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Mutagenesis, Vol. 15, No. 6, 503-506, November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press

N-Methyl-N-nitrosourea-induced DNA damage detected by the comet assay in Vicia faba nuclei during all interphase stages is not restricted to chromatid aberration hot spots

Merten Menke, Armin Meister and Ingo Schubert1

Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genotoxic effect of the monofunctional alkylating agent N-methyl-N-nitrosourea (MNU) on root-tip nuclei of the field bean, Vicia faba, has been tested by comparative application of three protocols of the comet assay. While the alkaline denaturation/alkaline electrophoresis (A/A) procedure proved to be most sensitive at low doses, the alkaline denaturation/neutral electrophoresis (A/N) procedure yielded an optimal dose–response curve within a wider dose range. With the neutral electrophoresis without alkaline denaturation (N/N) procedure only minimal response was found. MNU-mediated single-strand breaks occurred in nuclei of all interphase stages. Detection of tandemly repeated FokI elements on comets by fluorescence in situ hybridization showed an average involvement of these heterochromatin-specific sequences in MNU-mediated single-strand breaks. This, together with previous results, suggests that the pronounced clustering of chromosomal aberrations in heterochromatic regions after treatment with S phase-dependent mutagens is mainly due to an error-prone interference of recombinative repair and replication in damaged basic repeats of large tandem repeat arrays.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
DNA damage induced by genotoxicity can be detected in plants by the comet assay, in which nuclei isolated from various tissues are exposed to electrophoresis (e.g. Koppen and Verschaeve, 1996; Navarrete et al., 1997; Gichner and Plewa, 1998; Koppen and Angelis, 1998; Angelis et al., 1999). DNA moving towards the anode becomes visible as the `comet tail', while the remaining nuclear scaffold appears as the `comet head'. The average proportion of DNA in the tail is taken as a measure of DNA damage. Variations of the comet assay protocol have been introduced to detect different kinds of lesions (double-strand breaks, single-strand breaks and abasic sites) and to alter the sensitivity of the assay (Angelis et al., 1999Go; Menke et al., 2000Go). The comet assay is a quick and easy way of screening for environmentally and experimentally induced DNA damage and may complement other systems for testing genotoxicity. By comparing different endpoints of genotoxicity, addressed by various test systems, conclusions as to the types of DNA damage and the mechanisms by which they are processed can be drawn.

Here we provide data as to comet formation after treatment of Vicia faba roots with different doses of the monofunctional alkylating agent N-methyl-N-nitrosourea (MNU) applying the neutral electrophoresis without prior DNA denaturation (N/N), the alkaline denaturation, neutral electrophoresis (A/N) and the alkaline denaturation, alkaline electrophoresis (A/A) procedure for the comet assay. In addition, we compared the DNA damage in control and MNU-treated nuclei during different cell cycle stages and investigated the involvement in MNU-induced damage of tandemly repeated sequences (FokI elements) located within heterochromatic chromosome regions of V.faba which previously were found to be hot spots for MNU-induced chromosomal aberrations (Baranczewski et al., 1997Go). This, in comparison with published data, allowed us to draw conclusions about the sensitivity of the variants of the comet assay to MNU-induced damage and the processing of such damage into chromatid-type aberrations.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
MNU treatment and comet assay of V.faba roots
Seeds of V.faba (karyotype ACB; see Fuchs et al., 1994) were germinated in the dark for 4 days on wet paper at 23°C, then seedlings were transferred to Hoagland solution for 18 h. Main roots (2–3 cm in length) were incubated for 1 h at 23°C in 0.001–8 mM MNU (CAS 684-93-5, Sigma N4766, Steinheim, Germany) dissolved in distilled water. Immediately after treatment, 0.5 cm of the root tips were cut off on ice, frozen in liquid nitrogen and stored at –80°C. The comet assay protocols are based on those described by Angelis et al. (1999). In brief, five root tips were chopped with a fresh razor blade in phosphate-buffered saline (PBS) on ice. The resulting suspension was filtered through a 30 µm mesh and 30 µl of the suspension obtained was mixed with 90 µl of 0.5% normal melting point (NMP) agarose (Biozyme, Oldendorf, Germany) at 42°C. From this mixture, two comet gels were made by pipetting two 60 µl drops on one slide and covering each drop with a 22x22 mm coverslip. When the gels had solidified, 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 and then either equilibrated for 3x5 min in 1x TBE at room temperature and electrophoresed for 8 min at 1 V/cm, 15 mA in TBE at pH 8.4 (N/N variant) or denatured in alkaline solution (0.3 M NaOH, 5 mM EDTA, pH 13.5) for 2x15 min before electrophoresis in TBE for 6 min (A/N variant). For the A/A variant of the comet assay, electrophoresis was performed for 20 min at 0.72 V/cm, 300 mA in a chamber cooled by ice in the same alkaline solution as used for denaturation. After electrophoresis, gels were dehydrated in ethanol and air-dried. Comets were analysed after staining of the DNA with 15 µl of 5 µg/ml ethidium bromide in water with a Zeiss Axioskop equipped with an analogue CCD video camera and Lucia image analysis software (LIM, Prague). The comet assay was repeated at least twice. In total, for each experimental point at least four individual gels were evaluated, each providing a median value for the percentage of DNA in the tail of 25 comets.

