Mutation Research, 281 (1992) 277-282
277
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00645
Effects of benzyladenine on prokaryotic and eukaryotic cells M i r j a n a Pavlica, Dra~,ena Pape,~, J a s n a F r a n e k i 6
a
and Biserka Nagy
Department of Molecular Biology, Faculty of Science, Unit~ersityof Zagreb and a Faculty of Food Technology and Biotechnology, Laboratory of Biology and Microbial Genetics, 41000 Zagreb, Croatia (Yugo.dacia) (Received 16 October 1991) (Revision received 22 November 1991) (Accepted 25 November 1991)
Keywords: Benzyladenine; Chromosome aberrations; Cytotoxicity; Mutagenicity
Summary Comparative studies of the effect of benzyladenine (BA) on the yeast Saccharomyces cerevisiae, the bacterium Salmonella typhimurium, the shallot Allium ascalonicum and Chinese hamster fibroblast cells were performed. The tested substance had no mutagenic activity on yeast, bacteria and cultured fibroblast cells. Changes in mitotic activity and cell division abnormalities were observed after BA treatment in shallot root-tip cells.
Synthetic cytokinins such as benzyladenine (BA) are plant growth regulators used for establishing plant cell and tissue cultures. Cytokinins are implicated in the control of cell division in cultured cells as well as in meristems of intact plants (Skoog et al., 1965). They are also involved in the regulation of the cell cycle and function of the root meristem (Torrey, 1976). BA (CAS No. 1214-39-7), which is widely used in plant tissue culture, passively enters into the tissue at an optimum temperature of 25°C (Vogelman et al., 1984). The administration of BA to the intact plants inhibits root growth (Stenlid, 1982). Also, a decrease in mitotic activ-
Correspondence: Dr. M. Pavlica, Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, P.O. Box 933, 41001 Zagreb, Croatia (Yugoslavia).
ity and an increase in the number of nuclei in G 2 after treatment with BA in pea root meristems could be observed (Levi et al., 1987). BA induced endoreduplication in epidermal cells of bean primary leaves, and also increased the amount of DNA (Naito et al., 1978). But it is known that cell division is far more sensitive to the cytokinin level than DNA synthesis (Bernier et al., 1977). The aim of this work was to analyze the effect of BA on mitotic activity and chromosome aberration induction in shallot (Allium ascalonicum L.) root tip cells. The study was supplemented with cytotoxicity and mutagenicity testing of BA at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells. The Salmonella/mammalian microsome test on the bacterium Salmonella typhimurium, and the test with the yeast Saccharomyces cerevisiae for detection of gene mutations and gene conversion were also used.
278 Materials and methods
Plant test material In our experiments equal-sized bulbs from a diploid (2n = 16) population of shallot A. ascalonicum L. (syn. A. cepa var. aggregatum) were used. Bulbs were grown for 7 days in BA (Sigma) water solutions, at concentrations of 0.125, 1.25, 12.5 and 125/~M. BA solutions were changed every 24 h. Tap water was used as a control (Fiskesj6, 1988). The roots were incubated at room temperature (26 + I°C), exposed to artificial light for 16 h per day at a light intensity of 17 W / m 2. For microscopic studies root tips were cut evcry 24 h during the 7-day experiment, while roots grown in 125 /.tM BA were cut after 7 days of treatment. Material was fixed in ethanol:acetic acid (3: 1) for 24 h, and slides were prepared using the Feulgen squash technique (Sharma and Sharma, 1972). Permanent slides were prepared using liquid carbon dioxide. After 1 day drying, they were mounted in Euparai mounting medium ('Gurr'). In order to analyze microscopic parameters (Fiskesj6, 1985) at least 2000 ceils were scored (about 100 per slide) per treatment and concentration. During the 21-day experiment root elongation was measured and searched for any morphological changes.
Anhnal cells and culture conditions Chinese hamster V79 fibroblast cells were maintained as stock cultures in a-minimal essential medium (a-MEM, Gibco) with 10% fetal calf serum (Flow Laboratories) in a humidified atmosphere containing 5% CO 2 in air at 37°C. All experiments were performed in triplicate with exponentially growing cells. Exponentially growing cultures of V79 cells were treated with various concentrations of BA (0.125, 12.5, 125 and 1250 p,M) for 60 min. After treatment all cultures were washed twice with PBS (8.1 mM NazHPO4-1.5 mM KHzPO4-0.14 M NaCI-2.6 mM KCi) and trypsinized. Cell survival was determined by plating an appropriate number of cells to give about 200 colonies per dish, 6 dishes per experimental point (Nagy et al., 1986).
