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Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations
Mutation Research 383 Ž1997. 243–252
Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations D. Slamenova ˇ ´ a
a,)
, A. Gabelova ´ ´ a, L’. Ruzekova ˇ ´ a, I. Chalupa a, E. Horvathova ´ ´ a, b ˇˇ T. Farkasova ˇ ´ a, E. Bozsakyova´ a, R. Stetina
ˇ ´ Cancer Research Institute of SloÕak Academy of Sciences, Spitalska 21, 812 32 BratislaÕa, SloÕak Republic b Institute of Experimental Medicine, Õ Orlickych Czech Republic ´ 14, 517 83 Olesnice, ˇ ¸ horach Received 9 July 1996; revised 23 December 1996; accepted 23 December 1996
Abstract Human cells ŽVH10 or Hep G2. and hamster cells V79 were exposed to different concentrations of N-methyl-N X-nitroN-nitrosoguanidine ŽMNNG. and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 mgrml, alkaline elution of DNA at 1 mgrml and DNA unwinding at 1–2 mgrml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations ) 1 mgrml in VH10 cells Žshort-term treatment.; ) 0.2 mgrml in V79 cells Žlong-term treatment. and ) 8 mgrml in human lymphocytes Žlong-term treatment.. In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside ŽAra C. and hydroxyurea ŽHU. which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 = 10y5 M Ara C and 2 = 10y3 M HU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG Ž- 2 mgrml.. Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined. Keywords: Mammalian cell VH10; Hep G2; V79; Human lymphocyte; DNA unwinding technique; Alkaline elution of DNA; Comet assay; Chromosomal aberration; Single strand breaks of DNA; DNA damage
1. Introduction
)
Corresponding author.
Exposure of cells to mutagens produces chemical changes in DNA which may be either irreversible or
0921-8777r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 8 7 7 7 Ž 9 7 . 0 0 0 0 7 - 4
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repaired by various DNA repair enzymes. Among the survivors, however, some cells show mutations, which were generated by errors that occurred in DNA replication during the recovery period w1–3x. Because replication errors in damaged DNA are likely to be directly involved in inducing mutations, it would be useful to have some rapid biochemical methods for measuring DNA damage and repair to help evaluate the possible mutagenic effects of radiation or chemicals. The most available category of DNA lesions for detection by biochemical methods comprises DNA strand breaks. These result from a number of different types of reactions Žbase and nucleotide excision repair; direct scission of the DNA backbone by chemical or radical attack; scission following the binding of intercalating agents; alkalilabile DNA adducts; endonuclease and topoisomerase action; DNA hydrolase release from lysosomes.. The biochemical methods of DNA unwinding w4,5x and alkaline elution of DNA w6x are among the most convenient ways of detecting DNA strand breaks resulting from a number of different types of reactions. Cytogenetic detection of chromosomal aberrations has been considered a good criterion for induction of mutagenic lesions in DNA of mutagen treated cells w7x. However, since the effects of environmental toxicants are often tissue and cell type specific it was important to develop a technique which can detect DNA damage in individual cells. For the direct visualization of DNA damage in indi¨ vidual cells, Ostling and Johanson w8x developed a microgel electrophoresis technique which depends on the migration of DNA from nucleus to anode, when breaks are present. For validation of the comet assay against other methods we used human cells VH10 and Hep G2, human lymphocytes and hamster cells V79. As a model carcinogen and mutagen we chose the monofunctional alkylating agent N-methyl-N X-nitroN-nitrosoguanidine ŽMNNG.. Monofunctional alkylating agents are primarily not DNA strand-breaking agent w9x, they do not induce direct scission of the DNA backbone. MNNG-induced DNA strand breaks evaluated under alkaline conditions result mainly from endonuclease action through base and partly nucleotide excision repair and from alkali-labile DNA adducts. MNNG can methylate all oxygen and most nitrogen atoms of DNA. Fornace and Little w10x
suggested that MNNG produced a several hundredfold greater number of alkylations than do similar concentrations of other monofunctional alkylating agents. Methylated DNA is poorly repaired by the UVRABC nucleotide excision repair pathway w11x; however, some recognition does occur in vitro w12x and in vivo w13,14x.
2. Materials and methods 2.1. Cell lines Cells VH10, human fibroblasts, derived from foreskin of 10 years old healthy boy, non-transformed were obtained from Dr. A. Kolman, Laboratory of Radiobiology, University of Stockholm, Sweden. Quasidiploid Chinese hamster V79 cells were obtained from Dr. A. Abbondandolo, University of Genoa, Italy. Hep G2 cells, human hepatoma cells were obtained from Dr. A. Collins, The Rowett Research Institute, Aberdeen, Scotland. VH10 cells were cultured in MEM Eagle q 10% foetal calf serum, V79 cells in MEM Eagle mediumq 6% foetal calf serum. Hep G2 cells were cultured in Williams mediumq 10% foetal calf serum. To media was added: penicillin Ž200 Urml., streptomycin Ž100 mgrml. and kanamycin Ž100 mgrml.. Human lymphocytes were incubated in MEM E medium supplemented with 20% of foetal calf serum and phytohaemagglutinin Ž2%., L-glutamine Ž294.1 mgrml., and antibiotics. All cell lines were cultured on glass or plastic Petri dishes or small glass vials in CO 2 incubator, at 378C. 2.2. Chemicals and treatment of cells MNNG s N-methyl-N X-nitro-N-nitrosoguanidine ŽServa diluted 1:1 with H 2 O ŽFig. 1 and Fig. 2., Aldrich ŽFigs. 3–7.. The stock solution of this Nnitrosoamide in DMSO Ž1 mgrml. was kept at y208C and diluted immediately before use in PBS or MEM medium. Ara C Ž1-Ž b-D-arabinofuranosyl.cytosine. and HU Žhydroxyurea. ŽSigma.: Stock solutions of Ara C Ž2 = 10y2 M. and HU Ž0.4 M. were dissolved in tridistilled water and kept at y208C. These solutions
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10y3 M or 2 = 10y2 M. were applied either after or during MNNG treatment. 2.3. Alkaline DNA-unwinding and HTP chromatography
Fig. 1. The level of DNA strand breaks detected by alkaline elution of DNA in MNNG-treated VH10 cells Žleft side. and Hep G2 cells Žright side.. Cells were treated with MNNG either in the presence or in the absence of Ara C Ž2=10y5 M or 2=10y4 M. and HU Ž2=10y3 M or 2=10y2 M.. Control `; Controlqlower conc. of Ara C and HU v; Controlqhigher conc. of Ara C and HU \; MNNG Ž2.5 mgrml. %; MNNG Ž2.5 mgrml.qlower conc. of Ara C and HU I; MNNG Ž2.5 mgrml.qhigher conc. of Ara C and HU B.