Flow sorting of isolated nuclei
To investigate defined cell cycle stages, nuclei from MNU-treated and from control roots were stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) and sorted according to their DNA content (based on the fluorescence intensity) into G1, S and G2 fractions before the comet assay. For DNA measurement and sorting of nuclei, we used a FACStar Plus Flow Cytometer and Cell Sorter (Becton Dickinson, San Jose, CA, USA) with a Sort Enhancement Module (SEM) and an argon-ion laser (INNOVA 90C-5, Coherent, Palo Alto, CA, USA), emitting UV light with 200 mW output power controlled by a Macintosh Computer with Cell Quest Software. The gates for flow sorting were determined according to the histogram of DAPI-stained nuclear suspensions (Figure 1Go) obtained from roots chopped in PBS/5 mM EDTA on ice.



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  		Fig. 1. . Histogram of V.faba meristematic nuclei used for sorting and subsequent comet assay. The nuclear DNA content is reflected by DAPI fluorescence intensity; sorting gates are represented by bars.

 
Fluorescence in situ hybridization (FISH) on comets
The protocol used for FISH on comet nuclei was as described (Menke et al., 2000Go). Briefly, probes were obtained and labelled with digoxigenin by PCR using primers specific for the 59 bp FokI element (Kato et al., 1984Go). The hybridization mixture contained 2–4 µl of the PCR sample per comet gel, 30% deionized formamide, 2x SSC and 10% dextran sulfate. Hybridization was done at 37°C overnight. Slides were then washed (3x5 min) in 25% formamide, 2x SSC at 30°C and (3x5 min) in 2x SSC at 37°C. After blocking with 3% bovine serum albumin in 4x SSC, the FISH signals were visualized with a sheep anti-digoxigenin–fluorescein isothiocyanate (FITC) conjugate followed by two rounds of amplification employing rabbit anti-sheep–FITC and anti-rabbit–FITC antibodies (Boehringer). The comet assay module of the Lucia image analysis system, which provides a double-staining mode, was used for simultaneous measurement of DNA content (ethidium bromide signal) and FISH signals (FITC) in the head and tail of comets (Menke et al., 2000Go).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Dose–response comparison for different variants of the comet assay after MNU treatment
The effect of MNU doses between 0.04 and 8 mM on DNA migration immediately after treatment of V.faba root tips was compared under N/N, A/N and A/A conditions by the comet assay (Table IGo and Figure 2Go). With the N/N procedure, which detects mainly double-strand breaks (Menke et al., 2000Go), only a very small increase in the proportion of DNA in the tail was found, independent of the dose applied, when compared with the control value. Using the A/N procedure, which detects single- and double-strand breaks (Angelis et al., 1999Go; Menke et al., 2000Go), a nearly linear relationship with dose was observed. The A/A procedure, which detects DNA breaks as well as `alkali-labile sites', yielded the heaviest damage, although control values were high. The capacity to detect DNA damage applying the A/A protocol was already saturated at a dose of 0.4 mM.


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  		Table I. . Percentage of DNA in comet `tail' (±SE) after MNU treatment (1 h) of V.faba roots observed by the comet assay in isolated nuclei under N/N, A/N and A/A conditions
 


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  		Fig. 2. . Mean proportion DNA in the comet `tail' after 1 h MNU treatment of V.faba root tips and different comet assay variants: neutral electrophoresis without alkaline denaturation (N/N), alkaline denaturation/neutral electrophoresis (A/N) and alkaline denaturation/alkaline electrophoresis (A/A).