For detection of H G P R T mutants cells were plated into 150-cm 2 T-flasks at a cell density of 2 × 106 cells per flask. After 24 h of growth cells were treated with BA according to the previously described protocol. Cells were then washed, trypsinized and plated. Mutation induction was assayed by seeding at least 106 surviving ceils per experimental point. Cells were grown in nonselective medium for 6 days to allow mutants to be expressed (Thacker et al., 1976). The plating efficiency was determined by seeding 200 ceils per plate in standard growth medium. Mutation induction was determined by seeding 105 ceils into each of I0 plates containing standard growth medium and 6-thioguanine (6-TG) at a concentration of 5 /~g/ml. Following 7 days of growth, the resulting mutant colonies were stained with 0.5% methylene blue and counted. Mutation frequencies are expressed as the number of 6-TGresistant cells per 10 6 surviving ceils, corrected for spontaneous background frequencies.
Detection of genetic effects on Saccharomyces cerecisiae The test was performed on diploid yeast strain D7 (Zimmermann, 1975) with the genotype ade2-
40lade2-119;
trp5-12/trp5-27;
ih'l-92/ih;1-92.
Yeast cells in mid-log phase were harvested and exposed to various concentrations of benzyladenine (1.25, 12.5, 125 and 1250/~M) in phosphate buffer, pH 7.4, for 2 h at 30°C. Ceils were washed, centrifuged and adequate dilutions were spread on complete medium, tryptophan-free medium and isoleucine-free medium. The yield of colonies counted on these 3 media allows us to calculate cell survival and the frequency of spontaneous or chemically induced gene convertants and mutants.
Mutagenicity murium
assay in his-
Salmonella
typhi-
The mutagenicity of BA in the Salmonella test was examined according to the standard plate-incorporation test procedure with tester strains TA100 (hisG46, rfa, AUL,rB, pKMI01) and TA98 (hisD3052, rfa, AucrB, pKMI01) (Maron and Ames, 1983). BA solutions were adjusted to pH 3.3-3.7, incubation time was 1 h at 37°C. Concentrations of BA were the same as above. After
279 8
incubation each mixture was neutralized with PBS, pH 7.4, prior to adding to the top agar. For metabolic activation $9 mix containing 10% liver $9 fraction from male Aroclor-treated rats (protein concentration 37.8 m g / m l ) was added to the top agar as well as to the bacterial cells and the compound tested. All results are expressed as means + standard deviations of triplicate plates. Aliquots (50/zl) of each sample were used for the genotoxicity assay.
O M,I. (%) • aberrations(%)
°i 0
Results
0
2
4
6
8
Days
Shallot root-tip cells
Fig. 1. Mitotic activity and total number o f chromosomal aberrations in shallot root tips treated with 0.125 k~M BA.
Significant decreases in mitotic index (MI) after 2 and 5 days of treatment with the lowest concentration of BA (0.125 /~M) were observed (Fig. 1), while after 3 and 4 days of treatment mitotic activity was rather constant. A significant increase in mitotic activity was noticed after 6 days of treatment (Fig. 1). A similar pattern can be seen after treatment with a 100 × higher concentration (Fig. 2). Unexpected results were obtained after 2 days of treatment with 1.25/zM BA when mitotic activity increased, but afterwards the same pattern can be seen as mentioned above (Fig. 3). After 7 days of treatment with all concentrations of BA, the MI was significantly lower than in control (Table 1). The total number of chromosomal aberrations represents changes on chromosomes and the mitotic spindle. The most prominent spindle abnormalities were c-mitoses ob-
8
0 MI. (%)
0
2
4
n
6
8
Days Fig. 2. M i t o t i c activity and total number o f chromosomal aberrations in shallot root tips treated with 12.5 ~.M BA.
TABLE1 M I T O T I C I N D E X A N D TYPES O F C H R O M O S O M A L A B E R R A T I O N S I N D U C E D BY B E N Z Y L A D E N I N E A F T E R 7 DAYS O F T R E A T M E N T Concentration
MI
Effects on spindle (%)
Effects on chromosomes (%)
(~M)
(%)
c-mitosis
disturbed anaphase
bridges
fragments and
0 0.13 0.15 0.43 0
0.09 0.15 0.34 0.74 0.09
0 0.09 0.15 0.21 0
Total
Number
micronuclei
aberrations
of cells examined
0 0.09 0 0.17 0.25
0.09 0.46 0.64 1.55 0.34
laggards 0 0.125 1.25 12.5 125.0
10.28 6.56 3.00 7.07 3.23
* p < 0.05 by )f2 test.
* * * *
0 0 0.07 0.43 0
(%) 2138 2162 2031 2162 2 007
280 TABLE 2 C Y T O T O X I C 1 T Y A N D M U T A G E N I C I T Y O F B E N Z Y L A D E N I N E ON V79 CELLS Concentration
Survival
N u m b e r of cells
Plate count
Mutation frequency
Mutation frequency a
(~M)
(%)
plated
(X)
x 10 -6 cells
per 106 survivors
0 0.125 125.0 1 250.0
100 98 39 29
1.5 x 9.5 x 9.6 x 1.(I X
1.1 1.0 1.2 1. I
7.2 3.7 2.0 3.7
7.2 _+0.66 3.8 _+0.12 5.2 4- 0.36 13.0 _+0.89
105 104 104 105
Mean _+standard deviation.
