- Email: [email protected]
DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay
Mutation Research 377 Ž1997. 279–286
DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay Rainer Helbig 1, Gunter Speit ¨
)
UniÕersitat ¨ Ulm, Abteilung Medizinische Genetik, D-89069 Ulm, Germany Received 28 January 1997; revised 12 March 1997; accepted 12 March 1997
Abstract Using the alkaline comet assay Žsingle cell gel electrophoresis., we studied the induction and persistence of DNA damage induced by methyl methanesulfonate ŽMMS. and neocarzinostatin ŽNCS. in the repair-deficient Chinese hamster cell lines V-E5 and XR-V15B. Effects in the comet assay were analyzed directly after treatment as well as after a postincubation period in mutagen-free medium to gain insight into the DNA repair capacities of the mutant cell lines in relation to different primary DNA lesions. Both mutagens caused a concentration-related increase in DNA strand breakage in both mutant cell lines and in the normal parental cell lines. Repair of MMS-induced DNA damage during postincubation was similar in normal and mutant cell lines, while diminished repair was seen after NCS treatment in XR-V15B cells. Our data show that XR-V15B cells only repaired about 30% of NCS-induced DNA damage within 1 h, while the parental V79 cell line repaired about 70%. Since this cell line is defective in the repair of DNA double-strand breaks ŽDSB., the results indicate that NCS-induced DSB significantly contribute to the genotoxic effects seen in the comet assay. However, compared to previously studied induction of gene mutations and chromosome aberrations, detection of NCS-induced DNA effects with the comet assay was less sensitive and increased DNA migration only occurred under strong cytotoxic conditions. Keywords: Single cell gel ŽSCG. assay; DNA strand break; Genotoxicity; Cytotoxicity; Neocarzinostatin; Methyl methanesulfonate
1. Introduction Various mutant rodent cell lines with hypersensitivity to DNA damage resulting from a genetic defect in cellular response to the damage have been characterized Žfor review see w1,2x.. These mutant cell lines deficient in specific pathways of DNA repair have contributed to a better understanding of the relationship between induced DNA lesions and ) Corresponding author. Tel.: q49 Ž731. 502-3429; Fax: q49 Ž731. 502-3438; E-mail: [email protected] 1 Present address: CCR Cytotest Cell Research GmbH and Co. KG, D-64380 Robdorf, Germany.
observed biological effects. The comet assay Žsingle-cell gel electrophoresis. is a genotoxicity test which has rapidly developed during the past few years. It is a sensitive electrophoretic technique for detecting the presence of DNA strand breaks and alkali-labile damage in individual cells. It has already been used in both in vitro and in vivo studies to investigate DNA damage and repair induced by various agents in a variety of mammalian cells Žfor review see w3,4x.. The comet assay was now used to further evaluate the induction and repair of DNA damage in the repair-deficient Chinese hamster cell lines XR-V15B and V-E5. XR-V15B belongs to a group of X-ray-sensitive
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 0 8 7 - 0
280
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
mutants which were found to be defective in the repair of DNA double-strand breaks ŽDSB. w5x. Biochemical analysis of XR-V15B cells revealed a decreased ability to rejoin X-ray-induced DSB as measured by neutral elution w6x. This deficiency is also reflected by an increased sensitivity to radiomimetic mutagens such as bleomycin and neocarzinostatin w6,7x and an impaired VŽD.J recombination w8–11x. XR-V15B cells also showed a higher rate of spontaneous hprt mutations with a higher incidence of deletions w7x. Molecular analysis revealed that the genetic defect of XR-V15B results in the lack of the 80-kDA subunit of the Ku protein w12,13x. The Ku protein binds to free double-stranded DNA ends, thus suggesting an involvement in DNA repair w11x. The mutant cell line V-E5 was also isolated on the basis of its hypersensitivity to X-rays w14x. It represents one of the first rodent cell mutants which shows unique phenotypic characteristics strongly resembling cells derived from patients suffering from the genomic instability syndrome ataxia telangiectasia ŽAT. w14,15x. V-E5 cells are characterized by radioresistant DNA synthesis, chromosomal instability and cross-sensitivities to different radiomimetic agents w15–17x. However, they exhibit normal DNA strand-break repair and VŽD.J recombination capacity w8,16x. In contrast to XR-V15B, no definite molecular cause of repair deficiency in V-E5 was detected so far. We investigated the genotoxic and cytotoxic effects of two chemical mutagens with different DNA-damaging properties. Neocarzinostatin ŽNCS. is a radiomimetic mutagen which acts via a free diradical mechanism, thereby causing DSB and SSB w18,19x. Methyl methanesulfonate ŽMMS. is a monofunctional alkylating agent which acts directly on oxygen and nitrogen atoms in DNA bases and on oxygen moieties of the phosphate backbone. Thereby a broad spectrum of DNA lesions is formed, consisting of alkylated bases, phophotriesters and abasic sites which can lead to SSB w20x. Effects in the comet assay were analyzed according to two different protocols. DNA damage was assayed directly after treatment as well as after a postincubation period in mutagen-free medium. The investigation of the induction and repair of various types of DNA lesions in repair-deficient cells should provide further information about the significance of different
DNA lesions for genotoxic effects in the comet assay and their relationship to mutagenicity and cytotoxicity.