 
Comparison of DNA damage in different cell cycle stages
In order to compare the extent of DNA damage in different cell cycle stages, nuclei from untreated and from MNU-treated root tips were isolated and sorted into fractions of G1, S or G2 phase nuclei before preparation of comets according to the A/N protocol (Table IIGo). All interphase stages showed DNA damage after MNU treatment. However, significantly more DNA damage was detected in S phase nuclei than in G1 and G2 nuclei in controls and after treatment with 1 mM MNU. This is probably due to discontinuities of DNA strands during replication. This difference was no longer detectable when roots were treated with a higher dose (5 mM) of MNU. The sorting procedure by itself must have resulted in DNA damage, as shown by the control values being highter in sorted nuclei than in unsorted nuclei (Table IIGo). One possible explanation for this is that the interaction of the high-energy laser beam with the fluorochrome–DNA complex could induce DNA damage.


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  		Table II. . Percentage of DNA in tail (±SE) after MNU treatment (1 h) of V.faba roots observed by the A/N variant of the comet assay in isolated G1, S and G2 nuclei
 
FISH with a heterochromatin-specific DNA probe on comets
FokI elements, which are located in most of the interstitial heterochromatic regions of V.faba chromosomes (Fuchs et al., 1994Go), were digoxigenin-labelled and hybridized to comets obtained after the A/N assay from nuclei of roots treated with 1 mM MNU as well as from control nuclei. The proportion of total DNA in the tail and of FISH signal in the tail was measured for each comet. Control nuclei showed 5.8 ± 0.8% of total DNA and 7.8 ± 1.2% of FokI sequence-specific signals in the tail. After MNU treatment, on average 22.8 ± 1.6% of DNA and 23.4 ± 1.4% of the FISH signals were found in the tail. Groups of six median values compared pairwise for DNA and FISH signal in the tail showed no significant differences between the two parameters (P = 0.28 for controls and P = 0.66 for treated nuclei).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
MNU-induced DNA damage in plant cells can be quantified within a biologically significant dose range by the A/N and A/A variants but not the N/N variant of the comet assay
The comet assay was first described as a microelectrophoretic technique for the detection of X-ray-induced DNA breakage under neutral pH conditions (Östling and Johanson, 1984Go) and later modified by introduction of alkaline conditions before and during electrophoresis to extend the sensitivity of the assay to single-strand breaks and alkali-labile sites (Singh et al., 1988Go). Protocols for efficient detection of different types of DNA damage caused by a broad spectrum of genotoxic effects have been introduced (e.g. Angelis et al., 1999). Only exceptionally has the comet assay failed to detect DNA damage after treatment with agents that cause gene mutations and chromosomal aberrations in the same system (Gichner et al., 2000Go). Our data show that MNU-induced DNA damage in plants is detectable by the comet assay and confirm previous observations made for MMS, another monofunctional alkylating agent (Angelis et al., 1999Go), that the A/A assay detects DNA damage only within a narrow range of (low) mutagen doses. The high control values obtained with this assay variant probably result from the ability of strong alkali treatment to damage DNA (Yendle et al., 1997Go). The A/N procedure is more suitable for studying dose–effect relationships within a broader range of doses. This range includes doses that induce other endpoints of genotoxicity in V.faba root-tip meristems [<=25% of metaphases with chromosomal aberrations were found 18 h after treatment with 1 mM MNU for 1 h (Baranczewski et al., 1997Go)]. The lowest MNU dose detectable by the comet assay is within the range associated with a significant increase in the frequency of sister chromatid exchange (0.2 mM MNU for 30 min; Schubert and Heindorff, 1989).

Since MNU does not cause DNA breaks directly and mediates single-strand breakage only during the course of repair, it is reasonable that the effect observed under N/N conditions was negligible.