were diluted immediately before use in culture medium. Exponentially growing cells VH10 or Hep G2 were treated with MNNG for 30 or 60 min in PBS or 120 min in MEM. DNA-polymerase inhibitors Ara C Ž2 = 10y5 M or 2 = 10y4 M. and hydroxyurea Ž2 =
We used the method described by Ahnstrom ¨ and Erixon w4x. Our modification has already been described w15x. Briefly: Cells were lysed at room temperature in 1 ml of 0.03 M NaOH q 0.15 M NaCl for 30 min in the dark. Then the cells were neutralized by adding of 2 ml 0.02 M NaH 2 PO4 , the molecular weight of the DNA was reduced by ultrasonic treatment and sodium dodecyl sulfate was added to each sample Ž0.09% vrv.. Samples were kept at y208C. Single-strand DNA was separated by HTP chromatography with 2.5 ml of 0.09 M and 2.5 ml of 0.1 M potassium phosphate ŽKP. buffer and double-strand DNA with 2.5 ml of 0.25 M and 2.5 ml of 0.5 M KP buffer ŽpH 6.8. at 608C. The radioactivity of individual samples was measured on a liquid scintillation counter, Beckman LS 1801 and the percentage of single-stranded DNA was calculated in each sample. The proportion of singlestranded DNA was converted to the frequency of DNA breaks using a standard curve derived from alkaline unwinding experiments with cells containing
Fig. 2. The level of DNA strand breaks detected by DNA unwinding in VH10 cells. Left side: VH10 cells treated with MNNG for 30 min in PBS buffer v; 60 min in PBS buffer \; and 120 min in MEMmedium `. Right side: VH10 cells treated with MNNG for 120 min `; treated with MNNG for 120 min and then for 60 min with Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v; treated with MNNG for 30 min and then for 90 min with Ara C and HU \; and treated for 120 min with MNNGq Ara C q HU %.
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known numbers of breaks introduced by X-irradiation. 2.4. Alkaline elution of DNA We adapted the method described by Kohn et al. w6x. Differences from the original method: Ži. proteinase K was substituted by pronase E; Žii. DNA in individual eluates was precipitated by trichloroacetic acid Ž5% TCA vrv., and collected by filtration through nitrocellulose filters SYNPOR 6 Ž0.44 mm., washed, and after drying the radioactivity of membranes measured on a Beckman LS 1801 liquid scintillation counter; Žiii. for all samples, the filter holder and tubing were flushed with 6 ml of 0.4 N NaOH, the content of DNA in 0.4 N NaOH was determined in the same way as in eluting solutions, and the radioactivity measured was added to the radioactivity of DNA retained; Živ. the DNA retained on filters was measured in the following way: filters were carefully taken from filter holders, placed in 5% TCA Ž08C. and kept there for approx. 20 min before being washed, dried, and their radioactivity measured. In preliminary experiments a comparison was made between the original method and our modification and the results were very similar. 2.5. Chromosome aberrations Human peripheral blood lymphocytes were incubated for 48 h before adding MNNG in culture medium. After a further 22 h, colchicine was added at a final concentration of 0.75 Žmgrml for 2 h. V79 cells were grown Žinoculum of 6 = 10 5 cellsrdish, diameter 60 mm. for 24 h, until they reached the exponential stage of growth. Then MNNG was added and cultures were incubated for a further 18 h. Colchicine was added 1 h prior to harvesting. VH10 cells were grown Žinoculum of 9 = 10 5 cellsrdish, diameter 90 mm. for 24 h. Then MNNG was added for 2 h. Then the cells were rinsed twice with PBS and fresh grown medium was added. The cultures were incubated for a further 120 h. Three hours before the end of cultivation colchicine was added. The concentrations of MNNG used were selected on the basis of preliminary cytotoxicity tests. Slides were prepared with standard air drying procedure.
Chromosome aberrations were scored in conventional Giemsa stained preparations. The classification of aberrations was as described by Venitt and Parry w16x and ISCN w7x. The metaphases were analysed for chromatid gaps and breaks. The results were statistically evaluated by the chi-square test. 2.6. Comet assay The procedure of Singh et al. w17x was followed with minor modifications. A base layer of 100 ml of 0.75% NMP agarose in PBS buffer ŽCa2q- and Mg 2q-free. was placed on microscope slides. Instead of fully frosted microscope slides we used plain slides covered by a thin layer of 1% NMP agarose in water and dried at 508C. Human or hamster cells Žwith or without MNNG treatment respectively with post-MNNG-treatment., were suspended in 0.75% LMP agarose and 2 = 10 4 cells in 85 ml spread on base layer on each triplicate of slides. A cover slip was added and the agarose was allowed to solidify. The cover slips were removed and the slides were placed in lysis mixture Ž2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris, pH 10 and 1% Triton X-100. at 48C for 1 h to remove cellular proteins. Slides were then transferred to an electrophoresis box containing an alkaline solution Ž300 mM NaOH, 1 mM Na 2 EDTA.. Slides were kept in this solution for 40 min unwinding time, at 48C. A current of 25 V Ž300 mA. was then applied for 30 min. The slides were removed, neutralised with 3 = 5 min washes with Tris Ž0.4 M, pH 7.5., stained with 20 ml DAPI Ž1 mgrml. and the cover slip returned. DAPI stained nucleotides were examined with a Zeiss Jenalumar fluorescence microscope and one hundred comets on each slide were scored visually as belonging to one of five classes according to tail intensity and given a value of 0, 1, 2, 3 or 4 Žfrom undamaged, 0, to maximally damaged, 4.. Thus, the total score for 100 comets could range from 0 Žall undamaged. to 400 Žall maximally damaged.. This visual scoring was suggested by Collins et al. w18x. Results obtained by this method were confirmed by computerised image analysis ŽKinetic Imaging, Liverpool, UK.. Arbitrary units were converted to the frequency of DNA breaks using a standard curve derived from comet assay experiments with cells containing known numbers of breaks introduced by X-irradiation.