2. Materials and methods 2.1. Cell cultures V79 Chinese hamster cells ŽV79-B and V79-LE. and the derived mutants XR-V15B and V-E5 were kindly provided by Dr. M. Zdzienicka, Leiden w6,14x. Cells were maintained in minimal essential medium ŽMEM. with Earle’s salts, supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics. Cells were cultivated in a humidified incubator at 378C with 5% CO 2 at pH 7.2 and harvested with 0.15% trypsin and 0.08% EDTA. Under these conditions, the population doubling time of the 4 cell lines was 12–15 h and the absolute plating efficiency was between 70 and 80%. 2.2. Chemicals MMS and fluorescein diacetate ŽFDA. were purchased from Sigma ŽMunich, Germany.. NCS was a gift from Kayaku Co., Ltd., Japan. NCS and MMS were dissolved immediately before use in Hank’s balanced salt solution. Agarose was supplied by Roth ŽKarlsruhe, Germany., low melting agarose ŽLMA, Sea plaque. was purchased from Biozym ŽHameln, Germany.. Cell culture media were obtained from Biochrom ŽBerlin, Germany.. 2.3. Fluorochrome-mediated Õiability assay To investigate whether DNA effects detected in the comet assay are accompanied by cytotoxic effects, cell viability was determined using the FDArethidium bromide assay according to Strauss w21x. A freshly prepared solution consisting of FDA Ž30 mgrml. and ethidium bromide Ž8 mgrml. was used. Twenty-five microliters of the cell suspension was mixed with 25 ml of the staining solution, spread on a microscope slide and covered with a coverslip. The viability was checked immediately after the treatment. Viable cells appeared green-fluorescent, whereas orange-stained nuclei indicated dead cells. At least 200 cells were counted per data point.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
2.4. Plating efficiency assay Relative plating efficiency as a measure for longterm survivability has been determined previously in the course of HPRT mutation tests w7,17x. Briefly, after mutagen treatment, 250 cells were plated into each of 4 replicate Petri dishes Ždiameter, 60 mm. and cultivated for 7 days. Colonies were fixed, stained, counted and related to the corresponding control. 2.5. Comet assay For the comet assay, 2 = 10 5 cells were seeded into multiwells Ž6 wells, of 36 mm diameter., cultivated overnight and then treated with MMS or NCS for 1 h. At the end of the treatment, cells were washed with ice-cold PBS and trypsinized with 50 ml trypsin Ž0.15%.. After 2 min, the cells were gently resuspended in 50 ml complete medium and 15 ml of the cell suspension were immediately used for the test. For postincubation experiments, cells were rinsed twice with Hank’s salt solution and further cultivated in complete medium for 1 h. The comet assay was performed as described by Singh et al. w22x with minor modifications w23x. Three hundred microliters of agarose Ž0.75%. diluted in Ca- and Mg-free PBS buffer were added to fully frosted microscope slides ŽLabcraft, Houston, TX, USA., immediately covered with coverslips and kept in a refrigerator to solidify. Coverslips were removed and 15 ml of the single-cell suspension mixed with 85 ml of LMA Ž0.5%. were added to the slides. The slides were covered again with a coverslip and placed in a refrigerator for another 5 min to allow solidification of the LMA and were then covered with a top layer of 100 ml LMA Ž0.5%.. Afterwards, slides were carefully immersed in lysing solution Ž100 mM Na-EDTA, 10 mM Tris, 2.5 M NaCl, 1% Triton X-100 and 10% DMSO, 1% Na-lauroyl-sarcosinate, pH 10.. The slides were kept at 48C for at least 1 h to lyse the cells. After lysis, the slides were placed in a horizontal gel electrophoresis chamber with alkaline buffer Ž1 mM Na-EDTA, 300 mM NaOH, pH 13.. The cells were exposed to alkali for 40 min to permit DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed for 20 min at 25 V Ž0.86 Vrcm. and 300 mA. All of these steps were conducted under dim light to prevent the occur-
281
rence of additional DNA damage. After electrophoresis, the slides were rinsed with neutralization buffer Ž0.4 M Tris, pH 7.5.. Finally, they were stained with 70 ml ethidium bromide Ž20 mgrml. and covered with a coverslip. Images of 50 randomly selected cells Ž25 cells from each of two replicate slides. were analyzed using a fluorescence microscope Ž200 = magnification. with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Measurements were made by image analysis ŽColourmorph Comet Assay 5.0, Perceptive Instruments, Haverhill, Suffolk, UK., determining the mean tail moment Žpercentage of DNA in the tail times tail length. of the 50 cells. All experiments were repeated twice in independent tests. Thus, the data in the results section represent the mean " SEM of 150 cells per dose level. Differences between the control and the other values were tested for significance Ž p - 0.01. using Student’s t-test. 3. Results Using the comet assay, the initial rate of DNA strand breaks induced by NCS and their persistence
Fig. 1. Effects of NCS on DNA migration Žtail moment. in XR-V15B and V79-B directly after treatment Žv, V79-B; ', XR-V15B. and after mutagen-free postincubation Ž`, V79-B; n, XR-V15B.. Mean"SEM of 150 cells.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
282
Table 1 Cytotoxicity induced by neocarzinostatin ŽNCS. and amount of persisting DNA damage in V79 cell lines Concentration
XR-V15B
Viability ŽFDA. 0.25 mgrml 97% 0.5 mgrml 96% Relative survival ŽPE. a 0.25 mgrml -1% 0.5 mgrml -1% Persisting DNA damage b 0.25 mgrml 68% 0.5 mgrml 70%
V79-B
V-E5
V79-LE
98% 97%
87% 89%
94% 94%
-10% -10%
-1% -1%
-10% -10%
33% 24%
37% 37%
51% 37%
a
Data taken from HPRT mutation experiments w7,17x. Relative tail moment value after 1 h of postincubation in mutagen-free medium. b
after 1 h of postincubation in mutagen-free medium was analyzed in XR-V15B cells and the parental cell line V79-B. The results are shown in Fig. 1. In both
cell lines, NCS caused a concentration-dependent increase of the tail moment. The initial DNA damage directly after treatment was significantly higher in XR-V15B than in the parental V79-B cell line. The results for the two cell lines also differed with respect to the persistence of NCS-induced DNA lesions. While in V79-B only 24–33% of the DNA lesions remained after an 1-h postincubation period, in XR-V15B about 70% of the strand breaks persisted ŽTable 1.. Fig. 2 illustrates the distribution of DNA damage among the cells in 4 experiments Ža–d.. It can be seen that after NCS treatment, the majority of cells exhibited increased DNA migration in both cell lines ŽFig. 2a,c.. Furthermore, after postincubation of XR-V15B cells, DNA damage is still equally distributed among the cells, indicating persistence of lesions in the whole cell population ŽFig. 2d.. Viability as measured with the fluorochrome-mediated viability assay ŽFDA. and the
Fig. 2. Effects of NCS on the distribution of DNA damage Žtail moment. among single cells in V79-B Ža,b. and XR-V15B Žc,d. directly after treatment Ža,c. and after mutagen-free postincubation Žb,d..
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
283
Table 2 Cytotoxicity induced by methyl methanesulfonate ŽMMS. and amount of persisting DNA damage in V79 cell lines Concentration
XR-V15B
Viability ŽFDA. 0.5=10 -4 M 98% 1=10 -4 M 98% Relative survival ŽPE. a 0.5=10 -4 M )90% 1=10 -4 M 89% Persisting DNA damage b 0.5=10 -4 M 33% 1=10 -4 M 30%
V79-B
V-E5
V79-LE
98% 99%
94% 97%
95% 91%
)85% 83%
)80% 76%
)85% 82%
27% 33%
36% 26%
30% 24%
a
Data taken from HPRT mutation experiments w7,17x. Relative tail moment value after 1 h of postincubation in mutagen-free medium.
b
Fig. 3. Effects of MMS on DNA migration Žtail moment. in XR-V15B and V79-B directly after treatment Žv, V79-B; ', XR-V15B. and after mutagen-free postincubation Ž`, V79-B; n, XR-V15B.. Mean"SEM of 150 cells.