0 ML (%) .£
3 £
2 -
1
0 2
0
4
6
8
Days
Fig. 3. Mitotic activity and total number of chromosomal aberrations in shallot root tips treated with 1.25 ~ M BA.
I.'79 cells Table 2 contains data demonstrating the effect of BA on Chinese hamster cells, including toxicity
O 0.125 uM • 1.25 uM
•
served after treatment with 12.5 /zM BA and disturbed anaphases, the frequency of which increased linearly with concentration (Table 1). Fragments and laggards, ana- and telo-phase bridges and micronuclei were also observed (Table 1). Treatment with the highest concentration of BA (125 /~M) significantly decreased mitotic activity so far that the total amount of chromosomal aberrations was very low (Table 1). Stickiness, a phenomenon which is not especially taken into account, was very frequent and accompanied all the instabilities of the spindle and chromosomes. Results on measuring root elongation during the 21-day experiment showed that all tested BA concentrations (0.125, 1.25 and 12.5 ~M) inhibited root growth (Fig. 4). Morphological changes on roots were not observed.
12.5uM
TABLE 3 MUTAGENIC
ACTIVITY
OF
BA
IN
Salmonella
phimurium Concentration of test substance ( t.t M / p l a t e )
A ~
A A
.
A
0 z=_; 0
.
A
A
.
10
A .
A .
~ .
A
A .
~ .
20
oa~
Fig. 4. Root elongation during the treatment with BA.
:30
0 1.25 12.5 125.0 1250.0
Number of his* revertants Strain TAI00
Strain TA98
- $9
+ $9
- $9
+ $9
85__+ 4 92_+ 2 96+ 7 99+ 5 117±13
97+ 2 94:t:: 9 129__+ 5 139_+ 5 130±10
26:t:4 26__+2 21_+3 22_+4 22-1-2
37+ 3 325-10 37_+ 2 36+ 6 41::1:: 4
Results are expressed as mean-+ standard deviation.
ty-
281 TABLE 4 G E N O T O X I C I T Y O F BA IN Saccharomyces ceret:isme D7 Concentration of test substance (p.M)
T R P convertants per 105 survivors
ILV recombinants per 106 survivors
0 1.25 12.5 125.0 1 250.0
1.19 1.25 1.54 1.68 1.60
0.10 0.13 0.16 0.14 O. 17
data (survival), plate counts for mutant plates and mutation frequencies corrected for spontaneous background frequencies. H G P R T mutation induction was not evident for any of the concentrations tested, which is obvious from plate counts (X) for mutant plates compared to control. Treatment of exponentially growing Chinese hamster cells with 0.125, 125 and 1250/.tM BA resulted in corresponding changes in survival ranging from 98% to 29%. The mutagenic activity of BA on S. typhimurium strains TA100 and TA98 is shown in Table 3. No significant difference between control and BA-treated values was observed either with or without rat liver $9. Thus, this compound is not genotoxic in S. typhimurium. The same was true for the mutagenic efficiency of BA in the yeast S. ceret~isiae D7 (Table 4). Addition of 1.25-1250 /zM of BA suspended in buffer did not increase mutagenicity measured by two different genetic endpoints. Discussion
Our results showed that after 48 h of treatment with 0.125 and 12.5 /zM BA the mitotic activity in shallot root tips decreased. It is well known that cytokinin synthesis takes place in meristematic tissue, mainly in root-tip ceils and quiescent center cells (Short and Torrey, 1972). The decrease in mitotic activity after administration of BA to the intact roots could arise as a result of interaction between endogenous and exogenous cytokinins. After 6 days of treatment, we noticed a significant increase in mitotic activity at all concentrations of BA used. This could
be a consequence of cell division induction by exogenous cytokinin, since endogenous cytokinin has been transported from the root, which is the site of its synthesis, to leaves and other aerial parts. Our results on measuring root elongation during 21 days showed that BA inhibited root growth. A similar observation was made by Stenlid (1982). Treatment with BA caused irregularities of mitotic divisions, especially effects on the mitotic spindle in the form of disturbed anaphase. The same was noticed in European black pine root tips treated with BA (Papeg et al., 1991). Also, laggards and fragments were observed after BA treatment (Ogura, 1982). The presence of micronuclei could be a sign of chromosome breakage or elimination. The mechanism of chromosome aberration induction is still not very well understood. BA is a derivative of purine, and there is a possibility of its integration into the DNA molecule, but so far we have no evidence of that. The control of replicon-origin activation by cytokinins is essential for their promoting effect on cell division (Houssa et al., 1990). Also, plant cells contain a protein which is a target for cytokinins. This protein is part of a protein group that appears to be involved in the initiation of DNA replication (Houssa et al., 1990). The specific protein which is also a possible target for cytokinins is found on ribosomes, so cytokinins could be involved in the regulation of protein synthesis (Maas and Klambt, 1977). Mutagenicity assays on bacteria, yeast and V79 ceils did not show mutagenic effects of BA. The battery of test systems used in our study enabled us to establish possible effects of BA at different levels of the genome (BagiE-Zaninovi6, 1991). Our results showed that BA did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. The same was true for the mutagenic efficiency of BA in S. cereeisiae. Also, H G P R T mutation induction was not evident in V79 Chinese hamster ceils. However, in A. ascalonicum somatic cells BA had influence on the mitotic cycle as well as on mitotic aberration induction. Our experiments showed that BA caused different effects on chromosome structure, spindle components and mitotic process. The results indi-
282 cate that BA is not a p o t e n t genotoxin, but it is genetically active. It seems that BA acts in a r a t h e r complex way, c a u s i n g a variety of different genetic activity in plants (Bagi6-Zaninovi6, 1991). T h e reason for the difference observed b e t w e e n in vivo a n d in vitro genotoxicity of BA is not known. Theoretically, the discrepancy b e t w e e n the in vitro a n d in vivo situations could be d u e to the loss of enzymatic activity in the cells d u r i n g their p r e p a r a t i o n (Smith a n d O r r e n i u s , 1984). F u r t h e r study is necessary to clarify the genotoxic effects of B A a n d o t h e r synthetic growth regulators.