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3. Results and discussion Although most DNA breaks occur as a result of metabolic participation by the cell, determination of single strand breaks in mammalian cells is probably the most commonly used approach for the rapid and sensitive monitoring of genotoxic events which may originate in the environment. In the framework of an EC Copernicus project we evaluated by different techniques the level of ss breaks of DNA in different mammalian cell lines damaged by MNNG as well as the influence of DNA repair inhibitors Ara C and HU w19x on the level of DNA breaks induced by MNNG. In addition to measuring DNA breaks chromosome aberrations were examined as an indicator of damage at the level of nuclear organisation. Fig. 1 represents results obtained by alkaline elution of DNA. The number of breaks in DNA is inversely related to the retention of DNA on the filter. The left side of the figure represents the results obtained with human cells ŽVH10.. We tested the effects of two concentrations of Ara C Ž2 = 10y5 M or 2 = 10y4 M. and HU Ž2 = 10y3 M or 2 = 10y2 M. on the level of ss breaks induced by MNNG. Lower concentrations of inhibitors were as effective as 10-times higher concentrations; therefore in further experiments we used only this lower concentra-
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tion of inhibitors. A similar experiment was performed with the human hepatoma cell line Hep G2 Žright side of the Fig. 1.. In this case we used only the lower concentrations of inhibitors Ara C and HU, which, however, evidently increased the level of DNA breaks not only in MNNG-treated cells but also in control cells. Alkaline elution of DNA is a sensitive technique for studying strand breaks of DNA; its disadvantage is the time taken to process samples and the limited number of samples that can be accommodated at one time. Different kinds of MNNG treatment of mammalian cells have been described by different authors Žduration of 30, 60, 120, and 180 min or 24 h in PBS, complete medium, or serum-free medium; different MNNG sources.. We have therefore measured the level of strand breaks Ždosereffect. in MNNG-treated VH10 cells Žconcentrations 0–2 mgrml. either in PBS buffer Ž30 and 60 min. or in complete medium Ž120 min.. The results are presented in the left side of Fig. 2. The method used in these experiments was the alkaline unwinding of cellular DNA with separation of unwound single-stranded DNA fragments from the intact double-stranded DNA by elution from hydroxyapatite with phosphate solutions of different ionic strength. The amount of unwound DNA is proportional to the number of strand breaks. As the
Fig. 3. The level of DNA strand breaks detected by DNA unwinding in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side.. Cells treated for 120 min with MNNG `; and cells treated simultaneously with MNNG and Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v.
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level of strand breaks per 10 9 daltons measured by DNA unwinding was in all three cases rather similar, in further experiments we used only 120 min MNNG-treatment of cells in complete MEM. The levels of strand breaks in VH10 cells treated for 120 min with MNNG alone and with different combinations of Ara C, HU and MNNG are shown on the right side of Fig. 2. Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. increased the level of ss breaks of DNA in all three kinds of application, the most effective seemed to be the simultaneous treatment of cells with MNNG and inhibitors. Similar effects were observed in human cell line XP12 and hamster cells V79 Ždata not shown.. When we repeated simultaneous treatment of different cells lines ŽVH10, Hep G2, V79. with higher concentrations of MNNG Ž0–7 mgrml, Aldrich. q DNA repair inhibitors ŽFig. 3., however, we found only a small ŽHep G2. or no ŽVH10, V79. effect of DNA repair inhibitors on the level of DNA breaks in MNNG treated cells. It could be that at higher levels of damage the repair system was saturated, i.e., the polymerase could not keep up with the continuing production of strand breaks and inhibitors didn’t add to the breakage. It is possible that accumulation of strand breaks at higher concentrations of MNNG requires higher concentrations of Ara C and HU. In view of the higher level of DNA breaks in control
cells ŽFig. 1. we deliberately used only the lower concentrations of Ara C and HU. A great advantage of DNA unwinding is the higher number of samples which can be processed in the same experiment. Fig. 4 represents the results which were obtained by SCGE. The level of DNA breakage is indicated by the extent of movement of DNA into the comet tail evaluated by visual scoring. As was shown with the DNA unwinding technique, the influence of AraC and HU was observed only in combination with very low MNNG concentrations. If we compare the results obtained by DNA unwinding technique and by the comet assay, e.g., in human VH10 cells it is evident that the comet assay is very sensitive to low levels of damage; but it has a limitation that it saturates at a fairly low level of damage compared with alkaline unwinding. This is what we would expect, since as soon as all the DNA loops contain breaks, i.e., the tail is full of DNA, there is no possibility of further expansion of DNA into the tail, and the level at which this occurs is determined by the number of loops. Although the comet assay is fairly restricted in the range of damage it can detect, it does as a result show a steep dose-response over that range, which makes it particularly presize as well as sensitive. In addition to methylation of all bases of DNA Žmainly the 6-oxygen of guanine, producing O 6-
Fig. 4. The level of DNA strand breaks detected by SCGE in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side.. Cells treated for 120 min with MNNG `; and cells treated simultaneously with MNNG and Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v.
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Table 1 Induction of chromosomal aberrations in human peripheral lymphocytes treated with MNNG for 22 h Sample
a
NC SC MNNG 0.08 MNNG 0.8 MNNG 2.0 MNNG 4.0 MNNG 8.0 MNNG 10.0 ) a b
Aberrant metaphases
3 1 4 4 5 4 10 7
)) )
Aberrations per 100 metaphases
b
Chromatid
Isochromatid
Exchange
Total no.
brf
brf
dic
dmin
qr
tr
3 1 1 4 4 3 3 5
– – 1 – – – 2 –
– – – – – – 1 1
– – 2 – 1 1 4 1
– – – – – – – –
– – – – – – – –
3 1 4 4 5 4 10 7
)) c
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.08–10.0 mgrml.. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial.
methyldeoxyguanosine which causes mutations., MNNG gives rise to MNNG and OH radicals w20,21x. Radical formation in biological systems, especially of the active oxygen species, is of considerable interest as a cause of mutation, including chromosomal damage. Osada et al. w22,23x reported that the light emission induced by S9-activated BwaxP is related to active oxygen species. Kimura et al. w24x investigated the possible role of radical formation by
MNNG by means of light emission measurements and chromosomal aberration testing. The production of chromosomal aberrations in Chinese hamster fibroblasts was correlated with the light emission intensity. In an Oy 2 generating system, chromatid type aberrations are induced, including chromatid gaps and breaks w25,26x. We evaluated the effect the level of chromosomal aberrations in treated peripheral human lymphocytes ŽPHL; Table 1., hamster cells V79
Table 2 Induction of chromosomal aberrations in hamster cells V79 treated with MNNG for 18 h Sample
a
NC SC MNNG 0.05 MNNG 0.2 MNNG 0.4 MNNG 0.6 MNNG 0.8 MNNG 2.0 MNNG 4.0 MNNG 6.0 MNNG 8.0 ) a b c
Aberrant metaphases
2 2 4 8 6 8 7 12 12 12 14
)
)
)) )) )) )))
b
Aberrations per 50 metaphases
c
Chromatid
Isochromatid
Exchange
brf
brf
dic
dmin
qr
tr
r
Total no.
2 – 1 2 3 5 3 11 3 12 5
– – 1 3 – 1 3 1 6 – 6
– – – – 2 1 – – – – –
– – 3 2 – 1 4 – 4 1 4
– 2 – 1 2 – – 1 1 2 –
– – – – 1 1 – 1 – – –
– – – – – – – – – – –
2 2 5 8 8 9 10 14 14 15 15
) ) ) ) ))) ))) ))) )))
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.05–8.0 mgrml.. 50 metaphases were scored in each sample. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial; r, ring chromosome.