relative survival Žplating efficiency, PE. are summarized in Table 1. To induce significant effects in the comet assay, NCS concentrations had to be applied which resulted in a relative plating efficiency of less than 10%. In contrast, the viability, even at the highest NCS concentration Ž5 mgrml., was above 95%. Viability did not decrease during postincubation Ždata not shown.. The comet assay revealed a clear and concentration-dependent increase in DNA damage in XR-V15B and V79-B at MMS concentrations which only caused little cytotoxicity as analyzed with both methods of cytotoxicity testing ŽFDA and PE.. The initial damage as well as the persistence of DNA effects were nearly the same in XR-V15B and V79-B ŽFig. 3.. Survival ŽPE. was above 83% and viability determined with the FDA assay was above 89% ŽTable 2.. In both cell lines, about 30% of the initial DNA damage persisted after 1 h postincubation ŽTable 2.. In the cell line V-E5 and its parental cell line V79-LE, NCS and MMS treatment led to a concentration-related increase in DNA migration ŽFigs. 4 and 5.. In both cell lines, about 30–40% of the induced strand breaks persisted after a 1-h postincu-
bation period. However, NCS induced DNA damage could be seen in the comet assay only at concentrations which resulted in a relative plating efficiency of less than 10%, whereas viability ŽFDA. was above 95% ŽTable 2.. Viability did not decrease during postincubation Ždata not shown.. In the case of MMS treatment, it was possible to detect DNA damage
Fig. 4. Effects of NCS on DNA migration Žtail moment. in V-E5 and V79-LE directly after treatment ŽB, V79-LE; l, V-E5. and after mutagen-free postincubation ŽI, V79-LE; e, V-E5.. Mean "SEM of 150 cells.
284
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
Fig. 5. Effects of MMS on DNA migration Žtail moment. in V-E5 and V79-LE directly after treatment ŽB, V79-LE; l, V-E5. and after mutagen-free postincubation ŽI, V79-LE; e, V-E5.. Mean "SEM of 150 cells.
with the comet assay at concentrations which only resulted in a weak cytotoxicity in the FDA assay as well as in a high PE ŽTable 2..
4. Discussion The comet assay is a genotoxicity test, which detects DNA strand breaks and alkali-labile sites in individual cells. It has already been used in both in vitro and in vivo studies to investigate DNA damage and repair induced by various agents in a variety of mammalian cells w3,4,23,24x. We now studied the induction and persistence of DNA damage by MMS and NCS in normal and repair deficient V79 Chinese hamster cells. With respect to MMS-induced effects, no fundamental difference was observed between all cell lines used. Induction of DNA effects was found in the same range of MMS concentrations. Genotoxic effects of MMS were detected with the comet assay at significantly lower concentrations compared to the induction of gene- and chromosome mutations in the same cell lines w7,17x. Persistence of DNA damage
after postincubation was similar in normal and mutant cell lines, indicating similar repair capacities for MMS-induced DNA lesions that cause DNA migration in the comet assay. NCS also induced DNA migration in the 4 cell lines. However, a significantly higher tail moment was found in XR-V15B than in the parental V79-B cells after treatment with NCS. Furthermore, persistence of NCS-induced DNA damage was much higher in XR-V15B. These results can be explained by the impaired DSB repair in this cell line. Reduced DSB repair capacity in XR-V15B already leads to an accumulation of DNA strand breaks during the mutagen treatment period. Consequently, higher tail moment values are observed in XR-V15B cells directly at the end of the treatment. During the postincubation period, less DSB are repaired, leading to enhanced lesion persistence compared with the repairproficient parental cells. It can be concluded from our data that XR-V15B cells only repaired about 30% of the induced DNA damage during the postincubation period, while in V79-B about 70% of the strand breaks were repaired. Deficient DSB rejoining has also been reported for the human B-lymphoblast line TK6 and this deficiency was confirmed using the neutral comet assay w25x. In TK6 cells, 38% of DSB rejoining was found 60 min after exposure to a high dose of ionizing radiation Ž50 Gy. in comparison to approximately 80% in repair-proficient cells. Recently, reduced DSB rejoining after ionizing radiation has also been shown with the neutral comet assay in the radiosensitive CHO mutant xrs-6, which belongs to the same complementation group as XRV15B w26x. Taken together, these results point to a significant contribution of DSB to the DNA effects seen in both versions of the comet assay under the experimental conditions used. AT-like V-E5 cells, which show hypersensitivity to radiation and NCS w7,14x, showed similar levels of strand break induction and rejoining as normal V79 cells in our experiments. These results indicate that the comet assay can only determine the amount of break induction and rejoining, but cannot measure the fidelity of repair of those breaks. Our findings with V-E5 cells are in accordance with earlier results for AT cells which, in spite of their significant radiosensitivity, showed unaltered levels of strand break induction and rejoining w27x.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
Our results also show that the comet assay seems to be rather insensitive for NCS-induced DNA lesions compared to other genotoxicity tests. NCS-induced gene mutations and chromosome aberrations at lower concentrations in the same cell lines w7,17x. A genotoxic effect of NCS in the comet assay was only found under extreme cytotoxic conditions. Long-term cell survival Žplating efficiency. was reduced to less than 10% in the parental cells and to nearly zero in the repair-deficient cells. Interestingly, cell viability at the end of the treatment as measured with the FDA assay was only slightly reduced. However, the viability assay only indicates the presence of dead cells with disintegrated membranes at the end of the treatment. Determination of survivability is more relevant with respect to the biological significance of test results since genotoxicity is more important if it occurs in cells capable of surviving the damage. The comparatively low sensitivity of the comet assay for the detection of NCS-induced damage might be explained by a high portion of DSB in the spectrum of induced DNA lesions. It has been estimated that overall about 20% of NCS-induced lesions are bistranded, a quarter of these being direct DSB w19,28x. DSB are the most important DNA lesion with respect to cytotoxicity w18,29x and it has been suggested that a single unrepaired DSB in the yeast genome can represent a lethal DNA lesion w30x. DSB are also directly involved in the formation of chromosome aberrations and deletion mutations w7,17,31x. Possibly, strand breaks induced at lower NCS-concentrations are rapidly repaired, but misrepair of some DSB can lead to chromosome aberrations and deletion mutations. Under these conditions, the amount of breaks obviously is not high enough to cause DNA migration in the comet assay. The present results as well as previously published data w25,26,32x indicate that the investigation of DSB-related effects with the comet assay requires extreme treatment conditions. Remarkably, the neutral version of the comet assay, which is supposed to trace predominantly DSB, was also shown to detect radiation-induced DNA damage in Chinese hamster cells at extremely high doses ranging from 10 to 100 Gy w26,32x. Such doses were shown to induce cytotoxicity resulting in survival rates of less than 2% at 10 Gy w6x. Taken together, the comet assay seems to be a
285
useful tool for studying the induction and persistence of DNA strand breaks. DSB obviously lead to DNA migration in the alkaline as well as in the neutral version of the comet assay. However, the comet assay does not seem to be a very sensitive genotoxicity test for the detection of genotoxic agents producing DSB as the predominant DNA lesion. Since DSB are important lesions with respect to cytotoxicity and mutagenicity, the importance of the amount of damage produced by a drug and detected by the comet assay has to be weighed in relation to the nature of the damage being detected.
