Mutagenesis, Vol. 15, No. 5, 385-389,
September 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Maleic hydrazide induces genotoxic effects but no DNA damage detectable by the Comet assay in tobacco and field beans
Gichner2
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce 1a, 160 00 Prague 6, Czech Republic and 1 Institute of Plant Genetics and Crop Plant Research (IPK), D-06466, Gatersleben, Germany
This article is dedicated to Professor Rigomar Rieger on the occasion of his seventieth birthday
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Abstract |
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The plant growth regulator and herbicide maleic hydrazide (MH) induced a high frequency of somatic mutations in leaves of tobacco (Nicotiana tabacum var. xanthi) and a high yield of chromosome aberrations in roots of field beans (Vicia faba, karyotype ACB). In contrast, no significant increase in MH-induced DNA damage, as measured by the Comet assay, could be demonstrated in either plant species. The absence of DNA migration induced by MH was not effected in tobacco by either pH of the MH solution, the sampling time after MH treatment or continuous MH treatment for 14 days. To our knowledge, MH represents the first agent which has proved to be highly mutagenic and clastogenic but does not cause DNA damage as measured by the Comet assay in the same experimental system.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The Comet assay, applied to nuclei of leaves, roots and cell suspensions of tobacco (Nicotiana tabacum), proved to be a sensitive method for detection of DNA damage (Gichner and Plewa, 1998
; Stavreva et al., 1998; Gichner et al., 1999). Using monofunctional alkylating agents [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea] linear concentration–response characteristics for tail moment values and somatic mutations were observed when tobacco seedlings were treated under identical conditions. Nuclei of field bean (Vicia faba) root tips were also found to be suitable for the study of induced DNA damage and DNA repair by the Comet assay (Koppen and Angelis, 1998; Angelis et al., 1999; Menke et al., 2000). In this paper we report that no significant increase in DNA migration from tobacco and field bean nuclei was detectable by the Comet assay after treatment with the plant growth regulator and herbicide maleic hydrazide (MH) under conditions that caused high mutagenic effects in tobacco or clastogenic effects in field beans. The experiments with tobacco were conducted at the Institute of Experimental Botany (Prague, Czech Republic) and the experiments with beans at the Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany).
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Materials and methods |
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Chemicals and media
EMS (CAS no. 62-50-0), MH (CAS no. 123-33-1), the plant growth medium (Phytagel, MS salts) reagents for electrophoresis, normal melting point (NMP) and low melting point (LMP) agarose and general laboratory reagents were purchased from Sigma Chemical Co. (St Louis, MO).
Experiments with tobacco
Nicotiana tobacum growth and mutagenic treatment conditions.
Seedlings of Nicotiana tabacum var. xanthi (a1+/a1; a2+/a2) (Dulieu and Dalebroux, 1975) were cultivated in a plant growth chamber at 26°C with a 18 h photoperiod to the 4–5 leaf stage. The roots of seedlings were carefully rinsed in water and immersed in glass vials containing 22 ml of a defined concentration of MH or EMS in distilled water or citrate/phosphate buffers. The plants were treated in the dark at 26°C for 24 h. A detailed description of plant growth conditions was previously published (Gichner and Plewa, 1998; Gichner et al., 1999).
Somatic mutation assay.
The tobacco seedlings were treated as described above. For each experiment eight seedlings treated with MH were individually cultivated in glass vials with 50% Hoagland's solution in a growth chamber at 22–26°C with a 18 h photoperiod for 2–3 weeks. The mutant sectors were scored on leaves 6 and 7 as counted after the cotyledons. Three types of mutant sectors were identified on the pale green leaves: (i) green; (ii) yellow; (iii) green/yellow double sectors (Dulieu and Dalebroux, 1975). The mutant frequencies were calculated from the total number of mutation events per leaf. Each experiment was repeated twice.
Comet assay. After seedling treatment, individual tobacco leaves were placed in a 60 mm Petri dish kept on ice and spread with 300 µl of cold modified Sörensen buffer [50 mM sodium phosphate, pH 6.8. 0.1 mM EDTA, 0.5% dimethyl sulfoxide (DMSO)]. Using a fresh razor blade, each leaf was gently sliced. The plate was kept tilted in the ice so that the isolated nuclei would collect in the buffer. All operations were conducted under dim or yellow light.