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Table 3 Induction of chromosomal aberrations in human cells VH10 treated with MNNG for 2 h Sample
a
NC SC MNNG 0.1 MNNG 0.5 MNNG 1.0 MNNG 5.0 MNNG 10.0 MNNG 20.0 MNNG 40.0 ) a b c
Total no. of metaphases scored
Aberrant metaphases
26 25 47 7 20 9 28 16 NE
1 2 2 – 3 4 10 7
) ) ))
Aberrations
b
Chromatid
Isochromatid
Exchange
Total no.
brf
brf
dic
dmin
qr
tr
r
1 1 1 – 3 4 4 29
– – 1 – – – 2 6
– – – – 2 1 5 3
– 1 – – 13 3 1 14
– – – – – – 20 14
– – – – – 1 7 11
– – – – 1 1 – 3
1 2 2 – 19 10 39 80
))) ))) ))) )))
c
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.1–40.0 mgrml.. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial; r, ring chromosome. NE, not evaluable.
ŽTable 2. and VH10 cells ŽTable 3.. Obtained results confirmed that MNNG-induced chromosomal damage in all cell types studied. It is evident that MNNG damages not only molecular but also nuclear organisation of mammalian cells. Fig. 5 shows the kinetics of DNA rejoining in three cell lines, assayed by DNA unwinding. The highest decrease of ss breaks of DNA with time of incubation Žindicating repair. was observed in human
VH10 cells. DNA rejoining in Hep G2 and V79 cells was less effective. The kinetics of DNA rejoining in MNNG-treated VH10 cells and Hep G2 cells measured by the comet assay are shown in Fig. 6. The last figure ŽFig. 7. represents DNA rejoining measured by alkaline elution of DNA in MNNG-treated V79 cells. On the basis of the obtained results, we suggest that the comet assay is able to recognize sites of incomplete repair of DNA breaks which are
Fig. 5. The kinetics of DNA rejoining in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side. assayed by DNA unwinding. The level of DNA strand breaks was detected immediately after MNNG-treatment `; 2 h after treatment v; and 4 h after treatment \.
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Fig. 6. The kinetics of DNA rejoining in VH10 cells Žleft side. and Hep G2 cells Žright side. assayed by SCGE. The level of DNA strand breaks was detected immediately after MNNG-treatment `; 4 h after treatment v; and 24 h after treatment \.
to a lesser degree recognized by alkaline elution of DNA or by DNA unwinding. Though the O 6 position of guanine and, to a lesser extent, the O 4 position of thymidine which can be repaired by direct dealkylation by methyltransferases Žwithout creation of strand breaks. are viewed as the sites of major importance with respect to alkylation mutagenesis, determination of strand
breaks and alkali-labile lesions of DNA not only represents a very good indicator of mutagenic potential of the studied compound but can be used as well for determination of DNA repair during the postMNNG-treatment. The ability of Ara C and HU to increase the level of ss DNA breaks induced by low concentrations of MNNG ŽFig. 1 and Fig. 2. as well as investigation of ss breaks of DNA during 2–24 h
Fig. 7. The kinetics of DNA rejoining in V79 cells assayed by alkaline elution of DNA. The level of DNA strand breaks was detected immediately after MNNG-treatment Žleft side., 4 h after treatment Žmiddle. and 24 h after treatment Žright side.. Control `; 1 mg of MNNGrml \; 2.5 mg of MNNGrml I; 5 mg of MNNGrml ^; 10 mg of MNNGrml e.
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of post-MNNG treatment ŽFigs. 5–7. enables more information to be obtained about the overall DNA repair in MNNG-treated mammalian cells. Acknowledgements The authors express their appreciation to Dr. A.R. Collins ŽThe Rowett Research Institute, Aberdeen, Scotland. for his helpful comments. This study was supported by EC Grant ŽCopernicus. CIPA-CT940129. References w1x Witkin, E.M. Ž1969. Ultraviolet-induced mutation and DNA repair, Annu. Rev. Genet., 3, 525–552. w2x Witkin, E.M. and D.L. George Ž1973. Ultraviolet mutagenesis. In: POLA and UVRA POLA derivatives of Escherichia coli BrR: evidence for an inducible error prone repair system, Genetics, Suppl. 73, 9–108. w3x Cleaver, J.E. Ž1984. Methods for studying excision repair of eukaryotic DNA damaged by physical and chemical mutagens, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel ŽEds.., Handbook of mutagenicity test procedures, Elsevier Science Publishers BV, pp. 33–69. w4x Ahnstrom, ¨ G. and K. Erixon Ž1981. Measurement of strand breaks by alkaline denaturation and hydroxylapatite chromatography, in: E.C. Friedberg, P.C. Hanawalt ŽEds.., DNA Repair. A Laboratory Manual of Research Procedures, Vol. 1, Part B, Basel, pp. 403–418. w5x Bolcsfoldi, G. Ž1995. The DNA alkaline unwinding genotoxicity test, in: S. O’Hare and C.K. Atterwill ŽEds.., Methods in Molecular Biology, Vol. 43, Humana Press Inc., Totowa, NJ, pp. 257–266. w6x Kohn, K.W., R.A.G. Ewig, L.C. Erickson and L.A. Zwelling Ž1981. Measurement of strand breaks and crosslinks by alkaline elution, in: E.C. Friedberg and P.C. Hanawalt ŽEds.. DNA Repair. A Laboratory Manual of Research Procedures, Vol. 1, Part A, New York, pp. 379–401. w7x ISCN Ž1985. An international system for human cytogenetic nomenclature Ž1978., Cytogenet. Cell Genet., 21, 309–404. ¨ w8x Ostling, O. and J. Johanson Ž1984. Microelectrophoretic studies of radiation-induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun., 123, 291–298. w9x Singer, B. and D. Grunberger Ž1983. Molecular Biology of ¨ Mutagens and Carcinogens, Plenum Press, New York and London. w10x Fornace, A.J. Jr. and J.B. Little Ž1979. DNA-protein crosslinking by chemical carcinogens in mammalian cells, Cancer Res., 39, 704–710. w11x Van Houten, B. and A. Sancar Ž1987. Repair of N-methylX N -nitro-N-nitrosoquanidine-induced DNA damage by ABC exinuclease, J. Bacteriol., 169, 540–545.