Acknowledgements We wish to thank Dr. Margaret Zdzienicka for the V79 cell lines, and we are especially grateful to Dr. Ulla Plappert and Dr. Andreas Hartmann for their valuable contributions and helpful discussions. This study was financially supported by the Deutsche Forschungsgemeinschaft.
References w1x A.R. Collins, Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristics, Mutation Res. 239 Ž1993. 99–118. w2x M.Z. Zdzienicka, Mammalian mutants defective in the response to ionizing radiation-induced DNA damage, Mutation Res. 336 Ž1995. 203–213. w3x V.J. McKelvey-Martin, M.H.L. Green, P. Schmezer, B.L. Pool-Zobel, M.P. De Meo, A. Collins, The single cell gel electrophoresis assay Žcomet assay.: a European review, Mutation Res. 288 Ž1993. 7–63. w4x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutation Res. 339 Ž1995. 37–59. w5x P.A. Jeggo, Studies on mammalian mutants defective in rejoining double-strand breaks in DNA, Mutation Res. 239 Ž1990. 1–16. w6x M.Z. Zdzienicka, Q. Tran, G.P. van der Schans, J.W.I.M. Simons, Characterization of an X-ray-hypersensitive mutant of Chinese hamster cells, Mutation Res. 194 Ž1988. 239–249. w7x R. Helbig, M.Z. Zdzienicka, G. Speit, The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XRV15B, Radiation Res. 143 Ž1995. 151–157. w8x F. Pergola, M.Z. Zdzienicka, M.R. Lieber, VŽD.J recombina-
286
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286 tion in mammalian cell mutants defective in DNA double strand break repair, Mol. Cell. Biol. 13 Ž1993. 3464–3471. G.E. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P.A. Jeggo, F.W. Alt, Impairment of VŽD.J recombination in doublestrand break repair mutants, Science 260 Ž1993. 207–210. G.E. Taccioli, T.M. Gottlieb, T. Blunt, A. Priestley, J. Demengeot, R. Mizuta, A.R. Lehman, F.W. Alt, S. Jackson, P.A. Jeggo, Ku80: product of the XRCC5 gene and its role in DNA repair and VŽD.J recombination, Science 265 Ž1994. 1442–1445. V. Smider, W.K. Rathmell, M.R. Lieber, G. Chu, Restoration of X-ray resistance and VŽD.J recombination in mutant cells by Ku cDNA, Science 266 Ž1994. 288–291. M. Hafezparast, G.P. Kaur, M.Z. Zdzienicka, R.S. Athwal, A.R. Lehmann, P.A. Jeggo, Subchromosomal localization of a gene ŽXRCC5. involved in double strand break repair to the region 2q34-36, Somat. Cell. Mol. Genet. 19 Ž1993. 413–421. D.J. Chen, B.L. Marrone, T. Nguyen, M. Stackhouse, Y. Zhao, M.J. Siciliano, Regional assignment of a human DNA repair gene ŽXRCC5. to 2q35 by X-ray hybrid mapping, Genomics 21 Ž1994. 423–427. M.Z. Zdzienicka, N.G.J. Jaspers, G.P. van der Schans, A.T. Natarajan, J.W.I.M. Simons, Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair, Cancer Res. 49 Ž1989. 1481–1485. W. Jongmans, G.W.C.T. Verhaegh, K. Sankaranarayanan, P.H.M. Lohman, M.Z. Zdzienicka, Cellular characteristics of Chinese hamster cell mutants resembling ataxia-telangiectasia cells, Mutation Res. 294 Ž1993. 207–214. Zdzienicka, M.Z., J.W.I.M. Simons, P.H.M. Lohman, Chinese hamster cell lines defective in DNA repair, in: M.W. Lambert and J. Laval ŽEds.., DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells, Plenum, New York, 1985, pp. 535–545. R. Helbig, E. Gerland, M.Z. Zdzienicka, G. Speit, The pattern of mutations induced by neocarzinostatin and methyl methanesulfonate in the ataxia telangiectasia-like Chinese hamster cell line V-E5, Mutation Res. 336 Ž1995. 307–316. I.H. Goldberg, Mechanism of neocarzinostatin action: role of DNA microstructure in determination of chemistry of bistranded oxidative damage, Acc. Chem. Res. 24 Ž1991. 191– 198. L.F. Povirk, DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes, Mutation Res. 355 Ž1996. 71–89. R. Mirzayans, M. Liuzzi, M.C. Paterson, Methylmethanesulfonate-induced DNA damage and its repair in cultured
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x w32x
human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum Žgroup A. cells, Carcinogenesis 9 Ž1988. 2257–2263. G.H.S. Strauss, Non-random cell killing in cryopreservation: implications for performance of the battery of leukocyte tests ŽBLT. I. Toxic and immunotoxic effects, Mutation Res. 252 Ž1991. 1–15. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantification of low levels of DNA damage in individual cells, Exp. Cell Res. 175 Ž1988. 184– 191. G. Speit, A. Hartmann, The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test Žcomet assay., Mutagenesis 10 Ž1995. 555–559. A. Hartmann, G. Speit, Genotoxic effects of chemicals in the single cell gel ŽSCG. test with human blood cells in relation to the induction of sister-chromatid exchanges ŽSCE., Mutation Res. 346 Ž1995. 49–56. H.H. Evans, M. Ricanati, M.-F. Horng, Q. Jiang, J. Mencl, P. Olive, DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines, Radiation Res. 134 Ž1993. 307–315. G.M. Ross, J.J. Eady, N.P. Mithal, C. Bush, G.G. Steel, P.A. Jeggo, T.J. McMillan, DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene, Cancer Res. 55 Ž1995. 1235–1238. A.J. Ward, P.L. Olive, A.H. Burr, M.P. Rosin, Response of fibroblast cultures from ataxia-telangiectasia patients to reactive oxygen species generated during inflammatory reactions, Environ. Mol. Mutagen. 24 Ž1994. 103–111. L.F. Povirk, C.W. Houlgrave, Effect of apurinicrapyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands, Biochemistry 27 Ž1988. 3850–3857. A. Giaccia, R. Weinstein, J. Hu, T.D. Stamato, Cell cycle-dependent repair of double-strand DNA breaks in a g-ray-sensitive Chinese hamster cell, Somat. Cell. Mol. Genet. 11 Ž1985. 485–491. D. Frankenberg, M. Frankenberg-Schwager, D. Bloecher, R. Harbich, Evidence for DNA double-strand breaks as the critical lesions in yeast cells irradiated with sparsely or densely ionising radiation under oxic or anoxic conditions, Radiation Res. 88 Ž1981. 524–532. J.W. Phillips, W.F. Morgan, DNA double-strand breaks in mutagenesis, Environ. Mol. Mutagen. 22 Ž1993. 214–217. P.L. Olive, D. Wlodek, R.E. Durand, J.P. Banath, Factors ` influencing DNA migration from individual cells subjected to gel electrophoresis, Exp. Cell. Res. 198 Ž1992. 259–267.
