The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

The effect of sodium fluoride on DNA synthesis, mitotic indices and chromosomal aberrations in human leukocytes treated with trenimon in vitro

253 Mutation Research, 37 (1976) 253--266 © Elsevier/North-Holland Biomedical Press THE EFFECT OF SODIUM FLUORIDE ON DNA SYNTHESIS, MITOTIC INDICES...

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253

Mutation Research, 37 (1976) 253--266

© Elsevier/North-Holland Biomedical Press

THE EFFECT OF SODIUM FLUORIDE ON DNA SYNTHESIS, MITOTIC INDICES AND CHROMOSOMAL ABERRATIONS IN HUMAN LEUKOCYTES TREATED WITH TRENIMON IN VITRO

RENATE SLACIK-ERBENand GUNTER OBE Institut fiir Genetik, Freie Universit~'t Berlin, Arnimallee 5--7, D-IO00 Berlin 33 (Germany)

(Received February 26th, 1976) (Revision received May 10th, 1976) (Accepted June 8th, 1976) Summary Human leukocyte cultures were set up with Ham's F-10 medium and stimulated with PHA-M. Treatment of the cells in G, from 15--20 h with 0.5 × 10 -6 M Trenimon resulted in a considerable cell cycle delay, as measured by [3H]TdR autoradiography and determination of mitotic indices. Under these conditions only few cells incorporated the tracer at the same time as most cells did in untreated cultures. However, this did n o t lead to a mitotic activity at the same time as obtained in controls. Most of the treated cells started their DNA synthesis and mitotic activities with a delay of around 20 h, as compared with the controls. Continuous treatment of the cells with 10 -3 M NaF had no effect on [3H]TdR labelling or mitotic indices in otherwise untreated cultures, but led to an impressive effect on DNA synthesis in Trenimon-treated cultures, without a considerable effect on the mitotic indices. This finding could be explained as due to a lower alkylation in cellular DNA in the presence of NaF. More cells can start with their DNA synthesis, although they are, like Trenimontreated cultures, incapable of completing it normally. Analyses of the effect of NaF on chromosome aberrations induced by Trenimon revealed that pre-, simultaneous and post-treatments significantly enhanced the frequency of undamaged mitoses. Continuos fluoride treatment also protected the cells from Trenimon-induced damage, but the effect was not significant, possibly because of heavily damaged mitoses which appeared under these conditions. We interpret our findings as an indication of a real anti-mutagenic activity of NaF.

Introduction The human leukocyte culture is a very complex system which is dependent on many factors [ 18]. Cytogenetic work using this system leads to reproducible

254 results only if the experimental conditions are strictly controlled. This is especially important in investigations on combined effects of different agents, such as the analysis of antimutagenic agents and chemical mutagens. In such investigations, at first only post-treatment mitoses should be analyzed. Mixed populations of first, second and even third post-treatment mitoses result in a very complex picture of primary and derived aberrations as can be seen in 3-day leukocyte cultures treated with X-rays or chemical mutagens [21,22]. For example, influences on cell cycle parameters may mimic an anti-mutagenic effect in such systems. Another difficulty in the analysis of anti-mutagenic activities could be that the "anti-mutagen" may influence the phytohaemagglutinininduced stimulatory processes of the lymphocytes. Reducing agents, such as Lcystein, enhance the response of l y m p h o c y t e s to PHA [8]. Reaction products formed between the mutagen and the "anti-mutagen", as between Trenimon and cystein [17], may also influence the results. Cystein has been reported to be antimutagenic to the chromosome-breaking activity of chemical mutagens in three
Materials, methods and results L e u k o c y t e cultures were set up as follows. Ham's F-10 medium was complemented with 10% foetal bovine serum. 10 ml of this medium contained 0.24 ml PHA M, 2.0 mg dihydrostreptomycin, 200 IU penicillin and 0.8 ml female venous blood. The cultures were incubated in plastic syringes (Expts. A, B), in plastic centrifuge tubes (Expt. E) or in glass bottles (Expts. C, D) at 37 ° C. Determination of DNA synthesis and mitotic indices Expt. A (controls) Cultures containing 2.0 ml medium-blood mixture were started on the first day of the experiment at 17.00 h (part (1) of the experiment) and on the second day at 9.00 h (part (2) of the experiment). Each run consisted of 8 cultures. On the third day of *.,he experiment one culture was terminated every 2 h, and preparations were made from 9.00 h (24 h culture time for part (2) and 40 h for part (1)) up to 23.00 h (38 h culture time for part (2) and 54 h for part (1)). This experimental procedure provided preparations every 2 h from