w12x Voigt, J.M., B. Van Houten, A. Sancar and M.D. Topal Ž1989. Repair of O 6 -methylguanine by ABC exinuclease of Escherichia coli in vitro, J. Biol. Chem., 264, 5172–5176. w13x Samson, L., J. Thomale and M.F. Rajewsky Ž1988. Alternative pathways for the in vivo repair of O 6-alkylguanine and O 4-alkylthymine in Escherichia coli: The adaptive response and nucleotide excision repair, EMBO J., 7, 2261–2267. w14x Rossi, S.C., M. Conrad, J.M. Voigt and M.D. Topal Ž1989. Excision repair of O 6-methylguanine synthesized at the rat H-ras N-methyl-N-nitrosourea activation site and introduced into Escherichia coli, Carcinogenesis, 10, 373–377. w15x Slamenova, ˇ ´ D., T. Bohusova ˇ ´ and C. Oravec Ž1990. Inhibitory effect of theophylline on repair of potentially lethal MMS-induced damages to DNA in V79 cells, Mutation Res., 242, 127–134. w16x Venitt, S. and J.M. Parry Ž1984. Mutagenicity testing: a practical approach, IRL Press, Oxford, Washington DC, 353 pp. w17x Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider Ž1988. A simple technique for quantification of low levels of DNA damage in individual cells, Exp. Cell Res., 175, 184– 191. w18x Collins, A.R., Ma Ai-guo and S.J. Duthie Ž1995. The kinetics of repair of oxidative DNA damage Žstrand breaks and oxidised pyrimidine dimers. in human cells, Mutation Res., 336, 69–77. w19x Collins, A.R. and R.T. Johnson Ž1984. The inhibition of DNA repair, Adv. Radiat. Biol., 11, 71–129. w20x Nagata, C., M. Nakadate, Y. Ioki and A. Imamura Ž1972. Electron spin resonance study on the free radical production from N-methyl-N X-nitro-N-nitrosoquanidine, GANN, 63, 471–481. w21x Mikuni, T., M. Tatsuta and M. Kamachi Ž1985. Production of hydroxyl-free radical by reaction of hydrogen peroxide X with N-methyl-N -nitro-N-nitrosoguanidine, Cancer Res., 45, 6442–6445. w22x Osada, K., Y. Furukawa, M. Komai and S. Kimura Ž1990. Detection of ultra weak chemiluminescence from benzowaxpyrene and 3-methylcholanthrene, J. Clin. Nutr., 8, 185–192. w23x Osada, K., Y. Furukawa, M. Komai, M. Kimura, K. Hishinuma, C. Saitoh, H. Inaba and S. Kimura Ž1991. Correlation between chemiluminescence intensity from mutagens and their mutagenicity, Mutation Res., 252, 102–103. w24x Kimura, M., P. Roschger, M. Kobayshi, S. Kimura and H. X Inaba Ž1992. N-methyl-N -nitro-N-nitrosoguanidine-induced light emission in Chinese hamster cell cultures: correlation with enhancement of chromosomal aberrations, Mutation Res., 281, 215–220. w25x Nicotera, T.M., A.W. Block, Z. Gibas and A.A. Sandberg Ž1985. Induction of superoxide dismutase, chromosomal aberration and sister-chromatid exchanges by paraquat in Chinese hamster fibroblasts, Mutation Res., 151, 263–268. w26x Sofuni, T. and M. Ishidate Jr. Ž1988. Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture, Mutation Res., 197, 127–132.
Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations D. Slamenova ˇ ´ a
a,)
, A. Gabelova ´ ´ a, L’. Ruzekova ˇ ´ a, I. Chalupa a, E. Horvathova ´ ´ a, b ˇˇ T. Farkasova ˇ ´ a, E. Bozsakyova´ a, R. Stetina
ˇ ´ Cancer Research Institute of SloÕak Academy of Sciences, Spitalska 21, 812 32 BratislaÕa, SloÕak Republic b Institute of Experimental Medicine, Õ Orlickych Czech Republic ´ 14, 517 83 Olesnice, ˇ ¸ horach Received 9 July 1996; revised 23 December 1996; accepted 23 December 1996
Abstract Human cells ŽVH10 or Hep G2. and hamster cells V79 were exposed to different concentrations of N-methyl-N X-nitroN-nitrosoguanidine ŽMNNG. and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 mgrml, alkaline elution of DNA at 1 mgrml and DNA unwinding at 1–2 mgrml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations ) 1 mgrml in VH10 cells Žshort-term treatment.; ) 0.2 mgrml in V79 cells Žlong-term treatment. and ) 8 mgrml in human lymphocytes Žlong-term treatment.. In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside ŽAra C. and hydroxyurea ŽHU. which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 = 10y5 M Ara C and 2 = 10y3 M HU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG Ž- 2 mgrml.. Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined. Keywords: Mammalian cell VH10; Hep G2; V79; Human lymphocyte; DNA unwinding technique; Alkaline elution of DNA; Comet assay; Chromosomal aberration; Single strand breaks of DNA; DNA damage
1. Introduction
)
Corresponding author.
Exposure of cells to mutagens produces chemical changes in DNA which may be either irreversible or
0921-8777r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 8 7 7 7 Ž 9 7 . 0 0 0 0 7 - 4
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repaired by various DNA repair enzymes. Among the survivors, however, some cells show mutations, which were generated by errors that occurred in DNA replication during the recovery period w1–3x. Because replication errors in damaged DNA are likely to be directly involved in inducing mutations, it would be useful to have some rapid biochemical methods for measuring DNA damage and repair to help evaluate the possible mutagenic effects of radiation or chemicals. The most available category of DNA lesions for detection by biochemical methods comprises DNA strand breaks. These result from a number of different types of reactions Žbase and nucleotide excision repair; direct scission of the DNA backbone by chemical or radical attack; scission following the binding of intercalating agents; alkalilabile DNA adducts; endonuclease and topoisomerase action; DNA hydrolase release from lysosomes.. The biochemical methods of DNA unwinding w4,5x and alkaline elution of DNA w6x are among the most convenient ways of detecting DNA strand breaks resulting from a number of different types of reactions. Cytogenetic detection of chromosomal aberrations has been considered a good criterion for induction of mutagenic lesions in DNA of mutagen treated cells w7x. However, since the effects of environmental toxicants are often tissue and cell type specific it was important to develop a technique which can detect DNA damage in individual cells. For the direct visualization of DNA damage in indi¨ vidual cells, Ostling and Johanson w8x developed a microgel electrophoresis technique which depends on the migration of DNA from nucleus to anode, when breaks are present. For validation of the comet assay against other methods we used human cells VH10 and Hep G2, human lymphocytes and hamster cells V79. As a model carcinogen and mutagen we chose the monofunctional alkylating agent N-methyl-N X-nitroN-nitrosoguanidine ŽMNNG.. Monofunctional alkylating agents are primarily not DNA strand-breaking agent w9x, they do not induce direct scission of the DNA backbone. MNNG-induced DNA strand breaks evaluated under alkaline conditions result mainly from endonuclease action through base and partly nucleotide excision repair and from alkali-labile DNA adducts. MNNG can methylate all oxygen and most nitrogen atoms of DNA. Fornace and Little w10x
suggested that MNNG produced a several hundredfold greater number of alkylations than do similar concentrations of other monofunctional alkylating agents. Methylated DNA is poorly repaired by the UVRABC nucleotide excision repair pathway w11x; however, some recognition does occur in vitro w12x and in vivo w13,14x.