DNA effects in repair-deficient V79 Chinese hamster cells studied with the comet assay Rainer Helbig 1, Gunter Speit ¨
)
UniÕersitat ¨ Ulm, Abteilung Medizinische Genetik, D-89069 Ulm, Germany Received 28 January 1997; revised 12 March 1997; accepted 12 March 1997
Abstract Using the alkaline comet assay Žsingle cell gel electrophoresis., we studied the induction and persistence of DNA damage induced by methyl methanesulfonate ŽMMS. and neocarzinostatin ŽNCS. in the repair-deficient Chinese hamster cell lines V-E5 and XR-V15B. Effects in the comet assay were analyzed directly after treatment as well as after a postincubation period in mutagen-free medium to gain insight into the DNA repair capacities of the mutant cell lines in relation to different primary DNA lesions. Both mutagens caused a concentration-related increase in DNA strand breakage in both mutant cell lines and in the normal parental cell lines. Repair of MMS-induced DNA damage during postincubation was similar in normal and mutant cell lines, while diminished repair was seen after NCS treatment in XR-V15B cells. Our data show that XR-V15B cells only repaired about 30% of NCS-induced DNA damage within 1 h, while the parental V79 cell line repaired about 70%. Since this cell line is defective in the repair of DNA double-strand breaks ŽDSB., the results indicate that NCS-induced DSB significantly contribute to the genotoxic effects seen in the comet assay. However, compared to previously studied induction of gene mutations and chromosome aberrations, detection of NCS-induced DNA effects with the comet assay was less sensitive and increased DNA migration only occurred under strong cytotoxic conditions. Keywords: Single cell gel ŽSCG. assay; DNA strand break; Genotoxicity; Cytotoxicity; Neocarzinostatin; Methyl methanesulfonate
1. Introduction Various mutant rodent cell lines with hypersensitivity to DNA damage resulting from a genetic defect in cellular response to the damage have been characterized Žfor review see w1,2x.. These mutant cell lines deficient in specific pathways of DNA repair have contributed to a better understanding of the relationship between induced DNA lesions and ) Corresponding author. Tel.: q49 Ž731. 502-3429; Fax: q49 Ž731. 502-3438; E-mail: [email protected] 1 Present address: CCR Cytotest Cell Research GmbH and Co. KG, D-64380 Robdorf, Germany.
observed biological effects. The comet assay Žsingle-cell gel electrophoresis. is a genotoxicity test which has rapidly developed during the past few years. It is a sensitive electrophoretic technique for detecting the presence of DNA strand breaks and alkali-labile damage in individual cells. It has already been used in both in vitro and in vivo studies to investigate DNA damage and repair induced by various agents in a variety of mammalian cells Žfor review see w3,4x.. The comet assay was now used to further evaluate the induction and repair of DNA damage in the repair-deficient Chinese hamster cell lines XR-V15B and V-E5. XR-V15B belongs to a group of X-ray-sensitive
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 0 8 7 - 0
280
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
mutants which were found to be defective in the repair of DNA double-strand breaks ŽDSB. w5x. Biochemical analysis of XR-V15B cells revealed a decreased ability to rejoin X-ray-induced DSB as measured by neutral elution w6x. This deficiency is also reflected by an increased sensitivity to radiomimetic mutagens such as bleomycin and neocarzinostatin w6,7x and an impaired VŽD.J recombination w8–11x. XR-V15B cells also showed a higher rate of spontaneous hprt mutations with a higher incidence of deletions w7x. Molecular analysis revealed that the genetic defect of XR-V15B results in the lack of the 80-kDA subunit of the Ku protein w12,13x. The Ku protein binds to free double-stranded DNA ends, thus suggesting an involvement in DNA repair w11x. The mutant cell line V-E5 was also isolated on the basis of its hypersensitivity to X-rays w14x. It represents one of the first rodent cell mutants which shows unique phenotypic characteristics strongly resembling cells derived from patients suffering from the genomic instability syndrome ataxia telangiectasia ŽAT. w14,15x. V-E5 cells are characterized by radioresistant DNA synthesis, chromosomal instability and cross-sensitivities to different radiomimetic agents w15–17x. However, they exhibit normal DNA strand-break repair and VŽD.J recombination capacity w8,16x. In contrast to XR-V15B, no definite molecular cause of repair deficiency in V-E5 was detected so far. We investigated the genotoxic and cytotoxic effects of two chemical mutagens with different DNA-damaging properties. Neocarzinostatin ŽNCS. is a radiomimetic mutagen which acts via a free diradical mechanism, thereby causing DSB and SSB w18,19x. Methyl methanesulfonate ŽMMS. is a monofunctional alkylating agent which acts directly on oxygen and nitrogen atoms in DNA bases and on oxygen moieties of the phosphate backbone. Thereby a broad spectrum of DNA lesions is formed, consisting of alkylated bases, phophotriesters and abasic sites which can lead to SSB w20x. Effects in the comet assay were analyzed according to two different protocols. DNA damage was assayed directly after treatment as well as after a postincubation period in mutagen-free medium. The investigation of the induction and repair of various types of DNA lesions in repair-deficient cells should provide further information about the significance of different
DNA lesions for genotoxic effects in the comet assay and their relationship to mutagenicity and cytotoxicity.
