosomal aberrations. The actual relationship between the cytostatic and mutagenic effects of FT is not entirely clear but results indicate that, for a better understanding of such effects, different cytogenetic and biochemical methods should be adopted.
305 References Blokhina, N.G., and E.K. Voznyj (1972) Results of t r e a t m e n t of malignant t u m o u r s w i t h Ftorafur, Cancer, 3 0 , 3 9 0 - - 3 9 2 . Bochkov, N.P., R.J. Sram, N.P. Kuleshov and V.S. Zhurkov (1976) System for the evaluation of the risk from chemical mu tagens for man: Basic principles and practical r e c o m m e n d a t i o n s , Mut a t i on Res., 38, 191--202. Cichy, A., L. Jurga and M. Klvana (1974) Experience with furamidyl fluorouracil in advanced t u m o u r s of breast, Neoplasma, 2 1 , 7 2 3 - - 7 3 2 . Fujita, H., K. Ogava and T. Sawabe (1972a) In vivo distribution of Nl-(2r-tetrahydroxyfuryl)-5-finorouracil (FT-207), Jpn. J. Cancer Clin., 18, 911--916. Fujita, H., K. Ogawa and T. Sawabe (1972b) Metabolism of N l -(2t -t e t ra hydroxyfuryl )-5-fl uoroura c i l (FT-207), Jpn. J. Cancer Clin., 18, 917--922. Garibjanian, B.T., R.K. Johnson, Ira KHne, Srikrishna Vadlamudi, M. Gang, J.M. V e n d i t t i and A. Goldin (1976) Comparison of 5-fluorouracfl and Ftorafur, II. Therapeutic response and d e v e l o p m e n t of resistance in m~rine tumours, Cancer Treat. Rep., 60, 1347--1361. Goetz, P., R.J. Sram and J. Dohnalov~ (1975) Relationship b e t w e e n e xpe ri me nt a l results in m a m m a l s and m an, I. Cytogenetic analysis of bone marrow injury induced by a single dose of c y c l o p h o s p h a m i d e , M utatio n Res., 3 1 , 2 4 7 - - 2 5 4 . Goetz, P., R.J. Sram, I. Kodytkova, J. Dohnalova, O. Dostalova and J. Bartova (1976) Relationship between experimental results in m a m m a l s and man, II. Cytogenetie analysis of bone m a r r o w cells after t r e a t m e n t of C y t e m b e n a and c y c l o p h o s p h a m i d e - - C y t e m b e n y c o m b i n a t i o n , Mutation Res., 41, 143-152. Hattori, T., H. Furue and K. F u k u r a w a (1973) Clinical experience with FT-207, Jpn. J• Cancer Clin., 19, 50---53. Hiller, S.A., R.A. Zhuk and M.Y. Lidak (1967) Analogs of pyri mi di ne nucleosides, I. N l - ( ~ - t e t r a h y d r o x y furyl) derivatives of natural pyrimidine bases and their antimetabolites, Dokl. Akad. Nauk (S.S.S.R.), 176,332--335. Hrsak, I., and S. Pavicic (1974) Comparison of the effects of 5-fluorouracil and F t ora fur on he ma t opoi e s i s in mice, Biomed. J., 21, 164--167. Johnson, R.R., B.T. Garibjanian, D.P. Houchens, I. Kline, M.R. Gaston, A.B. S yrki n and A•G. Idin (1976) Comparison of 5-fluorouracll and Ftorafur, I. Quantitative and qualitative differences in t o x i c i t y t o mice, Cancer Treat. Rep., 60, 1335--1345. Karev, N.I., N.G. Blokhina, E.K. Voznyj and M.D. Pershin (1972) Experience w i t h F t o r a f u r t r e a t m e n t in breast cancer, Neoplasma, 19, 347--350. L~onard, A., G.B., Gerber, D.G. Papworth, G. Decat, E.D. Leonard and Gh. D e k n u d t (1976) The radiosensitivities of l y m p h o c y t e s from pig, sheep, goat and cow, Mut a t i on Res., 36, 319--332. Lu, K., T.L. Loo and J.A. Benvenuto (1975) Pharmacological disposition and m e t a b o l i s m of Ftorafur, Pharmacologist, 1 7 , 2 0 2 . Meixena, D.V., and A.K. Belousova (1974) On the mechanism of action of Ftorafur, a new a n t i t u m o u r agent, Vop. Med. Khim., 1 8 , 2 8 8 - - 2 9 3 . Okazaki, N., N. Hatto ri and T. Ono (1974) T r e a t m e n t of liver cancer with oral a d m i n i s t r a t i o n of