2. Materials and methods 2.1. Cell lines Cells VH10, human fibroblasts, derived from foreskin of 10 years old healthy boy, non-transformed were obtained from Dr. A. Kolman, Laboratory of Radiobiology, University of Stockholm, Sweden. Quasidiploid Chinese hamster V79 cells were obtained from Dr. A. Abbondandolo, University of Genoa, Italy. Hep G2 cells, human hepatoma cells were obtained from Dr. A. Collins, The Rowett Research Institute, Aberdeen, Scotland. VH10 cells were cultured in MEM Eagle q 10% foetal calf serum, V79 cells in MEM Eagle mediumq 6% foetal calf serum. Hep G2 cells were cultured in Williams mediumq 10% foetal calf serum. To media was added: penicillin Ž200 Urml., streptomycin Ž100 mgrml. and kanamycin Ž100 mgrml.. Human lymphocytes were incubated in MEM E medium supplemented with 20% of foetal calf serum and phytohaemagglutinin Ž2%., L-glutamine Ž294.1 mgrml., and antibiotics. All cell lines were cultured on glass or plastic Petri dishes or small glass vials in CO 2 incubator, at 378C. 2.2. Chemicals and treatment of cells MNNG s N-methyl-N X-nitro-N-nitrosoguanidine ŽServa diluted 1:1 with H 2 O ŽFig. 1 and Fig. 2., Aldrich ŽFigs. 3–7.. The stock solution of this Nnitrosoamide in DMSO Ž1 mgrml. was kept at y208C and diluted immediately before use in PBS or MEM medium. Ara C Ž1-Ž b-D-arabinofuranosyl.cytosine. and HU Žhydroxyurea. ŽSigma.: Stock solutions of Ara C Ž2 = 10y2 M. and HU Ž0.4 M. were dissolved in tridistilled water and kept at y208C. These solutions
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10y3 M or 2 = 10y2 M. were applied either after or during MNNG treatment. 2.3. Alkaline DNA-unwinding and HTP chromatography
Fig. 1. The level of DNA strand breaks detected by alkaline elution of DNA in MNNG-treated VH10 cells Žleft side. and Hep G2 cells Žright side.. Cells were treated with MNNG either in the presence or in the absence of Ara C Ž2=10y5 M or 2=10y4 M. and HU Ž2=10y3 M or 2=10y2 M.. Control `; Controlqlower conc. of Ara C and HU v; Controlqhigher conc. of Ara C and HU \; MNNG Ž2.5 mgrml. %; MNNG Ž2.5 mgrml.qlower conc. of Ara C and HU I; MNNG Ž2.5 mgrml.qhigher conc. of Ara C and HU B.
were diluted immediately before use in culture medium. Exponentially growing cells VH10 or Hep G2 were treated with MNNG for 30 or 60 min in PBS or 120 min in MEM. DNA-polymerase inhibitors Ara C Ž2 = 10y5 M or 2 = 10y4 M. and hydroxyurea Ž2 =
We used the method described by Ahnstrom ¨ and Erixon w4x. Our modification has already been described w15x. Briefly: Cells were lysed at room temperature in 1 ml of 0.03 M NaOH q 0.15 M NaCl for 30 min in the dark. Then the cells were neutralized by adding of 2 ml 0.02 M NaH 2 PO4 , the molecular weight of the DNA was reduced by ultrasonic treatment and sodium dodecyl sulfate was added to each sample Ž0.09% vrv.. Samples were kept at y208C. Single-strand DNA was separated by HTP chromatography with 2.5 ml of 0.09 M and 2.5 ml of 0.1 M potassium phosphate ŽKP. buffer and double-strand DNA with 2.5 ml of 0.25 M and 2.5 ml of 0.5 M KP buffer ŽpH 6.8. at 608C. The radioactivity of individual samples was measured on a liquid scintillation counter, Beckman LS 1801 and the percentage of single-stranded DNA was calculated in each sample. The proportion of singlestranded DNA was converted to the frequency of DNA breaks using a standard curve derived from alkaline unwinding experiments with cells containing
Fig. 2. The level of DNA strand breaks detected by DNA unwinding in VH10 cells. Left side: VH10 cells treated with MNNG for 30 min in PBS buffer v; 60 min in PBS buffer \; and 120 min in MEMmedium `. Right side: VH10 cells treated with MNNG for 120 min `; treated with MNNG for 120 min and then for 60 min with Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v; treated with MNNG for 30 min and then for 90 min with Ara C and HU \; and treated for 120 min with MNNGq Ara C q HU %.
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known numbers of breaks introduced by X-irradiation. 2.4. Alkaline elution of DNA We adapted the method described by Kohn et al. w6x. Differences from the original method: Ži. proteinase K was substituted by pronase E; Žii. DNA in individual eluates was precipitated by trichloroacetic acid Ž5% TCA vrv., and collected by filtration through nitrocellulose filters SYNPOR 6 Ž0.44 mm., washed, and after drying the radioactivity of membranes measured on a Beckman LS 1801 liquid scintillation counter; Žiii. for all samples, the filter holder and tubing were flushed with 6 ml of 0.4 N NaOH, the content of DNA in 0.4 N NaOH was determined in the same way as in eluting solutions, and the radioactivity measured was added to the radioactivity of DNA retained; Živ. the DNA retained on filters was measured in the following way: filters were carefully taken from filter holders, placed in 5% TCA Ž08C. and kept there for approx. 20 min before being washed, dried, and their radioactivity measured. In preliminary experiments a comparison was made between the original method and our modification and the results were very similar. 2.5. Chromosome aberrations Human peripheral blood lymphocytes were incubated for 48 h before adding MNNG in culture medium. After a further 22 h, colchicine was added at a final concentration of 0.75 Žmgrml for 2 h. V79 cells were grown Žinoculum of 6 = 10 5 cellsrdish, diameter 60 mm. for 24 h, until they reached the exponential stage of growth. Then MNNG was added and cultures were incubated for a further 18 h. Colchicine was added 1 h prior to harvesting. VH10 cells were grown Žinoculum of 9 = 10 5 cellsrdish, diameter 90 mm. for 24 h. Then MNNG was added for 2 h. Then the cells were rinsed twice with PBS and fresh grown medium was added. The cultures were incubated for a further 120 h. Three hours before the end of cultivation colchicine was added. The concentrations of MNNG used were selected on the basis of preliminary cytotoxicity tests. Slides were prepared with standard air drying procedure.