255 24 up to 54 h after the initiation of culture. 2 pCi of [3H]TdR (Biochemical Centre, Amersham; specific activity 2 Ci/mmol) were added to the cultures 15 min before termination of their growth. The preparations were made in a routine way (hypotonic treatment with 0.075 M KC1, acetic--alcohol fixation, air drying) and stained with 2% orcein in 65% acetic acid. Autoradiographs were made with Kodak nuclear track emulsion NTB 3 and exposed for two weeks. The percentages of labelled interphase nuclei were calculated from 200 to 1000 analyzed cells per sample. The mitotic indices were calculated after the analysis of 1000 cells for each entry. The whole experiment was performed in duplicate. The pooled data of the two parallel experiments are given in Fig. 1. Maxima for DNA synthesis were found around 36 h (labelling index, LI = 41.74%) and 44 h (LI = 43.26%). First mitoses occurred at 32 h, a first peak was seen at about 42 h (mitotic index, MI = 1.2%) and a second one at about 48 h (MI = 1.95%).

Expt. B (NaF) Expt. B was carried out in duplicate in the same way as Expt. A, except for the addition of NaF to a final concentration of 10 -3 M at the initiation of culture. The pooled data of the two parallel experiments are given in Fig. 2. The results are similar to that of Expt. A. DNA synthesis maxima were found around 38 h (LI = 50.0%) and 46 h (LI = 48.14%) and mitotic peaks at around 42 h (MI = 1.65%) and 48 h (MI = 1.7%).

Expt. C (Trenimon) This experiment was performed to determine the effect of Trenimon on the DNA synthesis and the mitotic indices at every hour from 23 up to 74 h culture time. Eight cultures were set up, each containing 13.5 ml medium-blood mixture. Trenimon was added to a final concentration of 0.5 × 10 -6 M from 15 to 20 h after initiation of culture, i.e. in the G~ phase of the cell cycle [7]. The Trenimon medium was removed by washing the cells once with Hank's balanced salt solution. The cell pellet was resuspended in conditioned medium from untreated cultures growing in parallel. All handlings were performed at 37°C. Preparations and autoradiographs were made from 1-ml samples taken from the pooled cultures at the appropriate times. This procedure allows the hourly determination of the percentage of cells undergoing DNA synthesis and of the mitotic indices from two parallel series [7,21]. The percentages of the labelled interphase nuclei were calculated after 300 up to 1000 cells had been counted for each h and parallel series. The mitotic indices were calculated after 1000 cells had been analyzed for each entry. The results of this experiment are shown in Fig. 3. DNA synthesis started 26h after initiation of the cultures. A first peak was found in the period of 33 to 36 h with about 15% of the interphases labelled. Thereafter up to 50 h almost no DNA synthesis occurred. Later, about 57 h after the culture had been started the percentage of [3H]TdR-labelled interphase nuclei increased up to 20%. The results of the two parallel series are similar. The first mitoses were observed around 54 h after initiation of the culture. Sub-populations were not recognizable.

Expt. D (NaF and Trenimon) Expt. D was carried out as Expt. C except that NaF was added to a final con-

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Controls NaF 0--53 h

Series I Trenimon (TR) 15--20 h TR 15--20 h + NaF 0--15 TR 15--20 h + NaF 15--20 TR 15--20 h + NaF 20--65 TR 15--20 h + NaF 0--65

S e r i e s II Trenimon (TR) 15--20 h TR 15--20 h + NaF 0--15 TR 15--20 h + NaF 15--20 TR 15--20 h + NaF 20--74 TR 15--20 h + NaF 0--74