2. Materials and methods 2.1. Cell cultures V79 Chinese hamster cells ŽV79-B and V79-LE. and the derived mutants XR-V15B and V-E5 were kindly provided by Dr. M. Zdzienicka, Leiden w6,14x. Cells were maintained in minimal essential medium ŽMEM. with Earle’s salts, supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics. Cells were cultivated in a humidified incubator at 378C with 5% CO 2 at pH 7.2 and harvested with 0.15% trypsin and 0.08% EDTA. Under these conditions, the population doubling time of the 4 cell lines was 12–15 h and the absolute plating efficiency was between 70 and 80%. 2.2. Chemicals MMS and fluorescein diacetate ŽFDA. were purchased from Sigma ŽMunich, Germany.. NCS was a gift from Kayaku Co., Ltd., Japan. NCS and MMS were dissolved immediately before use in Hank’s balanced salt solution. Agarose was supplied by Roth ŽKarlsruhe, Germany., low melting agarose ŽLMA, Sea plaque. was purchased from Biozym ŽHameln, Germany.. Cell culture media were obtained from Biochrom ŽBerlin, Germany.. 2.3. Fluorochrome-mediated Õiability assay To investigate whether DNA effects detected in the comet assay are accompanied by cytotoxic effects, cell viability was determined using the FDArethidium bromide assay according to Strauss w21x. A freshly prepared solution consisting of FDA Ž30 mgrml. and ethidium bromide Ž8 mgrml. was used. Twenty-five microliters of the cell suspension was mixed with 25 ml of the staining solution, spread on a microscope slide and covered with a coverslip. The viability was checked immediately after the treatment. Viable cells appeared green-fluorescent, whereas orange-stained nuclei indicated dead cells. At least 200 cells were counted per data point.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
2.4. Plating efficiency assay Relative plating efficiency as a measure for longterm survivability has been determined previously in the course of HPRT mutation tests w7,17x. Briefly, after mutagen treatment, 250 cells were plated into each of 4 replicate Petri dishes Ždiameter, 60 mm. and cultivated for 7 days. Colonies were fixed, stained, counted and related to the corresponding control. 2.5. Comet assay For the comet assay, 2 = 10 5 cells were seeded into multiwells Ž6 wells, of 36 mm diameter., cultivated overnight and then treated with MMS or NCS for 1 h. At the end of the treatment, cells were washed with ice-cold PBS and trypsinized with 50 ml trypsin Ž0.15%.. After 2 min, the cells were gently resuspended in 50 ml complete medium and 15 ml of the cell suspension were immediately used for the test. For postincubation experiments, cells were rinsed twice with Hank’s salt solution and further cultivated in complete medium for 1 h. The comet assay was performed as described by Singh et al. w22x with minor modifications w23x. Three hundred microliters of agarose Ž0.75%. diluted in Ca- and Mg-free PBS buffer were added to fully frosted microscope slides ŽLabcraft, Houston, TX, USA., immediately covered with coverslips and kept in a refrigerator to solidify. Coverslips were removed and 15 ml of the single-cell suspension mixed with 85 ml of LMA Ž0.5%. were added to the slides. The slides were covered again with a coverslip and placed in a refrigerator for another 5 min to allow solidification of the LMA and were then covered with a top layer of 100 ml LMA Ž0.5%.. Afterwards, slides were carefully immersed in lysing solution Ž100 mM Na-EDTA, 10 mM Tris, 2.5 M NaCl, 1% Triton X-100 and 10% DMSO, 1% Na-lauroyl-sarcosinate, pH 10.. The slides were kept at 48C for at least 1 h to lyse the cells. After lysis, the slides were placed in a horizontal gel electrophoresis chamber with alkaline buffer Ž1 mM Na-EDTA, 300 mM NaOH, pH 13.. The cells were exposed to alkali for 40 min to permit DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed for 20 min at 25 V Ž0.86 Vrcm. and 300 mA. All of these steps were conducted under dim light to prevent the occur-
281
rence of additional DNA damage. After electrophoresis, the slides were rinsed with neutralization buffer Ž0.4 M Tris, pH 7.5.. Finally, they were stained with 70 ml ethidium bromide Ž20 mgrml. and covered with a coverslip. Images of 50 randomly selected cells Ž25 cells from each of two replicate slides. were analyzed using a fluorescence microscope Ž200 = magnification. with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Measurements were made by image analysis ŽColourmorph Comet Assay 5.0, Perceptive Instruments, Haverhill, Suffolk, UK., determining the mean tail moment Žpercentage of DNA in the tail times tail length. of the 50 cells. All experiments were repeated twice in independent tests. Thus, the data in the results section represent the mean " SEM of 150 cells per dose level. Differences between the control and the other values were tested for significance Ž p - 0.01. using Student’s t-test. 3. Results Using the comet assay, the initial rate of DNA strand breaks induced by NCS and their persistence
Fig. 1. Effects of NCS on DNA migration Žtail moment. in XR-V15B and V79-B directly after treatment Žv, V79-B; ', XR-V15B. and after mutagen-free postincubation Ž`, V79-B; n, XR-V15B.. Mean"SEM of 150 cells.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
282
Table 1 Cytotoxicity induced by neocarzinostatin ŽNCS. and amount of persisting DNA damage in V79 cell lines Concentration
XR-V15B
Viability ŽFDA. 0.25 mgrml 97% 0.5 mgrml 96% Relative survival ŽPE. a 0.25 mgrml -1% 0.5 mgrml -1% Persisting DNA damage b 0.25 mgrml 68% 0.5 mgrml 70%
V79-B
V-E5
V79-LE
98% 97%
87% 89%
94% 94%
-10% -10%
-1% -1%
-10% -10%
33% 24%
37% 37%
51% 37%
a
Data taken from HPRT mutation experiments w7,17x. Relative tail moment value after 1 h of postincubation in mutagen-free medium. b
after 1 h of postincubation in mutagen-free medium was analyzed in XR-V15B cells and the parental cell line V79-B. The results are shown in Fig. 1. In both
cell lines, NCS caused a concentration-dependent increase of the tail moment. The initial DNA damage directly after treatment was significantly higher in XR-V15B than in the parental V79-B cell line. The results for the two cell lines also differed with respect to the persistence of NCS-induced DNA lesions. While in V79-B only 24–33% of the DNA lesions remained after an 1-h postincubation period, in XR-V15B about 70% of the strand breaks persisted ŽTable 1.. Fig. 2 illustrates the distribution of DNA damage among the cells in 4 experiments Ža–d.. It can be seen that after NCS treatment, the majority of cells exhibited increased DNA migration in both cell lines ŽFig. 2a,c.. Furthermore, after postincubation of XR-V15B cells, DNA damage is still equally distributed among the cells, indicating persistence of lesions in the whole cell population ŽFig. 2d.. Viability as measured with the fluorochrome-mediated viability assay ŽFDA. and the
Fig. 2. Effects of NCS on the distribution of DNA damage Žtail moment. among single cells in V79-B Ža,b. and XR-V15B Žc,d. directly after treatment Ža,c. and after mutagen-free postincubation Žb,d..
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
283
Table 2 Cytotoxicity induced by methyl methanesulfonate ŽMMS. and amount of persisting DNA damage in V79 cell lines Concentration
XR-V15B
Viability ŽFDA. 0.5=10 -4 M 98% 1=10 -4 M 98% Relative survival ŽPE. a 0.5=10 -4 M )90% 1=10 -4 M 89% Persisting DNA damage b 0.5=10 -4 M 33% 1=10 -4 M 30%
V79-B
V-E5
V79-LE
98% 99%
94% 97%
95% 91%
)85% 83%
)80% 76%
)85% 82%
27% 33%
36% 26%
30% 24%
a
Data taken from HPRT mutation experiments w7,17x. Relative tail moment value after 1 h of postincubation in mutagen-free medium.
b
Fig. 3. Effects of MMS on DNA migration Žtail moment. in XR-V15B and V79-B directly after treatment Žv, V79-B; ', XR-V15B. and after mutagen-free postincubation Ž`, V79-B; n, XR-V15B.. Mean"SEM of 150 cells.