N-(2 sfuranidyl)-5-fluorouracil (FT-207), Jpn. J. Gastroentero]., 7 1 , 5 9 7 - - 6 0 1 . Pallavicini, M.G., and A.M. Cohen (1975) The a n t l t u m o u r effect of Ftorafur against l e u k e m i a s and mammary turnouts in mice, Pharmacologist, 1 7 , 2 0 2 • Pallavicini, M.G., A.M. Cohen, L.A. Dethlefsen and J.W. Gray (1979) In vivo effects of 5-fluorouracll a nd F t o r a f u r [1-(tetrahydroxyfuran-2-yl)-5-fluorouracfl] on murine m a m m a r y t u m o r s and s~aall intestine, Cell Tissue Kinet., 12, 177--189. Smart, C.R., L.B. Towsend and W.J. Rusho (1975) Phase I study of Ftorafur, an analog of 5-fluorouracil, Cancer, 3 6 , 1 0 3 - - 1 0 6 . Sokova, O.I. (1978) Ch romosome aberrations and sister c h r o m a t i d exchanges in Chinese h a m s t e r cells treated with Fto rafur and 5-fluorouracll, Genetika, 14, 1818--1823. Sokova O.I., and G.M. Volgareva (1977) The effect of Ftorafttr and 5-fluorouraeil on c h r o m o s o m e s of nor mal and t u m o r cells in vivo, Vopr. Onkol., 23, 94--97. Sokova O.I., and G.M. Volgareva (1979) Effect of Ftorafur and 5-fluorouxacll on the c h r o m o s o m e s of h u m a n t u m o r cells in vitro, Genetika, 15, 855--861. Sokova O.I., G.M. Volg~reva and E.E. Pogosianz (1976) Effect of Ftoraf~Lr on c h r o m o s o m e s of n o r m a l and malignant Djungarian hamster cells, Genetika, 12, 156--159. Suxkova, N.I., and A.M. Malashenko (1975a) Cytogenetic effect of m i t o m y c i n C and c yt os i n arabinoside on mice of different genotypes, Genetika, 11, 38--45. Surkova N.I., and A.M. Malashenko (1975b) Mutagenic effect of t h i o t e p a in l a b o r a t o r y mice, IV. Influence of g enotype and sex on the flcequency of induced c h r o m o s o m a l aberrations in bone ma rrow cells, Genetika, 11, 66--77.
306 V a l d i v i e s o , M., G.P. B o d e y a n d J . A . G o t t l i e b ( 1 9 7 6 ) Clinical e v a l u a t i o n o f F t o r a f u r , C a n c e r R e s . , 3 6 , 1821--1824. V o l g a r e v a , G . M . , O.I. S o k o v a a n d E . E . P o g o s i a n z ( 1 9 7 7 ) S t u d y o f t h e m e c h a n i s m o f c h r o m o s o m e - b r e a k ing effect of Ftorafur, Genetika, 13, 461--467. Volgareva, G.M., O.I. Sokova and E.E. Pogosianz (1978) Effect of Ftorafur on the chromosomes of norr e a l a n d t u m o r cells o f D j u n g a r i a n h a m s t e r i n v i t r o , G e n e t i k a , 1 4 , 4 4 4 - - 4 4 7 . Volgv.reva, G . M . , O . I . S o k o v a a n d E . E . P o g o s i a n z ( 1 9 7 9 ) E f f e c t o f F t o r a f u r o n c h r o m o s o m e s o f h u m a n n o r m a l a n d t u m o r cells in v i t r o , G e n e t i k a , 1 5 , 1 5 7 - - 1 6 1 .
Mutation Research, 88 (1981) 301--306 © Elsevier/North-Holland Biomedical Press
CLASTOGENIC EFFECTS OF FTORAFUR II. CYTOGENETIC ANALYSIS OF BONE MARROW IN MICE
P. GOETZ and BO~ENA NOVOTNA
Research Institute of Child Development, Genetical Department, Faculty of Pediatrics, Department for Congenital Defects, Institute of Experimental Medicine, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 28 May 1980) (Revision received 23 September 1980) (Accepted 29 September 1980)
Summary Ftorafur was administered i.p. to C57 B16 mice in single doses of 40, 80 and 160 mg/kg b.w. Bone-marrow cells were examined 6, 12, 24 and 48 h after administration. The highest frequency of chromosomal aberrations was present after 24 h, as well as a dose--effect relationship, both in the frequency of abnormal metaphases and the frequency of breaks, mainly chromatid ones. The cytostatic and clastogenic mechanism of action is discussed.