Chromosome aberrations were scored in conventional Giemsa stained preparations. The classification of aberrations was as described by Venitt and Parry w16x and ISCN w7x. The metaphases were analysed for chromatid gaps and breaks. The results were statistically evaluated by the chi-square test. 2.6. Comet assay The procedure of Singh et al. w17x was followed with minor modifications. A base layer of 100 ml of 0.75% NMP agarose in PBS buffer ŽCa2q- and Mg 2q-free. was placed on microscope slides. Instead of fully frosted microscope slides we used plain slides covered by a thin layer of 1% NMP agarose in water and dried at 508C. Human or hamster cells Žwith or without MNNG treatment respectively with post-MNNG-treatment., were suspended in 0.75% LMP agarose and 2 = 10 4 cells in 85 ml spread on base layer on each triplicate of slides. A cover slip was added and the agarose was allowed to solidify. The cover slips were removed and the slides were placed in lysis mixture Ž2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris, pH 10 and 1% Triton X-100. at 48C for 1 h to remove cellular proteins. Slides were then transferred to an electrophoresis box containing an alkaline solution Ž300 mM NaOH, 1 mM Na 2 EDTA.. Slides were kept in this solution for 40 min unwinding time, at 48C. A current of 25 V Ž300 mA. was then applied for 30 min. The slides were removed, neutralised with 3 = 5 min washes with Tris Ž0.4 M, pH 7.5., stained with 20 ml DAPI Ž1 mgrml. and the cover slip returned. DAPI stained nucleotides were examined with a Zeiss Jenalumar fluorescence microscope and one hundred comets on each slide were scored visually as belonging to one of five classes according to tail intensity and given a value of 0, 1, 2, 3 or 4 Žfrom undamaged, 0, to maximally damaged, 4.. Thus, the total score for 100 comets could range from 0 Žall undamaged. to 400 Žall maximally damaged.. This visual scoring was suggested by Collins et al. w18x. Results obtained by this method were confirmed by computerised image analysis ŽKinetic Imaging, Liverpool, UK.. Arbitrary units were converted to the frequency of DNA breaks using a standard curve derived from comet assay experiments with cells containing known numbers of breaks introduced by X-irradiation.
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3. Results and discussion Although most DNA breaks occur as a result of metabolic participation by the cell, determination of single strand breaks in mammalian cells is probably the most commonly used approach for the rapid and sensitive monitoring of genotoxic events which may originate in the environment. In the framework of an EC Copernicus project we evaluated by different techniques the level of ss breaks of DNA in different mammalian cell lines damaged by MNNG as well as the influence of DNA repair inhibitors Ara C and HU w19x on the level of DNA breaks induced by MNNG. In addition to measuring DNA breaks chromosome aberrations were examined as an indicator of damage at the level of nuclear organisation. Fig. 1 represents results obtained by alkaline elution of DNA. The number of breaks in DNA is inversely related to the retention of DNA on the filter. The left side of the figure represents the results obtained with human cells ŽVH10.. We tested the effects of two concentrations of Ara C Ž2 = 10y5 M or 2 = 10y4 M. and HU Ž2 = 10y3 M or 2 = 10y2 M. on the level of ss breaks induced by MNNG. Lower concentrations of inhibitors were as effective as 10-times higher concentrations; therefore in further experiments we used only this lower concentra-
247
tion of inhibitors. A similar experiment was performed with the human hepatoma cell line Hep G2 Žright side of the Fig. 1.. In this case we used only the lower concentrations of inhibitors Ara C and HU, which, however, evidently increased the level of DNA breaks not only in MNNG-treated cells but also in control cells. Alkaline elution of DNA is a sensitive technique for studying strand breaks of DNA; its disadvantage is the time taken to process samples and the limited number of samples that can be accommodated at one time. Different kinds of MNNG treatment of mammalian cells have been described by different authors Žduration of 30, 60, 120, and 180 min or 24 h in PBS, complete medium, or serum-free medium; different MNNG sources.. We have therefore measured the level of strand breaks Ždosereffect. in MNNG-treated VH10 cells Žconcentrations 0–2 mgrml. either in PBS buffer Ž30 and 60 min. or in complete medium Ž120 min.. The results are presented in the left side of Fig. 2. The method used in these experiments was the alkaline unwinding of cellular DNA with separation of unwound single-stranded DNA fragments from the intact double-stranded DNA by elution from hydroxyapatite with phosphate solutions of different ionic strength. The amount of unwound DNA is proportional to the number of strand breaks. As the
Fig. 3. The level of DNA strand breaks detected by DNA unwinding in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side.. Cells treated for 120 min with MNNG `; and cells treated simultaneously with MNNG and Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v.
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level of strand breaks per 10 9 daltons measured by DNA unwinding was in all three cases rather similar, in further experiments we used only 120 min MNNG-treatment of cells in complete MEM. The levels of strand breaks in VH10 cells treated for 120 min with MNNG alone and with different combinations of Ara C, HU and MNNG are shown on the right side of Fig. 2. Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. increased the level of ss breaks of DNA in all three kinds of application, the most effective seemed to be the simultaneous treatment of cells with MNNG and inhibitors. Similar effects were observed in human cell line XP12 and hamster cells V79 Ždata not shown.. When we repeated simultaneous treatment of different cells lines ŽVH10, Hep G2, V79. with higher concentrations of MNNG Ž0–7 mgrml, Aldrich. q DNA repair inhibitors ŽFig. 3., however, we found only a small ŽHep G2. or no ŽVH10, V79. effect of DNA repair inhibitors on the level of DNA breaks in MNNG treated cells. It could be that at higher levels of damage the repair system was saturated, i.e., the polymerase could not keep up with the continuing production of strand breaks and inhibitors didn’t add to the breakage. It is possible that accumulation of strand breaks at higher concentrations of MNNG requires higher concentrations of Ara C and HU. In view of the higher level of DNA breaks in control
cells ŽFig. 1. we deliberately used only the lower concentrations of Ara C and HU. A great advantage of DNA unwinding is the higher number of samples which can be processed in the same experiment. Fig. 4 represents the results which were obtained by SCGE. The level of DNA breakage is indicated by the extent of movement of DNA into the comet tail evaluated by visual scoring. As was shown with the DNA unwinding technique, the influence of AraC and HU was observed only in combination with very low MNNG concentrations. If we compare the results obtained by DNA unwinding technique and by the comet assay, e.g., in human VH10 cells it is evident that the comet assay is very sensitive to low levels of damage; but it has a limitation that it saturates at a fairly low level of damage compared with alkaline unwinding. This is what we would expect, since as soon as all the DNA loops contain breaks, i.e., the tail is full of DNA, there is no possibility of further expansion of DNA into the tail, and the level at which this occurs is determined by the number of loops. Although the comet assay is fairly restricted in the range of damage it can detect, it does as a result show a steep dose-response over that range, which makes it particularly presize as well as sensitive. In addition to methylation of all bases of DNA Žmainly the 6-oxygen of guanine, producing O 6-
Fig. 4. The level of DNA strand breaks detected by SCGE in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side.. Cells treated for 120 min with MNNG `; and cells treated simultaneously with MNNG and Ara C Ž2 = 10y5 M. and HU Ž2 = 10y3 M. v.