18.50 15.50 11.50 20.00 22.00

24.00 16.50 15.50 14.00 27.50

9.50 10.66

0.240 0,205 0,145 0.275 0.286

0.375 0.205 0,230 0.230 0.485

0.105 0.123

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29.50 19.50 22.50 22.00 24.50

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4.50 5.00

0.315 0.185 0.235 0.265 0.273

0.340 0.235 0.275 0.270 0.290

0.045 0.050

Number per cell

Per c e n t of cells

Per c e n t of cells

Number per cell

Chromatid breaks (B')

Achromatic lesions (AL)

28.00 18.00 22.00 20.50 21.00

31.50 16.50 19.50 20.50 27.50

1.00 0.33

Per c e n t of cells

with four chromosomes

= 6, w i t h f i v e c h r o m o s o m e s

= 8; R B ' B "

= 3.

12.50 4.00 4.50 5.50 8.50

11.50 8.50 9.00 8.50 13.50

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Per c e n t of cells

0.145 0,045 0.050 0.070 0.103

0.160 0.090 0.095 0.090 0.165

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Number per cell

Chromatid translocations (RB' B")

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0.030 0.035 0.015 0.025 0.030

0.035 0.015 -0.010 0.01 5

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1.425 0.715 0.845 1.045 1.103

1.600 0.835 0.885 0.935 1.440

0.065 0.057

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S u m t o t a l of all breakage events per cell a

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7.00 3.00 -1.00 1.50

Per c e n t of cells

Dicentric and Ring chromosomes

o f c u l t u r e s a t 6 5 h, C o l e e m i d t r e a t m e n t

TREATMENT W I T H N a F ( 1 0 - 3 M), B Y T R E A T WITH BOTH SUBSTANCES

= 2; R B ' w i t h t w o c h r o m o s o m e s

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Treatments

as i n S e r i e s I.

o f c u l t u r e s a t 5 3 h, C o l c e m i d t r e a t m e n t

LEUKOCYTES IN VITRO PRODUCED BY CONTINUOUS ( 0 . 5 ) < 1 0 - 6 M) A N D B Y C O M B I N A T I O N A L TREATMENTS

t e r m i n a t i o n o f c u l t u r e s a t 7 4 h, C o l e e m i d t r e a t m e n t

Controls and NaF treatment:

CHROMOSOMAL ABERRATIONS IN H U M A N M E N T I N G 1 F R O M 15---20 h W I T H T R E N I M O N

TABLE I

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261 centration of 10 -3 M at time 0. After Trenimon treatment the cells were reincubated in conditioned medium from NaF-treated cultures growing in parallel. Fig. 4 shows the results of this experiment. DNA synthesis started around 29 h after initiation of the culture and the percentage of labelled interphase nuclei increased slowly up to about 35% around 56 h after initiation of the culture. The results of the two parallel series are similar. The first mitoses occurred around 47 h in one, and around 55 h in the other, series. Sub-populations were not recognizable.

Effect of NaF on chromosomal aberrations produced by Trenimon Expt. E After the addition of Trenimon and NaF, each culture contained 5.0 ml fluid. The Trenimon treatment was performed as described in Expts. C and D. In the combination series, NaF to a final concentration of 10 -3 M was applied simultaneously (NaF, 15--20 h, series I and II), before (NaF, 0--15 h, series I and II) and after (NaF, 20--65 h, series I; NaF, 20--74 h, series II) Trenimon treatment. In one series NaF was present from the start of culture till its termination (NaF, 0 - 6 5 h, series I; NaF, 0--74 h, series II). For each combination experiment, as well as for the series treated with Trenimon alone, 4 cultures were set up: 2 cultures were stopped at 65 h (series I) and 2 at 74 h (series II) culture time, with a 9-h Colcemid treatment included. Chromosomes were prepared as usual and stained with Giemsa stain. Preparations from control cultures and from cultures treated with NaF alone were made 53 h after inoculation, with a 5-h Colcemid treatment included. In .these two series, 200 and 300 mitoses were analyzed. Except in the one combinational series (NaF 0--74 h) where 300 mitoses were analyzed, in all combinational series and in the series treated with Trenimon alone 200 mitoses were analyzed (100 mitoses for each

T A B L E II PERCENTAGE A N D II.