Acknowledgement This work was partially s u p p o r t e d by the Ministry of Science, T e c h n o l o g y a n d Informatics, Republic of Croatia (Nos. 1-08-272; 1-08-273 a n d 1-08-101).
References Ba.~i~5-Zaninovi6,T., D. Pape.~ and J. Franeki6 (1991) Cycloheximide genotoxicity in in vitro and in vivo test systems, Mutation Res., 263, 203-210. Bernier, G., J.-M. Kinet, A. Jacqmard, A. Havelange and M. Bodson (1977) Cytokinin as a possible component of the floral stimulus in Sinapis alba, Plant Physiol., 60, 282-285. Fiskesj6, G. (1985) The Allium test as a standard in environmental monitoring, Hereditas, 102, 99-112. Fiskesj6, G. (1988) The Allium test - an alternative in environmental studies: the relative toxicology of metal ions, Mutation Res., 197, 243-291. Houssa, C., A. Jacqmard and G. Bernier (1990) Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis, Planta, 181,324-326. Levi, M., D. Colombo, S. Sgorbati, D. Chiatante and E. Sparvoli (1987) Effect of benzyladenine on cell cycle in intact roots: a cytofluorimetric approach, J. Plant Physiol., 131,385-391. Maas, H., and D. Klambt (1977) Cytokinin effect on protein synthesis in vivo in higher plants, Planta, 133, 117-120.
Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Nagy, B., P.J. Dale and D.J. Grdina (1986) Protection against c/s-diamminedichloroplatinum cytotoxicity and mutagenicity in V79 cells by 2-[(aminopropyl)amino]ethanethiol, Cancer Res., 46, 1132-1135. Naito, K., H. Tsuji and 1. Hatakeyama (1978) Effects of benzyladenine on DNA, RNA, protein and chlorophyll contents in intact bean leaves: differential responses to benzyladenine according to leaf age, Physiol. Plant., 43, 367-371. Ogura, H. (1982) Studies on the genetic instability of cultured tissues and regenerated plants - effects of auxins and cytokinins on mitosis of 14cia faba cells, Proc. 5th Int. Cong. Plant Tissue and Cell Culture, pp. 433-434. Paper, D., V. Besendorfer and M. Pavlica (1991) Chromosome changes in European black pine seedlings caused by growth regulators, Acta Bot. Croat., 50, 31-36. Sharma, A.K., and A. Sharma (1972) Chromosome Techniques - Theory and Practice, University Park Press, Baltimore, MD, 56 pp. Short, K.C., and J.G. Torrey (1972) Cytokinins in seedling roots of pea, Plant Physiol., 49, 155-160. Skoog, F., F.M. Strong and C.O. Miller (1965) Cytokinins, Science, 148, 532-533. Smith, M.T., and S. Orrenius (1984) Studies on drug metabolism and drug toxicity in isolated mammalian cells, in: J.R. Mitchell and M.G. Horning (Eds.), Metabolism and Drug Toxicity, Raven, New York, pp. 71-98. Stenlid, G. (1982) Cytokinins as inhibitors of root growth, Physiol. Plant., 56, 500-506. Thacker, J., M.A. Stephens and A. Strech (1976) Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35, 465-478. Torrey, J.G. (1976) Root hormones and plant growth, Annu. Rev. Plant Physiol. 27, 435-459. Vogelman, T.C., C.H. Bornman and P. Nissen (1984) Uptake of benzyladenine in explants of Picea abies and Pinus .~ylcestris, Physiol. Plant., 61,513-517. Zimmermann, F.K. (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381388.