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Table 1 Induction of chromosomal aberrations in human peripheral lymphocytes treated with MNNG for 22 h Sample
a
NC SC MNNG 0.08 MNNG 0.8 MNNG 2.0 MNNG 4.0 MNNG 8.0 MNNG 10.0 ) a b
Aberrant metaphases
3 1 4 4 5 4 10 7
)) )
Aberrations per 100 metaphases
b
Chromatid
Isochromatid
Exchange
Total no.
brf
brf
dic
dmin
qr
tr
3 1 1 4 4 3 3 5
– – 1 – – – 2 –
– – – – – – 1 1
– – 2 – 1 1 4 1
– – – – – – – –
– – – – – – – –
3 1 4 4 5 4 10 7
)) c
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.08–10.0 mgrml.. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial.
methyldeoxyguanosine which causes mutations., MNNG gives rise to MNNG and OH radicals w20,21x. Radical formation in biological systems, especially of the active oxygen species, is of considerable interest as a cause of mutation, including chromosomal damage. Osada et al. w22,23x reported that the light emission induced by S9-activated BwaxP is related to active oxygen species. Kimura et al. w24x investigated the possible role of radical formation by
MNNG by means of light emission measurements and chromosomal aberration testing. The production of chromosomal aberrations in Chinese hamster fibroblasts was correlated with the light emission intensity. In an Oy 2 generating system, chromatid type aberrations are induced, including chromatid gaps and breaks w25,26x. We evaluated the effect the level of chromosomal aberrations in treated peripheral human lymphocytes ŽPHL; Table 1., hamster cells V79
Table 2 Induction of chromosomal aberrations in hamster cells V79 treated with MNNG for 18 h Sample
a
NC SC MNNG 0.05 MNNG 0.2 MNNG 0.4 MNNG 0.6 MNNG 0.8 MNNG 2.0 MNNG 4.0 MNNG 6.0 MNNG 8.0 ) a b c
Aberrant metaphases
2 2 4 8 6 8 7 12 12 12 14
)
)
)) )) )) )))
b
Aberrations per 50 metaphases
c
Chromatid
Isochromatid
Exchange
brf
brf
dic
dmin
qr
tr
r
Total no.
2 – 1 2 3 5 3 11 3 12 5
– – 1 3 – 1 3 1 6 – 6
– – – – 2 1 – – – – –
– – 3 2 – 1 4 – 4 1 4
– 2 – 1 2 – – 1 1 2 –
– – – – 1 1 – 1 – – –
– – – – – – – – – – –
2 2 5 8 8 9 10 14 14 15 15
) ) ) ) ))) ))) ))) )))
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.05–8.0 mgrml.. 50 metaphases were scored in each sample. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial; r, ring chromosome.
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Table 3 Induction of chromosomal aberrations in human cells VH10 treated with MNNG for 2 h Sample
a
NC SC MNNG 0.1 MNNG 0.5 MNNG 1.0 MNNG 5.0 MNNG 10.0 MNNG 20.0 MNNG 40.0 ) a b c
Total no. of metaphases scored
Aberrant metaphases
26 25 47 7 20 9 28 16 NE
1 2 2 – 3 4 10 7
) ) ))
Aberrations
b
Chromatid
Isochromatid
Exchange
Total no.
brf
brf
dic
dmin
qr
tr
r
1 1 1 – 3 4 4 29
– – 1 – – – 2 6
– – – – 2 1 5 3
– 1 – – 13 3 1 14
– – – – – – 20 14
– – – – – 1 7 11
– – – – 1 1 – 3
1 2 2 – 19 10 39 80
))) ))) ))) )))
c
Significantly different from the solvent control Ž p - 0.05.. X NC, negative control; SC, solvent control Ždimethylsulfoxide.; MNNG, N-methyl-N -nitro-N-nitrosoguanidine Ž0.1–40.0 mgrml.. Abbreviations: brf, break andror fragment; dic, dicentric; dmin, double minute; qr, quadriradial; tr, triradial; r, ring chromosome. NE, not evaluable.
ŽTable 2. and VH10 cells ŽTable 3.. Obtained results confirmed that MNNG-induced chromosomal damage in all cell types studied. It is evident that MNNG damages not only molecular but also nuclear organisation of mammalian cells. Fig. 5 shows the kinetics of DNA rejoining in three cell lines, assayed by DNA unwinding. The highest decrease of ss breaks of DNA with time of incubation Žindicating repair. was observed in human
VH10 cells. DNA rejoining in Hep G2 and V79 cells was less effective. The kinetics of DNA rejoining in MNNG-treated VH10 cells and Hep G2 cells measured by the comet assay are shown in Fig. 6. The last figure ŽFig. 7. represents DNA rejoining measured by alkaline elution of DNA in MNNG-treated V79 cells. On the basis of the obtained results, we suggest that the comet assay is able to recognize sites of incomplete repair of DNA breaks which are
Fig. 5. The kinetics of DNA rejoining in VH10 cells Žleft side., Hep G2 cells Žmiddle. and V79 cells Žright side. assayed by DNA unwinding. The level of DNA strand breaks was detected immediately after MNNG-treatment `; 2 h after treatment v; and 4 h after treatment \.
D. SlamenoÕa ˇ ´ et al.r Mutation Research 383 (1997) 243–252
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Fig. 6. The kinetics of DNA rejoining in VH10 cells Žleft side. and Hep G2 cells Žright side. assayed by SCGE. The level of DNA strand breaks was detected immediately after MNNG-treatment `; 4 h after treatment v; and 24 h after treatment \.
to a lesser degree recognized by alkaline elution of DNA or by DNA unwinding. Though the O 6 position of guanine and, to a lesser extent, the O 4 position of thymidine which can be repaired by direct dealkylation by methyltransferases Žwithout creation of strand breaks. are viewed as the sites of major importance with respect to alkylation mutagenesis, determination of strand
breaks and alkali-labile lesions of DNA not only represents a very good indicator of mutagenic potential of the studied compound but can be used as well for determination of DNA repair during the postMNNG-treatment. The ability of Ara C and HU to increase the level of ss DNA breaks induced by low concentrations of MNNG ŽFig. 1 and Fig. 2. as well as investigation of ss breaks of DNA during 2–24 h
Fig. 7. The kinetics of DNA rejoining in V79 cells assayed by alkaline elution of DNA. The level of DNA strand breaks was detected immediately after MNNG-treatment Žleft side., 4 h after treatment Žmiddle. and 24 h after treatment Žright side.. Control `; 1 mg of MNNGrml \; 2.5 mg of MNNGrml I; 5 mg of MNNGrml ^; 10 mg of MNNGrml e.
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