Ftorafur (FT), synthesized by Hiller et al. (1967) as the analogue of 5-fluorouracil (5-FU), showed an anti-tumour activity both in experimental tumours (Pallavicini and Cohen, 1975; Garibjanian et al., 1976) and in tumours in man, particularly in breast and gastro-intestinal turnouts (Blokhina and Voznyj, 1972; Karev et al., 1972; Hattori et al., 1973; Cichy et al., 1974; Okazaki et al., 1974; Valdivieso et al., 1976). A lower toxicity of FT as compared with that of 5-FU found in mice with the same anti-turnout activity (Hrsak and Pavicic, 1974; Johnson et al., 1976), suggested the possibility of different effects on t u m o u r and non-turnout cells. This hypothesis was confirmed by the results of clastogenic activity of FT in human and hamster tumour and non-turnout cells in vitro, even with regard to the difference between the turnout karyotype and the karyotype of normal tissues (Volgareva et al., 1977, 1978, 1979; Sokova, 1978; Sokova and Volgareva, 1979). Nevertheless, the studies in vivo did not indicate different frequencies of chromosomal aberrations in turnout and non-turnout Djungarian hamster cells after the application of FT (Sokova and Volgareva, 1977). In the present study,
302
therefore, we focused our interest on the FT effects on bone-marrow cells in C57 B16 mice with the aim of characterizing its clastogenic effect on nontumour proliferating tissue. Material and methods Ftorafur (Medeexport USSR series 950678, a 10-ml ampoule of 4% solution), was freshly diluted with physiological saline and given intra-peritoneally in single doses of 40, 80 and 160 mg/kg b.w. to C57 Bl6 mice. Bone-marrow cells were examined 6, 12, 24 and 48 h after the administration. Each experimental and control groups consisted o f 5 animals; 50 cells were analysed in all concentrations and from each animal, according to the recommendation of Bochkov et al. (1976). Gaps, breaks and exchanges were evaluated. Cells showing any of these abnormalities were considered abnormal. Statistical assessment was done by an arcsin transformation t test. Results The results of the cytogenetic analysis are shown in Table 1. The highest frequencies of aberrant metaphases were present 24 h after the application of FT, with doses of 40 and 80 mg/kg b.w. With both doses, the difference between intervals of 6 and 12 h after the application was not significant. Both doses resulted in a decrease of the frequency of abnormal metaphases 48 h after the
TABLE 1 FTORAFUR,
Interval after appHcation
BONE MARROW,
Dose in mg/kg
N
Aberrant cells
Gaps %
%
Control
M I C E C 5 7 BI6
A b e r r a n t cells e x c l u d i n g gaps G/C
abs.
Breaks %
%
Exchanges B/C
%
E/C
--
--
abs.
250
1.6
4
1.2
0.012
0.4
1
0.4
0.004
40 80
250 250
7.6 13.6
19 34
6.4 8.8
0.068 0.088
1.2
3 18
1.2
0.012
--
--
7.2
7.2
0.076
--
--
40 80
250 250
8.4 18.0
21 45
5.6 8.0
0.056 0.080
2.8
7
2.8
0.032
--
--
10.8
27
10.8
0.112
--
--
24 h
40 80 160
250 250 250
19.2 44.8 55.2
48 112 138
11.2 22.8 20.0
0.116 0.28 0.26
11.2 32.0 79.2
28 80 198
11.2 28.8 55.2
0.136 0.48 1.056
--2.8
--0.028
48 h
40
250
14.0
35
8.4
0.088
8.0
20
8.0
0.112
--
--
80
250
25.2
63
12.8
0.132
13.6
34
14.0
0.172
--
--
6h
12 h
G / C , gaps per cell.
B / C , b r e a k s per cell. E / C , e x c h a n g e s per cell.