Regular microscope slides were dipped in a solution of 1% NMP agarose prepared with water at 50°C, dried overnight at room temperature and kept dry in slide boxes until use. To each slide were added 50 µl of the nuclear suspension and 50 µl of 1% LMP agarose prepared with PBS at 37°C. The nuclei and the LMP agarose were gently mixed by repeated pipetting using a cut micropipette tip and a coverslip was placed on the mixture. The slide was placed on ice for a minimum of 5 min. Thereafter the coverslip was removed and a final layer of 90 µl of 0.5% LMP agarose was placed on the slide. A coverslip was placed upon the LMP agarose and the slide kept at 4°C for 5 min. Then the coverslips were removed and the slides were immersed overnight in a lysing solution consisting of 2.5 M NaCl, 1% sodium sarcosinate, 100 mM Na2EDTA and 10 mM Tris, pH 10, with 1% Triton X-100 and 10% DMSO added freshly at 4°C. After lysing, the slides were placed in a horizontal gel electrophoresis tank containing freshly prepared cold electrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH, pH > 13). The nuclei were incubated for 15 min to allow the DNA to unwind prior to electrophoresis at 0.72 V/cm (26 V, 300 mA) for 30 min at 4°C. After electrophoresis the slides were rinsed three times with 400 mM Tris, pH 7.5, stained with 70 µl of ethidium bromide (20 µg/ml) for 5 min, dipped in ice-cold water to remove the excess ethidium bromide and covered with a coverslip. For each slide 25 randomly chosen nuclei were analyzed using a fluorescence microscope with a BP 546/10 nm excitation filter and a 590 nm barrier filter. A computerized image analysis system (Komet v.3.1; Kinetic Imaging, Liverpool, UK) was employed. The tail moment (TM) (integrated value of DNA density multiplied by the migration distance) was used as the primary measure of DNA damage. Three slides were evaluated per treatment and each treatment was repeated at least twice. From the repeated experiments, the averaged median TM value was calculated for each treatment group from the median TM value of each slide (Lovell et al., 1999).
Experiments with Vicia faba
Seeds of Vicia faba karyotype ACB were germinated in the dark for 4 days on wet paper at 24°C, then seedlings were transferred to Hoagland's solution for 18 h.
To score metaphases with chromatid aberrations, 2–3 cm long root tips were treated with 0.4 or 0.8 mM MH dissolved in distilled water for 30 min. After recovery in running tap water for 12, 15, 18 or 21 h, the roots were exposed to 0.05% colchicine for 2 h and fixed overnight in ethanol:acetic acid (3:1) prior to hydrolysis in 1 N HCl for 11 min at 60°C, Feulgen staining, squashing and mounting in Euparal. Two hundred complete metaphases were evaluated per experimental point (50 per slide) for the presence of chromatid/isochromatid breaks, reciprocal chromatid translocations, duplication deletions and intercalary deletions. Gaps were omitted.
For the Comet assay treatment was done by incubating the 2–3 cm long roots for 24 h at 24°C in 0.04–8 mM MH dissolved in distilled water (pH 5.4) and to 10 mM EMS dissolved in distilled water as a positive control. Immediately after treatment, 0.5 cm of the root tips were cut on ice, frozen in liquid nitrogen and stored at –80°C. Five root tips were chopped with a fresh razor blade in PBS containing 5 mM EDTA on ice. The resulting suspension of nuclei was cleaned of debris by filtering through a 20 µm mesh. Thirty microliters of this suspension was mixed with 90 µl of 0.5% NMP agarose at 42°C. From this mixture two Comet gels were established by twice pipetting 60 µl onto one slide and covering each drop with a 22x22 mm coverslip. After solidifying the gels on ice, the nuclei were lysed in high salt solution (2.5 M NaCl, 10 mM Tris–HCl, pH 7.5, 100 mM EDTA) for 20 min at room temperature. Slides were washed three times for 5 min each in 1x TBE at room temperature, followed by denaturation in alkaline solution (0.3 M NaOH, 5 mM EDTA, pH 13.5) twice for 15 min each and electrophoresed in the same alkaline solution for 20 min at 0.72 V/cm, 300 mA in a tank cooled with ice, followed by neutralization with Tris buffer, pH 7.5. After electrophoresis, gels were dehydrated in ethanol and air dried. The analysis of comet formation was done after staining with 15 µl of 5 µg/ml ethidium bromide in water with a Zeiss Axioskop microscope equipped with an analog CCD video camera and the image analysis software Lucia (LIM, Prague). The Comet assay was repeated three times. For each repetition four individual gels from two slides were evaluated, providing 12 median TM values of 25 comets each for statistical analysis.