Communicated by M. Ala~:evi(~
277
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00645
Effects of benzyladenine on prokaryotic and eukaryotic cells M i r j a n a Pavlica, Dra~,ena Pape,~, J a s n a F r a n e k i 6
a
and Biserka Nagy
Department of Molecular Biology, Faculty of Science, Unit~ersityof Zagreb and a Faculty of Food Technology and Biotechnology, Laboratory of Biology and Microbial Genetics, 41000 Zagreb, Croatia (Yugo.dacia) (Received 16 October 1991) (Revision received 22 November 1991) (Accepted 25 November 1991)
Keywords: Benzyladenine; Chromosome aberrations; Cytotoxicity; Mutagenicity
Summary Comparative studies of the effect of benzyladenine (BA) on the yeast Saccharomyces cerevisiae, the bacterium Salmonella typhimurium, the shallot Allium ascalonicum and Chinese hamster fibroblast cells were performed. The tested substance had no mutagenic activity on yeast, bacteria and cultured fibroblast cells. Changes in mitotic activity and cell division abnormalities were observed after BA treatment in shallot root-tip cells.
Synthetic cytokinins such as benzyladenine (BA) are plant growth regulators used for establishing plant cell and tissue cultures. Cytokinins are implicated in the control of cell division in cultured cells as well as in meristems of intact plants (Skoog et al., 1965). They are also involved in the regulation of the cell cycle and function of the root meristem (Torrey, 1976). BA (CAS No. 1214-39-7), which is widely used in plant tissue culture, passively enters into the tissue at an optimum temperature of 25°C (Vogelman et al., 1984). The administration of BA to the intact plants inhibits root growth (Stenlid, 1982). Also, a decrease in mitotic activ-
Correspondence: Dr. M. Pavlica, Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, P.O. Box 933, 41001 Zagreb, Croatia (Yugoslavia).
ity and an increase in the number of nuclei in G 2 after treatment with BA in pea root meristems could be observed (Levi et al., 1987). BA induced endoreduplication in epidermal cells of bean primary leaves, and also increased the amount of DNA (Naito et al., 1978). But it is known that cell division is far more sensitive to the cytokinin level than DNA synthesis (Bernier et al., 1977). The aim of this work was to analyze the effect of BA on mitotic activity and chromosome aberration induction in shallot (Allium ascalonicum L.) root tip cells. The study was supplemented with cytotoxicity and mutagenicity testing of BA at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells. The Salmonella/mammalian microsome test on the bacterium Salmonella typhimurium, and the test with the yeast Saccharomyces cerevisiae for detection of gene mutations and gene conversion were also used.
278 Materials and methods
Plant test material In our experiments equal-sized bulbs from a diploid (2n = 16) population of shallot A. ascalonicum L. (syn. A. cepa var. aggregatum) were used. Bulbs were grown for 7 days in BA (Sigma) water solutions, at concentrations of 0.125, 1.25, 12.5 and 125/~M. BA solutions were changed every 24 h. Tap water was used as a control (Fiskesj6, 1988). The roots were incubated at room temperature (26 + I°C), exposed to artificial light for 16 h per day at a light intensity of 17 W / m 2. For microscopic studies root tips were cut evcry 24 h during the 7-day experiment, while roots grown in 125 /.tM BA were cut after 7 days of treatment. Material was fixed in ethanol:acetic acid (3: 1) for 24 h, and slides were prepared using the Feulgen squash technique (Sharma and Sharma, 1972). Permanent slides were prepared using liquid carbon dioxide. After 1 day drying, they were mounted in Euparai mounting medium ('Gurr'). In order to analyze microscopic parameters (Fiskesj6, 1985) at least 2000 ceils were scored (about 100 per slide) per treatment and concentration. During the 21-day experiment root elongation was measured and searched for any morphological changes.
Anhnal cells and culture conditions Chinese hamster V79 fibroblast cells were maintained as stock cultures in a-minimal essential medium (a-MEM, Gibco) with 10% fetal calf serum (Flow Laboratories) in a humidified atmosphere containing 5% CO 2 in air at 37°C. All experiments were performed in triplicate with exponentially growing cells. Exponentially growing cultures of V79 cells were treated with various concentrations of BA (0.125, 12.5, 125 and 1250 p,M) for 60 min. After treatment all cultures were washed twice with PBS (8.1 mM NazHPO4-1.5 mM KHzPO4-0.14 M NaCI-2.6 mM KCi) and trypsinized. Cell survival was determined by plating an appropriate number of cells to give about 200 colonies per dish, 6 dishes per experimental point (Nagy et al., 1986).