relative survival Žplating efficiency, PE. are summarized in Table 1. To induce significant effects in the comet assay, NCS concentrations had to be applied which resulted in a relative plating efficiency of less than 10%. In contrast, the viability, even at the highest NCS concentration Ž5 mgrml., was above 95%. Viability did not decrease during postincubation Ždata not shown.. The comet assay revealed a clear and concentration-dependent increase in DNA damage in XR-V15B and V79-B at MMS concentrations which only caused little cytotoxicity as analyzed with both methods of cytotoxicity testing ŽFDA and PE.. The initial damage as well as the persistence of DNA effects were nearly the same in XR-V15B and V79-B ŽFig. 3.. Survival ŽPE. was above 83% and viability determined with the FDA assay was above 89% ŽTable 2.. In both cell lines, about 30% of the initial DNA damage persisted after 1 h postincubation ŽTable 2.. In the cell line V-E5 and its parental cell line V79-LE, NCS and MMS treatment led to a concentration-related increase in DNA migration ŽFigs. 4 and 5.. In both cell lines, about 30–40% of the induced strand breaks persisted after a 1-h postincu-
bation period. However, NCS induced DNA damage could be seen in the comet assay only at concentrations which resulted in a relative plating efficiency of less than 10%, whereas viability ŽFDA. was above 95% ŽTable 2.. Viability did not decrease during postincubation Ždata not shown.. In the case of MMS treatment, it was possible to detect DNA damage
Fig. 4. Effects of NCS on DNA migration Žtail moment. in V-E5 and V79-LE directly after treatment ŽB, V79-LE; l, V-E5. and after mutagen-free postincubation ŽI, V79-LE; e, V-E5.. Mean "SEM of 150 cells.
284
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
Fig. 5. Effects of MMS on DNA migration Žtail moment. in V-E5 and V79-LE directly after treatment ŽB, V79-LE; l, V-E5. and after mutagen-free postincubation ŽI, V79-LE; e, V-E5.. Mean "SEM of 150 cells.
with the comet assay at concentrations which only resulted in a weak cytotoxicity in the FDA assay as well as in a high PE ŽTable 2..
4. Discussion The comet assay is a genotoxicity test, which detects DNA strand breaks and alkali-labile sites in individual cells. It has already been used in both in vitro and in vivo studies to investigate DNA damage and repair induced by various agents in a variety of mammalian cells w3,4,23,24x. We now studied the induction and persistence of DNA damage by MMS and NCS in normal and repair deficient V79 Chinese hamster cells. With respect to MMS-induced effects, no fundamental difference was observed between all cell lines used. Induction of DNA effects was found in the same range of MMS concentrations. Genotoxic effects of MMS were detected with the comet assay at significantly lower concentrations compared to the induction of gene- and chromosome mutations in the same cell lines w7,17x. Persistence of DNA damage
after postincubation was similar in normal and mutant cell lines, indicating similar repair capacities for MMS-induced DNA lesions that cause DNA migration in the comet assay. NCS also induced DNA migration in the 4 cell lines. However, a significantly higher tail moment was found in XR-V15B than in the parental V79-B cells after treatment with NCS. Furthermore, persistence of NCS-induced DNA damage was much higher in XR-V15B. These results can be explained by the impaired DSB repair in this cell line. Reduced DSB repair capacity in XR-V15B already leads to an accumulation of DNA strand breaks during the mutagen treatment period. Consequently, higher tail moment values are observed in XR-V15B cells directly at the end of the treatment. During the postincubation period, less DSB are repaired, leading to enhanced lesion persistence compared with the repairproficient parental cells. It can be concluded from our data that XR-V15B cells only repaired about 30% of the induced DNA damage during the postincubation period, while in V79-B about 70% of the strand breaks were repaired. Deficient DSB rejoining has also been reported for the human B-lymphoblast line TK6 and this deficiency was confirmed using the neutral comet assay w25x. In TK6 cells, 38% of DSB rejoining was found 60 min after exposure to a high dose of ionizing radiation Ž50 Gy. in comparison to approximately 80% in repair-proficient cells. Recently, reduced DSB rejoining after ionizing radiation has also been shown with the neutral comet assay in the radiosensitive CHO mutant xrs-6, which belongs to the same complementation group as XRV15B w26x. Taken together, these results point to a significant contribution of DSB to the DNA effects seen in both versions of the comet assay under the experimental conditions used. AT-like V-E5 cells, which show hypersensitivity to radiation and NCS w7,14x, showed similar levels of strand break induction and rejoining as normal V79 cells in our experiments. These results indicate that the comet assay can only determine the amount of break induction and rejoining, but cannot measure the fidelity of repair of those breaks. Our findings with V-E5 cells are in accordance with earlier results for AT cells which, in spite of their significant radiosensitivity, showed unaltered levels of strand break induction and rejoining w27x.
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286
Our results also show that the comet assay seems to be rather insensitive for NCS-induced DNA lesions compared to other genotoxicity tests. NCS-induced gene mutations and chromosome aberrations at lower concentrations in the same cell lines w7,17x. A genotoxic effect of NCS in the comet assay was only found under extreme cytotoxic conditions. Long-term cell survival Žplating efficiency. was reduced to less than 10% in the parental cells and to nearly zero in the repair-deficient cells. Interestingly, cell viability at the end of the treatment as measured with the FDA assay was only slightly reduced. However, the viability assay only indicates the presence of dead cells with disintegrated membranes at the end of the treatment. Determination of survivability is more relevant with respect to the biological significance of test results since genotoxicity is more important if it occurs in cells capable of surviving the damage. The comparatively low sensitivity of the comet assay for the detection of NCS-induced damage might be explained by a high portion of DSB in the spectrum of induced DNA lesions. It has been estimated that overall about 20% of NCS-induced lesions are bistranded, a quarter of these being direct DSB w19,28x. DSB are the most important DNA lesion with respect to cytotoxicity w18,29x and it has been suggested that a single unrepaired DSB in the yeast genome can represent a lethal DNA lesion w30x. DSB are also directly involved in the formation of chromosome aberrations and deletion mutations w7,17,31x. Possibly, strand breaks induced at lower NCS-concentrations are rapidly repaired, but misrepair of some DSB can lead to chromosome aberrations and deletion mutations. Under these conditions, the amount of breaks obviously is not high enough to cause DNA migration in the comet assay. The present results as well as previously published data w25,26,32x indicate that the investigation of DSB-related effects with the comet assay requires extreme treatment conditions. Remarkably, the neutral version of the comet assay, which is supposed to trace predominantly DSB, was also shown to detect radiation-induced DNA damage in Chinese hamster cells at extremely high doses ranging from 10 to 100 Gy w26,32x. Such doses were shown to induce cytotoxicity resulting in survival rates of less than 2% at 10 Gy w6x. Taken together, the comet assay seems to be a
285
useful tool for studying the induction and persistence of DNA strand breaks. DSB obviously lead to DNA migration in the alkaline as well as in the neutral version of the comet assay. However, the comet assay does not seem to be a very sensitive genotoxicity test for the detection of genotoxic agents producing DSB as the predominant DNA lesion. Since DSB are important lesions with respect to cytotoxicity and mutagenicity, the importance of the amount of damage produced by a drug and detected by the comet assay has to be weighed in relation to the nature of the damage being detected.
Acknowledgements We wish to thank Dr. Margaret Zdzienicka for the V79 cell lines, and we are especially grateful to Dr. Ulla Plappert and Dr. Andreas Hartmann for their valuable contributions and helpful discussions. This study was financially supported by the Deutsche Forschungsgemeinschaft.