Statistical analyses
Data from the experiments were transferred to Microsoft Excel 7 spreadsheets (Microsoft, Redmond, WA) and analyzed using the statistical and graphical functions of SigmaPlot 4.0 and SigmaStat 2.0 (SPSS, Chicago, IL). The averaged median TM values and the average frequency of mutant sectors per leaf obtained from repeated experiments were used for analysis with a one-way analysis of variance test. If a significant F value of P < 0.05 was obtained, a Dunnett's multiple comparison versus the control group was conducted.
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Results |
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Nicotiana tabacum
MH-induced somatic mutation frequency in tobacco leaves. In Figure 1
the data of the mutagenic activity of MH in the chlorophyll-deficient tester strain N.tabacum var. xanthi are presented. Concentrations of 0.005–0.05 mM induced from 10.1 ± 1.6 to 284.3 ± 25.3 mean (± SE) mutant sectors per leaf. A significant increase over the control was observed above 0.0075 mM MH (F5,76 = 79.8, P < 0.001). The controls showed 1.2 ± 0.3 mutant sectors per leaf. Concentrations >0.05 mM could not be applied since they inhibited growth of the apical meristems and no new leaves were formed.
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TM values of tobacco leaf nuclei after MH treatment. With concentrations of MH from 0.005 to 6 mM the TM values ranged from 2.3 to 5.4 µm and except for the 5 mM value they were not significantly different (F11,71 = 2.137, P = 0.312) from the average median (± SE) control TM value (2.8 ± 0.3 µm) (Figure 1
). Treatment with 4–6 mM MH was lethal to seedlings within 72 h.
Somatic mutations and DNA damage evaluated on the same leaves.
The above presented data were obtained at two different points: TM values immediately after the end of MH treatment and somatic mutation frequency 2–3 weeks after MH treatment. To analyze the possible relationship between somatic mutations and DNA migration following treatment with MH at the same time in the same leaf, we treated tobacco seedlings with 0.05 mM MH for 24 h at 26°C and after treatment cultivated the seedlings in 50% Hoagland's solution for an additional 3 weeks. From eight seedlings we selected leaf 6 and analyzed the number of mutant sectors on one half of the leaf. The second half was used to determine the DNA damage. As demonstrated in Table I, the frequency of somatic mutations after MH treatment ranged from 103 to 247 and of the controls from 0.5 to 2 sectors per leaf. In contrast, DNA migration, expressed as the average median TM value after MH treatment, ranged from 1.2 ± 0.3 to 2.8 ± 0.3 µm and was not significantly different from the control TM value (2.4 ± 0.4 µm). These data clearly demonstrate that the high frequency of somatic mutations is not associated with a high level of DNA damage in the same leaf.
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Effect of pH of the MH solution. It was reported that the chromosome breaking ability of MH in V.faba roots is higher at pH 4 than at pH 6 (Heindorff and Rieger, 1984
). The same effect of pH was demonstrated for MH-induced DNA damage in human lymphocytes evaluated by the Comet assay (Ribas et al., 1995). Thus, we determined whether or not pH of the MH solution could influence DNA migration in tobacco leaves after MH treatment.
As demonstrated in Figure 2, different pH values (pH 4, 5 or 6) of the 1 mM MH solutions did not significantly increase the TM values as compared with the TM values in nuclei of seedlings treated with buffer alone (F5,39 = 0.619, P = 0.686). The different pH values also had no significant effect (F2,15 = 1.161, P = 0.340) on DNA damage after EMS treatment, used as a positive control.