For detection of H G P R T mutants cells were plated into 150-cm 2 T-flasks at a cell density of 2 × 106 cells per flask. After 24 h of growth cells were treated with BA according to the previously described protocol. Cells were then washed, trypsinized and plated. Mutation induction was assayed by seeding at least 106 surviving ceils per experimental point. Cells were grown in nonselective medium for 6 days to allow mutants to be expressed (Thacker et al., 1976). The plating efficiency was determined by seeding 200 ceils per plate in standard growth medium. Mutation induction was determined by seeding 105 ceils into each of I0 plates containing standard growth medium and 6-thioguanine (6-TG) at a concentration of 5 /~g/ml. Following 7 days of growth, the resulting mutant colonies were stained with 0.5% methylene blue and counted. Mutation frequencies are expressed as the number of 6-TGresistant cells per 10 6 surviving ceils, corrected for spontaneous background frequencies.
Detection of genetic effects on Saccharomyces cerecisiae The test was performed on diploid yeast strain D7 (Zimmermann, 1975) with the genotype ade2-
40lade2-119;
trp5-12/trp5-27;
ih'l-92/ih;1-92.
Yeast cells in mid-log phase were harvested and exposed to various concentrations of benzyladenine (1.25, 12.5, 125 and 1250/~M) in phosphate buffer, pH 7.4, for 2 h at 30°C. Ceils were washed, centrifuged and adequate dilutions were spread on complete medium, tryptophan-free medium and isoleucine-free medium. The yield of colonies counted on these 3 media allows us to calculate cell survival and the frequency of spontaneous or chemically induced gene convertants and mutants.
Mutagenicity murium
assay in his-
Salmonella
typhi-
The mutagenicity of BA in the Salmonella test was examined according to the standard plate-incorporation test procedure with tester strains TA100 (hisG46, rfa, AUL,rB, pKMI01) and TA98 (hisD3052, rfa, AucrB, pKMI01) (Maron and Ames, 1983). BA solutions were adjusted to pH 3.3-3.7, incubation time was 1 h at 37°C. Concentrations of BA were the same as above. After
279 8
incubation each mixture was neutralized with PBS, pH 7.4, prior to adding to the top agar. For metabolic activation $9 mix containing 10% liver $9 fraction from male Aroclor-treated rats (protein concentration 37.8 m g / m l ) was added to the top agar as well as to the bacterial cells and the compound tested. All results are expressed as means + standard deviations of triplicate plates. Aliquots (50/zl) of each sample were used for the genotoxicity assay.
O M,I. (%) • aberrations(%)
°i 0
Results
0
2
4
6
8
Days
Shallot root-tip cells
Fig. 1. Mitotic activity and total number o f chromosomal aberrations in shallot root tips treated with 0.125 k~M BA.
Significant decreases in mitotic index (MI) after 2 and 5 days of treatment with the lowest concentration of BA (0.125 /~M) were observed (Fig. 1), while after 3 and 4 days of treatment mitotic activity was rather constant. A significant increase in mitotic activity was noticed after 6 days of treatment (Fig. 1). A similar pattern can be seen after treatment with a 100 × higher concentration (Fig. 2). Unexpected results were obtained after 2 days of treatment with 1.25/zM BA when mitotic activity increased, but afterwards the same pattern can be seen as mentioned above (Fig. 3). After 7 days of treatment with all concentrations of BA, the MI was significantly lower than in control (Table 1). The total number of chromosomal aberrations represents changes on chromosomes and the mitotic spindle. The most prominent spindle abnormalities were c-mitoses ob-
8
0 MI. (%)
0
2
4
n
6
8
Days Fig. 2. M i t o t i c activity and total number o f chromosomal aberrations in shallot root tips treated with 12.5 ~.M BA.
TABLE1 M I T O T I C I N D E X A N D TYPES O F C H R O M O S O M A L A B E R R A T I O N S I N D U C E D BY B E N Z Y L A D E N I N E A F T E R 7 DAYS O F T R E A T M E N T Concentration
MI
Effects on spindle (%)
Effects on chromosomes (%)
(~M)
(%)
c-mitosis
disturbed anaphase
bridges
fragments and
0 0.13 0.15 0.43 0
0.09 0.15 0.34 0.74 0.09
0 0.09 0.15 0.21 0
Total
Number
micronuclei
aberrations
of cells examined
0 0.09 0 0.17 0.25
0.09 0.46 0.64 1.55 0.34
laggards 0 0.125 1.25 12.5 125.0
10.28 6.56 3.00 7.07 3.23
* p < 0.05 by )f2 test.
* * * *
0 0 0.07 0.43 0
(%) 2138 2162 2031 2162 2 007
280 TABLE 2 C Y T O T O X I C 1 T Y A N D M U T A G E N I C I T Y O F B E N Z Y L A D E N I N E ON V79 CELLS Concentration
Survival
N u m b e r of cells
Plate count
Mutation frequency
Mutation frequency a
(~M)
(%)
plated
(X)
x 10 -6 cells
per 106 survivors
0 0.125 125.0 1 250.0
100 98 39 29
1.5 x 9.5 x 9.6 x 1.(I X
1.1 1.0 1.2 1. I
7.2 3.7 2.0 3.7
7.2 _+0.66 3.8 _+0.12 5.2 4- 0.36 13.0 _+0.89
105 104 104 105
Mean _+standard deviation.