References w1x A.R. Collins, Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristics, Mutation Res. 239 Ž1993. 99–118. w2x M.Z. Zdzienicka, Mammalian mutants defective in the response to ionizing radiation-induced DNA damage, Mutation Res. 336 Ž1995. 203–213. w3x V.J. McKelvey-Martin, M.H.L. Green, P. Schmezer, B.L. Pool-Zobel, M.P. De Meo, A. Collins, The single cell gel electrophoresis assay Žcomet assay.: a European review, Mutation Res. 288 Ž1993. 7–63. w4x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutation Res. 339 Ž1995. 37–59. w5x P.A. Jeggo, Studies on mammalian mutants defective in rejoining double-strand breaks in DNA, Mutation Res. 239 Ž1990. 1–16. w6x M.Z. Zdzienicka, Q. Tran, G.P. van der Schans, J.W.I.M. Simons, Characterization of an X-ray-hypersensitive mutant of Chinese hamster cells, Mutation Res. 194 Ž1988. 239–249. w7x R. Helbig, M.Z. Zdzienicka, G. Speit, The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XRV15B, Radiation Res. 143 Ž1995. 151–157. w8x F. Pergola, M.Z. Zdzienicka, M.R. Lieber, VŽD.J recombina-
286
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
R. Helbig, G. Speit r Mutation Research 377 (1997) 279–286 tion in mammalian cell mutants defective in DNA double strand break repair, Mol. Cell. Biol. 13 Ž1993. 3464–3471. G.E. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P.A. Jeggo, F.W. Alt, Impairment of VŽD.J recombination in doublestrand break repair mutants, Science 260 Ž1993. 207–210. G.E. Taccioli, T.M. Gottlieb, T. Blunt, A. Priestley, J. Demengeot, R. Mizuta, A.R. Lehman, F.W. Alt, S. Jackson, P.A. Jeggo, Ku80: product of the XRCC5 gene and its role in DNA repair and VŽD.J recombination, Science 265 Ž1994. 1442–1445. V. Smider, W.K. Rathmell, M.R. Lieber, G. Chu, Restoration of X-ray resistance and VŽD.J recombination in mutant cells by Ku cDNA, Science 266 Ž1994. 288–291. M. Hafezparast, G.P. Kaur, M.Z. Zdzienicka, R.S. Athwal, A.R. Lehmann, P.A. Jeggo, Subchromosomal localization of a gene ŽXRCC5. involved in double strand break repair to the region 2q34-36, Somat. Cell. Mol. Genet. 19 Ž1993. 413–421. D.J. Chen, B.L. Marrone, T. Nguyen, M. Stackhouse, Y. Zhao, M.J. Siciliano, Regional assignment of a human DNA repair gene ŽXRCC5. to 2q35 by X-ray hybrid mapping, Genomics 21 Ž1994. 423–427. M.Z. Zdzienicka, N.G.J. Jaspers, G.P. van der Schans, A.T. Natarajan, J.W.I.M. Simons, Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair, Cancer Res. 49 Ž1989. 1481–1485. W. Jongmans, G.W.C.T. Verhaegh, K. Sankaranarayanan, P.H.M. Lohman, M.Z. Zdzienicka, Cellular characteristics of Chinese hamster cell mutants resembling ataxia-telangiectasia cells, Mutation Res. 294 Ž1993. 207–214. Zdzienicka, M.Z., J.W.I.M. Simons, P.H.M. Lohman, Chinese hamster cell lines defective in DNA repair, in: M.W. Lambert and J. Laval ŽEds.., DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells, Plenum, New York, 1985, pp. 535–545. R. Helbig, E. Gerland, M.Z. Zdzienicka, G. Speit, The pattern of mutations induced by neocarzinostatin and methyl methanesulfonate in the ataxia telangiectasia-like Chinese hamster cell line V-E5, Mutation Res. 336 Ž1995. 307–316. I.H. Goldberg, Mechanism of neocarzinostatin action: role of DNA microstructure in determination of chemistry of bistranded oxidative damage, Acc. Chem. Res. 24 Ž1991. 191– 198. L.F. Povirk, DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes, Mutation Res. 355 Ž1996. 71–89. R. Mirzayans, M. Liuzzi, M.C. Paterson, Methylmethanesulfonate-induced DNA damage and its repair in cultured
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x w32x
human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum Žgroup A. cells, Carcinogenesis 9 Ž1988. 2257–2263. G.H.S. Strauss, Non-random cell killing in cryopreservation: implications for performance of the battery of leukocyte tests ŽBLT. I. Toxic and immunotoxic effects, Mutation Res. 252 Ž1991. 1–15. N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantification of low levels of DNA damage in individual cells, Exp. Cell Res. 175 Ž1988. 184– 191. G. Speit, A. Hartmann, The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test Žcomet assay., Mutagenesis 10 Ž1995. 555–559. A. Hartmann, G. Speit, Genotoxic effects of chemicals in the single cell gel ŽSCG. test with human blood cells in relation to the induction of sister-chromatid exchanges ŽSCE., Mutation Res. 346 Ž1995. 49–56. H.H. Evans, M. Ricanati, M.-F. Horng, Q. Jiang, J. Mencl, P. Olive, DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines, Radiation Res. 134 Ž1993. 307–315. G.M. Ross, J.J. Eady, N.P. Mithal, C. Bush, G.G. Steel, P.A. Jeggo, T.J. McMillan, DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene, Cancer Res. 55 Ž1995. 1235–1238. A.J. Ward, P.L. Olive, A.H. Burr, M.P. Rosin, Response of fibroblast cultures from ataxia-telangiectasia patients to reactive oxygen species generated during inflammatory reactions, Environ. Mol. Mutagen. 24 Ž1994. 103–111. L.F. Povirk, C.W. Houlgrave, Effect of apurinicrapyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands, Biochemistry 27 Ž1988. 3850–3857. A. Giaccia, R. Weinstein, J. Hu, T.D. Stamato, Cell cycle-dependent repair of double-strand DNA breaks in a g-ray-sensitive Chinese hamster cell, Somat. Cell. Mol. Genet. 11 Ž1985. 485–491. D. Frankenberg, M. Frankenberg-Schwager, D. Bloecher, R. Harbich, Evidence for DNA double-strand breaks as the critical lesions in yeast cells irradiated with sparsely or densely ionising radiation under oxic or anoxic conditions, Radiation Res. 88 Ž1981. 524–532. J.W. Phillips, W.F. Morgan, DNA double-strand breaks in mutagenesis, Environ. Mol. Mutagen. 22 Ž1993. 214–217. P.L. Olive, D. Wlodek, R.E. Durand, J.P. Banath, Factors ` influencing DNA migration from individual cells subjected to gel electrophoresis, Exp. Cell. Res. 198 Ž1992. 259–267.