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Effect of sampling time after MH treatment. Several papers have reported the first appearance of DNA damage after a certain post-treatment period. Miyamae et al. (1997) described the onset of DNA damage, expressed as DNA migration, after a 3 h treatment of L5178Y mouse lymphoma cells with various mutagens. The treated cells were sampled 0, 21 and 45 h after treatment. DNA damage induced by MMS, MNU and bleomycin was observed just after treatment. In contrast, DNA damage after treatment with the crosslinking agent mitomycin C was detected first 45 h after treatment.
The purpose of this experiment was to find out if various sampling times after termination of MH treatment could influence DNA migration after 0.5 and 2 mM MH treatment. As demonstrated in Figure 3, DNA migration was not significantly different from the control values (F8,45 = 1.186, P = 0.329) for all sampling times (0, 24 and 48 h) after the end of MH treatment. The different sampling times also had no significant effect on the TM values after EMS treatment, as a positive control (F2,15 = 0.225, P = 0.801).
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Continuous MH treatment of tobacco seedlings for up to 14 days. Tobacco seedlings were treated with 0.0075 and 0.05 mM MH for 1–14 days. The MH solution was changed every second day. On days 1, 7 and 14 the lower leaves were taken for DNA damage analysis. As demonstrated in Figure 4
, even continuous MH treatment did not result in a significant increase in TM values above the negative controls (F8,45 = 1.464, P = 0.197). Continuous treatment with 0.05 mM MH led to inhibition of growth of the apical meristem and no new leaves were formed. In contrast, application of 0.0075 mM MH did not prevent formation of new leaves. Thus, cell division took place during this MH exposure. We have analyzed DNA migration in these `new leaves', however, no increase in TM values above the control was observed (data not shown). Continuous treatment with 0.04 mM EMS as a positive control led to a significant increase in the TM value dependent on the time of treatment (F2,11 = 90.542, P < 0.001), although this increase was not linear.
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Vicia faba
Chromatid aberration data. After treatment of root tips with 0.4 or 0.8 mM MH for 30 min at 24°C, an increasing proportion of cells with chromatid type aberrations was observed in the first post-treatment mitosis (Figure 5
). The highest yield of metaphases with chromatid aberrations (56.5%) was induced by 0.8 mM MH following a 15 h recovery after treatment. The control roots showed between 0 and 2% metaphases with chromatid aberrations. Only chromatid aberrations were observed.
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Comet assay data and plant growth inhibition. The tail moment (mean median ± SE) values were 21.0 ± 3.6 µm for untreated controls, 27.9 ± 4.2 µm after 0.4 mM MH, 26.5 ± 3.5 µm after 4 mM MH and 35.7 ± 6.4 µm after 8 mM MH treatment. These values were not significantly different from the control TM value (F4,55 = 1.986, P = 0.109). After 10 mM EMS the TM value was 104.5 ± 7.7 µm (Table II
).
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Seedlings which were kept on wet blotting paper at 24°C in the dark after treatment showed a dose-dependent growth reduction caused by MH. After 0.04 mM MH and 10 mM EMS a weak growth reduction of both the stems and roots was observed. A dose of 8 mM MH led to complete growth repression of the green parts of the seedlings. In addition, the formation of lateral roots was inhibited after 0.4 mM MH and the roots became necrotic after 4 and 8 mM MH.
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Discussion |
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Mutagenic and clastogenic activity of MH in various organisms
The plant growth regulator and herbicide MH (1,2 dihydro-pyridazine-3,6-dione, a structural isomer of uracil) is mutagenic in tobacco (B
íza et al., 1984), Tradescantia (Gichner et al., 1982) and some other plant species (Swietlinska and Zuk, 1978), however, not in Arabidopsis thaliana (Gichner et al., 1994). It also proved to be a S phase-dependent clastogenic agent in V.faba and in barley (McLeish, 1953; Michaelis and Rieger, 1968; Nicoloff et al., 1979). In V.faba exposure to 0.01 mM MH for 2 h yielded a doubling of sister chromatid exchanges (Schubert et al., 1979). For mammals the results are contradictory. None or only a few chromosomal aberrations are caused by MH in mammalian cells (for a review see Heindorff and Rieger, 1984) and no increase in chromosomal aberrations and sister chromatid exchange (SCE) frequency was found after treatment with 1 mM MH by Perry and Evans (1975) in CHO cells. However, Takehisa et al. (1982) reported on a dose-dependent increase (up to 70 SCE/cell) in CHO cells at concentrations between 0.5 and 10 mM MH.