OF MITOSES WITHOUT ANY ABERRATION

( A L I N C L U D E D ) F O U N D IN S E R I E S I

P r o b a b i l i t i e s (P) for d i f f e r e n c e s b e t w e e n t h e T r e n i m o n t r e a t m e n t s as c o m p a r e d w i t h t h e T r e n i m o n + N a F t r e a t m e n t s as r e v e a l e d b y t h e X 2 t e s t . Treatments

Mitoses without a b e r r a t i o n s (%)

P

Series I Trenimon (TR) 15--20 h TR 15--20 h + NaF 0--15 h TR 15--20 h + NaF 15--20 h T R 1 5 - - 2 0 h + N a F 20---65 h TR 15--20 h + NaF 0--65h

28.50 49.00 45.50 49.00 36.00

0.00003 0.0007 0.0003 0.11

Series II Trenimon (TR) 15--20 h TR 15--20 h + NaF 0--15h TR 15--20 h + NaF 15--20 h TR 15--20 h + NaF 20--74 h TR 15--20h+ NaF 0--74h

35.00 53.00 52.50 48.50 41.33

0.0003 0.0004 0.006 0.05

262 culture). The following types of aberration were found: achromatic lesions (AL), chromatid breaks (B'), isochromatid breaks (B"), chromatid translocations (RB'; tri-radials, RB'B"), dicentric chromosomes and ring chromosomes. The results of these experiments are shown in Tables I and II. NaF alone had no effect on the chromosomes. Trenimon induced chromatid aberrations and a few dicentric and ring chromosomes. With few exceptions, the frequencies of all types of aberration were suppressed in the NaF-Trenimon series as compared with the series treated with Trenimon alone. The sum totals of all breaking events per cell were lowered in all NaF-Trenimon series as compared with the Trenimon series (Table I). The frequencies of undamaged mitoses were higher in the NaF-Trenimon series as compared with the Trenimon series. However, this effect is only significant in the differential NaF treatments (Table II). Discussion The results of the cell cycle analyses demonstrate that 10 -3 M NaF has no effect on the PHA-induced stimulatory processes (Figs. 1 and 2). In the untreated cultures and in the cultures treated with NaF alone, DNA synthesis results in two maxima around 36 and 44 h (NaF-free cultures) and around 38 and 46 h (NaF-treated cultures), and in two maxima of mitotic indices (in both series around 42 and 48 h). In the same culture system, however, with blood from another donor, DNA synthesis maxima were found at 34 and 40 h and maxima of mitotic indices at 44 and 48 h [7]. The differences may well be an outcome of the use of different blood samples [1,4,29]. Our results give a further indication of at least two sub-populations of cells being present in PHAstimulated leukocyte cultures [2--4,13--15,19,30--32]. PHA stimulates T lymphocytes predominantly [10,12,16,25] and the sub-populations may be similar to those of T lymphocytes, which are known to occcur in mice [24]. Treatment of the cells in G~ with Trenimon results in a considerable cell cycle delay (Fig. 3), a well-known phenomenon in mammalian cells treated with alkylating agents [27,28]. The few cells that incorporate [3H]TdR during the same time as the untreated cells may represent cells with only few alkylations which can start DNA synthesis, however, w i t h o u t completing it. Such cells do not give rise to mitoses at the time when untreated cells would (Figs. 1--3). In the presence of NaF, more cells of this type can be seen without any significant effect on the mitotic indices (Fig. 4), indicating that NaF may have an effect on the extent of alkylations in cellular DNA. Papers concerning the anti-mutagenic activity of NaF in Drosophila [5,33] and in human leukocyte chromosomes [23] and unpublished data from our laboratory on leukocyte chromosomes indicate that cross-links are the target of the NaF action. A chemical modification of Trenimon by NaF can be excluded [33]. Possibly, NaF influences the induction of cross-links, or their repair which is known to occur in mammalian cells [11]. The chromosome analyses show that pre-, simultaneous and post-treatment with NaF suppresses the frequencies of the different types of aberration and leads to a significant reduction of the number of damaged mitoses in cultures treated with Trenimon in G1 as compared with cultures treated with Trenimon alone (Tables I and II). The cell cycle analyses indicate that only first post-treatment mitoses had been analyzed in this series, at