303 application, probably due to a rapid elimination of the substance from the organism and the elimination or repair of damaged cells. The same frequency changes as in abnormal metaphases could be seen in the number of breaks related to the cell. With both doses the maximal frequency of chromosomal breaks occurred 24 h after the application; after 48 h the decrease appeared. The value of chromosomal breaks 6 h after application of the dose of 40 mg/kg b.w. did n o t differ significantly from the control level; after 12 h the difference was at the 5% significance level. After the dose of 40 mg/kg b.w. the numbers of breaks after 6 and 12 h did n o t differ significantly from each other. When the dose of 80 mg/kg b.w. was applied, 6 h after application the number of breaks was significantly higher when compared with the controls, b u t no significant difference between the intervals of 6 and 12 h was proved. The highest frequency of chromosomal aberrations was found 24 h after the application. This interval was also found to be most suitable for follow-up of the dose--effect relationship. Both in the frequency of abnormal metaphases and the frequency of chromosomal breaks a significant dose--effect relationship was found. Discussion On the basis of our results, it is obvious that FT is a substance that induces chromosomal aberrations in C57 B16 mice with a maximal frequency 24 h after the application as happens with the majority of chemical clastogens. A significant increase of the frequency of breaks, mostly chromatid ones, occurred in (11% of cells) 24 h after the application even when the dose of 40 mg/kg b.w. was applied. This dosage is the therapeutic dose currently used in man, and a single application represents a b o u t 1/20 of the LD 50 for mice. The dosage of 80 mg/kg b.w. induced breaks in 32% of cells. For man this dose is semi-lethal or even lethal, as was confirmed by the death from t h r o m b o c y t o penia and leukopenia of 2 patients, after the application of a dose of 80 mg FT/kg b.w. in the infusion for a period of 8 days (Smart et al., 1975). Chromosomal rearrangements in our material were found only after the application o f 160 mg FT/kg b.w. and in 3% of cells only. Sokova et al. (1976) did not find increased frequencies of aberrant metaphases in bone-marrow cells of Djungarian hamsters after a single i.p. administration of FT at doses of 200 and 300 mg/kg b.w. when compared with controls. The difference between this finding and our results could be explained by a different species sensitivity to the mutagen. Different frequencies of chromosomal aberrations after identical doses of radiation and/or chemical mutagens were reported both for various species (Goetz et al., 1975, 1976; Ldonard et al., 1976) and in different strains of mice (Surkova and Malashenko, 1975a, b). Different sensitivity of Djungarian hamsters and mice C57 to FT is also indicated by different values of the LD 50. In hamsters, the value stated by Sokova et al. (1976) is 240 mg/kg b.w.; in C57 mice, Johnson et al. (1976) gave the value of 880 mg/kg b.w. The doses applied to hamsters seem to have such cytotoxic effects that they would make the detection of chromosomal aberrations almost impossible.
304 The clastogenic effect of FT on the cells of proliferating tissues in vivo can be presumed from its metabolic conversion and cytostatic effects. In the organism, FT acts as the depot form of 5-FU. The cytological effect is ensured by its metabolite 5-fluoro-2'-deoxyuridine-5-monophosphate which inhibits the activity of thymidylate synthetase; that is, it inhibits the incorporation of thymine into DNA and its synthesis (Pallavicini et al., 1979). The importance of the metabolic activation in vivo was proved by a low inhibition of deoxyuridine incorporation into DNA in vitro, and by the acceleration of the FT-5-FU conversio.n after the addition of the homogenates of various tissues, particularly of liver (Fujita et al., 1972; Meirena and Belousova, 1974). The conversion of FT to 5-FU is species-specific, and it occurs most rapidly in mice (Fujita et al., 1972; Lu et al., 1975). Pallavicini et al. (1979) proved that the inhibition of t u m o u r growth in mice influenced by FT and 5-FU depends on the extent of suppression of the deoxyuridine incorporation into DNA. Changes of the incorporation of deoxyuridine into t u m o u r cells and proliferating cells of the small intestine were similar after the application of FT. If one supposes that chromosomal damage is a consequence of blockage of DNA synthesis by FT metabolites (Volgareva et al., 1977) an increase in the frequency of abnormal metaphases, particularly with trials in vivo, may be expected, and that both in the proliferating cells of bone marrow as shown by our own results, as well as in t u m o u r cells (Volgareva et al., 1977, 1978). Very low frequencies of chromosomal aberrations in non-tumour cells cultivated in vitro in culture media with different FT concentrations (Sokova et al., 1976; Sokova and Volgareva, 1979; Volgareva et al., 1978, 1979) might well add support to the hypothesis that an extension of mutagenic activity of FT is caused by metabolic activation in vivo. However, the arrangement of such experiments is itself of paramount importance. For example, when media (FT) were removed at different intervals before the fixation of embryonal fibroblasts from Djungarian hamsters, an increased frequency of chromosomal aberrations was also found (Volgareva et al., 1979). This finding, together with the positive clastogenic effects of FT manifested in different t u m o u r lines, in vitro (Sokova et al., 1976; Sokova and Volgareva, 1979; Volgareva et al., 1978, 1979) shows that the mutagenic mechanism does n o t have to be fully dependent upon the metabolic activation in vivo. Moreover, on the basis of cytological and metabolic variations, the t u m o u r cells in particular may have different sensitivities altogether. According to Volgareva et al. (1978), a more intensive antimetabolic effect of FT on non-tumour cells causes a blockage of the mitotic cycle, so that cells bearing chromosomal aberration do n o t reach the stage of metaphase. A different reactivity of t u m o u r cells was also reported by Pallavicini et al. (1979), who proved the presence'of moderate rates of DNA synthesis within S10 2F m a m m a r y t u m o u r cells and an almost complete inhibition of thymidylate synthetase activity caused by 5-FU. Incomplete inhibition of the mitotic cycle of t u m o u r cells could explain the increased frequencies of metaphases with chromosomal aberrations. The actual relationship between the cytostatic and mutagenic effects of FT is not entirely clear but results indicate that, for a better understanding of such effects, different cytogenetic and biochemical methods should be adopted.
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