Potential mechanism of mutagenic and clastogenic activity of MH
Of the putative degradation products of MH (maleic acid diamide, succinic acid, maleic acid, lactic acid and hydrazine; see Biswas et al., 1967) only hydrazine caused a few chromosomal aberrations in V.faba, however at much higher doses. Additionally, the chromosomal distribution of the induced chromatid aberrations differed significantly from that observed for MH (Heindorff et al., 1984). Therefore, MH does not evoke clastogenicity via these metabolites.
Although autoradiographic studies with [14C]MH (Callaghan and Grun, 1961) indicated preferential labeling of interphase nuclei, nucleoli and mitotic chromosomes, no labeled DNA was found after exposure of plant cells to [14C]MH (Coupland and Peel, 1971; Aurich and Schubert, unpublished results). Therefore, neither MH nor its metabolites become directly incorporated into DNA.
MNU treatment of in vitro cultivated V.faba embryos resulted in a dose-dependent decrease in DNA molecular weight up to 20 h after treatment, due to randomly distributed single-strand breaks and alkali-labile sites, as judged from the profile of alkaline sucrose gradients. In contrast, MH treatment produced DNA fragments of replicon size independent of the applied dose (0.1–0.5 mM) immediately after treatment, which became joined during the 20 h after treatment to form fragments corresponding in size to that of untreated controls. In pulse–chase experiments with [3H]thymidine, MH-mediated DNA fragmentation occurred only in labeled DNA. This was interpreted as specific impairment of the joining of replicon termini by MH (Angelis et al., 1986). Thus it could be hypothesized that these MH-induced replicon sized DNA fragments disappear during lysis and/or electrophoresis in the Comet assay protocol. As a result, no breaks are detectable in the parental DNA.
Mutagenic and clastogenic activity of MH is not correlated with comet formation in tobacco and field beans
In previous papers (Gichner and Plewa,1998; Gichner et al., 1999) we demonstrated a close correlation between induced DNA damage, as evaluated by the Comet assay, and yield of somatic mutations in tobacco seedlings after treatment with EMS and other monofunctional alkylating agents. The Pearson product moment correlations (r) were, for example, for EMS 0.99 and for MMS 0.96.
In contrast, after treatment of tobacco seedlings with MH no such correlation was found. While the frequency of somatic mutations was extremely high (at high concentrations nearly 10 times higher than could be reached with monofunctional alkylating agents; see Gichner and Plewa, 1999), the DNA damage was at the control level.
Similarly, in V.faba root tip meristems in which treatment with 0.8 mM MH for 30 min induced 56.5% metaphases with chromatid aberrations, 24 h treatment with doses between 0.04 and 8 mM MH did not significantly increase DNA migration as measured by the Comet assay. Thus, MH does not cause significant numbers of DNA breaks (or alkali-labile sites) in plant nuclei under conditions yielding strong mutagenic/clastogenic effects. This is surprising because genotoxic treatments usually result in increased DNA migration, due to direct or repair-mediated DNA breakage. Even crosslinking activities are detectable, either as increased DNA migration several hours after treatment (Miyamae et al., 1997) or as decreased DNA migration immediately after simultaneous treatment with crosslinking and other genotoxic agents (Merk and Speit, 1999).
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Acknowledgments |
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This research was funded by the Grant Agency of the Czech Republic, grant no. 521/99/0532 (T.G.), and the Deutsche Forschungsgemeinschaft (Schu 951/5-1).
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Notes |
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2 To whom correspondence should be addressed. Tel: + 4202 2431 0109; Fax: + 4202 24310113; Email: gichner{at}ueb.cas.cz
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References |
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Received on February 23, 2000; accepted on April 14, 2000.
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