263 least in the control experiments and in the cultures treated with NaF alone. There are further lines of evidence indicating that this may also be true in the series treated with Trenimon. (1) An analysis of the frequencies of micronuclei in one series of Expt. C (treatment with Trenimon and NaF) revealed no time-effect relationships as expected if second and further post-treatment interphases had been accumulated. The lack of micronucleus-derived premature chromosome condensations is in agreement with this interpretation [20--22]. (2) The dicentric c h r o m o s o m e s - the very infrequent ring chromosomes are not discussed h e r e - found in the Trenimon series could represent derivatives of aberrations occurring in second or further post-treatment mitoses. However, most dicentrics are associated with fragments and should represent first post-treatment events [4]. However, few are free of fragments at both fixation times (65 and 74 h). The combined data show that the ratio of fragmentless to fragment-associated dicentrics is the same in both series (fixation time 65 h, 0.32; fixation time 74 h, 0.30), indicating that there are at best no more derived aberrations in the 65-h as compared with the 74-h series. The series treated differentially with NaF gave results different from those obtained from series continuously treated. This could be interpreted as meaning that differential NaF treatment, unlike continuous treatment, influences the cell cycle. A simulated anti-mutagenic activity in cell cultures differentially treated with NaF could be the result of Trenimon-damaged cells which are delayed under the influence of NaF. This would imply that the additional delay has to be more than 9 h, because with two fixation times at 65 and 74 h, the same results were obtained. The observation that continuous NaF treatment had no effect on the cell cycle makes this interpretation highly unlikely. The remote possibility that the anti-mutagenic activity of the differential NaF treatment is influenced by an interaction between centrifugation and the NaF treatm e n t is also highly unlikely. We interpret the o u t c o m e of the chromosome analyses as an indication of a real anti-mutagenic activity of NaF. The result of the continuous NaF treatment is unexpected. Possibly the anti-mutagenic activity of the NaF is obscured here by mitoses with heavy damage that are n o t detectable in short time treatments with NaF under our experimental conditions. The anti-mutagenic activity of NaF was more pronounced in experiments described earlier [23]. This may in part be the result of the type of culture used (see Introduction). The results of the post-treatment series indicate that NaF may influence repair processes rather than the induction of cross-links per se. The fact that, irrespective of the impressive influence of NaF on the Trenimon-influenced DNA synthesis, mitoses cannot be observed earlier in the NaF-Trenimon cultures as compared with the cultures treated with Trenimon alone, may indicate that some rate-limiting processes are not influenced by the NaF. Intra- and inter-strand cross-links are repaired by different mechanisms [6,26] and possibly only one of these repair mechanisms is influenced by NaF. Acknowledgement We thank Dr. A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, for valuable discussion. Part of this work

264

was performed in fulfillment of the PhD thesis of R.S.-E. and was sponsored by a post-graduate scholarship of the GraduiertenfSrderungsgesetz of the FRG. References 1 A o k i , Y. a n d G.E. M o o r e , C o m p a r a t i v e s t u d y of m i t o t i c stages of cells d e r i v e d f r o m h u m a n p e r i p h e r a l b l o o d , E x p . Cell Res., 59 ( 1 9 7 0 ) 2 5 9 - - 2 6 6 . 2 B e e k , B. and G. Obe, T h e h u m a n l e u k o c y t e test s y s t e m . II. D i f f e r e n t sensitivities of s u b - p o p u l a t i o n s to a c h e m i c a l m u t a g e n , M u t a t i o n Res., 24 ( 1 9 7 4 ) 3 9 5 - - 3 9 8 . 3 B e e k , B. a n d G. Obe, T h e h u m a n l e u k o c y t e test s y s t e m IV. T h e R N A - s y n t h e s i s p a t t e r n in the first G lp h a s e of the cell cycle a f t e r s t i m u l a t i o n w i t h P H A , M u t a t i o n Res., 29 ( 1 9 7 5 ) 1 6 5 - - 1 6 8 . 4 B e n d e r , M.A. a n d J . G . 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