0 ML (%) .£
3 £
2 -
1
0 2
0
4
6
8
Days
Fig. 3. Mitotic activity and total number of chromosomal aberrations in shallot root tips treated with 1.25 ~ M BA.
I.'79 cells Table 2 contains data demonstrating the effect of BA on Chinese hamster cells, including toxicity
O 0.125 uM • 1.25 uM
•
served after treatment with 12.5 /zM BA and disturbed anaphases, the frequency of which increased linearly with concentration (Table 1). Fragments and laggards, ana- and telo-phase bridges and micronuclei were also observed (Table 1). Treatment with the highest concentration of BA (125 /~M) significantly decreased mitotic activity so far that the total amount of chromosomal aberrations was very low (Table 1). Stickiness, a phenomenon which is not especially taken into account, was very frequent and accompanied all the instabilities of the spindle and chromosomes. Results on measuring root elongation during the 21-day experiment showed that all tested BA concentrations (0.125, 1.25 and 12.5 ~M) inhibited root growth (Fig. 4). Morphological changes on roots were not observed.
12.5uM
TABLE 3 MUTAGENIC
ACTIVITY
OF
BA
IN
Salmonella
phimurium Concentration of test substance ( t.t M / p l a t e )
A ~
A A
.
A
0 z=_; 0
.
A
A
.
10
A .
A .
~ .
A
A .
~ .
20
oa~
Fig. 4. Root elongation during the treatment with BA.
:30
0 1.25 12.5 125.0 1250.0
Number of his* revertants Strain TAI00
Strain TA98
- $9
+ $9
- $9
+ $9
85__+ 4 92_+ 2 96+ 7 99+ 5 117±13
97+ 2 94:t:: 9 129__+ 5 139_+ 5 130±10
26:t:4 26__+2 21_+3 22_+4 22-1-2
37+ 3 325-10 37_+ 2 36+ 6 41::1:: 4
Results are expressed as mean-+ standard deviation.
ty-
281 TABLE 4 G E N O T O X I C I T Y O F BA IN Saccharomyces ceret:isme D7 Concentration of test substance (p.M)
T R P convertants per 105 survivors
ILV recombinants per 106 survivors
0 1.25 12.5 125.0 1 250.0
1.19 1.25 1.54 1.68 1.60
0.10 0.13 0.16 0.14 O. 17
data (survival), plate counts for mutant plates and mutation frequencies corrected for spontaneous background frequencies. H G P R T mutation induction was not evident for any of the concentrations tested, which is obvious from plate counts (X) for mutant plates compared to control. Treatment of exponentially growing Chinese hamster cells with 0.125, 125 and 1250/.tM BA resulted in corresponding changes in survival ranging from 98% to 29%. The mutagenic activity of BA on S. typhimurium strains TA100 and TA98 is shown in Table 3. No significant difference between control and BA-treated values was observed either with or without rat liver $9. Thus, this compound is not genotoxic in S. typhimurium. The same was true for the mutagenic efficiency of BA in the yeast S. ceret~isiae D7 (Table 4). Addition of 1.25-1250 /zM of BA suspended in buffer did not increase mutagenicity measured by two different genetic endpoints. Discussion
Our results showed that after 48 h of treatment with 0.125 and 12.5 /zM BA the mitotic activity in shallot root tips decreased. It is well known that cytokinin synthesis takes place in meristematic tissue, mainly in root-tip ceils and quiescent center cells (Short and Torrey, 1972). The decrease in mitotic activity after administration of BA to the intact roots could arise as a result of interaction between endogenous and exogenous cytokinins. After 6 days of treatment, we noticed a significant increase in mitotic activity at all concentrations of BA used. This could
be a consequence of cell division induction by exogenous cytokinin, since endogenous cytokinin has been transported from the root, which is the site of its synthesis, to leaves and other aerial parts. Our results on measuring root elongation during 21 days showed that BA inhibited root growth. A similar observation was made by Stenlid (1982). Treatment with BA caused irregularities of mitotic divisions, especially effects on the mitotic spindle in the form of disturbed anaphase. The same was noticed in European black pine root tips treated with BA (Papeg et al., 1991). Also, laggards and fragments were observed after BA treatment (Ogura, 1982). The presence of micronuclei could be a sign of chromosome breakage or elimination. The mechanism of chromosome aberration induction is still not very well understood. BA is a derivative of purine, and there is a possibility of its integration into the DNA molecule, but so far we have no evidence of that. The control of replicon-origin activation by cytokinins is essential for their promoting effect on cell division (Houssa et al., 1990). Also, plant cells contain a protein which is a target for cytokinins. This protein is part of a protein group that appears to be involved in the initiation of DNA replication (Houssa et al., 1990). The specific protein which is also a possible target for cytokinins is found on ribosomes, so cytokinins could be involved in the regulation of protein synthesis (Maas and Klambt, 1977). Mutagenicity assays on bacteria, yeast and V79 ceils did not show mutagenic effects of BA. The battery of test systems used in our study enabled us to establish possible effects of BA at different levels of the genome (BagiE-Zaninovi6, 1991). Our results showed that BA did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. The same was true for the mutagenic efficiency of BA in S. cereeisiae. Also, H G P R T mutation induction was not evident in V79 Chinese hamster ceils. However, in A. ascalonicum somatic cells BA had influence on the mitotic cycle as well as on mitotic aberration induction. Our experiments showed that BA caused different effects on chromosome structure, spindle components and mitotic process. The results indi-
282 cate that BA is not a p o t e n t genotoxin, but it is genetically active. It seems that BA acts in a r a t h e r complex way, c a u s i n g a variety of different genetic activity in plants (Bagi6-Zaninovi6, 1991). T h e reason for the difference observed b e t w e e n in vivo a n d in vitro genotoxicity of BA is not known. Theoretically, the discrepancy b e t w e e n the in vitro a n d in vivo situations could be d u e to the loss of enzymatic activity in the cells d u r i n g their p r e p a r a t i o n (Smith a n d O r r e n i u s , 1984). F u r t h e r study is necessary to clarify the genotoxic effects of B A a n d o t h e r synthetic growth regulators.