Repair-mediated DNA breaks detectable at all cell cycle stages after MNU treatment do not preferentially involve heterochromatin-specific sequences; therefore MNU-induced chromatid-type aberrations within the heterochromatin apparently originate from recombinative mis-repair of damage within large tandem repeat arrays during S phase
Olive and Banath (1993a,b) observed longer tail moments for various untreated mammalian cell lines during S phase than during G1 or G2 phase. This has been confirmed by our investigation of plant cells when untreated nuclei or nuclei exposed to a low dose of MNU were tested (Table IIGo). The greater mobility of DNA from S-phase nuclei is probably due to the presence of free DNA ends during replication.

Usually, only cells exposed to alkylating agents during S phase show chromosomal aberrations at the first post-treatment mitosis, while single-strand breaks are detectable at all cell cycle stages. From these observations we infer that DNA repair, which occurs throughout the cell cycle, is mainly error-free during G1 and G2 but error-prone (yielding chromatid-type aberrations) during S phase, possibly because of interaction of DNA discontinuities mediated by repair with those mediated by replication.

MNU, like other S-phase-dependent clastogens, causes various types of chromatid aberration in V.faba, with breakpoints preferentially located within the heterochromatic regions of the V. faba genome. Sister chromatid exchanges caused by the same mutagens probably represent mainly error-free recombinative circumvention of replication-blocking lesions (Lindenhahn and Schubert, 1983Go) and are more or less randomly distributed (reviewed in Schubert et al., 1986). The heterochromatic `hot spot' regions are known to consist of large arrays of tandem repeats of which the most prominent is the so-called FokI element, characteristic of ~75% of the heterochromatic regions in V.faba (Fuchs et al., 1994Go).

The accumulation of chromatid aberrations within the heterochromatin is neither the consequence of a preferential induction of the clastogenic primary lesion O6-methylguanine within FokI elements nor an indication of preferential removal of this MNU-caused lesion from the remaining DNA as shown by immuno-slot-blot analysis (Baranczewski et al., 1997Go).

Previously, we have developed a method to detect preferential DNA damage within the heterochromatin-specific FokI elements of V.faba by FISH on comets using labelled FokI elements as a hybridization probe. This has shown that, after treatment of nuclei with the restriction endonuclease FokI, FokI elements are preferentially damaged (Menke et al., 2000Go). FISH with FokI elements on comets caused by MNU treatment did not reveal a disproportional involvement of FokI elements in MNU-mediated DNA damage. Thus, FokI elements are randomly involved in repair of MNU-induced DNA damage. This supports the hypothesis that O6-methylguanine—and possibly other clastogenic primary lesions such as N3-methyl- adenine (Angelis et al., 2000Go)—induced by MNU result in formation of aberrations only when subjected to recombinative misrepair. The latter seems to involve preferentially damaged repeats of extended tandem repeat arrays during S phase. This inference supports a previously suggested interpretation for the generally observed clustering of aberrations in heterochromatic domains which consist of extended tandem repeat arrays. According to this hypothesis, clustering of aberrations within heterochromatin is due to an increased probability of errors during recombinative repair within tandem repeat arrays when interfering with replication (Schubert et al., 1994Go; Baranczewski et al., 1997Go).


   Acknowledgments
 
We thank Martina Kühne and Joachim Bruder for excellent technical assistance, and Rigomar Rieger, Karel Angelis and Tomas Gichner for critical reading of the manuscript and for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (Schu 951/5-1) and by the Fonds der Chemischen Industrie (A.M., I.S.).


   Notes
 
1 To whom correspondence should be addressed. E-mail: schubert{at}ipk-gatersleben.de Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    Angelis,K.J., Dusinská,M. and Collins,A.R. (1999) Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity. Electrophoresis, 20, 2133–2138.[ISI][Medline]

    Angelis,K.J., McGuffie,M., Menke,M. and Schubert,I. (2000) Adaptation to alkylating damage in DNA measured by the comet assay. Environ. Mol. Mutagen., 36, in press.

    Baranczewski,P., Nehls,P., Rieger,R., Pich,U., Rajewsky,M.F. and Schubert,I. (1997) Formation and repair of O6-methylguanine in recombination hot spots of plant chromosomes. Environ. Mol. Mutagen., 29, 394–399.[ISI][Medline]

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    Kato,A., Yakura,K. and Tanifuji,S. (1984) Sequence analysis of Vicia faba repeated DNA, the FokI repeat element. Nucleic Acids Res., 12, 6415–6426.[Abstract/Free Full Text]

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Received on April 14, 2000; accepted on August 3, 2000.


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