Acknowledgement This work was partially s u p p o r t e d by the Ministry of Science, T e c h n o l o g y a n d Informatics, Republic of Croatia (Nos. 1-08-272; 1-08-273 a n d 1-08-101).
References Ba.~i~5-Zaninovi6,T., D. Pape.~ and J. Franeki6 (1991) Cycloheximide genotoxicity in in vitro and in vivo test systems, Mutation Res., 263, 203-210. Bernier, G., J.-M. Kinet, A. Jacqmard, A. Havelange and M. Bodson (1977) Cytokinin as a possible component of the floral stimulus in Sinapis alba, Plant Physiol., 60, 282-285. Fiskesj6, G. (1985) The Allium test as a standard in environmental monitoring, Hereditas, 102, 99-112. Fiskesj6, G. (1988) The Allium test - an alternative in environmental studies: the relative toxicology of metal ions, Mutation Res., 197, 243-291. Houssa, C., A. Jacqmard and G. Bernier (1990) Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis, Planta, 181,324-326. Levi, M., D. Colombo, S. Sgorbati, D. Chiatante and E. Sparvoli (1987) Effect of benzyladenine on cell cycle in intact roots: a cytofluorimetric approach, J. Plant Physiol., 131,385-391. Maas, H., and D. Klambt (1977) Cytokinin effect on protein synthesis in vivo in higher plants, Planta, 133, 117-120.
Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Nagy, B., P.J. Dale and D.J. Grdina (1986) Protection against c/s-diamminedichloroplatinum cytotoxicity and mutagenicity in V79 cells by 2-[(aminopropyl)amino]ethanethiol, Cancer Res., 46, 1132-1135. Naito, K., H. Tsuji and 1. Hatakeyama (1978) Effects of benzyladenine on DNA, RNA, protein and chlorophyll contents in intact bean leaves: differential responses to benzyladenine according to leaf age, Physiol. Plant., 43, 367-371. Ogura, H. (1982) Studies on the genetic instability of cultured tissues and regenerated plants - effects of auxins and cytokinins on mitosis of 14cia faba cells, Proc. 5th Int. Cong. Plant Tissue and Cell Culture, pp. 433-434. Paper, D., V. Besendorfer and M. Pavlica (1991) Chromosome changes in European black pine seedlings caused by growth regulators, Acta Bot. Croat., 50, 31-36. Sharma, A.K., and A. Sharma (1972) Chromosome Techniques - Theory and Practice, University Park Press, Baltimore, MD, 56 pp. Short, K.C., and J.G. Torrey (1972) Cytokinins in seedling roots of pea, Plant Physiol., 49, 155-160. Skoog, F., F.M. Strong and C.O. Miller (1965) Cytokinins, Science, 148, 532-533. Smith, M.T., and S. Orrenius (1984) Studies on drug metabolism and drug toxicity in isolated mammalian cells, in: J.R. Mitchell and M.G. Horning (Eds.), Metabolism and Drug Toxicity, Raven, New York, pp. 71-98. Stenlid, G. (1982) Cytokinins as inhibitors of root growth, Physiol. Plant., 56, 500-506. Thacker, J., M.A. Stephens and A. Strech (1976) Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35, 465-478. Torrey, J.G. (1976) Root hormones and plant growth, Annu. Rev. Plant Physiol. 27, 435-459. Vogelman, T.C., C.H. Bornman and P. Nissen (1984) Uptake of benzyladenine in explants of Picea abies and Pinus .~ylcestris, Physiol. Plant., 61,513-517. Zimmermann, F.K. (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381388.
Communicated by M. Ala~:evi(~
